Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
  • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
  • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams

Definitions

• the invention relates to reduction processes of ezetimibe intermediates to obtain ezetimibe or a derivative thereof.
• Ezetimibe l-(4-fluorophenyl)- 3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone, is a selective inhibitor of intestinal cholesterol and related phytosterol absorption.
• the empirical formula for ezetimibe is C 24 H 2 1F2NO3, and its molecular weight is 409.4.
• Ezetimibe is a white, crystalline powder that is freely to very soluble in ethanol, methanol, and acetone and practically insoluble in water. Ezetimibe has the following chemical structure:
• Ezetimibe is the active ingredient in the drug sold under the brand name
• ZETIA ® which is manufactured by Merck/Schering-Plough Pharmaceuticals. ZETIA ® has been approved by the United States Food and Drug Administration for use in patients with high cholesterol to reduce low density lipoprotein (“LDL”) cholesterol and total cholesterol.
• LDL low density lipoprotein
• Ezetimibe can be prepared by reducing (3R,4S)-4-((4-benzyloxy)phenyl)-l-(4- fluorophenyl)-3-(3-(4-fluorophenyl)-3-oxopropyl)-2-azetidinone ("Compound 1”) with borane dimethyl sulfide complex or borane tetrahydrofuran complex in tetrahydrofuran in the presence of Corey's reagent and subsequently deprotecting the benzyl group, as shown in Scheme 1 below.
• the process is disclosed in US patent nos. 5,631,365 (“the '365 patent”) and 6,627 ',757 ', which are incorporated herein by reference.
• the starting material, Compound 1 or a similar compound can be prepared by processes known in the art, for example, those disclosed in the '365 patent.
• CBS (R)-(+)-2-methyl-CBS-oxazaborolidine
• BMS borohydride dimethylsulfide complex
• U.S. patent No. 6,133,001 refers to a process for stereoselective microbial reduction of ezetimibe-ketone to ezetimibe, as illustrated below.
• PCT publication No. WO 2005/066120 refers to an enantioselective reduction of ezetimibe-ketone to ezetimibe with (-)-B-chlorodiisopinocampheylborane ("DIP-Cl").
• the invention encompasses a process for preparing a compound of formula I
• ketoreductase is selected from the group consisting of the predominant enzyme in each of KRED-NADH- 105, KRED- NADH-107, KRED-116, KRED-118, KRED-119, KRED-120, KRED-128, KRED-133, and mixtures thereof.
• the invention encompasses a process for preparing a compound of formula I
• ketoreductase enzyme a co-factor
• the invention encompasses a process for purifying ezetimibe from EZT-ketone comprising crystallizing ezetimibe from MIBK.
• the present invention relates to a process for preparing a compound of formula I using enzymes as catalysts and optionally involves the use of water-miscible organic solvents.
• yield refers to percentage of a synthesized compound with respect to the original amount of starting material. It is determined by the area percentage under the peak of the synthesized compound relative to the total area in the HPLC chromatogram. Yield is typically measured during or immediately following a reaction.
• purity refers to the percentage of a compound with respect to other compounds. It is determined by the area percentage under the peak of the compound relative to the total area in the HPLC chromatogram. Purity is typically measured after a purification process.
• diastereometric excess refers to diastereometric excess, defined as:
• Ezetimibe has a S,R,S configuration, as shown below:
• the R,R,S-diastereomer differs in that it has an (R) configuration at the chiral center on the third carbon in the propyl chain.
• EZT refers to ezetimibe, or l-(4-fluorophenyl)-
• EZT RRS-isomer refers to l-(4-fluorophenyl)-3(R)-[3-(4- fluorophenyl)-3(R)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone.
• ezetimibe-ketone or “EZT-ketone” refers to l-(4-fluorophenyl)-3(R)-[3-(4- fluorophenyl)-3-oxopropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone, having the following chemical structure:
• Compound 1 refers to (3R,4S)-4-((4- benzyloxy)phenyl)- 1 -(4-fluorophenyl)-3-(3-(4-fluorophenyl)-3-oxopropyl)-2-azetidinone;
• Compound 2a refers to (3R,4S)-4-((4-benzyloxy)phenyl)-l-(4-fluorophenyl)-3-((S)-3- (4-fluorophenyl)-3-hydroxypropyl)-2-azetidinone;
• Compound 2b refers to (3R,4S)-4-((4-benzyloxy)phenyl)-l-(4-fluorophenyl)-3-((R)-3-(4-fiuorophenyl)-3- hydroxypropyl)-2-azetidinone.
