IE832732L - Butyric acid derivatives convertible to l-carnitine - Google Patents

Abstract

A process for preparing L- carnitine which comprises exposing gamma - substituted acetoacetic acid esters or amides to the fermentative enzymatic action of a microorganism which elaborates L- beta -hydroxyacyl CoA dehydrogenase [EC1.1.1.35], recovering the resulting, optically active, corresponding gamma -substituted- beta - hydroxybutyric acid derivative and converting said derivative to L- carnitine. An improvement in the process is also disclosed which comprises reacting a 4-chloro-3(R)- hydroxybutyrate with sodium iodide or bromide to produce the corresponding 4-iodo- or 4-bromo-3(R)- hydroxybutyrate, converting the 4- iodo or 4-bromo-3(R)-hydroxybutyrate to the trimethylamino-3(R)- hydroxybutyrate salt, then converting the trimethylamino-3(R)- hydroxybutyrate salt into L-carnitine inner salt. Novel chemical intermediates prepared in the processes are also disclosed; namely the 3(R) forms of <IMAGE> where R represents specified esterifying or amidifying groups and X is Cl, Br, I or OH. [GB2132614A]

Description

The present invention relates to processes for producing L-carnitine. Specifically, it relates to a process for microbiologically reducing y-substituted-acetoacetic esters or amides into their respective L- fi -hydroxy- f -substituted-butyric acid derivatives, which derivatives can be readily converted into L-carnitine chloride. It also relates to novel chemical intermediates employed in the process.

As is well known, carnitine ( -hydroxy- y -trimethyl-amino 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 carry 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 esterifi- 3 cation with L-carnltlne has taken place. The carrier function of L-carnltlne is exerted both by transporting active long-chain fatty acids from the sites of their bio-synthesis, for the example the microsomes, to the 5 mitochondria where they are oxidized, and by transporting acetyl CoA from the mitochondria, wherein it. is formed, to the extramltochondrial sites whore the synthesis of long-chain fatty acids occurs, e.g., in the microsomes wherein acetyl CoA can be utilized for synthesizing 10 cholesterol and fatty acids.

While it has been established that the laevorotatory isomer (L-carnltlne) exclusively is the biologic form (D-carnitlne has never been detected so far in mammalian tissues), the D,L-carnltlne racemate has been used for a 15 number of years for different indications. For example, D,L-carnitine is sold in Europe as an appetite stimulant, and it has been reported that the material has an effect on the growth rate of children; see e.g., Borniche et al., Cllnica Chemlca Acta, 5, 171-176, 1960 and Alexander 20 et al., "Protldes in the Biological Fluids", 6th Colloquim, Bruges, 1959, 306-310. U.S. Patent No. 3,830,931 describes improvements in myocardial contractility and systolic rhythm in congestive heart failure which can often be obtained through administration of D.L-carnl-25 tine. U.S. Patent No. 3,968,241 describes the use of D,L-carnitine in cardiac arrhythmias. U.S. Patent No. 3,810,994 discloses the use of D,L-carnltlne in the treatment of obesity.

Recently, however, there has been an increasing 30 emphasis on the importance of utilizing exclusively the carnitine laevorotatory isomer for at least some therapeutic applications. It has, in fact, been shown that O-carnitine is a competitive inhibitor of carnitine-1inked enzymes such as carnitine acetyl transferase (CAT) 35 and carnitine palmityl transferase (PTC). Moreover, •* recent evidence suggests that D-carnitine can deplete L-carnitine from the heart tissue. Consequently, it is essential that L-carnitine exclusively be administered to patients under medical treatment for heart diseases or lowering of blood lipids.

Several processes have been proposed for producing carnitine on an industrial scale. The chemical synthesis of carnitine unavoidably leads, however, to a racemic mixture of the D and L isomers. Consequently, resolution methods have to be employed to obtain the separate optical antipodes from the racemate. These resolution methods are, however, cumbersome and expensive.

It is an object of this invention to produce L-carnitine in good yield through a combination of microbiological and chemical processes.

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

Another object of the present invention is to disclose the preparation of novel, useful optically-active intermediates for the synthesis of L-carnitine and its salts or esters.

Another object of the present invention is to provide processes for preparing L-carnitine via the trimethylamine displacement of the halo group of a 4-halo-3(R)-hydroxybutyrate.

Still another object of the present invention is to provide a proces.s for producing 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 proceeds.

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

That the j9-keto function in the 3-position in the y -substituted-acetoacetic acid derivatives can be reduced by hydrogenation over Pt/C is known (e.g., U.S. Patent No. 3,969,406). However, the hydroxy compound resulting from such method is racemic. In contrast, by employing the fermentative action of a microorganism in accordance with the process of the present invention, the hydrogenation of the oxo-function at the 3-position can be accomplished stereoselectively to yield optically active f--substituted -hydroxybutyric acid derivatives.

In particular, upon suitable selection (as hereinbelow described) of the substrate to be exposed to the fermentative action of the microorganisms in accordance with the process of this invention, the 3(R) or L epimeric configuration is obtained. This configuration is required for the conversion into the natural L-carnitine.

Broadly, this invention comprises the use of the microbial reductase enzyme, L- j9 -hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] , to catalyze stereoselective hydrogenation of y-substituted acetoacetic acid derivatives as hereinbelow defined.

