Synthesis and evaluation of novel azoles as potent antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2013.11.037

Using a rational approach to the design of antifungal agents, a series of azole agents with 1,3,4-oxadiazole side chains were designed and synthesized. The results of preliminary in vitro antifungal tests with eight human pathogenic compounds showed that all of the title compounds exhibited excellent activities against all of the tested fungi except Aspergillus fumigatus. Compounds 11e and 11f were found to be the most effective, with a minimum inhibitory concentration of 0.0039 μg/mL, followed by voriconazole, which has a MIC of 0.0625 μg/mL. The 1,3,4-oxadiazole side chain is not the major contributor but plays a role in eliciting the observed antifungal activity.

Full text of DOI:10.1016/j.bmcl.2013.11.037

Accepted Manuscript
Synthesis and evaluation of novel azoles as potent antifungal agents
Liangjing Li, Hao Ding, Baogang Wang, Shichong Yu, Yan Zou, Xiaoyun Chai,
Qiuye Wu
PII:
S0960-894X(13)01316-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.11.037
BMCL 21064
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 July 2013
23 October 2013
16 November 2013
Please cite this article as: Li, L., Ding, H., Wang, B., Yu, S., Zou, Y., Chai, X., Wu, Q., Synthesis and evaluation
of novel azoles as potent antifungal agents, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/
10.1016/j.bmcl.2013.11.037
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Synthesis and evaluation of
novel azoles as potent antifungal agents
Liangjing Li a,b,†, Hao Ding a,†, Baogang Wanga, Shichong Yua, Yan Zoua, Xiaoyun
Chaia,*, Qiuye Wua,*
aDepartment of Organic Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, China
bCadet Company 2nd, Faculty of Naval Medicine, Second Military Medical University,
800 Xiangyin Road, Shanghai 200433, China
Abstract: Using a rational approach to the design of antifungal agents, a series of
azole agents with 1,3,4-oxadiazole side chains were designed and synthesised. The
results of preliminary in vitro antifungal tests with eight human pathogenic
compounds showed that all of the title compounds exhibited excellent activities
against all of the tested fungi except Aspergillus fumigatus. Compounds 11e and 11f
were found to be the most effective, with a minimum inhibitory concentration of
0.0039 µg/mL, followed by voriconazole, which has a MIC of 0.0625 µg/mL. The
1,3,4-oxadiazole side chain is not the major contributor but plays a role in eliciting the
observed antifungal activity.
Fungal infections, which are an increasing healthcare concern worldwide, are
associated with significant costs, morbidity, and mortality.1-3 Although the number
and types of antifungal drugs have markedly increased, only a few of the identified
drugs can be used in the clinic.4 The commonly used antifungal agents are azoles,
polyenes, and echinocandins.3,5 Among these antifungal drugs, the gold standard for
*Corresponding authors. Tel: +86 21 81871228 (Xiaoyun Chai), Tel./fax: +86 21 81871225 (Qiuye Wu).
E-mail addresses: chaixy1207@163.com (Xiaoyun Chai), wuqy640319@163.com (Qiuye Wu).
† These two authors contributed equally to this work.
2

