Synthesis and in vitro evaluation of novel tetrazole embedded 1,3,5-trisubstituted pyrazoline derivatives as Entamoeba histolytica growth inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.03.049

A series of pyrazoline derivatives (1a-15a) was synthesized by cyclization of chalcones (1-15) with 2-[5-(4-methoxyphenyl)-1H-tetrazol-1-yl]acetohydrazide under basic conditions and were screened in vitro, to find out effect on the growth of HM1: IMSS strain of Entamoeba histolytica. The compounds 3a, 4a, 11a, 13a and 14a showed encouraging results with IC₅₀ value in the range of 0.86-1.28 μM. However compound 13a showed most promising results with IC₅₀ = 0.86 μM which is half of the metronidazole, the standard drug used for protozoal infection. Cell viability test in human hepatocellular carcinoma cell line (HepG2) revealed non-toxic nature of new synthesized compounds. Safety index calculations prevailed compound 13a as highly antiamoebic and least cytotoxic (S.I. = >116.28), almost twice than metronidazole.

Full text of DOI:10.1016/j.ejmech.2012.03.049

European Journal of Medicinal Chemistry 54 (2012) 845e854
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and in vitro evaluation of novel tetrazole embedded 1,3,5-trisubstituted
pyrazoline derivatives as Entamoeba histolytica growth inhibitors
,
Mohmmad Younus Wani a, Abdul Roouf Bhat b, Amir Azam c, Dae Hyung Lee d, Inho Choi b
**
,
,
Fareeda Athar a
*
a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Maulana Ali Jauhar Marg, New Delhi-110025, India
b School of Biotechnology, Yeungnam University, Gyeongshan 712-749, South Korea
c Department of Chemistry, Jamia Millia Islamia, Maulana Ali Jauhar Marg, New Delhi-110025, India
d Department of Obstetrics and Gynecology, Yeungnam School of Medicine, Daegu, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazoline derivatives (1ae15a) was synthesized by cyclization of chalcones (1e15) with 2-[5-
(4-methoxyphenyl)-1H-tetrazol-1-yl]acetohydrazide under basic conditions and were screened in vitro,
to find out effect on the growth of HM1: IMSS strain of Entamoeba histolytica. The compounds 3a, 4a, 11a,
Received 21 February 2012
Received in revised form
23 March 2012
Accepted 26 March 2012
Available online 3 April 2012
13a and 14a showed encouraging results with IC50 value in the range of 0.86e1.28
mM. However
compound 13a showed most promising results with IC50 ¼ 0.86
m
M which is half of the metronidazole,
the standard drug used for protozoal infection. Cell viability test in human hepatocellular carcinoma cell
line (HepG2) revealed non-toxic nature of new synthesized compounds. Safety index calculations pre-
vailed compound 13a as highly antiamoebic and least cytotoxic (S.I. ¼ >116.28), almost twice than
metronidazole.
Keywords:
Pyrazoline
Tetrazole
Antiamoebic
MTT assay
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
drug resistant to parasites [14e16]. Considering these facts, an
unmet need to search new molecules arises, which will show
Protozoan diseases stand as a major health issue in developing
countries, affecting hundreds of millions of people around the
world [1]. The eukaryotic origin of protozoa impedes the devel-
opment of effective and selective drug, as they share many
common features with their mammalian host. Amoebiasis,
a protozoal disease caused by Entamoeba histolytica is the third
prominent cause of death [2,3]. It is acquired by ingestion of E.
histolytica cysts, which develops into motile trophozoites in ileo-
cecal region of the intestine. The major clinical manifestations of
E. histolytica infection are amoebic colitis and amoebic liver
abscesses. Metronidazole (2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethanol), has long been used for the treatment of this disease.
However reports show that metronidazole is ecotoxic [4], induces
encephalopathy [5,6], neurotoxic [7], genotoxic, carcinogen [8e12]
and causes spermatozoid damage [13]. Furthermore, failures in the
treatment of several intestinal protozoan parasites are results of
better therapeutic activity and have least side effects. Serving the
purpose we have synthesized a new series of pyrazoline derivatives
bearing a tetrazole tail and screened the compounds for anti-
amoebic evaluation.
Pyrazoline skeleton has received progressive attention of many
researchers because of its diverse biological properties such as
antitumor, immunosuppressive, antibacterial, anti-inflammatory,
anticancer, antidiabetic, and antidepressant properties [17e25].
On the other hand, tetrazole rings are shared by a number of
modern drugs owing to their potential biological activities [26e33].
The recent success of pyrazoline-based antiamoebic compounds
reported by our group has further highlighted the importance of
these heterocycles [34].
Prompted by the above-mentioned important properties of
pyrazolines and tetrazoles it was speculated that designing and
synthesis of a new series of tetrazole embedded pyrazoline deriv-
atives would be worthwhile, as an E. histolytica growth inhibitors
(Fig. 1). Furthermore, after extensive literature search, it was
observed that, till date no effort has been made to combine these
vital moieties as a single molecular scaffold and to study its anti-
amoebic activity.
* Corresponding author. Tel.: þ91 11 26981717x4492; fax: þ91 11 26980164.
** Corresponding author. Tel.: þ52 53 8103024; fax: þ82 53 8104769.
E-mail addresses: inhochoi@ynu.ac.kr (I. Choi), fathar@jmi.ac.in (F. Athar).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.049

846
M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
polar nature of
b carbon is controlled by inductive effect of
substituted aromatic ring. The substituted halo and nitro groups in
the aromatic ring increase the partial electron deficiency of
b
carbon, leading to quick completion of the reaction. However
electron donating alkyl and alkoxy groups contributed to slow
reaction.
A variety of methods has been reported for the synthesis of
1,3,5-trisubstituted pyrazolines [38e42]. Among them, simplest
and more efficient methods reported by E. Fischer et al. [38],
involves condensation of
a, b-unsaturated ketone with an acid
hydrazide in glacial acetic acid (bp. 117 ꢀC) under reflux. However,
people have reported a significantly improve in the yield by using
n-butanol (bp. 115 ꢀC) as a reaction medium. In the present inves-
tigation, we found that n-butanol as a solvent not only decreased
the progress of the reaction, but also froze the reaction in some
cases. The reaction was refluxed in methanol and 5% NaOH, which
was found to be the best solvent. Structure of compounds 1e15 and
1ae15a were confirmed by NMR, IR and MS techniques. All of the
Pyrazoline derivatives possess similar basic skeletal structure.
Proton NMR signals were assigned by comparing the spectra of the
products (1ae15a) with their corresponding chalcones (1e15).
Fig. 1. General structure of 1,3,5-trisubstituted pyrazoline derivatives with a tetrazole
tail.
2. Results and discussion
2.1. Chemistry
Signals around
d value 3.9 and 3.6 ppm recorded as doublet of
Present study was undertaken to synthesize some novel tetra-
zole embedded Pyrazoline derivatives and to investigate their
probable antiamoebic effect. Target compounds (1ae15a) were
obtained in four-step reaction process, outlined in Scheme 1e4. In
the first step, synthesis of chalcones (1e15) was carried out by
ClaiseneSchmidt reaction and products were purified by recrys-
tallization from methanol (70e85% yield). In the second step, 5-(4-
methoxyphenyl)-1H-tetrazole (2) was synthesized starting from p-
methoxy benzaldehyde via an oxime and nitrile intermediate
[35,36] (Scheme 2). 2-[5-(4-methoxyphenyl)-1H-tetrazol-1-yl]
acetohydrazide (3) was synthesized from 5-(4-methoxyphenyl)-
1H-tetrazole (2) following a reported reaction procedure [37]
(Scheme 3). The target compounds (1ae15a) were obtained by
the reaction of chalcones (1e15) with 2-[5-(4-methoxyphenyl)-1H-
tetrazol-1-yl]acetohydrazide (3) in refluxing methanol and 5%
doublets (dd) was assigned to 4-Ha and 4-Hb protons. The coupling
constant “J” was found approximately 18 and 5 Hz for 3.9 ppm and
18 and 11 Hz for 3.6 ppm. 5-H proton (5.5 ppm) of pyrazoline ring
showed a ‘dd’ pattern with “J” values 11 and 5 Hz. The coupling
occurs most likely due to scalar or indirect coupling with 4-Ha and
4-Hb protons. This coupling strongly sports the conformational
differences between Ha and Hb protons.
2.2. Pharmacology
The synthesized chalcones (1e15) and their corresponding
pyrazoline derivatives (1ae15a) were screened in vitro against
HM1:IMSS strain of E. histolytica by microdilution method [43]. All
experiments were carried out in triplicate for each concentration
level and repeated thrice. Cytotoxicity of all the compounds has
been studied by cell viability test of human hepatocellular carci-
noma cell line (HepG2). The results of antiamoebic activity and
cytotoxicity are summarized in Tables 1and 2 and Fig. 2.
