FI66840B - ALFA-FLUORMETHYL-ALFA-AMINOALKANSYROR FOERFARANDE AV NYA THERAPEUTIC ANVAENDBARA - Google Patents

Description

--- m re, „„ ANNOUNCEMENT,, A, _ ^ ^ utlAggningsskrift 66840 n ^ W 'C 101/72, 101/20, 101/21 ,, FINLAND — FINLAND (* i) 781674 (22) HakMiIMNl-AMBkniivd ^ 26.05.78 '(23) AtoplM— GlMgteadag 26.05.78 (41) Tallat julktooksl - RJMt affmtSg ^ 12 78 ^ Piston and register halti · (44) NUitMkripanon M listen + iOota · »pvm. -

Patent- och regfsterstyralean 7 AnBkan utta * d od »ucUkrftwi peMcarad 31 .08.84 (32) (33) (31) • Vr1 · ** / etuoikeu * —e * f» rd priority 01.06.77 01.06.77 USA (US) 802391, 802389 (71) Merck ε Co., I ric. , 126 E. Lincoln Avenue, Rahway, New Jersey, USA (72) Janos Kollonitsch, Westfield, NJ, Arthur Allan Patchett, Cranford, NJ, Stephen Marburg, Metuchen, NJ, USA (7 * 0 Oy Kolster Ab (5 **) Method for the preparation of new therapeutically useful c * <fluoromethyl-T ^ -amino amino Aging acids - Förfarande för framställ-Ning av nya therapeutically used vC-fluoromethyl-o ^ -aminoalkansyror

The invention relates to a process for the preparation of novel substituted α-fluoromethyl-ού-aminoalkanoic acids and their pharmaceutically acceptable acid addition salts. The formula for the new compounds is

CH ~ F

I 2

R - C ---- COOR. I

NH 2 wherein R 1 is H or C 1 -C 4 alkyl and R is 2,668,410 ho? “-O-0 · * 2 · 'H2N ~ (CH2,2 ~ CH2' · HO ^ ch2, ηο_ <ΓΛ_ch2-,

NH

H 2 N-c-nh ~ CH2-CH2-CH2. tax hooc-ch2-ch2-.

These compounds have a biological effect, such as decarboxylase inhibitory activity.

Unsubstituted α-fluoromethyl-.beta.-aminoalkanoic acid, namely 2-fluoromethylalanine of the formula

pF

CH3-C -COOH

nh2 (A) is a known compound / jiollonitsch et al., J. Org. Chem. 40, 3808-9 (1975) 1 /. No specific biological effect is shown for this compound. This compound (A) is prepared by fluoridehydroxylation of the corresponding 2-hydroxymethylalanine.

δC-methylamino acids such as 1-oC-methyl-3,4-dihydroxyphenylalanine (oO-methyldopa, an antihypertensive agent) are known to have decarboxylate inhibitory activity (Goodman, 3,66840 et al. The Pharmacological Basis of Therapeutics, Mac Millan Company, New York, New York 1970, p. 577; Canadian Patent 737,907).

It has now been found that the novel substituted oC-fluoromethyl- (X-aminoalkanoic acids) of the formula I have a significantly greater decarboxylase inhibitory effect than the oC-methylamino acid.

Pharmaceutically acceptable acid addition salts generally consist of a base of formula I and a suitable organic or inorganic acid. Preferred inorganic acid salts are hydrogen halides, e.g. hydrochlorides, hydroiodides, hydrobromides, sulphates and phosphates. Most preferred are halogenated hydrogens and especially hydrochlorides.

The compounds of formula I have a chiral center and can exist in optically active forms, i. as optical isomers. These isomers are conventionally denoted by the symbols L and D, + ja-, 1 jad, S and R or combinations thereof. Unless the name or formula of a compound indicates an isomer, mixtures and racemates of their individual isomers are included within the name or formula.

Compounds having the S-isomer configuration are generally preferred.

Examples of suitable alkyl groups include methyl, octadecyl, 2-ethylhexyl, t-butyl, hexyl, isopropyl, ethyl, undecyl and the like alkyl groups; C 1 -C 4 alkyl is preferred and ethyl is particularly preferred. is most preferably H.

Preferred compounds of formula I are those of formula

CH F

H0 ~ / - \ I

HO - (/ y CH2-c-COOR2 NH2 Of these compounds, those in which R1 is hydrogen or ethyl and which have the S-isomer configuration are more preferred.

Preferred compounds of formula I are also those of formula _ - τ 4 ch f 66840 H | 2

Jj_ CH9-C-COOR, <J k and in particular the S-isomer with hydrogen.

Particularly preferred compounds of formula I are those of the formulas

HO ^ j'H2F

// "> -CH9 - C - COOH,

\ = J 2 I

nh2 ch2f

HO —CH2-C — COOH

NH 2

CH-F

I 2 HOOC-CH2-CH2-C COOH, and NH2

NH H | H2F

h2n— i: - n- (ch2) 3 — c - cooh nh2 5 66840

Compounds of formula I and their acid addition salts may be prepared by: a) a compound of formula

CH90H

R -C -COOH II

nh2 wherein R is as defined above is fluorodehydroxylated, or b) a compound of formula p

R '--C -COOH III

nh2 wherein R 'is HOOC-CH2-CH2- or H2N- (CH2) 2-CH2-, is fluorofluorated, or c) α-fluoromethyl-ornithine of formula

2 F

H2NCH2CH2CH2-C -COOH 1 'NH2 is reacted with S-methylisothiourea to give? -Fluoromethyl-arginine; and, if desired, the resulting compound of formula I wherein R 1 is H is esterified to give a compound of formula I wherein R 1 is C 1 -C 10 alkyl, and / or the compound of formula I is converted into a pharmaceutically acceptable acid addition salt.

