KRED catalyzed ketoreduction of a cyclobutanone carbamic ester

Abstract

Disclosed, inter alia, are processes of preparing a compound of formula II (II), comprising reducing a compound of formula I (I), in the presence of a ketoreductase to form the compound of formula II. Also disclosed are processes of preparing a compound of formula D1, which is useful for preparing pharmaceutically active compounds such as androgen receptor (AR) degraders.

Description

KRED CATALYZED KETOREDUCTION OF A CYCLOBUTANONE CARBAMICESTERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 638,039, filedApril 24, 2024, which is incorporated by reference herein in its entirety for all purposes.TECHNICAL FIELD

[0002] The present disclosure relates, for example, to an enzymatic process for reducing a 3- amino-2,2,4,4-tetramethylcyclobutan-l-one which is protected with an amine protecting group to a trans-3-amino-2,2,4,4-tetramethylcyclobutan-l-ol protected with an amine protecting group.BACKGROUND

[0003] A trans-3-amino-2,2,4,4-tetramethylcyclobutan-l-ol protected with an amine protecting group is a versatile intermediate which can be used in the synthesis of a variety of pharmaceutically active ingredients (APIs), e.g. for selective androgen receptor (AR) degrader molecules, which have a potential for being used in the cancer treatment. See, e.g., U.S. Patent No. 11,767,312, as well as International Patent Application Publication No. WO / 2021 / 249534, both of which are incorporated by reference herein for all purposes.

[0004] There is a need in the art for improved processes for making a trans-3-amino-2,2,4,4- tetramethylcyclobutan-l-ol protected with an amine protecting group or in its unprotected form with high conversion and high selectivity towards the desired stereoisomer. Provided herein are processes that address this and other needs in the art.SUMMARY

[0005] The present disclosure provides processes for the enzymatic reduction of a ketone of formula Ito form the protected trans amino alcohol of formula IIwherein Prot is an amine protecting group. In embodiments, reducing the ketone of formula I is performed in the presence of a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%. In embodiments, reducing the ketone of formula I is performed in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose.In embodiments, Prot is tert-butoxycarbonyl (Boc), the compound of formula I is a compound of formula lb, and the compound of formula II is a compound of formula lib.

[0006] In another aspect, provided is a orocess for preparing a compound of formula libcomprising:(1) reacting a compound of formula DI -2(Dl-2) with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form a compound of formula lb(2) reducing the compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose;to form the compound of formula lib. In embodiments, the process of preparing a compound of formula lib further comprises reacting a compound of formula Dl-1(Dl-1) with hydroxylamine to form the compound of formula DI -2.

[0007] In another aspect, provided is a orocess for preparing a compound of formula libcomprising:(1) reacting a compound of formula(Dl-1) with hydroxylamine to form a compound of formula DI -2(Dl-2);(2) reacting a compound of formula DI -2 with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form a compound of formula lb(3) reducing the compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula lib.

[0008] The present disclosure further provides processes for preparing a compound of formula DIor salt thereof, comprising:(1) preparing a compound of formula lib as disclosed herein(lib);(2) reacting the compound of formula lib with 2-chloro-4-fluorobenzonitrile in the presence of a base to form a compound of formula DI -7(3) deprotecting the compound of formula Dl-7 in the presence of an acid to form the compound of formula DI or salt thereof. In embodiments, preparing the compound of formula lib comprises reducing a compound of formula lbin the presence of a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%. In embodiments, preparing the compound of formula lib comprises reducing a compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula lib.

[0009] The present disclosure further provides processes for preparing a compound of formula DIor salt thereof, comprising:(1) obtaining a compound of formula libcomprising less than about 4 wt% of an impurity of formula Illb(2) reacting the compound of formula lib with 2-chloro-4-fluorobenzonitrile in the presence of a base to form a compound of formula DI -7(3) deprotecting the compound of formula DI -7 in the presence of an acid to form the compound of formula DI or salt thereof.

[0010] The present disclosure further relates to the use of the enzymatic process for the synthesis of pharmaceutically active ingredients, which carry the structure element of the protected trans amino alcohol of formula II.DETAILED DESCRIPTION OF EMBODIMENTS

[0011] The present application will be described in detail below with reference to embodiments, but it does not mean that there is any unfavorable limitation to the present application. The present application has been described in detail herein, and the specific embodiments thereof are also disclosed. For those skilled in the art, it would be obvious to make various changes and improvements to the specific embodiments of the present application without departing from the spirit and scope of the present application.Definitions

[0012] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered to be indeterminate or unclear without specific definitions, but should be understood in its ordinarymeaning. When a trade name appears herein, it is intended to refer to a corresponding commercial product or an active ingredient thereof.

[0013] The “conversion” of the enzymatic processes disclosed herein is calculated by, e.g. dividing the sum of the mole quantities of the product compounds (the compounds of formula II (trans) and formula III (cis)) by the sum of the mole quantities of the product compounds and residual substrate (the compound of formula I).

[0014] The term “ketoreductase” refers to a subclass of enzymes belonging to the group of oxidoreductases, i.e. enzymes catalyzing redox reactions allowing the transfer of an electron from an electron donor molecule to an electron acceptor molecule. The subclass of ketoreductases have the specific capability of catalyzing the stereoselective conversion of desired ketones to their corresponding secondary alcohol. During this reduction reaction, a hydride and a proton are transferred to the keto group (C=O) leading to the addition of a hydride to the carbon of the keto group and the proton to the oxygen. This reaction generally requires electron donors as cofactors, such as NADH or NADPH, which may be regenerated in-situ with the help of cofactor regeneration systems such as the glucose dehydrogenase (GDH) cofactor regeneration system.

[0015] In the GDH cofactor regeneration system, the cofactor NAD(P) is used by the enzyme GDH to catalyze the oxidation of glucose. During this reaction, NAD(P) is converted to its reduced form NADPH or NADH.

