DK148322B - METHOD OF ANALOGUE FOR THE PREPARATION OF ALFA-MONO OR-DIFLUOROMETHYLORNITHINE OR-ARGININE OR-ORNITHINAL COOLESTERS, THEIR PHARMACEUTICAL ACCEPTABLE ACID OR BASIC OR OPTICAL ISOMERS - Google Patents

Description

in 148322 o

The present invention relates to an analogous process for the preparation of novel α-mono- or difluoromethylornithine or arginine or alkyl esters of the ornithine compounds of the general formula

x- (ch2) 3-c-cor1 I

nh 2 where Y is FCH, - or F ~ CH-, X is amino or guanidyl and 1 Δ Δ 1 R is hydrogen, or, when X is amino, R is also a straight or branched alkoxy group of 1-8 carbon atoms, or pharmaceutically acceptable acid or base salts or individual optical isomers thereof. The method according to the invention is characterized by the method of claim 1.

Examples of alkoxy groups having 1-8 carbon atoms as used in Formula I are methoxy, ethoxy, propoxy, butoxy, pentyloxy and octyloxy.

Examples of pharmaceutically acceptable salts of the compounds prepared by the process of the invention include nontoxic acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as methanesulfonic acid, salicylic acid, maleic acid, tartaric acid, citric acid, cyclamic acid and ascorbic acid, as well as non-toxic salts formed with inorganic or organic bases such as alkali metals, e.g. sodium, potassium and lithium, alkaline earth metals, e.g. calcium and magnesium, light metals from main group 3 e.g. aluminum, organic amines such as primary, secondary or tertiary amines, e.g. cyclohexylamine, ethylamine, pyridine, methylaminoethanol, ethanolamine and piperazine. The salts are prepared in the usual manner.

Preferred compounds are those of formula I wherein R 1 is hydroxy.

The compounds of formula I are inhibitors of decarboxylase enzymes which assist in polyamide formation, making these compounds valuable pharmacological agents.

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agents. Polyamines, especially putrescine, spermidine and spermine, are present in plant and animal tissues and in certain microorganisms. Although the exact physiological role of polyamines has not been clearly delineated, there is evidence to suggest that polyamines are instrumental in cell division and growth.

(HG Williams-Ashman et al., The Italian J. Biochem. 25, 5-32 (1976), A. Raina and J. Janne, Med. Biol. 53, 121-147 (1975) and DH Russell, Life Sciences 13, 1635-1647 (1973)). Polyamines are important growth factors in cells or contribute to the growth processes of certain microorganisms, e.g.

E. coli, Enterobacter, Klebsiella, Staphylococcus aureus, C. cadaveris, Salmonella typhosa and Haemophilus parainfluenza. Polyamines are associated with both normal and neoplastic rapid growth, with an increase in the synthesis and accumulation of polyamines following the stimulus that induces cell proliferation. It is also known that the level of polyamines is high in embryo systems, in tests and in patients with rapidly growing tissues. It is known that there is a correlation between ornithine, S-adenosylmethionine, arginine and lysine decarboxylase enzyme activity and the formation of polyamine.

Putrescins, spermidins and spermine biosyntheses are interrelated. Putrescin is the decarboxylation product of ornithine catalyzed by ornithine decarboxylase. Putrescine formation can also occur by decarboxylating arginine to form agmatine which is hydrolyzed to give putrescine and urea. Arginine also contributes to ornithine formation through the action of the enzyme arginase. Activation of methionine by S-adenosylmethionine synthetase forms S-adenosylmethionine which is decarboxylated, after which the propylamine moiety of activated methionine can be transferred to putrescin to form spermidine or the polyamine moiety can be transferred to spermidine to form spermine. Therefore, putrescin acts as a precursor for spermidine and spermine and has further been shown to have a pronounced regulatory effect on the biosynthetic pathway of the polyamine in that it has been found that increased synthesis of putrescin is the first sign that a tissue will be subject to renewed growth processes.

