BG62227B2 - Polycyclic ether antibiotics - Google Patents

Abstract

The antibiotic and its cation salts are active againstdifferent microorganisms and are used in controlling thecoccidiosis, enterites and dysentery in pigs, as well as forincreasing the growth and/or improving the food assimilation inpigs and in ruminants. The antibiotic has the formula4 claims.

Description

Technical field

The invention relates to novel antibiotics from the group of acidic polycyclic ethers, representing a class of compounds characterized biologically by their effect on cationic transport through cell membranes.

BACKGROUND OF THE INVENTION

Said group of antibiotics includes well known agents such as monensin, nigericin, grisorixin, dianemicin, meduramycin, narasin, salinomycin, lasalocid, mutalomycin, ionomycin and leuseramycin. These are described by Westley in Polyether Antibiotics, Adv.Appl.Microbiol., 22, 177, 1977.

The polycyclic ether antibiotics cited above are active against gram-positive bacteria, fungi and protozoa. In particular, these antibiotics exhibit strong anticoccidial activity. Therefore, they have been used with varying success to treat different infections in animals.

The well-known protozoal disease, coccidiosis, continues to be a serious problem, and keeping it under control is an economic problem for veterinary science, especially poultry. Coccidiosis is the result of infections by one or more species of Eimeria or Isospora (see Land and Far, Diseases of Poultry, 5th ed., Biester and Schwarte, Ed., Iowa State University Press, Ames, la., 1965, pp. 10561096).

There are six types of coccidia, Eimeria tenella, E. necatrix, E.brunetti, E.maxima and E.muvati, which cause easily detectable disease in susceptible chicks, causing destruction of the epithelial cells of the digestive tract directly, or causing damage indirectly, producing toxins . There are three other types of protozoa that belong to the same genus and are considered relatively safe. However, E.mitis, E.hagani and E.rgaesoh are able to reduce weight gain, reduce food absorption and adversely affect egg production and egg production.

In view of the great economic losses due to coccidiosis, the demand for new anticoccidial agents continues.

Enterites are another disease that can cause serious economic losses for livestock producers. The enteritis occurs in chickens, pigs, cattle and sheep and is mainly due to anaerobic bacteria and in particular Clostridium perfringens and viruses. Enterotoxemia in ruminants, an example of which is "overeating disease" in sheep, is a condition caused by C.perfringens infections.

Swine dysentery is one of the most commonly diagnosed diseases in the United States. The disease is widespread in many other countries and causes significant losses of pig breeders worldwide annually. It has recently been discovered that a large spirochete is the cause of the disease. This organism, Treponema hyodysenteriae, is now isolated and has been shown to be capable of causing the disease. / Harris D.L. and co-authors of Vet.Med/SAC, 67, 61-64, 1972 /.

The test data below is derived from experiments performed with this organism. However, it should be noted that it is not known whether T.hyodysenteriae is the sole cause of swine fever. From the available data, it can be concluded that it is one of the primary sources of infection.

Improving results (such as increasing weight gain and / or improving food absorption) in ruminants such as cattle and monogastric animals such as pigs is another economically desirable goal in veterinary science. It is of particular interest to improve weight gain as a result of increasing food efficiency. The mechanism of utilization of nutritional nutrients in ruminant foods is well known. Microorganisms in the abdomen of an animal break down carbohydrates to produce monosaccharides, and then convert these monosaccharides into pyruvic compounds. Pyruvates are metabolized by microbiological processes to form acetates, butyrates or protonates, commonly known as volatile fatty acids. For more details see: Lang, Physiology of Digestion and Metabolism in the Ruminant, Phillipson et al., Eds., Oriel Press, Newcastle-upon-Tyne, England, 1970, pp. 408-410.

The relative efficacy of the use of volatile fatty acids is described by McCooloff in Feedstuffs, June 19, 1971, page 19, Esqueland et al. In J.An.Sci., 33, 282, 1971, and by Church et al., Digestive Physiology and Nutrition of Ruminants ”, Vol. 2, 1971, pp. 622-625. Although acetates and butyrates are used, propionates are used more efficiently. Moreover, when very few propionates are present, animals can develop ketosis. Therefore, 5 a useful compound stimulates animals to produce a higher proportion of propionates than carbohydrates, while increasing carbohydrate utilization while reducing the risk of ketosis. The present invention relates to a novel acidic polycyclic ether antibiotic. EPA0169011 describes an acidic polycyclic ether antibiotic designated UK with the formula:

in which R and R [both are hydrogen, which is formed by aerobic propagation of an organically immersed microorganism Actinomadura roseorufa Huang sp. nov. ATCCC 39697 isolated from soil sample from Japan. It has now been found that upon hydrolysis of UK-58 852, under carefully controlled conditions, cleavage of one of the linked glucon rings results in a new compound, which is very effective as a broad-spectrum anticoccidial agent and has advantages over UK-58 852 , because of its better toxicological properties.

