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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 29 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 29 year tenure till date Aug 2016, Around 30 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 25 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 13 lakh plus views on New Drug Approvals Blog in 212 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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FDA approves new combination treatment for acute myeloid leukemia, Rydapt (midostaurin)


MIDOSTAURIN

04/28/2017
The U.S. Food and Drug Administration today approved Rydapt (midostaurin) for the treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) who have a specific genetic mutation called FLT3, in combination with chemotherapy. The drug is approved for use with a companion diagnostic, the LeukoStrat CDx FLT3 Mutation Assay, which is used to detect the FLT3 mutation in patients with AML.

April 28, 2017

Release

The U.S. Food and Drug Administration today approved Rydapt (midostaurin) for the treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) who have a specific genetic mutation called FLT3, in combination with chemotherapy. The drug is approved for use with a companion diagnostic, the LeukoStrat CDx FLT3 Mutation Assay, which is used to detect the FLT3 mutation in patients with AML.

AML is a rapidly progressing cancer that forms in the bone marrow and results in an increased number of white blood cells in the bloodstream. The National Cancer Institute estimated that approximately 19,930 people would be diagnosed with AML in 2016 and 10,430 were projected to die of the disease.

“Rydapt is the first targeted therapy to treat patients with AML, in combination with chemotherapy,” said Richard Pazdur, M.D., acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research and director of the FDA’s Oncology Center of Excellence. “The ability to detect the gene mutation with a diagnostic test means doctors can identify specific patients who may benefit from this treatment.”

Rydapt is a kinase inhibitor that works by blocking several enzymes that promote cell growth. If the FLT3 mutation is detected in blood or bone marrow samples using the LeukoStrat CDx FLT3 Mutation Assay, the patient may be eligible for treatment with Rydapt in combination with chemotherapy.

The safety and efficacy of Rydapt for patients with AML were studied in a randomized trial of 717 patients who had not been treated previously for AML. In the trial, patients who received Rydapt in combination with chemotherapy lived longer than patients who received chemotherapy alone, although a specific median survival rate could not be reliably estimated. In addition, patients who received Rydapt in combination with chemotherapy in the trial went longer (median 8.2 months) without certain complications (failure to achieve complete remission within 60 days of starting treatment, progression of leukemia or death) than patients who received chemotherapy alone (median three months).

Common side effects of Rydapt in patients with AML include low levels of white blood cells with fever (febrile neutropenia), nausea, inflammation of the mucous membranes (mucositis), vomiting, headache, spots on the skin due to bleeding (petechiae), musculoskeletal pain, nosebleeds (epistaxis), device-related infection, high blood sugar (hyperglycemia) and upper respiratory tract infection. Rydapt should not be used in patients with hypersensitivity to midostaurin or other ingredients in Rydapt. Women who are pregnant or breastfeeding should not take Rydapt because it may cause harm to a developing fetus or a newborn baby. Patients who experience signs or symptoms of lung damage (pulmonary toxicity) should stop using Rydapt.

Rydapt was also approved today for adults with certain types of rare blood disorders (aggressive systemic mastocytosis, systemic mastocytosis with associated hematological neoplasm or mast cell leukemia). Common side effects of Rydapt in these patients include nausea, vomiting, diarrhea, swelling (edema), musculoskeletal pain, abdominal pain, fatigue, upper respiratory tract infection, constipation, fever, headache and shortness of breath.

The FDA granted this application Priority Review, Fast Track (for the mastocytosis indication) and Breakthrough Therapy (for the AML indication) designations.

The FDA granted the approval of Rydapt to Novartis Pharmaceuticals Corporation. The FDA granted the approval of the LeukoStrat CDx FLT3 Mutation Assay to Invivoscribe Technologies Inc.

MIDOSTAURIN

(9S,10R,11R,13R)-2,3,10,11,12,13-Hexahydro-10-methoxy-9-methyl-11-(methylamino)-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3′,2′,1′-lm]pyrrolo[3,4-j][1,7]benzodiamzonine-1-one

N-[(9S,10R,11R,13R)-2,3,10,11,12,13-Hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3′,2′,1′-lm]pyrrolo[3,4-j][1,7]benzodiazonin-11-yl]-N-methylbenzamide

N-((9S,10R,11R,13R)-2,3,9,10,11,12-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9H-diindolo(1,2,3-gh:3′,2′,1′-lm)pyrrolo(3,4-j)(1,7)benzodiazonin-11-yl)-N-methyl-,

N-[(2R,4R,5R,6S)-5-methoxy-6-methyl-18-oxo-29-oxa-1,7,17-triazaoctacyclo[12.12.2.12,6.07,28.08,13.015,19.020,27.021,26]nonacosa-8,10,12,14(28),15(19),20(27),21(26),22,24-nonaen-4-yl]-N-methylbenzamide hydrate

N-benzoyl staurosporine

NOVARTIS ONCOLOGY ORIGINATOR

Chemical Formula: C35H30N4O4

Exact Mass: 570.22671

Molecular Weight: 570.63710

Elemental Analysis: C, 73.67; H, 5.30; N, 9.82; O, 11.22

Tyrosine kinase inhibitors

PKC 412。PKC412A。CGP 41251。Benzoylstaurosporine;4′-N-Benzoylstaurosporine;Cgp 41251;Cgp 41 251.

120685-11-2 CAS

PHASE 3

  • 4′-N-Benzoylstaurosporine
  • Benzoylstaurosporine
  • Cgp 41 251
  • CGP 41251
  • CGP-41251
  • Midostaurin
  • PKC 412
  • PKC412
  • UNII-ID912S5VON

Midostaurin is an inhibitor of tyrosine kinase, protein kinase C, and VEGF. Midostaurin inhibits cell growth and phosphorylation of FLT3, STAT5, and ERK. It is a potent inhibitor of a spectrum of FLT3 activation loop mutations.

it  is prepared by acylation of the alkaloid staurosporine (I) with benzoyl chloride (II) in the presence of diisopropylethylamine in chloroform.Production Route of Midostaurin

Midostaurin is a synthetic indolocarbazole multikinase inhibitor with potential antiangiogenic and antineoplastic activities. Midostaurin inhibits protein kinase C alpha (PKCalpha), vascular endothelial growth factor receptor 2 (VEGFR2), c-kit, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) tyrosine kinases, which may result in disruption of the cell cycle, inhibition of proliferation, apoptosis, and inhibition of angiogenesis in susceptible tumors.

MIDOSTAURIN

Derivative of staurosporin, orally active, potent inhibitor of FLT3 tyrosine kinase (fetal liver tyrosine kinase 3). In addition Midostaurin inhibits further molecular targets such as VEGFR-1 (Vascular Endothelial Growth Factor Receptor 1), c-kit (stem cell factor receptor), H-and K-RAS (Rat Sarcoma Viral homologue) and MDR (multidrug resistance protein).

Midostaurin inhibits both wild-type FLT3 and FLT3 mutant, wherein the internal tandem duplication mutations (FLT3-ITD), and the point mutation to be inhibited in the tyrosine kinase domain of the molecule at positions 835 and 836.Midostaurin is tested in patients with AML.

Midostaurin, a protein kinase C (PKC) and Flt3 (FLK2/STK1) inhibitor, is in phase III clinical development at originator Novartis for the oral treatment of acute myeloid leukemia (AML).

Novartis is conducting phase III clinical trials for the treatment of aggressive systemic mastocytosis or mast cell leukemia. The National Cancer Institute (NCI) is conducting phase I/II trials with the drug for the treatment of chronic myelomonocytic leukemia (CMML) and myelodysplastic syndrome (MDS).

Massachusetts General Hospital is conducting phase I clinical trials for the treatment of adenocarcinoma of the rectum in combination with radiation and standard chemotherapy.

MIDOSTAURIN

Midostaurin (PKC412) is a multi-target protein kinase inhibitor being investigated for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). It is a semi-synthetic derivative of staurosporine, an alkaloid from the bacterium Streptomyces staurosporeus, and is active in patients with mutations of CD135 (FMS-like tyrosine kinase 3 receptor).[1]

After successful Phase II clinical trials, a Phase III trial for AML has started in 2008. It is testing midostaurin in combination with daunorubicin and cytarabine.[2] In another trial, the substance has proven ineffective in metastatic melanoma.[3]

Midostaurin has also been studied at Johns Hopkins University for the treatment of age-related macular degeneration (AMD), but no recent progress reports for this indication have been made available. Trials in macular edema of diabetic origin were discontinued at Novartis.

In 2004, orphan drug designation was received in the E.U. for the treatment of AML. In 2009 and 2010, orphan drug designation was assigned for the treatment of acute myeloid leukemia and for the treatment of mastocytosis, respectively, in the U.S. In 2010, orphan drug designation was assigned in the E.U. for the latter indication.

MIDOSTAURIN

References

  1.  Fischer, T.; Stone, R. M.; Deangelo, D. J.; Galinsky, I.; Estey, E.; Lanza, C.; Fox, E.; Ehninger, G.; Feldman, E. J.; Schiller, G. J.; Klimek, V. M.; Nimer, S. D.; Gilliland, D. G.; Dutreix, C.; Huntsman-Labed, A.; Virkus, J.; Giles, F. J. (2010). “Phase IIB Trial of Oral Midostaurin (PKC412), the FMS-Like Tyrosine Kinase 3 Receptor (FLT3) and Multi-Targeted Kinase Inhibitor, in Patients with Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome with Either Wild-Type or Mutated FLT3”. Journal of Clinical Oncology 28 (28): 4339–4345. doi:10.1200/JCO.2010.28.9678PMID 20733134edit
  2.  ClinicalTrials.gov NCT00651261 Daunorubicin, Cytarabine, and Midostaurin in Treating Patients With Newly Diagnosed Acute Myeloid Leukemia
  3.  Millward, M. J.; House, C.; Bowtell, D.; Webster, L.; Olver, I. N.; Gore, M.; Copeman, M.; Lynch, K.; Yap, A.; Wang, Y.; Cohen, P. S.; Zalcberg, J. (2006). “The multikinase inhibitor midostaurin (PKC412A) lacks activity in metastatic melanoma: a phase IIA clinical and biologic study”British Journal of Cancer 95 (7): 829–834. doi:10.1038/sj.bjc.6603331PMC 2360547PMID 16969355.
    1. Midostaurin product page, Fermentek
    2.  Wang, Y; Yin, OQ; Graf, P; Kisicki, JC; Schran, H (2008). “Dose- and Time-Dependent Pharmacokinetics of Midostaurin in Patients With Diabetes Mellitus”. J Clin Pharmacol 48 (6): 763–775. doi:10.1177/0091270008318006PMID 18508951.
    3.  Ryan KS (2008). “Structural studies of rebeccamycin, staurosporine, and violacein biosynthetic enzymes”Ph.D. Thesis. Massachusetts Institute of Technology.

Bioorg Med Chem Lett 1994, 4(3): 399

US 5093330

EP 0657164

EP 0711556

EP 0733358

WO 1998007415

WO 2002076432

WO 2003024420

WO 2003037347

WO 2004112794

WO 2005027910

WO 2005040415

WO 2006024494

WO 2006048296

WO 2006061199

WO 2007017497

WO 2013086133

WO 2012016050

WO 2011000811

8-1-2013
Identification of potent Yes1 kinase inhibitors using a library screening approach.
Bioorganic & medicinal chemistry letters
 
3-1-2013
Evaluation of potential Myt1 kinase inhibitors by TR-FRET based binding assay.
European journal of medicinal chemistry
2-23-2012
Testing the promiscuity of commercial kinase inhibitors against the AGC kinase group using a split-luciferase screen.
Journal of medicinal chemistry
 
1-26-2012
VX-322: a novel dual receptor tyrosine kinase inhibitor for the treatment of acute myelogenous leukemia.
Journal of medicinal chemistry
1-1-2012
H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling.
PloS one
10-27-2011
Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase.
Journal of medicinal chemistry
 
6-1-2011
Discovery, synthesis, and investigation of the antitumor activity of novel piperazinylpyrimidine derivatives.
European journal of medicinal chemistry
3-1-2010
Colony stimulating factor-1 receptor as a target for small molecule inhibitors.
Bioorganic & medicinal chemistry
7-18-2012
Staurosporine Derivatives as Inhibitors of FLT3 Receptor Tyrosine Kinase Activity
6-13-2012
Crystal form of N-benzoyl-staurosporine
12-14-2011
COMPOSITIONS FOR TREATMENT OF SYSTEMIC MASTOCYTOSIS
7-6-2011
Staurosporine derivatives as inhibitors of flt3 receptor tyrosine kinase activity
7-6-2011
Staurosporine Derivatives for Use in Alveolar Rhabdomyosarcoma
12-10-2010
Pharmaceutical Compositions for treating wouds and related methods
11-5-2010
COMBINATIONS OF JAK INHIBITORS
7-23-2010
COMBINATIONS COMPRISING STAUROSPORINES
3-5-2010
COMBINATION OF IAP INHIBITORS AND FLT3 INHIBITORS
1-29-2010
ANTI-CANCER PHOSPHONATE ANALOGS
1-13-2010
Therapeutic phosphonate compounds
11-20-2009
Use of Staurosporine Derivatives for the Treatment of Multiple Myeloma
7-17-2009
KINASE INHIBITORY PHOSPHONATE ANALOGS
6-19-2009
Organic Compounds
3-20-2009
Use of Midostaurin for Treating Gastrointestinal Stromal Tumors
11-21-2008
PHARMACEUTICAL COMPOSITIONS COMPRISING A POORLY WATER-SOLUBLE ACTIVE INGREDIENT, A SURFACTANT AND A WATER-SOLUBLE POLYMER
11-19-2008
Anti-cancer phosphonate analogs
9-12-2008
Multi-Functional Small Molecules as Anti-Proliferative Agents
9-5-2008
Sensitization of Drug-Resistant Lung Caners to Protein Kinase Inhibitors
8-29-2008
Organic Compounds
8-27-2008
Kinase inhibitory phosphonate analogs
4-25-2008
Treatment Of Gastrointestinal Stromal Tumors With Imatinib And Midostaurin
12-28-2007
Pharmaceutical Uses of Staurosporine Derivatives
12-7-2007
Kinase Inhibitor Phosphonate Conjugates
8-17-2007
Combinations comprising staurosporines
10-13-2006
Staurosporine derivatives for hypereosinophilic syndrome
7-15-2005
Phosphonate substituted kinase inhibitors
10-20-2004
Staurosporin derivatives

MIDOSTAURIN HYDRATE

Midostaurin according to the invention is N-[(9S,10R,11R,13R)-2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3′,2′,1′-lm]pyrrolo[3,4-j][1,7]benzodiazonin-11-yl]-N-methylbenzamide of the formula (II):

Figure US20090075972A1-20090319-C00002

or a salt thereof, hereinafter: “Compound of formula II or midostaurin”.

Compound of formula II or midostaurin [International Nonproprietary Name] is also known as PKC412.

Midostaurin is a derivative of the naturally occurring alkaloid staurosporine, and has been specifically described in the European patent No. 0 296 110 published on Dec. 21, 1988, as well as in U.S. Pat. No.  5093330 published on Mar. 3, 1992, and Japanese Patent No. 2 708 047.

………………….

https://www.google.co.in/patents/EP0296110B1

The nomenclature of the products is, on the complete structure of staurosporine ([storage]-NH-CH ₃derived, and which is designated by N-substituent on the nitrogen of the methylamino group

Figure imgb0028

Example 18:

     N-Benzoyl-staurospor

  • A solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (0.38 mmol) of N, N-diisopropylethylamine in 2 ml of chloroform is added at room temperature with 0.035 ml (0.3 mmol) of benzoyl chloride and 10 stirred minutes.The reaction mixture is diluted with chloroform, washed with sodium bicarbonate, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride / ethanol 30:1), mp 235-247 ° with brown coloration.
  • cut paste may not be ok below

Staurosporine the formula [storage]-NH-CH ₃ (II) (for the meaning of the rest of [storage] see above) as the basic material of the novel compounds was already in 1977, from the cultures of Streptomyces staurosporeus AWAYA, and TAKAHASHI

O ¯

Figure imgb0003

MURA, sp. nov. AM 2282, see Omura, S., Iwai, Y., Hirano, A., Nakagawa, A.; awayâ, J., Tsuchiya, H., Takahashi, Y., and Masuma, R. J. Antibiot. 30, 275-281 (1977) isolated and tested for antimicrobial activity. It was also found here that the compound against yeast-like fungi and microorganisms is effective (MIC of about 3-25 mcg / ml), taking as the hydrochloride = having a LD ₅ ₀ 6.6 mg / kg (mouse, intraperitoneal). Stagnated recently it has been shown in extensive screening, see Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y, Morimoto, M. and Tomita, F.: Biochem. and Biophys. Research Commun. 135 (No. 2), 397-402 (1986) that the compound exerts a potent inhibitory effect on protein kinase C (rat brain)

…………………

https://www.google.co.in/patents/US5093330

EXAMPLE 18 N-benzoyl-staurosporine

0.035 ml (0.3 mmol) of benzoyl chloride is added at room temperature to a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (0.38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform and the whole is stirred for 10 minutes. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulphate and concentrated by evaporation. The crude product is chromatographed on silica gel (eluant:methylene chloride/ethanol 30:1); m.p. 235

…………………….

Bioorg Med Chem Lett 1994, 4(3): 399

http://www.sciencedirect.com/science/article/pii/0960894X94800049

Full-size image (2 K)

……………………

http://www.google.com/patents/WO1998007415A2

A variety of PKC inhibitors are available in the art for use in the invention. These include bryostatin (U.S. Patent 4,560,774), safinogel (WO 9617603), fasudil (EP 187371), 7- hydoxystaurosporin (EP 137632B), various diones described in EP 657458, EP 657411 and WO9535294, phenylmethyl hexanamides as described in WO9517888, various indane containing benzamides as described in WO9530640, various pyrrolo [3,4-c]carbazoles as described in EP 695755, LY 333531 (IMSworld R & D Focus 960722, July 22, 1996 and Pharmaprojects Accession No. 24174), SPC-104065 (Pharmaprojects Accession No. 22568), P-10050 (Pharmaprojects Accession No. 22643), No. 4432 (Pharmaprojects Accession No. 23031), No. 4503 (Pharmaprojects Accession No. 23252), No. 4721 (Pharmaprojects Accession No. 23890), No. 4755 (Pharmaprojects Accession No. 24035), balanol (Pharmaprojects Accession No. 20376), K-7259 (Pharmaprojects Accession No. 16649), Protein kinase C inhib, Lilly (Pharmaprojects Accession No. 18006), and UCN-01 (Pharmaprojects Accession No. 11915). Also see, for example, Tamaoki and Nakano (1990) Biotechnology 8:732-735; Posada et al. (1989) Cancer Commun. 1:285-292; Sato et al. (1990) Biochem Biophys. Res. Commun. 173:1252-1257; Utz et al. (1994) Int. J. Cancer 57:104-110; Schwartz et al. (1993) J. Na . Cancer lnst. 85:402-407; Meyer et al. (1989) Int. J. Cancer 43:851-856; Akinaga et al. (1991) Cancer Res. 51:4888-4892, which disclosures are herein incorporated by reference. Additionally, antisense molecules can be used as PKC inhibitors. Although such antisense molecules inhibit mRNA translation into the PKC protein, such antisense molecules are considered PKC inhibitors for purposes of this invention. Such antisense molecules against PKC inhibitors include those described in published PCT patent applications WO 93/19203, WO 95/03833 and WO 95/02069, herein incorporated by reference. Such inhibitors can be used in formulations for local delivery to prevent cellular proliferation. Such inhibitors find particular use in local delivery for preventing rumor growth and restenosis.

N-benzoyl staurosporine is a benzoyl derivative of the naturally occurring alkaloid staurosporine. It is chiral compound ([a]D=+148.0+-2.0°) with the formula C35H30R1O4 (molecular weight 570.65). It is a pale yellow amorphous powder which remains unchanged up to 220°C. The compound is very lipophilic (log P>5.48) and almost insoluble in water (0.068 mg/1) but dissolves readily in DMSO.

……………………….

staurosporine

Staurosporine (antibiotic AM-2282 or STS) is a natural product originally isolated in 1977 from the bacterium Streptomyces staurosporeus. It was the first of over 50 alkaloids to be isolated with this type of bis-indole chemical structure. The chemical structure of staurosporine was elucidated by X-ray analysis of a single crystal and the absolute stereochemical configuration by the same method in 1994.

Staurosporine was discovered to have biological activities ranging from anti-fungal to anti-hypertensive. The interest in these activities resulted in a large investigative effort in chemistry and biology and the discovery of the potential for anti-cancer treatment

Synthesis of Staurosporine

Staurosporine is the precursor of the novel protein kinase inhibitor midostaurin(PKC412). Besides midostaurin, staurosporine is also used as a starting material in the commercial synthesis of K252c (also called staurosporine aglycone). In the natural biosynthetic pathway, K252c is a precursor of staurosporine.

Indolocarbazoles belong to the alkaloid sub-class of bisindoles. Of these carbazoles the Indolo(2,3-a)carbazoles are the most frequently isolated; the most common subgroup of the Indolo(2,3-a)carbazoles are the Indolo(2,3-a)pyrrole(3,4-c)carbazoles which can be divided into two major classes – halogenated (chlorinated) with a fully oxidized C-7 carbon with only one indole nitrogen containing a β-glycosidic bond and the second class consists of both indole nitrogen glycosilated, non-halogenated, and a fully reduced C-7 carbon. Staurosporine is part of the second non-halogenated class.

The biosynthesis of staurosporine starts with the amino acid L-tryptophan in its zwitterionic form. Tryptophan is converted to an imineby enzyme StaO which is an L-amino acid oxidase (that may be FAD dependent). The imine is acted upon by StaD to form an uncharacterized intermediate proposed to be the dimerization product between 2 imine molecules. Chromopyrrolic acid is the molecule formed from this intermediate after the loss of VioE (used in the biosynthesis of violacein – a natural product formed from a branch point in this pathway that also diverges to form rebeccamycin. An aryl aryl coupling thought to be catalyzed by a cytochrome P450enzyme to form an aromatic ring system occurs

Staurosporine 2

This is followed by a nucleophilic attack between the indole nitrogens resulting in cyclization and then decarboxylation assisted by StaC exclusively forming staurosporine aglycone or K252c. Glucose is transformed to NTP-L-ristoamine by StaA/B/E/J/I/K which is then added on to the staurosporine aglycone at 1 indole N by StaG. The StaN enzyme reorients the sugar by attaching it to the 2nd indole nitrogen into an unfavored conformation to form intermediated O-demethyl-N-demethyl-staurosporine. Lastly, O-methylation of the 4’amine by StaMA and N-methylation of the 3′-hydroxy by StaMB leads to the formation of staurosporine

US4107297 * 28 Nov 1977 15 Aug 1978 The Kitasato Institute Antibiotic compound
US4735939 * 27 Feb 1987 5 Apr 1988 The Dow Chemical Company Insecticidal activity of staurosporine
ZA884238A * Title not available
////////FDA 2017, acute myeloid leukemia, Rydapt, midostaurin, Novartis Pharmaceuticals Corporation, LeukoStrat CDx FLT3 Mutation Assay,  Invivoscribe Technologies Inc, Priority Review, Fast Track, (for the mastocytosis indication, Breakthrough Therapy
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FDA approves first drug Ingrezza (valbenazine) to treat tardive dyskinesia


Valbenazine.svg

Valbenazine

  • Molecular FormulaC24H38N2O4
  • Average mass418.569 Da
(2R,3R,11bR)-3-Isobutyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl L-valinate
(2R,3R,11bR)-9,10-dimethoxy-3-(2-methylpropyl)-1,3,4,6,7,11b-hexahydro-2H-benzo[a]quinolizin-2-yl L-valinate
1025504-45-3 cas
L-Valine, (2R,3R,11bR)-1,3,4,6,7,11b-hexahydro-9,10-dimethoxy-3-(2-methylpropyl)-2H-benzo[a]quinolizin-2-yl ester
NBI-98854
Image result for valbenazine
Valbenazine ditosylate. RN: 1639208-54-0. UNII: 5SML1T733B, Molecular Formula, C24-H38-N2-O4.2C7-H8-O3-S, Molecular Weight, 762.9806

(2R,3R,11bR)-9,10-Dimethoxy-3-(2-methylpropyl)-1,3,4,6,7,11b-hexahydro-2H-benzo(a)quinolizin-2-yl L-valinate bis(4-methylbenzenesulfonate)

and

Valbenazine dihydrochloride
1639208-51-7

04/11/2017
The U.S. Food and Drug Administration today approved Ingrezza (valbenazine) capsules to treat adults with tardive dyskinesia. This is the first drug approved by the FDA for this condition.

April 11, 2017

Release

The U.S. Food and Drug Administration today approved Ingrezza (valbenazine) capsules to treat adults with tardive dyskinesia. This is the first drug approved by the FDA for this condition.

Tardive dyskinesia is a neurological disorder characterized by repetitive involuntary movements, usually of the jaw, lips and tongue, such as grimacing, sticking out the tongue and smacking the lips. Some affected people also experience involuntary movement of the extremities or difficulty breathing.

“Tardive dyskinesia can be disabling and can further stigmatize patients with mental illness,” said Mitchell Mathis, M.D., director of the Division of Psychiatry Products in the FDA’s Center for Drug Evaluation and Research. “Approving the first drug for the treatment of tardive dyskinesia is an important advance for patients suffering with this condition.”

Tardive dyskinesia is a serious side effect sometimes seen in patients who have been treated with antipsychotic medications, especially the older medications, for long periods to treat chronic conditions, such as schizophrenia and bipolar disorder. Tardive dyskinesia can also occur in patients taking antipsychotic medications for depression and certain medications for gastrointestinal disorders and other conditions. It is unclear why some people who take these medications develop tardive dyskinesia yet others do not.

The efficacy of Ingrezza was shown in a clinical trial of 234 participants that compared Ingrezza to placebo. After six weeks, participants who received Ingrezza had improvement in the severity of abnormal involuntary movements compared to those who received placebo.

Ingrezza may cause serious side effects including sleepiness and heart rhythm problems (QT prolongation). Its use should be avoided in patients with congenital long QT syndrome or with abnormal heartbeats associated with a prolonged QT interval. Those taking Ingrezza should not drive or operate heavy machinery or do other dangerous activities until it is known how the drug affects them.

The FDA granted this application Fast Track, Priority Review and Breakthrough Therapy designations.

The FDA granted approval of Ingrezza to Neurocrine Biosciences, Inc.

Valbenazine (INN,[1]:114 proposed trade name Ingrezza) is the first drug approved by the FDA[2] for use in the treatment of tardive dyskinesia.[3][4] Clinical trials are underway to evaluate its efficacy in the treatment of Tourette’s syndrome.[5][6] It acts as a vesicular monoamine transporter 2 (VMAT2) inhibitor.[7]

Pharmacology

Mechanism of action

Valbenazine is known to cause reversible reduction of dopamine release by selectively inhibiting pre-synaptic human vesicular monoamine transporter type 2 (VMAT2). In vitro, valbenazine shows great selectivity for VMAT2 and little to no affinity for VMAT1 or other monoamine receptors.[8] Although the exact cause of tardive dyskinsia is unknown, it is hypothesized that it may result from neuroleptic-induced dopamine hypersensitivity.[9] By selectively reducing the ability of VMAT2 to load dopamine into synaptic vesicles,[10] the drug reduces overall levels of available dopamine in the synaptic cleft, ideally alleviating the symptoms associated with dopamine hypersensitivity. The importance of valbenazine selectivity inhibiting VMAT2 over other monoamine transporters is that VMAT2 is mainly involved with the transport of dopamine, and to a much lesser extent other monoamines such as norepinephrine, serotonin, and histamine. This selectivity is likely to reduce the likelihood of “off-target” adverse effects which may result from the upstream inhibition of these other monoamines.[11]

Society and culture

Commercial aspects

Valbenazine is produced by Neurocrine Biosciences, a company based in San Diego. In addition to the late-stage clinical trials studying valbenazine, Neurocrine Biosciences (partnered with AbbVie Inc.) also has another product, elagolix (a hormone antagonist), undergoing clinical trials.[12] Following the initiation of these trials, on 5 May 2016 Neurocrine reported revenues of $15 million for the first quarter of 2016.[13] The company now focuses on filing the valbenazine new drug application as they prepare for the commercial launch of the drug for the treatment of tardive dyskinesia.Neurocrine’s expenses have risen steadily since May 2015, primarily due to the pre-commercialization activities for valbenazine. [14]

Intellectual property

While Neurocrine Biosciences does not currently hold a final patent for valbenazine or elagolix, they do hold a patent for the VMAT2 inhibitor [9,10-dimethoxy-3-(2-methylpropyl)-1H,2H,3H,4H,6H,7H,11bH-pyrido-[2,1-a]isoquinolin-2-yl]methanol and related compounds, which includes valbenazine.[15]

ChemSpider 2D Image | Valbenazine | C24H38N2O4

References

  1.  “International Nonproprietary Names for Pharmaceutical Substances (INN). Recommended International Nonproprietary Names: List 71” (PDF). World Health Organization. Retrieved 18 November 2016.
  2.  Newswire, MultiVu – PR. “Neurocrine Announces FDA Approval of INGREZZA TM (valbenazine) Capsules as the First and Only Approved Treatment for Adults with Tardive Dyskinesia (TD)”. Multivu. Retrieved 2017-04-11.
  3.  Ben Adams (Aug 30, 2016). “Neurocrine submits valbenazine NDA early, set for 2017 approval”. fiercebiotech.com.
  4.  “Safety and Tolerability Study of NBI-98854 for the Treatment of Tardive Dyskinesia – Full Text View – ClinicalTrials.gov”. clinicaltrials.gov. Retrieved 2016-11-13.
  5. Jump up^ “Tourette Syndrome Clinical Trials | Neurocrine Biosciences”. http://www.neurocrine.com. Retrieved 2016-11-13.
  6. Jump up^ “Safety and Efficacy Study of NBI-98854 in Adults With Tourette Syndrome – Full Text View – ClinicalTrials.gov”. clinicaltrials.gov. Retrieved 2016-11-13.
  7. Jump up^ O’Brien, C. F.; Jimenez, R; Hauser, R. A.; Factor, S. A.; Burke, J; Mandri, D; Castro-Gayol, J. C. (2015). “NBI-98854, a selective monoamine transport inhibitor for the treatment of tardive dyskinesia: A randomized, double-blind, placebo-controlled study”. Movement Disorders. 30 (12): 1681–7. doi:10.1002/mds.26330. PMC 5049616Freely accessible. PMID 26346941.
  8. Jump up^ “NBI-98854 – VMAT2 Inhibitor | Tics in Children Treatment | Neurocrine Biosciences”. http://www.neurocrine.com. Retrieved 2016-11-13.
  9. Jump up^ “tardive-dyskinesia”. http://www.priory.com. Retrieved 2016-11-13.
  10. Jump up^ Purves, Dale, et al. Neuroscience. Sinauer Associates. 087893646
  11.  “NBIX: NDA for Valbenazine in Tardive Dyskinesia to be Filed in 2016…”. Retrieved 2016-11-13.
  12.  “Endocrine & Movement Disorder R&D | About | Neurocrine Biosciences”. http://www.neurocrine.com. Retrieved 2016-11-14.
  13.  “NBIX: NDA for Valbenazine in Tardive Dyskinesia to be Filed in 2016…”. Retrieved 2016-11-20.
  14.  “Press Release | Neurocrine Biosciences, Inc.”. phoenix.corporate-ir.net. Retrieved 2016-11-20.
  15.  “[9,10-dimethoxy-3-(2-methylpropyl)-1h,2h,3h,4h,6h,7h,11bh-pyrido-[2,1-a]isoquinolin-2-yl]methanol And Compounds, Compositions And Methods Relating Thereto”. Retrieved 2016-11-20.
1 to 3 of 3
Patent ID Patent Title Submitted Date Granted Date
US8039627 SUBSTITUTED 3-ISOBUTYL-9, 10-DIMETHOXY-1, 3, 4, 6, 7, 11B-HEXAHYDRO-2H-PYRIDO[2, 1-A]ISOQUINOLIN-2-OL COMPOUNDS AND METHODS RELATING THERETO 2008-07-10 2011-10-18
US8357697 Substituted 3-isobutyl-9, 10-dimethoxy-1, 3, 4, 6, 7, 11b-hexahydro-2H-pyrido[2, 1-A]isoquinolin-2-ol compounds and methods relating thereto 2011-09-20 2013-01-22
US2016068526 BENZOQUINOLONE INHIBITORS OF VMAT2 2014-01-28 2016-03-10
Valbenazine
Valbenazine.svgImage result for valbenazine
Clinical data
ATC code
  • none
Legal status
Legal status
  • Investigational
Identifiers
Synonyms NBI-98854
CAS Number
PubChem CID
ChemSpider
UNII
KEGG
ChEMBL
Chemical and physical data
Formula C24H38N2O4
Molar mass 418.58 g·mol−1
3D model (Jmol)
////////fda 2017, Ingrezza, valbenazine, tardive dyskinesia, Fast Track, Priority Review ,  Breakthrough Therapy designations, 1025504-45-3, NBI-98854, 

Deflazacort


Deflazacort structure.svgChemSpider 2D Image | Deflazacort | C25H31NO6

Deflazacort

  • CAS 14484-47-0
  • Molecular Formula C25H31NO6
  • Average mass 441.517 Da
(11b,16b)-21-(Acetyloxy)-11-hydroxy-2′-methyl-5’H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione
11b,21-Dihydroxy-2′-methyl-5’bH-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione 21-acetate
2-[(4aR,4bS,5S,6aS,6bS,9aR,10aS,10bS)-5-Hydroxy-4a,6a,8-trimethyl-2-oxo-2,4a,4b,5,6,6a,9a,10,10a,10b,11,12-dodecahydro-6bH-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]oxazol-6b-yl]-2-oxoethyl acetate
  • 5’βH-Pregna-1,4-dieno[17,16-d]oxazole-3,20-dione, 11β,21-dihydroxy-2′-methyl-, 21-acetate (8CI)
  • (11β,16β)-21-(Acetyloxy)-11-hydroxy-2′-methyl-5’H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione
  • 2H-Naphth[2′,1′:4,5]indeno[1,2-d]oxazole, 5’H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione deriv.
  • Azacort
  • Azacortinol
  • Calcort
  • DL 458IT
  • Deflan
Optical Rotatory Power +62.3 ° Conc: 0.5 g/100mL; Solv: chloroform (67-66-3); Wavlength: 589.3 nm

…………..REF, “Drugs – Synonyms and Properties” data were obtained from Ashgate Publishing Co. (US)Hoechst Marion Roussel (now Aventis Pharma) has developed and launched Deflazacort (Dezacor; Flantadin; Lantadin; Calcort) a systemic corticosteroid developed for the treatment of a variety of inflammatory conditions .

In March 1990, the drug was approved in Spain, and by January 2013, the drug had been launched by FAES Farma . By the end of 1999, the product had been launched in Germany, Italy, Belgium, Switzerland and South Korea

Deflazacort is a corticosteroid first launched in 1985 by Guidotti in Europe for the oral treatment of allergic asthma, rheumatoid arthritis, arthritis, and skin allergy.

In 2017, an oral formulation developed at Marathon Pharmaceuticals was approved by the FDA for the treatment of Duchenne’s muscular dystrophy in patients 5 years of age and older.

Deflazacort (trade name Emflaza or Calcort among others) is a glucocorticoid used as an anti-inflammatory and immunosuppressant.

In 2013, orphan drug designation in the U.S. was assigned to the compound for the treatment of Duchenne’s muscular dystrophy. In 2015, additional orphan drug designation in the U.S. was assigned for the treatment of pediatric juvenile idiopathic arthritis (JIA) excluding systemic JIA.

Also in 2015, deflazacort was granted fast track and rare pediatric disease designations in the U.S. for the treatment of Duchenne’s muscular dystrophy.

Deflazacort is a glucocorticoid used as an anti-inflammatory and immunosuppressant. It was approved in February, 2017 by the FDA for use in treatment of Duchenne muscular dystrophy (trade name Emflaza).
  • Aventis Pharma (Originator), Lepetit (Originator), Guidotti (Licensee), Shire Laboratories (Licensee)

Image result for deflazacort

February 9, 2017 FDA approved

The U.S. Food and Drug Administration today approved Emflaza (deflazacort) tablets and oral suspension to treat patients age 5 years and older with Duchenne muscular dystrophy (DMD), a rare genetic disorder that causes progressive muscle deterioration and weakness. Emflaza is a corticosteroid that works by decreasing inflammation and reducing the activity of the immune system.

Corticosteroids are commonly used to treat DMD across the world. This is the first FDA approval of any corticosteroid to treat DMD and the first approval of deflazacort for any use in the United States.

Image result for Deflazacort

“This is the first treatment approved for a wide range of patients with Duchenne muscular dystrophy,” said Billy Dunn, M.D., director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research. “We hope that this treatment option will benefit many patients with DMD.”

DMD is the most common type of muscular dystrophy. DMD is caused by an absence of dystrophin, a protein that helps keep muscle cells intact. The first symptoms are usually seen between 3 and 5 years of age and worsen over time. The disease often occurs in people without a known family history of the condition and primarily affects boys, but in rare cases it can affect girls. DMD occurs in about one of every 3,600 male infants worldwide.

People with DMD progressively lose the ability to perform activities independently and often require use of a wheelchair by their early teens. As the disease progresses, life-threatening heart and respiratory conditions can occur. Patients typically succumb to the disease in their 20s or 30s; however, disease severity and life expectancy vary.

The effectiveness of deflazacort was shown in a clinical study of 196 male patients who were 5 to 15 years old at the beginning of the trial with documented mutation of the dystrophin gene and onset of weakness before age 5. At week 12, patients taking deflazacort had improvements in a clinical assessment of muscle strength across a number of muscles compared to those taking a placebo. An overall stability in average muscle strength was maintained through the end of study at week 52 in the deflazacort-treated patients. In another trial with 29 male patients that lasted 104 weeks, deflazacort demonstrated a numerical advantage over placebo on an assessment of average muscle strength. In addition, although not statistically controlled for multiple comparisons, patients on deflazacort appeared to lose the ability to walk later than those treated with placebo.

The side effects caused by Emflaza are similar to those experienced with other corticosteroids. The most common side effects include facial puffiness (Cushingoid appearance), weight gain, increased appetite, upper respiratory tract infection, cough, extraordinary daytime urinary frequency (pollakiuria), unwanted hair growth (hirsutism) and excessive fat around the stomach (central obesity).

Other side effects that are less common include problems with endocrine function, increased susceptibility to infection, elevation in blood pressure, risk of gastrointestinal perforation, serious skin rashes, behavioral and mood changes, decrease in the density of the bones and vision problems such as cataracts. Patients receiving immunosuppressive doses of corticosteroids should not be given live or live attenuated vaccines.

The FDA granted this application fast track designation and priority review. The drug also received orphan drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.

The sponsor is receiving a rare pediatric disease priority review voucher under a program intended to encourage development of new drugs and biologics for the prevention and treatment of rare pediatric diseases. A voucher can be redeemed by a sponsor at a later date to receive priority review of a subsequent marketing application for a different product. This is the ninth rare pediatric disease priority review voucher issued by the FDA since the program began.

Emflaza is marketed by Marathon Pharmaceuticals of Northbrook, Illinois.

Medical uses

The manufacturer lists the following uses for deflazacort:[1]

In the United States, deflazacort is only FDA-approved for the treatment of Duchenne muscular dystrophy in people over the age of 5.

Image result for DeflazacortImage result for Deflazacort

Image result for DeflazacortImage result for Deflazacort

Adverse effects

Deflazacort carries the risks common to all corticosteroids, including immune suppression, decreased bone density, and endocrine insufficiency. In clinical trials, the most common side effects (>10% above placebo) were Cushing’s-like appearance, weight gain, and increased appetite.[2]

Pharmacology

Mechanism of action

Deflazacort is an inactive prodrug which is metabolized rapidly to the active drug 21-desacetyldeflazacort.[3]

Relative potency

Deflazacort’s potency is around 70–90% that of prednisone.[4] A 2017 review found its activity of 7.5 mg of deflazacort is approximately equivalent to 25 mg cortisone, 20 mg hydrocortisone, 5 mg of prednisolone or prednisone, 4 mg of methylprednisolone or triamcinolone, or 0.75 mg of betamethasone or dexamethasone. The review noted that the drug has a high therapeutic index, being used at initial oral doses ranging from 6 to 90 mg, and probably requires a 50% higher dose to induce the same demineralizing effect as prednisolone. Thus it has “a smaller impact on calcium metabolism than any other synthetic corticosteroid, and therefore shows a lower risk of growth rate retardation in children and of osteoporosis” in the elderly, and comparatively small effects on carbohydrate metabolism, sodium retention, and hypokalemia.[5]

History

In January 2015, the FDA granted fast track status to Marathon Pharmaceuticals to pursue approval of deflazacort as a potential treatment for Duchenne muscular dystrophy, a rare, “progressive and fatal disease” that affects boys.[6] Although deflazacort was approved by the FDA for use in treatment of Duchenne muscular dystrophy on February 9, 2017,[7][8] Marathon CEO announced on February 13, 2017 that the launch of deflazacort (Emflaza) would be delayed amidst controversy over the steep price Marathon was asking for the drug – $89,000-a-year. In Canada the same drug can be purchased for around $1 per tablet.[9] Marathon has said that Emflaza is estimated to cost $89,000/year which is “roughly 70 times” more than it would cost overseas.[10] Deflazacort is sold in the United Kingdom under the trade name Calcort;[4] in Brazil as Cortax, Decortil, and Deflanil; in India as Moaid, Zenflav, Defolet, DFZ, Decotaz, and DefZot; in Bangladesh as Xalcort; in Panama as Zamen; Spain as Zamene; and in Honduras as Flezacor.[11]

SYNTHESIS

Worlddrugtracker drew this

1 Protection of the keto groups in pregna-1,4-diene derivative  with NH2NHCOOMe using HCOOH, yields the corresponding methyl ester.

2 Cleavage of epoxide  with NH3 in DMAc/DMF gives amino-alcohol,

3 which on esterification with acetic anhydride in the presence of AcOH furnishes acetate.

4 Cyclization of amine using NaOH, Na2CO3 or K2CO3 produces oxazoline derivative ,

5 which is finally deprotected with HCl to afford Deflazacort 

SYNTHESIS FROM CHEMDRUG

The cyclization of 17alpha-azido-3beta,16alpha-acetoxy-5alpha-pregnane-11,20-dione (I) by hydrogenation with H2 over Pt in methanol, followed by a treatment with 10% HCl gives 3beta-hydroxy-5alpha-pregnane-11,20-dione-[17alpha,16alpha-d]-2′-methyloxazoline (II), which is converted into the semicarbazone (III) by treatment with semicarbazide hydrochloride (A) and pyridine in refluxing methanol. The reduction of one ketonic group of (III) with NaBH4 in refluxing ethanol yields the dihydroxy-semicarbazone (IV), which is hydrolyzed with 10% HCl in refluxing methanol to afford the ketodiol (V). The oxidation of (V) with cyclohexanone and aluminum isopropoxide in refluxing toluene gives 11beta-hydroxy-5alpha-pregnane-3,20-dione-[17alpha,16alpha-d]-2′-methyloxazoline (VI). The dehydrogenation of (VI) by treatment with Br2 in dioxane-acetic acid, followed by treatment with Li2CO3 in DMF at 140 C yields the corresponding 1,4-diene derivative (VII). Finally, the reaction of (VII) with I2 by means of azobisisobutyronitrile in CH2Cl2 affords the corresponding 21-iodo compound, which is then acetylated with triethylammonium acetate in refluxing acetone.

The monoacetylation of (V) with acetic anhydride and pyridine at 100 C gives the 3-acetoxy-11-hydroxy compound (IX), which is dehydrated by treatment with methanesulfonyl chloride and then with sodium acetate yielding 3beta-acetoxy-5alpha-pregn-9(11)-ene-20-one-[17alpha,16alpha-d]-2′-methyloxazoline (X). The hydrolysis of (X) with KOH in refluxing methanol affords the corresponding hydroxy compound (XI), which is acetoxylated by treatment with I2 and AZBN as before giving the iodo derivative (XII), and then with triethylammonium acetate also as before, yielding 3beta-hydroxy-21-acetoxy-5alpha-pregn-9(11)-ene-20-one-[17alpha,16alpha-d]-2′-methyloxazoline (XIII). The oxidation of (XIII) with CrO3 in acetone yields the 3,20-diketone (XIV), which by treatment with Br2 and Li2CO3 as before is dehydrogenated affording the 1,4,9(11)-pregnatriene (XV). Finally, the reaction of (XV) with N-bromoacetamide in THF yields 9alpha-bromo-11beta-hydroxy-21-acetoxy-5alpha-pregna-1,4-dieno-3,20-dione-[17alpha,16alpha-d]-2′-methyloxazoline (XVI), which is then debrominated by reaction with chromous acetate and butanethiol in DMSO.

PAPER

Journal of Medicinal Chemistry (1967), 10(5), 799-802

Steroids Possessing Nitrogen Atoms. III. Synthesis of New Highly Active Corticoids. [17α,16α,-d]Oxazolino Steroids

J. Med. Chem., 1967, 10 (5), pp 799–802
DOI: 10.1021/jm00317a009

PATENT

CN 105622713

PATENT CN 106008660

MACHINE TRANSLATED FROM CHINESE may seem funny

Description of the drawings

[0007] Figure 1 is a map of the traditional method of the combination process;

Figure 2 is a two-step method of the present invention.

detailed description

[0008] In order to more easily illustrate the gist and spirit of the present invention, the following examples illustrate:

Example 1

A: Preparation of hydroxylamine

In a 100 ml three-necked flask, 20 g of 16 (17) a-epoxy prednisolone, 30 ml of DMF, 300 ml of chloroform was added and incubated at 30-35 ° C with 8 g of ammonia gas at 1-2 atmospheres Reaction 16 ~ 20 hours, TLC detection reaction end point, after the reaction, the vacuum exhaust ammonia gas, add 3x100ml saturated brine washing 3 times, plus 10ml pure water washing times, then, under reduced pressure to chloroform to dry, add 200ml Ethyl acetate, Ig activated carbon, stirring reflux 60-90 minutes, cooling to 50-55 degrees, hot filter, l-2ml ethyl acetate washing carbon, combined filtrate and lotion, and then below 500C concentrated under pressure 95 % Of ethyl acetate, the system cooled to -5-0 ° C, stirring crystallization 2 ~ 3 hours, filter, 0.5-lml ethyl acetate washing, lotion and filtrate combined sets of approved; filter cake below 70 ° C Drying, get hydroxylamine 18.2g, HPLC content of 99.2%, weight loss of 91%.

[0009] B: Preparation of terracavir

Add 10 g of hydroxylamine, 150 ml of glacial acetic acid and 150 ml of acetic anhydride in a 100 ml three-necked flask. Add 5 g of concentrated sulfuric acid under stirring at room temperature. The reaction was carried out at 30-35 ° C for 12-16 hours. TLC confirmed the end of the reaction. Add 500ml of pure water, and adjust the pH of 7.5.5 with liquid alkali, cool to 10 ~ 15 ° C, stirring crystallization 2-3 hours, filtration, washing to neutral, combined filtrate and lotion, pretreated into Waste water treatment tank, filter cake below 70 V drying, Texaco can be special crude 112.5g, HPLC content of 98.2%, the yield of 112.5% ο the above terracotta crude dissolved in 800ml of alcohol, add 5g activated carbon, Decolorization 1-1.5 hours, hot filter, 10ml alcohol detergent cake, lotion and filtrate combined, atmospheric pressure recovery of about 90% of the alcohol, and then cooled to -5-0 ° C, frozen crystal 2-3 hours, Filtration, filter cake with 4-5ml alcohol washing, 70 ° C below drying, digoxin special product 89.2g, melting point 255.5-256.0 degrees, HPLC content of 99.7%, yield 89.2%. The mother liquor is recycled with solvent and crude.

[0010] Example II

A: Preparation of hydroxylamine

In a 100 ml three-necked flask, 20 g of 16 (17) a-epoxy prednisolone, 120 ml of toluene was added and incubated at 30-35 ° C with 8 g of ammonia and 16 to 20 at atmospheric pressure The reaction was carried out in the presence of 3 x 50 ml of saturated brine and 50 ml of pure water was added. Then, the toluene was dried under reduced pressure to dryness, and 200 ml of ethyl acetate, Ig activated carbon was added, and the mixture was stirred. Reflux 60-90 minutes, cool to 50-55 ° C, hot filter, l2ml ethyl acetate wash carbon, combined filtrate and lotion, and then below 500C under reduced pressure 95% ethyl acetate, the system cooling To 5-0C, stirring crystallization 2 ~ 3 hours, filter, 0.5-lml ethyl acetate washing, lotion and filtrate combined sets of the next batch; filter cake 70 ° C below drying, hydroxylamine 18.0g, HPLC content 99.1%, 90% by weight.