The compounds of formula I are inhibitors 5 of ornithine decarboxylase. The compounds of formula I wherein X is guanidino are also inhibitors of arginine decarboxylase. Therefore, as inhibitors of the aforementioned decarboxylase enzymes, the compounds of formula I are valuable anti-infective agents which are effective in controlling 10 microorganisms, e.g. E. coli, Enterobacter,

Klebsiella, Staphylococcus aureus, C. cadaveris, viruses such as H. parainfluenza, picornavirus, e.g. encephalo-myocarditis, Herpes simplex, poxvirus and arbovirus such as Semliki forest. The compounds of formula I must, for the same reason, be considered suitable for the control of certain rapid growth processes such as by inhibiting spermatogensis and embryogenesis and as abortifacients, also by inhibiting immune response ie. as immunosuppressors and to control neoplastic growth, e.g. solid 20 tumors, leukemia and lymphomas, as well as inhibitors of prostatic hypertrophy of scalp growth (occurring in scaling) and as inhibitors of abnormal skin cell growth (case of psoriasis). The utility of the compounds of formula I as irreversible inhibitors of ornithine decarboxylase enzymes in vivo has been demonstrated by an aqueous solution of a selected compound of formula I administered parenterally to male rats. The animals are sacrificed 5 hours after administration of the compound and the ventral lobes of the prostate are removed and homogenized and the ornithine decarboxylase enzyme activity is measured as described by E.A. Pegg and H.G. Williams-Ashman in Biochem. J. 108, 533-539 (1968) and by J. Janne and H.G. Williams. Ashman in Biochem. and Biophys. Res. Comm.

42, 222-228 (1971), cf. the biological test listed below. The compounds of formula I and their pharmaceutically acceptable salts and isomers can be administered alone

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Or in the form of pharmaceutical preparations either orally or parenterally and topically to warm-blooded animals, ie. birds and mammals, e.g. cats, dogs, cows, sheep, horses and humans. For oral administration, the compounds 5 may be administered in the form of tablets, capsules or pills or in the form of elixirs or suspensions. For parenteral administration, the compounds can best be used in the form of a sterile aqueous solution which may contain other dissolved constituents, thus sufficient saline or glucose to make the solution isotonic.

As pharmacologically useful agents, the compounds of formula I can be administered in various ways to the patient to be treated to achieve the desired effect. The compounds may be administered alone or in combination in the form of a pharmaceutical composition which may be administered orally, parenterally, e.g. intravenously, intraperitoneally or subcutaneously or topically. The amount of compound administered can vary over a wide range and can be any effective amount. Depending on the patient to be treated, the disorder to be treated, and the mode of administration, the effective amount of compound administered may vary from about 50 to about 100%. 0.1 mg / kg to 500 mg / kg body weight per per unit dose and will preferably be approx. 10 mg / kg to approx. 100 mg / kg body weight per day. unit dose. Thus, a typical unit dosage form may be a tablet containing 10-300 mg of a compound of formula I which may be administered to the patient to be treated 1-4 times daily to achieve the desired effect.

As used herein, the term "patient" is intended to include 30 warm-blooded animals such as mammals, e.g. cats, dogs, rats, mice, guinea pigs, horses, cows, sheep or humans.

The compounds of formula I wherein hydroxy and X are amino are prepared by treating an alkyl ester derivative of ornithine, wherein the formation of the carbanion intermediate is reacted with an appropriate halo methyl haloalkylation reagent in an aprotic solvent 5

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143322 such as dimethylsulfoxide, dimethylformamide, dimethylacetamide, benzene, toluene, ethers such as tetrahydrofuran, diethyl ether or dioxane, optionally in the presence of hexamethylphosphoric triamide when Y is not F -120 to 120 ° C, preferably approx. 25-50 ° C, for 1/2 to 48 hours, followed by acid or base hydrolysis as depicted in the following reaction scheme.

