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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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FDA expands approval of Blincyto (blinatumomab) for treatment of a type of leukemia in patients who have a certain risk factor for relapse


Image result for blinatumomab

FDA expands approval of Blincyto for treatment of a type of leukemia in patients who have a certain risk factor for relapse

Blincyto (blinatumomab)

The U.S. Food and Drug Administration granted accelerated approval to Blincyto (blinatumomab) to treat adults and children with B-cell precursor acute lymphoblastic leukemia (ALL) who are in remission but still have minimal residual disease (MRD). MRD refers to the presence of cancer cells below a level that can be seen under the microscope. In patients who have achieved remission after initial treatment for this type of ALL, the presence of MRD means they have an increased risk of relapse.Continue reading.

 

March 29, 2018

Release

The U.S. Food and Drug Administration granted accelerated approval to Blincyto (blinatumomab) to treat adults and children with B-cell precursor acute lymphoblastic leukemia (ALL) who are in remission but still have minimal residual disease (MRD). MRD refers to the presence of cancer cells below a level that can be seen under the microscope. In patients who have achieved remission after initial treatment for this type of ALL, the presence of MRD means they have an increased risk of relapse.

“This is the first FDA-approved treatment for patients with MRD-positive ALL,” 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. “Because patients who have MRD are more likely to relapse, having a treatment option that eliminates even very low amounts of residual leukemia cells may help keep the cancer in remission longer. We look forward to furthering our understanding about the reduction in MRD after treatment with Blincyto. Studies are being conducted to assess how Blincyto affects long-term survival outcomes in patients with MRD.”

B-cell precursor ALL is a rapidly progressing type of cancer in which the bone marrow makes too many B-cell lymphocytes, an immature type of white blood cell. The National Cancer Institute estimates that approximately 5,960 people in the United States will be diagnosed with ALL this year and approximately 1,470 will die from the disease.

Blincyto works by attaching to CD19 protein on the leukemia cells and CD3 protein found on certain immune system cells. Bringing the immune cell close to the leukemia cell allows the immune cells to attack the leukemia cells better. The FDA first approved Blincyto under accelerated approval in December 2014 for the treatment of Philadelphia chromosome (Ph)-negative relapsed or refractory positive B-cell precursor ALL. Full approval for this indication was granted in July 2017, and at that time, the indication was also expanded to include patients with Philadelphia chromosome-positive ALL.

The efficacy of Blincyto in MRD-positive ALL was shown in a single-arm clinical trial that included 86 patients in first or second complete remission who had detectable MRD in at least 1 out of 1,000 cells in their bone marrow. Efficacy was based on achievement of undetectable MRD in an assay that could detect at least one cancer cell in 10,000 cells after one cycle of Blincyto treatment, in addition to the length of time that the patients remained alive and in remission (hematological relapse-free survival). Overall, undetectable MRD was achieved by 70 patients. Over half of the patients remained alive and in remission for at least 22.3 months.

The side effects of Blincyto when used to treat MRD-positive B-cell precursor ALL are consistent with those seen in other uses of the drug. Common side effects include infections (bacterial and pathogen unspecified), fever (pyrexia), headache, infusion related reactions, low levels of certain blood cells (neutropenia, anemia), febrile neutropenia (neutropenia and fever) and low levels of platelets in the blood (thrombocytopenia).

Blincyto carries a boxed warning alerting patients and health care professionals that some clinical trial participants had problems with low blood pressure and difficulty breathing (cytokine release syndrome) at the start of the first treatment, experienced a short period of difficulty with thinking (encephalopathy) or other side effects in the nervous system. Serious risks of Blincyto include infections, effects on the ability to drive and use machines, inflammation in the pancreas (pancreatitis), and preparation and administration errors—instructions for preparation and administration should closely be followed. There is a risk of serious adverse reactions in pediatric patients due to benzyl alcohol preservative; therefore, the drug prepared with preservative free saline should be used for patients weighing less than 22 kilograms.

This new indication for Blincyto was approved under the accelerated approval pathway, under which the FDA may approve drugs for serious conditions where there is unmet medical need and a drug is shown to have certain effects that are reasonably likely to predict a clinical benefit to patients. Further study in randomized controlled trials is required to verify that achieving undetectable MRD with Blincyto improves survival or disease-free survival in patients with ALL.

The FDA granted this application Priority Review and it received Orphan Drugdesignation, which provides incentives to assist and encourage the development of drugs for rare diseases.

The FDA granted the approval of Blincyto to Amgen Inc.

 

//////amgen, fda 2018,  Priority Review m  Orphan Drug designation, Blincyto, blinatumomab,

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AMG 176


str1 

AMG 176

C33 H41 Cl N2 O5 S, 613.21
str2
14E/8’E
Spiro[5,7-etheno-1H,11H-cyclobut[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-2(3H),1′(2′H)-naphthalen]-8(9H)-one, 6′-chloro-3′,4′,12,13,16,16a,17,18,18a,19-decahydro-16-methoxy-11,12-dimethyl-, 10,10-dioxide, (1′S,11R,12S,14E,16S,16aR,18aR)-
(1S,3’R,6’R,7’S,8’E,1 l’R,12’R)-6-CHLORO-7′-METHOXY-l 1′-METHYL- 12′-( 1 -METHYL)-3 ,4-DIHYDRO-2H, 15 Ή-SPIRO [NAPHTHALENE- 1 ,22′- [20]OXA[13]THIA[1,14]DIAZATETRACYCLO[14.7.2.036.01924]PENTACOS A[8,16,18,24]TETRAEN]-15′-ONE 13 ‘,13 ‘-DIOXIDE
E FORM 1883727-34-1
.
.
.
14Z/8’Z
Spiro[5,7-etheno-1H,11H-cyclobut[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-2(3H),1′(2′H)-naphthalen]-8(9H)-one, 6′-chloro-3′,4′,12,13,16,16a,17,18,18a,19-decahydro-16-methoxy-11,12-dimethyl-, 10,10-dioxide, (1′S,11R,12S,14Z,16S,16aR,18aR)-
(1S,3’R,6’R,7’S,8’Z,1 l’R,12’R)-6-CHLORO-7′-METHOXY-l 1′-METHYL- 12′-( 1 -METHYL)-3 ,4-DIHYDRO-2H, 15 Ή-SPIRO [NAPHTHALENE- 1 ,22′- [20]OXA[13]THIA[1,14]DIAZATETRACYCLO[14.7.2.036.01924]PENTACOS A[8,16,18,24]TETRAEN]-15′-ONE 13 ‘,13 ‘-DIOXIDE
Z FORM 1883727-35-2
 str3

PHASE 1,  Amgen, Mcl-1 inhibitor,  tumors

  • Class Antineoplastics; Small molecules
  • Mechanism of Action MCL1 protein inhibitors
  • Phase I Multiple myeloma
  • 01 Jun 2016 Phase-I clinical trials in Multiple myeloma (Second-line therapy or greater) in USA, Australia (IV) (NCT02675452)
  • 12 Feb 2016 Amgen plans a first-in-human phase I trial for Multiple myeloma (Second-line therapy or greater) in USA, Germany and Australia (IV) (NCT02675452)
  • 22 Dec 2015 Preclinical trials in Multiple myeloma in USA (IV) before December 2015

Inventors Sean P. Brown, Yunxiao Li, Mike Elias Lizarzaburu, Brian S. Lucas, Nick A. Paras, Joshua TAYGERLY, Marc Vimolratana, Xianghong Wang, Ming Yu, Manuel Zancanella, Liusheng Zhu, Buenrostro Ana Gonzalez, Zhihong Li
Applicant Amgen Inc.

Synthesis

1 Kang catalyst used, ie Pyridine, 2,6-bis[(4R)-5,5-dibutyl-4,5-dihydro-4-phenyl-2-oxazolyl]-

2 Martin’s reagent to get CHO group

3 Hydrolysis or Hydrogenolysis of Carboxylic Esters :p-MeC6H4SO3H

4 R:(Me3Si)2NH •Li,

5 Hydrolysis of Acetals CF3SO3H

6 Fe, AcOH CYCLIZATION

7 l-Camphor-SO3H, Na+ •(AcO)3BH-,

8 SOCl2, MeOH ESTERIFICATION

9 OXIDATION

CONTD………..

10 GRIGNARD BuLi, Me(CH2)4Me,

11 Hydrogenolysis of Carboxylic Esters

12 Acylation INVOLVING NITROGEN ATOM

13 CYCLIZATION , Ruthenium, [1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphine)-, (SP-5-41)-

14 METHYL IODIDE, Alkylation TO GET AMD 176

AMG 176

str1 str2

One common characteristic of human cancer is overexpression of Mcl-1. Mcl-loverexpression prevents cancer cells from undergoing programmed cell death (apoptosis), allowing the cells to survive despite widespread genetic damage.

Mcl-1 is a member of the Bcl-2 family of proteins. The Bcl-2 family includes pro-apoptotic members (such as BAX and BAK) which, upon activation, form a homo-oligomer in the outer mitochondrial membrane that leads to pore formation and the escape of mitochondrial contents, a step in triggering apoptosis. Antiapoptotic members of the Bcl-2 family (such as Bcl-2, Bcl-XL, and Mcl-1) block the activity of BAX and BAK. Other proteins (such as BID, BIM, BIK, and BAD) exhibit additional regulatory functions.

Research has shown that Mcl- 1 inhibitors can be useful for the treatment of cancers. MCl-1 is overexpressed in numerous cancers. See Beroukhim et al. (2010) Nature 463, 899-90. Cancer cells containing amplifications surrounding the Mcl-1 and Bcl-2-1-1 anti-apoptotic genes depend on the expression of these genes for survival. Beroukhim et al. Mcl- 1 is a relevant target for the re-iniation of apoptosis in numerous cancer cells. See G. Lessene, P. Czabotar and P.

Colman, Nat. Rev. Drug. Discov., 2008, 7, 989-1000; C. Akgul Cell. Mol. Life

Sci. Vol. 66, 2009; and Arthur M. Mandelin II, Richard M. Pope, Expert Opin. Ther. Targets (2007) l l(3):363-373.

New compositions and methods for preparing and formulating Mcl-1 inhibitors would be useful.

PATENT

WO 2016033486

https://www.google.com/patents/WO2016033486A1?cl=ru

GENERAL SYNTHETIC SCHEMES

General Procedure 1

Intermediates III can be prepared using standard chemistry techniques. For example, cyclobutane carbaldehyde II was combined with oxazepine I in an appropriate solvent at a temperature below RT, preferably about 0°C. Sodium cyanoborohydride was added, and the mixture was added to NaOH solution, to provide compound III.

General Procedure 2

Intermediate AA Intermediate EE IV

Intermediates IV can be prepared using standard peptide like chemistry. For example, DMAP was added to carboxylic acid Intermediate AA and Intermediate EE in an appropriate solvent at a temperature below RT, preferably about 0°C, followed by the addition of EDC hydrochloride. The mixture was warmed to ambient temperature, to provide carboxamide IV.

General Procedure 3

EXAMPLE A

Example A intermediates can be prepared using standard chemistry techniques. For example, carboxamide IV was combined with DCM followed by the addition of Hoveyda-Grubbs II. The mixture was cooled to ambient temperature to provide Example A.

General Procedure 4

Intermediate AA Intermediate EE

Intermediates V can be prepared using standard chemistry techniques. For example, Intermediate AA was combined with Intermediate EE in an appropriate solvent followed by the addition of Hoveyda-Grubbs II to provide compound V.

General Procedure 5

Example A intermediates can be prepared using standard chemistry techniques. For example, N,N-dimethylpyridin-4-amine was combined with compound VI in an appropriate solvent at a temperature below RT, preferably about 0°C, followed by the addition of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride. The resulting mixture warmed to ambient temperature to provide Example A.

General Procedure 6

Example B intermediates can be prepared using standard chemistry techniques. For example, sodium hydride was added to a solution of Example A at a temperature below RT, preferably about 0°C, followed by the addition of Mel. The resulting mixture warmed to ambient temperature to provide Example B.

General Pr

Intermediates such as Example C can be prepared using standard chemistry techniques. For example, Example A and/or B and/or VII and platinum (IV) oxide were combined in an appropriate solvent at ambient temperature to provide Example C.

Compounds of the present invention generally can be prepared combining and further elaborating synthetic intermediates generated from commercially available starting materials. The syntheses of these intermediates are outlined below and further exemplification is found in the specific examples provided.

EXAMPLE 4. (1S,3’R,6’R,7’S,8’E,1 l’S,12’R)-6-CHLORO-7′-METHOXY-11′, 12 ‘-DIMETHYL-3 ,4-DIHYDRO-2H, 15 ‘H-SPIRO [NAPHTHALENE- 1 ,22’-[20]OXA[13]THIA[1,14]DIAZATETRACYCLO[14.7.2.036.01924]PENTACOS A[8, 16, 18,24]TETRAEN]-15′-ONE- 13 ‘, 13 ‘-DIOXIDE

To a slurry of (1 S,3’R,6’R,7’S,8’E, 1 l’S, 12’R)-6-chloro-7′-hydroxy-l l’,12′-dimethyl-3,4-dihydro-2h, 15’h-spiro[naphthalene-l,22′- [20]oxa[13]thia[l, 14]diazatetracyclo[14.7.2.036.01924]pentacosa[8,16, 18,24]tetra en]-15′-one 13 ‘, 13 ‘-dioxide (Example 2; 32.6 g, 49.1 mmol) (containing 9.8% toluene, starting material was not completely soluble in Me-THF) and Mel (15.2 mL, 245 mmol) in Me-THF (820 mL) was added KHMDS (1.0 M in THF, 167 mL, 167 mmol) dropwise for 30 min while maintaining reaction temperature between – 44°C and – 38°C under N2. After the mixture was stirred at – 44°C for 30 min, the reaction was allowed to warm to rt and stirred for 1.5 h (LC/MS confirmed the reaction was complete). The reaction mixture was cooled to 5°C, quenched (170 mL of sat. aqueous NH4C1 and 170 mL of FLO) while maintaining temperature between 5°C and 14°C, and acidified (340 mL of 10% aqueous citric acid). The organic layer was separated and the aqueous layer was back-extracted with EtOAc (500 mL). The combined organic layers were washed with brine (3 x 500 mL), dried (MgS04), and concentrated under reduced pressure to provide a crude target compound (30.1 g, 49.1 mmol, quantitatively) (purity >98% with no over 1% major impurity from HPLC) as a bright yellow solid. After the same scale reaction was repeated four times, all the crude products (4 x 49.1 mmol = 196 mmol) were dissolved in EtOAc, combined, and concentrated under reduced pressure. Then the combined crude product was recrystallized as follows:

ethanol (800 mL) was added to the crude product and the resulting slurry solution was shaken while heating the solution for 20 min. H20 (250 mL) was added dropwise for 30 min at rt and the slurry was cooled down to 0°C. After the slurry was kept in an ice bath for 4 h, the solid product was filtered through filter paper. The filter cake was rinsed with ice-cold 30% FLO in EtOH (300 mL) and air-dried for 2 days. The product was further dried under high vacuum at 40°C for 4 days to provide the pure target compound (1 15 g, 188 mmol, 96 % yield) as a

white solid. XH NMR (600 MHz, DMSO-i¾) δ 11.91 (s, 1 H), 7.65 (d, J= 8.6 Hz, 1 H), 7.27 (dd, J= 8.5, 2.3 Hz, 1 H), 7.17 (d, J= 2.4 Hz, 1 H), 7.04 (dd, J= 8.2, 2.0 Hz, 1 H), 6.90 (d, J= 8.2 Hz, 1 H), 6.76 (d, J= 1.8 Hz, 1 H), 5.71 (ddd, J= 15.1, 9.7, 3.5 Hz, 1 H), 5.50 (ddd, J= 15.2, 9.2, 1.1 Hz, 1 H), 4.08 (qd, J= 7.2, 7.2, 7.2, 1.5 Hz, 1 H), 4.04 (d, J= 12.3 Hz, 1 H), 3.99 (d, J= 12.3 Hz, 1 H), 3.73 (d, J= 14.9 Hz, 1 H), 3.56 (d, J= 14.1 Hz, 1 H), 3.53 (dd, J= 9.1, 3.3 Hz, 1 H), 3.19 (d, J= 14.1 Hz, 1 H), 3.09 (s, 3 H), 3.03 (dd, J= 15.4, 10.4 Hz, 1 H), 2.79 (dt, J= 17.0, 3.5, 3.5 Hz, 1 H), 2.69 (ddd, J= 17.0, 10.7, 6.3 Hz, 1 H), 2.44-2.36 (m, 1 H), 2.24-2.12 (m, 2 H), 2.09 (ddd, J= 15.5, 9.6, 2.3 Hz, 1 H), 1.97 (dt, J = 13.6, 3.6, 3.6 Hz, 1 H), 1.91-1.80 (m, 4 H), 1.80-1.66 (m, 3 H), 1.38 (td, J= 12.3, 12.3, 3.5 Hz, 1 H), 1.33 (d, J= 7.2 Hz, 3 H), 0.95 (d, J= 6.8 Hz, 3 H); [cc]D (24°C, c = 0.0103 g/mL, DCM) = – 86.07 °; m.p. 222.6 – 226.0°C; FT-IR (KBr): 3230 (b), 2931 (b), 1688 (s), 1598 (s), 1570 (s), 1505 (s), 1435 (s), 1384 (s), 1335 (s), 1307 (s), 1259 (s), 1155 (s), 1113 (s), 877 (s), 736 (s) cm“1; Anal. Calcd. for C33H41CIN2O5S: C, 64.64; H, 6.74; N, 4.57; CI, 5.78; S, 5.23. Found: C, 64.71; H, 6.81; N, 4.65; CI, 5.81; S, 5.11; HRMS (ESI) m/z 613.2493 [M + H]+ (C33H41CIN2O5S requires 613.2503).

The mother liquor was concentrated under reduced pressure and further purification of the residue by flash column chromatography (200 g S1O2, 10% and 10% to 45% and 45% EtO A/Hex w/ 0.3% AcOH, gradient elution) provided additional pure product (3.1 g, 5.1 mmol, 2.6%) as an off-white solid.

EXAMPLE 5. (1S,3’R,6’R,7’S,8’Z,1 l’S,12’R)-6-CHLORO-7′-METHOXY-11 ‘, 12 ‘-DIMETHYL-3 ,4-DIHYDRO-2H, 15 Ή-SPIRO [NAPHTHALENE- 1 ,22’-[20]OXA[13]THIA[1,14]DIAZATETRACYCLO[14.7.2.036.01924]PENTACOS A[8, 16, 18,24]TETRAEN]- 15′-ONE 13 ‘, 13’-DIOXIDE

To a solution of (1S,3’R,6’R,7’S,8’Z,1 l’S,12’R)-6-chloro-7′-hydroxy-i r,12′-dimethyl-3,4-dihydro-2h,15’h-spiro[naphthalene-l,22′-[20]oxa[13]thia[l,14]diazatetracyclo[14.7.2.036.01924]pentacosa[8,16,18,24]tetra en]-15′-one 13 ‘,13 ‘-dioxide (Example 3; 34 mg; 0.057 mmol) in THF cooled to 0°C was added sodium hydride (60% dispersion in mineral oil; 22.70 mg, 0.567 mmol). The reaction mixture was stirred at 0 °C for 20 min, and then Mel (0.018 mL, 0.284 mmol) was added. The reaction mixture was stirred at ambient temperature for 1 h, then quenched with aqueous NH4CI, and diluted with

EtOAc. The organic layer was dried over MgS04 and concentrated. Purification of the crude material via column chromatography eluting with 10-40 % EtOAc (containing 0.3% AcOH)/heptanes provided (lS,3’R,6’R,7’S,8’Z,l l’S,12’R)-6-chloro-7′-methoxy-l l’,12′-dimethyl-3,4-dihydro-2h,15’h-spiro[naphthalene-l,22′-[20]oxa[13]thia[l,14]diazatetracyclo[14.7.2.036.01924]pentacosa[8,16,18,24]tetra en]-15′-one 13 ‘,13 ‘-dioxide (34 mg, 0.054 mmol, 95% yield). ¾ NMR (400MHz, CD2C12) δ 8.29 (s, 1H), 7.71 (d, J=8.4 Hz, 1H), 7.17 (dd, J=2.2, 8.5 Hz, 1H), 7.09 (d, J=2.3 Hz, 1H), 7.01 (dd, J=1.6, 7.8 Hz, 1H), 6.92 (d, J=8.2 Hz, 1H), 6.88 (s, 1H), 5.90 – 5.80 (m, 1H), 5.54 (t, J=10.2 Hz, 1H), 4.14 – 4.04 (m, 3H), 3.87 – 3.79 (m, 2H), 3.73 (d, J=14.7 Hz, 1H), 3.32 (d, J=14.5 Hz, 1H), 3.23 (s, 3H), 3.28 -3.19 (m, 1H), 2.82 – 2.73 (m, 2H), 2.62 (t, J=10.6 Hz, 1H), 2.55 – 2.44 (m, 1H), 2.29 – 2.21 (m, 1H), 2.10 – 1.97 (m, 4H), 1.97 – 1.80 (m, 4H), 1.75 (dd, J=8.9, 18.7 Hz, 1H), 1.48 (d, J=7.4 Hz, 3H), 1.43 (br. s., 1H), 1.08 (d, J=6.5 Hz, 3H). MS (ESI, +ve ion) m/z 613.3 (M+H)+.

//////////////AMG 176, PHASE 1,  Amgen, Mcl-1 inhibitor,  tumors

Last talk in AM 1st time disclosures is from Sean Brown of @Amgen on an Mcl-1 inhibitor to treat tumors

str1

Clc5cc6CCC[C@@]4(CN2C[C@H]1CC[C@H]1[C@H](OC)C=CC[C@@H](C)[C@H](C)S(=O)(=O)NC(=O)c3cc2c(cc3)OC4)c6cc5

AMG-3969


Image result for amg 3969

AMG-3969

M.Wt: 522.46
Cas : 1361224-53-4 , MF: C21H20F6N4O3S

WO 2012027261 PRODUCT PATENT

Inventors Kate Ashton, Michael David Bartberger, Yunxin Bo, Marian C. Bryan, Michael Croghan, Christopher Harold Fotsch, Clarence Henderson Hale, Roxanne Kay Kunz, Longbin Liu, Nobuko Nishimura, Mark H. Norman, Lewis Dale Pennington, Steve Fong Poon, Markian Myroslaw Stec, Jean David Joseph St., Jr., Nuria A. Tamayo, Christopher Michael Tegley, Kevin Chao Yang
Applicant Amgen Inc.

2-[4-[(2S)-4-[(6-Amino-3-pyridinyl)sulfonyl]-2-(1-propyn-1-yl)-1-piperazinyl]phenyl]-1,1,1,3,3,3-hexafluoro-2-propanol)

(S)-2-(4-(4-((6-Aminopyridin-3-yl)sulfonyl)-2-(prop-1-yn-1-yl)piperazin-1-yl)phenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol,

mp 113–123 °C;
[α]D20 = +75.1 (c = 2.2, MeOH).
Agents for Type 2 Diabetes,  PRECLINICAL

AMG-3969, a novel and stable small-molecule disruptor of glucokinase (GK) and glucokinase regulatory protein (GKRP) interaction by the optimization of initial screening hit and AMG-1694. AMG-3969 potently induced the dissociation of the GK-GKRP complex and promoted GK translocation both in-vitro and in-vivo. In rodent model of diabetes, AMG-3969 reduced blood glucose levels without affecting euglycemic animals. The study represents the first successful discovery of a small molecule that targets the GK-GKRP complex as a novel pathway for managing blood glucose levels with reduced hypoglycemic risk.

Image result for AMGEN

 Kate Ashton

Kate Ashton

Senior Scientist at Amgen, Inc

Amgen
Thousand Oaks, United States
Dr. Kate Ashton received a Masters in Chemistry with Industrial Experience from the University of Edinburgh. She conducted her PhD thesis research on the synthesis and structure elucidation of Reidispongiolide A with Prof. Ian Paterson at the University of Cambridge, and her postdoctoral work on SOMO catalysis with Prof. David W. C. MacMillan at both Caltech and Princeton. She has been at Amgen for 6 years and has worked on indications for cancer, Alzheimer’s and diabetes.Dr Fecke works in the area of industrial early drug discovery since 1996. He is currently Group Leader in the Primary Pharmacology department at UCB Pharma (UK) and is involved in the identification and characterization of NCE and NBE drugs in molecular interaction assays for both immunological and CNS diseases. Prior to joining UCB, he worked for Novartis and Siena Biotech in the areas of transplant rejection, neurodegeneration and oncology. He obtained his PhD at the Heinrich-Heine-University Dusseldorf in Germany in 1994.

Image result for amg 3969

(S)-2-(4-(4-((6-Aminopyridin-3-yl)sulfonyl)-2-(prop-1-yn-1-yl)piperazin-1-yl)phenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol, AMG-3969

Glucokinase (GK) is a member of a family of four hexokinases that are critical in the cellular metabolism of glucose. Specifically GK, also known as hexokinase IV or hexokinase D, facilitates glucose induced insulin secretion from pancreatic β-cells as well as glucose conversion into glycogen in the liver. GK has a unique catalytic activity that enables the enzyme to be active within the physiological range of glucose (from 5mM glucose to lOmM glucose).

Genetically modified mouse models support the role of GK playing an important role in glucose homeostasis. Mice lacking both copies of the GK gene die soon after birth from severe hyperglycemia, whereas mice lacking only one copy of the GK gene present with only mild diabetes. Mice that are made to overexpress the GK gene in their livers are hypoglycemic.

Numerous human mutations in the GK gene have been identified, with the vast majority of them resulting in proteins with impaired or absent enzymatic activity. These loss-of-function mutations are thought to contribute to the hyperglycemia seen with maturity-onset diabetes of the young type II (MODY-2). A small fraction of these mutations result in a GK with increased catalytic function. These individuals present with moderate to severe hypoglycemia.

GK activity in the liver is transiently regulated by glucokinase regulatory protein (GKRP). GK catalytic activity is inhibited when GK is bound to GKRP. This interaction is antagonized by increasing concentrations of both glucose and fructose -1 -phosphate (F1P). The complex of the two proteins is localized primarily to the nuclear compartment of a cell. Post prandially as both glucose and fructose levels rise, GK released from GKRP translocates to the cytoplasm. Cytoplasmic GK is now free of the inhibitory effects of GKRP and able to kinetically respond to glucose. Evidence from the Zucker diabetic fatty rat (ZDF) indicates that their glucose intolerance may be a result of this mechanism failing to function properly.

A compound that acts directly on GKRP to disrupt its interaction with GK and hence elevate levels of cytoplasmic GK is a viable approach to modulate GK activity. Such an approach would avoid the unwanted hypoglycemic effects of over stimulation of GK catalytic activity, which has been seen in the

development of GK activators. A compound having such an effect would be useful in the treatment of diabetes and other diseases and/or conditions in which GKRP and/or GK plays a role.

CLIP

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors
Nature 2013, 504(7480): 437

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SYNTHESIS

Figure

aReagents and conditions: (a) 1-propynylmagnesium bromide, THF, 0 °C, 99%; (b) TFA, DCM, then NaBH(OAc)3 77%; (c) NH4OH, EtOH, 120 °C, 88%; (d) chiral SFC, 38%………..Nature 2013,504, 437440

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2012027261

EXAMPLE 241 : 2-(4-(4-((6-AMINO-3-PYRIDINYL)SULFONYL)-2-(l-PROP YN- 1 – YL)- 1 -PIPERAZINYL)PHENYL)- 1,1,1 ,3 ,3 ,3 -HEXAFLUORO-2-PROPANOL

STEP 1 : 4-BENZYL 1 -TERT-BUTYL 2-0X0-1,4-PIPERAZINEDICARBOXYLATE

A 2-L Erlenmeyer flask was charged with 2-piperazinone (36.5 g, 364 mmol, Sigma- Aldrich, St. Louis, MO), sodium carbonate (116 g, 1093 mmol), 600 mL of dioxane, and 150 mL of water. To this was slowly added benzyl chloroformate (62.1 g, 364 mmol, Sigma-Aldrich, St. Louis, MO) at room temperature over 20 min. After the addition was complete, the mixture was stirred for 2 h and then diluted with water and extracted with EtOAc (2 L). The combined organic extracts were dried (MgS04), filtered, and concentrated to give a white solid. To this solid was added 500 mL of DCM, triethylamine (128 mL, 911 mmol), DMAP (4.45 g, 36.4 mmol), and di-tert-butyl dicarbonate (119 g, 546 mmol, Sigma-Aldrich, St. Louis, MO). After 1 h at room temperature, the mixture was diluted with water and the organics were separated. The organics were dried (MgS04), filtered, and concentrated to give a brown oil. To this oil was added 100 mL of DCM followed by 1 L of hexane. The resulting white solid was collected by filtration to give 4-benzyl 1-tert-butyl 2-oxo-l,4-piperazinedicarboxylate (101 g).

