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IACS -9571

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IACS-9571

TRIM24/BRPF1 bromodomain inhibitor

IACS-9571; IACS 9571; IACS9571.

Molecular Formula:	C₃₂H₄₂N₄O₈S
Molecular Weight:	642.76288 g/mol

N-[6-[3-[4-(dimethylamino)butoxy]-5-propoxyphenoxy]-1,3-dimethyl-2-oxobenzimidazol-5-yl]-3,4-dimethoxybenzenesulfonamide

BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM

IACS-9571 is a potent and selective inhibitor TRIM24 and BRPF1. The bromodomain containing proteins TRIM24 (Tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). IACS-9571 has low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), IACS-9571 is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo. (J Med Chem. 2015 Jun 10. [Epub ahead of print] )

PAPER

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b00405

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

^†Institute for Applied Cancer Science, and ^‡Core for Biomolecular Structure and Function, The University of Texas MD Anderson Cancer Center, 1881 East Road, Unit 1956, Houston, Texas 77054, United States

^§ Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center,

1515 Holcombe Boulevard

, Houston, Texas 77030, United States

J. Med. Chem., 2016, 59 (4), pp 1440–1454

DOI: 10.1021/acs.jmedchem.5b00405

Publication Date (Web): June 10, 2015

*E-mail: wpalmer@mdanderson.org. Telephone: (001) 713-745-3022. Fax: (001) 713-745-8865.

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC K_d = 31 nM and ITC K_d = 14 nM, respectively). With its excellent cellular potency (EC₅₀ = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.

NMR

IACS -9571

STR2

N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate

CLICK ON IMAGE

ABSTRACT

251st ACS National Meeting & Exposition

13–17 March 2016

San Diego, United States

MEDI 5

Discovery and development of a potent dual TRIM24/BRPF1 bromodomain inhibitor, IACS -9571, using structure- based drug design Wylie S. Palmer 1 , wpalmer@mdanderson.org, Guillaume Poncet -Montagne 1 , Gang Liu 1 , Alessia Petrocchi 1 , N aphtali Reyna 1 , Govindan Subramanian 1 , Jay Theroff 1 , Maria Kost -Alimova 1 , Jennifer Bardenhagen 1 , Elisabetta Leo 1 , Hannah Sheppard 1 , Trang Tieu 1 , Shi Xi 1 , Yanai Zhan 1 , Shuping Zhao 1 , Michelle Barton 2 , Giulio Draetta 1 , Carlo Toniatti 1 , Philip Jones 1 , Mary Ge ck Do 1 , Jannik Andersen 1 . (1) Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States (2) Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

Bromodomains are an important class of chromatin remodeling proteins that recognize acetylated lysine residues on histone tails. As epigenetic targets they regulate gene transcription and offer a new way to treat diseas es, particularly in inflammation and oncology. The bromodomain and extra- terminal (BET) family has emerged as an important and druggable example of this class of proteins with the successful entry of small- molecule inhibitors into the clinic. Other families of bromodomains are only starting to be explored, such as the Tripartite Motif -containing 24 protein (TRIM24) and bromodomain- PHD finger protein 1 (BRPF1). Both proteins contain a dual PHD -bromo motif which have a role in recognizing specific histone mar ks. TRIM24 recognizes the dual histone marks of unmodified H3K4 and acetylated- H3K23 within the same histone tail. TRIM24 is a potent co- activator of ER -alpha and overexpression of TRIM24 has been linked to poor survival rates in breast cancer patients.

This presentation will describe the structure guided development of a series of N,N- dimethyl -benzimidazolones through the iterative use of X -ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor (IACS -9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC 50 = 50 nM) and favorable pharmacokinetic properties, IACS -9571 is a high- quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo

PATENT

WO-2016033416-A1

Synthesis of Intermediates:

N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide (Intermediate 1):

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Step 1 : 5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 4-nitrobenzene- 1 ,2-diamine (44 g, 285 mmol) in 80 mL of DMF was added l, l’-carbonyldiimidazole (70 g, 428 mmol). The reaction mixture was stirred at RT for 4 h, then water (250 mL) was added. The resulting suspension was filtered, and the collected solids were washed with water (200 mL) and dried to give 5-nitro-lH- benzo[d]imidazol-2(3H)-one as a yellow solid (45 g, 88%). MS (ES+) C7H5N3O3 requires: 179, found: 180 [M+H]⁺.

Step 2: l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a solution of 5-nitro-lH-benzo[d]imidazol-2(3H)-one (55 g, 309 mmol) in 150 mL of DMF was added K2CO3 (85 g, 618 mmol), the reaction mixture was cooled to 0 °C, then iodomethane (109 g, 772 mmol) was slowly added. The reaction mixture was stirred at RT overnight, then water was added to the reaction mixture. The resulting suspension was filtered and the collected solids were washed with water (200 mL) and dried to give 1,3- dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one as a yellow solid (55 g, 86%). MS (ES+) C9H9N3O3 requires: 207, found: 208 [M+H] ⁺.

Step 3: 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a solution of l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one (50 g, 240 mmol) in 200 mL of EtOAc under an inert atmosphere was added 10% palladium on activated carbon (5 g, 24 mmol). The reaction mixture was then charged with hydrogen and stirred at RT under an ¾ atmosphere overnight. The reaction mixture was filtered through a pad of celite then concentrated to give 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)- one as a yellow solid (32 g, 68%). MS (ES+) C9H11N3O requires: 177, found: 178 [M+H]⁺.

Step 4: 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 5-amino-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (4 g, 22.6 mmol) in 25 mL of CHCI3 and 25 mL of AcOH was slowly added drop wise bromine (3.5 g, 22.6mmol). The mixture was stirred at RT for 30 min, then concentrated and purified by silica gel chromatography (1 : 1 EtOAc/ hexanes) to afford 5-amino-6-bromo-l ,3-dimethyl- lH-benzo[d]imidazol-2(3H)-one as a yellow solid (3.2 g, 69%). MS (ES+) C₉HioBrN₃0 requires: 256, found: 257 [M+H]⁺.

Step 5: N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide:

To a 0 °C solution of 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (1.50 g, 5.9 mmol) in DCM (45 ml) was added DMAP (72 mg, 0.59 mmol), triethylamine (1.63 ml, 11.7 mmol) and trifluoroacetic anhydride (0.91 ml, 6.4 mmol). The reaction mixture was stirred for 2 h and warmed to RT. The reaction mixture was then quenched with water and the organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated to give N-(6-bromo-l,3-dimethyl-2-oxo-2,3-dihydro-lH- benzo[d]imidazol-5-yl)-2,2,2-trifluoroacetamide (Intermediate 1) as a yellow solid (2.20 g, 100%). MS (ES+) CiiH₉BrF₃N302 requires: 352, found 353 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route A):

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To a mixture of 5-amino-6-(3-(benzyloxy)phenoxy)-l,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one (400 mg, 1.07 mmol) in DCM (20 mL) at -78 °C was added tribromoborane (5.3 mL, 5.3 mmol). The mixture was warmed up to room temperature gradually, then quenched by methanol dropwise, concentrated, and purified by column chromatography (20-100% EtOAc/hexanes and then 0-40% methanol/EtOAc) to give 5- amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one as a solid (240 mg, 79%). MS (ES+) C15H15N3O3 requires: 285, found: 286 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route B):

Step 2

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Step 1: 3-[(ieri-butyldimethylsilyl)oxy]phenol:

A mixture of lH-imidazole (2.25 g, 33.1 mmol), ieri-butylchlorodimethylsilane (3.83 g, 25.4 mmol) and resorcinol (5.6 g, 51 mmol) in THF (30 ml) was stirred at 80 °C for 5 h. The resulting suspension of the cooled reaction mixture was filtered and the collected filtrate was concentrated and purified by silica-gel chromatography (20:80 to 0:100, EtOAc/hexanes) to give 3-((ieri-butyldimethylsilyl)oxy)phenol (2.78 g, 49%). MS (ES+) C12H20O2S1 requires: 224, found 225 [M+H]⁺.

Step 2: 5-amino-6-(3-((ier^butyldimethylsilyl)oxy)phenoxy)-l ,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one:

A mixture of 3-((ieri-butyldimethylsilyl)oxy)phenol (1.39 g, 6.20 mmol), quinolin-8-ol (79 mg, 0.55 mmol), copper(I) chloride (20 mg, 0.21 mmol), potassium phosphate (526 mg, 2.48 mmol) and 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (529 mg, 2.07 mmol) in diglyme (20 ml) in a 100 mL round-bottom flask was degassed under a nitrogen atmosphere and heated to 120 °C for 24 h. To the cooled reaction mixture was added silica gel, stirred for 2 min, then the mixture was filtered through a pad of silica gel. The collected filtrate was concentrated and purified by column chromatography (20:80 to 0: 100, EtOAc/hexanesthen 0: 100 to 40:60, MeOH/EtOAc) to give 5-amino-6-(3- ((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (521 mg, 63%). MS (ES+) C21H29N3O3S1 requires: 399, found 400 [M+H]⁺.

Step 3: 5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 5-amino-6-(3-((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3- dimethyl-lH-benzo[d]imidazol-2(3H)-one (623 mg, 1.56 mmol) in THF was added a solution of ieira-butylammonium fluoride (0.90 mL, 3.1 mmol) in THF, the reaction mixture was allowed to warm up to RT and then stirred for 1-2 h. The reaction mixture was quenched with 1 M hydrogen chloride (0.10 mL, 3.1 mmol) and then partitioned between EtOAc and water. The seperated organic layer was washed with water twice, then concentrated and purified by column chromatography (20-80% EtOAc/hexanes and 0-40% MeOH/DCM) to give 5-amino-6-(3-hydroxyphenoxy)-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (120 mg, 27%) as a solid. MS (ES+) C15H15N3O3 requires: 285, found 286 [M+H]⁺.

EXAMPLE 10: N-(6-(3-(4-(dimethylamino)butoxy)-5-propoxyphenoxy)-l,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2-

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To a solution of N-(6-(3-(4-aminobutoxy)-5-propoxyphenoxy)-l ,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2- trifluoroacetate (180 mg, 0.247 mmol) in methanol (3.0 ml) was added triethylamine (0.034 ml, 0.25 mmol), acetic acid (0.028 ml, 0.49 mmol), formaldehyde (0.054 ml, 2.0 mmol), and sodium triacetoxyborohydride (131 mg, 0.618 mmol). The reaction mixture was stirred at room temperature and checked by LCMS every 30 minutes. After 3 h the reaction was complete by LCMS. The reaction was quenched with a few drops of TFA and concentrated under reduced pressure. The residue was purified by prep-HPLC using a gradient of 20-60% ACN/water containing 0.1% TFA to afford N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate (106 mg, 57%) as a white solid. MS (ES+) C32H42N4O8S requires: 642, found 643 [M+H]⁺. ¾ NMR (600 MHz, DMSO-ifc) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, 7 = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, 7 = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, 7 = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12-3.05 (m, 2H), 2.78 (d, 7 = 4.7 Hz, 6H), 1.77-1.63 (m, 6H), 0.95 (t, 7 = 7.3 Hz, 3H)

References

1: Palmer WS, Poncet-Montange G, Liu G, Petrocchi A, Reyna N, Subramanian G, Theroff J, Yau A, Kost-Alimova M, Bardenhagen JP, Leo E, Shepard HE, Tieu TN, Shi X, Zhan Y, Zhao S, Draetta G, Toniatti C, Jones P, Geck Do M, Andersen JN. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. J Med Chem. 2015 Jun 10. [Epub ahead of print] PubMed PMID: 26061247.

US-20160060260-A1

Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

The University of Texas MD Anderson Cancer Center | University of Texas System

The new Institute for Applied Cancer Science will be located at the south campus of M.D.

Draetta arrived at MD Anderson in 2011 to direct the Institute for Applied Cancer Science. He oversees the moon shots platforms

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

///////IACS-9571, TRIM24, BRPF1 bromodomain inhibitor, IACS-9571, IACS 9571, IACS9571, BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM
CAS BASE 1800477-30-8
CAS OF 1:1 TRIFLUOROACETATE 1883598-69-3

c1(cc(cc(c1)OCCC)Oc3cc2N(C(N(c2cc3NS(=O)(=O)c4cc(c(cc4)OC)OC)C)=O)C)OCCCCN(C)C

CCCOC1=CC(=CC(=C1)OC2=C(C=C3C(=C2)N(C(=O)N3C)C)NS(=O)(=O)C4=CC(=C(C=C4)OC)OC)OCCCCN(C)C

Tianagliflozin IND filed by Tianjin Institute of Pharmaceutical research

March 30, 2016 3:20 am / Leave a comment

Tianagliflozin,

taigeliejing, 6-deoxydapagliflozin

Molecular Formula:	C₂₁H₂₅ClO₅
Molecular Weight:	392.8732 g/mol

Tianjin Institute Of Pharmaceutical Research,

(3R,4S,5S,6R)-2-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-6-methyloxane-3,4,5-triol

1-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-1,6-dideoxy-b-D-glucopyranose

D-Glucitol, 1,5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-6-deoxy-, (1S)-

1–[4–Chloro–3–(4–ethoxybenzyl)phenyl]–1,6–dideoxy–β–d–glucopyranose

6-deoxydapagliflozin

https://pubchem.ncbi.nlm.nih.gov/compound/67891767#section=Top

A SGLT-2 inhibitor potentially for the treatment of type 2 diabetes.

