Biocatalytic synthesis of (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid compounds

Abstract

Improved processes for the preparation of (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid and intermediates thereto are described. These compounds are useful as intermediates for the preparation of carbamoyloxymethyltriazole cyclohexyl acid LPA antagonists.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This invention claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 269,370, filed March 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present invention relates to an improved process for producing (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid and intermediates thereto. [Background technology]

[0003] Carbamoyloxymethyltriazole cyclohexyl acid LPA (particularly LPA1) antagonists have been described, which are useful for treating fibrosis.See, for example, WO2017 / 223016 (US 2017 / 0360759).However, there is a need for further improved methods of producing intermediate (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid, which are practical, allow large-scale synthesis, and have improved product quality, efficiency and safety. Summary of the Invention

[0004] The present invention provides an improved method for producing (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid. A significantly optimized overall process including a one-pot enzyme cascade is also described, which produces the target compound with improved quality and efficiency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] The features and advantages of the present invention may be more readily understood by those skilled in the art upon reading the following detailed description. It should also be understood that, for reasons of clarity, certain features of the present invention that are described above and below in the context of separate embodiments may also be combined to form a single embodiment. Conversely, for reasons of brevity, various features of the present invention that are described above and below in the context of a single embodiment may also be combined to form subcombinations thereof.

[0006] Those skilled in the art will appreciate that some chemical structures described herein may be represented on paper in one or more other resonance forms; or may exist in one or more other tautomeric forms, and even when referred to kinetically, those skilled in the art will appreciate that such tautomeric forms represent only a small sample of such compounds. Although such compounds are expressly contemplated within the scope of the present disclosure, such resonance forms or tautomers are not explicitly depicted herein.

[0007] In a first aspect, the present invention provides a compound of formula (I): [ka] 1. A method for producing a compound of formula (deoxy-functionalization / carbonylation), comprising the steps of: (1) Formula (II): [ka] with a halogenating or sulfonylating reagent in a polar aprotic solvent or solvent mixture, with or without an inorganic or organic base, for a time and temperature sufficient to effect deoxy-functionalization to provide a compound of formula (III): [ka] wherein R is Cl, Br, I, OMs, OTs, or OTf. to produce a compound represented by the formula: (2) With or without isolating the compound of formula (III), the process comprises contacting the compound of formula (III) with a palladium catalyst, with or without a phosphine ligand, and an inorganic or organic base, in water and a polar aprotic solvent, purged with carbon monoxide, for a time, temperature and pressure sufficient to subject the compound of formula (III) to carbonylation to produce a compound of formula (I).

[0008] In a second aspect, the present invention provides a compound of formula (I): [ka] 1. A method for producing a compound of formula (deoxy-functionalization / carbonylation), comprising the steps of: (1) Formula (II): [ka] with a halogenating reagent selected from oxalyl iodide, oxalyl bromide, and oxalyl chloride, or a sulfonylation reagent selected from p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, N-phenyl-bis(trifluoromethanesulfonimide), and N-(5-chloropyridin-2-yl)-N-(methanesulfonyl)methanesulfonamide, in a polar aprotic solvent selected from EtOAc, 2-MeTHF, CHCN, DMAc, DMF, THF, toluene, and mixtures thereof, with or without an inorganic or organic base selected from KCO, N,N-diisopropylethylamine, triethylamine, t-butyltetramethylguanidine, tetramethylguanidine, KHCO, DBU, NaCO, NaCO*10H0, and KOAc, for a time and temperature sufficient to provide deoxy-functionalization to obtain a compound of formula (III): [ka] wherein R is Cl, Br, I, OMs, OTs, or OTf. to produce a compound represented by the formula: (2) With or without isolating the compound of formula (III), the compound of formula (III) is reacted with a palladium catalyst selected from Pd(A-taPhos)2Cl2, Pd(DPEPhos)Cl2, Pd(PPh3)2Cl2, Pd(DCEPhos)Cl2, Pd(OAc)2, PdCl2(CH3CN)2 and Pd(DPPP)Cl2 in a polar aprotic solvent selected from water and EtOAc, 2-MeTHF, CH3CN, DMAc, DMF, THF, toluene and mixtures thereof, with 1,3-bis(dicyclohexylphosphino)propane, 1,3-bis(diphenylphosphino)propane, rac-BINAP, with a phosphine ligand selected from Xantphos, Josiphos SL-J001-1, Josiphos SL-J009-1-G3 palladacycle, and BIPHEP; and an inorganic or organic base selected from K2CO3, N,N-diisopropylethylamine, triethylamine, t-butyltetramethylguanidine, tetramethylguanidine, KHCO3, DBU, Na2CO3, Na2CO3*10H2O, and KOAc, for a time, temperature, and pressure sufficient to purge with carbon monoxide and subject to carbonylation to produce a compound of formula (I).

[0009] In a third aspect, the present invention provides a compound of formula (I): [ka] A process for producing a compound of the formula (deoxy-chlorination / carbonylation), comprising the steps of: (1) Formula (II): [ka] with an acid chloride in a polar aprotic solvent or solvent mixture selected from 2-MeTHF and DMF for a time and temperature sufficient to effect deoxy-chlorination to provide a compound of formula (IIIa): [ka] to produce a compound of; (2) With or without isolating the compound of formula (IIIa), the method includes contacting the compound of formula (IIIa) with Pd(A-taPhos)2Cl2, Pd(PPh3)2Cl2, Pd(DCEPhos)Cl2 or PdCl2(CH3CN)2, in a polar aprotic solvent selected from water and 2-MeTHF, CH3CN, toluene, and mixtures thereof, together with 1,3-bis(dicyclohexylphosphino)propane, and an organic or inorganic base selected from DIPEA, K2CO3, Na2CO3, and Na2CO3*10H2O, with a carbon monoxide purge, for a time, temperature, and pressure sufficient to effect carbonylation to produce a compound of formula (I).

