WO2009152051A1

WO2009152051A1 - Synthesis of a macrocyclic hcv protease inhibitor

Info

Publication number: WO2009152051A1
Application number: PCT/US2009/046426
Authority: WO
Inventors: Peng Wang
Original assignee: Phenomix Corporation
Priority date: 2008-06-12
Filing date: 2009-06-05
Publication date: 2009-12-17

Abstract

The present invention provides a method of synthesis of a macrocyclic compound known as a potent inhibitor of a protease produced by the hepatitis C virus (HCV). Inhibition of the viral protease blocks assembly of mature viral particles in an infected mammalian host. The method of synthesis involves a ring-closing metathesis step using a ruthenium catalyst. Hydrogenation and boronate deprotection provide the HCV protease-inhibitory product.

Description

SYNTHESIS OF A MACROCYCLIC HCV PROTEASE INHIBITOR

Cross-Referenee to Related Application

This application claims the priority of U.S. Ser. No. 61/060,876, filed June 12, 2008, the disclosure of which is incorporated herein by reference in its entirety.

Background

Hepatitis C virus ("HCV") is the causative agent for hepatitis C, a chronic infection characterized by jaundice, fatigue, abdominal pain, loss of appetite, nausea, and darkening of the urine. HCV, belonging to the hepacivirus genus of the Flaviviriae family, is an enveloped, single-stranded positive-sense RNA-containing virus. The long-term effects of hepatitis C infection as a percentage of infected subjects include chronic infection (55-85%), chronic liver disease (70%), and death (1-5%). Furthermore, HCV is the leading indication for liver transplant. In chronic infection, there usually presents progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma.

The HCV genome (Choo et al., Science 1989, 244, 359-362; Simmonds et al., Hepatology 1995, 21, 570-583) is a highly variable sequence exemplified by GenBank accession NC_004102 as a 9646 base single-stranded RNA comprising the following constituents at the parenthetically indicated positions: 5' NTR (i.e., non-transcribed region) (1-341); core protein (i.e., viral capsid protein involved in diverse processes including viral morphogenesis or regulation of host gene expression) (342-914); El protein (i.e., viral envelope) (915-1490); E2 protein (i.e., viral envelope) (1491-2579); p7 protein (2580- 2768); NS2 protein (i.e., non-structural protein 2) (2769-3419); NS3 protease (3420-5312); NS4a protein (5313-5474); NS4b protein (5475-6257); NS5a protein (6258-7601); NS5b RNA-dependent RNA polymerase (7602-9372); and 3' NTR (9375-9646). Additionally, a 17-kDalton -2/+1 frameshift protein,

"protein F", comprising the joining of positions (342-369) with (371-828) may provide functionality originally ascribed to the core protein. The NS3 (i.e., non-structural protein 3) protein of HCV exhibits serine protease activity, the N-terminus of which is produced by the action of a NS2- NS3 metal-dependent protease, and the C-terminus of which is produced by auto-proteolysis. The HCV NS3 serine protease and its associated cofactor, NS4a, process all of the other non-structural viral proteins of HCV. Accordingly, the HCV NS3 protease is essential for viral replication.

Several compounds have been shown to inhibit the hepatitis C serine protease, but all of these have limitations in relation to the potency, stability, selectivity, toxicity, and/or pharmacodynamic properties. Such compounds have been disclosed, for example, in published U.S. Patent Application Nos. 2004/0266731, 2002/0032175, 2005/0137139, 2005/0119189, and 2004/0077600A1, and in published PCT patent applications WO 2005/037214 and WO 2005/035525. Macrocyclic inhibitors of HCV have been disclosed by the inventors herein in patent application U.S. Serial No. 60/883,946.

In PCT application number PCT/US2007/086530, by the inventors herein, various macrocyclic inhibitors of HCV protease are disclosed and claimed.

Summary

The present invention is directed to methods of synthesis of a macrocyclic inhibitor of HCV protease, the structure of which is disclosed and claimed in PCT application number PCT/US2007/086530, by the inventors herein, and which is designated compound 12 in the present application.