Brief description of the invention

Thus, according to the invention, a compound designated as Antibiotic UK61 689 of formula is obtained

and its pharmaceutically acceptable cationic salts. Examples of such salts are sodium and potassium.

Also according to the invention is a method of producing Antibiotic UK61 689 or a cationic salt thereof, which consists in the controlled hydrolysis of compound UK-58 852 or a cationic salt thereof.

Detailed description of the invention

The hydrolysis of UK-58 852 is preferably carried out using paratoluenesulfonic acid in an acetonitrile / water solvent. The preferred acetonitrile / aqueous composition contains between 5 and 0.5% water. The ratio of para-toluenesulfonic acid to the sodium salt of UK-58 852 is preferably 1.1 mol equivalents per 1 mol of salt.

Other solvent systems that may be used are pure acetonitrile or tert-butanol / water or acetone / water. Other acids that may find application are mineral acids such as hydrochloric and acetic acid or strong acid resins. Typically, the reaction is carried out at room temperature and monitored by thin layer chromatography until yields of the desired product appear to be optimal. The reaction time is usually about 1-3 h for para-toluenesulfonic acid and the acetonitrile / aqueous hydrolysis system, but will vary depending on the acid and solvent system used. After the reaction was practically complete, the reaction mixture was neutralized with excess sodium bicarbonate, concentrated and extracted with diethyl ether or dichloromethane and then purified using standard silica gel chromatography. The final product may be crystallized as the free acid or the corresponding cationic salt.

Alternatively, the antibiotic UK-61 689 can be obtained from the crude fermentation extract containing UK-58 852. Thus, the methyl 10 isobutyl ketone (MIBK) fermentation extract is concentrated and dissolved in acetonitrile / water and treated with para-tolulol sulfonic acid. The preferred acetonitrile 5 aqueous composition contains 5% water and the ratio of crude fermentation oil to para-toluenesulfonic acid is about 9: 1. These ratios are indicative and may vary depending on the composition of the fermentation extract. Other solvents that may be used are methanol / water, but hydrolysis can be carried out by treating the crude MIBK extract with para-toluolosulfonic acid in the absence of added solvent. Alternative acids are mineral acids such as hydrochloric acid.

The antibiotic UK-61 689 inhibits the growth of numerous gram-positive microorganisms. Table 1 below shows the results of the in vitro test. In this test, each organism was inoculated into a series of test tubes containing the culture medium and different concentrations of the antibiotic UK61 689 to determine the minimum concentration of the compound in pg / ml that holds the growth of the microorganism for 24 h (MIC). .

Table 1.

Antibacterial activity

Microorganism Strain # MIC pg / ml Clostridium perfringens 10A006 25 10A009 3.12 Actinomyces pyogenes 14D002 0.39 14D008 0.39 14D011 0.39 Treponema hyodysenteriae 94A001 6.25 94A002 6.25 94A007 3.12 94A008 3.12

Data on the efficacy of the antibiotic UK-61 689 and its salts against coccidiosis infection in chickens were collected as follows: Groups of 3-5 day-old male pathogens-free white Leghorn chickens are fed a nutrient mixture containing contains the antibiotic UK-61 689 or its sodium or potassium salt, evenly distributed in the food. After being fed for 24 hours, each of the chicks was inoculated by mouth with oocysts of the particular Eimeria species to be tested. Another group of 3-5 daily chickens is fed the same nutrient mixture, however, which does not contain the antibiotic UK-61 689 or its salts. They also become infected after 24 hours and serve as an infected control. Another group of 3-5 daily chickens were fed the same nutrient mixture, without the UK-61 689 antibiotic, and were not infected with coccidia. They serve for normal control. Treatment results are reported after five days in the case of E. acervulina and after six days in all other types of infectious agents.

The criteria used to measure anticoccidiosis activity consist in the determination of 0 to 4 degrees of impairment for E.tenella by JE Lynch, “A New Method for the Primary Evaluation of Anticoccidal Activity”, Am.J.Vet. Really. 22, 324-326, 1961 and 0 to 3 degrees of impairment in accordance with the system recommended by J. Johnson and W. H. Reid, Anticoccidial Drugs. Lesion Scoring Techniques in Battery and Floor Pen Experiments in Chicks, Exp.Parasit 28, 30-36, 1970, for other Eimeriaa species. A constant ratio is established by dividing the degrees of damage for each of the treated groups by the degree of damage to the infected control.

