CN117500806A - Synthesis of BTK inhibitors and intermediates thereof - Google Patents

Abstract

The present invention relates to a highly efficient synthetic method useful for preparing a BTK inhibitor of compound a, formula (I), or a pharmaceutically acceptable salt thereof, comprising the preparation of an intermediate useful for preparing compound a or a pharmaceutically acceptable salt thereof.

Description

Synthesis of BTK inhibitors and intermediates thereof

Sequence listing

The present application contains a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created at 25 months 05 of 2022, named 25247-WO-PCT_SL.txt, and was 24009 bytes in size.

Technical Field

The present invention relates to BTK inhibitors useful in the preparation of compound a, formula (I):

or a pharmaceutically acceptable salt thereof, which comprises the preparation of an intermediate for the preparation of compound a or a pharmaceutically acceptable salt thereof.

Background

Bruton's Tyrosine Kinase (BTK) is a member of the Tec family of tyrosine kinases and plays an important role in regulating early B cell development and mature B cell activation and survival. BTK acts downstream of a variety of receptors such as growth factors, B cell antigens, chemokines and innate immune receptors, initiating a number of cellular processes including cell proliferation, survival, differentiation, motility, angiogenesis, cytokine production and antigen presentation.

The BTK deficient mouse model has been shown to play a role in allergic disorders and/or autoimmune diseases and/or inflammatory diseases. The expression of BTK in osteoclasts, mast cells and monocytes has been shown to be important for the function of these cells. For example, in mice and humans, impaired IgE-mediated mast cell activation and reduction of TNF- α produced by activated monocytes are associated with BTK deficiency.

Thus, inhibition of BTK with small molecule inhibitors provides treatment for hematologic malignancies, immune disorders, cancer, cardiovascular diseases, viral infections, inflammatory, metabolic/endocrine functional disorders and neurological disorders. While compound a may be useful in the treatment of a disease or disorder, such as hematological malignancy, the existing route to compound a requires a multi-step process. In particular key intermediate 4'

Known syntheses of (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-amine salts (including the free base) from carbohydrate starting materials (N.M. A.J. Kriek et al Eur.J. Org. chem. (2003) 2003 (13): 2418-27; C.E.Lu nse et al ACS chem. Biol. (2011) 6 (7): 675-78; F.Amann et al org. Process Res. Dev. (2016) 20 (2): 446-51) or L-serine (J. -C.Gauvin et al WO/105154 A2 and M.J. Dunn et al J.org. Chem. (1995) 60 (7): 2210-15) are inefficient and complex because multiple synthetic steps are required, including several protecting group manipulations. In view of the difficult and tedious synthetic options developed so far to produce compound 4', a minimum number of synthetic steps and the use of protecting groups are required to prepare the synthetic route of compound 4' in order to obtain compound a in a more sustainable manner.

Disclosure of Invention

The present invention relates to compounds useful in the synthesis of compound A

Or a pharmaceutically acceptable salt thereof, which comprises the preparation of an intermediate for the preparation of compound a. The process of the present invention provides advantages over previously known procedures and includes a more efficient route for preparing intermediates useful in the preparation of compound a.

Other embodiments, aspects, and features of the present invention will be further described in, or will be apparent from, the ensuing description, examples, and appended claims.

Detailed description of the preferred embodiments

The present disclosure provides enzymatic methods for the preparation of intermediates that produce compound a, or a pharmaceutically acceptable salt thereof.

In a first embodiment of the invention, a process for preparing compound 3

Or a pharmaceutically acceptable salt thereof, comprising the steps of:

a) Combining cofactors in a buffer or water with isopropylamine and a transaminase to produce a reagent mixture; and

b) Addition of Compound 2 to reagent mixture

To produce a solution comprising compound 3.

In another aspect of the first embodiment, the present invention relates to a process for preparing compound 3 comprising the further step of heating the solution to about 25 ℃ to about 70 ℃ after adding compound 2 in step c) above to produce a solution comprising compound 3.

In another aspect of the first embodiment, the buffer is an aqueous solution of sodium tetraborate having a pH of about 6 to about 12.

In another aspect of the first embodiment, the present invention relates to a process for preparing compound 3 comprising, in step b above, after the addition of compound 2, maintaining the pH between about 7 and about 9 by the addition of an inorganic base to produce a solution comprising compound 3.

In another aspect of the first embodiment, the method for preparing compound 3 comprises the further step of removing acetone by-product generated during the process by applying vacuum or nitrogen purge to produce a solution comprising compound 3 after adding compound 2 in step (c) above.

In a second embodiment, the invention relates to a process for the preparation of compound 3',

wherein HX is a pharmaceutically acceptable acid comprising the steps of:

a) Adding an inorganic base to adjust the pH of the solution comprising compound 3 to about 12 to about 14;

b) Adding a solvent and an inorganic salt to produce a resulting two-phase mixture comprising compound 3, said resulting two-phase mixture comprising an organic layer and an aqueous layer;

c) Separating the organic layer from the resulting two-phase mixture with a solvent;

d) Combining the organic layer of the resulting two-phase mixture with a solution of an acid in a solvent to produce a slurry comprising compound 3'; and

e) The slurry was filtered to obtain compound 3' as a solid.

In a further aspect of the second embodiment, the present invention relates to a process for the preparation of compound 3', further comprising the step of filtering the resulting two-phase mixture of step (b) above to remove transaminase.

In another aspect of the second embodiment, the present invention relates to a process for preparing compound 3', further comprising the steps of:

a) After separation of the organic layer in step (c) above, isopropylamine is removed by concentrating the organic layer to produce a resulting solution comprising compound 3; and

b) The resulting solution of compound 3 is combined with a solution of acid in a solvent to produce a slurry comprising compound 3'.

In another aspect of the second embodiment, the present invention relates to a process for preparing compound 3', comprising adding water to the organic layer comprising compound 3 to adjust the water content to about 0 to about the water saturation point of the organic layer, before adding the solution of acid in the solvent in step d) above.

In another aspect of the second embodiment, the invention relates to a process for preparing 3' of a compound wherein HX is a pharmaceutically acceptable acid selected from p-toluene sulfonic acid, benzene sulfonic acid or hydrochloric acid.

In another aspect of the second embodiment, HX is para-toluenesulfonic acid and compound 3' is represented as compound 3a

And the process for preparing compound 3a further comprises the steps of:

a) Combining a solution of p-toluene sulfonic acid in a solvent with an organic layer of the resulting two-phase mixture to produce a slurry comprising 3 a; and

b) The slurry was filtered to obtain 3a as a solid.

In a third embodiment, the present invention relates to a process for preparing compound 3', comprising the steps of:

a) Adding an inorganic base to adjust the pH of the solution comprising compound 3 to about 12 to about 14;

b) Distilling the solution comprising compound 3' to remove isopropylamine;

c) Adding a solution of a base and di-tert-butyl dicarbonate in a solvent to produce a resulting two-phase mixture comprising compound 3c

The resulting two-phase mixture comprises an aqueous layer and an organic layer;

d) Separating the organic layer comprising the resulting two-phase mixture of compound 3c by adding a solvent;

e) Adding a solution of an acid in a solvent to the organic layer to produce a slurry comprising compound 3'; and

f) The slurry was filtered to give compound 3' as a solid.

In a further aspect of the third embodiment, the present invention relates to a process for preparing compound 3', comprising the further step of filtering the resulting two-phase mixture comprising compound 3c produced in step (c) above to remove transaminase.

In another aspect of the third embodiment, compound 3' is compound 3a, and the method for preparing compound 3a further comprises the steps of:

a) Adding a solution of p-toluenesulfonic acid in a solvent to the organic layer comprising compound 3c in step e) above to produce a slurry comprising compound 3 a; and

b) The slurry was filtered to give compound 3a as a solid.

In a fourth embodiment of the present invention, a process for preparing compound 3' comprises the steps of:

a) Combining a transaminase with a buffer or water and a solid support, followed by incubation to prepare an immobilized transaminase;

b) Washing the immobilized transaminase with a buffer or water;

c) Washing with an aminotransferase solvent (transamination solvent);

d) Combining compound 2 with isopropylamine and a transamination solvent to provide a reaction stream;

e) Combining the reaction stream with an immobilized transaminase to produce a slurry comprising compound 3;

f) Separating the slurry comprising compound 3 from the immobilized transaminase to produce a solution comprising compound 3;

g) Combining a solution comprising compound 3 with a solution of an acid in an aminotransferase solvent to produce a slurry comprising compound 3'; and

h) The slurry was filtered to give compound 3' as a solid.

In another aspect of the fourth embodiment, the method for preparing compound 3' further comprises combining a cofactor with a transaminase and a buffer in step (a) above to produce a mixture of transaminases.

In another aspect of the fourth embodiment, the buffer in step (a) above is a buffer having a pH of from about 4 to about 11.

In another aspect of the fourth embodiment, the buffer is an aqueous potassium phosphate solution and the pH of the buffer is from about 6 to about 8.

In a further aspect of the fourth embodiment, the immobilized transaminase in step b) is washed with water in step b) above.

In another aspect of the fourth embodiment, the process for preparing compound 3' further comprises the step of washing the immobilized transaminase with water and then washing the immobilized transaminase with an isopropanol:PEG-400:water mixture.

In another aspect of the fourth embodiment, the method for preparing compound 3' is as described above, wherein the aminotransferase solvent in step c) above is an aqueous aminotransferase solvent.

In another aspect of the fourth embodiment, the process for preparing compound 3' further comprises the step of drying the immobilized transaminase prior to combining it with the reaction stream.

In another aspect of the fourth embodiment, the process for preparing compound 3' further comprises the step of combining compound 2 with isopropylamine in an aqueous transamination solvent to prepare the reaction stream in step (d) above.

In another aspect of the fourth embodiment, the method for preparing compound 3' further comprises the step of heating the solution to about 25 ℃ to about 70 ℃ after combining the reaction stream with the immobilized transaminase in step e) above to produce a slurry comprising compound 3.

In another aspect of the fourth embodiment, the immobilized transaminase can be reused in a new reaction stream comprising compound 2 to produce a slurry comprising compound 3.

In another aspect of the fourth embodiment, the process for preparing compound 3' comprises performing steps b), c) and e) above in a continuous reaction system, wherein the buffer or water, the transamination solvent and the reaction stream are continuously subjected to the immobilized transaminase.

In another aspect of the fourth embodiment, the method for preparing compound 3' comprises the steps of:

a) Mixing a transaminase with a buffer or water to produce a solution comprising a transaminase;

b) Mixing a solution comprising a transaminase with a solid support in a continuous reaction system, wherein the solution is continuously passed through the solid support to produce an immobilized transaminase; and

c) Steps b), c) and e) of the fourth embodiment are carried out in a continuous reaction system, wherein the buffer or water, the transamination solvent and the reaction stream are continuously immobilized transaminase.

In another aspect of the fourth embodiment, the continuous reaction system is a Packed Bed Reactor (PBR).

In another aspect of the fourth embodiment, the method for preparing compound 3' further comprises the step of distilling the solution comprising compound 3 to remove isopropylamine.

In another aspect of the fourth embodiment, the present invention relates to a process for preparing compound 3', comprising the further steps of:

a) Distilling the solution comprising compound 3 to remove isopropylamine to produce a resulting solution comprising compound 3; and

b) Water is added to the resulting solution comprising compound 3 to adjust the water content to about 0 to about the water saturation point of the transamination solvent.

In another aspect of the fourth embodiment, compound 3' is compound 3a, and the process for preparing compound 3a further comprises the steps of:

a) Combining a solution comprising compound 3 with a solution of p-toluene sulfonic acid in a transamination solvent to produce a slurry comprising compound 3 a; and

b) The slurry was filtered to isolate 3a as a solid.

In another aspect of the fourth embodiment, compound 3' is compound 3b, and the process for preparing compound 3b further comprises the steps of:

a) Combining the solution comprising compound 3 with hydrochloric acid to produce a slurry comprising compound 3 b; and

b) The slurry was filtered to isolate 3b as a solid.

In a fifth embodiment of the invention, a process for preparing compound 4

Wherein HX is a pharmaceutically acceptable acid comprising the steps of:

a) Adding compound 3' to a weakly coordinating solvent;

b) Adding a silane or borane reducing agent and a lewis acid and heating to about 30 ℃ to about 70 ℃ to produce a resulting solution;

c) Adding an alcohol to produce a solution comprising compound 4';

d) Cooling the solution to produce a slurry comprising compound 4'; and

e) The slurry was filtered to give compound 4' as a solid.

In another aspect of the fifth embodiment, compound 4' is compound 4a

And the process for preparing compound 4a additionally comprises the step of adding compound 3a to the weakly coordinating solvent in step a) above.

In another aspect of the fifth embodiment of the present invention, the weakly coordinating solvent in step a) above is a mixture of anisole and sulfolane.

In a further aspect of the fifth embodiment, 2, 3-dihydrothiophene 1, 1-dioxide, also known as 2-cyclobutanesulfone, or 2, 5-dihydrothiophene 1, 1-dioxide, also known as 3-cyclobutanesulfone, is added in step a) above.

In another aspect of the fifth embodiment of the present invention, the silane reducing agent in step b) above is triethylsilane, the lewis acid in step b) above is boron trifluoride diethyl etherate, and the ratio of triethylsilane to boron trifluoride diethyl etherate is less than 3:1.

In another aspect of the fifth embodiment, an antisolvent is added in step d) above to obtain a slurry comprising compound 4'.

In another aspect of the fifth embodiment of the present invention, the process for preparing compound 4a is carried out in a sealed reactor or a reactor capable of controlling the reaction pressure. In another aspect of the fifth embodiment, the process for preparing compound 4a in step b) comprises adding triethylsilane and boron trifluoride diethyl etherate and heating to about 30 to about 70 ℃ in a reactor to produce a resulting solution, wherein the reactor is a sealed reactor or a reactor capable of controlling the reaction pressure.

In a sixth embodiment of the present invention, a process for preparing compound 4b,

The method comprises the following steps:

a) Adding compound 3a to a weakly coordinating solvent;

b) Adding a silane reducing agent and a lewis acid and heating to about 30 ℃ to about 70 ℃ to produce a resulting solution;

c) Adding an alcohol to produce a solution comprising compound 4 a;

d) Adding a hydrochloric acid solution to produce a slurry comprising compound 4 a; and

e) The slurry was filtered to obtain compound 4b as a solid.

In a seventh embodiment of the present invention, the process for preparing compound 4b comprises the steps of:

a) Adding compound 3' to an organic solvent;

b) Adding an organic base to produce a reaction mixture;

c) Adding a silane reducing agent and trimethylsilyl triflate to the reaction mixture to produce a resulting solution;

d) Adding water to the resulting solution to produce a bilayer mixture comprising compound 4b, the bilayer mixture having a top layer and a bottom layer, and separating the bottom layer phase from the bilayer mixture;

e) Cooling the bottom phase to produce a resulting slurry; and

f) The slurry was filtered to obtain compound 4b as a solid.

In another aspect of the seventh embodiment for preparing compound 4b, the silane reducing agent is chlorodimethylsilane.

