NL8304190A - PROCESS FOR PREPARING L-CARNITINE AND INTERMEDIATE CHEMICALS USED THEREOF - Google Patents

Description

N.0. 32,179 «. a ^ *

Process for the preparation of L-camitine and chemical intermediates used therein_____

The present invention relates to methods of preparing L-carnitine. In particular, the invention relates to a method for the microbiological reduction of / -substituted acetoacetic acid esters or amides to their respective 5 L-ft-hydroxy- / -substituted butyric acid vessels, which derivatives are readily convertible to L-carnitine chloride. The invention also relates to new chemical intermediates used in the process.

As is known, carnitine (S-hydroxy - / - trimethylamino-butyric acid) contains a center of asymmetry and therefore carnitine exists in two stereoisomeric forms, the D and the L forms.

L-carnitine is normally present in the body, where it functions to transport activated long chain free fatty acids through the mitochondrial membrane. Since the mitochondrial membrane is impermeable to acyl-CoA derivatives, long chain free fatty acids can enter only when esterification with L-carnitine has occurred. The carrier function of L-carnitine is effected both by transporting long-chain active fatty acids from the sites of their biosynthesis, for example the microsomes to the mitochondria, where they are oxidized, and by transporting acetyl-CoA from the mitochondria, where it is formed, to the ex-tram microchondrial sites, where long-chain fatty acid synthesis takes place, for example, in the microsomes, where the acetyl-CoA can be used to synthesize cholesterol and fatty acids .

While it has been established that the counterclockwise lsomer (L-carnitine) is exclusively the biological form (D-carnitine has never been determined in mammalian tissues so far), the D, L-carnitine racemate has been used for various indications for several years. For example, D, L-carnitine is sold in Europe as an appetite stimulant and the product has been reported to have an effect on the growth rate of children; see, e.g., Borniche et al., Clinica Chemica Acta, (1960) 171-176 and Alexander et al., "Protides in the Biological Fluids", 6th Colloquim, Bruges, 1958, 306-310. U.S. Patent 3,830,931 describes improvements in cardiac contractility and systolic rhythm in congestive heart failure, which can often be obtained by administering 6,94190 2 F "4 D, L-camitine. U.S. Patent 3,968 241 discloses the use of D, L-carnitine in cardiac arrhythmias U.S. Patent 3,810,994 describes the use of D, L-carnitine in the treatment of corpulence.

Recently, however, there has been an increasing emphasis on the importance of using only the counterclockwise carnitine isomer for at least some therapeutic applications. In fact, D-carnitine has been shown to be a competitive inhibitor of carnitine-linked enzymes, such as carnitine acetyl transferase (CAT) and carnitine ne-palmityl transferase (PTC). In addition, recent evidence suggests that D-carnitine can drain L-carnitine from the heart tissue. As a result, it is essential that L-carnitine be administered only to patients under medical treatment for heart disease or for lowering lipids in the blood.

Several methods have been proposed for the production of carnitine on an industrial scale. However, the chemical synthesis of carnitine inevitably leads to a racemic mixture of the D and L isomers. As a result, separation methods must be used to obtain the separated racemate optical antipodes. However, these separation methods are cumbersome and expensive.

It is an object of the present invention to produce L-camitine in good yield by a combination of microbiological and chemical processes.

An object of the present invention is to provide an improved process for the synthesis of L-carnitine from readily available raw materials of moderate cost.

Another object of the present invention is to describe the preparation of new, suitable optical active intermediates for the synthesis of L-carnitine and its salts or esters.

Another object of the present invention is to provide methods for the preparation of L-carnitine via the replacement of the halogen group of a 4-halogen-3CR) hydroxybutyrate with the trimethyl amine group.

Yet another object of the present invention is to provide a process for preparing 4-iodo or 4-bromo-3 (R) -hydroxybutyrates from 4-chloro-3 (R) -hydroxybutyrates.

These and other objects of the invention will become more apparent as the description thereof continues.

The advantages of the present invention will be apparent to those skilled in the art from the following detailed description.

8 3 0 4 1-9 0- 3 • -, «V *

It is known that the β-keto group in position 3 in the--substituted acetoacetic acid derivatives can be reduced by hydrogenation over Pt / C (for example, U.S. Patent 3,969,406). However, the hydroxy compound resulting from such a process is chemical. In contrast, by using the fermentative activity of a microorganism according to the method of the present invention, the hydrogenation of the oxo group at position 3 can be accomplished stereoselectively to form optically active / substituted β-hydroxybutyric acid derivatives.

In particular, after a suitable selection (as described below) of the substrate to be exposed to the fermentative activity of the microorganisms according to the method of the present invention, the 3 (R) or L epimeric configuration is obtained. This configuration is required for conversion to the natural L-camitine.

In a broad sense, the present invention encompasses the use of the

microbial reductase enzyme, Ι-β-hydroxyacyl CoA dehydrogenase [EC

I. 1.1.35], to stereoselectively catalyze the hydrogenation of / -substituted acetoacetic acid derivatives as defined below.