[0011] B: Preparation of terracavir

Add 10 g of hydroxylamine, 500 ml of chloroform and 150 ml of acetic anhydride in a 100 ml three-necked flask, add 5 g of p-toluenesulfonic acid under stirring at room temperature, and incubate at 30-35 ° C for 12-16 hours. TLC confirms the reaction end, After the addition of 500ml of pure water, and with the liquid alkali pH 7.55, down to 10 ~ 15 ° C, stirring 0.5_1 hours, separate the water layer, washed to neutral, combined with water and lotion, pretreated into Waste water treatment tank, organic layer under reduced pressure concentrated chloroform to near dry, adding 200ml hexane, reflux 0.5-1 hours, slowly cooling to -5 ~ O0C, stirring crystallization 2-3 hours, filter, filter cake with 4-5ml Alcohol washing, the filtrate and lotion combined apply to the next batch, the filter cake below 70 ° C drying, Texaco can crude 110.5g, HPLC content of 98.4%, the yield of 110.5%. The above-mentioned diltiazem crude product dissolved in 800ml alcohol, add 5g activated carbon, temperature reflux bleaching 1-1.5 hours, hot filter, 10ml alcohol washing cake, lotion and filtrate combined, atmospheric pressure recovery of about 90% of the alcohol And then cooled to -500C, frozen crystallization for 2-3 hours, filtration, filter cake with 4-5ml alcohol washing, 70 ° C the following drying, digester can special products 88.6g, melting point 255.0-256.0 degrees, HPLC content of 99.5%, the yield of 88.6%. The mother liquor is recycled with solvent and crude.

[0012] Example 3

A: Preparation of hydroxylamine

Add 20 g of 16 (17) a-epoxy prednisolone to 120 ml of ethanol in a 100 ml three-necked flask and incubate at 30-35 ° C with stirring to give Sg ammonia at 16 to 20 hours , TLC test reaction end point, after the reaction, vacuum exhaust ammonia gas, concentrated ethanol to the near dry, cooling, adding 300ml chloroform, stirring dissolved residue, and then add 3x100ml saturated brine washing, plus 10ml pure water washing, washing And then concentrated to reduce the chloroform to dry, add 200ml of ethyl acetate, Ig activated carbon, stirring reflux 60-90 minutes, cooling to 50-55 ° C, hot filter, l2ml ethyl acetate washing carbon, combined filtrate and lotion And then concentrated below 50 ° C to 95% ethyl acetate under reduced pressure. The system was cooled to -5-0 0C, stirred for 2 to 3 hours, filtered, 0.5-l of ethyl acetate, washed and filtrate The filter cake was dried at 70 ° C, 18.6 g of hydroxylamine, 99.5% of HPLC, and 93% by weight.

[0013] B: Preparation of terracavir

In a 100ml three-necked flask, add 10g of hydroxylamine, 500ml toluene, 150ml acetic anhydride, stirring at room temperature by adding 5g concentrated sulfuric acid, insulation at 30-35 degrees stirring reaction 12-16 hours, TLC confirmed the end of the reaction, after the reaction, Add 500ml of pure water, and liquid pH adjustment pH 7.5, cooling to 1 ~ 15 ° C, stirring 0.5-1 hours, the water layer, washed to neutral, combined with water and lotion, pretreated into the wastewater The cells were dried and the organic layer was concentrated to dryness under reduced pressure. 200 ml of hexane was added and refluxed

0.5-1 hours, slowly cool to -5 ~ O0C, stirring crystallization 2-3 hours, filtration, filter cake with 4-5ml hexane, the filtrate and lotion combined apply to the next batch, filter cake below 70 ° C Drying, digoxin crude 112.5g, HPLC content of 97.4%, the yield of 112,5% ο will be the above terracotta crude dissolved in 800ml of alcohol, add 5g activated carbon, heating reflux bleaching 1-1.5 hours, while Hot filter, 10ml alcohol detergent cake, lotion and filtrate combined, atmospheric pressure recovery of about 90% of the alcohol, and then cooled to -500C, frozen crystallization for 2-3 hours, filter, filter cake with 4-5ml alcohol Washing, 70 ° C below the dry, Diges can special products 86.2g, melting point 255.5-256.0 degrees, HPLC content of 99.8%, the yield of 86.2%. The mother liquor is recycled with solvent and crude.

PATENT

https://www.google.com/patents/CN101418032A?cl=en

Example 1

21- bromo -ll (3- hydroxy – pregna–l, 4- diene -3, 20-dione [170, 16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask was added 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), N- bromosuccinimide (9.79 g; Fw: 178.00; 55 mmol), 150 ml of ether; then ammonium acetate (0.39 g; Fw: 77.08; 0.005 mmol) added to the system. System continues to stir at 20 ° C 0.5 h, the reaction is complete. After completion of the reaction was filtered to remove the white precipitate cake was washed with 50 mL of dichloromethane, and the combined organic Xiangde pale yellow clear liquid, the solvent was evaporated under reduced pressure to give a pale yellow solid 21.27 g, yield: 92%, HPLC content of greater than 95%.

Example 2

21- bromo -lip- hydroxy – pregna–l, 4- diene -3, 20-dione [17 “16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask were added sequentially 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), N- bromosuccinimide (9.79 g; Fw : 178.00; 55 mmol), 150 ml of toluene; then ammonium acetate (0.39 g; Fw: 77.08; 0.005 mmol) added to the system. System continues to stir at 110 ° C 5 h, the reaction is complete. After completion of the reaction was cooled to room temperature, the white precipitate was removed by filtration cake was washed with 50 mL of dichloromethane, and the combined organic Xiangde pale yellow clear liquid, concentrated under reduced pressure to remove the solvent to give a pale yellow solid 19.65 g, yield: 85%, HPLC content greater than 95%.

Example 3

21 Jie bromo -11 – hydroxy – pregna-1,4-diene -3, 20-dione [17a, 16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask were added sequentially 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), 1,3- dibromo-5,5-dimethyl- Hein (35.74 g; Fw: 285.94; 125 mmol), 150 ml of ether; then ammonium acetate (0.39 g; Fw: 77.08; 0.005 mmol) added to the system. System Stirring was continued at reflux for 3 h, the reaction was completed. After completion of the reaction a white precipitate was removed by filtration and the cake was washed with 50 mL of diethyl ether, and the combined organic Xiangde pale yellow clear liquid, concentrated under reduced pressure to remove the solvent to give a pale yellow solid 16.18 g, yield: 70%, HPLC content greater than 92%.

Example 4

21- bromo -11 Jie – hydroxy – pregna-1,4-diene -3, 20- dione [17c, 16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask were added sequentially 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), 1,3- dibromo-5,5-dimethyl- Hein (35.74 g; Fw: 285.94; 125 mmol), 150 ml dichloromethane; followed by ammonium acetate (0.039 g; Fw: 77.08; 0.0005 mmol) added to the system. System Stirring was continued at reflux for 24 h, the reaction was completed. After completion of the reaction a white precipitate was removed by filtration and the cake was washed with 50 mL of diethyl ether, and the combined organic Xiangde pale yellow clear liquid, concentrated under reduced pressure to remove the solvent to give a pale yellow solid 16.41 g, yield: 71%, HPLC content of greater than 92. / 0.

Example 5

Deflazacort Preparation:

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of sodium acetate (8.20g; Fw: 82.03; lOOmmol), 50 mL methanol was added to the system.

Then tetrabutylammonium bromide (O. 81g; Fw: 322.38; 2.5 mmol). Warmed to 50 ° C with stirring

48 h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase washed with 10% aqueous sodium carbonate paint 3 times, saturated sodium chloride once. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, purified ethyl acetate to give the product 9.93g, yield 90%, HPLC content> 990/0.

Example 6

Deflazacort Preparation –

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of anhydrous potassium acetate (3.68g; Fw: 98.14; 37.5 mmol), 50 mL acetone was added to the system. Followed by tetrabutylammonium iodide (0.10g; Fw: 369.37; 0.25 mmol). Heated to reflux with stirring 2h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase was washed 3 times with 10% aqueous sodium carbonate , washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, ethyl acetate was purified to give the product 10.93 g, yield 99%, HPLC content> 99%.

Example 7

Deflazacort Preparation:

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of anhydrous potassium acetate (3.68g; Fw: 98.14; 37.5 mmol), 50 mL acetonitrile was added to the system. Followed by tetrabutylammonium iodide (0.10g; Fw: 369.37; 0.25 mmol). Heated to reflux with stirring 2h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase was washed 3 times with 10% aqueous sodium carbonate , washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, ethyl acetate was purified to give the product 10.93 g, yield 99%, HPLC content> 99%.

Example 8

Deflazacort Preparation:

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of anhydrous potassium acetate (2.45g; Fw: 98.14; 25 mmol), the N, N- dimethylformamide, 50 mL added to the system. Followed by tetrabutylammonium iodide (O.IO g; Fw: 369.37; 0.25 mmol). Warmed to 120. C stirring 2h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase was washed 3 times with 10% aqueous sodium carbonate , washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, ethyl acetate was purified to give the product 10.93 g, yield 99%, HPLC content> 99o / q.

PATENT

https://www.google.com/patents/WO1997021722A1?cl=zh

compound (llβ,16β)-21-(acetyloxy)-11- hydroxy-2 ‘ -methyl-5 ‘H-pregna-1, -dieno[17 , 16-d Joxazole- 3,20-dione, also known, and hereinafter referred to, with the INN (International Nonproprietary Name) deflazacort. Deflazacort is represented by the following formula I

Figure imgf000003_0001

Deflazacort is employed in therapy aince some years as a calcium-sparing corticoid agent. This compound belongs to the more general class of pregneno-oxazolines, for which anti-inflammatory, glucocorticoid and hormone-like pharmacological activities are reported. Examples of compounds of the above class, comprising deflazacort, are disclosed in US 3413286, where deflazacort is referred to as llβ-21-dihydroxy-2 ‘ -methyl-5 ‘ βH-pregna-1,4-dieno.17 , 16- d]oxazole-3,20-dione 21-acetate.

According to the process disclosed by US 3413286, deflazacort is obtained from 5-pregnane-3β-ol-ll , 20- dione-2 ‘-methyloxazoline by 2 , -dibromination with Br2– dioxane, heating the product in the presence of LiBr- iC03 for obtaining the 1,4-diene, and converting this latter into the 21-iodo and then into the desired 21- acetyloxy compound. By hydrolysis of deflazacort, the llβ-21-dihydroxy-2 ‘ -methyl-5 ‘βH-pregna-1, -dieno[ 17 , 16- d-]oxazoline-3, 20-dione of formula II is obtained:

Figure imgf000004_0001

The compound of formula II is preferably obtained according to a fermentation process disclosed in

EP-B-322630; in said patent, the compound of formula II is referred to as llβ-21-dihydroxy-2 ‘-methyl-5 ‘ βH- pregna-1,4-dieno[17,16-d-]oxazoline-3,20-dione.

The present invention provides a new advantageous single-step process for obtaining deflazacort, by acetylation of the compound of formula II.

CLIP

Image result for Deflazacort NMR

tructure of deflazacort and its forced degradation product (A), chromatogram plot of standard deflazacort (B), contour plot of deflazacort (C). Deflazacort was found to be a stable drug under stress condition such as thermal, neutral and oxidative condition. However, the forceddegradation study on deflazacort showed that the drug degraded under alkaline, acid and photolytic conditions.

Mass fragmentation pathway for degradant product of deflazacort.

PATENT

CN 103059096

Figure CN103059096AD00051

Example 1: Protective reaction To the reaction flask was added 20 g of 1,4-diene-11? -hydroxy-16,17-epoxy_3,20-dione pregnone (Formula I) 20% of the aqueous solution of glacial acetic acid 300g, stirring 5 minutes, temperature 10 ° C ~ 15 ° C, adding ethyl carbazate 14g, temperature control 30 ° C reaction 6 hours; TLC detection reaction is complete, cooling to 0 ° C ~ 5 ° C for 2 hours, until dry, washed to neutral; 60 ° C vacuum dry to dry creatures 20. 5g; on P, oxazoline ring reaction The above protective products into the reaction bottle, add 41ml Of the DMAC dissolved, temperature 25 ~ 30 ° C, access to ammonia, to keep the reaction bottle micro-positive pressure, the reaction of 32 hours, atmospheric pressure exhaust ammonia and then decompression pumping ammonia for 30 minutes; 5 ° C, temperature 5 ~ 0 ° C by adding 5ml glacial acetic acid, then add 21ml acetic anhydride, heated to 35 ° C reaction 4 hours, the sample to confirm the reaction completely; slowly add 5% sodium hydroxide solution 610ml and heated to 60 ~ 70 ° C reaction 2 hours; point plate to confirm the end of the reaction, cooling to 50 ° C, half an hour by adding refined concentrated hydrochloric acid 40ml, insulation 50 ~ 55 ° C reaction 10 hours; to the end of the reaction temperature to room temperature, chloroform Extraction, drying and filtration, concentration of at least a small amount of solvent, ethyl acetate entrained twice, leaving a small amount of solvent, frozen crystallization filter high purity [17a, 16a-d] terfu Kete intermediate. Example 2: Protective reaction 20 g of 1,4-diene-l1-la-hydroxy-16,17-epoxy_3,20_dione progestin (Formula I) was added to the reaction flask and 15% Formic acid solution 300g, stirring for 5 minutes, temperature 10 ~ 15 ° C, adding methyl carbazate 12g, temperature control 30 ° C reaction 5 hours to test the end of the reaction, cooling to O ~ 5 ° C stirring 2 hours crystallization, Suction to dry, washed to neutral; 60 ° C vacuum drying to dry protection of 20g; on P, oxazoline ring reaction The protection of the reaction into the reaction flask, add 30ml of DMF dissolved, temperature control 25 ~ 30 ° C, access to ammonia, keep the reaction bottle in the micro-positive pressure, reaction 30 hours, atmospheric pressure exhaust ammonia and then decompression pumping ammonia for 30 minutes, ice water cooled to 5 ° C, temperature 5 ~ 10 ° C add 5ml of glacial acetic acid, then add 20ml acetic anhydride, heated to 30 ° C reaction for 5 hours to confirm the reaction is complete; slowly add 20% sodium carbonate aqueous solution 500ml and heated to 60 ~ 70 ° C reaction 4 hours, the point plate to confirm the reaction The temperature of 55 ~ 60 ° C for 10 hours; to be the end of the reaction temperature to room temperature, chloroform extraction, drying and filtration, concentration of a small amount of solvent, acetic acid isopropyl The ester was entrained twice, leaving a small amount of solvent, frozen and crystallized to obtain high purity [17a, 16a-d] oxazoline residues. [0024] Example 3: Protective reaction 20 g of I, 4-diene-16,17-epoxy-3,11,20-triketone pregnone (Formula I) was added to the reaction flask and 20% Formic acid solution 300g, stirring for 5 minutes, temperature 10 ~ 15 ° C, adding hydrazine carbamate 15g, temperature control 30 ° C reaction 5 hours to test the end of the reaction, cooling to O ~ 5 ° C stirring 2 hours crystallization, To the dry, washed to neutral; 60 ° C vacuum drying to dry protection of 22g; on P, oxazoline ring reaction of the protection of the reaction into the bottle, add 30ml of DMAC dissolved temperature control 35 ~ 40 ° C, access to ammonia, keep the reaction bottle in the micro-positive pressure, reaction 40 hours, atmospheric pressure exhaust ammonia and then decompression pumping ammonia for 30 minutes, ice water cooling to 5 ° C, temperature 5 ~ 10 ° C add 5ml of glacial acetic acid, then add 20ml acetic anhydride, heated to 40 ° C reaction 5 hours to confirm the reaction is complete; slowly add 20% potassium carbonate aqueous solution 500ml and heated to 60 ~ 70 ° C reaction 7 hours, the point plate to confirm the reaction The temperature of the reaction to the end of the temperature to room temperature, chloroform extraction, drying filter, concentrated to a small amount of solvent, acetic acid isopropyl The ester was entrained twice, leaving a small amount of solvent, frozen and crystallized to obtain high purity [17a, 16a-d] oxazoline residues.

PATENT

CN 102936274

Figure CN102936274BD00041

xample 1

[0028] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 15 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10-15 ° C), 30 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give product 30.6 g, 102% mass yield, product by HPLC , a purity of 95.2%.

[0029] Example 2

[0030] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mL of pyridine were mixed, added pressure reactor, stirring ammonia gas to the reactor pressure to 0. 15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 15 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give product 28.6 g, yield 95% by mass, product by HPLC , a purity of 94.8%.

[0031] Example 3

[0032] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction.Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction for 6 hours. The reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 31.2 g, yield 104% quality products by HPLC , a purity of 95.4%.

[0033] Example 4

[0034] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.5 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 31. I g, 102% mass yield, product by by HPLC, the purity was 95.2%.

[0035] Example 5

[0036] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 60 mL of acetic acid, 15 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 29. 5 g, yield 98% by mass, the product of by HPLC, purity of 95%.

[0037] Example 6

[0038] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. The reaction was complete, the material was transferred to a glass reaction flask until the material temperature drops below 10 ° C, plus acetic acid to adjust the pH to 5 to 6, the solvent was removed under reduced pressure; the reaction flask was added 30 mL of acetic acid, 30 g of maleic dianhydride, the reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 30 g, 100% mass yield, product by HPLC purity of 95.2%.

[0039] Example 7

[0040] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of propionic anhydride, The reaction temperature was controlled at 30 ° C, the reaction for 6 hours. The reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 27.6 g, 92% yield of quality products by HPLC , a purity of 93.5%.

[0041] Example 8

[0042] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature is controlled at 50 ° C, the reaction for 6 hours. The reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 29.8 g, 99% yield of quality products by HPLC , a purity of 94.8%.

References

  1. Jump up^ “Refla: deflazacort” (PDF).
  2. Jump up^http://www.accessdata.fda.gov/drugsatfda_docs/label/2017/208684s000,208685s000lbl.pdf
  3. Jump up^ Möllmann, H; Hochhaus, G; Rohatagi, S; Barth, J; Derendorf, H (1995). “Pharmacokinetic/pharmacodynamic evaluation of deflazacort in comparison to methylprednisolone and prednisolone”. Pharmaceutical Research. 12 (7): 1096–100. PMID 7494809.
  4. ^ Jump up to:a b “Calcort”. electronic Medicines Compendium. June 11, 2008. Retrieved on October 28, 2008.
  5. Jump up^ Luca Parente (2017). “Deflazacort: therapeutic index, relative potency and equivalent doses versus other corticosteroids”. BMC Pharmacol Toxicol. doi:10.1186/s40360-016-0111-8.
  6. Jump up^ Ellen Jean Hirst (January 19, 2015), Duchenne muscular dystrophy drug could get OK for U.S. sales in 2016, The Chicago Tribune, retrieved February 13, 2017,has been shown to prolong lives … a progressive and fatal disease that has no drug treatment available in the US
  7. Jump up^ “FDA approves drug to treat Duchenne muscular dystrophy”. http://www.fda.gov. 2017-02-09. Retrieved 2017-02-10.
  8. Jump up^ “Marathon Pharmaceuticals to Charge $89,000 for Muscular Dystrophy Drug”. http://www.wsj.com. 2017-02-10. Retrieved 2017-02-10.
  9. Jump up^ Clifton Sy Mukherjee (February 10, 2017). “Brainstorm Health Daily”. Retrieved February 13, 2017.
  10. Jump up^ Joseph Walker and Susan Pulliam (February 13, 2017), Marathon Pharmaceuticals to Charge $89,000 for Muscular Dystrophy Drug After 70-Fold Increase, The Wall Street Journal, retrieved February 13, 2017,FDA-approved deflazacort treats rare type of disease affecting boys
  11. Jump up^ “Substâncias: DEFLAZACORT” (in Portuguese). Centralx. 2008. Retrieved on October 28, 2008.
Deflazacort
Deflazacort structure.svg
Clinical data
Trade names Emflaza, Calcort, others
AHFS/Drugs.com International Drug Names
Routes of
administration
By mouth
ATC code
Legal status
Legal status
Pharmacokinetic data
Protein binding 40%
Metabolism By plasma esterases, to active metabolite
Biological half-life 1.1–1.9 hours (metabolite)
Excretion Renal (70%) and fecal (30%)
Identifiers
CAS Number
PubChem CID
DrugBank
ChemSpider
UNII
KEGG
ChEMBL
ECHA InfoCard 100.034.969
Chemical and physical data
Formula C25H31NO6
Molar mass 441.517 g/mol
3D model (Jmol)
CN102746358A * Apr 22, 2011 Oct 24, 2012 天津金耀集团有限公司 Novel technology for synthesis of pregnane 21-bit bromide
CN102746358B * Apr 22, 2011 Feb 10, 2016 天津金耀集团有限公司 一种合成孕甾21位溴化物的工艺
CN102936274A * Nov 12, 2012 Feb 20, 2013 浙江仙居君业药业有限公司 Preparation method for [17alpha, 16alpha-d] methyl oxazoline
CN102936274B * Nov 12, 2012 Apr 1, 2015 江西君业生物制药有限公司 Preparation method for [17alpha, 16alpha-d] methyl oxazoline

///////FDA 2017, Emflaza, Calcort, Deflazacort, orphan drug designation, FAST TRACK

[H][C@@]12C[C@@]3([H])[C@]4([H])CCC5=CC(=O)C=C[C@]5(C)[C@@]4([H])[C@@]([H])(O)C[C@]3(C)[C@@]1(N=C(C)O2)C(=O)COC(C)=O

Evofosfamide, эвофосфамид , إيفوفوسفاميد , 艾伏磷酰胺 ,


str1

TH-302.svg

Evofosfamide, HAP-302 , TH-302, TH 302

эвофосфамид ,  إيفوفوسفاميد ,  艾伏磷酰胺 ,

  • Molecular Formula C9H16Br2N5O4P
  • Average mass 449.036 Da

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl-N,N’-bis(2-bromethyl)phosphorodiamidat
918633-87-1

TH-302 is a nitroimidazole-linked prodrug of a brominated derivative of an isophosphoramide mustard previously used in cancer drugs

  • Originator Threshold Pharmaceuticals
  • Developer Merck KGaA; Threshold Pharmaceuticals
  • Class Antineoplastics; Nitroimidazoles; Phosphoramide mustards; Small molecules
  • Mechanism of Action Alkylating agents
  • Orphan Drug Status Yes – Soft tissue sarcoma; Pancreatic cancer
  • On Fast track Pancreatic cancer; Soft tissue sarcoma
  • Suspended Glioblastoma; Leukaemia; Malignant melanoma; Multiple myeloma; Non-small cell lung cancer; Solid tumours
  • Discontinued Pancreatic cancer; Soft tissue sarcoma

Most Recent Events

  • 01 Aug 2016 Threshold plans a clinical trial for Solid tumours
  • 01 Aug 2016 Threshold announces intention to submit NDA to the Pharmaceuticals and Medical Device Agency in Japan
  • 16 Jun 2016 Merck KGaA terminates a phase II trial in Soft tissue sarcoma (Combination therapy, Inoperable/Unresectable, Metastatic disease, Late-stage disease) in Japan (IV) due to negative results from the phase III SARC021 trial (NCT02255110)

Evofosfamide (first disclosed in WO2007002931), useful for treating cancer.

Image result for Evofosfamide

Threshold Pharmaceuticals and licensee Merck Serono are codeveloping evofosfamide, the lead in a series of topoisomerase II-inhibiting hypoxia-activated prodrugs and a 2-nitroimidazole-triggered bromo analog of ifosfamide, for treating cancer, primarily soft tissue sarcoma and pancreatic cancer (phase 3 clinical, as of April 2015).

In November 2014, the FDA granted Fast Track designation to the drug for the treatment of previously untreated patients with metastatic or locally advanced unresectable soft tissue sarcoma.

Evofosfamide (INN,[1] USAN;[2] formerly known as TH-302) is an investigational hypoxia-activated prodrug that is in clinical development for cancer treatment. The prodrug is activated only at very low levels of oxygen (hypoxia). Such levels are common in human solid tumors, a phenomenon known as tumor hypoxia.[3]

Evofosfamide is being evaluated in clinical trials for the treatment of multiple tumor types as a monotherapy and in combination with chemotherapeutic agents and other targeted cancer drugs.

Dec 2015 : two Phase 3 trials fail, Merck will not apply for a license

Collaboration

Evofosfamide was developed by Threshold Pharmaceuticals Inc. In 2012, Threshold signed a global license and co-development agreement for evofosfamide with Merck KGaA, Darmstadt, Germany (EMD Serono Inc. in the US and Canada), which includes an option for Threshold to co-commercialize evofosfamide in the United States. Threshold is responsible for the development of evofosfamide in the soft tissue sarcoma indication in the United States. In all other cancer indications, Threshold and Merck KGaA are developing evofosfamide together.[4] From 2012 to 2013, Merck KGaA paid 110 million US$ for upfront payment and milestone payments to Threshold. Additionally, Merck KGaA covers 70% of all evofosfamide development expenses.[5]

Mechanism of prodrug activation and Mechanism of action (MOA) of the released drug[edit]

Evofosfamide is a 2-nitroimidazole prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM). Evofosfamide is activated by a process that involves a 1-electron (1 e) reduction mediated by ubiquitous cellular reductases, such as the NADPH cytochrome P450, to generate a radical anion prodrug:

  • A) In the presence of oxygen (normoxia) the radical anion prodrug reacts rapidly with oxygen to generate the original prodrug and superoxide. Therefore, evofosfamide is relatively inert under normal oxygen conditions, remaining intact as a prodrug.
  • B) When exposed to severe hypoxic conditions (< 0.5% O2; hypoxic zones in many tumors), however, the radical anion undergoes irreversible fragmentation, releasing the active drug Br-IPM and an azole derivative. The released cytotoxin Br-IPM alkylates DNA, inducing intrastrand and interstrand crosslinks.[6]

Evofosfamide is essentially inactive under normal oxygen levels. In areas of hypoxia, evofosfamide becomes activated and converts to an alkylating cytotoxic agent resulting in DNA cross-linking. This renders cells unable to replicable their DNA and divide, leading to apoptosis. This investigational therapeutic approach of targeting the cytotoxin to hypoxic zones in tumors may cause less broad systemic toxicity that is seen with untargeted cytotoxic chemotherapies.[7]

The activation of evofosfamide to the active drug Br-IPM and the mechanism of action (MOA) via cross-linking of DNA is shown schematically below:

Activation of eofosfamide to the active drug Br-IPM, and mechanism of action via cross-linking of DNA

Drug development history

Phosphorodiamidate-based, DNA-crosslinking, bis-alkylator mustards have long been used successfully in cancer chemotherapy and include e.g. the prodrugs ifosfamide andcyclophosphamide. To demonstrate that known drugs of proven efficacy could serve as the basis of efficacious hypoxia-activated prodrugs, the 2-nitroimidizole HAP of the active phosphoramidate bis-alkylator derived from ifosfamide was synthesized. The resulting compound, TH-281, had a high HCR (hypoxia cytotoxicity ratio), a quantitative assessment of its hypoxia selectivity. Subsequent structure-activity relationship (SAR) studies showed that replacement of the chlorines in the alkylator portion of the prodrug with bromines improved potency about 10-fold. The resulting, final compound is evofosfamide (TH-302).[8]

Synthesis

Evofosfamide can be synthesized in 7 steps.[9][10]

  1. CPhI.cn: Synthetic routes to explore anti-pancreatic cancer drug Evofosfamide, 22 Jan 2015
  2.  Synthetic route Reference: International patent application WO2007002931A2

Formulation

The evofosfamide drug product formulation used until 2011 was a lyophilized powder. The current drug product formulation is a sterile liquid containing ethanol,dimethylacetamide and polysorbate 80. For intravenous infusion, the evofosfamide drug product is diluted in 5% dextrose in WFI.[11]

Diluted evofosfamide formulation (100 mg/ml evofosfamide, 70% ethanol, 25% dimethylacetamide and 5% polysorbate 80; diluted to 4% v/v in 5% dextrose or 0.9% NaCl) can cause leaching of DEHP from infusion bags containing PVC plastic.[12]

Clinical trials

Overview and results

Evofosfamide (TH-302) is currently being evaluated in clinical studies as a monotherapy and in combination with chemotherapy agents and other targeted cancer drugs. The indications are a broad spectrum of solid tumor types and blood cancers.

Evofosfamide clinical trials (as of 21 November 2014)[13] sorted by (Estimated) Primary Completion Date:[14]


Both, evofosfamide and ifosfamide have been investigated in combination with doxorubicin in patients with advanced soft tissue sarcoma. The study TH-CR-403 is a single arm trial investigating evofosfamide in combination with doxorubicin.[35] The study EORTC 62012 compares doxorubicin with doxorubicin plus ifosfamide.[36] Doxorubicin and ifosfamide are generic products sold by many manufacturers.Soft tissue sarcoma

The indirect comparison of both studies shows comparable hematologic toxicity and efficacy profiles of evofosfamide and ifosfamide in combination with doxorubicin. However, a longer overall survival of patients treated with evofosfamide/doxorubicin (TH-CR-403) trial was observed. The reason for this increase is probably the increased number of patients with certain sarcoma subtypes in the evofosfamide/doxorubicin TH-CR-403 trial, see table below.

However, in the Phase 3 TH-CR-406/SARC021 study (conducted in collaboration with the Sarcoma Alliance for Research through Collaboration (SARC)), patients with locally advanced unresectable or metastatic soft tissue sarcoma treated with evofosfamide in combination with doxorubicin did not demonstrate a statistically significant improvement in OS compared with doxorubicin alone (HR: 1.06; 95% CI: 0.88 – 1.29).

Metastatic pancreatic cancer

Both, evofosfamide and protein-bound paclitaxel (nab-paclitaxel) have been investigated in combination with gemcitabine in patients with metastatic pancreatic cancer. The study TH-CR-404 compares gemcitabine with gemcitabine plus evofosfamide.[39] The study CA046 compares gemcitabine with gemcitabine plus nab-paclitaxel.[40] Gemcitabine is a generic product sold by many manufacturers.

The indirect comparison of both studies shows comparable efficacy profiles of evofosfamide and nab-paclitaxel in combination with gemcitabine. However, the hematologic toxicity is increased in patients treated with evofosfamide/gemcitabine (TH-CR-404 trial), see table below.

In the Phase 3 MAESTRO study, patients with previously untreated, locally advanced unresectable or metastatic pancreatic adenocarcinoma treated with evofosfamide in combination with gemcitabine did not demonstrate a statistically significant improvement in overall survival (OS) compared with gemcitabine plus placebo (hazard ratio [HR]: 0.84; 95% confidence interval [CI]: 0.71 – 1.01; p=0.0589).

Drug development risks

Risks published in the quarterly/annual reports of Threshold and Merck KGaA that could affect the further development of evofosfamide (TH-302):

Risks related to the formulation

The evofosfamide formulation that Threshold and Merck KGaA are using in the clinical trials was changed in 2011[43] to address issues with storage and handling requirements that were not suitable for a commercial product. Additional testing is ongoing to verify if the new formulation is suitable for a commercial product. If this new formulation is also not suitable for a commercial product another formulation has to be developed and some or all respective clinical phase 3 trials may be required to be repeated which could delay the regulatory approvals.[44]

Risks related to reimbursement

Even if Threshold/Merck KGaA succeed in obtaining regulatory approvals and bringing evofosfamide to the market, the amount reimbursed for evofosfamide may be insufficient and could adversely affect the profitability of both companies. Obtaining reimbursement for evofosfamide from third-party and governmental payors depend upon a number of factors, e.g. effectiveness of the drug, suitable storage and handling requirements of the drug and advantages over alternative treatments.

There could be the case that the data generated in the clinical trials are sufficient to obtain regulatory approvals for evofosfamide but the use of evofosfamide has a limited benefit for the third-party and governmental payors. In this case Threshold/Merck KGaA could be forced to provide supporting scientific, clinical and cost effectiveness data for the use of evofosfamide to each payor. Threshold/Merck KGaA may not be able to provide data sufficient to obtain reimbursement.[45]

Risks related to competition

Each cancer indication has a number of established medical therapies with which evofosfamide will compete, for example:

  • If approved for commercial sale for pancreatic cancer, evofosfamide would compete with gemcitabine (Gemzar), marketed by Eli Lilly and Company; erlotinib (Tarceva), marketed by Genentech and Astellas Oncology; protein-bound paclitaxel (Abraxane), marketed by Celgene; and FOLFIRINOX, which is a combination of generic products that are sold individually by many manufacturers.
  • If approved for commercial sale for soft tissue sarcoma, evofosfamide could potentially compete with doxorubicin or the combination of doxorubicin and ifosfamide, generic products sold by many manufacturers.[46]

Risks related to manufacture and supply

Threshold relies on third-party contract manufacturers for the manufacture of evofosfamide to meet its and Merck KGaA’s clinical supply needs. Any inability of the third-party contract manufacturers to produce adequate quantities could adversely affect the clinical development and commercialization of evofosfamide. Furthermore, Threshold has no long-term supply agreements with any of these contract manufacturers and additional agreements for more supplies of evofosfamide will be needed to complete the clinical development and/or commercialize it. In this regard, Merck KGaA has to enter into agreements for additional supplies or develop such capability itself. The clinical programs and the potential commercialization of evofosfamide could be delayed if Merck KGaA is unable to secure the supply.[47]

History

Date Event
Jun 2005 Threshold files evofosfamide (TH-302) patent applications in the U.S.[48]
Jun 2006 Threshold files an evofosfamide (TH-302) patent application in the EU and in Japan[49]
Sep 2011 Threshold starts a Phase 3 trial (TH-CR-406) of evofosfamide in combination with doxorubicin in patients with soft tissue sarcoma
Feb 2012 Threshold signs an agreement with Merck KGaA to co-develop evofosfamide
Apr 2012 A Phase 2b trial (TH-CR-404) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer meets primary endpoint
Jan 2013 Merck KGaA starts a global Phase 3 trial (MAESTRO) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer
Dec 2015 two Phase 3 trials fail, Merck will not apply for a license

CLIP

CLIP

Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

*Corresponding authors
aDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, UK
E-mail: stuart.conway@chem.ox.ac.uk
bCancer Research UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, UK
Org. Chem. Front., 2015,2, 1026-1029

DOI: 10.1039/C5QO00211G

http://pubs.rsc.org/en/content/articlelanding/2015/qo/c5qo00211g/unauth#!divAbstract

http://www.rsc.org/suppdata/c5/qo/c5qo00211g/c5qo00211g1.pdf

Hypoxia, regions of low oxygen, occurs in a range of biological environments, and is involved in human diseases, most notably solid tumours. Exploiting the physiological differences arising from low oxygen conditions provides an opportunity for development of targeted therapies, through the use of bioreductive prodrugs, which are selectively activated in hypoxia. Herein, we describe an improved method for synthesising the most widely used bioreductive group, 2-nitroimidazole. The improved method is applied to an efficient synthesis of the anti-cancer drug Evofosfamide (TH-302), which is currently in Phase III clinical trials for treatment of a range of cancers.

Graphical abstract: Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

Image result for Evofosfamide

(1-Methyl-2-nitro-1H-imidazol-5-yl)-N,N–bis(2-bromoethyl) phosphordiamidate (TH- 302)

The residue was then purified by semi-preparative HPLC on a Phenomenex Luna (C18(2), 10 µm, 250 × 10 mm) column, eluting with H2O and methanol (50 – 70% methanol over 10 min, then 1 min wash with methanol, 5 mL/min flow rate) to afford TH-302 as a yellow gum: vmax (solid) cm-1 : 3212 (br), 1489 (m), 1350 (m), 1105 (m), 1004 (s); δH (DMSO-D6, 400 MHz) 7.25 (1H, s, CH), 5.10–4.90 (2H, m, NHCH2CH2Br), 4.98 (2H, d, J 7.8, CH2O), 3.94 (3H, s, CH3), 3.42 (4H, t, J 7.0, NHCH2CH2Br), 3.11 (4H, dt, J 9.8, 7.2, NHCH2CH2Br); δC (DMSO-D6, 126 MHz) 146.1, 134.2 (d, J 7.5, OCH2CN), 128.2, 55.6 (d, J 4.6, CH2O), 42.7, 34.2 (d, J 26.4, CH2Br), 34.1; δP (DMSO-D6, 202 MHz) 15.4; HRMS m/z (ESI− ) [found; (M-H)− 447.9216, C9H16 79Br81BrN5O4P requires (M-H)− 447.9213]; m/z (ESI+ ) 448.0 ([M-H]− , 60%, [C9H15 79Br81BrN5O4P] − ), 493.9 ([M+formate] − , 100%, [C10H17 79Br81BrN5O6P] − ). These data are in good agreement with the literature values.4

4 J.-X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J. W. Evans, L. Lan, G. Lorente, M. Banica, D. Jung, J. Wang, H. Ma, X. Li, Z. Yang, R. M. Hoffman, W. S. Ammons, C. P. Hart and M. Matteucci, J. Med. Chem., 2008, 51, 2412–2420.

J. Med. Chem., 2008, 51, 2412–2420/……………….1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N-bis(2-bromoethyl)
phosphordiami-date (3b). Compound 3b was synthesized by a procedure similar to that described for 3a and obtained as an off-white solid in 47.6% yield.

1H NMR (DMSO-d6) δ: 7.22 (s, 1H), 5.10–5.00 (m, 2H), 4.97 (d, J ) 7.6 Hz, 2H), 3.94 (s, 3H), 3.42 (t, J ) 7.2 Hz, 4H), and 3.00–3.20 (m, 4H).

13C NMR (DMSOd6)δ: 146.04, 134.16 (d, J ) 32 Hz), 128.17, 55.64, 42.70, 34.33,and 34.11 (d, J ) 17.2 Hz).

31P NMR (DMSO-d6) δ: -11.25.
HRMS: Calcd for C9H16N5O4PBr2, 446.9307; found, 446.9294.

CLIP

Synthesis Route reference WO2007002931A2

Med J.. Chem. 2008, 51, 2412-2420

From compound S-1 starting aminoacyl protection is S-2 , a suspension of NaH grab α -proton, offensive, ethyl, acidification, introduction of an aldehyde group, S-3followed by condensation with the amino nitrile, off N- acyl ring closure, migration rearrangement amino imidazole compound S-. 8 , the amino and sodium nitrite into a diazonium salt, raising the temperature, nitrite anion nucleophilic attack diazonium salt obtained nitro compound S-9, under alkaline conditions ester hydrolysis gives acid S-10 , followed by NEt3 under the action of isobutyl chloroformate and the reaction mixed anhydride formed by of NaBH 4 reduction to give the alcohol S-. 11 , [use of NaBH 4 reduction of the carboxyl group is another way and the I 2 / of NaBH 4 ] , to give S-11 later, the DIAD / PPh3 3 under the action via Mitsunobu linking two fragments obtained reaction Evofosfamide

Image result for Evofosfamide.

PATENT

http://www.google.co.in/patents/WO2015051921A1?cl=en

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (Rt = 7,7 min): 97,9% (a/a).

HPLC data was obtained using Agilent 1100 series HPLC from agilent technologies using an Column: YMC-Triart CI 8 3μ, 100 x 4,6 mm Solvent A: 950 ml of ammonium acetate/acetic acid buffer at pH = 6 + 50 ml acetonitril; Solvent B: 200 ml of ammonium acetate/acetic acid buffer at pH = 6 + 800 ml acetonitril; Flow: 1,5 ml/min; Gradient: 0 min: 5 % B, 2 min: 5 % B, 7 min: 20 % B, 17 min: 85% B, 17, 1 min: 5% B, 22 min: 5% B.

PATENT

WO2007002931

http://www.google.com/patents/WO2007002931A2?cl=en

Example 8

Synthesis of Compounds 25, 26 [0380] To a solution of 2-bromoethylammmonium bromide (19.4 g) in DCM (90 mL) at – 1O0C was added a solution OfPOCl3 (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14.1 mL) in DCM (25 mL). The reaction mixture was filtered, the filtrate concentrated to ca. 30% of the original volume and filtered. The residue was washed with DCM (3×25 mL) and the combined DCM portions concentrated to yield a solid to which a mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary evaporator, the resulting solution chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:

Figure imgf000127_0001

Isophosphoramide mustard

Figure imgf000127_0002

can be synthesized employing the method provided in Example 8, substituting 2- bromoethylammmonium bromide with 2-chloroethylammmonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et al., supra).

The phosphoramidate alkylator toxin:

Figure imgf000127_0003

was transformed into compounds 24 and 25, employing the method provided in Example 6 and the appropriate Trigger-OH.

Example 25

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

Figure imgf000143_0002

A suspension of the nitro ester (39.2 g, 196.9 rnmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HCl and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over MgS O4 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid (“nitro acid”) as a light brown solid (32.2 g, 95%). Example 26

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

Figure imgf000144_0001

A mixture of the nitro acid (30.82 g, 180.23 mmol) and triethylamine (140 niL, 285 mmol) in anhydrous THF (360 mL) was stirred while the reaction mixture was cooled in a dry ice-acetonitrile bath (temperature < -20 0C). Isobutyl chloroformate (37.8 mL, 288 mmol) was added drop wise to this cooled reaction mixture during a period of 10 min and stirred for 1 h followed by the addition of sodium borohydride (36 g, 947 mmol) and dropwise addition of water during a period of 1 h while maintaining a temperature around or less than O0C. The reaction mixture was warmed up to O0C. The solid was filtered off and washed with THF. The combined THF portions were evaporated to yield l-N-methyl-2- nitroimidazole-5-methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.

PATENT

WO-2015051921

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (Rt = 7,7 min): 97,9% (a/a).

PATENT

WO 2016011195

http://google.com/patents/WO2016011195A1?cl=en

Figure 1 provides the differential scanning calorimetry (DSC) data of crystalline solid form A of TH-302.

Figure 2 shows the 1H-NMR of crystalline solid form A of TH-302.

Figure 5 shows the Raman Spectra of TH-302 (Form A)

Scheme 1 illustrates a method of preparing TH-302.

Scheme 1: Process for the Preparation of TH-302

NaOH (RGT)

Step 1. Imidazole Purified water (SLV)

Carboxylic Acid IPC: NMT 1.0% SM by HPLC

HCI (RGT)

IPC: pH 1.0 ± 0.5

IPC: NMT 1.0% water by KF

TH-302

MW = 449.0

SM = Starting Material INT = Intermediate IPC = In-process Control RGT = Reagent SLV = Solvent MW = Molecular Weight LOD = Loss on drying NMT = Not more than NLT = Not less than

TH-302 can be prepared by hydro lyzing (l-methyl-2-nitro-lH-imidazol-5-yl) ethyl ester above for example under aqueous conditions with a suitable base catalyst (e.g. NaOH in water at room temperature). The imidazole carboxylic acid prepared by this method can be used without further purification. However, it has been found that treating the dried crude intermediate product with a solvent such as acetonitrile, ethyl acetate, n-heptane, acetone, dimethylacetamide, dimethylformamide, 1, 4-dioxane, ethylene glycol, 2-propanol, 1-propanol, tetrahydrofuran (1 : 10 w/v) or combinations thereof in a vessel with heating, followed by cooling and filtration through a filtration aid with acetone decreased the number and levels of impurities in the product. The number and levels of impurities could be further reduced by treating the dried crude product with water (1 :5.0 w/v) in a vessel with heating followed by cooling and filtration through a filtration aid with water.

The carboxylic acid of the imidazole can then be reduced using an excess of a suitable reducing agent (e.g. sodium borohydride in an appropriate solvent, typically aqueous. The reaction is exothermic (i.e. potentially explosive) releasing borane and hydrogen gases over several hours. It was determined that the oxygen balance of the product imidazole alcohol is about 106.9, which suggests a high propensity for rapid decomposition. It has been found that using NaOH, for example 0.01M NaOH followed by quenching the reaction with an acid. Non-limiting examples of acids include, but are not limited to water, acetic acid, hydrobromic acid, hydrochloric acid, sodium hydrogen phosphate, sulfuric acid, citric acid, carbonic acid, phosphoric acid, oxalic acid, boric acid and combinations thereof. In some embodiments, the acid may diluted with a solvent, such as water and/or tetrahydrofuran. In some embodiments, acetic acid or hydrochloric acid provide a better safety profile, presumably because it is easier to control the temperature during the addition of the reducing agent and the excess reducing agent is destroyed after the reaction is complete. This also results in improved yields and fewer impurities, presumably due to reduced impurities from the reducing agent and decomposition of the product. Using this process, greater than 98.5% purity could be achieved for this intermediate. The formation of ether linkage can be accomplished by treating the product imidazole alcohol with solution of N,N’-Bis(2-bromoethyl)phosphorodiamidic acid (Bromo IPM), a trisubstituted phosphine and diisopropyl azodicarboxylate in tetrahydrofuran at room temperature to afford TH-302. It has been found that by recrystallizing the product from a solvents listed in the examples, one could avoid further purfication by column chromatography, which allowed for both reduced solvent use especially on larger scales.

Scheme 2 illustrates an alternative method of preparing TH-302.

Scheme 2: Process for the Preparation of TH-302

(SM)

ethylamine mide (SM) 04.9 ) SLV) , RGT) ter by KF

NT)

MW = 449.0

Example 1: Synthesis of TH-302

Step 1 – Preparation intermediate imidazole carboxylic

I T)

Crude imidazole carboxylic acid ethyl ester (1 : 1.0 w/w) was taken in water (1 : 10.0 w/v) at 25± 5°C and cooled to 17± 3°C. A 2.5 N sodium hydroxide solution (10 V) was added slowly at 17±3°C. The reaction mass was warmed to 25±5°C and monitored by HPLC. After the completion of reaction, the reaction mass was cooled to 3±2°C and pH of the reaction mass adjusted to 1=1=0.5 using 6 M HC1 at 3±2°C. The reaction mass was then warmed to 25±5°C and extracted with ethyl acetate (3 x 10 V). The combined organic layers

were washed with water (1 x 10 V) followed by brine (1 x 10 V). The organic layer was dried over sodium sulfate (3 w/w), filtered over Celite and concentrated. n-Heptane (1.0 w/v) was added and the the reaction mixture was concentrated below 45°C to 2.0 w/v. The reaction mass was cooled to 0±5°C. The solid was filtered, and the bed was washed with n-heptane (1 x 0.5 w/v) and dried at 35±5°C. In a vessel, acetone (1 : 10 w/v) was added. Dry crude imidazole carboxylic acid (ICA) from 1.12 was added to the acetone. The mixture was warmed to 45±5°C and was stirred for 30 minutes. The mass was cooled to 28±3°C and filtered through a Celite bed. The filter bed was washed with 1 : 1.0 w/v of acetone. Water (1 :5.0 w/v) was added to the filtrate and the mixture was concentrated. The concentrated mass was cooled to 5±5°C and stirred for 30 minutes. The material was filtered and the solid was washed 2 x 1 : 1.0 w/v of water at 3±2°C. The product was dried for 2 hours at 25±5°C and then at 45±5°C. As can be seen below, the number and levels of impurities are decreased.

Table I: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Imidazole alcohol:

CI^Oi-Bu

T

o

Imidazole carboxylic acid (1.0 w/w) was taken in tetrahydrofuran (10 w/v) under nitrogen atmosphere at 25±5°C. The reaction mass was cooled to -15±5°C. Triethylamine (1 : 1.23 w/v) was added slowly over a period of 1 hour maintaining the temperature at – 15±5°C. The reaction mass was stirred at -15±5°C for 15-20 min. Isobutylchloroformate (1 : 1.14 w/v) was added slowly over a period of 1 hour maintaining the temperature at – 15±5°C. The reaction mass was stirred at -15±5°C for 30-40 min. A solution of sodium borohydride (1 : 1.15 w/w) in 0.01 M aqueous sodium hydroxide (2.2 w/v) was divided into 6 lots and added to the above reaction mass while maintaining the temperature of the reaction mass between 0±10°C for 40-60 min for each lot. The reaction mass was warmed to 25±5°C and stirred until imidazole carboxylic acid content < 5.0 % w/w. The reaction mass was filtered and the bed was washed with tetrahydrofuran (1 :2.5 w/v). The filtrate was quenched with 10 % acetic acid in water at 25±5°C. Reaction mass stirred for 50-60 minutes at 25±5°C. The filtrate was concentrated below 45°C until no distillate was observed. The mass was cooled to 5±5°C and stirred for 50-60 minutes. The reaction mass was filtered and the solid was taken in ethanol (1 :0.53 w/v). The reaction mass was cooled 0±5°C and stirred for 30-40 min. The solid was filtered and the bed was washed ethanol (1 :0.13 w/v). The solid was dried at 40±5 °C.

Step 3 – Synthesis of intermediate Br-IPM:

P

o

M
W = 286.7 MW = 204.9 Purified water (SLV, RGT)

Acetone (SLV)

IPC: NMT 1.0% water by KF

2-Bromoethylamine hydrobromide (1 : 1.0 w/w) and POBr^ (1 :0.7 w/w) were taken in DCM (1 :2 w/v) under nitrogen atmosphere. The reaction mixture was cooled to -70±5°C. Triethylamine (1 : 1.36 w/v) in DCM (1 :5 w/v) was added to the reaction mass at -70±5°C. The reaction mass was stirred for additional 30 min at -70±5°C. Reaction mass was warmed to 0±3°C and water (1 :1.72 w/v) was added. The reaction mixture was stirred at 0±3°C for 4 hrs. The solid obtained was filtered and filter cake was washed with ice cold water (2 x 1 :0.86 w/v) and then with chilled acetone (2 x 1 :0.86 w/v). The solid was dried in at 20±5°C.