In another aspect of the seventh embodiment, compound 3' is compound 3a and the method for preparing compound 4b further comprises the step of adding compound 3a to the organic solvent in step a) above.

In another aspect of the seventh embodiment, an antisolvent is added in step (e) above to obtain a slurry comprising compound 4 b.

In an eighth embodiment of the invention, the invention relates to a process for the preparation of compound 7,

which comprises the following steps:

a) Adding a solution of a first base to compound 5

In a slurry in a first aprotic solvent to produce a resulting mixture;

b) Combining a solution of a second base with the resulting mixture;

c) Addition of Compound 6

A solution in a second aprotic solvent to produce a solution comprising compound 7;

d) Combining the aqueous solution with a solution comprising compound 7 to produce a two-phase mixture comprising compound 7, the two-phase mixture comprising an aqueous layer and an organic layer;

e) Separating the organic layer from the two-phase mixture comprising compound 7;

f) Adding an alcohol, water or alcohol-water mixture to the organic layer to produce a resulting slurry comprising compound 7; and

g) The slurry was filtered to obtain compound 7 as a solid.

In another aspect of the eighth embodiment, the method for preparing compound 7 comprises the further step of adding lithium bromide in step (a) above.

In another aspect of the eighth embodiment, the process for preparing compound 7 comprises the further step of combining the resulting mixture with a solution of the second base and compound 6 in a continuous stirred tank reactor.

In another aspect of the eighth embodiment, the method for preparing compound 7 comprises the further step of:

a) Adding a solution of a first base to a solution of compound 5 and lithium bromide in a first aprotic solvent to produce a resulting mixture; and

b) The resulting mixture is combined with 1) a solution of the second base and 2) a solution of compound 6 in a second aprotic solvent in a Plug Flow Reactor (PFR) to produce a solution comprising compound 7.

In another aspect of the eighth embodiment, the method for preparing compound 7 comprises the further step of treating the organic layer from the two-phase mixture with activated carbon; the mixture was then filtered.

In another aspect of the eighth embodiment, the method for preparing compound 7 further comprises the step of distilling the organic layer prior to performing step f) above.

In a ninth embodiment of the present invention, the method for preparing compound A

Or a pharmaceutically acceptable salt thereof, comprising the steps of:

a) To a reaction solvent containing compound 7

And Compound 4'

Adding a base to a slurry of the mixture of (1), wherein HX is a pharmaceutically acceptable acid, to produce a resulting mixture;

b) Heating the resulting mixture to produce a solution comprising compound a;

c) Adding a crystallization solvent to produce a slurry comprising compound a; and

d) The slurry was filtered to obtain compound a as a solid.

In another aspect of the ninth embodiment, the process for preparing compound a comprises the step of using compound 4a in step a) above

In another aspect of the ninth embodiment, the process for preparing compound a further comprises the step of using N, N-Diisopropylethylamine (DIPEA) as a base.

In another aspect of the ninth embodiment, the process for preparing compound a comprises the step of heating the resulting mixture of step (a) above to about 40 to about 85 ℃ to produce a solution comprising compound a.

In another aspect of the ninth embodiment, the method for preparing compound a comprises the further step of:

a) Adding water as a crystallization solvent to produce a slurry comprising compound a;

b) Cooling the slurry and adding acetic acid to adjust the pH to about 11 to about 4; and

c) The slurry was filtered to obtain compound a as a solid.

In embodiments of the present invention, the methods of the present disclosure may be performed in a single vessel, as a "one-pot" method, or the steps may be performed sequentially. For the sake of clarity, it should be noted that the steps and reactions of the present invention may be performed simultaneously or sequentially, unless otherwise specifically indicated. In embodiments, the intermediate product may optionally be isolated. It should also be noted that when a term is used multiple times, such as with organic solvents, each case is defined independently of the previous selections. For example, the same or different organic solvents may be selected for each step of the process independently of previous selections.

Definition of the definition

Certain technical and scientific terms are specifically defined as follows. Unless specifically defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. That is, the terms used herein have their ordinary meaning, independent at each occurrence. Nevertheless, and unless otherwise indicated, the following definitions apply throughout the specification and claims. Chemical names, common names, and chemical structures may be used interchangeably to describe the same structure. If both chemical structure and chemical name are used to refer to a compound, and there is ambiguity between the structure and name, the structure is subject to control. Unless otherwise indicated, these definitions apply regardless of whether the terms are used alone or in combination with other terms.

The term "e.g." or any example subsequent to "such as" is not intended to be exhaustive or limiting.

As used herein and throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

as used herein, the expressions "compound of formula (I)", "compound (I)" and "compound a" refer to the same compound and are used interchangeably.

As used herein, including the appended claims, the singular forms "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

As used herein, the term "at least one" item or "one or more" items each includes a single item selected from a list and a mixture of two or more items selected from a list.

All ranges cited herein are inclusive unless explicitly stated to the contrary; that is, a range includes values of the upper and lower limits of the range, as well as all values therebetween. All ranges are also intended to include all sub-ranges subsumed, although not explicitly stated. As an example, temperature ranges, percentages, equivalent ranges, and the like described herein include any of the upper and lower limits of the ranges, as well as the continuum therebetween. The numerical values provided herein, as well as the use of the term "about," may include variations of + -1%, + -2%, + -3%, + -4%, + -5% and + -10% and numerical equivalents thereof. When "about" is used to modify a numerically-defined parameter (e.g., the temperature or length of time of a reaction as described herein), it means that the parameter can vary by less than or up to 10% from the recited value for that parameter; where appropriate, the parameters may be rounded to the nearest integer. For example, the temperature of about 30 ℃ may vary between 25 ℃ and 35 ℃. Furthermore, the term "or" as used herein designates alternatives which may be combined where appropriate; that is, the term "or" includes each alternative listed individually.

As used herein, compound 2 may also be referred to herein as Cyrene or (1 s,5 r) -6, 8-dioxabicyclo [3.2.1] oct-4-one. Compound 3 may also be referred to herein as (1 s,4r,5 r) -6, 8-dioxabicyclo [3.2.1] oct-4-amine. Compound 3a may also be referred to herein as (1 s,4r,5 r) -6, 8-dioxabicyclo [3.2.1] oct-4-amine 4-toluene-1-sulfonate. Compound 3b may also be referred to herein as (1 s,4r,5 r) -6, 8-dioxabicyclo [3.2.1] oct-4-amine hydrochloride. Compound 4a may also be referred to herein as (3 r,6 s) -6- (hydroxymethyl) oxa-3-amine 4-toluene-1-sulfonate. Compound 4b may also be referred to herein as (3 r,6 s) -6- (hydroxymethyl) oxacyclohexane-3-amine hydrochloride. Compound 5 may also be referred to herein as 5-bromo-4-chloro-7H-pyrrolo [2,3-d ] pyrimidine. Compound 6 may also be referred to herein as methyl 2-chloro-4-phenoxybenzoate. Compound 7 may also be referred to herein as (2-chloro-4-phenoxyphenyl) (4-chloro-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) methanone. The compound of formula I, compound A, may also be referred to herein as (2-chloro-4-phenoxyphenyl) (4- { [ (3R, 6S) -6- (hydroxymethyl) oxacyclohexan-3-yl ] amino } -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) methanone.

For use in medicine, salts of the compounds described herein will be pharmaceutically acceptable salts. However, other salts may be used to prepare the compounds according to the invention or pharmaceutically acceptable salts thereof. When the compounds of the present invention are acidic, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases (including inorganic and organic bases). Examples of inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include Primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and salts of basic ion exchange resins, e.g., arginine, betaine caffeine, choline, N ¹ Dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine (hydrabamine), isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine, and the like.

When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like. Other examples of such acids include aryl sulfonic acids such as, but not limited to, p-toluene sulfonic acid, 3-methyl-toluene sulfonic acid, 2-methyl-toluene sulfonic acid, benzene sulfonic acid, 2-naphthalene sulfonic acid, 2, 6-naphthalene sulfonic acid, and hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, phenylacetic acid, trimethylacetic acid, tetrafluoroboric acid, tetraphenylboric acid, maleic acid, fumaric acid, oxalic acid, or camphorsulfonic acid. Specific examples are citric acid, hydrobromic acid, p-toluenesulfonic acid, benzenesulfonic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid. P-toluenesulfonic acid, benzenesulfonic acid and hydrochloric acid are preferred.

Berg et al, "Pharmaceutical Salts," j.pharm.sci.,1977:66:1-19 describe more fully the preparation of the above pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts.

One or more compounds herein may be present in unsolvated as well as solvated forms along with pharmaceutically acceptable solvents such as water, ethanol, and the like, and the present disclosure is intended to cover both solvated and unsolvated forms. "solvate" means a compound with one or morePhysical association of solvent molecules. The physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances of this aspect, the solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "solvate" includes both solution phases and separable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. "hydrate" is where the solvent molecule is H ₂ Solvates of O.

The present disclosure also includes all isolated forms of compounds and synthetic intermediates thereof. For example, an identified compound is intended to encompass all forms of the compound, such as any solvate, hydrate, stereoisomer, and tautomer thereof.

Those skilled in the art will recognize that certain compounds, particularly those containing certain heteroatoms and double or triple bonds, may be tautomers, which are structural isomers that readily interconvert. Common pairs of tautomers are: keto-enols, amide-nitriles, lactam-lactams, amide-imidic acid tautomerism in heterocycles (e.g., in nucleobases such as guanine, thymine, and cytosine), amine-enamines, and enamine-imines. The (pyrrolopyrimidinyl) methanone- (pyrrolopyrimidinyl) methanone tautomeric pair is included in the present application:

those skilled in the art will recognize that chiral compounds, such as those described herein, may be drawn in a variety of different equivalent ways.

The term "alkyl" refers to a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, free of unsaturation. Unless otherwise indicated, alkyl groups may contain one to ten carbon atoms (e.g., C ₁ -C ₁₀ Alkyl). In other embodiments, the alkyl group comprises one to eight carbon atoms (e.g., C ₁ -C ₈ Alkyl). In other embodiments, the alkyl group comprises one to four carbon atoms (e.g., C ₁ -C ₆ Alkyl). In other embodiments, the alkyl is selected from methyl, ethyl, propyl, butyl, or pentyl. In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1-dimethylethyl (tert-butyl) or 1-pentyl (n-pentyl). In other embodiments, the alkyl is selected from methyl, ethyl, propyl, or butyl. In other embodiments, the alkyl group is methyl.

As used herein, the term "aryl" is intended to refer to any stable mono-or bi-cyclic carbocycle of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracyl or acenaphthylenyl (acenaphthyl). In an embodiment of the invention, aryl is phenyl or naphthyl. In one embodiment, the aryl group is phenyl.

As used herein, the term cofactor refers to a non-protein compound that operates in combination with a transaminase. Cofactors suitable for the engineered transaminases described herein include compounds from the vitamin B6 family such as, but not limited to, pyridoxal 5 '-phosphate (PLP) or pyridoxamine 5' -phosphate (PMP). In some embodiments, the cofactor is pyridoxal 5' -phosphate monohydrate.

As used herein, buffer refers to a solution that, when added to a liquid mixture, functions to maintain the pH of the liquid mixture at a constant value. In an embodiment of the invention, the buffer is independently selected from aqueous sodium tetraborate, aqueous tris (hydroxymethyl) aminomethane ("tris"), aqueous bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane ("bis-tris"), aqueous Triethanolamine (TEOA), aqueous potassium phosphate, aqueous 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES), or aqueous 2- [ [1, 3-dihydroxy-2- (hydroxymethyl) propan-2-yl ] amino ] ethane sulfonic acid (TES). In further embodiments, the buffer is independently selected from aqueous sodium tetraborate or aqueous potassium phosphate.

The aminotransferase described herein is the product of directed evolution from a commercial aminotransferase 1 as shown in SEQ ID NO. 1 herein (described in Yasuda, N.; cleactor, E.; kosjek, B.; yin, J.; xiang, B. Et al org. Process Res. Dev.2017,21,1851-1858; PCT publications WO2010/099501 and WO2013/036861, and U.S. Pat. No. 9,109,209). Enzyme 1 (SEQ ID NO: 1) is commercially available as a lyophilized cell-free lysate from Codexis, inc., redwood City, california. Transaminases are capable of catalyzing the stereoselective reduction of ketones to amines. As used herein, the transaminase may be a lyophilized cell-free lysate, a crude lysate, a whole cell protein, a cell-free lysate, or a purified enzyme.

In embodiments, the amino acid sequences of the aminotransferases described herein may have one or more amino acid differences compared to a reference aminotransferase amino acid sequence (enzyme 1-SEQ ID NO: 1). The amino acid sequence of the aminotransferase used in the present invention has substantial identity to SEQ ID NO. 1. In some embodiments, the amino acid sequence of the transaminase has 90% or more sequence identity to SEQ ID NO. 1. The aminotransferases described herein include, but are not limited to, those enzymes 1, 2, 3, 4, 5, 6, 7 or 8 having SEQ ID NO 1, 2, 3, 4, 5, 6, 7 or 8, respectively. Enzymes 2 through 8 are described in separate, commonly owned patent applications filed concurrently herewith on the same day, the entire contents of which are incorporated herein in their entirety.

Further examples of aminotransferases useful in the present invention are also described in PCT publications WO2010/099501, WO2012024104 and WO2013036861, in particular SEQ ID NOs 74 and 102 from WO2010/099501 and SEQ ID NO 206 from WO 2012/0241104, as well as other sequences encompassed by the disclosure. In some embodiments of the invention, the transaminase is selected from enzyme 1 (SEQ ID NO: 1) or enzyme 6 (SEQ ID NO: 6). In an embodiment of the invention, the aminotransferase described herein comprises enzyme 1 having the amino acid sequence set forth in SEQ ID No. 1:

MAFSADTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISIFDQGFYTSDATYTVFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAIVWVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFAAGDLIRAIQETHDRGFELPLLLDFDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAILADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:1)

in embodiments, the aminotransferase described herein includes enzyme 2 having the amino acid sequence set forth in SEQ ID No. 2:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISIFDQGFYTSDATYTVFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAIVWVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFAAGDLIRAIQETHDRGFELPLLLDFDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAILADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQYSEQ ID NO:2)

in embodiments, the aminotransferase described herein includes enzyme 3 having the amino acid sequence set forth in SEQ ID No. 3:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISVFDQGFYTSDATYTVFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVWVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFAAGDLIRAIQETHDRGFELPLLLDHDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIARSLGHEAILADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:3)

in embodiments, the aminotransferase described herein includes enzyme 4 having the amino acid sequence set forth in SEQ ID No. 4:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISVFDQGFYTSDATYTVFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVWVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFASIDLIRAIQETHDRGFELPLLLDHDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAMLADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:4)

in embodiments, the aminotransferase described herein includes enzyme 5 having the amino acid sequence set forth in SEQ ID No. 5:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISVFDQGFYTSDATYTAFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVWVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFASIDLIRAIQETHDRGFELPLLLDHDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAMLADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:5)

in embodiments, the aminotransferase described herein includes enzyme 6 having the amino acid sequence set forth in SEQ ID No. 6:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPVSEARISVFDQGFYASDATYTAFHVWNGNAFRLGDHIERLWSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVGVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFASIDLIRAIQETHDRGFELPLLLDHDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIARSLGHEAMLADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:6)

In embodiments, the aminotransferase described herein includes enzyme 7 having the amino acid sequence set forth in SEQ ID No. 7:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPVSEARISVFDQGFYASDATYTAFHVWNGNAFRLGDHIERLWSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVGVVITRGYSSTPLERDVTKHRPQVYMYAIPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFASIDLIRAIQETHDRGFELPLLLDHDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIARSLGHEAMLADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:7)

in embodiments, the aminotransferase described herein includes enzyme 8 having the amino acid sequence set forth in SEQ ID No. 8:

MAFSLDTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISVFDQGFYTSDATYTAFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVWVAITRGYSSTPLERDVTKHRPQVYMYAVPYQWIVPFDRIRDGVHLMVAQSVRRTPRSSIDPQVKNFAAGDLIRAIQETHDRGFELPLLLDHDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIARSLGHEAILADITPAELRDADEVLGCSTAGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSCLLTPVQY(SEQ ID NO:8)

in an embodiment of the invention, the inorganic base is independently selected from lithium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate or potassium phosphate. In further embodiments, the inorganic base is independently selected from sodium hydroxide or potassium hydroxide.