Therefore, in accordance with the broadest aspect, the method of the present invention for the preparation of optically active / substituted β-hydroxybutyric acid derivatives of the formula II wherein X is selected from Cl, Br, J and OR and R includes a group straight chain, branched chain or cyclic configuration is selected from the group consisting of alkoxy groups of 1 to about 15 carbon atoms, alkylamino groups of about 5 to about 15 carbon atoms, cycloalkoxy groups and cycloalkylamino groups of about 5 to about 12 carbon atoms, phenoxy and phenylalkoxy groups of 7 to about 14 carbon atoms, phenylamino and phenylalkylamino groups of formulas 12 and 13, wherein Y and Z are selected from F, an alkyl group of 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH- }, Cl and Br from corresponding / -substituted aceto-acetic acid esters or amides, subjecting said / -substituted acetoacetic acid esters or amides to the fermentative enzymatic action of a microorganism producing L-β-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] and recovering the desired optically active / substituted-40 β-hydroxybutyric acid derivatives.

£ 8304190 * 4

In particular, in order to prepare the optically active / -substituted 3 (R) -hydroxybutyric acid derivatives of the formula 2 with the 3 (R) configuration, the method comprises subjecting compounds of the formula 1, wherein X and R have the above meanings provided that when R is an alkoxy group it contains 5 to about 15 carbon atoms, to the fermentative enzymatic activity of a microorganism producing L-O-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] and recovering the desired optical active 4-substituted 3 (R) -hydroxybutyric acid derivatives from the fermentative reaction mixture.

It has been found that any microorganism producing the desired enzyme is able to function as a catalyst of the stereoselective reduction. Particularly suitable are the microorganisms of the class Ascomycetes, the order Endomycetales, Mucorales, Moniliales and 15 Eurotiales and the genus Saccharomyces. Saccharomyces Cerevisiae is particularly preferred.

For the preparation of optically active 4-substituted 3 (R) -hydroxybutyrate esters containing 1-4 carbon atoms, it is necessary to use purified L-6-hydroxy CoA dehydrogenase [EC 1.1.1.35], such as that of pig heart, because the intact microorganism has interfering oxido reductases of opposite configuration. Therefore, microbial reduction of, for example, 4-chloroacetoacetic acid esters with 1-4 carbon atoms produces 4-chloro-3-hydroxybutyrates of unsatisfactory optical purities.

Therefore, the present invention also provides a process for preparing optically active / substituted 3 (R) -hydroxybutyric acid derivatives of the formula 2 having the 3 (R) configuration, wherein X is Cl, Br, J or OH and R is an alkoxy group of 1 to 4 carbon atoms which subject compounds of formula 1, wherein X and R have the above meaning to the enzymatic action of L-8-hydroxyacyl Coa dehydrogenase [EC 1.1.1.35] purified form and recovering the desired optically active / substituted 3 (R) -hydroxybutyric acid derivatives from the enzymatic reaction mixture.

The present invention therefore provides compounds of formula 2 with the 3 (R) configuration, wherein X is selected from Cl, Br, J and OH and R is a straight chain, branched chain or cyclic configuration group selected from the group consisting of alkoxy groups of 1 to about 15 carbon atoms, 40 alkylamino groups of about 5 to about 15 carbon atoms, <3 7 -f. 4 Λ Λ v v -} -5? y «« 5 cycloalkoxy groups and cycloalkylamino groups of about 5 to about 12 carbon atoms, phenoxy and phenylalkoxy groups of 7 to about 14 carbon atoms, phenylamlno and phenylalkylamino groups of formulas 12 and 5 wherein Y and Z are selected from H, an alkyl group having 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH 3, Cl and Br.

The optically active / -substituted-L-8-hydroxybutyric acid derivatives can then be reacted with trimethylamine to form the corresponding β-trimethylammonium-LS-hydroxybutyric acid derivative, which can be easily converted to L-carnitine by treatment with aqueous acids. Figure 1 gives a schematic representation of the reaction steps of this process.

It has been found that the reaction 1 ......) 2 takes place more easily when X is Cl. However, since the subsequent reaction takes place 2-> · 3 in better yields when X is iodine or bromine, it is preferable to first prepare the chlorine derivative and then convert it to the corresponding iodine or bromine derivative.

The present invention also relates to an improved process comprising first conversion of 4-chloro-3 (R) -hydroxybutyrate ester to the corresponding 4-iodo or 4-bromo-3 (R) -hydroxybutyrates. For the sake of simplicity, reference will be made hereafter to the iodine derivative. The iodohydrin (Formula 5) can be easily reacted with trimethylamine at room temperature to form the compound of Formula 6, which is easily converted to L-carnitine according to the reaction scheme of Figure 2.

This method, as suggested by the equation, is susceptible to numerous variations. Regardless of which form is made available, the ester is reacted with sodium iodide in a suitable solvent such as 2-butanone, acetone, butanol, etc. The main reaction desired at this point in the reaction with sodium iodide is a replacement reaction which forms the iodohydrin of formula 5 without interfering with the chiral center at the adjacent carbon atom. This reaction requires at least enough sodium iodide to replace all of the chloride from the compound of formula II. Generally speaking, a small excess of sodium iodide is used.

The reaction of the compound of formula 5 with trimethylamine can be performed at a mild temperature (e.g. 25 ° C) (see SG Boots and MR Boots, J. Pharm, Sci., 64, 1262 (1975) in various solvents such as methanol or ethanol, which contain an excess of Λ M 9 0 it * 6 trimethylamine. It is noteworthy that depending on the alcoholic solution used, an ester exchange takes place. For example, when methanol is used as the solvent, the L- carnitine methyl ester obtained in the reaction This exchange reaction is advantageous because it is known that L-carnitine methyl ester can be directly converted to the free base form of L-carnitine by passing the ester through an ion exchange column (OH ”) [see E. Strack and J. Lorenz, J. Physiol. Chem. 344, (1966) 276].

