JPH0432052B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION This invention relates to certain α-substituted amino acids useful for inhibiting protozoan growth in animals and particularly poultry. [Prior Art] Polyamines are deeply involved in many aspects of cell division. Attenuating the biosynthesis of polyamines with enzyme inhibitors appears to reduce cell proliferation in mammals. Although the physiological role of polyamines is not completely clear, there is evidence of a link with cell division and growth. HG Williams-
Ashman) et al., The Italian J.Biochem.25, 5~
32 pages (1976); A.Raina and J.Janne, Med.Biol.53, 121~
147 (1975) and DHR Russell, Life Sciences 13, pp. 1635-1647 (1973). Polyamines are produced by certain microorganisms, such as Escherichia coli,
It is also known to be an essential growth factor for Enterobacter, Klebsiella, Staphylococcus aureus, C. cadaveris, S. tybus, and H. parainfluenzae. There is evidence that polyamines are involved in normal and neoplastic cell growth in mammals, with increased synthesis and accumulation of polyamines following stimuli that cause cell proliferation. It is also known that there is a correlation between polyamine formation and ornithine, S-adenosylmethionine, arginine, and lysine decarboxylase enzyme activity. The term polyamine is
Uses include the diamine putrescine and the polyamines spermidine and spermine. Putrescine is a decarboxylation product of ornithine catalyzed by ornithine decarboxylase. Putrescine formation also occurs when arginine is decarboxylated to form agmatine, which is hydrolyzed to form putrescine and urea.
Arginine is also involved in ornithine formation through the action of the enzyme arginase. Activation of methionine by the enzyme S-adenosylmethionine synthetase forms S-adenosylmethionine, which is decarboxylated. The propylamine moiety of the activated methionine is then transferred to putrescine to form spermidine. Instead,
Transferring the propylamine moiety to spermidine forms spermine. Putrescine therefore serves as a precursor for spermidine and spermine. In addition, putrescine was shown to have a significant regulatory effect on the polyamine biosynthesis pathway.
It was also shown that an increase in putrescine synthesis is an early sign that the tissue is undergoing a new growth process. Cadavuerin, a decarboxylation product of lysine, has been shown to stimulate the activity of S-adenosylmethionine decarboxylase;
It is known to be essential for the growth process of many microorganisms, such as H. parainfluenza. The rationale for polyamine metabolism was proposed by Cohen, Science 205, 964 (1979). Due to the apparently unique role of polyamine metabolism in trisanosomes and their dependence on ornithine decarboxylase as a source of putrescine, certain ornithine decarboxylase inhibitors of polyamine synthesis may be highly effective in inhibiting protozoan growth. Our finding that there is is further support. SUMMARY OF THE INVENTION We have discovered that certain compounds belonging to the class of irreversible inhibitors of ornithine decarboxylase are useful in inhibiting the growth of protozoa. Additionally, the subphylum Sarcomastigophora and Sporozoa
This inhibition occurs across a wide range of protozoa, such as More specifically, the classes of compounds described below refer to members of the mastigophora super-class, specifically Trypanosoma brucei.
brucei brucei) and members of the Telosporea subclass, specifically Eimeria tenella, an organism that causes coccidiosis in poultry. . [Means for Solving the Problems] Compounds useful in the practice of the present invention have the general formula α-substituted α-amino acids or their salts and individual optical isomers. When administered in vivo to animals with active protozoal infections, compounds of the formula can be used to treat such animals by inhibiting further growth of the infected protozoa. Alternatively, the compounds can be administered prophylactically to prevent the occurrence of such protozoal infections. Examples of salts of the compounds of the invention are inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and methanesulfonic acid, salicylic acid, maleic acid, malonic acid,
Includes non-toxic acid addition salts formed with organic acids such as tartaric acid, citric acid, cyclamic acid and ascorbic acid. In addition to the salts mentioned above, the term salts is intended to include internal salts or Zwitzter ions of the compounds of the above formula which are amphoteric in nature. Furthermore, although the optical configuration of the compounds described herein is not specifically specified, it is recognized that the α-carbon atom has an asymmetric center and that individual optical isomers of these compounds exist. It will be done. Both the d- and l-enantiomers as well as racemic mixtures are therefore considered to be within the scope of this invention. Compounds useful in this invention are 2,5-diamino-2
-(difluoromethyl)pentanoic acid. Compounds of the formula are made by treating an ester derivative of ornithine with a strong base, when the amino group is suitably protected, to form a carbanion intermediate. Dimethyl sulfoxide,
dimethylformamide, dimethylacetamide,
With a suitable halomethyl-haloalkylating agent in an aprotic solvent such as benzene, toluene, ethers such as tetrahydrofuran, diethyl ether or dioxane at a temperature of about -120°C to 120°C, preferably about 25°C to 50°C. It takes about 30 minutes
Allow to react for 48 hours, followed by acid or base hydrolysis. This can be expressed by the following reaction order. In the above reaction sequence, Y is _F2CH2- ^, _R2 is _a lower alkyl group, such as methyl, ethyl, isopropyl, n-propyl or n-butyl, and _R3
is hydrogen, phenyl, a straight or branched alkyl group of 1 to 8 carbon atoms, methoxy or ethoxy; R ₄ is phenyl or a straight or branched chain of 1 to 8 carbon atoms; is an alkyl group, or
Taking R ₃ and R ₄ together gives an alkylene group of 5 to 7 carbon atoms, i.e. -CH ₂ -(CH ₂ ) _n -CH ₂ -(m
is an integer from 3 to 5). Examples of straight-chain or branched alkyl groups of 1 to 8 carbon atoms which R ₃ and R ₄ may represent include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl or triethylmethyl group. The symbol is Z ₁

