MXPA00007811A - Method for enzymatic enantiomer-separation of 3(r)- and 3(s)-hydroxy-1- methyl-4-(2,4, 6-trimethoxyphenyl)-1, 2,3,6- tetrahydro-pyridine or its carboxylic acid esters - Google Patents

Abstract

The invention relates to a methodfor producing optically pure compounds of 3(R)- and 3(S)-hydroxy-1- methyl-4-(2,4, 6-trimethoxyphenyl)-1, 2,3,6- tetrahydro-pyridine or its carboxylic acid esters by reacting the enantiomer mixtures stereoselectively, using an enzyme.

Description

METHOD FOR THE ENZYMATIC SEPARATION OF BIRTHDAY 3 (R) AND 3 (S) HYDROXY-L-METI-4- (2,4,6-TRIMETOXYPENYL) -1,2,3,6-TETRAHYDROPYRIDINE OR ITS CARBOXYLIC ACID STEROIDS The invention relates to a process for the preparation of optically pure compounds of the formula (I) by reaction of stereodifferentiation of the mixtures of enantiomers with the aid of an enzyme. 3 (S) - and 3 (R) -hydroxy-1-methyl-4- (2,4,6-trimethoxy-phenyl) -1,2,3,6-tetrahydropyridine (compounds of the formula (I) wherein R = H) or its ester derivatives (compounds of the formula (I) wherein R = COR1) are central units or precursors of the synthesis of flavopiridol (HMR 1275 or L 86 8275) described in the patent application HMR 98 / L 001 ("Process for the preparation of (-) cis-3-hydroxy-1-methyl-4 (R) - (2,4,6-trimethoxy-phenyl) piperidine") (Process for the preparation of (-) cis -3-hydroxy-l-methyl-4 (R) - (2,4,6-trimethoxy-phenyl) piperidine), of the first potent inhibitor of cyclin-dependent protein kinase (see for example Sedlacek, Hans Harald; Czech, Joerg Naik, Ramachandra, Kaur, Gurmeet, Worland, Peter, Losiewicz, Michael, Parker, Bernard, Carlson, Bradley, Smith, Adaline, and collaborators Flavopiridol (L 86 8275; NSC 649890), a new kinase inhibitor for tumor therapy , Int. J. Oncol. (1996), 9 (6), 1143-1168 or Czech, Joerg; Hoffmann, Di ether, Naik, Ramachandra; Sedlacek, Hans-Harald; Antitumor activity of flavone L 86 8275 (Antitumor activity of flavone L 86 8275) Int. J. Oncol. (1995), 6 (1), 31-36). A resolution of racemates or separation of enantiomers of the compounds of formula (I) is not known. It has now been found that compounds of the formula (I) can be obtained in optically pure form from the mixtures of enantiomers by cleavage of enzymatic ester (hydrolysis or alcoholysis). The present invention is thus related to a method for the kinetic resolution of racemates of compounds of the formula (I), (I) which comprises subjecting enantiomeric mixtures or racemic mixtures of compounds of the formula (I), wherein R is COR 1 wherein R 1 = (C 1-6) alkyl-, (C 2 -C 16) alkenyl- or alkynyl (with 3 to 16 carbon atoms) - CnH2n- cycloalkyl wherein n = 1-16, which may be branched or unbranched and which may be substituted by 1-3 substituents of the group F, Cl, Br, 1, CF3 , CN, N02, hydroxyl, methoxy, ethoxy and COOR2, wherein R2 = alkyl (with 1 to 4 carbon atoms) - and alkenyl (with 2 to 4 carbon atoms) -, which may be branched or unbranched and which it can be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, in aqueous or inorganic media, aqueous homogeneous or heterogeneous in the presence of an enzyme, for example a lipase or esterase, for example livers of mammals or pancreases or microbial origin such as, for example, Candida, Pseudomonas and Aspergillus, or a protease, for example from Bacillus, to a hydrolysis or stereoselective alcoholysis at a temperature of 10-80 ° C, if appropriate in the presence of co-solvents and a buffer, the reaction mixture preferably contains 2-50% by weight of ester, and after the reaction was carried out, separate the unreacted ester (compound of the formula (I) wherein R = COR1) and the alcohol formed (composed of the formula (I) wherein R = H) - and thus the two enantiomers. The process according to the invention is economic, simple and fast. The reaction does not require any equimolar amounts of optically pure auxiliaries, any expensive reagents, any disproportionately large amounts of solvents and cost-intensive process steps. After finishing the reaction, the separation of the products or the enantiomers can be carried out by simple measures, for example by extraction. Preferably, in the compounds of the formula (I) R is COR1 wherein R1 = alkyl (with 1 to 12 carbon atoms) -, alkenyl (with 2 to 12 carbon atoms) - or alkynyl (with 3 to 12 carbon atoms) -, CnH2n-cycloalkyl wherein n = 1-12, which is branched or unbranched and which may be substituted by 1- 3 substituents of the group consisting of F, Cl, Br, CF3, CN, N02, hydroxyl, methoxy, ethoxy and COOR2, wherein R2 = methyl, ethyl and vinyl, which may be substituted by 1-3 substituents of the group consisting of F, Cl, CF3. Particularly preferably, in the compounds of the formula (I): R is COR1 wherein R1 = alkyl (with 1 to 10 carbon atoms) -, alkenyl (with 2 to 10 carbon atoms) - or alkynyl (with 3 to 10 carbon atoms) -, CnH2n-cycloalkyl wherein n = 1-10, which is branched or unbranched and which may be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, CN, N02 , methoxy, and COOR2, wherein R2 = methyl, ethyl and vinyl, which may be substituted by 1-3 substituents of the group consisting of F, Cl, CF3. Very particularly preferably in the compounds of the formula (I) R is COR 1 wherein R = (C 1 -C 10) alkyl-, (C 2 -C 10) alkenyl- or alkynyl (with 3 to 10 C) carbon atoms) -, which may be branched or unbranched and which may be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, and methoxy. A process of preference is used in the process in which an ester of the formula (I), for example R = COR1 wherein R1 = C3H7 or C8H17, is treated with a lipase, esterase or protease in a solution containing water- or alcohol- and shake. It may be advantageous to buffer said solution, for example with phosphate or TRIS [= tris (hydroxymethyl) -methylamine] as a buffer. Hearing can be, for example, 0.01-1.0 molar. A suitable buffer range is pH 5-9. In addition it can be advantageous to add cosolvent.

