HK1118270A - Process for the preparation of sulfonamide derivatives - Google Patents

Description

Process for preparing sulfonamide derivatives

Technical Field

The invention relates to a process for the preparation of compounds of formula (I)

Wherein Q¹As defined below; or, where appropriate, a pharmaceutically acceptable salt thereof and/or an isomer, tautomer, solvate or isotopic variant thereof, as well as intermediates used in the process, or, where appropriate, a salt thereof and/or an isomer, tautomer, solvate or isotopic variant thereof.

Background

The compound of formula (I) is beta₂Agonists of the receptor exhibit excellent efficacy, especially when administered by inhalation, and are therefore particularly useful in the treatment of the transbeta₂Mediated diseases and/or conditions.

Disclosure of Invention

The invention relates to a process for the preparation of compounds of formula (I),

wherein Q¹Is a group selected from:

and group^*-NR⁶-Q²-A, wherein symbol^*Represents the point of attachment to the carbonyl group, p is 1 or 2, Q²Is C optionally substituted by one hydroxy group₁To C₄Alkylene radical, R⁶Is H or C₁To C₄Alkyl, and A is pyridyl optionally substituted with OH, C optionally substituted with OH₃To C₇Cycloalkyl radicals, or the following groups

Wherein R is¹、R²、R³、R⁴And R⁵Same or different and selected from H, C₁To C₄Alkyl, OR⁷、SR⁷Halogen, CN, CF₃、OCF₃、COOR⁷、SO₂NR⁷R⁸、CONR⁷R⁸、NR⁷R⁸、NHCOR⁷And phenyl optionally substituted by 1 to 3 substituents selected from OR⁷Halogen and C₁To C₄Radical substitution of alkyl, wherein R⁷And R⁸Are identical or different and are selected from H or C₁To C₄An alkyl group;

or, where appropriate, a pharmaceutically acceptable salt thereof and/or an isomer, tautomer, solvate or isotopic variant thereof.

The invention relates to a process for the preparation of compounds of formula (I)

Wherein Q¹As defined above, which comprises using a compound of the formula

Preferably, the above process comprises reacting the compound of formula (7) with a compound of formula (5),

or the step of reacting the compound of formula (6)

Wherein PG²As a suitable phenol protecting group, PG³Is a suitable hydroxy protecting group, LG is a suitable leaving group, and R⁹Is H or SO₂CH₃。

Preferably, the process comprises a deprotection step to obtain a compound of formula (I).

Preferably, the method comprises the step of isolating the compound of formula (I).

In a preferred embodiment, the process comprises reacting a compound of formula (7) with a compound of formula (5)

Wherein R is⁹Is H, to obtain a compound of formula (3)

Preferably, the compound of formula (3) is then deprotected to obtain the compound of formula (I).

Preferably, two deprotection steps are performed to remove PG²And PG³And obtaining the compound of formula (I).

Preferably, a first deprotection step is performed to remove PG³To obtain the compound of formula (2)

Or a salt thereof.

Preferably the compound of formula (3) is not isolated and is directly subjected to a first deprotection step.

Preferably a salt of the compound of formula (2) is prepared and used in the next step. A preferred salt of the compound of formula (2) is the dibenzoyl- (L) -tartrate salt.

A second deprotection step is preferably carried out to remove PG²And obtaining the compound of formula (I).

In another preferred embodiment, the compound of formula (7) is reacted with a compound of formula (5)

Wherein R is⁹Is SO₂CH₃To obtain a compound of formula (3a)

Preferably, said compound of formula (3a) is then deprotected to obtain a compound of formula (I).

Preferably, 3 deprotection steps are performed to remove SO₂CH₃Radical, PG²And PG³. Preferably, a first deprotection step is performed to remove PG³And obtaining the compound of formula (4)

Preferably, a second deprotection step is performed to remove SO₂CH₃Radical and obtaining a compound of formula (2)

Or a salt thereof.

A third deprotection step is preferably carried out to remove PG²And obtaining the compound of formula (I). In another preferred embodiment, the compound of formula (7) is reacted with a compound of formula (6)

Wherein PG²Is a suitable phenol protecting group to obtain a compound of formula (4)

Preferably, said compound of formula (4) is then deprotected to obtain a compound of formula (I).

Preferably, two deprotection steps are performed to remove SO₂CH₃And PG²And obtaining the compound of formula (I).

Preferably, a first deprotection step is performed to remove SO₂CH₃Radical and obtaining a compound of formula (2)

Or a salt thereof.

Preferably, a second deprotection step is performed to remove PG²And obtaining the compound of formula (I).

Preferably, LG is bromide.

Preferably, PG³Is TBDMS.

Preferably, PG²Is benzyl.

In a preferred embodiment, the compound of formula (7) is prepared by the following method: reacting a compound of formula (10)

Wherein PG¹Is a suitable amino protecting group, with Q¹-H or a salt thereof (wherein Q)¹As defined above) to obtain a compound of formula (8)

Preferably, a deprotection step is performed to remove PG¹And obtaining said compound of formula (7).

Preferably, the compound of formula (10) is prepared by hydrolysis of a compound of formula (11)

Preferably, the compound of formula (11) is prepared by protecting a compound of formula (12)

Preferably, PG¹Boc, trichloroacetyl or chloroacetyl.

In another preferred embodiment, the compound of formula (8) is prepared by reacting a compound of formula (19) with an alkyl or aryl nitrile, preferably trichloroacetonitrile or chloroacetonitrile,

preferably, by reacting a compound of formula (15) with Q¹-H or a salt thereof (wherein Q)¹As defined above) to said compound of formula (19)

The compound of formula (16), which is a precursor of the compound of formula (12), may be prepared by hydrolysis in the presence of an enzyme.

In a preferred embodiment, the compound of formula (16)

By hydrolysing a compound of formula (18) in the presence of an enzyme selected from lipase, esterase or protease

And then the product is obtained.

Preferably, the enzyme is selected from the group consisting of Mucor Miehei (Mucor Miehei) esterase, Rhizomucor Miehei (Rhizomucor Miehei) lipase, Thermomyces lanuginosus (Thermomuceses Languinosus) lipase, Penicillin (Penicillin) acylase.

More preferably, the enzyme is Thermomyces lanuginosus lipase.

Preferably, the hydrolysis of the compound of formula (18) is carried out in water at a pH between 5 and 9 and at a temperature between 10 ℃ and 40 ℃, in the presence of a suitable buffer and optionally in the presence of a suitable base.

The invention also relates to intermediates useful in the process of the invention.

In a preferred embodiment, the present invention relates to the following intermediates:

wherein Q¹As defined above, R¹⁰Is H or PG²Wherein PG is²As a suitable phenol protecting group, R⁹Is H or PG³Wherein PG is³Is a suitable hydroxy protecting group, and R¹¹Is H, PG¹Wherein PG is¹Are suitable amino protecting groups.

Preferred intermediates are:

2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (dimethylsulfonyl) amino ] phenyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide;

2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (dimethylsulfonyl) amino ] phenyl } -2-hydroxyethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide;

tert-butyl- [2- (3- { [ (4' -hydroxybiphenyl-3-ylmethyl) -carbamoyl ] -methyl } -phenyl) -1, 1- (dimethyl) ethyl ] carbamate;

2, 2, 2-trichloro-N- [2- (3- { [ 4' -hydroxybiphenyl-3-ylmethyl) carbamoyl ] -methyl } -phenyl) -1, 1-dimethylethyl ] acetamide;

2-chloro-N- {2- [3- (2- { [ (4' -hydroxybiphenyl-3-yl) methyl ] amino } -2-oxoethyl) phenyl ] -1, 1-dimethylethyl } acetamide;

2- [3- (2-amino-2-methylpropyl) -phenyl ] -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] acetamide and

n- [ (R) -2-benzyloxy-5-oxiranyl-phenyl ] -dimethanesulfonamide.

In the above general formula (I), C₁To C₄Alkyl represents a straight or branched chain group containing 1,2, 3 or 4 carbon atoms. If it has substituents or occurs as substituents of other groups, e.g. in O- (C)₁To C₄) Alkyl, S- (C)₁To C₄) The definition also applies when … appears in alkyl and the like. Suitably (C)₁To C₄) Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl …. Suitably (C)₁To C₄) Examples of alkoxy are methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy and t-butoxy ….

Halogen represents a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine, in particular fluorine or chlorine.

Term C₃To C₇Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

Suitable hydroxy protecting groups include tert-butyl (dimethyl) silyl (TBDMS), triethylsilyl, tert-butyl (diphenyl) silyl, tri (isopropyl) silyl, tetrahydropyranyl, methoxymethyl, benzyloxymethyl, 1-ethoxyethyl and benzyl. Preferred hydroxy protecting groups are tert-butyl (dimethyl) silyl or triethylsilyl.

Suitable phenol protecting groups include benzyl, methyl, methoxymethyl, benzyloxymethyl, TBDMS, 4-methoxybenzyl and 4-chlorobenzyl. The preferred phenol protecting group is benzyl.

Suitable amino protecting groups include tert-butoxycarbonyl (Boc), chloroacetyl, trichloroacetyl, acetyl, trifluoroacetyl, benzyloxycarbonyl, formyl, phenylacyl, allyloxycarbonyl, 2- (trimethylsilyl) ethoxycarbonyl or 2, 2, 2-trichloroethoxycarbonyl. Preferred amino protecting groups are Boc, chloroacetyl or trichloroacetyl.

Suitable leaving groups include bromide, 4-bromobenzenesulfonyl, chloride, iodide, methanesulfonyl, 4-nitrobenzenesulfonyl, p-toluenesulfonyl and trifluoromethanesulfonyl. Preferred leaving groups are bromide, chloride or p-toluenesulfonyl.

In the compounds of formula (I) and in the intermediates used for their preparation, Q¹Preferably is

Preferably, R¹、R²、R³、R⁴And R⁵Same or different and selected from H, C₁To C₄Alkyl, OR⁶、SR⁶Halogen (preferably chlorine), CF₃、OCF₃、SO₂NR⁷R⁸、CONR⁷R⁸、NR⁷R⁸、NHCOR⁷Provided that R is¹To R⁵At least 2 of which are H;

wherein R is⁷And R⁸Are identical or different and are selected from H or C₁To C₄An alkyl group.

Preferably, R¹、R²、R³、R⁴And R5 are the same or different and are selected from H, OH, CH₃、OCH₂-CH₃、SCH₃Halogen (preferably chlorine), CF₃、OCF₃Provided that R is¹To R⁵At least two of which are H.

Preferably, R¹、R²、R³、R⁴And R⁵Identical or different and selected from H or halogen, preferably chlorine, with the proviso thatR¹To R⁵At least two of which are H.

Preferably, R²And R³Is chlorine, and R¹、R⁴And R⁵Is H.

Preferably, R¹To R⁵One of them is OH.

Preferably, R¹、R²、R³、R⁴And R⁵One of them is phenyl substituted by OH, and the others are H.

Preferably, R²Is 4-hydroxy-phenyl, and R¹、R³、R⁴And R⁵Is H.

Preferably, the process of the invention is used to prepare the following compounds:

n- [ (4' -hydroxybiphenyl-4-yl) methyl ] -2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) acetamide;

2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) acetamide;

n- [ (4' -hydroxybiphenyl-3-yl) methyl ] -2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) acetamide;

n- (3, 4-dichlorobenzyl) -2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) acetamide;

2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (6-hydroxy-2-naphthyl) methyl ] acetamide;

2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (2-hydroxy-1-naphthyl) methyl ] acetamide, and

2- (3- {2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ 3-hydroxy-5- (trifluoromethyl) benzyl ] acetamide.

In a preferred embodiment, the present invention relates to a process for the preparation of compounds of formula (I) wherein the carbon atom substituted by a hydroxyl group is in the R configuration:

wherein Q¹As defined above, and intermediates for their preparation.

In a preferred embodiment, the present invention relates to a process for the preparation of a compound of formula (Ia):

wherein R is¹To R⁵As defined above, and intermediates for their preparation.

The process of the invention is illustrated by the following scheme:

scheme 1

Q¹As defined above.

PG¹Are suitable amino protecting groups. Preferably, PG¹Boc, chloroacetyl or trichloroacetyl.

PG²As a suitable phenol protecting group. Preferably, PG²Is benzyl.

PG³Are suitable hydroxy protecting groups. Preferably, PG³Is TBDMS.

LG is a suitable leaving group. Preferably, LG is bromide.

Preferably, in the above scheme, by hydroxy or OPG³The carbon atom substituted by the group is in the R configuration.

Q¹-H is selected from

And HNR⁶-Q²-A, wherein p, Q²、A、R¹To R⁵And R⁶As defined above.

