FR2537130A1 - B-HYDROXYBUTYRIC ACID DERIVATIVES, PROCESS FOR THE PREPARATION THEREOF AND THEIR USE FOR THE PREPARATION OF L-CARNITINE - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR PREPARING L-CARNITINE. THIS PROCEDURE CONSISTS OF SUBMITTING ESTERS OR AMIDS OF ACETOACETIC ACIDS SUBSTITUTED IN GA THE ENZYMATIC ACTION OF FERMENTATION OF A MICROORGANISM WHICH ELABORATES LB-HYDROXY-ACYL-COA-DEHYDROGENASE EC 1.1.1.35, TO RECUPERATE THEIDE B-HYDROXYBUTYRIC SUBSTITUTE IN CORRESPONDING G, OPTICALLY ACTIVE RESULTING, AND TO TRANSFORM ITS DERIVATIVE IN L-CARNITINE. THE INVENTION ALSO DESCRIBES AN IMPROVEMENT OF THE PROCESS WHICH CONSISTS OF REACTING A 4-CHLORO-3 (R) -HYDROXYBUTYRATE WITH IODIDE OR SODIUM BROMIDE TO OBTAIN 4-IODO-OR 4-BROMO-3 (R) -HYDROXYBUTYRATE CORRESPONDING, TO BE TRANSFORMED TO 4-IODO- OR 4-BROMO-3 (R) -HYDROXYBUTYRATE TO TRIMETHYLAMINO-3 (R) -HYDROXYBUTYRATE, THEN TO TRANSFORMED TRIMETHELYNE-3DROX (LAMINO-3 (R)) -CARNITINE.

Description

25371 3

  The present invention relates to methods

  of L-carnitine production It relates more

  particularly to a process for the microbiological reduction of acetoacetic esters or amides substituted for their respective derivatives of substituted Lp-hydroxybutyric acids.

  end Y, which derivatives can easily be trans-

  L-carnitine chloride The invention also relates to novel chemical intermediates used in

this process.

  As we know, carnitine (p-acid)

  hydroxytrimethylamino-butyric acid) contains a

  asymmetry and, as a result, exists under two

  my stereoisomers, forms D and L. L-carnitine is normally present in the body where it has the role of transporting long-chain activated free fatty acids across the membrane

  mitochondria Since the membrane of mitochondria

  chondria is impermeable to acyl-Co A derivatives, long-chain free fatty acids can not penetrate

  when the esterification with L-carnitine took place

  The role of transporter of L-carnitine is exerted

  both for the transport of long chain fatty acids

  sites of their biosynthesis, for example microsomes, at the mitochondria where they are oxidized, and for the transport of acetyl-Co A mitochondria where it is formed, at sites outside the mitochondria where the synthesis of fatty acids takes place. long chain, for example in microsomes where acetyl-Co A can be used for

  synthesis of cholesterol and fatty acids.

  Although it is established that the levorotatory isomer (L-carnitine) exclusively is the biological form (D-carnitine has never been detected until now in mammalian tissues), the racemate of D, L- carnitine

  has been used for a number of years to

  For example, D, L-carnitine is sold in Europe as an appetite stimulant and has been reported to have an effect on the growth rate of children; see, for example, Clinica Chemica Acta, 5, 171-176, 1960 by Borniche et al. and "Protects in the Biological Fluids", 6th Symposium, Bruges, 1958, 306-310 of Alexander et al.

  United States of America No. 3,830,931 discloses improvements in

  rations of myocardial contractility and rhythm

  systolic in the case of congestive heart failure

  which can often be obtained by adminis-

  of D, L-carnitine The United States Patent of

  No. 3,968,241 discloses the use of D, L-carnitine in cardiac arrhythmias. The United States Patent

  No. 3,810,994 discloses the use of D, L-

  carnitine in the treatment of obesity.

  Recently, however, there has been a growing focus on the importance of exclusive use

  of the levorotatory isomer of carnitine for at least

  In fact, it has been de-

  showed that D-carnitine is a competitive inhibitor

  carnitine-related enzymes such as carnitine

  acetyl transferase (CAT) and carnitine palmityl-

  In addition, recent evidence suggests that D-carnitine can displace L-carnitine from the tissue.

  Therefore, it is essential that the L-

  carnitine is exclusively administered to patients under

  medical treatment for cardiac disorders or lowering

blood lipid levels.

  Various methods have been proposed to produce

  carnitine on an industrial scale However, the synthesis of

  The chemical structure of carnitine inevitably leads to a racemic mixture of the D and L isomers.

  must use methods of duplication to obtain

  separate optical antipodes separated from the racemate.

  These methods of duplication are however tedious

and expensive.

  The present invention proposes to produce L-carnitine in a satisfactory yield by a combination of microbiological and chemical processes. The present invention proposes to provide an improved process for the synthesis of L-carnitine from starting materials. inexpensive

readily available.

  Another object of the present invention is the preparation of optically active intermediates, new and useful for the synthesis of L-carnitine and its

salts or esters.

  Another object of the present invention is to provide processes for the preparation of L-carnitine via displacement by trimethylamine

  halo group of a 4-halo-3 (R) -hydroxybutyrate.

  Another object of the present invention is to

  provide a process for the production of 4-iodo or 4-bromo

  3 (R) -hydroxy-butyrates from 4-chloro-3 (R) -hydro-

  xybutyrates. These and other goals will emerge from

the following description.

  The fact that the p-ketonic function posi-

  3, substituted acetoacetic acid derivatives can be reduced by hydrogenation on Pt / C is known.

  (eg United States Patent No. 3,969,406).

  However, the hydroxylated compound resulting from such a procedure

  On the contrary, by using the fermentation action of a microorganism according to the process of the present invention, the hydrogenation of the oxo function

  in position 3 can be performed in a stereoselective

  by giving derivatives of P-hydroxybutyric acids

substituted in eoptically active.

