Pyrazole-pyrazoline as promising novel antimalarial agents: A mechanistic study

Article abstract of DOI:10.1016/j.ejmech.2018.01.082

A series of pyrazole-pyrazoline substituted with benzenesulfonamide were synthesized and evaluated for their antimalarial activity in vitro and in vivo. The compounds were active against both chloroquine (CQ) sensitive (3D7) and CQ resistant (RKL-9) strains of Plasmodium falciparum. Seven compounds (7e, 7i, 7j, 7l, 7m, 7o and 7p) exhibiting EC₅₀ less than 2 μM. A mechanistic study of compound 7o revealed that these compound act through the inhibition of β-hematin. The study indicated that these compounds can serve as lead compounds for further development of potent antimalarial drugs.

Full text of DOI:10.1016/j.ejmech.2018.01.082

Accepted Manuscript
Pyrazole-pyrazoline as promising novel antimalarial agents: A mechanistic study
Gautam Kumar, Omprakah Tanwer, Jitender Kumar, Mymoona Akhter, Supriya
Sharma, C.R. Pillai, Md Mumtaz Alam, M.S. Zama
PII:
S0223-5234(18)30101-6
10.1016/j.ejmech.2018.01.082
EJMECH 10159
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 November 2017
Revised Date: 17 January 2018
Accepted Date: 25 January 2018
Please cite this article as: G. Kumar, O. Tanwer, J. Kumar, M. Akhter, S. Sharma, C.R. Pillai, M.M.
Alam, M.S. Zama, Pyrazole-pyrazoline as promising novel antimalarial agents: A mechanistic study,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.082.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Pyrazole-Pyrazoline as Promising Novel Antimalarial Agents: A
Mechanistic Study
Gautam Kumara1, Omprakah Tanwera, Jitender Kumara, Mymoona Akhtera2*, Supriya
Sharmab, C.R. Pillaib, Md. Mumtaz Alama, M. S. Zamaa
aDrug Design and Medicinal Chemistry Lab, Department of Pharmaceutical Chemistry,
School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi
bNational Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi, 110077, India
1Currently at National Institute of Pharmaceutical Education and Research, Mohali,
Chandigarh
2Bioinformatics Infrastructure Facility (BIF), Jamia Hamdard, New Delhi, 110062, India
Correspondence
Dr. Mymoona Akhter
Associate Professor and Dy. Coordinator, Bioinformatics Infrastructure Facility (BIF),
Drug Design and Medicinal Chemistry Lab
Department of Pharmaceutical Chemistry
School of Pharmaceutical Education and Research
Jamia Hamdard, New Delhi, 110062, India

ACCEPTED MANUSCRIPT
ABSTRACT:
A series of pyrazole-pyrazoline substituted with benzenesulfonamide were synthesized
and evaluated for their antimalarial activity in vitro and in vivo. The compounds were
active against both chloroquine (CQ) sensitive (3D7) and CQ resistant (RKL-9) strains
of Plasmodium falciparum. Seven compounds (7e, 7i, 7j, 7l, 7m, 7o and 7p
)
exhibiting EC50 less than 2µM. A mechanistic study of compound 7o revealed that
these compound act through the inhibition of β-hematin. The study indicated that these
compounds can serve as lead compounds for further development of potent
antimalarial drugs.
Key words: Plasmodium falciparum, antimalarial, β-hematin, heme binding, pyrazole,
pyrazoline

ACCEPTED MANUSCRIPT
1. Introduction
Malaria remains one of the most challenging infectious diseases in the world especially in
third world and developing countries like India. Due to the development of resistance to the
existing drug regimen the problem has increased too many folds [1]. Identification and
development of new antimalarial agents specifically against new validated targets are
therefore an urgent requirement [2]. Among the potential new targets for the development of
antimalarial drugs directed against P. falciparum are proteases. For protein synthesis,
erythrocytic malaria parasites acquire amino acids by degradation of haemoglobin. Multiple
enzymes appear to participate in this process [3, 4] including the cysteine proteases falcipain-
2 [5] and falcipain-3 [6]. The cysteine protease inhibitors have been shown to arrest the
erythrocytic life cycle of P. falciparum, by blocking the hydrolysis of the host hemoglobin,
causing abnormally swollen food vacuoles and also some of these molecules have led to the
cure of malaria in mice. [7, 8].
One of the most widely explored pathways for development of antimalarial agent includes
inhibition of hemozoin/β-hematin formation. Parasite converts toxic heme into a non-toxic
hemozoin by a mechanism known as hemozoin formation and this process is considered to be
the most important mechanism for heme detoxification in Plasmodium sp. [9]. Several types
of antimalarial agents are reported to exhibit antimalarial activity by enhancing free heme
toxicity through the inhibition of hemozoin formation. Compounds that show antimalarial
activity via inhibition of hemozoin are presented in figure 1. Benzimidazole derivatives (1)
[10], known antifungal agent miconazole (2) [11], clotrimazole (3) [12], dibromocryptolepine
(4) [13], floxacrine (5) [14], along with other compounds like RO 22-8014 (6) [15], N1-(4-
(benzyloxy)benzyl)-N3-(1-(2,2-diphenylethyl)piperidin-4-yl)propane-1,3-diamine (7), [14]
and pyrazole (8) [16] are reported as inhibitors of hemozoin formation. Efforts, therefore,
continue to discover newer antimalarial agents through different mechanisms and targets. We
here in present the potential of pyrazole-pyrazoline based compounds for their antimalarial
activity through β-hematin binding studies.
2. RESULTS AND DISCUSSION

ACCEPTED MANUSCRIPT
The title compounds were synthesized successfully and their structure was elucidated on the
basis of spectral analysis. All the synthesized compounds (7a-q) were tested for their
antimalarial activity and most active compound was selected for the mechanistic study.
2.1.Chemistry
The synthesis of title compounds was carried out by the method outlined in Scheme 1.
Pyrazole carbaldehydes (3a-3e) were synthesized by reported method [17] in 63-79% yield.
The aldehydes (3a-3e) were made to react with substituted acetophenone by Claisen-Schmidt
reaction in presence of sodium hydroxide in ethanol to obtain different chalcones (5a-5q)
with 56-79% of yield. The bipyrazol-benzenesulfonamide derivatives (7a-7q) were prepared
by cyclocondensation reaction of chalcones (5a-5q) with 4-hydrazinylbenzenesulfonamide in
presence of HCl in ethanol with a yield of 54-67%. The compounds (7a-7q) were obtained as
racemic mixtures and were fully characterized by IR, NMR, and MS. The IR spectra
absorption bands at 3429-3335 cm-1 and 3292-3247 cm-1 supported presence of primary NH2,
absorption bands in the region of 1592-1596 cm-1 and 1491-1503 cm-1 indicated azomethine
1
C= N and aromatic C=C stretching. The H NMR spectra showed peak at 8.25-8.38 ppm
which could be assigned to pyrazole protons whereas pyrazoline stereogenic proton and
methylenic protons both couple and appear as double-doublets at (5.73-5.60) ppm, with
J5’−4a’=11.6-12.2 Hz, and J5’−4b’=6.0- 6.8 Hz, and methylenic protons at δ= 4.02-4.18 ppm
with J4a’−4b’=12.4-17.4 Hz, and, J4a’−5’=12-13.2 Hz, and 4b’-H at δ=3.30-3.36 ppm, J4b’-
4a’=16-17.6 Hz, and J4b’-5’=6-6.4 Hz which indicates ‘trans’ conformation. The –NH2 peak
could be located between 6.95-7.02 ppm. The 13C NMR spectra show the absence of carbonyl
carbon peak and presence of pyrazoline chiral carbon at 55.20-55.90 ppm, and pyrazoline
methylenic carbon at 42.40-45.37 ppm. The mass spectra of the compound shows M+, (M-
H)+, (M+2) peaks respectively. The synthesized compound were found pure beyond 95% by
HRMS and HPLC data (Supplimentray data). Compound 7q showed two peaks of slight
difference in height and retention time indicating that it may be present in recemic mixture.
The X-ray crystallographic studies of the compound 7e confirmed that the conformation of
the compound at chiral centre is ‘trans’ (Fig. 2). Crystallographic data for the structure 7e
have been deposited with the Cambridge Crystallographic Data Center (CCDC) under the
number CCDC- 1475330.