Suitable cosolvents are, for example, dimethoxyethane, acetone, THF, dioxane, hexane, tert-butyl methyl ether and tert-butanol. The proportion of cosolvents in the solution of preference is 10-80%. The enzymes used are preferably lipases and esterases, such as, for example, cholesterol esterase (EC 3.1.1.13) of bovine pancreas (Sigma Chemical Co.), porcine liver esterase (PLE, Sigma Chemical Co.), pancreatin (Fluka and Sigma). Chemical Co.), acetone powder, cattle pancreas (Sigma Chemical Co.), horse liver acetone powder (Sigma Chemical Co.) and porcine pancreas lipase (PPL, Sigma Chemical Co.), Candida rugosa lipase (Meito Sangyo) and lipase AP-6 from Aspergillus niger (Amano Pharmaceuticals). Each of the mentioned enzymes can be used in free or immobilized form (Immobilized Biocatalysts, 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 (for example free or immobilized) and is easy to determine by simple preliminary experiments.

The reaction mixture preferably contains 2-50% by weight of ester, particularly preferably 5-20%. The reaction temperature is 10-80 ° C, preferably 20-60 ° C, particularly preferably 20-40 ° C. The preparation of the esters (compounds of the formula (I) wherein R = COR1) is carried out rapidly from the alcohol (compound of the formula I where R = H) according to known methods of esterification (Haslam , Tetrahedron 1980, 36, 2409, Hófle, Steglich, Vorbrüggen, Angew, Chem. 1978, 90, 602) or as described in the patent application HMR 98 / L 001 ("Process for the preparation of (-) cis- 3-hydroxy-l-methyl-4 (R) - (2,4,6-trimethoxy-phenyl) piperidine "(" Process for the preparation of (-) cis-3-hydroxy-l-methyl-4 (R) - (2, 4, 6-trimethoxy-phenyl) piperidine ")). The products resulting from or remaining in the process can be separated into a simple form, for example by extraction or chromatographic methods. The remaining ester is obtained, for example, by dividing the reaction solution between water and n-heptane and concentrating the organic phase. The resulting alcohol can then be extracted from the aqueous phase with ethyl acetate. The enzyme can be recovered by freeze drying. The separation (and if appropriate subsequent reuse) of the enzyme can be facilitated by immobilization.

