WO2010143150A2

WO2010143150A2 - Process for stereoselective preparation of an intermediate of protease inhibitors

Info

Publication number: WO2010143150A2
Application number: PCT/IB2010/052585
Authority: WO
Inventors: Hariharan Sivaramakrishnan; Mita Roy; Abhay Atmaram Upare; Sushil Kumar Mishra; Santosh Crasta; Karuna Wankhede; Sandesh Vishwasrao
Original assignee: Piramal Healthcare Limited
Priority date: 2009-06-12
Filing date: 2010-06-10
Publication date: 2010-12-16
Also published as: WO2010143150A3

Abstract

The present invention provides a process for stereoselective preparation of N-[(1S,2R)-2- hydroxy-3-nitro-l-(phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroalcohol) comprising asymmetric reduction of the carbonyl group in [(1S)-3-nitro-2- oxo-1-(phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroketone) using sodium borohydride as the reducing agent in a solvent mixture of an alcohol and a halogenated solvent at a temperature ranging from -15°C to 00C to yield nitroalcohol in > 78 % yield and > 99 % chiral purity.

Description

PROCESS FOR STEREOSELECTIVE PREPARATION OF AN INTERMEDIATE OF PROTEASE INHIBITORS

FILED OF INVENTION

The present invention relates to a process for the preparation of N-[(lS,2R)-2-hydroxy-3- nitro-l-(phenylmethyl)propyl]carbamic acid 1 , 1 -dimethylethyl ester (hereinafter referred as Nitroalcohol), represented herein by formula I. More particularly, the present invention relates to an improved process for stereoselective preparation of the nitroalcohol of formula I, which is a key intermediate of protease inhibitors Amprenavir, Fosamprenavir and Darunavir.

BACKGROUND OF THE INVENTION

Amprenavir [(35')-tetrahydro-3-furyl-N-[(15',2i?)-3-(4-amino-N-isobutyl-benzene sulfonamido)-l-benzyl-2-hydroxypropyl] carbamate], having the following structure, is a protease inhibitor, which is used in the treatment of HIV (Human Immunodeficiency Virus) infection.

Amprenavir

A prodrug of Amprenavir, namely Fosamprenavir [[[(2i?,35')-l-[N-(2-methylpropyl)(4- aminobenzene)sulfonamido]-3-([[(35')-oxolan-3-yloxy]carbonyl]amino)-4-phenylbutan- 2-yl]oxy]phosphonic acid] is made commercially available by GlaxoSmithKline. Fosamprenavir calcium is an active ingredient of Lexiva® and Telzir®, the product currently used in the treatment of HIV infection. Fosamprenavir is structurally represented as follows:

Fosamprenavir

Darunavir, [N-[3-[N-(4-aminophenylsulfonyl)-N-isobutylamino]-l(S)-benzyl-2(R)- hydroxypropyl]carbamic acid (3Z?,3«S,6ai?)-perhydrofuro[2,3-b]furan-3-yl _est_er] is used in the treatment of HIV infection. Darunavir is an active ingredient of Prezista®, developed by Tibotec. Prezista is an OARAC (Office of AIDS Research Advisory Council) recommended treatment option for treatment-naϊve and treatment-experienced adults and adolescents. Darunavir is structurally represented as follows:

Darunavir

N-[(lS,2R)-2-hydroxy-3-nitro-l-(phenylmethyl)propyl]carbamic acid 1 , 1 -dimethylethyl ester, (nitroalcohol) represented by the following formula I, is a known intermediate of the protease inhibitors, Amprenavir, Fosamprenavir and Darunavir.

Formula I

There are several methods known in the art for the preparation of stereochemically pure nitroalcohol of formula I. Generally, preparation of stereochemically pure nitroalcohol involves asymmetric reduction of the carbonyl group in [(lS)-3-nitro-2-oxo-l- (phenylmethyl)propyl]carbamic acid 1 , 1 -dimethylethyl ester (nitroketone) represented by the following formula II, to obtain stereochemically pure nitroalcohol.

Formula II However, asymmetric synthesis may require expensive reagents or have other limitations, for instance, difficulty in separation of the undesired diastereomers from the resulting product for obtaining the desired diastereomer having good chiral purity and/or in good yield.

In Tetrahedron Letters, 1994, vol. no. 35, issue 33, page no. 6123-6126, a process for the preparation of nitroalcohol of formula I is disclosed. The process involves reaction of nitroaldol with nitromethane using optically active rare earth Li-BINOL catalyst, such as La-Li-(R)-BINOL complex, in the presence of tetrahydrofuran as the solvent at a temperature of -40⁰C for 72 hours to obtain nitroalcohol of formula I with 81% yield in a diastereomeric ratio of 96 : 4 [(1S,2R) : (1S,2S)] and with 90% diastereomeric excess. The process is schematically represented as follows:

complex , h

(lS,2R) Nitroalcohol (lS,2S) Nitroalcohol

Formula I

Although, the process provides the desired (IS, 2R) diastereomer of nitroalcohol in good yield and in a good diastereomeric excess, the process is disadvantageous, as: (i) it involves use of a large excess of nitromethane i.e. 20 equivalent, (ii) the process also involves use of optically active rare earth Li-BINOL catalyst, such as La-Li-(R)-BINOL complex, which is an expensive catalyst, thereby rendering the process costly and hence, is not a commercially viable process.