  In particular, after a suitable choice (see below) of the substrate to be exposed to the fermentation action of the microorganisms according to the process of the present invention, the epimer configuration is obtained.

  3 (R) or L This configuration is necessary for the trans-

natural L-carnitine formation,

  Roughly speaking, this inyention includes the use

  microbial reductase, L-p-hydroxyacyl-

  Co A-dehydrogenase, to catalyze the hydro-

  stereoselective generation of acetoacetic acid derivatives

  substituted in Y as defined below.

  * Therefore, according to its widest aspect,

  the process of the present invention for the preparation of

  6-substituted optically active P-hydroxybutyric acid derivatives of the formula:

OH O

  Wherein X is selected from C 1, Br, I and OH and R is a straight chain, branched, branched, or cyclic radical selected from alkoxy radicals having from 1 to about 15 carbon atoms. carbon; alkylamino radicals having from about 5 to about 15 carbon atoms; cycloalkoxy and cycloalkylamino radicals having from about 5 to about 12 carbon atoms; phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms; phenylamino and phenylalkylamino radicals having the formulas

Y Y Z

  N-0-A; and -N-CH-O-A o Y and Z are selected from H, an alkyl group of 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH 3, Cl and Br from the esters or acetoacetic acid amides

  correspondents substituted in Y, consists in submitting

  so-called esters or amides of substituted acetoacetic acids

  k in the enzymatic fermentation action of a microorganism

  which produces L-hydroxyacyl-Co A-dehydrogenase

2537 13 O

  JEC 1 1 ol 35 J, and to recover the 3-hydroxy acid derivatives.

  substituted xybutyric substituted active.

  In particular, to prepare Yoptically active substituted 3 (R) -hydroxybutyric acid derivatives having the formula and the 3 (R) configuration.

OH H O

X A "/} |

  The process comprises subjecting compounds of the formula:

0 O

he

XCH 2-C-CH 2 CR

  X and R have the meanings indicated above, provided that if R is an alkoxy radical, it comprises from 5 to about 15 carbon atoms, with the action

  enzymatic fermentation of a microorganism that

  boron L-p-hydroxyacyi-Co A-dehydrogenase ZEC 1 1 1 35), and to recover 3 (R) -hydroxybutyric acid derivatives

  4 optically active substituents desired from the

reaction mixture of fermentation.

  It has been found that any microorganism that pro-

  the desired enzyme is able to catalyze the reduction of

  Stereoselective microorganisms of the class Ascomycetes, the order Endomycetales, Mucorales, Moniliales and Eurotiales and the genus Saccharomyces are particularly suitable.

Saccharomyces cerevisiae.

  To prepare the 3 (R) -hydro-

  xybutyric substituted in 4 optically active, containing 1 to 4 carbon atoms, it is necessary to use

  purified L-p-hydroxyacyl-Co A-dehydrogenase CEC 1 1.

  1,357 such as that of pork heart, because a microorgan-

  intact has uncomfortable oxidoreductases of con-

  opposite representation As a result, microbial reduction,

  for example, 4-chloroacetoacetic esters of 1 to 4 carbon atoms

  carbon monoxide produces 4-chloro-3-hydroxybutyrates

unsatisfactory optical purity.

  Therefore, the present invention also provides a process for the preparation of optically active substituted 3 (R) -hydroxy-butyric acid derivatives having the formula and the 3 (R) configuration.

OH O

X

R

  in which X represents Ci, Br, I or OH and R is an alkoxy radical of 1 to 4 carbon atoms, which consists in subjecting compounds of formula:

O C

XCH 2-C-CH C-R

  in which X and R have the meanings previously

  indicated for the enzymatic action of L -) - hydroxyacyl-

  Co A-dehydrogenase / ZC 1 1 1 35 in purified form, and recovering the desired optically active 3 (R) -hydroxybutyric acid derivatives desired from the mixture

enzymatic reaction.

  The present invention therefore provides compounds having the formula and the configuration 3 (R)

OH H

  Wherein X is selected from C 1, Br, I and OH and R is a straight chain, branched chain or cyclic chain radical selected from

  a class comprising alkoxy radicals having from 1 to about

  15 carbon atoms; alkylamino radicals having from about 5 to about 15 carbon atoms; cycloalkoxy and cycloalkylamino radicals having from about 5 to about 12 carbon atoms; phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms; phenylamino and phenylalkylamino radicals having the formulas: y Y z Z y -N-O-A; andN-CH-O-A o Y and Z are selected from H, an alkyl group of 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH 3, Cl and Br. Lp-hydroxybutyric substituted <optically active can then be reacted

  with trimethylamine to give the acid derivative "-

  trimethylammonium-L-p-hydroxybutyric acid which can then be easily converted to L-carnitine by treatment with aqueous acids.

  diagram of the reaction steps of this process.

x

X I R

I OH

Microphone-

-organism I

or L-B-hydroxyacyi-

  Co A-dehydrogenase X = C 1, Br, I, 3 OH CE 3 OH

HC -N c 2 H 4 -

ca 3

X C 3 V

IV

3 O H OH

Oor

R 3 C 1 R

  C 3 III L-carcinates S O -, R II CU 3 CH 1

C c 3-

I VI

253713 D

  It has been found that the above reaction occurs more easily if X = C 1 However, since the subsequent reaction Ir> III takes place in better yields when X = iodine or bromine, it is preferable to prepare first of all the derivative C1 and then the

  shooting in the derivative I or Br corresponding.

  The present invention also provides an improved process of transforming everything

  first the 4-chloro-3 (R) -hydroxybutyric ester in the 4-

  icdo or 4-bromo-3 (R) -hydroxybutyrates corresponding.

  For the sake of simplicity, reference will be made hereinafter to

  derivative I Iodohydrin (V) can be reacted doubly

  with trimethylamine at room temperature

  to give VI which is easily transformed into L-carniti-

  according to the following reaction sequence:

HO 0, O Hey A -

Cl SO Evil>

C RI 'OR

  R 5 H or ester H H c H 3 CH 3 CH 3 OH O CH 3 OHH o

CH 3-5 C Dowex-1.

