FI81115B - FOERFARANDE FOER FRAMSTAELLNING AV OPTISKT AKTIVA -SUBSTITUERADE -HYDROXIBUTANSYRADERIVAT. - Google Patents

Description

1 81115

Process for the preparation of optically active γ-substituted β-hydroxybutanoic acid derivatives

The invention relates to a process for the preparation of optically active γ-substituent β-hydroxybutanoic acid derivatives. These derivatives are intermediates in the manufacture of L-carnitine.

As is known, carnitine (β-hydroxy-γ-trimethylaminobutanoic acid) contains a center of asymmetry and therefore exists in two stereoisomeric forms, the L and D forms.

L-carnitine is normally present in the body where it acts as a transporter of activated long chain free fatty acids across the mitochondrial membrane. Because mitochondrial membranes are impermeable to acyl-CoA derivatives, long-chain free fatty acids can cross the membrane only when esterification with L-carnitine has occurred. L-carnitine acts as a transporter, both by transporting active long-chain fatty acids from their biosynthetic site, e.g. microsomes, to the mitochondria where they are oxidized, and by transporting acetyl CoA from the mitochondrial acetyl-CoA can be used to synthesize cholesterol and fatty acids.

Although the levo isomer alone (L-Carnitine) has been shown to be a biological form (D-carnitine has not yet been found in mammalian tissues), the racemate of D, L-carnitine has been used for several years in various indications. For example, D, L-carnitine is sold in Europe as an appetite stimulant and has been shown to have an effect on children's growth rate; see. e.g., Borniche et al., Clinica Chemica Acta, 5, 171-176, 1960 and Alexander et al., "Protides in the Biological Fluids", 6th Colloquim, Bruges, 1958, 306-2 81115 310. U.S. Patent 3,830 931 describes an improvement in myocardial contractility and systolic rhythm in congestive heart failure, which can often be caused by D, L-Carnitine. U.S. Patent 3,968,241 describes the use of D, L-carnitine in cardiac arrhythmias. U.S. Patent 3,810,994 discloses the use of D, L-carnitine in the treatment of obesity.

Recently, however, the importance of using the levo isomer of carnitine alone for at least some therapeutic applications has been increasingly emphasized. In fact, D-carnitine has been shown to be a competitive inhibitor of carnitine-bound enzymes such as carnitine acetyltransferase (CAT) and carnitine palmityltransferase (PTC). Further, recent findings indicate that D-carnitine can displace L-carnitine from cardiac tissue. For this reason, it is essential that L-carnitine alone be administered to patients with heart disease or a decrease in blood lipids.

Several processes for producing carnitine are shown on an industrial scale. However, chemical synthesis of carnitine necessarily results in a racemic mixture of D and L isomers. Therefore, separation methods must be used to separate the optical antipodes from the racemate. However, these separation methods are cumbersome and expensive.

The object of the present invention is to produce L-carnitine in good yields by combining microbiological and chemical processes and using readily available, relatively inexpensive raw materials.

In particular, the invention relates to the preparation of new optically active intermediates for the synthesis of L-carnitine and its salts or esters.

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

3,81115

The fact that the β-keto group in the 3-position of γ-substituted acetoacetic acid derivatives can be reduced by hydrogenation of Pt / C as a catalyst is known from the prior art (e.g. U.S. Patent 3,969,406). However, the hydroxy compound obtained from this method is racemic. Conversely, by using fermentation with a microorganism in conjunction with the process of this invention, hydrogenation of the 3-position oxo group can be accomplished stereoselectively to give optically active γ-substituted β-hydroxybutanoic acid derivatives.

In particular, by appropriately selecting (as described below) the substrate of the fermentation microorganism to be performed in the process of this invention, a 3 (R) or L-epimeric configuration is obtained. This configuration is required to obtain natural L-carnitine.

More broadly, this invention encompasses the use of a microbial reductase enzyme, L 2 -hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), to catalyze the stereoselective hydrogenation of γ-substituted acetoacetic acid derivatives as defined below.