Therefore, in accordance with its broadest aspect, the process of the present invention for preparing optically active /-substituted j3-hydroxybutyric acid derivatives having the formula OH 0 R 6 is selected from CI, Br, I and OH and is a radical in straight chain, branched chain or cyclic configuration selected from alkoxy radicals having from 1 to 15 carbon atoms; alkylamino radicals having from 5 to 15 carbon atoms; cycloalkoxy radicals and cycloalkylamino radicals hating from 5 to 12 carbon atoms; phenoxy and phenylalkoxy radicals having from 7 to 14 carbon atoms; phenylamino and phenylalkylamino radicals having the formulas Y Y Z -N-0-A; and -I H-0-A where Y and Z are selected from H, an alkyl group having from 1 to 8 carbon atoms, phenyl or benzyl and A is selected from H, CH^, CI and Br from corresponding y-substituted acetoacetic acid esters or amides, comprises subjecting said J»-substituted acetoacetic acid esters or amides to the fermentative enzymatic action of a microorganism which elaborates L- ff -hydroxyacyl CoA dehydrogenase [EC 1.1.1. and recovering the desired optically active f-substituted- 0-hydroxybutyric acid derivatives 7 In particular, in order to prepare optically active y-substituted 3(RMiydroxybutyric acid derivatives having the formula and 3(R) configuration the process comprises subjecting compounds having the formula X XCH2-C-CH2CR wherein X and R have the above-identified meanings provided that if R is em alkoxy radical it has from 5 to 15 carbon atoms, to the fermentative enzymatic action of a microorganism which elaborates L- j? -hydroxyacyl CoA dehydrogenase (EC 1.1.1.35] , and recovering the desired optically active 4-substituted 3(R)--hydroxybutyric acid derivatives from the fermentative 15 reaction mixture.

It has been found that any microorganism which produces the desired enzyme is capable of functioning to catalyze the stereoselective reduction. Particularly suitable are those microorganisms of the class Ascomycetes, the orders 20 Endomycetales, Hucorales, Moniliales and Eurotiales, and the genus Saccharomyces. Particularly preferred is Saccha-romyces cerevisiae. 8 To prepare optically-active 4-substituted 3(R)--hydroxybutyrate esters containing 1-4 carbon atoms, it is necessary to use purified L- J3 -hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] such as that of porcine heart, because intact microorganism possess interfering oxido-reductases of opposing configuration. Hence, microbial reduction of e.g. 4-chloro-acetoacetic esters of 1-4 carbons produce 4-chloro-3-hydroxy-butyrates of unsatisfactory optical purities.

Therefore, the present invention also provides a process for preparing optically active /-substituted 3(R) hydroxybutyric acid derivatives having the formula and 3(R) configuration wherein X is CI, Br, I or OH and R is an alkoxy radical having from 1 to 4 carbon atoms which comprises subjecting compounds having the formula I i XCH2-C-CH2C-R wherein X and R have the above-identified meaning to the enzymatic action of L- -hydroxyacyl CoA dehydrogenase {EC 1.1.1.35] in purified form, and recovering the desired optically active f -substituted 3(R)-hydroxybutyric acid derivatives from the enzymatic reaction mixture. 9 The present invention thus provides compounds having the formula and 3( R) configuration wherein X is selected from CI, Br, I and OH and R is a radical in straight chain, branched chain or cyclic configuration selected from alkoxy radicals having from 1 to 15 carbon atoms; alkylamino radicals having from 5 to 15 carbon atoms; cycloalkoxy radicals and cycloalkylamino radicals having from 5 to 12 carbon atoms phenoxy and phenylalkoxy radicals having f,rom 7 to 14 carbon atoms phenylamino and phe'nylalkylamino radicals having the formulae Y Y Z -N-0-A; and -N-CH-0-A where Y and Z are selected from H, an alkyl group having from 1 to 8 carbon atxms, phenyl or benzyl and A is selected from H, CH^, CI and Br.

More specifically. R may for Instance be a straight-chain alkoxy radical having from 1 to 10 carbon atoms, e.g. 0Ci0H2i« 0C8H17' 0C7H15' 0C6H13' or a lower alkoxy radical having from 1 to 4 carbon atoms.

Also, in the case of the fourth of the above-listed sub-classes of values of R. namely that comprising phenoxy and phenylalkoxy radicals having from 7 to 14 carbon atoms, it is possible for these radicals to be " modified in that, instead of being unsubstituted, they 10 may be substituted with lower alkyl, halo or nitro group. 11 The optically-active y-substituted-L-j}-hydroxybutyric acid derivatives may then be reacted with trimethylamine to yield the corresponding y-trimethylammonium-L- ^-hydroxybutyric acid derivative, which can be readily converted Into Ir-carnitlne by hydrolysis. The following is a schematic of the reaction steps of this process.

■JU °v Micro- jt jf H o«wni«n y or L-IJ-hydroxyacyl' X CoA dehydrogenase XI X-d, Br, I, OH *3 SB H -co-o CH-g "{ IV I.-Carnitine IXX It has been found that the foregoing reaction I —f II 10 takes place more easily if X = CI. However, since the subsequent reaction II —-*> III occurs with better yields when X = iodine or bromine, it is preferred to prepare first the Cl-derivative and then convert it into the corresponding I- or Br-derivative.

IS The present invention also relates to an improved process which comprises first converting 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 hereinbelow made to the I-derivative. The iodohydrin (V) may be reacted smoothly with trimethylamine at room temperature to yield VI which is readily converted to L-carnitine according to the following reaction sequence: n R "H or «st«r & 2* a 0 L-carnitine The foregoing process as exemplified by the equation is subject to numerous variations. Regardless of which form is then made available, the ester is reacted with sodium iodide in a suitable solvent such as, far example, 2-butancne, acetone and butanol. The principal reaction.desired at lr »„ M 0 feOH resin OH" form f3 fC«3 «3 «3 OH H o ICH- 3 VI 13 this point ia the reaction with aodiua iodide ia a dls-placaaent reaction which foras the iodohydrin V without disturbing the chiral center on the adjacent carbon atcau For this reaction at least enough sodlua iodide ia required to displace all chloride from IX. Generally speaking, a alight excesa of aodiua iodide la used.