In step (1a), the amine of formula (12) is reacted with a protecting agent, for example di-tert-butyl dicarbonate or benzyl chloroformate, in the presence of an amine, such as 4-dimethylaminopyridine or triethylamine, in a suitable solvent, such as Tetrahydrofuran (THF). Other suitable protective agents are described in the textbook "protective Groups in Organic Synthesis" (t.w.greene and p.g.m.wuts). Typical conditions include reaction of 1.0 equivalent of compound (12), 1 to 3 equivalents of di-tert-butyl dicarbonate and 0.05 to 2 equivalents of 4-dimethylaminopyridine in a suitable solvent, such as tetrahydrofuran, at 10 to 50 ℃ for 12 to 48 hours.

In step (1b), the ester of formula (11) is hydrolyzed to the carboxylic acid of formula (10) using standard procedures as described in the textbook "Protective Groups in organic synthesis" (t.w.greene and p.g.m.wuts). Typical conditions include reaction of 1.0 equivalent of compound (11) with 2 to 5 equivalents of sodium hydroxide in a suitable solvent, such as a mixture of water and tetrahydrofuran or ethanol, at 10 to 50 ℃ for 12 to 48 hours.

In step (1c), in a suitable base, for example triethylamine or diisopropylethylamine, and the appropriate baseCoupling agents, such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, carbonyldiimidazole, pivaloyl chloride or isobutyl chloroformate, optionally in the presence of suitable additives, such as 1-hydroxybenzotriazole or N-hydroxysuccinimide, in a suitable solvent, such as dimethylformamide, propionitrile, acetonitrile or pyridine, for example¹Or a primary or secondary amine (or a salt thereof). Typical conditions comprise 1.0 equivalent of compound (10), 1.0 to 1.5 equivalents of formula H-Q in a suitable solvent, such as propionitrile, dimethylformamide or acetonitrile, at 10 to 40 deg.C¹The compound, 1 to 5 equivalents of base and 1.05 to 2 equivalents of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are reacted for 1 to 24 hours.

In step (1d), standard methods as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.G.M.Wuts) can be used to remove PG¹. When PG is used¹In the case of tert-butoxycarbonyl, typical conditions comprise reacting 1.0 equivalent of compound (8) with 1 to 10 equivalents of hydrochloric or trifluoroacetic acid in a suitable solvent, e.g. dichloromethane or a mixture of ethanol and 1, 4-dioxane, at 10 to 50 ℃ for 12 to 100 hours.

In step (1e), the amine of formula (7) is reacted with the activated compound of formula (5a) in a suitable solvent such as propionitrile, butyronitrile, 1-methyl-2-pyrrolidone, n-propyl acetate, n-butyl acetate or 4-methyl-2-pentanone, optionally in the presence of a base such as sodium bicarbonate, triethanolamine, dipotassium hydrogen phosphate or diisopropylethylamine at a temperature between 50 ℃ and 150 ℃ for 12 to 48 hours. Typical conditions include reaction of 1.0 equivalent of compound (7), 0.5 to 2.0 equivalents of compound (5a) and 2 to 5 equivalents of sodium bicarbonate in acetonitrile or n-butyl acetate at 110 to 120 ℃ for 24 to 48 hours.

In step (1f), standard methods as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.G.M.Wuts) can be used to remove PG³. When PG is used³In the case of tert-butyldimethylsilyl, the reaction mixture is, in a suitable solvent,for example tetrahydrofuran, ethanol, methanol or propionitrile, it is possible to use deprotecting agents, for example tetrabutylammonium fluoride, HF or triethylamine trihydrofluoride. Typical conditions comprise of 1.0 equivalent of compound (3), and 1-5 equivalents of triethylamine trihydrofluoride in a suitable solvent such as methanol, tetrahydrofuran, a mixture of butyronitrile and methanol or a mixture of n-butyl acetate, ethyl acetate and methanol at 25 to 40 ℃ for 1 to 24 hours.

In step (1g), the amine of formula (7) is reacted with the epoxide of formula (6) in a suitable solvent, such as propionitrile, butyronitrile or n-butanol, at a temperature of 80 ℃ and 150 ℃ for 12 to 60 hours. Typical conditions comprise of 1.0 equivalent of compound (7) with 0.5 to 2 equivalents of compound (6) in a suitable solvent (e.g. butyronitrile or n-butanol) at 100 to 130 ℃ for 12 to 48 hours.

In step (1h), the compound of formula (4) is reacted with a suitable deprotecting agent, such as sodium hydroxide, potassium hydroxide, tetrabutylammonium fluoride or potassium carbonate, in the presence of a suitable solvent, such as tetrahydrofuran or a mixture of water and a water-miscible alcohol, such as ethanol or methanol, at 10 to 50 ℃ for 3 to 100 hours. Typical conditions include reacting 1.0 equivalent of compound (4) with 4 to 10 equivalents of sodium hydroxide in a mixture of ethanol and water at 25 to 40 ℃ for 12 to 100 hours.

In step (1i), PG can be removed using standard methods as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.Wutz)². When PG is used²For benzyl, typical conditions include 25 to 60 deg.C under 40 to 80psi of hydrogen over a suitable catalyst (e.g., 20% Pd (OH)₂/C or 5% Pd/C) in a suitable solvent, for example ethanol, aqueous ethanol, tetrahydrofuran, aqueous tetrahydrofuran, ethylene glycol, propylene glycol or dimethylformamide, 1.0 equivalent of compound (2) is reacted for 2 to 54 hours.

Alternatively, deprotection step (1i) can be performed before deprotection step (1f), as illustrated in the scheme below.

In this embodiment, standard methods as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.Wutz) can be used to remove PG²And PG³Both of which are described below. When PG is used²For benzyl, typical conditions for step (1i) include 25 to 60 ℃ under 40 to 80psi of hydrogen over a suitable catalyst (e.g., 20% Pd (OH))₂/C or 5% Pd/C) in a suitable solvent, for example ethanol, tetrahydrofuran, ethyl acetate or a mixture of ethyl acetate and n-butyl acetate, 1.0 equivalent of compound (3) is reacted for 2 to 48 hours. When PG is used³In the case of tert-butyldimethylsilyl group, typical conditions for step (1f) include reaction of 1.0 equivalent of compound (3a) and 1.0 to 10.0 equivalents of ammonium fluoride in a suitable solvent such as aqueous methanol, aqueous ethanol or aqueous acetonitrile at 10 to 40 ℃ for 1 to 48 hours.

Preferably, in the above compounds, the compound is substituted by hydroxy or OPG³The carbon atom substituted by the radical is in the R configuration.

Alternatively, the deprotection step (1i) may be performed before the deprotection step (1 h).

Alternatively, step (1e) may be replaced by the following step, using a compound of formula (5 b).

The conditions for steps (1j) and (1k) are the same as those disclosed for steps (1e) and (1h), respectively. Preferably, in the above compounds, the compound is substituted by hydroxy or OPG³The carbon atom substituted by the group is in the R configuration.

In step (1j), the amine of formula (7) is reacted with the activated compound of formula (5b) in the presence of a suitable solvent such as propionitrile, butyronitrile, 1-methyl-2-pyrrolidone, n-propyl acetate, n-butyl acetate or 4-methyl-2-pentanol, optionally in the presence of a base such as sodium bicarbonate, triethanolamine, dipotassium hydrogen phosphate or diisopropylethylamine at a temperature between 50 ℃ and 150 ℃ for 12 to 48 hours. Typical conditions include reaction of 1.0 equivalent of compound (7), 0.5 to 2.0 equivalents of compound (5b) and 2 to 5 equivalents of sodium bicarbonate in butyronitrile at 110 to 120 ℃ for 24 to 48 hours.

In step (1k), a compound of formula (3a) is treated with a suitable deprotecting agent, such as sodium hydroxide, potassium hydroxide, tetrabutylammonium fluoride or potassium carbonate, in the presence of a suitable solvent, such as tetrahydrofuran or a mixture of water and a water-miscible alcohol, such as ethanol or methanol, at 10 to 50 ℃ for 3 to 100 hours. Typical conditions include reacting 1.0 equivalent of compound (3a) with 4 to 10 equivalents of sodium hydroxide in a mixture of ethanol and water at 25 to 40 ℃ for 12 to 100 hours.

Alternatively, the order of deprotection steps used to convert a compound of formula (3a) to a compound of formula (I) may be varied such that PG may be removed in any order²、PG³And methane sulfonamide.

A compound of formula (5) wherein PG²Is benzyl, PG³Is TBDMS and LG is bromide, and can be prepared as disclosed in the following scheme:

details of the preparation of compound (5a) are disclosed in the examples.

Preferably, in the above compounds, the carbon atom substituted by a hydroxyl or OTBDMS group is in the R configuration.

The compounds of formulae (5a) and (6) may be prepared by a process according to scheme 2:

scheme 2

PG²、PG³And LG is as defined above.

Preferably, in the above compounds, the compound is substituted by hydroxy or OPG³The carbon atom from which the group is taken is in the R configuration.

The R isomer of the compound of formula (6) is also preferred:

in step (2a), a compound of formula (5a) is treated with methanesulfonyl chloride in the presence of a suitable base such as diisopropylethylamine, triethylamine, sodium hydride, lithium diisopropylamide or n-butyllithium in a suitable solvent such as acetonitrile, propionitrile, tetrahydrofuran, dichloromethane, 1, 4-dioxane or dimethylformamide at a temperature between-80 ℃ and 80 ℃ for 1 to 24 hours. Typical conditions include reaction of 1.0 equivalent of compound (5a), 2 to 5 equivalents of diisopropylethylamine and 1 to 5 equivalents of methanesulfonyl chloride in a suitable solvent, such as acetonitrile, at 5 to 25 ℃ for 1 to 5 hours.

In step (2b), standard methods as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.G.M.Wuts) can be used to remove PG³. When PG is used³In the case of tert-butyldimethylsilyl, a deprotecting agent such as tetrabutylammonium fluoride, HF or triethylamine trihydrofluoride may be used in the presence of a suitable solvent such as tetrahydrofuran, methanol, ethanol or propionitrile. Typical conditions include reaction of 1.0 equivalent of compound (5b) and 1 to 5 equivalents of triethylamine trihydrofluoride in a suitable solvent such as methanol or tetrahydrofuran at 25 to 40 ℃ for 12 to 48 hours.

In step (2c), the compound of formula (13) is reacted with a suitable base (e.g., potassium carbonate, triethylamine, sodium hydride, sodium carbonate, diisopropylethylamine) in the presence of a suitable solvent such as tetrahydrofuran, methanol, ethanol, dichloromethane, water at 10 to 40 ℃ for 2 to 24 hours. Typical conditions comprise of 1.0 equivalent of compound (13) with 1 to 5 equivalents of potassium carbonate in a suitable solvent, e.g. a mixture of methanol and tetrahydrofuran, at 20 to 25 ℃ for 12 to 18 hours.

Formula H-Q¹The compounds are commercially available or can be prepared from commercially available materials by conventional methods well known to those skilled in the art (e.g., reduction, oxidation, alkylation, transition metal mediated coupling, protection, deprotection, etc.). Examples of such preparations are disclosed in WO 2004/032921, WO2004/108676, WO 2004/108675 and WO 2004/100950.

The compound of formula (12) may be prepared according to the method of scheme 3 below.

Scheme 3

Details of the preparation of the compound of formula (12) are disclosed in the examples.

Alternatively, step (3a) may be replaced by the following steps:

scheme 4

In step (4a), the diester of formula (18) is prepared by esterification of a dibasic acid of formula (17) according to any method known to those skilled in the art for preparing esters from acids without modification of the remainder of the molecule. Typical conditions include reacting 1.0 equivalent of a dibasic acid of formula (17) with an alcoholic solvent, preferably ethanol, in the presence of an acid catalyst, such as hydrogen chloride or sulfuric acid, at a temperature between 10 ℃ and 100 ℃ for 6 to 24 hours.

In step (4b), the diester of formula (18) is hydrolysed to the monoester of formula (16) in the presence of a suitable enzyme known in the art, for example a lipase, esterase or protease, preferably a lipase. Preferred enzymes are Mucor miehei esterase, Rhizomucor lipase, Thermomyces lanuginosus lipase, penicillin acylase. More preferably, the reaction is carried out with Lipolase in water at a pH between 5 and 9 and a temperature between 10 ℃ and 40 ℃ in the presence of a suitable buffer, such as calcium acetate, dipotassium hydrogen phosphate or triethanolamine, and optionally in the presence of a suitable base, such as sodium hydroxide, potassium hydroxide or lithium hydroxide^®(Thermomyces lanuginosus lipase, (EC No. 3.1.1.3)). Typical conditions include 1.0 equivalent of a diester of formula (18) with 5 to 200 ml of Lipolase in a calcium acetate buffer solution at a temperature between 20 ℃ and 40 ℃ and maintaining the pH between 5.5 and 6.8 by addition of a base, such as sodium hydroxide or potassium hydroxide^®(liquid preparation) the reaction was continued for 12 to 24 hours.

