Synthesis and antimalarial evaluation of 1, 3, 5-trisubstituted pyrazolines

Article abstract of DOI:10.1016/j.ejmech.2009.10.023

A series of 1,3,5-trisubstituted pyrazolines were synthesized and evaluated for in vitro antimalarial efficacy against chloroquine sensitive (MRC-02) as well as chloroquine resistant (RKL9) strains of Plasmodium falciparum. The activity was at nano molar concentration. β-hematin formation inhibition activity (BHIA₅₀) of the pyrazolines were determined and correlated with antimalarial activity. A reasonably good correlation (r = 0.62) was observed between antimalarial activity (IC₅₀) and BHIA₅₀. This suggests that antimalarial mode of action of this class of compounds appears to be similar to that of chloroquine and involves the inhibition of hemozoin formation. Some of the compounds were showing better antimalarial activity than chloroquine against resistant strain of P. falciparum and were also found active in the in vivo experiment.

Full text of DOI:10.1016/j.ejmech.2009.10.023

European Journal of Medicinal Chemistry 45 (2010) 430–438
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimalarial evaluation of 1, 3, 5-trisubstituted pyrazolines
Badri Narayan Acharya a, Deepika Saraswat a, Mugdha Tiwari a, Asish Kumar Shrivastava a,
,
Ramarao Ghorpade a, Saroj Bapna b, Mahabir Parshad Kaushik a
*
a Defence Research & Development Establishment, Jhansi Road, Gwalior 474002, India
b Hafkine Institute for Training, Research and Testing, Parel, Mumbai 400012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,3,5-trisubstituted pyrazolines were synthesized and evaluated for in vitro antimalarial
efficacy against chloroquine sensitive (MRC-02) as well as chloroquine resistant (RKL9) strains of Plas-
Received 23 March 2009
Received in revised form
30 July 2009
Accepted 15 October 2009
Available online 28 October 2009
modium falciparum. The activity was at nano molar concentration.
b-hematin formation inhibition
activity (BHIA50) of the pyrazolines were determined and correlated with antimalarial activity. A
reasonably good correlation (r ¼ 0.62) was observed between antimalarial activity (IC50) and BHIA50. This
suggests that antimalarial mode of action of this class of compounds appears to be similar to that of
chloroquine and involves the inhibition of hemozoin formation. Some of the compounds were showing
better antimalarial activity than chloroquine against resistant strain of P. falciparum and were also found
active in the in vivo experiment.
Keywords:
Pyrazoline
Plasmodium falciparum
Haem detoxification
Antimalarial
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
possess potential antiviral activity against flavivirus [24] and HIV
[25]. Our literature survey revealed that these classes of
Haem detoxification is a crucial biochemical process of malaria
parasite [1]. The survival of the parasite is highly dependent up on
proteolysis of hemoglobin of the host during its erythrocyte cycle
[2, 3]. Digestion of hemoglobin releases toxic iron II ferroproto-
porphyrin (FeII FPIX) moiety that subsequently oxidizes to FeIII FPIX
or hematin [4]. Hematin then polymerized forming inert crystals of
hemozoin [5] or malaria pigment. The polymerization process is
a mechanism of detoxification and can be targeted for antimalarial
therapy [6]. Considerable data now support the hypothesis that
antimalarial quinolines like chloroquine inhibit parasite growth
by binding to hematin and preventing its aggregation to hemozoin
[6–10]. But chloroquine resistance appears to arise from changes in
a food vacuole membrane protein PfCRT of the parasite, rather than
changes in detoxification pathway [11]. This suggests that, if a novel
chemical class of drug is being discovered against the same target
then that could have immense clinical value.
compounds are yet to be explored for their possible antimalarial
activities. As a part of the continuing effort towards antimalarial
drug discovery, we identified an 1,3,5-trisubstituted pyrazoline
(Fig. 1) as potential antimalarial agent targeting haem detoxifica-
tion pathway of malaria parasite. This identification was done on
the basis of mapping (Fig. 2) of the 1,3,5-trisubstituted pyrazoline
with our earlier reported pharmacophore model [26]. The phar-
macophore model contains ring aromatic (RA), positive ionisable
(PI), hydrogen bond acceptor (HBA) and aliphatic hydrophobic
(HY-ALI) features. Fig. 2 shows the mapping of 1,3,5-trisubstituted
pyrazoline for all the pharmacophoric features except PI.
Recently Ali et al., have reported similar 1,3,5-trisubstituted
pyrazolines for anti HIV activity involving one additional methyl
group on ring A and isoniotinyl group at 1 position of pyrazolyl ring
[25]. Molecules selected for this work contain nicotinyl group at 1
position of pyrazolyl ring without any methyl group on ring A. On
the basis of pharmacophore mapping, we hypothesized that this
type of 1,3,5-trisubstituted pyrazolines may show potential anti-
malarial activity targeting haem detoxification pathway of malaria
parasite. In order to validate the hypothesis experimentally, herein
we report the antimalarial activity of 1,3,5-trisubstituted pyrazo-
lines by synthesizing a series of ten molecules (1a–j) and evaluating
them against chloroquine sensitive (MRC-02) as well as chloro-
quine resistant (RKL9) strains of P. falciparum. In this study, only the
HY-ALI feature associated with ‘B’ ring of the 1, 3, 5-trisubstituted
Pyrazolines and their derivatives have been found to possess
a broad spectrum of biological activities such as antibacterial
[12–14], antidepressant [15] anticonvulsant [16–18] antihyperten-
sive [19] antioxidant [20] antitumor [21] and anticancer activities
[22,23]. Recently these classes of compounds are reported to
* Corresponding author. Tel.: þ91 751 2343972; fax: þ91 751 2341148.
E-mail address: mpkaushik@rediffmail.com (M.P. Kaushik).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.023

B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
431
the reaction. Haloginated chalcones (1a, 1f and 1i) took 8 h for
complete conversion followed by unsubstituted (1b, 12 h), methyl
substituted (1c and 1d, 16 h) and alkoxy substituted (1e and 1g,
24 h) chalcones. Compound 1h contains both halogen and alkoxy
groups, took 18 h for complete conversion. The generally accepted
interpretation of this reaction, involves the initial formation of an
aryl hydrazone with subsequent nucleophilic attack of nitrogen
upon the carbon-carbon double bond at
electropositive nature of carbon may control the overall rate of
the reaction. The electropositive nature of carbon is controlled by
b position. Hence the
b
b
the aromatic ring directly connected to it. Halogens being electron
withdrawing in nature significantly increase the positive character
of b carbon lead to faster reaction while electron donating alkyl and
alkoxy groups contributed for slower reaction. However this
phenomenon was not observed in case of nitro group (1j). The
possible explanation might be the meta-directing nature of nitro
group. In compound 1j, nitro group is placed at meta position of the
phenyl ring and by virtue of its meta-directing nature electron
Fig. 1. Structure of 1,3,5-trisubstituted pyrazoline.
pyrazoline moiety was changed by keeping the basic skeleton
intact. The compounds were also evaluated for b-hematin forma-
density at
b carbon increases and its electropositivity decreases
leading to longer time for completion of reaction.
A variety of methods have been reported for the synthesis of
1,3,5-trisubstituted pyrazolines [27–31]. Among the simplest
tion inhibition activity and correlated with their antiplasmodial
activity against CQ sensitive as well as resistant strains of malaria
parasite. From the correlations, the mode of action of this class of
compounds was proposed to follow haem detoxification inhibition
pathway. Three compounds (1a, 1f and 1g) found most active in
vitro against CQ resistant malaria parasite were also tested in vivo
against P. berghei.
methods, condensation of an a,b-unsaturated ketone with an acid
hydrazide in glacial acetic acid (bp 117 ꢀC) under reflux was
reported by Fischer and Kno¨venagel [27]. Comparing similar type
of molecules reported in literature [25], we found that the use of
n-butanol (bp 115 ꢀC) as reaction medium has significantly
improved the yield. This is essentially because acetic acid proton-
ates the pyrazoline and pyridine rings resulting improved solubility
in water. Hence during processing, significant quantities of the
products were washed away by water. N-butanol being very low
acidic in nature didn’t protonate the products and thus improved
the yield quantitatively.
