Synthesis and antifungal activity of (-)-(Z)-dysidazirine

Article abstract of DOI:10.1021/ol802065d

(Chemical Equation Presented) A short, flexible synthesis of the marine natural product (2R)-(Z)-dysidazirine (-)-1 has been completed. (-)-1 shows significant antifungal activity across a panel of seven human pathogens, whereas the structural analogue (-)-2, featuring a terminal tert-butyl group, is essentially inactive.

Full text of DOI:10.1021/ol802065d

ORGANIC
LETTERS
2008
Vol. 10, No. 22
5269-5271
Synthesis and Antifungal Activity of
(-)-(Z)-Dysidazirine
Colin K. Skepper,† Doralyn S. Dalisay,† and Tadeusz F. Molinski*,†,‡
Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and
Pharmaceutical Sciences, UniVersity of California San Diego,
9500 Gilman DriVe MC0358, La Jolla, California 92093
tmolinski@ucsd.edu
Received September 3, 2008
ABSTRACT
A short, flexible synthesis of the marine natural product (2R)-(Z)-dysidazirine (-)-1 has been completed. (-)-1 shows significant antifungal
activity across a panel of seven human pathogens, whereas the structural analogue (-)-2, featuring a terminal tert-butyl group, is essentially
inactive.
Invasive fungal infections, particularly those associated
with Candida sp., pose a serious and increasing threat to
human health. Widespread use of fluconazole since the mid-
1990s has led to the rise of resistant Candida strains (e.g.,
Candida glabrata and Candida albicans) and attendant
increased mortality among immunocompromised individu-
als.1,2 New antifungal agents are urgently needed, and marine
natural products represent an underexploited source of
potential leads.2
(2R)-(E)-Dysidazirine, (-)-3, one of a few rare naturally
occurring azacyclopropenes (2H-azirines), was isolated in
1987 from the marine sponge Dysidea fragilis (collected in
Fiji).3 Compound (-)-3 exhibited potent antifungal activity
against C. albicans and Saccharomyces cereVisiae (4 µg/
disk, disk-diffusion assay). More recently, brominated analo-
gues 4 and (+)-6 (antazirines) were isolated along with (Z)-
dysidazirine (1) and (2S)-(E)-dysidazirine [(+)-3] from the
same sponge species collected in Micronesia.4 Antifungal
activity for 4 and (+)-6 were not reported,4 and they were
found to be inactive against a panel of bacteria.
We recently isolated three new antazirine analogues [(+)-
5, (+)-7, and (-)-8] from a sample of D. fragilis that also
provided the two known antazirines [(+)-4 and (+)-6].5 All
five antazirines proved to be inactive against a panel of
Candida and Cryptococcus spp., in stark contrast with (-)-
3. This surprising result led us to hypothesize that terminal
substitution (e.g., Br, Cl, Me) attenuates antifungal activity
in long-chain 2H-azirines. In order to explore this hypothesis,
we have developed a short, flexible synthesis of (-)-1 and
structural analogue (-)-2 that features a terminal tert-butyl
group.
There is only one published synthesis of a long-chain 2H-
azirine, that of E-dysidazirine, completed in 1995 by Davis
and co-workers.6 In this case, the key azirine ring was formed
by treating an appropriately substituted N-(p-tolylsulfinyl)-
† Department of Chemistry and Biochemistry.
‡ Skaggs School of Pharmacy and Pharmaceutical Sciences.
(1) Nguyen, M. H.; Peacock, J. E.; Morris, A. J.; Tanner, D. C.; Nguyen,
M. L.; Snydman, D. R.; Wagener, M. M.; Rinaldi, M. G.; Yu, V. L. Am. J.
(4) Salomon, C. E.; Williams, D. H.; Faulkner, D. J. J. Nat. Prod. 1995,
58, 1463–1466. (b) Neither [R]D nor optical purity were reported for natural
(Z)-antazirine (4) or (Z)-dysidazirine (1). See also ref 5.
Med. 1996, 100, 617–623
.
(2) (a) Vicente, M. F.; Basilio, A.; Cabello, A.; Pela´ez, F. Clin.
Microbiol. Infect. 2003, 9, 15–32. (b) Bernan, V. S.; Greenstein, M.; Carter,
G. T. Curr. Med. Chem.: Anti-Infect. Agents 2004, 3, 181–195. (c) Molinski,
T. F. Curr. Med. Chem.: Anti-Infect. Agents 2004, 3, 197–220. (d) Li, H-y.;
(5) Skepper, C. K.; Molinski, T. F. J. Org. Chem. 2008, 73, 2592–2597.
All naturally occurring long-chain azirines from marine sponges appear to
be heterochiral (non-racemic mixtures of enantiomers).
(6) (a) Davis, F. A.; Reddy, G. V.; Liu, H. J. Am. Chem. Soc. 1995,
117, 3651–3652. (b) Davis, F. A.; Liu, H.; Liang, C.-H.; Reddy, G. V.;
Zhang, Y.; Fang, T.; Titus, D. D. J. Org. Chem. 1999, 64, 8929–8935.
Matsunaga, S.; Fusetani, N. Curr. Org. Chem. 1998, 2, 649–682
.
(3) Molinski, T. F.; Ireland, C. M. J. Org. Chem. 1988, 53, 2103–2105.
10.1021/ol802065d CCC: $40.75
Published on Web 10/21/2008
2008 American Chemical Society

