HK1035001B - Method for enzymatic enantiomer-separation of 3(r)-and 3(s)-hydroxy-1-methyl-4-(2,4,6-trimethoxyphe nyl)-1,2,3,6-tetrahydro-pyridine or its carboxylic acid esters - Google Patents

Description

Method for the enzymatic separation of enantiomers of 3(R) -and 3(S) -hydroxy-1-methyl-4- (2, 4, 6-trimethoxyphenyl) -1, 2, 3, 6-tetrahydropyridine or its carboxylic esters

The invention relates to a process for the preparation of optically pure compounds of formula (I) by stereospecific reaction of a mixture of enantiomers by means of an enzyme.

3(S) -and 3(R) -hydroxy-1-methyl-4- (2, 4, 6-trimethoxyphenyl) -1, 2, 3, 6-tetrahydropyridine (compound of formula (I) wherein R ═ H) or an ester derivative thereof (compound of formula (I) wherein R ═ COR)¹Compounds of (ii) are important units or precursors for the synthesis of: the first potent inhibition of flavopiridol (HMR 1275 or L868275), cyclin-dependent protein kinase, described in patent application HMR 98/L001 ("(-) cis-3-hydroxy-1-methyl-4 (R) - (2, 4, 6-trimethoxyphenyl) piperidine preparation methodFormulations (see, for example, Sedlacek, Hans Harald; Czech, Joerg; Naik, Ramachandra; Kaur, Gurmet; Worland, Peter; Losiewicz, Michael; Parker, Bernard; Carlson, Bradley; Smith, Adaline et al, "Flavopiridol (L868275; NSC 649890), a novel kinase inhibitor for tumor therapy" [ International journal of Oncology ] (int.J.Oncol.) (1996), 9(6), 1143. f-1168 or Czeg, Joerg; Hoffmann, Dieter; Naik, Ramachandra; Sedlacek, Hans-Harald "[ antitumor Activity of flavone L868275 ] [ International journal of Oncology ] (1995), (6, 31-36) ].

The racemate resolution or the enantiomeric separation of the compounds of the formula (I) is unknown.

It has now been found that optically pure forms of the compounds of the formula (I) can be obtained from enantiomeric mixtures by enzymatic ester cleavage (hydrolysis or alcoholysis).

The invention therefore relates to a process for the dynamic resolution of racemates of compounds of formula (I),

wherein

R is COR¹Wherein R is¹＝(C₁-C₁₆) Alkyl radicals, (C)₂-C₁₆) -alkenyl or (C)₃-C₁₆) -alkynyl, C_nH_2n-cycloalkyl, wherein n ═ 1-16, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from F, Cl, Br, I, CF₃、CN、NO₂Hydroxy, methoxy, ethoxy and COOR²Wherein R is²＝(C₁-C₄) -alkyl and (C)₂-C₄) Alkenyl, which may be branched or unbranched and which may be substituted by 1 to 3 substituents selected from F, Cl, Br, CF₃，

The process comprises subjecting an enantiomeric or racemic mixture of a compound of formula (I), preferably containing from 2 to 50% by weight of an ester, to stereoselective hydrolysis or alcoholysis at a temperature of from 10 to 80 ℃ in a homogeneous or heterogeneous, aqueous/organic or organic medium in the presence of an enzyme, for example a lipase or esterase, for example from mammalian liver or pancreas or microbial sources, for example from Candida, Pseudomonas and Aspergillus, or a protease, for example from Bacillus, with or without co-solvents and buffers,

after the reaction has taken place, the unreacted ester (R ═ COR in formula (I)) is isolated¹Compound(s) and the alcohol formed (compound of formula (I) wherein R ═ H), and thus the two enantiomers.

The process according to the invention is economical, simple and rapid. The reaction does not require any equimolar amounts of optically pure auxiliary, expensive reagents, disproportionately large amounts of solvents and any costly work-up steps. After the reaction is complete, simple measures can be taken to effect separation of the product or the enantiomer, for example extraction.