Therefore, the process of the present invention provides optically active γ-substituted β-hydroxybutanoic acid derivatives of the formula wherein X is Cl, Br or OH and R is a straight-chain, branched or cyclic configuration radical selected from the group consisting of: an alkoxy radical having 5 to 15 carbon atoms; a phenylalkoxy radical containing phenoxy or 7 to 14 carbon atoms; and a phenylamino radical, for the preparation of «81115 from the corresponding γ-substituted acetoacetic acid esters or amides, comprises subjecting the microorganism to fermentation of said γ-substituted acetoacetic acid esters or amides with the microorganism producing L 1 -hydroxyacylase .1.35), and recovery of the desired optically active γ-substituted β-hydroxybutanoic acid derivatives.

In particular, with a view to preparing optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives of the formula OH H 0 and 3 (R) -configuration, the process comprises the formula O 0 nn xch2-c-ch2cr in which the substituents R and X have the meanings defined above, subjecting the compounds of claim 1 to Lp-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) to undergo fermentative enzymatic activity and recovering the desired optically active 4-substituted 3 (R) -hydroxybutanoic acid derivatives from the fermentation reaction mixture.

It has been found that any microorganism that produces the desired enzyme is capable of catalyzing stereoselective reduction. Particularly suitable are Endomycetales, Mucorales, Moniliales and Eurotiales of the order Ascomycetes, and microorganisms of the genus Saccharomyces. Particularly preferred is Saccharomyces cerevisiae.

For the preparation of optically active 4-substituted 3 (R) -hydroxybutyrate esters with 1 to 4 5 81115 carbon atoms in the ester moiety, it is necessary to use purified L-β-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) as obtained from porcine heart because intact micro organisms have interfering oxy-reductases of opposite configuration. As a result, for example, microbial reduction of 4-chloroacetoacetic acid esters having 1 to 4 carbon atoms in the ester moiety yields 4-chloro-3-hydroxybutyrates having unsatisfactory optical purities.

Therefore, the present invention also includes a process for the preparation of optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives having the 3 (R) configuration and the formula wherein X is Cl, Br or OH and R is an alkoxy radical having 1 to 4 carbon atoms. , which method comprises the formula 0 0

Il II

subjecting the compounds of xch2-c-ch2c-r, wherein X and R are as defined above, to the enzymatic action of Lp-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in purified form, and to the desired optically active γ-substituted 3 Recovery of (R) -hydroxybutanoic acid derivatives from an enzymatic reaction mixture.

The invention thus provides compounds of the 3 (R) configuration and of the formula OH_H 0 6 81115 wherein X is Cl, Br or OH and R is a straight chain, branched or cyclic configuration radical selected from the group consisting of: 1-15 carbon atoms containing alkoxy radicals; phenylalkoxy radicals containing phenoxy or 7 to 14 carbon atoms; and phenylamino radicals.

The optically active γ-substituted L-β-hydroxybutanoic acid sub-derivatives can then be reacted with trimethylamine to give the corresponding γ-trimethylammonium-L-β-hydroxybutanoic acid derivative, which derivative can be immediately converted to L-carnitine by treatment with aqueous acids. The following is a diagram of the reaction steps in this process.

0 0 OH ^ H 0

Micro-

organism_. R

or L-p-hydroxyacyl-CoA-

I dehydrogenase II

X = C1, Br, OH CH3

Cho-N

i I

ch3 ch3 ch3

1 OH H I OH H O

h3c-n + sCO2h h + h3c-n + \ r I

X "ch3 * X" ch3 ^ r

IV III

L-carnitine

It has been found that the above reaction I -> II occurs more easily if X = Cl. However, since the next reaction II -> III proceeds in better yields when X * is bromine, it is preferable to first prepare a Cl derivative and then convert it to the corresponding Br derivative.

7 81115

Microorganisms having the desired oxidido reductase activity are well known in microbiology, and any such microorganism can be used in the process of this invention [cf. K. Kieslich, "Microbial Transformations of Non-Steroid Cyclic Compounds" (Georg Thieme Publishers, Stuttgart, 1967)] with any genus of microorganism which has been described as particularly suitable in this context. Easily available and inexpensive microorganisms of the genus Saccharomyces, such as brewer's yeast, baker's yeast and wine yeast (Saccharomyces Vini), have been found to produce L-β-hydroxylacetyl-CoA dehydrogenase (EL 1.1.1.35) and to be remarkably advantageous in carrying out the process of the invention. The enzyme is described by S. J. Wakil and E. M. Barnes Jr. in Comprehensive Biochemistry Vol. 185 (1971) pp. 57-104.