Step 4 Synthesis ofTH-302

TH-302

MW = 449.0

Imidazole alcohol (IA) (1 : 1.0 w/w), Bromo-IPM (1 :2.26 w/w) and

triphenylphosphine (1 :2.0 w/w) were added to THF (1 : 13.5 w/v) at 25±5°C. The reaction

mass was cooled to 0±5°C and DIAD (1.5 w/v) was added. The reaction mixture warmed to 25±5°C and stirred for 2 hours. Progress of the reaction was monitored by HPLC. Solvent was removed below 50°C under vacuum. Solvent exchange with acetonitrile (1 :10.0 w/v) below 50°C was performed. The syrupy liquid was re-dissolved in acetonitrile (1 : 10.0 w/v) and the mixture was stirred at -20±5°C for 1 hour. The resulting solid was filtered and the filtrate bed was washed with chilled acetonitrile (1 : 1.0 w/v). The acetonitrile filtrate was concentrated below 50°C under vacuum. The concentrated mass was re-dissolved in ethyl acetate (1 : 10.0 w/v) and concentrated below 50°C under vacuum. The ethyl acetate strip off was repeated two more times. Ethyl acetate (1 : 10.0 w/v) and silica gel (230-400 mesh, 1 :5.3 w/w) were added to the concentrated reaction mass. The mixture was concentrated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was charged to the above mass and the mixture was evaporated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was again added to the above mass and the solid was filtered and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in a mixture oftoluene (1 :7.1 w/v) and n-heptane (1 :21.3 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane

(1 : 1.0 w/v). The solid was re-suspended in a mixture of toluene (1 : 10.6 w/v) and n-heptane (1 : 10.6 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in acetone (1 : 19.0 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with acetone (1 : 1.0 w/v). The acetone washes were repeated 3 more times. Filtrates from the above acetone washings were combined and concentrated below 40°C under vacuum. The residue dissolved in ethyl acetate (1 : 10.0 w/v) and concentrated below 40°C under vacuum. The ethyl acetate strip off was repeated one more time. The residue was re-dissolved in ethyl acetate (1 :5.5 w/v), cooled to 0±3°C and stirred at 0±3°C for 2 h and then at -20±5°C for 2 h. The solid was filtered and the solid was washed with ethyl acetate (1 :0.10 w/v). The solid was dissolved in ethyl acetate (1 : 10.0 w/v) at 50±5°C and the resulting solution was filtered through a cartridge filter. The filtrate was concentrated to ~4.0 w/w and stirred at 0±3°C for 4 hours. The solid was filtered and washed with ethyl acetate (1 :0.10 w/v). The crystallization from ethyl acetate was repeated and TH-302 was dried at 25±5°C. Table 2 shows how the process reduces solvent use.

Table 2: Solvent and Silica Gel Usage for 10 kg Column and 10 kg Column-free Purification

“Amounts are estimated from a 5 kg batch

b Amounts are estimated

Example 2: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel, water (1 :7.0 w/v) was added. Dry crude ICA was added to the water. The reaction mixture was heated to 85±5°C until a clear solution was obtained. The reaction mass was cooled to 20±5°C and filtered through a Celite bed. The filter bed was washed with 2 x 5.0 of n-heptane. The material was dried for 2 hours at 25±5°C and then 45±5°C. As can be seen below, the number and levels of impurities decreased.

Table 3: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 3: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel

ethanol (1 :30.0 w/v) and ICA (1 : 1.0 w/w) were mixed. The reaction mixture was stirred at

25±5°C for 30 minutes and filtered. Water (1 :50.0 w/v) was added and the mixture was

stirred at 50±5°C for 30 minutes. The reaction mass was cooled to 20±5°C and filtered. The isolated solid was dried at 25±5°C for 24 hours. As can be seen below, the number and levels

of impurities generally decreased.

Table 4: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 4: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel

acetonitrile (1 :20.0 w/v) and ICA (1 : 1.0 w/w) were mixed at 25±5°C for one hour. The

reaction mixture was filtered and the solution was concentrated to ~ 6 volumes. The mixture

was then cooled to 0±5°C, stirred at this temperature for one hour and filtered. The isolated

solid was dried at 25±5°C for 24 hours. As can be seen below the number of impurities

decreased and except for TH-2717, the amounts also decreased.

Table 5: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 5: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with dimethylacetamide and water.

Example 6: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with dimethylforamide and water.

Example 7: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0109] Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a 1,4-dioxane and water mixture.

Example 8: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a mixture of ethylene glycol and water.

Example 9: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with 2-propanol and water.

Example 10: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0112] Crude ICA is prepared according to the method described in Example 1 and purified by treatment with 1-propanol and water.

Example 11: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0113] Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a mixture of tetrahydrofuran and water.

Example 12: Synthesis ofTH-302 using alternative procedure to quench IA:

[0114] The reduction of ICA to IA was carried out according to Example 1 except that after reaction completion and filtration of the inorganics, the filtrate was quenched with 1.5 M hydrochloric acid.

Example 13: Synthesis ofTH-302 using alternative procedure to quench IA:

[0115] The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with 1.5 M

hydrobromic acid.

Example 14: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with

hydrobromic acid in acetic acid.

Example 15: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was treated with sodium

hydrogen phosphate.

Example 16: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with 10% acetic

acid in tetrahydrofuran.

Example 17: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with water.

Example 18: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with sulfuric acid.

Example 19: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with citric acid.

Example 20: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is treated with carbonic acid.

Example 21: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is treated with phosphoric

acid.

Example 22: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with oxalic acid.

Example 23: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after reaction completion and filtration of the inorganics, the filtrate is quenched with boric acid.

Example 24: Synthesis ofTH-302 using alternative procedure to purify TH-302:

[0126] Coupling of bromo-IPM and IA was performed according to Example 1 except that after concentration of the reaction mixture, ethyl acetate (1 : 10 w/v) was added to the concentrated mass. The mixture was stirred at -55±5°C for 2 hours. The resulting solid was filtered and washed with chilled EtOAc (1 :2.0 w/v). The solid was reslurried in ethyl acetate (1 : 10 w/v) at -55±5°C for 2 hours, filtered and the solid was washed with chilled ethyl acetate (1 : 1.0 w/v). The filtrates from both filtrations were combined and treated with silica gel (1 :5.3 w/w) of silica gel (230-400 mesh). The mixture was concentrated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was again added to the above mass and the solid was filtered and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in a mixture of toluene (1 :7.1 w/v) and n-heptane (1 :21.3 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was re-suspended in a mixture of toluene (1 : 10.6 w/v) and n-heptane (1 :10.6 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in acetone (1 : 19.0 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with acetone (1 : 1.0 w/v). The acetone washes were repeated 3 more times. Filtrates from the above acetone washings were combined and concentrated below 40°C under vacuum. The residue dissolved in ethyl acetate (1 :5.5 w/v), cooled to 0±3°C and stirred at 0±3°C for 2 h and then at -20±5°C for 2 h. The solid was filtered and the solid was washed with ethyl acetate (1 :0.10 w/v). The solid was dissolved in ethyl acetate (1 :27 w/v), stirred at 50±5°C and filtered through Celite. The filtrate was concentrated to ~4.0 w/w and stirred at 0±5°C for 4 hours. The recrystallization from ethyl acetate was repeated and TH- 302 was dried at 25±5°C. Table 4 shows how the process reduced solvent use.

Table 4: Estimated Solvent and Silica Gel Usage for Column and 10 kg Column-free

(EtOAc) Purification

References

  1.  WHO Drug Information; Recommended INN: List 73
  2.  Adopted Names of the United States Adopted Names Council
  3.  Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M; et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q.PMID 18257544.
  4. Jump up^ Threshold Pharmaceuticals and Merck KGaA Announce Global Agreement to Co-Develop and Commercialize Phase 3 Hypoxia-Targeted Drug TH-302 – Press release from 3 February 2012
  5. Jump up^ Threshold Pharmaceuticals Form 8-K from 3 Nov 2014
  6. Jump up^ Weiss, G.J., Infante, J.R., Chiorean, E.G., Borad, M.J., Bendell, J.C., Molina, J.R., Tibes, R., Ramanathan, R.K., Lewandowski, K., Jones, S.F., Lacouture, M.E., Langmuir, V.K., Lee, H., Kroll, S., Burris, H.A. (2011) Phase 1 Study of the Safety, Tolerability, and Pharmacokinetics of TH-302, a Hypoxia-Activated Prodrug, in Patients with Advanced Solid Malignancies. Clinical Cancer Research 17, 2997–3004.doi:10.1158/1078-0432.CCR-10-3425
  7.  J. Thomas Pento (2011). “TH-302”. Drugs of the Future. 36 (9): 663–667.doi:10.1358/dof.2011.036.09.1678337.
  8. Jump up^ Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M; et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q.PMID 18257544.
  9. Jump up^ CPhI.cn: Synthetic routes to explore anti-pancreatic cancer drug Evofosfamide, 22 Jan 2015
  10.  Synthetic route Reference: International patent application WO2007002931A2
  11. Jump up^ FDA Advisory Committee Briefing Materials Available for Public Release, TH-302: Pediatric oncology subcommittee of the oncologic drugs advisory committee (ODAC) meeting, December 4, 2012
  12. Jump up^ AAPS 2014 – Measurement of Diethylhexyl Phthalate (DEHP) Leached from Polyvinyl Chloride (PVC) Containing Plastics by Infusion Solutions Containing an Organic Parenteral Formulation – Poster W4210, Nov 5, 2014
  13. Jump up^ ClinicalTrials.gov
  14.  The Primary Completion Date is defined as the date when the final subject was examined or received an intervention for the purposes of final collection of data for the primary outcome.
  15. Jump up^ Detailed Results From Positive Phase 2b Trial of TH-302 in Pancreatic Cancer at AACR Annual Meeting – Press release from 30 March 2012
  16. Jump up^ TH-302 Plus Gemcitabine vs. Gemcitabine in Patients with Untreated Advanced Pancreatic Adenocarcinoma. Borad et al. Presentation at the European Society for Medical Oncology (ESMO) 2012 Congress, September 2012. (Abstract 6660)
  17. Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  18.  Updated Phase 2 Results Including Analyses of Maintenance Therapy With TH-302 Following Induction Therapy With TH-302 Plus Doxorubicin in Soft Tissue Sarcoma – Press release from 15 November 2012
  19.  TH-302 Maintenance Following TH-302 Plus Doxorubicin Induction: The Results pf a Phase 2 Study of TH-302 in Combination with Doxorubicin in Soft Tissue Sarcoma. Ganjoo et al. Connective Tissue Oncology Society (CTOS) 2012 Meeting, November 2012
  20. Jump up^ Chawla, S.P., Cranmer, L.D., Van Tine, B.A., Reed, D.R., Okuno, S.H., Butrynski, J.E., Adkins, D.R., Hendifar, A.E., Kroll, S., Ganjoo, K.N., 2014. Phase II Study of the Safety and Antitumor Activity of the Hypoxia-Activated Prodrug TH-302 in Combination With Doxorubicin in Patients With Advanced Soft Tissue Sarcoma. Journal of Clinical Oncology 32, 3299–3306.doi:10.1200/JCO.2013.54.3660
  21. Jump up^ Follow-Up Data From a Phase 1/2 Clinical Trial of TH-302 in Solid Tumors – Press release from 12 October 2010
  22.  TH-302 Continues to Demonstrate Promising Activity in Pancreatic Cancer Phase 1/2 Clinical Trial – Press release from 24 January 2011
  23. Jump up^ TH-302, a tumor selective hypoxia-activated prodrug, complements the clinical benefits of gemcitabine in first line pancreatic cancer. Borad et al. ASCO Gastrointestinal Cancers Symposium, January 2011
  24. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  25. Jump up^ Borad et al., ESMO Annual Meeting, October 2010
  26. Jump up^ Video interview of Stefan Oschmann, CEO Pharma at Merck – Merck Serono Investor & Analyst Day 2014 – 18 Sept 2014 – 2:46 min – Youtube
  27. Jump up^ The Phase 3 Trial of TH-302 in Patients With Advanced Soft Tissue Sarcoma Will Continue as Planned Following Protocol-Specified Interim Analysis – Press release from 22 September 2014
  28. Jump up^ Threshold Pharmaceuticals’ Partner Merck KGaA, Darmstadt, Germany, Completes Target Enrollment in the TH-302 Phase 3 MAESTRO Study in Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma – Press release from 3 November 2014
  29.  Data From Ongoing Phase 1/2 Trial of TH-302 Plus Bevacizumab (Avastin(R)) in Patients With Recurrent Glioblastoma – Press release from 30 May 2014
  30. Jump up^ Phase 1/2 Study of Investigational Hypoxia-Targeted Drug, TH-302, and Bevacizumab in Recurrent Glioblastoma Following Bevacizumab Failure. Brenner, et al. 2014 ASCO, 7 – 30 May 2014
  31. Jump up^ Phase 1/2 Interim Data Signaling Activity of TH-302 Plus Bevacizumab (Avastin(R)) in Patients With Glioblastoma – Press release from 17 November 2014
  32. Jump up^ Threshold Pharmaceuticals’ Partner Merck KGaA, Darmstadt, Germany, Completes Target Enrollment in the TH-302 Phase 3 MAESTRO Study in Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma – Press release from 3 November 2014
  33. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  34. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  35. Jump up^ Chawala SP, et al. J Clin Oncol. 2014 (54) 3660 doi:10.1200/JCO.2013.54.3660
  36. Jump up^ Judson I, et al. Lancet Oncol. 2014 Apr;15(4):415-23doi: 10.1016/S1470-2045(14)70063-4
  37. Jump up^ Judson I, et al. Lancet Oncol. 2014 Apr;15(4):415-23doi: 10.1016/S1470-2045(14)70063-4
  38. Jump up^ Chawala SP, et al. J Clin Oncol. 2014 (54) 3660 doi:10.1200/JCO.2013.54.3660
  39. Jump up^ Borad, M. J. et al. Randomized Phase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients With Advanced Pancreatic Cancer. Journal of Clinical Oncology (2014). doi: 10.1200/JCO.2014.55.7504
  40. Jump up^ Von Hoff, D. D. et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 369, 1691–1703 (2013). doi:10.1056/NEJMoa1304369
  41. Jump up^ Von Hoff, D. D. et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 369, 1691–1703 (2013). doi:10.1056/NEJMoa1304369
  42. Jump up^ Borad, M. J. et al. Randomized Phase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients With Advanced Pancreatic Cancer. Journal of Clinical Oncology (2014). doi: 10.1200/JCO.2014.55.7504
  43. Jump up^ Threshold Pharmaceuticals 10-K Annual report 2011 from 15 Mar 2012
  44. Jump up^ Threshold Pharmaceuticals 10-Q Quarterly report Q3/2014 from 3 Nov 14
  45. Jump up^ Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  46. Jump up^ Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  47.  Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  48.  Phosphoramidate alkylator prodrugs US8003625B2,US8507464B2, US8664204B2
  49.  Phosphoramidate alkylator prodrugs EP1896040B1and JP5180824B2
WO2007002931A2 * Jun 29, 2006 Jan 4, 2007 Threshold Pharmaceuticals, Inc. Phosphoramidate alkylator prodrugs
WO2008083101A1 * Dec 21, 2007 Jul 10, 2008 Threshold Pharmaceuticals, Inc. Phosphoramidate alkylator prodrugs for the treatment of cancer
WO2010048330A1 * Oct 21, 2009 Apr 29, 2010 Threshold Pharmaceuticals, Inc. Treatment of cancer using hypoxia activated prodrugs
WO2015051921A1 * Oct 10, 2014 Apr 16, 2015 Merck Patent Gmbh Synthesis of 1-alkyl-2-amino-imidazol-5-carboxylic acid ester via calpha-substituted n-alkyl-glycine ester derivatives
Reference
1 * DUAN, J.-X. ET AL.: “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs“, JOURNAL OF MEDICINAL CHEMISTRY, vol. 51, 2008, pages 2412 – 2420, XP008139620, DOI: doi:10.1021/jm701028q
Evofosfamide
TH-302.svg
Names
IUPAC name

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate
Other names

TH-302; HAP-302
Identifiers
918633-87-1 Yes
ChemSpider 10157061 Yes
Jmol-3D images Image
PubChem 11984561
Properties
C9H16Br2N5O4P
Molar mass 449.04 g·mol−1
6 to 7 g/l

///////////Orphan Drug Status, soft tissue sarcoma,  Pancreatic cancer, Fast track,  TH-302, TH 302, эвофосфамид ,  إيفوفوسفاميد ,  艾伏磷酰胺 , Evofosfamide, 918633-87-1, PHASE 3

O=[N+]([O-])c1ncc(COP(=O)(NCCBr)NCCBr)n1C

AVORALSTAT


2D chemical structure of 918407-35-9

Avoralstat, BCX4161,

CAS  918407-35-9
UNII: UX17773O15

513.5513, C28-H27-N5-O5

2-Pyridinecarboxylic acid, 3-(2-(((4-(aminoiminomethyl)phenyl)amino)carbonyl)-4-ethenyl-5-methoxyphenyl)-6-(((cyclopropylmethyl)amino)carbonyl)-

3-(2-((4-Carbamimidoylphenyl)carbamoyl)-4-ethenyl-5-methoxyphenyl)-6-((cyclopropylmethyl)carbamoyl)pyridine-2-carboxylic acid

Hereditary angioedema (HAE)

Kallikrein inhibitor

BioCryst Pharmaceuticals

Biocryst Logo

BioCryst is also investigating second-generation plasma kallikrein inhibitors to avoralstat, for treating HAE (in February 2016, this program was listed as being in preclinical development).

2D chemical structure of 918407-35-9

Prevent acute attacks in patients with hereditary angioedema (HAE); Treat hereditary angioedema (HAE)

U.S. – Fast Track (Treat hereditary angioedema (HAE));
U.S. – Orphan Drug (Prevent acute attacks in patients with hereditary angioedema (HAE))

26 Feb 2016Clinical trials in Hereditary angioedema (Prevention) in USA (PO, Hard-gelatin capsule) before February 2016

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in France (PO, Soft-gelatin capsule)

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in Germany (PO, Soft-gelatin capsule)

Conditions Interventions Phases Recruitment Sponsor/Collaborators
Hereditary Angioedema|HAE Drug: BCX4161|Drug: Placebo Phase 2|Phase 3 Recruiting BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161|Drug: Placebo Phase 2 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals

Avoralstat, also known as BCX-4161, is a potent and orally active Kallikrein inhibitor and Bradykinin inhibitor. Avoralstat may be potentially useful for treatment for Hereditary angioedema. Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

Selective inhibitor of plasma kallikrein that subsequently suppresses bradykinin production

Hereditary angioedema (HAE) is a serious and potentially life-threatening rare genetic illness, caused by mutations in the C1-esterase inhibitor (C1 INH) gene, located on chromosome 11q. HAE is inherited as an autosomal dominant condition, although one quarter of diagnosed cases arise from a new mutation. HAE has been classed as an orphan disease in Europe, with an estimated prevalence of 1 in 50,000. Individuals with HAE experience recurrent acute attacks of painful subcutaneous or submucosal edema of the face, larynx, gastrointestinal tract, limbs or genitalia which, if untreated, may last up to 5 days. Attacks vary in frequency, severity and location and can be life-threatening. Laryngeal attacks, with the potential for asphyxiation, pose the greatest risk. Abdominal attacks are especially painful, and often result in exploratory procedures or unnecessary surgery. Facial and peripheral attacks are disfiguring and debilitating.

HAE has a number of subtypes. HAE type I is defined by C1 INH gene mutations which produce low levels of C1 -inhibitor, whereas HAE type II is defined by mutations which produce normal levels of ineffective C1 protein. HAE type III has separate pathogenesis, being caused by mutations in the F12 gene which codes for the serine protease known as Factor XII. Diagnostic criteria for distinguishing the subtypes of HAE, and distinguishing HAE from other angioedemas, can be found in Ann Allergy Asthma Immunol 2008; 100(Suppl 2): S30-S40 and J Allergy Clin Immunol 2004; 114: 629-37, incorporated herin by reference.

Current treatments for HAE fall into two main types. Older non-specific treatments including androgens and antifibrinolytics are associated with significant side effects, particularly in females. Newer treatments are based on an understanding of the molecular pathology of the disease, namely that C1 INH is the most important inhibitor of kallikrein in human plasma and that C1 INH deficiency leads to unopposed activation of the kallikrein-bradykinin cascade, with bradykinin the most important mediator of the locally increased vascular permeability that is the hallmark of an attack.

Approved therapies include purified plasma-derived C1 INH (Cinryze®, Berinert), the recombinant peptide kallikrein inhibitor ecallantide (Kalbitor®), and the bradykinin receptor B2 inhibitor iticabant (Firazyr®). All of the currently available targeted therapies are administered by intravenous or subcutaneous injection. There is currently no specific targeted oral chronic therapy for HAE.

There are many delivery routes for active pharmaceutical ingredients (APIs). Generally, the oral route of administration is favored. Oral administration provides a number of advantages, such as, but not limited to, patient convenience, flexibility of timing of administration, location of administration and non-invasiveness. Oral administration also provides more prolonged drug exposure compared with intermittent intravenous infusion, which may be important for drugs with schedule-dependent efficacy. For example, a drug with a short half-life can achieve a greater exposure time by either continuous infusion or by continuous oral dosing. The use of oral therapy further has the potential to reduce the cost of healthcare resources for inpatient and ambulatory patient care services.

In the pharmaceutical arts, it is known that a number of APIs cannot be administered effectively by the oral route. The main reasons why these compounds cannot be administered by the oral route are: a) rapid enzymatic and metabolic degradation; b) chemical and/or biological instability; c) low solubility in aqueous medium; and/or d) limited permeability in the gastrointestinal tract. For such compounds, non-oral routes of delivery, such as parenteral administration, mainly via intramuscular or subcutaneous injections, may be developed. However, non-oral administration poses a disadvantage for the patient as well as healthcare providers, and for this reason, it is important to develop alternative routes of administration for such compounds, such as oral routes of administration.

While the oral route of administration is the most convenient for the patient and the most economical, designing formulations for administration by the oral route involves many complications. Several methods are available to predict the ease by which an API may be formulated into a formulation suitable for administration by the oral route. Such methods include, but are not limited to, and Lipinski rule (also referred to as the Rule of Five) and the Biopharmaceutical Drug Disposition Classification System (BDDCS).

The BDDCS divides APIs into four classifications, depending on their solubility and permeability. Class I APIs have high solubility and high permeability; Class II APIs have low solubility and high permeability; Class III APIs have high solubility and low permeability; and Class IV APIs have low solubility and low permeability. APIs in higher classes in the BDDCS face greater challenges in formulating into an effective, pharmaceutically acceptable product than those in lower classes. Of the four classes, APIs falling into Class IV are the most difficult to formulate into a formulation for administration by the oral route that is capable of delivering an effective amount of the API as problems of both solubility and permeability must be addressed (note the BDDCS does not inherently address chemical stability). The role of BDDCS in drug development is described generally in L.Z. Benet J Pharm Sci. 2013, 102(1), 34-42.

Lipinski’s rule (described in Lipinski et al. Adv. Drug Deliv. Rev. 46 (1-3): 3-26) states, in general, that in order to develop a successful formulation for administration by the oral route, an API can have no more than one violation of the following criteria:

i) not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms)

ii) not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms) iii) a molecular mass less than 500 daltons

iv) an octanol-water partition coefficient log P not greater than 5.

J. Zhang et al. Medicinal Chemistry, 2006, 2, 545-553, describes a number of small molecule amidine compounds which have activity as inhibitors of kallikrein. The molecules described in this document fall into Class IV of the BDDCS as described above. The compounds are poorly soluble in aqueous and physiological fluids, and are poorly permeable as demonstrated by oral dosing in rats and in vitro experiments with Caco-2 cells.

Furthermore, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, one of the compounds described in Zhang et al., is a Class IV API and violates criteria iii) and iv) as set forth in the Lipinski Rule.

Furthermore, the compounds described in Zhang et al., including 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, exhibit poor stability with respect to oxidation in air, to light

(photodegradation) and in aqueous and physiological fluids, as well as to elevated temperatures.

Therefore, the compounds described by Zhang et al. including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, not only exhibit poor solubility and permeability characteristics, but also poor stability characteristics. As a result, such compounds are predicted to be especially difficult to formulate into an effective, orally deliverable

pharmaceutical composition that is capable of delivering an effective amount of the compound to a subject.

Polymorphism, the occurrence of different crystal forms, is a property of some molecules. A single molecule may give rise to a variety of polymorphs having distinct crystal structures and physical properties, such as, but not limited to, melting point, thermal behaviors (e.g. measured by thermogravimetric analysis (TGA), or differential scanning calorimetry (DSC), x-ray diffraction pattern, infrared absorption fingerprint, and solid state NMR spectrum. One or more of these techniques may be used to distinguish different polymorphic forms of a compound.

Discovering new polymorphic forms and solvates of a pharmaceutical product can provide alternate forms of the compound that display a number of desirable and advantageous properties, such as, but not limited to, ease of handling, ease of processing, ease of formulation, storage stability, and/or ease of purification. Further, new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof may further provide for improved pharmaceutical products, by providing compounds that are more soluble in a set of pharmaceutical excipients. Still further, the provision of new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof enlarges the repertoire of compounds that a formulation scientist has available for formulation optimization, for example by providing a pharmaceutical product with different properties, such as, but not limited to, improved processing characteristics, improved handling characteristics, improved solubility profiles, improved dissolution profile and/or improved shelf-life. Therefore, there is a need for additional polymorphs of pharmaceutically useful compounds, such as, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid and the compounds disclosed herein.

In one aspect, the present invention provides an oral formulation that is capable of delivering an effective amount of the amidine compounds described by Zhang et al. to a subject. In particular, the present invention provides an oral formulation that is capable of delivering an effective amount of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid to a subject. In one specific aspect, the 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid is present in a particular crystal form designated Form A. In light of the art suggesting the difficulties in formulating such an oral formulation, this result was unexpected.

As described herein, the amidine compounds described in Zhang et al., including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (specifically including particular crystal Form A), may now be conveniently used in oral administration and further used in oral administration for the treatment of a number of diseases and conditions in a subject, such as, but not limited to, HAE as described herein.

Avoralstat & next generation kallikrein inhibitors for HAE

Avoralstat

Avoralstat is being developed as an oral prophylactic treatment for patients suffering from Hereditary Angioedema (HAE). Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

In May 2014 BioCryst, announced that the OPuS-1 (OralProphylaxiS-1) Phase 2a proof of concept clinical trial met its primary efficacy endpoint, several secondary endpoints and all other objectives established for the trial. OpuS-1 enrolled 24 HAE patients with a history of HAE attack frequency of at least 1 per week. Treatment with avoralstat demonstrated a statistically significant mean attack rate reduction of 0.45 attacks per week versus placebo, p<0.001. The mean attack rate per week was 0.82 on BCX4161 treatment, compared to 1.27 on placebo.

In December 2014, BioCryst initiated enrollment in OPuS-2 (Oral ProphylaxiS-2). OPuS-2 is a blinded, randomized, 12-week, three-arm, parallel cohort design trial evaluating the efficacy and safety of two different dose regimens of avoralstat administered three-times daily, 300 mg and 500 mg, compared with placebo. The primary efficacy endpoint for the trial will be the mean angioedema attack rate, which will be reported for each avoralstat dose group compared to placebo. The trial is being conducted in the U.S., Canada and Europe. On October 8, 2015, announced that it has completed enrollment of approximately 100 HAE patients with a history of moderately frequent to very frequent attacks in OPuS-2. BioCryst expects to report the OPuS-2 trial results in early 2016.

PATENT

WO200234711

http://www.google.com/patents/WO2002034711A1?cl=en

PATENT

WO2015134998

PATENT

WO2016029214

Examples

Example 1 – Synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl- phenyll-6-(cvclopropylmethyl-carbarnoyl)-pyridine-2-carboxylic acid

The synthesis of the above compound and intermediates is described below. In this section, the following abbreviations are used:

The synthesis of starting material, (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) is described in Scheme 1.

f 0HCY ° ΒΓΥΥ°

Preparation of 6-bromobenzofdl[1,3ldioxole-5-carbaldehvde (1b)

1a 1b

To a mixture of piperonal (1a) (498 g, 3.32 mol) in glacial acetic acid (1000 mL) was added a solution of bromine (200 mL, 3.89 mol) in glacial acetic acid (500 mL) over a period of 30 min and stirred at room temperature for 24h. The reaction mixture was poured into water (2000 mL) and the solid that separated was collected by filtration. The solid was dissolved in boiling ethanol (4000 mL) and cooled to room temperature. The solid obtained on cooling was collected by filtration to furnish 6-bromobenzo[d][1 ,3]dioxole-5-carbaldehyde (lb) (365 g, 48 %) as a white solid, MP 126 °C; HNMR (300 MHz, DMSO-d6): δ 10.06 (s, 1 H), 7.42 (s,1 H), 7.29 (s, 1 H), 6.20 (d, J=12.3, 2H); IR (KBr) 3434, 2866, 1673,1489, 1413, 259, 1112, 1031 , 925 cm“1; Analysis calculated for CeH5BrO3.O 25H C, 41.15; H, 2.37; Found: C, 41.07; H, 2.11.

Preparation of 2-bromo-5-hvdroxy-4-methoxybenzaldehyde (1c)

1c

A solution of potassium tert-butoxide (397 g, 3.36 mol) in DMSO (1.5 L) was heated at 50 °C for 30 min. Methanol (1.5 L) was added to it and continued heating at 50 °C for additional 30 min. To the hot reaction mixture was added 6-bromo-benzo[d][1,3]dioxole-5-carbaldehyde (1 b) (350g, 1.53 mol) and continued heating at 50 °C for 30 min. The reaction mixture was cooled to room temperature and quenched with water (2.3 L) and sodium hydroxide (61.2 g, 1.53 mol). The reaction mixture was washed with ether (2 x 1.5 L), acidified to pH 2 using cone. HCI and extracted with ethyl acetate ( 1 L). The ethyl acetate layers were combined and concentrated under vacuum to dryness. The residue obtained was treated with water (1.5 L) and ethyl acetate (1 L). The solid obtained was collected by filtration to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (97 g, 27.5% as a first crop). The layers from the filtrate were separated and aqueous layer was extracted with ethyl acetate (200 ml_). The ethyl acetate layers were combined dried over MgS04 and concentrated under vacuum to dryness to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (192 g, 54.4%, second crop) as an orange solid, MP 108 °C; ‘HNMR (300MHz, DMSO-cfe): S 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H); IR (KBr) 3477, 2967, 2917,

2837, 2767, 2740, 1657, 1595, 1428, 1270, 1210, 1164, 1022 cm‘; Analysis calculated for C8H7Br03.H20: C, 38.58; H, 3.64: Found: C, 38.60; H, 3.60.

Preparation of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehvde ( d)

To a solution 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (120 g, 520 mmol) in DMF (1000 mL) was added potassium carbonate (79 g, 572 mmol) and benzyl bromide (68 mL, 572 mmol). The reaction mixture was stirred at room temperature overnight and quenched with water (3000 mL). The solid obtained was collected by filtration, washed with ether and dried under vacuum to furnish 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (113.19 g, 67.9%) as a white solid, MP 144 °C;1HNMR (300 MHz, DMSO-c/6): δ 10.06 (s, 1H), 7.47-7.34 (m, 7H), 5.17 (s, 2H), 3.92 (s, 3H); IR (KBr) 2898, 2851 , 1673, 1592, 1502, 1437, 1402, 1264, 1210, 1158, 1017, 754 cm“1; Analysis calculated for C 5H13Br03: C, 56.10; H, 4.08; Found: C, 55.44; H, 4.08.

Preparation of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e)

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1d 1e

To a solution of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (100 g, 311 mmol) in

ethanol (1500 mL) was added triethyl orthoformate (103 mL, 622 mmol), ammonium nitrate

(7.5 g, 93.3 mmol) and stirred at room temperature overnight. The reaction mixture was

treated with ether (1200 mL) and stirred for 15 min before filtration. The filtrate was

concentrated under vacuum to dryness to give 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (134 g) as a brown syrup; The product was used in the next step

without further purification; 1H N R (300 MHz, DMSO-cf6) δ 7.45 – 7.37 (m, 4H), 7.36 – 7.33

(m, 1 H), 7.17 – 7.14 (m, 1 H), 7.10 (s, 1 H), 5.10 (s, 2H), 3.80 (s, 3H), 3.58 – 3.33 (m, 5H),

1.13 – 1.07 (m, 6H); IR (KBr) 2974, 2879, 1601 , 1503, 1377, 1260, 1163, 1060 cm“1;

Analysis calculated for C19H23Br04: C, 57.73; H, 5.86; Found: C, 57.21 ; H, 5.94.

acid (1fi

To a solution of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (120 g,

300 mmol) in dry ether (1000 mL) at -78 °C was added n-butyllithium (1.6 M solution in

hexanes, 244 mL, 390 mmol) over a period of 30 min and further stirred at -78 °C for 30 min.

A solution of tri-n-butylborate (110 mL, 405 mmol) in dry ether (300 mL) was added to this

solution at -78 °C over a period of 30 min. The reaction mixture was further stirred for 2 h at -78 °C and warmed to 0 °C. The reaction mixture was quenched with 3N HCI (300 mL) at 0

°C and heated at reflux for 1 h. After cooling to room temperature, the solid obtained was

collected by filtration washed with water (250 mL) dried in vaccum to afford (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (30.85 gm, 37.6% as a white solid. The organic

layer from above filtrate was extracted with 1.5 N NaOH (3 x 200 mL). The combined basic

extracts were acidified with cone. HCI (pH about 4). The solid obtained was collected by

filtration, washed with water and dried under vacuum to furnish a second crop of (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (22.3 g, 26%) as a light orange solid

MP 158 °C; 1H NMR (300 MHz, DMSO-cfe) δ 10.08 (s, 1 H), 7.52 (s, 1 H), 7.48 – 7.33 (m, 5H),

7.24 (s, 1H), 5.18 (s, 2H), 3.89 (s, 3H); 1H NMR (300 MHz, DMSO-d6/D20) δ 10.06 (s, 1H),

7.52 (s, 1H), 7.49 – 7.32 (m, 5H), 7.23 (s, 1 H), 5.18 (s, 2H), 3.89 (s, 3H); MS (ES+) 309.1 (M+Na); IR (KBr) 3335, 2937, 1647, 1545, 1388, 1348, 1268, 1146, 1095 cm-1; Analysis calculated for C15H15BO5.0.25H2O: C, 62.00; H, 5.38; Found: C, 61.77; H, 5.19.

Synthesis of methyl-6-(cvclopropylmethylcarbamoyl¾-3-ftrifluoromethylsulfonyloxyVpicolinate

The synthesis of the intermediate methyl 6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethyl sulfonyloxy)picolinate (2h) is described in Scheme 2.

Preparation of 2-bromo-3-hvdroxy-6-methylpyridine (2b)


H3C N Br

2a 2b

To a solution of 3-hydroxy-6-methylpyridine (2a) (3000 g, 27.5 mol) in pyridine (24 L) cooled to 15 °C was added a solution of bromine (4.83 kg, 1.55 L, 30.2 mol) in pyridine (3 L) over a period of 50 min maintaining the internal temperature between 20 to 25 DC. After stirring for 19 h at room temperature the solvent was removed under vacuum and the residue was triturated with water. The solid separated was collected by filtration, washed with water and dried under vacuum to give 2-bromo-3-hydroxy-6-methylpyridine (2b) (3502 g, 67.7 %) as a light brown solid which was used as such without further purification; 1H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1H), 7.18 (d, J = 8.0 Hz, 1 H), 7.08 (d, J

MS (ES+) 188.35, 186.36 (M+1).

(2c)

2b 2c

A mixture of 2-bromo-3-hydroxy-6-methylpyridine (2b) (3000 g, 15.96 mol), anhydrous potassium carbonate (3308 g, 23.94 mol), and iodomethane (2.491 kg, 1.09 L, 17.556 mol) in 30 L of acetone was heated at 40 °C overnight. The reaction mixture was cooled to room temperature and filtered through Celite. Evaporation of the solvent followed by silica gel chromatography (Hexane: ethyl acetate = 7:3) afforded the desired compound, 2-bromo-3-methoxy-6-methylpyridine (2c) which was used as such for the next step; 1H NMR (300 MHz, DMSO-cfe) δ 7.42 (dd, J = 8.3, 1.5 Hz, 1H), 7.29 – 7.19 (m, 1H), 3.84 (d, J = 1.6 Hz, 3H), 2.37 (d, J = 1.7 Hz, 3H).

2c

2d

To a solution of 2-bromo-3-methoxy-6-methylpyridine (2c) (310 g, 1.53 mol) in 6000 mL of water at 60 °C was added KMnO, (725 g, 4.59 mol) in small portions over a 90 min period with vigorous mechanical stirring. A dark purple solution resulted. This solution was kept at 90 °C for a further 3 h and filtered through Celite while still hot to give a colourless filtrate.

After cooling, the aqueous solution was acidified to pH 1-2 by adding 6 N HCI. The white solid obtained was collected by filtration to give on drying 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (302g, 85%) of product, which was used as such in the next reaction without further purification. An analytical sample was obtained by recrystallization from methanol to give 6-bromo-5-methoxy-2-pyridinecarboxylic acid; 1H NMR (300 MHz, DMSO-tfe) δ 7.40 – 7.28 (m, 1H), 7.17 (d, J = 8.3 Hz, 1 H), 3.83 (d, J = 1.7 Hz, 3H).

Preparation of 6-bromo-N-(cvclopropylmethyl)-5-methoxypicolinamide (2e)

To a solution of 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (12 g, 52 mol) in pyridine (70 mL) was added EDCI (11.5 g, 59 mmol) and cyclopropylmethylamine (3.6 g, 52 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated under vacuum. The reaction mixture was diluted with water (100 mL) and ethyl acetate (100 mL). The organic layer was separated and the water layer was extracted with ethyl acetate (2 x 100 mL). The organic layers were combined and washed with water (2 x 50 mL), brine (500 mL), dried over magnesium sulphate, filtered and concentrated under vacuum to furnish 10.43g of crude product. The crude product was converted into a slurry (silica gel 20 g) and purified by flash column chromatography (silica gel 230 g, eluting with 0-100% ethyl acetate in hexane) to yield compound 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (8.02 g, 54%) as off white solid, mp 67-70 °C; 1HNMR (300 MHz, DMSO-d6) δ 8.51 (t, J = 5.8, 1 H), 8.02 (d, J = 8.4, 1 H), 7.65 (d, J = 8.5, 1 H), 3.96 (s, 3H), 3.14 (t, J = 6.5, 2H), 1.11 -0.99 (m, 1 H), 0.47 – 0.36 (m, 2H), 0.27 – 0.20 (m, 2H); MS (ES+) 307.0, 309.0 (100%

M+Na)

Preparation of methyl 6-(cvclopropylmethylcarbamoyl)-3-methoxypicolinate (2f)

To a solution of 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (7.5 g, 27.6 mol) in methanol (300 mL) in a 2-L stainless steel bomb was added Pd(OAc)2(750 mg), 1 ,1-bis(diphenylphosphino)-ferrocene (750 mg), and triethylamine (3.9 mL, 27.6 mmol). The reaction mixture was vacuum flushed and charged with CO gas to 150 psi. The reaction mixture was and heated with stirring at 150°C overnight and cooled to room temperature. The catalyst was filtered through a pad of celite, and concentrated to dryness to furnish crude product. The crude was purified by flash column chromatography (silica gel 150 g,

eluting with, 0%, 5%, 10%, 20%, 30%, 50% ethyl acetate/hexanes (250 mL each) as eluents to give methyl 6-(cyclopropylmethyl-carbamoyl)-3-methoxypicolinate (2f) (6.29 g, 86.1 %) as a salmon coloured solid, MP 107 °C; 1HNMR (300 MHz, DMSO-cfe) δ 8.28 (t, J = 6.0, 1H), 7.91 (d, J = 8.8, 1H), 7.55 (d, J = 8.8, 1 H), 3.68 (s, 3H), 3.64 (s, 3H), 2.90 (t, J = 6.5, 2H), 0.89 – 0.68 (m, 1 H), 0.26 – 0.09 (m, 2H), 0.08 – 0.00 (m, 2H); MS (ES+) 287.1 (M+Na); IR (KBr) 3316, 2921 , 1730, 1659, 1534, 1472, 1432, 1315, 1272, 1228, 1189, 1099, 1003, 929, 846, 680 cm“1; Analysis calculated for C13H16 204: C, 59.08; H, 6.10; N, 10.60; Found: C, 58.70; H, 5.97; N, 10.23.

Preparation of 6-(cvclopropylmethylcarbamoyl 3-hvdroxypicolinic acid (2q)

2f 2g

Aluminium chloride method:

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-methoxypicolinate (2f) (0.16 mmol) in dichloromethane (840 mL) was added AICI3 (193 g, 1.5 mol). The reaction mixture was heated at reflux for 12 h under nitrogen. After slowly adding ~2L of 1 N HCI, the organic layer was separated. The aqueous layer was re-extracted several times with ethyl acetate/DME. The combined organic layer was washed with brine, dried (MgSO.4), and evaporated in vacuo to furnish crude 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid. To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid was added a solution of acetyl chloride (1 10 mL) in methanol (1.1 L). The reaction mixture was stirred for 12 h at room temperature and then concentrated to dryness in vacuo. After co-evaporating once with methanol, the compound was purified by flash-column chromatography (silica gel, 500 g, eluted with chloroform and 3% methanol in chloroform) to furnish 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g).

Boron tribromide method:

To a stirring solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-ethoxypicolinate (2f) (58.0 g, 208 mmol) was added BBr3 (79 mL, 834 mmol) in CH2CI2 (1.3 L) at 0-5 °C. The reaction mixture was allowed to warm to room temperature and stirred for 18h. The reaction mixture was evaporated to dryness and anhydrous methanol (1 L) was added to the light yellowish solid residue. Insoluble solid was collected by filtration (36 g). Mother liquor was evaporated and co-evaporated with MeOH (2 x 200 mL). The insoluble solid (36 g) was treated with MeOH (500 mL) and acetyl chloride (50 mL) and stirred at room temperature for 18 h (at this point reaction mixture was clear). The mixture was evaporated to dryness and diluted with water and extracted with EtOAc. White solid that separated out from EtOAc layer was collected by filtration, washed with water (2 x 20 mL), dried in vacuo at 50 °C to afford 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (5.36 g, 10 %) as a white solid, MP 92-95 °C. 1HNMR (DMSO-cfe) δ 11.04 (s, 1 H, exchangeable with D20), 8.37 (t, J = 6.0, 1 H, exchangeable with D20), 8.12 (d, J = 8.7 Hz, 1 H), 7.57 (d, J = 8.7 Hz, 1 H), 3.90 (m, 3 H), 3.15 (m, 2 H), 1.04 ( m, 1 H), 0.41 (m, 2 H), 0.24 (m, 2 H). IR (KBr): 3346, 3205, 1684 cm“1; MS (ES+): 251.1 (M+1); Analysis calculated for C12H14N2O4.0.1 H2O: C, 57.18; H, 5.67; N, 11.14; Found: C, 57.11 ; H, 5.61; N, 11.09.

Preparation of methyl-6-(cvclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy) picolinate (2h

To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (28 mmol) in DMF (200 mL) were added triethylamine (12 mL, 84 mmol) and N-phenyl-bis(trifluoromethanesulfonimide) (12 g, 34 mmol). The reaction mixture was stirred for 1.5 h at room temperature and then poured into ice. After diluting with water and extracting with ethyl acetate, the aqueous phase was re-extracted, and then the combined organic layer was washed with water and concentrated under vacuum to give methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)picolinate (2h), which was used in the next step without purification.

1H NMR (300 MHz, CDCI3) δ 8.50 (d, J = 8.6, 1 H), 8.07 (s, 1 H), 7.88 (d, J = 8.6, 1 H), 4.09 (d, J = 12.6, 3H), 3.48 – 3.24 (m, 2H), 1.18 – 1.01 (m, 1 H), 0.69 – 0.44 (m, 2H), 0.42 – 0.20 (m, 2H). MS (ES*): 405.17, 100%, M+Na.

Synthesis of 3-f2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid:

The synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i) is described as shown in Scheme 3.

3-f4-Benzyloxy-2-formyl-5-methoxy-phenylV6-(cvcloDroDvlmethvl-carbarnovn-pyridine-2-carboxylic acid methyl ester (3a)

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3a

To a solution of methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)

picolinate (2h) (24.3g, 63 mmol) in DME (225 mL) were added water (25 mL), (4- (benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (27.3 g, 95 mmol), NaHC03(15.9 g,

5 189 mmol), and bis(triphenylphosphine)palladium(ll) chloride (0.885 g). The reaction

mixture was stirred at 70°C overnight under nitrogen. After extracting with ethyl acetate, the organic layer was washed with water and brine and dried (MgSO^), and then concentrated

under vacuum. The compound was purified by flash-column chromatography (silica gel, 300 g, eluting with 10%, 20%, 30% and 40% ethyl acetate in hexane) to furnish 3-(4-benzyloxy- 10 2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

methyl ester (3a) (25 g, 83%) as off white solid, MP 48-50°C: 1H NMR (300 MHz, DMSO-cfe) δ 9.61(s, 1 H), 8.40 (d, J= 7.9 Hz, 1H), 8.14 (t, J= 5.0 Hz, 1H), 7.87 (d, J= 8.1 Hz, 1 H), 7.58

(s, 1H), 7.54-7.30 (m, 5H), 6.71 (s, 1 H), 5.24 (s, 2H), 3.93 (s, 3H), 3.70 (s, 3H), 3.45-3.34 (m,

2H), 1.19-1.05 (m, 1 H), 0.64-0.54 (m, 2H), 0.37-0.30 (m, 2H); IR ( Br) 1735, 1678, 1594,

15 1513, 1437, 1283, 1217, 1141, 1092 cm“1; MS (ES+) 497.29 (M+Na); Analysis calculated for

C27H2eN206: C, 68.34; H, 5.52; N, 5.90; Found; C, 68.16; H, 5.62; N, 5.80.

2-(6-(Cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-vn-4-methoxy-5- vinylbenzoic acid (3b)

To a solution of 3-(4-benzyloxy-2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl- carbamoyl)-pyridine-2-carboxylic acid methyl ester (3a) (24g, 50.6 mmol) in acetonitrile (50

mL), 2-methyl-2-propanol (350 mL), and water (125 mL) were added sodium dihydrogen

phosphate (12.5 g) and 2-methyl-2-butene (55 mL, 519 mmol). The reaction mixture was cooled in an ice bath and then sodium chlorite (28 g) was added. After stirring for 1 h, the reaction mixture was extracted with ethyl acetate and washed with water. The aqueous layer was re-extracted and then the combined organic layers were dried (MgS04). The solvent was evaporated in vacuo to furnish 5-(benzyloxy)-2-(6- ((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (29 g) which was used for the next step. MS (ES+): 513.24, (M+Na(; (ES ): 489.26, M-1.

Methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxytoarbonyltohenyl)-6-(cvclopropylmethylcarbamovnpicolinate (3c)

To a mixture of 5-(benzyloxy)-2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxy-carbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (31 g, 63.2 mmol), and triethylamine (17.7 mL, 126.4 mmol) in dichloromethane (300 mL), was added MEM-chloride (9.03 mL, 79 mmol), and stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, 40 g) to furnish methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 89%) as a thick gum; H NMR (300 MHz, CDCI3) δ 8.35 (d, J = 8.0 Hz, 1 H), 8.15 (t, J = 5.7 Hz, 1 H), 7.78 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.49 (d, J = 6.8 Hz, 2H), 7.36 (ddd, J = 7.5, 14.8, 22.4 Hz, 3H), 6.66 (s, 1 H), 5.37-5.13 (m, 4H), 3.90 (s, 3H), 3.69 (s, 3H), 3.60-3.49 (m, 2H), 3.49 (s, 2H), 3.39 (dd, J = 4.4, 8.4 Hz, 2H), 3.34 (s, 3H), 1.19-1.00 (m, 1H), 0.57 (q, J = 5.8 Hz, 2H), 0.38-0.25 (m, 2H). MS (ES+): 601.24 (M+Na); (ES): 577.27 (M-1);1H NMR (300 MHz, DMSO-cfe) δ 8.69 (t, 7 = 6.1 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1 H), 7.63 (s, 1H), 7.41 (m, 5H), 6.92 (s, 1 H), 5.20 (m, 4H), 3.83 (s, 3H), 3.57 (s, 3H), 3.44 (m, 2H), 3:33 (m, 2H), 3.21 (m, 5H), 1.14 (m, 1H), 0.44 (m, 2H), 0.27 (m, 2H). IR (KBr):

1732, 1671 cm“1. MS (ES+): 601.1(M+Na); Analysis calculated for C31H 2Oe: C, 64.35; H, 5.92; N, 4.84; Found: C, 64.27; H, 6.04; N, 4.79.

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(4-hvdroxy-5-methoxy-2-(((2-methoxyethoxy¾methoxy)carbonyl)phenyl)picolinate (3d)

3c 3d

To a solution of methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)-carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 56.68 mmol) in ethanol (650 mL) was added 10% Pd/C (4 g) and hydrogenated at 45 psi for 5 h. The catalyst was removed by filtration through Celite and the filtrate was concentrated under vacuum to yield methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)picolinate (3d) (31.87 g, 86%), which was pure enough to be used as such for the next step. An analytical sample of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) was obtained by purification of 350 mg of above crude using flash column chromatography (silica gel, eluting with ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethyl-carbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-phenyl)picolinate (3d) as a clear gum; 1HNMR (300 MHz, DMSO-d6) δ 9.74 (s, 1 H), 8.68 (t, J = 6.1 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1 H), 7.95 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.83 (s, 1H), 5.19 (s, 2H), 3.77 (m, 3H), 3.58 (s, 3H), 3.44 (m, 2H), 3.34 (m, 2H), 3.21 (m, 5H), 1.04 (m, 1 H), 0.44 (m, 2H), 0.27 (m, 2H); IR (KBr): 1731 , 1664 cm‘1. MS (ES*): 489.0 (M+1); Analysis calculated for C^e^O,,: C, 59.01; H, 5.78; N, 5.73; Found: C, 58.92; H, 6.15; N, 5.29.