Preferably, in the compounds of formula (I),

r is COR¹Wherein R is¹＝(C₁-C₁₂) Alkyl radicals, (C)₂-C₁₂) -alkenyl or (C)₃-C₁₂) -alkynyl, C_nH_2nCycloalkyl, where n ═ 1-12, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from the group consisting of F, Cl, Br, CF₃、CN、NO₂Hydroxy, methoxy, ethoxy and COOR²Wherein R is²Methyl, ethyl and vinyl, which may be substituted with 1 to 3 substituents selected from the group consisting of F, Cl, CF₃。

Particularly preferably, in the compounds of the formula (I),

r is COR¹Wherein R is¹＝(C₁-C₁₀) Alkyl radicals, (C)₂-C₁₀) -alkenesRadical or (C)₃-C₁₀) -alkynyl, C_nH_2nCycloalkyl, where n ═ 1 to 10, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from the group consisting of F, Cl, Br, CF₃、CN、NO₂Methoxy, and COOR²Wherein R is²Methyl, ethyl and vinyl, which may be substituted with 1 to 3 substituents selected from the group consisting of F, Cl, CF₃。

Very particularly preferably, in the compounds of the formula (I),

r is COR¹Wherein R is¹＝(C₁-C₁₀) Alkyl radicals, (C)₂-C₁₀) -alkenyl or (C)₃-C₁₀) Alkynyl, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from F, Cl, Br, CF₃And a methoxy group.

Preferably used is the procedure of treating the ester of formula (I) in which for example R ═ COR, with a lipase, esterase or protease in aqueous or alcoholic solution and stirring¹Wherein R is¹＝C₃H₇Or C₈H₁₇. It is advantageous to buffer the solution, for example with phosphate or TRIS (hydroxymethyl) methylamine) buffer. The amount of addition may be, for example, 0.01 to 1.0 mol. A suitable buffer range is pH 5-9.

It may also be advantageous to add a co-solvent. Suitable co-solvents are, for example, dimethoxyethane, acetone, THF, dioxane, hexane, tert-butyl methyl ether and tert-butanol. The proportion of co-solvent in the solution is preferably from 10 to 80%.

The enzymes used are preferably lipases and esterases, for example cholesterol esterase from bovine pancreas (EC3.1.1.13) (Sigma Chemical Co.), pig liver esterase (PLE, Sigma Chemical Co.), pancreatin (Fluka and Sigma Chemical Co.), pancreas acetone powder from bovine pancreas (Sigmachemical Co.), liver acetone powder from equine (Sigma Chemical Co.) and lipase from porcine pancreas (PPL, Sigma Chemical Co.), lipase OF from Candida rugosa (Meito gyo), lipase AP-6 from Aspergillus niger (Amano Pharmaceuticals).

The various enzymes can be used in free form or in immobilized form (immobilized biocatalyst W.Hartmeier, Springer Verlag Berlin, 1988). The amount of enzyme is freely chosen, depending on the reaction rate or the desired reaction time and the nature of the enzyme (e.g.free or immobilized), and is readily determinable by simple preliminary experiments.

The reaction mixture preferably contains from 2 to 50% by weight of ester, particularly preferably from 5 to 20%. The reaction temperature is 10 to 80 ℃, preferably 20 to 60 ℃, particularly preferably 20 to 40 ℃.

Esters (formula (I) R ═ COR)¹Compounds of (ii)) are suitably prepared according to known esterification methods (Haslam tetrahedron 1980, 36, 2409; hoefle, Steglich, vorberreggen, applied chemistry (angelw. chem.)1978, 90, 602) from alcohols (compounds of formula (I) where R ═ H), or as described in patent application HMR 98/L001 ("(-) cis-3-hydroxy-1-methyl-4 (R) - (2, 4, 6-trimethoxyphenyl) piperidine preparation").

The product obtained or the remaining product of the process can be isolated in a simple manner, for example by extraction or chromatography. For example, the reaction solution is partitioned between water and n-heptane, and the organic phase is concentrated to give the remaining ester. The resulting alcohol can then be extracted from the aqueous phase with ethyl acetate. The enzyme may be recovered by freeze-drying. The isolation of the enzyme (and possible subsequent reuse) can be facilitated by immobilization.

By carrying out the reaction appropriately, it is always possible to obtain at least one optically pure enantiomer. If it is desired to obtain an optically pure ester, the conversion should exceed (or be equal to) 50%, and if it is desired to obtain an optically pure alcohol, the conversion should be less than (or be equal to) 50%. The conversion by enzymatic hydrolysis or alcoholysis is determined by HPLC (RP 18 LiChrosorb ) and the optical purity is determined by HPLC (Chiralpak AD). The ester produced by the racemate resolution process or the remaining ester can be converted into the corresponding alcohol by known ester cleavage methods (S.J.Salomon, E.G.Mata, O.A.Mascaretti, tetrahedron, 1993, 49, 3691-one 3748) without inversion or racemization. Instead, the resulting alcohol can be converted into the corresponding ester by known esterification methods (Haslam, tetrahedron, 1980, 36, 2409) without inversion or racemization.

