US3860703A

US3860703A - Amides of methobottromycin

Info

Publication number: US3860703A
Application number: US235286A
Authority: US
Inventors: Frank J Wolf; William J Miller
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1968-03-14
Filing date: 1972-03-16
Publication date: 1975-01-14
Anticipated expiration: 1992-01-14

Abstract

Amides of methobottromycin and amethobottromycin and mixtures thereof and processes for preparing these compounds are disclosed. The compounds of this application are active antibiotic agents useful in the treatment of chronic respiratory disease of chickens and infectious sinusitis of turkeys.

Description

United States Patent Wolf et al. Jan. 14, 1975 AMIDES OF METHOBOTTROMYCIN [56] References Cited [75] Inventors: Frank J. Wolf, Westfield; William J. OTHER PUBLICATIONS Somerset both of Derwent Farm Doc No. 25,374, Abstracting NE [73] Assignee: Merck & Co., Inc., Rahway, NJ. Published [22] Flled: 1972 Primary Examiner-Jerome D. Goldberg [21] Appl. No.: 235,286

Related US. Application Data ABSTRACT [63] Continuation-impart of Ser. No. 7l3,256, March 14, Amides of methobottromycin and amethobottromycin I968, abandoned, which is a continuation-in-part of d i t th f d processes f preparing 480,040 these compounds are disclosed. The compounds of this application are active antibiotic agents useful in [52] US. Cl 424/121, 424/115, 424/122 the treatment of chronic respiratory disease of chick [51] Int. Cl A61k 21/00 ens and infectious sinusitis of k [58] Field of Search 424/117, 121,122 4 4 Claims, 2 Drawing Figures AMIDES OF METHOBOTTROMYCIN CROSS-REFERENCES TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. Ser. No. 713,256, filed Mar. 14, 1968, now abandoned, which was a continuation-in-part of US. Ser. No. 480,040, filed Aug. 16, 1965, now abandoned.

This invention relates to new antibiotic agents and their use. More particularly, the present invention is concerned with novel, highly active antibiotic compounds that are molecular modifications of new compounds known as methobottromycin and amethobottromycin and mixtures thereof, and their use in the treatment of chronic respiratory disease of chickens and infectious sinusitis of turkeys.

The discovery of remarkable antibiotic properties of penicillin and similar substances has stimulated great interest in the field of antibiotic compounds such as: streptomycin, gramicidin, subtilin, bacitracin, chlortetracycline, oxytetracycline, cycloserine, colistin, fervenulin, streptozotocin, novobiocin and the like. In general, such antibiotics are particularly active against certain gram positive bacteria. Others are active against certain gram negative bacteria and some are active against both gram negative and gram positive bacteria. However, the activity of these known antibiotics is usually limited to a few pathogenic microorganisms and work has been conducted in this field in an attempt to find additional antibiotic substances which would be effective against other pathogens.

In addition, many bacteria which, at one time, were controlled by known antibiotics, have developed increasing resistance over the years to these antibiotic substances. As a result, although some of these antibiotics have been found to be invaluable in the treatment of various diseases, it has been discovered that certain strains of some pathogens develop a resistance to various particular antibiotics and, consequently, these antibiotics are no longer active against such strains of pathogens or the activity of these antibiotics has been reduced to such a degree so as to make their use against such pathogens of little consequence.

Many antibiotics appear to be highly active in laboratory tests in vitro and in ova but are destroyed or lose much of their effectiveness when administered inside the body, in vivo.

Accordingly, the deficiencies of the known antibiotics have stimulated further research to find other antibiotics which will be highly active against a wider range of pathogens as well as those strains of various microorganisms which are resistant to other antibiotics. This is true not only with disease-producing bacteria which attack humans but also for disease-producing bacteria which attack animals and poultry.

Chronic respiratory disease is a disease of chickens and turkeys, caused by a certain group of microorganisms known as PPLO or pleuropneumonia-like organisms, which have been classified as Mycoplasma. This is referred to in the art as PPLO infection. In chickens the disease may be complicated by a secondary invader, at which time the disease is known as chronic respiratory disease complex. In turkeys this disease appears in two forms. It is called infectious sinusitis when it is in the form that affects the upper respiratory tract, and air sac disease when it affects the lower respiratory areas. For the purposes of simplicity, these diseases will be referred to herein as infectious sinusitis.

In chickens the chronic respiratory diseasesymptoms may be like those of any other respiratory disease such as Newcastle disease, infectious bronchitis, laryngotracheitis, fungus infection, etc. The usually observed symptoms are nasal discharge and a slight swelling below the eye. Coughing, sneezing, and a hoarse throat rattle or rale may accompany these signs. The symptoms of the disease in turkeys are often demonstrated by swollen sinuses with gelatinous exudate, watery eyes and coughing with chcesey or cloudy air sacs.

The economic loss that accompanies chronic respiratory disease is a drop in egg production by at least 10 to 40 percent, which affects the birds for several weeks or months. Poor hatchability of fertile eggs laid by infected hens can cause additional losses. Mycoplasma (PPLO) caused infection results in the death of a high percentage of embryos. Loss of weight in a large percentage of birds is also evident. There is, in addition, a significant amount of mortality in birds beginning at about four weeks of age.

