FORM2
THE PATENT ACT 1970 (39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
An enzymatic process for the preparation of Ramelteon
2. APPLICANT (S)
(a) NAME: Enaltec Labs Pvt. Ltd.
(b) NATIONALITY:
An Indian Company incorporated under the Indian Companies ACT 1956
(c) ADDRESS:
Enaltec Labs Pvt. Ltd., 17th Floor, Kesar Solitaire, Plot No. 5, Sector 19, Sanpada, Navi Mumbai- 400705, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

Technical field of the invention:
The present invention relates to an enzymatic process for the preparation of Ramelteon, compound represented by structural formula (I).

Background of the invention:
Ramelteon is chemically known as (S)-N-[2-(l,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]propionamide. It is Melatonin Receptor Agonists indicated for the treatment of the treatment of primary insomnia. Commercially sold under the brand name ROZEREM® in US and was first disclosed in U.S. patent number 6,034,239 and is represented by structural formula I.

U.S. patent number 6,034,239 discloses the formation of chiral intermediates (S)-(-)-
N-[2-(l,6,7,8,-tetrahydro-2H-indeno[5,4-b]furan-8-yl)ethylamine hydrochloride;
compound represented by structural formula (III) by the catalytic asymmetric hydrogenation of 2-(l,2,6,7,-tetrahydro-8H-indeno[5,4-b]furan-8-ylidene)ethylamine;

compound represented by structural formula (II) in the presence of a catalytic amount of BINAP-ruthenium complex. The yield of the compound represented by structural formula (III) is obtained in approximately 89% enantiomeric excess. Further, the compound represented by structural formula (III) is purified by preparing acid salts and acylated with propionyl chloride to obtain Ramelteon; the compound represented by structural formula (I) which is further purified using column chromatography to obtain Ramelteon; compound represented by structural formula (I) in its pure (S) isomer form.

The chemical process disclosed in said patent involves use of costlier ruthenium complex including use of other reagent and solvent. This creates need for development of cost effective, environment friendly method for synthesis of Ramelteon. Further the process disclosed in the said prior art reference involves the use of hazardous chemicals and tedious column chromatography hence prior art method for the synthesis of the compound represented by structural formula I from the compound represented by structural formula II is economically not viable.
Journal of Bioscience and Bioengineering (2002), 93(1), 44-47 discloses the preparation of amine intermediate of Ramelteon; compound represented by structural formula (VIII) having S-configuration by enzymatic resolution using 'Bacillus sp. SUI-12' enzyme subsequently Ramelteon compound represented by structural formula (I) is prepared.


This process requires longer period of time of 18 hours for completion and product required purification by column chromatography which makes process tedious and economically not viable.
PCT publication WO2008062468 discloses the process for preparation of Ramelteon involves separation of the enantiomers of intermediate compound represented by structural formula (VII) by optical resolution of the racemic amine intermediate compound represented by structural formula (VII) by preparing acid salts with chirally pure acids; or by chromatographic techniques using chiral and/or achiral stationary phases for batch process, super critical or sub critical chromatography and/or continuous process chromatography. WO'468 discusses the possible use of optical resolution with chirally pure acids, but does not discuss about this method. However, WO'468 provides detailed descriptions of chromatographic methods for separating the isomers of intermediate compound represented by structural formula (VII). But preparative chromatography is time consuming and expensive. There is need of highly sophisticated instrumentation for preparative chromatography. Therefore the said process is not commercially viable.


U.S. patent application US2011207949 discloses the preparation of (S)-Ramelteon via resolution of (S)-N-2-(l,6,7,8-tetrahydro-2H-indeno 5,4-b furan-8-yl)ethylamine using (S)-(+)-2-(4-isobutylphenyl) propionic acid in solvent further which is converted into S isomer of Ramelteon compound represented by structural formula (I). However US'949 is silent on yield of resolution step which was found to be on lower side. Hence process of US'949 is not commercially viable.

PCT publication WO2015144902 discloses the conversion of the amine to its corresponding amide by acylating the amine with an acylating agent by method involving use of organocatalyst and lipase enzyme.
Resolution of racemic mixtures via reaction with optically active acids and the subsequent crystallization of the resulting salts is preferably employed when the chiral carbon of the racemic compound is an alpha carbon to the functional group