UK-61 689 and its cationic salts have been found to exhibit excellent activity against coccidiosis in birds. When mixed with chicken feed at a concentration of 15 to 120 ppm, these compounds effectively control infections due to Eimeria tenella, E.acervulina, E.maxima, E.brunetti and E.necatrix.

The value of animal feed is generally determined by feeding the animal. GB 1 197 826 provides a detailed description of an in vitro abdominal examination technique whereby the occurring changes in food caused by microorganisms are measured with great precision in the evaluation of animal feed. This technique uses an apparatus by which the digestive processes in animals are conducted and investigated in vitro. Animal foods, ruminant inoculum inoculum, and various growth promoters are introduced and withdrawn from a laboratory device under carefully controlled conditions, and changes occurring are examined critically and consistently during food consumption by microorganisms. Increasing the propionic acid content of the abdominal fluid indicates that the desired result in the abdominal functions has been achieved due to the growth promoter in the food mix. The change in propionic acid content is expressed as a percentage of the propionic acid found in the control abdominal fluid. Prolonged live feeding experiments have been used to show a sure correlation between the increase in propionic acid in abdominal fluid and improved animal development.

The abdominal fluid is collected from a cow's fistula, which feeds on a ready-made commercial fattening diet plus hay. The abdominal fluid was filtered immediately through cheesecloth and 10 ml of it was added to a 400 mg standard substrate (68% corn starch + 17% cellulose + 15% extracted soybean meal), 10 ml pH 6.8 buffer and the test compound in 50 ml. conical flask. The flask was saturated with oxygen-free nitrogen for about 2 min and incubated in a shaking water bath at 39 ° C for about 16 h. Each of the experiments is performed in triplicate.

After incubation, 5 ml of the sample is mixed with 1 ml of 25% metaphosphoric acid. After 10 minutes, 0.25 ml of formic acid was added and the mixture was centrifuged at 1500 rpm for 10 minutes. The samples were then analyzed by gas-liquid chromatography by the method of D.W.Kellog, J.Dairy Science, 52, 1690, 1969. The peak heights of acetic, propionic and butyric acids were measured in the samples from treated and untreated incubated flasks.

In the in vitro test, the antibiotic UK-61 689 at 20 pg / ml gave an increase of about 80% of the propionic acid obtained above that obtained in the control solution, which did not contain UK-61 689. The compounds that stimulate the production of RPA (propionic acid in the abdomen), are known to improve food use in ruminants such as cattle and sheep and can also have a similar effect in monogastric animals such as pigs. The antibiotic UK-61 689 may be administered to the animal by mixing it in the composition of the food, either as free acid or in the form of a salt, for example sodium or potassium salt or a mixture thereof. Alternatively, the raw form or dry fermentation broth containing the antibiotic UK-61 689 may be mixed in the food at the desired concentration. Antibiotic UK-61 689 may also be administered in the form of a sustained release formulation which releases a constant amount of the drug substance.

When the compound of the present invention is used to treat coccidiosis in birds, it is orally administered in a suitable vehicle. It is convenient to place the drug in drinking water or in bird feed so that a therapeutic dose of the agent is taken with the water or food ingested daily. The agent can be placed directly into the drinking water, preferably in the form of a liquid, a water-soluble concentrate (such as a water-soluble sodium salt or other water-soluble salt) or added directly to the food, either as a premix or concentrate. The premix or concentrate of the therapeutic agent is the commonly used way of incorporating the agent into the food. Suitable carriers are water and various foods such as soy, lentils, corn cobs, mineral mixtures, etc., which are most commonly used for bird feeding and can be liquid or solid. Particularly effective carrier is the bird feed itself, that is to say small amounts of food. The carrier assists in the even distribution of the active substance in the finished food with which the premix is mixed. This is important because only small amounts of this powerful agent are needed. It is important that the compound is well homogenized in the premix and then in the feed. In this aspect, the agent may be dispersed or dissolved in a suitable oily carrier such as soybean, corn and cottonseed oil and the like or in volatile organic solvents and then mixed with the food. It should be understood that the proportions of the active substance in the concentrate can vary greatly, as the amount of the substance in the finished food can be adjusted by mixing the appropriate ratio with the premix and the food so as to obtain the desired content of the therapeutic agent. .

