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ORGANIC SPECTROSCOPY

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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 30 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, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, 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 Open superstar 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 30 year tenure till date Dec 2017, Around 35 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, 50 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 19 lakh plus views on New Drug Approvals Blog in 216 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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RIDINILAZOLE


ChemSpider 2D Image | Ridinilazole | C24H16N6
Ridinilazole.svg

RIDINILAZOLE

SMT19969

  • Molecular FormulaC24H16N6
  • Average mass388.424 Da
  • ридинилазол [Russian] [INN]ريدينيلازول [Arabic] [INN]利地利唑 [Chinese] [INN]
  • リジニラゾール;

10075
2,2′-Di(4-pyridinyl)-3H,3’H-5,5′-bibenzimidazole
308362-25-6[RN]6,6′-Bi-1H-benzimidazole, 2,2′-di-4-pyridinyl-

Summit Therapeutics (formerly Summit Corp ) is developing ridinilazole the lead compound from oral narrow-spectrum, GI-restricted antibiotics, which also include SMT-21829, for the treatment of Clostridium difficile infection and prevention of recurrent disease.

Ridinilazole (previously known as SMT19969) is an investigational small molecule antibiotic being evaluated for oral administration to treat Clostridioides difficile infection (CDI). In vitro, it is bactericidal against C. difficile and suppresses bacterial toxin production; the mechanism of action is thought to involve inhibition of cell division.[1] It has properties which are desirable for the treatment of CDI, namely that it is a narrow-spectrum antibiotic which exhibits activity against C. difficile while having little impact on other normal intestinal flora and that it is only minimally absorbed systemically after oral administration.[2] At the time ridinilazole was developed, there were only three antibiotics in use for treating CDI: vancomycinfidaxomicin, and metronidazole.[1][2] The recurrence rate of CDI is high, which has spurred research into other treatment options with the aim to reduce the rate of recurrence.[3][4]

As of 2019, two phase II trials have been completed and two phase III trials comparing ridinilazole to vancomycin for CDI are expected to be completed in September 2021.[2][5][6] Ridinilazole was designated as a Qualified Infectious Disease Product (QIDP) and was granted Fast Track status by the U.S. FDA.[2] Fast Track status is reserved for drugs designed to treat diseases where there is currently a gap in the treatment, or a complete lack thereof.[7] The QIDP designation adds five more years of exclusivity for ridinazole upon approval.[8]

str1-1

PATENT

WO-2021009514

Process for preparing ridinilazole useful for treating Clostridium difficile infection. Also claimed is the crystalline form of a compound.

The present invention relates to processes for the preparation of 2,2′-di(pyridin-4-yl)-1/-/,T/-/-5,5′-bibenzo[d]imidazole (which may also be known as 5,5’-bis[2-(4-pyridinyl)-1/-/-benzimidazole], 2,2′-bis(4-pyridyl)-3/-/,3’/-/-5,5′-bibenzimidazole or 2-pyridin-4-yl-6-(2-pyridin-4-yl-3/-/-benzimidazol-5-yl)-1/-/-benzimidazole), referenced herein by the INN name ridinilazole, and pharmaceutically acceptable derivatives, salts, hydrates, solvates, complexes, bioisosteres, metabolites or prodrugs thereof. The invention also relates to various crystalline forms of ridinilazole, to processes for their preparation and to related pharmaceutical preparations and uses thereof (including their medical use and their use in the efficient large-scale synthesis of ridinilazole).

WO2010/063996 describes various benzimidazoles, including ridinilazole, and their use as antibacterials (including in the treatment of CDAD).

WO 2011/151621 describes various benzimidazoles and their use as antibacterials

(including in the treatment of CDAD).

W02007056330, W02003105846 and W02002060879 disclose various 2-amino benzimidazoles as antibacterial agents.

W02007148093 discloses various 2-amino benzothiazoles as antibacterial agents.

W02006076009, W02004041209 and Bowser et at. (Bioorg. Med. Chem. Lett., 2007, 17, 5652-5655) disclose various substituted benzimidazole compounds useful as anti-infectives that decrease resistance, virulence, or growth of microbes. The compounds are said not to exhibit intrinsic antimicrobial activity in vitro.

US 5,824,698 discloses various dibenzimidazoles as broad-spectrum antibiotics, disclosing activity against both Gram-negative and Gram-positive bacteria, including Staphylococcus spp.and Enterococcus spp. However, this document does not disclose activity against anaerobic spore-forming bacteria and in particular does not disclose activity against any Clostridioides spp. (including C. difficile).

US 2007/0112048 A1 discloses various bi- and triarylimidazolidines and bi- and

triarylamidines as broad-spectrum antibiotics, disclosing activity against both Gram negative and Gram-positive bacteria, including Staphylococcus spp., Enterococcus spp. and Clostridioides spp. However, this document does not disclose compounds of formula (I) as described herein.

Chaudhuri et al. (2007) J.Org. Chem. 72, 1912-1923 describe various bis-2-(pyridyl)-1 H-benzimidazoles (including compounds of formula I as described herein) as DNA binding agents. This document is silent as to potential antibacterial activity.

Singh et al. (2000) Synthesis 10: 1380-1390 describe a condensation reaction for producing 2,2′-di(pyridin-4-yl)-1/-/,T/-/-5,5′-bibenzo[d]imidazole using 4-pyridine

carboxaldehyde, FeCI3, 02, in DMF at 120°C.

Bhattacharya and Chaudhuri (2007) Chemistry – An Asian Journal 2: 648-655 describe a condensation reaction for producing 2,2′-di(pyridin-4-yl)-1/-/,T/-/-5,5′-bibenzo[d]imidazole using 4-pyridine carboxaldehyde and nitrobenzene at 120°C.

WO2019/068383 describes the synthesis of ridinilazole by metal-ion catalyzed coupling of 3,4,3’,4’-tetraaminobiphenyl with 4-pyridinecarboxaldehyde in the presence of oxygen, followed by the addition of a complexing agent.

PATENT

WO2010063996

claiming antibacterial compounds. Bicyclic heteroaromatic compounds, particularly bi-benzimidazole derivatives.

WO2007056330, WO2003105846 and WO2002060879 disclose various 2-amino benzimidazoles as antibacterial agents.

WO2007148093 discloses various 2-amino benzothiazoles as antibacterial agents.

WO2006076009, WO2004041209 and Bowser et al. (Bioorg. Med. Chem. Lett., 2007, 17, 5652-5655) disclose various substituted benzimidazole compounds useful as anti-infectives that decrease resistance, virulence, or growth of microbes. The compounds are said not to exhibit intrinsic antimicrobial activity in vitro.

US 5,824,698 discloses various dibenzimidazoles as broad-spectrum antibiotics, disclosing activity against both Gram-negative and Gram-positive bacteria, including Staphylococcus spp.and Enterococcus spp. However, this document does not disclose activity against anaerobic spore-forming bacteria and in particular does not disclose activity against any Clostridium spp. (including C. difficile).

US 2007/0112048 A1 discloses various bi- and triarylimidazolidines and bi- and triarylamidines as broad-spectrum antibiotics, disclosing activity against both Gram-negative and Gram-positive bacteria, including Staphylococcus spp., Enterococcus spp.

and Clostridium spp. However, this document does not disclose compounds of general formula (I) as described herein.

Chaudhuri et al. (J.Org. Chem., 2007, 72, 1912-1923) describe various bis-2-(pyridyl)-1 H-benzimidazoles (including compounds of formula I as described herein) as DNA binding agents. This document is silent as to potential antibacterial activity.

PATENT

Product PATENT, WO2010063996 ,

protection in the EP until 2029 and expire in the US in December 2029.

PAPER

https://www.frontiersin.org/articles/10.3389/fmicb.2018.01206/full

PAPER

Synthesis (2000), (10), 1380-1390.

https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-2000-7111

PAPERT

Chemistry – An Asian Journal (2007), 2(5), 648-655.

https://onlinelibrary.wiley.com/doi/abs/10.1002/asia.200700014

Studies of double‐stranded‐DNA binding have been performed with three isomeric bis(2‐(n‐pyridyl)‐1H‐benzimidazole)s (n=2, 3, 4). Like the well‐known Hoechst 33258, which is a bisbenzimidazole compound, these three isomers bind to the minor groove of duplex DNA. DNA binding by the three isomers was investigated in the presence of the divalent metal ions Mg2+, Co2+, Ni2+, Cu2+, and Zn2+. Ligand–DNA interactions were probed with fluorescence and circular dichroism spectroscopy. These studies revealed that the binding of the 2‐pyridyl derivative to DNA is dramatically reduced in the presence of Co2+, Ni2+, and Cu2+ ions and is abolished completely at a ligand/metal‐cation ratio of 1:1. Control experiments done with the isomeric 3‐ and 4‐pyridyl derivatives showed that their binding to DNA is unaffected by the aforementioned transition‐metal ions. The ability of 2‐(2‐pyridyl)benzimidazole to chelate metal ions and the conformational changes of the ligand associated with ion chelation probably led to such unusual binding results for the ortho isomer. The addition of ethylenediaminetetraacetic acid (EDTA) reversed the effects completely.

PAPER

 Journal of Organic Chemistry (2007), 72(6), 1912-1923.

https://pubs.acs.org/doi/10.1021/jo0619433

Three symmetrical positional isomers of bis-2-(n-pyridyl)-1H-benzimidazoles (n = 2, 3, 4) were synthesized and DNA binding studies were performed with these isomeric derivatives. Like bisbenzimidazole compound Hoechst 33258, these molecules also demonstrate AT-specific DNA binding. The binding affinities of 3-pyridine (m-pyben) and 4-pyridine (p-pyben) derivatized bisbenzimidazoles to double-stranded DNA were significantly higher compared to 2pyridine derivatized benzimidazole o-pyben. This has been established by combined experimental results of isothermal fluorescence titration, circular dichroism, and thermal denaturation of DNA. To rationalize the origin of their differential binding characteristics with double-stranded DNA, computational structural analyses of the uncomplexed ligands were performed using ab initio/Density Functional Theory. The molecular conformations of the symmetric head-to-head bisbenzimidazoles have been computed. The existence of intramolecular hydrogen bonding was established in o-pyben, which confers a conformational rigidity to the molecule about the bond connecting the pyridine and benzimidazole units. This might cause reduction in its binding affinity to double-stranded DNA compared to its para and meta counterparts. Additionally, the predicted stable conformations for p-, m-, and o-pyben at the B3LYP/6-31G* and RHF/6-31G* levels were further supported by experimental pKa determination. The results provide important information on the molecular recognition process of such symmetric head to head bisbenzimidazoles toward duplex DNA.

Patent

US 8975416

PATENT

WO 2019068383

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019068383

Clostridium difficile infection (CDI) is the leading cause of infectious healthcare-associated diarrhoea. CDI remains a challenge to treat clinically, because of a limited number of antibiotics available and unacceptably high recurrence rates. Because of this, there has been significant demand for creating innovative therapeutics, which has resulted in the development of several novel antibiotics.

Ridinilazole (SMT19969) is the INN name of 5,5’bis[2-(4-pyridinyl)-lH-benzimidazole], which is a promising non-absorbable small molecule antibiotic intended for oral use in the treatment of CDI. It has been shown to exhibit a prolonged post-antibiotic effect and treatment with ridinilazole has resulted in decreased toxin production. A phase 1 trial demonstrated that oral ridinilazole is well tolerated and specifically targets Clostridia whilst sparing other faecal bacteria.

Ridinilazole has the following chemical structure:

Bhattacharya & Chaudhuri (Chem. Asian J., 2007, No. 2, 648-655) report performing double-stranded DNA binding with three benzimidazole derivatives, including ridinilazole. The compounds have been prepared by dissolving the reactants in nitrobenzene, heating at 120°C for 8- 1 Oh and purifying the products by column chromatography over silica gel. The compounds were obtained in 65-70% yield. Singh et al., (Synthesis, 2000, No. 10, 1380-1390) describe a catalytic redox cycling approach based on Fe(III) and molecular oxygen as co-oxidant for providing access to benzimidazole and

imidazopyridine derivatives, such as ridinilazole. The reaction is performed at high temperatures of 120°C and the product is isolated in 91% yield by using silica flash chromatography.

Both processes are not optimal, for example in terms of yield, ease of handling and scalability. Thus, there is a need in the art for an efficient and scalable preparation of ridinilazole, which overcomes the problems of the prior art processes.

Example 1 : Preparation of crude ridinilazole free base

A solution of 3,4,3′,4′-tetraaminobiphenyl (3.28 g, 15.3 mmol) and isonicotinaldehyde (3.21 g, 30.0 mmol) in DMF (40 mL) was stirred at 23 °C for one hour. Then anhydrous ferric chloride (146 mg, 0.90 mmol), water (0.10 mL, 5.4 mmol) and additional DMF (2 mL) were added and fresh air was bubbled into the solution during vigorous stirring for 5 hours at room temperature. Next, water (80 mL) and EDTA (0.29 g) were added resulting in a brownish suspension, which was stirred overnight. The product was isolated by filtration, washed with water, and dried in a desiccator in vacuo as a brown powder (5.56 g; 95%). The addition of EDTA had held iron in solution and the crude ridinilazole contained significantly lower amounts of iron than comparative example 1.

Example 12: Formation of essentially pure ridinilazole free base

To a suspension von ridinilazole tritosylate (1 10 mg, 0.12 mmol) in water (35 mL) featuring a pH value of about 4.5 stirring at 70 °C sodium bicarbonate (580 mg, 6.9 mmol) were added and caused a change of color from orange to slightly tan. The mixture, now at a pH of about 8.5, was cooled down to room temperature and the solids were separated by filtration, washed with water (1 ML) and dried in vacuo providing 40 mg (85%) essentially pure ridinilazole as a brownish powder.

Spectroscopic analysis:

¾ NMR (DMSO-de, 300 MHz): δ 7.55 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.88 (s, 2H), 8.13 (d, J = 5.8 Hz, 4H), 8.72 (d, J = 5.8 Hz, 4H) ppm.

13C NMR (DMSO-d6, 75 MHz): δ 1 13.4 (2C), 1 16.4 (2C), 120.4 (4C), 121.8 (2C), 135.7 (2C), 138.7 (2C), 140.7 (2C), 141.4 (2C), 150.3 (4C), 151.1 (2C) ppm.

IR (neat): v 3033 (w), 1604 (s), 1429 (m), 1309 (m), 1217 (m), 1 1 15 (w), 998 (m), 964 (m), 824 (m), 791 (s), 690 (s), 502 (s) cm .

UV-Vis (MeOH): 257, 341 nm.

The sharp peaks in the ¾ NMR indicated that iron had been efficiently removed.

Comparative example 1 : Preparation of ridinilazole

A solution of 3,4,3′,4′-tetraaminobiphenyl (0.69 g, 3.2 mmol) and isonicotinaldehyde (0.64 g, 6.0 mmol) in DMF (20 mL) was stirred at 80°C for one hour. Then ferric chloride hexahydrate (49 mg, 0.18 mmol), water (0.10 mL, 5.4 mmol) and additional DMF (2 mL) were added and fresh air was bubbled into the solution during vigorous stirring for 10 hours at 120 °C. After cooling to room temperature water (50 mL) and the mixture was stirred for one hour. A black crude product was isolated by filtration and comprised ridinilazole and iron.

References

  1. Jump up to:a b Cho JC, Crotty MP, Pardo J (March 2019). “Clostridium difficile infection”Annals of Gastroenterology32 (2): 134–140. doi:10.20524/aog.2018.0336PMC 6394264PMID 30837785.
  2. Jump up to:a b c d Carlson TJ, Endres BT, Bassères E, Gonzales-Luna AJ, Garey KW (April 2019). “Ridinilazole for the treatment of Clostridioides difficile infection”Expert Opinion on Investigational Drugs28 (4): 303–310. doi:10.1080/13543784.2019.1582640PMID 30767587.
  3. ^ Bassères E, Endres BT, Dotson KM, Alam MJ, Garey KW (January 2017). “Novel antibiotics in development to treat Clostridium difficile infection”Current Opinion in Gastroenterology33 (1): 1–7. doi:10.1097/MOG.0000000000000332PMID 28134686These tables highlight the increased drug development directed towards CDI due to the rise in prevalence of infections and to attempt to reduce the number of recurrent infections.
  4. ^ Vickers RJ, Tillotson G, Goldstein EJ, Citron DM, Garey KW, Wilcox MH (August 2016). “Ridinilazole: a novel therapy for Clostridium difficile infection”International Journal of Antimicrobial Agents48 (2): 137–43. doi:10.1016/j.ijantimicag.2016.04.026PMID 27283730there exists a significant unmet and increasing medical need for new therapies to treat CDI, specifically those that can reduce the rate of disease recurrence.
  5. ^ Clinical trial number NCT03595553 for “Ri-CoDIFy 1: Comparison of Ridinilazole Versus Vancomycin Treatment for Clostridium Difficile Infection” at ClinicalTrials.gov
  6. ^ Clinical trial number NCT03595566 for “Ri-CoDIFy 2: To Compare Ridinilazole Versus Vancomycin Treatment for Clostridium Difficile Infection” at ClinicalTrials.gov
  7. ^ “Fast Track”. U.S. Food and Drug Administration. 2018-11-03.
  8. ^ “”HHS spurs new antibiotic development for biodefense and common infections””Public Health Emergency. U.S. Department of Health and Human Services. Retrieved 2020-12-04.
Clinical data
Other namesSMT19969
ATC codeNone
Identifiers
IUPAC name[show]
CAS Number308362-25-6
PubChem CID16659285
ChemSpider17592423
UNII06DX01190R
KEGGD11958
Chemical and physical data
FormulaC24H16N6
Molar mass388.42 g/mol
3D model (JSmol)Interactive image
SMILES[hide]c6cc(c5nc4ccc(c3ccc2nc(c1ccncc1)[nH]c2c3)cc4[nH]5)ccn6

/////////RIDINILAZOLE, SMT19969, SMT 19969, ридинилазол , ريدينيلازول , 利地利唑 , リジニラゾール , Qualified Infectious Disease Product, QIDP,  Fast Track , PHASE 3,  Clostridioides difficile infection , 

Devimistat


Devimistat Chemical Structure
DEVIMISTAT
6,8-Bis(benzylthio)octanoic acid.png

Devimistat

CPI-613

Molecular Weight388.59
FormulaC₂₂H₂₈O₂S₂
CAS No.95809-78-2
SMILESO=C(O)CCCCC(SCC1=CC=CC=C1)CCSCC2=CC=CC=C2

phase III, hematological cancer

6,8-Bis(benzylsulfanyl)octanoic acid

Octanoic acid, 6,8-bis[(phenylMethyl)thio]-

Octanoic acid, 6,8-bis((phenylmethyl)thio)-

Rafael Pharmaceuticals (formerly Cornerstone Pharmaceuticals), a subsidiary of Rafael Holdings, is developing devimistat, the lead candidate from a program of thioctans and their derivatives that act as pyruvate dehydrogenase and alpha-ketoglutarate inhibitors and stimulators of pyruvate dehydrogenase kinase (PDK), using the company’s proprietary Altered Energy Metabolism Directed (AEMD) platform, for the iv treatment of hematological cancer [phase III, January 2021].

Devimistat (INN; development code CPI-613) is an experimental anti-mitochondrial drug being developed by Rafael Pharmaceuticals.[1] It is being studied for the treatment of patients with metastatic pancreatic cancer and relapsed or refractory acute myeloid leukemia (AML).

Devimistat’s mechanism of action differs from other drugs, operating on the tricarboxylic acid cycle and inhibiting enzymes involved with cancer cell energy metabolism. A lipoic acid derivative different from standard cytotoxic chemotherapy, devimistat is currently being studied in combination with modified FOLFIRINOX to treat various solid tumors and heme malignancies.

Regulation

The U.S. Food and Drug Administration (FDA) has designated devimistat as an orphan drug for the treatment of pancreatic cancer, AML, myelodysplastic syndromes (MDS), peripheral T-cell lymphoma, and Burkitt’s lymphoma, and given approval to initiate clinical trials in pancreatic cancer and AML.

Clinical trials

Clinical trials of the drug are underway including a Phase III open-label clinical trial[2] to evaluate efficacy and safety of devimistat plus modified FOLFIRINOX (mFFX) versus FOLFIRINOX (FFX) in patients with metastatic adenocarcinoma of the pancreas.

Developed as part of Rafael’s proprietary Altered Metabolism Directed (AMD) drug platform, CPI-613® was discovered at Stony Brook University. CPI-613® is designed to target the mitochondrial tricarboxylic acid (TCA) cycle, an indispensable process essential to tumor cell multiplication and survival, selectively in cancer cells.

The attacks of CPI-613® on the TCA cycle also substantially increases the sensitivity of cancer cells to a diverse range of chemotherapeutic agents. This synergy allows for combinations of CPI-613® with lower doses of these generally toxic drugs to be highly effective with lower patient side effects. Combinations with CPI-613® represent a diverse range of potential opportunities to substantially improve patient benefit in many different cancers.

The U.S. Food and Drug Administration (FDA) has given Rafael approval to initiate pivotal clinical trials in pancreatic cancer and acute myeloid leukemia (AML), and has designated CPI-613® as an orphan drug for the treatment of pancreatic cancer, AML, Myelodysplastic syndromes (MDS), peripheral T-cell lymphoma and Burkitt’s lymphoma. The EMA has granted orphan drug designation to CPI-613® for pancreatic cancer and AML.


Learn more about recent developments involving CPI-613®CPI-613® (devimistat) Fact Sheet

he FDA granted a Fast Track designation to devimistat for the treatment of patients with acute myeloid leukemia.

The FDA has granted a Fast Track designation to devimistat (CPI-613) for the treatment of patients with acute myeloid leukemia (AML), Rafael Pharmaceuticals, announced in a press release.1

“This designation underscores the pressing need to find new ways to combat this aggressive disease,” said Jorge Cortes, MD, director of the Georgia Cancer Center at Augusta University, and principal investigator on the phase 3 clinical trial, in a statement. “It brings hope not only to clinicians, but to patients who hear that they have been diagnosed.”

