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5-O-Mycarosyltylactone has been isolated as a predominant factor from fermentation broths of a Streptomyces fradiae mutant. The relative configurations of mycarose and tylactone (protylonolide) have been determined by X-ray crystal structure analysis. Hydrolysis of 5-O-mycarosyltylactone yielded (-)-tylactone and L-(-)-mycarose. Taken together, these two experiments establish the absolute configuration of (-)-tylactone. Bioconversion of (-)-tylactone to tylosin by tyl G mutants of S. fradiae proves the absolute configuration of tylosin. Physicochemical data for tylactone and a unique component piece of tylactone are also reported.

Although a substantial number of 16-membered macrolides related to tylosin have now been isolated and evaluated as antibiotics, none appeared to be superior to tylosin in treating bacterial or mycoplasmal infections caused by sensitive organisms. Nevertheless, this comparison of the antibiotic activity of 16-membered macrolides clearly indicates that novel antibiotics with potentially useful activity can be obtained from mutant strains which have been blocked at various steps in their biosynthesis of antimicrobial agents. The novel compounds thus produced may also be used as starting materials for additional chemical and microbiological modification. Furthermore, the mutant strains which produced these novel compounds should be useful recipients for interspecific genetic recombination by protoplast fusion or gene cloning to yield hybrid antibiotics. Even greater exploitation of these methods will be required in the continuing search for new antibiotics and improved methods for producing them.

A large series of C-23-modified derivatives of 5-O-mycaminosyltylonolide were synthesized, in which the C-23 hydroxyl group was replaced by halo, aryl ether or thioether, azido, amino or dialkylamino substituents via SN2 displacement reactions. The majority of derivatives possessed excellent in vitro activity against a variety of aerobic and anaerobic bacteria. While some of the compounds treated experimental infections in rodents by parenteral administration, none showed any significant efficacy or bioavailability after oral dosing. Novel rearrangement products were obtained from some of the reactions; these were identified as 13,23-cyclopropyl-12,22-exomethylene and 13,23-cyclopropyl-12-alkoxy derivatives.

Three series of semi-synthetic derivatives of tylosin-related macrolides were evaluated for utility in veterinary medicine. 23-Modified derivatives of 5-O-mycaminosyltylonolide (OMT) possessed potent activity in vitro against species of Pasteurella and Mycoplasma. An experimental infection in chicks caused by Pasteurella multocida was utilized to evaluate efficacy; several of these derivatives of OMT effectively treated the infection when given subcutaneously, but none were effective after oral administration in drinking water. Macrolides retaining the 4'-O-mycarosyl moiety (tylosin, DMT) had relatively poor activity against Pasteurella in vitro. Certain 20-modified derivatives of desmycosin demonstrated good oral bioavailability in chicks and a lead compound with oral efficacy in the Pasteurella infection model was discovered.

A large number and wide variety of acyl derivatives of the tylosin-related macrolides 23-demycinosyltylosin (DMT), 23-demycinosyloxytylosin (DMOT) and 5-O-mycaminosyltylonolide (OMT) were synthesized and evaluated. This encompassed conversion of the hydroxyl groups at 2',4' and 23 of the appropriate macrolides to the corresponding esters, in which a variety of different substitution patterns were examined. A wide range of acyl substituents was investigated, particularly for 23-O-acyl derivatives of OMT, since these were substantially more active in vitro than OMT itself. However, the acyl derivatives which were prepared demonstrated no substantial improvement in oral efficacy or bioavailability over the parent macrolides.

A series of 20-deoxo-20-cyclic (alkylamino) derivatives of tylosin, desmycosin, macrocin and lactenocin was prepared by reductive amination of the C-20 aldehyde group. The majority of the compounds were prepared using metal hydrides (sodium cyanoborohydride or sodium borohydride) as the reducing agents and a suitable cyclic alkylamine. Subsequently, a more convenient procedure was developed using formic acid as a reducing agent. The C-20 amino derivatives prepared from desmycosin exhibited good in vitro antimicrobial activity against Pasteurella haemolytica and Pasteurella multocida (MIC range of 0.78 approximately 6.25 micrograms/ml) as well as Mycoplasma species (MIC range of 0.39 approximately 6.25 micrograms/ml). Several derivatives showed excellent oral efficacy against infections caused by P. multocida in chicks. One of these derivatives, 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmicosin or EL-870) was selected for development as a therapeutic agent for pasteurellosis in calves and pigs.

Modification of the aldehyde group in tylosin and related macrolide antibiotics dramatically enhanced the oral efficacy of the derivatives against experimental infections caused by susceptible bacteria in laboratory animals. A large number and wide variety of aldehyde-modified macrolide derivatives were prepared, utilizing the Mitsunobu reaction and other chemical transformations. Evaluation of in vitro and in vivo antimicrobial activity indicated that derivatives of demycarosyltylosin (desmycosin) combined the broadest spectrum of antimicrobial activity with the best efficacy and bioavailability after oral administration.

A large group of ester derivatives of tylosin-related macrolides was prepared in which the hydroxyl groups at C-3 and C-4'' were acylated by either chemical or biochemical methods. Most of the derivatives exhibited excellent in vitro antimicrobial activity. However, only the 3,4''-diacyl derivatives of tylosin and macrocin showed any significant improvements of in vivo efficacy against experimental infections in rodents.