FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
THE PATENTS (AMENDMENT) RULES, 2006
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
One Pot Process For Preparing N-Protected L-Tert-Leucine Or Its Corresponding Salt
2. APPLICANT
NAME : Frichem Private Limited
NATIONALITY : IN
ADDRESS : 12, Concord, Bullock Road, Band Stand, Bandra West, Mumbai-400 050,
India
3. PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of the Invention
The present invention relates to a process for preparing enantio-pure N-protected L-tert-leucine or its salts by means of reductive animation. More particularly, the present invention relates to one pot process for preparation of N-protected-L-tert-leucine and its salts by means of enzymatic reductive amination of trimethyl pyruvic acid.
Background of the Invention
The preparation of enantiomerically pure non-natural L-tert leucine is of wide importance due to its pharmaceutical application as intermediates for synthesizing the corresponding antiretroviral drug of the protease inhibitor like Atazanavir and Telepravir. A. S. Bommarius et. al. (J. Mol Cat. B: Enzymatic 1998, 5, 1-11) provides examples of uses of L-tert-leucine as a building block for pharmaceutically active compounds.
The preparation of L-tert leucine by means of a reductive amination has been achieved in the presence of synthetic catalysts as well as enzymes. However, synthetic routes are still rare. In patent and non-patent literature there are numerous examples disclosed which relate to the enzymatic reductive amination of trimethyl pyruvic acid to get corresponding L-tert-leucine and then converting the same to its corresponding N-protected L-tert-leucine or its salts. All these conventional methods involve the isolation of L-tert lecuine from reactive biomass and then its conversion to corresponding N-protected compound or its salts.
The general reaction concept of reductive amination is depicted in below scheme 1.

Scheme 1
In the above reaction scheme, reductive amination of corresponding a-keto acid in the presence of two isolated enzymes, namely leucine dehydrogenase and formate dehydrogenase has been achieved. The reductive amination step under consumption of the cofactor NADH is catalyzed by a leucine dehydrogenase, forming the desired L-tert-

leucine, as well as the oxidized cofactor form, NAD+. This oxidized cofactor form, NAD+, is subsequently reduced into NADH by means of a formate dehydrogenase catalyzed oxidation of formate. This continuous recycling of the cofactor NADH, required as the reducing agent, enables its use in catalytic amounts only, which is of importance due to its high price. This enzymatic reaction has already proven its technical feasibility on large scale.
Further, with time, "second-generation process" for the enantioselective synthesis of L-tert-leucine has been developed, realizing the first whole celL-catalyzed approach to this pharmaceutically interesting, bulky amino acid. The synthesis of this non-natural amino acid proceeds highly efficiently via a reductive amination of the corresponding a-keto acid in presence of a recombinant whole cell, bearing a leucine dehydrogenase and formate dehydrogenase.
EP 0692538; Wandrey, Bioprocess Engineering 1996, 14, 291-297; J. Mol Cat. B: Enzymatic 1998, 5, 1-11; 7. Biotechnol 1997, Industrial Bioreductiveformations, Wiley-VCH, Weinheim, 2000, p. 125f. and Biocatalysis 1994, 10, 37-47 discloses the industrially established method for preparing optically active L-tert-leucine on the ton scale in which a leucine dehydrogenase and a formate dehydrogenase has been used for reducing the 2-ketocarboxylic acids while regenerating cofactor in situ.
Subsequently, Chem. Comm., Vol 42, 1977; pp. 1069-1076 discloses the process for preparing benzyloxycarbonyl-L-tert-leucine and its salts using the L-tert-leucine isolated from whole-cell catalyst system. The process comprises the reaction of isolated L-tert-leucine with aqueous, solution of sodium hydroxide followed by introduction of benzyloxycarbonyl chloride to attain benzyloxycarbonyl L-tert-leucine which is isolated and then converted to its salts.
Despite the conversion and enantioselectivity, however, there are drawbacks like isolation of L-tert-leucine from aqueous layer which consequently affects on the total yield, purity and cost of the product. Currently available processes for the preparation of N-protected L-tert-leucine or its salts tend to be inefficient and low yielding because intermediates are isolated after each reaction step. Given the commercial importance of N-protected L-tert-