She reaction of V with trlaethylaalna can be carried out at talld temperature (e.g., 2S°C) (Sea S. O. Boots and H. R. Boots, J. Fhara. Scl.. 64. 1262, 197S), la a variety of solvents such as Methanol or ethaaol containing an excess of trlaathylaalae. It Is noteworthy that depending oa the alcoholic solvaat used, thara is ester exchange taking place. For exaaple, when aethaaol is used aa solvent, L-carnitiae aethyl ester is obtained ia the reaction. Thla exchange reaction is advantageous because it la knowa that L-caraitiaa aethyl aster caa be trans-formed directly to the free base foxa of L-carnitine by passing through aa ion-exchange coluaa (OH-) [see B.

Strack aad J. boreas, J. Physiol. Chen. (1966) 3*4. 276].

Xt caa be aeea froa the daacrlptloa of tha foregoing processes that a nuaber of aaw sad highly useful optically active latetaedlates are fosaad. Especially uaaful are the 4-iodo-3(R)-hydroxybutyric acid alkyl esters where tha alkyl groupa have froa six to tea carbon atoaa each. She octyl ester Is particularly preferred.

Hlcroorgaalsas which have the desired oxido-reductase activity are well kaowa la the Microbiological art aad aay of such nlcroorgaalsas eaa be eoployed ia coaductlag tha process of the preseat laveatioa (See, X. Xieslich, "Microbial Transformations of Noa-Sterold Cyclic Coa-pouada" (Ceorg Thieae Publishers, Stuttgart, 1976)) with any of the genera of alcroorganiaas specifically described herein being particularly applicable. Readily available and inexpensive nicroorganisas of the genus Saccharomycas, 9.9., brewer'a yeaat, baker's yeast aad winamakar'a yeast (Saceharomyees vial) bava baoa feuad to produca tha L-0-hydroxy1acy1 CoA dehydrogenase [EL 1.1.1.35] aad to ba eminently advantageous la carrying oat tha proeaas of tha invention. tha enzyme la described by S.J. Hakil aad E.M. Barnes Jr. ia Comprehensive Biochemistry Vo. 185(1971) p 57-104.

Xhe 4-substltuted-aeetoaeetlc substrata eaa ba incorporated la a nutrient medium of staadard composition ia which such organ! sw are cultivated aad tha usual conditions of fermentation eaa than be employed to effect tha reductive transformation. Alternatively, tha active principle caa be removed from tha growing culture of tha microorganism, for instance by lysis of tha cells to release tha pnzymes, or by suspension of the resting calls ia a freah aqueous system. Za any of these techniques tha B-kato fuaction will be selectively reduced, so long aa the active enzyme elaborated by tha mlcroorgsnlsms is present la tha medium. Of course, the temperature, time aad preaaura conditions under which the contact of the 4-substltutad-acetoacetle derivative with the reductive enzyme ia carried out are laterdapeadeat as will be apparent"to those skilled la the art. For iastaace, with gentle heading aad at atmospheric pressure the time required to effect the reductive conversion will be less than If it progreaaea at room temperature uader conditions otherwise tha same. Of course, neither temperature, nor pressure, nor time, should be so great that it reaults in the substrata being degraded. Mhere a growing culture of the organism is being used, the process condi-tloaa should also be sufficiently gentle so the organiam is not killed before it elaboratea sufficient hydrolytie enzymes to permit the reaction to proeaed. Generally, at atmospheric pressure, the temperature can range from about 10"C to about 35*0, and tin tlae froa about 12 hours to about 10 days.

In the following tuuusplas which are presented to Illustrate this invention and are not to be construed as Uniting the scope of the appended claims, the v-halo acetoacetic acid derivative substrates to be subjected to nlcrobiologlcai reduction were prepared froa dlketone according to the general Method of C. D. Surd and B. X>. Aberaethy (J. Aa. Chen. Soc.. 62. 1147, 1940) for the T-chloro-acetoacMtlc derivatives and V. Chick, M. T. N. Mllsnore [J. Chen. Soc., 1979 (1910)] for the Y-broao-acetoacetic derivatives via the following re-action seq^encet Alternatively, if desired, the Y-halo acetoacetic acid derivatives can be prepared froa Y-halo acetic esters via a conventional Crlgnard reaction. For ex-Map la, Y-chloro acetoacetic octyl ester was readily prepared by refluxing y-chloro octyl ester with two equivalents of nagnesiua in ether for 48 hours. After removal of the solvent the acetoacetic octyl ester was recovered in about 70% yield. where X ■ CI or Br * » H or alkyl R ■ as defined previously 16 t-Hydroxy acetoacetic acid derivatives were prepared froa their corresponding! Y-bromoacetoacetic acid derivatives by stirring la a dloxana-watar (Is 1) solution containing CaCOj at 25 *C for 12 hours.

Baeh of tha products produced in aecordaaee with tha following examples was Identified as to structure through tha use of nuclear magnetic resonance {we), infrared spectra, aad by thla layer chromatographic mobilities, tha optical purity and tha absolute ceaflguratoa of the products were established by thalr conversion into L-carnltlne as weXX ss by conversion Into tbalr astars which are readily aaalysed by aar spectrometry, aad optical rotation.