【formula】

【formula】 or

[Formula], where R ₃ and R ₄ are the same and have the meanings defined above, and
Each of R ₅ and R ₆ is phenyl, benzyl or 1 to 4
a straight-chain or branched lower alkyl group of carbon atoms, such as methyl, ethyl or isopropyl, Z ₂ is H ₂ N(CH ₂ ) ₃ ^- ,

【formula】 or

[Formula] (R ₅ and R ₆ have the meanings defined above). Suitable strong bases that may be used in the above reaction sequence to create the carbanion intermediate are those that extract a proton from the alpha carbon atom for the carboxy group, such as alkyllithiums such as butyllithium or phenyl Lithium, lithium dialkylamides such as lithium diisopropylamide, lithium amide, tertiary potassium butyrate, sodium amide, metal hydrides such as sodium hydride or potassium hydride, tertiary amines such as triethylamine, lithium acetylide or dilithium acetylide It is. Lithium acetylide, dilithium acetylide, sodium hydride and lithium diisopropylamide are particularly preferred bases. Suitable alkylating reagents that can be used in the above reaction sequence are, for example, chlorofluoromethane, bromofluoromethane, fluoroiodomethane, chlorodifluoromethane, bromodifluoromethane, difluoroiodomethane, bromotrifluoromethane,
Chlorotrifluoromethane, trifluoroiodomethane, bromochloromethane, dichloromethane,
These are chloroiodomethane, bromodichloromethane, and dichloroiodomethane. These alkylating reagents are well known in the art. Removal of the protecting groups for the amine and carboxylic acid functions is accomplished in one step by treating compound 2 with an aqueous acid solution such as hydrochloric acid or toluenesulfonic acid at a temperature of about 0° to 100° C. for about 4 to 24 hours. achieved, yielding compounds of the general formula. When the amine function of compound 2 is protected as a Schiff salt, it may be treated with a dilute aqueous solution of an acid, such as hydrochloric acid, or in a solvent such as a lower alcohol such as methanol or ethanol, ethers, chlorinated hydrocarbons, benzene and water. Compound 2 was first prepared by treating compound 2 with hydrazine or phenylhydrazine.
It is preferred to remove the protecting group for the amine function of . Removal of the carboxylic acid functionality and the protecting group on the amine group when the amine group is protected other than as a Schiff base can be achieved by treating compound 3 with a concentrated aqueous solution of an acid such as hydrobromic acid.
at a temperature of about 0° to 100° C. or in an aqueous base such as ammonium hydroxide. Amine-protected ester derivatives i.e. R ₃
Compound 1, when is other than methoxy or ethoxy, is prepared by treating a suitable amino acid ester with a carbonyl-containing compound to generate a Schiff base by a generally known method, but specifically as follows: It is. (a) When R ₃ is hydrogen, benzaldehyde or a straight or branched alkanol having 1 to 9 carbon atoms, such as 1-propanal, 1-butanal, 2,2-dimethylpropan-1-al; or 2,2-diethylbutane-1
- Treat the appropriate amino acid ester with R.
(b) When R ₃ is phenyl, a suitable amino acid ester is substituted with benzophenone or
Phenyl alkyl ketones when linear or branched with ~8 carbon atoms, such as phenyl methyl ketone, phenylethyl ketone, phenyl isopropyl ketone, phenyl n-butyl ketone or phenyl tert-butyl ketone It will be processed. (c)
When R ₃ is a straight-chain or branched alkyl group having 1 to 8 carbon atoms, a suitable amino acid ester is substituted with the above-mentioned phenyl alkyl ketone or each alkyl moiety has 1 to 8 carbon atoms. with a dialkyl ketone, straight or branched, such as dimethyl ketone, diethyl ketone, methyl isopropyl ketone, di-n-butyl ketone or methyl tert-butyl ketone. Carbonyl-containing compounds are known in the art or made by procedures well known in the art. When R ₃ is methoxy or ethoxy in compound 1, benzoyl halide or alkanoic acid is 1-
Straight or branched alkanoic acid halides having 9 carbon atoms, such as acetyl chloride, propionyl chloride, butyryl chloride, tert-butyryl chloride, 2,
2-diethylbutyric acid or valeryl chloride is reacted with an appropriate amino acid ester derivative. ether,
The reaction is carried out at 0° C. in an organic solvent such as methylene chloride, dimethylformamide, dimethylacetamide or chlorobenzene in the presence of an organic base such as triethylamine or pyridine. After the reaction, the reaction mixture is warmed to about 25° C. for 1 hour. The resulting amide derivatives are treated as methyl fluorosulfonate, dimethyl sulfate, methyl iodide, methyl p-toluenesulfonate or trimethyloxonium hexafluorophosphate (R ₃ is methoxy) or triethyloxonium tetrafluoroborate (R ₃ is ethoxy). methylene chloride, at about 25 °C with a suitable alkylating agent.
Combine in a chlorinated hydrocarbon solvent such as chlorobenzene or chloroform. The reaction mixture is approx.
Reflux for ~20 h, cool to ~25 °C, and add an organic base such as triethylamine or pyridine before
The product is isolated by extracting the solution with brine. When R ₃ and R ₄ are taken together to form an alkylene group having 5 to 7 carbon atoms in compound 1, the corresponding amino acid ester derivatives can be converted into amino acid esters by procedures generally known in the art. is obtained by treating with a cyclic alkanone to form a Schiff base. Cyclic alkanones that can be used include cyclopentanone, cyclohexanone and cycloheptanone. The symbol Z ₁ of compound 1 is

[Formula] or

In the case of [formula], 1 to 6 amino acids are added in boiling water with an excess amount of copper salt such as copper carbonate.
By processing time,

[Formula] and

A protecting group is added to the corresponding free amino acid, ornithine. After cooling to room temperature,
The undissolved material is filtered and dissolved in a suitable acid halide (Z ₁ is