The first-in-class agent devimistat targets enzymes that are involved in cancer cell energy metabolism. This therapy substantially increases the sensitivity of cancer cells to a diverse range of chemotherapies, and this synergy allows for potential combinations that could be more effective with devimistat and lower doses of drugs that are generally toxic.

“Receiving Fast Track designation, especially during a pandemic that has created significant challenges for many trials across the globe, is a testament to the dedicated work of the Rafael team,” stated Sanjeev Luther, president and CEO of Rafael Pharmaceuticals, Inc.

Devimistat combinations appear promising with a diverse range of potential opportunities to improve benefit in patients with various cancer types. Two pivotal phase 3 clinical trials, including the AVENGER 500 study in pancreatic cancer (NCT03504423) and ARMADA 2000 for AML (NCT03504410), have been approved for initiation by the FDA.

The primary end point of the multicenter, open-label, randomized ARMADA 2000 study is complete response (CR), and secondary end points include overall survival and CR plus CR with partial hematologic recovery rate. To be eligible to enroll to the study, patients must be aged ≥50 years with a documented AML diagnosis that has relapsed from or became refractory to previous standard therapy. Patients must have an ECOG performance status of 0 to 2 and an expected survival longer than 3 months.

Five hundred patients are expected to be enrolled and randomized in the study. To enroll, patients could not have received prior radiotherapy or cytotoxic chemotherapy for their current AML. Those with active central nervous system involvement, active uncontrolled bleeding, history of other malignancy, or known hypersensitivity to study drugs are ineligible to enroll to the trial as well.

This study aims to determine the safety and efficacy of devimistat in combination with high-dose cytarabine and mitoxantrone in older patients with relapsed/refractory AML compared with high-dose cytarabine and mitoxantrone therapy alone. Other control groups include patients treated with mitoxantrone, etoposide, and cytarabine and the combination of fludarabine, cytarabine, and filgrastim. The addition of devimistat is expected to improve the CR rate in patients who are aged 50 years or older with relapsed/refractory AML.

In a prior phase 1 study of devimistat plus high-dose cytarabine and mitoxantrone in patients with relapsed/refractory AML, the addition of devimistat sensitized AML cells to chemotherapy treatment.2

The objective response rate was 50% including CRs in 26 of 62 evaluable patients. Median overall survival was 6.7 months. In patients above age 60, the CR or CR with incomplete hematologic recovery rate was 47% and the median survival was 6.9 months.

This designation for this experimental anti-mitochondrial agent follows news of another Fast Track designation granted to devimistat for the treatment of patients with metastatic pancreatic cancer in November 2020, as well as an Orphan Drug designation granted in October 2020 for the treatment of patients with soft tissue sarcoma.

References

1. Rafael Pharmaceuticals Receives FDA Fast Track Designation for CPI-613® (devimistat) for the treatment of acute myeloid leukemia (AML). News Release. Rafael Pharmaceuticals, Inc. December 15, 2020. Accessed December 15, 2020. https://bit.ly/34g6YsR

2. Pardee TS, Anderson RG, Pladna KM, et al. A Phase I Study of CPI-613 in Combination with High-Dose Cytarabine and Mitoxantrone for Relapsed or Refractory Acute Myeloid Leukemia. Clin Cancer Res. 2018;24(9):2060-2073. doi:10.1158/1078-0432.CCR-17-2282 P[APERJournal of the American Chemical Society (1954), 76, 4109-12.https://pubs.acs.org/doi/abs/10.1021/ja01645a016
PAPERJournal of the American Chemical Society (1955), 77, 416-19.https://pubs.acs.org/doi/abs/10.1021/ja01607a057PAPERJustus Liebigs Annalen der Chemie (1958), 614, 66-83.https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/jlac.19586140108PATENTWO 2009123597WO 2009110859WO 2010110771PATENTCN 111362848

PATENT

WO-2021011334

Deuterated derivatives of 6,8-bis(benzylsulfanyl)octanoic acid (CPI-613 or devimistat ) or its salts for treating cancer.

CPI-613 (6,8-bis(benzylsulfanyl)octanoic acid) is a first-in-class investigational small-molecule (lipoate analog), which targets the altered energy metabolism unique to many cancer cells. CPI-613 is currently being evaluated in two phase III clinical trials, and has been granted orphan drug designation for the treatment of pancreatic cancer, acute myeloid leukemia (AML), peripheral T-cell lymphoma (PTCL), Burkitt lymphoma and myelodysplastic syndromes (MDS).

[0004] One limitation to the clinical utility of CPI-613 is its very rapid metabolism. After IV dosing the half-life of 6,8-bis(benzylsulfanyl)octanoic acid is only about 1-2 hours (Pardee,

T.S. et al, Clin Cancer Res. 2014, 20, 5255-64). The short half-life limits the patient’s overall exposure to the drug and necessitates administration of relatively high doses. For safety reasons, CPI-613 is administered via a central venous catheter as an IV infusion over 30-120 minutes, with higher doses requiring longer infusion times.

The terms“6,8-bis(benzylsulfanyl)octanoic acid” and“ 6,8-bis-benzylthio-octanoic acid” refer to the compound known as CPI-613 or devimistat, having the chemical structure

PATENT

WO2020132397

claiming the use of CPI-613 in combination with an autophagy inhibitor eg chloroquine for treating eg cancers.

CPI-613 (6,8-bis-benzylthio-octanoic acid) is a first-in-class investigational small-molecule (lipoate analog), which targets the altered energy metabolism that is common to many cancer cells. CPI-613 has been evaluated in multiple phase I, I/II, and II clinical studies, and has been granted orphan drug designation for the treatment of pancreatic cancer, acute myeloid leukemia (AML), peripheral T-cell lymphoma (PTCL), Burkitt lymphoma and myelodysplastic syndromes (MDS).

PAPER

https://pubs.acs.org/doi/10.1021/op200091t

An Efficient, Economical Synthesis of the Novel Anti-tumor Agent CPI-613

Cite this: Org. Process Res. Dev. 2011, 15, 4, 855–857

Publication Date:May 2, 2011
https://doi.org/10.1021/op200091t

An efficient and practical synthesis of the novel anti-tumor compound 6,8-dithiobenzyl octanoic acid, CPI-613 (2), was developed and executed on a practical scale. CPI-613 can be made in a single vessel from (±)-lipoic acid (1) via reductive opening of the disulfide ring followed by benzylation of the sulfhydryls with benzyl bromide. CPI-613 was isolated by simple crystallization in high yield and purity. The process is scaleable and has been demonstrated at up to 100 kg.CPI-613 (2) was isolated [4.7 kg (90%)] with an HPLC purity of 99.8 area %. Mp 66–67 °C. IR: 3050, 1710, 1400, 668 cm–11H NMR (400 MHz, CDCl3) δ 7.40–7.20 (m, 10 H), 3.80–3.60 (m, 4 H), 2.60–2.50 (m, 2 H), 2.44 (t, J = 8.7, 2 H), 2.23 (t, J = 8.1, 2 H) 2.03–1.30 (m, 8 H). Anal. Calc for C22H28O2S2: C, 68.00; H, 7.26; S, 16.50. Found: C, 67.99; H, 7.31; S, 16.37. 

References

  1. ^ “CPI-613”. Rafael Pharmaceuticals.
  2. ^ Philip PA, Buyse ME, Alistar AT, Rocha Lima CM, Luther S, Pardee TS, Van Cutsem E (October 2019). “A Phase III open-label trial to evaluate efficacy and safety of CPI-613 plus modified FOLFIRINOX (mFFX) versus FOLFIRINOX (FFX) in patients with metastatic adenocarcinoma of the pancreas”Future Oncology15 (28): 3189–3196. doi:10.2217/fon-2019-0209PMC 6854438PMID 31512497.
Clinical data
Other namesCPI-613
Legal status
Legal statusInvestigational
Identifiers
IUPAC name[show]
CAS Number95809-78-2
PubChem CID24770514
DrugBank12109
ChemSpider28189062
UNIIE76113IR49
ChEMBLChEMBL3186849
CompTox Dashboard(EPA)DTXSID70914807
ECHA InfoCard100.231.125 
Chemical and physical data
FormulaC22H28O2S2
Molar mass388.58 g·mol−1
3D model (JSmol)Interactive image
SMILES[hide]C1=CC=C(C=C1)CSCCC(CCCCC(=O)O)SCC2=CC=CC=C2

//////////devimistat, CPI-613, CPI 613, phase 3, hematological cancer , Fast Track designation, ORPHAN DRUG, 

LYS 228


2D chemical structure of 1810051-96-7

LYS228

BOS-228
LYS-228

Molecular Formula, C16-H18-N6-O10-S2

Molecular Weight, 518.4783

(3S,4R)-3-((Z)-2-(2-Ammoniothiazol-4-yl)-2-((1-carboxycyclopropoxy)imino)acetamido)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)azetidine-1-sulfonate

RN: 1810051-96-7
UNII: 29H7N9XI1B

Unii-005B24W9YP.png

UNII-005B24W9YP

005B24W9YP

Lys-228 trihydrate

2091840-43-4

Yclopropanecarboxylic acid, 1-(((Z)-(1-(2-amino-4-thiazolyl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxo-3-oxazolidinyl)methyl)-1-sulfo-3-azetidinyl)amino)ethylidene)amino)oxy)-, hydrate (1:3)

1-[(Z)-[1-(2-amino-1,3-thiazol-4-yl)-2-oxo-2-[[(3S,4R)-2-oxo-4-[(2-oxo-1,3-oxazolidin-3-yl)methyl]-1-sulfoazetidin-3-yl]amino]ethylidene]amino]oxycyclopropane-1-carboxylic acid;trihydrate

BOS-228 (LYS-228) is a monobactam discovered at Novartis and currently in phase II clinical development at Boston Pharmaceuticals for the treatment of complicated urinary tract infection and complicated intraabdominal infections in adult patients.

The compound has been granted fast track and Qualified Infectious Disease Product (QIDP) designation from the FDA.

In October 2018, Novartis licensed to Boston Pharmaceuticals worldwide rights to the product.

Paper

https://pubs.acs.org/doi/10.1021/acs.oprd.9b00330

Patent

US 20150266867

PATENT

WO 2017050218

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017050218&tab=FULLTEXT

Compound X: 1- ( ( (Z) – (1- (2-aminothiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) -1-sulfoazetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylic acid.

[0126]
Step 1: Benzhydryl 1- ( ( (Z) – (1- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylate. To a solution of (Z) -2- ( (1- ( (benzhydryloxy) carbonyl) cyclopropoxy) imino) -2- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) acetic acid (854 mg, 1.59 mmol) prepared according to published patent application US2011/0190254, Intermediate B (324 mg, 1.75 mmol) and HATU (785 mg, 2.07 mmol) in DMF (7.9 mL) , DIPEA was added (832 μL, 4.77 mmol) . After 1 h of stirring, it was poured into water and extracted with EtOAc. Brine was added to the aqueous layer, and it was further extracted with ethyl acetate (EtOAc) (3x) . The combined organic layers were dried over Na 2SO 4 and concentrated in vacuo. The crude residue was purified via silica gel chromatography (0-10%MeOH-DCM) to afford the title compound (1.09 g, 97%) as a beige foam. LCMS: R t = 0.97 min, m/z =705.3 (M+1) Method 2m_acidic.

[0127]
Instead of HATU, a variety of other coupling reagents can be used, such as any of the typical carbodiimides, or CDMT (2-chloro-4, 6-dimethoxy-1, 3, 5-triazine) and N-methylmorpholine to form the amide bond generated in Step 1.

[0128]
Step 2: (3S, 4R) -3- ( (Z) -2- ( (1- ( (benzhydryloxy) carbonyl) cyclopropoxy) imino) -2- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) acetamido) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidine-1-sulfonic acid. Benzhydryl 1- ( ( (Z) – (1- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylate (1.00 g, 1.42 mmol) in DMF (7.0 mL) at 0 ℃ was treated with SO 3·DMF (448 mg, 2.84 mmol) . After 2 h of stirring at rt, the solution was poured into ice-cold brine and extracted with EtOAc (3x) . The combined organic layers were dried over Na 2SO 4 and concentrated in vacuo, affording the title compound (assumed quantitative) as a white solid. LCMS: Rt =0.90 min, m/z = 785.2 (M+1) Method 2m_acidic.

[0129]
Step 3: 1- ( ( (Z) – (1- (2-aminothiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) -1-sulfoazetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylic acid.

[0130]

[0131]
To a solution of (3S, 4R) -3- ( (Z) -2- ( (1- ( (benzhydryloxy) carbonyl) cyclopropoxy) imino) -2- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) acetamido) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidine-1-sulfonic acid (1.10 g, 1.40 mmol) in DCM (1.5 mL) at 0℃, TFA (5.39 mL, 70.0 mmol) was added, and after 10 minutes, the ice bath was removed. Additional TFA (3.24 mL, 42.0 mmol) was added after 1 hr at rt and the solution was diluted with DCM and concentrated in vacuo after an additional 30 min. Optionally, anisole may be added to the TFA reaction to help reduce by-product formation, which may increase the yield of desired product in this step. The crude residue was purified by reverse phase prep HPLC (XSelect CSH, 30 x 100 mm, 5 μm, C18 column; ACN-water with 0.1%formic acid modifier, 60 mL/min) , affording the title compound (178 mg, 23%) as a white powder. LCMS: R t = 0.30 min, m/z = 518.9 (M+1) Method 2m_acidic; 1H NMR (400 MHz, DMSO-d 6) δ 9.27 (d, J = 9.0 Hz, 1H) 6.92 (s, 1H) 5.23 (dd, J = 9.1, 5.7 Hz, 1H) 4.12-4.23 (m, 3H) 3.72-3.62 (m, 2H assumed; obscured by water) 3.61-3.52 (m, 1H assumed; obscured by water) 3.26 (dd, J = 14.5, 5.9 Hz, 1H) 1.36 (s, 4H) . 1H NMR (400 MHz, D 2O) δ 7.23 (s, 1H) , 5.48 (d, J = 5.8 Hz, 1H) , 4.71-4.65 (m, 1H) , 4.44 (t, J = 8.2 Hz, 2H) , 3.89-3.73 (m, 3H) , 3.54 (dd, J = 14.9, 4.9 Hz, 1H) , 1.65-1.56 (m, 2H) , 1.56-1.46 (m, 2H) . The product of this process is amorphous. Compound X can be crystallized from acetone, ethanol, citrate buffer at pH 3 (50 mM) , or acetate buffer at pH 4.5 (50 mM) , in addition to solvents discussed below.

PAPER

Bioorganic & Medicinal Chemistry Letters (2018), 28(4), 748-755.

https://www.sciencedirect.com/science/article/pii/S0960894X18300064

PATENT

WO 2019026004

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019026004&tab=PCTDESCRIPTION

Over the past several decades, the frequency of antimicrobial resistance and its association with serious infectious diseases have increased at alarming rates. The increasing prevalence of resistance among nosocomial pathogens is particularly disconcerting. Of the over 2 million (hospital-acquired) infections occurring each year in the United States, 50 to 60% are caused by antimicrobial-resistant strains of bacteria. The high rate of resistance to commonly used antibacterial agents increases the morbidity, mortality, and costs associated with nosocomial infections. In the United States, nosocomial infections are thought to contribute to or cause more than 77,000 deaths per year and cost approximately $5 to $10 billion annually.

Important causes of Gram-negative resistance include extended-spectrum 13- lactamases (ESBLs), serine carbapenemases (KPCs) and metallo-13-lactamases (for example NDM-1 ) in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis, high-level third-generation cephalosporin (AmpC) 13-lactamase resistance among Enterobacter species and Citrobacter freundii, and multidrug-resistance genes observed in Pseudomonas, Acinetobacter, and Stenotrophomonas. The problem of antibacterial resistance is compounded by the existence of bacterial strains resistant to multiple antibacterials. For example, Klebsiella pneumonia harboring NDM-1 metallo-13- lactamase carries frequently additional serine-13-lactamases on the same plasmid that carries the NDM-1 .

Thus there is a need for new antibacterials, particularly antibacterial compounds that are effective against existing drug-resistant microbes, or are less susceptible to development of new bacterial resistance. Monobactam antibiotic, which is referred to herein as Compound X, is primarily effective against Gram-negative bacteria, including strains that show resistance to other monobactams.

The present invention relates to a process for the preparation of monobactam antibiotic Compound X and intermediates thereof.

More particularly, the present invention relates to a process for the preparation of Compound X

Compound X

also referred to as 1 -(((Z)-(1 -(2-aminothiazol-4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)-1 -sulfoazetidin-3-yl)amino)ethylidene)amino)oxy)cyclopropanecarboxylic acid, or a salt thereof, or a solvate including hydrate thereof.

Patent application number PCT/US2015/02201 1 describes certain monobactam antibiotics. Compound X may be prepared using the method disclosed in PCT/US2015/02201 1 , in particular example 22, and in PCT/CN2016/099482.

A drawback from these processes is that they exhibit a large number of process steps and intermediate nitrogen protection/deprotection steps, reducing the overall yield and efficiency. Furthermore, these processes require several chromatographic purification steps to be carried out in course of the processes. We have found that the preparation of Compound X, as previously prepared on a manufacturing scale, possesses a number of disadvantages, in particular poor handling characteristics.

It would thus be beneficial to develop alternative or improved processes for the production of Compound X that do not suffer from some or all of these disadvantages.

Compound x Compound x

Scheme 1

Preparation of Compound X from Intermediates 22 and 2A

Scheme 3

Examples

The Following examples are merely illustrative of the present disclosure and they should not be considered as limiting the scope of the disclosure in any way, as these examples and other equivalents thereof will become apparent to those skilled in the art in the light of the present disclosure, and the accompanying claims.

Synthesis of Compound 8 (R = benzyl)

1 .50kg oxazolidin-2-one (7b) was charged into the reactor. 7.50kg THF was charged and the stirring started. The mixture was cooled to 10~20°C. 2.18kg potassium fert-butoxide was charged intol 2.00kg THF and stirred to dissolve.

The potassium fert-butoxide solution was added dropwise into the reactor while maintaining the temperature at 10-20 °C. The reaction was stirred for 1 ~2hrs at 10-20 °C after the addition. The solution of 2.36kg methyl-2-chloroacetate (7a) in 3.00kg of THF was added to the reactor while maintaining the temperature at 10-20 °C. The reaction mixture was stirred for 16-18 h at 20-25 °C. The IPC (in process control) showed completion of the reaction. The mixture was centrifuged and the wet cake was washed with 7.50kg THF. The filtrate was concentrated and the crude 7 was provided as reddish brown liquid, which was used for the next step without further purification,

1H NMR (400 MHz, CHLOROFORM- /) δ ppm 3.65 – 3.71 (m, 2 H) 3.74 (s, 3 H) 4.02 (s, 2 H) 4.34 – 4.45 (m, 2 H).

The dried reactor was exchanged with N2 three times. 3.71 kg LiHMDS solution in THF/Hep (1 M) and 1 .30kg THF were charged under nitrogen protection. The stirring was started and the solution was cooled to -70—60 °C. The solution of 0.71 kg benzyl acetate (6) in 5.20 kg THF was added dropwisely at -70— 60 °C, and the resulted mixture was stirred for 1 -1 .5 h after the addition. The solution of 0.65kg 7 in 3.90kg THF was added dropwise while maintaining the temperature at -70—60 °C, then stirred for 30-40 minutes. The reaction mixture was warmed to 20-25 °C and stirring was continued for 0.5-1 .0 h. IPC showed 6 was less than1 .0% (Otherwise, continue the reaction till IPC passes). The reaction mixture was poured into 13.65 kg aqueous citric acid below 10 °C. The mixture was stirred for 15-20 minutes after the addition. Phases were separated and the organic layer was collected. The aqueous layer was extracted with EA (6.50kg * 2). The organic layer was combined, washed by 6.50 kg 28% NaCI solution and dried with 0.65

kg anhydrous MgSC . The mixture was filtered and the wet cake was washed with 1 .30kg EA. The filtrate was concentrated under vacuum to provide crude 8. The crude 8 was stirred in 2.60 kg MTBE at 20-25 °C for 1 -1 .5 h. The mixture was cooled to 0-10 °C and stirred for 1 .5-2.0 h and filtered. The filter cake was washed with 0.65kg pre-cooled MTBE and dried under vacuum (<-0.096Mpa) at 20-25 °C for 12~16hrs till a constant weight to give 513 g of 8 as a white solid, Yield: 45%, HPLC purity 96.4%,1 H NMR (400 MHz, CHLOROFORM-c δ ppm 3.48 – 3.55 (m, 1 H) 3.56 – 3.63 (m, 2 H) 3.66 – 3.74 (m, 1 H) 4.17 – 4.26 (m, 2 H) 4.31 – 4.44 (m, 2H) 5.12 – 5.24 (m, 2 H) 7.30 – 7.44 (m, 5 H).

Synthesis of Compound 9 (R = benzyl)

The dried reactor was charged with 3.75kg HOAc and 1 .50 kg 8. The stirring was started and the reaction mixture was cooled to 0-5 °C. 3.53kg aqueous NaN02 was added dropwise at 0-10 °C, and the reaction mixture was stirred for 15-30 minutes after the addition. IPC showed 8 was less than 0.2%. The reaction mixture was treated with 7.50kg EA and 7.50 kg water. Phases were separated and the organic layer was collected. The aqueous layer was extracted with EA (7.50kg * 2). The organic layers were combined, washed with 7.50 kg 28% NaCI solution, and concentrated under vacuum to provide crude 9. The crude 9 was slurried with 5.25 kg water at 10-20 °C for 3~4hrs, and filtered. The wet cake was washed with 1 .50kg water. The solid was dried under vacuum (<-0.096 Mpa) at 45-50 °C for 5-6 h till a constant weight to give 1 .44 Kg of 9, yield: 86.9%, HPLC purity 92.9%,1H NMR (400 MHz, CHLOROFORM- /) δ ppm 3.60 – 3.76 (m, 2 H) 4.44 (t, J=8.07 Hz, 2 H) 4.60 (s, 2 H) 5.25 – 5.41 (m, 2 H) 7.30 – 7.43 (m, 5 H) 1 1 .62 (br s, 1 H).

