Synthesis, QSAR and anticandidal evaluation of 1,2,3-triazoles derived from naturally bioactive scaffolds

Article abstract of DOI:10.1016/j.ejmech.2015.02.007

In the present study, we used eight natural precursors (1a-h) with most of them having promising antimicrobial activities and synthesised their novel 1,2,3-triazole derivatives (3a-h). In the reaction sequences, the precursor compounds (1a-h) were convert

Full text of DOI:10.1016/j.ejmech.2015.02.007

European Journal of Medicinal Chemistry 93 (2015) 246e254
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis, QSAR and anticandidal evaluation of 1,2,3-triazoles derived
from naturally bioactive scaffolds
,
Mohammad Irfan a b, Babita Aneja a, Umesh Yadava c, Shabana I. Khan d,
,
*
Nikhat Manzoor b, Constantin G. Daniliuc e, Mohammad Abid a
a Medicinal Chemistry Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India
b Medical Mycology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India
c Department of Physics, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur, UP 273009, India
d National Center for Natural Products Research (NCNPR), School of Pharmacy, University of Mississippi, MS 38677, USA
e Organisch-Chemisches Institut, Westfalische Wilhelm-Universitat Munster, 48149, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we used eight natural precursors (1a-h) with most of them having promising
antimicrobial activities and synthesised their novel 1,2,3-triazole derivatives (3a-h). In the reaction se-
quences, the precursor compounds (1a-h) were converted to their respective alkyne (2a-h) followed by
addition of benzyl azide freshly prepared by the reaction of benzyl bromide with sodium azide using
[3 þ 2] azide-alkyne cycloaddition strategy. Structural elucidation of all the triazole derivatives was done
using FT-IR, 1H, 13C NMR, mass and elemental analysis techniques. The single crystal X-ray diffraction for
3d was also recorded. The result of in vitro anticandidal activity performed against three different strains
of Candida showed that compound 3e was found superior/comparable to fluconazole (FLC) with IC50
Received 31 August 2014
Received in revised form
2 February 2015
Accepted 6 February 2015
Available online 7 February 2015
Keywords:
Candida
QSAR
values of 0.044
m
g/mL against Candida albicans (ATCC 90028), 12.022
mg/mL against Candida glabrata
(ATCC 90030), and 3.60
m
g/mL against Candida tropicalis (ATCC 750). Moreover, at their IC50 values,
Triazole
compounds 3e and 3h showed <5% hemolysis which indicates the non-toxic behaviour of these in-
hibitors. Cytotoxicity assay was also performed on VERO cell line and all the derivatives were found non-
toxic up to the concentration of 10.0 mg/mL. The in silico technique of 3D-QSAR was applied to establish
structure activity relationship of the synthesized compounds. The results reveal the molecular fragments
that play an essential role in improving the anticandidal activity.
X-ray diffraction
Hemolysis
Cytotoxicity
Fluconazole
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
flucytosine. Among them, azole drugs have an important class of
triazole antifungal agents like fluconazole, itraconazole, vor-
Fungal infections pose a continuous and serious threat to human
health. Among them, the incidents of Candida infection have
increased substantially in recent years especially in immune-
compromised patients and those with human immunodeficiency
virus infections. According to a report of CDC 2013, Candida is the
fourth most common cause of healthcare-associated bloodstream
infections in the United States. It is estimated that approximately
30% of patients with bloodstream infections (candidemia) with
drug-resistant Candida die during their hospitalization [1]. Anti-
fungal drugs currently used for the treatment of Candida infections
include polyenes, azoles, echinocandins, allyamines, and
iconazole and posaconazole (Fig. 1) which becomes the group of
most widely used therapeutic antifungal agents with low toxicity,
oral bioavailability, and broad spectrum [2]. However, the incidents
of drug resistance, specially the front line antifungal drug flucon-
azole, have increased with a very rapid rate. Therefore, there is an
urgent need to develop antifungal agents with novel structure and
mode of action.
The triazole based compounds are well known for their wide
range of biological activity such as antineoplastic [3], antimicrobial
[4], analgesic [5], anti-inflammatory [6], local anesthetic [7], anti-
convulsant [8], antimalarial [9] and anti-HIV agents [10]. Prompted
by these observations and in continuation our efforts to develop
structurally diverse organic scaffolds [11,12], we designed the
synthesis of new series of 1,2,3-triazole derivatives from biologi-
cally active natural scaffolds (Fig. 2). The main reason for the
* Corresponding author.
E-mail address: mabid@jmi.ac.in (M. Abid).
http://dx.doi.org/10.1016/j.ejmech.2015.02.007
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
247
Fig. 1. Structure of azole based antifungal drugs.
Fig. 2. Structure of precursor molecules (1a-h) used in the study.
synthesis was the diverse array of bioactivities possessed individ-
ually by them and to study the effect on the biological activity after
their conversion into 1,2,3-triazole derivatives.
purified by silica gel chromatography using 10% methanol in
dichloromethane as eluent to provide the title compounds (3a-h) in
good to excellent yields. The proposed structures were confirmed
by elemental analysis, FT IR, 1H, 13C NMR & Mass spectral data
(Table 1). The single crystal X-ray diffraction for 3d was also
recorded.
2. Results and discussion
2.1. Chemistry
2.1.1. IR analysis
In the IR spectra of all the compounds (3a-h), the main evidence
for their formation came from the disappearance of absorption
band at 2090 cmꢁ1corresponding to azido functionality indicating
that azide had been completely consumed in the reaction and the
peak corresponding to alkyne ≡CH and C^C which appeared in the
range 3250e3376 cmꢁ1 and 2112e2137 cmꢁ1 respectively was also
absent from their spectra. All the synthesized compounds (3a-h)
gave characteristic peak of triazole ring in the region
3065e3189 cmꢁ1 as broad absorption bands. All the other ab-
sorption bands also appeared at the expected regions.
