DESC:The following specification particularly describes the invention and the manner in which it is to be performed.

Introduction
The present invention relates to the stereoselective process for preparation of the compound viz., (1R,2R,4R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-hydroxycyclopentane-1-carboxylic acid and its esters, which is an intermediate for HCV compounds like Simeprevir. In particular, the invention is directed to the stereoselective enzymatic reduction process for the preparation of alkyl (1R,2R,4R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-hydroxy- cyclopentane-1-carboxylate, more specifically for the preparation of ethyl (1R,2R,4R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-hydroxycyclopentane-1-carboxylate. The invention further provides the use of a ketoreductase derived from Streptomyces coelicolor having oxidoreductase activity for said stereoselective reduction process.
Background
The compound having the adopted name “Simeprevir” having the chemical name (2R,3aR,10Z,11aS,12aR,14aR)-N-(cyclopropylsulfonyl)-2[[2-(4-isopropyl-1,3-thiazol-2-yl)-7-methoxy-8-methyl-4-quinolinyl]oxy]-5-methyl-4,14 dioxo2,3,3a, 4,5,6,7,8,9, 11a,12,13,14,14atetradecahydrocyclopenta[c]cyclopropa[g] [1,6] diazacyclotetradecine-12a(1H)-carboxamide (Formula I) has been developed by Janssen for the treatment of HCV infection in adults.

Compound of formula II is a key intermediate in the preparation of Simeprevir.

wherein R1 is H, C1-6 alkyl, aralkyl, substituted or unsubstituted aryl, acylalkyl aroylalkyl, and aralkyl silyl.
The presence of multiple chiral centers in the compound of Formula I and its predecessors like Formula II poses particular challenges in achieving the chiral purity that is essential to have a product acceptable for the therapeutic use. The intermediate compound of Formula II has three chiral centers and achieving the required stereochemistry at all the three centers during the synthesis of the said compound is a challenging task. The processes for preparing a compound of Formula II should result in products of acceptable chiral purity without the use of cumbersome purification procedures that result in loss of substantial amounts of desired stereoisomeric forms.
US patent documents viz., US8148399, US8212043, US8927722, US20130005976A1 and US20140228574A1 reported different processes for the preparation of Simeprevir or its intermediates.
All the above references involved chemical reduction method at precursor stage followed by lactonization and ring opening with the desired amine to afford the compound of Formula II. As mentioned above, the compound of Formula II is having three chiral centers and thus obtaining it in high yield and quality with right stereochemistry at all the three centers is a big challenge. The methods for its preparation in literature experience major drawback of multi-step synthesis to achieve desired configuration. In addition, reported methods may result in the formation of inorganic salts and product of inferior quality that may further affect the yield of next amidation product. Since, high quality and yield are the pre-requisites for any preferred process in a pharmaceutical industry, there remains a need to provide commercially viable process for the preparation of the intermediate compound of Formula II of Simeprevir while overcoming the drawbacks presented by processes described in the art.
With the advent of chiral catalyst, chiral reducing agents and biotechnology; it has been possible to develop stereoselective processes to obtain diastereomerically pure compound. Particularly, chiral reducing agents and enzymes can have a unique stereoselective property of producing preferably or selectively one isomer with good purity.
Further, the enzymatic reduction processes in which the enzyme acts as a reduction catalyst are environmentally advantageous as compared to the methods of synthesis described in prior art.
We herein particularly disclose stereoselective process for the preparation of optically pure or substantially pure compound of Formula II. Further, enzymatic process is disclosed for the preparation of optically pure or substantially pure compound of Formula II. We herein further disclose the novel intermediate compounds of Formulae III, IIIa, IIIa’, IIa and their use for preparation of anti-HCV molecules like Simeprevir (Formula I).

The processes of the present invention are advantageous in that they are suitable for large scale production. Cumbersome purification steps, in particular chromatography, are avoided.
The use of a compound of Formula II prepared by stereoselective route as a starting material for preparation of Simeprevir of Formula I is also advantageous because the purity of starting material plays an important role in getting the Simeprevir of Formula I, in high yield and required purity, thus overcoming the problems associated with the processes reported in prior art.
Summary
In the first embodiment, the present application provides a process for preparing a diastereomerically enriched compound of Formula II, comprising;
a) reacting a compound of Formula IV with a compound of Formula V or a salt thereof to afford a compound of Formula IIIa,

wherein R1 is selected from H, C1-6 alkyl, aralkyl, alkoxyalkyl, substituted or unsubstituted aryl, acylalkyl, aroylalkyl, and aralkyl silyl;
b) stereoselectively converting the compound of Formula IIIa to a diastereomerically enriched compound of Formula II,

wherein R1 is as defined above,
c) isolating the diastereomerically enriched compound of Formula II.
In the second embodiment, the present application provides a process for preparing a diastereomerically enriched compound of Formula II, comprising;
a) stereoselectively converting a compound of Formula IIIa to a diastereomerically enriched compound of Formula II,

wherein R1 is selected from H, C1-6 alkyl, aralkyl, alkoxyalkyl, substituted or unsubstituted aryl, acylalkyl and aroylalkyl, aralkyl silyl.
b) isolating the diastereomerically enriched compound of Formula II.
In the third embodiment, the present application provides a process for preparing a diastereomerically enriched compound of Formula II, comprising;
a) stereoselective reduction of a compound of Formula IIIa with an enzyme and/or variant thereof to afford a diastereomerically enriched compound of Formula II,

wherein R1 is selected from H, C1-6 alkyl, aralkyl, alkoxyalkyl, substituted or unsubstituted aryl, acylalkyl, aroylalkyl and aralkyl silyl.
b) optionally maintaining the pH of about 5-10 during the reaction.
c) isolating the diastereomerically enriched compound of Formula II.
In the fourth embodiment, the present application provides a process for preparing a compound of Formula IIIa, comprising;
a) reacting a compound of Formula IV with a compound of Formula V or a salt thereof to afford the compound of Formula IIIa,
b) isolating the compound of Formula IIIa.
In the fifth embodiment, the present application provides novel compounds of Formulae III, IIIa , IIIa’ and IIa.
In the sixth embodiment, the present application provides a process for preparation of Simeprevir of Formula I from a compound of Formula IIIa.
In the seventh embodiment, the present application provides a process for the preparation of Simeprevir of Formula I from a compound of Formula II that has been prepared by a process of the present application. In one variant, the compound of Formula II can be converted to Simeprevir of Formula I by methods reported in the US patent document US 8148399.
Detailed Description
In the first embodiment, the present application provides a process for preparing a diastereomerically enriched compound of Formula II, comprising;
a) reacting a compound of Formula IV with a compound of Formula V or a salt thereof to afford a compound of Formula IIIa,