Synthesis of Compound 9a (R = benzyl)

9

The dried reactor was charged with 0.58 kg Zn, 4.72kg (Βο Ο, 6.00 kg water, 1 .20 kg NH4CI and 6.00kg THF. The reaction mixture was stirred and heated to 50-55 °C. The solution of 0.60 kg 9 in 4.20kg THF

was added dropwisely while maintaining the temperature at 50-55 °C. The reaction mixture was stirred for 0.5-1 .Ohrs after the addition. IPC showed 9 was less than 0.1 %. The reaction mixture was treated withl .50 kg ethyl acetate and stirred for 15-20 minutes. Phase was separated and the water layer was extracted by1 .50 kg ethyl acetate. The organic layers were combined, washed with 6.00 kg 28% NaCI solution and concentrated under vacuum to provide crude 9a. The crude 9a was stirred with 3.60kg*2 n-heptane to remove excess (Βο Ο. The residue was purified by silica gel chromatography column eluted with ethyl acetate: Heptane= 1 :1 to provide crude 9a solution. The solution was concentrated under reduced pressure to obtain crude 9a. The crude 9a was slurried with 1 .80 kg MTBE for 2.0-3. Ohrs, filtered, and the wet cake was washed with MTBE. The solid was dried under vacuum (<-0.096 Mpa) at 50-55 °C for 16-18 h till a constant weight to give 392 g of 9a as a white solid, Yield: 51 %, HPLC purity 98.1 %,1H NMR (400 MHz, DMSO-cfe) δ ppm 1.17 – 1 .57 (m, 9 H) 3.39 – 3.61 (m, 2 H) 4.20 – 4.45 (m, 3 H) 5.10 – 5.32 (m, 3 H) 5.75 (s, 1 H) 7.38 (br s, 5 H) 7.75 – 7.99 (m, 1 H).

Synthesis of compound (VII) (R = benzyl, X = CI)

9a VII

The dried reactor was charged with 13.0kg HCI in IPA and the stirring was started. 1 .33 kg 9a was charged in portions at 20-25 °C. The mixture was stirred at 20-25 °C for 3-4 h. IPC showed 9a was less than 0.1 %. The reaction solution was concentrated under vacuum 40-45 °C. The residue was treated with 21 .58kg MTBE at 20-25 °C for 3-4 h. The mixture was filtered and the wet cake was washed with 2.60kg MTBE. The solid was dried under vacuum (<-0.096 Mpa) at 45-50 °C for 5-6 h till a constant weight to give 1 .045 Kg of compound VII (R = benzyl, X = CI) as a yellow solid, Yield: 93.7%, HPLC purity 99.2%,1 H NMR (400 MHz, DMSO-cfe) δ ppm 3.16 – 3.74 (m, 3 H) 4.10 – 4.35 (m, 4 H) 5.09 – 5.39 (m, 2 H) 7.27 – 7.60 (m, 5 H) 8.72 (br s, 2 H).

Synthesis of compound (Vile) (R = benzyl)

VII Vile

To an autoclave (3L) were added VII (R = benzyl, X = CI) (100 g, 304.2 mmol, 1 .0 equiv.), DCM (2650 g, 26.5 equiv., w/w) and (S-BINAP)RuCl2 (2.4 g, 3.04 mmol, 0.01 equiv.), successively. Air in the autoclave was replaced with N2 5 times. N2 in the autoclave was was replaced with H2 5 times. The solution was stirred with 250-260 r/min and H2 (2.1 ±0.1 MPa) at 40±5°C for 24 h. The reaction mixture was filtered, and the filter cake was washed with DCM (400 g, 4.0 equiv., w/w). The filter cake was slurried with IPA (785 g, 7.85 equiv., w/w) and H2O (40 g, 0.4 equiv., w/w) overnight (18-20 h). The mixture was filtered. The filter cake was washed with IPA (200 g, 2.0 equiv., w/w) and dried at 45±5°C overnight (18-20 h). Vile (R = benzyl) was obtained as off-white solid, 80.4 g, 79.9% yield, 95.5% purity, 97.6% de, >99.5% ee. 1H NMR (400 MHz, DMSO-cfe) δ ppm 3.34-3.38 (m, 2 H) 3.50-3.52 (m, 1 H) 3.60-3.62 (m, 1 H) 4.18-4.24 (m, 4 H) 5.23 (s, 2H) 6.16 (s, 1 H) 7.32 (m, 5H) 8.74 (s, 1 H).

Alternative synthesis of compound 9a (R = benzyl)

5b

Mg(OtBu)2

To a flask was added 5a (1 .88 g, 12.93 mmol), THF (40 mL), and CDI (2.20 g, 13.58 mmol) at 25 °C. The mixture was stirred for 3 h. To the reaction mixture was added 5b (2.00 g, 6.47 mmol), and Mg(OfBu)2 (2.21 g, 12.93 mmol). The reaction mixture was stirred at 25 °C for 24 h. The reaction mixture was concentrated under vacuum to remove most of the THF solvent. To the concentrated solution was added MTBE (40 mL), followed by addition of an aqueous solution of HCI (1 M, 60mL) to adjust to pH = 2-3. Two phases were separated, and the water phase was extracted with MTBE (20 mL). The combined organic phase was washed with aqueous NaHCC (5%, 50 mL) and brine (20%, 40 mL). The organic phase was concentrated to a weight of -19 g, and a lot of white solid was obtained in the concentration process. The suspension was cooled to 0 °C, and filtered. The filter cake was washed with cold MTBE (5 mL) and dried under vacuum to obtain product 9a (1 .6g, 63% yield).

Synthesis of compound (Vile) (R = benzyl, PG = Cbz)

Vile Vile

To a flask (5 L) were added Vile (R = benzyl) (140 g, 423.2 mmol, LOequiv.), H20 (1273 g, 9.09 equiv., w/w) and toluene (2206 g, 15.76 equiv., w/w). The solution was stirred and cooled to 0-5 °C with ice bath. Then NaHCOa (78.4 g, 933 mmol, 2.22 equiv.) was added and CbzCI (89.6 g, 527 mmol, 1 .24 equiv.) was dropped into the stirring solution, respectively. The solution was stirred at 30±5 °C overnight (18-20 h). Heptane (3612 g, 25.8 equiv., w/w) was added dropwise to the stirring solution over 1 h at 20-30 °C. The mixture was filtered. The filter cake was washed with heptane (280 g, 2.00 equiv., w/w) and MTBE (377 g, 2.69 equiv., w/w), respectively. The filter cake was dried at 45±5°C overnight (18-20 h). Vile (R = benzyl, PG = Cbz) was obtained as an off-white solid, 169.4 g, 93% yield, 96.7% purity, 98% de, >99.5% ee, 1 H NMR (400 MHz, DMSO-cfe) δ ppm 3.23-3.24 (m, 1 H) 3.30 (m, 1 H) 3.51 -3.55 (m, 2 H) 3.99 (s, 1 H) 4.17-4.21 (m, 3 H) 5.02-5.03 (m, 2H) 5.12 (s, 2H) 5.46-5.48 (d, 1 H) 7.33-7.36 (m, 10H) 7.75-7.73 (d, 1 H).

Synthesis of compound (IV) (PG = Cbz)

Vile IV

Vile (R = benzyl) (220 g, 513.5 mmol, 1 .0 equiv.) was dissolved in THF (1464g, 6.65 equiv., w/w). The solution was filtered. The filter cake was washed with THF (488g, 2.22 equiv., w/w). The filtrate (Vile) was collected. To an autoclave (3L) were added the filtrate (Vile). The reactor was cooled down to -75 – -65 °C with dry-ice/EtOH bath, and bubbled with NH3 for not less than 4 h. Then the solution was stirred at 25±5 °C with NH3 (0.5-0.6 MPa) for 24 h. The autoclave was deflated to release NH3. The reaction solution was concentrated with a rotary evaporator to remove THF until the residue was around 440 g. The residue was slurried with EA (2200 g, 10 equiv., w/w) at 70±2 °C, then cooled to 25±5 °C and stirred for 16-18 h. The mixture was filtered. The filter cake was washed with EA (440 g). The filter cake was slurried with EA (1320 g, 6.00 equiv. w/w), and the temperature was raised to 70±2 °C, then cooled to 25±5 °C and stirred for 16-20 h. The mixture was filtered. The filter cake was washed with EA, and dried at 50±5 °C overnight (18-20 h). IV (PG = Cbz) was obtained as off-white solid, 141 g, 81 .5% yield, 99.1 % purity, >99.5% assay, 1H NMR (400 MHz, DMSO-cfe) δ ppm 3.12 – 3.23 (m, 2 H) 3.31 (br s, 1 H) 3.56 (t, J=8.01 Hz, 2 H) 3.88 (quin, J=6.02 Hz, 1 H) 3.93 – 4.03 (m, 1 H) 4.20 (t, J=8.01 Hz, 2 H) 5.02 (s, 2 H) 5.27 (d, J=5.87 Hz, 1 H) 7.12 (s, 1 H) 7.22 – 7.45 (m, 5 H).

Synthesis of compound (III) (PG = Cbz, LG = S02CH3)

IV III

To a flask was added IV (PG = Cbz) (14.00 g, 41 .50 mmol, 1 .00 equiv), and dry 1 , 2-dimethoxyethane (300 mL) under N2. The mixture was stirred at -5°C ~ 0°C for 1 h to obtain a good suspension. MsCI (7.89 g, 68.89 mmol, 5.33 mL, 1 .66 eq) in 1 , 2-dimethoxyethane (20.00 mL) was added dropwise during 30 min, and Et3N (12.60 g, 124.50 mmol, 17.26 mL, 3.00 eq) in 1 , 2-dimethoxyethane (20.00 mL) was added dropwise during 30 min side to side. The reaction mixture was stirred for additional 5 min at -5°C ~ 0°C, and was quenched with water (6 mL). The reaction mixture was concentrated to remove DME. The solid was slurried in water (250 mL) and MTBE (125 mL) for 1 h. The solid was collected by filtration, and then slurried in water (250 mL) for 1 hr. The solid was collected by filtration, and washed with water (25 mL) to give white solid. The solid was slurried in EA (150 mL) and dried in vacuum at 60°C for 24 h to give III (PG = Cbz, LG = SO2CH3) (15.00 g, 36.1 1 mmol, 87.01 % yield), 1H NMR (400 MHz, DMSO-cfe) δ ppm 3.17 (s, 3 H) 3.26 (br d, J=15.04 Hz, 1 H) 3.47 – 3.57 (m, 1 H) 3.64 (br d, J=6.36 Hz, 2 H) 4.22 (br dd, J=17.79, 8.50 Hz, 2 H) 4.50 (br s, 1 H) 4.95 – 5.17 (m, 3 H) 7.21 – 7.56 (m, 5H) 7.43 (s, 1 H) 7.63 – 7.89 (m, 2 H).

Synthesis of compound II (PG = Cbz, LG = SO2CH3, M+ = NBu4+)

O OMs o CISO3H, 2-picoline – ° O ?yO

HN Bu4NHS04< NHCbz

“Cbz

III II

To a flask was added 2-picoline (1 1 .50 g, 12.23 mL) and DMF (10 mL). The solution was cooled to 5 SC, followed by slow addition of chlorosulfonic acid (7.20 g, 4.14 mL). The temperature was increased to 20 SC. Ill (PG = Cbz, LG = SO2CH3) (5.13 g, 12.35 mmol) was added to the reaction mixture. The reaction mixture was heated to 42 SC for 18h. IPC (in process control) showed complete conversion of starting material. The reaction was cooled to 20 SC and dropwise added to a solution of tetrabutylammonium hydrogen sulfate (4.6 g, 13.6 mmol) in the mixed solvents of dichloromethane (100 mL) and water (100 mL) at 5SC. The phases were separated and the water phase was extracted with dichloromethane (2*50mL). The combined organic phase was washed with water (5*100mL). The organic phase was concentrated to dryness and purified by column chromatography (dichloromethane/methanol = 15/1 v/v) to afford II (PG = Cbz, LG = SO2CH3, M+ = NBii4+) (8.4 g, 92.30%), 1 H NMR (400 MHz, CHLOROFORM-c/) δ ppm 0.99 (t, J=7.34 Hz, 12 H) 1 .36 – 1 .50 (m, 8 H) 1 .54 – 1 .76 (m, 8 H) 3.15 (br d, J=8.31 Hz, 2 H) 3.21 – 3.35 (m, 8 H) 3.47 (br dd, J=14.73, 7.27 Hz, 1 H) 3.54 – 3.65 (m, 1 H) 3.67 – 3.81 (m, 2 H) 4.17 – 4.32 (m, 1 H) 4.39 – 4.62 (m, 1 H) 4.74 (br s, 1 H) 5.1 1 (s, 3 H) 5.32 – 5.50 (m, 1 H) 6.47 (br s, 1 H) 7.29 – 7.47 (m, 5 H) 8.69 – 8.94 (m, 1 H).

Synthesis of compound (IA)

A solution of II (PG = Cbz, LG = SO2CH3, M+ = NBu4+) (4.0 g) in dichloromethane (38 mL) was pumped to tube A at rate of 2.0844 mL/min, and a solution of KHCO3 (3.0 g) in water (100 mL) was pumped to tube B at a rate of 1 .4156 mL/min side to side. These two streams were mixed in a cross-mixer then flowed to a tube coil that was placed in an oil bath at 100 °C. The residence time of the mixed stream in the coil was 2 min. The reaction mixture flowed through a back-pressure regulator that was set at ~ 7 bars, and was collected to a beaker. After completion of the collection, two phases was separated. The organic phase was concentrated to dryness. The residue was slurried in ethyl acetate (5 mL). The solid was filtered and the filter cake was dried to give IA (2.6 g, 75%),

1H NMR (400 MHz, CHLOROFORM-c/) δ ppm 1.00 (t, J=7.27 Hz, 12 H) 1 .42 (sxt, J=7.31 Hz, 8 H) 1 .62 (quin, J=7.83 Hz, 8 H) 3.13 – 3.39 (m, 8 H) 3.54 – 3.69 (m, 2 H) 3.81 (dd, J=14.98, 2.51 Hz, 1 H) 3.96 – 4.13 (m, 1 H) 4.22 – 4.47 (m, 3 H) 4.99 – 5.23 (m, 3 H) 6.42 (br d, J=9.29 Hz, 1 H) 7.26 – 7.44 (m, 5 H).

Synthesis of compound 2A

Step 1

To a stirring solution of compound 16b (2 g, 10.14mmol, 1 .0 eq) in DMF (20 ml_) was added CS2CO3 (5.29g, 16.22 mmol, 1 .6 eq), then the resulting solution was stirred at room temperature for 10mins, then compound 16a (5.27g, 20.28mmol, 2eq) was added dropwise to the mixture for 2 minutes, then the resulting solution was stirred for another 2 hours. TLC showed the starting material was consumed completely. The mixture was added with water (60mL) and extracted with MTBE (20mL*3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude was slurried in heptane to give 1 .65 g 16 as a white solid (Yield: 57%), 1H NMR (400 MHz, DMSO-cfe) δ ppm 7.48-7.28 (m, 10 H), 5.00-4.96 (t, J=6.0 Hz, 1 H), 3.81 (s, 3H), 3.44-3.42 (m, 2H), 2.40-2.37 (m, 2H).

Compound 16 (1 g, 2.66mmol, 1 eq) was dissolved in THF (20mL) under Nitrogen, and cooled to -40 °C. NaHMDS (1 .6mL, 2.0M THF solution, 1 .2 eq) was added dropwise. The reaction was stirred for 1 h at -40 °C. HPLC indicated the reaction was finished. The reaction was quenched with 10% Citric acid, extracted with MTBE (25 ml_ x 2). The combined organic layers were washed with brine (30 ml_), dried with Na2S04, filtered and concentrated to give 17 as a yellow solid, which was used for the next step without purification (assay yield: 65%); 1H NMR (400 MHz, DMSO-cfe) δ ppm 7.27-7.13 (m, 10 H), 3.46 (s, 3H), 1 .21 -1 .17(dd, J=7.2, 10.4 Hz, 2H ); 1 .14-1 .1 1 (dd, J=7.2, 10.4 Hz, 2H).

Step 3

Compound 17 (100 mg) was dissolved in methanol (5 mL) and 2.0 M HCI IPAC solution (5 mL). The solution was heated at 45 °C for 3 days. HPLC indicated the reaction was finished. The reaction was cooled to room temperature and was diluted with 10 mL water. The reaction mixture was washed with MTBE (10 mL x 2), organic layer was discarded and the aqueous layer was concentrated to give compound 2A HCI (32 mg, 62% yield), 1 H NMR (400 MHz, DMSO-cfe) δ ppm 3.80-3.44 (br, 4H), 1 .56 (s, 2H), 1 .38 (s, 2H).

Step 4

To a solution of 2A HCI (0.70 g, 4.57 mmol) in methanol (5 mL) was added triethylamine (1 .26 mL, 9.14 mmol) at room temperature. The solution was stirred for 20 min, and the solvent was removed under vacuum. To the residue was added IPAC (10 mL) leading to precipitation. The solid was filtered, and the filtrate was concentrated to provide 2A (0.50g, 94% yield) containing ca. 6 wt% Et3N-HCI.

Synthesis of Compound X from compound of formula (I), (IA)

Compound x

To a flask was charged 21 (1 .00 g, 68.43 wt%, 2.50 mmol) and DMF (10 mL). The suspension was cooled to -20 °C, to which was added diphenylphosphinic chloride (0.52 mL, 2.75 mmol). The solution was stirred at -20 °C for 30 min, followed by addition of a mixed solution of (IA) (1 .52g, 3.00 mmol) and triethylamine (0.52 mL, 3.76 mmol) in DMF (2mL). The reaction mixture was stirred at 20 °C for 20 h, followed by addition of MTBE (20 mL). The reaction mixture was adjusted to pH = 2-3 using aqueous HCI solution (37%). To the mixture was added isopropanol (100 mL). The resulting mixture was stirred for 4 h to obtain a suspension. The suspension was filtered and the filter cake was dried under vacuum to afford crude 22 (1 .17 g). The crude 22 was slurried in a combined solvent of THF/H2O (= 12 mL / 3mL), and filtered to afford 22 (0.744 g, 75 wt% by Q-NMR, 53.3% yield). 1H NMR (400 MHz, DMSO-cfe) δ ppm 3.47 – 3.55 (m, 2 H) 3.59 – 3.63 (m, 2 H) 4.13 – 4.21 (m, 3 H ) 5.05 (dd, J=8.8, 5.6 Hz, 1 H) 8.22 (s, 1 H) 9.73 (d, J=8.7 Hz, 1 H).

To a suspension of 22 (580 mg, 75 wt%, 1 .037 mmol) in DMAC (1 .5 mL) was added 2A (214.3 mg, 85 wt%, 1 .556 mmol). The reaction was stirred at 25 °C for 3 days, and in process control showed 22, Compound X = 4/96, and Z/E = 91 /9. the mixture was slowly added into 15ml acetone to precipitate yellowish solid. The reaction mixture was filtered to afford Compound X (0.7 g, 34 wt% by QNMR, 44% yield).

Synthesis of compound 3 (R2 = CH(Ph)2)

R2 = CH(Ph)2

2-(2-aminothiazol-4-yl)-2-oxoacetic acid (Y) (10.00 g, 47.93 mmol) and compound W (R2 = CH(Ph)2) (13.31 g, 46.98 mmol) were suspended in DMAC (40 mL), followed by addition of triethylamine (5.01 mL, 35.95 mmol). The reaction mixture was stirred at 20 °C for 5 h. HPLC showed completion of the reaction, and Z/E

= 97/3. To the reaction mixture was added water (120 mL) with stirring. The mixture was stirred for 20 min to obtain a suspension. The suspension was filtered and the filter cake was washed with water (50 mL).

The filter cake was slurried in a combined solvent of THF/ethyl acetate (50 mL / 50 mL) at 60 °C and cooled to 20 °C. The solid was filtered and dried at 50 °C for 3 h to get 3 (R2 = CH(Ph)2) (19.5 g, 88% yield). 1H

NMR (400 MHz, DMSO-cfe) δ ppm 1.37 -1 .42 (m, 2 H) 1 .44 – 1 .49 (m, 2 H) 6.87 (s, 1 H) 6.94 (s, 1 H) 7.22

– 7.30 (m, 6 H) 7.45 – 7.49 (m, 4 H).

Alternative Synthesis of Compound X from compound of formula (I), (IA)

Compound x

IA (40.14 g, 62.63 mmol) was dissolved in methanol (200 ml_), followed by addition of Pd/C (10%, 1 .1 g). The reaction mixture was maintained under hydrogen atmosphere (1 -2 bar) at 20 °C for 24 h. In process control showed completion of the reaction. The reaction mixture was filtered. The filtrate was concentrated to give an oil of IB (M+ = NBu4+) (58.20 g, 55 wt% by Q-NMR, 100% yield). 1 H NMR (400 MHz, DMSO-cfe) δ ppm 0.93 (t, J=7.3 Hz, 12 H) 1 .23 – 1 .36 (m, 8 H) 1 .57 (m, 8 H) 2.99 – 3.28 (m, 8 H) 3.37 (dd, J=14.3, 7.5 Hz, 1 H) 3.65 – 3.70 (m, 3 H) 3.84 – 3.88 (m, 1 H) 4.08 (d, J=5.6 Hz, 1 H) 4.18 – 4.22 (m, 2 H).

