New azoles with antifungal activity: Design, synthesis, and molecular docking

Article abstract of DOI:10.1016/j.bmcl.2010.12.006

In order to search for many target compounds with excellent activities, a series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluoro-phenyl)-3-[(4-substituted phenyl)-piperazin-1-yl]-propan-2-ols were designed, synthesized, and evaluated as antifungal agents. Re

Full text of DOI:10.1016/j.bmcl.2010.12.006

Bioorganic & Medicinal Chemistry Letters 21 (2011) 686–689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New azoles with antifungal activity: Design, synthesis, and molecular docking
Xiaoyun Chai a, Jun Zhang c, Yongbing Cao b, Yan Zou a, Qiuye Wu a, , Dazhi Zhang a,
⇑
Yuanying Jiang b, Qingyan Sun a,
⇑
a Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, People’s Republic of China
b Drug Research Center, School of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, People’s Republic of China
c Overseas Education Faculty of the Second Military Medical University, Xiangyin Road 800, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to search for many target compounds with excellent activities, a series of 1-(1H-1,2,4-triazol-1-
yl)-2-(2,4-difluoro-phenyl)-3-[(4-substituted phenyl)-piperazin-1-yl]-propan-2-ols were designed,
synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against eight
human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with
broad spectrum. Moreover, a molecular model for the binding between 5a and the active site of CACYP51
was provided based on the computational docking results.
Received 8 November 2010
Revised 29 November 2010
Accepted 1 December 2010
Available online 7 December 2010
Keywords:
Azole
Ó 2010 Elsevier Ltd. All rights reserved.
Synthesis
Antifungal activity
CYP51
Molecular docking
Fungal infections pose a continuous and serious threat to human
health and life especially to immunocompromised patients.1–3
Many fungal infections are caused by opportunistic pathogens that
may be endogenous (Candida infections) or acquired from the envi-
ronment ( Cryptococcus, Aspergillus infections). However, besides
these known fungal species, new emerging fungal pathogens appear
every year as the cause of morbidity and life-threatening infections
in the immunocompromised hosts.1,4
Nowadays, numerous antifungal drugs with various structures
and scaffolds spring up.5 However, their clinical uses have been
limited by the emergence of drug resistance, high risk of toxicity,
insufficiencies in their antifungal activity and undesirable side ef-
fects. Hence, there is still a need to develop and extend the safe
and efficient chemotherapeutic agents with potent antifungal
activities.6
Researches indicated that the structurally and functionally
important regions, such as the heme group, the hydrophilic H-bond-
ing region, the substrate access channel, and the active site have
been recognized accurately. The binding mode of azoles with CA-
CYP51 has been investigated by flexible molecular docking.10–12
The molecular modeling, which gives the utilization of structural
information of fungal CYP51s can accelerate the discovery of novel
antifungal agents. In our letter, we used the strategy of structure-
based rational drug design and find a series of new azoles with
excellent in vitro antifungal activity and broad antifungal spectrum.
The general synthetic methodology for the preparation of title
compounds
1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluoro-phenyl)-3-
[(4-substitutedphenyl)-piperazin-1-yl]-propan-2-ols (5a–q, 6a–q)
is outlined in Scheme 1. As a key intermediate of our designed tri-
azole antifungals, the oxirane compound 1 was synthesized by the
reported procedure.13 And compound 2 were synthesized accord-
ing to the literature.14 The title compound 3 was synthesized by
ring-open reaction of oxirane 1 with compound 2. The good yield
was obtained when the reaction was performed in a protic solvent
ethanol in the presence of triethylamine as a base at 80 °C. Then
the nitro group on the phenyl ring of compound 3 was reduced
to an amino group in the presence of Ranney Ni and hydrazine hy-
drate. In the presence of DMAP (4-dimethylaminopyridine) and
EDCI (1-ethyl-3-(3-dimethylaminopropyl) carbodii-mide HCl) in
dichloromethane at room temperature, the aniline 4 was converted
to title compounds by reacting with various substituted cinnamic
acids. All the new compounds (5a–q, 6a–q) described above were
characterized by IR, ESI, and NMR spectroscopic analysis.15
One of the most common classes of antifungal agents is azoles.
For over a decade, azoles have been a mainstay of the antifungal
armamentarium. Azoles inhibit the synthesis of ergosterol, the
bulk sterol in fungal membranes, by binding to the heme cofactor
located in the active site of the cytochrome P450 14a-demethylase
(CYP51).7 Unfortunately, the broad use of azoles has led to
development of severe resistance, which significantly reduced
their efficacy.8,9 So the discovery of novel and potent antifungal
azoles is the best way to overcome resistance and develop effective
therapies.
⇑
Corresponding authors. Tel.: +86 21 81871228 15; fax: +86 21 81871225 (Q.S.).
E-mail addresses: wuqy6439@sohu.com (Q. Wu), sqy_2000@163.com (Q. Sun).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.006

