IE46983B1 - Fluorinated amino acids - Google Patents

Abstract

Novel alpha -fluoromethyl- alpha -amino acids and esters have the following formula in which R is a substituted C1-C4-alkyl group and R1 has the meaning of H or C1-C18-alkyl. The compounds can be present as acid addition salts. The compounds of the formula I, as well as their pharmaceutically acceptable acid addition salts, are used as active compounds in medicaments which have a decarboxylase-inhibiting effect. Processes for preparing the novel compounds are described.

Description

The present invention is concerned with substituted α-fluoromethyl-a-amino alkanoic acids.

An unsubstituted α-fluoromethyl-a-amino alkanoic acid, namely 2-fluoromethylalanine, which has the formula: CH,F ( & CH3 - C - COOH nh2 (A) is known /Kollonitsch et al, J. Org. Chem. 40, 3808-9 (1975// No specific biological activity for this compound is suggested. This compound (A) is prepared by fluorodehydroxylation of the corresponding 2—hydroxymethylalanine. α-Methyl amino acids, such a L-a-methyl-3,4-dihydroxyphenylalanine (α-methyldopa, an antihypertensive agent), are known to have decarboxylase-inhibiting activity (Goodman, et al., The Pharmacological Basis of Therapeutics, MacMillan Company, New York, New York 1970, p. 577; Canadian Patent 737,907).

The present invention is based on the discovery of certain novel substituted α-fluoromethyl-a-amino alkanoic acids, at least some of which have decarboxylase-inhibiting activity significantly greater than that of α-methyl amino acids.

The present invention provides compounds having the formula: CH t R - C - COOR1 (I) I nh2 where R is a substituted alkyl group and R^ is H or C^-C^g alkyl. Ccmpounds that are jharmaceutically acceptable acid4 6 9 8 3 addition salts of the formula I compounds are also included. In general, the salts are those of the formula I base with a suitable organic or inorganic acid. Preferred inorganic acid salts are the hydrohalides, e.g., hydrochlorides, hydroiodides and hydrobromides? the sulfates, and the phosphates. The hydrohalides, and especially the hydrochlorides, are especially preferred.

The formula I compounds have a chiral center and may occur in optically active forms i.e., as optical isomers. These isomers are designated conventionally by the symbols L and D, + and -, 1 and d, S and R or combinations thereof. Where the compound name or formula has no isomer designation, the name or formula includes the individual isomer mixtures and the racemates.

The compounds having the S-isomer configuration are, in general, preferred.

R is a substituted alkyl group exemplified by where R2 is H or C2~Cg alkanoyl, e.g., CH^-CO, CH3(CH2)4-CO or (CH3)3C-CO; H 9 8 3 HOOC-CH2 ! NH2 HOOC-CH2-CH2 ; HOOC-CH—CH2~CH2CH2 ; H2N-CH2-CH2-CH-j; H2N-CH2CH2CH2CH^ ; HO-CH2 ; NH H2N-C-N-CH2-CH2-CH2; and h3cs-ch2-ch2is H, which is preferred or C^-C^g alkyl. Examples of suitable alkyl groups are methyl, octadecyl, 2-ethylhexyl, t-butyl, hexyl, isopropyl, ethyl, and undecyl; C^-Cg alkyl is preferred and ethyl is especially preferred.

Preferred compounds of formula I are those where R is G 9.8 3 H2N-(CH2)j , HOOC-CH2-CH5 , NH, I 2 HOOC-CH-CH2~CH2-CH3 H°~y22y~cH2 r H2N-CH2-CH2-CH2 , H2N-CN-CH2-CH2-CH2-, and H H3CS-CH2-CH2- , especially where R^ is hydrogen.

Compounds that are particularly preferred have the formula nh2 XI 6 9 8 3 especially who -e is hydrogen and is hydrogen or ethyl. Especially preferred formula II compounds are those where R^ and R2 are hydrogen, with the S-isomer configuration being particularly preferred Another particularly preferred compound has the formula especially where R^ is hydrogen.

The S-isomer of formula III is particularly preferred. 10 Especially preferred compounds are those of the formulae HO CH-F NH H ™2F H2N— -N-(CH2)3— C —COOH NH2 The compounds of the present invention have physiological or chemotherapeutic uses. In most cases, the biological activities of these compounds are in large measure a consequence of their potent decarboxylase inhibiting activities Decarboxylases are enzymes which act on a-amino acid substrates, effecting decarboxylation to produce the corresponding amine. This action is illustrated as follows: L-CH-CO,H Decarboxylase I 2 / 2 NH2 nh2 L=Alkyl or aralkyl croup α,-amino acid substrate amine By inhibiting this decarboxylation, the biosynthetic pathway to a number of biologically significant amines can be modulated or inhibited with physiologically useful consequences. For ex5 ample, n-fluoromethyl dopa inhibits dopa decarboxylase and can be used in combination with dopa to potentiate the latter's usefulness in the treatment of Parkinson's disease, aFluoromethyl-histidine inhibits biosynthesis of histamine via decarboxylation of histidine (Εϋ^θ in mice — 0.4 mg/kg) . Consequently, it and combinations with histamine antagonists have utilities in the prevention of gastric lesions and in treating allergic conditions. a-Fluoromethyl ornithine by virtue of its ornithine decarboxylase inhibition interrupts polyamine biosynthesis and is of utility in the treatment of some neoplasms. α-Fluoromethyl-arginine is an effective antibacterial. α-Fluoromethyl-glu :amic acid is a CNS stimulant.