From the description of the foregoing methods, it can be seen that a number of new and very useful optically active intermediates are being formed. Particularly suitable are the 4-iodo-3 (R) -hydroxybutyric acid alkyl esters, wherein the alkyl groups each contain 6 to 10 carbon atoms. The octyl ester is particularly preferred.

Micro-organisms which have the desired oxido-reductase activity are well known in the microbiological art and any of these micro-organisms can be used to practice the method of the present invention (see. K. Kieslich, "Microbial Transformations of Non-Steroid Cyclic Compounds "(Georg Thierae 20 Publishers, Stuttgart, 1976)), wherein any of the genera of microorganisms specifically described therein are particularly suitable. Easily available and inexpensive microorganisms of the genus Saccharomyces, for example brewer's yeast, baker's yeast and winemaker's yeast (Saccharomyces vini), have been shown to produce the L - $ - hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] and are highly beneficial in the implementation of the method according to the invention. The enzyme has been described by S.J. Wakil and E.M. Barnes Jr. in Comprehensive Biochemistry 185 (1971) 57-104.

The 4-substituted acetoacetic acid substrate can be incorporated into a standard composition feed medium in which such organisms are grown and the usual fermentation conditions can then be used to carry out the reductive rearrangement. Also, the active principle can be removed from the growing culture of the microorganism, for example, by lysis of the cells to release the enzymes, or by suspending the supernatant cells in a fresh water-containing system. In either of these techniques, the S-keto group will be selectively reduced as long as the active enzyme generated by the microorganisms is present in the environment. Of course, the temperature, time, and pressure conditions under which the contact of the 4-substituted acetoacetic acid 8304190 7 J * derivative with the reductive enzyme is performed are interdependent, as will be apparent to those skilled in the art. For example, with gentle heating and at atmospheric pressure, the time required to effect the reductive conversion will be less than if the conversion at room temperature proceeds under otherwise the same conditions. Obviously, neither the temperature, the pressure, nor the time should be so high or long that it results in degradation of the substrate. When a growing culture of the organism is used, the process conditions should also be sufficiently gentle so that the organism is not killed before it has produced enough hydrolytic enzymes to proceed the reaction.

Generally, at atmospheric pressure, the temperature can range from about 10 ° C to about 35 ° C and the time from about 12 hours to about 10 days.

In the following examples, which are given to illustrate the present invention and which are not set forth to limit the scope of the claims, the α-haloacetoacetic acid derivative substrates subjected to the microbiological reduction were prepared from diketene according to the general method from CD Hurd and H.L.

Abemethy (J. Am. Chem. Soc., 61_ (1940) 1147 for the ch-chloroacetoacetic acid derivatives and F. Chick, NTM Wilsmore [J. Chem. Soc., 1978 (1910)] for the /-bromoacetic acid derivatives via the reaction scheme of Figure 3, wherein X is Cl or Br, YH or alkyl and R as defined above.

If desired, the / -haloacetoacetic acid derivatives can also be prepared from) -haloacetic acid esters via a conventional Grignard reaction. For example,--chloroacetoacetic acid octyl ester was readily prepared by refluxing ch-chloroctyl ester with two equivalents of magnesium in ether for 48 hours.

After removal of the solvent, the acetoacetic acid octyl ester was obtained in a yield of about 70%.

γ-hydroxyacetoacetic acid derivatives were prepared from their corresponding γ-bromoacetoacetic acid derivatives by stirring in a dioxane water (1: 1) solution containing CaCOg at 25 ° C for 12 hours.

Each of the products produced according to the following examples was structurally identified using nuclear magnetic resonance (NMR), infrared spectra, and thin layer chromatography motility. The optical purity and absolute configuration of the products were determined by their conversion to L-carnitine, as well as conversion to its esters, which are readily analyzed by NMR, spectrometry and optical rotation.

Example I (yeasts) 5 (+) 4-chloro-3 (R) -hydroxybutyric acid octyl ester was prepared according to the reaction equation of Figure 4.

A. Fermentation. Surface growth of a week-old diagonally arranged agar of Candida keyfr. NRRL Y-329, grown on agar of the following composition: 10

Number of grams of agar 20 glucose 10 yeast extract 2.5 15 K2Hpo4 1 distilled water, q.s. 1 liter (sterilized at 140 kPa for 15 min) was suspended in 5 ml of 0.85% saline. 1 ml aliquots of this suspension were used to inoculate a 250 ml Erlenmeyer flask (step F-1) containing 50 ml of the following medium (Vogel medium):

Number of grams 25 yeast extract 5 casamino acids 5 dextrose 40

Na3-citrate-5 1/2 H 2 O 3 g KH2PO4 5 g 30 NH4NO3 2 g

CaCl2-2H20 0.1 g

MgSOA.7H20 0.2 g trace element solution 0.1 ml distilled water, q.s. 1 liter of 35 pH 5.5 (sterilized at 210 kPa for 15 min).