forming the acid addition salt. As the distance between the chiral carbon of the racemic compound to the functional group of the racemic compound increases to beta & gamma, the resolution of the diastereomeric salt using optically active acids becomes more difficult.
Ramelteon has a chiral center at the gamma carbon, which makes the separation of the isomer with an optically active acid quite a difficult task. Similarly, N-[2-(l,6,7,8,-tetrahydro-2H-indeno [5,4-b]furan-8-yl)]ethylamine; compound represented by structural formula VII, an intermediate useful in the production of Ramelteon has a chiral center at the gamma carbon which would lead a skilled artisan to believe that optical resolution with an optically active acid could prove difficult.
Processes disclosed in prior art involves the use of expensive catalyst and give poor enantioselectivity. Further, these processes are lengthy and expensive due to the need to perform multiple purifications steps in order to achieve a greater enantiomeric purity of the desired isomer.
Accordingly there is a need in the art to develop a process of preparing Ramelteon compound represented by structural formula (I), which is simple, economic, safe and industrially viable providing substantially pure Ramelteon with high yield. There is need to develop process including enzymatic transformation to obtain Ramelteon as enzymes are perfectly stereoselective and stereospecific which provides the final product with maximum purity according to its absolute configuration. Enzymes are more stereoselective than chemical catalysts and therefore desired stereochemistry of the product is much better with enzymes.

Object of the invention:
i) An object of the present invention is to provide a new, simple, economic,
safe and industrially viable enzymatic process for preparation of Ramelteon; compound represented by structural formula I.

ii) Another object of present invention is to provide enzymatic process for preparation of compound represented by structural formula XIV having enantiomeric purity of 99.5% which is further transformed to Ramelteon; the compound represented by structural formula I

Summary of the invention:
First aspect of the present invention is to provide a new, simple, economic, safe and industrially viable enzymatic process for preparation of Ramelteon; compound represented by structural formula (I).
Second aspect of the present invention a process for preparation of Ramelteon; the compound represented by structural formula (I):


comprises,
reacting a compound represented by structural formula (VII)

with ester in presence of enzyme in solvent.
Fourth aspect of the present invention is to provide a novel process for preparation of Ramelteon; compound represented by structural formula (I) comprising, enzymatic enantioselective amidation of compound represented by structural formula (VII) using enzyme to obtain Ramelteon; compound represented by structural formula (I) in its pure (S) form.
Detailed description of the invention:
According to first aspect of the present invention is to provide a new, simple, economic, safe and industrially viable enzymatic process for preparation of Ramelteon; compound represented by structural formula (I).

According to Second aspect of the present invention is to provide a process for preparation of Ramelteon; the compound represented by structural formula (I):

comprises
reacting a compound represented by structural formula (VII)

with ester in presence of enzyme in solvent.
The compound represented by structural formula (VII) may be prepared as per processes disclosed in PCT publications WO2010092107 and WO2008062468.
In accordance with an embodiment of the present invention, the ester used is selected from group consisting of vinyl acetate, vinyl propionate, and vinyl butyrate.
In accordance with an embodiment of the present invention, wherein reaction of the compound represented by structural formula (VII) with an ester selected from vinyl propionate is carried out in presence of enzyme in a solvent to obtain Ramelteon; compound represented by structural formula (I) in the single step.


In accordance with an embodiment of the present invention wherein;
a) reaction of the compound represented by structural formula (VII) with an ester
selected from vinyl acetate, vinyl butyrate in presence of enzyme is carried
out in a solvent to obtain the compound represented by structural formula
(XIV);

b) hydrolyzing the compound represented by structural formula (XIV) in
presence of an acid to obtain a compound represented by structural formula
(VIII); and


c) reacting the compound represented by structural formula (VIII) with a propionic anhydride to obtain Ramelteon; compound represented by structural formula (I).

In accordance with an embodiment of the present invention; the enzyme used is selected from Lipase, Amylase and Protease.
In accordance with an embodiment of the present invention; the solvent used is selected from organic solvents such as ethers selected from the group consisting of diethyl ether, methyl tert-butyl ether, di-isopropyl ether and alcohols selected from the group consisting of methanol, ethanol, isopropanol and butanol.
In accordance with an embodiment of the present invention; wherein reaction is carried out at a temperature in the range of 20-40°C.
In accordance with an embodiment of the present invention; wherein reaction is carried out for 6 to 48 hours.
In accordance with an embodiment of the present invention; wherein compound represented by structural formula (XIV) is isolated by steps of filtration, purification, centrifugation, drying or combination thereof.
In accordance with an embodiment of the present invention; wherein the compound represented by structural formula (XIV) can be optionally purified in alcohol solvent

selected from the group consisting of methanol, ethanol, isopropanol and butanol or ester solvent selected from the group consisting of ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, tertiary butyl acetate and pentyl acetate.
In accordance with an embodiment of the present invention; wherein the acid used in the steps b) is selected from group consisting of hydrochloric acid, sulfuric acid and nitric acid.
In accordance with an embodiment of the present invention; wherein the reaction
of step b) is carried out at a temperature in the range of 40-80°C for a period of 5-10
hours.
In accordance with an embodiment of the present invention; wherein the product of
step b) compound represented by structural formula (VIII) is optionally isolated or
instead preferably used for step c) in organic layer without isolation.
In accordance with an embodiment of the present invention; wherein the reaction of step c) the compound represented by structural formula (VIII) is reacted with propionic anhydride is carried out at a temperature in the range of-5°C to 10°C for a period of 30 minutes to 2 hours.
The product of step c) i.e. Ramelteon; compound represented by structural formula (I) is isolated by steps of filtration and purification in alcohols selected from the group consisting of methanol, ethanol, isopropanol and butanol or ester solvent selected from the group consisting of ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, tertiary butyl acetate and pentyl acetate.

