New triazole derivatives as antifungal agents: Synthesis via click reaction, in vitro evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.bmcl.2012.02.042

A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-substituted- 2-propanols (5a-5y) which are analogues of fluconazole, have been designed and synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition on the basis of computational docking experiments to the active site of the cytochrome P450 14α-demethylase (CYP51). The in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi. Compound 5l showed the best antifungal activities.

Full text of DOI:10.1016/j.bmcl.2012.02.042

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2959–2962
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New triazole derivatives as antifungal agents: Synthesis via click reaction,
in vitro evaluation and molecular docking studies
Yan Zou a, , Qingjie Zhao a, , Jun Liao a, Honggang Hu a, Shichong Yu a, Xiaoyun Chai a, Mingjuan Xu b,
,
⇑
Qiuye Wu a,
⇑
a Department of Organic Chemistry, College of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, People’s Republic of China
b Department of Gynecology and Obstetrics, Changhai Hospital, Second Military Medical University, 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:
A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols (5a–5y) which are
Received 13 October 2011
Revised 5 February 2012
Accepted 15 February 2012
Available online 23 February 2012
analogues of fluconazole, have been designed and synthesized via Cu(I)-catalyzed azide–alkyne cycload-
dition on the basis of computational docking experiments to the active site of the cytochrome P450 14a-
demethylase (CYP51). The in vitro antifungal activities of all the target compounds were evaluated
against eight human pathogenic fungi. Compound 5l showed the best antifungal activities.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Triazole
Antifungal activity
Cycloaddition
CYP51
Molecular docking
Fungal infections pose a continuous and serious threat to human
health and life especially to immunocompromised patients.1–3 Clin-
ically, candidosis, aspergillosis and cryptococcosis are three major
fungal infections in immunocompromized patients.4,5 Currently,
triazole agents (fluconazole, itraconazole, voriconazole and posaco-
nazole,Fig. 1) are the most frequently used antifungals in the clinic.6
However, fluconazole is not effective against invasive aspergillosis
and has suffered severe drug resistance.7,8 This situation highlights
the need for new triazole derivatives possessing broader antifungal
spectra and higher therapeutic indexes.
in the narrow hydrophobic cleft were also very important. We in-
tended to alter the side chains to find potent systemic antifungal
compounds with a broad antifungal spectrum and less potential
to develop resistance.
According to the above results, we designed a series of 1-(1H-
1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propa-
nols (5, Fig. 2) containing a triazole ring, a difluorophenyl group, a
hydroxyl group and a side chain. In our design, we systematically
altered the structure of fluconazole as a platform and tried to insert
1,2,3-triazolyl group into the side chain via Cu(I)-catalyzed azide–
alkyne cycloaddition.
Compounds 5a–5y were synthesized according to a very effi-
cient and straightforward synthetic route outlined in Scheme 1.
After the key intermediate 3 was synthesized by a known proce-
dure,15 Compound 4 was synthesized by Nucleophilic addition
reaction of 3 with propargyl bromide in the presence of Zn in
DMF at room temperature.16 The click reaction approach toward
the synthesis of the novel 1,2,3-triazolyl linked triazole antifungal
derivatives 5a–5z was achieved by Cu(I)-catalyzed 1,3-dipolar
cycloaddition with substituted azidomethyl benzene.17 All the
new compounds described above were characterized by 1H NMR,
ESI-MS and HR-MS spectroscopic analysis.18
Azole antifungals act by competitive inhibition of CYP51, the en-
zyme that catalyzes the oxidative removal of the 14a-methyl group
of lanosterol to give
D
14,15-desaturated intermediates in ergosterol
biosynthesis.9 In general, the active site of CYP51 for ligand binding
can be divided into three subsites: the hydrophilic H-bonding re-
gion, the hydrophobic region, and the narrow hydrophobic cleft
formed by the residues in the helix b0-meander 1 loop and N-termi-
nus of helix I.10
Some studies11–14 have revealed a pharmacophore of antifungal
triazoles, which contains a triazole ring linked to a dihalophenyl
ring through a two carbon chain. In addition, the carbon alpha to
the phenyl ring bears a hydroxyl group. But the side chains located
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).19
The in vitro antifungal activities of all the target compounds were
⇑
Corresponding authors. Tel./fax: +86 21 8187 3592 (M.X.); tel./fax: +86 21 8187
1225 (Q.W.).
E-mail addresses: mjxu68@gmail.com (M. Xu), smmuwuqy@gmail.com (Q. Wu).
These two authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.042