[0010] In a fourth aspect, the present invention provides a compound of formula (I): [ka] A method for producing a compound of the formula: (1) Formula (II): [ka] is contacted with oxalyl chloride in a mixture of 2-MeTHF and DMF at 0-50° C. for 1-3 hours to obtain a compound of formula (IIIa): [ka] to produce a compound of; (2) With or without isolating the compound of formula (IIIa), the compound of formula (IIIa) is contacted with Pd(A-taPhos)2Cl2 and Na2CO3 in water and 2-MeTHF at 20-50°C and 30-35 psi for 16-20 hours with a carbon monoxide purge to produce the compound of formula (I).

[0011] In a fifth aspect, the present invention provides a compound of formula (IV): [ka] A method for producing a compound of formula (I): [ka] with an enone reductase (ERED) and a keto reductase (KRED), in the presence of an aqueous polar protic solvent, NADPH and a co-substrate, with or without an organic co-solvent, for a time and at a temperature sufficient to produce a compound of formula (IV).

[0012] In a sixth aspect, the present invention provides a compound of formula (IV): [ka] A method for producing a compound of formula (I): [ka] with an ERED selected from ERED-309 and ERED-310, and a KRED selected from KRED-456, KRED-457 and KRED-P2-B07, in the presence of phosphate, an aqueous buffer selected from TRIS, HEPES, ACES, BES, MOPS and tricene, GDH, NADPH and glucose, with or without an organic co-solvent such as DMSO, IPA, dioxane or acetone, for a time and temperature sufficient to produce a compound of formula (IV).

[0013] In a 6a aspect, the present invention provides a compound of formula (IV): [ka] A method for producing a compound of formula (I): [ka] with an ERED selected from ERED-309 and ERED-310, and a KRED selected from KRED-456, KRED-457, and KRED-P2-B07, in the presence of an aqueous phosphate buffer, GDH, NADPH, and glucose, with or without an organic co-solvent such as DMSO, IPA, dioxane, or acetone, for a time and temperature sufficient to produce a compound of formula (IV).

[0014] In a seventh aspect, the present invention provides a compound of formula (IV): [ka] A method for producing a compound of formula (I): [ka] with ERED-310, and KRED-457, in the presence of phosphate, an aqueous buffer selected from TRIS, HEPES, ACES, BES, MOPS and tricene, GDH, NADPH and glucose, with or without an organic co-solvent such as DMSO, IPA, dioxane or acetone, for at least 16 hours and at 25-35° C. sufficient to produce a compound of formula (IV).

[0015] In a seventh aspect, the present invention relates to a compound of formula (IV): [ka] A method for producing a compound of formula (I): [ka] with ERED-310, and KRED-457, in the presence of aqueous phosphate buffer, GDH, NADPH, and glucose, with or without an organic co-solvent, such as DMSO, IPA, dioxane, or acetone, for at least 16 hours and at 25-35° C. sufficient to produce a compound of formula (IV).

[0016] In an eighth aspect, the present invention provides a method for preparing a compound of formula (IV), comprising steps (1) and (2) from any of the second to fourth aspects, followed by a step from any of the fifth to seventh aspects, wherein all formulae and variables are as defined in the second to seventh aspects.

[0017] In a ninth aspect, the present invention provides a method of preparing a compound of formula (IV), comprising steps (1) and (2) of the third or fourth aspect, followed by the step of the sixth or seventh aspect, wherein all formulae and variables are as defined in the third, fourth, sixth or seventh aspect.

[0018] In a tenth aspect, the present invention provides a method for preparing a compound of formula (IV), comprising steps (1) and (2) of the fourth aspect followed by the step of the seventh aspect, wherein all formulae and variables are as defined in the fourth and seventh aspects.

[0019] definition The presence of reaction and / or process impurities may be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear magnetic resonance spectroscopy, mass spectroscopy, and / or infrared spectroscopy.

[0020] Other embodiments include those described in the detailed description and / or claims. In order to facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures of organic chemistry, medicinal chemistry, and pharmacology described herein are those commonly used procedures well known in the art. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0021] The term "halo" or "halogen" refers to fluoro (F), chloro (Cl), bromo (Br) or iodo (I). The term "cycloalkyl" as used herein includes saturated cyclic hydrocarbon groups having 3 to 10 carbons, preferably 3 to 8 carbons, more preferably 3 to 6 carbons, where the cycloalkyl group may be optionally substituted. Preferred cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl.

[0022] The term "Brönsted acid" refers to the charge of a proton (H + ) donor. The term "Lewis acid" refers to a chemical species that can accept an electron pair from an electron donor compound. The term "Brønsted base" refers to a proton (H + ) receptor. The term "Lewis base" refers to a chemical species that can donate an electron pair to an electron acceptor compound.

[0023] The term "transition metal catalyst" refers to a coordination complex with any of a variety of metallic elements, such as palladium and nickel, that has two shells of valence electrons instead of only one shell, and that when added to a chemical reaction increases the rate of the reaction. Examples of palladium catalysts include, but are not limited to, Pd(A-taPhos)2Cl2, Pd(DPEPhos)Cl2, Pd(PPh3)2Cl2, Pd(DCEPhos)Cl2, or Pd(OAc)2, PdCl2(CH3CN)2, or Pd(DPPP)Cl2.