In an embodiment of the invention, the solvent is independently selected from 2-methyl THF, THF, MTBE, CPME, toluene, anisole, ethyl acetate, isopropyl acetate (IPAc) orC ₅ -C ₁₀ Alkyl alcohols such as n-butanol. In further embodiments, the solvent is selected from 2-methyl THF, MTBE or isopropanol. In some embodiments, the solvent is 2-methyl THF.

In some embodiments of the invention, the inorganic salt is selected from potassium carbonate, potassium phosphate, sodium carbonate, sodium sulfate, sodium hydroxide, or potassium hydroxide. In further embodiments, the inorganic salt is potassium carbonate or potassium phosphate.

In some embodiments of the invention, the acid is independently selected from aryl sulfonic acids such as, but not limited to, p-toluene sulfonic acid, 3-methyl-toluene sulfonic acid, 2-methyl-toluene sulfonic acid, benzene sulfonic acid, 2-naphthalene sulfonic acid, 2, 6-naphthalene sulfonic acid, and hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, phenylacetic acid, trimethylacetic acid, tetrafluoroboric acid, tetraphenylboric acid, maleic acid, fumaric acid, oxalic acid, or camphorsulfonic acid. In further embodiments, the acid is independently selected from p-toluene sulfonic acid or hydrochloric acid.

In some embodiments of the invention, the hydrochloric acid (in an organic solvent) is independently selected from, but not limited to, hydrochloric acid in 1, 4-dioxane, hydrochloric acid in diethyl ether, hydrochloric acid in CPME, or 37% aqueous hydrochloric acid. Alternatively, hydrochloric acid may also be prepared in situ by combining trimethylsilyl chloride or acetyl chloride with methanol or ethanol in an organic solvent.

In an embodiment of the invention, the base is independently selected from N, N-Diisopropylethylamine (DIPEA), triethylamine, DBU, DBN, DABCO, pyridine and pyridine derivatives such as 2, 6-lutidine and 2-methylpyridine, sodium hydroxide, potassium carbonate, potassium phosphate, potassium acetate, sodium carbonate or sodium acetate. In some embodiments, the base is DIPEA.

As used herein, the term "immobilized" or "immobilized" refers to covalent or non-covalent interactions between an enzyme and a solid support, encapsulation of the enzyme within a porous matrix or polymer, crosslinking of the enzyme to form insoluble aggregates, and similar techniques known to those skilled in the art. Such interactions include hydrogen bonding, ionic or electrostatic interactions, hydrophobic interactions, van der Waals interactions, electrostatic interactions, pi-pi interactions, hydrophilic interactions, coordination interactions, biospecific or affinity interactions, covalent interactions, combinations thereof, and the like.

The term "solid support" or "resin" refers to a solid material composition to which the support is immobilized. The solid material may be organic or inorganic. Physical properties and forms of solid materials include, but are not limited to, the following features: porosity, shape or morphology, shape factor (particles, monomers), bulk density, crosslink density, particle size, pore size distribution, particle or shape distribution, or other properties well known in the art

When the material for the solid support is inorganic, it forms a solid and consists of suitable minerals, ceramics, metals or other inorganic materials well known in the art. For example, inorganic solid materials include, but are not limited to: glass, silicon dioxide, hydroxyapatite, activated alumina, diatomaceous earthMagnesium silicate->Titanium dioxide, iron oxide, aluminosilicates, mixtures of these, and combinations thereof.

When the material for the solid support is organic, it forms a solid and consists of suitable organic polymers well known in the art. For example, organic solid materials include, but are not limited to: polymethacrylates, polyacrylates, polymethacrylamides, polyvinyl alcohols, polyacrylamides, polystyrenes, polypropylenes, polydivinylbenzenes, polymers formed from vinyl monomers, copolymers of hydroxyethyl methacrylate and divinylbenzene, copolymers of styrene and divinylbenzene, copolymers of acrylamide groups and vinyl monomers, copolymers of methacrylates and divinylbenzene, copolymers of phenol-formaldehyde, agarose, chitosan, cellulose, dextran, activated carbon, mixtures of these, and combinations thereof.

In other cases, the solid support material composition mayContains a combination of both organic and inorganic components and is formed as a solid comprised of inorganic and organic materials well known in the art. For example, such materials include, but are not limited to: glass, silicon dioxide, hydroxyapatite, activated alumina, diatomaceous earthMagnesium silicateTitanium dioxide, iron oxide, aluminosilicates, polymethacrylates, polyacrylates, polymethacrylamides, polyvinyl alcohols, polyacrylamides, polystyrenes, polypropylenes, polydivinylbenzenes, polymers formed from vinyl-based monomers, copolymers of hydroxyethyl methacrylate and divinylbenzene, copolymers of styrene and divinylbenzene, copolymers of acrylamido and vinyl monomers, copolymers of methacrylates and divinylbenzene, copolymers of phenol-formaldehyde, agarose, cellulose, dextran, activated carbon, mixtures of these, and combinations thereof.

The composition of the solid support may contain zero, one or more additional reactive species or ligands that impart the same or different functionalities to the resin surface to facilitate covalent or non-covalent interactions between the enzyme and the resin. For example, the reactive species or ligand includes, but is not limited to, at least one functional group selected from the group consisting of: strong ion exchangers, weak ion exchangers, multimodal ligands, modifiers and hydrophobic modifiers, and mixtures thereof. In more specific examples, the at least one ligand is selected from the group consisting of amines, quaternary ammonium, sulfonic acid, carboxylic acid, sulfopropyl, methylsulfonate, diethylaminoethyl, carboxymethyl, hexylamine, ethylamine, iminodiacetic acid, nitrilotriacetic acid, tricarboxymethylethylenediamine, (C) ₁ -C ₈ ) Alkyl, octadecyl, (C) ₃₀ ) Alkyl, butyldimethyl, biphenyl, pentafluoropropyl, cyanopropyl, aminopropyl, aryl, biotin, desthiobiotin, thiol, amide, alkoxy, acetal, ketal, ester, anhydride, carbonyl, nitrile, epoxy, carboxamide, ammonium, iodine, phenolImidazolyl, morpholinyl, pyridyl, phenyl, sulfide, disulfide, thioketone, acid chloride, imine, nitrile, anilino, nitro, halo, hydroxy, maleimide, iodoacetyl, triazine, sulfonate, alkylamine, diol, hydrazide, hydrazine, azlactone, aldehyde, diazo, carboxylate, azide, vinyl sulfone, epoxide, and oxirane groups, combinations thereof, and the like. In an even more specific example, at least one ligand selected from the previous group is linked to the resin by a homobifunctional or heterobifunctional spacer arm that is included to impart the same or different functionalities to the ligands of the previous group. Spacer arms are well known in the art and include, but are not limited to, (C ₂ -C ₂₀ ) Alkylene groups, aromatic groups, alkylaromatic groups, amide groups, amino groups, urea groups, urethane groups, ether groups, thioether groups, and the like, and combinations thereof. In even more specific examples, the spacer is selected from ethylenediamine, 1, 3-diamino-2-propanol, diaminodipropylamine (DADPA), cystamine, 1, 6-diaminohexane, O- (2-aminopropyl) -O' - (2-methoxyethyl) polypropylene glycol such as Jeffamine ^TM ED-600, non-hindered diamines such as Jeffamine ^TM EDR-148 polyetheramine, 4,7, 10-trioxa-1, 13-tridecanediamine, boc-N-amido-dPEG ₁₁ -amine, boc-N-amide-dPEG ₃ -amine, beta-alanine, aminocaproic acid, amino-PEG _n Carboxylic acid ester compounds (where N is between 2 and 20), succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, diglycolic acid, diglycolic anhydride, thioglycolic acid, N-succinimidyl S-acetylthioacetate, N-succinimidyl S-acetylthiopropionate, N-acetylhomocysteine thiolactone, 8-mercaptooctanoic acid, alpha-lipoic acid, lipoamide-PEG _n -carboxylate compounds (where n is between 2 and 20), thiol-PEG _n -carboxylate compounds (where n is between 2 and 20), NHS-PEG _n -one or more of acetylated thiol compounds, dithiothreitol (DTT) (where n is between 2 and 20), tetra (ethylene glycol) dithiol, hexa (ethylene glycol) dithiol, poly (ethylene glycol) dithiol, 2-mercaptoethylamine, adipic acid dihydrazide and carbohydrazide.

In one aspect, the transaminase is immobilized on or within a solid support.

In some embodiments, the transaminase is immobilized on the polymeric resin by a non-covalent bond. The polymeric resin may include, but is not limited to, polymethacrylates, polyacrylates, polystyrene-divinylbenzene or methacrylate-divinylbenzene, and the like. The resin may be selected from HPp2mgl、/>SP2mgl、/>PAD950、/>ECR1090F、/>XAD7HP、/>IB-ADS-1、/>HP-20, and the like.

In one embodiment, the transaminase is immobilized by non-covalent bonds to a resin comprising at least one reactive ligand or functional group selected from the group consisting of weak ion exchangers and strong ion exchangers. As described herein, the reactive ligand is selected from: quaternary ammonium groups, ammonium chloride, ammonium hydroxide, triethylammonium groups, dimethylammonium groups, primary amine groups, secondary amine groups, tertiary amine groups, sulfonic acid, carboxylic acid, diethylaminomethyl, carboxymethyl, quaternary ammonium, sulfopropyl, methylsulfonate, diethylaminoethyl, carboxymethyl, hexylamine, and ethylamine, or linear aliphatic primary amines. In one embodiment, the reactive ligand is selected from: hexylamine or ethylamine. In other cases, the resinSelected from:ECR8304、/>ECR8309、/>ECR8315、/>ECR8404、/>ECR8409、/>ECR8415、/>ECR1508、/>EC-HG、/>EC-EA、/>EC-HA、/>QA、/>HFA113、/>HFA403、/>EA403、/>HA403、/>QA403。

in some embodiments, the transaminase is immobilized on at least one resin comprising at least one chelating ligand selected from iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tricarboxymethylethylenediamine (TED), and mixtures thereof. In one embodiment, the at least one chelating ligand is NTA. In another embodiment, the transaminase is immobilized on at least one resin comprising at least one chelating ligand comprising at least one element selected from the group consisting of Fe ²⁺ 、Cu ²⁺ 、Mg ²⁺ 、Zn ²⁺ 、Co ²⁺ And Ni ²⁺ Is a metal ion of (a).

In one embodiment, the transaminase is immobilized via a non-covalent bond to a resin comprising at least one active ligand or functional group selected from the following: a non-ionizable ligand, an ionizable ligand, a hydrophobic ligand, a hydrophilic ligand, an aromatic ligand, a heterocyclic group, or a combination thereof. In one embodiment, the exposed ligand comprises a functional group ligand or functional group selected from the group consisting of: hydroxyl, hydrocarbyl, methyl, ethylbutyl, octyl, octadecyl, cyanopropyl, pentyl, hexyl, aryl, octadecyl, t-butyl, carboxylic acid, sulfonic acid, amide, alkyl mercaptan or amine, pyridyl, imidazolyl, or combinations thereof. For example, ligands include, but are not limited to: alkylamine, alpha, omega-diaminoalkane, phenylalkylamine, 2-amino-1-phenyl-1, 3-propanediol, N-benzyl-N-methylethanolamine, 4-mercaptoethylpyridine, 2-aminomethylpyridine, mercaptomethylimidazole, 2-mercaptobenzimidazole, tryptamine, 5-aminoindole, aminoalkylcarboxylic acid, N- (3-carboxypropionyl) aminodecylamine, N-pyromellitic-aminodecylamine, 2-benzamido-4-mercaptobutyric acid, 2-mercapto-5-benzimidazole sulfonic acid, 6-amino-4-hydroxy-2-naphthalene sulfonic acid, 2, 5-dimercapto-1, 3, 4-thiadiazole, hexylamine, and the like, and combinations thereof. In other cases, the resin is selected from: ECR8806、/>ECR1030、/>RB 1、/>RB2、/>RB3、/>BU113、BU114、/>PH400、/>EC-BU、/>IB-ADS-4, etc.

In some embodiments, the transaminase is immobilized via a covalent bond to at least one resin comprising at least one further reactive exposed ligand. In one embodiment, the exposed ligand comprises a functional group ligand or functional group selected from the group consisting of: aryl, biotin, desthiobiotin, thiol, amine, amide, alkoxy, acetal, ketal esters, anhydrides, carbonyl groups, nitriles, epoxy groups, carboxamides, ammonium, iodine, phenol, imidazolyl groups morpholinyl, pyridyl, phenyl, sulfide, disulfide, sulfhydryl ketone, acyl chloride imine, nitrile, anilino, nitro, halo, alkyl, hydroxy, maleimide,Iodoacetyl, triazine, sulfonate, alkylamine, diol, hydrazide, hydrazine, azlactone, aldehyde, diazo, carboxylate, azide, vinyl sulfone, epoxide, and oxirane groups, and combinations thereof. In other cases, the resin is selected from:IB-COV-1、IB-COV-2、/>ECR8204、/>EC-EP、/>EC-HFA、/>HFA403, etc. In another embodiment, the ligand may further react with a homobifunctional or heterobifunctional spacer to impart the same or different functionalities. Spacer arms are well known in the art and include, but are not limited to, (C ₂ -C ₂₀ ) Alkylene groups, aromatic groups, alkylaromatic groups, amido groups, amino groups, urea groups, urethane groups, ether groups, thioether groups, and the like, and combinations thereof. In one embodiment, the spacer arm is one or more selected from the group consisting of: ethylenediamine, 1, 3-diamino-2-propanol, diaminodipropylamine (DADPA), cystamine, 1, 6-diaminohexane, O- (2-aminopropyl) -O' - (2-methoxyethyl) polypropylene glycol such as Jeffamine ^TM ED-600, non-hindered diamines such as Jeffamine ^TM EDR-148 polyetheramine, 4,7, 10-trioxa-1, 13-tridecanediamine, boc-N-amido-dPEG ₁₁ -amine, boc-N-amide-dPEG ₃ -amine, beta-alanine, aminocaproic acid, amino-PEG _n -carboxylic acidsEster compounds (where N is between 2 and 20), succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, diglycolic acid, diglycolic anhydride, thioglycollic acid, N-succinimidyl S-acetylthioacetate, N-succinimidyl S-acetylthiopropionate, N-acetylhomocysteine thiolactone, 8-mercaptooctanoic acid, alpha-lipoic acid, lipoamide-PEG _n -carboxylate compounds (where n is between 2 and 20), thiol-PEG _n -carboxylate compounds (where n is between 2 and 20), NHS-PEG _n Acetylated thiol compounds, dithiothreitol (DTT) (where n is between 2 and 20), tetra (ethylene glycol) dithiol, hexa (ethylene glycol) dithiol, poly (ethylene glycol) dithiol, 2-mercaptoethylamine, adipic acid dihydrazide and carbohydrazide.