[Formula]) or a suitable alkyl or aryl haloformate (when Z ₁ is

Treat the filtrate with [Formula]). This treatment is followed by hydrogen sulfide treatment. Examples of acid halides that can be used include acetyl chloride, propionyl chloride, benzoyl chloride or 2-phenylacetyl chloride. Examples of halofluormates that can be used include benzyl chloroformate, phenyl chloroformate, methyl chloroformate or ethyl chloroformate. When R ₁ is carboxy, the lactam of the general formula compounds has the structural formula is made from the corresponding amino acid ester with In the formula, Y has the meaning defined in the formula, R ₇
is a straight-chain or branched alkoxy group of 1 to 8 carbon atoms, such as methoxy, ethoxy, isopropoxy, butoxy or hexyloxy. Lactams can be added to a suitable base such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium methoxide, potassium tert-butoxide, sodium amide, or a trialkylamine such as triethylamine. methanol, ethanol, isopropyl alcohol, n-
They are made by treating amino acid esters in solvents such as lower alcohols such as butanol, water, dimethylformamide, dimethylsulfoxide, hexamethylphosphortriamide, or mixtures thereof. The reaction is about 0° to 120°C
for 30 minutes to 24 hours, optionally under a nitrogen atmosphere. Compounds of general formula It is obtained by treating amino acids with alcohol. Individual optical isomers of compounds of the formula can be prepared using (+) or (-) binaphthyl phosphate according to the procedure of R. Viterbo et al., Tetrahedron Letters 48, 4617 (1971). Used to be split. Other resolving agents such as (+)syn-10-sulfonic acid may also be used. Instead, resolution is achieved via lactams of the above compounds. The acids and amines thus resolved can be used in the same manner as described above for racemic mixtures. The compounds described herein are useful for inhibiting the growth of protozoa in animals. The term "animal" is intended to include mammals such as rats, mice, guinea pigs, rabbits, weasels, dogs, cats, cows, horses, and primates, including humans, among others. The term animal also includes fish and birds. The term "bird" is intended to include any type of male or female bird, including parrots and canaries, but is primarily intended to include poultry raised commercially for eggs and meat. be. Thus, the term bird is intended to include hens, roosters, and roosters, chickens, chicks, turkeys, and ducks. The term "protozoa" is intended to encompass members of the phylum Sarcomastigophora and the subphylum Sprozoa. More particularly, the term "protozoa" as used herein is intended to include the genus of parasitic protozoa that are important to humans for causing disease in humans and livestock. Most of these genera are classified as Mastigophora in the subphylum Sarcomastigophora, according to Baker (1969).
It is classified in the superclass Telosporea of the subphylum Sporozoa. Examples of these parasitic protozoan genera are Histomonas, Trypanosoma, Gia-dia,
Includes Trichomonas, Eimeria, Isopora, Toxoplasma and Plasmodium. Excluded from the superclass Mastigophora is the genus Leishmania, some of which cause a tropical disease called leishmaniasis in humans. Also, specifically excluded from the trypanosoma genus used in the present invention is Trypanosoma cruzi, which causes Chagas disease in humans.
species, and Trypanosoma lewisi species. It has been found that the compounds described herein are not particularly effective against these species. On the other hand, compounds of the formula are particularly effective in inhibiting the growth of Trypanosoma brucei, the pathogen of Nagana and Tsetse fly disease in cattle and horses in Central Africa. The compounds described herein are derived from the protozoan species Eimeria tenella that causes coccidiosis in birds.
It is also extremely effective in inhibiting the growth of. Indeed, a preferred embodiment of the invention is the use of these compounds to inhibit the growth of intestinal coccidia in commercial poultry. The economic importance of intestinal coccidia is of great significance. That is,