[0024] The term "phosphine ligand" refers to a trivalent phosphorus compound capable of binding to a metal atom. Examples of phosphine ligands include, but are not limited to, 1,3-bis(dicyclohexylphosphino)propane, 1,3-bis(diphenylphosphino)propane, rac-BINAP, Xantphos, Josiphos SL-J001-1, Josiphos SL-J009-1-G3 palladacycle, and BIPHEP.

[0025] A protic solvent is a solvent in which a hydrogen atom is bonded to an oxygen (in the case of a hydroxyl group) or a nitrogen (in the case of an amine group). Aprotic solvents refer to solvents that are not hydrogen bond donors. Polar solvents refer to solvents that have a large dipole moment or partial charge; they contain bonds between atoms of widely differing electronegativities, such as oxygen and hydrogen. A solvent mixture refers to a combination of two or more solvents.

[0026] The term "halogenating reagent" refers to a reagent capable of introducing a halogen atom into a molecule. The term "sulfonylating reagent" refers to a reagent capable of introducing a sulfonate moiety into a molecule. Examples of sulfonate moieties include, but are not limited to, mesylates (OMs), tosylates (OTs), and trifluoromethanesulfonate (triflate or OTf).

[0027] As used herein, "enone reductase", "ene reductase", and "ERED" are used interchangeably herein and refer to a polypeptide capable of reducing α,β-unsaturated compounds to the corresponding saturated compounds. More specifically, enone reductases have the ability to reduce α,β-unsaturated ketones, aldehydes, nitriles, olefins, and esters. For example, ERED-309 and ERED-310 are available from Codexis, Inc.

[0028] As used herein, "ketoreductase" and "KRED" are used herein to refer to a class (EC 1.1.1.184) of polypeptides useful for synthesizing optically active alcohols from the corresponding prochiral ketone substrates and for stereoselective reduction of the corresponding racemic aldehyde substrates. KREDs typically convert ketone and aldehyde substrates to the corresponding alcohol products, but can also catalyze the reverse reaction, the oxidation of alcohol substrates to the corresponding ketone / aldehyde products. For example, KRED-456, KRED-457, and KRED-P2-B07 are available from Codexis, Inc.

[0029] In addition, atoms constituting the compounds of the embodiments of the present invention are intended to include all isotopic forms of such atoms. As used herein, isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, isomers of carbon include 13 C and 14 C is an example.

[0030] Working Example The following examples are presented to fully disclose and explain to one of ordinary skill in the art how to make and use the present invention, and are not intended to limit the scope of what the inventor regards as his invention, nor are they intended to represent that the experiments described below have been performed, or that they are all experiments that may be performed. It should be understood that the illustrative descriptions presented in the present tense have not necessarily been performed, but rather that the descriptions may be performed to obtain the data, etc. of the characteristics described therein. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0031] The starting materials used in the synthetic routes of the present invention are known, prepared by known methods, or commercially available. Those skilled in the art will also understand that the conditions and reagents described herein can be interchanged with equivalent conditions and reagents that are recognized as alternative art. For example, in some reactions, hydrochloric acid can be replaced with other acids such as hydrobromic acid, sulfuric acid, etc. Those skilled in the art will appreciate that the following methods may be used to characterize the compounds described herein, for example: 1 A variety of analytical methods will be appreciated, including H nuclear magnetic resonance spectroscopy (NMR), heteronuclear NMR, mass spectrometry (MS), liquid chromatography (LC), and infrared (IR) spectroscopy. The above list is a non-limiting list of characterization methods available to those skilled in the art.

[0032] To further illustrate the above, the following non-limiting exemplary synthetic schemes are included. Variations of these examples that fall within the scope of the claims are within the scope of the skilled artisan and are considered to be within the scope of the invention described and claimed herein. The reader will recognize that a skilled artisan provided with this disclosure and the art will be able to make and use the invention even without the exhaustive examples.

