FI57745C - FREQUENCY REQUIREMENT FOR THERAPEUTIC USE OF THERAPEUTIC 3-FLUOR-D-ALANIN ELLER DESS DEUTEROANALOG - Google Patents

Description

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VfiK W (, 1> UTLÄGGNINOSSKIlCFT 5 774 5 ς (1 $) Patent oySnne-tty 10 10 IvSO, Patent ceddelat T (51) tbMCfmtja? C 07 C 101/10 FINLAND —FINLAND (21) Ρ «· ηηΐΙ ^ ί · ηιυι - Patmtamatolni 1587/72 (22) H »k * mlipllv» - Anrtknlngrtig 06.06.72 ^ ^ (23) Alku pilvi - Glltl | h «t $ dag 06.06.72 (41) Tullut julklMksi - Bllvlt offmtllg 19.12.72

Patent · J * reki Sten hali itua (44) Date of diminutive and public publication—

Patent- och registerstyrelsen '7 Amektn uti «* d och uti. * Krtft * n puUfcwed 30.06.80 (32) (33) (31) Requested« tuolkuu * —B «| lrt prlorltet 18.06.71 Ο3.Ο2.72 USA (US) 15 ^ 695, 22335 ^ Verified-Styrkt ,, (71) Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey, USA (72) Janos Kollonitsch, Westfield, New Jersey, USA (US) (7 ^) Oy Kolster Ab (5 * 0 Method for the preparation of a therapeutically useful 3-fluoro-D-alanine or deutero analogue thereof - For the preparation of a therapeutically analogous 3-fluoro-D-Alan or deutero analogue of this The invention relates to a process for the preparation of a therapeutically useful 3-fluoro-D-alanine or a deutero analogue thereof, having the formula Ϊ2? 1.

F-C - C-C00H

r3 nh2 wherein R1, R2 and R2 are hydrogen or deuterium. 3-Fluoro-D-alanine and its deutero analogs are effective bactericidal agents and are valuable in inhibiting the growth of both gram-positive and gram-negative pathogenic bacteria.

57745

The process according to the invention is characterized in that a) a mixture of salts of 3-fluoro-DL-alanine of the formula I or a deutero analogue thereof and an optically active base is subjected to fractional crystallization to obtain a salt of 3-fluoro-D-alanine in substantially pure form, and that the salt is treated with a strong acid to liberate the compound of formula I from its salt, or b) subjecting the mixture of 3-fluoro-DL-alanine of formula I or a deutero analogue thereof and the salts of the optically active acid to fractional crystallization to give the 3-fluoro-D-alanine salt in substantially pure form, and that the salt is treated with a strong base to liberate the compound of formula I from its salt, or c) a mixture of an amino-protected derivative of a 3-fluoro-DL-alanine of formula I or a deutero analogue thereof and an optically active acid is subjected to fractional crystallization to give the corresponding amino-protected 3-fluoro-D -alanine salt in virtually pure form that the resulting salt is treated with a strong acid to liberate an amino-protected derivative of a compound of formula I from its salt, and the amino group is finally cleaved off, or d) amino protection of 3-fluoro-D-alanine or its deutero analogue from a derivative of the formula? 2 * 1 FC - C-COOH r 3 NHR 4 wherein R 1, R 8 and R 2 are as defined above and R 2 is carbobenzoxy, benzoyl, chloroacetyl or menthoxyacetyl, the protecting group is cleaved by hydrogenation or hydrolysis, or e) 3-fluoro-D- -alanine or an amino-protected derivative of its deutero analogue of the formula * 2 y,

F-C - C-COOH

R 3, R 5 and R 6 are as defined above, and either R 1 is hydrogen and R 8 is carbobenzoxy, benzyl, n-methylbenzyl or N-carboxybenzyl, or R 5 and R 6 are each benzyl, the protecting group is cleaved R 8 by catalytic hydrogenation, or f) 3-fluoro-D-alanine or a carboxyl-protected derivative of a deutero analog thereof having the formula 57745 RC - C-COR and R 3 NH 2 wherein R 1, R 2 and R 2 are as defined above, and R 2 , is an ester, amide or cyano group, the protecting group R7 is cleaved off by hydrolysis.

Carboxyl-protected groups are usually amides and substituted amides, ester substituents, e.g., alkyl esters such as methyl, ethyl and t-butyl esters, aryl esters, aralkyl esters such as benzyl ester, p-nitrobenzyl ester and the like.

Amino-protected groups commonly used include acyl-type substituents such as formyl, acetyl, benzoyl, phthaloyl, trifluoroacetyl, 1-methoxyacetyl, toluenesulfonyl, dibenzylphosphoryl, 2-nitrobenzenesulfonyl, tritylsulfenoxy, o-nitrophenyl or urethane protecting substituents such as benzyloxycarbonyl (carbobenzoxy) methyloxycarbonyl, ethyloxycarbonyl, p-methoxycarbobenzoxy, p-nitrocarbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl 2-propyloxycarbonyl, isonicotinyloxycarbonyl, diisopropylmethyloxycarbonyl and the like, or substituents of the alkyl or aralkyl type, such as benzyl, dibenzyl, N-phenylethyl (? -Methylbenzyl), triphenylmethyl, trimethylsilyl, trialkylsilyl, like.