  3 Resin CH 3 CH 3 form OH ICH 3 I YI-L-carnitine The above process, as illustrated by the equation, is subject to many variations. Whatever the form in which it is obtained, the 'ester

  is reacted with sodium iodide in a soil

  appropriate reaction such as 2-butanone, acetone, butanol, etc. The main reaction desired at this point of the reaction with sodium iodide is a reaction reaction with sodium iodide is a displacement reaction which forms iodohydrin V without disturbing the chiral center of the adjacent carbon atom. This reaction requires at least enough sodium iodide to displace all of the chloride from II. Generally, a slight excess of iodide is used.

sodium.

  The reaction of V with trimethylamine can be carried out at a moderate temperature (eg C) (see SG Boots and MR Boots, J Pharma Sci, 64, 1262, 1975), in various solvents such as methanol or ethanol containing an excess of trimethylamine It is

  interesting to note that according to the alcoholic solvent used

  If, for example, methanol is used as the solvent, then an exchange of ester groups takes place.

  obtain the methyl ester of L-carnitine This reaction

  exchange is advantageous because it is known that the ester

  Methyl L-carnitine can be converted directly

  in the free base form of L-carnitine by passing through an ion exchange column (OH-) see E.

  Strack and J Lorenz, J Physiol Chem (1966) 344, 2762.

  We can see from the description of the processes

  Above all, a number of novel and highly useful optically active intermediates are formed. Alkyl esters of 4-iodo-3 (R) -hydroxybutyric acid in which the alkyl groups have from six to ten carbon atoms each.

  The octyl ester is particularly preferred.

  than. Microorganisms that perform the activity

  oxido-reductase are well known in the art.

  and any of these microorganisms can be used to carry out the process of the present invention (see K Kieslich "Microbial Transformations of Non-Stezold Cyclic Compounds" (Georg Thieme Publishers, Stuttgart, 1976)). Any of the kinds of microorganisms described in particular herein being particularly applicable. It has been found that readily available and inexpensive microorganisms of the genus Saccharomyces, for example, brewer's yeast, baker's yeast and wine-making yeast (saccharomyces).

  charomyces vini) produced L-p-hydroxylacyl-Co A-

  dehydrogenase lEL 1 1 1 357 and were particularly

  advantageous in the implementation of the process of the invention.

  The enzyme is described by SJ Wakil and E M Barnes Jr in Comprehensive Biochemistry, Volume 185 (1971),

pages 57-104.

  The acetoacetic substrate substituted in 4

  can be incorporated into a nutrient medium of

  a classical reaction in which such microorganisms are

  cultivated and the usual fermentation conditions can be

  then be used to carry out the transformation

  In a variant, the active ingredient can be

  removed from the growing culture of the micro-

  such as lysis of cells to release

  enzymes, or by suspending the cells

  in a fresh aqueous system In one or the other

  of these techniques, the p-ketonic function is selected

  significantly reduced as long as the active enzyme is

  by the microorganisms is present in the medium.

  Naturally, the temperature, time and pressure conditions at which the contacting of the 4-substituted acetoacetic derivative with the reducing enzyme is carried out is interdependent, as will be appreciated by the specialist. For example, in the case of heating at the atmospheric pressure, the time necessary to carry out the reducing transformation will be less than in the case where one operates at ambient temperature, the other conditions being the same Naturally, the temperature, the pressure and the time must not to be sufficiently high to result in degradation of the substrate When using a growth culture of the microorganism the operating conditions must be

  to be sufficiently-moderate, so that the micro-

  the organization is not destroyed before it develops enough

  hydrolytic enz-ymes to allow the reaction to

  In general, at atmospheric pressure, the temperature may be between about 10 C and

  about 35: C, and the time between about 12 hours and about

10 days.

  In the following examples, which are intended only to illustrate the present invention and are not intended to be limiting, the substrates consisting of r-haloacetoacetic acid derivatives to be microbiologically reduced are prepared from diketene in accordance with the general method of C D. Hurd and HL Abernethy (J Am Chem Soc., 62, 1147, 1940) for Y-chloro-acetoacetic derivatives and F Chick,

  N.TM Wilsmore IJ Chem Soc, 1978 (1910) 1 for the de-

  Y-bromoacetoacetic rivets via the following reaction sequence: o H 2 CC <-2 'Xa 2 I 12 c-c = o C ccac

C-C = O- /

  dke'etene R OR Xl 2 C = 2 Cm X {2 Cz 2 COR o X = C 1 or Br Y = H or alkyl R = as previously defined

  Alternatively, if desired, Y-derivatives

  haloacetoacetic agents can be prepared from

  f'-haloacetic acids by a classical reaction of

  Grignard For example the octyl ester of the acid-chloro

  Acetoacetic acid can be easily prepared by refluxing the Y-chlorooctyl ester with two equivalents of magnesium in ether for 48 hours. After removing the solvent, the octyl ester of the acid is recovered.

  acetoacetic acid in a yield of about 70%.

  The f-hydroxyacetoacetic acid derivatives are

  prepared from their Y-bromo-acetoacetic acid derivatives

  corresponding acetic acid by agitation in a solution

  dioxane-water (1: 1) containing CaCO 3 at 25 C for 12 hours.

  Each of the products obtained according to the following examples is identified as to its structure using

  nuclear magnetic resonance (NMR), the spectra

  frarouges and by the mobilities in chromatography

  The optical purity and the absolute configuration of

  products are established by their conversion rate to L-

  carnitine as well as by their conversion rate into their esters which are easily analyzed by NMR spectrometry,

and optical rotation.

Example 1 (Yeasts)

  The octyl ester of the acid is prepared as follows.