CAS N. 1461750-27-5

The structures of dapagliflozin and 6-deoxydapagliflozin (1)

,deletion of the 6-OH in the sugar moiety of dapagliflozin led to the discovery of a more potent SGLT2 inhibitor, 6-deoxydapagliflozin (1, ). In an in vitro assay, 1 was a more active SGLT2 inhibitor, with IC ₅₀ = 0.67 nM against human SGLT2 (hSGLT2), as compared with 1.1 nM for dapagliflozin, leading to the identification of 1 as the most active SGLT2 inhibitor discovered so far in this field. Also in an in vivo assay, 1 also introduced more urinary glucose in a rat urinary glucose excretion test (UGE) and exhibited more potent blood glucose inhibitory activity in a rat oral glucose tolerance test (OGTT) than dapagliflozin.

Given the fact that 6-dexoydapagliflozin (1) is a very promising SGLT2 inhibitor that could be used to treat type 2 diabetes, led to preclinical trials

 Tianjin Institute Of Pharmaceutical Research,天津药物研究院

SPECTRAL DATA of Tianagliflozin

1 as a white solid (3.65 g, 93 %). R _f = 0.35 (EtOAc);

m.p.: 148–149 °C;

¹H NMR (400 MHz, DMSO-d ₆): δ = 7.35 (d, 1H, J = 8.4 Hz), 7.25 (s, 1H), 7.18 (d, 1H, J = 8.0 Hz), 7.08 (d, 2H, J = 8.4 Hz), 6.81 (d, 2H, J = 8.4 Hz), 4.95 (d, 1H, J = 5.2 Hz, OH), 4.90 (d, 1H, J = 4.4 Hz, OH), 4.79 (d, 1H, J = 5.6 Hz, OH), 3.92–4.01 (m, 5H), 3.24–3.29 (m, 1H), 3.18–3.22 (m, 1H), 3.09–3.15 (m, 1H), 2.89–2.95 (m, 1H), 1.29 (t, 3H, J = 7.0 Hz, CH₂ CH ₃), 1.15 (d, 3H, J = 6.0 Hz, CHCH ₃) ppm;

¹³C NMR (100 MHz, DMSO-d ₆): δ = 156.85, 139.65, 137.82, 131.83, 131.16, 130.58, 129.52, 128.65, 127.14, 114.26, 80.71, 77.98, 75.77, 75.51, 74.81, 62.84, 37.55, 18.19, 14.62 ppm;

IR (KBr): v¯¯¯ = 3,564 (w), 3,385 (s), 2,981 (s), 2,899 (s), 2,861 (s), 1,613 (m), 1,512 (s), 1,477 (m), 1,247 (s), 1,102 (s), 1,045 (s), 1,012 (s) cm⁻¹;

HR–MS: calcd for C₂₁H₂₉ClNO₅ ([M + NH₄]⁺) 410.1729, found 410.1724.

PATENT

CN 103864737

http://www.google.com/patents/CN103864737A?cl=en

PATENT

WO 2014094544

http://www.google.com/patents/WO2014094544A1?cl=en

Figure imgf000032_0001

-27-

1 D1 -6 Optionally, the step (7 ‘) is the step (7’) in place:

LS l- [4 – D (I- Dl- 6)

(DMSO-d _6, 400 MHz), δ 7.35 (d, 1H, J = 8.0 Hz), 7.28 (d, 1H, J ‘. 2.0 Hz), 7.17 (dd, IH, / = 2.0 Hz and 8.4 Hz), 7.05 (d, 2H, _J: 8.8 Hz), 6.79 (d, 2H, 8.8 Hz): 4.924,95 (m, 2H), 4,81 (d, IH, 6,0 Hz), 3.93- 3.99 (m, 5H), 3,85 (d, 1H, J = 10,4 Hz), 3,66 (dd, IH, 5,2 Hz and 11,6 Hz), 3.17-3,28 (m, 3H), 3.02-3.08 _(m: IH), 1.28 (t, 3H, J = 7,0 Hz), 0,80 (s, 9H), -0.05 (s, 3H), -0.09 (s, 3H) .

PATENT

CN 104045614

[0066] The added 100mL dried over anhydrous methanol 0. 5g of sodium metal, nitrogen at room temperature with stirring, until the sodium metal disappeared. Followed by addition of 5. 2g (10mmol) of compound 6, stirring was continued at room temperature for 3 hours. To the reaction system was added 5g strong acid cation exchange resin, stirred at room temperature overnight, the reaction mixture until pH = 7. The resin was removed by suction, and the filtrate evaporated to dryness on a rotary evaporator, the residue was further dried on a vacuum pump to give the product I-D1-6, as a white foamy solid.

PATENT

WO 2014139447

http://link.springer.com/article/10.1007%2Fs40242-014-4043-9#/page-1

Med Chem. 2015;11(4):317-28.

Design of SGLT2 Inhibitors for the Treatment of Type 2 Diabetes: A History Driven by Biology to Chemistry.

Cai W, Jiang L, Xie Y, Liu Y, Liu W, Zhao G¹.

Abstract

A brief history of the design of sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors is reviewed. The design of O-glucoside SGLT2 inhibitors by structural modification of phlorizin, a naturally occurring O-glucoside, in the early stage was a process mainly driven by biology with anticipation of improving SGLT2/SGLT1 selectivity and increasing metabolic stability. Discovery of dapagliflozin, a pioneering C-glucoside SGLT2 inhibitor developed by Bristol-Myers Squibb, represents an important milestone in this history. In the second stage, the design of C-glycoside SGLT2 inhibitors by modifications of the aglycone and glucose moiety of dapagliflozin, an original structural template for almost all C-glycoside SGLT2 inhibitors, was mainly driven by synthetic organic chemistry due to the challenge of designing dapagliflozin derivatives that are patentable, biologically active and synthetically accessible. Structure-activity relationships (SAR) of the SGLT2 inhibitors are also discussed.

http://www.ncbi.nlm.nih.gov/pubmed/25557661

Paper

Discovery of 6-Deoxydapagliflozin as a Highly Potent Sodium-dependent Glucose Cotransporter 2 (SGLT2) Inhibitor for the Treatment of Type 2 Diabetes

Authors: Zhang, Lingyu; Wang, Yuli; Xu, Huaqiang; Shi, Yongheng; Liu, Bingni; Wei, Qunchao; Xu, Weiren; Tang, Lida; Wang, Jianwu; Zhao, Guilong

Source: Medicinal Chemistry, Volume 10, Number 3, May 2014, pp. 304-317(14)

http://www.ingentaconnect.com/content/ben/mc/2014/00000010/00000003/art00009?crawler=true

CLIP

A facile synthesis of 6-deoxydapagliflozin

Keywords. Carbohydrates Drug research Hydrogenolysis Dapagliflozin SGLT2 inhibitor

http://link.springer.com/article/10.1007/s00706-013-1053-0/fulltext.html

The synthetic route to the target compound 1 is shown in Scheme 3. The starting material methyl 2,3,4-tri-O-benzyl-6-deoxy-6-iodo-α–d-glucopyranoside (3) was prepared from commercially available methyl α–d-glucopyranoside (2) according to a known method [5, 6].

Iodide 3 was reductively deiodinated to give 4 in 91 % yield under hydrogenolytic conditions using 10 % Pd/C as catalyst in the presence of Et₃N as base in THF/MeOH at room temperature.

when the iodide 3 was treated with Barton–McCombie reagent (n-Bu₃SnH/AIBN) [7] in toluene at room temperature no reaction occurred; however, when the reaction was carried out at elevated temperatures, such as reflux, a complex mixture formed with only a trace amount (3 %, entry 1) of the desired product 4.

When the iodide 3 was treated with LiAlH₄ in THF at 0 °C to room temperature, another complex mixture was produced with only a trace amount (2 %, entry 2) of 4.

When Pd(OH)₂ was used as the hydrogenolysis catalyst instead of 10 % Pd/C, the desired 4 was indeed formed (14 %, entry 4), but most of the starting material was converted to a few more polar byproducts, which were believed to result from the cleavage of at least one of the benzyl groups.

pdf available

Monatshefte für Chemie – Chemical Monthly

December 2013, Volume 144, Issue 12, pp 1903-1910

http://download.springer.com/static/pdf/721/art%253A10.1007%252Fs00706-013-1053-0.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs00706-013-1053-0&token2=exp=1458808857~acl=%2Fstatic%2Fpdf%2F721%2Fart%25253A10.1007%25252Fs00706-013-1053-0.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs00706-013-1053-0*~hmac=bd1c3c2bdc3712f5540267c99f732b2f7588020a868aa23021792a2a2a58d65e

References

1.Washburn WN (2009) J Med Chem 52:1785
2.Hardman TC, Dubrey SW (2011) Diabetes Ther 2:133
3.Meng M, Ellsworth BA, Nirschl AA, McCann PJ, Patel M, Girotra RN, Wu G, Sher PM, Morrison EP, Biller SA, Zahler R, Deshpande PP, Pullockaran A, Hagan DL, Morgan N, Taylor JR, Obermeier MT, Humphreys WG, Khanna A, Discenza L, Robertson JM, Wang A, Han S, Wetterau JR, Janovitz EB, Flint OP, Whaley JM, Washburn WN (2008) J Med Chem 51:1145
4.Nomura S, Sakamaki S, Hongu M, Kawanishi E, Koga Y, Sakamoto T, Yamamoto Y, Ueta K, Kimata H, Nakayama K, Tsuda-Tsukimoto M (2010) J Med Chem 53:6355
5.Lee JC, Francis S, Georg GI, Dutta D, Gupta V, Tash JS, Yang Y, Zhu J-Y, Schoenbrunn E (2012) J Org Chem 77:3082
6.Bartoszewicz A, Kalek M, Stawinski J (2008) Tetrahedron 64:8843
7.Zhao GL, Lou YY, Zhang LS, Shao H, Xu WR, Tang LD, Zou MX (2012) Synth Commun 42:2885
8.Koto S, Morishima N, Miyata Y, Zen S (1976) Bull Chem Soc Jpn 49:2639
9.Kakinuma H, Oi T, Hashimoto-Tsuchiya Y, Arai M, Kawakita Y, Fukasawa Y, Iida I, Hagima N, Takeuchi H, Chino Y, Asami J, Okumura-Kitajima L, Io F, Yamamoto D, Miyata N, Takahashi T, Uchida S, Yamamoto K (2010) J Med Chem 53:3247
10.Xu J, Egger A, Bernet B, Vasella A (1996) Helv Chim Acta 79:2004
11.Lee SH, Song K-S, Kim JY, Kang M, Lee JS, Cho S-H, Park H-J, Kim J, Lee J (2011) Bioorg Med Chem 19:5813
12.Song K-S, Lee SH, Kim MJ, Seo HJ, Lee J, Lee S-H, Jung ME, Son E-J, Lee MW, Kim J, Lee J (2011) ACS Med Chem Lett 2:182
13.Lee J, Lee S-H, Seo HJ, Son E-J, Lee SH, Jung ME, Lee MW, Han HK, Kim J, Kang J, Lee J (2010) Bioorg Med Chem 18:2178
14.Kim MJ, Lee SH, Park SO, Kang H, Lee JS, Lee KN, Jung ME, Kim J, Lee J (2011) Bioorg Med Chem 19:5468
15.Skaanderup PR, Poulsen CS, Hyldtoft L, Jørgensen MR, Madsen R (2002) Synthesis 1721
Monatshefte fuer Chemie (2013), 144(12), 1903-1910
Medicinal Chemistry (Sharjah, United Arab Emirates) (2014), 10(3), 304-317
Chinese Journal of Structural Chemistry (2014), 33(1), 85-89
Xiandai Yaowu Yu Linchuang (2013), 28(6), 838-841
Chemical Research in Chinese Universities (2014), 30(5), 785-793

////////, SGLT-2 inhibitor, type 2 diabetes, Tianagliflozin, taigeliejing, 6-deoxydapagliflozin, 1461750-27-5

Clc1c(cc(cc1)C2[C@@H]([C@H]([C@@H]([C@H](O2)C)O)O)O)Cc3ccc(cc3)OCC

CCOC1=CC=C(C=C1)CC2=C(C=CC(=C2)C3C(C(C(C(O3)C)O)O)O)Cl

c1(c(cc(cc1)C2OC(C(C(C2O)O)O)C)Cc3ccc(cc3)OCC)Cl

Avoralstat

March 4, 2016 3:57 am / Leave a comment

Avoralstat, BCX4161,

CAS 918407-35-9
UNII: UX17773O15

513.5513, C28-H27-N5-O5

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

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

Hereditary angioedema (HAE)

Kallikrein inhibitor

BioCryst Pharmaceuticals

Biocryst Logo

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

2D chemical structure of 918407-35-9

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

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

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

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

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

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

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

Selective inhibitor of plasma kallikrein that subsequently suppresses bradykinin production

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Avoralstat

May 16 is HAE awareness day
See BioCryst’s video regarding HAE to learn more

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

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

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

PATENT

WO200234711

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

PATENT

WO2015134998

PATENT

WO2016029214

Examples

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

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

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

f ^0HCY ° ^ΒΓΥΥ°

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

1a 1b

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

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

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

2837, 2767, 2740, 1657, 1595, 1428, 1270, 1210, 1164, 1022 cm^“‘; Analysis calculated for C₈H₇Br03_.H₂0: C, 38.58; H, 3.64: Found: C, 38.60; H, 3.60.