Scheme 2. Synthesis of 14,14-Dimethylpentadecyne (18)
Partial hydrogenation of (-)-13 using Lindlar’s catalyst
at ambient temperature in EtOH proved difficult due to facile
reduction of the product alkene and the azirine ring within
minutes. An improvement was found by lowering the
temperature of hydrogenation (0 °C, hexanes) to give (Z)-
dysidazirine [(-)-1] in 52% yield and optical purity (59%
ee) comparable to the related natural products.5
In order to examine the effect of terminal substitution on
antifungal activity, analogue (-)-2 was synthesized starting
with Wittig reaction between phosphonium bromide 14 and
3,3-dimethylbutanal, giving alkene 15 as a mixture of double
bond isomers. Hydrogenation of 15 (H2, Pd/C, EtOAc, 1.5 h)
gave saturated alcohol 16, which was oxidized under Swern
conditions to give the corresponding aldehyde.8 Treating the
crude aldehyde immediately with PPh3/CBr4 (CH2Cl2, 0 °C,
1 h)9 afforded dibromoalkene 17, which was subsequently
converted to terminal alkyne 18 (n-BuLi, THF, -78 °C f
rt) in 86% overall yield from 16. Addition of the lithiated
acetylide ion of 18 to 10 proceeded reliably to give 19, albeit
in moderate yield (45%).
Keto-ester 19 was converted to the corresponding oxime-
tosylate 20 (71%), which underwent quinidine-mediated
cyclization to give the desired azirine (-)-21 in 80% yield.
Cyclization of 20 proceeded even more slowly than the
corresponding reaction with 12 and required brief warming
to 10 °C to ensure complete consumption of starting material.
Lindlar reduction of (-)-21 gave (-)-(R)-17,17-dimethyl-
(Z)-dysidazirine [(-)-2] in 58% yield.
Figure 1. Structures of (Z)- and (E)-dysidazirine [(-)-1 and (-)-
3], antazirines [(+)-4-8], and synthetic (Z)-dysidazirine analogue
[(-)-2].
2-carbomethoxyaziridine with LDA at -78 °C. While this
approach allowed for synthesis of (-)-3 with high optical
purity, the necessity to preserve the Z configuration in (-)-1
required an alternate approach. We chose a strategy based
on a variant of the asymmetric cinchona base-catalyzed
Neber reaction of O-tosyl-oximes first reported by Zwanen-
burg and co-workers.7
Synthesis of (2R)-(Z)-dysidazirine [(-)-1] began with
addition of the lithio-anion of pentadecyne (9) to methyl
malonyl chloride (10, Table 1). The addition gave poor,
variable yields when the deprotonation of 9 was effected with
n-BuLi; using EtMgBr, however, led to clean formation of
11 in a reproducible 70% yield.
Keto-ester 11 was converted to the corresponding oxime
(NH2OH·HCl, pyridine, EtOH, 55 °C, 30 min), which was
tosylated immediately (p-toluenesulfonic anhydride, pyridine,
DMAP, 1.5 h) giving 12 in two steps (72% yield). Treatment
of 12 with quinidine (0 °C, 48 h) lead to clean, albeit slow,
formation of the desired 2H-azirine ring.7 While the product
azirine was formed with only modest enantioselectivity, this
methodology is notable for its practical simplicity and high
chemical yield.
Scheme 1. Synthesis of (-)-(Z)-Dysidazirine 1
Scheme 3. Synthesis of 17,17-Dimethyl-(Z)-dysidazirine
a % ee for (-)-1 ) 59% (determined by chiral HPLC, Chiralpak AD,
90:10 hexanes/i-PrOH).
a % ee for (-)-2 ) 60% (HPLC, Chiralpak AD, 10:90, i-PrOH /hexanes).
Org. Lett., Vol. 10, No. 22, 2008
5270