3 (R2 = CH(Ph)2) (0.95 g, 2.17 mmol) was dissolved in THF (20 ml_). To the solution was added /V-methyl morpholine (0.77 g, 7.60 mmol) and 2-chloro-4,6-dimethoxy-1 ,3,5-triazine (0.57 g, 3.26 mmol). The reaction mixture was stirred at 20 °C for 1 h followed by addition of IB (M+ = NBu +) (2.70 g, 48.98 wt%, 2.61 mmol). The reaction was stirred at 20 °C for 5 h. In process control showed completion of the reaction. To the reaction mixture was added ethyl acetate (20 ml_). The organic phase was washed with brine (10 ml_). Solvent was removed. Acetone (40ml) was added to dissolve residue. TFA (1 .24 g, 10.86 mmol) dissolved in acetone (3 ml) was added slowly. The white solid was filtered and washed by acetone (10 ml) two times. Dried at 40 °C for 5h to get compound 4 (R2 = CH(Ph)2). 1 H NMR (400 MHz, DMSO-cfe) δ ppm 1 .49 – 1 .55 (m, 4 H) 3.27 (dd, J=14.4, 6.2 Hz, 1 H) 3.49 – 3.65 (m, 2 H) 3.71 (dd, J=14.4, 6.2 Hz, 1 H) 4.04 – 4.10 (m, 1 H) 4.07 (dd, J=16.0, 8.6 Hz, 1 H) 4.17 (dd, J=1 1 .8, 6.0 Hz, 1 H) 5.28 (dd, J=9.0, 5.7 Hz, 1 H) 6.88 (s, 1 H) 7.03 (s, 1 H) 7.18 – 7.32 (m, 6 H) 7.43 (m, 4 H) 9.45 (d, J=9.0 Hz, 1 H).

Crude 4 (R2 = CH(Ph)2) (2.13 g) was dissolved in dichloromethane (20 ml_). The solution was cooled to 0 °C. To the solution was added anisole (0.68 ml_, 6.24 mmol) and trifluoroacetic acid (2.16 ml_, 28.08 mmol). The reaction was warmed to 20 °C, and stirred for 15 h. In process control showed completion of the

reaction. The aqueous phase was separated and added to acetone (40 mL) to obtain a suspension. The suspension was filtered to afford Compound X (0.98 g, 54.5% yield over two steps). 1 H NMR (400 MHz, DMSO-c/e) δ ppm 1.40 (m, 4 H) 3.26 (dd, J=14.4, 6.0 Hz, 1 H) 3.54 – 3.69 (m, 3 H) 4.14 – 4.21 (m, 3 H) 5.25 (dd, J= 8.9, 5.7 Hz, 1 H) 7.02 (s, 1 H) 9.38 (d, J=9.0 Hz, 1 H).

REF

Synthesis and optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228
57th Intersci Conf Antimicrob Agents Chemother (ICAAC) (June 1-5, New Orleans) 2017, Abst SATURDAY-297

//////////////LYS228, LYS 228, BOS-228, LYS-228, monobactam, Novartis, phase II,  Boston Pharmaceuticals, complicated urinary tract infection, complicated intraabdominal infections,  fast track, Qualified Infectious Disease Product, QIDP,

Nc1nc(cs1)\C(=N\OC2(CC2)C(=O)O)\C(=O)N[C@H]3[C@@H](CN4CCOC4=O)N(C3=O)S(=O)(=O)O

FDA approves novel treatment Oxbryta (voxelotor) to target abnormality in sickle cell disease


Today, the U.S. Food and Drug Administration granted accelerated approval to Oxbryta (voxelotor) for the treatment of sickle cell disease (SCD) in adults and pediatric patients 12 years of age and older.
“Today’s approval provides additional hope to the 100,000 people in the U.S., and the more than 20 million globally, who live with this debilitating blood disorder,” said Acting FDA Commissioner Adm. Brett P. Giroir, M.D. “Our scientific investments have brought us to a point where we have many more tools available in the battle against sickle cell disease, which presents daily challenges for those living with it. We remain committed to raising the profile of this disease as a public health priority and to approving new therapies that are proven to be safe and effective. Together with improved provider education, patient empowerment, and improved care delivery systems, these newly approved drugs have the potential to immediately impact people living with SCD.”

Sickle cell disease is a lifelong, inherited blood disorder in which red blood cells are abnormally shaped (in a crescent, or “sickle” shape), which restricts the flow in blood vessels and limits oxygen delivery to the body’s tissues, leading to severe pain and organ damage. It is also characterized by severe and chronic inflammation that worsens vaso-occlusive crises during which patients experience episodes of extreme pain and organ damage. Nonclinical studies have demonstrated that Oxbryta inhibits red blood cell sickling, improves red blood cell deformability (ability of a red blood cell to change shape) and improves the blood’s ability to flow.

“Oxbryta is an inhibitor of deoxygenated sickle hemoglobin polymerization, which is the central abnormality in sickle cell disease,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Oncologic Diseases in the FDA’s Center for Drug Evaluation and Research. “With Oxbryta, sickle cells are less likely to bind together and form the sickle shape, which can cause low hemoglobin levels due to red blood cell destruction. This therapy provides a new treatment option for patients with this serious and life-threatening condition.”

Oxbryta’s approval was based on the results of a clinical trial with 274 patients with sickle cell disease. In the study, 90 patients received 1500 mg of Oxbryta, 92 patients received 900 mg of Oxbryta and 92 patients received a placebo. Effectiveness was based on an increase in hemoglobin response rate in patients who received 1500 mg of Oxbryta, which was 51.1% for these patients compared to 6.5% in the placebo group.

Common side effects for patients taking Oxbryta were headache, diarrhea, abdominal pain, nausea, fatigue, rash and pyrexia (fever).
Oxbryta was granted Accelerated Approval, which enables the FDA to approve drugs for serious conditions to fill an unmet medical need based on a result that is reasonably likely to predict a clinical benefit to patients. Further clinical trials are required to verify and describe Oxbryta’s clinical benefit.
The FDA granted this application Fast Track designation. Oxbryta also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases. The FDA granted the approval of Oxbryta to Global Blood Therapeutics.

/////////fda 2019, Fast Track designation,  Oxbryta, Orphan Drug designation, voxelotor, Global Blood Therapeutics, sickle cell disease

http://s2027422842.t.en25.com/e/es?s=2027422842&e=279688&elqTrackId=376c7bc788024cd5a73d955f2e3dcbdc&elq=28d3a5d8573843f29b6ae4275802f1c8&elqaid=10429&elqat=1

Selinexor


Skeletal formula of selinexor

Selinexor.png

Selinexor

セリネクソル

KPT-330

UNII-31TZ62FO8F

(Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N‘-pyrazin-2-ylprop-2-enehydrazide

Formula
C17H11F6N7O
CAS
1393477-72-9
Mol weight
443.306

FDA, APPROVED 2019/7/3, Xpovio

CAS : 1393477-72-9 (free base)   1421923-86-5 (E-isomer)   1621865-82-4 (E-isomer)   Unknown (HCl)

Treatment of cancer, Antineoplastic, Nuclear export inhibitor

Selinexor (INN, trade name Xpovio; codenamed KPT-330) is a selective inhibitor of nuclear export used as an anti-cancer drug. It works by quasi-irreversibly binding to exportin 1 and thus blocking the transport of several proteins involved in cancer-cell growth from the cell nucleus to the cytoplasm, which ultimately arrests the cell cycle and leads to apoptosis.[1] It is the first drug with this mechanism of action.[2][3]

Selinexor was granted accelerated approval by the U.S. Food and Drug Administration in July 2019, for use as a drug of last resort in people with multiple myeloma. In clinical trials, it was associated with a high incidence of severe side effects, including low platelet counts and low blood sodium levels.[3][4]

Selinexor is an orally available, small molecule inhibitor of CRM1 (chromosome region maintenance 1 protein, exportin 1 or XPO1), with potential antineoplastic activity. Selinexor modifies the essential CRM1-cargo binding residue cysteine-528, thereby irreversibly inactivates CRM1-mediated nuclear export of cargo proteins such as tumor suppressor proteins (TSPs), including p53, p21, BRCA1/2, pRB, FOXO, and other growth regulatory proteins. As a result, this agent, via the approach of selective inhibition of nuclear export (SINE), restores endogenous tumor suppressing processes to selectively eliminate tumor cells while sparing normal cells. CRM1, the major export factor for proteins from the nucleus to the cytoplasm, is overexpressed in a variety of cancer cell types.

Selinexor has been used in trials studying the treatment of AML, Glioma, Sarcoma, Leukemia, and Advanced, among others.

 Selinexor, also known as KPT-330, is an orally bioavailable, potent and selective XPO1/CRM1 Inhibitor. Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia. Selinexor potentiates the antitumor activity of gemcitabine in human pancreatic cancer through inhibition of tumor growth, depletion of the antiapoptotic proteins, and induction of apoptosis. Selinexor has strong activity against primary AML cells while sparing normal stem and progenitor cells.

SYN

Medical uses

Selinexor is restricted for use in combination with the steroid dexamethasone in people with relapsed or refractory multiple myelomawhich has failed to respond to at least four or five other therapies (so-called “quad-refractory” or “penta-refractory” myeloma),[5] for whom no other treatment options are available.[3][4] It is the first drug to be approved for this indication.[6]

Adverse effects

In the clinical study used to support FDA approval, selinexor was associated with high rates of pancytopenia, including leukopenia(28%), neutropenia (34%, severe in 21%), thrombocytopenia (74%, severe in 61% of patients), and anemia (59%).[4][7] The most common non-hematological side effects were gastrointestinal reactions (nausea, anorexia, vomiting, and diarrhea), hyponatremia (low blood sodium levels, occurring in up to 40% of patients), and fatigue.[7][8] More than half of all patients who received the drug developed infections, including fatal cases of sepsis.[7] However, these data are from an open-label trial, and thus cannot be compared to placebo or directly attributed to treatment.

Mechanism of action

Schematic illustration of the Ran cycle of nuclear transport. Selinexor inhibits this process at the nuclear export receptor (upper right).

Like other so-called selective inhibitors of nuclear export (SINEs), selinexor works by binding to exportin 1 (also known as CRM1). CRM1 is a karyopherin which performs nuclear transport of several proteins, including tumor suppressorsoncogenes, and proteins involved in governing cell growth, from the cell nucleus to the cytoplasm; it is often overexpressed and its function misregulated in several types of cancer.[1] By restoring nuclear transport of these proteins to normal, SINEs lead to a buildup of tumor suppressors in the nucleus of malignant cells and reduce levels of oncogene products which drive cell proliferation. This ultimately leads to cell cycle arrest and death of cancer cells by apoptosis.[1][2][7] In vitro, this effect appeared to spare normal (non-malignant) cells.[1][8]

Because CRM1 is a pleiotropic gene, inhibiting it affects many different systems in the body, which explains the high incidence of adverse reactions to selinexor.[2] Thrombocytopenia, for example, is a mechanistic and dose-dependent effect, occurring because selinexor causes a buildup of the transcription factor STAT3 in the nucleus of hematopoietic stem cells, preventing their differentiation into mature megakaryocytes (platelet-producing cells) and thus slowing production of new platelets.[2]

Chemistry

Selinexor is a fully synthetic small-molecule compound, developed by means of a structure-based drug design process known as induced-fit docking. It binds to a cysteine residue in the nuclear export signal groove of exportin 1. Although this bond is covalent, it is not irreversible.[1]

History

Selinexor was developed by Karyopharm Therapeutics of Newton, Massachusetts, a pharmaceutical company devoted entirely to the development of drugs that target nuclear transport. It was approved by the FDA on July 3, 2019, on the basis of a single uncontrolled clinical trial. The decision was controversial, and overruled the previous recommendation of an FDA Advisory Panel which had voted 8–5 against approving the drug, due to concerns about efficacy and toxicity.[3]

Research

Under the codename KPT-330, selinexor was tested in several preclinical animal models of cancer, including pancreatic cancerbreast cancernon-small-cell lung cancerlymphomas, and acute and chronic leukemias.[9] In humans, early clinical trials (phase I) have been conducted in non-Hodgkin lymphomablast crisis, and a wide range of advanced or refractory solid tumors, including colon cancerhead and neck cancermelanomaovarian cancer, and prostate cancer.[9] Compassionate use in patients with acute myeloid leukemia has also been reported.[9]

The pivotal clinical trial which served to support approval of selinexor for people with relapsed/refractory multiple myeloma was an open-label study of 122 patients known as the STORM trial.[7] In all of the enrolled patients, selinexor was used as fifth-line or sixth-line therapy after conventional chemotherapytargeted therapy with bortezomibcarfilzomiblenalidomidepomalidomide, and a monoclonal antibody (daratumumab or isatuximab)[5]; nearly all had also undergone hematopoietic stem cell transplantation to no effect.[7] The overall response rate was 25%, and no patients had a complete response.[7] However, the response rate was higher in patients with high-risk myeloma (cytogenetic abnormalities associated with a worse prognosis).[5] The median time to progression was 2.3 months overall and 5 months in patients who responded to the drug.[2]

As of 2019, phase I/II and III trials are ongoing,[3][9] including the use of selinexor in other cancers and in combinations with other drugs used for multiple myeloma.[2]

PATENT

WO 2013019561

WO 2013019548

US 9079865

PATENT

WO 2016025904 A

https://patents.google.com/patent/WO2016025904A1/tr

International Publication No. WO 2013/019548 describes a series of compounds that are indicated to have inhibitory activity against chromosomal region maintenance 1 (CRM1, also referred to as exportin 1 or XPO1) and to be useful in the treatment of disorders associated with CRM1 activity, such as cancer. (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N’-(pyrazin-2-yl)acrylohydrazide (also referred to as selinexor) is one of the compounds disclosed in International Publication No. WO 2013/019548. Selinexor has the chemical structure shown in Structural Formula I:

Example 1. Preparation of Selinexor Lot No.1305365 (Form A).

[00274] Selinexor for Lot No. 1305365 was made in accordance with the following reaction scheme:

[00275] A solution of propane phosphonic acid anhydride (T3P®, 50% in ethyl acetate, 35Kg) in THF (24.6Kg) was cooled to about -40 °C. To this solution was added a solution of KG1 (13.8Kg) and diisopropylethylamine (12.4Kg) in tetrahydrofuran (THF, 24.6Kg). The resulting mixture was stirred at about -40°C for approximately 2.5 hours.

[00276] In a separate vessel, KJ8 (4.80Kg) was mixed with THF (122.7Kg), and the resulting mixture cooled to about -20°C. The cold activated ester solution was then added to the KJ8 mixture with stirring, and the reaction was maintained at about -20°C. The mixture was warmed to about 5°C, water (138.1Kg) was added and the temperature adjusted to about 20°C. After agitating for about an hour, the lower phase was allowed to separate from the mixture and discarded. The upper layer was diluted with ethyl acetate (EtOAc). The organic phase was then washed three times with potassium phosphate dibasic solution (~150Kg), then with water (138.6Kg).

[00277] The resulting organic solution was concentrated under reduced pressure to 95L, EtOAc (186.6Kg) was added and the distillation repeated to a volume of 90L. Additional EtOAc (186.8Kg) was added and the distillation repeated a third time to a volume of 90L. The batch was filtered to clarify, further distilled to 70L, then heated to about 75°C, and slowly cooled to 0 to 5°C. The resulting slurry was filtered and the filter cake washed with a mixture of EtOAc (6.3Kg) and toluene (17.9Kg) before being dried in a vacuum oven to provide selinexor designated Lot No. 1305365 (Form A).

Example 2. Preparation of Selinexor Lot No.1341-AK-109-2 (Form A).

[00278] The acetonitrile solvate of selinexor was prepared in accordance with Example 6.

[00279] The acetonitrile solvate of selinexor (2.7g) was suspended in a mixture of isopropanol (IPA, 8mL) and water (8mL), and the resulting mixture heated to 65 to 70 °C to effect dissolution. The solution was cooled to 45 °C, and water (28mL) was added over 15 minutes, maintaining the temperature between 40 and 45 °C. The slurry was cooled to 20 to 25 °C over an hour, then further cooled to 0 to 5 °C and held at that temperature for 30 minutes before being filtered. The filter cake was washed with 20% v/v IPA in water and the product dried under suction overnight, then in vacuo (40°C).

Example 3. Preparation of SelinexorSelinexorSelinexor Lot No. PC-14-005 (Form A).

[00280] The acetonitrile solvate of selinexor (Form D) was prepared in accordance with the procedure described in Example 6.

[00281] The acetonitrile solvate of selinexor (1.07Kg) was suspended in a mixture of IPA (2.52Kg) and water (3.2Kg) and the mixture heated to 70 to 75 °C to dissolve. The temperature was then adjusted to 40 to 45 °C and held at that temperature for 30 minutes. Water (10.7Kg) was added while maintaining the temperature at 40 to 45 °C, then the batch was cooled to 20 to 25 °C and agitated at that temperature for 4 hours before being further cooled to 0 to 5 °C. After a further hour of agitation, the slurry was filtered and the filter cake washed with a cold mixture of IPA (0.84Kg) and water (4.28Kg) before being dried.

Example 4. Preparation of SelinexorSelinexorSelinexor Lot No. PC-14-009 (Form A).

[00282] The acetonitrile solvate of selinexor (Form D) was prepared in accordance with the procedure described in Example 6.

[00283] The acetonitrile solvate of selinexor (1.5Kg) was suspended in IPA (3.6Kg) and water (4.5Kg) and warmed to 37 to 42 °C with gentle agitation. The suspension was agitated at that temperature for 4 hours, and was then cooled to 15 to 20 °C over 1 hour. Water (15.1Kg) was added, maintaining the temperature, then the agitation was continued for 1 hour and the batch was filtered. The filter cake was washed with a mixture of IPA (1.2Kg) and water (6Kg), then dried under a flow of nitrogen.

Example 5. Preparation of Selinexor Lot Nos.1339-BS-142-1, 1339-BS-142-2 and PC-14-008 (Form A).

[00284] A reactor, under nitrogen, was charged with KG1 (1Kg, 1.0 Eq), KJ8 (0.439 Kg, 1.4 Eq) and MeTHF (7L, 7 parts with respect to KG1). Diisopropylethylamine (0.902Kg, 2.45 Eq with respect to KG1) was added to the reaction mixture at -20 °C to -25 °C with a MeTHF rinse. To the reaction mixture, 50% T3P® in ethyl acetate (2.174Kg, 1.2 Eq with respect to KG1) was then charged, maintaining the temperature at -20 °C to -25 °C with a MeTHF rinse. After the completion of the addition, the reaction mixture was stirred briefly

and then warmed to 20 °C to 25 °C. Upon completion, the reaction mixture was washed first with water (5L, 5 parts with respect to KG1) and then with dilute brine (5L, 5 parts with respect to KG1). The organic layer was concentrated by vacuum distillation to a volume of 5 L (5 parts with respect to KG1), diluted with acetonitrile (15L, 15 parts with respect to KG1) at approximately 40 °C and concentrated again (5L, 5 parts with respect to KG1). After solvent exchange to acetonitrile, the reaction mixture was then heated to approximately 60 °C to obtain a clear solution. The reaction mixture was then cooled slowly to 0-5 °C, held briefly and filtered. The filter cake was washed with cold acetonitrile (2L, 5 parts with respect to KG1) and the filter cake was then dried under a stream of nitrogen to provide the acetonitrile solvate of selinexor (Form D) as a slightly off-white solid.

[00285] Form D of selinexor (0.9Kg) was suspended in IPA (2.1Kg, 2.7L, 3 parts with respect to Form D) and water (2.7Kg, 2.7L, 3 parts with respect to Form D) and warmed to approximately 40 °C. The resulting suspension was agitated for about 4 hours, selinexor, cooled to approximately 20 °C, and diluted with additional water (9Kg, 10 parts with respect to Form D). The mixture was stirred for a further 4-6 hours, then filtered, and the cake washed with a mixture of 20% IPA and water (4.5L, 5 parts with respect to Form D). The filter cake was then dried under vacuum to provide selinexor designated Lot No. PC-14-008 as a white crystalline powder with a >99.5% a/a UPLC purity (a/a=area to area of all peaks; UPLC-ultra performance HPLC).

Example 6. Preparation of Selinexor Lot No.1405463 (Form A).

[00286] Selinexor Lot No. 1405463 was prepared in accordance with the following reaction scheme:

 .

[00287] A reactor was charged with KG1 (15.8Kg), KJ8 (6.9Kg) and MeTHF (90Kg). Diisopropylethylamine (14.2Kg) was added to the reaction mixture over approximately 35 minutes at about -20 °C. Following the addition of the diisopropylethylamine, T3P® (50%

solution in EtAOc, 34.4Kg) was added maintaining the temperature at -20 °C. The mixture stirred to complete the reaction first at -20 °C, then at ambient temperature.

[00288] Upon completion of the reaction, water (79Kg) was added over about 1 hour. The layers were separated and the organic layer was washed with a mixture of water (55Kg) and brine (18Kg), The mixture was filtered, and the methyl-THF/ethyl acetate in the mixture distillatively replaced with acetonitrile (volume of approximately 220L). The mixture was warmed to dissolve the solids, then slowly cooled to 0 to 5 °C before being filtered. The filter cake was washed with acetonitrile to provide the acetonitrile solvate of

selinexorSelinexorSelinexor (Form D).

[00289] The acetonitrile solvate of selinexorSelinexorSelinexor was dried, then mixed with isopropanol (23Kg) and water (55Kg). The slurry was warmed to about 38 °C and held at that temperature for approximately 4 hours before being cooled to 15 to 20 °C. Water (182Kg) was added. After a further 5 hours of agitation, the mixture was filtered and the filter cake washed with a mixture of isopropanol (14Kg) and water (73Kg), before being dried under vacuum (45 °C). The dried product was packaged to provide

selinexorSelinexorSelinexor Lot No. 1405463 (Form A).

Example 7. Polymorphism Studies of Selinexor.

[00290] A comprehensive polymorphism assessment of selinexor was performed in a range of different solvents, solvent mixtures and under a number of experimental conditions based on the solubility of selinexor. Three anhydrous polymorphs of

selinexorSelinexorSelinexor were observed by XRPD investigation, designated Form A, Form B and Form C. Form A is a highly crystalline, high-melting form, having a melting point of 177 °C, and was observed to be stable from a physico-chemical point of view when exposed for 4 weeks to 25 °C/97% relative humidity (RH) and to 40 °C/75% RH. A solvated form of selinexor was also observed in acetonitrile, designated Form D. A competitive slurry experiment confirmed Form A as the stable anhydrous form under the conditions investigated, except in acetonitrile, in which solvate formation was observed. It was further found that in acetonitrile, below 50 °C, only Form D is observed, at 50 °C both Form A and Form D are observed, and at 55 °C, Form A is observed .