In another aspect, the transaminase is cross-linked with another enzyme. In particular cases, the transaminase is covalently crosslinked with another transaminase. In a more specific example, the crosslinking is mediated by an enzyme reacting with a homobifunctional or heterobifunctional spacer to impart the same or different functionalities. Spacer arms are well known in the art and include, but are not limited to, (C ₂ -C ₂₀ ) Alkylene groups, aromatic groups, alkylaromatic groups, amide groups, amino groups, urea groups, urethane groups, ether groups, thioether groups, and the like, and combinations thereof. In one embodiment, the spacer arm is one or more selected from the group consisting of: ethylenediamine, 1, 3-diamino-2-propanol, diaminodipropylamine (DADPA), cystamine, 1, 6-diaminohexane, O- (2-aminopropyl) -O' - (2-methoxyethyl) polypropylene glycol such as Jeffamine ^TM ED-600, non-hindered diamines such as Jeffamine ^TM EDR-148 polyetheramine, 4,7, 10-trioxa-1, 13-tridecanediamine, boc-N-amido-dPEG ₁₁ -amine, boc-N-amide-dPEG ₃ -amine, beta-alanine, aminocaproic acid, amino-PEG _n Carboxylic acid ester compounds (where N is between 2 and 20), succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, diglycolic acid, diglycolic anhydride, thioglycolic acid, N-succinimidyl S-acetylthioacetate, N-succinimidyl S-acetylthiopropionate, N-acetylhomocysteine thiolactone, 8-mercaptooctanoic acid, alpha-lipoic acid, lipoamide-PEG _n -carboxylate compounds (where n is between 2 and 20), thiol-PEG _n -carboxylate compounds (where n is between 2 and 20), NHS-PEG _n Acetylated thiol compounds, dithiothreitol (DTT) (where n is between 2 and 20), tetra (ethylene glycol) dithiol, hexa (ethylene glycol) dithiol, poly (ethylene glycol) dithiol, 2-mercaptoethylamine, adipic acid dihydrazide and carbohydrazide.

As used herein, an aminotransferase solvent refers to an organic solvent used to assist in the reduction of a ketone to an amine catalyzed by a transaminase. In an embodiment of the invention, the aminotransferase solvent is independently selected from 2-methyl THF, THF, MTBE, CPME, toluene, ethyl acetate, IPAc, DMSO, IPA, acetonitrile, DMF, NMP, or DMAc. In some embodiments, the aminotransferase is independently selected from 2-methyl THF or IPAc.

The aqueous aminotransferase solvent refers to an aminotransferase solvent to which water is added to increase the water content of the solvent. In some embodiments, the aqueous aminotransferase solvent is independently selected from aqueous 2-methyl THF or IPAc.

As used herein, the phrase "water saturation point" refers to the point at which a given solvent cannot absorb or dissolve more water. For example, for an organic solvent as a homogeneous solution, if the water saturation point is exceeded, two phases, an aqueous phase and an organic phase, will be observed.

In an embodiment of the present invention, a continuous reaction system is used. Examples of continuous reaction systems include, but are not limited to, packed Bed Reactors (PBR), fixed bed reactors, moving bed reactors, rotating bed reactors, fluidized bed reactors, slurry reactors, batch stirred tank reactors, continuous stirred tank reactors, membrane reactors, tube-in-tube reactors, monolithic reactors, microstructured reactors, fluidized bed reactors, and the like.

Immobilization may be performed using a variety of methods known to those skilled in the art. These methods include combining a transaminase and a solid support in a reactor configuration, including but not limited to a batch reactor or a continuous reaction system as defined above.

As used herein, weakly coordinating solvent refers to a solvent that may contain a weakly lewis basic heteroatom. In an embodiment of the invention, the weakly coordinating solvent or mixture thereof is independently selected from sulfolane, acetonitrile, DME, ethylene glycol or propylene carbonate. In particular embodiments, if sulfolane is used, the organic solvent is independently selected from anisole, toluene, chlorobenzene, dichloromethane, or 1, 2-dichloroethane.

In embodiments of the present invention, sulfolane may be replaced by other sulfones including, but not limited to, dialkyl sulfones, alkylaryl sulfones, or diaryl sulfones, such as isopropyl methyl sulfone, diisopropyl sulfone, di-n-butyl sulfone, diphenyl sulfone, bis (4-methylphenyl) sulfone.

As used herein, a silane reducing agent refers to a silane compound that can be used as a reducing agent. In an embodiment of the invention, the silane reducing agent is selected from the group consisting of triethylsilane, chlorodimethylsilane, phenylsilane, ethoxydimethylsilane, diethoxymethylsilane, triethoxysilane and 1, 3-tetramethyldisiloxane. In further embodiments, the silane reducing agent is selected from the group consisting of chlorodimethylsilane, phenylsilane, ethoxydimethylsilane, diethoxymethylsilane, and 1, 3-tetramethyldisiloxane. In other embodiments, the silane reducing agent is selected from chlorodimethylsilane, phenylsilane, or triethylsilane. In particular embodiments, the silane reducing agent is selected from chlorodimethylsilane or triethylsilane.

As used herein, a borane reducing agent refers to a borane compound that can be used as a reducing agent. In an embodiment of the invention, the borane reducing agent is selected from the group consisting of borane-tetrahydrofuran complex, borane-dimethyl sulfide complex, borane-N, N-diethylaniline complex, sodium borohydride (NaBH) ₄ ) Sodium borohydride with trifluoroacetic acid and sodium cyanoborohydride.

In an embodiment of the invention, the lewis acid is selected from boron trifluoride diethyl ether, boron trifluoride tetrahydrofuran complex, aluminum trichloride and trimethylsilyl triflic acid. In a further embodiment, the lewis acid is selected from boron trifluoride diethyl ether or trimethylsilyl triflic acid.

In an embodiment of the invention, the alcohol is independently selected from methanol, ethanol or isopropanol. In further embodiments, the alcohol is methanol.

As used herein, an antisolvent refers to a solvent that reduces the solubility of a solute. In an embodiment of the invention, the antisolvent is independently selected from the group consisting of 2-methyl THF, MTBE, CPME, THF, acetonitrile and C ₃ -C ₁₀ Alkyl alcohols such as, but not limited to, isopropyl alcohol, propyl alcohol, or n-butyl alcohol.

As used herein, a sealed reactor refers to a reaction vessel, such as an autoclave reactor, in which chemical reactions can be performed under pressure. Reactors capable of controlling reaction pressure include, but are not limited to, reactors equipped with pressure control valves or manual or automatic back pressure regulators.

In an embodiment of the present invention, the pressure is controlled between 3.5psig and 80 psig. Those skilled in the art will recognize that the reaction pressure depends on the vessel fill in the reactor, the reactor volume occupied by the reaction mixture.

In an embodiment of the invention, the organic solvent or mixture of organic solvents is independently selected from sulfolane, acetonitrile, DME, 2-methyl THF, THF, CPME, DCM, DCE or a combination thereof. In a specific embodiment, the organic solvent is DME.

In an embodiment of the present invention, the organic base is independently selected from tertiary amine bases such as, but not limited to, DIPEA, triethylamine, DABCO, DBU, and DBN. In a specific embodiment, the base is DIPEA.

In an embodiment of the invention, the first base is selected from methyllithium in diethyl ether, methyllithium in diethoxymethane, methyllithium-lithium bromide complex. In a specific embodiment, the first base is methyllithium in diethoxymethane.

In an embodiment of the invention, the second base is selected from n-butyllithium in hexane, n-butyllithium in heptane, n-butyllithium in toluene, n-butyllithium in cyclohexane, n-hexyllithium in hexane. In further embodiments, the second base is selected from n-butyllithium in hexane and n-hexyllithium in hexane.

As used herein, aprotic solvents refer to organic solvents in which acidic protons are absent. In an embodiment of the invention, the first and second aprotic solvents are independently selected from THF, 2-methyl THF, MTBE, diethyl ether, CPMe, DCM or DCE. In a specific embodiment, the first and second aprotic solvents are THF.

In an embodiment of the invention, the aqueous solution is independently selected from water, aqueous hydrochloric acid, aqueous hydrobromic acid, aqueous acetic acid, aqueous ammonium chloride, aqueous sodium chloride or a mixture of aqueous sodium chloride containing acetic acid. In further embodiments, the aqueous solution is selected from an aqueous acetic acid solution, an aqueous sodium chloride solution containing acetic acid, or an aqueous ammonium chloride solution.

As used herein, the reaction solvent refers to an organic solvent for assisting the reaction for preparing compound a. In an embodiment of the invention, the reaction solvent is independently selected from the group consisting of alcohol, 2-methyl THF, THF, MTBE, CPME, acetonitrile, dichloromethane, 1, 2-dichloroethane, DMF, NMP, or DMAc. In some embodiments, the reaction solvent is an alcohol independently selected from methanol, ethanol, isopropanol, or t-amyl alcohol. In further embodiments, the reaction solvent is ethanol.

As used herein, crystallization solvent refers to a solvent that reduces the solubility of solute compound a. In an embodiment of the invention, the crystallization solvent is independently selected from water, toluene, anisole, 2-methyl THF, THF, MTBE, CPME, DCM, hexane, heptane or alcohol. In some embodiments, the crystallization solvent is water.

In a first embodiment of the invention, the cofactor is PLP and the buffer is aqueous sodium tetraborate, and the transaminase is selected from the group consisting of enzyme 1 (SEQ ID NO: 1) and enzyme 6 (SEQ ID NO: 6).

In a second embodiment of the invention, the inorganic base is selected from sodium hydroxide or potassium hydroxide, the solvent is independently selected from 2-methyl THF or IPAc, the inorganic salt is potassium carbonate or potassium phosphate, and the acid is p-toluene sulfonic acid or hydrochloric acid.

In a third embodiment of the invention, the inorganic base is selected from sodium hydroxide or potassium hydroxide, the base is sodium hydroxide, the solvent is independently selected from 2-methyl THF and MTBE, and the acid is p-toluene sulfonic acid or hydrochloric acid.

In a fourth embodiment of the invention, the transferThe ammonia enzyme is selected from enzyme 1 or enzyme 6, the buffer solution is potassium phosphate aqueous solution, and the solid support is selected fromHp2mgL、/>SP2mgL、/>ECR8415F、/>ECR8415M polymeric resin, the aminotransferase solvent is independently selected from aqueous 2-methyl-THF or IPAc, and the acid is p-toluene sulfonic acid or hydrochloric acid.

In a fifth embodiment of the invention, the weakly coordinating solvent is a mixture of anisole and sulfolane, the silane reducing agent is selected from triethylsilane or phenylsilane, the lewis acid is boron trifluoride diethyl ether, and the alcohol is methanol.

In a fifth embodiment, after the addition of the alcohol in step c), a pharmaceutically acceptable acid may be added to obtain the desired pharmaceutically acceptable salt of compound 4.

In a sixth embodiment of the invention, the weakly coordinating solvent is a mixture of anisole and sulfolane, the silane reducing agent is selected from triethylsilane or phenylsilane, the lewis acid is boron trifluoride diethyl ether, and the alcohol is methanol.

In a seventh embodiment of the invention, the organic solvent is DME, the organic base is DIPEA, and the silane reducing agent is chlorodimethylsilane.

In an eighth embodiment of the invention, the first base is methyllithium in diethoxymethane, the aprotic solvent is THF, the second base is selected from n-butyllithium in hexane or n-hexyllithium in hexane, the aqueous solution is selected from aqueous acetic acid, aqueous sodium chloride solution or aqueous ammonium chloride solution containing acetic acid, and the alcohol is ethanol.

In a ninth embodiment of the invention, the base is DIPEA, the reaction solvent is ethanol, and the crystallization solvent is water.

In some cases, the transaminase is based on the amino acid sequence of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, etc., and may comprise an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference sequence of SEQ ID NOs 1, 2, 3, 4, 6, 7, or 8. These differences may be amino acid insertions, deletions, substitutions or any combination of such changes. In some cases, the amino acid sequence differences may comprise non-conservative, and combinations of non-conservative and conservative amino acid substitutions.

In some embodiments, such transaminase polypeptides are also capable of converting a substrate to a product at a diastereomeric ratio of at least 15:1. In some embodiments, such transaminase polypeptides are also capable of converting a substrate to a product in a diastereomeric ratio of at least 25:1. In some embodiments, such transaminase polypeptides are also capable of converting a substrate to a product at a diastereomeric ratio of at least 70:1.

In some embodiments, the transaminase polypeptide is highly stereoselective in that the polypeptide can reduce a substrate to a product at a diastereomeric ratio of greater than about 50:1, 60:1, and 70:1.

"amino acids" are referred to herein by their well-known three-letter symbols or one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Likewise, nucleotides may also be referred to by their commonly accepted single-letter codes.

Abbreviations used to genetically encode amino acids are conventional and are shown below: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).

"protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide linkage, whether in length or post-translational modification (e.g., glycosylation, lipidation, myristoylation, phosphorylation, ubiquitination, etc.). Included in this definition are D-and L-amino acids, as well as mixtures of D-and L-amino acids, and polymers comprising D-and L-amino acids, as well as mixtures of D-and L-amino acids.

When used in reference to, for example, a cell, nucleic acid or polypeptide, "recombinant" refers to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that does not occur in nature, or is the same as it, but is produced or derived from a synthetic material and/or is produced or derived by manipulation using recombinant techniques. Non-limiting examples include, inter alia, recombinant cells expressing genes not found in the native (non-recombinant) form of the cell or recombinant cells expressing native genes expressed at different levels.

