DESC:The following specification particularly describes the invention and the manner in which it is to be performed:
CHEMO-ENZYMATIC PREPARATION OF AMINO ACID DERIVATIVE

FIELD OF INVENTION
The present application relates to a chemo-enzymatic process for the preparation of amino acid derivative of formula (A) using protease enzyme. The present application specifically relates to a chemo-enzymatic process for the preparation of (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid (1) from 3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid ester (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde.

BACKGROUND
Schichl, D. A. et al. (Eur. J. Org. Chem., 2008, 3506-12) discloses dynamic kinetic resolution of phenylalanine and tyrosine esters in presence of 3,5-dinitrosalicyaldehyde using alcalase enzyme.

Chen et al. (J. Org. Chem., 1994, 59, 7580-81) teaches a process for the conversion of racemic amino acid to its L-isomer by alcalase catalyzed resolution of amino acid ester in a mixture of 2-methyl-2-propanol/water (19:1), simultaneously with pyridoxal 5-phosphate catalyzed racemization of the unhydrolyzed antipode.

Zimmerman et al., (Org. Proc. Res. & Dev., 2006, 622-27) discloses the reaction course of a dynamic kinetic resolution of amino acid derivatives.

Felten et al., (Org. Lett, 2010, 1916-19) teaches dynamic kinetic resolution of benzyl ester of phenyl glycine in presence of picolinaldehyde and zinc acetate.

SUMMARY
One aspect of the present application relates to a chemo-enzymatic process for the preparation of amino acid derivative of formula (A) from an amino acid ester derivative of formula (B) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is C1-C6 alkyl ester or benzyl ester; n is 0 or 1; and R2 is halogen or aromatic group optionally substituted with halogen, alkyl, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile or trifluoromethyl.

Yet another aspect of the present application relates to a chemo-enzymatic process for the preparation of amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein R1 is as defined above.

Still another aspect of the present application relates to use of compound of formula (1), as prepared by the process of the present application, for the preparation of sacubitril.

Another aspect of the present application relates to use of compound of formula (1), as prepared by the process of the present application, for the preparation of LCZ-696.

Yet another aspect of the present application relates to chemo-enzymatic process for the preparation of sacubitril comprising:
(a) chemo-enzymatic process for the preparation of amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is as defined above; and
(b) converting the amino acid derivative of formula (1) to sacubitril.

Still another aspect of the present application relates to a chemo-enzymatic process for the preparation of LCZ-696 comprising:
(a) chemo-enzymatic process for the preparation of amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is as above; and
(b) converting the amino acid derivative of formula (1) to LCZ-696.

DETAILED DESCRIPTION
The chemo-enzymatic process of the present application may be performed using any protease enzyme known in the art that is capable of preferentially cleaving an ester group of a substrate to form an acid. The enzyme may be of microbial origin or from plants or animals. Such enzymes may be commercially available or prepared by methods known in the art. Specifically, the chemo-enzymatic process of the present application may be performed using alcalase enzyme. More specifically, the chemo-enzymatic process of the present application may be performed using alcalase enzyme procured from Novozyme (Alcalase ® 2.4 L FG).

The chemo-enzymatic process of the present application may be carried out in a solvent selected from a group of water, an organic solvent and mixture thereof. The organic solvent may include but not limited to ethers such as tetrahydrofuran and the like; polar aprotic solvents such as dimethyl sulfoxide, dimethyl formamide and the like; ketones such as acetone and the like, nitriles such as acetonitrile and the like; alcohols such as methanol, ethanol, tert-butanol and the like. Specifically, the chemo-enzymatic process of the present application may be carried out in a mixture of water and acetonitrile. Alternatively, the chemo-enzymatic process of the present application may be carried out in a mixture of water and tert-butanol.

The chemo-enzymatic process of the present application may be carried out in presence of a buffer. The buffer may be selected from a group of potassium phosphate, magnesium sulfate, tris(hydroxymethyl)aminomethane and mixture thereof. Optionally, thiol may be added along with buffer.

The chemo-enzymatic process of the present application may be carried out in presence an aldehyde or a metal complex of aldehyde which is capable of racemizing the undesired enantiomer of amino acid ester of compound of formula (B) or compound of formula (2). Specifically, the aldehyde may include but not limited to 3,5-dinitrosalicylaldehyde and picolinaldehyde. Alternatively, a mixture of zinc salt such as zinc acetate and aldehyde may be used for racemizing the undesired enantiomer of amino acid ester of compound of formula (B) or compound of formula (2).