Table 1. Optimization of Pentadecyne Addition to Methyl Malonyl Chloride
entry
base
solvent
T1 (°C)
time1 (min)
equiv 10
T2 (°C)
time2 (min)
yield (%)
1
2
3
4
5
n-BuLi
n-BuLi
EtMgBr
EtMgBr
EtMgBr
THF
Et2O
Et2O
THF
THF
0
-20
0 f rt
0 f rt
0 f rt
90
60
80
120
150
0.5
0.5
0.5
0.8
0.5
-78f0
-60f0
0
0
0
150
120
40
50
60
0a
14a
0b
19
70
a Estimated from crude NMR. b Compound 11a was isolated in 35% yield.
Both (-)-1 and (-)-2 were screened for antifungal activity
against a panel of Candida and Cryptococcus spp. (Table
2). Synthetic (Z)-dysidazirine [(-)-1] showed strong activity
against each fungal strain (MIC50 2-8 µg/mL) with the
exception of C. krusei (MIC50 16 µg/mL). This appears to
be consonant with the activity originally reported for isomeric
(-)-E-3 in 1988.3 In contrast, compound (-)-2 with the
bulky tert-butyl chain terminus was effectively inactive
across the entire panel.
however, argues for a more specific effect that involves
metering of the lipid chain.10 We postulate that (-)-1 engages
its target protein in a two-point binding motif in which the
lipid chain occupies a hydrophobic pocket with stringent
steric requirements while the electrophilic R,ꢀ-unsaturated
azirine carboxylate terminus binds at a distal site. The
specificity of this interaction with respect to chain length
remains to be investigated, however it is notable that
dysidazirine shares a common chain length (C18) with several
sphingolipids important for fungal cell growth, such as
phytosphingosine and ceramide.2 This suggests dysidazirine
may be an “antimetabolite” that interdicts fungal cell lipid
metabolism, perhaps sphingolipid biosynthesis. A more
extensive study of SAR and mechanism of action of (-)-1
is underway, and our results will be reported in due course.
In summary, we have completed the first synthesis of the
marine natural product (-)-(Z)-dysidazirine4 and a structural
analogue (-)-2 that features a terminal tert-butyl group.
Compound (-)-1 shows significant antifungal activity toward
a range of Candida and Cryptococcus species, but (-)-2 is
almost completely inactive. This surprising result suggests
that the activity of (Z)-dysidazirine is modulated by two-
point binding to an as-yet unidentified target protein in fungal
cells. Interaction between (-)-1 and its putative target likely
involves sterically stringent interaction between the lipid side
chain and a hydrophobic domain.
Table 2. Antifungal activity of (-)-1 and (-)-2a
MIC50 (µg/mL)
(-)-1
(-)-2
C. albicans ATCC 14503
C. albicans UCD-FR1b
C. albicans 96-489b
8
8
4
>64
>64
>64
16
C. glabratab
4
C. krusei
16
2
2
>64
9
>64
Cryptococcus neoformans var. grubii
Cryptococcus neoformans var. gatti
a The in Vitro susceptibility of each compound was determined by the
broth micro dilution method according to the guidelines of the National
Committee for Clinical Laboratory Standards (NCCLS). b Fluconazole-resistant
(MIC > 64 µg/mL).
This surprising result points to a subtle complexity in
mechanism of action for (-)-1. The target of (-)-1 in fungi
is unknown; however, it is likely to involve essential
metabolism of lipids.
The electrophilic properties of the strained R,ꢀ-unsaturated
azacyclopropene suggests, at first glance, that the antifungal
activity of (-)-1 might arise from nonspecific Michael
addition to nucleophilic amino acid residues (e.g., cysteine,
lysine). The differential activity observed for (-)-1 and (-)-2
Acknowledgment. We are grateful to Kathy Holten
(University of Texas Medical Center) and Dr. Angie Gelli
(UC Davis School of Medicine) for clinical isolates of
Candida spp. and Cryptococcus neoformans, respectively.
MS analysis was performed by the UCSD Chemistry/
Biochemistry Mass Spectrometry Facility. We are grateful
for support for this research from the NIH (AI 039987).
Supporting Information Available: Full experimental
details and characterization of synthetic intermediates; copies
(7) Verstappen, M. M. H.; Ariaans, G. J. A.; Zwanenburg, B. J. Am.
Chem. Soc. 1996, 118, 8491–8492.
1
of H and 13C NMR spectra for all new compounds. This
(8) Leopold, E. J. Org. Synth. 1986, 64, 164.
material is available free of charge via the Internet at
http://pubs.acs.org.
(9) Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 36, 3769–3772.
(10) It is unlikely the effects are due to differential cell wall permeability
since the c log10 P of (-)-1 and (-)-2 are similar (6.8 and 7.6, respectively,
ChemDraw Ultra).
OL802065D
Org. Lett., Vol. 10, No. 22, 2008
5271