PROCESS FOR PREPARING AN INTERMEDIATE OF BOC-CORE VIA
ASYMMETRIC REDUCTION

FILED OF THE INVENTION

The present application relates a process for the preparation of an intermediate of Boc-core or a salt thereof via asymmetric reduction.
BACKGROUND Boc-core is an abbreviation for a compound having a chemical name (2S,3S,5S)-2-amino-3-hydroxy-5-(t-butyloxycarbonyl)amino)-1,6-diphenylhexane, and represented by structural formula (I).

(I) Compounds of formula (I), and salts thereof, are useful as intermediates for the manufacture of HIV protease inhibitors, such as lopinavir and ritonavir.
Ritonavir, the active ingredient in products sold as NORVIR™ by Abbott
Laboratories, has a chemical name 10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]3-6-dioxo-8,11 -bis(phenylmethyl)-2,4,7,12-tetraazatridecan-
13-oic acid, 5-thiazolylmethyl ester, [5S-(5R*,8R*,10R*,11R*)], and the following structural formula:

Lopinavir, an active ingredient (in combination with ritonavir) in products sold as KALETRA™ by Abbott Laboratories, has a chemical name [1S-[1R*,(R*).3R*,4R*]]-N-[4-[[(2,6-dimethylphenoxy)acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H)-pyrimidine acetamide, and has the following structural formula:

U.S. Patent No. 5,616,776 discloses a process for the preparation of a substituted 2, 5-diamino-3-hydroxyhexane, such as Boc-core, by reducing (S)-2-t-butyloxycarbonylamino-5-N,N-dibenzylamino-1,6-diphenyl-4-oxo-2-hexene, followed by deprotecting the resultant intermediate to give Boc-core. International Application Publication No. WO 2006/090264 A1 discloses an in situ process for the preparation of 2-amino-5-protected amino-3-hydroxy-1,6-diphenylhexane compounds, or acid addition salts thereof.

There remains a need to provide simple, cost effective, and commercially viable processes for the preparation of Boc-core.

SUMMARY

In an aspect, the present application provides processes for the preparation of a compound of formula (I) or a salt thereof, embodiments of which include one or more of the following steps, individually or in the sequence recited:

a) reacting a compound of formula (II) with a compound of formula (III) in the presence of a reagent to provide nitro ketone of formula (IV):

or a salt thereof; wherein R is a N-protecting group such as formyl, acetyl, t-butylacetyl, pivaloyl, benzoyl, trifluoroacetyl, 4-nitrobenzoyl, benzyl, benzyloxymethyl, benzyloxycarbonyl, t-butyloxycarbonyl (Boc), fluorenyl-9-methoxycarbonyl, or any other suitable protecting group;

b) asymmetric reduction of a compound of formula (IV) or a salt thereof to give nitro aldol of formula (V):

or a salt thereof, wherein R is as described previously; and

c) converting a compound of formula (V) or a salt thereof to provide a compound of formula (I):

or a salt thereof, wherein R is as described previously.

The compound of formula (I), or a salt thereof, prepared according to a process described in the present application is useful as an intermediate for the manufacture of HIV protease inhibitors, such as lopinavir and ritonavir.

DETAILED DESCRIPTION

In an aspect, the present application provides processes for the preparation of a compound of formula (I) or a salt thereof, embodiments of which include one or more of the following steps, individually or in the sequence recited:

a) reacting a compound of formula (II) with a compound of formula (III) in the presence of a reagent to provide nitro ketone of formula (IV):

or a salt thereof; wherein R is a N-protecting group such as formyl, acetyl, t-butylacetyl, pivaloyl, benzoyl, trifluoroacetyl, 4-nitrobenzoyl, benzyl, benzyloxymethyl, benzyloxycarbonyl, t-butyloxycarbonyl (Boc), fluorenyl-9-methoxycarbonyl, or any other suitable protecting group;

b) asymmetric reduction of a compound of formula (IV) or a salt thereof to
give nitro aldol of formula (V):

(V) or a salt thereof, wherein R is as described previously; and

c) converting a compound of formula (V) or a salt thereof to provide a compound of formula (I):

(I) or a salt thereof, wherein R is as described previously.

