Novel pyrazole-pyrazoline hybrids endowed with thioamide as antimalarial agents: Their synthesis and 3D-QSAR studies

Article abstract of DOI:10.3109/14756366.2014.958081

One of the most viable options to tackle the growing resistance to the antimalarial drugs is hybrid molecules. It involves combination of different scaffolds in one frame that may lead to compounds with diverse biological profiles. In this context, new hybrids of three different scaffolds viz pyrazole, pyrazoline and thiosemicarbazone moiety were incorporated into one single compound and evaluated for their in vitro schizontocidal activity against the CQ-sensitive 3D7 strain of Plasmodium falciparum. Compounds with significant in vitro antimalarial activity were further evaluated for cytotoxicity against VERO cell lines. The best active compound 48 exhibited an IC₅₀ of 1.13 μM. The in vitro results were further validated by quantitative structure-activity relationship (QSAR).

Full text of DOI:10.3109/14756366.2014.958081

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.958081
!
RESEARCH ARTICLE
Novel pyrazole–pyrazoline hybrids endowed with thioamide as
antimalarial agents: their synthesis and 3D-QSAR studies
Akranth Marella, Mohammad Shaquiquzzaman, Mymoona Akhter, Garima Verma, and Mohammad Mumtaz Alam
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Abstract
Keywords
One of the most viable options to tackle the growing resistance to the antimalarial drugs is
hybrid molecules. It involves combination of different scaffolds in one frame that may lead to
compounds with diverse biological profiles. In this context, new hybrids of three different
scaffolds viz pyrazole, pyrazoline and thiosemicarbazone moiety were incorporated into one
single compound and evaluated for their in vitro schizontocidal activity against the CQ-sensitive
3D7 strain of Plasmodium falciparum. Compounds with significant in vitro antimalarial activity
were further evaluated for cytotoxicity against VERO cell lines. The best active compound 48
exhibited an IC50 of 1.13 mM. The in vitro results were further validated by quantitative
structure–activity relationship (QSAR).
Antimalarial, cytoxicity, hybrid molecule,
P. falciparum (3D7 strain), pyrazoline
History
Received 3 June 2014
Revised 9 August 2014
Accepted 17 August 2014
Published online 17 October 2014
Introduction
anticancer9, CNS depressant10, etc. Lately, several researchers
have highlighted the antimalarial penchant of pyrazolines.
Wanare et al.11 reported pyrazoline derivatives (IV) as growth
inhibitors for P. falciparum. Similarly, Acharya et al.12 and
Insuasty et al.13 also developed pyrazoline analogues (V–VI) and
assessed their antimalarial activity (Figure 2).
Recently, there has been a great interest in thiosemicarbazones
as potential antimalarial agents14–16. The pioneering work on
acetylpyridine thiosemicarbazones17 (VII) as antimalarials paved
way for Pingaew et al.18 to develop benzoyl thiosemicarbazone
analogues of isoquinoline (VIII) as antimalarials. Khanye et al.19
also developed gold compounds containing thiosemicarbazone
ligands (IX) as antimalarials (Figure 2).
Parasite borne diseases like malaria, sleeping sickness, amoebia-
sis, dysentery, etc. are rife, predominantly in the developing
countries and are responsible for immense distress1. Malaria,
caused by P. falciparum, continues to be a grim health concern in
Africa, South America and many parts of Asia2. Curbing this
enervating disease has been sternly compromised by the emer-
gence and spread of resistance to a majority of existing
antimalarial agents3. In view of this, there is exigency for the
breakthrough of new, affordable and safe antimalarial agents4.
Pyrazole ring is a biologically active motif, which is of much
importance to medicinal chemists owing to its pharmacological
diversity. It is also an integral part of a number of well-established
commercially available drugs like, zoniporide, celecoxib,
pyrazomycin, lonazolac, fezolamine, difenamizole, mepirizole,
etc. (Figure 1).
Taking this as cue, some researchers have also highlighted the
antimalarial propensity of this ring. Cunico et al.5 synthesized
and evaluated the antimalarial activity of chloroquine–pyrazole
analogues (I) and highlighted the critical role of aromatic
functionality of pyrazole for activity. Qirante et al.6 evaluated
antimalarial activity of ligands and complexes derived from
pyrazole (II). Bekhit et al.7 also developed some pyrazole
derivatives (III) with antimalarial activity (Figure 2).
Currently World Health Organisation recommends the use of
two or more agents as the best way to minimize treatment failures
and the spread of drug resistance20. The presence of more than
one structurally active moiety in a single molecule may show an
enhanced activity21. In the design of new drugs, the development
of hybrid molecules through the combination of different
scaffolds in one frame may lead to compounds with interesting
biological profiles. Therefore, a molecule containing more than
one biologically active scaffold, each with different mode of
action could be favourable for the treatment of malaria. Therefore,
three different scaffolds were selected viz pyrazole, pyrazoline
and thiosemicarbazone moiety and incorporated into one single
compound. From our ongoing efforts on finding novel
hybrid antimalarials22 we, herein report the design, synthesis
and antimalarial activity of novel hybrid molecules based on
pyrazole moiety linked to pyrazoline moiety having thiosemi-
carbazone as a part of pyrazoline moiety (Figure 2). The synthetic
steps involved the formation of pyrazole carbaldehydes through
Vilsmeier-Haack reaction23, followed by Claisen-Schmidt con-
densation of these aldehydes with acetophenones. Resulting
chalcones on treatment with thiosemicarbazide gave the title
Pyrazolines, the reduced form of pyrazoles, also have several
applications in the field of pharmaceutical research. They have a
wide range of biological activities such as, antimicrobial8,
Address for correspondence: Mohammad Mumtaz Alam, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard,
New Delhi 110062, India. Tel: +91 11 26059681. Fax: +91 11
26059686. E-mail: drmmalam@hotmail.com
compounds24,25
.

2
A. Marella et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
HN
Figure 1. Commercially available drugs
bearing pyrazole nucleus.
O
NH
N
H
2
Me
(CH ) N(CH )
2 3
3 2
NH
OH
N
N
HO
N
N
CONH
N
2
N
F C
3
N
C H CI
6
4
OH
HO
COOH
N
SO NH
2
2
Pyrazomycin
Lonazolac
Zoniporide
Celecoxib
(CH ) N(CH )
MeO
Me
Ph
N
2 3
3 2
H
N
N
N
N
O
N
N
N
N
MeO
Ph
Ph
Ph
NMe
2
Me
Me
Fezolamin
Difenamizole
Mepirizole
R
Figure 2. Title compounds bearing the
structural motifs.
N
N
N
CH
N
N
CF
3
3
HN
N
O
O
C H
6
5
N
H
S
O
O
Cl
N
VII
I
IV
HO
R
R
2
1
N
R
3
N
N
Pd
N
Cl
2
N
N
R
O
HN
R
4
5
NMe
S
NH
2
N
II
V
VIII
NH
Cl-
O
R
2
OH
R
R
N
N
S
N
HN
N
N
1
N
Au
N
Cl
O
H
Cl
H
N+
IX
R
III
VI
N
N
R
2
R
1
N
N
S
NH
2
The synthesized compounds were evaluated for in vitro into the relationship existing between the chemical structure and
schizontocidal activity against the CQ-sensitive 3D7 strain of pharmacological activity, QSAR of the synthesised compounds
29
P. falciparum using modification of Trager and Jenson method26 as was performed using the PHASE module of Schrodinger .
¨
described by Dua et al.27. The compounds having IC50 less than The generated model gave statistically significant results, with
5 mg/mL were further evaluated for their cytotoxicity by Mosmann r2 of 0.9992, and the structural features spread across the entire
method28 with certain modifications. Finally, to provide an insight molecule. This further validated our in vitro results.

