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. Author manuscript; available in PMC: 2011 Mar 15.
Published in final edited form as: Bioorg Med Chem Lett. 2010 Feb 1;20(6):2029–2032. doi: 10.1016/j.bmcl.2010.01.068

Synthesis and Chain-Dependent Antifungal Activity of Long-Chain 2H-Azirine-Carboxylate Esters Related to Dysidazirine

Colin K Skepper a, Doralyn S Dalisay a, Tadeusz F Molinski a,b,*
PMCID: PMC2834865  NIHMSID: NIHMS176247  PMID: 20171099

Abstract

Analogues of the antifungal marine natural product (E)-dysidazirine were prepared and evaluated in broth ro-dilution assays against a panel of fungal pathogens. A simple structure-activity relationship was developed which provides insight into the mechanism of action of long-chain 2H-azirine carboxylates.

Keywords: antifungal, oxazole, azole, marine natural product

2H-Azirines are highly strained, weakly aromatic heterocycles rarely found in Nature. In fact, only nine natural products containing the 2H-azirine ring have been described to date. Azirinomycin1 (1) was isolated from Streptomyces aureus while the other compounds constitute a small family of long-chain 2H-azirine carboxylates. The first of this family, (E)-dysidazirine (2), was isolated in 1988 from the marine sponge Dysidea fragilis, collected in Fiji, and shown to exhibit potent antifungal activity against Candida albicans and Saccharomyces cerevisiae (4 μg/disk, disk diffusion assay).2 Faulkner and coworkers later characterized (Z)-dysidazirine (3) and two ω-dibromovinylidene azirine-2-carboxylate methyl esters, 4 and 5 (E- and Z-antazirines) from D. fragilis and reported them as inactive “against a standard panel of roorganisms”.3 Motualevic acid F, the parent carboxylic acid of ent-4, was reported by Bewley and coworkers along with related amides from Siliquariaspongia sp. with antibacterial activity against Staphylococcus aureus and MRSA.4 In our search5 for antifungal compounds from marine organisms with efficacy against Fluconazole-resistant strains of Candida and other pathogenic yeast,6 we found three new antazirines (6–8) along with 4 and 5 from a sample of Dysidea fragilis collected in Pohnpei, Micronesia that showed cytotoxicity in a crude screen.7 Compounds 4–8 were moderately cytotoxic (HCT-116 cells), but to our surprise they were completely inactive against a panel of fungal pathogens including Candida albicans (ATCC, UCD-FR1 and 96–489), less susceptible, non-albicans species such as C. glabrata and C. krusei, and two strains of Cryptococcus neoformans, var. grubii and gatti.

In order to elucidate the relationship between structure and antifungal activity of long-chain 2H-azirine carboxylates we carried out the first synthesis of (Z)-dysidazirine8 and a short series of structural analogues ((−)-9→13, Figure 2), along with (E)-dysidazirine. We report here the structure-activity relationships for (−)-2, (−)-3, (±)-3 and (−)-9→13 that reveal structural determinants for antifungal activity, including terminal branching [(−)-9], chain length [(−)-10 and (−)-11], C4–C5 unsaturation [(−)-12, (−)-13] and C2 configuration [(±)-3].

Figure 2.

Figure 2

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Targeted synthetic dysidazirine analogues.

The synthesis of (−)-(Z)-dysidazirine and analogues (−)-9 and (−)-12 has been described previously.8 Photocheal isomerization (500W sunlamp, Pyrex) of synthetic (−)-3 (59% ee) provided (−)-2 (Scheme 1) in low yield but without epimerization at C2. Natural dysidazirine and congeneric compounds have all been isolated as non-race mixtures of enantiomers. Neat, natural dysidazirine spontaneously epimerizes slowly in the dark (t1/2 ~12y, −20 °C),7 and the lack of racemization of 2 and 3 in the presence of light excludes a photocheal mechanism.

Scheme 1.

Scheme 1

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Photochemical isomerization of (−)-(Z)-dysidazirine to (−)- (E)-dysidazirine.

The shorter chain analogues (−)-10 and (−)-11 were prepared in an analogous fashion to (−)-3 (Scheme 2). Addition of the lithio-acetylide derived from alkyne 14 to methyl malonyl chloride gave β-ketoester 15 in reasonable yield (55%). Treatment of 15 with NH2OH•HCl/pyridine led to the corresponding oxime which was converted to oxime tosylate 16 without purification. Cyclization in the presence of quinidine under Zwanenburg's conditions9 provided 2H-azirine 17 in excellent yield. Partial hydrogenation with Lindlar's catalyst provided the truncated dysidazirine analogue (−)-11. The same sequence applied to alkyne 18 gave analogue (−)-10.

Scheme 2.

Scheme 2

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Synthesis of truncated analogues (−)-10 and (−)-11.

Synthesis of (−)-13, the 4,5-dihydro analogue of 3, began with generation of the enolate of dioxolane 19 (LDA, HMPA, −40 °C) followed by alkylation with 1-bromotetradecane (20) to provide 21 in low yield (18%) along with an equivalent amount of the product from α-alkylation (Scheme 3). Thermolysis of 21 (rowave) gave the incipient ketene that was captured with methanol to afford O-methyl β-ketoester 22 that was subsequently converted to oxime tosylate 23 in two steps as before. Treatment of 23 with quinidine led directly to (−)-13 in 91% yield.

Scheme 3.