In 1972, the estimated loss to the poultry industry from coccidial infections in the United States was approximately $47 million. Because drug resistance develops rapidly in coccidia, and because some of the drugs used to treat coccidial disease are relatively highly toxic, non-toxic drugs prevent intestinal coccidia from developing rapid drug resistance. There is a need for such effective coccidia inhibitors. It is not entirely clear how the compounds of the present invention can inhibit the growth of protozoa. In particular, the compounds described herein are irreversible inhibitors of ornithine decarboxylase and S-adenoxylmethionine decarboxylase. As irreversible inhibitors of these enzymes, these compounds block the formation of polyamines required for protozoan cell division. In any case, the practice of the present invention is not limited to any particular mode of action or theory of action that the compounds of the present invention can effectively inhibit the growth of protozoa. The effect of the compounds of the general formula () on the growth of protozoa, especially on the growth of coccidia, can be demonstrated using Eimeria tenella and two-week-old male white leghorn chicken chicks as test animals.
Chickens were kept in chicken boxes, and infected and uninfected chickens were placed in separate rooms to ensure chickens that had never been infected with coccidia. Each experimentally infected chicken is fed 100,000 sporulated oocysts. Test compounds are administered at the desired specific dose via drinking water and drug-free mash is provided ad libitum.
To determine the effect of the active ingredient on E. tenella infection, chickens are generally killed with carbon dioxide and dissected on the fifth day after the start of the study, and the blind end section is examined. Preventing the growth of protozoa is effective against Trypanosoma brucei, the pathogen of trypanosoma disease (nagana) in cattle in Africa.
brucei brucei). The related species Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense are the pathogens of African sleeping sickness in humans. Generally, drug activity is tested against infections set up with polymorphic EATRO110 isolates of Tbb brucei in rats. 5x 24 hours before the exam
Infect test animals with 10 ⁵ parasites. Control animals thus infected generally die 5 days after inoculation. The compound to be tested is administered to the test animals via drinking water at various doses. Animals that recover from infection remain parasite-free for more than 30 days after death of control animals, as shown by examination of blood smears. The compounds described herein are used in an amount effective to inhibit protozoan growth. These amounts will, of course, depend on a variety of factors, such as the type and nature of the protozoan infection, the activity of the particular compound, the age, sex, and species of the animal being treated, and whether the treatment is prophylactic or therapeutic. Generally, the compounds described herein can be administered orally or parenterally in daily doses ranging from 5 mg/Kg to 7 g/Kg. For trypanozole infections, the dosage range is from about 600 mg/Kg to about 2
g/Kg. In the case of Eimeria infections, the dosage can be lowered to a range of about 15 mg/Kg to about 1 g/Kg. Due to the low toxicity of the compounds described herein, the compounds can optionally be safely administered via the drinking water of test animals for the treatment of coccidiosis in chickens. Generally, the active ingredient concentration will suitably range from about 0.01% to about 2%, depending primarily on the nature of the protozoal infection to be treated, whether prophylactic or therapeutic, the extent of the infection, and the duration of treatment. . Thus, for example, the compound 2-difluoromethyl-2,5-diaminopentanoic acid can be effectively administered to chickens as a 2% solution for the treatment of coccidiosis one day before infection. Instead, 2-2-
Prophylactic treatment is available 8 days prior to infection using difluoromethyl-2,5-diaminopentanoic acid. Preventive concentrations of 0.06% to about 1.0% are preferred. This prophylactic treatment to inhibit protozoan growth provides one of the major advantages over the use of the decarboxylase inhibitors described herein. Thus, in the case of coccidia infections in chickens and chicks, for example, Eimeria tenella grows intracellularly in the epithelial cells of the caecum as a trophozoite stage. These cells then undergo a form of multiple mitosis to form multiple merozoites. These merozoids are released when host cells lyse and serve to widely infect new cells. As a result, the cecal wall becomes severely damaged, leading to significant loss of blood and body fluids, and ultimately death. Additionally, resistant oocysts are produced during the life cycle of E. tenella and excreted in the faeces of chickens and chicks. Because chickens and chicks are coprophagous, diseases can spread rapidly through contamination of the plant supply. Therefore, when coccidial infections occur in commercial herds, they are treated epidemically with large doses of currently available chemotherapeutic agents, which are primarily lethal. As a result, medicated feed is now routinely used in commercial herds to prevent coccidiosis outbreaks.