[0016] The term trans refers to the cis-trans isomerism, which is a type of stereoisomerism in which the atoms or groups of atoms are arranged differently around a double bond or a ring structure. In the context of the protected trans amino alcohol of formula II, it refers to the arrangement of substituents on the opposite side (trans) of the ring structure, while the arrangement on the same side means is a (cis) isomerism. This difference in arrangement results in different physical and chemical properties of the isomers.

[0017] The term “amine protecting group” or “amino protecting group” refers to substituent of the amino group, which is conventionally used to hinder the reactivity of the amino group. Suitable acid or Lewis acid sensitive amino protecting groups are described in Green T., “Protective Groups in Organic Synthesis”, 4th Ed. by Wiley Interscience, 2007, Chapter 7, 696 ff.. Suitable amino protecting groups for Prot can therefore be selected from Boc (tertbutoxycarbonyl), benzyl, 4-methoxybenzyl, benzhydryl, Fmoc (fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Moz (p-methoxybenzyl carbonyl), Alloc (allyloxycarbonyl), Troc (2,2,2-trichloroethoxycarbonyl), Teoc (2-(Trimethylsilyl)ethoxycarbonyl), Adoc (adamantoxycarbonyl), formyl, acetyl or from cyclobutoxycarbonyl. In embodiments, Prot is Boc.

[0018] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value.

[0019] Unless otherwise stated, a wedged solid bond (and a wedged dotted bond ( ■ "") are used to represent an absolute configuration of a stereocenter.

[0020] The solvents used in the present invention are commercially available.

[0021] Compounds were named according to conventional nomenclature in the art or by using ChemDraw® software, and commercially available compounds were named in supplier catalogs.Process of preparing a compound of formula (II)

[0022] In one aspect, provided is a process of preparing a compound of formula (II)comprising reducing a compound of formula Iin the presence of a ketoreductase to form the compound of formula II, wherein Prot is an amine protecting group. In embodiments, the process achieves a conversion of at least 90%. In embodiments, the ketoreductase is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%. In embodiments, process is performed in the presence of an aqueous buffer. In embodiments, the reducing is performed in the presence of an aqueous buffer containing the cofactor NADP and a cofactor regeneration system. In embodiments, the reducing is performed in the presence of an organic co-solvent and an aqueous buffer containing the cofactor NADP and a cofactor regeneration system. In embodiments, the reducing is further performed in the presence of an organic co-solvent. In embodiments, the reducing is further performed in the presence of an organic co-solvent yielding a biphasic mixture.

[0023] In embodiments, Prot is tert-butoxycarbonyl (Boc), the compound of formula I is a compound of formula lb, and the compound of formula II is a compound of formula lib.embodiments, the process of preparing a compound of formula lib further comprises reacting a compound of formula DI -2with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form the compound of formula lb.

[0025] In embodiments, the process of preparing a compound of formula lib further comprises reacting a compound of formula Dl-1with hydroxylamine to form the compound of formula DI -2.

[0026] In another aspect,is a processa compound of formula libcomprising:(1) reacting a compound of formula DI -2with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form a compound of formula lb(2) reducing the compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; andc) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula lib. In embodiments, the process of preparing a compound of formula lib further comprises reacting a compound of formula Dl-1(Dl-1) with hydroxylamine to form the compound of formula DI -2.

[0027] In another aspect, provided is a process for preparing a compound of formula libcomprising:(1) reacting a compound of formula(Dl-1) with hydroxylamine to form a compound of formula DI -2(2) reacting a compound of formula DI -2 with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form a compound of formula lb(3) reducing the compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula lib.

[0028] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the ketoreductase is capable to convert the compound of formula I (or formula lb) into the compound of formula II (or formula lib) in a conversion of at least about 95%, at least about 98%, or at least about 99%. In embodiments, the ketoreductase is a commercially available ketoreductase, e.g. KP00078 from Evoxx (Alcohol dehydrogenase 440; evo-1.1.440.S), KP00099 from Almac (CRED-A641), or and KP00189 from Seqens (E4336). In embodiments, the ketoreductase is a ketoreductase known in the literature, e.g. KPOOO88 (Ralstonia sp.; DSM 6428).

[0029] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the ketoreductase is capable to transform the compound of formula I (or formula lb) into the compound of formula II (or formula lib) with a trans to cis isomeric ratio of at least about 15, at least about 30, or at least about 45.

[0030] In embodiments, the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib) yields the compound of formula II (or formula lib) with a trans to cis isomeric ratio of at least about 15, at least about 30, at least about 45, at least about 50, at least about 55, or at least about 60, or any ratio therebetween.

[0031] In embodiments, the cofactor NADP is present in a concentration of below about 4.0 wt% relative to the substrate of formula I, e.g. below about 2.0 wt%, or below or equal to about lwt%.

[0032] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the cofactor regeneration system comprises glucose dehydrogenase and glucose. In embodiments, the glucose dehydrogenase is a commercially available glucose dehydrogenase, e.g. GDH-105 from Codexis. In embodiments, the concentration of the glucose dehydrogenase is below about 4.0 wt% relative to the substrate of formula I, e.g. below about 1.0 wt%, or below or equal to 0.2 wt%. In embodiments, the amount of glucose is more than 1.0 equivalent relative to the substrate of formula I, e.g., between about 1.05 eq. and about 1.5 eq.

[0033] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the buffer is selected from standard biochemical buffers, e.g., phosphate buffer, TRIS buffer (tris(hydroxymethyl)aminomethane), MES buffer (2-(N-morpholino)ethanesulfonic acid), citrate buffer, or acetate buffer. In embodiments, the buffer contains 25 mM to 250 mM of the buffering agent. In embodiments, the buffer contains 50 mM to 100 mM of the buffering agent. In embodiments, the buffering agent is MES buffer. In embodiments, the buffering agent is phosphate buffer. In embodiments, the buffering agent is acetate buffer, e.g. sodium acetate or magnesium acetate.

[0034] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the buffer contains Mg2+in a concentration of from about 1 mM to about 100 mM. In embodiments, the buffer contains Mg2+in a concentration of from about 2 mM to about 50 mM.

