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The antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene (TMPN) were examined. TMPN was fungicidal for the majority of 132 reference strains and clinical isolates tested, including those resistant to fluconazole, ketoconazole, amphotericin B or flucytosine. Minimum fungicidal concentration/minimum inhibitory concentration (MFC/MIC) ratios were < or = 2 for 96% of Cryptococcus neoformans clinical isolates and 71% of Candida albicans clinical isolates. TMPN was fungicidal for a variety of other basidiomycetes, endomycetes and hyphomycetes, and its activity was unaffected by alterations in media pH. The frequency of occurrence of fungal spontaneous mutations to resistance was <10(-6). Kill-curve analyses confirmed the fungicidal action of TMPN, and demonstrated that killing was concentration- and time-dependent. At sub-MIC exposure to TMPN, C. albicans did not exhibit yeast/hyphae switching. TMPN was slightly cytotoxic for murine and human cancer cell lines (GI50=1-4 microg ml(-1)), and weakly inhibited mammalian tubulin polymerization (IC50=0.60 microg ml(-1)).

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The pentapeptide dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester (auristatin PHE) is a derivative of the anticancer drug dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine). Broth microdilution assays with a wide variety of yeast and filamentous fungal species demonstrated the specificity of auristatin PHE for Cryptococcus neoformans and several species of Trichosporon. The duration of the postantifungal effect (PAFE) for C. neoformans was determined for exposure times ranging from 30 min to 2 h. For the derivative, a PAFE was detectable after 45 min of exposure. The effect plateaued after 1 h of exposure, with a PAFE of approximately 6.5 h at four or eight times the auristatin PHE MIC. In contrast, there was no measurable PAFE after 1 h of exposure to dolastatin 10. Human serum greatly prolonged the PAFE of auristatin PHE at eight times the MIC. Auristatin PHE arrested C. neoformans in the budding stage, possibly due to a tubulin-inhibitory action. Auristatin PHE has potential as a narrow-spectrum fungicidal agent and as a probe that can be used to study cryptococcal cell division.

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Human immune serum recognition of outer membrane components from commensal and pathogenic Neisseria cultured under neutral and acidic conditions was investigated. Acid stress caused no detectable alterations in lipooligosaccharide migration and (or) staining, in outer membrane protein profiles, or in immune serum recognition of outer membrane components from Neisseria mucosa or Neisseria sicca. There was also no difference in the lipoologosaccharide electrophoretic pattern of acid- and neutral-grown Neisseria lactamica, but there were differences in outer membrane protein expression. The outer membrane protein alterations induced by acid stress in N. lactamica were not the same as those seen in isolates from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection. Many differences were detected in the immune serum recognition of outer membrane components from acid- and neutral-cultured N. lactamica and from the clinical isolates of Neisseria gonorrhoeae, and these should be considered in vaccine design.

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The original synthesis of combretastatin A-2 (1a) was modified to provide an efficient scale-up procedure for obtaining this antineoplastic stilbene. Subsequent conversion to a useful prodrug was accomplished by phosphorylation employing in situ formation of dibenzylchlorophosphite followed by cleavage of the benzyl ester protective groups with bromotrimethylsilane to afford the phosphoric acid intermediate 11. The latter was immediately treated with sodium methoxide to complete a practical route to the disodium phosphate prodrug (2a). The phosphoric acid precursor (11) of phosphate 2a was employed in a parallel series of reactions to produce a selection of metal and ammonium cation prodrug candidates. Each of the phosphate salts (2a-q) was evaluated with respect to relative solubility behavior, cancer cell growth inhibition and antimicrobial activity.

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The in vitro activity of the steroidal amide 3beta-acetoxy-17beta-(L-prolyl)amino-5alpha-androstane against 179 gram-positive clinical isolates was examined. The minimum bactericidal concentration (MBC)/MIC ratios were < or = 2 for 73% of methicillin-resistant Staphyllococcus aureus, 59% of vancomycin-resistant Enterococcus spp. and 88% of penicillin-resistant Streptococcus pneumoniae. The androstane derivative was bactericidal for a variety of other gram-positive genera, including Nocardia, Corynebacterium and Listeria. Variation in MICs is pH 6-8 media was slight. The frequency of occurrence of bacterial spontaneous mutations to resistance ranged from 10(-6) to 10(-9). Kill curve analysis confirmed the bactericidal nature of the steroidal amide, and demonstrated that killing was time dependent but not concentration dependent for all organisms. The ability of 3beta-acetoxy-17beta-(L-prolyl)amino-5alpha-androstane to inhibit human cancer cell growth was also evaluated. The concentration required to inhibit 50% of cell growth (GI50) was < 2.5 mg/l for all cell lines examined. In single-dose murine toxicity evaluations, the androstane derivative was non-toxic at doses up to 400 mg/kg.