wherein R1 is selected from H, C1-6 alkyl, aralkyl, alkoxyalkyl, substituted or unsubstituted aryl, acylalkyl and aroylalkyl, aralkyl silyl.
Step a) can be materialized under amide forming reaction conditions which comprises reacting the starting materials with an amide-coupling reagent in a suitable inert solvent, optionally in the presence of a base and a catalyst.
Suitable solvents that can be used include but not limited to halogenated hydrocarbons such as dichloromethane (DCM) or chloroform, ethers such as tetrahydrofuran (THF) or 2-methyltetrahydrofuran (MeTHF), alcohols such as methanol or ethanol, aromatic hydrocarbon solvents such as toluene or xylene, dipolar aprotic solvents such as DMF, DMA, acetonitrile or mixtures thereof. Preferred solvents are methanol, toluene, DMF, dichloromethane, THF or mixtures thereof.
Amide coupling agents that can be used comprise agents such as N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDI or EDCI) as well as its hydrochloride salt, 1,1’-Carbonyldiimidazole (CDI), N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)uranium hexafluorophosphate (HATU), benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (commercially available asPyBOP), 1,3-diisopropylcarbodiimide,O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU) and the like.
A catalyst may optionally be added. The said catalyst includes 1-hydroxybenzotriazole (HOBt) or 4-dimethylaminopyridine (DMAP).
The reaction can be usually conducted in the presence of a base, in particular an organic base such as tertiary amine, e.g. triethylamine, N-methylmorpholine, N,N-diisopropylethylamine (DIPEA). Preferably, DIPEA is employed.
b) stereoselectively converting the compound of Formula IIIa to a diastereomerically enriched compound of Formula II,

wherein R1 is as defined above,
Step b) involves stereoselective reduction of carbonyl group in the compound of Formula IIIa to afford the compound of Formula II. The said stereoselective reduction can be carried out with the aid of chemical reagents or by microbial methods.
In a chemical method, suitable reducing agents will include those which are able to stereoselectively reduce the carbonyl group to the hydroxyl group and give a diastereomeric excess of the compound of Formula II.
The said stereoselective reduction by chemical methods can be carried out using suitable reduction methods, for example, catalytic hydrogenation, or reduction in the presence of one or more chiral catalyst.
Suitable reducing agents that may be used include but not limited to, lithium aluminum hydride, sodium borohydride, lithium borohydride, potassium borohydride, NaCNBH3, diisobutyl aluminium hydride (DIBAL), borane-dimethyl sulfide (BMS), boranetetrahydrofuran (BTHF), a combination thereof, or any other suitable reducing
agent known in the art.
Suitable chiral catalysts that may be used for asymmetric reduction include, but are not limited to, chiral oxazaborolidine catalysts, such as (R)- or (S)-MeCBS (tetrahydro-l-methyl-3, 3- diphenyl-lH, 3H-pyrrolo [1, 2-c] [1, 3, 2] oxazaborole, or the like; (-)-DIP-chloride [(-)-diisopinocampheylchloroborane] ; L- or K-Selectride®; binol-metal complexes, or the like; a complex of C6-C14aryl or C6-C14aryl substituted boronic acid with tartaric acid, such as chiral boronic esters, , (+)-B-fluorodiiso-2-ethylapopinocampheylborane, (+)-B-bromodiiso-2-ethylapopino-campheyl-borane,bis(10-methylisopinocampheyl) chloroborane, borane-THF, borane-dioxane, borane-diethyl aniline or mixtures thereof.
Chiral reduction can optionally be carried out in presence of a suitable chiral ligand. Suitable chiral ligands that can be used include, but are not limited to, [(R) HexaPHEMP RuCl2 (R,R )-DACH], [(R)-HexaPHEMP RuCl2 (R,R)-DPEN], [(R)-PhanePhos RuCl2 (S,S)-DACH], [(S)-PhancPhos RuCl; (R,R)-DPEN], [(S)-MeO-Xylyl-PhanePhos RuCl2 (R,R)-DPEN], [(R)-BINAP RuCl2 (R)-DAIPEN], [R,R-TsDPEN (Ru) (p-cymene) CI], and [S,S-TsDPEN (Ru) (p-cymene) CI]. An optically active reducing agent or chiral ligand can be used to achieve desired stereoselectivity selectivity and to afford the desired stereoisomer in higher yield and purity.
In one variant of the first embodiment, a suitable reducing agent is oxazaborolidine which may be formed by mixing of trimethoxyborane and S-diphenyl prolinol, followed by addition of borane dimethylsulfide. A suitable solvent which is inert to the reaction conditions can be employed.
In a microbial method, suitable enzymes or microbes can be used for said stereoselective reduction of carbonyl to hydroxy group of Formula II.
In a preferred variant of the first embodiment, the microbial method is employed for said stereoselective reduction.
Suitable enzymes which can be used are selected from suitable oxidoreductases. Examples of such oxido-reductase include, but not limited to, aldose-reductase, aldehyde reductase, carbonyl reductase and ketoreductase. In a preferred variant, suitable enzymes that can be used are selected from aldo-keto reductases. In a preferred variant, suitable enzymes are selected from ketoreductases.
The enzyme which can be used in the process of the present application may be formulated either in the form of a cell paste or in the form of a cell free extract or lyophilized or spray-dried powder or the use of recombinant host cell, cell free extract/crude lysate obtained from recombinant host cell, isolated desired enzyme which is isolated from cell free extract/crude lysate or from the suitable organism.
In a preferred variant of the first embodiment, the instant step is carried out in presence of a co-factor. The co-factor that can be used may be any co-factor known in the art. Specifically, the co-factor may be NAD or NADP or NADH or NADPH.
In yet another variant of the first embodiment, a co-factor regeneration system may be optionally added to the process of the present application. The co-factor regeneration system that can be used may be selected from a group of glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, phosphite/phosphite dehydrogenase and isopropanol recycle system. Specifically, the co-factor regeneration system for the process of the present application may be glucose/glucose dehydrogenase.
The substrate concentration in enzymatic method ranges from about 5 g/l to about 200 g/l. In a preferred variant, it ranges from about 10 g/l to about 50 g/l.
The enzyme loading in the process of the present application ranges from about 50 to about 1500 units per gram of the substrate. Specifically, enzyme loading ranges from about 70 to about 1300 units per gram of the substrate. More specifically, the enzyme loading ranges from about 100 to about 1200 units per gram of the substrate.
The enzymatic reduction of the compound of Formula IIIa to yield the compound of Formula II according to the process of the present application is carried out at a suitable temperature. Specifically, the suitable temperature ranges from about 5 ºC to about 50 ºC. More specifically, the said enzymatic reduction is carried out at a temperature from about 20 ºC to about 35 ºC. Most specifically, the said enzymatic reduction is carried out at a temperature from about 25 ºC to about 30 ºC.
In addition, these vectors may further contain a gene encoding an enzyme which can regenerate the co-factors such as NAD, NADP, NADH, NADPH.
According to such embodiment the oxidoreductase is derived from Streptomyces coelicolor, and may be or is coupled with the cofactor selected from NAD(P)H/NAD(P) to produce the optically pure or substantially pure compound of Formula II, by reduction of the compound of Formula IIIa wherein the cofactor is either added externally in reaction medium or obtained by enzyme/substrate coupled regeneration system.
The enzymatic reduction of the compound of Formula IIIa to yield the compound of Formula II according to the process of the present application is carried out at a suitable pH. Specifically, suitable pH for said reaction ranges from about 5 to about 10. More specifically, the said reaction is carried out at a pH of about 7 to about 9. In a preferred variant, it is carried out at pH of about 8.
A buffer may optionally be added to the reaction mixture. The buffer that can be added to the reaction mixture may be any buffer known in the art. Specifically, the buffer that can be used is selected from a group of potassium phosphate buffer, tris/HCl buffer and triethanolamine buffer. More specifically, the buffer is potassium phosphate buffer.
To stabilize or activate the enzyme, metallic ions like zinc ions, magnesium ions, calcium ions may optionally be added to the buffer.
Optionally, a stabilizer of oxidoreductase/dehydrogenase may also be added to the reaction mixture. Suitable stabilizers that can be used are, for example, glycerol, sorbitol, 1,4-DL-dithiothreitol (DTT) or dimethylsulfoxide (DMSO).
The enzymatic reaction steps can be carried out in an aqueous solution in combination with organic solvents. Such aqueous solutions include buffers having buffer capacity at a neutral pH and are selected from above mentioned list. Alternatively, no buffer is required when the use of acid or alkali can keep the pH change during the reaction within a desired range. Organic solvents that can be used in this step include but not limited to n-butanol, Isopropyl alcohol, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, ethanol, acetone, dimethyl sulfoxide, and acetonitrile etc. In a preferred variant of the first embodiment, the reaction is performed without buffer in presence of an acid or an alkali which maintains the pH change during the reaction within a desired range. Alternatively, the reaction can be carried out in a mixed solvent system consisting of water miscible solvents such as ethanol, acetone, dimethyl sulfoxide, and acetonitrile.
The stereoselective reduction step can be carried out, for example, in a closed reaction vessel made of glass or metal. For this purpose, the components are transferred individually into the reaction vessel and stirred under an atmosphere of, e.g., nitrogen or air.
c) isolating the diastereomerically enriched compound of Formula II.
At the end of the reaction when the product is formed, thereafter the product is isolated from the reaction mixture by conventional techniques known in the art.
The process of the present application produces compound of Formula II in a diastereomeric purity of more than about 85%. Specifically, the process of the present application produces compound of Formula II in a diastereomeric purity of more than about 90%. More specifically, the process of the present application produces the compound of Formula II in a diastereomeric purity of more than about 95%. Most specifically, the process of the present application produces the compound of Formula II in a diastereomeric purity of more than about 99%.
In the second embodiment, the present application provides a process for preparing a diastereomerically enriched compound of Formula II, comprising;
a) stereoselectively converting a compound of Formula IIIa to a diastereomerically enriched compound of Formula II,