A series of novel 1,2,3-triazole derivatives (3a-h) was synthe-
sized as shown in Scheme 1. The precursor molecules (1a-h) were
converted into 1,2,3-triazoles via copper catalyzed [3 þ 2] azide-
alkyne cycloaddition reaction. The hydroxyl group present in pre-
cursor compounds (1a-h) on treatment with propargyl bromide
and K2CO3 in anhyd. DMF at 0 ꢀC to room temperature gave the
corresponding alkyne derivatives (2a-h). The reaction was
completed in 18e24 h depending upon the structure of precursor
molecule. In the other set of reaction, benzyl bromide was treated
with sodium azide (NaN3) in anhyd. DMF under reflux condition to
give benzyl azide in excellent yield. Finally, azide-alkyne cyclo-
addition in ter. BuOH: H2O (1:2) mixture and catalytic amount of
copper sulphate and sodium ascorbate at room temperature for
48e72 h gave the title compounds (3a-h). The crude obtained was
2.1.2. 1H NMR analysis
In the 1H NMR spectra, these compounds showed a sharp singlet
in the region
d 7.18e7.67 ppm assigned to triazole ring, thus

248
M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
Br
K2CO3
,
O
HO R
R
DMF, 0o C - RT, 18-24 hrs
t-Butanol:Water (1:2),
RT, CuSO4.5H2O,
Br
NaN3, DMF
N3
Sod. ascorbate, 48- 72 hrs
reflux, 80o C, 18 hrs
N
N
N
O
R
Scheme 1. General route of synthesis for triazole derivatives (3a-h).
confirming its formation. The characteristic singlets at
5.54e4.66 ppm and 5.50e4.36 ppm were also observed for OCH2
and C6H5CH2 protons of triazole derivatives (3a-h) respectively.
While a sharp singlet corresponding to ≡CH group which appeared
rings N1eN2eN3eC2eC1, C11eC12eC13eC14eC15eC16 and
C21eC122eC23eC24eC25eC26 are planar [maximum deviations
of atoms from there least squares planes are 0.001(1)Å, 0.006(1)Å
and 0.014(1)Å, respectively]. The planes containing aromatic rings
C11eC16 and C21eC26 are inclined at an angle of 79.7(1)º while the
ring N1eC1 form a bridge between them. The unit cell contains four
molecules of similar conformations. In the crystal structure, the
neighboring two molecules are arranged in anti-parallel manner in
such a way that they form dimmer through intermolecular CeH …
d
d
in the region
d 2.49e2.57 ppm was absent from the spectra. In
addition, all the aromatic and aliphatic protons appeared at the
expected chemical shifts and integral values.
2.1.3. 13C NMR analysis
The cyclization of all alkynes with benzyl azide to triazole ring
was further reconfirmed by 13C NMR spectral data in which both
the carbons of triazole ring appeared in the range
p
interactions (Fig. 4). There are also weak intermolecular CeH/N
hydrogen bonding interactions involving C4 and N1 atom at sym-
metry position ꢁ1
C4eH4B/N1 ¼ 170.0].
þ
X, Y,
Z
[d(H4BeN1)
¼
2.590,
d
120.23e127.34 ppm and
of OCH2 and C6H5CH2 groups were resonated at
and 54.25e55.99 ppm respectively, while all the other carbons
d
138.32e145.92 ppm. The carbon signals
d
62.16e65.23 ppm
d
2.3. Anticandidal evaluation
gave peaks at their expected values.
The inhibitory potential of synthesized 1,2,3-triazole derivatives
(3a-h) was evaluated against three different strains of Candida: C.
albicans ATCC 90028, C. glabrata ATCC 90030 and Candida tropicalis
ATCC 750 and the results were compared with their precursors (1a-
2.1.4. Mass analysis
The mass spectra of all the compounds (3a-h) showed [MþH]þ
peak which is in accordance with the molecular formula of the
compounds thus further reconfirming their formation. Some of the
compounds showed [2 M þ Na]þ peak in the mass spectra which
suggests the dimerization of the molecular ion peak. In the mass
spectra of compound 3b, molecular ion peak was obtained with
very low intensity and a fragment peak 133.2 corresponding to [M-
h) as well as standard drug fluconazole (FLC). The IC50 values in mg/
mL and minimum fungicidal concentrations (MFC) were estimated
and the results are summarized in Table 3. The objective of this
study was to see the effect of structural transformation on the
original anticandidal activity attributed to these natural precursors.
The results showed that the original bioactivity of precursor mol-
ecules (1a-h) was lost except in case of 1e and 1h which showed
many fold enhanced activity after structural transformation into
triazoles i.e. 3e and 3h, respectively. Compounds 1a, 1c, and 1g
were found to be good inhibitors of C. tropicalis with IC50 values of
C
10H10N3O]þ was observed as the base peak.
2.2. X-ray crystallographic analysis
Crystal structure analysis of compound 3d reveals that it crys-
tallizes in monoclinic crystal system with space group P21/c. The
data collection and structure refinement details are described in
Table 2. 17347 reflections collected were collected out of which
2628 reflections were independent with Rint ¼ 0.041. The number of
69.50
their structural transformed 3a, 3c, and 3g were found less active
with IC50 values of 493.17 g/mL, 105.12 g/mL, and 734.00 g/mL
against same organism. Compounds 1b showed very good anti-
candidal activity against C. glabrata with IC50 value of 7.09 g/mL as
compare to its triazole derivative 3b which showed poor IC50 value
of 316.15 g/mL. Similarly, compounds 1d and 1f exhibited mod-
erate anticandidal activity against all tested strains with IC50 value
<350 g/mL and <130 g/mL, respectively but their structural de-
rivatives 3d and 3f showed less effect on the growth of Candida spp.
mg/mL, 67.60 mg/mL, and 92.68 mg/mL, respectively while
m
m
m
observed reflections having I > 2
s
(I) was 2386. Finally, after
m
refinement, a reasonably good R factor was found (R ¼ 0.036). The
hydrogen atom at C2 was refined freely but others were calculated
and refined as riding atoms. The ORTEP diagram [13] of the com-
pound 3d with ellipsoids drawn at 50% probability level along with
atomic numbering scheme is shown in Fig. 3. All the three aromatic
m
m
m

M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
249
Table 1
Table 2
Structure of 1,2,3-triazole derivatives (3a-h).
Crystal data and structure refinement details for 3d.