Alternatively, step (3d) may be replaced by the following steps as illustrated in scheme 5:

scheme 5

In step (5a), the ester of formula (14a) is prepared by esterification of the acid of formula (14) according to any method known to those skilled in the art for preparing esters from acids without modification of the remainder of the molecule. Typical conditions include reacting 1.0 equivalent of an acid of formula (14) with an alcoholic solvent, preferably ethanol, at a temperature between 20 ℃ and 100 ℃ in the presence of an acid catalyst, such as hydrogen chloride or sulfuric acid, for 1 to 12 hours.

In step (5b), the amide of formula (14a) is deprotected using standard procedures as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.G.M.Wuts). Typical conditions comprise of 1.0 equivalent of chloroacetamide of formula (14a) with 1 to 3 equivalents of thiourea in a suitable solvent, e.g. a mixture of ethanol and acetic acid, at a temperature between 50 ℃ and 120 ℃ for 12 to 24 hours.

Alternatively, compounds of formula (7) can be prepared according to scheme 6 below:

scheme 6

In scheme 6, PG¹Preferably trichloroacetyl or chloroacetyl. More preferably, PG¹Is trichloroacetyl.

In step (6a), the tertiary alcohol of formula (15) is treated with an alkyl or aryl nitrile and an acid catalyst to give the amide of formula (10). Preferably, the tertiary alcohol of formula (15) is reacted with trichloroacetonitrile or chloroacetonitrile in the presence of an acid, e.g. sulfuric acid, acetic acid, trifluoroacetic acid, to give the protected amide of formula (20). Typical conditions include the addition of 1 to 3 ml of concentrated (98%) sulfuric acid per gram of alcohol of formula (15) to a solution of 1.0 equivalent of the alcohol of formula (15) and 1 to 2 equivalents of trichloroacetonitrile in a suitable solvent, such as acetic acid, at a temperature between 0 ℃ and 25 ℃ for 1 to 8 hours.

In step (6b), a carboxylic acid of formula (10) is reacted with a carboxylic acid of formula H-Q in the presence of a suitable base, such as triethylamine or diisopropylethylamine, and a suitable coupling agent, such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, carbonyldiimidazole, pivaloyl chloride or isobutyl chloroformate, optionally in the presence of a suitable additive, such as 1-hydroxybenzotriazole or N-hydroxysuccinimide, in a suitable solvent, such as ethyl acetate, dimethylformamide, propionitrile, acetonitrile or pyridine¹By reaction of primary or secondary amines or salts thereofShould be used. Typical conditions comprise 1.0 equivalent of a compound of formula (10), 0.8 to 1.2 equivalents of a compound of formula H-Q in a suitable solvent, e.g. ethyl acetate, propionitrile, dimethylformamide, at 20 to 60 deg.C¹The compound, 1 to 5 equivalents of base, 1 to 2 equivalents of 1-hydroxybenzotriazole and 1.05 to 2 equivalents of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are reacted for 12 to 36 hours.

In step (6c), PG is removed using standard procedures as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.G.M.Wuts) or other procedures well known to those skilled in the art¹. When PG is used¹In the case of trichloroacetyl, typical conditions include reacting 1.0 equivalent of compound (8) with 2 to 10 equivalents of a suitable base (e.g. potassium hydroxide or sodium hydroxide) in a suitable solvent, such as water, ethanol or methanol or preferably a mixture of water and ethanol, at a temperature between 30 ℃ and 80 ℃ for 16 to 36 hours.

Alternatively, compounds of formula (7) can be prepared according to scheme 7 below:

scheme 7

In scheme 7, PG¹Preferably trichloroacetyl or chloroacetyl. More preferably, PG¹Is chloroacetyl.

In step (7a), a carboxylic acid of formula (15) is reacted with a carboxylic acid of formula H-Q in the presence of a suitable base, such as triethylamine or diisopropylethylamine, and a suitable coupling agent, such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, carbonyldiimidazole, pivaloyl chloride or isobutyl chloroformate, optionally in the presence of a suitable additive, such as 1-hydroxybenzotriazole or N-hydroxysuccinamide, in a suitable solvent, such as dichloromethane, ethyl acetate, dimethylformamide, propionitrile, acetonitrile or pyridine¹Primary or secondary amines ofOr a salt thereof. Typical conditions comprise 1.0 equivalent of a compound of formula (15), 0.8 to 1.2 equivalents of a compound of formula H-Q in a suitable solvent such as dichloromethane, ethyl acetate, propionitrile, dimethylformamide at 20 to 60 deg.C¹The compound, 1 to 5 equivalents of base, 0.4 to 2 equivalents of 1-hydroxybenzotriazole and 1 to 2 equivalents of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are reacted for 1 to 24 hours.

In step (7b), the tertiary alcohol of formula (19) is treated with an alkyl or aryl nitrile and an acid catalyst to give an amide of formula (8). Preferably, the tertiary alcohol of formula (19) is reacted with trichloroacetonitrile or chloroacetonitrile in the presence of an acid, e.g. sulfuric acid, acetic acid, trifluoroacetic acid, to give the protected amide of formula (8). Typical conditions include the addition of 2 to 5 ml of trifluoroacetic acid to 1.0 equivalent of an alcohol of formula (19) per gram of alcohol of formula (19) and 2 to 5 ml of chloroacetonitrile per gram of alcohol of formula (19) for 1 to 8 hours at a temperature between 0 ℃ and 75 ℃. The compound of formula (8) may be isolated prior to performing step (7 c).

In step (7c), PG is removed using standard procedures as described in "Protective Groups in organic Synthesis" (T.W.Greene and P.G.M.Wuts) or other procedures well known to those skilled in the art¹. When PG is used¹In the case of chloroacetyl, typical conditions include reacting 1.0 equivalent of compound (8) and 2 to 8 equivalents of thiourea in a suitable solvent, e.g. acetic acid, isopropanol, ethyl acetate, isopropyl acetate, preferably acetic acid, at a temperature between 50 ℃ and 120 ℃ for 1 to 36 hours.

Detailed Description

The process of the present invention is illustrated by the following examples.

Example 1: n- (4' -hydroxy-biphenyl-3-ylmethyl) -2- (3- {2- [ 2-hydroxy-2- (4-hydroxy-3-) Methyl-phenyl) -ethylamino]Preparation of (E) -2-methyl-propyl-phenyl-acetamide

Preparation example 1: 2, 2' - (1, 3-phenylene) diacetic acid diethyl ester

Concentrated sulfuric acid (1.82 l) was added to a suspension of 2, 2' - (1, 3-phenylene) diacetic acid (45.55 kg; 234.6 mol) in ethanol (455.5 l). The resulting thin suspension was heated to reflux for 20 hours. The reaction was cooled to room temperature and the ethanol was removed at atmospheric pressure and then replaced with toluene (136.5 l). The toluene solution was washed with 5% aqueous sodium bicarbonate (1 × 91 l), then concentrated to about 1 ml/g toluene solution and subjected to the next step. An aliquot concentrated to dryness under vacuum was analyzed, showing-100% yield.

¹H NMR(CD₃OD, 400MHz) δ: 1.21(t, 6H), 3.59(s, 4H), 4.10(q, 4H), 7.15 to 7.27(m, 4H) ppm.

MS (electrospray): m/z 251[ M + H]⁺

Preparation example 2: [3- (2-oxo-propyl) -phenyl ] -acetic acid ethyl ester

Adding Lipolase^®(Thermomyces lanuginosus lipase solution; 9.4 liters) to a 0.2M solution of calcium acetate in water (117.5 liters) and the homogeneous solution was stirred at ambient temperature for 30 minutes. A solution of the product from preparative example 1 (29.35 kg, 117.3 moles) in toluene was added and the reaction stirred at ambient temperature. The pH was checked every 15 minutes and maintained between 5.5 and 6.8 by adding an aliquot of 1M aqueous sodium hydroxide solution. The reaction was completed after 48 hours. The pH was adjusted to 3 to 4 using 1M aqueous hydrochloric acid, and ethyl acetate (117 l) was added. The biphasic mixture was filtered through a Gauthier filter to remove denatured enzyme. The mixture was then separated and the aqueous layer was extracted with ethyl acetate (2 × 117 liters). The combined organic layers were extracted with saturated aqueous sodium bicarbonate (3 × 149.69 l). The combined sodium bicarbonate extracts were adjusted to pH 2 using 2M aqueous hydrochloric acid and the resulting solution was extracted with toluene (2 × 147 l). The toluene extract was then concentrated to about 1 ml/g toluene solution for the next step. Analysis was concentrated to dryness under vacuum to give an aliquot of the title compound, showing a yield of 19.68 kg; 75.6 percent.

¹H NMR(CD₃OD, 400MHz) δ: 1.25(t, 3H), 3.60(m, 2H), 3.63(m, 2H), 4.15(q, 2H), 7.18 to 7.32(m, 4H) ppm.

MS (electrospray): m/z 245[ M + Na ]]⁺

Alternative Process for the preparation of the product of preparation 2

Absolute ethanol (85.41 g; 1.85 mol) and 37% aqueous hydrochloric acid (30 ml) were added to a suspension of 2, 2' - (1, 3-phenylene) diacetic acid (300.0 g; 1.54 mol) in THF (3.0 l), which was almost completely soluble. The resulting thin suspension was heated to 50 ℃ until the reaction was complete (monitored by HPLC). Once the reaction was complete, the solvent was removed and replaced with toluene (1.5 l), and the resulting suspension was stirred vigorously for 15 minutes before being filtered under vacuum. The precipitate was washed with fresh toluene (300 ml) and then discarded (the precipitate was the starting 2, 2' - (1, 3-phenylene) diacetic acid). The toluene solution was extracted with saturated aqueous sodium bicarbonate (1.35 l +2 × 300 ml). The combined sodium bicarbonate extracts were adjusted to pH 5 to 6 using a combination of 37% hydrochloric acid and 2M hydrochloric acid, and the resulting pale cream solution was extracted with tert-butyl methyl ether (1.2 l +2 × 600 ml). The combined tert-butyl methyl ether extracts were washed with demineralized water (600 ml) over MgSO₄Dried over methyl acetate and concentrated to dryness under vacuum to give the title compound as a pale yellow oil (134.1 g).

Preparation example 3: [3- (2-hydroxy-2-methyl-propyl) -phenyl ] -acetic acid

A toluene solution of the product of preparation 2 (3.59 kg; solvent corrected; 16.15 mol) was dissolved in anhydrous tetrahydrofuran under nitrogen and cooled to 0 to 5 ℃. Methylmagnesium bromide (56.53 liters of a 1M solution in tetrahydrofuran, 56.53 moles) was added to the solution at a rate that maintained the temperature below 15 ℃. Once the addition was complete, the reaction was warmed to ambient temperature and stirred until completion. The reaction mixture was then cooled to between 0 and 5 ℃ and demineralized water (17.95 liters) was added maintaining the temperature below 15 ℃. Once the addition was complete, the pH was adjusted to between 1 and 2.5 by the addition of 5M hydrochloric acid. The mixture was extracted with isopropyl acetate (2 × 17.95 l), the combined organic extracts were washed with water (3 × 17.95 l), and the isopropyl acetate was then distilled and replaced with toluene until a toluene concentration of about 5 ml/g was reached. The toluene solution was cooled to 5 ℃ and the resulting slurry was granulated for 2 hours. The product was isolated by filtration and washed with toluene (3.59 l) to give the title compound as an off-white solid (2.29 kg, 68%).

¹H NMR(CDCl₃400MHz) δ: 1.22(6H, s), 2.75(2H, s), 3.63(2H, s), 7.12 to 7.30(4H, m).

MS(ESI)：m/z 209[M+H]⁺

Preparation example 4: {3- [2- (2-chloro-acetylamino) -2-methyl-propyl ] -phenyl } -acetic acid

2-Chloroacetonitrile (1.63 kg, 21.62 mol) was added to a solution of the alcohol from preparation 3 (3.00 kg, 14.41 mol) in dichloromethane (15 l). The resulting solution was treated with acetic acid (2.6 kg, 43.23 moles) and maintained at a temperature between 5 ℃ and 10 ℃. The resulting solution was treated with concentrated sulfuric acid (2.83 kg, 28.82 mol) and maintained at a temperature between 5 ℃ and 10 ℃. The mixture was warmed to 20 ℃ and after 90 minutes the reaction mixture was added to cold water (30 litres) and the temperature was maintained below 10 ℃. The mixture was stirred at 5 to 10 ℃ for 30 minutes and then at 20 ℃ for 30 minutes. The layers were separated and the aqueous layer was further extracted with dichloromethane (15 l). The combined dichloromethane layers were reduced to a volume of 8 liters by distillation at atmospheric pressure. The concentrate was treated with n-heptane (27 l) and toluene (3 l) and concentrated in vacuo to remove residual dichloromethane. The resulting slurry was granulated at 20 ℃ for 2 hours and the solid precipitate was isolated by filtration and washed with n-heptane (2X 3L) to give the title compound as an off-white solid (3.76 kg).