NaOH. Factors such as polarizing nature of substituents on
a,
b
unsaturated carbon moiety profoundly influenced the rate of
reaction. Halogen and nitro substituted chalcones took 10e12 h for
complete conversion followed by unsubstituted (15 h), methyl
substituted (16e20 h) and alkoxy substituted (24 h) groups. The
detailed mechanism of the reaction involves initial formation of an
aryl hydrazone with subsequent nucleophilic attack of nitrogen on
2.2.1. In vitro antiamoebic activity
Preliminary experiments were carried out to determine the
in vitro antiamoebic activity of compounds (1e15 and 1ae15a) by
microdilution method, using HM1:IMSS strain of E. histolytica. The
results are summarized in Tables 1and 2. The data is presented in
terms of percent growth inhibition relative to untreated controls,
and plotted as probit values as a function of drug concentration. The
antiamoebic activity of the synthesized compounds was compared
with the most widely used antiamoebic medication metronidazole
the carbonecarbon double bond at
reaction is determined by nucleophile attack unto
directly proportional to the electropositive nature of
b
position. The rate of the
carbon and is
carbon. The
b
b
(control) with 50% inhibitory concentration (IC50) of 1.80
mM in our
experiments. IC50 and 95% confidence limits were interpolated in
the corresponding dose response curve. The antiamoebic activity of
the test compounds was found to be substituent dependent. As
depicted in Tables 1and 2 compounds exhibit an interesting inhi-
bition pattern on HM1:IMSS strain of E. histolytica. The chalcone
derivatives (1e15) showed IC50 value in the range of 4.19e12.86
much higher than control drug. A sudden decrease in IC50
(0.86e5.32 M) was observed, when chalcones (1e15) were con-
mM,
m
verted into their corresponding pyrazoline derivatives (1ae15a).
However the interesting inhibitory behavior of these compounds
is relatively dependent on electronic nature of substituents on
phenyl ring. Compound 1 bearing unsubstituted phenyl ring,
showed 50% inhibition at a concentration of 12.86
mM. Presence of
Scheme 1. Synthesis of different substituted chalcones (1e15).
substituents overall increased the activity of the tested compounds.

M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
847
Scheme 2. Synthesis of 5-(4-methoxyphenyl)-1H-tetrazole (2).
Compounds bearing electron releasing groups, showed better
activity than compounds bearing electron withdrawing groups. The
than the methoxy group, which is strongly activating. The higher
inhibition activity of methoxy group can be speculated with the
effect of pi-electron cloud on lone pair of electrons than the
inductive effect of the electronegative oxygen. Among all, the
compounds bearing a methyl or methoxy group showed better
activity than those with chloro or nitro group at para position of the
phenyl rings. Presence of electron releasing and electron with-
drawing group in a same compound also resulted in decreased
activity. The compound 13a bearing two methyl groups at para
position (6 and 60) of the phenyl rings showed excellent anti-
pyrazoline derivatives (1ae15a) showed
a similar inhibitory
pattern, where activity was guided by the presence of electron
releasing and electron withdrawing substituents. The results
showed that compounds bearing electron withdrawing groups at
para position were less active in habiting the growth of E. histolytica
than compounds bearing electron donating/releasing groups.
However, it is interested to mention that growth inhibition activity
depends also on relative inductive effect of the substituents. Pres-
ence of weakly activating groups like methyl showed less activity
amoebic activity with IC50 value of 0.86 mM. The activity however,
Scheme 3. Synthesis of 2-[5-(4-methoxyphenyl)-1H-tetrazol-1-yl]acetohydrazide (3).

848
M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
Scheme 4. Synthesis of pyrazoline derivatives bearing a tetrazole tail (1ae15a).
was decreased when one methyl group was replaced with methoxy
group in compound 14a. The decrease in activity with electron
donating substituents (OCH3) is attributed to increased electron
density, which causes more difficult diffusion through the cell
membrane and substantial loss in activity [44,45]. Thus for
Table 2
In vitro antiamoebic activity of compounds (1ae15a) against HM1: IMSS strain of
Entamoeba histolytica and toxicity profile. Compounds with bold font (IC50 value) are
more active than metronidazole.
Table 1
In vitro antiamoebic activity of compounds (1e15) against HM1: IMSS strain of
Entamoeba histolytica and toxicity profile.
Compound
R1
R2
Antiamoebic activity
Toxicity profile
Compound
R1
R2
Antiamoebic activity
Toxicity profile
IC50
(
mM)
S.Da. (ꢁ)
IC50
(m
M)
Safety
index (SI)
IC50
(m
M)
S.Da. (ꢁ)
IC50
(
m
M)
Safety
index (SI)
1
2
3
4
5
6
7
8
H
H
H
H
H
Cl
Cl
Cl
H
Cl
CH3
OCH3
NO2
H
12.86
7.32
4.38
5.93
6.02
6.96
5.86
5.32
5.10
6.18
4.83
5.22
4.19
4.76
5.08
1.80
0.18
0.16
0.12
0.20
0.13
0.10
0.16
0.20
0.18
0.13
0.14
0.16
0.12
0.14
0.10
0.12
>100
97.2
>100
>100
>7.78
13.27
>22.83
>16.86
10.74
13.60
14.13
17.40
19.05
6.50
>20.70
14.21
23.87
21.01
16.85
>55.55
1a
2a
3a
4a
5a
6a
7a
8a
H
H
H
H
H
Cl
Cl
Cl
H
Cl
CH3
OCH3
NO2
H
5.32
3.02
1.28
1.16
3.68
3.15
4.65
2.96
2.63
4.89
1.20
2.84
0.86
1.08
2.63
1.80
0.09
0.12
0.10
0.16
0.10
0.14
0.10
0.14
0.18
0.14
0.18
0.16
0.16
0.10
0.13
0.12
85.3
80.4
>100
>100
79.8
90.6
80.3
92.6
>100
86.5
>100
98.0
>100
16.03
26.62
>78.12
>86.20
21.68
28.76
17.26
31.28
>38.02
17.68
>83.33
34.50
>116.27
>92.59
>38.02
>55.55
64.7
94.7
82.8
92.6
97.2
40.2
>100
74.2
>100
>100
85.6
>100
Cl
Cl
CH3
OCH3
NO2
H
CH3
OCH3
NO2
H
9
Cl
Cl
9a
Cl
Cl
10
11
12
13
14
15
MNZ
10a
11a
12a
13a
14a
15a
MNZ
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
CH3
OCH3
NO2
CH3
OCH3
NO2
>100
>100
>100
a
a
The value obtained in at least three separate assay done in triplicate, S.Da. (ꢁ)
The value obtained in at least three separate assay done in triplicate, S.Da. (ꢁ)
Standard deviation. MNZ stands for Metronidazole.
Standard deviation. MNZ stands for Metronidazole.

M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
849
Fig. 2. Percentage of viable cells after 48 h pre-treatment of HepG2 cell line with metronidazole, compounds 3a, 4a, 11a, 13a and 14a as evaluated by MTT assay.
a compound an optimum electron density is inevitable so as to gain
a significant activity. The compounds 2a, 5a, 7a, 8a, 9a, 10a, 12a and
15a having electron withdrawing chloro and nitro group at para
position, showed a moderate activity however, it was higher
than the compound 1a containing an unsubstituted phenyl ring
all the compounds showed a different patterns of cytotoxicity. At
a concentration of 25 M all the compounds showed viability in the
range of 72e90%. On increasing the concentration range up to 50
and 100 M the compounds showed moderate to least cytotoxicity
m
m
(viability 40e75%). It is also interesting to mention that the highly
antiamoebic compounds (3a, 4a, 11a,13a and 14a) did not show any
remarkable cytotoxicity against the HepG2 cell line. All these
compounds showed a viability of ꢂ68% at a concentration of
(IC50 ¼ 5.32
mM).
As discussed above, more optimization of substitution at ortho
and meta positions of the phenyl rings by electron releasing/
donating or electron withdrawing groups is needed to get
detailed insight in structure activity relationship. From the results
it can be inferred that the compounds 3a, 4a, 11a, 13a and 14a
showed encouraging results with IC50 value in the range of
100 mM as depicted in Fig. 2. To further investigate the selectivity of
the compounds, the “safety index” (SI), defined as the toxicity IC50
/
protozoal IC50, was calculated. This allows estimating the efficacy of
compounds. The results are summarized in Tables 1and 2. From the
results of antiamoebic activity and cytotoxicity it can be inferred
that all the tested compounds 3a, 4a, 11a, 13a and 14a are least
cytotoxic and excellent E. histolytica growth inhibitors as compared
to the reference drug metronidazole. These results also showed
that the compound 13a despite of being highly antiamoebic did not
show any marked toxicity on human cell line and has safety index
value of >116.28 which is better than metronidazole (>55.55).
0.86e1.28
promising results with lower IC50 (0.86
half of the reference drug metronidazole (IC50 ¼ 1.80
m
M, out of which compound 13a showed the most
M), which is less than
M). The
m
m
results were statistically evaluated by analysis of variance. The
null hypothesis was tested using t-test. The significance of the
difference between the IC50 values of metronidazole and the
compound 13a was evaluated by t-test. The values of the calcu-
lated T were found higher than the Table value of T at 5% level,
thus concluding that the character under study is said to be
significantly influenced by the treatment.