STEP 2: BENZYL (2-((TERT-BUTOXYCARBONYL)AMINO)ETHYL)(2-OXO-3 -PENTYN- 1 -YL)CARBAMATE

A 150-mL round-bottomed flask was charged with 4-benzyl 1-tert-butyl

2- oxo-l,4-piperazinedicarboxylate (1.41 g, 4.22 mmol) and THF (5 mL). 1-Propynylmagnesium bromide (0.5 M in THF, 20.0 mL, 10.0 mmol, Sigma-Aldrich, St. Louis, MO) was added at 0 °C slowly. The mixture was stirred at 0 °C for 2 h. Saturated aqueous NH4C1 (40 mL) was added and the aqueous phase was extracted with EtOAc (200 mL, then 2 x 100 mL). The combined organic phases were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography (50 g of silica, 0 to 50% EtOAc in hexanes) to afford benzyl (2-((tert-butoxycarbonyl)amino)ethyl)(2-oxo- 3- pentyn-l-yl)carbamate (1.55 g) as a clear oil.

STEP 3: BENZYL 3-(l-PROPYN-l-YL)-l-PIPERAZINECARBOXYLATE

A 3-L round-bottomed flask was charged with 2-((tert-butoxycarbonyl)amino)ethyl)(2-oxo-3-pentyn-l-yl)carbamate (82.2 g, 219 mmol) and 300 mL of DCM. After cooling to -10 °C, TFA (169 mL, 2195 mmol) was added and the resulting dark solution was stirred at room temperature for 15 min. Sodium triacetoxyborohydride (186 g, 878 mmol, Sigma-Aldrich, St. Louis, MO) was then added portion- wise over 10 min. After 2 h, the mixture was

concentrated, diluted with EtOAc (1 L), and neutralized with 5 N NaOH. The layers were separated and the organic extracts were washed with brine, dried (MgS04), filtered and concentrated. The resulting orange oil was purified via column chromatography (750 g of silica gel, 0 to 4.5 % MeOH/DCM) to give benzyl 3-(l-propyn-l-yl)-l-piperazinecarboxylate (43.7 g) as a brown foam.

STEP 4: BENZYL 3-(l-PROPYN-l-YL)-4-(4-(2,2,2-TRIFLUORO-l-HYDROXY- 1 -(TRIFLUOROMETHYL)ETHYL)PHENYL)- 1 -PIPERAZINECARBOXYLATE

A 150-mL reaction vessel was charged with benzyl 3-(prop-l-yn-l-yl)piperazine-l-carboxylate (2.88 g, 11.2 mmol), 2-(4-bromophenyl)-l, 1,1, 3,3,3-hexafluoropropan-2-ol (4.36 g, 13.5 mmol, Bioorg. Med. Chem. Lett. 2002, 12, 3009), dicyclohexyl(2′,6′-diisopropoxy-[ 1 , 1 ‘-biphenyl]-2-yl)phosphine, RuPhos (0.530 g, 1.14 mmol, Sigma- Aldrich, St. Louis, MO), RuPhos Palladacycle (0.417 g, 0.572 mmol, Strem Chemical Inc, Newburyport, MA), sodium tert-butoxide (2.73 g, 28.4 mmol, Strem Chemical Inc, Newburyport, MA) and toluene (35 mL). The mixture was degassed by bubbling Ar through the solution for 10 min. The vessel was sealed and heated at 100 °C for 1.5 h. The reaction mixture was cooled to room temerature and water (100 mL) was added. The aqueous phase was extracted with EtOAc (3 x 100 mL) and the combined organic phases were washed with saturated aqueous sodium chloride (150 mL). The organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography (100 g of silica, 0 to 50% EtOAc in hexanes) to afford benzyl 3-(l-propyn-l-yl)-4-(4-(2,2,2-trifluoro- 1 -hydroxy- 1 -(trifluoromethyl)ethyl)phenyl)- 1 -piperazinecarboxylate as a yellow solid.

STEP 5: 2-(4-(4-((6-CHLORO-3-PYRIDINYL)SULFONYL)-2-(l-PROPYN-l-YL)- 1 -PIPERAZIN YL)PHENYL)- 1,1,1 ,3 ,3 ,3 -HEXAFLUORO-2-PROPANOL

A 500-mL round-bottomed flask was charged with benzyl 3-(l-propyn-l-yl)-4-(4-(2,2,2-trifluoro- 1 -hydroxy- 1 -(trifluoromethyl)ethyl)phenyl)- 1 -piperazinecarboxylate (3.13 g, 6.25 mmol) and TFA (40 mL).

Trifluoromethanesulfonic acid (1.25 mL, 14.1 mmol, Acros/Fisher Scientific, Waltham, MA) was added dropwise at room temperature. After 5 min, additional TfOH (0.45 mL, 5.1 mmol) was added. After an additional 10 min, solid

NaHC03 was carefully added in potions. Saturated aqueous NaHC03 (250 mL) was added slowly to bring pH to approximately 7. The aqueous phase was extracted with EtOAc (100 mL). At this time, more solid NaHC03 was added to the aqueous phase and extracted again with EtOAc (100 mL). The combined organic phases were washed with water (200 mL) and saturated aqueous sodium chloride (200 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo to afford 3.10 g of tan solid.

A 500-mL round-bottomed flask was charged with this material, triethylamine (5.00 mL, 35.9 mmol) and CH2CI2 (30 mL). 6-Chloropyridine-3-sulfonyl chloride (1.58 g, 7.43 mmol, Organic Process Research & Development 2009, 13, 875) was added in potions at 0 °C. The brown mixture was stirred at 0 °C for 10 min. The volume of the reaction mixture was reduced to approximately 10 mL in vacuo then the mixture was purified twice by column chromatography (100 g of silica, 0 to 50% EtOAc in hexanes) to afford 2-(4-(4-((6-chloro-3-pyridinyl)sulfonyl)-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)- 1,1,1,3,3,3-hexafluoro-2-propanol (3.46 g) as an off-white solid.

STEP 6: 2-(4-(4-((6-AMINO-3-PYRIDINYL)SULFONYL)-2-(l-PROPYN-l-YL)- 1 -PIPERAZIN YL)PHENYL)- 1,1,1 ,3 ,3 ,3 -HEXAFLUORO-2-PROPANOL

A 20-mL sealed tube was charged with 2-(4-(4-((6-chloro-3-pyridinyl)sulfonyl)-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)- 1,1,1,3,3,3-hexafluoro-2-propanol (0.340 g, 0.627 mmol), concentrated ammonium hydroxide (5.00 mL, 38.5 mmol) and EtOH (5 mL). The reaction mixture was heated in an Initiator (Biotage, AB, Uppsala, Sweden) at 120 °C for 1 h. The reaction mixture was further heated in a heating block at 110 °C for 5 h. The reaction mixture was concentrated and purified by column chromatography (25 g of silica, 30 to 80% EtOAc in hexanes) to afford 2-(4-(4-((6-amino-3-pyridinyl)sulfonyl)-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)- 1,1,1,3,3,3-hexafluoro-2-propanol (0.289 g) as a mixture of two enantiomers.

1H NMR (400 MHz, CDC13) δ ppm 8.49 (br. s., 1 H), 7.80 (dd, J= 2.3, 8.8 Hz, 1 H), 7.59 (d, J= 8.8 Hz, 2 H), 6.97 (d, J= 9.0 Hz, 2 H), 6.55 (d, J= 8.8 Hz, 1 H), 5.05 (s, 2 H), 4.46 (br. s., 1 H), 3.85 – 3.72 (m, 2 H), 3.54 (br. s., 1 H), 3.50 – 3.34 (m, 2 H), 2.83 (dd, J= 3.3, 11.0 Hz, 1 H), 2.69 (dt, J= 3.4, 11.0 Hz, 1 H), 1.80 (s, 3 H). m/z (ESI, +ve ion) 523.1 (M+H)+. GK-GKRP IC50 (Binding) = 0.003 μΜ

The individual enantiomers were isolated using chiral SFC. The method used was as follows: Chiralpak® ADH column (21 x 250 mm, 5 μιη) using 35% methanol in supercritical C02 (total flow was 70 mL/min). This produced the two enantiomers with enantiomeric excesses greater than 98%.

2-(4-((2S)-4-((6-amino-3-pyridinyl)sulfonyl)-2-(l -propyn- 1-yl)- 1 -piperazinyl)phenyl)- 1,1,1 ,3 ,3 ,3 -hexafluoro-2-propanol and 2-(4-((2R)-4-((6-amino-3 -pyridinyl)sulfonyl)-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)- 1,1,1,3,3,3-hexafluoro-2-propanol.

FIRST ELUTING PEAK (PEAK #1)

1H NMR (400 MHz, CDC13) δ 8.48 (d, J= 2.3 Hz, 1 H), 7.77 (dd, J= 2.5, 8.8 Hz, 1 H), 7.57 (d, J= 8.8 Hz, 2 H), 6.95 (d, J= 9.2 Hz, 2 H), 6.52 (d, J= 8.8 Hz, 1 H), 4.94 (s, 2 H), 4.44 (br. s., 1 H), 3.82 – 3.71 (m, 2 H), 3.58 – 3.33 (m, 3 H), 2.81 (dd, J= 3.2, 11.1 Hz, 1 H), 2.67 (dt, J= 3.9, 11.0 Hz, 1 H), 1.78 (d, J = 2.2 Hz, 3 H). m/z (ESI, +ve ion) 523.2 (M+H)+. GK-GKRP IC50 (Binding) = 0.002 μΜ.

SECOND ELUTING PEAK (PEAK #2)

1H NMR (400 MHz, CDC13) δ 8.49 (d, J= 1.8 Hz, 1 H), 7.78 (dd, J= 2.3, 8.8 Hz, 1 H), 7.59 (d, J= 8.6 Hz, 2 H), 6.97 (d, J= 9.0 Hz, 2 H), 6.54 (d, J= 8.8 Hz, 1 H), 4.97 (s, 2 H), 4.46 (br. s., 1 H), 3.77 (t, J= 11.7 Hz, 2 H), 3.67 (br. s., 1 H), 3.51 – 3.33 (m, 2 H), 2.82 (dd, J= 3.3, 11.0 Hz, 1 H), 2.68 (dt, J= 3.9, 11.1 Hz, 1 H), 1.79 (d, J= 2.0 Hz, 3 H). m/z (ESI, +ve ion) 523.2 (M+H)+. GK-GKRP IC50 (Binding) = 0.342 μΜ.

Alternative procedure starting after Step 4.

STEP 5 : 2-(4-(4-((6-AMINO-3-PYRIDINYL)SULFONYL)-2-(l-PROPYN-l-YL)- 1 -PIPERAZIN YL)PHENYL)- 1,1,1 ,3 ,3 ,3 -HEXAFLUORO-2-PROPANOL

Alternatively, 2-(4-(4-((6-amino-3-pyridinyl)sulfonyl)-2-( 1 -propyn- 1 -yl)-l-piperazinyl)phenyl)-l,l,l,3,3,3-hexafluoro-2-propanol was synthesized from benzyl 3-( 1 -propyn- 1 -yl)-4-(4-(2,2,2-trifluoro- 1 -hydroxy- 1 -(trifluoromethyl)ethyl)phenyl)- 1 -piperazinecarboxylate as follows.

A 2-L round-bottomed flask was charged with benzyl 3 -(1 -propyn- 1-yl)-4-(4-(2,2,2-trifluoro- 1 -hydroxy- 1 -(trifluoromethyl)ethyl)phenyl)- 1 -piperazinecarboxylate (21.8 g, 43.5 mmol, step 5) and TFA (130 mL).

Trifluoromethanesulfonic acid (11.6 mL, 131 mmol, Acros/Fisher Scientific, Waltham, MA) was added slowly at rt resulting orange cloudy mixture. After stirring at rt for 10 min, the volume of the reaction mixture was reduced to half in vacuo. Solid NaHC03 was added in potions until the mixture became sludge. Saturated aqueous NaHC03(800 mL) was added slowly until the pH was about

8. The aqueous phase was extracted with EtOAc (3 x 250 mL). The combined organic phases were washed with water (500 mL) and saturated aqueous NaCl (500 mL). The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. This material was dissolved into DCM (200 mL) and triethylamine (31.0 mL, 222 mmol) was added. Then 6-aminopyridine-3-sulfonyl chloride (9.40 g, 48.8 mmol, published PCT patent application no. WO

2009/140309) was added in potions over 10 min period. The brown mixture was stirred at room temperature for 10 min. The reaction mixture was washed with water (300 mL) and saturated aqueous NaCl (300 mL). The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography (780 g of total silica, 30 to 90% EtOAc in hexanes) to afford 2-(4-(4-((6-amino-3-pyridinyl)sulfonyl)-2-(l-propyn-l-yl)-l-piperazinyl)phenyl)-l,l,l,3,3,3-hexafluoro-2-propanol (19.4 g) as a mixture of two enantiomers.

Paper

Nonracemic Synthesis of GK–GKRP Disruptor AMG-3969

Therapeutic Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
Amgen Inc. 360 Binney Street, Cambridge, Massachusetts 02142, United States
J. Org. Chem., 2014, 79 (8), pp 3684–3687

Abstract Image

A nonracemic synthesis of the glucokinase–glucokinase regulatory protein disruptor AMG-3969 (5) is reported. Key features of the synthetic approach are an asymmetric synthesis of the 2-alkynyl piperazine core via a base-promoted isomerization and a revised approach to the synthesis of the aminopyridinesulfonamide with an improved safety profile.

(S)-2-(4-(4-((6-Aminopyridin-3-yl)sulfonyl)-2-(prop-1-yn-1-yl)piperazin-1-yl)phenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol, AMG-3969 (5)

(S)-2-(4-(4-((6-aminopyridin-3-yl)sulfonyl)-2-(prop-1-yn-1-yl)piperazin-1-yl)phenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (5) (64.0 g, 49% yield) as white solid. The enanatiomeric excess was found to be >99.5% by chiral SFC (see Supporting Information):
1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1 H), 7.79 (d, J = 8.6 Hz, 1 H), 7.59 (d, J = 8.2 Hz, 2 H), 6.97 (d, J = 8.6 Hz, 2 H), 6.55 (d, J = 8.8 Hz, 1 H), 5.06 (br s, 2 H), 4.45 (br s, 1 H), 3.96 (br s, 1 H), 3.77 (t, J = 12.1 Hz, 2 H), 3.50–3.35 (m, 2 H), 2.82 (d, J = 11.0 Hz, 1 H), 2.68 (t, J = 10.9 Hz, 1 H), 1.79 (s, 3 H);
13C NMR (101 MHz, CD3OD) δ 163.8, 152.0, 150.1, 138.2, 129.0, 124.7 (q), 123.9, 121.1, 117.5, 109.3, 82.8, 78.3 (m), 75.5, 52.0, 47.2, 44.9, 3.2;
 
HRMS (ESI-TOF) m/z [M + H]+calcd for C21H21F6N4O3S 523.1239, found 523.1229;
 
mp 113–123 °C;
 
[α]D20 = +75.1 (c = 2.2, MeOH).
 

Clip

AMG-3969 is a disruptor of the glucokinase (GK)–glucokinase regulatory protein (GKRP) protein–protein interaction. Bourbeau and co-workers at Amgen describe their efforts towards an asymmetric synthesis of this compound ( J. Org. Chem. 2014, 79, 3684). The discovery route to this compound involved seven steps (14% overall yield), had certain safety concerns and relied upon SFC separation of the API enantiomers. The new route requires five steps (26% overall yield) and delivers the API in excellent enantiomeric excess (99% ee). A key feature of the synthetic approach was an asymmetric synthesis of the 2-alkynylpiperazine core via a base-promoted isomerization. It was found that the strongly basic conditions employed for the “alkyne-walk” did not erode the previously established stereocenter. Also, safety concerns around a late-stage amination of a 2-chloropyridine intermediate in the discovery route were alleviated by starting with a Boc-protected diaminopyridine instead.
PATENT

INTERMEDIATE A: TERT-EUTYL (5-(CHLOROSULFONYL)-2-PYRIDINYL)CARBAMATE

0,N

STEP 1 : TERT-BUTY (5-NITRO-2-PYRIDINYL)CARBAMATE

A 3-L round-bottomed flask was charged with 5-nitro-2-pyridinamine (75.0 g, 539 mmol, Alfa Aesar, Ward Hill, MA) and 500 mL of DCM. To this was added triethylamine (82 g, 810 mmol), di-tert-butyl dicarbonate (129 g, 593 mmol, Sigma-Aldrich, St. Louis, MO), and N,N-dimethylpyridin-4-amine (32.9 g, 270 mmol, Sigma-Aldrich, St. Louis, MO). After stirring at rt for 18 h, the mixture was diluted with water and the solid was collected by filtration. The yellow solid was washed with MeOH to give tert-butyl (5-nitro-2-pyridinyl)carbamate (94.6 g) as a light yellow solid.

STEP 2: TERT-BUTY (5 – AMINO-2-P YRIDINYL)C ARB AM ATE

A 3-L round-bottomed flask was charged with tert-butyl (5-nitro-2-pyridinyl)carbamate (96.4 g, 403 mmol), 500 mL of MeOH, 500 mL of THF, and 100 mL of sat aq NH4Cl. Zinc (105 g, 1610 mmol, Strem Chemical Inc, Newburyport, MA) was slowly added (over 10 min) to this solution. The mixture was stirred at room temperature for 12 h, then filtered. The filtrate was concentrated and then diluted with EtOAc and washed with water. The organic extracts were dried over MgS04, filtered, and concentrated. The resulting solid was recrystallized from MeOH to give tert-butyl(5-amino-2-pyridinyl)carbamate (38.6 g) as a light-yellow solid.

STEP 3: TERT-BUTYL (5-(CHLOROSULFONYL)-2-PYRIDINYL)CARBAMATE

A 3-L round-bottomed flask was charged with sodium nitrite (15.3 g, 221 mmol, J. T. Baker, Philipsburg, NJ), 100 mL of water and 500 mL of MeCN. After cooling to 0 °C, cone, hydrochloric acid (231 mL, 2770 mmol) was slowly added keeping the internal temperature below 10 °C. After stirring at 0 °C for 10 min, tert-butyl (5-amino-2-pyridinyl)carbamate (38.6 g, 184 mmol) was added as a suspension in MeCN (200 mL). The mixture was stirred for 30 min, then 150 mL of AcOH, copper(ii) chloride (12.4 g, 92.2 mmol, Sigma-Aldrich, St. Louis, MO), and copper(i) chloride (0.183 g, 1.85 mmol, Strem Chemical Inc,

Newburyport, MA) were added. S02 gas (Sigma-Aldrich, St. Louis, MO) was bubbled through the solution for 15 min. The mixture was stirred at 0 °C for 30 min, then about 500 mL of ice-cold water was added. The resulting precipitate was collected by filtration and dried over MgS04 to give tert-butyl (5-(chlorosulfonyl)-2-pyridinyl)carbamate (15.5 g) as a white solid.

1H NMR (400MHz, CDC13) δ ppm 8.93 (br s, 1 H), 8.63 – 8.42 (m, 1 H), 8.35 -7.94 (m, 2 H), 1.58 (s, 9 H).

INTERMEDIATE B: (3S)-l-BENZYL-3-(l-PROPYN-l-YL)PIPERAZINE

STEP 1 : (3S)-l-BENZYL-3-(2-PROPYN-l-YL)-2,5-PIPERAZINEDIONE

A 1-L round-bottoemd flask was charged with (S)-2-((tert-butoxycarbonyl)amino)pent-4-ynoic acid (42.0 g, 197 mmol, AK Scientific, Union City, CA), ethyl 2-(benzylamino)acetate (40.0 g, 207 mmol, Sigma-Aldrich, St. Louis, MO), HATU (90 g, 240 mmol, Oakwood Products, West Columbia, SC) and 200 mL of DMF. To this was added N-ethyl-N-isopropylpropan-2-amine (51.5 ml, 296 mmol, Sigma-Aldrich, St. Louis, MO). After 15 min of stirring at rt, the mixture was diluted with water 300 mL and extracted with 1 L of 20% EtOAc in diethyl ether. The layers were separated and the organic was washed with 2 M HCl, water, sat. aq. NaHC03 and brine. The extracts were dried and concentrated to give an off-white solid. To this was added 200 mL of DCM and TFA (152 ml, 1970 mmol, Sigma-Aldrich, St. Louis, MO). After stirring at rt for 30 min, the mixture was concentrated and then azetroped with 100 mL toluene (twice). To the brown oil obtained was added ammonia (2 M in MeOH, 394 ml, 789 mmol, Sigma-Aldrich, St. Louis, MO). The mixture was stirred at rt for 30 min. The mixture was concentrated, dissolved in EtOAc, and washed with water. The organics were dried (MgS04), filtered, and concentrated to give a white solid that was triturated with diethyl ether to give (S)-l-benzyl-3-(prop-2-yn-l-yl)piperazine-2,5-dione (37.3 g) as a white solid.

STEP 2: (3S)-l-BENZYL-3-(2-PROPYN-l-YL)PIPERAZINE

A 1-L round-bottomed flask was charged with (S)-l-benzyl-3-(prop-2-yn-l-yl)piperazine-2,5-dione (37.3 g, 154 mmol) and 150 mL of THF. To this was slowly added aluminum (III) lithium hydride (1M in THF, 539 ml, 539 mmol, Sigma-Aldrich, St. Louis, MO). After the addition was complete the mixture was heated at 80 °C for 12 h. The mixture was then cooled to 0 °C and solid sodium sulfate decahydrate was added until bubbling ceased. The mixture was filtered and the filtrate was concentrated to give (S)-l-benzyl-3-(prop-2-yn-l-yl)piperazine (18.1 g) as a yellow oil.

STEP 3: (35)-l-BENZYL-3-(l-PROPYN-l-YL)PIPERAZINE

To a solution of (35)-l-benzyl-3-(2-propyn-l-yl)piperazine (2.3 g, 11 mmol) in THF (50 mL) was added potassium t-butoxide (2.41 g, 21.5 mmol, Sigma-Aldrich, St. Louis, MO). The reaction mixture was stirred at rt for 30 min, then quenched with water (200 mL) and EtOAc (300 mL) was added. The organic phase was dried over sodium sulfate, filtered and concentrated under a vacuum to give a solid that was purified by silica gel column chromatography (0 to 10% MeOH in CH2CI2) and then recrystallized from hexanes to afford (35)- 1-benzyl-3-(l-propyn-l-yl)piperazine (2.16 g) as an off-white solid.

1H NMR (400MHz, CD3OD) δ ppm 7.42 – 7.21 (m, 5 H), 3.59 – 3.49 (m, 3 H), 2.93 (td, J= 2.9, 12.4 Hz, 1 H), 2.86 – 2.73 (m, 2 H), 2.68 (d, J= 11.3 Hz, 1 H), 2.22 – 2.04 (m, 2 H), 1.80 (d, J= 2.3 Hz, 3 H).

INTERMEDIATE C: N,N-BIS(4-METHOXYBENZYL)-5-(((35)-3-(l-PROPYN- 1 – YL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDIN AMINE

STEP 1 : (35)-l-((6-CHLORO-3-PYRIDINYL)SULFONYL)-3-(l-PROPYN-l-YL)PIPERAZINE

To a stirred solution of benzyl (35)-3-(l-propyn-l-yl)-l-piperazinecarboxylate (2.51 g, 9.71 mmol, Intermediate E) in TFA (20 mL) in 250-mL round-bottomed flask, trifluoromethanesulfonic acid (2.59 mL, 29.1 mmol, Alfa Aesar, Ward Hill, MA) was added slowly at rt. After stirring at room temperature for 3 min, the reaction mixture was concentrated to dryness under a vacuum. DCM (20 mL) was added to the residue followed by triethylamine (13.5 mL, 97 mmol). After the material went into solution, the mixture was cooled to 0 °C and 6-chloro-3-pyridinesulfonyl chloride (2.06 g, 9.73 mmol, Organic Process Research & Development 2009, 13, 875) was added portion-wise. After 5 min of stirring at 0 °C, water (40 mL) was added at that temperature and the layers were separated. The aqueous phase was extracted with DCM (2 x 50 mL). The combined organic phases were washed with saturated aqueous sodium chloride (60 mL). The organic phase was dried over sodium sulfate, filtered and concentrated under a vacuum. The crude product was purified by column chromatography (100 g of silica, 30 to 90% EtOAc in hexanes) to afford (35)- 1-((6-chloro-3-pyridinyl)sulfonyl)-3-(l-propyn-l-yl)piperazine (2.61 g) as an off-white solid.

STEP 2: N,N-BIS(4-METHOXYBENZYL)-5-(((35)-3-(l-PROPYN-l-YL)-l-PIPERAZINYL)SULFONYL)-2-PYRIDIN AMINE

A mixture of (35)-l-((6-chloro-3-pyridinyl)sulfonyl)-3-(l-propyn-l-yl)piperazine (2.6 g, 8.7 mmol), N-(4-methoxybenzyl)-l-(4-methoxyphenyl)methanamine (2.40 g, 9.33 mmol, WO2007/109810A2), and DIPEA (2.4 mL, 14 mmol) in z-BuOH (8.0 mL) was heated at 132 °C using a microwave reactor for 3 h. This reaction was run three times (total starting material amount was 7.2 g). The mixtures from the three runs were combined and partitioned between EtOAc (200 mL) and aqueous NaHC03 (half saturated, 50 mL). The organic layer was washed with aqueous NaHC03 (3 x 50 mL), dried over Na2S04, filtered, and concentrated. The residue was purified (5-times total) by chromatography on silica using MeOH:DCM:EtOAc:hexane

(4:20:20:60) as eluent to give N,N-bis(4-methoxybenzyl)-5-(((3S)-3-(l-propyn-i-yl)-l-piperazinyl)sulfonyl)-2-pyridinamine (6.6 g) as a white foam.

1H NMR (400MHz ,CDC13) δ ppm 8.55 (d, J= 2.3 Hz, 1 H), 7.64 (dd, J= 2.5, 9.0 Hz, 1 H), 7.13 (d, J= 8.6 Hz, 4 H), 6.91 – 6.81 (m, 4 H), 6.47 (d, J= 9.0 Hz, 1 H), 4.75 (s, 4 H), 3.80 (s, 6 H), 3.68 – 3.61 (m, 1 H), 3.57 (d, J= 11.2 Hz, 1 H), 3.41 (d, J= 11.3 Hz, 1 H), 3.07 (td, J= 3.3, 12.1 Hz, 1 H), 2.87 (ddd, J= 2.9, 9.7, 12.2 Hz, 1 H), 2.63 – 2.47 (m, 2 H), 1.80 (d, J= 2.2 Hz, 3 H). One exchangeable proton was not observed, m/z (ESI, +ve ion) 521.2 (M+H)+.