Infection of birds may occur in a number of ways. Birds may be infected by contact with other infected birds, usually by an inhalation of nasal exudate from a sneezing bird. In fact, infected chickens or turkeys may become sick, and they may become carriers in which they appear to be healthy but are, in fact, infected with pathogenic strains of Mycoplasma (PPLO). In addition, birds may be infected through contaminated litter, manure, water and feed, breeding hens or contaminated hatcheries. Transmission of the disease via the infected embryonated egg contributes largely to an infected flock.

Chemotherapeutic control of these diseases has been successful with a very limited number of compounds. With one exception, the agents which have been found satisfactory are known antibiotics used clinically for other diseases, principally human diseases. The exception is the antibiotic tylosin. Although tylosin is used fairly broadly, strains of PPLO resistant to it have been encountered, and the antibiotic has been shown to be toxic in use with turkeys.

Other antibiotics useful for controlling chronic respiratory disease in chickens and infectious sinusitis in turkeys are erythromycin and chlortetracycline or oxytetracycline. However, the dosage levels of these antibiotics required to obtain good results are quite high, which results in an economic barrier to the user. Other antibiotics known to have anti-PPLO activity usually require a dosage level too close to the toxic level to be of practical value. Included in this group are neomycin, kanamycin, and chloramphenicol.

Many other antibacterial antibiotics which are used for other infections have been found to be without effect on the PPLO. Examples of these would include penicillin and its many derivatives, cycloserine, novobiocin, and many others. As can be seen, this group includes agents with a wide spectrum of activity, hence their inactivity against Mycoplasma shows how these microbes are a unique and specialized type of bacterium.

It is an object of the present invention to provide useful antibiotic substances which are highly effective in controlling the primary etiologics of the chronic respiratory disease of chickens and infectious sinusitis of turkeys.

An additional object of the present invention is to produce new and useful antibiotic substances which may be used in higher concentrations than those presently available without the resultant danger of toxicity.

Another object of the present invention is to provide antibiotics that have an acceptable oral absorption for treating chronic respiratory disease of chickens and infectious sinusitis of turkeys.

A further object of the present invention is to provide antibiotics that may be applied in relatively low dosages in the treatment of chronic respiratory disease of chick ens and infectious sinusitis of turkeys.

Another additional object of the present invention is to provide antibiotics that are active against a wide range of strains of mycoplasmas, including those belonging to the species M. gallisepticum (PPLO), in the treatment of chronic respiratory disease of chickens and infectious sinusitis of turkeys.

A still further object of the present invention is to provide antibiotic substances that not only demonstrate significant antibiotic activity in vitro and in ova but also show significant antibiotic activity in vivo.

Another object of the present invention is to provide antibiotics that develop protective antibodies during treatment which prevent later reinfection.

A still further object of the present invention is to provide a process of preparing these novel antibiotic substance.

Other additional objects of the present invention will become apparent to those skilled in the art by reading the following specification.

The new antibiotic substances of the present invention are produced by molecular modification of newly discovered species of microorganism. The microorganism was isolated from the fermentation broth of a soil actinomycete collected from Canada. This new microorganism has been designated Streptomyces canadensis MA-959 in the culture collection of Merck & Co., Inc., Rahway, New Jersey. A culture thereof has been deposited with the fermentation section of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., and added to its permanent culture collection as ATCC 17776. The new products of the new species of microorganism, known as methobottromycin and amethobottromycin, are disclosed and claimed in U.S. Pat. Nos. 3,683,073 and 3,683,072.

The morphological and cultural characteristics of Streptomyces canadensis MA-959 are set forth in the following:

STREPTOMYCES CANADENSIS MA-959 Morphology Biverticillate. Straight chains of 8-10 spores, a few chains longer. Spores cylindrical (950 X Avg. size 1.0 X 1.7).

Czapek Dox Agar (Sucrose Nitrate) Growth light. Aerial mycelium scant, white. Vegetative growth colorless. Reverse colorless. No soluble pigment. Sporulation good.

GlycerolAspargine agar Growth good. Aerial mycelium medium gray. Vegetative growth brown to reddish-brown. Reverse brown to reddish-brown. Soluble pigment brown to reddish-brown (pink in early stage of growth 1 week). No sporulation observed.

Tomato paste-oatmeal agar growth good. Aerial mycelium medium gray with white tufts and pink exudate appearing after 3 weeks. Vegetative growth brown. Reverse dark brown. Soluble pigment brown. Some sporulation.

Emersons agar Growth moderate. Aerial mycelium scant, light gray. Vegetative growth brown. Reverse brown. Soluble pigment light brown (pink in early stage).

Potato plug Growth good. Colonies smooth, cream to grayish-brown. Aerial mycelium light gray (appears only in drier portion of plug). Soluble pigment medium brown (pink in early stage).

Starch agar Growth good. No aerial mycelium. Vegetative growth light brown. Reverse light brown. Soluble pigment light brown (pink in early growth stage). Hydrolysis.

Nutrient Gelatin plate Growth good. No aerial mycelium. Vegetative growth light brown. Reverse light brown. Soluble pigment light brown. Liquefaction. Gelatin stab Soluble pigment dark greenish-brown. One-third liquefaction.

Calcium Malate agar Growth good. Aerial mycelium scant, pinkish white. Vegetative growth yellowish brown. Slight browning of medium along growth streak.

Tyrosine agar Growth moderate. Aerial mycelium pale pinkish-white. Vegetative growth very light brown. Slight browning of medium.