PATENT

CN 106831731

https://patents.google.com/patent/CN106831731A/en

Selinexor is an orally bioavailable selective nuclear export inhibitors, 2012 for the first time in clinical, so far carried out a total of 21 trials, indications include chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphatic leukemia, prostate cancer, melanoma, non-small cell lung cancer, glioma, neuroblastoma into, gynecological cancer, diffuse large B-cell lymphoma, squamous cell carcinoma, colorectal cancer and the like. May 2014, FDA granted orphan drug designation Selinexor treatment of acute myeloid leukemia and diffuse large B-cell lymphoma, in June 2014, EMA is also granted orphan drug designation Selinexor treatment of both diseases. January 2015, received FDA orphan drug to treat multiple myeloma identified.

[0003] Currently, the synthesis process has been disclosed, the following reaction equation:

Figure CN106831731AD00041

[0006] wherein the compound is 5 Selinexor drug.

[0007] In this method, however, easy to produce Intermediate 1-2 double bond is easily reversed when synthetically produced from trans impurities, in addition to more difficult to impact yield; Intermediate 3 Intermediate 4 Synthesis APIs 5 when required ultra-low temperature, and the product was purified by column required, only a yield of 20%.

SUMMARY

[0008] The object of the present invention to provide a novel compound Selinexor drug synthesis of 5, in order to solve technical problems.

[0009] – novel synthetic method of Se species I inexor drug, comprising the steps of:

Synthesis [0010] A, Compound 7

[0011] Compound 6, dichloromethane and ethyl acetate mixture, stirred and dissolved, compound 4, T3P (n-propyl phosphoric anhydride) and DIPEA (N, N- diisopropylethylamine) at a low temperature; the reaction was stirred for 25-35min at a low temperature, dichloromethane and water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0012] B, Synthesis of Compound 8

[0013] the compound obtained in Step 7, and mixed sodium iodide acetic acid, warmed to 110-120 ° C, the reaction 2.5-3.5h; After completion of the reaction, the system cooled to room temperature, water and dichloromethane were added, stirred for 8 after -15min, standing layered organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, was dissolved in DMF (dimethyl fumarate) to give compound in DMF 8;

Synthesis [0014] C, of Compound 5

[0015] Compound 1, DBAC0 (triethylenediamine), the DMF mixed and dissolved with stirring, dropwise adding to the reaction system of the compound obtained in DMF step 8, after the addition was complete, stirring was continued for 3-4 hours; the reaction after completion, water and ethyl acetate were added to the system, the organic phase is evaporated to dryness and petroleum ether and recrystallized from ethyl acetate to give compound 5.

[0016] Preferably, said step A, the low temperature is 0-2 ° C.

[0017] Preferably, said step B in DMF, the crude compound 8 concentration of less than 1%.

[0018] The novel synthetic methods of the present invention Selinexor drug, the chemical equation is as follows:

Figure CN106831731AD00051

[0020] The present invention has the following advantages: novel synthetic method Selinexor drug of the present invention to overcome the conventional synthesis process, is easy to produce trans impurities, more difficult in addition, the influence the yield and the need for ultra-low temperature, and the product requires problems purified by column, the yield is very low, reducing the synthetic steps, increased yield, there is provided a new process for the synthesis of the drug Selinexor.

[0021] In addition to the above-described objects, features and advantages of the present invention as well as other objects, features and advantages. Below the invention will be described in further detail present.

Example 1

[0024] – novel synthetic method of Se species I inexor drug, comprising the steps of:

Synthesis [0025] A, Compound 7

[0026] 50ml three □ flask, 15ml of dichloromethane and 0.2g compound 6,15ml ethyl acetate, stirred and dissolved, was added 0.3g of compound 4 and 3gT3P, 0.75gDIPEA at 0 ° C; the system at 0 ° C the reaction was stirred for 30min, 50ml of dichloromethane and 30ml of water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0027] B, Synthesis of Compound 8

[0028] 50ml three-necked flask, added the compound obtained in Step 7,40ml of glacial acetic acid and 1.38g of sodium iodide was heated to 115. (:, The reaction 3H; After completion of the reaction, cooled to room temperature system, the system will be transferred to 500ml flask, 50ml of water was added and IOOml dichloromethane, after stirring IOmin, standing separation, the organic phase was washed with saturated sodium bicarbonate and saturated washed with sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, was dissolved in IOmL DMF to give DMF solution of compound 8;

Synthesis [0029] C, of Compound 5

[0030] After 50ml 3-necked flask was added 0.2g compound 1,0.24gDBAC0,20mlDMF, dissolved with stirring, dropwise adding to the reaction system in DMF compound obtained in Step 8, after the addition was complete, stirring continued for 3.5 hours; after completion of the reaction, 20ml water was added to the system and 50ml ethyl acetate, the organic phase is evaporated to dryness and petroleum ether to ethyl acetate to give 0.158g of compound 5, yield 50.9%.

[0031] Example 2

[0032] – new type Se Iinexor drug synthesis, comprising the steps of:

Synthesis [0033] A, Compound 7

[0034] 50ml three □ flask, 15ml of dichloromethane and 0.2g compound 6,15ml ethyl acetate, stirred and dissolved, was added 0.3g of compound 4 and 3gT3P, 0.75gDIPEA at 1 ° C; system at 1 ° C the reaction was stirred for 35min, 50ml of dichloromethane and 30ml of water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0035] B, Synthesis of Compound 8

Three-neck flask [0036] 50ml of addition of the compound obtained in Step 7,40ml glacial acetic acid and 1.38g of sodium iodide was heated to 120. (:, The reaction for 2.5 h; After completion of the reaction, cooled to room temperature system, the system will be transferred to 500ml flask, 60ml water and 120ml dichloromethane was added, after stirring for 15min, allowed to stand for separation, the organic phase was washed with saturated sodium bicarbonate and washed with saturated sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, 12mLDMF was dissolved in DMF to give a solution of compound 8;

Synthesis [0037] C, of Compound 5

[0038] After 50ml 3-necked flask was added 0.2g compound 1,0.24gDBAC0,20mlDMF, dissolved with stirring, dropwise adding to the reaction system of the compound obtained in DMF step 8, after the addition was complete, stirring continued for 3 hours; after completion of the reaction, 25ml of water and 50ml of ethyl acetate was added to the system, the organic phase is evaporated to dryness and petroleum ether to ethyl acetate to give 0.152g of compound 5, yield 49.0% billion

[0039] Example 3

[0040] – novel synthetic method of Se species I inexor drug, comprising the steps of:

Synthesis [0041] A, Compound 7

Three [0042] 50ml of flask, 15ml of dichloromethane and 0.2g compound 6,15ml ethyl acetate, stirred and dissolved, was added 0.3g of compound 4 and 3gT3P, 0.75gDIPEA at 2 ° C; system from 0 ° C the reaction was stirred for 25min, 40ml of dichloromethane and 35ml of water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0043] B, Synthesis of Compound 8

Three-neck flask [0044] 50ml of addition of the compound obtained in Step 7,35ml glacial acetic acid and 1.38g of sodium iodide was heated to 110. (:, The reaction for 3.5 h; After completion of the reaction, cooled to room temperature system, the system will be transferred to 500ml flask, 50ml of water was added and dichloromethane IOOml After Smin of stirring, standing separation, the organic phase was washed with saturated sodium bicarbonate and washed with saturated sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, was dissolved in IOmL DMF to give DMF solution of compound 8;

Synthesis [0045] C, of Compound 5

[0046] 50ml three-neck flask was added 0.2g compound 1,0.24gDBA⑶, 20mlDMF, and dissolved with stirring, dropwise adding to the reaction system of the compound obtained in DMF step 8, after the addition was complete, stirring was continued for 4 hours; after completion of the reaction, 20ml of water and 40ml ethyl acetate were added to the system, the organic phase is evaporated to dryness and petroleum ether to ethyl acetate to give 0.155g of compound 5, yield 49.9% billion

PATENT

WO 2017118940

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017118940&tab=PCTDESCRIPTION

The drug compound having the adopted name “Selinexor” has chemical name:(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-IH-l,2,4-triazol-1 -yl)-N’-(pyrazin-2yl) acrylohydrazide as below.

Figure imgf000003_0001

Selinexor (KPT-330) is a first-in-class, oral Selective Inhibitor of Nuclear Export / SINE™ compound. Selinexor functions by binding with and inhibiting the nuclear export protein XP01 (also called CRM1 ), leading to the accumulation of tumor suppressor proteins in the cell nucleus. This reinitiates and amplifies their tumor suppressor function and is believed to lead to the selective induction of apoptosis in cancer cells, while largely sparing normal cells. Over 1 ,200 patients have been treated with Selinexor in company and investigator-sponsored Phase 1 and Phase 2 clinical trials in advanced hematologic malignancies and solid tumors. Karyopharm has initiated four later-phase clinical trials of Selinexor, including one in older patients with acute myeloid leukemia (SOPRA), one in patients with Richter’s transformation (SIRRT), one in patients with diffuse large B-cell lymphoma (SADAL) and a single-arm trial of Selinexor and lose-dose dexamethasone in patients with multiple myeloma (STORM). Patients may receive a twice-weekly combination of Selinexor in combination with low dose dexamethasone. Randomized 1 :1 , Selinexor will be dosed either at 60mg + dexamethasone or at 100 mg + dexamethasone.

US 8999996 B2 discloses Selinexor and a pharmaceutically acceptable salt thereof, pharmaceutical compositions and use for treating disorders associated with CRM1 activity. Further, it discloses preparative methods for the preparation of compounds disclosed therein including Selinexor by reacting (Z)-3-(3- (3,5-

bis(trifluoromethyl)phenyl)-IH-l,2,4-triazol-l-yl)acrylic acid in 1 :1 CH2CI2: AcOEt with 2-Hydrazinopyrazine at -40 °C followed by addition of T3P[Propylphosphonic anhydride] (50%) and DIPEA. After 30 minutes, the reaction mixture was concentrated and the crude oil was purified by preparative TLC using 5% MeOH in CH2CI2 as mobile phase (under ammonia atmosphere) to afford 40 mg of Selinexor with purity: 95.78%. However, it is not disclosed about the nature of the compound obtained therein.

WO 2016025904 A1 discloses various crystalline forms of Selinexor namely Form A, Form B, Form C, Form D, compositions and MoU thereof for the treatment of disorder associated with CRM1 activity and their preparative processes.

Prior art process for the preparation of Selinexor suffers from disadvantages interms of process such as the use of lengthy procedures to practice and resulting in low yields, which may not be viable at industrial scale. Synthetic product obtained therein has very low purity and contains significant amounts of unreacted starting materials and trans-isomer of Selinexor, which are further purified by time consuming and expensive chromatographic separations leading to loss of yield. Hence, there remains a need for improved process for the preparation of Selinexor which is industrially viable and reproducible. Particularly, it is desirable to have a process avoiding purification steps still meeting desired pharmaceutical quality.

EXAMPLES

Example-1 : Preparation of isopropyl (Z)-3-(3-(3,5-bis(trifluoromethyl) phenyl)-1 H- -triazol-1 -yl)acrylate

Figure imgf000061_0001

3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazole (250 g) was dissolved in tetrahydrofuran (2 I) under nitrogen atmosphere at 27°C and cooled to -5°C. 1 ,4- diazabicyclo[2.2.2]octane (DABCO, 1 99.5 g) was added to the reaction mixture at -5°C and stirred at the same temperature for 40 minutes. Isopropyl (Z)-3- iodoacrylate (234.8 g in 500 mL of tetrahydrofuran) was added drop wise to the reaction mixture in 1 hour 1 0 minutes at -5°C and stirred at the same temperature for 2 hours. After the completion of the reaction, the reaction mixture was added to ice cold water (2 I) and separated the organic layer. The aqueous layer was extracted with ethyl acetate (2 x 1 I). The combined organic layer was washed with brine solution (1 I) and dried over sodium sulphate. The dried solution was evaporated completely under vacuum at 40°C to obtain crude product with HPLC purity of 93.53% The crude product was triturated with hexane (700 mL) and stirred for 20 minutes at -30°C and filtered the solid. Trituration of crude product with hexane was repeated for three times and dried under vacuum to obtain the title compound with HPLC purity of 97.46% and trans-isomer content of 0.66%. Yield: 297 g Example-2: Preparation of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4- triazol-1 -yl)acr lic acid.

Figure imgf000062_0001

To a mixture of tetrahydrofuran (300 mL) and water (300 mL), Isopropyl (Z)-3-(3- (3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylate (30 g) was added and cooled to 0°C. Lithium hydroxide monohydrate (16.03 g) under cooling condition at 0°C was added to the reaction mixture and stirred the reaction mixture at same temperature for 7 hours. After completion of the reaction, 2 N HCI (180 mL) was added to adjust the pH of the reaction mixture to 2 and extracted it with ethyl acetate (300 mL). Organic layer was dried over sodium sulphate and evaporated under vacuum at 40°C. The crude compound was stirred with hexane (150 mL) and filtered the solid. Dried the compound under vacuum at 40°C for 0.5 hour to obtain the title compound with HPLC purity of 97.25% with trans-isomer content of 3 %. Yield: 24 g

Example-3: Purification of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4- tria

Figure imgf000062_0002

A mixture of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylic acid (24 g) and acetone (240 mL) was stirred for complete dissolution at 30°C. Dicyclohexyl amine (1 5 mL) was added drop wise for 20 minutes under stirring at the same temperature. Acetone (50 mL) was added to the reaction mixture and stirred for 2 hours at 27°C. Filtered the solid and washed with hot acetone (150 mL) and dried in vacuum drier at 30°C for 1 hour to obtain the Dicyclohexyl amine salt of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylic acid. To the above salt, dichloromethane (150 mL) and water (1 00 mL) was added and stirred for complete dissolution at 30and adjusted the pH of the solution with 2 N sulphuric acid (100 mL) to 2. Filtered the reaction mixture and washed the product with water (1 00 mL) and then with hexane (150 mL). The solid was dried under vacuum at 40°C for 0.5 hour to obtain title compound with HPLC purity 99.98% with no detectable content of trans-isomer. Yield: 17 g

Example-4: Preparation of Selinexor

Figure imgf000063_0001

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylic acid (10 g) was combined with a mixture of acetonitrile (1 00 mL) and ethyl acetate (50 mL) then added the 2-hydrazinylpyrazine (3.76 g) and stirred for 5 min. Reaction mixture was cooled to 0°C and diisopropyl ethyl amine (16.63 ml) and then Propylphosphonic anhydride (T3P, 33.31 mL) was added at 0°C and stirred the reaction mixture for 2.5 hours at the same temperature. After completion of the reaction, the reaction mixture was quenched with cold water (100 mL) and extracted the product with ethyl acetate (2 x 150 mL). The combined organic layer was dried over sodium sulphate and evaporated the solvent under vacuum at 40°C to obtain the crude product as yellow syrup. The obtained crude product was combined with dichloromethane (1 00 mL) and filtered the solid and washed with dichloromethane (2 x 50 mL). The solid was dried under vacuum at 40°C to obtain the title compound with purity by HPLC of 99.86%. Yield : 7 g

PATENT
WO 2018129227

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7: Azmi AS, Li Y, Muqbil I, Aboukameel A, Senapedis W, Baloglu E, Landesman Y, Shacham S, Kauffman MG, Philip PA, Mohammad RM. Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration. Oncotarget. 2017 Jul 17;8(47):82144-82155. doi: 10.18632/oncotarget.19285. eCollection 2017 Oct 10. PubMed PMID: 29137251; PubMed Central PMCID: PMC5669877.

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9: Body S, Esteve-Arenys A, Miloudi H, Recasens-Zorzo C, Tchakarska G, Moros A, Bustany S, Vidal-Crespo A, Rodriguez V, Lavigne R, Com E, Casanova I, Mangues R, Weigert O, Sanjuan-Pla A, Menéndez P, Marcq B, Picquenot JM, Pérez-Galán P, Jardin F, Roué G, Sola B. Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells. Sci Rep. 2017 Oct 24;7(1):13946. doi: 10.1038/s41598-017-14222-1. PubMed PMID: 29066743; PubMed Central PMCID: PMC5654982.

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11: Soung YH, Kashyap T, Nguyen T, Yadav G, Chang H, Landesman Y, Chung J. Selective Inhibitors of Nuclear Export (SINE) compounds block proliferation and migration of triple negative breast cancer cells by restoring expression of ARRDC3. Oncotarget. 2017 May 18;8(32):52935-52947. doi: 10.18632/oncotarget.17987. eCollection 2017 Aug 8. PubMed PMID: 28881784; PubMed Central PMCID: PMC5581083.

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Selinexor
Skeletal formula of selinexor
Clinical data
Trade names Xpovio
Pregnancy
category
  • Known to cause fetal harm
Routes of
administration
Oral
Legal status
Legal status
Pharmacokinetic data
Protein binding 95%
Metabolism Hepatic oxidation, glucuronidation, and conjugation, by CYP3A4UGTand GST
Elimination half-life 6–8 h
Identifiers
CAS Number
PubChem CID
DrugBank
UNII
Chemical and physical data
Formula C17H11F6N7O
Molar mass 443.313 g·mol−1
3D model (JSmol)

Karyopharm’s Selinexor Receives Fast Track Designation from FDA for the Treatment of Patients with Penta-Refractory Multiple Myeloma

NEWTON, Mass., April 10, 2018 (GLOBE NEWSWIRE) — Karyopharm Therapeutics Inc. (Nasdaq:KPTI), a clinical-stage pharmaceutical company, today announced that the U.S. Food and Drug Administration (FDA) has granted Fast Track designation to the Company’s lead, oral Selective Inhibitor of Nuclear Export (SINE) compound selinexor for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy.  The FDA’s statement, consistent with the design of Karyopharm’s Phase 2b STORM study, noted that the three prior lines of therapy include regimens comprised of an alkylating agent, a glucocorticoid, Velcade® (bortezomib), Kyprolis® (carfilzomib), Revlimid® (lenalidomide), Pomalyst® (pomalidomide) and Darzalex® (daratumumab).  In addition, the patient’s disease must be refractory to at least one proteasome inhibitor (Velcade or Kyprolis), one immunomodulatory agent (Revlimid or Pomalyst), glucocorticoids and to Darzalex, as well as to the most recent therapy.  The Company expects to report top-line data from the STORM study at the end of April 2018.

ChemSpider 2D Image | selinexor | C17H11F6N7O

The FDA’s Fast Track program facilitates the development of drugs intended to treat serious conditions and that have the potential to address unmet medical needs.  A drug program with Fast Track status is afforded greater access to the FDA for the purpose of expediting the drug’s development, review and potential approval.  In addition, the Fast Track program allows for eligibility for Accelerated Approval and Priority Review, if relevant criteria are met, as well as for Rolling Review, which means that a drug company can submit completed sections of its New Drug Application (NDA) for review by FDA, rather than waiting until every section of the NDA is completed before the entire application can be submitted for review.

“The designation of Fast Track for selinexor represents important recognition by the FDA of the potential of this anti-cancer agent to address the significant unmet need in the treatment of patients with penta-refractory myeloma that has continued to progress despite available therapies,” said Sharon Shacham, PhD, MBA, Founder, President and Chief Scientific Officer of Karyopharm.  “We are fully committed to working closely with the FDA as we continue development of this potential new, orally-administered treatment for patients who currently have no other treatment options of proven benefit.”

About the Phase 2b STORM Study

In the multi-center, single-arm Phase 2b STORM (Selinexor Treatment oRefractory Myeloma) study, approximately 122 patients with heavily pretreated, penta-refractory myeloma receive 80mg oral selinexor twice weekly in combination with 20mg low-dose dexamethasone, also dosed orally twice weekly.  Patients with penta-refractory disease are those who have previously received an alkylating agent, a glucocorticoid, two immunomodulatory drugs (IMiDs) (Revlimid® (lenalidomide) and Pomalyst® (pomalidomide)), two proteasome inhibitors (PIs) (Velcade® (bortezomib) and Kyprolis® (carfilzomib)), and the anti-CD38 monoclonal antibody Darzalex® (daratumumab), and their disease is refractory to at least one PI, at least one IMiD, Darzalex, glucocorticoids and their most recent anti-myeloma therapy.  Overall response rate is the primary endpoint of the study, with duration of response and clinical benefit rate being secondary endpoints.  All responses will be adjudicated by an Independent Review Committee (IRC).

About Selinexor

Selinexor (KPT-330) is a first-in-class, oral Selective Inhibitor of Nuclear Export (SINE) compound. Selinexor functions by binding with and inhibiting the nuclear export protein XPO1 (also called CRM1), leading to the accumulation of tumor suppressor proteins in the cell nucleus. This reinitiates and amplifies their tumor suppressor function and is believed to lead to the selective induction of apoptosis in cancer cells, while largely sparing normal cells. To date, over 2,300 patients have been treated with selinexor, and it is currently being evaluated in several mid- and later-phase clinical trials across multiple cancer indications, including in multiple myeloma in a pivotal, randomized Phase 3 study in combination with Velcade® (bortezomib) and low-dose dexamethasone (BOSTON), in combination with low-dose dexamethasone (STORM) and as a potential backbone therapy in combination with approved therapies (STOMP), and in diffuse large B-cell lymphoma (SADAL), and liposarcoma (SEAL), among others. Additional Phase 1, Phase 2 and Phase 3 studies are ongoing or currently planned, including multiple studies in combination with one or more approved therapies in a variety of tumor types to further inform Karyopharm’s clinical development priorities for selinexor. Additional clinical trial information for selinexor is available at www.clinicaltrials.gov.

About Karyopharm Therapeutics

Karyopharm Therapeutics Inc. (Nasdaq:KPTI) is a clinical-stage pharmaceutical company focused on the discovery, development and subsequent commercialization of novel first-in-class drugs directed against nuclear transport and related targets for the treatment of cancer and other major diseases. Karyopharm’s SINE compounds function by binding with and inhibiting the nuclear export protein XPO1 (or CRM1). In addition to single-agent and combination activity against a variety of human cancers, SINE compounds have also shown biological activity in models of neurodegeneration, inflammation, autoimmune disease, certain viruses and wound-healing. Karyopharm, which was founded by Dr. Sharon Shacham, currently has several investigational programs in clinical or preclinical development.