The 4-substituted acetoacetic acid substrate can be combined with media of standard composition used in culturing such organisms, and standard fermentation conditions can then be used to effect reductive conversion. Alternatively, the active ingredient can be removed from the culture medium of the microorganism, for example, by dissolving the cells to release the enzyme or by suspending the remaining cells in a fresh aqueous system. In all of these techniques, the β-keto group is selectively reduced as long as the active enzyme produced by the microorganisms is present in the medium. Of course, the temperature, time and pressure conditions under which the contact of the 4-substituted acetoacetic acid derivative with the reducing enzyme 8 81115 is carried out are not insignificant as will be apparent to those skilled in the art. For example, by gently heating at atmospheric pressure, the time required for reductive conversion is less than if the progression occurs at room temperature under otherwise the same conditions. Of course, the temperature, pressure, and time should not be so great that the substrate decomposes.

When using the growth medium of an organism, the process conditions should be sufficiently gentle so that the organism does not die until it produces sufficient hydrolytic enzyme to promote the reaction. Generally, at atmospheric pressure, the temperature can be from about 10 to about 35 ° C and the time from about 12 hours to about 10 days.

In the following example, which is presented to illustrate the invention and which does not limit the scope of the appended claims, the substrates of the γ-haloacetoacetic acid derivative used for microbiological reduction were prepared from diketene C.D. Hurd and H.L. According to the general method of Abernethy (J.Am.Chem.Soc., 62, 1147, 1940) to γ-chloroacetoacetic acid derivatives and according to F.Chick and NTMWilsmore /J.Chem.Soc., 1978 (1910 | _7 to γ-bromoacetoacetic acid derivatives according to the following reaction scheme: 0 0

x2 II II

H, C = C-CH, -XCH-C-CH-CX

2 j | 2 2 2 0-C = 0

ηΗ ^ HOR

0 o Y

1 Ιϊ * M?

XCH2CCH2C-NR XCH2CCH2COR

wherein X = Cl or Br Y = H or alkyl R = as previously defined.

Alternatively, if desired, γ-haloacetoacetic acid derivatives can be prepared from γ-haloacetic acid esters by conventional 9,81115

The Grignard reaction. For example, γ-chloroacetoacetic acid octyl ester was prepared directly by refluxing γ-chlorooctyl ester with two equivalents of magnesium in ether for 48 hours. After removal of the solvent, the octyl ester of acetoacetic acid was recovered in about 70% yield.

The γ-hydroxyacetoacetic acid derivatives were prepared from their corresponding γ-bromoacetoacetic acid derivatives by stirring in aqueous dioxane (1: 1) containing CaCO 3 at 25 ° C for 12 hours.

The structures of the products prepared in the following examples were analyzed using NMR, IR spectra and thin layer chromatography. The optical purity and absolute configuration of the products were determined by converting them to L-carnitine as well as converting them to their esters, which can be directly analyzed by NMR spectrometry, and by optical rotation.

Example 1 (Yeast) (+) 4-Chloro-3 (R) -hydroxybutanoic acid octyl ester was prepared as follows: O 0 OH H o CVA ^ koc - * C10-10x C8H17 OC8H17 A. Fermentation. On an agar medium having the following composition: g

Agar 20

Glucose 10

Yeast extract 2.5 k2hpo4 1

Distilled water, q.s. 1 liter (sterilized for 15 min at 140 kPa) 10,81115 grown yeast Candida keyfr. NRRL Y-329 one week old surface culture was suspended in 5 ml of 0.85% saline. One ml of this suspension was inoculated into a 250 ml Erlemneyer flask (step F-1) containing 50 ml of the following medium (Vogel's medium): g

Yeast extract 5

Casamino acids 5

Dextrose 40

Na 2 -Citrate-5 1/2 1 ^ 0 3 g KH 2 PO 4 5 g NH.NO- 2 g 4 3

CaCl 2 -2H 2 O 0.1 g

MgSO 4 * 7H 2 O 0.2 g

Trace element solution 0.1 ml

Distilled water, q.s. 1 liter pH 5.6 (sterilized for 15 min at 210 kPa)