10 Γ "/ \ y1 N (CH2) 3-CH-COOR6 ^ 0¾ ^ -0" 00011! ° "r7 N = C-R7 Strong base! °“ R n = CR7 j R8 .3 - | R>: R; K 1 L. _ _i

Conn. 1.

Alkylating agent Y +

Y

Y dilute aqueous 1

20N (CH2) o-C-C00R6. acid / hydrazine N-C-COOR

1 'N = C-R7 NH 2 r; 8

Conn. 3

Conn. H2 O (acid / base)

Y

v

H 2 N (CH 2) 3 -C-COOH

30 NH2 compound 4 35 6 6 148322

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In the above reaction scheme, R is an alkyl group such as methyl, ethyl, isopropyl, n-propyl or n-butyl, 7 R is hydrogen, phenyl, a straight or branched alkyl group of 1-8 carbon atoms, or R and R can be together form an alkylene group of 5-7 carbon atoms, i.

-CH2 (CH2) m -CH2_f where m is an integer from 3 to 5. Examples of straight-chain or branched alkyl groups having 7 to 1-8 carbon atoms, which R6 may be R, are e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl or triethylmethyl, or R and R are hydrogen or carbonylphenyl or benzyl, respectively.

0

11 -NH-C-R

or 15 0 1 12 -NH-COR, 11 12 wherein R and R are phenyl, benzyl or alkyl or 7 8 alkoxy. Preferably, however, R and R are the same.

Suitable strong bases which can be used in the above reaction order to form the carbanion flour product are those which will move a proton from the α-carbonator network to the carboxy group such as alkyl lithium, e.g. butyllithium or phenyllithium, lithium di-alkylamide, e.g. lithium diisopropylamide, lithium amide, tertiary potassium butylate, sodium amide, metal hydrides, e.g. sodium hydride or potassium hydride, tertiary amines such as triethylamine, lithium acetylide or dilithium acetylide. Lithium acetylide, dilithium acetylide, sodium hydride and lithium diisopropylamide are particularly preferred bases.

Suitable alkylation reagents which can be used in the above reaction order are e.g. chlorofluoromethane, bromofluoromethane, fluorine iodomethane, chloridefluoromethane, bromodifluoromethane, difluoroiodemethane. The alkylation reagents are known.

Removal of the protecting groups for amino and carboxylic function can be done in one step by treating the compounds 2 with aqueous acid, e.g. hydrochloric acid 148322 7

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or toluene sulfonic acid, at a temperature of approx. 0-160 ° C for approx. 4-24 hours giving compounds 4. It is preferred to first remove the amino protecting groups 1 compounds 2, when these functions are protected as a Schiff's base, by treating compounds 2 with dilute aqueous acid, e.g. hydrochloric acid, or with hydrazine or phenylhydrazine in solvents such as lower alcohols, e.g. methanol or ethanol, ethers, chlorinated hydrocarbons, benzene and water. Removal of the protecting groups for the carboxylic functions and the amino groups, when protected otherwise than as a Schiff's base, is done by treating the compounds 2 with concentrated aqueous acids, e.g. hydrochloric acid, at a temperature of approx. 0 to 160 ° C or in aqueous bases, e.g. ammonium hydroxide.

The amino-protected ester derivatives, i.e. the compounds 1 are prepared when R is not methoxy or ethoxy by treating a suitable amino acid ester with a carbonyl-bearing compound to form a Schiff's base in a conventional manner, especially (a) when R is hydrogen, by treating an appropriate amino acid ester with benzaldehyde or an alkanal having 1 to 9 carbon atoms that are straight or branched, e.g. 1-propanal, 1-butanal, 2,2-dimethylpropan-1-al 7 or 2,2-diethylbutan-1-al; (b) when R is phenyl, by treating an appropriate amino acid ester with benzophenone or phenylalkyl ketone, wherein the alkyl moiety has from 1 to 8 carbon atoms and is straight or branched, e.g. phenylmethyl ketone, phenylethyl ketone, phenylisopropyl ketone, phenyl-n-butyl ketone 7 or phenyl tert-butyl ketone, and (c) when R is a straight chain or branched alkyl group of 1-8 carbon atoms, by treating a suitable amino acid ester with a phenylalkyl ketone or with a dialkyl ketone wherein each alkyl moiety has from 1 to 8 carbon atoms and is straight or branched e.g. dimethyl ketone, diethyl ketone, methyl isopropyl ketone, di-n-butyl ketone or methyl tert-butyl ketone. The carbonyl-bearing compounds are known or can be prepared by known methods.

