Synthesis, in vitro evaluation and molecular docking studies of new triazole derivatives as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2011.06.008

On the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51), a series of 1-(2-(2,4-difluorophenyl)-2-hydroxy-3- (1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one derivatives were synthesized as fluconazole analogs. Results of the preliminary antifungal tests against eight human pathogenic fungi in vitro showed that these compounds exhibited activities to some extent, and some displayed excellent antifungal activities against C. albicans than reference drug fluconazole. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the target compounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.

Full text of DOI:10.1016/j.bmcl.2011.06.008

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4471–4475
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, in vitro evaluation and molecular docking studies of new
triazole derivatives as antifungal agents
Yongwei Jiang a, , Jun Zhang c, , Yongbing Cao b, Xiaoyun Chai a, 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, PR China
b Drug Research Center, School of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, PR China
c Overseas Education Faculty of the Second Military Medical University, Xiangyin Road 800, Shanghai 200433, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of the active site of lanosterol 14a-demethylase from Candida albicans (CACYP51), a series of
Received 27 January 2011
Revised 29 May 2011
Accepted 2 June 2011
Available online 14 June 2011
1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one deriv-
atives were synthesized as fluconazole analogs. Results of the preliminary antifungal tests against eight
human pathogenic fungi in vitro showed that these compounds exhibited activities to some extent, and
some displayed excellent antifungal activities against C. albicans than reference drug fluconazole. Flexible
molecular docking was used to analyze the structure–activity relationships (SARs) of the target com-
pounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and
hydrogen-bonding interactions.
Keywords:
Triazole
Synthesis
Antifungal activity
CYP51
Ó 2011 Elsevier Ltd. All rights reserved.
Molecular docking
During the past two decades, the life-threatening infections
caused by pathogenic fungi have become increasingly common
especially in the immuno-compromised hosts suffering from
tuberculosis, cancer, AIDS and organ transplant cases.1–3 The
common antifungal agents (Fig. 1) used in clinic are azoles (such
as fluconazole, itraconazole and voriconazole),4 polyenes (such as
amphotericin B5,6 and nystatin),7 echinocandins (such as caspofun-
gin and micafungin)8 and allylamines (such as naftifine and
terbinafine).9 Among those, azoles have fungistatic, orally active
and broad-spectrum activities against most yeasts and filamentous
fungi and were widely used in antifungal chemotherapy.10 How-
ever, their clinical application value has been hampered by their
relatively high risk of toxicity, the emergence of drug resistance,
pharmacokinetic deficiencies and/or insufficiencies in their
antifungal activities. So there is still an urgent need for novel
broad-spectrum and low-toxicity antifungal agents.
and accumulation of lanosterol and other 14-methyl sterols lead-
ing to the growth inhibition of fungal cells.11 Currently, the crystal
structure of CYP51 of fungi has not been obtained, eukaryotic
CYP51s are membrane-associated proteins, and solving their crys-
tal structures remains a challenge. Thereby, we have constructed a
3D model of CYP51 from Mycobacterium tuberculosis (MTCYP51)
through homology modeling on the basis of the crystal structure
of MTCYP51 (M. tuberculosis 14a-demethylase) which is the only
crystal structure for the CYP51 family reported by Podust et al.
in 2001.12 The active site of the model divided into four sections:
a coordination bond with iron of the heme group, the hydrophilic
H-bonding region, the hydrophobic region, and the narrow hydro-
phobic cleft formed by the residues in the helix B0-meander 1 loop
and N-terminus of helix 1.13
Research indicated that the triazole ring in the scaffold of tria-
zole antifungals was positioned perpendicularly to the porphyrin
plane with a ring nitrogen atom coordinated to the heme iron of
CYP51 and was of key importance for the antifungal activity. The
halogenated phenyl group was deep in the same hydrophobic
binding cleft in the active site of the enzyme CYP51 and long side
chains surpassed the active site and interacted with residues in the
substrate access channel.
Azole antifungals function by competitive inhibition of the
lanosterol 14
the oxidative removal of the 14
give
14,15-desaturated intermediates in ergosterol biosynthesis.
a
-demethylase(CYP51), the enzyme that catalyzes
a-methyl group of lanosterol to
D
Selective inhibition of CYP51 would cause depletion of ergosterol
Here we designed
2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-4-substituted phenyl-
1H-1,2,4-triazol-5(4H)-one compounds, which containing
a series of 1-(2-(2,4-difluorophenyl)-
⇑
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).
These authors contributed equally to this work.
a
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.008