Trace element solution ct / 100 ml

Citric acid-1H 2 O 5

ZnSO 4 * 7 H 2 O 7

Fe (NH4) 2 (SO4) 2.6H2O 1

CuSO 4 -5H 2 O 0.25

MnSO 4 .1H 2 O 0.05 H 3 BO 3 0.05

NaH2MoO4-2H2O 0.05

The flask was incubated at 25 ° C on a rotary shaker (250 rpm to a radius of about 5 cm) for 24 hours, after which 10% by volume was transferred to another 250 ml Erlenmeyer flask (F-2 step) containing 50 ml of Vogel medium. After 24 hours of incubation on a rotary shaker, 150 mg of γ-chloroacetic acid octyl ester in 0.1 ml of 10% Tween 80 was added. The F-2 stage flask was incubated for another 24 hours under the same conditions as for the F-1 stage flasks.

n 81115 B. Insulation

Twenty-four hours after the addition of octyl ester of γ-chloroacetoacetic acid, the cells were removed by centrifugation. The liquid was removed by extraction three times with 50 ml of ethyl acetate. The ethyl acetate was dried over Na 2 SO 4 and evaporated to an oily residue (186 mg). The residue was dissolved in 0.5 ml of mobile phase and applied to a silica gel column (1 x 25 cm, MN-Kieselgel 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 a crystalline residue. Recrystallization from ethyl acetate-hexane gave 107 mg of 4-chloro-3 (R) -hydroxybutanoic acid octyl ester, δ-q_723 +13.3 ° (c, 4.45) (CHCl 3); nmr (5CDCl 3) 0.88 / 3H, tr. rotation, CH 2 - (CH 2) n - /; 1.28 / 10H, s, 1.65 (2H, m, -CH 2 -CH 2 -CH 2 O-C-); 2.62 (2H, d, J = 6Hz, -CH-CH 2 -COOR);

0 OH

3.22 (1H, br, -OH); 3.60 (2H, d, J = 6Hz, C1CH - CH-R); 4.20 (3H,

OH

-CH2 (CH-CH2 and -C-O-CH2CH2). Anal Calcd for C 12 H 23 O 3 Cl: OH 0 C 57, 47; H 9.25. Found: C, 57.52; H 9.07. / TLC Rf - 0.5, - Ju

Brinkman silica gel plate, 0.25 cm E.M; Skelly B: ethyl acetate (5: 1) J

Example 2 Residual cells. One hundred grams of commercial fresh baker's yeast Saccharomyces cerevisiae (Red Star) was suspended in 250 ml of tap water, to which were added 10 g of sucrose and 3.6 g of γ-chloroacetoacetic acid octyl ester. After incubating the contents at 25 ° C on a rotary shaker (250 rpm to a radius of about 5 cm) for 24 hours, an additional 10 g of sucrose was added to the flask and the reaction was allowed to proceed for another 24 hours. Cells were 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 with ethyl acetate. The ethyl acetate layer was dried over Maso and evaporated to an oily residue which was chromatographed on a silica gel column to give 2.52 g of 4-chloro-3 (R) -hydroxybutanoic acid ethyl ester as a low melting solid; + 13.2 ° (c, 4.0, chCl 3).

Example 3 Benzyl ester of (+) 4-chloro-3 (R) hydroxybutylic acid was prepared as follows:

O 0 OH H O

\ i i - »ci Jl OCH20 A. Fermentation. With an agar medium having the following composition: g

Malt extract 20

Glucose 20

Peptone 1

Agar 20

Distilled water, q.s. One week (sterilized for 15 min at 140 kPa) of the yeast Gliocladium virens ATCC 13362 one week old surface culture was suspended in 5 ml of 0.35% saline. One ml of this suspension was inoculated into a 250 ml Erlenmeyer flask (step F-1) containing 50 ml of the following medium (soybean dextrose medium):

Soy flour 5 g

Dextrose 20 g

NaCl 5 g KHoHPO. 5 g 2 4

Yeast 5 g

Water 1 1 pH adjusted to 7, 0 Autoclaved at 105 kPa for 15 min.

i3 81115

The flask was incubated at 25 ° C on a rotary shaker (250 rpm to a radius of about 5 cm) for 24 hours, after which 10% by volume was transferred to another 250 ml Erlenmeyer flask (F-2 step) containing 50 ml of soybean dextrose medium. After 24 hours of incubation on a rotary shaker, 150 mg of γ-chloroacetoacetic acid benzyl ester in 0.1 ml of 10% Tween 80 was added. The F-2 stage flask was incubated for another 24 hours under the same conditions as the F-1 stage flasks.