8304130 9 «9

Trace element solution g / 100 ml citric acid-lH20 5 • ZnSO4.7H20 7 5 Fe (NH4) 2 (S04) 2.6F20 1

CuSO4.5H20 0.25

MnSO4.1H20 0.05 H3BO3 0.05

NaH2MoO4.2H20 0.05 10

The flask was incubated at 25 ° C for 24 hours on a rotary shaker (250 cycles / min - radius 5 cm), after which a 10 vol% transfer to another 250 ml Erlenmeyer flask (step F-2) , which contained 50 ml of environment according to Vogel, was done. After incubation on a rotary shaker for 24 hours, 150 mg of γ-chloroacetacetic acid octyl ester in 0.1 ml of 10 percent Tween 80 was added.

The flask from step F-2 was then incubated for another 24 hours under the conditions used in the incubation of the flasks from step F-1.

20 B. Insulation. The cells were removed by centrifugation twenty-four hours after the addition of the--chloro-acetoacetic acid octyl ester. The supernatant was extracted three times with 50 ml of ethyl acetate. The ethyl acetate was dried over sodium sulfate and evaporated to give an oily residue (186 mg). The residue was dissolved in 0.5 ml of the mobile phase and added to a column (1 x 25 cm) of silica gel (MN silica gel 60). The column was eluted with Skelly B: ethyl acetate (8: 1) and 14 ml fractions were collected. Fractions 6 and 7 containing the desired product were combined and concentrated to dryness to give 120 mg of crystalline-line residue. Recrystallization from ethyl acetate-hexane gave 4-chloro-3 (R) -hydroxybutyric acid octyl ester [α] 3 + 13.3 ° (c, 4.45) (CHCl 3); pmr G ^ CDC ^ 0.88 [3H, tr. twisting, CH3 "(CH2) n ~]; 1.28 [10H, s, - (CH2) 5 µl; 1.65 (2H, m, 35 -CH2-CH2-CH2O-C-); 2.62 (2H, d, J-6Hz, -CH-CH 2 -COOR);

0 OH

3.22 (1H, br., -OH); 3.60 (2H, d, J = 6Hz, 3304190 C1CH2-CH-R); 4.20 3H, -CH2 “CH-CH2 and -0-0-¾¾).

OH OH O 5 Analysis calculated for 0 ^ 2 ^ 23 ^ 3 ^ 1: C 57.47, H 9.25.

Found: C 57.52, H 9.07. [BLO Rf - 0.5, silica gel plate according to Brinkmann, 0.25 cm EM; Skelly B-ethyl acetate (5: 1)].

Example II

Remaining cells. One hundred grams of fresh baker's yeast 10 Saccharomyces cerevisiae (Red Star) were suspended in 250 ml of tap water, to which was added 10 g of sucrose and 3.6 g of γ-chloroacetoacetic acid octyl ester. After incubating the contents on a rotary shaker (250 cycles / minute - radius 5 cm) at 25 ° C for 24 hours, another 10 g of sucrose was added to the flask and the reaction was continued for another 24 hours. The cells were then removed by filtration through a pad of Celite. The cells were washed with water and ethyl acetate. The washings were combined with the filtrate and extracted exhaustively with ethyl acetate. The ethyl acetate layer was dried over MgSO 4 and evaporated to give an oily residue, which was chromatographed on a silica gel column to give 2.52 g of 4-chloro-3 (R) -hydroxybutyric acid octyl ester as a solid. low melting point; [o] 23 + 13.2 ° (C, 4.0, C 1 Cl 3).

Example III

25 (+) 4-chloro-3 (R) hydroxybutyric acid benzyl ester was prepared according to the reaction equation of Figure 5.

A. Fermentation. Surface growth of a one week old diagonally disposed agar of Gliocladium virens ATCC 13362 grown on agar of the following composition: 30

Number of grams of malt extract 20 glucose 20 peptone 1 35 agar 20 distilled water, q.s. 1 liter (sterilized at 140 kPa for 15 min) was suspended in 5 ml of 0.85% saline. 1 ml aliquots of this suspension were used to make a Erlenmeyer flask. - * * 11 250 ml (step Fl) containing 50 ml of the following medium (soybean dextrose medium) to be inoculated: soybean meal 5 g 5 dextrose 20 g STaCl 5 g KR2HP04 5 g yeast 5 g water 1 1 10 pH set to 7.0

Autoclave for 15 minutes at 105 kPa.

The flask was incubated on a rotary shaker (250 cycles / min - radius 5 cm) for 24 hours at 25 ° C, after which a transfer of 10% by volume to another 250 ml Erlenmeyer flask (step F -2), containing 50 ml of soybean dextrose medium, was run. After a 24 hour incubation on a rotary shaker, 150 mg / chloroacetoacetic acid benzyl ester in 0.5 ml 10% Tween 80 was added. The flask from step F-2 was then incubated for another 24 hours under the conditions used in the incubation of the flasks from step F-1.

B. Insulation. The mycelium was removed by filtration twenty four hours after the addition of the X-chloroacetoacetic acid benzyl ester.