wherein R1 is as defined in abovementioned embodiment.
As mentioned above the instant step is carried out by using either chemical reagents or enzymes to materialize the stereoselective reduction of carbonyl group to afford desired alcohol i.e. compound of Formula II.
b) isolating the diastereomerically enriched compound of Formula II.
The products are isolated by conventional techniques known in the art.
In the third embodiment, the present application provides a process for preparing a diastereomerically enriched compound of Formula II, comprising;
a) stereoselective reduction of a compound of Formula IIIa with an enzyme and/or variant thereof to afford a diastereomerically enriched compound of Formula II,

wherein R1 is as defined in abovementioned embodiment.
The suitable reaction conditions employed in this step can be selected from the aforementioned embodiment of the present application. In a most preferred variant, NAD(P)+ dependent ketoreductase is selected from Streptomyces coelicolor.
b) optionally maintaining the pH of about 5-10 during the reaction.
The suitable reaction conditions employed in this step can be selected from the aforementioned embodiment of the present application. In a preferred variant, it is carried out at pH of about 8.
c) isolating the diastereomerically enriched compound of Formula II.
At the end of the reaction when the product is formed, thereafter the product is isolated from the reaction mixture by conventional techniques known in the art.
The process of the present application produces diastereomerically enriched compound of Formula II having a diastereomeric purity of more than about 85%. Specifically, the process of the present application produces compound of Formula II in a diastereomeric purity of more than about 90%. More specifically, the process of the present application produces the compound of Formula II in a diastereomeric purity of more than about 95%. Most specifically, the process of the present application produces the compound of Formula II in a diastereomeric purity of more than about 99%.
In the fourth embodiment, the present application provides a process for preparing a compound of Formula IIIa, comprising;
a) reacting a compound of Formula IV with a compound of Formula V or a salt thereof to afford the compound of Formula IIIa,
The suitable reaction conditions employed in this step can be selected from the aforementioned embodiment of the present application. In a preferred variant, it is carried in presence of DIPEA.
b) isolating the compound of Formula IIIa.
The product obtained in this step is isolated from the reaction mixture by conventional techniques known in the art.
In the fifth embodiment, the present application provides novel compounds of Formulae III, IIIa , IIIa’ and IIa.