Japanese Patent Application No. 9249622 discloses a reaction of nitroketone of formula II with sodium borohydride as the reducing agent in the presence of titanium tetrachloride (TiCl₄) in 1,2-dimethoxyethane, as the solvent to yield 26% of the desired (1S,2R) diastereomer of nitroalcohol of formula I with 99% diastereomeric purity. Although, the process provides good diastereomeric purity, the yield of the desired (IS, 2R) diastereomer of nitroalcohol is very low. As the process uses titanium tetrachloride along with the solvent, the resulting nitroalcohol may contain traces of titanium. Moreover, titanium tetrachloride is highly hygroscopic in nature and thus, handling of titanium tetrachloride at an industrial scale is not feasible. Also, titanium tetrachloride being very expensive reagent, its use renders the process costly thereby making the process for the preparation of nitroalcohol commercially not viable.

US Patent No. 5,599,994 describes reduction of the carbonyl group in nitroketone of formula II using sodium borohydride as the reducing agent, in the presence of methanol as the solvent at a temperature of 0⁰C to obtain a white residue. The white residue is then dissolved in a mixture of water and ethyl acetate to form a mixture of aqueous and organic phases. The aqueous phase of the reaction mixture is then acidified using potassium hydrogen sulfate (KHSO₄) and the two phases formed are then separated by means of a separatory funnel to yield crude nitroalcohol of formula I. The crude nitroalcohol is a mixture of (1S,2R) and (1S,2S) diastereomers [also referred to as erythro and threo isomers respectively], which are then separated with the aid of flash chromatography using hexane/ethyl acetate, as the solvents to obtain the desired (IS, 2R) diastereomer as a minor fraction, with an yield of about 13% only and the undesired (1S,2S) diastereomer as a primary fraction, with the yield of about 37%. Moreover, the process requires cumbersome technique such as, flash chromatography for the separation of diastereomers, which results in only 13% yield of the desired (1S,2R) diastereomer of nitroalcohol and therefore commercially not viable.

US Patent No. 6,462,221 describes a process for the preparation of l-nitro-3-substituted- 3-amino-2-propanol (nitroalcohol) of formula I from l-nitro-3-substituted-3-amino-2- propanone (nitroketone) of formula II. The process comprises the steps of: (i) reduction of the carbonyl group in nitroketone using sodium borohydride as the reducing agent in the presence of methanol as the solvent at a temperature of 0⁰C to 5⁰C, (ii) the reaction mass is then acidified with 10% HCl to adjust pH of the reaction mass to 3, (iii) methanol is evaporated from the reaction mass, (iv) water is then added to the reaction mass followed by extraction with ethyl acetate to obtain crude l-nitro-3-substituted-3- amino-2-propanol diastereomers and (v) the desired (IS, 2R) diastereomer is separated from the crude product using simulated moving bed chromatography. This patent utilizes a single solvent specifically methanol in the process involving reduction of the carbonyl group in nitroketone using sodium borohydride. The process also necessitates use of simulated moving bed chromatography for the separation of two diastereomers, which is not only complex but also it, incurs high cost and therefore not viable for commercial application.

Chinese Patent Application No. 1891698 describes a process for the preparation of nitroalcohol of formula I, wherein the process involves reduction of the carbonyl group in nitroketone of formula II using sodium borohydride, as the reducing agent in the presence of methanol, as the solvent to yield nitroalcohol. It should be noted that all the aforementioned prior art documents teach a method involving reduction of the carbonyl group in nitroketone of formula II using sodium borohydride, as the reducing agent and a single solvent, such as methanol or 1,2- dimethoxyethane to obtain the corresponding nitroalcohol of formula I. It is evident from the above discussion of the prior art that the processes involving use of a single solvent such as methanol or 1,2-dimethoxyethane for reduction of the carbonyl group in nitroketone provides the desired (IS, 2R) diastereomer of nitroalcohol in poor yield and/or chiral purity. Moreover, all the processes employ cumbersome techniques for the separation of the desired (IS, 2R) diastereomer of nitroalcohol from the mixture of (1S,2R) and (1S,2S) diastereomers of nitroalcohol.

In addition to the afore cited prior art references, in a journal reference, Synthetic Communication, 1998, vol. 28, issue 3, page no. 395-401, preparation of nitroalcohol of formula I through reduction of the carbonyl group in nitroketone of formula II, is reported. The process described in the journal reference involves reduction of the carbonyl group in nitroketone using sodium borohydride, as the reducing agent in the presence of a solvent mixture of methanol and tetrahydrofuran at a temperature of 0⁰C, followed by acidification using 10% potassium hydrogen sulfate (KHSO₄) to obtain a mixture of (1S,2R) and (1S,2S) diastereomers of nitroalcohol in a diastereomeric ratio of 83 : 17 [(1S,2R) : (1S,2S)]. The mixture of diastereomers of nitroalcohol was separated by means of silica gel chromatography using n-hexane/ethyl acetate, as the solvents and subsequently recrystallized from isopropyl alcohol (IPA) to yield merely 12.3% of pure (1S,2R) diastereomer of nitroalcohol. Whereas, the yield of the undesired (1S,2S) diastereomer of nitroalcohol is 69%. Eventhough the process teaches use of a mixture of solvents, such as methanol and tetrahydrofuran during the reduction of the carbonyl group in nitroketone, the yield of the desired (IS, 2R) diastereomer of nitroalcohol is considerably low i.e. only 12.3%. Also the process is disadvantageous as it necessitates use of silica gel chromatography for separation of the diastereomers, which requires large volume of solvents. Therefore, the use of such a process which provides the desired (IS, 2R) diastereomer of nitroalcohol in poor yield and also involving time consuming method for the separation of diastereomers is certainly a drawback for industrial application.