Example 1 (Yeasts) (♦)4-C&loro-3(R)-hydroxybutyric acid octyl astar was prepared as follows: trn 0CiPn A. Farmeatatlon. Surface growth from a one weak old agar sXant of Candida torft. MRSZ. Y-329, grown oa agar of tha following composition: Agar 20 Glucose 10 Yeast extract 2.5 *2®°4 1 Distilled water, q.s. 1 liter (Sterilised 15 mln at 20 p.s.i.) it? was suspended la 5 al of aa 0.85% saline solution. 0m al portions of thla suspension wars usod to laoculate a 250 al Erleaaeyer flask (F-l stage) eoatalalaff 50 al of tha following aodiua (Vocal's aodiua): S5S Tmoc attnct S Casaaiao acids 5 Dextrose 40 Hij-citntfS 1/2 BjO 3 g n^P04 5 g W4M03 2 9 CaCl2.2820 0.1 g ugao^.TB^o 0.2 g Xraee element solution 0.1 al Distilled water, q.s. 1 liter pH 5.5 (sterilized for 15 aia at 30 p.s.i.) Trace element nolution Qa/100 al Citric acid-lHjO S ZaSO^.TBjO 7 fa(SH4)2(804)2.8H20 1 CuS04.5H20 0.25 MbS04.1^0 0.05 83803 0.05 MaB2Ho04.2B20 0.05 The flask was incubated at 25"C oa a rotary abakor (250 cycles/Ma - 2" radius) for 24 feours, aftor which a 10% by voluae transfer was aad* to aaothar 250 al Erleaaeyer flask (F-2 stage) containing 50 al 30 of Vogel's medium. After 24 hours of incubation oa a rotary shaker, 150 mg of y-chloroacetoaceti<; acid octyl ester in 0.1 al of 10% Tween*80 was added. *Trade Hark 18 Tha F-2 stage flask was than incubated for an additional 24 hours under the conditions used in the incubation of the F-l stage flasks.

Isolation. Twenty-four hours after the addition of the Y-ehloroacetoacetic acid octyl ester, the cells ware reaoved by catntri ligation. The supernatant was exhaustively extracted with SO ml of ethyl acetate three tlaas. The ethyl acetate was dried over ItajSO^ and evaporated to afford an oily residue (186 ag). The residue was dissolved la 0.5 al of the aobile phase and added onto a coluan (1 x 25 cat) of silica gel (Ntf-kleselgal 60). The coluan was eluted with Shelly Btothyl acetate (8:1) and 14 al fractions ware collected. Tractions 6 and 7 containing ' the desired product ware pooled and concentrated to dryness yielding 120 ag of crystalline residua. Recrystalllzatlon froa ethyl acetate-hexane afforded 107 ag of 4-chloro-3(R)-hydroxybutyric acid octyl ester, [a]23 *13.3" (c, *.45) (CBClg); par (ftCDCl-j) 0.88 [3B, tr. dlstortlonal, Cgg^CE^-]; 1.28 [10H, s, -(CHjJg-J; 1.65 (28, a, -C^-Cgj-CHjO-C-); 2.62 (2H. d, J a 6 Hz, -^H-g^-COOR); 3.22 (IB, br., -OH); 3.60 (2B, d, J ® 6 Hz, CICE,-CB-R); 4.20 (3B, -CHj-^-CHj and -g-O-CHjCHj). Anal. Calcd for C12H23°3C1: C* 57•47•' B' 9-2S- 7ou&d: C, 57.52; B, 9.07. [TLC Kg - 0.5, Brlnkaann silica gel plate, 0.25 cm EH; Skelly 8:athyl acetate (5:1).] 19 Examala 2 Ratting Calls. On* hundred grams of commercial fr*sh baker* a yaut Saccharoavcea ccrevisiae (Red Star) vaa suspended la 2S0 al of tap water ta which was addad 10 9 of sucroaa aad 3.6 9 eg y-chloroacetoacetic Octyl aatar. After tha coatasta war* incubated at 25°C oa a rotary shaker (2S0 cyclaa/feinuta - 2" radius) for 24 hours, aa additional 10 9 of sucroaa waa addad to the flaslt aad tha raactlea waa allowed to proceed for aaothar 24 hours. Tha calls ware thea removed by filtration through a pad of eellta. tha cells vara washed with water and athyl acetate. Tha washings war* combined with tha filtrate aad exhaustively extracted with ethyl acetate. Tha athyl acetata layer was dried over NgSO^ aad evaporatad to give aa oily residua, which was chromatographad over a silica gel column to yiald 2.S2 g of 4-chloro-3(R)-hydroxybutyric acid octyl ester, as a low malting solid; [a]23+13.2* (c, 4.0, CSC13).