In the sixth embodiment, the present application provides a process for preparation of Simeprevir of Formula I from a compound of Formula IIIa.
In the seventh embodiment, the present application provides a process for the preparation of Simeprevir of Formula I from a compound of Formula II that has been prepared by a process of the present application. In one variant, the compound of Formula II can be converted to Simeprevir of Formula I by methods reported in the US patent document US 8148399.
The chemical/biochemical transformations described throughout the specification may be carried out using substantially stoichiometric amounts of reactants, though certain reactions may benefit from using an excess of one or more of the reactants. Additionally, many of the reactions disclosed throughout the specification, may be carried out at room temperature, but particular reactions may require the use of higher or lower temperatures, depending on reaction kinetics, yields, and the like.
Room temperature as used herein refers to ‘the temperatures of the thing close to or same as that of the space, e.g., the room or fume hood, in which the thing is located’. Typically, room temperature can be from about 20°C to about 30°C, or about 22°C to about 27°C, or about 25°C.
The reactions of the processes described herein can be carried out in air or under an inert atmosphere. Typically, reactions containing reagents or products that are substantially reactive with air can be carried out using air-sensitive synthetic techniques that are well known to the person skilled in art.
Some of the stages may benefit from being conducted in pressurized, sealed vessels to prevent loss of gaseous reagents. Furthermore, many of the chemical transformations may employ one or more compatible solvents, which may influence the reaction rates and yields. Depending on the nature of the reactants, one or more solvents may be polar protic solvents, polar aprotic solvents, non-polar solvents, or any of their combinations.
The compounds obtained by the chemical/biochemical transformations of the present application can be used for subsequent steps without further purification, or can be effectively separated and purified by employing a conventional method well known to those skilled in the art, such as recrystallization, column chromatography, by transforming them into a salt, or by washing with an organic solvent or with an aqueous solution, and eventually adjusting pH. Compounds at various stages of the process may be purified by precipitation or slurrying in suitable solvents, or by commonly known recrystallization techniques. The suitable recrystallization techniques include, but are not limited to, steps of concentrating, cooling, stirring, or shaking a solution containing the compound, combination of a solution containing a compound with an anti-solvent, seeding, partial removal of the solvent, or combinations thereof, evaporation, flash evaporation, or the like. An anti-solvent as used herein refers to a liquid in which a compound is poorly soluble. Compounds can be subjected to any of the purification techniques more than one time, until the desired purity is attained.
Compounds may also be purified by slurrying in suitable solvents, for example, by providing a compound in a suitable solvent, if required heating the resulting mixture to higher temperatures, subsequent cooling, and recovery of a compound having a high purity. Optionally, precipitation or crystallization at any of the above steps can be initiated by seeding of the reaction mixture with a small quantity of the desired product. Suitable solvents that can be employed for recrystallization or slurrying include, but are not limited to: alcohols, such as, for example, methanol, ethanol, and 2-propanol; ethers, such as, for example, diisopropyl ether, methyl tert-butyl ether, diethyl ether, 1,4-dioxane, tetrahydrofuran (THF), and methyl THF; esters, such as, for example, ethyl acetate, isopropyl acetate, and t-butyl acetate; ketones, such as acetone and methyl isobutyl ketone; halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and the like; hydrocarbons, such as toluene, xylene, and cyclohexane; nitriles, such as acetonitrile and the like; water; and any mixtures of two or more thereof.
Compounds of Formula III, Formula IIIa, Formula IIIa’ and Formula IIa manufactured by the present invention is substantially free from impurities. Typically, the level of impurities may be less than about 10%, 5%, 2%, 1% or 0.5%, by weight, as determined by using high performance liquid chromatography (HPLC).
Definitions
The following definitions are used in connection with the present application unless the context indicates otherwise.
As used herein, the term "enzyme" refers to a polypeptide sequence encoded by a polynucleotide sequence which shows desirable enzymatic activity. The term 'enzyme' used anywhere in the specification would also include its suitable 'variants' as defined below, unless specified otherwise.
The term “Ketoreductase,” “ketoreductase enzyme,” or KRED refers to an enzyme that catalyzes the reduction of a ketone to form the corresponding alcohol in a stereoselective manner, optionally with the aid of co-factor. Such enzymes are given various names in addition to ketoreductase, including, but not limited to, alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, sorbitol dehydrogenase.
As used herein, the terms "oxidoreductase," or "oxidoreductase enzyme" refer to an enzyme that catalyzes the reduction of a ketone to form the corresponding alcohol in a stereoselective manner, optionally with the aid of co-factor.
The term "variants" refers to polypeptides derived from the above nucleotide sequence by the addition, deletion, substitution or insertion of at least one nucleotide.
As used herein, 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, 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"), and any derivatives or analogs thereof.
The expression vector pET26b(+) is commercially available from Novagen.
Celite® is flux-calcined diatomaceous earth. Celite® is a registered trademark of World Minerals Inc.
Hyflow is flux-calcined diatomaceous earth treated with sodium carbonate. Hyflo Super Cel™ is a registered trademark of the Manville Corp.
As used herein, "comprising" means the elements recited, or their equivalents in structure or function, plus any other element or elements which are not recited. The terms "having" and "including" are also to be construed as open ended unless the context suggests otherwise. Terms such as "about," "generally," "substantially," and the like are to be construed as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify, as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error, or instrument error for a given technique used to measure a value.
All percentages and ratios used herein are by weight of the total composition and all measurements made are at about 25°C and about atmospheric pressure, unless otherwise designated. All temperatures are in degrees Celsius unless specified otherwise. As used herein, the terms “comprising” and “comprises” mean the elements recited, or their equivalents in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended. All ranges recited herein include the endpoints, including those that recite a range between two values. Whether so indicated or not, all values recited herein are approximate as defined by the circumstances, including the degree of expected experimental error, technique error, and instrument error for a given technique used to measure a value.
The term “optional” or “optionally” is taken to mean that the event or circumstance described in the specification may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Unless specified otherwise, the word "pure" as used herein means that the material is at least about 99% pure. In general, this refers to purity with regard to unwanted residual solvents, reaction by-products, impurities, and unreacted starting materials. "Substantially pure" as used herein means at least about 98% pure and, likewise, "essentially pure" as used herein means at least about 95% pure. In the case of stereoisomers, "pure" as used herein also means 99% of one enantiomer or diastereomer, as appropriate. "Substantially pure" as used herein means at least about 98% pure and, likewise, "essentially pure" as used herein means at least about 95% pure.
"Substantially free of one or more of its corresponding impurities" as used herein, unless otherwise defined refers to the compound that contains less than about 2%, or less than about 1 %, or less than about 0.5%, or less than about 0.3%, or less than about 0.2%, or less than about 0.1 %, or less than about 0.05%, or less than about 0.03%, or less than about 0.01 %, by weight, of each individual.
Certain specific aspects and embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the disclosure in any manner.
EXAMPLES