When 3-fluoro-D-alanine compounds are prepared by decomposing a 3-fluoro-DL-alanine derivative (not a salt) with an optically active group such as 1-menthoxyacetyl, or by asymmetric synthesis, e.g., by reacting a fluoroacetaldehyde with an optically active with an amine such as D - <* - methylbenzylamine, R (D) -phenylglycine, 1-amino-C5-Z1R1-1-hydroxyethyl-indoline and the like, or by administering an amino-protected o <-amino-β-fluoroacrylic acid, such as o <-benzamido / 3-fluoroacrylic acid to react with hydrogen or deuterium in the presence of an asymmetric catalyst, or when 3-fluoro-D-alanine compounds are prepared by asymmetric reaction of 2- (azidocarbonyl or aminocarbonyl) -3-fluoropropionic ester or nitrile. by rearrangement, or by preparation of N-acyl-3-fluoro-D-alanine by selective enzymatic Dease alkylation of the L-isomer, optionally an amino-protected and / or carboxyl-protected derivative of 3-fluoro-D-alanine is obtained, e.g. 1-Methoxy xiacetyl-3-fluoro-D-alanine, N- (D-o-methylbenzyl) -3-fluoro-D-alanine, N- (Do <-carboxybenzyl) -3-fluoro-D-alanine, N-benzyl-3 -fluoro-D-alanine, N-carbonyl-3-fluoro-D-alanine methyl ester, N-ethoxycarbonyl-3-fluoro-D-alanine, N-chloroacetyl-3-fluoro-D-alanine and the like.

57745

In the preparation of 3-fluoro-D-alanine compounds, cleavage of the salts of the optically active base and the amino-protected derivative of 3-fluoro-DL-alanine finally gives (after acidification) the corresponding amino-protected derivative of 3-fluoro-D-alanine, such as N-carbobenzoxy. 3-fluoro-D-alanine, N, N-di-benzyl-3-fluoro-D-alanine and the like.

In the preparation of 3-fluoro-D-alanine compounds, cleavage of the salts of the optically active acid and the carboxyl-protected derivative of 3-fluoro-DL-alanine gives the corresponding carboxyl-protected derivative of 3-fluoro-D-alanine, such as (after decomposition using tartaric acid, camphor-10-sulfuric acid, dibenzoyl-d-tartaric acid and the like as a disintegrant) 3-fluoro-D-alanine benzyl ester, D-2-amino-3-fluoropropioninitrile, D-2-amino-3-fluoropropionamide and the like.

These protected derivatives can all be converted to the free 3-fluoro-D-alanine compound by reaction with a cleavage agent. For example, in the following amino-protected derivatives listed above: N-1-menthoxyacetyl, N-benzoyl, N-carbonyl, N-ethoxycarbonyl and N-chloroacetyl, it is usually preferable to use an acid hydrolysing agent such as aqueous hydrochloric acid, hydrobromic acid and the like; basic hydrolysing agents are usually undesirable because they can react with the 3-fluoro substituent.

In connection with the other amino-protected derivatives already mentioned, namely N- (Do <-methylbenzyl), N- (D-α-carboxybenzyl), N-carbobenzoxy and N, N-dibenzyl derivatives, it is preferable to use a hydrogenating agent as a cleavage agent. , such as hydrogen, in the presence of a palladium-carbon catalyst or a palladium hydroxide-carbon catalyst. The hydrogenation is usually carried out in the case of relatively easily cleavable groups, such as a carbobenzoxy group, at approximately atmospheric pressure, and in the case of less easily cleavable groups, such as Ν, Ν-dibenzyl, N- (D-® * -methylbenzyl), N- ( D 2 O-carboxybenzyl) and the like, up to 2.8 kg / cm g overpressure when using a palladium hydroxide catalyst and up to TO kg / cm g overpressure when using a palladium on carbon catalyst.

In the case of the above carboxyl-protected derivatives such as, for example, benzyl ester and D-2-amino-3-fluoropropionamide (and nitrile), it is preferable to use an aqueous acid hydrolysing agent such as aqueous hydrochloric acid and the like as a cleavage agent.

5,57745

The present invention also encompasses the resolution of 3-fluoro-DL-alanine, its deutero analogs and amino-protected derivatives using optically active bases such as D + L-oHThenethylamine, quinine, brucine, quinidine, cinchonine, L-lysine, cinquinidine, strychnine, , morphine, 1-menthylamine, L-arginine, d-2-amino-1-hydroxyhydrindene, ephedrine, abiethylamine, amphetamine, D + L-threo-1-p-nitrophenyl-2-amino-1,3-propanediol, D + L-threo-1-phenyl-2-amino-1,3-propanediol, D + LN-dimethyl-o-phenethylamine, de-hydroabiethylamine, D + L-1- (1-naphthyl) ethylamine, D + L -1- (2-naphthyl) -ethylamine, D + L-tyrosine hydrazide, D + L-α-phenylamine, D + L-threo-2,2-dimethyl-U-phenyl-5-amino-1,3 -dioxane, optically active basic polymers such as dimethyl- (1-phenethyl) -polystyrene-divinylbenzene quaternary ammonium compound, as well as 3-fluoro-DL-alanine, its deutero analogues and its carboxyl-protected derivatives using optically active acids such as d + 1-camphor-10-sulfonic acid, 3-bromocamphor-8-sulfonic acid, D + L-tartaric acid, D + L-camphor-10-sulfonic acid, 3-bromocamphor-8-sulfonic acid, D + L-dibenzoyltartaric acid, D + L-ditoluoyltartaric acid, D + L-tartranilic acid, D + LN- (1-phenethyl) succinic acid, D + LN- (1-phenylethyl) phthalamic acid, N-acetylalanine -acetyl-leucine, pyroglutamic acid, glutamic acid, 1-methoxyacetic acid, mandelic acid, malic acid, LN-acetyldibromothyrosine, D + LN-acetyl-o <-methyl-3, U-dimethoxyphenylalanine, D + LN-acetyl N-methylenedioxyphenylalanine and the like.

The racemic compound 3-fluoro-D, L-alanine is a known compound described by Lettre et al., Ann. Chem. 708, 75 ”Ö (1967). However, optical isomers have not been described prior to this invention and the anti-bacterial activity of the racemate has not been studied.

- Surprisingly, it was found that only specific in vitro antibacterial testing methods showed the inhibitory effect of 3-fluoro-D-alanine. In this case, it was possible to show that the antibacterial activity of the optical isomers is equivalent in vitro. And yet, in vivo, the isomers differ considerably. The D-isomer retains its antibacterial properties and protects * the infected animal, while the L-isomer appears to lack the ability to protect bacterially infected animals and is indeed lethal to such animals when used only in moderate doses.