The process of producing nitroalcohol of formula I, the key intermediate of protease inhibitors, Amprenavir, Fosamprenavir and Darunavir, can be improved particularly in terms of cost, by providing a stereoselective synthesis that would result in substantially stereochemically pure (IS, 2R) diastereomer of nitroalcohol with good yield. As described in the cited prior art references, the processes for the preparation of nitroalcohol utilize either single solvent or a mixture of solvents coupled with complicated methods for the separation of diastereomers. Although useful, such processes are costly as they require large volume of solvents and are time consuming. Moreover, the prior art processes result in low yield of the desired diastereomer of nitroalcohol.

The inventors of the present invention have now found that nitroalcohol of formula I can be obtained in good yield and substantial diastereomeric purity from nitroketone of formula II through an improved process, which although involves use of a mixture of solvents, avoids cumbersome techniques for the separation of the diastereomers. Thus, the present invention provides a simple, cost-effective and industrially viable process for the preparation of nitroalcohol, a key intermediate of Amprenavir, Fosamprenavir and Darunavir, the protease inhibitors useful in the treatment of HIV infections.

OBJECTS OF THE INVENTION

An object of the present invention is to provide a process for the stereoselective preparation of N-[(lS,2R)-2-hydroxy-3-nitro-l-(phenylmethyl)propyl]carbamic acid 1,1- dimethylethyl ester (nitroalcohol) of formula I from [(lS)-3-nitro-2-oxo-l- (phenylmethyl)propyl]carbamic acid 1 , 1 -dimethylethyl ester (nitroketone) of formula II.

Another object of the present invention is to provide an improved process for the stereoselective preparation of nitroalcohol of formula I in > 78 % yield and > 99 % chiral purity. Yet another object of the present invention is to provide a process for the stereoselective preparation of nitroalcohol of formula I which utilizes a simple purification method.

Further object of the present invention is to provide a simple, cost-effective and industrially applicable process for the stereoselective preparation of nitroalcohol of formula I.

Yet further object of the present invention is to provide a process for the stereoselective preparation of nitroalcohol of formula I substantially free of the undesired (IS, 2S) diastereomer and enantiomers.

STATEMENT OF INVENTION

In accordance with the objects of the present invention, there is provided a process for the stereoselective preparation of N-[(lS,2R)-2-hydroxy-3-nitro-l-(phenylmethyl)propyl] carbamic acid 1,1-dimethylethyl ester (nitroalcohol) of formula I, comprising the steps of:

(a) asymmetric reduction of the carbonyl group in [(lS)-3-nitro-2-oxo-l- (phenylmethyl)propyl] carbamic acid 1,1-dimethylethyl ester (nitroketone) of formula II using sodium borohydride as the reducing agent in a solvent mixture of an alcohol and a halogenated solvent at a temperature ranging from -15°C to 0⁰C to obtain the nitroalcohol,

(b) purification of the nitroalcohol obtained in step (a) using a halogenated solvent to yield substantially pure nitroalcohol. The process of the present invention is depicted in the following scheme, Step (a)

Formula II

(Nitroketone)

( ^VIS,^> 2S) ^/ diastereomer _r [_Pure dji-as .tereomer o ,f , N,i.,troa ,lco ,ho ,l_π]

%

In accordance with another aspect of the present invention, the desired (IS, 2R) diastereomer of nitroalcohol of formula I is obtained in substantially pure form i.e. having a chiral purity of > 99 % and with yield of > 78 %.

In accordance with yet another aspect of the present invention, the process of the present invention overcomes the disadvantages associated with the processes described in the cited prior art, which concerns with the use of cumbersome purification techniques such as, silica gel chromatography, flash chromatography and simulated moving bed chromatography for the separation of the desired (IS, 2R) diastereomer of nitroalcohol of formula I from the mixture of (1S,2R) and (1S,2S) diastereomers of nitroalcohol, formed during asymmetric reduction of the carbonyl group in nitroketone of formula II.

In accordance with yet another aspect of the present invention, the process for the preparation of nitroalcohol of formula I provides the desired (IS, 2R) diastereomer of nitroalcohol with good chiral purity and good yield without the use of expensive and lengthy separation techniques, thereby making the process for the preparation of nitroalcohol simple, cost-effective and industrially applicable.