B. Isolation. Twenty-four hours after the addition of β-chloroacetoacetic acid, the mycelium was removed by filtration. The filtrate was extracted into 50 ml of ethyl acetate three times. The ethyl acetate layer was dried over MgSO 4 and concentrated in vacuo to give a residue (160 mg). The residue was chromatographed on a silica gel column (MN-Kieselgel 60, 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 products were collected and concentrated to dryness to give 115 mg of 4-chloro-3 (R) -hydroxybutanoic acid benzyl ester, m.p. + 8.7 ° (c, 5.26; CHCl); pmr (δ CDCl 3) 2.65 (2H, d, J = 6 Hz, -CH-CH-C 10 R), 3.20 (1H, br, -OH); 3.54 • ^

OH

(2H, d, J = 6Hz, C1-CH2CH); 4.20 (1H, m, -CH 2 -CH-CH 2 -),

OH OH

5.12 (2H, s, -C-O-CH2Cl2H2); 7.31 (5H, s, five aromatic pro-O

shove). Anal Calcd for C 12 H 12 N 2 O 2: C, 57.77; H 5.73. Found: C, 57.64; H 5.67. / TLC silica gel EM Brinkmann plate, 0.25 cm, Rf = 0.43, Skelly B-ethyl acetate (5: 1) ^ 7

Examples 4-23

The procedure of Example 1 was repeated for each of the organisms shown in Table 1, except that octyl ester of γ-chloroacetoacetic acid was added at a concentration of 1 mg / ml. Conversion to the desired product, (+) - 4-chloro-3 (R) -hydroxybutylic acid octyl ester was obtained. The procedures of these examples were repeated continuously with the addition of substrate to the yeast medium. The weight ratio i4 81115 substrate / yeast was about 1: 1.5 with excellent conversion to the desired product.

Examples 24-48

The procedure of Example 3 was repeated for each of the organisms shown in Table 2 except that γ-chloroacetoacetic acid octyl ester (1 mg / ml) was used. Conversion to the desired compound, (+) 4-chloro-3 (R) -hydroxybutanoic acid octyl ester, was accomplished.

Examples 49-68

The procedure of Example 1 was repeated for each of the organisms listed in Table 1 except that benzyl ester of v-chloroacetoacetic acid (1 mg / ml) was used as a substrate. Conversion to the desired product, benzyl ester of (+) 4-chloro-3 (R) -hydroxybutanoic acid, was accomplished.

Examples 69-93

The procedure of Example 3 was repeated for each organism listed in Table 2 using γ-chloroacetoacetic acid benzyl ester (1 mg / ml) as substrate. Conversion to the desired compound (()) 4-chloro-3 (R) -hydroxybutanoic acid benzyl ester was accomplished.

Example 94 (+) 4-Chloro-3 (R) -hydroxybutanoic acid anilide was prepared according to the procedure of Example 2 except that 4-chloroacetoacetanilide was used at a concentration of 1 mg / ml as a substrate. to convert it to the desired optically active product, m.p. 110-111 ° C; / et / 22 + 17.5 ° (c, 3.0, CHCl 3); 0 tl pmr (δ CD3CCD3) 2.67 (2H, d, J = 6Hz, -HOHHCH2-CONHR), 3.66 (2H, d, J = 6Hz, C1CH2CHOH-R), 4.43 (1H, m, -CH 2 -CHOH-CH 2 -), 7.03-7.44 (3H, m, aromatic protons, meta and para), 7.69 (2H, d, J -6Hz, aromatic protons, ortho), 9.24 (1H, br, -CN-O). Anal.

M

O

Calcd for C10H12NO2Cl: C, 56.21; H 5.66. Found: C, 56.17; H 5.47.

Examples 95-114

The procedure of Example 1 was repeated for each of the organisms listed in Table 1 except that γ-chloroacetoacetanilide was added at a concentration of 1 mg / ml. In all cases, conversion to the desired product (+) 4-chloro-3 (R) -hydroxybutanoic acid anilide was achieved.