Identification code
Empirical formula
Formula weight
3d
C
17 H17 N3 O2
R
295.34
223(2) K
O
Temperature
N
N
N
Wavelength
Crystal system, space group
Unit cell dimensions
0.71073 Å
monoclinic, P21/c
a ¼ 5.6192(1) Å, b ¼ 15.4234(1) Å,
c ¼ 17.2628(1) Å,
1492.03(3) Å3
4, 1.315 Mg/m3
0.715 mmꢁ1
624
b
¼ 94.236(1)ꢀ
Compound
R
Structure of compound
Mol.
formula
Reaction
time
Volume
Z, calculated density
Absorption coefficient
F(000)
3a
1a
C19H21N3O
18 h
Crystal size
Theta range for data collection
Limiting indices
0.15 ꢂ 0.12 ꢂ 0.07 mm
3.85e67.32ꢀ
O
N
N
N
0 ꢃ h<¼6, 0 ꢃ k<¼18, ꢁ20 ꢃ l<¼20
17347/2628 [R(int) ¼ 0.041]
97.8%
Reflections collected/unique
Completeness to theta ¼ 67.32
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
R indices (all data)
3b
3c
1b
1c
C20H23N3O
72 h
Semi-empirical from equivalents
0.9517 and 0.9003
O
N
Full-matrix least-squares on F2
2628/0/204
N
N
1.067
C20H19N3O4 40 h
O
R1 ¼ 0.0368, wR2 ¼ 0.0940
R1 ¼ 0.0402, wR2 ¼ 0.0975
0.116 and ꢁ0.177 e.Åꢁ3
OH
Largest diff. peak and hole
O
N
N
N
N
N
N
OCH3
N
N
The MFC values favoured the fungistatic nature of precursor com-
pounds (1a-h) and their 1,2,3-triazole derivatives (3a-h).
3d
3e
3f
1d
1e
1f
C17H17N3O2 50 h
O
O
O
O
N
OCH3
2.4. Synergistic studies
C19H16N4O
69 h
The in vitro synergistic anticandidal activity of most potent de-
rivative 3e in combination with FLC was determined for all the
three strains of Candida (Table 4). The results showed that com-
pound 3e alone showed good inhibitory activities against all tested
Candida species. The fractional inhibitory concentration index (FICI)
values of compound 3e were 0.74 for C. albicans, 0.48 for C. glabrata
and 0.74 for C. tropicalis, respectively. The results of synergistic
studies indicated that compound 3e showed no synergistic activity
in combination with FLC against C. albicans and C. tropicalis but
showed moderate synergy against C. glabrata.
N
N
N
C20H21N3O2 64 h
C20H21N3O2 18 h
C18H17N3O3 64 h
N
OCH3
OCH3
N
N
3g
3h
1g
1h
N
N
O
2.5. Cytotoxicity assays
H
2.5.1. Hemolysis
O
On the basis of in vitro anticandidal activity results, compounds
3e, 3h and their precursors (1e and 1h) were selected to check their
cytotoxic effect on human red blood cells (hRBCs) by hemolytic
assay. The toxicity of standard drug fluconazole was also deter-
N
OCH3
N
Very interestingly, precursor compound 1e, containing a quinoline
ring with IC50 value of 13.80 g/mL against C. glabrata, showed
m
mined for reference. At the concentration of 500
mg/mL, com-
many fold increase in the anticandidal activity after its conversion
into triazloe derivative 3e. The derivative 3e emerged as most
pounds 1e, 1h, 3e and 3h showed 14.97, 5.45, 4.76 and 33.5%
hemolysis while standard drug fluconazole showed 68.63% hemo-
lysis (Fig. 5). At their IC50 values, compounds 1e, 1h, 3e and 3h
showed <5% hemolysis which indicates that these compounds have
very low or no cytotoxic effect on human RBCs.
potent inhibitor with IC50 values of 0.044
12.02 g/mL against C. glabrata, and 3.60
m
g/mL against C. albicans,
m
mg/mL against C. tropicalis
and the results were superior/comparable to standard drug FLC.
Another naturally bioactive precursor 1h showed increased inhib-
itory activity after structural conversion into triazole derivative 3h.
Compound 3h with free eCHO group and 1,2,3-triazole ring
2.5.2. Cytotoxicity by cell proliferation assay
To examine the toxicity of all triazole derivatives (3a-h) on cell
proliferation, they were screened for cytotoxicity against monkey
kidney fibroblasts (VERO) and the results are shown in Table 3. A
subconfluent population of VERO cells was treated with increasing
concentration of active compounds and the number of viable cells
was measured after 48 h by MTT cell viable assay. The IC50 values
were calculated and Doxorubicin was used as control. None of the
showed good to moderate activity with IC50 value of 44.67
mg/mL
against C. albicans, 215.77 g/mL against C. glabrata, and 92.68
m
mg/
mL against C. tropicalis. The study suggested that anticandidal ac-
tivity of precursors (1a-h) was mainly due to their free eOH group
which is lost after conversion into 1,2,3 triazole derivatives (3a-h).
The enhanced anticandidal activity of compounds 3e and 3h might
be occurred due to presence of quinoline ring and free aldehyde
group, respectively along with 1,2,3 triazole ring in their structures.
compound was found cytotoxic upto the concentration of 10
mL.
mg/

250
M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
Fig. 3. ORTEP diagram of compound 3d with atomic labeling scheme (Displacement ellipsoids are drawn at 50% probability level).
Compounds 1b, 1c, 3b and 3c were chosen for test set while others
for training set. Among 163 descriptors, that were considered in
generating the QSAR model, few descriptors such as partial positive
surface area, partial negative surface area, total molecular surface
area, XY or YZ shadow, average information content, total number
of ‘O’ or ‘N’ atoms, number of aromatic rings etc. play important
roles in developing the QSAR model. Five descriptors were used to
develop the QSAR model. The calculated statistical parameters
(R2 ¼ 0.995 and F ¼ 255.179) based on the Heuristic method, were
found to be quite good to be considered as the best QSAR model. In
addition, the randomization test is also good indicating the better
model. Also, for the validation of the QSAR model, we went through
the test set via the training set. The results obtained for the test set
is R2
¼ 0.999, Ftest ¼ 3692.1509. RMSEs are found to be 0.5 and
test
0.6% respectively for training and test sets respectively. The squared
correlation coefficient for the test set and training set near ‘1’ and
very small values of RMSEs prove the validity of the generated
QSAR model.
2.6.2. QSAR model 2
In the second QSAR model the compounds which were chosen
for test set are 1b, 1h, 3e and 3h. Other compounds were used as
the training set. QSAR model developed using heuristic method,
based on the inhibition activities of triazole derivatives against
C. glabrata indicate that the squared cross correlation factor and
Fischer values are 0.958 and 27.327 for training set of 12 com-
pounds while 0.997 and 1868.48 for test set of 4 compounds. A total
of 163 descriptors were calculated which include geometrical,
constitutional, topological, electrostatic, quantum mechanical and
thermodynamic descriptors. The five descriptors which play
important role in developing QSAR model are minimum partial
charge for C-atom, weighted partial positive surface area, number
of ‘O’ atoms, average information content and HACA-1. RMSEs as
obtained through calculations are found to be 8.6% and 0.9%
respectively. The validation test on the model clearly demonstrate
that the developed model represent the valid model in which
electrostatic descriptors play important roles.
Fig. 4. Unit cell diagram in the crystal packing of compound 3d showing intermo-
lecular CeH …
p hydrogen bonding.