DESCRIPTION OF THE INVENTION

The present invention relates to a process for the stereoselective preparation of N- [(lS,2R)-2-hydroxy-3-nitro-l-(phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroalcohol) of formula I

OH

Formula I

comprising the steps of: a) asymmetric reduction of the carbonyl group in [(lS)-3-nitro-2-oxo-l-

(phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroketone) of formula II using sodium borohydride as the reducing agent in a solvent mixture of an alcohol and a halogenated solvent at a temperature ranging from -15°C to 0⁰C to obtain the nitroalcohol;

Formula II

b) purification of nitroalcohol obtained in step (a) using a halogenated solvent to yield substantially pure nitroalcohol. In an embodiment of the present invention, the nitroketone of formula II is reacted with sodium borohydride to reduce the carbonyl group in the nitroketone to hydroxy group.

In accordance with the present invention, sodium borohydride is used in an amount of 0.25 to 1.0 molar equivalents based on the nitroketone of formula II. Preferably, 0.4 to 0.8 molar equivalents of sodium borohydride is used with respect to the nitroketone.

In another embodiment of the present invention, the step (a) in the process involving asymmetric reduction of the carbonyl group in nitroketone of formula II is carried out using a mixture of solvents selected from an alcohol and a halogenated solvent.

In accordance with the present invention, the alcohol used in step (a) of the process is methanol.

In accordance with the present invention, in step (a) of the process 2.5 volume of the alcohol is used based on the weight of nitroketone of formula II.

In accordance with the present invention, the halogenated solvent used in the step (a) of the process is selected from methylene dichloride and ethylene dichloride. Preferably, methylene dichloride is used as the halogenated solvent.

In accordance with the present invention, in step (a) of the process 2.5 to 5 volume of the halogenated solvent is used based on the weight of nitroketone of formula II.

In accordance with the present invention, the asymmetric reduction of the carbonyl group in nitroketone of formula II is carried out in a mixture of an alcohol and a halogenated solvent in a volume ratio of about 1 : 1 to about 1 : 2. Preferably, the mixture of alcohol and halogenated solvent is used in a volume ratio of 1: 1.4.

In accordance with the present invention, the asymmetric reduction of the carbonyl group in nitroketone of formula II is preferably carried out at a temperature of -10⁰C to -5°C. In accordance with the present invention, a substantially pure nitroalcohol of formula I relates to the nitroalcohol having > 99 % chiral purity.

Moreover, the nitroalcohol of formula I obtained using the process of the present invention contains < 0.1 % of the undesired (1R,2R) and (1R,2S) enantiomers of the nitroalcohol. Thus, the process is advantageous as it would further aid in providing the final product, i.e. Amprenavir, Fosamprenavir or Darunavir, the protease inhibitors, in higher purity starting from the substantially pure nitroalcohol containing < 0.1 % of the undesired (1R,2R) and (1R,2S) enantiomers of the nitroalcohol.

The inventors of the present invention performed numerous experiments for the preparation of nitroalcohol of formula I from the nitroketone of formula II involving asymmetric reduction of the carbonyl group in nitroketone using sodium borohydride as the reducing agent in the presence of a mixture of two solvents taken in varying ratios at various temperature conditions. Results of the experiments are presented in Table- 1, which is included in the experimental section. Table- 1 specifically presents results of the effect of different mixtures of two solvents such that one of the solvent is an alcohol e.g. methanol, used in varying ratios in reduction of the carbonyl group in nitroketone. From the results presented in the Table- 1, it can be seen that the best results in terms of the in- process diastereomeric ratio of 85 : 15 [(1S,2R) : (1S,2S)] of nitroalcohol is obtained when the reduction of the carbonyl group in nitroketone is carried out in a mixture of methanol and methylene dichloride in the volume ratio of 1 : 1.4 at a temperature of -10⁰C to -5°C. On the basis of this result it can be said that the process of the present invention for the preparation of nitroalcohol is advantageous over the known methods disclosed in prior art.

In another embodiment of the present invention, the nitroalcohol of formula I obtained in step (a) is further subjected to purification as indicated in step (b). Purification of the nitroalcohol results in further improvement of the diastereomeric ratio and chiral purity of the desired (1S,2R) diastereomer of nitroalcohol. In accordance to the present invention, the purification step (b) is carried out using a halogenated solvent selected from methylene dichloride and ethylene dichloride. Preferred halogenated solvent used in the purification step is methylene dichloride.

In accordance with the present invention, the starting material nitroketone of formula II is dissolved in a mixture of an alcohol such as methanol and a halogenated solvent such as methylene dichloride or ethylene dichloride to obtain a clear solution. The reaction mixture is then cooled to a temperature ranging from -15°C to 0⁰C. To this cooled reaction mixture, the reducing agent sodium borohydride is charged to reduce the carbonyl group in the nitroketone to hydroxy group. During addition of sodium borohydride, hydrogen gas is evolved making the reaction exothermic, as a result the temperature of reaction increases. Therefore, sodium borohydride is charged in lots maintaining the temperature of the reaction mass below -5⁰C over a period of 2 to 3 hours. At this stage of the reaction, conversion of nitroketone to nitroalcohol of formula I is monitored using HPLC analysis method 2 - achiral column, which is included in the experimental section. The in-process diastereomeric ratio is in the range of 82 : 18 to 89 : U [(1S,2R) : (1S,2S)].