the treatment of fungal infections is Amphotericin B (polyenes), which has been
widely used in clinical practice and is effective against a large spectrum of fungi;
however, its adverse side effects, such as infusional toxicity, nephrotoxicity, and low
blood potassium levels, are serious.6,7 Recently, resistance to echinocandin drugs for
the treatment of fungal infections has increasingly emerged. Moreover, the expensive
price of echinocandins also limits their application.8,9 The above mentioned
limitations emphasise the pressing need for novel antifungal agents. Azoles play a
leading role in the treatment of invasive fungal infections. Based on their spectrum of
activity, safety profiles, costs, potential toxicity, and some other aspects, azoles,
which are the most commonly used antifungal drugs, present significant superiority
and have broad development prospects. In addition, it is worthwhile for us to conduct
further research.
Azoles target ergosterol biosynthesis through the inhibition of the fungal
cytochrome P450 14α-demethylase (CYP51) via a mechanism in which the N-4 of the
azole binds to the sixth coordination of the heme iron atom of the porphyrin in the
substrate binding site of the enzyme. Thereby, ergosterol is depleted, and this
depletion leads to the inhibition of either fungal cell death or growth.3,10-12 Based on
the structure of the active site of CYP51 and the results of structure-activity
relationship studies, the 1,2,4-triazole ring and the 2,4-difluorophenyl group and
hydroxyl groups are essential for this type of antifungal drugs. In addition, the side
chain is also very important to achieve improved activities.13
Based on the abovementioned facts, we designed and synthesised a series of
1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{4-[3-((5-substituted phenyl-1,3,4
-oxadiazol-2-yl)thio)propyl]piperazin-1-yl}-propan-2-ols, which retained the classic
skeleton of azole agents, and altered the side chains to find potent systemic antifungal
agents with higher antifungal activity.
The synthetic route designed for the intermediates 7a-t is outlined in Scheme 1.
Compounds 7a-t were synthesised from substituted benzoic acid through six steps.14
Various substituted benzoic acids (1a-t) were treated with SOCl2 to obtain
compounds 2a-t, which were reacted with CH3OH and EtN3 in CH2Cl2 at 0°C to yield
3

compounds 3a-t. Compounds 4a-t were prepared by the reaction of compounds 3a-t
with hydrazine hydrate in CH3OH under reflux conditions for approximately 1 s.
Subsequently, compounds 5a-t were obtained by the reaction of compounds 4a-t with
CS2 and KOH in CH3OH. Compounds 6a-t were obtained by the cyclization reaction
of compounds 5a-t in the presence of HCl at 0-5°C. Finally, treatment of compounds
6a-t with 1,3-dibromopropane in the presence of K2CO3 in CH3CN afforded
compounds 7a-t in moderate yields.
<Scheme 1.>
The chemical synthesis of the target compounds is outlined in Scheme 2. The
coupling reaction between compound 8 and N-Boc-piperazine afforded compound 9,
which was stirred with CH3COOF in CH2Cl2 to yield the key intermediate 10. The
target compounds 11a–t were synthesised through the treatment of compound 10 and
compounds 7a-t in the presence of K2CO3, KI, and CH3CN at 80°C. All of the novel
compounds described above were characterised by ESI-MS and NMR spectroscopic
analyses.
<Scheme 2.>
The in vitro minimal inhibitory concentrations (MICs) of the compounds were
determined through the micro-broth dilution method in 96-well micro test plates
according to the methods defined by the National Committee for Clinical Laboratory
Standards (NCCLS).15 The MIC80 was defined as the first well with an approximate
80% reduction in growth compared to the growth observed in the drug-free well. For
the assays, the title compounds to be tested were dissolved in dimethyl sulfoxide
(DMSO), serially diluted in growth medium, inoculated, and incubated at 35°C. The
growth MIC was determined at 24 h for Candida albicans and at 72 h for
Cryptococcus neoformans. The data presented are the means of three replicate tests
performed with each antifungal agent. All of our susceptibility tests were performed
three times for each antifungal agent.
<Table 1.>
The in vitro antifungal activities of the newly synthesised azole derivatives
11a-11t and the reference drugs fluconazole (FCZ), ketoconazole (KCZ), itraconazole
4

