JPS6131112B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel adenine derivative and a preventive and therapeutic agent for coccidiosis containing the same. Coccidiosis is an infectious disease caused by a parasitic protozoan that causes diarrhea and malnutrition in poultry and livestock. For example, chickens, quail, turkeys, rabbits, goats, sheep, and cattle are often severely affected. suffer. For example, nine types of parasitic protozoa of the genus Eimeria (hereinafter abbreviated as "E") have been recognized in domestic chickens. The main ones among these are E. tenella, E. acervulina, E. maxima, E. necatrix, E. brunetsutei. brunetti), especially Eimeria tenella and Eimeria necatorix, which cause the most fatal coccidia symptoms and, if left untreated, can lead to weight loss, reduced feed efficiency, and even death. leading to. Thus, for example, coccidiasis in chickens causes significant losses to the poultry industry, and coccidiasis prevention,
Various compounds have been developed and marketed for treatment. However, currently available drugs have drawbacks such as being highly toxic and giving off an unpleasant odor to chicken meat, and furthermore, coccidia protozoa that are resistant to these drugs have emerged, so they cannot be expected to be sufficiently effective. Therefore, there is a strong demand for the development of new drugs that are effective against coccidia parasites in general. In view of these points, the present inventors have carried out extensive research and have discovered a novel product that has a reliable effect on the preventive treatment of coccidia disease and has an excellent action that can overcome the shortcomings of the existing preventive and therapeutic agents for coccidia disease. Discovered an adenine derivative. The present invention was completed based on this knowledge and as a result of further research. That is, the present invention provides: (1) General formula [In the formula, R ¹ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ² is an alkyl group or an allyl group having 1 to 3 carbon atoms, and R ³ and R ⁴ are halogen atoms] The present invention relates to an adenine derivative or a salt thereof, and (2) an agent for preventing or treating coccidiosis, which contains an adenine derivative or a salt thereof represented by the general formula (). In the above general formula (), examples of the alkyl group having 1 to 3 carbon atoms represented by R ¹ and R ² include a methyl group, an ethyl group, a propyl group, an isopropyl group, and the like. Examples of the halogen atoms represented by R ³ and R ⁴ include chlorine atoms and fluorine atoms, and these may be the same or different. Examples of the above adenine derivatives () include the compounds shown below. 9-(2-chloro-6-fluorobenzyl)-6
-Methylaminopurine 9-(2,6-dichlorobenzyl)-6-methylaminopurine 9-(2-chloro-6-fluorobenzyl)-6
-dimethylaminopurine 9-(2,6-dichlorobenzyl)-6-dimethylaminopurine 9-(2-chloro-6-fluorobenzyl)-6
-ethylaminopurine 9-(2,6-dichlorobenzyl)-6-ethylaminopurine 9-(2-chloro-6-fluorobenzyl)-6
-diethylaminopurine 9-(2,6-dichlorobenzyl)-6-diethylaminopurine 9-(2,6-dichlorobenzyl)-6-isopropylaminopurine 6-allylamino-9-(2,6-dichlorobenzyl)purine These Among the adenine derivatives (),
Compounds in which R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, R ³ is a chlorine atom, and R ⁴ is a chlorine atom or a fluorine atom are preferable, and especially R ¹ is a hydrogen atom and R ² is a methyl group. group, R ³ is a chlorine atom, R ⁴ is a fluorine atom, 9-(2-chloro-6
-fluorobenzyl)-6-methylaminopurine is preferred. The adenine derivative () may be in the form of a salt, and such salts include, for example, acid addition salts such as hydrochloride and sulfate. The adenine derivative () of the present invention is available from, for example, Journal of Medicinal Chemistry,
Using 6-chloro-9-(2,6-dichlorobenzyl)purine described in Vol. 14, p. 809 (1971) as a raw material compound, amines corresponding to the following formula (e.g., methylamine , dimethylamine, ethylamine, diethylamine, propylamine, isopropylamine, allylamine). [In the formula, R ¹ , R ² , R ³ and R ⁴ have the same meanings as above,
represents a halogen atom or a methylthio group. ] The above reaction is usually carried out at a reaction temperature of about 60 to 200°C in a solvent that does not interfere with the reaction, such as alcohols (eg, methanol, ethanol), dimethylformamide, or the like. The amount of () to be used relative to () is usually about 1 to 5 equivalents, and if necessary, an excess of () may be used. Further, the compound () in which R ¹ is a hydrogen atom and R ² is a methyl group is, for example, 6-amino-9-(2-chloro-6-