[0035] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the organic co-solvent is selected from isopropyl acetate, ethyl acetate, n-heptane, n-dodecane, cyclohexane, methyl-cyclohexane, toluene and tert-butyl methyl ether. In embodiments, the organic co- solvent is isopropyl acetate or tert-butyl methyl ether. In embodiments, the organic co-solvent is isopropyl acetate. In embodiments, the organic co-solvent is tert-butyl methyl ether. In embodiments, the organic co-solvent is present in the reaction mixture at a concentration of 10 v% to 40 v% of the reaction mixture, e.g. about 10 v% or 20 v%.

[0036] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the concentration of the compound of formula I (or formula lb) in the reaction mixture is between 0.5 wt% and 10.0 wt% of the reaction mixture, e.g. between 2.0 wt% and 10.0 wt%.

[0037] In embodiments, in the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), the ketoreductase is present at a concentration of lower than 25.0 wt% relative to the compound of formula I in the reaction mixture, e.g. at or lower than about 20.0 wt%, 5.0 wt%, 2.0 wt%, or 1.0 wt%.

[0038] In embodiments, the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), is carried out at a temperature between about 20°C and about 50°C, e.g. between about 25°C and about 45°C. In embodiments, the process is carried out at a pH between about 5.5 and about 7.0, e.g. between about pH 5.5 and about pH 6.0. In embodiments, the process is carried out at a constant pH.

[0039] In embodiments, the step of reducing the compound of formula I (or formula lb) to form the compound of formula II (or formula lib), is carried out for between about 10 h and about 30 h.

[0040] In embodiments, the process further comprises isolating the compound of formula II (or formula lib). In embodiments, isolating the compound of formula II (or formula lib) comprises extracting the compound of formula II (or formula lib) into a suitable extraction solvent. In embodiments, the extraction solvent is an organic solvent. In embodiments, the extraction solvent is selected from isopropyl acetate, ethyl acetate, n-heptane, n-dodecane, cyclohexane, methyl-cyclohexane, toluene and tert-butyl methyl ether. In embodiments, the extraction solvent is isopropyl acetate or tert-butyl methyl ether. In embodiments, the extraction solvent is isopropyl acetate. In embodiments, the extraction solvent is tert-butylmethyl ether. In embodiments, the extraction solvent is the same as the co-solvent. In embodiments, drying and evaporation of the extraction solvent yields the crude protected trans amino alcohol of formula II (or formula lib) in quantitative yields of greater than 90% with a trans / cis ratio of at least about 45.

[0041] In embodiments, the process further comprises crystallizing the compound of formula II (or formula lib). In embodiments, the crystallizing comprises a solvent swap from the extraction solvent to a crystallization solvent. In embodiments, the crystallization solvent is an organic solvent selected from apolar hydrocarbons, e.g. n-heptane. In embodiments, the process further comprises isolating the crystalline compound of formula II (or formula lib) by filtration. In embodiments, the crystallization further increases the trans to cis isomeric ratio.

[0042] In embodiments of reacting a compound of formula DI -2 with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form the compound of formula lb, the reducing agent is hydrogen in the presence of a hydrogenation catalyst. In embodiments, the hydrogenation catalyst is Raney nickel. In embodiments, reacting a compound of formula DI -2 with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form the compound of formula lb is performed in a solvent. In embodiments, the solvent is THF, water, or a mixture thereof.

[0043] In embodiments of reacting a compound of formula Dl-1 with hydroxylamine to form the compound of formula DI -2, the hydroxylamine is hydroxylamine hydrochloride. In embodiments, the hydroxylamine is provided as a 50% solution in water. In embodiments, reacting the compound of formula Dl-1 with hydroxylamine is performed in the presence of a base. In embodiments, the base is sodium acetate. In embodiments, reacting the compound of formula Dl-1 with hydroxylamine is performed in the presence of an acid. In embodiments, the acid is acetic acid. In embodiments, reacting a compound of formula Dl-1 with hydroxylamine is performed in the presence of a solvent. In embodiments, the solvent is THF. In embodiments, the solvent is ethanol.Compounds

[0044] In embodiments, provided is a compound of formula II or lib.In embodiments the compounds of formula II or lib comprise less than about 4 wt% of an impurity of formula III or IllbIn embodiments, the compound of formula II or lib comprises less than about 3 wt% of an impurity of formula III or Illb. In embodiments, the compound of formula II or lib comprises less than about 2.5 wt% of an impurity of formula III or Illb.Process of preparing a compound of formula DI

[0045] In embodiments, provided is a process for preparing a compound of formula DIor salt thereof comprising (1) obtaining a compound of formula lib comprising less than about4 wt% of an impurity of formula Illb, and (2) reacting the compound of formula lib with 2- chloro-4-fluorobenzonitrile in the presence of a base to form a compound of formula DI -7In embodiments, obtaining the compound of formula lib comprising less than about 4 wt% of an impurity of formula Illb comprises preparing a compound of formula lib as disclosed herein. In embodiments, the base is, e.g., lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, sodium tert-pentoxide, potassium tert-pentoxide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, or sodium hydride. In embodiments, the base is sodium hydride. In embodiments, the base is sodium tert-butoxide. In embodiments, reacting the compound of formula lib with 2-chloro-4- fluorobenzonitrile in the presence of a base is performed in a solvent, e.g., DMSO, acetonitrile, NMP, DMF, DMA, THF, 2-MeTHF, toluene, or a combination thereof. In embodiments, the solvent is THF. In embodiments, the solvent is DMF. In embodiments, the solvent is NMP.

[0046] In embodiments, provided is a process of preparing a compound of formula DIor salt thereof comprising(1) preparing a compound of formula lib as described herein, and(2) reacting the compound of formula lib with 2-chloro-4-fluorobenzonitrile in the presence of a base to form a compound of formula DI -7In embodiments, the base is, e.g., lithium tert-butoxide, sodium tert-butoxide, potassium tert- butoxide, sodium tert-pentoxide, potassium tert-pentoxide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, or sodium hydride. In embodiments, the base is sodium hydride. In embodiments, the base is sodium tert-butoxide. In embodiments, reacting the compound of formula lib with 2-chloro-4-fluorobenzonitrile in the presence of a base is performed in a solvent, e.g., DMSO, acetonitrile, NMP, DMF, DMA, THF, 2-MeTHF, toluene, or a combination thereof. In embodiments, the solvent is THF. In embodiments, the solvent is DMF. In embodiments, the solvent is NMP.