INTERMEDIATE D: rEi?r-BUTYL(5-(((35)-3-(l-PROPYN-l-YL)-4-(4-(2-(TRIFLUOROMETHYL)-2-OXIRANYL)PHENYL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDINYL)CARBAMATE

step 1 step 2

STEP 1 : l-BR0M0-4-(l-(TRIFLU0R0METHYL)ETHENYL)BENZENE

To a 1-L round-bottomed flask was added methyl phenylphosphonium bromide (25.4 g, 71.1 mmol, Sigma- Aldrich, St. Louis, MO) and toluene (75 mL). The resulting mixture was stirred for 5 min then concentrated and dried under high vacuum for 30 min. To this residue was added THF (300 mL) followed by n-butyllithium (2.5 M in hexanes, 29.0 mL, 71.1 mmol, Aldrich, St. Louis, MO) dropwise via an addition funnel. After being stirred for 1 h at rt, a solution of l-(4-bromophenyl)-2,2,2-trifluoroethanone (15.0 g, 59.3 mmol, Matrix Scientific, Columbia, SC) in THF (20 mL) was added to the reaction mixture dropwise via an addition funnel. The reaction mixture was stirred at rt for 2 h. The reaction was quenched with saturated aqueous NH4C1 and the mixture was concentrated. The residue was partitioned between diethyl ether (150 mL) and saturated aqueous NH4C1 (80 mL). The organic layer was washed with water and brine, dried over MgS04, filtered, and concentrated. The resulting crude product was purified by column chromatography (330 g of silica gel, 2 to 5% EtOAc in hexanes) to afford l-bromo-4-(l-(trifluoromethyl)ethenyl)benzene (14.0 g) as a brown liquid.

STEP 2: 2-(4-BROMOPHENYL)-3,3,3-TRIFLUORO-l,2-PROPANEDIOL

To a solution of l-bromo-4-(l-(trifluoromethyl)ethenyl)benzene (13.5 g, 53.8 mmol) in acetone (100 mL) and water (100 mL) was added NMO (6.90 g, 59.2 mmol, Sigma- Aldrich, St. Louis, MO) and osmium tetroxide (0.140 mL, 2.70 mmol, Sigma-Aldrich, St. Louis, MO). The resulting mixture was stirred at rt for 6 h. The reaction mixture was filtered and the filtrate was concentrated. The residue was partitioned between EtOAc (100 mL) and water (30 mL). The aqueous layer was extracted with EtOAc (2 x 75 mL). The combined organic layers were dried over MgS04, filtered, and concentrated. The resulting product was purified by column chromatography (330 g of silica gel, 0 to 8% MeOH in DCM) to afford 2-(4-bromophenyl)-3,3,3-trifluoro-l,2-propanediol (14.5 g) as an off-white solid.

STEP 3: 4-(4-BROMOPHENYL)-2,2-DIMETHYL-4-(TRIFLUOROMETHYL)-1,3-DIOXOLANE

To a solution of 2-(4-bromophenyl)-3,3,3-trifluoro-l,2-propanediol (14.5 g, 51.0 mmol) in acetone (200 mL) was added 2,2-dimethoxypropane (19.0 mL, 153 mmol, Sigma-Aldrich, St. Louis, MO) and /?-toluenesulfonic acid (0.485 g, 2.54 mmol, Sigma-Aldrich, St. Louis, MO). The resulting mixture was stirred at rt for 20 h. Additional 2,2-dimethoxypropane (19.0 mL, 153 mmol, Sigma-Aldrich, St. Louis, MO) and /?-toluenesulfonic acid (0.485 g, 2.54 mmol, Sigma-Aldrich, St. Louis, MO) were added and the reaction was stirred for another 20 h. The reaction was quenched with saturated aqueous NaHC03 (10 mL). The reaction mixture was concentrated and the residue was partitioned between

EtOAc (100 mL) and saturated aqueous NaHC03 (60 mL). The aqueous layer was extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over MgS04, filtered, and concentrated. The resulting product was purified by column chromatography (330 g of silica gel, 0 to 8% EtOAc in hexanes) to afford 4-(4-bromophenyl)-2,2-dimethyl-4-(trifluoromethyl)-l,3-dioxolane (15.7 g) as a colorless liquid.

STEP 4: BENZYL (3S)-4-(4-(2,2-DIMETHYL-4-(TRIFLUOROMETHYL)-l,3-DIOXOLAN-4-YL)PHENYL)-3-(l -PROPYN- 1 -YL)- 1 -PIPERAZINECAPvBOXYLATE

To a 20-mL vial was added benzyl (3S)-3-(l -propyn- l-yl)-l-piperazinecarboxylate (1.0 g, 3.87 mmol, Intermediate E), RuPhos Palladacycle (0.250 g, 0.310 mmol, Strem Chemical, Newburyport, MA), 4-(4-bromophenyl)-2,2-dimethyl-4-(trifluoromethyl)-l,3-dioxolane (2.50 g, 7.74 mmol), dioxane (15.0 mL), and sodium t-butoxide (0.740 g, 7.74 mmol, Sigma-Aldrich, St.

Louis, MO). The reaction mixture was degassed by bubbling N2 through the solution for 5 min, then the vial was capped. The reaction mixture was heated at 80 °C for 30 min then allowed to cool to rt and partitioned between EtOAc (70 mL) and water (40 mL). The aqueous layer was extracted with EtOAc (1 x 50 mL). The combined organic layers were dried over MgS04, filtered, and concentrated. The crude product was purified by column chromatography (80 g of silica, 5% to 30% EtOAc in hexanes) to afford benzyl (35)-4-(4-(2,2-dimethyl-4-(trifluoromethyl)- 1 ,3-dioxolan-4-yl)phenyl)-3-(l -propyn- 1 -yl)- 1 -piperazinecarboxylate (1.6 g) as a yellow foam.

STEP 5: rEi?r-BUTYL(5-(((35)-3-(l-PROPYN-l-YL)-4-(4-(2,2,2-TRIFLUORO- 1 -HYDROXY- 1 -(HYDROXYMETH YL)ETHYL)PHENYL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDINYL)CARBAMATE

To a 150-mL round-bottomed flask was added benzyl (3S)-4-(4-(2,2-dimethyl-4-(trifluoromethyl)- 1 ,3 -dioxolan-4-yl)phenyl)-3 -( 1 -propyn- 1 -yl)- 1 -piperazinecarboxylate (1.60 g, 3.18 mmol) and TFA (20 mL, Sigma-Aldrich, St. Louis, MO). After the substrate was completely dissolved in TFA,

trifluoromethanesulfonic acid (0.850 mL, 9.55 mmol, Alfa Aesar, Ward Hill,

MA) was added and the resulting mixture was stirred at rt for 1.5 h. The reaction mixture was slowly poured into a 300-mL beaker which contained 100 mL ice water. The resulting mixture was stirred while NaOH pellets (11.0 g) were slowly added to adjust the pH to 7. The solution was extracted with EtOAc (2 x 70 mL) and 10% IPA in CHCI3 (2 x 40 mL). The combined organic layers were dried over MgS04, filtered, and concentrated. The resulting intermediate was redissolved in DCM (60 mL). Triethylamine (2.20 mL, 16.0 mmol, Sigma-Aldrich, St. Louis, MO) and tert-butyl (5-(chlorosulfonyl)-2-pyridinyl)carbamate (1.04 g, 3.60 mmol, Intermediate A) were added. The reaction mixture was stirred at rt for 1 h then partitioned between DCM (70 mL) and water (30 mL). The aqueous layer was extracted with DCM (2 x 40 mL). The combined organic layers were dried over MgS04, filtered, and concentrated. The crude product was purified by column chromatography (120 g of silica, 10% to 40% acetone in hexanes) to afford tert-butyl (5-(((35)-3-(l-propyn-l-yl)-4-(4-(2,2,2-trifiuoro-l-hydroxy- 1 -(hydroxymethyl)ethyl)phenyl)- 1 -piperazinyl)sulfonyl)-2-pyridinyl)carbamate (1.0 g) as a yellow foam.

STEP 6: rEi?r-BUTYL(5-(((35)-3-(l-PROPYN-l-YL)-4-(4-(2-(TRIFLUOROMETHYL)-2-OXIRANYL)PHENYL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDINYL)CARBAMATE

To a solution of tert-butyl (5-(((35)-3-(l-propyn-l-yl)-4-(4-(2,2,2-trifiuoro- 1 -hydroxy- 1 -(hydroxymethyl)ethyl)phenyl)- 1 -piperazinyl)sulfonyl)-2-pyridinyl)carbamate (0.300 g, 0.513 mmol) in DCM (5 mL) was added triethylamine (0.400 mL, 2.88 mmol, Sigma-Aldrich, St. Louis, MO) and p-toluenesulfonyl chloride (0.108 g, 0.564 mmol, Sigma-Aldrich, St. Louis, MO). The resulting mixture was heated at reflux (50 °C) under N2 for 2 h. The reaction mixture was cooled to rt and partitioned between sat. NaHCOs (30 mL) and DCM (70 mL). The aqueous layer was extracted with DCM (2 x 40 mL). The combined organic layers were dried over MgS04, filtered, and concentrated. The crude product was purified by column chromatography (40 g of silica, 10 to 40%> acetone in hexanes) to afford tert-butyl (5-(((35)-3-(l-propyn-l-yl)-4-(4-(2-(trifluoromethyl)-2-oxiranyl)phenyl)- 1 -piperazinyl)sulfonyl)-2-pyridinyl)carbamate (0.240 g) as an off-white solid.

1H NMR (400MHz, CDC13) δ ppm 8.66 (dd, J= 0.6, 2.3 Hz, 1 H), 8.20 – 8.10 (m, 1 H), 8.04 (dd, J= 2.2, 8.9 Hz, 1 H), 7.63 (s, 1 H), 7.41 (d, J= 8.6 Hz, 2 H), 6.94 (d, J= 8.8 Hz, 2 H), 4.42 (d, J= 2.2 Hz, 1 H), 3.89 – 3.67 (m, 2 H), 3.38 (d, J = 5.3 Hz, 3 H), 2.97 – 2.83 (m, 2 H), 2.80 – 2.60 (m, 1 H), 1.78 (dd, J= 0.8, 2.0 Hz, 3 H), 1.55 (s, 9 H). m/z (ESI, +ve ion) 567.2 (M+H)+.

ALTERNATIVE ROUTE TO 2-(4-BROMOPHENYL)-3,3,3-TRIFLUORO-l,2-PROPANEDIOL (INTERMEDIATE D STEP 2):

F3

step 1

STEP 1 : 2-(4-BROMOPHENYL)-2-(TRIFLUOROMETHYL)OXIRANE

To a flame-dried, 50-mL, round-bottomed flask was added potassium t-butoxide (0.450 g, 4.01 mmol, Sigma- Aldrich, St. Louis, MO), DMSO (5.0 mL) and trimethylsulfoxonium iodide (1.00 g, 4.54 mmol, Sigma- Aldrich, St. Louis, MO). The resulting mixture was stirred at rt for 40 min. To this reaction mixture was added l-(4-bromophenyl)-2,2,2-trifluoroethanone (1.0 g, 4.0 mmol, Matrix Scientific, Columbia, SC) in DMSO (5.0 mL) dropwise via an addition funnel. The reaction mixture was stirred at rt for 30 min then quenched with water (1 mL) and partitioned between EtOAc (70 mL) and water (30 mL). The organic layer was washed with water (4 x 30 mL), dried over MgS04, filtered, and concentrated. The crude product was purified by column chromatography (40 g of silica, 10 to 20% acetone in hexanes) to afford 2-(4-bromophenyl)-2-(trifluoromethyl)oxirane (0.610 g) as a pale-yellow liquid.

STEP 2: 2-(4-BROMOPHENYL)-3,3,3-TRIFLUORO-l,2-PROPANEDIOL

To a 20-mL vial was added 2-(4-bromophenyl)-2-(trifluoromethyl)oxirane (0.200 g, 0.750 mmol), dioxane (2.0 mL), and water (3.0 mL). The resulting mixture was heated at 85 °C for 24 h. The reaction mixture was cooled to rt and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over MgS04, filtered and concentrated. The crude product was purified by column chromatography (40 g of silica, 10 to 30% acetone in hexanes) to afford 2-(4-bromophenyl)-3,3,3-trifluoro-l,2-propanediol (2.0 g) as a white solid.

INTERMEDIATE E: BENZYL (3S)-3-(l-PROPYN-l-YL)-l-PIPERAZINECARBOXYLATE

-Cbz

STEP 1 : 4-BENZYL 1 – TER Γ-BUT YL 2-0X0-1,4-PIPERAZINEDICARBOXYLATE

A 2-L Erlenmeyer flask was charged with 2-piperazinone (36.5 g, 364 mmol, Sigma-Aldrich, St. Louis, MO), sodium carbonate (116 g, 1090 mmol, J. T. Baker, Philipsburg, NJ), 600 mL of dioxane, and 150 mL of water. To this was slowly added benzyl chloroformate (62.1 g, 364 mmol, Sigma-Aldrich, St. Louis, MO) at rt over 20 min. After the addition was complete, the mixture was stirred for 2 h and then diluted with water and extracted with EtOAc (2 L). The combined organic extracts were dried (MgS04), filtered, and concentrated to give a white solid. To this solid was added 500 mL of DCM, triethylamine (128 mL, 911 mmol, Sigma-Aldrich, St. Louis, MO), DMAP (4.45 g, 36.4 mmol, Sigma-Aldrich, St. Louis, MO), and di-tert-butyl dicarbonate (119 g, 546 mmol, Sigma-Aldrich, St. Louis, MO). After stirring at room temperature for 1 h, the mixture was diluted with water and the organics were separated. The organics were dried (MgS04), filtered, and concentrated to give a brown oil. To this oil was added 100 mL of DCM followed by 1 L of hexane. The resulting white solid was collected by filtration to give 4-benzyl 1-tert-butyl 2-oxo-l,4-piperazinedicarboxylate (101 g).

STEP 2: BENZYL (2-((7¾’i?J,-BUTOXYCARBONYL)AMINO)ETHYL)(2-OXO-3 -PENT YN- 1 – YL)C ARB AMATE

A 150-mL round-bottomed flask was charged with 4-benzyl 1-tert-butyl 2-oxo- 1 ,4-piperazinedicarboxylate (1.41 g, 4.22 mmol) and THF (5 mL). 1-Propynylmagnesium bromide (0.5 M in THF, 20.0 mL, 10.0 mmol, Sigma-Aldrich, St. Louis, MO) was added at 0 °C slowly. The mixture was stirred at 0 °C for 2 h. Saturated aqueous NH4C1 (40 mL) was added and the aqueous phase was extracted with EtOAc (200 mL, then 2 x 100 mL). The combined organic phases were dried over sodium sulfate, filtered and concentrated under a vacuum. The crude product was purified by column chromatography (50 g of silica, 0 to 50% EtOAc in hexanes) to afford benzyl (2- tert-butoxycarbonyl)amino)ethyl)(2-oxo-3-pentyn-l-yl)carbamate (1.55 g) as a clear oil.

STEP 3: BENZYL 3-(l-PROPYN-l-YL)-l-PIPERAZINECARBOXYLATE

A 3-L round-bottomed flask was charged with 2-((tert-butoxycarbonyl)amino)ethyl)(2-oxo-3-pentyn-l-yl)carbamate (82.17 g, 219 mmol) and 300 mL of DCM. After cooling to -10 °C, TFA (169 mL, 2200

mmol) was added and the resulting dark solution was stirred at rt for 15 min.

Sodium triacetoxyborohydride (186 g, 878 mmol, Sigma- Aldrich, St. Louis, MO) was then added portion- wise over 10 min. After 2 h, the mixture was

concentrated, diluted with EtOAc (1 L), and neutralized with 5 N NaOH. The layers were separated and the organic extracts were washed with brine, dried (MgS04), filtered and concentrated. The resulting orange oil was purified via column chromatography (750 g of silica gel, 0 to 4.5 % MeOH/DCM) to give benzyl 3 -(l-propyn-l-yl)-l -piperazmecarboxylate (43.67 g) as a brown foam.

STEP 4: 4-BENZYL 1 – TER Γ-BUT YL 2-(l -PROP YN-l-YL)- 1,4-PIPERAZINEDICARBOXYLATE

A 20-mL vial was charged with benzyl 3-(l-propyn-l-yl)-l-piperazinecarboxylate (0.616 g, 2.38 mmol), di-tert-butyl dicarbonate (0.979 g, 4.49 mmol, Sigma-Aldrich, St. Louis, MO), DMAP (0.0287 g, 0.235 mmol, Sigma-Aldrich, St. Louis, MO), TEA (0.90 mL, 6.5 mmol) and DCM (8 mL). The mixture was stirred at rt for 30 min. The reaction mixture was partitioned between water (20 mL) and EtOAc (20 mL). The aqueous phase was extracted with EtOAc (20 mL). The organic phase was washed with saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered, and concentrated under a vacuum. The crude product was purified by column chromatography (25 g of silica, 0 to 50% EtOAc in hexanes) to afford 4-benzyl 1-tert-butyl 2-(l-propyn-l-yl)-l,4-piperazinedicarboxylate (0.488 g) as a colorless oil.

STEP 5: 4-BENZYL 1 – TER Γ-BUT YL (2S)-2-( 1 -PROP YN-l-YL)- 1,4-PIPERAZINEDICARBOXYLATE

The individual enantiomers of 4-benzyl 1-tert-butyl 2-(l-propyn-l-yl)-1 ,4-piperazinedicarboxylate were isolated using chiral SFC. The method used was as follows: Chiralpak® ADH column (Daicel Inc., Fort Lee, NJ) (30 x 250 mm, 5 μιη) using 12% ethanol in supercritical C02 (total flow was 170 mL/min).

This separated the two enantiomers with enantiomeric excesses greater than 98%. The first eluting peak was subsequently identified as 4-benzyl 1-tert-butyl (2S)-2-(l-propyn-l-yl)-l,4-piperazinedicarboxylate and used in the next step.

STEP 6: BENZYL (3S)-3-(l-PROPY -l-YL)-l-PIPERAZINECAPvBOXYLATE

A 100-mL round-bottomed flask was charged with 4-benzyl 1-tert-butyl (25)-2-(l-propyn-l-yl)-l,4-piperazinedicarboxylate (0.145 g, 0.405 mmol), TFA (1.0 mL, 13 mmol) and DCM (2 mL). The mixture was stirred at rt for 40 min. The mixture was concentrated and solid NaHC03 was added followed by saturated aqueous NaHC03. The aqueous phase was extracted with EtOAc (2 x 20 mL). The combined organic phases were washed with IN NaOH (40 mL), saturated aqueous NaHC03 (40 mL), water (40 mL) and saturated aqueous sodium chloride (40 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated under a vacuum to afford benzyl (35)-3-(l-propyn-l-yl)-l-piperazinecarboxylate (0.100 g) as a pale yellow clear oil which solidified upon standing to give a pale yellow solid.

1H NMR (400MHz, MeOD) δ ppm 7.47 – 7.13 (m, 5 H), 5.27 – 5.00 (m, 2 H), 3.88 – 3.58 (m, 3 H), 3.48 – 3.33 (m, 2 H), 3.22 – 3.02 (m, 1 H), 2.89 – 2.63 (m, 1 H), 1.80 (s, 3 H). m/z (ESI, +ve ion) 259.1 (M+H)+.

XAMPLE 23: 5-(((3S)-3-(l-PROPYN-l-YL)-4-(4-(l,2,2,2-TETRAFLUORO-1 -(TRIFLUOROMETHYL)ETHYL)PHENYL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDIN AMINE

STEP 1 : 2-(4-((2S)-4-BENZYL-2-(l-PROPYN-l-YL)-l-PIPERAZINYL)PHENYL)-1 , 1 ,1 ,3,3,3-HEXAFLUORO-2-PROPANOL

A 20-mL vial was charged with (3S)-l-benzyl-3-(l-propyn-l-yl)piperazine (2.143 g, 10 mmol, Intermediate B), 2-(4-bromophenyl)-1,1,1, 3,3, 3-hexafluoropropan-2-ol (3.09 g, 11.5 mmol, Bioorg. Med. Chem. Lett. 2002, 12, 3009), sodium 2-methylpropan-2-olate (1.92 g, 20.0 mmol, Sigma-Aldrich, St. Louis, MO), dioxane (5 mL), RuPhos palladacycle (0.364 g, 0.500 mmol, Strem Chemical Inc., Newburyport, MA), and RuPhos (0.233 g, 0.500 mmol, Strem Chemical Inc., Newburyport, MA). The vial was sealed and heated at 100 °C for 1 h. The mixture was allowed to cool to rt, and diluted with water and extracted with EtOAc. The combined organic phases were dried over sodium sulfate, filtered and concentrated under a vacuum to give a solid that was purified by silica gel column chromatography (0 to 40% EtOAc in hexanes) to afford 2-(4-((2S)-4-benzyl-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)- 1,1,1,3,3,3-hexafluoro-2-propanol (1.75 g) as a slightly yellow oil.

STEP 2: l,l,l,3,3,3-HEXAFLUORO-2-(4-((2S)-2-(l-PROPYN-l-YL)-l-PIPERAZINYL)PHENYL)-2-PROPANOL

A 250 mL round-bottomed flask was charged with 2-(4-((2S)-4-benzyl-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)- 1,1,1 ,3 ,3 ,3-hexafluoro-2-propanol (1.75 g, 4.35 mmol), potassium carbonate (2.40 g, 17.4 mmol, Sigma-Aldrich, St. Louis, MO), CH2CI2 (25 mL), and 1-chloroethyl chlorocarbonate (1.88 mL, 17.4 mmol, Sigma-Aldrich, St. Louis, MO). After 30 min at rt, the reaction was filtered and the filtrate was concentrated. To the resulting oil was added MeOH (25 mL). This mixture was heated at 75 °C for 1.5 h then concentrated. The residue was triturated with diethyl ether to give l,l,l,3,3,3-hexafluoro-2-(4-((2S)-2-(l-propyn-l-yl)-l-piperazinyl)phenyl)-2-propanol (1.44 g) as a white solid.

STEP 3: TERT-BUTYL (5-(((3S)-3-(l-PROPYN-l-YL)-4-(4-(2,2,2-TRIFLUORO- 1 -HYDROXY- 1 -(TRIFLUOROMETHYL)ETHYL)PHENYL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDINYL)CARBAMATE

A 250-mL round-bottomed flask was charged with 1,1,1,3,3,3-hexaf uoro-2-(4-((2S)-2-( 1 -propyn- 1 -yl)- 1 -piperazinyl)phenyl)-2-propanol (18.9 g, 51.6 mmol) and DCM (150 mL) and cooled to 0 °C. TEA was added (14.4 mL, 103 mmol, Sigma-Aldrich, St. Louis, MO) followed by tert-butyl (5- (chlorosulfonyl)pyridin-2-yl)carbamate (15.9 g, 54.2 mmol, Intermediate A) portionwise. After 10 min, the reaction mixture was diluted with water (100 mL) and the organic layer was separated, dried over Na2S04, filtered and concentrated under a vacuum to give a solid that was purified by silica gel column

chromatography (0 to 50% EtO Ac in hexanes) to afford tert-butyl (5 -(((3 S)-3 -( 1 -propyn- 1 -yl)-4-(4-(2,2,2-trifluoro- 1 -hydroxy- 1 -(trifluoromethyl)ethyl)phenyl)- 1 -piperazinyl)sulfonyl)-2-pyridinyl)carbamate (19.9 g) as a tan foam.

STEP 4: 5-(((3S)-3-(l-PROPYN-l-YL)-4-(4-(l,2,2,2-TETRAFLUORO-l- (TRIFLUOROMETHYL)ETHYL)PHENYL)- 1 -PIPERAZINYL)SULFONYL)-2-PYRIDIN AMINE

A 500-mL round-bottomed flask was charged with tert-butyl (5-(((3S)-3-(1 -propyn- 1 -yl)-4-(4-(2,2,2-trifluoro- 1 -hydroxy- 1 – (trifluoromethyl)ethyl)phenyl)-l-piperazinyl)sulfonyl)-2-pyridinyl)carbamate (19.7 g, 31.6 mmol) and DCM (300 mL) and cooled to 0 °C.

(Diethylamino)sulfur trifluoride (4.18 mL, 31.6 mmol, Matrix Scientific, Columbia, SC) was added, and after 10 min, the reaction was diluted with water (250 mL) and DCM (200 mL). The organic layer was separated, dried over

Na2S04, filtered and concentrated under a vacuum. The resultant foam was taken up in DCM (200 mL) and cooled to 0 °C. TFA (100 mL, 1298 mmol) was added and the reaction mixture was warmed to rt for 1.5 h. The reaction was then re-cooled to 0 °C and solid sodium bicarbonate was added slowly until gas evolution ceased. The mixture was diluted with water (250 mL) and DCM (300 mL) and the organic layer was separated, dried over Na2S04, filtered and concentrated under a vacuum to give a solid that was purified by silica gel column chromatography (0 to 100% EtOAc in hexanes) to afford 5-(((3S)-3-(l-propyn- 1 -yl)-4-(4-( 1 ,2,2,2-tetrafluoro- 1 -(trifluoromethyl)ethyl)phenyl)- 1 -piperazinyl)sulfonyl)-2-pyridinamine (11.05 g) as a single enantiomer.

1H NMR (400MHz, CD3OD) δ ppm 8.31 (d, J= 2.2 Hz, 1 H), 7.74 (dd, J= 2.4, 8.9 Hz, 1 H), 7.47 (d, J = 8.8 Hz, 2 H), 7.12 (d, J = 9.0 Hz, 2 H), 6.63 (d, J= 8.8 Hz, 1 H), 4.76-4.70 (m, 1 H), 3.76 (dd, J= 1.9, 11.2 Hz, 2 H), 3.66 – 3.52 (m, 1 H), 3.29 – 3.20 (m, 1 H), 2.79 – 2.72 (m, 1 H), 2.66 – 2.53 (m, 1 H), 1.76 (d, J = 2.2 Hz, 3 H). m/z (ESI, +ve ion) 525.2 (M+H)+. GK-GKRP IC50 (Binding) = 0.187 μΜ.

PAPER

Small Molecule Disruptors of the Glucokinase–Glucokinase Regulatory Protein Interaction: 2. Leveraging Structure-Based Drug Design to Identify Analogues with Improved Pharmacokinetic Profiles

Department of Therapeutic Discovery—Medicinal Chemistry, Department of Therapeutic Discovery—Molecular Structure and Characterization, §Department of Metabolic Disorders, Department of Pharmacokinetics and Drug Metabolism, Department of Pathology, #Department of Pharmaceutics Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California, 91320 and 360 Binney Street, Cambridge, Massachusetts, 02142, United States
J. Med. Chem., 2014, 57 (2), pp 325–338
DOI: 10.1021/jm4016747
Abstract Image

In the previous report, we described the discovery and optimization of novel small molecule disruptors of the GK-GKRP interaction culminating in the identification of 1 (AMG-1694). Although this analogue possessed excellent in vitro potency and was a useful tool compound in initial proof-of-concept experiments, high metabolic turnover limited its advancement. Guided by a combination of metabolite identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor (27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.

2-(4-((2S)-4-((6-Amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (27)

1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 2.3 Hz, 1 H), 7.77 (dd, J = 2.5, 8.8 Hz, 1 H), 7.57 (d, J = 8.8 Hz, 2 H), 6.95 (d, J = 9.2 Hz, 2 H), 6.52 (d, J = 8.8 Hz, 1 H), 4.94 (s, 2 H), 4.44 (br s, 1 H), 3.82–3.71 (m, 2 H), 3.58–3.33 (m, 3 H), 2.81 (dd, J = 3.2, 11.1 Hz, 1 H), 2.67 (dt, J = 3.9, 11.0 Hz, 1 H), 1.78 (d, J = 2.2 Hz, 3 H).
m/z (ESI, +ve ion) 523.2 (M + H)+.
REFERENCES
St Jean, D.J. Jr.; Ashton, K.; Andrews, K.; et al.
Small molecule disruptors of the glucokinase-glucokinase regulatory protein (GK-GKRP) interaction
34th Natl Med Chem Symp (May 18-21, Charleston) 2014, Abst 4
Small molecule disruptors of the GK-GKRP interaction as potential antidiabetics
247th Am Chem Soc (ACS) Natl Meet (March 16-20, Dallas) 2014, Abst MEDI 214
Use of non-traditional conformational restriction in the design of a novel, potent, and metabolically stable series of GK-GKRP inhibitors
248th Am Chem Soc (ACS) Natl Meet (August 10-14, San Francisco) 2014, Abst MEDI 267
Small molecule inhibitors for glucokinase-glucokinase regulatory protein (GK-GKRP) binding: Optimization for in vivo target assessment of type II diabetes
248th Am Chem Soc (ACS) Natl Meet (August 10-14, San Francisco) 2014, Abst MEDI 268

MAKING CONNECTIONS Aleksandra Baranczak (right), a fourth-year grad student in Gary A. Sulikowski’s lab at Vanderbilt University, discusses her efforts to synthesize the core of the diazo-containing natural product lomaiviticin A with Kate Ashton, a medicinal chemist at Amgen
Dr. Kate Ashton

Mark Norman

Mark Norman

Michael Bartberger

Michael Bartberger

Chris Fotsch

Chris Fotsch

David St. Jean

David St. Jean

Klaus Michelsen

Klaus Michelsen

///////////1361224-53-4, AMGEN, AMG 3969, Type 2 Diabetes,  PRECLINICAL
O=S(=O)(c1ccc(N)nc1)N2C[C@H](C#CC)N(CC2)c3ccc(cc3)C(O)(C(F)(F)F)C(F)(F)F

AMG 900, An aurora kinase (ARK) inhibitor potentially for the treatment of leukemia and solid tumours


AMG-900

N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine.