The present compounds are also substantially specific in their decarboxylase inhibition activity, that is an α-fluoromethyl-a-amino acid generally inhibits the decarboxylation of the corresponding non α-fluoromethyl acid. For example, α-fluoromethyl dopa inhibits the decarboxylation of dopa; α-fluoromethyl histidine will inhibit the decarboxylation of histidine, etc.

Because of this specificity and potency as decarboxylase inhibitors, the present compounds are also useful as diagnostic tools to determine the presence and importance of the corresponding decarboxylase in relation to diseases or to the functioning of biological systems. For ex35 ample, the importance of γ-amino-butyric acid in the central nervous system (CNS) may be studied by inhibiting its biosynthesis using an a-fluoro-methyl glutamic acid . This diagnostic utility is aided by the potent and in many instances irreversible decarboxylase inhibiting activity of the present a-fluoromethyl amino acids.

Representative compounds have been determined to have decarboxylase-inhibiting activity using conventional in-vitro assays. a-Fluoromethyl-3,4-dihydroxyphenylalanine , a-fluoromethyl tyrosine, and a-fluoromethylmeta-tyrosine have also been found to have antihypertensive activity. This activity is determined by observing the antihypertensive effect (blood pressure reduction) on (peroral or parenteral) administration of the compounds to a spontaneously hypertensive (SH) rat. This observed effect indicates that the compounds are effective as antihypertensive agents, when conventionally adminis20 tered in suitable amounts in an appropriate pharmaceutical dosage form to a hypertensive human. The pharmaceutical dosage form is conventionally prepared and generally includes pharmaceutically acceptable diLuents.

The compounds of the present invention may be prepared usiug any convenient method.

One such useful process involves the reaction of an α-hydroxymethyl-a-amino acid with SF4 in liquid HF, as illustrated by the following equation: CH-OH J 2 R — - COOH SF^/HF » NHCH-F I 2 R- C —COOH ita2 - 10 The reaction is generally carried out at temperatures in the range —8O°C to 2O°C.

This general reaction is also referred to as fluorodehydroxylation and is described in the Journal of Organic Chemistry 40, 3809-10 (1975).

BF^ may he used to promote the reaction.

It has now been discovered that the fluoro dehydroxylation of certain aryl-substituted ahydroxymethyl-a-amino acids is substantially im10 proved by utilizing BF^ or AlCl^ as a co-reactant with SF^. Specifically, this is an improved process for preparing a compound having the formula CH,F I 2 R’ — CH,— C—COOH ik2 (IV) where R is an aryl group that comprises reaching a compound having the formula ch2oh R' — ch2C — COOH L2 with a) SF^ temperatures and b) BF^ or A1C1,, in liquid HF at in the range -80 C to 2O°C.

(V) R' is an aryl group exemplified by preferably or and H H H I Preferred R' groups are HO and H The compounds of formulae IV and V preferably have the S-isomer configuration.

This present process is preferably carried out at atmospheric pressure although pressures above atmospheric may be used. The reaction temperature ranges from -80°C to 20°, -80°C to 0°C being preferred.

The present process may conveniently be carried out by introducing the SF^ and BF^ or AlCl^ into the Formula V/HF reaction system initially. The process may also be carried out by first adding the SF^ to the reaction system, allowing the reaction to proceed for a period of time and then adding the BF^ or AlCl^ and allowing the reaction to go to completion.

The use of BF^ or AlCl^ in the SF^/HF reaction system substantially improves the yield of Formula IV product.

Another method for preparation of the substituted α-fluoromethyl-a-amino alkanoic acids involves the application of photofluorination. For a description of this method, see Journal of the American Chemical Society, 92, 7494 (1970) and ibid., 98, 5591 (1976). For example. 6 9®3 - 12 α-fluoromethyl-glutamic acid is prepared: CH, CH,F I 3 I 2 HOOC-C-CH,CH,COOH ,-,HOOC-C-CH,CH,COOH I 22 fluorination ι 2 2 NH2 nh2 Both optical isomers of α-methylglutamic acid are known; thus this method is useful for preparation of both optical isomers of α-fluoromethylglutamic acid.

Similarly, α-fluoromethyl-ornithine is prepared by photofluorination of α-methyl-ornithine: CH3 hooc-c-ch2ch2ch2nh2 nggg~tion<?H2F i!ih, > HOOC-C-CH2CH2CH2NH2 Jih, Since both optical isomers of α-methylornithine are 10 available, this method of synthesis can deliver both of the two optical isomers of α-fluoromethyl-ornithine. a-Fluoromethyl-ornithine is a suitable starting material for synthesis of α-fluoromethyl-arginine by reaction with S-methylisothiourea: ?H2F HOOC-C-CH2CH2CH2NH Ah, -3» HOOC-C-CH2CH2CH2-NH-C-NH2 CH,F I 2 II NH An acid-addition salt of the present invention may be prepared by conventional treatment of the free α-amino acid with a useful acid generally in a suitable solvent.

A single enantiomer of the present compounds may also be obtained by (1) resolving the fluorinated amino acid racemate using conventional resolution techniques or (2) resolving the precursor α-hydroxymethyl-a-amino acid using conventional resolution techniques and then fluorodehydroxylating the precursor enantiomer. A conventional resolution technique involves form15 ing a salt of the α-amino acid with an optically active base and subsequently recovering the specific enantiomer from the salt.

Compounds of the formula CH- — C—COOH k2 where R2 is C2-Cg alkanoyl are prepared by acylating the corresponding compound where R2 is hydrogen. Conventional acylating agents and conditions are used.

Compounds of the formula CH,F I 2 C- COOR.

