FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10, Rule 13]
A NOVEL PROCESS FOR SYNTHESIS OF A
SUBSTITUTED CYCLOPROPANE
INTERMEDIATE AND A PROCESS FOR SYNTHESIS OF PREGABALIN FROM THE SAME;
PHARMED MEDICARE PVT. LTD., A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS 141 WALCHAND HIRACHAND MARG, MUMBAI -400 001, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION
DESCRIBES THE INVENTION.
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TECHNICAL FIELD
The present invention is directed to new "substituted cyclopropane" intermediate and a method for stereoselective bioconversion to the "S" isomer of y-amino acids.
DESCRIPTION
Pregabalin is a Gamma-aminobutyric acid (GABA) analogue that is reported to prevent and treat a range of diseases in various patents.
Application of pregabalin has been disclosed in US 5563175 in treating seizure disorders, in US 6127418 to dramatically reduce the gastrointestinal damage caused by drugs and alcohol, treat the conditions resulting from ethanol withdrawal syndrome, and Gl disorders characterized as inflammatory bowel disorders, functional bowel disorders, dyspepsia, visceral pain; in US 6194459 for treating gastrointestinal damage and disorders and a composition comprising a GABA analog and a non-steroidal antiinflammatory drug; in US 6194459 for treating physiological conditions associated with the use, or sequelae of use, of psychomotor stimulants including cocaine, amphetamine and like addictive drugs/substances; in US 6242488 for preventing or treating pain comprising a GABA analog and a non-steroidal anti-inflammatory drug such as a composition comprising pregabalin and naproxen sodium; in US 6306910 for treating insomnia; in US 6326374 for eliciting an enhanced analgesic response comprising administering an analgesically effective amount of a GABA analog; and caffeine; in US 6329429 for preventing and treating inflammatory diseases; in US 6359005 for treating mania and treating and preventing bipolar disorder; in US 6372792 for preventing or treating alcoholism, irritable bowel disorder or irritable bowel syndrome; in US 6451857 as an anti-epileptic compound; in US 6566400 for treating physiological conditions associated with the use, or sequelae of use, of psychomotor stimulants including cocaine; in US 6593368 for treating acute pain by a combination, comprising a synergistic amounts of gabapentin and celecoxib; in US 6620829 for treating non-
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inflammatory cartilage damage; in US 6680343 for treating renal colic; in US 6887902 for treating inflammatory diseases arthritis and rheumatoid arthritis; in US 6992109, for treating urinary incontinence; US 7026505 for treating tinnitus. Pregabalin is also shown to be effective in combination with NMDA receptor antagonists (US 6942876), in buccal sprays (US 6977070); as lactose conjugates (US 7022678) in combination with 2-(7-chloro-1,8-naphthyridin-2-yl)-3-(5-methyl-2-oxo-hexyl)-1-isoindolino-ne or 2-(7-chloro-1,8-naphthyridine-2-yl)-3-(5-methyl-5-hydroxy-2-oxohexyl- )-1 -isoindolinone for obsessive-compulsive disorder (US 7026332); and can be gave as an ingredient of a liquid pharmaceutical composition. Many more examples not mentioned here exist and may be added in future on applications of pregabalin.
The therapeutic effects of pregabalin including seizure suppressive effect while avoiding the undesirable side effect of ataxia is attributed to the S-enantiomer, the racemic compound (.+-.)-3-(aminomethyl)-5-methylhexanoic acid (also known as racemic isobutyl-GABA or also known as Pregabalin),. With the S-enantiomer having better anticonvulsant activity than the R-enantiomer, the commercial utility of pregabalin requires an efficient method for directly preparing the S-enantiomer substantially free of the R-enantiomer or comprises following up of the synthesis process that results in a recemic mixture with a method of resolution of the two enantiomers from each other.
Pregabalin, 3-(aminomethyl)-5-methylhexanoic acid, is a compound having chemical Structure (I)