Example 3 (*)4-Chloro-3(R)hydroxybutyric acid benzyl astar was prepared as follows: A. Fermentation. Surface growth froa a.oaa weak old agar slaat of Gllocladlum virena AXCC 13362, grown oa agar of tha following composition: so cm Malt extract 20 Glucoae 20 Peptone. 1 Agar 20 Distilled water, q.a. 1 liter (Sterilized 15 ain at 20 p.a.l.) waa auspeaded la 5 al of aa 0.85% saline aolution. Ona al portions of thla auspanaloa wens used to Inoeulata a 250 al Erleaaeyer flask (F-l stags) containing 50 ail of tha following aadiua (Soybean dextrose aadiua): Soybean aaal 5 g Dextrose 20 g MaCl .5 g KH2HP04 5 g Yeast 5 g Hater 1 1 pH adjusted to 7.0 Autoclave at 15 p.s.l. for 15 ainutaa She flask was incubated at 25 °C oa a rotary shaker (250 cycles/Bin - 2" radius) for 24 hours, after which a 10% by volume transfer was made to another 250 al Erleaaeyer flask (F-2 stage) containing 50 al of aoybean dextrose medium. After 24 hours of incubation on a rotary shaker, 150 mg of T-chloroacatoacatic benzyl ester in 0.1 al of 10% Tween 80 was added. She F-2 stage flask was then incubated for an additional 24 hours under the conditions used in the incubation of the F-l stage flasks. 21 B. Igelation. Tvanty-four hours after tha addition o£ tha Y-chloroacatoacetic benzyl aotar, tha aycelia wars removed by filtration. Tha filtrate waa exhaustively extracted with 50 ml of ethyl acetate thraa times. Tha athyX aeatata layar waa dried over MgSO^ and concentrated ia vacuo to yield a residue (160 mg). She residua was chromatographad over a silica gel (MM-Rieselgel 60) column (1 x 25 cm). Tha coluan waa eluted with Shelly B and athyl acetate (10 > 1) and 12 ml fractions ware collected. Fractions 11-16 containing the desired product were pooled and concentrated to dryness to afford 115 mg of 4-chloro-3(R)-hydroxybutyric acid benzyl ester, {a]£3+e.7* {c, 5.26; C8CI3); pmr (6 CSC13 ) 2.65 (28, d, J » 6 BS, -g-CHjCoor), 3.20 (IB, be, -OH); 3.54 (28, d, J » 6 BS, Cl-CH^B); 4.20 (IB, m, -CRj-^g-CHj-), .12 (28, s, -^-O-C^CgBg); 7.31 (5B, s, five aromatic protons). Anal.caled.gor C^B^OjCli C, 57.77; 8, 5.73. Found: C, 57.64; 8, 5.67. [TLC silica gel EM Brinkmann plate, 0.25 cm. Kg m 0.43, Skelly 8-athyl acetate (Sil).] Examples 4-23 The procedure of Example 1 waa repeated with each of tha organisms listed in Table 1 except that -chloro-acetoacetlc acid octyl ester was added at a concentration of 1 mg/mi. Conversion to the desired product (+)4-chloro-3 Exaaplaa 49-66 Tha proeadora of Sxaaple 1 was repeated with aach of tha orgaalsns listed in Tabla 1 axe apt that y-chloro-acatoacatie aeid bonsyl aster (1 ag/ml) was usad as tha substrata. Conversion to tha desired product (+)4-ehloro~ 3 (R)-hydroxybutyric acid benzyl ester was obtained.

Examles 69-93 The procedure of Exanple 3 was repeated with aaeh of the organ! nan listed la Tabla 2 using y-ehloroacetoaeatie aeid bensyl aster (1 ag/al) as subetrate. Traasfornatloa to tha dealred eoapound (♦)4-chloro-3(R)-hydroxybutyric aeid bensyl eatar waa obtained.

Example 94 (+)4-Chloro-3(R)-hydroxybutyric aeid aallida was prepared la accordance with the procedure of Exasple 2 except that 4-ehloroacetoacetanillde was usad at a concentration of 1 mg/nl. avU^i S3 aa tha substrata for tha conversion into tha desired optically-active product, m.p. 110-111'C; [a]23+17.5* (c, 3.0, CaCI3){ pur (6 CD3§C3>3) 2.67 (2B, d. J • 6 BS, -BOHCgj-CONHR), 3.66 (2B, d, J'tBS, ClC^CHQB-ft), 4.43 5 (1H. n, -CB2-Ca0a-Cs2-), 7.03-7.44 (3q, a. aromatic protons, mats and para), 7.69 (28, d, 3 ■ 6 BS, aroaatic protons, ortho), 9.24 (IB, br, -g-MH0). Anal, calcd for Cj^gB^MOjCl: C, 56.21; 8, 5.66. Found: C, 56.17; 8, 5.47. gxaaolea 95-114 Tha procadura of Example 1 was rapaatad with aaeh of tha organisms liatad ia Table 1 except that Y-chloro-acetoacataailide waa addad at a concentration of 1 ag/ml. Ia all eaaaa conversion to tha desired product, 15 (♦)4-chloro-3(R)-hydroxybutyric acid aallida was obtained.

SHSBBlS2_115=122 The procedure of Exaaple 3 was repeated with the orgaaiama listed ia Tabla 2. Y-Chloroacetoacataailida waa iatrodueed at a concentration of 1 ng/al. Za these 20 cases, eonvarsioa to tha desired (+)4-ehloro-3(R)-hydroxybutyric acid aailide was achieved.

Examples 140-159 She procedure of Example 1 was repeated with the organisms listed ia Table 1 except that Y-bromoaceto-25 acetic acid octyl ester (1 mg/al) was used as tha substrate. Conversion to the desired product, (♦)4-bromo-3(S)-hydroxybutyric acid octyl ester was obtained. 24 Examples 160-184 The procedure of Exaaple 3 was repeated with the organisms listed la Table 2 except that Y-broaoeceto-acetic aeid octyl ester (1 mg/ml) was used. Conversion to the desired product, (*)4-bro«o-3(R)-hydroxybutyric acid octyl ester was obtained.

Examples 185-204 The procedure of.Example 1 waa rapeatad with the organisms listed in Table 1 except that Y-bromoaceto-acetle acid bensyl ester (1 mq/ml) waa used as the substrate. Conversion to the desired product, (+)4-broao-3(R)-hydroxybutyric acid bensyl eatar was obtained.

Examelaa 205-229 The procedure of Exaaple 3 was repeated with the organisms listed in Table 2 except tbat Y-bromoacetoacatlc acid benzyl ester (1 mg/ml) was used. Conversion to the desired product, (*)4-bromo-3(R)-hydroxybutyric acid benzyl ester was obtained.

Examples 230-249 The procedure of Example 1 waa repeated with the organisms listed in Table 1 except that y-broaoacetanilide (1 mg/ml) waa used aa the substrate. Conversion to the desired (+)4-broao-3(R)-hydroxybutyric aeid anllide was obtained. 85 Examples 250-274 The proeadora of Exsapla 3 was repeated with tha orgnnlnaa Listad la Tabla 2 axcept that Y-broaoacatanilida (1 eg/ml) waa uaad aa the substrate. Conversion to tha daaired (♦)4-bromo-3 (R) -hydroxybutyric acid aallida was obtained.