All commercial poultry are almost always medicated. The fact that the decarboxylase inhibitors described herein can be administered prophylactically prevents subsequent natural or artificially induced infection in the host via enzymatic, rather than lethal action. Allows blocking mechanisms to be overcome. For this reason
In the case of E. tenella infection, infection is reduced in a way that allows the host to utilize its own defense mechanisms. The antibodies generated following such a controlled infection serve to further permanently immunize the host from subsequent E. tenella infections. Pharmaceutical compositions particularly suitable for the prevention or treatment of protozoan infections in birds include the above-mentioned α-substituted amines or α-substituted amines in combination with a pharmaceutically acceptable carrier.
Consists of substituted α-amino acids. Advantageously, the active ingredient is combined with an inert carrier material to form the antigenic veterinary composition. Typical carriers are talc, clay,
Pumice, silica, chalk, diatomaceous earth, walnut shell powder,
and their equivalents. Instead,
The active ingredients may be incorporated into commercially available foods or vitamin and mineral premixes specifically adapted for use. In most cases, concentrated aqueous solutions of active ingredients are used for the management and treatment of coccidiosis in birds. The compounds described in most cases are highly soluble, especially in the form of salts. Such solutions advantageously contain preservatives such as parabens, benzyl alcohol, phenol or thimerosal. In addition, isotonic agents, sugars, stabilizers, or buffers can be effectively used. The compounds of the formula can be used in combination with many of the known agents currently used for chemotherapy and chemoprevention of diseases caused by parasitic protozoa.
Generally, this has the effect of reducing the amount of enzyme inhibitor administered. Such agents include, among others: Anthricide, quinapyramine, berenyl, diminazene aceturate, pentamidisoisethionate, primaquine, tripalsamide, amicarbalide, amprolium, amphotericin B, quinine, monensin, minocycline, 7-dimethylamino-6-demethyl-6-
Deoxyteracycline, clindamycin, 7
-deoxy-7(S)-chlorolincomycin, buquinolate, robenidine and nicarbazine. In some cases, compounds may actually enhance or synergize the effects of these drugs. Of particular interest in this regard are the compounds 2,5-diamino
-difluoromethylpentanoic acid. Therefore, 2,5-diamino-2-difluoromethylpentanoic acid concentrations can be reduced approximately 4-fold when used in combination with subtherapeutic doses (less than 1.0 mg/Kg) of these agents. Additionally, the compounds of the formula can be used in combination with other known cytotoxin agents for chemotherapy and chemoprevention of parasitic diseases, especially trypanosomal diseases. Such cytotoxin agents include the tumor growth inhibitor antibiotic bleomycin as well as other well-known cytotoxin agents such as cyclophosphamide, methotrexate, prednisone, 6-
Mercaptopurine, procarbozine, daunorubicin, vincristine, vindesine, vinblastine, chlorambucil, cytosine arabinoside, 6-thioguanine, thioTEPA, 5-fluorouracil, 5-fluoro-2-deoxyuridine, 5-azacytidine, nitrogen mustard, 1,3-bis(2-chloroethyl)-1-
Includes nitrosoyuria (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosoyuria (CCNU), busulfan or adriamycin. Of particular interest for the treatment of trypanosomiasis in general and bovine nagana in particular is the use of the enzyme inhibitor 2,5-diamino-2-difluoromethylpentanoic acid in combination with the antitumor antibiotic bleomycin. This specific enzyme inhibitor is believed to act synergistically with bleomycin. Thus, mice infected with Trypanosoma brucei are cured after 3 days by intraperitoneal administration of bleomycin at a daily dose of 7 mg/Kg. Similarly, trypanosome infections in rats are cured by administration of a 1% 2,5-diamino-2-difluoromethylpentanoic acid solution in the drinking water for 3 days. The results of several combination experiments showed that bleomycin in combination with 0.5% 2,5-diamino-2-difluoromethylpentanoic acid administered via drinking water
It shows that 0.5 mg/Kg consistently results in cure. Instead, bleomycin combined with only 0.25% of 2,5-diamino-2-difluoromethylpentanoic acid in drinking water