3. Conclusion
We attempted to synthesize a novel series of pyrazoline
2.2.2. Cell viability test
derivatives (1ae15a) from
potential of the new class of tetrazole embedded pyrazoline
M)
derivatives
M) was studied. Through a preliminary SAR
campaign, we found that presence of substituents play a dominant
role on activity of the compounds, where activity seems to be guided
by the electron donating and withdrawing nature of the substitu-
ents. Compound 13a bearing methyl group at para position of the
phenyl rings happened to be the most potent of all the reported
compounds. The replacement of methyl group with a stronger
electron releasing methoxy group resulted in decrease in activity.
The results revealed that an optimum electron density is inevitable
for a compound to gain a significant activity. Compounds 3a, 4a,11a,
a, b unsaturated ketones (1e15). The great
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) is reduced by the succinate dehydrogenase system of mito-
chondrial living cells to produce water insoluble purple formazan
crystals [46,47], which after solubilization, can be measured spec-
trophotometrically. Since the amount of formazan produced is
directly proportional to the number of active cells in the culture,
MTT has long been used to assess the cell viability in cell prolifer-
ation and cytotoxicity tests [48e50].
A series of synthesized compounds were screened for their
antiamoebic activity and then evaluated for their effect on growth
of Human hepatocellular carcinoma cell line (HepG2) to ensure their
toxic effect. Metronidazole was used as a reference drug. A sub-
confluent population of HepG2 cells was treated with increasing
compounds as innovative antiamoebic leads (IC50 ¼ 0.86e5.32
m
as
compared
to
m
their
parent
chalcone
(IC50 ¼ 4.19e12.86
(3.13e100
m
M) concentration of test compounds and the number of
and 14a also showed better activity (IC50 ¼ 1.08e1.28
mM) than the
M). Cytotoxicity
viable cells was measured after 48 h by MTT cell viability assay. The
viable cells (%) obtained after an exposure to the compounds for
48 h are depicted in Fig. 2. The toxicity of compounds was found to
be concentration-dependent. All the compounds including the
reference compound metronidazole showed viability in the range
reference compound metronidazole (IC50 ¼ 1.80
m
studies on human hepatocellular carcinoma cell line HepG2 also
revealed non-toxic nature of these active compounds. The most
promising results were observed forcompound 13a (1-(4,5-dihydro-
3,5-dip-tolylpyrazol-1-yl)-2-(5-(4-methoxyphenyl)-1H-tetrazol-1-
of 95e100% at the concentration of 3.13
active antiamoebic compounds showed a viability of 100% at this
concentration as depicted in Fig. 2. On increasing the concentration,
m
M, however the most
yl)ethanone) with potent antiamoebic activity (IC50 ¼ 0.86
cytotoxicity (IC50 ¼ >100 M) and safety index value of >116.28,
which is better than metronidazole (>55.55).
mM), least
m

850
M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
4. Experimental protocol
4.1.1.4. (2E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (4). White;
Yield 82.8% (3.52 g); mp. 60e63 ꢀC; Anal. Calc. For C16H14O2: C 80.65, H
4.1. Chemistry
5.92%; found: C 80.54, H 5.84%; IR nmax (cmꢃ1): 3054 (AreH), 2938
(CeH), 1656 (C]O), 1446 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.04 (d, 2H,
Solvents and organic reagents were purchased from Sigma
Aldrich, Merck (Germany) and Loba Chemie (India) and were used
without further purification. Melting points (mp) were performed
using a Mel-temp instrument, and the results are uncorrected.
Elemental analyses were performed on HeraeusVario EL III analyzer
at Central Drug Research Institute, Lucknow, India. The results were
within ꢁ0.4% of the theoretical values. IR spectra were recorded on
PerkineElmer model 1600 FT-IR RX1 spectrophotometer as KBr
discs/ATR mode. 1H NMR and 13C NMR spectra were recorded on
Bruker AVANCE 300 (300.13) MHz spectrometer using CDCl3/
DMSO-d6 as solvent with TMS as internal standard. Splitting
patterns are designated as follows; s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet. Chemical shift values
are given in ppm. ESI-MS was recorded on a MICROMASS QUATTRO
II triple quadrupole mass spectrometer. Reactions were monitored
using thin-layer chromatography (TLC) using commercially avail-
able precoated plates (Merck Kieselgel 60 F254 silica). Visualization
was achieved with UV light at 254 nm or I2 vapor staining.
J ¼ 8.1 Hz, H-20, H-60), 7.84 (d, 1H, J ¼ 15.9 Hz, H-
b), 7.64e7.27 (m, 7H,
AreH), 6.97 (d,1H, J ¼ 15.9 Hz, H-
a
), 3.88 (s, 3H, OCH3); 13C NMR (CDCl3)
d
(ppm): 190.64 (C]O), 161.70 (C-
b), (144.75, 138.51, 132.60,
130.27, 128.59, 127.62), 119.78 (C-
a), 55.45 (OCH3); ESI-MS m/z:
M þ Hþ ¼ 238.09.
4.1.1.5. (2E)-3-(4-nitrophenyl)-1-phenylprop-2-en-1-one (5). Orange;
Yield 87.7% (3.95 g); mp. 138e140 ꢀC; Anal. Calc. For C15H11NO3: C
71.14, H 4.38, N 5.53%; found: C 71.26, H 4.35, N 5.69%; IR nmax (cmꢃ1):
3055 (AreH), 2923 (CeH), 1668 (C]O), 1460 (C]C), 1547, 1365
(eNO2); 1H NMR (CDCl3)
8.07 (d, 1H, J ¼ 8.4 Hz, H-
J ¼ 14.7 Hz, H-
); 13C NMR (CDCl3)
d
(ppm): 8.31 (d, 2H, J ¼ 8.7 Hz, H-20, H-60),
b
), 7.86e7.62 (m, 7H, AreH), 7.57 (d, 1H,
a
d
(ppm): 190.10 (C]O), 145.66
(C-b), (136.28, 134.25, 132.06, 128.82, 126.72), 122.08 (C-a); ESI-MS
m/z: [M þ H]þ ¼ 254.08.
4.1.1.6. (2E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one (6). White;
Yield 83% (2.80 g); mp. 81e83 ꢀC; Anal. Calc. For C15H11OCl: C 74.23, H
4.57%; found: C 74.36, H 4.63%; IR nmax (cmꢃ1): 3060 (AreH), 2931
(CeH), 1662 (C]O), 1465 (C]C), 793 (CeCl); 1H NMR (CDCl3)
4.1.1. General method for synthesis of chalcones (1e15)
To the solution of a ketone (Acetophenone, p-chloro acetophe-
none, and p-methyl acetophenone 5 mmol) in 5 mL of methanol on
an ice bath, freshly prepared 2 N methanolic NaOH solution (30 mL)
was added and stirred for 10 min. To this 5 mmol of appropriate
aldehyde was added and the reaction mixture was stirred at room
temperature for 10e24 h. The reaction mixture was cooled on an
ice bath and neutralized with dilute hydrochloric acid. The
precipitate appeared was separated by filtration and washed three
times with 50 mL distilled water to give the crude product. The
product so obtained was recrystallized from methanol. The purity
of the products was checked on TLC (Merck Silica gel 60 F254), using
mixture of hexane and ethylacetate as mobile phase (7:3 v/v).
d
(ppm): 7.98 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 7.89 (d, 1H, J ¼ 15.6 Hz,
H-
(CDCl3)
130.68, 128.42), 123.18 (C-
b
), 7.66 (d, 1H, J ¼ 15.6 Hz, H-
a
), 7.51e7.16 (m, 7H, AreH); 13C NMR
d
(ppm): 188.89 (C]O), 143.47 (C-b), (136.46, 135.03, 134.31,
a
); ESI-MS m/z: [M þ H]þ ¼ 243.05.
4.1.1.7. (2E)-1,3-bis(4-chlorophenyl)prop-2-en-1-one
(7). White;
Yield 90.3% (3.45 g); mp. 96e98 ꢀC; Anal. Calc. For C15H10OCl2: C
65.01, H 3.64%; found: C 65.18, H 3.50%; IR nmax (cmꢃ1): 3058
(AreH), 2930 (CeH), 1665 (C]O), 1455 (C]C), 778 (CeCl); 1H NMR
(CDCl3)
J ¼ 15.6 Hz, H-
d
(ppm): 7.99 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 7.81 (d, 1H,
b
), 7.61 (d, 1H, J ¼ 15.6 Hz, H-
a), 7.51e7.49 (m, 2H, H-
30, H-50), 7.45e7.24 (m, 4H, H-2, H-3, H-5, H-6); 13C NMR (CDCl3)
d
(ppm): 189.65 (C]O),145.47 (C-b), (140.43, 139.24,136.46, 131.03,
4.1.1.1. (2E)-1,3-diphenylprop-2-en-1-one (1). Cream; Yield: 85.3%
(3.20 g); mp. 48e50 ꢀC; Anal. Calc. For C15H12O: C 86.51, H 5.81%;
found: C 86.38, H 5.69%; IR nmax (cmꢃ1): 3064 (AreH), 2939 (CeH),
129.68, 128.42), 120.18 (C-
a
); ESI-MS m/z: [M þ H]þ ¼ 277.01.