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of 4-(5-(1,3-diphenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazol-1-yl)
benzenesulfonamide Derivatives (7a-7q)
R
O
N
H
N
a
N
H2N
A
R1
O
1
2
3a-3e
Yield: 63-79%
N
N
O
N
N
R
b
R
R1
O
O
R1
3a-3e
4
5a-5q
Yield: 56-79%
B
N
N
NH2
NH
R
A
N
N
c
O
S
N
D
NH2
N
O
C
O
R1
O
S
O
R
NH2
6
5a-5q
7a-7q
R1
Yield: 54-67%
Reagents and conditions: (a) POCl3, DMF; NaHCO3, reflux; (b) MeOH, NaOH, reflux;
(c) MeOH, HCl, reflux
2.2.Biological activity
2.2.1. In vitro and in vivo antimalarial activity
Compounds (7a -7q) were evaluated for their in vitro antimalarial activity against
chloroquine CQS3D7 and CQRRKL-9 pf strain by reported method [16, 18] and in vivo
antimalarial activity by Peter’s 4-day suppressive test [16, 19] by using P. berghei NK65
strain. Cytotoxicity was also determined in VERO cells by reported procedure [20].The

ACCEPTED MANUSCRIPT
results of in vitro antimalarial activity against CQS 3D7 and CQR RKL-9 strains of P.
falciparum of synthesized compounds (7a -7q) is presented in Table 1. Most of the final
compounds showed potent antimalarial activity against CQS 3D7 with seven compounds (7e,
7i, 7j, 7l, 7m, 7o and 7p) exhibiting EC50 less than 2 µM which is better than CQ. These
compounds also exhibited good activity against CQRRKL-9 strains (EC50 1.31 -2.12 µM)
compared to the reference drug (EC50 11.25 µM). Compounds exhibited good tolerance as
indicated by SI index. Most potent compound 7o was selected for in vivo antimalarial studies
against P. berghei mouse model. The results obtained were encouraging as the parasitemia
suppression of 82.49% was observed at a dose 50 mg/kg body weight on day 4 and the mean
survival days were 19.8 days compared to 7.2 days for the negative control group (Table 2).
Results are expressed as mean ± SD, Statistical evaluation was performed using ANOVA
followed by Dunnet test for comparison against standard.
2.2.2. Mechanistic studies: hematin binding studies
Pyrazole based compounds are reported to show antimalarial activity from our lab earlier also
[21, 22]. Also in our previous work reported pyrazole based compounds were found to act
through the inhibition of β-hematin [16] therefore to draw a plausible mechanism for the
synthesized compounds, compounds 7o was selected for mechanistic. The hematin binding
studies of compound 7o was performed at pH 7.4, 5.6 and 5.8 (pH of food vacuole). An
absorption band (Soret band) at 402 nm indicated the presence of hematin (FP-Fe(III)), under
the conditions used (0.02 M HEPES buffer, pH 7.4 and 0.02 M MES buffer, pH 5.6). On
addition of increased concentration of 7o into a constant concentration of monomeric heme
(5.0 µM), a decrease in the intensity of the Soret band was observed with no shift in the
absorption maxima (Fig. 3). The stoichiometry ratio of the most stable complexes of
compound 7o with hematin at pH 5.6 and 7.4 was inferred from the Job’s plot and was found
to be 1:1. The absorbance at 402 nm got to maximum when mole fraction of the compound
was approximately 0.5. Thus 1:1 ratio was established for the association of compound
hematin at both the pH values (Fig. 4). Since CQ and its derivatives also bind to heme dimer,
the study of the binding of compound 7o with the heme dimer at pH 5.8 the physiological pH
of plasmodium food vacuole was also studied. In aqueous NaOH solution, heme exists as
dimer and shows an absorption band at 362 nm. The addition of compound 7o (0−20 µM) to
a solution of the dimer (10 µM) in 20 mM phosphate buffer at pH 5.8 resulted in a decrease

ACCEPTED MANUSCRIPT
in intensity of absorbance (Soret band) at 362 nm. The Job’s plot indicated a 1:1
stoichiometry for the most stable complex formed between heme dimer and compound 7o
(Fig. 5). From the data shown in Table 3, it is apparent that the compound 7o binds strongly
with monomeric heme as well as with heme dimer and the observed results are comparable to
the standard CQ (log K 5.58). Thus the formation of complexes between soluble hematin and
compound 7o suggests the inhibition of formation of β-hematin, as one of the possible
mechanisms of antimalarial action of these compounds. The hematic binding studies
indicated that it is not the only mode of action of these derivatives as the antimalarial activity
is excellent there must be another mode of action.
2.2.3. Structure activity relationship (SAR)
Pyrazole pyrazoline based compounds resulted in potent antimalarial agents against both
chloroquine sensitive and chloroquine resistant stains of P. falciparum. From the activity of
the series it could be observed that compounds with electron donating group like –OCH3,
CH3, OH, on para position of ring C were favourable for activity. For example the most
potent compound contained either –OCH3, CH3 (compound 7o and 7e). On ring A, no
substitution or small electron donating group on para position were favourable for the
activity. It was observed that the highest activity was shown by compound (7o) with CH3
group on ring A rather than –OCH3 group and the reason for this may increase in bulk of –
OCH3 group. This SAR is in agreement with the literature reports about the SAR of
antimalarial compounds.
3. Conclusion:
The syntheses of new pyrazolo-pyrazoline substituted compounds (7a-q) were successfully
attained and all of them were characterized by spectral analysis. All the compounds showed
antimalarial activity against CQS (3D7) and selected compounds were also found active
against CQR (RLK-9) strain of P. falciparum. Five compounds (7e, 7i, 7m, 7o, 7p) showed
comparable activity compared to chloroquine in CQS strain and were found better than CQR
Plasmodium strain. In vivo screening of compound 7o in mouse model of malaria also
showed promising results with good mean survival days. Compound 7o also showed good
inhibition of hematin at both the pH studied. The compounds seem to act through inhibition
of β-hematin in addition to other unknown mechanisms. These results indicate that the title