/////////Selinexor, FDA 2019, セリネクソル  ,KPT-330, KPT 330 , KPT330,  AML, Glioma, Sarcoma, Leukemia, Fast Track, CANCER

FDA approves first treatment Ruzurgi (amifampridine) for children with Lambert-Eaton myasthenic syndrome, a rare autoimmune disorder


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FDA approves first treatment Ruzurgi (amifampridine)  for children with Lambert-Eaton myasthenic syndrome, a rare autoimmune disorder

The U.S. Food and Drug Administration today approved Ruzurgi (amifampridine) tablets for the treatment of Lambert-Eaton myasthenic syndrome (LEMS) in patients 6 to less than 17 years of age. This is the first FDA approval of a treatment specifically for pediatric patients with LEMS. The only other treatment approved for LEMS is only approved for use in adults.

“We continue to be committed to facilitating the development and approval of treatments for rare diseases, particularly those in children,” said Billy Dunn, M.D., director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research. “This approval will provide a much-needed treatment option for pediatric patients with LEMS who have significant weakness and fatigue that can often cause great difficulties with daily activities.”

LEMS is a rare autoimmune disorder that affects the connection between nerves and muscles and causes weakness and other symptoms in affected patients. In people with LEMS, the body’s own immune system attacks the neuromuscular junction (the connection between nerves and muscles) and disrupts the ability of nerve cells to send signals to muscle cells. LEMS may be associated with …

May 06, 2019

The U.S. Food and Drug Administration today approved Ruzurgi (amifampridine) tablets for the treatment of Lambert-Eaton myasthenic syndrome (LEMS) in patients 6 to less than 17 years of age. This is the first FDA approval of a treatment specifically for pediatric patients with LEMS. The only other treatment approved for LEMS is only approved for use in adults.

“We continue to be committed to facilitating the development and approval of treatments for rare diseases, particularly those in children,” said Billy Dunn, M.D., director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research. “This approval will provide a much-needed treatment option for pediatric patients with LEMS who have significant weakness and fatigue that can often cause great difficulties with daily activities.”

LEMS is a rare autoimmune disorder that affects the connection between nerves and muscles and causes weakness and other symptoms in affected patients. In people with LEMS, the body’s own immune system attacks the neuromuscular junction (the connection between nerves and muscles) and disrupts the ability of nerve cells to send signals to muscle cells. LEMS may be associated with other autoimmune diseases, but more commonly occurs in patients with cancer such as small cell lung cancer, where its onset precedes or coincides with the diagnosis of cancer. LEMS can occur at any age. The prevalence of LEMS specifically in pediatric patients is not known, but the overall prevalence of LEMS is estimated to be three per million individuals worldwide.

Use of Ruzurgi in patients 6 to less than 17 years of age is supported by evidence from adequate and well-controlled studies of the drug in adults with LEMS, pharmacokinetic data in adult patients, pharmacokinetic modeling and simulation to identify the dosing regimen in pediatric patients and safety data from pediatric patients 6 to less than 17 years of age.

The effectiveness of Ruzurgi for the treatment of LEMS was established by a randomized, double-blind, placebo-controlled withdrawal study of 32 adult patients in which patients were taking Ruzurgi for at least three months prior to entering the study. The study compared patients continuing on Ruzurgi to patients switched to placebo. Effectiveness was measured by the degree of change in a test that assessed the time it took the patient to rise from a chair, walk three meters, and return to the chair for three consecutive laps without pause. The patients that continued on Ruzurgi experienced less impairment than those on placebo. Effectiveness was also measured with a self-assessment scale for LEMS-related weakness that evaluated the feeling of weakening or strengthening. The scores indicated greater perceived weakening in the patients switched to placebo.

The most common side effects experienced by pediatric and adult patients taking Ruzurgi were burning or prickling sensation (paresthesia), abdominal pain, indigestion, dizziness and nausea. Side effects reported in pediatric patients were similar to those seen in adult patients. Seizures have been observed in patients without a history of seizures. Patients should inform their health care professional immediately if they have signs of hypersensitivity reactions such as rash, hives, itching, fever, swelling or trouble breathing.

The FDA granted this application Priority Review and Fast Track designations. Ruzurgi also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.

The FDA granted the approval of Ruzurgi to Jacobus Pharmaceutical Company, Inc.

https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-children-lambert-eaton-myasthenic-syndrome-rare-autoimmune-disorder?utm_campaign=050619_PR_FDA%20approves%20first%20treatment%20for%20children%20with%20LEMS&utm_medium=email&utm_source=Eloqua

/////////////////FDA 2019, Ruzurgi, amifampridine,  Lambert-Eaton myasthenic syndrome, LEMS,  RARE DISEASES, CHILDREN, Jacobus Pharmaceutical Company, Priority Review,  Fast Track designations, Orphan Drug designation

Beperminogene perplasmid, ベペルミノゲンペルプラスミド


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4851ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
4901agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
4951aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
5001cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
5051tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
5101gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
5151ccttttgctg gccttttgct cacatgttct t

Beperminogene perplasmid

ベペルミノゲンペルプラスミド

HGF plasmid

  • DNA (human hepatocyte growth factor plasmid pVAX1 cDNA)
  • DNA (plasmid pVAX1HGF/MGBI)
  • AMG-0001
    DS-992

Nucleic Acid Sequence

Sequence Length: 51811342 a 1223 c 1314 g 1302 t

APPROVED, japan 2019, Collategene, 2019/3/29

Antiparkinsonian, Angiogenesis inducing agent

CAS: 627861-07-8

  • Originator AnGes MG
  • Developer AnGes MG; Osaka University Hospital
  • Class Antiparkinsonians; Gene therapies; Ischaemic heart disorder therapies; Vascular disorder therapies
  • Mechanism of Action Angiogenesis inducing agents; Gene transference; Hepatocyte growth factor expression stimulants
  • Available For Licensing Yes – Ischaemic heart disorders; Lymphoedema; Parkinson’s disease
  • Registered Peripheral arterial disorders
  • Phase I/II Lymphoedema
  • No development reported Arteriosclerosis obliterans; Ischaemic heart disorders; Parkinson’s disease; Thromboangiitis obliterans
  • 26 Mar 2019 Registered for Peripheral arterial disorders in Japan (IM)
  • 21 Feb 2019 The Pharmaceutical Affairs and Food Sanitation Council recommends conditional and time-limited approval of beperminogene perplasmid for the improvement of ulcers associated with chronic peripheral arterial disease
  • 21 Feb 2019 AnGes plans a clinical study to assess the efficacy of beperminogene perplasmid in improvement of pain at rest in chronic peripheral arterial disorders
  • In 2010, the product received fast track designation in the U.S. for the treatment of critical limb ischemia

HGF Plasmid (Beperminogene Perplasmid)Critical Limb Ischemia (Arteriosclerosis Obliterans & Buerger’s Disease) AMG0001 Injection, JAPAN AND US  ALLIANCE Mitsubishi Tanabe Pharma

PATENT

WO 2017126488

US 20170283446

Expert Review of Cardiovascular Therapy (2014), 12(10), 1145-1156.

////////////Beperminogene perplasmid,  japan 2019, ベペルミノゲンペルプラスミド , AnGes MG, Osaka University Hospital, Critical Limb Ischemia, Arteriosclerosis Obliterans,  Buerger’s Disease, AMG0001, AMG-0001, DS-992 , HGF plasmid ,  fast track designation

Cavosonstat (N-91115)


Cavosonstat.png

Cavosonstat (N-91115)

CAS 1371587-51-7

C16H10ClNO3, 299.71 g/mol

UNII-O2Z8Q22ZE4, O2Z8Q22ZE4, NCT02589236; N91115-2CF-05; SNO-6

3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

Treatment of Chronic Obstructive Pulmonary Diseases (COPD), AND Cystic fibrosis,  Nivalis Therapeutics, phase 2

The product was originated at Nivalis Therapeutics, which was acquired by Alpine Immune Sciences in 2017. In 2018, Alpine announced the sale and transfer of global rights to Laurel Venture Capital for further product development.

In 2016, orphan drug and fast track designations were granted to the compound in the U.S. for the treatment of cystic fibrosis.

  • Originator N30 Pharma
  • Developer Nivalis Therapeutics
  • Class Small molecules
  • Mechanism of Action Cystic fibrosis transmembrane conductance regulator modulators; Glutathione-independent formaldehyde dehydrogenase inhibitors; Nitric oxide stimulants
  • Orphan Drug Status Yes – Cystic fibrosis
  • 20 Jul 2018 Laurel Venture Capital acquires global rights for cavosonstat from Alpine Immune Sciences
  • 20 Jul 2018 Laurel Venture Capital plans a phase II trial for Asthma
  • 24 Jun 2018 Biomarkers information updated

 Cavosonstat, alos known as N91115) an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators.

cavosonstat-n91115Cavosonstat (N91115) was an experimental therapy being developed by Nivalis Therapeutics. Its primary mechanism of action was to inhibit the S-nitrosoglutathione reductase (GSNOR) enzyme and to stabilize cystic fibrosis transmembrane regulator (CFTR) protein activity. A press release published in February announced the end of research for this therapy in cystic fibrosis (CF) patients with F508del mutations. The drug, which did not meet primary endpoints in a Phase 2 trial, had been referred to as the first of a new class of compounds that stabilizes the CFTR activity.

History of cavosonstat

During preclinical studies, N91115 (later named cavosonstat) demonstrated an improvement in cystic fibrosis transmembrane regulator (CFTR) stability.

Phase 1 study was initiated in 2014 to evaluate the safety, tolerability, and pharmacokinetics (how a drug is processed in the body) of the drug in healthy volunteers. Later that year, the pharmacokinetics of the drug were assessed in another Phase 1 trial involving CF patients with F508del mutation suffering from pancreatic insufficiency. Results were presented a year later by Nivalis, revealing good tolerance and safety in study participants.

A second, much smaller Phase 2 study (NCT02724527) assessed cavosonstat as an add-on therapy to ivacaftor (Kalydeco). This double-blind, randomized, placebo-controlled study included 19 participants who received treatment with cavosonstat (400 mg) added to Kalydeco or with placebo added to Kalydeco. The primary objective was change in lung function from the study’s start to week 8. However, the treatment did not demonstrate a benefit in lung function measures or in sweat chloride reduction at eight weeks (primary objective). As a result, Nivalis decided not to continue development of cavosonstat for CF treatment.

The U.S. Food and Drug Administration (FDA) had granted cavosonstat both fast track and orphan drug designations in 2016.

How cavosonstat works

The S-nitrosoglutathione (GSNO) is a signaling molecule that is present in high concentrations in the fluids of the lungs or muscle tissues, playing an important role in the dilatation of the airways. GSNO levels are regulated by the GSNO reductase (GSNOR) enzyme, altering CFTR activity in the membrane. In CF patients, GSNO levels are low, causing a loss of the airway function.

Cavosonstat’s mechanism of action is achieved through GSNOR inhibition, which was presumed to control the deficient CFTR protein. Preclinical studies showed that cavosonstat restored GSNO levels.

PATENT
WO 2012083165

The chemical compound nitric oxide is a gas with chemical formula NO. NO is one of the few gaseous signaling molecules known in biological systems, and plays an important role in controlling various biological events. For example, the endothelium uses NO to signal surrounding smooth muscle in the walls of arterioles to relax, resulting in vasodilation and increased blood flow to hypoxic tissues. NO is also involved in regulating smooth muscle proliferation, platelet function, and neurotransmission, and plays a role in host defense. Although NO is highly reactive and has a lifetime of a few seconds, it can both diffuse freely across membranes and bind to many molecular targets. These attributes make NO an ideal signaling molecule capable of controlling biological events between adjacent cells and within cells.

[0003] NO is a free radical gas, which makes it reactive and unstable, thus NO is short lived in vivo, having a half life of 3-5 seconds under physiologic conditions. In the presence of oxygen, NO can combine with thiols to generate a biologically important class of stable NO adducts called S-nitrosothiols (SNO’s). This stable pool of NO has been postulated to act as a source of bioactive NO and as such appears to be critically important in health and disease, given the centrality of NO in cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA, 89:7674-7677 (1992)). Protein SNO’s play broad roles in the function of cardiovascular, respiratory, metabolic, gastrointestinal, immune, and central nervous system (Foster et al., Trends in Molecular Medicine, 9 (4): 160-168, (2003)). One of the most studied SNO’s in biological systems is S-nitrosoglutathione (GSNO) (Gaston et al., Proc. Natl. Acad. Sci. USA 90: 10957-10961 (1993)), an emerging key regulator in NO signaling since it is an efficient trans-nitrosating agent and appears to maintain an equilibrium with other S-nitrosated proteins (Liu et al., Nature, 410:490-494 (2001)) within cells. Given this pivotal position in the NO-SNO continuum, GSNO provides a therapeutically promising target to consider when NO modulation is pharmacologically warranted.

[0004] In light of this understanding of GSNO as a key regulator of NO homeostasis and cellular SNO levels, studies have focused on examining endogenous production of GSNO and SNO proteins, which occurs downstream from the production of the NO radical by the nitric oxide synthetase (NOS) enzymes. More recently there has been an increasing understanding of enzymatic catabolism of GSNO which has an important role in governing available concentrations of GSNO and consequently available NO and SNO’s.

[0005] Central to this understanding of GSNO catabolism, researchers have recently identified a highly conserved S-nitrosoglutathione reductase (GSNOR) (Jensen et al., Biochem J., 331 :659-668 (1998); Liu et al., (2001)). GSNOR is also known as glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), alcohol dehydrogenase 3 (ADH-3) (Uotila and Koivusalo, Coenzymes and Coƒactors., D. Dolphin, ed. pp. 517-551 (New York, John Wiley & Sons, (1989)), and alcohol dehydrogenase 5 (ADH-5). Importantly GSNOR shows greater activity toward GSNO than other substrates (Jensen et al., (1998); Liu et al., (2001)) and appears to mediate important protein and peptide denitrosating activity in bacteria, plants, and animals. GSNOR appears to be the major GSNO-metabolizing enzyme in eukaryotes (Liu et al., (2001)). Thus, GSNO can accumulate in biological compartments where GSNOR activity is low or absent (e.g. , airway lining fluid) (Gaston et al., (1993)).

[0006] Yeast deficient in GSNOR accumulate S-nitrosylated proteins which are not substrates of the enzyme, which is strongly suggestive that GSNO exists in equilibrium with SNO-proteins (Liu et al., (2001)). Precise enzymatic control over ambient levels of GSNO and thus SNO-proteins raises the possibility that GSNO/GSNOR may play roles across a host of physiological and pathological functions including protection against nitrosative stress wherein NO is produced in excess of physiologic needs. Indeed, GSNO specifically has been implicated in physiologic processes ranging from the drive to breathe (Lipton et al., Nature, 413: 171-174 (2001)) to regulation of the cystic fibrosis transmembrane regulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001)), to regulation of vascular tone, thrombosis, and platelet function (de Belder et al., Cardiovasc Res.; 28(5):691-4 (1994)), Z. Kaposzta, et al., Circulation; 106(24): 3057 – 3062, (2002)) as well as host defense (de Jesus-Berrios et al., Curr. Biol., 13: 1963-1968 (2003)). Other studies have found that GSNOR protects yeast cells against nitrosative stress both in vitro (Liu et al., (2001)) and in vivo (de Jesus-Berrios et al., (2003)).

[0007] Collectively, data suggest GSNO as a primary physiological ligand for the enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizes GSNO and

consequently reduces available SNO’s and NO in biological systems (Liu et al., (2001)), (Liu et al., Cell, 116(4), 617-628 (2004)), and (Que et al., Science, 308, (5728): 1618-1621 (2005)). As such, this enzyme plays a central role in regulating local and systemic bioactive NO. Since perturbations in NO bioavailability has been linked to the pathogenesis of numerous disease states, including hypertension, atherosclerosis, thrombosis, asthma, gastrointestinal disorders, inflammation, and cancer, agents that regulate GSNOR activity are candidate therapeutic agents for treating diseases associated with NO imbalance.

[0008] Nitric oxide (NO), S-nitrosoglutathione (GSNO), and S-nitrosoglutathione reductase (GSNOR) regulate normal lung physiology and contribute to lung pathophysiology. Under normal conditions, NO and GSNO maintain normal lung physiology and function via their anti-inflammatory and bronchodilatory actions. Lowered levels of these mediators in pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD) may occur via up-regulation of GSNOR enzyme activity. These lowered levels of NO and GSNO, and thus lowered anti-inflammatory capabilities, are key events that contribute to pulmonary diseases and which can potentially be reversed via GSNOR inhibition.

[0009] S-nitrosoglutathione (GSNO) has been shown to promote repair and/or regeneration of mammalian organs, such as the heart (Lima et al., 2010), blood vessels (Lima et al., 2010) skin (Georgii et al., 2010), eye or ocular structures (Haq et al., 2007) and liver (Prince et al., 2010). S-nitrosoglutathione reductase (GSNOR) is the major catabolic enzyme of GSNO. Inhibition of GSNOR is thought to increase endogenous GSNO.

[0010] Inflammatory bowel diseases (IBD’s), including Crohn’s and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal (GI) tract, in which NO, GSNO, and GSNOR can exert influences. Under normal conditions, NO and GSNO function to maintain normal intestinal physiology via anti-inflammatory actions and maintenance of the intestinal epithelial cell barrier. In IBD, reduced levels of GSNO and NO are evident and likely occur via up-regulation of GSNOR activity. The lowered levels of these mediators contribute to the pathophysiology of IBD via disruption of the epithelial barrier via dysregulation of proteins involved in maintaining epithelial tight junctions. This epithelial barrier dysfunction, with the ensuing entry of micro-organisms from the lumen, and the overall lowered anti-inflammatory capabilities in the presence of lowered NO and GSNO, are key events in IBD progression that can be potentially influenced by targeting GSNOR.

[0011] Cell death is the crucial event leading to clinical manifestation of

hepatotoxicity from drugs, viruses and alcohol. Glutathione (GSH) is the most abundant redox molecule in cells and thus the most important determinant of cellular redox status. Thiols in proteins undergo a wide range of reversible redox modifications during times of exposure to reactive oxygen and reactive nitrogen species, which can affect protein activity. The maintenance of hepatic GSH is a dynamic process achieved by a balance between rates of GSH synthesis, GSH and GSSG efflux, GSH reactions with reactive oxygen species and reactive nitrogen species and utilization by GSH peroxidase. Both GSNO and GSNOR play roles in the regulation of protein redox status by GSH.

[0012] Acetaminophen overdoses are the leading cause of acute liver failure (ALF) in the United States, Great Britain and most of Europe. More than 100,000 calls to the U.S. Poison Control Centers, 56,000 emergency room visits, 2600 hospitalizations, nearly 500 deaths are attributed to acetaminophen in this country annually. Approximately, 60% recover without needing a liver transplant, 9% are transplanted and 30% of patients succumb to the illness. The acetaminophen-related death rate exceeds by at least three-fold the number of deaths due to all other idiosyncratic drug reactions combined (Lee, Hepatol Res 2008; 38 (Suppl. 1):S3-S8).

[0013] Liver transplantation has become the primary treatment for patients with fulminant hepatic failure and end-stage chronic liver disease, as well as certain metabolic liver diseases. Thus, the demand for transplantation now greatly exceeds the availability of donor organs, it has been estimated that more than 18 000 patients are currently registered with the United Network for Organ Sharing (UNOS) and that an additional 9000 patients are added to the liver transplant waiting list each year, yet less than 5000 cadaveric donors are available for transplantation.

[0014] Currently, there is a great need in the art for diagnostics, prophylaxis, ameliorations, and treatments for medical conditions relating to increased NO synthesis and/or increased NO bioactivity. In addition, there is a significant need for novel compounds, compositions, and methods for preventing, ameliorating, or reversing other NO-associated disorders. The present invention satisfies these needs.

Schemes 1-6 below illustrate general methods for preparing analogs.

[00174] For a detailed example of General Scheme 1 see Compound IV-1 in Example 1.

[00175] For a detailed example of Scheme 2, A conditions, see Compound IV-2 in Example 2.

[00176] For a detailed example of Scheme 2, B conditions, see Compound IV-8 in Example 8.

[00177] For a detailed example of Scheme 3, see Compound IV-9 in Example 9.

[00178] For a detailed example of Scheme 4, Route A, see Compound IV-11 in Example 11.

[00179] For a detailed example of Scheme 4, Route B, see Compound IV-12 in Example 12.

[00180] For a detailed example of Scheme 5, Compound A, see Compound IV-33 in Example 33.

[00181] For a detailed example of Scheme 5, Compound B, see Compound IV-24 in Example 24.

[00182] For a detailed example of Scheme 5, Compound C, see Compound IV-23 in Example 23.

Example 8: Compound IV-8: 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

[00209] Followed Scheme 2, B conditions:

[00210] Step 1: Synthesis of 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid:

[00211] A mixture of 2-chloro-6-methoxyquinoline (Intermediate 1) (200 mg, 1.04 mmol), 4-carboxy-2-chlorophenylboronic acid (247 mg, 1.24 mmol) and K2CO3(369 mg, 2.70 mmol) in DEGME / H2O (7.0 mL / 2.0 mL) was degassed three times under N2 atmosphere. Then PdCl2(dppf) (75 mg, 0.104 mmol) was added and the mixture was heated to 110 °C for 3 hours under N2 atmosphere. The reaction mixture was diluted with EtOAc (100 mL) and filtered. The filtrate was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid (150 mg, yield 46%) as a yellow solid, which was used for the next step without further purification.