The reaction mixture is then acidified using a suitable acid to reduce the pH of the reaction mixture to a sufficiently low value to prevent significant enolate formation and to avoid cleavage of the protecting group, for example tert-butyloxycarbonyl (BOC). In a preferred embodiment, previously cooled 5% aqueous solution of potassium hydrogen sulfate (KHS O₄) is used to acidify the reaction mixture. The reaction mixture is then stirred for about 30 minutes at a temperature of about 5⁰C to 10⁰C to precipitate nitroalcohol of formula I. The resulting nitroalcohol is then filtered and washed with the previously cooled solution of 20% methylene dichloride in heptane to obtain the nitroalcohol in a diastereomeric ratio of about 95 : 5 to 98 : 2 [(1S, 2R) : (1S,2S)] and chiral purity > 97 %, using HPLC analysis method 1 - chiral column. And when HPLC analysis is carried out using method 2 - achiral column, the diastereomeric ratio is in the range of about 94 : 4 to 97 : 3 [(1S,2R) : (1S,2S)]. The nitroalcohol of formula I as obtained above is sufficiently pure to use it as such in the next step, but if the desired (1S,2R) diastereomer of nitroalcohol is required with a chiral purity > 99 %, then it is desirable to include an additional step of purification to obtain the desired (IS, 2R) diastereomer of nitroalcohol having desired purity. The resulting nitroalcohol is stirred with a halogenated solvent such as methylene dichloride to obtain a slurry. The reaction mixture is then heated to reflux temperature and maintained at this temperature for another 1 hour. After 1 hour the reaction mixture is cooled to 25⁰C to 30⁰C and simultaneously further cooled to 0⁰C to 15°C, the reaction mixture is maintained at this temperature for another 30 to 45 minutes. The product obtained is filtered and then washed with previously cooled heptane to yield substantially pure nitroalcohol having chiral purity > 99 %.

It is thus possible by the way of the present invention to achieve the much desired synthesis for the preparation of nitroalcohol of formula I enriched in its desired (IS, 2R) diastereomer.

The starting material of the process, [(lS)-3-nitro-2-oxo-l-

(phenylmethyl)propyl]carbamic acid 1 , 1 -dimethylethyl ester (nitroketone) of formula II is a known compound and can be prepared by a person skilled in the art by following methods described in the literature. For example, N-tert-butyloxycarbonyl-L- phenylalanine and a solution of l,l'-carbonyldiimidazole in dry tetrahydrofuran is stirred at room temperature to obtain carbonyldiimidazole tert-butoxycarbonylamino- phenylalanine solution, which on further treatment with nitromethane in the presence of potassium tert-butoxide yields nitroketone, as described in Synthetic Communication, 1998, vol. no. 28, issue 3, page no. 395-401, which is incorporated herein by reference.

As previously discussed, the compound of formula I, N-[(lS,2R)-2-hydroxy-3-nitro-l- (phenylmethyl)propyl]carbamic acid 1,1 -dimethylethyl ester (Nitroalcohol), which is product of the present invention is finally converted into Amprenavir [(SS^-tetrahydro-S- furyl-N-[(15',2i?)-3-(4-amino-N-isobutylbenzenesulfonamido)-l-benzyl-2-hydroxypropyl] carbamate], Fosamprenavir [[(2i?,35')-l-[N-(2-methylpropyl)(4-aminobenzene) sulfonamido]-3-([[(35')-oxolan-3-yloxy]carbonyl]amino)-4-phenylbutan-2-yl]oxy] phosphonic acid], or Darunavir [N-[3-[N-(4-aminophenylsulfonyl)-N-isobutylamino]- l(S)-benzyl-2(R)-hydroxypropyl]carbamic acid (3R,3aS,6aR)-perhydmfum[2,3-b]fumn- 3-yl ester], a protease inhibitor and antiretro viral drug. The nitroalcohol obtained by the process of the present invention may be converted to Amprenavir by following the process described in the cited prior art, Chinese Patent Application No. 1891698, which is incorporated herein by reference. Mainly the nitroalcohol may be treated with a reducing agent, followed by reaction with isobutyl chloride and 4-nitrobenzenesulfonyl chloride to obtain an intermediate, which on treatment with (S)-3-hydroxytetrahydrofuran and catalytic reduction of the resulting compound, may yield Amprenavir.

Further, the nitroalcohol of formula I obtained by the process of the present invention may be converted to another protease inhibitor, Darunavir by following one or more processes known in the prior art. For instance, the process for the preparation of Darunavir may involve the steps of : (i) reducing the nitroalcohol of formula I may be to the corresponding aminoalcohol using an appropriate reducing agent e.g., palladium on charcoal, palladium hydroxide or Raney Nickel, (ii) the resulting aminoalcohol may then be treated with isopropyl amine to obtain 3S-[N-(t-butoxycarbonyl)amino]-l-(2- methylpropyl)amino-4-phenylbutan-2R-ol, as per the method described in US Patent No. 6372778; and (iii) the resulting compound may then be condensed with p-methoxybenzenesulfonyl chloride in the presence of sodium bicarbonate and dichloromethane to yield an intermediate, which may be further condensed with (3i?,3a5',6a/?)-3-hydroxyhexahydrofuro[2,3-b]furanyl succinimidyl carbonate using 30% trifluoroacetic acid in dichloromethane solution to obtain Darunavir, as per the method described in the published International Patent Publication No. WO 2008/133734.