I - 14 where is alkyl are prepared by esterifying the corresponding compound where R^ is hydrogen. Again, conventional esterification reagents and conditions are employed.

The following examples illustrate preparation of representative compounds of the present invention. All temperatures are in °C.

The fluorodehydroxylation reactions described in the examples were performed in reactors made of KEL-F. Melting points are determined in open capillary and are uncorrected. The words “Kel-F Dowex“, Colite and Darco are Trade Marks.

EXAMPLE 1 Preparation of R,S-Alpha-(Fluoromethyl)-315 Hydroxy-Tyrosine_ 1.5 g of R,S, a-(hydroxymethyl)-3hydroxytyrosine hydrochloride (a-hydroxymethylDOPA HCl) was dissolved in 50 ml of anhydrous hydrogen fluoride, while being cooled in a dryice-acetone bath. The HF solvent was then evaporated after removal of the cooling bath with a stream of nitrogen gas. This operation transformed the HCl salt into the HF salt of the start25 ing material. (Alternatively 1.3 g of the free amino acid may be used as starting material, thus eliminating the need for the above operation.) The HF salt thus obtained was redissolved by pass4 6383 - 15 ing into the reactor a stream of HF gas after cooling it in a dry-ice-acetone bath, until 30 ml of liquid HF had been collected in the reactor. Sulfur tetrafluoride gas (1.2 ml, measured in liquid state at -78°C) was then passed in and the dry-ice-acetone cooling bath was then removed and replaced by a cooling bath kept at -12°C.

After 15 hours of aging, the solvent was evaporated with a stream of N2, the residue was dissolved in 50 ml of 2.5M aqueous HCl, evaporated to dryness in vacuo and subjected to amino acid analysis on Spinco-Beckman amino acid analyser. This analysis indicated the formation of o.-fluoromethyl-3-hydroxy-tyrosine. The product R^S-alpha-fluoromethyl-3-hydroxy-tyrosine is isolated by ion-exchange chromatography in the same manner as it is described in Example 2 for Salpha-fluoromethyl-3-hydroxy-tyro sine.

EXAMPLE 2 Preparation of S-alpha-Fluoromethyl-3-HydroxyTyrosine A.) Preparation of R-a-hydroxymethyl-3hydroxy-tyro sine_______ g of 3-/3',4'-diacetoxypheny1/-2acetamido-2-acetoxymethyl-propionic acid is added to 204 ml of 4M aqueous KOH with stirring. After 1 hour of stirring (under nitrogen), the solution contains the potassium salt of 3-(3',4'd ihydroxyphenyl)-2-ac etamido-2-hydroxymethylpropionic acid, formed in essentially quantitative yield. Without isolation, by methylation with dimethyl sulfate, this compound is trans46983 - 16 formed into 3-(31,4'-dimethoxyphenyl)-2-acetamido2-hydroxymethyl-propionic acid. This operation is performed at room temperature under N2 gas by dropwise addition with vigorous stirring of dimethyl sulfate (about 64 ml) and 4m aqueous KOH solution (about 148 ml) over a period of about 1 hour.

The reaction mixture was stirred for another hour, then left standing overnight.

Acidification (at 5-10°C with 55 ml of cone, aqueous HCl), extraction with ethyl acetate (12 X 300 ml), drying over Na2SO4 and evaporation In vacuo gave R,S-3-(3',4'-dimethoxyphenyl2-acetamido-2-hydroxymethyl-propionic acid. It was purified by recrystallization from 1325 ml of acetonitrile, m.p. 154-6°C (dec). 29.1 g of strychnine was suspended in 1.12 1 of ethanol and heated to reflux, then 26.1 g of R,S-3(3',4'-dimethoxy20 phenyl)-2-acetamido-2-hydroxymethyl-propionic acid was added. The solution thus obtained was allowed to cool down and left standing overnight at room temperature. Crystals of the strychnine salt of antimer, A separate; m.p. 193-194°C (HM).

The mother liquor of the above named precipitation was evaporated in vacuo to dryness and recrystallized from 270 ml of ethanol; the hot solution is allowed to cool to room temperature and left standing at room temperature for about 3 hours, then kept in the refrigerator for about 4 hours. The crystals formed were collect ed on a filter and after drying, recrystallized Ί6ί)83 - 17 from acetonitrile to give the strychnine salt of antimer B of 3—(3·, 4,-diInethoxy-phenyl)-2-acetamido-2hydroxymethyl-propionic acid, m.p. 130-132°C (dec.), yield 17.5 g. g of this strychnine salt was decomposed by dissolving it first in 160 ml of water; 31 ml of 1M aq. NaOH solution was added. The strychnine separated was removed by filtration and the solution evaporated to a small volume in vacuo and applied onto a small ion-exchange resin column (150 ml of AG-X2 cation exchange Dowex 50 resin, 200/400 mesh).

Elution with water, followed by evaporation in vacuo of the fractions which showed absorption, as indicated by an LKB UV absorption monitor (UVICORD II - 8300). This compound, antimer B of 3-(31 ,4'-dimethoxyphenyl)-2-acetamido2-hydroxymethyl-propionic-acid showed [a] 78.3 + 0.5° (C, 1.425 in 0.1M aq. NaOH).

Transformation of the above compound into the corresponding stereo-isomer of a-hydroxymethyl-3-hydroxytyrosine: 4.43 g of antimer B of 3-(3',4'-dimethoxyphenyl)-2acetamido-2-hydroxymethylpropionic acid is dissolved in 100 ml cone. HCl and sealed and heated for 90 minutes in a Fisher-Porter tube immersed into an oil bath of 130°C. The solvent is evaporated in vacuo and the above HCl treatment repeated. The residue thus obtained represents R-a-hydroxymethyl-3-hydroxy-tyrosine hydrochloride.