N-(4-(3-(2-Aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine

Phase I

Amgen Inc. INNOVATOR

Inventors Victor J. Cee, Holly L. Deak, Bingfan Du,Stephanie D. Geuns-Meyer, Brian L. Hodous,Hanh Nho Nguyen, Philip R. Olivieri, Vinod F. Patel, Karina Romero, Laurie Schenkel,Less «
Applicant Amgen Inc.

An aurora kinase (ARK) inhibitor potentially for the treatment of leukemia and solid tumours.

CAS No. 945595-80-2

In 2014, orphan drug designation was assigned in the U.S. for the treatment of ovarian cancer

Molecular Formula: C28H21N7OS
Molecular Weight: 503.57764 g/mo
AMG 900; AMG-900; 945595-80-2; AMG900; UNII-9R2G075611; N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine;

AMG 900 is a small-molecule inhibitor of Aurora kinases A, B and C with potential antineoplastic activity. Aurora kinase inhibitor AMG 900 selectively binds to and inhibits the activities of Aurora kinases A, B and C, which may result in inhibition of cellular division and proliferation in tumor cells that overexpress these kinases. Aurora kinases are serine-threonine kinases that play essential roles in mitotic checkpoint control during mitosis and are overexpressed by a wide variety of cancer cell types. Check for active clinical trials or closed clinical trials using this agent

AMG 900 is a potent and highly selective pan-Aurora kinases inhibitor for Aurora A/B/C with IC50 of 5 nM/4 nM /1 nM;  >10-fold selective for Aurora kinases than p38α, Tyk2, JNK2, Met and Tie2.
IC50 Value: 5 nM(Aurora A); 4 nM(Aurora B); 1 nM(Aurora C)
Target: pan-Aurora
in vitro: AMG 900 is a novel class of ATP-competitive phthalazinamine small molecule inhibitors of aurora kinases. In HeLa cells, AMG 900 inhibits autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser, a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division without a prolonged mitotic arrest, which ultimately results in cell death. AMG 900 inhibits the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations (about 2- 3 nM). Furthermore, AMG 900 is active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L) [1].
in vivo: Oral administration of AMG 900 blocks the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. AMG 900 is broadly active in multiple xenograft models, including 3 multidrugresistant xenograft models, representing 5 tumor types [1]. AMG 900 exhibits a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 hours. AMG 900 is well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake has an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption. The clearance and volume of distribution at steady state in humans are predicted to be 27.3 mL/h/kg and 93.9 mL/kg, respectively. AMG 900 exhibits acceptable PK properties in preclinical species and is predicted to have low clearance in humans [2].

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.

MG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser10, a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes. (Source: Cancer Res; 70(23); 9846–54.)

Clinical Information of AMG 900

Product Name Sponsor Only Condition Start Date End Date Phase Last Change Date
AMG 900 Amgen Inc Leukemia 31-JUL-11 31-JUL-14 Phase 1 14-SEP-13
Amgen Inc Advanced solid tumor 30-APR-09 30-JUN-13 Phase 1 10-SEP-13

AMG 900.png

PATENT

WO 2007087276

http://www.google.co.in/patents/WO2007087276A1?cl=en

PATENT

WO 2015084649

https://google.com/patents/WO2015084649A1?cl=en

The compound, N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)- 4-(4-methyl-2-thienyl)-l-phthalazinamine, also chemically named as 4-((3-(2-amino- pyrimidin-4-yl)-pyridin-2-yl)oxy)phenyl-(4-(4-methyl-thiophen-2-yl)-phthalazin-l- yl)amine, and is referred to herein as “AMG 900” has a chemical structure of

AMG 900 is an ATP competitive small molecule Aurora kinase inhibitor that is highly potent and selective for Aurora kinases A, B and C. AMG 900 is disclosed in US patent publication no. 20070185111, which published on August 9, 2007 and issued as U.S. Patent No. 7,560,551. AMG 900 is further disclosed in US patent publication no.

20090163501, now US patent no 8,022,221. Various uses and applications of AMG 900 are described in patent publications US20120028917 and WO2013149026. AMG 900 is being clinically evaluated primarily for its safety, tolerability and pharmacokinetic (PK) profile in human phase I trials for (1) advanced solid tumors (US Clinical Trial Id No. NCT00858377), and (2) for acute leukemias (US Clinical Trial Id No. NCT1380756).

Different solid state forms of a given compound are typically investigated to determine whether or not a particular form possesses and/or exhibits desirable properties allowing that compound to be clinically and/or commercially developed. Such beneficial and advantageous properties, by way of example, include without limitation, crystallinity, improved thermodynamic stability, non-hygroscopicity, high purity, minimal to total absence of moisture and/or residual solvents, chemical stability, high yielding synthetic process and/or manufacturability and reproducibility, desirable biopharmaceutical properties including improved dissolution characteristics and increased bioavailability, absence or reduced toxicities due to reduced or limited exposure, rate of exposure or release, or related to counter ions, good bulk and formulation properties including good flow, bulk density, desirable particle size and the like, or a combination of the aforementioned characteristic attributes.

Generally when a compound, also referred to herein as drug substance (DS), has been identified as a developmental candidate, the DS is screened to identify potentially beneficial polymorphic, crystalline or solid state forms of the compound and/or a pharmaceutically acceptable salt thereof. X-ray diffraction, Raman, solid state NMR and a melting point temperature and/or a melting point temperature range have been typically used to monitor or screen and identify the different polymorphic form of the DS.

Different polymorphic forms of a given DS can have an impact on that compound’s solubility, stability and bioavailability. Also, it is important to monitor possible changes in polymorphic forms of the DS during stability studies.

AMG 900 was previously isolated and identified as a free base compound. This compound exhibited rather lack-luster pharmacokinetic (PK) and/or pharmacodynamic (PD) properties, including poor aqueous solubility, poor bioavailability, poor absorption, poor target exposure and overall, a not-so-attractive in-vivo efficacy profile. Thus, there is a need to address and solve the technical problem of identifying alternative forms of AMG 900 to achieve substantially the same effect or an improved effect, including improved PK and PD profiles, as that of AMG 900 known in the art.

Example 1

Synthesis of N-(4-((3-(2-amino-4-pyrimidinylN)-2-pyridinylN)oxyN)phenylN)-4-(4-methyl-2-thienvD-l-phthalazinamine (AMG 900)

Step 1 : 4-(2-chloropyridin-3-yl)pyrimidin-2 -amine

In an argon purged 500 mL round bottom flask placed in an isopropanol bath, was added sodium metal (3.40g, 148mmol) slowly to methanol (180mL). The mixture was stirred at room temperature (RT) for about 30 minutes. To this was added guanidine hydrochloride (12.0 mL, 182 mmol) and the mixture was stirred at RT for 30 minutes, followed by addition of (E)-l-(2-chloropyridin-3-yl)-3-(dimethylamino)prop-2-en-l-one (12.0 g, 57.0 mmol), attached air condenser, moved reaction to an oil bath, where it was heated to about 50 °C for 24 hr. Approximately half of the methanol was evaporated under reduced pressure and the solids were filtered under vacuum, then washed with saturated sodium bicarbonate (NaHCO and H^O, air dried to yield 4-(2-chloropyridin-3-yl)pyrimidin-2-amine as off white solid. MS m/z = 207 [M+l]+. Calc’d for C9H7C1N4: 206.63.

Step 2: 4-(2-(4-aminophenoxy)pyridin-3-yl)pyrimidin-2-amine

To a resealable tube was added 4-aminophenol (1.3 g, 12 mmol), cesium carbonate (7.8 g, 24 mmol), and DMSO (16 ml, 0.75 M). The mixture was heated to 100 °C for 5 minutes, and then 4-(2-chloropyridin-3-yl)pyrimidin-2 -amine (2.5 g, 12 mmol) was added, and the reaction mixture was heated to 130 °C overnight. Upon completion, as judged by LCMS, the reaction mixture was allowed to cool to RT and diluted with water. The resulting precipitate was filtered, and the solid washed with water and diethyl ether. The solid was then taken up in 9: 1 CH2Cl2:MeOH and passed through a pad of silica gel with 9:1 CH2Cl2:MeOH as eluent. The solvent was concentrated in vacuo to provide the desired product, 4-(2-(4-aminophenoxy)pyridin-3-yl)pyrimidin-2-amine. MS m/z = 280

[M+l]+. Calc’d for Ci5H13N50: 279.30.

Step 3: l-Chloro-4-(4-methylthiophen-2-yl)phthalazine

1 ,4-Dichlorophthalazine (1.40 g, 7.03 mmol), 4-methyltmophen-2-ylboronic acid (999 mg, 7.03 mmol), and PdCl2(DPPF) (721 mg, 985 μιηοΐ) were added into a sealed tube. The tube was purged with Argon. Then sodium carbonate (2.0 M in water) (7.74 ml, 15.5 mmol) and 1,4-dioxane (35.2 ml, 7.03 mmol) were added. The tube was sealed, stirred at RT for 5 min, and placed in a preheated oil bath at 110 °C. After 1 hr, LC-MS showed product and byproduct (double coupling), and starting material

dichlorophthalazme. The reaction was cooled to RT, filtered through a pad of celite with an aid of ethyl acetate (EtOAc), concentrated, and loaded onto column. The product was purified by column chromatography using Hex to remove the top spot, then 80:20 hexanes:EtOAc to collect the product. The product, 1 -chloro-4-(4-methylthiophen-2-yl)phthalazine was obtained as yellow solid. LC-MS showed that the product was contaminated with a small amount of dichlorophthalazme and biscoupling byproduct. MS m/z = 261 [M+l]+. Calcd for Ci3H9ClN2S: 260.12.

Step 4: N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-(4-methyl-2-thienyl)- 1 -phthalazinamine

To 4-(2-(4-aminophenoxy)pyridin-3-yl)pyrimidin-2 -amine and l-chloro-4-(4-methyl-2-thienyl)phthalazine was added tBuOH. The resulting mixture was heated at 100 °C in a sealed tube for 16 hours. The rection was diluted with diethyl ether and saturated sodium carbonate and vigorously shaken. The resulting solids were filtered and washed with water, diethyl ether and air dried to yield N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-(4-methyl-2-thienyl)-l -phthalazinamine as an off-white solid. MS m/z = 504 [M+H]+. Calc’d for C28 H21 N7 O S: 503.58.

Example 2

Alternative Synthesis of N-(4-((3-(2-amino-4-pyrimidinylN)-2-pyridinylN)oxyN)phenylN)-4-(4-methyl-2-thienvD-l-phthalazinamine (AMG 900)

Step 1 : 4-(2-chloropyridin-3-yl)pyrimidin-2 -amine

In an argon purged 500 mL round bottom flask placed in an isopropanol bath, was added sodium metal (3.40g, 148mmol) slowly to methanol (180mL). The mixture was stirred at room temperature (RT) for about 30 minutes. To this was added guanidine hydrochloride (12.0 mL, 182 mmol) and the mixture was stirred at RT for 30 minutes, followed by addition of (E)-l-(2-chloropyridin-3-yl)-3-(dimethylamino)prop-2-en-l-one (12.0 g, 57.0 mmol), attached air condenser, moved reaction to an oil bath, where it was heated to about 50 °C for 24 hr. Approximately half of the methanol was evaporated under reduced pressure and the solids were filtered under vacuum, then washed with saturated sodium bicarbonate (NaHCO and H^O, air dried to yield 4-(2-chloropyridin-3-yl)pyrimidin-2-amine as off white solid. MS m/z = 207 [M+l]+. Calc’d for C9H7C1N4: 206.63.

Step 2: 4-(2-(4-aminophenoxy)pyridin-3-yl)pyrimidin-2-amine

To a reaction vessel at ambient temperature was added 4-aminophenol (571 g, 5.25 mol, 1.05 equiv) followed by 4-(2-chloropyridin-3-yl)pyrimidin-2-amine (1064g, 97 wt%, 5.00 mol, 1.0 equiv) and DMSO (7110 ml, 7820 g, 7x the volume of 4-(2-chloropyridin-3-yl)pyrimidin-2 -amine). The reaction mixture was agitated and sparged with nitrogen gas for at least 10 minutes. Then a 50 weight % aqueous KOH solution (593 g, 5.25 mol, 1.05 equiv.) was added to the mixture while keeping the reaction

mixture temperature below about 40°C. The mixture was sparged with nitrogen gas for more than 5 minutes, the sparging tube was removed, and the reaction mixture was heated to 110 +/- 10 °C for at least 1.5 hrs. Upon completion, as judged by HPLC, the reaction mixture was allowed to cool to RT and diluted with 6N HC1 (42 mL, 0.25 mol, 0.05 equiv). The desired product, 4-(2-(4-aminophenoxy)pyridin-3-yl)pyrimidin-2 -amine was not isolated. Rather, it was formed in-situ and combined with the product of step 3 below, in step 4 to form the desired product.

Step 3: l-Chloro-4-(4-methylthiophen-2-yl)phthalazine

A separate reaction vessel was fitted with a reflux condenser and an addition funnel, and 4-(4-methylthiophen-2-yl)phthalazin-l(2H)-one (1,537 mg, 6.34 mol, 1.0 equivalent) was added to the reaction vessel. Acetonitrile (7540 mL, 5859 g, 5 V), was added and the reaction vessel was agitated to allow the starting material to dissolve. The vessel was then charged with phosphorus oxychloride (709 ml, 1166 g, 7.44 mol, 1.2 equivalents) and the reaction was heated to about 75 +/- 5 °C for a least 4 hrs. The reaction was cooled to about about 25 +/- 5 °C and held there for more than 24 hrs. N,N-diisopropylethylamine (3046 g, 4100 mL, 3.8 equivalents) was added to the reaction vessel and the temperature was maintained at <30°C. Pyridine (97g, 1.24 mol, 0.2 equiv) was added in a single portion followed by water (4100 g, 2.7V) over more than 30 minutes. The reaction mixture was stirred at ambient temperature ofr about 24 hrs. the mixture was filtered through a <25uM polypropylene filter and the rsulting mother liquor was diluted with 1 : 1 ACN:water (9000 mL total) and stirred for a minimum of 2 minutes. Filter off product solids as they precipitate. Collect mother liquor and washes to obtain additional product. Dry the filter cake, and additional product crops, under a constant stream of nitrogen gas for at least 14 hrs. Unlike the previous method, the present method avoids contamination of impurities, such as dichlorophthalazine and biscoupling byproduct, as seen via LC-MS. Yield: 1537 g (97.2 weight %). MS m/z = 261 [M+l]+. Calcd for Ci3H9ClN2S: 260.12.

Step 4: N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-(4-methyl-2-thienyl)- 1 -phthalazinamine

To the reaction mixture was added l-chloro-4(4-methylthiophen-2-yl)phthalazine

(1450g, 97.2 wt%, 5.40 mol, 1.08 equiv) rinding the addition port with DMSO (520 ml, 572 g, 0.5x the volume of 4-(2-chloropyridin-3-yl)pyrimidin-2-amine). The reaction mixture was again agitated and sparged with nitrogen gas for at least 10 minutes. The sparging tube was removed, and the reaction mixture was heated to 80 +/- 20 °C for at least 2 hrs. Upon completion, as judged by HPLC, the reaction mixture was allowed to cool to RT and N,N-diisopropylethylamine (776 g, 1045 mL, 6.0 mol, 1.2 equiv) was added and the mixture was kept at about 80 +/- 10°C. Filter the mixture at about 80oC into a separate reactor vessel rinsing with DMSO (1030 mL, 1133 g, 1 V). Then adjust the raction mixture temperature to about 70+/-5 °C and add 2-propanol (13200 mL, 10360 g, 12.75 V) over more than 15 minutes at about 70°C. As the reaction mistreu cools, the product begins to precipitate out of solution. Add more 2-propanol (8780 mL, 6900 g, 8.5V) to the solution slowly over more then 60 minutes at about 70°C. The reactor vessel was cooled to about 20°C over more than 60 minutes and let sit for over 2 hrs. The product was filtered through an Aurora filter with a >25uM polypropylene filter cloth. Additional crops were obtained from the mother liquors by diluting with additional 2-propanol. The filter cakes were dried under a constant stream of nitrogen gas for at least 14 hrs to provide the desired product, N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-(4-methyl-2-thienyl)-l-phthalazinamine as an off-white solid. Yield: 2831 g (88.8%); purity 99.7%. MS m/z = 504 [M+H]+. Calc’d for C28 H21 N7 O S: 503.58.

The starting material 1 used/shown in Example 2 was prepared as follows:

and starting material 3, thienyl substituted phthalazinone, shown in Example 2 was prepared as follows:

Starting material 3

Synthesis of 4-(5-methylthiophen-2-yl)phthalazin-l(2//)-one

Step 1 : 2-(Dimethylamino)isoindoline-1.3-dione

A solution of isobenzofuran-l,3-dione (5.00 g, 34 mmol) and N,N-dimethylhydrazine (2.9 ml, 37 mmol) in toluene (75 ml, 34 mmol) was added p-TsOH H20 (0.32 g, 1.7 mmol). The Dean-Stark apparatus and a condenser were attached. The mixture was refluxed. After 4 hr, LCMS showed mainly product. The reaction was cooled to rt. Toluene was removed under reduced pressure the crude was dissolved in DCM, washed with sat NaHC03, water, and brine. The organic was dried over MgS04, filtered, and concentrated. Light yellow solid was obtained. !H NMR showed mainly product, 2-(dimethylamino)isoindoline-l,3-dione. MS Calcd for C10H10N2O2: [M]+ = 190. Found: [M+H]+ = 191.

Step 2 : 2-(Dimethylamino)-3 -hydroxy-3 -(5 -methylthiophen-2 -vDisoindolin- 1 -one

A solution of 2-bromo-5-methylthiophene (0.60 mL, 5.3 mmol) in THF (11 mL) was purged with nitrogen and cooled to -78 °C. «-Butyllithium (2.2 mL, 5.5 mmol; 2.5 M in THF) was added and the mixture was stirred under nitrogen for 30 min. This solution was cannulated into a flask containing a solution of 2-(dimethylamino)isoindoline-l,3-dione (1.5 g, 7.9 mmol) in THF (16 mL) at -78 °C under nitrogen. The reaction was allowed to warm to -30 °C over an hour, at which point LCMS showed complete conversion of 2-bromo-5-methylthiophene to product. The reaction was quenched by careful addition of saturated aqueous NH4C1. The reaction mixture was diluted with dichloromethane and water, and the layers were separated. The aqueous portion was extracted with additional dichloromethane, and the combined organic layers were dried with MgS04, filtered, concentrated, and purified by silica gel chromatography eluting with 0-2% MeOH in dichloromethane to provide intermediate A, as a light yellow solid, 2-(dimethylamino)-3-hydroxy-3-(5-methylthiophen-2-yl)isoindolin-l-one (1.2 g, 80% yield). !H NMR (400 MHz, DMSO-4) δ 7.68-7.65 (m, 1H). 7.63-7.59 (m, 1H), 7.57-7.51 (m, 1H), 7.37 (d, 1H, J=8), 7.09 (s, 1H), 6.69-6.66 (m, 1H), 6.65-6.62 (m, 1H), 2.81 (s, 6H), 2.40 (s, 3H). 13C NMR (400 MHz, DMSO-de) δ 165.0, 147.3, 141.6, 139.3, 132.7, 129.49, 129.46, 125.0, 124.7, 123.0, 122.1, 88.4, 44.7, 14.9. FT-IR (thin film, cm ) 3347, 3215, 1673. MS Calcd for Ci2H7ClN2S: [M]+ = 288. Found: [M+H]+= 289.

HRMS Calcd for Ci5H16N202S: [M+H]+= 288.1005, [M+Na]+ = 311.0825. Found:

[M+H]+ = 289.1022, [M+Na]+= 311.0838. mp = 138-140 °C.

Step 3: 4-(5-Methylthiophen-2-yl)phthalazin-l(2//)-one

2-(Dimethylamino)-3 -hydroxy-3 -(5 -methylthiophen-2-yl)isoindolin- 1 -one (1.1 g, 0.40 mmol), EtOH (4.0 mL), and hydrazine (0.19 mL, 59 mmol) were added into a RBF fitted with a reflux condenser. A nitrogen balloon was attached on top of the condenser. After refluxing overnight, the reaction was cooled to room temperature. An off-white solid precipitated. After cooling to 0 °C, water was added. The solid was filtered off with an aid of water and dried under vacuum to afford a white solid, 4-(5-methylthiophen-2-yl)phthalazin-l(2//)-one (0.82 g, 85% yield).

!H NMR (400 MHz, CDC13) δ 10.57 (s, 1H), 8.50-8.39 (m, 1H), 8.14-8.04 (m, 1H), 7.83- 7.69 (m, 2H), 7.20-7.17 (m, 1H), 6.82-6.71 (m, 1H), 2.47 (s, 3H). 13C NMR (400 MHz,

CDC13) 8 159.9, 142.5, 141.1, 134.3, 133.7, 131.7, 129.4, 128.8, 128.3, 127.1, 126.6,

125.8, 15.4. FT-IR (thin film, cm“1) 2891, 1660, 1334. MS Calcd for Ci3H10N2OS: [M]+

= 242. Found: [M+H]+= 243. HRMS Calcd for Ci3H10N2OS: [M+H]+= 243.0587. Found:

[M+H]+ = 243.0581. mp = 191-194 °C.

Alternatively, starting material 3 was prepared as follows:

The above scheme depicts the process by which intermediate-scale synthesis of thiophene-phthalazinone 5 (shown above) was prepared. Treatment of 50 grams of 3-methylthiophene with z-PrMgCl at 66 °C in the presence of catalytic TMP-H resulted in 98% conversion to the reactive species lb with a >40:1 regioisomeric ratio. After cooling to 20 °C, this mixture was added dropwise to a -20 °C slurry of phthalic anhydride in THF to provide keto acid 3 in 94% assay yield. While this intermediate could be crystallized from toluene/heptane, the crude reaction mixture was taken directly in a through -process conversion to the phthalazinone 5. To that end, removal of THF, MTBE, and residual 3-methylthiophene was accomplished through a distillative solvent switch into ethanol. The resulting solution of 3 was exposed to aqueous hydrazine at 80 °C. After 18 hours, the reaction was cooled and the precipitated product was filtered directly at 20 °C. This process provided 82.7 grams of 98.6 wt % thiophene-phthalazinone 5 in a weight-adjusted 85% yield over the two steps.

LCMS Method:

Samples were run on a Agilent model- 1100 LC-MSD system with an Agilent Technologies XDB-C8 (3.5 μ) reverse phase column (4.6 x 75 mm) at 30 °C. The flow rate was constant and ranged from about 0.75 mL/min to about 1.0 mL/min.

The mobile phase used a mixture of solvent A (H2O/0.1% HO Ac) and solvent B

(AcCN/O.1 HOAc) with a 9 min time period for a gradient from 10%> to 90%> solvent B. The gradient was followed by a 0.5 min period to return to 10% solvent B and a 2.5 min 10% solvent B re-equilibration (flush) of the column.

Other methods may also be used to synthesize AMG 900. Many synthetic chemistry transformations, as well as protecting group methodologies, useful in synthesizing AMG 900, are known in the art. Useful organic chemical transformation literature includes, for example, R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis, John Wiley and Sons (1994); A. Katritzky and

A. Pozharski, Handbook of Heterocyclic Chemistry, 2nd edition (2001); M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis, Springer- Verlag, Berlin Heidelberg (1984); J. Seyden-Penne, Reductions by the Alumino- and Borohydrides in Organic Synthesis, 2nd edition, Wiley- VCH, (1997); and L. Paquette, editor, Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995).

AMG 900 was tested for its ability to reduce or inhibit tumor progression in various cell lines (in-vitro) and multiple solid tumor types (in-vivo), some of which have previously been exposed to and developed resistance to standard-of-care antimitotic agents, including taxanes and vinca alkaloids, as well as to other chemotherapeutic agents. The following Examples and resulting data will illustrate the ability of AMG 900 to treat cancer, including cancer resistant to the presently standard-of-care therapies, including antimitotic agents, such as paclitaxel, and other drugs used in conjunction with chemotherapy, such as doxorubicin. Unless otherwise indicated, the free base form of AMG 900 was used in the Examples described hereinbelow.

The following Examples describe the efforts of identifying and characterizing various crystalline solid state forms of various salts of AMG 900. Some attempts at forming a solid state crystalline form of a given salt failed, as shown in table 1 hereinbelow. To this end, synthesizing and/or forming &isolating a crystalline solid state form of AMG 900 was not, in any way, straightforward or routine. Rather, the ability to prepare and identify a crystalline solid state form of AMG 900 depended upon the particular salt of AMG 900 and/or the crystallization conditions employed.

PAPER

Journal of Medicinal Chemistry (2015), 58(13), 5189-5207

Discovery of N-(4-(3-(2-Aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine (AMG 900), A Highly Selective, Orally Bioavailable Inhibitor of Aurora Kinases with Activity against Multidrug-Resistant Cancer Cell Lines

Departments of Medicinal Chemistry, Pharmaceutical Research and Development, §Pharmacokinetics and Drug Metabolism, Molecular Structure, and Oncology Research, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States, and Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
J. Med. Chem., 2015, 58 (13), pp 5189–5207
DOI: 10.1021/acs.jmedchem.5b00183
*Phone: 617-444-5041. E-mail: MeyerS@amgen.com.