DOI: 10.3109/14756366.2014.958081
Pyrazole–pyrazoline hybrids
3
Methods
5-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazole-
1-carbothioamide (29)
Chemistry
Yield: 64%; Rf: 0.32; m.p.: 245–246 ꢀC; FT-IR (cmꢁ1): 3413,
3269 (NH2), 1587 (C ¼ N), 1363 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.22 (dd, 1H, J ¼ 3.6 & 17.6 Hz), 3.91 (dd,
1H, J ¼ 11.2 & 17.8 Hz), 6.03 (dd, 1H, J ¼ 3.6 & 11.4 Hz),
7.24 (t, 1H, H-40, J ¼ 7.2 Hz), 7.38–7.49 (m, 8H,
H-3,4,5,30,50,300,400,500), 7.78 (d, 2H, J ¼ 7.6 Hz, H-200,600), 7.83–
7.86 (m, 5H, H-2,6,20,60 & one proton of NH2), 8.07 (bs, 1H, NH2,
D2O exchangeable), 8.13 (s, 1H, pyrazole ring); 13C-NMR
(d ppm, DMSO-d6, 100 MHz): 42.81, 56.16, 118.57, 124.76,
126.29, 126.64, 127.66, 128.37, 128.44, 129.05, 129.95, 130.93,
131.56, 133.53, 139.80, 149.68, 155.24, 176.52; Mass (m/z): 424
(M++1). Anal Calcd. for C25H21N5S: C, 70.90; H, 5.00; N, 16.54;
Found: C, 70.92; H, 4.98; N, 16.55.
All chemicals and solvents were purchased from SD-fine
chemical and used without further purification. Melting points
were measured by open-end capillary method and are uncor-
rected. TLC was performed with silica gel 60 F254 (Merck) using
methanol: chloroform (1:9) as the solvent system.
Elemental analyses were performed using CHNS Elementar
(Vario EL-III). The IR spectra were recorded using a Bruker alpha-
T spectrophotometer. 1H-NMR spectra were recorded on a Bruker
Avance-400 MHz in DMSO-d6 with tetramethylsilane (TMS) as
the internal standard. The 13C-NMR of compounds was recorded
on Bruker Avance-100 MHz instrument in DMSO-d6. Chemical
shifts (d) are reported in parts per million, and coupling constants
(J) in hertz. High-resolution mass spectral data were obtained on an
Agilent 6540 UHD Accurate Mass (Q-ToF) Mass Spectrometer
(LTQ XL/LTQ Orbitrap Discovery) coupled to Agilent 1290 series
LC system (Agilent PDA detector, autosampler, and thermostat,
thermostated column compartment, binary pump).
3-(4-Chlorophenyl)-5-(1,3-diphenyl-1H-pyrazol-4-yl)-4,5-dihy-
dropyrazole-1-carbothioamide (30)
Yield: 60%; Rf: 0.24; m.p.: 252–253 ꢀC; FT-IR (cmꢁ1): 3422,
3254 (NH2), 1582 (C ¼ N), 1364 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.21 (dd, 1H, J ¼ 3.6 & 17.8 Hz), 3.88 (dd,
1H, J ¼ 11.6 & 18.0 Hz), 6.02 (dd, 1H, J ¼ 3.6 & 11.2 Hz), 7.23
(t, 1H, H-40, J ¼ 7.6 Hz), 7.36–7.46 (m, 5H, H-3,4,5,30,50), 7.48
(d, 2H, J ¼ 8.4 Hz), H-300,500), 7.75 (d, 2H, J ¼ 7.2 Hz, H-2,6), 7.82
General procedure for the synthesis of 1-Phenyl-2-(1-
phenylethylidene)hydrazines 1–4
GAA (0.024 mole) and phenylhydrazine (0.05 mole) were added
to a solution of substituted acetophenone (0.05 mole) in 30 mL
methanol. The mixture was refluxed at 80 ꢀC until the completion
of reaction. The reaction was monitored through TLC. After
completion, the reaction vessel was cooled to 0 ꢀC. The precipi-
tate was filtered, washed with cold methanol and dried30.
(d, 2H, J ¼ 8.0 Hz, H-20,60), 7.86 (d, 3H, J ¼ 8.4 Hz, H-200,600
&
one proton of NH2 merged), 8.09 (bs, 1H, NH2, D2O exchange-
able), 8.14 (s, 1H, pyrazole ring); Mass (m/z): 458 (M++1), 459
(M++2). Anal Calcd. for C25H20ClN5S: C, 65.56; H, 4.40; N,
15.29; Found: C, 70.88; H, 4.41; N, 15.30.
General procedure for the synthesis of 1,3-diphenyl-1H-pyrazole-
4-carbaldehyde 5–8
5-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-dihydropyrazole-
1-carbothioamide (31)
POCl3 (0.09 mole) was added drop wise through dropping funnel
to previously cooled DMF (0.09 mole) at 0–5 ꢀC and stirred for
few minutes. A solution of compounds 1–4 (0.03 mole) in DMF
was added drop wise to the reaction mixture. This was then kept at
room temperature for few minutes and refluxed at 70–80 ꢀC
until the completion of reaction. The reaction was monitored
through TLC30.
Following completion, the reaction mixture was cooled to
room temperature, quenched with crushed ice and basified with
sodium hydroxide to obtain a precipitate. The precipitate was
filtered, washed thoroughly with water, dried and recrystallized
from ethanol.
Yield: 56%; Rf: 0.46; m.p.; 259–260 ꢀC; FT-IR (cmꢁ1): 3422,
3252 (NH2), 1580 (C ¼ N), 1365 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 2.32 (s, 3H, CH3), 3.19 (dd, 1H, J ¼ 3.6 &
17.8 Hz), 3.88 (dd, 1H, J ¼ 11.2 & 17.6 Hz), 6.02 (dd, 1H, J ¼ 3.6
& 11.2 Hz), 7.23 (d, 2H, J ¼ 8.0 Hz, H-300,500), 7.25 (t, 1H,
J ¼ 7.6 Hz, H-40), 7.38–7.49 (m 5H, H-3,4,5,30,50), 7.73 (d, 2H,
J ¼ 8.4 Hz, H-2,6), 7.78 (d, 3H, J ¼ 7.2 Hz, H-200,600 & one proton
of NH2 merged), 7.82 (d, 2H, J ¼ 8.0 Hz, H-20,60), 8.02 (bs, 1H,
NH2, D2O exchangeable), 8.10 (s, 1H, pyrazole ring); Mass (m/z):
438 (M++1). Anal Calcd. for C26H23N5S: C, 71.37; H, 5.30; N,
16.01; Found: C, 71.39; H, 5.29; N, 16.00.
5-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-(4-methoxyphenyl)-4,5-dihy-
dropyrazole-1-carbothioamide (32)
General procedure for the synthesis of 3-(1,3-diphenyl-1H-
pyrazol-4-yl)-1-phenylprop-2-en-1-one 9–28
Yield: 48%; Rf: 0.35; m.p.: 242–243 ꢀC; FT-IR (cmꢁ1): 3422,
3251 (NH2), 1586 (C ¼ N), 1359 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.19 (dd, 1H, J ¼ 3.2 & 17.8 Hz), 3.78
(s, 3H, OCH3), 3.86 (dd, 1H, J ¼ 11.6 & 17.6 Hz), 6.01 (dd, 1H,
J ¼ 3.2 & 11.2 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz, H-300,500), 7.24 (t, 1H,
J ¼ 7.2 Hz, H-40), 7.38–7.49 (m, 5H, H-3,4,5,30,50), 7.78 (d, 4H,
J ¼ 8.4 Hz, H-2,6,200,600), 7.83 (d, 2H, J ¼ 8.0 Hz, H-20,60), 7.76
(bs, 1H, NH2, D2O exchangeable), 8.00 (bs, 1H, NH2, D2O
exchangeable), 8.10 (s, 1H, pyrazole ring); Mass (m/z): 454
(M++1). Anal Calcd. for C26H23N5OS: C, 68.85; H, 5.11; N,
15.44; Found: C, 68.83; H, 5.09; N, 15.42.