2960
Y. Zou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2959–2962
Figure 1. Triazole antifungal agents used in clinical therapy.
antifungal activity of compounds (5j–5l, 5x–5y) was better than
that of the other compounds. The MIC80 value of compound 5l is
128 times lower than that of FCZ against C. alb. SC5314 in vitro
O
O
N
N
Cl
F
F
F
N
a
c
(with the MIC80 value of 0.0039
than that of FCZ against C. kef. (with the MIC80 value of
0.0156 g/mL). The MIC80 value of compound 5u is 16 times
lg/mL), and 256 times lower
b
l
F
F
F
3
lower than that of FCZ against C. tro and 64 times lower than
that of FCZ against C. kef in vitro (with the MIC80 value of
2
1
0.0625 lg/mL).Compounds 5l, 5t, 5y and 5z are worthy of further
study, and are expected to be developed into new antifungal
drugs.
OH
N
N
OH
N
N
N
N
N
R
N
N
To explain the results, we proposed a hypothetical binding
mode for 5l to the active site of CYP51 based on computational
docking results (Fig. 3). As usual, the triazole interacts with iron
of the heme group, while the 2,4-difluorophenyl group in the de-
signed compound could be placed into the hydrophobic pocket
formed by Gly114, Phe126, Leu139, Met140, Phe145, Ile304
and Met306. The side chain incorporate to adjust the overall
physical-chemical properties of the molecules and orientation
of aromatic rings. It would also be oriented to interact with a
hydrophobic pocket formed by Leu121, Thr122, Phe228,
Thr311, Pro375, Leu376, His377, Ser378, Met508, Val509 and
Val510. The 1,2,3-triazole group in the side chain would gener-
F
F
d
F
4
F
5a-5y
Scheme 1. Synthetic routes of the target compounds. Reagents and conditions: (a)
ClCH2COCl, AlCl3, 50 °C, 5 h, 82%; (b) 1H-1,2,4-triazole, NaHCO3, toluene, reflux, 5 h,
62%; (c) Zn, propargyl bromide, DMF/THF, 60 °C, 6 h; 95%; (d) NaN3, substituted
benzyl bromide, ascorbate sodium, CuSO4, DMSO, 84%.
ate
p–p stacking interactions with the Tyr118. Finally, the
N
N
OH
N
N
substituted benzyl could interact with a hydrophobic pocket
formed by Ala114, Phe126, Gln142 and Phe145, it could also
generate
N
R
N
F
p–p stacking interactions with Phe380.
In addition, the side chains were the pharmacophores, and
the spatial orientations of the pharmacophores were just ori-
ented in the hydrophobic pocket. The side chains were very
important. They played a role in adjusting the physical-chemical
properties of the whole molecule to avoid some negative side ef-
fects and improved their pharmacokinetic and pharmacodynamic
behavior.
In conclusion, an efficient method using the click reaction has
been developed for the synthesis of diversified novel triazole
derivatives. Results of preliminary antifungal tests against eight
human pathogenic fungi in vitro showed that these analogs
exhibited excellent activities with a broad spectrum. The ob-
tained results indicated that for antifungal activity of these novel
triazole derivatives it is very helpful to introduce the 1,2,3-triaz-
olyl group and the substituted benzyl groups as side chains. This
research has led to the discovery of a series of compounds for
further optimization.
F
Figure 2. General structure of the designed fluconazole analogues.
evaluated against eight human pathogenic fungi (C. alb.SC5314, C.
alb.Y0109, C. neo., C. par., C. tro., T. rub., C. kef., A. fum.) which are of-
ten encountered clinically and are summarized in Table 1. The MIC
values (in lg/mL) against different pathogenic fungi, in comparison
with itraconazole (ICZ), ketoconazole (KCZ), amphotericin B (AMB),
voriconazole (VCZ) and fluconazole (FCZ).
The results of antifungal activities in vitro showed that all of
the 25 target compounds (5a–5y) were active against nearly all
fungi tested to some extent (Table 1). Most of the target com-
pounds exhibited higher activities against C. alb Y0109, C. alb
SC5314 and C. kef than all five positive controls. Obviously, the