ACS Editors’ Choice – This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Abstract

Abstract Image

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

N-(4-(3-(2-Aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine (23r)

Applying similar SNAr conditions as for 23b, reaction of 22r and 20a in 2-butanol provided the title compound (2.08 g, 49%) as an off-white solid; mp (DSC) 216 °C.
1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (s, 1 H) 8.64–8.69 (m, 1 H) 8.41–8.44 (m, 1 H) 8.36–8.40 (m, 1 H) 8.35 (d, J = 5.2 Hz, 1 H) 8.23 (dd, J = 4.8, 2.0 Hz, 1 H) 8.00–8.10 (m, 2 H) 7.91–7.97 (m, 2 H) 7.52 (d, J = 1.0 Hz, 1 H) 7.26–7.33 (m, 3 H) 7.16–7.22 (m, 2 H) 6.74 (br s, 2 H) 2.34 (br s, 3 H).
13C NMR (150 MHz, DMSO-d6) δ 163.81, 160.72, 160.67, 158.68, 151.64, 148.50, 148.36, 147.14, 139.86, 139.24, 137.72, 137.10, 132.61, 131.74, 130.24, 125.27, 124.89, 122.92, 122.83, 122.44, 121.56, 121.52, 119.11, 118.23, 109.93, 15.6
HRMS m/z [M + H]+ Calcd for C28H21N7OS: 504.1601. Found: 504.1607.
Table 1. Aurora Kinase Inhibitors with Known Structures That Have Entered Clinical Trials

ID, compd name AKa AK cell assayb (nM) most potently inhibited non-AKs (nM)c [total kinases in panel] admin route
1 (MK-0457/VX-680/tozasertib)(7) A/B MDA-MB-231 p-HH3(12) 43 FLT3 (6), PLK4 (9), ABL (13), MLCK (15), RET (28); [317](16) IV
2 (PHA-739358/danusertib)(8) A/B MDA-MB-231 p-HH3(12) 49 ABL (25), RET (31), TrkA (31), FGFR1 (47); [35](17) IV
3a(AZD1152/barasertib)d,(9) B MDA-MB-231 p-HH3(12) 16 FLT3 (8), cKIT (17), PDGFRA (38), PDGFRB (41), RET (80); [317](16) IV
4 (AT9283)(18) A/B HCT-116 DNA ploidy ∼30 JAK2 (1), JAK3 (1) Abl (T315I) (4), 9 others ≤10 nM; [230] IV
5 (SNS-314)(19) A/B HCT-116 DNA ploidy(20) 9 TrkB (5), TrkA (12), FLT4 (14), Fms (15), DDR2 (82), Axl (84); [219] IV
6 (GSK1070916)(21) B HCT-116 p-HH3(22) 20 FLT1 (42), TIE2 (59), SIK (70), FLT4 (74), FGFR (78); [328](22) IV
7 (ENMD-2076)(23) A HCT-116 p-AurA 130 FLT3 (2), RET (10), FLT4 (16), SRC (20), TrkA (24), Fms (25); [100] PO
8 (CYC116)(24) A/B A549 p-HH3 480 VEGFR2 (44), FLT3 (44), CDK2 (390); [23] PO
9 (ABT-348)(25) A/B HCT-116 p-HH3 21 VEGFR1 (1), FLT3 (1), VEGFR2 (2), CSF-1R (3), PDGFR-α (11); [128] PO
10 (AS703569/R763)(26) A/B A549 p-HH3 14 cell-based assays: VEGFR2 (11), FLT3 (27), AMPK (201); [10] PO
11 (PF-03814735)(27) A/B MDA-MB-231 p-HH3 ∼50 FLT1 (10), FAK (22), TrkA (30), 17 others ≥90% inh@100 nM; [220] PO
12 (MK-5108)(28) A HeLa S3 ↑p-HH3+ cells <1000 TrkA (2), ABL (8), FLT4 (12), TrkB (13), VEGFR2 (30); [233] PO
13a (MLN8054)(29) A HCT-116 p-AurA 34 DRAK2 (8), BLK (68), DRAK1 (190), FGR (220); [317](16) PO
13b (MLN8237/alisertib)(30) A HeLa p-AurA 7 %inh@1 μM: EphA2 (111), FGR (97), CAMK2A (95), EphA4 (94); [220] PO

a

AK = Aurora kinase family member(s) inhibited (AurA and/or AurB; AurC potency not listed).

b

Cell line; substrate or phenotype detected.

c

Kinase activities of greatest potency listed in published literature.

d

Listed enzyme and cellular potency data is for 3b, the parent of prodrug 3a.

References on AMG 900

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US2016008316 2016-01-14 USE OF DIANHYDROGALACTITOL AND ANALOGS OR DERIVATIVES THEREOF IN COMBINATION WITH PLATINUM-CONTAINING ANTINEOPLASTIC AGENTS TO TREAT NON-SMALL-CELL CARCINOMA OF THE LUNG AND BRAIN METASTASES
US2016009785 2016-01-14 NOVEL FUSION MOLECULES AND USES THEREOF
US2015266868 2015-09-24 PHARMACEUTICALLY ACTIVE COMPOUNDS
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US2015072988 2015-03-12 USE OF N-(4-((3-(2-AMINO-4-PYRIMIDINYL)-2-PYRIDINYL)OXY)PHENYL)-4-(4-METHYL-2-THIENYL)-1-PHTHALAZINAMINE IN COMBINATION WITH HISTONE DEACETYLASE INHIBITORS FOR TREATMENT OF CANCER
US8921367 2014-12-30 Use of AMG 900 for the treatment of cancer
US2014163052 2014-06-12 FUSED TRICYCLIC DUAL INHIBITORS OF CDK 4/6 AND FLT3
US2014127271 2014-05-08 BLOCK COPOLYMERS FOR STABLE MICELLES
US2014113879 2014-04-24 BLOCK COPOLYMERS FOR STABLE MICELLES
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US2014066430 2014-03-06 AURORA KINASE MODULATORS AND METHOD OF USE
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US2012028917 2012-02-02 Use Of N-(4-((3-(2-Amino-4-Pyrimidinyl)-2-Pyridinyl)Oxy)Phenyl)-4-(4-Methyl-2-Thienyl)-1-Phthalazinamine In The Treatment Of Antimitotic Agent Resistant Cancer
US2011263530 2011-10-27 Aurora Kinase Modulators and Method of Use
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US7560551 2009-07-14 Aurora kinase modulators and method of use
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///////////945595-80-2, AMG 900,  aurora kinase (ARK) inhibitor,  treatment of leukemia and solid tumours, AMGEN, 2014, orphan drug designation,  U.S. for the treatment of ovarian cancer

CC1=CSC(=C1)C2=NN=C(C3=CC=CC=C32)NC4=CC=C(C=C4)OC5=C(C=CC=N5)C6=NC(=NC=C6)N

AM 2394


str1

AM 2394

1-(6′-(2-hydroxy-2-methylpropoxy)-4-((5-methylpyridin-3-yl)oxy)-[3,3′-bipyridin]-6-yl)-3-methylurea

Urea, N-[6′-(2-hydroxy-2-methylpropoxy)-4-[(5-methyl-3-pyridinyl)oxy][3,3′-bipyridin]-6-yl]-N‘-methyl-

CAS 1442684-77-6
Chemical Formula: C22H25N5O4
Exact Mass: 423.1907

Array Biopharma Inc., Amgen Inc. INNOVATORS

AM-2394 is a potent and selective Glucokinase agonist (GKA), which catalyzes the phosphorylation of glucose to glucose-6-phosphate. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes

Type 2 diabetes mellitus (T2DM) is a disease characterized by elevated plasma glucose in the presence of insulin resistance and inadequate insulin secretion. Glucokinase (GK), a member of the hexokinase enzyme family, catalyzes the phosphorylation of glucose to glucose-6-phosphate in the presence of ATP.

img

str1

Glucokioase i exok ase IV or D> is a glycolytic enssyiris that plays, an importaat. role irt blood sugar regulation .related to glucose utifeattoti a»d metabolism in the liver and pancreatic beta cells. Serving as a glucose sessor, gtoeokiuase controls lasma glucose, levels. Glucokinaae plays a doal rob in .reducing plasma glucose levels; glucose-mediated activation of the en¾ymc in hepatocytes facilitates hepatic giocose npiafcc aad glycogen synthesis, while that la pancreatic beta ceils ultimately induces ins lin seeretio«. Both of these effects in turn reduce plasma glucose levels.

Clinical evidence has shown that, glueokitiase variants with, decreased, and increased activities are associated with mature easel, diabetes of the y ung { O0Y2) and persistent: hyperinsul nemic hypoglycemia &( infancy (PHHI), respectively. lso, aoo n.sulin dependent diabetes rneilitos (NIDDM) patients have been reported to have inappropriately lo giueokaiase activity; Ftirtherrnare. overexpressioa of glucokiuase it* dietary or gesetie animal models of diabetes either prevents, aoKiiorafes, or reverses the progress of pathological. symptoms in the disease. For these reasons, compounds that activate gfecokiaase have been sought by the pitasaaceatjeai liidustry.

International patent application, Publication No. WO 2 7/OS3345, which was published on May 10, 200?, discloses as giocokinase act ators certain 2-an«.aopyridiiie derivatives bearing at the 3 -position a meihyieneoxy-dkrked aromatic group a d on. the ammo group a heteroaryl ring, such as dna/oly! or i A4-lmadiazoiyl

it has .now been found that pyridyl ureas are useful as glneokirtase activators. Cettain of these •compounds have been, found to have an outstanding combination of properties that especially adapts them, for oral use to control plasma glucose levels.

 

 

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394

Departments of Therapeutic Discovery, Metabolic Disorders, and Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
Departments of Metabolic Disorders, Comparative Biology and Safety Sciences and Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
§ Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, United States
ACS Med. Chem. Lett., Article ASAP
DOI: 10.1021/acsmedchemlett.6b00140

http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.6b00140

 

Abstract Image

Glucokinase (GK) catalyzes the phosphorylation of glucose to glucose-6-phosphate. We present the structure–activity relationships leading to the discovery of AM-2394, a structurally distinct GKA. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes.

PATENT

WO 2013086397

 http://www.google.com/patents/WO2013086397A1?cl=en

 COPYING ERROR

Example. 1734 t¾^Jtiyi¾rea

Figure imgf000643_0007

Step A: In 100 mL of DMA were corafeiaed 1 ^545miSO- -ll«omp ridinr2-yl)-3-i«e hir8a- (17.5 g, 70,5 ii!-!to!). 5-o:ieS:t}yI yiidlii~3- ). (9,24 g, S4.7 ΪΗΪΪΪΟ!}, sad CO · (10.1 g, 77.6 mmo!) mid heated to 90 *C for 5 days. After that time, the reaction was om lete a d to it was added water arid DCM and stirred vigorously for 3 hr. The resulting solid was isolated via vacuum .filtratiott nd the cake was wasted mill rater and DCM. The DCM in tli aqueous rime was dried vdth a stream of aidogeji aad vigorous sbrriug. Use resulting solid was then collected via vacuum filtration aad these solids were

Stirred vig rousl in f 0% MeOH irt EtOAc arid die res dtipg solid was colleeied. via vactiiars fiirfati m.

Trie two batches wen i coiiibiaed to yield I-(5-bmmo-4 5^»ie†fey pyiidin-3-yl xy)p Tidin-2- d 3~ metbySurea (I S J g, 5 3.7 om»)i, 76% yield).

S e .8: In 2 niL ofc ioxane

Figure imgf000644_0001

yI) iyridMJ-2-yios:y)pf¾ps3i-2-oI (0,098 g, 0.33 «ΜΠΟΪ), -i5-bs¾tao-4-{5-a3fidiy I py f idia-3 – ylosy)f5yridia-2-yl)-3-raethyl«rea (0.075 g, 0.22 tn ol.. t, and.2M poiass.ua» carbonate (0.33 ml, 0.67 m oi} artd tfets was s parged wi h At .for 10 mia before PdC§4dppl)*DCM (0.01 g g, 0.022 msttol) was added and dre reae!io a was sparged for aaotber 5 ma-, ir efore a was sealed and heated to 100 oversight The react! art was then loaded directly onto s ilica gel (50% acetone to PCM w4i. }%

MH40H) to afford i – (6′-(2diydioxy-2i-H5eth:ylpropCis:y) -4-{ 5″i:t re th y Ipy r i d i rt -3- io s y ) -3 ,3 : -bipyr id i rt -6- yl)-3-aie5¾ylt)rea φ.? 42 , 0.096 m ol, 43 % yield). !1 1 HMR (400 Mife, CDCij) 3 ppm 9.06 is,. !H),

S.33 is, 1H>, 8,27 (rs 2H), 8. Π (s, I H): K. (s, IHU 82 (dd, j-S.fi, 5.9 H HI), 1.21 (S !H), 6,«8

(d, Hz, i i i ). 6. ,4 (s:. m>, 4.25 (s, 2H), 2,87 (dj =4,3 Hz„ 3H) 2,37 (s, 3H>. 1 .33 is, <SH). Mass speetram (apci) tar/, : – 423.9 (M÷H).

REFERENCES

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394
Paul J. Dransfield, Vatee Pattaropong, Sujen Lai, Zice Fu, Todd J. Kohn, Xiaohui Du, Alan Cheng, Yumei Xiong, Renee Komorowski, Lixia Jin, Marion Conn, Eric Tien, Walter E. DeWolf Jr., Ronald J. Hinklin, Thomas D. Aicher, Christopher F. Kraser, Steven A. Boyd, Walter C. Voegtli, Kevin R. Condroski, Murielle Veniant-Ellison, Julio C. Medina, Jonathan Houze, and Peter Coward
Publication Date (Web): May 23, 2016 (Letter)
DOI: 10.1021/acsmedchemlett.6b00140

/////////Glucokinase activator,  GKA,  AM-2394, 1442684-77-6, AM 2394, Amgen

O=C(NC)NC1=CC(OC2=CC(C)=CN=C2)=C(C3=CC=C(OCC(C)(O)C)N=C3)C=N1

AMG 337


str1.

PIC CREDIT.BETHANY HALFORD

str1

 

Name: AMG-337(AMG337; AMG 337)
Cas 1173699-31-4
Formula: C23H22FN7O3
M.Wt: 463.46
Chemical Name: 6-[(1R)-1-[8-fluoro-6-(1-methylpyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl]ethyl]-3-(2-methoxyethoxy)-5-methylidene-1,6-naphthyridine

(R)-6-(1-(8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one

(R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one

6-{ (lR)-l-[8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)[l,2,4]triazolo[4,3-a]pyridin-3-yl]ethyl}-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (“Compound M”),

PHASE 2 CANCER OF ESOPHAGUS

AMG-337 is a potent and highly selective small molecule ATP-competitive MET kinase inhibitor. AMG 337 inhibits MET kinase activity with an IC50 of < 5nM in enzymatic assays.
IC50 value: < 5nM [1]
Target: MET
in vitro: AMG-337 demonstrates exquisite selectivity for MET when profiled against a diverse panel of over 400 protein and lipid kinases in a competitive binding assay. In cellular assays, AMG 337 inhibits HGF-dependent MET phosphorylation with an IC50 of < 10 nM. [1] AMG 337 is a selective inhibitor of Met, which inhibits multiple mechanisms of Met activation. [2]
in vivo: AMG-337 demonstrates robust activity in MET-dependent cancer models. Oral administration of AMG 337 results in robust dose-dependent anti-tumor efficacy in MET amplified gastric cancer xenograft models, with inhibition of tumor growth consistent with the pharmacodynamic modulation of MET signaling

AMG 337 is a potent and highly selective small molecule ATP-competitive MET kinase inhibitor that demonstrates robust activity in MET-dependent cancer models. In enzymatic assays, AMG 337 inhibited MET kinase activity with an IC50 less than 5 nM. AMG 337 demonstrated exquisite selectivity for MET when profiled against a diverse panel of over 400 protein and lipid kinases in a competitive binding assay. In cellular assays, AMG 337 inhibited HGF-dependent MET phosphorylation with an IC50 of less than 10 nM [1].

AMG 337 was profiled in cell viability assays using a diverse panel of over 200 cancer cell lines where on treatment with AMG 337 affected the viability of only two gastric cancer cell lines (SNU-5 and Hs746T), both of which harbor amplification of the MET gene. The AMG 337 IC50 in the two sensitive cell lines was less than 50 nM, and greater than 10 µM in all other tested cell lines.

The receptor tyrosine kinase c-Met and its natural ligand, hepatocyte growth factor (HGF), are involved in cell proliferation, migration, and invasion and are essential for normal embryonic development. Deregulation of c-Met/HGF signaling can lead to tumorigenesis and metastasis and has been implicated in a variety of cancers. Several mechanisms lead to deregulation, including overexpression of c-Met and/or HGF, amplification of the MET gene, or activating mutations of c-Met, all of which have been found in human cancers.

AMG 337 is a potent and highly selective inhibitor of wild-type and some mutant forms of MET. In a competitive binding assay conducted on 402 human kinases, AMG 337 bound only to MET. In a cell viability study, the only cell lines that responded to an AMG 337 analog were gastric cancer cells harboring MET gene amplification. None of the other cell lines were sensitive to the AMG 337 analog and none harbored MET gene amplification. In secondary pharmacology assays with transporters, enzymes, ion channels, and receptors, binding to the adenosine transporter was the only activity inhibited.

In vivo, oral administration of AMG 337 resulted in robust dose-dependent anti-tumor efficacy in MET amplified gastric cancer xenograft models, with inhibition of tumor growth consistent with the pharmacodynamic modulation of MET signaling. Further studies in an expanded panel of additional cancer cell lines derived from gastric, NSCLC, and esophageal cancer confirmed that the in-vitro anti-proliferative activity of AMG 337 correlated with amplification of MET. In those cell lines, treatment with AMG 337 inhibited downstream PI3K and MAPK signaling pathways, which translated into growth arrest as evidenced by an accumulation of cells in the G1 phase of the cell cycle, a concomitant reduction in DNA synthesis, and the induction of apoptosis [1].

In a small subset of patients with MET-amplified gastrointestinal (GI) tumors, monotherapy with the investigational agent AMG 337 produced a “dramatic” response. Of the 13 patients with MET-amplified gastric and esophageal cancers, eight experienced a response. The overall response rate in this group of patients was 62%. Response was rapid, with time to response being 4 weeks in most cases. Patients achieved tumor shrinkage and symptomatic improvement. One patient achieved a complete response and is still on treatment at 155 weeks; the others achieved partial responses or stable disease. This has led to further trials, including Phase II trials MET amplified gastric/esophageal adenocarcinoma or other solid tumors.

PAPER

Discovery of (R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET with High Unbound Target Coverage and Robust In Vivo Antitumor Activity.

Boezio, A.A.Copeland, K.W.Rex, K.K Albrecht, B.Bauer, D.Bellon, S.F.Boezio, C.Broome, M.A.Choquette, D.Coxon, A.Dussault, I.Hirai, S.Lewis, R.Lin, M.H.Lohman, J.Liu, J.Peterson, E.A.Potashman, M.Shimanovich, R.Teffera, Y.Whittington, D.A.Vaida, K.R.Harmange, J.C.

(2016) J.Med.Chem. 59: 2328-2342

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b01716

Abstract Image

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

(R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (23)

Step 1: Coupling 9c and 13c in MeCN for 30 min at room temperature resulted in 86% yield. LRMS (ESI): m/z (M + H) 482.2. Step 2: THF for 50 min at room temperature resulted in 48% yield. The racemate was purified by supercritical fluid chromatography (SFC) by repeating 0.75 mL injections of a 30 mg/mL solution onto a Chiralpak AS-H, 2 cm × 15 cm (i.d. × length) column, eluting with 20% i-PrOH and 80% CO2 at a flow rate of 50 mL/min to provide 120 mg peak 1 (23) with >99% ee and 150 mg of peak 2 (ent-23) with >99% ee.(29) 1H NMR (400 MHz, Chloroform-d): δ 8.72 (d, J = 2.93 Hz, 1H), 8.31 (d, J = 0.78 Hz, 1H), 8.15 (d, J = 2.84 Hz, 1H), 7.72 (s, 1H), 7.61 (s, 1H), 7.42 (d, J = 7.82 Hz, 1H), 7.09 (dd, J = 0.73, 10.61 Hz, 1H), 7.05 (q, J= 7.00 Hz, 1H), 6.82 (d, J = 7.82 Hz, 1H), 4.26–4.37 (m, 2H), 3.97 (s, 3H), 3.80–3.88 (m, J = 3.80, 5.10 Hz, 2H), 3.49 (s, 3H), 2.15 (d, J = 7.14 Hz, 3H). HRMS (ESI): m/z (M + H) calcd, 464.1859; found, 464.1841. The solid was recrystallized in EtOH followed by the addition of H2O to form crystalline free base monohydrate form I with a dehydration event at 40–55 °C followed by a melt at 151–153 °C. The solid could also be recrystallized in EtOH under anhydrous conditions to form crystalline anhydrous free base form I with a melting point of 151–153 °C.

PATENT

WO 2009091374

http://www.google.com/patents/WO2009091374A2?cl=en

Example 515

(SV6-(l-f8-fluoro-6-(3-methvIisoxazol-5-vn-|l,2,41triazoIo[4,3-a1pyridin-3-vncthvn-3-(f2- methoxyethoxy)methv.)-l,6-naphthyridin-5(6HVone Synthesized in the same general manner as that previously described for example 509 using General Method N. Chiral separation by preparative SFC (Chiralpak® AD-H (20 x 150 mm, 5Dm), 25% MeOH, 75% CO2, 0.2% DEA; 100 bar system pressure; 75 mL/min; tr 4.75min). On the basis of previous crystallographic data and potency recorded for related compound in the same program, the absolute stereochemistry has been assigned to be the S enantiomer. M/Z – 465.2 [M+H], calc 464.16 for C23H2iFN6O4

Figure imgf000165_0002

Example 516 ri?)-6-ri-(8-fluoro-6-(l-methyl-lH-pyrazol-4-vn-H.2.41triazolo[4,3-alpyridin-3-yl)ethyl)- 3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one The title compound was synthesized using General Method N. Chiral separation by preparative SFC (Chiralpak® AS-H (20 x 150 mm, 5 Dm), 20% iPrOH, 80% CO2; 100 bar system pressure, 50 mL/min; tr 1.67 min). On the basis of previous crystallographic data and potency recorded for related compound in the same program, the absolute stereochemistry has been assigned to be the R enantiomer. M/Z = 464.2 [M+H], calc 463.18 for C23H22FN7O3. 1H NMR (400 MHz, CHLOROFORM-^ D ppm 2.15 (d, J=7.14 Hz, 3 H) 3.49 (s, 3 H) 3.80 – 3.90 (m, 2 H) 3.97 (s, 3 H) 4.27 – 4.39 (m, 2 H) 6.83 (d, J=7.73 Hz, 1 H) 7.00 – 7.13 (m, 2 H) 7.42 (d, J=7.82 Hz, 1 H) 7.61 (s, 1 H) 7.72 (s, 1 H) 8.15 (d, J=2.84 Hz, 1 H) 8.31 (s, 1 H) 8.72 (d, J=3.03 Hz, 1 H).

Figure imgf000166_0001
PATENT
WO 2015161152

6-{ (lR)-l-[8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)[l,2,4]triazolo[4,3-a]pyridin-3-yl]ethyl}-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (“Compound M”), which is a selective inhibitor of the c-Met receptor, and useful in the treatment, prevention, or amelioration of cancer:

PATENT

https://www.google.com/patents/WO2014210042A2?cl=en

The overall scheme for the preparation of Compound A is shown below. The optical purity of Compound A is controlled during the synthetic process by both the quality of the incoming starting materials and the specific reagents used for the transformations. Chiral purity is preserved during both the coupling reaction (the second step) and the dehydration reaction (the third step).

NAPH (S)-halopropionic NAPA

acid/ester

PREPARATION OF COMPOUND A

In one aspect, provided herein is a method for preparing Compound A, salts of Compound A, and the monohydrate form of Compound A. Compound A can be prepared from the NAPH, PYRH, and S-propionic acid/ester starting materials in three steps. First, NAPH and ^-propionic acid/ester undergo an S 2 alkylation reaction to result in (R)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoic acid/ester. The ^-propionic acid starting material produces (R)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoic acid (“NAPA”) in one step. The ^-propionic ester starting material first produces the ester analog of NAPA, and is subsequently hydrolyzed to form NAPA. During workup, the acid can optionally form a salt (e.g., HC1 or 2-naphthalenesulfonic acid).

Step 1:

NAPH (S)-2-halopropionic

acid/ester

1 2

wherein R is Br, CI, I, or OTf; and R is COOH or Ci-salkyl ester, and

when R is Ci^alkyl ester, the method of forming the NAPA or salt thereof further comprises hydrolyzing the Ci-salkyl ester to form an acid.

Second, NAPA and PYRH are coupled together to form (R)-N’-(3-fluoro-5-(lmethyl-lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo- l,6-naphthyridin- 6(5H)yl)propanehydrazide (“HYDZ”).

Step 2:

Third, HYDZ is dehydrated to form Compound A.

The free base form of Compound A can be crystallized as a salt or a monohydrate.

Step 1: Alkylation of NAPH to form NAPA

The first step in the preparation of Compound A is the alkylation of NAPH to form NAPA. The NAPA product of the alkylation reaction is produced as a free base and is advantageously stable.

Thus, one aspect of the disclosure provides a method for preparing NAPA comprising admixing 3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (“NAPH”):

Me

1 R2 , and a base, under conditions sufficient to form NAPA:

wherein R1 is Br, CI, I, or OTf; and

R2 is COOH or C^alkyl ester;

and when R2 is Ci_3alkyl ester, the method of forming the NAPA or salt thereof further comprises hydrolyzing the Ci-3alkyl ester to form an acid.

Me

The compound, R1 R2 , represents an (^-propionic acid and/or (S)- propionic ester

Me

(“(S)-propionic acid/ester”). When R1 R2 is an acid (i.e., R2 is COOH), NAPA is formed in one step:

-prop on c ac

Me

When R1 R2 is an ester (i.e., R2 is C1-3 alkyl ester), then the NAPA ester analog is formed, which can be hydrolyzed to form NAPA.

The SN2 alkylation of NAPH to form NAPA occurs with an inversion of

EXAMPLE 1

SYNTHESIS OF (R)-2-(3-(2-METHOXYETHOXY)-5-OXO-l,6-NAPHTHYRIDIN-6(5H)- YL)PROPANOIC ACID NAPHTHALENE-2-SULFONATE (NAPA)

Scheme 1: Synthesis of naphthyridinone acid 2-napsylate (NAPA)

NAPA was synthesized according to Scheme 1 by the following procedure. A jacket reactor (60 L) was charged with 3000 g (1.0 equivalent) of 3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one and 4646 g (2.0 equivalents) of magnesium ie/t-butoxide. 12 L (4.0 Vol) tetrahydrofuran was added to the reactor and an N2sweep and stirring were initiated. 2213 g (1.5 equivalents) of S-2-bromopropionic acid was added over at least 30 min, controlling the addition such that the batch temperature did not rise above 30 °C. The charge port was rinsed with tetrahydrofuran (0.5 Vol) after addition. The batch was then aged for at least 5 min at 25 °C. 1600 g (1.05 equivalents) of potassium iert-butoxide was added to the reactor in four portions (approximately equal) such that the batch temperature did not rise above 30 °C. The charge port was again rinsed with tetrahydrofuran (1.5 L, 0.5 Vol). The batch temperature was adjusted to 35+5 °C and the batch was aged for at least 12 h.

A separate 100 L reactor was charged with 6 L of 2-Metetrahydrofuran (2-MeTHF) (2.0 Vol), 8.4 L of water (1.5 Vol) and 9.08 L (4.0 equivalents) of 6 N HC1. The mixture from the 60 L reactor was pumped into the 100 L reactor, while maintaining the batch temperature at less than 45 °C.

The batch temperature was then adjusted to 20+5°C. The pH of the batch was adjusted with 6N HC1 (or 2N NaOH) solution until the pH was 1.4 to 1.9. The aqueous layer was separated from the product-containing organic layer. The aqueous layer was extracted with 2-MeTHF (2 Vol), and the 2-MeTHF was combined with the product stream in the reactor. The combined organic stream was washed with 20% brine (1 Vol). The organic layer was polish-filtered through a < ΙΟμιη filter into a clean vessel.

In a separate vessel, 1.1 equivalents of 2-Naphthalenesulfonic acid hydrate was dissolved in THF (2 Vol). The solution was polish-filtered prior to use. The 2-naphthalenesulfonic acid hydrate THF solution was added into the product organic solution in the vessel over at least 2 h at 25+5 °C. The batch temperature was adjusted to 60+5 °C and the batch was aged for 1+0.5 h. The batch temperature was adjusted to 20+5 °C over at least 2 h. The batch was filtered to collect the product. The collected filter cake was washed with THF (5.0 Vol) by displacement. The product cake was dried on a frit under vacuum/nitrogen stream until the water content was < lwt% by LOD.

The yield of the product (R)-2-(3-(2-methoxyethoxy)-5-oxo- l,6-naphthyridin-6(5H)-yl)propanoic acid naphthalene-2-sulfonate, was 87%. The chiral purity was determined using chiral HPLC and was found to be 98-99% ee. The purity was determined using HPLC, and was found to be > 98%.

Thus, Example 1 shows the synthesis of NAPA according to the disclosure.