¹H NMR(CDCl₃400MHz) δ: 1.36(s, 6H), 3.02(s, 2H), 3.62(s, 2H), 3.95(s, 2H), 6.19(m, 1H), 7.06 to 7.31(m, 4H) ppm.

MS (electrospray): m/z 282[ M-H]^-

Preparation example 5: [3- (2-amino-2-methyl-propyl) -phenyl ] -acetic acid ethyl ester

A mixture of the amide from preparation 4 (151.4 g, 534 mmol), thiourea (48.7 g, 640 mmol) and acetic acid (303 ml) in ethanol (1.5 l) was heated to reflux under a nitrogen atmosphere for 5 h. The reaction mixture was allowed to cool to room temperature and the suspension was concentrated using vacuum. The residue was azeotroped with toluene (2 × 900 ml) and then treated with ethanol (1.5 l) and stirred for one hour. The solid precipitate was removed by filtration and the filtrate was cooled in an ice bath, treated with 98% sulfuric acid (227 ml) and stirred at ambient temperature for one hour. The solution was concentrated using vacuum to remove most of the ethanol and adjusted to pH 9 using aqueous sodium bicarbonate. The solid precipitate was removed by filtration and washed with water (300 ml) followed by ethyl acetate (1.0 l). The combined biphasic filtrate and washings were separated and the aqueous layer was extracted with further ethyl acetate (1.0 l +500 ml). The combined ethyl acetate extracts were dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound as a brown oil (89.5 g).

¹H NMR(DMSO-d₆，400MHz)δ：0.99(s，6H)、1.16(t，3H)、2.59(s，2H)、3.61(s，2H)、4.06(q，2H)、7.06(m，3H)、7.21(m，1H)

Preparation example 5 a: [3- (2-amino-2-methyl-propyl) -phenyl ] -acetic acid ethyl ester, di-p-toluoyl-L-tartrate salt

A solution of the amine from preparation 5 (assumed to be 9.45 moles) in acetonitrile (24.8 liters) was treated with a solution of di-p-toluoyl-L-tartaric acid (3.65 kg, 9.45 moles) in acetonitrile (18.6 liters). The resulting slurry was stirred at 20 ℃ for 15 h, and the solid precipitate was isolated by filtration and washed with acetonitrile (2X 6.2L) to give the title compound (5.72 kg) as a white solid.

¹H NMR(DMSO-d₆，400MHz)δ：1.13(s，6H)、1.17(t，3H)、2.34(s，6H)、2.78(s，2H)、3.63(s，2H)、4.06(q，2H)、5.61(s，2H)、7.02(d，2H)、7.15(d，1H)、7.25(m，5H)、7.80(d，4H)

Preparation example 5: [3- (2-amino-2-methyl-propyl) -phenyl ] -acetic acid ethyl ester

A solution of potassium carbonate (6.232 kg, 45.1 moles) in water (35.04 l) was added to a suspension of the salt from preparation 5a (7.008 kg, 11,272 moles) in propionitrile (35.04 l) and stirred until all solids had dissolved. The phases were then separated and the propionitrile phase was washed with water (17.52 l). The volume of the solution was reduced to about 3.70 kg under reduced pressure to give the title compound as a propionitrile solution. A sample (20 ml) was removed and concentrated to dryness to obtain a weight/weight assay; the yield is shown to be 92%.

¹H NMR(DMSO-d₆，400MHz)δ：0.99(s，6H)、1.16(t，3H)、2.59(s，2H)、3.61(s，2H)、4.06(q，2H)、7.06(m，3H)、7.21(m，1H)

Alternative procedure for the preparation of the product of preparation 5

A solution of the amide from preparation 14 (3.10 kg, 9.945 mol) in ethanol (34.1 l) was treated with thiourea (0.91 kg, 11.93 mol) and acetic acid (6.2 l) and heated at reflux for 4 h. The mixture was cooled and the solid precipitate was removed by filtration and then washed with ethanol (3.1 l). The combined filtrate and washings were concentrated in vacuo to a volume of 8 l and azeotroped with toluene (31.0 l and 24.8 l) in vacuo to 8 l. The resulting mixture was treated with water (9.3 l) and 2M aqueous sodium carbonate (7.5 l) and extracted with dichloromethane (31.0 l and 15.5 l). The combined dichloromethane extracts were concentrated to a volume of 8 liters at atmospheric pressure, treated with acetonitrile (12.4 liters) and concentrated in vacuo to a volume of 8 liters. The concentrate was diluted with acetonitrile (24.8 l) and used directly in preparation 5 a.

Preparation example 6: n- {2- (benzyloxy) -5- [ (1R) -2-bromo-1-hydroxyethyl ] phenyl } methanesulfonamide

Pyridine (18.4 ml; 227.2 mmol) was added to a solution of (1R) -1- [ 3-amino-4- (benzyloxy) phenyl ] -2-bromoethanol (org. Process Research and Development, 1998, 2, 96) (36.59 g; 113.6 mmol) in THF (160 ml). Methanesulfonyl chloride (10.5 ml; 136.3 mmol) was then added and the reaction mixture was stirred at 20 to 25 ℃ for 2 hours. The reaction was quenched with 1M hydrochloric acid (180 ml) and then extracted with toluene (180 ml). The toluene solution was then washed with water (2 × 90 ml). The toluene solution was then concentrated to 110 ml at 45 ℃ under reduced pressure, the solution was then cooled to room temperature (20 to 25 ℃) and stirred for one hour, and then the mixture was cooled to 10 to 15 ℃ and stirred for one hour. The precipitate was collected by filtration and washed with toluene (2 × 10 ml) to give the title compound as a pink solid (37.95 g).

¹H NMR(DMSO-d₆400MHz) δ: 2.93(s, 3H), 3.52 to 3.66(m, 2H), 4.74(m, 1H), 5.19(s, 2H), 7.11(d, 1H), 7.19 to 7.22(m, 1H), 7.33 to 7.36(m, 2H), 7.40 to 7.43(m, 2H), 7.56(d, 2H), 8.95(s, 1H) ppm.

MS (electrospray): m/z 398/400[ M-H]^-

Alternative procedure for the preparation of the product of preparation 6

The product of preparation 6 can be prepared by stereoselective enzymatic reduction of N- [ 2-benzyloxy-5- (2-bromo-acetyl) -phenyl ] -methanesulfonamide (Journal of Medicinal Chemistry, 1967, 10, 462 and Journal of Medicinal Chemistry, 1980, 23, 738) as described in the Journal of the American Oil Chemistry' Society 1998, 75, 1473 and in the examples below.

Biotransformation can be carried out by those skilled in the art by contacting the material to be transformed, and other necessary reactants, with enzymes from a variety of living organisms under conditions suitable for chemical interactions to occur. Thereafter, the products of the reaction are isolated and the desired products are purified to understand their chemical structure and physical and biological properties. The enzyme may be present as a purification reagent, in a crude extract or lysate, or in intact cells and may be present in solution, in suspension (e.g., intact cells), may be covalently linked to a support surface, or embedded in a permeable matrix (e.g., agarose or alginate beads). The substrate and other necessary reactants (e.g., water, air, cofactors) are supplied according to the chemical indication. Generally, the reaction is carried out in the presence of one or more liquid phases (aqueous and/or organic) to facilitate mass transfer of reactants and products. The reaction may be carried out under sterile or non-sterile conditions. The conditions for monitoring the progress of the reaction and the separation of the reaction products may vary depending on the physical properties of the reaction system and the chemistry of the reactants and products.

For whole cell biocatalysis, nutrient media (e.g., IOWA medium: dextrose, yeast extract, dipotassium phosphate, sodium chloride, soy flour, water; adjusted to neutral pH) are added to one or more culture vessels (e.g., fermentation tubes or bottles), which are then steam sterilized. The containers are inoculated aseptically under growth from an agar culture, a suspension of washed cells or spores, or a broth of a liquid nutrient medium culture derived from the bioconverted microorganism. The container is mounted on a shaker designed for fermentation and vibrated at a suitable temperature (e.g., 20 to 40 ℃) (e.g., rotating at 100 to 300 rpm) for a time long enough to encourage the microorganisms to grow to a suitable population size (e.g., 1 to 3 days). The compound to be converted (i.e., the substrate) is dissolved in water or a suitable water-miscible solvent (e.g., dimethyl sulfoxide, dimethylformamide, ethanol, methanol). The resulting solution is added aseptically to each of the bioconversion containers to obtain the desired concentration of the substrate. The dosed container is mounted on a shaker and shaken as described above until the substrate is converted to the product by microbial metabolism (e.g., 1 to 10 days).

The isolated enzyme may be mixed with any desired cofactor and the substrate by suitable agitation in a suitable buffer, such as potassium phosphate, with or without an organic solvent at a temperature (25 to 37 ℃) and for a duration suitable for carrying out the biocatalysis. Many enzymes can be screened immediately in a microtiter plate. The enzyme is dissolved in a suitable buffer and distributed in the wells of the microtiter plate. The enzyme can be frozen (-80 ℃) or used immediately. For screening, additional buffer is added to each well along with the substrate and any cofactors (e.g., NADPH) required for enzyme function. The plates are then mixed as before (e.g., using an Eppendorf thermal mixer).

The contents of the biochemical conversion vessel may be mechanically treated (e.g., by filtration or centrifugation) to separate solids from the aqueous phase and/or extracted at a pH optimal for extraction of the desired compound (water-immiscible organic solvents include, but are not limited to, dichloromethane or ethyl acetate). The sample may be analyzed by HPLC or other suitable techniques.

The following are two examples of laboratory-scale screening methods (which can be performed by one skilled in the art) for carrying out biotransformations to produce compounds of interest.

Alternative method 1 for the synthesis of preparation 6: stereoselective microbial reduction of N- [ 2-benzyloxy-5- (2-bromo-acetyl) -phenyl ] -methanesulfonamide to the corresponding (R) -alcohol

Incubations were performed in 2.5 ml IOWA medium (anhydrous dextrose, 20 g; yeast extract, 5 g; dipotassium hydrogen phosphate, 5 g; sodium chloride, 5 g; soy flour, 5 g; distilled water, 1 l; adjusted to pH 7.0 using 1N hydrochloric acid, steam sterilized at 15psig and 121 ℃ for 15 minutes) in a 16 x 125 mm glass tube with a stainless steel Morton hood. The tubes were aseptically inoculated with 0.025 ml of a stock solution of mycelium of Candida magnoliae (Candida lignoliae) ATCC 56463 stored at low temperature (-80 ℃). The inoculated tubes were mounted on a rotary shaker (2 inch swing) at a small angle and shaken at 210rpm for two days at 29 ℃. N- [ 2-benzyloxy-5- (2-bromo-acetyl) -phenyl ] -methanesulfonamide (i.e., the substrate) was dissolved in dimethyl sulfoxide (10 mg/ml). Substrate was added to each tube to give an initial substrate concentration of 0.1 mg/ml up to 1 mg/ml. The tube of the feedstock was shaken at 210rpm for an additional 6 days at 29 ℃. At the end of the 6 day bioconversion period, the contents of the bioconversion tube were extracted with 4 ml ethyl acetate. The organic phase was concentrated under nitrogen. The residue was reconstituted in the appropriate amount of methanol for chiral HPLC analysis. At a substrate concentration of 1 mg/ml, the reaction gives a yield of (R) -alcohol of 33%, > 99% ee.

Chiral HPLC analysis:

the instrument comprises the following steps: a Waters 2695 HPLC system with 996 photodiode array detector.

Column: chiralpak AD-H, 4.6X 150 mm.

Mobile phase: methanol to ethanol [ 1: 1] at 1 ml/min

And (3) detection: PDA maximum curve (maxplot): 210 to 400 nanometers.

The (R) -alcohol eluted at 2.95 minutes; the substrate was eluted at 6.02 minutes.