Peptone-Iron Yeast Extract slant Growth good. No aerial mycelium. Vegetative growth gray. Soluble pigment blue-black at 2 days. Medium brown at 3 weeks. Skim Milk agar Growth good. Aerial mycelium pinkish white. Vegetative growth light brown. Soluble pigment very light brown. No hydrolysis.

Reduction of nitrates Negative under test conditions in organic and synthetic media.

Temperature Good growth at 28C. No growth at 50C. Micro-aerophilic growth (yeast extract-- -dextrose stab) Heavy surface growth and along twothirds of stab line.

Milk Peptronization complete at 3 weeks. No coagulation. Heavy brownish growth ring with sparse aerial mycelium (light gray). Soluble pigment medium grayish-brown. Alkaline reaction (pH 7.9).

Litmus Milk Peptonization complete in three weeks. No coagulation. Alkaline reaction.

The above description of the microorganismproducing methobottromycin and amethobottromycin is given as illustrative of suitable strains of Streptomyces which can be used in the production of methobottromycin and amethobottromycin, but it is understood that the information herein described is not to be limited to organisms answering this particular description. The present invention also contemplates the use of other species of Streptomyces or mutants of the described organisms such as those obtained by natural selection or those produced by mutating agents, for example, x-ray irradiation, ultraviolet irradiation, nitrogen mustards, and the like.

New antibiotics of the present invention are molecular modifications of the basic compounds which form salts with acids, both inorganic and organic, such as hydrochloric, tartaric, salicylic, etc., and other compounds. The free base forms of methobottromycin and amethobottromycin possess the following physical and chemical properties:

Methobottromycin a. Crystallizes from ethyl acetate in the form of white prisms at a temperature of from about 166 to 167C.

b. Easily soluble in alcohols, esters, ethers, chlorinated solvents and benzene. c. Partly soluble in water. d. Insoluble in petroleum ether, hexane and the like. e. Has a specific rotation of [a],," in a 5 percent solution of 95 percent ethanol. Amethobottromycin a. Has a melting point in the range of from about 154 to about 163C. b. Easily soluble in alcohols, esters, ethers, chlorinated solvents and benzene. c. Partly soluble in water. d. Insoluble in petroleum ether, hexane and the like. e. Has a specific rotation of [011 in a 5 percent solution of 95 percent ethanol. Unfortunately, in contrast to other antibiotics both methobottromycin and amethobottromycin are found to be exceedingly complex compounds having molecular weights of about 800. Consequently, thus far it has not been possible to determine the exact complete composition of either methobottromycin or amethobottromycin. It has been found that these compositions contain the elements carbon, hydrogen, nitrogen, sulfur and oxygen. The found percentages of these elements are as follows:

Methobottromycin Amethobottromycin C 59.50% C 58.40% H 7.52% H 7.44% N 13.50% N 13.42% S 3.90% S 4.77% O 15.58% (by difference) 0 15.97% (by difference) Total 100.00% Total 100.00%

These data suggest molecular structures C.,,H,, N O S and C ,H N,,O S for methobottromycin and amethobottromycin respectively but other similar molecular formulae are possible within the experimental error of these determinations.

The infrared absorption spectrum of the antibiotics methobottromycin and amethobottromycin in chloroform using sodium chloride prism is illustrated in the accompanying drawings. The more significant of the characteristic peaks occur at the following wave lengths expressed in reciprocal centimeters: Methobottromycin 3300, 2950, 1730, 1630-1650, 1490,

1360, 1300, 1242, 1160, 1132, 1119, 1102, 980, 806; Amethobottromycin 3290, 2980, 1739, 1640-1685, 1500,1440,1378,1240,1181,1122,1092,1061,995, 980.

The above infrared spectrum readings can be more clearly seen in the attached drawings.

1n the drawings:

FIG. 1 shows the infrared spectrum of amethobottromycin. FIG. 11 shows the infrared spectrum of methobottromycin.

Methobottromycin and amethobottromycin exhibit characteristic R; values in the following solvent systerns:

Ametho- Methobottrobottromycin mycin n-butyl alcohol saturated with 1% aqueous acetic acid 0.85 085 n-butyl alcohol saturated with 2% aqueous pyridine 0.84 0.84

ethyl acetate saturated with 1% aqueous acetic acid 0.37 0.37

ethyl acetate saturated with 0.1M phosphate buffer (pl-l 7) 0.83 0.83

benzene:hexane:methanol:5% aqueous acetic acid (7:6:10:6) 0.0 0.0

benzene:hexane:methanol:5% aqueous pyridine (7:6:1016) 0.5 0.5

benzene saturated with 1% aqueous acetic acid 0.0 0.0

benzene saturated with 2% aqueous pyridine 0.5 0.5

capryl alcohol saturated with 0.1M: pH 6 phosphate (reverse phase) 030 0.19

Characteristics of the antibiotics from which the molecular modifications of the present invention may also include thin layer chromatography. Thin layer chromatographic plates containing silica gel are developed in 94 percent chloroform and 6 methanol, dried and placed in a chamber containing iodine vapor. A brown stain indicates the presence of these antibiotics. The R, of the methobottromycin zone is 0.64 and the amethobottromycin 'zone is 0.60.