2. Results and discussion
2.1. Chemistry
1,3,5-trisubstituted pyrazolines (1a–j) were synthesized
according to the method shown in Scheme. In the first step,
syntheses of chalcones were carried out by the well-known
Claisen-Schmidt reaction and products were purified by recrystal-
lization from methanol (60–70% yield). In the second step, chalcone
and nicotinic acid hydrazide were refluxed in n-butanol in order to
synthesize the desired product. Factors such as the structure and
position of the substituents have profoundly influenced the rate of
Structures of compounds 1a–j were confirmed by MS and NMR
techniques. All of the 1,3,5-trisubstituted pyrazolines possesses
similar basic skeletal structure. Proton NMR signals were assigned
by comparing the spectra of the products (1a–j) with their corre-
sponding chalcones. Signals around
d value 3.1 and 3.9 ppm recor-
ded as doublet of doublets (dd) were assigned to 4-Ha and 4-Hb
protons. The J values were calculated for above signals and found to
be around 17 Hz and 5 Hz for signal around 3.1 ppm and 17 Hz and
11 Hz for signal around 3.9 ppm respectively. 5-H proton (d around
5.9 ppm) of pyrazoline ring showed a ‘dd’ pattern of J values around
11 Hz and 5 Hz respectively and most likely interacting with 4-Ha
and 4-Hb protons. In the deuterium exchange, protons at
10 ppm was exchanged which was assigned as OH proton.
d value
2.2. In vitro antiplasmodial activity
In vitro antiplasmodial activity (Table 1) of 1a–b and 1f–h were
M)
found comparable with chloroquine diphosphate (IC50 ¼ 0.021
m
against chloroquine sensitive strain (MRC-02) of P. falciparum.
Compounds 1a, 1c, 1d, 1f and 1g of this series have shown better
activity than chloroquine diphospahte (IC50 ¼ 0.177
chloroquine resistant strain (RKL9) of parasite. Compound 1g was
found to be most potent against resistant (IC50 ¼ 0.0425 M) as
M) strains of P. falciparum. For
mM) against
m
well as sensitive (IC50 ¼ 0.0265
m
compound 1g, R2, R3 and R4 are methoxy groups. Interestingly
compound 1b, for which R1–R5 are all hydrogen, also showed
antiplasmodial efficacy close to 1g against chloroquine sensitive
strain (IC50 ¼ 0.0304
compound 1b (IC50 ¼ 0.1305
M). This indicates that HY-ALI feature plays
an important role for antimalarial activity.1a,1f and 1i are the three
halogen containing compounds showed very good activity against
m
M). However, against resistant strain,
mM) was found to be 3 fold less active
Fig. 2. Pharmacophore mapping of 1,3,5-trisubstituted pyrazoline. The blue contour
represents the hydrophobic aliphatic feature (HY-ALI), the orange contour represents
ring aromatic feature (AR), the green contour represents hydrogen bond acceptor
feature (HBA) and the red contour represents positive ionizable feature (PI).
than 1g (IC50 ¼ 0.0425
m

432
B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
Scheme. Synthesisa of 1, 3, 5-trisubstituted pyrazolines.
both chloroquine sensitive and resistant strains. Methyl group in
place of R1 (1c) and R3 (1d) also delivered good activities. 1e and 1h
are the two compounds containing two HY-ALI features each but
showed moderate to poor in vitro antiplasmodial activity. In this
series only the HY-ALI feature was changed and all other features
were kept intact. 1b and 1j are lacking in HY-ALI feature were
predicted as poorly active, which was found valid for both of the
compounds.
2.3. In vitro cytotoxicity
The maximum concentration of DMSO in any well was 0.1% and
did not affect cell growth. All the ten molecules of this series were
found to be at least 10 times more toxic than chloroquine (Table 1).
Yet for all the compounds, the cytotoxic/antiplasmodial ratios were
>1, indicating better selectivity against P. falciparum even though
a long cell-drug exposure time (72 h) was given for cytotoxicity
test. Selectivity ratios of two most potent antimalarial compounds
1f and 1g were 59.56 and 19.48 for resistant strain of P. falciparum.
In case of sensitive strain those values were 140.14 and 31.24
respectively. The most potent 1,3,5-tritrisubstituted pyrazoline 1g
was found to be most toxic and second best active 1f was found to
be least toxic among the ten compounds under study. Selectivity
ratio of 1f was found best for both sensitive and resistant strains of
malaria parasite.
Table 1
Cytotoxicity, in vitro and in vivo antimalarial activity of 1,3,5-trisubstituted
pyrazolines.
Compounds Cytotox.a Activity vs
M)
MRC-02 strainb antimal.
M) ratio
33.33
Cytotox./ Activity vs
RKL9 strainc
M)
Cytotox./
antimal.
ratio
(
m
(
m
(m
1a
1b
1c
1d
1e
1f
1.250
1.875
1.812
2.187
3.562
3.812
0.828
2.660
3.062
3.750
0.0375 ꢁ 0.005
0.0304 ꢁ 0.006
0.0625 ꢁ 0.022
0.0555 ꢁ 0.016
0.0755 ꢁ 0.024
0.0272 ꢁ 0.005
0.0265 ꢁ 0.005
0.0495 ꢁ 0.011
0.0260 ꢁ 0.011
0.1065 ꢁ 0.056
0.0680 ꢁ 0.019 18.38
0.1305 ꢁ 0.031 14.36
0.1019 ꢁ 0.002 17.78
0.0887 ꢁ 0.005 24.65
0.2140 ꢁ 0.049 16.64
0.0640 ꢁ 0.003 59.56
0.0425 ꢁ 0.005 19.48
0.2420 ꢁ 0.089 10.99
0.0786 ꢁ 0.003 38.95
61.67
28.99
39.40
47.49
140.14
31.24
53.73
117.76
35.21
2.4. In vivo antiplasmodial activity
The three most active compounds (1a, 1f and 1g) found in the
in vitro antiplasmodial assays, were tested in mice infected with
P. berghei by oral administration (Table 2). Compound 1a showed
69% average suppression at 50 mg/kg/day in comparison to 100%
suppression by CQ at 8 mg/kg/day. The HPLC retention time (Rt) of
1a, 1f and 1g was 12.67, 11.64 and 11.87 respectively. This signifies
that with two chlorines 1a is most hydrophobic followed by 1f and
1g. Their in vivo antimalartial activity followed the same trend and
showed a correlation. The mean survival time (MST) was found in
accordance with the inhibition data.
1g
1h
1i
1j
0.4235 ꢁ 0.041
8.85
CQd
66.233
0.0210 ꢁ 0.003 3153.95
0.177 ꢁ 0.004 374.19
a
Cytotoxixity (maximum nontoxic dose, MNTD) against HeLa cells.
b
Antiplasmodial activity against chloroquine sensitive (MRC-02) strain of P. falci-
parum, IC50
,
m
M ꢁ SD; results of two separate determinations.
Antiplasmodial activity against chloroquine resistant (RKL9) strain of P. falci-
parum.IC50
M ꢁ SD; results of two separate determinations.
Chloroquine diphosphate.
c
,
m
d
Table 2
In vivo antimalarial activity against P. berghei ANKA strain in swiss mice.