[0033] The following abbreviations have the following meanings: ACES = N-(2-acetamido)-2-aminoethanesulfonic acid aq = aqueous BES = N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid BIPHEP = 2,2'-bis(diphenylphosphino)biphenyl CH3CN = Acetonitrile CO = carbon monoxide d=double term D2O=heavy water DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene DIPEA = N,N-diisopropylethylamine DMAc = Dimethylacetamide DMF = Dimethylformamide DMSO = dimethyl sulfoxide equiv=equivalent ERED = enone reductase or ene reductase ESI = electrospray ionization EtOAc = ethyl acetate g = grams GDH = glucose dehydrogenase h=time HEPES = (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) HCl = Hydrogen Chloride (usually in solution) H2O = Water HPLC = High Pressure Liquid Chromatography IPA = Isopropyl alcohol Josiphos SL-J001-1 = (2R)-1-[(1R)-1-(dicyclohexyl phosphino)ethyl]-2-(diphenylphosphino)ferrocene Joshihos SL-J009-1-G3 Paradacycle = {(R)-1-[(Sp)-2 -(Dicyclohexylphosphino)ferrocenyl]ethyl di-tert-butyl phosphine}[2-(2'-amino-1,1'-biphenyl)]palladium(II) Methanesulfonate K2CO3 = Potassium Carbonate KHCO3 = Potassium Bicarbonate KH2PO4 = Potassium phosphate monobasic KF = Karl Fischer titration KOAc = Potassium Acetate kg = kilograms KRED = ketoreductase L = liters LCAP = Liquid Chromatography Area Percent m=multiplet M = moles MOPS = 3-(N-morpholino)propanesulfonic acid mg = milligrams MeOH or CH3OH = Methanol D4-MeOH = deuterated methanol MeTHF or 2-MeTHF = 2-methyltetrahydrofuran MHz = Megahertz min=minutes mL = milliliters mmol = millimolar MS=mass spectroscopy MSA = methanesulfonic acid N2 = dinitrogen NaCl = Sodium chloride Na2CO3 = Sodium Carbonate Na2CO3*H2O = Sodium carbonate decahydrate NADPH = Nicotinamide adenine dinucleotide hydrogen phosphate NaOH = Sodium hydroxide NaOMe = sodium methoxide Na2SO4 = Sodium sulfate NH4Cl = Ammonium chloride NMT = At ​​most Pd(A-taPhos)2Cl2 = bis(di-tert-butyl(4-dimethylamino) Phenyl)phosphine)dichloropalladium(II) PdCl2(CH3CN)2 = Bis(acetonitrile)dichloropalladium(II) Pd(DCEPhos)Cl2 = dichloro[bis(dicyclohexylphosphino Phenyl)ether]palladium(II) Pd(DPEPhos)Cl2 = dichloro{bis[2-(diphenylphosphino) Phenyl]ether}palladium(II) Pd(DPPP)Cl2 = [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(OAc)2 = Palladium(II) acetate Pd(PPh3)2Cl2 = Bis(triphenylphosphine)palladium(II) Dichloride ppm = parts per million q=quartet rac-BINAP = racemic (2,2'-bis(diphenylphosphino)-1,1' -Binaphthyl) RT=retention time s=singlet t=triplet td = triplet of doublet THF = tetrahydrofuran Tol = Toluene Tricene = N-[tris-(hydroxymethyl)methyl]glycine TRIS = tromethamine ℃ = degrees Celsius UPLC / MS = Ultra High Pressure Liquid Chromatography Mass Spectrometer vol=capacity wt=weight Xantphos = [5-(diphenylphosphino)-9,9-dimethyl- 9H-Xanthen-4-yl](diphenyl)-phosphine

[0034] Several embodiments of the invention are described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0035] Experimental Method Deoxy-chlorination / carbonylation [ka]

[0036] Example 1. Preparation of sodium 3-oxocyclohex-1-ene-1-carboxylate Step 1a: A 250 mL jacketed Chemglass reactor was charged with the dione (10.0 g, 1.0 equiv), MeTHF (150.0 mL, 15.0 L / kg) and DMF (0.69 mL, 0.1 equiv). The jacket temperature was set to 5.0° C. and the reaction was cooled until the internal temperature was <7.5° C. After cooling, oxalyl chloride (8.41 mL, 1.1 equiv) was added via syringe pump over 30 minutes (Caution: gas evolution; Caution: observe exotherm; internal temperature did not exceed 10.0° C.). After the addition was complete, the jacket temperature was set to 20.0° C. and the reaction was allowed to warm to 20.0° C. After 90 minutes, the reaction was sampled for its conversion and found to be completely consumed starting dione (NMT1.0RAP starting dione). The jacket temperature was set to 5.0°C and the reaction was cooled to an internal temperature of <7.5°C. After cooling, 5 wt% aqueous NH4Cl (30 mL, 3.0 L / kg) was added in no less than 30 minutes (Caution: gas evolution; Caution: exothermic). After addition, the jacket temperature was set to 20.0°C and the reaction was allowed to warm to 20.0°C. After 10 minutes, agitation was stopped and the phases were allowed to settle. The phases were split and the aqueous layer was sent to waste. Agitation was resumed and the stream was treated with 15 wt% aqueous NaCl (20 mL, 2.0 L.kg). After 10 minutes, agitation was stopped and the phases were allowed to settle. The phases were split and the aqueous layer was sent to waste. The reactor pressure was reduced to 150 Torr and the jacket temperature was set to 55.0°C. The reaction was concentrated to 4.0 L / kg, then the jacket temperature was set to 20.0° C. and the pressure was increased to 760 Torr. After cooling the internal temperature to 20.0° C., the process stream was subjected to a polishing filter.

[0037] Step 1b: A cooled (inerted) 300 mL HEL reactor was charged with sodium carbonate (12.3 g, 2.0 equiv.), PdCl2(A-taphos)2 (651.0 mg, 0.01 equiv.), MeTHF (100.0 mL, 10.0 L / kg), vinyl chloride reaction solution (see above), and water (8.0 mL, 5.0 equiv.). The reactor was sealed, pressurized to 20 psi with N2, and vented to ambient pressure (purging procedure repeated 3 times). The vessel was pressurized to 30 psi with CO, and vented to ambient pressure (purging procedure repeated 3 times). The reactor was pressurized to 35 psi with CO, and the contents were heated to 50° C. (Note: Use vigorous stirring to help suspend inorganic base and promote good mixing of gas and liquid). After 16 hours, the vessel was cooled to 20°C and vented to ambient pressure. The vessel was then pressurized to 20 psi with N2 and vented to ambient pressure (purging procedure repeated 3x). The reaction was sampled for conversion and found to be completely consumed of the intermediate vinyl chloride (NMT1.0RAP of vinyl chloride remaining).