Step a) involves reacting a compound of formula (II) with a compound of formula (III) in the presence of a reagent to provide nitro ketone of formula (IV), or a salt thereof. Step a) may be carried out in the presence of a reagent. Suitable reagents that may be used in step

a) include, but are not limited to, coupling agents, such as, for example,
carbonyldiimidazole, dicyclohexylcarbodiimide, or any other suitable coupling reagent. Step a) may be carried out in presence of a base. Suitable bases that may be used in step a) include, but are not limited to: alkali metal alkoxides, such as, for example, potassium tertiary butoxide, (1-cyano-2-tertiary butoxy-2-oxoethyl) sodium, sodium methoxide, sodium ethoxide, potassium isopropoxide, lithium methoxide, or the like.
Step a) may be optionally carried out in a suitable solvent. Suitable solvents that may be used include, but are not limited to: ethers such as, for example, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, or the like; aliphatic or alicyclic hydrocarbons such as, for example, hexanes, heptanes, pentanes, cyclohexane, or the like; halogenated hydrocarbons such as, for example, dichloromethane, chloroform, or the like; aromatic hydrocarbons such as, for example, toluene, xylenes, or the like; polar aprotic solvents such as, for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide, or the like; or any mixtures thereof.

Suitable temperatures that may be used in step a) may be less than about 100°C, less than about 80°C, less than about 60°C, less than about 40°C, less than about 30°C, less than about 20°C, less than about 10°C, less than about 0°C or any other suitable temperatures. The salt of compound of formula (IV) include, but are not limited to: inorganic acid addition salts such as hydrochloride, hydrobromide, sulphate, phosphate; or the like; organic acid addition salts such as formate, acetate, fumarate, oxalate, maleate, succinate, citrate, tartarate, glutarate, or the like. Optionally, the reaction mixture containing the compound of formula (IV) obtained in step a), before or after conventional work-up, may be carried forward to step b) without isolating the compound.

Step b) involves asymmetric reduction of a compound of formula (IV) or a salt thereof to give nitro aldol of formula (V), or a salt thereof. Asymmetric reduction of a compound of formula (IV) may be carried out by stereoselective enzymatic reduction to provide the required isomer of nitro aldol of formula (V) with major stereo selectivity and the other possible stereoisomers are (Va), (Vb) & (Vc).

Suitable enzymatic catalysts that may be used in step b) include, but are not limited to, ketoreductases such as NADH dependent ketoreductases or NADPH-dependent ketoreductases. Suitable ketoreductases enzymes such as, for example, Codexis Inc's products with catalog numbers KRED-101, KRED-151, KRED-111, KRED-112, KRED-113, KRED-121, KRED-130, KRED-114, KRED-123, KRED-115, KRED-125, KRED-NADH-129, KRED-NADH-109, KRED-NADH-110, KRED-NADH-112, KRED-NADH-106, KRED-164, or KRED-171; Enzysource Inc's products with catalog numbers ES-KRED-104, ES-KRED-112, ES-KRED-149, ES-KRED-120, ES-KRED-121, ES-KRED-134, or ES-KRED-125; Almac Sciences products with catalog numbers CRED-A131, CRED-A401, or CRED-A231; Daicel Chemical Industries products with catalog numbers Chiralscreen E007 or Chiralscreen E008; Evo-Catal GmbH product with catalog number Evo-1.1-200; or combinations thereof.

Step (b) may be carried out in the presence of a co-factor. The term "co-factor" refers to an organic compound that operates in combination with an enzyme which catalyzes the reaction of interest. Co-factors include, but are not limited to, for example, nicotinamide co-factors such as, nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), any derivatives thereof, or analogs thereof.

Optionally, in step (b), the reaction mixture further comprises a co-factor regeneration system. A co-factor regeneration system comprises a substrate and a dehydrogenase. The reaction between the substrate and dehydrogenase enzyme regenerates the co-factor. The co-factor regeneration system comprises a substrate/dehydrogenase pair selected from the group consisting of D- glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, phosphite/phosphite dehydrogenase, and 2-propanol and ketoreductase/hydrogenase; secondary alcohols such as secondary butanol, 2-pentanol, or 3-pentanol; or any other suitable co-factor regeneration system.

Step (b) may be carried out in the presence of a buffer. The buffer has pH of from about 4 to about 9, or from about 4 to about 8, or from about 5 to about 8, or from about 6 to about 8, or from about to 5 about 7. The buffer is a solution of a salt. The salt is potassium phosphate, magnesium sulfate, or mixtures thereof.