The culture producing methobottromycin and amethobottromycin produces generally two types of substances: a netropsin-type antibiotic and a bottromycintype antibiotic. The bottromycin group of antibiotics from which methobottromycin and amethobottromycin are extracted, is readily separated from the netropsin group by extraction with chloroform from aqueous solutions. The chloroform extract, after purification, shows the presence of five antibiotic substances on bioautograph of paper strips. The paper strip system utilized for this test consists of paper impregnated with capryl alcohol and developed downflow with buffer, wherein the R; of methobottromycin is 0.19 and amethobottromycin is 0.30. The five components have been designated components A through E in order of decreasing polarity. Table A below lists bioactivity of all of the components of bottromycin. The first column Staph. MIC is a tube dilution assay which measures the minimal inhibitory concentration of the antibiotic in a broth culture of the test microorganism, Mycoplasma gallisepticum (PPLO). The second column In Ova ED is an in ova assay which measures the effec- Table A Staph. PPLO Egg Test Component MIC ug/ml ED ,ug/egg A 3.0 720 B 0.23 13 (Amethobottromycin) C 0.04 (Methobottromycin) D 0.16 E 0.23 5

Table B shows that ninhydrin-producing substances are liberated from the five components on acid hydrolysis and paper chromatography.

percentage present too small to separate As can be seen from Table B, amethobottromycin contains proline but does not contain methyl proline and methobottromycin does not contain proline but does contain methyl proline.

In an aqueous solution at pH greater than 10, these antibiotics are unstable. However, at pH from 3 to 9 these products are stable for 24 hours at room temperature.

The molecular modifications of the present invention are derived from antibiotics which are produced by the aerobic fermentation of S treptomyces canadensis MA- 959 in a suitable aqueous medium. Aqueous mediums such as those employed for the production of other antibiotics are suitable for the production of methobottromycin and amethobottromycin. Such mediums contain sources of carbon and nitrogen, assimilable by the microorganism, and inorganic salts. In addition, the fermentation mediums contain traces of metalnecessary for the growth of the microorganism which are usually present in complex sources of carbon and nitrogen in the medium.

In general, carbohydrates such as sugars, for example, dextrose, sucrose, dextrin and the like, are suitable sources of assimilable carbon. The exact quantity of the carbon source will depend, in part, upon the other ingredients of the medium, but it is usually found that an amount of carbohydrate between about 1 and 6 percent by weight of the medium is satisfactory. These carbon sources can be used individually, or several such sources may be combined in the medium.

Various nitrogen sources such as casein hydrolysates, amino acids, for example, asparagine, glycine, arginine, digests of soybean meal, soybean meal, distillers solubles, and the like are readily assimilated by the methobottromycin and amethobottromycin producing microorganism and can be used in fermentation mediums for the production of these antibiotics. In general, we find that organic sources of nitrogen, particularly soybean meal, are very satisfactory for the production of the new antibiotics. The various organic and inorganic sources of nitrogen can be used either alone or in combination in amounts ranging from about 0.2 to about 6 percent by weight of the aqueous medium.

The following example illustrates a method of preparing the antibiotic from which the molecule modifications of the present invention are derived, but it is to be understood that it is given for purposes of illustration and not of limitation.

Preparation of Methobottromycin and Amethobottromycin A. Fermentation A medium containing 1% dextrose, 0.3% meat extract, 1.0% tryptic digest of casein, and 0.5% sodium chloride was made up in water and adjusted to pH 7.0 with sodium hydroxide sterilized and aseptically added to a slant culture of Streptomyces canadensis MA-959 (ATCC 17776) and the spores scraped into suspension. About 3 ml. of this spore suspension was aseptically added to a stoppered 2 liter bafflcd Erlenmeyer flask containing 500 ml. of sterile aqueous medium consisting of 1% dextrose, 0.3% meat extract, 1.0% tryptic digest of casin, and 0.5% sodium chloride and the pH again adjusted to 7.0. The flask was incubated at 28C. on a rotary shaker at a speed of 120 RPM with a 2-inch throw for a period of 48 hours.

This vegetative culture was then aseptically added to a SO-gallon stainless steel fermenter containing about 30 to 40 gallons of sterile medium having a composition comprising 1.5% yeast autolysate, 1% dextrose, 0.25% sodium chloride with the pH adjusted to 7.5. The inoculated medium was incubated at 28C. for 40 hours during which time it was agitated with sterile air being passed through the medium at a rate of about 3 cubic feet per minute. About 8.4 percent of this vegetative culture was employed to inoculate a ISO-gallon stainless steel fermenter containing about 120 gallons of a medium having the composition comprising 1.5% yeast autolysate, 0.5% sodium chloride, and 3% dextrose at pH 7.0 previously sterilized with steam at about 120C. for 15 minutes. The culture was incubated at 28C. with agitation and aeration at a rate of 10 CFM until maxium antibiotic yield was obtained.