[0038] The reaction was quenched with water (50.0 mL, 5.0 L / kg) and acidified with 6N HCl (25.4 mL, 2.0 equiv.) (Note: before acidification, the reaction is heterogeneous; after acidification, the stream is two-phase homogeneous). The phases were split and the aqueous layer was sent to waste (Note: sampling of the organic layer typically shows in-process yields of >90%; Note: KF is typically 3.5 wt%; Note: pH is 1.0-1.5). After splitting, the organic layer was polished filtered and sent to a clean reactor. The reactor pressure was reduced to 150 Torr and the jacket temperature was set to 55.0°C. The reaction was concentrated to 5.0 L / kg, then the jacket temperature was set to 20.0°C and the pressure was increased to 760 Torr. After cooling to an internal temperature of 20.0°C, the process stream was polished filtered and sent to a clean reactor. The transfer line and filter were rinsed with MeTHF (30.0 mL, 3.0 L / kg) and the rinse was charged to the clean reactor containing the process stream (Note: KF was typically 1.6 wt%). A 25 wt% solution of NaOMe in MeOH (17.5 mL, 1.0 equiv.) was charged dropwise over 90 min (Note: pH should not exceed 6.5). The resulting slurry was allowed to stand for 2 h before filtering. The reactor and filter cake were washed with 4:1 v / v MeTHF:MeOH (30 mL, 3.0 L / kg). The filter cake was washed with MeTHF (30 mL, 3.0 L / kg). The wet cake was dried at 45.0° C. under a nitrogen sweep in an oven vacuum for no less than 7 h. The sodium carboxylate product was an off-white solid (>75%).

[0039] 1 H NMR(500MHz,D2O) δ 6.34(1H,s), 2.54(2H,td), 2.45(2H,t), 2.02(2H,q) MS: molecular formula, expected exact mass; Found: as C7H8O3 (free acid), expected exact mass 140.0473: high resolution mass spectrometry data not yet collected. HPLC method conditions: Column: Zorbox Eclipse Plus C8, 150x4.6mm, 3.5μm; Mobile phase A: 0.05% MSA in water:acetonitrile 98:2 + 10mM KH2PO4; Mobile phase B: 0.05% MSA in water:acetonitrile 10:90; Temperature: 50°C; Gradient: 0 min (2% B), 4.0 min (20% B), 8.0 min (30% B); 10.0 min (90% B); 12.0 min (90% B); 12.1 min (2% B), 17.0 min (2% B); Flow: 0.8mL / min; 210nm; HPLC RT 7.0 min

[0040] Alternative Experimental Procedure: Step 1a: A glass-lined reactor was charged with MeTHF (10.0 L / kg), DMF (0.1 equiv), and 1,3-cyclohexanedione (1.0 equiv). The jacket temperature was set to maintain an internal temperature of 10-20 °C (target 15 °C). Oxalyl chloride (1.0 equiv) was charged through a metered adder (20-40 kg / h) (Caution: gas evolution; Caution: observe exotherm). After 1.0 h, the reaction was sampled for conversion of the starting dione (NMT0.5RAP starting dione). Additional oxalyl chloride (0.1 equiv) was charged if dione remained, and after another 1.0 h, the reaction was sampled again. The pH was adjusted with DIPEA (approximately 1.0 equiv) to pH 6.0-7.0. Water (1.5 L / kg) was charged and the resulting biphasic mixture was agitated for 0.5-1.0 h. After agitation was stopped, the phases were allowed to separate for NLT 1.0 h. The phases were split and the aqueous layer was sent to waste. Agitation was resumed and the process stream was treated with 15 wt% aqueous NaCl solution (2.0 L / kg). The resulting biphasic mixture was agitated for 0.5-1.0 h. Agitation was stopped and the phases were allowed to separate for NLT 1.0 h. The phases were split and the aqueous layer was sent to waste.

[0041] Step 1b: An autoclave reactor was charged with MeTHF (5.0 L / kg) and the process stream from step 1a. The process streams were combined and sampled for water content. The KF of the process streams was adjusted as necessary. > 1.2 equivalents of water were present. The process stream was treated with Na2CO3 (1.2 equivalents) and Pd(A-taPhos)2Cl2 (0.01 equivalents). The atmosphere of the autoclave was exchanged for nitrogen and carbon monoxide. The vessel was pressurized with carbon monoxide (0.2-0.3 MPa) and the reaction was allowed to warm to 30-40°C (target 35°C). After 3.0 hours the reaction was sampled for conversion of the starting vinyl chloride. The remaining vinyl chloride was <At 80.0 RAP, the reaction was then warmed to 45-55°C (target 50°C) and left for an additional 16.0 hours. After this time, the process stream was sampled for conversion of starting vinyl chloride (NMT1.0 RAP starting vinyl chloride). The reaction was cooled to 0-5°C (target 2.5°C). The process stream was quenched with water (10.0 L / kg) and the resulting biphasic mixture was agitated for 1.0-2.0 hours. The biphasic mixture was filtered through a Natche filter into a glass-lined reactor. The autoclave reactor and Natche filter were rinsed with water (10.0 L / kg) and the rinse was placed into the same glass-lined reactor. The pH of the mixture was adjusted to 1.0-2.0 (target 1.5) with 6N HCl and the biphasic mixture was agitated for NLT0.5 hours. After agitation was stopped, the phases were allowed to separate for NLT 1.0 h. The phases were split and the aqueous layer was sent to waste. Agitation was resumed and the process stream was treated with 15 wt% NaCl in water (1.5 L / kg). The resulting two-phase mixture was agitated for 0.5-1.0 h. Agitation was stopped and the phases were allowed to separate for NLT 1.0 h. The phases were split and the aqueous layer was sent to waste. Agitation was resumed and the process stream was treated with 15 wt% NaCl in water (1.5 L / kg). The resulting two-phase mixture was agitated for 0.5-1.0 h. After agitation was stopped, the phases were allowed to separate for NLT 1.0 h. The phases were split and the aqueous layer was sent to waste. The process stream was treated with activated carbon (0.1 g / g) and agitated for NLT 1.0 h. The process stream was filtered and the filter cake was washed with MeTHF (3.0 L / kg). The reactor pressure was reduced and the jacket temperature was set to 55.0°C. The reaction was concentrated to 4.0-5.0 L / kg and then the jacket temperature was set to -15.0 to 5.0°C (target -10°C). The stream was diluted with MeTHF (3.5 L / kg) and the KF of the stream was adjusted to 1.0-2.0 wt%. 20 wt% NaOMe in MeOH was metered in until the pH was 6.0-7.0. The resulting slurry was agitated for an additional 2.0 h.After standing, the slurry was filtered and washed with 4:1 v / v MeTHF:MeOH (2.0 L / kg) and MeTHF (2.0 L / kg). The wet cake was dried under oven vacuum for NLT 7.0 hours at 45.0° C. The sodium carboxylate product was isolated as an off-white solid (>75%).