It can be produced by the following reaction using raw materials such as (fluorobenzyl) purine. [In the formula, R ³ and R ⁴ have the same meanings as above. ] The adenine derivative () and its salt of the present invention have excellent anti-coccidial effects and extremely low toxicity, and are useful as preventive and therapeutic agents for coccidiosis in poultry, livestock (e.g., chickens, turkeys, cows), etc. be. Such coccidiosis preventive and therapeutic agents can be prepared by diluting the compound () and its salt with a solid or liquid diluent or not diluting it into a powder, dust, granule, tablet, liquid, capsule, etc. It is produced by adding directly or once dispersed in a diluent to feed, drinking water, etc. Any diluent may be used as long as it is physiologically harmless in itself, and it is more desirable to use a diluent that can be used as feed or a component of feed. As solid carriers, for example,
Barley flour, wheat flour, corn flour, soybean meal, soybean flour, rapeseed meal, rice husks, rice bran, defatted rice bran, corn starch, potato starch, tofu cake, starch, lactose, sugar, glucose, fructose, yeast, waste Examples of the liquid carrier include yeast and fishmeal, and examples of the liquid carrier include water, physiological saline, and physiologically harmless organic solvents. Other appropriate auxiliary agents such as emulsifiers, dispersants, suspending agents, wetting agents, thickening agents, gelling agents, and solubilizing agents may be added as appropriate. Furthermore, preservatives, bactericidal agents, antibiotics, enzyme preparations, and lactic acid bacteria preparations may be added, and other anti-coccidial agents, sulfur agents, vitamin preparations, etc. may also be added to these compositions. The preventive and therapeutic agent for coccidiosis containing an adenine derivative () or a salt thereof of the present invention exhibits extremely excellent effects on the prevention and treatment of coccidia in poultry and domestic animals, and has low toxicity, so the dosage thereof is as follows: For example, it can be appropriately determined depending on the type of poultry or livestock, body weight, age, administration method, purpose of administration, etc. For example, when the purpose is to prevent or treat coccidiosis in chickens, the amount of the compound () is about 0.4 to 100 mg/Kg/day, preferably 0.8 to 100 mg/Kg/day.
It is best to administer at a dose of about 30mg/Kg/day. As a practical method, for example, the agent for preventing and treating coccidiosis of the present invention is administered as a compound () in the feed or drinking water of poultry in such a manner that it is contained in an amount of about 0.0004 to 0.1% by weight, preferably about 0.0008 to 0.03% by weight. It is convenient. As seen in the following experimental examples, the coccidiosis prevention and treatment agent of the present invention completely suppresses bleeding, infection death, and intestinal lesions even when administered at low concentrations, and also causes significant weight gain. It has excellent effects. Examples of experiments using chicken chicks to demonstrate the effects of the preventive and therapeutic agent for coccidiosis of the present invention are listed below. As used below, "non-infected control" refers to "non-infected, non-medicated control", and "infected control" refers to "infected, non-medicated control". Experimental example 1 Test materials and test methods (1) Test compounds: Compound 1: 9-(2-chloro-6-fluorobenzyl)-6-methylaminopurine Compound 2: 9-(2,6-dichlorobenzyl) −
6-dimethylaminopurine (2) Mixing ratio of test drug to feed: 0.009% by weight of Compound 1 in a compound feed for day-old chicks that does not contain anticoccidial agents (see Table 2 for the composition); 2 was mixed to a concentration of 0.0125% by weight. (3) Test method: Three 9-day-old white Leghorn male chicks were tested.
They were raised as a group on a basal diet containing the above test compound, and 24 hours after the start of drug administration, each bird was
50000 Eimeria tenella mature oocysts were directly inoculated into chicken pouches. Separately, an infected control group was established in which three birds were placed in one group, fed a basal diet containing no drug, and infected with coccidia at the same time as above. Furthermore, a non-infected control group was established in which three birds were placed in one group and fed only with basal feed containing no drugs, and no oocyst inoculation was performed. Judgment was based on the following method. (a) The chicks were examined for signs of bleeding on the 4th, 5th, 6th, and 7th days after infection, and determined by the number of drops. (b) Life and death were observed on days 5, 6, 7, and 8 after infection. (C) On the 7th day after infection, the weight gain ratio per group (test group weight gain/uninfected control group weight gain x 100) was measured. (d) On the 8th day, the chicks were dissected, the cecum was macroscopically observed, and the lesions were detected in Experimental Paragitology, Vol. 28.
Judgment was made according to the method of Johnsson and Reed, described on page 30 (1970), using a five-step method from (-) to (ⓓ). The results are shown in Table-1.