Compounds
% Suppression on day 4a
Mean survival timea
% Suppression on day 4b
Mean survival timeb
Rt (min)e
Purity (%)e
(MST in days) ꢁ SE
(MST in days) ꢁ SE
1a
1f
1g
CQ
68.93
47.52
43.70
100c
0d
13.22 ꢁ 1.19
10.33 ꢁ 1.98
11.00 ꢁ 0.78
All alive
60.55
34.98
32.78
–
11.55 ꢁ 1.02
12.67
11.64
11.87
–
98.72
98.91
98.83
–
9.00 ꢁ 1.06
9.00 ꢁ 1.55
–
–
Control
6.44 ꢁ 0.97
–
–
–
a
At 50 mg/kg/day.
At 25 mg/kg/day.
At 8 mg/kg/day.
Without drug.
b
c
d
e
HPLC.

B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
433
h
h
i
h
i
2 9
Table 3
Hþ
Hþ
ꢀ
ꢀ
=
Relationship between pka, normalized antiplasmodial IC50 values, and inhibition of
v
e
v
1 þ
1 þ
þ
þ
b
-hematin formation for 1,3,5-trisubstituted pyrazolines and chloroquine
;
Ka2
Ka1Ka2
diphosphate.
Qv
Qe
VAR ¼
¼
(1)
i
h
i
2 9
=
e
Compounds pKa2, pKa1 VARa
ab
IC50
ꢂ
ac IC50
ꢂ
ad BHIA50
e
Hþ
Ka2
Hþ
1a
1b
1c
1d
1e
1f
1g
1h
1i
9.08, 6.65 1001.20 0.79 0.0295
8.95, 6.78 1254.89 1.00 0.0300
8.89, 7.08 2049.04 1.63 0.1020
9.01, 6.80 1303.19 1.03 0.0576
8.93, 6.84 1464.13 1.16 0.0879
9.11, 7.20 2457.64 1.95 0.0532
8.98, 6.95 1676.66 1.33 0.0354
9.10, 6.90 1551.03 1.23 0.0611
9.01, 6.90 1545.72 1.23 0.0320
0.0542
0.1305
0.1663
0.0921
0.1524
0.1253
0.0567
0.2991
0.0968
0.2046
0.8307
1.53 ꢁ 0.025
1.58 ꢁ 0.454
2.43 ꢁ 0.010
1.57 ꢁ 0.142
2.39 ꢁ 0.023
1.82 ꢁ 0.462
1.70 ꢁ 0.002
1.89 ꢁ 0.002
2.06 ꢁ 0.034
2.07 ꢁ 0.630
1.89 ꢁ 0.008
;
Ka1Ka2
The calculated VAR and the VAR relative to 1b (
Table 3. Compound 1b was taken as reference molecule to calculate
because this molecule was not substituted at B ring. VAR values
for the compounds of this series are calculated in the range of 606
(1j) to 2458 (1f). For chloroquine diphosphate the value was 5890.
a
) are shown in
a
1j
8.95, 6.38
606.30 0.48 0.0512
9.61, 8.55 5889.85 4.69 0.0985
A
significant difference between VAR values were observed
CQf
between 1f (VAR ¼ 2458) and 1i (VAR ¼ 1546) in which bromine is
present at ortho and meta position respectively. In case of 1h
(VAR ¼ 1551) bromine is present at meta position and its VAR value
a
Vacuolar accumulation ratio (VAR) calculated using Eq. (1) and assuming
vacuolar pH of 5.5 and external pH of 7.4.
b
Vacuolar accumulation ratio relative to 1b.
Normalized antiplasmodial activity against chloroquine sensitive (MRC-02)
c
was found closer to 1i. The relative VAR (a) values for all the
compounds were calculated in the range 0.48–1.95 and that for
chloroquine diphospahte was 4.69. Due to higher pKa values,
chloroquine diphosphate achieved 2.5–9 times better accumulation
in the parasite food vacuole in comparison to the 1,3,5-trisubsti-
strain of P. falciparum.
d
Normalized antiplasmodial activity against chloroquine resistant (RKL9) strain
of P. falciparum.
e
b-hematin inhibitory activity, IC50
,
mM ꢁ SD; results of two separate
determinations.
f
tuted pyrazolines (1a–j). Normalized IC50 values (IC50
obtained by multiplying experimentally obtained antimalarial IC50
with . Normalized IC50 values are the theoretically expected IC50
ꢂ
a) were
Chloroquine diphosphate.
a
2.5. Correlation of antiplasmodial activities with
formation inhibition
b
-hematin
values, if the compounds accumulate in the vacuole to the same
extent as 1b. Having adjusted for the differential accumulation of
the compounds, the normalized antiplasmodial IC50 values and the
To establish the mode of action, vacuolar accumulation ratio
(VAR) of all the compounds were theoretically calculated from
experimentally determined pKa values (Table 3) using Eq. (1) [32].
The two pKa values of CQ diphosphate were determined experi-
mentally and were found close to the previously reported values
[32]. In Eq. (1), VAR represents the ratio of drug inside and outside
of the parasite food vacuole. pHv ¼ pH inside food vacuole
(assumed to be pH 5.5) and pHe ¼ pH externally (assumed to be
pH 7.4).
inhibition of b-hematin formation (BHIA50) should show a clear
correlation. Hawley et al. [9] and Kaschula et al. [32] reported direct
proportionality between normalized antiplasmodial activity and
inhibition of b-hematin formation of quinoline antimalarials using
chloroquine sensitive strains of P. falciparum. In this study we have
taken both chloroquine sensitive (MRC-02) and chloroquine resis-
tant (RKL9) strains of P. falciparum.
The quantities of the compounds required to inhibit the
formation of b-hematin by 50% (BHIA50) were found in a narrow
Fig. 3. Plot of the log of the normalized antiplasmodial IC50 (IC50
sensitive (MRC-02) strain without chloroquine diphosphate. (B) Study involving chloroquine sensitive (MRC-02) strain with chloroquine diphosphate. (C) Study involving chlo-
roquine resistant (RKL9) strain without chloroquine diphosphate. (D) Study involving chloroquine sensitive (RKL9) strain with chloroquine diphosphate.
ꢂ
a) versus the log of the activity against the inhibition of b-hematin formation. (A) Study involving chloroquine

434
B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
range 1.53 mM (1a) to 2.43 mM (1c). Un-substituted 1b showed
comparable -hematin formation inhibition activity (1.58 mM).
HPLC system equipped with Waters 1525 binary gradient pump
and 2487 dual absorbance UV detector. Separations were carried
b
Exceptionally, compound 1c containing a methyl group showed
better BHIA50 than 1g (1.70 mM), which contains three methoxy
groups. In case of 1f and 1i, unlike VAR, the position of bromine
did not make any significant difference between their BHIA50
values. Chloroquine diphosphate showed a comparable value of
out on Waters Exterra C8 MS column (2.1 ꢂ 30 mm, 3.5
m) with
binary linear gradient of 5% methanol in water to 100% methanol in
20 min and wavelength at 254 nm.
4.2. General method for synthesis of chalcones (4a–4j)
1.89 mM. Although chloroquine showed lower b-hematin inhibi-
tion activity, due to better VAR value it was found to be more
potent than 1g against chloroquine sensitive P. falciparum. The
To the solution of (8 mmole, 1.1 g) of 2-hydroxy acetophenone
(2) 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 8 mmole of appropriate aldehyde (3a–j) was added
and the reaction mixture was stirred at room temperature for 24 h.
The reaction mixture was cooled on an ice bath and neutralized
with dilute hydrochloric acid. The precipitate appeared was sepa-
rated 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 60F254) by using mixture of eth-
ylacetate and hexane as mobile phase.
normalized IC50 (IC50
chloroquine diphosphate were calculated for both sensitive and
resistant strains. A plot of log (IC50 ) against log BHIA50 (Fig. 3)
showed some correlation present between the normalized IC50
and the ability of the compounds to inhibit -hematin formation.
ꢂ
a) values for all the compounds including
ꢂ
a
b
Against chloroquine sensitive and resistant strains, reasonably
good correlations (r ¼ 0.62 and 0.63 respectively) were observed.