The process can be carried out, for example, by reacting an N-hydroxymethyl <* amino acid with SF 1 in liquid HF, according to the following reaction equation: 6 CH 2 OH CH 2 F 66840

R - C - COOH SF - / HF \ R -C - COOH

NH2 NH2

The reaction is generally carried out at a temperature of about -80 ° C to about 20 ° C. This reaction is termed fluoride dehydroxylation and is described in Journal of Organic Chemistry 40, 3809-10 (1975). BF 2 can be used to promote the reaction.

Fluoride dehydroxylation of certain aryl-substituted o6-hydroxymethyl-vC-amino acids can be substantially promoted by using as a co-reagent in BF or Ale with SF4.

This is particularly suitable for the preparation of compounds of formula

2 F

R - CH --- C -COOH

2 I

nh2 (Ia) wherein R 'is an aryl group, wherein the compound of formula ch2oh

R '- CH2 - C ---- COOH

nh 2 (Ha) is reacted with a) SF 2 and b) BF 3 or AlCl 2 in liquid HF at a temperature of about -80 ° C to about 20 ° C.

Preferred R 'groups are

HO II

Ha'ja ^ *

The fluoride dehydroxylation is preferably carried out at atmospheric pressure, although pressures higher than atmospheric pressure may be used. The reaction temperature is about -80 ° C to about 20 ° C, preferably -80 ° C.

li 66840

The process can be conveniently carried out by adding SF 2 and BF 3 or AlCl 3 to a basic reaction mixture of a compound of formula IIa and HF. The process can also be carried out by first adding SF 4 to the reaction mixture, allowing the reaction to continue for some time and then adding BF 3 or AlCl 3 and completing the reaction.

The use of BF 3 or AlCl 2 in the SF 2 / HF reaction system substantially improves the yield of the product of formula Ia.

Substituted N-fluoromethyl-α-aminoalkanoic acids of formula I can also be prepared using the fluorofluorination described in Journal of the American Chemical Society, 92, 7494 (1970) and ibid, 98, 5591 (1976).

For example, <X> -fluoromethylglutamic acid is prepared as follows:

CH0 CH „F

I 3 I 2

HOOC-C-CH-CH COOH light-_HOOC-C-CH CH COOH

and fluorination ' nh2 Both optical isomers of nh2-methylglutamic acid are known; thus, this method is useful for the preparation of both optical isomers of <* »- fluoromethylglutamic acid.

oC-fluoromethyl-ornithine is prepared in a similar way by photofluorination of σό-methyl-ornithine:

CH-, CH „F

J | Z

H00C-C-CH2 CH2NHo light- \ HOOC-C-CH2CH „CH„ NH „j 2 2 f fluorine | 2 2 2 2 nh2 nh2

Since both optical isomers of -methylornithine are available, each of these two optical isomers of β-fluoromethyl-ornithine can be produced by this synthetic method.

oC-f. fluoromethyl-ornithine is a suitable starting material for the synthesis of O-fluoromethyl-arginine by reaction with S-methylisothiourea: 66840 8

E

CH2F CH2F

HOOC-C-CH2CH2CH2NH2 -HOOC-C-CH2CH2CH2-NH-C-NH2 NH2 nh2 nh

The acid addition salts of the compounds of formula I may be prepared by treating the free β-amino acid with a suitable acid in a suitable solvent in a conventional manner.

One enantiomer of the compounds of formula I may also be obtained by (1) resolution of the fluorinated amino acid racemate using a conventional resolution technique or (2) resolution of the preceding α-hydroxymethylamino acid using a conventional resolution technique and fluoride dehydroxylation. enantiomer. Conventional resolution techniques involve forming a salt of an oC amino acid with an optically active base and recovering the desired enantiomer from the salt.

Compounds of formula I having C 1 -C 4 alkyl are prepared by esterification of the corresponding compound with hydrogen. In this case, too, the usual esterification reagents and conditions are used.

The compounds of the formula I have physiological and chemotherapeutic applications. In most cases, the biological effects of these compounds are due in large part to their potent decarboxylase inhibitory effects. Decarboxylases are enzymes that act on <X, -amino substrates, causing decarboxylation to form the corresponding amine. This effect is illustrated by the following equation: L-CH2-CO2H Decarboxylase L-CH2 L-alkyl or NH2 NH2 aralkyl group amino acid substrate amine 66840 9

By preventing this decarboxylation, the biosynthetic chain of events into a number of biologically significant amines can be modulated or inhibited with physiologically beneficial consequences. For example, α-fluoromethyl-dopa inhibits dopa-decarboxylase (dopa = dihydroxyphenylalanine) and can be used in conjunction with dopa to enhance the utility of the latter in the treatment of Parkinson's disease. oC-fluoromethyl-histidine inhibits histamine biosynthesis through histidine decarboxylation (ED ^ q in mice ^ 0.4 mg / kg). Consequently, it and its combinations with histamine antagonists have utility in preventing the development of gastric lesions and in the treatment of allergic conditions. Due to its ability to inhibit ornithine decarboxylase, N-fluoromethyl-ornithine disrupts polyamine biosynthesis and can be used in the treatment of some tumors, N-fluoromethyl-arginine is a potent bactericidal agent. oC-fluoromethyl-glutamic acid is a central nervous system stimulant.

The decarboxylase inhibitory activity of the compounds of formula I is also substantially specific, i.e. β-fluoromethyl-β-amino acid generally inhibits the decarboxylation of the corresponding non-β-fluoromethyl acid. For example, N-fluoromethyl-dopa inhibits the decarboxylation of dopa; Ά-fluoromethyl-histidine inhibits histidine decarboxylation, etc.