EXAMPLE 2

SYNTHESIS OF (R)-N’-(3-FLUORO-5-(l-METHYL-lH-PYRAZOL-4-YL)PYRIDIN-2- YL)-2-(3-(2-METHOXYETHOXY)-5-OXO-l,6-NAPHTHYRIDIN-6(5H)- YL)PROPANEHYDRAZIDE (HYDZ)

Scheme 2: Synthesis of (R)-N’-(3-fluoro-5-(l-methyl- lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanehydrazide

HYDZ was synthesized according to Scheme 2 by the following procedure. A 60 L jacket reactor was charged with 2805.0 g (1.0 equivalent) of (R)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoic acid 2-napsylate (NAPA) and N,N-dimethylacetamide (DMAC) (4.6 mL DMAC per gram of NAPA). Stirring and an N2 sweep were initiated. 1.05 equivalents of N,N-diisopropylethylamine (DIPEA) was added while maintaining the batch temperature at less than 35°C. Initially the NAPA dissolves. A white precipitate formed while aging, but the precipitate had no impact on the reaction performance. 2197 g (1.10 equivalents) of 3-fluoro-2-hydrazinyl-5-(l-methyl- lH-pyrazol-4-yl)pyridine (PYRH) was added to the batch. The batch temperature was adjusted to 10+5 °C. 2208 g (1.2 equivalents) of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) was added in four portions (approximately equal) over at least 1 h (about 20 min interval per portion) at 10+5 °C.

The batch was aged until the amide conversion target was met. If the amide conversion target was not reached within 2 h, additional EDC was added until the conversion target was met. Once the target was met, the batch was heated to 55 °C until the solution was homogeneous. The batch was filtered through a <20 μ in-line filter into a reactor. The vessel and filter were rinsed with DMAC (0.2 mL DMAC/g of NAPA). The batch temperature was adjusted to 45+5 °C.

The reactor was charged with a seed slurry of (R)-N’-(3-fluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanehydrazide (HYDZ) (0.01 equivalents) in water (0.3 mL/g).

The batch was aged at 50+5 °C for at least 30 min. The batch temperature was adjusted to 20+5°C over at least 2 h. The batch was aged at 20+5°C for at least 30 min. 2.90 mL water per g was added at 25+5 °C over at least 2 h. The batch was aged at 20+5 °C for at least 1 h. The batch slurry was filtered to collect the product. The product was washed with 30% DMAC/H20 (0.5 Vol) by displacement. The product cake was washed with water (3 Vol) by displacement. The product cake was dried on the frit under vacuum/nitrogen stream until the water content was < 0.2 wt% as determined by Karl Fischer titration (KF). The product was a white, crystalline solid. The yield was about 83-84%. The ee was measured by HPLC and was found to be > 99.8%ee. The purity was determined by HPLC and was found to be >99.8 LCAP (purity by LC area percentage).

Thus, Example 2 demonstrates the synthesis of HYDZ according to the disclosure.

EXAMPLE 3

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE HYDROCHLORIDE SALT (COMPOUND A-HCL) – ROUTE 1

Scheme 3 Route 1 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl- lH-pyrazol-4-yl)- [l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride salt (Compound A-

HC1) was synthesized according to Scheme 3, Route 1 by the following procedure. A 15 L reactor, Reactor 1, was charged with 750 g HYDZ and the reactor jacket temperature was adjusted to 20+5 °C. A nitrogen sweep was initiated in Reactor 1 and the condenser coolant (at 5+5 °C) was started. Acetonitrile (3.4 L, 4.5 Vol) was added to Reactor 1 and stirring was initiated. 420 g (2.5 equivalents) of 2,6-lutidine was added to the reactor.

A solution of diphenylphosphinyl chloride Ph2P(0)(Cl) was prepared by combining 850 g (2.3 equivalents) of Ph2P(0)(Cl) and 300 g acetonitrile in an appropriate container. The contents of the PH2P(0)(C1) solution were added to Reactor 1. The jacket temperature was adjusted over 60+30 min until the reflux temperature of the batch (approximately 85 °C) was reached. The reaction was stirred for 14+6 h. The batch temperature was reduced to 75+5 °C and the batch was sampled for IPT analysis. The expected result was < 2% HYDZ remaining. If the target was not met, the heating at reflux temperature was continued for 9+6 h. Sampling, analysis, and heating was repeated until a satisfactory conversion assay result was obtained (< 10% HYDZ was considered satisfactory, < 1% was actually achieved). The final sample was assayed for optical purity by HPLC, and was found to be > 99.5% ee.

A K2CO3/KCI quench solution (5.0 Vol) was prepared in advance by combining 555 g (3.1 equivalents) of potassium carbonate with 335 g (2.9 equivalents) of potassium chloride and 3450 g of water in an appropriate container. The quench solution was added to Reactor 1 over at least 15 min, maintaining the batch temperature at 60+5 °C. As the aqueous base reacted with excess acid some bubbling (C02) occurred. 3.0 L (4.0 Vol) of toluene was added to Reactor 1 at 65+5 °C. A sample of the batch was taken for IPT analysis. The lower (aqueous) phase of the sample was assayed by pH probe (glass electrode). The pH was acceptable if in the range of pH 8-11. The upper (organic) phase of the sample was assayed by HPLC.

The batch was agitated for 20+10 min at 65+5 °C. Stirring was stopped and the suspension was allowed to settle for at least 20 min. The aqueous phase was drained from Reactor 1 via a closed transfer into an appropriate inerted container. The remaining organic phase was drained from Reactor 1 via a closed transfer to an appropriate inerted container. The aqueous phase was transferred back into Reactor 1.

An aqueous cut wash was prepared in advance by combining 2.3 L (3.0 Vol) acetonitrile and 2.3 L(3.0 Vol) toluene in an appropriate container. The aqueous cut wash was added to Reactor 1. The batch was agitated for 20+10 min at 65+5 °C. The stirring was stopped and the suspension was allowed to settle for at least 20 min. The lower (aqueous) phase was drained from Reactor 1 via a closed transfer into an appropriate inerted container. The organic phase was drained from Reactor 1 via a closed transfer to the inerted container containing the first organic cut. The combined mass of the two organic cuts was measured and the organic cuts were transferred back to Reactor 1. Agitation was initiated and the batch temperature was adjusted to 60+10 °C. A sample of the batch was taken and tested for Compound A content by HPLC. The contents of Reactor 1 were distilled under vacuum (about 300-450 mmHg) to approximately 8 volumes while maintaining a batch temperature of 60+10 °C and a jacket temperature of less than 85 °C. The final volume was between 8 and 12 volumes.

The nitrogen sweep in Reactor 1 was resumed and the batch temperature adjusted to 70+5 °C. A sample of the batch was taken to determine the toluene content by GC. If the result was not within 0-10% area, the distillation was continued and concomitantly an equal volume of 2-propanol, up to 5 volumes, was added to maintain constant batch volume. Sampling, analysis, and distillation was repeated until the toluene content was within the 0-10% area window. After the distillation was complete, 540 g (450 mL, 3.5 equivalents) of hydrochloric acid was added to Reactor 1 over 45+15 min while maintaining a batch temperature at 75+5 °C.

A Compound A-HC1 seed suspension was prepared in advance by combining 7.5 g of Compound A-HC1 and 380 mL (0.5 Vol) of 3 propanol in an appropriate container. The seed suspension was added to Reactor 1 at 75+5 °C. The batch was agitated for 60+30 min at 75+5 °C. The batch was cooled to 20+5 °C over 3+1 h. The batch was agitated for 30+15 min at 20+5 °C. 2.6 L (3.5 Vol) of heptane was added to the batch over 2+1 h. The batch was then agitated for 60+30 min at 20+5 °C. A sample of the batch was taken and filtered for IPT analysis. The filtrate was assayed for Compound A-HC1. If the amount of Compound A-HC1 in the filtrate was greater than 5.0 mg/mL the batch was held at 20 °C for at least 4 h prior to filtration. If the amount of Compound A-HC1 in the filtrate was in the range of 2-5 mg.ML, the contents of Reactor 1 were filtered through a < 25 μιη PTFE or PP filter cloth, sending the filtrate to an appropriate container.

A first cake wash was prepared in advance by combining 1.5L (2.0 Vol) of 2-propanol and 1.5L (2.0 Vol) of heptane in an appropriate container. The first cake wash was added to Reactor 1 and the contents were agitated for approximately 5 min at 20+5 °C. The contents of Reactor 1 were transferred to the cake and filter. A second cake wash of 3.0L (4.0 Vol) of heptane was added to Reactor 1 and the contents were agitated for approximately 5 min at 20+5 °C. The contents of Reactor 1 were transferred to the cake and filter. The wet cake was dried under a flow of nitrogen and vacuum until the heptane content was less than 0.5 wt% as determined by GC. The dried yield was 701g, 85% as a yellow powder. The dried material was assayed for chemical purity and potency by HPLC and for residual solvent content by GC. The isolated product was 88.8% Compound A-HC1, having 99.8% ee and 0.6% water.

Thus, Example 3 shows the synthesis of Compound A-HCL according to the disclosure.

EXAMPLE 4

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE HYDROCHLORIDE SALT (COMPOUND A-HCL) – ROUTE 2

HYDZ A HCI

Scheme 4: Route 2 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl- lH-pyrazol-4-yl)- [l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride salt was synthesized according to Scheme 4, Route 2, by the following procedure. A clean and dry 60 L reactor was fitted with a reflux condenser, nitrogen inlet, and vented to a scrubber (Reactor 1). The jacket temperature of Reactor 1 was set to 20 °C. A scrubber was set up to the vent of Reactor 1, and aqueous bleach solution was charged to the scrubber. The circulating pump (commercial 5.25% NaOCl) was initiated. The scrubber pump was turned on and N2 sweep on Reactor 1 was started. Reactor 1 was charged with 2597 g (0.52 equivalents) of Lawesson’s reagent. Reactor 1 was then charged with 6000 g (1.0 equivalent) of HYDZ and 30 L (5.0 vol) acetonitrile (MeCN). Agitation of Reactor 1 was initiated. The reactor was heated to 50+5 °C and aged until an LC assay showed consumption of HYDZ (> 99% conversion).

The jacket temperature of a second clean and dry reactor, Reactor 2, was set to 50 °C. The contents of Reactor 1 were transferred to Reactor 2 through a 5 micron inline filter. Reactor 1 was rinsed with MeCN, and the rinse was transferred through the inline filter to Reactor 2. Reactor 2 was charged with toluene. (31.7 Kg)

In a separate container a solution of 16.7% K2C03 was prepared by adding 7200 g K2C03 and 36 L water to the container and shaking the container well until all the solid was dissolved. Half of the contents of the K2C03 solution was added to Reactor 2 over at least 10 min. The batch temperature of Reactor 2 was adjusted to 50+5 °C. The batch in Reactor 2 was agitated at 50+5 °C for at least 1 h. The agitation was stopped and the batch in Reactor 2 was allowed to phase separate. The aqueous phase was removed. The remaining contents of the K2C03 solution was added to Reactor 2 over at least 10 min. The batch temperature in Reactor 2 was adjusted to 50+5 °C. The batch in Reactor 2 was agitated at 50+5 °C for at least 1 h. The agitation was stopped and the batch in Reactor 2 was allowed to phase separate. The aqueous phase was removed.

The jacket temperature of a clean and dry reactor, Reactor 3, was set to 50 °C. The contents of Reactor 2 were transferred to Reactor 3 through a 5 micron in-line filter. The contents of Reactor 3 were distilled at reduced pressure. Isopropyl alcohol (IP A, 23.9 kg) was charged to Reactor 3 and then the batch was distilled down. IPA (23.2 kg) was again added to Reactor 3. The charge/distillation/charge cycle was repeated. The batch temperature in Reactor 3 was adjusted to 70+15 °C. Reactor 3 was then charged with DI water (1.8 L). Concentrated HC1 (1015 mL) was added to Reactor 3 over at least 15 min at 70+15 °C.

A seed of the Compound A-HCl was prepared by combining a seed and IPA in a separate container. The Compound A-HCl seed was added to Reactor 3 as a slurry. The batch in Reactor 3 was aged at 70+15 °C for at least 15 min to ensure that the seed held. The batch in Reactor 3 was cooled to 20+5 °C over at least 1 h. Heptane (24.5 kg) was added to Reactor 3 at 20+5 °C over at least 1 h. The batch was aged at 20+5 °C for at least 15 min. The contents of Reactor 3 were filtered through an Aurora filter fitted with a <25 μιη PTFE or PP filter cloth. The mother liquor was used to rinse Reactor 3.

A 50% v/v IP A/heptane solution was prepared, in advance, in a separate container by adding the IPA and heptane to the container and shaking. The filter cake from Reactor 3 was washed with the 50% IP A/heptane solution. If needed, the IP A/heptane mixture, or heptane alone, can be added to Reactor 3 prior to filtering the contents through the Aurora filter. The cake was washed with heptane. The cake was dried under nitrogen and vacuum until there was about < 0.5 wt% heptane by GC analysis. The product was analyzed for purity and wt% assay by achiral HPLC, for wt% by QNMR, for water content by KF, for form by XRD, for chiral purity by chiral HPLC, and for K and P content by ICP elemental analysis.

Compound A-HCl had a purity of 99.56 area% and 88.3 wt% assay by achiral HPLC, and 89.9 wt% by QNMR. The water content was 0.99 wt% as determined by KF. The chiral purity was 99.9%ee as determined by chiral HPLC. The P and K content was found to be 171 ppm and 1356 ppm, respectively, as determined by ICP elemental analysis.

Thus, Example 4 shows the synthesis of Compound A-HCl according to the disclosure.

EXAMPLE 5

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE (COMPOUND A) – ROUTE 3

Scheme 5: Route 3 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one (compound A)

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one was synthesized according to Scheme 5, Route 3, by the following procedure. 0.760 g (1.6 mmol) N’-iS-fluoro-S-il-methyl-lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo- l,6-naphthyridin-6(5H)-yl)propanehydrazide (HYDZ) and 0.62 g (2.4 mmol) triphenylphosphine were taken up in 16 mL THF. 0.31 mL (2.4 mmol) trimethylsilyl (TMS)-azide was added, followed by addition of 0.37 mL (2.4 mmol) DEAD, maintaining the reaction temperature below 33 °C. The reaction was stirred at room temperature for 50 minutes. The reaction mixture was concentrated in vacuo.

The crude material was taken up in dichloromethane and loaded onto silica gel. The crude material was purified via medium pressure liquid chromatography using a 90: 10: 1 DCM : MeOH : NH4OH solvent system. 350 mg, (48% yield) of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one was collected as a tan solid. The (S) isomer was also collected. The product had a purity of 97% by HPLC.

Thus, Example 5 shows the synthesis of enantiomerically pure Compound A according to the disclosure.

EXAMPLE 6

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6-NAPHTHYRIDIN-5(6H)-ONE (COMPOUND A) AND THE HYDROCHLORIDE SALT- ROUTE 3

Scheme 6: Route 3 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (compound A) and the hydrochloride salt

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one was synthesized according to Scheme 6, Route 3, by the following procedure. Benzothiazyl disulfide (3.31 g, 9.97 mmol), HYDZ (4.0 g, 8.31 mmol), and a stir bar were added to a 50 mL 3-neck flask fitted with a reflux condenser topped with a nitrogen inlet, a thermocouple and a septum. The flask headspace was purged with nitrogen, and the solids were suspended in MeCN (20.00 mL, 5 mL/g) at ambient conditions. The flask contents were heated to 50 °C on a heating mantle. Finally,

trimethylphosphine, solution in THF (9.97 ml, 9.97 mmol) was added dropwise by syringe pump with stirring over 1 h. An ice pack was affixed to the side of the flask in lieu of a reflux condenser. After about 0.5 h from addition, the resulting suspension was sampled and analyzed by, showing about 99% conversion of penultimate, and about 94% Compound A vs.

benzothiazole-2-thiol (“BtSH”) adduct selectivity.

After about 0.75 h from addition, the yellow reaction mixture was cooled to 0 °C in an ice bath, and 30% hydrogen peroxide in water (2.037 mL, 19.94 mmol) was added dropwise over 2 hours. The reaction solution was allowed to warm to room temperature overnight.

The suspension was heated to 30 °C, held at that temperature for 3 h and then cooled to room temperature. After cooling was complete, an aliquot was filtered and the filtrate was analyzed by liquid chromatography, showing 99% Compound A vs. BtSH adduct (91% purity for Compound A overall).

A Celite filtration pad about 0.5″ thick was set up on a 50 mL disposable filter frit and wetted with toluene (32.0 mL, 8 mL/g). The reaction suspension was transferred to the Celite pad and filtered to remove BtSH-related byproducts, washing with MeCN (2.000 mL, 0.5 mL/g). The filtrate was transferred to a 100 mL round bottom flask, and treated with 30 mL (7.5 Vol) of an aqueous quench solution consisting of sodium bicarbonate (7.5 ml, 8.93 mmol) and sodium thiosulfate (3.75 ml, 4.74 mmol) at overall about 5 wt% salt. The suspension was stirred for about 15 min and then the layers were allowed to separate. Once the layers were cut, the aqueous waste stream was analyzed by LC, showing 8% loss. The organic stream was similarly analyzed, showing 71% assay yield, implying about 20% loss to waste cake.

The organic cut was transferred to a 3-neck 50 mL round bottom flask with magnetic stir bar, thermocouple, and a shortpath distillation head with an ice-cooled receiving flask. The boiling flask contents were distilled at 55 °C and 300 torr pressure. The volume was reduced to 17 mL. The distillation was continued at constant volume with concomitant infusion of IPA (about 75 mL). The resulting thin suspension was filtered into a warm flask and water (0.8 mL) was added. The solution was heated to 80 °C. After this temperature had been reached, hydrochloric acid, 37% concentrated (0.512 ml, 6.23 mmol) was added, and the solution was seeded with about 30 mg (about 1 wt%) Compound A-HC1 salt. The seed held for 15 min. Next the suspension was cooled to 20 °C over 2 h. Finally heptane (17 mL, 6 Vol) was added over 2 h by syringe pump. The suspension was allowed to stir under ambient conditions overnight.

The yellow-green solid was filtered on an M-porosity glass filter frit. The wet cake was washed with 1: 1 heptane/IPA (2 Vol, 5.5 mL) and then with 2 Vol additional heptane (5.5 mL). The cake was dried by passage of air. The dried cake (3.06 g , 78.5 wt%, 94 LC area% Compound A, 62% yield) was analyzed by chiral LC showing optical purity of 99.6% ee.

Thus, Example 6 shows the synthesis of enantiomerically pure Compound A and the hydrochloric salt thereof, according to the disclosure.

EXAMPLE 7

RE-CRYSTALLIZATION OF COMPOUND A

A-HCI A monohydrate

Scheme 7: Re-crystallization of Compound A

Compound A-HCI was recrystallized to Compound A. A (60 L) jacketed reactor, Reactor 1, with a jacket temperature of 20 °C was charged with 5291 g, 1.0 equivalent of Compound A-HCI. 2 Vol (10.6 L) of IPA and 1 Vol (5.3 L) of water were added to Reactor 1 and agitation of Reactor 1 was initiated.

An aqueous NaHC03 solution was prepared in advance by charging NaHC03 (1112 g) and water (15.87 L, 3 Vol) into an appropriate container and shaking well until all solids were dissolved. The prepared NaHC03 solution was added to Reactor 1 over at least 30 min, maintaining the batch temperature below 30 °C. The batch temperature was then adjusted to about 60 °C. The reaction solution was filtered by transferring the contents of Reactor 1 through an in-line filter to a second reactor, Reactor 2, having a jacket temperature of 60+5 °C. Reactor 2 was charged with water (21.16 L) over at least 30 min through an in-line filter, maintaining the batch temperature at approximately 60 °C. After the addition, the batch temperature was adjusted to approximately 60 °C.

A seed was prepared by combining Compound A seed (0.01 equivalents) and IP A/water (20:80) in an appropriate container, in an amount sufficient to obtain a suspension. The seed preparation step was performed in advance. Reactor 2 was charged with the seed slurry. The batch was aged at 55-60 °C for at least 15 min. The batch was cooled to 20+5 °C over at least 1 h. The batch from Reactor 2 was recirculated through a wet mill for at least 1 h, for example, using 1 fine rotor stator at 60 Hz, having a flow rate of 4 L/min, for about 150 min.

The reaction mixture was sampled for particle size distribution during the milling operation. The solids were analyzed by Malvern particle size distribution (PSD) and

microscopic imaging. At the end of the milling operation a sample of the reaction mixture was again analyzed. The supernatant concentration was analyzed by HPLC, and the solids were analyzed by Malvern PSD and microscopic imaging to visualize the resulting crystals.

The batch temperature was adjusted to 35+5 °C and the batch was aged for at least 1 h. The batch was cooled to 20+5 °C over at least 2 h. The reaction mixture was sampled to determine the amount of product remaining in the supernatant. The supernatant concentration was analyzed by HPLC for target of <5 mg/mL Compound A in the supernatant. The contents of Reactor 2 were filtered through an Aurora filter fitted with a <25 μιη PTFE or PP filter cloth.

A 20% v/v IP A/water solution was prepared and the filter cake from Reactor 2 was washed with the 20% IP A/water solution. The cake was then washed with water. If needed, the IP A/water solution, or water alone, can be added to Reactor 2 prior to filtering to rinse the contents of the reactor. The cake was dried under moist nitrogen and vacuum until target residual water and IPA levels were reached. The product had 3.2-4.2% water by KF analysis. The product was analyzed by GC for residual IPA (an acceptable about less than or equal to about 5000 ppm). The yield and purity were determined to be 100% and 99.69% (by HPLC), respectively.

Thus, Example 6 shows the recrystallization of Compound A from the HC1 salt, Compound A-HC1, according to the disclosure.

EXAMPLE 8

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE (COMPOUND A)

HYDZ A

Scheme 8 Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one was synthesized according to Scheme 8 by the following procedure. A clean and dry 60 L reactor was fitted with a reflux condenser, nitrogen inlet, and vented to a scrubber (Reactor 1). The jacket temperature of Reactor 1 was set to 20 °C. A scrubber was set up to the vent of Reactor 1, and aqueous bleach solution was charged to the scrubber. The circulating pump (commercial 5.25% NaOCl) was initiated. The scrubber pump was turned on and N2 sweep on Reactor 1 was started. Reactor 1 was charged with 1599.5 g (0.52 equivalents) of Lawesson’s reagent. Reactor 1 was then charged with 24.4 L acetonitrile (MeCN). Agitation of Reactor 1 was initiated. 3664.7 g (1.0 equivalent) of HYDZ was added to the reactor in portions over 1+0.5 h, using acetonitrile (5 L) as rinse. The reactor was heated to 50+5 °C and aged until an LC assay shows consumption of HYDZ (> 99% conversion).

The reactor was cooled to 20 °C and the reaction was assayed by HPLC for

Compound A. The assay showed a 99% crude yield of Compound A.

The contents of Reactor 1 were transferred to second reactor, Reactor 2, through a 1 micron inline filter. Reactor 2 was charged with 2 L of water. Reactor 2 was connected to a batch concentrator and vacuum distilled until a final volume of about 10 L. The jacket temperature was 50 °C during distillation and the pot temperature was maintained below 50 °C. The batch was then cooled to 20 °C.

In a separate container a solution of 10% K2CO3 was prepared by adding 1160 g K2CO3 and 10450 mL water to the container and shaking the container well until all the solid was dissolved. The K2CO3 solution was added to Reactor 2 through an in-line filter (5 μηι). 13 kg of purified water was added to the reactor through the in-line filter (5 μηι).

A Compound A seed was added to the reactor through an addition port. The resulting slurry was aged for one hour during which crystallization was observed. The reactor was placed under vacuum and charged with 16 L of water. The resulting slurry was aged at 20 °C overnight. The product slurry was filtered through a 25 μιη filter cloth and washed with 10 L of a 10% MeCN in water solution, followed by 12 L of water. The product was dried on a frit under a stream of ambient humidity filtered air.

Compound A was isolated as a monohydrate crystalline solid which reversibly dehydrates at < 11% RH. After drying, there was 3.9 wt.% water present in constant weight solid as determined by KF. 3.317 kg, 89% yield, of Compound A was isolated as a pale yellow solid. The product had a purity of 99.4 wt.% as determined by LCAP.

EXAMPLE 9

SYNTHESIS OF NAPH – ROUTE 1

CuBr (5-10%)

ethyl 5-bromo-2- Bromonaphthyridinone Naphthyridinone ether methylnicotinate

Scheme 9: Synthesis of NAPH – Route 1

The NAPH starting material for the synthesis of Compound A was synthesized according to Scheme 9, Route 1 by the following procedure. The jacket temperature of a 6 L jacketed reactor, Reactor 1, was set to 22 °C. 2409 g (1.0 equiv) of ethyl 5-bromo-2-methylnicotinate, 824 g (1.0 equivalent) of triazine, and 3.6 L dimethyl sulfoxide (DMSO) were added to the reactor. The jacket temperature was adjusted to 45 °C. The reactor was agitated until a homogenous solution resulted. Once complete dissolution has occurred (visually) the jacket of Reactor 1 was cooled to 22 °C.

A second, 60 mL reactor, Reactor 2, was prepared. 8.0 L of water was charged to a scrubber. 4.0 L of 10 N sodium hydroxide was added to the scrubber and the scrubber was connected to Reactor 2. The cooling condenser was started. 6411.2 g of cesium carbonate and 12.0 L of DMSO were added to Reactor 2. Agitation of Reactor 2 was initiated. The batch temperature of Reactor 2 was adjusted to 80 °C. The solution from Reactor 1 was added slowly over 1 h at 80 °C, while monitoring the internal temperature. 1.2 L of DMSO was added to Reactor 1 as a rinse. The DMSO rinse was transferred from Reactor 1 to Reactor 2 over 6 min. Reactor 2 was agitated for more than 1 h and the conversion to 3-bromo-l,6-naphthyridin-5(6H)-one was monitored by HPLC until there was < 1.0% ethyl 5-bromo-2-methylnicotinate remaining. When the reaction was complete the batch temperature was adjusted to 60 °C. 24.0 L (10V) of water was added to Reactor 2 over 2 h, maintaining a reaction temperature of 60+5 °C, using a peristaltic pump at 192 mL/min. Reactor 2 was cooled to 22 °C over 1 h 10 min. Stirring was continued at 22+5 °C until the supernatant assays for less than 3mg/mL of 3-bromo-l,6-naphthyridin-5(6H)-one (analyzed by HPLC). The crystallized product was filtered through an Aurora filter fitted with 25 μιη polypropylene filter cloth. The reactor and filter cake were washed with a 75 wt% H20-DMSO solution (3 Vol made from 1.6 L DMSO and 5.6 L water), followed by water (7.2 L, 3 Vol), and finally toluene (7.2 L, 3 Vol). The product cake was dried on the aurora filter under vacuum with a nitrogen stream at ambient temperature. The product was determined to be dry when the KF was < 2.0 wt% water. 2194 g of 3-bromo-l,6-naphthyridin-5(6H)-one was isolated as a beige solid. The chemical purity was 99.73%. The adjusted yield was 2031.6 g (91.9%).

The jacket temperature of a 100 L reactor, Reactor 3, was set to 15+5 °C. 6.45 L of 2-methoxyethanol was added to the reactor and agitation was initiated. (8107 g) lithium tert-butoxide was added portion- wise to the reactor, maintaining the reactor temperature in a range of 15 °C to 24 °C. 3795 g of 3-bromo-l,6-naphthyridin-5(6H)-one was added to the reactor. 4 mL of 2-methoxyethanol was added to rinse the solids on the wall of the reactor. The reactor contents were stirred for at least 5 min. The reaction mixture was heated to distillation to remove i-BuOH and water, under 1 atm of nitrogen (jacket temperature 145 °C). Distillation continued until the pot temperature reached 122+3 °C. The reactor contents were sampled and analyzed for water content by KF. The reaction mixture was cooled to less than 35 °C. 243 g CuBr was added to the reactor. The reaction mixture was de-gassed by applying vacuum to 50 torr and backfilling with nitrogen three times. The batch was heated to 120+5 °C while maintaining the jacket temperature below 150 °C. The batch was agitated (174 RPM) for 15.5 h. A sample of the reaction was taken and the reaction progress was monitored by HPLC. When the remaining 3-bromo-l,6-maphthyridin-5(6H)-one was less than 1%, the jacket temperature was cooled down to 25 °C.

An Aurora filter was equipped with a 25 μιη PTFE cloth and charged with Celite®. The reactor content was transferred onto the filter cloth and the filtrate was collected in the reactor. 800 mL of 2-methoxyethanol was added to the reactor and agitated. The reactor contents were transferred onto the filter and the filtrate was collected in the reactor. 5.6 L of acetic acid was added to the reactor to adjust the pH to 6.5, while maintaining the temperature at less than 32 °C. The batch was then heated to 80 °C. The reaction mixture was concentrated to 3.0+5 Vol (about 12 L) at 80+5 °C via distillation under vacuum.