[0047] In embodiments, the process of preparing a compound of formula DI further comprises (3) deprotecting the compound of formula Dl-7 in the presence of an acid to form the compound of formula DI or salt thereof. In embodiments, the acid is hydrochloric acid. In embodiments, deprotecting the compound of formula Dl-7 in the presence of an acid is performed in a solvent. In embodiments, the solvent is 1-propanol or 1-butanol. In embodiments, the compound of formula DI is an HC1 salt of the formula:

[0048] In embodiments, the process of preparing a compound of formula DI or salt thereof further comprises treating the compound of formula DI with an acid to form the salt of formula DI. In embodiments, the acid is HC1 and the compound of formula DI or salt thereof is the HC1 salt of the compound of formula DI.Other Processes

[0049] In another embodiment, provided is the use of the processes disclosed herein for the synthesis of pharmaceutically active ingredients, which carry the structure element of the protected trans amino alcohol of formula II, i.e.:In embodiments, the processes disclosed herein are useful for preparing AR degrader compounds such as those disclosed in U.S. Patent No. 11,767,312, as well as International Patent Application Publication No. WO / 2021 / 249534, both of which are incorporated by reference herein for all purposes, including N-((lr,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-((4-(2-((S)-2,6-dioxopiperidin-3-yl)- 1-oxo- 1,2-dihy drophthalazin- 6-yl)piperazin- 1 -yl)methyl)piperidin- 1 -yl)benzamide (Compound 1 ) .EXAMPLESAbbreviations:Example 1. Ketoreductase screening1.1 Ketoreductase screening at reduced enzyme loadings and 1% substrate loading

[0050] A reaction mixture composed of 5 mg ketone of formula lb (1 wt% relative to reaction mixture) dissolved in 450 pL PPB 100 mM, pH 7.0 containing 2 mM MgCh , GDH-105 [Codexis], lot A10036 s / e 500 (10 pg, 0.2 wt% relative to ketone), NADP s / c 50 (100 pg, 2 wt% relative to ketone), Glucose 6.7 eq. (25 mg), and cyclohexane (10 v% relative to reaction mixture) was exposed 19-20 h to varying enzyme loadings from 1 wt% to 20 wt% (relative to ketone) at room temperature.

[0051] Table 1 (KRED screening at reduced enzyme loadings)awt% relative to ketonebCalculated using exact, non-rounded valuescPercentage of peak interval corresponding to cis isomer1.2, Temperature screening at 1% substrate loading

[0052] A reaction mixture composed of 5 mg ketone of formula lb (1 wt% relative to reaction mixture) dissolved in 450 pL PPB 100 mM, pH 7.0 containing 2 mM MgCh, KRED KP00078 s / e 200 (25 pg, 0.5 wt% relative to ketone), GDH-105 [Codexis], lot A10036 s / e 500 (10 pg, 0.2 wt% relative to ketone), NADP s / c 50 (100 pg, 2 wt% relative to ketone), Glucose 6.7 eq. (25 mg), and cyclohexane (10% v% relative to reaction mixture) was exposed 20 h to different temperatures.

[0053] Table 2: Temperature ScreeningaCalculated using exact, non-rounded valuesbPercentage of peak interval corresponding to cis isomer1,3 Biphasic co-solvents screening at 1% substrate loading

[0054] A reaction mixture composed of 5 mg ketone of formula lb (1 wt% relative to reaction mixture) dissolved in 450 pL PPB 100 mM, pH 7.0 containing 2 mM MgCh, KRED KP00078s / e 100 (10 pg, 1 wt% relative to ketone), GDH s / e 500 (10 pg, 0.2 wt%), NADP s / c 50 (100 pg, 2 wt% relative to ketone), Glucose 6.7 eq. (25 mg) and varying co-solvents (10 v% relative to reaction mixture) was incubated for 20 h at room temperature.

[0055] Table 3: Biphasic co-solvents impact on KP00078’s activity and selectivityConversion achieved applying the organic co- solvent divided by conversion achieved without solvent (none)bCalculated using exact, non-rounded values (cis content normalized to trans + cis = 100%)Ctrans / cis ratio achieved applying the organic co-solvent divided by trans / cis ratio achieved without solvent (none)1,4 pH screening at 1% substrate loading

[0056] A reaction mixture composed of 5 mg ketone of formula lb (1 wt% relative to reaction mixture) dissolved in 450 pL of various buffer solutions (100 mM) at pH 5.0 to 9.0, containing 2 mM MgCh, various KREDs s / e 150 (33.3 pg, 0.67 wt%), GDH-105 [Codexis], lot A10036 s / e 500 (10 pg, 0.2 wt%), NADP s / c 50 (100 pg, 2 wt%), Glucose 6.7 eq. (25 mg) and cyclohexane (10 v% relative to reaction mixture) was incubated for 20 h at room temperature.

[0057] Table 4: pH impact on activity and selectivity“Calculated using exact, non-rounded values (cis content normalized to trans + cis = 100%)1.5 Magnesium ion concentration screening at 5% substrate loading

[0058] A reaction mixture composed of 1.25 g ketone of formula lb (5 wt% relative to reaction mixture) dissolved in 18mL of varying buffers 50 mM at pH 5.9 containing 2 to 20 mM MgCh, KRED KP00078 s / e 100 (12.5 mg, 1 wt% relative to ketone) and GDH-105 [Codexis], lot A10036 s / e 500 (2.5 mg, 0.2 wt% relative to ketone), NADP s / c 100 (12.5 mg, 1 wt% relative to ketone), Glucose 1.2 eq. (1.15 g) and 5 mL (20 v% relative to reaction mixture) of isopropyl acetate at 30°C. During the reaction time of about 21 h the pH was kept constant by the addition of about 5.1 mL (~1 eq.) NaOH.

