HK1005723B - Improved process for manufacturing l-(-)-carnitine from a waste product having opposite configuration - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to an improved process for manufacturing L-(-)-carnitine from a starting compound containing an asymmetrical carbon atom having a configuration opposite to that of L-(-)-carnitine. The process of the present invention overcomes the drawbacks of conventional processes which first convert a starting compound into an achiral intermediate, generally crotonobetaine or gamma-butyrobetaine, and then convert the achiral intermediate to L-(-)-carnitine. The process of the present invention uses D-(+)-carnitinamide as starting compound.

Discussion of the Background

Carnitine contains a single center of asymmetry and therefore exists as two enantiomers, designated D-(+)-carnitine and L-(-)-carnitine. Of these, only L-(-)-carnitine is found in living organisms, where it functions as a vehicle for transporting fatty acids across mitochondrial membranes. Whilst L-(-)-carnitine is the physiologically-active enantiomer, racemic D,L-carnitine has conventionally been used as a therapeutic agent. It is now recognized, however, that D-(+)-carnitine is a competitive inhibitor of carnitine acyltransferases, and that it diminishes the level of L-(-)-carnitine in myocardium and skeletal muscle.

It is therefore essential that only L-(-)-carnitine be administered to patients undergoing haemodialysis treatment or treatment for cardiac or lipid metabolism disorders. The same requirement applies to the therapeutic utilization of acyl derivatives of carnitine for treating disorders of the cerebral metabolism, peripheral neuropathies, peripheral vascular diseases and the like. These disorders are typically treated with acetyl L-(-)-carnitine and propionyl L-(-)-carnitine, which are obtained by acylating L-(-)-carnitine.

Various chemical procedures have been proposed for the industrial-scale production of carnitine. Unfortunately, these procedures are not stereospecific and produce racemic mixtures of D-(+)- and L-(-)-isomers. It is thus necessary to apply resolution methods in order to separate the enantiomeric constituents of the racemate.

Typically, the D,L-racemic mixture is reacted with an optically active acid (e.g. D-(-)-tartaric acid, D-(+)-camphorsulfonic acid, (+)-dibenzoyl-D-(-)-tartaric acid, N-acetyl-L-(+)-glutamic acid and D-(+)-camphoric acid) to obtain two diastereoisomers which can be separated from each other. In the classic process disclosed in U.S. Patent 4,254,053, D-(+)-camphoric acid is used as the resolution agent of a racemic mixture of D,L-carnitinamide, obtaining D-(+)-carnitinamide as a by-product, and L-(-)-carnitinamide which, by hydrolysis, gives L-(-)-carnitine.

However, these resolution procedures are complex and costly, and in all cases result in the production of equimolar quantities of L-(-)-carnitine and D-(+)-carnitine or a precursor thereof as by-product, having configuration opposite to that of L-(-)-carnitine. Several microbiological processes have recently been proposed for producing L-(-)-carnitine via stereospecific transformation of achiral derivatives obtained from the huge amounts of D-(+)-carnitine (or of a precursor thereof, such as D-(+)-carnitinamide) which are generated as by-products in the industrial production of L-(-)-carnitine.

These processes are generally predicated upon the stereospecific hydration of crotonobetaine to L-(-)-carnitine, and differ principally by virtue of the particular microorganism employed to accomplish the biotransformation of interest. See, for example, the processes disclosed in: EP 0 12 1444 (HAMARI), EP 0 122 794 (AJINOMOTO), EP 0 148 132 (SIGMA-TAU), JP 275689/87 (BIORU) , JP 61067494 (SEITETSU), JP 61234794 (SEITETSU), JP 61234788 (SEITETSU), JP 61271996 (SEITETSU), JP 61271995 (SEITETSU), EP 0 410 430 (LONZA), EP 0 195 944 (LONZA), EP 0 158 194 (LONZA), and EP 0 457 735 (SIGMA-TAU).