In a separate container labeled as HEDTA Solution, 589.9 g of N-(2-hydroxyethyl)ethylenediaminetriacetic acid trisodium salt hydrate and 7660 mL water were mixed to prepare a clear solution. The HEDTA solution was slowly added to the reactor while maintaining the temperature of the batch at about 80-82 °C. The batch was then cooled to 72 °C.

An aqueous seed slurry of NAPH (31.3g) in 200 mL of water was added to the reactor. The slurry was aged for 30+10 min. 20 L of water was slowly added to the reactor to maintain the temperature at 65+5 °C. The batch was aged at 65+5 °C for 30 min. The batch was cooled to 20 °C over 1 h. The reactor contents were purged with compressed air for 1 h, and then the batch was further cooled to – 15 °C and aged for 12.5 h. The batch was filtered through a centrifuge fitted with 25 μιη PTFE filter cloth. 5.31 Kg of wet cake was collected (60-62 wt ). The wet cake was reslurried in 6V HEDTA solution and filtered through the centrifuge. The collected wet cake was dried in the centrifuge, and transferred to an Aurora filter for continued drying.

2.82 kg (76% isolated yield) of NAPH was collected having a 2.7% water content by KF.

Thus, Example 8 shows the synthesis of NAPH according to the examples.

EXAMPLE 10

SYNTHESIS OF NAPH – ROUTE 2

Scheme 10: Synthesis of NAPH via Route 2

The NAPH starting material for the synthesis of Compound A was synthesized according to Scheme 10, Route 2, by the following procedure.

Preparation of protected 2-methoxy-pyridin-4ylamine. A 1600 L reactor was flushed with nitrogen and charged with 120 L of N,N-dimethylacetamide, 100.0 kg 2-methoxy-pyridin-4-ylamine, and 89.6 kg triethylamine, maintaining the temperature of the reactor at less than 20 °C. In a separate container, 103.0 kg pivaloyl chloride was dissolved in 15.0 L of N,N-dimethylacetamide and cooled to less than 10 °C. The pivaloyl chloride solution was added to the reactor using an addition funnel over 3.2 hours while maintaining the reactor temperature between 5 °C and 25 °C. The addition funnel was washed with 15.0 L of N,N-dimethylacetamide, which was added to the reactor. The reaction was stirred for 2.3 hours at 20-25 °C. A sample of the reaction was taken and analyzed for 2-methoxy-pyridin-4ylamine by TLC. No 2-methoxy-pyridin-4ylamine remained in the solution and the reaction was aged at 20-25 °C under nitrogen over night. 1200 L of deionized water was added to the reaction over 2

hours at while the reaction was maintained at 5-15 °C. The resulting mixture was stirred at 15 °C for 2 hours and then cooled to 5 °C. The reaction was centrifugated at 700-900 rpm in 3 batches. Each batch was washed 3 times with deionized water (3x 167 L) at 800 rpm. The wet solids obtained were dried under vacuum at 55 °C for 18 hours in 2 batches, sieved and dried again under vacuum at 55 °C for 21 hours until the water content was < 0.2% as determined by KF. 80.4 kg (89.7% yield) of the protected 2-methoxy-pyridin-4ylamine was collected as a white solid.

Preparation of protected 3-formyl-4-amino-2-methoxypyridine. A 1600 L reactor was flushed with nitrogen and charged with 1000 L of THF and 70.5 kg of the protected 2-methoxy-pyridin-4ylamine. The reaction was stirred for 10 min at 15-25°C. The reaction was cooled to -5 °C and 236.5 kg of w-hexyllithium (solution in hexane) was added over 11.5 hours while maintaining the temperature of the reaction at <-4°C. The reaction was maintained at <-4°C for 2 hours. A sample of the reaction was quenched with D20 and the extent of the ortho-lithiation was determined by 1H NMR (98.2% conversion). 61.9 kg dimethylforaiamide (DMF) was added at <-4°C over 3.2 h. After stirring 7.5 hours at <-4°C, a sample of the reaction was assayed for conversion by HPLC (98.5% conversion).

A 1600 L reactor, Reactor 2, was flushed with nitrogen and charged with 145 L THF and 203.4 kg of acetic acid. The resulting solution was cooled to -5 °C. The content of the first reactor was transferred to Reactor 2 over 2.5 hours at 0 °C. The first reactor was washed with 50 L THF and the washing was transferred into Reactor 2. 353 L deionized water was added to Reactor 2 while maintaining the temperature at less than 5 °C. After 15 min of decantation, the aqueous layer was removed and the organic layer was concentrated at atmospheric pressure over 5 hours until the volume was 337 L. Isopropanol (350 L + 355 L) was added and the reaction was again concentrated at atmospheric pressure until the volume was 337 L. Distillation was stopped and 90 L of isopropanol was added to the reactor at 75-94 °C. 350 L of deionized water was added to the reactor at 60-80 °C over 1 h (the temperature was about 60-65 °C at the end of the addition). The reaction was cooled to 0-5 °C. After 1 hour, the resulting suspension was filtered. Reactor 2 was washed twice with deionized water (2x 140L). The washings were used to rinse the solid on the filter. The wet solid was dried under vacuum at 50 °C for 15 h. 71.0 kg (80% yield) of the protected 3-formyl-4-amino-2-methoxypyridine was produced. The purity of the formyl substituted pyridine was found to be 92.7% by LCAP.

A 1600 L reactor, Reactor 3, was flushed with nitrogen and successively charged with 190 L ethanol, 128.7 kg of protected 3-formyl-4-amino-2-methoxypyridine, 144 L of deionized water and 278.2 kg of sodium hydroxide. The batch was heated to 60-65°C and 329.8 kg of the bisulfite adduct was added over 1 h. After lh of stirring, a sample was taken for HPLC analysis which showed 100% conversion. The batch was aged 2 hours at 60-65 °C, then was allowed to slowly cool down to 20-25 °C. The batch was aged 12 h at 20-25 °C. The batch was filtered and the reactor was washed with water (2x 125 L). The washings were used to rinse the solid on the filter. The wet solid was transferred to the reactor with 500 L deionized water and heated to 45-50 °C for 1 h. The batch was allowed to return to 20-25 °C (24 h). The solid was filtered and the reactor was washed with deionized water (2x 250 L). The washings were used to rinse the solid on the filter. 112.5 kg of wet white solid was obtained (containing 85.1 Kg (dry) of the naphthyridine, 72.3% yield, greater than 97% purity as determined by HPLC). The wet product was used directly in the next step, without drying.

A 1600 L reactor was flushed with nitrogen and charged with 417 L of deionized water and 112.5 kg of the wet napthyridine. The scrubber was filled with 700 L of water and 92.2 kg monoethanolamine. A solution of hydrochloric acid (46.6 kg diluted in 34 L of deionized water) was added to the reactor at 15-20 °C over 10 minutes. The batch was heated to 60-65 °C for 3 h. A sample of the batch was taken and contained no remaining starting material as determined by TLC. A solution of concentrated sodium hydroxide (58.2 kg in 31 L of deionized water) was added to the reactor at 60-65 °C. 65% of the solution was added over 15 min and then the batch was seeded with crystallized NAPH. Crystallization was observed after 2.5 h and then the remaining35% of the sodium hydroxide solution was added (pH – 11.1). The batch was cooled to 25-30 °C and a solution of sodium phosphate monobasic (1.8 kg in 2.9 L of deionized water) was added over 25 min at 25-30 °C) (pH = 6.75). The batch was stirred at 15-20 °C for 12 hours and filtered. The reactor was washed twice with deionized water (2x 176 L). The washings were used to rinse the solid on the filter. The wet solid was dried under vacuum at 50 °C until the water content was < 5% (by KF), to give 78.1 kg (73.8% yield, > 95%)) of NAPH as a beige powder.

Thus, Example 9 shows the synthesis of NAPH according to the disclosure.

EXAMPLE 11

SYNTHESIS OF (R)-2-(3-(2-METHOXYETHOXY)-5-OXO-l,6-NAPHTHYRIDIN-6(5H)- YL)PROPANOIC ACID NAPHTHALENE-2-SULFONATE (NAPA)

6N HCI/ THF 80C

Scheme 11: Synthesis of NAPA, Route 3

NAPA was synthesized according to Scheme 11, Route 3 by the following procedure. 4.75 g of 3-(2-Methoxyethoxy)-l,6-naphthyridin-5(6H)-one was suspended in 45 mL of DMF. 2.58 mL (s)-methyl lactate and 9.05 g triphenylphosphine were added to the suspension. The reaction mixture was cooled to 0 °C. 5.12 mL diethyl azodicarboxylate (DEAD) was added dropwise via syringe. The mixture was stirred at 0 °C for 1 h. A sample of the reaction was taken and the reaction was determined to be complete by LCMS. The reaction mixture was concentrated under vacuum to give crude material as a yellow oil.

1 g of the crude material was loaded in dichloromethane onto a silia gel pre-column. The sample was purified using the Isco Combi-Flash System; column 40 g, solvent system hexane/ethyl acetate, gradient 0-100% ethyl acetate over 15 minutes. Product eluted at 100% ethyl acetate. The product fractions were combined and concentrated under vacuum. 256 mg of (R)-methyl 2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoate was collected as a pale yellow oil.

The remaining residue was partitioned between benzene and 6N aq hydrochloric acid (35.9 mL). The acidic layer was extracted with benzene (3x), diethyl ether (2x), ethyl acetate (2x) and dichloromethane (lx). The dichloromethane layer was back extracted with 6N aq. Hydrochloric acid (2x). The aqueous layer was diluted with THF (80 mL). The mixture was heated at 80 °C for 3 h. The reaction mixture was concentrated to remove the THF. The remaining acidic water layer was extracted with ethyl acetate and dichloromethane. The aqueous layer was concentrated under vacuum. The remaining solid was triturated with methanol. The mixture was filtered to remove the solid (naphthyridone). The methanol layer was concentrated under vacuum. The remaining solid was dried overnight on a freeze drier. 10.2 g of material was collected as a yellow solid. NAPA made up 72% of the material as determined by HPLC.

1.0 g of the crude material was dissolved in minimal hot iPrOH then filtered and cooled to RT. Crystallization didn’t occur; therefore the solution was cooled in the freezer overnight. A yellow precipitate formed. The solid was collected on a glass frit and was washed with minimal iPrOH. 171 mg of yellow solid was collected, which was NAPA with a small amount of naphthyridone by LC-MS and 1H NMR.

Acid-base extraction. About 1 g of the crude material was dissolved in saturated aqueous sodium bicarbonate. The crude material was extracted with dichloromethane. The pH of the aqueous layer was adjusted to 6-7 with acetic acid then extracted with dichloromethane. 11 mg of the product was isolated; the majority of the product remained in the aqueous layer. The pH was reduced to approximately 4-5 with additional acetic acid. The aqueous layer was extracted with dichloromethane, ethyl acetate, and 15% methanol/dichloromethane. The organic layers were concentrated under vacuum to yield 260 mg of NAPA as the free base, as determined by LC-MS.

Thus, Example 10 shows the synthesis of NAPA according to the disclosure.

EXAMPLE 12

SYNTHESIS OF BISULFITE ADDUCT

DMSO

(COCI)2

MeCX ,ΟΗ Et3N

O

aqueous solution

Scheme 12: Synthesis of bisulfite adduct

Method 1

The bisulfite adduct was synthesize according to Method 1 of Scheme 12 by the following procedure. A 2L round-bottom flask (RBF) was purged with nitrogen and charged with 73.1 mL of reagent grade oxalyl chloride and 693 mL methylene chloride. The batch was cooled to less than -40 °C. 88 mL of dimethyl sulfoxide was added to the flask via an addition funnel at less than -40 °C. After the addition, the batch was stirred for 10 in at -60 °C. 97 mL diethylene glycol monomethyl ether was added to the flask at less than -50 °C over 10 min. The resulting white slurry was stirred at -60 °C for 30 min. 229 mL triethylamine was added to the flask via an addition funnel at less than -30 °C over 1 h. The batch was warmed to RT. 300 mL MTBE was added to the flask and the batch was stirred for 15 min. The slurry was filtered through a fritted funnel and the cake was washed with 300 mL MTBE. The filtrate was concentrated to 350-400g and then filtered again to remove triethylamine-HCl salt, and the solid was rinsed with MTBE, resulting in 357.7 g of a slightly yellow filtrate solution. The solution was assayed by QNMR and comprised 19 wt (68 g) of the desired aldehyde (70% crude yield). The solution was concentrated to 150.2 g.

A 500 mL RBF was charged with 60.0 g sodium bisulfite and 150 mL of water to give a clear solution. The concentrated aldehyde solution was added to the aqueous bisulfite solution over 5 min. An exothermic temperature rising was observed up to 60 °C from 18 °C. The solution was rinsed with 15 mL water. The resulting yellow solution was cooled to RT and was stirred under a sweep of nitrogen overnight.. A QNMR of the solution was taken. The solution contained 43 wt.% of the bisulfite adduct (300 g, 70% yield).

Method 2

The bisulfite adduct was synthesized according to Method 2 of Scheme 12 by the following procedure. A 2500 L reactor was flushed with nitrogen and charged with 657.5 L of 2-methoxyethanol. 62.6 kg of lithium hydroxide monohydrate was added to the reactor while maintaining the temperature at less than 30 °C. The reactor was heated to 113+7 °C. 270 L of solvent were distilled over 1 h and then the reactor temperature was adjusted to 110 °C. 269.4 kg of bromoacetaldehyde diethyl acetal was added over 16 minutes, maintaining the temperature between 110 and 120 °C. The reaction was heated to reflux (115-127°C) for 13 hours. A sample of the reaction was assayed and conversion to 2-(2-methoxyethoxy)acetaldehyde was found to be 98.3%. The reaction was cooled to 15-20°C and 1305 L of methyl ie/t-butyl ether (MTBE) and 132 L of deionized water was added to the reactor. The reaction was stirred for 20 min and then was decanted. The aqueous layer was transferred into a 1600 L reactor and the organic layer was kept in the first reactor. The aqueous layer was extracted with 260 L of MTBE for 10 min. After 10 min decantation, the aqueous layer was removed and the organic layer was transferred to the first reactor. The mixed organic layers were washed twice, 15 min each, with a mixture of concentrated sodium hydroxide solution (2x 17.3 kg) diluted in deionized water (2x 120 L). The aqueous layers were removed, and the organic layer was concentrated at atmospheric pressure at 60-65 °C until the volume was 540 L. The organic layer was cooled down to 15-20 °C to give 2-(2-methoxyethoxy)acetaldehyde as an orange liquid solution (417.4 kg) containing 215.2 kg of pure product (87.3% yield) as determined by 1H NMR and HPLC assay.

A 1600 L reactor, Reactor 3, was flushed with nitrogen and charged with 595 L deionized water followed by 37.8 kg sulfuric acid over 25 minutes via addition funnel, while maintaining the temperature below 25 °C. The addition funnel was washed with 124 L of deionized water and the washing was added to Reactor 3.

A 2500 L reactor, Reactor 4, was flushed with nitrogen and charged with 417.4 kg of the solution of the 2-(2-methoxyethoxy)acetaldehyde. The content of Reactor 3 was transferred into Reactor 4 over 25 min while maintaining the temperature of Reactor 4 below 35 °C. The batch was aged at 30-35 °C for 3 hours. A sample of the batch was taken and assayed for 2-(2- methoxyethoxy)acetaldehyde. No 2-(2-methoxyethoxy)acetaldehyde remained. The batch was aged 5 h then cooled to 15-20 °C.

A solution of sodium carbonate (39.2 kg) in deionized water (196 L) was prepared in Reactor 3. The sodium carbonate solution was transferred to Reactor 4 over 25 min while maintaining the temperature of Reactor 4 below 30 °C. The pH of the resulting mixture was pH 5-6. 1.0 kg sodium carbonate was added by portion until the pH was about 7-8. A solution of sodium bisulfite (116.5 kg) in deionized water (218 L) was prepared in Reactor 3. The sodium bisulfite solution was transferred to Reactor 4 over 20 min while maintaining the temperature of Reactor 4 below 30 °C. Reactor 3 was washed with deionized water (15 L) and the washing was added to Reactor 4. The batch was stirred for 1.2 hours. 23.3 kg sodium bisulfite was added to Reactor 4 and the batch was aged overnight. The batch was concentrated under vacuum at 30-50 °C over 6.5 hours until precipitation was observed. The batch was cooled to 0-10°C at atmospheric pressure. After 30 min at 0-10 °C, the suspension was filtered on 2 filters. Reactor 4 was washed with deionized water (2x 23 L). The first washing was used to rinse the solid on the first filter and the second washing was used to rinse the solid on the second filter. Filtrates were joined to give 473.9 kg of an aqueous solution of the bisulfite adduct (202.5 kg of pure product, 76.3% yield) as a yellow liquid.

Thus, Example 11 shows the synthesis of the bisulfite adduct according to the invention.

EXAMPLE 13

SYNTHESIS OF 2,3-DIFLUORO-5-(l-METHYL-lH-PYRAZOL-4-YL)PYRIDINE

Scheme 13: Synthesis of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine, precursor to PYRH

2,3-Difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was synthesized according to Scheme 13 by the following procedure. A boronic-ate complex slurry was prepared in a first 3-neck-2-L round-bottom flask (RBF #1). RBF #1 was charged with 141 g (66.4 wt%, 0.9 equivalents based on boronic ester) of lithium 2-hydroxy-4,4,5,5-tetramethyl-2-(l-methyl-lH-pyrazol-4-yl)-l,3,2-dioxaborolan-2-uide. 120 mL (1.6 Vol relative to 5-chloro-2,3-difluoropyridine) of nitrogen- sparged (2 h) 2-BuOH and 120 mL (1.6 Vol) nitrogen-sparged (2 h) water were added to RBF #1. Agitation and N2 sweep were initiated. The reaction was aged at 20 °C for at least 30 min (reactions aged to 24 h were also successful).

] A second 3-neck-2-L round-bottom flask (RBF #2) was charged with 1.48 g (0.004 equivalents) of Xphos-palladacycle and 450 mL (6 Vol relative to 5-chloro-2,3-difluoropyridine) of nitrogen- sparged (2 h) 2-BuOH. Vacuum/N2 flush was cycled through RBF #2 three times to inert the RBF with N2. The batch in RBF #2 was heated to 80 °C. 75 g (1.0 equivalents) of 5-chloro-2,3-difluoropyridine was added to RBF #2.

The slurry of boronic-ate complex was transferred from RBF #1 to a 500 mL dropping funnel. RBF #1 was rinsed with 30 mL (0.4 Vol) 2-BuOH. Using the dropping funnel, the slurry of boronic-ate complex was added over 1 h to the hot solution mixture in RBF #2. After 1 h, 95% conversion was observed. If greater than 90% conversion was not observed, additional boronic-ate complex slurry was added (0.1 equivalents at a time with 1.6 Vol of 1: 1 2-BuOH/water relative to boronic-ate complex). After the conversion was complete, the batch was cooled to 50 °C. While cooling, 600 mL (8 Vol) of toluene was added to RBF #2. 300 mL (4 Vol) of 20% w/v NaHS03 in water was added to RBF #2 and the batch was stirred at 50 °C for at least 1 h. The batch was polish filtered using a 5 micron Whatman filter at 50 °C, into a 2-L Atlas reactor. RBF #2 was rinsed with 30 mL (4.0 Vol) of a 1: 1 2-BuOH:toluene solution. The temperature of the batch was adjusted to 50 °C in the Atlas reactor while stirring. The stirring was stopped and the phases were allowed to settle for at least 15 min while maintaining the batch at 50 °C. The bottom, aqueous layer was separated from the batch. The Atlas reactor was charged with 300 mL (4 Vol) of a 20% w/v NaHS03 solution and the batch was stirred at 50°C for 1 h. The agitation was stopped and the phases were allowed to settle for at least 15 min at 50 °C. The bottom, aqueous layer was removed. Agitation was initiated and the Atlas reactor was charged with 200 mL (4 Vol) of 0.5 M KF while keeping the batch at 50 °C for at least 30 min. The agitation was stopped and the phases were allowed to settle for at least 15 min at 50 °C. The bottom, aqueous layer was removed. Agitation was initiated and the reactor was charged with 300 mL (4 Vol) of water. The batch was aged at 50 °C for at least 30 min. Agitation was stopped and the phases were allowed to settle for at least 15 min at 50 °C. The bottom, aqueous later was removed.

The organic phase was concentrated by distillation under reduced pressure (180 torr, jacket temp 70°C, internal temp about 50 °C) to a minimal stir volume (about 225 mL). 525 mL (7 Vol) of 2-BuOH was added to the Atlas reactor. The organic batch was again concentrated using reduced pressure (85-95 torr, jacket temp 75 °C, internal temp about 55 °C) to a minimal stir volume (about 125 mL). The total volume of the batch was adjusted to 250 mL with 2-BuOH.

525 mL (7 Vol) heptane was added to the slurry mixture in the Atlas reactor. The jacket temperature was adjusted to 100 °C and the batch was aged for more than 15 min, until the batch became homogeneous. The batch was cooled to 20 °C over at least 3 h. A sample of the mixture was taken and the supernatant assayed for 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine. If the concentration was greater than 10 mg/mL, the aging was continued for at least 1 h until the supernatant concentration was less than 10 mg/mL. The batch was filtered using a medium frit. The filter cake was washed with 150 mL (2 Vol) 30% 2-BuOH/heptane solution followed by 150 mL (2 Vol) heptane. The filter cake was dried under N2/vacuum. 76.64 g of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was isolated as a white solid (87% yield).

A 60 L jacketed reactor was fitted with a reflux condenser. The condenser cooling was initiated at 0+5 °C. The reactor was charged with 2612 g (1 equivalent) of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine and placed under an atmosphere of nitrogen. 31.7 L (12.2 Vol) water was added to the reactor and the resulting slurry was nitrogen sparged for 1 h with agitation. 7221 mL (6 equivalents) of hydrazine (35 wt% in water) was added to the reactor under a nitrogen atmosphere. The reactor was heated to 100 °C for 2+2 h until reaction was complete by HPLC analysis. The reactor was cooled to 20 °C over 2+1 h at a rate of 40°C/h. The reactor contents were stirred for 10+9 hours until the desired supernatant assay (< 2mg/mL PYRH in mother liquor). The reactor contents were filtered through an Aurora filter fitted with 25 μιη polypropylene filter cloth. The collected filter cake was washed with 12.0 L (4.6 V) of water in three portions. The filter cake was dried on the Aurora filter for 4-24 h at 22+5 °C, or until the product contained less than 0.5% water as determined by KF. The dry product was collected. 2.69 kg (97% yield) 2,3-Difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was collected as a white crystalline solid. The solid had a water content of 12 ppm as determined by KF.

Thus, Example 12 shows the synthesis of 2,3-Difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine, a precursor to PYRH, according to the disclosure.

EXAMPLE 14

SYNTHESIS OF PYRH – ROUTE 2

Scheme 14: Synthesis of 3-fluoro-2-hydrazinyl-5-(l-methyl-lH-pyrazol-4-yl)-pyridine (PYRH)

3-fluoro-2-hydrazinyl-5-(l-methyl-lH-pyrazol-4-yl)-pyridine was synthesized according to Scheme 14 by the following procedure. A 60 L jacketed reactor was fitted with a 5 L addition funnel and the jacket temperature was set to 20+5 °C. 36.0 L (15 Vol) of 2-methyltetrahydrofuran was added to the reactor via a 20 μιη inline filter with vacuum using polypropylene transfer lines. The solution was sparged by bubbling nitrogen through a dipstick in the solution for 1+0.5 h with agitation. After 1 h the dipstick was removed but the nitrogen sweep continued. 1.55 kg of sparged 2-MeTHF was removed to be used as rinse volumes. 36.7 g of Pd2dba3, 75.6 g X-Phos, 259 g of tetrabutylammonium bromide, and 7397 g of potassium phosphate tribasic were added to the reactor. The manhole was rinsed with 0.125 kg of sparged 2-MeTHF. The reactor was agitated and the nitrogen sweep continued for 1+0.5 h. Then the nitrogen sweep was stopped and the reaction left under a positive pressure of nitrogen.

3.6 L (1.5 Vol) of sparged water was prepared in advance by bubbling nitrogen through a 4 L bottle of water for 1+0.5 h. The nitrogen sparged water was transferred to the 5 L addition funnel via a 20 μηι inline filter with vacuum using polypropylene transfer lines, then slowly added to the reaction while maintaining the internal temperature at 20+5 °C. The 5 L addition funnel was replaced with a 2 L addition funnel. 2412 g of 5-chloro-2,3-difluoropyridine was added to the 2 L addition funnel. The 5-chloro-2,3-difluoropyridine was then added to the reaction through the 2 L addition funnel. The 2L addition funnel was rinsed with 0.060 kg of sparged 2-MeTHF. 83.8 g (1.15 equivalents) of l-methylpyrazole-4-boronic acid, pinacol ester was added to reactor, the reactor was swept with nitrogen for 1+0.5 h, then left under a positive pressure of nitrogen. The internal temperature of the reactor was adjusted to 70+5 °C. The batch was agitated at 70+5 °C for at least 4 hours after the final reagent was added. A sample was taken from the reaction and the reaction progress assayed for conversion. The progress of the reaction was checked every 2 hours until the reaction was completed (e.g., greater than 99% conversion). The batch was cooled to 20+5 °C.

A 20% w/v sodium bisulfite solution (12.0 L, 5 Vol) was prepared by charging 12.0 L of water then 2411 g sodium bisulfite to an appropriate container and agitating until

homogeneous. The 20% sodium bisulfite solution was transferred into the reactor and agitated for 30 minutes. The agitation was stopped, the phases allowed to settle, and the aqueous phase was removed. A 0.5 M potassium fluoride solution (12.0 L, 5 Vol) was prepared by charging 12.0 L of water and 348 g of potassium fluoride to an appropriate container and agitating until homogenous. The 0.5 M potassium fluoride solution was transferred into the reactor and agitated for 30 min. The agitation was stopped, the phases were allowed to settle, and the aqueous phase was removed. A 25% w/v sodium chloride solution (12.0 L, 5 Vol) was prepared by charging an appropriate container with 12.0 L of water and 2999 g of sodium chloride and agitating until homogeneous. The 25% sodium chloride solution was transferred into the reactor and agitated for 30 min. The agitation was stopped, the phases were allowed to settle, and the aqueous phase was removed from the reactor.

The organic phase was distilled at constant volume (36 L, 15 Vol) while maintaining the internal temperature of the reactor at 50+5 °C by adjusting the vacuum pressure until no more than 0.3% of water remained. 2-Methyltetrahydrofuran was added to the reactor as needed to

maintain constant volume. The batch was cooled to 20 °C and transferred into drums. The batch was transferred using a polish filter (using a 5 μιη inline filter) into a 60 L jacketed reactor with a batched concentrator attached. 1.2 L of 2-MeTHF was used to rinse the drums. The batch was concentrated to about 9 Vol while maintaining the internal temperature of the vessel at 50+5 °C by adjusting the vacuum pressure. The batch was then distilled at constant volume (22.0 L, 9Vol) while maintaining the internal temperature of the vessel at 50+5 °C by adjusting the vacuum pressure. Heptane was added with residual vacuum until a 15% 2-MeTHF:heptane supernatant mixture was obtained. The pressure was brought to atmospheric pressure under nitrogen. The reactor was cooled to 20+5 °C over 2+2 h. The batch was agitated at 20+5 °C until an assay of the supernatant indicated that the amount of product was 7 mg/mL 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine.

A 10% 2-MeTHF:heptane (7.2 L, 3 Vol) wash solution was prepared by mixing 720 mL of 2-MeTHF and 6.5 L of heptane. The batch slurry was filtered through an Aurora filter fitted with a 25 μιη polypropylene filter cloth, resulting in heavy crystals that required pumping with a diaphragm pump using polypropylene transfer lines through the top of the reactor while stirring. The mother liquor was recycled to complete the transfer. The reactor and filter cake were washed with two portions of the 10% 2-MeTHF:heptane wash solution (3.6 L each). The product cake was dried on a frit under a nitrogen stream at ambient temperature. The 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was determined to be dry when the 1H NMR assay was < 0.05+0.05. 2.635 kg was isolated as an off white crystalline solid (85% yield).