• the term “isolated” refers to an enzyme separated or removed from its native environment. Use of the term “isolated” indicates that a naturally occurring or recombinant enzyme has been removed from its normal cellular environment. Preferably, the isolated enzyme is in a cell-free system. The isolated enzyme can be crude or highly purified depending on the effort put in removing other materials. The term “isolated” does not imply that the enzyme is the only enzyme present, but that it is the predominant enzyme present (at least 10-20% more than any other enzyme). As used herein, when applied to an enzyme, the term “synthesized” refers to an enzyme that is prepared by chemical synthesis, recombinant means, or the combination thereof. As used herein, when applied to an enzyme, the term “purified” refers to an enzyme that is essentially free (at least about 90-95% pure) of non-enzymatic material or other enzymes.
• dehydrogenase or “dehydrogenase enzyme” refers to an enzyme that oxidizes a substrate by transferring one or more protons and a pair of electrons to an acceptor.
• dehydrogenase include alcohol dehydrogenase, glucose dehydrogenase, formate dehydrogenase, and phosphite dehydrogenase.
• Glucose dehydrogenase include, for example, those classified under the Enzyme Commission (“EC") number of 1.1.1.47 and the Chemical Abstract Service (“CAS”) number 9028-53-9, and are commercially available, for example, from Codexis, Inc.
• Formate dehydrogenase include, for example, those classified under the EC number of 1.2.1.2 and the CAS number of 9028-85-7, and are commercially available, for example, from Codexis, Inc. under the catalog number FDH-101.
• Phosphite dehydrogenase include, for example, those classified under the EC number of 1.20.1.1 and CAS number of 9031-35-0, and are commercially available, for example, from Codexis, Inc. under the catalog number PDH-101.
• ketooreductase As used herein, the term "ketoreductase,” “ketoreductase enzyme,” or
• Ketoreductase enzymes include, for example, those classified under the EC numbers of 1.1.1. Such enzymes are given various names in addition to ketoreductase, including, but not limited to, alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, hydroxyisocaproate dehydrogenase, ⁇ - hydroxybutyrate dehydrogenase, steroid dehydrogenase, sorbitol dehydrogenase, aldoreductase, and the like.
• NADPH-dependent ketoreductases are classified under the EC number of 1.1.1.2 and the CAS number of 9028-12-0.
• NADH-dependent ketoreductases are classified under the EC number of 1.1.1.1 and the CAS number of 9031 -72-5.
• Ketoreductases are commercially available, for example, from Codexis, Inc. under the catalog numbers KRED- 100 to KRED- 177.
• co-factor refers to a non-protein 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 ('TSfAD”), reduced nicotinamide adenine dinucleotide (“NADH”), nicotinamide adenine dinucleotide phosphate ("NADP + "), reduced nicotinamide adenine dinucleotide phosphate (“NADPH”), and any derivatives or analogs thereof.
• nicotinamide co-factors such as nicotinamide adenine dinucleotide ('TSfAD), reduced nicotinamide adenine dinucleotide (“NADH”), nicotinamide adenine dinucleotide phosphate ("NADP + "), reduced nicotinamide adenine din
• MeOH refers to methanol
• EtOAc refers to ethyl acetate
• IPA isopropyl alcohol
• DMSO dimethyl sulfoxide
• MIBK methyl isobutyl ketone
• DCM dichlormethane
• MTBE methyl tert-butyl ether
• DTT dithiotreitol
• room temperature or "RT” refers the ambient temperature of a typical laboratory, which is usually about 15 0 C to about 3O 0 C.
• vacuum refers to a pressure of about to 2 mmHg to about 100 mmHg.
• the invention encompasses a process for preparing a compound of formula I
• the ketoreductase is selected from the group consisting of the predominant enzyme in each of KRED-NADH- 105, KRED- NADH-107, KRED-116, KRED-118, KRED-119, KRED-120, KRED-128, KRED-133, and mixtures thereof.
• the invention encompasses a process for preparing a compound of formula I
• a biocatalyst and a buffer, wherein R is H or a hydroxyl protecting group.
• the invention encompasses a process for preparing a compound of formula I
• a compound of formula II a ketoreductase enzyme, a co-factor, and a buffer having a pH of about 4 to about 8, wherein R is H or a hydroxy 1 protecting group.
• R is H
• the compound of formula I is ezetimibe and the compound of formula II is ezetimibe-ketone.