On the other hand, JP 62044189 (SEITETSU) discloses a process for stereoselectively producing L-(-)-carnitine starting from gamma-butyrobetaine, which is in turn obtained enzymically from crotonobetaine.

All of these processes have several drawbacks. First, D-(+)-carnitine must first be converted to an achiral compound (crotonobetaine, gamma-butyrobetaine) before it can be used as the starting compound in all of the aforesaid microbiological processes.

In addition, the microbiological procedures proposed to date have not proven practicable for manufacturing L-(-)-carnitine on an industrial scale for one or more of the following reasons:

• (i) the yield of L-(-)-carnitine is extremely low;
• (ii) the microorganisms must be cultivated in a costly nutritive medium;
• (iii) the microorganism can only tolerate low concentrations [up to 2-3% (w/v)] of crotonobetaine;
• (iv) side reactions occur, such as the reduction of crotonobetaine to gamma-butyrobetaine or the oxidation of L-(-)-carnitine to 3-dehydrocarnitine. These side reactions reduce the final yield of L-(-)-carnitine.

In order to overcome all of the aforesaid drawbacks of the known processes, in the Italian patent application RM 92 A 000 915 filed on December 21, 1992 (CORRESPONDS TO EP-A-609843) in the name of the same applicants as the present application, not available to the public inspection at the filing date of this application, a process has been disclosed which allows high yields of L-(-)-carnitine to be obtained starting from a by-product having configuration opposite to that of L-(-)-carnitine (such as D-(+)-carnitinamide) with no need to first convert the starting by-product into an achiral intermediate.

This process which is illustrated in the following reaction scheme 1: comprises hydrolyzing a D-(+)-carnitinamide salt 1 to D-(+)-carnitine 2 and esterifying 2 into ester 3 (via known methods) wherein R1 is preferably arylalkoxy, e.g. benzyloxy.

The ester 3 is then converted to the acyl derivative 4 wherein Y, which can be the same as X, is preferably a counterion, e.g. perchlorate, imparting solubility to 4. OR is a leaving group wherein R is preferably an alkylsulfonyl group having 1-12 carbon atoms, e.g. mesyl.

The acylation of 3 to 4 is carried out preferably in pyridine by reacting the ester 3 with an acylating agent RY wherein Y is halogen and R is an acyl group as defined above. Preferably RY is the chloride of the selected acyl group.

The ester group -COR₁ of 4 (R₁ =benzyloxy) is hydrogenated to carboxyl group thus giving acyl D-(+)-carnitine 5 which is converted to the lactone 6 of L-(-)-carnitine. The lactonization is suitably carried out in an aqueous basic environment: either with NaHCO₃ (ratio 1:1) or with an AMBERLITE IRA-402 basic resin activated in HCO $\frac{\text{-}}{\text{3}}$ form or with an LA2 resin. The lactone is isolated by evaporating the aqueous solution or precipitating it as a salt (for example, as tetraphenylborate or reineckate).

Finally, lactone 6 is suitably converted to L-(-)-carnitine inner salt 7. The lactone is dissolved in water and the resulting solution treated with a base such as NaHCO₃ (ratio 1:1), for 8-24 hours.

L-(-)-carnitine can suitably be purified from the salts which are formed from the X^- anion, from the excess, if any, of the acyl halogenide, from pyridine, and the like, by chromatographing the aqueous solution on a strongly acidic resin such as IR 120, eluting with water and then with NH₄OH, or alternatively eluting first on a strongly basic resin such as AMBERLITE IRA 402 activated in OH form and thereafter on a weakly acid resin such as AMBERLITE IRC-50.

The process of the present invention which is illustrated in the following reaction scheme 2 constitutes a remarkable improvement over the previous process.