By means of a convenient conduit to the reaction, it is always possible to obtain at least one optically pure enantiomer. If optically pure ester is desired, the conversion should be about (or equal to) 50%, if an optically pure alcohol is desired, the conversion should be smaller (or equal to) 50%. The conversion of the alcoholysis or enzymatic hydrolysis was determined using HPLC (RP 18 LiChrosorb ™) and the determination of the optical purity was carried out by HPLC (Chiralpak AD). The esters that result or remain in the racemate solution process can be converted to the corresponding alcohol without inversion or racemization by known methods of ester cleavage (S.J. Solomon, E.G. Mata, O.A. Mascaretti, Tetrahedron 1993, 49, 3691-3748). In contrast, the resulting alcohol can be converted to the corresponding ester without inversion or racemization by known methods of esterification (Haslam, Tetrahedron 1980, 36, 2409). The products resulting from or remaining in the process can be racemized and reused in the resolution of racemate, according to known methods, for example by metal-catalyzed rearrangements (LE Overman, Angew, Chem. 1984, 96, 565-573 and the aforementioned literature). This increases the yield to more than 50%. For example, the compounds of the formula (I) wherein R = COR1 can be directly racemarked and those of the formula (I) wherein R = H can be racemarked, for example after conversion to convenient derivatives, such as described in L.E. OvermanAngew Chem. 1994, 96, 565-573. Metal catalysts which can be used are, for example, compounds of Hg (II), Pd (0) or Pd (II) or their salts. The present invention is intended to be illustrated in more detail by means of the following examples. Examples: All isolated products or mixtures of crude products were identified by NMR ^ H and mass spectrum or by HPLC. The optical purity of the products is determined by HPLC, for example Chiralpak AD 250 X 4.6 (Daicel). Example 1: 10 mg of the acetic acid ester [compound of the formula I wherein R1 = COR2 and R2 = COCH3] are introduced into 1 ml of potassium phosphate buffer (0.1M, pH = 7.0) / dimethoxyethane (5: 1) ). 5 mg of pancreatin are added. The mixture is stirred at 20-25 ° C until the conversion reaches approximately 40% (HPLC). It was then filtered, concentrated to dryness and the resulting mixture was investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 5 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee of remaining (R) -acetic acid: 63%; ee of (S) -alcohol: 85%. Example 2: 10 Mg of the butyric acid ester [compound of the formula I wherein R x = COR 2 and R 2 = CO (CH 2) 2 CH 3] are introduced into 1 ml of potassium phosphate buffer (0.1 M, pH = 7.0) / dimethoxyethane (5: 1). 5 Mg of PPL (porcine pancreas lipase, Sigma Chemical Co.) were added. The mixture is stirred at 30 ° C until the conversion reaches approximately 48% (HPLC). It was then filtered, concentrated to dryness and the resulting mixture is investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ee of the ester of (R) -butyric acid: 90%; ee of (S) -alcohol: 97%. Example 3: 1.0 Mg (2.86 mmol) of the butyric acid ester [composed of the formula I where R1 = COR2 and R2 = CO (CH2) 2CH3] were introduced into 8 ml of dimethoxyethane and 40 ml of potassium phosphate buffer (0.1M, pH = 7.0), 90Mg of pancreatin were added. The mixture is stirred at 22-25 ° C until the conversion exceeded 50%. It was then concentrated in vacuo, mixed with water and extracted six times with approximately 50 ml of n-heptane. After drying (Na2SO4), concentrated in vacuo, 450 mg (45%) of the ester of (R) -butyric acid are obtained; ee (HPLC): = 99%. After extraction of the remaining aqueous phase with ethyl acetate, dried (Na2SO4) and concentrated in vacuo. 190 Mg (23.8%) of (S) -alcohol are obtained; ee (HPLC): 97%. Example 4: 10 Mg of the butyric acid ester [compound of the formula I wherein R1 = COR2 and R2 = C0 (CH2) 2CH3] are introduced into 1 ml of potassium phosphate buffer (0.1M, pH = 7.0) / dimethoxyethane (5: 1) 5 Mg of PPL are added. The mixture is stirred at 30 ° C until a conversion of approximately 48% (HPLC) is reached. It was then filtered, concentrated to dryness and the resulting mixture was investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee of (R) -butyric acid: 90%; ee of (S) -alcohol: 97%. Example 5: 10 Mg of the butyric acid ester [compound of the formula I wherein R1 = COR2 and R2 = CO (CH2) 2CH3] are introduced into 1 ml of potassium phosphate buffer (0.1M, pH = 7.0) / dimethoxyethane (5: 1) 5 mg of PLE (porcine liver esterase, Sigma Chemical Co.) are added. The mixture is stirred at 30 ° C until a conversion of approximately 47% (HPLC) is reached. It was then filtered, concentrated to dryness and the resulting mixture was investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee of (R) -butyric acid: 88%; ee of (S) - alcohol: 97%. Example 6: 10 Mg of the caproic acid ester [compound of the formula I wherein R 1 = COR 2 and R 2 = CO (CH 2) 4 CH 3] are introduced into 1 ml of potassium phosphate buffer (0.1 M, pH = 7.0) / dimethoxyethane (5: 1) 5 mg of PLE are added. The mixture is stirred at 30 ° C until a conversion of approximately 40% (HPLC) is reached. It was then filtered, concentrated to dryness and the resulting mixture was investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee of (R) -caproic acid; 66%; ee of (S) - alcohol: 96%. Example 7: 10 Mg of the caproic acid ester [compound of the formula I wherein R1 = COR2 and R2 = CO (CH2) 4CH3] are introduced into 1 ml of potassium phosphate buffer (0.1M, pH = 7.0) / dimethoxyethane (5: 1) 5 mg of bovine pancreatic esterase esterase were added. The mixture is stirred at 30 ° C until a conversion of approximately 50% (HPLC) is reached. It was then filtered, concentrated to dryness and the resulting mixture was investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee of (R) -caproic acid: > 99.8%; ee of (S) -alcohol: = 99.8%. Example 8: 10 Mg of the capric acid ester [compound of the formula I wherein R1 = COR2 and R2 = CO (CH2) 8CH3] are introduced in 1 ml of potassium phosphate buffer (0.1 M, pH = 7.0) / dimethoxyethane (5: 1) 5 mg of PPL were added. The mixture is stirred at 30 ° C until a conversion of about 10% (HPLC) is reached. It is then filtered, concentrated to dryness and the resulting mixture is investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee (R) -capric acid: >; eleven %; ee of (S) - alcohol: 95%. Example 9: 10 Mg of butyric acid ester [compound of formula I wherein R1 = COR2 and R2 = CO (CH2) 2CH3] are introduced into 1 ml of potassium phosphate buffer (0.1M, pH = 7.0) / dimethoxyethane (5: 1) 5 mg of acetone-horse liver powder were added. The mixture is stirred at 30 ° C until a conversion of about 46% (HPLC) is reached. It is then filtered, concentrated to dryness and the resulting mixture is investigated by HPLC (Chiralpak AD 250 x 4.6, n-hexane + EtOH 6 + 1, flow 1 ml / min, 25 ° C, 220/240 nm): ester ee of (R) -butyric acid: 82%; ee of (S) -alcohol: 96%.