4472
Y. Jiang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4471–4475
O
OH
F
O
N
N
N
N
N
N
F
N
N
CH2O
N
N
N
N
O
N
N
Cl
Cl
O
fluconazole
itraconazole
OH
OH
O
F
CH3
OH
OH
OH
N
N
F
HO
O
OH OH
OH OH
N
N
N
H
O
OHNH2
O
F
voriconazole
amphotericin B
O
OH
Figure 1. Antifungal agents used in clinical therapy.
triazole ring, a difluorophenyl group and a triazolone side chain,
aiming to find potent systemic antifungal agents that have a broad
antifungal spectrum.
Letter we applied a ‘one-pot’ method to synthesize the intermediate
2. Here in our method, hydrazinocarboxylate and trimethyl ortho-
formate was added to a solution of p-nitroaniline in methanol. After
the reaction mixture was refluxed for 2 h, 25% sodium methoxide
was added dropwise to it which continued to reflux for another
3 h. The resulting mixture was concentrated under reduced pres-
sure, then added 30 ml water and adjusted pH value to 1 with con-
centrated hydrochloric acid. Filtration of the latter mixture gave the
intermediate 2.16 The yield was 76.4–85.6%. Apparently, the former
method needs three steps, while ours is a one step procedure. It is
more succinct and is of more efficiency. Treatment of 2 with excess
1 in the presence of potassium carbonate in DMF gave an important
intermediate, which reduced with 10% Pd–C under H2 atmosphere
led to the synthesis of compound 3. Target compounds 4a–4t and
5a–5p were obtained by using different aromatic acid or substi-
tuted cinnamic acid to react with 3 in the presence of N-(3-dimeth-
ylamino-propyl)-N0-ethylcarbodiimide hydrochloride (EDCꢀHCl)
Synthetic routes of the target compounds 4a–4t and 5a–5p were
outlined in Scheme 1. According to a known procedure, the oxirane
intermediate 1 was obtained by Corey–Chaykovsky expoxidation in
the presence of trimethylsulfoxonium iodide and NaOH.14 Interme-
diate 2 was originally synthesized by a three-step procedure out-
lined in Scheme 2. First, phenyl chloroformate was added
dropwise to a stirred mixture of p-nitroaniline, pyridine and EtOAc
at 0 °C. The mixture was stirred for 3 h at room temperature to give
compound i. The yield was 97.7%. Second, hydrazine hydrate was
added to a solution of compound i in dimethoxyethane, the result-
ing reaction mixture was stirred for 24 h at room temperature to
give compound ii. The yield was 81.3%. Finally, treatment of com-
pound ii with formamidine acetate in DMF at room temperature
for 30 min gave the intermediate 2.15 The yield was 82.2%. In this
O
N
N
N
O
N
F
F
F
N
N
CH3SO3H
F
F
1
F
O
HN
N
a
NH2NHCOOCH3
N
NO2
+
H2N
NO2
2
O
OH
N
N
F
N
N
NH2
c
d
b
N
N
1
+
2
F
3
O
O
OH
N
R
N
F
N
N
NH
N
n
N
4a-4t: n=0
5a-5p: n=1
F
Scheme 1. Synthetic routes of the target compounds. Reagents and conditions: (a) HC(OCH3)3, CH3ONa, CH3OH, p-toluenesulfonic acid, reflux, 5 h; (b) DMF, K2CO3, 80 °C, 6 h;
(c) Pd/C, H2, 4 h, rt; (d) RCOOH, EDCꢀHCl/DMAP, CH2Cl2, rt, 6 h.