The products obtained or the remaining products of the process can be racemized and reused in the racemate resolution according to known methods, for example metal-catalyzed rearrangement (L.E.Overman, applied Chemicals 1984, 96, 565-573 and the documents already cited). Thus, the yield can be improved to more than 50%. For example, in formula (I) R ═ COR¹The compounds of formula (I) wherein R ═ H can be racemized directly, for example after conversion to the appropriate derivative, as described in l.e. overlaman, applied chemistry, 1994, 96, 565-. Metal catalysts which may be used are, for example, Hg (II), Pd (O) or Pd (II) compounds or salts.

The invention will be illustrated in detail by the following examples.

Example (b):

all separated products or crude product mixtures¹H-NMR and mass spectrometry or HPLC identification.

The optical purity of the product is determined by HPLC, for example Chiralpak AD 250X 4.6 (Daicel).

Example 1:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of acetate (R ═ COR in formula I)¹、R¹＝CH₃The compound of (1). 5mg of pancreatin were added. The mixture was stirred at 20-25 ℃ until a conversion of about 40% (HPLC) was reached. Then filtered, concentrated to dryness and the mixture obtained is assayed by HPLC (Chiralpak AD 250X 4.6, n-hexane + EtOH5+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of the remaining (R) -acetate: 63%; ee of (S) -alcohol: 85 percent.

Example 2:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of butyrate ester (R ═ COR in formula I)¹、R¹＝(CH₂)₂CH₃The compound of (1). 5mg PPL (Lipase from porcine pancreas, Sigma Chemical Co.) was added. The mixture was stirred at 30 ℃ until a conversion of about 48% (HPLC) was reached. Then filtered, concentrated to dryness and the resulting mixture determined by HPLC (Chiralpak AD 250X 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -butyric acid ester: 90 percent; ee of (S) -alcohol: 97 percent.

Example 3:

to 8ml of dimethoxyethane and 40ml of potassium phosphate buffer (0.1M, pH 7.0) was added 1.0g (2.86mmol) of butyric acid ester (R ═ COR in formula I)¹、R¹＝(CH₂)₂CH₃The compound of (1). 90mg of pancreatin were added. The mixture was stirred at 22-25 ℃ until the conversion exceeded 50%. It is then concentrated in vacuo, mixed with water and extracted six times with about 50ml of n-heptane. Drying (Na)₂SO₄) After this time, the mixture was concentrated in vacuo. 450mg (45%) of (R) -butyric acid ester was obtained; ee (HPLC): not less than 99 percent. The remaining aqueous phase was extracted with ethyl acetate and dried (Na)₂SO₄) Concentration in vacuo gave 190mg (23.8%) of (S) -alcohol; ee (HPLC): 97 percent.

Example 4:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of butyrate ester (R ═ COR in formula I)¹、R¹＝(CH₂)₂CH₃The compound of (1). 5mg of PPL was added. The mixture was stirred at 30 ℃ until a conversion of about 48% (HPLC) was reached. Then filtered, concentrated to dryness and the resulting mixture is purified by HPLC: (Chiralpak AD 250 × 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -butyric acid ester: 90 percent; ee of (S) -alcohol: 97 percent.

Example 5:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of butyrate ester (R ═ COR in formula I)¹、R¹＝(CH₂)₂CH₃The compound of (1). 5mg PLE (pig liver esterase, Sigma Chemical Co.) was added. The mixture was stirred at 30 ℃ until a conversion of about 47% (HPLC) was reached. Then filtered, concentrated to dryness and the resulting mixture determined by HPLC (Chiralpak AD 250X 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -butyric acid ester: 88 percent; ee of (S) -alcohol: 97 percent.

Example 6:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of hexanoate (R ═ COR in formula I)¹、R¹＝(CH₂)₄CH₃The compound of (1). 5mg of PLE were added. The mixture was stirred at 30 ℃ until a conversion of about 40% (HPLC) was reached. Then filtered, concentrated to dryness and the resulting mixture determined by HPLC (Chiralpak AD 250X 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -hexanoate ester: 66 percent; ee of (S) -alcohol: 96 percent.