When chloroquine was included, the
r values were found
decreased to 0.54 and 0.40 for sensitive and resistant strains
respectively. In case of resistant strain the value of r significantly
decreased from 0.63 to 0.40 because chloroquine is less active
against resistant strain. Low difference between r values was
observed when chloroquine diphosphate was included against
sensitive strain of parasite. This indicates, chloroquine and 1,3,5-
trisubstituted pyrazolines (1a–j) have similar antiplasmodial
mode of action. This finding further strengthens the hypothesis
that haem polymerization inhibition may be a possible mode of
action for this series of compounds (1a–j).
4.2.1. 3-(2,4-Dichloro-phenyl)-1-(4-hydroxy-phenyl)-
propenone (4a)
Prepared by above method from 2 (8 mmol, 1.1 g) and 2,4-
dichlorobenzaldehyde (8 mmol,1.42 g); Yield: 1.42 g, 61%; Rf ¼ 0.64
in EtOAc/hexane, 3:7; Off white crystalline solid. mp:192–193 ꢀC;
ESI-MS (M þ H): C15H10Cl2O2, calcd.: 293.0136, found: 293.0222;
FTIR (cmꢃ1): 3129, 1591, 1549, 1223; 1H NMR (400 MHz, DMSO-d6)
3. Conclusions
d
/ppm 6.89 (2H, d, J ¼ 8 Hz), 7.53 (1H, dd, J ¼ 8.8 Hz, 2 Hz), 7.74 (1H,
d, J ¼ 2 Hz), 7.92 (1H, d, J ¼ 15.6 Hz), 7.98 (1H, d, J ¼ 15.2 Hz), 8.07
(2H, d, J ¼ 8.8 Hz), 10. 39 (1H, s, OH).
We reported the synthesis and evaluation of 1,3,5-trisubstituted
pyrazolines as potential antimalarial agents targeting haem
detoxification pathway of malaria parasite. The molecules were
found active against in vitro culture of both chloroqine sensitive as
well as chloroquine resistant strains of P. falciparum. Compound 1g
was found to be most active as well as most cytotoxic. The second
best active compound 1f was found to be least cytotoxic among the
ten compounds. Three compounds (1a, 1f and 1g) were tested
against P. berghei in mouse model found active. Good correlation
4.2.2. 1-(4-Hydroxy-phenyl)-3-phenyl-propenone (4b)
Prepared by above method from 2 (8 mmol, 1.1 g) and benzal-
dehyde (8 mmol, 0.89 g); Yield: 1.21 g, 67%, Rf ¼ 0.45 in EtOAc/
hexane, 3:7. Colourless crystalline solid. mp: 180–181 ꢀC; ESI-MS
(M þ H): C15H12O2, calcd.: 225.2625, found: 225.2032; FTIR (cmꢃ1):
3115, 1646, 1598, 1565, 1337, 1219; 1H NMR (400 MHz, DMSO-d6)
d/
was observed between normalized antimalarial activity (IC50
ꢂ
a)
ppm 6.89 (2H, d, J ¼ 8 Hz), 7.43 (3H, m), 7.65 (1H, d, J ¼ 16), 7.85 (2H,
and -hematin formation inhibition (BHIA50) against both sensitive
b
m), 7.88 (1H, d, J ¼ 16 Hz), 8.05 (2H, d, J ¼ 8), 10. 46 (1H, s, OH).
and resistant strains. When chloroquine was included, a drastic
drop in correlation was observed in case of resistant strain only.
These observations indicated that the 1,3,5-trisubstituted pyrazo-
lines are antimalarial and their mode of action may be through
inhibition of haem detoxification process.
4.2.3. 1-(4-Hydroxy-phenyl)-3-o-tolyl-propenone (4c)
Prepared by above method from 2 (8 mmol, 1.1 g) and 2-methyl-
benzaldehyde (8 mmol, 1.12 g); Yield: 1.20 g, 63%, Rf ¼ 0.53 in EtOAc/
hexane, 3:7; Paleyellow crystalline solid. mp: 171–172 ꢀC; ESI-MS
(M þ H): C16H14O2, calcd.: 239.1072, found: 239.2142; FTIR (cmꢃ1):
4. Experimental protocols
3127, 1647, 1600, 1560, 1386, 1218; 1H NMR (400 MHz, DMSO-d6)
d/
4.1. Chemistry
ppm 2.37 (3H, s, CH3), 6.83 (2H, d, J ¼ 8.2 Hz), 7.22 (3H, m), 7.70 (1H, d,
J ¼ 15.5 Hz), 7.85 (1H, d, J ¼ 14.9 Hz), 7.89 (1H, s), 7.99 (2H, d,
J ¼ 8.6 Hz), 10. 39 (1H, s, OH).
Nicotinic acid hydrazide, 2-hydroxy acetophenone and all the
aromatic aldehydes were obtained from Sigma–Aldrich. Melting
points were determined on Electrothermal 9100 apparatus and
uncorrected. Mass spectra were acquired on a Micromass Q-ToF
high resolution mass spectrometer equipped with electrospray
ionization (ESI) on Masslynx 4.0 data acquisition system. ESI was
used in þve ionization mode. Vibrational spectra were recorded on
a Bruker Tensor 27 FTIR spectrometer. 1H NMR spectra were
acquired on Bruker DPX 400 FT NMR spectrometer at 400 MHz. 13C
NMR spectra were acquired on Bruker AV 400 FT NMR at 100 MHz.
All the NMR spectra were acquired in DMSO-d6 at 27 ꢀC. Purity of
chalcones was checked on TLC (Merck Silica gel 60F254). Purity of
the 1,3,5-trisubstituted pyrazolines were checked by HPLC. Waters
4.2.4. 1-(4-Hydroxy-phenyl)-3-p-tolyl-propenone (4d)
Prepared by above method from 2 (8 mmol, 1.1 g) and 4-meth-
ylbenzaldehyde (8 mmol, 1.15 g); Yield: 1.14 g, 60%, Rf ¼ 0.56 in
EtOAc/hexane, 3:7; Yellow crystalline solid. mp: 197–198 ꢀC; ESI-
MS (M þ H): C16H14O2, calcd.: 239.1072, found: 293.1841; FTIR
(cmꢃ1): 3117, 1646, 1596, 1553, 1226; 1H NMR (400 MHz, DMSO-d6)
d
/ppm 2.28 (3H, s, CH3), 6.82 (2H, d, J ¼ 8.6 Hz), 7.19 (2H, d,
J ¼ 7.9 Hz), 7.51 (1H, d, J ¼ 15.6 Hz), 7.68 (2H, d, J ¼ 8.0 Hz), 7.77 (1H,
s), 7.99 (2H, d, J ¼ 8.6 Hz), 10.37 (1H, s, OH).

B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
435
4.2.5. 3-(4-Ethoxy-3-methoxy-phenyl)-1-(4-hydroxy-phenyl)-
1650, 1600, 1559, 1516, 1344, 1223; 1H NMR (400 MHz, DMSO-d6)
d/
propenone (4e)
ppm 6.82 (2H, d, J ¼ 7.9 Hz), 7.75 (2H, m), 8.10 (2H, d, J ¼ 8.1 Hz),
8.14 (1H, m), 8.23 (1H, d, J ¼ 8.4 Hz), 8.30 (1H, d, J ¼ 8 Hz), 8.74 (1H,
s), 10.53 (1H, s, OH).