leucine salt, the present invention provides one pot enzymatic process for the preparation of N-protected L-tert-leucine and its salts.
Summary of the Invention
It is an object of the present invention to provide simple, cost effective and eco-friendly one pot enzymatic process for preparation of N-protected-L-tert leucine or its salts whereby the reaction is carried out by means of reductive amination.
It is an object of the invention to provide one pot enzymatic process for preparation of N-protected-L-tert-leucine and its salt whereby resultant product is accomplished with a high yield and purity.
It is an object of the present invention to provide one pot process for producing N-protected L-tert-leucine or its salts comprising the steps of:
(a) converting trimethyl pyruvic acid of formula (IV) into L-tert-leucine of formula (III) by means of reductive amination;
(b) converting L-tert-leucine of formula (III) into N-protected L-tert-leucine of formula (II); and
(c) converting the protected compound of formula (II) into its corresponding pharmaceutically acceptable salts of formula (I).
The above and other objects of the present invention are attained according to following preferred embodiments of the present invention. However the scope of the invention is not restricted to the particular embodiments discussed herein after.
Detailed Description of the Invention
It has now been found that to attain the object of high yield of the resultant biochemical molecule, it is required to have its intermediate in high purity with optimum yield. Accordingly, the present invention provides one pot process for preparation of N-protected L-tert-leucine or its corresponding salts. These salts can be used as an intermediate for preparation of an anti-retroviral drug of the protease inhibitor like Atazanavir and Telaprevir.

As per the invention, one pot process for preparing N-protected L-tert-leucine or its corresponding salts comprises of three steps which is listed below:
(a) reductive animation, which involves the conversion of a-keto acid of formula (IV) into L-tert leucine of formula (III) by means of an whole cell catalyst enzyme or cell lysate enzyme;
(b) amino group protection, which involves the conversion of L-tert leucine of formula (III) into N-protected L-tert-leucine of formula (II) without isolating from the reaction mass; and
(c) optionally isolating N-protected L-tert-leucine of formula (II);
(d) salt formation, which involves the conversion of N-protected L-tert-leucine of formula (II) into its pharmaceutically acceptable salt of formula (I) with high yield and purity.
The detailed description of above mentioned three steps are as given below.
Step (A) - Reductive Animation
Synthesis of L-tert-leucine in a very good yield can be carried out by means of reductive amination in the presence of amino group donors. For this, enzymes from numerous organisms capable of converting trimethyl pyruvic acid into non-natural L-tert-leucine are
used.
Prior to reductive amination, selective microorganism expressed in E. coli capable of generating formate dehydrogenase (pURL2) and leucine dehydrogenase (pURL5) are advantageously cultured in a nutrient medium, optimum for their growth, under appropriate favorable temperature and aeration conditions up to a dry weight of about 4 to 60 g/1 of nutrient solution. For reductive amination step, cell lysate pURL2 & pURL5 is used. In place of cell lysate, whole cell can also be used herein. Further whole cell pURL2 and pURL5 can optionally be adsorbed on celite to make it immobilized which will increase the efficiency and stability of the enzyme used for reductive amination and adsorbed or immobilized enzyme is known as URL-IM 105. '
During reductive amination reaction, trimethyl pyruvic acid and whole cells or cell lysate and the amino group donor all together are allowed to react in an aqueous medium. The

reactants can be used herein for the reaction either in the form of aqueous solution or as solid substances and can be added to the reaction mass simultaneously, in one portion or in multiple portions.
Whole cell or cell lysate used herein bearing a formate dehydrogenase (pURL2) & leucine dehydrogenase (pURL5) is suspended in a physiological phosphate buffer. During reductive amination step, under the consumption of the cofactor, NADH is catalyzed by a leucine dehydrogenase, forming the desired L-tert leucine, as well as the oxidized cofactor form, NAD+. This oxidized cofactor form, NAD+, is subsequently reduced under formation of NADH by means of a formate dehydrogenase catalyzed oxidation of formate.
The cell lysates bearing a leucine dehydrogenase (pURL5) & formate dehydrogenase (pURL2) enzymes are commercially available from animal-derived, plant-derived, or microorganism-derived sources. Examples of microorganisms capable of producing leucine dehydrogenase belong to the genera: Brevibacterium, Rhodococcus, Sporosarcina, Thermoactinomyces, Microbacterium, Halomonas, Clostridium, Bacillus, Neurospora, Escherichia, and Aerobacter. The microorganisms capable of producing formate dehydrogenase belong to the genera: Candida, Kloeckera, Pichia, Lipomyces, Pseudomonas, Moraxella, Hyphomicrobium, Paracoccus, Thiobacillus, and Ancylobacter, and the like.
Trimethyl pyruvic acid serving as the substrate of the reductive amination is dissolved or suspended in water at a loading concentration of 0.1% (w/v) to 90% .(w/v). A preferred loading concentration is 1% to 60% w/v. Further, the amino group donor can be selected from ammonia, ammonium formate and the like.
Addition of a cofactor such as NAD to the above-mentioned reductive amination is expected to improve the efficiency of the reaction. The cofactor such as NAD is added desirably at a concentration of 0.000001 equivalents to 2 equivalents with respect to the substrate. A preferred concentration is 0.00001 to 0.1 equivalents.
The reaction is allowed to proceed with a temperature kept in a suitable range of from 0°C to 80°C while the reaction fluid is allowed to stand or stirred for a certain period of time.