  (+) 4-chloro-3 (R) -hydroxybutyric acid:

> C

  0 C 8 A 17 O CSH 17 A Fermentation A culture grown on a one-week agar slant of Candida Keyfr NRRL Y-329 grown on agar having the following composition: Grams Agar 20 Glucose 10 Yeast Extract 2.5

K 2 HPO 4 1

  Distilled water, qs 1 liter (sterilized for 15 minutes at a pressure of 140 kPa) is suspended in 5 ml of 0.85% saline solution. 1 ml portions of this suspension are used to inoculate a 250 ml Erlenmeyer flask (stage F-1)

  containing 50 ml of the following medium (Vogel's medium) -

  Yeast Extract 5 Casamino-Acids Dextrose 40 Na-Citrate 3-5 1/2 H 3 g KH 2 PO 4 5 g NH 4 NO 3 2 g Ca C 12 2 H 20 0.1 g

2

  Mg SO 47 H 20 0.2 g Trace element solution 0.1 ml Distilled water, q s 1 liter p H 5.6 (sterilized for 15 minutes at 210 k Pa) Trace element solution Gm / 100 ml

-

  Citric acid 5 Zn SO 4 7 H 20 5 Fe (NH 4) 2 (504) 6 H 20 1 Cu SO 45 H 20 0.25 Mn SO 41 H 20 0.05 HBO

39, H 3 803 0.05

  Na H 2 Mo 04 2 H 20 0.05 The flask is incubated at 25 ° C on a rotary shaker (250 cycles / min radius of 5 cm) for 24 hours, after which 10% by volume is transferred to another flask. 250 ml Erlenmeyer flask (stage 1-2) containing 50 ml of medium

  de Vogel After 24 hours of incubation on a rotary shaker

In addition, 15 mg of octyl ester of t-chloroacetoacetic acid are added.

  acetic acid in 0.1 ml of 10% Tween 80.

  the F-2 stage balloon for an additional 24 hours under the conditions used in the incubation of balloons

stage F-1.

  B Isolation Twenty-four hours after the addition of the ester

  octyl of the I-chloroacetic acid, it eliminates the

  by centrifugation, the extract is thoroughly extracted three times

  Supernatant with 50 ml of ethyl acetate The ethyl acetate is dried over Na2O4 and evaporated to give an oily residue (186 mg). The residue is dissolved in 0.5 ml of the mobile phase and the residue is dissolved. add in a column (1 x 25 cm) of gel

  silica (MN-kieselgel 60), the column is eluted with a

  lange Skelly B: ethyl acetate (8: 1) and fractions of 14 ml are collected Fractions 6 and 7 containing

  the desired product, and concentrate them to dryness to obtain

  mg of crystalline residue by recrystallization in a -

  mixture of ethyl acetate and hexane gives 107 mg of 4-chloro-3 (R) -hydroxybutyric acid, octyl ester, (a) 23 + 13.30

  (c 4.45) (CHCl 3); RMP (b CDC 13) 0.88 (3H, distortion of tr.

  CH 3 (CH 2) n-; 1.28 n OH, s, -c (CH 2) 5-; 1.65 (2H, m, -CH 2-

  CH 2 -CH 20); 2.62 (2H, d, J = 6Hz, -H-CH 2 -COOR); 3.22 (1H, broad, OH); 3.60 (2H, d, J = 6Hz, C 1 CH 2 H-R); 4.20

  (3H, -CH 2 -CH-CH 2 and -O-CH 2 CH 2).

  Analysis: Calculated for C 12 H 2303 Cl C% H%

57.47 9.25

  Found: 57.52 9.07 lCCM Rf 0.5, Brinkmann silica gel plate 0.25 cm EM;

  Skelly B: ethyl acetate (5: 1) 7.

  E> e 1 e 2 Remaining cells 100 g of fresh commercial bakery yeast Saccharomyces cerevisiae (Re (Star) are suspended in 250 ml of tap water with 10 g of

  saccharose and 3.6 g of octyl ester of Y-chlorace

  After incubating the contents at 25 ° C. on a rotary shaker (250 cycles / minute radius of 5 cm) for 24 hours, an additional 10 g of sucrose is added to the flask and the reaction is allowed to proceed for another 24 hours. The cells are then removed by filtration on a "Celite" pad. The cells are washed with water and with ethyl acetate.

  filtrate and extract them thoroughly with ethyl acetate.

  Derate the acetate phase over MgSO 4 and evaporate to obtain an oily residue which is chromatographed on a column of silica gel to obtain 2.52 g of octyl ester of 4-chloro-3 acid (R. ) -hydroxybutyric acid, in the form of a solid of

  low melting point; 23 + 13.20 (c, 4.0, CHCl 3).

Example '3

  The benzyl ester of the (+) 4- acid is prepared

  chloro-3 (R) hydroxybutyric acid as follows: Ci CR 2 C 2

OCH 20

  Fermentation A culture obtained on the agar surface grown on agar one week old of Gliocladium virens ATCC 13362, grown on agar having the following composition: Malt extract 20 Glucose 20 Peptone 1 Agar 20 Distilled water, qs 1 liter (sterilized 15 minutes at 140 k Pa)

  Denesion scion is in 5 ml of u-ne 0.85% saline solution.

  1 ml portions of this suspension are used to inoculate a 250 ml Erlenmeyer flask (stage F-1) containing ml of the following medium (dextrose and soybean medium): Soy flour 5 g Dextrose 20 g NaCl 5 g KH 2 HPO 4 5 g 2 4 Yeast 5 g Water 1 1 p H adjusted to 7.0 Autoclave under pressure of 105 k Pa

for 15 minutes.

  The flask is incubated at 25 ° C on a rotary shaker (250 cycles / minute 5 cm radius) for 24 hours after which 10% volume transfer is carried out in another 250 ml Erlenmeyer flask (stage F-2). ) containing ml of dextrose and soybean medium After 24 hours

  incubation on a rotary shaker, 150 mg of

  Acid benzyl-chloroacetoacetic acid in 0.1 ml of 10% Tween 80 The F-2 flask is then incubated for a further 24 hours under the conditions used.

  in the incubation of F-1 balloons.

