MACROLIDE ANTIBIOTICS, THEIR PREPARATION AND VETERINARY COMPOSITIONS CONTAINING THEM

Abstract: Novel macrolides of formula (I): where Q is -CHO or -CH₂OH and Y is hydroxyl or a myca­ rosyloxy group; and salts or esters thereof; can be prepared by the sub­ merged aerobic cultivation of Streptomyces fradiae ATCC 31669 and are of value in the chemotherapy of microbiai infec­ tions in warm blooded animals and as intermediates in the pre­ paration of other closely related macrolide antibiotics.

Description: MACROLIDE ANTIBIOTICS, THEIR PREPARATION AND VETERINARY COMPO.SITIONS CONTINING THEM This invention relates to macrolide antibiotics, in particular to compounds similar to the well-known therapeutic agent ty'osin (see, for example, Tetrahedron Letters, 2339 (1970)). Despite tylosin's great value, there extsts a constant need to develop new antibiotics, inter alia, in view of the possibility of emergence of resistant strains. Further, structural modification can lead to changes in the spectrum of susceptible microorganisms. Unfortunately, chemical modification of the structure of tylosin-like macrolides has proven to be extra- ordinarily difficult. For instance, in J. Org. Chew. 44 (12)-, 2050-2 (1979) there is an indication of the problems involved in the cleavage of the mycinosyl glycoside linkage of tylosin by chemical means. Indeed, in the overwhelming majority of cases, research workers in this area, intent on investigating novel structures, have been forced to search for new microorganisms, either naturally-occurring or synthetic, hoping that their cultivation will yield related structures. The Applicants have now discovered that compounds similar to tylosin can be prepared by the submerged aerobic fermentation of certain Streptomyces fradiae strains. According to the present invention there is provided a new macrolide antibiotic having the struc- tural formula t EMI2.1 where Q is -CHO or -CH2OH and Y is hydroxyl or a mycarosyloxy group; or a salt or ester thereof. The compounds of formula (I) are 2'''-Odemethylmacrocin, hereinafter referred to as "demethylmacrocin or DO.YM" and having the structural formula (II) : EMI2.2 the dihydro-derivative of DOMM, i.e. 20-dihydro-2"'- o-demethylmacrocin, hereinafter referred to as dihydro- DOMM" and having the structural formula (III): EMI3.1 2"'-de-O-methylactenocin, hereinafter referred to as "DOML", and having the structural formula (IV): EMI3.2 and 20-dihydro-2' ' 1-de-O-methylactenocin, hereinafter referred to as "dihydro-DOML", and having the structural formula (V): EMI4.1 and salts and esters thereof. The stereochemistry of the compounds of the invention is identical to that of the well-known macrolide tylosin--see, for instance, Tetrahedron Letters, 4737, (1970). The amino sugar is mycaminose, the left hand neutral sugar is 6-deoxy-D-allose and the neutral sugar in structures (II) and (III) is mycarose. The hydroxyl groups of DOMM and dihydro-DOMM at the 2', 4", 3", 2''', 3''', 4''' and 3-positions can be esterified to form useful acyl ester derivatives. The hydroxyl groups of DOML and dihydro-DOML at the 2', 4', 2''', 3''', 4' " and 3-positions can be esterified to form useful acyl ester derivatives. In addition, dihydro-DOMM and dihydro-DOML can be esterified on the 20-hydroxyl group. Esterification of the 2'-hydroxyl group of DOMM and dihydro-DONM is facile. The 2'- and 4'-hydroxyl groups of DOML and dihydro-DOML are readily esterified. Typical esters are those of a monocarboxylic acid or hemi-esters of a dicarboxylic acid having from 2 to 18 carbon atoms. DOMM, dihydro-DOMM, DOML, dihydro-DOML, and their acyl ester derivatives are basic compounds which, when combined with acids, are converted to acid addition salts. These addition salts are also part of this invention. To simplify discussions of utility, the term "DOMM compound" is used and refers to DOMM, dihydro-DOMM, DOML, dihydro-DOML a specified acyl ester derivative of these compounds, or a pharmaceutically acceptable acid addition salt of DOMM, dihydro-DOMM, DOML, dihydro-DOML or of their acyl ester derivatives. In a further aspect of the invention there is provided a method of preparing a novel macrolide of formula (I) which comprises culturing a strain of Streptomyces fradiae under submerged aerobic conditions until a substantial level of antibiotic activity is produced, provided that if it LS desired to produce a compound of formula (IV) or (V) then the cultivation should be effected