X. Chai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 686–689
687
Scheme 1. Conditions: (a) CH3CH2OH, Et3N, 80 °C, 5 h; (b) Ranney Ni, NH2NH2ꢀH2O, CH3CH2OH, 80 °C, 3.5 h; (c) substituted cinnamic acids, DMAP, EDCI, CH2Cl2, 8 h.
The in vitro minimal inhibitory concentrations (MICs) of the
compounds were determined by the micro-broth dilution method
in 96-well microtestplates according to the methods defined by
the National Committee for Clinical Laboratory Standards
(NCCLS).16 The MIC80 was defined as the first well with an approx-
imate 80% reduction in growth compared to the growth of the drug-
free well. For assays, the title compounds to be tested were dis-
solved in dimethyl sulfoxide (DMSO), serially diluted in growth
medium, inoculated and incubated at 35 °C. Growth MIC was deter-
mined at 24 h for Candida albicans and at 72 h for Cryptococcus
neoformans. Fluconazole (FLC), itraconazole (ICZ), and voriconazole
(VCZ) served as the positive control were obtained from their
respective manufacturers. The results of assays are summarized
in Table 1. The data points from the mean of replicates. All of our
susceptibility tests were performed three times by each antifungal
agent.
Table 1
Antifungal activities of the title compounds in vitro (MIC80, lg/mL)
Compound
R
R0
C. albicans
C. parapsilosis
C. tropicalis
C. neoformans
T. rubrum
F. com.
M. gypseum
A. fumigatus
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
3,4-Cl
3-Br
0.0156
0.0039
0.0156
0.0625
0.0039
0.0625
0.0625
0.0156
0.0625
0.0625
0.0625
0.0156
0.0156
0.0156
0.0156
0.0625
0.0156
0.25
0.25
0.25
0.25
1
1
0.25
1
1
0.0625
0.0625
0.25
0.25
0.25
1
0.25
0.0625
0.25
0.25
0.25
0.0625
0.25
0.25
0.0625
1
0.25
0.0625
0.0625
0.0156
0.0156
0.25
0.25
0.0156
0.25
0.25
0.0625
1
0.0625
0.0156
0.25
0.25
0.0625
0.25
0.25
0.0625
0.25
0.25
0.25
0.0625
0.0625
0.0625
0.25
0.25
0.25
0.0625
0.0625
0.25
0.0156
0.25
0.0625
0.0625
0.0625
0.0625
0.0156
0.0625
1
0.25
0.0625
0.25
0.25
0.25
1
1
0.25
1
0.25
1
0.25
0.25
1
0.25
1
1
0.0156
0.25
0.25
0.25
1
0.25
0.25
1
0.0156
0.0156
4
1
1
0.0039
0.25
0.25
0.0039
0.0156
1
0.0625
1
1
1
1
64
4
0.25
1
1
16
1
4
4
1
16
16
0.25
1
0.25
0.25
1
0.25
16
>64
4
0.0625
4
64
64
4
16
4
64
4
>64
>64
4
4
>64
1
4
4
1
4
1
1
4
4
0.0625
0.0156
0.0625
0.0156
0.0156
0.25
0.25
1
0.25
0.25
0.25
1
0.25
0.25
0.0625
0.0625
0.25
0.25
0.25
0.25
0.25
0.0625
0.0625
0.25
0.25
0.0625
0.25
0.25
1
0.25
0.0156
0.0625
0.0156
0.0156
0.25
5j
5k
5l
4-Br
4-CH3
3,4-OCH3
2,5-OCH3
2-NO2
3-NO2
4-NO2
3-CN
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
3,4-Cl
3-Br
4-Br
4-CH3
3,4-OCH3
2,5-OCH3
2-NO2
3-NO2
4-NO2
3-CN
5m
5n
5o
5p
5q
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
FCZ
ICZ
VCZ
0.25
0.0625
0.0625
0.0156
1
0.0625
0.0625
0.25
0.0625
0.25
0.25
0.25
0.0625
0.25
0.0625
0.25
4
0.0625
0.25
0.0625
0.0625
4
0.25
0.0152
0.0039
H
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
1
0.25
0.25
1
1
1
0.25
0.25
0.25
0.0625
0.25
0.0039
0.0156
0.25
4
1
1
1
0.25
1
1
0.0625
0.25
1
1
0.25
1
4
>64
>64
64
>64
>64
>64
64
1
0.25
1
0.0625
0.25
1
1
0.25
0.25
64
16
0.0625
1
64
16
0.25
0.0152
1
1
0.0625
1
1
1
1
0.0625
0.25
4
0.25
0.0152
0.0039
0.0152
0.0152
>64