When R is methoxy or ethoxy, the ornithines react the tert derivative with benzoyl halide, e.g. chloride, or an alkanoic acid halide, e.g. chloride, wherein the alkanoic acid has from 1 to 9 carbon atoms and may be straight or branched, such as acetyl chloride, propionyl chloride, butyryl chloride, tert-butyryl chloride, 2,2-diethylbutyric acid chloride or valeryl chloride, at 0 ° C in ethers, methylene chloride, dimethyl, dimethylacetamide or chlorobenzene in the presence of an organic base such as triethylamine or pyridine, after which the reaction mixture is allowed to warm to ca. 25 ° C for one hour. The resulting amide derivative is combined with an alkylating reagent such as methyl fluorosulfonate, dimethyl sulfate, methyl iodide, methyl p-toluenesulfonate, 7 or trimethyloxonium hexafluorophosphate when R is methoxy, 7 or triethyloxonium tetrafluoroborane when R is about 25 ° C in a chlorinated hydrocarbon solvent such as methyl chloride, chlorobenzene or chloroform, and the reaction mixture is refluxed for 12-20 hours. The mixture is then cooled to ca. 25 ° C and an organic base such as triethylamine or pyridine is added, after which the solution is extracted with brine and the product is isolated.

8 When R and R in compounds 1 form an alkylene group of 5-7 carbon atoms, the amino acid ester derivatives are obtained by treating the amino acid ester with a cyclic alkanone which is cyclopentanone, cyclohexanone or cycloheptanone to form a Schiff base by known methods.

The 0CR - and -COR protecting groups are applied to the free amino acid, ie. ornithine for treating the amino acid with an excess of copper salt, e.g. copper carbonate in boiling water for approx. 1-6 hours and after cooling to room temperature, the insoluble materials are filtered off, and the filtrate is treated with an appropriate acid halogen when 9A8322

0 O

II 11 the protected group is -NH-CR, or an appropriate alkyl or aryl halogen formate when it is 0 "-12 -NH-COR, for example in acetone in the presence of a base such as sodium carbonate or sodium hydroxide, followed by Examples of acid halides that can be used are acetyl chloride, propionyl chloride, benzyl chloride or 2-phenylacetyl chloride. Examples of halogenated substances which can be used are benzyl chloroformate, phenyl chloroformate, methyl chloroformate or ethyl chloroformate.

The ornithine ester is formed by known methods, e.g. the amino acid is treated with a suitable alcohol such as methanol, ethanol or n-butanol saturated with HCl gas.

The compounds of formula I wherein X is guanidino are prepared from the corresponding ornithine derivative, where Y is FCH2_ or F2CH-, i.e. compound

Y

on '

H2N (CH2) 3-C-COOH V

nh2 by treatment with an alkyl isothiouronium salt, e.g. ethyl isothioronium hydrobromide, by known methods e.g.

Organic Synthesis III, page 440 (1955). The reaction is carried out in the presence of a base, e.g. aqueous sodium hydroxide or potassium hydroxide at pH ca. 8-12 and at a temperature of approx. 0 to 100 ° C for approx. 6 hours to 8 days, after which the reaction mixture is neutralized with concentrated hydrochloric acid and the product is isolated.