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

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

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

15 046578

146

1d 1e

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

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

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

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

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

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

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

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

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

acid (1fi

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

H₃C N Br

2a 2b

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

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

(2c)

2b ^2c

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

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

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

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

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

M+Na)

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

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

eluting with, 0%, 5%, 10%, 20%, 30%, 50% ethyl acetate/hexanes (250 mL each) as eluents to give methyl 6-(cyclopropylmethyl-carbamoyl)-3-methoxypicolinate (2f) (6.29 g, 86.1 %) as a salmon coloured solid, MP 107 °C; ¹HNMR (300 MHz, DMSO-cfe) δ 8.28 (t, J = 6.0, 1H), 7.91 (d, J = 8.8, 1H), 7.55 (d, J = 8.8, 1 H), 3.68 (s, 3H), 3.64 (s, 3H), 2.90 (t, J = 6.5, 2H), 0.89 – 0.68 (m, 1 H), 0.26 – 0.09 (m, 2H), 0.08 – 0.00 (m, 2H); MS (ES+) 287.1 (M+Na); IR (KBr) 3316, 2921 , 1730, 1659, 1534, 1472, 1432, 1315, 1272, 1228, 1189, 1099, 1003, 929, 846, 680 cm^“1; Analysis calculated for C₁₃H_{16 2}0₄: C, 59.08; H, 6.10; N, 10.60; Found: C, 58.70; H, 5.97; N, 10.23.

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

2f 2g

Aluminium chloride method:

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

Boron tribromide method:

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

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

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

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

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

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

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

5 046578

153

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

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

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

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

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

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

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

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

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

C₂₇H_2eN₂0₆: C, 68.34; H, 5.52; N, 5.90; Found; C, 68.16; H, 5.62; N, 5.80.

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

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

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

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

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

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

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

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

3c 3d

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

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

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

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

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

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

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

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

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

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

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

46578

159

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

3h 3i

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

PATENT

WO 2016029216

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

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

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

O y OHCk n ^Br^ ^OCH₃

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

OHC^^^{^}O ” _Br^\^0 MeOH ” OHC

1a 1b 66%

1d 95% ^{1 e}

Scheme A



3h 31

Scheme C

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

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

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

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

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

The process is also illustrated in Fig. 1.

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

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

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

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

The process is also illustrated in Fig. 2.

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

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

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

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

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

The process is also illustrated in Fig. 3.

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

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

The process is also illustrated in Fig. 4.

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

5b 5c

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

The process is also illustrated in Fig. 5.

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

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

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

The process is also illustrated in Fig. 6.

6582

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

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

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

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

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

The process is also illustrated in Fig. 7.

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

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

The process is also illustrated in Fig. 8.

46582

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

6a 6b

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

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

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

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

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

The process is also illustrated in Fig. 10.

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

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

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

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

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

2015/046582

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

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

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

The process is also illustrated in Fig. 12.

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

(3i) ^{,a 3i}

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

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

The process is also illustrated in Fig. 13.

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

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

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

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

The process is also illustrated in Fig. 14.

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

6d 8a

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

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

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

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

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

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

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

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

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

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

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

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

46582

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

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

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

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

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

SKLB 1028, a novel oral multikinase inhibitor of EGFR, FLT3 and Abl,

January 12, 2016 5:45 am / Leave a comment

SKLB 1028

A multi-targeted inhibitor potentially for the treatment of leukemia and non small cell lung cancer.

SKLB-1028

Si Chuan University, 四川大学

CAS 1350544-93-2

9-isopropyl-N²-(4-(4-methylpiperazin-1-yl)phenyl)-N⁸-(pyridin-3-yl)-9H-purine- 2,8-diamine

2-N-[4-(4-methylpiperazin-1-yl)phenyl]-9-propan-2-yl-8-N-pyridin-3-ylpurine-2,8-diamine

9-Isopropyl-N2-[4-(4-methylpiperazin-1-yl)phenyl]-N8-(3-pyridyl)-9H-purine-2,8-diamine, 443.5474, C24H29N9, Preclinical

9-isopropyl-N²-(4-(4-methylpiperazin-1-yl)phenyl)-N⁸-(pyridin-3-yl)-9H-purine- 2,8-diamine. Yield 65.6 %. HPLC>98.6%. ¹H NMR(400 MHz, DMSO-d₆)： δ 9.22(s, 1H), 9.05(s, 1H), 8.94(d, J=2.8Hz, 1H), 8.39(s, 1H), 8.34(d, J=8.4Hz, 1H), 8.20(m, 1H), 7.63(d, J=8.8Hz, 2H), 7.37(m, 1H), 6.88 (d, J=8.8Hz, 2H), 4.88(m, 1H), 3.05(m, 4H), 2.45(m, 4H), 2.22(s, 3H), 1.69(s, 3H), 1.68(s, 3H)ppm。HRMS (ESI) m/z [M-H]- calcd for C₂₄H₂₉N₉: 443.2546, found: 442.2538………..Leukemia (2012), 26(8)

PATENT

WO 2011147066

Synthetic route is as follows:

Example reaction is as follows:

str1

Preparation of chloro-4-amino-5-nitro pyrimidine of Example 12-

Was added dropwise 2,4-dichloro-5-nitro-pyrimidine (lO Aqueous ammonia (8.0ml) and Ν, Ν- diisopropylethylamine (13.2ml) was dissolved in 150ml dichloromethane, 0 ° C when .Og) in dichloromethane (30ml) solution, after dropwise, maintaining the temperature of the reaction one hour, the precipitate was filtered off, the filter cake was recrystallized to give a yellow solid 8.1g, yield 90.1%

Product 1HNMR (400MHz, DMSO-i¾): δ 9.20 (s, 1H), 9.02 (s, 1H), 8.60 (s, lH) ppm

Preparation of pyrimidine

Isopropylamine (4.5ml) and Ν, Ν- diisopropylethylamine (13.2ml) was dissolved in 150ml of dichloromethane, was added dropwise 2,4-dichloro-5-nitro-pyrimidine at 0 ° C ( lO.Og) in dichloromethane (30ml) solution, after dropwise, maintaining the reaction temperature for half an hour, and purified by column chromatography to give a light yellow solid was 10.1g, 90.4% yield of product 1H NMR (400 MHz, CDCl ₃ ): [delta] 9.03 (s, 1H), 8.24 (s, 1H), 4.53 (m, 1H), 1.34 (d, J = 6.8 Hz, 6H) ppm ₀

Example 16, 4-amino-2- (4- (4-methyl-piperazin-1-yl) anilino) -5-nitro-pyrimidin embodiment

4- (4-methylpiperazine) aniline (3.8g) was added to the compound 2-l (3.5g) in n-butanol (150ml) solution, the reaction for 4.5 hours at 90 ° C, cooled to room temperature, filtered , washed, and dried to give a red solid (5.2g), a yield of 79.5%. Product ‘H NMR (400 MHz, CDCl ₃ ): [delta] 9.07 (s, 1H), 8.52 (s, 2H), 8.40 (s, 1H), 7.57 (s, 1H), 7.51 (s, 1H), 7.10 (m, 2H), 3.3 l (t, J = 4.8Hz, 4H), 2.81 (t, J = 4.8Hz, 4H), 2.30 (s, 3H) ppm.

Example 90,

9-isopropyl-2- (4- (4-methyl-piperazin-1-yl) anilino) -8- (pyridin-3-yl) -9H- purine

The compound 5- 7 (2.05g) was dissolved in dichloromethane (90ml), were added sequentially EDCI (2.3g), Ν, Ν- diisopropylethylamine (4.9ml), 3- pyridyl isothiocyanate ester (1.0g), stirred at room temperature for half an hour, then refluxed for 10 hours, TLC monitoring completion of the reaction the raw material 5-7 was cooled and purified by column chromatography to give a light red solid, yield 65.7%.

Product ESI-MS (m / z,%) 442.26 (MH) -. Ή NMR (400 MHz, DMSO-d ₆ ): [delta] 9.38 (s, IH), 9.13 (s, IH), 8.99 (s, IH), 8.40 (s, IH), 8.36 (d, J = 8.4 Hz, IH), 8.20 (d, J = 4.4Hz, IH), 7.70 (d, J = 8.8Hz, 2H), 7.37 (m, IH), 6.96 (d, J = 8.8Hz, 2H), 4.97-4.92 ( m, IH), 3.35 (s, 6H), 2.80 (s, 3H): 2.53 (s, 2H), 1.69 (s, 6H) ppm.

/////////SKLB 1028,

CN1CCN(CC1)c5ccc(Nc3nc4n(C(C)C)c(Nc2cccnc2)nc4cn3)cc5

Shanghai Hengrui’s potent inhibitors of Human Uric Acid Transporter 1 (hURAT1)

January 10, 2016 5:22 am / Leave a comment

CID 86294127.png

MF	C ₁₄ H ₁₂ BrNO ₂ S
MW	338.21958 g / mol

1- (6-bromoquinolin-4-yl) sulfanylcyclobutane-1-carboxylic acid

CAS…….1638327-48-6

Cyclobutanecarboxylic acid, 1-[(6-bromo-4-quinolinyl)thio]-

COMING ………….

Image loading ...

MS m / z (ESI): 338.0 [M + l]

1H NMR (400 MHz, DMSO) δ 13.17 (s, 1H), 8.75-8.79 (m, 1H), 8.24 (s, 1H), 7.87-7.98 (m, 2H), 7.21-7.25 (m, 1H), 2.83-2.95 (m, 2H), 2.30-2.41 (m, 2H), 2.16-2.27 (m, 1H), 1.97-2.08 (m, 1H)

WO-2014183555-A1 / 2014-11-20

http://www.google.co.in/patents/WO2014183555A1?cl=en

PROCEDURE

6-bromo-quinoline-4-thiol

A mixture of 6-bromo-4-chloro-quinoline 3a (260 mg, 1.1 mmol, using known methods “Bioorganic &

Medicinal Chemistry Letters, 2012, 22 (4), 1569-1574 “prepared to give) and sodium sulfide (100 mg, 1.3 mmol) was added to 4 mL of N, N- dimethyl formamide, plus complete, heated 80 ° C, the reaction was stirred for 2 hours. To the reaction mixture was added 50 mL of water, 1 M hydrochloric acid was added dropwise to the reaction solution to pH 5-6, extracted with ethyl acetate (50 mL X 3), the combined organic phases, with no over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give the title product 6-bromo-quinolin-4-thiol 3b (257 mg, yellow oil), it was used directly in the next reaction.

The second step

L – ((6-bromo-quinolin-4-yl) thio) cyclobutyl carboxylate

Under an argon atmosphere, 6-bromo-quinolin-4-thiol 3b (257 mg, 1.1 mmol), 1- bromo-cyclobutyloxy embankment carboxylate (266 mg, 1.3 mmol) and cesium carbonate (371 mg, 1.1 mmol) were sequentially added to 5 mL of N, N- dimethylformamide and heated to 60 ° C, the reaction was stirred for 2 hours. The reaction solution was filtered, the filter cake washed with ethyl acetate (10 mL X 3) and the filtrate was concentrated under reduced pressure to give the title product l – ((6-bromo-quinolin-4-yl) thio) ethyl cyclobutyl 3c ( 300 mg, brown oil). Yield: 77%.

MS m / z (ESI): 368.2 [M + l]

1H MR (400 MHz, CDCl ₃ ) [delta] 8.67 (d, = 4.77 Hz, IH), 8.31 (d, = 2.13 Hz, IH), 7.94 (d, = 8.91Hz, IH), 7.78 (dd, = 9.03, 2.13Hz, IH), 7.15 (d, = 4.89Hz, IH), 4.16 (q, = 7.15Hz, 2H), 2.86-3.04 (m, 2H), 2.39-2.51 (m, 2H), 2.25-2.37 ( m, IH), 2.00-2.15 (m, IH), 1.16 (t, = 7.09Hz, 3H)

third step

L – ((6-bromo-quinolin-4-yl) thio) cyclobutyl acid

L – ((6-bromo-quinolin-4-yl) thio) ethyl cyclobutyl 3c (100 mg, 0.27 mmol) and lithium hydroxide monohydrate (23 mg, 0.55 mmol) was dissolved in 6 mL of tetrahydrofuran, ethanol and water (^ = 4: 1: 1) mixed solvent, the reaction was stirred for 3 hours. 1M hydrochloric acid was added dropwise to the reaction solution pH of 5 to 6, liquid separation, the aqueous phase was extracted (10 mL X 3) with dichloromethane, the combined organic phases, the organic phase was washed with a saturated sodium chloride solution (10 mL XI), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, the resulting A by thin layer chromatography in a developing solvent system, and the residue was purified to give the title product l – ((6-bromo-quinolin-4-yl) thio) cyclobutyl acid 3 (20 mg, white solid), yield: 22%.

