Preparation and structure-activity relationships of simplified analogues of the antifungal agent cilofungin: a total synthesis approach.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.

The discovery of enfumafungin, a novel antifungal compound produced by an endophytic Hormonema species biological activity and taxonomy of the producing organisms.

In a screening of natural products with antifungal activity derived from endophytic fungi, we detected a potent activity in a culture belonging to the form-genus Hormonema, isolated from leaves of Juniperus communis. The compound is a new triterpene glycoside, showing an antifungal activity highly potent in vitro against Candida and Aspergillus and with moderate efficacy in an in vivo mouse model of disseminated candidiasis. The agent is especially interesting since its antifungal spectrum and its effect on morphology of Aspergillus fumigatus is comparable to that of the glucan synthase inhibitor pneumocandin B,,, the natural precursor of the clinical candidate MK-0991 (caspofungin acetate). An additional search for other Hormonema isolates producing improved titers or derivatives resulted in the isolation of two more strains recovered from the same plant host showing identical activity. The producing isolates were compared with other non-producing Hormonema strains by DNA fingerprinting and sequencing of the rDNA internal transcribed spacers. Comparison of rDNA sequences with other fungal species suggests that the producing fungus could be an undetermined Kabatina species. Kabatina is a coelomycetous genus whose members are known to produce Hormonema-like states in culture.

L-671,329, a new antifungal agent. III. In vitro activity, toxicity and efficacy in comparison to aculeacin.

L-671,329 is a novel, echinocandin-like natural product that possesses potent anti-Candida activity, including activity against Candida parapsilosis. The in vitro MICs of L-671,329 were comparable to aculeacin against 18 yeasts and three filamentous fungi in an agar dilution assay. L-671,329 lysed mouse red blood cells (RBCs) at a concentration of 400 micrograms/ml, but not at 50 or 12.5 micrograms/ml. Aculeacin lysed RBCs at 400 and 50 micrograms/ml. L-671,329 significantly prolonged survival of mice infected with Candida albicans (ED50 3.38 mg/kg) following twice-daily intraperitoneal dosing for five consecutive days. The prolongation observed was greater than that seen with aculeacin therapy (ED50 6.44 mg/kg). No acute or chronic toxicities of L-671,329 or aculeacin (as measured by mortality) were detected at a concentration of 100 mg/kg following intraperitoneal administration (TD50 greater than 100 mg/kg). Both L-671,329 and aculeacin eradicated cells of C. albicans from the kidneys of infected mice. L-671,329 eradicated the yeast at therapeutic concentrations of 12.5 to 100 mg/kg. Aculeacin eradicated yeast cells at therapy concentrations of 25 to 100 mg/kg. L-671,329 has potential as an anti-Candida compound.

Pneumocandins from Zalerion arboricola. IV. Biological evaluation of natural and semisynthetic pneumocandins for activity against Pneumocystis carinii and Candida species.

A series of lipopeptide compounds co-produced during the fermentation of pneumocandin A0 (L-671,329) and related semisynthetic compounds were evaluated in vivo against Pneumocystis carinii pneumonia and systemic candidiasis. In addition, they were tested in vitro against a panel of pathogenic Candida species and in a Candida membrane 1,3-beta-D-glucan synthesis assay. The results of these studies demonstrate that pneumocandin A0 and pneumocandin B0 (L-688,786) are the most potent compounds when considering both antipneumocystis and anticandida activity. Other compounds in the series are selectively more potent against P. carinii or Candida albicans suggesting a diverging structure-activity relationship. Evaluation of these compounds for their ability to inhibit C. albicans 1,3-beta-D-glucan synthesis in vitro demonstrates that they inhibit this process. A positive correlation between 1,3-beta-D-glucan synthesis inhibition and in vitro antifungal activity was also demonstrated for some of the pneumocandins.

L-658,310, a new injectable cephalosporin. III. Experimental chemotherapeutics and pharmacokinetics in laboratory animals.

The therapeutic activity of L-658,310 was demonstrated in experimental bacteremias in normal, diabetic and neutropenic mice. Especially potent activity was shown against the usually difficult to control pathogens, Enterobacter cloacae and Pseudomonas aeruginosa, that were resistant to ceftazidime and/or gentamicin. Pharmacokinetic studies in mice showed a linear dose response in serum after the 20 and 50 mg/kg subcutaneous dose and urinary recoveries of administered dose of about 60% in 6 hours. Excretion was mainly by glomerular filtration. In a crossover design in rhesus monkeys, the pharmacokinetics of L-658,310 were similar to those of ceftazidime and suggest a moderately long half-life in serum of humans.

