Synthesis and structure-activity relationships of bengazole A analogs

Article abstract of DOI:10.1016/j.bmcl.2009.04.069

Analogs of the potent antifungal agent, bengazole A, were prepared and evaluated against Candida spp. in both microbroth dilution and disk diffusion assays.

Full text of DOI:10.1016/j.bmcl.2009.04.069

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2928–2930
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of bengazole A analogs
Roger J. Mulder c, , Cynthia M. Shafer c,à, Doralyn S. Dalisay a, Tadeusz F. Molinski a,b,c,
*
a Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
b Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
c Department of Chemistry, University of California Davis, 1 Shields Avenue, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs of the potent antifungal agent, bengazole A, were prepared and evaluated against Candida spp. in
both microbroth dilution and disk diffusion assays.
Received 16 March 2009
Revised 13 April 2009
Accepted 17 April 2009
Available online 22 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal
Oxazole
Natural product
Synthesis
Bengazoles A (1) and B (2) are homologous bis-oxazole natural
products first reported by Crews and co-workers from the sponge
Jaspis sp. with anthelmintic activity against the nematode Nippo-
strongulus braziliensis.1
and voriconazole) inhibit 14-
a-demethylase in yeast by tight bind-
ing of one of the 1,2,4-triazole rings to the Fe-heme active site.8
A
cursory comparison of the structures of 1 and 4 suggested one of
the oxazole rings in 1 might play a similar role, although this is
not certain and other structural elements also appear to be impor-
tant (Fig. 1).
Bengazole 1 exhibits ergosterol-dependent in vitro activity
against C. albicans similar to amphotericin B (AmB). When grown
on agar plates containing Sabouraud media and defined concentra-
tions of ergosterol or cholesterol, C. albicans showed diminished
susceptibility that was dose-dependent upon 1, but inversely
dependent upon the sterol concentration.9 Conversely, the activity
of miconazole, exhibited no ergosterol-dependence.9b Interestingly,
In 1990 we re-isolated 1 and 2, together with five additional
homologs, bengazoles C–G (3a–e), from Jaspis sp. collected on the
Great Barrier Reef,2 during a bioassay-guided screening of marine
organisms for antifungal activity and found both compounds
exhibited exceptionally potent in vitro activity against Candida
albicans (e.g., MIC 1 l
g/mL, C. albicans ATCC 14503),3 a pathogenic
fungus that is responsible for systemic infections in immunocom-
promised individuals, including AIDS patients. The absolute stereo-
structure of 1 was determined using a combination of chemical
degradation, synthesis of model compounds and comparisons of
the CD spectra of perbenzoyl derivatives of both models and the
natural product.2 Only total syntheses of bengazoles have been re-
ported; bengazole A (1) by our group in 19994 and, more recently,
1 and 2 by Ley,5 and ‘bengazole Z’ (4) by Shioiri and co-workers.6
The bis-oxazole, digonazole from Jaspis digonoxea and its homo-
logs,1b lack the C10 acyloxy group of 1–3,7 and have not been made
or evaluated.
OH OH OH
N
N
N
O
CH3
2
6
N
N
OH
OH
O
10
N
N
OR
N
F
The mechanism of action of 1 is unknown and only limited of
structure–activity relationships (SAR) are available.7 The clinically
important ‘azole’ antifungal drugs (e.g., fluconazole (5), miconazole
F
1: R = (C=O)C10H20-n-Pr
5
2: R = (C=O)C10H20-i-Bu, C10H22-sec-Bu (5:1)
3a: R = (C=O)C10H20-Et
3b: R = (C=O)C10H20-i-Pr
3c: R = (C=O)C10H20-n-Bu
3d: R = (C=O)C11H22-sec-Bu
3e: R = (C=O)C11H22-n-Bu
4: R = H
O
* Corresponding author. Tel.: +1 858 534 7115; fax: +1 858 822 0386.
E-mail address: tmolinski@ucsd.edu (T.F. Molinski).
Present address: CSIRO Molecular and Health Technologies, Clayton, Vic 3169,
Australia.
N
6
à
Present address: Novartis Biosciences, Emeryville, CA, USA.