Due to this specificity and efficacy as decarboxylase inhibitors, these compounds are also useful as diagnostic agents in attempting to detect the presence and importance of the corresponding decarboxylase in relation to diseases or the functioning of biological systems. For example, the role of T-aminobutyric acid in the central nervous system (CNS) can be studied by inhibiting its biosynthesis using N-fluoromethyl-glutamic acid, etc.

This diagnostic utility is enhanced by the potent and in many cases permanent decarboxylase inhibitory activity of the -fluoromethyl amino acids of formula I.

Using conventional in vitro assays, typical compounds have been shown to have decarboxylase inhibitory activity.

_____ - r 10 66840-Fluoromethyl-3,4-dihydroxyphenylalanine,? -C-fluoromethyltyrosine and 6-fluoromethyl-meta-tyrosine have also been found to have antihypertensive activity. This effect has been determined by observing the antihypertensive effect (reduction in blood pressure) when the compounds are administered (orally parenterally) to spontaneously hypertensive (SH) rats. This observed effect indicates that the compounds are effective antihypertensive agents when administered in the usual manner in appropriate amounts in a suitable pharmaceutical dosage form to a human suffering from hypertension. The pharmaceutical dosage form is prepared in a conventional manner and generally contains conventional, pharmaceutically acceptable diluents.

The following examples illustrate the invention. All temperatures are in degrees Celsius. The fluorohydroxylation reactions described in the examples were performed in reaction vessels prepared from KEL-E®. Melting points are determined in open Kapil bay tubes and are uncorrected.

Example 1 Preparation of R, S-alpha- (fluoromethyl) -3-hydroxy-tyrosine

CH9F

i

HO - CH2 — c COOH

ho y I

NH 2 1.5 g of R, S, oC- (hydroxymethyl) -3-hydroxytyrosine hydrochloride (06-hydroxymethyl-DOPA HCl) was dissolved in 50 ml of dry hydrogen fluoride by cooling in a dry ice-acetone bath. After removal from the cooling bath, the HF solvent was then evaporated off with a stream of nitrogen gas. In this step, the HCl salt is converted to the HF salt of the starting material. (Alternatively, 1.3 g of the free amino acid can be used as the starting material, eliminating the above step. The HF salt thus obtained was redissolved by introducing HF gas into the reaction vessel after cooling in an ice-cold acetone bath until 30 ml of liquid 11 66840 HF Sulfur tetrafluoride gas (1.2 mL, measured as a liquid at -78 ° C) was then introduced, then the carbon dioxide ice-Toni cooling bath was removed and replaced with a cooling bath maintained at -12 ° C. then the solvent was evaporated off under a stream of N 2, the residue was dissolved in 50 ml of 2.5 M aqueous HCl, evaporated to dryness in vacuo and the amino acid analysis was performed on a Spinco-Beckman amino acid analyzer. f The fluoromethyl-3-hydroxy-tyrosine The product R, S-alpha-fluoromethyl-3-hydroxytyrosine is isolated using ion exchange chromatography in the same manner as described e in Example 2 for S-alpha-fluoromethyl-3-hydroxy-tyrosine.

Example 2 Preparation of S-alpha-fluoromethyl-3-hydroxy-tyrosine A.) Preparation of R-oQ-hydroxymethyl-S-hydroxy-tyrosine 50 g of 3 / 3'4'-diacetoxyphenyl / -2-acetamino-2-acetoxymethyl-propionic acid is added with stirring to 204 ml of 4-mol. KOH aqueous solution. After stirring for 1 hour (protected under nitrogen), the solution contains the potassium salt of 3- (3'4'-dihydroxyphenyl) -2-acetamino-2-hydroxymethylpropionic acid, which has formed mainly in quantitative yields. Without isolation, by methylation with dimethyl sulfate, this compound is converted to 3- (3,4'-dimethoxyphenyl) -2-acetamino-2-hydroxymethyl-propionic acid. This work is carried out at room temperature under shielding gas with the addition of dimethyl sulfate (about 64 ml) dropwise with vigorous stirring and 4 mol. Aqueous KOH (ca. 148 mL) over about an hour.

The reaction mixture was stirred for an additional hour, then allowed to stand overnight. Acidification (55 ml of concentrated aqueous HCl at 5-10 ° C), extraction with ethyl acetate (12X300 ml), drying over Na 2 SO 4 and evaporation to dryness in vacuo gave R, S-3 (3141-dimethoxyphenyl-2-acetamino). 2-Hydroxymethyl-propionic acid, which was purified by recrystallization from 1325 ml of acetonitrile, mp 154-156 ° C (dec.).

29.1 g of strychnine were suspended in 1.12 liters of ethanol 2BA, heated to boiling, followed by the addition of 26.1 g of R / S-3- (3'4I-dimethoxyphenyl) -2-acetamino-2-hydroxymethylpropionic acid.

The solution thus obtained was allowed to cool and left overnight at room temperature. Distinguishes optical antipode "A" Stryk-so-salt crystals, m.p. 193-194 ° C ("MH").

The mother liquor of the above precipitate was evaporated to dryness in vacuo and recrystallized from 270 ml of ethanol 2BA; the hot solution was allowed to cool to room temperature and left in place at room temperature for about 3 hours, then kept in the refrigerator for 4 hours. The formed crystals were collected on a filter and, after drying, recrystallized from acetonitrile to give the Strykin salt of the optical antipode "B" of 3- (314'-dimethoxyphenyl) -2-acetamino-2-hydroxymethylpropionic acid, m.p. 130-132 ° C (dec.). Yield 17.5 g.

