Enzymatic process for the resolution of enantiomeric mixtures of β-lactams

This application claims the benefit of U.S. Provisional Application No. 60/160,103 filed Oct. 18, 1999.

The present invention relates to enzymatic processes for the resolution of enantiomeric mixtures of β-lactams useful in the preparation of taxanes.

The taxane family of terpenes, of which taxol and docetaxel are members, has attracted considerable interest in both the biological and chemical arts. Such taxanes may be prepared through a variety of semi-synthetic routes. In one, a β-lactam is coupled to a derivative of 10-deacetylbaccatin III to form a sidechain at the C-13 position of the derivative. As the stereochemistry of these taxanes may affect their pharmaceutical activity, methods allowing efficient stereospecific preparation of the intermediate β-lactam, as well as the final taxane products, have been the subject of investigation.

Brieva et al. (Brieva, R.; Crich, J. Z. and Sih, C. J., J. Org. Chem . 1993,58, 1068) reported that racemic β-lactam underwent selective kinetic hydrolysis with several Pseudomonas lipases and two penicillinases. Pseudomonas lipases used by Brieva et al. include P-30, AK and K-10.

Similarly, Patel reported in U.S. Pat. No. 5,879,929, that enantiomeric mixtures of certain β-lactams and, in particular, racemic mixtures of certain β-lactams, can be resolved by a stereoselective hydrolysis using a variety of lipases and enzymes. Lipases identified by Patel include Amano PS-30 ( Pseudomonas cepacia ), Amano GC-20 ( Geotrichum candidum ), Amano APF ( Aspergillus niger ), Amano AK (Pseudomonas sp.), Pseudomonas fluorescens lipase (Biocatalyst Ltd.), Amano Lipase P-30 (Pseudomonas sp.), Amano P ( Pseudomonas fluorescens ), Amano AY-30 ( Candida cylindracea ), Amano N ( Rhizopus niveus ), Amano R (Penicillium sp.), Amano FAP ( Rhizopus oryzae ), Amano AP-12 ( Aspergillus niger ), Amano MAP ( Mucor meihei ), Amano GC-4 ( Geotrichum candidum ), Sigma L-0382 and L-3126 (porcine pancreas), Lipase OF (Sepracor), Esterase 30,000 (Gist-Brocarde), KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano), Sigma L-3001 (Wheat germ), Sigma L-1754 ( Candida cylindracea ), Sigma L-0763 ( Chromobacterium viscosum ) and Amano K-30 ( Aspergillus niger ). Enzymes identified by Patel include enzymes derived from animal tissue such as esterase from pig liver, α-chymotrypsin and pancreatin from pancreas such as Porcine Pancreatic Lipase (Sigma). While these enzymes may be used in the stereoselective hydrolysis of β-lactams, the required purification of the enzyme can significantly increase the cost of the preparation of the β-lactam.

Whitesell et al. (Whitesell, J. K. Lawrence, R. M. Chimia , 1986, 40, 315) and Basavaiah et al. (Basavaiah,, D. and Rao, P. Tetrahedron. Asym ., 1994, 5, 223-234) reported successful application of pig liver acetone powder (PLAP), bovine liver acetone powder (BLAP) and chicken liver acetone powder (CLAP), in the resolution of numerous chiral secondary alcohols. Experimental evidence obtained to date, however, suggests that the use of these materials results in a product having relatively low optical purity. While the reason for this is not entirely clear, it is believed that this is the result of incomplete reaction rather than the enzyme's lack of selectivity which, in turn, is likely a consequence of inconsistent amounts of active enzyme present in different batches of the liver acetone powder.

Among the objects of the present invention, therefore, is the provision of enzymatic processes for the resolution of enantiomeric mixtures of β-lactams useful in the preparation of taxanes which offers improved reproducibility as compared to processes which employ acetone powders of animal livers and which compares favorably in cost to processes which employ purified lipases and other enzymes.

Briefly, therefore, the present invention is directed to a process for the resolution of a racemic mixture of β-lactams which contain an ester. The process comprises selectively hydrolyzing the ester of one of the enantiomers by combining the mixture with homogenized liver.

Other objects and features of this invention will be in part apparent and in part pointed out hereinafter.

A. Starting Materials

In general, the β-lactam enantiomers in the mixture have the following structural formula:

wherein

X 1 is —OX 6 ;

X 2 is hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclo;

X 3 is hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclo;

X 4 is hydrocarbyl, substituted hydrocarbyl, or heterocyclo;

X 5 is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX 10 , —COOX 10 , or —CONX 8 X 10 ;

X 6 is acyl;

X 8 is hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclo; and

X 10 is hydrocarbyl, substituted hydrocarbyl, or heterocyclo.

Preferably, X 2 and X 3 are hydrogen and the mixture contains the 3S,4R and 3R,4S enantiomers, and more preferably a racemic mixture of these enantiomers. Still more preferably, X 2 and X 3 are hydrogen; X 5 is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX 10 , or —COOX 10 ; X 10 is alkyl, aryl or heterocyclo; and the mixture contains the 3S,4R and 3R,4S enantiomers, preferably a racemic mixture of these enantiomers. In a particularly preferred embodiment, X 2 and X 3 are hydrogen; X 5 is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX 10 , or —COOX 10 ; X 10 is alkyl, aryl or heterocyclo; X 4 is 2-furyl, 3-furyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, substituted phenyl (substituted with any of the substituents identified elsewhere herein as hydrocarbyl substituents such as halo or nitro with the phenyl being mono or poly substituted in one or more of the ortho, meta or para positions), cycloalkyl, or alkenyl, and the mixture contains the 3S,4R and 3R,4S enantiomers, preferably a racemic mixture of these enantiomers.

