NL8102863A - METHODS FOR INHIBITING PROTOON GROWTH. - Google Patents

Description

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Methods for inhibiting protozoan growth.

!

The invention relates "to" certain α-sub-! substituted amines and α-substituted amino acids, which can be used to inhibit protozoan growth in animals, especially poultry.

Polyamines are involved in cell division in many ways. It is believed that interference with the biosynthesis of polyamines by enzyme inhibitors causes a decrease in mammalian cell multiplication. Although the physiological role of polyamines is not clearly defined, it can apparently be assumed that they are involved in cell division! and growth, H.G. Williams-Ashman et al., The Italian J. Biochem.

25, 5-32 (19-6), A. Raina and J. Janne, Med. Biol. J53, 121-1 UT 15 (1975) and D.H. Russell, Life Sciences 3, 1635-161 + 7 (1973).

Polyamines are also known to be essential growth factors for certain microorganisms, for example for E.coli, Enterobacter, Klebsiella, Staphylococcus aureus, C. cadaveris, Salmonella typhosa and Haemophilus parainfluenza. Apparently, polyamines may be believed to be associated with both normal and neoplastic cell growth in mammals, since there is an increase in both the synthesis and collection of polyamines following a stimulus cell proliferation. It is also known that there is a correlation between polyamine-formation and the activity of the decarboxylase enzymes of ornithine, S-adenosylmethionine, arginine and lysine. The term polyamine is meant to include the diamine putrescine and the polyamines spermidine and spermine. Putrescine is the product of ornithine decarboxylase-catalyzed decarboxylation of ornithine. Putrescine formation can also occur by decarboxylation of arginine to form agmatine, which is hydrolyzed to putrescine and urea. Arginine is also involved with j

ornithine formation by action of the enzyme arginase. Activation of methionine by the enzyme S-adenosylmethionine synthetase

j .....................; ........................... _........._ ............ ...... _ I

8102863-2Se forms S-adenosylmethionine, which is decarboxylated. The propylamine residue of the activated methionine can then be transferred to putrescine are transferred to form spermidine. Optionally, the propylamine residue can be transferred to spermidine to form spermine. Thus putrescine serves as a starting material for spermidine and spermine. In addition, it has been shown that putrescine has a remarkable regulatory effect on the polyamine synthesis pathway. It has also been shown that an increase in putrescine synthesis is an early indication that a tissue is undergoing renewed growth processes. Cadaverine, which is the decarboxylation product of lysine, has been shown to stimulate the activity of S-adenosylmethionine decarboxylase and is known to be essential for the growth processes of many microorganisms, e.g., H. parainfluenza.

The rationale of polyamine metabolism has been suggested by Cohen, Science 205, 96b (1979). The apparently unique role of polyamine metabolism in trysanosomes and the dependence of trysanosomes on ornithine decarboxylase as a source of putrescine further supports the current observations that certain specific ornithine decarboxylases as inhibitors of polyamine synthesis are highly effective in inhibiting protozoa.

It has now been found that certain compounds belonging to a class of irreversible inhibitors of ornithine decarboxylase can be used to inhibit protozone growth. Moreover, this inhibition occurs over a wide spectrum of protozoa, for example in members of the subphylum Sarcomastigophora and Sporozoa. The class of compounds yet to be described is particularly suitable for inhibiting the growth of members of the superclass of Mastigofora, in | especially Trypanosoma brucei brucei and members of the class | from Telosporea, in particular Eimeria tenella, the organism, "! ... '.'v ·. · j causing coecidiosi | in poultry.

| The compounds of the invention are α-substituted 35 amines or α-substituted-α-amino acids with the general ones 81 02 8 63 - 3 - ί ί! formula 1, wherein hydrogen or carboxyl represents, Y CH 2 F,. ; CHF ^, CF ^ or C = CH, Z represents HgN-1CHg ^ ï H ^ N-CH- (CH ^) 2 or j 'CH3 H2N-CH2CH = CH, provided that when R1 represents hydrogen Y cannot be CF2 and Z must be H2N-CH- (CHg) 2 and the | CH3 | salts and individual optical isomers thereof. j

When administered in vivo to animals with active protozoan infections, the compounds of formula II can be used to treat such animals by inhibiting further growth of the protozoan infections. Optionally, the above-described compounds can be administered profilactically in order to prevent the occurrence of such protozoan infections.

In the above general formula 1, the symbol represents either hydrogen or a carboxyl group. When the symbol R 1 represents hydrogen, the compounds are α-substituted amines. When the symbol represents a carboxyl group, the compounds are α-substituted α-amino acids.

The symbol Y represents either an ethnic group, i or a fluoromethyl group. Possible fluoromethyl groups are monofluoromethyl, difluoromethyl and trifluoromethyl.

The symbol Z represents either a 3-aminopropyl group, a 3-amino-3-methyl group, or a 3-amino-1-propene-25 group. The saturated groups, that is, the 3-aminopropyl group and the 3-amino-3-methylpropyl group, are the preferred side chains. The limitation "provided that" | j has been included in the definition of formula 1 in order to exclude certain diamine classes from the compounds of the invention. Thus, by this limitation are excluded α-substituted diamines, in which the symbol Z represents a 3-aminopropyl group or a 3-amino-1-propene group, i.e. the compounds ij of the general formulas 2 and 3, in which the symbol Y above- j has the meaning.

"35 Ia Particularly excluded from the remaining 81 02 8 63

• V

u-α-substituted diamines is the species in which the symbol Y represents a trifluoromethyl group. Thus, the compound of the formula 1 + -methyl-1-trifluoromethyl-1,1 + -butanediamine is specifically excluded from the group of compounds which can be used successfully.

The group of compounds which can be used includes α-substituted amino acids of the formula 5, 6 and 7

In these formulas, Y has the above meaning.

The α-substituted amines of the invention are defined by the general formula 8, wherein Y 'represents CH 1 F, CHF 1, or C = CH, but where, since this is a diamine, the CF 1 group of the meaning Y is excluded.

Good examples of the salts of the compounds of the invention are non-toxic acid addition salts formed with inorganic acids such as hydrochloric acid, hydrochloric acid, sulfuric and phosphoric acid, and organic acids such as methanesulfonic acid, salicylic acid, maleic acid, malonic acid, tartaric acid, citric acid, cyclamic acid and ascorbic acid.

A preferred group of compounds of the invention are the compounds wherein Y represents a difluoromethyl group.

Another preferred group of compounds are those wherein Z represents a 3-aminopropyl residue or 3-amino-3-methylpropyl residue.

In addition to the aforementioned salts, the term salts is also understood to refer to those internal salts of zwitterions of these compounds of formula 1 which are of amphoteric nature. Although the optical configuration of the | Compounds described herein are not specifically designated, the α-carbon naturally has an asymmetric center and, moreover, separate optical isomers of these compounds exist. Thus, both the optical d and 1 isomers and the chemical mixtures are included in the invention.

Lactam formation can occur when R ^ is a carboxylic acid group and Z represents a 3-aminopropyl radical or 3-amino-3-methyl-1 | represents propyl radical as shown in the general formula 91-35. In formula 9 Y has the above meaning. In addition, when Z represents a 3-amino-J- | ____________________________________________________________________________ ________ j 8102863-5-j 3-methylpropyl radical, the (CH2) eroeP in formula 9j may be substituted with a 3-methyl group.

| Good examples of compounds to be used according to the invention are: | 2.5-diamino-2- (fluoromethyl) -pentanoic acid, 2.5-diamino-2- (difluoromethyl) pentanoic acid, 2.5-diamino-2- (trifluoromethyl) pentanoic acid, 2.5-dimino-2- (ethynyl) pentanoic acid, 2.5 - diamino-2-fluoromethyl-5-methylpentanoic acid, 2,5-diamino-2-difluoromethyl-5-methylpentanoic acid, 2,5-diamino-2-trifluoromethyl-5-methylpentanoic acid, 2,5-diamino-2-ethynyl-5-methylpentanoic acid, 2.5-diamino-2-fluoromethyl-3-pentenoic acid, 2.5-diamino-2-difluoromethyl-3-pentenoic acid, 15 2,5-diamino-2-trifluoromethyl-3-pentenoic acid, 2.5-diamino-2-ethynyl-3-pentenoic acid 1-fluoromethyl-t-methyl-1, t-butanediamine, 2-di-fluoromethyl-1-methyl-1, U-butanediamine, and 1-ethylnyl-U-methyl-1, E-butanediamine.

20

The compounds of the general formula I, in which Z has the meaning Y, the meaning CH 2 F, CHF 2 and CF 2 and R 2, the meaning carboxyl, are prepared by an ester derivative of ornithine, in which the amino groups are suitably protected, respectively. with a strong base to form the intermediate carbanion. This is reacted with a suitable halomethyl haloalkylating agent in an aprotic solvent, eg dimethyl sulfoxide, dimethylformamide, dimethylacetamide, benzene, toluene, ethers such as tetrahydrofuran, diethyl ether or dioxane and in the presence of a hexamethylphosphoric triamide, when Y has a meaning other than F CE-, at a temperature from -120 ° C to 120 ° C, preferably 25 to 50 ° C, for half an hour to + 8 hours, followed by acid or basic hydrolysis. This can be displayed with | '35 attached reaction scheme A.