Examples 275-294 Tha procadura of Exaaple 1 waa rapeatad with tha orgaalsas listed la Table 1 except tbat y-hydroxyaca-10 toacetlc octyl eater (1 mg/ml) waa usad as tha substrate. Conversion to the desired 4-hydroxy-3(R)-hydroxybutyric acid octyl ester waa obtained.

Examples '295-319 the procedure of Example 3 waa repeated with tha 15 organ!awn listed in Table 2 except that Y-hydroxyace- toacetle octyl ester (1 mg/ml) was used aa tha substrata. Conversion to the desired 4-hydroxy-3(R)-hydroxybutyric acid octyl ester was obtained.

Examples 320-339 The procedure of Exaaple 1 was repeated with the orgaalsas listed In Table 1 except that y-hydroxyace-toacetanlllde (1 mg/ml) was usad aa the substrata. Conversion to the desired 4-hydroxy-3-(&)-hydroxybutyric acid anllide was obtained. 26 Examal'aa 340-364 , Tha procedure of Example 3 waa repeated with tha organisms Xiatad in Tabla 2 axeopt that y-hydroxyace- i teaeatanilida (1 mg/ml) was used as tha substrata.

Conversion to tha desirad 4-hydroxy-3(R)-hydroxybutyric acid aallida was obtained.

Exaaple 365 Mathvl-4-chloro-3m-hvdroxvbutvrata (Villi Mathyl-4-ehloroaeatoaeatata (VII) (100 ag) was Incubated with 29 units of porcine ha art (EC 1.1.1.35). 0-hydroxyacylCoA dehydrogenase (Sigma. B4626), and 1.36 g of HUM (Sigma* 90%) ia 30 al of 0.1 M sodium phosphate buffer, pB 6.5.

After 30 hours at 25*C, tha raaetiea mixture was ex tracted four times with 30 ml of ethyl aeatata. Tha organic layer waa dried over sodium sulfate and was evaporated to dryness under reduced pressure. The residue (90 ag) waa chromatographad over a ailiea gal (12 g) 20 column (1.3 x 34 cm). Tha column was eluted with a solvent system consisting of Skelly B-ethyl acetate (8:1) and 20 ml fractions ware collected. Fractions 9-11 contained the desirad methyl-4-chloro-3(R)-hydroxybutyrata (VIII) as revealed by TLC, [a]23 + 23.5" (c, 5.2 CSCl^) were pooled. 25 *Trade Mark 37 Exaaple 366 The procedure of exaaple 365 was repeated using ethyl-4-chloroacetoacet*te aa tbe substrate to afford ethyl-4-chloro-3(R)-hydroxybutyrate, [a]^® ♦ 22.7* (c, 5 4.7 C&C13). ?67 • The procedure of exaaple 365 waa repeated using n-propyl-4-chloroacetoacetate as tiie substrate to afford a-propyl-4-chloro-3(R)-hydroxybutyrata, [a]^3 ♦ 21.3® (c, 10 5.0, CSC13).

Hxamele 366 The procedure of exaaple 365 was repeated using n-butyl-4-cbloroacetoacetate as the substrate to afford n-buty1—4-ehloro-3(R)-bydroxybwtyrate, (Alp3 ♦ 20.1" (e, 15 3.1, CBC13).

General procedure for tha conversion of 4-halo-3(Rl-frrttyrr-fautTrtc fsters «n Exaaale 369 A Mixture of 4-chloro-3(R) -hydroxybutyric acid octyl 20 eatar (1.5 g), ethane! (3 ml) and trimethylandne (25 wt % solution) in water (3 al) was heated at 80-90°C for about 2 hours. Tbe solvents and excess trimethylanine were evaporated to dryneaa in vacuo to yield 1.8 g of crude 28 residua. The crude product (If) was heated at 80-90°C ia a solution of 10% HC1 (7 ml) for 1.5 hours. After evaporation of tha solvents under reduced pressure, the crude product was extracted twice with absolute ethanol (10 al) and the ethanol was evaporated in vacuo. The crystalline residua was dissolved in a snail quantity of ethanol and th» L-carnitine chloride was precipitated by the addition of ether in good yield (320 no), a.p. 142" (dec.); MI - 23.7" (c, 4.5, HjO).

Tha L-carnitina chloride can be readily converted to the pharaaceutically preferred L-carnitine inner salt by ion exchange means as is well known in the art.

A mixture of octyl-4-chloro-3(&)-hydroxybutyrate (II) (1.426 g), and anhydrous Nal (1.2g) in 15 ml of methyl ethyl ketone waa refluxed for 24 hours. The mixture was rotor evaporated and reacted with ether (100 ml) aad water (50 ml). The organic phase was separated and washed with 10% sodium thiosulfate solution (150 ml), brine (ISO ml) aad dried over anhydrous scdium sulfate. 13m solveat was evaporated under reduced pressure to afford 1.762 g of IX as a pale-yellow oil; IS (thin m 37Q Octvl-4-iodo-3 fKl-hvdroxvbutvrate (IX) XI XX 2 9 film) 3460 cm _1 (OH) aad 1730 cm"1 (eater 00); BUR (C0C13) _ 3.93-4.27 (a, 3H), 3.17 (d, 2H), 2.50 (d. 2H), 1.50-1.87 (a. 2H). 1.30 (be, 12H). 0.93 (a, 3B).

T"rrf"T—of octvl-4-lodo-3(R)-hvdroxybutvrate (IX) To a solution of XX (1.593 g) in methanol (15 al) was addad at 25% solution of aqueous triaethylnaine (8 al). tha mixture was stirred at 27*C for 20 hours. Tha solvents aad tha excess trimsthy1amine wars evaporated off under reduced pressure to afford a semi-crystalline solid, X. this residue was washed with saall amounts of ether to remove the octanol aad then dissolved in water and passed over a Dowesf l-x4 [OH- form - 50-100 aosh, coluan voluae (2.5 x 15 em). The column was washed with distilled water. Removal of the solvent in vacuo *Trade Hark X XX X L-Carnitine froa the first 200 al of the eluate gave L-earaitiae as a white crystalline solid (490 aa, 63% yield) (a]23 - 29.2* (c, 6.5 HjO).