Chalcones were synthesised by Claisen Schmidt reaction31.
Aldehyde (0.05 mole) and substituted acetophenone (0.05 mole)
were dissolved in 35 mL methanol. To this reaction mixture was
added sodium hydroxide (0.075 mole), then the mixture was
refluxed with stirring for 4–5 h. The reaction was monitored
through TLC. After completion, the reaction mixture was poured
in ice-cold water, acidified with 4 N HCl and the solid was
filtered, dried, and recrystallized from ethanol.
General procedure for the synthesis of 5-(1,3-diphenyl-1H-
pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazole-1-carbothioamide
29–48
3-(4-Aminophenyl)-5-(1,3-diphenyl-1H-pyrazol-4-yl)-4,5-dihy-
dropyrazole-1-carbothioamide (33)
Equimolar quantities (0.01 mole) of chalcone and thiosemicarba-
zide were refluxed in presence of sodium hydroxide (0.025 mole)
in 25 mL ethanol for 3 h. White solid separation occurs in the Yield: 54%; Rf: 0.52; m.p.: 249–250 ꢀC; FT-IR (cmꢁ1): 3457,
1
reaction vessel itself. After the completion of reaction, the 3276 (NH2), 3391, 3329 (NH2), 1588 (C ¼ N), 1361 (C ¼ S); H-
precipitated solid was filtered, washed with water, and dried24,25
.
NMR (d ppm, DMSO-d6, 400 MHz): 3.08 (dd, 1H, J ¼ 3.2 &

4
A. Marella et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
17.2 Hz), 3.78 (dd, 1H, J ¼ 11.2 & 17.0 Hz), 5.68 (bs, 2H, NH2, exchangeable), 8.14 (s, 1H, pyrazole ring); Mass (m/z): 488
D2O exchangeable), 5.98 (dd, 1H, J ¼ 3.2 & 11.0 Hz), 6.52 (d, 2H, (M++1), 489 (M++2). Anal Calcd. for C26H22ClN5OS: C, 63.99;
J ¼ 7.6 Hz, H-300,500), 7.23 (t, 1H, J ¼ 7.6 Hz, H-40), 7.37–7.54 H, 4.54; N, 14.35; Found: C, 64.00; H, 4.55; N, 14.33.
(m, 8H, H-3,4,5,30,50,200,600, one proton of NH2), 7.78 (d, 2H,
J ¼ 8.0 Hz, H-2,6), 7.81 (d, 3H, J ¼ 8.8 Hz, H-20,60& one proton of
(M++1). Anal Calcd. for C25H22N6S: C, 68.47; H, 5.06; N, 19.16;
Found: C, 68.45; H, 5.04; N, 19.14.
3-(4-Aminophenyl)-5-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-
4-yl)-4,5-dihydropyrazole-1-carbothioamide (38)
NH2 merged), 8.02 (s, 1H, pyrazole ring); Mass (m/z): 439
Yield: 56%; Rf: 0.33; m.p.: 252–253 ꢀC; FT-IR (cmꢁ1): 3455,
3273 (NH2), 3391, 3323 (NH2), 1590 (C ¼ N), 1360 (C ¼ S);
1H-NMR (d ppm, DMSO-d6, 400 MHz): 3.09 (dd, 1H, J ¼ 2.4 &
5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-
dihydropyrazole-1-carbothioamide (34)
17.6 Hz), 3.78 (dd, 1H, J ¼ 10.8 & 17.4 Hz), 5.70 (bs, 2H, NH2,
D2O exchangeable), 5.96 (dd, 1H, J ¼ 2.8 & 10.8 Hz), 6.52 (d, 2H,
J ¼ 8.0 Hz, H-300,500), 7.25 (t, 1H, J ¼ 7.2 Hz, H-40), 7.42 (t, 2H,
J ¼ 7.6 Hz, H-30,50), 7.49–7.54 (m, 5H, H-3,5,200,600 and proton of
NH2), 7.80–7.84 (m, 4H, H-2,6,20,60), 7.87 (bs, 1H, NH2), 8.07
(s, 1H, pyrazole ring); 13C-NMR (d ppm, DMSO-d6, 100 MHz):
42.22, 55.09, 113.12, 117.68, 118.11, 124.52, 125.82, 126.27,
128.61, 128.74, 129.48, 129.52, 131.86, 132.65, 139.16, 147.92,
151.35, 155.58, 174.82. Mass (m/z): 473 (M++1), 474 (M++1).
Anal Calcd. for C25H21ClN6S: C, 63.48; H, 4.48; N, 17.77;
Found: C, 63.49; H, 4.46; N, 17.79.
Yield: 49%; Rf: 0.28; m.p.: 271–272 ꢀC; FT-IR (cmꢁ1): 3406,
3236 (NH2), 1589 (C ¼ N), 1376 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.23 (dd, 1H, J ¼ 3.6 & 17.8 Hz), 3.90
(dd, 1H, J ¼ 11.2 & 17.8 Hz), 6.01 (dd, 1H, J ¼ 3.6 &
11.2 Hz),7.24 (t, 1H, J ¼ 7.2 Hz, H-40), 7.42–7.45 (m, 5H,
H-300,400,500,30,50), 7.51 (d, 2H, J ¼ 8.4 Hz, H-3,5), 7.79 (d, 2H,
J ¼ 8.4 Hz, H-20,60), 7.82–7.85 9m, 5H, H-2,6,200,600 & one proton
of NH2), 8.09 (bs, 1H, NH2, D2O exchangeable), 8.17 (s, 1H,
pyrazole ring); Mass (m/z): 458 (M++1), 459 (M++2). Anal
Calcd. for C25H20ClN5S: C, 65.56; H, 4.40; N, 15.29; Found:
C, 65.55; H, 4.38; N, 15.30.
3-Phenyl-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-dihydropyr-
azole-1-carbothioamide (39)
3-(4-Chlorophenyl)-5-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-
4-yl)-4,5-dihydropyrazole-1-carbothioamide (35)
Yield: 60%; Rf: 0.55; m.p.: 242–243 ꢀC; FT-IR (cmꢁ1): 3407,
3267 (NH2), 1591 (C ¼ N), 1368 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 2.34 (s, 3H, CH3), 3.19 (dd, 1H, J ¼ 3.6 &
17.8 Hz), 3.89 (dd, 1H, J ¼ 11.2 & 17.8 Hz), 6.03 (dd, 1H, J ¼ 3.6
& 11.2 Hz), 7.23 (t, 1H, J ¼ 7.6 Hz, H-40), 7.26 (d, 2H, J ¼ 7.6 Hz,
H-3,5), 7.41–7.45 (m, 5H, H-30,50,300,400,500), 7.66 (d, 2H,
J ¼ 8.0 Hz, H-200,600), 7.81–7.85 (m,5H, H-2,6,20,60 & one proton
of NH2 merged), 8.08 (bs, 1H, NH2, D2O exchangeable), 8.10
(s, 1H, pyrazole ring); Mass (m/z): 438 (M++1). Anal Calcd.
for C26H23N5S: C, 71.37; H, 5.30; N, 16.01; Found: C, 71.35;
H, 5.29; N, 16.00.