The following examples which fully illustrate the practice of the preferred embodiments of the present invention are intended to be for illustrative purpose only and should not be construed in anyway to limit the scope of the present invention. EXAMPLES

Example 1 : Preparation of Nitroalcohol

To a 5 liter round bottom flask, 320 gm (1.039 moles) of nitroketone, in 800 ml of methanol and 1120 ml of methylene dichloride was charged and stirred to obtain a clear solution. The reaction mixture was cooled to -10⁰C to -5°C. To this cooled solution 25.56 gm (0.68 moles) of sodium borohydride was charged in lots maintaining the temperature of the reaction below -5⁰C over a period of 2 hours. (At this stage reaction completion is monitored using HPLC analysis method 2 - achiral column. The in-process diastereomeric ratio is 85 : 15 [(1S,2R) : (1S,2S)]). The reaction mixture was then added to the previously cooled 5% potassium hydrogen sulfate solution under stirring and further stirred for 15-30 minutes. Then the reaction mixture was cooled to 5°C. The resulting solid precipitate was filtered and washed with 20% methylene dichloride solution in heptane. The product was dried under vacuum to yield 249.6 gm of nitroalcohol. The diastereomeric ratio and chiral purity of nitroalcohol are monitored using HPLC. Yield - 78 %

Using HPLC analysis method 1 - chiral column Diastereomeric ratio - 98.08 : 1.98 [(1S,2R) : (1S.2S)] Chiral purity - 97.93 %

Using HPLC analysis method 2 - achiral column Diastereomeric ratio - 97 : 3 [(1S,2R) : (1S,2S)]

Example 2 : Preparation of Nitroalcohol To a 5 liter round bottom flask, 320 gm (1.039 moles) of nitroketone in 800 ml of methanol and 1120 ml of methylene dichloride was charged and stirred to obtain a clear solution. The reaction mixture was cooled to -10⁰C to -5°C. To this cooled solution 27.52 gm (0.73 moles) of sodium borohydride was charged in lots maintaining the temperature of the reaction below -5⁰C over a period of 2 hours. (At this stage reaction completion is monitored using HPLC analysis method 2 - achiral column. The in-process diastereomeric ratio is 84 : 16 [(1S, 2R) : (1S,2S)]). The reaction mixture was then added to the previously cooled 5% potassium hydrogen sulfate solution under stirring and further stirred for 15-30 minutes. Then the reaction mixture was cooled to 5°C. The resulting solid precipitate was filtered and washed with 20% methylene dichloride solution in heptane. The product was dried under vacuum to yield 253 gm of nitroalcohol. The diastereomeric ratio and chiral purity of nitroalcohol are monitored using HPLC. Yield - 79.1 %

Using HPLC analysis method 1 - chiral column Diastereomeric ratio - 95.02 : 4.98 [(1S,2R) : (1S,2S)] Chiral purity - 93 %

Using HPLC analysis method 2 - achiral column Diastereomeric ratio - 94 : 6 [(1S,2R) : (1S,2S)]

Reference Example 3 : Preparation of Nitroalcohol The inventors of the present invention carried out the process for the preparation of nitroalcohol of formula I from nitroketone of formula II as per the method described in Synthetic Communication, 1998, vol. no. 28, issue 3, page no. 395-401, cited herein as reference. However, the inventors found that by following the procedure as described in said reference, the nitroalcohol was obtained with in-process diastereomeric ratio of 75 : 25 [(1S,2R) : (1S,2S)], using HPLC analysis method 2 - achiral column. Said process carried out by the inventors is illustrated below:

(2S,3S)-3-tert-butoxycarbonylamino-2-hydroxy-l-nitro-4-phenylbutane (6a) and

(2R,3S)-isomer (6b) (as per reference Synthetic Communication, 1998, vol. no. 28, issue 3, page no. 395-401): The compound 6b corresponds to (1S,2R) diastereomer of nitroalcohol of formula I of the present invention:

To a solution of (3S)-3-tert-butoxycarbonylamino-l-nitro-2-oxo-4-phenylbutane (5 gm) in methanol (25 ml) and tetrahydrofuran (5 ml), sodium borohydride (0.1623 gm) was added dropwise at 0⁰C over 1 hour. The reaction mixture was stirred for 1 hour at 0⁰C and then 10% KHSO₄ (potassium hydrogen sulfate) (23 ml) was added. The organic layer was extracted with AcOEt (ethyl acetate) (27 ml), washed with brine and dried with MgSO₄ (magnesium sulfate). The solvent was evaporated to give 6 [6 is a mixture of (2S,3S) and (2S,3R) diastereomer] . The diastereomeric ratio using HPLC analysis method 2 - achiral column is 75 : 25 [(1S.2R) : (1S.2S)].

In the above described reference example 3, the diastereomeric ratio [(1S,2R) : (1S,2S)] of the nitroalcohol of formula I after its isolation from the reaction mixture remains the same i.e. 75 : 25 [(1S,2R) : (IS, 2S)] as that of the in-process diastereomeric ratio of the nitroalcohol before its isolation from the reaction mixture.

In the above described process of said reference, 6a corresponds to the undesired (IS, 2S) diastereomer of nitroalcohol of formula I of the present invention and 6b corresponds to the desired (IS, 2R) diastereomer and 6 corresponds to a mixture of (IS, 2R) and (IS, 2S) diastereomers of nitroalcohol.