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It is also titled as CI-1008 assigned by Park and Davis. It has y-aminobutyric acid functionality, which is considered to be responsible for the desired activity. S-pregabalin having structure II, given in the following,

is useful as an anticonvulsant because it has been found to activate GAD (L-glutamic acid decarboxylase) promoting the production of gamma-aminobutyric acid (GABA), one of the brain's major inhibitory neurotransmitters. It is expected to show several times higher activity than Neurontin. Pregabalin has analgesic as well as anxiolytic activity.
Several processes for synthesis of (S)-(+) pregabalin are known, some of which are given in DRUGS OF FUTURE, 24 (8), 862-870(1999). One such process includes 1,3-isobutyl glutaric acid conversion into corresponding anhydride, by treatment with refluxing acetic anhydride. The monoamide formed after NH4OH treatment is resolved and subjected for Hoffmann degradation provides (S)-(+) pregabalin. This process differs from this invention that comprises cyclopropane ring'formation as intermediate and followed by ring opening by reduction to yield racemic pregabalin while the bioconversion yields the desired active isomer.
US patent No. 5563175, U.S. 5,599,973, US 5,563,175, U.S.5,684,189, U.S. 6,197,819, U.S.2003225161, WO9209560 and W09323383 disclosed synthesis of pregabalin using azide as an intermediate. The enantioselectivity of the pregabalin is achieved by 4-methyl 5-phenyl oxazolidinone ring formation. The process disclosed comprises the steps of forming an acid chloride of an acid of the formula HOC(= 0)CH(Ri)(R2) (where wherein R-i is a straight or branched alkyl of from 1 to 6 carbon atoms, phenyl, or
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cycloalkyl having from 3 to 6 carbon atoms; R2 is hydrogen, methyl, or carboxyl) the corresponding acid chloride having the formula CIC(= 0)CH(Ri)(R2), adding the acid chloride to a solution of (4R,5S)-(+)-4-methyl-5-phenyl-2-oxazolidinone and n-butyllithium at -78.degree. C. under argon to produce an oxazolidinone derivative, treating the oxazolidinone derivative with benzyl .alpha.-bromoacetate to produce an ester, treating the ester with hydrogen peroxide and lithium oxide followed by treatment with sodium metabisulfite to produce a compound, treating the compound with borane dimethyl sulfide complex to produce the corresponding tosylate from the compound, forming an azide from the corresponding tosoylate and reducing the azide to the amine. In general, this method can be represented by following route for steriospecific pregabalin synthesis;



Phs

CH,



HO

this method involves nine steps in the synthesis and enantioselectivity is obtained by using chiral reagent like oxazolidinone along with some hazardous reagents like N-BuLi (n-butyllithium), LiOH. However, this process is limited to small scale and is not
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practical for large-scale synthesis because it employs costly reagents which are difficult to handle.
In another prior art method, 3-alkyl-4-aminobutanoic acid is prepared via Michael addition between nitromethane and 2-alkenoic ester followed reduction of nitro group to amine with 10% palladium on carbon (Andruszkiewicz et al., Synthesis, 1989, 953) by following reaction.

This prior art differs from the process of this invention that comprises preparation of nitroalkene and Michael addition type reaction of sulfur ylide.
According to the US patent no. 5637767 and 5840956, 3-(aminomethyl)-5-methylhexanoic acid is prepared in four steps from isovaleraldehyde that include condensation, cyanide addition, selective monocarboxylation and reduction of cyanide to amine followed by resolution with (S)-mandelic acid. This prior art method comprises use the hazardous metal cyanides as amine source. The process of this invention avoids use of the toxic metal cyanide as it proceeds through cyclopropane ring opening metathesis using nitromethane as amine source.
Yet another process disclosed in patent U.S. 5629447 and U.S. 5616793 follows the same path that includes opening of cyclic anhydride with ammonia followed by resolution and then Hoffmann rearrangement lead to (S)-(+)-3-aminomethyl-5-methylhexanoic acid as per following route of reaction:

The process of this invention, as distinct from the prior art process, does not involve Hoffmann rearrangement and resolution but the bioconversion of 2-aminomethyl-5-methylhexanecyanide will directly gives the pure S-enantiomer.
According to the patent WO 03/093220, pregabalin was prepared from highly substituted butyrolactams. Butyrolactams were prepared from reductive amination of mucobromic acid by ammonium formate. Route of reactions used is shown in the following :

This prior art process is very much different than the process of this invention that
comprises reductive cyclopropane ring opening. In yet another process for
enantioselective synthesis of pregabalin is an asymmetric hydrogenation process. The
process mentioned in U.S. 6891059 uses chiral catalyst [(R,R)-
MeDuPHOS]Rh(COD)BF4]for asymmetric hydrogenation of ethyl-3-cyano-5-methylhex-3-enoate to afford enantiomerically pure isomer of pregabalin. This patent also mentions the use of sponge Ni for stereoselective reduction of cyanohexenoate ester. Route of reaction used is as follows:

,CN

vCO,K

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Process of this invention, as distinct from this prior art, comprises reduction of nitro group by simple Raney Ni, and stereochemistry is imparted by bioconverion of nitrile group to acid.
Burk et al., in WO 99/31041 and WO 99/52852, describes the asymmetric hydrogenation of .beta.-substituted and .beta.,.beta.-disubstituted itaconic acid derivatives to provide enantiomerically enriched 2-substituted succinic acid derivatives which is intermediate precursor for pregabalin.
The patent U.S.7141695 discloses the synthesis and use of substituted acrylic acid esters for synthesis of pregabalin as well as gabapentin. The process proceeds through lactam intermediate followed by ring opening by hydrolysis. This process provides racemic pregabalin and the process is shown in the following:

C02Et
H3C

?H,

.NO,
X02Et

N07

H3C

Patent US 6488964 refers to the manufacture of coated pregabalin.
The method mentioned in patent WO 2003/104184 discloses method for synthesis of
acyloxy
derivatives of GABA analogues.
A process published by G. M. Sammis, et al., J. Am. Chem. Soc. 125(15), 4442-3(2003)
takes advantages of the asymmetric catalysis of cyanide conjugate addition reactions.
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The method discloses the application of aluminium salen catalyst to the conjugate addition of hydrogen cyanide to a,b-unsaturated imides . Reportedly TMSCN is useful source of cyanide that can be used in place of hydrogen cyanide. The route of reaction is given below:


TMSCN, i PrOH, cat.

O O
Ph N H

CN



-^

Recent studies (Li, et al., J. Am. Chem. Soc, 126(32), 9906-07 (2004). have indicated that cinchona alkaloids are broadly effective in chiral organic chemistry. A range of nitro alkenes were reportedly treated with dimethyl or diethyl malonate in tetrahydrofuran in presence of cinchona alkaloids to provide high enantiomeric selectivity as shown below:

R3'

,

Where R3 may be several alkyl; aryl groups

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The above mentioned prior art processes use (a) chiral auxiliary such as oxazolidinone and proceed through azide intermediate with several steps, or (b) make use of asymmtric reduction of cyano hexenoate derivative, or (c) proceed thorough anhydride formation from glutaric acid followed by ring opening with ammonia and Hoffmann rearrangement and resolution.
The method of this invention proceeds through an entirely different route that comprises formation of a di-substituted cyclopropane ring, shown as structure III in the following, which in this invention is used for the first time as a precursor of pregabalin:

III
wherein, R1=Alkyl, Aryl, allyl, heterocycle, straight chain or branch chain, X= may be -CN, or-COOR wherein R may be straight or branch chain alkyl, aryl, groups
When X = -CN, the compound is a new chemical entity.
First step of the novel route to prepare pregabalin comprises reductive ring opening of compound II to, form a compound of structure IV; the said reductive ring opening being achieved using reducing agent like Raney Ni/ H2 or Pd/C or Zn/HCOONH4 or Zn/HCOOH or Fe/CH3COOH or Fe/HCI at 25 °C in polar solvent for the first time:
reduction

III IV
When R1 = isopropyl group and X = -COOEt, the result of the reaction is formation of a recemic mixture of pregabalin.
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When R1 = isopropyl group and X= -CN , the reaction also results into racemic pregabalin
The compound of structure IV leads to two routes of further conversion to pregabalin.
In a chemical process, when X = -COOEt, and R1 = isopropyl group the compound of structure IV that is formed by a chemical process of reductive ring opening described above is a recemic mixture of pregabalin. When X = -CN, and R1 = isopropyl group either one more step of conversion of -CN to -C(0)NH2 (nitrile to amide) followed by reduction and hydrolysis of the amide group to carboxylic acid group or direct conversion of nitrile to acid also leads to racemic pregabalin. The alternative routes available for such a production of recemic mixture from a compound of structure III are summarized below:

COOH
Reduction
& Hydrolysis
X = -COOR
NH,

CH3 ^

The recemic mixture formed by chemical process can be subjected to a prior art process of resolving them to get (S)-(+) pregabalin, including use of agent for resolution like (S)-(+)-mandelic acid in IPA /Water or (+)-a-phenyl ethylamine in ethanol/chloroform or/-ephedrine:
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-'NH2
COOH u ^/\^\^COOH
QH3 ■£ Resolution

When X= -CN, and R1 = isopropyl group compound of structure IV, this invention opens up and shall include in it, another possibility of subject the compound of structure IV having nitrile group at first carbon atom to the action of nitrilase that may regioselctively as well as sterioselectively convert nitrile group to carboxyl group by enzymatic action of nitrilase leading to production of (S)-(+) pregabalin as the only isomer as shown below:

-NH2 ^NH2
Bioconversion r
^ Rl.
IV I,
Work on standardization of this component of the invention is in progress and shall be completed before filing of complete specification.
A further embodiment of this invention comprises use of enzyme catalyst isolated from microorganisms. Yet further embodiment of this invention comprises isolation of the said enzyme from a microorganism of one or more of a group including, but not limited to, Rhodococcus species, Pseudomonas species, Arthrobactor species, Bacillus species and Aspargillus nigar.
There are several methods available in prior art for achieving synthesis of asymmetric cyclopropane ring (Marian Mikolajczyk, Pure and applied chemistry 2005, 77; Armando Cordova ef a I, (Advanced Synthesis and Catalysis, 2007, 349, 1028-1032; Jurgen Zindel et a/, (Tetrahedron Letters, 1993, 34, 1917-1920); G. H. Kulkarni, SYNTHESIS 1995, 1545-1548, G. B. Payne, J. Am. Chem. Soc. 1967, 32, 3351-3355). The method
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of preference in this invention for achieving synthesis of asymmetric cyclopropane ring formation comprises its formation from nitroalkene of structure V and sulphurylide through Michael type addition reaction as shown in the following:
RI /-c^ NO R2YCH2X |Base \^ ^-^
V III
Wherein,
R2 = Alkyl, aryl
Y=PorS.
X is as defined as above.
The nitroalkene of Structure (V) when treated with sulphur or phoshphorus ylide under basic condition at reflux temperature in chlorinated solvent preferably in dichloromethane affords the compound of structure II!.
Sulphur or phosphorus ylide can be prepared preferably by a prior art process comprising mixing ethyl bromoacetate and dimethylsulfide with or without solvents, more preferably without solvent. Thereafter neutralizing the sulfonium salt by using saturated potassium carbonate and NaOH solution at 0-25°C.
Preferably the compound of Structure V is prepared by known standard procedure as mentioned by Kulkarni (1995) (Synthesis 1995, p. 1545). Under basic condition preferably 50% aqueous. NaOH or more preferably sodium metal in ethanol, isovaleraldehyde and nitromethane were stirred for three hour. Yield of the nitroalkene can be achieved by acylation followed by elimination of compound structure (VI) as shown in the following reaction:
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-N02
RK X + CHjN02 tee , K'^-^-N°2 + R..
H HCI
V VI
In a further embodiment of this invention the compound of structure VI is converted to compound of structure V by acylation followed by elimination. Acyl chloride or anhydride especially acetyl chloride is the acylating reagent in chlorinated solvents like chloroform, dichloromethane, ethylene dichloride. Base selected for elimination consist of anhydrous sodium acetate or sodium carbonate in dry solvents like THF.