*«"Pla 371 The proeadure of exaaple 370 was repeated using hexyl-4-chloro-3(R)-hydroxybutyrate to yield hexyl-4-iodo-3 (R) -hydroxybutyrata, which was than converted to L-carnltlne.

Exaaple 373 The procedure of exaaple 370 was repeated ualaig heptyl-4-chloro-3(R)-hydroxybutyrate to yield heptyl-4-iodo-3(R)-hydroxybutyrate, which was then converted to L-carnitina.

Example 373 The procedure of exluple 370 waa repeated using decyl-4-chloro-3(R)-hydroxybutyrata to yield decyl-4-lodo-3(R)-hydroxybutyrata, which was then converted to L-caraitiae.

Exaaple 374 The procedure of exaaple 370 was repeated using aethyl-4-chloro-3(R)-hydroxybutyrate (VIII) to give aethyl-4-iodo-3(R)-hydroxybutyrate, which was then converted into L-caraitiae. 31 gxaaela 375 tha procedure of exaaplo 370 ma repeatod using athyl-4-chloro-3(R)-hydroxybutyrata to give athyl-4-iodo-3(R)-hydroxybutyrata, which was tfaaa converted into 5 L-caraitiae.

Exaaola 376 the procedure of oxaaple 370 waa repeated uaing n-propyl-4-chloro-3(R)-hydroxybutyrato to gin a-propyl-4-iodo-3(R)-hydroxybutyrata, which ms than coovertad 10 into L-c*rnitina.

SSSSiftJIZ She proeadure of axample 370 was rapeatad using n-buty1-*-chloro-3(R)-hydroxybutyrata to give n-butyl-4-iodo-3(R)-hydroxybutyrata, which waa then converted 15 into L-caraitlna.

Reprasahtativa yeasts that produco the daairad enzyme are listed ia Table 1 and representative fungi are Hated la Tabla 2. 32 Tabla 1 (Yeaata) 1. Candida llaolvtica MBSL Y-1095 2. Candida paaudotrooicalia M8SL Y-1264 3. Mvcodarma caraviaiaa HRHL Y-1615 4. Torula lactoaa MBBIi Y-329 . Gaotrichun candldun MSBL Y-532 6. Hanaanula anoaala MBHT. 7-366 7. Hanaanula aubaalllculoga MSSL Y-1683 8. Pichla alcoholoafalla NBSti T-2026 9. Saccharowvcaa caravleiaa MBSZt Y-12,632 . Saccharoaycaa lactia MBU Y-1140 11. ZvtToaaccharoavcaa prlorianua MRRL Y-12,624 12. Saccfaagoarycga acidifaclana NHHL Y-7253 13. Kloackara corticia ATCC 20109 14. Crvptococua naacarana ATCC 24194 . Rhodotoxrula bp. ATCC 20254 16. Candida alblcana ATCC 752 17. Dipodaacua albldua ATCC 12934 18. Saccharontvcaa caraviaiaa (caauarcial Had Star) 19. Rhodotorula rubra MRU Y-1592 . Ooapora lactia ATCC 14318 MSRIi - Northern Raglonal Rasaarch Lab. at Paeria, Illinoia. ATCC - American Type Culture Collection at Boekville, Maryland. 33 Tabla 2 (Fungi! W 1. Ollocladiua virana ATCC 13362 $4 2. Caldarionvcaa fuaaco ATCC 16373 3. Lindarlna ounilaopora ATCC 12442 4. Aaoarqillua ochraewia NXBL 405 . Trichodarma lionorum ATCC 8678 6. g»t»roei«AT|.» ""IT ATCC 16328 7. Eatomopfathora coronata MRHL 1912 8. Scopularlopala conatantlnl HHBL 1860 10 9. Zvqorfavnchua botorowamua ATCC 6743 . gcopularlopala bravlcaulia NHRX. 2157 U- Hhiaopua arrhlaia M88L 2286 12. Panieillli"" NRHI. 2077 13. i*"*" fcfiy (-) Mttt 4088 15 14. Bvaaochlaanra aivea ATCC 12550 . Panicilliun oatulua NRBL 1952 16- Hatarrhlsf™ AICC 24942 17. Fonlcllllw AICC 10127 18. ftTm4i"1*"iolla alaoana ATCC 10028a 19 ■ AICC 11585a . Aaparolllw AICC 16907 21. Aaporqillwa aMa*»i«a«iiH nrbl 90 22. gliocladiua roaeua ATCC 10521 23. Aapowrtllua aioantaua AICC 10059 25 24. Abaldia blakalaaana AICC 10148b . PanlclllliMi roouaforti UStRL 849a

Claims (30)

1. A compound having the formula and 3(R) configuration ■R wherein X is selected from CI, Br,'I and OH and R is a radical in straight chain, branched chain or cyclic configuration selected from alkoxy radicals having from 1 to 15 carbon atoms; alJcylamino radicals having from 5 to 15 carbon atoms; cycloalkoxy radicals and cycloalkylamino radicals having from 5 to 12 carbon atoms phenoxy and phenylalkoxy radicals having from 7 to 14 carbon atoms phenylamino and phenylalkylamino radicals having the formulae Y Y Z -U-0-A; and - H-0-A where Y and Z are selected from H, an alkyl group having from 1 to 8 carbon atoms, phenyl or benzyl and a is selected from H, CH3, CI and Br a 5

2. The compound of claim 1, wherein 1 is a straight-chain alkoxy radical having from 1 to 10 carbon atoms.

3. The compound of claim 2, wherein R is 0C1QH21.

4. The compound of claim 2, wherein R is OCgH,^.

5. 5. The compound of claim 2, wherein R is OC^^.