17 g of this strychnine salt were decomposed by first dissolving in 160 ml of water, 31 ml of 1 mol. Aqueous NaOH. The separated strychnine was removed by filtration and the solution was evaporated to low volume in vacuo and applied to a small ion exchange resin column (150 ml AG-X2 cation exchanger Dowex 50 resin, 200/400 mesh). Elution with water followed by evaporation to dryness in vacuo showed fractions showing absorption as indicated by an LKB UV absorption monitor (UVICORD 11-8300). The optical antipode "B" -q of this compound, 3- (3'''-dimethoxyphenyl) -2-acetamino-2-hydroxymethyl-propionic acid, was 78.3 ± 0.5 ° (C, 1.425 in 0.1 mol aqueous NaOH).

Conversion of the above compound to the corresponding stereoisomer of o6-hydroxymethyl-3-hydroxytyrosine: 4.43 g of the optical antipode "B" of 3- (3'4'-dimethoxyphenyl) -2-acetamino-2-hydroxymethylpropionic acid are dissolved in 100 ml: concentrated HCl and kept sealed and heated for 90 minutes in a Fisher-porter tube immersed in a 130 ° C oil bath. The solvent was evaporated in vacuo and the above HCl treatment was repeated. The residue thus obtained is R-, χ, -hydroxymethyl-3-hydroxy-tyrosine hydrochloride.

B.) Fluoride Dehydroxylation 8 g of R-TO-hydroxymethyl-3-hydroxytyrosine .HCl are placed in a 1 liter reaction vessel. The reaction vessel is immersed in a dry ice-acetone bath and 80 ml of liquid HF is condensed on the substrate. To remove the HCL present, the cooling bath is removed and the HF solvent is removed by introducing N 2 gas into the vessel. The reaction vessel is re-immersed in a cooling bath and HF gas is introduced into the vessel until about 250 ml of liquid has accumulated in it. 6.2 ml of SF 2 (17.6 mmol / ml: 109109 mmol) are then bubbled into the vessel, the solution is left to stand for about an hour, the cooling bath is changed to an ethylene glycol bath kept at -16 ° C and the solution is kept here. at a temperature of about 22 hours. Boron trifluoride gas is introduced into the vessel until the solution is saturated and the solution is again kept at 16 ° C for 46 hours. The cooling bath is removed and the solvent is evaporated off by passing a strong stream of N2® gas through it. The residue is rapidly cooled by immersion in about 100 ml of ice-cold aqueous HCl solution (2.5 mol), evaporated to dryness in vacuo, the residue is dissolved in water and applied to a column containing cation exchange resin. Here, 2.2 liters of AG-50-X-8 resin (200/400 mesh) was used. Elution was 0.25 mol. Aqueous HCl with 5% methanol; 7.2 liters of this solvent are pumped through the column in about 8.5 hours. 7.2 liters of 0.4 mol are then pumped. Aqueous HCl with 7.5% methanol over 8.5 hours, and then finally 0.6 mol. Aqueous HCl with 10% methanol. In this case, 22 ml fractions are collected, 10 tubes per rack. In racks no. Tubes 45-66 contained the desired compound. Evaporation to dryness in vacuo gave the HCl salt of the S-isomer of O-fluoromethyl-3-hydroxy-tyrosine.

To liberate the free amino acid, 4.826 g of this compound was dissolved in 90 ml of isopropanol, filtered through Celite. To the filtrate was added 6.2 ml of propylene dioxide and the suspension was kept at room temperature for 3.5 hours, then at 55 ° C for another 2.5 hours. The S -? - 6-fluoromethyl-3-hydroxy-tyrosine thus formed was collected by filtration, washed with isopropanol and dried overnight in vacuo at 76 ° C. [α] 25 D: + 9.3 ° - 0.5. (c, 1.82 in a 1:11 mixture of trifluoroacetic acid and water.

Example 3 Preparation of R-Ki-fluoromethyl-3-hydroxy-tyrosine

To prepare the above compound, the strychnine salt of the optical anti-pode A of 3- (3'4'-dimethoxy-phenyl) -2-acetamino-2-hydroxymethyl-propionic acid (Example 2 "HM") was treated in similar steps as in Example 2. The final product of the successive steps was the skeleton. style 3-hydroxy-tyrosine; -9 ° (c, 2.5 in a 1: 1 mixture of H 2 O and trifluoroacetic acid).

_ - - Γ ..........

14

Example 4 66840 R, S- ex-fluoromethyl-tyrosine 1.05 g (0.005 mol) of R, S- <X-hydroxymethyl-tyrosine are placed in a reaction vessel. The reaction vessel is immersed in a dry ice-acetone bath and 50 ml of liquid HF is collected in the vessel by introducing HF gas. SF 3 gas (4 ml, measured as a liquid at -78 ° C) is continuously introduced into the vessel, followed by BF 3 gas until the solution is saturated at -78 ° C (stirring with a magnetic stirrer). The deep red solution thus obtained is kept overnight at -78 ° C, then the cooling powder is removed and the solvent is evaporated off by blowing dry nitrogen gas through the vessel.

The residue is dissolved in 20 ml of 2.5 mol. Aqueous HCl and evaporated to dryness in vacuo. The residue is dissolved in water and applied to a strongly acidic cation exchange resin column formed from 100 ml of AG-50X-8 resin (200/400 mesh). The column is washed first with water (1.8 L), then with 0.5 M aqueous HCl. Collect the 20 ml eluate fractions and monitor the elution progress with a LKB Model UVICORD II UV monitor. The fractions corresponding to the main peak of the UV curve are combined and evaporated to dryness in vacuo to give the hydrochloride salt of R, S-fluoromethyl-tyrosine. 400 mg of this salt are dissolved in 6 ml of water; after a few minutes R, S-fluoromethyl-tyrosine begins to crystallize. After overnight at 5 ° C, the product is filtered off, washed with water, ethanol and diethyl ether and dried in vacuo at 76 ° C to give R, S-N-fluoromethyl-tyrosine.