Figure 1. Bengazoles A–G (1–3), ÀZ (4), fluconazole (5) and oxazole (6).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.069

R. J. Mulder et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2928–2930
2929
incorporation of cholesterol or synthetic ent-cholesterol9c in the
alcohols
MeS
OH
R1
media elicited dose-dependent, differential suppression of suscep-
tibility of C. albicans towards AmB, but no enantiospecific differ-
ences were observed with (–)- and (+)-cholesterol and 2.9a
Here, we report the synthesis of new bengazole A analogs and
an advanced SAR study of the new and known compounds that re-
veal the importance of structural features in 1 for anti-Candida
activity; a long-hydrophobic side-chain, the polyol side chain,
and at least one oxazole in the heterocyclic core.
The fatty acyl side chain of 1 is important for antifungal activity.
We observed that 1 was prone to spontaneous solvolysis in the
presence of protic solvents, or more rapidly, by ammoniolysis
(NH3, MeOH).2 The des-acyl product, ‘bengazole Z’ (4),10 showed
no anti-Candida activity in disk diffusion assays.
Ra-Ni
t-BuLi, TMEDA
O
O
MeS
THF, -78 ºC,
N
N
RCH=O or R(C=O)Cl
14 R1 =
17
OH
R1
a
b
c
d
e
f
Ph
O
O
p-MeO-Ph
p-Br-Ph
2-naphth.
2-furyl
X
n-C5H11
S
n-C4H9
N
N
O
16
15
g
t-Bu
X =
O
O
ketones
OH
O
OTBS
CH3
O
O
R2
6
R1
In order to examine the role of the heterocyclic core of 1, ana-
logs were prepared with replacement of the polyol side-chain with
a simple 1-hydroxy-1-butyl group (the parent, 1,3-oxazole (6) is
too volatile, bp 69–70 °C, for bioassay).
N
O
N
20a: C-6-β-OH
14h R2 = SMe
15h R2 = H
20b: C-6-α-OH
Compounds 7a–c were prepared by a variant of the ‘oxazole
grafting’ approach employed for the synthesis of 1 (Scheme
1).4,11 Aldehyde 8 (prepared by DIBAL reduction of ethyl oxazole-
4-carboxylate) was treated with n-PrMgBr and the product 9 was
protected as the silyl ether 10 (TBSCl, imidazole, THF). Deprotona-
tion of 10 using the Vedejs’ protocol12 (i: À78 °C, THF/hexanes, ii:
BH3ÁTHF), followed by separate additions of three aldehydes (oxa-
zole-5-carboxaldehyde, 11a,13 furfural, 11b and benzaldehyde,
11c), gave 2,4-substituted oxazole adducts 12a–c.14 Diastereo-
meric mixtures of the secondary carbinols 12a–c were acylated
in good yield (myristoyl chloride, Et3N, DMAP) to give the long-
chain esters 13a–c that were deprotected (TBAF, THF) to afford
bengazole A analogs 7a–c. This small library, 7a–c, 12a–c and
13a–c, was tested in paper disk diffusion assays against Candida
albicans (96–489, a patient clinical isolate, and ATCC 14503), C.
glabrata and C. krusei. To our surprise, none were active at doses
esters
O(CO)n-C13H27
R1
n-C13H27COOH
EDCI,
OH
R1
O
R2
N
O
R2
DMAP, CH2Cl2
N
18 R2= SMe
19 R2= H
14 R2= SMe
15 R2= H
Scheme 2. 2-Methylthio-, 5- and 2,5-disubstituted oxazoles. See Ref. 15 for 14a–g,
and 15a, d, g, Ref. 4 for 18a:18b, and Ref. 15b for 16.
pounds were found to be less potent that 1 or AmB. The most ac-
tive compounds were 14c (9–17 mm zones of inhibition) and
ketones 14h (8–12 mm) and 15h (9–12 mm). 2-Methylthio-5-
oxazolyl analog 14a appeared to be slightly less active than its
5-monosubstituted counterpart 15a. Surprisingly, myristate esters
18a,c,g and 19a,g lost activity compared to their active parent alco-
hols, suggesting the activities of the compounds were limited by
solubility. This contrasts with 1 where the fatty acyl chain is re-
quired for activity. Intermediates 20a and 20b, employed in the
synthesis of 1,4 (Table 1) showed modest activity (9–10 mm)
of up to 300 lg/disk.