8102863 - 6 -!

In this reaction scheme Y has the meaning "FCH ^ -, F ^ CH-, or F ^ C-, R ^ is a lower alkyl group, for example methyl, ethyl, isopropyl, n-propyl or n-butyl, R ^ is hydrogen, phenyl, a straight or branched alkyl group of 1 to 8 carbon atoms, methoxy or ethoxy, R 1 phenyl or a straight or branched alkyl 5 group of 1 to 8 carbon atoms, or R 1 and R 2 together are an alkylene group of 5 to 7 carbon atoms, i.e. -CH 2 - (CH 2) m -CH 2 in which an integer is from 3 to 5. Good examples of straight or branched chain alkyl groups of 1 to 8 carbon atoms, which are represented by R 2 and R 2 can be represented are methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, neopentyl and triethylmethyl.The symbol Z represents R -C = N (CH) -, 3 9 i? R ^ -CffiUCHg) ^ - or Rg-OCNHiCHg) ^ -, R ^ and R ^ are the same and have the above meanings and R ^ and Rg each represent phenyl, benzyl or a lower alkyl group having 1 to ^ carbon atoms, which are straight or branched, for example, methyl, ethyl or isopropyl, for. sets 0 0 II,. (I, R -CHH (CEL or R ^ -0CM (CEL for which R, _ and R ^ above-mentioned are meaningful).

Suitable strong bases which can be used in the above reaction series to form the carbanion are those which remove a proton from the carbon atom in the α-position relative to the carboxyl group, for example an alkyl lithium as butyl lithium or phenyl lithium, a lithium dialkylamide as lithium diisopropylamide lithium amide, tertiary potassium butylate, sodium amide, metal hydrides such as 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. above

Suitable alkylating agents which can be used in the said reaction series are chlorofluoromethane, bromofluorine. Methane, fluoroiodomethane, chlorodifluoromethane, bromodifluoromethane! 8102863 -Τ

Ι ........ ....... “................................. .............................. ........ "........... ....... '"" ............... "........................ .......................... I

thane, difluoroiodomethane, bromotrifluoromethane, chlorotrifluoro- methane, trifluoroiodomethane, hydrochloromethane, dichloromethane,! chloroiodomethane, bromodichloromethane and dichloroiodomethane.

These alkylating agents are generally "known.

Removal of the protecting groups from the amine and carboxylic acid function can be accomplished in one step by treating compound 2 with aqueous acid, for example hydrochloric acid or toluenesulfonic acid, at a temperature of 0 to 100 ° C for k to hours to obtain compounds with general formula 5 · It is preferable to first remove the protecting groups of the amine function (s) of compounds 2, when these functions are protected as a base of Schiff, by compounds 2 with dilute aqueous acid for example with hydrochloric acid or with hydrazine or phenylhydrazine in solvents such as lower alcohols, for example methanol or ethanol, ethers, chlorohydrocarbons, benzene and water. Removal of protecting groups from the carboxylic acid function and the amino groups, when the amino groups are protected other than as a Schiff base, is carried out by treatment of Bonds 3 with concentrated aqueous acids, for example bromo-hydrochloric acid at a temperature of 0 to 100 ° C or in an aqueous base, for example ammonium hydroxide.

The ester derivatives with protected amino group, that j. . . . . I

ie compounds 1, wherein R is not methoxy or ethoxy,! 25 are prepared by treating an appropriate amino acid ester; with a carbonyl compound to form a base of Schiff j in a well known manner, in particular: (a) when R 1 is hydrogen j, by treating the appropriate amino acid ester with bezdehyde or an alkanal of 1 to 9 carbon atoms, straight or branched, for example propanal-1, butanal-1, 2,2-dimethylpro- | panal-1 or 2,2-di: ethylbutanol-1, (b) when R 1 is phenyl, by treatment of the appropriate amino acid ester with benzophenone or phenylalkyl ketone, wherein the alkyl radical contains 1 to 8 carbon atoms and is straight or branched, for example phenylmethyl ketone, phenylethyl; I 35 ketone, phenyl isopropyl ketone, phenyl η-butyl ketone or phenyl t-butyl | 8102863

V

- 8 -

I I

Ketone and (c) as R_ a straight or branched chain alkyl group having 1 to 8 carbon atoms, by treatment of the appropriate amino acid with a phenyl alkyl ketone described above or with a dialkyl ketone, wherein each alkyl radical contains 1 to 8 carbon atoms. and straight or branched, for example dimethyl chain, diethyl ketone, | methyl sopropyl ketone, di-n-butyl ketone or methyl t-butyl ketone. The carbonyl compounds are well known or can be prepared in well known ways.

If R_ in compound 1 is methoxy or ethoxy, let /! - a suitable amino acid ester derivative is reacted with benzoyi halide or an alkanoic acid halide, the alkanoic acid of which has 1 to 9 carbon atoms and may be straight or branched, for example | acetyl chloride, propionyl chloride, butyryl chloride, t-butyryl chloride, 2,2-diethyl butyric chloride or valeryl chloride. The reaction is carried out at 0 ° C in an organic solvent, for example ether, methylene chloride, dimethylformamide, dimethyl acetamide or chlorobenzene in the presence of an organic base such as triethylamine or pyridine. After the reaction, the reaction mixture is allowed to warm to 25 ° C for 1 hour. The resulting amide derivative is combined at 25 ° C in a chlorinated hydrocarbon solvent, for example methylene chloride, chlorobenzene or chloroform, with an alkylating agent, for example methyl fluorosulfonate, dimethyl sulfate, methyl diodide, methyl p-toluenesulfonate or trimethyloxonephthoxyphenol (oxironium hexafluoromethyl) triethyloxonium tetrafluoroborate (when R 1 is ethoxy). The reaction mixture is heated to a reflux condenser for 12 to 20 hours, cooled to 25 ° C and an organic base such as triethylamine or pyridine is added, the solution is extracted with brine and the product is recovered.

When R 2 and R 2 in compounds 1 together form an alkylene

group of 5 to 7 carbon atoms, the corresponding amino acid ester derivatives are obtained by the amino acid ester I.

to be treated with a cyclic alkanone to form a base! I by Schiff in well known ways. Cyclic alkanones which can be used are, for example, cyclopentanone, cyclohexanone! 8102863-9 and cycloheptanone. ^ i! As in compounds 1 R ΟΝΗίΟΗ. ^) ^ - or; 0 n II ï R OCNH (CH.) - represents, the protecting groups - $ Rj- and 6 d 3 ^

5 Q

-CORg to the corresponding free amino acid, i.e., ornithine, added by this amino acid in boiling water for j | 1 to 6 hours to be treated with an excess of a copper salt,! for example copper carbonate. After cooling to room temperature, the insolubles are filtered off and the filtrate is treated with a suitable acid halide (when Z ^ R ^ CNHiCH ^) ^ -) or an appropriate alkyl or aryl haloformate (when

Is RgO 2 NHiCHg) in a solvent such as acetone in the presence of a base, for example sodium bicarbonate or sodium hydroxide.

This treatment is followed by treatment with hydrogen sulphide. Good examples of acid halides to be used are acetyl chloride, propionyl chloride, benzoil chloride and 2-phenyl-acetyl chloride. Good examples of the haloformates to be used are benzyl chloroformate, phenyl chloroformate, methyl chloroformate and ethyl chloroformate.

The lactams of the compounds of the general formula 1, wherein R 1 is carboxyl, are prepared from the corresponding amino acid esters of the formula 10, wherein Y has the same meaning as in formula 1 and R 1 a straight or branched chain alkoxy group having 1 to 8 represents carbon atoms, for example, methoxy, ethoxy, isopropoxy, butoxy or hexyloxy. The lactams are prepared by these amino acid esters with a suitable base, for example sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methanolate, potassium methanolate, potassium t-butoxide, sodium amide or an organic amine. as a trialkylamine, for example triethylamine, in a solvent such as a lower alcohol, for example methanol, [ethanol, isopropyl alcohol, n-butanol, water, dimethylformamide, Dimethyl sulfoxide, hexamethylphosphoric triamide or a mixture thereof.

81 02 8 6 3 -10-

The reaction is carried out at a temperature of 0 to 120 ° C for half an hour to 2 hours, optionally under a nitrogen atmosphere. The compounds of the general formula (10) are obtained in a known manner, for example by using the corresponding amino acid and treating this amino acid with a suitable alcohol, for example methanol, ethanol, isopropyl alcohol, n-butanol or n-heptanol, saturated with HCl gas.

The compounds of the general formula 1, wherein Z has the meaning, Y has the meaning CH ^ F, CHF ^ or ch3 CF ^ and R ^ is carboxyl, are prepared by methods analogous to those just described.

Compounds of general formula 1, wherein Z 15 H 2 I-GH (CH 2) 2 or), Y has the meaning C = CH and R 1 is carboxyl, are prepared by treating a suitable protected propaigylamine derivative, for example a silyl derivative, with a strong base to form a protected propagyl amine carbanion as an intermediate. This carbanion is reacted with an alkylating agent of the formula RgX, wherein X represents a halogen as chlorine or bromine and R 6 is PhHC = U (CH) -, where n is the integer 3. The alkylated protected propargylamine derivative thus formed is then treated with a strong base to form an alkylated, protected propargylamine carbanion. The second carbanion is reacted with an acylating agent and the protecting groups are then removed by acidic or basic hydrolysis as shown in reaction scheme B.