Example 5 R, S-oC-fluoromethyl-histidine (FM HIST)

H CH2OH

N— / ^ __ / ° ¾ ^ N '^ 2 2) CuCO3 1 ^ jJ ^ CH C H 2) H-CO Jrj tr U2L6 5 2 CH2C6H5 (L) (D, L)

I II

ci ^ oh ch2f

CR, -C-CO, / CH -CHXX3H

t \ j / 1 I * HF / SF.BF,, N — Λ

Na / NH, v || NR, —4—3—- u \\ NH2 ----- 3-u> L nM -2 \ N /

i H

(D, L) FM-HIST

III IV

15 6 6 8 4 0 A) Racemic N (im) benzyl-histidine

Thirty grams of N (im) benzyl L-histidine is dissolved in 600 ml of H 2 O and the solution is heated with shaking in a high pressure autoclave at 200 ° C for 8 hours. The autoclave is cooled to room temperature, the clear supernatant is evaporated to dryness in vacuo to give R, chloromethyl-histidine as a colorless crystalline substance.

B) R, S-cK-hydroxymethyl-N (im) benzyl-histidine (II)

Twenty grams of ras.-N (im) benzyl-histidine is dissolved in 1 liter of hot water, then 40 g of basic copper (2) carbonate are added portionwise and the mixture is boiled with stirring for one hour. The mixture is filtered hot and the filtrate is evaporated to dryness 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% H 2 CO), 3.1 ml of pyridine and 2.13 g of Na 2 CO 2 is heated to 70 ° C with stirring, then 20 g of the above-mentioned Cu chelate are added and the mixture is stirred at 75 ° for 90 minutes. . Evaporation to dryness in vacuo gives a blue solid residue. This is dissolved in a mixture of 50 ml of H 2 O and 50 ml of concentrated NH 4 OH and applied to a cation exchange resin column (Dowex 50-X-8, 300 ml of resin in NH 4 form) and eluted with 2 mol. With aqueous NH 4 OH. The eluate is monitored by LKB UVICORD

II on a UV absorption monitor and a 1.1 liter batch of eluate showing UV absorption are combined and evaporated in vacuo to a solid. The residue is dissolved in a mixture of 60 ml of H 2 O and 5 ml of conc. Aqueous NH 4 OH and applied to an anion exchange resin column (300 ml of Dowex 1-X-2 resin in OH form). The column is washed with water (2 liters) and eluted with 2 mol. With aqueous HCl, monitoring the UV absorption of UVICORD II. The eluate fractions showing ultraviolet absorption were combined and evaporated to dryness to give essentially pure HCl salt (II) of N (im) benzyl-ci-hydroxymethyl-histidine (new compound). This compound is converted to β-hydroxymethyl-histidine (III) as follows: 12.5 g of compound II are dissolved in 200 ml of liquid NH 4 (3-necked flask equipped with a cold-finger condenser filled with carbon dioxide ice). -acetone), then sodium (5.5 g, cut into small pieces) is added until the blue color persists for 10 minutes. NH 3 Cl is then added to consume excess Na (appears as a colorless change) and the NH 4 solvent is allowed to evaporate under a stream of 16 66840. The product III thus obtained is purified by chromatography on a cation exchange resin column (2.2 Dowex-50-X-8 resin, 200/400 mesh) The crude compound III is dissolved in 100 ml of IVO and applied to a resin column, washed first with water (4 liters), then developed with aqueous HCl solution (1 liter). , 5 molar, then 2 molar) Collect 20 ml fractions at a flow rate of 600 ml / h.

Fraction No Pauly reaction 1-400 1.5-mol. HCl 401-670 2-mol. HCl 671 & later +

Fractions 671-760 are combined and evaporated to dryness in vacuo to give compound III: R, S-A-hydroxymethyl-histidine. 2HCl (new compound).

C) R, S-ds-fluoromethyl-histidine (IV) 2.73 g of R, S-hydroxymethyl-histidine. 2 HCl (III) is dissolved in 70 ml of liquid HF and then evaporated to dryness by introducing a stream of N2. The residue thus obtained is the hydrofluoride salt of ε-hydroxymethyl-histidine. It is redissolved in 200 ml of liquid HF (carbon dioxide-ice-acetone cooling bath), after which 9 ml of SF (measured as a liquid at -78 ° C) are introduced into the vessel. The solution is kept overnight in a cooling bath at -12 ° C. The solution is then saturated with BF 4 gas, allowed to stand for 5 hours, resaturated at -12 ° C and again allowed to stand at the same temperature for 66 hours. The cooling bath is then removed and the solvent is evaporated off by passing a stream of N2 into the vessel. Residue and mainly λ, -fluoromethyl-histidine HBF ^ salt. This is dissolved in 100 ml of 2.5 mol. Aqueous HCl, evaporated to dryness and converted to the HCl salt as follows: the substance is redissolved in H 2 O and applied to a cation exchange resin column (100 ml resin AG50-X-2, 200/400 mesh), eluting with H 2 O until the eluate is neutral and F-ion-free. The product is then extracted from the column with 3M aqueous HCl, evaporated to dryness in vacuo to give a residue which is mainly the dihydrochloride of compound IV. For final purification, this is rechromatographed on a second AG-50-X-2 column (900 ml of resin).

