Comparison of aminoglycoside antibiotics with respect to uptake and lethal activity in Escherichia coli.

Forty-five aminoglycoside antibiotics and related compounds were compared for their ability to induce the accumulation of dihydrostreptomycin in Escherichia coli K12. The common aminoglycosides and a streptothricin antibiotic all induced enhanced uptake within a relatively narrow concentration range. These concentrations were lethal to the bacteria. Comparison of aminoacyl derivatives of tobramycin and apramycin, the latter synthesized utilizing transition-metal cations to selectively control the site of substitution, revealed that 1-N-aminoacyl modifications resulted in an increased ability to induce enhanced uptake. 2'-N-Aminoacyl modifications were also effective at inducing enhanced uptake, albeit without noticeable improvement over parent. The findings from this structure-activity comparison support the proposition that aminoglycosides share a common critical target (most likely the ribosome), which, when acted upon, results in both drug accumulation and killing.

Synthesis and in vitro evaluation of new wortmannin esters: potent inhibitors of phosphatidylinositol 3-kinase.

New C-11 esters of the fermentation product wortmannin have been synthesized, with some of them further derivatized at C-17. The new esters show greater inhibition of isolated phosphatidylinositol 3-kinase and increased cell cytotoxicity in a rapidly proliferating leukemia cell line, when compared to wortmannin. Reduction of the C-17 ketone caused a slight increase in activity, while acylation of this new alcohol caused severe loss of activity. With their increased activity, the new C-11 esters may be good candidates to explore the in vivo antitumor effects of phosphatidylinositol 3-kinase inhibitors.

Synthesis and structure-activity relationships of new 9-N-alkyl derivatives of 9(S)-erythromycylamine.

A series of new 9-N-alkyl derivatives of 9(S)-erythromycylamine has been synthesized by reductive alkylation of erythromycylamine with aliphatic aldehydes and sodium cyanoborohydride. Alternative syntheses employing hydrogenation methods have also been developed. These new 9-N-alkyl derivatives possess excellent antimicrobial activity in vitro and in vivo, especially when administered orally to treat experimental infections in mice. From structure-activity studies, 9-N-(1-propyl)erythromycylamine (LY281389) was selected as the most efficacious derivative. These methods have also been extended to the synthesis of some 9-N,N-dialkyl derivatives of erythromycylamine.

Synthesis and evaluation of tylosin-related macrolides modified at the aldehyde function: a new series of orally effective antibiotics.

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.

The antibacterial and NMDA receptor activating properties of aminoglycosides are dissociable.

The use of aminoglycoside antibiotics is limited by side effects, the most critical of which are vestibular and cochlear toxicity. Recent evidence indicates that these effects result from an excitotoxic process mediated, at least in part, through a polyamine-like activation of NMDA receptors. This study investigated whether these positive modulatory effects of aminoglycosides at NMDA receptors are dissociable from their antibacterial properties. A group of structurally related apramycin derivatives was evaluated for the ability to enhance [3H]dizocilpine binding to rat brain membranes, and for the ability to augment agonist responses on recombinant (NR1A/2B) NMDA receptors expressed in Xenopus oocytes. Based on the antibacterial potencies of these derivatives against Staphylococcus aureus and Escherichia coli, it is concluded that there is no correlation between the ability of an aminoglycoside to produce a positive modulation of NMDA receptors and minimum inhibitory antibacterial concentrations. These findings indicate that it may be possible to develop an aminoglycoside antibiotic with reduced potential for ototoxicity.

Semi-synthetic derivatives of erythromycin.

Semi-synthetic derivatives of erythromycin have played an important role in antimicrobial chemotherapy. First generation derivatives such as 2'-esters and acid-addition salts significantly improved the chemical stability and oral bioavailability of erythromycin. A second generation of erythronolide-modified derivatives: roxithromycin, clarithromycin, azithromycin, dirithromycin and flurithromycin, have been synthesized and have exhibited significant improvements in pharmacokinetic and/or microbiological features. In addition, erythromycin itself has expanded its utility as an effective antibiotic against a variety of newly emerged pathogens. As a result of these developments, macrolide antibiotics have enjoyed a resurgence in clinical interest and use during the past half-dozen years, and semi-synthetic derivatives of erythromycin should continue to be important contributors to this macrolide renaissance. Despite these recent successes, other useful niches for macrolide antibiotics will remain unfilled. Consequently, the search for new semi-synthetic derivatives of erythromycin possessing even better antimicrobial properties should be pursued.

