Synthesis, in vitro antifungal activity and in silico study of 3-(1,2,4-triazol-1-yl)flavanones

Article abstract of DOI:10.1016/j.ejmech.2013.06.008

A series of novel 3-(1,2,4-triazol-1-yl)flavanones were synthesized based on the N -phenethylazole pharmacophore of azole antifungals. The results of antifungal assay revealed that 4′-fluoroflavanone derivative 4c exhibited the best profile of activity ag

Full text of DOI:10.1016/j.ejmech.2013.06.008

European Journal of Medicinal Chemistry 66 (2013) 480e488
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro antifungal activity and in silico study of
-(1,2,4-triazol-1-yl)flavanones
3
Saeed Emami a,*, Shahaboddin Shojapour , Mohammad Ali Faramarzi , Nasrin Samadi ,
Hamid Irannejad
a
a
b
c
a
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari,
Iran
b
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Quality Assurance Research Center, Tehran University of Medical Sciences,
c
Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 January 2013
Received in revised form
A series of novel 3-(1,2,4-triazol-1-yl)flavanones were synthesized based on the N-phenethylazole
pharmacophore of azole antifungals. The results of antifungal assay revealed that 4 -fluoroflavanone
derivative 4c exhibited the best profile of activity against Candida and Saccharomyces strains. Compound
0
18 March 2013
4c was 4e16 times more potent than reference drug fluconazole against Candida albicans and Saccha-
Accepted 6 June 2013
Available online 17 June 2013
romyces cerevisiae. The molecular docking study with lanosterol 14 -demethylase, in silico toxicity risks
a
and drug-likeness predictions were used to better define of title compounds as antifungal agents. The
favorable drug-like property of compound 4c makes 3-(1,2,4-triazol-1-yl)flavanone prototype as a
promising lead for the future development of azole antifungal agents.
Keywords:
Azole antifungals
Antifungal activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1,2,4-Triazole
Flavanones
Docking study
Drug-likeness
1. Introduction
posaconazole (Fig. 1) represent an interesting class of azole anti-
fungal agents. These compounds have become the most rapidly
In recent decades, the rising in the number of immunocom-
promised hosts, such as patients suffering from AIDS, tuberculosis,
diabetes, and undergoing organ transplantations and cancer
chemotherapy has been complicated with opportunistic fungal
infections and become a major cause of morbidity and mortality in
these patients [1]. Although several antifungal agents have recently
been introduced to the clinical chemotherapy, the number of
available drugs to treat life-threatening fungal infections is still
limited and more new alternative drugs with improved efficacy are
needed [2,3]. The majority of available chemotherapeutic agents
suffer from drug-related toxicity, hazardous drugedrug in-
teractions and pharmacokinetics deficiencies. However, triazole
antifungals including fluconazole, itraconazole, voriconazole and
expanding group of antifungals with the advantages of low toxicity,
high oral bioavailability, and broad spectrum of activity against
several fungi [4]. Thus, the triazole class of antifungal agents
currently plays a leading role in the field of drug development.
These antifungal agents inhibit the cytochrome P450-dependent
lanosterol 14a-demethylase (CYP51) by a mechanism in which
the N-4 of triazole ring binds to the heme iron atom in the binding
site of the enzyme [5,6].
As a part of our works on azole antifungal agents [7e10],
recently we have designed conformationally constrained azole
antifungals, namely 3-imidazolylflavanones (Fig. 2) [11]. Based on
the N-phenethylazole pharmacophore of azole antifungals, these
compounds possess pharmacophoric backbone from two sides. In
this series, compounds containing a substituent on 2-phenyl ring
showed significant antifungal activity. Moreover, modification of
the 4-oxo group to oxime resulted in the changes of antifungal
activity profile [11]. These encouraging results prompted us to
prepare triazole analogs of 3-azolylflavanones and investigate their
structureeactivity relationships. Triazole antifungals have several
*
Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
macy, Mazandaran University of Medical Sciences, Sari, Iran. Tel.: þ98 151 3543082;
fax: þ98 151 3543084.
E-mail address: sd_emami@yahoo.com (S. Emami).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.008

S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
481
Fig. 1. Triazole antifungal agents.
advantages over imidazole agents. For example, they have good oral
bioavailability and are metabolized more slowly, making them
longer acting than imidazoles. Moreover, triazoles have higher
selectivity for human CYP450 enzymes, resulting to lesser side ef-
fects and drugedrug interactions [12].
Considering the advantages of triazole-containing antifungal
drugs, we describe herein, synthesis, antifungal activity and in silico
study of 3-(1,2,4-triazol-1-yl)flavanones (Fig. 2).
appropriate benzaldehyde derivative produced trans-triazolyl-
flavanones 4. This reaction was occurred in 2-propanol, in the pres-
ence of piperidine as a catalyst and under reflux conditions. In the H
NMR spectra of triazolylflavanones 4, two doublet signals related to
the H-2 and H-3 of chroman ring appear at ca. 6.01e6.65 ppm with a
coupling constant J2,3 > 12 Hz, which indicated that the relative
configuration of these compounds is 2,3-trans.
To obtain triazolylflavanone oxime derivatives, we primarily
used a mild reaction condition which has been reported for
imidazolylflavanone oximes [11]. Accordingly, compound 4 was
treated with hydroxylamine hydrochloride in the presence of
potassium carbonate in methanol at room temperature. Sur-
prisingly, using these very mild conditions the reaction resulted
in a mixture of unknown degraded products. Thus, the reaction
was attempted by using the excess of hydroxylamine hydro-
chloride in refluxing methanol in the absence of potassium car-
bonate. For completion of the reaction, it should be refluxed for
long time. However, the corresponding oxime was produced in
good yield.
1
2
. Chemistry
As depicted in Scheme 1, the synthesis of compounds 4 and 5 was
started from 2-hydroxyphenacyl bromide (1). The reaction of com-
pound 1 with 4-amino-1,2,4-triazole in refluxing 2-propanol gave
aminotriazolium bromide 2. In this reaction, 2-hydroxyphenacyl
substituent was regioselectively connected to N-1 of 1,2,4-triazole.