The chemo-enzymatic process of the present application may be carried out at a suitable pH. Specifically, the pH of chemo-enzymatic process of the present application may be from about 4 to about 9; more specifically from about 5 to about 8 and most specifically from about 6 to about 7.5.

The chemo-enzymatic process of the present application may be carried out at a suitable temperature. Specifically, the temperature may be of about 10 °C to about the boiling point of the solvent; more specifically of about 20 °C to about 40 °C and more specifically of about 25 °C to about 30 °C.

The concentration of substrate for the chemo-enzymatic process of the present application may be from about 0.1 g/L to about 1000 g/L. The loading of the enzyme, for the chemo-enzymatic process of the present application, may be from about 0.1 wt% to about 100 wt% with respect to the weight of substrate. Specifically, the loading of the enzyme, for the chemo-enzymatic process of the present application, may be less than about 10 wt% with respect to the weight of substrate.

The chemo-enzymatic process of the present application may be carried out in presence of a stabilizer. Any stabilizer known in the art may be used for the above chemo-enzymatic process of the present application.

The chemo-enzymatic process of the present application may be carried out in a suitable equipment, for example, in a closed reaction vessel made of glass or metal. The chemo-enzymatic process of the present application may be carried out under an atmosphere of nitrogen or air.

The present application also relates to a process for the enhancement of enantiomeric excess of compound of formula (1) by suspending the compound of formula (1) in a mixture of organic solvents and isolating by known techniques. One of the organic solvents which may be used but not limited to an ether solvent such as diethyl ether, methyl tert-butyl ether, tetrahydrofuran and 1,2-dimethoxy ethane. The other organic solvent in the mixture may be methane sulfonic acid. The compound of formula (1) may be isolated from the suspension by methods known in the art.

One aspect of the present application relates to a chemo-enzymatic process for the preparation of sacubitril comprising:
(a) chemo-enzymatic process for the preparation of amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is defined as above;
(b) enhancement of enantiomeric excess of compound of formula (1) by suspending the compound of formula (1) in a mixture of an ether and methane sulfonic acid; and
(c) converting the compound of formula (1) to sacubitril.

The process for conversion of compound of formula (1) to sacubitril may be followed involving the use of any method known in the art. Specifically, compound of formula (1) may be converted to sacubitril by following the method as disclosed in US patent number 5217996.

Sacubitril, obtained by the process of the present application may be converted to LCZ-696 by a process known in the art.

The process of the present application produces amino acid derivative of formula (1) in an enantiomeric purity of more than about 85%. Specifically, the process of the present application produces amino acid derivative of formula (1) in an enantiomeric purity of more than about 90%. More specifically, the process of the present application produces amino acid derivative of formula (1) in an enantiomeric purity of more than about 95%. Most specifically, the process of the present application produces amino acid derivative of formula (1) in an enantiomeric purity of more than about 99%.

DEFINITIONS
The following definitions are used in connection with the present disclosure unless the context indicates otherwise.

“Chemo-enzymatic” is defined as a chemical reaction wherein an enzyme is used as a catalyst.

“Halogen” is defined as non-metallic elements found in group VII of the periodic table and is selected from fluorine, bromine, chlorine and iodine.

“Hydroxy” is defined as the group –OH.

“Nitrile” is defined as the group –CN.

“Trifluoromethyl” is defined as the group –CF3.

“Alkyl” is defined as straight or branched chain alkyl having 1-8 carbon atoms. Examples of alkyl chain are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.

“Alkenyl” is defined as an alkyl group, as defined above, containing one or more double bonds.

“Alkynyl” is defined as an alkyl group, as defined above, containing one or more triple bonds.

“Aryl” is defined as monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" also refers to heteroaryl ring systems. The term “heteroaryl” refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members.

“Alkoxy” is defined as an alkyl group or an alkenyl group or an alkynyl group, as previously defined, attached to the principal carbon chain through oxygen.

“Aralkyl” is defined as an aryl group, as previously defined, attached to the principal carbon chain through an alkyl group.

“Aralkoxy” is defined as is defined as an aryl group, as previously defined, attached to the principal carbon chain through oxygen.

“Aryloxyalkyl” is defined as an aryl group, as previously defined, attached to an oxygen atom which in turn is attached to the principal carbon chain through an alkyl group or an alkenyl group or an alkylnyl group, as defined above.

“Dynamic kinetic resolution” is a kinetic resolution that results in a pure enantiomer starting from a racemic compound.