Claims (5)

1. CLAIMS 1. A procedure for the kinetic resolution of racemates of compounds of the formula (I), which comprises subjecting enantiomeric mixtures or racemic mixtures of compounds of the formula (I), wherein: R is COR1 wherein R1 = alkyl (with 1 to 6 carbon atoms) -, alkenyl (with 2 to 16 carbon atoms) - or alkynyl (with 3 to 16 carbon atoms) - CnH2n-cycloalkyl wherein n = 1-16, which may be branched or unbranched and which may be substituted by 1-3 substituents of the group F, Cl, Br, 1, CF3, CN, N02, hydroxyl, methoxy, ethoxy and COOR2, wherein R2 = alkyl (with 1 to 4 carbon atoms) - and alkenyl (with 2 to 4 carbon atoms) -, which may be branched or unbranched and which may be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, in aqueous or inorganic media, aqueous homogeneous or heterogeneous in the presence of an enzyme, a hydrolysis or stereoselective alcoholysis at a temperature of 10-80 ° C, if appropriate in the presence of co-solvents and a buffer, the reaction mixture of preferably contains 2-50% by weight of an ester, and after the reaction has been carried out, separate the unreacted ester (compound of the formula (I) wherein R = COR1), and the alcohol formed (compound of the formula (I) where R = H) - and in this way the two enantiomers.
2. 2. The procedure for the kinetic resolution of racemates of compounds of the formula (I), according to claim 1, characterized in that: R is COR1 wherein R1 = alkyl (with 1 to 12 carbon atoms) -, alkenyl ( with 2 to 12 carbon atoms) - or alkynyl (with 3 to 12 carbon atoms) -, CnH2n-cycloalkyl wherein n = 1-12, which is branched or unbranched and which may be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, CN, N02, hydroxyl, methoxy, ethoxy and COOR2, wherein R2 = methyl, ethyl and vinyl, which may be substituted by 1-3 substituents of the group consisting of F, Cl, CF3.
3. 3. The process for the kinetic resolution of racemates of compounds of the formula (I), according to claim 1 or 2, characterized in that R is COR1 wherein R1 = (C1-C10) alkyl-, alkenyl (with 2 to 10 carbon atoms) - or alkynyl (with 3 to 10 carbon atoms) -, CnH2n-cycloalkyl where n = 1-10, which is branched or unbranched and which may be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, CN, N02, methoxy, and COOR2, wherein R2 = methyl, ethyl and vinyl, which may be substituted by 1-3 substituents of the group consisting of F, Cl, CF3.
4. 4. The process for the kinetic resolution of racemates of compounds of the formula (I) according to claims 1 to 3, characterized in that R is COR1 wherein R1 = alkyl (with 1 to 10 carbon atoms) -, alkenyl ( with 2 to 10 carbon atoms) - or alkynyl (with 3 to 10 carbon atoms) -, which may be branched or unbranched and which may be substituted by 1-3 substituents of the group consisting of F, Cl, Br, CF3, and methoxy.
5. 5. The process for the kinetic resolution of racemates or compounds of the formula (I) according to claims 1 to 4, characterized in that the enzyme used is a lipase, esterase or protease.