. In reaction scheme B, RD and X have the above j

o I

Meaning, Ph represents phenyl, R 1 is hydrogen, methoxy or ethoxy, R 1 is phenyl, t-butyl or triethylmethyl, R 1 represents straight or branched chain alkyl group having 1 to b carbon atoms, R 2 represents a carboxyl ion,: a carboxylic acid ester, a carboxamide, a nitrile or other group, which can be hydrolyzed to a carboxylic acid function ......... 35 for and is Z j ___________________ __________________________ i 8102863 i -11- * h2n-c- (ce2) 2 or h2n (ch2 ) 3.

OH

Suitable strong bases which can be used in the above reaction to form any carbanion are those, | 5 which remove a proton from the neighboring carbon atom relative to the ethyl radical, for example an alkyl lithium. Suitable alkyl lithium compounds to be used are butyl lithium or phenyl lithium, lithium diisopropylamide, lithium amide, t-potassium butylate and sodium amide. ! ! The alkylating agents, RgX, used in the above reaction are well known and can be prepared by well known standard methods. Thus, for example, the reagent PhCH = N (CH2) n can be prepared by reacting 3-bromo-n-propylamine hydrochloride or U-bromo-n-butylamine hydrochloride with benzaldehyde in the presence of an organic amine, for example triethylamine. a suitable solvent. Solvents to be used are, for example, diethyl ether, tetrahydrofuran, dioxane, chloroform and dichloromethane.

Suitable acylating agents to be used in the above reaction are haloformates such as chloromethyl formate or chloroethyl formate, azido-t-butyl formate, cyanogen bromide, carbon dioxide, diethyl carbonate, phenyl isocyanate, triethoxymethyl-methyl-methyl-2-methyl-dimethyl, Ν, Ν-methyl-tetrachloride ethylene carbonate and ethylene trithiocarbonate. When 2-methylthio-1,3-dithiolinium iodide is used, an additional alcoholysis step using a lower alcohol, for example ethanol or isopropyl alcohol, which precedes the hydrolysis of the protecting groups is necessary.

The alkylation reaction can be easily carried out in the presence of an aprotic solvent such as benzene, toluene, ether, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dimethyl acetamide, or hexamethylphosphoric triamide. The reaction temperature ranges from -120 ° C to 25 ° C, with a preferred one; reaction temperature is -70 ° C at half an hour to | "35 2 ^ hour varying reaction period. J 81 02 8 6 3« -12-

Removal of the protecting groups as shown in reaction scheme B at the step, which involves the conversion of compounds 6 to compounds of formula 11, is accomplished by treatment of compounds 6 with an aqueous

Acid, for example hydrochloric acid or toluenesulfonic acid. I

Using an aqueous base such as sodium hydroxide or potassium hydroxide, or hydrazine or phenylhydrazine.

The propargylamine derivatives of compounds 11, wherein R1n represents hydrogen, are prepared by addition of

10 '... I

Protecting groups on the ethylene function and the nitrogen function of propargylamine. Protection of the nitrogen function of propargylamine is obtained by forming a base of Schiff with a non-enisolisable carbonyl-bearing compound, for example, benzaldehyde, 2,2-dimethylpropanal or 2,2-diethylbutanol. Protection of the ethylene function is obtained by reacting Schiff's base described above with a trialkylsilyl chloride, wherein the alkyl radical contains 1 to 1+ carbon atoms and is straight or branched. Trialkylsilyl chlorides which can be used. trimethylsilyl chloride and triethylsilyl chloride.

The propargyl derivatives of compounds U, in which is methoxy or ethoxy, are prepared by reacting propargyl amines, in which the ethyl function is protected by a trialkylsilyl group, with benzoyl chloride, pivalic acid chloride or 2,2-di- ethyl butyric acid chloride at 0 ° C in a suitable solvent. Ge- | Suitable solvents are diethyl ether, dioxane, tetrahydrofuran, chloroform, methylene chloride, dimethylformamide, dimethyl acetamide and chlorobenzene. The reaction is carried out in the presence of an organic base, for example triethylamine or pyridine.

after which the reaction mixture is allowed to warm to 25 ° C in an hour. I

The resulting amide derivative is treated with an alkylating agent, for example methyl fluorosulfonate, dimethyl sulfate, methyl iodide, methyl p-toluenesulfonate or trimethyl oxonium hexafluorophosphate, | I {j when R · is methoxy, or triethyloxonium tetrafluoroborate, j | when R 1 is ethoxy, combined at about 25 ° C in a | ; ...... 35 chlorinated hydrocarbon solvent, for example methyl 81 0 2 8 6 3 * -13-lene chloride, chlorobenzene or chloroform. The reaction mixture is heated to reflux for 12 to 20 hours, cooled to 25 ° C and an organic base, for example triethylamine, is added.

J

j or pyridine. The resulting solution is extracted with brine and the desired product is recovered therefrom.

The protected starting propargylamine I is obtained by treatment of a 3-trialkylsilylpropyn-2-yl-1-ininobenzyl derivative, i.e. compounds U, wherein R 2 is hydrogen and is phenyl, with hydrazine or phenylhydrazine at 25 ° C for half an hour, after which the mixture is diluted with, for example, petroleum ether, benzene or toluene and the amine is recovered. Optionally, the imine is hydrolyzed with 0.5 to 1 If HCl solution and the aqueous phase evaporated to obtain the amine as hydrochloride.

15 Compounds of the formula 1, where Z is the mean

niche H has N-CH (CH), Y has the meaning CH F or CHF and R.

hydrogen, are prepared by reduction of a ketone of the formula 12, R. 'R10 113 113 20 wherein Z' phthaloyl-NCH (CH2) ^ benzoyl-NHCH (CH2) ^ alkanoyl-f13 ^ 13 MCH (CH ^ ) Represents 2 -, alkoxycarbonyl-HHCH (CH2) ^ - or benzyloxyearbonyl (NHCtUCH ^) ^ -, wherein the alkanoyl residue contains 2 to 5 carbon-25 atoms and is straight or branched, the alkoxy residue contains 1 to 4 carbon atoms and is straight or branched, Y has the meaning CH ^ F or CHFg and is methyl. The ketones are reduced to the corresponding alcohol, which is treated with one equivalent of an imide, for example phthalic imide, succinic imide or maleic imide, 1.1 equivalent of phosphine, e.g. picture triphenylphosphine or a trialkylphosphine, for example tri-n-butylphosphine and 1.1 equivalent diethyl azodicarboxylate in a suitable solvent. Suitable solvents include ethers, for example diethyl ether, tetrahydrofuran or p-dioxane, benzene or dimethoxyethane. The reaction is carried out at 0 to 100 ° C, i ............................ ........... ........ _; 81 02 8 6 3 ♦ -I- • i "preferably 25 ° C, carried out under an inert atmosphere, for example nitrogen or argon, for a period of from half an hour to 2 hours. The imido derivative thus obtained is then converted into the free amine hydrolyzed.

The compounds of the general formula 12, wherein | Y represents FCH 2 - are prepared by a compound of formula 13, wherein Z 'has the above meaning and represents a suitable removing group, for example chlorine, bromine or iodine, mesylate, tosylate, triflate or trifluoro-10 acetate, to be treated with a suitable fluorinating agent, for example, potassium fluoride, silver fluoride, cesium fluoride, thallium fluoride or tetrabutylammonium fluoride in a suitable solvent. Suitable solvents include dimethoxyethane, dimethyl sulfoxide, dimethylformamide, ethylene glycol, acetic acid nitrile, acetone, benzene and hydrogen fluoride. The reaction is carried out at a temperature of 0 to 200 ° C for a period of 2 to 8 hours. The removing group R1 can also be a diazo group, in which case the fluorinating agent used is fluorine hydrogen / pyridine. Suitable solvents for the reaction when R 1 is a diazo group are aprotic solvents such as diethyl ether, tetrahydrofuran and pentane. The reaction time ranges from 30 minutes to 2b hours at a temperature ranging from -20 ° to 65 ° C. Thus, for example, a compound of the formula 17 as defined above, wherein R 1 represents a diazo-25 group, is added in a suitable aprotic solvent to a solution of hydrogen fluoride / pyridine and cool to -10 ° C. The reaction mixture is stirred vigorously at -10 ° C for 1 hour, warmed to 25 ° C in two hours and then poured over ice. The! | organic phase is separated, washed with a base, eg sodium bicarbonate, dried over magnesium sulfate and | concentrated under vacuum to obtain the appropriate fluoromethyl ketone derivative of the formula 12. The diazoketone derivatives, that is, the compounds of the formula 13, wherein R 1 represents a diazo group, | 35 can be obtained via the corresponding acid halide, i.e. a compound of the formula 18, wherein halogen- the example may be chloride and Z 'has the same meaning as in the formula 12. The acid halide, which is contained in an aprotic solvent, for example diethyl ether, tetrahydrofuran, pentane, hexane, benzene, dimethoxyethane or dioxane, is added to a solution of diazomethane in ether, which is was cooled to 20 ° C, followed by vigorous stirring at 25 ° C for 1 to 2 hours. The diazoketone derivative thus obtained can be recovered by standard methods, for example, by int. Evaporation of the solvent with subsequent purification by recrystallization or chromatography. Alternatively, the reaction mixture can be treated as described above with a suitable fluorinating agent without insulation.