17 66840

EluointLin is used: 0/5-mol. Aqueous HCl - 1 liter.

1.0- mol. Aqueous HCl - 1.5 liters.

1.5 mol. Aqueous HCl - 3.3 liters (collection then begins in 20 ml fractions) 2.0 mol. Aqueous HCl - 8.00 liters.

The desired product IV was localized using the Pauly experiment. Fractions 390-470 were combined and evaporated to dryness in vacuo to give pure dihydrochloride of compound IV. Recrystallization from water-isopropanol (1: 9 v / v) gives the crystalline monohydrochloride salt of N-fluoromethyl-histidine, m.p. 226-227 ° C (dec.) Example 6 Synthesis of R, S-tj-v-fluoromethyl-ornithine A) R, S-hydroxymethyl- (= E-N-benzoyl-ornithine) R, S-N-benzoyl-ornithine copper chelate (7.995 g) is added in small portions at 70 ° C with mechanical stirring to a mixture of formalin (38% H 2 CO; 12.45 mL), pyridine (1.25 mL) and sodium carbonate (0.81 g). ). After stirring for a further 90 minutes at 75 ° C, the mixture is evaporated to dryness in vacuo, the dark blue residue is dissolved in a mixture of 30 ml of H90 and 30 ml of conc. Aqueous NH 3 and applied to a cation exchange ^ + 2+ resin column (130 mL Dowex 50-X-8, NH 4 form) to remove the Cu ion. The column is eluted with 250 ml of 2-mol. The aqueous NH 4 solution and eluate are evaporated to dryness in vacuo. The residue is redissolved in H 2 O and applied to an anion exchange resin column (Dowex 1-X-2, OH, 130 mL of resin). The column is washed with H 2 O (250 mL) and eluted with 3 M aqueous HCl. The HCl eluate is concentrated in vacuo to give R, S - N - hydroxymethyl - <S -N-benzoyl-ornithine.

B) R, S-cA-hydroxymethyl-ornithine dihydrochloride 3.5 g of the product obtained in a) are dissolved in 40 ml of 6-mol. Aqueous HCl and boil for 21 hours. The solution is extracted with toluene (2 x 40 ml) and the aqueous phase is evaporated to dryness in vacuo to give R, S- λ-λ-hydroxymethyl-ornithine dihydrochloride (new compound).

C) R, S-ck-fluoromethyl-ornithine 1.1 g of the product obtained in b) are placed in a reaction vessel, the vessel is immersed in a dry ice-acetone bath and the vessel is charged with HF-18.

6 6 δ 4 O

gas until the volume of the HF solution in the reaction vessel is 25 ml. The cooling bath is removed and the solvent is evaporated off by passing a stream of N2 into the vessel. The residue thus obtained is the HF salt of R, S-c> 1-hydroxymethyl-ornithine. This residue is redissolved in HF liquid by cooling the reaction vessel in a dry ice-acetone bath and introducing HF gas into the vessel until the volume of HF liquid is 50 ml. The vessel is charged with SF4 gas (4 ml liquid measured at -78 ° C). the dry ice-acetone cooling bath is removed and replaced with a bath maintained at -15 ° C. After 16 hours at -15 ° C, BF 2 gas is bubbled in until the mixture is saturated. After 5 hours of storage, the cooling bath is removed and the solvent is evaporated off by passing a stream of mixture into the mixture. The residue is dissolved in 6 mol. Aqueous HCl, evaporated to dryness in vacuo and redissolved in water (10 mL). This solution is applied to a column containing Dowex 50-X-8 cation exchange resin (400 ml resin, 200/400 mesh, H + form). The column is first washed with water (800 ml); eluted with 2 mol. With aqueous HCl and collect 15 ml fractions. Flow rate 600 ml / h. One in five fractions is sampled on a chromatography plate and developed with a ninhydrin spray. Fractions Nos 171-220 are combined and evaporated to dryness in vacuo to give a mixture of amino acids, the main component being R, S-ci-fluoromethyl-ornithine.

2 HCl. For further purification, this product is rechromatographed on a second column loaded with Dowex 50-X-8 cation exchange resin (200/400 mesh). For development, the column is first washed with water, then eluted with 1.5 mol. With aqueous HCl at a flow rate of 0.6 l / h. Collect 20 ml fractions. The residue obtained by evaporating to dryness fractions Nos. 521 to 540 is pure R, S-eZ-fluoromethyl-ornithite dihydrochloride.

Example 7 Synthesis of S-oZ-fluoromethyl-tyrosine A) Preparation of copper chelate of tyrosine-methyl ether

Twenty-five grams of R, S-tyrosine methyl ether (128 mmol) was dissolved in 646 ml of 0.2 N NaOH at 80 ° C and this solution was added to a solution of 16.1 g of copper sulphate pentahydrate dissolved in 1600 ml of water. A precipitate formed immediately and the solution was allowed to cool overnight, then filtered to give 28.9 g of copper chelate of R, S-tyrosine methyl ether.