6. The compound of claim 2, wherein R is 0C,H,,. o I 3

7. The compound of claim 2, wherein R is a lower alkoxy radical having from 1 to 4 carbon atoms.

8. The ocnpound of claim 1 modified in that R is selected 10 from phenoxy and phenylalkoxy substituted with lower alkyl, halo or nitro group.

9. The compound of claim 8, wherein R is benzyloxy and X is selected from CI and Br.

10. The compound of claim 1, wherein R is phenylamino 15 and X is selected from CI and Br. 3 6

11. A process for preparing optically active /-substituted fj-hydroxybutyric acid derivatives having the formula wherein X is selected from CI, Br, I and OH and S is a radical in straight chain, branched chain or cyclic configuration selected From alkoxy radicals having from i to 15 carbon atoms; alkylamino radicals having from 5 to 15 carbon atoms; cycloalkoxy radicals and cycloalkylamino radicals having from 5 to 12 carbon aboms phenoxy and phenylalkoxy radicals having from 7 to 14 carbon atoms phenylamlno and phenylalkylamino radicals having the formulae Y HZ and -1-CHHJ-A where Y and Z are selected from H, an alkyl group having from 1 to 8 carbon atoms, phenyl or bensyl' and A is selected from H, CH3, CI and Br 37 15 20 from corresponding /-substituted acetoacetic acid esters or amides, which comprises subjecting said /-substituted acetoacetic acid esters or amides to the fermentative enzymatic action of a microorganism which elaborates L- ft-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] • and recovering the desired optically active /-substituted- -hydroxybutyric acid derivatives.

12. The process of claim 11 for preparing optically active /-substituted 3(R)—hydroxybutyric acid derivatives having the formula and 3(K) configuration wherein X and R have the above-identified meanings, provided that if R is an alkoxy radical it has from 5 to about 15 carbon atoms, which comprises subjecting compounds having the formula wherein X and R have the above-identified meanings to the fermentative enzymatic action of a microorganism which elaborates L- {} -hydroxyacyl CoA dehydrogenase [EC 1.1 .1.35! • and R 38 recovering the desired optically active 4-substituted 3-( R)-hydroxybutyric acid derivatives from the ferment- * ative reaction mixture. t

13. The method of cla£m 12,wherein the microorganism is 5 selected from the claas Ascomycetes.

14. The method of claim 12,wherein the microorganism is selected from the orders Endomycetales, Mtucorales, Moniliales or Eurotiales.

15. The method of claim 12,wherein the microorganism is 10 selected from the genus Saccharomyees.

16. . The method of claim 1% wherein the microorganism is Saccharomyces cerevisiae.

17. The method of claim 12,wherein the /-substituted acetoacetic acid derivative subjected to fermentative 15 enzymatic action is y-chloro-acetoacetic acid octyl ester.

18. The method of claim 12,wherein the /-substituted acetoacetic acid derivative subjected to fermentative enzymatic action is /-chloro-acetoacetic acid'benzyl ester. 20

19. The method of claim M,wherein the /-substituted acetoacetic acid derivative subjected tc fermentative enzymatic action is /-chloro-acetoacetanilide. r 10 39

20. The method of claim 1 % wherein the microorganism is Saccharomices cerevisiae.

21. The method of claim 18, wherein the microorganism is Saccharomyces cerevisiae.

22. The method of claim 19, wherein the microorganism is Saccharomyces cerevisiae.

23. The process of claim 11 for preparing optically active /-substituted 3(E) hydroxybutyric acid derivatives having the formula and 3(R) configuration wherein X has the above-identified meanings and S is an alkoxy radical having from 1 to 4 carbon atoms which comprises subjecting compounds having the formula 0 0 JLCH 1-fi is xch2-c-ch2c-r wherein x and r have the above-identified meaning to the enzymatic action of L- p-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35] in purified form, and recovering the desired optically active /-substituted 20 3(R/-hydroxybutyric acid derivatives from the enzymatic reaction mixture. * 1 40

24. Tha process of claim 23. wherein said L- -hydroxyacyl CoA dehydrogenase (EC i.1.1.35] in purified form is that isolated from porcine heart.

25. A process for preparing L-carnitine inner salt which comprises 5 reacting a 4-substituted -3(R)-hydroxybutyric acid derivative of claim 1 sequentially with trimethylamine and hydrochloric acid, extracting the L-carnitine chloride, and subjecting said chloride to ion exchange and recpvering the inner salt of L-carnitine; 10

26. A process for the preparation of a 4-iodo or 4-bromo--3( R ^-hydroxybutyric acid derivative which comprises reacting a 4-chloro-3(R)-hydroxybutyric acid derivative with sodium iodide or sodium bromide in a solvent at a temperature of 50"C to 100°C to form tha corresponding 15 4-iodo or 4-bromo-3(R)-hydroxybutyric acid derivative.

27. A process for the preparation of L-carnitine inner salt which comprises: a) reacting a 4-iodo or 4-brom©-3(R)-hydroxybutyrate alkyl ester containing 1-10 carbon atoms with tri- 20 methylamine in methanol or ethanol to form a L-carnitine methyl or ethyl ester salt, and b) converting said L-carnitine ester salt to L-carnitine inner salt by hydrolysis under alkaline conditions. i r-

28. A process as claimed in claim 11, substantially as described in any of the foregoing Examples 1 to 364.

29. A Y-substituted B-hydroxybutyric acid derivative prepared by a process as claimed in any of claims 11 to 24 or claim 28.

30. L-carnitine inner salt prepared by the process of claim 25 or 27. F. R. KELLY * CO., AGENTS FOR THE APPLICANTS.