At lower temperatures, the enzyme reaction proceeds increasingly slowly, whereas the enzyme is progressively deactivated at higher temperatures. A preferred temperature range for carrying out the reaction is 20 to 60°C.
During the reaction, the pH is desirably adjusted in a range of from 4 to 12. More preferably, the pH is adjusted in a range of 6 to 11. The pH can be adjusted by adding an acid or a base. The L-tert leucine produced through the reductive amination is further utilized for step (B) without isolation.
(B) Amino group protection
With respect to Step (B), the compound of formula (III), L-tert leucine, in reaction mixture of step (A) is further reacted with compound of formula (X) in presence of a base to get compound of formula (II), N-protected L-tert leucine.
In compound of formula (X), R is selected from the group comprising alkyl having CI- C5 carbon; aryl having benzyl, tritlyl; alkylaryl and the like. Base employed during step (B) is selected from alkali or alkaline earth metal salts wherein the salts are hydroxide, carbonate, sulphate and the like. The temperature of the reaction step is between ranges of 0°C to 100°C. The reaction is carried at a pH range of 1 to 2.5.
Further the compound of formula (II) is extracted in water immiscible solvent selected from ethyl acetate, methylene dichloride, toluene, methyl isobutyl ketone (MIBK), methyl tert-butyl ether (MTBE) and the like.
It will be apparent to those skilled in the art that in general, sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods.
(C) Formation of pharmaceutically acceptable salts
The term "pharmaceutically acceptable salts" refers to the salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like.

Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines like dicyclohexyl amine.
The pharmaceutically acceptable salts of N-protected L-tert leucine is prepared by dissolving N-protected L-tert leucine obtained from the step (B) in solution of basejn solvent.
The base for formation of salts is as depicted above and the solvent employed for is selected from ethyl acetate, methylene dichloride, toluene. The temperature range for this reaction is about 10°C to 50°C, more preferably 15°C to 35°C.
The above described process can be synthetically represented by the following scheme.

In the following section preferred embodiments are described by way of examples to illustrate the process of the invention. However, these are not intended in any way to limit the scope of the present invention.

Example 1: Preparation of N-methoxycarbonyL-L-tert-leucine dicyclohexylamine salt
[A] Preparation of L-tert leucine
Trimethyl pyruvic acid (129 gm, 80% soln) was added to a solution of sodium hydroxide
(32.17g) in water. Ammonium formate (49.94g), cofactor NAD (0.53g) and formate dehydrogenase (pURL2) and leucine dehydrogenase (pURL5) was then added to the solution at room temperature. The reaction mixture was stirred for 15-24 hours and pH was maintained between 7.5 to 8.5. The reaction mixture was further centrifuged to separate out inactive enzyme and the remaining reaction mass is allowed to provide L-tert leucine (90gm).
[B] Preparation of L- MOC- tert leucine
Methyl chloroformate (144 g) was introduced to the reaction mass from step [A] with
stirring between 0-7°C. The reaction mass was then heated at 60-65°C for at least 4-5 hours and pH of reaction mass was adjusted to 2 to 2.5 by means of hydrochloric acid. The L-MOC-tert-leucine in the reaction mass was extracted in methylene dichloride and distilled out by addition of cyclohexane.
[C] Preparation of L-MOC-tert-leucine dicyclohexyl amine salt
Dicyclohexyl amine in ethyl acetate was subjected to reaction mass of step [B] with
stirring for at least one hour at RT. The solid thus obtained was filtered and further purified to get highly pure L-MOC- tert-leucine dicyclohexyl amine salt. [Yield: 1.15 w/w]; [Purity: > 99.0%, Chiral Purity > 99.9%]
Example 2: Preparation N-caboxybenzyl L-tert leucine dicyclohexylamine salt
[A] Preparation of L-tert leucine
Trimethyl pyruvic acid (129 gm, 80% soln) was added to a solution of sodium hydroxide
(32.17g) in water. Ammonium formate (49.94g), cofactor NAD (0.53g) and formate
dehydrogenase (pURL2) and leucine dehydrogenase (pURL5) was then added to the
solution at room temperature. The reaction mixture was stirred for 15-24 hours and pH
was maintained between 7.5 & 8.5. The reaction mixture was further centrifuged to
separate out inactive enzyme and the remaining reaction mass is allowed to provide L-tert
leucine (90gm).