Prepared by above method from 2 (8 mmol, 1.1 g) and 4-Ethoxy-
3-Methoxybenzaldehyde (8 mmol, 1.45 g); Yield: 1.54 g, 65%,
Rf ¼ 0.51 in EtOAc/hexane, 4:6; Yellow crystalline solid. mp: 212–
214 ꢀC; ESI-MS (M þ H): C18H18O4, calcd.: 299.1283, found:
299.1613; FTIR (cmꢃ1): 3075, 2968, 1643, 1593, 1503, 1250; 1H NMR
4.3. General method for synthesis of 1, 3, 5- trisubstituted
pyrazolines (1a–1j)
(400 MHz, DMSO-d6)
d
/ppm 1.28 (3H, t, J ¼ 6.9 Hz, CH3), 3.81 (3H, s,
To the solution of (4 mmole) of the appropriate chalcone (4a–
j) in 10 mL of n-butanol, (4 mmole, 0.55 g) of nicotinicacidhy-
drazide was added and the reaction mixture was refluxed for 8–
24 h. Conversion was monitored in every 60 min interval on pre-
coated silica TLC plates (Merck, 60F254) by using mixture of
acetone and petroleum ether (40:60 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. Purity of the prod-
ucts was checked by HPLC.
CH3), 4.02 (2H, q, J ¼ 6.9 Hz, CH2), 6.84 (2H, d, J ¼ 8.6 Hz), 6.94 (1H,
d, J ¼ 8.3 Hz), 7.27 (1H, d, J ¼ 8.3 Hz), 7.46 (1H, s), 7.57 (1H, d,
J ¼ 15.4 Hz), 7.72 (1H, d, J ¼ 15.5 Hz), 8.01 (2H, d, J ¼ 8.6 Hz), 10.35
(1H, s, OH).
4.2.6. 3-(2-Bromo-phenyl)-1-(4-hydroxy-phenyl)-propenone (4f)
Prepared by above method from 2 (8 mmol, 1.13 g) and 2-Bro-
mobenzaldehyde (8 mmol, 1.1 g); Yield: 1.55 g, 64%; Rf ¼ 0.59 in
EtOAc/hexane, 3:7; Paleyellow crystalline solid. mp: 168–170 ꢀC;
ESI-MS (M þ Na): C15H11BrO2, calcd.: 325.1325, found: 325.1485;
FTIR (cmꢃ1): 3201, 1641, 1601, 1560, 1342, 1220; 1H NMR (400 MHz,
DMSO-d6)
d
/ppm 6.90 (2H, d, J ¼ 8.7 Hz), 7.39 (1H, m), 7.38 (1H, m),
4.3.1. [5-(2,4-Dichloro-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-
pyrazol-1-yl]-pyridin-3-yl-methanone (1a)
7.48 (1H, d, J ¼ 7.9 Hz), 7.94 (2H, d, J ¼ 2.5 Hz), 8.08 (2H, d,
J ¼ 8.9 Hz), 8.16 (d, 1H, J ¼ 7.8 Hz), 10.39 (1H, s, OH).
Prepared by above method from 4a (4 mmol, 1.17 g) and 5
(4 mmol, 0.55 g) after 8 h reflux; Yield: 1.61 g, 98%; White amor-
phous solid. mp: 220–221 ꢀC; HPLC Rt (min), purity: 12.67, 98.72%;
ESI-MS (M þ H): C21H16Cl2N3O2, calcd.: 412.0620, found: 412.0801;
IR (KBr, cmꢃ1): 3200, 2967, 1664, 1596, 1502, 1465, 1260, 1092, 798;
4.2.7. 1-(4-Hydroxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-
propenone (4g)
Prepared by above method from 2 (8 mmol, 1.1 g) and 3,4,5-
trimethoxydenzaldehyde (8 mmol, 1.58 g); Yield: 1.65 g, 66%,
Rf ¼ 0.48 in EtOAc/hexane, 4:6; Yellow crystalline solid. mp: 236–
238 ꢀC; ESI-MS (M þ H): C18H18O5, calcd.: 315.1232, found: 315.1901.
FTIR (cmꢃ1): 3115, 1645, 1595, 1282; 1H NMR (400 MHz, DMSO-d6)
1H NMR (DMSO-d6,
d
): 3.10 (1H, dd, J ¼ 17.9 Hz, 5.2 Hz, 4-Ha), 3.91
(1H, dd, J ¼ 17.8 Hz, 12.0 Hz, 4-Hb), 5.92 (1H, dd, J ¼ 11.7 Hz, 6.6 Hz,
5-H), 6.81 (2H, d, J ¼ 8.3 Hz, 14-H and 16-H), 7.28 (1H, d, J ¼ 8.3 Hz,
11-H), 7.39 (1H, d, J ¼ 8.3 Hz, 10-H), 7.54 (1H, m, 23-H), 7.55 (2H, d,
J ¼ 8.1 Hz, 13-H and 17-H), 7.70 (1H, s, 8-H), 8.27 (1H, d, J ¼ 7.8 Hz,
24-H), 8.71 (1H, d, J ¼ 4.4 Hz, 22-H), 9.07 (1H, s, 20-H), 10.05 (1H, s,
OH).
d
/ppm 3.64 (3H, s, CH3), 3.80 (6H, s, CH3), 6.84 (2H, d, J ¼ 8.5 Hz),
7.14 (2H, s), 7.55 (1H, d, J ¼ 15.5 Hz), 7.78 (1H, d, J ¼ 15.5 Hz), 8.03
(2H, d, J ¼ 8.5 Hz), 10.37 (1H, s, OH).
4.2.8. 3-(5-Bromo-2-methoxy-phenyl)-1-(4-hydroxy-phenyl)-
4.3.2. [3-(4-Hydroxy-phenyl)-5-phenyl-4,5-dihydro-pyrazol-1-yl-
propenone (4h)
]-pyridin-3-yl-methanone (1b)
Prepared by above method from 2 (8 mmol, 1.1 g) and 5-Bromo-
2-methoxybenzaldehyde (8 mmol, 1.75 g); Yield: 1.61 g, 60%,
Rf ¼ 0.64 in EtOAc/hexane, 4:6; Redish-yellow crystalline solid. mp:
199–200 ꢀC; ESI-MS (M þ H): C16H13BrO3, calcd.: 333.0126, found:
332.9661; FTIR (cmꢃ1): 3166, 1648, 1603, 1255; 1H NMR (400 MHz,
Prepared by above method from 4b (4 mmol, 0.89 g) and 5
(4 mmol, 0.55 g) after 12 h reflux; Yield: 1.34 g, 98%; Light yellow
amorphous solid. mp: 202–204 ꢀC; HPLC Rt (min), purity: 10.90,
98.68%; ESI-MS (M þ H): C21H18N3O2, calcd.: 344.1399, found:
344.0312; IR (KBr, cmꢃ1): 3420, 1685, 1599, 1502, 1270; 1H NMR
DMSO-d6)
d
/ppm 3.88 (3H, s,CH3), 6.87 (2H, d, J ¼ 8 Hz), 7.20 (1H, d,
(DMSO-d6,
d
): 3.12 (1H, dd, J ¼ 17.9 Hz, 5.2 Hz, 4-Ha), 3.85 (1H, dd,
J ¼ 7.8 Hz), 7.56 (1H, d, J ¼ 16 Hz), 7.91 (2H, m), 8.07 (2H, d,
J ¼ 17.9 Hz,11.9 Hz, 4-Hb), 5.73 (1H, dd, J ¼ 11.5 Hz, 5.1 Hz, 5-H), 6.82
(2H, d, J ¼ 8.4 Hz, 14-H and 16-H), 7.25 (5H, m, 7-H, 8-H, 9-H, 10-H
and 11-H), 7.56 (2H, d, J ¼ 8.1 Hz,13-H and 17-H), 7.53 (1H, m, 23-H),
8.24 (1H, d, J ¼ 7.8 Hz, 24-H), 8.69 (1H, d, J ¼ 4.4 Hz, 22-H), 9.04 (1H,
s, 20-H), 10.04 (1H, s, OH).
J ¼ 8.4 Hz), 8.17 (1H, s), 10.35 (1H, s, OH).