Alternative method 2 for the synthesis of preparation 6: stereoselective enzymatic reduction of N- [ 2-benzyloxy-5- (2-bromo-acetyl) -phenyl ] -methanesulfonamide to the corresponding (R) -alcohol

50 mg of KRED-130 from BioCatalytics (Pasadena, CA) was dissolved in 1.5 ml of buffer (50mM potassium phosphate buffer, 0.1M potassium chloride, 0.5mM dithiothreitol, pH 6.0) and 0.030 ml was distributed in the wells as part of the ketoreductase screening plate. The plates of the polypropylene lid were frozen at-80 ℃ and one was thawed before being used in the experiment. 0.42 ml of buffer (described above) and 0.1 mg of substrate (0.01 ml of 10 mg/ml DMSO stock solution) were added to each well along with NADPH (0.040 ml of 100 mg/ml water stock solution). The plates were incubated on an Eppendorf thermal mixer R at 30 ℃ and 750rpm for 24 hours. The wells were extracted with 0.8 ml of ethyl acetate and then centrifuged (Damon IEC centrifuge (CRU5000), 2200rpm, 3 min). From each well, 0.7 ml was transferred to a new microtiter plate. The organic phase was dried under nitrogen and then reconstituted in methanol for HPLC analysis (above). The expected (R) -alcohol is obtained in a yield of 57% and ee > 99%.

Preparation example 7: n- [2- (benzyloxy) -5- ((1R) -2-bromo-1- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) phenyl ] methanesulfonamide

The bromide solution of preparation 6 (10 g; 25.0 mmol) was dissolved in dichloromethane (20 ml) and imidazole (4.58 g; 37.5 mmol) and tert-butyldimethylsilyl chloride (5.27 g; 35.0 mmol) were added successively. The reaction mixture was heated to reflux for one hour and then cooled to 30 ℃. The mixture was diluted with isopropyl acetate (80 ml) and the reaction was quenched with 2M hydrochloric acid (50 ml) and stirred vigorously for 10 minutes. The phases were separated and the organic phase was washed with water (50 ml). The volume of the organic phase is then reduced to 25 to 30 ml at 45 ℃ under reduced pressure. The solution was then cooled to room temperature and a suspension formed quickly, which was then stirred at room temperature for 30 minutes. Heptane (20 ml) was then added over 10 minutes and the suspension was cooled to 5 to 10 ℃ and then stirred for one hour. The suspension was then filtered and washed with heptane (2 × 10 ml) on filter paper to give the title compound (11.05 g) as a white solid.

¹H NMR(CDCl₃400MHz) δ: -0.07(s, 3H), 0.11(s, 3H), 0.89(s, 9H), 2.91(s, 3H), 4.80 to 4.83(m, 1H), 6.80(bs, 1H), 6.98(d, 1H), 7.12(d, 1H), 7.36 to 7.44(m, 5H), 7.52 to 7.54(m, 1H) ppm.

Preparation example 8: n- [2- (benzyloxy) -5- ((1R) -2-bromo-1- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) phenyl ] -dime-thylsulfonamide

N- [2- (benzyloxy) -5- ((1R) -2-bromo-1- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) phenyl ] methanesulfonamide (20.0 g; 39.2 mmol), prepared as described in preparation 7, and diisopropylethylamine (24 ml; 138 mmol) were combined in acetonitrile (100 ml) and cooled to about 5 ℃. Methanesulfonyl chloride (9.0 ml; 118.8 mmol) was added over about 10 minutes and the resulting mixture was stirred at 5 ℃ for about one hour. Water (300 ml) was added and the resulting slurry was granulated for 15 minutes, then filtered and dried at 40 ℃ under vacuum to give the title compound (23.3 g) as a pale yellow solid.

¹H NMR(CDCl₃400MHz) δ: -0.06(s, 3H), 0.12(s, 3H), 0.90(s, 9H), 3.31(s, 6H), 3.40 to 3.50(m, 2H), 4.83(dd, 1H), 5.14(s, 2H), 7.05(d, 1H), 7.32 to 7.42(m, 5H), 7.46 to 7.50(m, 2H) ppm.

Preparation example 9: n- [2- (benzyloxy) -5- ((1R) -2-bromo-1-hydroxyethyl) phenyl ] -dime-thylsulfonamide

The silyl ether from preparation 8 (19.2 g; 32.4 mmol) was suspended in a mixture of tetrahydrofuran (40 ml) and methanol (2 ml). Triethylamine trihydrofluoride (9 ml; 55.2 mmol) was added and the resulting solution was stirred at ambient temperature for 30 h. The reaction was quenched with aqueous ammonia (35%, 20 ml) and the product extracted in ethyl acetate (2 × 30 ml). The combined organic phases were washed with saturated aqueous sodium bicarbonate and water, over anhydrous MgSO₄Dried, filtered and concentrated to dryness. The residue was then slurried in ethyl acetate (40 ml)Two hours later, the product was isolated by filtration and washed with ethyl acetate (10 ml) and tert-butyl methyl ether (20 ml) to give the title compound as a white solid (11.3 g).

¹H NMR(CDCl₃400MHz) δ: 3.33(s, 6H), 3.51(dd, 1H), 3.63(dd, 1H), 4.90(dd, 1H), 5.16(s, 2H), 7.08(d, 1H), 7.33 to 7.45(m, 5H), 7.46 to 7.50(m, 2H) ppm.

Preparation example 10: n- [ (R) -2-benzyloxy-5-oxiranyl-phenyl ] -dime thylsulfonamide

Potassium carbonate (2.25 g; 16.3 mmol) was added to a solution of bromohydrin (6.0 g; 12.5 mmol) from preparative example 9 in a mixture of methanol (30 ml) and tetrahydrofuran (30 ml) and the resulting mixture was stirred at ambient temperature for about 18 hours. The reaction was quenched in water (60 ml) and extracted with propionitrile (2 × 60 ml). The combined propionitrile layers were washed with water (100 ml) and anhydrous MgSO₄Dried, filtered and concentrated to give the title compound (4.98 g) as a pale yellow solid which was used without purification.

¹H NMR(CDCl₃400MHz) δ: 2.76(dd, 1H), 3.13(dd, 1H), 3.31(s, 3H), 3.33(s, 3H), 3.83(m, 1H), 5.15(s, 2H), 7.06(d, 1H), 7.22(d, 1H), 7.31 to 7.44(m, 4H), 7.46 to 7.50(m, 2H) ppm.

Preparation example 11: (3-bromobenzyl) carbamic acid tert-butyl ester

Triethylamine (6.57 l; 46.7 mol) was added to a solution of 3-bromobenzylamine hydrochloride (9.9 kg; 44.5 mol) in ethyl acetate (39.6 l) and the resulting mixture was stirred at 20 to 25 ℃ for 30 minutes and then cooled to 0 ℃. A solution of di-tert-butyl dicarbonate (10.7 kg; 49 mol) in ethyl acetate (19.8 l) is then added over 30 minutes, at a rate such as to maintain the temperature between 0 ℃ and 20 ℃. The reaction mixture was then stirred at 20 to 25 ℃ for two hours, followed by addition of water (29.7 l) and vigorous stirring of the mixture for 10 minutes, after which the phases were separated. The ethyl acetate phase was distilled and replaced with heptane at 35 to 45 ℃ under reduced pressure to a final volume of about 40 liters, after which the solution was cooled to 0 ℃ over 2 hours. The resulting suspension was stirred at 0 ℃ for 12h, then the product was collected by filtration and washed with heptane (2 × 3.37 l) to give the title compound as a white solid (10.26 kg).

¹H NMR(400MHz，CDCl₃) δ: 1.46(s, 9H), 4.25 to 4.32(m, 2H), 4.75 to 4.90(bs, 1H), 7.16 to 7.22(m, 2H), 7.39(dt, 1H), 7.43(bs, 1H) ppm.

Preparation example 12: [ (4' -Hydroxybiphenyl-3-yl) methyl ] carbamic acid tert-butyl ester

Nitrogen was bubbled through a stirred solution of the bromide from preparation 11 (5.12 kg; 17.9 mol), 4-hydroxyphenylboronic acid (2.71 kg; 19.7 mol) and sodium carbonate (2.848 kg; 26.8 mol) in a mixture of 1, 4-dioxane (25.6 l) and demineralized water (25.6 l) at 20 to 25 ℃ for one hour. 1, 1' -bis (diphenylphosphino) ferrocenylpalladium (II) chloride (14.6 g; 0.0179 mol) was then added to the mixture and nitrogen bubbling continued for a further 30 minutes. Thereafter, the reaction was heated at 65 to 70 ℃ under a nitrogen blanket for 2 hours. The reaction was cooled to 20 to 25 ℃, ethyl acetate (41 l) was added and the resulting mixture was stirred vigorously for 10 minutes, followed by separation of the phases. The organic phase was washed with a solution of citric acid (1.9 kg) in demineralised water (18.9 l) followed by a solution of sodium chloride (3.15 kg) in demineralised water (18.9 l). The ethyl acetate solution was treated with activated charcoal (Darco KB 100 mesh, wet powder; 5.12 kg) and stirred for 12 hours. The resulting slurry was then filtered through Arbocel and the filter cake was washed with methanol (25.6 liters). The combined filtrates were distilled and replaced with toluene at 40 to 50 ℃ under reduced pressure to a final volume of about 15 liters. The solution was then cooled to 10 ℃ over 2 hours and the resulting suspension was stirred at 10 ℃ for 12 hours. The product was isolated by filtration and washed with cyclohexane (2 × 2.56 l) to give the title compound (4.26 kg) as a white solid.

¹H NMR(400MHz，CDCl₃) δ: 1.47(s, 9H), 4.33 to 4.41(m, 2H), 4.87 to 4.94(bs, 1H), 6.89(d, 2H), 7.21(d, 1H), 7.37(dd, 1H), 7.43 to 7.45(m, 4H) ppm.

MS (electrospray) M/z 298 [ M-H ]]^-、322 [M+Na]⁺

Preparation example 13: 3' - (aminomethyl) biphenyl-4-olate hydrochloride

A solution of hydrogen chloride in 1, 4-dioxane/water (4M, 64.7 l; 135 mol) was added over 20 minutes to a solution of the phenol from preparation 12 (8.09 kg; 27 mol) in 1, 4-dioxane (16.15 l) and the resulting mixture was stirred at 20 to 25 ℃ for one hour. The suspension is concentrated to about 40 l at 40 to 45 ℃ under reduced pressure and stirred for 12 hours at 20 to 25 ℃. The precipitate was collected by filtration and washed with 1, 4-dioxane (2X 4.05L). The resulting filter cake was added to acetonitrile (80.9 liters) and heated at reflux for 2 hours. The precipitate was isolated by filtration and washed with acetonitrile (2 × 4.05 l) to give the title compound as a white solid (3.65 kg; 57%).

The aqueous 1, 4-dioxane liquid was distilled and replaced with fresh 1, 4-dioxane until the vapor temperature was greater than 100 ℃ and the reaction volume was-40 liters. The reaction mixture was cooled to 20 to 25 ℃, granulated for 18 hours, and the crude product was isolated by filtration. The resulting filter cake was added to acetonitrile (40 liters) and heated at reflux for 2 hours. The precipitate formed was isolated by filtration and washed with acetonitrile (2 × 4.05 l) to give the title compound in a second yield as a pale brown solid (2.36 kg; 37%).

¹H NMR(400MHz，CD₃OD) δ: 4.17(s, 2H), 6.87(d, 2H), 7.34(d, 1H), 7.45 to 7.50(m, 3H), 7.61(d, 1H), 7.65(s, 1H) ppm.

MS (electrospray) M/z 198[ M-H]^-、200[M+H]⁺

Preparation example 14: {3- [2- (2-chloro-acetylamino) -2-methyl-propyl ] -phenyl } -acetic acid ethyl ester

A solution of the acid from preparation 4 (3.76 kg, 13.24 mol) in ethanol (30.1 l) was treated with concentrated sulfuric acid (130 g, 1.31 mol) and heated at reflux for 90 minutes. The cooled solution was adjusted to-pH 5 using 1.0M aqueous sodium bicarbonate (2.0 kg). The mixture was concentrated to a volume of 8 l under vacuum, diluted with toluene (11.7 l) and concentrated to a volume of 12 l under vacuum. The concentrate was diluted with toluene (25.8 l), washed with water (22.6 l) and the aqueous layer was further re-extracted with toluene (15.0 l). The combined toluene layers were concentrated to 8 liters under vacuum. The concentrate was maintained at 35 ℃ and treated with n-heptane (15.0 l) to maintain the temperature above 30 ℃. The mixture is cooled and the resulting slurry is granulated at 20 ℃ for 2 hours. The solid precipitate was isolated by filtration and washed with n-heptane (2 × 3.76 l) to give the title compound as a white solid (3.15 kg).