Deamination of compound 2 was occurred in diluted hydrochloric
acid in the presence of sodium nitrite to afford 1-(2-hydroxy
phenacyl)-1,2,4-triazole (3) [13]. The reaction of compound 3 with
N
N
X
X
N
N
N
O
O
R
R
X= O, NOH
X= O, NOH
R = H, Cl, F, MeO, Me, 2,4-Cl2
Imidazolylflavanones
Triazolylflavanones
Fig. 2. Imidazolylflavanones possessing N-phenethylazole pharmacophore of azole antifungals and their triazole analogs as new antifungal agents.

482
S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
Scheme 1. Synthesis of 3-(1,2,4-triazol-1-yl)flavanone derivatives.
0
3
. Biology
ATCC 9763. Among them, 4 -halogenated flavanones 4b and 4c with
MIC values of 3.9 mg/ml were the most potent compounds. Also,
The antifungal activity of synthesized compounds 4 and 5 was
evaluated against Candida albicans ATCC 10231, Candida glabrata
ATCC 90030, Saccharomyces cerevisiae NCYC 694, S. cerevisiae ATCC
compounds 4g and 4k showed noticeable activity against former
strain of S. cerevisiae. All triazole compounds 4aek and 5a,b as well
as fluconazole were inactive against A. niger (MICs > 62.5
However, the MIC of ketoconazole (an imidazole antifungal) against
A. niger was 15.6 g/ml.
mg/ml).
9
763 and Aspergillus niger ATCC 16404. The minimum inhibitory
concentrations (MICs) of compounds were determined by micro-
dilution method [14]. A stock solution of compounds was trans-
ferred intothe plates with96U-shaped wells and serially dilutedwith
Sabouraud dextrose broth in subsequent wells. Then, fungal sus-
m
From the in vitro antifungal activity data of unsubstituted tri-
azolylflavanone 4a and halo-substituted compounds 4b, 4c, 4f and
4g against C. albicans, it is revealed that the presence of halogen
on the 3- or 4-position of 2-phenyl ring is beneficial for activity. In
contrast, the introduction of chlorine atom at the position 2
diminished the growth inhibitory activity against C. albicans
(compound 4i vs. compound 4a). Also, the observed MIC values of
2,4-dichloro derivative 4k and 4-chloro compound 4b against
C. albicans demonstrated the negative effect of chlorine substitu-
tion on the 2-position. Although, 4-chloro analog 4b and 4-fluoro
derivative 4c showed same activity against C. albicans, but 3-
chloro-substituted compound 4f was more potent than 3-fluoro
analog 4g. The results of antifungal bioassay against other yeasts
C. glabrata and S. cerevisiae revealed that the type and the position
of the substitutions may have different effects, related to the type
of strains. For example, the introduction of second chlorine atom
in the 4-chlorophenyl derivative decreased the activity against
C. albicans and S. cerevisiae ATCC 9763 (compound 4k vs. 4b),
while this modification enhanced the potency against S. cerevisiae
NCYC 694.
pension was added to each well to reach the final inoculum size of
3
0
.5 ꢀ 10 CFU/mL. After incubation, the plates were observed for the
absence or presence of visible growth in comparison with that of the
drug-free control well. The endpoint MIC was the lowest concentra-
tion of the compound at which the test strain does not demonstrate
visible growth. The MICs of tested compounds in comparison with
fluconazole as a standard azole antifungal agent were listed inTable 1.
4
. Results and discussion
4
.1. Antifungal activity
The MIC values of target compounds against C. albicans revealed
0
0
that 4 -halogenated flavanones 4b and 4c, and 3 -halogenated fla-
vanones 4f and 4g showed the highest inhibitory activity against
this fungi (MICs ¼ 3.9e15.6
mg/ml). Their activities were 2e8 times
more than that of fluconazole as standard drug. In addition, the
antifungal activity of compound 4a against C. albicans was com-
0
In general, 4 -fluoroflavanone derivative 4c exhibited the best
profile of activity against yeasts. The conversion of ketone to oxime
0
parable to fluconazole. The 4 -fluoroflavanone derivative 4c
(compounds 5 vs. compounds 4) could not improve antifungal ac-
exhibited remarkable activity against C. glabrata as potent as flu-
tivity. Among the ketones, compounds containing methyl or
methoxy substituents were inactive while 3- or 4-chloro and 3- or
conazole (MIC ¼ 31.3
mg/ml). However, the rest of compounds
showed no activity against this microorganism. In the case of
4-fluoro derivatives showed antifungal activity.
S. cerevisiae (NCYC 694), compounds 4c and 4f with MIC values of
0
.98e1.95 mg/ml were more potent than fluconazole. Their activ-
ities were 8e16 times better than that of fluconazole. Moreover,
compound 4k had inhibitory activity at the concentration of
4.2. Docking study
31.3
mg/ml against S. cerevisiae (NCYC 694). Most of compounds
The molecular basis of the antifungal activity of fluconazole and
showed significant growth inhibitory activity against S. cerevisiae
related azole antifungals is the inhibition of ergosterol biosynthesis,

S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
mg/ml) of compounds 4aek and 5a,b.
483
Table 1
The minimum inhibitory concentrations (MICs,
N
X
N
N
O
R
Compound
X
R
C. albicans ATCC 10231
C. glabrata ATCC 90030
S. cerevisiae NCYC 694
S. cerevisiae ATCC 9763
A. nigera ATCC 16404
4
4
4
4
4
4
4
4
4
4
4
5
5
a
b
c
d
e
f
O
O
O
O
O
O
O
O
O
O
O
NOH
NOH
H
4-Cl
4-F
4-Me
4-OMe
3-Cl
3-F
3-OMe
2-Cl
31.3
3.9
3.9
>62.5
>62.5
31.3
>62.5
>62.5
62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
31.3
>125
>125
0.98
>125
>125
1.95
>125
>125
>125
>125
31.3
>125
>125
15.6
125
3.9
3.9
>125
125
>125
15.6
>125
125
>125
31.3
125
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>125
>125
>125
3.9
15.6
>125
>125
>125
125
g
h
b
i
j
b
2-OMe
2,4-Cl
H
k
a
b
2
>125
>125
31.3
2,4-Cl
2
62.5
15.6
Fluconazole
The significance of bold values in comparison with control group was less than 0.05 (P < 0.05).
a
The MIC value of ketoconazole against A. niger was 15.6 mg/ml.