688
X. Chai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 686–689
Figure 1. Computed binding geometry of the new inhibitor 5a in the active site of CYP51.
In vitro antifungal activity assay (Table 1) indicates that all the
hydrophobic residues such as Tyr64, Gly65, Leu87, Leu88, Met92,
Ala117, Tyr118, Pro230, Ile231, Phe233, Val234, Leu376, His377,
Ser378, Ile379, Phe380, Met508. Furthermore, the phenyl group at-
tached to the piperazinyl of the side chain interacts with the phe-
synthesized compounds (5a–q, 6a–q) show moderate to excellent
activity against nearly all the tested fungal pathogens. The MIC80
values indicate that the series of 5a–q showed more excellent anti-
fungal activities against C. albicans than that of the 6a–q series.
Most of the compounds are more excellent than fluconazole and
nol group of Phe380 through the formation of
interaction.
p-p face-to-edge
itraconazole with their minimal inhibitory concentration (MIC80
)
In addition, all of the side chains were of the pharmacophores,
and the spatial orientations of the pharmacophores were just ori-
ented in the hydrophobic pocket. The side chains of inhibitors were
not the determinants for activity, but were very important. They
played a role in adjusting the physico-chemical properties of the
whole molecule to avoid some dissatisfying side effects and im-
prove their pharmacokinetic and pharmacodynamic behaviors.
In summary, a highly efficient and versatile synthetic method
was developed for the synthesis of new azoles. Antifungal avtivity
studies of the synthetic compounds suggest that the piperazinyl
side chain greatly enhanced the antifungal activity of these analogs
against Candida species. This observation was explainable by a
molecular model resulting from the computational docking simu-
lation, which showed that 5a could fit into the hydrophobic pocket
of CACYP51. The piperazinyl side chain of the compounds is
oriented into substrate access channel 2 (FG loop) and forms
hydrophobic and van der waals interactions with surrounding
hydrophobic residues. Effort aimed at further optimization, as well
as in-depth biological investigations, of the identified lead com-
pounds is continuing in our laboratories, and results will be
reported in due course.
values in the range of 0.0039–1 g/mL. Noticeably, the MIC value
l
of compounds 5b and 5e is 1024 times lower than that of fluconaz-
ole against C. albicans in vitro and also showed higher activities
than that of the other positive controls. Cryptococcus neoformans
has a worldwide distribution and is the most common cause of
life-threatening fungal infections. All the target compounds show
higher inhibitory activity against C. neoformans than fluconazole
and itraconazole with their MIC80 values in the range of 0.0156–
1 lg/mL. Especially, the inhibitory activity of compounds 6a, 6i,
and 6j is 64-fold higher than that of fluconazole and voriconazole.
Fluconazole is not effective against Aspergillus fumigatus, while our
compounds show moderate activity. The MIC80 values of com-
pounds 6a, 6d, 6f, 6g, 6j, and 6k against A. fumigatus are only
1 lg/mL. Moreover, the designed compounds also show good
activity against dermatophytes (Trichophyton rubrum and Micros-
porum gypseum). For example, compounds 5a and 5d (with the
MIC80 value of 0.0039
lg/mL) are 64 fold more potent against T. ru-
brum than fluconazole.
To clarify the binding mode of our synthesized compounds,
compound 5a was docked into the active site of CACYP51 by the
Builder module within InsightII 2000 software package (Fig. 1).
The docking results revealed that the compound binds to the active
site of CACYP51 through the formation of a coordination bond with
iron of heme group. The difluorophenyl group is located in the
hydrophobic binding cleft lined with Phe126, Ile304, Met306,
Gly307, and Gly308. The long side chain of the compound 5a is ori-
ented into substrate access channel 2 (FG loop) and forms hydro-
phobic and van der waals interactions with surrounding
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Nos. 30300437 and 20772153), the Eleventh Five
Year Military Medicine and Public Health Research Projects (Nos.
06MB206) and by Shanghai Leading Academic Discipline Project
Number: B906.