2.6. QSAR studies
The data set of 16 compounds was divided randomly into
training set of 12 compounds and test set of 4 compounds (75 and
25% respectively of the total number of compounds). When these
compounds were subjected to QSAR analysis, in order to develop
QSAR models, various statistically significant two parametric
models were obtained. For each type of descriptors, the best multi-
linear regression equations were obtained by the stepwise selec-
tion methods of Heuristic subroutine of the software. The squared
correlation coefficient (R2), standard error of estimate (r2_se) and
Fisher's value (F) which represents the F-ratio between the variance
of actual and predicted activity, were employed to judge the val-
idity of the model. The results of regression models are shown.
2.6.3. QSAR model 3
On the basis of experimental activities of compounds for the
inhibition of C. tropicalis, QSAR model is developed using Heuristic
method. Four compounds chosen for test set are 1e, 1c, 3f and 3g
while rests were used for training set. Developed QSAR model on
training set show that the cross correlation factor and Fischer
values are reasonably good (R2 ¼ 0.923, F ¼ 31.793). Among 163
calculated descriptors, five descriptors namely; fractional HDSA-1,
2.6.1. QSAR model 1
In the first model, QSAR study was carried out on the activities of
a series of substituted triazole derivatives for C. albicans inhibitors.

M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
251
Table 3
Anticandidal evaluation of 1,2,3-triazoles and their precursor molecules.
Compound
Anticandidal properties
Cytotoxicity
VERO
C. albicans ATCC 90028
C. glabrata ATCC 90030
IC50 S.D. ( g/mL)
C. tropicalis ATCC 750
IC50 S.D. ( g/mL)
IC50 S.D. (
m
g/mL)
MFC (
m
g/mL)
m
MFC (
m
g/mL)
m
MFC (
mg/mL)
IC50 (mg/mL)
1a
1b
1c
1d
1e
1f
1g
1h
3a
3b
3c
3d
3e
3f
181.55 0.33
81.47 1.10
210.37 0.69
179.06 0.18
18.47 0.06
82.98 0.89
236.59 1.18
321.36 0.60
549.67 1.34
324.49 0.46
891.25 0.68
514.63 0.94
0.044 0.11
480.83 1.23
1094.4 1.87
44.67 0.76
15.62 0.65
ND
500
1000
>1000
>1000
500
153.46 0.76
7.09 0.64
>1000
500
>1000
>1000
500
69.50 0.56
44.77 0.08
67.60 0.78
201.37 1.02
22.90 0.91
127.73 0.73
92.68 0.59
123.40 0.79
493.17 0.87
169.43 0.83
105.12 0.07
165.12 0.70
3.60 0.07
>1000
1000
>1000
>1000
500
ND
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
NA
NA
NA
NA
NA
ND
>5
616.59 0.55
335.73 0.92
13.80 0.70
71.61 0.66
215.77 0.81
483.06 1.21
1063.8 1.74
316.15 0.94
416.48 0.57
369.83 0.94
12.02 0.02
292.76 0.93
847.23 1.32
215.77 1.41
7.81 0.88
1000
500
1000
>1000
>1000
1000
>1000
>1000
ND
>1000
>1000
1000
>1000
>1000
>1000
125
>1000
>1000
500
>1000
>1000
1000
>1000
>1000
ND
62.5
>1000
ND
62.5
ND
154.00 0.85
734.00 1.37
92.68 0.83
8.55 1.59
3g
3h
FLC
D
>1000
>500
ND
500
ND
ND
ND
ND
ND
ND
ND
FLC ¼ Fluconazole, D ¼ Doxorubicin, NA ¼ no activity upto 10
m
g/mL, ND ¼ not done.
Table 4
designed and synthesized as potent inhibitors of candida species.
Compound 3e and 3h showed many fold increase in the anti-
candidal activity in comparison to their precursors and were also
found non-toxic by hemolytic assay as well on cell proliferation
assay on VERO cells. QSAR models were developed using Heuristic
method on the studied compounds. The FICI results indicated that
there is no synergistic activity in compound 3e in combination with
FLC against C. albicans and C. tropicalis but showed moderate syn-
ergy against C. glabrata. Further structural optimization, in vivo
studies and their mode of action may lead to the development of
these prototype molecules into potential antifungal agents.
In vitro Synergistic anticandidal activity of compound 3e.
Organism
MIC alone
3e FLC
MIC in combination FICIa Mode of
interaction
3e
FLC
C. albicans ATCC 90028 15.62 31.25 7.81
C. glabrata ATCC 90030 31.25 15.62 7.81
C. tropicalis ATCC 750
7.81
3.90
3.90
0.74 Indifferent
0.48 Synergy
0.74 indifferent
15.62 15.62 7.81
a
Synergy and antagonism were defined by FICI ꢃ0.5 and >4, respectively.
Indifferent was defined by 0.5 < FICI ꢃ4.
4. Experimental section
4.1. Chemistry
All the reagents and solvents purchased from Sigma-Aldrich, S.D.
Fine, SRL and Hi Media, were used without further purification.
Reaction was monitored by thin-layer chromatography (TLC) using
pre coated aluminium sheets (Silica gel 60 F254, Merck Germany)
and spots were visualized under UV light. The IR spectra of the
compounds were recorded on Agilent Cary 630 FT-IR spectrometer.
1H NMR and 13C NMR spectra were obtained in CDCl3with reference
to TMS as internal standard using Bruker Spectrospin DPX-300
spectrometer at 300 MHz and 75 MHz respectively. Splitting pat-
terns are designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. Chemical shift (d) values are given in parts per million
Fig. 5. Hemolysis activity of compounds of 1e, 1h, 3e, 3h and FLC.
(ppm) and coupling constants (J) in Hertz (Hz). Mass spectra were
carried out on Agilent LC- 1200 series MS/MS-6410 spectrometer.
CHN analysis was recorded on Elemental Vario analyzer. Melting
points were recorded on a digital Buchi melting point apparatus
(Mꢁ560) and were reported uncorrected. Purification of the com-
pounds was done by column chromatography using silica gel
(230e400 mesh size) with Petroleum Ether/Ethyl acetate (8:2) as
eluent for alkynes and 10% methanol in DCM for triazole derivatives.
fractional PPSA, relative number of rings, number of ‘O’ atoms and
HDCA-1 were used for developing the model. To validate the model,
we went through the test set via training set. The results obtained
are R2test ¼ 0.835, Ftest ¼ 10.133. The validation tests on the model
demonstrate 13.8 and 19.1% root mean square error for training and
test sets respectively.