6-(Cvclopropylmethylcarbamovn-3-(5-methoxy-2-(((2-methoxyethoxy^methoxy)-carbonyl)-4- (trifluoromethylsulfonyloxy)phenyl)picolinate (3e)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2- methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) (14.3 g, 29.3 mmol) in dichloromethane (150 mL) were added pyridine (12 mL, 146 mmol) and triflic anhydride (7.5 mL g, 44 mmol). After stirring overnight at room temperature under N2. the reaction mixture was poured into ice water and then extracted twice with dichloromethane. After washing the combined organic extracts with water and drying (MgS0 ), the solvent was evaporated in vacuo. The compound was purified by flash chromatography over silica gel column using ethyl acetate: hexane to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)-carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (1 g, 93%); H NMR (300 MHz, CDCy a 8.41 (d, J = 8.0, 1H), 8.17 (s, 1H), 8.03 (s, 1H), 7.79 (d, J = 8.0, 1 H), 6.82 (s, 1H), 5.32 (q, J = 6.1, 2H), 3.97 (s, 3H), 3.74 (s, 3H), 3.67 – 3.57 (m, 2H), 3.55 – 3.45 (m, 2H), 3.41 (dd, J = 8.2, 14.5, 2H), 3.34 (s, 3H), 1.36 – 1.17 (m, 1H), 0.58 (d, J = 7.1 , 2H), 0.33 (d, J = 5.1 , 2H).

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(5-methoxy-2-f((2-methoxyethoxy)- methoxy)carbonvn-4-vinylphenyl)picolinate (3f)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (37.4

g, 60.30 mmol) and potassium vinyltrifluoroborate (16.87 g, 120.6 mmol) in DMF (450 mL) and water (45 mL) was bubbled N2 for 5 min. To this mixture was added NaHC03 (20.26 g, 241.2 mmol) and dichloro-bis(triphenylphosphine)palladium (II) (6.34 g, 9.0 mmol). The reaction mixture was stirred at 70 °C for 20 h under N2(reaction progress was checked by 1H N R because product and starting material had same Rf in TLC). The reaction mixture was cooled down to room temperature and diluted with ethyl acetate. The organic layer was separated, washed with water, brine, dried ( gS04) and filtered. The filtrate was concentrated under vacuum to yield crude methyl 6-(cyclopropylmethyl-carbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-4-vinylphenyl)-picolinate (3f). The crude product was purified by flash column chromatography (silica gel, 1 kg, eluting with 0-100% ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate [31) (26.54 g, 88%) as an amber gum; H NMR (300 MHz, DMSO-c¾ δ 8.70 (t, J = 6.1 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1 H), 8.12 (s, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 6.98 (m, 2H), 5.94 (dd, J = 1.2, 17.8 Hz, 1H), 5.43 (d, J = 12.5 Hz, 1 H), 5.21 (d, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.64 (s, 3H), 3.48 (d, J = 3.1 Hz, 2H), 3.35 (m, 5H), 3.22 (m, 2H), 1.11 (s, 1H), 0.44 (dt, J = 4.9, 5.5 Hz, 2H), 0.28 (q, J = 4.8 Hz, 2H). IR (KBr); 1732, 1670 cm“1. MS (ES+) 499.1 (M+1).

2-(6-(cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzolc acid (3g)

A mixture of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate (3f) (27.4 mmol) in DME (160 mL) and 6N HCI (40 mL) was stirred at room temperature for 6 h or till TLC showed complete conversion. The solvent was removed under vacuum. The residue obtained was suspended in water, the solid separated out was collected by filtration, washed with water and dried under vacuum to give 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (7.0 g, 63%) as a white

solid MP 40 – 42 °C; H NMR (300 MHz, DMSO-de) δ 8.69 (t, J= 6.0 Hz, 1H, NH), 8.20 (d, J= 7.9 Hz, 1H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1H), 6.97 (dd, J= 18.0, 11.3 Hz, 1H), 6.88 (s, 1H), 5.92 (d, J= 7.9 Hz, 1H), 5.38 (d, J= 11.1 Hz, 1H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); IR (KBr): 3084, 1728, 1650, 1533, 1212, 1143 cm-1; MS (ES+) 433.26 (M+Na); (ES-): 409.28 (M-1); Analysis calculated for θ22Η22Ν2Ο6.0.25Η2Ο; C, 63.68; H, 5.47; N, 6.75; Found C, 63.75; H, 5.56; N, 6.65

Methyl-3-(2-(4-carbamimidoylprienylcarbamoyl)-5-metrioxy-4-vinylphenyl)-6- (cvclopropylmethylcarbamoyl)picolinate (3h)

To a solution of 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (2.35 g, 5.7 mmol) and 4-aminobenzimidamide dihydrochloride (3j) (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 mL) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS04, filtered and purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 in CMA 50) yielding methyl-3-(2-(4-carbamimidoylphenyl-carbamoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (2.2 g, 65%) as a white solid MP 266 °C; 1H NMR (300 MHz, DMSO-c/6) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 – 7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for
C, 58.93; H, 5.63; N,11.85; Found: C, 58.75; H, 5.65; N, 11.92.

46578

159

3-r2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy -vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i)

3h 3i

To a solution of methyl-3-(2-(4-carbamirriidoylphenylcarbarnoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (1 g, 1.9 mmol) in methanol (10 mL) and THF

(10 mL) was added 2 N NaOH (10 mL). The reaction mixture was stirred at room

temperature for 3 h, and concentrated in vacuo to remove methanol and THF. The aqueous layer was acidified with 6N HCI to pH 6-7 and the solid obtained was collected by filtration

washed with water and ether to furnish on drying 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

(3i)(0.775 g, 80%) as the hydrochloride salt as an off white solid.

1H NMR (300 MHz, DMSO-d6) δ 12.67 (s, 1 H), 9.11 (s, 2H), 8.97 (s, 2H), 8.74 (s, 1 H), 7.90

(d, J = 7.8, 1 H), 7.80 (s, 1 H), 7.72 – 7.58 (m, 4H), 6.99 (dd, J = 11.3, 17.7, 1 H), 6.78 (s, 1H),

5.95 (d, J = 17.2, 1H), 5.38 (d, J = 11.9, 1H), 3.82 (s, 3H), 3.18 (s, 2H), 1.06 (s, 1 H), 0.43 (d,

J = 7.9, 2H), 0.25 (d, J = 4.7, 2H); MS (ES+) 514.0 (M+1 ); Analysis calculated for

C2eH27N5O5.HCI.H2O: C, 59.21; H, 5.32; N, 12.33; Found: C, 59.43; H, 5.21; N, 12.06.

Example 1A- Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride in Form

C

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 to -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1.6 kg) and water (0.57 kg). The mixture was heated to 46 °C. Smopex-234 (21 g) and Acticarbone 2SW (10 g) were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 “C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature ❤ °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The

suspension was stirred at 0-3.0 “for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (Tjackel= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 11 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C.

Example-1 B: Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbartiovQpyridine-2-carboxylic acid hydrochloride in Form A

The procedure was carried out in an identical manner to Example 1 A, with the exception that after the final filtration the filter cake was rinsed with 2.87 kg methyl ierf-butyl ether instead of 2.87 kg water, and pulled dry. The product was dried at 40-43 °C and 50 mbar to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) as Form A.

PATENT

WO 2016029216

Methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (compound 6a) is (I) (pages 85 and 86). Avoralstat hydrochloride (compound of formula XVIII) is (II) (claim 40, page 109). A Markush structures is presented (claim 1, page 99).

The synthesis of (II) via intermediate (I) is described (example 1, pages 80-93).

A synthesis of the compound 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (Compound 3i) is described in Schemes A-C.

O y OHCk n Br^ ^OCH3

B Brr22,, AAccOOHH Y^ V” \ \ tt–BBuuOOKK

OHC^^^O ” Br^\^0 MeOH ” OHC

1a 1b 66%

1d 95% 1 e

1f

Scheme A

3h 31

Scheme C

Examples. In this section, the following abbreviations are used:

Example-1 : Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b)

7b

Step (1): Preparation of 6-Bromobenzo 1 ,3]dioxole-5-carbaldehyde (1 b):

1b

A solution of bromine (33.0 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to a solution of piperonal (1a) (29.9 kg, 199.16 mol) in acetic acid (105 L) at room

temperature over a period of 50 min and the reaction mixture was stirred at room temperature for 14.2 h. Additional solution of bromine (33 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to the reaction mixture over a period of 2 h and the reaction mixture was stirred for 22 h. The reaction mixture was quenched by addition of ice water (500 L) with stirring over a period of 6 h and continued stirring for additional 1.25 h. The mixture was allowed to settle and most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (600 L) was added to the solid, stirred, mixture was allowed to settle and then most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (100 L) was added to the decanted mixture, stirred for 15 min and the solid obtained was collected by filtration using a centrifuge. The solid was washed with water (2 x 100 L) and air-dried in a tray drier for 3.75 h to afford the crude product 1 b (52 kg). The crude product (51.2 kg) was stirred in n-hexane (178 L) for 3 h, collected by filtration, washed with n-hexane (25 L) and dried to afford 6-bromobenzo[1 ,3]dioxole-5-carbaldehyde (1b) (40.1 1 kg, 87.9%) as a light brown solid. MP: 109-112°C. 1H NMR (300 MHz, CDCI3) δ 10.21 (s, 1 H), 7.37 (s, 1 H), 7.07 (s, 1 H), 6.10 (s, 2H); HNMR (DMSO-cf6): δ 10.06 (s, 1 H), 7.42 (s, 1 H), 7.29 (s, 1 H), 6.20 (d, J =12.3 Hz, 2H)

The process is also illustrated in Fig. 1.

Average yield of isolated 1 b from step-1 is 78 – 88%.

Step (2): Preparation of 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c)

A solution of potassium terf-butoxide (10.7 kg, 95.36 mol) in DMSO (49 L) was stirred at 50 °C for 30 min. Methanol (49 L) was added slowly over a period of 4.25 h and stirred at 50 °C for 30 min. 6-Bromobenzo[1 ,3]dioxole-5-carbaldehyde (1 b) (9.91 kg, 43.27 mol) was added to the reaction mixture in small portions over a period of 45 min and stirred at 50 °C for 1 h. The reaction mixture was cooled to room temperature and split into two equal portions. Each portion was quenched with water (50.9 L) and basified with 50% aqueous NaOH solution (2.4 L). Each portion was extracted with MTBE (4 x 36 L) to remove impurities. The aqueous layer was acidified with cone. HCI to pH ~ 3 to obtain

product as a yellow solid. The solid was collected by filtration using a centrifuge, washed with water (2 x 35 L) and air-dried to afford 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) (4.37 kg, 40.7%, contains 7 % water); Mp: 100-102°C; 1HNMR (300MHz, DMSO-d6): δ 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H).

The process is also illustrated in Fig. 2.

Average yield of isolated product 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) from step-2 is 40-50%.

Step (3): 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-y benzaldehyde (4a)

2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) [1.3 kg (93%, 7% water content), 5.25 mol] was dissolved in toluene (13 L) in a reaction flask equipped with a Dean Stark apparatus. The solution was heated at reflux with stirring to distil off about 25% of the toluene along with water (90 ml_). The solution was cooled to 90 °C then

bis(pinacolato)diboron (1.5 kg, 5.82 mol), KOAc (772.6 g, 7.87 mol) and Pd(PPh3) (24.3 g, 0.02 mol) were added and the reaction mixture was heated at reflux for 10h. After confirming the completion of reaction by TLC (mobile phase: 100% DCM), the reaction mixture was cooled to room temperature and was kept standing overnight. The reaction mixture was filtered through celite and the celite cake was washed with toluene (4 L). The filtrate of this batch was mixed with the filtrate of another batch (batch size 1.3 kg obtained from an identical reaction). The mixed filtrate was washed with water (17.5 L), brine (17.5 L), dried over Na2S04, filtered and the solution was passed through a pad of silica gel (2 kg, mesh size 230-400). The silica gel pad was washed with toluene. The combined filtrate and washing was concentrated under reduced pressure and the residual crude product was stirred with n-hexane (23 L) for 1 h to obtain a solid product. The solid was collected by filtration, washed with n-hexane (5 L) and dried to afford 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-yl)benzaldehyde (4a) (2.47 kg, 84.6%). H NMR (300 MHz, CDCI3) δ 10.54 (s, 1 H), 7.57 (s, 1 H), 7.33 (s, 1 H), 5.89 (s, 1 H), 4.01 (s, 3H), 1.37 (s, 12H); 1H NMR (300 MHz, DMSO-d6) δ 10.35 (s, 1 H), 9.95 (s, 1 H), 7.33 (s, 1 H), 7.23 (s, 1 H), 3.87 (s, 3H), 1.33 (s, 12H); MS (ES+) 301.1 (M+Na); 579.1 (2M+Na); Analysis calculated for C14H19B05: C, 60.46; H, 6.89; Found: C, 60.60; H, 6.87

The average yield of 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) from step (3) is 78 – 90%.

The process is also illustrated in Fig. 3.

Step (4): Preparation of 3-Bromo-2,6-dimethylpyridine (5b)

2,6-lutidine (5a) (115 kg, 1073.3 mol) was added into pre-chilled oleum (20-23%, 1015 kg, 2276.7 mol) at 0 °C over a period of 4.5 h (temperature r6ached 14 °C during the addition). Bromine (88.18 kg, 1103.6 mol) was then added at 5-10 °C over a period of 1 h. The reaction mixture was slowly heated to 150 °C over a period of 12h. TLC analysis indicated about 40-50% conversion to product and the formation of a dimer by-product (5%). The reaction mixture was cooled to room temperature and then additional bromine (88.18 kg, 1103.6 mol) was added slowly. The reaction mixture was slowly heated to maintain a temperature of 65-75 °C over a period of 15h. TLC analysis indicated a 65-70 % conversion to product and the formation of 5% dimer by product. The reaction mixture was quenched by addition of water (500L) while maintaining the reaction temperature below 20 °C. The mixture was basified with 6.6 M NaOH (3800 L) while maintain the temperature at < 40 °C. EtOAc (220 L) was added and the mixture was stirred for 1 h then allowed to settle over a period of 2 h. The layers were separated and the aqueous layer was treated with NaOH (10 kg) in water (10 L) and extracted with EtOAc (160 L). The organic extracts were combined washed with brine (100 L), dried over Na2S04 (50.0 kg), filtered and the solvent was evaporated under atmospheric pressure. The residue was vacuum distilled and the desired product 3-bromo-2,6-dimethylpyridine (5b) was collected at 58-60 °C, 2 mmHg (98.45 kg, 49.2 %) as a colorless liquid.

The process is also illustrated in Fig. 4.

Step (5): Preparation of 3-Bromopyridine-2,6-dicarboxylic acid (5c)

5b 5c

To a stirred solution of 3-bromo-2,6-dimethylpyridine (5b) (98 kg, 5326 mol) in water (1310 L) was added KMn0 (225 kg, 1423.6 mol) in 5 equal portions in 1 h intervals at 70 °C. After stirring for 1 h at 70 °C, additional KMn04 (225 Kg, 1423.6 mol) was added in 5 equal portion in 1 h intervals at 90 °C. The reaction mixture was stirred for 12 h at 90 °C. The suspension was filtered hot through celite to obtain a clear solution. The solvent was distilled off to remove about 30% of the total volume. The remaining concentrated solution was chilled to 0 °C and made acidic (to pH 3-4) by the addition of cone. HCI (120 L). The white precipitate obtained was collected by filtration and dried at 70 °C to afford 3-bromopyridine-2,6-dicarboxylic acid (5c) as a white solid (109 kg, 84%).

The process is also illustrated in Fig. 5.

Step (6): Preparation of Dimethyl 3-Bromopyridine-2,6-dicarboxylate (5d)

To a stirred solution of 3-bromopyridine-2,6-dicarboxylic acid (5c) (20.0 kg, 81.29 mol) in methanol (100 L) was added cone. H2S04 (4.4 L) over a period of 30 min. The reaction mixture was heated to 65 °C and maintained at that temperature for 5 h (the reaction was monitored by TLC analysis to determine completion of reaction). The reaction mixture was cooled to room temperature basified by careful addition of aqueous NaHC03 solution (prepared from 10 kg NaHC03 in 120 L of water) and further diluted with water (120 L). The white solid obtained was collected by filtration, washed with plenty of water and then oven-dried at 40 °C to obtain dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (9.2 kg, 41.3%) as a white solid; 1HNMR (300 MHz, DMSO-cf6) δ 8.47 (d, J = 8.4, 1 H), 8.08 (dd, J = 4.5, 8.4, 1 H), 3.95 (s, 3H), 3.91 (s, 3H); MS (ES+) 570.6 (2M+Na); Analysis calculated for C9H8BrN04: C, 39.44; H, 2.94; Br, 29.15 N, 5. 1 ;

Found: C, 39.52; H, 2.92; Br, 29.28; N, 5.03.

The process is also illustrated in Fig. 6.

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Step (7): Preparation of Methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (

To a stirred solution of dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (27 kg, 98.52 mol) in ierf-butanol (135 L) was added at room temperature cyclopropylmethanamine (7.83 kg, 110.1 mol). The reaction mixture was heated at 65 °C for 17 h. The progress of reaction was monitored by TLC and HPLC (HPLC analysis showed the formation of 74% of the product 5e after 17 h. The reaction mixture was cooled to room temperature and then cone. HCI (2.7 L) was added slowly and the mixture was stirred for 15 min. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in hot /-PrOH (54 L) filtered through a celite pad. The filtrate was cooled with stirring to 10 °C to obtain a white precipitate. The solid obtained was collected by filtration, washed with cold

i-PrOH (13 kg), n-hexane (15 L) and dried to provide pure methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e) (15.7 kg, 50.9%). The filtrate was concentrated under reduced pressure and the crude product can be purified by silica gel column chromatography eluting with tert-butanol in hexanes to furnish additional 10% methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e). HNMR (300 MHz, DMSO-cf6) δ 8.83 (t, J = 5.9, 1 H), 8.47 – 8.41 (m, 1 H), 8.06 (d, J = 8.4, 1 H), 3.96 (s, 3H), 3.16 (t, J = 6.5, 2H), 1.14 – 0.99 (m, 1 H), 0.42 (m, 2H), 0.30 -0.19 (m, 2H); MS (ES+) 337.0 (M+23), 650.8 (2M+23); Analysis calculated for

C12H13BrN203: C, 46.03; H, 4.18; N, 8.95; Br, 25.52; Found: C, 46.15; H, 4.17; N, 8.72; Br, 25.26.

The average isolated yield for step (7) is 50% to 60%.

The process is also illustrated in Fig. 7.

Step (8): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a)

2

6a

THF (37.5 L) was charged to a 100 L reactor followed by ethyl 3-bromo-6- (cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate (5e) (2.5 kg, 7.98 mol) under a nitrogen atmosphere. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) (2.88 kg, 10.36 mol) was added, followed by the addition of PPh3 (53.13 g, 0.20 mol), PdCI2(PPh3)2 (120.4 g, 0.17 mol) and a solution of Na2C03(2.12 kg, 20.00 mol) in demineralized water (10.0 L) under nitrogen atmosphere. The reaction mixture was degassed again two times by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 6.5 h, cooled to room temperature and filtered through a Celite bed. Water (75 L) was added to the filtrate and the product was extracted with ethyl acetate (75 L). The aqueous layer was back extracted with ethyl acetate (2 χ 60 L). The combined ethyl acetate extract was divided into two equal portions and each portion was washed with brine (37 L), dried over Na2S04, filtered and concentrated under reduced pressure to give crude methyl 6- ((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) as a reddish viscous material (-4.5 Kg) which was used as such for the next step without further purification. An analytical sample was prepared by purification of a small sample by flash column chromatography (silica gel, eluting with 0-100% ethyl acetate in hexane) to furnish methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)-picolinate (6a) as an off-white solid; HNMR (300 MHz, DMSO-d6) δ 9.89 (s, 1 H), 9.52 (s, 1 H), 8.79 (t, J = 6.1 Hz, 1 H), 8.23 (d, J = 8.0 Hz, 1 H), 8.09 (d, J = 8.0 Hz, 1 H), 7.34 (s, 1 H), 6.90 (s, 1 H), 3.85 (s, 3H), 3.62 (s, 3H), 3.22 (m, 2H), 1.16 -1.02 (m, 1 H), 0.49 – 0.38 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 791.0 (2M+Na), (ES-) 382.7 (M-1), 767.3 (2M-1); Analysis calculated for C20H20N2O6.0.25 H20: C, 61.77; H, 5.31 ; N, 7.20; Found: C, 61.54; H, 5.13; N, 7.05.

The process is also illustrated in Fig. 8.

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Step (9): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b)

6a 6b

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) (2.11 kg, estimated about 3.83 mol from step-8) in dichloromethane (16.0 L) and pyridine (1.4 L, 17.4 mol) cooled to -10°C and maintained at that temperature for 1 h was added a solution of triflic anhydride (980.0 ml_, 5.8 mol) in dichloromethane (6.0 L) drop wise over a period of 3 h at -10 °C. The reaction mixture was stirred at -5°C for 1.3 h, quenched with saturated aqueous NaHCO3(10.4 L) and stirred for 30 mins. The organic layer was separated, washed successively with saturated aqueous NaHC03 (10.4 L), 1 HCI (2 x 16.6 L), water (13.2 L), brine (13.2 L), dried over MgS04, filtered and concentrated under reduced pressure to give the crude product. The crude product was stirred with 15% ethyl acetate in n-hexane (7.0 L) for 1 h. The solid obtained was collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (7.0 L) for 1 h, was collected by filtration and washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (8.0 L) for 1 h, collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was dried to afford methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)-oxy)phenyl)picolinate (6b) as a light brown solid (1.7 kg, 86% yield, for combined steps 8 & 9). Average isolated yield for combined steps 8 and 9 was 70% to 86%; Ή NMR (300 MHz, DMSO-cf6): δ 9.64 (s, 1 H), 8.78 (t, J = 6.1 , 1 H), 8.29 (d, J = 8.0, 1 H), 8.16 (d, J = 8.0, 1 H), 8.03 (s, 1H), 7.39 (s, 1 H), 4.00 (s, 3H), 3.63 (s, 3H), 3.22 (m, 2H), 1.11 (m, 1 H), 0.52 – 0.39 (m, 2H), 0.28 (m, 2H); MS (ES+) 538.9 (M+Na). The process is also illustrated in Fig. 9.

Step (10): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c)

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4- (((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b) (12 kg, 23.24 mol) in DME (106 L) was charged into reactor under nitrogen. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. Potassium trifluoro(vinyl)borate (3.9 kg, 29.1 1 mol), PdCI2(PPh3)2 (815 g, 1.13 mol), KHC03 (4.65 g, 46.44 mol) and demineralized water (12 L) was then added under a N2 atmosphere. The reaction mixture was degassed by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 5 h. The reaction mixture was cooled to room temperature and then filtered through a Celite bed. Demineralized water (118 L) was added to the filtrate followed by ethyl acetate (124 L). The mixture was stirred for 20 min and then the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 95 L). The combined organic extract was washed with brine (95 L), dried over Na2S04, and filtered. The solvent was evaporated under reduced pressure to give the crude product. The crude product was purified by column chromatography (silica gel, 120 kg, 230-400 mesh size, eluting with ethyl acetate in n-hexane) to obtain methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (6 kg, 72%). 1H NMR (300 MHz, CDCI3): δ (ppm) 9.64 (s, 1 H), 8.35 (d, J = 7.8 Hz, 1 H), 8.06-8.03 (m, 2H), 7.78(d, J = 7.8 Hz, 1 H), 7.02-6.92 (m, 1 H), 6.61 (s, 1 H), 5.86 (d, J = 17.7 Hz, 1 H), 5.38 (d, J = 1 1.4 Hz, 1 H), 3.84 (s, 3H), 3.67 (s, 3H), 3.35-3.29 (m, 2H),1.08-1.03 (m, 1H), 0.55-0.49 (m, 2H), 0.29-0.2 4(m, 2H). 1HNMR (300 MHz, DMSO-d6) 6 9.68 (s, 1 H), 8.77 (t, J = 6.1 , 1 H), 8.35 – 8.21 (m, 1 H), 8.16 – 8.01 (m, 2H), 7.14 -6.87 (m, 2H), 6.01 (dd, J = 1.2, 17.8, 1 H), 5.45 (dd, J = 1.1 , 1 1.3, 1 H), 3.91 (s, 3H), 3.64 (s, 3H), 3.23 (m, 2H), 1.21 – 1.01 (m, 1H), 0.51 – 0.40 (m, 2H), 0.34 – 0.20 (m, 2H). MS

(ES+) 417.0 (M+Na); Analysis calculated for C22H22N205: C, 66.99; H, 5.62; N, 7.10;

Found: C, 66.75; H, 5.52; N, 7.06.

The process is also illustrated in Fig. 10.

Step (1 1): Preparation of 2-(6-((cyclopropylmethyl)carbamoyl)-2- (methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d)

To a stirred solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (1.57 kg, 3.80 mol) in acetonitrile (15.4 L) was added ferf-butyl alcohol (22.2 L), demineralized water (3.2 L) and sodium dihydrogen phosphate monohydrate (323.74 g, 2.346 mol). The reaction mixture was cooled to 0 °C and added 2-methyl-2-butene (5.3 L, 50.0 mol) and stirred at 0 °C for 30 min. A solution of 80% sodium chlorite (1.36 kg, 12.0 mol) in demineralized water (5.2 L) was added to the reaction mixture over a period of 2.5 h at 0 °C [temperature rises to 7 °C during the addition]. The reaction mixture was stirred at 0 °C for 2 h, diluted with water (40 L) and ethyl acetate (24 L). After stirring the mixture, it was allowed to settle and the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 20 L) then acidified with 5.9 % aqueous acetic acid (2 L) and extracted once with ethyl acetate (10 L). The organic extracts were combined washed with water (2 x 20 L), a solution of acetic acid (125 mL) in water (20.0 L), brine (2 χ 20 L), dried over Na2S04, filtered and concentrated under reduced pressure (vapor temperature below 40 °C). The residue obtained was dissolved in acetone (7 L) (residue didn’t dissolve completely). The solution was poured slowly into a reactor containing stirred n-hexane (70.0 L) to precipitate the solid product and the mixture was stirred for 2 h. The solid obtained was collected by filtration, washed with 10% acetone in n-hexane (6.3 L), AJ-hexane (6.3 L), dried to afford 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4- methoxy-5-vinylbenzoic acid (6d) as an off-white solid (1.29 Kg, yield: 79.0%). Average isolated yield for step 1 1 is 74% to 84%. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.50 (brs, 1 H), 8.69(t, J= 6.0 Hz, 1 H, NH), 8.20 (d, J= 7.9 Hz, 1 H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1 H), 6.97 (dd, J= 18.0, 1 1.3 Hz, 1 H), 6.88 (s, 1 H), 5.92 (d, J= 7.9 Hz, 1 H), 5.38 (d, J= 1 1.1 Hz, 1 H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); MS (ES+) 433.26, (M+Na); (ES-) 409.28 (M-1). The process is also illustrated in Fig. 1 1.

Step (12): Preparation of Methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate methanesulfonate (7a

Pyridine (3.8 L, 47.17 mol) and EDCI (5.31 kg, 27.66 mol) were sequentially added to a cooled solution (0 °C) of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (9 kg, 21.92 mol) and 4-aminobenzamidine dihydrochloride (5.13 kg, 24.65 mol) in /-PrOH (90 L). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. TLC analysis indicated incomplete reaction. Additional EDCI (1.08 kg, 5.6 mol) was added and the reaction mixture was stirred for 8 h. The reaction was still incomplete as indicated by TLC analysis, additional EDCI (0.54 kg, 2.8 mol) was added and the reaction mixture was stirred for 5 h. TLC analysis indicated there was trace amount of unreacted starting material remaining. The reaction mixture was cooled to 0 °C and a solution of

methanesulfonic acid (MSA) (9.13 kg, 95 mol) in MeOH (38.7 L) was added to the cooled mixture over a period of 4 h. The reaction mixture was allowed to warm to room temperature and stirred for 15 h. The product was collected by filtration, washed with a mixture of /-PrOH and MeOH (4:1 , 45 L). The wet cake was slurried in a mixture of /-PrOH and MeOH (2:1 , 135 L) stirred for 1 h and the product was collected by filtration and washed with a mixture of /-PrOH and MeOH (4:1 , 46.8 L). The product was dried in

2015/046582

a vacuum oven at 45 °C to afford methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) as a pink-colored solid (12.71 kg, 93%). Average isolated yield for this step: >90%.

1H NMR (300 MHz, DMSO-c/6) δ 10.71 (s, 1 H), 9.16 (s, 2H), 8.80 (s, 2H), 8.68 (t, J = 6.1 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1 H), 7.93 (s, 1H), 7.84 – 7.72 (m, 4H), 7.12 – 6.97 (m, 2H), 6.04 (dd, J = 17.8, 1.3 Hz, 1 H), 5.45 (d, J = 12.6 Hz, 1H), 3.91 (s, 3H), 3.60 (s, 3H), 3.25 – 3.16 (m, 2H), 2.32 (s, 3H), 1.10 – 1.01 (m, 1 H), 0.48 – 0.37 (m, 2H), 0.30 – 0.22 (m, 2H); MS (ES+) 528.0 (M+1); Analysis calculated for

C29H29N5O5.CH3SO3H.2H2O. C, 54.62; H, 5.65; N, 10.62; S, 4.86; Found: C, 54.95; H, 5.55; N, 10.61 ; S, 4.87.

The process is also illustrated in Fig. 12.

Step (13): Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-rnethoxy-4- vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate

(3i) ,a 3i

A pre-cooled (0-5 °C) aq. NaOH solution [prepared from solid NaOH (4 kg, 100 mol) in water (86 L)] was added to a suspension of methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) (28.7 kg, 46 mol) in acetonitrile (86 L) cooled to 0 to 5 °C over a period of 25 mins. The reaction mixture was stirred at 0 to 5 °C for 2.5 h (TLC analysis showed the reaction was complete). The reaction mixture was filtered through a sparkler filter, washed with a mixture of 1 :1 CH3CN / H20 ( 57.4 L). Acetic acid (3.2 L, 55.9 mol) in water (56 L) was added to the filtrate at room temperature over a period of 25 mins and the resulting mixture was stirred at room temperature for 2.5 h. The solid product obtained was collected by filtration, washed with a 1 :4 mixture of CH3CN / H20 (57.5 L). The solid was dried at 45°C in a vacuum oven to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate (3i) as an off-white solid (12,77 kg, 54.1%). Average yield for this step is 50% to 75%. Mp: >200°C; H NMR (300 MHz, DMSO-d6): δ 13.49 (s, 1 H), 8.94 (bs, 4H), 8.56 (t, 1 H), 7.82 – 7.71 (m, 2H), 7.67 -7.56 (m, 4H), 7.51 (d, J = 7.8, 1 H), 6.98 (dd, J = 11.3, 17.8, 1 H), 6.68 (s, 1 H), 5.92 (d, J = 16.6, 1 H), 5.36 (d, J = 12.4, 1 H), 3.80 (s, 3H), 3.16 (m, 2H), 1.05 (m, 1 H), 0.43 (m, 2H), 0.24 (m, 2H); MS (ES+) 514.1 (M+1), 536.1 (M+Na), (ES-) 512.1 ; Analysis calculated for C28H27N5O5.3H2O: C, 59.25; H, 5.86; N, 12.34; Found C, 59.50; H,

5.75; N, 12.05. If needed this material can be crystallized from a mixture of acetone and water.

The process is also illustrated in Fig. 13.

Step 14: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b

A pre-cooled (5-8 °C) aqueous NaOH solution (prepared from solid NaOH (1.97 kg, 49.25 mol) in demineralized water (41 L) was added to a pre-cooled (0-5 °C) suspension of (3i) (13.8 kg, 26.9 mol) in acetonitrile (41 L). The reaction mixture was stirred at 0-5 °C for 30 min (until the reaction mixture becomes homogeneous). The reaction mixture was filtered through a sparkler filter washed with 50% acetonitrile in demineralized water (4.4 L). The filtrate was charged into a reactor and cooled to 0-5 °C. Aqueous HCI [prepared from cone. HCI (9.3 L) in demineralized water (36 L)] was added slowly with stirring to keep the reaction temperature at or below 15 °C, the resulting mixture was stirred at 10-15 °C for 13 h. The reaction mixture was cooled to 0-5 °C and stirred for 1 h. The solid obtained was collected by filtration and washed with demineralized water (36 L). The solid product was suspended in water (69 L) stirred for 30 mins and collected by filtration washed twice with water (20 L each). The solid product was dried in a vacuum oven at 45°C to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethyl carbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (1 1.21 Kg, 75.77%). Mp: >200°C; 1H NMR (300 MHz, DMSO-ci6): δ 12.98 (br s, 1 H), 10.86 (s, 1 H), 9.24 (s, 3H), 9.04 (s, 2H), 8.22 (d, J = 7.8 Hz, 1 H), 7.96 (d, J = 5.7 Hz, 2H), 7.78 (s, 4H), 7.09-6.99 (m, 2H), 6.07 (d, J = 17.7 Hz, 1 H), 5.45(d, J = 11.4 Hz, 1 H), 3.88 (s, 3H), 3.26-3.24 (m, 2H), 1.09 (m, 1 H), 0.47 (m, 2H), 0.28 (m, 2H).

Average isolated yield for this step varies from 63% to 80%.

The process is also illustrated in Fig. 14.

Example-2: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

6d 8a

To a solution of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (2.35 g, 5.7 mmol) and 4-aminobenzamidine dihydrochloride (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 ml_) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The

reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS04, filtered and concentrated in vacuum. The residue obtained was purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 and 0-100% chloroform in CMA 50) to furnish methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)-carbamoyl)picolinate hydrochloride (8a) (2.2 g, 65%) as a white solid; MP 266 °C; 1HNMR (300 MHz, DMSO-d6) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 -7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1 H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1 H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for C29H29N505 (H20)1 5 (HCI): C, 58.93; H, 5.63; N, 1 1.85; Found: C, 58.75; H, 5.65; N, 1 1.92.

Step-2: preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

8a 8b j0 a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml), was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with a solution of sulfuric acid (0.483 ml, 9.00 mmol) in water (5 mL) and stirred for 10 min at room temperature. To this cold water (5 ml) was added and stirred at room temperature until product crystallized out. Cold water (5 mL) was added to the slurry and stir for additional 20 min, additional cold water (5 mL) was added prior to filtration of solid. The solid obtained was collected by filtration washed with water (5 mL and 2.5 mL), dried under vacuum overnight to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b) (1.103 g, 90 % yield) as a white solid; MP 221.7 °C; H NMR (300 MHz, DMSO-d6) δ 12.30 – 10.91 (bs, 1 H, D20 exchangeable), 10.69 (bs, 1 H, D20 exchangeable), 9.24 (t, J = 6.0 Hz, 1 H), 9.16 (s, 2H, D2O exchangeable), 8.78 (s, 2H, D2O exchangeable), 8.24 (d, J = 8.0 Hz, 1 H), 8.04 – 7.91 (m, 2H), 7.84 – 7.67 (m, 4H), 7.13 – 6.94 (m, 2H), 6.03 (dd, J = 17.8, 1 .4 Hz, 1 H), 5.51 – 5.37 (m, 1 H), 3.88 (s, 3H), 3.24 (t, J = 6.4 Hz, 2H), 1.16 – 1.01 (m, 1 H), 0.52 – 0.41 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for: C28H27N605 1.0H2SO4 1.5H20: C, 52.66; H, 5.05; N, 10.97; S, 5.02; Found: C, 52.81 ; H, 4.95; N, 10.94; S, 4.64.

Example-3: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane s

To a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml) was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with methanesulfonic acid (0.584 ml, 9.00 mmol) and stirred for 1 h at room temperature. Cold water (5.00 ml) was added to the reaction mixture and stirred at room temperature until product crystallized out. To the slurry was added water (5 ml) of water stirred for additional 20 min, followed by the addition of water (5 ml) prior to filtration. The solid obtained was collected by filtration washed with water (5 ml and 2.5 ml), dried under vacuum to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane sulfonate salt (8c)

(1 .138 g, 1.867 mmol, 93 % yield) as a white solid; MP 221.2 °C; 1 H NMR (300 MHz,

DMSO-d6) δ 12.89 (s, 1 H, D2O exchangeable), 10.69 (s, 1 H, D2O exchangeable), 9.24

(t, J = 6.0 Hz, 1 H), 9.16 (s, 2H,), 8.85 (s, 2H), 8.24 (d, J = 8.0 Hz, 1 H), 8.06 – 7.91 (m, 2H), 7.86 – 7.70 (m, 4H), 7.15 – 6.96 (m, 2H), 6.03 (dd, J = 17.8, 1.4 Hz, 1 H), 5.52 – 5.35 (m, 1 H), 3.88 (s, 3H), 3.25 (t, J = 6.3 Hz, 2H), 2.34 (s, 3H), 1.17 – 1.01 (m, 1 H), 0.53 -0.43 (m, 2H), 0.32 – 0.23 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for:

CzeH^NsOsCHsSOsH 1.5H20: C, 54.71 ; H, 5.38; N, 11.00; S, 5.04; Found: C, 54.80; H, 5.14; N, 10.94; S, 4.90.

Example-4: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) in Form C (Compound XX)

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled

46582

to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 – -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1 .6 kg) and 0.57 kg water. The mixture was heated to 46 °C. 21 g of Smopex-234 and 10 g Acticarbone 2SW were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 °C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature ❤ °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The suspension was stirred at 0-3.0 °for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (Tjacke,= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 1 17 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C (Compound XX).

/////avoralstat, BCX4161, Fast Track, Treat hereditary angioedema (HAE), Orphan Drug, PRECLINICAL

COc1cc(c(cc1C=C)C(=O)Nc2ccc(cc2)C(=N)N)c3cc(ncc3C(=O)O)C(=O)NCC4CC4

Avoralstat


Avoralstat, BCX4161,

CAS  918407-35-9
UNII: UX17773O15

513.5513, C28-H27-N5-O5

2-Pyridinecarboxylic acid, 3-(2-(((4-(aminoiminomethyl)phenyl)amino)carbonyl)-4-ethenyl-5-methoxyphenyl)-6-(((cyclopropylmethyl)amino)carbonyl)-

3-(2-((4-Carbamimidoylphenyl)carbamoyl)-4-ethenyl-5-methoxyphenyl)-6-((cyclopropylmethyl)carbamoyl)pyridine-2-carboxylic acid

Hereditary angioedema (HAE)

Kallikrein inhibitor

BioCryst Pharmaceuticals

Biocryst Logo

BioCryst is also investigating second-generation plasma kallikrein inhibitors to avoralstat, for treating HAE (in February 2016, this program was listed as being in preclinical development).

2D chemical structure of 918407-35-9

Prevent acute attacks in patients with hereditary angioedema (HAE); Treat hereditary angioedema (HAE)

U.S. – Fast Track (Treat hereditary angioedema (HAE));
U.S. – Orphan Drug (Prevent acute attacks in patients with hereditary angioedema (HAE))

26 Feb 2016Clinical trials in Hereditary angioedema (Prevention) in USA (PO, Hard-gelatin capsule) before February 2016

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in France (PO, Soft-gelatin capsule)

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in Germany (PO, Soft-gelatin capsule)

Conditions Interventions Phases Recruitment Sponsor/Collaborators
Hereditary Angioedema|HAE Drug: BCX4161|Drug: Placebo Phase 2|Phase 3 Recruiting BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161|Drug: Placebo Phase 2 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals

Avoralstat, also known as BCX-4161, is a potent and orally active Kallikrein inhibitor and Bradykinin inhibitor. Avoralstat may be potentially useful for treatment for Hereditary angioedema. Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

Selective inhibitor of plasma kallikrein that subsequently suppresses bradykinin production

Hereditary angioedema (HAE) is a serious and potentially life-threatening rare genetic illness, caused by mutations in the C1-esterase inhibitor (C1 INH) gene, located on chromosome 11q. HAE is inherited as an autosomal dominant condition, although one quarter of diagnosed cases arise from a new mutation. HAE has been classed as an orphan disease in Europe, with an estimated prevalence of 1 in 50,000. Individuals with HAE experience recurrent acute attacks of painful subcutaneous or submucosal edema of the face, larynx, gastrointestinal tract, limbs or genitalia which, if untreated, may last up to 5 days. Attacks vary in frequency, severity and location and can be life-threatening. Laryngeal attacks, with the potential for asphyxiation, pose the greatest risk. Abdominal attacks are especially painful, and often result in exploratory procedures or unnecessary surgery. Facial and peripheral attacks are disfiguring and debilitating.

 

 

HAE has a number of subtypes. HAE type I is defined by C1 INH gene mutations which produce low levels of C1 -inhibitor, whereas HAE type II is defined by mutations which produce normal levels of ineffective C1 protein. HAE type III has separate pathogenesis, being caused by mutations in the F12 gene which codes for the serine protease known as Factor XII. Diagnostic criteria for distinguishing the subtypes of HAE, and distinguishing HAE from other angioedemas, can be found in Ann Allergy Asthma Immunol 2008; 100(Suppl 2): S30-S40 and J Allergy Clin Immunol 2004; 114: 629-37, incorporated herin by reference.

Current treatments for HAE fall into two main types. Older non-specific treatments including androgens and antifibrinolytics are associated with significant side effects, particularly in females. Newer treatments are based on an understanding of the molecular pathology of the disease, namely that C1 INH is the most important inhibitor of kallikrein in human plasma and that C1 INH deficiency leads to unopposed activation of the kallikrein-bradykinin cascade, with bradykinin the most important mediator of the locally increased vascular permeability that is the hallmark of an attack.

Approved therapies include purified plasma-derived C1 INH (Cinryze®, Berinert), the recombinant peptide kallikrein inhibitor ecallantide (Kalbitor®), and the bradykinin receptor B2 inhibitor iticabant (Firazyr®). All of the currently available targeted therapies are administered by intravenous or subcutaneous injection. There is currently no specific targeted oral chronic therapy for HAE.

There are many delivery routes for active pharmaceutical ingredients (APIs). Generally, the oral route of administration is favored. Oral administration provides a number of advantages, such as, but not limited to, patient convenience, flexibility of timing of administration, location of administration and non-invasiveness. Oral administration also provides more prolonged drug exposure compared with intermittent intravenous infusion, which may be important for drugs with schedule-dependent efficacy. For example, a drug with a short half-life can achieve a greater exposure time by either continuous infusion or by continuous oral dosing. The use of oral therapy further has the potential to reduce the cost of healthcare resources for inpatient and ambulatory patient care services.

In the pharmaceutical arts, it is known that a number of APIs cannot be administered effectively by the oral route. The main reasons why these compounds cannot be administered by the oral route are: a) rapid enzymatic and metabolic degradation; b) chemical and/or biological instability; c) low solubility in aqueous medium; and/or d) limited permeability in the gastrointestinal tract. For such compounds, non-oral routes of delivery, such as parenteral administration, mainly via intramuscular or subcutaneous injections, may be developed. However, non-oral administration poses a disadvantage for the patient as well as healthcare providers, and for this reason, it is important to develop alternative routes of administration for such compounds, such as oral routes of administration.

While the oral route of administration is the most convenient for the patient and the most economical, designing formulations for administration by the oral route involves many complications. Several methods are available to predict the ease by which an API may be formulated into a formulation suitable for administration by the oral route. Such methods include, but are not limited to, and Lipinski rule (also referred to as the Rule of Five) and the Biopharmaceutical Drug Disposition Classification System (BDDCS).

The BDDCS divides APIs into four classifications, depending on their solubility and permeability. Class I APIs have high solubility and high permeability; Class II APIs have low solubility and high permeability; Class III APIs have high solubility and low permeability; and Class IV APIs have low solubility and low permeability. APIs in higher classes in the BDDCS face greater challenges in formulating into an effective, pharmaceutically acceptable product than those in lower classes. Of the four classes, APIs falling into Class IV are the most difficult to formulate into a formulation for administration by the oral route that is capable of delivering an effective amount of the API as problems of both solubility and permeability must be addressed (note the BDDCS does not inherently address chemical stability). The role of BDDCS in drug development is described generally in L.Z. Benet J Pharm Sci. 2013, 102(1), 34-42.

Lipinski’s rule (described in Lipinski et al. Adv. Drug Deliv. Rev. 46 (1-3): 3-26) states, in general, that in order to develop a successful formulation for administration by the oral route, an API can have no more than one violation of the following criteria:

i) not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms)

ii) not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms) iii) a molecular mass less than 500 daltons

iv) an octanol-water partition coefficient log P not greater than 5.

J. Zhang et al. Medicinal Chemistry, 2006, 2, 545-553, describes a number of small molecule amidine compounds which have activity as inhibitors of kallikrein. The molecules described in this document fall into Class IV of the BDDCS as described above. The compounds are poorly soluble in aqueous and physiological fluids, and are poorly permeable as demonstrated by oral dosing in rats and in vitro experiments with Caco-2 cells.

Furthermore, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, one of the compounds described in Zhang et al., is a Class IV API and violates criteria iii) and iv) as set forth in the Lipinski Rule.

Furthermore, the compounds described in Zhang et al., including 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, exhibit poor stability with respect to oxidation in air, to light

(photodegradation) and in aqueous and physiological fluids, as well as to elevated temperatures.

Therefore, the compounds described by Zhang et al. including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, not only exhibit poor solubility and permeability characteristics, but also poor stability characteristics. As a result, such compounds are predicted to be especially difficult to formulate into an effective, orally deliverable

pharmaceutical composition that is capable of delivering an effective amount of the compound to a subject.

Polymorphism, the occurrence of different crystal forms, is a property of some molecules. A single molecule may give rise to a variety of polymorphs having distinct crystal structures and physical properties, such as, but not limited to, melting point, thermal behaviors (e.g. measured by thermogravimetric analysis (TGA), or differential scanning calorimetry (DSC), x-ray diffraction pattern, infrared absorption fingerprint, and solid state NMR spectrum. One or more of these techniques may be used to distinguish different polymorphic forms of a compound.

Discovering new polymorphic forms and solvates of a pharmaceutical product can provide alternate forms of the compound that display a number of desirable and advantageous properties, such as, but not limited to, ease of handling, ease of processing, ease of formulation, storage stability, and/or ease of purification. Further, new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof may further provide for improved pharmaceutical products, by providing compounds that are more soluble in a set of pharmaceutical excipients. Still further, the provision of new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof enlarges the repertoire of compounds that a formulation scientist has available for formulation optimization, for example by providing a pharmaceutical product with different properties, such as, but not limited to, improved processing characteristics, improved handling characteristics, improved solubility profiles, improved dissolution profile and/or improved shelf-life. Therefore, there is a need for additional polymorphs of pharmaceutically useful compounds, such as, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid and the compounds disclosed herein.

In one aspect, the present invention provides an oral formulation that is capable of delivering an effective amount of the amidine compounds described by Zhang et al. to a subject. In particular, the present invention provides an oral formulation that is capable of delivering an effective amount of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid to a subject. In one specific aspect, the 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid is present in a particular crystal form designated Form A. In light of the art suggesting the difficulties in formulating such an oral formulation, this result was unexpected.

As described herein, the amidine compounds described in Zhang et al., including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (specifically including particular crystal Form A), may now be conveniently used in oral administration and further used in oral administration for the treatment of a number of diseases and conditions in a subject, such as, but not limited to, HAE as described herein.

Avoralstat & next generation kallikrein inhibitors for HAE

Avoralstat

Avoralstat is being developed as an oral prophylactic treatment for patients suffering from Hereditary Angioedema (HAE). Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

In May 2014 BioCryst, announced that the OPuS-1 (OralProphylaxiS-1) Phase 2a proof of concept clinical trial met its primary efficacy endpoint, several secondary endpoints and all other objectives established for the trial. OpuS-1 enrolled 24 HAE patients with a history of HAE attack frequency of at least 1 per week. Treatment with avoralstat demonstrated a statistically significant mean attack rate reduction of 0.45 attacks per week versus placebo, p<0.001. The mean attack rate per week was 0.82 on BCX4161 treatment, compared to 1.27 on placebo.

In December 2014, BioCryst initiated enrollment in OPuS-2 (Oral ProphylaxiS-2). OPuS-2 is a blinded, randomized, 12-week, three-arm, parallel cohort design trial evaluating the efficacy and safety of two different dose regimens of avoralstat administered three-times daily, 300 mg and 500 mg, compared with placebo. The primary efficacy endpoint for the trial will be the mean angioedema attack rate, which will be reported for each avoralstat dose group compared to placebo. The trial is being conducted in the U.S., Canada and Europe. On October 8, 2015, announced that it has completed enrollment of approximately 100 HAE patients with a history of moderately frequent to very frequent attacks in OPuS-2. BioCryst expects to report the OPuS-2 trial results in early 2016.

PATENT

WO200234711

http://www.google.com/patents/WO2002034711A1?cl=en

PATENT

WO2015134998

PATENT

WO2016029214

Examples

Example 1 – Synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl- phenyll-6-(cvclopropylmethyl-carbarnoyl)-pyridine-2-carboxylic acid

The synthesis of the above compound and intermediates is described below. In this section, the following abbreviations are used:

The synthesis of starting material, (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) is described in Scheme 1.

f 0HCY ° ΒΓΥΥ°

Preparation of 6-bromobenzofdl[1,3ldioxole-5-carbaldehvde (1b)

1a 1b

To a mixture of piperonal (1a) (498 g, 3.32 mol) in glacial acetic acid (1000 mL) was added a solution of bromine (200 mL, 3.89 mol) in glacial acetic acid (500 mL) over a period of 30 min and stirred at room temperature for 24h. The reaction mixture was poured into water (2000 mL) and the solid that separated was collected by filtration. The solid was dissolved in boiling ethanol (4000 mL) and cooled to room temperature. The solid obtained on cooling was collected by filtration to furnish 6-bromobenzo[d][1 ,3]dioxole-5-carbaldehyde (lb) (365 g, 48 %) as a white solid, MP 126 °C; HNMR (300 MHz, DMSO-d6): δ 10.06 (s, 1 H), 7.42 (s,1 H), 7.29 (s, 1 H), 6.20 (d, J=12.3, 2H); IR (KBr) 3434, 2866, 1673,1489, 1413, 259, 1112, 1031 , 925 cm“1; Analysis calculated for CeH5BrO3.O 25H C, 41.15; H, 2.37; Found: C, 41.07; H, 2.11.