Nitrate salt.
b
which is necessary to maintain fungal cell membrane integrity and
permeability. Indeed, azole antifungals inhibit the cytochrome P-
Generally, in the binding model of azole antifungals with CYP51,
the maximal effective distance between the N-4 atom of the tri-
azole ring and the Fe atom of heme was 3 A [16]. Based on our
ꢀ
4
50 lanosterol 14
a
-demethylase (CYP51) which is responsible for
-methyl group of lanosterol [15].
the oxidative removal of the 14
a
docking experiment, distance between the triazole nitrogen of
ꢀ
Accordingly, the preliminary molecular docking calculations of the
representative compound 4c were performed to provide more
insight into the interactions as well as to rationalize the observed
antifungal activity of title compound as lanosterol 14a-demethy-
lase inhibitor. Because no experimental data are available for the
(RR)-enantiomer and Fe atom of heme (1.9 A) is less than that of
ꢀ
(SS)-enantiomer (2.3 A), indicating stronger coordination bond.
Moreover, the free energy of binding (
with the highest score indicated that (RR)-enantiomer with
G ¼ ꢁ38 kJ/mol should have a better interaction than the (SS)-
enantiomer (
DG) for the best docking pose
D
target enzyme Candida P450 DM in the Protein Data Bank, the
D
G ¼ ꢁ17 kJ/mol). It should be noted that many of
crystallographic structure of Cytochrome P450 14
a
-sterol deme-
marketed azole antifungals such as miconazole, bifonazole, serta-
conazole, ketoconazole, itraconazole and terconazole are racemic
thylase from Mycobacterium tuberculosis (MTCYP51) was used for
the docking study. The docking and subsequent scoring were per-
formed using the default parameters of the FlexX program
implanted in LeadIT 2.0.2. The active site of the enzyme was defined
ꢀ
to include residues within 6.5 A radius around bound inhibitor
(fluconazole). Fluconazole was re-docked into the MTCYP51 active
site in order to check and validate the accuracy of our docking
protocol (Fig. 3).
As discussed in chemistry section, trans-triazolylflavanones 4
have two stereogenic centers C2 and C3. The method for
obtaining trans-triazolylflavanones 4 was non-enantioselective.
Thus, the isolated compound 4c might be mixture of (RR)- and
(
(
SS)-enantiomers. Docking experiments on both the (RR)- and
SS)-enantiomers of the target compound 4c indicated that p-
fluorophenyl ring of both enantiomers has been situated in a very
lipophilic pocket made by hydrophobic amino acids and interacts
with aromatic rings or aliphatic side chains of amino acids
Leu321, His259, Phe255, Val434, Met79 and Tyr76 through hy-
drophobic interactions (Figs. 4 and 5). Rotation of the p-fluo-
rophenyl ring of (SS)-isomer in the docked conformation makes
an additional hydrophobic interaction with aromatic ring of
Phe78. The aromatic part of chromanone ring in both enantio-
mers constitutes hydrophobic interactions with aromatic or
aliphatic parts of Phe83, Leu100 and Met99 (Figs. 4 and 5).
Meanwhile, the N-4 atom of the triazole ring in both enantio-
mers is positioned above the porphyrin plane and coordinated to
the heme iron.
Fig. 3. Conformation of fluconazole in the active site of Cytochrome P450 14a-sterol
demethylase after docking calculations. For clarity only amino acids within 7 A distant
from the docked ligand are shown.
ꢀ

484
S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
Fig. 4. Docking pose view (2D map) for (RR)- and (SS)-enantiomers of 4c. Only important amino acids for interactions are shown.
mixtures. However, our docking study is promising to separate the
enantiomers of 4c which might have different antifungal activity.
Although, azoles are known to inhibit mainly CYP51 enzymes,
and also our in silico study on the target compound 4c, indicated its
interactions with MTCYP51, but we cannot rebut other mechanisms
which are responsible for antifungal activity. Only experiments on
the isolated CYP51 could confirm our theoretical study.
4.3. Drug-like properties
The concept of drug-likeness helps us to optimize pharmaco-
kinetics and physicochemical properties, and to predict the overall
potential of designed compounds to be qualified as a drug [17]. In
the present work, OSIRIS Property Explorer program (http://www.
organic-chemistry.org/prog/peo/) was used to predict the
fragment-based drug-likeness and drug-score of title compounds
in comparison with commercial triazole antifungals fluconazole
and voriconazole. As summarized in Table 2, the analysis of theo-
retical toxicity risks (mutagenic, tumorigenic, irritant and repro-
ductive effects) for these series of compounds using Osiris program
showed that all title compounds have any potential of mutagenic,
tumorigenic and irritant effects. 3-Methoxy analog 4h showed high
risk of reproductive effect, while remaining compounds as well as
standard drugs fluconazole and voriconazole showed medium risk
of reproductive effect. The estimated drug-likeness values of
compounds revealed that all compounds with the exception of 4g,
had higher values compared to those of fluconazole and vor-
iconazole. As depicted in Fig. 6, the drug-likeness and drug score
values of compound 4c (the most potent compound) were signifi-
cantly higher than those of fluconazole and voriconazole. These
results could be a measure of compound’s potential to meet the
criteria of a possible drug candidate.
5. Conclusion
Fig. 5. (A) (RR)-Enantiomer of 4c in the active site of Cytochrome P450 14a-sterol
Considering the leading role of triazole antifungals in the field of
drug development, a series of novel 3-(1,2,4-triazol-1-yl)flavanone
derivatives were synthesized based on the N-phenethylazole
demethylase. (B) Binding mode of both (RR)- and (SS)-enantiomers of compound 4c in
ꢀ
the active site of enzyme. For clarity only amino acids within 7 A distant from the
docked ligand are shown.

S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
485
Table 2
Drug-likeness properties of compounds 4aek and 5a,b predicted by Osiris Property Explorer tool.