The novel compounds of the present invention and their salts are antibacterial agents useful in inhibiting the growth of pathogenic bacteria of both gram-positive and gram-negative genera, such as Streptococcus, Escherichia, Staphylococcus, Salmonella, Pseudomonas, Diplococcus, Klebsiella, Proteus, Mycobacterium, Vibrio, Pasteurella and Serratia, as well as their antibiotic-resistant species. Typical such pathogens are Escherichia coli, Salmonella 3chottmueller,

Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. These new compounds and their salts can be used as antiseptics to remove suspicious organisms from pharmaceutical, dental and medical devices, and can also be used in other fields that are susceptible to infection by such an organism.

The novel D-isomers and their salts are also valuable in the treatment of diseases caused by the above organisms in humans and animals and can be administered in a variety of therapeutic doses in conventional carriers, for example orally in capsule or tablet form or dissolved or suspended in a liquid. Suitable formulation formulations include diluents, granulating agents, preservatives, binders, flavors, and coatings well known to those skilled in the art, and the dosage of the products can be varied widely, with an upper limit of the autoantagonic threshold for 3-fluoro-D-alanine in the becomes the target of the expected antibacterial immediate effect. Doses may, for example, be from about 250 mg to about 500 mg of active ingredient 2-U times a day in the case of an adult person weighing TO kg. When growth-enhancing therapy is given to growing animals in the feeding group, they should receive approximately 100 g / ton of complete diet. Alternatively, they may be administered parenterally by injection in an inert carrier.

The uniqueness of the in vitro antibacterial assay referred to above is due to the unusual property of 3-fluoro-D-alanine to be autoantagonistic at high concentrations. Therefore, standard antibacterial screens designed to recover activities of very low order of magnitude, and in cases where relatively high concentrations of test compound are used, may lose 3-fluoro-D-alanine activity.

7 57745 This autoantagonistic property does not detract from its usefulness, as it only occurs in, for example, in vivo experiments at doses greater than about 10 times the average effective amount of the healing agent.

When 3-fluoro-L-alanine is rapidly and completely metabolized with toxic consequences, 3-fluoro-D-alanine undergoes much slower degradation in vivo. A potential contributing factor is the enzyme D-amino acid oxidase, which is abundant in humans and rats, but to a much lesser extent in mice and some domestic animals that eat plants. However, the rate of metabolism does not, as it may affect the amounts of therapeutic agent in the blood, and the by-products of this metabolism do not counteract the use of the compound at the dosage level intended for humans or animals.

In addition, it has been found that the deutero forms are significantly less sensitive to the action of D-amino acid oxidase and still retain the antibacterial activity of the parent compound. In this case, the in vivo activity increases and is maintained.

Efficacy of 3-fluoro-D-alanine compared to other antibiotics in the treatment of infected mice.

Female C.D. 1 mice weighing an average of 21.0 g was injected intraperitoneally with 0.5 ml of a suitably diluted 16-hour broth culture containing the pathogens in question (the severity of the infection risk is represented in each case by a multiple dilution of the broth culture, which must be killed for only $ 50 untreated test). animals, which means the LD ^ Q dose). The therapist was rapidly administered a volume of 0.5 mm in one of the following ways: either subcutaneously on the dorsal surface (S.C.), intraperitoneally (i.P.), or by gavage (orally, P.O.). In the case of mice infected with Streptococcus or Diplococcus, a second dose of therapist was administered 6 hours after infection. The results of these experiments are expressed as the statistically interpolated dose required to protect 50% of infected experimental animals for 7 days after infection and after treatment, (deaths for treatment deficiency and non-medicated control animals occurred in all cases during the first 3 days.) Abbreviations for control compounds are as follows: 3FDA = 3-fluoro-D-alanine CYC * D-cycloserin TET = tetracycline CLM = chloramphenicol.

8 57745

Organism Therapeutic efficacy (ED 2) mg

Method of administration 3FDA CYCLO TET CLM

Escherichia coli 2017 I.P. 0.331 --- 0.008 0.01 + 0 7-io ld5Q s.c. 0.361 2.50 0.0Wt 0.52 P.0, 0.377 2.36 0.600 0.1 + U6

Klebsiella pneumoniae "B" S.C. 0.162 0.332 0.190 0.31 + 0 10-30 ld5Q P.O. 0.109 1.51 1, 1 + 2 0.1 + 1 + 0

Pseudomonas aeruginosa S.C. 0.897 --- 2.1+ 7.66 26.6 9 “50

Streptococcus pyogenes S.C. (x2) 0.023 1.25 0.02 0.71 3009 »“ 50

Staphylococcus aureus S.C. 0.538 0.726 0.0U6 1.5 2919 3 “50

Diplococcus pneumoniae S.C. (x2) 0.10U> 10 0.081+ 1, 780 1-37 ™ 50

Salmonella schottmuelleri P.O. 0.291+ 2.5 1, 217 0.1 + 1 + 0 30.0

Significant superiority in efficacy in the treatment of various bacterial infections is achieved with 3-fluoro-D-alanine. In particular, when treated orally, 3-fluoro-D-alanine produces a degree of potency equal to or greater than that of the antibiotics Chloramphenicol and tetracycline used.

Comparison of safety and efficacy of 3-fluoro-L-alanine (3-FLS) and 3-fluoro-D-alanine (3FDA) in mice

Groups of 5 female CD1 mice with a mean weight of 21 g were used either as uninfected experimental animals or as targets of antibacterial therapy given by intraperitoneal injection of 0.5 ml of a diluted broth culture of Escherichia coli 2017, representing 7 LDtjo bacterial pathogens. Therapist was administered at the indicated doses in a volume of 0.5 ml orally in each case, 9,57745

$: Number of surviving animals

Dose mg Non-infected control mice Infected mice

3-FLA 3-FDA 3-FLA 3-FDA

0 100 100 00 0.078 - 00 0.312 100 100 0 20 1,250 100 100 0 100 5 0 100 0 80 20 100 - -1 * 0 - 100 - -

The tolerance of mice to 3-FDA exceeds that of 3-FLA by at least a factor of 10. Below the maximum tolerated dose, 3-FLA has no therapeutic effect. In contrast, reasonable doses of 3-FDA were completely protective, with higher amounts indicating autoantagonism in vivo as well as in vitro.