• R is a hydroxyl protecting group
• the compound of formula II can be further deprotected to obtain ezetimibe. The deprotection can be done by known methods, for example those described in Example 6 of the '365 patent.
• reaction is an enzymatic process as illustrated below:
• ketoreductase stereoselectively reduces the carbonyl group.
• a co-factor such as NADH or NADPH is oxidized.
• the hydroxyl protecting group is selected from the group consisting of benzyl, tert-butyloxycarbonyl, acyl, and silyl groups.
• suitable silyl protecting groups include, but are not limited to, (R a )(R b )(R c )-Si-, wherein R a , R b and R c are each independently selected from the group consisting of Ci to C 6 alkyl, phenyl, benzyl, acetyl, or the like.
• the ketoreductase is isolated.
• the ketoreductase can be separated from any host, such as mammals, filamentous fungi, yeasts, and bacteria.
• the isolation, purification, and characterization of a NADH-dependent ketoreductase is described in, for example, in Kosjek et al, Purification and Characterization of a Chemotolerant Alcohol Dehydrogenase Applicable to Coupled Redox Reactions, Biotechnology and Bioengineering, 86:55-62 (2004).
• the ketoreductase is synthesized.
• the ketoreductase can be synthesized chemically or using recombinant means.
• ketoreductases The chemical and recombinant production of ketoreductases is described in, for example, in European patent no. EP 0918090.
• the ketoreductase is synthesized using recombinant means in Escherichia coli.
• the ketoreductase is purified, preferably with a purity of about 90% or more, more preferably with a purity of about 95% or more.
• the ketoreductase is substantially cell-free.
• the ketoreductase is one that capable of producing ezetimibe with a d.e. of about 90% or higher in the processes of the invention.
• the ketoreductase is one that capable of producing ezetimibe with a yield of about 50% or higher in the processes of the invention.
• the ketoreductase is selected from the group consisting of NADH- dependent ketoreductases and NADPH-dependent ketoreductases.
• Suitable ketoreductases include, but are not limited to, a) Codexis Inc's products with catalog numbers KRED- NADH- 105, KRED-NADH- 107, KRED-116, KRED-118, KRED-119, KRED- 120, KRED- 128, KRED-133, equivalent products thereof, and mixtures thereof; and b) the predominant enzyme in each of Codexis Inc's products with catalog numbers KRED-NADH- 105, KRED- NADH-107, KRED-116, KRED-118, KRED-119, KRED-120, KRED-128, KRED-133, equivalent enzymes thereof, and mixtures thereof.
• the term "equivalent” refers to an enzyme or product with similar or identical enzymatic activity.
• the ketoreductase is selected from the group consisting of the predominant enzyme in each of Codexis Inc's products with catalog numbers KRED-NADH- 105, KRED-NADH- 107, KRED-116, KRED-118, KRED-119, KRED-120, KRED-128, KRED-133, and mixtures thereof.
• the ketoreductase is selected from the group consisting the predominant enzyme in each of KRED-NADH- 105, KRED-116, KRED-118, KRED-119, KRED-128, and mixtures thereof. More preferably, the ketoreductase is selected from the group consisting of the predominant enzyme in each of KRED-118, KRED-119, KRED-128, and mixtures thereof.
• the process of the invention further comprises combining a co-factor with the ketoreductase.
• the co-factor is selected from the group consisting of NADH, NADPH, NAD + , NADP + , salts thereof, and mixtures thereof.
• the ketoreductase is NADH-dependent
• the co-factor is selected from the group consisting of NADH, NAD + , salts thereof, and mixtures thereof. More preferably, the co-factor is NADH or a salt thereof.
• the ketoreductase is NADPH- dependent
• the co-factor is selected from the group consisting of NADPH, NADP + , salts thereof, and mixtures thereof. More preferably, the co-factor is NADPH or a salt thereof.
• the process of the invention is carried out in a buffer.
• the buffer has a pH of from about 4 to about 9, more preferably from about 4 to about 8, more preferably from about 5 to about 8, most preferably from about 6 to about 8 or about 5 to about 7.
• the buffer is a solution of a salt.
• the salt is selected from the group consisting of potassium phosphate, magnesium sulfate, and mixtures thereof.
• the buffer comprises a thiol.
• the thiol is DTT.
• the thiol reduces the disulfide bond in the enzyme.
• the process of the invention is carried out at a temperature of about 1O 0 C to about 5O 0 C.
• the process is carried out at room temperature, at a temperature of about 24 0 C to about 28 0 C, or at about 25 0 C to about 35 0 C.
• the process is carried out at a temperature of about 25 0 C to about 3O 0 C.