Yield: 52%; Rf: 0.42; m.p.: 260–261 ꢀC; FT-IR (cmꢁ1): 3426,
3264 (NH2), 1577 (C ¼ N), 1358 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.22 (dd, 1H, J ¼ 3.6 & 18.0 Hz), 3.87
(dd, 1H, J ¼ 11.6 & 17.6 Hz), 6.00 (dd, 1H, J ¼ 3.6 & 11.4 Hz),
7.25 (t, 1H, J ¼ 7.2 Hz, H-40), 7.42 (t, 2H, J ¼ 8.0 Hz, H-30,50),
7.49–7.51 (d, 2H, J ¼ 8.4 Hz, H-300,500), 7.50–7.52 (d, 2H,
J ¼ 8.0 Hz, H-3,5), 7.77 (d, 2H, J ¼ 8.4 Hz, H-20,60), 7.83
(d, 2H, J ¼ 8.0 Hz, H-200,600), 7.85 (d, 2H, J ¼ 8.8 Hz, H-2,6),
7.91 (bs, 1H, NH2, D2O exchangeable), 8.12 (bs, 1H, NH2, D2O
exchangeable), 8.19 (s, 1H, pyrazole ring); Mass (m/z): 492
(M++1), 493 (M++2). Anal Calcd. for C25H19Cl2N5S: C, 60.98;
H, 3.89; N, 14.22; Found: C, 61.00; H, 3.87; N, 14.23.
3-(4-Chlorophenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-
dihydropyrazole-1-carbothioamide (40)
Yield: 60%; Rf: 0.35; m.p.: 269–270 ꢀC; FT-IR (cmꢁ1): 3418,
3260 (NH2), 1572 (C ¼ N), 1361 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 2.32 (s, 3H, CH3), 3.21 (dd, 1H, J ¼ 4.0 &
11.2 Hz), 3.88 (dd, 1H, J ¼ 10.8 & 17.0 Hz), 6.01 (dd, 1H, J ¼ 4.0
& 11.2 Hz), 7.22 (t, 1H, J ¼ 7.6 Hz, H-40), 7.24 (d, 2H, J ¼ 8.4 Hz,
H-3,5), 7.41 (t, 2H, J ¼ 8.0 Hz, H-30,50), 7.46 (d, 2H, J ¼ 8.8 Hz,
H-300,500), 7.80 (d, 2H, J ¼ 8.8 Hz, H-20,60), 7.83–7.85 (m, 5H, H-
200,600, 2,6 and one proton of NH2 merged), 8.01 (bs, 1H, NH2),
8.06 (s, 1H, pyrazole ring); Mass (m/z): 472 (M++1), 473
(M++2). Anal Calcd. for C26H22ClN5S: C, 66.16; H, 4.70; N,
14.84; Found: C, 66.15; H, 4.68; N, 14.86.
5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-
dihydropyrazole-1-carbothioamide (36)
Yield: 54%; Rf: 0.56; m.p.: 272–273 ꢀC; FT-IR (cmꢁ1): 3428,
3268 (NH2), 1577 (C ¼ N), 1361 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 2.32 (s, 3H, CH3), 3.20 (dd, 1H, J ¼ 3.6 &
17.8 Hz), 3.87 (dd, 1H, J ¼ 11.6 & 17.6 Hz), 5.99 (dd, 1H, J ¼ 3.6
& 11.4 Hz), 7.23–7.28 (m, 3H, H-40,300,500), 7.41 (t, 2H,
J ¼ 8.0 Hz, H-30,50), 7.51 (d, 2H, J ¼ 8.4 Hz, H-3,5), 7.73
(d, 2H, J ¼ 8.0 Hz, H-20,60), 7.79 (d, 3H, J ¼ 8.4 Hz, H-200,600 &
one proton of NH2 merged), 7.83 (d, 2H, J ¼ 8.0 Hz, H-2,6), 8.05
(bs, 1H, NH2, D2O exchangeable), 8.15 (s, 1H, pyrazole ring);
Mass (m/z): 472 (M++1), 473 (M++2). Anal Calcd. for
C26H22ClN5S: C, 66.16; H, 4.70; N, 14.84; Found: C, 66.15; H,
4.68; N, 14.85.
5-(1-Phenyl-3-p-tolyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-dihydropyr-
azole-1-carbothioamide (41)
Yield: 52%; Rf: 0.30; m.p.: 259–260 ꢀC; FT-IR (cmꢁ1): 3428,
3266 (NH2), 1578 (C ¼ N), 1363 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 2.27 (s, 3H, CH3), 2.32 (s, 3H, CH3), 3.16
(dd, 1H, J ¼ 4.0 & 11.2 Hz), 3.86 (dd, 1H, J ¼ 10.8 & 17.0 Hz),
6.01 (dd, 1H, J ¼ 4.0 & 11.2 Hz), 7.22 (d, 2H, J ¼ 8.4 Hz,
H-300,500), 7.26 (d, 2H, J ¼ 7.6 Hz, H-3,5), 7.22–7.28 (1H, H-40
merged), 7.41 (t, 2H, J ¼ 8.0 Hz, H-30,50), 7.65 (d, 2H, J ¼ 7.6 Hz,
H-2,6), 7.73 (d, 2H, J ¼ 7.6 Hz, H-200,600), 7.79 (bs, 1H, NH2),
7.81(d, 2H, J ¼ 8.0 Hz, H-20,60), 8.04 (bs, 1H, NH2),8.08 (s, 1H,
pyrazole ring); Mass (m/z): 452 (M++1). Anal Calcd. for
C27H25N5S: C, 71.81; H, 5.58; N, 15.51; Found: C, 71.80;
H, 5.60; N, 15.50.
5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(4-methoxy-
phenyl)-4,5-dihydropyrazole-1-carbothioamide (37)
Yield: 53%; Rf: 0.38; m.p.: 262–263 ꢀC; FT-IR (cmꢁ1): 3426,
3260 (NH2), 1585 (C ¼ N), 1363 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.19 (dd, 1H, J ¼ 3.6 & 17.8 Hz), 3.78
(s, 3H, OCH3), 3.86 (dd, 1H, J ¼ 11.6 & 18.0 Hz), 5.98 (dd, 1H,
J ¼ 3.6 & 11.4 Hz), 6.97 (d, 2H, J ¼ 8.8 Hz, H-300,500), 7.25 (t, 1H,
J ¼ 7.6 Hz, H-40), 7.42 (t, 2H, J ¼ 8.0 Hz, H-30,50), 7.51
(d, 2H, J ¼ 8.4 Hz, H-3,5), 7.75 (bs, 1H, NH2), 7.78–7.80 (d,
2H, J ¼ 8.8 Hz, H-20,60), 7.79–7.81 (d, 2H, J ¼ 8.4 Hz, H-200,600),
7.83 (d, 2H, J ¼ 8.0 Hz, H-2,6), 8.00 (bs, 1H, NH2, D2O

DOI: 10.3109/14756366.2014.958081
Pyrazole–pyrazoline hybrids
5
3-(4-Methoxyphenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-
dihydropyrazole-1-carbothioamide (42)
6.00 (dd, 1H, J ¼ 3.2 & 11.4 Hz), 7.00 (d, 2H, J ¼ 8.4 Hz, H-3,5),
7.22 (3H, H-40,300,500), 7.40 (t, 2H, J ¼ 7.6 Hz, H-30,50), 7.69 (d,
2H, J ¼ 8.8 Hz, H-2,6), 7.72 (d, 2H, J ¼ 8.4 Hz, H-200,600), 7.80 (d,
2H, J ¼ 8.0 Hz, H-20,60), 7.79 (bs, 1H, NH2), 8.02 (bs, 1H,
NH2),8.06 (s, 1H, pyrazole ring); Mass (m/z): 468 (M++1). Anal
Calcd. for C27H25N5OS: C, 69.35; H, 5.39; N, 14.98; Found:
C, 69.36; H, 5.40; N, 14.96.