4.1.1. General procedure for the synthesis of alkynes (2a-h)
A round bottom flask with stir bar was charged with natural
precursor (1eq) and anhyd. DMF (10 mL) and the reaction flask was
cooled to 0 ꢀC. To this solution, potassium carbonate (1eq) was
added, followed by slow addition of propargyl bromide (1.2 eq). The
3. Conclusion
In summary, a novel series bearing 1,2,3-triazole ring (3a-h) was

252
M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
reaction mixture was allowed to warm to room temperature and
stirred overnight under argon. After completion of the reaction as
confirmed by TLC, it was concentrated and water was added to the
residue. The compound was extracted with ethyl acetate, dried over
anhydrous sodium sulphate and concentrated under vacuum. The
crude was purified by column chromatography using 20% ethyl
acetate in petroleum ether to give the desired alkynes (2a-h) in
good to excellent yields [14].
(C13H14O2) calc. C 77.20 H 6.98, found: C 77.18 H 6.96%. IR (neat): n
(cmꢁ1) 3313, 2920, 2851, 2125, 1656, 1560, 1510, 1486, 1378, 1266,
1235,1143,1125,1033, 996, 912, 799, 722. 1H NMR (300 MHz, CDCl3)
(
d
, ppm): 6.96 (d, 1H, J ¼ 8.7 Hz, AreH), 6.73- 6.67 (m, 2H, AreH),
6.00e5.89 (m,1H, ¼CH), 5.11e5.05 (m, 2H, ¼CH2), 4.73 (s, 2H, OCH2),
3.86 (s, 3H, OCH3), 3.34 (d, 2H, J ¼ 6.6 Hz, CH2), 2.49 (s, 1H, ≡CH).
4.1.1.8. 3-Methoxy-4-(prop-2-ynyloxy)benzaldehyde (2h). Off white
solid, yield: 98%,Rf (Ethyl acetate/Pet. ether, 20:80) ¼ 0.54, Anal
4.1.1.1. 1-Isopropyl-4-(prop-2-ynyloxy)benzene (2a). Pale yellow oil,
yield: 92%, Rf (Ethyl acetate/Pet. ether, 20:80) ¼ 0.54, Anal
(C11H10O3) calc. C 69.46 H 5.30, found: C 69.43 H 5.27%. IR (neat):
n
(cmꢁ1) 3250, 2987, 2926, 2859, 2738, 2116, 1693, 1592, 1510, 1456,
1382,1337,1272,1223,1127,1036,1013, 927, 860, 814, 782, 720, 694.
(C12H14O) calc. C 82.72 H 8.10, found: C 82.69 H 8.08%. IR (neat):
n
(cmꢁ1) 3293, 2981, 2872, 1613, 1587, 1512, 1460, 1330, 1290, 1264,
1H NMR (300 MHz, CDCl3) (
d, ppm): 9.92 (s,1H, CHO), 7.49e7.44 (m,
1218, 1182, 1030, 990, 830, 672. 1H NMR (300 MHz, CDCl3) (
d
, ppm):
2H, AreH), 7.15 (d, 1H, J ¼ 8.1 Hz, AreH), 4.87 (s, 2H, CH2), 3.95 (s,
7.19 (d, 2H, J ¼ 8.1 Hz, AreH), 6.94 (d, 2H, J ¼ 8.1 Hz, AreH), 4.67 (s,
2H, OCH2), 2.94e2.88 (m, 1H, CH), 2.54 (s, 1H, ≡CH), 1.26 (d, 6H,
J ¼ 6.9 Hz, CH3).
3H, OCH3), 2.57 (s, 1H, ≡CH).
4.1.2. Procedure for the synthesis of benzyl azide
A mixture of benzyl bromide (1 eq) and sodium azide (3 eq) in
anhyd. DMF (10 mL) was stirred overnight at 70 ꢀC. After comple-
tion of the reaction as monitored by TLC, the reaction was quenched
with water. The crude was extracted with ethyl acetate, washed
with brine, dried over anhydrous sodium sulphate and concen-
trated under vacuum. The oily residue was used as such without any
further purification [15].
4.1.1.2. 1-Isopropyl-4-((prop-2-ynyloxy)methyl)benzene
Pale yellow oil, yield: 73%, Rf (Ethyl acetate/Pet. ether,
(2b).
20:80) ¼ 0.65, Anal (C13H16O) calc. C 82.94 H 8.57, found: C 82.91 H
8.55%. IR (neat):
n
(cmꢁ1) 3339, 2981, 2931, 2131, 1672, 1524, 1475,
1418, 1387, 1359, 1254, 1212, 1062, 1021, 854, 815, 745, 710. 1H NMR
(300 MHz, CDCl3) (
d, ppm): 7.43 (d, 2H, J ¼ 8.7 Hz, AreH), 7.31 (d,
2H, J ¼ 8.7 Hz, AreH),4.61 (s, 2H, OCH2), 4.32 (s, 2H, OCH2),
2.92e2.89 (m, 1H, CH), 2.52 (s,1H, ≡CH), 1.23 (d, 6H, J ¼ 7.9 Hz, CH3).
4.1.3. General procedure for the synthesis of triazole derivatives
(3a-h)
4.1.1.3. (E)-3-(3-methoxy-4-(prop-2-ynyloxy)phenyl)acrylic
acid
(2c). Off-white solid, yield: 98%, Rf (Ethyl acetate/Pet. ether,
50:50) ¼ 0.54, Anal (C13H12O4) calc. C 67.23 H 5.21, found: C 67.20 H
Equimolar amounts of alkyne (2a-h) and benzyl azide were
dissolved in ter-butanol and water (1:2) mixture. To this reaction
mixture, copper sulphate (0.05 eq) and sodium ascorbate (0.01 eq)
were added and stirred at room temperature till the disappearance
of starting materials as indicated by TLC. The reaction was
quenched with saturated brine and crude was extracted with ethyl
acetate, dried over anhydrous Na2SO4 and concentrated under
vacuum. The crude was purified by column chromatography using
10% methanol in DCM as eluent to give 1,2,3-triazole derivatives in
good to excellent yields [16].
5.19%. IR (neat):
n
(cmꢁ1) 3376, 3298, 2959, 2926, 2855, 2125, 1710,
1623, 1596, 1511, 1487, 1413, 1310, 1257, 1143, 1023, 954, 724. 1H
NMR (300 MHz, CDCl3) (
d
, ppm): 7.68 (d, 1H, J ¼ 15.9 Hz, ¼CH),
7.06e7.02 (m, 2H, AreH), 6.92 (d, 1H, J ¼ 8.1 Hz, AreH), 6.31 (d, 1H,
J ¼ 15.9 Hz, ¼CH), 4.81 (s, 2H, OCH2), 3.92 (s, 3H, OCH3), 2.55 (s, 1H,
≡CH).