Reference Examples 4 to 10

It has been indicated herein above that the inventors of the present invention performed numerous experiments (reference examples) to obtain nitroalcohol of formula I involving reduction of nitroketone of formula II using sodium borohydride, as the reducing agent. The examples are referred to herein as reference examples 4 to 10 which are carried out by following the procedure as described in Example 1 above. The reduction reaction pertaining to the reference examples was carried out under different reaction conditions particularly, using different mixtures of solvents at varying ratios and at a different temperature conditions. The reaction mixture is monitored using HPLC analysis method 2 - achiral column to check in-process diastereomeric ratio of the nitroalcohol of formula I.

The results are presented in the following Table- 1. For the sake of comparison, the results of the present invention, Example 1 and 2 and reference Example 3 (from the cited prior art reference, Synthetic Communication, 1998, vol. no. 28, issue 3, page no. 395-401) are also presented in Table 1. Table-1

Ex. No. - Example No. Vol. - Volume ratio of solvents dr ratio - In-process diastereomeric ratio [(1S,2R) : (IS, 2S)]

The results presented in the above Table 1 are discussed herein below:

• As indicated in Example No. 4, when the reduction of nitroketone of formula II is carried out at the reaction temperature of 0⁰C using only methanol as the solvent, it does not result in significant improvement in the in-process diastereomeric ratio

(with respect to the reference example no. 3).

• As indicated in Example No. 5, the reduction of nitroketone of formula II when carried out at a reaction temperature of 0⁰C in a solvent mixture of methanol and tetrahydrofuran such that, the volume ratio of methanol to tetrahydrofuran is 1 : 5 i.e. the volume of methanol in the mixture is decreased and the volume of tetrahydrofuran is increased (with reference to reference example no. 3), it results in significant decrease in the in-process diastereomeric ratio (with respect to the reference example no. 3). • As described in Example No. 6, when the reduction of nitroketone of formula II is carried out at a reaction temperature of 0⁰C in a solvent mixture of methanol and tetrahydrofuran such that the volume ratio of methanol to tetrahydrofuran is 5 : 2 i.e. the volume of tetrahydrofuran is increased trivially, whereas the volume of methanol in the mixture is maintained (with respect to the reference example no.

3), it does not result in considerable increase in the in-process diastereomeric ratio (with respect to reference example no. 3).

• As indicated in Example No. 7, the reduction of nitroketone of formula II when carried out at a decreased temperature of -10⁰C to -5°C in a solvent mixture of methanol and tetrahydrofuran such that the volume ratio of methanol to tetrahydrofuran is maintained to 5 : 1 (with respect to reference example no. 3), it results in marginal increase in the in-process diastereomeric ratio (with respect to reference example no. 3).

• On the other hand, when the reduction of nitroketone of formula II is carried out at an increased temperature of about 10⁰C to 15⁰C in a solvent mixture of methanol and tetrahydrofuran such that the volume ratio of methanol to tetrahydrofuran is maintained to 5 : 1 (with respect to reference example no. 3), it results in marginal increase in the in-process diastereomeric ratio, as indicated in Example No. 8 (with respect to the reference example no. 3). • As indicated in Example No. 9, when the reduction of nitroketone of formula II is carried out at an increased temperature of about 25⁰C to 30⁰C in a solvent mixture of methanol and tetrahydrofuran such that the volume ratio of methanol to tetrahydrofuran is maintained to 5 : 1 (with respect to reference example no. 3), it does not result in any change in the in-process diastereomeric ratio (with respect to reference example no. 3).

• As indicated in Example No. 10, when the reduction of nitroketone of formula II is carried out at a temperature of 0⁰C in a solvent mixture of methanol and toluene such that the volume ratio of methanol to toluene is 1 : 5 i.e. from the mixture of methanol and tetrahydrofuran (with respect to reference example no. 5) the solvent tetrahydrofuran is replaced with toluene and the volume of methanol in the mixture is maintained (with respect to reference example no. 5), it does not result in significant improvement in the in-process diastereomeric ratio (with respect to reference example no. 5).

• It is evident from the example no. 1 and 2, that when the reduction of nitroketone of formula II is carried out at a decreased reaction temperature of -10⁰C to -5⁰C in a solvent mixture of methanol and methylene dichloride such that in the solvent mixture of methanol and tetrahydrofuran (with respect to example no. 3), the solvent tetrahydrofuran is replaced with a halogenated solvent such as methylene dichloride and further varying the volume ratio of methanol to methylene dichloride in the solvent mixture to 1 : 1.4 (with respect to reference Example No. 3), it results in significant increase in the in-process diastereomeric ratio i.e. 85 :

15 [(1S, 2R) : (1S,2S)] of nitroalcohol of formula I (with respect to reference

Example No. 3).