MS m / z (ESI): 338.0 [M + l]

1H NMR (400 MHz, DMSO) δ 13.17 (s, 1H), 8.75-8.79 (m, 1H), 8.24 (s, 1H), 7.87-7.98 (m, 2H), 7.21-7.25 (m, 1H), 2.83-2.95 (m, 2H), 2.30-2.41 (m, 2H), 2.16-2.27 (m, 1H), 1.97-2.08 (m, 1H)

L – ((6-bromo-quinolin-4-yl) thio) cyclobutyl acid

First step

6-bromo-quinoline-4-thiol

A mixture of 6-bromo-4-chloro-quinoline 3a (260 mg, 1.1 mmol, a known method of “Bioorganic &

The second step

L – ((6-bromo-quinolin-4-yl) thio) ethyl cyclobutyl

Under an argon atmosphere, 6-bromo-quinolin-4-thiol 3b (257 mg, 1.1 mmol), 1- bromo-cyclobutyloxy embankment carboxylate (266 mg, 1.3 mmol) and cesium carbonate (371 mg, 1.1 mmol) were added to 5 mL of N, N- dimethylformamide and heated to 60 ° C, the reaction was stirred for 2 hours. The reaction mixture was filtered, the filter cake washed with ethyl acetate (10 mL X 3) and the filtrate was concentrated under reduced pressure to give the title product l – ((6-bromo-quinolin-4-yl) thio) ethyl cyclobutyl 3c ( 300 mg, brown oil). Yield: 77%.

MS m / z (ESI): 368.2 [M + l]

1H MR (400 MHz, CDC1 ₃₎ δ 8.67 (d, = 4.77Hz, IH), 8.31 (d, = 2.13Hz, IH), 7.94 (d, = 8.91Hz, IH), 7.78 (dd, = 9.03, 2.13Hz, IH), 7.15 (d, = 4.89Hz, IH), 4.16 (q, = 7.15Hz, 2H), 2.86-3.04 (m, 2H), 2.39-2.51 (m, 2H), 2.25-2.37 ( m, IH), 2.00-2.15 (m, IH), 1.16 (t, = 7.09Hz, 3H) Step

L – ((6-bromo-quinolin-4-yl) thio) cyclobutyl acid

L – ((6-bromo-quinolin-4-yl) thio) ethyl cyclobutyl 3c (100 mg, 0.27 mmol) and lithium hydroxide monohydrate (23 mg, 0.55 mmol) was dissolved in 6 mL of tetrahydrofuran, ethanol and water (^ = 4: 1: 1) mixed solvent, the reaction was stirred for 3 hours. 1M hydrochloric acid was added dropwise to the reaction solution pH of 5 to 6, liquid separation, the aqueous phase was extracted (10 mL X 3) with dichloromethane, the combined organic phases, the organic phase was washed with a saturated sodium chloride solution (10 mL XI), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, to the resulting thin layer chromatography using a developing solvent system A and the residue was purified to give the title product l – ((6-bromo-quinolin-4-yl) thio) cyclobutyl acid 3 (20 mg, white solid), yield: 22%. MS m / z (ESI): 338.0 [M + l]

1H NMR (400 MHz, DMSO) δ 13.17 (s, 1H), 8.75-8.79 (m, 1H), 8.24 (s, 1H), 7.87-7.98 (m, 2H), 7.21-7.25 (m, 1H), 2.83-2.95 (m, 2H), 2.30-2.41 (m, 2H), 2.16-2.27 (m, 1H), 1.97-2.08 (m, 1H)

SHANGHAI HENGRUI PHARM CO LTD

CYCLOALKYL ACID DERIVATIVE, PREPARATION METHOD THEREOF, AND PHARMACEUTICAL APPLICATION THEREOF

Discovery of potent and orally bioavailable inhibitors of Human Uric Acid Transporter 1 (hURAT1) and binding mode prediction using homology model

Jianbiao Peng ^, ,

Shanghai Hengrui Pharmaceutical Co. Ltd, 279 Wenjing Rd., Shanghai 200245, China

This Letter describes the Discovery of a series of potent inhibitors of Human Uric Acid Transporter 1 (hURATl). Lead generation via 3D pharmacophore Analysis and Optimization resulted in compound 41 . With an IC ₅₀ of 33.7 nM, 41 Also Demonstrated good Oral Bioavailability in RAT (74.8%) and displayed a consistent PK profile across all species tested (rat, dog and monkey).

http://www.sciencedirect.com/science/article/pii/S0960894X1530353X

doi: 10.1016 / j.bmcl.2015.12.040

Bioorganic & Medicinal Chemistry Letters

Volume 26, Issue 2 , 15 January 2016, Pages 277-282

Map of Shanghai Hengrui Pharmaceutical Co. Ltd

//////// Shanghai Hengrui, inhibitors of Human Uric Acid Transporter 1 (hURAT1), 1- (6-bromoquinolin-4-yl) sulfanylcyclobutane-1-carboxylic acid

c13cc (ccc3nccc1SC2 (C (= O) O) CCC2) Br

BMS 955829

January 9, 2016 1:39 pm / Leave a comment

(4R,5R)-5-(2,5-difluorophenyl)-4-(5-(phenylethynyl)pyridin-3-yl)oxazolidin-2-one

(4R,5R)-5(2,5-Difluorophenyl)-4-(5-(phenylethynyl)-3-pyridinyl)-1,3-oxazolidin-2-one

(4R,5R)-5-(2,5-difluorophenyl)- 4-(5-(phenylethynyl)pyridin-3-yl)oxazolidin-2-one.

cas 1375751-08-8
Chemical Formula: C22H14F2N2O2
Exact Mass: 376.1023

Bristol-Myers Squibb Company INNOVATOR

BMS-955829 is a Positive allosteric modulators (PAMs). BMS-955829 shows high functional PAM potency, excellent mGluR5 binding affinity, low glutamate fold shift, and high selectivity for the mGluR5 subtype. BMS-955829 is a potent mGluR5 PAM (EC50 = 2.6 ± 1.0 nM; n = 6), devoid of inherent mGluR5 agonist activity (EC50 > 30μM). The measured binding Ki of BMS-955829 was found to be 1.6 nM, which was in good agreement with its functional potency.

SYNTHESIS AND INTERMEDIATES…….https://www.google.co.in/patents/WO2012064603A1?cl=en

Intermediate 73

Diethyl 2,5-difluorobenzylphosphonate. A mixture of 2-(bromomethyl)-l,4- difluorobenzene (3 g, 14.49 mmol) and triethyl phosphite (7.72 ml, 43.5 mmol) was heated to 160 °C with stirring for 4 hours, cooled to ambient temperature and concentrated under high vacuum to remove most triethyl phosphite. The resulting residue was purified by column chromatography (20% to 30 % EtO Ac/Toluene) providing diethyl 2,5-difluorobenzylphosphonate (3.76 g, 13.52 mmol, 93 % yield) as colorless oil. ¾ NMR (500MHz, DMSO-d₆) δ 7.30 – 7.10 (m, 3H), 4.05 – 3.91 (m, 4H), 3.31 – 3.20 (m, 2H), 1.18 (t, J=7.0 Hz, 6H). MS Anal. Calcd. for [M+H]⁺ CiiHieFzOsP: 265.2; found 265.3.

Intermediate 74

(E)-3-Bromo-5-(2,5-difluorostyryl)pyridine. To a stirred solution of diethyl 2,5-difluorobenzylphosphonate (63.5 g, 240 mmol) and 5-bromonicotinaldehyde (50.7 g, 264 mmol) in tetrahydrofuran (1923 ml) was added potassium tert-butoxide in tetrahydrofuran (312 ml, 312 mmol) at -10 °C. After three hours, the reaction mixture was allowed to warm to ambient temperature and stirring was continued for another 16 hours at which time the reaction mixture was diluted with ether (800 mL) and washed with H₂O. The organic layer was dried over anhydrous magnesium sulfate, filered and concentrated to provide a yellow wax to which was added 300 mL of hexane and after sonication filtered to provide (is)-3-bromo-5-(2,5- difluorostyryl)pyridine (54 g, 173 mmol, 72.1%) as a white solid. ^XH NMR

(500MHz, DMSO-d₆) δ 8.78 (d, J=1.8 Hz, IH), 8.63 (d, J=2.1 Hz, IH), 8.44 (t, J=2.0 Hz, IH), 7.67 (ddd, J=9.4, 6.0, 3.2 Hz, IH), 7.56 – 7.48 (m, IH), 7.46 – 7.40 (m, IH), 7.34 (td, J=9.6, 4.6 Hz, IH), 7.24 (tt, J=8.3, 3.6 Hz, IH). MS Anal. Calcd. for [M+H]⁺ Ci₃H₉BrF₂N: 296.0; found 298.1

Intermediate 75

Tert-butyl (lR,2R)-l-(5-bromopyridin-3-yl)-2-(2,5-difluorophenyl)-2- hydroxyethylcarbamate. A solution of tert-butyl carbamate (4.18 g, 35.0 mmol) in propanol (39 ml) was sequentially treated with sodium hydroxide (1.376 g, 34.4 mmol) in water (72 ml) and tert-butyl hypochlorite (3.88 ml, 34.4 mmol). After 5 min of stirring, the reaction mixture was cooled to 0 °C. A solution of

(DHQD)₂PHAL (0.555 g, 0.677 mmol) in propanol (39 ml), a solution of (E)-3- bromo-5-(2,5-difluorostyryl)pyridine (3.34 g, 11.28 mmol) in propanol (68 ml) , and potassium osmate dihydrate (0.166 g, 0.451 mmol) were sequentially added. The reaction mixture was stirred for three additional hours at 0 °C, warmed to ambient temperature and after an additional 16 hours the light yellow homogenous solution was quenched with saturated aqueous sodium sulfite (100 mL). The aqueous phase was extracted with ethyl acetate( 2 X 50 mL), the combined organic phases were washed with brine (100 mL), dried over anhydrous magnesium sulfate and concentrated to afford a residue which was purified via column chromatography (25% to 40 % EtO Ac/Hex) to provide tert-butyl (7R,2R)-l-(5-bromopyridin-3-yl)-2- (2,5-difluorophenyl)-2-hydroxyethylcarbamate (2.2991 g, 5.09 mmol, 45.1 % yield) as an optically enriched mixture of enantiomers. ^XH NMR (500MHz, DIVISOR) δ 8.56 (d, J=1.8 Hz, IH), 8.40 (s, IH), 8.03 (s, IH), 7.52 (d, J=9.5 Hz, IH), 7.25 (br. s., IH), 7.10 (t, J=5.6 Hz, 2H), 5.89 (d, J=4.9 Hz, IH), 5.03 (t, J=5.0 Hz, IH), 4.83 (dd, J=8.9, 5.2 Hz, IH), 1.40 – 1.34 (m, 9H), MS Anal. Calcd. for [M+H]⁺

Ci₈H₂oBrF₂ 20₃: 429.1; found 431.3.

Intermediate 77

(lR,2R)-2-Amino-2-(5-bromopyridin-3-yl)-l-(2,5-difluorophenyl)ethanol To a stirred solution of tert-butyl tert-butyl (7R,2R,)-l-(5-bromopyridin-3-yl)-2-(2,5- difluorophenyl)-2-hydroxyethylcarbamate (2.30 g, 5.09 mmol) in methylene chloride (30 mL) was added HC1 in dioxane (30 ml, 120 mmol). The reaction mixture was placed in an oil bath set to 50 °C. After three hours, the reaction mixture was concentrated providing (7R,2R^-2-amino-2-(5-bromopyridin-3-yl)-l-(2,5- difluorophenyl)ethanol 2HC1 salt (2.10 g, 4.97 mmol, 98 % yield) as an optically enriched yellow wax. ^XH NMR (500MHz, DMSO-d₆) δ 8.95 (d, J=3.7 Hz, 2H), 8.64 (d, J=2.4 Hz, 1H), 8.45 (d, J=1.5 Hz, 1H), 8.31 (t, J=2.0 Hz, 1H), 7.47 – 7.09 (m, 3H), 7.04 (td, J=9.2, 4.4 Hz, 1H), 5.29 (d, J=9.2 Hz, 1H), 4.57 (dd, J=9.0, 5.3 Hz, 1H). Anal. Calcd. for [M+H]⁺ Ci₃H₁₂BrF₂N₂0: 329.0; found 331.2.

Intermediate 78

(4R,5R)-4-(5-Bromopyridin-3-yl)-5-(2,5-difluorophenyl)oxazotidin-2-one. To optically enriched (7R,2R)-2-amino-2-(5-bromopyridin-3-yl)-l-(2,5- difluorophenyl)ethanol, 2 HC1 (2.019 g, 4.82 mmol) in tetrahydrofuran (98 ml) was added diisopropylethylamine (2.95 ml, 16.87 mmol) and the resultant solution was stirred for ten mintues at ambient temperature, cooled to 0 °C and

carbonyldiimidazole (1.094 g, 6.75 mmol) was added. After an additional three hours at 0 °C the reaction mixture was warmed to ambient temperature and allowed to stir for another 16 hours. 2M ¾ in methanol (5ml) was added and after ten mintues the suspension was filtered and concentrated to a pink oil which was purified by column chromatography (25% to 40 % EtO Ac/Hex) providing (4R,5R)-4-(5- bromopyridin-3-yl)-5-(2,5-difluorophenyl)oxazolidin-2-one (1.353 g, 3.62 mmol, 75 % yield) as an optically enriched white solid. ¾ NMR (500MHz, DMSO-d₆) δ 8.80 – 8.68 (m, 1H), 8.55 (d, J=2.1 Hz, 2H), 8.16 (t, J=2.1 Hz, 1H), 7.46 – 7.28 (m, 3H), 5.71 – 5.58 (m, 1H), 5.02 (d, J=6.7 Hz, 1H). MS Anal. Calcd. for [M+H]⁺ Ci₄H₁₀BrF₂ 2O2: 355.0; found 357.2.

