PROCESSES FOR THE PREPARATION OF NELFINAVIR

Field of the Invention

The field of the invention relates to a novel crystalline form of nelfmavir mesylate and processes for its preparation. More particularly, it relates to the preparation of crystalline form of nelfmavir mesylate designated as Form I. The invention also relates to pharmaceutical compositions that include the crystalline Form I of nelfmavir mesylate and use of said compositions for treatment of HTV-I infections. The invention also relates to a process for the preparation of nelfmavir or salts thereof.

Background of the Invention

Nelfmavir is chemically, [35 ^f -[2(25*,35*),3α ₎ 4αp,8αβ]]-iV-(l ₎ l- dimethylethyl)decahydro-2-[2-hydroxy-3-[(3-hydroxy-2-methylb enzoyl)amino]-4- (phenylthio)butyi]-3-isoquinoline carboxamide. It is commercially available in the form of its mono-methanesulfonate salt of structural Formula I.

FORMULA I

Nelfmavir belongs to the class of HIV-protease inhibitors indicated for the treatment of HIV infection when antiretro viral combination therapy is warranted. It was originally used in combination with antiretroviral nucleoside analogues for the treatment of HIV-I in adults, adolescents and children. Nelfmavir is a member of the group of drugs referred to as protease

inhibitors (Pis). This class of drag combats viral replication of HIV by blocking HIV's protease protein. This protein or enzyme is used by HIV to break up large viral proteins into smaller particles from which new HIV particles can be formed. Protease inhibitors ensure that these new particles are immature and incapable of infecting new cells, thus inhibiting the HIV replication process.

Several processes have been reported for the preparation of nelfmavir for example, in International (PCT) Publication Nos. WO 95/09843; 98/09951; 98/09952; and 01/29013; and European Patent No. 983,999. All the processes provide amorphous nelfmavir mesylate.

Summary of the Invention The present inventors have found a new crystalline form of nelfmavir mesylate and have developed a process for preparation of the crystalline form. The new crystalline form of nelfmavir mesylate is designated as Form I.

In one general aspect there is provided a crystalline Form I of nelfmavir mesylate.

The Form I of nelfmavir mesylate may have the X-ray diffraction pattern of Figure 1, differential scanning calorimetry thermogram of Figure 2, and infrared spectrum of Figure 3.

In one general aspect there is provided a process for the preparation of Form I of nelfmavir mesylate. The process includes obtaining a solution of nelfmavir in one or more organic solvents; contacting the solution with methanesulphonic acid; and isolating the crystalline Form I of nelfinavir mesylate by the removal of the solvents. Removing the solvents may include, for example, one or more of filtration, filtration under vacuum, decantation, centrifugation, distillation and distillation under vacuum.

The process may include further drying the product obtained.

In another general aspect there is provided a pharmaceutical composition that includes a therapeutically effective amount of the crystalline Form I of nelfinavir mesylate; and one or more pharmaceutically acceptable carriers, excipients or diluents.

In another general aspect there is provided a method for treating HIV-I infections in a warm-blooded animal. The method includes providing a pharmaceutical composition to the

warm-blooded animal, the pharmaceutical composition comprising Form I of nelfmavir mesylate.

The present inventors also have developed a one-pot reaction for the preparation of nelfmavir. The present process does not involve the isolation of any intermediate, thereby reducing the work-up time as well as the cost of production. By following the present process, the yield of the final product nelfmavir, is also considerably improved.

In one general aspect there is provided a process for the preparation of nelfmavir or a salt thereof. The process includes: a) reacting a compound of Formula II or a salt thereof,

FORMULA II wherein R ₁ and R ₂ are independently hydrogen or amino protecting group with a proviso that both R ₁ and R ₂ are not hydrogen or R ₁ and R ₂ do not combine together to form a divalent amino protecting group, with 3-t-butylcarboxamide perhydroisoquinoline of Formula III,

FORMULA III get a compound of Formula IV,

FORMULA IV wherein R ₁ and R ₂ are as described above; b) deprotecting the compound of Formula IV to get a compound of Formula V;

FORMULA V

C) condensing the compound of Formula V with a compound of Formula VI,

FORMULA VI wherein R ₃ represents a hydroxy protecting group, to get protected nelfϊnavir;

d) deprotecting the hydroxy protecting group of the protected nelfmavir; and e) isolating the nelfmavir or a salt thereof, wherein all the above steps are carried out in- situ without isolation of any intermediate.