"percent (%) sequence identity", "percent identity" and "percent identity" are used herein to refer to a comparison between polynucleotide sequences or polypeptide sequences, and are determined by comparing the two optimally aligned sequences within a comparison window, wherein for optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence within the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence. The percentage is calculated by determining the number of positions in the two sequences where the same nucleobase or amino acid residue is present, or where the nucleobase or amino acid residue is aligned with a notch to give the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to give the percentage of sequence identity. Optimal alignment and determination of percent sequence identity were performed using BLAST and BLAST 2.0 algorithms (see, e.g., altschul et al, 1990, J. Mol. Biol.215:403-410; and Altschul et al, 1977,Nucleic Acids Res.3389-3402). Software for performing BLAST analysis is publicly available through the national center for biotechnology information website.

Many other algorithms are available that provide a percentage identity of two sequences that function similarly to BLAST. Optimal alignment of sequences for comparison can be performed by, for example, the local homology algorithm of Smith and Waterman,1981, ADV.APPL.MATH.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J.MOL.BIOL.48:443, by the similarity search of Pearson and Lipman,1988,N USA 85:2444, by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in GCG Wisconsin Software Package) or by visual inspection (see generally, current Protocols in Molecular Biology, edited by F.M.Ausubel et al, current Protocols, a joint venture between Greene Publishing Associates, inc and John Wiley & Sons, inc., (supplement 1995) (Ausubel)). Alternatively, sequence alignment and determination of percent sequence identity may employ the BESTFIT or GAP program in GCG Wisconsin Software package (Accelrys, madison Wis.) using default parameters provided.

"substantial identity" means that a polynucleotide or polypeptide sequence has at least 80% sequence identity, preferably at least 85% sequence identity, more preferably at least 89% sequence identity, more preferably at least 95% sequence identity, and even more preferably at least 99% sequence identity over a comparison window of at least 20 residue positions, typically over a window of at least 30-50 residues, as compared to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence comprising deletions or additions, which total is 20% or less of the reference sequence over the comparison window. In particular embodiments applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80% sequence identity, preferably at least 89% sequence identity, more preferably at least 95% sequence identity or more (e.g., 99% sequence identity) when optimally aligned, e.g., by the programs GAP or BESTFIT using default GAP weights. Preferably, the different residue positions differ by conservative amino acid substitutions.

As used in the context of numbering of a given amino acid or polynucleotide sequence, "corresponding to," "referring to," or "relative to" refers to the numbering of residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is specified relative to a reference sequence, rather than by the actual numbered position of the residue within the given amino acid or polynucleotide sequence. For example, the residue matching between two sequences can be optimized by introducing gaps, thereby aligning a given amino acid sequence with a reference sequence. In these cases, the numbering of residues in a given amino acid or polynucleotide sequence is relative to the reference sequence to which it is aligned, although gaps exist.

"stereoselectivity" refers to the formation of one stereoisomer over another in a chemical or enzymatic reaction. The stereoselectivity may be partial, where one stereoisomer forms better than the other, or it may be complete, where only one stereoisomer forms. When stereoisomers are enantiomers, the stereoselectivity is referred to as the enantioselectivity, i.e., the fraction of one enantiomer (usually expressed as a percentage) in the sum of the two enantiomers. It is generally reported in the art alternatively as an Enantiomeric Excess (EE) (typically expressed as a percentage) calculated according to the formula [ primary enantiomer-secondary enantiomer ]/[ primary enantiomer + secondary enantiomer ]. When a stereoisomer is a diastereomer, the stereoselectivity is referred to as diastereoselectivity, i.e., the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), usually alternatively reported as Diastereomeric Excess (DE). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess.

"highly stereoselective" refers to a chemical or enzymatic reaction capable of converting a substrate to the corresponding product having a stereomeric excess of at least about 85%.

"conversion" refers to the enzymatic conversion of a substrate to the corresponding product. "percent conversion" refers to the percentage of substrate that converts to product over a period of time under the indicated conditions. Thus, for example, the "enzymatic activity" or "activity" of a polypeptide can be expressed as a "percent conversion" of a substrate to a product.

Immobilized enzyme formulations have a number of recognized benefits. For example, they may impart storage stability to the enzyme formulation, may increase the stability of the enzyme in organic solvents, and may assist in the removal of proteins from the reaction stream. "stable" refers to the ability of an immobilized enzyme to retain its structural conformation and/or activity under given conditions, e.g., in a solvent system comprising an organic solvent. In solvent systems containing organic solvents, the stable immobilized enzyme loses less than 10% of its activity per hour. In solvent systems containing organic solvents, the stable immobilized enzyme loses less than 9% of its activity per hour. Preferably, the stabilized immobilized enzyme loses less than 8% activity per hour in a solvent system comprising an organic solvent. Preferably, the stabilized immobilized enzyme loses less than 7% activity per hour in a solvent system containing an organic solvent. Preferably, the stabilized immobilized enzyme loses less than 6% of its activity per hour in a solvent system comprising an organic solvent. Preferably, the stabilized immobilized enzyme loses less than 5% activity per hour in a solvent system comprising an organic solvent. Preferably, the activity of the stabilized immobilized enzyme is less than 4% per hour in a solvent system comprising an organic solvent. Preferably, the stabilized immobilized enzyme loses less than 3% activity per hour in a solvent system comprising an organic solvent. Preferably, the stabilized immobilized enzyme loses less than 2% of its activity per hour in a solvent system comprising an organic solvent. Preferably, the stabilized immobilized enzyme loses less than 1% of activity per hour in a solvent system comprising an organic solvent.

As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides that are covalently linked together. Polynucleotides may consist entirely of ribonucleotides (i.e., RNA), entirely of 2 'deoxyribonucleotides (i.e., DNA), or consist of a mixture of ribonucleotides and 2' deoxyribonucleotides, and may include DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof. Although nucleosides are typically linked together by standard phosphodiester linkages, polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or the polynucleotide may include both single-stranded and double-stranded regions. Furthermore, while a polynucleotide typically consists of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and/or synthetic nucleobases, such as inosine, xanthine, hypoxanthine, and the like. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

As used herein, the terms "biocatalysis," "bioconversion," and "biosynthesis" refer to the use of enzymes to chemically react organic compounds.

As used herein, "deletion" refers to modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total amino acids, or up to 20% of the total amino acids making up the reference enzyme, while maintaining the enzymatic activity and/or maintaining the improved properties of the evolved enzyme. Deletions may be directed to the internal and/or terminal portions of the polypeptide. In various embodiments, the deletions may comprise contiguous segments or may be discontinuous. Deletions in the amino acid sequence are generally indicated by "-".

As used herein, "insertion" refers to modification of a polypeptide by adding one or more amino acids from a reference polypeptide. The insertion may be at an internal portion of the polypeptide, or at the carboxy or amino terminus. Insertions as used herein include fusion proteins known in the art. The insertions may be consecutive fragments of an amino acid or separated by one or more amino acids in the naturally occurring polypeptide.

The term "amino acid substitution set" or "substitution set" refers to a set of amino acid substitutions in a polypeptide sequence as compared to a reference sequence. The substitution set may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions.

As used herein, an "isolated polypeptide" refers to a polypeptide that is substantially isolated from other contaminants (e.g., proteins, lipids, and polynucleotides) with which it is naturally associated. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or by in vitro synthesis). The recombinant polypeptide may be present within the cell, in a cell culture medium, or prepared in various forms, such as a lysate or an isolated preparation. Thus, in some embodiments, the recombinant polypeptide may be an isolated polypeptide.

Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting. The transaminases are used in the form of lyophilized cell-free lysate powder. Solvents and reagents are commercially available and used as such unless otherwise indicated.

Scheme for the production of a semiconductor device

The general procedure for the synthesis of (2-chloro-4-phenoxyphenyl) (4- { [ (3R, 6S) -6- (hydroxymethyl) oxazin-3-yl ] amino } -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) methanone or compound A of the present invention is summarized in scheme 1 below. The overall process is a highly pooled synthesis, wherein compound a is prepared from the reaction between two intermediates: (2-chloro-4-phenoxyphenyl) (4-chloro-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) methanone, referred to herein as compound 7; and (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-amine, herein referred to as compound 4'.

Scheme one.

Wherein X is TsO or Cl

The present invention describes a non-protecting group synthetic route starting from (1 s,5 r) -6, 8-dioxabicyclo [3.2.1] oct-4-one, herein referred to as Cyrene or compound 2 (a bio-renewable material readily available by biomass pyrolysis) to give the appropriate salt of compound 4'.

It has been found that when isopropylamine is used as an amino donor, the recombinant transaminase is able to catalyze the conversion of compound 2 to (1 s,4r,5 r) -6, 8-dioxabicyclo [3.2.1] oct-4-amine (referred to herein as compound 3) with good diastereoselectivity.

These transamination reactions can be carried out in aqueous solution, as described herein, followed by isolation of the water-soluble compound 3 as a salt by a suitable work-up method. The ammonia transfer reaction in aqueous solution by removing the acetone byproduct by applying vacuum or nitrogen purge can increase the yield and purity of compound 3 because aldol side reactions between Cyrene (2) and acetone byproduct can be minimized by acetone removal.

Alternatively, the present invention describes a process for the preparation of compound 3 using an immobilized transaminase in an organic solvent. This approach greatly simplifies protein removal from water-soluble compound 3 and separation of water-soluble compound 3. Instead of cumbersome extraction and enzymatic denaturation procedures, compound 3 can be isolated directly as a salt from an organic solvent.

The transamination reaction using the immobilized transaminase can be carried out batchwise, in which case the immobilized transaminase can be filtered off after the reaction has been completed. Reactions using immobilized transaminase can also be carried out in rotating bed or rotating basket reactors, where the immobilized transaminase remains in the basket and the product can be collected without excessive filtration. Alternatively, the transamination reaction can be carried out in a continuous reaction system, in which the reaction stream is passed continuously over the immobilized transaminase and the product is collected. The latter approach further simplifies the handling of the immobilized transaminase and increases the efficiency of the transamination reaction by increasing the amount of product available per kg of enzyme.

The transamination reaction described herein yields compound 3 as the primary product, and (1S, 4S, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine (3 d) (cis-diastereomer of compound 3) as the secondary product

Those skilled in the art will recognize that the diastereomer ratio of compounds 3 and 3d depends on the selection of the transaminase, the selection of the transaminase solvent, the reaction temperature and the reaction time. The diastereomeric ratio of compounds 3 and 3d also depends, for a given enzyme, on whether the transaminase is used in an aqueous solvent or is immobilized on a solid support. For the transamination reactions described herein, the diastereomer ratio decreases when a given transaminase is immobilized on a solid support. This effect is demonstrated by, but not limited to, example 1A versus example 1C. During the separation of compound 3 as salt 3', a further increase in the diastereomer ratio is obtained. During the separation of compound 3 as salt 3', the addition of water can further increase the diastereomer ratio.

In another aspect, the invention describes the conversion of compound 3 or a salt thereof to compound 4'. This unprecedented conversion was found to occur by combining compound 3' with a borane or silane reducing agent and a lewis acid. In particular, the present invention describes a process for the preparation of compound 4' using triethylsilane and boron trifluoride diethyl etherate in the presence of sulfolane as an organic solvent. The combination of the reagent and solvent was found to produce diborane, the reactive reducing agent and triethylsilyl fluoride in situ. Due to the gaseous nature of the active reducing agent, it is beneficial to allow pressure build up during the 4' formation by conducting the reaction in a sealed reactor or reactor vessel capable of controlling the reaction pressure.

In addition, a process for preparing compound 7 from commercially available 5-bromo-4-chloro-7H-pyrrolo [2,3-d ] pyrimidine (referred to herein as compound 5) and methyl 2-chloro-4-phenoxybenzoate (referred to herein as compound 6) is described. The process for preparing compound 7 may be carried out batchwise as is well known to the person skilled in the art. As described herein, the process for preparing compound 7 can be carried out in a continuous reaction system to avoid the need for low temperatures.

Abbreviations (abbreviations)

Measurement:

g/L

mg

min

mL, mL mL

mM millimoles, 1mM is a concentration of one thousandth of a mole per liter

mmol millimoles, one thousandth of moles (amount of any chemical equivalent to the number of atoms in 12 g C-12).

N equivalent concentration, the gram-equivalent weight of the solution in solution, i.e. its molar concentration divided by the equivalence factor.

rpm revolution/min

uL, uL, uL microliters

CPME cyclopentyl methyl ether

DABCO 1, 4-diazabicyclo [2.2.2] octane

DBN 1, 5-diazabicyclo [4.3.0] non-5-ene

DBU 1, 8-diazabicyclo [5.4.0] undec-7-ene

DIPEA N, N-diisopropylethylamine

DCM dichloromethane

DCE 1, 2-dichloroethane

DMAc N, N-dimethylacetamide

DME 1, 2-dimethoxyethane

DMF N, N-dimethylformamide

iPrNH ₂ Isopropylamine

LB broth Luria-Bertani broth, commercially available nutrient-rich medium for bacterial culture and growth

NMP N-methyl-2-pyrrolidone

Boc t-Butoxycarbonyl group

IPA isopropyl alcohol

IPAc acetic acid isopropyl ester

KOH potassium hydroxide

2-MethylTHF 2-methyltetrahydrofuran

MTBE methyl tert-butyl ether

NaOH sodium hydroxide

Optical Density at OD600 600nm

PBR packed bed reactor

pCK110900 escherichia coli recombinant protein expression system

PEG polyethylene glycol

Ph phenyl

PLP 5' -pyridoxal phosphate

PMP 5' -pyridoxamine phosphate

TB Terrific broth, commercially available nutrient-rich medium for bacterial culture and growth

THF tetrahydrofuran

TMS trimethylsilyl group

TsOH para-toluene sulfonic acid

Throughout this disclosure, additional abbreviations may be defined.

Examples

Example 1A: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-toluene-1-sulfonate (3a)

Pyridoxal 5' -phosphate monohydrate (1 g) was dissolved in 500mL of buffer (0.1M sodium tetraborate with 1.56M isopropylamine, pH 9.5). Enzyme 1 (SEQ ID NO: 1) (10 g,20 wt%) was then added and dissolved at room temperature. (enzyme 1 is commercially available from CodexiS, inc. As a lyophilized cell-free lysate). (1S, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-one (2) (49.9 g,389 mmol) was then added and the mixture was heated to 33-37℃for 27h. During the reaction, vacuum and nitrogen flow were applied to remove the acetone formed. The pH was adjusted during the reaction to maintain the pH between 7.9 and 8.6 to give a solution of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine (3) (15:1 d.r.).