Intermediate 79

(4R,5R)-4-(5-Bromopyridin-3-yl)-5-(2,5-difluorophenyl)oxazotidin-2-one. Method – 2 A mixture of tert-butyl ((lR,2R)-l-(54oromopyridin-3-yl)-2-(2,5- difluorophenyl)-2-hydroxyethyl)carbamate and tert-butyl ((lR,2R)-2-(5- bromopyridin-3-yl)-l-(2,5-difluorophenyl)-2-hydroxyethyl)carbamate (about 6: 1 ratio) (101 g, 236 mmol) in tetrahydrofuran (590 mL) was cooled to -7 °C with a methanol/ice bath. To this mixture was added a solution of 1 M potassium tert- butoxide in tetrahydrofuran (590 mL, 590 mmol) via an addition funnel while maintaining the internal temperature < 3 °C. The reaction mixture was stirred with a cooling bath for 30 min and then allowed to warm up to room temperature. After 20 h, the reaction was deemed complete by LC/MS. The reaction mixture was concentrated to dryness to give crude product. Another identical scale reaction was performed. The crude products of the two batches were combined to work up together. They were treated with ethyl acetate (1.75 L) and water (1.75 L). The layers were separated. The organic layer was washed with brine (1.75 L), dried (sodium sulfate), and evaporated to give 161.5 g of crude product as a brown solid. This was purified by ISCO to give 67.1 g (42% yield). LC/MS (ES+) 355/357 (M+H, 100; Br isotope pattern); ^XH NMR (400MHz, CDCl₃) δ 8.75 (d, J=2.2 Hz, 1H), 8.53 (d, J=1.8 Hz, 1H), 7.97 (t, J=2.0 Hz, 1H), 7.29 – 7.23 (m, 1H), 7.18 – 7.09 (m, 2H), 6.40 (s, 1H), 5.56 (d, J=5.7 Hz, 1H), 4.84 (d, J=5.5 Hz, 1H); Calcd for

Ci₄H₉N₂BrF₂0₂: C, 47.34; H, 2.55; N, 7.86; Br, 22.50; F, 10.69. Found: C, 47.29; H, 2.61; N, 7.87; Br, 22.40; F, 10.37. Note: Chiral HPLC of the above sample showed 4.7% of the enantiomer. The (4S, 55) enantiomer can be purged by recrystallization from methanol to give > 99.9 ee with 67% recovery.

WO2012064603

https://www.google.co.in/patents/WO2012064603A1?cl=en

Scheme 1.

Pd(0)/Cu(l)/ TBAF Scheme 2.

cheme 4.

R’ = H, alkyl

Scheme 8.

cheme 11.

Scheme 12.

Scheme 14.

Scheme 15.

R” = H, alkyl R” = alkyl

cheme 16.

R’ = alky I

R” = alkyl

Scheme 17.

R’ = H, alkyl

R” = H, alkyl

Scheme 18.

R’ = H, alkyl R’ = H, alkyl

P T/US2011/059339

COMPD IS 185

Figure imgf000226_0001

Example 185

(4R, 5R)-5-(2, 5-difluorophenyl)-4-(5-(phenylethynyl)-3-pyridinyl)-l, 3-oxazolidin-2- one.

To a stirred solution of optically enriched (4R,5R)-4-(5-bromopyridin-3-yl)-5- (2,5-difluorophenyl)oxazolidin-2-one (1.25 g, 3.25 mmol) in triethylamine (70 mL) was added ethynylbenzene (0.592 mL, 5.28 mmol), copper(I) iodide (67 mg, 0.352 mmol), and triphenylphosphine (653 mg, 2.464 mmol). Nitrogen was bubbled through the mixture for 10 mintues before adding dichlorobis(triphenylphosphine)- palladium(II) (202 mg, 0.282 mmol) with continued nitrogen gas bubbling. After an additional 10 mintues the reaction mixtrue was heated to reflux for 16 hours, cooled to ambient temperature, diluted with EtOAc, washed with water (3X), brine, dried over magnesium sulfate, and concentrated in vacuo. Column chromatography (25% – -> 40% EtO Ac/Hex) provided optically enriched (4R,5R)-5-(2,5-difluorophenyl)-4- (5-(phenylethynyl)pyridin-3-yl)oxazolidin-2-one which was separated by chiral SFC chromatography (Chiralcel OJ-H preparative column, 30 x 250mm, 5μιη, Mobile Phase: 40% MeOH (0.1%DEA) in C0₂ @ 150Bar, Temp: 35°C, Flow rate: 70.0 mL/min. for 16 min, UV monitored @ 280 nM . tR = 9.23 min) to provide (1.38 g, 2.99 mmol, 85 % yield) of pure single enantiomer (4R,5R)-5-(2,5-difluorophenyl)- 4-(5-(phenylethynyl)pyridin-3-yl)oxazolidin-2-one.

‘H NMR (500 MHz, DMSO-i¾) δ ppm 8.77 (d, J=2.21 Hz, 1 H) 8.57 (s, 1 H) 8.56 (d, J=2.20 Hz, 1 H) 8.07 (t, J=2.05 Hz, 1 H) 7.58 – 7.66 (m, 2 H) 7.44 – 7.52 (m, 3 H) 7.39 – 7.45 (m, 1 H) 7.28 – 7.39 (m, 2 H) 5.67 (d, J=6.62 Hz, 1 H) 5.04 (d, J=6.62 Hz, 1 H). ¹³C NMR (126 MHz,

DMSO-i¾) δ ppm 157.28; 157.24 (d, J=240.70 Hz) 155.92 (d, J=245.20 Hz) 151.63; 147.70; 136.78; 135.02; 131.57; 129.43; 128.89; 126.63 (dd, J=14.99, 7.72 Hz) 121.51; 119.47; 117.83 (dd, J=23.60, 9.10 Hz) 117.50 (dd, J=24.50, 8.20 Hz); 114.60 (dd, J=26.34, 4.54 Hz); 92.86; 85.76; 78.12; 59.43;

LCMS (ESI) m/z calcd for C₂₂H₁₅F₂N₂0₂: 377.11, found 377.20[M+H]⁺;

HRMS (ESI) m/z calcd for

C₂₂H₁₅F₂N₂0₂: 377.1096, found 377.1096 [M+H]+.

SEE

WO2015054103, OXAZOLIDINONES AS MODULATORS OF MGLUR5

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=15257519640294865E18C0BA057EADF3.wapp1nA?docId=WO2015054103&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

PAPER

Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5) are of interest due to their potential therapeutic utility in schizophrenia and other cognitive disorders. Herein we describe the discovery and optimization of a novel oxazolidinone-based chemotype to identify BMS-955829 (4), a compound with high functional PAM potency, excellent mGluR5 binding affinity, low glutamate fold shift, and high selectivity for the mGluR5 subtype. The low fold shift and absence of agonist activity proved critical in the identification of a molecule with an acceptable preclinical safety profile. Despite its low fold shift, 4 retained efficacy in set shifting and novel object recognition models in rodents.

Discovery and Preclinical Evaluation of BMS-955829, a Potent Positive Allosteric Modulator of mGluR5

Bristol-Myers Squibb Research & Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States

ACS Med. Chem. Lett., Article ASAP

DOI: 10.1021/acsmedchemlett.5b00450

Publication Date (Web): January 4, 2016

*Tel: 1-203-677-7675. Fax: 1-203-677-7702. E-mail: fukang.yang@bms.com.

http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.5b00450

http://pubs.acs.org/doi/suppl/10.1021/acsmedchemlett.5b00450/suppl_file/ml5b00450_si_001.pdf

SEE…………http://orgspectroscopyint.blogspot.in/2016/01/bms-955829.html

///////BMS 955829, mGluR5, positive allosteric modulator, schizophrenia, cognition, neurotoxicity, Bristol-Myers Squibb

FC1=CC=C(C=C1[C@H]([C@@H](C2=CC(C#CC3=CC=CC=C3)=CN=C2)N4)OC4=O)F

AZD 2716

January 9, 2016 8:50 am / Leave a comment

AZD2716

CAS 1845753-81-2

MF C₂₄ H₂₃ N O_{3, MW 373.44}

[1,1′-Biphenyl]-3-propanoic acid, 2′-(aminocarbonyl)-α-methyl-5′-(phenylmethyl)-, (αR)-

Antiplaque candidate drug

AstraZeneca INNOVATOR

(R)-7(AZD2716) a novel, potent secreted phospholipase A₂ (sPLA₂) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in vivo efficacy, and minimized safety risk. Based on accumulated profiling data, (R)-7 was selected as a clinical candidate for the treatment of coronary artery disease.

Chiral HPLC using a Chiralcel OJ 5 μm 20×250 mm
column with heptane/EtOH/formic acid ((10:90:0.1; 15 ml/min, 40 °C, 260 nm) as mobile
phase to yield (S)-7 and (R)-7

(R)-7:tR=5.8 min [α]D20 15.4 (c 0.5, ACN), 99.7 %ee. desired

(S)-7: tR=9.2 min. 99.0 % ee. undesired

LINK

http://pubs.acs.org/doi/suppl/10.1021/acsmedchemlett.6b00188

SYNTHESIS

¹H NMR (400 MHz, DMSO-d₆): δ 1.04 (d, J = 6.6 Hz, 3H), 2.55–2.68 (m, 2H), 2.95 (dd, J = 6.1, 12.8 Hz, 1H), 4.00 (s, 2H), 7.13–7.37 (m, 13H), 7.49–7.54 (m, 1H), 12.2 (s, br, 1H).

¹³C NMR (151 MHz, DMSO): δ 16.7, 39.1, 40.7, 41.0, 126.3, 126.4, 127.3, 127.8, 128.0, 128.2, 128.7, 128.9, 129.2, 130.3, 135.3, 139.2, 139.5, 140.5, 141.2, 142.7, 171.3, 177.1.

HRMS (ESI): [M + H]⁺ m/z calcd for C₂₄H₂₄NO₃ 374.1751, found 374.1748.

1H NMR

13C NMR

An Enantioselective Hydrogenation of an Alkenoic Acid as a Key Step in the Synthesis of AZD2716

Staffan Karlsson^*^†, Henrik Sörensen^†, Sören M. Andersen^†, Angele Cruz^†, and Per Ryberg^‡

^† CVMD iMed, Medicinal Chemistry, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden

^‡SP Process Development, Box 36, SE-151 21 Södertälje, Sweden

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00382………..http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00382

http://pubs.acs.org/doi/suppl/10.1021/acs.oprd.5b00382/suppl_file/op5b00382_si_001.pdf

A classical resolution of a racemic carboxylic acid through salt formation and an asymmetric hydrogenation of an α,β-unsaturated carboxylic acid were investigated in parallel to prepare an enantiomerically pure alkanoic acid used as a key intermediate in the synthesis of an antiplaque candidate drug. After an extensive screening of rhodium- and ruthenium-based catalysts, we developed a rhodium-catalyzed hydrogenation that gave the alkanoic acid with 90% ee, and after a subsequent crystallization with (R)-1-phenylethanamine, the ee was enriched to 97%. The chiral acid was then used in sequential Negishi and Suzuki couplings followed by basic hydrolysis of a nitrile to an amide to give the active pharmaceutical ingredient in 22% overall yield.

Paper

Expedited structure-based optimization of the initial fragment hit 1 led to the design of (R)-7(AZD2716) a novel, potent secreted phospholipase A₂ (sPLA₂) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in vivo efficacy, and minimized safety risk. Based on accumulated profiling data, (R)-7 was selected as a clinical candidate for the treatment of coronary artery disease.

Discovery of AZD2716: A Novel Secreted Phospholipase A₂ (sPLA₂) Inhibitor for the Treatment of Coronary Artery Disease

^†Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit Departments of Medicinal Chemistry, ^‡Bioscience, ^§DMPK, ^∥Discovery Sciences Departments of Structure & Biophysics, ^⊥Reagents and Assay Development, and ^#Screening Sciences and Sample Management, Astrazeneca, Mölndal, Pepparedsleden 1, SE-431 83 Mölndal, Sweden

ACS Med. Chem. Lett., Article ASAP

DOI: 10.1021/acsmedchemlett.6b00188

*(F.G.) Phone: +1-212-4780-822. E-mail: fabrizio.giordanetto@deshawresearch.com., *(D.P.) Phone: +46 31 7065 663. E-mail:daniel.pettersen@astrazeneca.com.

http://pubs.acs.org/doi/full/10.1021/acsmedchemlett.6b00188

STR1

str2

akenoic acid as a key step in the sysnthesis of AZD2716. Org. Proc. Res. Dev. 2016, 20(2),
262-269).