Embodiments of this process may include the deprotection being carried out in the presence of inorganic or organic bases. Removal of the hydroxy protecting group provides nelfmavir, which can be then converted to its salt.

The term "protecting group" in the present invention refers to those used in the art and serves the function of blocking the amino or hydroxyl groups while the reactions are carried out at other sites of the molecule. Suitable amino and hydroxy protecting groups are known to a person skilled in the art. Examples of hydroxyl and amino protecting groups include, but not limited to, lower alkylsilyl groups, lower alkoxymethyl groups, aralkyl groups, acyl groups, lower alkoxycarbonyl groups, alkenyloxycarbonyl groups and aralkyloxycarbonyl groups.

The details of one or more embodiments of the inventions are set forth in the description below. Other features, objects and advantages of the inventions will be apparent from the description and claims.

Description of the Drawings

Figure 1 is an X-Ray Diffraction Pattern of crystalline Form I of nelfmavir mesylate. Figure 2 is a Differential Scanning Calorimetric (DSC) thermogram of crystalline Form I of nelfϊnavir mesylate. Figure 3 is a Fourier Transform Infrared (FTIR) spectrum of crystalline Form I of nelfmavir mesylate.

Detailed Description of the Invention

The inventors have developed a process for the preparation of a new crystalline form of nelfmavir mesylate. More particularly, the inventors have developed a process for the preparation of a crystalline Form I of nelfmavir mesylate.

The term "Form I" of nelfinavir mesylate refers to a crystalline form of nelfinavir mesylate having X-ray diffraction pattern as depicted in Figure 1.

A first aspect of the invention provides a process for the preparation of crystalline Form I of nelfinavir mesylate wherein the process includes the steps of: a) obtaining a solution of nelfinavir in one or more organic solvents: b) contacting the solution with methanesulphonic acid; and c) isolating the crystalline Form I of nelfinavir mesylate by the removal of the solvents.

The inventors also have developed pharmaceutical compositions that contain the crystalline Form I of nelfinavir mesylate in admixture with one or more solid or liquid pharmaceutical diluents, carriers, and/or excipients.

In general, the solution of nelfinavir may be obtained by dissolving nelfinavir in a suitable solvent. Alternatively, such a solution may be obtained directly from a reaction in which nelfinavir is formed. The solvent containing nelfinavir may be heated to obtain a solution. It may be heated from about 3O ⁰ C to about reflux temperature of the solvent used, for example from about 5O ⁰ C to about 100 ⁰ C. It may be heated from about 10 minutes to about 24 hours. More particularly, it maybe heated for about 1-2 hours.

The nelfinavir which is used as the starting material can be prepared according to the process of the present invention or by method disclosed in any of the known processes, for example, processes as disclosed in International (PCT) Publication Nos. WO 95/09843; 98/09951; 98/09952; and 01/29013; and European Patent No. 983,999.

The term "obtaining" includes dissolving, slurrying, stirring or a combination thereof.

The term "suitable solvents" includes any solvent or solvent mixture in which nelfinavir can be solubilized, including, for example, alkanols, ketones, nitriles, chlorinated hydrocarbons, polar aprotic solvents, esters, ethers, or mixtures thereof. The alkanol may include one or more of methanol, ethanol, n-propanol, isopropanol and butanol. The ketone

may include one or more of acetone, ethyl methyl ketone, methyl isobutyl ketone, and diisobutyl ketone.

In general, the solution may be cooled to obtain Form I of nelfinavir mesylate. It may ^■ be cooled from about 5O ⁰ C to about 1O ⁰ C, for example from about 4O ⁰ C to about 2O ⁰ C. In one aspect, the solution may be seeded with crystals of Form I resulting in the precipitation of the Form I of nelfinavir mesylate and removing the solvent there from by filtration, filtration under vacuum, decantation or centrifugation.

The product obtained may be further or additionally dried to achieve the desired moisture values. For example, the product may be further or additionally dried in a tray drier, dried under vacuum and/or in a Fluid Bed Dryer.