Semi-synthetic derivatives of 16-membered macrolide antibiotics.

The fermentation-derived 16-membered and 14-membered macrolides have been equally productive sources of semi-synthetic derivatives which have significantly extended the utility of the macrolide class as important antibiotics. New derivatives, prepared by both chemical and biochemical methods, have exhibited a variety of improved features, such as an expanded antimicrobial spectrum, increased potency, greater efficacy, better oral bioavailability, extended chemical and metabolic stability, higher and more prolonged concentrations in tissues and fluids, lower and less frequent dosing, and/or diminished side-effects [302]. However, even more improvements are both achievable and necessary if problems such as resistance to existing antibiotics continue to rise [303, 304]. Newer semi-synthetic macrolides which satisfy these important needs should be anticipated as the contributions from new fields such as genetic engineering of macrolide-producing organisms and more powerful computational chemistry are combined with the more traditional disciplines of chemical synthesis, bioconversions, and screening fermentation broths.

Conversion of spinosyn A and spinosyn D to their respective 9- and 17-pseudoaglycones and their aglycones.

Forosamine at the 17-position of spinosyns A and D was hydrolyzed under mild acidic conditions to give the corresponding 17-pseudoaglycones. The tri-O-methylrhamnose at the 9-position of the 17-pseudoaglycone of spinosyn A was hydrolyzed under more vigorous acidic conditions to give the aglycone of spinosyn A. However, these conditions led to decomposition of the 17-pseudoaglycone of spinosyn D, presumably due to more facile protonation of the 5,6-double bond to produce a tertiary carbonium ion which undergoes further rearrangements. Spinosyns J and L (3'-O-demethyl spinosyn A and D, respectively) obtained from fermentation of biosynthetically-blocked mutant strains of Saccharopolyspora spinosa, were oxidized to give the corresponding 3'-keto-derivatives and the resultant keto-sugars were then beta-eliminated under basic conditions to give the 9-pseudoaglycones of spinosyns A and D respectively. Forosamine at the 17-position of the 9-pseudoaglycone of spinosyn D was then readily hydrolyzed to yield the aglycone of spinosyn D.

Synthesis and insecticidal activity of spinosyn analogs functionally altered at the 2'-,3'- and 4'-positions of the rhamnose moiety.

In an effort to increase the insecticidal activity of the spinosyn family of naturally occurring macrolides, the 2'-, 3'- and 4'-O-desmethyl-O-acetyl analogs and the 2'-, 3'-, and 4'-O-desmethoxy analogs have been synthesized. These analogs were prepared synthetically from the minor spinosyn factors H, J, K, L and Q either via direct acylation of the corresponding factor or deoxygenation of an intermediate xanthate. The acylated analogs were all more potent insecticides against Heliothis virescens larvae than their respective parent factors, but not as potent as spinosyns A or D. The deoxy analogs were also more potent insecticides than their respective parent factors. The 2'-desmethoxy analogs showed, for the first time, analogs with insecticidal potency against H. virescens greater than that of spinosyns A and D, indicating that polarity is not well tolerated in the rhamnose moiety of spinosyn A.

Synthesis, antimicrobial activity and in vivo fluorine NMR of a hexafluorinated derivative of tilmicosin.

A new fluorinated analog of tilmicosin was synthesized by the reductive amination of desmycosin with 3,5-bis(trifluoromethyl)piperidine. Despite an apparently small change in structure, the fluorinated analog had much less in vitro antimicrobial activity than tilmicosin and it failed to protect 3-day old chicks against a Pasteurella multocida challenge at 64 mg/kg sc. In a preliminary in vivo fluorine NMR experiment in a female Sprague-Dawley rat, a 19F NMR signal was detected in the liver one hour after ip administration of the fluorinated compound. Therefore, although this fluorinated derivative had less antimicrobial activity than tilmicosin, it may nevertheless provide a suitable model of tilmicosin for pharmacokinetic studies using in vivo fluorine NMR.