ACCEPTED MANUSCRIPT
compounds are promising lead for development of activity based agents and provide a
significant model for further structural and biological optimization.
4. Experimental Section
4.1.Chemistry
All chemicals were purchased from commercial suppliers. Melting points were determined
by an open capillary method by digital melting point apparatus by VEEGO and are
1
uncorrected. H NMR spectra were recorded on a Bruker Avance 400MHz spectrometer,
using CDCl3 and DMSO-d6 as solvent and TMS as internal standard.The 13C NMR spectra of
compounds were recorded on Bruker Avance-100 MHz instrument in DMSO-d6. The
chemical shift in NMR is reported as delta (δ) values. Infrared (IR) spectra were recorded on
a Shimadzu FT-IR 8400S infrared spectrophotometer using the ATR accessory. MS analysis
was performed on an Agilent 6410 LC-MS (Agilent Technologies, USA) triple-stage
quadrupole mass spectrometer equipped with electrospray ionization (ESI) interface.
Electrospray ionization (ESI) was run in following operating conditions: Source and Mode;
ESI, Polarity; positive and negative, Collision gas; nitrogen, Capillary voltage; ±3000 V, Ion
source temperature; 3000C, Gas flow; 10L/min, Dwell time; 10ms, Resolution (Q1 and Q3);
unit. High Resolution Mass Spectra (HRMS) were recorded on Agilent Technologies 6540
instrument. Reaction progress was monitored with the help of pre-coated TLC plates [0.25
mm Merck silica-gel plates (60 F254)] and spots were visualized under UV light and by
exposing them to iodine vapors. The reactant pyrazolecarbaldehyde (3a-3e) was prepared by
reported method. [19] These were then reacted to substituted acetophenone by Claisen
Schmidt reaction to obtain chalcones which were cyclized to final pyrazoline derivatives by
treating with 4-hydrazinylbenzenesulfonamide. The purity of the data was confirmed by
HRMS and HPLC data. HPLC was performed on Shimadzu CLASS-VP V6.14 SP1 using HPLC
grade acetonitrile and methanol in 9:1 ratio.
4.2. General procedure for preparation of pyrazole carbaldehyde (3a-3e)
Cooled POCl3 (0.075 mole) was added dropwise through dropping funnel to previously
cooled DMF (0.075 mole at 0-5°C) and stirred for 15 minutes. A solution of hydrazone
(0.025 mole) in DMF was added drop-wise to the DMF- POCl3 mixture. The mixture was
then warmed to room temperature and heated at 75-80°C for 5 hour. The completion of

ACCEPTED MANUSCRIPT
reaction was monitored with the help of TLC. After the completion of reaction the mixture
was cooled to room temperature and basified with saturated solution of NaHCO3. The
precipitate was filtered, thoroughly washed with distilled water, dried and crystallized in the
ethanol
1
4.2.1. 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (3a) white crystal (74%): mp: 122°C; H
NMR (400MHz, CDCl3): δ=10.03 (s, 1H), 8.52 (s, 1H), 7.83-7.80 (m, 2H), 7.78-7.75 (m,
2H), 7.51-7.43 (m, 5H), 7.38 ppm (t, J=6.84 Hz, 1H); 13C NMR (100 MHz, CDCl3)
δ=119.69, 122.49, 127.92, 128.74, 128.95, 129.27, 129.64, 131.02, 131.33, 138.98, 154.70,
185.14 ppm; IR (KBr): ν˜== 2836, 1671, 1596, 1525 Cm-1.
4.2.2. 3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3b) white crystal (67%):
1
mp: 150°C; H NMR (400MHz, CDCl3): δ=10.03 (s, 1H), 8.53 (s, 1H), 7.88-7.85 (m, 2H),
7.80-7.77 (m, 2H), 7.54 (t, J=7.44 Hz, 2H), 7.42 (t, J=7.4 Hz, 1H), 7.21 ppm (t, J=7.6 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ=115.67, 115.88, 119.74, 122.43, 127.49, 127.52,
128.07, 129.73, 130.77, 130.85, 131.81, 138.94, 153.49, 162.28, 164.75, 184.62 ppm; IR
(KBr): ν˜==2837, 1672, 1597, 1522 Cm-1.
4.2.3. 3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3c) white crystal (79%):
1
mp: 124°C; H NMR (400MHz, CDCl3): δ=10.03 (s, 1H), 8.52 (s, 1H), 7.84-7.82 (m, 2H),
7.81-7.76 (m, 2H), 7.53-7.42 (m, 4H), 7.42-7.39 ppm (m, 1H); 13C NMR (100 MHz, CDCl3)
δ= 119.72, 122.50, 128.11, 128.93, 129.74, 129.84, 130.19, 132.03, 135.43, 138.88, 153.18,
184.46 ppm; IR (KBr): ν˜==2830, 1672, 1600, 1521 Cm-1.
4.2.4. 1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde (3d) white crystal (71%): mp:
99°C; 1H NMR (400MHz, CDCl3): δ=10.04 (s, 1H), 8.52 (s, 1H), 7.79-7.77 (m, 2H), 7.72 (d,
J=7.24 Hz, 2H), 7.51 (t, J=6.52 Hz, 2H), 7.40 (t, J=6.68 Hz, 1H), 7.31 (d, J=7.92 Hz, 2H),
2.46 ppm (s, 3H); 13C NMR (100 MHz, CDCl3) δCppm 21.38, 119.74, 122.46, 127.90,
128.46, 128.85, 129.48, 129.66, 130.87, 139.05, 139.35, 154.89, 185.34 ppm; IR (KBr):
ν˜==2834, 1671, 1596, 1518 Cm-1.
4.2.5. 3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3e) white crystal(63%):
1
mp: 118°C; H NMR (400MHz, CDCl3): δ=10.03 (s, 1H), 8.51 (s, 1H), 7.80-7.76 (m, 4H),
7.51 (t, J=6.48 Hz, 2H), 7.39 (t, J=7.36 Hz, 1H), 7.03 (d, J=6.68 Hz, 2H), 3.86 ppm (s, 3H);
13C NMR (100 MHz, CDCl3) δ=55.37, 114.19, 119.68, 122.35, 123.84, 127.85, 129.65,

ACCEPTED MANUSCRIPT
130.26, 131.22, 139.04, 154.46, 160.54, 185.15 ppm; IR (KBr): ν˜==2854, 1671, 1595, 1525
Cm-1.
4.3.General procedure for preparation of bipyrazol-benzenesulfonamide (7a-7q)
To a mixture of ethanol (35 ml) and 0.5 ml HCl, chalcones (0.001 mol) and 4-
hydrazinylbenzenesulfonamide (0.0025 mol) were added. The mixture was stirred vigorously
at 75°C and the completion ofthe reaction was monitored with the help of TLC. After
completion of the reaction, the reaction mixture was cooled to room temperature and
quenched by pouring the mixture into the crushed ice. The icy mixture was neutralized with
NaHCO3 solution to get the precipitated product. The precipitate was filtered, dried using a
vacuum and crystallized in the mixture of ethanol and chloroform (1: 0.25).
4.3.1. 4-(5-(2,4-dichlorophenyl)-1',3'-diphenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7a):
Yellow crystal (60%): mp: 200°C; 1H NMR (400MHz, DMSO) δ=8.38 (s, 1H), 7.82 -7.77 (m,
3H), 7.71 (t, J=7.6 Hz, 3H), 7.58 (d, J=8.8 Hz, 2H), 7.50-7.41 (m, 7H), 7.28 (t, J=7.6 Hz,
1H), 7.04 (s, 1H), 7.02 (s, 2H), 5.73 (dd, J=12 Hz, J=6.4 Hz, 1H), 4.18 (dd, J=17.4 Hz,
J=12.4 Hz, 1H), 3.45 ppm (dd, J=17.4 Hz, J=6.4 Hz, 1H); 13C NMR (100 MHz, DMSO)
δ=45.3, 55.9, 112.9, 118.8, 121.5, 126.3, 126.7, 127.3, 127.5, 128.1, 128.5, 128.9, 129.5,
129.6, 130.6, 131.1, 132.7, 132.8, 133.9, 134.8, 139.5, 146.0, 147.1, 150.3 ppm; MS m/z
588.00 (M+H)+, 590.09 (M+2)+; IR (KBr): ν˜=3423, 3292, 1592, 1502 Cm-1.
4.3.2. 4-(5-(4-chlorophenyl)-1',3'-diphenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7b)
White crystal (64%): mp: 198°C; 1H NMR (400MHz, DMSO) δ= 8.33 (s, 1H), 7.82 (d, J=7.6
Hz, 2H), 7.77 (t, J=8.8 Hz, 4H), 7.57 (d, J=8.8 Hz, 2H), 7.51 (t, J=7.6 Hz, 4H), 7.44 (t, J=7.6
Hz, 3H), 7.27 (t, J=7.2 Hz, 1H), 7.04 (d, J=8.8 Hz, 2H), 6.96 (s, 2H), 5.71 (dd, J=12 Hz,
J=6.4 Hz, 1H), 4.08 (dd, J=17.4 Hz, J=12.4 Hz, 1H), 3.36 ppm (dd, J=17.6 Hz, J=6.4 Hz,
13
1H); C NMR (100 MHz, DMSO) δ=42.4, 55.0, 112.9, 118.2, 121.4, 126.4, 127.0, 127.1,
127.73, 128.02, 128.29, 128.69, 129.45, 130.80, 132.53, 133.49, 133.63, 139.12, 145.89,
148.69, 149.86 ppm; MS m/z 554.00 (M+H), 555.17 (M+2) ; IR (KBr): ν˜=3363, 3247, 1594,
1491 Cm-1.