In vivo evaluation of three acid-stable azalide compounds, L-701,677, L-708,299 and L-708,365 compared to erythromycin, azithromycin and clarithromycin.

L-701,677, L-708,299 and L-708,365 are novel azalide derivatives of erythromycin that exhibit improved acid stability over erythromycin, azithromycin and clarithromycin. The half-life in aqueous solution at pH = 2.1 of these compounds ranged from 0.3 hour for erythromycin to 16.2 hours for L-708,299. The rank order of half-life in acid solution from most to least stable was L-708,299 > L-701,677 > L-708,365 > azithromycin = clarithromycin > erythromycin. In a disseminated Streptococcus pyogenes mouse infection model, azithromycin and L-708,365 were slightly more efficacious than clarithromycin, L-701,677 and L-708,299; all 5 compounds being more active than erythromycin. In a Klebsiella pneumoniae pulmonary challenge mouse model, azithromycin, L-701,677, L-708,299 and L-708,365 were all equal in efficacy and at least four-fold more active than clarithromycin and erythromycin. Clarithromycin, L-708,365 and interestingly erythromycin, showed greater bacterial clearance than azithromycin, L-701,677 and L-708,299 in a localized infection model that measured clearance of Staphylococcus aureus from mouse thigh tissues. Our results indicate that L-701,677, L-708,299 and L-708,365 exhibit improved acid stability and were at least equally efficacious as presently marketed macrolide/azalide antibiotics.

L-658,310, a new injectable cephalosporin. I. In vitro antibacterial properties.

The in vitro antibacterial spectrum of L-658,310, a new semisynthetic cephalosporin, was compared with ceftazidime, aztreonam and piperacillin against a wide variety of randomly selected human clinical isolates. The compound was found to be a broad spectrum bactericidal agent that was more potent than any of the comparison drugs against glucose nonfermenting bacteria. It has especially potent activity against Pseudomonas aeruginosa including multiply-resistant strains. The superior activity of L-658,310 against this group of organisms is attributed to the presence of the dihydroxy substituents on the 2-methylisoindoline moiety of the compound. L-658,310 is not cross-resistant with either imipenem, ceftazidime or piperacillin (representatives of three different classes of beta-lactam compounds) against P. aeruginosa. The lack of cross-resistance with ceftazidime extends to other glucose nonfermenters and several strains of Enterobacteriaceae as well. The compound is active against bacteria known to possess either R-plasmid- or chromosomally-mediated beta-lactamases.

L-656,575 (OCP-9-176): a novel oxacephem. In vitro activity against aerobic and anaerobic clinical bacterial isolates.

L-656,575 (OCP-9-176) is a novel oxacephem superior to ceftazidime in in vitro activity against clinical isolates of Enterobacter species, methicillin-susceptible Staphylococcus aureus and Staphylococcus epidermidis, and multiply-resistant Pseudomonas aeruginosa. Our results suggest that L-656,575 has a high affinity for penicillin binding proteins of Pseudomonas and may bind preferentially to PBP-3 in this organism. L-656,575 is active against beta-lactamase derepressed Enterobacteriaceae and ceftazidime-resistant P. aeruginosa.

Restricticin, a novel glycine-containing antifungal agent.

Restricticin (1) is a naturally-occurring antifungal agent which contains triene, pyran and glycine ester functionalities and is unrelated to any previously known family of natural products. This unstable compound, as well as its corresponding N,N-dimethyl derivative (2), have been produced and isolated from both solid and liquid fermentations of Penicillium restrictum. The desglycyl hydrolysis product, restrictinol (3), was produced via the hydrolysis of pure restricticin and as an artifact of the isolation of restricticin.

Chemiluminescence as a tool for the evaluation of antimicrobial agents: a review.