Yield: 55%; Rf: 0.27; m.p.: 236–237 ꢀC; FT-IR (cmꢁ1): 3423,
3249 (NH2), 1586 (C ¼ N), 1365 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 2.29 (s, 3H, CH3), 3.75 (s, 3H, OCH3),
3.21 (dd, 1H, J ¼ 4.0 & 11.2 Hz), 3.86 (dd, 1H, J ¼ 10.8 &
17.0 Hz), 6.03 (dd, 1H, J ¼ 4.0 & 11.2 Hz), 6.94 (d, 2H,
J ¼ 8.8 Hz, 300,500), 7.21 (t, 1H, J ¼ 7.6 Hz, H-40), 7.26 (d, 2H,
J ¼ 7.6 Hz, H-3,5), 7.41 (t, 2H, J ¼ 8.0 Hz, H-30,50), 7.72 (d, 2H,
J ¼ 7.6 Hz, H-2,6), 7.75 (bs, 1H, NH2), 7.77 (d, 2H, J ¼ 7.6 Hz,
H-200,600), 7.80 (d, 2H, J ¼ 8.0 Hz, H-20,60), 7.88 (bs, 1H, NH2),
8.03 (s, 1H, pyrazole ring); Mass (m/z): 468 (M++1). Anal Calcd.
for C27H25N5OS: C, 69.35; H, 5.39; N, 14.98; Found: C, 69.33;
H, 5.41; N, 15.00.
3-(4-Methoxyphenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyra-
zol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (47)
Yield: 61%; Rf: 0.43; m.p.: 234–235 ꢀC; FT-IR (cmꢁ1): 3424,
3250 (NH2), 1587 (C ¼ N), 1365 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.15 (dd, 1H, J ¼ 3.2 & 17.6 Hz), 3.78
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.83 (dd, 1H, J ¼ 11.2 &
17.8 Hz), 5.99 (dd, 1H, J ¼ 3.2 & 11.2 Hz), 6.96 (d, 2H,
J ¼ 8.4 Hz, H-3,5), 7.01 (d, 2H, J ¼ 8.8 Hz, H-300,500), 7.22
(t, 1H, J ¼ 7.2 Hz, H-40), 7.40 (t, 2H, J ¼ 8.0 Hz, H-30,50), 7.70
(d, 2H, J ¼ 8.4 Hz, H-20,60), 7.75 (bs, 1H, NH2), 7.77 (d, 2H,
J ¼ 8.8 Hz, H-200,600), 7.80 (d, 2H, J ¼ 8.4 Hz, H-2,6), 7.99 (bs, 1H,
NH2), 8.06 (s, 1H, pyrazole ring); 13C-NMR (d ppm, DMSO-d6,
100 MHz): 42.78, 55.63, 55.86, 56.09, 114.49, 114.54, 118.46,
124.02, 124.40, 125.96, 126.07, 126.47, 129.38, 129.62, 129.93,
139.84, 149.51, 155.19, 159.57, 161.63, 176.14; Mass (m/z): 484
(M++1). Anal Calcd. for C27H25N5O2S: C, 67.06; H, 5.21;
N, 14.48; Found: C, 67.04; H, 5.20; N, 14.50.
3-(4-Aminophenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-
dihydropyrazole-1-carbothioamide (43)
Yield: 48%; Rf: 0.59; m.p.: 241–242 ꢀC; FT-IR (cmꢁ1): 3449,
3271 (NH2), 3382, 3317, (NH2), 1591 (C ¼ N), 1359 (C ¼ S);
1H-NMR (d ppm, DMSO-d6, 400 MHz): 2.35 (s, 3H, CH3), 3.04
(dd, 1H, J ¼ 3.2 & 17.6 Hz), 3.76 (dd, 1H, J ¼ 10.8 & 17.4 Hz),
5.68 (bs, 2H, NH2, D2O exchangeable), 5.97 (dd, 1H, J ¼ 3.6 &
11.2 Hz), 6.51 (d, 2H, J ¼ 8.8 Hz, H-300,500), 7.22 (t, 1H,
J ¼ 7.6 Hz, H-40), 7.26 (d, 2H, J ¼ 8.4 Hz, H-3,5), 7.40 (t, 2H,
J ¼ 8.0 Hz, H-30,50), 7.48 (d, 2H, J ¼ 8.8 Hz, H-200,600), 7.54
(bs, 1H, NH2), 7.66 (d, 2H, J ¼ 8.0 Hz, H-2,6), 7.80 (d, 2H,
J ¼ 7.6 Hz, H-20,60), 7.84 (bs, 1H, NH2), 7.98 (s, 1H, pyrazole
ring); Mass (m/z): 453 (M++1). Anal Calcd. for C26H24N6S:
C, 69.00; H, 5.35; N, 18.57; Found: C, 68.98; H, 5.33; N, 18.55.
3-(4-Aminophenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-
4-yl)-4,5-dihydropyrazole-1-carbothioamide (48)
Yield: 52%; Rf: 0.40; m.p.: 255–256 ꢀC; FT-IR (cmꢁ1): 3483,
1
3204 (NH2), 3418, 3308 (NH2), 1565 (C ¼ N), 1347 (C ¼ S); H-
NMR (d ppm, DMSO-d6, 400 MHz): 3.05 (dd, 1H, J ¼ 3.2 &
17.6 Hz), 3.75 (dd, 1H, J ¼ 11.2 & 17.4 Hz), 3.79 (s, 3H, OCH3),
5.68 (bs, 2H, NH2, D2O exchangeable), 5.96 (dd, 1H, J ¼ 3.2 &
10.8 Hz), 6.51 (d, 2H, J ¼ 8.4 Hz, H-300,500), 7.01 (d, 2H,
J ¼ 8.8 Hz, H-3,5), 7.22 (t, 1H, J ¼ 7.2 Hz, H-40), 7.40 (t, 2H,
J ¼ H-30,50), 7.48 (d, 2H, J ¼ 8.4 Hz, H-200,600), 7.54 (bs, 1H,
NH2), 7.70 (d, 2H, J ¼ 8.8 Hz, H-2,6), 7.79 (d, 2H, J ¼ 8.0 Hz, H-
20,60), 7.81 (bs, 1H, NH2), 7.97 (s, 1H, pyrazole ring); Mass (m/z):
469 (M++1). Anal Calcd. for C26H24N6OS: C, 66.64; H, 5.16;
N, 17.94; Found: C, 66.66; H, 5.13; N, 17.95.
5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-
dihydropyrazole-1-carbothioamide (44)
Yield: 50%; Rf: 0.49; m.p.: 255–256 ꢀC; FT-IR (cmꢁ1): 3423,
3268 (NH2), 1592 (C ¼ N), 1365 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.79 (s, 3H, OCH3), 3.19 (dd, 1H, J ¼ 4.0
& 11.2 Hz), 3.86 (dd, 1H, J ¼ 10.8 & 17.0 Hz), 6.04 (dd, 1H,
J ¼ 4.0 & 11.2 Hz), 7.03 (d, 2H, J ¼ 8.4 Hz, H-3,5), 7.23 (t, 1H,
J ¼ 7.2 Hz, H-40), 7.40–7.45 (m, 5H, H-30,50,300,400,500), 7.73–7.77
(m, 4H, H-2,6,200,600), 7.80–7.82 (m, 3H, H-20,60 and one proton of
NH2 merged), 7.98 (bs, 1H, NH2), 8.09 (s, 1H, pyrazole ring);
Mass (m/z): 454 (M++1). Anal Calcd. for C26H23N5OS: C, 68.85;
H, 5.11; N, 15.44; Found: C, 68.83; H, 5.09; N, 15.46.
Pharmacology
In vitro antimalarial activity
In vitro drug sensitivity of the synthesized compounds was
assessed using Trager and Jensen26 method as described by
Dua et al.27. Chloroquine sensitive 3D7 P. falciparum strains
were used for the study. Culture was maintained in A+ erythro-
cytes using RPMI 1640 medium supplemented with AB+ human
serum (10%), sodium bicarbonate (0.2%), HEPES buffer (25 mM)
and gentamycin (50 mg mLꢁ1). The culture was treated with
different drug concentrations. After 72 h of incubation, blood
smears were prepared and stained with JSB I and JSB II.