EXAMPLE 1: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (0.1M, 1000mL), magnesium acetate (5.36 g) and lipozyme CAL-B (6.5 mL) at 5°C. To this mixture, di-ester (49 g) is slowly added at the same temperature and the pH was maintained at 6.2 using 1M sodium hydroxide (84 mL) overnight. pH of aqueous layer is adjusted to 9 using 1M sodium hydroxide 60 mL followed by addition of ethyl acetate (3x300 mL) and maintenance for 10 minutes. The mixture is filtered using Hy-flow and organic layer is processed for recovery of undesired isomer as diester. pH of the aqueous layer is adjusted to 2.5 using 5M hydrochloric acid (25 mL) and extracted with ethyl acetate (3x300 mL). The organic solvent is subjected to distillation under vacuum to afford the title compound.
EXAMPLE 2: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (0.1M, 3000 mL), magnesium acetate (16.08 g) and di-ester (150 g) at 15°C. To this mixture, lipozyme CAL-B (20 mL) is added at the same temperature. The pH 6.2 of the mixture was maintained overnight with continues addition of 2M sodium hydroxide (220 mL). pH of aqueous layer is adjusted to 9 using 2M sodium hydroxide (90 mL) followed by addition of ethyl acetate (3x1000 mL). The organic layer is processed for recovery of undesired isomer as diester. Then pH of aqueous layer is adjusted to 2.5 using 5M hydrochloric acid (75 mL) and extracted with ethyl acetate (3x1000 mL). The organic solvent is subjected to distillation under vacuum to afford the title compound.
EXAMPLE 3: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (0.1M, 3000 mL), magnesium acetate (16.08 g) and lipozyme CAL-B (20 mL) at about 10°C. Then, di-ester (147 g) is added to this mixture at the same temperature. The mixture is allowed to stir at the room temperature for 24 h. The pH 6.2 of the mixture was maintained overnight with continues addition of 2M sodium hydroxide (180 mL). pH of aqueous layer is adjusted to 10.2 using 2M sodium hydroxide (150 mL) followed by addition of ethyl acetate (3x1000 mL). The organic layer is processed for recovery of undesired isomer as diester. Then pH of the aqueous layer is adjusted to 2.9 using 5M hydrochloric acid (131 mL) and extracted with ethyl acetate (3x1000 mL). The organic solvent is subjected to distillation under vacuum to afford the title compound.
EXAMPLE 4: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (0.1M, 3000mL), magnesium acetate (16.08 g) and lipozyme CAL-B (20 mL) at about 15°C. Then, di-ester (150 g) is slowly added to this mixture at the same temperature. The mixture is allowed to stir at the 10°C and the pH 6.2 of the mixture was maintained overnight with continues addition of 1M sodium hydroxide (410 mL). pH of aqueous layer is adjusted to 9 using 2M sodium hydroxide(270 mL) followed by addition of ethyl acetate (3x1000 mL). The organic layer is processed for recovery of undesired isomer as diester. Then pH of the aqueous layer is adjusted to 2.9 using 5M hydrochloric acid 130.5 mL) and extracted with ethyl acetate (3x1000 mL). The organic solvent is subjected to distillation under vacuum to afford the title compound.
EXAMPLE 5: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (50mM, 3900 mL) and immobilized Novozyme 435 (9.8 g) at about 25°C. Then, di-ester (195 g) is slowly added to this mixture at the same temperature. The pH 6.5 of the mixture was maintained 27 hours with continues addition of 2M sodium hydroxide (280 mL). pH of aqueous layer is adjusted to 9 using 2M sodium hydroxide (80 mL) followed by addition of ethyl acetate (3x1000 mL). The combined organic layer is dried and then processes for recover of diester having undesired stereochemistry. The pH of the aqueous layer is adjusted to 3 using 5M hydrochloric acid (100 mL) followed by extraction with ethyl acetate (3x1000 mL). The combined organic layer is dried with sodium sulphate and then subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 6: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (50 mM, 2600 mL) at about 25°C. Then, di-ester (130 g) is slowly added to this mixture followed by addition of immobilized Novozyme 435 (6.7 g) at the same temperature. The pH (6.5) of the mixture was maintained overnight with continues addition of 1M sodium hydroxide (295 mL). pH of aqueous layer is adjusted to 7.5 using 2M sodium hydroxide (70 mL) followed by extraction with ethyl acetate (3x1000 mL). The aqueous extract is acidified with 5M hydrochloric acid (60 mL) to pH 3 followed by extraction with ethyl acetate (3x1000 mL). The ethyl acetate layer is dried with sodium sulphate and then subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 7: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
A flask is charged with potassium phosphate buffer (50 mM, 4000 mL) at about 25°C. Then, di-ester (200 g) is slowly added to this mixture followed by addition of Novozyme 435 (10 g) at the same temperature. The pH 6.5 of the mixture was maintained overnight with continues addition of 1 M sodium hydroxide (520 mL). pH of aqueous layer is adjusted to 8.0 using 1 M sodium hydroxide followed by extraction with ethyl acetate (3x1000 mL).The aqueous extract is acidified with 5M hydrochloric acid (100 mL) to pH 3 followed by extraction with ethyl acetate (3x1000 mL). The ethyl acetate layer is dried with sodium sulphate and then subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 8: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
The recombinant lipase/esterase producing strains were inoculated in 100 mL auto-induction media. The organisms (R. gluberulus; P. aeruginosa penny lipase; P. aeruginosa Lipase 14; DAPD esterase; Nokardia esterase; BSG1 esterase) were maintained for 48 hours at 37°C and 220 rpm in an orbital shaker. After 48 hours the media containing biomass was centrifuged at 5000 rpm for 10 min to obtain the wet cell paste. The cell pallets were washed with 0.05 M phosphate buffer pH 6.5. To a clean Erlenmeyer flask (50 ml) containing 10.0 mL 0.05M phosphate buffer cell paste (~1.0 g), diester (200.0 µL) and Magnesium acetate (50.0 mg) was added. The reaction was incubated for 72 hours in orbital shaker at 37°C and 220 rpm. The reaction pH was adjusted to 2.5 by using 5.0M HCl (100 µL) and the reaction mass centrifuged at 9000 rpm for 10min at 10°C. The pellet was washed with ethyl acetate. The product was extracted by using ethyl acetate (20 mL) and finally the combined ethyl acetate layers were subjected to distillation under vacuum to give mixture of substrate and title compound.
EXAMPLE 9: PREPARATION OF (1R,2R)-4-OXO-CYCLOPENTANE-1,2-DICARBOXYLIC ACID MONOETHYL ESTER
The recombinant lipase producing strain P.aeruginosa Penny lipase was grown in 1L auto-induction media. The organism was maintained for 48 hours at 37°C and 220 rpm in an orbital shaker. After 48 hours the media containing biomass was centrifuged at 5000 rpm for 10 min to obtain the wet cell paste (13.0 g). The cell pallets were washed with 0.05 M phosphate buffer pH 6.5. To a clean flask (250 ml) containing 50.0 mL 0.05M phosphate buffer cell paste (~13.0 g), diester (1.0 g) and Magnesium acetate (500.0 mg) was added. The reaction was incubated for 72 hours in orbital shaker at 37°C and 220 rpm. The reaction pH was adjusted to 2.5 by using 5.0M HCl (1mL) and the reaction mass centrifuged at 9000 rpm for 10min at 10°C. The pellet was washed with ethyl acetate (50 mL). The product was extracted by using ethyl acetate (200 mL) and finally the combined ethyl acetate layers were subjected to complete distillation to give mixture of substrate and title compound.
EXAMPLE 10: PREPARATION OF ETHYL (1R,2R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-OXOCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with (1R,2R)-2-(ethoxycarbonyl)-4-oxocyclopentane-1-carboxylic acid (1 g), DMF (8 mL) under nitrogen atmosphere. The mixture is cooled to 0oC followed by addition of ethyl N,N-dimethylaminopropyl carbodiimide hydrochloride (1.16 g) and HOBt (0.8 g) at the same temperature. To the mixture, a solution of N-methyl hex-5-en-1-amine (0.68 g) in DMF (2 mL) is added at 0-5oC followed by drop-wise addition of diisopropylethyl amine (2.6 g) over a period of 10 minutes. The reaction mixture is stirred at 0-5oC for 30 minutes and then allowed to attain room temperature. The mixture is stirred at room temperature for 16 hours and completion of the reaction is monitored by TLC. After completion of reaction, mixture is poured into cold water (20 mL) and extracted with ethyl acetate (2x15 mL). The organic layers are combined, washed with brine (15 mL), dried over sodium sulfate and then subjected to distillation under reduced pressure to afford the crude compound. The crude compound is purified by column chromatography using ethyl acetate:hexane to afford the title compound.