Scheme 3

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Synthesis of 4,5-dihydrodysidazirine [(−)-13].

The antifungal s of the synthetic and natural azirines are compared in Figure 4.10 Each yeast strain was susceptible to (−)-3 and some synthetic analogues with the notable exception of Fluconazole-resistant C. krusei. The other Fluconazole-resistant strains (C. glabrata, C. albicans UCSD-FR1 and 96–489) were all susceptible. Strains of Cryptococcus neoformans (var. grubii and var. gatti) were the most susceptible overall.

Figure 4.

Figure 4

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Antifungal activity of (−)-2, (−)-3, (±)-3 and (−)-9→13 against: (a) Candida albicans, three strains: ATCC 14503, UCD-FR1 and 96–489 (see ref. 18). (b) Candida glabrata and Candida krusei (c) Cryptococcus neoformans var. grubii and gatti. Compounds with off-scale 50s were inactive up to 64 μg/mL.

Both (−)-2 and (−)-3 were consistently amongst the most active compounds against all strains, with 50 values in the range of 2–8 μg/mL. For Candida spp., (−)-3 was notably more active than (−)-2 (Figure 4a). Shorter chain analogues (−)-10 and (−)-11 were comparable in activity to (−)-3, although Fluconazole-resistant C. albicans UCD-FR1 was susceptible to (−)-10, but not to (−)-11.

Alkynyl analogue (−)-12 retains most of the activity of the natural products (−)-2 and (−)-3, however the 4,5-dihydro analogue (−)-13 lost activity across the entire panel of fungal cells. The C2 configuration appears to be less important. Race Z-dysidazirine [(±)-3] shows good activity against all strains, comparable to or slightly better than the optically enriched homologue (−)-3.

As previously observed,8 the tert-butyl terminus analogue (−)-9 is essentially inactive. Clearly terminal substitution or branching abrogates antifungal activity, consistent with the lack of activity for ω-halo-alkenyl antazirines (4–8).3,7

The foregoing results suggest specific structural attributes determine antifungal activity rather than non-specific toxicity. Our original hypothesis regarding a putative sub-cellular target of dysidazirine, based on data obtained from (−)-3 and (−)-9, is supported by the wider range of analogues presented here.8 We favor a model for activity of long-chain azirines involving interaction with a putative protein target through a 2-point binding motif and propose the following. We envision the lipid chain occupies a tightly constrained hydrophobic pocket that is intolerant of bulky ω-substitution, which helps to explain the loss of activity seen for 4–8 and (−)-9 despite logPs and molecular size that are comparable to (−)-3. The polar, electrophilic azirine terminus – a potent hael acceptor capable of covalent modification of nucleophilic sites (e.g. cysteine, lysine) – binds at a distal site, possibly interacting with one or more nucleophilic amino acid residues. The absolute requirement of C4–C5 unsaturation for high antifungal activity supports the latter hypothesis. (Z)-Dysidazirine [(−)-3)], which is expected to be a superior hael acceptor to (−)-2, shows enhanced antifungal activity against Candida albicans strains.

The structure of dysidazirine bears a resemblance to phytosphingosine (24) and sphingosine (25) (Figure 5) – both 24 and 25 share the same carbon chain length (C18) as dysidazirine. The long-chain base 24 occurs in plant and fungal cells and acts in multiple capacities, both as a cellular structural component and a signaling molecule.11 Sphingosine (25) is the primary long-chain base of mammalian cells; it also plays roles in cell structure and signaling, and exhibits modest antifungal activity (30 μg/mL, C. glabrata).12

Figure 5.

Figure 5

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Structures of sphingoid bases and antifungal long-chain aminoalcohol marine natural products.

Several long-chain vicinal aminoalcohol marine natural products (Figure 5) also exhibit antifungal activity,5 including crucigasterins 275 (26) and 277 (27),13 (R)-1-aminotridecen-2-ol14 (28–30) and oceanapiside (31),15 an unusual `two-headed' sphingoid base.16

Natural products dysidazirine (−)-3, other long-chain azirines and 31 resemble sphingosine or its immediate precursor sphinganine (4,5-dihydrosphingosine) and may act as antimetabolites that competitively inhibit sphingolipid metabolism.

The biosynthesis of 24 and 25 in yeast follows the canonical sphingolipid pathway that begins with pyridoxal phosphate-dependent condensation of palmitoyl CoA thioester with serine. Long-chain azirines may compete for natural substrate in the condensation reaction or downstream reactions that tailor 24 and 25. It is notable that the antifungal properties of 3 and other sphingoid base analogues are not solely determined by the presence of a long hydrophobic tail since simple lipids are inactive and other antifungal long-chain fatty acid conjugates appear to act by distinctly different mechanisms.17

In summary, we have completed the synthesis of a series of dysidazirine analogues and evaluated each for antifungal activity against a panel of clinically-relevant yeasts. Several generalizations can be made: 1) C4–C5 unsaturation is required for high activity. 2) Activity is not strongly dependent on minor changes in chain length or the configuration at C2. 3) Terminal branching of the lipid chain abolishes activity. Further investigations are underway in our laboratories to identify high-affinity binding targets of (−)-3.

Figure 1.

Figure 1

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Naturally occurring 2H-azirines.

Acknowledgements

The NSF CRIF program (CHE0741968) is acknowledged for acquisition of the 500 MHz NMR spectrometers. We are grateful to the NIH for generous funding of this research (AI 039987).

Footnotes

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