Step b) may optionally be carried out in a suitable co-solvent. Suitable co-solvents that may be used in step b) include, but are not limited to: alcohols such as, for example, methanol, ethanol, 2-propanol, n-propanol, n-butanol, 2-methoxyethanol, secondary butanol, tertiary butanol, or the like; ethers such as, for example, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, dimethoxyethane, or the like; esters such as, for example, ethyl acetate or butyl acetate; aliphatic or alicyclic hydrocarbons such as, for example, hexanes, heptanes, pentanes, cyclohexane, or the like; halogenated hydrocarbons such as, for example, dichloromethane, chloroform, or the like; aromatic hydrocarbons such as, for example toluene, xylenes, or the like; polar aprotic solvents such as, for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide, or the like; or any mixtures thereof.

Suitable recycle mixes that may be used for enzymatic reduction of the present application, to provide for the regeneration and recovery of the enzymes are summarized in Table-1:

Table-1

Stereo selective enzymatic reduction of a compound of formula IV with different enzymatic catalysts, using above procedures, to provide a compound of formula (V) with major selectivity and the results are summarized in Table -2.

Table-2

Step b) may be carried out in the presence of a metal-based catalyst and a hydrogen source. Suitable metal based catalysts that may be used in step b) include, but are not limited to: a compound of formula VI:

wherein when M = Ru, then R1 is optionally substituted C6-C10yl;
when M = Rh or Ir, then R1 is pentamethylcyclopentadienyl;
R2 is optionally substituted C6-C10yl or di(C1-C6alkylamino)-;
R3 and R4 are either both phenyl or R3 and R4 together are (CH2)4;
R5 is H, C1-C6alkyl, (C1-C4alkoxy)-CH2-, (C1-C3alkoxy)-CH2CH2-) (C1-C2alkoxy)- CH2CH2CH2-, CH3OCH2CH2CH2CH2-, di(C1-C4alkylamino)-CH2-, di(C1- C3alkylamino)-CH2CH2-, di(Ci-C2alkylamino)-CH2CH2CH2- or (CH3)2NCH2CH2CH2CH2-; X is chlorine or trifluoromethanesulfonate;
or any other suitable metal based complex.

In one embodiment R1 is phenyl, mesitylene or p-cymene.

In one embodiment, R2 is phenyl, 4-methylphenyl, or 2,3,4,5,6-pentafluorophenyl.

In one embodiment, R2 is dimethylamino, diethylamino, diisopropylamino, piperidyl, pyrrolidinyl, or morpholinyl.

Hydrogen source that may be used in step (b) include, but are not limited to: 2-propanol, 3-pentanol, hydrogen, formic acid in presence of an amine, such as triethyl amine, tributylamine, isopropyldiethylamine, N-methylmorpholine, N-methylpyrrolidine, DBU, or other suitable hydrogen source. Suitable temperatures that may be used in step b) may be less than about 80°C, less than about 60°C, less than about 40°C, less than about 30°C, less than about 20°C, less than about 10°C, or any other suitable temperatures. The salts of compound of formula (V) include, but are not limited to: inorganic acid addition salts such as hydrochloride, hydrobromide, sulphate, phosphate, or the like; organic acid addition salts such as formate, acetate, fumarate, oxalate, maleate, succinate, citrate, tartarate, or glutarate;, or the like.

Optionally, the mixture containing a compound of formula (V) obtained in step b), before or after conventional work-up, may be carried forward to step c) without isolating the compound.

Step c) involves converting a compound of formula (V) or a salt thereof to provide a compound of formula (I), or a salt thereof by any process known in the art. For example a compound of formula (I) or a salt thereof by a process as described in co-pending India Patent Application No. 1092/CHE/2009.

A HPLC method for the conversion of a compound of formula (IV) to a compound of formula (V) of the present application involves the use of Phenomenex Luna® C18 column or equivalent. Other parameters of the method are shown in Table-3.
Table-3

The compound of formula (I), or a salt thereof, prepared according to the processes described in the present application is useful as an intermediate for the manufacture of HIV protease inhibitors such as lopinavir and ritonavir.

DEFINITIONS

The following definitions are used in connection with the present disclosure unless the context indicates otherwise. In general, the number of carbon atoms present in a given group is designated "Cx-Cy", where x and y are the lower and upper limits, respectively. For example, a group designated as "C1-C6" contains from 1 to 6 carbon atoms. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like. Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming from left to right the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycabonyl" refers to the group (C6-Ci4aryl)-(Ci-C6alkyl)-0-C(0)-. Terms not defined herein have the meaning commonly attributed to them by those skilled in the art.

"Alkoxy-" refers to the group R-O- where R is an alkyl group, as defined below. Exemplary C16alkoxy- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1-propoxy, n-butoxy and t-butoxy.

"Alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, for example, a C1C12kyl group may have from 1 to 12 (inclusive) carbon atoms in it. Examples of C1C6alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl. Examples of C1C8alkyl groups include, but are not limited to, methyl, propyl, pentyl, hexyl, heptyl, 3-methylhex-1-yl, 2,3-dimethylpent-2-yl, 3-ethylpent-1-yl, octyl, 2-methylhept-2-yl, 2,3-dimethylhex-1-yl, and 2,3,3-trimethylpent-l-yl.

"Aryl" refers to an aromatic hydrocarbon group. Examples of a C6-C14aryl group include, but are not limited to, phenyl, a-naphthyl, p-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenanaphthyl. Examples of a C6-C10aryl group include, but are not limited to, phenyl, a-naphthyl, p-naphthyl, biphenyl, and tetrahydronaphthyl. An aryl group can be unsubstituted or substituted with one or more groups, including: C1-C6alkyl, halo, haloalkyl-, hydroxyl, hydroxyl(C1-C6alkyl)-, -NH2, aminoalkyl-, dialkylamino-, -COOH, -C(O)O-(C1-C6alkyl), -OC(O)(C1-C6alkyl), N-alkylamido-, -C(O)NH2, (C1-C6alkyl)amido-, or -NO2.
"Coupling agent" refers to a compound, molecule, or substance, capable of activating carboxylic acids with respect to nucleophilic attack. In some embodiments, the carboxyl-activating agents are capable of activating carboxylic acids where the attacking nucleophile is an acidic carbon atom, resulting in C-C bond formation. Non-limiting examples of such coupling agents include carbodiimide compounds (e.g. N.N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCHCI), or the like). Carbodiimide compounds may be either used alone or in combination with HOAt, HOBt, or HODhbt. Further examples of coupling agents include alkyl chloroformate compounds (e.g. ethyl chloroformate, isobutyl chloroformate, or the like) that are generally used with a tertiary amine like triethyl amine, diethyl azodicarboxylate (DEAD) with triphenylphosphine (the Mitsunobu reaction), various chlorosilanes, chlorosulfonyl isocyanate, N,N'-carbonyldiimidazole (CDI), phosphonium reagents (e.g. BOP, AOP, PyBOP, PyAOP, BroP, PyBroP, CF3-NO-PyBOP or the like), in situ acid fluoride generators (e.g. TFFH, BTFFH, DAST, cyanuric fluoride, or the like), aminium reagents (e.g. HBTU, HATU, HBPyU, HAPyU, or the like) phosphinyls (e.g. DPPA, DEPC, or the like), pentafluorophenyl active ester generators (e.g. PfTU, PfPyU, FDPP, PFP-trifluoroacetate, FPFOH plus DCC, or the like), mixed carbon anhydrides (e.g. EEDQ, IIDQ, or the like), CIP, or BOP-CI.

"Di(alkylamino)-" refers to a nitrogen atom attached to two alkyl groups, as defined above. Each alkyl group can be independently selected. Representative examples of an di(C1C6alkylamino)- group include, but are not limited to, -N(CH3)2, -N(CH2CH3)(CH3), -N(CH2CH3)2, -N(CH2CH2CH3)2, -N(CH2CH2CH2CH3)2, -
N(CH(CH3)2)2, -N(CH(CH3)2)(CH3), -N(CH2CH(CH3)2)2, -NH(CH(CH3)CH2CH3)2| -N(C(CH3)3)2, -N(C(CH3)3)(CH3), and -N(CH3)(CH2CH3). The two alkyl groups on the nitrogen atom, when taken together with the nitrogen to which they are attached, can form a 3- to 7- membered nitrogen containing heterocycle wherein up to two of the carbon atoms of the heterocycle can be replaced with -N(H)-, -N(C1-C6alkyl)-, -N(C3-C8cycloalkyl)-, -N(C6-C14aryl)-, -N(C1-C9heteroaryl)-, -N(amino(C1-C6alkyl))-, -N(C6-Cuarylamino)-, -O-, -S-, -S(O)-, or -S(O)2-.

"Halo" or "halogen" refers to -F, -CI, -Br and -I.