using mildly acidic conditions. The preferred Streptomyces fradiae microorganism is that deposited in the American Type Culture Collection, Rockville, Maryland, U.S.A., under ATCC 31669 on 21st July 1980. DOMM is soluble in water and in most polar organic solvents, such as acetone, methanol, ethanol, chloroforn, dimethylformamide and methyl sulfcxide. DObX acid addition salts are more soluble in water than is DOMM base. DOMM can be distinguished from tylosin by paper and thin-layer chromatography. The approximate Rf and Rx values of DOMM and tylosin are summarized in Tables 1 and 2. In Table 2 Rx value is the rate of movement expressed relative to that of tylosin, which was given a value of 1.0. Bioautography with Bacillus subtilis was used for detection. Table 1 Thin-Layer Chromatography of DOMMa Rf Value Compound Ab B C Tylosin 0.53 0.53 0.67 DOMM 0.05 0.44 0.44 medium: Merck, Darmstadt - Silica Gel 60 bSolvent: A = ethyl acetate:diethylamine (96:4) B = acetone:ethanol (2:1) C = chloroform:methanol (3:1) Table 2 Paper Chromatography of DOMMa Rx Compound Db E Tylosin 1.0 1.0 DOMM 0.17 0.88 apaper : Whatman No. 1 treated with 0.75 M KH2PO4 buffer at pH 4.0 and dried bSclvent: D = ethyl acetate saturated with water E = n-butanol saturated with water The dihydro derivative of DOMM can be obtained by chemical reduction or by fermentation. When preparing dihydro-DOMM by chemical reduction, known procedures such as, for example, treating DOMM with van approximately stoichiometric amount of sodium borohydride in an alcoholic solvent, may be used. Dihydro-DOMM is also produced by the S. fradiae ATCC -31669 of this invention under controlled fermentation conditions. This invention further relates to methods of preparing DOML and dihydro-DOML by mild acid hydrolysis of DOMM and dihydro-DOMM, respectively. Dihydro-DOML can also be prepared by 1) mild acid hydrolysis of DOMM to give DOML followed by 2) reduction of DOML to give dihydro-DOML. Mild acid hydrolysis conditions are known In the art. Appropriate aqueous solutions having a pH of about four or below can be used to accomplish the hydrolysis. Temperatures of about 200C to about 1000C can be used in this method. The reaction time needed to carry out the hydrolysis varies, depending upon the pH of the reaction mixture and the temperature used. At higher pH levels the reaction rate is slower, and at higher temperatures the reaction rate is faster. At about pH 2 and at room temperature, the reaction time required is from about 6 to about 24 hours. The reaction is carried out by treating either DOMM or dihydro-DOMM with a mild acid solution for a time sufficient to effect removal of the mycarosyl group to give DOML or dihydro-DOML, respectively. Alternatively, and sometimes preferably, DOML or dihydro-DOML can be prepared by treating DOMM or dihydro-DOMM in the fermentation broth in which it is produced, using mild acidic conditions as above described for a time sufficient to convert the DOMM or dihydro DOMM to DOML or dihydro-DOML, respectively. DOML or dihydro-DOML thus prepared can be isolated from the fermentation broth using techniques known in the art. DOMM and dihydro-DOMM can be esterified at the 2', 4", 3", 2''', 3''', 4111 and 3-hydroxyl groups to give acyl ester derivatives by treatment with acylating agents using methods known in the art. DGML and dihydro-DOML can be esterified at the 2', 4', 2"', 3''', 4"' and 3-hydroxyl groups in a similar manner. In addition, dihydro-DOMM and dihydro-DOML can be esterified at the 20-position. Esterification of the 2'-hydroxyl group of DOMM and dihydro-DOMM is facile. The 2'- and 4'-hydroxyl groups of DOML and dihydro DOML are readily esterified. Typical acylating agents include anhydrides, halides (usually in combination with a base or other acid scavenger) and active esters of organic acids. Acylation can also be achieved by using a mixture of an organic acid and a dehydrating agent such as N,N'-dicyclohexylcarbodiimide. Acylations can also be carried out enzymatically as described by Okamoto et al. in U.S. Patent No. 4,092,473. Once formed, the acyl derivatives can be separated and purified by known techniques. The 2'-monoester derivatives of DOMM and dihydro-DOMM can be prepared by selective esterizi- cation techniques generally known in the art, such as, for example, treatment of the antibiotic with a stoichiometric quantity (or a slight excess) of an acylating agent, such as an acyl anhydride, at from about