4.1.1.8. (2E)-1-(4-chlorophenyl)-3-(4-methylphenyl)prop-2-en-1-one
(8). White; Yield 86.5% (3.08 g); mp. 158e160 ꢀC; Anal. Calc. For
C16H13OCl: C 74.85, H 5.10%; found: C 74.79, H 5.18%; IR nmax (cmꢃ1):
3028 (AreH), 2916 (CeH), 1657 (C]O), 1448 (C]C), 793 (CeCl); 1H
1658 (C]O), 1460 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.04e7.50 (m,
), 7.31 (d, 1H, J ¼ 12 Hz, H- );
(ppm): 190.59 (C]O), 144.89 (C- ), (136.90,
); ESI-MS m/z:
10H, AreH), 7.45 (d, 1H, J ¼ 12 Hz, H-
b
a
13C NMR(CDCl3)
d
b
132.84, 130.59, 128.67, 127.49), 122.09 (C-
a
NMR (CDCl3)
d
(ppm): 8.05 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 7.90 (d, 1H,
[M þ H]þ ¼ 209.09.
J ¼ 15.6 Hz, H-
b
), 7.63 (d, 1H, J ¼ 15.6 Hz, H-
a), 7.56e7.49 (m, 4H,
AreH), 7.40e7.29 (m, 2H, AreH) 2.47 (s, 3H, CH3); 13C NMR (CDCl3)
(ppm): 189.31 (C]O), 145.50 (C- ), (141.39, 139.10, 136.64, 131.96,
), 21.60 (methyl); ESI-MS m/z:
4.1.1.2. (2E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (2). White;
Yield 87.4% (3.76 g); mp. 83e85 ꢀC; Anal. Calc. For C15H11OCl: C 74.23,
H 4.57%; found: C 74.13, H 4.48%; IR nmax (cmꢃ1): 3060 (AreH), 2931
(CeH), 1662 (C]O), 1465 (C]C), 779 (CeCl); 1H NMR (CDCl3)
d
b
a
129.78, 128.93, 128.58), 120.46 (C-
[M þ H]þ ¼ 257.07.
d
(ppm): 8.05 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 7.81 (d, 1H, J ¼ 15.6 Hz,
4.1.1.9. (2E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-
one (9). White; Yield 98.4% (3.70 g); mp.118e120 ꢀC; Anal. Calc. For
C16H13O2Cl: C 70.46, H 4.80%; found: C 70.66, H 4.93%; IR nmax
(cmꢃ1): 3064 (AreH), 2934 (CeH), 1677 (C]O), 1442 (C]C), 790
H-
b
), 7.64e7.50 (m, 7H, AreH), 7.43 (d,1H, J ¼ 15.6 Hz, H-
a
); 13C NMR
(CDCl3)
134.28,
d
(ppm): 189.53 (C]O), 143.47 (C-b), (136.45, 135.40,
132.41, 130.52, 128.08), 123.54 (C-a); ESI-MS m/z:
[M þ H]þ ¼ 243.05.
(CeCl); 1H NMR (CDCl3)
d
(ppm): 7.99 (d, 2H, J ¼ 12.9 Hz, H-20, H-
60), 7.84 (d, 1H, J ¼ 15.6 Hz, H-
b), 7.64e7.47 (m, 4H, AreH), 7.41 (d,
4.1.1.3. (2E)-3-(4-methylphenyl)-1-phenylprop-2-en-1-one (3). White;
Yield 95.4% (3.81 g); mp. 67e69 ꢀC; Anal. Calc. For C16H14O: C 86.45, H
6.35%; found: C 86.33, H 6.24%; IR nmax (cmꢃ1): 3058 (AreH), 2932
1H, J ¼ 15.6 Hz, H- ), 6.98 (d, 2H, J ¼ 8.7 Hz, H-3, H-5), 3.88 (s, 3H,
b
OCH3); 13C NMR (CDCl3)
d
(ppm): 189.22 (C]O), 145.25 (C-
b),
(138.96, 136.80, 130.37, 129.85, 128.51, 127.42), 119.13 (C-
a), 55.46
(CeH), 1659 (C]O), 1447 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.05
b), 7.63e7.49
(OCH3); ESI-MS m/z: [M þ H]þ ¼ 273.06.
(d, 2H, J ¼ 7.5 Hz, H-20, H-60), 7.85 (d, 1H, J ¼ 15.6 Hz, H-
(m, 7H, AreH), 7.28 (d,1H, J ¼ 11.7 Hz, H-
a
), 2.42 (s, 3H, CH3); 13C NMR
4.1.1.10. (2E)-1-(4-chlorophenyl)-3-(4-nitrophenyl)prop-2-en-1-one
(10). Orange; Yield 82.5% (3.27 g); mp. 138e140 ꢀC; Anal. Calc. For
C15H10NO3Cl: C 62.62, H 3.50, N 4.87%; found: C 62.78, H 3.43, N
4.68%; IR nmax (cmꢃ1): 3064 (AreH), 2934 (CeH), 1677 (C]O), 1442
(CDCl3)
d
(ppm): 189.98 (C]O), 144.54 (C-
b), (134.34, 130.12, 129.42,
128.22, phenyl), 123.12 (C-
a), 22.48 (methyl); ESI-MS m/z:
[M þ H]þ ¼ 223.11.

M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
851
(C]C), 1540, 1362 (eNO2), 789 (CeCl); 1H NMR (CDCl3)
d
(ppm):
), 7.82
dehyde, following a standard protocol [36]. Nitrile (2.66 g,
8.32 (d, 2H, J ¼ 12.9 Hz, H-20, H-60), 8.02 (d,1H, J ¼ 15.6 Hz, H-
b
20 mmol), sodium azide (1.43 g, 22 mmol) and zinc bromide (4.50 g,
20 mmol), were put in 60 mL of water. 5 mL of isopropanol was also
added to stop the formation of clumps. The reaction mixture was
refluxed for 24 h and monitored by TLC; vigorous stirring is
essential. After 24 h HCl (3 N, 30 mL) and ethyl acetate (100 mL)
were added, and vigorous stirring was continued until no solid was
present and the aqueous layer had a pH of 1. If necessary, additional
ethyl acetate was added. The organic layer was isolated and the
aqueous layer extracted with 2 ꢄ 100 mL of ethyl acetate. The
combined organic layers were evaporated, 200 mL of 0.25 N NaOH
was added, and the mixture was stirred for 30 min, until the original
precipitate was dissolved and a suspension of zinc hydroxide was
formed. The suspension was filtered, and the solid washed with
20 mL of 1 N NaOH. To the filtrate was added 40 mL of 3 N HCl with
vigorous stirring causing the tetrazole to precipitate. The tetrazole
was filtered and washed with 2 ꢄ 20 mL of 3 N HCl and dried in
a drying oven to furnish the tetrazole as a white powder.
(d, 1H, J ¼ 15.6 Hz, H-
a
), 7.64e7.45 (m, 6H, AreH); 13C NMR (CDCl3)
(ppm): 188.37 (C]O),142.06 (C-b), (135.81,130.02,129.20,129.04,
d
125.09), 124.29 (C-
a
); ESI-MS m/z: [M þ H]þ ¼ 288.04.
4.1.1.11. (2E)-1-(4-methylphenyl)-3-phenylprop-2-en-1-one (11). White;
Yield 82.2% (2.88 g); mp. 58e60 ꢀC; Anal. Calc. For C16H14O: C 86.45, H
6.35%; found: C 86.63, H 6.48%; IR nmax (cmꢃ1): 3058 (AreH), 2932
(CeH), 1659 (C]O), 1447 (C]C); 1H NMR (CDCl3)
d
(ppm): 7.97 (d, 2H,
J ¼ 7.5 Hz, H-20, H-60), 7.85 (d, 1H, J ¼ 15.6 Hz, H-
b), 7.67e7.53 (m, 4H,
AreH), 7.44 (d, 1H, J ¼ 15.6 Hz, H-
a), 7.34e7.27 (m, 3H, AreH), 2.46 (s,
(ppm): 189.29 (C]O),144.47 (C-
3H, CH3). 13C NMR (CDCl3)
d
b), (134.70,
130.02, 129.53, 128.54, phenyl), 122.58 (C-
a), 22.40 (CH3); ESI-MS m/z:
[M þ H]þ ¼ 223.11.
4.1.1.12. (2E)-3-(4-chlorophenyl)-1-(4-methylphenyl)prop-2-en-1-
one (12). White; Yield 88.0% (3.60 g); mp. 140e142 ꢀC; Anal. Calc.