B. Recovery The antibiotics of the present invention were recovered from the fermentation broth by adjusting the pH of the broth to 4.8 with hydrochloric acid and filtering. The filtered broth was passed through a Dowex 50 X 2 sodium cycle resin (5 gallons) at a rate of 0.5 gallons per minute. The resin was washed with 10 gallons of water and eluted with 50 gallons of methanol, 30% IN ammonia at a rate of 025 gallons per minute. Ten S-gallon cuts were taken and each was neutralized to pH 7 with 5% hydrochloric acid. The cuts were assayed and the active cuts were evaporated to 7.5 gallons of water. The concentrate was adjusted to pH 8 and extracted three times with an equal volume of chloroform and the extracts were dried over sodium sulfate. The rich chloroform was passed through a column containing a mixture of 70% Florisil, 30% Celite 545 at a 10 minute contact time. The absorption was followed with gallons of chloroform wash followed by 50% chloroform-acetone elution at the same rate. V2 gallon cuts of the eluate were taken and the cuts were assayed. The active cuts were combined and evaporated to 0.5 gallons of chloroform. The chloroform concentrate was dried to a syrup and taken up in about 160 ml. of methanol. Ten volumes of ethyl ether were added and the insolubles filtered. A 1.2 N methanolic hydrochloric acid solution was added to the ether filtrate with stirring until no further precipitation occured. The precipitate was filtered, washed with ether and dried.

Methobottromycin and amethobottromycin were isolated by partition chromatography with 0.1 M pH 6.0 phosphate buffer as the eluent. Celite impregnated with capryl alcohol is used as the stationary phase. The stationary phase is made by wetting 250 lbs. of acid washed celite (diatomaceous earth) with a solution of 6 gallons of capryl alcohol and 24 gallons of acetone. The celite is air dried to remove acetone and packed in o a :7! me n wherein M is des-carbomethoxy methobottromycin, descarbomethoxy amethobottromycin or mixtures thereof; R and R can be the same or different hydro gen, alkyl radical, aryl radical or alkaryl radical said alkyl groups containing 1 to about 12 carbon atoms. A reaction for the preparation of the molecular modifications of the present invention takes place by reacting methobottromycin and amethobottromycin with varia satisfactory column in thin layers with tamping, to inous amines is shown as follows:

sure uniformity. After packing, the column is washed with one-half volume of 0.1 M pH 6.0 phosphate buffer. About grams of the crude hydrochloride described above is dissolved in 8 gallons of pH 6.0 buffer n-cou o c u-c-unn' 21-10 11' MCNRR' ca oir wherein M, R and R are the same as above.

Another method for the preparation of the molecular modifications of the present invention is illustrated as follows:

and placed on the column. The column was developed wherein M, R-and R are the same as above.

with pH 6.0 buffer taking 5 gallon cuts. Each fraction is assayed and examined by paper chromatography. The rich fractions were worked up by concentration to about one-fifth volume, adjusted to pH 8.5 and extracted two times with an equal volume of chloroform. The chloroform solutions were evaporated to dryness. The residual solids were dissolved in a small amount of methanol (about 500 ml.) and diluted with 20 volumes of ethyl ether and filtered. To the filtrate was added 1.2 N methanolic hydrochloric acid until further addition caused no precipitation.

The antibiotic hydrochlorides were collected and dried in vacuo with the following results:

The following examples illustrate methods of preparing the antibiotic molecular modifications of the present invention. It is to be understood, however, that they are given for the purposes of illustration and not of limitation.

EXAMPLE 1 Preparation of Methobottromycin Methylamide Methobottromycin (2.5 g.) is dissolved in 30 ml. of a methanolic solution containing 10 percent by weight of methylamine. The mixture is sealed in a Carius tube, and heated at 50C. for 20 hours. The reaction mixture is evaporated to dryness and the last trace of methylamine is removed. The residue is crystallized from ethyl acetate to yield 1.9 g., m.p. 225228C. dec. [(11, 23(C l, EtOH). Anal. Calcd. for C H N O S: C,

61.3; H,7.68; N, 15.33. Found: C, 61.0; H, 7.68; N, 15.39.

The hydrochloride is prepared by precipitation from ether by the addition of methanolic hydrogen chloride. Anal. Calcd. for C., H N O S.HC1.2H O: C, 56.5; H, 7.7; N, 14.15. Found: C, 56.20; H, 7.6; N, 14.00.

The infrared absorption spectrum of methobottromycin methylamide indicates the more significant of the characteristic peaks occur at the following wave lengths expressed in reciprocal centimeters (cm): 1690, 1662, 1632, 1620, 1510, 1450, 1428, 1372, 1356, 1332, 1278, 1255, 1232, 1140, 1010, 711 and 700.

Less prominent but distinguishable bands are observed as follows: 1651, 1530, 1495, 1410, 1308, 1221, 1208,1195,1170,1115,1l00, 768 and 755 cm.

EXAMPLE 2 Preparation of Amethobottromycin Methylamide 2.5 Grams of amethobottromycin is dissolved in 30 ml. of a methanolic solution containing 10 percent by weight of methylamine. The mixture is heated to 50C. for 20 hours in a sealed tube. Amethobottromycin methylamide is recovered and isolated according to the process described in Example 1.

EXAMPLE 3 Preparation of Mixture of Methobottromycin and Amethobottromycin Methylamide 2.5 Grams of a 70-30 percent mixture of methobottromycin and amethobottromycin is dissolved in 30 ml. of a methanolic solution containing 10 percent by weight of methylamide. The mixture is heated to 50C. for 20 hours in a sealed tube. The mixture of methobottromycin and amethobottromycin methylamide is recovered and isolated according to the process described in Example 1.