N
X
N
N
O
R
Compound
X
R
Log S
CLog P
MW
Toxicity risksa
Drug-likeness
Drug-score
Mb
Tc
Id
Re
4
4
4
4
4
4
4
4
4
4
4
5
5
a
b
c
d
e
f
g
h
i
O
O
O
O
O
O
O
O
O
O
O
NOH
NOH
H
4-Cl
4-F
4-Me
4-OMe
3-Cl
3-F
3-OMe
2-Cl
ꢁ2.95
ꢁ3.69
ꢁ3.27
ꢁ3.3
2.66
3.27
2.72
2.97
2.55
3.27
2.72
2.55
3.27
2.55
3.89
2.89
4.12
ꢁ0.21
1.11
291
325
309
305
321
325
309
321
325
321
359
306
374
306
349
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(þ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
(ꢂ)
3.15
5.21
2.57
1.62
0.4
2.38
ꢁ0.26
2.33
2.96
2.41
5.29
2.06
4.35
ꢁ1.13
0.42
0.68
0.62
0.62
0.61
0.55
0.60
0.48
0.49
0.61
0.66
0.53
0.63
0.48
0.46
0.55
ꢁ2.97
ꢁ3.69
ꢁ3.27
ꢁ2.97
ꢁ3.69
ꢁ2.97
ꢁ4.43
ꢁ3.33
ꢁ4.80
ꢁ2.18
ꢁ3.23
j
2-OMe
2,4-Cl
H
k
a
b
2
2,4-Cl
2
Fluconazole
Voriconazole
a
Ranked according to: (ꢁ) no bad effect, (ꢂ) medium bad effect, (þ) bad effect.
b
c
M: mutagenic effect.
T: tumorigenic effect.
I: irritating effect.
d
e
R: reproductive effect.
pharmacophore of azole antifungals. The trans-isomer of target
compounds was selectively prepared from the reaction of 1-(2-
hydroxyphenacyl)-1,2,4-triazole with appropriate benzaldehyde
in the presence of piperidine as a catalyst. The results of antifungal
6. Experimental protocols
6.1. Chemistry
0
evaluation tests revealed that 4 -fluoroflavanone derivative 4c
The 1-(2-hydroxyphenyl)ethanone derivatives 1e3 were pre-
pared using the literature methods [13]. The purity of synthesized
compounds were checked by thin-layer chromatography using
silica gel 60 F254 plastic sheets (Merck), and UV light (254 nm) was
used for visualization. Yields are based on isolated material and
were not optimized. Melting points were determined in open glass
capillaries using Bibby Stuart Scientific SMP3 apparatus (Stuart
Scientific, Stone, UK) and are uncorrected. The IR spectra were
obtained on a PerkineElmer FT-IR spectrophotometer using KBr
disks. The NMR spectra were recorded using a Bruker 500 spec-
exhibited the best profile of activity against Candida and Saccha-
romyces strains. Compound 4c was 4e16 times more potent than
standard drug fluconazole against C. albicans and S. cerevisiae.
Finally, compound 4c prototype with promising activity and useful
properties can be considered as lead compound in the design and
development of new azole antifungal agents.
trometer and chemical shifts are expressed as
d (ppm) with tetra-
methylsilane (TMS) as internal standard. The mass spectra were
obtained using a HP 5937 Mass Selective Detector (Agilent
technologies).
6
4
.1.1. General procedure for the synthesis of trans-3-(1H-1,2,
-triazol-1-yl)-flavanones 4aek
A
solution of 1-(2-hydroxyphenyl)-2-(1H-1,2,4-triazol-1-yl)
ethanone (3, 609 mg, 3.0 mmol) and appropriate benzaldehyde
(
(
3.15 mmol) in 2-propanol (5 mL) containing piperidine
1.05 mmol) was heated under reflux for 4e6 h. The excess of
solvent was evaporated under reduced pressure. After cooling, the
mixture was left in the fridge overnight. The precipitated product 4
was collected by filtration and washed with cooled 2-propanol and
0
diethyl ether, respectively. In the cases of 2 -substituted flavanone
derivatives 4i and 4j, after concentration of the reaction mixture,
water was added. Then, the aqueous phase was decanted and the
oily residue was dissolved in diethyl ether (5 mL), and treated with
Fig. 6. Drug-likeness and drug score values of the most active compound 4c compared
to fluconazole and voriconazole as standard azole antifungals.
3
65% HNO (0.2 mL). The mixture was cooled at a fridge to complete

486
S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
0
0
the precipitation of the nitrate salts 4i or 4j, which were collected
by filtration and washed with diethyl ether.
J ¼ 8.65 Hz, H-2 and H-6 ), 7.70 (dt, 1H, J ¼ 7.72 and 1.36 Hz, H-7),
7.88 (dd, 1H, J ¼ 7.84 and 1.50 Hz, H-5), 7.93 (s, 1H, triazole H-5),
13
8
6
.41 (s, 1H, triazole H-3). C NMR (125 MHz, DMSO-d ) d 55.97,
6
.1.1.1. trans-2,3-Dihydro-2-phenyl-3-(1H-1,2,4-triazol-1-yl)-4H-1-
65.87, 81.96, 114.64, 119.06, 120.40, 123.22, 127.97, 128.15, 130.12,
138.09, 146.36, 152.59, 160.69, 161.83, 188.55. Anal. Calcd for
C H N O : C, 67.28; H, 4.71; N, 13.08. Found: C, 67.31; H, 4.79; N,
18 15 3 3
ꢃ
ꢁ1
benzopyran-4-one (4a). Yield 60%; mp 120e121 C; IR (KBr, cm
)
n
max: 1707 (C]O); 1H NMR (500 MHz, DMSO-d
6
)
d
6.07 (d, 1H,
J ¼ 12.00 Hz, H-3), 6.45 (d, 1H, J ¼ 12.5 Hz, H-2), 7.19 (d, 1H,
J ¼ 8.50 Hz, H-8), 7.22 (dt, 1H, J ¼ 7.75 and 0.50 Hz, H-6), 7.32e7.37
12.91.
0
0
0
0
0
(
m, 3H, H-2 , H-4 and H-6 ), 7.43e7.47 (m, 2H, H-3 , H-5 ), 7.71 (dt,
6.1.1.6. trans-2-(3-Chlorophenyl)-2,3-dihydro-3-(1H-1,2,4-triazol-1-
yl)-4H-1-benzopyran-4-one (4f). Yield 68%; mp 126e127 C; IR
(KBr, cm
ꢃ
1
5
H, J ¼ 8.00 and 2.00 Hz, H-7), 7.88 (dd, 1H, J ¼ 8.00 and 2.00 Hz, H-
13
ꢁ1
1
), 7.92 (s, 1H, triazole H-5), 8.40 (s, 1H, triazole H-3). C NMR
65.50, 81.83, 118.67, 120.12, 122.96, 127.56,
28.28, 128.90, 129.85, 135.87, 137.31, 146.10, 153.34, 161.33, 188.00.