Effect of 3 ~ FDA and 3-FLA concentration gradients on bacterial growth reflecting autoantagonism

1 * was applied on top of a 2 mm thick layer of nutrient agar in a petri dish. 2 10 organisms / cm Escherichia coli 2017 culture followed by paper discs 7 mm in diameter carrying the indicated amounts of 3-FDA or 3-FLA, after 16 hours of incubation at 37 ° C , circular zones free of bacterial growth surrounding the discs were measured. It is unique that in the case where 3-FDA was used, and even then only with large amounts of drug, a growth ring associated with the paper disc was observed, followed by a bacterial-free agar ring. The inner growth zone, specifically in the high concentration range of the concentration gradient provided by the diffusible drug, is shown as the zone of autoantagonism.

S

\ '· 10 57745

Diameter of the zones around the discs, e.g.

Quantity on disc 3 ~ FLA 3-FDA

} ig prevention autoanthesion prevention autoantagonism gonism 25 2b 0 30 0 50 28 0 33 11 100 32 0 35 13 200 3b 0 38 15 1 + 00 37 0 1 + 1 17

The following examples illustrate the method of the invention.

Example 1

To a solution of 20 g of 3-fluoro-DL-alanine in 200 ml of water and 56 ml of 2.5 N sodium hydroxide solution is added 62.0 g of carbobenzoxychloride in five portions every 15 minutes while stirring vigorously and cooling in an ice bath. Stirring and cooling are continued for a further 3 hours. During the reaction, the pH is maintained at 10-11 by adding 2.5 N aqueous sodium hydroxide solution. The reaction mixture is extracted with ethyl acetate and the extract is discarded. The aqueous phase is adjusted to pH 2 with dilute aqueous hydrochloric acid and the resulting solution is extracted several times with ethyl acetate. The ethyl acetate extracts are dried over anhydrous magnesium sulfate and evaporated to dryness. The residue is crystallized from 1 + 00 ml of moist carbon tetrachloride and dried to give about 33 g of N-carbobenzoxy-3-fluoro-DL-alanine.

A mixture of 22.3 g of L-tyrosine hydrazide and 2 + +, 1 g of N-carbobenzoxy-3-fluoro-DL-alanine is dissolved while heating in a 50% aqueous solution of ethanol and the solution is evaporated to dryness under reduced pressure to give diastereoisomeric L- a mixture of tyrosine hydrazide salts which is then separated by partitioning from ethanol.

The L-tyrosine hydrazide-N-carbobenzoxy-3-fluoro-D-alanine salt is suspended in dilute hydrochloric acid and the suspension is changed several times with ethyl acetate. The ethyl acetate is evaporated to give N-carbobenzoxy-3-fluoro-D-alanine.

xioin 1 g of N-carbobenzoxy-3-fluoro-D-alanine is dissolved in 50 ml of methanol and the resulting solution is reacted with hydrogen at atmospheric pressure in the presence of 1 g of 5% palladium-on-carbon catalyst. The catalyst is filtered off and the methanol is evaporated from the filtrate in vacuo. The residue is crystallized from water to give substantially pure 3-fluoro-D-alanine.

Example 2

To a rapidly stirred solution of 20.1 g of 3-fluoro-DL-alanine in 200 ml of water and 56 ml of 2.5N aqueous sodium hydroxide solution is added dropwise 28.0 g of levorotatory menthoxyacetyl chloride. An additional 2.5N aqueous sodium hydroxide solution is added to maintain the pH in the range of 10-11. The reaction solution is then slowly poured into a dilute aqueous solution of hydrochloric acid containing ice. The precipitated material is collected by filtration, and the mixed diastereoisomers thus obtained are separated by fractional crystallization from aqueous ethanol.

The N-1-menthoxyacetyl-3-fluoro-D-alanine stereoisomer thus obtained is heated under reflux in a 6N aqueous hydrochloric acid solution containing a small amount of ethanol for solution purposes. After refluxing for 18 hours, the solution is partially evaporated and extracted twice with ether. The aqueous solution is then evaporated to dryness to remove excess hydrochloric acid. The remaining 3-fluoro-D-alanine hydrochloride is dissolved in as little water as possible and the pH is adjusted to 1, 8 by adding concentrated aqueous ammonium hydroxide solution. The resulting mixture is cooled to 0 ° C and optically pure 3-fluoro-D-alanine is recovered by filtration.

Alternatively, 3-fluoro-D-alanine hydrochloride is dissolved in water and the solution is passed through a strong acidic cation exchange resin (H + step). The column is washed with water to remove chloride ions; the column is eluted with dilute aqueous ammonium hydroxide solution and the eluate is evaporated, whereupon 3-fluoro-D-alanine precipitates and is removed by filtration and dried.

Example 3

A suspension of 20.7 S of 3-fluoro-DL-alanine in 200 ml of benzyl alcohol a is heated to 100 ° C while bubbling dry hydrogen chloride through the mixture. The reaction mixture is kept at 100 ° C until a clear solution is obtained. The benzyl alcohol is then evaporated off under reduced pressure and the residual oil is dissolved in 50 ml of water and 200 ml of methylene chloride are added. Add solid sodium carbonate while stirring until the effervescence ceases.

12,57745

The methylene chloride layer is separated and washed with water. The methylene chloride layer is then evaporated in vacuo to give 3-fluoro-DL-alanine benzyl ester as a viscous oil.

About 10 g of the benzyl ester of 3-fluoro-DL-alanine prepared above and 9.0 g of dibenzoyl-D-tartaric acid are combined in 100 ml of ethanol and the mixture is slightly heated to obtain a solution. The solution is then evaporated to dryness. The residue is dissolved in as little hot ethanol as possible and the mixed diastereoisomers are separated by fractional crystallization.