[0042] Enone reductase / keto reductase cascade [ka]

[0043] Example 2: Biocatalytic synthesis of (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid: To a 100 mL reactor, ERED-310 (80 mg, 8 wt%), GDH-105 (2 mg, 0.002 wt%), NADP+ (10 mg, 0.1 wt%) and KRED-457 (40 mg, 4 wt%) were added. 0.25 M sodium phosphate buffer (pH 7.0, 10 mL, 10 L / Kg) was then added and agitation was set to 300 rpm. The reaction was allowed to sit for 30 minutes to allow the biocatalyst to dissolve. The pH was then adjusted to pH 7.0 using 2.5 M aqueous NaOH, followed by the addition of D-glucose (2.4 g, 2.16 equiv). At this point, the temperature was set to 32.5° C. and the pH of the reaction was maintained at 7.0 using a pH stat. A separate vessel was charged with sodium 3-oxocyclohex-1-ene-1-carboxylate (1.0 g, limiting reagent), 0.25 M sodium phosphate buffer (pH 7.0, 5 mL, 5 L / Kg), glucose (0.6 g, 0.54 equiv.) and stirred for 10 minutes. The pH of this solution was then adjusted to pH 7.0 by adding 2.5 M NaOH. This solution was then charged to the biocatalyst solution via syringe pump over 6 hours. After 16 hours, the reaction was deemed complete (>99 LCAP).

[0044] After the reaction was complete, cellulose (2.0 g, 200 wt%) was added to the reaction mixture and 6N HCl (aq) was added to adjust the pH to 1-2. The resulting slurry was stirred at room temperature for no more than 1 hour before filtration. The filter cake was washed thoroughly with MeTHF (20 mL, 20 L / Kg) to minimize yield loss. The resulting biphasic filtrate was charged with Na2SO4 solid (3 g, 300 wt%) and stirred until all Na2SO4 was dissolved. The phases were split and the aqueous layer was taken to waste. The organic layer was transferred to a clean vial. The stream was concentrated under 100 Torr at 35.0°C. After the reaction mixture was concentrated to 2.5 L / kg, MeTHF (5 mL, 5.0 L / Kg) was added and the reaction mixture was again concentrated to 2.5 L / Kg. The process was repeated until the KF of the stream was NMT 0.5 wt%. The process stream was polished filtered and placed in a clean vial. Toluene / heptane mixture (1 / 3, 3 mL, 3 L / Kg) was charged dropwise over 90 minutes. The resulting slurry was allowed to stand for 3 hours and additional toluene / heptane mixture (1 / 3, 7 mL, 7 L / Kg) was added slowly over 3 hours. The resulting slurry was allowed to stand at room temperature for 4 hours and then cooled to 0° C. and allowed to stand for 3 hours. The slurry was then filtered. The vial and filter cake were washed with toluene / heptane (1 / 3, 2 mL, 2.0 L / kg). The wet cake was dried at 50.0° C. in a vacuum oven under a nitrogen sweep for no more than 8 hours. The product (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid was obtained as an off-white solid (70-80%).

[0045] 1 H NMR (400MHz, d4-MeOH) δ 3.93-4.00(m,1H), 2.67-2.75(m,1H), 1.65-1.85(m,4H), 1.45-1.63(m,4H) 13 C NMR: (100MHz, d4-MeOH) δ 179.64, 66.88, 39.11, 36.51, 33.75, 29.27, 21.05 MS: molecular formula, expected exact mass; measured value 144.08 g / mol LC / MS(DCI)[C7H 12 O3+H] + Calculated value = 145.086, measured value: 145.0865

[0046] Analysis: Working concentration 5mg / mL, Injection volume: 5μL; Column: Ascentis Express C18, PCP, 4.6x150mm, 2.7μm, Flow: 0.8mL / min Column temperature: 25°C, Mobile phase A 0.1% formic acid in water, Mobile phase B 0.1% formic acid in MeCN, UV wavelength (210nm), Gradient 98%A (0.0min), 80%A (4min), 50%A (8min), 10%A (10min), 98%A (10.1min), 98%A (15min) RT(UPLC): 5.0min

[0047] Alternative Experimental Procedure: To a 100 mL reactor, ERED-310 (80 mg, 8 wt%), GDH-105 (2 mg, 0.2 wt%), NADP+ (10 mg, 1 wt%) and KRED-457 (30 mg, 3 wt%) were added. 0.1 M sodium phosphate buffer pH 7.0 was then added along with 2 mM MgCl2 (12 mL, 12 L / Kg) and agitation was set to 300 rpm. The reaction was left for 30 minutes to allow the biocatalyst to dissolve. The pH was then adjusted to pH 7.0 using 2.5 M aqueous NaOH, followed by the addition of D-glucose (2.4 g, 2.16x). At this point, the internal temperature was set to 30.0° C. and a pH stat was utilized to maintain the reaction pH=7.0.