)
n
max: 1699 (C]O); H NMR (500 MHz, DMSO-d
6
)
d
6.14
(
125 MHz, DMSO-d
6
)
d
(d, 1H, J ¼ 12.39 Hz, H-3), 6.47 (d, 1H, J ¼ 12.39 Hz, H-2), 7.21 (d, 1H,
1
J ¼ 8.08 Hz, H-8), 7.24 (t, 1H, J ¼ 7.36 Hz, H-6), 7.34e7.37 (m, 2H, H-
þ
0
0
0
0
MS (m/z, %): 291 (M , 8), 256 (17), 222 (100), 194 (10), 170 (51), 145
29), 120 (37), 92 (33), 63 (16). Anal. Calcd for C17 : C, 70.09;
H, 4.50; N, 14.42. Found: C, 69.88; H, 4.46; N, 14.66.
4 and H-6 ), 7.42 (m, 1H, H-5 ), 7.59 (br s, 1H, H-2 ), 7.72 (dt, 1H,
(
H
13
N
3
O
2
J ¼ 7.77 and 1.66 Hz, H-7), 7.90 (dd, 1H, J ¼ 7.83 and 1.53 Hz, H-5),
13
7.97 (s, 1H, triazole H-5), 8.46 (s, 1H, triazole H-3). C NMR
(
125 MHz, DMSO-d
127.60, 128.01, 129.85, 130.87, 133.55, 137.84, 138.14, 146.11, 152.36,
161.16, 187.67. Anal. Calcd for C17 12ClN : C, 62.68; H, 3.71; N,
12.90. Found: C, 62.61; H, 3.99; N, 12.86.
6
) d 65.27, 80.91, 118.69, 119.96, 123.09, 126.94,
6
.1.1.2. trans-2-(4-Chlorophenyl)-2,3-dihydro-3-(1H-1,2,4-triazol-1-
ꢃ
yl)-4H-1-benzopyran-4-one (4b). Yield 71%; mp 156e157 C; IR
H
3 2
O
ꢁ
1
1
(
KBr, cm
)
n
max: 1709 (C]O); H NMR (500 MHz, DMSO-d
6
)
d
6.12
(
d, 1H, J ¼ 12.40 Hz, H-3), 6.45 (d, 1H, J ¼ 12.41 Hz, H-2), 7.20 (d, 1H,
J ¼ 8.34 Hz, H-8), 7.23 (t, 1H, J ¼ 7.60 Hz, H-6), 7.42 (d, 2H,
6.1.1.7. trans-2,3-Dihydro-2-(3-fluorophenyl)-3-(1H-1,2,4-triazol-1-
0
0
0
0
ꢃ
J ¼ 8.48 Hz, H-2 and H-6 ), 7.48 (d, 2H, J ¼ 8.48 Hz, H-3 and H-5 ),
.72 (dt, 1H, J ¼ 7.76 and 1.61 Hz, H-7), 7.89 (dd, 1H, J ¼ 7.86 and
.49 Hz, H-5), 7.95 (s, 1H, triazole H-5), 8.43 (s, 1H, triazole H-3). 13
NMR (125 MHz, DMSO-d 65.80, 81.39, 119.06, 120.42, 123.43,
28.00, 129.43, 130.47, 134.86, 135.22, 138.18, 146.46, 152.73, 161.82,
yl)-4H-1-benzopyran-4-one (4g). Yield 54%; mp 122e123 C; IR
(KBr, cm
ꢁ
1
1
7
1
6
) nmax: 1707 (C]O); H NMR (500 MHz, DMSO-d ) d 6.14
C
(d, 1H, J ¼ 12.39 Hz, H-3), 6.45 (d, 1H, J ¼ 12.37 Hz, H-2), 7.16e7.27
0
0
0
0
6
)
d
(m, 4H, H-5 , H-6 , H-6 and H-8), 7.34e7.41 (m, 2H, H-2 , H-4 ), 7.72
(dt, 1H, J ¼ 7.80 and 1.72 Hz, H-7), 7.90 (dd, 1H, J ¼ 7.91 and 1.67 Hz,
1
1
þ
13
88.12. MS (m/z, %): 325 (M , 6), 277 (5), 256 (100), 228 (10), 205
H-5), 7.96 (s, 1H, triazole H-5), 8.45 (s, 1H, triazole H-3). C NMR
(
C
38), 179 (22), 151 (14), 120 (45), 92 (32), 63 (14). Anal. Calcd for
(125 MHz, DMSO-d
6
)
d
65.75, 81.37,115.32 (d, JC,F ¼ 22.25 Hz),117.18
17
H12ClN
3
O
2
: C, 62.68; H, 3.71; N, 12.90. Found: C, 62.61; H, 3.60;
(d, JC,F ¼ 20.75 Hz), 119.09, 120.38, 123.48, 124.83 (d, JC,F ¼ 2.50 Hz),
128.00, 131.45 (d, JC,F ¼ 8.13 Hz), 138.22, 138.85 (d, JC,F ¼ 7.38 Hz),
N, 13.15.
3 2
146.50, 152.74, 161.56, 161.74, 188.04. Anal. Calcd for C17H12FN O :
6
.1.1.3. trans-2,3-Dihydro-2-(4-fluorophenyl)-3-(1H-1,2,4-triazol-1-
C, 66.02; H, 3.91; N, 13.59. Found: C, 65.94; H, 3.90; N, 14.07.