“Enantiomeric purity” of a compound is measured in terms of “enantiomeric excess” (e.e.) of the compound. “Enantiomeric excess” denotes the degree to which a compound contains one enantiomer in greater amounts than the other. The enantiomeric excess (e.e.) of a compound is calculated by the following formula:
e.e. = ((R-S)/(R+S)) × 100;
wherein, R and S stand for the individual optical isomer in the mixture (and R + S = 1).

Certain specific aspects and embodiments are further described by the following examples, being provided only for purposes of illustration, and the scope of the disclosure is not intended to be limited by the examples.

EXAMPLES
Example 1: Preparation of (S)-4-bromophenyl alanine
Ethyl ester of 4-bromo phenylalanine (300 mg) was dissolved in a mixture of acetonitrile (12.5 mL) and water (12.5 mL). The pH of the reaction mixture was adjusted to 7.5. To the reaction mixture and 3,4-dinitrosalicylaldehyde (10 mg) was added. The reaction mixture was heated to 35 °C for 5 minutes and alcalase enzyme (100 mL) has been charged into the reaction mixture. It was noticed after 15 minutes that the pH of the reaction mixture was dropped and it was adjusted to 7.5 again. The reaction mixture was stirred for about 6 hours and 30 minutes and was left overnight at about 20 °C. The pH of the reaction mixture was then adjusted to 5.6 and the solvent was evaporated in rotavapor at 50 °C. To the residue, 1,2-dimethoxy ethane (5 mL) was added, heated to 50 °C for about 90 minutes and then cooled to about 20-25 °C. A solid was precipitated out, which was filtered and dried to provide the title compound.
Enantiomeric Excess: 97.5 %.

Example 2: Preparation of amino acid derivative of formula (1)
Ethyl 3-([1,1'-biphenyl]-4-yl)-2-aminopropanoate hydrochloride salt (2A, 1 g) was dissolved in a mixture of acetonitrile (10 mL) and water (10 mL). The pH of the reaction mixture was adjusted to 7.5. To the reaction mixture and 3,4- dinitrosalicylaldehyde (19 mg) was added. The reaction mixture was heated to 35 °C for 5 minutes and alcalase enzyme (200 mL) has been charged into the reaction mixture. The pH of the reaction mixture was checked periodically and adjusted to 7.5. The reaction mixture was stirred for about 6 hours and 30 minutes and was left overnight at about 20 °C. The pH of the reaction mixture was then adjusted to 5.6 and the solvent was evaporated in rotavapor at 50 °C. To the residue, 1,2-dimethoxy ethane (15 mL) was added, heated to 50 °C for about 90 minutes and then cooled to about 20-25 °C. A solid was precipitated out, which was filtered and dried to provide the title compound.
Yield: 850 mg
Enantiomeric Excess: 97.1 %.

Example 3: Preparation of amino acid derivative of formula (1)
Ethyl 3-([1,1'-biphenyl]-4-yl)-2-aminopropanoate hydrochloride salt (2A, 2.7 g) was dissolved in a mixture of acetonitrile (25 mL) and water (25 mL). The pH of the reaction mixture was adjusted to 7.5. To the reaction mixture and 3,4- dinitrosalicylaldehyde (51 mg) was added. The reaction mixture was heated to 35 °C for 5 minutes and alcalase enzyme (1 mL) has been charged into the reaction mixture. The pH of the reaction mixture was checked periodically and adjusted to 7.5. The reaction mixture was stirred for about 24 hours. The pH of the reaction mixture was then adjusted to 5.5 and the solvent was evaporated in rotavapor at 50 °C. To the residue, 1,2-dimethoxy ethane (15 mL) was added, heated to 50 °C for about 90 minutes and then cooled to about 20-25 °C. A solid was precipitated out, which was filtered and dried to provide the title compound.
Yield: 2.5 g
Enantiomeric Excess: 99.3 %.

Example 4: Preparation of amino acid derivative of formula (1)
Ethyl 3-([1,1'-biphenyl]-4-yl)-2-aminopropanoate hydrochloride salt (2A, 8 g) was dissolved in a mixture of acetonitrile (100 mL) and water (100 mL). To the reaction mixture 3,4- dinitrosalicylaldehyde (145 mg) and alcalase enzyme (2 mL) was added. The pH of the reaction mixture was adjusted to 7.5. The reaction mixture was stirred at room temperature for 24 hours. The pH of the reaction mixture was checked periodically and adjusted to 7.5. The reaction mixture was stirred for about 24 hours. The pH of the reaction mixture was then adjusted to 5.56 and the solvent was evaporated in rotavapor at 55 °C. To the residue, 1,2-dimethoxy ethane (30 mL) was added, stirred for some time and filtered to provide a solid. Water (30 mL) was added and the pH was adjusted to 5.54. The reaction mixture was distilled at 55 °C. To the residue, 1,2-dimethoxy ethane (30 mL) was added and stirred for 10 minutes. A solid was precipitated out, which was filtered, washed with 1,2-dimethoxy ethane (30 mL) and dried to provide the title compound.
Enantiomeric Excess: 98.1 %.