In various embodiments, the present invention provides a method of preparing a compound of Formula (I):

BocHN,

(I)

comprising contacting a compound of formula (II):

(H) and a Ring-Closure-Metathesis (RCM) catalyst to provide the compound of Formula (I), wherein the RCM catalyst comprises a compound of

Formula (III):

(HI) under conditions effective to bring about formation of the compound of Formula (I).

S In various embodiments, the invention further comprises a method wherein the compound of Formula (II) is prepared by contacting a compound of Formula (IV):

(IV) and a compound of Formula (V):

(V) or a salt thereof, under conditions effective to bring about formation of the compound of Formula (II).

In various embodiments, the invention further comprises, after formation of the compound of Formula (I), contacting the compound of Formula (I) and hydrogen gas in the presence of a catalyst under condition effective to bring about formation of a compound of Formula (VI):

(VI).

In various embodiments, the invention further comprises contacting the compound of Formula (VI) and phenylboronic acid or oct-7-enylboronic acid under conditions effective to hydrolyze a boronate ester bond to provide a compound of Formula (VII):

(VII).

A compound of Formula (VII) is disclosed and claimed in related patent application number PCT/US2007/086530, by the inventors herein. This compound is a potent inhibitor of HCV protease, and is designated compound 12 in the present application.

Detailed Description The terms "HCV NS3 serine protease", "HCV NS3 protease", "NS3 serine protease", and "NS3 protease" denote all active forms of the serine protease encoded by the NS3 region of the hepatitis C virus, including all combinations thereof with other proteins in either covalent or noncovalent association. For example, other proteins in this context include without limitation the protein encoded by the NS4a region of the hepatitis C virus. Accordingly, the terms "NS3/4a" and "NS3/4a protease" denote the NS3 protease in combination with the HCV NS4a protein.

All chiral, diastereomeric, racemic forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds used in the present invention include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention.

A double bond that can be either of the cis or the trans configuration is indicated by a crossed double bond, as depicted below in the compound of Formula (I) of the present invention:

wherein the newly formed bond can be of either configuration.

BocHN,

(I)

comprising contacting a compound of formula (II):

Formula (III):

(HI) under conditions effective to bring about formation of the compound of Formula (I). The olefinic bond formed in the RCM reaction can be either of the cis or the trans configuration, as is indicated by the crossed double bond depiction in Formula (I). The compound of Formula (III) is also known as Zhan Catalyst- IB catalyst. Other catalysts tested, that were ineffective or less effective in catalyzing the transformation, include Strem ruthenium-based catalysts numbers 44-0063, 44-7775, 44-7777, and 44-7778 as well as 1^st generation and 2^nd generation Hoveyda-Grubbs catalyst. More specifically, the conditions can comprise contacting the compound of Formula (II) and the RCM catalyst of Formula (III) in solution in a solvent within a temperature range of about 46-11O⁰C. For example, the solvent can include isopropyl acetate, toluene, or dichloromethane, or a combination thereof.

More specifically, the reaction can be carried out within a reaction temperature range of about 70-85⁰C. More specifically, the reaction can be carried out in isopropyl acetate.

More specifically, after formation of the compound of Formula (I), 2- mercaptonicotinic acid can be added to the solution to assist in removal of residual ruthenium catalyst.

More specifically, after formation of the compound of Formula (I), residual ruthenium catalyst can be removed from the solution by contacting with Ecosorb C-906 carbon. In various embodiments, the invention further comprises a method wherein the compound of Formula (II) is prepared by contacting a compound of Formula (IV):

and a compound of Formula (V):

(V) or a salt thereof, under conditions effective to bring about formation of the compound of Formula (II). More specifically, the salt of the compound of

Formula (V) can be a tosylate salt. It was found that trifluoracetic acid (TFA) and p-toluenesulfonic acid (tosylate, TsOH) salts can both be isolated as crystals, and purity improvements were realized with crystallization of these two salts.