4.2.9. 3-(3-Bromo-phenyl)-1-(4-hydroxy-phenyl)-propenone (4i)
Prepared by above method from 2 (8 mmol, 1.1 g) and 3-Bro-
mobenzaldehyde (8 mmol, 1.49 g); Yield: 1.61 g, 67%, Rf ¼ 0.61 in
EtOAc/hexane, 3:7; Paleyellow crystalline solid. mp: 174–175 ꢀC;
ESI-MS (M þ Na): C15H11BrO2Na, calcd.: 325.1325, found: 325.4017;
FTIR (cmꢃ1): 3201, 1641, 1601, 1560, 1342, 1220; 1H NMR (400 MHz,
4.3.3. [3-(4-Hydroxy-phenyl)-5-o-tolyl-4,5-dihydro-pyrazol-1-yl]-
pyridin-3-yl-methanone (1c)
Prepared by above method from 4c (4 mmol, 0.95 g) and 5
(4 mmol, 0.55 g) after 16 h reflux; Yield: 1.38 g, 97%; Light yellow
amorphous solid. mp: 146–148 ꢀC; HPLC Rt (min), purity: 11.40,
98.75%; ESI-MS (M þ H): C22H20N3O2, calcd.: 358.1556, found:
358.0750; IR (KBr, cmꢃ1): 3153, 2800, 1657, 1597, 1506, 1277, 1160;
DMSO-d6)
d
/ppm 6.83 (2H, d, J ¼ 8.0 Hz), 7.68 (1H, d, J ¼ 16 Hz), 7.98
(3H, m), 8.03 (2H, m), 8.07 (2H, d, J ¼ 8 Hz), 10.51 (1H, s, OH).
4.2.10. 1-(4-Hydroxy-phenyl)-3-(3-nitro-phenyl)-propenone (4j)
1H NMR (DMSO-d6,
d
): 2.44 (3H, s, CH3), 3.00 (1H, dd, J ¼ 17.8 Hz,
Prepared by above method from 2 (8 mmol, 1.1 g) and enzal-
dehyde3-nitro (8 mmol,1.2 g); Yield: 1.31 g, 61%, Rf ¼ 0.53 in EtOAc/
hexane, 4:6; Offwhite solid. mp: 222–223 ꢀC; ESI-MS (M þ H):
C15H11NO4, calcd.: 270.2601, found: 270.1700; FTIR (cmꢃ1): 3142,
4.8 Hz, 4-Ha), 3.89 (1H, dd, J ¼ 17.8 Hz, 11.7 Hz, 4-Hb), 5.85 (1H, dd,
J ¼ 11.6 Hz, 4.7 Hz, 5-H), 6.81 (2H, d, J ¼ 8.1 Hz, 14-H and 16-H), 7.06
(1H, m, 8-H), 7.16 (2H, m, 9-H and 10-H), 7.22 (1H, m, 11-H), 7.56
(2H, d, J ¼ 7.6 Hz, 13-H and 17-H), 7.54 (1H, m, 23-H), 8.27 (1H, d,

436
B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
J ¼ 7.9 Hz, 24-H), 8.70 (1H, d, J ¼ 4.6 Hz, 22-H), 9.06 (1H, s, 20-H),
10.03 (1H, s, OH).
7.64 (1H, m, 23-H), 8.35 (1H, d, J ¼ 7.7 Hz, 24-H), 8.75 (1H, s, 22-H),
9.08 (1H, s, 20-H), 10.09 (1H, s, OH). 13C NMR (DMSO-d6,
): 42.1(2-
d
C), 55.9 (m-OCH3), 60.0 (p-OCH3), 60.7 (3-C), 102.6 (5-C,9-C), 115.7
(12-C, 14-C), 121.6 (10-C), 124.0 (18-C), 128.9 (17-C), 131.3 (11-C, 15-
C), 136.5 (7-C), 137.8 (4-C), 138.7 (19-C), 148.7 (20-C), 149.9 (6-C, 8-
C), 153.2 (22-C), 156.6, 159.9 (13-C) and 162.9 (16-C).
4.3.4. [3-(4-Hydroxy-phenyl)-5-p-tolyl-4,5-dihydro-pyrazol-1-yl]-
pyridin-3-yl-methanone (1d)
Prepared by above method from 4d (4 mmol, 0.95 g) and 5
(4 mmol, 0.55 g) after 16 h reflux; Yield: 1.38 g, 97%; Straw yellow
crystaline solid. mp: 188–190 ꢀC; HPLC Rt (min), purity: 11.62,
98.56%; ESI-MS (M þ H): C22H20N3O2, calcd. 358.1556, found:
358.5118; IR (KBr, cmꢃ1): 3435, 2993, 1645, 1598, 1507, 1286, 1241,
4.3.8. [5-(5-Bromo-2-methoxy-phenyl)-3-(4-hydroxy-phenyl)-4,5-
dihydro-pyrazol-1-yl]-pyridin-3-yl-methanone (1h)
Prepared by above method from 4h (4 mmol, 1.32 g) and 5
(4 mmol, 0.54 g) after 18 h reflux. Yield; 1.73 g, 96%; Brickred-yellow
solid. mp: 140–142 ꢀC; HPLC Rt (min), purity: 12.24, 98.12%; ESI-MS
(M þ H): C22H19BrN3O3, calcd.: 452.0610, found: 452.4197; IR (KBr,
827; 1H NMR (DMSO-d6,
d): 2.27 (3H, s, CH3), 3.09 (1H, dd,
J ¼ 17.9 Hz, 4.8 Hz, 4-Ha), 3.82 (1H, dd, J ¼ 17.8 Hz, 11.7 Hz, 4-Hb),
5.68 (1H, dd, J ¼ 11.3 Hz, 4.7 Hz, 5-H), 6.82 (2H, d, J ¼ 8.1 Hz, 14-H
and 16-H), 7.18 (4H, m, 7-H, 8-H, 10-H and 11-H), 7.51 (1H, m, 23-H),
7.54 (2H, d, J ¼ 7.6 Hz, 13-H and 17-H), 8.22 (1H, d, J ¼ 7.9 Hz, 24-H),
8.69 (1H, d, J ¼ 4.6 Hz, 22-H), 9.02 (1H, s, 20-H), 10.04 (1H, s, OH).
cmꢃ1): 3183, 2946, 1651, 1600, 1482, 1286; 1H NMR (DMSO-d6,
d):
3.03 (1H, dd, J ¼ 17.8 Hz, 5.0 Hz, 4-Ha), 3.83 (3H, s, OCH3), 3.86 (1H, m,
4-Hb), 5.79 (1H, dd, J ¼ 11.6 Hz, 4.9 Hz, 5-H), 6.81 (2H, d, J ¼ 8.6 Hz,
14-H and 16-H), 7.04 (1H, d, J ¼ 8.7 Hz, 8-H), 7.17 (1H, d, J ¼ 2.0 Hz,11-
H), 7.34 (1H, m, 23-H), 7.43 (1H, dd, J ¼ 8.7 Hz, 2.3 Hz, 9-H), 7.55 (2H,
d, J ¼ 8.4 Hz, 13-H and 17-H), 8.25 (1H, d, J ¼ 7.8 Hz, 24-H), 8.70 (1H,
d, J ¼ 3.7 Hz, 22-H), 9.04 (1H, s, 20-H), 10.05 (1H, s, OH).