[0048] In a separate vessel, sodium 3-oxocyclohex-1-ene-1-carboxylate (1.0 g, limiting reagent), 0.1 M sodium phosphate buffer (pH 7.0, 3 mL, 3 L / Kg) containing 2 mM MgCl2, 0.6 g glucose (0.6 g / g), and NADP+ (5 mg, 0.5 wt%) were charged and stirred for 10 minutes. The pH of this solution was then adjusted to pH 7.0 by adding 2.5 M NaOH or 2.5 M HCl. This solution was then charged to the biocatalyst solution via a syringe pump over a period of 8-10 hours. The pH of the reaction was maintained at 7.0 during the additions using a pH stat.

[0049] After 16 hours, the reaction was judged complete (>99% conversion of sodium 3-oxocyclohex-1-ene-1-carboxylate). After the reaction was complete, cellulose (1.0 g, 1x) was added to the reaction mixture, which was heated to 50-60°C and stirred for 1 hour. The mixture was cooled to 15-30°C and its pH was adjusted to 1-2 by adding 6N HCl (ag). The resulting slurry was stirred at room temperature for NLT 1 hour and filtered. The filter cake was washed thoroughly with MeTHF (20 mL, 20 L / Kg) to minimize yield loss. The resulting biphasic filtrate was charged with solid Na2SO4 (3 g, 3x) and stirred until all Na2SO4 was dissolved. The phases were split and the aqueous layer was taken to waste. The organic layer was transferred to a clean vial. The stream was distilled under 100 torr at 35.0°C. The reaction mixture was concentrated to 5 L / kg, then MeTHF (5 mL, 5.0 L / Kg) was added and the reaction mixture was concentrated again to 5 L / Kg. The process was repeated (usually 2 puts and takes) until the stream had a KF of NMT 0.5 wt%. The process stream was polished filtered and placed in a clean vial. The reaction was then concentrated again to 2.5 L / kg. The concentrated solution was charged dropwise with toluene / heptane mixture (1 / 3, 3 mL, 3 L / Kg) over 90 minutes. The resulting slurry was allowed to stand for 3 hours and additional toluene / heptane mixture (1 / 3, 7 mL, 7 L / Kg) was added slowly over 3 hours. The resulting slurry was allowed to stand at room temperature for 2 hours, then cooled to 0° C. and allowed to stand for 1 hour. The slurry was then filtered. The reactor and filter cake were washed with toluene / heptane (1 / 3, 2 mL, 2.0 L / kg). The wet cake was dried in a vacuum oven at 50° C. under a nitrogen sweep for NLT 8 h. The product, (1S,3S)-3-hydroxycyclohexane-1-carboxylic acid, was obtained as an off-white solid (70-80%).

[0050] It should be understood that the Detailed Description section is to be used to interpret the claims, and not the Abstract and Summary sections. The Abstract and Summary sections describe one or more exemplary aspects of the disclosure as contemplated by the inventors, but are not all-inclusive, and thus are not intended to be limiting of the scope of the disclosure and the appended claims in any way. The present disclosure has been described above with the aid of functional building blocks that demonstrate implementation of certain functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for convenience of description. Alternative boundaries may be defined so long as the certain functions and relationships thereof are appropriately implemented.

[0051] The above description of the specific embodiments fully reveals the general characteristics of the present disclosure, so that a third party may easily modify and / or adapt such specific embodiments to various applications by applying knowledge within the skill of the art without undue experimentation and without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the expressions or terms used herein are for the purpose of explanation and not for the purpose of limitation, and thus the terms or terms used herein should be interpreted by those skilled in the art in light of this teaching and guidance. The breadth and scope of the present disclosure should not be limited by any of the exemplary embodiments described above, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. Formula (I): 【Chemistry 1】 A method for producing a compound of the formula (1) The following formula (II): 【Chemistry 2】 with a halogenating or sulfonylating reagent in a polar aprotic solvent or solvent mixture, with or without an inorganic or organic base, for a time and temperature sufficient to effect deoxy-functionalization, to produce a compound of formula (III): 【Chemistry 3】 wherein R is Cl, Br, I, OMs, OTs, or OTf. to produce a compound represented by the formula: (2) With or without isolating the compound of formula (III), contacting the compound of formula (III) with a palladium catalyst, in water and a polar aprotic solvent, with or without a phosphine ligand, and an inorganic or organic base, purged with carbon monoxide, for a time, temperature and pressure sufficient to subject the compound of formula (III) to carbonylation to produce a compound of formula (I). The method includes:

2. A process for preparing a compound of formula (I), comprising the steps of: (1) The compound of formula (II) is reacted with a halogenating agent selected from oxalyl iodide, oxalyl bromide, and oxalyl chloride, or a sulfonylating agent selected from p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, N-phenyl-bis(trifluoromethanesulfonimide), and N-(5-chloropyridin-2-yl)-N-(methanesulfonyl)methanesulfonamide in EtOAc, 2-MeTHF, CH ３ in a polar aprotic solvent selected from CN, DMAc, DMF, THF, toluene, and mixtures thereof; ２ CO ３ , N,N-diisopropylethylamine, triethylamine, t-butyltetramethylguanidine, tetramethylguanidine, KHCO ３ , D.B.U., Na ２ CO ３ , Na ２ CO ３ *10H ２ with or without an inorganic or organic base selected from O and KOAc for a time and temperature sufficient to effect deoxy-functionalization to produce a compound of formula (III) where R is Cl, Br, I, OMs, OTs, or OTf; (2) With or without isolating the compound of formula (III), the compound of formula (III) is reacted with Pd(A-taPhos) ２ C ２ , Pd(DPEPhos)Cl ２ , Pd(PPh ３ ) ２ C ２ , Pd(DCEPhos)Cl ２ , Pd(OAc) ２ , PdCl ２ (CH ３ C.N. ２ and Pd(DPPP)Cl ２ A palladium catalyst selected from the group consisting of water and EtOAc, 2-MeTHF, CH ３ CN, DMAc, DMF, THF, toluene, and mixtures thereof, in a polar aprotic solvent selected from 1,3-bis(dicyclohexylphosphino)propane, 1,3-bis(diphenylphosphino)propane, rac-BINAP, Xantphos, Josiphos SL-J001-1, Josiphos SL-J009-1-G3 palladacycle, and BIPHEP; and K ２ CO ３ , N,N-diisopropylethylamine, triethylamine, t-butyltetramethylguanidine, tetramethylguanidine, KHCO ３ , D.B.U., Na ２ CO ３ , Na ２ CO ３ *10H ２ 2. The method of claim 1, comprising contacting with carbon monoxide, with or without an inorganic or organic base selected from O and KOAc, for a time, temperature and pressure sufficient to subject the compound to carbonylation to produce the compound of formula (I).

3. A process for preparing a compound of formula (I), comprising the steps of: (1) contacting a compound of formula (II) with an acid chloride in a polar aprotic solvent or solvent mixture selected from 2-MeTHF and DMF for a time and at a temperature sufficient to subject the compound to deoxy-chlorination to produce a compound of formula (IIIa): 【Chemistry 4】 to produce a compound represented by the formula: (2) With or without isolating the compound of formula (IIIa), the compound of formula (IIIa) is reacted with Pd(A-taPhos) ２ C ２ , Pd(PPh ３ ) ２ C ２ , Pd(DCEPhos)Cl ２ or PdCl ２ (CH ３ C.N. ２ and water, 2-MeTHF, CH ３ 1,3-bis(dicyclohexylphosphino)propane, and DIPEA, K, in a polar aprotic solvent selected from CN, toluene, and mixtures thereof. ２ CO ３ , Na ２ CO ３ and Na ２ CO ３ *10H ２ and purging with carbon monoxide at a temperature and pressure sufficient to subject the compound to carbonylation to produce a compound of formula (I). The method of claim 2 comprising:

4. A process for preparing a compound of formula (I), comprising the steps of: (1) contacting a compound of formula (II) with oxalyl chloride in a mixture of 2-MeTHF and DMF for 1-3 hours at 0-50° C. to produce a compound of formula (IIIa); (2) With or without isolating the compound of formula (IIIa), the compound of formula (IIIa) is reacted with Pd(A-taPhos) ２ C ２ , and Na ２ CO ３ in water and 2-MeTHF at 20-50° C. and 30-35 psi for 16-20 hours with a carbon monoxide purge to produce a compound of formula (I). The method of claim 3 , comprising:

5. Formula (IV): 【Chemistry 5】 The present invention relates to a method for producing a compound of formula (I): 【Chemistry 6】 with an enone reductase (ERED) and a keto reductase (KRED) in the presence of an aqueous polar protic solvent, NADPH and a co-substrate, with or without an organic co-solvent, for a time and at a temperature sufficient to produce a compound of formula (IV).

6. 6. The method of claim 5, comprising contacting a compound of formula (I) with an ERED selected from ERED-309 and ERED-310, and a KRED selected from KRED-456, KRED-457 and KRED-P2-B07, in the presence of phosphate, an aqueous buffer selected from TRIS, HEPES, ACES, BES, MOPS and Trisene; GDH, NADPH and glucose, with or without an organic co-solvent such as DMSO, IPA, dioxane or acetone, for a time and temperature sufficient to produce a compound of formula (IV).

7. 7. The method of claim 6, comprising contacting a compound of formula (I) with ERED-310, and KRED-457, in the presence of an aqueous buffer selected from phosphate, TRIS, HEPES, ACES, BES, MOPS and Trisene; GDH, NADPH and glucose, with or without an organic co-solvent such as DMSO, IPA, dioxane or acetone, for at least 16 hours and at 25-35° C. sufficient to produce a compound of formula (IV).

8. A process for preparing a compound of formula (IV), comprising steps (1) and (2) from any one of claims 2 to 4, followed by a step from any one of claims 5 to 7, wherein all formulae and variables are as defined in claims 2 to 7.

9. A process for preparing a compound of formula (IV), comprising steps (1) and (2) from claim 3 or claim 4, followed by a step from claim 6 or claim 7, wherein all formulae and variables are as defined in claim 3, 4, 6 or 7.

10. A process for preparing a compound of formula (IV), comprising steps (1) and (2) from claim 4 followed by a step from claim 7, wherein all formulae and variables are as defined in claims 4 and 7.

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