Preparation of 2-bromo-5-hvdroxy-4-methoxybenzaldehyde (1c)

1c

A solution of potassium tert-butoxide (397 g, 3.36 mol) in DMSO (1.5 L) was heated at 50 °C for 30 min. Methanol (1.5 L) was added to it and continued heating at 50 °C for additional 30 min. To the hot reaction mixture was added 6-bromo-benzo[d][1,3]dioxole-5-carbaldehyde (1 b) (350g, 1.53 mol) and continued heating at 50 °C for 30 min. The reaction mixture was cooled to room temperature and quenched with water (2.3 L) and sodium hydroxide (61.2 g, 1.53 mol). The reaction mixture was washed with ether (2 x 1.5 L), acidified to pH 2 using cone. HCI and extracted with ethyl acetate ( 1 L). The ethyl acetate layers were combined and concentrated under vacuum to dryness. The residue obtained was treated with water (1.5 L) and ethyl acetate (1 L). The solid obtained was collected by filtration to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (97 g, 27.5% as a first crop). The layers from the filtrate were separated and aqueous layer was extracted with ethyl acetate (200 ml_). The ethyl acetate layers were combined dried over MgS04 and concentrated under vacuum to dryness to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (192 g, 54.4%, second crop) as an orange solid, MP 108 °C; ‘HNMR (300MHz, DMSO-cfe): S 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H); IR (KBr) 3477, 2967, 2917,

2837, 2767, 2740, 1657, 1595, 1428, 1270, 1210, 1164, 1022 cm‘; Analysis calculated for C8H7Br03.H20: C, 38.58; H, 3.64: Found: C, 38.60; H, 3.60.

Preparation of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehvde ( d)

To a solution 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (120 g, 520 mmol) in DMF (1000 mL) was added potassium carbonate (79 g, 572 mmol) and benzyl bromide (68 mL, 572 mmol). The reaction mixture was stirred at room temperature overnight and quenched with water (3000 mL). The solid obtained was collected by filtration, washed with ether and dried under vacuum to furnish 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (113.19 g, 67.9%) as a white solid, MP 144 °C;1HNMR (300 MHz, DMSO-c/6): δ 10.06 (s, 1H), 7.47-7.34 (m, 7H), 5.17 (s, 2H), 3.92 (s, 3H); IR (KBr) 2898, 2851 , 1673, 1592, 1502, 1437, 1402, 1264, 1210, 1158, 1017, 754 cm“1; Analysis calculated for C 5H13Br03: C, 56.10; H, 4.08; Found: C, 55.44; H, 4.08.

Preparation of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e)

15 046578

146

1d 1e

To a solution of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (100 g, 311 mmol) in

ethanol (1500 mL) was added triethyl orthoformate (103 mL, 622 mmol), ammonium nitrate

(7.5 g, 93.3 mmol) and stirred at room temperature overnight. The reaction mixture was

treated with ether (1200 mL) and stirred for 15 min before filtration. The filtrate was

concentrated under vacuum to dryness to give 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (134 g) as a brown syrup; The product was used in the next step

without further purification; 1H N R (300 MHz, DMSO-cf6) δ 7.45 – 7.37 (m, 4H), 7.36 – 7.33

(m, 1 H), 7.17 – 7.14 (m, 1 H), 7.10 (s, 1 H), 5.10 (s, 2H), 3.80 (s, 3H), 3.58 – 3.33 (m, 5H),

1.13 – 1.07 (m, 6H); IR (KBr) 2974, 2879, 1601 , 1503, 1377, 1260, 1163, 1060 cm“1;

Analysis calculated for C19H23Br04: C, 57.73; H, 5.86; Found: C, 57.21 ; H, 5.94.

acid (1fi

To a solution of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (120 g,

300 mmol) in dry ether (1000 mL) at -78 °C was added n-butyllithium (1.6 M solution in

hexanes, 244 mL, 390 mmol) over a period of 30 min and further stirred at -78 °C for 30 min.

A solution of tri-n-butylborate (110 mL, 405 mmol) in dry ether (300 mL) was added to this

solution at -78 °C over a period of 30 min. The reaction mixture was further stirred for 2 h at -78 °C and warmed to 0 °C. The reaction mixture was quenched with 3N HCI (300 mL) at 0

°C and heated at reflux for 1 h. After cooling to room temperature, the solid obtained was

collected by filtration washed with water (250 mL) dried in vaccum to afford (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (30.85 gm, 37.6% as a white solid. The organic

layer from above filtrate was extracted with 1.5 N NaOH (3 x 200 mL). The combined basic

extracts were acidified with cone. HCI (pH about 4). The solid obtained was collected by

filtration, washed with water and dried under vacuum to furnish a second crop of (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (22.3 g, 26%) as a light orange solid

MP 158 °C; 1H NMR (300 MHz, DMSO-cfe) δ 10.08 (s, 1 H), 7.52 (s, 1 H), 7.48 – 7.33 (m, 5H),

7.24 (s, 1H), 5.18 (s, 2H), 3.89 (s, 3H); 1H NMR (300 MHz, DMSO-d6/D20) δ 10.06 (s, 1H),

7.52 (s, 1H), 7.49 – 7.32 (m, 5H), 7.23 (s, 1 H), 5.18 (s, 2H), 3.89 (s, 3H); MS (ES+) 309.1 (M+Na); IR (KBr) 3335, 2937, 1647, 1545, 1388, 1348, 1268, 1146, 1095 cm-1; Analysis calculated for C15H15BO5.0.25H2O: C, 62.00; H, 5.38; Found: C, 61.77; H, 5.19.

Synthesis of methyl-6-(cvclopropylmethylcarbamoyl¾-3-ftrifluoromethylsulfonyloxyVpicolinate

The synthesis of the intermediate methyl 6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethyl sulfonyloxy)picolinate (2h) is described in Scheme 2.

Preparation of 2-bromo-3-hvdroxy-6-methylpyridine (2b)


H3C N Br

2a 2b

To a solution of 3-hydroxy-6-methylpyridine (2a) (3000 g, 27.5 mol) in pyridine (24 L) cooled to 15 °C was added a solution of bromine (4.83 kg, 1.55 L, 30.2 mol) in pyridine (3 L) over a period of 50 min maintaining the internal temperature between 20 to 25 DC. After stirring for 19 h at room temperature the solvent was removed under vacuum and the residue was triturated with water. The solid separated was collected by filtration, washed with water and dried under vacuum to give 2-bromo-3-hydroxy-6-methylpyridine (2b) (3502 g, 67.7 %) as a light brown solid which was used as such without further purification; 1H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1H), 7.18 (d, J = 8.0 Hz, 1 H), 7.08 (d, J

MS (ES+) 188.35, 186.36 (M+1).

(2c)

2b 2c

A mixture of 2-bromo-3-hydroxy-6-methylpyridine (2b) (3000 g, 15.96 mol), anhydrous potassium carbonate (3308 g, 23.94 mol), and iodomethane (2.491 kg, 1.09 L, 17.556 mol) in 30 L of acetone was heated at 40 °C overnight. The reaction mixture was cooled to room temperature and filtered through Celite. Evaporation of the solvent followed by silica gel chromatography (Hexane: ethyl acetate = 7:3) afforded the desired compound, 2-bromo-3-methoxy-6-methylpyridine (2c) which was used as such for the next step; 1H NMR (300 MHz, DMSO-cfe) δ 7.42 (dd, J = 8.3, 1.5 Hz, 1H), 7.29 – 7.19 (m, 1H), 3.84 (d, J = 1.6 Hz, 3H), 2.37 (d, J = 1.7 Hz, 3H).

2c

2d

To a solution of 2-bromo-3-methoxy-6-methylpyridine (2c) (310 g, 1.53 mol) in 6000 mL of water at 60 °C was added KMnO, (725 g, 4.59 mol) in small portions over a 90 min period with vigorous mechanical stirring. A dark purple solution resulted. This solution was kept at 90 °C for a further 3 h and filtered through Celite while still hot to give a colourless filtrate.

After cooling, the aqueous solution was acidified to pH 1-2 by adding 6 N HCI. The white solid obtained was collected by filtration to give on drying 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (302g, 85%) of product, which was used as such in the next reaction without further purification. An analytical sample was obtained by recrystallization from methanol to give 6-bromo-5-methoxy-2-pyridinecarboxylic acid; 1H NMR (300 MHz, DMSO-tfe) δ 7.40 – 7.28 (m, 1H), 7.17 (d, J = 8.3 Hz, 1 H), 3.83 (d, J = 1.7 Hz, 3H).

Preparation of 6-bromo-N-(cvclopropylmethyl)-5-methoxypicolinamide (2e)

To a solution of 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (12 g, 52 mol) in pyridine (70 mL) was added EDCI (11.5 g, 59 mmol) and cyclopropylmethylamine (3.6 g, 52 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated under vacuum. The reaction mixture was diluted with water (100 mL) and ethyl acetate (100 mL). The organic layer was separated and the water layer was extracted with ethyl acetate (2 x 100 mL). The organic layers were combined and washed with water (2 x 50 mL), brine (500 mL), dried over magnesium sulphate, filtered and concentrated under vacuum to furnish 10.43g of crude product. The crude product was converted into a slurry (silica gel 20 g) and purified by flash column chromatography (silica gel 230 g, eluting with 0-100% ethyl acetate in hexane) to yield compound 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (8.02 g, 54%) as off white solid, mp 67-70 °C; 1HNMR (300 MHz, DMSO-d6) δ 8.51 (t, J = 5.8, 1 H), 8.02 (d, J = 8.4, 1 H), 7.65 (d, J = 8.5, 1 H), 3.96 (s, 3H), 3.14 (t, J = 6.5, 2H), 1.11 -0.99 (m, 1 H), 0.47 – 0.36 (m, 2H), 0.27 – 0.20 (m, 2H); MS (ES+) 307.0, 309.0 (100%

M+Na)

Preparation of methyl 6-(cvclopropylmethylcarbamoyl)-3-methoxypicolinate (2f)

To a solution of 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (7.5 g, 27.6 mol) in methanol (300 mL) in a 2-L stainless steel bomb was added Pd(OAc)2(750 mg), 1 ,1-bis(diphenylphosphino)-ferrocene (750 mg), and triethylamine (3.9 mL, 27.6 mmol). The reaction mixture was vacuum flushed and charged with CO gas to 150 psi. The reaction mixture was and heated with stirring at 150°C overnight and cooled to room temperature. The catalyst was filtered through a pad of celite, and concentrated to dryness to furnish crude product. The crude was purified by flash column chromatography (silica gel 150 g,

eluting with, 0%, 5%, 10%, 20%, 30%, 50% ethyl acetate/hexanes (250 mL each) as eluents to give methyl 6-(cyclopropylmethyl-carbamoyl)-3-methoxypicolinate (2f) (6.29 g, 86.1 %) as a salmon coloured solid, MP 107 °C; 1HNMR (300 MHz, DMSO-cfe) δ 8.28 (t, J = 6.0, 1H), 7.91 (d, J = 8.8, 1H), 7.55 (d, J = 8.8, 1 H), 3.68 (s, 3H), 3.64 (s, 3H), 2.90 (t, J = 6.5, 2H), 0.89 – 0.68 (m, 1 H), 0.26 – 0.09 (m, 2H), 0.08 – 0.00 (m, 2H); MS (ES+) 287.1 (M+Na); IR (KBr) 3316, 2921 , 1730, 1659, 1534, 1472, 1432, 1315, 1272, 1228, 1189, 1099, 1003, 929, 846, 680 cm“1; Analysis calculated for C13H16 204: C, 59.08; H, 6.10; N, 10.60; Found: C, 58.70; H, 5.97; N, 10.23.

Preparation of 6-(cvclopropylmethylcarbamoyl 3-hvdroxypicolinic acid (2q)

2f 2g

Aluminium chloride method:

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-methoxypicolinate (2f) (0.16 mmol) in dichloromethane (840 mL) was added AICI3 (193 g, 1.5 mol). The reaction mixture was heated at reflux for 12 h under nitrogen. After slowly adding ~2L of 1 N HCI, the organic layer was separated. The aqueous layer was re-extracted several times with ethyl acetate/DME. The combined organic layer was washed with brine, dried (MgSO.4), and evaporated in vacuo to furnish crude 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid. To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid was added a solution of acetyl chloride (1 10 mL) in methanol (1.1 L). The reaction mixture was stirred for 12 h at room temperature and then concentrated to dryness in vacuo. After co-evaporating once with methanol, the compound was purified by flash-column chromatography (silica gel, 500 g, eluted with chloroform and 3% methanol in chloroform) to furnish 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g).

Boron tribromide method:

To a stirring solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-ethoxypicolinate (2f) (58.0 g, 208 mmol) was added BBr3 (79 mL, 834 mmol) in CH2CI2 (1.3 L) at 0-5 °C. The reaction mixture was allowed to warm to room temperature and stirred for 18h. The reaction mixture was evaporated to dryness and anhydrous methanol (1 L) was added to the light yellowish solid residue. Insoluble solid was collected by filtration (36 g). Mother liquor was evaporated and co-evaporated with MeOH (2 x 200 mL). The insoluble solid (36 g) was treated with MeOH (500 mL) and acetyl chloride (50 mL) and stirred at room temperature for 18 h (at this point reaction mixture was clear). The mixture was evaporated to dryness and diluted with water and extracted with EtOAc. White solid that separated out from EtOAc layer was collected by filtration, washed with water (2 x 20 mL), dried in vacuo at 50 °C to afford 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (5.36 g, 10 %) as a white solid, MP 92-95 °C. 1HNMR (DMSO-cfe) δ 11.04 (s, 1 H, exchangeable with D20), 8.37 (t, J = 6.0, 1 H, exchangeable with D20), 8.12 (d, J = 8.7 Hz, 1 H), 7.57 (d, J = 8.7 Hz, 1 H), 3.90 (m, 3 H), 3.15 (m, 2 H), 1.04 ( m, 1 H), 0.41 (m, 2 H), 0.24 (m, 2 H). IR (KBr): 3346, 3205, 1684 cm“1; MS (ES+): 251.1 (M+1); Analysis calculated for C12H14N2O4.0.1 H2O: C, 57.18; H, 5.67; N, 11.14; Found: C, 57.11 ; H, 5.61; N, 11.09.

Preparation of methyl-6-(cvclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy) picolinate (2h

To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (28 mmol) in DMF (200 mL) were added triethylamine (12 mL, 84 mmol) and N-phenyl-bis(trifluoromethanesulfonimide) (12 g, 34 mmol). The reaction mixture was stirred for 1.5 h at room temperature and then poured into ice. After diluting with water and extracting with ethyl acetate, the aqueous phase was re-extracted, and then the combined organic layer was washed with water and concentrated under vacuum to give methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)picolinate (2h), which was used in the next step without purification.

1H NMR (300 MHz, CDCI3) δ 8.50 (d, J = 8.6, 1 H), 8.07 (s, 1 H), 7.88 (d, J = 8.6, 1 H), 4.09 (d, J = 12.6, 3H), 3.48 – 3.24 (m, 2H), 1.18 – 1.01 (m, 1 H), 0.69 – 0.44 (m, 2H), 0.42 – 0.20 (m, 2H). MS (ES*): 405.17, 100%, M+Na.

Synthesis of 3-f2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid:

The synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i) is described as shown in Scheme 3.

3-f4-Benzyloxy-2-formyl-5-methoxy-phenylV6-(cvcloDroDvlmethvl-carbarnovn-pyridine-2-carboxylic acid methyl ester (3a)

5 046578

153

3a

To a solution of methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)

picolinate (2h) (24.3g, 63 mmol) in DME (225 mL) were added water (25 mL), (4- (benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (27.3 g, 95 mmol), NaHC03(15.9 g,

5 189 mmol), and bis(triphenylphosphine)palladium(ll) chloride (0.885 g). The reaction

mixture was stirred at 70°C overnight under nitrogen. After extracting with ethyl acetate, the organic layer was washed with water and brine and dried (MgSO^), and then concentrated

under vacuum. The compound was purified by flash-column chromatography (silica gel, 300 g, eluting with 10%, 20%, 30% and 40% ethyl acetate in hexane) to furnish 3-(4-benzyloxy- 10 2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

methyl ester (3a) (25 g, 83%) as off white solid, MP 48-50°C: 1H NMR (300 MHz, DMSO-cfe) δ 9.61(s, 1 H), 8.40 (d, J= 7.9 Hz, 1H), 8.14 (t, J= 5.0 Hz, 1H), 7.87 (d, J= 8.1 Hz, 1 H), 7.58

(s, 1H), 7.54-7.30 (m, 5H), 6.71 (s, 1 H), 5.24 (s, 2H), 3.93 (s, 3H), 3.70 (s, 3H), 3.45-3.34 (m,

2H), 1.19-1.05 (m, 1 H), 0.64-0.54 (m, 2H), 0.37-0.30 (m, 2H); IR ( Br) 1735, 1678, 1594,

15 1513, 1437, 1283, 1217, 1141, 1092 cm“1; MS (ES+) 497.29 (M+Na); Analysis calculated for

C27H2eN206: C, 68.34; H, 5.52; N, 5.90; Found; C, 68.16; H, 5.62; N, 5.80.

2-(6-(Cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-vn-4-methoxy-5- vinylbenzoic acid (3b)

To a solution of 3-(4-benzyloxy-2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl- carbamoyl)-pyridine-2-carboxylic acid methyl ester (3a) (24g, 50.6 mmol) in acetonitrile (50

mL), 2-methyl-2-propanol (350 mL), and water (125 mL) were added sodium dihydrogen

phosphate (12.5 g) and 2-methyl-2-butene (55 mL, 519 mmol). The reaction mixture was cooled in an ice bath and then sodium chlorite (28 g) was added. After stirring for 1 h, the reaction mixture was extracted with ethyl acetate and washed with water. The aqueous layer was re-extracted and then the combined organic layers were dried (MgS04). The solvent was evaporated in vacuo to furnish 5-(benzyloxy)-2-(6- ((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (29 g) which was used for the next step. MS (ES+): 513.24, (M+Na(; (ES ): 489.26, M-1.

Methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxytoarbonyltohenyl)-6-(cvclopropylmethylcarbamovnpicolinate (3c)

To a mixture of 5-(benzyloxy)-2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxy-carbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (31 g, 63.2 mmol), and triethylamine (17.7 mL, 126.4 mmol) in dichloromethane (300 mL), was added MEM-chloride (9.03 mL, 79 mmol), and stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, 40 g) to furnish methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 89%) as a thick gum; H NMR (300 MHz, CDCI3) δ 8.35 (d, J = 8.0 Hz, 1 H), 8.15 (t, J = 5.7 Hz, 1 H), 7.78 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.49 (d, J = 6.8 Hz, 2H), 7.36 (ddd, J = 7.5, 14.8, 22.4 Hz, 3H), 6.66 (s, 1 H), 5.37-5.13 (m, 4H), 3.90 (s, 3H), 3.69 (s, 3H), 3.60-3.49 (m, 2H), 3.49 (s, 2H), 3.39 (dd, J = 4.4, 8.4 Hz, 2H), 3.34 (s, 3H), 1.19-1.00 (m, 1H), 0.57 (q, J = 5.8 Hz, 2H), 0.38-0.25 (m, 2H). MS (ES+): 601.24 (M+Na); (ES): 577.27 (M-1);1H NMR (300 MHz, DMSO-cfe) δ 8.69 (t, 7 = 6.1 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1 H), 7.63 (s, 1H), 7.41 (m, 5H), 6.92 (s, 1 H), 5.20 (m, 4H), 3.83 (s, 3H), 3.57 (s, 3H), 3.44 (m, 2H), 3:33 (m, 2H), 3.21 (m, 5H), 1.14 (m, 1H), 0.44 (m, 2H), 0.27 (m, 2H). IR (KBr):

1732, 1671 cm“1. MS (ES+): 601.1(M+Na); Analysis calculated for C31H 2Oe: C, 64.35; H, 5.92; N, 4.84; Found: C, 64.27; H, 6.04; N, 4.79.

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(4-hvdroxy-5-methoxy-2-(((2-methoxyethoxy¾methoxy)carbonyl)phenyl)picolinate (3d)

3c 3d

To a solution of methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)-carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 56.68 mmol) in ethanol (650 mL) was added 10% Pd/C (4 g) and hydrogenated at 45 psi for 5 h. The catalyst was removed by filtration through Celite and the filtrate was concentrated under vacuum to yield methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)picolinate (3d) (31.87 g, 86%), which was pure enough to be used as such for the next step. An analytical sample of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) was obtained by purification of 350 mg of above crude using flash column chromatography (silica gel, eluting with ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethyl-carbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-phenyl)picolinate (3d) as a clear gum; 1HNMR (300 MHz, DMSO-d6) δ 9.74 (s, 1 H), 8.68 (t, J = 6.1 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1 H), 7.95 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.83 (s, 1H), 5.19 (s, 2H), 3.77 (m, 3H), 3.58 (s, 3H), 3.44 (m, 2H), 3.34 (m, 2H), 3.21 (m, 5H), 1.04 (m, 1 H), 0.44 (m, 2H), 0.27 (m, 2H); IR (KBr): 1731 , 1664 cm‘1. MS (ES*): 489.0 (M+1); Analysis calculated for C^e^O,,: C, 59.01; H, 5.78; N, 5.73; Found: C, 58.92; H, 6.15; N, 5.29.

6-(Cvclopropylmethylcarbamovn-3-(5-methoxy-2-(((2-methoxyethoxy^methoxy)-carbonyl)-4- (trifluoromethylsulfonyloxy)phenyl)picolinate (3e)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2- methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) (14.3 g, 29.3 mmol) in dichloromethane (150 mL) were added pyridine (12 mL, 146 mmol) and triflic anhydride (7.5 mL g, 44 mmol). After stirring overnight at room temperature under N2. the reaction mixture was poured into ice water and then extracted twice with dichloromethane. After washing the combined organic extracts with water and drying (MgS0 ), the solvent was evaporated in vacuo. The compound was purified by flash chromatography over silica gel column using ethyl acetate: hexane to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)-carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (1 g, 93%); H NMR (300 MHz, CDCy a 8.41 (d, J = 8.0, 1H), 8.17 (s, 1H), 8.03 (s, 1H), 7.79 (d, J = 8.0, 1 H), 6.82 (s, 1H), 5.32 (q, J = 6.1, 2H), 3.97 (s, 3H), 3.74 (s, 3H), 3.67 – 3.57 (m, 2H), 3.55 – 3.45 (m, 2H), 3.41 (dd, J = 8.2, 14.5, 2H), 3.34 (s, 3H), 1.36 – 1.17 (m, 1H), 0.58 (d, J = 7.1 , 2H), 0.33 (d, J = 5.1 , 2H).

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(5-methoxy-2-f((2-methoxyethoxy)- methoxy)carbonvn-4-vinylphenyl)picolinate (3f)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (37.4

g, 60.30 mmol) and potassium vinyltrifluoroborate (16.87 g, 120.6 mmol) in DMF (450 mL) and water (45 mL) was bubbled N2 for 5 min. To this mixture was added NaHC03 (20.26 g, 241.2 mmol) and dichloro-bis(triphenylphosphine)palladium (II) (6.34 g, 9.0 mmol). The reaction mixture was stirred at 70 °C for 20 h under N2(reaction progress was checked by 1H N R because product and starting material had same Rf in TLC). The reaction mixture was cooled down to room temperature and diluted with ethyl acetate. The organic layer was separated, washed with water, brine, dried ( gS04) and filtered. The filtrate was concentrated under vacuum to yield crude methyl 6-(cyclopropylmethyl-carbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-4-vinylphenyl)-picolinate (3f). The crude product was purified by flash column chromatography (silica gel, 1 kg, eluting with 0-100% ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate [31) (26.54 g, 88%) as an amber gum; H NMR (300 MHz, DMSO-c¾ δ 8.70 (t, J = 6.1 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1 H), 8.12 (s, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 6.98 (m, 2H), 5.94 (dd, J = 1.2, 17.8 Hz, 1H), 5.43 (d, J = 12.5 Hz, 1 H), 5.21 (d, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.64 (s, 3H), 3.48 (d, J = 3.1 Hz, 2H), 3.35 (m, 5H), 3.22 (m, 2H), 1.11 (s, 1H), 0.44 (dt, J = 4.9, 5.5 Hz, 2H), 0.28 (q, J = 4.8 Hz, 2H). IR (KBr); 1732, 1670 cm“1. MS (ES+) 499.1 (M+1).

2-(6-(cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzolc acid (3g)

A mixture of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate (3f) (27.4 mmol) in DME (160 mL) and 6N HCI (40 mL) was stirred at room temperature for 6 h or till TLC showed complete conversion. The solvent was removed under vacuum. The residue obtained was suspended in water, the solid separated out was collected by filtration, washed with water and dried under vacuum to give 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (7.0 g, 63%) as a white

solid MP 40 – 42 °C; H NMR (300 MHz, DMSO-de) δ 8.69 (t, J= 6.0 Hz, 1H, NH), 8.20 (d, J= 7.9 Hz, 1H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1H), 6.97 (dd, J= 18.0, 11.3 Hz, 1H), 6.88 (s, 1H), 5.92 (d, J= 7.9 Hz, 1H), 5.38 (d, J= 11.1 Hz, 1H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); IR (KBr): 3084, 1728, 1650, 1533, 1212, 1143 cm-1; MS (ES+) 433.26 (M+Na); (ES-): 409.28 (M-1); Analysis calculated for θ22Η22Ν2Ο6.0.25Η2Ο; C, 63.68; H, 5.47; N, 6.75; Found C, 63.75; H, 5.56; N, 6.65

Methyl-3-(2-(4-carbamimidoylprienylcarbamoyl)-5-metrioxy-4-vinylphenyl)-6- (cvclopropylmethylcarbamoyl)picolinate (3h)

To a solution of 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (2.35 g, 5.7 mmol) and 4-aminobenzimidamide dihydrochloride (3j) (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 mL) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS04, filtered and purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 in CMA 50) yielding methyl-3-(2-(4-carbamimidoylphenyl-carbamoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (2.2 g, 65%) as a white solid MP 266 °C; 1H NMR (300 MHz, DMSO-c/6) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 – 7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for
C, 58.93; H, 5.63; N,11.85; Found: C, 58.75; H, 5.65; N, 11.92.

46578

159

3-r2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy -vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i)

3h 3i

To a solution of methyl-3-(2-(4-carbamirriidoylphenylcarbarnoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (1 g, 1.9 mmol) in methanol (10 mL) and THF

(10 mL) was added 2 N NaOH (10 mL). The reaction mixture was stirred at room

temperature for 3 h, and concentrated in vacuo to remove methanol and THF. The aqueous layer was acidified with 6N HCI to pH 6-7 and the solid obtained was collected by filtration

washed with water and ether to furnish on drying 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

(3i)(0.775 g, 80%) as the hydrochloride salt as an off white solid.

1H NMR (300 MHz, DMSO-d6) δ 12.67 (s, 1 H), 9.11 (s, 2H), 8.97 (s, 2H), 8.74 (s, 1 H), 7.90

(d, J = 7.8, 1 H), 7.80 (s, 1 H), 7.72 – 7.58 (m, 4H), 6.99 (dd, J = 11.3, 17.7, 1 H), 6.78 (s, 1H),

5.95 (d, J = 17.2, 1H), 5.38 (d, J = 11.9, 1H), 3.82 (s, 3H), 3.18 (s, 2H), 1.06 (s, 1 H), 0.43 (d,

J = 7.9, 2H), 0.25 (d, J = 4.7, 2H); MS (ES+) 514.0 (M+1 ); Analysis calculated for

C2eH27N5O5.HCI.H2O: C, 59.21; H, 5.32; N, 12.33; Found: C, 59.43; H, 5.21; N, 12.06.

Example 1A- Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride in Form

C

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 to -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1.6 kg) and water (0.57 kg). The mixture was heated to 46 °C. Smopex-234 (21 g) and Acticarbone 2SW (10 g) were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 “C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature ❤ °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The

suspension was stirred at 0-3.0 “for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (Tjackel= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 11 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C.

Example-1 B: Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbartiovQpyridine-2-carboxylic acid hydrochloride in Form A

The procedure was carried out in an identical manner to Example 1 A, with the exception that after the final filtration the filter cake was rinsed with 2.87 kg methyl ierf-butyl ether instead of 2.87 kg water, and pulled dry. The product was dried at 40-43 °C and 50 mbar to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) as Form A.

 

PATENT

WO 2016029216

Methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (compound 6a) is (I) (pages 85 and 86). Avoralstat hydrochloride (compound of formula XVIII) is (II) (claim 40, page 109). A Markush structures is presented (claim 1, page 99).

The synthesis of (II) via intermediate (I) is described (example 1, pages 80-93).

A synthesis of the compound 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (Compound 3i) is described in Schemes A-C.

O y OHCk n Br^ ^OCH3

B Brr22,, AAccOOHH Y^ V” \ \ tt–BBuuOOKK

OHC^^^O ” Br^\^0 MeOH ” OHC

1a 1b 66%

1d 95% 1 e

1f

Scheme A

3h 31

Scheme C

Examples. In this section, the following abbreviations are used:

Example-1 : Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b)

7b

Step (1): Preparation of 6-Bromobenzo 1 ,3]dioxole-5-carbaldehyde (1 b):

1b

A solution of bromine (33.0 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to a solution of piperonal (1a) (29.9 kg, 199.16 mol) in acetic acid (105 L) at room

temperature over a period of 50 min and the reaction mixture was stirred at room temperature for 14.2 h. Additional solution of bromine (33 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to the reaction mixture over a period of 2 h and the reaction mixture was stirred for 22 h. The reaction mixture was quenched by addition of ice water (500 L) with stirring over a period of 6 h and continued stirring for additional 1.25 h. The mixture was allowed to settle and most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (600 L) was added to the solid, stirred, mixture was allowed to settle and then most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (100 L) was added to the decanted mixture, stirred for 15 min and the solid obtained was collected by filtration using a centrifuge. The solid was washed with water (2 x 100 L) and air-dried in a tray drier for 3.75 h to afford the crude product 1 b (52 kg). The crude product (51.2 kg) was stirred in n-hexane (178 L) for 3 h, collected by filtration, washed with n-hexane (25 L) and dried to afford 6-bromobenzo[1 ,3]dioxole-5-carbaldehyde (1b) (40.1 1 kg, 87.9%) as a light brown solid. MP: 109-112°C. 1H NMR (300 MHz, CDCI3) δ 10.21 (s, 1 H), 7.37 (s, 1 H), 7.07 (s, 1 H), 6.10 (s, 2H); HNMR (DMSO-cf6): δ 10.06 (s, 1 H), 7.42 (s, 1 H), 7.29 (s, 1 H), 6.20 (d, J =12.3 Hz, 2H)

The process is also illustrated in Fig. 1.

Average yield of isolated 1 b from step-1 is 78 – 88%.

Step (2): Preparation of 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c)

A solution of potassium terf-butoxide (10.7 kg, 95.36 mol) in DMSO (49 L) was stirred at 50 °C for 30 min. Methanol (49 L) was added slowly over a period of 4.25 h and stirred at 50 °C for 30 min. 6-Bromobenzo[1 ,3]dioxole-5-carbaldehyde (1 b) (9.91 kg, 43.27 mol) was added to the reaction mixture in small portions over a period of 45 min and stirred at 50 °C for 1 h. The reaction mixture was cooled to room temperature and split into two equal portions. Each portion was quenched with water (50.9 L) and basified with 50% aqueous NaOH solution (2.4 L). Each portion was extracted with MTBE (4 x 36 L) to remove impurities. The aqueous layer was acidified with cone. HCI to pH ~ 3 to obtain

product as a yellow solid. The solid was collected by filtration using a centrifuge, washed with water (2 x 35 L) and air-dried to afford 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) (4.37 kg, 40.7%, contains 7 % water); Mp: 100-102°C; 1HNMR (300MHz, DMSO-d6): δ 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H).

The process is also illustrated in Fig. 2.

Average yield of isolated product 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) from step-2 is 40-50%.

Step (3): 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-y benzaldehyde (4a)

2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) [1.3 kg (93%, 7% water content), 5.25 mol] was dissolved in toluene (13 L) in a reaction flask equipped with a Dean Stark apparatus. The solution was heated at reflux with stirring to distil off about 25% of the toluene along with water (90 ml_). The solution was cooled to 90 °C then

bis(pinacolato)diboron (1.5 kg, 5.82 mol), KOAc (772.6 g, 7.87 mol) and Pd(PPh3) (24.3 g, 0.02 mol) were added and the reaction mixture was heated at reflux for 10h. After confirming the completion of reaction by TLC (mobile phase: 100% DCM), the reaction mixture was cooled to room temperature and was kept standing overnight. The reaction mixture was filtered through celite and the celite cake was washed with toluene (4 L). The filtrate of this batch was mixed with the filtrate of another batch (batch size 1.3 kg obtained from an identical reaction). The mixed filtrate was washed with water (17.5 L), brine (17.5 L), dried over Na2S04, filtered and the solution was passed through a pad of silica gel (2 kg, mesh size 230-400). The silica gel pad was washed with toluene. The combined filtrate and washing was concentrated under reduced pressure and the residual crude product was stirred with n-hexane (23 L) for 1 h to obtain a solid product. The solid was collected by filtration, washed with n-hexane (5 L) and dried to afford 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-yl)benzaldehyde (4a) (2.47 kg, 84.6%). H NMR (300 MHz, CDCI3) δ 10.54 (s, 1 H), 7.57 (s, 1 H), 7.33 (s, 1 H), 5.89 (s, 1 H), 4.01 (s, 3H), 1.37 (s, 12H); 1H NMR (300 MHz, DMSO-d6) δ 10.35 (s, 1 H), 9.95 (s, 1 H), 7.33 (s, 1 H), 7.23 (s, 1 H), 3.87 (s, 3H), 1.33 (s, 12H); MS (ES+) 301.1 (M+Na); 579.1 (2M+Na); Analysis calculated for C14H19B05: C, 60.46; H, 6.89; Found: C, 60.60; H, 6.87

The average yield of 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) from step (3) is 78 – 90%.

The process is also illustrated in Fig. 3.

Step (4): Preparation of 3-Bromo-2,6-dimethylpyridine (5b)

2,6-lutidine (5a) (115 kg, 1073.3 mol) was added into pre-chilled oleum (20-23%, 1015 kg, 2276.7 mol) at 0 °C over a period of 4.5 h (temperature r6ached 14 °C during the addition). Bromine (88.18 kg, 1103.6 mol) was then added at 5-10 °C over a period of 1 h. The reaction mixture was slowly heated to 150 °C over a period of 12h. TLC analysis indicated about 40-50% conversion to product and the formation of a dimer by-product (5%). The reaction mixture was cooled to room temperature and then additional bromine (88.18 kg, 1103.6 mol) was added slowly. The reaction mixture was slowly heated to maintain a temperature of 65-75 °C over a period of 15h. TLC analysis indicated a 65-70 % conversion to product and the formation of 5% dimer by product. The reaction mixture was quenched by addition of water (500L) while maintaining the reaction temperature below 20 °C. The mixture was basified with 6.6 M NaOH (3800 L) while maintain the temperature at < 40 °C. EtOAc (220 L) was added and the mixture was stirred for 1 h then allowed to settle over a period of 2 h. The layers were separated and the aqueous layer was treated with NaOH (10 kg) in water (10 L) and extracted with EtOAc (160 L). The organic extracts were combined washed with brine (100 L), dried over Na2S04 (50.0 kg), filtered and the solvent was evaporated under atmospheric pressure. The residue was vacuum distilled and the desired product 3-bromo-2,6-dimethylpyridine (5b) was collected at 58-60 °C, 2 mmHg (98.45 kg, 49.2 %) as a colorless liquid.

The process is also illustrated in Fig. 4.

Step (5): Preparation of 3-Bromopyridine-2,6-dicarboxylic acid (5c)

5b 5c

To a stirred solution of 3-bromo-2,6-dimethylpyridine (5b) (98 kg, 5326 mol) in water (1310 L) was added KMn0 (225 kg, 1423.6 mol) in 5 equal portions in 1 h intervals at 70 °C. After stirring for 1 h at 70 °C, additional KMn04 (225 Kg, 1423.6 mol) was added in 5 equal portion in 1 h intervals at 90 °C. The reaction mixture was stirred for 12 h at 90 °C. The suspension was filtered hot through celite to obtain a clear solution. The solvent was distilled off to remove about 30% of the total volume. The remaining concentrated solution was chilled to 0 °C and made acidic (to pH 3-4) by the addition of cone. HCI (120 L). The white precipitate obtained was collected by filtration and dried at 70 °C to afford 3-bromopyridine-2,6-dicarboxylic acid (5c) as a white solid (109 kg, 84%).

The process is also illustrated in Fig. 5.

Step (6): Preparation of Dimethyl 3-Bromopyridine-2,6-dicarboxylate (5d)

To a stirred solution of 3-bromopyridine-2,6-dicarboxylic acid (5c) (20.0 kg, 81.29 mol) in methanol (100 L) was added cone. H2S04 (4.4 L) over a period of 30 min. The reaction mixture was heated to 65 °C and maintained at that temperature for 5 h (the reaction was monitored by TLC analysis to determine completion of reaction). The reaction mixture was cooled to room temperature basified by careful addition of aqueous NaHC03 solution (prepared from 10 kg NaHC03 in 120 L of water) and further diluted with water (120 L). The white solid obtained was collected by filtration, washed with plenty of water and then oven-dried at 40 °C to obtain dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (9.2 kg, 41.3%) as a white solid; 1HNMR (300 MHz, DMSO-cf6) δ 8.47 (d, J = 8.4, 1 H), 8.08 (dd, J = 4.5, 8.4, 1 H), 3.95 (s, 3H), 3.91 (s, 3H); MS (ES+) 570.6 (2M+Na); Analysis calculated for C9H8BrN04: C, 39.44; H, 2.94; Br, 29.15 N, 5. 1 ;

Found: C, 39.52; H, 2.92; Br, 29.28; N, 5.03.

The process is also illustrated in Fig. 6.

6582

Step (7): Preparation of Methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (

To a stirred solution of dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (27 kg, 98.52 mol) in ierf-butanol (135 L) was added at room temperature cyclopropylmethanamine (7.83 kg, 110.1 mol). The reaction mixture was heated at 65 °C for 17 h. The progress of reaction was monitored by TLC and HPLC (HPLC analysis showed the formation of 74% of the product 5e after 17 h. The reaction mixture was cooled to room temperature and then cone. HCI (2.7 L) was added slowly and the mixture was stirred for 15 min. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in hot /-PrOH (54 L) filtered through a celite pad. The filtrate was cooled with stirring to 10 °C to obtain a white precipitate. The solid obtained was collected by filtration, washed with cold

i-PrOH (13 kg), n-hexane (15 L) and dried to provide pure methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e) (15.7 kg, 50.9%). The filtrate was concentrated under reduced pressure and the crude product can be purified by silica gel column chromatography eluting with tert-butanol in hexanes to furnish additional 10% methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e). HNMR (300 MHz, DMSO-cf6) δ 8.83 (t, J = 5.9, 1 H), 8.47 – 8.41 (m, 1 H), 8.06 (d, J = 8.4, 1 H), 3.96 (s, 3H), 3.16 (t, J = 6.5, 2H), 1.14 – 0.99 (m, 1 H), 0.42 (m, 2H), 0.30 -0.19 (m, 2H); MS (ES+) 337.0 (M+23), 650.8 (2M+23); Analysis calculated for

C12H13BrN203: C, 46.03; H, 4.18; N, 8.95; Br, 25.52; Found: C, 46.15; H, 4.17; N, 8.72; Br, 25.26.

The average isolated yield for step (7) is 50% to 60%.

The process is also illustrated in Fig. 7.

Step (8): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a)

2

6a

THF (37.5 L) was charged to a 100 L reactor followed by ethyl 3-bromo-6- (cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate (5e) (2.5 kg, 7.98 mol) under a nitrogen atmosphere. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) (2.88 kg, 10.36 mol) was added, followed by the addition of PPh3 (53.13 g, 0.20 mol), PdCI2(PPh3)2 (120.4 g, 0.17 mol) and a solution of Na2C03(2.12 kg, 20.00 mol) in demineralized water (10.0 L) under nitrogen atmosphere. The reaction mixture was degassed again two times by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 6.5 h, cooled to room temperature and filtered through a Celite bed. Water (75 L) was added to the filtrate and the product was extracted with ethyl acetate (75 L). The aqueous layer was back extracted with ethyl acetate (2 χ 60 L). The combined ethyl acetate extract was divided into two equal portions and each portion was washed with brine (37 L), dried over Na2S04, filtered and concentrated under reduced pressure to give crude methyl 6- ((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) as a reddish viscous material (-4.5 Kg) which was used as such for the next step without further purification. An analytical sample was prepared by purification of a small sample by flash column chromatography (silica gel, eluting with 0-100% ethyl acetate in hexane) to furnish methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)-picolinate (6a) as an off-white solid; HNMR (300 MHz, DMSO-d6) δ 9.89 (s, 1 H), 9.52 (s, 1 H), 8.79 (t, J = 6.1 Hz, 1 H), 8.23 (d, J = 8.0 Hz, 1 H), 8.09 (d, J = 8.0 Hz, 1 H), 7.34 (s, 1 H), 6.90 (s, 1 H), 3.85 (s, 3H), 3.62 (s, 3H), 3.22 (m, 2H), 1.16 -1.02 (m, 1 H), 0.49 – 0.38 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 791.0 (2M+Na), (ES-) 382.7 (M-1), 767.3 (2M-1); Analysis calculated for C20H20N2O6.0.25 H20: C, 61.77; H, 5.31 ; N, 7.20; Found: C, 61.54; H, 5.13; N, 7.05.

The process is also illustrated in Fig. 8.

46582

Step (9): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b)

6a 6b

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) (2.11 kg, estimated about 3.83 mol from step-8) in dichloromethane (16.0 L) and pyridine (1.4 L, 17.4 mol) cooled to -10°C and maintained at that temperature for 1 h was added a solution of triflic anhydride (980.0 ml_, 5.8 mol) in dichloromethane (6.0 L) drop wise over a period of 3 h at -10 °C. The reaction mixture was stirred at -5°C for 1.3 h, quenched with saturated aqueous NaHCO3(10.4 L) and stirred for 30 mins. The organic layer was separated, washed successively with saturated aqueous NaHC03 (10.4 L), 1 HCI (2 x 16.6 L), water (13.2 L), brine (13.2 L), dried over MgS04, filtered and concentrated under reduced pressure to give the crude product. The crude product was stirred with 15% ethyl acetate in n-hexane (7.0 L) for 1 h. The solid obtained was collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (7.0 L) for 1 h, was collected by filtration and washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (8.0 L) for 1 h, collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was dried to afford methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)-oxy)phenyl)picolinate (6b) as a light brown solid (1.7 kg, 86% yield, for combined steps 8 & 9). Average isolated yield for combined steps 8 and 9 was 70% to 86%; Ή NMR (300 MHz, DMSO-cf6): δ 9.64 (s, 1 H), 8.78 (t, J = 6.1 , 1 H), 8.29 (d, J = 8.0, 1 H), 8.16 (d, J = 8.0, 1 H), 8.03 (s, 1H), 7.39 (s, 1 H), 4.00 (s, 3H), 3.63 (s, 3H), 3.22 (m, 2H), 1.11 (m, 1 H), 0.52 – 0.39 (m, 2H), 0.28 (m, 2H); MS (ES+) 538.9 (M+Na). The process is also illustrated in Fig. 9.

Step (10): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c)

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4- (((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b) (12 kg, 23.24 mol) in DME (106 L) was charged into reactor under nitrogen. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. Potassium trifluoro(vinyl)borate (3.9 kg, 29.1 1 mol), PdCI2(PPh3)2 (815 g, 1.13 mol), KHC03 (4.65 g, 46.44 mol) and demineralized water (12 L) was then added under a N2 atmosphere. The reaction mixture was degassed by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 5 h. The reaction mixture was cooled to room temperature and then filtered through a Celite bed. Demineralized water (118 L) was added to the filtrate followed by ethyl acetate (124 L). The mixture was stirred for 20 min and then the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 95 L). The combined organic extract was washed with brine (95 L), dried over Na2S04, and filtered. The solvent was evaporated under reduced pressure to give the crude product. The crude product was purified by column chromatography (silica gel, 120 kg, 230-400 mesh size, eluting with ethyl acetate in n-hexane) to obtain methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (6 kg, 72%). 1H NMR (300 MHz, CDCI3): δ (ppm) 9.64 (s, 1 H), 8.35 (d, J = 7.8 Hz, 1 H), 8.06-8.03 (m, 2H), 7.78(d, J = 7.8 Hz, 1 H), 7.02-6.92 (m, 1 H), 6.61 (s, 1 H), 5.86 (d, J = 17.7 Hz, 1 H), 5.38 (d, J = 1 1.4 Hz, 1 H), 3.84 (s, 3H), 3.67 (s, 3H), 3.35-3.29 (m, 2H),1.08-1.03 (m, 1H), 0.55-0.49 (m, 2H), 0.29-0.2 4(m, 2H). 1HNMR (300 MHz, DMSO-d6) 6 9.68 (s, 1 H), 8.77 (t, J = 6.1 , 1 H), 8.35 – 8.21 (m, 1 H), 8.16 – 8.01 (m, 2H), 7.14 -6.87 (m, 2H), 6.01 (dd, J = 1.2, 17.8, 1 H), 5.45 (dd, J = 1.1 , 1 1.3, 1 H), 3.91 (s, 3H), 3.64 (s, 3H), 3.23 (m, 2H), 1.21 – 1.01 (m, 1H), 0.51 – 0.40 (m, 2H), 0.34 – 0.20 (m, 2H). MS

(ES+) 417.0 (M+Na); Analysis calculated for C22H22N205: C, 66.99; H, 5.62; N, 7.10;

Found: C, 66.75; H, 5.52; N, 7.06.

The process is also illustrated in Fig. 10.

Step (1 1): Preparation of 2-(6-((cyclopropylmethyl)carbamoyl)-2- (methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d)

To a stirred solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (1.57 kg, 3.80 mol) in acetonitrile (15.4 L) was added ferf-butyl alcohol (22.2 L), demineralized water (3.2 L) and sodium dihydrogen phosphate monohydrate (323.74 g, 2.346 mol). The reaction mixture was cooled to 0 °C and added 2-methyl-2-butene (5.3 L, 50.0 mol) and stirred at 0 °C for 30 min. A solution of 80% sodium chlorite (1.36 kg, 12.0 mol) in demineralized water (5.2 L) was added to the reaction mixture over a period of 2.5 h at 0 °C [temperature rises to 7 °C during the addition]. The reaction mixture was stirred at 0 °C for 2 h, diluted with water (40 L) and ethyl acetate (24 L). After stirring the mixture, it was allowed to settle and the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 20 L) then acidified with 5.9 % aqueous acetic acid (2 L) and extracted once with ethyl acetate (10 L). The organic extracts were combined washed with water (2 x 20 L), a solution of acetic acid (125 mL) in water (20.0 L), brine (2 χ 20 L), dried over Na2S04, filtered and concentrated under reduced pressure (vapor temperature below 40 °C). The residue obtained was dissolved in acetone (7 L) (residue didn’t dissolve completely). The solution was poured slowly into a reactor containing stirred n-hexane (70.0 L) to precipitate the solid product and the mixture was stirred for 2 h. The solid obtained was collected by filtration, washed with 10% acetone in n-hexane (6.3 L), AJ-hexane (6.3 L), dried to afford 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4- methoxy-5-vinylbenzoic acid (6d) as an off-white solid (1.29 Kg, yield: 79.0%). Average isolated yield for step 1 1 is 74% to 84%. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.50 (brs, 1 H), 8.69(t, J= 6.0 Hz, 1 H, NH), 8.20 (d, J= 7.9 Hz, 1 H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1 H), 6.97 (dd, J= 18.0, 1 1.3 Hz, 1 H), 6.88 (s, 1 H), 5.92 (d, J= 7.9 Hz, 1 H), 5.38 (d, J= 1 1.1 Hz, 1 H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); MS (ES+) 433.26, (M+Na); (ES-) 409.28 (M-1). The process is also illustrated in Fig. 1 1.

Step (12): Preparation of Methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate methanesulfonate (7a

Pyridine (3.8 L, 47.17 mol) and EDCI (5.31 kg, 27.66 mol) were sequentially added to a cooled solution (0 °C) of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (9 kg, 21.92 mol) and 4-aminobenzamidine dihydrochloride (5.13 kg, 24.65 mol) in /-PrOH (90 L). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. TLC analysis indicated incomplete reaction. Additional EDCI (1.08 kg, 5.6 mol) was added and the reaction mixture was stirred for 8 h. The reaction was still incomplete as indicated by TLC analysis, additional EDCI (0.54 kg, 2.8 mol) was added and the reaction mixture was stirred for 5 h. TLC analysis indicated there was trace amount of unreacted starting material remaining. The reaction mixture was cooled to 0 °C and a solution of

methanesulfonic acid (MSA) (9.13 kg, 95 mol) in MeOH (38.7 L) was added to the cooled mixture over a period of 4 h. The reaction mixture was allowed to warm to room temperature and stirred for 15 h. The product was collected by filtration, washed with a mixture of /-PrOH and MeOH (4:1 , 45 L). The wet cake was slurried in a mixture of /-PrOH and MeOH (2:1 , 135 L) stirred for 1 h and the product was collected by filtration and washed with a mixture of /-PrOH and MeOH (4:1 , 46.8 L). The product was dried in

2015/046582

a vacuum oven at 45 °C to afford methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) as a pink-colored solid (12.71 kg, 93%). Average isolated yield for this step: >90%.