Percentage maturation of schizonts against control was deter-
mined. Chloroquine was used as the standard reference. The
inhibitory concentration value which kills 50% of the parasites
(IC50) was considered for anti-plasmodial activity. IC50 values
were determined using HN-NonLin V1.1.
3-(4-Chlorophenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyra-
zol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (45)
Yield: 54%; Rf: 0.38; m.p.: 232–233 ꢀC; FT-IR (cmꢁ1): 3428,
3265 (NH2), 1594 (C ¼ N), 1373 (C ¼ S); 1H-NMR (d ppm,
DMSO-d6, 400 MHz): 3.18 (dd, 1H, J ¼ 3.2 & 17.6 Hz), 3.77
(s, 3H, OCH3), 3.85 (dd, 1H, J ¼ 11.6 & 17.8 Hz), 6.00 (dd, 1H,
J ¼ 3.6 & 11.2 Hz), 6.99 (d, 2H, J ¼ 8.0 Hz, H-3,5), 7.22 (m, 3H,
H-300,500,40), 7.40 (t, 2H, J ¼ 7.2 Hz, H-30,50), 7.69 (d, 2H,
J ¼ 8.0 Hz, H-2,6), 7.72 (d, 2H, J ¼ 8.0 Hz, H-20,60), 7.78 (bs,
1H, NH2), 7.80 (d, 2H, J ¼ 8.0 Hz, H-200,600), 8.02 (bs, 1H, NH2),
8.06 (s, 1H, pyrazole ring); Mass (m/z): 488 (M++1), 489(M++2).
Anal Calcd. for C26H22ClN5OS: C, 63.99; H, 4.54; N, 14.35;
Found: C, 63.98; H, 4.55; N, 14.30.
Compounds with IC50 less than 5 mM were further tested for
cytotoxicity study to find out the selectivity index (SI) of the
synthesised compounds.
5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-
dihydropyrazole-1-carbothioamide (46)
Yield: 60%; Rf: 0.31; m.p.: 245–246 ꢀC; FT-IR (cmꢁ1): 3433,
3273 (NH2), 1581 (C ¼ N), 1358 (C ¼ S); 1H-NMR (d ppm,
Cytotoxicity assay
DMSO-d6, 400 MHz): 2.31 (s, 3H, CH3), 3.15 (dd, 1H, J ¼ 3.2 & Cytotoxicity of the compounds was carried out with Vero cell line
17.0 Hz), 3.78 (s, 3H, OCH3), 3.84 (dd, 1H, J ¼ 11.2 & 17.2 Hz), using Mosmann method28 with certain modifications. The cells

6
A. Marella et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Table 1. IC50 and SI values of the synthesised compounds.
N
N
R
2
R
1
N
N
S
NH
2
a
Substitution
IC50
Compound No.
R1
R2
mg/mL
mM
SIb
Activity pIC50
Predicted activity pIC50
Fitness
29^^
H
H
H
H
H
4-Cl
4-Cl
4-Cl
H
Cl
4-CH3
4-OCH3
4-NH2
H
26.036
30.101
15.209
5.883
61.47
65.73
31.17
12.06
3.22
66.54
77.83
19.63
7.86
ND
ND
ND
ND
33
ND
ND
ND
ND
22
ND
ND
ND
ND
16
ND
ND
ND
40
4.584
4.521
4.818
5.230
5.822
4.485
4.448
5.050
5.449
6.010
4.605
4.919
5.112
5.557
6.146
4.684
4.996
5.333
5.720
6.263
4.47
4.63
4.82
5.26
5.62
4.68
4.85
5.07
5.47
5.86
4.73
4.90
5.02
5.47
6.18
5.00
5.18
5.20
5.70
6.43
2.93
2.94
2.48
2.91
2.94
2.96
2.97
2.51
2.49
2.96
2.96
2.97
2.97
2.95
2.96
2.99
3.00
3.00
2.98
2.98
30^^
31
32^^
33^^
34^
1.508
32.768
35.643
8.905
3.559
0.977
24.845
12.043
7.721
2.772
0.715
20.713
10.091
4.642
1.904
0.546
35*^
36^^
37*^^
38^*^^
39^^
40^^
41
Cl
4-CH3
4-OCH3
4-NH2
H
4-Cl
4-Cl
2.09
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
52.53
27.46
16.36
6.14
Cl
4-CH3
4-OCH3
4-NH2
H
42^^
43^^
44*^
45*^^
46^^
47^^
48^^
1.53
43.88
23.06
10.61
4.20
Cl
4-CH3
4-OCH3
4-NH2
1.13
12
aCalculated using HN-NonLin V1.1 Software.
bSI: IC50 values of cytotoxic activity/IC50 values of antimalarial activity.
*Compounds used as test.
Ligands of the inactive set.
^
^^Ligands of the active set.
were incubated with different dilutions of selected compounds for using a tree-based partitioning technique, which groups together
72 h using MTT as reagent for the detection of cytotoxicity. Fifty similar pharmacophores according to their intersite distances.
percent cytotoxic concentration (CC50) values represent the After the generation of pharmacophores their scoring, clustering
concentration of compound required to kill 50% of the fibroblast and ranking was done. The purpose of scoring was to identify the
cells. The SI was calculated using the formula
suitable hypothesis, which can explain the overall aspects of the
structural requirements of the receptor site. Highest scoring
hypothesis was subsequently used for the development of 3D
QSAR models. For the present study, ADHRR.9 hypothesis,
containing one H-acceptor, one H-donor, an hydrophobic core and
two aromatic ring systems, was selected (Figure 3). This
hypothesis was chosen as it had structural features spread across
the entire molecule.
The 3D-QSAR models were generated by combining
the generated hypothesis with their schizontocidal activity
data, to identify the overall aspects of molecular requirements
which govern the biological activity. For generating the
3D-QSAR model, the dataset was divided into training set
(70%) and test set (30%) by using random selection criteria.
The training set molecules were further taken to generate
3D-QSAR models using an atom based QSAR method with a
SI ¼ CC50=IC50
QSAR
In order to get better insights into the structural requirements of
the title compounds to act as antimalarials QSAR of the
compounds was performed using PHASE module of
29
¨
Schrodinger . The structures were drawn on maestro interface.
The IC50 of the compounds were converted to their molar values
and subsequently calculated to free energy-related terms,
i.e. pIC50 (Table 1). Activity threshold was set to 4.75 to
discriminate between active and inactive ligands. The standar-
dized structures were then subjected to conformational search by
OPLS 2005 force field using the confogen module of the
¨
Schrodinger software. After generating the conformers, various
pharmacophoric features were defined.
˚
grid spacing of 1.0 A. QSAR models were validated by predicting
the activities of the test set. A three component (PLS factor)
model was found to be the best statistical parameter to obtain an
optimum model.
Active analogue approach was used to identify common
pharmacophore hypotheses (CPHs). Common pharmacophores
were selected from the conformations of the set of active ligands

DOI: 10.3109/14756366.2014.958081
Pyrazole–pyrazoline hybrids
7
Results and discussion
Chemistry
Synthetic pathway which leads to the synthesis of title compounds
(29–48), is outlined in Scheme 1. The synthesis involves four-step
reaction. In the first step, various substituted acetophenones were
reacted with phenylhydrazine to yield corresponding hydrazine
derivatives (1–4). The pyrazole aldehydes (5–8), an intermediate
required for chalcone (9–28) formation, were obtained by using
Vilsmeier Haack formylation reaction23. The key chalcone
intermediates (9–28) were then synthesized through Claisen-
Schmidt condensation of acetophenone derivatives in alcoholic
solution containing sodium hydroxide31. For preparation of
pyrazoline, a variety of methods have been applied, among
which refluxing of chalcone and thiosemicarbazide in methanol
using alkaline condition (sodium hydroxide; 25 mmol) was found
to be the best method with greater yields and shorter reaction
time24,25. The purity of the new synthesized compounds was
checked by TLC. The structures of title compounds were
determined on the basis of elemental analysis and various
spectroscopic methods. Elemental analysis values were within
the range of 0.4% of the theoretical value. In IR spectra,
absorptions were observed at 1347–1376 cmꢁ1 (C ¼ S), 1572–
1594 cmꢁ1 (C ¼ N) and 3204–3276 cmꢁ1 and 3406–3483 cmꢁ1
1
(NH2). In H-NMR spectra, the three protons HA, HM and HX of
pyrazoline ring appeared as doublets of doublets (Figure 4).