/////////atherosclerosis, coronary artery disease, fragment screening, fragment-based drug discovery, Secreted phospholipase A₂, sPLA₂, AZD2716, AZD-2716, AZD 2716, PRECLINICAL

c1c(cc(c(c1)C(=O)N)c2cccc(c2)CC(C(=O)O)C)Cc3ccccc3

Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists

January 7, 2016 5:33 am / Leave a comment

MW 422.79, MF C₁₈ H₁₄ Cl F₃ N₆ O

cas 1627748-32-6

1,2,4-Triazolo[4,3-a]pyrazin-8(5H)-one, 7-[[2-chloro-3-(trifluoromethyl)phenyl]methyl]-6,7-dihydro-6-methyl-3-(2-pyrazinyl)-, (6S)-

(6S)-7-[[2-chloro-3-(trifluoromethyl)phenyl]methyl]-6-methyl-3-pyrazin-2-yl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8-one

(6S)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one

Janssen Pharmaceutica Nv INNOVATOR

Michael K. Ameriks, Jason C. Rech, Brad M. Savall

(6S)-7-[[2-chloro-3-(trifluoromethyl)phenyl]methyl]-6-methyl-3-pyrazin-2-yl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8-one

PAPER

Image for unlabelled figure

The synthesis, SAR, and preclinical characterization of a series of substituted 6,7-dihydro[1,2,4]triazolo[4,3]pyrazin-8(5H)-one P2X7 receptor antagonists are described. Optimized leads from this series comprise some of the most potent human P2X7R antagonists reported to date (IC₅₀s < 1 nM). They also exhibit sufficient potency and oral bioavailability in rat to enable extensive in vivo profiling. Although many of the disclosed compounds are peripherally restricted, compound 11d is brain penetrant and upon oral administration demonstrated dose-dependent target engagement in rat hippocampus as determined by ex vivo receptor occupancy with radiotracer 5 (ED₅₀ = 0.8 mg/kg).

Volume 26, Issue 2, 15 January 2016, Pages 257–261

Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists

Michael K. Ameriks , et al

Janssen Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States

doi:10.1016/j.bmcl.2015.12.052

http://www.sciencedirect.com/science/article/pii/S0960894X15303656

Scheme 3.

Synthesis of compounds 11d and 11l–t. Reagents and conditions: (a) Boc₂O, NaOH, H₂O/MeOH, 0 °C→rt (42%); (b) 2-chloro-3-trifluoromethylbenzaldehyde, Na(OAc)₃BH, DCE, rt (85%); (c) methyl chlorooxoacetate, Et₃N, CH₂Cl₂, 0 °C→rt (97%); (d) 4 N HCl/dioxane, rt, then Et₃N, CH₂Cl₂, rt (100%); (e) Et₃O⁺BF₄⁻, DCM, rt, or Lawesson’s reagent, THF, 55 °C (67–99%); (f) RCONHNH₂, 1-butanol, 130 °C (27–90%).

PATENT

US 20140275096

http://www.google.com/patents/US20140275096

Step A. tert-butyl 3-ethoxy-5,6-dihydropyrazine-1(2H)-carboxylate

To a solution of tert-butyl 3-oxopiperazine-1-carboxylate (1 g, 5 mmol) in DCM (15 mL) was added triethyloxonium tetrafluoroborate (2.9 g, 15 mmol). Stirred for 2 h and neutralized with sat. aq NaHCO₃. Layers separated and aqueous layer extracted with DCM. Combined organic layers dried over Na₂SO₄, filtered, and concentrated to give the title compound, which was used directly without further purification.

Step B. tert-butyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate

To a solution of tert-butyl 3-ethoxy-5,6-dihydropyrazine-1(2H)-carboxylate (1.14 g, 5 mmol) in 1-butanol (30 mL) was added pyrazine-2-carbohydrazide (685 mg, 5 mmol). The reaction mixture was heated at reflux for 16 h. After cooling to rt, the reaction mixture was concentrated and purified by chromatography (SiO2; 2.5% MeOH in DCM) to afford the desired product as a white solid (700 mg, 50% over 2 steps). MS (ESI): mass calcd. for C₁₄H₁₈N₆O₂, 302.2; m/z found, 303.2 [M+H]⁺.
1H NMR (500 MHz, CDCl3) d 9.57 (d, J=1.4 Hz, 1H), 8.62 (d, J=2.5 Hz, 1H), 8.59-8.54 (m, 1H), 4.94 (s, 2H), 4.63-4.50 (m, 2H), 3.89 (t, J=5.4 Hz, 2H), 1.51 (s, 9H).

Step C. 3-(pyrazin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine

To a solution of tert-butyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate (9.3 g, 30 mmol) in DCM (100 mL) was added 1.25M HCl in EtOH (30 mL, 37.5 mmol). After 3 h, the reaction mixture was concentrated, and the resulting solid was purified by chromatography (SiO₂; 10% MeOH in DCM) to provide the desired product as a white solid (3.7 g, 61%). MS (ESI): mass calcd. for C₉H₁₀N₆, 202.1; m/z found, 203.1 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD) δ 9.35 (d, J=1.4 Hz, 1H), 8.72 (dd, J=2.5, 1.6 Hz, 1H), 8.66 (d, J=2.6 Hz, 1H), 4.50 (t, J=5.6 Hz, 2H), 4.22 (s, 2H), 3.24 (t, J=5.6 Hz, 2H).

Step D. 2-(trimethylsilyl)ethyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate

To a solution of 3-(pyrazin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine (1.0 g, 5.0 mmol) and N,N-diisopropylethylamine (1.7 mL, 9.9 mmol) in DMF (15 mL) was added 1-[2-(trimethylsilyl)ethoxycarbonyloxy]pyrrolidin-2,5-dione (1.5 g, 5.9 mmol). Stirred for 18 h and poured into ice cold brine (150 mL). Precipitate filtered and washed successively with water and ether to afford the desired product as a white solid (1.5 g, 89%). MS (ESI): mass calcd. for C₁₅H₂₂N₆O₂Si, 346.2; m/z found, 347.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 9.50 (d, J=1.4 Hz, 1H), 8.56 (d, J=2.5 Hz, 1H), 8.52-8.48 (m, 1H), 4.91 (s, 2H), 4.60-4.45 (m, 2H), 4.25-4.14 (m, 2H), 3.87 (t, J=5.3 Hz, 2H), 1.07-0.92 (m, 2H), 0.01-0.04 (m, 9H).

Step E. 2-(trimethylsilyl)ethyl 8-oxo-3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate

To a vigorously stirred solution of 2-(trimethylsilyl)ethyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate (172 mg, 0.5 mmol) in 1:1 CHCl₃:MeCN (3.8 mL) was added a solution of ruthenium (IV) oxide hydrate (9.8 mg, 0.07 mmol) and sodium metaperiodate (504 mg, 2.3 mmol) in water (4.7 mL). After 4 h, the reaction mixture was diluted with water and extracted with CHCl₃(×3). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford a green oil. Purification by chromatography (SiO₂; EtOAc—10% IPA/EtOAc) provided the desired product as a white solid (663 mg, 63%).
[0140]

MS (ESI): mass calcd. for C₁₅H₂₀H₆O₃Si, 360.1; m/z found, 361.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 9.59 (d, J=1.5 Hz, 1H), 8.63 (d, J=2.5 Hz, 1H), 8.55 (dd, J=2.5, 1.6 Hz, 1H), 4.88-4.75 (m, 2H), 4.47-4.33 (m, 2H), 4.33-4.24 (m, 2H), 1.18-1.04 (m, 2H), 0.04-(−0.02) (m, 9H).

Step F. 3-(pyrazin-2-yl)-6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one

To a solution of 2-(trimethylsilyl)ethyl 8-oxo-3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate (1.0 g, 2.9 mmol) in DCM (29 mL) was added TFA (5.7 mL, 75 mmol). After 1 h, the reaction mixture was concentrated. The crude residue was diluted with EtOAc, sonicated, and filtered to provide the desired product as a white solid (1.2 g, 95%). MS (ESI): mass calcd. for C₉H₈N₆O, 216.1; m/z found, 217.1 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 9.39 (d, J=1.1 Hz, 1H), 8.77 (q, J=2.6 Hz, 2H), 8.56 (s, 1H), 4.73-4.60 (m, 2H), 3.67-3.55 (m, 2H).

Intermediate 3 was made in a manner analogous to Intermediate 1 substituting (±)-tert-butyl 2-methyl-5-oxopiperazine-1-carboxylate for tert-butyl 3-oxopiperazine-1-carboxylate in Step A. MS (ESI): mass calcd. for C₁₀H₁₀N₆O, 230.1; m/z found, 231.1 [M+H]⁺.

Intermediate 4. (6S)-1-(2-chloro-3-(trifluoromethyl)benzyl)-6-methylpiperazine-2,3-dione

[0146]

Step A. (S)-tert-butyl(2-aminopropyl)carbamate

To a solution of (S)-1,2-diaminopropane dihydrochloride (16 g, 109 mmol) in MeOH (64 mL) and water (16 mL) was added di-tert-butyl dicarbonate (28.5 g, 131 mmol) in MeOH (16 mL). The resulting solution was cooled in an ice bath, and 4N NaOH (35 mL, 140 mL) was added dropwise over 2 h. The mixture was allowed to warm to rt and stirred for a total of 20 h. The reaction was filtered, and the filtrate concentrated to remove MeOH. 200 mL EtOAc, 200 mL water, and 16 mL 1M HCl were added sequentially. The layers were separated and the aqueous layer washed with EtOAc (200 mL). The combined organic extracts were washed with 0.04M HCl (208 mL). The organic phase was separated and discarded. The aqueous phases were combined, adjusted to pH=14 with 10N NaOH (20 mL), and extracted with DCM (400 mL×2). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford the desired product as a clear oil (8.0 g, 42%). MS (ESI): mass calcd. for C₈H₁₈N₂O₂, 174.1; m/z found, 175.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 5.01 (br s, 1H), 3.24-3.09 (m, 1H), 3.09-2.95 (m, 1H), 2.92-2.84 (m, 1H), 1.45 (s, 9H), 1.35-1.19 (m, 2H), 1.07 (d, J=6.4 Hz, 3H).

Step B. (6S)-tert-butyl(2-((2-chloro-3-(trifluoromethyl)benzyl)amino)propyl) carbamate

A solution of (S)-tert-butyl(2-aminopropyl)carbamate (4.0 g, 23 mmol) and 2-chloro-3-trifluoromethylbenzaldehyde (4.8 g, 23 mmol) in DCE (100 mL) was stirred at rt for 2 h. Sodium triacetoxyborohydride (7.3 g, 34 mmol) was added at once and stirring continued overnight. Saturated aqueous NaHCO₃was added, and the resulting mixture was extracted with DCM (×2). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford a clear oil. Purification by chromatography (SiO₂; hex—60% EtOAc/hex) provided the desired product as a clear oil (7.2 g, 85%). MS (ESI): mass calcd. for C₁₆H₂₂ClF₃N₂O₂, 366.1; m/z found, 367.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.72-7.56 (m, 2H), 7.35 (t, J=7.7 Hz, 1H), 4.94 (s, 1H), 3.99 (d, J=14.1 Hz, 1H), 3.90 (d, J=14.1 Hz, 1H), 3.29-3.14 (m, 1H), 3.11-2.99 (m, 1H), 2.84 (dd, J=11.1, 6.2 Hz, 1H), 1.44 (s, 9H), 1.11 (d, J=6.4 Hz, 3H).

Step C. (6S)-methyl 2-((1-((tert-butoxycarbonyl)amino)propan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate

To an ice cold solution of (6S)-tert-butyl(2-((2-chloro-3-(trifluoromethyl)benzyl)amino)propyl) carbamate (7.2 g, 20 mmol) and triethylamine (2.8 mL, 21 mmol) in DCM (121 mL) was added methyl chlorooxoacetate (1.9 mL, 21 mmol) dropwise. The resulting mixture was warmed to rt and stirred overnight. After diluting with brine, the layers were separated, and the aqueous layer washed with DCM. The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford the desired product as a white solid (8.5 g, 97%). ¹H NMR (400 MHz, CDCl₃) δ 7.72-7.56 (m, 1H), 7.49-7.32 (m, 2H), 4.83 (d, J=17.1 Hz, 1H), 4.79-4.62 (m, 1H), 4.51 (d, J=17.1 Hz, 1H), 4.11-3.97 (m, 1H), 3.93 (s, 3H), 3.24-3.13 (m, 2H), 1.44 (s, 9H), 1.16-1.12 (m, 3H).

Step D. (6S)-methyl 2-((1-aminopropan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate hydrochloride

To a solution of 4M HCl in dioxane (75 mL) was added (6S)-methyl 2-((1-((tert-butoxycarbonyl)amino)propan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate (7.5 g, 16.7 mmol). After 30 minutes, the reaction mixture was concentrated and the product was used in the next step without further purification (6.5 g, 100%). MS (ESI): mass calcd. for C₁₄H₁₆ClF₃N₂O₃, 352.1; m/z found, 353.1 [M+H]⁺.