X. Chai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 686–689
689
10. Ji, H.; Zhang, W.; Zhou, Y.; Zhang, M.; Zhu, J.; Song, Y.; Lü, J.; Zhu, J. J. Med. Chem.
2000, 43, 2493.
Supplementary data
11. Sheng, C.; Zhang, W.; Ji, H. Chin. Chem. Lett. 2004, 15, 404.
12. Sheng, C.; Zhang, W.; Ji, H.; Zhang, M.; Song, Y.; Xu, H.; Zhu, J.; Miao, Z.; Jiang,
Q.; Yao, J.; Zhou, Y.; Lü, J. J. Med. Chem. 2006, 49, 2512.
13. Aoyama, Y.; Yoshida, Y.; Sonoda, Y.; Sato, Y. J. Biol. Chem. 1987, 262, 1239.
14. Sang, W. K.; Jung, G. L.; Eun, J. L.; Hea-Young, P. C.; Chung, Y. Y.; Dae, Y. L.;
Kyoung, R. R.; Kim, E. K. J. Comb. Chem. 2004, 6, 851.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.006.
References and notes
15. Representative analytical data for compound 5a: (E)-N-(4-{4-[2-(2,4-difluoroph-
enyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)-propyl]-piperazin-1-yl}-phenyl)-
cinna-mamide. Mp: 196–198 °C; 1H NMR (300 MHz, CDCl3) d: 6.50–7.74 (12H,
m, Ar-H), 7.79, 8.15 (2H, ss, triazole-H), 6.83, 7.52 (2H, ss, C@C–H), 4.51–4.60
(2H, dd, triazole-CH2–), 2.52–3.04 (8H, m, piperazine-H), 2.75–3.14 (2H, dd,
CH2-piperazine–); IR (KBr): 3404, 3284, 3125, 3057, 2822, 1736, 1676, 1616,
1513, 1234, 1137 cmꢁ1. ESI, calcd for C30H30F2N6O2, [M+H]+ 545.24, found
545.56.
16. 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.
1. Walsh, T. J.; Groll, A.; Hiemenz, J.; Fleming, R.; Roilides, E.; Anaissie, E. Clin.
Microbiol. Infect. 2004, 10, 48.
2. Georgopapadakou, N.; Tkacz, J. Trends Microbiol. 1995, 3, 98.
3. Nagiec, M.; Nagiec, E.; Baltisberger, J.; Wells, G.; Lester, R.; Dickson, R. J. Biol.
Chem. 1997, 272, 9809.
4. Pfaller, M. A.; Diekema, D. J. J. Clin. Microbiol. Rev. 2007, 20, 133.
5. Kleeman, A.; Engel, J.; Kutscher, B.; Reichert, D. Bioseparation 1999, 8, 336.
6. Johnson, D. S.; Li, J. J. Art Drug Synth. 2007, 71.
7. Vanden, B. H.; Willemsens, G.; Marichal, P. Crit. Rev. Microbiol. 1987, 15, 57.
8. Casalinuovo, I. A.; Di Francesco, P.; Garaci, E. Eur. Rev. Med. Pharmacol. Sci. 2004,
8, 69.
9. Hoffman, H. L.; Ernst, E. J.; Klepser, M. E. Expert Opin. Invest. Drugs 2000, 9, 593.