1H NMR (300 MHz, DMSO-c/6) δ 10.71 (s, 1 H), 9.16 (s, 2H), 8.80 (s, 2H), 8.68 (t, J = 6.1 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1 H), 7.93 (s, 1H), 7.84 – 7.72 (m, 4H), 7.12 – 6.97 (m, 2H), 6.04 (dd, J = 17.8, 1.3 Hz, 1 H), 5.45 (d, J = 12.6 Hz, 1H), 3.91 (s, 3H), 3.60 (s, 3H), 3.25 – 3.16 (m, 2H), 2.32 (s, 3H), 1.10 – 1.01 (m, 1 H), 0.48 – 0.37 (m, 2H), 0.30 – 0.22 (m, 2H); MS (ES+) 528.0 (M+1); Analysis calculated for

C29H29N5O5.CH3SO3H.2H2O. C, 54.62; H, 5.65; N, 10.62; S, 4.86; Found: C, 54.95; H, 5.55; N, 10.61 ; S, 4.87.

The process is also illustrated in Fig. 12.

Step (13): Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-rnethoxy-4- vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate

(3i) ,a 3i

A pre-cooled (0-5 °C) aq. NaOH solution [prepared from solid NaOH (4 kg, 100 mol) in water (86 L)] was added to a suspension of methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) (28.7 kg, 46 mol) in acetonitrile (86 L) cooled to 0 to 5 °C over a period of 25 mins. The reaction mixture was stirred at 0 to 5 °C for 2.5 h (TLC analysis showed the reaction was complete). The reaction mixture was filtered through a sparkler filter, washed with a mixture of 1 :1 CH3CN / H20 ( 57.4 L). Acetic acid (3.2 L, 55.9 mol) in water (56 L) was added to the filtrate at room temperature over a period of 25 mins and the resulting mixture was stirred at room temperature for 2.5 h. The solid product obtained was collected by filtration, washed with a 1 :4 mixture of CH3CN / H20 (57.5 L). The solid was dried at 45°C in a vacuum oven to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate (3i) as an off-white solid (12,77 kg, 54.1%). Average yield for this step is 50% to 75%. Mp: >200°C; H NMR (300 MHz, DMSO-d6): δ 13.49 (s, 1 H), 8.94 (bs, 4H), 8.56 (t, 1 H), 7.82 – 7.71 (m, 2H), 7.67 -7.56 (m, 4H), 7.51 (d, J = 7.8, 1 H), 6.98 (dd, J = 11.3, 17.8, 1 H), 6.68 (s, 1 H), 5.92 (d, J = 16.6, 1 H), 5.36 (d, J = 12.4, 1 H), 3.80 (s, 3H), 3.16 (m, 2H), 1.05 (m, 1 H), 0.43 (m, 2H), 0.24 (m, 2H); MS (ES+) 514.1 (M+1), 536.1 (M+Na), (ES-) 512.1 ; Analysis calculated for C28H27N5O5.3H2O: C, 59.25; H, 5.86; N, 12.34; Found C, 59.50; H,

5.75; N, 12.05. If needed this material can be crystallized from a mixture of acetone and water.

The process is also illustrated in Fig. 13.

Step 14: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b

A pre-cooled (5-8 °C) aqueous NaOH solution (prepared from solid NaOH (1.97 kg, 49.25 mol) in demineralized water (41 L) was added to a pre-cooled (0-5 °C) suspension of (3i) (13.8 kg, 26.9 mol) in acetonitrile (41 L). The reaction mixture was stirred at 0-5 °C for 30 min (until the reaction mixture becomes homogeneous). The reaction mixture was filtered through a sparkler filter washed with 50% acetonitrile in demineralized water (4.4 L). The filtrate was charged into a reactor and cooled to 0-5 °C. Aqueous HCI [prepared from cone. HCI (9.3 L) in demineralized water (36 L)] was added slowly with stirring to keep the reaction temperature at or below 15 °C, the resulting mixture was stirred at 10-15 °C for 13 h. The reaction mixture was cooled to 0-5 °C and stirred for 1 h. The solid obtained was collected by filtration and washed with demineralized water (36 L). The solid product was suspended in water (69 L) stirred for 30 mins and collected by filtration washed twice with water (20 L each). The solid product was dried in a vacuum oven at 45°C to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethyl carbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (1 1.21 Kg, 75.77%). Mp: >200°C; 1H NMR (300 MHz, DMSO-ci6): δ 12.98 (br s, 1 H), 10.86 (s, 1 H), 9.24 (s, 3H), 9.04 (s, 2H), 8.22 (d, J = 7.8 Hz, 1 H), 7.96 (d, J = 5.7 Hz, 2H), 7.78 (s, 4H), 7.09-6.99 (m, 2H), 6.07 (d, J = 17.7 Hz, 1 H), 5.45(d, J = 11.4 Hz, 1 H), 3.88 (s, 3H), 3.26-3.24 (m, 2H), 1.09 (m, 1 H), 0.47 (m, 2H), 0.28 (m, 2H).

Average isolated yield for this step varies from 63% to 80%.

The process is also illustrated in Fig. 14.

Example-2: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

6d 8a

To a solution of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (2.35 g, 5.7 mmol) and 4-aminobenzamidine dihydrochloride (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 ml_) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The

reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS04, filtered and concentrated in vacuum. The residue obtained was purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 and 0-100% chloroform in CMA 50) to furnish methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)-carbamoyl)picolinate hydrochloride (8a) (2.2 g, 65%) as a white solid; MP 266 °C; 1HNMR (300 MHz, DMSO-d6) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 -7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1 H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1 H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for C29H29N505 (H20)1 5 (HCI): C, 58.93; H, 5.63; N, 1 1.85; Found: C, 58.75; H, 5.65; N, 1 1.92.

Step-2: preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

8a 8b j0 a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml), was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with a solution of sulfuric acid (0.483 ml, 9.00 mmol) in water (5 mL) and stirred for 10 min at room temperature. To this cold water (5 ml) was added and stirred at room temperature until product crystallized out. Cold water (5 mL) was added to the slurry and stir for additional 20 min, additional cold water (5 mL) was added prior to filtration of solid. The solid obtained was collected by filtration washed with water (5 mL and 2.5 mL), dried under vacuum overnight to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b) (1.103 g, 90 % yield) as a white solid; MP 221.7 °C; H NMR (300 MHz, DMSO-d6) δ 12.30 – 10.91 (bs, 1 H, D20 exchangeable), 10.69 (bs, 1 H, D20 exchangeable), 9.24 (t, J = 6.0 Hz, 1 H), 9.16 (s, 2H, D2O exchangeable), 8.78 (s, 2H, D2O exchangeable), 8.24 (d, J = 8.0 Hz, 1 H), 8.04 – 7.91 (m, 2H), 7.84 – 7.67 (m, 4H), 7.13 – 6.94 (m, 2H), 6.03 (dd, J = 17.8, 1 .4 Hz, 1 H), 5.51 – 5.37 (m, 1 H), 3.88 (s, 3H), 3.24 (t, J = 6.4 Hz, 2H), 1.16 – 1.01 (m, 1 H), 0.52 – 0.41 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for: C28H27N605 1.0H2SO4 1.5H20: C, 52.66; H, 5.05; N, 10.97; S, 5.02; Found: C, 52.81 ; H, 4.95; N, 10.94; S, 4.64.

Example-3: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane s

To a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml) was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with methanesulfonic acid (0.584 ml, 9.00 mmol) and stirred for 1 h at room temperature. Cold water (5.00 ml) was added to the reaction mixture and stirred at room temperature until product crystallized out. To the slurry was added water (5 ml) of water stirred for additional 20 min, followed by the addition of water (5 ml) prior to filtration. The solid obtained was collected by filtration washed with water (5 ml and 2.5 ml), dried under vacuum to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane sulfonate salt (8c)

(1 .138 g, 1.867 mmol, 93 % yield) as a white solid; MP 221.2 °C; 1 H NMR (300 MHz,

DMSO-d6) δ 12.89 (s, 1 H, D2O exchangeable), 10.69 (s, 1 H, D2O exchangeable), 9.24

(t, J = 6.0 Hz, 1 H), 9.16 (s, 2H,), 8.85 (s, 2H), 8.24 (d, J = 8.0 Hz, 1 H), 8.06 – 7.91 (m, 2H), 7.86 – 7.70 (m, 4H), 7.15 – 6.96 (m, 2H), 6.03 (dd, J = 17.8, 1.4 Hz, 1 H), 5.52 – 5.35 (m, 1 H), 3.88 (s, 3H), 3.25 (t, J = 6.3 Hz, 2H), 2.34 (s, 3H), 1.17 – 1.01 (m, 1 H), 0.53 -0.43 (m, 2H), 0.32 – 0.23 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for:

CzeH^NsOsCHsSOsH 1.5H20: C, 54.71 ; H, 5.38; N, 11.00; S, 5.04; Found: C, 54.80; H, 5.14; N, 10.94; S, 4.90.

Example-4: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) in Form C (Compound XX)

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled

46582

to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 – -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1 .6 kg) and 0.57 kg water. The mixture was heated to 46 °C. 21 g of Smopex-234 and 10 g Acticarbone 2SW were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 °C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature ❤ °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The suspension was stirred at 0-3.0 °for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (Tjacke,= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 1 17 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C (Compound XX).

/////avoralstat, BCX4161, Fast Track, Treat hereditary angioedema (HAE), Orphan Drug, PRECLINICAL

COc1cc(c(cc1C=C)C(=O)Nc2ccc(cc2)C(=N)N)c3cc(ncc3C(=O)O)C(=O)NCC4CC4

MK 7655, RELEBACTAM, a β-Lactamase inhibitor


MK 7655, RELEBACTAM

(1R,2S,5R)-7-Oxo-N-(4-piperidinyl)-6-(sulfooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide

(1R,2S,5R)-7-oxo-2-((piperidin-4-yl)carbamoyl)-1,6-diazabicyclo(3.2.1)octan-6-yl hydrogen sulfate monohydrate

Sulfuric acid, mono((1R,2S,5R)-7-oxo-2-((4-piperidinylamino)carbonyl)-1,6-diazabicyclo(3.2.1)oct-6-yl) ester, hydrate (1:1)

MF C12H22N4O7S
MW 366.39068 g/mol

CAS 1174020-13-3

β-Lactamase inhibitor

MK-7655 is a beta-lactamase inhibitor in phase III clinical studies at Merck & Co for the treatment of serious bacterial infections…….See clinicaltrials.gov, trial identifier numbers NCT01505634 and NCT01506271.

In 2014, Qualified Infectious Disease Product (QIDP) and Fast Track designations were assigned by the FDA for the treatment of complicated urinary tract infections, complicated intra-abdominal infections and hospital-acquired bacterial pneumonia/ventilator-associated bacterial pneumonia.

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PAPER

A concise synthesis of a beta-lactamase inhibitor
Org Lett 2011, 13(20): 5480

http://pubs.acs.org/doi/abs/10.1021/ol202195n

http://pubs.acs.org/doi/suppl/10.1021/ol202195n/suppl_file/ol202195n_si_001.pdf

 

Abstract Image

MK-7655 (1) is a β-lactamase inhibitor in clinical trials as a combination therapy for the treatment of bacterial infection resistant to β-lactam antibiotics. Its unusual structural challenges have inspired a rapid synthesis featuring an iridium-catalyzed N–H insertion and a series of late stage transformations designed around the reactivity of the labile bicyclo[3.2.1]urea at the core of the target.

H NMR (400 MHz, DMSO-d6): δ 8.30 (br s, 2H), 8.20 (d, J = 7.8 Hz, 1H), 4.01 (s, 1H), 3.97-3.85 (m, 1H), 3.75 (d, J = 6.5 Hz, 1H), 3.28 (dd, J = 12.9, 2.5 Hz, 2H), 3.05-2.93 (m, 4H), 2.08-1.97 (m, 1H), 1.95-1.79 (m, 3H), 1.73-1.59 (m, 4H);

13C NMR (DMSO-d6, 100 MHz) δ 169.7, 166.9, 59.8, 58.3, 46.9, 44.3, 42.9, 28.5, 28.3, 20.8, 18.9;

HRMS calculated for C12H20N4O6S (M+H): 349.1182, found: 349.1183.

[α]D 25 = -23.3 (c = 1.0, CHCl3)

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PATENT

WO 2009091856

http://www.google.com/patents/WO2009091856A2?cl=en

EXAMPLE IA

(2S ,5 R)-7-Oxo-N-piperidin-4-yl-6-(sulfooxy)- 1 ,6-diazabicyclo [3.2.1 ]octane-2-carboxamide

Figure imgf000063_0001

Step 1 : tert-butyl 4-({[(2S,5R)-6-(benzyloxy)-7-oxo-l,6-diazabicyclo[3.2.1]oct-2- yljcarbonyl } amino)piperidine- 1 -carboxylate : To a solution of (2S,5R)-6-(phenylmethoxy)-7-oxo-l,6-diazabicyclot3.2.1]octane-

2-carboxylic acid (1.484 g, 5.37 mmol) in dry dichloromethane (60 ml) was added triethylamine (1.88 ml, 13.49 mmol), 2-chloro-l-methylpyridinium iodide (1.60 g, 6.26 mmol), and 4-amino-l- BOC-piperidine (1.30 g, 6.49 mmol) sequentially at room temperature under nitrogen. The reaction was then heated to 500C for 1 hour. The reaction mixture was concentrated under vacuum and purified by silica gel chromatography on an Isco Combiflash (40 g silica gel, 40 mL/min, 254 nM, 15% to 100% EtOAc/hexane over 14 column volumes then 100% EtOAc for 4 column volumes; title compuond eluted at 65% ethyl acetate/hexane) to afford the title compound as a pale orange solid.

Step 2: tert-butyl 4-({[(2S,5R)-6-hydroxy-7-oxo-l ,6-diazabicyclo[3.2.1]oct-2- yl] carbonyl } amino)piρeridine- 1 -carboxylate:

Palladium on carbon (394 mg; 10% Pd/C) was added to a solution of the product of step 1 (1.81 g, 3.95 mmol) in methanol (50.6 mL) and the resulting mixture was stirred under hydrogen (balloon) overnight. LC/MS analysis indicated the reaction was not complete. Acetic acid (6 drops) and additional catalyst (159 mg of 10% Pd/C) were added to the reaction and the resulting mixture was stirred under hydrogen (balloon) for an additional 90 minutes. Additional catalyst (0.2085 g of 10% Pd/C) was added to the reaction and stirring under hydrogen was continued for an additional 2.5 hours at which time the reaction was judged complete by LC-MS analysis. The reaction was filtered through a celite pad and the collected solid was washed well wtih MeOH. The filtrate was concentrated under vacuum to afford the title compound as a colorless oil which was used without purification in the next step.

Step 3 : tert-butyl-4-({ [(2S,5R)-7-oxo-6-(sulfooxy)- 1 ,6-diazabicyclo[3.2.1 ]oct-2- yl] carbonyl } amino)ρiperidine- 1 -carboxylate:

To a solution of the product of step 2 (1.455 g, 3.95 mmol; theoretical yield of step 2) in dry pyridine (30 mL) was added sulfur trioxide pyridine complex (3.2 g, 20.11 mmol) at room temperature under nitrogen. The resulting thick mixture was stirred over the weekend.

The reaction was filtered and the white insoluble solids were washed well with dichloromethane. The filtrate was concentrated in vacuo. The residue was further azeotroped with toluene to remove excess pyridine to afford the title compound which was used without purification in the next step.

Step 4: (2S,5R)-7-oxo-N-piperidin-4-yl-6-(sulfooxy)-l,6-diazabicyclo[3.2.1]octane-2- carboxamide:

To a mixture of the product of step 3 (1.772 g, 3.95 mmol; theoretical yield of step 3) in dry dichloromethane (30 ml) at 00C under nitrogen was slowly added trifluoroacetic acid (6.1 ml, 79 mmol). Immediately the reaction became a solution. After 1 hour, additional trifluoroacetic acid (8 ml) was added to the reaction. The reaction was stirred at 00C until judged complete by LC-MS analysis then concentrated in vacuo. The residue was triturated with ether (3X) to remove excess TFA and organic impurities. The resulting white insoluble solid was collected via centrifugation, dried in vacuo, then purified by preparative HPLC (250X21.2 mm Phenomenex Synergi Polar-RP 80A column; 10 micron; 35 mL/min.; 210 nM; 0% to 30% methanol/water over 15 minutes; title compound eluted at 10% methanol/water). Fractions containing the title compound were combined and Iyophilized overnight to afford the title compound as a white solid. LC-MS (negative ionization mode) m/e 347 (M-H).

PAPER

Discovery of MK-7655, a beta-lactamase inhibitor for combination with Primaxin
Bioorg Med Chem Lett 2014, 24(3): 780

http://www.sciencedirect.com/science/article/pii/S0960894X13014856

Image for unlabelled figure

PATENT

WO 2014200786

http://www.google.dj/patents/WO2014200786A1?cl=en

 

 

 

Exemplary Scheme

– 50% isolated yield overall from 1 to 5

O via crystallization

XAMPLE 1

(2S,5R)-7-oxo-N-piperidin-4-yl-6-(sulfooxy)- 1 ,6-diazabicyclo[3.2.1 ]octane-2-carboxamide

Preparation of (15′,45)-5-((2-nitrophenyl)sulfonyl)-2-oxa-5-azabicyclo[2.2.2]octan-3 one (2)

To a reactor (R-1) equipped with an additional funnel, nitrogen inlet and agitator was charged (2S,5S)-5-hydroxypiperidine-2-carboxylic acid (77.3 wt%) (50.0 g, 344 mmol), and water (150 mL). Agitation was begun, the pH adjusted to 10-11 by addition of 10 N NaOH (~ 46.5 mL) and the reactor charged with acetone (50.0 mL).

In a separate reactor (R-2) equipped with an agitator and nitrogen inlet was charged 2-nitrobenzene-l-sulfonyl chloride (97%) (106.0 g, 478 mmol) and acetone (80 mL). The contents of R-2 were transferred to R-1 at 23-30 °C while the pH of the solution was maintained at 10-11 by simultaneously addition of 10 N NaOH. After 15 to 30 min, the pH was adjusted to about 6 by addition of 12 N HC1. The solution was charged with EtOAc (500 mL) and the pH adjusted to 3.0 by addition of 12 N HC1. The layers were separated and the aqueous back-extracted with EtOAc (150 mL x 2).

To a separate reactor (R-3) was charged product la in the combined organic layers, 2-nitrobenzene-l-sulfonyl chloride (73.0 g, 329 mmol), and triethylamine (130 mL). The batch in R-3 was agitated at 20-28°C for 30 min. The solution was charged with water (100 mL), the layers separated, and the aqueous back extracted with EtOAc (150 mL x 2). The combined EtOAc layer was washed with 10% NaHC03 (100 mL) and brine (100 mL). The organic phase was concentrated to 150 mL upon which a crystalline slurry was formed. The concentrated solution was agitated at 13-18°C for 2-3 hours followed by filtration of crystalline solids. The resulting wet cake was washed with EtOAc (60 mL) and then dried under vacuum oven at 25-30°C to afford 2 (65.6 g, 79% yield), m.p. 126.0-126.7 °C. 1H NMR (CDC13, 400 MHz) δ: 8.02 (m, 1 H), 7.80-7.71 (m, 2 H), 7.66 (m, 1 H), 4.88 (m, 1 H), 4.55 (dd, J= 3.8, 2.7 Hz, 1 H), 3.78 (dt, J= 11.2, 3.0 Hz, 1 H), 3.66 (dd, J = 11.2, 1.1 Hz, 1 H), 2.44 (m, 1 H), 2.11 (m, 2 H), 1.91 (m, 1 H); 13C NMR (CDC13, 100 MHz) δ: 168.4, 148.3, 134.4, 132.1, 131.0, 130.7, 124.2, 73.5, 51.4, 48.0, 25.1, 23.2

Preparation oftert-butyl 4-((25*,55)-l-((2-nitrophenyl)sulfonyl)-5-(((2- nitrophenyl)sulfony l)oxy)piperidine-2-carboxamido)piperidine- 1 -carboxylate (3)

To a reactor (R-l) was charged lactone 2 (65.5 g, 210 mmol), THF (131 mL) and tert-butyl 4-aminopiperidine-l -carboxylate (44.5 g, 222 mmol). The stirred solution was heated to reflux (typical temperature 72 °C) for ~18 hr. The reaction was cooled to 25-35 °C and then charged with THF (325 mL) and 4-dimethylaminopyridine (40.1 g, 328 mmol) followed by agitation for 30 minutes.

To a separate reactor (R-2) was charged 2-nitrobenzene-l-sulfonyl chloride (60.9 g,

275 mmol) and THF (200 mL). The contents of R-2 were added to R-l over the course of 45 to 75 minutes maintaining batch temperature of 20 to 30°C. The batch in R-l was agitated for 2 to 4 hours at a temperature of 20 to 30°C.

To a separate reactor (R-3) was charged water (600 mL) and methanol (600 mL). The contents of R-3 were charged to the main batch over the course of 45 to 75 minutes with agitation while maintaining temperature of 20 to 30°C. The batch was cooled to 5 to -5°C and then agitated at 5 to -5°C for at least 4 hours. The solids were filtered and then washed twice with methanol (130 mL x 2). The wet cake was dried in a vacuum oven at 40 to 50°C to afford 3 (144.0 g, 98% yield), m.p. 131.8-133.1 °C. 1H NMR (CDC13, 400 MHz) δ: 8.14 (m, 2 H), 7.83-7.74 (m, 6 H), 6.50 (d, J= 7.9 Hz, 1 H), 4.69 (m, 1 H), 4.43 (s, 1H), 4.11 (dd, , J= 13.7, 4.9 Hz, 1H), 3.95 (m, 2H), 3.83 (m, 1H), 3.47 (s, 1H), 3.10 (dd, J= 13.7, 11.0 Hz, 1H), 2.81 (m, 2H), 2.51 (m, 1H), 2.12 (m, 1H), 1.85-1.72 (m, 4H), 1.45 (s, 9H), 1.26 (m, 1H); 13C NMR (CDC13, 100 MHz) δ: 166.9, 154.6, 148.2, 147.6, 135.2, 134.8, 132.6, 132.5, 131.9, 131.6, 131.4, 129.7, 124.9, 124.7, 79.8, 76.5, 55.0, 47.1, 46.0, 31.8, 31.5, 28.4, 27.3, 24.4.

Preparation of N-4-nitrobenzene sulfonyl-O-benzylhydroxylamine

To a reactor (R-l) was charged O-benzylhydroxylamine hydrochloride (61.0g, 382 mmol) and pyridine (400 mL). The solution cooled to 5 to -5°C.

To a separate reactor (R-2) was charged 4-nitrobenzenesulfonyl chloride (89.0 g, 402 mmol) and pyridine (200 mL). The contents of R-2 were transferred to R-l at a rate to maintain temperature range of -5 to -5°C. The batch in R-l was agitated at 5 to -5 °C for 15 to 45 minutes then warmed to 20 to 30°C for 45 to 75 minutes. Water (250 mL) was then added at a rate to maintain 20 to 30°C and agitated 5 to 15 minutes. The solids were filtered and the wet cake washed with water (100 mL x 3). The wet cake was dried in vacuum oven at 50°C to afford N-4-nitrobenzenesulfonyl-O-benzylhydroxylamine (113.3 g, 96% yield), m.p. 128.4-130.0 °C. 1H NMR (CDCls, 400 MHz) δ: 8.36 (d, J = 8.9 Hz, 2 H), 8.11 (d, J = 8.9 Hz, 2 H), 7.36 (m, 5H), 7.11 (s, 1H), 5.02 (s, 2H); 13C NMR (CDC13, 100 MHz) δ: 151.0, 142.5, 134.9, 130.2, 129.7, 129.3, 128.9, 124.5, 80.2.

Step C. Preparation of tert-butyl 4-((2S,5R)-5-((benzyloxy)amino)piperidine -2-carboxamido)piperidine- 1 -carboxylate (4)

Boc

To a reactor (R-l) was charged tert-butyl 4-((2R,5R)-l-((2-nitrophenyl)sulfonyl)-5-(((2-nitrophenyl)sulfonyl)oxy)piperidine-2-carboxamido)piperidine-l -carboxylate (3) (110 g, 158 mmol), N-4-nitrobenzene sulfonyl-O-benzylhydroxylamine (58 g, 188 mmol), potassium carbonate (25.9 g, 187 mmol) and dimethylacetamide (440 mL). The stirred solution was heated to 60 to 70°C for 24 – 32 hours. The batch was cooled to 20 to 30°C and charged with toluene (660 mL). The batch was extracted with 1 N sodium hydroxide (3×220 mL) then washed with water (220 mL).

The toluene solution was azotropically distilled at ~50°C to about 1/3 volume. The solution was solvent-switched to MeOH at 45-55°C, adjusted to 237 mL.

The batch was cooled to 20-25°C, charged with thioglycolic acid (57.9 g, 629 mmol) at 10 °C, and then charged with K2CO3 anhydrous (172.0 g, 1225 mmol). The batch was agitated at 10-15°C for 0.5 h, warmed to 20-25°C, agitated at 20-25°C for 10-15 h, and heated at 48-53°C for 3-6 h.

The batch was charged with 10 wt% sodium chloride (1.10 L) and toluene (880 mL) at about 40°C. The layers were separated and the aq. layer back-extracted with toluene (3 x440 mL). The combined organic layer was washed with 10% NaHC03 (2 x220 mL). The batch was concentrated at 40-50°C to 165 mL, then cooled to 35-40°C. The batch was charged with seed (50 mg) and agitated for 1 h at 35-40°C. The batch was charged with heptanes (110 mL) at 35-40°C over 1 h, then slowly cooled to 15-20°C over 1 h. The batch was agitated for 3 h and the solids filtered. The wet cake was washed with toluene/heptanes (137.5 mL) then dried in vacuum oven at 30 °C for 3-8 h to affored 4. (47.3 g, 70% overall yield from 3), m.p. 117.5-118.0 °C. 1H NMR (CDC13, 500 MHz) δ: 7.37-7.29 (m, 5 H), 6.64 (d, J= 8.2 Hz, 1 H), 5.36 (brs, 1 H), 4.67 (s, 2 H), 4.00 (m, 2 H), 3.90 (m, 1 H), 3.28 (ddd, J= 11.8, 4.0, 1.7 Hz, 1 H), 3.12 (dd, J= 10.2, 3.2 Hz, 1 H), 2.95 (m, 1 H), 2.86 (m, 2 H), 2.46 (dd, J= 11.8, 9.5 Hz, 1 H), 2.10 (m, 1 H), 1.93-1.83 (m, 3 H), 1.58 (brs, 1 H), 1.45 (s, 9 H), 1.41 (m, 1 H), 1.35-1.23 (m, 3 H); 13C NMR (CDC13, 125 MHz) δ: 172.8, 154.7, 137.7, 128.4 (4 C), 127.9, 79.6, 76.9, 59.8, 57.0, 49.2, 46.1, 42.8 (br, 2 C), 32.0 (2 C), 28.4 (3 C), 28.3, 27.2.

Step D: Preparation of tert-butyl 4-((lR,2S,5R)-6-(benzyloxy)-7-oxo-l,6-diazabicyclo[3.2.1 ]octane-2-carboxamido)piperidine- 1 -carboxylate (5)

To a reactor (R-l) was charged tert-butyl 4-((2S,5R)-5-((benzyloxy)amino)piperidine-2-carboxamido)piperidine-l-carboxylate (4) (46.3 g, 107 mmol), dichloromethane (463 mL), and Hunig’s base (58.0 mL). The batch was cooled to -18°C and then charged with triphosgene in four portions (25.1 g total; 85 mmol) at <-8°C. The batch was agitated at -5 to 0°C for 0.5 h then charged with 11.4 wt% aqueous H3P04 at -5 to 0 °C (347 g, 3541 mmol). The batch was agitated at 20-25°C for 15-20 h then phase cut. The aqueous layer was back-extracted with dichloromethane (138 mL). The combined organic layer was washed with 10% NaHC03 (115 mL), then water (115 mL). The organic solution was concentrated at atmospheric pressure to ~80

mL, then charged with MTBE (347 mL) at 35-45 °C over 0.5 h, then concentrated at 35-45 °C to 231 mL two times to form a slurry.

The slurry was charged with heptanes (139 mL) at 35-45 °C over 2 h, then slowly cooled to 15-20°C over 1 h. The batch was agitated at 15-20°C for 6-8 h. Solids were filtered and the wet cake washed with MTBE/heptanes (1.4 : 1 , 185 mL) then dried under vacuum at 25-30°C for 5-10 hours to afford 5 (43.7 g, 92% yield), m.p. 161.3-161.8 °C. 1H NMR (CDC13, 500 MHz) δ: 7.45-7.32 (m, 5 H), 6.55 (d, J= 8.2 Hz, 1 H), 5.05 (d, J= 11.6 Hz, 1 H), 4.90 (d, J= 11.6 Hz, 1 H), 4.02 (m, 2 H), 3.90 (m, 2 H), 3.30 (m, 1 H), 2.99 (dt, J= 11.7, 1.1 Hz, 1 H), 2.86 (m, 2 H), 2.64 (d, J = 11.7 Hz, 1 H), 2.37 (dd, J= 14.6, 6.9 Hz, 1 H), 2.04-1.82 (m, 4 H), 1.58 (m, 1 H), 1.45 (s, 9 H), 1.30 (m, 2 H); 13C NMR (CDC13, 125 MHz) δ: 168.3, 167.5, 154.7, 135.6, 129.2 (2 C), 128.8, 128.6 (2 C), 79.7, 78.3, 60.4, 57.8, 47.5, 46.8, 42.5 (br, 2 C), 32.0, 31.7, 28.4 (3 C), 20.8, 17.2.

Step E: Preparation of tert-butyl 4-((2S,5R)-6-hydroxy-7-oxo-l,6-diazabicyclo[3.2.1|octane- 2-carboxamido) iperidine- 1 -carboxylate

tert-butyl 4-((2S,5R)-6-hydroxy-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carboxamido)piperidine-l -carboxylate (9.2 g, 20.1 mmol) was charged to a glass bottle, and the solids were dissolved in THF (150 mL). The solution was then charged to a hydrogenation reactor along with Pd/Al203 (10 wt%, 1.5 g). The reaction was purged three times with hydrogen and then set to a hydrogen pressure of 50 psi. The reaction temperature was adjusted to 25°C and the reaction was allowed to agitate for 22 hours. After the reaction was complete as determined by HPLC analysis, the solution was filtered through SOLKA-FLOC® (Interational Fiber Corporation, North Tonawanda, NY) to remove the catalyst and the filter cake was washed with THF. The filtrate and washes were then solvent switched by vacuum distillation to iPrOAc to a final volume of 40 mL. The resulting iPrOAc slurry was aged at room temperature for 1 hour. The solids were then filtered and washed with iPrOAc (20 mL) and dried under vacuum and N2 at 40°C to afford the title product (6.62 g., 17.97 mmol, 90% isolated yield). Spectral data matched the reference compound.

Preparation of (2S,5R)-7-oxo-N-piperidin-4-yl-6-(sulfooxy)- 1 ,6-diazabicyclo[3.2.1 ]octane-2-carboxamide

tert-butyl 4-((2S,5R)-6-hydroxy-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carboxamido)piperidine-l-carboxylate (20 g, 54.3 mmol), THF (200 mL), 2-picoline (10.9 mL, 309 mmol) and pyridine-S03 complex (30.2 g, 190 mmol) were charged to a flask under nitrogen. The heterogeneous mixture was allowed to stir overnight (~15 h). The reaction mixture was cooled to -10°C then DCM (200 mL) was added. 0.5 M K2HP04 (168 mL, 84 mmol) was added over 10 minutes. Bu4NHS04 (19.4 g, 57 mmol) was then added over 10 minutes. The biphasic mixture was stirred for 30 minutes, phase cut and the water layer was back extracted with 40 ml of DCM. The combined DCM solution was washed with water (120 ml), phase cut and the organic solution was solvent-switched to MeCN (320 ml) by vacuum distillation with 3 bed volumes of MeCN (total 1.0 L) and used as is in the next step. The solution of Bu4N+ OSO3 salt 7 in MeCN solution was used with an assumed yield of 100% (37.5 g, 54.3 mmol). The reaction mixture was cooled in an ice bath, and TMSI (10.26 ml, 70.7 mmol) was added via addition funnel over 30 minutes between 0°C and 5°C. The resulting mixture was agitated for 1-2 h and then quenched with H20:MeCN (1 :1, 6 ml) to afford a slurry. The slurry was warmed to room temperature and agitated for 12 h and after this time the pH of the supernatant was about 3.0. Tetrabutylammonium acetate (13.6 ml, 13.59 mmol) was slowly added over 30 min. The slurry was agitated for 1 h and pH of the supernatant was about 4.0. Solids were collected by filtration. The solid was washed with 60 mL of aqueous MeCN to afford 19.5 g of the crude product 8 in a 93% isolated yield from compound 6 .

At this stage, all byproducts (including hydro lyzation products of TMS-carbonate) and impurities were soluble in the organic phase.

The product was dissolved back into 140 ml of MeCN:H20 (1 :2) at room temperature. 1-Butanol (390 ml) as antisolvent was slowly added into the solution to afford a slurry. The slurry was agitated overnight. The white crystalline solid was filtered and washed with 3:1 IPA: water (40 ml) and dried under vacuum and nitrogen at room temperature to afford the title product in the form of a crystalline hydrate. (Yield = 16.3 g, 82%). Spectral data matched reference compound.

Preparation of (2S,5R)-7-oxo-2-(piperidin- 1 -ium-4-ylcarbamoyl)- 1 ,6-diazabicyclo[3.2.1 ]octan-6-yl sulfate (1).

tert-Butyl 4-( {[(25*,5i?)-6-hydroxy-7-oxo- 1 ,6-diazabicyclo[3.2.1 ]oct-2-yl]carbonyl}amino)piperidine-l-carboxylate 16 (0.54 g, 1.5 mmol), THF (5.4 mL), 2-picoline (0.29 mL, 2.9 mmol) and pyridine-S03 complex (0.70 g, 4.4 mmol) were charged to a vial under nitrogen. The heterogeneous mixture was allowed to stir overnight (~15 hr). The reaction mixture was cooled to -10°C then dichloromethane (5.4 mL) was added. 0.5 M K2HPO4 (4.5 mL, 2.3 mmol) was added over 10 minutes. BU4NHSO4 (0.53 g, 1.54 mmol) was then added over 10 min. The biphasic mixture was stirred for 30 min, phase cut and the water layer was back extracted with 1 ml of DCM. The combined DCM solution was washed with water (2.0 mL), phase cut and the organic solution was solvent-switched to MeCN (3.2 mL) by vacuum distillation with 3 bed volumes of MeCN. The product was used as is in the next step (water content less than 1000 ppm).

The solution of Bu4N+S04~~ salt 8 in MeCN solution was used with an assumed yield of 100% (1.0 g, 1.47 mmol). The reaction mixture was cooled in an ice bath, and Ν,Ο-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (0.4 lg, 1.59 mmol) was added into the reaction and was allowed to stir for 10 min. TMSI (0.06g, 0.27 mmol) was added between 0°C and 5°C. The resulting mixture was allowed to agitate for 2 hr and then quenched with H2O (0.07g, 4.1 mmol) and acetic acid (0.08g, 1.5 mmol) to afford a slurry. The slurry was warmed to room temperature and agitated for 12 hr. Filter to collect the solid. The solid was washed with MeCN/water (94:6, 1 mL X 4) to afford the crystalline product 1 (0.38 g) in a 75% yield.

If NO-bis(trimethylsilyl)acetamide (BSA) (0.32g, 1.59 mmol) was applied, the reaction needed 24 hr to achieve full conversion.

Patent

WO2015033191

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015033191&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Scheme 1.

Formula (V)

Formula (VI)

Formula (I)

Scheme – 1

Example -1

Preparation of (2S, 5R)-Sulfuric acid mono-{2-[N’-(4-aminopiperidinyl)-carbonyl]-7-oxo- l,6-diaza-bicyclo[3.2.1]oct-6-yl} ester (I).

Step-1: Preparation of (2S, 5R)-tert-butyl { (6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate (IV):

To a 250 ml round bottom flask equipped with magnetic stirrer was charged a solution of (2S, 5R)-sodium 6-benzyloxy-7-oxo-l,6-diaza-bicyclo [3.2.1] octane-2-carboxylate (11.1 gm, 0.037 mol, prepared using a method disclosed in Indian Patent Application No 699/MUM/2013) in water (180 ml) followed by l-tert-butoxycarbonyl-4-amino-piperidine (7.8 gm, 0.039 mol), EDC hydrochloride (11 gm, 0.055 mol) and 1 -hydro ybenzotriazole (4.8 gm, 0.037 mol) at 30°C successively under stirring. The reaction mixture was stirred for 24 hours at 30°C to provide a suspension. The suspension was filtered under suction and washed with 45°C warm water (40 ml) to provide (2S, 5R)-tert-butyl { (6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate in 12.7 gm quantity in 74% yield after drying under vacuum.

Analysis

NMR: (CDC13,) = 7.36-7.44 (m, 5H), 6.56 (d,lH), 5.06 (d,lH), 4.91 (d, 1H), 4.03 (br s, 1H), 3.88-3.97 (m, 2H), 3.29 (s, 1H), 3.00 (d, 1H), 2.86 (t, 2H), 2.64 (d, 1H), 2.37 (dd, 1H), 1.85-2.01 (m, 4H), 1.54-1.62 (m, 2H), 1.45 (s, 9H), 1.25-1.36 (m, 2H).

MS (ES+) C24H34N405 = 459.5 (M+l).

Step-2: Preparation of (2S, 5R)-tert-butyl { (6-hydroxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate (V):

To a 100 ml single neck round bottom flask equipped with magnetic stirrer was charged a solution of (2S, 5R)-tert-butyl { (6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate (9 g, 19.5 mmol) in methanol (90 ml) followed by 10% palladium on carbon (2.7 g) at 35°C. The reaction mixture was stirred under 1 atm hydrogen pressure at 35°C for 2 hours. The catalyst was removed by filtering the reaction mixture under suction over a celite bed. The celite bed was washed with dichloromethane (50 ml). The combined filtrate was evaporated under vacuum below 35°C to provide (2S, 5R)-tert-butyl {(6-hydroxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate in 8.45 g quantity; it was used as such for the next reaction.

Analysis

NMR: (CDC13,) = 6.60 (d, 1H), 3.88-4.10 (m, 4H), 3.78 (s, 1H), 3.20 (d, 1H), 3.90 (t, 2H), 2.80 (d, 1H), 2.46 (dd, 1H), 2.1-2.2 (m, 1H), 2.85-2.20 (m, 4H), 1.70-1.80 (m, 1H), 2.47 (s, 9H), 1.30-1.41 (m, 3H).

MS (ES+) C17H28N405 = 369.4 (M+l).

Step-3: Preparation of Tetrabutyl ammonium salt of (2S, 5R)-tert-butyl {(6-sulfooxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate (VI):

To a 100 ml single neck round bottom flask equipped with magnetic stirrer was charged a solution of (2S, 5R)-tert-butyl {(6-hydroxy-7-oxo-l,6-diaza-bicyclo [3.2.1 ]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate (6.40 g, 7.6 mmol) in dichloromethane (90 ml), triethyl amine (9.3 ml), followed by pyridine – sulfur trioxide complex (5.4 g, 34.2 mmol) at 35°C under stirring. The reaction mixture was stirred for additional 4 hours at 35°C. The solvent was evaporated under vacuum below 40°C to provide a residue. The residue was stirred with 0.5N aqueous potassium dihydrogen phosphate solution (90 ml) for 1 hour. The resulting solution was extracted with dichloromethane (2 x 100 ml) to remove impurities. To the aqueous layer was added tetrabutyl ammonium hydrogen sulfate (6.9 g, 20.52 mmol) and the reaction mixture was stirred for 14 hours at 35°C. It was extracted with dichloromethane (3 x 30 ml). Combined organic layer was dried over sodium sulfate and evaporated under vacuum to provide tetrabutyl ammonium salt of (2S, 5R)-tert-butyl {(6-sulfooxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate in 8.0 g quantity in 62% yield.

Analysis

NMR: (CDC13,) – 6.64 (d, 1H), 4.36 (br s, 1H), 4.05(br s, 2H), 3.90-4.00 (m, 1H), 3.87 (d, 1H), 2.28-3.34 (m, 10H), 3.80-3.95 (m, 2H), 3.74 (d, 1H), 2.42 (dd, 1H), 2.15-2.24 (m, 1H), 1.82-1.97 (m, 4H), 1.61-1.74 (m, 14 H), 1.41-1.52 (m, 10 H), 1.02 (t, 12H).

MS (ES-) C17H27N408S. N(C4H9)4 = 447.4 (M-l) as a free sulfonic acid.

Step-4: Synthesis of (2S, 5R)- Sulfuric acid mono-{ [(4-aminopiperidin-4-yl) carbonyl]-7-oxo-l,6-diaza-bicyclo[3.2.1]-oct-6-yl} ester (I):

To a 100 ml round bottom flask equipped with magnetic stirrer was charged a solution of tetrabutyl ammonium salt of (2S, 5R)-tert-butyl {(6-sulfooxy-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-2-yl-carbonyl) amino} piperidine-l-carboxylate (6.0 g) in dichloromethane (15 ml). The solution was cooled to -10°C under stirring and to it was added trifluoro acetic acid (15 ml) drop wise. The reaction mixture was stirred at -10°C for 1 hour. Solvents were evaporated under vacuum below 30°C to its 1/3 volume to provide a thick residue. The thick residue was stirred twice with diethyl ether (60 ml each time) to provide a precipitation. The solid obtained was filtered at suction and suspended in acetone (90 ml). To the suspension was added 10% solution of sodium-2-ethyl-hexanoate in acetone to adjust pH between 4.5 to 5.5. The suspension was stirred for 10 minutes and filtered under suction. The wet cake was washed with acetone and dried under vacuum below 40°C to provide 3 gm crude compound. The crude compound was stirred with aqueous isopropanol (3ml water: 21 ml iospropanol) for overnight to purify further. The resulting suspension was filtered under suction and washed with aqueous isopropanol (1 ml water: 7 ml IPA mixture). Finally the cake was dried under vacuum below 40°C to provide the title compound as a off-white solid in 1.8 g quantity in 65% yield.

Analysis

H1NMR (DMSO-d6, D20 exchange) = 8.19 (d, exchanges with D20), 3.99 (s, 1H), 3.82-3.92 (m, 1H), 3.72 (d, 1H), 2.24 (br d, 3H), 2.90-3.04 (m, 5H), 1.96-2.06 (m, 1H), 1.80-1.94 (m, 3H), 1.58-1.72 (m, 4H).

MS (ES+) C12H20N4O6S = 349.2 (M+l) as a free sulfonic acid;

Purity by HPLC: 99.2%

Specific rotation: [a] D -45.25 °, (c 0.3%, water)

SEE BACTAM SERIES…………..http://apisynthesisint.blogspot.in/p/bactam-series.html

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C1CC(N2CC1N(C2=O)OS(=O)(=O)O)C(=O)NC3CCNCC3.O

FINAFLOXACIN IN PHASE II for the treatment of ear infections


FINAFLOXACIN

(S-cyano-1-cyclopropyl-ό-fluoro-T-^aS, 7aS)-hexahydropyrrolo [3,4- b]-1,4-oxazin-6(2H)-yl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid)

7-[(4aS,7aS)-3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazin-6-yl]-8-cyano-1-cyclopropyl-6-fluoro-4-oxoquinoline-3-carboxylic acid |

BAY-35-3377
BY-377

CAS Registry Number: 209342-40-5

HYD SALT

(-)-(4aS,7aS)-8-Cyano-1-cyclopropyl-6-fluoro-4-oxo-7-(perhydropyrrolo[3,4-b]-1,4-oxazin-6-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride

209342-41-6,

C20 H19 F N4 O4 . Cl H
 MW 434.849

Synonyms: Finafloxacin, UNII-D26OSN9Q4R,

MerLion Pharmaceuticals (Singapore)…POSTER…….http://www.merlionpharma.com/sites/default/files/file/PPS/F1-2036_Wohlert.pdf

H. pylori, Broad-Spectrum

Finafloxacin is a novel fluoroquinolone being developed by MerLion Pharmaceuticals. Under neutral pH conditions (pH 7.2–7.4), the compound has shown in vitro activity equivalent to that of ciprofloxacin. However, under slightly acidic pH5.8 the compound shows enhanced potency.

Other marketed fluoroquinolones, such as ciprofloxacin, levofloxacin and moxifloxacin, exhibit reduced activity at slightly acidic pH 5.0–6.5. This feature of finafloxacin makes the compound suitable for use in the treatment of infections in acidic foci of infections such as urinary tract infections

Finafloxacin hydrochloride, a novel highly potent antibiotic, is in phase III clinical trials at Alcon for the treatment of ear infections. MerLion Pharmaceuticals is evaluating the product in phase II clinical trials at for the treatment of Helicobacter pylori infection and for the treatment of lower uncomplicated urinary tract infections in females.

A quinolone, finafloxacin holds potential for the treatment of Helicobacter pylori infection and urinary tract infection. Unlike existing antibiotics, finafloxacin demonstrates a unique acid activated activity whereby it becomes increasingly active under acidic conditions.

In 2009, a codevelopment agreement was signed between Chaperone Technologies and MerLion Pharmaceuticals. In 2011, finafloxacin hydrochloride was licensed to Alcon by MerLion Pharmaceuticals in North America for the treatment of ear infections.

MerLion Pharmaceuticals has announced that the FDA has granted a Qualified Infectious Disease Product Designation and Fast Track Status for finafloxacin. The company is currently recruiting patients for the Phase II clinical trial of the compound for the treatment of complicated urinary tract infections (cUTI) and/or acute pyelonephritis compared to ciprofloxacin

Finafloxacin and derivatives thereof can be synthesized according to the methods described in U.S. Patent No. 6,133,260 to Matzke et al., the contents of which are herein incorporated by reference in their entirety. The compositions of the invention are particularly directed toward treating mammalian and human subjects having or at risk of having a microbial tissue infection. Microbial tissue infections that may be treated or prevented in accord with the method of the present invention are referred to in J. P. Sanford et al., “The Sanford Guide to Antimicrobial Therapy 2007” 37 Edition (Antimicrobial Therapy, Inc.). Particular microbial tissue infections that may be treatable by embodiments of the present invention include those infections caused by bacteria, protozoa, fungi, yeast, spores, and parasites.

 

SYNTHESIS

WO1998026779A1

http://www.google.sc/patents/WO1998026779A1   COPY PASTE ON BROWSER

 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5 ,8-di-azabicyclo [4.3.0] non-8-yl)-l, 4-dihydro-4-oxo-3-quinolinecarboxylic acid.

The compounds, which are suitable for use in the invention are known already to some extent in EP-A-0350733, EP-A-0550903 as well as from DE-A-4329600 or can be prepared according to the processes described in .

If, for example 9,10-difluoro-3 ,8-dimethyl-7-oxo-2 ,3-dihydro-7H-pyrido [l ,2,3-d, e] [l, 3,4] benzoxadiazine-6 -carboxylic acid and 2-oxa-5 ,8-diazabicyclo [4.3.0] nonane, the reaction can be represented by the following equation:

Figure imgf000012_0001

The 7-halo-quinolonecarboxylic acid derivatives used for preparing the compounds of Fomel (I) of the invention are known or can be prepared by known methods. Thus, the 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid, or of the 7-chloro-8-cyano-l-cyclopropyl-6-fluoro- l been ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester described in EP-A-0 276 700th The corresponding 7-fluoro derivatives can be, for example, via the following reaction sequence to build:

 

Figure imgf000012_0002

An alternative process for preparing the intermediate compound 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride as the starting material for the preparation of 7-chloro-

8-cyano-1-cyclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid is used (EP-A-0276700) and in the 3-cyano-2 ,4,5-trifluoro- benzoyl can be converted, is based on 5-fluoro-l ,3-xylene, 5-fluoro-l ,3-xylene, in the presence of a catalyst under ionic conditions in the nucleus disubstituted to 2,4-dichloro-5-fluoro-l ,3-dimethylbenzene, and this is subsequently chlorinated chlorinated under free radical conditions in the side chains of 2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloro-methylbenzene. This is the 2,4-dichloro-5-fluoro-3-dichloromethyl-benzoic acid to give 2,4-dichloro-5-fluoro-3-formyl-benzoic acid, and then hydrolyzed to 2,4-dichloro-5-fluoro-3 N-hydroxyiminomethyl acid implemented. By treatment with thionyl chloride, 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride is obtained, which can still be ,4,5-trifluoro-ben-zoylfluorid converted by a chlorine / fluorine exchange on-3-cyano-2 .