¹H NMR(DMSO-d₆，400MHz)δ：1.14(t，3H)、1.19(s，6H)、2.95(s，2H)、3.59(s，2H)、3.94(s，2H)、4.07(q，2H)、7.00(m，2H)、7.09(d，1H)、7.20(t，1H)、7.59(s，1H)

Preparation example 15: [3- (2-tert-Butoxycarbonylamino-2-methylpropyl) phenyl ] acetic acid ethyl ester

The amine from preparation 5 (48.0 g; 204 mmol) was added to a solution of di-tert-butyl dicarbonate (55.0 g; 252 mmol) and 4-dimethylaminopyridine (1.5 g; 12.3 mmol) in THF (50 mL) over about 30 minutes and the resulting solution was stirred at ambient temperature under nitrogen for 23 hours. The reaction mixture was then partitioned between ethyl acetate (100 ml) and hydrochloric acid (1.5M, 150 ml) and the phases were separated. The organic phase was washed with water (100 ml) and brine (50 ml) over anhydrous MgSO₄Dry above and concentrate to give the title compound as a dark brown oil (65.8 g), which was used without further purification.

¹H NMR(CDCl₃400MHz) δ: 1.22 to 1.24(m, 9H), 1.47(s, 9H), 2.96(s, 2H), 3.57(s, 2H), 4.13(q, 2H), 4.27(s, 1H), 7.05(m, 2H), 7.15(m, 1H), 7.22(m, 1H) ppm

Preparation example 16: [3- (2-tert-Butoxycarbonylamino-2-methylpropyl) phenyl ] acetic acid

Sodium hydroxide (16.0 g; 400 mmol) and water (100 ml) were added to the solution obtained fromThe ethyl ester of preparation 15 (64.7 g; 193 mmol) in a cooled solution of THF (100 ml) and the resulting solution stirred at ambient temperature for about 16 hours. The solution was then acidified to pH 1 using hydrochloric acid and the product was extracted in ethyl acetate (2 × 200 ml). The combined organic extracts were washed with water and brine over anhydrous MgSO₄The mixture was dried and concentrated to give the title compound as a thick brown oil (57.3 g). Recrystallization from toluene/heptane gave the product as an off-white solid.

¹H NMR(CDCl₃，400MHz)δ：1.25(s，6H)、1.47(s，9H)、2.96(s，2H)、3.61(s，2H)、7.07(m，2H)、7.15(m，1H)、7.23(m，1H)ppm。

Alternative Process for the preparation of the product of preparation 16

Diisopropylethylamine (210 ml; 1.21 mol) was added to a suspension of the salt from preparation 5a (250 g; 0.40 mol) in propionitrile (1.0 l) to give a pale yellow solution. A solution of di-tert-butyl dicarbonate (97 g; 0.44 mol) in propionitrile (250 ml) is added and the resulting pale yellow solution is stirred at ambient temperature for 21 hours. Water (250 ml) was added and the mixture was stirred for 30 minutes. The phases were separated and the organic phase was washed successively with 10% aqueous citric acid (500 ml), water (300 ml), saturated aqueous sodium bicarbonate (500 ml) and brine (500 ml). The organic phase was then concentrated to a dark orange oil and dissolved in a mixture of tetrahydrofuran (250 ml) and water (250 ml). Sodium hydroxide (80 g; 2.0 mol) was added and the resulting mixture was stirred at ambient temperature for 91 hours. Toluene (400 ml) was added and the mixture was stirred for 30 minutes, then the phases were separated. The organic phase was extracted with a mixture of water (200 ml) and saturated aqueous sodium bicarbonate (100 ml). The combined aqueous phases were then adjusted to pH 1 with concentrated hydrochloric acid and extracted with ethyl acetate (2 × 250 ml). The combined ethyl acetate extracts were washed with water (2 × 200 ml) and then concentrated to dryness. The resulting oil was dissolved in refluxing toluene (100 ml) and heptane (-400 ml) was added. The mixture was cooled to ambient temperature and granulated for 3 hours. The solid was isolated by filtration, washed with heptane (2 × 200 ml), and dried in a vacuum oven at 40 ℃ to give the title compound as a pale yellow solid (110.9 g; 90%).

Preparation example 17: [2- (3- { [ (4' -hydroxybiphenyl-3-ylmethyl) -carbamoyl ] -methyl } -phenyl) -1, 1- (dimethyl) ethyl ] carbamic acid tert-butyl ester

A mixture of the acid from preparation 16 (25 g; 81.3 mmol), the amine hydrochloride from preparation 13 (18.2 g; 77.3 mmol), 4-dimethylaminopyridine (100 mg; 0.81 mmol) and diisopropylethylamine (22.1 g; 170.8 mmol) in acetonitrile (125 ml) was stirred at ambient temperature under nitrogen, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (17.15 g; 89.5 mmol) was added and the mixture was stirred at ambient temperature for 18 hours. Water (190 ml) was added and the resulting suspension was stirred for 1.5 hours. The solid was isolated by filtration, washed with water (100 ml) and dried using suction for 20 minutes. The wet cake was slurried in 10% aqueous citric acid (100 ml) for one hour. The solid was isolated by filtration and washed with water (100 ml) to give the title compound as a white solid (31.0 g; 82%).

¹H NMR(DMSO-d₆400MHz) δ: 1.11(s, 6H), 1.39(s, 9H), 2.85(s, 2H), 3.43(s, 2H), 4.30(d, 2H), 6.25(s, 1H), 6.82(d, 2H), 6.98(d, 1H), 7.03(s, 1H), 7.09-7.20 (m, 3H), 7.30(t, 1H), 7.35-7.42 (m, 4H), 8.50(s, 1H), 9.50(s, 1H).

Preparation example 18: {3- [2- (2, 2, 2-trichloro-acetylamino) -2-methyl-propyl ] -phenyl } -acetic acid

Trichloroacetonitrile (20 g, 0.14 mol) was added to a solution of the alcohol from preparation 3 (20 g, 0.09 mol) in acetic acid (40 ml). The resulting solution was cooled to 0 ℃, treated with concentrated sulfuric acid (98%; 30 ml), and the reaction mixture was gradually warmed to room temperature. After 4 hours, the reaction mixture was poured into ice/water (400 ml) and the solution was extracted with isopropyl acetate (2 × 200 ml). The combined organic layers were washed with demineralized water (120 ml) and then concentrated in vacuo to give a viscous brown oil. The oil was then treated with toluene (100 ml) and concentrated. The residue was then treated with heptane (100 ml) and filtered under vacuum to give the title product as an off-white solid (28.32 g).

¹H NMR(CD₃OD, 400MHz) δ: 1.39(s, 6H), 3.07(s, 2H), 3.56(s, 2H), 7.07 to 7.45(m, 1H), ppm.

Preparation example 19: 2, 2, 2-trichloro-N- [2- (3- { [ 4' -hydroxy-biphenyl-4-ylmethyl ] -carbamoyl } -methyl) -phenyl ] -1, 1-dimethyl-ethyl ] acetamide

The product of preparation 13 (19.8 g, 0.085 mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (24.45 g, 0.13 mol), 1-hydroxybenzotriazole (17.2 g, 0.13 mol), triethylamine (42.9 g, 0.42 mol) and the product from preparation 18 (30 g, 0.085 mol) were suspended in ethyl acetate and heated at 40 ℃ for 20 hours. The ethyl acetate solution was washed with water (4 × 150 ml), and then concentrated to give the title compound (33.7 g) as a brown solid.

¹H NMR(CD₃OD，400MHz)δ：1.34(s，6H)、3.01(s，2H)、3.53(s, 2H), 4.40(s, 2H), 6.82 to 7.41(m, 12H) ppm.

Preparation example 20: 2- [3- (2-amino-2-methylpropyl) -phenyl ] -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] acetamide

A suspension of Boc protected amine (28.0 g; 57.3 mmol) from preparation 17 in ethanol (100 mL) was treated with hydrochloric acid (4M in dioxane, 35 mL; 80 mmol) and the reaction stirred at ambient temperature for about 100 h. The reaction mixture was poured into a mixture of aqueous ammonia (35%, 30 ml) and water (200 ml). The product was then extracted with propionitrile (2X 50 mL) and n-butanol (100 mL). The combined organic phases were washed with water, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was slurried in acetone (100 ml) for about 18 hours and the resulting suspension was filtered and dried to give the title compound as an off-white solid (13.4 g).

¹H NMR(CD₃OD, 400MHz) δ: 1.09(s, 6H), 2.66(s, 2H), 3.56(s, 2H), 4.41(s, 2H), 6.82(d, 2H), 7.08 to 7.15(m, 3H), 7.20 to 7.42(m, 8H).

MS (electrospray) M/z 389 [ M + H ]]⁺，372[M-H₂O]⁺

Alternative Process for preparing the product of preparation 20

Alternative method 1:

a suspension of the protected amine from preparation 17 (31.0 g; 63.4 mmol) in dichloromethane (150 ml) was stirred under an inert atmosphere while trifluoroacetic acid (50 ml; 649 mmol) was added. The resulting light orange-brown solution was stirred for 1.5 hours and then concentrated under reduced pressure to give a thick brown oil. The oil was treated with a mixture of water and concentrated aqueous ammonia (9: 1, 250 ml) until a pH of 12 was reached, and the mixture was then extracted with a mixture of ethyl acetate and methanol (9: 1, 2X 150 ml). The combined organic extracts were washed with water and then concentrated under reduced pressure. The resulting foam was refluxed in acetone (500 ml) for one hour, then cooled to ambient temperature and pelletized overnight. The solid was isolated by filtration, washed with acetone, and dried in a vacuum oven at 40 ℃ to give the title compound as a white solid (13.42 g; 54%).

An alternative method 2:

the product from preparation 19 (33 g, 0.06 mol) was dissolved in a mixture of 4M aqueous potassium hydroxide (78.6 ml) and ethanol (78.6 ml) and stirred at 50 ℃ for 24 h. The mixture was partially concentrated under vacuum (to about 80 ml) and then extracted with ethyl acetate (4 × 40 ml). The organic extracts were combined and concentrated in vacuo to give the crude title product as a yellow oil (24.11 g). The material was suspended in acetone (120 ml), heated to reflux and the solution was cooled to room temperature over 10 hours and granulated at 5 ℃ for one hour then filtered under vacuum and washed with acetone (25 ml) to give the title compound (7 g) as a white solid.

Alternative method 3:

1-hydroxybenzotriazole hydrate (11.93 g; 0.08 mol), amine hydrochloride from preparation 13 (45.78 g; 0.19 mol) and triethylamine (35.73 g; 0.35 mol) were added successively to a solution of the alcohol from preparation 3 (36.77 g; 0.18 mol) in dichloromethane (368 ml), the solution was stirred for one hour, then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (33.84 g; 0.18 mol) was added and the mixture was stirred at ambient temperature for 2 hours. Tetrahydrofuran (184 ml) was added and the resulting solution was washed successively with water (2 × 184 ml), 1M aqueous hydrochloric acid (2 × 184 ml), and 1M aqueous potassium bicarbonate (2 × 184 ml). The organic solution was distilled and replaced with chloroacetonitrile (132 ml). Trifluoroacetic acid (331 ml) was added to the chloroacetonitrile solution, and the resulting mixture was heated to 50 ℃ for 2 hours. Dichloromethane (331 ml) was added and the organic phase was washed with water (2 x 662 ml) followed by 1M aqueous potassium bicarbonate (2 x 331 ml). The organic solution was then distilled and replaced with acetic acid (404 ml). Thiourea (44 g; 0.58 mol) was added to an aliquot of this solution (250 ml) and the resulting suspension was heated to 100 ℃ for 3 hours. The suspension was filtered and the filter cake was washed with acetic acid (54 ml). The acetic acid solution was diluted with water (774 ml) and the aqueous layer was washed with a mixture of dichloromethane and methanol (9: 1,2 × 242 ml). Methanol (53 ml) was added to the aqueous phase and the pH was adjusted to > 9 using concentrated ammonia (-230 ml) keeping the temperature below 15 ℃. Dichloromethane (480 ml) was added and the mixture was stirred for 30 minutes. The phases were then separated and the organic phase was distilled and replaced with acetone (-440 ml). The resulting suspension was cooled to ambient temperature and stirred for 18 hours, then granulated at 5 ℃ for 2 hours. The product was collected by filtration and washed with acetone (2 × 45 ml) to give the title compound (11.39 g) as a pale yellow solid.

Preparation example 21: 2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (dimethylsulfonyl) amino ] phenyl } -2-hydroxyethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide

A mixture of the amine from preparation 20 (500 mg; 1.29 mmol) and the epoxide from preparation 10 (670 mg; 1.69 mmol) in butyronitrile (2 ml) was heated at reflux under an inert atmosphere for 20 hours. The mixture was then cooled to ambient temperature and chromatographed directly on silica gel (40 g) eluting with methanol-dichloromethane (1: 19 to 1: 9) to give the title compound as a waxy oil (543 mg).