OOC to about room temperature for from about 1 to about 24 hours until esterification is substantially complete. The 2'-monoester can be isolated from the reaction mixture by standard procedures such as extraction, chromatography and crystallization. Useful esters are those of organic acids including aliphatic, cycloaliphatic, aryl, aralkyl, heterocyclic carboxylic, sulfonic and alkoxycarbonic acids of from 2 to 18 carbon atoms, and of inorganic acids, such as sulfuric and phosphoric acids. Representative suitable esters include those derived from acids such as acetic, chioroacetic, propionic, butyric, isovaleric, alkoxycarbonic, stearic, cyclopropanecarboxylic, cyclohexanecarboxylic, D-cyclo- hexylpropionic, l-adamantanecarboxylic, benzoic phenylacetic, phenoxyacetic, mandelic and 2-thienyi- acetic acids, and alkyl-, aryl-, and aralkyl-sulfonic acids, the aryl- and aralkyl- acids optionally bearing substituents such as halogen, nitro, lower alkoxy and the like on the aromatic moiety. Suitable esters also include hemi-esters derived from dicarboxylic acids such as succinic, maleic, fumaric, malonic and phthalic acids. Pharmaceutically acceptable ester derivatives are a preferred group, such esters do not exhibit any significant toxic effects towards the warm-blooded animals to be treated. Other ester derivatives are useful, however, as intermediates. DOMM, dihydro-DOMM, DOML, dihydro-DOML and their specified derivatives form acid addition salts. The acid addition salts of DOMM, dihydro-DOMM, DOML, dihydro-DOML and of their acyl derivatives are also part of this invention. Such salts are useful, for example, for separating, purifying and crystallizing DOMM, dihydro-DOMM, DOML, dihydro-DOML, and their acyl derivatives. In addition, the salts have an improved solubility in water. Representative suitable salts include those salts formed by standard reactions with both organic and inorganic acids such as, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumÅaric, palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic and the like acids. Pharmaceutically acceptable acid addlticn salts are an especially preferred group of salts of this invention. µ'Pharmaceutically acceptable" salts are salts which do not exhibit any significant toxic effects towards the warm-blooded animals to be treated. The compounds of this invention will usually be administered as veterinary formulations, incorporating conventional excipients and carriers, i.e., pharmaceutically-acceptable organic or inorganic carrier substances suitable for enteral, parenteral or topical application which do not deleteriously react with the active compounds. Suitable, pharmaceutically acceptable carriers, include but are not limited to water, salt solutions, alcohols, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, paraffins, perfume oil, fatty acid monoglycerides and diglycerides and hydroxy methylcellulose. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, buffers, coloring or flavoring substances which do not deleteriously react with the active compounds. The compounds of this invention can also be employed in feed additive forms suchas feed premix in conventional formulation methods. In a further aspect of the invention there is provided a veterinary formulation comprising from 0.1 to 90% by weight of a compound of formula (1), or a pharmaceutically acceptable salt or ester thereof, associated with one or more pharmaceutically-acceptable carriers or excipients therefor. This invention also provides additional methods of preparing 5-O-mycaminosylty:onolide (OMT) and 20-dihydro-5-O-mycaminosyltylonolide (dihydro-OMT). OMT and dihydro-OMT are described by Gorman et al. in U.S. Patent No. 3,459,853. In U.S. Patent No. 3,459,853, OMT and dihydro-OMT are prepared by removing the neutral sugars by controlled acid hydrolysis of tylosin, desmycosin, macrocin, and lactenocin and their dihydroderivatives. This invention provides new starting materials from which OMT and dihydro-OMT can be prepared. Thus, using the method described in U.S. Patent No. 3,459,853, DOMM and DOML can be used to prepare OMT; and dihydro-DOMM and dihydrcrOML can be used to prepare dihydro-OMT. DO and dihydro-DOMM can be prepared by culturing a strain of Streptomyces fradiae which produces these compounds under submerged aerobic conditions in a suitable'culture medium until substantial antibiotic activity is produced. As will be appreciated by those skilled in the