A concentration of 0.25mg/Kg provides cure. The combination of 0.1 mg/Kg of bleomycin and 0.1% 2,5-diamino-2-difluoromethylpentanoic acid has no effect. In this way, the combination of curative doses is used in combination with a dose that is less than 1/2 to 1/4 of the curative dose of 2,5-diamino-2-difluoromethylpentanoic acid. This reflects that when administered, the dose of this drug is reduced to 1/2 to 1/28 of the therapeutic dose of the bleomycin drug used alone. The invention described and claimed herein is further illustrated by the following examples which specifically illustrate how the compounds of the invention are made and utilized. Preparation of the compound 2-difluoromethyl-2,5-diaminopentanoic acid A solution of 2M butyllithium in hexane (500 ml) under nitrogen was dissolved in tetrahydrofuran 1.5 at -78 °C.
Add to the stirred solution of 143.1 ml of diisopropylamine in the solution. After this, tetrahydrofuran
Add 261 g (0.81 mol) of ornithine dibenzaldimine methyl ester in 1.5. After the addition is complete, the reaction temperature is raised to 40°C and held between 40° and 50°C for 3 hours, during which time chlorodifluoromethane gas is bubbled through the mixture while stirring. The reaction mixture is then treated with saturated sodium chloride solution. 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. Stir the oil with 1.5 1N HCl for 3 hours,
The mixture is extracted several times with chloroform and the aqueous solution is evaporated to dryness. Remove oily residue with 12N hydrochloric acid
After refluxing with 1.5 for 16 hours, the cooled solution is purified by chloroform extraction, then concentrated, decolorized (charcoal) and further concentrated to about 750 ml. solution
The PH is adjusted to 3.5 by addition of triethylamine and the solution is treated again with charcoal, then concentrated to approximately 500 ml and diluted with acetone 7-8. The precipitated product is filtered off and washed with ethanol. The crude product is recrystallized by dissolving it in about 150 ml of hot water and treating the solution with 450 ml of hot ethanol.
After cooling, 71 g (37%) of 2-difluoromethyl-2,5-diaminopentanoic acid hydrochloride hydrate crystals precipitate out. Melting point: 183℃. Formulation Example 1 Granules suitable for addition to poultry drinking water are prepared as follows. 2-Difluoromethyl-2,5-diaminopentanoic acid 33.0g Corn starch 18.5g Lactose 48.2g Zinc stearate 0.3g 100.0g 2-Difluoromethyl-2,5-diaminopentanoic acid mixed with about 6-9g lactose Particle sizes of 1 to 25 microns are obtained when subjected to a fluid energy mill or micronizer. Corn starch approx.
Add 35 ml of water to 2.0 g and mix to make 5% starch paste. Thoroughly blend the finely ground 2-difluoromethyl-2,5-diaminopentanoic acid/lactose powder with the remaining lactose and remaining corn starch. Add starch paste and mix, and mix the resulting mixture for 12
Hang on the number screen. The resulting granules were dried at 38°C to a moisture content of approximately 3% and
Grind through a number sieve, zinc stearate 0.3
Lubricate by mixing with g. Formulation Example 2 A 10% stock solution used in the treatment of coccidiosis is made by dissolving 37.5 g of 2-difluoromethyl-2,5-diaminopentanoic acid in 1 gallon of water at room temperature. One part of this stock solution diluted with nine parts of water makes a 1% drug-spiked drinking water solution for poultry, useful for the prevention of coccidiosis in poultry. Formulation Example 3 A medicated animal feed suitable for poultry can be made using the following ingredients: The chickens are allowed to eat the medicated feed ad libitum. Weight % Ground Yellow Corn 60.3 Soybean Oil Meal 33.0 Alpha Alpha Drug Powder 1.0 Dicalcium Phosphate 3.0 Calcium Carbonate 1.0 Iodized Salt 0.2 2-difluoromethyl-2,5-diaminopentanoic acid 0.33 Provides the following ingredients per 100 pounds of feed: Vitamin-mineral-amino acid antibiotic mixture Oxytetracycline 0.5g Penicillin (as procaine salt) 0.25g Manganese sulfate 8g DL-Methionine 22.7g Riboflavin 130mg DL-Calcium pantothenate 930mg Niacin 1400mg Pyridoxine 130mg Vitamin B ₁₂ 1mg Choline chloride 22.7g Vitamin A 300,000 units Vitamin D ₃ 25,000 units Example 1 The following shows the effect of 2-difluoromethyl-2,5-diaminopentanoic acid on Trypanosoma brucei brucei infection in mice. Groups of 5 Hatsukanesumi each weighing 20-25g.
Tb brucei (EATRO110 isolate, ⁵ x 105 individuals/1 rat) are inoculated. Compounds are administered ad libitum via drinking water 24 hours post-infection. The results are expressed as the average number of days of survival since death of the control group animals based on the average number of days of survival of the control group animals over a 5-day period. Berenyl (diminazene acetylate) is included as a control trypanoside. The results are shown in Table 1 below.