The HA proton (d 3.26–3.30) appeared as doublet of doublet
due to cisoid coupling to the HX proton and geminally to HM
proton. The HM proton, deshielded to some extent by aryl group
(d 3.80–3.86), was coupled transoid to HX proton and geminally to
HA proton. The third, HX proton (d 5.63–5.68) was more
Figure 3. Active alignment of the data set.
O
C
(i)
H
C
N
N
CH
3
R
CH
1-4
1
3
R
1
(ii)
N
(iii)
N
N
N
R
1
R
5-8
1
O
C
OHC
HC
9-28
CH
R
2
N
N
(iv)
R
1
29: R =H, R =H; 30: R =H, R =4-Cl;31: R =H, R =4-CH ;
1
2
1
2
1
2
3
32: R =H, R =4-OCH ; 33: R =H, R =4-NH ; 34: R =4-Cl, R =H;
1
2
3
1
2
2
1
2
35: R =4-Cl, R =4-Cl; 36: R =4-Cl, R =4-CH ; 37: R =4-Cl, R =4-OCH ;
1
2
1
2
3
1
2
3
N
38: R =4-Cl, R =4-NH ; 39: R =4-CH , R =H; 40: R =4-CH , R =4-Cl;
1
2
2
1
3
2
1
3
2
S
N
29-48
41: R =4-CH , R =4-CH ; 42: R =4-CH , R =4-OCH ;43: R =4-CH , R =4-NH ;
R
1
3
2
3
1
3
2
3
1
3
2
2
2
44: R =4-OCH , R =H; 45: R =4-OCH , R =4-Cl;46: R =4-OCH , R =4-CH ;
1
3
2
1
3
2
1
3
2
3
NH
47: R =4-OCH , R =4-OCH ; 48: R =4-OCH , R =4-NH ;
2
1
3
2
3
1
3
2
2
Reagents and conditions: (i) Phenyl hydrazine, GAA, methanol, reflux; (ii) DMF, POCl ; (iii) Aromatic ketone, NaOH,
3
methanol, reflux; (iv) Thiosemicarbazide, ethanol, reflux
Scheme 1. Reagents and conditions: (i) Phenyl hydrazine, GAA, methanol, reflux; (ii) DMF, POCL3; (iii) Aromatic ketone, NaOH, methanol, reflux;
(iv) Thiosemicarbazide, ethanol, reflux.

8
A. Marella et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Table 2. Statistical parameters of the best model.
A
Hypothesis
ADHRR.9
SD
r2
RMSE
0.2507
q2
Pearson-r
N
N
0.1299
0.9612
0.7982
0.9992
H
N
SD: standard deviation, r2: determination coefficient on the training set,
RMSE: root mean squared error on the test set, q2: determination
coefficient on the test set, r-Pearson: Pearson’s correlation coefficient
on the test set.
A
H
B
R
M
2
H
x
R
C
1
N
S
NH
2
Figure 4. Title compounds showing AMX pattern and the different rings.
deshielded due to effect of aryl group and electronegative nitrogen
and was coupled transoid to the HM proton and cisoid to the HA
proton. The two protons of CSNH2 appeared at different chemical
shift values (d 7.76–7.82 and 8.04–8.06) due to anisotropic effect
exerted by thiocarbomyl group.
Pharmacology
In vitro antimalarial activity of the synthesized compounds was
assessed using Trager and Jensen26 method as modified by Dua
et al.27 against the CQ-sensitive 3D7 strain of P. falciparum
(Table 1).
Keeping all the aromatic features constant (Figure 4), the
compounds bear H, Cl, CH3 and OCH3 on the one of the phenyl
ring (B) attached to the pyrazole ring and H, Cl, CH3, OCH3 and
NH2 on the phenyl ring (C) attached to pyrazoline ring. The
in vitro studies indicate that compounds with amino substitution
on ring C gave the best inhibitory profiles, as shown by
compounds 33, 38, 43 and 48 with IC50 values of 3.22, 2.09,
1.53 and 1.13 mM, respectively. The most active compound, i.e. 48
bears amino at one end and methoxy group at the other.
Compounds 37, 42 and 47 having methoxy substitution on ring
C also showed good activity with IC50 values of 7.86, 6.14 and
4.20 mM. The replacement of the methoxy by a methyl group and
chloro atom decreases the antimalarial activity. The compounds
with chloro substitution at both ring B and C elicit the least
in vitro schizontocidal activity (Table 1).
The compounds 33, 38, 43, 47 and 48 with IC50-value less than
5 mM were further tested for cytotoxicity study on VERO cells
using Mosmann method28 to find out their selectivity. The
compounds showed SI ranging from 12 to 40. The most active
compound 48 with IC50-value 1.13 mM also showed the highest SI
amongst the tested compounds indicating that compounds
belonging to this prototype would be less toxic (Table 1).
Following conclusions can be made on the basis of in vitro
results:
Figure 5. Fitness plot of the model.
pharmacophore hypotheses, ADHRR.9 was selected. Active
alignment of the data set is shown in Figure 3.
The statistical parameters of the 3D-QSAR analysis are
summarized in Table 2.
The model expresses 99% correlation (r2 ¼ 0.9992). Observed
and predicted activities show a close agreement with the
regression line as indicated by the fitness graph (Figure 5). This
further validated the significance of the model. Validity of the
model was also expressed by a determination coefficient on the
test set (q2) of 0.79.The results of QSAR characteristics are given
in Figure 6(a–c).
The 3D-QSAR results covering the electron withdrawing,
hydrogen bond donor (HBD) and hydrophobic characteristics
were generated. The hypothesis ADHRR.9 includes one hydrogen
bond acceptor, one HBD, one hydrophobic core and two aromatic
ring features. The 3D-QSAR model is in agreement with the
in vitro results. The blue cubes of the 3D pharmacophore regions
refer to ligand regions in which the specific feature is vital for
activity, while the red cubes indicate that the presence of that
particular feature will reduce the activity.
Figure 6(a) represents the electron withdrawing characteristics
of the data set. The QSAR shows that presence of electron
withdrawing groups at position-4 of Ring C reduces the activity.
Further, substantiating the in vitro results, which show that
compounds 30, 35 and 40 having chloro substitution at this
position have poor inhibitory profiles.
ꢂ Substitution with electron releasing group showed good
activity; however, electropositive group with hydrogen bond-
ing ability were found to be most active.
ꢂ The replacement of electropositive group by electronegative
group decreases the activity.
ꢂ The least activity is elicited by compound with electronegative
substitution at both rings B and C.
ꢂ The compounds having in vitro antimalarial activity also
showed less cytotoxicity.
QSAR
By taking into account the antimalarial activity of our synthesized
compounds, a systemic pharmacophore modelling was performed
and 3D-QSAR model was generated by PHASE module of
Figure 6(b) represents the HBD characteristics of the data set.
HBD at position-4 of Ring C improves the activity, as is evident
¨
Schrodinger, to support the assumption of flexible substituents
responsible for better inhibition. Out of the different

DOI: 10.3109/14756366.2014.958081
Pyrazole–pyrazoline hybrids
9
Figure 6. Electron withdrawing, Hydrogen bond donor and hydrophobic characteristics of the hypothesis ADHRR.9.