Indeed:

• (1) D-(+)-carnitinamide 1 is directly converted to ester 2 (without previous conversion to D-(+)-carnitine);
• (2) acylation (particularly, mesylation) of 2 to 3 can be carried out in the absence of solvents, particularly pyridine the use of which brings about serious drawbacks;
• (3) the ester group of acyl derivatives 3 is converted into the carboxyl group of acyl derivative 4 via simple acid hydrolysis, thus avoiding the drawbacks of hydrogenation reduction, which are particularly serious when the process is conducted on an industrial scale.

In detail, with reference to the reaction scheme 2, D-(+)-carnitinamide 1 is converted into ester 2 via conventional procedures, in the presence of an excess of alcohol, preferably an alkanol having 1-4 carbon atoms, by acid catalysis, e.g. with gaseous HCl or concentrated H₂SO₄.

X^- is for instance a halogenide, (preferably chloride); sulphate; phosphate; perchlorate: metaperiodate; tetraphenylborate; an alkylsulphonate having from 1 carbon atom (methansulphonate) to 12 carbon atoms (dodecylsulphonate) : trifluoroacetate; tetrahalogenborate; fumarate or alkylsulphate having 10-14 carbon atoms.

Suitable esters 2 include those esters wherein R1 is a straight or branched alkyl group having 1-11 carbon atoms, preferably n-butyl or isobutyl.

The ester 2 is then converted to the acyl derivative 3 wherein R is an alkylsulfonyl group having 1-12 carbon atoms, formyl or trifluoroacetyl. Preferably, the alkylsulfonyl group is selected from methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), p-bromobenzenesulfonyl (brosyl), p-nitrobenzenesulfonyl (nosyl), trifluoro-methanesulfonyl (triflyl), nonafluoromethanesulfonyl (nonaflyl) and 2,2,2-trifluoroethanesulfonyl (tresyl). Mesyl is particularly preferred.

The acylation of 2 to 3 is carried out by reacting the ester 2 with R₂O, the anhydride of the selected acid wherein R is an acyl group as defined above.

The acylation reaction is carried out in inert anhydrous solvents, such as methylene chloride or acetonitrile or directly in a molten mixture of the two reactants, without any solvent. The acylating agent is added at ratios ranging from 1:1 to 1:5, preferably 1:3, at temperatures comprised between 40°C and 80°C, for 8-48 hours.

The compound 3 can be isolated (it is not mandatory to isolate the compound 3, as will be shown below), via precipitation with a suitable solvent, such as ethyl ether or hexane. The compound is then purified via crystallization or by eluting its aqueous solution on a weak basic resin such as AMBERLITE IR 45 (Rohm and Haas) or shaking the aqueous solution with a LA-2-type weak basic resin diluted in hexane, and finally lyophilizing or concentrating the aqueous solution.

The ester group -COOR₁ of 3 converted to the carboxyl of acyl D-(+)-carnitine 4 via acid hydrolysis with conventional procedures.

Conversion of acyl D-(+)-carnitine 4 to lactone 5 and the conversion of this latter compound to L-(-)-carnitine 6 are carried out as disclosed in the previously cited Italian patent application RM92A000915.

It should be understood that, whereas the process disclosed above has been described, for the sake of clarity, as a sequence of five distinct operating steps, the corresponding industrial process consists of three steps only. When the process of the present invention is carried out as an industrial process, the acyl D-(+)-carnitine ester 3 can be directly converted to L-(-)-carnitine inner salt 6 without isolating either the acyl D-(+)-carnitine 4 or the lactone 5.

In fact, the ester of acyl D-(+)-carnitine 3 is hydrolized in an acid environment, then the resulting aqueous solution is concentrated and the concentrate is brought to pH 7-9, preferably 8-9 and kept at this pH value for 30-50 hours yielding L-(-)-carnitine.

In the following example which describes one embodiment of the process of the invention, the intermediate compounds 2, 3 and 4 were isolated so as to exhaustively characterize them from a physico-chemical standpoint.