(ICZ), and voriconazole (VCZ) against eight common clinical fungi are reported in
Table 1 . In general, the newly synthesised azole derivatives showed good antifungal
activities except against Aspergillus fumigatus. The target compounds showed
excellent activity against Candida albicans., which has a worldwide distribution and
is the most common cause of life-threatening fungal infections. Compounds 11c, 11e,
11f, and 11g (with MIC80 values of 0.0039 µg/mL) are 64-fold more potent than ICZ
(with an MIC80 value of 0.25 µg/mL) and 16-fold more potent than VCZ (with an
MIC80 value of 0.0625 µg/mL). Compound 11i showed the highest activity against
Candida glabrata (with an MIC80 value of 0.0039 µg/mL) and was 16-, 32-, 256-, and
2051-fold more potent than VCZ, ICZ, FCZ, and KCZ, respectively. As a rule, the
compounds with a halogen group (11b-11j) were found to be more potent than those
compounds with alkyl, alkoxy, nitro, and cyano groups (11k-11t). We hypothesised
that this decrease in activity could be ascribed to the electronic effects and the spatial
configuration of the substituent.
This study was undertaken to evaluate the effects on the antifungal properties of
novel azoles containing1,3,4-oxadiazole side chains. The data shown in Table 1
indicate that almost all of the target compounds have strong activities against eight
common clinical fungi, with the exception of Aspergillus fumigatus. Compared with
the intermediate 10, the title compounds show markedly improved antifungal activity.
This finding demonstrates that the 1,3,4-oxadiazole side chains are responsible for the
antifungal activity of the title compounds and that the 1,3,4-oxadiazole side chain is
an ideal supplemental group that presumably contributes to enzyme binding. In this
context, the antifungal activity data presented in this manuscript suggest a potential
role of the new compounds for the treatment of Candida infections and contribute to
the active and challenging research on the development of novel antifungal agents.
Compared with some first-line drugs, this series of novel antifungal compounds
demonstrated improved antifungal activity in in vitro studies; thus, it is worthwhile
for us to undertake further studies to identify the best compound. This study has laid a
solid foundation for further lead compound optimisation through systematic chemical
modifications of this class of compounds.
5

Acknowledgments
This work was supported by the National Natural Science Foundation of China
(Nos. 20972188), a grant from Science & Technology Commission of Shanghai (Nos.
09dZ1976700), and by Shanghai Leading Academic Discipline Project Number:
B906.
References and notes
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of Investigators. JAMA. 2009, 302, 2323.
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J. Med. Chem. 2011, 46, 101.
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5. Kathiravan, M. K.; Salake, A. B.; Chothe, A. S.; Dudhe, P. B.; Watode, R.
P.; Mukta, M. S.; Gadhwe, S. Bioorg Med Chem. 2012, 20, 5678.
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7. Laniado-Laborín, R.; Cabrales-Vargas, M. N. Rev Iberoam Micol. 2009, 26, 223.
8. Garcia-Effron, G.; Kontoyiannis, D. P.; Lewis, R. E.; Perlin, D. S. Antimicrob.
Agents Chemother. 2008, 52, 4181.
9. Pfaller, M. A.; Diekema, D. J.; Ostrosky-Zeichner, L.; Rex, J. H.; Alexander, B.
D.; Andes, D.; Brown, S. D.; Chaturvedi, V.; Ghannoum, M. A.; Knapp, C.
C.; Sheehan, D. J.; Walsh, T. J. J. Clin. Microbiol. 2008, 46, 2620.
10. Mohr, J.; Johnson, M.; Cooper, T.; Lewis, J. S.; Ostrosky-Zeichner, L.
Pharmacotherapy, 2008, 28, 614.
11. Hitchcock, C. A.; Dickinson, K.; Brown, S. B.; Evans, E. G.; Adams, D. J.
Biochem.J. 1990, 266, 475.
12. Aher, N. G.; Pore, V. S.; Mishra, N. N.; Kumar, A.; Shukla, P. K.; Sharma,
6

A.; Bhat, M. K. Bioorg Med Chem Lett. 2009, 19, 759.
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Sun, Q. Y. Bioorg Med Chem Lett. 2011, 21, 759.
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D. Y. Bioorg Med Chem. 2008, 16, 3632.
15. National Committee for Clinical Laboratory Standards, Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Yeasts Approved standard.
Document M27-A2, National Committee for Clinical Laboratory Standards,
Wayne, PA, 2002.
7

Scheme 1. Synthetic route to intermediates 7a-t. Conditions and reagents: (a) SOCl2, reflux, 5h; (b)
CH3OH, EtN3, CH2Cl2, 0℃; (c) NH2NH2·H2O, CH3OH, reflux, 5h; (d) CS2, CH3OH, KOH,
rt-reflux; (e) HCl, 0-5℃; (f) BrCH2CH2CH2Br, K2CO3, CH3CN, rt, 8h.
Scheme 2. Synthetic route to the target compounds 11a-t. Conditions and reagents: (a)
N-Boc-piperazine, Et3N, C2H5OH, reflux, 8h; (b) CF3COOH, CH2Cl2, rt; (c) compounds 7a-t,
K2CO3, KI, CH3CN, 80℃.
8