Examples 115-139

The procedure of Example 3 was repeated for the organisms listed in Table 2. γ-chloroacetoacetanilide was added to a concentration of 1 mg / ml. In these cases, conversion to the desired (+) 4-chloro-3 (R) -hydroxybutanoic acid anilide was achieved.

Examples 140-159

The procedure of Example 1 was repeated for the organisms listed in Table 1 except that γ-bromoacetoacetic acid octyl ester (1 mg / ml) was used as substrate. Conversion to the desired product, (+) 4-bromo-3 (R) -hydroxybutanoic acid octyl ester, was accomplished.

Examples 160-184

The procedure of Example 3 was repeated with the organisms listed in Table 2 except that γ-bromoacetoacetic acid octyl ester (1 mg / ml) was used. Conversion to the desired product, (+) 4-bromo-3 (R) -hydroxybutanoic acid octyl ester, was accomplished.

ie 81115

Examples 185-204

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

Examples 205-229

The procedure of Example 3 was repeated for the organisms listed in Table 2 except that benzyl ester of γ-bromoacetoacetic acid was used. Conversion to the desired product, (+) - 4-bromo-3 (R) -hydroxybutanoic acid benzyl ester was accomplished.

Examples 230-249

The procedure of Example 1 was repeated for the organisms listed in Table 1 except that v-bromoacetanilide (1 mg / ml) was used as substrate. Conversion to the desired (+) 4-bromo-3 (R) -hydroxybutanoic acid anilide was accomplished.

Examples 250-274

The procedure of Example 3 was repeated for the organisms listed in Table 2 except that γ-bromoacetanilide (1 mg / ml) was used as substrate. Conversion to the desired (+) 4-bromo-3 (R) -hydroxybutanoic acid anilide was accomplished.

Examples 275-294

The procedure of Example 1 was repeated for the organisms listed in Table 1 except that γ-hydroxyacetoacetic acid octyl ester (1 mg / ml) was used as substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutanoic acid octyl ester was accomplished.

Examples 295-319

The procedure of Example 3 was repeated for the organisms listed in Table 2 except that γ-hydroxyacetoacetic acid octyl ester (1 mg / ml) was used as substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutanoic acid octyl ester was accomplished.

17 81115

Examples 320-339

The procedure of Example 1 was repeated for the organisms listed in Table 1 except that γ-hydroxyacetoacetanilide (1 mg / ml) was used as substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutanoic acid anilide was accomplished.

Examples 340-364

The procedure of Example 3 was repeated with the organisms listed in Table 2 except that γ-hydroxyacetoacetanilide (1 mg / ml) was used as substrate. Conversion to the desired 4-hydroxy-3 (R) -hydroxybutanoic acid anilide was accomplished.

Example 365

Methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) 0 0 H0 ^ H 0

0CH3 OCH

J 3

VII VIII

Methyl 4-chloroacetoacetate (VII) (100 mg) was incubated with 29 units of porcine heart isolate (EC 1.1.1.35), 8-hydroxyacyl-CoA dehydrogenase (Sigma, H4626) and 1.36 g of NAHDra (Sigma, 90%) in 30 ml of 0.1 M sodium phosphate buffer, pH 6.5.

After 30 hours at 25 ° C, the reaction mixture was extracted four times with 30 ml of ethyl acetate. 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 containing Skelly B-ethyl acetate (8: 1) and 20 ml fractions were collected. Fractions 9-11 containing the desired methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) as interpreted by thin layer chromatography were combined at + 23.5 ° (c, 5.2 CHCl 3).

Example 366

The procedure of Example 365 was repeated using 18,81115 ethyl 4-chloroacetoacetate as substrate to give ethyl 4-chloro-3 (R) -hydroxybutyrate, [α] 25 D = 22.7 ° (c, 4.7 CHCl 3) ·

Example 367

The procedure of Example 365 was repeated using n-propyl 4-chloroacetoacetate as substrate to give n-propyl 4-chloro-3 (R) -hydroxybutyrate, m.p. + 21.5 ° (c, 5.0, CHCl3). .

Example 368

The procedure of Example 365 was repeated using n-butyl 4-chloroacetoacetate as substrate to give n-butyl 4-chloro-3 (R) -hydroxy-23 ° butyrate, /. qJp + 20.1 ° (c, 3.1, CHCl 3).

