Synthesis of 1,3,5-trisubstituted pyrazolines as potential antimalarial and antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2017.02.025

A series of novel 1,3,5-trisubstituted pyrazolines derivatives have been synthesized from chalcones and nicotinic acid hydrazide in two steps. In first step, chalcones were prepared by treatment of 4-hydroxy acetophenone with different substituted benzald

Full text of DOI:10.1016/j.bmc.2017.02.025

Accepted Manuscript
Synthesis of 1,3,5-trisubstituted pyrazolines as potential antimalarial and anti-
microbial agents
Vikash K Mishra, Mitali Mishra, Varsha Kashaw, Sushil K Kashaw
PII:
S0968-0896(17)30291-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.025
BMC 13554
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Bioorganic & Medicinal Chemistry
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9 February 2017
10 February 2017
Please cite this article as: Mishra, V.K., Mishra, M., Kashaw, V., Kashaw, S.K., Synthesis of 1,3,5-trisubstituted
pyrazolines as potential antimalarial and antimicrobial agents, Bioorganic & Medicinal Chemistry (2017), doi:
http://dx.doi.org/10.1016/j.bmc.2017.02.025
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Synthesis of 1,3,5-trisubstituted pyrazolines as potential antimalarial and
antimicrobial agents
Vikash K Mishraa, Mitali Mishraa, Varsha Kashawb and Sushil K Kashawa,c*
aDepartment of Pharmaceutical Sciences, Dr. Harisingh Gour University (A Central University),
Sagar (MP), India
bSVN Institute of Pharmaceutical Sciences, SVN University, Sagar (M.P.), India
cUse‐ inspired Biomaterials & Integrated Nano Delivery (U‐ BiND) Systems Laboratory,
Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan, USA
*Corresponding author
Dr. Sushil Kumar Kashaw
Assistant Professor
Department of Pharmaceutical Sciences
Dr. Harisingh Gour University
Sagar (M.P.)-470003, India
E-mail: sushilkashaw@gmail.com
Phone: 09425655720
www.dhsgsu.ac.in
1

Abstract
A series of novel 1,3,5-trisubstituted pyrazolines derivatives have been synthesized from
chalcones and nicotinic acid hydrazide in two steps. In first step, chalcones were prepared by
treatment of 4-hydroxy acetophenone with different substituted benzaldehyde by Claisen-
Schimidt Condensation. In second step, various pyrazoline derivatives were prepared by reflux
reaction of chalcones with nicotinic acid hydrazide in ethanolic solution. Compounds were
confirmed by elemental analyses, IR, 1H NMR and 13C NMR spectral data and were evaluated for
antimalarial and antibacterial activity. Compounds 5n (IC50=0.022 μM for MRC-2 and IC50=0.192
μM for RKL-9) displayed better antiplasmodial activity than the chloroquine (CQ) against
chloroquine-sensitive (MRC-2) and chloroquine-resistant (RKL-9) P. falciparum strains. The in
vitro cytotoxicity study conducted on the human HepG2 cell line (>30μM) and selectivity index
(100-220) indicate that this series presents an interesting selective antiplasmodial profile. Further,
in vitro heme crystallization inhibition assay showed compound 5e inhibited formation of β-
hematin more efficiently than CQ. In addition, antibacterial and antifungal evaluations were
conducted, compounds 5c, 5i and 5j displayed better antibacterial activity against S. aureus, B.
subtilis, E. coli and P. aeruginosa than ciproafloxacin. Antifungal activity of compound 5l against
A. niger (MIC-3.25μg/ml) and C. albicans (MIC-6.5μg/ml) was found to be better than the
standard drug fluconazole.
Keywords: Pyrazolines, Claisen-Schmidt condensation, antimicrobial, antimalarial, antifungal,
cytotoxicity assay, heme crystallization inhibition assay
2

1. Introduction
Malaria continues to present a major health challenge in many resource-limited countries, with an
estimated 212 million cases of malaria worldwide and 429 000 deaths estimated in 20151,2. Most deaths
result from infection with Plasmodium falciparum, although Plasmodium vivax also contributes
substantially to the overall morbidity3. In absence of effective vaccines, treatment of malaria only relies
on chemotherapy4. The current antimalarial chemotherapy has various drawbacks such as drug
resistance, toxicity, and higher cost which heightened alarms about malaria in the international health
community5. Frontline drugs (Chloroquine, mefloquine etc.) are becoming futile due to the emergence
and spread of drug resistance6. The newer chemotherapeutic agents such as artesunate and arteether
suffer with its lower abundance also get trapped by the resistance problem7,8. The dearth of new
affordable drugs has not only intricate the clinical management of malaria in endemic areas, but has
also resulted increased mortality rate. This state emphasizes the need for urgent discovery of new
chemical entities.
Considering all the scaffolds of heterocyclic ring, pyrazoline among the various 5-
membered heterocyclic compound derivatives have received significant attention in the recent years
due to their diverse pharmacological and biological activities such as antifungal9,10
,
antidepressant11,12, anticonvulsant12,13, anti-inflammatory14,15, antibacterial16-18, antitubercular18,
anticancer19-21, human acyl-CoA: cholesterol acyltransferase inhibitors22 and analgesic properties23.
In the last five years there has been an extensive focus of research towards the investigation of
antiplasmodial potential of the pyrazoline ring with promising results24-28. Quite recently, based on
pharmacophore mapping studies Acharya et al.28,29 explored antimalarial potential of 1,3,5-
trisubstituted pyrazoline by targeting haem detoxification pathway of malaria parasite.
In order to carry on with investigation of the antiplasmodial potential of new pyrazoline
derivatives, we focused on evaluation of influence of substitution of position 5 of the pyrazoline
ring toward this activity. All the synthesized compounds were evaluated for in vitro antiplasmodial
activity against chloroquine-sensitive (MRC-2) and chloroquine-resistant (RKL-9) P. falciparum
strains. In order to assess compounds selectivity, cytotoxicity study was performed on human
HepG2 cell line. The compounds were also evaluated for β-hematin formation inhibition activity to
known possible mechanism of action. In parallel, to investigate potential of pyrazoline scaffold in
other pharmacological profile, synthesized compounds have been screened for antimicrobial and
antifungal activity.
3