¹H NMR(CD₃OD，400MHz)δ：1.00(s，3H)、1.03(s，3H)、2.66(dd，2H)、2.82(m，2H)、3.31(s, 6H), 3.55(s, 2H), 4.40(s, 2H), 4.69(dd, 1H), 5.16(s, 2H), 6.82(d, 2H), 7.03 to 7.54(m, 18H).

MS (electrospray) M/z 786 [ M + H ]]⁺

Preparation example 22: 2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (methylsulfonyl) amino ] phenyl } -2-hydroxyethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide

A solution of sodium hydroxide (500 mg; 12.5 mmol) in water (5 ml) was added to a solution of the bis-sulfonamide from preparative example 21 (500 mg; 0.64 mmol) in ethanol (5 ml) and the resulting yellow solution was stirred at ambient temperature for 14 days. The mixture was then diluted with water (10 ml) and washed with dichloromethane (10 ml). The aqueous phase was adjusted to pH 1 using hydrochloric acid and extracted with propionitrile (2 × 20 ml). The combined propionitrile extracts were washed with water and anhydrous MgSO₄Dried, filtered and concentrated to give the title compound (272 mg) as a pale yellow glassy solid.

¹H NMR(CD₃OD, 400MHz) δ: 1.03(s, 3H), 1.05(s, 3H), 2.68(dd, 2H), 2.78 to 2.90(m, 4H), 3.34(s, 3H), 3.54(s, 2H), 4.40(s, 2H), 4.66(dd, 1H), 5.18(s, 2H), 6.81(m, 2H), 7.01 to 7.40(m, 16H), 7.43 to 7.48(m, 2H).

MS (electrospray) M/z 708[ M + H ]]⁺

Alternative Process for preparing the product of preparation 22

Alternative method 1:

the crude silyl ether from preparation 24 (1.24 g; assumed to be 1.7 mmol) was dissolved in a mixture of THF (5 mL) and methanol (1 mL). Additive for foodTriethylamine trihydrofluoride (0.5 ml; 3.1 mmol) was added and the mixture was stirred at ambient temperature for 8 h. The reaction was quenched with aqueous ammonia (35%, 10 ml) and extracted with propionitrile (2 × 20 ml). The combined propionitrile extracts were washed with water and anhydrous MgSO₄Dried, filtered and concentrated to give a brown foam. This was chromatographed on silica gel eluting with methanol-dichloromethane (1: 9) to give the title compound as an off-white foam (474 mg).

An alternative method 2:

the crude silyl ether from preparation 25 (3.0 g; assuming 3.6 mmol) was dissolved in THF (15 mL). Triethyl trihydrofluoride (1.5 ml; 9.2 mmol) was added and after 10 minutes ethanol (0.5 ml) was added. The light orange solution was stirred at ambient temperature for 3 hours, then aqueous ammonia (35%, 10 ml) was added and the product was extracted in propionitrile (2 × 20 ml). The combined organic phases were washed with water, over anhydrous MgSO₄Dry, filter and concentrate to give the title compound as a light brown foam (2.6 g, 70% pure).

Alternative method 3:

a mixture of protected bromohydrin from preparative example 7 (13.21 g; 25.7 mmol), amine from preparative example 20 (9.50 g; 24.4 mmol) and sodium bicarbonate (4.11 g; 48.9 mmol) in n-butyl acetate (29 ml) was refluxed under nitrogen for 24 h. The mixture was cooled to ambient temperature and diluted with water (30 ml) and ethyl acetate (114 ml). The phases were separated and the organic phase was washed successively with 1M (L) -aqueous tartaric acid (25 ml), water-concentrated aqueous ammonia (3: 1, 40 ml) and water (19 ml). Methanol (19 ml) and triethylamine trihydrofluoride (4.5 ml; 27.6 mmol) were added successively and the resulting mixture was stirred at ambient temperature under nitrogen. After one hour, another aliquot of methanol (9.5 ml) was added. After 6 hours, the reaction was quenched with a mixture of water and concentrated aqueous ammonia (3: 1, 40 ml) and stirred for 15 minutes. The phases were separated and the organic phase was washed with water (47.5 ml) and ethyl acetate was then distilled under reduced pressure to give an n-ethyl acetate solution of the title compound which was used directly in preparation 22 a.

Preparation 22 a: 2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (methylsulfonyl) amino ] phenyl } -2-hydroxyethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide, dibenzoyl- (L) -tartrate

A solution of dibenzoyl- (L) -tartaric acid (8.74 g; 24.4 mmol) in 2-butanone (19 ml) was added to a solution of the amine from preparation 22 (alternative method 3) in n-butyl acetate to give a thick gum. The mixture was then diluted with 2-butanone (76 ml) and warmed to 40 ℃ to give an orange solution. The orange solution was then cooled to ambient temperature and added to tert-butyl methyl ether (285 ml) under vigorous stirring at ambient temperature for 15 minutes, washed with 2-butanone (2X 9.5 ml) and the resulting slurry granulated at ambient temperature for 18 hours. The solid was isolated by filtration and washed with tert-butyl methyl ether (2 × 95 ml) to give the title compound (24.64 g) (estimated as an approximately 4: 3 mixture of amine and acid components and containing a portion of tert-butyl methyl ether) as an off-white solid.

¹H NMR(DMSO-d₆400MHz) δ: 1.02(s, 6H), 2.70 to 3.10(m, 6H), 3.40(s, 3H), 4.25(d, 2H), 4.65(br.d, 1H), 5.18(s, 2H), 5.60(s, 1.5H), 6.81(d, 2H), 6.90 to 7.60(m, 23.5H), 7.90(m, 2H), 8.55(t, 1H).

Alternative Process for preparing the product of preparation 22a

A solution of dibenzoyl- (L) -tartaric acid (9.0 g; 25.1 mmol) in 2-butanone (50 ml) was added to a solution of the product of preparation 22 (16.9 g) in butyronitrile (35 ml). An additional 50 ml of 2-butanone was added to completely dissolve all material. The solution was then added to tert-butyl methyl ether (500 ml) under vigorous stirring at ambient temperature for 10 minutes and washed with 2-butanone (20 ml). Tert-butyl methyl ether (100 ml) was added to the slurry and then aged at ambient temperature for 3 hours. The solid was isolated by filtration and washed with tert-butyl methyl ether (200 ml) to give the title compound (20.86 g) as a yellow solid.

Preparation example 23: 2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (dimethylsulfonyl) amino ] phenyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide

A mixture of protected bromohydrin (1.0 g; 1.7 mmol) from preparation 8, amine (650 mg; 1.67 mmol) from preparation 20 and sodium bicarbonate (560 mg; 6.7 mmol) in butyronitrile (2 ml) was heated under reflux under an inert atmosphere for 31 h. The reaction mixture was then cooled to ambient temperature and diluted with propionitrile (10 ml) and water (10 ml). Separating the phases; the organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to give the title compound (1.54 g), which was used directly in the next step without purification.

Preparation example 24: 2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (methylsulfonyl) amino ] phenyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide

The crude bis-sulfonamide from preparation 23 (1.54 g; assuming 1.7 mmol) was dissolved in ethanol (5 ml). Water (5 ml) and sodium hydroxide (600 mg; 15 mmol) were added and the resulting mixture was stirred at ambient temperature for 72 h. The mixture was then acidified to pH 1 with concentrated hydrochloric acid, followed by neutralization with aqueous ammonia (35%) (to pH 10). The product was then extracted in propionitrile (2X 20 ml). The combined organic extracts were dried over anhydrous magnesium sulfate, filtered and the solution concentrated in vacuo to give the title compound as a yellow oil (1.24 g), which was used in the next step without purification.

Preparation example 25: 2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (methylsulfonyl) amino ] phenyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide

A mixture of protected bromohydrin from preparative example 7 (2.0 g; 3.92 mmol), amine from preparative example 20 (1.5 g; 3.86 mmol) and sodium bicarbonate (1.0 g; 11.9 mmol) in butyronitrile (4 mL) was refluxed under an inert atmosphere for about 30 hours. The cooled reaction mixture was then diluted with propionitrile (20 ml), washed with water (2 × 10 ml), dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to give the title product (3.05 g, 80% pure) which was used in the next step without further purification.

Preparation example 26: n- [ (4' -hydroxybiphenyl-3-yl) methyl ] -2- (3- (2- [ ((2R) -2-hydroxy-2- { 4-hydroxy-3- [ (methylsulfonyl) amino ] phenyl } ethyl) amino ] -2-methylpropyl } phenyl) acetamide

Palladium hydroxide (20 wt% on carbon; 60 mg) was added to a solution of the benzyl ether from preparation 22 (613 mg; 0.87 mmol) in a mixture of ethanol (4.5 ml) and water (1.5 ml). The mixture was placed under a hydrogen atmosphere (60psi) and stirred at 60 ℃ for 18 hours. The reaction mixture was then purged with nitrogen, diluted with a solution of ammonia (35%) in ethanol (1: 9,. about.15 ml), filtered through Celite (Celite), and washed with a solution of ammonia (35%) in ethanol (1: 9,. about.15 ml) and ethanol (. about.10 ml). The liquid was concentrated to a residue, which was then dissolved in a mixture of ammonia (35%) and THF (1: 19,. -10 ml) and filtered through a pad of silica, and washed with ammonia (35%)/THF (1: 19,. -250 ml). The liquid was concentrated to a residue, slurried in refluxing methanol (10 ml), then cooled to ambient temperature and stirred for 18 hours. The precipitate was collected by filtration and washed with methanol to give the title compound (296 mg) as an off-white solid.

¹H NMR(DMSO-d₆400MHz) δ: 0.88(s, 3H), 0.90(s, 3H), 2.54(s, 2H), 2.62(m, 2H), 2.88(s, 3H), 3.44(s, 2H), 4.30(d, 2H), 4.41(dd, 1H), 6.81(m, 3H), 6.98(m, 2H), 7.05 to 7.18(m, 5H), 7.25 to 7.42(m, 5H), 8.49(t, 1H) ppm.

Alternative Process for the preparation of the product of preparation 26

Alternative method 1:

a mixture of the salt from preparation 22a (6.7 g; 6.74 mmol), tetrahydrofuran (67 ml) and concentrated aqueous ammonia (10 ml) was stirred vigorously for 15 minutes. The phases were separated and the organic phase was washed with a mixture of water (10 ml) and saturated brine (10 ml). The tetrahydrofuran solution was then distilled at a constant volume (50 to 60 ml) and additional tetrahydrofuran was added if necessary until a total of 60 ml distillate had been collected. The solution was then diluted with tetrahydrofuran (total volume about 84 ml) and water (18 ml) and palladium on carbon (5%, 50% water wet; 670 mg) was added, then the resulting mixture was hydrogenated at 40 ℃/50psi hydrogen pressure for 31 hours and the catalyst (500 mg and 600 mg) was added after 8 hours and 24 hours, respectively. The mixture was removed from the hydrogenation reactor and Arbocel (5 g) was added and the mixture was stirred for 20 minutes. The resulting slurry was filtered through an Arbocel pad and washed with tetrahydrofuran/water (9: 1, ca. 50 ml). The filtrate was then diluted with acetonitrile (85 ml) and tetrahydrofuran was removed by distillation. Once the vapor temperature reached 76 ℃, 20 ml more acetonitrile was added and then 20 ml more distillate was collected. The resulting slurry was cooled to ambient temperature and allowed to cure for 16 hours. The solid was collected by filtration, washed with acetonitrile-water (9: 1, 40 ml) and dried under vacuum for 20 minutes. The wet cake was then slurried in methanol-water (9: 1, 40 ml), initially at 50 ℃ for one hour, and then at ambient temperature for 16 hours. The precipitate was isolated by filtration and washed with methanol-water (8: 2, 40 ml) to give the title compound as an off-white solid (2.25 g; 54%).