For C16H13OCl: C 74.85, H 5.10%; found: C 74.70, H 5.18%; IR nmax
(cmꢃ1): 3028 (AreH), 2916 (CeH), 1657 (C]O), 1448 (C]C), 790
Yield 84%; mp. 180e182 ꢀC; Anal. Calc. For C8H8N4O: C 54.54, H
4.58, N 31.80%; found: C 54.74, H 4.34, N 32.09%; IR nmax (cmꢃ1):
3285 (NH), 3010 (CeH, Ar), 1712 (C]O), 1630 (C]N), 1584 (C]C,
(CeCl); 1H NMR (CDCl3)
7.79 (d, 1H, J ¼ 15.6 Hz, H-
J ¼ 15.6 Hz, H-
NMR (CDCl3)
d
(ppm): 7.96 (d, 2H, J ¼ 8.1 Hz, H-20, H-60),
b
), 7.60e7.46 (m, 4H, AreH), 7.42 (d, 1H,
Ar), 1285 (CeO); 1H NMR (DMSO)
d
(ppm): 8.45 (1H, NH, br s),
a
), 7.34e7.28 (m, 2H, AreH), 2.46 (s, 3H, CH3); 13C
(ppm): 184.97 (C]O), 145.44 (C- ), (139.12, 138.15,
), 16.97 (CH3); ESI-MS
7.54e6.84 (4H, m), 3.58 (s, 3H, OCH3); 13C NMR (DMSO)
d
(ppm):
d
b
159.8 (C]N), 150.5, 129.4, 121.6, 118.5, 54.9; ESI-MS m/z:
[Mþþ1] ¼ 177.03.
131.55, 130.70, 128.75, 124.82), 117.72 (C-
a
m/z: [M þ H]þ ¼ 257.07.
4.1.3. Synthesis of 2-[5-(4-methoxyphenyl)-1H-tetrazol-1-yl]
acetohydrazide (3)
4.1.1.13. (2E)-1,3-bis(4-methylphenyl)prop-2-en-1-one (13). White;
Yield 87.0% (3.30 g); mp. 120e123 ꢀC; Anal. Calc. For C17H16O: C
86.40, H 6.82%; found: C 86.32, H 6.96%; IR nmax (cmꢃ1): 3032
(AreH), 2908 (CeH), 1650 (C]O), 1440 (C]C); 1H NMR (CDCl3)
2-[5-(4-methoxyphenyl)-1H-tetrazol-1-yl]acetohydrazide was
prepared by a reported method [37].
Off white; Yield 65%; mp. 193e195 ꢀC; Anal. Calc. For
C10H12N6O2: C 48.38, H 4.87, N 33.85%; found: C 48.22, H 4.64, N
33.68%; IR nmax (cmꢃ1): 3285 (NH), 3025 (CeH, Ar), 1720 (C]O),
d
(ppm): 7.97 (d, 2H, J ¼ 8.1 Hz, H-20, H-60), 7.84 (d, 1H, J ¼ 15.6 Hz,
H-
b
), 7.58e7.49 (m, 4H, AreH), 7.33 (d, 1H, J ¼ 7.8 Hz, H-
a),
7.28e7.23 (m, 2H, AreH), 2.45 (s, 6H, CH3); 13C NMR (CDCl3)
(ppm): 190.16 (C]O), 144.53 (C- ), (143.54, 140.98, 135.15, 132.25,
), 21.71 (CH3); ESI-MS m/z:
1630 (C]N),1594 (C]C, Ar),1292 (CeO); 1H NMR (DMSO)
d
(ppm):
8.85 (1H, NH, s), 7.24e6.72 (4H, m), 4.42 (2H, CH2, s), 3.58 (s, 3H,
OCH3), 2.76 (2H, s, NH2); 13C NMR (DMSO)
(ppm): 162.0 (C]N),
d
b
129.71, 128.64, 128.47), 121.07 (C-
a
d
[M þ H]þ ¼ 237.12.
152.8, 128.5, 124.8, 121.6, 118.5, 68.5, 54.9, 48.2; ESI-MS m/z:
[Mþþ1] ¼ 249.10.
4.1.1.14. (2E)-3-(4-methoxyphenyl)-1-(4-methylphenyl)prop-2-en-1-
one (14). White; Yield 85.0% (3.50 g); mp. 98e100 ꢀC; Anal. Calc.
For C17H16O2: C 80.93, H 6.39%; found: C 80.84, H 6.44%; IR nmax
(cmꢃ1): 3025 (AreH), 2920 (CeH), 1650 (C]O), 1445 (C]C); 1H
4.1.4. General method for synthesis of pyrazolines (1ae15a)
To a solution of (5 mmol) of appropriate chalcone (1e15) in
10 mL of methanol and 5% NaOH, (5 mmol, 1.24 g) of 2-[5-(4-
methoxyphenyl)-1H-tetrazol-1-yl]acetohydrazide was added and
the reaction mixture refluxed for 10e24 h. Conversion was moni-
tored in every 60 min interval on precoated silica TLC plates (Merck,
60F254) by using mixture of Hexane and EtOAc (70:30 v/v) as
mobile phase. The excess of solvent was removed under reduced
pressure and the reaction mixture was cooled on an ice bath. The
products precipitated out at low temperature were washed five
times with 50 mL distilled water, reconstituted in minimum
amount of methanol and dried under reduced pressure. In some
cases the products were purified by column chromatography using
hexane: ethyl acetate (70:30 v/v) as eluent.
NMR (CDCl3)
J ¼ 15.6 Hz, H-
J ¼ 15.6 Hz, H-
(s, 3H, OCH3), 2.45 (s, 3H, CH3); 13C NMR (CDCl3)
(C]O), 145.04 (C- ), (138.42, 138.05, 132.65, 130.15, 128.55, 124.02),
d
(ppm): 7.96 (d, 2H, J ¼ 8.1 Hz, H-20, H-60), 7.83 (d, 1H,
b
), 7.64 (d, 2H, J ¼ 8.7 Hz, AreH), 7.47 (d, 1H,
a), 7.33e7.28 (m, 2H, AreH), 6.97 (d, 2H, AreH), 3.88
d
(ppm): 188.64
b
120.82 (C-a), 54.07 (OCH3), 21.87 (CH3); ESI-MS m/z:
[M þ H]þ ¼ 253.12.
4.1.1.15. (2E)-1-(4-methylphenyl)-3-(4-nitrophenyl)prop-2-en-1-one
(15). Orange; Yield 85.0% (3.50 g); mp. 149e151 ꢀC, Anal. Calc. For
C16H13NO3: C 71.90, H 4.90, N 5.24%; found: C 72.10, H 4.82, N 5.13%;
IR nmax (cmꢃ1): 3015 (AreH), 2906 (CeH), 1651 (C]O), 1445 (C]C),
1548, 1360 (eNO2); 1H NMR (CDCl3)
d
(ppm): 8.30 (d, 2H, J ¼ 8.7 Hz,
4.1.4.1. 1-(4,5-dihydro-3,5-diphenylpyrazol-1-yl)-2-(5-(4-methoxyphenyl)-
H-20, H-60), 7.98 (d, 1H, J ¼ 8.1 Hz, H-
b
), 7.85e7.78 (m, 4H, AreH),
1H-tetrazol-1-yl)ethanone
(1a). White; Yield
45.0%; mp.
7.69 (d, 1H, J ¼ 15.6 Hz, H-
a
), 7.33 (d, 2H, AreH), 2.43 (s, 3H,
(ppm): 189.12 (C]O), 148.48 (C- ),
), 21.77
290e292 ꢀC; Anal. Calc. For C25H22N6O2: C 68.48, H 5.06, N 19.17%;
Found: C 68.34, H 5.14, N 19.01%; IR nmax (cmꢃ1): 3035 (AreH), 2928
(CH2), 1683 (C]O), 1592 (C]N), 1446, (C]C), 1238 (CeN); 1H NMR
CH3); 13C NMR (CDCl3)
d
b
(144.43, 141.19, 134.99, 129.55, 128.92, 125.76), 124.22 (C-
a
(CH3); ESI-MS m/z: [M þ H]þ ¼ 268.09.
(CDCl3) d (ppm): 8.55e7.18 (m, 14H, AreH), 5.94 (s, 2H, CH2), 5.58
[dd, 1H, J ¼ 11.5, 4.8 Hz, Hx (pyrazoline ring)], 3.86 [dd, 1H, J ¼ 18.2,
4.1.2. Synthesis of 5-(4-methoxyphenyl)-1H-tetrazole (2)
5.0 Hz, Ha (pyrazoline ring)], 3.42 [dd, 1H, J ¼ 18.0, 11.7 Hz, Hb
5-(4-methoxyphenyl)-1H-tetrazole was synthesized from 4-
methoxybenzonitrile by following a reported procedure [35].
4-methoxybenzonitrile was obtained from p-methoxy benzal
(pyrazoline ring)], 3.20 (s, 3H, OCH3); 13C NMR (CDCl3)
d (ppm):
169.45 (C]O), 154.12 (C]N), 152.85 (C-3, pyrazoline ring), (149.83,
141.35, 132.65, 128.63, 127.68, 127.76, 123.33, 120.34, 114.18, phenyl

852
M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
ring), 55.45 (C-5, pyrazoline ring), 54.56 (OCH3), 48.51 (CH2), 42.54
58.0%; mp. 310e312 ꢀC; Anal. Calc. For C25H21ClN6O2: C 63.49, H
4.48, N 17.77%; found: C 63.35, H 4.54, N 17.83%; IR nmax (cmꢃ1):
3033 (AreH), 2928 (CH2), 1682 (C]O), 1585 (C]N), 1437, (C]C),
(C-4, pyrazoline ring); ESI-MS m/z: [M þ H]þ ¼ 439.16.