EXAMPLE 11: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with potassium phosphate buffer (0.1M, 30 mL), AKR066 (alcohol dehydrogenase, 15 mg), CDX901 (GDH, 7.5 mg), glucose (2.1 g) and NADP (30 mg) at 25oC. To the mixture, substrate (1.5 g) is slowly added and mixture is maintained for about 20 hours, during the reduction pH is maintained at 8.0 using 1.0 M Sodium Hydroxide (5 mL). The product was extracted by using ethyl acetate (2x50 mL) and then organic layer is subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 12: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with potassium phosphate buffer (0.1M, 20 mL), AKR066 (alcohol dehydrogenase, 15 mg), CDX901 (GDH, 7.5mg), D-glucose (1.4 g) and NADP (30 mg) at 25oC. To the mixture, ethyl (1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate (1g) is slowly added and mixture is maintained for overnight. The pH was maintained at 8 using 0.5 M sodium hydroxide (13.0 mL). The product was extracted by using ethyl acetate (2x40 mL) and then organic layer is subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 13: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with potassium phosphate buffer (0.1M, 1000 mL), AKR066 (alcohol dehydrogenase, 500 mg), CDX901 (GDH, 250mg), D-glucose (70 g) and NADP (1000 mg) at 25oC. To the mixture, ethyl (1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate (45g) is slowly added and mixture is maintained for overnight, during the reduction pH is maintained at about 7.5-8.0 using 1 M sodium hydroxide (150 mL). The product was extracted by using ethyl acetate (3x600 mL) and then organic layer is subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 14: PREPARATION OF DIETHYL CYCLOHEX-4-ENE-1,2-DICARBOXYLATE
A flask is charged with butadiene sulfone (240.17 g), diethyl fumarate (250 g) and hydroquinone (1.918 g) and ethanol (500 mL). The reaction mixture is heated to 100oC and maintained at the same temperature for 18 hours. Progress of the reaction is monitored by TLC. After completion of the reaction, mixture is cooled to room temperature and reaction mass is quenched with 10% sodium bicarbonate solution (2500 mL) followed by extraction with dichloromethane (3x700 mL). The combined organic layer is washed with brine solution (1000 mL) and then subjected to complete distillation under vacuum at 40oC to afford the title compound.
EXAMPLE 15: PREPARATION OF 3,4-BIS(ETHOXYCARBONYL)HEXANEDIOIC ACID
A flask is charged with potassium permanganate (332 g) and water (2250 mL) and mixture is stirred at room temperature for 20 minutes followed by cooling of the mixture to 0oC. To this mixture, a solution of diethyl cyclohex-4-ene-1,2-dicarboxylate (150 g) in acetone (225 mL) is drop-wise added at 0-5oC. Then the mixture is allowed to attain room temperature and stirred at same for 4 hours followed by portion wise addition of sodium bisulfite and further maintenance of reaction mixture at room temperature for 20 minutes. Then reaction mixture is cooled to 10-15oC and then acidified with conc. hydrochloric acid to pH of about 2-2.5. Aqueous layer is extracted with mixture of ethyl acetate and tetrahydrofuran (1:1, 2500 mL) and then organic layer is subjected to complete distillation under vacuum at 45oC to afford title compound as off-white solid.
EXAMPLE 16: PREPARATION OF TRANS-DIETHYL (1R*,2R*)-4-OXOCYCLOPENTANE-1,2-DICARBOXYLATE
A flask is charged with 3,4-bis(ethoxycarbonyl)hexanedioic acid (170 g), acetic anhydride (884 mL), sodium acetate (43.23 g) and maintained at room temperature for 10 minutes. The mixture is slowly heated to 140oC and maintained at the same temperature for 2hours, progress of the reaction is monitored by TLC. After completion of the reaction, mixture is cooled to room temperature and quenched with ice cold water (3000 mL). The reaction mass is extracted with ethyl acetate (3x1000 mL). The combined organic layer is washed with saturated sodium bicarbonate solution (2x1500 mL) followed by brine solution (1000 mL). After drying the organic layer with sodium sulphate (150 g), it is subjected to complete distillation under vacuum at 50oC to afford the crude compound which is purified by column chromatography using 10% ethyl acetate and hexane as eluents to afford the title compound.
EXAMPLE 17: PREPARATION OF (1R,2R)-2-(ETHOXYCARBONYL)-4-OXOCYCLOPENTANE-1-CARBOXYLIC ACID
A flask is charged with phosphate buffer (1000 mL, 0.1M), magnesium acetate tetrahydrate (5.354 g) and mixture is cooled to 5oC. Then sequentially CAL-B (6 mL) and trans-diethyl (1R*,2R*)-4-oxocyclopentane-1,2-dicarboxylate (50 g) are added to the mixture at 5oC. The pH of the mixture is adjusted to 6.2 with 1M sodium hydroxide and reaction mixture is maintained at 5oC for 24 hours. Then pH is adjusted to 9 with 2M sodium hydroxide solution (50 mL) at below 10oC. The reaction mixture is extracted with methyl tert-butyl ether (2x300 mL). Aqueous layer is isolated and cooled to 10oC and then acidified with 5M hydrochloric acid (40 mL) to pH 2-2.5 and further extracted with methyl tert-butyl ether (2x300 mL). The organic layers are combined and subjected to complete distillation under vacuum at 45oC to afford the title compound.
EXAMPLE 18: PREPARATION OF (1R,2R)-4-OXOCYCLOPENTANE-1,2-DICARBOXYLIC ACID
A flask is charged with (1R,2R)-2-(ethoxycarbonyl)-4-oxocyclopentane-1-carboxylic acid (28 g), water (112 mL) and conc. hydrochloric acid solution (112 mL) at room temperature. The reaction mixture is heated to reflux and maintained at the same temperature for 16 hours, progress of the reaction is monitored by TLC. After completion of the reaction, mixture is cooled to room temperature followed by addition of charcoal (2.8 g). The reaction mixture is stirred for 10 minutes and then filtered on celite bed, washed with water (56 mL). The filtrate is subjected to complete distillation under vacuum at 50-55oC. Then hexane (150 mL) is added to the obtained solid, stirred for 10 minutes and filtered under vacuum followed by drying of solid product at 50-55oC for 4 hours.
EXAMPLE 19: PREPARATION OF (1R,2R)-4-HYDROXYCYCLOPENTANE-1,2-DICARBOXYLIC ACID
A flask is charged with (1R,2R)-4-oxocyclopentane-1,2-dicarboxylic acid (19.4 g) and methanol (194 mL) under nitrogen atmosphere at room temperature. The reaction mixture is cooled to 0-5oC, then aqueous sodium hydroxide solution (9.24 g in 38.8 mL) is added followed by portion wise addition of sodium borohydride (4.26 g). The reaction mixture is allowed to attain room temperature and stirred for 16 hours. After completion of the reaction, mixture is cooled to 0-5oC and its pH is adjusted to about 3 by addition of 6N hydrochloric acid. The reaction mixture is subjected to complete distillation under reduced pressure to afford crude solid. To the crude solid, tetrahydrofuran (50 mL) is added, mixture is heated to 50oC for 10 minutes and supernatant THF is collected. This operation is repeated once with second lot of tetrahydrofuran (50 mL). The THF layers are combined and subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 20: PREPARATION OF (1R,4R,5R)-3-OXO-2-OXABICYCLO[2.2.1]HEPTANE-5-CARBOXYLIC ACID
A flask is charged with (1R,2R)-4-hydroxycyclopentane-1,2-dicarboxylic acid (20 g), acetone (400 mL) and tetrahydrofuran (400 mL) under nitrogen atmosphere. The mixture is cooled to 10oC and triethyl amine (12 g) is added followed by further cooling of the mixture to 0oC. Then ethyl chloroformate (12.8 g) is drop-wise added to the mixture at 0oC and reaction is maintained for 30 minutes. Then powdered molecular sieves are added to the mixture and it is allowed to attain room temperature. The reaction mixture is stirred at room temperature for 16 hours under nitrogen atmosphere. After completion of reaction, mixture is filtered on celite bed and washed with THF (60 mL). The filtrate is subjected to complete distillation under vacuum at 45oC. To the residue obtained, water (100 mL) is added and pH is adjusted to about 3 using 2N hydrochloric acid followed by extraction with ethyl acetate (3x100 mL). The combined organic layers are washed with brine solution (100 mL) and concentrated under reduced pressure to afford the title compound.
EXAMPLE 21: PREPARATION OF (1R,4R,5S)-N-(HEX-5-EN-1-YL)-N-METHYL-3-OXO-2-OXABICYCLO[2.2.1]HEPTANE-5-CARBOXAMIDE
A flask is charged with (hex-5-enyl)(methyl)amine (16.5 g), dimethyl formamide ( mL) and HATU (55.5 g) under nitrogen atmosphere. Then mixture is cooled to 0oC followed by drop-wise addition of a solution of (1R,4R,5R)-3-oxo-2-oxabicyclo[2.2.1]heptane-5-carboxylic acid (19 g) in DMF (114 mL) and subsequently diisopropylethylamine (47.2 g) at 0oC. The reaction mixture is stirred at 0oC for 40 minutes and then allowed to attain room temperature. The reaction mixture is further stirred at RT for 5 hours under nitrogen atmosphere and progress of the reaction is monitored by TLC. After completion of reaction, cold water (760 mL) is added and mixture is extracted with ethyl acetate (2x220 mL). The organic layers are combined and washed with cold water (220 mL) and brine solution (220 mL). The organic layers are subjected to complete distillation under vacuum to afford crude compound which is purified by column chromatography using 30-35% ethyl acetate/hexane as eluents to afford the title compound.
EXAMPLE 22: PREPARATION OF (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLIC ACID
A flask is charged with (1R,4R,5S)-N-(hex-5-en-1-yl)-N-methyl-3-oxo-2-oxabicyclo[2.2.1]heptane-5-carboxamide and cooled to 10oC. Then an aqueous solution of lithium hydroxide monohydrate (2g in 24 mL) is added to the reaction mixture at 10oC and mixture is stirred for 30 minutes. The reaction mixture is allowed to attain room temperature and stirred for 1hour at the same temperature. After completion of reaction, ethyl acetate (30 mL) and water (10 mL) is added to the mixture. The aqueous layer is acidified to pH of about 2-3 using 6N hydrochloric acid solution. The aqueous layer is extracted with ethyl acetate (3x50 mL) and combined organic layer is subjected to complete distillation under vacuum to afford the title compound.