art, DOMM is produced first in the fermentation process. Dihydro-DOMM is produced when the fermentation is carried out for a longer time, thus permitting the DOMM present to be reduced enzymatically. The culture medium used to grow Streptomyces fradiae ATCC 31669 can be any one of a number of media. For economy in production, optimal yield, and ease of product isolation, however, certain culture media are preferred. Thus, for example, preferred carbon sources in large-scale fermentation include carbohydrates such as dextrin, glucose, starch, and corn meal, and oils such as soybean oil. Preferred nitrogen sources include corn meal, soybean meal, fish meal, amino acids and the like. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, potassium, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, and like ions. EssentIal trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism. It may be necessary to add small amounts (i.e. 0.2 ml/r) of an antifoam agent such as polypropylene glycol (M.W. about 2000) to large-scale fermentation media if foaming becomes a problem. For production of substantial quantities of DOMM or dihydro-DOMM, submerged aerobic fermentation in tanks is preferred. Small quantities of DOMM or dihydro-DOMM may be obtained by shake-flask culture. Because of the time lag in antibiotic production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larder tank. The medium used for the vegetative inoculum can be the same as that used for larger fermentations, but other media can also be used. S. fradiae ATCC 31669 can be grown at temperatures between about 100C and about 370C. Optimum antibiotic production appears to occur at temperatures of about 280C. As is customary in aerobic submerged culture processes, sterile air is bubbled through the culture medium. For efficient antibiotic production the per cent of air saturation for tank production should be about 30% or above (at 280C and one atmosphere of pressure). Antibiotic production can be followed during the fermentation by testing samples of the broth against organisms known to be sensitive to these antibiotics. One useful assay organism is Staphylococcus aureus ATCC 9144. The bioassay is conveniently performed by an automated turbidometric method. In addition, antibiotic production can be readily monitored by ultraviolet absorption of the extracted broth. Highperformance liquid chromatography with UV detection can also be used to monitor production Csee for example, J.H. Xennedy in J. Chromatographic Science, 16, 492-295 (1978)). Following its production under submerged aerobic fermentation conditions, DCMM or dihydro-DOMM can be recovered from the fermentation medium by methods used in the fermentation art. Recovery of DOMM or dihydro-DOMM is accomplished by an initial fiitration of the fermentation broth. The filtered broth can then be further purified to give the desired antibiotic. A variety of techniques may be used in this purification. A preferred technique for purification of the filtered broth involves adjusting the broth to about pH 9; extracting the broth with a suitable solvent such as ethyl acetate, amyl acetate, or methyl isobutyl ketone; extracting the organic phase with an aqueous acidic solution; and precipitating the antibiotic by making the aqueous extract basic. Further purification involves the use of extraction, adsorption and/or crystallization techniques. As is the case with other organisms, the characteristics of Streptomyces fradiae ATCC 31669 are subject to variation. For example, artificial variants and mutants of the ATCC 31669 strain may be obtained by treatment with various known physical and chemical mutagens, such as ultraviolet rays, X-rays, gamma rays, and N-methyl-N'-nitro-N-nitrosoguanidine. All natural and artificial variants, mutants and recombinants of Streptomyces fradiae ATCC 31669 which retain the characteristic of production of DOtAM may be used in this invention. The DOMM compounds inhibit the growth of pathogenic bacteria, especially gram-positive bacteria and Mycoplasma species. Accordingly, in one aspect of the invention there is provided a method of preventing, controlling or treating microbial infections, particularly those caused by gram-positive bacteria and mycoplasma, with the novel compounds of the invention. Table 3 summarizes the minimal inhibitory concentrations (MIC), as measured by standard agardilution assays, at which DOM (free base) inhibits certain bacteria. Table 3 In Vitro Activity of DOMM Free Base Organism MIC (;g/ml) Streptococcus pyogenes C203 0.25 Streptococcus pneumoniae Park I 0.25 Streptococcus sp. (Group D) 282 4.0 Pasteurella multocida 12.5 Pasteurella hemolytica 50.0 Mycoplasma gallisepticum 0.78 Mycoplasma hyopneumoniae 0.78 Mycopiasma hyorhinis 3.12 The DOMM-compounds have shown in vivo antimicrobial activity against experimental bacterial infections. When two doses of test compound were administered to mice in experimental infections, the activity observed was measured as an ED50 value [effective dose in mg/kg to protect 50% of the test animals: see Warren WicX, et al., J. Bacteriol. 