  B Isolation Twenty-four hours after the addition of the ester

  Y-chloroacetoacetic acid benzyl, the fungus is separated

  Lium by filtration The filtrate is extracted three times

  with 50 ml of ethyl acetate The acetyl phase is dehydrated

  ethyl acetate over MgSO 4 and concentrated under vacuum to obtain

  a residue (160 mg) is chromatographed the residue on a

  1 liter (1 x 25 cm) of silica gel (MN-Kieselgel 60) The column was eluted with Skelly B and ethyl acetate (10: 1) and 12 ml fractions were collected. -16 containing the desired product and concentrated to dryness for

  obtain 115 mg of benzyl ester of 4-chloro-3 (R) -hydro-

  235 xybutyric, + 8.7 (, 5.26, CHC 3); RMP (CDC 13) 2.65 xybutyric, Z "TD + 8.70 (c, 5.26, CHCl 3) RMP (CDCl 3) 2.65 (2H, d, J = 6Hz, H-CH 2) Coor), 3.20 (1H, broad, -OH); 3.54 (2H, d, J = 6Hz, C 1 -CH 2 H) 4, 20 (1H, m, -CH 2 CH); 2-) 5,12 (2H, s, -O-CH 2 C 6 H 5); 7.31 (5H, s, five aromatic protons Analysis: Calculated for C 1 H 1303 Cl C% H%

57.77 5.73

  Found: 57.64 5.67 / TLC on EM Brinkmann silica gel plate, 0.25 cm, Rf

  = 0.43, Skelly B-ethyl acetate {5: 1) 7.

  Examples 4 to 23 The procedure of Example 1 is repeated with each of the microorganisms listed in Table 1 at

  the difference is that the octyl ester of

  Acetoacetic acid at a concentration of 1 mg / ml gives the conversion to the desired product, the octyl ester of (+) 4-chloro-3 (R) hydroxybutyric acid. The procedures of these Examples are repeated by adding continuously substrate to yeast media The substrate / yeast weight ratio is about 1: 1.5 while giving an excellent:

  transformation into the desired product.

Examples 24-48

  The procedure of Example 3 is repeated with each of the microorganisms listed in Table 2 at the same time.

  The octyl ester of Y-chloro acetoacetic acid (1 mg / ml) is used. The conversion to the compound is obtained.

  octyl ester of (+) 4-chloro-3 (R) -hydro-

xybutyrique.

Examples 49-68

  The procedure of Example 1 is repeated with each of the microorganisms listed in Table 1 except that the benzyl ester of r-chloroacetoacetic acid is used as the substrate.

  into the desired product, the benzyl ester of (+) 4- acid

chloro-3 (R) -hydroxybutyric acid.

Examples 69-93

  The procedure of Example 3 is repeated with each of the microorganisms listed in Table 2 in use.

  reading as substrate the benzyl ester of Y-chloro

  Acetoacetic acid (1 mg / ml) is converted to the desired compound, the benzyl ester of (+) 4-chloro-3

(R) -hydroxybutyric

Example 94

  The (+) 4-chloro-3 (R) -acid acid anilide is prepared

  hydroxybutyric acid according to the procedure of Example 2, except that 4-chloroacetoacetate is used.

  nilide at a concentration of 1 mg / ml.

Oh this is C_

NO

  as a substrate for transformation into the optical product

  ndan 10-110 ° C; Active desired element melting at 110-111 C; + 17.50 (c, 3 O, CHCl 3); RMP (SCD 3 CD 3) 2.67 (2H, d, J = 6Hz, -HOHCH 2-CONHR),

  3.66 (2H, d, J = 6Hz, CH 2 CHOH-R), 4.43 (1H, m, -CH 2 -CHOH-

  CH 2 O), 7.03 7.44 (3H, m, aromatic protons, meta and para), 7.69 (2H, d, J = 6HZ, aromatic protons, ortho), 9.24 (1H), H, broad, N-0) Analysis: C% H% Calculated for CH 12 NO 2 C 156.21 5.66 Found: 56.17 5.47 Examples 95-114 The procedure of Example 1 is repeated with each of the microorganisms listed in Table 1, with the difference that Y-chloroacetoacetanilide is added at a concentration of 1 mg / ml. In all cases, the conversion to the desired product, the anilide, is obtained. (+) 4-chloro-3 (R) -hydroxybutyric acid, Examples 115-139 The procedure of Example 3 is repeated with the microorganisms listed in Table 2.

  > -chloroacetoacetanilide at a concentration of 1 mg / ml.

  In this case, a transformation into the anilide of a-

  desired (+) 4-chloro-3 (R) -hydroxybutyric acid.

  Examples 140-159 The procedure of Example 1 is repeated with

  the microorganisms listed in Table 1, with the difference

  the octyl ester of the

  e-bromoacetoacetic acid (1 mg / ml). The transfor-

  to the desired product, the octyl ester of the acid

(+) 4-bromo-3 (R) -hydroxybutyric acid.

  Examples 160-184 The procedure of Example 3 is repeated with the microorganisms listed in Table 2 except

  octyl ester of <-bromoacetic acid is used

  (1 mg / ml) The transformation into the product is obtained

  desired, octyl ester of (+) 4-bromo-3 (R) -hydroxy-

  butyric. Examples 185-204 The procedure of Example 1 is repeated with the microorganisms indicated in Table 1, except that the benzyl ester of Y-bromoacetoacetic acid (1 mg / ml) is used as the substrate. gets the transformation

  into the desired product, the benzyl ester of (+) 4- acid

bromo-3 (R) -hydroxybutyric acid.