ꢃ
yl)-4H-1-benzopyran-4-one (4c). Yield 38%; mp 120e121 C; IR
ꢁ
1
1
(
(
(
(
(
KBr, cm
)
n
max: 1709 (C]O); H NMR (500 MHz, DMSO-d
6
)
d
6.11
6.1.1.8. trans-2,3-Dihydro-2-(3-methoxyphenyl)-3-(1H-1,2,4-triazol-
ꢃ
d, 1H, J ¼ 12.44 Hz, H-3), 6.45 (d, 1H, J ¼ 12.42 Hz, H-2), 7.15e7.20
1-yl)-4H-1-benzopyran-4-one (4h). Yield 79%; mp 163e164 C; IR
0
0
ꢁ1
1
m, 3H, H-2 , H-6 and H-8), 7.23 (t, 1H, J ¼ 7.25 Hz, H-6), 7.50e7.54
(KBr, cm
)
n
max: 1711 (C]O); H NMR (500 MHz, DMSO-d
6
) d 3.72
0
0
m, 2H, H-3 and H-5 ), 7.72 (dt, 1H, J ¼ 7.79 and 1.70 Hz, H-7), 7.89
dd, 1H, J ¼ 7.85 and 1.63 Hz, H-5), 7.94 (s, 1H, triazole H-5), 8.42 (s,
(s, 3H, OCH
3
), 6.04 (d, 1H, J ¼ 12.37 Hz, H-3), 6.43 (d, 1H,
0
J ¼ 12.39 Hz, H-2), 6.90 (dd, 1H, J ¼ 8.26 and 2.43 Hz, H-4 ), 6.99 (d,
H, triazole H-3). 13C NMR (125 MHz, DMSO-d
)
d
65.47, 81.03,
1H, J ¼ 7.59 Hz, H-6 ), 7.05 (br s, 1H, H-2 ), 7.20 (d,1H, J ¼ 8.52 Hz, H-
0
0
1
6
0
1
15.88 (d, JC,F ¼ 21.25 Hz), 118.66, 120.00, 122.99, 127.59, 130.54 (d,
C,F ¼ 8.75 Hz), 132.14, 137.78, 146.01, 152.30, 161.27, 162.86 (d,
12FN : C, 66.02; H,
8), 7.21e7.26 (m, 2H, H-5 and H-6), 7.71 (dt, 1H, J ¼ 7.76 and
J
J
1.53 Hz, H-7), 7.89 (dd, 1H, J ¼ 7.82 and 1.25 Hz, H-5), 7.95 (s, 1H,
13
C,F ¼ 243.75 Hz), 187.88. Anal. Calcd for C17
H
3
O
2
triazole H-5), 8.41 (s, 1H, triazole H-3). C NMR (125 MHz, DMSO-
3
.91; N, 13.59. Found: C, 66.13; H, 3.78; N, 13.60.
d
6
)
d
55.58, 65.42, 81.69, 113.69, 115.21, 118.70, 119.98, 120.36,
1
22.95, 127.58, 130.05, 137.30, 137.78, 146.07, 152.25, 159.55, 161.30,
þ
6
.1.1.4. trans-2,3-Dihydro-2-(4-methylphenyl)-3-(1H-1,2,4-triazol-1-
187.94. MS (m/z, %): 321 (M , 9), 252 (100), 221 (9), 200 (40), 175
(13), 132 (36), 92 (33), 63 (10). Anal. Calcd for C18 : C, 67.28;
H, 4.71; N, 13.08. Found: C, 67.50; H, 4.63; N, 12.79.
ꢃ
yl)-4H-1-benzopyran-4-one (4d). Yield 69%; mp 166e167 C; IR
15 3 3
H N O
ꢁ
1
1
(
(
KBr, cm
6
) nmax: 1709 (C]O); H NMR (500 MHz, DMSO-d ) d 2.27
s, 3H, Methyl), 6.03 (d, 1H, J ¼ 12.43 Hz, H-3), 6.42 (d, 1H,
0
0
J ¼ 12.42 Hz, H-2), 7.14 (d, 2H, J ¼ 7.97 Hz, H-2 and H-6 ), 7.17 (d,1H,
J ¼ 8.38 Hz, H-8), 7.22 (dt, 1H, J ¼ 7.52 and 0.75 Hz, H-6), 7.35 (d, 2H,
6.1.1.9. trans-2-(2-Chlorophenyl)-2,3-dihydro-3-(1H-1,2,4-triazol-1-
ꢃ
yl)-4H-1-benzopyran-4-one nitrate (4i). Yield 55%; mp 124e126 C;
0
0
ꢁ1
1
J ¼ 8.03 Hz, H-3 and H-5 ), 7.70 (dt, 1H, J ¼ 7.78 and 1.69 Hz, H-7),
IR (KBr, cm
)
n
max: 1714 (C]O); H NMR (500 MHz, DMSO-d
6
)
7
8
6
1
C
.89 (dd, 1H, J ¼ 7.87 and 1.57 Hz, H-5), 7.92 (s, 1H, triazole H-5),
d
6.48 (d, 1H, J ¼ 12.55 Hz, H-3), 6.65 (d, 1H, J ¼ 12.58 Hz, H-2), 7.19
13
.41 (s, 1H, triazole H-3). C NMR (125 MHz, DMSO-d
5.84, 82.13, 119.06, 120.41, 123.26, 127.98, 128.58, 129.86, 133.26,
38.11, 139.65, 146.39, 152.60, 161.79, 188.45. Anal. Calcd for
: C, 70.81; H, 4.95; N, 13.76. Found: C, 70.73; H, 5.08; N,
6
)
d
21.65,
(d, 1H, J ¼ 8.34 Hz, H-8), 7.25 (t, 1H, J ¼ 7.64 Hz, H-6), 7.36e7.45 (m,
0
0
0
3H, H-4 , H-5 and H-6 ), 7.72 (dt, 1H, J ¼ 7.75 and 0.94 Hz, H-7),
0
7.88e7.95 (m, 3H, H-3 , H-5 and triazole H-5), 8.59 (br s, 1H, triazole
13
18
H
15
N
3
O
2
H-3). C NMR (125 MHz, DMSO-d
123.24, 127.70, 128.14, 130.15, 130.34, 131.34, 132.70, 133.94, 137.90,
46.08, 151.95, 160.93, 187.03. Anal. Calcd for C17 13ClN : C,
52.52; H, 3.37; N, 14.41. Found: C, 52.79; H, 3.25; N, 14.40.
6
) d 64.43, 77.82, 118.53, 119.99,
14.00.