Also it was found that the relative stability of the salts is TsOH>TFA>HCl. The TsOH salt is stable at room temperature for at least 1 week and is stable at 4 degree for 2 months. The TFA salt is stable at -20 degrees but degraded slowly at room temperature upon exposure to air and water. The HCl salt failed to crystallize at all.

More specifically, the conditions can include contacting the compound of Formula (IV) and the compound of Formula (V) and a carbonyl activating reagent in a solvent. For example, the carbonyl activating reagent can be carbonyl diimidazole. More specifically, the solvent can include an ester, for example isopropyl acetate.

(VI).

More specifically, the catalyst can be platinum or palladium metal. For example, the catalyst can be 10% palladium on carbon. More specifically, the hydrogenation reaction can be carried out in an ester solvent. An example is isopropyl acetate. The method can further include recrystallization of the compound of Formula (VI).

(VII).

More specifically, the hydrolysis reaction can include contacting at a pH of about 4. More specifically, the hydrolysis reaction can be carried out in a two-phase solvent mixture including water, acetonitrile and a hydrocarbon. An example of a suitable hydrocarbon is heptane. The water phase of the two-phase solvent for the hydrolysis reaction can also include a buffer at a pH of about 4. The heptane phase is removed and fresh heptane added to remove reaction

iα byproducts; this operation can be repeated several times. The method can further include recrystallizing the compound of Formula (VII). Compound 12, as shown below in the Examples, is a compound of Formula (VII).

Examples

Synthesis of HCV Protease-Inhibitory Macrocyclic Compound 12

1. Synthesis of Compound 6

Compound 3: Crystalline oct-7-enylboronic acid 2 (21 Kg) is treated with (+)- pinanediol (22.9 Kg) in t-butyl methyl ether (78 Kg) at room temperature. After water is removed, the reaction solution is subjected to the next step reaction without further purification.

Compound 4: To a solution of oct-7-enyl-(+)-pinanediol boronic ester (39.1 Kg) 3 in t-butyl methyl ether (78 Kg) is added dichloromethane (57.2 Kg). The solution is cooled to -50° C and kept at that temperature. A solution of lithium diisopropylamide (1.25 equiv) is slowly added. After two hours of reaction, the solution is warmed up to -40⁰C. A zinc chloride solution (32.1 Kg prepared at 50° C) in t-butyl methyl ether (48 Kg) and THF (142 Kg) is slowly added while keeping the temperature at -4O⁰C to -45⁰C. After 1 hour, the reaction solution is warmed up to 10⁰C and quenched with 25% sulfuric acid (79.3 Kg). Layers are split and the aqueous is removed. The organic layer is washed with water (7 x 98 Kg) until pH is >5.5. Solvent is removed and the oil dissolved in t-butyl methyl ether (135 Kg) and passed through a bed of molecular sieves (35.7 Kg) until the Karl Fisher (KF) water content is < 0.05%. The product solution is carried over to the next step of the process without isolation.

Compound 5: The solution of lithium bis(trimethylsilyl)amide (87.3 Kg solution, 132 moles) in THF is diluted with MTBE (91 Kg). To this is added a solution of chloroboronate 4 in t-butyl methyl ether from Step 2 over 2.5 h. The reaction mixture is stirred at -15 ⁰C until the reaction is completed (1 h). The reaction mixture is warmed to 20 - 25 ⁰C and added Celite subsequently. Solvents are removed. The slurry is suspended in isooctane (57 Kg) and filtered through a bed of Celite and is taken on to the next step of the process without isolation.

Compound 6: To a chilled slurry (0 ⁰C) of p-toluenesulfonic acid (5.86 Kg, 1.2 equiv.) in MTBE (14 L) is added a solution of bis(trimethlysilyl)amino- pinanediol boronic ester 5 in isooctane (33 kg) over 4 h. Solution temperature and pH maintained at below 0 °C and 2, respectively, throughout the addition. Product 6 precipitated out about 2 h into addition. Stirring is continued for 2 h at 5 °C to complete product crystallization. The product is isolated by filtration and rinsed with isooctanes (3 x 4 L), dried at 40 ⁰C for 48 h to constant weight. The product 6 (10.3 Kg) is obtained as an off-white crystal.