The filtrate was extracted extensively three times with 50 ml of ethyl acetate. The ethyl acetate layer was dried over MgSO 4 and concentrated under reduced pressure to give a residue (160 mg). The residue was chromatographed on a silica gel (MN silica gel 60) column (1 x 25 cm). The column was eluted with Skelly B and ethyl acetate (10: 1) and 12 ml fractions were collected. Fractions 11-16 containing the desired product were combined and concentrated to dryness to give 115 mg of 4-chloro-23 3 (R) -hydroxybutyric acid benzyl ester, [α] 25 + 8.7 ° (c. 5, 26; CHCl3); pmr (i5 "CDCl3) 2.65 (2H, 8304 1 0.0 12 d, J * 6 Hz, -CH-CH2COOR), 3.20 (1H, br, -OH); 3.54 (2H, d, J - 6 Hz,

OH

CI-CH2CH); 4.20 (1H, m, -CH2-CH-CH2-), 5.12 (2F, s,

5 OH OH

-C-O-CH ^ C ^ He;); 7.31 (5F, s, five aromatic protons.

H

Anal, ber. for C11 H13 O3 Cl: C 57.77, H 5.73.

Found: C 57.64, H 5.67. [TLC silica gel EM Brinkmann plate, 0.25 cm, = 0.43, Skelly B ethyl acetate (5: 1)].

Examples IV-XXIII

The method of Example I was repeated with each of the organisms listed in Table A, except that ch-chloroacetoacetic acid octyl ester 15 was added at a concentration of 1 mg / ml. Conversion to the desired product (+) 4-chloro-3 (R) -hydroxybutyric acid octyl ester was obtained. The methods of these examples were repeated by continuously adding the substrate to the yeast mediums. The substrate / yeast weight ratio was about 1: 1.5 with excellent conversion to the desired product.

Examples mV-XlVIII

The method of Example III was repeated with each of the organisms listed in Table B, except that / -chloroacetoacetic acid octyl ester (1 mg / ml) was used. Conversion to the desired compound 25 (+) 4-chloro-3 (R) -hydroxybutyric acid octyl ester was obtained.

Examples XLIX-LXVIII

The method of Example I was repeated with each of the organisms listed in Table A, except that ch-chloroacetoacetic acid benzyl ester (1 mg / ml) was used as the substrate. Conversion to the desired product (+) 4-chloro (R) -hydroxybutyric acid benzyl ester was obtained. Examples LXIX-XCIII

The procedure of Example III was repeated with each of the organisms listed in Table B, using--chloroacetoacetic acid benzyl ester (1 mg / ml) as a substrate. Conversion to the desired compound (+) 4-chloro-3 (R) -hydroxybutyric acid benzyl ester was obtained. Examples XCIV

(+) 4-Chloro-3 (R) -hydroxybutyric anilide was prepared according to the method of Example II, except that 4-chloroacetoacetanilide at a concentration of 1 mg / ml was used (see Figure 6) as the substrate 40 for the conversion to the desired optically active product, m.p.

8304190 13 110-111 ° C [α] 23 + 17.5 °

O

(c, 3.0, CHCl3; pmr (é CD3CCD3) 2.67 (2F, d, J »6 Hz, 5-HOHCH2-CONHR), 3.66 (2H, d, J s 6 Hz, CICH2CFOH-R ), 4.43 flF, m, ~ CH2 “CHOH-CF2-), 7.03-7.44 (3F, m, aromatic protons, meta and para), 7.69 (2H, d, J = Hz, aromatic protons, ortho), 9.24 (1H, br, -CN-O). Anal, her, for C122122: 0 56.21, H 5.66, 10 Found: C 56.16, F 5.47.

Examples XCV-CXIV

The method of Example I was repeated with each of the organisms listed in Table A, except that / -chloroacetoacetanilide was used at a concentration of 1 mg / ml. In all cases conversion to the desired product, (+) 4-chloro-3 (R) -hydroxybutyric anilide was obtained.

Examples CXCV-CXXXIX

The method of Example III was repeated with the organisms listed in Table B, ch-chloroacetoacetanilide was added at a concentration of 1 mg / ml. In these cases, conversion to the desired (+) 4-chloro-3 (R) -hydroxybutyric anilide was achieved.

Examples CXL-CLIX

The method of Example I was repeated with the organisms listed in Table A, except that b-bromoacetoacetic acid octyl ester (1 mg / ml) was used as the substrate. Conversion to the desired product, (+) 4-bromo-3 (R) -hydroxybutyric acid octyl ester was obtained.

Examples CLX-CLXXXIV

The method of Example III was repeated with the organisms listed in Table B, except that b-bromoacetoacetic acid octyl ester (1 mg / ml) was used. Conversion to the desired product, (+) 4-bromo-3 (R) -hydroxybutyric acid octyl ester was obtained.

Examples CLXXXV-CCIV

The method of Example I was repeated with the organisms listed in Table A, except that (-bromoacetoacetic acid benzyl ester 1 mg / ml) was used as the substrate. Conversion to the desired product, (+) 4-bromo-3 (R) -hydroxybutyric acid benzyl ester was obtained.

Examples CCV-CCXXIX

The method of Example III was repeated with the organisms listed in Table B, except that dgt / bromoacetoacetic acid benzyl ester (1 83 Cl 2 SO 14 mg / ml) was used. Conversion to the desired product, (+) 4-bromo-3 (R) -hydroxybutyric acid benzyl ester was obtained.

Examples CCXXX-CCXLIX

The method of Example I was repeated with the organisms listed in Table A except that ^-bromoacetanilide (1 mg / ml) was used as the substrate. Conversion to the desired (+) 4-bromo-3 (R) -hydroxybutyric anilide was obtained.

Examples CCL-CCLXXIV

The method of Example III was repeated with the organisms listed in Table B, except that b-bromoacetanilide (1 mg / ml) was used as the substrate. Conversion to the desired (+) 4-bromo-3 (R) -hydroxybutyric anilide was obtained.