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The compounds of formula I wherein R is a straight or branched alkoxy group of 1-8 carbon atoms are prepared by converting the corresponding compound wherein R α-mono- or difluoroornithine for the acid halide e.g. by treatment with thionyl chloride followed by alcoholysis with an alcohol of the formula R 1 OH, by known methods. Optionally, compounds of formula I wherein R 1 is a straight or branched alkoxy group of 1-8 carbon atoms can be prepared from the corresponding derivative, which is hydroxy, by treating the derivative with an alcohol of formula R 5 above saturated with HCl for approx. 30 minutes to 12 hours at a temperature of approx. 25 ° C to the boiling point of the alcohol.

The individual optical isomers of compounds of formula I are obtained in the same way as the racemate, just starting with the optical ornithine analog.

The individual optical isomers of the compounds of formula I wherein R 1 - is hydroxy and X is amino are also obtained from the racemate via the lactam of formula NH 2 Y-C - C = 0

(CH2) 3-NH

Using an (+) or (-) binaphthylphosphoric acid salt by that of R. Viterbo et al. method described in Tetrahedron Letters 48, 4617 (1971). Other optical solvents such as (+) camphor-10-sulfonic acid may also be used.

The following examples illustrate the process of the invention.

Example 1 2-Difluoromethyl-2,5-diaminopentanoic acid

Under nitrogen, add 500 ml of 2 molar butyl lithium in hexane to a stirred solution of 143.1 ml of diisopropylamine in 1.5 liters of tetrahydrofuran at -78 ° C, then add 261 g or 0.81 mol of ornithine dibenzaldimine methyl ester in 1.5 liter of tetrahydro furan. After the addition is complete, the reaction temperature is raised to 40 ° C and maintained between 40 and 50 ° C for 3 hours while bubbling chlorodifluoromethane gas through the mixture with stirring. The reaction mixture is then treated with a saturated solution of sodium chloride. The organic material is extracted with ether and the ether extract is washed several times with sodium chloride solution, dried over magnesium sulfate and evaporated to give a viscous oil. The oil is stirred with 1.5 liters of 1N HCl for 3 hours, the mixture is extracted several times with chloroform and the aqueous solution is evaporated to dryness. The oily residue is refluxed with 1.5 liters of 12N hydrochloric acid for 16 hours, the cooled solution is clarified by chloroform extraction before evaporation, decolourization (charcoal) and further evaporation to ca. 750 ml.

10 The pH of the solution is adjusted to 3.5 by the addition of triethylamine. The solution is again treated with charcoal before evaporation to approx. 500 ml and dilution with 7-8 liters of acetone. The precipitated product is filtered off and washed with ethanol. The crude product is recrystallized at 15 solution for approx. 150 ml of warm water and treatment of the solution with 450 ml of hot ethanol. After cooling, 71 g of crystals (37%) of 2-difluoromethyl-2,5-diaminopenoic acid hydrochloride monohydrate, m.p. 183 ° C, are separated. Example 2 2-Difluoromethyl-2-amino-5-guanidinopentanoic acid

To a solution of 5 g or 21.12 mmol of 2-difluoromethyl-2,5-diaminopentanoic acid monohydrate monohydrochloride (prepared according to Example 1) in 8.5 ml of 25 NaOH 2 molar is added at once 7.82 g or 42.26 mmol ethylthiouronium hydrobromide. The pH of the solution is adjusted to 10-, 5 with 2 molar NaOH and maintained at this value for 4 days. Then, the reaction mixture is neutralized to pH = 7 with 1 molar HCl and evaporated under reduced pressure. The residue is passed on an Amberlite column IR 120 H + form. Elution with 2 molar NH 4 OH gives 2.3 g of the title compound which is recrystallized from H 2 O / EtOH, mp 257 ° C.

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148322 12

Example 3

Division into optical isomeric 3-Amino-3-difluoromethyl-2-piperidone

To a solution of 2.7 g of methyl 2-difluoromethyl-5 -2,5-diaminopentanoate dihydrochloride prepared from the 2-difluoromethyl-2,5-diaminopentanoic acid of Example 1 by treatment with methanol saturated with HCl 2 ml of dry methanol are added under nitrogen 2 equivalents of sodium methylate in methanol (0.46 g sodium 10 in 20 ml methanol). The reaction mixture is stirred for 3 hours at room temperature, then solvents are evaporated under reduced pressure. The residue is extracted with ether to give the title lactam compound which is purified either by crystallization from CHCl 3 / pentane 15 (mp 149 ° C) or by distillation (boiling point 135 ° C / 0.05 mm Hg).