Rl

-N02 l.RCOClor(RCO)20/CHCl3 R] ^ NQ
0H 2. THF/NaOAc V
VI

2

Thus, this invention, for the first time provides a new process for synthesis of recemic as well as (S+) pregabalin starting from a compound of structure V.
The following examples are not to be construed as limiting the scope of invention but are to be construed as illustrative only in nature. Additional variations within the concept of this invention that shall be further standardized during course of work for a complete specification and those ones that will be clear to a person skilled in the art and such variations are also included as embodiments of this invention.
Example 1: Preparation of 4-methyl-1-nitropent-1-ene
To 200 ml of ethanol 4.01 gm of sodium was added slowly to produce sodium ethoxide. To this vigorously stirred solution was added 15 gm (0.174 moles) of isovaleraldehyde and 10.6 gm (0.174 moles) of nitromethane was added drop wise for 1 hour. Soon solid
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separated out. After 1 hour 50 ml of excess of ethanol was added to make a slurry, the resultant slurry was filtered and the cake obtained was then neutralized by using 10 ml of cone. HCI and extracted with 2x100 ml of chloroform. The organic layer was then washed with water, brine and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure to give crude 4-methyl-1-nitropent1-ene in 50% yield. 1H NMR ( 60 MHz, CDCI3) d = 0.92-0.98 (d, 6H), 1.82 (m, 1H ), 2.13-2.15( m, 2H) , 6.95-7.23 ( m, 2H) ppm. 13C NMR( 125 MHz, CDCI3) d = 141.5, 140.1, 37.3, 29.7, 27.8, 22.1 ppm. IR ( Neat): 1350, 1554, 1635 cm"!.
Example 2 : Preparation of Ethyl (Dimethylsulphuranylidene ) acetate :
A. Carboethoxymethyl dimethylsulphonium bromide:
A solution of ethyl bromoacetate (20 gm, 0.119 moles) and dimehtyl sulphide ( 7.44 gm, 0.12 moles) was stirred at room temperature for 7 hours without using any solvent. White salt was diluted with 50 ml of chloroform and collected by filtration. Sulphonium bromide salt was obtained quantitatively as a white crystalline solid in 98% yield.
B. Ethyl (dimehtylsulphuranylidene) acetate : A solution of sulphonium bromide (15
gm, 65.5 mmole) in chloroform (100 ml) was vigorously stirred at 5-10 °C with ice
cooling and treated in one portion with mixture of saturated potassium carbonate
solution (40 ml) and 12.5 N sodium hydroxide solution (5.20 ml). The reaction mixture
was warmed to 15-20 °C and was held for 15 min. After removal of salt by filtration, the
filtrate was separated and the upper layer of chloroform was dried for 2 hrs. over
anhydrous potassium carbonate. Removal of solvent under vacuum at 25 °C gave the
product 7.8 gm as a pale yellow oil in 81%
Saturated potassium carbonate solution was prepared by dissolving 130 gm of K2C03 in 100 ml of water stirring for 2 hour. As sulfur methylide degrades at room temperature, it was totally used for next reaction
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Example 3: Preparation ethyl-2-isobutyl-3-nitrocyclopropanecarboxylate : To a solution of 4-methyl-1-nitropentene (6.45 gm, 50 mmol) in dry CH2CI2 ( 50 ml) was added a freshly prepared solution of EDSA ethyl(dimethylsulphuralidene)acetate. (7.8 gm, 52.7 mmol) in dry CH2CI2 and the mixture was refluxed for six hours. After removal of solvent, residue was purified by column chromatography (Petroleum ether) to give cyclopropane carboxylate as a liquid 5.3 gm (50%) yield. 1HNMR (300 MHz, CDCI3) 5 = 0.90-0.98 (m, 9H), 1.26-1.31 (t, 3H), 1.57-1.71 (m, 2H), 2.13-2.18 (t, 1H), 2.86-2.91 (dd, 1H), 4.15-4.23 (q, 2H), 4.52-4.55(t, 1H) ppm.13C NMR( 125 MHz, CDCI3) 5 = 14.1 22.0, 22.1, 28.0, 29.9, 30.4, 33.1, 61.4, 63.5, 63.9, 167.3 ppm. IR: 1028, 1188, 1368, 1547, 1759 cm1
Example 4: Preparation of 2-isobutyl-3-nitrocycloppropane-1-carboxylic acid :
5 gm (23.2 mmol) of ethyl-2-isobutyl-3-nitrocyclopropanecarboxylate was added to a solution of 1 mol equivalent of NaOH (0.93 gm) in methanol (40 ml). The reaction mixture was allowed to stir for additional 4 hour at room temperature. After completion of reaction, the mixture was neutralized by cone. HCI and extracted with Chloroform (2 x 50 ml). The organic layer was washed with water, brine and dried over anhydrous sodium sulphate. After removal of solvent under reduced pressure, sticky mass was obtained. After purification by column chromatography, pure acid was obtained in 3.26 gm 75% yield.
Result: white solid, m.p. : 108-110 °C, 1HNMR (300 MHz, CDCI3) 5 = 0.95-0.98 (d, 6H), 1.56-1.70 (m, 3H), 2.45(m, 1H), 2.90-2.94 (t, 1H), 4.56-4.622 (t, 1H), 9.816 (bs, 1H) ppm.13C NMR( 125 MHz, CDCI3) 8 = 22.0, 22.2, 28.0, 29.5, 31.1, 33.1, 64.2, 173.9 ppm. IR: 951, 1220, 1371, 1546, 1693 cm"1
Example 5: Reductive ring opening of 2-isobutyl-3-nitrocyclopropane-1-carboxylic acid
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To a solution of Raney Ni (Kaloat 8030) in methanol (50ml) was added 2 gm (10.6 mmol) of cyclopropane carboxylic acid and the reaction was allowed to proceed for 5 hour under hydrogen pressure. Reaction was monitored by TLC. After completion of reaction, the mixture was filtered. Methanol was removed under vacuum to give residue in 0.500 gm (30% yield).
Results : White solid. IR (KBr): umax = 2931, 2625, 1612, 1550 cm"1.
Dated this 15th day of October, 2007.

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