  Examples 205-229 The procedure of Example 3 is repeated with the microorganisms listed in Table 2 except

  benzyl ester of Y-bromoacetoacetic acid is used

  tick (1 mg / ml) The transformation into the product is obtained

  benzyl ester of (+) 4-bromo-3 (R) -hydro-

  xybutyrique. The procedure of Example 1 is repeated with the microorganisms listed in Table 1 except that Y-bromoacetanilide (1 mg / ml) is used as the substrate. transformation into the anilide of

  the desired (+) 4-bromo-3 (R) -hydroxybutyric acid.

  EXAMPLES 250-274 The procedure of Example 3 is repeated with the microorganisms listed in Table 2 except that X-bromoacetoanilide (1 mg / ml) is used as the substrate.

  the desired (+) 4-bromo-3 (R) -hydroxybutyric acid.

Examples 275-294

  The procedure of Example 1 is repeated with

  the microorganisms listed in Table 1, except that the octyl ester of hydroxyacetoacetic acid (1 mg / ml) is used as the substrate. The conversion to the octyl ester of 4-hydroxy-3-acid (R. Example 295319 The procedure of Example 3 is repeated with the microorganisms listed in Table 2 except that the octyl ester of γ-hydroxyacetoacetic acid (1 mg / ml) is used as the substrate. The conversion to the desired octyl ester of 4-hydroxy-3 (R) hydroxybutyric acid is obtained. EXAMPLES 320-339 The procedure of Example 1 is repeated with the microorganisms listed in Table 1, except that Y-hydroxyacetoacetanilide (1 mg / ml) is used as the substrate. acid anilide

  4-hydroxy-3 (R) -hydroxybutyric desired.

  Examples 340-364 The procedure of Example 3 is repeated with

  the microorganisms listed in Table 2 in the

  use of Y-hydroxyacetoacetic acid as a substrate

  tanilide (1 mg / ml) is converted into anilide

  desired 4-hydroxy-3 (R) -hydroxybutyric acid.

Example 365

  Methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) c

VII VIII

V I

  1 mg of 4-chloroacetoacetate is incubated:

  of methyl (VII-) with 29 units of porcine heart (EC -r 1 1 -35), hydroxyacyl-Co A-dehydrogenase (Sigma, H 4626) and 1.36 g of NADH (Sigma, 90%) in 30 ml of 0.1 M sodium phosphate

buffer, pH 6.5.

  After 30 hours at 25 ° C., the mixture is extracted

  reaction mixture four times with 30 ml of ethyl acetate -

  The organic phase is dehydrated over sodium sulphate and evaporated to dryness under reduced pressure. The residue is chromatographed (90 mg) on a column (1.3 × 34 cm) of silica gel.

  (12 g) The column is eluted with a solvent system con-

  Stored in Skelly B-ethyl acetate (8: 1) and collected

  Fractions of 20 ml were pooled.

  hold the desired methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) as evidenced by TLC 73 + 23.50 (c 5.2;

CHC 13).

Example 366

  The procedure of Example 365 is repeated using ethyl 4-chloroacetoacetate as substrate to obtain ethyl 4-chloro-3 (R) hydroxybutyrate (3 + 22.70).

(c 4.7, CHC 13).

Example 367

  The procedure of Example 365 is repeated using n-propyl 4-chloroacetoacetate as substrate 53713 '0 to obtain n-propyl 4-chloro-3 (R) -hydroxybutyrate A 723 + 21.50 (c 5 CHC 13),

Example 368

  The procedure of Example 365 is repeated using n-butyl 4-chloroacetoacetate as substrate to obtain n-butyl 4-chloro-3 (R) -hydroxybutyrate.

23 + 20.10 (c 3; 1; CHCl 3).

  General process for converting 3-halogen-3 (R) -hydroxybutyrous esters and amides to L-carnitine: Example 369 Heated at 80-90 ° C for about 2 hours

  a mixture of octyl ester of 4-chloro-3 (R) -hydro-

  xybutyric (1.5 g), ethanol (3 ml) and trimethylamine

  (25% by weight solution) in water (3 ml). Evaporated.

  solvents and excess trimethylamine to dryness to give 1.8 g of crude residue. The crude product (1 g) was heated at 80-90 ° C in a 10% solution of HCI (7 ml).

  during 1.5 hours After evaporation of the solvents under

  reduced ratio; we extract: the product, two times with de; absolute ethanol (10 ml) and the ethanol was evaporated in vacuo The crystalline residue was dissolved in a small amount of ethanol and the L-carnitine chloride was precipitated by the addition of ether in good yield (320.degree. mg), point of

  merger 142 & C (dec); 23.7 "(c 4.5, H 20).

  The L-carnitine chloride can be readily converted to the pharmaceutically preferred inner salt of L-carnitine by ion exchange.

as we know in practice.

Example 370

  4-iodom, 3 (R) octyl hydroxybutyrate (IX)

CE -

  A mixture of octyl (II) 4chloro-3 (R) -hydroxybutyrate (1.426 g) was refluxed for 24 hours.

  anhydrous sodium iodide (1.2 g) in 15 ml of methyl ethyl

  Ketone The mixture is evaporated on a rotary evaporator and reacted with ether (100 ml) and water (50 ml).

  The organic phase is separated and washed with a solution -

  10% sodium thiosulfate (150 ml), brine (150 ml) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give 1.762 g.

  compound IX as a pale yellow oil; IR (pel-

  thin licilum) 3460 cm (OH) and 1730 cm (ester C = O); PMR (CDCl3) 3.93-4.27 (m, 3H), 3.17 (d, 2H), 2.50 (d, 2H),

  1.50-1.87 (m, 2H), 1.30 (s, 12H), 0.93 (m, 3H).

  Transformation of octyl 4-iodo-3 (R) -hydroxybutyrate (IX)

in L-carnitine.

o H 3 o

OS O N-O 3

r- I \> 2 \ O C 8 r 17C Eo X 3

C 3 '3

XX: X

CE 3 OH

c 3 f

L-Carnittne -

  To a solution of compound IX (1.593 g) in methanol (15 ml) is added a 25% solution of trimethylamine 3 O

  Aqueous (8 ml) The mixture is stirred at 27 ° C. for 20 hours.