4.1.1.4. 1-Methoxy-2-(prop-2-ynyloxy)benzene (2d). Yellow oil,
yield: 98%, Rf (Ethyl acetate/Pet. ether, 20:80) ¼ 0.62, Anal
(C10H10O2) calc. C 74.06 H 6.21, found: C 74.03 H 6.18%. IR (neat):
n
4.1.3.1. 1-Benzyl-4-((4-isopropylphenoxy)methyl)-1H-1,2,3-triazole
(3a). White solid, M.pt. 90e92 ꢀC, yield: 90%, Rf (Ethyl acetate/Pet.
ether, 30:70) ¼ 0.48, Anal (C19H21N3O) calc. C 74.24 H 6.89 N 13.67,
(cmꢁ1) 3286, 3070, 2936, 2840, 2125, 1674, 1596, 1503, 1462, 1376,
1331, 1253, 1212, 1182, 1127, 1026, 929, 847, 743. 1H NMR (300 MHz,
CDCl3) (d, ppm): 7.35e7.32 (m, 2H, AreH), 7.24e7.21 (m, 2H, AreH),
4.92 (s, 2H, OCH2), 3.87 (s, 3H, OCH3), 2.52 (s, 1H, ≡CH).
found: C 74.20 H 6.85 N 13.64%. IR (neat):
n
(cmꢁ1) 3126, 3070,
3041, 2962, 2869, 1614, 1588, 1514, 1462, 1383, 1365, 1305, 1287,
1249, 1220, 1182, 1134, 1115, 1063, 1030, 1007, 851, 832, 769, 702,
4.1.1.5. 8-(Prop-2-ynyloxy)quinoline (2e). Dark brown solid, yield:
66%, Rf (Ethyl acetate/Pet. ether, 20:80) ¼ 0.21, Anal (C12H9NO) calc.
680. 1H NMR (300 MHz, CDCl3) (
d, ppm): 7.59 (s, 1H, triazole ring),
7.38e7.36 (m, 3H, AreH), 7.28e7.26 (m, 2H, AreH), 7.13 (d, 2H,
J ¼ 8.1 Hz, AreH), 6.89 (d, 2H, J ¼ 8.4 Hz, AreH), 5.53 (s, 2H, OCH2),
5.16 (s, 2H, CH2), 2.90e2.81 (m, 1H, CH), 1.21 (d, 6H, J ¼ 6.9 Hz, CH3).
C 78.67 H 4.95 N 7.65, found: C 78.65 H 4.93 N 7.62%. IR (neat):
n
(cmꢁ1) 3319, 2925, 2854, 2112, 1855, 1573, 1503, 1473, 1410, 1369,
1316, 1264, 1182, 1104, 1019, 996, 825, 795, 754, 728, 713, 665. 1H
13C NMR (75 MHz, CDCl3) (
d, ppm): 156.29, 141.71, 134.45, 129.15,
NMR (300 MHz, CDCl3) (
(d, 1H, J ¼ 7.5 Hz, AreH), 7.56e7.47 (m, 3H, AreH), 7.32 (d, 1H,
J ¼ 8.1 Hz, AreH), 5.41 (s, 2H, OCH2), 2.56 (s, 1H, ≡CH).
d
, ppm): 8.82 (d, 1H, J ¼ 8.7 Hz, AreH), 8.12
128.84, 128.16, 127.34, 114.60, 62.16, 54.43, 33.27, 24.19. LC-MS (m/
z): 308.3[MþH] þ, 637.4 [2 M þ Na]þ.
4.1.1.6. (E)-2-methoxy-4-(prop-1-enyl)-1-(prop-2-ynyloxy)benzene
(2f). Off-white solid, yield: 98%, Rf (Ethyl acetate/Pet. ether,
20:80) ¼ 0.68, Anal (C13H14O2) calc. C 77.20 H 6.98, found: C 77.18 H
4.1.3.2. 4-((4-Isopropylbenzyloxy)methyl)-1-benzyl-1H-1,2,3-triazole
(3b). Yellow oil, yield: 76%, Rf (Ethyl acetate/Pet. ether,
30:70) ¼ 0.61, Anal (C20H23N3O) calc. C 74.74 H 7.21 N 13.07, found:
6.97%. IR (neat):
n
(cmꢁ1) 3285, 3019, 2916, 2937, 2121, 1674, 1587,
C 74.71 H 7.19 N 13.05%. IR (neat): n
(cmꢁ1) 3189, 2961, 2929, 2872,
1510, 1452,1415,1378,1300,1257,1218,1140,1024, 964, 924, 910, 786
1516, 1460, 1423, 1385, 1365, 1257, 1207, 1059, 1017, 843, 817, 752,
.1H NMR (300 MHz, CDCl3) (
d, ppm): 6.96e6.86 (m, 3H, AreH), 6.36
700. 1H NMR (300 MHz, CDCl3) (
d, ppm): 7.57 (s, 1H, triazole ring),
(d, 1H, J ¼ 15.6 Hz, ¼CH), 6.17e6.10 (m, 1H, ¼CH), 4.76 (s, 2H, OCH2),
3.89 (s, 3H, OCH3), 2.51 (s, 1H, ≡CH), 1.88 (d, 3H, J ¼ 6.6 Hz, CH3).
7.43e7.24 (m, 9H, AreH), 4.66 (s, 4H, OCH2), 4.36 (s, 2H, CH2),
2.99e2.90 (m, 1H, CH), 1.28 (d, 6H, J ¼ 6.9 Hz, CH3). 13C NMR
(75 MHz, CDCl3) (d, ppm): 148.48, 138.32, 135.38, 128.88, 128.35,
4.1.1.7. 4-Allyl-2-methoxy-1-(prop-2-ynyloxy)benzene
Yellowoil, yield: 79%, Rf (Ethyl acetate/Pet. ether, 20:80) ¼ 0.56, Anal
(2g).
128.26, 127.24, 126.66, 65.23, 54.82, 33.90, 29.74, 24.05. LC-MS (m/
z): 322.2[MþH]þ,133.2 [M-C10H10N3O]þ.