Examples CCLXXV-CCXOIV

The method of Example I was repeated with the organisms listed in Table A, except that Jf-hydroxyacetoacetic acid octyl ester (1 mg / ml) was used as the substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutyric acid octyl ester was obtained.

Examples CCCXCV-CCCXIX

The method of Example III was repeated with the organisms listed in Table B, except that Jf-hydroxyacetoacetic acid octyl ester (1mg / ml) was used as the substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutyric acid octyl ester was obtained.

Examples CCCXX-CCCXXXIX

The method of Example I was accomplished with the organisms listed in Table A, except that hyd-hydroxyacetoacetanilide (1mg / ml) was used as the substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutyric anilide was obtained.

Examples CCCXL-CCCLXIV

The method of Example III was repeated with the organisms listed in Table B, except that γ-hydroxyacetoacetanilide (1 mg / ml) was used as the substrate. Conversion to the 4-hydroxy-3 (R) -hydroxybutyric anilide was obtained.

Example CCCLXV

Methyl 4-chloro-3 (R) -hydroxybutyrate (compound of formula 8) (see the reaction equation with figure 7).

100 mg of methyl 4-chloroacetoacetate (compound of formula 7) were incubated with 29 units of pig heart (EC 1.1.1.35), B-hydro-xyacyl CoA dehydrogenase (Sigma, H4626) and 1.36 g of NADH (Sigma, 90% ) in 30 ml of a 0.1 molar sodium phosphate buffer, pH 6.5.

After 30 hours at 25 ° C, the reaction mixture was added four times with 30 ml

8 3 0 4 1 ^ Q

15] ethyl acetate extracted. The organic layer was dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue (90 mg) was chromatographed on a silica gel (12 g) column (1.3 x 34 cm). The column was eluted with a solvent system consisting of Skelly B ethyl acetate (8: 1) and 20 ml fractions were collected. Fractions 9-11 containing the desired methyl 4-chloro-3 (R) -hydroxybutyrate (compound of the formula 8) as shown by TLC, fct] + 23.5 ° (c, 5.2 CHCl 3) ) were united.

10 Example CCCLXVI

The method of Example CCCLXV was repeated using ethyl 4-chloroacetoacetate as the substrate to form ethyl 4-chloro-3 (R) -hydroxybutyrate [111 + 22.7 ° (c, 4.7 Cl 3).

Example CCCLXVII

The method of Example CCCLXV was repeated using n-propyl 4-chloroacetoacetate as the substrate to form 23 n-propyl 4-chloro-3 (R) -hydroxybutyrate fa + 21.5 ° (c, 5.0 , CHCl3).

Example CCCLXVIII

The procedure of Example CCCLXV was repeated using n-butyl 4-chloroacetoacetate as the substrate to form n-butyl 4-chloro-3 (R) -hydroxybutyrate [α] 20 + 20.1 ° (c, 3.1, (CHCl3).

General method for the conversion of 4-halogen-3 (R) -hydroxybutyric acid esters and amides to L-carnitine.

Example CCCLXIX

A mixture of 1.5 g of 4-chloro-3 (R) -hydroxybutyric acid octyl ester, 3 ml of methanol and 3 ml of a 25% by weight solution of trimethylamine-30 in water was heated at 80-90 ° C for about 2 hours. . The solvents and excess trimethylamine were evaporated to dryness under reduced pressure to give 1.8 g of an impure residue. The crude product (1 g) was heated to 80-90 ° C in 7 ml of a 10% HCl solution for 1.5 hours. After evaporation of the solvents under a reduced pressure, the crude product was extracted twice with 10 ml of absolute ethanol and the ethanol was evaporated under a reduced pressure. The crystalline residue was dissolved in a small amount of ethanol and the L-carnitine chloride was precipitated in good yield (320 mg) by the addition of ether, m.p. 142 ° 40 (dec.); [α] 23.7 ° (c, 4.5, H2o).

8 3 0 4;: o 16

The L-carnitine chloride can be easily converted to an internal salt of L-carnitine, which is pharmaceutically preferred, by an ion exchange agent, as is known in the art. Example CCCLXX

Octyl 4-iodo-3 (R) -hydroxybutyrate (compound of formula 9) (see the reaction equation with figure 8)

A mixture of 1,426 g of octyl 4-chloro-3 (R) -hydroxybutyrate (compound of the formula 2) and 1.2 g of anhydrous NaJ in 15 ml of methyl ethyl ketone was refluxed for 24 hours. The mixture was evaporated on a rotary evaporator and reacted with 100 ml ether and 50 ml water. The organic phase was separated and washed with 150 ml of a 10% sodium thiosulfate solution and 150 ml of brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give 1.762 g of the compound of formula 9 as a pale yellow oil; IR

(thin film) 3460 cm -1 (OH) and 1730 cm -1 (ester 0 = 0); PMR (CDCI3) - 3.93-4.27 (m, 3H), 3.17 (d, 2H), 2.50 (d, 2H), 1.50-1.87 (m, 2H), 1 .30 (bs, 12H), 0.93 (m, 3H).

Conversion of octyl 4-iodo-3 (R) -hydroxybutyrate (compound of the formula 9) to L-carnitine (see the reaction equation with figure 9).