[B] Preparation of N-Caboxybenzyl L-tert leucine
Benzyl chlorformate (177gm) was introduced to the reaction mass from step [A] in
presence of NaOH in water with stirring at temperature range of 0°C to 5°C. The reaction mixture was stirred at 5-10°C for at least 3 hours. The pH of reaction mass was adjusted to 1.5 to 2.0 by addition of hydrochloric acid and the said reaction mass was extracted with ethyl acetate.
[C] Preparation of N-caboxybenzyl L-tert leucine dicyclohexyl amine salt
Dicyclohexyl amine (125.3gm) in ethyl acetate was subjected to the ethyl acetate layer of
step [B] at room temperature. The solid thus obtained was filtered and further purified to
get highly pure N-caboxybenzyl-L-tert leucine dicyclohexylamine salt. [Yield: 2.2 w/w];
[Purity: > 99.0%, Chiral Purity > 99.9%]
Example 3: Preparation of N-Caboxybenzyl L-tert leucine dicyclohexyl amine salt using immobilized enzyme URL-IM 105
[A] Preparation of L-tert-Leucine:
Trimethyl pyruvic acid (12.9 Kg, 80% soln) was added to a solution of sodium hydroxide (12.92 kg) in water and adjust the pH 8.5 by 40 % of sodium hydroxide solution at 25 to 30°C. Ammonium formate (5.2 Kg) and NAD (52 g) was added and again adjust the pH 8.0 by 15% liq. ammonia. Thereafter immobilized enzyme URL-IM 105 was added into the solution at 25 to 30°C. The reaction mixture was stirred for 20 to 24 hrs and pH was maintained between 7.8 to 8.3 by 15 % liq ammonia. After completion of the reaction it was acidified with 30 % sulfuric acid and was further centrifuged to separate out inactive enzymes and celite and the remaining supernatant liquid was taken for derivatization
[B] Preparation of N-Carboxylbenzyl L-tert leucine
Sodium hydroxide (12.3 kg) was added into the step [A] supernatant and mix it well, there after benzyl chloroformate (17.7 kg) was added into the mixture with stirring at temperature range 0 to 5°C. This reaction mixture was stirred at 5 to 10°C for 3 hrs. After completion of reaction the pH was adjusted to 1.5 to 2.0 by addition of 50 % hydrochloric acid and the said reaction mixture was extracted with ethyl acetate (3X50 lit) and combine all the ethyl acetate layer and proceed for next stage for salt formation.

[C] Preparation of N-Caboxybenzyl L-tert leucine dicyclohexyl amine salt Dicyclohexyl amine (12.5 kg) was added in to ethyl acetate layer of step [B] at room . temperature. After completion of addition temperature was arise upto 70 to 80°C and maintain it for 15 to 20 min. The reaction mixture was slowly cooled to RT and maintained at this temperature for 1 to 2 hrs. The solid was centrifuged and obtained the pure N-Caboxybenzyl-L-tert leucine dicyclohexyl amine salt (25 kg). [Yield: 2.5 w/w]; [Purity: > 99.0%, Chiral Purity > 99.9%]
While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those skilled in the art without departing from the scope of this invention.

We Claim:
1. A one pot process for preparing N-protected L-tert-leucine or its
corresponding salt, comprising the steps of:
(a) converting trimethyl pyruvic acid of formula (IV) into L-tert leucine of formula (III) by reductive amination;
(b) converting the L-tert leucine of formula (III) from step (a) into N-protected L-tert-leucine of formula (II);
(c) optionally isolating N-protected L-tert leucine of formula (II); and
(d) converting the N-protected L-tert leucine of formula (II) into its corresponding salt of formula (I).

2. The one pot process as claimed in claim 1, wherein reductive amination is carried out by means of enzyme pURL5 & pURL 2 as cell lysates or whole cell, expressed in E-coli.
3. The one pot process as claimed in claim 2, wherein enzyme pURL2 and pURL5 as a whole cell is optionally adsorbed on celite before use.
4. The one pot process as claimed in claim 1, wherein reductive amination is carried out employing amino group donor compound and co-factor NAD.
5. The one pot process as claimed in claim 4, wherein the amino donor group is selected from ammonia, and ammonium formate and a co-factor NAD is used at a concentration of from 0.000001 equivalents to 2 equivalents with respect to the substrate.
6. The one pot process as claimed in claim 1, wherein the L-tert leucine of formula (III) is converted into N-protected-L-tert leucine by reacting L-tert leucine of formula (III) with a compound of formula (X),

in presence of base in water.

7. The one pot process as claimed in claim 6, wherein R in compound of formula (X) is alkyl, aryl or aralkyl.
8. The one pot process as claimed in claim 6, wherein base is selected from alkali or alkaline earth metal salts wherein salts include hydroxides, carbonates, sulphates and bicarbonates.
9. The one pot process as claimed in claim 1, wherein the compound N-protected L-tert-leucine of formula (II) obtained from step (b) or step (c) is converted to its corresponding dicyclohexyl amine salt in presence of solvent selected from ethyl acetate, methylene dichloride, toluene.
10. The N-protected L-tert-leucine or its salts obtained by the process as claimed in claim 1, for use in preparation of antiretroviral drug of the protease inhibitor like Atazanavir, Telapravir.