Y. Jiang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4471–4475
4473
O
O
NHNH2
O
b
a
NH
NH2
NH
i
O2N
O2N
O2N
ii
O
c
NH
N
N
O2N
2
Scheme 2. Original synthesis route of intermediate 2. Reagents and conditions: (a) phenyl chloroformate, pyridine, EtOAc, rt, 3 h; (b) Hydrazine hydrate, dimethoxyethane, rt,
24 h; (c) formamidine acetate, DMF, rt, 30 min.
Table 1
a
In vitro antifungal activities of the target compounds (MIC80
l
g mlꢁ1
)
Compound no.
R
C. alb
C. tro
C. par
C. kef
C. neo BLS108
T. nru
A. fumi
SC5314
Y0109
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4-F
2-F
4-Cl
3-Cl
2,4-Cl
3-Br
2-Br
4-OCH3
3-OCH3
4-OCF3
4-CH3
3-CH3
2-CH3
3,4-CH3
2,4-CH3
4-CH2CH2CH3
4-(CH2)3CH3
4-(CH2)4CH3
4-C (CH3)3
4-CH (CH3)2
4-F
0.0625
0.0625
1
0.00024
0.0625
4
1
1
4
1
0.0625
0.0625
1
1
0.25
16
4
4
1
64
1
1
1
0.25
1
0.0625
0.0156
1
0.00024
0.0156
16
4
1
1
4
4
64
>64
16
16
>64
16
>64
>64
4
64
64
16
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.0625
0.0039
>64
1
0.0625
16
>64
>64
16
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.0625
0.0039
1
>64
>64
>64
>64
>64
>64
>64
4
>64
>64
>64
>64
>64
>64
>64
>64
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
0.00024
0.0625
16
4
1
1
1
1
0.0625
0.25
0.0625
0.0625
0.25
0.25
0.0625
0.0039
0.0156
4
0.0625
4
>64
16
1
4
4
1
1
1
1
4
1
1
0.0625
1
0.0625
1
0.0625
0.0625
0.0625
0.25
0.0625
1
0.0156
>64
1
4
>64
16
1
16
4
4
1
4
4
>64
1
0.25
0.0625
0.0156
0.25
0.0625
4
0.0625
>64
0.0625
4
>64
16
4
>64
64
1
64
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
0.25
>64
0.25
4
>64
>64
4
>64
4
>64
1
4s
4t
5a
5b
5c
5d
5e
5f
5g
5h
5i
3-F
2-F
4-Cl
3,4-Cl
4-Br
3-Br
16
4-CH3
2,3-CH3
4-OCH3
3,4-OCH3
2,5-OCH3
4-NO2
3-NO2
2-NO2
3-CN
—
—
—
—
4
16
>64
>64
>64
4
1
1
0.0039
1
16
0.0625
0.0039
0.25
0.25
5j
5k
5l
>64
1
>64
>64
0.25
1
16
5m
5n
5o
5p
ICZ
VCZ
FCZ
AMB
>64
0.00024
1
>64
0.00024
1
>64
>64
0.0625
0.0039
4
>64
>64
0.0625
0.0156
1
16
16
64
0.0625
0.0039
0.25
0.25
0.0625
0.0039
0.25
0.25
0.0625
0.0039
1
1
0.25
1
4
a
Abbreviations: C. alb. Candida albicans; C. tro. Candida tropicalis; C. par. Candida parasilosis; C. kef. Candida kefyr; C. neo. Cryptococcus neoformans; T. nru. Trichophyton
rubrum; A. fumi. Aspergillus fumigates. FCZ: fluconazole; VCZ: voriconazole; ICZ: itraconazole; AMB: amphotericin B.
and 4-dimethyl-aminopyridine (DMAP) in CH2Cl2 at ambient
temperature.
BLS108, and Aspergillus fumigatus).C. albicans andC. neoformans were
provided by Shanghai Changzheng Hospital; C. tropicalis, C. parapsi-
losis,C. kefyr,C. neoformans,T. rubrum andA. fumigatus were provided
by Shanghai Changhai Hospital. C. albicans and C. neoformans were
purchased from ATCC, and other strains were clinic isolates. C. albi-
cans (SC5314 and Y0109) and C. neoformans (BLS108) were used as
the quality-controlled strains, and tested in each assay. C. kefyr
was flucnazole resistant strain. Fluconazole, itraconazole,
voriconazole and amphortericin B which served as positive controls
were obtained from their respective manufacturers. The in vitro
The in vitro minimal inhibitory concentrations (MIC80) of the
compounds were determined by the micro-broth dilution method
in 96-well microtest plates according to the methods defined by
the NationalCommittee for Clinical Laboratory Standards (NCCLS).17
The tested fungi species included eight pathogenic fungi, which
were found in dermatomycoses (Trichophyton rubrum) and systemic
mycoses (Candida albicans (SC5314 and Y0109), Candida tropicalis,
Candida parapsilosis, Candida kefyr, Cryptococcus neoformans