To a solution of 1.593 g of the compound of formula 9 in 15 ml of methanol was added 8 ml of a 25% aqueous solution of triethyl amine. The mixture was stirred at 27 ° C for 20 hours. The solvents and excess trimethylamine were evaporated under reduced pressure to give a semi-crystalline solid of the formula 10. This residue was washed with small amounts of ether to remove the octanol and then dissolved in water and over a Dowex 1 x 4 column guided [OH shape - particle size 0.15-0.3 mm, column volume (2.5 x 15 cm). The column was washed with distilled water. Removal of the solvent under reduced pressure from the first 200 ml of the eluate gave L-carnitine as a white crystalline solid (490 mg, yield 65%) [ojp3 - 29.2 ° (c, 6.5 H2 O).

Example CCCLXXI

The method of Example CCCLXX was repeated using

hexyl 4-chloro-3 (R) -hydroxybutyrate to form hexyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-camitine. Example CCCLXXII

The procedure of Example CCCLXX was repeated using 40 heptyl 4-chloro-3 (R) -hydroxybutyrate to form heptyl 4-iodine- 8 1 Π Λ 4 «Λ UYY> yy ny 17 3 (R) -hydroxybutyrate, which then converted to L-carnitine. Example CCCLmil

The procedure of Example CCCLXX was repeated using decyl 4-chloro-3 (F) -hydroxybutyrate to form decyl 4-iodo-5 (R) -hydroxybutyrate, which was then converted to L-carnitine. Example GCCLXXIV

The procedure of Example CCCLXX was repeated using methyl 4-chloro-3 (R) -hydroxybutyrate (compound of the formula 8) to give methyl 4-iodo-3 (R) -hydroxybutyrate, which was then 10 L-carnitine was converted.

Example CCCLXXV

The procedure of Example CCCLXX was repeated using ethyl 4-chloro-3 (R) -hydroxybutyrate to form ethyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

15 Example CCCLXXVI

The procedure of Example CCCLXX was repeated using n-propyl 4-chloro-3 (R) -hydroxybutyrate to form n-propyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Example CCCLXXVII

The procedure of Example CCCLXX was repeated using n-butyl-4-chloro-3 (R) -hydroxybutyrate to form n-butyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Representative yeasts which produce the desired enzyme

Listed in Table A and representative fungi are listed in Table B.

8304190 18

Table A (yeasts) 1. Candida lipolytica NRRL Y-1095 2. Candida pseudotropicalis NRRL Y-1264 3. Mycoderma cerevisiae NRRL Y-1615 4. Torula lactosa NRRL Y-328 5. Geotrichum candidum NRRL Y-552 6. Hansenula anomala NRRL Y-366 7. Hansenula subpelliculosa NRRL Y-1683 8. Pichia alcoholphila NRRL Y-2026 9. Saccharomyces cerevisiae NRRL Y-12,632 10. Saccharomyces lactis NRRL Y-1140 11. Zygosaccharomyces priorianus NRRL Y-12,624 12. Saccharomyces acidifaciens NRRL Y- 7253 13. Kloeckera corticis ATCC 20109 14. Cryptococus mascerans ATCC 24194 15. Rhodotorula sp. ATCC 20254 16. Candida albicans ATCC 752 17. Dipodascus albidus ATCC 12934 18. Saccharomyces cerevisiae (Red Star from the market) 19. Rhodotornula rubra NRRL Y-1592 20. Oospora lactis ATCC 14318 NRRL - Northern Regional Research Lab. at Peoria, Illinois.

ATCC - American Type Culture Collection in Rockville, Maryland.

* 8304190 19

Table B (fungi) 1. Gliocladium virens ATCC 13362 2. Caldariomyces Fumago ATCC 16373 3. Linderina pennissopora ATCC 12442 4. Aspergillus ochraceus NRRL 405 5. Trichoderma lignorum ATCC 8678 6. Heterocephalum autantiacum ATCC 16328 7. Entata phrthora constantini NRRL 1860 9. Zygorhynchus heterogamus ATCC 6743 10. Scopulariopsis brevicaulis NRRL 2157 11. Rhizopus arrhizus NRRL 2286 12. Penicillium thomii NRRL 2077 13. Mucor hiemalis (-) NRRL 4088 14. Byssochlamys nivea ATCC 12550 NR 19. Metarrhizium anisopliae ATCC 24942 17. Penicillium islandicum ATCC 10127 18. Cunninghamella elegans ATCC 10028a 19. Cunninghamella echinulata ATCC 11585a 20. Aspergillus fumigatus ATCC 16907 21. Aspergillus amstelodami NRRL 90 22. Gliocladium roseum ATCCus 10521 23. Aspergillia ATCC 10148b 25. Penicillium roquefort! NRRL 849a 8 3 0 4! 9 0

Claims (27)