4.3.5. [5-(4-Ethoxy-3-methoxy-phenyl)-3-(4-hydroxy-phenyl)-4,5-
dihydro-pyrazol-1-yl]-pyridin-3-yl-methanone (1e)
Prepared by above method from 4e (4 mmol, 1.19 g) and 5
(4 mmol, 0.55 g) after 24 hr reflux; Yield: 1.57 g, 95%; Purified by
preparative HPLC; Bright yellow crystalline solid. mp: 198–200 ꢀC;
HPLC Rt (min), purity: 11.78, 98.69%; ESI-MS (M þ H): C24H24N3O4,
calcd. 418.1667, found: 418.2295; IR (KBr, cmꢃ1): 3066, 2977, 1595
4.3.9. [5-(3-Bromo-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-
pyrazol-1-yl]-pyridin-3-yl-methanone (1i)
Prepared by above method from 4i (4 mmol, 1.21 g) and 5
(4 mmol, 0.55 g) after 8 h reflux; Yield: 1.65 g, 98%; Off white
amorphous solid. mp: 144–146 ꢀC; HPLC Rt (min), purity: 12.09,
98.43%. ESI-MS (M þ H): C21H17BrN3O2, calcd.: 422.0504, found:
422.0550; IR (KBr, cmꢃ1): 3202, 1608, 1519, 1476, 1281, 1198;. 1H
(C]O), 1515, 1442, 1258, 1138, 1029, 836; 1H NMR (DMSO-d6,
d):
1.30 (3H, t, J ¼ 6.84, CH3), 3.14 (1H, dd, J ¼ 13.5 Hz, 4.3 Hz, 4-Ha),
3.75(3H, s, OCH3), 3.85 (1H, m, 4-Hb), 3.96 (2H, q, J ¼ 6.8 Hz, OCH2),
5.67 (1H, dd, J ¼ 11.2 Hz, 4.2 Hz, 5-H), 6.82 (2H, d, J ¼ 8.2 Hz, 14-H,
16-H), 6.78 (1H, m, 7-H), 6.89 (2H, m, 10-H, 11-H), 7.51 (1H, m, 23-
H), 7.56 (2H, d, J ¼ 8.2 Hz, 13-H, 17-H), 8.23 (1H, d, J ¼ 6.6 Hz, 24-H),
8.72 (1H, s, 22-H), 9.03 (1H, s, 20-H), 10.03 (1H, s, OH).
NMR (DMSO-d6,
d
): 3.24 (1H, dd, J ¼ 18.1 Hz, 5.1 Hz, 4-Ha), 3.90 (1H,
dd, J ¼ 18.0 Hz, 11.7 Hz, 4-Hb), 5.91 (1H, dd, J ¼ 11.6 Hz, 5.1 Hz, 5-H),
6.83 (2H, d, J ¼ 8.7 Hz, 14-H and 16-H), 7.53 (1H, m, 23-H), 7.58 (2H,
d, J ¼ 8.5 Hz, 13-H and 17-H), 7.69 (1H, m, 10-H), 7.79 (1H, d,
J ¼ 7.7 Hz, 11-H), 8.15 (1H, d, J ¼ 8.1 Hz, 9-H), 8.19 (1H, s, 7-H), 8.26
(1H, d, J ¼ 7.8 Hz, 24-H), 8.70 (1H, d, J ¼ 4.5 Hz, 22-H), 9.05 (1H, s,
20-H), 10.08 (1H, s, OH).
4.3.6. [5-(2-Bromo-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-
pyrazol-1-yl]-pyridin-3-yl-methanone (1f)
Prepared by above method from 4f (4 mmol, 1.20 g) and 5
(4 mmol, 0.55 g) after 8 h reflux; Yield: 1.66 g, 98%; Offwhite solid.
mp: 138–140 ꢀC. HPLC Rt (min), purity: 11.64, 98.91%; ESI-MS
(M þ H): C21H17BrN3O2, calcd.: 422.0504, found: 422.2609; IR (KBr):
4.3.10. [3-(4-Hydroxy-phenyl)-5-(3-nitro-phenyl)-4,5-dihydro-
pyrazol-1-yl]-pyridin-3-yl-methanone (1j)
3185 (br), 1603, 1517, 1440, 1339, 1277; 1H NMR (DMSO-d6,
d
): 3.02
Prepared by above method from 4j (4 mmol, 1.07 g) and 5
(4 mmol, 0.54 g) after 16 h reflux; Yield: 1.47 g, 95%; Brown amor-
phous solid. mp: 230–232 ꢀC; HPLC Rt (min), purity: 10.59, 98.37%;
ESI-MS (M þ H): C21H17N4O4, calcd.: 389.1250, found: 389.1574. IR
(KBr, cmꢃ1): 3202, 1604, 1525, 1442, 1344, 1277, 1094; 1H NMR
(1H, dd, J ¼ 17.8 Hz, 5.0 Hz, 4-Ha), 3.92 (1H, dd, J ¼ 17.8 Hz, 1.8 Hz, 4-
Hb), 5.92 (1H, dd, J ¼ 11.8 Hz, 4.9 Hz, 5-H), 6.81 (2H, d, J ¼ 8.6 Hz, 14-
H and 16-H), 7.22 (2H, m, 9-H and 10-H), 7.34 (1H, m, 23-H), 7.53
(1H, m, 8-H), 7.55 (2H, d, J ¼ 8.6 Hz, 13-H and 17-H), 7.67 (1H, d,
J ¼ 7.9 Hz,11-H), 8.28 (1H, d, J ¼ 7.9 Hz, 22-H), 8.71 (1H, d, J ¼ 3.8 Hz,
(DMSO-d6,
d
): 3.29 (1H, dd, J ¼ 18.0 Hz, 5.1 Hz, 4-Ha), 3.95 (1H, dd,
24-H), 9.09 (1H, s, 20-H), 10.05 (1H, s, OH). 13C NMR (DMSO-d6,
d
):
J ¼ 18.0 Hz, 11.7 Hz, 4-Hb), 5.96 (1H, dd, J ¼ 11.5 Hz, 5.0 Hz, 5-H),
6.89 (2H, d, J ¼ 8.5 Hz, 14-H and 16-H), 7.58 (1H, m, 23-H), 7.63 (2H,
d, J ¼ 8.5 Hz, 13-H and 17-H), 7.74 (1H, m, 10-H), 7.84 (1H, d,
J ¼ 7.6 Hz, 11-H), 8.19 (1H, d, J ¼ 8.1 Hz, 9-H), 8.24 (1H, s, 7-H), 8.31
(1H, d, J ¼ 7.8 Hz, 24-H), 8.75 (1H, d, J ¼ 3.7 Hz, 22-H), 9.11 (1H, s,
20-H), 10.15 (1H, s, OH).
40.7 (2-C), 60.3 (3-C), 115.5 (12-C, 14-C), 115.6 (18-C), 121.1 (5-C),
121.5 (10-C), 123.1 (8-C), 128.4 (11-C, 15-C), 128.8 (17-C), 129.4 (9-C),
130.3 (7-C), 133.0 (6-C), 137.2 (4-C), 140.3 (19-C), 150.1(20-C),
151.3(22-C), 156.1 (1-C), 159.9 (13-C), 163.1 (16-C).