The suitably substituted diazo-ketone derivative described above can be used to prepare compounds of the formula wherein R 1 is, for example, halogen, mesylate, tosylate, triflate, or trifluoroacetate, using well known methods. To obtain compounds of the general formula 13, wherein R 1 is halogen, 20, for example chlorine, bromine or iodine, msi treats the corresponding compound of the formula 13, wherein R 1 is a diazo group, in a suitable aprotic solvent, with aqueous hydrogen chloride, hydrogen bromide or hydrogen iodine. To obtain compounds of the formula, wherein R 1 is mesylate, tosylate, triflate or trifluoroacetate, the corresponding diazoketone derivative, wherein R 1 is a diazo group, is dissolved in a suitable aprotic solvent and treated with dilute sulfuric acid to give the corresponding benzyl methanol ketone derivative. Finally, the benzyl methanol ketone is esterified with an appropriate acid chloride or anhydride using methanesulfonic acid, p-toluenesulfonic acid, trifluoromethylsulfonic acid or trifluoroacetic acid. j

The acid halides, that is to say compounds of the formula 18, as described above, are known compounds which can be prepared from the corresponding acids. Thus result 'L ............................................. ................. ... ..... .............. ...

81 02 8 6 3 * * -16-, for example, treatment of the corresponding acid with thionyl chloride in an aprotic solvent, for example diethyl ether, tetrahydrofuran, benzene or dichloromethane, at a temperature of 0 ° C to the reflux temperature of the 5 solvent for 1 to 2 hours in the formation of the corresponding acid halide Optionally, treatment of the corresponding acid with oxalyl chloride in one of the above-described aprotic solvents at a temperature of 0 to + 0 ° C also for 1 to 2 hours to prepare the corresponding acid halide.

The compounds of the general formula (12), wherein R 1, R 1, 11 Y FCHg- and Z 'does not represent benzoyl-1HHC (Cl 2) 2 or alkanoyl-HHC (CH 2) gj, can also be obtained by treatment of a Compound of the formula 14, wherein Zg phthaloyl-N-R, R 10, R 13, 13, 13, 13, CH (CH 2) -, alkoxycarbonyl-CHC (CH 2) 2, benzyloxycarbonyl-MHC (CH 2) 3, - methylthioethyl or β-benzylthioethyl, is methyl and R 1 is chlorine, bromine or iodine, mesylate or tosylate. Thus, a compound of formula 1b is reacted with triphenylphosphine or tri (lower) alkylphosphine, for example 3-n-butylphosphine, in a solvent such as benzene, toluene, methanol, ethanol, acetic nitrile, tetrahydrofuran, diethyl ether or dimethoxyethane.

The reaction is carried out at a temperature ranging from 25 ° C to the reflux temperature of the solvent for 10 minutes to H8 hours. After cooling, the precipitate formed /

washed with solvent and recrystallized to obtain the corresponding phosphonium salt. The triphenylphosphonium or trialkylphosphonium salt is added to an excess (up to I

i | 25%) metallic sodium or lithium, dissolved in liquid amine monk, which has a catalytic amount of ferric nitrate

will be added. Stirring is continued for 10 minutes to 3 hours, after which the ammonia is evaporated under an inert atmosphere, for example nitrogen or argon. A suitable solvent is added, for example benzene, toluene, diethyl ether, tetrahydro-

81 02 8 6 3 <-17-furan or dimethoxyethane and 'collects the resulting sub- | I substituted methylidene phosphoran. The methylidene phosphoran is | treated with a lower alkyl ester of monofluoroacetic acid in a solvent such as benzene, toluene, diethyl ether, tetrahydrofuran 5 or dimethoxyethane. The reaction is carried out under an inert atmosphere, for example nitrogen or argon, at a temperature from 0 ° C to the reflux temperature of the solvent for 30 minutes to 2 hours. The reaction mixture is concentrated by distillation to yield an olefin. The olefin is treated with an inorganic acid in water, for example hydrochloric acid! or hydrobromic acid, or an organic acid, for example tri-fluoroacetic acid or p-toluenesulfonic acid, in the presence of a | co-solvent, for example tetrahydrofuran, diethyl ether or benzene, at 0 ° C for 30 minutes to 2b hours to the reflux temperature of the solvent. The amount of acid used can vary from a catalytic amount to a concentrated acid solution.

As in the general formula 1U, the expression 13 king phthaloyl-N-CH (CH - refers to the group of the formula [19], the expression alkoxycarbonyl-HCHCH2] - refers to the 0 RH_ | 113 group of the formula alkyl-OC-HHCHCH2CH - and the term 25 (13-benzyloxycarbonyl-LHCHCHCH2) - refers to the group of formula 20, wherein R ^ has the same meaning as in formula III and alkyl is a straight or branched group is with 1 to U carbon atoms.

Compounds of the general formula 12, wherein Y is FgCH-, are obtained by treatment of] / (ethylsulfinyl) methyl / thio / methane or (ethylsulfinyl) methyl / thio /. ethane with a suitable strong base, followed by alkylation with a suitable derivative of the formula: wherein Z 'has the same meaning as in formula 12 and R 2 chlorine, bromine, iodine, 81 02 8 6 3 -18- "" "......... i is mesylate or tosylate. The Z 'substituted sulfinyl derivative thus formed is treated with a suitable strong base, followed by alkylation with a suitable halomethyl compound, namely chlorodifluoromethane, bromodifluoromethane or difluoro-iodomethane The alkylation reaction is followed by hydrolysis using an acidic aqueous solution.

Suitable strong bases, which can be used in | the preparation of the difluoromethyl-substituted ketone derivatives j | as described above are sodium hydride, dilithium acetylide, lithium diisopropylamide, butyl lithium, potassium t-butoxide, | sodium t-butoxide, lithium t-butoxide, phenyl lithium, methyl! . . . . . . . ♦ i lithium, sodium amide, lithium amide and potassium hydride.

The alkylation reaction described in the preparation of the difluoromethyl ketone derivatives is carried out in a suitable solvent, for example tetrahydrofuran, diethyl ether, hexamethylphosphoric triamide, dimethyl sulfoxide or benzene. The reaction is carried out at -78 to 65 ° C for 30 minutes to 2 hours. Preferably, a temperature of about 1 + 0 ° C is used for the alkylation to difluoromethyl compound. The alkylated sulfinyl compounds which act as intermediates are recovered by quenching with a brine solution, followed by extraction with diethyl ether, dichloromethane or benzene.

These alkylated sulfinyl derivatives are recovered from the combined extracts.

Hydrolysis of the alkylated sulfonyl derivatives to the ketone is effected with an aqueous inorganic acid solution, for example hydrochloric acid, hydrobromic acid, perchloric acid or sulfuric acid in a solvent such as tetrahydrofuran, acetic acid nitrile, diethyl ether or benzene. The hydrolysis is carried out at -20 ° to 105 ° C, preferably at 25 ° C for 30 minutes to 2b hours, preferably for 2 hours. In general, a solution of 0.3 equivalents of mineral acid in 1.5 is used. i

% water. The examples below provide further explanation! to the preparation of the difluoromethyl ketone derivatives of the formula - 12. I

i i ................. ..................._ .. ..... .... .................................................. .................................... .............. .....! 81 02 8 6 3 -19-

The compounds of formulas and wherein R and are halogen are well known or can be prepared from a corresponding derivative of acetic acid of formula 1 Rig I13 I13; 5 16, wherein Z ^ phthaloyl-MCH (CH ^) benzoyl-NHCH (CH ^) alkanoyl-R-10 R R-iq I13 M3 I13 KHCH (CH2) -, alkoxycarbonyl-NHCH (CH ^) -, benzyloxycarbonyl-WHCH (CH 2) -, methylthiomethyl or benzylthiomethyl. These acids are well known or can be obtained in a known manner from the corresponding unprotected amino acids. The compounds of formulas 1H and 15, wherein R 1 and R 2 are mesylate or tosylate, can be prepared by treating the corresponding derivatives, wherein R 1 and R 3 are halogen, with a metal salt of 15-metal sulfonic acid or p-toluenesulfonic acid. For example, one can use the sodium salt of methanesulfonic acid or p-toluenesulfonic acid.