[Table] Example 2 The following example shows the concentration of 2 in the drinking water of chickens infected with Eimeria tenella oocysts.
-Illustrating the effect of a 2% solution of difluoromethyl-2,5-diaminopentanoic acid. On the first day, 20 chickens were orally infected with 100,000 E. tenella oocysts. 2-difluoromethyl-2,5-diaminopentanoic acid 2%
Provide drinking water containing the solution to 10 chicks. The remaining chicks will be used as a control group. All animals in the control group show clinical signs of disease until day 3. On the seventh day, all animals are sacrificed and the blind end lesions are visually inspected and graded as follows. 0 = Damage cannot be detected with the naked eye. +1=slightly scattered petechiae on the cecal wall. There is no wall thickening. Normal cecal contents are present. +2 = There are multiple lesions and loss of cecal contents is observed. The cecal wall is slightly thickened. +3 = Large amount of blood and tissue debris present. i.e. cecal cores. The cecal wall is greatly thickened, and there is little, if any, normal cecal content. +4=The cecal wall is widened due to the presence of a large amount of blood or a cecal core. Cecal fragments are absent or included in the core. (Dead chicks are also included in +4).

[Table] Example 3 Following essentially the same procedure as in the previous example, six chicken chicks were treated with a 2% solution of 2-difluoromethyl-2,5-diaminopentanoic acid (DFMO) in drinking water for 3 days. to be administered. On the first day, in addition to this group of six birds, two groups of 10 birds each were given 100,000 E. tenera oocysts per bird.
Orally infect individuals. One group of 10 birds served as a control group, while the other group of 10 birds received a standard dose of Amprolium in their drinking water for the next 5 days. Currently, amprolium is the coccidia inhibitor of choice. On the fifth day, all animals are sacrificed and examined for evidence of disease using the lesion scoring index described in the previous example. We get the following result:

[Table] Solution Example 4 The following example shows the use of 2-difluoromethyl-2,5 against Eimeria tenella infection in chickens and chicks.
- shows the effect of various doses of diaminopentanoic acid. Following essentially the same procedure as in Example 2, the dosage of 2-difluoromethyl-2,5-diaminopentanoic acid (DFMO) is varied as shown in Table 4 below. DFMO treatment begins on day −1. Chicks are infected orally on day 0 and treatment continues for 5 days thereafter, or 6 days in total. Chicks are killed at 5 days and examined for evidence of disease using the lesion scoring index described in Example 10.

Table: Example 5 The following example demonstrates the effectiveness of low prophylactic doses against Eimeria tenella lesions in chicks. Following essentially the same procedure as described in Example 2,
Varying the 2-difluoromethyl-2,5-diaminopentanoic acid (DFMO) dose and duration, the following results are obtained.

Table: Example 6 The following example demonstrates the acquisition of permanent immunity against Eimeria tenella infection in chickens. 2 from -8 days to +5 days from infection
Chickens pretreated with -difluoromethyl-2,5-diaminopentanoic acid at a low concentration of 0.5% were challenged with pathogens one week following the end of treatment as shown in Table 6 below. These results indicate that low-dose prophylactic therapy allows adequate development of the parasite without causing a disease state, resulting in the development of immunity against subsequent E. tenella infection.

【table】

Claims (1)

Claims: 1. An antiprotozoal agent comprising protozoan-inhibiting amounts of 1 2-difluoromethyl-2,5-diaminopentanoic acid or salts thereof, the individual optical isomers. 2 The animal is a bird, and 2-difluoromethyl-
Claim 1, wherein 2,5-diaminopentanoic acid is administered to drinking water at a concentration of 0.01% to 2.0%.
The antigenic protozoan agent described in section. 3 2-difluoromethyl-2,5-diaminopentanoic acid is administered at a concentration of 0.06% to 1.0%,
The antigenic protozoan agent according to claim 1. 4. The antigen according to claim 1, which is a combination of a protozoan-inhibiting amount of 2-difluoromethyl-2,5-diaminopentanoic acid or a salt thereof, each optical isomer, and a suitable carrier. Live animal agents. 5 The animal is a bird, the carrier is water, and 2,5
-diamino-2-difluoromethylpentanoic acid
Antiprotozoan agent according to claim 4, present in a concentration of 0.01% to 2%. 6 2-difluoromethyl-2,5-diaminopentanoic acid or salts thereof, individual optical isomers, and an antiprotozoan agent selected from the group consisting of anthricide, pentamidine isethionate, amicarbalide, and bleomycin. A synergistically effective amount of an antiprotozoal agent.