EXAMPLE 4 Preparation of Methobottromycin Ethylamide Methobottromycin (0.5 g.) is dissolved in 30 m1. of a methanolic solution containing 10 percent by weight of ethylamine. The mixture is sealed in a Carius tube, and heated at 50C. for 20 hours. The reaction mixture is evaporated to dryness. The residue is recrystallized from ethyl acetate to yield methobottromycin ethylamide, m.p. 174-180C. dec., [01],, 16(C 0.1, 95% EtOH). Anal. Calcd. for C,, H N O S.2H O: C, 59.20; H, 7.9; N, 14.41; S, 3.7. Found: C, 59.20; H, 7.63; N, 14.1; S, 4.00.

EXAMPLE 5 Preparation of Amethobottromycin Ethylamide 0.5 Gram of amethobottromycin is dissolved in 30 ml. of a methanolic solution containing percent by weight of ethylamine. The mixture is heated to 50C. for hours in a sealed tube. Amethobottromycin ethylamide is recovered and isolated according to the procedure described in Example 4.

EXAMPLE 6 Preparationof Mixture of Methobottromycin and Amethobottromycin n-propylamides 1.0 Gram of a 80-20 percent mixture of methobottromycin and amethobottromycin is dissolved in 30 ml. of a methanolic solution containing 10 percent by weight of n-propylamine. The mixture is heated to 50C. for 20 hours in a sealed tube. The mixture of the methobottromycin and amethobottromycin npropylamides is recovered and isolated according to the procedure described in Example 1.

EXAMPLE 7 Preparation of Methobottromycin B-phenylethylamide 1.0 Gram of methobottromycin is dissolved in 10 ml. of a methanolic solution containing 20 percent by weight of B-phenylethylamine. The mixture is heated to 50C. for 20 hours in a sealed tube. Methobottromycin B-phenylethylamide is recovered and isolated according to the procedure described in Example 1.

EXAMPLE 8 Preparation of Amethobottromycin 1-naphthyl methylamide 1.0 Gram of amethobottromycin is dissolved in 20 ml. of a methanolic solution containing 10 percent by weight of l-naphthylmethylamine. The mixture is heated to 50C. for 20 hours in a sealed tube. Amethobottromycin l-naphthylmethylamide is recovered and isolated according to the procedure described in Example 1.

EXAMPLE 9 Preparation of Methobottromycin Cyclohexylamide 1.0 Gram of methobottromycin is dissolved in 10 ml. of a methanolic solution containing 20 percent by weight of cyclohexylamine. The mixture is heated to 50C. for 20 hours in a sealed tube. Methobottromycin cyclohexylamide is recovered and isolated according to the procedure described in Example 1.

EXAMPLE 10 Preparation of Methobottromycin Benxylamide EXAMPLE 1 1 Preparation of Methobottromycin 2,3Propandiol- 1 -Amide 1.0 Gram of methobottromycin is dissolved in 10 ml. of a methanolic solution containing 20 percent by weight of 2,3-propandiol-lamine. The mixture is heated to 50C. for 20 hours in a sealed tube. Methobottromycin 2,3-propandiol-l-amide is recovered and isolated according to the procedure described in Example 1.

EXAMPLE 12 Preparation of Methobottromycin Ethanolamide 1.0 Gram of methobottromycin is dissolved in 10 ml. of a methanolic solution containing 20 percent by weight of ethanolamine. The mixture is heated to 50C. for 20 hours in a sealed tube. Methobottromycin ethanolamide is recovered and isolated according to the procedure described in Example 1.

EXAMPLE 13 Preparation of t-butylamide of Methobottromycin A solution of 2.0 grams of methobottromycin dissolved in 100 ml. of acetone and 100 ml. of 0.1 N sodium hydroxide is stirred at C. for 3 hours. Hydrochloric acid is added to adjust the pH to 6.3 and the solution concentrated to about 50 ml. and allowed to cool. The free acid methobottromycin crystallizes on cooling and is collected by filtration and washed with water. After drying at 80C. at 1 mm for 1 hour, 1.85 grams of the free acid of methobottromycin is obtained, m.p. 195-200C.

450 Milligrams of the free acid is dissolved in ml. of dimethylformamide and 219 mg. of bis-(2,4-dinitrophenyl) carbonate is added. 0.14 Milliliters of triethylamine is then added and the mixture is stirred at 0C. for 45 minutes and then at room temperature for minutes. To this mixture is added 2 ml. of tbutylamine. The mixture is heated at 50-60C. for 30 minutes, diluted to about 25 ml. with chloroform and filtered. The filtrate is concentrated to near dryness under reduced pressure. The residue is dissolved in chloroform and then washed with saturated sodium chloride solution containing 5 percent ammonia until the aqueous layer is colorless. The organic layer is dried over sodium sulfate and then concentrated to 390 mg. of a yellow glass. Preparative tlc yields 197 mg. of the t-butylamide of methobottromycin. Anal. Calcd. for C., H N O S: C, 62.54; H, 8.05; N, 14.59; S, 3.17. Found: C, 62.33; H, 8.11; N, 14.35; S, 3.61. The infrared absorption spectrum of methobottromycin tbutylamide indicates that the more significant of the characteristic peaks occur at the following wave lengths expressed in reciprocal centimeters (cm): 1655, 1648, 1530, 1495, 1460. 1388, 1374, 1362, 1305, 1255, 1220, 1140, 1115, 718 and 700. Less prominent but distinguishable bands are observed as follows: 1508, 1340, 1195, 1178 and 752 cm.

EXAMPLE 14 Preparation of Aniline Amide of Methobottromycin 8O Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand overnight. To this mixture 10 ml. of methylene chloride is added, the mixture is then washed with water, and the water washes discarded. The methylene chloride is evaporated and the aniline amide of methobottromycin is recovered in a yield of about 70 percent.