ACCEPTED MANUSCRIPT
4.3.3. 4-(5-(4-fluorophenyl)-1',3'-diphenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7c)
Yellow crystal (58%): mp: 210°C; 1H NMR (400MHz, DMSO) δ= 8.33 (s, 1H), 7.82-7.69 (m,
6H), 7.60 (d, J=8.8 Hz, 2H), 7.52 (t, J=7.2 Hz, 2H), 7.44 (t, J=8 Hz, 3H), 7.28 (t, J=8.8 Hz,
3H), 7.05 (d, J=8.8 Hz, 2H), 6.99 (s, 2H), 5.69 (dd, J=12.2 Hz, J=6.4 Hz, 1H), 4.08 (dd,
13
J=15.4 Hz, J=12.4 Hz, 1H), 3.37 ppm (dd, J=14 Hz, J=6.4 Hz, 1H); C NMR (100 MHz,
DMSO) δ=42.64, 55.26, 111.98, 112.19, 114.10, 115.53, 115.74, 118.22, 122.03, 122.20,
124.43, 126.39, 127.07, 127.11, 127.70, 128.03, 128.23, 128.29, 128.53, 128.71, 129.45,
132.56, 132.92, 133.33, 139.14, 146.10, 146.34, 148.90, 149.73, 149.87, 161.41, 163.87 ppm;
MS m/z 536.10 (M-H)+ ; IR (KBr): ν˜=3429, 3279, 1594, 1501 Cm-1. HRMS m/z: 538.1577
4.3.4. 4-(5-(4-hydroxyphenyl)-1',3'-diphenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7d)
1
Yellow crystal (58%): mp: 264°C; H NMR (400MHz, DMSO) δ=9.81 (s, 1H), 8.27 (s, 1H),
7.82 (d, J=8 Hz, 2H), 7.79 (d, J=7.6 Hz, 2H), 7.62 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.8 Hz, 2H),
7.53 (t, J=7.2 Hz, 2H), 7.45-7.40 (m, 3H), 7.27 (t, J=7.2 Hz, 1H), 7.01 (d, J=8.8 Hz, 2H),
6.95 (s, 2H), 6.84 (d, J=8.4 Hz, 2H), 5.62 (dd, J=11.8 Hz, J= 6.4 Hz, 1H), 4.05 (dd, J=17 Hz,
J=12.4 Hz, 1H), 3.31 ppm (dd, J=17.4 Hz, J=6.4 Hz, 1H); 13C NMR (100 MHz, DMSO)
δ=42.85, 54.99, 111.88, 115.50, 118.21, 122.27, 122.86, 126.37, 126.96, 127.05, 127.85,
127.98, 128.27, 128.73, 129.45, 132.59, 132.71, 139.15, 146.44, 149.80, 150.09, 158.75 ppm;
MS m/z 534.00 (M-H); IR (KBr): ν˜=3483, 3335, 3243, 1595, 1503 Cm-1.
4.3.5. 4-(5-(4-methoxyphenyl)-1',3'-diphenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7e)
1
Yellow crystal (61%): mp: 192°C; H NMR (400MHz, DMSO) δ=8.30 (s, 1H), 7.82 (d, J=8
Hz, 2H), 7.77 (d, J=7.2 Hz, 2H), 7.71 (d, J=8.8 Hz, 2H), 7.55 (t, J=8.8 Hz, 2H), 7.50 (d,
J=7.6 Hz, 2H), 7.45-7.39 (m, 3H), 7.26 (t, J=7.6 Hz, 1H), 6.99 (d, J=9.2 Hz, 6H), 5.63 (dd,
J=12 Hz, J=6.4 Hz, 1H), 4.07 (dd, J=17.4 Hz, J=12.4 Hz, 1H), 3.77 (s, 3H), 3.33 ppm (dd,
J=17.2 Hz, J=6 Hz, 1H); 13C NMR (100 MHz, DMSO) δ=43.28, 55.51, 55.76, 112.44,
114.60, 118.68, 122.70, 124.89, 126.90, 127.55, 128.22, 128.80, 129.98, 133.04, 133.31,
139.61, 146.77, 150.24, 150.27, 160.70 ppm; MS m/z 550.20 (M+H)+; IR (KBr): ν˜=3382,
3258, 1592, 1502 Cm-1.

ACCEPTED MANUSCRIPT
4.3.6. 4-(5-(4-bromophenyl)-3'-(4-fluorophenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)benzenesulfonamide (7f)
1
White crystal (76%): mp: 204°C; H NMR (400MHz, DMSO) δ=8.35 (s, 1H), 7.81 (d, J=8
Hz, 2H), 7.75-7.71 (m, 2H), 7.68 (d, J=8.4 Hz, 2H), 7.63 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.8
Hz, 2H), 7.43 (t, J=7.2 Hz, 2H), 7.31-7.23 (m, 3H), 7.04 (d, J=8.8 Hz, 2H), 6.99 (s, 2H), 5.70
(dd, J=12 Hz, J=6 Hz, 1H), 4.05 (dd, J=17.2 Hz, J=13.2 Hz, 1H), 3.34 ppm (dd, J=15.4 Hz,
J=6.4 Hz, 1H); 13C NMR (100 MHz, DMSO) δ=42.70, 55.69, 112.82, 115.97, 116.19,
118.71, 122.34, 122.86, 126.96, 127.57, 127.77, 128.45, 129.51, 129.98, 130.66, 130.74,
+
131.58, 132.09, 134.00, 139.56, 146.31, 149.28, 149.46 ppm; MS m/z 617.90 (M+H) ,
618.24 (M+2)+; IR (KBr): ν˜=3367, 3251, 1595, 1498 Cm-1. HRMS m/z: 616.0772
4.3.7. 4-(3',5-bis(4-fluorophenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7g)
1
White crystal (68%): mp: 212°C; H NMR (400MHz, DMSO) δ=8.35 (s, 1H), 7.81 (m, 6H),
7.57 (d, J=8.8 Hz, 2H), 7.44 (t, J=7.6 Hz, 2H), 7.32-7.24 (m, 5H), 7.04 (d, J=8.8 Hz, 2H),
7.00 (s, 2H), 5.68 (dd, J=12 Hz, J= 6.4 Hz, 1H), 4.06 (dd, J=17.4 Hz, J=12.4 Hz, 1H), 3.34
13
ppm (dd, J=17.4 Hz, J=6.4 Hz, 1H); C NMR (100 MHz, DMSO) δ=42.98, 55.61, 112.69,
115.98, 116.05,116.19, 116.27, 118.70, 122.43, 126.95, 127.57, 128.72, 128.81, 128.97,
129.52, 129.99, 130.65, 130.74, 133.76, 139.56, 146.52, 149.43, 149.46, 161.39, 161.90,
163.83, 164.35 ppm; MS m/z 556.12 (M+H)+;IR (KBr): ν˜=3370, 3257, 1596, 1502 Cm-1.
HRMS m/z: 555.1579
4.3.8. 4-(3'-(4-fluorophenyl)-1'-phenyl-5-(p-tolyl)-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7h)
1
White crystal (62%): mp: 224°C; H NMR (400MHz, DMSO) δ=8.32 (s, 1H), 7.81(d, J=8.0
Hz, 2H), 7.77-7.74 (m, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.8 Hz, 2H), 7.43 (t, J=7.6
Hz, 2H), 7.32 (t, J=8.8 Hz, 2H), 7.25 (t, J=8.0 Hz, 3H), 7.02 (d, J=8.8 Hz, 2H), 6.96 (s, 2H),
5.65 (dd, J=12.2 Hz, J=6 Hz, 1H), 4.05 (dd, J=17.2 Hz, J=12.00 Hz, 1H), 3.30 (dd, J=16.00
Hz, J=6.4 Hz, 1H), 2.46 ppm (s, 3H); 13C NMR (100 MHz, DMSO) δ=21.48, 55.48,
112.61,115.99, 116.20, 118.71, 122.54, 126.94, 127.56, 129.72, 129.98, 130.62, 130.70,
133.60, 139.46, 139.57, 150.37 ppm; MS m/z 550.00 (M-H)+; IR (KBr): ν˜=3369, 3253,
1596, 1502 Cm-1.