66840 19 B) R, S- <A-hydroxymethyl-tyrosine methyl ether Twenty-nine grams of copper (Cu ++) chelate of tyrosine methyl ether (0.064 mol) was added at 70 ° C with stirring to a solution of 3.9 g of sodium carbonate, 52 ml. 37% aqueous formaldehyde and 5.2 ml pyridine (nitrogen protection). At the end of the addition, another 18 ml of formaldehyde solution and 1.6 ml of pyridine were added. After the mixture was heated at 70 ° C for 3.5 hours and the solution was allowed to cool to room temperature over 1.5 hours, the solution was left at room temperature overnight. In the morning, abundant blue crystals were visible, which were filtered off and the filtrate was evaporated to dryness in vacuo. After the residue was dissolved in water and re-evaporated to dryness, it was dissolved in 90 ml of 4N HCl. After filtration, the solution was used to dissolve the above-mentioned 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 and concentrated to give 40 g of crude product. This was applied to a strongly acidic cation exchange resin column (0.5% Dowex 50 X 8), eluted with 4 liters of water and then with 2N aqueous ammonia solution. The eluate was monitored with UVICORD II (stored ultraviolet spectrophotomer) and the UV absorbing fraction was evaporated to dryness in vacuo to give 22.16 g of pure R, S-? N-hydroxymethyl-tyrosine methyl ether.

C) R, SN-Acetyl-Δ-hydroxymethyl-tyrosine methyl ether 19.7 g of R, S-o-hydroxymethyl-tyrosine methyl ether (87.5 mmol) were suspended in 200 ml of dry pyridine, followed by the addition of 68 ml. etikkahappoanhdyridiä. After standing overnight at room temperature, the solution was evaporated to dryness in vacuo and azeotroped with 2 x 50 ml of toluene. The residue was dissolved in 118 ml of methanol and 130 ml of 2.5-norm. Aqueous NaOH and stirred for 3.5 hours at room temperature. Acidifying with 30 mL of conc. Hi and then extraction with 4 x 200 mL of ethyl acetate followed by drying and concentration gave 21 g of crude product. This was recrystallized from 75 ml of acetonitrile to give 9.35 g of R, S-N-acetyl-N-hydroxymethyl-tyrosine methyl ether, m.p. 151-152 ° C with decomposition.

20 66840 D). Optical resolution of R, S-N-acetyl- (N-hydroxymethyl-tyrosine methyl ether)

Ten grams of R, S-N-acetyl-cA-hydroxymethyl-tyrosine methyl ether and 6.18 g of d-ephedrine were dissolved in 50 ml of methanol. The solution was evaporated to dryness in vacuo and the residue was then redissolved in 50 ml of warm acetonitrile. Crystallization gave 7.34 g of the d-ephedrine salt of R-N-acetyl-N-hydroxymethyl-tyrosine methyl ether, m.p. 125-131 ° C (product A).

Product A was recrystallized from 40 ml of acetonitrile to give 4.78 g of product B, m.p. 130-134 °. The mother liquors of Asn and Bsn were combined, evaporated to dryness, the residue was dissolved in 22.4 ml of 2.5-norm. NaOH to 50 mL of IVO. The aqueous solutions were extracted with 2 x 75 mL of s 11a ethyl acetate. The aqueous solutions were cooled and acidified with 5 mL of s 11a concentrated HCl and the resulting solution was extracted with 3x70 mL of s 11a ethyl acetate. The dried organic solution was evaporated to dryness to 7.73 g of product (product C). Product C and 4.7 g of 1-ephedrine were dissolved in 50 ml of methanol and concentrated to 12.39 g of product (product D). This was recrystallized from 50 ml of acetonitrile to give 5.06 g of the 1-ephedrine salt of S-N-acetyl-C-hydroxymethyl-tyrosine methyl ether (product E), m.p. 131.5 133.5 ° C (dec.) Product E was recrystallized from 27 ml of acetonitrile to give product F, 4.72 g, m.p. 130.5-134.5 ° C (dec.). The mother liquors of products F and E were combined and evaporated to dryness to give 7.31 g of product G. Product G was converted back to the free acid using the method used to obtain product C to give 3.0 g of product H. This was treated as the original R, S crude with 1.9 gsn of d-ephedrine. Recrystallization of the salt from 17 ml of acetonitrile gave 2.4 g of product J, m.p. 127-130 ° C. Product J was recrystallized to 2.06 grams of product K, m.p. 130-134 ° C (dec.).

The combined products B and K (6.52 g) were recrystallized from 40 ml of acetonitrile to give 6.06 g of the d-eferin salt of R-N-acetyl-β-hydroxymethyl-tyrosine methyl ether (75.8% in total).

The free acid was regenerated in the same manner as the combined mother liquors of products A and B were converted to product C to give 3.50 g of RN-acetyl- (A-hydroxymethyl-tyrosine-methyl ether / Δ 7 = + 92 ° (C, 1.35, 0.27 -normal NaOH).

D

II

21 66840 E) R-N-hydroxymethyl-tyrosine 3.3 g of R-N-acetyl-c-hydroxymethyl-tyrosine methyl ether were dissolved in 100 ml of conc. HCl and heated in a pressure tube at 130 ° C for 2 hours. The solution was evaporated to dryness, the residue was dissolved in 35 ml, filtered and treated with 1 ml of pyridine. 2.11 g of pure R, N-hydroxymethyl-tyrosine (81%) = 0.86 ° (C, 1.15, 50% aqueous trifluoroacetic acid) crystallized out. The circular dichroism (CD) spectrum corresponded to the CD spectrum of S -? - methyl tyrosine.

F) S - ^ - fluoromethyl-tyrosine

Following the procedure of Example 4, S-cK-fluoromethyl-tyrosine was prepared from R-c4-hydroxymethyl-tyrosine.