General procedure for the conversion of esters and amides of 4-halo-3 (R) -hydroxybutanoic acid to L-carnitine_

Example 369 A mixture of 4-chloro-3 (R) -hydroxybutanoic acid octyl ester (1.5 g), ethanol (3 ml) and water (3 ml) of trimethylamine (25% w / w) was heated to 80-90 ° C. for about 2 hours. The solvents and excess trimethylamine were evaporated to dryness in vacuo to a crude residue of 1.8 g. The crude product (1 g) was heated to 80-90 ° C in 10% HCl (7 mL) for 1.5 h. After evaporation of the solvents under reduced pressure, the crude product was extracted twice with absolute ethanol (10 ml) and the ethanol was evaporated in vacuo. The crystalline residue was dissolved in a small amount of ethanol-1 and L-carnitine chloride precipitated upon addition of ether in good yield (320 mg), m.p. 142 ° (dec.); 1 H - 23.7 ° (c, 4.5, H 2 O).

L-carnitine chloride can be converted directly to a pharmaceutically preferred internal salt of L-carnitine by ion exchange techniques. . as is known in the art.

Example 370

Octyl 4-iodo-3 (R) -hydroxybutyrate (IX) ° H H? OH H °

-xXX * ö <X

„OC8« 17 A / ^^ OC8 «17 i9 81115

A mixture of octyl 4-chloro-3 (R) -hydroxybutyrate (II) (1.426 g) and anhydrous Nairn (1.2 g) in 15 ml of methyl ethyl ketone was refluxed for 24 hours. The mixture was evaporated on a rotary evaporator and reacted with ether (100 ml) and water (50 ml). The organic phase was separated and washed with 10% sodium thiosulfate solution (150 ml) and brine (150 ml), and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give 1.762 g of compound IX as a pale yellow oil; IR (thin layer) 3460 cm-1 (OH) and 1730 cm-1 (ester C = 0); PMR (CDCl 3) - 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 (IX) to L-carnitine__ CH-.

OH 0 IiI "CH3 - H n 1 CH, 1 V * 0 CH3 I 3 OH h 0 'Vs / ^^^ OC8H17 CH3OH ^ V” 1 CHt. 3 CH3 ^

OH H O

“F-XA, · CH3 L-carnitine

To a methanol solution (15 ml) of compound IX (1.593 g) was added a 25% aqueous solution of trimethylamine (8 ml). The mixture was stirred at 27 ° C for 20 hours. The solvents and excess trimethylamine were evaporated under reduced pressure to give a semicrystalline solid X. This residue was washed with small amounts of ether to remove octanol and then dissolved in water and passed through a Dowex 1-x4 resin / OH form - 50-100 mesh, column volume (2.5 x 15 cm -1). The column was washed with distilled water (20 x 81 cm -1). Removal of the solvent in vacuo from the first 200 mL of eluate gave L-carnitine as a white crystalline 23 ° solid (490 mg, 65% yield) “29.2 (c, 6.5 H 2 O).

Example 371

The procedure of Example 370 was repeated using hexyl 4-chloro-3 (R) -hydroxybutyrate to give hexyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-Carnitine.

Example 372

The procedure of Example 370 was repeated using heptyl 4-chloro-3 (R) -hydroxybutyrate to give heptyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Example 373

The procedure of Example 370 was repeated using Decyl 4-chloro-3 (R) -hydroxybutyrate to give Decyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Example 374

The procedure of Example 370 was repeated using methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) to give methyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Example 375

The procedure of Example 370 was repeated using ethyl 4-chloro-3 (R) -hydroxybutyrate to give ethyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Example 376

The procedure of Example 370 was repeated using n-propyl 4-chloro-3 (R) -hydroxybutyrate to give n-propyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

2i 81115

Example 377

The procedure of Example 370 was repeated using n-butyl 4-chloro-3 (R) -hydroxybutyrate to give n-butyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Typical yeasts that produce the desired enzyme are listed in Table 1 and typical fungi are listed in Table 2.