M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
253
4.1.3.3. (E)-3-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)-3-
5.40 (s, 2H, OCH2), 5.04e4.97 (m, 2H, ¼CH2), 4.66 (s, 2H, CH2), 3.80
(s, 3H, OCH3), 3.27 (t, 2H, J ¼ 6.9 Hz, CH2). 13C NMR (75 MHz, CDCl3)
methoxyphenyl)acrylic acid (3c). Off-white solid, M.pt. 83e85 ꢀC,
yield: 90%, Rf (Ethyl acetate/Pet. ether, 30:70)
(C20H19N3O4) calc. C 65.74 H 5.24 N 11.50, found: C 65.72 H 5.22 N
11.53%. IR (neat):
(cmꢁ1) 3093, 3037, 2925, 2854, 1734, 1707, 1637,
1599, 1514, 1458, 1428, 1313, 1264, 1145, 1056, 1007, 989, 814, 724, 702,
668.1H NMR (300 MHz, CDCl3)(
, ppm):7.63 (s,1H, triazolering), 7.59
¼
0.25, Anal
(d, ppm): 149.59, 145.92, 137.52, 134.47, 133.91, 129.11, 128.77,
128.14, 122.84, 120.54, 115.72, 114.28, 112.34, 111.14, 63.56, 55.82,
n
54.22, 39.82. LC-MS (m/z):336.3 [MþH] þ, 693.4 [2M þ Na]þ.
d
4.1.3.8. 4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-3-
methoxybenzaldehyde (3h). Off white solid, M.pt. 79e81 ꢀC, yield:
83%,Rf (Ethyl acetate/Pet. ether, 30:70) ¼ 0.18, Anal (C18H17N3O3)
calc. C 66.86 H 5.30 N 13.00, found: C 66.85 H 5.27 N 12.96%. IR
(d, 1H,J ¼ 5.7 Hz, ¼CH), 7.36e7.27 (m, 5H, AreH), 7.03 (d, 2H,
J ¼ 11.4 Hz, AreH), 6.90 (d, 1H, J ¼ 8.1 Hz, AreH), 6.30 (d, 1H,
J ¼ 10.8 Hz, ¼CH), 5.52 (s, 2H, OCH2), 5.31 (s, 2H, CH2), 3.83 (s, 3H,
OCH3). 13C NMR (75 MHz, CDCl3) (
d, ppm): 166.97, 149.79, 148.28,
(neat):
1425,1397,1341,1261, 1160,1136, 1030, 994, 855, 810, 784, 758, 720,
700. 1H NMR (300 MHz, CDCl3) (
, ppm): 9.86 (s, 1H, CHO), 7.59 (s,
1H, triazole ring), 7.46e7.37 (m, 5H, AreH), 7.29e7.22 (m, 3H,
AreH), 5.54 (s, 2H, OCH2), 5.37 (s, 2H, CH2), 3.91 (s, 3H, OCH3). 13
NMR (75 MHz, CDCl3) ( , ppm): 190.90, 153.03, 149.95, 143.62,
n
(cmꢁ1)3150, 2959, 2937, 2857, 1678, 1585, 1508, 1460,
146.89, 145.28, 134.40, 129.16, 128.84, 128.17, 126.74, 123.65, 122.52,
115.53,113.72,110.17, 63.04, 55.88, 54.26. LC-MS(m/z):366.2 [MþH]þ
.
d
4.1.3.4. 1-Benzyl-4-((2-methoxyphenoxy)methyl)-1H-1,2,3-triazole
(3d). Brown crystalline solid, M.pt. 119e121 ꢀC, yield: 78%, Rf (Ethyl
acetate/Pet. ether, 30:70) ¼ 0.18, Anal (C17H17N3O2) calc. C 69.14 H
C
d
134.27, 130.64, 129.17, 128.89, 128.18, 126.68, 123.10, 112.69, 109.27,
5.80 N 14.23, found: C 69.11 H 5.77 N 14.21%. IR (neat):
n
(cmꢁ1
)
62.98, 55.99, 54.32. LC-MS (m/z):324.3 [MþH] þ, 669.3 [2 M þ Na]þ.
3131, 2972, 2931, 2877, 2834, 1685, 1588, 1497, 1452, 1434, 1389,
1313, 1251, 1216, 1181, 1121, 1059, 1028, 998, 918, 860, 808, 745, 672.
4.2. X-ray crystallographic analysis
1H NMR (300 MHz, CDCl3) (
d, ppm): 7.57 (s, 1H, triazole ring),
7.38e7.36 (m, 3H, AreH), 7.28e7.26 (m, 2H, AreH), 7.05 (d, 1H,
J ¼ 7.5 Hz, AreH), 6.92 (d, 1H, J ¼ 8.1 Hz, AreH), 6.89e6.86 (m, 2H,
The structure of the compound 3d was unequivocally estab-
lished by X-ray crystallographic analysis. Single crystal of 3d was
obtained through the slow evaporation of its ethanol solution. A
suitable crystal (C17H17N3O2, compound 3d) was selected and
analysed on a Nonius Kappa CCD APEXII diffractometer equipped
with rotating anode generator, the radiation was monochromated
AreH), 5.52 (s, 2H, OCH2), 5.28 (s, 2H, CH2), 3.84 (s, 3H, OCH3). 13
C
NMR (75 MHz, CDCl3) (d, ppm): 149.67, 147.62, 144.74, 134.51,
129.10, 128.75, 128.13, 122.88, 121.94, 120.89, 114.60, 111.87, 63.33,
55.81, 54.17. LC-MS (m/z):296.3 [MþH] þ, 613.3 [2 M þ Na]þ.
with a Montel mirror (Mo K
collected at 223K using ɸ and
a
¼ 0.71073 Å). Intensity data were
4.1.3.5. 8-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)quinoline (3e).
Dark brown solid, M.pt. 82e84 ꢀC, yield: 90%, Rf (Ethyl acetate/Pet.
ether, 30:70) ¼ 0.22, Anal (C19H16N4O) calc. C 72.13 H 5.10 N 17.71,
u
scans. No significant loss in in-
tensities was observed during data collection. Multi-scan absorp-
tion corrections were applied to the intensity data empirically
using Denzo [17]. Data collection, reduction, and refinement were
performed using Collect [18] and Denzo-SMN [19] software. Crystal
structure was solved by direct methods using SHELXS-97 [20] and
refined with full-matrix least-squares based on F2 using SHELXL-97
[20]. All non-hydrogen atoms were refined anisotropically. Hy-
drogens were first located in the Fourier difference map, then
positioned geometrically and allowed to ride on their respective
parent atoms. The molecular graphics and crystallographic illus-
trations were prepared using PLATON [21] and MERCURY [22].