The reaction was adjusted to pH 13.4 with 50 wt% NaOH solution (28 mL). The mixture was cooled to 10-15 ℃ and potassium carbonate (83.3 g) was slowly added, keeping the temperature below 25 ℃. 2-MeTHF (300 mL) was added and the resulting mixture was stirred at room temperature overnight to denature the enzyme. The denatured protein solid was then removed by filtration and the filter cake was washed with 2-MeTHF (3X 50 mL). The aqueous filtrate was extracted with 2-MeTHF (75 mL). The combined organic layers were concentrated under reduced pressure to remove isopropylamine. 2-methyl THF is then added to the resulting (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine (3) solution to adjust the total volume to 200mL. Water was then added to adjust the water content to 3-3.6 wt.%. P-toluenesulfonic acid monohydrate (73.6 g,387 mmol) in 2-MeTHF (150 mL) was then added dropwise over 4 hours at 40 ℃. The resulting slurry was cooled to room temperature and aged for 2 hours. The batch was filtered. The filter cake was washed with wet (2 wt% water) 2-MeTHF (50 mL) and dry 2-MeTHF (50 mL). The wet cake was dried overnight at 50℃to give 3a (92.1 g,79% yield, 127:1 dr) as a solid. mp 227 ℃ (DSC); ¹ H NMR(DMSO-d ₆ ,500MHz)δ7.96(br s,3H),7.49(d,2H,J＝8.0Hz),7.13(d,2H,J＝8.0Hz),5.40(s,1H),4.61(s,1H),3.98(d,1H,J＝7.3Hz),3.68(m 1H),3.12(br m,1H),2.30(s,3H),2.04(m,2H),1.57(m,1H),1.43(m,1H)ppm； ¹³ C NMR(DMSO-d ₆ ,125MHz)δ145.6,138.5,128.7,125.9,99.1,73.5,67.5,47.9,23.7,21.3,19.1ppm。

example 1B: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-toluene-1-sulfonate (3a)

Pyridoxal 5' -phosphate monohydrate (500 mg) was dissolved in 250mL of buffer (0.1M sodium tetraborate with 1.56M isopropylamine, pH 9.8). Enzyme 1 (SEQ ID NO: 1) (3.75 g,15 wt%) was then added and dissolved at room temperature. (1S, 5R) -6, 8-dioxabicyclo [3.2.1 is then added]Octan-4-one (2) (25 g,195 mmol) and the mixture was heated to 35℃for 29h. In the opposite directionIn the process, vacuum and air flow are applied to remove the acetone formed. The stream was then cooled to room temperature and 10ml of 50 wt% NaOH was added to adjust the pH to 12. The reaction mixture was then concentrated under vacuum using a nitrogen sweep to remove isopropylamine while maintaining the pH at 12 using 50 wt% NaOH. To the reaction mixture at pH 12 was added a solution of di-tert-butyl dicarbonate (42.6 g,195 mmol) in THF (62 mL) by syringe pump at 25℃over 2.5 h. During the addition, the pH was monitored and another 50 wt% NaOH was added to maintain the pH above 10. The reaction was aged at 22-25 ℃ overnight. MTBE (250 mL) was then loaded and the mixture was stirred for 2h. The batch was filtered and the solids were washed with MTBE. The layers of the filtrate separated and the aqueous layer was extracted with MTBE (150 mL). The combined organic layers were washed with 15 wt% NaCl (100 mL) and concentrated. 2-MeTHF was added and the solution was filtered to remove solids to give a 2-MeTHF solution of compound 3c (82.7 g). Then, 2-MeTHF (320 mL) was added followed by p-toluenesulfonic acid monohydrate (100 g,581 mmol) and the reaction was heated to 35℃and aged overnight. The resulting slurry was then cooled to 21 ℃ and aged for 4h. The slurry was filtered, washed with 2-MeTHF (2 x 100 ml) and dried in vacuo to give 3a (43.5 g,74.0% yield, >30:1d.r.)。mp 227℃(DSC)； ¹ H NMR(DMSO-d ₆ ,500MHz)δ7.96(br s,3H),7.49(d,2H,J＝8.0Hz),7.13(d,2H,J＝8.0Hz),5.40(s,1H),4.61(s,1H),3.98(d,1H,J＝7.3Hz),3.68(m 1H),3.12(br m,1H),2.30(s,3H),2.04(m,2H),1.57(m,1H),1.43(m,1H)ppm； ¹³ C NMR(DMSO-d ₆ ,125MHz)δ145.6,138.5,128.7,125.9,99.1,73.5,67.5,47.9,23.7,21.3,19.1ppm。

As used herein, d.r. is used to designate "diastereomeric ratio". The first digit indicates the fraction of the product as (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine and the second digit indicates the fraction of the product as (1S, 4S, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine.

Example 1C: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-methylbenzene-1-sulfonate (3a)

Pyridoxal 5' -phosphate monohydrate (13.7 g) was added to 100mM potassium phosphate buffer (1.67L) pH 7.0, and the mixture was cooled to 4 ℃. The pH was adjusted to pH 6.95 using 1N KOH (58 g). Enzyme 1 (334.6 g) was then loaded over 1.25 hours. Then adding resinHP2MGL (2.50 kg, hydrated) and the mixture incubated at 4℃for 60h. The mixture was then diluted with 1.75L of water and stirred for 15min. />

The immobilized transaminase slurry is packed into a 1.7L jacketed column cooled to 5 ℃. The column was washed with water at a line speed of 2.5cm/min for 140min and then with an isopropanol/PEG-400/water mixture (88:10:2 wt%) at a line speed of 1.5-2.5cm/min for 150min. The column was warmed to 20 ℃ and an isopropanol-PEG-400:water mixture was flowed through the column until the effluent reached 18 ℃. The water saturated isopropyl acetate was then passed through the column at a linear velocity of 2.5cm/min for 135min. The column was heated to 60 ℃. (1S, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-one (2) (2.0 kg,15.6 mol), isopropylamine (1.153 kg,19.5mol,1.25 eq.) and 16.8L of water-saturated isopropyl acetate were combined at room temperature to prepare a reaction stream. The mixture was passed through the column with a residence time of 3h. The first 15h (5 column volumes) was split and then the product stream was collected for 72h to give a solution of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine (3) (13.7 kg,8.06 wt% 3,13:1 d.r.).

The mixture was then concentrated to about 3L (3 volumes relative to starting material) under reduced pressure, then rinsed with 5.5L isopropyl acetate to a final volume of 3L. The solution was then warmed to 35℃and a solution of p-toluenesulfonic acid monohydrate (1.65 kg,8.67 mol) in 2-MeTHF (6.5L) was added over 1.5 h. The resulting slurry was aged at 37-40 ℃ for 30min and then stirred at room temperature overnight. The slurry was filtered and the wet cake was washed with 2-MeTHF (2X 6L). The filter cake was put under vacuum/N ₂ Drying under purge for 18 hours to give 3a as a solid (2.4 kg,71% yield based on flow over 72 hours)The amount of compound 2, 35:1 dr). mp 227 ℃ (DSC); ¹ H NMR(DMSO-d ₆ ,500MHz)δ7.96(br s,3H),7.49(d,2H,J＝8.0Hz),7.13(d,2H,J＝8.0Hz),5.40(s,1H),4.61(s,1H),3.98(d,1H,J＝7.3Hz),3.68(m 1H),3.12(br m,1H),2.30(s,3H),2.04(m,2H),1.57(m,1H),1.43(m,1H)ppm； ¹³ C NMR(DMSO-d ₆ ,125MHz)δ145.6,138.5,128.7,125.9,99.1,73.5,67.5,47.9,23.7,21.3,19.1ppm。

example 1D: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-methylbenzene-1-sulfonate (3a)

Pyridoxal 5' -phosphate monohydrate (0.716 g) was added to 100mM potassium phosphate buffer (102 mL) pH 7.0, and the mixture was cooled to 4 ℃. The pH was adjusted to pH 6.80 using 1N KOH (4.63 g). Enzyme 6 (SEQ ID NO: 6) (17.9 g) was then added in 4 portions and the resulting mixture was aged for 1h. Then adding resinHP2MGL (150 g, hydrated) and the mixture incubated at 4 ℃ for 60h.

The immobilized transaminase slurry is then packed into a 10mm x 300mm glass jacketed column cooled to 4 ℃. The column was washed with 118mL of water at a linear speed of 2cm/min, then 175mL of an isopropanol/PEG-400/water mixture (86:10:4 wt%) at a linear speed of 2 cm/min. The column was heated to 20℃and then passed through it with 118mL of water saturated with 2-MeTHF at a linear velocity of 2 cm/min. The column was heated to 60 ℃. The reaction stream was prepared by combining (1S, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-one (2) (100 g,0.78 mol), isopropylamine (28.8 g,0.48mol,1.25 eq.) and 840mL of water saturated 2-MeTHF at room temperature. The mixture was passed through the column with a retention time of 90 minutes. The stream 71h was collected to give a solution of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine (3) (296.3 g,12.6 wt% 3, 34:1d.r.).

The mixture was then concentrated under reduced pressure to about 190mL (phaseFor starting material-5 volumes), then rinsed with 260mL of 2-MeTHF to a final volume of 190mL. Then 2.85mL of water (1.5%) was added followed by 75mL of 2-MeTHF. The wet solution was then heated to 35 ℃ and a solution of p-toluenesulfonic acid monohydrate (65.9 g, 277 mmol) in wet 2-MeTHF (190 mL) was added over 1.5 h. The resulting slurry was aged at 37-40 ℃ for 30min and then stirred at room temperature overnight. The slurry was filtered and the wet cake was washed with 2-MeTHF. The filter cake was put under vacuum/N ₂ Drying under purge for 18h gave 3a (79.1 g,76% yield based on the amount of compound 2 flowing through over 71 hours, 385:1 dr) as a solid. mp 227 ℃ (DSC); ¹ H NMR(DMSO-d ₆ ,500MHz)δ7.96(br s,3H),7.49(d,2H,J＝8.0Hz),7.13(d,2H,J＝8.0Hz),5.40(s,1H),4.61(s,1H),3.98(d,1H,J＝7.3Hz),3.68(m 1H),3.12(br m,1H),2.30(s,3H),2.04(m,2H),1.57(m,1H),1.43(m,1H)ppm； ¹³ C NMR(DMSO-d ₆ ,125MHz)δ145.6,138.5,128.7,125.9,99.1,73.5,67.5,47.9,23.7,21.3,19.1ppm。

example 1E: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-methylbenzene-1-sulfonate (3a)

Pyridoxal 5' -phosphate monohydrate (0.18 g) was added to 100mM potassium phosphate buffer (27.3 mL) at pH 6.7, and the mixture was cooled to 4 ℃. Enzyme 6 (SEQ ID NO: 6) (4.50 g) was then loaded and the resulting mixture was aged for 1h. Resin ECR8415M (37.8 g, hydrated) was then added and the mixture incubated at 4 ℃ for 60h.

The immobilized transaminase slurry is packed into a 10mm x 300mm glass jacketed column cooled to 4 ℃. The column was washed with 118mL of water at a linear speed of 2cm/min, then 170mL of an isopropanol/PEG-400/water mixture (86:10:4 wt%) at a linear speed of 2 cm/min. The column was heated to 20℃and then passed through it with 118mL of water saturated with 2-MeTHF at a linear velocity of 2 cm/min. The column was heated to 60 ℃. The reaction stream was prepared by combining (1S, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-one (2) (200 g,1.56 mol), isopropylamine (115 g,1.95 mol) and 1.68L of water saturated 2-MeTHF at room temperature. The mixture was allowed to flow through the column with a retention time of 45 minutes. The stream was collected for 95h to give a solution of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine (3) (255 g,10.2 wt% 3, 30:1d.r.).

The solution (108 g) was then concentrated to about 55mL (5 volumes relative to starting material) under reduced pressure, then rinsed with 77mL of 2-MeTHF to a final volume of 55mL. Then 0.82mL of water (1.5%) was added followed by 22mL of 2-MeTHF. The wet solution was then heated to 35 ℃ and a solution of p-toluenesulfonic acid monohydrate (19.44 g,102 mmol) in a mixture of 2-MeTHF (55 mL) and water (1.65 mL) was added over 1.5 h. The resulting slurry was aged at 37-40 ℃ for 30min and then stirred at room temperature overnight. The slurry was filtered and the wet cake was washed with 2-MeTHF. The filter cake was put under vacuum/N ₂ Drying under purge for 18 hours gave 3a (23.0 g,77% yield based on the amount of compound 2 flowing through over 95 hours, 94:1 dr) as a solid. mp 227 ℃ (DSC); ¹ HNMR(DMSO-d ₆ ,500MHz)δ7.96(br s,3H),7.49(d,2H,J＝8.0Hz),7.13(d,2H,J＝8.0Hz),5.40(s,1H),4.61(s,1H),3.98(d,1H,J＝7.3Hz),3.68(m 1H),3.12(br m,1H),2.30(s,3H),2.04(m,2H),1.57(m,1H),1.43(m,1H)ppm； ¹³ C NMR(DMSO-d ₆ ,125MHz)δ145.6,138.5,128.7,125.9,99.1,73.5,67.5,47.9,23.7,21.3,19.1ppm。

example 1F: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1] oct-4-amine hydrochloride (3 b)

Pyridoxal 5' -phosphate monohydrate (0.96 g,3.62 mmol) was added to 100mM potassium phosphate buffer (130.4 mL) pH 6.9, and the mixture was aged at 4℃for 0.25h. The solution pH was then readjusted to 6.9 by the addition of 1N KOH (6.58 mL). Enzyme 1 (24.19 g) was then loaded into the same vessel over about 0.5 hours, then rinsed with an additional 100mM potassium phosphate buffer, pH 6.9 (5.98 mL), and the mixture stirred for 2h. Then adding resin HP2MGL (171.77 g, hydrated) was then rinsed with 100mM potassium phosphate buffer pH 6.9 (11.97 mL) and the mixture was aged at 4℃for 48h. The resulting immobilized transaminase slurry mixture is diluted with cold (4-8 ℃) water (150 mL), stirred at 4℃for 15 minutes, and a portion is transferred to a sintered filter funnel. Subsequently, the mother liquor was removed by vacuum filtration to give a wet cake (about 30 g) of the immobilized enzyme resin. The wet cake was then slurry washed as follows: cold water was added to the filter and stirred for about 3 minutes, followed by removal of the mother liquor by filtration. This process was repeated three times, with a first wash with 120mL of water followed by a water wash with 90mL of water. Next, the immobilized transaminase was similarly slurry washed three times with 90mL of cold isopropanol/PEG-400/water mixture (88:10:2 wt%). Three similar slurry washes were then performed with 90mL of water saturated IPAc. Subsequently, the excess water-saturated IPAC was removed by gentle filtration to give a wet cake of immobilized transaminase resin, a portion of which was used in the subsequent reaction.

Immobilized transaminase resin (15.02 g) was reacted with (1S, 5R) -6, 8-dioxabicyclo [3.2.1]A mixture of octyl-4-ketone (2) (10.04 g,78 mmol), isopropyl amine (5.77 g,98 mmol) and water saturated isopropyl acetate (60 mL) was combined. Removing the internal retention web S2 RBR was placed in the reactor and rotated stepwise to about 400rpm to load resin into the RBR, with rotation maintained throughout the reaction. The vessel was heated to 60℃and aged for about 75 hours. At the end of the reaction, the reaction stream was cooled to room temperature and recovered by filtration to give a solution of compound 3 (68.97 g,10.2 wt% 3,10.5:1 d.r.).