EXAMPLE Preparation of p fluoro Aniline Amide of Methobottromycin 80 Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitriphenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of p-fluoro aniline is then added and the mixture is allowed to stand overnight. To this mixture 10 ml.-of methylene chloride is added, the mixture is then washed with water and the water washes discarded. The methylene chloride is evaporated and the p-fluoro aniline amide of methobottromycin is recovered in a yield of about 60 percent.

EXAMPLE 16 Preparation of N,N-dimethyl Amide of Methobottromycin Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitropheny] carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of dimethylamine is then added and the mixture is allowed to stand overnight. To this mixture 10 ml. of methylene chloride is added, the mixture is then washed with water and the water washes discarded. The methylene chloride is evaporated and the N,N- dimethyl amide of methobottromycin is recovered in a yield of about 56 percent.

EXAMPLE 17 Preparation of 2-aminobenzimidazole Amide of Amethobottromycin 1.0 Gram of amethobottromycin is dissolved in 50 ml. of acetone and 50 ml. of 0.1 N sodium hydroxide is added and let stand for 2 hours. Hydrochloric acid is added to adjust to pH 6.2 and the solution is concentrated to one-half volume and allowed to cool. About 0.8 grams of the free acid of amethobottromycin crystallizes from this solution. 80 Milligrams of the free acid of amethobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4- dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of 2- aminobenzimidazole is then added and the mixture is allowed to stand overnight. To this mixture 10 ml. of methylene chloride is added, the mixture is then washed with water and the water washes discarded. The methylene chloride is evaporated and the 2- aminobenzimidazole amide of amethobottromycin is recovered in a yield of about 50 percent.

EXAMPLE 18 Preparation of Morpholine Amide of Methobottromycin 80 Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of morpholine is then added and the mixture is allowed to stand overnight. To this mixture 10 ml. of methylene chloride is added, the mixture is then washed with water, and the water washes discarded. The methylene chloride is evaporated and the morpholine amide of methobottromycin is recovered in a yield of about 65 percent.

EXAMPLE 19 Preparation of N,N-dimethylamino Ethyl Amide of Methobottromycin 80 Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of N,N-dimethylamino ethylamine is then added and the mixture is allowed to stand overnight. To this mixture, l ml. of methylene chloride is added, the mixture is then washed with water, and the water washes discarded. The methylene chloride is evaporated and the N,N-dimethylamino ethyl amide of methobottromycin is recovered in a yield of about 60 percent.

EXAMPLE 2O Preparation of Methyl Ester of Phenylalanine Amide of Methobottromycin 8O Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of methyl ester of phenylalanine is then added and the mixture is allowed to stand overnight. To this mixture, 10 ml. of methylene chloride is added, the mixture is then washed with water, and the water washes discarded. The methylene chloride is evaporated and the methyl ester of phenylalanine amide of methobottromycin is recovered in a yield of about 58 percent.

EXAMPLE 21 Preparation of Dodecyl Amide of Methobottromycin 80 Milligrams of the free acid of methobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. l0 Milligrams of dodecylamine is then added and the mixture is allowed to stand overnight. To this mixture 10 ml. of methylene chloride is added, the mixture is then washed with water and the water washes discarded. The methylene chloride is evaporated and the dodecyl amide of methobottromycin is recovered in a yield of about 62 percent.

EXAMPLE 22 Preparation of Imidazole Amide of Amethobottromycin 80 Milligrams of the free acid of amethobottromycin is dissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is then added and the mixture is allowed to stand for 30 minutes. 10 Milligrams of imidazole is then added and the mixture is allowed to stand overnight. To this mixture 10 ml.'of methylene chloride is added, the mixture is then washed with water and the water washes discarded. The methylene chloride is evaporated and the imidazole amide of amethobottromycin is recovered in a yield of about 59 percent.

The molecular modification amides of amethobottromycin, methobottromycin, and mixtures thereof are valuable antibacterial agents which, as has been pointed out above, are active in inhibiting the growth of various gram positive organisms. However, these, antibiotics are extremely useful in the treatment of chronic respiratory disease of chickens and infectioussinusitis of turkeys. In this embodiment of the present invention it has been found that chronic respiratory disease of chickens and infectious sinusitis of turkeys may be effectively inhibited by the use of molecular modification amides of the present invention which may be administered by either the subcutaneous or the oral route. Furthermore, it has been found that the molecular modification amides of the present invention are effective in controlling chronic respiratory disease in chickens and infectious sinusitis in turkeys when administered in dosages ranging from about 0.1 mg./kg. to about 250 mg./kg. of body weight ofthe bird without the resulting danger of toxicity and preferably from 0.5 mg./kg. to I50 mg./kg. of body weight of the birds, depending on the route of therapy.

In order to illustrate the activity and the antibiotic advantages of the present invention in utilizing the molecular modification amides of amethobottromycin, methobottromycin, and mixtures thereof, the following test examples are given. It is understood, however, that they are given merely for the purpose of illustration and in no way are they to be taken as limiting.