4.1.4.2. 1-(5-(4-chlorophenyl)-4,5-dihydro-3-phenylpyrazol-1-yl)-2-
(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (2a). White; Yield
45.8%; mp. 238e240 ꢀC; Anal. Calc. For C25H21ClN6O2: C 63.49, H
4.48, N 17.77%; found: C 63.58, H 4.34, N 17.81%; IR nmax (cmꢃ1):
3028 (AreH), 2930 (CH2), 1685 (C]O), 1585 (C]N), 1440, (C]C),
1236 (CeN), 793 (CeCl); 1H NMR (CDCl3)
d (ppm): 8.23e7.28 (m,
13H, AreH), 5.90 (s, 2H, CH2), 5.67 [dd, 1H, J ¼ 11.4, 5.0 Hz, Hx
(pyrazoline ring)], 3.85 [dd, 1H, J ¼ 18.2, 5.1 Hz, Ha (pyrazoline
ring)], 3.36 [dd, 1H, J ¼ 11.8, 14.4 Hz, Hb (pyrazoline ring)], 3.24 (s,
3H, OCH3); 13C NMR (CDCl3)
d (ppm): 169.45 (C]O), 156.22 (C]N),
1235 (CeN), 790 (CeCl); 1H NMR (CDCl3)
d
(ppm): 8.04e6.74 (m,
151.18 (C-3, pyrazoline ring), (148.29, 142.12, 131.25, 128.77, 127.02,
126.33, 123.15, 120.15, 114.98, phenyl ring), 55.38 (C-5, pyrazoline
ring), 54.10 (OCH3), 48.50 (CH2), 46.45 (C-4, pyrazoline ring); ESI-
MS m/z: [M þ H]þ ¼ 473.14.
13H, AreH), 5.90 (s, 2H, CH2), 5.67 [dd, 1H, J ¼ 11.8, 5.8 Hz, Hx
(pyrazoline ring)], 3.86 [dd, 1H, J ¼ 18.1, 5.1 Hz, Ha (pyrazoline
ring)], 3.38 [dd, 1H, J ¼ 18.0, 11.8 Hz, Hb (pyrazoline ring)], 3.25 (s,
3H, OCH3); 13C NMR (CDCl3)
d (ppm): 168.75 (C]O), 155.58 (C]N),
152.80 (C-3, pyrazoline ring), (149.79, 142.90, 133.75, 128.45,
127.25, 127.65, 123.30, 120.46, 114.65, phenyl ring), 56.45 (C-5,
pyrazoline ring), 52.10 (OCH3), 49.55 (CH2), 42.85 (C-4, pyrazoline
ring); ESI-MS m/z: [M þ H]þ ¼ 473.14.
4.1.4.7. 1-(3,5-bis-(4-chlorophenyl)-4,5-dihydropyrazol-1-yl)-2-(5-
(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (7a). Cream; Yield
61.5%; mp. 199e201 ꢀC; Anal. Calc. For C25H20Cl2N6O2: C 59.18, H
3.97, N 16.56%; found: C 59.26, H 3.87, N 16.43%; IR nmax (cmꢃ1):
3025 (AreH), 2932 (CH2), 1680 (C]O), 1582 (C]N), 1435, (C]C),
4.1.4.3. 1-(4,5-dihydro-3-phenyl-5-p-tolylpyrazol-1-yl)-2-(5-(4-
methoxyphenyl)-1H-tetrazol-1-yl) ethanone (3a). White; Yield
52.5%; mp. 300e302 ꢀC; Anal. Calc. For C26H24N6O2: C 69.01, H 5.35,
N 18.57%; found: C 69.21, H 5.44 N 18.43%; IR nmax (cmꢃ1): 3035
(AreH), 2930 (CH2), 1682 (C]O), 1585 (C]N), 1448, (C]C), 1235
1230 (CeN), 790 (CeCl); 1H NMR (CDCl3)
d (ppm): 7.83e6.85 (m,
12H, AreH), 5.95 (s, 2H, CH2), 5.54 [dd, 1H, J ¼ 11.5, 4.5 Hz, Hx
(pyrazoline ring)], 3.87 [dd, 1H, J ¼ 18.0, 4.8 Hz, Ha (pyrazoline
ring)], 3.42 [dd, 1H, J ¼ 11.4, 14.6 Hz, Hb (pyrazoline ring)], 3.28 (s,
3H, OCH3); 13C NMR (CDCl3)
d (ppm): 167.35 (C]O), 154.28 (C]N),
(CeN); 1H NMR (CDCl3)
d
(ppm): 7.96e6.91 (m, 13H, AreH), 5.89 (s,
152.10 (C-3, pyrazoline ring), (148.25, 142.05, 131.20, 128.64, 127.60,
126.11, 123.10, 120.82, 114.45, phenyl ring), 56.45 (C-5, pyrazoline
ring), 54.02 (OCH3), 49.65 (CH2), 43.25 (C-4, pyrazoline ring); ESI-
MS m/z: [M þ H]þ ¼ 507.10.
2H, CH2), 5.69 [dd, 1H, J ¼ 11.0, 5.4 Hz, Hx (pyrazoline ring)], 3.94
[dd, 1H, J ¼ 18.0, 5.1 Hz, Ha (pyrazoline ring)], 3.34 [dd, 1H, J ¼ 11.8,
14.6 Hz, Hb (pyrazoline ring)], 3.24 (s, 3H, OCH3), 2.82 (s, 3H, CH3);
13C NMR (CDCl3)
d (ppm): 169.45 (C]O), 154.88 (C]N), 150.44 (C-
3, pyrazoline ring), (148.09, 142.34, 133.05, 128.24, 127.15, 126.45,
123.38, 120.25, 114.25, phenyl ring), 56.35 (C-5, pyrazoline ring),
54.80 (OCH3), 48.55 (CH2), 44.65 (C-4, pyrazoline ring), 22.85 (CH3);
ESI-MS m/z: [M þ H]þ ¼ 453.20.
4.1.4.8. 1-(3-(4-chlorophenyl)-4,5-dihydro-5-p-tolylpyrazol-1-yl)-2-
(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (8a). White; Yield
55.0%; mp. 208e210 ꢀC; Anal. Calc. For C26H23ClN6O2: C 64.13, H
4.76, N 17.26%; found: C 64.25, H 4.65, N 17.10%; IR nmax (cmꢃ1):
3032 (AreH), 2935 (CH2), 1682 (C]O), 1588 (C]N), 1445, (C]C),
4.1.4.4. 1-(4,5-dihydro-5-(4-methoxyphenyl)-3-phenylpyrazol-1-yl)-
1230 (CeN), 779 (CeCl); 1H NMR (CDCl3)
d (ppm): 8.20e6.91 (m,
2-(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone
(4a). White;
12H, AreH), 5.99 (s, 2H, CH2), 5.45 [dd, 1H, J ¼ 11.6, 4.9 Hz, Hx
(pyrazoline ring)], 3.94 [dd, 1H, J ¼ 18.0, 5.1 Hz, Ha (pyrazoline
ring)], 3.40 [dd, 1H, J ¼ 18.0, 11.6 Hz, Hb (pyrazoline ring)], 3.25 (s,
Yield 48.0%; mp.198e200 ꢀC; Anal. Calc. For C26H24N6O3: C 66.65, H
5.16, N 17.94%; found: C 66.53, H 5.08, N 17.83%; IR nmax (cmꢃ1):
3025 (AreH), 2924 (CH2), 1685 (C]O), 1580 (C]N), 1442, (C]C),
3H, OCH3), 2.17 (s, 3H, CH3); 13C NMR (CDCl3)
d (ppm): 169.45 (C]
1230 (CeN); 1H NMR (CDCl3)
d
(ppm): 8.27e7.15 (m, 13H, AreH),
O), 156.22 (C]N), 151.18 (C-3, pyrazoline ring), (148.29, 142.12,
131.25, 128.77, 127.02, 126.33, 123.15, 120.15, 114.98, phenyl ring),
58.38 (C-5, pyrazoline ring), 52.10 (OCH3), 48.50 (CH2), 44.85 (C-4,
pyrazoline ring), 22.65 (CH3); ESI-MS m/z: [M þ H]þ ¼ 487.16.
5.90 (s, 2H, CH2), 5.67 [dd, 1H, J ¼ 11.6, 5.0 Hz, Hx (pyrazoline
ring)], 3.91 [dd, 1H, J ¼ 18.8, 5.4 Hz, Ha (pyrazoline ring)], 3.36 [dd,
1H, J ¼ 18.0, 11.5 Hz, Hb (pyrazoline ring)], 3.23 (s, 3H, OCH3), 3.25
(s, 3H, OCH3); 13C NMR (CDCl3)
d (ppm): 169.46 (C]O), 156.22 (C]
N), 151.18 (C-3, pyrazoline ring), (148.29, 142.12, 131.25, 128.77,
127.02, 126.33, 123.15, 120.15, 114.98, phenyl ring), 54.38 (C-5,
pyrazoline ring), 52.16 (OCH3), 49.50 (CH2), 44.45 (C-4, pyrazoline
ring); ESI-MS m/z: [M þ H]þ ¼ 469.19.