【table】

【table】

[Table] *Product name = Takeda Pharmaceutical Company Limited
Company (Japan) Sales〓
Experimental Example 2 Test drug and test method (1) Test compound: Compound 1: 9-(2-chloro-6-fluorobenzyl)-6-methylaminopurine Control drug: 6-amino-9-(2-chloro -6
-fluorobenzyl)purine
No. 29394] (2) Mixing ratio of test chemicals to feed: For the feed shown in Table 2, the test compounds were mixed individually at 0.01, 0.009, 0.008, 0.007,
They were mixed to a concentration of 0.006% by weight. (3) Test method: Testing was carried out in the same manner as in Experimental Example 1, except that 9 chicks were used in each group. The results are shown in Table-3.

[Table] Experimental example 3 Test materials and test methods (1) Test compound: Compound 1: 9-(2-chloro-6-fluorobenzyl)-6-methylaminopurine (2) Addition of test drug to feed Mixing ratio: The mixing ratio shown in Experimental Example 2 was used. (3) Test method: Testing was carried out in the same manner as in Experimental Examples 1 and 2, except that the effectiveness was determined using the presence or absence of oocyst excretion from the test chicks and the sporulation rate of oocysts. To test for the presence or absence of oocyst excretion, the caecum of the test chicks was sampled on the 8th day after infection, the cecal mucosa and contents were smeared on each individual, and the presence or absence of oocysts was confirmed by microscopic examination. If no oocysts are found, the cecal contents are further suspended in a saturated saline solution, centrifuged at 2000 rpm for 5 minutes, and the supernatant is examined microscopically to check for the presence of oocysts. The proportion of chicks in which oocyst excretion was observed among the test chicks was determined.
As for the sporulation rate, the collected oocysts were suspended in 40 ml of a 2% potassium dichromate aqueous solution for each group in which oocyst discharge was observed, and left in a Schierer at room temperature for 3 days. Then, one drop of the solution was randomly placed on a plankton counting board, and the number of sporulated oocysts and non-spore-formed oocysts were counted at least three times under a microscope. If only a few oocysts were observed, measurements were performed six times. The percentage of the number of sporulating oocysts to the total number of measured oocysts was determined and used as the sporulation rate. The results are shown in Table 4.

[Table] Experimental example 4 Test materials and test methods (1) Test compound: Compound 3: 9-(2-chloro-6-fluorobenzyl)-6-dimethylaminopurine Control drug: 6-amino-9-( 2-chloro-6
-fluorobenzyl)purine
No. 29394] (2) Test method: The test was carried out using the method shown in Experimental Example 1 and Experimental Example 3. The results are shown in Table-5.