2. Synthesis of Compound 7

or CDI

Overall yield 38% for 5 steps

Step 1: Preparation of isoindolidine

Phthalimide is charged into the reactor followed by THF (1 volume) and cooled to 10 ⁰C. 4.0 equivalents of 1 M BH₃ THF is charged over the course of 30 minutes. The reactor is heated to reflux for 48 hours. Upon reaction completion, the reactor is cooled to 10 ⁰C and is quenched with 6 equivalents of MeOH. 5 equivalents of cone. HCl is subsequently charged and the reaction is heated to 55 ⁰C for 12 h. Upon completion of the quench, the reaction mixture is concentrated to remove the MeOH. The residual oil is diluted with water (5 volumes) and dichloromethane (5 volumes). The reaction mixture is adjusted to pH = 8 to 9 with 25% NaOH. The reaction mixture is filtered to remove precipitated borate salts. The pH is adjusted to 12-13 with 25% NaOH. The layers are separated. The aqueous layer is washed with dichloromethane (2 x 3 volumes). The combined organic layers are added water (5 volumes). The pH is adjusted to 3-4 with cone. HCl. The layers are separated (product is in aqueous phase). The aqueous layer is washed with dichloromethane (2x 3 volumes) to remove side-products. The pH of the aqueous layer is adjusted to 12 with 25% NaOH. The aqueous layer is extracted with dichloromethane (3 x 2 volumes). The combined dichloromethane layers are washed with brine, dried over MgSO₄, and filter and concentrated to an oil. The oil is dissolved in acetonitrile (5 volumes) and cooled to ~10°C. HCl gas is bubbled until solution become acidic (pH <2).

The resultant slurry is stirred at 10 °C for 1 h. The solid is collected by filtration and rinsed with acetonitrile (1 to 2 volumes) and dried at 45 ⁰C overnight under vacuum.

Step 2 and Step 3: Synthesis of isoindolidinylcarbonyl 3-hydroxyproline methyl ester

1.00 equivalents of isoindoline from the previous reaction (28.9 g), suspended in isopropyl acetate (60 mL) is warmed to 30 ⁰C and added into a suspension of 1.02 equivalents of carbonyldiimidazole (CDI, 19.3 g) in isopropylacetate (60 mL) in a separate flask over 30 minutes and stirred for 3 h. After the activation is completed, 1.05 equivalents of isoindoline-HCl salt (19.2 g) and 2 equivalents of N-methylmorpholine (NMM, 23.6 g) suspended in isopropylacetate (60 mL) in a separate flask is added over 1 h and stirred for 16 h at 30 °C. After the coupling reaction is completed, the reaction slurry was chilled to 10 ⁰C and washed with 2 equivalents 25% H₂SO₄ (225 mL). The aqueous phase was separated, and the organic phase re-washed repeatedly with slightly acidic water until the aqueous phase was almost colorless (3 x). The organic phase was washed with fresh water until pH of aqueous phase become 6 - 7. The organic was dried over MgSO₄, filtered, concentrated at 45 °C to a thin oil. The oil was azeotropically dried with acetonitrile (2 x 100 mL) twice to a thin oil. The organic phase is chilled to 10 ⁰C and acidified with 1.3 equivalents gaseous HCl (~5.5 g). The solution is warmed to 30 ⁰C and stirred overnight. The slurry was chilled to 10 ⁰C and filtered to give an off-white solid. The solids were re- slurried with cold acetonitrile for 1 h and then filtered, dried overnight at 45 ⁰C under vacuum.