[0059] Table 5: Magnesium ion concentration screeningaCalculated using exact, non-rounded values (cis content normalized to trans + cis = 100%)Example 2. Preparative synthesis of compound of formula lib2,1 Preparative reaction at 5% substrate loading

[0060] In a 200 mL reactor 5.0 g (5 wt%, 20.72 mM, 1.00 eq.) N-(3-keto-2,2,4,4-tetramethyl- cyclobutyl) carbamic acid tert-butyl ester (ketone of formula lb) was added under moderate stirring at room temperature to 10 mL isopropyl acetate. Then 85.0 ml of the MES (50 mM) buffer solution, containing 2 mM MgCh, 4.6 g (1.2 eq.) D-Glucose, 50.0 mg (1 wt%) NADP and 10 mg (0.2 wt%) GDH (glucose dehydrogenase; GDH-105 [Codexis]; lot A10036), was added and the pH was adjusted to pH 6.0 at 30°C. The addition of 50 mg (1 wt%) ketoreductase KP00078 (evo-1.1.440.S [EVOXX]; Lot: 11440-E201108.01) started the reduction. The biphasic suspension was stirred at 30°C while the pH was kept constant at 6.0 by the addition of 1 N NaOH for 20 h until reaction completion.

[0061] The alcohol of formula lib was extracted twice with 100 mL ethyl acetate. The separated organic phases were combined and dried over sodium sulfate and evaporated.

[0062] 5.0 g white crystals of tert-butyl N-(trans-3-hydroxy-2,2,4,4-tetramethyl- cyclobutyl)carbamate (alcohol of formula lib) were obtained after drying under HV, corresponding to a yield of 98.0% and 97.8 A% GC-purity (cis-isomer 2.07 A% and ketone lb 0.1 A%, corresponding to a trans / cis ratio of 47.2 : 1).2,2 Preparative reaction at 10% substrate loading

[0063] In a 200 mL reactor 10.0 g (10 wt%, 41.44 mM, 1.00 eq.) of N-(3-keto-2, 2,4,4- tetramethyl-cyclobutyl) carbamic acid tert-butyl ester (ketone of formula lb) was added under moderate stirring at room temperature to 20 mL isopropyl acetate. Then 65.0 ml of the sodium acetate (50 mM) buffer solution, containing 5 mM MgCh, 9.0 g (1.2 eq,) D-Glucose, 0.1 g (1 wt%) NADP and 0.02 g (0.2 wt%) GDH (glucose dehydrogenase; GDH-105 [Codexis]; lot A10036), was added and the pH was adjusted to pH 5.8 at 31°C. The addition of 133 mg (1.3 wt%) ketoreductase KP00078 (evo-1.1.440.S [EVOXX]; Lot: 11440-E201108.01), dissolved in 5.0 mL of the acetate buffer solution started the reduction. The biphasic suspension was stirred at 31 °C while the pH was kept constant at 5.8 by the addition of 1 N NaOH for 21 h until reaction completion.

[0064] The alcohol of formula lib was extracted with additional 100 mL isopropyl acetate. The organic phase was separated and dried over sodium sulfate and evaporated to 20 g (100 mbar and 40°C). 60 mL n-heptane was added and the mixture was evaporated to 19 g (100 mbar and 40°C) yielding a colourless oil. By adding 60 mL n-heptane the crystallization started. After stirring overnight the suspension was cooled down to ~0°C and allowed to warm to room temperature.

[0065] 8.7 g white crystals of tert-butyl N-(trans-3-hydroxy-2,2,4,4-tetramethyl- cyclobutyl)carbamate (alcohol of formula lib) were obtained after filtration and drying under HV, corresponding to a yield of 85.9% and 98.7 A% GC-purity (cis-isomer 1.27 A% andketone lb 0.05 A%, corresponding to a trans / cis ratio of 77.7 : 1 (prior crystallisation 56.9 : 1 (end of the reaction)).Example 3. Preparation of compound of formula libD1-1 D1-2 D1-8 D1-6(compound of formula lb) (compound of formula lib)Step 1: Preparation of 3-(hydroxyimino)-2,2,4,4-tetramethylcyclobutan-l-one (Dl-2)

[0066] A I L double-jacketed reactor was charged with 2,2,4,4-tetramethyl-l,3- cyclobutanedione (150 g, 1.07 mol), THF (450 mL), and acetic acid (6.40 g, 0.11 mol). The internal temperature was set to 60°C and hydroxylamine, 50% solution in water (70.7 g, 1.07 mol) was added over 3 h at 60°C. Subsequently, the reaction mixture was aged for 1 h at 60°C. The solvent was switched to n-hcptanc / THF (~95:5-v / v) by distillation under atmospheric pressure and dosage of / -hcptanc (ca. 880 mL). The product suspension was cooled to 25°C, aged for 30 min at 25 °C and filtered. The filter cake was washed in displacement with n- heptane / THF (95:5-v / v, 150 mL). The filter cake was dissolved from the Nutsche filter with THF (375 mL) and the keto-oxime Dl-2 product solution in THF (515 mL) was directly telescoped into the hydrogenation.