1. Compounds of formula 2 with the 3 (R) configuration, wherein X is selected from Cl, Br, J and OH and R is a straight chain, branched chain or cyclic configuration group selected from the group consisting of alkoxy groups of 1 to about 15 carbon atoms, alkylamino groups of about 5 to about 15 carbon atoms, cycloalkoxy groups and cycloalkylamino groups of about 5 to about 12 carbon atoms, phenoxy and phenylalkoxy groups of 7 to about 14 carbon atoms, phenylamino and phenylalkylamino groups of the formulas 12 and 13, wherein Y and Z are selected from H, an alkyl group having 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH 3, Cl and Pr. The compound of claim 1, wherein R is a straight chain alkoxy group of 1 to 10 carbon atoms. The compound of claim 2, wherein R is OC ^ qF ^ j. The compound of claim 2, wherein R is OCgHjj. A compound according to claim 2, wherein R is OCyH ^ ^. The compound of claim 2, wherein R is OCgFjj. A compound according to claim 2, wherein R is a lower alkyloxy group of 1 to 4 carbon atoms. 8. G 4 1 9 0 8. A compound according to claim 1, wherein R is selected from phenoxy and phenylalkoxy substituted with a lower alkyl, halogen or nitro group. The compound of claim 8, wherein R is benzyloxy and X is selected from Cl and Br. The compound of claim 1, wherein R is phenylamino and X is selected from Cl and Br. 11. A method of preparing optically active / -substituted 8-hydroxybutyric acid derivatives of the formula 11 wherein X is selected from Cl, Br, J and OH and R is a straight chain, branched chain or cyclic configuration group selected from the group consisting of 35 alkoxy groups of 1 to about 15 carbon atoms, alkylamino groups of about 5 to about 15 carbon atoms, cycloalkoxy groups and cycloalkylamino groups of about 5 to about 12 carbon atoms, phenoxy and phenylalkoxy groups of 7 to about 14 carbon atoms, phenylamino and phenylalkylamino groups with formula 12 and "f; Λ 1 0 AVV 'J" ï I j U Γ> = Λ 13, wherein Υ and Ζ are selected from Η, an alkyl group with 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CHg, Cl and Br from the corresponding / -substituted acetoacetic acid esters or amides, characterized in that / -substituted acetoacetic acid esters or amides are subjected to the fermentative enzymatic action of a microorganism producing L-8-hydroxyacyl CoA dehydrogenase [FC 1.1.1.35] and recovering the desired optically active / substituted β-hydroxybutyric acid derivatives. A process according to claim 11 for the preparation of optically active / substituted 3 (R) -hydroxybutyric acid derivatives of the formula 2 having the 3 (R) configuration, wherein X and R have the above meanings, provided that when R is an alkoxy group containing from 5 to about 15 carbon atoms, characterized in that a compound of formula 1, wherein X and R have the above meanings, is subjected to the fermentative enzymatic activity of a microorganism containing L- fJ-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] and recovering the desired optically active 4-substituted 3 (R) -hydroxybutyric acid derivatives from the fermentative reaction mixture. Method according to claim 12, characterized in that the micro-organism is selected from the class Ascomycetes. Method according to claim 12, characterized in that the microorganism is selected from the orders of Endomycetales, Mucorales, Honiliales or Eurotiales. Method according to claim 12, characterized in that the micro-organism is selected from the genus Saccharomyces. The method according to claim 15, characterized in that the microorganism is Saccharomyces cerevisiae. A method according to claim 12, characterized in that the 30-substituted acetoacetic acid derivative subjected to the fermentative enzymatic action is chloroacetoacetic acid octyl ester. 18. Process according to claim 12, characterized in that the / -substituted acetoacetic acid derivative, which is subjected to the fermentative enzymatic action, is / -chloroacetoacetic acid benzyl ester. Process according to claim 12, characterized in that the / -substituted acetoacetic acid derivative, which is subjected to the fermentative enzymatic action, is / -chloroacetoacetanilide 40. The method according to claim 17, characterized in that the microorganism is Saccharomyces cerevisiae. Method according to claim 18, characterized in that the microorganism is Saccharomyces cerevisiae. Method according to claim 19, characterized in that the microorganism is Saccharomyces cerevisiae. A process according to claim 11 for the preparation of optically active / substituted 3 (R) hydroxybutyric acid derivatives of the formula 2 with the 3 (R) configuration, wherein X has the above meanings 10 and R is an alkoxy group of 1 to 4 carbon atoms, characterized in that compounds of formula 1, wherein X and R have the above meaning, are subjected to the enzymatic action of L-O-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] in purified form and the desired optically active / - substituted 3 (R) -hydroxybutyric acid derivatives from the enzymatic reaction mixture. Process according to claim 23, characterized in that said L-p-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] in purified form is the product isolated from pig heart. Process for the preparation of an internal salt of L-carnitine, characterized in that a 4-substituted 3 (R) -hydroxybutyric acid derivative according to claim 1 is reacted successively with trimethylamine and hydrogen chloride, the L-camitine chloride is extracted and ion-exchanging this chloride and recovering the internal salt of L-carnitine. 26. Process for the preparation of a 4-iodo or 4-bromo-3 (R) -hydroxybutyric acid derivative, characterized in that a 4-chloro-3 (R) -hydroxybutyric acid derivative with sodium iodide or sodium bromide in a solvent at a temperature from 50 ° C to 100 ° C to form the corresponding 4-iodo or 4-bromo-3 (R) -hydroxybutyric acid derivative. 27. Process for the preparation of an internal salt of L-carnitine, characterized in that a) a) a 4-iodo or 4-bromo-3 (R) -hydroxybutyrate alkyl ester, wherein the alkyl group contains 1-10 carbon atoms, with trimethylamine in methanol or ethanol to form an L-carnitine methyl or ethyl ester salt and b) converting this L-carnitine ester salt to an internal salt of L-carnitine. 40 -l-H- l · l "l" H l · 8 3 0 4 1 9 o