 

Figure imgf000013_0001

 

Figure imgf000013_0002

 

Figure imgf000013_0003

The amines used for the preparation of compounds of formula (I) according to the invention are known from EP-A-0550903, EP-A-0551653 as well as from DE-A-4 309 964th

An alternative to the synthesis of lS, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane-dihydro-drobromid or the free base 1 S, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0 ] nonane and the corresponding IR, 6R enantiomer provides the following path represents:

Starting material for this synthesis is the cis-l ,4-dihydroxy-2-butene, which is converted to the bis-mesylate with mesylation tosylamide for 1-tosylpyrrolidine. This is converted into the epoxide m-chloroperbenzoic. The ring opening of the epoxide by heating in isopropanol with ethanolamine to trans-3-hydroxy-4 – (2-hydroxy-ethylamino)-l-(toluene-4-sulfonyl)-pyrrolidine in 80% yield. Tetrahydrofuran is then in pyridine / reacted with tosyl chloride, with cooling to Tris-tosylate, which as a crude product in a mixture with some tetra-tosyl derivative with basichen reaction conditions to give the racemic trans-5 ,8-bis-tosyl-2-oxa-5, 6 – diazabicyclo [4.3.0] nonane is cylisiert. At this stage occurs with high selectivity of a chromatographic resolution kieselgelgebundenem poly (N-methacryloyl-L-leucine-d menthylamide) as the stationary phase. The desired enantiomer, (lS, 6S) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane, is of a purity of

> 99% ee. Cleavage of the p-tosyl protecting groups is carried out with HBr-acetic acid to the lS, 6S-2-Oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide, the one with a base such as sodium or potassium hydroxide or with the aid of ion exchanger can be converted into the free base. The analogous sequence may be used for the preparation of lR, 6R-2-Oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide.

 

Figure imgf000014_0001
Figure imgf000015_0001

HBr / AcOH

 

Figure imgf000015_0002

Synthesis of lS, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane

Examples of compounds of the invention are mentioned in addition to the compounds listed in the preparation examples, the compounds listed in Table 1 below, which can be used both in racemic form as well as enantiomerically pure or diastereomerically pure compounds. Table 1:

 

Figure imgf000016_0001

 

Figure imgf000016_0002

Example 1 Z

8-cyano-1-cyclopropyl-6 ,7-difluoro-1 ,4-dihydro-4-oxo-3-quinoline-carboxylic acid ethyl ester

 

Figure imgf000020_0001

a 3-bromo-2 ,4,5-trifluoro-benzoate

To a mixture of 1460 ml of methanol and 340 g of triethylamine, 772 g of 3-bromo-2 ,4,5-trifluoro-benzoyl fluoride was added dropwise under ice cooling. There is one

Stirred for an hour at room temperature. The Reaktionsgemsich is concentrated, the residue dissolved in water and methylene chloride, and the aqueous phase was extracted with methylene chloride. After drying the organic phase over sodium sulfate, concentrated, and the residue was distilled in vacuum. This gives 752.4 g of 3-bromo-2 ,4,5-trifluoro-benzoic acid methyl ester of boiling point 122 ° C/20 mbar.

b. 3-Cyano-2 ,4,5-trifluoro-benzoic acid methyl ester:

269 ​​g of 3-bromo-2 ,4,5-trifluoro-benzoic acid methyl ester and 108 g of copper cyanide are heated to reflux in 400 ml of dimethylformamide for 5 hours. , All volatile components of the reaction mixture are then distilled off in vacuo. The distillate was then fractionated on a column. This gives 133 g of 3-cyano-2 ,4,5-trifluoro-benzoate of boiling point 88-89 ° C / 0.01 mbar.

c. 3-Cyano-2 ,4,5-trifluoro-benzoic acid

A solution of 156 g of 3-cyano-2 ,4,5-trifluoro-benzoate in 960 ml of glacial acetic acid, 140 ml of water and 69 ml concentrated sulfuric acid is heated for 8 hours under reflux. Then the acetic acid is distilled off under vacuum and the residue treated with water. Of failed-ne solid is filtered off, washed with water and dried. Obtained

118.6 g of 3-cyano-2 ,4,5-trifluoro-benzoic acid as a white solid, mp 187-190 ° C.

d 3-cyano-2 ,4,5-trifluoro-benzoyl chloride:

111 g of 3-cyano-2 ,4,5-trifluoro-benzoic acid and 84 g of oxalyl chloride are stirred in 930 ml of dry methylene chloride with the addition of a few drops of dimethylformamide for 5 hours at room temperature. The methylene chloride is evaporated and the residue distilled in vacuo. This gives 117.6 g of 3-cyano-2 ,4,5-trifluoro-benzoyl chloride as a yellow oil.

e 2 – (3-cyano-2 ,4,5-trifluoro-benzoyl)-3-dimethylamino-acrylic acid ethyl ester:

To a solution of 36.5 g of 3-dimethylamino-acrylate and 26.5 g of triethylamine in 140 ml toluene, a solution of 55 g 3-cyano-2, 4,5 – trifluoro-benzoyl chloride are added dropwise in 50 ml of toluene so that the temperature 50-55 ° C remains. Then stirred for 2 hours at 50 ° C.

The reaction mixture is concentrated in vacuo and used without further

Processing used in the next step. f 2 – (3-cyano-2 ,4,5-trifluoro-benzoyl)-3-cyclopropylamino-acrylic acid ethyl ester:

To the reaction product of step e 30 g of glacial acetic acid are added dropwise at 20 ° C. A solution of 15.75 g of cyclopropyl amine in 30 ml of toluene is added dropwise. The mixture is stirred at 30 ° C for 1 hour. Are then added 200 ml of water, stirred 15 minutes, the organic phase is separated off and shakes it again with 100 ml of water. The organic phase is dried over sodium sulfate and concentrated in vacuo. The crude product thus obtained is a set-without further purification in the next step.

g 8-cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester:

The reaction product from stage f and 27.6 g of potassium carbonate are stirred in 80 ml dimethylformamide for 16 hours at room temperature. The reaction mixture is then poured into 750 ml ice water, the solid filtered off with suction and washed with 80 ml cold methanol. After drying, 47 g of 8 – cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinoline carboxylic acid ethyl ester, mp 209-211 ° C.

Example 2 Z

2,4-dichloro-5-fluoro-l ,3-dimethylbenzene

 

Figure imgf000023_0001

a solvent-free

In 124 g of 3,5-dimethyl-fluorobenzene 1 g of anhydrous iron (III) chloride are pre-loaded and launched with the speed of chlorine (about 4 h), with which the reaction. This is initially slightly exothermic (temperature increase from 24 to 32 ° C) and is maintained by cooling below 30 ° C. After addition of 120 g of chlorine, the mixture is determined. According to GC analysis are 33.4% monochloro compound, formed 58.4% desired product and 5%> overchlorinated connections. The hydrogen chloride is removed and the reaction mixture is then distilled in a column in a water jet vacuum:

In the run 49 g of 2-chloro-5-fluoro-l ,3-dimethylbenzene obtained at 72-74 ° C/22 mbar. After 5 g of an intermediate fraction proceed at 105 ° C/22 mbar 75 g of 2,4 – dichloro-5-fluoro-l ,3-dimethylbenzene via, Melting range: 64 – 65 ° C.

b in 1,2-dichloroethane

1 kg of 3,5-dimethyl-fluorobenzene and 15 g of anhydrous iron (III) chloride are placed in 1 1 1 ,2-dichloroethane and chlorine is introduced in the same extent as the reaction proceeds (about 4 h). The reaction is initially exothermic (temperature rise from 24 to 32 ° C) and is kept below 30 ° C by cooling. After the introduction of 1200 g of chlorine are according to GC analysis 4% monochloro compound, 81.1% and 13.3% desired product overchlorinated connections emerged. After distilling off the solvent and the hydrogen chloride is distilled in a column in a water jet vacuum:

In the run 40 g of 2-chloro-5-fluoro-l ,3-dimethylbenzene receive. After some intermediate run going at 127-128 ° C/50 mbar 1115 g of 2,4-dichloro-5-fTuor-l ,3-dimethyl-ethylbenzene over.

Example 3 Z

2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloromethylbenzene

 

Figure imgf000024_0001

In a photochlorination using chlorine inlet and outlet for the hydrogen chloride to a scrubber and a light source in the vicinity of the chlorine inlet tube, 1890 g of 2,4-dichloro-5-fluoro-l ,3-dimethylbenzene pre-loaded and at 140 to 150 ° C. Chlorine metered. Within 30 hours 3850 g of chlorine are introduced. The content of the desired product according to GC analysis is 71.1% and the proportion of connections minderchlorierten 27.7%. The DestiUaton a 60 cm column with Wilson spirals provides a flow of 1142 g, which can be reused in the chlorination. The main fraction at 160-168 ° C / 0.2 mbar gives 2200 g of 2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloro-methyl benzene having a melting range of 74-76 ° C. After one recrystallization

Sample from methanol, the melting point 81-82 ° C.

Example Z 4

2,4-dichloro-5-fluoro-3-formyl-benzoic acid

 

Figure imgf000025_0001

In a 2500 ml stirred apparatus with gas discharge are presented 95% sulfuric acid at 70 ° C. and under stirring, 500 g of molten added dropwise 2,4-dichloro-5-fluoro-3-dichloromethyl-1 trichloromethylbenzene. It is after a short while hydrochloric development. Is metered during a 2 h and stirred until the evolution of gas after. After cooling to 20 ° C., the mixture is discharged ice to 4 kg and the precipitated solid is filtered off with suction. The product is after-washed with water and dried.

Yield: 310 g, melting range: 172-174 ° C

Example Z 5

2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl-benzoic acid

 

Figure imgf000026_0001

In a stirred reactor 80 g of hydroxylamine hydrochloride in 500 ml of ethanol are charged and added dropwise 200 ml of 45% strength sodium hydroxide solution and then with 40 – 200 g of 2,4-dichloro-5-fluoro-3-formyl-benzoic acid added 45.degree.The reaction is slightly exothermic and it is stirred for 5 h at 60 ° C. After cooling to

Room temperature is provided by the dropwise addition of hydrochloric acid to pH <3, the product taken up in tert-butyl methyl ether, the organic phase separated and the solvent distilled off. The residue obtained 185 g of 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl benzoic acid, melting range: 190 – 194 ° C.

Example No. 6

2,4-dichloro-3-cyano-5-benzoyl-fιuor

 

Figure imgf000026_0002

In a stirred vessel with metering and gas outlet via a reflux condenser to a scrubber 600 ml of thionyl chloride are introduced and registered at 20 ° C. 210 g of 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl benzoic acid in the proportion as hydrochloric developed and sulfur dioxide. After the addition the mixture is heated until the gas evolution under reflux. Mixture is then distilled, and boiling in the range of 142-145 ° C/10 mbar, 149 g of 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride (98.1% purity by GC) Melting range: 73-75 ° C.

Example No. 7

3-Cyano-2 ,4,5-trifluoro-benzoyl

 

Figure imgf000027_0001

50 g of potassium fluoride are suspended in 120 ml of tetramethylene sulfone and at 15 mbar for drying distilled (ca. 20 mL).Then, 50.4 g of 2,4 – dichloro-3-cyano-5-fluoro-benzoyl chloride was added and stirred at an internal temperature with exclusion of moisture for 12 hours at 180 ° C. Are removed by vacuum distillation to 32.9 g of 3-cyano-2 ,4,5-trifluoro-benzoyl fluoride in the boiling range of 98 –

Obtain 100 ° C/12 mbar.

Example No. 8

3-Cyano-2 ,4,5-trifluoro-benzoyl chloride

 

Figure imgf000027_0002

76.6 g of 3-cyano-2 ,4,5-trifluoro-benzoyl fluoride together with 1 g of anhydrous

Aluminum chloride introduced at 60-65 ° C and then added dropwise 25 g of silicon tetrachloride gas in the course of development. After the evolution of gas at 65 ° C is distilled in a vacuum. Boiling range 120-122 ° C/14 mbar, 73.2 g of 3 – cyano-2 ,4,5-trifluoro-benzoyl chloride over.

Example No. 9

1 – (toluene-4-sulfonyl-pyrroline

 

Figure imgf000028_0001

In a 20 1 HC4-HWS boilers are 2.016 kg (17.6 mol)

Submitted methanesulfonyl chloride in dichloromethane and 12 1 at -10 ° C internal temperature under strong cooling (-34 ° C) solution of 705 g (8.0 mol) of 2-butene-l ,4-diol in 1.944 kg (2.68 1 , 19.2 mol) of triethylamine was added dropwise over 30 minutes. A yellow suspension stirred for 1 hour at -10 ° C and then treated with 4 1 of water, the temperature rises to 0 ° C.The suspension is warmed to room temperature, stirred for 10 minutes at room temperature and then fed in a 30 1 separating funnel. The phases are stirred separately (good phase separation) and the aqueous phase extracted with 2 1 of dichloromethane. The combined dichloromethane phases are presented in a pre-cooled 20 1 HC4 vessel and kept at 0 ° C.

In another 20-1 HC4 boiler distillation 1.37 kg (8.0 mol) toluenesulfonamide be submitted in 6 1 toluene. It is mixed with 3.2 kg of 45% sodium hydroxide solution, 0.8 1 of water and 130.5 g Tetrabutylammomiimhydrogensulfat, heated to 40 ° C maximum temperature inside and creates a vacuum. Then, the previously obtained

Dichloromethane (15.2 1) was added dropwise over 1.5 hours while the dichloromethane was removed by distillation at 450 mbar (bath temperature: 60 ° C). During the distillation is foaming. In the end, a solution is available at an internal temperature of 33-40 ° C. After the addition of dichloromethane is distilled off, until barely distillate is (duration: about 85 minutes; internal temperature 40 ° C at 60 ° C bath temperature at the end). The vessel contents will be warm transferred to a separating funnel and rinsed the tank with water and 5 1 2 1 toluene at 50 ° C. Before phase separation, the solids are extracted in the intermediate phase and washed with 0.5 1 of toluene. The organic phase is extracted with 2.4 1 of water, separated and evaporated to dryness on a rotary evaporator. The solid residue (1758 g) is suspended in 50 ° C bath temperature in 1.6 1 of methanol, the suspension is transferred into a 10 1-flanged flask and the flask rinsed with diisopropyl 2,4 1. The mixture is heated to reflux temperature (59 ° C) and stirred for 30 minutes under reflux. The suspension is cooled to 0 ° C., stirred at 0 ° C for 1 hour and extracted with 0.8 1 of a cold mixture of ether Methanol/Diisopropyl-: washed (1 1.5). The crystals are dried under a nitrogen atmosphere at 50 ° C/400 mbar.

Yield: 1456 g (81.5% of theory)

Example Z 10

3 – (toluene-4-sulfonylV6-oxa-3-aza-bicvclo [3.1.0] hexane

o “|” h “CH3

334.5 g (1.5 mol) of l-(toluene-4-sulphonyl)-pyrroline are dissolved in 1.5 1 of dichloromethane at room temperature and over 15 minutes with a suspension of 408 g (approx. 1.65 to 1, 77 mol) of 70-75% m-chloroperbenzoic acid in 900 ml of dichloromethane (cools added in manufacturing from). The mixture is heated under reflux for 16 hr (test for

Peroxide with KI / starch paper shows yet to peroxide), the suspension was cooled to 5 ° C, sucks the precipitated m-chlorobenzoic acid and washed with 300 ml of dichloromethane (peroxide with Precipitation: negative; precipitate was discarded). The filtrate is to destroy excess peroxide with 300 ml of 10% sodium sulfite solution, washed twice (test for peroxide runs now negative), extracted with 300 ml of saturated sodium bicarbonate solution, washed with water, dried with sodium sulfate and about a quarter of the volume evaporated. Again on test peroxide: negative. The mixture is concentrated and the solid residue is stirred with ice cooling, 400 ml of isopropanol, the precipitate filtered off and dried at 70 ° C in vacuum.

Yield: 295 g (82.3%),

Mp: 136-139 ° C,

TLC (dichloromethane methanol 98:2): 1 HK (Jodkammer)

Example CLOSED

trans-3-Hydroxy-4-(2-hydroxy-ethylamino-l-(‘toluene-4-sulfonyl’) pyrrolidine

 

Figure imgf000030_0001

643.7 g (2.65 mol) 3 – (Toluoι-4-sulfonyl)-6-oxa-3-aza-bicyclo [3.1.0] hexane to 318.5 ml with ethanolamine in 4 1 of isopropanol at reflux for 16 hours cooked. After TLC monitoring, further 35.1 ml (total 5.86 mol) of ethanolamine added to the mixture and boiled again until the next morning. The mixture is filtered hot with suction and the filtrate concentrated on a rotary evaporator to 3.5 ltr. After seeding and stirring at room temperature for 3.5 1 diisopropyl ether are added, and stirred at 0 ° C for 6 hours. The precipitated crystals are filtered off, with 250 ml of a mixture of isopropanol / diisopropyl ether (1: 1) and washed 2 times with 300 ml of diisopropyl ether and dried overnight under high vacuum.

Yield: 663.7 g (83% of theory), content: 96.1% (area% by HPLC). Example Z 12

trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l-(toluene-4-sulfonyl)-pyrrolidin-3-yl] – ftoluol-4-sulfonyl)-amino]-ethyl ester)

 

Figure imgf000031_0001

552 g (1.837 mol) of trans-3-hydroxy-4-(2-hydroxy-ethylamino)-l-(toluene-4-sulfonyl) – pyrrolidine are dissolved under argon in 1.65 1 tetrahydrofuran and 0.8 1 of pyridine dissolved and at -10 ° C in portions 700 g (3.675 mol) p-toluenesulfonyl chloride are added thereto. The mixture is then stirred at this temperature for 16 hours. The work is done by adding 4.3 18.5 1% aqueous hydrochloric acid, extraction twice with dichloromethane (3 1, 2 1), washing the combined organic phases with saturated Natriurnhydrogencarbonatlösung (3 1, 2 1), drying over sodium sulfate, extracting and distilling off the solvent in vacuo. The residue is dried overnight at the oil pump and crude in the next reaction. There were 1093 g as a hard foam (content [area% by HPLC]: 80% Tris-tosyl-product and 13% tetra-tosyl-product, yield see next step). Example Z 13

rac. trans-5 ,8-bis-tosyl-2-oxa-5 .6-diazabicyclor4 .3.01 nonane

 

Figure imgf000032_0001

1092 g of crude trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l-(toluene-4-sulfonyl) – pyrrolidin-3-yl] – (toluene-4-sulfonyl)-amino]-ethyl} were dissolved in tetrahydrofuran and 9.4 1 at 0-3 ° C with 1.4 1 of a 1.43 molar solution of sodium hydroxide in

Methanol reacted. After half an hour at this temperature, 2.1 1 of water and 430 ml of diluted (2:1) was added to the mixture and acetic acid with previously isolated crystals of trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l – (toluene-4-sulfo-phenyl)-pyrrolidin-3-yl] – (toluene-4-sulfonyl)-amino] ethyl}-seeded. The suspension is stirred overnight at 0 to -4 ° C. The next morning, the crystals are filtered off, washed twice with 400 ml of cold mixture of tetrahydrofuran / water (4:1) and dried at 3 mbar at 50 ° C overnight.

Yield: 503 g of white crystals (62.7%> of theory over 2 steps), content: 99.7% (area% by HPLC). Example Z 14

Preparative chromatographic resolution of racemic rac. trans-5.8-bis-tosyl-2-oxa-5.6-diazabicyclor4.3.0] nonane

The chromatography of the racemate at room temperature in a column (inner diameter 75 mm), which with 870 g of a chiral stationary phase (kie-selgelgebundenes poly (N-methacryloyl-L-leucine-d menthylamide) based on the mer captomodifizierten silica Polygosil 100 , 10 microns; see EP-A 0 379 917) is filled (bed height: 38 cm). Detection is carried out using a UV detector at 254 nm

For the sample application using a solution of a concentration of 100 g of rac. trans-5 ,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane in 3000 ml of tetrahydrofuran. A Trenncyclus is carried out under the following conditions: with the aid of a pump is required for 2 min at a flow of 50 ml / min, a part of the sample solution and the same time at a flow rate of 50 ml / min, pure n-heptane to the column.

Thereafter eluted at a flow rate of 100 ml / min 18 minutes with a mixture of n-Heptan/Tetrahydrofuran (3/2 vol / vol). This is followed for 3 minutes at a flow of 100 ml / min elution with pure tetrahydrofuran. Thereafter, further eluted with n-Heptan/Tetrahydro-furan (3/2 vol / vol). This cycle is repeated several times.

The first eluted enantiomer is the (lS, 6R) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo-[4.3.0] nonane, which is isolated by concentration. The eluate of the more retarding enantiomers is largely evaporated in vacuo, and the precipitated crystals are filtered off with suction and dried. From the separation of 179 g of racemate in this

As 86.1 g (96.2% of theory) of the enantiomer (lS, 6S) -5,8-bis-tosyl-2-oxa-5, 6 – diazabicyclo [4.3.0] nonane having a purity of> 99 % ee. Example Z 15

(LR, 6R-2-oxa-5.6-diazabicvclo [4.3.0] nonane dihydrobromide

 

Figure imgf000034_0001

38.3 g (87 mmol) of (lS, 6R) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane in 500 ml of 33 -% HBr / glacial acetic acid 10 g added anisole and heated for 4 hours at 60 ° C (bath). After standing overnight, the suspension is cooled, the precipitate filtered, with

100 ml of abs. Ethanol and dried at 70 ° C under high vacuum.

Yield: 23.5 g (93%) of white solid product, mp 309-310 ° C (dec.), DC (dichloromethane/methanol/17% aq ammonia 30:8:1.): 1 HK

[Α] D: + 0.6 ° (c = 0.53, H 2 O) (fluctuating).

Example Z 16

(LS.6S-2-oxa-5.6-diazabicvclor4.3.01nonan-Dihvdrobromid

 

Figure imgf000034_0002

Z is analogous to Example 15 from (lS, 6S) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] no-nan (1S, 6S)-2-oxa-5, 6-diazabicyclo [4.3.0] nonane dihydrobromide receive. Example Z 17

(1 R.6R-2-oxa-5.8-diazabicvclo [4.3.Olnonan

 

Figure imgf000035_0001

1 Method: 5,8 g (20 mmol) of (lS, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydro-drobromid are suspended in 100 ml of isopropanol at room temperature with 2.4 g ( 42.9 mmol) and powdered potassium hydroxide while leaving about 1 hour in an ultrasonic bath. The suspension is cooled in an ice bath, filtered, washed with isopropanol and the undissolved salt, the filtrate was concentrated and distilled in a Kugelrohr oven at 150-230 ° C oven temperature and 0.7 mbar. Obtained 2.25 g (87.9% of theory) of a viscous oil which crystallizes. [Α] D -21.3 ° (c = 0.92, CHC1 3) Accordingly, this reaction can be carried out in ethanol.

2 Method: A homosexual genie catalyzed mixture of (lR, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide and 620 mg (11 mmol) of powdered potassium hydroxide is dry in a Kugelrohr apparatus at 0.2 mbar and increasing oven temperature to 250 ° C distilled. Obtained 490 mg (76.6% of theory) of (lR, 6R) -2 – oxa-5 ,8-diazabicyclo [4.3.0] nonane as a viscous oil which slowly crystallized.

3 Method: 100 g of moist, pretreated cation exchanger (Dowex 50WX, H + – form, 100-200 mesh, capacity: 5.1 meq / g of dry or 1.7 meq / mL) are charged into a column with about 200 ml 1 N HC1 activated and washed neutral with water 3 1. A solution of 2.9 g (10 mmol) of (lS, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane

Dihydrobromide in 15 ml of water is added to the ion exchanger, and then washed with 2 1 water, and eluted with approximately 1 1 1 N ammonia solution. The eluate is evaporated. concentrated. Yield: 1.3 g of a viscous oil (quantitative), DC (dichloromethane/methanol/17% NH 3 30:8:1): 1 HK, GC: 99.6% (area).

Example Z 18

(LS.6SV2-oxa-5.8-diazabicvclor4.3.01nonan

 

Figure imgf000036_0001

Z is analogous to Example 17 from (lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane-di-hydrobromide the free base (lS, 6S)-2-oxa-5 ,8-diazabicyclo [ 4.3.0] nonane made.

Example Z 19

2 – (2,4-dichloro-3-cyano-5-fluoro-benzoyl)-3-dimethylamino-acrylic acid ethyl ester

 

Figure imgf000036_0002

To a solution of 626 g (4.372 mol) of 3-dimethylamino-acrylate and 591 g (4.572 mol) of ethyl-diisopropyl-amine (Hunigs base) in 1060 ml of dichloromethane, a solution of 1075 g starting at room temperature 2,4-dichloro -3-cyano-5-fluoro-benzoyl chloride (94% pure, corresponding to 1010.5 g = 4.00 mol) was dropped in 850 ml of dichloromethane. The temperature rises to 50-55 ° C (dropwise addition about 90 minutes). Then stirred for 2 hours at 50 ° C and the reaction mixture was used without further purification in the next step.

Example Z 20

2 – (2,4-dichloro-3-Cyano-5-fluoro-benzoyl-3-cvclopropylamino-acrylate

 

Figure imgf000037_0001

To the reaction mixture from the above step 306 g (5.1 mol) of glacial acetic acid are added dropwise under cooling at about 15 ° C. Then, with further cooling at 10-15 ° C. 267.3 g (4.68 mol) of cyclopropyl amine is added dropwise. Immediately after which the reaction mixture is mixed with 1300 ml of water under ice-cooling and 15 minutes stirred well. The dichloromethane layer was separated and used in the next step.

Example 21 Z

7-chloro-8-cyano-1-cyclopropyl-6-fluoro-1.4-dihydro-4-oxo-3-chinolincarbonsäureethyl ester

 

Figure imgf000038_0001

To a heated to 60-70 ° C suspension of 353 g (2.554 mol) of potassium carbonate in 850 ml of N-methylpyrrolidone, the dichloromethane phase is dropped from the precursor (about 90 minutes). During the addition of the dichloromethane at the same time

Reaction mixture was distilled off. Then the reaction mixture for 5 Vz hours at 60-70 ° C is well stirred. The mixture is cooled to about 50 ° C. and distilled under a vacuum of about 250 mbar residual dichloromethane from. At room temperature is added dropwise 107 ml 30% hydrochloric acid under ice cooling, then to obtain a pH of 5-6 is set. Then, 2,200 ml of water are added under ice cooling. The reaction mixture is thoroughly stirred for 15 minutes, the solid was then filtered off and washed on the filter twice with 1000 ml of water and extracted three times with 1000 ml of ethanol and then dried in a vacuum oven at 60 ° C.

Yield: 1200 g (89.6% of theory).

This product can be purified, if desired by, the solid is stirred in 2000 ml of ethanol for 30 minutes at reflux. You filtered hot with suction, washed with 500 ml of ethanol and dried at 60 ° C in vacuum. Melting point: 180-182 ° C.

Η-NMR (400 MHz, CDC1 3): d = 1.2 to 1.27 (m, 2H), 1.41 (t, 3H), 1.5-1.56 (m, 2H), 4, 1 to 4.8 (m, 1H), 4.40 (q, 2H), 8.44 (d, J = 8.2 Hz, H), 8.64 (s, 1H) ppm.

Example Z 22

7-chloro-8-cyano-1-cvclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid

 

Figure imgf000039_0001

33.8 g (0.1 mol) of 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylate dissolved in a mixture of 100 ml of acetic acid, 20 ml water and 10 ml concentrated sulfuric acid was heated for 3 hours under reflux. After cooling, the mixture is poured onto 100 ml of ice water, the precipitate filtered off, washed with water and ethanol and dried at 60 ° C in vacuum.

Yield: 29.6 g (96% of theory),

Mp 216-21 C. (with decomposition)

Example 1

 

Figure imgf000040_0001

A 8-Cyano-l-cvclopropyl-6-fluoro-7-((lS.6S-2-oxa-5.8-diazabicvclo [4.3.0] non-8-yl – 1 ,4-dihydro-4-oxo-3 -quinoline carboxylic acid

1.00 g (3.26 mmol) of 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid are heated with 501 mg (3.91 mmol) of ( lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane and 0.9 ml of triethylamine in 30 ml of acetonitrile was stirred at 40-45 ° C under argon for 25 hours. All volatile components in vacuo. removed and the residue recrystallized from ethanol. Yield: 1.22 g (94%)

Melting point: 294 ° C. (with decomposition)

B) 8-Cyano-l-cyclopropyl-6-fluoro-7-(‘(lS.6S-2-oxa-5 ,8-diazabicvclo [4.3.01nonan-8-YLV 1.4-dihydro-4-oxo-3- quinoline carboxylic acid Hvdrochlorid

200 mg (0.63 mmol) of 8-cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester to be 97 mg (0.75 mmol) of (lS, 6S)-2-oxa-5, 8 – diazabicyclo [4.3.0] nonane and 0.17 ml of triethylamine in 3 ml of acetonitrile was stirred at 40-45 ° C for 2 hours under argon. All volatile components in vacuo. removed, the residue treated with water, insolubles filtered off and the filtrate was extracted with dichloromethane. The organic phase is dried over sodium sulfate and then concentrated under reduced pressure. a. The resulting residue is dissolved in 6 ml of tetrahydrofuran and 2 ml of water and 30 mg (0.72 mmol) of lithium hydroxide monohydrate was added. After 16 hours of stirring at room temperature, acidified with dilute hydrochloric acid and the resulting precipitate was filtered off with suction and dried. Yield: 155 mg (57%) Melting point:> 300 ° C

C) 8-Cyano-l-cvclopropyl-6-fluoro-7-((lS, 6S-2-oxa-5.8-diazabicvclo [4.3.01non-8 yiyi.4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride

1 g (2.5 mmol) of 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] non-8-yl )-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid is suspended in 20 ml of water was added to the suspension, 10 ml hydrochloric acid and stirred for In at room temperature for 3 hours. The resulting precipitate is filtered off, washed with ethanol and dried at 80 ° C under high vacuum.

Yield: 987 mg (90.6% of theory), Melting point: 314-316 ° C. (with decomposition).

D) 8-Cyano-l-cvclopropyl-6-fluoro-7-(iS, 6S)-2-oxa-5.8-diazabicyclo [4.3.0] non-8-YLV 1 ,4-dihydro-4-oxo-3 -quinoline carboxylic acid hydrochloride

86.4 g (217 mmol) of 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5, 8 – diazabicyclo [4.3.0] non-8-yl) – l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid are dissolved at room temperature in 963 ml of water and 239 ml of 1 N aqueous sodium hydroxide solution. After filtration and washing with 200 ml of water is added to 477 ml in aqueous hydrochloric acid and the precipitated crystals placed at 95 ° C to 100 ° C in solution. The solution is cooled overnight, the precipitated crystals are filtered off with suction and washed three times with 500 ml of water and dried in vacuum.

Yield 90 g (94.7% of theory), content:> 99% (area% by HPLC) 99.6% ee. [] D 23: -112 ° (c = 0.29, N NaOH).

 

……………….

Tetrahedron Lett 2009, 50(21): 2525

A novel approach to Finafloxacin hydrochloride (BAY35-3377)

Pages 2525-2528
Jian Hong, Zonghua Zhang, Huoxing Lei, Haiying Cheng, Yufang Hu, Wanliang Yang, Yinglin Liang, Debasis Das, Shu-Hui Chen, Ge Li

 

Graphical abstract

 

image

Finafloxacin hydrochloride, an important clinical compound was synthesized by a novel synthetic approach. An active intermediate ethyl 7-chloro-8-cyano-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate 19 was prepared by a new route. The chiral (S,S′)-N-Boc 10 was derived from protected pyrrolidine and the absolute stereochemistry was established by X-ray analysis.

http://www.sciencedirect.com/science/article/pii/S0040403909005875

……………….

 

 

 

  1. Durata Therapeutics, Inc. Finafloxacin for the treatment of cUTI and/or acute pyelonephritis. Available online: http://www.clinicaltrials.gov/ct2/show/NCT01928433 (accessed on 28 September 2013).
  2. Merlion Pharma. A multi-dose, double-blind, double-dummy, active control, randomized clinical (Phase II) study of two dosing regimens of finafloxacin for the treatment of cUTI and/or acute pyelonephritis.Available online: http://www.clinicaltrialsregister.eu/ctr-search/trial/2011–006041–14/PL/ (accessed on 14 April 2013).
  3. Pharma, M. FDA Grants Qualified Infectious Disease Product Designation and Fast Track Status for MerLion Pharma’s Lead Antibacterial Candidate Finafloxacin; Merlion Pharma: Singapore, 2013; Volume 2013.
  4. Lemaire, S.; van Bambeke, F.; Tulkens, P.M. Activity of finafloxacin, a novel fluoroquinolone with increased activity at acid pH, towards extracellular and intracellular Staphylococcus aureusListeria monocytogenes and Legionella pneumophilaInt. J. Antimicrob. Agents 201138, 52–59, doi:10.1016/j.ijantimicag.2011.03.002.
  5. Finafloxacin hydrochlorideDrugs Fut 2009, 34(6): 451
  6. A novel approach to finafloxacin hydrochloride (BAY35-3377)Tetrahedron Lett 2009, 50(21): 2525
  7. New fluoroquinolone finafloxacin HCI (FIN): Route of synthesis, physicochemical characteristics and activity under neutral and acid conditions48th Annu Intersci Conf Antimicrob Agents Chemother (ICAAC) Infect Dis Soc Am (IDSA) Annu Meet (October 25-28, Washington DC) 2008, Abst F1-2036

 

WO2011003091A1 * 2 Jul 2010 6 Jan 2011 Alcon Research, Ltd. Compositions comprising finafloxacin and methods for treating ophthalmic, otic, or nasal infections
US7723524 29 Sep 2004 25 May 2010 Daiichi Pharmaceutical Co., Ltd. 8-cyanoquinolonecarboxylic acid derivative
US8536167 2 Jul 2010 17 Sep 2013 Alcon Research, Ltd. Methods for treating ophthalmic, otic, or nasal infections
DE4329600A1 * 2 Sep 1993 9 Mar 1995 Bayer Ag Pyrido [1,2,3-d,e] [1,3,4] benzoxadiazinderivate
EP0276700A1 * 15 Jan 1988 3 Aug 1988 Bayer Ag 8-Cyano-1-cyclopropyl-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids, process for their preparation, and antibacterial agents containing them
EP0350733A2 * 30 Jun 1989 17 Jan 1990 Bayer Ag 7-(1-Pyrrolidinyl)-3-quinolone- and -naphthyridone-carboxylic-acid derivatives, method for their preparation and for substituted mono- and bi-cyclic pyrrolidine intermediates, and their antibacterial and feed additive compositions
EP0550903A1 * 28 Dec 1992 14 Jul 1993 Bayer Ag Quinolone- and naphthyridone carboxylic acid derivatives as antibacterial agents
EP0603887A2 * 23 Dec 1993 29 Jun 1994 Daiichi Pharmaceutical Co., Ltd. Bicyclic amine derivatives
EP0676199A1 * 23 Mar 1995 11 Oct 1995 Pfizer Inc. Use of trovafloxacin or derivatives thereof for the manufacture of a medicament for the treatment of H. pylori infections
GB2289674A * Title not available

BELINOSTAT, FAST TRACK, ORPHAN DRUG, A hydroxamate-type inhibitor of histone deacetylase.


File:Belinostat.svg

Belinostat (PXD101)

PHASE 2, FAST TRACK FDA , ORPHAN STATUS

  • PDX101
  • PX 105684
  • PXD-101
  • PXD101
  • UNII-F4H96P17NZ

Belinostat (PXD101) is a novel HDAC inhibitor with IC50 of 27 nM, with activity demonstrated in cisplatin-resistant tumors.

CLINICAL TRIALS…http://clinicaltrials.gov/search/intervention=Belinostat+OR+PXD101

Belinostat inhibits the growth of tumor cells (A2780, HCT116, HT29, WIL, CALU-3, MCF7, PC3 and HS852) with IC50 from 0.2-0.66 μM. PD101 shows low activity in A2780/cp70 and 2780AD cells. Belinostat inhibits bladder cancer cell growth, especially in 5637 cells, which shows accumulation of G0-G1 phase, decrease in S phase, and increase in G2-M phase. Belinostat also shows enhanced tubulin acetylation in ovarian cancer cell lines. A recent study shows that Belinostat activates protein kinase A in a TGF-β signaling-dependent mechanism and decreases survivin mRNA.

MW 318.07
MF C15H14N2O4S

414864-00-9  cas no

866323-14-0

(2E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]acrylamide

A novel HDAC inhibitor

…………………………

BELINOSTAT

Belinostat (PXD101) is experimental drug candidate under development byTopoTarget for the treatment of hematological malignancies and solid tumors. It is a histone deacetylase inhibitor.[1]

A hydroxamate-type inhibitor of histone deacetylase.

NCI: A novel hydroxamic acid-type histone deacetylase (HDAC) inhibitor with antineoplastic activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cellular differentiation, and inhibiting angiogenesis. This agent may sensitize drug-resistant tumor cells to other antineoplastic agents, possibly through a mechanism involving the down-regulation of thymidylate synthase

In 2007 preliminary results were released from the Phase II clinical trial of intravenous belinostat in combination with carboplatin and paclitaxel for relapsedovarian cancer.[2] Final results in late 2009 of a phase II trial for T cell lymphomawere encouraging.[3] Belinostat has been granted orphan drug and fast trackdesignation by the FDA.[4]

 

The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al., 1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida and Beppu, 1988), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999).

Trichostatin A (TSA)

Figure imgf000005_0001

Suberoylanilide Hydroxamic Acid (SAHA)

Figure imgf000005_0002

Cell cycle arrest by TSA correlates with an increased expression of gelsolin (Hoshikawa et al., 1994), an actin regulatory protein that is down regulated in malignant breast cancer (Mielnicki et al., 1999). Similar effects on cell cycle and differentiation have been observed with a number of deacetylase inhibitors (Kim et al., 1999). Trichostatin A has also been reported to be useful in the treatment of fibrosis, e.g., liver fibrosis and liver cirrhosis. See, e.g., Geerts et al., 1998.

Recently, certain compounds that induce differentiation have been reported to inhibit histone deacetylases. Several experimental antitumour compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been reported to act, at least in part, by inhibiting histone deacetylase (see, e.g., Yoshida et al., 1990; Richon et al., 1998; Kijima et al., 1993). Additionally, diallyl sulfide and related molecules (see, e.g., Lea et al., 1999), oxamflatin (see, e.g., Kim et al., 1999), MS-27-275, a synthetic benzamide derivative (see, e.g., Saito et al., 1999; Suzuki et al., 1999; note that MS-27-275 was later re-named as MS-275), butyrate derivatives (see, e.g., Lea and Tulsyan, 1995), FR901228 (see, e.g., Nokajima et al., 1998), depudecin (see, e.g., Kwon et al., 1998), and m-carboxycinnamic acid bishydroxamide (see, e.g., Richon et al., 1998) have been reported to inhibit histone deacetylases. In vitro, some of these compounds are reported to inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases, and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines (see, e.g., Richon et al, 1996; Kim et al., 1999; Yoshida et al., 1995; Yoshida & Beppu, 1988). In vivo, phenybutyrate is reported to be effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid (see, e.g., Warrell et al., 1998). SAHA is reported to be effective in preventing the formation of mammary tumours in rats, and lung tumours in mice (see, e.g., Desai et al., 1999).

The clear involvement of HDACs in the control of cell proliferation and differentiation suggest that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukaemias (APL). In most APL patients, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARσ (retinoic acid receptor). In some cases, a different translocation (t(11 ;17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation. The RARα fusion proteins occurring in APL patients are no longer responsive to physiological levels of RA and they interfere with the expression of the RA- inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL patients. (See, for example, Kitamura et al., 2000; David et al., 1998; Lin et al., 1998).

BELINOSTAT

Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, coloreetal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).

Psoriasis is a common chronic disfiguring skin disease which is characterised by well-demarcated, red, hardened scaly plaques: these may be limited or widespread. The prevalence rate of psoriasis is approximately 2%, i.e., 12.5 million sufferers in the triad countries (US/Europe/Japan). While the disease is rarely fatal, it clearly has serious detrimental effects upon the quality of life of the patient: this is further compounded by the lack of effective therapies. Present treatments are either ineffective, cosmetically unacceptable, or possess undesired side effects. There is therefore a large unmet clinical need for effective and safe drugs for this condition. Psoriasis is a disease of complex etiology. Whilst there is clearly a genetic component, with a number of gene loci being involved, there are also undefined environmental triggers. Whatever the ultimate cause of psoriasis, at the cellular level, it is characterised by local T-cell mediated inflammation, by keratinocyte hyperproliferation, and by localised angiogenesis. These are all processes in which histone deacetylases have been implicated (see, e.g., Saunders et al., 1999; Bernhard et al, 1999; Takahashi et al, 1996; Kim et al , 2001 ). Therefore HDAC inhibitors may be of use in therapy for psoriasis. Candidate drugs may be screened, for example, using proliferation assays with T-cells and/or keratinocytes.

 ………………………………………………………………………..

PXD101/Belinostat®

(E)-N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide, also known as PXD101 and Belinostat®, shown below, is a well known histone deacetylate (HDAC) inhibitor. It is being developed for treatment of a range of disorders mediated by HDAC, including proliferative conditions (such as cancer and psoriasis), malaria, etc.

Figure US20100286279A1-20101111-C00001

PXD101 was first described in WO 02/30879 A2. That document describes a multi-step method of synthesis which may conveniently be illustrated by the following scheme.

Figure US20100286279A1-20101111-C00002
Figure US20100286279A1-20101111-C00003

…………………………………..

GENERAL SYNTHESIS

WO2002030879A2

IGNORE 10

Figure imgf000060_0002

ENTRY 45 IS BELINOSTAT

Scheme 1

Figure imgf000101_0001

By using amines instead of aniline, the corresponding products may be obtained. The use of aniline, 4-methoxyaniline, 4-methylaniline, 4-bromoaniline, 4-chloroaniline, 4-benzylamine, and 4-phenethyamine, among others, is described in the Examples below.

In another method, a suitable amino acid (e.g., ω-amino acid) having a protected carboxylic acid (e.g., as an ester) and an unprotected amino group is reacted with a sulfonyl chloride compound (e.g., RSO2CI) to give the corresponding sulfonamide having a protected carboxylic acid. The protected carboxylic acid is then deprotected using base to give the free carboxylic acid, which is then reacted with, for example, hydroxylamine 2-chlorotrityl resin followed by acid (e.g., trifluoroacetic acid), to give the desired carbamic acid.

One example of this approach is illustrated below, in Scheme 2, wherein the reaction conditions are as follows: (i) RSO2CI, pyridine, DCM, room temperature, 12 hours; (ii) 1 M LiOH or 1 M NaOH, dioxane, room temperature, 3-48 hours; (iii) hydroxylamine 2-chlorotrityl resin, HOAt, HATU, DIPEA, DCM, room temperature, 16 hours; and (iv) TFA/DCM (5:95, v/v), room temperature, 1.5 hours.

Scheme 2

Figure imgf000102_0001

Additional methods for the synthesis of compounds of the present invention are illustrated below and are exemplified in the examples below.

Scheme 3A

Figure imgf000102_0002

Scheme 3B

Figure imgf000103_0001

Scheme 4

Figure imgf000104_0001
Figure imgf000105_0001

Scheme 8

Figure imgf000108_0002

Scheme 9

Figure imgf000109_0001

……………………………………………………………………..

SYNTHESIS

WO2002030879A2

Example 1

3-Formylbenzenesulfonic acid, sodium salt (1)

Figure imgf000123_0001

Oleum (5 ml) was placed in a reaction vessel and benzaldehyde (2.00 g, 18.84 mmol) was slowly added not exceeding the temperature of the reaction mixture more than 30°C. The obtained solution was stirred at 40°C for ten hours and at ambient temperature overnight. The reaction mixture was poured into ice and extracted with ethyl acetate. The aqueous phase was treated with CaC03 until the evolution of C02 ceased (pH~6-7), then the precipitated CaSO4was filtered off and washed with water. The filtrate was treated with Na2CO3 until the pH of the reaction medium increased to pH 8, obtained CaCO3 was filtered off and water solution was evaporated in vacuum. The residue was washed with methanol, the washings were evaporated and the residue was dried in desiccator over P2Oβ affording the title compound (2.00 g, 51%). 1H NMR (D20), δ: 7.56-8.40 (4H, m); 10.04 ppm (1 H, s).

Example 2 3-(3-Sulfophenyl)acrylic acid methyl ester, sodium salt (2)

Figure imgf000124_0001

Sodium salt of 3-formylbenzenesulfonic acid (1) (1.00 g, 4.80 mmol), potassium carbonate (1.32 g, 9.56 mmol), trimethyl phosphonoacetate (1.05 g, 5.77 mmol) and water (2 ml) were stirred at ambient temperature for 30 min., precipitated solid was filtered and washed with methanol. The filtrate was evaporated and the title compound (2) was obtained as a white solid (0.70 g, 55%). 1H NMR (DMSO- dβl HMDSO), δ: 3.68 (3H, s); 6.51 (1 H, d, J=16.0 Hz); 7.30-7.88 (5H, m).

Example 3 3-(3-Chlorosulfonylphenyl)acrylic acid methyl ester (3)

Figure imgf000124_0002

To the sodium salt of 3-(3-sulfophenyl)acrylic acid methyl ester (2) (0.670 g, 2.53 mmol) benzene (2 ml), thionyl chloride (1.508 g, 0.9 ml, 12.67 mmol) and 3 drops of dimethylformamide were added and the resultant suspension was stirred at reflux for one hour. The reaction mixture was evaporated, the residue was dissolved in benzene (3 ml), filtered and the filtrate was evaporated to give the title compound (0.6’40 g, 97%).

Example 4 3-(3-Phenylsulfamoylphenyl)acrylic acid methyl ester (4a)

Figure imgf000125_0001

A solution of 3-(3-chlorosulfonylphenyl)acrylic acid methyl ester (3) (0.640 g, 2.45 mmol) in dichloromethane (2 ml) was added to a mixture of aniline (0.465 g, 4.99 mmol) and pyridine (1 ml), and the resultant solution was stirred at 50°C for one hour. The reaction mixture was evaporated and the residue was partitioned between ethyl acetate and 10% HCI. The organic layer was washed successively with water, saturated NaCl, and dried (Na2S0 ). The solvent was removed and the residue was chromatographed on silica gel with chloroform-ethyl acetate (7:1 , v/v) as eluent. The obtained product was washed with diethyl ether to give the title compound (0.226 g, 29%). 1H NMR (CDCI3, HMDSO), δ: 3.72 (3H, s); 6.34 (1H, d, J=16.0 Hz); 6.68 (1 H, br s); 6.92-7.89 (10H, m).

Example 5 3-(3-Phenylsulfamoylphenyl)acrylic acid (5a)

Figure imgf000125_0002

3-(3-Phenylsulfamoylphenyl)acrylic acid methyl ester (4a) (0.220 g, 0.69 mmol) was dissolved in methanol (3 ml), 1N NaOH (2.08 ml, 2.08 mmol) was added and the resultant solution was stirred at ambient temperature overnight. The reaction mixture was partitioned between ethyl acetate and water. The aqueous layer was acidified with 10% HCI and stirred for 30 min. The precipitated solid was filtered, washed with water and dried in desiccator over P2Os to give the title compound as a white solid (0.173 g, 82%). Example 6 3-(3-Phenylsulfamoylphenyl)acryloyl chloride (6a)

Figure imgf000126_0001

To a suspension of 3-(3-phenylsulfamoylphenyl)acrylic acid (5a) (0.173 g, 0.57 mmol) in dichloromethane (2.3 ml) oxalyl chloride (0.17 ml, 1.95 mmol) and one drop of dimethylformamide were added. The reaction mixture was stirred at 40°C for one hour and concentrated under reduced pressure to give crude title compound (0.185 g).

Example 7

N-Hydroxy-3-(3-phenylsulfamoylphenyl)acrylamide (7a) (PX105684) BELINOSTAT

Figure imgf000126_0002

To a suspension of hydroxylamine hydrochloride (0.200 g, 2.87 mmol) in tetrahydrofuran (3.5 ml) a saturated NaHCOβ solution (2.5 ml) was added and the resultant mixture was stirred at ambient temperature for 10 min. To the reaction mixture a 3-(3-phenylsulfamoylphenyl)acryloyl chloride (6a) (0.185 g) solution in tetrahydrofuran (2.3 ml) was added and stirred at ambient temperature for one hour. The reaction mixture was partitioned between ethyl acetate and 2N HCI. The organic layer was washed successively with water and saturated NaCl, the solvent was removed and the residue was washed with acetonitrile and diethyl ether.

The title compound was obtained as a white solid (0.066 g, 36%), m.p. 172°C. BELINOSTAT

1H NMR (DMSO-d6, HMDSO), δ: 6.49 (1 H, d, J=16.0 Hz); 7.18-8.05 (10H, m); 9.16 (1 H, br s); 10.34 (1 H, s); 10.85 ppm (1 H, br s).

HPLC analysis on Symmetry C18column: impurities 4% (column size 3.9×150 mm; mobile phase acetonitrile – 0.1 M phosphate buffer (pH 2.5), 40:60; sample concentration 1 mg/ml; flow rate 0.8 ml/ min; detector UV 220 nm).

Anal. Calcd for C154N204S, %: C 56.59, H 4.43, N 8.80. Found, %: C 56.28, H 4.44, N 8.56.

……………………………………………………………………….

SYNTHESIS

US20100286279

Figure US20100286279A1-20101111-C00034

…………………………………………………….