found: C 72.11 H 5.06 N 17.68%. IR (neat): n
(cmꢁ1) 3183, 3062, 1572,
1501, 1473, 1428, 1369, 1315, 1182, 1102, 994, 927, 823, 791, 750,709,
672. 1H NMR (400 MHz, CDCl3) (
AreH), 8.13 (d, 1H, J ¼ 8.32 Hz, AreH), 7.67 (s, 1H, triazole ring),
7.46e7.39 (m, 3H, AreH), 7.35e7.33 (m, 4H, AreH), 7.26e7.24 (m,
2H, AreH), 5.54 (s, 2H, OCH2), 5.50 (s, 2H, CH2). 13C NMR (75 MHz,
d
, ppm): 8.91 (d, 1H, J ¼ 2.52 Hz,
CDCl3) (d, ppm): 153.81, 149.30, 144.43, 140.19, 136.08, 134.34,
129.48, 128.78, 128.17, 126.49, 123.27, 121.93, 121.02, 120.23, 109.45,
62.96, 54.25. LC-MS (m/z): 317.2[MþH]þ, 655.3 [2 M þ Na]þ.
4.1.3.6. (E)-1-benzyl-4-((2-methoxy-4-(prop-1-enyl)phenoxy)
methyl)-1H-1,2,3-triazole (3f). Off-white solid, M.pt. 100e102 ꢀC,
yield: 75%, Rf (Ethyl acetate/Pet. ether, 30:70) ¼ 0.28, Anal
(C20H21N3O2) calc. C 71.62 H 6.31 N 12.53, found: C 71.59 H 6.28 N
4.3. In vitro anticandidal activity
A broth dilution technique was employed to determine the IC50
value for all the synthesized 1,2,3 triazole derivatives (3a-3h) and
their precursor molecules (1a-h) against three strains of Candida.
The test compounds were dissolved in DMSO with less than 4%
concentration of DMSO in the final test volume. Various concen-
12.51%. IR (neat):
1460,1423,1380,1343,1296,1262,1225,1162,1140,1061,1035,1018,
964, 855, 836, 737, 717, 678.1H NMR (300 MHz, CDCl3) (
, ppm): 7.54
n
(cmꢁ1)3065, 2924, 2851, 1721, 1605, 1587, 1512,
d
(s, 1H, triazole ring), 7.37e7.35 (m, 3H, AreH), 7.26e7.24 (m, 2H,
AreH), 6.95e6.80 (m, 3H, AreH), 6.32 (d, 1H, J ¼ 15.9 Hz, ¼CH),
6.12e6.07 (m, 1H, ¼CH), 5.51 (s, 2H, OCH2), 5.25 (s, 2H, CH2), 3.84 (s,
trations (1000 … 7.8 mg/mL) of test compounds were dispensed into
wells, then inoculated with test organism with approx. 2.5 ꢂ 103
cells/mL and incubated at 37 ꢀC for 24 h. After incubation period,
the optical density was measured at 600 nm by using Thermo
Scientific Multiskan Go plate reader. The IC50 value was defined as
the concentration of the test compound that causes 50% decrease in
absorbance compared with that of the control (no test compound).
The MFC values were determined as the lowest concentration
3H, OCH3), 1.89 (d, 3H, J ¼ 6.3 Hz, CH3). 13C NMR (75 MHz, CDCl3) (
d,
ppm): 149.57, 146.62, 144.75, 134.44, 132.17, 130.50, 129.12, 128.78,
128.16, 124.31, 122.86, 118.62, 114.41, 108.94, 63.38, 55.78, 54.22,
18.41. LC-MS (m/z):336.3 [MþH] þ, 693.4 [2M þ Na]þ.
4.1.3.7. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-benzyl-1H-1,2,3-
triazole (3g). Pinkish white solid, M.pt. 78e80 ꢀC, yield: 90%, Rf
(Ethyl acetate/Pet. ether, 30:70) ¼ 0.29, Anal (C20H21N3O2) calc. C
resulting in no growth on the subculture of 100 mL aliquot from
wells without turbidity on YEPD agar after 48 h of incubation
period at 37 ꢀC for Candida cells [23]. All the experiments were
done in triplicate in separate time.
71.62 H 6.31 N 12.53, found: C 71.59 H 6.28 N 12.50%. IR (neat):
n
(cmꢁ1) 3131, 3037, 3090, 2959, 2931, 2915, 2831, 1639, 1592, 1514,
1462, 1445, 1385, 1287, 1262, 1231, 1145, 1041, 1018, 924, 853, 836,
4.4. FIC index
812, 717, 700. 1H NMR (300 MHz, CDCl3) (
d, ppm): 7.18 (s, 1H, tri-
azole ring), 6.90 (d, 1H, J ¼ 8.7 Hz, AreH), 6.77 (d, 1H, J ¼ 8.7 Hz,
The fractional inhibitory concentration (FIC) index is defined as
the sum of the MIC of each drug when used in combination divided
AreH), 6.67- 6.62 (m, 4H, AreH), 5.92- 5.83 (m, 3H, AreH, ¼CH),

254
M. Irfan et al. / European Journal of Medicinal Chemistry 93 (2015) 246e254
by the MIC of the drug used alone. Synergy and antagonism were
defined by FIC indices of ꢃ0.5 and >4, respectively. An FIC index
result of >0.5 but ꢃ4 was considered indifferent [24]. Both, com-
pound 3e and FLC were serially diluted in growth medium upto the
measurement of statistical parameters.
Acknowledgement
concentration of 0.48 mg/mL and 0.24 mg/mL, respectively. The MIC
values of compound alone and in combination with FLC were
determined by broth micro dilution method in 96 well plate.
Mohammad Abid gratefully acknowledges the funding support
in the form of Young Scientist from Science & Engineering Research
Board (Grant No. SR/FT/LS-03/2011), New Delhi, INDIA and Uni-
versity Grant Commission (UGC), Govt. of India (Grant No. 41e277/
2012 (SR)). MI and BA would like to acknowledge UGC, INDIA for
non-NET fellowship.
4.5. Cytotoxicity assays
4.5.1. Hemolysis
The hemolytic activities of the test compounds 1e,1h, 3e, 3h and
fluconazole were determined on human red blood cells [25]. Hu-
man blood from healthy individual was collected in tubes con-
taining EDTA as anti-coagulant. The erythrocytes were harvested by
centrifugation for 10 m at 2000 rpm and 20 ꢀC, washed three times
in PBS. To the pellet, PBS was added to yield a 10% (v/v) erythro-
cytes/PBS suspension. The 10% suspension was then diluted 1:10 in
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.02.007.
Conflict of interest
No conflict of interest.
References
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