Step E. (6S)-1-(2-chloro-3-(trifluoromethyl)benzyl)-6-methylpiperazine-2,3-dione

To a solution of (6S)-methyl 2-((1-aminopropan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate hydrochloride (7.3 g, 18.9 mmol) in DCM (90 mL) was added triethylamine (7.9 mL, 57 mmol) at once. After 2 h, 1N HCl was added and the layers were separated. The aqueous layer was extracted with DCM (×2). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford the desired product as a white solid (5.9 g, 98%). MS (ESI): mass calcd. for C₁₃H₁₁ClF₃N₂O₂, 320.1; m/z found, 321.1 [M+H]⁺. ¹H NMR (600 MHz, CDCl₃) δ 8.24 (d, J=3.6 Hz, 1H), 7.68 (dd, J=7.8, 1.1 Hz, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 5.22 (d, J=15.7 Hz, 1H), 4.52 (d, J=15.7 Hz, 1H), 3.82-3.73 (m, 1H), 3.69-3.61 (m, 1H), 3.31 (ddd, J=13.2, 5.2, 2.3 Hz, 1H), 1.46-1.38 (m, 3H).
Example 14

(±)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one………..

……………………(±) FORM

Example 14 was made in a manner analogous to Example 2 substituting Intermediate 3 for Intermediate 1 and 1-(bromomethyl)-2-chloro-3-(trifluoromethyl)benzene for 1-(bromomethyl)-2,3-dichlorobenzene to provide the desired compound as a white solid (102 mg, 63%). MS (ESI): mass calcd. for C₁₈H₁₄ClF₃N₆O, 422.1; m/z found, 423.1 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) 89.48 (d, J=1.2 Hz, 1H), 8.84-8.82 (m, 2H), 7.85-7.82 (m, 2H), 7.56 (t, J=7.8 Hz, 1H), 5.20 (d, J=16.5 Hz, 1H), 4.98 (dd, J=13.8, 2.2 Hz, 1H), 4.80 (dd, J=13.8, 4.6 Hz, 1H), 4.56 (d, J=16.6 Hz, 1H), 4.23-4.10 (m, 1H), 1.23 (d, J=6.7 Hz, 3H).

Example 15

(6R)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one

Chiral SFC separation of (±)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one on a CHIRALCEL OD-H column (5 μM, 250×20 mm) using 70% CO₂/30% MeOH provided 39 mg of the title compound as the first eluting enantiomer. [α]=+40° (c 2.2, CHCl₃).
MS (ESI): mass calcd. for C₁₈H₁₄ClF₃N₆O, 422.1; m/z found, 423.1 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 9.66 (d, J=1.5 Hz, 1H), 8.68 (d, J=2.5 Hz, 1H), 8.59 (dd, J=2.5, 1.5 Hz, 1H), 7.76-7.72 (m, 1H), 7.69 (dd, J=7.9, 1.6 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 5.44 (d, J=15.5 Hz, 1H), 5.17 (dd, J=13.9, 2.1 Hz, 1H), 4.62-4.54 (m, 2H), 4.08-4.02 (m, 1H), 1.36 (d, J=6.8 Hz, 3H).

Example 16

Chiral SFC separation of (±)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one on a CHIRALCEL OD-H column (5 μM, 250×20 mm) using 70% CO₂/30% MeOH provided 40 mg of the title compound as the second eluting enantiomer.
[α]=−44° (c 2.2, CHCl₃).
MS (ESI): mass calcd. for C₁₈H₁₄ClF₃N₆O, 422.1; m/z found, 423.1 [M+H]⁺.
¹H NMR (500 MHz, CDCl₃) δ 9.66 (d, J=1.5 Hz, 1H), 8.68 (d, J=2.5 Hz, 1H), 8.59 (dd, J=2.5, 1.5 Hz, 1H), 7.76-7.72 (m, 1H), 7.69 (dd, J=7.9, 1.6 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 5.44 (d, J=15.5 Hz, 1H), 5.17 (dd, J=13.9, 2.1 Hz, 1H), 4.62-4.54 (m, 2H), 4.08-4.02 (m, 1H), 1.36 (d, J=6.8 Hz, 3H).

Patent	Submitted	Granted
P2X7 MODULATORS [US2014275096]	2014-03-14	2014-09-18

see,,,,,,,,,http://worlddrugtracker.blogspot.in/2016/01/preclinical-characterization-of.html

//////////////P2X7, 6,7-Dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one, Autoradiography, Depression, CNS, Preclinical characterization, substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one, P2X7 receptor antagonists, Janssen Pharmaceutical Research & Development L.L.C, 1627748-32-6

FC(F)(F)c4cccc(CN1C(=O)c2nnc(n2C[C@@H]1C)c3cnccn3)c4Cl

CC1CN2C(=NN=C2C(=O)N1CC3=C(C(=CC=C3)C(F)(F)F)Cl)C4=NC=CN=C4

Merck’s Novel Indoline Cholesterol Ester Transfer Protein Inhibitors (CETP)

January 6, 2016 11:52 am / Leave a comment

Indoline 7 as in ACS MEDCHEM LETTERS, DOI: 10.1021/acsmedchemlett.5b00404

and

eg 10 as in WO2015054088

(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.

1H-Indole-1-ethanol, 2,3-dihydro-3-[3-(trifluoromethoxy)phenyl]-3-[[3-(trifluoromethoxy)phenyl]methyl]-α-(trifluoromethyl)-, (αR)-

cas 1699732-96-1 R ISOMER

MF C₂₆ H₂₀ F₉ N O_3,MW 565.43

Merck Sharp & Dohme Corp. INNOVATOR

Using the collective body of known (CETP) inhibitors as inspiration for design, a structurally novel series of tetrahydroquinoxaline CETP inhibitors were discovered. An exemplar from this series, compound 5, displayed potent in vitro CETP inhibition and was efficacious in a transgenic cynomologus-CETP mouse HDL PD (pharmacodynamic) assay. However, an undesirable metabolic profile and chemical instability hampered further development of the series. A three-dimensional structure of tetrahydroquinoxaline inhibitor 6 was proposed from ¹H NMR structural studies, and this model was then used in silico for the design of a new class of compounds based upon an indoline scaffold. This work resulted in the discovery of compound 7, which displayed potent in vitro CETP inhibition, a favorable PK–PD profile relative to tetrahydroquinoxaline 5, and dose-dependent efficacy in the transgenic cynomologus-CETP mouse HDL PD assay.

chemical compounds that inhibit cholesterol ester transfer protein (CETP) and are expected to have utility in raising HDL-C, lowering LDL-C, and in the treatment and prevention of atherosclerosis.

see………….http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.5b00404

http://pubs.acs.org/doi/suppl/10.1021/acsmedchemlett.5b00404/suppl_file/ml5b00404_si_001.pdf

Discovery of Novel Indoline Cholesterol Ester Transfer Protein Inhibitors (CETP) through a Structure-Guided Approach

^†Department of Medicinal Chemistry and ^‡Department of Structural Chemistry, Merck Research Laboratories, Merck & Co, Inc., P.O. Box 2000, Rahway, New Jersey 07065, United States

^§Department of Pharmacology, ^∥Department of Drug Metabolism and Pharmacokinetics, and ^⊥Department of Biology, Merck Research Laboratories, Merck & Co, Inc., P.O. Box 2000, Kenilworth, New Jersey 07033, United States

ACS Med. Chem. Lett., Article ASAP

DOI: 10.1021/acsmedchemlett.5b00404

Publication Date (Web): January 4, 2016

*E-mail: jonathan_wilson@merck.com.

PATENT

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=5D4A5D79894298F5BC0A664CF54B61E9.wapp2nB?docId=WO2013028382&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Atherosclerosis and its clinical consequences, including coronary heart disease

(CHD), stroke and peripheral vascular disease, represent a truly enormous burden to the health care systems of the industrialized world. In the United States alone, approximately 13 million patients have been diagnosed with CHD, and greater than one half million deaths are attributed to CHD each year. Further, this toll is expected to grow over the next quarter century as an epidemic in obesity and diabetes continues to grow.

It has long been recognized that in mammals, variations in circulating lipoprotein profiles correlate with the risk of atherosclerosis and CHD. The clinical success of HMG-CoA reductase inhibitors, especially the statins, in reducing coronary events is based on the reduction of circulating low density lipoprotein cholesterol (LDL-C), levels of which correlate directly with an increased risk for atherosclerosis. More recently, epidemiologic studies have

demonstrated an inverse relationship between high density lipoprotein cholesterol (HDL-C) levels and atherosclerosis, leading to the conclusion that low serum HDL-C levels are associated with an increased risk for CHD.

Metabolic control of lipoprotein levels is a complex and dynamic process involving many factors. One important metabolic control in man is the cholesteryl ester transfer protein (CETP), a plasma glycoprotein that catalyzes the movement of cholesteryl esters from HDL to the apoB containing lipoproteins, especially VLDL (see Hesler, C.B., et. al. (1987) Purification and characterization of human plasma cholesteryl ester transfer protein. J. Biol. Chem. 262(5), 2275-2282)). Under physiological conditions, the net reaction is a heteroexchange in which CETP carries triglyceride to HDL from the apoB lipoprotein and transports cholesterol ester from HDL to the apoB lipoprotein.

In humans, CETP plays a role in reverse cholesterol transport, the process whereby cholesterol is returned to the liver from peripheral tissues. Intriguingly, many animals do not possess CETP, including animals that have high HDL levels and are known to be resistant to coronary heart disease, such as rodents (see Guyard-Dangremont, V., et. al, (1998)

Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 120(3), 517-525). Numerous epidemiologic studies correlating the effects of natural variation in CETP activity with respect to coronary heart disease risk have been performed, including studies on a small number of known human null mutations (see Hirano, K.-L, Yamashita, S. and Matsuzawa, Y. (2000) Pros and cons of inhibiting cholesteryl ester transfer protein, Curr. Opin. Lipidol. 11(6), 589-596). These studies have clearly demonstrated an inverse correlation between plasma HDL-C concentration and CETP activity (see Inazu, A., et. al. (2000) Cholesteryl ester transfer protein and atherosclerosis, Curr. Opin. Lipidol. 11(4), 389-396), leading to the hypothesis that pharmacologic inhibition of CETP lipid transfer activity may be beneficial to humans by increasing levels of HDL-C while lowering LDL-C.

Despite the significant therapeutic advance that statins such as simvastatin and atorvastatin represent, statins only achieve a risk reduction of approximately one-third in the treatment and prevention of atherosclerosis and ensuing atherosclerotic disease events.

Currently, few pharmacologic therapies are available that favorably raise circulating levels of HDL-C. Certain statins and some fibrates offer modest HDL-C gains. Niacin provides an effective therapy for raising HDL-C but suffers from patient compliance issues, due in part to side effects such as flushing. Drugs that inhibit CETP (CETP inhibitors) have been under development with the expectation that they will effectively raise HDL cholesterol levels and also reduce the incidence of atherosclerosis in patients. Torcetrapib was the first drug that was tested in a long-term outcomes clinical trial. The clinical trial of torcetrapib was terminated early due to a higher incidence of mortality in patients to whom torcetrapib and atorvastatin were administered concomitantly compared with patients who were treated with atorvastatin alone. The cause of the increased mortality is not completely understood, but it is not believed to be associated with the CETP inhibiting effects of the drug.

Two other drug candidates, dalcetrapib and anacetrapib, are currently being tested in Phase III clinical trials, including large scale outcomes trials. Data from the recently completed DEFINE Phase III trial of anacetrapib are promising. Patients who were being treated with anacetrapib along with baseline statin therapy showed an increase of HDL-C of 138% and a decrease of LDL-C of 40%> compared with patients who were treated with just a statin. See: N. Engl. J. Med. 2010: 363: 2406-15. The data in the DEFINE trial were sufficient to indicate that an increase in mortality for patients treated with anacetrapib is unlikely. Additional drug candidates are still being sought that may have properties that are advantageous compared with the CETP inhibitors that have so far been studied or are currently being studied. Such properties may include, for example, higher potency, reduced off-target activity, better pharmacodynamics, higher bioavailability, or a reduced food effect compared with many of the highly lipophilic compounds that have so far been studied. “Food effect” refers to the variability in exposure to the active drug that occurs depending on when the patient had last eaten, whether or not the drug is administered with food, and the fat content of the food.

Example 18 as in patent

(R)- 1,1, 1 -trifluoro-3-((R)-4-(3-trifluoromethoxy)benzyl)-2-(3-(l, 1 ,2,2,-tetrafluoroethoxy)phenyl)-3,4- dihydroquinoxalin- 1 (2H)-yl)propan-2-ol

SPA: 15 nM

Example 18 was prepared from 2-bromo-l-(3-(l , 1 ,2,2,-tetrafluoroethoxy)phenyl)ethanone in three steps, using the reactions detailed in Schemes A6, A2 and Al . Spectral data are as follows: 1H NMR (400 MHz, CDC1₃) £2.70 (bd, J=4.1 Hz, IH), 3.24 (dd, J=l 1.3, 3.4 Hz, IH), 3.34 (dd, J=15.5, 9.7 Hz, IH), 3.58 (dd, J=l 1.3, 3.3 Hz, IH), 3.86 (d, J=15.4 Hz, IH), 4.20 (d, J=15.7 Hz, IH), 4.40 (d, J=15.8 Hz, IH), 4.46 (m, IH), 4.927 (t, J=3.3 Hz, IH), 5.90 (tt, J=53.1 , 2.7 Hz, IH), 6.59 (d, J= 7.9 Hz, IH), 6.72 (m, 2H), 6.84 (m, 2H), 6.92 (d, J=7.6 Hz, IH), 7.20 (m, 2H), 7.35 (t, J=7.9 Hz, IH), MS m/z = 613.03.