B.) Fluorodehydroxylation g of R-a-hydroxymethyl-3-hydroxy46983 - 18 tyrosine.HCl is charged to a 1 1. reactor.

The reactor is immersed in a dry-ice-acetone bath and 80 ml of liquid HF is condensed on top of the substrate. To remove the HCl present, the cooling bath is removed and the HF solvent removed by passing in a stream of N2 gas. The reactor is immersed in the cooling bath again and a stream of HF gas is passed in until a liquid volume of about 250 ml collects. 6.2 ml of SFg (17.6 mmol/ml: about 109 mmol) is then bubbled in, the solution aged for about 1 hour, the cooling bath exchanged for an ethylene-glycol bath kept at -16°C and the solution aged for about 22 hours. Boron trifluoride gas is passed in until satura15 tion and the solution aged again at -16° C for 46 hours. The cooling bath is removed and the solvent evaporated by passing through it a vigorous steam of N2 gas. The residue is quenched in about 100ml of ice-cold aqueous HCl (2.5M), evapor20 ated in vacuo, the residue dissolved in water and added onto a column of 2.21 of Dowex AG-50-X-8 cation-exchange resin (200/400 mesh).

Elution with 0.2514 aq. HCl, containing 5% methanol; in~·8.5 hours, 7.2 1 of this sol25 vent is pumped through the column. This is followed by 7.2 1 of O.ttaq. HCl with 7.5% methanol in 8.5 hours, then concluding with 0.6m aq.

HCl with 10% methanol. 22-ml fractions are collected, 10 tubes per rack. Tubes in racks No 45-66 contained the desired compound. Evaporation in vacuo gave the HCl salt of the S-isomer of a.-fluoromethyl-3-hydroxy-tyrosine.

For liberation of the free amino acid, 4.826 g of this compound was dissolved in 90 ml 4691 - 19 of isopropanol and filtered through Celite. 6.2 ml of propylene oxide was added to the filtrate and the suspension kept at room temperature for 3.5 hours, then at about 5°C for another 2.5 hours. The S-a-fluoromethyl-3-hydroxy-tyrosine thus formed was collected by filtration, washed with isopropanol and dried overnight in vacuo at 76°C. [α]θ: +9.3°+ 0.5, c, 1.82 in 1:1 mixture of trifluoracetic acid and water.

EXAMPLE 3 Preparation of R-g-Fluoromethyl-3-Hydroxy-Tyrosine For preparation of the above named compound, the strychnine salt of antimer A of 3-(31,4'-dimethoxyphenyl)-2-acetamido-2-hydroxymethyl-propionic acid (Example 2 “HM) was carried through steps analogous to those in Example 2.

The final product of the sequential steps was Ra-fluoromethyl-3-hydroxy-tyrosine, with [a]D: -9° (c, 2.5 in a 1:1 mixture of ^O-trifluoroacetic acid).

EXAMPLE 4 R,S-a - Fluoromethyl-Tyrosine 1.05 g (0.005 mol) of R,Sα-hydroxymethyl-tyrosine is charged into a reactor. The reactor is immersed in a dry-iceacetone bath and about 50 ml of liquid HF is collected by passing in a stream of HF gas. Under continuing cooling, SF4 gas (4 ml, measured in liquid state at -78°C) is passed in, then BF^ gas until saturation at -78°C. (Stirring with magnetic stirrer). The deep-red solution thus obtained is aged overnight at -78°C; the cooling bath is removed then, and the solvent evaporated by blowing a dry stream of nitrogen gas through it. 4ΰί)θ3 The residue is dissolved in 20 ml of 2.5m aq.

HCl and evaporated to dryness in vacuo. The residue is dissolved in water and applied to a strong acid cation-exchange resin column, prepared with 100 ml of Dowex AG5O-X-8 resin (2OD/4OO mesh). The column is first washed with water (1.8 1), followed by 0.5M aq. HCl. 20 ml fractions of the effluent are collected and the course of the elution is followed by an UV monitor of LKB, Model UVICORD II.

- The fractions corresponding to the main peak in the UV curve are combined and evaporated to dryness in vacuo, to yield the hydrochloride salt of R,S-fluoromethyl-tyrosine. 400 mg of this salt is dissolved in 6 ml of water; after a few minutes, crystallization of R,S-u-fluoromethyl-tyrosine begins. After standing overnight at 5°C, the product is . filtered, washed with water, ethanol and diethyl ether and dried in vacuo at 76°C, to give R,Sα-fluoromethyl-tyrosine.

EXAMPLE 5 R,S-a-Fluoromethyl-Histidine (FM HIST) H 3) H2C=O (L) 1) Racemization 2) CuCO3 (D,L) II - 21 46983 hf/sf4bf3 CH-F I CH--C-COOH (D,L) FM-HIST III IV A) Racemic N(im)Benzyl-Histidine 30 g of N(im)Benzyl-L-histidine is dissolved in 600 ml H20 and the solution 5 heated in a high-pressure autoclave at 200°C for 8 hours with shaking. The autoclave is cooled to room temperature, the clear supernatant solution evaporated in vacuo to dryness to give the racemic N(im)benzyl-histidine as a colorless crystal.