Reduction of the ketones of formula 12 to the corresponding alcohols is obtained chemically using 1 to 10 equivalents of metal hydride, for example lithium borohydride, sodium borohydride, sodium cyanoborohydride or lithium aluminum hydride. In addition, the ketones can be reduced with boran or dimethylthioboran or catalytically reduced using, for example, Raney 25 nickel, rhodium, palladium on carbon or platinum oxide. In general, the reaction time ranges from 10 minutes to 2 hours and the temperature at which the reduction is carried out can vary from - 0 ° C to 100 ° C depending on the particular reducing agent used. When reduction with hydride or boran 30 is used, the reaction is carried out in a suitable solvent for 10 minutes to 2 hours at -0 ° C to 65 ° C. Suitable for the reduction of compounds of the general formula II Solvents to be used are lower alcohols such as methanol or ethanol, or ethers such as diethyl ether or tetrahydrofuran. When using catalytic reduction, the reaction time varies from 1! 81 02 8 6 3 -20 to 2b hours and the reaction temperature from 25 to 100 ° C, while | the reaction pressure can vary from 1 to 120 atmospheres.

| Hydrolysis to the amine and removal of any any distal amine protecting group is effected j 5 with a strong mineral acid such as hydrochloric acid, hydrobromic acid j | or sulfuric acid or an organic acid such as toluene sulfonic acid or trifluoroacetic acid. The hydrolysis is carried out in water or an aqueous solvent at the reflux temperature for 1 * to 8 hours. Optionally, 1 to 3 equivalents of hydrazine, 10-methylhydrazine or methylamine can be used at a temperature of 25 ° C to the reflux temperature of the solution for 1 to 12 hours, followed by treatment with a strong inorganic or organic acid as described above.

Compounds of formula 1, wherein ZH represents N-CH (CH) 15 & 3, R 1 is hydroxyl and YC = CH, or as mentioned below represents an alkynyl group, are prepared by hydrolysis of the alkylated compounds described above 5 · The necessary alkyl RgX teaching aids which are used can be prepared according to well known methods. It is thus possible to prepare the reagent of formula 21 by reacting 3-bromo-n-propylamine hydrochloride with benzaldehyde and an organic trialkylamine, for example triethylamine, in a solvent such as diethyl ether, tetrahydrofuran, dioxane, chloroform or dichloromethane. The reagent of formula 22 is prepared by reacting 3-aminobutyl bromide hydrobromide with benzaldehyde and an organic amine such as triethylamine. The compound 3-aminobutyl bromide hydrobromide is a known compound which can be prepared from the corresponding alkanol by treatment with concentrated HBr at 25 ° C | 30 to 110 ° C for 1 to 12 hours. The γ-aminoalkanol derivative j! is obtained by treatment of a corresponding β-j

ketoalkanoic acid ester of the formula 23. I

i

The β-ketoalkanoic acid ester is treated with hydroxyl amine to form the corresponding oxime which is reduced with lithium aluminum hydride in ether or tetrahydrofuran for 1 to 12 hours at a temperature of 25 ° to 50 ° C. Subsequent hydrolysis of the ester residue leads to the formation of | the γ-aminoalkanol.

The alkylation reaction can be carried out in an aprotic solvent, for example benzene, toluene, ether, tetrahydrofuran, dimethyl sulfoxide or hexamethylphosphoric triamide. The reaction temperature ranges from -100 ° to 25 ° C and is preferably -70 ° C and the reaction time ranges from half an hour to S 2 h hours. | Removal of the protecting groups as shown in the reaction scheme at the step going from compounds 5 to. the desired amines is accomplished by treatment with aqueous acid, for example hydrochloric acid, followed by treatment with aqueous base, for example sodium hydroxide or potassium hydroxide, or treatment with phenylhydrazine, hydroxylamine or hydrazine and then with aqueous base.

The individual optical isomers of compounds of formula 1, wherein R 1 is carboxyl or hydrogen, are cleaved using a (+) or (-) binaphthyl phosphoric acid salt according to the method of R. Viterbo et al., Tetrahedron Letters 8, 1T (1971). Other splitting agents such as (+) camphor-10-sulfonic acid may also be used. Optionally, when Z has the meaning H N-CH- (CH) or H N (CH), cleavage is obtained via 2 CH 2 2 2 2 2 n the lactam of these compounds. The acids and amines thus split can be used in the same manner as described above for the racemic mixtures.

The present compounds can be used to inhibit protozone growth in animals. The term "animals" refers here, inter alia, to mammals such as mice, rats, guinea pigs, rabbits, ferrets, dogs, cats, cows, horses and primates, including humans. The term animals also includes both fish and poultry. The term "poultry" is intended to include birds of all species I35 and both sexes, including parrots and canaries, but mainly poultry which is of commercial interest in connection with eggs and meat. Thus, the term "poultry" particularly refers to hens, I roosters and drakes of chickens, turkeys and ducks. j | 5 The term "protozoon" is considered to be members! include the subphila Sarcomastigophora and Sprozoa of the phylum Protozoa. The term "protozoa" as used herein refers in particular to the genera of parasitic protozoa which are important to humans because they cause diseases in humans or their pets. These genera are mainly classified in the superclass of Mastigophora of the subphilum Sarcomastigophora and the class of Telosporea of the subphilum Sporozoa in the classification according to Baker (1969). Good examples of genera of these parasitic protozoa are Histomonas, Trypanosoma, Giardia, Trichomonas, Eimeria, Isopora, Toxoplasma and Plasmodium.

Excluded from the superclass of Mastigophora is the genus Leishmania, of which certain species cause the tropical disease Leishmaniasis in humans. Also notably excluded from the genus Trypanosoma, as intended according to the invention, are the species Trypanosoma cruzi, which cause Chagas disease in humans and the species Trypanosoma lewisi. The present compounds have not been found to be particularly effective against these species.

On the other hand, the compounds of the formula I can be used particularly well to inhibit the growth of Trypanosoma brucei, the causative agent of nagana, or the tsetse-fly disease in horses and corn cattle in Central Africa. The present compounds are also remarkably effective. in the inhibition of Eiméria tenella, a protozoan species, which causes coccidiosis in birds. j

A preferred embodiment of the present; In fact, the invention is the use of these compounds in particular growth of intestinal coccidia in commercial plume; 35 cattle. The economic importance of intestinal coccidia is of great importance 81 02 8 6 3 -23- meaning. For example, in 1972 the estimated loss to the poultry industry in the United States, due to coccidial infections, was approximately $ 17 million. Because of i

I I

j the rapid development of drug resistance in | Coccidia and because of the relatively high toxicity of some drugs used in the treatment of coccidiosis. . . There is a need for effective coccidiostats, which are non-toxic and to which intestinal coccidia do not develop rapid resistance.

[10] It is not entirely clear how the compounds of the invention can inhibit protozone growth. The present compounds include irreversible inhibitors of ornithine decarboxylase and S-adenoxylmethoinine decarboxylase.

As irreversible inhibitors of these enzymes, these compounds inhibit polyamine formation, which may be required for protozoan cell division. In any event, the present invention is not limited to any particular conception or theory of action by which the compounds of the invention can effectively inhibit protozone growth.

The effect of the compounds of general formula 1 on protozoan growth and more particularly on the growth of coccidia can be demonstrated with Eimeria tenella and two-week-old male white leghorn chicks as test animals. The animals are kept in batteries and both the infected and non-infected birds are housed in separate rooms to ensure that they retain coccidia-free birds.

Each experimentally infected bird receives 100,000 splayed oocysts through the throat probe. The connection to be tested is administered at the determined desired dose via the drinking water and drug free compound feed is provided ad libitum. To evaluate the effect of the active ingredient on E. tenella infections, the chickens are killed with carbon dioxide and generally examined on the fifth day of the study, evaluating the ceca lesions.

The inhibition of protozoan growth can also be assayed with Trypanosoma brucei brucei, which is the pathogen of bovine trypanosomiasis (nagana) in Africa. The related species Trypanosoma brucei rhodesiense and Typanosoma brucei gambiense are the causative agents of African sleeping sickness in humans.

In general, drug activity is tested on established infections of a pleomorphic EATRO 110 iso! let of T. b. brucei with the mouse. Laboratory animals are infected I 5 i i with 5 x 10 parasites, 20 hours before the test. Thus gemfek- | j j | laboratory animals generally die 5 days after vaccination, j

| The compound to be tested is presented to the test animals in different I

•. Only doses are administered via the drinking water. Animals cured from the infection remain parasite-free for more than 30 days after the control animals die, as shown in a blood smear study.

The present compounds are used in effective amounts by inhibiting protozoan growth.

These amounts, of course, depend on various factors, such as the type and nature of the protozoan infection, the activity of the particular compound, the age, sex and species of the treated animals, and whether the treatment is prophylactic or is therapeutic. In general, the present compounds can be administered orally or parenterally in one. daily dose from 5 mg / kg to 7 g / kg. Preferably in case of Trypanosoma infections the dose range is 600 mg / kg to 2 g / kg. In case of Eimeria infections, the dose can be reduced, ranging from 15 mg / kg to 1 g / kg.

Because of the low toxicity of the present compounds, the compounds can be safely administered via the drinking water of the test animals in the treatment of coccidiosis in birds. Generally, concentrations are active 0.01 to 2% is appropriate, primarily depending on the nature of the protozoan infection to be treated, and whether the treatment is prophylactic or therapeutic, the severity of the infection and the duration of treatment.