(-) and (+) 2-Amino-3-difluoromethyl-2-piperidone hydrochloride To a solution of 1.27 g of (-) binaphthylphosphoric acid (BNPA) in 50 ml of hot ethanol is added a solution of 0.546 mg ( -) 3-Amino-3-difluoromethyl-2-piperidone in 5 ml of hot ethanol. On cooling, crystals are separated.

Then, the reaction mixture is left at 4 ° C overnight. The precipitate is filtered off, washed with ethanol and diethyl ether to give 0.54 g of (-) binaphthylphosphoric acid ([α] D = -409 ° C = 0.3, MeOH melting point: 300 ° C). Recrystallization of the mother liquor gives 0.15 g of (-) binaphthyl phosphorus salt. Evaporation of the filtrate gives 1.1 g of an adhesive which is treated with 3 molar HCl 30 at room temperature for 3 hours. (-) BNPA is filtered off and the filtrate is evaporated under reduced pressure. Recrystallization of the residue (320 mg) in ethanol gives (+) 3-amino-3-difluoromethyl-2-piperidone monohydrochloride (160 g) ([a] D = + 18 ° C, C = 1 (MeOH), m.p. 238 ° C ).

Treated under the same conditions, the (-) BNPA salt (436 g) (-) gives 3-amino-3-difluoromethyl-2-piperidone monohydrohydrochloride.

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Chloride (137 mg) recrystallized in ethanol (67 mg) ([α] D = -19 °, C = 1.02, MeOH, m.p. 240 ° C, dec.).

5 (-) and (+) 2-difluoromethyl-2,5-diaminopentanoic acid monohydro, chloride (-) 3-Difluoromethyl-3-amino-2-piperidone hydrochloride (60 g) are heated in 4 ml of HCl 6 molar at reflux for 12 hours. After evaporation under reduced pressure, the residue is dissolved in water and the pH of the solution is adjusted to 4.5 with a solution of NEt 2. Then, the solution is evaporated under reduced pressure and the residue is extracted many times with chloroform and then recrystallized from H 2 O / EtOH to give 54 mg (+) 2-difluoromethyl-2,5-diamino-pentanoic acid monohydrochloride ([α] D = + 6 ° C, C = 0.48, MeOH, m.p.> 240 ° C). In identical treatment, 96 mg (+) of 3-difluoromethyl-3-amino-2-piperidone hydrochloride gives 56 mg of (-) 2-difluoromethyl-2,5-diaminopentanoic acid monohydrochloride ([o] = -10 °, C = 0 , 7, MeOH, mp> 244 ° C).

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Biological testing

Three representative compounds, namely I α-monofluoromethylornithine hydrochloride II α-difluoromethylornithine hydrochloride 25 III α-difluoromethylarginine hydrochloride, respectively, are tested by administering as a single dose in aqueous isotonic solution intraperitoneally to male rats of the strain.

Sprague-Dawley divided into groups of 5 each. The animals are sacrificed 5 hours later by decapitation, and the ornithine carboxylase activity of the preparations is measured on the prostate ventral lobes 14 14 by determining the release from [1- Cjornithin according to that of Danzin et al. described in Life Sciences 24, 519 (1979). The following results express the remaining ornithine decarboxylase activity relative to an untreated control. Ie the inhibition of ornithine carboxylase is in the% subtraction from 100%.

o 14 148322

remaining

Dose of decarboxylase activity (mg / kg) (% relative to unprocessed control).

5 Control 0 100 - 17

Compound I 200 15.5-1.0

Compound II 200 4.5 - 1.5

Compound III 200 67 - 14