In order to determine the properties of mono-oxazole analogs of
1, selected 5- and 2,5-substituted oxazoles (Scheme 2) were tested
in disk diffusion assays against five species of Candida including
fluconazole-resistant C. glabrata, C. krusei and C. albicans (Table 1).
The preparation of alcohols 14a–g, 15a, d, g, ketones 14h and
15h, and 2-methylthio-5-butyl-oxazole (16), starting with 2-meth-
ylthiooxazole (17),16 have been reported previously15 and summa-
rized in Scheme 2. Selected alcohols 14 and 15 were O-acylated
(EDCI, DMAP, n-C13H27COOH) group to give the new myristate
esters 18 and 19. When assayed against fungi (Table 1), all com-
Table 1
Anti-Candida activity of analogs of 1: disk diffusion assays
Zones of inhibition @ 300
l
g/disk (mm)a
Compd
C. albicansc
C. glabrata
C. krusei
C. albicansd,e
C.albicanse
1b
50
10
8
13
7.5
8
8
0
8
10
10
0
9
8
0
0
10
8
22
15
0
7.5
10
0
7.5
7
0
7
0
0
0
10
0
0
0
0
35
9
0
15
9
50
9
14a
14b
14c
14d
14e
14f
14g
14h
15a
15d
15g
15h
16
0
12
8
7.5
17
9
9
n-BuLi,
O
OR
6.5
7.5
0
0
11
9
7.5
0
8
7
0
n-PrMgBr
BF3•THF, THF/hex,
0
0
0
8
7
0
N
O
N
O
THF, Et2O, rt
89%
-78 ºC, Ar-CH=O (11)
28-75%
10
12
8
12
12
8
9
R = H
TBSCl
8
imid, CH2Cl2,
96%
10 R = TBS
0
0
OR1
n-Pr
12
0
0
—
—
0
N
O
O
O
Ar
18a,c,g
19a,g
20a
20b
AmBf
a Ar =
c Ar = Ph
b Ar =
0
9
0
15
0
0
0
0
0
0
R2O
N
0
19
12
23
n-C13H27COCl
Et3N, DMAP
12: R1 = TBS, R2 = H
a
Sabouraud agar.’–‘, not tested.
10 lg/disk.
ATCC 14503.
96–489, patient isolate, Ref. 9b.
CDFR1, fluconazole-resistant (MIC >32
13: R1 = TBS, R2 = (C=O)n-C13H27
7: R1 = H, R2 = (C=O)n-C13H27
b
c
d
e
f
69
-
89%
TBAF, THF
l
g/mL), see Ref. 9b.
AmB, amphotericin B (5 lg/disk).
Scheme 1. Synthesis of truncated 2,4-disubstitued oxazole analogs of 1.

2930
R. J. Mulder et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2928–2930
Table 2
Partial recovery of susceptibility of Candida strains was ob-
served in some 5-monosubstituted oxazole analogs. These findings
will guide further development of antifungal analogs of 1, particu-
larly modifications of the lipophilic acyl and hydrophilic polyol
side-chains.17
Anti-Candida activity of analogs of 1: minimum inhibitory concentrations (MIC’s)a
Compd
C. albicansc
C. kruseib,d
C. albicanse
1
1
—
1
14c
14d
15d
18c
18d
19d
AmB
4
4
8
8
16
8
>64
>64
>64
>64
0.50
>64
>64
>64
>64
0.25
32
>64
>64
>64
0.50
Acknowledgements
We thank the Great Barrier Reef Authority for the permit to col-
lect of Jaspis sp. This work was funded by the NIH (AI 039987).
a
RPMI media, 37 °C, 24 h incubation.
48 h incubation.
See caption in Table 1 for strain identification.
b
References and notes
c–e
1. (a) Adamczeski, M.; Quiñoà, E.; Crews, P. J. Am. Chem. Soc. 1988, 110, 1598; (b)
Rodríguez, J.; Nieto, R. M.; Crews, P. J. Nat. Prod. 1993, 56, 2034.
2. Searle, P. A.; Richter, R. K.; Molinski, T. F. J. Org. Chem. 1996, 61, 4073.
although diastereomer 20a, matching the configuration of 1, was
slightly more active with a susceptibility profile similar to that of 1.
The most active compounds retained antifungal activity when
tested in microbroth dilution assays (Table 2, for example 14c,
MIC = 4 lg/mL against C. albicans and C. krusei), but again, myris-
tate esters 18c, d and 19d were inactive.