【table】

[Table] Example 1 (1) 2.22 g of 6-amino-9-(2-chloro-6-fluorobenzyl)purine was suspended in 24 ml of N.N-dimethylacetamide, and 2.4 ml of methyl iodide was added.
After stirring for 10 hours at room temperature in a dark place,
I left it overnight. Ethanol was added to the reaction solution, the precipitate was collected by filtration, and recrystallized from ethanol to give 9-(2-chloro-6-fluorobenzyl)-1.
- 2.09 g (yield, 62%) of colorless scale crystals of methyladenine hydroiodide were obtained. Melting point 300℃ or above Elemental analysis C ₁₃ H ₁₁ ClFN ₅・HI Calculated value C 37.21; H 2.88; N 16.69 Experimental value C 37.30; H 2.83; N 16.86 (2) 500 mg of the compound obtained in (1) above and 10% - Mix 200ml of sodium hydroxide methanol solution,
After stirring at room temperature for 5 hours, it was left for 3 days.
Methanol was distilled off under reduced pressure, water was added to the residue, the precipitate was collected by filtration, and recrystallized from ethanol to give 9-(2-chloro-6-fluorobenzyl)-
Colorless needle crystals of 6-methylaminopurine 251mg
(yield, 72%). Melting point 184-185℃ Elemental analysis C ₁₃ H ₁₁ ClFN ₅ Calculated value C 53.53; H 3.80; N 24.01 Experimental value C 53.54; H 3.63; N 24.06 Example 2 6-chloro-9-(2,6-dichlorobenzyl) Purine 940mg, 40% methylamine aqueous solution 1.17
Mix g and 75 ml of methanol and place in a sealed tube at 100 ~
Heated at 110°C for 3 hours. After cooling, it was concentrated to dryness under reduced pressure, water was added to the residue, insoluble matter was filtered off, and recrystallized from ethanol to obtain needle-like crystals of 9-(2,6-dichlorobenzyl)-6-methylaminopurine. 770mg
(yield, 83%). Melting point 224-225℃ Elemental analysis C ₁₃ H ₁₁ Cl ₂ N ₅ Calculated value C 50.67; H 3.60; N 22.73 Experimental value C 50.58; H 3.54; N 22.72 Example 3 (1) 6-Amino-9-(2. Example 1 from 588 mg of 6-dichlorobenzyl)purine, 0.7 ml of methyl iodide and 7 ml of N·N-dimethylacetamide
In the same manner as above, 401 mg (yield, 47%) of colorless needle-like crystals of 9-(2,6-dichlorobenzyl)-1-methyladenine hydroiodide were obtained. Melting point: 300°C or higher (2) 9-(2,6-dichlorobenzyl)- was prepared in the same manner as in Example 1 from 218 mg of the compound obtained in (1) above.
91 mg of needle-like crystals of 6-methylaminopurine (yield,
59%). Example 4 From 627 mg of 6-chloro-9-(2,6-dichlorobenzyl)purine, 2.2 g of 20% dimethylamine methanol solution and 50 ml of methanol Example 2
Similarly, 9-(2,6-dichlorobenzyl)-
Colorless needle crystals of 6-dimethylaminopurine 504mg
(yield, 79%). Melting point 174-175℃ Elemental analysis C ₁₄ H ₁₃ Cl ₂ N ₅ Calculated value C 52.19; H 4.08; N 21.74 Experimental value C 52.16; H 4.46; N 21.60 Example 5 6-chloro-9-(2,6-dichloro benzyl) purine 314mg, 70%-ethylamine aqueous solution 322
255 mg (yield, 79%) of needle-like crystals of 9-(2,6-dichlorobenzyl)-6-ethylaminopurine were obtained in the same manner as in Example 2 from 25 ml of methanol and 25 ml of methanol. Melting point 222-223℃ Elemental analysis C ₁₄ H ₁₃ Cl ₂ N ₅ Calculated value C 52.19; H 4.08; N 21.74 Experimental value C 51.95; H 3.91; N 21.46 Example 6 6-chloro-9-(2,6-dichloro In the same manner as in Example 2, 9-9-
212 mg (yield, 61%) of needle-like crystals of (2,6-dichlorobenzyl)-6-diethylaminopurine was obtained. Melting point 140-141℃ Elemental analysis C ₁₆ H ₁₇ Cl ₂ N ₅ Calculated value C 54.87; H 4.89; N 20.00 Experimental value C 55.17; H 4.88; N 19.94 Example 7 6-chloro-9-(2,6-dichloro 314 mg of benzyl)purine, 296 mg of isopropylamine and 25 ml of methanol were reacted in the same manner as in Example 2, and then recrystallized from aqueous ethanol to obtain 9-(2.
276 mg (yield, 79%) of colorless needle-like crystals of 6-dichlorobenzyl)-6-isopropylaminopurine were obtained. Melting point 109-110℃ Elemental analysis C ₁₅ H ₁₅ Cl ₂ N ₅・3/4H ₂ O Calculated value C 51.51; H 4.75; N 20.02 Experimental value C 51.59; H 4.52; N 19.85 Example 8 6-chloro-9- Using 314 mg of (2,6-dichlorobenzyl)purine, 286 mg of allylamine and 25 ml of methanol, 271 mg of colorless needle crystals of 6-allylamino-9-(2,6-dichlorobenzyl)purine (yield, 81%). Melting point 202.5-204℃ Elemental analysis C ₁₅ H ₁₃ Cl ₂ N ₅ Calculated value C 53.91; H 3.92; N 20.95 Experimental value C 53.80; H 3.77; N 20.98 Example 9 6-chloro-9-(2-chloro-6 -fluorobenzyl) purine (melting point 134-135°C) 2.97g, 40
9-9 in the same manner as in Example 2 from 7.03 g of % dimethylamine methanol solution and 100 ml of methanol.
2.49 g of colorless needles of (2-chloro-6-fluorobenzyl)-6-dimethylaminopurine (yield: 82
%) was obtained. Melting point 134-135℃ Elemental analysis C ₁₄ H ₁₃ ClFN ₅ Calculated value C 55.00; H 4.29; N 22.91 Experimental value C 55.05; H 4.12; N 23.17

Claims (1)

[Claims] 1. General formula [In the formula, R ¹ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ² is an alkyl group or an allyl group having 1 to 3 carbon atoms, and R ³ and R ⁴ are halogen atoms] Adenine derivatives or their salts. 2 General formula [In the formula, R ¹ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ² is an alkyl group or an allyl group having 1 to 3 carbon atoms, and R ³ and R ⁴ are halogen atoms] A prophylactic and therapeutic agent for coccidiosis comprising an adenine derivative or a salt thereof.