In order to illustrate the activity of the compounds of the present invention as antibiotic agents against chronic respiratory disease of chickens and infectious sinusitis of turkeys the following examples are given:

EXAMPLE 23 Activity of the Methyl Amide of Methobottromycin against Mycoplasma gallisepticum (PPLO) Infections in Chickens (Oral Administration) of Non- Avg. Wt. Infected Mortality Treatment Gain-gm Body Wt. Gain Dead/Total Non-Infected Control 212 0/6 Infected Control 139 67 10/21 Methyl Amide of Methobottromycin 25 mgJkg. I73 82 0/6 50 mgJkg. 208 98 3/6 I00 mgJkg. I69 0/6 In the above test the body weight gain in the birds treated by the method of the present invention showed a significant increase over those of the infected controls. In addition, where 11 out of 21 or 52 percent of the infected controls died, only 3 out of 18 or 17 percent of the birds treated by the method of the present invention died.

EXAMPLE 24 Activity of the Methyl amide of Methobottromycin against Mycoplasma gallisepticum (PPLO) Infections in Chickens (Subcutaneous Administration) of Non- Avg.Wt. Infected Mortality Treatment Gain-gm. Body Wt. Gain Dead/Total Non-Infected Control 216 /6 Infected Control 107 50 7/18 Methyl Amide of Methobottromycin 0.5 mgJkg. 157 73 0/6 1.0 mgJkg. 193 89 2/6 2.0 mgJkg. 196 91 2/6 Weeks-n .9 2 .9. 9L9.

In the above test the body weight gain in the birds treated by the method of the present invention showed a significant increase over those of the infected controls. In addition, were 7 out of 18 or 39 percent of the infected controls died, only 4 out of 24 or 17 percent of the birds treated by the method of the present invention died.

To further illustrate the antibiotic activity of the amide compositions of the present invention, these compositions were tested against PPLO microorganisms. The results of in vitro assays (agar diffusion method) are illustrated but not limited to the following example:

Example 25 Amide in Vitro Activity Zone of Inhibition: mm 250 'yglml Amide seo 'yg/ml 1.0 mglml Benzyl Amide of Methobottromycin Aniline Amide of Methobottromycin p-Fluoroaniline Amide of Methobottromycin Morpholine Amide of Methobottromycin Methyl B-phenylalanine amide of Methobottromycin Dodecyl Amide of Methobottromycin lmidazole Amide of Methobottromycin EXAMPLE 26 In Vivo Activity of Methobottromycin and Amethobottromycin Amides against Mycoplasma gallisepticum in Chickens Groups of 5-day old white Leghorn pullets were infected via the air sac route with a broth culture of Mycoplasma gallisepticum. Various amides of the present invention were administered in a water solution by the subcutaneous route with dosages given at 2 and 18 hours after infection in the amount of 10, 25, and 50 mgjkg. of body weight for each group of pullets. The birds were observed for development of symptoms of chronic respiratory disease, and the results were as follows:

Amides Results at Test Termination Disease evident Disease arrested Disease arrested Disease arrested Disease arrested Disease arrested Untreated Controls Methylamide Ethanolamide 2,3-propandiolamide p-Fluoroaniline amide Pyrolidineamide It is evident from the test data above that chickens that were infected with Mycoplasma gallisepticum had the infectious chronic respiratory disease arrested when treated with as little as 10 mgjkg. of the amides of the present invention. Whereas, the untreated chickens showed continued infection of chronic respiratory disease.

protect 50% of the infected mice.

The in vivo test, the results of which are listed above,

was carried out by giving the mice a two-dose treatment at O and 6 hours after infection and the test was concluded seven days after infection.

While specific embodiments of the present invention have been named and described, it will be apparent to those skilled in the art that changes may be made in the detail shown without departing from the spirit of the present invention or the scope intended. Any departure from the above description which conforms to the present invention is intended to be included within the scope 'of the claims.

What is claimed is:

1. Methobottromycin methylamide being characterized by the following properties:

a. melting at 225228C. with decomposition,

b. having a specific rotation of [07],, 23 (C l,

percent ethyl alcohol),

0. having the empirical formula C H N O S,

(1. containing 61.3% carbon, 7.68% hydrogen and 15.33% nitrogen, and

e. having an infrared absorption spectrum exhibiting characteristic peaks at the following wave lengths expressed in reciprocal centimeters: 1690, 1662, 1632,1620,1510,1450,1428,1372,1356,1332, 1278, 1255, 1232, 1140, 1010, 711 and 700.

2. Methobottromycin t-butylamide being characterized by the following properties:

a. having the empirical formula C H N O S,

b. containing 62.54% carbon, 805% hydrogen, 0

14.59% nitrogen and 3.17% sulfur, and 0. having an infrared absorption spectrum exhibiting the following characteristic peaks expressed in re-

Claims

2. Methobottromycin t-butylamide being characterized by the following properties: a. having the empirical formula C45H69N9O6S, b. containing 62.54% carbon, 8.05% hydrogen, 14.59% nitrogen and 3.17% sulfur, and c. having an infrared absorption spectrum exhibiting the following characteristic peaks expressed in reciprocal centimeters: 1655, 1648, 1530, 1495, 1460, 1388, 1374, 1362, 1305, 1255, 1220, 1140, 1115, 718 and 700.
3. A method of treating poultry infected with pleuropneumonia-like organisms which comprises administering to said infected poultry an effective dose of methobottromycin methylamide or methobottromycin t-butylamide.
4. The method of claim 3 wherein said effective dose is in the range of from about 0.1 mg./kg. to about 250 mg./kg. of body weight of said infected poultry.
700.