• the process is carried out at a temperature of about 3O 0 C.
• the reaction mixture further comprises a co-factor regeneration system.
• a co-factor regeneration system comprises a substrate and a dehydrogenase. The reaction between the substrate and dehydrogenase enzyme regenerates the co-factor.
• the co-factor regeneration system comprises a substrate/dehydrogenase pair selected from the group consisting of D-glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, and phosphite/phosphite dehydrogenase.
• the glucose dehydrogenase is selected from the group consisting of the predominant enzyme in each of Codexis Inc's products with catalog numbers GDH-102, GDH-103, GDH-104, and mixtures thereof.
• the glucose dehydrogenase is the enzyme in GDH-104.
• the formate dehydrogenase is the predominant enzyme in Codexis Inc's product with catalog number FDH-101.
• the phosphite dehydrogenase is the predominant enzyme in Codexis Inc's product with catalog number PDH-101.
• the process of the invention further comprises adding a solvent.
• the solvent is an organic solvent.
• the organic solvent is water-miscible.
• the water-miscible organic solvent is selected from the group consisting of alcohols and DMSO.
• the alcohol is a C1-C4 alcohol, more preferably methanol or IPA.
• the process comprises the following steps: (a) dissolving a compound of formula II in a solvent; and (b) combining the solution from (a) with a buffer containing a co-factor and a ketoreductase.
• the obtained mixture is stirred for a period of time sufficient to obtain the compound of formula I.
• the stirring is at a temperature of about 25 0 C to about 35 0 C or about 25 0 C to about 4O 0 C, more preferably at a temperature of about 24 0 C to about 28 0 C, about 25 0 C to about 3O 0 C, or about 3O 0 C.
• the stirring is for about 0.5 hours or more, about 1.5 hours or more, or about 2.5 hours or more.
• the stirring is for about 50 hours or less.
• the stirring is for about 3 hours to about 40 hours, more preferably for about 6 hours to about 24 hours or about 6 hours to about 16 hours.
• a water-immiscible organic solvent is added to the reaction mixture, preferably after the stirring.
• the reaction mixture is separated into an organic phase and an aqueous phase.
• the compound of formula I is recovered by evaporating the organic phase.
• water-immiscible organic solvents include, but are not limited to, C 2 -Cs ethers, C 3 -Cs esters such as EtOAc, C 4 -Cs ketones such as MIBK, and halogenated hydrocarbons such as DCM.
• the water-immiscible organic solvent is selected from the group consisting of EtOAc, MTBE, diethyl ether, and mixtures tehreof.
• the water- immiscible organic solvent is EtOAc.
• reaction mixture preferably after the stirring, is filtered to recover the solid product, which may optionally be further purified to obtain the compound of formula I.
• the aqueous phase may be treated to recycle the enzyme, co-factor, and/or the dehydrogenase in the co-factor regeneration system.
• the pH of the aqueous phase may be adjusted to obtain the desired pH.
• the aqueous phase is evaporated to remove organic solvent residue.
• the aqueous phase is reused in the process of the invention.
• the processes described above have high stereoselectivity toward ezetimibe.
• the d.e. of the ezetimibe obtained is preferably about 90% or higher, more preferably about 98% or higher, or about 99% or higher, and most preferably about 99.9% or higher.
• the processes described above has high yield.
• the yield of the compound of formula I obtained is preferably about 50% or higher, more preferably about 60% or higher, or about 70% or higher, or about 80% or higher and most preferably about 85% or higher.
• the invention encompasses a process for purifying ezetimibe from EZT-ketone comprising crystallizing ezetimibe from MIBK.
• the process comprises: a) dissolving a sample comprising ezetimibe and EZT-ketone in MIBK; b) cooling the solution from step a); and c) recovering ezetimibe.
• step a) is performed under heating.
• the heating is to a temperature of from about 5O 0 C to about reflux temperature, more preferably to about reflux temperature.
• the cooling is to about room temperature or less, preferably to about 1O 0 C.
• a slurry is obtained after the cooling step.
• ezetimibe is recovered from the slurry by filtering and optionally drying. The drying is preferably at about 4O 0 C to about 5O 0 C, preferably under vacuum.
• the ezetimibe obtained has a purity of about 98% or more, more preferably about 99% or more.
• KRED-128 (5mg, Codexis, Lot No. 090305CL, year of production: 2005) was dissolved in 5ml buffer (containing 25OmM potassium phosphate, 0.5mM DTT, 2mM magnesium sulfate, l.lmM NADP + , 8OmM D-glucose, 10 U/ml glucose dehydrogenase, pH 7.0).