It will be, however, apparent to any expert in organic synthesis that the industrial process comprises the following steps only:

• (a) conversion of D-(+)-carnitinamide 1 to the ester of D-(+)-carnitine 2;
• (b) acylating of the hydroxyl group of ester 2 with an anhydride R2O, wherein R has the previously defined meanings, with the resulting formation of a leaving group OR thus obtaining the ester 3 of acyl D-(+)-carnitine; and
• (c) conversion of 3 to L-(-)-carnitiine inner salt 6.

EXAMPLE 1

Preparation of D-carnitine isobutyl ester chloride 2.

Step (a)

D-carnitinamide chloride 1 (10 g; 0,05 moles) was suspended in 50 mL isobutanol. The solution was cooled to 4°C and gaseous HCl was added thereto till saturation. The reaction mixture was refluxed for 1 hour and then filtered while still hot, in order to remove NH₄Cl.

The alcohol solution was concentrated to dryness under vacuum, taken up twice with isobutanol and concentrated.

Acetone was added to the residue thus obtained and the solid product filtered off.

11.6 g of compound 2 were obtained. Yield 90% HPLC

• Column: nucleosil 5-SA 4.0 mm x 200 mm
• Temperature: 30°C
• Eluant: CH3CN-KH2PO4 50 mM 65-35 pH 3.5
• Flow rate: 0.75 mL/min
• Detector I.R.
• Retention time: 14.6 min

¹H NMR D₂O δ 4.7 (1H,m,CHOH); 4.0-3.9 (2H,m,COOCH₂-); 3.5 (2H,m,N⁺CH₂-); 3.2 (9H,s,(CH₃)₃N⁺); 2.7 (2H,m,CH₂COO); 2.0-1.9 (1H,m,CH(CH₃)₂); 0.9 (6H,d,(CH(CH ₃)₂)

	C%	H%	N%	CL%
Calculated	52.06	9.53	5.52	13.97
Found	49.89	10.26	6.23	14.88

H₂O 0.8% [α] $\frac{\text{25}}{\text{D}}$ = +15 (c=1% H₂O)

Preparation of methanesulfonyl D-carnitine isobutyl ester methanesulphonate 3.

Step (b)

A mixture of D-carnitine isobutyl ester chloride (2.5 g; 0.01 moles) and methanesulfonic anhydride (5.2 g; 0.03 moles) was heated at 80°C for 24 hours.

The molten mass was taken up with CH₂Cl₂ and precipitated with ethyl ether. This operation was repeated three times in order to remove the excess of methanesulfonic anhydride.

3.9 g of compound 3 were obtained.

• Yield: 100%
• HPLC
• Column: nucleosil 5-SA 4.0 mm x 200 mm
• Temperature: 30°C
• Eluant: CH3CN-KH2PO4 50 mM 65-35 pH 3.5
• Flow rate: 0.75 ml/min
• Detector: I.R.
• Retention time: 10.11 min

¹H NMR D₂O δ 5.5 (1H,m,-CH-O) ; 3.9 - 3.8 (3H,m,OCH₂,N+CH-H); 3.6 (1H,d,N+CH-H) 3.2 (3H,s,OSO₂CH₃) ; 3.1 (9H,s,(CH₃)₃N+); 3.0 (2H,dd,CH₂COO); 2.7 (3H,s,CH₃SO₃-); 1.8 (1H,m,CH(CH₃)₂); 0.8 (6H,d,CH(CH ₃)₂).

	C%	H%	N%	S%
Calculated	39.88	7.47	3.58	16.38
Found	39.45	7.43	3.75	16.24

[α] $\frac{\text{25}}{\text{D}}$ = + 24.7 (c = 1% H₂O) M.P. = 137-140 °C

Preparation of methanesulfonyl D-carnitine methanesulfonate 4.

Step (c)

Methanesulfonyl D-carnitine isobutyl ester methansulphonate 3 (3.9 g; 0.01 moles) was dissolved in 65 mL 2N HCl and the resulting solution was kept at 50°C for 20 hours.