[0067] (Dl-2): Mp. = 143-145°C. ^-NMR (400 MHz, DMSO-d6) 5 10.57 (s, 1H), 1.35 (s, 6H), 1.26 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 217.4, 161.9, 63.6, 61.3, 21.9, 19.9. HRMS (GC-MS) calculated for CsHuNCh [M+H]+m / z = 156.1019, found 156.1026.Step 2: Preparation of tert-butyl (2,2,4,4-tetramethyl-3-oxocyclobutyl)carbamate (Dl-8) (compound of formula lb)

[0068] A 2 L steel autoclave was charged with the keto-oxime product solution in THF (515 mL), THF (975 mL), and Raney-Ni, 50% in water (18.0 g). The autoclave was rendered inert with nitrogen and subsequently, the atmosphere was changed to hydrogen. A pressure of 15 bar hydrogen was applied and the batch was heated to an internal temperature of 60°C. Once the temperature of 60°C was reached, BOC2O, 70% in THF (267 g, 0.86 mol) was dosed into the autoclave over 4 h with a HPLC pump. Hydrogenation was continued for 1 h at 60°C after completed dosage. The reaction mixture was cooled to 25°C, the atmosphere was changed to nitrogen and the reaction mixture was discharged via a filter. The solvent was switched to n- heptane / THF (~95:5-v / v) by distillation under atmospheric pressure and dosage of / / -heptane (ca. 920 mL). The product suspension was cooled to 25 °C, aged for 30 min at 25 °C andfiltered. The filter cake was washed in displacement with zz-heptane / THF (95:5-v / v, 150 mL). Subsequently, the filter cake was slurry washed with z-PrOH / H O (l:l-v / v, 300 mL) followed by a displacement wash with z-PrOH / FLO (l:l-v / v, 150 mL). The wet product was dried at 45°C in vacuo (5-50 mbar). The product Dl-8 was obtained as a white solid (159 g, 62%).

[0069] (Dl-8): Mp. = 146-147°C. ^-NMR (400 MHz, DMSO-d6) 5 6.86-6.42 (m, 1H), 3.75-3.62 (m, 1H), 1.43 (s, 9H), 1.17 (s, 6H), 1.05 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 221.1, 156.7, 78.6, 59.0, 58.3, 28.6, 24.2, 18.5. HRMS (GC-MS) calculated for C13H24NO3 [M+H]+m / z = 242.1751, found 242.1752.Step 3: Preparation of tert-butyl (trans-3-hydroxy-2, 2,4,4- tetramethylcyclobutyl)carbamate (Dl-6) (compound of formula lib)

[0070] A 250 mL double-jacketed reactor was charged with deionized water (110 mL), sodium acetate (0.49 g, 6.00 mmol, 50.0 mM), magnesium chloride hexahydrate (0.12 g, 0.60 mmol, 5.00 mM), dipotassium phosphate (1.04 g, 6.00 mmol, 50.0 mM), D-glucose monohydrate (39.4 g, 199 mmol), and NADP disodium salt (0.40 g, 1.00%-w / w). The internal temperature was set to 25 °C and the pH was adjusted to 5.8 (±0.1) with acetic acid (ca. 0.2 g). Glucose dehydrogenase GDH-105 [Codexis], lot A10036 (0.08 g, 0.20%-w / w) dissolved in deionized water (5.00 mL) was added followed by addition of Dl-8 (40.0 g, 166 mmol) and MTBE (120 mL). The internal temperature was adjusted to 31 °C and if necessary, the pH was again set to 5.8 (±0.1). Subsequently, ketoreductase KP00078 (evo-1.1.440.S [EVOXX], 0.52 g, 1.30%-w / w) dissolved in deionized water (5.00 mL) was added. The reaction mixture was stirred for -14-16 h at 31°C while keeping the pH constant at 5.8 (±0.1) by addition of aqueous sodium hydroxide solution, 10% (66.3 g, 166 mmol). Vitacel FAC 200 (8.00 g, 20.0%-w / w) was added upon complete conversion followed by addition of aqueous sulfuric acid, 30% (43.4 g, 133 mmol). The mixture was stirred for 30 min at 25°C, filtered and the filter cake was washed with MTBE (40 mL). The phases were separated, and the organic layer was washed with deionized water (40 mL) and the crude product solution was directly telescoped into the next step. Typical assay yield for step 3 was approximately 94%.

[0071] (Dl-6) (compound of formula lib) Mp. = 95-97°C. ^-NMR (400 MHz, DMSO-d6) 5 6.39-5.95 (m, 1H), 4.67 (d, J= 4.8 Hz, 1H), 3.32-3.20 (m, 2H), 1.39 (s, 9H), 0.98 (s, 6H), 0.91 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 156.7, 78.6, 77.9, 59.9, 40.3, 28.7, 24.4, 24.1.Example 4. Preparation of compound DI(compound of formula lb),(compound of formula lib)Step 1: Preparation of 3-(hydroxyimino)-2,2,4,4-tetramethylcyclobutan-l-one (Dl-2)

[0072] A I L double-jacketed reactor was charged with 2,2,4,4-tetramethyl-l,3- cyclobutanedione (150 g, 1.07 mol), THF (450 mL), and acetic acid (6.40 g, 0.11 mol). The internal temperature was set to 60°C and hydroxylamine, 50% solution in water (70.7 g, 1.07 mol) was added over 3 h at 60°C. Subsequently, the reaction mixture was aged for 1 h at 60°C. The solvent was switched to n-hcptanc / THF (~95:5-v / v) by distillation under atmospheric pressure and dosage of / -hcptanc (ca. 880 mL). The product suspension was cooled to 25°C, aged for 30 min at 25 °C and filtered. The filter cake was washed in displacement with n- heptane / THF (95:5-v / v, 150 mL). The filter cake was dissolved from the Nutsche filter with THF (375 mL) and the keto-oxime Dl-2 product solution in THF (515 mL) was directly telescoped into the hydrogenation.

[0073] (Dl-2): Mp. = 143-145°C. ^-NMR (400 MHz, DMSO-d6) 5 10.57 (s, 1H), 1.35 (s, 6H), 1.26 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 217.4, 161.9, 63.6, 61.3, 21.9, 19.9. HRMS (GC-MS) calculated for CsHuNCh [M+H]+m / z = 156.1019, found 156.1026.Step 2: Preparation of tert-butyl (2,2,4,4-tetramethyl-3-oxocyclobutyl)carbamate (Dl-8) (compound of formula lb)