EXAMPLE 23: PREPARATION OF ETHYL (1R,2S)-1-((1R,2R,4S)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXAMIDO)-2-VINYLCYCLOPROPANE-1-CARBOXYLATE
A flask is charged with (1R,2R,4R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-hydroxycyclopentane-1-carboxylic acid (5.5 g) and DMF (66 mL) under nitrogen atmosphere. Then, ethyl (1R, 2S)-dehydrocoronomate (4.4 g) and HATU (9.3 g) is added to the reaction mixture. The reaction mixture is cooled to 0oC followed by drop-wise addition of diisopropyl ethylamine (3.96 g) at the same temperature. The mixture is stirred for 30 minutes at 0oC and then warmed to room temperature at which it is further maintained for 3 hours. After completion of reaction, cold water (250 mL) is added and mixture is extracted with ethyl acetate (3x60 mL). The organic layers are combined and sequentially washed with 0.5 N hydrochloric acid solution (100 mL), water (50 mL) and brine (50 mL). The organic layer is subjected to complete distillation under reduced pressure to afford the crude compound which is purified by column chromatography to afford the title compound.
EXAMPLE 24: PREPARATION OF ETHYL (1R,2S)-1-((1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-((2-(4-ISOPROPYLTHIAZOL-2-YL)-7-METHOXY-8-METHYLQUINOLIN-4-YL)OXY)CYCLOPENTANE-1-CARBOXAMIDO)-2-VINYLCYCLOPROPANE-1-CARBOXYLATE
A flask is charged with ethyl (1R,2S)-1-((1R,2R,4S)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-hydroxycyclopentane-1-carboxamido)-2-vinylcyclopropane-1-carboxylate (5 g) and dry tetrahydrofuran (150 mL) under nitrogen atmosphere. To the mixture, then 2-(4-isopropylthiazol-2-yl)-7-methoxy-8-methylquinolin-4-ol (3 g) and triphenylphosphine (9.6 g) are added and the mixture is cooled to 0oC. Then diisopropyl azodicarboxylate (7.4 g) is slowly added to the mixture at 0oC and mixture is stirred for 1 hour at the same temperature. The reaction mixture is allowed to attain the room temperature and stirred for 16 hours under nitrogen atmosphere. After completion of the reaction as monitored by TLC, ice cold water (100 mL) is added and mixture is extracted with ethyl acetate (80 mL). The organic layers are combined and washed with brine (100 mL) followed by complete distillation under vacuum to afford the crude compound. The crude is purified by column chromatography using ethyl acetate-hexane (4:6) as eluents to afford the title compound as yellow solid.
EXAMPLE 25: PREPARATION OF ETHYL (2R,3AR,11AS,12AR,14AR,Z)-2-((2-(4-ISOPROPYLTHIAZOL-2-YL)-7-METHOXY-8-METHYLQUINOLIN-4-YL)OXY)-5-METHYL-4,14-DIOXO-2,3,3A,4,5,6,7,8,9,11A,12,13,14,14A-TETRADECAHYDRO CYCLOPENTA[C]CYCLOPROPA[G][1,6] DIAZACYCLOTETRADECINE-12A(1H)-CARBOXYLATE
A flask is charged with ethyl (1R,2S)-1-((1R,2R,4R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-((2-(4-isopropylthiazol-2-yl)-7-methoxy-8-methylquinolin-4-yl)oxy)cyclopentane-1-carboxamido)-2-vinylcyclopropane-1-carboxylate (3 g) and 1,2-dichloroethane (2400 mL) under nitrogen atmosphere. The mixture is purged with nitrogen gas for 30 minutes and then degassed with vacuum under nitrogen. To the mixture, Hoveyda Grubb’s 2nd generation catalyst (0.19 g) is added under nitrogen followed by degassing of the mixture with vacuum and nitrogen. The reaction mixture is warmed to 75-80oC and stirred at the same temperature for 18 hours. After completion of the reaction as monitored by TLC, the mixture is cooled to room temperature followed by complete distillation under vacuum to afford the crude compound. The crude is purified by column chromatography using ethyl acetate:hexane (4:6) to afford the title compound in the form of solid.
EXAMPLE 26: PREPARATION OF (2R,3AR,11AS,12AR,14AR,Z)-2-((2-(4-ISOPROPYLTHIAZOL-2-YL)-7-METHOXY-8-METHYLQUINOLIN-4-YL)OXY)-5-METHYL-4,14-DIOXO-2,3,3A,4,5,6,7,8,9,11A,12,13,14,14A-TETRADECAHYDRO CYCLOPENTA[C]CYCLOPROPA[G][1,6] DIAZACYCLOTETRADECINE-12A(1H)-CARBOXYLIC ACID
A flask is charged with ethyl (2R,3aR,11aS,12aR,14aR,Z)-2-((2-(4-isopropylthiazol-2-yl)-7-methoxy-8-methylquinolin-4-yl)oxy)-5-methyl-4,14-dioxo-2,3,3a,4,5,6,7,8,9, 11a,12,13,14,14a-tetradecahydrocyclopenta[c]cyclopropa[g][1,6] diazacyclo tetradecine-12a(1H)-carboxylate, tetrahydrofuran (70 mL) and methanol (45 mL). To this, aqueous solution of lithium hydroxide monohydrate (3.65 g in 35 mL) is added at room temperature and mixture is stirred at the same temperature for 16 hours. After completion of the reaction as monitored by TLC, reaction mixture is quenched with saturated ammonium chloride solution (28 mL). The mixture is subjected to complete distillation under vacuum and then cooled to 10oC followed by acidification with 1N hydrochloric acid to pH of about 3 and then extracted with ethyl acetate (3x20 mL). The organic layers are combined and subjected to complete distillation under vacuum at 45oC to afford the title compound as off-white solid.
EXAMPLE 27: PREPARATION OF SIMEPREVIR
A flask is charged with (2R,3aR,11aS,12aR,14aR,Z)-2-((2-(4-isopropylthiazol-2-yl)-7-methoxy-8-methylquinolin-4-yl)oxy)-5-methyl-4,14-dioxo-2,3,3a,4,5,6,7,8,9,11a,12, 13,14,14a-tetradecahydrocyclopenta[c]cyclopropa[g][1,6] diazacyclotetradecine-12a(1H)-carboxylic acid (1.1 g) and dry THF (22 mL) under nitrogen atmosphere followed by addition of 1,1-carbonyl diimidazole (0.77 g) in one portion at room temperature. The mixture is heated to reflux and maintained at the same temperature for 2 hours. The reaction mixture is then cooled to room temperature and progress of the reaction is monitored by TLC. Then cyclopropyl sulfonamide (0.78 g) and DBU (0.57 g) are added to the mixture at room temperature. The mixture is warmed to 50oC and allowed to stir at the same temperature for 15 hours. After completion of the reaction, mixture is cooled to room temperature and subjected to distillation under vacuum to afford residue. To the residue, dichloromethane (40 mL) and 1N hydrochloric acid (15 mL) are added. The organic layer is separated and washed with brine (25 mL) and then subjected to complete distillation under vacuum to afford crude compound. The crude compound is purified by column chromatography using ethyl acetate:hexane (6:4) to afford the title compound as solid.
EXAMPLE 28: PREPARATION OF (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLIC ACID
A flask is charged with 412 mg (1.39 mmoL) of ethyl trans-(1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate and tetrahydrofuran (THF). A solution of lithium hydroxide (126 mg) in 1:1 mixture of water: methanol (6 mL) is then added to the above THF solution. The reaction is stirred at ambient temperature for 60 minutes and then mixture is subjected to complete distillation under reduced pressure. The resulting residue is diluted with 1M hydrochloric acid (10 mL) solution and then mixture is extracted with ethyl acetate (20 mL). The organic layer is separated and subjected to complete distillation under reduced pressure to afford title compound in 88% yield.
EXAMPLE 29: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with ethyl (1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate (150 mg), isopropanol (1mL), potassium phosphate buffer (0.1M, 4 mL), NADP (3.6 mg) and ketoreductase in the form of cell paste obtained from Streptomyces coelicolor ATCC BAA-471 at 25oC. The mixture is maintained at ambient temperature for 92 hours followed by extraction of mixture with ethyl acetate (10 mL). The ethyl acetate layer is subjected to complete distillation under vacuum to afford the title compound.
EXAMPLE 30: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with ethyl (1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate (1 g). To this, a mixture of NADP (20 mg), GDH (6 mg), ketoreductase (15 mg) obtained from Streptomyces coelicolor ATCC BAA-471 and a solution of D-glucose (1.4 g) in phosphate buffer (0.1 M, 20 mL, pH = 8) is added and mixture is maintained at 25oC. The pH of the mixture is maintained at 8 by addition of sodium hydroxide (1M) via a pH autotitrator. After 20 hours reaction mixture is extracted with ethyl acetate (3x20 mL). The combined organic layers are subjected to distillation under reduced pressure to afford the title compound in 95% (950 mg) yield having diastereomeric excess of 98.9%.
EXAMPLE 31: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with ethyl (1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate (22.5 g). To this, a mixture of NADP (450 mg), GDH (137 mg), ketoreductase (231 mg) obtained from Streptomyces coelicolor ATCC BAA-471 and a solution of D-glucose (31.5 g) in phosphate buffer (0.1 M, 450 mL, pH = 8) is added and mixture is maintained at 25oC. The pH of the mixture is maintained at 8 by addition of sodium carbonate (1M) solution (14% wt) via a pH autotitrator. Additionally, GDH (118 mg) is added to the reaction at 22 hour. Then after 28 hours of reaction, the mixture is extracted with ethyl acetate (2x450 mL). The combined organic layer is subjected to complete distillation under vacuum to afford the title compound in 91% yield (20.4 g) having diastereomeric excess of 97.4%.
EXAMPLE 32: PREPARATION OF ETHYL (1R,2R,4R)-2-(HEX-5-EN-1-YL(METHYL)CARBAMOYL)-4-HYDROXYCYCLOPENTANE-1-CARBOXYLATE
A flask is charged with ethyl (1R,2R)-2-(hex-5-en-1-yl(methyl)carbamoyl)-4-oxocyclopentane-1-carboxylate (200 mg), isopropanol (1mL), phosphate buffer (50 mM, 4 mL, pH = 8), NADP (10 mg) and ketoreductase (20 mg) obtained from Streptomyces coelicolor ATCC BAA-471.The mixture is maintained at 25oC for 77 hours followed by extracted of mixture with ethyl acetate (10 mL). The ethyl acetate layer is subjected to distillation under vacuum to afford 140 mg of title compound having diastereomeric excess of 97.4%.
,CLAIMS:We Claim:
1. A process for preparation of compound of Formula II, comprising,
a) reacting compound of Formula IIIa or its salt with a suitable enzyme and/or variant thereof that stereoselectively reduces the keto group under suitable reaction conditions to afford compound of Formula II,