• a solution of EZT-ketone in MeOH (lOmg in 0.25ml) was added. The mixture was stirred at 30 0 C for 40 hours and monitored by HPLC. EtOAc (5ml) was added and the phases were separated. The EZT in the organic phase was analyzed, (yield: 71.55%, d.e.: 99.9%).
• KRED- 133 (5mg, Codexis, Lot No. 113006MM, year of production: 2006) was dissolved in 5ml buffer (containing 25OmM potassium phosphate, 0.5mM DTT, 2mM magnesium sulfate, l.lmM NADP + , 8OmM D-glucose, 10 U/ml glucose dehydrogenase, pH 7.0).
• a solution of EZT-ketone in MeOH (lOmg in 0.25ml) was added. The mixture was stirred at 30 0 C for 40 hours and monitored by HPLC. EtOAc (5ml) was added and the phases were separated. The EZT in the organic phase was analyzed, (yield: 17.6%, d.e.: 99.3%).
• KRED-NADH -105 (5mg, Codexis) was dissolved in 5ml buffer (containing
• KRED-NADH -107 (5mg, Codexis) was dissolved in 5ml buffer (containing
• KRED-116 (5mg, Codexis) was dissolved in 5ml buffer (contains 25OmM potassium phosphate, 0.5mM "DTT, 2mM magnesium sulfate, 1.ImM NADP + , 8OmM D- glucose, 10 U/ml glucose dehydrogenase, pH 7.0).
• a solution of EZT-ketone in MeOH (20mg in 0.25ml) was added and the mixture was stirred at 30 0 C over night.
• EtOAc (5ml) was added and the phases were separated. The organic phase was evaporated. The EZT in the residue was analyzed, (yield: 57.97 %, d.e.: 99.9%).
• KRED- 118 (5mg, Codexis) was dissolved in 5ml buffer (contains 25OmM potassium phosphate, 0.5mM DTT, 2mM magnesium sulfate, l.lmM NADP + , 8OmM D- glucose, 10 U/ml glucose dehydrogenase, pH 7.0).
• a solution of EZT-ketone in MeOH (20mg in 0.25ml) was added and the mixture was stirred at 30 0 C over night.
• EtOAc (5ml) was added and the phases were separated. The organic phase was evaporated, (yield: 87.59%, d.e.: 99.9%).
• Example 7 Reduction of EZT-ketone with KRED-119
• KRED-119 (5mg, Codexis, Lot No. 100407WW, year of production: 2007) was dissolved in 5ml buffer (contains 25OmM potassium phosphate, 0.5mM DTT, 2mM magnesium sulfate, l.lmM NADP + , 8OmM D-glucose, 10 U/ml glucose dehydrogenase, pH 7.0).
• a solution of EZT-ketone in MeOH (20mg in 0.25ml) was added and the mixture was stirred at 30 0 C over night.
• EtOAc (5ml) was added and the phases were separated. The organic phase was evaporated. The EZT in the residue was analyzed, (yield: 83.19%, d.e.: 99.9%).
• KRED-120 (5mg, Codexis) was dissolved in 5ml buffer (contains 25OmM potassium phosphate, 0.5mM DTT, 2mM magnesium sulfate, l.lmM NADP + , 8OmM D- glucose, 10 U/ml glucose dehydrogenase, pH 7.0).
• a solution of EZT-ketone in MeOH (20mg in 0.25ml) was added and the mixture was stirred at 30 0 C over night.
• EtOAc (5ml) was added and the phases were separated. The organic phase was evaporated. The EZT in the residue was analyzed, (yield: 37.41%, d.e.: 99.9%).
• a solution of 600mg EZT-ketone in 4ml IPA was added to 20 ml 10OmM phosphate buffer solution (pH 6) containing: 800mg (0.25M) D-glucose, 40mg glucose dehydrogenase, 16mg (ImM) NADP + , and 20mg KRED-119 (Codexis, Lot No. 100407WW, year of production: 2007.
• the obtained milky mixture was stirred at room temperature for
• Example 11 Purification of ezetimibe [0061] 2g of mixture of ezetimibe and EZT-ketone (purity of ezetimibe: 82%; EZT- ketone content: 15.8%) was dissolved in 5ml MIBK at reflux temperature. The solution was cooled to room temperature and stirred overnight. The obtained slurry was filtered and dried to obtain 0.9g of white product (purity of ezetimibe: 99%).

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