3. Bengazole
A (1) shows no antibacterial activity against gram-positive
(Escherechia coli, Staphylococcus aureus) or gram-negative strains
(Pseudomonas aeruginosa).
4. (a) Mulder, R. J.; Shafer, C. M.; Molinski, T. F. J. Org. Chem. 1999, 64, 4995; (b)
Shafer, C. M.; Molinski, T. F. Tetrahedron Lett. 1998, 39, 2903.
5. (a) Bull, J. A.; Balskus, E. P.; Horan, R. A. J.; Langner, M.; Ley, S. V. Angew. Chem.,
Intl. Ed. 2006, 45, 6714; (b) Bull, J. A.; Balskus, E. P.; Horan, R. A. J.; Langner, M.;
Ley, S. V. Chem. Eur. J. 2007, 13, 5515.
Clearly, the antifungal activity of 1 is not accounted for by sim-
ple analogs with one or two oxazole rings alone. At the very least,
activity was correlated with the presence of a 5-monosubstituted
oxazole (Scheme 2). 2,4-Disubstituted oxazolyl carbinol analogs,
including their long-chain esters, were inactive. Interestingly, the
closest heterocyclic analog, 7a, to bengazole A (1), was inactive
suggesting abrogration of biological activity upon replacement of
the hydrophilic polyol side chain of 1 with a truncated n-propyl
carbinol. The fatty acyloxy chain at C-10 potentiates activity only
when the native polyol chain is present (e.g., 1) since highly lipo-
philic 18a,c,g and 19a,g are inactive.
Although lipophilicity plays a role in activity of bengazole ana-
logs, not all the effects can be explained by simple logP consider-
ations, and subtle effects appear to be conferred by both the
hydrophilic and fatty acyl chains.
In summary, several analogs of bengazole A (1) were prepared,
containing one or two 1,3-oxazole rings, and modified side chains.
Good activity was observed in some compounds but none were
more potent than 1.
6. Chittari, P.; Hamada, Y.; Shioiri, T. Heterocycles 2003, 59, 465.
7. Rudi, A.; Kashman, Y.; Benayahu, Y.; Schleyer, M. J. Nat. Prod. 1994, 57, 829.
8. Trinci, A. P. J.; Ryley, J. F. (Eds.), Mode of Action of Antifungal Agents, Cambridge
University Press, Cambridge, 1984.
9. (a) Richter, R. K.; Mickus, D. E.; Rychnovsky, S. D.; Molinski, T. F. Bioorg. Med.
Chem. Lett. 2004, 14, 115; (b) Molinski, T. F. J. Nat. Prod. 1993, 56, 1; (c) Mickus,
D. E.; Levitt, D. G.; Rychnovsky, S. D. J. Am. Chem. Soc. 1992, 114, 359.
10. Bengazole Z, a name coined by P. Crews for des-acyl 1, was found in methanol
1b
extracts of Jaspis sp. along with 1 and 2. See Ref.
.
11. Hodges, J. C.; Patt, W. C.; Connolly, C. J. J. Org. Chem. 1991, 56, 449.
12. Vedejs, E.; Monahan, S. D. J. Org. Chem. 1996, 61, 5192.
13. (a) Kende, A. S.; Kawamura, K.; DeVita, R. J. J. Am. Chem. Soc. 1990, 112, 4070;
(b) Schöllkopf, U.; Schröder, R. Angew. Chem., Int. Ed. Engl. 1971, 10, 333.
14. Generally, the diastereomers (drꢀ1:1, 1H NMR) we inseparable by TLC; no
attempt was made to purify the isomers. All compounds gave satisfactory
spectroscopic data.
15. (a) Shafer, C. M.; Molinski, T. F. J. Org. Chem. 1998, 63, 551; (b) Shafer, C. M.
Ph.D. Thesis, University of California, Davis, 1998.
16. Lacasse, G.; Muchowski, J. M. Can. J. Chem. 1972, 50, 3082.
17.
A recent report describes synthesis of ‘bengazole analogs’ and their
antimicrobial activity. The compounds are actually based on 2,5-disubstitued
furans; none of the analogs contain an oxazole ring: Kraus, J.; Kalkbrenner, S.;
Schuster, A.; Obainoke, A.; Bracher, F. Turk. J. Chem. 2008, 32, 125.