81, 233-235 (1961)l. The ED50 values observed for DOMM are given in Table 4. Table 4 Subcutaneous ED50 Values (mg/kg x 2) of DOMM Streptococcus Streptococcus Staphylococcus Compound pyogenes C203 pneumoniae Park I aureus 3055 DOMM 1.1 37.5 3.8 Bacterial 133 41.2 139 challenge (x LD50) The following non-limitative Examples are provided to more fully illustrate the invention. Example 1 A. Shake-flask Fermentation of DOMM A lyophilized pellet of Streptomyces fradiae ATCC 31669 was dispersed in 1-2 ml of sterilized water. A portion of this solution (0.5 ml) was used to inoculate a vegetative medium (150 ml) having the following composition: Ingredient Amount (%) Corn steep liquor 1.0 Yeast extract 0.5 Soybean grits 0.5 CaCO3 0.3 Soybean oil (crude) 0.45 Deionized water 97.25 Alternatively, a vegetative culture of c. fradiae ATCr 31669 preserved, in l-ml volumes, in liquid nitrogen Fmsrapidy thawed and used to inoculate the vegetative medium. The inoculated vegetative medium was incubated in a 500-ml Erlecmeyer flask at 29OC for about 48 hours on a closed-box shaker at 300 rpm. This incubated vegetative medium (0.5 ml? was used to inoculate 7 ml of a production medium having the following composition: Ingredient Amount (%) Beet molasses 2.0 Corn meal 1.5 Fish meal 0.9 Corn gluten 0.9 NaCl 0.1 (NH4)2HPO4 0.04 CaCO3 0.2 Soybean oil (crude) 3.0 Deionized water 91.36 The inoculated fermentation medium was incubated in a 50-ml bottle at 290C for about 6 days on a closed-box shaker at 300 rpm. B. Tank Fermentation of DOMM In order to provide a larger volume cf inoculum, 60 ml of incubated vegetative medium, prepared in a manner similar to that described in section A, was used to inoculate 40 liters of a second-stage vegetative growth medium having the following composition: Ingredient Amount (%) Corn steep liquor 1.0 Soybean oil meal 0.5 Yeast extract 0.5 CaCO3 0.3 Soybean oil (crude) 0.5 Lecithin (crude) 0.015 Water 97.185 The pH was adjusted to 8.5 with 50% NaOH solution. This second-stage vegetative medium was incubated in a 68-liter tank for about 40 hours at 290C, with adequate aeration and agitation. Incubated second-stage medium ( L) was used to inoculate 44 liters of sterile production medium having the following composition: Ingredient Amount (%) Fish meal 0.875 Corn meal 1.5 Corn gluten 0.875 CaCO3 0.2 NaC1 0.1 (NH4) 2HPO4 0.04 Beet molasses 2.0 Soybean oil (crude) 3.0 Lecithin 0.09 Water 91.32 The pE was adjusted to 7.2 with 50% NaOH solution. The inoculated production medium was allowed to ferment in a 68-liter tank for about four days at a temperature of 280C. The fermentation medium was aerated with sterile air to keep the dissolved oxygen level between about 30% and 50% and stirred with conventional agitators at about 250 rpm. Example 2 Isolation of DOMM Harvested whole broth (38 L), obtained as described in Example 1, was filtered with the use of a filter aid. The mycelial cake was washed with water; this water wash was then added to the filtrate. The pH of the filtrate was adjusted to pH 9.1, using a 25% aqueous solution of sodium hydroxide. The filtrate was extracted twice with ethyl acetate (9 and 5 L). Deionized water (3500 ml) was added to the ethyl acetate extract. The pH of this solution was adjusted to about pH 4.1, using a 28% phosphoric acid solution (2 parts water to one part concentrated phosphoric acid). After extraction, the aqueous phase was separated. The ethyl acetate extract was extracted again with water (3500 ml) using this procedure. The two aqueous extracts were combined. The combined aqueous extracts was adjusted to pE 8.5 with sodium hydroxide and extracted twice with chloroform (3000- and 1200-ml portions). The chloroform extracts were dried under vacuum to give about 40 g of crude DOMM base. This DOMM base was purified by crystallization from