  The solvents and excess trimethylamine are evaporated under reduced pressure to give a semi-crystalline solid, compound X. This residue is washed with small amounts of ether I to remove the octanol, and then dissolved in the water and the DD

  is passed on a column of Dowex l-x 4 b OH OH 0,1449-

  0.297 mm, (2.5 x 15 cm) 7, The column is washed with distilled water. By removing the solvent in vacuo from the first 200 ml of the eluate, L-carnitine is obtained in the form of a white crystalline solid (490 mg, yield

%) 23-29.2 (c 6.5, H 20).

Example 371

  The procedure of Example 370 is repeated using hexyl 4-chloro-3 (R) -hydroxybutyrate to give hexyl 4-iodo-3 (R) -hydroxybutyrate which is

then transforms into L-carnitine.

Example 372

  The procedure of Example 370 is repeated using heptyl 4-chloro-3 (R) -hydroxybutyrate to obtain heptyl 4-iodo-3 (R) hydroxybutyrate which is

then transforms into L-carnitine.

Example 373

  The procedure of Example 370 is repeated using decyl 4-chloro-3 (R) -hydroxybutyrate to give decyl 4-iodo-3 (R) hydroxybutyrate which is

then transforms into L-carnitine.

Example 374

  The procedure of Example 370 is repeated using methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) to obtain methyl 4-iodo-3 (R) hydroxybutyrate.

  which is then transformed into L-carnitine.

Example 375

  The procedure of Example 370 is repeated using ethyl 4-chloro-3 (R) -hydroxybutyrate to obtain ethyl 4-iodo-3 (R) -hydroxybutyrate which is

then transforms into L-carnitine.

Example 376

  The procedure of Example 370 is repeated using n-propyl 4-chloro-3 (R) -hydroxybutyrate to obtain n-propyl 4-iodo-3 (R) hydroxybutyrate.

Z 537130

  it is then transformed into L-carnitine.

Example 377

  The procedure of Example 370 is repeated using 4-chloro-3 (R) -hydroxybutyrated n-butyl to give 4-iodo-3 (R) -N-butyl hydroxybutyrate which is

then transforms into L-carnitine.

  Examples of yeasts that produce the enzyme

  desired are listed in Table i and examples of

  fungi are listed in Table 2.

  T-ableau i (Yeasts) 1. 2. 4. 5. 6. 7. a. '9 10. he. 13.

14.

  15. 16. 17. 18. 19. 20. NRRL ATCC Candrda 6-imo 1 vti-ca NRJRL Y-1095, candida vseudotropiaiiz NRRL Y-1264 Mycoderma cerevisiae NRRL Y-16 '15 Torula lactosa NPRRL Y-329 Geotricehum candidunm NRRL Y o 552 Hanseriula anomala NRRL Y-366 Hansenula sub-oelliculosa NRRL Y-1683 Pichia alcoholonhila NRIRL Y-2026 Saccharomyces cerevisiae NRRL -Y-12, 632 Saccharomnvees lactis NP-PL Y-1140

  Zygosaccharomyces priorianus NRRL Y-12,524 12.

  Saceh Acidifacial acids NP RL Y-7253 Kloeckera corticis ATCC 20109 Crv-totcoctis mascerans ATC-C 24194 Rhodotorula N / A ATCC 20254 Candida aibicans ATCC 752 Dimodascus albidus ATCC 12934 Erevisiaa Saccharomycese (Red Star Trade) Rhodotorula rubra NRRL Y-1592 Oosmora lacti S ATCC 14318 &

  Northeru Regional Research Laboratory in Peoria, Illinois.

  Axnerican Type Cultu-re Collection at Rockville, Maryland Tal: 'e ~ = u 2 (Chanloiçnons) 1. 2. 3. 4.

5.

6. 7. a. 9.

10.

he. 12. i 3. 14.

15.

16. 17. the. 19.

20.

21. 22. 23. 24.

25.

  ATCC 13362 Caldaricmyces flimal J 0, ATCC 16373 Linderizia pennisomora ATCIC 12442 As-pergillus'ochraceus NRRL 405 Trichodon na ligno, ATCC 8678 Heterocer, halum autantiacum AZCC 16328 Entomophthora coronata NRRL 1912 Scorulaosis const-antini In ZRL 1860 Zylâorhnchus heterogamus ATCC 6743

  Sco-oular-here Dsis brevica-ulis NRRL 2137-

  Rhiz -Pus arrhizus -NILU 2286 Penicillium thomii NRRL 2077 Mucor hiernalis C- NRRL 4088 Byssochiamys n-4 vea ATCC 12550

NRRL 1952

  Metarrhizium aniso-cliae ATCC 24942 Penicillium islandicum ATCC 10127 Cunnin Ehamella elegans ATCC 10028 a Cunnin 2 hamella echinulata ATCC 11585 a Aspergillus fumigatus ATCC 16907 Aspergillus amstelodami NRRL 90 Pink gliocladium = ATCC 10521 Asperl Zillus giganteus ATCC 10059 Absidia blakeleeana ATC 41 10148b Penicillium rocueforti NRIRL 849 a

253713 O

Claims (22)