"N-protecting group" refers to a radical when attached to a nitrogen atom in a target molecule is capable of surviving subsequent chemical reactions applied to the target molecule i.e. hydrogenation, reaction with acylating agents, alkylation etc. The amine-protecting group can later be removed. Amine protecting groups include, but are not limited to, fluorenylmethoxycarbonyl (FMOC), tert-butoxycarbonyl (t-BOC), benzyloxycarbonyl (Z), those of the acyl type (e.g., formyl, benzoyl, trifluoroacetyl, p-tosyl, aryl- and alkylphosphoryl, phenyl- and benzylsulfonyl, o-nitrophenylsulfenyl, o-nitrophenoxyacetyl), and of the urethane type (e.g. tosyloxyalkyloxy-, cyclopentyloxy-, cyclohexyloxy-, 1,1-dimethylpropyloxy, 2-(p-biphenyl)-2-propyloxy-.and benzylthiocarbonyl). Amine-protecting groups are made using a reactive agent capable of transferring an amine-protecting group to a nitrogen atom in the target molecule. Examples of an amine-protecting agent include, but are not limited to, C1-C6 aliphatic acid chlorides or anhydrides, C6-C14arylcarboxylic acid chlorides or anhydrides, t-butylchloroformate, di-tert-butyl dicarbonate, butoxycarbonyloxyimino-2-phenylacetonitrile, t-butoxycarbonyl azide, t-butylfluoroformate, fluorenylmethoxycarbonyl chloride, fluorenylmethoxycarbonyl azide, fluorenylmethoxycarbonyl benzotriazol-1-yl, (9-fluorenylmethoxycarbonyl) succinimidyl carbonate, fluorenylmethoxycarbonyl pentafluorophexoxide, trichloroacetyl chloride, methyl-, ethyl-, trichloromethyl- chloroformate, and other amine protecting agents known in the art. Examples of such known amine-protecting agents are found in pages 385-397 of T. W. Green, P. G. M. Wuts, "Protective Groups in Organic Synthesis, Second Edition", Wiley-lnterscience, New York, 1991.

Celite™ is flux-calcined diatomaceous earth. Celite™ is a registered trademark of World Minerals Inc. The term "ketoreductase" or "ketoreductase enzyme" or KRED refers to an enzyme that catalyzes the reduction of ketone to form the corresponding alcohol in a stereoselective manner, optionally with the aid of co-factor. DMSO is dimethylsulfoxide, TFA is trifluoroacetic acid, and MeCN is acetonitrile.

Certain specific aspects and embodiments of the present application will be explained in more detail with reference to the following examples, which are provided for the purpose of illustration only, and should not be construed as limiting the scope of the application in any manner.

Examples

Example 1: Preparation of (S)-3-((tert-butoxycarbonyl) amino)-4-phenylbutanoic acid. (S)-tert-Butyl (1-cyano-3-phenylpropan-2-yl)carbamate (30.0 g) and methanol (300 ml_) is charged into a round bottom flask and stirred at 25-35°C for 5 minutes. A solution of sodium hydroxide (15.0 g) in water (300 mL) is added to the reaction mixture at 25-35°C. The reaction mixture is heated to 70°C and maintained at reflux at 70°C for 15 hours. Water (200 mL) is added to the reaction mixture after cooling to 30°C and stirred for 10 minutes. The reaction mixture is washed with dichloromethane (2X100 mL) at 30°C. The pH of the mixture is_;adjusted to 3.5-4.0 with IN HCI solution and the mixture is..extracted with ethyl acetate (3X100 mL). Both layers are separated and the organic layer is washed with sodium sulphate at 30°C. The solvent from the organic layer is evaporated at 40°C under reduced pressure, to afford 29 g of the title compound. Purity by HPLC: 97.8%.

Example 2: Preparation of (2RS, 5S)-2-nitro-5-(tert-butyloxycarbonyl) amino) 1,6-diphenylhexane-3-one. Carbonyldiimidazole (7.0 g) and
tetrahydrofuran (5 mL) are charged into the round bottom flask under nitrogen atmosphere at 28°C. A solution of (S)-3-((tert-butoxycarbonyl)amino)-4-phenylbutanoic acid (10 g) in tetrahydrofuran (10 mL) is slowly added to the carbonyldiimidazole complex at 28°C and stirred for 3 hours.

Potassium tertiary butoxide (3.4 g) and tetrahydrofuran (35.0 mL) are charged into another round bottom flask under nitrogen atmosphere at 28°C. The reaction mixture is cooled to 0°C. A solution of 2-phenylnitroethane (4.9 g) in tetrahydrofuran (10 mL) is slowly added to the reaction mixture at 0°C over 60 minutes and stirred for 90-120 minutes. The carbonyldiimidazole complex from above is slowly added to the reaction mixture by drops at 0°C over 30 minutes. The reaction mixture temperature is slowly raised up to 25°C and maintained for 15 hours. The pH of the aqueous phase is adjusted to 3.9 with 1N HCI solution. Both layers are separated and the aqueous layer is extracted with ethyl acetate (3X100 ml_). Combine the organic layer and the organic layer is washed with brine solution (2X50 mL). The organic layer is dried with sodium sulfate. The solvent from the organic layer is evaporated at 40°C under reduced pressure, followed by column purification to afford 4.4 g of the title compound as a solid. Purity by HPLC: 90.0%.