[0074] A 2 L steel autoclave was charged with the keto-oxime product solution in THF (515 mL), THF (975 mL), and Raney-Ni, 50% in water (18.0 g). The autoclave was rendered inert with nitrogen and subsequently, the atmosphere was changed to hydrogen. A pressure of 15 bar hydrogen was applied and the batch was heated to an internal temperature of 60°C. Once the temperature of 60°C was reached, BOC2O, 70% in THF (267 g, 0.86 mol) was dosed into the autoclave over 4 h with a HPLC pump. Hydrogenation was continued for 1 h at 60°C after completed dosage. The reaction mixture was cooled to 25°C, the atmosphere was changed tonitrogen and the reaction mixture was discharged via a filter. The solvent was switched to n- heptane / THF (~95:5-v / v) by distillation under atmospheric pressure and dosage of zz-heptane (ca. 920 mL). The product suspension was cooled to 25 °C, aged for 30 min at 25 °C and filtered. The filter cake was washed in displacement with n-hc tanc / THF (95:5-v / v, 150 mL). Subsequently, the filter cake was slurry washed with z-PrOH / FLO (l:l-v / v, 300 mL) followed by a displacement wash with z-PrOH / FLO (l:l-v / v, 150 mL). The wet product was dried at 45°C in vacuo (5-50 mbar). The product Dl-8 was obtained as a white solid (159 g, 62%).

[0075] (Dl-8): Mp. = 146-147°C. ^-NMR (400 MHz, DMSO-d6) 5 6.86-6.42 (m, 1H), 3.75-3.62 (m, 1H), 1.43 (s, 9H), 1.17 (s, 6H), 1.05 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 221.1, 156.7, 78.6, 59.0, 58.3, 28.6, 24.2, 18.5. HRMS (GC-MS) calculated for C13H24NO3 [M+H]+m / z = 242.1751, found 242.1752.Step 3: Preparation of tert-butyl (trans-3-hydroxy-2, 2,4,4- tetramethylcyclobutyl)carbamate (Dl-6) (compound of formula lib)

[0076] A 250 mL double-jacketed reactor was charged with deionized water (110 mL), sodium acetate (0.49 g, 6.00 mmol, 50.0 mM), magnesium chloride hexahydrate (0.12 g, 0.60 mmol, 5.00 mM), dipotassium phosphate (1.04 g, 6.00 mmol, 50.0 mM), D-glucose monohydrate (39.4 g, 199 mmol), and NADP disodium salt (0.40 g, 1.00%-w / w). The internal temperature was set to 25 °C and the pH was adjusted to 5.8 (±0.1) with acetic acid (ca. 0.2 g). Glucose dehydrogenase GDH-105 [Codexis], lot A10036 (0.08 g, 0.20%-w / w) dissolved in deionized water (5.00 mL) was added followed by addition of Dl-8 (40.0 g, 166 mmol) and MTBE (120 mL). The internal temperature was adjusted to 31 °C and if necessary, the pH was again set to 5.8 (±0.1). Subsequently, ketoreductase KP00078 (evo-1.1.440.S [EVOXX], 0.52 g, 1.30%-w / w) dissolved in deionized water (5.00 mL) was added. The reaction mixture was stirred for -14-16 h at 31°C while keeping the pH constant at 5.8 (±0.1) by addition of aqueous sodium hydroxide solution, 10% (66.3 g, 166 mmol). Vitacel FAC 200 (8.00 g, 20.0%-w / w) was added upon complete conversion followed by addition of aqueous sulfuric acid, 30% (43.4 g, 133 mmol). The mixture was stirred for 30 min at 25°C, filtered and the filter cake was washed with MTBE (40 mL). The phases were separated, and the organic layer was washed with deionized water (40 mL). The product was telescoped directly into the next step as solution in MTBE (156 mL, 27-30%-w / w).

[0077] (Dl-6) (compound of formula lib) Mp. = 95-97°C. ^-NMR (400 MHz, DMSO-d6) 5 6.39-5.95 (m, 1H), 4.67 (d, J= 4.8 Hz, 1H), 3.32-3.20 (m, 2H), 1.39 (s, 9H), 0.98 (s, 6H), 0.91 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 156.7, 78.6, 77.9, 59.9, 40.3, 28.7, 24.4, 24.1.Step 4: Preparation of tert-butyl ((lr,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)carbamate (Dl-7)

[0078] A 250 mL double-jacketed reactor was charged with Dl-6 (122 g, 33%-w / w in MTBE) and the volume was reduced by normal pressure distillation to 100 mL. The remaining solution was dried by an azeotropic distillation at constant volume by addition of THF until the water content was below 0.15 wt%. After a polish filtration at 40°C, 2-chloro-4- fluorobenzonitrile (25.8 g, 166 mmol) was added at 25 °C. The mixture was cooled to 0°C and NaOt-Bu (87.6 g, 20 wt% in THF, 182 mmol) was added. After complete addition, the mixture was stirred for at least 1 h. Then, potassium carbonate (160 mL, 20 wt% in water) was added in one portion. The mixture was warmed to room temperature and the phases were separated. The organic phase was distilled under normal pressure at constant volume by addition of heptane (120 mL). Afterwards, the organic layer was washed two times with water (40 mL) and the volume was reduced by normal pressure distillation to 160 mL. The distillation was continued at constant volume by addition of n-hcptanc until an internal temperature of 93-94°C was reached. The solution was subsequently cooled to 60 °C and seeds were added. After stirring for 0.5 h, the suspension was cooled over 2 h to room temperature and stirred for 2 h. The precipitate was filtered, washed with n-hcptanc / THF (40 mL, 97:3), and dried under vacuum to obtain Dl-7 (52.4 g, 83%, 99.3 A%, 0.62 A% cis-isomer) as a white solid.