water-acetone to give about 30 g of DOMM base. DOMM is a white solid which crystallizes from acetone-water. DOMM softens at about 135-1400C and melts completely by about 1600C. Elemental analysis of DOMM indicates that it has the following approximate percentage composition: carbon, 59.58; hydrogen, 8%; nitrogen, 28; oxygen, 30.5%. DOMM has an empirical formula of C44H73NO17 and a molecular weight of about 888 (887, as determined by mass spectrometryl. The infrared absorption spectrum of DOMM (free base) in chloroform is shown in the accompanying drawing. Observable absorption maxima occur at the following frequencies (cm 1): 3679 (small), 3604 (shoulder), 3486 (broad), 3012 (shoulder), 2979 (intense), 2938 (intense), 2880 (shoulder), 2821 (shoulder), 2794 (v. small), 2735 (v. small), 1723 (intense), 1678 (medium), 1627 (small), 1593 (intense), 1477 (shoulder), 1459 (medium), 1409 (medium), 1373 (medium), 1333 (shoulder), 1316 (medium), 1273 (small), 1183 (shoulder), 1161 (intense), 1114 (medium), 1080 (intense), 1048 (intense), 1013 (medium), 996 (medium), 984 (shoulder), 960 (shoulder), 924 (v. small), 902 (small), 865 (v. small), and 841 (small). The ultraviolet absorption spectrum of DOMM in neutral ethanol exhibits an absorption maximum at 283 nm (E 22,550). DOMM (free base) has the following specific rotation : #-39.69% (c 1, CH3OH). Electrometric titratic of DOMM in 662 aqueous dimetnylformamide indicates the presence of a titratable group with a pK value of about 7.14. Example 3 Preparation of DOML DOMM, prepared as described in Example 2, was dissolved in a dilute hydrochloric acid solution (HC1 added to water until the pH of the solution was 1.8) The resulting solution was allowed to stand for about eight hours at room temperature and was then adjusted to pH 9.0 by the addition of sodium hydroxide. This basic solution was extracted with ethyl acetate, dichloromethane or chloroform. The extract was dried under vacuum to give DOML base (softens at 115-1200C and melts at about 134-1400C). Example 4 Preparation of Dihydro-DOMM DOMM (50 mg), prepared as described in Example 2, was dissolved in an aqueous isopropyl alcohol solution (approximately 40%; 25 ml). Sodium borohydride (20 mg) was dissolved in a 30% aqueous isopropyl alcohol solution (10 ml). The NaBH4 solution (1 ml) was added to the solution containing the DOMM. The resulting mixture was stirred for 5 minutes, adjusted to pH 7.5 with phosphoric acid, and concentrated under vacuum to remove the isopropyl alcohol. Water was added to the resulting aqueous concentrate to give a volume of 25 ml; chloroform (50 ml) was then added. The pH of the aqueous phase was adjusted to 7.5. After extraction, the chloroform was separated and evaporated to dryness under vacuum to give dihydro-DOMM. Example 5 Preparation of Dihydro-DOML Dihydro-DOMM, prepared as described in Example 4, was treated in the manner described in Example 3 to give dihydro-DOk. Example 6 Alternative Preparation of DOML DOML was prepared from DOMM by treating the DOMM in the fermentation broth in which it was produced with mild acid as described in Example 3. Isolation of DOML was accomplished by a procedure similar to that described for DOMM in Example 2. Example 7 Alternative Preparation of Dihydro-DOML DOML, obtained as described in Example 3 was reduced uslng the procedure described in Example 4 to give dihydro-DOML. Example 8 2'-O-Propionyl-DOMM DOMM was dissolved in acetone and treated with 1.2 equivalents of propionic anhydride at room temperature for about six hours to give 2'-O-propionyl DOMM. Examples 9-11 2'-O-Isovaleryl-DOMM, was prepared according to the procedure of Example 8, but using isovaleric anhydride. 2'-O-Benzoyl-DOMM, was prepared according to the procedure of Example 8 but using benzoic anhydride. 2'-O-(n-Butyryl)DOMM, was prepared according to the procedure of Example 8, but using n-butyric anhydride. Example 12 2', 4'-Di-O-Propionyl-DOML DOML was dissolved in acetone and treated with slightly more than two equivalents of propionic anhydride at room temperature for about six hours to give 2' ,4'-di-O-propionyl-DOML. Examples 13-15 2' ,4'-Di-O-isovaleryl-DOML, was prepared according to the method of Example 12, but using isovaleric anhydride. 2',4'-Di-O-benzoyl-DOML, was prepared according to the method of Example 12, but using benzoic anhydride. 2' ,4'-Di-O-(n-butyryl)DOML, was prepared according to the method of Example 12, but using n-butyric anhydride.