Example-3: Preparation of (2S, 3S, 5S)-2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl)-amino) 1,6-diphenylhexane. A 50 mM phosphate buffer solution is prepared from potassium phosphate monobasic (680 mg; 5.00 mmol) and water (100 mL). The pH of the buffer solution is adjusted to 7.0 with 8 molar sodium hydroxide solution. A solution of nicotinamide adenine dinucleotide phosphate (NADP\ 5 mg), D-glucose (180 mg), glucose dehydrogenase (1 mg) and KRED-130 (20 mg) in 50 mM pH 7 phosphate buffer (10mL) is charged into a jacketed reaction vessel and the temperature is adjusted to 30°C.

A solution of (2RS,5S)-2-nitro-5-(tert-butyloxycarbonyl)amino)1,6-diphenylhexane-3-one (100 mg) in DMSO (0.5 mL) is added to the reaction mixture. The resultant suspension is stirred at 30°C for 45 hours. Toluene (10 mL), water (10 mL) and brine solution (2 mL) are added to the reaction mixture and the -mixture is stirred for 10 minutes. The reaction mixture is filtered through a Celite® pad. Both layers are separated and the organic layer is washed with water (3X10 mL). The organic layer is dried with sodium sulfate. The solvent from the organic layer is evaporated to afford 83 mg of the title compound as a viscous, pale yellow liquid. The obtained compound is purified by flash chromatography on silica eluting with dichloromethane-ethyl acetate (95:5-90:10) to afford 27 mg of the title compound as a viscous, colorless oil.

Example-4: Preparation of (2S, 3S, 5S)-2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl)-amino) 1,6-diphenylhexane. A 50 mM phosphate buffer solution is prepared from potassium phosphate monobasic (680 mg; 5.00 mmol) and water (100 mL). The pH of the buffer solution is adjusted to 7.0 with 8 molar sodium hydroxide solution. A mixture of B-nicotinamide adenine dinucleotide (NAD+, 5 mg), KRED-NADH-112 (5 mg), and buffer solution (9.0 mL) are charged into a jacketed reaction vessel. A solution of (2RS,5S)-2-nitro-5-(tert-butyloxycarbonyl)amino)1,6-diphenylhexane-3-one (100 mg) in warm 2-propanol (1.0 mL) is added to the reaction mixture. The reaction mixture is warmed to 30°C and stirred for 20 hours at 30°C. Toluene (5 ml_) is added to the reaction mixture and the mixture is stirred for 10 minutes. The reaction mixture is filtered through a Celite® pad. Both layers are separated and the organic layer is dried with sodium sulfate. The solvent from the organic layer is evaporated under reduced pressure to afford 90 mg of the title compound as a viscous, pale yellow oil. Conversion by HPLC: 90.5 %; Isomer ratio (V: Va: Vb: Vc): 97.2: 2.4: 0.3: 0.

Example-5: Preparation of (2S, 3S, 5S)-2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl)-amino) 1,6-diphenylhexane. A 50 mM phosphate buffer solution is prepared from potassium phosphate monobasic (680 mg; 5.00 mmol) and water (100 mL). The pH of the buffer solution is adjusted to 7.0 with 8 molar sodium hydroxide solution. A mixture of B-nicotinamide adenine dinucleotide (NAD+, 5 mg), KRED-NADH-112 (5 mg), and buffer solution (9.0 mL) are charged into a jacketed reaction vessel. A solution of (2RS,5S)-2-nitro-5-(tert-butyloxycarbonyl)amino)1,6-diphenylhexane-3-one (100 mg) in warm 2-propanol (1.0 mL) is added to the reaction mixture. The reaction mixture is warmed to 30°C and stirred for 24 hours at 30°C. A small quantity of seed crystals of (2S,3S,5S)-2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl)-amino)1,6-diphenylhexane (< 1 mg) is added to the reaction mixture and the-reaetion mixture is stirred at 30°C for 16 hoursr The formed solid is collected by filtration, washed with water (3X2mL) and dried to afford 90 mg of the title compound as a white solid. Conversion by HPLC: 95.6%.