[00212] Step 2: Synthesis of Compound IV-8: To a suspension of 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid (150 mg, 0.479 mmol) in anhydrous CH2Cl2 (5 mL) was added AlCl3 (320 mg, 2.40 mmol). The reaction mixture was refluxed overnight. The mixture was quenched with saturated NH4Cl (10 mL) and the aqueous layer was extracted with CH2Cl2 / MeOH (v/v=10: l, 30 mL x3). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give the crude product, which was purified by prep-HPLC (0.1% TFA as additive) to give 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (25 mg, yield 18%). 1H NMR (DMSO, 400 MHz): δ 10.20 (brs, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.10-8.00 (m, 2H), 7.95 (d, J = 9.2 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.38 (dd, J = 6.4, 2.8 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), MS (ESI): m/z 299.9 [M+H]+.

PATENT
WO 2012048181
PATENT
WO 2012170371

REFERENCES

1: Donaldson SH, Solomon GM, Zeitlin PL, Flume PA, Casey A, McCoy K, Zemanick ET,
Mandagere A, Troha JM, Shoemaker SA, Chmiel JF, Taylor-Cousar JL.
Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic
fibrosis adults homozygous for F508DEL-CFTR. J Cyst Fibros. 2017 Feb 13. pii:
S1569-1993(17)30016-4. doi: 10.1016/j.jcf.2017.01.009. [Epub ahead of print]
PubMed PMID: 28209466.

//////////Cavosonstat, N-91115, Orphan Drug Status, NCT02589236, N91115-2CF-05,  SNO-6, PHASE 2, N30 Pharma, Nivalis Therapeutics, CYSTIC FIBROSIS, FAST TRACK

O=C(O)C1=CC=C(C2=NC3=CC=C(O)C=C3C=C2)C(Cl)=C1

IMETELSTAT


Image result for IMETELSTAT

Image result for IMETELSTAT

2D chemical structure of 868169-64-6

IMETELSTAT

CAS 868169-64-6, N163L

Molecular Formula, C148-H211-N68-O53-P13-S13, Molecular Weight, 4610.2379,

Nucleic Acid Sequence

Sequence Length: 135 a 1 c 4 g 3 tmodified

DNA d(3′-amino-3′-deoxy-P-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A) 5′-[O-[2-hydroxy-3-[(1-oxohexadecyl)amino]propyl] hydrogen phosphorothioate]

PHASE 3, GERON, Myelodysplasia

Image result for IMETELSTAT

ChemSpider 2D Image | Imetelstat sodium | C148H197N68Na13O53P13S13

IMETELSTAT SODIUM

CAS 1007380-31-5, GRN163L, GRN 163L Sodium Salt

Molecular Formula: C148H198N68Na13O53P13S13
Molecular Weight: 4895.941 g/mol

5′-(O-(2-hydroxy-3-((1-oxohexadecyl)amino)propyl)phosphorothioate)-d(3′-amino-3′-deoxy-p-thio)(t-a-g-g-g-t-t-a-g-a-c-a-a), sodium salt (13)

DNA, d(3′-amino-3′-deoxy-p-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A), 5′-(o-(2-hydroxy-3-((1-oxohexadecyl)amino)propyl) hydrogen phosphorothioate), sodium salt (1:13)

UNII-2AW48LAZ4I, Antineoplastic

In 2014, Geron entered into an exclusive worldwide license and collaboration agreement with Janssen Biotech for the treatment of hematologic cancers. However, in 2018, the agreement was terminated and Geron regained global rights to the product.

In 2015, imetelstat was granted orphan drug status in the U.S. for the treatment of myelodysplastic syndrome, as well as in both the U.S. and the E.U. for the treatment of myelofibrosis. In 2017, fast track designation was received in the U.S. for the treatment of adult patients with transfusion-dependent anemia due to low or intermediate-1 risk myelodysplastic syndromes (MDS) who are non-del(5q) and who are refractory or resistant to treatment with an erythropoiesis stimulating agent (ESA).

Imetelstat Sodium is the sodium salt of imetelstat, a synthetic lipid-conjugated, 13-mer oligonucleotide N3′ P5′-thio-phosphoramidate with potential antineoplastic activity. Complementary to the template region of telomerase RNA (hTR), imetelstat acts as a competitive enzyme inhibitor that binds and blocks the active site of the enzyme (a telomerase template antagonist), a mechanism of action which differs from that for the antisense oligonucleotide-mediated inhibition of telomerase activity through telomerase mRNA binding. Inhibition of telomerase activity in tumor cells by imetelstat results in telomere shortening, which leads to cell cycle arrest or apoptosis.

Imetelstat sodium, a lipid-based conjugate of Geron’s first-generation anticancer drug, GRN-163, is in phase III clinical trials at Geron for the treatment of myelodysplastic syndrome, as well as in phase II for the treatment of myelofibrosis. 

Geron is developing imetelstat, a lipid-conjugated 13-mer thiophosphoramidate oligonucleotide and the lead in a series of telomerase inhibitors, for treating hematological malignancies, primarily myelofibrosis.

Imetelstat, a first-in-class telomerase inhibitor and our sole product candidate, is being developed for the potential treatment of hematologic myeloid malignancies. Imetelstat is currently in two clinical trials being conducted by Janssen under the terms of an exclusive  worldwide collaboration and license agreement.

Originally known as GRN163L, imetelstat sodium (imetelstat) is a 13-mer N3’—P5’ thio-phosphoramidate (NPS) oligonucleotide that has a covalently bound 5’ palmitoyl (C16) lipid group. The proprietary nucleic acid backbone provides resistance to the effect of cellular nucleases, thus conferring improved stability in plasma and tissues, as well as significantly improved binding affinity to its target. The lipid group enhances cell permeability to increase potency and improve pharmacokinetic and pharmacodynamic properties. The compound has a long residence time in bone marrow, spleen and liver. Imetelstat binds with high affinity to the template region of the RNA component of telomerase, resulting in direct, competitive inhibition of telomerase enzymatic activity, rather than elicit its effect through an antisense inhibition of protein translation. Imetelstat is administered by intravenous infusion.

Preclinical Studies with Imetelstat

A series of preclinical efficacy studies of imetelstat have been conducted by Geron scientists and academic collaborators. These data showed that imetelstat:

  • Inhibits telomerase activity, and can shorten telomeres.
  • Inhibits the proliferation of a wide variety of tumor types, including solid and hematologic, in cell culture systems and rodent xenograft models of human cancers, impacting the growth of primary tumors and reducing metastases.
  • Inhibits the proliferation of malignant progenitor cells from hematologic cancers, such as multiple myeloma, myeloproliferative neoplasms and acute myelogenous leukemia.
  • Has additive or synergistic anti-tumor effect in a variety of cell culture systems and xenograft models when administered in combination with approved anti-cancer therapies, including radiation, conventional chemotherapies and targeted agents.

Clinical Experience with Imetelstat

Over 500 patients have been enrolled and treated in imetelstat clinical trials.

PHASE 1

Six clinical trials evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics both as a single agent and in combination with standard therapies in patients with solid tumors and hematologic malignancies:

  • Single agent studies of imetelstat were in patients with advanced solid tumors, multiple myeloma and chronic lymphoproliferative diseases. Combination studies with imetelstat were with bortezomib in patients with relapsed or refractory multiple myeloma, with paclitaxel and bevacizumab in patients with metastatic breast cancer, and with carboplatin and paclitaxel in patients with advanced non-small cell lung cancer (NSCLC).
  • Doses ranging from 0.5 mg/kg to 11.7 mg/kg were tested in a variety of dosing schedules ranging from weekly to once every 28 days.
  • The human pharmacokinetic profile was characterized in clinical trials of patients with solid tumors and chronic lymphoproliferative diseases. Single-dose kinetics showed dose-dependent increases in exposure with a plasma half-life (t1/2) ranging from 4-5 hours. Residence time in bone marrow is long (0.19-0.51 µM observed at 41-45 hours post 7.5 mg/kg dose).
  • Telomerase inhibition was observed in various tissues where the enzymes’s activity was measurable.

PHASE 2

Imetelstat was studied in two randomized clinical trials, two single arm proof-of-concept studies and an investigator sponsored pilot study:

  • Randomized trials were in combination with paclitaxel in patients with metastatic breast cancer and as maintenance treatment following a platinum-containing chemotherapy regimen in patients with NSCLC.
  • Single arm studies were as a single agent or in combination with lenalidomide in patients with multiple myeloma and as a single agent in essential thrombocythemia (ET) or polycythemia vera (PV).
  • An investigator sponsored pilot study was as a single agent in patients with myelofibrosis (MF) or myelodysplastic syndromes (MDS).

SAFETY AND TOLERABILITY

The safety profile of imetelstat across the Phase 1 and 2 trials has been generally consistent. Reported adverse events (AEs) and laboratory investigations associated with imetelstat administration included cytopenias, transient prolonged activated partial thromboplastin time (aPTT; assessed only in Phase 1 trials), gastrointestinal symptoms, constitutional symptoms, hepatic biochemistry abnormalities, and infusion reactions. Dose limiting toxicities include thrombocytopenia and neutropenia.

A Focus on Hematologic Myeloid Malignancies

Early clinical data from the Phase 2 clinical trial in ET and the investigator sponsored pilot study in MF suggest imetelstat may have disease-modifying activity by suppressing the proliferation of malignant progenitor cell clones for the underlying diseases, and potentially allowing recovery of normal hematopoiesis in patients with hematologic myeloid malignancies.

Results from these trials were published in the New England Journal of Medicine:

Current Clinical Trials

Imetelstat is currently being tested in two clinical trials: IMbark, a Phase 2 trial in myelofibrosis (MF), and IMerge, a Phase 2/3 trial in myelodysplastic syndromes (MDS).

IMbark

IMbark is the ongoing Phase 2 clinical trial to evaluate two doses of imetelstat in intermediate-2 or high-risk MF patients who are refractory to or have relapsed after treatment with a JAK inhibitor.

Internal data reviews were completed in September 2016, April 2017 and March 2018. The safety profile was consistent with prior clinical trials of imetelstat in hematologic malignancies, and no new safety signals were identified. The data supported 9.4 mg/kg as an appropriate starting dose in the trial, but an insufficient number of patients met the protocol defined interim efficacy criteria and new patient enrollment was suspended in October 2016. As of January 2018, median follow up was approximately 19 months, and median overall survival had not been reached in either dosing arm. In March 2018, the trial was closed to new patient enrollment. Patients who remain in the treatment phase of the trial may continue to receive imetelstat, and until the protocol-specified primary analysis, all safety and efficacy assessments are being conducted as planned in the protocol, including following patients, to the extent possible, until death, to enable an assessment of overall survival.

IMerge

IMerge is the ongoing two-part Phase 2/3 clinical trial of imetelstat in red blood cell (RBC) transfusion-dependent patients with lower risk MDS who are refractory or resistant to treatment with an erythropoiesis stimulating agent (ESA). Part 1 is a Phase 2, open-label, single-arm trial of imetelstat administered as a single agent by intravenous infusion, and is ongoing. Part 2 is designed to be a Phase 3, randomized, controlled trial, and has not been initiated.

Preliminary data as of October 2017 from the first 32 patients enrolled in the Part 1 (Phase 2) of IMerge were presented as a poster at the American Society of Hematology Annual Meeting in December 2017.

The data showed that among the subset of 13 patients who had not received prior treatment with either lenalidomide or a hypomethylating agent (HMA) and did not have a deletion 5q chromosomal abnormality (non-del(5q)), 54% achieved RBC transfusion-independence (TI) lasting at least 8 weeks, including 31% who achieved a 24-week RBC-TI. In the overall trial population, the rates of 8- and 24-week RBC-TI were 38% and 16%, respectively. Cytopenias, particularly neutropenia and thrombocytopenia, were the most frequently reported adverse events, which were predictable, manageable and reversible.

Based on the preliminary data from the 13-patient subset, Janssen expanded Part 1 of IMerge to enroll approximately 20 additional patients who were naïve to lenalidomide and HMA treatment and non-del(5q) to increase the experience and confirm the benefit-risk profile of imetelstat in this refined target patient population

PATENT

WO 2005023994

WO 2006113426
WO 2006113470

 WO 2006124904

WO 2008054711

WO 2008112129

US 2014155465

WO 2014088785

PATENT

WO 2016172346

http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PG01&p=1&u=/netahtml/PTO/srchnum.html&r=1&f=G&l=50&s1=20160312227.PGNR.

PATENT

WO2018026646

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2018026646

Patients of acute myeloid leukemia (AML) have limited treatment options at diagnosis; treatment typically takes the form of chemotherapy to quickly reduce the leukemic cell burden. Invasive leukapheresis procedures to remove large numbers of leukocytes (normal and diseased) may be applied in parallel to chemotherapy to temporarily lower tumor cell burden. Induction phase chemotherapy can be successful but, most healthy cells residing in patient bone marrow are also killed, causing illness and requiring additional palliative therapy to ward off infection and raise leukocyte counts. Additional rounds of chemotherapy can be used in an attempt to keep patients in remission; but relapse is common.

[0005] Telomerase is present in over 90% of tumors across all cancer types; and is lacking in normal, healthy tissues. Imetelstat sodium is a novel, first-in-class telomerase inhibitor that is a covalently-lipidated 13-mer oligonucleotide (shown below) complimentary to the human telomerase RNA (hTR) template region. Imetelstat sodium does not function through an anti-sense mechanism and therefore lacks the side effects commonly observed with such therapies. Imetelstat sodium is the sodium salt of imetelstat (shown below):

Imetelstat sodium

Unless otherwise indicated or clear from the context, references below to imetelstat also include salts thereof. As mentioned above, imetelstat sodium in particular is the sodium salt of imetelstat.

[0006] ABT-199/venetoclax (trade name Venclexta) is an FDA approved Bcl-2 inhibitor for use in chronic lymphocytic leukemia (CLL) patients with dell7p who are relapsed/refractory. ABT-199 is also known as ABT 199, GDC0199, GDC-0199 or RG7601. The chemical name for ABT-199 is 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-l-yl]methyl]piperazin-l-yl]-N-[3-nitro-4-(oxan-4-ylmethylamino)phenyl]sulfonyl-2-(lH-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide (Cas No. 1257044-40-8). Unless otherwise indicated or clear from the context, references below to ABT-199 also include pharmaceutically acceptable salts thereof. Specifically in the Examples however, ABT-199 was used in the free base form.

[0007] ABT-199, shown below in the free base form, is highly specific to Bcl-2, unlike other first generation inhibitors which show affinity for related Bel family members and induce greater side effects. Inhibition of Bcl-2 blocks the pro-apoptotic signals caused by damage to or abnormalities within cellular DNA and ultimately leads to programmed cell death in treated cells via the caspase cascade and apoptosis through the intrinsic pathway.

ABT-199 (shown in the free base form)

PATENT

WO-2019011829

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019011829&tab=PCTDESCRIPTION&maxRec=1000

Improved process for preparing imetelstat .  claiming use of a combination comprising a telomerase inhibitor, specifically imetelstat sodium and a Bcl-2 inhibitor, specifically ABT-199 for treating hematological cancer such as acute myeloid leukemia, essential thrombocythemia and polycythemia vera, specifically acute myeloid leukemia.

Imetelstat (SEQ ID NO: 1 ) is a N3′- P5′ thiophosphoramidate oligonucleotide covalently linked to a palmitoyl lipid moiety and has been described in WO-2005/023994 as compound (1 F). The sodium salt of imetelstat acts as a potent and specific telomerase inhibitor and can be used to treat telomerase-mediated disorders, e.g. cancer, including disorders such as myelofibrosis (MF), myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML).

The structure of imetelstat sodium is shown below :

The structure of imetelstat can also be represented as shown below

imetelstat

The LPT group represents the palmitoyi lipid that is covalently linked to the N3′- P5′ thiophosphor-amidate oligonucleotide. The base sequence of the thirteen nucleotides is as follows :

TAGGGTTAGACAA and is represented by the bases B1 to B13. The -NH-P(=S)(OH)-and -0-P(=S)(OH)- groups of the structure can occur in a salt form. It is understood that salt forms of a subject compound are encompassed by the structures depicted herein, even if not specifically indicated.

Imetelstat sodium can also be represented as follows

o H

LPT = CH3-(CH2)i4-C-N-CH2-(CHOH)-CH2-

The -NH-P(=S)(OH)- group and the thymine, adenine, guanine and cytosine bases can occur in other tautomeric arrangements then used in the figures of the description. It is understood that all tautomeric forms of a subject compound are encompassed by a structure where one possible tautomeric form of the compound is described, even if not specifically indicated.

Prior art

The synthetic scheme used in WO-2005/023994 to prepare imetelstat as compound (1 F) is described in Scheme 1 and Scheme 2. The synthesis of this oligonucleotide is achieved using the solid-phase phosphoramidite methodology with all reactions taking place on solid-phase support. The synthesis of imetelstat is carried out on controlled pore glass (LCAA-CPG) loaded with

3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol. The oligonucleotide is assembled from the 5′ to the 3′ terminus by the addition of protected nucleoside 5′-phosphor-amidites with the assistance of an activator. Each elongation cycle consists of 4 distinct, highly controlled steps : deprotection, amidite coupling, sulfurization and a capping step.

Scheme 1 : imetelstat synthetic scheme cycle 1

3. Sulfurization

In Scheme 1 the solid-phase supported synthesis starts with removal of the acid-labile 4,4-dimethoxy-trityl (DMT) protecting group from the palmitoylamidopropanediol linked to the solid-phase support. The first phosphoramidite nucleotide is coupled to the support followed by sulfurization of the phosphor using a 0.1 M solution of phenylacetyl disulfide (PADS) in a mixture of acetonitrile and 2,6-lutidine (1 : 1 ratio). Then a capping step is applied to prevent any unreacted solid-phase support starting material from coupling with a phosphoramidite nucleotide in the following reaction cycles. Capping is done using an 18:1 :1 mixture of THF / isobutyric anhydride / 2,6-lutidine.

After the first cycle on the solid-phase support, chain elongation is achieved by reaction of the 3′-amino group of the support-bound oligonucleotide with an excess of a solution of the protected nucleotide phosphoramidite monomer corresponding to the next required nucleotide in the sequence as depicted in Scheme 2.

Scheme 2 : imetelstat synthetic scheme cycle 2-13

In Scheme 2 the first cycle is depicted of the chain elongation process which is achieved by deprotection of the 3′-amino group of the support-bound oligonucleotide (a), followed by a coupling reaction of the 3′-amino group of the support-bound oligonucleotide (b) with an excess of a solution of a 5′-phosphoramidite monomer corresponding to the next required nucleotide in the sequence of imetelstat. The coupling reaction is followed by sulfurization of the phosphor of the support-bound oligonucleotide (c) and a capping step (see Scheme 3) to prevent any unreacted solid-phase support starting material (b) from coupling with a 5′-phosphoramidite nucleotide in the following reaction cycles. The reaction cycle of Scheme 2 is repeated 12 times before the solid-phase support-bound oligonucleotide is treated with a 1 :1 mixture of ethanol and concentrated ammonia, followed by HPLC purification to obtain imetelstat.

Scheme 3

The capping step using an 18:1 : 1 mixture of THF / isobutyric anhydride / 2,6-lutidine is done to convert after the coupling step any remaining solid-phase support bound oligonucleotide (b) with a primary 3′-amino group into oligonucleotide (e) with a protected (or ‘capped’) 3′-amino group in order to prevent the primary 3′-amino group from coupling with a phosphoramidite nucleotide in the next reaction cycles.

WO-01/18015 discloses in Example 3 with SEQ ID No. 2 a N3’^P5′ thiophosphoramidate oligonucleotide and a process for preparing this oligonucleotide encompassing a capping step.

Herbert B-S et al. discusses the lipid modification of GRN163 (Oncogene (2005) 24, 5262-5268).

Makiko Horie et al. discusses the synthesis and properties of 2′-0,4′-C-ethylene-bridged nucleic acid oligonucleotides targeted to human telomerase RNA subunit (Nucleic Acids Symposium Series (2005) 49, 171-172).

Description of the invention

The coupling reaction in the solid-phase support bound process disclosed in WO-01/18015 and WO-2005/023994 include a capping step to prevent any unreacted primary 3′ amino groups on the support-bound oligonucleotide from reacting during subsequent cycles.

It has now surprisingly been found that the use of a capping step as described in the prior art is superfluous and that imetelstat can be prepared using a 3-step cycle without an additional capping step with nearly identical yield and purity compared to the prior art 4-step cycle that uses a specific capping step. Eliminating the capping step from each cycle benefits the overall process by reducing the number of cycle steps by 22% (from 54 to 42 steps) and consequent reduction of process time. Also, the solvent consumption is reduced due to the reduction of cycle steps which makes for a greener process.

Wherever the term “capping step” is used throughout this text, it is intended to define an additional chemical process step wherein the primary free 3′-amino group on the solid-phase support bound oligonucleotide is converted into a substituted secondary or tertiary 3′-amino group that is not capable of participating in the coupling reaction with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer in the ensuing coupling step.