Step 4: Synthesis of Compound 7 methyl ester

7 methyl ester

1.15 equivalents of Boc-Allylglycine DCHA salt, 1.3 equivalents of HOBT, 1 equivalent of the product of the previous reaction, and 1.2 equivalents EDCl is charged in sequence to the reactor and cool reactor to 5⁰C. DCM (10 volumes) is charged followed by 0.87 equivalents NMM over the course of 20 minutes and keep the temperature below 10⁰C. The reaction mixture was stirred for 2 h or until completion. The DCHA hydrochloride is filtered off and rinsed with dichloromethane (1 volume). The combined dichloromethane layers are concentrated at 35⁰C in the presence of IPOAc (5 volumes). After all dichloromethane has been removed, the organic layer is washed with 5% NaHCO₃ solution (2 x 2 volumes), water (2 x 1 volume) and concentrated to an oil. The oil is dissolved in heptanes (1 volume) and concentrated to dryness. The residual is dissolved in MTBE (5 volumes) at 45⁰C and added heptanes (5 volumes). The reaction mixture is allowed to cool to room temperature and seeded with a crystal of pure product. The resultant slurry is stirred at room temperature for 2 h. The compound 7 methyl ester is collected by filtration as a white solid.

Step 5: Synthesis of compound 7.

7 methyl ester

The methyl ester is dissolved in MTBE (5 volumes) at 45 ⁰C and cooled to 25 ⁰C. The reaction mixture is added 1.5 equivalents LiOH in H₂O (2.5 volumes) over 20 minutes at 25⁰C and stir for 1 h. Upon completion, the layers are separated. The aqueous layer is added MTBE (5 volumes) and the pH is adjusted to 2 with 25% H₂SO_4. The organic layer is washed with brine (1 volume), dried over MgSO₄ and concentrated to an oil. The oil is triturated with heptanes (5 volumes) to provide compound 7 as an amorphous solid. . Synthesis of Compound 12

10

12

Compound 8: Compound 7 (6.52 Kg) is activated by carbonyl diimidazole (CDI, 2.57 Kg) in isopropylacetate (13 L) and further reacted with tosylate salt compound 6 (7.449 Kg) in isopropylacetate (65 L) . After the reaction is completed, the reaction mixture is washed with 1 N hydrochloride solution (77 L) followed by washing with saturated sodium bicarbonate solution (77 L) and water (2 x 39 L). Residual water is removed by azeotropic distillation. Inorganic salt is removed by filtration. The resultant solution is concentrated to dryness and subjected to the next step reaction without further purification.

Compound 9: The crude compound 8 (5.911 Kg) is diluted with isopropylacetate (591 L) , and degassed with nitrogen followed by addition of trifluoracetic acid (TFA, 55 g) at room temperature. The reaction solution is subsequently heated at 70 ⁰C and Zhan Catalyst- IB (84 g) is added to start the ring closing metathesis reaction. After heating for 1 h, another portion of the Zhan Catalyst- IB (56 g) is charged into the reactor and heated for 104 min to drive the reaction to completion. After the reaction is completed, the reaction solution is cooled to 60 ⁰C and added 2-mercaptonicotinic acid (148 g) and stirred for 18 h at room temperature. The reaction solution is washed with half saturated sodium bicarbonate solution (119 L) and saturated sodium chloride solution (1 19 L). Activated carbon (Ecosorb C-906, 3.815 Kg) is added and stirred for 4 h. The reaction mixture is filtered through a bed of Celite and rinsed with isopropylacetate (50 L). The filtrate is washed with saturated sodium bicarbonate solution (75 L) and water (119 L). The organic phase is concentrated to a 10% solution and subjected to the next step hydrogenation reaction without further purification.

Compound 10: The crude compound 9 (containing 9.5 Kg of compound 9 by LOD) is hydrogenated in the presence of 10% Pd on carbon (1.89 Kg) at 1 bar until all starting material has been consumed. The reaction mixture is filtered through a bed of Celite and rinsed with isopropylacetate (63 L+45 L). The filtrate is further treated with Norite carbon (1.89 Kg) and filtered through a bed of Celite and rinsed with isopropylacetate (64 L). Finally, the filtrate is concentrated to dryness under reduced pressure and recrystallized from acetonitrile (54 L) and water (48 L). The crude penultimate intermediate (6.8 Kg) is further purified on a short plug of silica gel (60.7 Kg).