Thus, for example, the compound 2-difluorom-8102863-25-2,5-di-aminopentanoic acid for the treatment of coccidiosis can be effectively administered to chickens 1 day before infection. If necessary, a prophylactic treatment! use split time 8 days before the infection using concentrations of 2-difluoromethyl-2,5-diaminopentanoic acid of only 0.015 ° C in the drinking water of chickens. Preferably, a prophylactic concentration of 0.06% to 1.0% is used. This prophylactic treatment to inhibit | protozoan growth means one of the major advantages of using the present decarboxylase inhibitors. So ! in coccidial infections in chickens Eimeria tenella grows intracellularly in the epithelial cells of the caecum as a trophozoic stage. These cells then undergo multiple mitosis to form a large number of merozoites. These mero-15 proteins are released as the host cell lyses and serve to extensively infect unaffected cells. The result is that the wall of the caecum is severely damaged, leading to severe blood and fluid loss and ultimately death. In addition, resistant oocytes are produced during the life cycle of E. tenel-20a, which are stored in the faeces of chickens. Because chickens are coprophagous in nature, the disease spreads quickly through contamination from their food source. Accordingly, coccidial infections in commercial colonies, when they occur, are treated epidemically with large doses of currently available chemotherapeutics, which are essentially killing. As a result, medicated feed is now regularly used in commercial colonies, so that all commercial poultry is now receiving almost constant drug to prevent the outbreak of coccidiosis.

The fact that the present decarboxylase inhibitors1! can be administered prophylactically allows the host can then enzymatically overcome a naturally or artificially generated infection through an inhibition mechanism, ; 35 instead of through a killing effect. Thus in case of! 81 02 8 63 -26- i; i! In an E. tenella infection, the infection is kept short so that the host can provide himself with his own body defense mechanism. The resulting antibodies, produced via such controlled infection, serve to further permanently immunize the host against future E. tenella.

| infections. I

The pharmaceutical preparations particularly suitable for the prophylaxis or treatment of poultry protozoan infections include the above-described α-substituted 10 amines or α-substituted-α-amino acids in combination | i with a pharmaceutically acceptable carrier. The anti-photo kissable compositions can be prepared well by mixing the active compound with an inert carrier. Good examples of carriers are talc, clay, pumice, silicon oxide, lime, diotomic earth, note-15 shell flower and equivalents thereof. Optionally, the active ingredient can be mixed with a commercially available powder or vitamin and mineral admixture, which is particularly suitable for birds.

In most cases, a concentrated solution of the active ingredient in water is used in the handling and treatment of coccidiosis in birds. Most of the present compounds are highly soluble, especially in the form of their salts. Such solutions may well contain preservative agents such as parabens, benzyl alcohol, phenol or thimerosal. In addition, one can make good use of isotonic agents, sugars, stabilizers and buffering agents.

The compounds of the formula I can be used in combination with other known drugs currently used for the chemotherapy and chemoprophylaxis of diseases caused by parasitic protozoa. Generally, this has the effect of decreasing the amount of enzyme inhibitors administered. Such drugs include: Antrycide ,, quinapyramine, Berenil, Diminazene eceturate, Penta- iriidine isethionate, Primaquine, Tryparsamide, Amicarbalide, j i ...... 35 Amprolium, Amphotericin B, quinine, Monensin, Minocycline, 7- di- | 81 02 8 6 3 -27-methylamino-6-demethyl-6-deoxytetracycline, Clindamycin, 7-deoxy-7 (S) -chlorlincomycin, Buquinolate, Robenidine and Nicarbazin. In some cases, the compounds of formula I even potentiate or potentiate the effects of these drugs.

5 Of particular significance in this respect is the compound 2,5-diamino-2-difluoromethylpentanoic acid, of which is | has been shown to act synergistically with the antiprotoenzoic agents Antrycide, quinapyramine, Pentamidine isethionate and Amicarbalide. j

Thus, the 2,5-diamino-2-difluoromethylpentanoic acid concentration j 10 can be reduced by about fourfold when used in bowl; combination with sub-curative doses (less than 1.0 mg / kg) of these drugs.

In addition, the compounds of formula I can be used in combination with other cytotoxic agents for the chemotherapy and chemoprophylaxis of parasitic diseases, in particular trypanosomiasis. Such cytotoxic agents include the antineoplastic antibiotic Bleomycin as well as other known cytotoxic agents, for example cyclophosphamide, methotrexate, prednisone, 6-mercaptopurine, procarbozine, dauno-20 rubicin, vincristine, vindesine, vinblastine, chlorambucil, cytosine arabinoside, 6-thiooguanide 5-fluorouracil, 5-fluoro-2-deoxyuridine, 5-azacytidine, nitrogen mustard, 1,3-bis (2r chloroethyl) -1-nitrosourea (BCFU), 1- (2-chloroethyl) -3-cyclohexyl-1 nitrosourea (CCNU), busulfan and adriamycin.

Of particular significance in the treatment of trypanosomiasis in general and in particular in the treatment of nagana in corn cattle is the use of the enzyme inhibitor 2,5-diamino-2-difluoromethylpentanoic acid in combination with the anti-tumor antibiotic Bleomycin. This particular enzyme inhibitor 30 appears to act synergistically with Bleomycin. Thus, mice infected with Trypanosoma brucei after three days are cured after daily intraperitoneal administration of Bleomycin in I at a dose of 7 mg / kg. Likewise, trypanozoom infections become! ! _ j! cured in the mouse by the administration of a 1% solution, Γ 35 of 2,5-diamino-2-difluoromethylpentanoic acid in the drinking water for three days.

The results of various combination tests indicate that permanent healing is obtained with 0.5 mg / kg of Bleomycin in combination with 0.5% 2,5-diamino-2-difluoromethylpentanoic acid administered via drinking water. Optionally, cure is also obtained with concentrations of 0.25 mg / kg of Bleomicin in kombina-! with only 0.25% 2,5-diamino-2-difluoromethylpentanoic acid in the drinking water. A combination of 0.1 mg / kg Bleomycin and 0.1% 2,5-diamino-2-difluoromethylpentanoic acid has no effect. Thus, the curative dose combinations reflect a reduction in Bleomicynia dose of 1/2 to 1/28 of the curative dose of the medicament used separately, when used in combination j with a subcurative dose of 1/2 to 1 / H of the curative dose of 2,5-diamino-2-difluoromethylpentanoic acid.

The following examples illustrate the invention.

Example I

2-Difluoromethyl-2,5-diaminopentanoic acid.

500 ml of a solution of 2M-20 butyl lithium in hexane are added with stirring to a solution of 1 ^ 3.1 ml of diisopropylamine in 1.5 liters of tetrahydrofuran at -78 ° C, followed by 261 g or 0.81 mol of ornithine dibenzaldimine methyl ester in 1.5 liters of tetrahydrofuran. After everything has been added, the reaction temperature is brought to 10 ° C and maintained between 0 and 50 ° C for 3 hours, during which time chlorine difluoromethane gas is bubbled through the mixture with stirring. The reaction mixture is then treated with a saturated sodium chloride solution.

The organic material is extracted with ether and the. ether extract washed several times with sodium chloride solution, dried over magnesium sulfate and evaporated to 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 heated with reflux condenser with 1.5 l of 12N hydrochloric acid for 16 hours and the cooled solution clarified

| 35 by chloroform extraction for concentration, decolorization (active I

8102863-29) and further concentration to 750 ml. The pH of the solution is adjusted to 3.5 by adding triethylamine and the active carbon solution again treated for concentration up to .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 by dissolving it in 150 ml of hot water and treat the solution with 1 + 50 ml hot ethanol.

; | ; On cooling, crystals separate from 2-difluoromethyl-2,5-! diaminopentanoic acid hydrochloride monohydrate, 71 g (37%), melting point 183 ° C.

Example II

q-Ethynyl-Qt, 6-diaminovaleric acid.

11.8 g or 0.0 ^ + 8 MI- (3-trimethylsilyl-A2-15 propynyl) -benzene carboximidate in 20 ml of tetrahydrofuran is added to lithium diisopropylamide prepared from 1.9 g or 6.78 ml 0.0 ^ 8 M di-isopropylamide in 60 ml of tetrahydrofuran and 23.6 ml of 2.05 M solution of n-butyl lithium at -70 ° C, after which 9.5 g or 0.0U2 of M (3-bromopropyl) benzylimine are added and the mixture is stirred at 20 -70 ° C for 5 hours. 23.6 ml of 2.05 M are added to the reaction mixture

n-butyl lithium solution, followed by addition of 1 +, 5 g or 3.67 ml of 0.01 + 8 M methyl chloroformate. After 30 minutes at -78 ° C, the mixture is treated with brine and the reaction product is recovered by ether extraction. The ether extract is evaporated and 300 ml of 3N HCl is added to the resulting residue and the mixture is refluxed for 7 hours. On cooling, the mixture is washed well with methylene chloride, made alkaline and washed again. The aqueous solution is acidified and concentrated to dryness. The residue is triturated with ethanol, filtered and the ethanol is evaporated. The residue is dissolved in water, the pH adjusted to 6 and the solution over j

4. # I

a column of Amberlite 120 H passed under eluermg with 1 M NH 2 OH, which, after recrystallization from ethanol-water, gives α-ethynyl-α, 6-diaminovaleric acid, mp 168-169 ° C under decomposition.

In the above process, N- (s-bromopropyl) benzylimine is prepared in a well known manner from 3-bromopropylamine and benzaldehyde.

i

Example III

1-Fluoromethyl-1-butanediamine dihydrochloride.