  Compounds characterized in that they correspond to the formula and have the configuration 3 (R): ## STR2 ## wherein X is selected from C 1, Br, I and OH and R is a straight chain configuration radical; branched or cyclic chain selected from: alkoxy radicals having 1 to about 15 carbon atoms   the alkylamino radicals having from about 5 to   about 15 carbon atoms; cycloalkoxy and cycloalkylamino radicals having from about 5 to about 12 carbon atoms; phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms; phenylamino and phenylalkylamino radicals having the formulas: Y Y Z 1, 121   N-0-A; and -N-CH-O-A o Y and Z are selected from H, an alkyl group of 1 to about 8 carbon atoms,   phenyl or benzyl and A is selected from H, CH 3, Cl and Br.   Compound according to claim 1, characterized in that R is a straight chain alkoxy radical having 1 to 10 carbon atoms.   Compound according to claim 2, characterized   in that R represents OC 1 o H 21.   Compound according to claim 2, characterized in that R is OC 8 H 17 8 17 '   Compound according to claim 2, characterized in that R represents OC 7 H 15. 7 15 253713-0   Compound according to claim 2, characterized in that R represents OC 6 H 13.   Compound according to claim 2, characterized in that R is a lower alkoxy radical having 1 to 4 carbon atoms. Compound according to claim 1, characterized   in that R is selected from phenoxy radicals and   nylalkoxy substituted with a lower alkyl group, geno or nitro.   Compound according to claim 8, characterized in that R is a benzyl group Xyloxy and X is selected from -R C 1 and Br.   Compound according to claim 1, characterized   rised in that R is a phenylamino group and X is chosen between Cl and Br.   Process for the preparation of optically active Y-substituted p-hydroxybutyric acid derivatives having the formula: OH H x X ^^, R   wherein X is selected from Cl, Br, I and OH and R is a straight-chain, branched-chain or cyclic-chain radical selected from:   alkoxy radicals having from 1 to about 15 atoms;   carbon mon of alkylamino radicals having about 5 to 15 carbon atoms; cycloalkoxy and cycloalkylamino radicals having from about 5 to about 12 carbon atoms; phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms   phenylamino and phenylalkylamino radicals having the mules: Y YZ = II I   N-0; and -N-CI 1 -NL-A, and -N-CH-O-A 0 Y and Z are chosen   among H, an alkyl group of 1 to about 8 carbon atoms.   bone, phenyl or benzyl and A is selected from H, CH 3, Cl   and Br from acetoacetic acid esters or amides.   substituted in i corresponding, characterized in that it comprises: to submit said esters or acid amides   V-substituted acetoacetic to the enzymatic action of   tion of a microorganism that produces L-hydroxy-   acyl-Co A-dehydrogenase-lEC 1 to 1 1 35 l, and to recover the   so-called substituted Y-substituted hydroxy-butyric acid derivatives actively active.   Process according to claim 11, for   to the preparation of 31 R) -hydroxybutyric acid derivatives -   Yoptically-substituted substituent having the formula and the configuration 3 (R) wherein X and R have the meanings given in claim 11, provided that if R is an alkoxy radical of from 5 to about 15 carbon atoms, characterized in that it consists in subjecting the compounds of formula XCH 2-C-CH 2 CR   in which X and R have the meanings given more   top to the enzymatic action of fermentation of a microorgano-   ganism that produces L-hydroxyacyl-Co A-dehydrogenase   lEC 1 1 1 35 l, and to recover the derivatives of acid 3- (R) -   hydroxybutyric substituted in 4 optically active de-   taken from the fermentation reaction mixture.   The method of claim 12, characterized   in that the microorganism is selected from the class: Ascomycetes.   Process according to claim 12, characterized   in that the microorganism is selected from the orders of Endomycetales, Mucorales, Monoliales or Eurotiales.   Process according to claim 12, characterized   in that the microorganism is selected from Saccharomyces genus.   The method of claim 15, characterized   in that the microorganism is Sacchar-omyces cerevia siae.   The method of claim 12, characterized   in that the Y-substituted acetoacetic derivative   enzymatic action of fermentation-is 1-oc-ethyl ester of g-chloroacetoacetic acid   The method of claim 12, characterized   in that the substituted acetoacetic acid derivative   in Y subjected to the enzymatic action of fermentation is   the benzyl ester of chloroacetoacetic acid -   The method of claim 12, wherein   in that the acetoacetic acid derivative substituted   killed in Y subjected to enzymatic fermentation action is chloroacetoacetanilide.   Process according to claim 17, characterized   in that the microorganism is Saccharomyces cerevis siae -   The method of claim 18, characterized   in that the microorganism is Saccharomyces cerevis siae.   The method of claim 19, wherein   in that the microorganism is Saccharomyces   revisiae. Method according to Claim 1 for the   preparation of 3 (R) -hydroxybutyric acid derivatives   optically active compound having the formula and configuration 3 (R) X a Hns O H T O x 1 2   in which X has the meanings given to the   11 and R is an alkoxy radical having 1 to 4 carbon atoms, characterized in that it consists in subjecting compounds of formula: 0 O XCH 2-C-CH 2 C-R   in which X and R have the meaning given above   the enzymatic action of L-hydroxyacyl-Co A-dehydro-   genase lEC 1 1 1 35 l in purified form, and to recover them   3 (R) -hydroxybutyric acid derivatives substituted in opti-   desired active ingredients from the enzyme reaction mixture matic.   The method of claim 23, characterized   L-, B-hydroxyacyl-Co A-dehydrogenase lEC 1 1.   1.35 l in purified form is that isolated from heart of pork.   Process for the preparation of the internal salt of L-carnitine, characterized in that it involves reacting   a substituted 3 (R) -hydroxybutyric acid derivative of 4   According to claim 1, successively with trimethyl   amine and hydrochloric acid, to extract the L-carnitine chloride, and to subject said chloride to an exchange   of ions and to recover the internal salt of L-carnitine.   Process for the preparation of a 4-iodo or 4-bromo-3 (R) hydroxybutyric acid derivative, characterized in that   it consists in reacting a 4-chloroacidic acid derivative   3 (R) -hydroxybutyric with sodium iodide or sodium bromide in a solvent at a temperature of 50 C to 100 C   to form the 4-iodo or 4-bromo- (R) -hydro- corresponding xybutyric.   Process for the preparation of the internal salt of L-carnitine, characterized in that it consists of: a) reacting an alkyl ester of 4-iodo or 4-bromo-3 (R) -hydroxybutyric acid containing 10 ataess carbon with trimethylamine in methanol or ethanol to form a methyl or ethyl ester of L-carnitine, and b) converting said L-carnitine ester to salt internal L-carnitine.