The dibenzoyl-D-tartaric acid salt of 3-fluoro-D-alanine benzyl ester thus obtained is added to a dilute aqueous hydrochloric acid solution, and the resulting mixture is extracted five times with 25 ml portions of methylene chloride to extract dibenzoyl-d-tartaric acid from an aqueous acid solution containing 3-fluoroacid. Benzyl ester of D-alanine hydrochloride. The aqueous acid solution is then heated to boiling for 5 hours, whereupon the benzyl ester hydrolyzes (decomposes). The reaction solution is cooled and then extracted with methylene chloride. The aqueous solution is evaporated to dryness to remove excess hydrochloric acid. The remaining 3-fluoro-D-alanine hydrochloride is dissolved in as little water as possible, the pH is adjusted to 8 by adding concentrated aqueous ammonium hydroxide solution, and then the precipitated 3-fluoro-D-alanine is collected by filtration.

Alternatively, after extracting dibenzoyl-D-tartaric acid, an aqueous acidic solution of the benzyl ester of 3-fluoro-D-alanine is hydrogenated using 1.0 g of 10 2 palladium on carbon to give 3-fluoro-D-alanine hydrochloride, which is converted to the free amino acid 3 "fluoro-D-alanine by treatment with concentrated aqueous ammonium hydroxide as described above.

Example k

About 39 x 8 6 dibenzylamine is added to a solution of about 17.1 g of 2-bromo-3-fluoropropionic acid in 50 ml of isopropanol. The mixture is allowed to stand at 25 ° C for 5 hours and then heated to reflux for a further 3 hours. The isopropanol is evaporated in vacuo and 200 ml of a 10% aqueous solution of sodium carbonate are added to the residue. The aqueous solution obtained is extracted five times with 100 ml portions of ether. The aqueous layer is then acidified with acetic acid and extracted twice with 50 ml portions of chloroform. The chloroform is evaporated in vacuo and the residue is crystallized from cyclohexane to give N, N-dibenzyl-3-fluoro-DL-alanine.

About 5.7 g of N, N-dibenzyl-3-fluoro-DL-alanine and 1.2 g of D (-) - threo-1β-nitrophenyl-2-aminopropane-1,3-diol are combined in 150 ml of hot 50 ml of ethanol. »Aqueous solution to form a mixture of diastereoisomeric salts. Partitioning of 13,57745 from aqueous ethanol gives the D (-) - threo-1-p-nitrophenyl-2-aminopropane-1,3'-diol salt of N, N-dibenzyl-3-fluoro-D-alanine. The salt is purified by recrystallization from a 50 L aqueous solution of ethanol.

2 g of the D (-) - threo-1β-nitrophenyl-2-aminopropane-1,3-diol salt of N, N-dibenzyl-3-fluoro-D-alanine are treated with 10 ml of 10% aqueous sodium carbonate solution and the precipitated D (-) - threo-1β-nitrophenyl-2-aminopropane-1,3-diol is filtered off. The filtrate is acidified with acetic acid and N, N-di-benzyl-3-fluoro-D-alanine is extracted into chloroform; evaporation of the chloroform extract gives N, N-dibenzyl-3-fluoro-D-alanine.

A solution of 1.0 g of N, N-dibenzyl-3-fluoro-D-alanine in 10 ml of ethanol and 10 ml of 6N hydrochloric acid is hydrogenated at 2.8 kg / cm using 1.0 g of 10%: palladium on carbon. When the absorption of hydrogen is complete, the catalyst is filtered off and the filtrate is evaporated to dryness to give the hydrochloride salt of 3-fluoro-D-alanine. This salt is dissolved in water and the aqueous solution is vigorously passed through the naphtha cation exchange resin (H + step). The column is washed with water to remove chloride ions and then the column is eluted with dilute aqueous ammonium hydroxide. The resulting eluate is evaporated to remove ammonia and the precipitate is collected and dried to give 3-fluoro-D-alanine.

Example 5

To a solution of 2 g of 3-fluoro-DL-alanine in 20 ml of water and 5.6 ml of 2.5N aqueous sodium hydroxide solution are added in five portions at intervals of 15 minutes about 6 g of carbobenzoxy chloride while stirring vigorously and cooling in an ice bath. Stirring and cooling are continued for a further 3 hours. During the reaction, the pH is maintained in the range of 10-11 by the addition of 2.5N aqueous sodium hydroxide solution. The reaction mixture is extracted with ethyl acetate and the aqueous phase is adjusted to pH 2 with dilute aqueous hydrochloric acid. The aqueous acidic solution is then extracted with ethyl acetate several times and the extracts are combined, dried over anhydrous magnesium sulphate and evaporated to dryness. The residue is crystallized from 10 ml of moist carbon tetrachloride and dried to give about 3 g of ✓ H-carbobenzoxy-3-fluoro-DL-alanine.

To a solution of 0.150 g of N-carbobenzoxy-3-fluoro-DL-alanine in 5 ml of ethyl acetate is added 0.09 U g of 1-6-phenylethylamine. The resulting mixture is evaporated to dryness, the residue is dissolved in 1 ml of ethyl acetate and the resulting mixture is fractionally crystallized to give the 1-o-phenylethylamine salt of N-carbobenzoxy-3-fluoro-D-alanine. About 0.100 g of this salt is dissolved in water, the solution is acidified to pH 2, the acidic solution is extracted with ethyl acetate and the ethyl 1,1,57745 acetate solution is evaporated to dryness to give N-carbobenzoxy-3-fluoro-D-alanine.

0.255 g of N-carbobenzoxy-3-fluoro-D-alanine are dissolved in 10 ml of methanol and 0.3 g of 5% palladium on carbon is added. Hydrogen gas is bubbled through the mixture at room temperature for 12 hours. The catalyst is removed by filtration and the filtered solution is evaporated to dryness. The residue is crystallized from water-isopropanol to give 3-fluoro-D-alanine.