4.1.4.9. 1-(3-(4-chlorophenyl)-4,5-dihydro-5-(4-methoxyphenyl)pyr-
azol-1-yl)-2-(5-(4-methoxy
phenyl)-1H-tetrazol-1-yl)ethanone
(9a). Yellowish; Yield 65.0%; mp. 315e317 ꢀC; Anal. Calc. For
C26H23ClN6O3: C 62.09, H 4.61, N 16.71%; found: C 62.31, H 4.47, N
16.78%; IR nmax (cmꢃ1): 3025 (AreH), 2932 (CH2), 1680 (C]O), 1582
(C]N), 1445, (C]C), 1233 (CeN), 778 (CeCl); 1H NMR (CDCl3)
4.1.4.5. 1-(4,5-dihydro-5-(4-nitrophenyl)-3-phenylpyrazol-1-yl)-2-
(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (5a). Yellowish;
Yield 54.8%; mp. 298e300 ꢀC; Anal. Calc. For C25H21N7O4: C 62.11, H
4.38, N 20.28%; found: C 62.32, H 4.26, N 20.37%; IR nmax (cmꢃ1):
3038 (AreH), 2921 (CH2), 1682 (C]O), 1580 (C]N), 1440, (C]C),
d
(ppm): 8.05e7.21 (m, 12H, AreH), 5.91 (s, 2H, CH2), 5.34 [dd, 1H,
J ¼ 11.5, 4.7 Hz, Hx (pyrazoline ring)], 3.91 [dd,1H, J ¼ 18.4, 5.4 Hz, Ha
(pyrazoline ring)], 3.60 [dd, 1H, J ¼ 18.0, 11.7 Hz, Hb (pyrazoline
ring)], 3.23 (s, 3H, OCH3), 3.19 (s, 3H, OCH3); 13C NMR (CDCl3)
1230 (CeN); 1H NMR (CDCl3)
d
(ppm): 8.10e6.83 (m, 13H, AreH),
d (ppm): 168.85 (C]O),156.45 (C]N),152.65 (C-3, pyrazoline ring),
5.95 (s, 2H, CH2), 5.52 [dd, 1H, J ¼ 11.4, 4.9 Hz, Hx (pyrazoline
ring)], 3.86 [dd, 1H, J ¼ 18.1, 4.8 Hz, Ha (pyrazoline ring)], 3.66 [dd,
1H, J ¼ 18.5, 11.7 Hz, Hb (pyrazoline ring)], 3.19 (s, 3H, OCH3); 13C
(148.25,143.65,133.20,128.54,127.33,125.89,123.45,120.25,114.28,
phenyl ring), 56.40 (C-5, pyrazoline ring), 50.65 (OCH3), 46.58 (CH2),
45.35 (C-4, pyrazoline ring); ESI-MS m/z: [M þ H]þ ¼ 503.15.
NMR (CDCl3)
d (ppm): 167.48 (C]O), 154.87 (C]N), 152.88 (C-3,
pyrazoline ring), (149.10, 142.76, 133.28, 129.80, 127.42, 126.21,
123.20, 120.10, 114.35, phenyl ring), 56.45 (C-5, pyrazoline ring),
54.65 (OCH3), 48.45 (CH2), 43.28 (C-4, pyrazoline ring); ESI-MS m/z:
[M þ H]þ ¼ 484.17.
4.1.4.10. 1-(3-(4-chlorophenyl)-4,5-dihydro-5-(4-nitrophenyl)pyrazol-1-
yl)-2-(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (10a). Orange
red; Yield 52.3%; mp. 187e190 ꢀC; Anal. Calc. For C25H20ClN7O4:
C 57.98, H 3.89, N 18.93%; found: C 57.83, H 3.94, N 18.80%; IR
nmax (cmꢃ1): 3035 (AreH), 2934 (CH2), 1685 (C]O),1580 (C]N), 1445,
1
4.1.4.6. 1-(3-(4-chlorophenyl)-4,5-dihydro-5-phenylpyrazol-1-yl)-2-
(C]C), 1230 (CeN), 792 (CeCl); H NMR (CDCl3)
d
(ppm): 8.27e7.15
(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (6a). White; Yield
(m, 12H, AreH), 5.90 (s, 2H, CH2), 5.51 [dd, 1H, J ¼ 11.5, 5.0 Hz, Hx

M.Y. Wani et al. / European Journal of Medicinal Chemistry 54 (2012) 845e854
853
(pyrazoline ring)], 3.86 [dd, 1H, J ¼ 17.8, 5.0 Hz, Ha (pyrazoline ring)],
3.67 [dd, 1H, J ¼ 17.8, 11.7 Hz, Hb (pyrazoline ring)], 3.30 (s, 3H, OCH3);
131.20, 128.11, 127.18, 125.04, 123.25, 120.15, 114.25, phenyl ring),
56.40 (C-5, pyrazoline ring), 54.16 (OCH3), 46.25 (CH2), 42.15 (C-4,
pyrazoline ring), 21.80 (CH3); ESI-MS m/z: [M þ H]þ ¼ 483.21.
13C NMR (CDCl3)
d (ppm): 169.20 (C]O), 154.98 (C]N), 151.35 (C-
3, pyrazoline ring), (149.55, 144.15, 131.28, 128.34, 127.03, 125.09,
123.65, 120.18, 114.28, phenyl ring), 56.48 (C-5, pyrazoline ring), 55.06
(OCH3), 48.24 (CH2), 43.08 (C-4, pyrazoline ring); ESI-MS m/z:
[M þ H]þ ¼ 518.13.
4.1.4.15. 1-(4,5-dihydro-5-(4-nitrophenyl)-3-p-tolylpyrazol-1-yl)-2-
(5-(4-methoxyphenyl)-1H-tetrazol-1-yl) ethanone (15a). Orange
red; Yield 46.5%; mp. 149e151 ꢀC, Anal. Calc. For C26H23N7O4: C
62.77, H 4.66, N 19.71%; found: C 62.54, H 4.74, N 19.57%; IR nmax
(cmꢃ1): 3035 (AreH), 2930 (CH2), 1680 (C]O), 1582 (C]N), 1441,
4.1.4.11. 1-(4,5-dihydro-5-phenyl-3-p-tolylpyrazol-1-yl)-2-(5-(4-
methoxyphenyl)-1H-tetrazol-1-yl) ethanone (11a). White; Yield
60.0%; mp. 302e305 ꢀC; Anal. Calc. For C26H24N6O2: C 69.01, H 5.35,
N 18.57%; found: C 69.13, H 5.19, N 18.48%; IR nmax (cmꢃ1): 3035
(AreH), 2932 (CH2), 1682 (C]O), 1580 (C]N), 1435, (C]C), 1230
(C]C), 1230 (CeN); 1H NMR (CDCl3)
d (ppm): 8.55e7.18 (m, 12H,
AreH), 5.94 (s, 2H, CH2), 5.57 [dd,1H, J ¼ 11.4, 5.0 Hz, Hx (pyrazoline
ring)], 3.91 [dd, 1H, J ¼ 17.8, 4.8 Hz, Ha (pyrazoline ring)], 3.34 [dd,
1H, J ¼ 18.0,11.7 Hz, Hb (pyrazoline ring)], 3.19 (s, 3H, OCH3), 2.18 (s,
(CeN); 1H NMR (CDCl3)
d
(ppm): 8.04e6.90 (m, 13H, AreH), 5.93 (s,
3H, CH3); 13C NMR (CDCl3)
d (ppm): 169.85 (C]O), 156.15 (C]N),
2H, CH2), 5.52 [dd, 1H, J ¼ 11.2, 4.8 Hz, Hx (pyrazoline ring)], 3.94
[dd, 1H, J ¼ 17.4, 4.7 Hz, Ha (pyrazoline ring)], 3.68 [dd, 1H, J ¼ 17.8,
11.2 Hz, Hb (pyrazoline ring)], 3.25 (s, 3H, OCH3), 2.45 (s, 3H, CH3);
152.45 (C-3, pyrazoline ring), (149.28, 142.26, 131.25,128.45, 127.25,
125.26, 123.25, 120.55, 114.20, phenyl ring), 58.28 (C-5, pyrazoline
ring), 55.50 (OCH3), 46.20 (CH2), 42.05 (C-4, pyrazoline ring), 24.11
(CH3); ESI-MS m/z: [M þ H]þ ¼ 498.18.
13C NMR (CDCl3)
d (ppm): 169.20 (C]O), 154.98 (C]N), 151.35 (C-3,
pyrazoline ring), (149.55, 144.15, 131.28, 128.34, 127.03, 125.09,
123.65, 120.18, 114.28, phenyl ring), 58.48 (C-5, pyrazoline ring),
55.45 (OCH3), 48.24 (CH2), 43.08 (C-4, pyrazoline ring), 20.45 (CH3);
ESI-MS m/z: [M þ H]þ ¼ 453.20.