An alternative method 2:

a mixture of the salt from preparation 22a (11.26 g; 11.6 mmol), 2-methyltetrahydrofuran (100 ml), concentrated aqueous ammonia (50 ml) and water (150 ml) was stirred vigorously for one hour. The phases were separated and the aqueous phase was back-extracted with 2-methyltetrahydrofuran (20 ml). The combined organic phases were washed with water (50 ml), then diluted with ethylene glycol (100 ml) and the 2-methyltetrahydrofuran was removed by distillation under reduced pressure. A palladium on carbon catalyst (5%, 50% water wet; 1100 mg) was added and the resulting mixture was hydrogenated at 40 ℃/50psi hydrogen pressure for 18 hours. The mixture was removed from the hydrogenation reactor and Arbocel (5 g) was added. The resulting mixture was stirred for 30 minutes, then filtered through a pad of Arbocel, washed with ethylene glycol (25 ml). Fresh palladium on carbon catalyst (5%, 50% water wet; 1100 mg) was added and the resulting mixture was hydrogenated at 40 ℃/50psi hydrogen pressure for 7 hours and then at 40 ℃/80psi for 16 hours. Additional palladium on carbon (5%, 50% water wet; 1000 mg) was then added and the resulting mixture was hydrogenated at 40 ℃/80psi hydrogen pressure for 4 hours. The mixture was removed from the hydrogenation reactor and Arbocel (10 g) was added. The resulting mixture was stirred for 30 minutes, then filtered through a pad of Arbocel, washed with ethylene glycol (25 ml). The glycol filtrate was then added to water (200 ml) with vigorous stirring for about 10 minutes, washed with additional glycol (20 ml) and water (100 ml), and the resulting light brown color was stirred at ambient temperature for 30 minutes. The solid was isolated by filtration, washed with water (100 ml) and dried in a vacuum oven at 40 ℃. The light brown solid formed was further purified by slurrying in methanol-water (9: 1, 54 ml), initially at 50 ℃ for 2 hours and then at ambient temperature for 16 hours. The precipitate was isolated by filtration and washed with methanol-water (8: 2, 15 ml) to give the title compound as an off-white solid (4.57 g; 64%).

Alternative method 3:

a mixture of protected bromohydrin (10.93 g; 21.2 mmol) from preparation 7, amine (7.50 g; 19.3 mmol) from preparation 20, and sodium bicarbonate (9.0 g; 107.1 mmol) in n-butyl acetate (55 mL) was refluxed under nitrogen for 53 hours. The mixture was cooled to ambient temperature and diluted with water (180 ml) and ethyl acetate (180 ml). The phases were separated and the organic phase was washed successively with 1M (L) -aqueous tartaric acid (55 ml), water-concentrated aqueous ammonia (3: 1, 60 ml) and water (55 ml). A palladium on carbon catalyst (5%, 50% water wet; 1300 mg) was added and the resulting mixture was hydrogenated at 60 ℃/60psi hydrogen pressure for 24 hours. The reaction mixture was removed from the hydrogenation reactor and Arbocel (13 g) was added and the resulting slurry was stirred for 30 minutes. The mixture was then filtered through a pad of Arbocel and the catalyst bed was washed with ethyl acetate (200 ml). The pale yellow filtrate was then concentrated under reduced pressure to remove ethyl acetate, then methanol (60 ml) was added and the mixture was concentrated to dryness under reduced pressure. The viscous orange-brown oil formed was dissolved in methanol (100 ml) and placed in a polypropylene container. Ammonium fluoride (2.1 g; 56.7 mmol) was added, washed with water (20 ml) and methanol (20 ml) and the resulting solution was stirred at ambient temperature for 65 hours. The precipitated solid was isolated by filtration, washed with methanol-water (8: 2, 100 ml), and dried under suction for 10 minutes, then dried in a vacuum oven at 40 ℃ for 4 hours. The light brown solid was then slurried in methanol-water (9: 1, 75 ml), initially at 50 ℃ for 2 hours and then at ambient temperature for 16 hours. The precipitate was isolated by filtration, washed with methanol-water (8: 2, 2 × 20 ml) and dried in a vacuum oven at 40 ℃, and the solid was further purified by slurrying in water (80 ml) at ambient temperature for 16 hours. The solid was isolated by filtration and washed with water (50 ml) to give the title compound as an off-white solid (6.01 g; 50%).

Claims (46)

1. A process for the preparation of a compound of formula (I),

wherein Q¹Is selected from

And group^*-NR⁶-Q²A radical of formula (A), wherein the symbols^*Represents the point of attachment to the carbonyl group, p is 1 or 2, Q²Is C optionally substituted by one hydroxy group₁To C₄Alkylene radical, R⁶Is H or C₁To C₄Alkyl, and A is pyridyl optionally substituted with OH, C optionally substituted with OH₃To C₇Cycloalkyl radicals, or the following groups

Wherein R is¹、R²、R³、R⁴And R⁵Same or different and selected from H, C₁To C₄Alkyl, OR⁷、SR⁷Halogen, CN, CF₃、OCF₃、COOR⁷、SO₂NR⁷R⁸、CONR⁷R⁸、NR⁷R⁸、NHCOR⁷And phenyl optionally substituted by 1 to 3 substituents selected from OR⁷Halogen and C₁To C₄Radical substitution of alkyl, wherein R⁷And R⁸Are identical or different and are selected from H or C₁To C₄An alkyl group;

or, where appropriate, a pharmaceutically acceptable salt thereof and/or an isomer, tautomer, solvate or isotopic variant thereof, which comprises the use of a compound of the formula

2. A method according to claim 1, comprising reacting the compound of formula (7) with a compound of formula (5),

or the step of reacting the compound of formula (6)

Wherein PG²As a suitable phenol protecting group, PG³Is a suitable hydroxy protecting group, LG is a suitable leaving group, and R⁹Is H or SO₂CH₃。

3. A process according to claim 2, which comprises reacting the compound of formula (7) with a compound of formula (5)

Wherein R is⁹Is H, to obtain a compound of formula (3)

4. A process according to claim 3, which comprises two deprotection steps.

5. A method according to claim 3 or 4, comprising a first deprotection step to remove PG³And obtaining the compound of formula (2)

Or a salt thereof.

6. A process according to claim 2, which comprises reacting the compound of formula (7) with a compound of formula (5)

Wherein R is⁹Is SO₂CH₃To obtain a compound of formula (3a)

7. The method according to claim 6, comprising 3 deprotection steps.

8. A process according to claim 6 or 7, comprising a deprotection step to remove PG³And a compound of formula (4) is obtained

9. A process according to any one of claims 6 to 8, comprising a deprotection step to remove SO₂CH₃Radical and obtaining a compound of formula (2)

Or a salt thereof.

10. A process according to claim 2, which comprises reacting the compound of formula (7) with a compound of formula (6)

Wherein PG²As a suitable phenol protecting group to obtain a compound of formula (4)Step (ii) of

11. A process according to claim 10, comprising two deprotection steps to remove SO₂CH₃Group and PG²。

12. A process according to claim 10 or 11, wherein the process comprises a first deprotection step to remove SO₂CH₃Radical and obtaining a compound of formula (2)

Or a salt thereof.

13. A process according to claim 5, 9 or 12 comprising a deprotection step to remove PG²And obtaining a compound of formula (I) as defined in claim 1.

14. A method according to any one of claims 2 to 9 wherein LG is bromide.

15. A process according to any one of claims 2 to 9 and 14, wherein PG is³Is TBDMS.

16. A method according to any one of claims 2 to 15, wherein PG is²Is benzyl.

17. A process according to any one of claims 1 to 16, wherein the compound of formula (10) is prepared by a process comprising reacting a compound of formula (10)

Wherein PG¹Is a suitable amino protecting group, with Q¹-H or a salt thereof, wherein Q¹As defined in claim 1, wherein the first and second groups are,

a process for the step of obtaining a compound of formula (8), preparing said compound of formula (7),

18. a process according to any one of claims 1 to 16, wherein the compound of formula (19) is prepared by a process comprising reacting a compound of formula (19)

A process for the step of reacting with an alkylnitrile or arylnitrile to obtain a compound of formula (8), a process for the preparation of said compound of formula (7),

19. a process according to claim 18, wherein the reaction is carried out by reacting a compound of formula (15)

And Q¹-H or a salt thereof, wherein Q¹A compound of formula (19) as defined in claim 1, is prepared.

20. A method according to claim 17 or 18 comprising a deprotection step to remove PG¹And obtaining said compound of formula (7).

21. A process according to claim 17, wherein the compound of formula (11) is hydrolyzed by a process comprising the step of hydrolyzing the compound of formula (11),

preparing the compound of formula (10).

22. The method according to claim 21, wherein the compound of formula (12) is protected by a method comprising the step of protecting the compound of formula (12),

preparing the compound of formula (11).

23. The method according to claim 17, wherein the compound of formula (15) is protected by a method comprising the step of protecting the compound of formula (15),

preparing the compound of formula (10).

24. A method according to claim 17, 18, 20 or 21, wherein PG is¹Is threeChloroacetyl or chloroacetyl.

25. The method of claim 17, wherein PG is¹Is trichloroacetyl.

26. The method of claim 18, wherein PG is¹Is chloroacetyl.

27. A method according to claim 17, 18, 20 or 21, wherein PG is¹Is Boc.

28. A process for preparing a compound of formula (16)

The method comprises the following steps: hydrolyzing the compound of formula (18) in the presence of an enzyme selected from the group consisting of lipase, esterase or protease

29. The method according to claim 28, wherein the enzyme is selected from the group consisting of Mucor miehei esterase, Rhizomucor lipase, Thermomyces lanuginosus lipase, penicillin acylase.

30. The method according to claim 28 or 29, wherein the enzyme is thermomyces lanuginosus lipase.

31. A process according to any one of claims 28 to 30, wherein the reaction is carried out at a pH between 5 and 9 and a temperature between 10 ℃ and 40 ℃ in the presence of a suitable buffer and optionally in the presence of a suitable base.

32. A method according to any one of claims 1 to 27 wherein Q is¹Is a group of the formula

Wherein R is¹To R⁵As defined in claim 1.

33. The method according to claim 32, wherein R¹、R²、R³、R⁴And R⁵Same or different and selected from H, C₁To C₄Alkyl, OR⁶、SR⁶Halogen, CF₃、OCF₃、SO₂NR⁶R⁷、CONR⁶R⁷、NR⁶R⁷、NHCOR⁷Provided that R is¹To R⁵At least two of which are H;

wherein R is⁶And R⁷Are identical or different and are selected from H or C₁To C₄An alkyl group.

34. The method according to claim 32, wherein R¹、R²、R³、R⁴And R⁵Identical or different and selected from H, OH, CH₃、OCH₂-CH₃、SCH₃Halogen, CF₃、OCF₃Provided that R is¹To R⁵At least two of which are H.

35. A method according to any one of claims 32 to 34, wherein R is¹To R⁵One of them is OH.

36. The method according to claim 32, wherein R¹、R²、R³、R⁴And R⁵One of them is phenyl substituted by OH, and the others are H.

37. The method according to claim 32, wherein R²Is 4-hydroxy-phenyl, and R¹、R³、R⁴And R⁵Is H.

38. The method according to claim 32, wherein R²And R³Is Cl, and R¹、R⁴And R⁵Is H.

39. A process according to any one of claims 33 to 37 wherein the hydroxy group or OPG is substituted³The asymmetric carbon substituted by the group is in the R configuration.

40. A compound of the formula

Wherein Q¹As defined in claim 1, R¹⁰Is H or PG²Wherein PG is²As a suitable phenol protecting group, R⁹Is H or PG³Wherein PG is³Is a suitable hydroxy protecting group, and R¹¹Is H or PG¹Or in which PG¹Are suitable amino protecting groups.

41. A compound of the formula according to claim 40,

wherein Q¹As defined in any one of claims 32 to 38.

42. A compound of the formula according to claim 41,

wherein is OR-ed⁹The substituted asymmetric carbon is in the R configuration.

43. A compound of the formula according to claim 40,

wherein PG²Is benzyl.

44. A compound according to claim 43, having the formula:

45. a compound of the formula according to claim 42,

wherein R is¹¹Is H.

46. A compound according to claim 40, selected from:

2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (dimethylsulfonyl) amino ] phenyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide;

2- (3- {2- [ ((2R) -2- { 4-benzyloxy-3- [ (dimethylsulfonyl) amino ] phenyl } -2-hydroxyethyl) amino ] -2-methylpropyl } phenyl) -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] -acetamide;

tert-butyl- [2- (3- { [ (4' -hydroxybiphenyl-3-ylmethyl) -carbamoyl ] -methyl } -phenyl) -1, 1- (dimethyl) ethyl ] carbamate;

2, 2, 2-trichloro-N- [2- (3- { [ 4' -hydroxy-biphenyl-4-ylmethyl) carbamoyl ] -methyl } -phenyl) -1, 1-dimethyl-ethyl ] acetamide;

2-chloro-N- {2- [3- (2- { [ (4' -hydroxybiphenyl-3-yl) methyl ] amino } -2-oxoethyl) phenyl ] -1, 1-dimethylethyl } acetamide;

2- [3- (2-amino-2-methylpropyl) -phenyl ] -N- [ (4' -hydroxybiphenyl-3-yl) methyl ] acetamide and

n- [ (R) -2-benzyloxy-5-oxiranyl-phenyl ] -dimethanesulfonamide.