ACCEPTED MANUSCRIPT
4.3.9. 4-(3'-(4-fluorophenyl)-5-(4-hydroxyphenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)benzenesulfonamide (7i)
1
White crystal (63%): mp: 228°C; H NMR (400MHz, DMSO) δ=9.82 (s, 1H), 8.29 (s, 1H),
7.81-7.75 (m, 4H), 7.59 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.8 Hz, 2H), 7.44 (t, J=7.6 Hz, 2H),
7.32-7.27 (m, 3H), 7.00 (d, J=8.8 Hz, 2H), 6.95 (s, 2H), 6.81 (d, J=8.8 Hz, 2H), 5.60 (dd,
J=11.8 Hz, J=6.4 Hz, 1H), 4.02 (dd, J=17.2 Hz, J=12 Hz, 1H), 3.28 ppm (dd, J=17.4 Hz, J=6
Hz, 1H); 13C NMR (100 MHz, DMSO) δ=43.21, 55.37, 112.38, 115.99, 116.21, 118.71,
122.62, 123.29, 126.92, 127.55, 128.33, 129.54, 129.57, 129.98, 130.60, 130.68, 133.20,
139.58, 146.88, 149.40, 150.60, 159.23, 161.38 ppm; MS m/z 552.00 (M-H)+.; IR (KBr):
ν˜=3466, 3401, 3292, 1594, 1501 Cm-1.
4.3.10. 4-(3'-(4-fluorophenyl)-5-(4-methoxyphenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)benzenesulfonamide (7j)
Yellow crystal (57%): mp: 170 °C; 1H NMR (400MHz, DMSO) δ=8.31 (s, 1H), 7.81-7.74 (m,
4H), 7.70 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.8 Hz, 2H), 7.43 (t, J=7.6 Hz, 2H), 7.32-7.23 (m,
3H), 7.00-6.95 (m, 6H), 5.62 (dd, J=11.8 Hz, J=6 Hz, 1H), 4.05 (dd, J=17.4 Hz, J=12 Hz,
13
1H), 3.78 (s, 3H), 3.30 ppm (m, 1H); C NMR (100 MHz, DMSO) δ= 43.00, 55.42, 55.78,
112.47, 114.60, 116.00, 116.21, 118.69, 122.60, 124.85, 127.56, 128.20, 129.99, 130.61,
130.70, 150.26, 160.71 ppm; MS m/z 568.10 (M+H)+; IR (KBr): ν˜=3370, 3261, 1595, 1503
Cm-1. HRMS m/z: 568.1776
4.3.11. 4-(3',5-bis(4-chlorophenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7k)
Yellow crystal (70%): mp: 232 °C; 1H NMR (400MHz, DMSO) δ=8.34 (s, 1H), 7.81 (d, J=8
Hz, 2H), 7.75-7.72 (m, 4H), 7.58 (d, J=9.2 Hz, 2H), 7.53-7.47 (m, 4H), 7.44 (t, J=8 Hz, 2H),
7.28 (t, J=7.2 Hz, 1H), 7.06 (d, J=8.8 Hz, 2H), 6.99 (s, 2H), 5.73 (dd, J=12.2 Hz, J=6 Hz,
13
1H), 4.06 (dd, J=17.4 Hz, J=12.4 Hz, 1H), 3.34 ppm (dd, J=16 Hz, J=6 Hz, 1H); C NMR
(100 MHz, DMSO) δ=42.75, 55.72, 112.84, 118.77, 122.55, 127.04, 127.58, 127.89, 128.23,
129.21, 129.99, 130.27, 131.22, 131.89, 133.56, 134.03, 134.17, 139.52, 146.35, 149.11,
149.22 ppm; MS m/z 589.10 (M+2)+; IR (KBr): ν˜=3367, 3268, 1593, 1497 Cm-1.
4.3.12. 4-(3'-(4-chlorophenyl)-5-(4-methoxyphenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)benzenesulfonamide (7l)