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The synthetic (E)-isomer (3b) of natural combretastatin A-1 (1a) isolated from the African bushwillow Combretum caffrum was the focus of chiral hydroxylation (Sharpless) reactions as part of a structure-activity relationship study. The resulting (R,R)- and (S,S, )-diols (6 and 7) and synthetic intermediates were evaluated against a series of cancer cell lines, microorganisms, and tubulin. Chiral diols 6 and 7 showed increased activity against the P-388 murine lymphocytic leukemia cell line with ED(50) values of 3.9 and 2.9 microg/mL, respectively, when compared to the precursor (E)-stilbene 3b. In contrast, (E)-stilbene 3b exhibited more potent antibiotic activity than the chiral diols (6 and 7). Both diols, (R,R)-6 and (S, S)-7, displayed less cancer cell growth inhibition and less antibiotic activity than did natural combretastatin A-1 (1a) (P-388 ED(50) 0.25 microg/mL).

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A structure-activity relationship (SAR) study of the South African willow tree (Combretum caffrum) antineoplastic constituent combretastatin A-4 (3b) led to the discovery of a potent cancer cell growth inhibitor designated phenstatin (5a). This benzophenone derivative of combretastatin A-4 showed remarkable antineoplastic activity, and the benzophenone derivative of combretastatin A-1 was therefore synthesized. The benzophenone, designated hydroxyphenstatin (6a), was synthesized by coupling of a protected bromobenzene and a benzaldehyde to give the benzhydrol with subsequent oxidation to the ketone. Hydroxyphenstatin was converted to the sodium phosphate prodrug (6e) by a dibenzyl phosphite phosphorylation and subsequent benzyl cleavage (6a --> 6d --> 6e). While hydroxyphenstatin (6a) was a potent inhibitor of tubulin polymerization with activity comparable to that of combretastatin A-1 (3a), the phosphorylated derivative (6e) was inactive.

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Continued investigation of cancer-cell growth-inhibitory constituents of the blue marine sponge Cribrochalina sp. has led to discovery of cribrostatins 3 (4a), 4 (5), and 5 (4b) in 10(-5) to 10(-7) % of the wet weight. The structure of cribrostatin 3 (4a) was determined by results of high field (500 MHz) (1)H and (13)C NMR and HRMS interpretations. The same general approach to the structures of cribrostatins 4 (5) and 5 (4b) was completed by X-ray crystal structure determinations. Cribrostatins 3, 4, and 5 provided significant cancer cell line inhibitory activities. Cribrostatins 1 and 2(2) and the newly isolated cribrostatins 3-5 displayed antibacterial and/or antifungal activities.

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A new and more efficient synthesis of combretastatin A-3 (2a) was completed (8.4% overall yield) starting from methyl gallate and isovanillin with aldehyde 5 and phosphonium salt 8 as key intermediates. Conversion of combretastatin A-3 (2a) to a series of diphosphate prodrugs (10a-l) was readily achieved. Both the diphosphate sodium (10a) and potassium salts (10c) displayed aqueous solubility in excess of 220 mg/ml at room temperature and good cancer cell line inhibitory activity.

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Spongistatin 1, a macrocyclic lactone from the marine sponge Hyrtios erecta, has broad-spectrum antifungal activity. Since this compound is a potent antimicrotubule agent in mammalian cells, we examined its effects on the filamentous fungus Aspergillus nidulans to determine if its antifungal effects are due to antimicrotubule activity. At 25 microg/ml (twice the MIC), spongistatin 1 caused a greater-than-twofold elevation of the chromosome and spindle mitotic indices. Immunofluorescence microscopy revealed that mitotic spindles were smaller and shorter than in control germlings. However, late-anaphase and telophase nuclei were seen occasionally, and this suggests that the spindles are capable of segregating chromosomes. Spongistatin 1 had more dramatic effects on cytoplasmic microtubules. At 30 min after initiation of treatment, 83% of germlings contained fragmented microtubules and after 2 h of treatment, microtubules had disappeared completely from 82% of germlings. In contrast, microtubules disappeared rapidly and completely from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in A. nidulans and that its mechanism of action may involve a novel microtubule-severing activity.