[0079] Mp. = 103-104°C. ^-NMR (400 MHz, DMSO-d6) 5 7.88 (d, 7 = 8.8 Hz, 1H), 7.17 (d, 7= 2.4 Hz, 1H), 6.97 (dd, 7= 8.8, 2.4 Hz, 1H), 6.74-6.17 (m, 1H), 4.17 (s, 1H), 3.65-3.37 (m, 1H), 1.41 (s, 9H), 1.13 (s, 6H), 1.05 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 163.1, 156.3, 137.3, 136.5, 117.3, 116.7, 115.1, 104.0, 84.4, 78.4, 60.1, 40.6, 28.7, 24.4, 23.7.Step 5: Preparation of 4-((lr,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2- chlorobenzonitrile, hydrochloric acid salt (DI, HC1 salt)

[0080] A 250 mL double-jacketed reactor was charged with Dl-7 (20 g, 52.8 mmol), n-PrOH (60 mL) and water (6 mL). The mixture was heated to 80 °C and hydrochloric acid (6.9 mL, 33 wt% in water, 73.9 mmol) was added dropwise. After complete addition, the mixture was stirred for 1 h. Then, 10 mL of solvent was removed by vacuum distillation at 65-70°C. The remaining solvent was dried by an azeotropic vacuum distillation at 65-70°C and constant volume by addition of n-PrOH until the water content was below 4.0 wt%. Then, heptane (20 mL) was added over 0.5 h. After complete addition, the solution was cooled over 2 h to 0 °C and stirred for 0.5 h at the same temperature. The precipitate was filtered, washed with n- PrOH / n-hcptanc (20 mL, 3:2) and subsequently with 20 mL water. The wet product was dried in the vacuum oven to obtain DI (15.6 g, 93%, 99.9 A%, 0.12 A% cis-isomer) as a white solid.

[0081] Mp. = 239-241°C (decomposition onset). ^-NMR (400 MHz, DMSO-d6) 5 8.56 (s, 3H), 7.89 (d, 7 = 8.7 Hz, 1H), 7.21 (d, 7 = 2.5 Hz, 1H), 6.99 (dd, 7 = 8.7, 2.5 Hz, 1H), 4.36 (s, 1H), 3.06 (s, 1H), 1.32 (s, 6H), 1.09 (s, 6H).13C-NMR (101 MHz, DMSO-d6) 5 162.7, 137.3, 136.6, 117.4, 116.7, 115.1, 104.4, 83.6, 59.5, 38.8, 23.7, 23.2.

Claims

CLAIMS1. A process for preparing a compound of formula IIcomprising reducing a compound of formula Iin the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co-solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula II, wherein Prot is an amino protecting group.

2. The process of claim 1, wherein the amino protecting group is tertbutoxycarbonyl (Boc), the compound of formula I has the formula lband the protected trans amino alcohol of formula II has the formula lib3. A process for preparing a compound of formula libcomprising:(1) reacting a compound of formula DI -2with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form a compound of formula lb(2) reducing the compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula lib.

4. A process for preparing a compound of formula libcomprising:(1) reacting a compound of formulawith hydroxylamine to form a compound of formula Dl-2(2) reacting a compound of formula Dl-2 with a reducing agent followed by protection of the resulting free amine using di-tert-butyl dicarbonate to form a compound of formula lb(3) reducing the compound of formula lb in the presence of: a) a ketoreductase which is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 90%; b) an organic co- solvent; and c) an aqueous buffer comprising NADP, glucose dehydrogenase, and glucose; to form the compound of formula lib.

5. The process of any one of claims 1 to 4, wherein the ketoreductase is capable to convert the ketone of formula I into the protected trans amino alcohol of formula II in a conversion of at least 95%.

6. The process of any one of claims 1 to 5, wherein the organic co- solvent is selected from the group consisting of isopropyl acetate, ethyl acetate, n-heptane, n-dodecane, cyclohexane, methyl-cyclohexane, toluene and tert-butyl methyl ether.

7. The process of claim 6, wherein the organic co-solvent is isopropyl acetate or tertbutyl methyl ether.

8. The process of any one of claims 1 to 7, wherein the aqueous buffer comprises 1 to 100 mM Mg2+.

9. The process of any one of claims 1 to 8, wherein the aqueous buffer comprises the cofactor NADP in a concentration of below about 4.0 wt% relative to the compound of formula I.

10. The process of any one of claims 1 to 9, wherein the aqueous buffer comprises the glucose dehydrogenase in a concentration below about 4.0 wt% relative to the compound offormula I, and comprises the glucose in an amount of more than 1.0 equivalent of the compound of formula I.

11. The process of any one of claims 1 to 10, wherein the process is performed at a temperature of between about 25°C and about 45°C at a constant pH between about 5.5 and about 7.0.

12. The process of any one of claims 1 to 11, wherein the concentration of the compound of formula I is between about 0.5 wt% and about 10.0 wt% relative to the reaction mixture.

13. The process of any one of claims 1 to 12, wherein the concentration of the ketoreductase in the reaction mixture is less than 25.0 wt% relative to the compound of formula I.

14. The process of any one of claims 3 to 13, wherein the reducing agent is hydrogen in the presence of Raney nickel.

15. Use of the process of any one of claims 1 to 14 for the preparation of a pharmaceutically active ingredient comprising the structural element of the protected trans amino alcohol of formula II.

16. A compound of formula IIwherein Prot is an amino protecting group.

17. The compound of claim 16, wherein the compound comprises less than about 4 wt% of an impurity of formula III18. The compound of claim 16, wherein the compound of formula II is a compound of formula lib19. The compound of claim 18, wherein the compound comprises less than about 4 wt% of an impurity of formula Illb20. A process of preparing a compound of formula DIor a salt thereof, comprising(1) preparing the compound of formula lib as provided in any one of claims 1-14;(2) reacting the compound of formula lib with 2-chloro-4-fluorobenzonitrile in the presence of a base to form a compound of formula DI -7(3) deprotecting the compound of formula DI -7 in the presence of an acid to form the compound of formula DI or salt thereof.

21. A process of preparing a compound of formula DIor a salt thereof, comprising(1) obtaining a compound of formula libcomprising less than about 4 wt% of an impurity of formula Illb(Illb);(2) reacting the compound of formula lib with 2-chloro-4-fluorobenzonitrile in the presence of a base to form a compound of formula DI -7(3) deprotecting the compound of formula DI -7 in the presence of an acid to form the compound of formula DI or salt thereof.

22. The process of claim 20 or 21, wherein the base is sodium hydride.

23. The process of claim 20 or 21, wherein the base is sodium tert-butoxide.

24. The process of any one of claims 20 to 23, wherein the acid is hydrdochloric acid and the compound of formula DI or salt thereof is

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