ACCEPTED MANUSCRIPT
1
White crystal (64%): mp: 202°C; H NMR (400MHz, DMSO) δ=8.378 (s, 1H), 7.81 (d, J=8
Hz, 2H), 7.77 (d, J=8 Hz, 2H), 7.70 (d, J=8.8 Hz, 2H), 7.56 (t, J=8.8 Hz, 4H), 7.44 (t, J=7.2
Hz, 2H), 7.27 (t, J=6.8 Hz, 1H), 7.01 (d, J=9.2 Hz, 3H) 6.97 (d, J=5.6 Hz, 3H) 5.65 (dd,
J=12 Hz, J=6.4 Hz, 1H), 4.02 (dd, J=12.4 Hz, J=5.2 Hz, 1H), 3.78 (s, 3H), 3.31 ppm (dd,
J=17.6 Hz, J=4.8 Hz, 1H); 13C NMR (100 MHz, DMSO) δ=43.15, 55.47, 55.79, 112.51,
114.60, 118.76, 122.81, 124.84, 127.02, 127.56, 127.76, 128.21, 129.23, 129.99, 130.22,
131.92, 133.41, 133.51, 139.53, 146.78, 149.04, 150.26, 160.73 ppm; MS m/z 584.10
(M+H)+; IR (KBr): ν˜==3417, 3278, 1587, 1499 Cm-1.
4.3.13. 4-(1'-phenyl-3',5-di-p-tolyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7m)
1
Yellow crystal (62%): mp: 208°C; H NMR (400MHz, DMSO) δ=8.28 (s, 1H), 7.80 (d, J=8
Hz, 2H), 7.66 (d, J=8 Hz, 4H), 7.55 (d, J=8.8 Hz, 2H), 7.42 (t, J=7.6 Hz, 2H), 7.31 (d, J=8
Hz, 2H), 7.25 (t, J=8 Hz, 3H), 7.00 (t, J=8.8 Hz, 4H) 5.63 (dd, J=12 Hz, J=6.4 Hz, 1H), 4.07
(dd, J=17.4 Hz, J=12 Hz, 1H), 3.31 (dd, J=19.6 Hz, J=13.6 Hz, 1H), 2.35 (s, 3H), 2.31 ppm
(s, 3H); 13C NMR (100 MHz, DMSO) δ=21.37, 21.48, 43.12, 55.58, 112.56, 118.64, 122.51,
126.59, 126.80, 127.40, 127.54, 128.38, 129.61, 129.71, 129.83, 129.95, 130.19, 133.54,
138.15, 139.43, 139.64, 146.66, 150.34 ppm; MS m/z 546.00 (M-H)+; IR (KBr): ν˜= 3365,
3247, 1592, 1497 Cm-1.
4.3.14. 4-(1',5-diphenyl-3'-(p-tolyl)-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7n)
1
White crystal (62%): mp: 198°C; H NMR (400MHz, DMSO) δ=8.30 (s, 1H), 7.81 (d, J=8
Hz, 2H), 7.76 (d, J=6.8 Hz, 2H), 7.65 (d, J=8 Hz, 2H), 7.55 (d, J=8.8 Hz, 2H), 7.44-7.38 (m,
5H), 7.31 (d, J=7.8 Hz, 2H), 7.26 (t, J=7.8 Hz, 1H), 7.02-6.96 (m, 4H), 5.66 (dd, J=12.2 Hz,
J=6.0 Hz, 1H), 4.09 (dd, J=17.4 Hz, J=12.4 Hz, 1H), 3.34 (dd, J=15.6 Hz, J=11.6 Hz, 1H),
2.35 ppm (s, 3H); 13C NMR (100 MHz, DMSO) δ=21.37, 43.01, 55.00, 112.64, 118.64,
122.46, 126.60, 126.81, 127.46, 127.55, 128.41, 129.12, 129.73, 129.82, 129.95, 130.18,
132.35, 138.17, 139.63, 146.55, 150.26, 150.35 ppm; MS m/z 532.0 (M-H)+; IR (KBr):
ν˜=3366, 3249, 1593, 1492 Cm-1.
4.3.15. 4-(5-(4-methoxyphenyl)-1'-phenyl-3'-(p-tolyl)-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7o)

ACCEPTED MANUSCRIPT
1
Yellow crystal (58%): mp: 178°C; H NMR (400MHz, DMSO) δ=8.27 (s, 1H,), 7.81 (d J=8
Hz, 2H,), 7.70 (d, J=8.4 Hz, 2H,), 7.67 (d, J=7.6 Hz, 2H), 7.56 (d, J=8.8 Hz, 2H), 7.42 (t,
J=7.6 Hz, 2H), 7.31 (d, J=8 Hz, 2H), 7.25 (t, J=7.2 Hz, 1H), 7.00-6.97 (m, 6H), 5.61 (dd,
J=11.8 Hz, J= 6.4 Hz, 1H), 4.05 (dd, J=17.4 Hz, J=12.4 Hz, 1H), 3.76 (s, 3H), 3.31 (dd,
13
J=17.4 Hz, J=6.4 Hz, 1H), 2.35 ppm (s, 3H); C NMR (100 MHz, DMSO) δ=21.37, 43.24,
55.54, 55.76, 112.43, 114.58, 118.63, 122.57, 124.91, 126.80, 127.37, 127.55, 128.21,
128.38, 129.84, 129.95, 130.20, 133.31, 138.16, 139.64, 146.78, 150.22, 150.29, 160.70
ppm; MS m/z 564.10 (M+H)+; IR (KBr): ν˜= 3501, 3239, 1593, 1503, Cm-1.
4.3.16. 4-(5-(3,4-dimethoxyphenyl)-1'-phenyl-3'-(p-tolyl)-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)benzenesulfonamide (7p)
White crystal (62%): mp: 192°C; 1H NMR (400MHz, DMSO) δ=8.25 (s, 1H), 7.81 (d, J=9.2
Hz, 2H), 7.67 (d, J=7.6 Hz, 2H), 7.53 (J=8.4 Hz, 2H), 7.42-7.38 (m, 3H), 7.32 (d, J=7.2 Hz,
2H), 7.25 (t, J=7.2 Hz, 2H), 6.98-6.95 (m, 5H), 5.62 (dd, J=11.6 Hz, J=6.8 Hz, 1H), 4.07 (dd,
J=17.6 Hz, J=12 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.35-3.31 ( dd, 1H), 2.46 ppm (s, 3H);
13C NMR (100 MHz, DMSO) δ=21.37, 43.28, 55.56, 56.06, 109.38, 111.95, 112.45, 118.64,
120.20, 122.60, 125.08, 126.81, 127.31, 127.52, 128.37, 129.85, 129.96, 130.19, 133.32,
138.16, 139.63, 146.68, 149.25, 150.40, 150.59 ppm; MS m/z 592.20 (M-H)+; IR (KBr): ν˜=
3344, 3291, 1593, 1501Cm-1. HRMS m/z: 594.213, 595.2159
4.3.17. 4-(3',5-bis(4-methoxyphenyl)-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)benzenesulfonamide (7q)
1
White crystal (54%): mp: 180°C; H NMR (400MHz, DMSO) δ=8.27 (s, 1H), 7.80 (d, J=8
Hz, 2H), 7.71-7.67 (m, 4H), 7.55 (d, J=9.2 Hz, 2H), 7.42 (t, J=7.6 Hz, 2H), 7.25 (t, J=7.6 Hz,
1H), 7.05 (d, J=8.8 Hz, 2H), 7.00-6.95 (m, 6H), 5.60 (dd, J=12 Hz, J=6.4 Hz, 1H), 4.05 (dd,
J=17.2 Hz, J=12 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.31 ppm (dd, J=16.6 Hz, J=6.4 Hz,
1H); 13C NMR (100 MHz, DMSO) δ=43.17, 55.54, 55.66, 55.78, 112.44, 114.59, 114.64,
118.57, 122.32, 124.92, 125.42, 126.72, 127.35, 127.54, 128.20, 129.81, 129.95, 133.30,
139.66, 146.78, 150.17, 150.25, 159.77, 160.70 ppm; MS m/z 580.10 (M+H)+; IR (KBr):
ν˜=3348, 3286, 1594, 1502 Cm-1..
5. Crystallographic analysis
The structure of compound 7o was resolved by the PanalyticalX’Pert Pro Multipurpose
Diffractometer (MPD). The instrument is a PANalyticalX'Pert Pro MPD, powered by a