Exampie-6: Preparation of (2S,3S,5S)-2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl)-amino)1,6-diphenylhexane. A 50 mM phosphate buffer solution is prepared from potassium phosphate monobasic (680 mg; 5.00 mmol) and water (100 mL). The pH of the buffer solution is adjusted to 7.0 with 8 molar sodium hydroxide solution. A mixture of B-nicotinamide adenine dinucleotide (NAD+, 2 mg), CRED-A131 (10 mg), and buffer solution (0.9 mL) are charged into a jacketed reaction vessel. A solution of (2RS,5S)-2-nitro-5-(tert-butyloxycarbonyl)amino)1,6-diphenylhexane-3-one (10 mg) in warm 2-propanol (100 uL) is added to the reaction mixture. The reaction mixture is warmed to 30°C and stirred for 24 hours at 30°C. Toluene (2 mL) is added to the reaction mixture and stirred for 10 minutes. The reaction mixture is filtered through a Celite® pad. Both layers are separated and the organic layer is dried with sodium sulfate. The solvent from the organic layer is evaporated under reduced pressure to afford 9 mg of the title compound as a viscous, pale yellow oil. Conversion by HPLC: 97.0 %.

Exampie 7: preparation of z-nitro-3-nyaroxy-5-{tert-DutyioxycarDonyi) amino)-1,6-diphenylhexane. (2RS, 5S)-2-nitro-5-(tert-butyloxycarbonyl) amino) 1,6-diphenylhexane-3-one (50 mg) and chloro(((1R,2R)-(-)2-amino-1,2-diphenylethyl)(4-toluenesulfonyl)amido)(p-cymene) ruthenium (II) (4 mg) are charged in a Schlenk flask. The reaction mixture is purged with nitrogen. Formic acid-triethylamine (5:2, 200 uL) and dichloromethane (100 uL) are added to the reaction mixture. The reaction mixture is stirred at room temperature for 5 hours. Toluene (2 mL) is added to the reaction mixture. The reaction mass is applied to a silica gel plug, which was eluted with dichloromethane-ethyl acetate (80:20). The solvent from the reaction mass is evaporated under reduced pressure to afford 49 mg of the title compound as a brown viscous oil. Conversion by HPLC: 75.6%; Isomer ratio (V: Va: Vb: Vc) by HPLC: 13.0: 57.5: 14.0: 15.5.

Example 8: Preparation of 2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl) amino)-1,6-diphenylhexane. (2RS, 5S)-2-nitro-5-(tert-butyloxycarbonyl) amino) 1,6-diphenylhexane-3-one (50 mg) and chloro (((1S,2S)-(-)2-amino-1,2-diphenylethyl)(4-toluenesulfonyl)amido)(p-cymene) ruthenium (II) (4 mg) are charged in a Schlenk flask.

The reaction mixture is purged with nitrogen. Formic acid-triethylamine (5:2, 200 uL)^and dichloromethane (100 uL) are added to thereaction , mixture. The reaction mixture is stir-red at room temperature for 5 hours. Toluene (2 mL) is added to the reaction mixture. The reaction mass is applied to a silica gel plug, which was eluted with dichloromethane-ethyl acetate (80:20). The solvent from the reaction mass is evaporated under reduced pressure to afford 49 mg of the title compound as a brown viscous oil. Conversion by HPLC: 91.4%; Isomer ratio (V: Va: Vb: Vc) by HPLC: 4.7: 2.9: 20.8: 71.6.

Example 9: Preparation of 2-nitro-3-hydroxy-5-(tert-butyloxycarbonyl) amino)-1,6-diphenylhexane. (2RS,5S)-2-Nitro-5-(tert-butyloxycarbonyl)amino)1,6-diphenylhexane-3-one (50 mg) and chloro(((1 R,2R)-(-)2-amino-1,2-diphenylethyl)(4-toluenesulfonyl)amido) (pentamethylcyclopentadienyl) rhodium (III) (4 mg) are charged in a Schlenk flask. The reaction mixture is purged with nitrogen. Formic acid-triethylamine (5:2, 200 uL) and dichloromethane (100 uL) is added to the reaction mixture. The reaction mixture is stirred at room temperature for 5 hours. Toluene (2 mL) is added to the reaction mixture. The reaction mass is applied to a silica gel plug, which was eluted with dichloromethane-ethyl acetate (80:20). The solvent from the reaction mass is evaporated under reduced pressure to afford 48 mg of the title compound as a brown viscous oil. Conversion by HPLC: 33.7%; Isomer ratio (V: Va: Vb: Vc) by HPLC: 15.2: 49.1: 20.5: 15.2.