Example:
The present invention is described in the examples given below; further these are provided only to illustrate the invention and therefore should not be construed to limit the scope of the invention.
Example 1: Preparation of Ramelteon (I)
To a solution of 2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethanamine (20 g) and vinyl propionate (100 g) in methyl tert-butyl ether (150 ml) was added lipase enzyme (2 g). The reaction mixture stirred at 25- 30°C for 36 hrs. Filtered the reaction mixture and filtrate was distilled out and the obtained Ramelteon was isolated by filtration. The isolated (S)-Ramelteon (I) was purified from ethanol or ethyl acetate.
Yield: 10.20 g, Purity: 99.7%, Enantiomeric purity: 99.8%
Example 2: Preparation of Ramelteon (I)
Step-a: Preparation of (S)-N-(2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethyl)acetamide
To a solution of 2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethanamine (20 g) and vinyl acetate (85 g) in methyl tert-butyl ether (150 ml) was added lipase enzyme (0.2 g). The reaction mixture stirred at 25-30°C for 40 hrs. Filtered the reaction mixture and filtrate was distilled out and obtained (S)-N-(2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethyl)acetamide was isolated by filtration. The isolated compound was purified from ethanol or diisopropyl ether. Yield: 10.6 g, Enantiomeric purity: 99.5%
Step-b: Preparation of (S)-2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethanamine

To a solution of (S)-N-(2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethyl)acetamide (10.5 g) in methanol (50 ml) was added cone. Hydrochloric acid (150 ml). The reaction mass was stirred at 60-70°C for 6 - 8 hrs. The resulting reaction mass was extracted with methyl tert-butyl ether (100 ml) and the organic layer containing (S)-2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethanamine (V) was used for step-C.
Step-c: Preparation of Ramelteon (I)
To the Methyl tert-butyl ether layer of (S)-2-(2,6,7,8-tetrahydro-lH-indeno[5,4-b]furan-8-yl)ethanamine was added propionic anhydride (10.6 g) at 0-5°C and stirred for 45 -90 minutes. The resulting reaction mixture was distilled out 50 ml methyl tert-butyl ether and then cooled to 0-10°C. Filtered the reaction mass to isolate (S)-Ramelteon (I). Isolated (S)-Ramelteon (I) was purified from ethanol or ethyl acetate. Yield: 9.69 g, Purity: 99.5%, Enantiomeric purity: 99.8%

We claim:
1. A process for preparation of Ramelteon; the compound represented by
structural formula (I):

comprising,
reacting a compound represented by structural formula (VII)

with ester in presence of enzyme in solvent.
2. The process as claimed in claim 1 wherein ester used is selected from group consisting of vinyl acetate, vinyl propionate, vinyl butyrate.
3. The process as claimed in claim 1; wherein ester selected from vinyl propionate to obtain Ramelteon; compound represented by structural formula (I) in a single step.
4. The process as claimed in claim 1; wherein ester selected from vinyl acetate, vinyl butyrate comprises the steps of,

a) reacting a compound represented by structural formula (VII) with an ester
selected from vinyl acetate, vinyl butyrate to obtain the compound
represented by structural formula (XIV);

b) hydrolyzing the compound represented by structural formula (XIV) in
presence of an acid to obtain a compound represented by structural
formula (VIII); and

c) reacting the compound represented by structural formula (VIII) with a
propionic anhydride to obtain Ramelteon; compound represented by
structural formula (I).


5. The process as claimed in claim 1; wherein enzyme is selected from the group consisting of Lipase, Amylase and Protease.
6. The process as claimed in claim 1; wherein solvent used is ether selected from the group consisting of diethyl ether, methyl tert-butyl ether, di-isopropyl ether or alcohol selected from the group consisting of methanol, ethanol, isopropanol and butanol.
7. The process as claimed in claim 1; wherein reaction is carried out at a temperature in the range of 20-40°C for a period of 6 to 48 hours.
8. The process as claimed in claim 4 wherein acid used in the steps b) is selected from group consisting of hydrochloric acid, sulfuric acid and nitric acid.
9. The process as claimed in claim 1 wherein Ramelteon; compound represented by structural formula (I) is isolated by steps of filtration and purification in alcohols selected from the group consisting of methanol, ethanol, isopropanol and butanol or ester solvent selected from the group consisting of ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, tertiary butyl acetate and pentyl acetate.
10. The process as claimed in claim 3 wherein the compound represented by structural formula (VII) reacted with vinyl acetate in methyl tert-butyl ether in presence of lipase enzyme.