Example 6

About 12 .mu.g of freshly prepared fluoroacetaldehyde is slowly distilled into 2 *, 3 g of D (+) -? -Ethylbenzylamine, which is cooled in an ice-methanol bath. About 50 g of anhydrous calcium sulfate is added to the mixture, and the resulting mixture is stirred overnight at room temperature. About 200 ml of ether are added and the calcium sulphate is filtered off. The ether filtrate is then evaporated in vacuo, the remaining N- (D- (E-methylbenzyl) -6-fluoroacetaldimine is dissolved in anhydrous ethanol, the ethanolic solution is cooled to -10 ° C and about 7.9 ml of hydrogen cyanide are added. about 200 hours and the ethanol is evaporated in vacuo to give 2- (Dimethylbenzylamino) -3-fluoro-D-propionitrile About 200 ml of cold concentrated aqueous hydrochloric acid are added to the cooled residual nitrile, the mixture is stirred overnight at room temperature, then heated overnight. Excess hydrochloric acid is removed from the residue by adding 100 ml of t-butanol and evaporating to dryness to give the hydrochloride salt of N- (Dimethylbenzyl) -3-fluoro-D-alanine, which is dissolved in absolute ethanol, dissolved in ethanol. and 16 ml of propylene oxide are added to liberate the amino acid from the hydrochloride salt; allow to stand at room temperature overnight to complete the reaction to give N- (D-T3-methylbenzyl) -3-fluoro-D-alanine, which is collected by filtration, washed with ethanol and crystallized from aqueous ethanol.

N- (D *? -Methylbenzyl) -3-fluoro-D-alanine is dissolved in ethanol 2 containing an equivalent amount of hydrogen chloride and hydrogenated at a pressure of 2.0 kg / cm in the presence of 10> palladium hydroxide on carbon, whereby «( The catalyst is filtered off and the filtrate is evaporated to dryness to give 3-fluoro-D-alanine hydrochloride, the remaining hydrochloride salt is dissolved in as little water as possible and the pH is adjusted to 0.8 with concentrated aqueous ammonium hydroxide solution. cooled to 0 ° C and the optically pure 3-fluoro-D-alanine is separated therefrom by filtration.

57745 15

Alternatively, the hydrochloride salt of 3-fluoro-D-alanine is dissolved in water and passed through a strongly acidic cation exchange resin (H + step). The column is washed with water to remove chloride ions and the column is eluted with dilute aqueous ammonium hydroxide. Upon evaporation, a precipitate forms which is collected by filtration and dried to give 3-fluoro-D-alanine.

Example 7

A cold solution of 12.1 g of D - *> fc: methylbenzylamine in 100 ml of ethanol is slowly added to a solution of 5-3 g of fluoropyruvic acid in 250 ml of ethanol, while maintaining the resulting solution at 0 ° C. The solution is allowed to warm ...... ........ . . To 25 ° C and maintained at this temperature for 1 hour. A solution containing 2- (N, N-methylbenzylimino) -3-fluoropropionic acid is then reacted with hydrogen at a pressure of 2.8 kg / cm 2 to 0 g of 10? Palladium on carbon, whereby hydrogenation is continued. until hydrogen absorption ceases. The catalyst is removed by filtration and the filtrate is evaporated to half volume. About 100 ml of water are added to form an aqueous ethanol solution containing N- (D-> 3-methylbenzyl) -3-fluoro-D-alanine, and the methylbenzyl group is hydrogenated at 2.8 kg / cm using 5> 0 g of 10 J » of palladium hydroxide on carbon. When the absorption of hydrogen is complete, the catalyst is removed by filtration, the filtrate is evaporated to dryness in vacuo and the residue is recrystallized from 50 ml of isopropanol-water and then from water to give essentially pure 3-fluoro-D-alanine.

Example 8

A cold solution of 12.1 g of D-o-methylbenzylamine in 100 ml of ethanol is slowly added to a solution of 5> 3 g of fluoropyruvic acid in 250 ml of ethanol, while maintaining the resulting solution at 0 ° C. The solution is allowed to warm to 25 ° C and maintained at this temperature for 1 hour. A solution of 2- (dimethylbenzylimino) -3-fluoropropionic acid is then reacted with deuterium at 2.8 kg / cm using 5.0 g of 10 palladium on carbon, whereby hydrogenation is continued until the absorption of deuterium ceases. The catalyst is removed by filtration and the filtrate is evaporated to half volume. About 100 ml of water is added to form an aqueous ethanol solution containing N- (D-γ-methylbenzyl) -2-deutero-3-fluoro-D-alanine, and the methylbenzyl group is hydrogenated with carbon. When the absorption of hydrogen is complete, the catalyst is removed by filtration, the filtrate is evaporated to dryness in vacuo and the residue is recrystallized from 50% isopropanol and then from water to give essentially pure 2-deutero-3-fluoro-D-anine.

16 57745

Example 9

About 0.1 millimoles of asymmetric diphosphine-rhodium catalyst (prepared as described in Chem. Comm 1971 p. U81) is dissolved in 50 ml of benzene-ethanol (1: 2), and 11.0 g of eC-benzamido is added to the solution. -γ-fluoro-2 acrylic acid. The mixture is reacted with hydrogen at a pressure of 7 kg / cm and a temperature of 25 ° C. The reaction solution is evaporated to dryness and the remaining substance is dissolved in chloroform. The chloroform extract is washed with water, treated with carbon, filtered and evaporated to dryness to give N-benzoyl-3'-fluoro-D-alanine. A concentrated aqueous hydrochloric acid solution is added and the mixture is heated under reflux for 8 hours, whereupon the benzoyl substituent is hydrolyzed.

The solution obtained is evaporated to dryness and the residue is dissolved in as little water as possible. The aqueous solution is adjusted to pI with 0.8 M concentrated ammonium hydroxide solution, cooled and the resulting precipitate is collected by filtration to give crude 3-fluoro-D-alanine, which is stirred with 5 volumes of water at 25 ° C for 2 h. Filtration gives substantially pure 3-fluoro-D-alanine.