Y. Zou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2959–2962
2961
Table 1
Antifungal activities of the target compounds in vitro
Compound No.
R
MICa
(lg/mL)
C. alb Y0109
C. alb SC5314
C. par
C. tro
C. kef
C. neo
T. nru
A. fum
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0156
0.0156
0.0039
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.125
0.0625
<0.125
<0.125
0.0625
<0.125
<0.125
0.0625
<0.125
0.0625
0.0625
0.0625
0.0156
<0.125
<0.125
<0.125
0.125
0.125
0.125
0.125
0.0625
0.125
0.125
0.0625
0.125
0.125
0.0625
0.0625
0.0625
<0.25
<0.25
0.25
0.0625
0.25
0.25
0.0625
0.25
0.5
0.0625
0.25
0.25
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.0625
0.0625
0.125
0.125
0.25
0.0625
0.0625
0.0625
0.125
0.0625
0.0625
0.0625
0.125
0.0625
0.0039
0.0156
0.0156
0.0625
0.0625
0.0625
<0.125
<0.125
<0.125
<0.125
0.0625
0.25
0.125
0.25
0.25
0.25
0.5
0.25
0.25
0.25
0.25
0.125
0.125
0.125
0.5
0.125
0.25
0.125
0.25
0.125
0.125
0.25
16
32
32
16
8
4
4
8
8
0.25
4-Br
0.125
0.0625
0.0625
0.0625
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.0625
0.25
0.25
<0.125
0.0625
0.0625
0.0625
0.0156
4
2-CH3
3-CH3
4-CH3
2-NO2
3-NO2
4-NO2
2-CN
3-CN
4-CN
H
4-tert-Butyl
3,4-F
3,4-Cl
3,5-Cl
3,4-OCH3
2,5-OCH3
—
2
4
2
32
32
16
16
32
32
16
4
16
32
32
2
0.5
0.5
0.5
1
0.25
0.25
0.25
0.125
0.125
0.125
0.0625
0.25
0.5
5s
5t
0.0625
0.125
0.125
0.125
0.25
0.0625
0.0625
0.0625
0.0039
0.25
<0.25
<0.125
1
1
1
0.0625
0.0625
0.0625
0.0156
1
5u
5v
5w
5x
5y
ICZ
KCZ
AMB
VCZ
FCZ
0.125
0.125
0.25
0.25
0.25
0.5
0.0156
0.0156
0.0625
0.0625
0.25
0.0625
0.0625
0.0625
0.0625
0.25
0.125
0.125
0.0625
0.0039
1
0.0625
0.0625
0.125
0.0625
0.25
2
0.0625
0.0156
1
0.0156
64
—
—
—
—
0.0625
0.5
0.0625
0.5
0.0039
0.5
0.0039
1
0.0625
4
0.0039
1
0.0156
1
Abbreviations: C. alb, Candida albicans; C. par, Candida parasilosis; C. tro, Candida tropicalis; C. kef, Candida kefyr; C. neo, Cryptococcus neoformans; T. nru, Trichophyton rubrum;
A. fumi, Aspergillus fumigatus; ICZ, itraconazole; KCZ, ketoconazole; VCZ, voriconazole; AMB, amphotericin B; FCZ, fluconazole.
a
Minimum inhibitory concentration for 80% inhibition of growth.
Figure 3. Computed binding geometry of the new inhibitor 5l in the active site of CYP51.

2962
Y. Zou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2959–2962
9. Aoyama, Y.; Yoshida, Y.; Sato, R. J. Biol. Chem. 1984, 259, 1661.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.042.
18. Representative analytical data for compound 5a: 1-(1H-1,2,4-triazol-1-yl)-2-
(2,4-difluorophenyl)-3-[1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl]-2-propanol.
White solid, mp 61.0–62.3 °C. 1H NMR (300 MHz, CDCl3) d: 8.04 (1H, s, triazole-
H), 7.87 (1H, s, triazole-H), 7.38–7.57 (1H, m, triazole-H), 6.76–7.29 (7H, m, Ar-
H), 5.41 (2H, s, CH2-Ar), 4.65–4.77 (2H, dd, J = 14.1 Hz, trizole-CH2), 3.08–3.49
(2H, dd, J = 14.7 Hz, trizole-CH2); IR (KBr): 3400, 3280, 3100, 2822, 1736, 1676,
1610, 1511, 1234, 1137 cmꢀ1; ESI-MS, calcd for C20H18F3N6O, [M+H]+ 415.15,
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