In general, the crystalline Form I of nelfinavir mesyalte may be characterized by X-ray diffraction peaks at about 7.74, 9.54, 10.84, 11.16, 12.00, 12.40, 12.90, 13.42, 13.56, 15.40, 16.54, 17.26, 17.66, 18.14, 18.60, 19.20, 19.60, 20.08, 20.86, 21.82, 22.16, 22.96, 23.46, 23.84, 24.42, 25.04, 25.24, 25.70, 26.08, 26.24, 26.70, 27.08, 27.42, 27.98, 28.20, 28.58, 29.28, 29.40, 30.08, 30.22, 30.78, 31.82, 32.34, 32.76, 32.92, 33.44, 33.62, 34.32, 34.62, 34.72, and 36.12.

The crystalline Form I of nelfinavir mesylate may also be characterized by Differential Scanning Calorimetric (DSC) thermogram as depicted in Figure II having characteristic endothermic peak between 118 ⁰ C and 128 ⁰ C. The resulting crystalline Form I is pure and stable at normal and accelerated stability conditions.

The Form I of nelfinavir mesylate may be formulated into ordinary dosage forms such as, for example, tablets, capsules, pills, solutions, etc. In these cases, the medicaments can be prepared by conventional methods with conventional pharmaceutical excipients. The crystalline Form I of nelfinavir mesylate can be administered for the treatment of

HIV- 1 infections in combination with other antiretroviral agents, in a warm-blooded animal.

For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom possessed of a homeostatic mechanism and includes mammals and birds.

A second aspect of the invention provides a process for the preparation of nelfinavir or a salt thereof. The process includes: a) reacting a compound of Formula II or a salt thereof,

FORMULA II wherein R ₁ and R ₂ are independently hydrogen or amino protecting group with the provisos that both R ₁ and R ₂ are not hydrogen or R ₁ and R ₂ do not combine together to form a divalent amino protecting group, with 3-t-butylcarboxamide perhydroisoquinoline of Formula III,

FORMULA III get a compound of Formula IV,

FORMULA IV wherein R ₁ and R ₂ are as described above; b) deprotecting the compound of Formula IV to get a compound of Formula V;

FORMULA V

C) condensing the compound of Formula V with a compound of Formula VI,

FORMULA VI wherein R ₃ represents hydroxy protecting group, to get protected nelfmavir;

d) deprotecting the hydroxy protecting group of protected nelfinavir; and e) isolating the nelfinavir or a salt thereof from the reaction mixture thereof, wherein all the above steps are carried out in-situ without isolation of any intermediate.

The inventors have developed a one pot process for the preparation of nelfinavir or salts thereof by reacting a compound of Formula II or a salt thereof with 3-t- butylcarboxamide perhydroisoquinoline of Formula III to get a compound of Formula IV; deprotecting the compound of Formula IV to get a compound of Formula V; condensing the compound of Formula V with a compound, of Formula VI to get protected nelfinavir; deprotecting the hydroxy protecting group of the protected nelfinavir; and isolating the nelfinavir or a salt thereof from the reaction mixture thereof.

A solution of (2S,3R)-l,2-epoxy-3-N-(protected)-amino-4-phenylthiobutane of Formula II and 3-t-butylcarboxamide perhydroisoquinoline of Formula III is stirred in a suitable solvent, including, for example alkanols, ketones, polar aprotic solvents, chlorinated hydrocarbons, non-polar aprotic solvents, esters, ethers and mixtures thereof. It may be stirred from about 1 hour to about 24 hours at about 3O ⁰ C to about 100 ⁰ C and the reaction is monitored for formation of compound of Formula IV. An aqueous solution of a suitable deprotecting agent is added after completion of the reaction to deprotect the protected-amino group in the compound of formula IV to get a compound of formula V. The resulting suspension is heated from about 3O ⁰ C to reflux temperature for about 1-30 hours. Water and a suitable organic solvent, including, for example chlorinated hydrocarbons, non-polar aprotic solvents, esters, ethers and mixtures thereof is added to the reaction mixture. The organic layer is separated and treated with a base. A solution of 3-O-protected-2-methyl benzoyl chloride (compound of formula VI) in an organic solvent, including, for example chlorinated hydrocarbons, non-polar aprotic solvents, esters or ethers is added to the reaction mixture at about 5 ⁰ C to about 3O ⁰ C and stirred for about 0.5-1 hour. Water is added to the reaction mixture and the organic layer is separated. The solvent may be evaporated under vacuum at about 25 ⁰ C to about 4O ⁰ C to obtain O-protected nelfinavir. A suitable organic solvent, including, for example alkanols, ketones, polar aprotic solvents, chlorinated hydrocarbons, non-polar aprotic solvents, esters, and ethers is added to the resulting residue and treated with