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A Montana soil actinomycete, Streptomyces anulatus, produced (1 x 10(-2)% yield) a new cancer cell growth inhibitory cyclooctadepsipeptide named montanastatin (1) accompanied by the potent anticancer antibiotic valinomycin (2) in very high (5.1%) yields. Valinomycin but not montanastatin inhibited growth of a number of pathogenic bacteria and fungi. Interpretation of high-field (500 MHz) NMR and high-resolution FAB mass spectral data allowed assignment of the structure cyclo-(D-Val-L-Lac-L-Val-D-Hiv) to montanastatin. Valinomycin (2) was also isolated from actinomycetes cultured from a tree branch and animal feces collected in Malaysia. Streptomyces exfoliatus, isolated from the tree branch, was found to contain valinomycin in 1.6% yield, while the fecal isolate, S. anulatus, gave valinomycin in 0.9% yield.

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The South African willow tree Combretum caffrum has yielded a number of potent cancer cell growth inhibitors. The present SAR studies of the antineoplastic agent combretastatin A-4 (1c) were focused mainly on the olefinic bridge to determine the effects on cancer cell growth and, potentially, to better define the combretastatin A-4 binding site on tubulin. The geometric trans-isomer 3a of combretastatin A-4 was converted to the (1S,2S)- and (1R,2R)-vicinal diols 4c and 4d, respectively, under Sharpless' asymmetric dihydroxylation conditions. Cancer cell line testing showed the (1S, 2S)-diol 4c to be more potent than its enantiomer 4d. Diol 4c weakly inhibited tubulin polymerization (IC50 = 22 microM, versus 1.2 microM for combretastatin A-4), while 4d was inactive (IC50 > 40 microM). Esterification of either stereoisomer at the diol and/or phenolic positions resulted in elimination of inhibitory activity.

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This study examined the response to acidic conditions of four gonococcal isolates -NRL38874 (Proto/IB-2), NRL38884 (Pro/IA-2), NRL38953 (Proto/IB-3) and NRL39029 (Pro/IA-3) - obtained from various sites in patients in whom a diagnosis of pelvic inflammatory disease had been made by laparoscopic examination. Acid tolerance of the clinical isolates was strain and growth phase dependent. Growth of the four strains on solid media was undetectable below pH 5.8. In liquid culture, strain NRL38884 did not survive below pH 5.2; strains NRL38874, NRL38953 and NRL39029 survived to pH 4.5. Between pH 4.2 and pH 5.1, the latter three strains exhibited a peak in survival at pH 4.6-4.7 during log phase, suggesting that there may be a distinct acid tolerance system operating at this pH. SDS-PAGE of whole-cell, total membrane and outer-membrane fractions of the four strains prepared from pH 7.2 and pH 6.1 plate cultures revealed numerous differences in protein composition. Acidic conditions reduced the expression of the reduction modifiable outer-membrane protein Rmp, and induced the expression of many membrane proteins, including gonococcal hsp63. Immunoblotting studies with matched serum samples and strains from patients with pelvic inflammatory disease indicated that IgG recognition of outer-membrane components from strains cultured in acidic and neutral conditions was quite different. The results suggest that the immune system interacts with unique outer-membrane constituents on gonococci colonising sites at different pH.

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The biosynthetic peptide dolastatin 10 is currently in phase I and II cancer clinical trials. We evaluated the antifungal spectrum of dolastatin 10 and four structural modifications. In broth macrodilution assays, the peptides were fungicidal for American Type Culture Collection strains and clinical isolates (including fluconazole-resistant strains) of Cryptococcus neoformans but no other yeasts or filamentous fungi examined. Specificity for C. neoformans was also demonstrated in the solid-phase disk diffusion assay, and fungicidal activity was confirmed in time-kill experiments. For a methyl ester modification, the MICs at which 50 and 90% of 19 clinical isolates were inhibited (MIC50 and MIC90, respectively) were 0.195 and 0.39 microg/ml, respectively. The MFC50 (50% minimum fungicidal concentration) for this peptide was 0.39 microg/ml, and the MFC90 was 0.78 microg/ml. MICs and MFCs were identical or lower in the presence of human serum but increased with lowered pH. These peptides should be pursued as potential chemotherapeutics for C. neoformans, a leading cause of infection and mortality in immunocompromised patients.