4.2. In vitro antiamoebic assay
All the test compounds (1e15 and 1ae15a) were screened
in vitro for antiamoebic activity against HM1:IMSS strain of E. his-
tolytica by microdilution method [43]. E. histolytica trophozoites
were cultured in 96-well microtiter plate by using Diamond TYIS-
33 growth medium [51]. The test compounds (1 mg) were dis-
4.1.4.12. 1-(5-(4-chlorophenyl)-4,5-dihydro-3-p-tolylpyrazol-1-yl)-2-
(5-(4-methoxyphenyl)-1H-tetrazol-1-yl)ethanone (12a). Yellowish;
Yield 47.0%; mp. 210e213 ꢀC; Anal. Calc. For C26H23ClN6O2: C
64.13, H 4.76, N 17.26%; found: C 64.27, H 4.62, N 17.18%; IR nmax
(cmꢃ1): 3032 (AreH), 2935 (CH2), 1680 (C]O), 1580 (C]N), 1445,
solved in DMSO (40
mL, level at which no inhibition of amoeba
occurs) [52,53]. The stock solutions of the compounds were
prepared freshly before use at a concentration of 1 mg mLꢃ1. Two-
fold serial dilutions were made in the wells of 96-well microtiter
plate (costar). Each test includes metronidazole as a standard
amoebicidal drug, control wells (culture medium plus amoebae)
and a blank (culture medium only). All the experiments were
carried out in triplicate at each concentration level and repeated
thrice. The amoeba suspension was prepared from a confluent
culture by pouring off the medium and adding 5 mL of fresh
medium, chilling the culture tube on ice to detach the organisms
from the side of the flask. The number of amoeba mLꢃ1 was esti-
mated with a haemocytometer, using trypan blue exclusion to
confirm the viability. The suspension was diluted to 105 cells mLꢃ1
(C]C), 1230 (CeN), 790 (CeCl); 1H NMR (CDCl3)
d (ppm):
8.07e6.87 (m, 12H, AreH), 5.91 (s, 2H, CH2), 5.52 [dd, 1H, J ¼ 11.4,
5.0 Hz, Hx (pyrazoline ring)], 3.86 [dd, 1H, J ¼ 17.8, 4.8 Hz, Ha
(pyrazoline ring)], 3.40 [dd, 1H, J ¼ 18.0, 11.8 Hz, Hb (pyrazoline
ring)], 3.24 (s, 3H, OCH3), 2.21 (s, 3H, CH3); 13C NMR (CDCl3)
d
(ppm): 169.28 (C]O), 154.06 (C]N), 152.30 (C-3, pyrazoline
ring), (149.25, 144.29, 131.25, 128.15, 127.16, 125.04, 123.45, 120.45,
114.45, phenyl ring), 58.25 (C-5, pyrazoline ring), 55.05 (OCH3),
48.20 (CH2), 42.64 (C-4, pyrazoline ring) 22.60 (CH3); ESI-MS m/z:
[M þ H]þ ¼ 487.16.
4.1.4.13. 1-(4,5-dihydro-3,5-dip-tolylpyrazol-1-yl)-2-(5-(4-methoxyphenyl)-
1H-tetrazol-1-yl)ethanone (13a). White; Yield 56.2%; mp.
149e151 ꢀC, Anal. Calc. For C27H26N6O2 : C 69.51, H 5.62, N 18.01%;
found: C 69.34, H 5.52, N 18.13%; IR nmax (cmꢃ1): 3025 (AreH), 2925
(CH2), 1682 (C]O), 1585 (C]N), 1445, (C]C), 1238 (CeN); 1H NMR
by adding fresh medium and 170 mL of this suspension was added to
the test and control wells in the plate so that the wells were
completely filled (total volume, 340
m
L). An inoculum of 1.7 ꢄ 104
organisms/well was chosen so that confluent, but not excessive
growth, took place in control wells. Plates were sealed and gassed
for 10 min with nitrogen before incubation at 35.5 ꢀC for 72 h. After
incubation, the growth of amoeba in the plate was checked with
a low power microscope. The culture medium was removed by
inverting the plate and shaking gently. Plate was then immediately
washed with sodium chloride solution (0.9%) at 35.5 ꢀC. This
procedure was completed quickly and the plate was not allowed to
cool in order to prevent the detachment of amoebae. The plate was
allowed to dry at room temperature and the amoebae were fixed
with methanol and when dried, stained with (0.5%) aqueous eosin
for 15 min. The stained plate was washed once with tap water, then
(CDCl3)
d (ppm): 8.04e7.01 (m, 12H, AreH), 5.89 (s, 2H, CH2), 5.52
[dd, 1H, J ¼ 11.5, 5.0 Hz, Hx (pyrazoline ring)], 3.86 [dd, 1H, J ¼ 18.1,
5.1 Hz, Ha (pyrazoline ring)], 3.36 [dd, 1H, J ¼ 17.4, 11.6 Hz, Hb
(pyrazoline ring)], 3.25 (s, 3H, OCH3), 2.75 (s, 3H, CH3), 2.59 (s, 3H,
CH3); 13C NMR (CDCl3)
d (ppm): 168.65 (C]O), 153.28 (C]N),
151.30 (C-3, pyrazoline ring), (149.25, 143.18, 131.25, 128.14, 127.63,
124.45, 123.25, 120.48, 114.20, phenyl ring), 56.40 (C-5, pyrazoline
ring), 54.85 (OCH3), 48.25 (CH2), 42.68 (C-4, pyrazoline ring), 22.40
(CH3); ESI-MS m/z: [M þ H]þ ¼ 467.21.
4.1.4.14. 1-(4,5-dihydro-5-(4-methoxyphenyl)-3-p-tolylpyrazol-1-yl)-
2-(5-(4-methoxyphenyl)-1H-tetrazole-1-yl)ethanone (14a). Cream;
Yield 45.0%; mp. 149e151 ꢀC, Anal. Calc. For C27H26N6O3: C 67.21, H
5.43, N 17.42%; found: C 67.28, H 5.40, N 17.34%; IR nmax (cmꢃ1):
3032 (AreH), 2925 (CH2), 1682 (C]O), 1580 (C]N), 1435, (C]C),
twice with distilled water and allowed to dry. A 200 mL portion of
0.1 N sodium hydroxide solution was added to each well to dissolve
the protein and release the dye. The optical density of the resulting
solution in each well was determined at 490 nm with a microplate
reader. The % inhibition of amoebal growth was calculated from the
optical densities of the control and test wells and plotted against
the logarithm of the dose of the drug tested. Linear regression
analysis was used to determine the best fitting line from which the
1232 (CeN); 1H NMR (CDCl3)
d (ppm): 7.98e6.98 (m, 12H, AreH),
5.81 (s, 2H, CH2), 5.57 [dd, 1H, J ¼ 11.6, 5.0 Hz, Hx (pyrazoline
ring)], 3.86 [dd, 1H, J ¼ 18.0, 5.1 Hz, Ha (pyrazoline ring)], 3.42 [dd,
1H, J ¼ 18.0, 11.5 Hz, Hb (pyrazoline ring)], 3.23 (s, 3H, OCH3), 3.19
(s, 3H, OCH3), 2.59 (s, 3H, CH3); 13C NMR (CDCl3)
d
(ppm): 169.27
IC50 value was found. The IC50 values in
Tables 1and 2.
mM are reported in
(C]O), 158.48 (C]N), 152.65 (C-3, pyrazoline ring), (149.44, 142.20,

854
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4.3.1. Cell culture
Human hepatocellular carcinoma cell line (HepG2) was cultured
in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum
(heat inactivated), 100 units mLꢃ1 penicillin, 100 g mLꢃ1 strepto-
m
mycin, and 2.5
m
g mLꢃ1 amphotericin B, at 37 ꢀC in a saturated
humidity atmosphere containing 95% air/5% CO2 [54]. The cell lines
were harvested when they reached 80% confluence to maintain
exponential growth.
4.3.2. MTT assay
The MTT assay is a standard colorimetric assay, in which mito-
chondrial activity is measured by splitting tetrazolium salts with
mitochondrial dehydrogenases in viable cells only [55]. For viability
testing, MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazo-
lium bromide, M2128 from Sigma) cell proliferation assay was
carried out. The cell monolayers in exponential growth were har-
vested using 0.25% trypsin and single-cell suspensions were
obtained by repeated pipetting. Only viable cells were used in the
assay. Exponentially growing cells were plated at 1.2 ꢄ 104 cells per
well into 96-well plates (Costar, Corning, NY, USA) and incubated for
48 h before the addition of drugs to achieve the maximum con-
fluency of the cells. Stock solutions were prepared by dissolving the
compounds in 10% (v/v) DMSO and further diluted with fresh
complete medium to achieve 1 M concentration. Cells were incu-
bated with different concentrations of metronidazole and test
compounds for 48 h at 37 ꢀC in 5% CO2 humidified incubator
together with untreated control sample. At appropriate time points,
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mL MTT solution
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