Example 10

About 0.1 millimoles of asymmetric diphosphine-rhodium catalyst (prepared as described in Chem. Comm 1971 p. H81) is dissolved in 50 ml of benzene-ethanol (1: 2), and 11.0 g of otrbenzamido- »-fluoro-acrylic acid. The mixture is reacted with deuterium at a pressure of 7 kg / cra and a temperature of 25 ° C. The reaction solution is evaporated to dryness and the remaining substance is dissolved in chloroform. The chloroform extract is washed with water, treated with charcoal, filtered and evaporated to dryness to give N-benzoyl-2,3-dideutero-3-fluoro-D-alanine. A concentrated aqueous hydrochloric acid solution is added and the mixture is heated under reflux for 8 hours, whereupon the benzoyl substituent is hydrolyzed. The solution obtained is evaporated to dryness and the residue is dissolved in as little water as possible. The aqueous solution is adjusted to pH h, 8 with concentrated aqueous ammonium hydroxide solution, cooled and the precipitate obtained is collected by filtration to give crude 2,3 "dideutero-3-fluoro-D-alanine, which is mixed with 5 volumes of water at 25 ° C for 2k. hours. Filtration gives substantially pure 2,3-dideutero-3'-fluoro-D-alanine.

Example 11

About 50 g of dimethyl methylene malonate (2-methoxycarbonylalkyl acid methyl ester) are added to 200 ml of anhydrous hydrogen fluoride at a temperature below -20 ° C. The hydrogen fluoride is evaporated and 500 ml of ether are added. The ether solution is washed with ice water, dried and filtered. The filtrate is evaporated to dryness and the residual oil is distilled in vacuo to give a substantially pure dimethyl ester of 3-fluoromethyl-malonic acid (methyl-2-methoxycarbonyl-3-fluoro-propionate).

Exactly 16 .mu.g of this methyl 2-methoxycarbonyl-3-fluoropropionate are dissolved in 100 ml of methanol, 10 ml of aqueous sodium hydroxide solution are added and the temperature is maintained at 0 [deg.] C. to give an aqueous methanolic solution containing methyl 2-carboxy- Sodium salt of 3-fluoropropionate. This solution is neutralized with dilute aqueous hydrogen chloride, extracted with chloroform, and the chloroform solution is dried and evaporated in vacuo to give methyl 2-carboxy-3-fluoropropionate.

About 15 g of this compound are dissolved in methanol-benzene, a stoichiometrically equivalent amount of L-o-methylbenzylamine is added and the diastereoisomeric salts of methyl 2-carboxy-3-fluoropropionate obtained are separated by fractional crystallization. The L-β-methylbenzylamine salt of methyl S-2-carboxy-3-fluoropropionate thus obtained is reacted in aqueous solution with exactly one stoichiometrically equivalent amount of aqueous hydrochloric acid, the aqueous solution obtained is extracted with chloroform and the chloroform extract is dried and evaporated in vacuo. To obtain 3-fluoropropionate.

About 3.0 g of methyl 3-2-carboxy-3-fluoropropionate are dissolved in 25 ml of acetone and the solution is treated at 0 [deg.] C. first with 2.02 g of triethylamine and then with an excess of ethyl chloroformate. To the resulting mixture is added 10 ml of water, and the solution is stirred at 0 ° C for about 30 minutes to form an aqueous acetone solution containing the corresponding carboxyethoxy ester mixed anhydride of methyl S-2-carboxy-3-fluoropropionate. To this solution is added 1.3 g of sodium azide and the mixture is stirred for a further 2 hours at -5 to 0 ° C. The reaction mixture is poured into ice-cold saturated aqueous sodium chloride solution and the mixture is extracted several times with ether. The ether solution containing methyl S-2-azido-carbonyl-3-fluoropropionate is dried over anhydrous sodium sulfate and filtered and 100 ml of dioxane are added. The ether is then evaporated and the resulting dioxane solution is gently heated to boiling point (behind the barrier), after which the azide decomposes and regroups to form methyl 5-2-isocyanato-3-fluoropropionate, i. H-carbonyl-3-fluoro-D-alanine-metyylies-ester.

To this isocyanate are added 100 ml of water and 50 ml of an aqueous hydrochloric acid solution, and the mixture is heated under reflux for 2 hours, during which time both the isocyanate and raethyl ester groups are hydrolyzed. The aqueous reaction solution is evaporated in vacuo to give crude 3-fluoro-D-alanine hydrochloride (S-3-fluoroalanine hydrochloride).

The hydrochloride salt of 3-fluoro-D-alanine is dissolved in water and the solution is passed through a strongly acidic cation exchange resin (H + step). The column is washed with water to remove chloride ion and the column is eluted with dilute aqueous ammonium hydroxide. The eluate is evaporated and the precipitate is collected by filtration, dried and crystallized from 50 n / a aqueous isopropanol to give essentially pure 3-fluoro-D-alanine (S-3 'fluoroalanine).

Alternatively, if methyl S-2-azidocarbonyl-3-fluoropropionate is decomposed in ethanol solution, the corresponding ethyl urethane of 3-fluoro-D-alanine methyl ester (ethyl urethane of 3-3-fluoroalanine methyl ester) is formed. The latter is then reacted with aqueous hydrochloric acid to form 3-fluoro-D-alanine hydrochloride, which is converted to 3'-fluoro-D-alanine as described above.

Optically active compounds in this example are sometimes denoted by "R" and "3" in the IUPAC Tentative Rules for the Nomenclature or Organic Chemistry; Section E Fundamental Stereochemistry, J. Org. Chem 35 s.

2063-7 (1970).