The solution was concentrated under reduced pressure to a final volume of 13mL. 2-methyl THF (50 mL) was added and the solution was concentrated again under reduced pressure to a final volume of 13mL. 2-methyl THF (50 mL) and water (127 uL) were added and the solution was heated to 40 ℃. Hydrochloric acid solution (3M in CPME) was then added over 2 h. After aging for an additional 2h at 40 ℃, the resulting slurry was cooled to room temperature and aged overnight. ThenThe slurry was filtered and the solid was washed with 2-methyl THF. In vacuum/N ₂ The solid was dried under a purge to give 3b (6.57 g,49.3% yield, 38:1 dr) as a solid. ¹ H NMR(DMSO-d ₆ ,500MHz)δ8.26(br s,3H),5.45(s,1H),4.59(s,1H),3.97(d,J＝7.3Hz,1H),3.73–3.58(m,1H),3.06(s,1H),2.12–1.99(m,2H),1.68–1.52(m,1H),1.45–1.42(m,1H)ppm； ¹³ C NMR(DMSO-d ₆ ,125MHz)δ98.6，73.0，66.9，47.3，23.2，18.6ppm。

Example 1G: preparation of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine hydrobromide (3 c)

9.86 wt.% of (1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]A solution of oct-4-amine 3a (87.9 g,67.1 mmol) in 2MeTHF was loaded into a round bottom flask and then concentrated under reduced pressure. 2-MeTHF (50 mL) was added and the process repeated to remove water and isopropylamine. 2MeTHF (70 mL) was added followed by slow addition of aqueous HBr (9.1 mL,81.0 mmol) at 40 ℃. The biphasic mixture was then concentrated at 40 ℃ and then 2-MeTHF (50 mL) was added and the process repeated to remove excess water. The residue was dissolved in 2-MeTHF (50 mL) and heated to 40 ℃. Methanol (5 mL) was added and the solution was cooled to room temperature overnight. The resulting slurry was filtered and washed with 2-MeTHF (2X 20 mL). The isolated solid was recrystallized from 2MeTHF (60 mL) and methanol (6 mL) at 40℃and then from methanol (25 mL) at 60 ℃. 2MeTHF (10 mL) was added at room temperature to increase recovery. The resulting solid was filtered, washed with 2-MeTHF (2X 20 mL) and dried to give compound 3c (7.86 g,100 wt%, >200d.r.)。 ¹ H NMR(500MHz,DMSO-d6)δ8.09(s,3H),5.42(s,1H),4.60(d,J＝4.6Hz,1H),3.98(dd,J＝7.3Hz,J＝0.6Hz,1H),3.74-3.60(m,1H),3.16-3.03(m,1H),2.19-1.83(m,2H),1.67-1.50(m,1H),1.49-1.36(m,1H)ppm； ¹³ C NMR(126MHz,DMSO-d6)δ98.52,73.02,67.00,47.36,23.24,18.54。

Example 2A: preparation of (3R, 6S) -6-Hydroxymethyl) oxacyclohexane-3-amine 4-methylbenzene-1-sulfonate (4 a)

(1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-methylbenzene-1-sulfonate (3 a) (1.5 kg,4.98mol, 96:4dr) was suspended in a mixture of sulfolane (3.00L) and anisole (4.50L) at room temperature, and the mixture was placed under a nitrogen atmosphere and stirred using an overhead stirrer. Triethylsilane (3.98L, 24.89 mol) was then added followed by boron trifluoride diethyl ether (1.26L, 9.95 mol) and the mixture was heated to 40℃for about 18 hours. The resulting homogeneous solution was quenched with MeOH (2.00L) at a rate that maintained the internal temperature below 45 ℃ and then the subsequent quenched solution was heated to 60 ℃ for about 2 hours. To this mixture was added (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-amine 4-methylbenzene-1-sulfonate (4 a) (15 g,1 mol%) and the resulting seedbed was aged at 60℃for about 1 hour, then cooled to 20℃over 6 hours, and then aged at that temperature for an additional 18 hours. The slurry was filtered and the wet cake was washed with a 2:1v/v 2-MeTHF: meOH (3.00L) solution followed by a 9:1v/v 2-MeTHF: meOH (2X 3.00L) solution. Using N ₂ The filter cake was dried under vacuum for about 18 hours by purging to give 4a (1.14 kg,76% yield, >500:1dr)。 ¹ H NMR(MeOH-d ₄ ,500MHz)δ7.71(d,J＝8.1Hz,2H),7.25(d,J＝8.0Hz,2H),4.10(ddd,J＝10.8,4.4,2.3Hz,1H),3.53–3.47(m,2H),3.39–3.34(m,1H),3.33(t,J＝10.8Hz,1H),3.17(tt,J＝11.0,4.4Hz,1H),2.37(s,3H),2.17(dt,J＝12.3,2.8Hz,1H),1.77–1.73(m,1H),1.60(app.qd,J＝12.5,4.2Hz,1H),1.47–1.38(m,1H)ppm。 ¹³ C NMR(MeOH-d ₄ ,125MHz)δ143.5,141.8,129.9,126.9,79.2,69.2,65.6,48.0,28.6,27.0,21.3ppm。

Example 2B: preparation of (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-amine 4-methylbenzene-1-sulfonate (4 a)

(1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-methylbenzene-1-sulfonate (3 a) (8 g,26.5mmol,>99:1dr) and 2, 3-dihydrothiophene 1, 1-dioxide (16 mg,0.2 wt%) were suspended in a mixture of sulfolane (16 mL) and anisole (24 mL), and the mixture was placed under a nitrogen atmosphere and stirred using an overhead stirrer. Triethylsilane (23.3L, 146 mmol) was then added followed by boron trifluoride diethyl ether (8.1 mL,63.7 mmol). The vessel was sealed and equipped with a Pressure Control Valve (PCV) set at 50psig, and the mixture was then heated to 40 ℃ for about 20 hours. The resulting homogeneous solution was quenched with MeOH (16 mL) at a rate such that the internal temperature was maintained below 45 ℃ and then the subsequent quenched solution was heated to 60 ℃ for about 2 hours. To this mixture was added (3 r,6 s) -6- (hydroxymethyl) oxacyclohexane-3-amine 4-methylbenzene-1-sulfonate (4 a) (80 mg,1 wt%) and the resulting seedbed was aged at 60 ℃ for about 1 hour, then cooled to 20 ℃ over 6 hours, then aged at that temperature for an additional 18 hours. The slurry was filtered and the wet cake was washed with 2:1 v/v 2-MeTHF in MeOH (16 mL) and then with 9:1 v/v 2-MeTHF in MeOH (2X 16 mL). Using N ₂ The filter cake was dried under vacuum for about 18 hours with a purge to give 4a (6.0 g,74% yield,>500:1dr)。

example 2C: preparation of (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-amine hydrochloride (4 b)

(1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Octyl-4-amine 4-methylbenzene-1-sulfonate (3 a) (10.0 g,33.2mmol, 96:4dr) was suspended in a mixture of sulfolane (20 mL) and anisole (30 mL), and the mixture was placed under nitrogen and stirred using an overhead stirrer. Triethylsilane (15.9 ml,100 mmol) was then added followed by boron trifluoride diethyl etherate (8.4 ml,66.4 mmol) and the mixture was heated to 50 ℃ for about 18 hours. The resulting homogeneous solution was treated with MeOH (10 mL) to maintain the internal temperature below 45℃at a rateQuench, then heat the subsequent quench solution to 60 ℃ for about 2 hours. HCl (4 m in dioxane, 12.5 ml) was slowly added, seeds of (3 r,6 s) -6- (hydroxymethyl) oxacyclohexane-3-amine hydrochloride (4 b) (112 mg,2 wt%) were added to the mixture, then slowly cooled to room temperature and aged for about 18 hours. The slurry was filtered and the wet cake was washed with 2-MeTHF (2X 20 mL). Using N ₂ The filter cake was dried under vacuum for about 18 hours, giving 4b (4.87 g,88% yield, >100:1dr)。 ¹ H NMR(MeOH-d ₄ ,500MHz)δ4.13(ddd,J＝10.8,4.4,2.3Hz,1H),3.55-3.48(m,2H),3.43-3.38(m,1H),3.37(t,J＝10.8Hz,1H),3.21(ddd,J＝15.3,11.0,4.1Hz,1H),2.21(dt,J＝12.2,2.8Hz,1H),1.79(app.dq,J＝13.4,3.5,3.0Hz,1H),1.64(app.qd,J＝12.5,4.2Hz,1H),1.51-1.42(m,1H)ppm。 ¹³ C NMR(MeOH-d ₄ ,125MHz)δ79.3,69.2,65.6,48.0,28.7,27.1ppm。

Example 2D: preparation of (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-amine hydrochloride (4 b)

(1S, 4R, 5R) -6, 8-dioxabicyclo [3.2.1]Oct-4-amine 4-methylbenzenesulfonate 3a (10 g,33.2mmol,>100:1 dr) was suspended in DME (50 mL), and the mixture was placed under nitrogen and stirred using a magnetic stirrer. N, N-diisopropylethylamine (5.80 mL,33.2mmol,1.0 eq) was then added in one portion and the batch was stirred at room temperature for about 5 minutes. Chlorodimethylsilane (14.74 mL,133mmol,4.0 eq.) was added slowly followed by trimethylsilyl triflate (12.59 mL,69.7mmol,2.1 eq.) and the mixture was heated to 30℃for about 20 hours. The resulting homogeneous solution was quenched with water (4.18 mL,232mmol,7 eq.) and the phases separated. The bottom layer was cooled to room temperature and then seeded with ((2 s,5 r) -5-aminotetrahydro-2H-pyran-2-yl) methanolic hydrochloride (100 mg,1 wt%). 2-methyltetrahydrofuran (30 mL) was then added to the resulting slurry over about 3 minutes, and the mixture was aged at room temperature for about 18 hours. The slurry was filtered and the wet washed with 2-MeTHF (2X 20 mL)And (5) a filter cake. Using N ₂ The filter cake was dried under vacuum for about 18 hours, giving 4b (3.13 g,56% yield, >100:1dr)。 ¹ H NMR(MeOH-d4,500MHz)δ4.13(ddd,J＝10.8,4.4,2.3Hz,1H),3.55-3.48(m,2H),3.43–3.38(m,1H),3.37(t,J＝10.8Hz,1H),3.21(ddd,J＝15.3,11.0,4.1Hz,1H),2.21(dt,J＝12.2,2.8Hz,1H),1.79(app.dq,J＝13.4,3.5,3.0Hz,1H),1.64(app.qd,J＝12.5,4.2Hz,1H),1.51-1.42(m,1H)。 ¹³ C NMR(MeOH-d4,125MHz)δ79.3,69.2,65.6,48.0,28.7,27.1。

3. Synthesis of Compound 7

A. Synthesis without lithium bromide additive

Example 3A: (2-chloro-4-phenoxyphenyl) (4-chloro-7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) methanone (7)

To 5-bromo-4-chloro-7H-pyrrolo [2,3-d ] at-35 DEG C]To a slurry of pyrimidine (5) (5.0 g,21.5 mmol) in THF (60 mL) was added methyllithium (1.6M, 14.1mL,22.6 mmol) in diethyl ether, maintaining the temperature below-30deg.C. The mixture was then cooled to below-65 ℃ and n-butyllithium (2.7 m,8.4ml,22.6 mmol) in hexane was added dropwise, the temperature was kept below-60 ℃ and the resulting slurry was stirred for 1 hour. A solution of methyl 2-chloro-4-phenoxybenzoate (6) (5.71 g,21.72 mmol) in THF (10 mL) was then added dropwise, maintaining the temperature below-60 ℃. The resulting mixture was stirred at this temperature for an additional 1.5 hours, then quenched by the addition of acetic acid (2.7 mL,47.3 mmol) and then warmed to room temperature. The mixture was washed with water (37.5 mL) and the layers separated. THF was removed under vacuum and replaced with ethanol (70 mL). The resulting slurry was aged at room temperature, filtered, and the solid was washed with ethanol (25 mL) and dried to give 7 (6.71 g,81% yield). ¹ H NMR(500MHz,DMSO-d ₆ )δ13.40(s,1H),8.74(s,1H),8.12(s,1H),7.59(d,J＝8.5Hz,1H),7.48(tt,J＝7.5Hz,J＝2.2Hz,2H),7.29–7.23(tt,J＝7.5Hz,J＝1.1Hz,1H),7.22–7.14(m,3H),7.01(dd,J＝8.5Hz,J＝2.4Hz,1H)ppm。 ¹³ CNMR(126MHz,DMSO-d ₆ )δ185.9,159.1,155.1,154.0,1511.9,151.8,138.4,134.0,132.1,131.8,130.4,124.8,119.7,119.2,116.2,115.4,113.9ppm。

B. Synthesis of Compound 7 with lithium bromide in a flow reactor

Example 3B: (2-chloro-4-phenoxyphenyl) (4-chloro-7H-pyrrolo [2, 3-d) ]Pyrimidin-5-yl) methanone (7)

Preparation of solution a: 5-bromo-4-chloro-7H-pyrrolo [2,3-d ] pyrimidine (5) (1.20 kg,5.16 mol) and lithium bromide (1.57 kg,18.12 mol) were dissolved in THF (27L). The mixture was stirred until a homogeneous solution was produced. The water content of the resulting solution was 250ppm (7.2 mol%). The mixture was cooled to-27℃and methyllithium (2.88M, 1.92L,5.53 mol) in diethoxymethane was added dropwise. The mixture was warmed to room temperature to give a 0.173M solution of 5.

Preparation of solution B: solution B contained n-butyllithium (1.435M) in commercially available hexane.

Preparation of solution C: methyl 2-chloro-4-phenoxybenzoate (6) (1.356 kg,5.162 mol) was dissolved in THF (9.183 kg).

Preparation of solution D: acetic acid (1.11L) was added to a 10 wt% aqueous NaCl solution (9.0L).

Solution A containing compound 5 (0.173M, 3.90 kg/h) and solution B containing n-butyllithium in hexane (1.435M, 5.91 g/min) were pre-cooled and combined in a 56.09mL stainless steel Plug Flow Reactor (PFR) at 27℃for a total of 0.727min. The resulting AB stream was then combined with solution C (0.4638M, 1.53 kg/h) containing compound 6 in 215mL PFR for 2.074min, then quenched with solution D. The resulting two-phase mixture was collected and the phases separated.