SYNTHESIS AND SPECTRAL DATA

Journal of Medicinal Chemistry, 2011 ,  vol. 54,  13  pg. 4694 – 4720

(E)-N-Hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (28, belinostat, PXD101).

http://pubs.acs.org/doi/full/10.1021/jm2003552

 http://pubs.acs.org/doi/suppl/10.1021/jm2003552/suppl_file/jm2003552_si_001.pdf

The methyl ester (27) (8.0 g) was prepared according to reported synthetic route,

(Watkins, C. J.; Romero-Martin, M.-R.; Moore, K. G.; Ritchie, J.; Finn, P. W.; Kalvinsh, I.;
Loza, E.; Dikvoska, K.; Gailite, V.; Vorona, M.; Piskunova, I.; Starchenkov, I.; Harris, C. J.;
Duffy, J. E. S. Carbamic acid compounds comprising a sulfonamide linkage as HDAC
inhibitors. PCT Int. Appl. WO200230879A2, April 18, 2002.)
but using procedure D (Experimental Section) or method described for 26 to convert the methyl ester to crude
hydroxamic acid which was further purified by chromatography (silica, MeOH/DCM = 1:10) to
afford 28 (PXD101) as off-white or pale yellow powder (2.5 g, 31%).

LC–MS m/z 319.0 ([M +H]+).

1H NMR (DMSO-d6)  12–9 (very broad, 2H), 7.90 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.70 (d, J

= 7.8 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 15.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 2H), 7.08 (d,
J = 7.8 Hz, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.50 (d, J = 15.8 Hz, 1H);

13C NMR (DMSO-d6)  162.1,
140.6, 138.0, 136.5, 135.9, 131.8, 130.0, 129.2, 127.1, 124.8, 124.1, 121.3, 120.4.

Anal.
(C15H14N2O4S) C, H, N

………………………………………………..

SYNTHESIS

WO2009040517A2

PXDIOI / Belinostat®

(E)-N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide, also known as PXD101 and Belinostat®, shown below, is a well known histone deacetylate (HDAC) inhibitor. It is being developed for treatment of a range of disorders mediated by HDAC, including proliferative conditions (such as cancer and psoriasis), malaria, etc.

Figure imgf000003_0001

PXD101 was first described in WO 02/30879 A2. That document describes a multi-step method of synthesis which may conveniently be illustrated by the following scheme.

Scheme 1

Not isolated

Figure imgf000003_0002

ed on (A)

on (D)

Figure imgf000003_0003

d on (H)

Figure imgf000004_0001

There is a need for alternative methods for the synthesis of PXD101 and related compounds for example, methods which are simpler and/or employ fewer steps and/or permit higher yields and/or higher purity product.

Scheme 5

Figure imgf000052_0001

DMAP, toluene

Figure imgf000052_0003
Figure imgf000052_0002
Figure imgf000052_0004

Synthesis 1 3-Bromo-N-phenyl-benzenesulfonamide (3)

Figure imgf000052_0005

To a 30 gallon (-136 L) reactor was charged aniline (2) (4.01 kg; 93.13 g/mol; 43 mol), toluene (25 L), and 4-(dimethylamino)pyridine (DMAP) (12 g), and the mixture was heated to 50-600C. 3-Bromobenzenesulfonyl chloride (1) (5 kg; 255.52 g/mol; 19.6 mol) was charged into the reactor over 30 minutes at 50-600C and progress of the reaction was monitored by HPLC. After 19 hours, toluene (5 L) was added due to losses overnight through the vent line and the reaction was deemed to be complete with no compound (1) being detected by HPLC. The reaction mixture was diluted with toluene (10 L) and then quenched with 2 M aqueous hydrochloric acid (20 L). The organic and aqueous layers were separated, the aqueous layer was discarded, and the organic layer was washed with water (20 L), and then 5% (w/w) sodium bicarbonate solution (20 L), while maintaining the batch temperature at 45-55°C. The batch was then used in the next synthesis.

Synthesis 2 (E)-3-(3-Phenylsulfamoyl-phenyl)-acrylic acid ethyl ester (5)

Figure imgf000053_0001

To the batch containing 3-bromo-N-phenyl-benzenesulfonamide (3) (the treated organic layer obtained in the previous synthesis) was added triethylamine (2.97 kg; 101.19 g/mol; 29.4 mol), tri(o-tolyl)phosphine (119 g; 304.37 g/mol; 0.4 mol), and palladium (II) acetate (44 g; 224.51 g/mol; 0.2 mol), and the resulting mixture was degassed four times with a vacuum/nitrogen purge at 45-55°C. Catalytic palladium (0) was formed in situ. The batch was then heated to 80-900C and ethyl acrylate (4) (2.16 kg; 100.12 g/mol; 21.6 mol) was slowly added over 2.75 hours. The batch was sampled after a further 2 hours and was deemed to be complete with no compound (3) being detected by HPLC. The batch was cooled to 45-55°C and for convenience was left at this temperature overnight.

The batch was then reduced in volume under vacuum to 20-25 L, at a batch temperature of 45-55°C, and ethyl acetate (20 L) was added. The batch was filtered and the residue washed with ethyl acetate (3.5 L). The residue was discarded and the filtrates were sent to a 100 gallon (-454 L) reactor, which had been pre-heated to 600C. The 30 gallon (-136 L) reactor was then cleaned to remove any residual Pd, while the batch in the 100 gallon (-454 L) reactor was washed with 2 M aqueous hydrochloric acid and water at 45-55°C. Once the washes were complete and the 30 gallon (-136 L) reactor was clean, the batch was transferred from the 100 gallon (-454 L) reactor back to the 30 gallon (-136 L) reactor and the solvent was swapped under vacuum from ethyl acetate/toluene to toluene while maintaining a batch temperature of 45-55°C (the volume was reduced to 20-25 L). At this point, the batch had precipitated and heptanes (10 L) were added to re-dissolve it. The batch was then cooled to 0-100C and held at this temperature over the weekend in order to precipitate the product. The batch was filtered and the residue was washed with heptanes (5 L). A sample of the wet-cake was taken for Pd analysis. The Pd content of the crude product (5) was determined to be 12.9 ppm.

The wet-cake was then charged back into the 30 gallon (-136 L) reactor along with ethyl acetate (50 L) and heated to 40-500C in order to obtain a solution. A sparkler filter loaded with 12 impregnated Darco G60® carbon pads was then connected to the reactor and the solution was pumped around in a loop through the sparkler filter. After 1 hour, a sample was taken and evaporated to dryness and analysed for Pd content. The amount of Pd was found to be 1.4 ppm. A second sample was taken after 2 hours and evaporated to dryness and analysed for Pd content. The amount of Pd had been reduced to 0.6 ppm. The batch was blown back into the reactor and held at 40-500C overnight before the solvent was swapped under vacuum from ethyl acetate to toluene while maintaining a batch temperature of 45-55°C (the volume was reduced to 20-25 L). At this point, the batch had precipitated and heptanes (10 L) were added to re-dissolve it and the batch was cooled to 0-100C and held at this temperature overnight in order to precipitate the product. The batch was filtered and the residue was washed with heptanes (5 L). The filtrate was discarded and the residue was dried at 45-55°C under vacuum for 25 hours. A first lot of the title compound (5) was obtained as an off-white solid (4.48 kg, 69% overall yield from 3-bromobenzenesulfonyl chloride (1)) with a Pd content of 0.4 ppm and a purity of 99.22% (AUC) by HPLC.

Synthesis 3 (E)-3-(3-Phenylsulfamoyl-phenyl)-acrvlic acid (6)

Figure imgf000054_0001

To the 30 gallon (-136 L) reactor was charged the (E)-3-(3-phenylsulfamoyl-phenyl)- acrylic acid ethyl ester (5) (4.48 kg; 331.39 g/mol; 13.5 mol) along with 2 M aqueous sodium hydroxide (17.76 L; -35 mol). The mixture was heated to 40-50°C and held at this temperature for 2 hours before sampling, at which point the reaction was deemed to be complete with no compound (5) being detected by HPLC. The batch was adjusted to pH 2.2 using 1 M aqueous hydrochloric acid while maintaining the batch temperature between 40-500C. The product had precipitated and the batch was cooled to 20-300C and held at this temperature for 1 hour before filtering and washing the cake with water (8.9 L). The filtrate was discarded. The batch was allowed to condition on the filter overnight before being charged back into the reactor and slurried in water (44.4 L) at 40-500C for 2 hours. The batch was cooled to 15-20°C, held for 1 hour, and then filtered and the residue washed with water (8.9 L). The filtrate was discarded. The crude title compound (6) was transferred to an oven for drying at 45-55°C under vacuum with a slight nitrogen bleed for 5 days (this was done for convenience) to give a white solid (3.93 kg, 97% yield). The moisture content of the crude material was measured using Karl Fischer (KF) titration and found to be <0.1% (w/w). To the 30 gallon (-136 L) reactor was charged the crude compound (6) along with acetonitrile (47.2 L). The batch was heated to reflux (about 80°C) and held at reflux for 2 hours before cooling to 0-10°C and holding at this temperature overnight in order to precipitate the product. The batch was filtered and the residue was washed with cold acetonitrile (7.9 L). The filtrate was discarded and the residue was dried under vacuum at 45-55°C for 21.5 hours. The title compound (6) was obtained as a fluffy white solid (3.37 kg, 84% yield with respect to compound (5)) with a purity of 99.89% (AUC) by HPLC.

Synthesis 4 (E)-N-Hvdroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (PXD101) BELINOSTAT

Figure imgf000055_0001

To the 30 gallon (-136 L) reactor was charged (E)-3-(3-phenylsulfamoyl-phenyl)-acrylic acid (6) (3.37 kg; 303.34 g/mol; 11.1 mol) and a pre-mixed solution of 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in isopropyl acetate (IPAc) (27 g in 30 L; 152.24 g/mol; 0.18 mol). The slurry was stirred and thionyl chloride (SOCI2) (960 mL; density ~1.631 g/mL; 118.97 g/mol; -13 mol) was added to the reaction mixture and the batch was stirred at 20-300C overnight. After 18.5 hours, the batch was sampled and deemed to be complete with no compound (6) being detected by HPLC. The resulting solution was transferred to a 100 L Schott reactor for temporary storage while the

30 gallon (-136 L) reactor was rinsed with isopropyl acetate (IPAc) and water. Deionized water (28.9 L) was then added to the 30 gallon (-136 L) reactor followed by 50% (w/w) hydroxylamine (6.57 L; -1.078 g/mL; 33.03 g/mol; -214 mol) and another charge of deionized water (1.66 L) to rinse the lines free of hydroxylamine to make a 10% (w/w) hydroxylamine solution. Tetrahydrofuran (THF) (6.64 L) was then charged to the

30 gallon (-136 L) reactor and the mixture was stirred and cooled to 0-100C. The acid chloride solution (from the 100 L Schott reactor) was then slowly charged into the hydroxylamine solution over 1 hour maintaining a batch temperature of 0-10°C during the addition. The batch was then allowed to warm to 20-300C. The aqueous layer was separated and discarded. The organic layer was then reduced in volume under vacuum while maintaining a batch temperature of less than 300C. The intention was to distill out 10-13 L of solvent, but this level was overshot. A larger volume of isopropyl acetate (IPAc) (16.6 L) was added and about 6 L of solvent was distilled out. The batch had precipitated and heptanes (24.9 L) were added and the batch was held at 20-30°C overnight. The batch was filtered and the residue was washed with heptanes (6.64 L). The filtrate was discarded and the residue was dried at 45-55°C under vacuum with a slight nitrogen bleed over the weekend. The title compound (PXD101) was obtained as a light orange solid (3.11 kg, 89% yield with respect to compound (6)) with a purity of 99.25% (AUC) by HPLC.

The title compound (PXD101) (1.2 kg, 3.77 mol) was dissolved in 8 volumes of 1:1 (EtOH/water) at 600C. Sodium bicarbonate (15.8 g, 5 mol%) was added to the solution. Water (HPLC grade) was then added at a rate of 65 mL/min while keeping the internal temperature >57°C. After water (6.6 L) had been added, crystals started to form and the water addition was stopped. The reaction mixture was then cooled at a rate of 10°C/90 min to a temperature of 0-10cC and then stirred at ambient temperature overnight. The crystals were then filtered and collected. The filter cake was washed by slurrying in water (2 x 1.2 L) and then dried in an oven at 45°C for 60 hours with a slight nitrogen bleed. 1.048 kg (87% recovery) of a light orange solid was recovered. Microscopy and XRPD data showed a conglomerate of irregularly shaped birefringant crystalline particles. The compound was found to contain 0.02% water.

As discussed above: the yield of compound (5) with respect to compound (1) was 69%. the yield of compound (6) with respect to compound (5) was 84%. the yield of PXD101 with respect to compound (6) was 89%.

……………….

FORMULATION

WO2006120456A1

Formulation Studies

These studies demonstrate a substantial enhancement of HDACi solubility (on the order of a 500-fold increase for PXD-101) using one or more of: cyclodextrin, arginine, and meglumine. The resulting compositions are stable and can be diluted to the desired target concentration without the risk of precipitation. Furthermore, the compositions have a pH that, while higher than ideal, is acceptable for use.

Figure imgf000047_0001

UV Absorbance

The ultraviolet (UV absorbance E\ value for PXD-101 was determined by plotting a calibration curve of PXD-101 concentration in 50:50 methanol/water at the λmax for the material, 269 nm. Using this method, the E1i value was determined as 715.7.

Methanol/water was selected as the subsequent diluting medium for solubility studies rather than neat methanol (or other organic solvent) to reduce the risk of precipitation of the cyclodextrin.

Solubility in Demineralised Water

The solubility of PXD-101 was determined to be 0.14 mg/mL for demineralised water. Solubility Enhancement with Cvclodextrins

Saturated samples of PXD-101 were prepared in aqueous solutions of two natural cyclodextrins (α-CD and γ-CD) and hydroxypropyl derivatives of the α, β and Y cyclodextrins (HP-α-CD, HP-β-CD and HP-γ-CD). All experiments were completed with cyclodextrin concentrations of 250 mg/mL, except for α-CD, where the solubility of the cyclodextrin was not sufficient to achieve this concentration. The data are summarised in the following table. HP-β-CD offers the best solubility enhancement for PXD-101.

Figure imgf000048_0001

Phase Solubility Determination of HP-β-CD

The phase solubility diagram for HP-β-CD was prepared for concentrations of cyclodextrin between 50 and 500 mg/mL (5-50% w/v). The calculated saturated solubilities of the complexed HDACi were plotted against the concentration of cyclodextrin. See Figure 1.

………………………..

Links

  1.  Plumb, Jane A.; Finn, Paul W.; Williams, Robert J.; Bandara, Morwenna J.; Romero, M. Rosario; Watkins, Claire J.; La Thangue, Nicholas B.; Brown, Robert (2003). “Pharmacodynamic Response and Inhibition of Growth of Human Tumor Xenografts by the Novel Histone Deacetylase Inhibitor PXD101”. Molecular Cancer Therapeutics 2 (8): 721–728. PMID 12939461.
  2.  “CuraGen Corporation (CRGN) and TopoTarget A/S Announce Presentation of Belinostat Clinical Trial Results at AACR-NCI-EORTC International Conference”. October 2007.
  3. Final Results of a Phase II Trial of Belinostat (PXD101) in Patients with Recurrent or Refractory Peripheral or Cutaneous T-Cell Lymphoma, December 2009
  4.  “Spectrum adds to cancer pipeline with $350M deal.”. February 2010.
  5. Helvetica Chimica Acta, 2005 ,  vol. 88,  7  PG. 1630 – 1657, MP 172
  6. WO2009/40517 A2, ….
  7. WO2006/120456 A1, …..
  8. Synthetic Communications, 2010 ,  vol. 40,  17  PG. 2520 – 2524, MP 172
  9. Journal of Medicinal Chemistry, 2011 ,  vol. 54,   13  PG. 4694 – 4720, NMR IN SUP INFO
US2008274120 11-7-2008 Histone Deacetylase (Hdac) Inhibitors (Pxd101) for the Treatment of Cancer Alone or in Combination With Chemotherapeutic Agent
US2008227845 9-19-2008 CYCLOOXYGENASE-2 INHIBITOR/HISTONE DEACETYLASE INHIBITOR COMBINATION
US2008213399 9-5-2008 Combination Therapies Using Hdac Inhibitors
US2008194690 8-15-2008 Pharmaceutical Formulations Of Hdac Inhibitors
US7407988 8-6-2008 Carbamic acid compounds comprising a sulfonamide linkage as HDAC inhibitors
US7402603 7-23-2008 Cyclooxygenase-2 inhibitor/histone deacetylase inhibitor combination
US7183298 2-28-2007 Carbamic acid compounds comprising a sulfonamide linkage as HDAC inhibitors
US2005107445 5-20-2005 Carbamic acid compounds comprising a sulfonamide linkage as HDAC inhibitors
US6888027 5-4-2005 Carbamic acid compounds comprising a sulfonamide linkage as hdac inhibitors
WO2002030879A2 Sep 27, 2001 Apr 18, 2002 Prolifix Ltd Carbamic acid compounds comprising asulfonamide linkage as hdac inhibitors
US7973181 7-6-2011 HYDROXAMIC ACID DERIVATIVES AS INHIBITORS OF HDAC ENZYMATIC ACTIVITY
US7928081 4-20-2011 Combined Use of Prame Inhibitors and Hdac Inhibitors
US2011077305 3-32-2011 5-LIPOXYGENASE INHIBITORS
US2011003777 1-7-2011 Methods of Treatment Employing Prolonged Continuous Infusion of Belinostat
US2010286279 11-12-2010 Methods of Synthesis of Certain Hydroxamic Acid Compounds
US2010190694 7-30-2010 Methods for identifying patients who will respond well to cancer treatment
US2010010010 1-15-2010 HDAC INHIBITORS
US2009312311 12-18-2009 COMBINATION OF ORGANIC COMPOUNDS
US2009192211 7-31-2009 CYCLOOXYGENASE-2 INHIBITOR/HISTONE DEACETYLASE INHIBITOR COMBINATION
US7557140 7-8-2009 CARBAMIC ACID COMPOUNDS COMPRISING A SULFONAMIDE LINKAGE AS HDAC INHIBITORS
WO1998038859A1 * Mar 4, 1998 Sep 11, 1998 Thomas E Barta Sulfonyl divalent aryl or heteroaryl hydroxamic acid compounds
WO1999024399A1 * Nov 12, 1998 May 20, 1999 Darwin Discovery Ltd Hydroxamic and carboxylic acid derivatives having mmp and tnf inhibitory activity
WO2000056704A1 * Mar 22, 2000 Sep 28, 2000 Duncan Batty Hydroxamic and carboxylic acid derivatives
WO2000069819A1 * May 12, 2000 Nov 23, 2000 Thomas E Barta Hydroxamic acid derivatives as matrix metalloprotease inhibitors
WO2001038322A1 * Nov 22, 2000 May 31, 2001 Methylgene Inc Inhibitors of histone deacetylase
EP0570594A1 * Dec 7, 1992 Nov 24, 1993 SHIONOGI &amp; CO., LTD. Hydroxamic acid derivative based on aromatic sulfonamide
EP0931788A2 * Dec 16, 1998 Jul 28, 1999 Pfizer Inc. Metalloprotease inhibitors
GB2312674A * Title not available
WO2002030879A2 Sep 27, 2001 Apr 18, 2002 Prolifix Ltd Carbamic acid compounds comprising a sulfonamide linkage as hdac inhibitors
WO2005063806A1 Dec 30, 2003 Jul 14, 2005 Council Scient Ind Res Arginine hydrochloride enhances chaperone-like activity of alpha crystallin
US4642316 May 20, 1985 Feb 10, 1987 Warner-Lambert Company Parenteral phenytoin preparations
WO2008090585A2 * Jan 25, 2008 Jul 31, 2008 Univ Roma Soluble forms of inclusion complexes of histone deacetylase inhibitors and cyclodextrins, their preparation processes and uses in the pharmaceutical field
WO2009109861A1 * Mar 6, 2009 Sep 11, 2009 Topotarget A/S Methods of treatment employing prolonged continuous infusion of belinostat
WO2010048332A2 * Oct 21, 2009 Apr 29, 2010 Acucela, Inc. Compounds for treating ophthalmic diseases and disorders
WO2011064663A1 Nov 24, 2010 Jun 3, 2011 Festuccia, Claudio Combination treatment employing belinostat and bicalutamide
US20110003777 * Mar 6, 2009 Jan 6, 2011 Topotarget A/S Methods of Treatment Employing Prolonged Continuous Infusion of Belinostat

………………………..

SPECTRUM

Tiny Biotech With Three Cancer Drugs Is More Alluring Takeover Bet Now
Forbes
The drug is one of Spectrum’s two drugs undergoing phase 3 clinical trials. Allergan paid Spectrum $41.5 million and will make additional payments of up to $304 million based on achieving certain milestones. So far, Raj Shrotriya, Spectrum’s chairman, 

http://www.forbes.com/sites/genemarcial/2013/07/14/tiny-biotech-with-three-cancer-drugs-is-more-alluring-takeover-bet-now/

……………………………..

Copenhagen, December 10, 2013
Topotarget announces the submission of a New Drug Application (NDA) for belinostat for the treatment of relapsed or refractory (R/R) peripheral T-cell lymphoma (PTCL) to the US Food and Drug Administration (FDA). The NDA has been filed for Accelerated Approval with a request for Priority Review. Response from the FDA regarding acceptance to file is expected within 60 days from the FDA receipt date.
read all this here
…………………….
 SEE COMPILATION ON SIMILAR COMPOUNDS AT …………..http://drugsynthesisint.blogspot.in/p/nostat-series.html

Phase 3 FDA -Acorafloxacin (Avarofloxacin) Granted QIDP and Fast Track Designation


Avarofloxacin .HCl/Acorafloxacin. HCl
cas no  1001162-01-1 
3-Quinolinecarboxylic acid, 7-[(3E)-3-(2-amino-1-fluoroethylidene)-1-piperidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-, hydrochloride (1:1)
2. 7-[(3E)-3-(2-amino-1-fluoroethylidene)piperidin-1-yl]-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid monohydrochloride
PHASE 2 FURIEX,FOR  acute bacterial skin and skin structure (ABSSSI)Infection, 
February 25, 2013
Avarofloxacin Granted QIDP and Fast Track Designation

JNJ-Q2, JNJ-32729463-AAA

CAS NO   878592-87-1 of base

7-[3-[2-Amino-1(E)-fluoroethylidene]piperidin-1-yl]-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

 

Figure imgf000063_0002

Furiex Pharmaceuticals Inc. announced that the FDA has granted Qualified Infectious Disease Product (QIDP) and Fast Track designation for avarofloxacin (JNJ-Q2). Avarofloxacin is a Phase 3-ready broad-spectrum fluoroquinolone antibiotic for the treatment of acute bacterial skin and skin-structure (ABSSSI) infections, community-acquired pneumonia and has proven to be effective in treating methicillin-resistant Staphylococcus aureus (MRSA) infections.

Avarofloxacin is an investigational novel fluoroquinolone antibiotic that has been shown to be effective in a Phase 2 study of ABSSSI infections. In this study, avarofloxacin demonstrated favorable efficacy for both early clinical response endpoints as well as all clinical cure endpoints for the intent to treat population.

Avarofloxacin has a low tendency for development of drug resistance and exhibits a broad range of antibacterial activities in vitro, including MRSA, fluoroquinolone-resistant Staphylococcus aureus, Streptococcus pneumoniae (including multi-drug resistant strains), gram positive, gram negative, atypical respiratory pathogens (such as legionella and mycoplasma) and anaerobic bacteria, which are often associated with abscesses of the skin and other organs.

The availability of IV and oral formulations for avarofloxacin differentiates it from a number of other products for MRSA infections which are only available for intravenous administration.

For more information call (919) 456-7800or visit http://www.furiex.com/

About Methicillin-Resistant Staphylococcus aureus (MRSA)

MRSA is a strain of the bacteria Staphylococcus aureus (staph) which commonly causes skin and soft tissue infections and is resistant to many antibiotics. Although MRSA had previously been primarily a hospital-acquired pathogen, its incidence has been rising in the community, and it has become the most frequent cause of skin and soft tissue infections presenting to emergency departments in the United States. There are a limited number of antibiotics approved to treat MRSA, and their frequent usage has led to emergence of multi-drug resistant bacteria. Thus, we believe there is significant unmet medical need for new antibiotics such as avarofloxacin that provide flexible (hospital and outpatient) treatment options for MRSA.

WO-2006/101603 describes 7-amino alkylidenyl- heterocyclic quinolones as antimicrobial compounds and the synthesis of 7-[(3E)-3-(2-amino-l-fluoroethylidene)-l- piperidinyl]- 1 -cyclopropyl-6-fluoro- 1 ,4-dihydro-8-methoxy-4-oxo 3-quinolinecarboxylic acid is disclosed as compound (303) in Table 1 on page 20. This compound is conveniently referred to as compound ‘A’ hereafter.

compound ‘A’

Figure imgf000002_0001

7-[(3E)-3-(2-amino-1 -fluoroethylidene)-1 -piperidinyl]-1 -cyclopropyl-6-fluoro- 1 ,4-dihydro-8-methoxy-4-oxo 3-quinolinecarboxylic acid

The in vitro antibacterial properties of compound ‘A’ are described by Morrow B.J. et al. in Antimicrobial Agents and Chemotherapy, vol. 54, pp. 1995 – 1964 (2010).

WO-2008/005670 discloses one-pot methods for the production of substituted allylic alcohols as well as extractive methods for the separation of certain isomeric alcohol products which are useful for preparing quinolones such as the antimicrobial compound 7-[(3E)-3-(2-amino- 1 -fluoroethylidene)- 1 -piperidinyl]- 1 -cyclopropyl-6-fluoro- 1 ,4- dihydro-8-methoxy-4-oxo 3-quinolinecarboxylic acid (i.e. compound ‘Α’). An important intermediate in the overall synthesis route of said antimicrobial compound ‘A’ is 2-[(2E)-2-fluoro-2-(3-piperidinylidene)ethyl]-lH-isoindole-l,3(2H)- dione and its hydrochloric acid salt thereof : compound (1 )

Figure imgf000003_0001

2-[(2E)-2-fluoro-2-(3-piperidinylidene)ethyl]-1 H-isoindole-1 ,3(2H)-dione compound (1 ) .HCI

Figure imgf000003_0002

Compound (1) introduces the desired E- stereochemistry into the overall synthesis route for the antimicrobial compound ‘Α’.

WO-2008/005670 discloses a synthesis route for compound (1) on page 38 as depicted below :

Figure imgf000003_0003

– highly enriched (E)

Figure imgf000004_0001

(Step 3a) O

The detailed reaction procedure for compound (1) is disclosed in WO-2008/005670 in Example 1 on pages 37 to 44 affording compound (1) in Method A with a E:Z ratio of 97:3 in an approximate overall yield of 18 % in Method A (step 1 for the first 3 heptane layers has a yield of 34 % with a ratio E:Z of 71 :29, step 2a has a yield of 53.4% with a ratio E:Z of 97:3, and step 3 has quantitave yield), or affording compound (1) in Method B with an approximate overall yield of 15% with a ratio E:Z of 94.4 : 5.6. WO-2008/005670 discloses a synthesis route for the hydrochloric acid addition salt of compound (1) on page 15 in Scheme 2 as depicted below :

Figure imgf000004_0002

into n-butanol,

Figure imgf000005_0001

1 ) 5/6 N HCl in IPA

2) heat to distill

3) add IPA

enriched E-isomer

Figure imgf000005_0002

compound (1 ) .HCl

The detailed reaction procedure to prepare the HCl salt of compound (1) is disclosed in WO-2008/005670 in Example 4 on pages 49 to 52 affording >95% of desired E-isomer with an overall yield of 18 – 22% starting from N-boc-3-piperidone.

The reaction procedures described in WO-2008/005670 for the preparation of compound (1) or its HCl salt are characterized by lack of selectivity of the Wadsworth- Emmons-Horner reaction which produces the undesired Z-isomer in large quantities. This undesired Z-isomer requires additional time consuming separation steps.

Hence there is a need for a more efficient and less waste-producing procedure for the preparation of compound (1) or its HCl salt. WO-2010/056633 discloses a synthesis scheme XIV on page 87 to prepare tert-butyl 4- (2-ethoxy-2-oxoethylidene)piperidinyl-l-carboxylate and a synthesis scheme XXVI on page 111 to prepare (l-benzyl-piperidin-4-ylidene)bromoacetic acid ethyl ester.

In a first embodiment the present invention relates to an improved process for preparing compounds of formula (III) having an improved ratio of the desired (E)-isomer over the undesired (Z)-isomer.

Figure imgf000006_0001

(I)

In a further embodiment the compound (E)-(III) is then converted in to compound (1) or its hydrochloric acid addition salt thereof.

…………………………..

WO 2008005670

http://www.google.com/patents/WO2008005670A2?cl=en

Scheme 1

Figure imgf000013_0001

3 eq NaBH4 OH

30 – 4O0C

Figure imgf000013_0002

2a 2b 2a

Figure imgf000013_0003

2b shows the preparation of alcohol 2

 

 

Scheme 2

Figure imgf000016_0001

1 ) HCI (5 eq )

Figure imgf000016_0002

aqueous layer to enriched E isomer pH 9-10 then extract into n-Butanol, discard aqueous layer

 

Preparation of

7-[3-(2-Amino-l-fluoroethylidene)piperidin-l-yl]-l-cyclopropyl- 6-fluoro-8-methoxy-4-oxo-l,4-dihydroquinoline-3-carboxylic acid (10) and its HCl salt (12)

Figure imgf000039_0001

7-[3-(2-Amino-1-fluoro-ethyhdene)-piperidin-1-yl]-1-cyclopropyl–fluoro-8-mΘthoxy-4-oxo-1 ,4-dιhydro-quιnolιne-3-carboxylιc acid (10)

Step 1: Preparation of 3-(l-fluoro-2-hydroxyethylidene)piperidine-l-carboxylic acid tert-butyl ester (2a)

Figure imgf000040_0001

A 22-L 4-neck round bottom flask, equipped with a thermocouple controller, overhead mechanical stirrer, condenser, nitrogen inlet adapter, and stopper, was charged with N-Boc-3-piperidone (663.36 g, 3.34 mol), 2-methoxyethanol (6.0 L) and 2-fluorotriethylphosphonoacetate (843.54 g, 3.49 mol). The mixture was stirred to obtain a homogeneous solution and then CS2CO3 was added in portions over 1.5 h. After the CS2CO3 addition was complete, NaBH4 was added in portions over 6 h; during most of this addition the reaction temperature was maintained between 35 0C to 40 0C. After the addition was complete, the reaction was allowed to stir overnight after which time HPLC analysis indicated that the reaction was complete. This run was combined with two additional runs of equal size and transferred to a stirred 100-L Hastalloy® reactor containing water (90 L). The aqueous mixture was extracted with heptane (4 x 20 L) followed by extraction with MTBE (methyl tert-butyl ether) (20 L). The first three heptane extracts provided 842 g of the allylic alcohol as 71:29 (E: Z) mixture (HPLC and NMR). The product mixture from the first three heptane extractions was carried on to the next step without any additional purification. The fourth heptane extract gave 114 g of product that was a 67:33 mixture of is: Z alcohols (NMR). MTBE extraction and concentration gave 1.1 Kg of product as a 33:67 mixture of E:Z alcohols (HPLC). The total overall yield for both isomers was 2.06 Kg (83%). 1H NMR of 2a (400 MHz, CDCl3): £ 1.45 (s, 9 H), 1.52 (m, 2 H), 2.40 (m, 2 H), 3.45 (m, 2 H), 3.90 (s, 2 H), 4.25 (d, 2 H). 1H NMR of 2b (400 MHz, CDCl3): δ 1.46 (s, 9 H), 1.65 (m, 2 H), 2.27 (m, 2 H), 3.45 (m, 2 H), 4.1 (s, 2 H), 4.25 (d, 2 H).

Step 2, Method A: Preparation of 3-is-[2-(l,3-dioxo-l,3-dihydroisoindol-2-yl)-l- fluoroethylidene]-piperidine-l-carboxylic acid tert-butyl ester (3-ϋ)

Figure imgf000041_0001

A 22-L 4-neck round bottom flask, equipped with a thermocouple controller, overhead mechanical stirrer, condenser, pressure-equalizing addition funnel, nitrogen inlet adapter, and stopper, was charged with E:Z alcohol mixture 2a and 2b (377.5 g, 1.296 mol corrected), 2-MeTHF (3.31 L), phthalimide (232.8 g, 1.581 mol), and Ph3P (411.3 g, 1.568 mol). The white suspension was stirred under N2 and cooled to -12 0C in an acetone/Dry-Ice bath, DIAD (309 mL, 1.49 mol) was added via the addition funnel over a 36-min period, while the reaction temperature was maintained at -15 0C to -10 0C. After the addition, the reaction was warmed to 20 0C in a water bath and stirred for 2 h. The reaction was cooled to 0 0C in an ice/water bath and quenched with cold 1.0 M HCl (950 mL). The aqueous phase was separated and EtOAc (1.70 L) was added to the organic phase. This phase was washed with cold 1.0 M HCl (0.95 L) (the aqueous phase was pH < 2) and then separated. The organic phase was next washed with cold 4 NNaOH (1.70 L), the alkaline aqueous phase (pH > 13) was separated and the EtOAc layer washed with brine (1.70 L). Concentration of the organic phase at 60 0C under house vacuum (-120 mm Hg) afforded 1,442.0 g of crude 3. This run was repeated on the same scale to provide an additional 1,431.0 g of crude material for a combined yield of 2,873 g (159%). HPLC analysis (area%) indicated crude 3 was a mixture of 3-E (29.4%), 3-Z (10.4 %), Ph3PO (51.0 %), and phthalimide (1.1 %). This was purified by recrystallization as described in step 2a.

Step 2a, Method A: Purification of 3-is-[2-(l,3-dioxo-l,3-dihydroisoindol-2-yl)-l- fluoroethylidene]-piperidine-l-carboxylic acid tert-butyl ester

Figure imgf000042_0001

A 22-L 4-neck round bottom flask equipped with a thermocouple controller, overhead mechanical stirrer, condenser, pressure-equalizing addition funnel, nitrogen inlet adapter and stopper was charged with the combined crude 3 (2,873 g) and MeOH (9.0 L). The solution was stirred under nitrogen and heated to 65 0C, while hot (60 0C) D.I. water (7.8 L) was added over a 15-min period. The solution was stirred at 65 0C for 5 min, and then the heating mantle was replaced with a water bath, and the mixture was gradually cooled to 0 0C over a 4-h period, and continued stirring for 1 h at 0 0C. The off-white solid was collected by filtration, and dried by air-suction at 60 0C for 20 h, this provided 1,172.6 g of a mixture of 3-E and 3-Z.

The partially purified product above was recrystallized a second time in the same manner using hot MeOH (7.2 L) and hot water (5.0 L) except that the water was added over a 10-min period to afford 515.6 g (53.4%) of 3-E as a 97:3 mixture of E:Z geometric isomers. This material was used in the next step without additional purification. . 1H NMR of 3-E (400 MHz, CDCl3): δ 1.48 (s, 9 H), 1.52-1.66 (m, 2 H), 2.28-2.38 (m, 2 H), 3.40-3.51 (m, 2 H), 4.18 (s, 2 H), 4.55 (d, J= 21.0 Hz, 2 H), 7.68- 7.77 (m, 2 H), 7.80-7.89 (m, 2 H). MS: 397 (M+Na)+, 771 (2M+Na)+.

3 -E-[2-( 1 ,3 -dioxo- 1 ,3-dihydroisoindol-2-yl)- 1 -fluoroethylidene]-piperidine- 1 – carboxylic acid tert-butyl ester was also prepared with Method B below: Step 2, Method B: Preparation of 3-£-[2-(l,3-dioxo-l,3-dihydroisoindol-2-yl)-l- fluoroethylidene]-piperidine-l-carboxylic acid tert-butyl ester (3-E)

Preparation of the methanesulfonate and chloride derivatives

Figure imgf000043_0001

2a

A 12-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, pressure-equalizing addition funnel, and a nitrogen inlet adapter was charged with 2a (297.0 g, 1.21 mol) and CH2Cl2 (3.9 L). The solution was cooled to 0 0C under N2 and EtsN (320 mL, 2.30 mol) was added via the addition funnel over a 10- min period. This was followed by methanesulfonyl chloride (115 mL, 1.49 mol) added over a 60-min period then the reaction was stirred for an additional 60-min at 0 0C. The mixture was poured into a mixture of deionized water (4.4 L) and saturated NaHCθ3 (0.78 L), the layers were separated, the aqueous layer was extracted with CH2Cl2 (2 x 2 L). All the CH2Cl2 layers were combined and washed with saturated NaHCθ3 (2 L). The CH2Cl2 was removed under vacuum at 40 0C to afford a mixture of the mesylate and chloride (342.3 g). This mixture was taken on to the next step without any purification.

Conversion of the methanesulfonate/chloride to phthalimide 3

Figure imgf000043_0002

A 5-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, pressure-equalizing addition funnel, and a nitrogen inlet adapter was charged with the mixture of the mesylate and chloride from above (342.2 g, 1.21 mol) and DMF (2.0 L) followed by potassium phthalimide (224.9 g, 1.21 mol). The mixture was stirred at 60 0C for 1-h then at 20 0C for 18 h. The mixture was poured into ice- water, allowed to stand for 30-min and filtered. The liquors from the filtration were allowed to stand at 0 0C over the weekend and filtered again. The combined solids were dissolved in acetone (4 L) and concentrated on the rotary evaporator, this process was repeated a second time to give the phthalimide derivative 3 as a mixture oiEIZ (79/31) isomers (263.2 g, 58.1 %).

Step 2a, Method B: Purification of 3-£-[2-(l,3-dioxo-l,3-dihydroisoindol-2-yl)-l- fluoroethylidene]-piperidine-l-carboxylic acid tert-butyl ester

Figure imgf000044_0001

A 12-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, pressure-equalizing addition funnel, and a nitrogen inlet adapter was charged with the crude phthalimide derivative 3 (263.1 g) and MeOH (2.74 L). The mixture was heated to 66 – 68 0C while water (2.1 L) was added over 20-min, the mixture was stirred at 68 0C for 5-min, then gradually cooled to 20 0C for 18-h. While the crystallization mixture was cooling it was seeded at 60 0C, 56 0C and 530C. This crystallization gave a white solid that was filtered and dried under vacuum at 50 0C to afford 3-E (118.8 g, 45.2%) as a mixture containing 94.4% E and 5.6% Z isomers (NMR analysis).

Step 3: Preparation of 2-[2-fluoro-2-(3-piperidinylidene)ethyl]-lH-isoindole-l,3)- dione (4)

Figure imgf000045_0001

A 12-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, pressure-equalizing addition funnel, and a nitrogen inlet adapter was charged with 3-E (578.0 g, 1.544 mol) and CH2Cl2 (4.5 L). The solution was stirred at 200C under N2 and TFA (476 mL, 6.18 mol) was added via the addition funnel over a 10-min period. The mixture was gently heated to 38 0C and stirred for 3 h. The solvent was removed under vacuum to give the TFA salt of 4 (962.6 g). This material was dissolved in CH2Cl2 (4.0 L) and washed with 2.5 NNa2CO3 (4.6 L)-followed by saturated NaHCO3 (4.6 L). The organic phase was dried (MgSO4), filtered, and condensed in vacuo. The off-white solid was dried at 40 0C under vacuum (20 mm Hg) for 20 h to afford 464.3 g of the free base of 4 as slightly yellowish foamy substance. 1H NMR of 4 TFA salt (400 MHz, CDCl3): δ 1.87-1.98 (m, 2 H), 2.42-2.55 (m, 2 H), 3.38-3.50 (m, 2 H), 4.08-4.18 (br s, 2 H), 4.50 (d, J= 21.0 Hz, 2 H), 7.69-7.78 (m, 2 H), 7.79-7.87 (m, 2 H), 7.98-8.23 (br s, 1 H), 12.48 (s, 1 H). MS: 275 (MH)+, 549 (2M+H)+.

Step 4: Preparation of l-Cyclopropyl-ό^-difluoro-S-methoxy^-oxo-l^- dihydroquinoline-3-carboxylic acid difluoroborate ester (6)

Figure imgf000045_0002

A 22-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, condenser, pressure equalizing addition funnel, and a nitrogen inlet adapter was charged with quinoline-3-carboxylic acid 5 (450.0 g, 1.524 mol), THF (5.40 L) and K2CO3 (247.2 g, 1.753 mol). This suspension was first stirred at 20 0C under N2 for 5 min, and BF3 »Et20 (259 mL, 2.04 mol) was added dropwise via the addition funnel to the stirred mixture over a 5-min period. After the addition, the mixture was heated to reflux (66 0C) for 6 h. The reaction was cooled to 10 0C, diluted with Et2O (9.0 L) and stirred for 10 min. The solid was filtered and washed with Et2O (200 mL x 2) and then dried at 50 0C under house vacuum (-160 mm Hg) for 20 h to afford 771.O g of crude difluoroborate ester 6. After this, the crude material was suspended in MeCN (8.0 L) and stirred at 20 0C for 20 min; the solid was collected by filtration. The filter cake was re-suspended and stirred in MeCN four more times (2.0 L x 4), and all filtrates were combined and concentrated at 60 0C under hi-vac (~10 mmHg). The resulting off- white solid was dried at 50 0C under house vacuum (-160 mmHg) for 20 h to afford 508.66 g (97.2% isolated yield, HPLC = 99.2% by area) of pure difluoroborate ester 6. 1H NMR of 6 (400 MHz, CD3CN): «51.17-1.28 (m, 2 H), 1.29-1.40 (m, 2 H), 4.19 (s, 3 H), 4.40-4.52 (m, 1 H), 8.16 (dd, J= 6.9, 7.0 Hz, 1 H), 9.17 (s, 1 H). MS: 344 (MH)+, 667 (2M-F)+.

Step 5: Preparation of intermediate 8

Figure imgf000046_0001
Figure imgf000046_0002

A 5-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, condenser, pressure-equalizing addition funnel and a nitrogen inlet adapter was charged with difluoroborate ester 6 (320.0 g, 0.933 mol), DMF (1.10 L) and piperidine 4 (289.0 g, 1.053 mole). This suspension was stirred at 20 0C under N2 for 5 min, EtsN (299 mL, 2.15 mol) was added to the stirred mixture via the addition funnel over an additional 5-min period. After this addition, the mixture was heated to 60 0C and stirred for 3 h, to give crude intermediate 7. HPLC analysis (area%) indicated crude 7 is a mixture of 7 (40.5%), 8 (1.7 %), 6 (24.1%), and the rest of unknowns (33.7%). MS: 598 (MH)+. The coupled crude product 7 was carried on to the next step without isolation.

Removal of the Fluoroborate Ester The above stirred reaction mixture containing 7 was treated in the same flask with EtOH (6.80 L) and Et3N (299 mL, 2.147 mol) under N2 at 60 0C. The amber solution was heated to reflux at 72 0C for 2 h and cooled to 20 0C. The reaction mixture was poured into a rapidly stirred 22-L 4-neck round bottom flask containing a 1 : 1 (v/v) ice-water mixture (8.0 L) over a 10-min period; stirring was continued for -10 min. Cold 1 NHCl (4.0 L) was added to the solution over 20 min to adjust the pH from 9-10 to 3; stirring was continued for an additional 20 min at 0 0C. The yellow solid was isolated by filtration and dried in a filter funnel by air-suction using house vacuum (-160 mm Hg) at 20 0C for 20 h to afford 1,889.0 g of crude 8 as a damp solid (HPLC = 33.6%, area%).

Purification of Intermediate 8

To a 22-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, pressure-equalizing addition funnel, and a nitrogen inlet adapter was charged with crude 8 (1889.0 g), MeCN (3.6 L) and EtOH (3.2 L). The suspension was heated to reflux (76 0C), while D.I. H2O (500 mL) was added over 10 min. The solution was stirred at 76 0C for 5 min, and then gradually cooled to 10 0C over 1 h; stirred for an additional hour. The yellow solid was collected by filtration, dried in a vacuum oven under house vacuum (-160 mm Hg) at 60 0C for 20 h to afford 229. Ig (45%) of 8, which was used in next step without further purification. 1H ΝMR of 8 (400 MHz, DMSO-d6): £ 1.02-1.10 (m, 2 H), 1.11-1.19 (m, 2 H), 1.67-1.79 (m, 2 H), 2.34-2.45 (m, 2 H), 3.38-3.49 (m, 2 H), 3.78 (s, 3 H), 4.10 (s, 2 H), 4.15-4.26 (m, 1 H), 4.54 (d, J= 21.0 Hz, 1 H), 7.72 (d, J= 9.1 Hz, 1 H), 7.81 (s, 4 H), 8.71 (s, I H), 14.98 (s, 1 H). MS: 550 (MH)+.

Step 6: Preparation of 7-[3-(2-amino-l-fluoro-ethylidene)-piperidin-l-yl]-l- cyclopropyl-6-fluoro-8-methoxy-4-oxo-l,4-dihydro-quinoline-3-carboxylic acid (10)

Figure imgf000048_0001

8 10 A

22-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, condenser, pressure-equalizing addition funnel and a nitrogen inlet adapter was charged with 8 (253.6 g, 0.462 mol) and MeOH (5.10 L). This suspension was stirred at 20 0C under N2 and H2NNH2 (86.9 mL, 2.796 mol) was added over a 5-min period. The yellow suspension was heated to 65 0C and refluxed for 1 h. The reaction was cooled to 60 0C and MeCN (3.84 L) was added. The mixture was heated to reflux for 5 min, and then cooled to 20 0C in a water bath. The light-yellow solid was collected by filtration and the filter cake was washed with MeCN (150 mL x 2). The combined filtrate was concentrated at 60 0C affording 322.0 g of crude product 10. This product was recrystallized from a mixture of MeOH (1.0 L) and water (1.195 L) to give 176.6 g (91.2%) of pure product 10 as a light yellow solid.

BASE FORM

1H NMR of 10 (400 MHz, DMSO- d6): £ 1.0-1.09 (m, 2 H), 1.10-1.19 (m, 2 H), 1.66-1.78 (m, 2 H), 2.30-2.41 (m, 2 H), 3.17 (s, 2 H), 3.35 (s, 1 H), 3.36-3. 47 (m, 2 H), 3.74 (s, 3 H), 3.89 (s, 2 H), 4.13-4.22 (m, 1 H), 5.35-6.18 (br, 2 H), 7.74 (d, J= 8.9 Hz, 1 H), 8.69 (s, 1 H).

MS: 420 (MH)+.

 

Example 3

Preparation of7-[3-(2-Amino-l-fluoro-ethylidene)-piperidin-l-yl]-l-cyclopropyl-

6-fluoro-8-methoxy-4-oxo-l,4-dihydro-quinoline-3-carboxylic acid hydrogen chloride salt (12)

Figure imgf000049_0001

7-[3-(2-amino-l-fluoro-ethylidene)-piperidin-l-yl]-l-cyclopropyl-6-fluoro-8- methoxy-4-oxo-l,4-dihydro-quinoline-3-carboxylic acid (10) was prepared as described in Step 6 of Example 1.

A 5-L 4-neck round bottom flask equipped with an overhead stirrer, thermocouple, condenser, pressure-equalizing addition funnel, and a nitrogen inlet adapter was charged with compound 10 (176.0 g, 0.4196 mol) and EtOH (2.40 L). The suspension was stirred under N2 and cooled to 10 0C with an ice/water bath. A solution of HCl in EtOH (1.25 M, 350 mL) was added via the addition funnel over a 20-min period. After the addition, the reaction was stirred at 10 0C for 5 min. The water bath was replaced with a heating mantle and the solution was heated to 76 0C and stirred for 5 min. The heating mantle was replaced with the water bath, the solution was cooled to 0 0C over 1 h and stirred at this temperature for an additional 1 h. The solid was collected by filtration, washed with ice-cold EtOH (100 mL x 2) and dried at 60 0C under vacuum (~4 mmHg) for 60 h. There was obtained 88.9 g (82%) of HCl salt 12 as an off-white to very light-yellow solid.

 

HCl SALT

1H NMR of HCl salt 12 (400 MHz, CD3CO2D): £ 1.10-1.19 (m, 2 H), 1.29-1.38 (m, 2 H), 1.81-1.93 (m, 2 H), 2.51-2.60 (m, 2 H), 3.48- 3.60 (m, 2 H), 3.86 (s, 3 H), 4.08 (s, 2 H), 4.18 (s, 1 H), 4.19-4.30 (m, 2 H), 7.92 (d, J= 8.6 Hz, 1 H), 8.98 (s, 1 H) 11.65 (s, 1 H).

MS: 420 (MH)+

 

………………………………………

http://www.google.com/patents/WO2013045599A1?cl=en

Experiment 6

Figure imgf000033_0001

(VI) compound (1 ) .HCI

Compound (1) .HCI salt from Compound (Via) :

9.8 ml (90.6 mmol) of 1-chloroethyl chloro formate are added slowly to a solution of 30 g (82.3 mmol) of compound (E)-(Va) in 165 ml of toluene kept at 0°C. The reaction mixture is stirred 1 hour at room temperature than 1 hour at 80°C and filtered. 24 ml of ethanol and 15.35 ml (90.6 mmol) of 6M HCI solution in isopropanol are added to the filtrate and the resulting mixture is refluxed for 4 hours then cooled to 0°C. The precipitate is filtered, washed with 16 ml of acetone and 16 ml of toluene and dried under vacuum to give 21.94 g of compound (1) . HCI salt. Yield: 86%>.

NMR and MS data are identical to those of the literature.

 

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    November 28, 2012. N12/130. STATEMENT ON A NONPROPRIETARY NAME ADOPTED BY THE USAN COUNCIL. USAN (ZZ-145). AVAROFLOXACIN.

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