Example 7:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of hexanoate (R ═ COR in formula I)¹、R¹＝(CH₂)₄CH₃The compound of (1). 5mg of cholesterol esterase from bovine pancreas was added. The mixture was stirred at 30 ℃ until a conversion of about 50% (HPLC) was reached. Then filtering, concentrating to dryness, and using the obtained mixtureHPLC (ChiralpakAD 250X 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -hexanoate ester: not less than 99.8%; ee of (S) -alcohol: not less than 99.8 percent.

Example 8:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of decanoate (R ═ COR in formula I)¹、R¹＝(CH₂)₈CH₃The compound of (1). 5mg of PPL was added. The mixture was stirred at 30 ℃ until a conversion of about 10% (HPLC) was reached. Then filtered, concentrated to dryness and the resulting mixture determined by HPLC (Chiralpak AD 250X 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -decanoate: more than or equal to 11 percent; ee of (S) -alcohol: 95 percent.

Example 9:

to 1ml of potassium phosphate buffer (0.1M, pH 7.0)/dimethoxyethane (5: 1) was added 10mg of butyrate ester (R ═ COR in formula I)¹、R¹＝(CH₂)₂CH₃The compound of (1). 5mg of horse liver acetone powder was added. The mixture was stirred at 30 ℃ until a conversion of about 46% (HPLC) was reached. Then filtered, concentrated to dryness and the resulting mixture determined by HPLC (Chiralpak AD 250X 4.6, n-hexane + EtOH 6+1, flow rate 1 ml/min, 25 ℃, 220/240 nm):

ee of (R) -butyric acid ester: 82%; ee of (S) -alcohol: 96 percent.

Claims (4)

1. A process for the kinetic resolution of racemates of compounds of formula (I),

wherein

R is COR¹Wherein R is¹＝(C₁-C₁₆) Alkyl radicals, (C)₂-C₁₆) -alkenyl or (C)₃-C₁₆) -alkynyl, C_nH_2nCycloalkyl, where n is 1 to 16, which may be branched or unbranched, andand may be substituted with 1 to 3 substituents selected from F, Cl, Br, I, CF₃、CN、NO₂Hydroxy, methoxy, ethoxy and COOR²Wherein R is²＝(C₁-C₄) -alkyl and (C)₂-C₄) Alkenyl, which may be branched or unbranched and which may be substituted by 1 to 3 substituents selected from F, Cl, Br, CF₃，

The process comprises stereoselective hydrolysis or alcoholysis of an enantiomeric or racemic mixture of a compound of formula (I) in the presence of a lipase, esterase or protease, with or without a cosolvent and a buffer, in a homogeneous or heterogeneous, aqueous/organic or organic medium at a temperature of from 10 to 80 ℃ wherein the reaction mixture contains from 2 to 50% by weight of an ester,

after the reaction has taken place, the unreacted R ═ COR in formula (I) is isolated¹And the resulting alcohol of formula (I) in which R ═ H, and thus the two enantiomers.

2. A process for the dynamic resolution of a racemate of a compound of formula (I) as claimed in claim 1,

wherein

R is COR¹Wherein R is¹＝(C₁-C₁₂) Alkyl radicals, (C)₂-C₁₂) -alkenyl or (C)₃-C₁₂) -alkynyl, C_nH_2nCycloalkyl, where n ═ 1-12, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from the group consisting of F, Cl, Br, CF₃、CN、NO₂Hydroxy, methoxy, ethoxy and COOR²Wherein R is²Methyl, ethyl and vinyl, which may be substituted with 1 to 3 substituents selected from the group consisting of F, Cl, CF₃。

3. A process for the dynamic resolution of racemates of compounds of formula (I) as claimed in claim 1 or 2, wherein

R is COR¹Wherein R is¹＝(C₁-C₁₀) Alkyl radicals, (C)₂-C₁₀) -alkenyl or (C)₃-C₁₀) -alkynyl, C_nH_2nCycloalkyl, where n ═ 1 to 10, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from the group consisting of F, Cl, Br, CF₃、CN、NO₂Methoxy and COOR²Wherein R is²Methyl, ethyl and vinyl, which may be substituted with 1 to 3 substituents selected from the group consisting of F, Cl, CF₃。

4. A process for the dynamic resolution of racemates of compounds of formula (I) as claimed in claim 1 or 2, wherein

R is COR¹Wherein R is¹＝(C₁-C₁₀) Alkyl radicals, (C)₂-C₁₀) -alkenyl or (C)₃-C₁₀) Alkynyl, which may be branched or unbranched and may be substituted by 1 to 3 substituents selected from F, Cl, Br, CF₃And a methoxy group.