The organic layer containing 1.38kg 7 was heated to 30 ℃ and concentrated to 11L under vacuum. The resulting slurry was heated to 45-50 ℃ and aged for 30 minutes. Ethanol was added over 3 hours: water (2/3, v/v) mixture (17.8L) while maintaining the internal temperature at 45-50 ℃. The slurry was allowed to cool to room temperature and stirred overnight. The mixture was filtered, the solid was washed three times with ethanol (4.1L) and dried under vacuum with a stream of nitrogen to give compound 7 (1.25 kg,75% yield, based on the amount of compound 5 flowing through, 63% yield, based on the amount of 5 loaded). ¹ H NMR(500MHz,DMSO-d ₆ )δ13.40(s,1H),8.74(s,1H),8.12(s,1H),7.59(d,J＝8.5Hz,1H),7.48(tt,J＝7.5Hz,J＝2.2Hz,2H),7.29–7.23(tt,J＝7.5Hz,J＝1.1Hz,1H),7.22-7.14(m,3H),7.01(dd,J＝8.5Hz,J＝2.4Hz,1H)ppm。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ185.9,159.1,155.1,154.0,1511.9,151.8,138.4,134.0,132.1,131.8,130.4,124.8,119.7,119.2,116.2,115.4,113.9ppm。

Example 4A: (2-chloro-4-phenoxyphenyl) (4- { [ (3R, 6S) -6- (hydroxymethyl) oxazin-3-yl]Ammonia Radical } -7H-pyrrolo [2,3-d ]]Pyrimidin-5-yl) methanones (A)

(2-chloro-4-phenoxyphenyl) (4-chloro-7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) methanone (7) (0.5 kg,1.3 mol) and (3R, 6S) -6- (hydroxymethyl) oxa-n-3-amine 4-methylbenzene-1-sulfonate (4 a) (433 g,1.43 mol) were slurried in ethanol (4L, 8V). N, N-Diisopropylethylamine (DIPEA) (420 g,3.25 mol) was added, and the reaction mixture was heated to 80℃and stirred for 12 hours. The reaction was cooled to 55-65℃and water (2L, 2V) was added. The solution was passed through a fine filter and then rinsed with ethanol/water=4/1 (volume/volume, 0.5l,1 v). The filtered solution was then cooled to 35±5 ℃, product seeds (1 g,0.2 wt%) were added, and the mixture was aged for at least 15min. Additional water (5.5L, 11V) was added over 10 hours and the mixture was aged at 35.+ -. 5 ℃ for 3-5 hours and then cooled to 20 ℃ over at least 1 hour. Acetic acid (37 mL,0.5 eq.) was added dropwise at 25℃until the pH reached 6-8. The slurry was then aged at 20 ℃ for at least 3-5 hours until the desired supernatant concentration was obtained. The slurry was filtered and the product was washed 3 times with 2:3 (v/v) ethanol: water (1L). Drying the wet filter cake under vacuum and nitrogen flow at a temperature of less than or equal to 50 ℃ to obtain the chemical Compound a (557 g,89% yield). ¹ H NMR(600MHz,DMSO-d ₆ )δ12.78(s,1H),8.63(d,J＝7.1Hz,1H),8.28(s,1H),7.65(s,1H),7.59(d,J＝8.4Hz,1H),7.52–7.45(m,2H),7.26(tt,J＝7.3,1.1Hz,1H),7.22–7.17(m,3H),7.03(dd,J＝8.4,2.4Hz,1H),4.69(s,1H),4.22–4.13(m,2H),3.48–3.42(m,1H),3.41–3.34(m,2H),3.18–3.11(m,1H),2.25–2.17(m,1H),1.79(dq,J＝15.1,3.2Hz,1H),1.60(qd,J＝12.3,3.9Hz,1H),1.41(tdd,J＝13.3,10.5,3.9Hz,1H)ppm。 ¹³ C NMR(151MHz,D DMSO-d ₆ )δ189.65,158.59,156.25,155.19,154.10,152.71,136.08,133.32,131.17,130.78,130.27,124.62,119.59,119.04,116.35,116.11,100.54,77.69,69.82,64.24,46.18,29.29,27.06ppm。

Example 4B: (2-chloro-4-phenoxyphenyl) (4- { [ (3R, 6S) -6- (hydroxymethyl) oxazin-3-yl]Ammonia Radical } -7H-pyrrolo [2,3-d ]]Pyrimidin-5-yl) methanones (A)

(2-chloro-4-phenoxyphenyl) (4-chloro-7H-pyrrolo [2, 3-d) under nitrogen atmosphere]Pyrimidin-5-yl) methanone (7) (1.0 g,2.6 mmol) and (3R, 6S) -6- (hydroxymethyl) oxacyclohexane-3-ammonium chloride (4 b) (0.48 g,2.86 mmol) were slurried in ethanol (10 mL, 10V). N, N-Diisopropylethylamine (DIPEA) (0.84 g,6.51 mmol) was added and the reaction mixture was heated to 80℃and stirred for 18 hours. The reaction was cooled to 45-55deg.C and water (2.5 mL, 2.5V) was added. The reaction was further cooled to 35±5 ℃. An aqueous solution (7.5 ml,7.5 v) of acetic acid (0.094 g,0.6 eq) was added over 5 hours and the mixture was aged at 35±5 ℃ for 1-2 hours and then cooled to 20 ℃ over at least 1 hour. The slurry was then aged at 20 ℃ overnight for 10-15 hours until the desired supernatant concentration was obtained. The slurry was filtered and the product was washed twice with 1/1 (v/v) ethanol: water (5 mL). The wet cake was dried under vacuum and nitrogen flow at 50℃or lower to give Compound A (1.055 g,85% yield). ¹ H NMR(600MHz,DMSO-d ₆ )δ12.78(s,1H),8.63(d,J＝7.1Hz,1H),8.28(s,1H),7.65(s,1H),7.59(d,J＝8.4Hz,1H),7.52–7.45(m,2H),7.26(tt,J＝7.3,1.1Hz,1H),7.22–7.17(m,3H),7.03(dd,J＝8.4,2.4Hz,1H),4.69(s,1H),4.22–4.13(m,2H),3.48–3.42(m,1H),3.41–3.34(m,2H),3.18–3.11(m,1H),2.25–2.17(m,1H),1.79(dq,J＝15.1,3.2Hz,1H),1.60(qd,J＝12.3,3.9Hz,1H),1.41(tdd,J＝13.3,10.5,3.9Hz,1H)ppm. ¹³ C NMR(151MHz,D DMSO-d ₆ )δ189.65,158.59,156.25,155.19,154.10,152.71,136.08,133.32,131.17,130.78,130.27,124.62,119.59,119.04,116.35,116.11,100.54,77.69,69.82,64.24,46.18,29.29,27.06ppm。

Example 5: enzyme preparation as lyophilized cell-free lysate powder

20. Mu.l of glycerol stock of E.coli W3110 strain cells carrying the plasmid encoding the aminotransferase in the pCK110900 vector were inoculated into 25mL of LB broth supplemented with 34. Mu.g/mL of chloramphenicol and 1% (W/v) of glucose. Cells were grown at 30 ℃/250RPM for 18 hours until saturated. The next day, a 2.8L flask containing 1L of TB supplemented with 34 micrograms/mL chloramphenicol and 0.1mM pyridoxine was sub-cultured with overnight saturated culture to an initial OD 600.05. The cultures were grown at 30℃at 250RPM for 2.5 hours until an OD600 of 0.6-0.8 was reached. Protein production was induced with 1mM IPTG at 30℃at 250rpm for 20 hours. Cells were pelleted by centrifugation and the supernatant discarded. The cell pellet was flash frozen in liquid nitrogen, thawed and resuspended in (per gram pellet) 5mL ice-cold 50mM triethanolamine-HCl buffer (pH 7.5, supplemented with 0.1mM PLP). The suspension was shaken at 20 ℃ for 30 min, then the cells were cooled on ice and then destroyed by high pressure homogenization (16,000PSI). The resulting lysate was then clarified by centrifugation at 10,000Xg for 45 minutes at 4 ℃. After centrifugation, the supernatant was frozen and lyophilized. This protocol can be followed to prepare any transaminase of SEQ ID NO. 1 to SEQ ID NO. 8.

It will be appreciated that various of the above-described and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. It will also be appreciated that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims (25)

1. A process for preparing compound 3, or a pharmaceutically acceptable salt thereof,

which comprises the following steps:

a) Mixing cofactors in a buffer or water with isopropylamine and a transaminase to produce a reagent mixture; and

b) Adding compound 2 to the reagent mixture

To produce a solution comprising compound 3.

2. The method of claim 1, further comprising the step of:

a) Adding an inorganic base to adjust the pH of the solution comprising compound 3 to about 12 to about 14;

b) Adding a solvent and an inorganic salt to produce a resulting two-phase mixture comprising compound 3, the two-phase mixture comprising an organic layer and an aqueous layer;

c) Separating said organic layer from said resulting biphasic mixture with a solvent;

d) Combining the organic layer of the resulting two-phase mixture with a solution of an acid in a solvent to produce a slurry comprising compound 3

Wherein HX is selected from pharmaceutically acceptable acids; and

e) The slurry was filtered to obtain compound 3' as a solid.

3. The process of claim 2, wherein HX is p-toluenesulfonic acid, further comprising, in step d, combining a solution of p-toluenesulfonic acid in a solvent with an organic layer of the resulting two-phase mixture to produce a slurry comprising compound 3 a; and the slurry was filtered to obtain compound 3a as a solid.

4. Method for preparing compound 3

Wherein HX is a pharmaceutically acceptable acid,

the method comprises the following steps:

a) Combining the transaminase with a buffer or water and a solid support, followed by incubation to prepare an immobilized transaminase;

b) Washing the immobilized transaminase with a buffer or water;

c) Washing with an aminotransferase solvent;

d) Combining compound 2 with isopropylamine and a transamination solvent to provide a reaction stream;

e) Combining the reaction stream with the immobilized transaminase to produce a slurry comprising compound 3;

f) Separating the slurry comprising compound 3 from the immobilized transaminase to produce a solution comprising compound 3;

g) Combining the solution comprising compound 3 with a solution of an acid in an aminotransferase solvent to produce a slurry comprising compound 3'; and

h) The slurry was filtered to obtain compound 3' as a solid.

5. The method of claim 4, further comprising the step of: the immobilized transaminase is washed with water and then with an isopropanol-PEG-400-water mixture.

6. The method of claim 4, further comprising the step of: combining compound 2 with isopropylamine in an aqueous transamination solvent to produce the reaction stream in step d).

7. The method of claim 4, further comprising performing steps b), c) and e) in a continuous reaction system, wherein the buffer or water, the transamination solvent, and the reaction stream are continuously passed through the immobilized transaminase.

8. The method of claim 7, wherein the continuous reaction system is a Packed Bed Reactor (PBR).

9. The method of claim 4, further comprising the step of:

a) Distilling the solution comprising compound 3 to remove isopropylamine and water to produce a resulting solution comprising compound 3; and

b) Water is added to the resulting solution comprising compound 3 to adjust the water content to about 0 to the approximate water saturation point of the transamination solvent.

10. The method of claim 4, wherein HX is p-toluene sulfonic acid, further comprising the steps of: combining the solution comprising compound 3 with a solution of p-toluene sulfonic acid in a transamination solvent to produce a slurry comprising compound 3a,

and filtering the slurry to isolate 3a as a solid.

11. A process for preparing compound 4',

wherein HX is a pharmaceutically acceptable acid,

the method comprises the following steps:

a) Adding compound 3' to a weakly coordinating solvent;

b) Adding a silane reducing agent or borane reducing agent and a lewis acid and heating to about 30 to about 70 ℃ to produce a resulting solution;

c) Adding an alcohol to produce a solution comprising compound 4';

d) Cooling the solution to produce a slurry comprising compound 4'; and is also provided with

e) The slurry was filtered to obtain compound 4' as a solid.

12. The method of claim 11, further comprising the step of: after the addition of the alcohol in step c), a pharmaceutically acceptable acid is added to obtain a pharmaceutically acceptable salt of compound 4'.

13. The method of claim 11, wherein HX is p-toluene sulfonic acid, further comprising the steps of: adding compound 3a to a weakly coordinating solvent in step a), and producing a solution comprising compound 4a in step c)

14. The process of claim 13, wherein the weakly coordinating solvent in step a) is a mixture of anisole and sulfolane, the silane reducing agent in step b) is triethylsilane, and the lewis acid in step b) is boron trifluoride diethyl ether, and the ratio of triethylsilane to boron trifluoride diethyl ether is less than 3:1.

15. The process according to claim 14, wherein 2, 3-dihydrothiophene 1, 1-dioxide or 2, 5-dihydrothiophene 1, 1-dioxide is also added in step a).

16. A process according to claim 14, wherein in step b) triethylsilane and boron trifluoride diethyl etherate are added and heated to about 30 to about 70 ℃ in a reactor to produce a resulting solution, wherein the reactor is a sealed reactor or a reactor capable of controlling reaction pressure.

17. A process for preparing compound 4b comprising the steps of:

a) Adding compound 3' to an organic solvent;

b) Adding an organic base to produce a reaction mixture;

c) Adding a silane reducing agent and trimethylsilyl triflate to the reaction mixture to produce a resulting solution;

d) Adding water to the resulting solution to produce a bilayer mixture comprising compound 4b, the bilayer mixture having a top layer and a bottom layer, and separating the bottom layer from the bilayer mixture comprising compound 4 b;

e) Cooling the bottom phase to produce a resulting slurry; and is also provided with

f) The slurry was filtered to obtain compound 4b as a solid.

18. A process for the preparation of compound 7,

which comprises the following steps:

a) A solution of a first base is added to a slurry of compound 5 in a first aprotic solvent,

to produce a resulting mixture;

b) Combining a solution of a second base with the resulting mixture;

c) A solution of compound 6 in a second aprotic solvent,

to produce a solution comprising compound 7;

d) Combining an aqueous solution with the solution comprising compound 7 to produce a two-phase mixture comprising compound 7, the two-phase mixture comprising an aqueous layer and an organic layer;

e) Separating the organic layer from the two-phase mixture comprising compound 7;

f) Adding an alcohol, water or alcohol-water mixture to the organic layer to produce a resulting slurry comprising compound 7; and is also provided with

g) The slurry was filtered to obtain compound 7 as a solid.

19. The method of claim 18, further comprising the step of adding lithium bromide in step a) above.

20. The method of claim 18, further comprising the step of: the resulting mixture is combined with a solution of the second base and compound 6 in a continuous stirred tank reactor.

21. The method of claim 18, further comprising the step of:

a) Adding a solution of a first base to a solution of compound 5 and lithium bromide in a first aprotic solvent to produce the resulting mixture; and

b) Combining the resulting mixture with 1) a solution of the second base and 2) a solution of compound 6 in a second aprotic solvent in a plug flow reactor to produce a solution comprising compound 7.

22. A process for the preparation of compound A, or a pharmaceutically acceptable salt thereof,

which comprises the following steps:

a) Comprising the compound 7 according to claim 16 into a reaction solvent

And compound 4 'according to claim 12'

Adding a base to a slurry of the mixture of (a) to produce a resulting mixture;

b) Heating the resulting mixture to produce a solution comprising compound a;

c) Adding a crystallization solvent to produce a slurry comprising compound a; and is also provided with

d) The slurry was filtered to obtain compound a as a solid.

23. The process according to claim 22, wherein compound 4a is used in step a) above,

24. the method of claim 22, wherein the base is N, N-Diisopropylethylamine (DIPEA).

25. The method of claim 22, further comprising the step of:

a) Adding water as a crystallization solvent to produce a slurry comprising compound a;

b) Cooling the slurry and adding acetic acid to adjust the pH to about 11 to about 4; and

c) The slurry was filtered to obtain compound a as a solid.