B) R,S-a-Hydroxymethyl-Histidine Dihydrochloride_ g of racemic N(im) benzyl-histidine is dissolved in 1 1 of hot water, then 40 g of basic cupric carbonate is added in portions and the mixture refluxed with stirring for 1 hour. The mixture is filtered while hot and the filtrate 6 9 8 3 - 22 is evaporated in vacuo to give the Cu chelate of racemic N(im)benzyl-histidine as a blue solid.

A mixture of 31 ml of formalin (38% H2CO), 3.1 ml of pyridine and 2.13 g of Na2CO3 is heated with stirring to 70°C, then 20 g of the above named Cu chelate is added and the system heated and stirred at 75°C for 90 minutes. Evaporation in yacuo gives a blue solid residue. This is dissolved in a mixture of 50 ml of Η-,Ο with 50 nil of cone. NH^OH and charged onto a cation-exchange resin column (Dowex 50-X-8, 300 ml resin in the NH^-form) and eluted with 2ΓΙ aq. NH^OH solution. The effluent is monitored with a LKB UVICORD II UV absorption monitor and 1.1 1. of the effluent is found to have OV absorption.

This portion is evaporated in vacuo to a solid. The residue is dissolved in a mixture of 60 ml of H2O with 5 ml of cone. aq.NH^OH and charged onto an anion-exchange resin column (300 ml of Dowex l-X-2 resin in the OH~ form). The column is washed with water (2 l.j , eluted with 2’i aq. HCl and monitored with a UVICORD II for UV absorption. The effluent fractions with ultraviolet absorption are combined and evaporated to dryness, to give substantially pure HCl salt of N(im)benzyl-a-hydroxy methyl-histidine (II) (new compound). This compound is transformed into a-hydroxymethyl-histidine (III) in the following way: 12.5 g of II is dissolved in 200 ml of liguid NH3(3-neck flask, equipped with cold-finger condenser filled with dry-ice-acetone) , then sodium is added (5.5 g, cut in small pieces) until the blue color persists for 10 minutes. NH^Cl is added then to consume the excess of Na (indicated by decolonization) and the NH3 solvent is allowed to evaporate under a stream A6983 - 23 of Ν2· The product III thus obtained is purified by chromatography on a cation-exchange resin column (2.2 1. of Dowex-50-X-8, 200/400 mesh). Crude III is dissolved in 100 ml of H20 and applied onto the resin column. The column is washed first with water ( 41.) and developed with ag. HCl (1.5 M, then 2 M). 20 ml fractions are collected, flow rate 600 ml/hr.

Fraction No. Pauly Reaction 1-400 1.5MHC1 401-670 2M HCl 671 & later + Fractions 671-760 are combined and evaporated in vacuo to dryness, to give R,S-g-hydroxymethylhistidine·2HC1 (new compound).

C) R^S-a-Fluoromethyl-Histidine (IV) 2.73 g of R,S-g-hydroxymethylhistidine-2 HCl is dissolved in 70 ml of liquid HF and then evaporated to dryness by passing in a stream of N2· The residue thus obtained represents the hydrofluoride salt of a-hydroxymethyl-histidine. It is redissolved in 200 ml of liquid HF (dry-ice-acetone cooling bath), then 9 ml SF4 is passed in (measured as liquid at -78°C). The solution is stored overnight, while being kept in a cooling bath of -12°C. The solution is saturated then with BF^ gas, left standing for 5 hours, saturated again at -12°C and left aging at the same temperature for 66 hours. The cooling-bath is then removed and the solvent evaporated by passing in a stream of N2· The residue represents mainly the HBF4 salt of a-fluoromethyl-histidine. This is dissolved in 100 ml 469 8 3 - 24 10 of 2.5M aq. HCl, evaporated to dryness and transformed into the HCl salt as follows: It is redissolved in H2O and applied onto a cationexchange resin column (100 ml of Dowex AG5O-X-2, 200/ 400 mesh) and eluted with HjO until effluent is neutral and free of F . The product is released from the column by 3M aq. HCl and evaporated to dryness in vacuo to result in a residue consisting mainly of the dihydrochloride of IV.

For final purification, this is rechromatographed on another AG-50-X-2 column (900 ml resin).

Elution with: 0.5M aq. HCl - 1 1. 1.0M aq. HCl - 1.5 1. 1.5M aq. HCl - 3,3 1 (collection begins here, -ml fractions) 2.0M aq. HCl - 8.00 1.

The desired product IV is located by Pauly test. Fractions 390-470 are combined and evaporated to dryness in vacuo to give pure dihydrochloride of IV. Recrystallization from water-isopropanol (1:9 v/v) gives the crystalline monohydrochloride salt of a-fluoromethyl-histidine, m.p. 226-7°C (dec.) .

EXAMPLE 6 Synthesis of R, S-cc-Fluoromethyl-Ornithine A) RJS-a-Hydroxymethyl-S-N-Benzoyl-Ornithine The copper chelate of R, S-J-H-benzoyl-orni30 thine (7.995 g) is added in small portions onto a mixture made of formalin (38% H2CO; 12.45 ml), pyridine (1.25 ml), and sodium carbonate (0.81 g) at 7O’C,a under mechanical stirring. After a /46983 - 25 further 90 minutes stirring at 75°C, it is evaporated to dryness in vacuo, the dark blue residue dissolved in a mixture of 30 ml of H2O and 30 ml of cone. aq. NH^ solution and charged to a cation-exchange resin column (130 ml of Dowex 50-X-8 in the NH^+form) to remove Cu2+. The column is eluted with 250 ml of 2M aq. NH^ and the effluent evaporated to dryness in vacuo.