Enantiomeric mixtures of the β-lactam starting materials may be obtained as described in Example 1 herein, or by methods analogous to those described in U.S. Pat. No. 5,229,526 which is incorporated herein by reference.

Homogenates of fresh or fresh frozen crude liver may be prepared using a high speed blender or other grinder. The liver is ground into pieces of a relatively small size and suspended in an aqueous solution. Preferably, about 1 pound (about 450 grams) of liver is combined with sufficient liquid to form about 0.5 to about 2 liters of homogenate, more preferably about 0.75 to about 1.25 liters, and most preferably about 1 liter of homogenate. In a preferred embodiment, the homogenate is buffered, preferably to a pH of about 8 with a phosphate or other suitable buffering agent.

Derivatives of homogenates are also included within the invention, such as refined fractions. To obtain refined fractions one may subject the homogenate to a series of fractionation procedures, the number of fractionation steps employed being dependent on the degree of purification desired. The series of fractionation steps could involve column chromatography such as a gel filtration column, centrifugation, heat treatment, precipitation, filtration or various other appropriate means of purification.

In general, the liver may be obtained from any animal. Preferably, the liver is avian or mammalian, more preferably chicken, turkey, pig or beef, and most preferably beef.

Stereoselective Hydrolysis

In general, a solution of β-lactam mixture in an organic solvent is combined with the homogenate to form a reaction mixture which contains about 1 gram of β-lactam to about 5 ml. to about 100 ml. of homogenate, more preferably about 1 gram of β-lactam to about 50 ml. of homogenate (with the homogenate containing about 450 grams of liver and sufficient liquid to form about 1 liter of homogenate). The reaction mixture is preferably adjusted to and maintained at about pH 7 to pH 8, preferably with a buffer, more preferably with a phosphate buffer.

The hydrolysis is preferably conducted in an aqueous, such as a buffered aqueous (e.g., phosphate buffer), medium or in an aqueous medium containing a miscible or immiscible organic solvent. For example, the reaction may be conducted in a biphasic solvent system comprising an organic phase, immiscible in water, and an aqueous phase.

Solvents for the organic phase of a biphasic solvent system may be any organic solvent immiscible in water, such as toluene, benzene, hexane, cyclohexane, xylene, trichlorotrifluoroethane, dichloromethane, ether and the like, and is preferably ether or toluene. Typically, the concentration of the β-lactam mixture will be about 0.1 to about 1 millimolar. The aqueous phase is water, preferably deionized water, or a suitable aqueous buffer solution, especially a phosphate buffer solution. The biphasic solvent system preferably comprises between about 10 to 90 percent by volume of organic phase and between about 90 to 10 percent by volume of aqueous phase.

The reaction time may be selected based on the homogenate, the temperature and the enzyme concentration. Temperatures of from about 4° C. to about 60° C. are preferably employed.

Separation

The products of the stereoselective conversions may be isolated and purified by methodologies such as extraction, distillation, crystallization, column chromatography, and the like.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describe organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, and aryl moieties. These moieties also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl and alkynaryl. Preferably, these moieties comprise 1 to 20 carbon atoms.

The “substituted hydrocarbyl” moieties described herein are hydrocarbyl moieties which are substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted with a hetero atom such as nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen atom. These substituents include halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protected hydroxy, keto, acyl, acyloxy; nitro, amino, amido, nitro, cyano, and thiol.

The alkyl groups described herein are preferably lower alkyl containing from one to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.

The alkenyl groups described herein are preferably lower alkenyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.

The alkynyl groups described herein are preferably lower alkynyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

The terms “aryl” or “ar” as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic groups containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl. Phenyl and substituted phenyl are the more preferred aryl.

The terms “halogen” or “halo” as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.

The terms “heterocyclo” or “heterocyclic” as used herein alone or as part of another group denote optionally substituted, fully saturated or unsaturated, monocyclic or bicyclic, aromatic or nonaromatic hydrocarbon groups having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring. The heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to the remainder of the molecule through a carbon or heteroatom. Exemplary heterocyclo include furyl, thienyl, pyridyl and the like. Exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, and thiol.

The acyl moieties described herein contain hydrocarbyl, substituted hydrocarbyl or heterocyclo moieties.

The term “stereoselective conversion,” as used herein, refers to the preferential reaction of one enantiomer relative to another, that is, asymmetric, enantioselective, reaction. Likewise, the terms “stereoselective hydrolysis”, refers to the preferential hydrolysis of one enantiomer relative to another.

The term “mixture,” as said term is used herein in relation to enantiomeric compounds, denotes mixtures having equal (racemic) or non-equal amounts of enantiomers.

The term “resolution” as used herein denotes partial, as well as, preferably, complete resolution.

The terms “hydroxyl protecting group” and “hydroxy protecting group” as used herein denote a group capable of protecting a free hydroxyl group (“protected hydroxyl”) which, subsequent to the reaction for which protection is employed, may be removed without disturbing the remainder of the molecule. A variety of protecting groups for the hydroxyl group and the synthesis thereof may be found in “Protective Groups in Organic Synthesis” by T. W. Greene, John Wiley and Sons, 1981, or Fieser & Fieser. Exemplary hydroxyl protecting groups include methoxymethyl, 1-ethoxyethyl, benzyloxymethyl, (.beta.-trimethylsilylethoxy)methyl, tetrahydropyranyl, 2,2,2-trichloroethoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl, trichloromethoxycarbonyl and 2,2,2-trichloroethoxymethyl.