Table 1. Antifungal activities of the title compounds in vitro (MIC80, µg/mL)
Compd.
R
C.alb
Y0109 SC5314
0.0156 0.0156 0.0625 0.0625 0.0625 0.25
C. alb
C. par C. neo C. gla
T. rub
A. fum M. gyp
H
>64
1
11a
11b
11c
11d
11e
11f
2-F
0.0156 0.0625 0.25
0.0625 0.0039 0.25
0.0625 0.0625 0.0625 >64
0.0625 0.0625 0.0625 >64
1
3-F
1
4-F
0.25
0.0156 0.25
0.25
0.25
0.25
0.25
0.0625 >64
0.25 >64
0.0625 >64
4
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
0.0039 0.0039 0.25
0.0039 0.0039 0.25
4
0.0625 0.25
1
0.0625 0.0039 0.0625 0.0625 0.0625 0.25
0.0625 0.0156 0.0625 0.0625 0.0625 0.25
>64
>64
>64
>64
>64
>64
>64
>64
4
11g
11h
11i
0.25
1
0.0625 0.0625 0.25
0.25
0.0039 0.25
0.0156 0.25
0.0625 0.0625 0.0625 0.25
0.25
0.25
1
11j
2-CH3 0.25
3-CH3 0.25
4-CH3 0.125
2-OCH3 0.125
3-OCH3 0.25
4-OCH3 0. 25
2-NO2 0.25
4-NO2 0.125
0.25
0.125
0.25
0.25
0.25
0.25
0.125
0.25
0.125
0.125
0.5
0.125
4
0.125
1
0.125
1
0.25
1
11k
11l
0.025
16
1
0.25
1
1
1
11m
11n
11o
11p
11q
11r
11s
4
0.25
16
0.25
0.25
0.25
1
0.25
0.25
0.125
0.125
0.25
0. 25
2
0.25
0.25
0.125
0.25
0.25
0.25
4
0.25
0.25
0.25
0.125
0.125
0.25
0.25
8
0.0625 >64
0.25
0.25
1
>64
>64
>64
>64
>64
>64
2
2-CN
0.125
0.125
1
0.25
0.25
1
1
3-CN
1
11t
-
-
-
-
-
16
2
10
8
4
8
4
8
KCZ
FCZ
ICZ
VCZ
0.5
0.5
1
1
1
>64
0.5
8
8
0.25
0.25
0.125
0.125
0.125
1
1
0.0625 0.0625 0.0625 0.0156 0.0625 0.25
0.0625 0.125
Abbreviations: C. alb., Candida albicans; C. par., Candida parapsilosis; C. neo., Cryptococcus
neoformans; C. gla., Candida glabrata; T. rub., Trichophyton rubrum; A.fum., Aspergillus
fumigatus; M. gyp., Microsporum gypseum.
9

Graphical abstract
Synthesis and evaluation of
novel azoles as potent antifungal agents
Liangjing Li a,b,†, Hao Dinga,†, Baogang Wanga, Shichong Yua, Yan Zoua, Xiaoyun
Chaia,*, Qiuye Wua,*
aDepartment of Organic Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, China
bCadet Company 2nd, Faculty of Naval Medicine, Second Military Medical University,
800 Xiangyin Road, Shanghai 200433, China
Twenty novel azoles have been designed and synthesized as potential antifungal agents. All
the title compounds exhibited excellent activity with a broad spectrum of antifungal activity.
*Corresponding authors. Tel: +86 21 81871228 (Xiaoyun Chai), Tel./fax: +86 21 81871225 (Qiuye Wu).
E-mail addresses: chaixy1207@163.com (Xiaoyun Chai), wuqy640319@163.com (Qiuye Wu).
† These two authors contributed equally to this work.
10