6. Qirante J, Ruiz D, Gonzalez A, et al. Platinum(II) and palladium(II)
complexes with (N,N0) and (C,N,N0)-ligands derived from pyrazole
as anticancer and antimalarial agents: Synthesis, characterization
and in vitro activities. J Inorg Biochem 2011;105:1720–8.
7. Bekhit AA, Hymete A, Asfaw H, Bekhit AEDA. Synthesis and
biological evaluation of some pyrazole derivatives as anti-malarial
agents. Archiv der Pharmazie 2012;345:147–54.
8. Wahab BFA, Latif EA, Mohamed HA, Awad GEA. Design and
synthesis of new 4-pyrazolin-3-yl-1,2,3-triazoles and 1,2,3-triazol-4-
yl-pyrazolin-1-ylthiazoles as potential antimicrobial agents. Eur J
Med Chem 2012;52:263–8.
9. Bashir R, Ovais S, Yaseen S, et al. Synthesis of some new
1,3,5-trisubstituted pyrazolines bearing benzene sulfonamide as
anticancer and anti-inflammatory agents. Bioorg Med Chem Lett
2011;21:4301–5.
10. Das N, Dash B, Dhanawat M, Shrivastava SK. Design, synthesis,
preliminary pharmacological evaluation, and docking studies of
pyrazoline derivatives. Chem Papers 2012;66:67–74.
from compounds 33, 38, 43 and 48. In the series, these
compounds have the best inhibitory profiles in the data set.
Figure 6(c) represents the hydrophobic characteristics of the
data set. It demonstrates that the presence of hydrophobic
substitutions at position-4 of Ring B increases the inhibitory
profiles of the compounds. For instance, compounds with
methoxy substitution at this position among all series are better
inhibitors.
Conclusion
In conclusion, 20 compounds were synthesised, characterized and
tested against 3D7 strain of P. falciparum. The active compounds
had an IC50 value in the range of 1.13–4.20 mM. The compounds
with IC50 less than 5 mM were further evaluated for their
cytotoxicity assay. The most active compound, 48 shows an
IC50 of 1.13 mM (0.546 mg/mL) with reduced cytotoxicity. In order 11. Wanare G, Aher R, Kawathekar N, et al. Synthesis of novel
a-pyranochalcones and pyrazoline derivatives as Plasmodium
falciparum growth inhibitors. Bioorg Med Chem Lett 2010;20:
4675–8.
to support the systemic pharmacophore model, a predictive
3D-QSAR model was generated, which further substantiated the
in vitro antimalarial results.
12. Acharya BN, Saraswat D, Tiwari M, et al. Synthesis and antimalarial
evaluation of 1, 3, 5-trisubstituted pyrazolines. Eur J Med Chem
2010;45:430–8.
Acknowledgements
13. Insuasty B, Montoya A, Becerra D, et al. Synthesis of novel analogs
of 2-pyrazoline obtained from [(7-chloroquinolin-4-yl)amino]chal-
cones and hydrazine as potential antitumor and antimalarial agents.
Eur J Med Chem 2013;67:252–62.
14. Chellan P, Nasser S, Vivas L, et al. Cyclopalladated complexes
containing tridentate thiosemicarbazone ligands of biological
significance: Synthesis, structure and antimalarial activity.
J Organomet Chem 2010;695:2225–32.
We are thankful to Jamia Hamdard, for providing research laboratory and
instrumentation facilities (NMR, mass and IR) at the institute.
Declaration of interest
The authors report no declarations of interest. Authors are thankful to
University Grants Commission, Government of India, New Delhi, for
financial support.
15. Chellan P, Shunmoogam-Gounden S, Hendricks DT, et al.
Synthesis, structure and in-vitro biological screening of
palladium(II) complexes of functionalised salicylaldimine thiosemi-
carbazones as antimalarial and anticancer agents. Eur J Inorg Chem
2010;2010:3520–8.
16. Khanye SD, Smith GS, Lategan C, et al. Synthesis and in vitro
evaluation of gold(I) thiosemicarbazone complexes for antimalarial
activity. J Inorg Biochem 2010;104:1079–83.
17. Klayman DL, Bartosevich JF, Griffin TS, et al. 2-Acetylpyridine
thiosemicarbazones. 1. A new class of potential antimalarial agents.
J Med Chem 1979;22:855–62.
18. Pingaew R, Prachayasittikul S, Ruchirawat S. Synthesis, cytotoxic
and antimalarial activities of benzoyl thiosemicarbazone analogs of
isoquinoline and related compounds. Molecules 2010;15:988–96.
19. Khanye SD, Wan B, Franzblau SG, et al. Synthesis and in-vitro
antimalarial and antitubercular activity of gold(III) complexes
containing thiosemicarbazone ligands. J Organomet Chem 2011;
696:3392–6.
References
1. Tomar V, Bhattacharjee G, Kamaluddin, et al. Synthesis of new
chalcone derivatives containing acridinyl moiety with potential
antimalarial activity. Eur J Med Chem 2010;45:745–51.
2. Mane UR, Li H, Huang J, et al. Pyrido[1,2-a]pyrimidin-4-ones as
antiplasmodial falcipain-2 inhibitors. Bioorg Med Chem 2012;20:
6296–304.
3. Cochin SD, Paunescu E, Maes L, et al. Synthesis and antimalarial
activity of new analogues of amodiaquine. Eur J Med Chem 2008;
43:252–60.
4. Hans RH, Guantai EM, Lategan C, et al. Synthesis, antimalarial and
antitubercular activity of acetylenic chalcones. Bioorg Med Chem
Lett 2010;20:942–4.
5. Cunico W, Cechinel CA, Bonacorso HG, et al. Antimalarial activity
of 4-(5-trifluoromethyl-1H-pyrazol-1-yl)-chloroquine analogues.
Bioorg Med Chem Lett 2006;16:649–53.

10 A. Marella et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
20. WHO Guidelines for the Treatment of Malaria. 2nd ed. Geneva: 1-N-substituted thiocarbamoyl-3,5-diphenyl-2-pyrazoline deriva-
WHO Press, World Health Organisation; 2010. tives. Eur J Med Chem 2007;42:544–51.
21. Capela R, Cabal GG, Rosenthal PJ, et al. Design and evaluation of 26. Trager W, Jensen JB. Human malaria parasites in continuous culture.
primaquine-artemisinin hybrids as a multistage antimalarial strategy.
Antimicrob Agents Chemother 2011;55:4698–706.
22. Marella A, Akhtar M, Shaquiquzzaman M, et al. Synthesis and
in-vitro antimalarial evaluation of pyrazoline: A new antimalarial
scaffold. J Pharm Res 2014;8:77–80.
23. Smith MB, March J. Aromatic substitution, electrophilic. In: March’s
advanced organic chemistry reactions, mechanisms, and structure.
6th ed. Hoboken (NJ): John Wiley & Sons, Inc.; 2007:724–5.
Science 1976;193:673–7.
27. Dua VK, Sinha SN, Biswas S, et al. Isolation and antimalarial
activity of peroxydisulfate oxidation products of primaquine. Bioorg
Med Chem Lett 2002;12:3587–9.
28. Mosmann TJ. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 1983;65:55–63.
29. Phase, version 3.5, Schrodinger. New York (NY): LLC; 2013.
24. Desai NC, Joshi VV, Rajpara KM, et al. Facile synthesis of novel 30. Vora JJ, Vasava SB, Parmar KC, et al. Synthesis, spectral and
fluorine containing pyrazole based thiazole derivatives and evalu-
ation of antimicrobial activity. J Fluorine Chem 2012;142:67–78.
microbial studies of some novel schiff base derivatives of
4-Methylpyridin-2-amine. Eur J Chem 2009;6:1205–10.
25. Budakoti A, Abid M, Azam A. Syntheses, characterization and 31. Kohler EP, Chadwell HM. Benzalacetophenone (Chalcone). Org
in-vitro antiamoebic activity of new Pd(II) complexes with Syntheses 1941;1:78.
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