In vitro and in vivo evaluation of C-20- and C-23-modified derivatives of tylosin against veterinary pathogens.

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.

The structure of A201A, a novel nucleoside antibiotic.

The structure of the novel nucleoside antibiotic A201A has been determined by a combination of chemical and spectroscopic methods. It is composed of 6-dimethylaminopurine, 3-amino-3-deoxyribose, p-hydroxy-alpha-methylcinnamic acid, a novel unsaturated hexofuranose and 3,4-di-O-methylrhamnose. Structures have also been assigned to several related minor factors simultaneously isolated from the fermentation broth. These unique nucleosides have very interesting similarities and differences in structure with the known antibiotics puromycin and hygromycin A.

Structure of the new spiroketal-macrolide A82548A.

A new member of the spiroketal-containing macrolide class of fermentation-derived natural products was isolated from mycelial extracts of Streptomyces diastatochromogenes. The principal component, A82548A, was shown to possess a 22-membered macrolide ring system onto which was incorporated both a spiroketal and a hemiketal moiety. Relative stereochemistry was established by single crystal X-ray diffraction studies. Absolute stereochemistry was determined via hydrolysis of the amino sugar glycosidically linked to the aglycone, which was identified as L-kedarosamine. The overall three-dimensional structure is closely related to that of the macrolides cytovaricin, rutamycin, and ossamycin.

Structure of the spiroketal-macrolide ossamycin.

Ossamycin is a cytotoxic agent of undetermined structure that was originally isolated in 1965 from culture broths of Streptomyces hygroscopicus var. ossamyceticus. Its overall structure and relative stereochemistry have now been determined by single crystal X-ray diffraction studies. Absolute stereochemistry was established according to the previously determined configuration of its aminosaccharide constituent, ossamine. The aglycone of ossamycin possesses a 24-membered macrolide ring system onto which is incorporated both a 6,6-spiroketal and 5-membered hemiketal ring system. The overall three-dimensional structure possesses features in common with the related macrocyclic antibiotics dunaimycin, cytovaricin, and A82548A.

Synthesis and characterization of a novel inhibitor of an aminoglycoside-inactivating enzyme.

A novel low-molecular weight inhibitor of an aminoglycoside-inactivating enzyme, initially isolated from fermentation broths of Streptomyces neyagawaensis, was determined to be 7-hydroxytropolone. Its structure was confirmed by synthesis. In vitro synergy was demonstrated between 7-hydroxytropolone and certain aminoglycosides against bacteria which were resistant to those aminoglycosides by virtue of a 2"-O-adenylyltransferase. The synthesis and characterization of some analogs of 7-hydroxytropolone is also described.

Novel fermentation products from Streptomyces fradiae: X-ray crystal structure of 5-O-mycarosyltylactone and proof of the absolute configuration of tylosin.

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.

Structure-activity studies among 16-membered macrolide antibiotics related to tylosin.

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.

Structure elucidation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098, are homologous compounds of molecular formulas C12H13NO6 and C13H15NO6. The molecular similarities of the two inhibitors were established by comparison of their 1H NMR, 13C NMR, and UV spectra. Catalytic hydrogenation of A58365A led to a tetrahydro-deoxy derivative, C12H17NO5; extensive 1H NMR decoupling studies at 360 MHz allowed all the non-exchangeable protons of the derivative to be connected in a continuous substructure. This fragment was combined with information from other spectroscopic methods to suggest the structures for A58365A (1) and A58365B (2); the conclusions were confirmed by an X-ray crystallographic analysis of A58365A-dimethyl ester.

Synthesis and antimicrobial evaluation of 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmicosin, EL-870) and related cyclic amino derivatives.

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.

Synthesis, antimicrobial evaluation and structure-activity relationships within 23-modified derivatives of 5-O-mycaminosyltylonolide.

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.