Compound 12:

ACN, buffer phenylboronic acid, heptane

10 12

The appropriate fractions from the short plug are collected and solvent is switched to acetonitrile. Phenylboronic acid (0.692 Kg + 0.069 Kg) is added along with pH 4 buffer (29 L) and heptanes (58 L). The reaction mixture is heated at 45 ⁰C for 2-3 h. The mixture is allowed to settle and the heptane layer is removed and replaced with fresh heptanes. After heating and replacing the heptanes layer three times, the phases are separated. The aqueous layer is subsequently concentrated under reduced pressure to 60% of its original volume, water (58 L) is added and the mixture is cooled to room temperature. The white solid compound 12 is collected by filtration and rinsed with mother liquor and heptanes, then dried under vacuum. Compound 12 can be further purified by slurrying in acetonitrile (33.5 L) and water (67 L) and dried at 45 ⁰C under vacuum to constant weight to give 3.4 Kg product as a white crystal.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a compound of Formula (I):

BocHN,

(I) comprising contacting a compound of formula (II):

B

(H)

2α and a Ring-Closure-Metathesis (RCM) catalyst to provide the compound of Formula (I), wherein the RCM catalyst comprises a compound of Formula (III):

(III) under conditions effective to bring about formation of the compound of Formula (I).

2. The method of claim 1 wherein the conditions comprise contacting the compound of Formula (II) and the RCM catalyst of Formula (III) in solution in a solvent within a temperature range of about 46-1 1O⁰C.

3. The method of claim 2 wherein the solvent comprises isopropyl acetate, toluene, or dichloromethane, or a combination thereof.

4. The method of claim 2 wherein the temperature range is about 70-85⁰C.

5. The method of claim 2 wherein, after formation of the compound of Formula (I), 2-mercaptonicotinic acid is added to the solution.

6. The method of claim 2 wherein, after formation of the compound of Formula (I), residual ruthenium is removed from the solution by contacting with Ecosorb C-906 carbon.

7. The method of claim 1 wherein the compound of Formula (II) is prepared by contacting a compound of Formula (IV):

(IV) and a compound of Formula (V):

8. The method of claim 7 wherein the salt of the compound of Formula (V) is a tosylate salt.

9. The method of claim 7 wherein the conditions comprise contacting the compound of Formula (IV) and the compound of Formula (V) and a carbonyl activating reagent in a solvent.

10. The method of claim 9 wherein the carbonyl activating reagent comprises carbonyl diimidazole.

11. The method of claim 9 wherein the solvent comprises an ester.

12. The method of claim 9 wherein the solvent comprises isopropyl acetate.

13. The method of claim 1 comprising, after formation of the compound of Formula (I), contacting the compound of Formula (I) and hydrogen gas in the presence of a catalyst under condition effective to bring about formation of a compound of Formula (VI):

(VI).

14. The method of claim 13 wherein the catalyst comprises platinum or palladium.

15. The method of claim 13 wherein the catalyst comprises 10% palladium on carbon.

16. The method of claim 13 wherein contacting the compound of Formula (I) and hydrogen gas is carried out in an ester solvent.

17. The method of claim 16 wherein the ester solvent comprises isopropyl acetate.

18. The method of claim 13 further comprising recrystallization or short plug chromatography, or both, of the compound of Formula (VI).

19. The method of claim 13 further comprising contacting the compound of Formula (VI) and phenylboronic acid or oct-7-enylboronic acid under conditions effective to hydrolyze a boronate ester bond to provide a compound of Formula (VII):

(VII).

20. The method of claim 19 wherein the contacting comprises contacting at a pH of about 4.

21. The method of claim 19 wherein the contacting is carried out in a two- phase solvent mixture comprising water, acetonitrile and a hydrocarbon.

22. The method of claim 21 wherein the hydrocarbon comprises heptane.

23. The method of claim 21 wherein the water of the solvent mixture further comprises a buffer at a pH of about 4.

24. The method of claim 19 further comprising recrystallizing the compound of Formula (VII) from a suitable solvent or combination of solvents.

25. The method of claim 24 wherein the solvent is water or the combination of solvents is water and acetonitrile.