The residue is redissolved in H2O and applied onto an anion exchange resin column (Dowex l-X-2, OH~ form, 130 ml resin). The column is washed with H20 (250 ml) and eluted with 3M aq. HCl.

The HCl effluent is concentrated in vacuo to give J^S-a-Hydroxymethyl-S-N-Benzoyl-Ornithine.

B) R,S-tt-Hydroxymethyl-0rnithine Dihydrochloride 3.5 g of the product obtained in A) is dissolved in 40 ml of 6M aq. HCl and refluxed for 21 hours. The solution is extracted with toluene (2 x 40 ml) and the aqueous phase evaporated in vacuo to dryness, to give R,S-a-hydroxymethyl-ornithine dihydroehloride (new compound).

C) R,S-a-Fluoromethyl-Ornithine 1.1 g of the product obtained under B) is placed in a reactor, the reactor immersed into a dry-ice-acetone bath and HF gas passed in until HF solution of 25 ml volume is formed in the reactor. The cooling bath is removed and the solvent evaporated by passing in a stream of N2. The residue thus obtained represents the HF salt of R,S-g-hydroxymethyl-ornithine. This residue is redissolved in HF, by cooling the reactor in the dry-ice-acetone bath 469 83 - 26 and passing in HF gas until 50 ml volume is reached. SF^ gas is passed in (4 ml as measured in liquid state at -78°C), the dry-ice-acetone cooling bath removed and replaced by a bath kept at -15°C. After aging for 16 hours at -15°C, BF3 gas is passed in for saturation.

After 5 hours further aging, the cooling bath is removed and the solvent evaporated by passing in a stream of N2· The residue is dissolved in 6M ag. HCl, evaporated to dryness in vacuo and redissolved in HjO (10 ml). This solution is applied onto a Dowex 50-X-8 cation-exchange resin column (400 ml resin, 200/400 mesh, H+ form).

The column is first washed with H2O (800 ml); elution with 2M aq. HCl, 15 ml fractions are collected. Flow rate 600 ml/h. Every 5th fraction is spotted on TLC plate and developed with ninhydrin spray. Fractions No. 171-220 are combined and evaporated to dryness in vacuo, to de20 liver a mixture of amino acids, the main component being R/S-u-fluoromethyl-ornithine^HCl. For further purification, this product is rechromatographed on another column, made of Dowex 50-X-8 cation exchange resin (200/400 mesh). For devel25 opment, the column is first washed with water, then eluted with 1.5 aq. HCl, flow rate 0.6 l./h. 20-ml fractions are collected. The residue obtained on evaporation of fractions No. 521-540 represents pure R,S-a.-fluoromethyl-ornithine dihydrochloride.

EXAMPLE 7 Synthesis of S-a-Fluoromethyl-Tyrosine A.) Preparation of The Copper Chelate of Tyrosine Methyl ether 9 of R,S-tyrosine methyl ether - 27 (128 mmol) was dissolved in 646 ml of 0.2N NaOH at 80°C and this solution was added to 16.1 g of copper sulfate pentahydrate dissolved in 1600 ml of water at 80°C. An immediate precipitate was formed and the solution was allowed to cool overnight, after which it was filtered affording 28.9 g of the copper chelate of R,S-tyrosine methyl ether.

B.) R,S-a-Hydroxymethyl tyrosine methyl ether 29 g of the copper (Cu++) chelate of tyrosine methyl ether (0.064 mole) was added at 0°C under stirring to a solution of 3.9 g sodium carbonate, 52 ml of 37% aqueous formaldehyde and 5.2 ml of pyridine (nitrogen blanket). After completion of addition, there was added another 18 ml of formalde15 hyde solution and 1.6 ml of pyridine. After heating at 70°C for 3.5 h and allowing the solution to cool to room temperature in an additional 1.5 h, the solution remained at room temperature overnight. In the morning, there appeared copious blue crystals which were filtered off and the filtrate concentrated to dryness in vacuo. After the residue had been dissolved in water and reconcentrated to dryness, it was dissolved in 90 ml of 4N HCl. After filtration the solution was used to dissolve the above blue crystals. This required an additional 300 ml of 4n HCl. The solution was then treated with hydrogen sulfide, filtered through a diatomaceous earth filter aid and concentrated to about 40 g of crude product. This was applied to a strong acid cation-exchange resin (0.5% of Dowex 50 X 8), eluted with 4 1 of water and then 2n aqueous ammonia.

The effluent was monitored with UVICORD II (recording ultraviolet spectrophotometer) and the UV absorbing fraction was concentrated in vacuo to 22.16 g of pure R,S-a-hydroxymethyl-tyrosine methyl ether. - 28 C. ) R,S-N-Aoetyl-a-hydroxymethyl-Tyrosine Methyl Ether______ 19.7 g of R,S-a-hydroxymethyl-tyrosine methyl ether (87.5 mmol) was sus5 pended in 200 ml of dry pyridine, then 68 ml of acetic anhydride was added. After aging overnight at room temperature, the solution was concentrated in vacuo to dryness and azeotroped with 2 X 50 ml toluene.

The residue was dissolved in 118 ml of methanol and 130 ml of aqueous 2.5N NaOH solution and stirred at room temperature for 3.5 h. Acidification with 30 ml of cone. HCl followed by extraction with 4 X 200 ml of ethyl acetate, and then drying and concentration afforded 21 g of crude product. This was recry15 stallized from 75 ml of acetonitrile yielding 9.35 g of R,S-N-acetyl-a-hydroxymethyl-tyrosine methyl ether, mp 151-152°C dec.