4.3.7. [3-(4-Hydroxy-phenyl)-5-(3,4,5-trimethoxy-phenyl)-4,5-
dihydro-pyrazol-1-yl]-pyridin-3-yl-methanone (1g)
4.4. In vitro antimalarial assay
Prepared by above method from 4g (4 mmol, 1.25 g) and 5
(4 mmol, 0.54 g) after 24 h reflux; Yield: 1.68 g, 97%; Dark brown
solid. mp: 184–186 ꢀC; HPLC Rt (min), purity: 11.87, 98.83%. ESI-MS
(M þ H): C24H24N3O5, calcd. 434.1716, found: 434.2260; IR (KBr):
P. falciparum strains MRC-02 (CQ sensitive) and RKL9 (CQ resis-
tant) were obtained from National Institute of Malaria Research, New
Delhi, India and maintained in a continuous culture using the stan-
dard method described by Trager and Jensen [33]. Parasites were
cultured in human B (þ) erythrocytes in RPMI-1640 media (GIBCO-
BRL, Paisely, Scotland) supplemented with 25 mM HEPES buffer, 10%
human AB (þ) serum and 0.2% sodium bicarbonate (Sigma) and
maintained at 5% CO2. Cultures containing predominantly early ring
3193,1596,1509,1454,1329,1281,1238; 1H NMR (DMSO-d6,
d): 3.15
(1H, dd, J ¼ 17.9 Hz, 5.0 Hz, 4-Ha), 3.63 (3H, s, p-OCH3), 3.72 (6H, s,
m-OCH3), 3.83 (1H, dd, J ¼ 11.7 Hz, 14.6 Hz, 4-Hb), 5.65 (1H, dd,
J ¼ 11.4 Hz, 4.9 Hz, 5-H), 6.57 (2H, s, 7-H and 11-H), 6.81 (2H, d,
J ¼ 8.6 Hz, 14-H and 16-H), 7.54 (2H, d, J ¼ 8.5 Hz, 13-H and 17-H),
stages were synchronized by addition of 5% D-sorbitol (Sigma) lysis

B.N. Acharya et al. / European Journal of Medicinal Chemistry 45 (2010) 430–438
437
[34], used for testing. Initial culture was maintained in small vials
with 10% haematocrit, i.e. 10 l erythrocytes containing 1.0% ring
stage parasite in 100 l complete media. The culture volume per well
for the assay was 100 l. Number of parasites for the assay was
by adding 100 mL of 0.5% acetic acid (V/V) in 50% ethanol to each
m
well followed by 10 min incubation. Absorbances of the resulting
solutions were read at 550 nm and cell survival was calculated as
the absorbance of the treated cells divided by the control (EMEM
plus DMSO). Results were expressed in terms of the MNTD
(maximum non toxic dose) values i.e the maximum concentration
of drug where 100% cell survival observed.
m
m
adjusted at 1–1.5% by diluting with fresh human B (þ) RBC. Assay
was done in 96 well microtitre flat-bottomed tissue culture plates
incubated at 37 ꢀC for 24 h in presence of two fold serial dilutions of
compounds and chloroquine diphosphate for their effect on schizont
maturation. Compounds were dissolved in ethanol and further
diluted with RPMI 1640 medium (the final ethanol concentration did
not exceed 0.5% which did not affect parasite growth). Chloroquine
diphosphate was dissolved in aqueous medium. Test was done in
duplicate wells for each dose of the drugs. Solvent control culture
containing the same concentrations of the solvent as present in the
test wells was done with RPMI-1640 containing 10% AB (þ) serum.
Parasite growth was found unaffected at the solvent concentrations.
Growth of the parasites from duplicate wells of each concentration
was monitored in Giemsa stained blood smears by counting number
of schizont per 100 asexual parasites. Percent schizont maturation
inhibition was calculated by the formula: (1-Nt/Nc) ꢂ 100 where, Nt
and Nc represent the number of schizont in the test and control well
respectively.
4.7. Determination of pKa values
Acid dissociation constants (pKas) were determined spectro-
photometrically as previously described [37] by using a Unicam UV
300 spectrometer of Thermo Spectronic. Absorbances of each
compound (0.025 mg mLꢃ1)dissolved in buffers of different pH
values ranging from 5.8 to 11.2 as well as in 0.1 M NaOH and 0.1 M
HCL were determined. Buffers from pH 5.8–8.0 were prepared from
0.1 M solutions of monobasic potassium phosphate (KH2PO4) and
dibasic potassium phosphate (K2HPO4) and from pH 8.4–11.2 were
prepared from 0.1 M glycine and 0.1 M NaOH. Analytical wave-
lengths (212 nm, 240 nm, 270 nm and 340 nm) were chosen at
which significant difference between the absorbances of molecules
in 0.1 M HCl and 0.1 M NaOH were observed. To reduce error, pKa
was determined in duplication for each compound.
4.5. In vivo antimalarial assay
4.8. Determination of inhibition of b-hematin formation
The in vivo efficacy was determined by Peter’s 4-Day suppres-
sive test [35] by using P. berghei ANKA strain and male white swiss
albino mice (18–20 gm of body weight). In brief, groups of five mice
each were inoculated intraperitonially with approximately 30% of P.
berghei infected erythrocytes from a donor mouse. Four hours
latter, selected test compounds were administered at a dose of
50 mg/kg/day and 25 mg/kg/day by oral route. Chloroquine (8 mg/
kg/day) was taken as standard for comparison. A total of four doses
were given on day 0, day 1, day 2 and day 3. Control groups received
the same amount of solvent used to suspend the compounds. The
tail blood smears were made on day 4 and day 7 stained with
Giemsa and examined microscopically. The % parasitemia was
determined and average %suppression of parasitemia in compar-
ison to control groups. Mean survival time (MST) was calculated for
the mice died during the 28 day observation period.
The quantitative BHIA (
based on the differential solubility of hematin and
DMSO and NaOH solution, respectively [38,39]. The method
determines a 50% inhibitory concentration for -hematin forma-
tion inhibition (BHIA50) of the compound. In a micro centrifuge
tube, 100 L of 6.4 mM solution of hematin freshly dissolved in
0.2 N NaOH solution; 50 L of compound dissolved in ethanol
(chloroquine diphosphate was dissolved in water); 200 L of 3 M
solution sodium acetate trihydrate (C2H3NaO2.3H2O); 50 L of
b-hematin inhibitory activity) assay is
b-hematin in
b
m
m
m
m
glacial acetic acid were incubated at 37 ꢀC for 24 h. Concentration
of compounds were varied from 6 mM to 0.3 mM range. Water
and ethanol were taken as controls for water soluble and ethanol
soluble compounds respectively. After incubation, the tubes were
centrifuged for 15 min at 3300g. The supernatant was discarded
and the pellet was reconstituted in DMSO and again centrifuged
4.6. In vitro cytotoxicity assay
for 15 min at 3300g. The supernatant was discarded and
hematin was obtained as a pellet. Following centrifugation to
isolate DMSO insoluble -hematin, the pellet was dissolved in
b-
HeLa (Human cervical epithelial) cells obtained from National
Centre for Cell Sciences (NCCS), Pune, India were maintained in our
laboratory. Cells were routinely maintained at 37 ꢀC in Eagles
Minimum Essential Medium (EMEM, Sigma, USA) supplemented
b
0.1 N NaOH and absorbance was recorded at 405 nM to calculate
BHIA50 as previously described [39].
with foetal bovine serum (10%), L-glutamine (2 mM), and genta-
Acknowledgment
micine (80 mg Lꢃ1). Cytotoxicity was assessed using the neutral red
(NR) dye uptake assay [36] using 96 well microtiter tissue (MT)
culture plates (Greiner, Germany). Briefly, 2 ꢂ 103 cells were seeded
in to each well of a 96 well MT plate and incubated overnight at
37 ꢀC in a humidified atmosphere containing 5% CO2. All the
compounds were dissolved in DMSO and subsequently serial two
fold dilution were prepared in EMEM to give a broad range of
concentration. Prior to assessment of cytotoxicity, the preformed
cell monolayer were washed with EMEM and replaced with drug
solutions in duplicate keeping cell control including corresponding
dilution of DMSO. The plates were incubated at 37 ꢀC and observed
microscopically daily for appearance of any morphological changes.
Following 72 h incubation, the cells were fixed with 5% glutaral-
dehyde solution prepared in PBS, kept at room temperature for
45 min. The fixed cells were washed with PBS, stained with 0.2% NR
dye aqueous solution and incubated for 1 h at room temperature.
After washing the stained cells were subjected to acid alcohol lysis
We thank Dr. R. Vijayaraghavan, Director DRDE, Gwalior for his
keen interest and encouragement in the present work. We espe-
cially thank Dr. Shri Prakash and Dr. M.M Parida for their helpful
advises and critical comments, Mr. Avik Mazumdar and Mr. Basant
Lal for NMR analysis.
Appendix. Supplementary data
Supplementary information related to this article can be found
at doi:10.1016/j.ejmech.2009.10.023.
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