4474
Y. Jiang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4471–4475
Figure 2. The docking conformation of compound 4d (A) and 5n (B) in the active site of CACYP51.
antifungal activities of all the target compounds were listed in Table
1. These data are the mean of three replicate tests performed with
each antifungal compound.
with iron of heme group. The difluorophenyl group is located in a
hydrophobic pocket and interacts with Phe126, Met306, Gly303,
Ile304, Gly307, and Gly308. The terminal triazolone side chain
was placed into a hydrophobic cleft formed by Gly65, Met92,
Phe380, Met508, Tyr118, and Ala117.
In addition, all of the side chains were of the pharmcophores,
and the spatial orientations of the pharmacophores oriented in
the hydrophobic pocket. The side chains of inhibitors played an
important role in adjusting the physic-chemical properties of the
whole molecule. Comparing 4a–4t with 5a–5p, the overall
in vitro antifungal activity of 4a–4t is better than 5a–5p, we as-
sume that the longer side chain influence their spatial orientations
in target enzyme which lead to their low antifungal activities. In
cinnamic acid series, the activity of 5n is an exception, we presume
that the meta-nitro substituent enhance the interaction between
three major active sites of CACYP51 with our inhibitor by interact-
ing with the remote residues of target enzyme.
The results of preliminary antifungal activities indicated that
most of the target compounds 4a–4t showed higher activities
against C. albicans (SC5314), C. albicans (Y0109), Candida tropicalis
and Candida parasilosis than the standard reference drug FCZ and
AMB. Compounds 4a, 4b, 4i, 4l, and 4o exhibited equivalent anti-
fungal activities of ICZ against C. albicans (SC5314), C. albicans
(Y0109) and C. parasilosis with the MIC80 value of 0.0625
Additionally, compound 4d exhibited excellent activities with
an MIC80 value of 0.00024
g mlꢁ1 against C. albicans (SC5314),
C. albicans (Y0109) and C. parasilosis, versus the MIC80 value of
VCZ, which is 0.0039
g mlꢁ1. Among compounds 4k and 4p–4r,
l .
g mlꢁ1
l
l
which contain an alkyl substituent in the para position, 4q exhib-
ited higher antifungal activity with the minimal inhibitory concen-
trations (MIC80) value in the range of 0.25–0.0156 l
g mlꢁ1. It
indicates that suitable length of the alkyl side chain is relative to
its activity. As for cinnamic acid series, including 5a–5p, their
activities were not desirable against all the tested fungal patho-
gens except 5n. The activities of 5n were excellent with the min-
imal inhibitory concentrations (MIC80) value in the range of
In summary, a series of novel triazole antifungal agents contain-
ing a triazolone side chain were successfully designed and synthe-
sized. Wherein, we adopted
a more convenient method to
synthesize the intermediate 2 and acquired higher yield. The anti-
fungal activities of target compounds were screened for eight hu-
man pathogenic fungi. In vitro antifungal activity assay indicated
that most of these compounds showed higher antifungal activities
against C. albicans than the reference drug FCZ and AMB. Com-
pounds 4d and 5n possessed excellent activities against C. albicans,
C. tropicalis and C. parasilosis, we also studied their structure–activ-
ity relationships (SARs) through molecular docking. All these
observations will be helpful in designing newer antifungal agents
for our further research.
0.25–0.00024
negative against C. neoformans, A. fumigates and T. rubrum except
4c with a MIC80 value of 0.0625
g mlꢁ1 against C. neoformans.
l
g mlꢁ1. Apparently, all the target compounds were
l
The MIC80 values of compound 4b and 4c were four times lower
than that of FCZ against C. kefyr.
The inactivity of these compounds against A. fumigatus was not
a surprise because it has been known that this species possesses an
intrinsic mechanism resistant to triazole antifungal,18 but there are
some papers in which compounds reported are active against A.
fumigatus.3 It gives us hopes to develop broad-spectrum and more
affordable antifungal agents.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 30300437 and 20772153), the Eleventh
Five Year Military Medicine and Public Health Research Projects
(Grant No. 06MB206) and by Shanghai Leading Academic
Discipline Project (Project No. B906).
All the target compounds were obtained as racemates. However,
in previous study, R isomers showed lower interaction energy with
CYP51 than S isomers, which indicated that the R configuration
might have better antifungal activity than the S isomers.15 Hence,
in the following discussion, all the docking conformations refer to
R configuration of the compounds. To clarify the binding mode of
our synthesized compounds, compounds 4d and 5n were docked
in the active site of CACYP51 by the Builder module within Insight-
ll2000 software package (Fig. 2). The triazole ring of the compounds
binds to CACYP51 through the formation of a coordination bond
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.008.

Y. Jiang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4471–4475
4475
11. Lamb, D. C.; Kelly, D. E.; Venkateswarlu, K.; Manning, N. J.; Bligh, H. F.; Schunck,
W. H.; Kelly, S. L. Biochemistry 1999, 38, 8733.
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