D. ) Optical Resolution of R,S-N-Acetyl-a-hydroxymethyl-tyrosine methyl ether_ g of R,S-N-acetyl-a-hydroxymethyl-tyrosine methyl ether and 6.18 g of jl-ephedrine were dissolved in 50 ml of methanol. The solution was concentrated to dryness in vacuo and then redissolved in 50 ml of warm acetonitrile. Crystallization afforded 7.34 g of the d-ephedrine salt of R-N-acetyl-u-hydroxymethyltyrosine methyl ether, mp 125-131°C (Crop A). Crop A was recrystallized from 40 ml of acetonitrile affording 4.78 g of Crop B, mp 130-134’C. The mother liquors from A and B were combined and concentrated, and the residues dissolved in 22.4 ml of 2.5N NaOH and 50 ml of H20The aqueous solutions were extracted with 2 X 75 ml ethyl acetate. The aqueous solutions were cooled and acidified with 5 ml of cone. HCl and the resultant solution extracted with 3 X 70 ml ethyl acetate.

The dried organic solution was concentrated to 7.73 g (Crop C). Crop C and 4.7 g of 1-ephedrine were di- ssolved in 50 ml of methanol and concentrated to - 29 12.39 g (Crop D). This was recrystallized from ml acetonitrile to yield 5.06 g of the 1-ephedrine salt of S-N-acetyl-a-hydroxymethyl-tyrosine methyl ether (Crop E) , mp 131.5-133.5’C dec. Crop E was recrystallized from 27 ml of acetonitrile to give Crop F, 4.72 g, mp 130.5-134.5“C dec. The mother liquors from Crop F and Crop E were combined and concentrated to 7.31 g (Crop G). Crop G was converted back to the free acid using the method used to obtain Crop C and there was obtained 3.0 g (Crop H). This was treated as was the initial R,S-material with 1.9 g of d-ephedrine. Recrystallization of the salt from 17 ml of acetonitrile afforded 2.4 g Crop J, mp 127130°C. Crop J was recrystallized to 2.06 g of Crop K, mp 130-134°C dec.

Combined Crops B and K (6.52 g) were recrystallized from 40 ml of acetonitrile affording 6.06 g of the d-ephedrine salt of R-N-acetyl-a-hydroxymethyltyrosine methyl ether (75.8% overall).

The free acid was regenerated in the same manner as that by which the combined mother liquors of Crops A and B were converted to Crop C and there was obtained 3.50 g of R-N-acetyl-a-hydroxymethyl-tyrosine methyl ether : [a)D= +92° (C, 1.35, 0.27NNaOH).

E.) R-a-Hydroxymethyl-Tyrosine 3.3 g of R-N-acetyl-a-hydroxymethyl -tyro sine methyl ether was dissolved in 100 ml of cone. HCl and heated in a pressure tube at 130°C for 2 hr. The solution was concentrated to dryness, the residue dissolved in 35 ml of H2O, filtered and treated with 1 ml of pyridine. 2.11 g of pure R-a-hydroxymethyltyrosine (81%) crystallized out: [α]θ= 0.86° (C, 1.15, 50% aqueous trifluoroacetic acid). The circular - 30 dichroism (CD) spectrum has the same sense as the CD of S-a-methyl-tyrosine.

F.) S-a-Fluoromethyl-Tyrosine Following the procedure of example 4, S— α-fluoromethyl-tyrosine was prepared from R-ghydroxymethyl-tyrosine.

EXAMPLE 8 (+)-g-Fluoromethylglutamic Acid 6.56 g of α-methylglutamic acid hemi10 hydrate was photofluorinated in liquid HF solution by the general technique described in Journal of the American Chemical Society, 92, 7494 (1970) and 98, 5591 (1976). The substrate was dissolved in 120 ml of liquid HF and irradiated with a 2500W ultraviolet light source under stirring while fluoroxytrifluoro-methane (CF^OF) gas (3.0 ml as measured in liquid form at -78 °C) was passed in the course of 80 min, under cooling in a dry-ice-acetane bath. After another 80-minute period with irradiation under similar conditions, an additional similar dose of CF^OF was added in 3 hours, continuing with the stirring, cooling cind irradiation. The mixture was kept overnight in the dry-ice-acetone bath, then It was further fluorinated (with 3 ml of CF^OF, added in 5 hours with irradiation). Nitrogen gas was blown through the solution for removal of the solvent and the residue was evaporated with 2.5n aq. HCl (2X) in vacuo.

The residue was dissolved in 40 ml of water. To 10 ml of this solution 10 ml of cone. HCl was added and the mixture was refluxed for about 68 hours. After treatment with DARCO G-60, the filtrate was evaporated in vacuo and the residue refluxed with 30 ml of cone. HCl for another 68-hour period. After 6 9 8 3 - 31 treatment with DARCO, the solution was evaporated to dryness, dissolved in 10 ml of cone. HCl and heated in a sealed glass tube for 24 hours in an oil-bath kept at 130-135°. Evaporation in vacuo to dryness gave a residue which was dissolved in HjO and subjected to elution chromatography on a cationexchange resin column, made of 360 ml cf Dowex AG5O-X12 resin (mesh 200/400). Eluants: 2.6 1 of H20, followed by O.JN aq. HCl (1.5 1), then by 0.15N aq. HCl. UV absorption of the effluent was monitored by a recording UV at 206 nm. 15-ml fractions of effluent were collected and 20 fractions, corresponding to the first ultraviolet absorbing peak, were combined and evaporated in vacuo to dryness, to give a-fluoro15 methyl-glutamic acid hydrochloride. For liberation of the amino acid, this was dissolved in isopropanol and filtered, and then propylene oxide was added. a-Fluoromethyl glutamic acid 0.7 H20 crystallizes out of the solution.