Table 1 (yeasts) 1. Candida lipolytica NRRL Y-1095 2. Candida pseudotropicalis NRRL Y-1264 3. Mycoderma cerevisiae NRRL Y-1615 4. Torula lactosa NRRL Y-329 5. Geotrichum candidum NRRL Y-552 6. Hansenula anomala NRRL Y-366 7. Hansenula subpelliculosa NRRL Y-1683 8. Pichia alcoholophila 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 NR- 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 (commercial Red Star) 19. Rhodotorula rubra NRRL Y-1592 20. Oospora lactis ATCC 14318 NRRL- Northern Regional Research Lab., Peoria, Illinois.

ATCC - American Type Culture Collection, Rockville, Maryland.

22 81 1 1 5

Table 2 (fungi) 1. Gliocladium virens ATCC 13362 2. Caldariomyces fumago ATCC 16373 3. Linderina pennisopora ATCC 12442 4. Aspergillus ochraceus NRRL 405 5. Trichoderma lignorum ATCC 8678 6. Heterocephalum autantiacum ATCC 16328 7. Entomopht Constantini NRRL 1860 9. Zygorhynchus heterogamus ATCC 6743 10. Scopulariopsis brevicaulis NRRL 2157 11. Rhizopus arrhizus NRRL 2286 12. PeniciIlium thomii NRRL 2077 13. Mucor hiemalis (-) NRRL 4088 14. Byssochlamys nivea ATCC 12550 15. Penicillium Pat. 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 amstelamia NRRL 90 22. Gliocladium roseum ATCC 105 g ATCC 10148b 25. Penicillium roqueforti NRRL 849a

Claims (12)

23 81 1 1 5 A process for the preparation of optically active γ-substituted β-hydroxybutanoic acid derivatives of the formula OH HO wherein X is Cl, Br or OH and R is a straight-chain, branched or cyclic configuration radical selected from the group consisting of: an alkoxy radical having 5 to 15 carbon atoms ; a phenylalkoxy radical having 7 to 14 carbon atoms; and a phenylamino radical, for the preparation of the corresponding γ-substituted acetoacetic acid esters or amides, characterized in that said γ-substituted acetoacetic acid esters or amides are subjected to fermentation of the microorganism by the microorganism producing Lp-hydroxyacyl-CoA , and the desired optically active γ-substituted β-hydroxybutanoic acid derivatives are recovered. Process for the preparation of optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives of the formula OH, H 0 in which X and R have the meanings given above and the 3 (R) configuration, known according to claim 1 that the compounds of formula 0 0 II II xch2-c-ch2cr 24 81 1 1 5 in which X and R have the meanings given above are subjected to enzymatic enzymatic activity of the microorganism with the production of Lp-hydroxyacyl by the microorganism -CoA dehydrogenase (EC 1.1.1.35) and the desired optically active 4-substituted 3 (R) -hydroxybutanoic acid derivatives are recovered from the fermentation reaction mixture. Method according to Claim 2, characterized in that the microorganism is selected from the class Ascomycetes. Method according to Claim 2, characterized in that the microorganism is selected from the orders Endomycetales, Mucorales, Moniliales or Eurotiales. Method according to Claim 2, characterized in that the microorganism is selected from the genus Saccharomyces. Method according to Claim 5, characterized in that the microorganism is Saccharomyces cerevisiae. Process according to Claim 2, characterized in that the γ-substituted acetoacetic acid derivative which has been subjected to a fermenting enzymatic action is the octyl ester of γ-chloroacetoacetic acid. Process according to Claim 2, characterized in that the γ-substituted acetoacetic acid derivative which has been subjected to a fermenting enzymatic action is a benzyl ester of γ-chloroacetoacetic acid. Process according to Claim 2, characterized in that the γ-substituted acetoacetic acid derivative which has been subjected to a fermenting enzymatic action is γ-chloroacetoacetanilide. 25 81 1 1 5 Method according to one of Claims 7 to 9, characterized in that the microorganism is Saccharomyces Cerevisiae. A process for the preparation of optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives of the formula HO and the 3 (R) configuration, wherein X is Cl, Br or OH and R is an alkoxy radical having 1 to 4 carbon atoms, characterized in that that formula Wherein X and R have the meanings given above, the compound is subjected to the enzymatic action of pure L 1 -hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and optically the active γ-substituted 3 (R) -hydroxybutanoic acid derivative is recovered from the enzymatic reaction mixture. A method according to claim 11, characterized in that said pure L-p-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) is isolated from porcine heart. 26 81 1 1 5