Example 10: Preparation of 2-nitro-3-hydroxy-5-(tert-
butyloxycarbonyl)am ino)-1,6-diphenylhexane. (2RS, 5S)-2-nitro-5-(tert-
butyloxycarbonyl) amino) 1,6-diphenylhexane-3-one (50 mg) and chloro(((1S,2S)-(-)2-amino-1,2-diphenylethyl)(4-toluenesulfonyl)amido) (pentamethylcyclopentadienyl) rhodium (III) (4 mg) are charged in a Schlenk flask. The reaction mixture is purged with nitrogen. Formic acid-triethylamine (5:2, 200 uL) and dichloromethane (100 uL) is added to the reaction mixture. The reaction mixture is stirred at room temperature for 5 hours. Toluene (2 mL) is added to the reaction mixture. The reaction mass is applied to a silica gel plug, which was eluted with dichloromethane-ethyl acetate (80:20). The solvent from the reaction mass is evaporated under reduced pressure to afford 48 mg of the title compound as a brown viscous oil. Conversion by HPLC: 50.7%; Isomer ratio (V: Va: Vb: Vc) by HPLC: 3.5:1.9: 30.1: 64.4.

Example 11: Preparation of (2S,3S,5S)-2-amino-3-hydroxy-5-(t-butyloxycarbonyl) amino)-1,6-diphenylhexane. (2S,3S,5S)-2-nitro-3-hydroxy-5-(t-butyloxycarbonyl)amino)-1,6-diphenylhexane (20 mg) in methanol (2 mL) is charged into a round bottom flask at 25°C, foJlowed by zinc (6 mg), and ammonium formate (3 mg) at 25°G. The mixture is maintained at 25°C for a period of 4-5 hours and filtered through a Celite® bed. The solvent from the filtrate is evaporated at 45°C and the residue is extracted with ethyl acetate (5 mL). The organic layer is washed with brine solution (5 mL) and finally washed with water (2 mL). The solvent from the organic layer is evaporated at 45°C and the residue is dried at 24°C under vacuum, to afford 15 mg of the
title compound.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover all such changes and modifications that are within the scope of this invention.

We claim:

1. A process for the preparation of a compound of formula (I), or a salt thereof, comprising:

a) reacting a compound of formula (II) with a compound of formula (III) in the
presence of a reagent to provide nitro ketone of formula (IV):

or a salt thereof; wherein R is a N-protecting group such as formyl, acetyl, t-butylacetyl, pivaloyl, benzoyl, trifluoroacetyl, 4-nitrobenzoyl, benzyl, benzyloxymethyl, benzyloxycarbonyl, t-butyloxycarbonyl (Boc), fluorenyl-9-methoxycarbonyl, or any other suitable protecting group;

b) stereoselective reduction of a compound of formula (IV) or a salt thereof,

to give nitro aldol of formula (V):

or a salt thereof, wherein R is as described previously; and

c) converting a compound of formula (V) or a salt thereof to provide a compound of formula (I):

or a salt thereof.

2. The process of claim 1, wherein asymmetric reduction in step (b) involves stereoselective reduction of a compound of formula (IV) with an enzyme and in the presence of a co-factor.

3. The process of claims 1-2, wherein the enzyme is a ketoreductase.

4. The process of claims 1-3, wherein the enzyme is selected from the group consisting of at least one of KRED-101, KRED-151, KRED-111, KRED-112, KRED-113, KRED-121, KRED-130, KRED-114, KRED-123, KRED-115, KRED-125,
KRED-NADH-129, KRED-NADH-109, KRED-NADH-110, KRED-NADH-112, KRED-NADH-106, KRED-164, KRED-171, ES-KRED-104, ES-KRED-112, ES-KRED-149, ES-KRED-120, ES-KRED-121, ES-KRED-134, ES-KRED-125, CRED-A131, CRED-A401,and CRED-A231.

5. The process of claims 1-2, wherein the co-factor is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and any mixtures thereof.

6. The process according to any preceding claim, wherein the reaction mixture in step (b) further comprises a co-factor regeneration system that comprises a substrate and a dehydrogenase.

7. The process of claim 6, wherein the substrate/dehydrogenase pair selected from the group consisting of D-glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, phosphite/phosphite dehydrogenase, and 2-propanol and ketoreductase/hydrogenase; secondary alcohols such as secondary butanol, 2-pentanol, or 3-pentanol; or any other suitable co-factor regeneration system.