In one embodiment, the present invention relates to a method of synthesizing an oligonucleotide N3′ – P5′ thiophosphoramidate of formula

imetelstat

the method comprises of

a) providing a first 3′-amino protected nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;

b) deprotecting the protected 3′-amino group to form a free 3′-amino group;

c) reacting the free 3′-amino group with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N- diisopropylaminophosphoramidite monomer of formula (B n) wherein n = 2 to form an internucleoside N3′- P5′-phosphoramidite linkage;

mer (B’n)

d) sulfurization of the internucleoside phosphoramidite group using an acyl disulfide to form a N3′- P5′ thiophosphoramidate;

e) repeating 1 1 times in successive order the deprotection step b), the coupling step c) with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer of formula (B n) wherein the protected nucleoside base B’ in monomer (B n) is successively the protected nucleobase B3 to B13 in the respective 1 1 coupling steps, and the sulfurization step d);

f) removing the acid-labile protecting group PG; and

g) cleaving and deprotecting imetelstat from the solid-phase support;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

In one embodiment, the present invention relates to a method of synthesizing the N3′ – P5′

thiophosphoramidate oligonucleotide imetelstat of formula

imetelstat

the method comprises of

a) providing a first 3′-amino protected nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;

b) deprotecting the protected 3′-amino group to form a free 3′-amino group;

c) reacting the free 3′-amino group with a protected 3′-aminonucleoside-5′-0-cyanoethyl- Ν,Ν-diisopropylaminophosphoramidite monomer of formula (B n), wherein B n with n = 2 is protected A, to form an internucleoside N3′- P5′-phosphoramidite linkage;

mer

d) sulfurization of the internucleoside phosphoramidite group using an acyl disulfide to form a N3′- P5′ thiophosphoramidate;

e) repeating 1 1 times in successive order the deprotection step b), the coupling step c) with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer of formula (B n) wherein the nucleoside base B’ of monomer (B n) is protected B except when B is thymine, and wherein Bn is successively nucleobase B3 to B13 in the respective 1 1 coupling steps, and the sulfurization step d);

f) removing the acid-labile protecting group PG; and

g) deprotecting and cleaving imetelstat from the solid-phase support;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

In one embodiment, the present invention relates to a method of synthesizing the N3′ – P5′

thiophosphoramidate oligonucleotide imetelstat of formula

imetelstat

thymine

adenine

guanine


cytosine

9 H

LPT =CH3-(CH2)i4-C-N-CH2-(CHOH)-CH2-

the method comprises of

a) providing a first protected 3′-amino nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;

b) deprotecting the PG-protected 3′-amino nucleotide to form a free 3′-amino nucleotide of formula (A’);

c) coupling the free 3′-amino nucleotide with a protected 3′-aminonucleoside-5′-0- cyanoethyl-N,N-diisopropylaminophosphoramidite monomer (B n), wherein B nwith n = 2 is protected A, to form an internucleoside N3′- P5′-phosphoramidite linkage;

monomer (B’n)

d) sulfurizing the N3′- P5′-phosphoramidite linkage using an acyl disulfide to form an internucleoside N3′- P5′ thiophosphoramidate linkage;

e) repeating 1 1 times in successive order:

the deprotecting step b);

the coupling step c) with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N- diisopropylamino-phosphoramidite monomer (B n) wherein the nucleoside base B’ of monomer (B n) is protected B except when B is thymine, and wherein Bn is successively nucleobase B3 to B13 in the respective 1 1 coupling steps; and

the sulfurizing step d);

to produce a protected N3′ – P5′ thiophosphoramidate oligonucleotide imetelstat attached to the solid-phase support;

f) removing the 3′-terminal acid-labile protecting group PG from the protected N3′ – P5′ thiophosphoramidate oligonucleotide imetelstat; and

g) deprotecting and cleaving the protected N3′ – P5′ thiophosphoramidate oligonucleotide imetelstat from the solid-phase support to produce imetelstat;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

A wide variety of solid-phase supports may be used with the invention, including but not limited to, such as microparticles made of controlled pore glass (CPG), highly cross-linked polystyrene, hybrid controlled pore glass loaded with cross-linked polystyrene supports, acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like.

The 3′-amino protected nucleotide attached to a solid-phase support of formula (A)

can be prepared as disclosed in WO-2005/023994 wherein a controlled pore glass support loaded with 3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol has been coupled with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer of formula (B^ )

monomer (B’-| ) wherein B’-| = T

wherein PG is an acid-labile protecting group. Suitable acid-labile 3′-amino protecting groups PG are, but not limited to, e.g. triphenylmethyl (i.e. trityl or Tr), p-anisyldiphenylmethyl (i.e. mono-methoxytrityl or MMT), and di-p-anisylphenylmethyl (i.e. dimethoxytrityl or DMT).

The protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomers of formula (B n) have a 3′-amino protecting group PG which is an acid-labile group, such as triphenylmethyl (i.e. trityl or Tr), p-anisyldiphenylmethyl (i.e. monomethoxytrityl or MMT), or di-p-anisylphenylmethyl (i.e. dimethoxytrityl or DMT). Furthermore the nucleoside base B’ is protected with a base-labile protecting group (except for thymine).

ed A ed C ed A ed A

B’s = protected A G = guanine

B’g = protected G C = cytosine

The nucleotide monomers and B’2 to B’13 are used successively in the 13 coupling steps starting from the provision of a solid-phase support loaded with 3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol and coupled to nucleotide monomer and the following cycle of 12 deprotection, coupling, and sulfurization reactions wherein the nucleotide monomers B’2 to B -I 3 are used.

The 3′-amino protecting group PG can be removed by treatment with an acidic solution such as e.g. dichloroacetic acid in dichloromethane or toluene.

The nucleoside base B’ in the protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropyl-aminophosphoramidite monomers of formula (B n) is protected with a base-labile protecting group which is removed in step g). Suitable base-labile protecting groups for the nucleoside base adenine, cytosine or guanine are e.g. acyl groups such as acetyl, benzoyl, isobutyryl, dimethyl-formamidinyl, or dibenzylformamidinyl. Under the reaction conditions used in oligonucleotide synthesis the thymine nucleoside base does not require protection. Such protected 3′- amino-nucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomers of formula (B N) having a 3′-amino protected with an acid-labile group protecting group PG and a nucleoside base B’ protected with a base-labile protecting group are commercially available or can be prepared as described in WO-2006/014387.

The coupling step c) is performed by adding a solution of protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer of formula (BN) and a solution of an activator (or a solution containing the phosphoramidite monomer (BN) and the activator) to the reaction vessel containing the free amino group of an (oligo)nucleotide covalently attached to a solid support. The mixture is then mixed by such methods as mechanically vortexing, sparging with an inert gas, etc. Alternately, the solution(s) of monomer and activator can be made to flow through a reaction vessel (or column) containing the solid-phase supported (oligo)nucleotide with a free 3′-amino group. The monomer and the activator either can be premixed, mixed in the valve-block of a suitable synthesizer, mixed in a pre-activation vessel and preequilibrated if desired, or they can be added separately to the reaction vessel.

Examples of activators for use in the invention are, but not limited to, tetrazole, 5-(ethylthio)-1 H-tetrazole, 5-(4-nitro-phenyl)tetrazole, 5-(2-thienyl)-1 H-tetrazole, triazole, pyridinium chloride, and the like. Suitable solvents are acetonitrile, tetrahydrofuran, dichloromethane, and the like. In practice acetonitrile is a commonly used solvent for oligonucleotide synthesis.

The sulfurization agent for use in step d) is an acyl disulfide dissolved in a solvent. Art know acyl disulfides are e.g. dibenzoyl disulphide, bis(phenylacetyl) disulfide (PADS), bis(4-methoxybenzoyl) disulphide, bis(4-methylbenzoyl) disulphide, bis(4-nitrobenzoyl) disulphide and bis(4-chlorobenzoyl) disulfide.

Phenylacetyl disulfide (PADS) is a commonly used agent for sulfurization reactions that it is best ‘aged’ in a basic solution to obtain optimal sulfurization activity (Scotson J.L. et al., Org. Biomol. Chem., vol. 14, 10840 – 10847, 2016). A suitable solvent for PADS is e.g. a mixture of a basic solvent such as e.g. 3-picoline or 2,6-lutidine with a co-solvent such as acetonitrile, toluene, 1-methyl-pyrrolidinone or tetrahydrofuran. The amount of the basic solvent to the amount of the co-solvent can be any ratio including a 1 :1 ratio. Depending upon the phosphite ester to be converted into its corresponding thiophospate, both ‘fresh’ and ‘aged’ PADS can be used however ‘aged’ PADS has been shown to improve the rate and efficiency of sulfurization. ‘Aged’ PADS solutions are freshly prepared PADS solutions that were maintained some time before usage in the sulfurization reaction. Aging times can vary from a few hours to 48 hours and the skilled person can determine the optimal aging time by analysing the sulfurization reaction for yield and purity.

For the preparation of imetelstat in accordance with the present invention, a PADS solution in a mixture of acetonitrile and 2,6-lutidine, preferably in a 1 :1 ratio, with an aging time of 4 to 14 hours is used. It has been found that when 2,6-lutidine is used, limiting the amount of 2,3,5-collidine (which is often found as an impurity in 2,6-lutidine) below 0.1 % improves the efficiency of sulfurization and less undesirable phosphor oxidation is observed.

In step g) imetelstat is deprotected and cleaved from the solid-phase support. Deprotection includes the removal of the β-cyanoethyl groups and the base-labile protecting groups on the nucleotide bases. This can be done by treatment with a basic solution such as a diethylamine (DEA) solution in acetonitrile, followed by treatment with aqueous ammonia dissolved in an alcohol such as ethanol.

The reaction steps a) to f) of the present invention are carried out in the temperature range of 10°C to 40°C. More preferably, these reactions are carried out at a controlled temperature ranging from 15°C to 30°C. In particular reaction step b) of the present invention is carried out in the temperature range of 15°C to 30°C; more in particular 17°C to 27°C. In particular reaction step d) of the present invention is carried out in the temperature range of 17°C to 25°C; more in particular 18°C to 22°C; even more in particular 19°C. The step g) wherein imetelstat is deprotected and cleaved from the solid-phase support is carried out at a temperature ranging from 30°C to 60°C. Depending upon the equipment and the specific reaction conditions used, the optimal reaction temperature for each step a) to g) within the above stated ranges can be determined by the skilled person.

After each step in the elongation cycle, the solid-phase support is rinsed with a solvent, for instance acetonitrile, in preparation for the next reaction.

After step g), crude imetelstat is obtained in its ammonium salt form which is then purified by a preparative reversed phase high performance liquid chromatography (RP-HPLC) by using either polymeric or silica based resins to get purified imetelstat in triethyl amine form. An excess of a sodium salt is added, and then the solution is desalted by diafiltration thereby yielding imetelstat sodium which is then lyophilized to remove water.

Experimental part

‘Room temperature’ or ‘ambient temperature’ typically is between 21-25 °C.

Experiment 1 (no capping step)

All the reagents and starting material solutions were prepared including 3% dichloroacetic acid (DCA) in toluene, 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile, 0.15 M of all 4 nucleotide monomers of formula (B n) in acetonitrile, 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine and 20% DEA (diethylamine) in acetonitrile.

The oligonucleotide synthesis was performed in the direction of 5′ to 3′ utilizing a repetitive synthesis cycle consisting of detritylation followed by coupling, and sulfurization performed at ambient temperature.

A column (diameter : 3.5 cm) was packed with a solid-support loaded with 3-palmitoylamido-1-0- (4, 4′-dimethoxytrityl)-2-0-succinyl propanediol (3.5 mmol based on a capacity of 400 μιηοΙ/g) that was coupled with the nucleotide monomer B Detritylation was achieved using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes in each detritylation step) and the solid-support bound nucleotide was washed with acetonitrile (amount: 5 column volumes). Coupling with the next nucleotide monomer of formula (B n) was achieved by pumping a solution of 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile and 0.15 M of the next nucleotide monomer of formula (B n) in the sequence, dissolved in acetonitrile, through the column. The column was washed with acetonitrile (amount : 2 column volumes). Then sulfurization was performed by

pumping a solution of 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine mixture through the column followed by washing the column with acetonitrile (amount : 5 column volumes).

The synthesis cycle of detritylation, coupling with the next nucleotide monomer of formula (B n) and sulfurization was repeated 12 times, followed by detritylation using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes).

Upon completion of the synthesis cycle, the crude oligonucleotide on the solid-support support was treated with a diethylamine (DEA) solution followed by treatment with ammonium hydroxide solution: ethanol (3: 1 volume ratio) at a temperature of 55°C. The reaction mixture was aged for

4 to 24 hours at 55°C, cooled to room temperature, and slurry was filtered to remove the polymeric support. The solution comprising imetelstat in its ammonium form was subjected to the HPLC analysis procedure of Experiment 3.

Experiment 2 (with capping step)

All the reagents and starting material solutions were prepared including 3% dichloroacetic acid (DCA) in toluene, 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile, 0.15 M of all 4 nucleotide monomers of formula (B n) in acetonitrile, 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine mixture, 20% N-methylimidazole (NMI) in acetonitrile as capping agent A, isobutryic anhydride in a 1 :1 mixture of acetonitrile and 2,6-lutidine mixture as capping agent B and 20% DEA in acetonitrile.

The oligonucleotide synthesis was performed in the direction of 5′ to 3′ utilizing a repetitive synthesis cycle consisting of detritylation followed by coupling, and sulfurization performed at ambient temperature.

A column (diameter : 3.5 cm) was packed with a solid-support loaded with 3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol (3.5 mmol based on a capacity of 400 μιηοΙ/g) that was coupled with the nucleotide monomer B Detritylation was achieved using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes in each detritylation step) and the solid-support bound nucleotide was washed with acetonitrile (amount : 5 column volumes). Coupling with the next nucleotide monomer of formula (B n) was achieved by pumping a solution of 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile and 0.15 M of the next nucleotide monomer of formula (B n) in the sequence, dissolved in acetonitrile, through the column. The column was washed with acetonitrile (amount : 2 column volumes). Then sulfurization was performed by pumping a solution of 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine mixture through the column followed by washing the column with acetonitrile (amount :

5 column volumes).

The sulfurization was followed by a capping step. Each capping in a given cycle used 37-47 equivalents (eq.) of the capping agent NMI, and 9-1 1 equivalents of the capping agent B isobutryic anhydride (IBA), and 1 .4-1.8 equivalents of 2,6 lutidine. Capping agents A and B were pumped through the column with separate pumps at different ratios such as 50:50, 35:65, 65:35.

The synthesis cycle of detritylation, coupling with the next nucleotide monomer of formula (B n) and sulfurization, and capping step was repeated 12 times, followed by detritylation using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes).

Upon completion of the synthesis cycle, the crude oligonucleotide on the solid-support support was treated with a diethylamine (DEA) solution followed by treatment with ammonium hydroxide solution: ethanol (3: 1 volume ratio) at a temperature of 55°C. The reaction mixture was aged for 4 to 24 hours at 55°C, cooled to room temperature, and slurry was filtered to remove the polymeric support. The solution comprising imetelstat in its ammonium form was subjected to the HPLC analysis procedure of Experiment 3.

Experiment 3 : comparision of no-capping vs. capping

Imetelstat obtained in Experiment 1 and Experiment 2 was analysed by HPLC. The amount of the desired full length oligonucleotide having 13 nucleotides was determined and listed in the Table below for Experiment 1 and Experiment 2. Also, the total amount of shortmer, specifically the 12mer, was determined and listed in the Table below for Experiment 1 and Experiment 2.

HPLC analysis method :

column type: Kromasil C18, 3.5 μιτι particle size, 4.6 X 150 mm

eluent:

A: 14.4 mM TEA/386 mM HFIP (hexafluoroisopropanol) /100 ppm(w/v) Na2EDTA in water B: 50% MeOH, 50% EtOH containing 5% IPA

Gradient :

Step Run time (minutes) %B

1 0 10

2 5 10

3 12 26 (linear)

4 35 45 (linear)

5 40 50 (linear)

6 42 50

7 44 10 (linear)

8 50 10

Table : capping vs. no-capping experiments (Experiment 1 was run twice and results are listed as Experiment 1a and 1 b).

The HPLC analysis of Experiment 1 and Experiment 2 demonstrates that yield and purity are comparable for the no-capping experiment vs. the capping experiment.

Main peak % includes Full length oligonucleotide + PO impurities + depurinated impurities.

PO impurities are impurities including one or more oxophosphoramidate internucleoside linkages instead of thiophosphoramidate internucleoside linkages.

Solvent use and reaction time

0.45 L of acetonitrile/mmol is used to prepare capping agent A and capping agent B reagents which corresponds to approximately 25 % of the overall acetonitrile use during the preparation of the reagents. Since each chemical reaction step is followed by a solvent wash, after each capping step too, a solvent wash takes place which is equivalent to about 40 column volumes of the solvent. Considering that about 212 column volumes of the solvent wash is done for a given synthesis run, about 19 % of the wash solvent is used for the capping steps. Each capping step takes between 3 – 6 minutes. This corresponds to about 8 % of the overall synthesis time including the 13 cycles and DEA treatment.

Experiment 4 (detritylation temperature)

The detritylation temperature has an impact in terms of controlling n-1 and depurinated impurities. The temperature of the deblocking solution at the entrance of the synthesizer was chosen between 17.5 and 27 °C (at 3.5 mmol scale) and the selected temperature was kept the same for all detritylation steps. The acetonitrile washing was also kept at the same temperature of the deblocking solution. The % depurinated impurities increased linearly with temperature while n-1 was higher at lower temperatures.

Temperature n-1 % Depurinated Impurity %

17.5 10.7 5.3

19 7.6 6.4

22 5.4 8.7

25 6.1 10.8

27 5.3 12.3

Experiment 5 (sulfurization step temperature)

In the experiments below, the temperature (RT means room temperature) of the PADS solution used in the sulfurization reactions was tested for the % of less favourable PO impurities (these are impurities where phosphor oxidation occurred instead of sulfurization). Lower temperature results in lower PO %.

SEQ ID NO:1 – imetelstat and imetelstat sodium

5′-R-TAGGGTTAGACAA-NH2-3′

wherein R represents palmitoyl [(CH2)1 CH3] amide is conjugated through an aminoglycerol linker to the 5′-thiophosphate group of an N3′ – P5′ thiophosphoramidate (NPS) -linked oligonucleotide.

///////////IMETELSTAT,  GRN163L, PHASE 3, orphan drug, FAST TRACK

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USFDA approval to Lumoxiti (moxetumomab pasudotoxtdfk) a new treatment for hairy cell leukemia


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USFDA approval to Lumoxiti is a new treatment for hairy cell leukemia

On September 13, 2018, the U.S. Food and Drug Administration approved Lumoxiti (moxetumomab pasudotoxtdfk) injection for intravenous use for the treatment of adult patients with relapsed or refractory Hairy Cell Leukemia (HCL) who have received at least two prior systemic therapies, including treatment with a purine nucleoside analog 1. Lumoxiti is a CD22-directed cytotoxin and is the first of this type of treatment for patients with HCL. The efficacy of Lumoxiti was studied in a single-arm, open-label clinical trial of 80 patients who had received prior treatment for HCL with at least two systemic therapies, including a purine nucleoside analog. The trial measured durable complete response (CR), defined as maintenance of hematologic remission for more than 180 days after achievement of CR. Thirty percent of patients in the trial achieved durable CR, and the overall response rate (number of patients with partial or complete response to therapy) was 75 percent. The FDA granted this application Fast Track and Priority Review designations. Lumoxiti also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases. The FDA granted the approval of Lumoxiti to AstraZeneca Pharmaceuticals. About Hairy Cell Leukemia HCL is a rare, slow-growing cancer of the blood in which the bone marrow makes too many B cells (lymphocytes), a type of white blood cells that fight infection. HCL is named after these extra B cells which look “hairy” when viewed under a microscope. As the number of leukemia cells increases, fewer healthy white blood cells, red blood cells and platelets are produced.

About Lumoxiti2 Lumoxiti (moxetumomab pasudotox) is a CD22-directed cytotoxin and a first-in-class treatment in the US for adult patients with relapsed or refractory hairy cell leukaemia (HCL) who have received at least two prior systemic therapies, including treatment with a purine nucleoside analog. Lumoxiti is not recommended in patients with severe renal impairment (CrCl ≤ 29 mL/min). It comprises the CD22 binding portion of an antibody fused to a truncated bacterial toxin; the toxin inhibits protein synthesis and ultimately triggers apoptotic cell death.

September 13, 2018

Release

The U.S. Food and Drug Administration today approved Lumoxiti (moxetumomab pasudotox-tdfk) injection for intravenous use for the treatment of adult patients with relapsed or refractory hairy cell leukemia (HCL) who have received at least two prior systemic therapies, including treatment with a purine nucleoside analog. Lumoxiti is a CD22-directed cytotoxin and is the first of this type of treatment for patients with HCL.

“Lumoxiti fills an unmet need for patients with hairy cell leukemia whose disease has progressed after trying other FDA-approved therapies,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “This therapy is the result of important research conducted by the National Cancer Institute that led to the development and clinical trials of this new type of treatment for patients with this rare blood cancer.”

HCL is a rare, slow-growing cancer of the blood in which the bone marrow makes too many B cells (lymphocytes), a type of white blood cell that fights infection. HCL is named after these extra B cells which look “hairy” when viewed under a microscope. As the number of leukemia cells increases, fewer healthy white blood cells, red blood cells and platelets are produced.

The efficacy of Lumoxiti was studied in a single-arm, open-label clinical trial of 80 patients who had received prior treatment for HCL with at least two systemic therapies, including a purine nucleoside analog. The trial measured durable complete response (CR), defined as maintenance of hematologic remission for more than 180 days after achievement of CR. Thirty percent of patients in the trial achieved durable CR, and the overall response rate (number of patients with partial or complete response to therapy) was 75 percent.

Common side effects of Lumoxiti include infusion-related reactions, swelling caused by excess fluid in body tissue (edema), nausea, fatigue, headache, fever (pyrexia), constipation, anemia and diarrhea.

The prescribing information for Lumoxiti includes a Boxed Warning to advise health care professionals and patients about the risk of developing capillary leak syndrome, a condition in which fluid and proteins leak out of tiny blood vessels into surrounding tissues. Symptoms of capillary leak syndrome include difficulty breathing, weight gain, hypotension, or swelling of arms, legs and/or face. The Boxed Warning also notes the risk of hemolytic uremic syndrome, a condition caused by the abnormal destruction of red blood cells. Patients should be made aware of the importance of maintaining adequate fluid intake, and blood chemistry values should be monitored frequently. Other serious warnings include: decreased renal function, infusion-related reactions and electrolyte abnormalities. Women who are breastfeeding should not be given Lumoxiti.

The FDA granted this application Fast Track and Priority Review designations. Lumoxiti also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.

The FDA granted the approval of Lumoxiti to AstraZeneca Pharmaceuticals.

1 https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm620448.htm

2 https://www.astrazeneca.com/media-centre/press-releases/2018/us-fda-approves-lumoxiti-moxetumomab-pasudotox-tdfk-for-certain-patientswith-relapsed-or-refractory-hairy-cell-leukaemia.html

/////////// Lumoxiti, moxetumomab pasudotoxtdfk, FDA 2018, Fast Track,  Priority Review ,  Orphan Drug, AstraZeneca

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