This compound is a time-dependent inhibitor of glu20 tamic acid decarboxylase.

Claims (38)

CLAIMS:

1. A compound having the formila R- C-COOR-l nh 2 wherein R is a substituted C^-C^ alkyl group 5 and R^ is H or C^-C^g alkyl.

2. Pharmaceutically acceptable acid addition salts of a compound as claimed in Claim 1.

3. A compound as claimed in Claim 1 having the S-isomer configuration. 10

4. A compound as claimed in Claim 1 in which the substituted alkyl group is CHj whereH in R 2 is H or C 2 ~Cg alkanoyl, N-p— CH^ HO CH HOOC-CH 2 , H 2 N-&-NH-CH 2 -CH 2 -CH 2 , H 2 N-(CH 2 ) 2 -CHj, HOOC-CH 2 -CH 2 , HO-CHg H - 33 ΝΗ, I 2 HOOC-CH-CH 2 -CH 2 CH 2 , H 2 N-CH 2 -CH 2 -CH 2 -CH ? , HO K 3 CS-CHjCH 2 or b- CHj / and Ρ. χ is H.

5. A compound as claimed in Claim 4 in which the substituted alkyl group is H N-t-CHt; *2 O -®-™2 ’ {'IT HO ro O cn 5 χν 2 H 2 N~(CH 2 ) 2 CH 2 , HO. NH h 2 n - c - nh-ch 2 -ch 2 ch 2 HOOC-CH 2 -CH 2 - , or CH,

6. A compound having the formula CH-F I 2 CH 2 -C- COOR-l 1h, where R^ and R 2 are as defined in Claim 1 and 4 respectively

7. A compound as claimed in Claim 6 in which R 2 is hydrogen and Rj^ is Η or ethyl. 409 83 - 34

8. A compound as claimed in Claim 7 in which is hydrogen.

9. A compound as claimed in Claim 8 having •the S-isomer configuration.

10. A compound as claimed in Claim 1 having the formula CH_F I 2 -CHj-C-COOR-, z | j. NH10

11. A compound as claimed in Claim 10 having the formula CH-F I z H ,ά-ρ-ΟΗ,-C — COOH /,1 NH 2

12. A compound as claimed in Claim 11 having the S-isomer configuration.

13. . A compound as claimed in Claim 1 having the formula CH-F I H 2 N-CH 2 -CH 2 -CH 2 -C COOH. . NH

14. A compound as claimed in Claim 13 having the_S-isomer configuration. 46083 - 35

15. A compound as claimed in Claim 1 having the formula ch 2 f HO—~ C t ~~ '—' NH, COOH

16. A compound as claimed in Claim 15 having 5 the S-isomer configuration.

17. A compound as claimed in Claim 1 having the formula HO . CH 2 F (f 'λ—CH 2 -C—COOH W nh 2

18. A compound as claimed in Claim 17 having 10 the S-isomer configuration.

19. Ά compound as claimed in Claim 1 having the formula HO- CH,F I -CH--C— COOH 2 I NH,

20. A compound as claimed in Claim 19 having 15 the S-isomer configuration.

21. A compound as claimed in Claim 1 having the formula NH H H,N-C-N-i H CH,F I 2 2„-v,-„-CH 2 -CH 2 -CH 2 — C— COOH NH, 46383 - 36

22. A compound as claimed in Claim 21 having the S-isomer configuration.

23. A compound as claimed in Claim 1 having the formula: CH.F I 2 HOOC CH O CH O -C COOH 2 2 | nk 2

24. A compound as claimed in Claim 23 having the S-isomer configuration.

25. A compound as claimed in Claim 1 having the formula:

26. A compound as claimed in Claim 25 having the S-isomer configuration. .27. A pharmaceutical composition containing a compound of Claim 1 or a pharmaceutically acceptable

15. Acid-addition salt thereof, together with a pharmaceutically acceptable diluent.

28. A composition as claimed in Claim 27 in which the seiid compound has the formula - 37 where R^ is H or C^-Cg alkyl and R 2 is H or C 2 ~Cg alkanoyl.

29. A composition as claimed in Claim 27 in which the said ccmpound has the formula where R^ is H or C 2 -Cg alkyl.

3 0. A composition as claimed in Claim 27 in which the said compound has the formula in CH-F I 2 COOH

31. A process for preparing a compound of Claim 1 in which R^ is H which comprises fluorodehydroxylation of a compound having the formula CH-OH I 2 R— C — COOH Ah 2 by treatment with sulfur tetrafluoride in hydrofluoric acid

32. A process as claimed in Claim 31 in which R is HO CH 2 - , H 2 N-CH 2 '

33. A process CH 2 -ch 2 ~ , or 32 in which R is . 34.

A process for preparing compounds having the formula CH,F I Z (XV) where R' is an aryl group, comprising reacting a compound having the formula CH,OHI 2 R'-CH,-C-COOH (V) 2 I NH 2 with SF 4 in liquid HF at temperatures ranging from -80°C to 20°C, in which BF.j or AlCl^ is present in the reaction system.

35. in Claim 34 in which A process as claimed and the added compound is BF^.

36. 35 in which A process as claimed in Claim

37. A process as claimed in Claim 35 in which the Formula IV and V compounds have the S-isomer configuration. 15

38. A compound as claimed in Claim 1, when prepared by a process substantially as hereinbefore described in any one of the foregoing Examples.