PURIFICATION OF NITROALCOHOL Example 11

In a 5 liter round bottom flask 240 gm (0.77 moles) of nitroalcohol (from example 2) in 1440 ml of methylene dichloride was charged. The resulting slurry was heated to reflux temperature for 1 hour. The reaction mixture was cooled to 25⁰C to 30⁰C and further cooled to 0⁰C to 5⁰C, and the reaction mixture was maintained at this temperature for another 45 minutes. The resulting product was filtered and washed with previously cooled heptane. The product was dried under vacuum to yield 226 gm of pure nitroalcohol. The diastereomeric ratio and chiral purity of nitroalcohol are monitored using HPLC. Yield - 94.2 %

Using HPLC analysis method 1 - chiral column Diastereomeric ratio - 98.47 : 1.53 [(1S.2R) : (1S.2S)] Chiral purity - 98.3 % Using HPLC analysis method 2 - achiral column Diastereomeric ratio - 98 : 2 [(1S.2R) : (1S.2S)] Example 12

In a 100 ml round bottom flask 5 gm (16.1 mmol) of nitroalcohol (from example 2) in 50 ml of methylene dichloride was charged, the resulting slurry was heated to reflux temperature for 1 hour. The reaction mixture was cooled to 25⁰C to 30⁰C and further cooled to 10⁰C to 15⁰C, and the reaction mixture was maintained at this temperature for another 30 minutes. The resulting product was filtered under vacuum and washed with previously cooled heptane. The product was dried under vacuum to yield 4 gm of pure nitroalcohol. The diastereomeric ratio and chiral purity of nitroalcohol are monitored using HPLC. Yield - 80 %

Using HPLC analysis method 1 - chiral column Diastereomeric ratio - 99.39 : 0.61 [(1S,2R) : (1S,2S)] Chiral purity - 99.17 % Using HPLC analysis method 2 - achiral column Diastereomeric ratio - 99.35 : 0.65 [(1S.2R) : (1S.2S)]

DETAILS FOR HPLC ANALYSIS: Method 1 - chiral column Column : Chiralcel OD-H, 250mm

Make : Diecel Chemical Industries

Mobile Phase:

Mixed solution A: Combine 800ml of n-Hexane, 200ml of tert-butyl methyl ether and 2ml of trifluoroacetic acid in a suitable container and mix well.

Final composition: Transfer 960ml of mixed solution A to a container and add 40 ml of ethanol, mix well. Allow to equilibrate to room temperature.

X_1113x :215 nm

Flow rate : 0.5 ml/min Temperature : Ambient

Run time : 30 minutes Injection volume : 20 μl

Diluent : Mobile phase

Method 2 - achiral column

Column : Eclipse XDB C8, (150mm x 4.6mm, 5μm)

Make : Agilent (Part No. - 993967-906) Mobile phase : A - Buffer

B - Acetonitrile Buffer solution - Dissolve 0.77g of ammonium acetate in 1000 ml of water and adjust pH 4.5 (+ 0.05) with acetic acid. X_1113x : 215 nm Flow rate : 1.0 ml/min Temperature : Ambient Run time : 32 minutes Injection volume : lOμl Diluent : Water : acetonitrile (1 : 1)

Claims

WE CLAIM:

1. A process for the stereoselective preparation of N-[(lS,2R)-2-hydroxy-3-nitro-l- (phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroalcohol) of formula I,

OH

Formula I comprising the steps of,

(a) asymmetric reduction of the carbonyl group in [(lS)-3-nitro-2-oxo-l- (phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroketone) of formula II,

Formula II using sodium borohydride as the reducing agent in a mixture of solvents selected from an alcohol and a halogenated solvent at a temperature ranging from -15°C to 0⁰C to obtain nitroalcohol of formula I; (b) purification of nitroalcohol obtained in step (a) using a halogenated solvent to yield substantially pure nitroalcohol of formula I.

2. The process as claimed in claim 1, wherein said alcohol and said halogenated solvent are used in a volume ratio of about 1 : 1 to 1 : 2.

3. The process as claimed in claim 1 or 2, wherein said alcohol and said halogenated solvent are used in a volume ratio of 1 : 1.4.

4. The process as claimed in any one of the preceding claims 1 to 3, wherein said alcohol is used in a volume of 2.5 based on the weight of nitroketone of formula II.

5. The process as claimed in any one of the preceding claim 1 to 3, wherein said halogenated solvent used in step (a) is used in a volume of 2.5 to 5 based on the weight of nitroketone of formula II.

6. The process as claimed in any one of the preceding claims 1 to 4, wherein said alcohol is methanol.

7. The process as claimed in any one of the preceding claims 1 to 3 and 5, wherein said halogenated solvent used in step (a) is selected from methylene dichloride and ethylene dichloride.

8. The process as claimed in claim 7, wherein said halogenated solvent used in step (a) is methylene dichloride.

9. The process as claimed in claim 1, wherein sodium borohydride is used in an amount of 0.25 to 1.0 molar equivalent based on the nitroketone of formula II.

10. The process as claimed in claim 9, wherein sodium borohydride is used in an amount of 0.4 to 0.8 molar equivalent based on the nitroketone of formula II.

11. The process as claimed in any one of the preceding claims 1 to 10, wherein step (a) is carried out at a temperature ranging from -10⁰C to -5°C.

12. The process as claimed in claim 1 , wherein said halogenated solvent used in step (b) is methylene dichloride.

13. A process for the stereoselective preparation of N-[(lS,2R)-2-hydroxy-3-nitro-l- (phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester (nitroalcohol) as herein described with reference to examples 1, 2, 11 and 12.