Chemiluminescence is a measure of the respiratory burst of phagocytic immune cells following stimulation by antigen. Measurement of chemiluminescence by scintillation counters set in the out of coincidence mode or by instruments designed specifically to measure chemiluminescence is an accurate, reproducible means of evaluating the immune potential of chemiluminescence emiting cell populations. One area under recent investigation has been the study of the effect of antimicrobial agents on the ability of various immune cell populations to emit chemiluminescence. This technique permits the investigator to test the effect of a drug on the activity of immune cell populations. The purpose of this review is to describe chemiluminescence, the basic kinetics of the response following stimulation with various antigens, and the reported effects of antimicrobial agents on chemiluminescence which have appeared in the literature.

Antibacterial efficacy of norfloxacin, trimethoprim-sulfamethoxazole, and gentamicin in experimentally-infected normal and streptozotocin-induced diabetic mice.

Systemic bacterial infections due to Escherichia coli MB 2884, Proteus mirabilis MB 3125 and Klebsiella pneumoniae MB 4005 were well controlled by treatment with norfloxacin both in normal and streptozotocin-induced diabetic mice. similar observations were made when trimethoprim-sulfamethoxazole was used against susceptible pathogens. Systemic infection due to Pseudomonas aeruginosa MB 4700 was well controlled by norfloxacin and gentamicin in normal mice; this infection was more refractory to treatment by both drugs in diabetic animals. These observations suggest that norfloxacin may be an effective drug in the treatment of bacterial infections which may occur under diabetic conditions, and further investigation is warranted.

In vitro effect of pH and glucose concentration on the antibacterial activity of norfloxacin in urine.

The in vitro effect of pH and glucose concentration on the antibacterial activity of norfloxacin in urine was studied. Norfloxacin effectively inhibited the growth of four gram-negative pathogens in urine in vitro at pH values of 6.0, 7.0, and 8.0. The antibacterial activity of norfloxacin in urine was reduced severalfold at pH 6, but minimum inhibitory concentrations (MICs) at this pH remained clinically significant. Glucose at concentrations of 200 mg/dl and 400 mg/dl (simulating glucosuria of diabetes) did not significantly affect the antibacterial activity of norfloxacin when tested against clinical isolates of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, or Pseudomonas aeruginosa. Norfloxacin appears to be a highly effective antibiotic in vitro under conditions which simulate normal and diabetic states.

Lack of cross-resistance between efrotomycin and antibacterial agents used in the therapy of human and animal infections.

Efrotomycin is an N-methylhydroxypyridone glycoside antibiotic with activity primarily against Gram-positive bacteria. It is intended for use as a feed additive for swine. Although efrotomycin is unrelated to any antibacterial drug used in human or veterinary medicine, the possibility of cross-resistance with other antibacterials is of concern. The minimum inhibitory concentrations (MICs) of efrotomycin were determined for a broad panel of bacterial isolates. In addition, the susceptibility of each isolate to 12-15 antibacterials was determined using a standardized disk susceptibility test. No evidence of cross-resistance between efrotomycin and any of the 12-15 antibacterial compounds was observed. When the MIC of efrotomycin for nine selected isolates was increased from 16- to greater than 100-fold by serial passage in subinhibitory concentrations of efrotomycin, no increased resistance to the 15 antibacterials was noted. Subinhibitory concentrations of efrotomycin had no effect on the conjugative transfer of antibacterial-resistance plasmids between K-12 strains of Escherichia coli. The data from this study suggest that if resistance to efrotomycin should occur, it is unlikely to result in the appearance of multiply-resistant bacterial populations.

Occurrence and characterization of plasmids in field isolates of Bordetella bronchiseptica.

Twenty-seven isolates of Bordetella bronchiseptica were examined for the presence of plasmid DNA. Eleven of the isolates contained plasmids. An attempt was made to correlate the presence of plasmids with resistance to a number of antibiotics or heavy metals or with production of bacteriocins. Eight of the isolates contained a plasmid of approximately 35 megadaltons molecular weight; these isolates were all resistant to 1 mM mercuric chloride. There were no other correlations between plasmid content and any of the phenotypes tested. The plasmids conferring resistance to mercuric chloride were transferred by conjugation to Escherichia coli K-12. The plasmid-bearing isolates were then compared with their isogenic plasmidless parent for resistance to a number of antibiotics. Isolates acquiring the plasmids demonstrated increased resistance to streptomycin and ampicillin, as well as to mercuric chloride. The 8 plasmids were also analyzed by restriction endonuclease digestion.

Efficacy of caspofungin against Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans.

The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-beta-D-glucan synthase from Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans. In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.