Formula IIIa Formula II
Wherein R1 is H, C1-C6 alkyl, benzyl, substituted or unsubstituted phenyl.
b) optionally, maintaining pH of about 7-9 during the reaction.
c) isolating the compound of Formula II.
2. The process of claim 1, wherein the suitable enzyme is selected from oxidoreductase, ketoreductase enzyme.
3. The process of claim 1 wherein the suitable enzyme is utilized as a whole cell or isolated enzyme form from either a wild type or a recombinant microorganism including but not limited to Streptomyces coelicolor, E.coli.
4. A process for the preparation of compound of Formula IIIa, comprising,
a) reacting a compound of Formula IV with a compound of Formula V or its salts under suitable reaction conditions to afford compound of Formula IIIa,

Formula IV Formula V

b) isolating compound of Formula IIIa.
5. The process of claim 4 wherein R1 is selected from hydrogen, methyl, ethyl, isopropyl, tert-butyl and like.
6. The process of claim 4 wherein step a) is conducted in the presence of amide coupling reagent in a reaction inert solvent, optionally in the presence of tertiary amine base.
7. The process of claim 6, wherein the amide forming agent comprises N-ethoxycarbonyl-2-ethoxy- 1 ,2-dihydroquinoline (EEDQ), N-isopropoxy- carbonyl-2-isopropoxy-l,2-dihydroquinoline (IIDQ), N,N,N,N"-tetramethyl-O-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (HATU), benzotriazol-1-yl- oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, CDI, 1-ethyl-3-(3-di-methylaminopropyl) carbodiimide (EDCI) or its hydrochloride, dicyclohexyl- carbodiimide (DCC), 1,3-diisopropylcarbodiimide, or O-benzotriazole-N,N,N',N'- tetramethyl-uronium-hexafluorophosphate (HBTU), optionally in the presence of a catalyst such as 1-hydroxybenzotriazole (HOBt) or 4-dimethylaminopyridine (DMAP).
8. A process for preparation of compound of Formula I, comprising
a) preparing compound of Formula II according to claims 1-4.
b) converting compound of Formula II to compound of Formula I.
9. A compound of Formula IIIa,

R1 is H, C1-6 alkyl, alkyl aryl and substituted or unsubstituted aryl.
10. A compound of Formula IIIa’,