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The remarkable anticancer drug dolastatin 10 (1a) from the Indian Ocean sea hare Dolabella auricularia is currently undergoing phase I clinical trials. Thirty-eight new structural modifications of this unusual peptide have been synthesized and evaluated against a variety of human and murine cancer cell lines, and for their ability to inhibit tubulin polymerization and vinblastine and GTP binding to tubulin. Dolastatin 10 and one structural modification was found to have antifungal activity, while one other structural modification of the parent compound exhibited antibacterial activity. Some of the new peptides approximated the antineoplastic potency of dolastatin 10, especially those based on replacement of the Doe unit with Met, Phe or an appropriately substituted phenylethylamide.

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A structure-activity relationship (SAR) study of the South African willow tree (Combretum caffrum) antineoplastic constituent combretastatin A-4 (1b) directed at maintaining the (Z)-stilbene relationship of the olefin diphenyl substituents led to synthesis of a potent cancer cell growth inhibitor designated phenstatin (3b). Initially phenstatin silyl ether (3a) was unexpectedly obtained by Jacobsen oxidation of combretastatin A-4 silyl ether (1c --> 3a), and the parent phenstatin (3b) was later synthesized (6a --> 3a --> 3b) in quantity. Phenstatin was converted to the sodium phosphate prodrug (3d) by a dibenzyl phosphite phosphorylation and subsequent hydrogenolysis sequence (3b --> 3c --> 3d). Phenstatin (3b) inhibited growth of the pathogenic bacterium Neisseriagonorrhoeae and was a potent inhibitor of tubulin polymerization and the binding of colchicine to tubulin comparable to combretastatin A-4 (1b). Interestingly, the prodrugs were found to have reduced activity in these biochemical assays. While no significant tubulin activity was observed with the phosphorylated derivative of combretastatin A-4 (1d), phosphate 3d retained detectable inhibitory effects in both assays.

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Dolastatin 15 (1), a potent antineoplastic constituent of the Indian Ocean shell-less mollusk Dolabella auricularia, was utilized as the lead substance for a series of structure-activity studies. The synthetic methods, in vitro evaluations against a variety of murine and human cancer cell lines, as well as a selection of bacteria and fungi, and inhibition of tubulin polymerization are described. Remarkably, all of the compounds studied, in which the C-terminal (S)-dolapyrrolidinone unit (Dpy) was replaced with a series of structurally diverse and more readily available amides, showed cancer cell growth inhibition activities generally quite comparable to those of the parent molecule (1). All analogues, however, were less potent than 1 as inhibitors of tubulin polymerization. The structurally modified peptides, like the parent compound, caused mitotic arrest in cultured cells, consistent with tubulin being the primary cellular target. The ability of dolastatin 15 and eight modifications or precursors thereof to inhibit the growth of a Gram-negative bacterium suggests that these compounds have an additional target distinct from tubulin.

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The (E)-stilbene isomer (2a) of the (Z)-combretastatin A-4 prodrug (1b) was efficiently prepared from (E)-combretastatin A-4 by a reaction sequence employing phosphorylation (dibenzyl chlorophosphite), cleavage (trimethyliodosilane) of the benzyl ester and reaction of the resulting phosphoric acid with sodium methoxide. The sodium phosphate product (2c) was also found to be an important side-product, presumably from iodine-catalyzed isomerization, when the analogous synthetic route was used to obtain the combretastatin A-4 prodrug (1b). The phosphoric acid precursor of prodrug 1b derived from (Z)-combretastatin A-4 (1a) was converted into a series of metal cation and ammonium cation salts to evaluate effects on human cancer cell growth, antimicrobial activities and solubility behavior.

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The wide ranging marine sponge Hyrtios erecta is the source of the spongistatins, a new class of macrocyclic lactone antineoplastic agents. Continuation of a detailed investigation of cancer cell growth inhibitory (P388 lymphocytic leukemia) fractions (trace) from H. erecta has revealed the presence (10(-5) to 10(-7)% yield) of cytotoxic pentacyclic sesterterpenes. Employing P388 leukemia and human tumor cell line-guided bioassay techniques, two new moderate inhibitors of cancer cells were isolated and named sesterstatins 4 (1a, P388 ED50 4.9 micrograms/mL) and 5 (1b, DU-145 prostate GI50 1.9 micrograms/mL). Similar to other sesterterpenes, sesterstatin 5 inhibited growth of a Gram-positive bacterium. High field (500 MHz) 2-D NMR techniques were primarily employed for initial structural assignments, and structural assignments were confirmed by X-ray crystal structure determination of sesterstatin 4 (1a) and 5 (1b).

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