To a solution of mmol0 mmol diazomethane in 110 ml ether, cooled to 0 ° C and stirred magnetically, a solution of 20 ml of 1-phthalimidobutyryl chloride in 75 ml is added dropwise under nitrogen over a period of 10 hours. ml of ether. Stirring is continued for 1 hour at 25 ° C, after which the reaction mixture is added to a solution of +0 ml HF / pyridine, which has been previously cooled to 0 ° C. The resulting heterogeneous mixture is stirred at 25 ° C for one hour and then poured onto ice water. The ether phase is separated, washed with a bicarbonate solution, then brine, and dried over magnesium sulfate. Concentration of the solvent under reduced pressure yields a solid which is recrystallized from diethyl ether / pentane to yield fluoromethyl-3-phthalimidopropyl ketone, mp 92 ° C.

To a solution of 550 mg or 2.2 mmol of fluoromethyl-3-phthalimidopropyl ketone in a mixture of 5 ml of tetrahydrofuran and 5 ml of methanol cooled at 420 ° C, a solution of 0.8 mmol of sodium borohydride is added in a mixture of 5 ml of tetra-25 hydrofuran and 5 ml of methanol previously cooled to -20 ° C.

The reaction mixture is stirred at -20 ° C for 15 minutes and then neutralized to pH 1 with 2M HCl. The solvents are evaporated under reduced pressure and the residue is partitioned between water and chloroform. The organic phase is washed with brine, dried over magnesium sulfate and concentrated to a residue which is re-mixed; crystallized from tetrahydrofurandi ethyl ether to obtain 1-fluoro-5-phthalimido-2-pentanol, mp 85 ° C. A mixture of 26U mg or 1.05 mmol 1-fluoro-5-phthalimido-2 pentanol! 170 mg or 1.05 mmol of the phthalimide, 302 mg or 1.05 mmol of tri-; Phenylphosphine and 201 mg or 1.15 mmol of diethylazodicarboxylate in 8102863-3 8 ml of tetrahydrofuran are added under nitrogen for 2 hours at 25 ° C | stirred J. The solvent is evaporated under reduced pressure and the residue is taken up in benzene. The insoluble matter is discarded and the residue obtained is recrystallized from tetrahydrofuran diethyl ether after concentration of the filtrate under Obtaining 1-fluoromethyl-1, k-butanediyl-bis-phthalimide, melting point 112 ° C. A suspension of 3.1 g of 1-fluoromethyl-1,1 + -butanediyl-bis-phthalimide in 1 ml of concentrated HCl is heated on a reflux condenser for 3 days. The phthalic acid, which precipitates upon cooling to k ° C, is filtered off. The filtrate is concentrated to 20 ml and cooled to k ° C. The remaining phthalic acid which separates is filtered off and the filtrate concentrated under reduced pressure. The residue is treated 3 times with 10 ml of boiling isopropyl alcohol and then recrystallized from absolute ethanol to give 1-fluoromethyl-1, U-butanediamine dihydrochloride, melting point 15 ° C.

Example IV

1-Fluoromethyl-methyl-1-butyl amine dihydro chlorine.

To a solution of Uo mmol diazomethane in 110 ml ether, cooled to 0 ° C and stirred magnetically, a solution of 20 ml U-phthalimido-U-methylbutyryl chloride is added dropwise under nitrogen over a period of 1 hour. in 75 ml ether. Stirring is continued for 1 hour at 25 ° C, after which the reaction mixture is added to a solution of ko ml HF / pyridine which has been previously cooled to 0 ° C. The resulting heteror-

i. . O

The resulting mixture is stirred at 25 ° C for 1 hour and then poured onto ice water. The ether phase is separated, washed with a bicarbonate solution and then with brine and dried over magnesium sulfate. Concentration of the solvent under reduced pressure yields a solid which is recrystallized from diethyl; ether / pentane to obtain fluoromethyl-3-phthalimido-3-methylpropyl ketone. j ί

With a solution of 550 mg or 2.2 mmol of fluoromethyl-i! 3-phthalimido-3-methylpropyl ketone in a mixture of 5 ml of tetrahydroj 8102863 -32-furan and 5 ml of methanol cooled to -20 ° C, a solution of 0.8 mmol sodium borohydride is added in a mixture of 5 ml of tetrahydrofuran and 5 ml of methanol pre-cooled to -20 ° C. The reaction mixture is stirred at -20 ° C for 15 minutes and then neutralized to pH 1 with 2M HCl. The solvents are evaporated under reduced pressure and the residue is partitioned between water and chloroform. The organic phase is washed with brine, dried over magnesium sulfate and concentrated to a residue, which is recrystallized from tetrahydrofuran-diethyl-10 ether to give 1-fluoro-5-phthalimido-5-methyl-2-pentonol. A mixture of 26mg or 1.05mmol 1-fluoro-5-phthalimido-5-methylpentanol, 170mg or 1.05mmol of the phthalimide, 302mg or 1.05mmol triphenylphosphine and 201mg or 1.15mmol diethyl azodicar boxylate in 8 ml of tetrahydrofuran is stirred under nitrogen at 25 ° C for 2 hours, the solvent is evaporated under reduced pressure and the residue is taken up in benzene. The insoluble matter is discarded and the residue obtained after crystallization of the filtrate recrystallized from tetrahydrofuran diethyl ether to give 1-fluoromethyl-1-methyl-1,4-butanediyl bisphthalimide. A suspension of 3.1 g of 1-fluoromethyl-U-methyl-1, w-butanediyl-bis-phthalimide in 1 ml of concentrated HCl is heated to a reflux condenser for 3 days. The phthalic acid which precipitates on cooling to 1 ° C is filtered off. The filtrate is concentrated to 20 ml and cooled to 4 ° C. The remaining phthalic acid, which separates, is filtered off and the filtrate is concentrated under reduced pressure. The residue is treated 3 times with ice-0 ml of boiling isopropyl alcohol and then recrystallized from absolute ethanol to give 1-itluoromethyl-methyl-1-U-butanediamine dihydrochloride.

30

Example V

1-Ethynyl-H-methyl-1,1-butanediamine.

To 10.8 g or 0.05 M 3-trimethylsilyl-A2 propynyl-1-iminobenzyl in 500 ml tetrahydrofuran, 0.05 M n-butyl lithium is added under nitrogen -35 atmosphere at -78 ° C. After 10 minutes, treat i_________________________________________ _____________________. .. ..........

The dark red carbanion is divided with 11.3 g or 0.05 M U-bromo-2-iminobenzylbutane in 20 ml of tetrahydrofuran. After 3 hours at -18 ° C, 50 ml of water are added and the tetrahydrofuran is evaporated, leaving a residue, which is heated under reflux with 100 ml of 6N hydrochloric acid for 5 hours under a nitrogen atmosphere.

After cooling, the aqueous solution is washed with methylene chloride, made alkaline with aqueous sodium hydroxide, and dissolved. newly extracted with methylene chloride. The methylene chloride extract is dried over magnesium sulfate, filtered, concentrated and distilled to give 1-ethynyl-A-methyl-1-butanediamine.

Example VI

Granules, suitable for addition to the drinking water of poultry, are prepared as follows:

Grams 2-difluoromethyl ~ 2,5-diaminopentanoic acid 33.0 corn starch 18.5 20 lactose b8.2 zinc stearate 0.3 100.0

The 2-difluoromethyl-2,5-diaminopentanoic acid is mixed with 6 to 9 g of lactose and passed through a fluid energy mill or micronizer to obtain a particle size of 1 to 25 microns. 35 ml of water are added to 2.0 g of the corn starch and mixed to give a 5% starch paste. The micronized 2-difluoromethyl-2,5-diaminopentanoic acid lactose powder, the remaining lactose and the remaining corn starch are mixed well. The starch paste is added and mixed and the resultant mixture is passed through a sieve with | mesh width 1.68 mm. The resulting granules are dried at 38 ° C to a moisture content of 3%, passed through a 1.68 mm mesh sieve and lubricated by mixing with 0.3 g of zinc stea. 35 comb.

8102863 -3b-

Example VII

A "10% stock solution for use" is prepared in the treatment of coccidiosis by dissolving 37.5 g of 2-difluoromethyl-2,5-diaminopentanoic acid in 3.78 L of chamber water. 5 temperature. Part of this stock solution, diluted with! 9 parts of water, resulting in the preparation of a 1% drug solution in poultry drinking water, which can be used

.... I

! for the prevention of coccidiosis in poultry. I

i! j. !

Example VIII

A drug-containing poultry feed is prepared using the following ingredients. The birds can be fed ad libitum with the drug-containing feed.

15

Wt. %

Corn flour 60.3

Soybean meal 33.0

Alpha leaf flour 1.0 20 Dicalcium phosphate 3.0

Calcium carbonate 1.0

Iodine-containing salt 0.2 2-difluoromethyl-2,5-diaminopentanoic acid 0.33

Vitamin-mineral-amino acid-antibiotic mixture to provide 25 of the following per ^ 5 kg of feed:

Oxytetracycline 0.5 g

Penicillin (as procaine salt) 0.25 g

Manganese sulfate 8 'g DL-methionine 22.7 g 30 Riboflavin 130 mg DL-calcium pantothenate 1930 mg j Niacin 1 ^ 00: mg I Pyridoxine 130 mg i | Vitamin B 1 mg

I Ί Z

| 35 Choline chloride 2 297 g 81 02 8 6 3

V

4 -35- ....... ...... ...................... ".......... . ........................ ......................... ............................................