The solution was then concentrated to dryness under vacuum. The oily residue was washed with acetone and the solid product which was filtered off.

3.3 g of compound 4 were obtained. Yield: 90%

• HPLC
• Column: nucleosil 5-SA 4.0 mm x 200 mm
• Temperature: 30°C
• Eluant: CH3CN-KH2PO4 50 mM 65-35 pH 3.5
• Flow rate: 0.75 ml/min
• Detector I.R.
• Retention time: 12.60 min

• 1H NMR D2O δ 5.5 (1H.m.CHOSO2CH3); 3.9 (1H,dd,N+CH-H);
• 3.6 (1H,dd,N+CH-H); 3.2 (3H,s,OSO2CH3); 3.1 (9H,s,(CH3)3N+);
• 2.9 (2H,m,CH2COOH); 2.7 (3H,s,CH3SO3-)

• [α]25D = + 22 (c = 1% H2O)
• M.P. = 148-150 °C

Claims (4)

1. A process for producing L-(-)-carnitine from D-(+)-carnitinamide, which comprises:

   (a) reacting D-(+)-carnitinamide 1 of the formula: wherein X^- is any counteranion, with an excess of a straight or branched chain alkanol having 1-11, preferably 1-4 carbon atoms, thereby forming ester 2 of the formula: wherein R₁ is a straight or branched alkyl group having 1-11, preferably 1-4 carbon atoms;

   (b) acylating ester 2 to an acyl derivative 3 of the formula: wherein R is an alkylsulfonyl having 1-12 carbon atoms, formyl or trifluoroacetyl group, by reacting ester 2 with an anhydride R₂O, wherein R has the above defined meaning in an inert solvent or mixture of solvents selected from methylene chloride and/or acetronile or preferably in a molten mixture of anhydride/ester 2 in a ratio of anhydride to ester 2 ranging from 1:1 to 1:5, at 40°C-80°C, for 8-48 hours;

   (c) acid hydrolizing the COOR₁ group of said acyl derivative 3 to a carboxylic acid group, thereby obtaining an acyl D-(+)-carnitine 4 of the formula:

   (d) reacting said acyl D-(+)-carnitine 4 with a base selected from NaHCO₃, AMBERLITE IRA-402 basic resin activated in HCO₃ form or LA2 resin thereby preparing lactone 5 of L-(-)-carnitine of the formula:

   and (e) converting said lactone 5 to L-(-)-carnitine by reacting lactone 5 with an aqueous basic solution, preferably NaHCO₃, ratio 1:1, for 8-24 hours and isolating L-(-)-carnitine inner salt by contacting the L-(-)-carnitine containing solution with an ion exchange resin.
2. The process of claim 1, wherein said steps (c), (d) and (e) are carried out as a single step, without isolating said intermediate compounds 3, 4 and 5.
3. The process of claim 1 or 2 wherein:

   X-   is halogenide, preferably chloride, sulphate, phosphate, perchlorate, metaperiodate, tetraphenylborate, or alkylsulfonate having 1-12 carbon atoms;

   R1   is n-butyl or isobutyl; and

   R   is methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), p-bromobenzenesulfonyl (brosyl), p-nitrobenzenesulfonyl (nosyl), trifluoromethanesulfonyl (triflyl), nonfluoromethanesulfonyl (nonaflyl) and 2,2,2-trifluoroethanesulfonyl (tresyl).
4. Ester of acyl D-(+)-carnitine having the general formula wherein X^- is any counterion, particularly an anion selected from halogenide, preferably chloride, sulphate, phosphate, perchlorate, metaperiodate, tetraphenylborate, alkylsulfonate having 1-12 carbon atoms;

   R₁   is n-butyl or isobutyl; and

   R   is an alkylsulfonyl group having 1-12 carbon atoms, preferably mesyl.