Similarly, the carboethoxy ester mixed anhydride of methyl S-2-carboxy-3-fluoropropionate is reacted with ammonia (instead of sodium azide) to form methyl S-2-aminocarbonyl-3-fluoropropionate, which is then reacted with sodium hypobromide to give the amido group. regroups to form methyl S-2-isocyanato-3-fluoropropionate using dioxane as solvent and ethyl urethane corresponding to 3-fluoro-D-alanine when using ethanol as solvent, i.e. N-ethoxycarbonyl-3-fluoro-D-alanine.

Alternatively, and according to the method described above, 2-cyanoacrylic acid is reacted with hydrogen fluoride to form 2-carboxy-3-fluoropropionitrile, which is separated into its stereoisomers using Ly-methylbenzylamine as a disintegrant to form R-2-carboxy-3 * fluoropropionitrile, which is converted to a mixture. anhydride and the latter is reacted with sodium azide as described above to form R-2-azidocarbonyl-3-fluoropropionitrile. This is heated to decompose the azide to form S-2-isocyanato-3'-fluoropropionitrile, which is then heated to reflux with aqueous hydrochloric acid (such as the hydrolysis of methyl S-2-isocyanato-3-fluoropropionate) to give both the isocyanato that the nitrile group is hydrolyzed to form 3-fluoro-D-alanine hydrochloride.

19 5 774 5

Alternatively, optically active D-2-amino-3-fluoropropiononitrile compounds are obtained by resolution of the corresponding DL-2-amino-3-fluoropropionitrile compound. For example, DL-2-amino-3'-fluoropropionitrile itself is decomposed using an optically active disintegrant such as tartaric acid, camphor-10-sulfonic acid and the like to form an optically active salt of D-2-amino-3'-fluoropropionitrile which, when treated with aqueous with ammonia converts to the free D-2-amino-3-fluoropropionitrile, which is extracted from aqueous solution with ether. D-2-amino-3-fluoropropionitrile is then reacted at a temperature below 0 ° C with concentrated (50% HCl) hydrochloric acid to form D-2-amino-3-fluoropropionamide. It is heated with concentrated aqueous hydrochloric acid under reflux to form the 3'-fluoro-D-alanine hydrochloride salt.

Various changes and modifications may be made to the present invention without departing from the basic idea of the invention.

e * 'j * ·

Claims (2)

A process for the preparation of a therapeutically useful 3-fluoro-D-alanine or deutero analogue thereof, having the formula I2 I1 F - C - C - COOH I '1 H 3 NH2 wherein R1, R8 and R3 are hydrogen or deuterium , characterized in that a) a mixture of salts of 3-fluoro-DL-alanine of the formula I or a deutero analogue thereof and an optically active base is subjected to fractional crystallization to obtain a salt of 3-fluoro-D-alanine in a substantially pure form, and that the salt is treated with a strong acid or b) a mixture of salts of 3-fluoro-DL-alanine of formula I or a deutero analogue thereof and an optically active acid is subjected to fractional crystallization to give a salt of 3-fluoro-D-alanine in substantially pure form, and that the salt treated with a strong base to liberate the compound of formula I from its salt, or c) 3-fluoro-DL-alanine of formula I or its d a mixture of the amino-protected derivative of the eutero analogue and the salts of the optically active acid is subjected to fractional crystallization to give the corresponding amino-protected 3-fluoro-D-alanine salt in substantially pure form, the resulting salt is treated with a strong acid to liberate the amino-protected derivative of the compound of formula I and its salt. finally cleaved off · or d) from 3-fluoro-D-alanine or an amino-protected derivative of a deutero analogue of formula RP R, R 1, R 8 and R 3 are as defined above, and R 1 is carbobenzoxy, benzoyl, chloroacetyl or menthoxyacetyl, the protecting group R 4 is cleaved off by hydrogenation or hydrolysis. e) 3-fluoro-D-alanine or an amino-protected support derivative thereof deutero analogue of the formula 21 57745 RP R, t2 i1 F - C - C - COOH II wherein R 1, R 2 and R 2 are as defined above, and either R 1 is hydrogen and Rg are carbobenzoxy, benzyl, ofH-methylbenzyl or N-carboxybenzyl, or R1 and R6 are each benzyl, the protecting group Rg is cleaved by catalytic hydrogenation, or f) protection of 3-fluoro-D-alanine or its deutero analogue carboxyl. a derivative of the formula * 2 «1 R - C - C - COR7: wherein R 1, R 8 and R 2 are as defined above, and R 2. is an ester, amide or cyano group, the protecting group Ry is cleaved off by hydrolysis. 22 57745 For the preparation of therapeutically active compounds with 3-fluoro-D-Alanine or a deutero analog, one of which is a compound of the form * 2? 1 FC - C-COOH R3 Figure a) Bleaching of 3-fluoro-DL-Alanine with formula I or a deutero analogue and an optical salt of the active base underlying crystal crystallization, for example, the site of 3-fluoro-D-Alanine is practically tagged. form, with or without the use of a starch in the form of a mixture with a formulation I or a site, or b) and a mixture of 3 “fluoro-DL-Alanine with the formulation of a deuteroanalog and an optically active a crystalline fraction of the underlying crystal is formed, for example, the image of the site of 3-fluoro-D-Alanine is practically tagged in the form, and the salt is treated with the residue from the site of formation of the site, or (c) is blended 3-Fluoro-DL-Alanine is formulated with amino acids Derived from the deuteroanalog and the salt of the optically active crystal underlying crystallization, for the image and the molten amino-amino acid site of the 3-fluoro-D-Alanine is practically tagged in the form, and when the salt is treated with the starch the addition of amino acids to the derivatives of formula I is carried out in the form of salt, and the amino acid groups are derived from the formulation, or d) the amino acids of the derivatives are derived from 3-fluoro-D-Alan or a deutero analogue, which is derived from the formula R2 R1 I * J 1 FC - C-COOH R3 NHRu of Rj, Rg and R ^ har ovannämnda betydelse, och R ^ är carbobenzoxy, benzoyl, chloroacetyl or menthoxyacetyl, open group of the R4 genome hydration or hydrolysis, or