Scheme A12

Methyl 3 – { 1 – [(R)-3 ,3 ,3 -trifluoro-2-hy droxypropyl] -4- [3 -(trifluoromethoxy) benzyl]-l,2,3,4-tetrahydroquinoxalin-2-yl}benzoate (700 mg, 1.262 mmol) is made as described in Example 16 but with one stereochemical center unresolved. The compound was dissolved in MeOH (12.6mL), lithium hydroxide monohydrate (530 mg, 12.62 mmol) was added, and the reaction mixture was heated to 60°C for 4 hours. The crude mixture was dissolved in saturated ammonium chloride solution and extracted into EtOAc, the organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and purified on a silica gel column with a 0-100% Hex/EtOAc gradient. The major peak was concentrated to afford 3-{l-[(R)-3,3,3-trifluoro-2-hydroxypropyl]-4-[3-(trifluoromethoxy)benzyl]-l,2,3,4-tetra-hydroquinoxalin-2-yl} benzoic acid. MS m/z = 541.09.

1H and 13C NMR spectra for compound 7

(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.

Patent

WO2015054088

http://google.com/patents/WO2015054088A1?cl=en

Scheme Al

Scheme A2

Scheme A3

R = Ar, NR_2l C0₂R, CN, S0₂Me

SEE EXAMPLE ………SIMILAR BUT NOT SAME

Example 1. (2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethyl)-phenyl)indolin-l-yl)propan-2-ol. This material was prepared according to Scheme Al, as described below.

3-(3-(trifluoromethyl)phenyl)indolin-2-one. Oxindole (1.598 g, 12 mmol), 3-bromo-a,a,a-trifluoromethyltoluene (2.009 ml, 14.40 mmol), potassium carbonate (3.32 g, 24.00 mmol), Pd₂dba₃ (0.220 g, 0.240 mmol), and 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropylbiphenyl (0.458 g, 0.960 mmol) were combined in THF (12 ml) and the mixture was degassed with nitrogen. The solution was then heated to 80 °C for 18h. The mixture was cooled to room temperature, filtered through silica eluting with ethyl acetate, and concentrated. The material was then purified by silica gel chromatography (Biotage lOOg SNAP cartridge, 0-50% ethyl acetate in hexanes) to provide 3-(3-(trifluoromethyl)phenyl)indolin-2-one as a white solid.

1H NMR (500 MHz) δ 8.58 (s, 1H), 7.61 (d, J=7 Hz, 1H), 7.53-7.45 (m, 3H), 7.33-7.29 (m, 1H), 7.16 (d, J=7 Hz, 1H), 7.10 (m, 1H), 7.01-6.90 (m, 1H), 4.73 (s, 1H).

3 -(3 -(trifluoromethoxy)benzyl)-3 -(3 -(trifluoromethyl)phenyl)indolin-2-one . 3 -Trifluoromethoxy-benzylbromide (0.204 ml, 1.255 mmol) was added to a mixture of 3-(3-(trifluoromethyl)-phenyl)indolin-2-one (290 mg, 1.046 mmol) and potassium carbonate (289 mg, 2.092 mmol) (sodium carbonate may be used in place of potassium carbonate) in DMA (2.5 ml). The mixture was stirred at r.t. for 16h. The reaction was diluted with ethyl acetate and washed with water (3×5 mL). The organic layer was dried with Na₂S0₄, filtered, and concentrated. The products were then purified by silica gel chromatography (Biotage 50g SNAP cartridge; 0-40%> ethyl acetate in hexanes) to provide 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)-phenyl)indolin-2-one .

1H NMR (500 MHz) δ 7.79 (s, 1H), 7.73 (d, J=7 Hz, 1H), 7.62-7.60 (m, 2H), 7.51 (t, J=7 Hz, 1H), 7.26- 7.22 (m, 2H), 7.14 (t, J=7.0 Hz, 1H), 7.11 (m, 1H), 6.97 (m, 1H), 6.92 (m, 1H), 6.78 (m, 1H), 6.73 (s, 1H), 3.77 (d, J=13 Hz, 1H), 3.49 (d, J=13 Hz, 1H).

LCMS m/z = 451.8 (M+H)

3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indoline. Borane tetrahydrofuran complex (1.673 ml, 1.673 mmol) was added to a solution of 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indolin-2-one (302 mg, 0.669 mmol) in THF (1.5 ml). The mixture was heated to 70 °C for 20h. The reaction was cooled to room temperature and quenched with saturated NH₄C1 solution, and this mixture was stirred vigorously for 20 minutes. The product was extracted with ethyl acetate. The extracts were dried over Na₂S0₄, filtered, and concentrated. The product was purified by silica gel chromatography (Biotage 25g SNAP cartridge, 0-50% ethyl acetate in hexanes) to provide 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indoline. This material may also be used without purification in the final step of the sequence, epoxide opening.

1H NMR (500 MHz) δ 7.66 (s, IH), 7.59 (d, J=7 Hz, IH), 7.53 (d, J=7 Hz, IH), 7.45 (t, J=8 Hz, IH), 7.18-7.13 (m, 2H), 7.04 (d, J=8 Hz, IH), 6.98 (d, J=7 Hz, IH), 6.81 (t, J=7.5 Hz, IH), 6.71 (m, 2H), 6.60 (s, IH), 3.83 (m, IH), 3.75-3.73 (m, 2H), 3.46 (d, J=13 Hz, IH), 3.41 (d, J=13 Hz, IH).

= 437.9 (M+H)

(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)-phenyl)indolin-l-yl)propan-2-ol. (S)-2-(trifluoromethyl)oxirane (81 μΐ, 0.933 mmol) was added to a solution of 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indoline (136 mg, 0.311 mmol) in l,l,l,3,3,3-hexafluoro-2-propanol (412 μΐ, 3.91 mmol). The reaction was stirred at room temperature overnight. The solvent was removed and the product was purified by silica gel chromatography (Biotage 25 g SNAP cartridge; 0-25% ethyl acetate in hexanes) to provide (2R)- 1 ,1,1 -trifluoro-3 -(3 -(3 -(trifluoromethoxy)benzyl)-3 -(3 -(trifluoromethyl)phenyl)indolin- 1 -yl)propan-2-ol.

1H NMR (500 MHz) (mixture of diastereomers) δ 7.72 (s, 0.5 H), 7.69 (s, 0.5 H), 7.65 (d, J=6.5 Hz, 0.5 H), 7.61 (d, J=7.5 Hz, 0.5 H), 7.56 (s, 1H), 7.50 (m, 1H), 7.25-7.17 (m, 2H), 7.07 (broad s, 2H), 6.91-6.89 (m, 1H), 6.79-6.75 (m, 1H), 6.53 (m, 2H), 4.00 (broad s, 1H), 3.83 (d, J= 9 Hz, 0.5H), 3.77 (d, J=9 Hz, 0.5H), 3.59-3.55 (m, 1H), 3.45-3.43 (m, 1H), 3.39-3.29 (m, 2H), 3.21-3.15 (m, 1H), 2.32 (m, 0.5H), 2.15 (m, 0.5H).

LCMS m/z = 549.8 (M+H)

Examples 1-25, in the table below, were prepared according to Scheme Al in a

SEE EG 10…….(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.

ABOUT AUTHOR

Jonathan Wilson

Associate Principal Scientist at Merck

Merck

https://www.linkedin.com/in/jonathan-wilson-23206523

Experience

Associate Principal Scientist

Merck

October 2013 – Present (2 years 4 months)

Senior scientist

Merck

May 2009 – October 2013 (4 years 6 months)

Postdoctoral researcher

Princeton University

October 2007 – May 2009 (1 year 8 months)

Associate Medicinal Chemist

Merck

2000 – 2002 (2 years)

Education

Massachusetts Institute of Technology

Ph.D., Organic Chemistry

2002 – 2007

Oberlin College

B. A., Chemistry

1996 – 2000

///////CETP inhibition, cholesterol ester transfer protein, HDL, indoline, tetrahydroquinoxaline, merck, discovery

c21ccccc1N(C[C@@]2(c3cccc(c3)OC(F)(F)F)Cc4cc(ccc4)OC(F)(F)F)C(C(F)(F)F)O

FC(F)(F)Oc1cccc(c1)C3(CN(C[C@@H](O)C(F)(F)F)c2ccccc23)Cc4cccc(OC(F)(F)F)c4

see…………http://worlddrugtracker.blogspot.in/2016/01/mercks-novel-indoline-cholesterol-ester.html

Fluorofenidone

January 4, 2016 4:03 am / Leave a comment

2(1H)-Pyridinone, 1-(3-fluorophenyl)-5-methyl-.png

Fluorofenidone

1- (3-fluorophenyl) -5-methyl – 2 (1H) pyridone

2(1H)-Pyridinone, 1-(3-fluorophenyl)-5-methyl-

1- (3_ fluorophenyl) -5_ methylpyridine _2 (IH) – one

C₁₂ H₁₀ F N O, 203.2123

An anti-inflammatory agent potentially for the treatment of organ fibrosis.

CAS No. 848353-85-5

Synthesis

PATENT

WO 2006108354

http://www.google.co.in/patents/WO2006108354A1?cl=en

PATENT

http://www.google.com/patents/CN102241625A?cl=zh

(Compound 1)

A. (3_ fluorophenyl) methyl pyridine _2 (IH) 1- -5_ – -one

9. 6gDMF, 45 0g (0 2mol.) Inter-fluoro-iodobenzene, 21 8g (0. 2mol) 5_ methylpyridine _2_ (IH) -.. -one, 28g of anhydrous potassium carbonate and 1. Og copper powder, 160 ° -170 °, the reaction was stirred at reflux for 20 hours, the natural cooling to 110~120 ° C, was slowly added to about 330ml 80~90 ° C hot water, cooled to 20 ° C. Suction filtered, the filter cake was washed with about 20ml of water, remove the cake, with about 300ml of ethyl acetate ultrasound 30min, suction filtered, the filter residue was washed with 20ml of ethyl acetate. The combined ethyl acetate, washed with water three times (50ml * 3), and the filtrate layers were separated and allowed to stand for 15min, ethyl acetate fraction was concentrated to a non-steamed, hot added under stirring for about 85ml of petroleum ether, cooling to 15~20 ° C insulation ~ 1.5 hours. Filtration, the filter cake was washed twice with petroleum ether (about 20ml * 2) used to give 34. 9g crude. Recrystallized from 20% ethanol to give the product 1- (3_ fluorophenyl) -5_ methylpyridine _2 (IH) – one as a white solid # 30. Ig0 Μ P.: 132 · 1 ~133 7 °.. C.

PATENT

http://www.google.co.in/patents/WO2009149188A1?cl=zh-CN

PATENT

CN 102241625

http://www.google.com/patents/CN102241625A?cl=zh

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2009111947

PAPER

http://www.mdpi.com/1420-3049/17/1/884

CN1386737A *	Jun 11, 2002	Dec 25, 2002	中南大学湘雅医学院	Antifibrosis pyridinone medicine and its prepaing process
CN1846699A	Apr 13, 2005	Oct 18, 2006	中南大学湘雅医院	Application of 1-(substituted phenyl)-5-methyl-2-(1H)-pyridone compound in preparing medicine for anti-other organifibrosis and tissue fibrosis except renal interstitial fibrosis
CN101235013A*	Mar 10, 2008	Aug 6, 2008	广东东阳光药业有限公司;张中能	Crystallized 1-(3-fluorophenyl)-5-methyl-2-(1H)pyridine and its preparation method composition and application
US20070203203	May 1, 2007	Aug 30, 2007	Tao Li J	Composition and Method for Treating Fibrotic Diseases


Patent	Submitted	Granted
COMPOUNDS AND METHODS FOR TREATING INFLAMMATORY AND FIBROTIC DISORDERS [US2009318455]	2009-12-24
COMPOSITION AND METHOD FOR TREATING PROTEINURIA [US2010099719]	2010-04-22
COMPOSITION AND METHOD FOR TREATING FIBROTIC DISEASES [US2009258911]	2009-10-15
Composition and Method for Treating Fibrotic Diseases [US2008319027]	2008-12-25
METHODS FOR TREATING ACUTE MYOCARDIAL INFARCTIONS AND ASSOCIATED DISORDERS [US2010190731]	2010-07-29
Methods for Treating Acute Myocardial Infarctions and Associated Disorders [US2011218515]	2011-09-08
METHODS OF TREATING HIV PATIENTS WITH ANTI-FIBROTICS [US2012014917]	2012-01-19
Composition and Method for Treating Fibrotic Diseases Composition and Method for Treating Fibrotic Diseases [US2009005424]	2007-08-30
Crystalline 1-(3-fluorophenyl)-5-methyl-2-(1H)pyridone, the preparation methods, compositions and applications thereof [US8232408]	2009-03-10	2012-07-31

/////////

CC1=CN(C(=O)C=C1)C2=CC(=CC=C2)F

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251st ACS National Meeting & Exposition

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SEE EXAMPLE ………SIMILAR BUT NOT SAME

SEE EG 10…….(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.

Jonathan Wilson

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Discovery of AZD2716: A Novel Secreted Phospholipase A₂ (sPLA₂) Inhibitor for the Treatment of Coronary Artery Disease