Example 8 (+) - R-fluoromethylglutamic acid 6.56 gcJL-methylglutamic acid hemihydrate is photofluorinated in a solution of liquid HF-N using the conventional technique described in Journal of the American Chemical Society, 92, 7494 (1970) and 98, 5591 (1976). ). The material was dissolved in 120 mL of liquid HF and irradiated with a 2500 W ultraviolet light source and stirred under a vessel cooled in a dry ice-acetone bath for 80 minutes with fluorooxy-trifluoromethane gas (CF 2 OF) ( 3.0 ml measured as a liquid at -78 ° C). After the irradiation was continued for another 80 minutes under the same conditions, a similar dose of CF 2 OF was added over 3 hours, continuing to stir, cool and irradiate. The mixture was kept overnight in a dry ice-acetone bath, after which it was further fluorinated (3 ml of CF-10F added over 5 hours and further irradiated). To remove the solvent, nitrogen gas was blown through the solution and the residue was evaporated to dryness in vacuo at 2.5-norm. With aqueous HCl (2x). The residue was dissolved in 40 ml of water. To 10 ml of this solution was added 10 ml of conc. HCl and the mixture were boiled for about 68 hours. After treatment with DARCO G-60, the filtrate was evaporated to dryness in vacuo and the residue was boiled with 30 ml of concentrated HCl for another 68 hours. After treatment with DARCO, the solution was evaporated to dryness, dissolved in 10 ml of conc.

HCl and heated in a sealed glass tube for 24 hours in an oil bath at ~22,66840 maintained at 130-135 ° C. Evaporation to dryness in vacuo gave a residue which was dissolved in H 2 O and chromatographed eluting on a cation exchange resin column with 360 ml of AG50-X12 resin (200/400 mesh). Eluents: 2.6 L of H 2 O followed by 0.1 N aqueous HCl (1.5 L) and then 0.15 N 2. Aqueous HCl. The UV absorption of the eluate was monitored by recording the UV at 206 nm. The eluate was collected in 15 ml fractions and 20 fractions corresponding to the first ultraviolet absorption peak were combined and evaporated to dryness in vacuo to give N, N-fluoromethylglutamic acid hydrochloride. To liberate the amino acid, this was dissolved in isopropanol, filtered and then propylene oxide was added.--Trifluoromethyl-glutamic acid-0.7 H 2 O crystallizes out of solution. This compound is a time-dependent inhibitor of glutamic acid decarboxylase.

Example 9 (R, S) -6-fluoromethylarginine R, S-δC-fluoromethylornithine dihydrochloride (400 mg, 1.69 mmol) was dissolved in 2.5 mL of 2N NaOH at room temperature and 2-methyl-2-thiopsendourea sulfate (236 g). , 0.85 mmol) was added with stirring and allowed to stand at room temperature for 20 hours. Thin layer chromatography (silica gel, methanol-conc. Ammonium hydrochloride 7: 3) showed 2 main spots, one corresponding to the starting material. The reaction mixture was evaporated to dryness in vacuo and purified by ion exchange chromatography (Dowex 50-X 8 resin, H +, 400 ml). After washing the column with 600 mL of H 2 O, eluting with 2.5N HCl to give 215 mg of o6-fluoromethylganganine dihydrochloride (46% yield) (single spot by TLC; after trimethylsilylation, the mass spectrum of the pentatrimethylsilylated compound , m / e 566; granidone group Sakaguchi test, positive; NMR consistent with structure).

Claims (7)

23 6 6 8 4 0 A process for the preparation of therapeutically useful oC-fluoromethyl-β-aminoalkanoic acids of formula I and their pharmaceutically acceptable acid addition salts, CH 2 F R - C COOR 1 I nh 2 wherein R 1 is H or C 1 -C 4 alkyl and R is HO H f / ~ / N_ — 1 CH2 ~ 'h2n- (ck2) 2-ch2-, H0 “W_CH2' \ A HO. CH2, HO2 ^ CH2-, NH M H2N-C-NH-CH2-CH2-CH2 or HOOC-CH2-CH2-, characterized in that a) a compound of the formula CH-OH I 2 R-C -COOH II NH2 24 6 6 8 4 0 wherein R is as defined above is fluorodehydroxylated, or b) a compound of formula CH-R'-C COOH III NH2 wherein R 'is HOOC-CH2-CH2- or E ^ N- ( CH2) 2-CH2-, photofluorinated, or c) α-fluoromethyl-ornithine of the formula cr2f H2NCH2CH2CH2-C COOH 1 'NH2 is reacted with S-methylisothiourea to give o <-fluoromethyl-arginine; and optionally esterifying the resulting compound of formula I wherein R 1 is H to give a compound of formula I wherein R 1 is -C 1-6 alkyl, and / or converting the compound of formula I to a pharmaceutically acceptable acid addition salt. Process for the preparation of S-od-fluoromethyl-3-hydroxytyrosine according to Claim 1, characterized in that R-od-hydroxymethyl-3-hydroxy-tyrosine is fluorodehydroxylated, after which the S-isomer is isolated. Process for the preparation of R 1 -N-fluoromethyl-3-hydroxy-tyrosine according to Claim 1, characterized in that R 1 -N-hydroxymethyl-3-hydroxytyrosine is fluorodehydroxylated, after which the R-isomer is isolated. Process for the preparation of R, S-c-fluoromethylhistidine according to Claim 1, characterized in that R, S-ck-hydroxymethyl-histidine is fluorodehydroxylated. Process for the preparation of R, S-X-fluoromethyl-ornithine according to Claim 1, characterized in that R, S-X-hydroxymethyl-ornithine is fluorodehydroxylated. Process for the preparation of S -? - fluoromethyl tyrosine according to Claim 1, characterized in that R-OO-hydroxymethyl-tyrosine is fluorodehydroxylated, after which the S-isomer is isolated. 25 66840 Process for the preparation of (-) - β-fluoromethylglutamic acid according to Claim 1, characterized in that the α-methyl-glutamic acid hemihydrate is fluorodehydroxylated. 26 66840