Claims: Claims 1. A novel macrolide of formula (I): EMI26.1 where Q is -CHO or -CH2OH and Y is hydroxyl or a mycarosyloxy group; or a salt or ester thereof. 2. A novel macrolide as claimed in claim 1, having the structural formula (II): EMI26.2 3. A novel macrolide as claimed in claim 1, having the structural formula (III): EMI27.1 4. A novel macrolide as claimed in claim 1, having the structural formula (IV): EMI27.2 5. A macrolide as claimed in claim 1, having the structural formula (V): EMI28.1 6. A method of preparing a macrolide of formula (I) as claimed in any one of claims I to 5, which comprises culturing a strain of Streptomyces fradiae under submerged aerobic conditions until a substantial level of antibiotic activity is produced, provided that if it is desired to produce a compound as claimed in claim 4 or 5, then the cultivation should be effected using mildly acidic conditions. 7. A veterinary formulation comprising from 0.1 to 90% by weight of a compound of formula (I) as claimed in any one of claims 1 to 5, or a pharmaceutically acceptable salt or ester thereof, associated with one or more pharmaceutically-acceptable carriers or excipients therefor. 8. A compound of formula (I) as claimed in any one of claims 1 to 5, or a pharmaceuticallyacceptable salt or ester thereof, for use in treating microbial infections in warm-blooded animals. 9. A method of preparing a compound cf formula (I) as claimed in claim 4 or 5, which comprises the mild acid hydrolysis of a compound as claimed in claim 2 or 3 respectively. 10. A method of preparing 5-O-mycaminosyltylonolide or 2 0-dihydro-5-O-mycaminosyltylonolide by the controlled acid hydrolysis of a compound of formula (IV) or (V) as claimed in claim 4 or 5. 11. Streptomyces fradiae ATCC 31669 12. A culture medium comprising Streptomyces fradiae -ATCC 31669 and assimilable sources of carbon, nitrogen and inorganic salts. Claims 1. A process for preparing a novel macrolide of formula (I): EMI30.1 where Q is -CHO or -CH2OH and Y is hydroxyl or a mycarosyloxy group; or a salt or ester thereof; by culturing a strain of Streptomyces fradiae under submerged aerobic conditions until a substantial level of antibiotic activity is produced, provided that if it is desired to produce a compound of formula (I) in which Y is hydroxyl, then the cultivation should be effected using mildly acidic conditions. 2. A process according to claim 1 in which the microorganism utilised is Streptomyces fradiae ATCC 31669. 3. Streptomyces fradiae ATCC 31669 4. A culture medium comprising Streptomyces fradiae ATCC 31669 and assimilable sources of carbon, nitrogen and inorganic salts 5. A novel macrolide produced by a process according to either of Claims 1 and 2.

Inventors: BALTZ, RICHARD HENRY, SENO, EUGENE THOMAS, WILD, GENE MURIEL
Applicants: ELI LILLY AND COMPANY
Application year: 1981
Main classification: A61K31
IPC codes: C12P19, A61K31, A61P31, C12N1, C07H17, C12R1, C12P17