1
H
4 5
O
6
1
.1.1.5. trans-2,3-Dihydro-2-(4-methoxyphenyl)-3-(1H-1,2,4-triazol-
ꢃ
-yl)-4H-1-benzopyran-4-one (4e). Yield 74%; mp 104e105 C; IR
ꢁ
1
1
(
KBr, cm
)
n
max: 1710 (C]O); H NMR (500 MHz, DMSO-d
6
)
d
3.72
6.1.1.10. trans-2,3-Dihydro-2-(2-methoxyphenyl)-3-(1H-1,2,4-
(
s, 3H, OCH
3
), 6.01 (d, 1H, J ¼ 12.69 Hz, H-3), 6.43 (d, 1H,
triazol-1-yl)-4H-1-benzopyran-4-one nitrate (4j). Yield 62%; mp
0
0
ꢃ
ꢁ1
1
J ¼ 12.44 Hz, H-2), 6.88 (d, 2H, J ¼ 8.65 Hz, H-3 and H-5 ), 7.17 (d,
H, J ¼ 8.34 Hz, H-8), 7.21 (t, 1H, J ¼ 7.52 Hz, H-6), 7.40 (d, 2H,
141e143 C; IR (KBr, cm
)
n
max: 1713 (C]O); H NMR (500 MHz,
), 6.32 (d, 1H, J ¼ 12.62 Hz, H-3), 6.58
1
DMSO-d 3.71 (s, 3H, OCH
6
)
d
3

S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
487
0
0
(
1
d, 1H, J ¼ 12.61 Hz, H-2), 6.87e6.98 (m, 2H, H-3 and H-5 ), 7.16 (d,
H, J ¼ 8.29 Hz, H-8), 7.21 (t, 1H, J ¼ 7.22 Hz, H-6), 7.32 (dt, 1H,
6.2. Micro-dilution method for MICs determination
0
0
J ¼ 7.86 and 1.52 Hz, H-4 ), 7.60 (dd, 1H, J ¼ 7.45 and 1.33 Hz, H-6 ),
.69 (dt, 1H, J ¼ 7.79 and 1.65 Hz, H-7), 7.89 (dd, 1H, J ¼ 7.85 and
.52 Hz, H-5), 7.94 (br s, 1H, triazole H-5), 8.59 (s, 1H, triazole H-3).
The MICs of compounds were determined by micro-dilution
method [14]. A stock solution of compounds was prepared in
7
1
Sabouraud dextrose broth (SDB)/DMSO (9:1). Then, 200
stock solution was transferred into the plates with 96 U-shaped
wells and serially diluted by mixing with 100 L of SDB in subse-
quent wells. Then, aliquot of 100 L of fungal suspension
mL of the
1
3
C NMR (125 MHz, DMSO-d
20.42, 121.27, 123.21, 123.33, 128.04, 130.23, 131.85, 138.02, 158.33,
61.98, 188.55. Anal. Calcd for C18 : C, 56.25; H, 4.20; N,
6
) d 56.55, 64.78, 77.17, 112.49, 119.01,
1
1
1
m
16
H N
4
O
6
m
3
4.58. Found: C, 56.11; H, 4.33; N, 14.46.
(1 ꢀ 10 CFU/mL) was added to each well to reach the final inoc-
3
ulum size of 0.5 ꢀ10 CFU/mL. After 24, 48 and 72 h of incubation at
ꢃ
6
.1.1.11. trans-2-(2,4-Dichlorophenyl)-2,3-dihydro-3-(1H-1,2,4-
35 C, the plates were observed for the absence or presence of
triazol-1-yl)-4H-1-benzopyran-4-one (4k). Yield 70%; mp 155e
visible growth in comparison with that of the drug-free control
well. The endpoint MIC was the lowest concentration of the com-
pound at which the test strain does not demonstrate visible growth.
ꢃ
ꢁ1
1
157 C; IR (KBr, cm
) nmax: 1714 (C]O); H NMR (500 MHz, DMSO-
6
d ) d
6.47 (d, 1H, J ¼ 12.56 Hz, H-3), 6.64 (d, 1H, J ¼ 12.54 Hz, H-2),
7.20 (d, 1H, J ¼ 8.31 Hz, H-8), 7.25 (t, 1H, J ¼ 7.41 Hz, H-6), 7.56 (dd,
1
1
0
0
H, J ¼ 8.39 and 1.62 Hz, H-5 ), 7.60 (d, 1H, J ¼ 1.72 Hz, H-3 ), 7.72 (t,
H, J ¼ 7.14 Hz, H-7), 7.90e7.94 (d and s, 2H, H-5 and triazole H-5),
6.3. Docking study
0
13
7
(
1
1
.97 (d, 1H, J ¼ 8.41 Hz, H-6 ), 8.59 (s, 1H, triazole H-3). C NMR
3D-structures of compounds were generated using HyperChem
and geometrically optimized using semiempirical AM1 method in
Gaussian 98. FlexX, a fully automated docking program available on
LeadIT 2.0.2 package was used to dock compounds into the active
site of the enzyme. FlexX considers ligand flexibility by changing
the conformations of the ligand in the active site while making the
protein rigid. The X-ray coordinate of MTCYP51 in complex with
fluconazole (PDB id: 1EA1) was retrieved from Protein Data Bank
(http://www.rcsb.org). All crystallographic water molecules were
removed. The docking and subsequent scoring were performed
using the default parameters of the FlexX program implanted in
LeadIT 2.0.2. The active site of the enzyme was defined to include
125 MHz, DMSO-d
28.83, 130.05, 132.16, 132.47, 135.29, 135.92, 138.20, 146.53, 152.57,
61.33, 187.79. Anal. Calcd for C17 : C, 56.69; H, 3.08; N,
6
) d 64.79, 77.77, 118.96, 120.49, 123.61, 128.08,
2 3 2
H11Cl N O
11.67. Found: C, 56.73; H, 2.97; N, 11.70.
6
1
.1.2. Synthesis of trans-2,3-dihydro-2-phenyl-3-(1H-1,2,4-triazol-
-yl)-4H-1-benzopyran-4-one oxime (5a)
A solution of compound 4a (146 mg, 0.5 mmol) and hydroxyl-
amine hydrochloride (104 mg, 1.5 mmol) in methanol (5 mL) was
heated under reflux for 24 h. After completion of the reaction, the
reaction mixture was concentrated under reduced pressure and
mixed with water (15 mL). The mixture was left in the fridge
overnight. The precipitated solid was collected by filtration and
washed with water (3 ꢀ 5 mL). The crude product was recrystal-
lized from methanolewater to give compound 5a. Yield 79%; mp
ꢀ
residues within 6.5 A radius around bound inhibitor. Final scores for
all FlexX solutions were calculated by a consensus scoring function
(CScore) and used for database ranking. Finally the best pose with
the highest score was selected for investigating the interactions,
ꢃ
ꢁ1
1
2
42e244 C; IR (KBr, cm
)
n
max: 3442 (OH), 1604 (C]NOH);
H
HYDE assessment and calculating the free energy of binding (
[18,19].