a suitable deprotecting agent to deprotect the O-protected nelfinavir. The pH of the reaction mixture is adjusted to about 7-8. Water is charged and the resulting mass is heated to about 4O ⁰ C to about 7O ⁰ C, filtered, washed and dried to obtain nelfinavir free base. Nelfinavir free base may be converted to its salts. The term "deprotecting agent" in the present invention refers to those used in the art and serves the function of removing the protecting groups. Suitable deprotecting agents are known to a person skilled in the art. Examples of deprotecting agents include, but not limited to, inorganic and organic bases such as hydroxides, alkoxides, carbonates, or bicarbonates of alkali and alkaline earth metals, ammonia, triethylamine, dicyclohexylamine amine, diisopropylamine, and the like.

While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

Example 1: Preparation of nelfinavir free base A solution of (2S,3R)- 1 ,2-epoxy-3N-(benzyloxycarbonyl)-amino-4-phenylthiobutane (50 g) and 3-t-butylcarboxamide perhydroisoquinoline (39.9 g) in ethanol (100 ml) was stirred at 4O ⁰ C for 15 hours and the progress of the reaction was monitored (Thin Layer Chromatography/High Performance Liquid Chromatography). After the completion of reaction, a solution of sodium hydroxide (12.1 g) in water (12.1 ml) was added. The resulting suspension was heated to reflux for 20 hours. Water (250 ml) and dichloromethane (250 ml) were added and the mixture was stirred for 10 minutes and allowed to settle. The lower dichloromethane layer was collected. Triethylamine (42 ml) was added to the dichloromethane layer under stirring. This mixture was added drop wise to a solution of 3- acetoxy-2 -methyl benzoyl chloride (32.3 g) in dichloromethane (100 ml) at 5-25 ⁰ C in 30 minutes. The reaction mixture was stirred for 30 minutes and monitored (Thin Layer

Chromatography/High Performance Liquid Chromatography). Water (250 ml) was charged followed by stirring for 10 minutes. This mixture was allowed to settle for 5 minutes and the dichloromethane layer was collected. The solvent was evaporated at 35 ⁰ C under vacuum. To

the resulting paste, ethanol (100 ml) and a solution of sodium hydroxide (12.1 g) in water (12.1 ml) were added and the mixture was stirred for 2 hours and the reaction monitored (Thin Layer Chromatography/High Performance Liquid Chromatography). The pH of the mixture was adjusted to 7.5 by adding a mixture of acetic acid (15 ml) in water (15 ml). Water (750 ml) was charged and the resulting white mass was heated to 6O ⁰ C and stirred for 1 hour. The white solid was filtered and slurry washed with water. The solid so obtained was dried in air at 8O ⁰ C to afford nelfϊnavir free base.

Purity = 80% by HPLC

Example 2: Preparation of crystalline Form I of nelfinavir mesylate Nelfϊnavir obtained in Example 1 was suspended in acetone (600 ml) and the suspension heated to 55 ⁰ C. Methane sulphonic acid (14.2 g) was added and the reaction mixture was stirred for 15 minutes at 55 ⁰ C. Active carbon was charged at 55 ⁰ C with stirring for 15 minutes followed by filtration through hyflo. The resulting clear filtrate was stirred for 2.0 hours at room temperature and heated to reflux for 1 hour. After this period, heating was stopped and the reaction mixture was allowed to attain a temperature of 4O ⁰ C, filtered and slurry washed with acetone (50 ml x 2). The solid so obtained was dried under vacuum at 8O ⁰ C for 24 hours to obtain crystalline Form I of nelfinavir mesylate. Yield = 65 g

Purity = 99.5% by HPLC While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention. For example, the compounds described herein can be formulated into dosage forms that are suitable for administering to patients in need of the compound for treating a medical condition for which the compound is indicated, approved, or otherwise beneficial. Specifically, the Form I of nelfinavir can be formulated with one or more pharmaceutically acceptable excipients and/or with one or more active ingredients into a dosage form and administered to treat HIV-I infections.