Example 5: Preparation of amino acid derivative of formula (1)
Ethyl 3-([1,1'-biphenyl]-4-yl)-2-aminopropanoate hydrochloride salt (2A, 1 g) was dissolved in a mixture of tetrt-butanol (16 mL) and water (0.8 mL). To the reaction mixture lithium carbonate (180 mg), zinc acetate (44 mg) and picolinaldehyde (40 mL) was added one after the other. Alcalase enzyme solution (4 mL) was added to the reaction mixture. The reaction mixture was stirred for about 24 hours at room temperature. Tert-butanol (15 mL) was added to the reaction mixture and the precipitated solid was filtered, dried to afford the title compound.
Yield: 800 mg
Enantiomeric Excess: 95 %.

Example 6: Preparation of amino acid derivative of formula (1)
Ethyl 3-([1,1'-biphenyl]-4-yl)-2-aminopropanoate hydrochloride salt (2A, 7.5 g) was dissolved in a mixture of acetonitrile (90 mL) and water (90 mL). The pH of the reaction mixture was adjusted to pH 7.5 by the addition of aqueous solution of sodium hydroxide (4N). To the reaction mixture 3,4- dinitrosalicylaldehyde (143 mg) and alcalase enzyme (1.5 mL) was added. The pH of the reaction mixture was checked periodically and adjusted to 7.5. The reaction mixture was stirred for about 24 hours. The pH of the reaction mixture was then adjusted to 5.56 and the solvent was evaporated in rotavapor at 55 °C. To the residue, 1,2-dimethoxy ethane (20 mL) was added and stirred for 10 minutes. The precipitated solid was filtered and washed with a mixture of 1,2-dimethoxy ethane (20 mL) and methane sulfonic acid (1 mL) to provide the title compound.
Yield: 6.5 g
Enantiomeric Excess: 97.6 %.

Example 7: Purification of Compound of formula (1)
To a suspension of compound of formula (1) (500 mg, enantiomeric excess: 99.26%) in 1,2-dimethoxy ethane (5 mL), methane sulfonic acid (200 mL) was added and the suspension was stirred for 5 minutes. The solid was filtered and washed with 1,2-dimethoxy ethane (5 mL) and dried to provide a pure compound of formula (1).
Enantiomeric Excess: 99.9 %.
,CLAIMS:WE CLAIM:
1. A chemo-enzymatic process for the preparation of amino acid derivative of formula (A) from an amino acid ester derivative of formula (B) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is C1-C6 alkyl ester or benzyl ester; n is 0 or 1; and R2 is halogen or aromatic group optionally substituted with halogen, alkyl, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile or trifluoromethyl.

2. A chemo-enzymatic process for the preparation of amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is C1-C6 alkyl ester or benzyl ester.
3. The chemo-enzymatic process of claim 1 or claim 2, wherein the enzyme is an alcalase enzyme.
4. The chemo-enzymatic process of claim 1 or claim 2, wherein the aldehyde may be selected from a group consisting of 5-dinitrosalicylaldehyde and picolinaldehyde.
5. The chemo-enzymatic process of claim 1 or claim 2, wherein the metal complex of aldehyde is zinc acetate and aldehyde.
6. The chemo-enzymatic process of claim 1 or claim 2, wherein the pH is from about 6 to about 7.5.
7. A process for the preparation of LCZ-696 comprising:
(a) chemo-enzymatic process for the preparation of amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in presence of an aldehyde or a metal complex of aldehyde

wherein, R1 is as above; and
(b) converting the amino acid derivative of formula (1) to LCZ-696.
8. The chemo-enzymatic process of claim 7, wherein the enzyme is an alcalase enzyme.
9. The chemo-enzymatic process of claim 7, wherein the aldehyde may be selected from a group consisting of 5-dinitrosalicylaldehyde and picolinaldehyde.
10. The chemo-enzymatic process of claim 7, wherein the metal complex of aldehyde is zinc acetate and aldehyde.