ACCEPTED MANUSCRIPT
Philips PW3040/60 X-ray generator and fitted with aX'Celerator detector. Diffraction data is
acquired by exposing powder samples to Cu-Kα X-ray radiation, which has a characteristic
wavelength (λ) of 1.5418 Å. X-rays were generated from a Cu anode supplied with 40 kV
and a current of 40 mA. The data were collected over a range of 5.0084 to 49.9904 ˚2θ with
a step size of 0.0170 ˚2θ and nominal time per step of 25.1954s, using the scanning
X’Celerator detector (hence the counting time per step). Fixed anti-scatter and divergence
slits of 0.4354˚ were used together with a beam mask of 10mm and all scans were carried out
in ‘continuous’ mode. Phase identification was carried out by means of the X'Pert
accompanying software program PANalytical High Score Plus in conjunction with the ICDD
Powder Diffraction File 2 Database (1999), ICDD Powder Diffraction File 4 - Minerals
(2012), the American Mineralogist Crystal Structure Database (March 2010) and the
Crystallography Open Database (February 2012; www.crystallography.net) . Crystallographic
data for the structure 7e have been deposited with the Cambridge Crystallographic Data
Center (CCDC) under the number CCDC- 1475330. Copies of the data can be obtained, free
of charge, on application to CCDC 12 Union Road, Cambridge CB2 1EZ,
UK [Fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or at www.ccdc.cam.ac.uk]
6. Biological Studies:
6.1. In-vitro anti-malarial activity
The in vitro antimalarial activity was performed by reported method [19]. Briefly the
parasitized blood was made by infecting red blood cells with CQS (3D7) strain and CQR
(RKL9) strain of P. falciparum separately with 2-3% parasitemia and 8% hematocrit in RPMI
1640 medium supplemented with human AB+ serum. Parasitized blood was added to the
wells of 96 - well plate containing 100 µl of test sample (1 mg/ml) diluted in medium at
different concentrations. After incubation of the plates at 37ºC for 24 hours the blood smear
was prepared from all wells and stained with giemsa stain. Schizonts stage (3 or more
merozoites containing) were counted and result was analysed using dose-response curves by
non-linear regression analysis using HN-NonLin regression analysis.
6.2. In-vivo antimalarial activity
The in vivo antimalarial efficacy was determined by Peter’s 4-day suppressive test [16, 19] by
using P. berghei NK65 strain and female Swiss albino mice (20-25 g of body weight). Briefly
animals were divided into five groups of five mice each. The animals were inoculated

ACCEPTED MANUSCRIPT
intraperitonially with approximately 10-15% of P. Berghei infected erythrocytes from a
donor mouse. Compound 7o was administered orally at a dose of 25 mg/kg body weight and
50 mg/kgbody weight after four hours of infection. Chloroquine (5 mg/kg body weight) was
administered to positive (standard) control group. Negative control group (5 mice) were
administered the same amount of solvent used to suspend the test compound. The compounds
were administered on day 0, 1, 2 and 3. On day 4, blood was taken from tail vein, smears
were made, fixed with methanol and stained with Giemsa and analysed for parasitemia
microscopically (1000 magnification). The percent parasitemia and average percent
suppression of parasitemia were determined and compared to negative control group. The
animals were observed for 30 days and mean survival days (MSD) was determined by
calculating the death of mice during 30 days observation period.
6.3.Mechanistic studies (heme binding studies)
6.3.1. Binding of compound 7o with monomeric heme:
The study was performed by reported method [16]. The working solutions of hemin were
prepared by mixing 20 µl stock solutions (1.2 mM) with 4 ml DMSO and 1 ml 0.2 M HEPES
buffer (pH 7.4). The final volume was made up to 10 ml with double distilled deionized
water. The increasing concentration (0-100 µM) of 7o solution in DMSO was used for
titration of heme (2.4 mM) and absorbance was recorded at 402 nm. The same procedure was
followed for conducting the experiment at pH 5.6, except that 2-[N-morpholino]
ethanesulphonate (MES) buffer (pH 5.4) was used in place of HEPES buffer.
6.3.2. Binding of 7o with dimeric heme
Heme stock solution (10 mM) was prepared by dissolving hemin chloride in 0.1 M NaOH
and sonicated for 30 min. Heme solution (60 mM) was prepared by diluting stock solution
with 20 mM phosphate buffer (pH 5.8). Compound 7o stock solution (2 mM) was prepared in
DMSO. Titrations were done by addition of stock solution of compound 7o to 60 mM heme
solution and absorbance was recorded at 362 nm.
.
Acknowledgements
The authors acknowledge the Vice Chancellor, Jamia Hamdard for his constant support and
motivation. Authors are also thankful to Prof. Renu Chaddha, University Department of

ACCEPTED MANUSCRIPT
Pharmaceutical Sciences, Panjab University and Mr. Vikram Bhardwaj, Technical Staff,
Indian Institute of Integrative Medicine (IIIM), CSIR, Jammu for their help in interpreting the
X-ray data and carrying out HRMS of the compounds. Acknowledged is financial support
from Department of Biotechnology (DBT), Govt. of India and for providing Bio
infrastructure facility lab via letter n: AS/MP (RES.)/JH-5/2013.
Conflict of interest
The authors report no declarations of interest.
Supplementary data: The supporting information includes spectral data details (NMR, IR
and Mass) and detailed procedures of some biological activities.
References:
1. W.H.O. (2015) World Malaria Report 2015. Available at: http://www.who.int/
malaria/publications/ world-malaria-report-2015/report/en/Accessed May 2017
2. P. L. Olliaro, Y. Yuthavong, An overview of chemotherapeutic targets for
antimalarial drug discovery, Pharmacol. Ther. 81 (1999) 91–110.
3. M. Klemba, D. E. Goldberg, Biological roles of proteases in parasitic protozoa. Annu.
Rev. Biochem. 71 (2002) 275–305.
4. P. J. Rosenthal, Hydrolysis of erythrocyte proteins by proteases of malaria parasites.
Curr. Opin. Hematol. 9 (2002) 140–145.
5. B. R. Shenai, P. S. Sijwali, A. Singh, P. J. Rosenthal, Characterization of native and
recombinant falcipain-2, a principal trophozoite cysteine protease and essential
hemoglobinase of Plasmodium falciparum, J. Biol. Chem. 275 (2000) 29000–29010.
6. P. S. Sijwali, B. R. Shenai, J. Gut, A. Singh, P. J. Rosenthal, Expression and
characterization of the Plasmodium falciparum haemoglobinase falcipain-3. Biochem.
J. 360 (2001) 481–489.
7. P. J. Rosenthal, J. H. McKerrow, M. Aikawa, H. Nagasawa, J. H. Leech, A malarial
cysteine proteinase is necessary for hemoglobin degradation by Plasmodium
falciparum. J. Clin. Investig. 82 (1988) 1560–1566
8. J. E. Olson, G. K. Lee, A. Semenov, P. J. Rosenthal, Antimalarial effects in mice of
orally administered peptidyl cysteine protease inhibitors, Bioorg. Med. Chem. 7
(1999) 633.

ACCEPTED MANUSCRIPT
9. Sherman, I.W., 1998. Malaria: Parasite Biology, Pathogenesis and Protection. ASM
Press, Washington, DC.
10. Kalicharan Sharma, Apeksha Shrivastava, Ram N. Mehra, Girdhar S. Deora,
Mohammad M. Alam, Mohammad S. Zaman Mymoona Akhter. Synthesis of novel
benzimidazole acrylonitriles for inhibition of Plasmodium falciparum growth by dual
target inhibition. Arch. Pharm. Chem. Life Sci. 2018; 351: e1700251
11. Chong, C.R., Sullivan Jr., D.J., 2003. Inhibition of heme crystal growth by
antimalarials and other compounds: implications for drug discovery. Biochem.
Pharmacol. 66 (11), 2201–2212.
12. Tiffert, T., Ginsburg, H., Krugliak, M., Elford, B.C., Lew, V.L., 2000. Potent
antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum.
Proc. Nat. Acad. Sci. United States of America 97 (1), 331–336.
13. Van Miert, S., Jonckers, T., Cimanga, K., Maes, L., Maes, B., Lemiere, G.,
Dommisse, R., Vlietinck, A., Pieters, L., 2004. In vitro inhibition of betahaematin
formation, DNA interactions, antiplasmodial activity, and cytotoxicity of synthetic
neocryptolepine derivatives. Exp. Parasitol. 108 (3–4), 163–168
14. Wiesner, J., Ortmann, R., Jomaa, H., Schlitzer, M., 2003. New antimalarial drugs.
Angewandte Chemie & Angewandte Chemie International Edition in English,
Supplement 42 (43), 5274–5293.
15. Brinner, K.M., Mi Kim, J., Habashita, H., Gluzman, I.Y., Goldberg, D.E., Ellman,
J.A., 2002. Novel and potent anti-malarial agents. Bioorg. Med. Chem. 10 (11), 3649–
3661.
16. A. K. Pandey, S. Sharma, M. Pandey, M. M. Alam, M. S. zaman, M. Akhter,4, 5-
Dihydrooxazole-pyrazoline hybrids: Synthesis and their evaluation as potential
antimalarial agents. Eur. J. Med. Chem. 123 (2016) 476-486
17. M. A. Kira, M.O. Abdel-Rahman and K.Z. Gadalla, The vilsmeier-haack reaction - III
Cyclization of hydrazones to pyrazoles. Tetrahedron Lett. 10 (1969) 109–110
18. J. B. Jensen, W. Trager, and J. Doherty, Plasmodium falciparum: Continuous
cultivation in a semiautomated apparatus. Exper. Parasitol. 48 (1979) 36-41
19. R. Maurya, A. Soni, D. Anand, M. Ravi, K. S. R. Raju, I. Taneja, N. K. Naikade, S.
K. Puri, S. Wahajuddin, Kanojiya, P. P. Yadav, Synthesis and antimalarial activity of