Vitamin A 300,000 units

Vitamin D ^ 25,000 units

I Example IX

I lil · ι ·· ι i. » »- - i 5 The following serves to explain the effect! i | of 2-difluoromethyl-2,5-diaminopentanoic acid on Trypanosoma brucei brucei infections in mice.

Groups of 5 mice of 20-25 g are inoculated with T. b. brucei (EATRO.110 isolate, 5 x 10 ^ organisms mouse).

The compound is administered ad libitum via drinking water, 2b hours after the infection. Results are expressed as mean survival (in days) after control animal death, based on an average control animal survival of 5 days. Berenil (diminazene aceturate) has been included as a control tripanocide. The 15 results are shown in Table A below.

Table A

Medicinal product Treatment method Total do- Average over- 20 _______ sis (mg) survival (days)

None 0 0 2-difluoromethyl-2,5-diaminopentanoic acid 2% in drinking water, 6 days 600 -> 30 ^ - „2% in drinking water, 3 days 300 -> 30 1% in drinking water, 6 days 300 -> 30 .1 % in drinking water, 3 days 150 -> 30 jai 0.5% in drinking water, 3 days 75 - 28.6 | a ! 0.1% in drinking water, 3 days 15 ~ 2; 2q 300 mg / kg p.o., 3 days a day 22.5 26.3 150 mg / kg p.o., 3 days a day 11.3 22.8

75 mg / kg p.o., daily 3 days 5.6 19.2 J

50 mg / kg p.o., 3 days a day 3.8 0 y yl ..... 25 2-methyl-2,5-diaminopentanoic acid; 8102863 -36- 2% in drinking water, 3 days 300 ^. 0 diminazene aceturate: i 2.5 mg / kg i.p. daily, 3 days 0.2> 30 y 5 _ y - Based on a daily intake of 5 ml water / 25 g mouse / day - Considered curative. The animals survived the control-10: run by more than 1 month. Blood smears were negative for parasites after 1 month i. Subinoculation of brain suspensions in uninfected animals remained negative after more than 30 days. !

i I

Example X.

The following example illustrates the effect of a 2% solution of 2-difluoromethyl-2,5-diaminopentanoic acid in the drinking water of chickens; infected with oocysts of Eimeria tenella.

Twenty chickens are infected per day with 100,000 oocysts of E. tenella per os. Ten of the animals received drinking water containing the 2% solution of 2-difluoromethyl-2,2-diamino-pentanoic acid. The remaining animals were used for control.

On day 3, all control animals showed clinical signs of the disease. On day 7, the animals were all sacrificed, and the ecological lesions were examined macroscopically and scored as follows: 0 = No observable macroscopic lesions.

+ 11 = Little spread petechiaè in the cecaal wall; no thickening of the wall and normal cecal content present.

+2 = Numerous lesions with perceptible loss in faecal content; cecal wall slightly thickened.

+3 = Large quantities: blood and tissue fragments i "35 present, ie cecal nuclei, cecal wall L ............................. .................................................. ...................................... 1 81 02 8 6 3 -37- highly thickened, little or no normal cecal content.

+1+ = Cecal wall strongly swollen with a lot of blood and cecal nuclei present. Cecale fragments absent 5 or contained in nuclei.

(Dead bird also falls under rating + ^.)

Table B

; 10 Injuries to individual animals. Average per group I jfontrole + h +3 +1 | +1+ + h +1 | +2 + h +1 | 3j6 (N = 10)

Treated with 2-difluoromethyl-2,5-diaminopentanoic acid; (N = 10) +1 +1 +0 +1 +1 +3 + k + k +1 +1 1.7 15

Example XI

Following essentially the same procedure as in the previous example, a 2% solution is administered to six chickens; of 2-difluoromethyl-2,5-diaminopentanoic acid (DFM0) in their drinking water 'PO. ; over a period of 3 days. On day 1 this group becomes | ! I of six chickens in addition to two groups of ten chickens per ox! infected with 100,000 oocysts EL tenella per chicken. One group of ten chickens served as a control test, the other group of ten chickens received a standard dose of Amprolium in drinking water for the following 5 days. Amprodium is now the cocci; diostat, which one chooses. On day 5, all animals are sacrificed and screened for disease using j | the same valuation of the injuries as in the previous example. The following results are obtained.

30

Table C;

Treatment Number of chickens Days of treatment Average treatment _______ _ Treatment assessment_ 35 Check 10 - 3.70 81 0 2 8 6 3 '1 -38-

2% DFMO

solution 6-30; 0.120 Amprolium i solution 10 5 0.3 I 5

Example XII

The following example illustrates the effect of different doses of 2-difluoromethyl-2,5-diaminopentanoic acid on chicken Eimeria tenella infections.

Following essentially the same procedure as in Example X, the dose of 2-difluoromethyl-2,5-diaminopentanoic acid (DEMO) is varied as shown in Table D below. Treatment with DFMO is started on day -1. The chickens are infected per day on day 0 and the treatment is continued for another 5 days, or 15 days in total. Animals are sacrificed on day 5 and examined for disease using the assessment system described in Example X.

i |

Table D

20!

I I

Treatment; 1; Number of chickens Days of average _________ _ action judgment_ j Control 7 6 3.29 | 2% DFMO 660 25 1.0% DFMO 6 6 0.5 0.5% DFMO 6 6 1.00 | Example XIII j i „i | The following example illustrates the effect of; a small prophylactic dose on Eimeria tenella lesions in chickens.

| Following essentially the same procedure as in the example; X, but varying the dose of 2-difluoromethyl-2,5-diamino-pentanoic acid administered and the period of administration, the following results are obtained.

81 02 8 6 3 f -39-

Table E

Treatment Number of chickens Days of treatment Average treatment -______ _______ operation judgment_

Control 9 - 3.33 5 2% DFMO 9 -1 to +5 0 1% DFMO 9 -1 to +5 0 0.5% DFMO 9 -1 to +5 0 0.25% DFMO 9 -8 to +5 0. ^ 1Q 0.125% DFMO 9 -8 to +5 0 0.0625% DFMO 9 -8 to +5 0.66

Example XIV

The following example relates to the acquisition of permanent immunity to Eimeria tenella infections in chickens.

Birds pre-treated with 2-difluoromethyl-2:, 5-diaminopentanoic acid in concentrations as low as 0.5% on days -8 to +5 relative to infection become 1 week I after completion of investigated the therapy as indicated in section 1! Table F. These results indicate that prophylactic therapy in small doses provides an adequate development of parasitic! allows in the absence of morbid conditions, thereby making it possible to develop an immunity to E. j | i 2 ^ tenella infections.

Table F

. j Treatment Number of chickens Days of average Average action final evaluation

30. at the evaluation grade_; _______gin ling _L

Control 9 - 0 2.50 1.0% DFMO 9 -8 to +5 0.33 0 0.5% DFMO 9 -8 to +5 0 0 35 8 102 863

Claims (10)

5. H2N- (CH2) 3, H2H-CH- (CH2) 2 or IIN-CH2CH = CH represents ™ 3 with the proviso that when hydrogen is Y cannot be CF_ and Z the meaning H H-CH- ( CH) o 2 Λττ 2 2 \ 3; 10 and their salts and individual optical isomers. 2. A compound according to claim 1, characterized in that R 1 represents hydrogen. ! Compound according to claim 1, characterized in; 15 which represents carboxyl. i +. Compound according to claim 1, characterized in that Z represents H. H-CH- (CH) or HJST- (CH). C.) | tt C. <- C. 2 · 3 3 Compound according to claim 1, characterized in that 20.7 is that Y has the meaning CHF2. ' 6. Solution in water, characterized in that it is one! α-substituted amino acid or α-substituted amine at a concentration of 0.01 to 2.0! Solution according to claim 6, characterized in! That they are 0 ', totβ to 1.0% α-substituted amino acid or α-substituted containing amine according to claim 1. Pharmaceutical or veterinary preparation for the prophylaxis or treatment of protozoan infections characterized in that it contains: I i 30 a. A protozoan-inhibiting amount of α-substituted amino acid or α-substituted amine of the formula 1: as defined in claim 1 or a salt or separate optical isomer thereof, optionally in combination with I ............... _ _; 81 02 8 6 3 -U1- t 'b. A suitable carrier. Preparation according to claim 8, characterized in that the α-substituted amino acid is 2,5-diamino-2-difluoromethyl-pentanoic acid. Preparation according to claim 8, characterized in that it is intended for administration to birds, the carrier is water and the α-substituted amino acid is 2,5-diamino-2-difluoromethyl-: pentanoic acid, present in a concentration of 0 , 01 to 2%. 11. A composition according to claim 8, characterized in that the α-substituted amino acid is 2,5-diamino-2-difluoromethyl-pentanoic acid in combination with a synergistically effective | amount of anti-protozoan agent, namely Anthycide, Pentamidine, Amicarbalide or Bleomycin: 15%; j | I j | j i i: i 81 02 8 6 3