A 60 L jacketed reactor was fitted with a reflux condenser. The condenser cooling was initiated at 0+5 °C. The reactor was charged with 2612 g (1 equivalent) of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine and placed under an atmosphere of nitrogen. 31.7 L (12.2 Vol) water was added to the reactor and the resulting slurry was nitrogen sparged for 1 h with agitation. 7221 mL (6 equivalents) of hydrazine (35 wt% in water) was added to the reactor under a nitrogen atmosphere. The reactor was heated to 100 °C for 2+2 h until reaction was complete by HPLC analysis. The reactor was cooled to 20 °C over 2+1 h at a rate of 40°C/h. The reactor contents were stirred for 10+9 hours until the desired supernatant assay was reached (< 2mg/mL PYRH in mother liquor). The reactor contents were filtered through an Aurora filter fitted with 25 μιη polypropylene filter cloth. The collected filter cake was washed with 12.0 L

(4.6 V) of water in three portions. The filter cake was dried on the Aurora filter for 4-24 h at 22+5 °C, or until the product contained less than 0.5% water as determined by KF. The dry product was collected. 2.69 kg was isolated as a white crystalline solid (97% yield). The water content was determined to be 12 ppm by KF.

WO2007075567A1 * Dec 18, 2006 Jul 5, 2007 Janssen Pharmaceutica, N.V. Triazolopyridazines as tyrosine kinase modulators
WO2007138472A2 * May 18, 2007 Dec 6, 2007 Pfizer Products Inc. Triazolopyridazine derivatives
WO2008008539A2 * Jul 13, 2007 Jan 17, 2008 Amgen Inc. Fused heterocyclic derivatives useful as inhibitors of the hepatocyte growth factor receptor
WO2008051805A2 * Oct 18, 2007 May 2, 2008 Sgx Pharmaceuticals, Inc. Triazolo-pyridazine protein kinase modulators
WO2008155378A1 * Jun 19, 2008 Dec 24, 2008 Janssen Pharmaceutica Nv Polymorphic and hydrate forms, salts and process for preparing 6-{difluoro[6-(1-methyl-1h-pyrazol-4-yl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl]methyl}quinoline

References:
1. Hughes, P. E.; et. al. Abstract 728: AMG 337, a novel, potent and selective MET kinase inhibitor, has robust growth inhibitory activity in MET-dependent cancer models. Cancer Res 2014, 74, 728.
2. Boezio, A. A.; et. al. Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors. Bioorg Med Chem Lett 2009, 19(22), 6307-6312.
3. ClinicalTrials.gov Phase 2 Study of AMG 337 in MET Amplified Gastric/Esophageal Adenocarcinoma or Other Solid Tumors. NCT02016534 (retrieved 10-06-2015)
4. ClinicalTrials.gov A Study of AMG 337 in Subjects With Advanced Solid Tumors. NCT01253707 (retrieved 10-06-2015)

/////////// AMG-337,  AMG337,  AMG 337,  1173699-31-4, AMGEN, ESOPHAGUS

O=C1C2=C(N=CC(OCCOC)=C2)C=CN1[C@@H](C3=NN=C4C(F)=CC(C5=CN(C)N=C5)=CN43)C

AMG-319


AMG-319

N-((1S)-1-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine, WO2008118468

(S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine

 CAS 1608125-21-8

Chemical Formula: C21H16FN7
Exact Mass: 385.14512

Phosphoinositide-3 kinase delta inhibitor

AMGEN, PHASE 2

PI3K delta isoform selective inhibitor that is being investigated in human clinical trials for the treatment of PI3K-mediated conditions or disorders, such as cancers and/or proliferative diseases

Useful for treating PI3K-mediated disorders such as acute myeloid leukemia, myelo-dysplastic syndrome, myelo-proliferative diseases, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkins lymphoma, B-cell lymphoma, or breast cancer.

Amgen is developing AMG-319, a small molecule PI3K-δ inhibitor, for treating lymphoid malignancies and solid tumors including, head and neck squamous cell carcinoma.

AMG-319 is a highly selective, potent, and orally bioavailable small molecule inhibitor of the delta isoform of the 110 kDa catalytic subunit of class IA phosphoinositide-3 kinases (PI3K) with potential immunomodulating and antineoplastic activities. PI3K-delta inhibitor AMG 319 prevents the activation of the PI3K signaling pathway through inhibition of the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), thus decreasing proliferation and inducing cell death. Unlike other isoforms of PI3K, PI3K-delta is expressed primarily in hematopoietic lineages. The targeted inhibition of PI3K-delta is designed to preserve PI3K signaling in normal, non-neoplastic cells.

PATENT

http://www.google.com/patents/WO2008118468A1?cl=en

 

 

PATENT

WO2013152150

http://www.google.com/patents/WO2013152150A1?cl=en

PATENT

WO-2015171725

 

Example 4: Method of making N-((lSM-(7-fluoro-2-(2-pyridinyl)- 3-quinolinyl)ethyl)-9H-purin-6-amine

N-((l S)- 1 -(7-Fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine (4) is synthesized in four steps beginning with (S)-l-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine hydrochloride (1). A nucleophilic aromatic substitution between coupling partners 1 and purine 5 affords the penultimate intermediate 2. Cleavage of the p-methoxybenzyl (PMB) group leads to the isolation of the desired butyl acetate solvate 3. A crystalline form change is induced through an aqueous-acetone recrystallization to afford the target hydrate 4.

Synthetic Scheme

Step 1. Preparation of PMB protected pyridylpurinamine tosylate (2)

(S)- 1 -(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine is prepared similar to that described in US20130267524. The (S)-l-(7-fluoro-2-(pyridin-2-

yl)quinolin-3-yl)ethanamine hydrochloride (1) is coupled to PMB-chloropurine (5, prepared similar to that described in J. Med. Chem. 1988,31, 606-612) in the presence of K2CO3 in IPA. Upon reaction completion the K2CO3 is removed via filtration and the product is crystallized by the addition of /?-toluenesulfonic acid (pTSA). Isolation of the PMB-protected pyridylpurinamine tosylate (2) is conducted via filtration.

Dry 100 L reactor under nitrogen. Set the temperature to 20 ± 5 °C. Charge (l S)-N-chloro-l-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine HCl salt (1) to the reactor. Then 9-(4-methoxybenzyl)-6-chloro-9H-purine (5) is added. Potassium carbonate is added to the reactor. Isopropyl alcohol is added to the reactor and the mixture is heated to 80 °C and stirred for 24 hours. Additional isopropyl alcohol is added to the reactor and the mixture is cooled to 20 °C. The mixture is filtered through Celite and the solid is washed with isopropyl alcohol and the isopropyl alcohol solutions containing 9-(4-methoxybenzyl)-N-((S)- 1 -(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine are collected.

The 9-(4-methoxybenzyl)-N-((S)- 1 -(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine isopropyl alcohol solution is heated to 50 °C. /^-Toluene sulfonic acid monohydrate is dissolved in isopropyl alcohol and added to the 9-(4-methoxybenzyl)-N-((S)-l-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine in portions. The mixture is slowly cooled to 20 ± 5 °C over 6 ± 2 hrs. The crystalline 9-(4-methoxybenzyl)-N-((S)- 1 -(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)- 9H-purin-6-amine toluene sulfonic acid salt is collected, rinsed with isopropyl alcohol and dried with vacuum.

Example 5: Method of Making the Crystalline Hydrate Form of N-((1S)-1- (7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine Step 1: Isolation of a Butyl Acetate (BuOAc) Solvate of N-((lS)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine (3)

To a 2 L jacketed reactor equipped with a condenser, a mechanical stirrer, and a bubbler, under an atmosphere of N2, was added N-((l S)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9-(4-methoxybenzyl)-9H-purin-6-amine (2, 100.0 g, 0.148 mol), followed by acetic acid (AcOH; 240 mL) and 1 -dodecanethiol (71.1 mL, 0.295 mol). The vessel was evacuated and back-filled with nitrogen three times. Methanesulfonic acid (MSA; 28.7 mL, 0.443 mol) was added to the vessel over 10 minutes. Then, the reaction was heated to 80 °C and stirred for 20 hrs. The reaction was then cooled to ambient temperature, after which toluene (1000 mL) and water (700 mL) were sequentially added. The solution was then stirred for 30 minutes. The phases were separated by removing the organic phase, adding another charge of toluene (1000 mL) to the aqueous phase, and the mixture was stirred for another 30 minutes. After removing the organic phase again, the aqueous phase was charged to a jacketed 5 L reactor equipped with a mechanical stirrer followed by n-butyl acetate (1500 mL,) and heated to 50 °C. The aqueous phase was neutralized to pH 6.3 with 10 N NaOH (350 mL). The organic (BuOAc) phase was azeotropically dried to 600 ppm water, while keeping a constant volume. The dried organic phase was polish filtered at 50 °C to remove salts, which were subsequently washed with hot BuOAc (285 mL). The BuOAc was charged back into the 2 L jacketed reactor equipped with a mechanical stirred and distillation apparatus, and then concentrated to 54 mg/g of N-((l S)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine in solution. The solution was then seeded with 1 wt% seed of the BuOAc solvate of N-((l S)- 1 -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. The slurry was further concentrated to 300 mL total volume and cooled to ambient temperature over 1 hour. Heptane (460 mL) was added dropwise to the solution, and the solution was aged overnight. The supernatant concentration was checked, and determined to be 5.3 mg/g of N-((l S)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. The supernatant was filtered and the resulting solid cake was washed with 1 : 1 BuOAc:heptane (280 mL), followed by heptane (280 mL). The washed cake was then

allowed to dry on the filter. The BuOAc solvate of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine was obtained as a white solid (59.5 g, 99.6 LCAP, 86.3 wt%, 90 % corrected yield). !H NMR (400 MHz, CDC13) δ 13.72 (s, 1H), 8.80 (s, 1H), 8.37 (s, 1H), 8.31 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 18.8 Hz, 2H), 7.76 (t, J = 1 1.6 Hz, 2H), 7.39 (s, 1H), 7.31 (td, J = 8.7, 2.5 Hz, 1H), 6.15 (s, 1H), 4.06 (t, J = 6.7 Hz, 1H), 2.04 (s, 1H), 1.65 – 1.44 (m, 3H), 1.39 (dt, J = 14.9, 7.4 Hz, 1H), 1.33 – 1.20 (m, 2H), 0.93 (t, J = 7.4 Hz, 1H), 0.88 (t, J = 6.8 Hz, 1H); 13C NMR (101 MHz, CDC13) δ 152.28 (s), 148.46 (s), 138.10 (s), 137.22 (s), 135.58 (s), 129.47 (s), 124.80 (s), 123.53 (s), 1 13.24 – 1 13.09 (m), 1 12.89 (d, J = 20.3 Hz), 64.40 (s), 48.60 (s), 31.91 (s), 30.67 (s), 29.05 (s), 22.72 (s), 19.15 (s), 14.15 (s); IR: 3193, 3087, 2967, 2848, 1738, 1609, 1493, 1267, 1242, 1 143, 933, 874, 763, 677, 646, 627, 606, 581 , 559, 474 cm“1; exact mass m/z calcd for C2iH16FN7, (M + H)+386.1451 , found 386.1529; MP = 144 °C.

Step 2: Isolation of the Crystalline Hydrate of N-((lS)-l-(7-fluoro-2-(2-pyridinvn-3-quinolinyl)ethyl)-9H-purin-6-amine 4

To a 100 L reactor with its jacket set to 20 °C, 1.206 kg butyl acetate solvate of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine 3 was charged, followed by 6.8 L of acetone and 6.8 L of water. The resulting mixture was stirred at 90 rpm under nitrogen for 13 minutes to ensure complete dissolution of all solids. During these charges, the reactor contents increased in temperature that maximized at 26 °C. The solution was then transferred to another clean 100 L reactor through a 5 μιη filter, and stirred at 85 rpm under nitrogen. The solution was heated to 45 °C, and water (14.8 L) was added to reach a water content (by Karl Fischer, KF) of 75 wt%. The reactor solution was assayed by HPLC and shown to contain 42 mg/g N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. The solution was seeded with a slurry of 1 13 g of the crystalline hydrate of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine in 1 L water, and the seed slurry was rinsed into the reactor with an additional 1 L water. The reactor contents were cooled to 0 °C over 16 h and held at that temperature for 1 h. The supernatant was then assayed, and found to contain 7.6 mg/g of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. Next, 10 L of water was added to the reactor over 38 min and aged for 1 h. The supernatant was assayed at 4.9 mg/g, and the solids were isolated by filtration. The solids were washed with an acetone/water solution (140 mL acetone in 2.7 L water), then 4 L water, and dried under nitrogen on the filter for 68 h. The crystalline hydrate of N-((l S)-l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine was isolated as an off-

white solid (1.12 kg, 616 ppm acetone, 3.73 wt% water, 99.56 LCAP, 95.88 wt%). This material was co-milled at 3900 rpm using a 0.024″ screen to yield an off-white powder (1.09 kg, 99.7 LCAP, 95.4 wt%, 75% yield). Calculated losses were 212 g (18%) to liquors, 5.5g (0.5%) to washes, and 23 g (2%) to fouling. ¾ NMR (400 MHz, DMSO) δ 12.86 (s, 1H), 8.69 (s, 1H), 8.64 (s, 1H), 8.27 (s, 1H), 8.10 (s, 1H), 8.06 – 7.91 (m, 4H), 7.76 (dd, J = 10.4, 2.4 Hz, 1H), 7.50 (ddd, J = 19.2, 9.5, 3.6 Hz, 2H), 6.03 (s, 1H), 3.38 (s, 2H), 1.63 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 163.58, 161.12, 158.36, 157.94, 151.99, 147.98, 146.49, 146.36, 136.82, 134.07, 130.24, 130.14, 124.69, 124.65, 123.30, 1 17.36, 1 17.1 1, 112.10, 1 1 1.90, 46.02, 22.01. HRMS m/z Calcd. for C2iH17FN7 (M + H): 386.15295. Found: 386.15161.

 

PAPER

1: Cushing TD, Hao X, Shin Y, Andrews K, Brown M, Cardozo M, Chen Y, Duquette J, Fisher B, Gonzalez-Lopez de Turiso F, He X, Henne KR, Hu YL, Hungate R, Johnson MG, Kelly RC, Lucas B, McCarter JD, McGee LR, Medina JC, San Miguel T, Mohn D, Pattaropong V, Pettus LH, Reichelt A, Rzasa RM, Seganish J, Tasker AS, Wahl RC, Wannberg S, Whittington DA, Whoriskey J, Yu G, Zalameda L, Zhang D, Metz DP. Discovery and in vivo evaluation of (S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (AMG319) and related PI3Kδ inhibitors for inflammation and autoimmune disease. J Med Chem. 2015 Jan 8;58(1):480-511. doi: 10.1021/jm501624r. Epub 2014 Dec 3. PubMed PMID: 25469863.

http://pubs.acs.org/doi/abs/10.1021/jm501624r

Abstract Image

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

(S)-N-(1-(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (1)

 1H NMR (400 MHz, [D6]DMSO) δ ppm 12.76 (1 H, br s), 8.69 (1 H, br s), 8.63 (1 H, s), 8.21 (1 H, br s), 7.96–8.12 (4 H, m), 7.93 (1 H, s), 7.76 (1 H, dd, J = 10.4, 2.5 Hz), 7.45–7.57 (2 H, m), 6.00 (1 H, d, J = 1.2 Hz), 1.61 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 386.0 (M + 1).

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C[C@H](NC1=C2N=CNC2=NC=N1)C3=CC4=CC=C(F)C=C4N=C3C5=NC=CC=C5

Novartis launches first US ‘biosimilar’ drug at 15 percent discount


Zarxio, filgrastim-sndz

 

Novartis launches first US ‘biosimilar’ drug at 15 percent discount

LONDON/ZURICH: Novartis kicked off a new era in U.S. medicine on Thursday with the launch of the first “biosimilar” copy of a biotechnology drug approved in the United States, at a discount of 15 percent to the original.

The Swiss drugmaker’s generics unit Sandoz said Zarxio, its form of Amgen’s white blood cell-boosting product Neupogen, would increase access to an important treatment by offering a “high-quality, more affordable version”.

U.S. biotech group Amgen had tried to stop the sale of Zarxio, also known as filgrastim-sndz, but the Washington-based appeals court rejected its attempt to block the launch…..http://www.channelnewsasia.com/news/health/novartis-makes-history-wi/2097550.html

On March 6, 2015, FDA approved the first biosimilar under the Biologics Price Competition and Innovation Act (BPCIA), Sandoz’s Zarxio®. Sandoz submitted Zarxio®as a highly similar, not interchangeable biosimilar, for the same indications as the referenced product. The BPCIA was signed into law in March 2010.

FDA designated “filgrastim-sndz” as the placeholder nonproprietary name rather than the innovator’s name, filgrastim. FDA said that this nonproprietary name “should not be viewed as reflective of the agency’s decision on a comprehensive naming policy for biosimilar and other biological products. While the FDA has not yet issued draft guidance on how current and future biological [biosimilar?] products marketed in the United States should be named, the agency intends to do so in the near future.”

Accompanying the news release was a document “Biosimilars: More Treatment Options Are on the Way”. The document includes various quotes and paraphrased statements by Leah Christl, Ph.D., Associate Director for Therapeutic Biologics, to help describe to consumers what biosimilar medications are. Below are some quotes and information from that document:

Biologics are medicines that generally come from living organisms, which can include humans, animals and microorganisms such as yeast and bacteria.

. . .

“Biologics are different from conventional medications. Conventional medications—drugs—are generally made from chemicals, or chemically synthesized, and therefore their structure can be relatively easily defined,” explains Christl.

Unlike conventional medications, biologics can’t be made by following a chemical “recipe.” “Biologics come from living organisms which are variable in nature. In addition, they are generally more complex and not as easy to define and characterize,” Christl explains. For that reason, manufacturing biologics is a far more complex process than manufacturing drugs.

Just as it does for drugs, FDA rigorously and thoroughly evaluates a biologic’s safety and effectiveness before granting it licensure (approval). Currently, biologics are among the fastest growing segments of the prescription product market.

. . .

Christl explains that a biosimilar is a type of biologic that is highly similar to another, already FDA-approved biologic (known as the reference product).

“It is important to note that a biosimilar is not just like a generic drug,” she adds. “Because of the differences in complexity of the structure of the biologic and the process used to make a biologic, biosimilars are not as easy to produce as generics, which are copies of brand name drugs.” A biosimilar is not an exact duplicate of another biologic; rather, a biosimilar is highly similar to the reference product.

Before approving a biosimilar, FDA experts must also first verify that there are no clinically meaningful differences between the biosimilar and its reference product. In other words, it will work the same way as the reference product for its approved indications.

Also, the biosimilar must have the same strength and dosage form (injectable, for example) and route of administration as the reference product. The biosimilar must be manufactured followingCurrent Good Manufacturing Practices.

“Patients can rest assured that they’ll be able to rely upon the safety and effectiveness of an FDA-approved biosimilar, just as they can rely on the reference product that the biosimilar was compared to,” Christl says. Like other biologics, biosimilars generally must be prescribed by a physician.

. . .

“Biosimilars are likely to create greater competition in the medical marketplace,” saysChristl. This could not only increase treatment options for patients, but also lead to less expensive alternatives to comparable products. With an increasing number of biosimilars on the market, consumers may expect to get equally safe and effective treatment, but at lower costs, she says.

Despite the significant achievement for FDA to approve the first biosimilar under the BPCIA, significant questions other than nonproprietary naming remain. First, Sandoz chose not to take advantage of the pre-approval patent exchange mechanism of the BPCIA, which could have addressed possible patent challenges that may prevent Sandoz from marketing Zarxio®until certain patents are invalidated, are found unenforceable, or have expired. Second, because this and other non-interchangeable versions of biosimilars are not expected to have automatic substitution based on the BPCIA, it remains unclear how ready physicians or patients will be to try a biosimilar version over its referenced product. Third, company representatives from Sandoz and other biosimilar manufacturers have not indicated at what price their biosimilar products will be sold, at times suggesting “at parity,” which may cause reimbursement issues. Fourth, many states have enacted rules that include special physician notification provisions, even when interchangeable biosimilars are dispensed to patients. And there are still issues surrounding pharmacovigilance and risk management when there are innovator and corresponding biosimilar versions marketed. Nevertheless, FDA proclaims that more biosimilars are on the way, as additional companies have indicated that they have submitted or FDA has filed their biosimilar applications. Sandoz’s Zarxio® then is just the tip of the iceberg of what is coming with more issues to be resolved along the way.

http://www.fdalife.com/2015/03/06/first-u-s-biosimilar-zarxio-filigrastim-sndz-approved-issues-remain/

 

ZARXIO (filgrastim-sndz) is a 175 amino acid human granulocyte colony-stimulating factor (G-CSF) manufactured by recombinant DNA technology.

ZARXIO is produced by Escherichia coli (E coli) bacteria into which has been inserted the human granulocyte colony-stimulating factor gene. ZARXIO has a molecular weight of 18,800 daltons. The protein has an amino acid sequence that is identical to the natural sequence predicted from humanDNA sequence analysis, except for the addition of an N-terminal methioninenecessary for expression in E coli. Because ZARXIO is produced in E coli, the product is non-glycosylated and thus differs from G-CSF isolated from a human cell.

ZARXIO injection is a sterile, clear, colorless to slightly yellowish , preservative-free liquid containing filgrastimsndz at a specific activity of 1.0 x 108 U/mg (as measured by a cell mitogenesis assay). The product is available in single-use prefilled syringes. The single-use prefilled syringes contain either 300 mcg/0.5 mL or 480 mcg/0.8 mL of filgrastim-sndz. See table below for product composition of each single-use prefilled syringe.

300 MCG/0.5 ML SYRINGE 480 MCG/0.8 ML SYRINGE
Filgrastim-sndz 300 mcg 480 mcg
Glutamic Acid 0.736 mg 1.178 mg
Polysorbate 80 0.02 mg 0.032 mg
Sorbitol 25 mg 40 mg
Sodium hydroxide q.s. qs.
Water for Injection
USP q.s. ad* ad 0.5 mL ad 0.8 mL

Japanese filing for Amgen’s PCSK9 inhibitor Repatha


Amgen has filed its closely watched PCSK9 inhibitor Repatha (evolocumab) in Japan for the treatment of high cholesterol.

Repatha is an investigational fully human monoclonal antibody that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that reduces the liver’s ability to remove low-density lipoprotein cholesterol (LDL-C), or ‘bad’ cholesterol, from the blood.

Evolocumab

Monoclonal antibody
Type Whole antibody
Source Human
Target PCSK9
Clinical data
  • Investigational
Subcutaneous injection
Identifiers
1256937-27-5
C10AX13
Chemical data
Formula C6242H9648N1668O1996S56
141.8 kDa

Evolocumab[1] (also known as compound number AMG-145 or AMG145)[2] is a monoclonal antibody designed for the treatment of hyperlipidemia.[3] Evolocumab is a fully human monoclonal antibody that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9).

PCSK9 is a protein that targets LDL receptors for degradation and thereby reduces the liver’s ability to remove LDL-C, or “bad” cholesterol, from the blood.

Evolocumab, being developed by Amgen scientists, is designed to bind to PCSK9 and inhibit PCSK9 from binding to LDL receptors on the liver surface. In the absence of PCSK9, there are more LDL receptors on the surface of the liver to remove LDL-C from the blood.

Clinical trials

Two trials have been in progress as at mid-2014:

On 23 January 2014 Amgen announced that the Phase 3 GAUSS-2 (Goal Achievement After Utilizing an Anti-PCSK9 Antibody in Statin Intolerant Subjects-2) trial evaluating evolocumab in patients with high cholesterol who cannot tolerate statins met its co-primary endpoints: the percent reduction from baseline in low-density lipoprotein cholesterol (LDL-C) at week 12 and the mean percent reduction from baseline in LDL-C at weeks 10 and 12. The mean percent reductions in LDL-C, or “bad” cholesterol, compared to ezetimibe were consistent with results observed in the Phase 2 GAUSS study.[4][5]

The GAUSS-2 trial evaluated safety, tolerability and efficacy of evolocumab in 307 patients with high cholesterol who could not tolerate effective doses of at least two different statins due to muscle-related side effects. Patients were randomly assigned to one of four treatment groups: subcutaneous evolocumab 140 mg every two weeks and oral placebo daily; subcutaneous evolocumab 420 mg monthly and oral placebo daily; subcutaneous placebo every two weeks and oral ezetimibe 10 mg daily; or subcutaneous placebo monthly and oral ezetimibe 10 mg daily.

Safety was generally balanced across treatment groups. The most common adverse events (> 5 percent in evolocumab combined group) were headache (7.8 percent evolocumab; 8.8 percent ezetimibe), myalgia (7.8 percent evolocumab; 17.6 percent ezetimibe), pain in extremity (6.8 percent evolocumab; 1.0 percent ezetimibe), and muscle spasms (6.3 percent evolocumab; 3.9 percent ezetimibe).

Cholesterol-lowering treatment with a statin as part of follow-up care can help reduce a patient’s risk after myocardial infarction, ischaemic stroke or TIA.

The FOURIER Phase 3 clinical study http://www.fourierstudy.com/ seeks to find out whether lowering cholesterol by an additional 50% might reduce this risk even further. Several sites in the UK are part of this very large clinical study, lasting up to five years, and it is hoped that the study will help guide future clinical practice.

Evolocumab (also formerly known as AMG145, from Amgen) binds to PCSK9, a natural protein produced by the liver. By binding to PCSK9, evolocumab allows the LDL receptor (a protein present in the liver) to move LDL-cholesterol out of the bloodstream more efficiently. This study is designed to see whether treatment of dyslipidemia with evolocumab in people who have experienced a prior myocardial infarction, ischaemic stroke or TIA, and who are taking a highly effective dose of a statin, reduces the risk of recurring or additional cardiovascular events. Participants in this study have clinically evident cardiovascular disease.

READ AT

https://newdrugapprovals.org/2014/03/19/amgen-drug-evolocumab-hits-endpoint-of-cholesterol-reduction/

MY EARLIER ARTICLE

DR ANTHONY MELVIN CRASTO Ph.DDR ANTHONY CRASTO

https://newdrugapprovals.org/

References

 1

Pierson, Ransdell (17 March 2014). “Amgen drug meets goal for those with high genetic cholesterol”. Associated Press. Retrieved 19 March 2014.

Amgen/Onyx file multiple myeloma drug in US, EU…..a supplemental New Drug Application has now been filed to support the conversion of this to full approval and expand target population.


Carfilzomib.svg

Carfilzomib

 

synthesis………https://newdrugapprovals.org/2014/08/05/amgens-multiple-myeloma-drug-shows-promise-in-phase-3-trial/

supplemental New Drug Application filed

Amgen and its subsidiary Onyx Pharmaceuticals have submitted filings for their multiple myeloma drug Kyprolis (carfilzomib) on both sides of the Atlantic.

The companies are seeking approval to market their drug for the treatment of patents with relapsed multiple myeloma who have received at least one prior therapy.

read all at…………http://www.pharmatimes.com/Article/15-01-28/Amgen_Onyx_file_multiple_myeloma_drug_in_US_EU.aspx

 

 

synthesis………https://newdrugapprovals.org/2014/08/05/amgens-multiple-myeloma-drug-shows-promise-in-phase-3-trial/

 

Carfilzomib.svg
Systematic (IUPAC) name
(S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide
Clinical data
Trade names Kyprolis
Licence data US FDA:link
Legal status
Routes Intravenous
Identifiers
CAS number 868540-17-4
ATC code L01XX45
PubChem CID 11556711
ChemSpider 9731489
KEGG D08880
ChEMBL CHEMBL451887
Synonyms PX-171-007
Chemical data
Formula C40H57N5O7 
Molecular mass 719.91 g mol

 

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