ACCEPTED MANUSCRIPT
3,3-spiroanellated 5,6-disubstituted 1,2,4-trioxanes. ACS Med. Chem. Lett. 4 (2013)
165−169
20. T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J Immunol. Methods. 65 (1983) 55–63.
21. A. Marella, M. Akhter, M. S. Zaman, O. Tanwar, G. Verma and M. M.
Alam.Synthesis, 3D-QSAR and docking studies of pyrimidine nitrile-pyrazoline: a
novel class of hybrid antimalarial agents. Med. Chem. Res. 24 (2015) 1018–1037
22. A. Marella, M. S. Zaman, M. Akhter, G. Verma and M. M. Alam, Novel pyrazole-
pyrazoline hybrids endowed with thioamide as antimalarial agents: their synthesis and
3D-QSAR studies, J. Enzyme Inh. Med. Chem. 30 (2015) 597-606

ACCEPTED MANUSCRIPT
Table Captions
Table 1: In-vitro anti-malarial activity studies of synthesized compounds (7a-7q and 8, 9a-d)
against CQS 3D7 and CQRstrain RLK-9 of P. falciparum
Table 2: In vivo antimalarial activity of 7o in mice infected with P. berghei
Table 3: Binding constants for compound 7o and chloroquine with heme
Figure Captions
Figure1: Reported antimalarial compounds acting via inhibition of hemazoin/β-haematin
formation
Figure 2: trans conformation of the compound 7e identified from crystal structure. The same is
deposited in Cambridge Crystallographic Data Centre (CCDC) under the number CCDC-
1475330.
Figure 3: a) Titration of compound 7owith monomeric heme at pH 5.6; b) Titration of
compound 7o with monomeric heme at pH 7.4
Figure 4: Job’s plot of monomeric heme complex formation with compound 7o; a) at pH 5.6;
b) at pH 7.4; X (mole fraction of compound 7o) = [compd. 7o]/[compd. 7o]+[
heme]; A0 is the absorbance, when x = 1 and A is the absorbance at respective
values of x
Figure 5: a) Titration of compound 7owith dimeric heme at pH 5.8; b) Job’s plot of dimeric
heme complex formation with compound 7o at pH 5.8

ACCEPTED MANUSCRIPT
Table 1: In-vitro anti-malarial activity of synthesized compounds (7a-7q and 8, 9a-d) against
CQS (3D7) and CQR (RLK-9) strains of P. falciparum
B
N
R
N
A
O
S
N
D
NH2
N
C
R1
O
7a-q
3D7 (EC50)
RLK-9 (EC50)
RI
--
µ
M
µM
Cpd
7a
7b
7c
R
R1
µ
g/ml
µg/ml
H
2,4- diCl
4-Cl
2.694±0.054** 4.58
1.858±0.013** 3.35
3.579±0.022** 6.67
1.629±0.003** 3.05
0.781±0.003** 1.42
1.908±0.003** 3.08
3.538±0.034** 6.36
1.227±0.005** 2.23
0.868±0.006** 1.57
0.941±0.006 ns 1.65
1.651±0.004** 2.80
0.941±0.007 ns 1.61
0.884±0.009** 1.43
1.641±0.012** 2.97
0.781±0.006** 1.38
NT
NT
NT
NT
NT
NT
NT
NT
H
--
H
4-F
--
H
4-OH
4-OCH3
4-Br
--
7d
7e
H
0.98±0.042** 1.78
1.25
--
4-F
4-F
4-F
4-F
4-F
4-Cl
4-Cl
4-CH3
4-CH3
4-CH3
4-CH3
NT
NT
NT
NT
NT
NT
7f
4-F
--
7g
7h
7i
4-CH3
4-OH
4-OCH3
4-Cl
--
1.12±0.036** 2.02
1.36±0.067** 2039
1.29
1.44
--
7j
NT
NT
7k
7l
4-OCH3
4-CH3
4-H
1.24±0.053** 2.12
0.9±0.047** 1.64
1.31
1.14
--
7m
7n
7o
7p
7q
CQ
NT
NT
4-OCH3
0.74±0.072** 1.31
0.92±0.051** 1.55
0.94
0.97
--
2,3- diOCH3 0.945±0.014 ns 1.61
1.315±0.002** 2.26
0.95 2.97
4-OCH3 4-OCH3
NT
NT
3.60
11.25
3.78
ns
Cpd: compound; NT: not tested; RI (Resistance Index) = IC50:PfRKL9/IC50: Pf3D7; non-significant compared to control;**
significant at P<0.01compared to control.

ACCEPTED MANUSCRIPT
Table 2: In vivo antimalarial activity of 7o in mice infected with P. berghei
% reduction
Mean
dose
parasitemia survival days
Compound
(mg/kg/day X 4)
Route
(day 4)
(MSD)
25
50
5
po
po
po
-
68.42
82.49
97.10
-
13.4
19.8
30
7o
7o
CQ
-
7.2
Negative Control

ACCEPTED MANUSCRIPT
Table 3: Binding constants for compound 7o and chloroquine (CQ) with heme
Monomeric heme
µ-oxo-heme
(Log K ± σ)
(Log K ± σ)
Compound
pH 5.6
pH 7.4
pH 5.8
5.58
4.72
1:1
5.12
5.23
1:1
5.19
5.68
1:1
7o
CQ
Stoichiometry

ACCEPTED MANUSCRIPT
Figure1: Reported antimalarial compounds acting via inhibition of hemazoin/β-haematin
formation

ACCEPTED MANUSCRIPT
Figure 2: trans conformation of the compound 7e identified from crystal structure. The
same is deposited in Cambridge Crystallographic Data Centre (CCDC) under
the number CCDC- 1475330.

ACCEPTED MANUSCRIPT
Figure 3. a) Titration of compound 7o with monomeric heme at pH 5.6; b) Titration of compound
7o with monomeric heme at pH 7.4

ACCEPTED MANUSCRIPT
0.4
0.35
0.3
0.4
0.35
0.3
0.25
0.2
0.25
0.2
0.15
0.1
0.15
0.1
0.05
0
0.05
0
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1
X
b
X
a
Figure 4. Job’s plot of monomeric heme complex formation with compound 7o; a) at pH 5.6; b) at
pH 7.4; X (mole fraction of compound 7o) = [Compd. 7o]/[Compd. 7o] + [heme]; A0 is
the absorbance, when x = 1 and A is the absorbance at respective values of x