Highly concentrated formulations may be mixed by the food manufacturer with a protein carrier such as soybean food and other foods as described above to obtain concentrated additives that are suitable for direct bird feeding. In these cases, the birds are left to consume the usual food. Alternatively, such concentrated additives may be added directly to the poultry feed to provide a nutritionally balanced prepared food containing a therapeutically effective amount of the compound of the invention. The mixtures are homogenized by conventional means, such as a twin shell mixer, to ensure a uniform distribution of the substance.

It will be appreciated by those skilled in the art that the amounts of the compound used will vary depending on the different conditions. Continuous administration of the compound for the first 6 to 12 weeks in chickens is an effective prophylactic measure. In the case of established infections, more abundant amounts may be needed to overcome the infection. The amounts used in the food will generally be in the order of 15 to 120 ppm. When added to drinking water, the amount should be such as to provide the same daily dose of medication, i. 15 to 120 ppm, a coefficient derived from the weight ratio of daily food intake to daily water intake.

The present invention is illustrated by the following examples. However, it should be borne in mind that the invention is not limited to the specific details of these examples.

Example 1. To a solution of 15.0 g (0.0147 mol) pure antibiotic UK-58-852 (prepared according to EPA 0169011) in acetonitrile / water (95: 5) 400 ml was added 3.07 g (0.0161 mol) paratoluenesulfonic acid. The reaction was monitored by thin layer chromatography until the yield of the desired product seemed optimal (about 3 h). The reaction mixture was treated with an excess of solid or aqueous sodium bicarbonate solution and concentrated to dryness in vacuo. The resulting solid was dissolved in diethyl ether and washed with saturated aqueous sodium bicarbonate solution. The bicarbonate washes were extracted with diethyl ether and all the diethyl ether extracts were combined and washed successively with water and saturated sodium chloride solution. The ether solution was dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The crude reaction product obtained contains UK-61 689 as the main product but is contaminated with varying amounts of starting UK-58 852 and other by-products. The crude product was purified by silica gel chromatography followed by recrystallization from isopropyl ether to give 4.2 g (32%) of UK-61 689 as the sodium salt, m.p. 175-176 ° C. (a), ²⁵ = + 19.3 ° (c = 0.5, MeOH), C-13 NMR (CDCl ₃ ): 179.15 ppm, 107.45, 103.18, 97.74, 96.96 , 86.92, 84.60, 84.20, 82.27, 82.01, 80.88, 80.20, 79.86, 74.55, 73.07, 70.06, 67.71, 66 , 89, 59.11, 56.81, 45.40, 39.82, 38.93, 36.46, 33.81, 33.71, 33.54, 33.42, 33.13, 32.44 , 32.26, 30.56, 27.58, 26.90, 26.84, 26.11, 23.23, 18.40, 17.53, 16.99, 12.13, 11.04, 10 , 42.

Example 2. The crude fermentation extract (MIBK) (1 L) containing the antibiotic UK58 852 (found 25 g) was concentrated in vacuo to 680 g. The dark oil was added to acetonitrile / water (95: 5) (5.6 l) and the resulting lactic emulsion was treated with p-toluenesulfonic acid (78.2 g). As the reaction progresses, the emulsion is gradually separated into two layers, which are vigorously stirred for a total of 1.25 hours. The reaction mixture was poured into a separatory funnel and the oily layer at the bottom removed and stirred with additional acetonitrile / water (95: 5). The acetonitrile / aqueous layers were collected and treated with saturated aqueous sodium bicarbonate solution (400 ml). The mixture was then concentrated to a thick syrup in vacuo, dissolved in diethyl ether / ethyl acetate (3: 1) 2 L and washed with saturated aqueous sodium bicarbonate solution. The organic layer was dried over sodium sulfate and concentrated to a thick brown oil (432 g). Purification is easiest by using continuous extraction techniques. The extraction used hexane: ethyl acetate (1: 1) as the non-polar phase and methanol: water (3: 2) as the polar aqueous phase. Evaporation of the organic extract gave a semi-solid mass which, upon friction with isopropyl ether, gave UK-61 689 as the sodium salt, 10 g, m.p. 169-1170 ° C. This product is identical to product UK-61 689 obtained by the method of Example 1, demonstrated by comparison by thin layer chromatography and NMR spectra.

Claims (4)

Claims An antibiotic of the formula or a pharmaceutically acceptable cationic salt thereof. The antibiotic of claim 1 in the form of its sodium salt. A bovine or porcine feed composition comprising an antibiotic according to claim 1 in an amount effective to increase the gain or improve the absorption efficiency of the bovine or porcine feed. A poultry feed composition comprising the antibiotic of claim 1 in sufficient quantity to provide effective control of the coccidiosis infection in the birds.