DG)
NMR (500 MHz, DMSO-d
6
)
d
5.63 (d, 1H, J ¼ 2.75 Hz, H-3), 6.18 (d,
1
2
1
H, J ¼ 2.83 Hz, H-2), 7.07e7.12 (m, 2H, H-6 and H-8), 7.13e7.18 (m,
0
0
0
0
0
H, H-2 and H-6 ), 7.24e7.29 (m, 3H, H-3 , H-4 and H-5 ), 7.39 (dt,
H, J ¼ 7.77 and 1.57 Hz, H-7), 7.79 (s, 1H, triazole H-5), 7.93 (dd, 1H,
Acknowledgments
J ¼ 8.24 and 1.56 Hz, H-5), 7.96 (s, 1H, triazole H-3), 11.83 (s, 1H,
This work was supported by a grant from the Research Council
of Mazandaran University of Medical Sciences, Sari, Iran. A part of
this work was related to the Pharm.D thesis of Shahaboddin Sho-
japour (Faculty of Pharmacy, Mazandaran University of Medical
Sciences).
NOH). 13C NMR (125 MHz, DMSO-d
22.90, 124.08, 126.85, 128.99, 129.05, 131.66, 136.59, 145.66,
6
) d 53.76, 79.28, 118.78, 119.14,
1
þ
1
45.99,152.01,156.37. MS (m/z, %): 306 (M ,15), 289 (11), 237 (100),
20 (15), 178 (31), 121 (11), 102 (13), 77 (22), 63 (10), 51 (11). Anal.
Calcd for C17 : C, 66.66; H, 4.61; N, 18.29. Found: C, 66.90; H,
2
14 4 2
H N O
4
.45; N, 18.18.
Appendix A. Supplementary data
6
.1.3. Synthesis of trans-2-(2,4-dichlorophenyl)-2,3-dihydro-3-(1H-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.008.
1,2,4-triazol-1-yl)-4H-1-benzopyran-4-one oxime (5b)
A solution of compound 4k (180 mg, 0.5 mmol) and hydrox-
ylamine hydrochloride (104 mg, 1.5 mmol) in methanol (5 mL)
was heated under reflux for 68 h. The excess of methanol was
evaporated under reduced pressure. The concentrated solution
was mixed with water (15 mL) and the mixture was left in the
fridge overnight. The precipitated solid was collected by filtration
and washed with water (3 ꢀ 5 mL). The crude product was
recrystallized from methanolewater to give compound 5b. Yield
References
[1] M.J. Rüping, J.J. Vehreschild, O.A. Cornely, Drugs 68 (2008) 1941e1962.
[2] M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S. Mukta,
S. Gadhwe, Bioorg. Med. Chem. 20 (2012) 5678e5698.
[3] D.W. Denning, C.C. Kibler, R.A. Barnes, Lancet Infect. Dis. 3 (2003) 230e240.
[4] M.A. Hossain, M.A. Ghannoum, Exp. Opin. Invest. Drugs 9 (2000) 1797e1813.
[5] H. Vanden Bossche, L. Koymans, Mycoses 41 (1998) 32e38.
[6] D.C. Lamb, D.E. Kelly, S.L. Kelly, FEBS Lett. 425 (1998) 263e265.
ꢃ
ꢁ1
8
1%; mp 204e206 C; IR (KBr, cm
) nmax: 3436 (OH), 1591 (C]
[
7] S. Emami, M. Kazemi-Najafabadi, S. Pashangzadeh, A. Foroumadi,
M.A. Faramarzi, N. Samadi, M. Falahati, R. Fateh, M. Ashrafi-Khozani, Chem.
Biol. Drug Des. 78 (2011) 979e987.
1
NOH); H NMR (500 MHz, DMSO-d
3
6
)
d
5.78 (d, 1H, J ¼ 2.62 Hz, H-
), 6.24 (d, 1H, 2.69 Hz, H-2), 6.85 (d, 1H, J ¼ 8.48 Hz, H-8), 7.10e
0
[8] S. Emami, M. Falahati, A. Banifatemi, M. Amanlou, A. Shafiee, Bioorg. Med.
Chem. 12 (2004) 3971e3976.
7
5
.19 (m, 2H, H-6 and H-6 ), 7.27 (dd, 1H, J ¼ 8.48 and 1.98 Hz, H-
0
), 7.41 (dt, 1H, J ¼ 7.83 and 1.41 Hz, H-7), 7.71 (d, 1H, J ¼ 2.06 Hz,
[9] S. Emami, M. Falahati, A. Banifatemi, A. Shafiee, Bioorg. Med. Chem. 12 (2004)
0
H-3 ), 7.82 (s, 1H, triazole H-5), 7.93 (d, 1H, J ¼ 7.74 Hz, H-5), 8.17
5881e5889.
[
10] S. Emami, A. Foroumadi, M. Falahati, E. Lotfali, S. Rajabalian, S.-A. Ebrahimi,
S. Farahyar, A. Shafiee, Bioorg. Med. Chem. Lett. 18 (2008) 141e146.
11] S. Emami, M. Behdad, A. Foroumadi, M. Falahati, E. Lotfali, S. Sharifynia, Chem.
Biol. Drug Des. 73 (2009) 388e395.
(
s, 1H, triazole H-3), 12.00 (s, 1H, NOH). Anal. Calcd for
C
3
17 2 4 2
H12Cl N O : C, 54.42; H, 3.22; N, 14.93. Found: C, 54.41; H,
.53; N, 15.01.
[

488
S. Emami et al. / European Journal of Medicinal Chemistry 66 (2013) 480e488
[
[
[
12] R. Kharb, P.C. Sharma, M.S. Yar, J. Enzym. Inhib. Med. Chem. 26 (2010) 1e21.
13] S. Emami, A. Shafiee, Heterocycles 55 (2001) 2059e2074.
14] National Committee for Clinical Laboratory Standards, Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard
M27-A2, Wayne, PA (2006).
[16] L. Zhao, D. Liu, Q. Zhang, S. Zhang, J. Wan, W. Xiao, FEMS Microbiol. Lett. 277
(2007) 37e43.
[17] G. Vistoli, A. Pedretti, B. Testa, Drug Discov. Today 13 (2008) 285e294.
[18] I. Reulecke, G. Lange, J. Albrecht, R. Klein, M. Rarey, Chem. Med. Chem. 3
(2008) 885e897.
[
15] H.M. Guardiola, L. Foster, D. Mushrush, A.D. Vaz, Biochem. Pharmacol. 61
[19] N. Schneider, S. Hindle,G. Lange, R. Klein,J. Albrecht, H. Briem, K. Beyer, H.Claussen,
M. Gastreich, C. Lemmen, M. Rarey, J. Comput. Aided Mol. Des. 26 (2012) 701e723.
(
2001) 1463e1470.