2. Results and discussion
2.1. Chemistry
The synthesis of 1,3,5-trisubstituted pyrazoline scaffolds were carried out as outlined in
Fig.1. In the first step, chalcones (3a-o) were synthesized by reacting 1-(4-Hydroxy-phenyl) -
ethanone with different substituted aldehydes by the well-known Claisen- Schmidt reaction and
products were purified by crystallization from methanol (60–80% yield). In the second step,
desirable 1,3,5-trisubstituted pyrazolines (5a–o) were prepared by refluxing chalcone and nicotinic
acid hydrazide in n-butanol.
During the synthesis of 1,3,5-trisubstituted pyrazolines, it has been observed that refluxing
time or moreover the rate of the reaction is greatly influenced by the structure and position of the
substituents. It is supposed that the formation of 2-pyrazolines by chalcone takes place via an initial
formation of an aryl hydrazone with a subsequent nucleophilic attack of nitrogen upon the carbon-
carbon double bond at β position. Hence, whatever the factors will improve the electropositive
character of β carbon will ultimately accelerate the rate of the reaction and consequently will reduce
the refluxing time for the 2-pyrazoline formation. Compounds 5h, 5i and 5l took least refluxing
time (8-9 h) in their preparation since they have been formed from halogenated chalcones (3h, 3i
and 3l). Compounds 5a, 5b, 5c, 5m and 5n took 16-18 h for complete conversion from alkylated
chalcones 3a, 3b, 3c, 3m and 3n followed by alkoxy substituted (5d, 5e, 5k and 5o) chalcones which
took 19-22 h for complete conversion into corresponding 2-pyrazolines. Apart from the structure
and position of the substituents, solvent used in the reaction also profoundly affect the course of the
reaction. Lower acidity of the n-butanol compared to other solvent like acetic acid used in
condensation between chalcone and an acid hydrazide, improve the yield quantitatively. It is due to
the alkaline nature of final products which get protonated by the solvent like acetic acid and washed
away by water.
Structures of compounds 3a–o were confirmed by IR, NMR data as well as their distinct Rf
values in TLC analysis. As the chalcones are formed, C=C is introduced adjacent to a carbonyl
group result in delocalization of the π electrons in the C=O and C=C bonds. This conjugation
increases the single bond character of the C=O and C=C bonds in the resonance hybrid and hence
lowers their force constants, resulting in a lowering of the frequencies of carbonyl and double-bond
absorption. Characteristics IR signals in the ranges 1685-1695 cm-1 for C=O and 1645-1655 cm-1
for C=C were observed for chalcones.
Structures of compounds 5a–o were confirmed by IR and NMR techniques. All of the 1,3,5-
trisubstituted pyrazolines possesses a similar basic skeletal structure. As soon as 2-pyrazolines are
4

formed C=O disappeared from the chalcones and pyrazoline ring is formed. Therefore, IR signals
were assigned by comparing the spectra of the products (5a–o) with their corresponding chalcones.
Proton NMR signals were assigned by comparing the spectra of the products (5a–o) with
their corresponding chalcones. Signals around 1.8-1.9 for 2H (CH2) and 4.7-4.9 for 1H, (CH) of
pyrazoline ring were assigned. These signals clearly showing the formation of pyrazoline ring.
Similarly, signal around 155, 40 and 39 were obtained for C3, C4, and C5 respectively, for
pyrazoline ring which further support the ring formation.
Fig 1. Scheme for the synthesis of 1, 3, 5-trisubstituted pyrazolines
5

2.2 Biological evaluation
2.2.1 In vitro antiplasmodial activity
All the synthesized compounds (5a to 5o) were tested in vitro on Chloroquine (CQ) -
sensitive isolates (MRC-2) and CQ-resistant isolates (RKL-9) of P. falciparum malaria parasite. For
each compound, the inhibitory concentration 50% (IC50) was determined, as presented in Table 1.
The inhibitory concentration (IC50) was ranged from 0.02 to 2.5 μM for Chloroquine (CQ) -
sensitive (MRC-2) and 0.19 to 2.9 μM for CQ-resistant (RKL-9) P. falciparum strains. Compounds
5d, 5e, 5m, 5n and 5o of this series have shown better activity than chloroquine (IC50 = 0.05 μM)
against chloroquine sensitive strain (MRC-2) of P. falciparum and except 5m (almost similar to
chloquine) rest four compounds have shown better activity than chloroquine (IC50 = 0.4 μM)
against chloroquine resistant strain (RKL-9) of parasite. Compound 5n was found to be most potent
against sensitive (IC50 = 0.022 μM) as well as resistant (IC50 = 0.192 μM) strains of P. falciparum.
For compound 5n, R4 is propyl, 5m, R4 is isopropyl, 5o R4 is alkoxy, 5e and 5d has methoxy group
at meta, para and ortho, meta position respectively. Interestingly, compounds with para substitution
showed better antiplasmodial activity except those with electron withdrawing groups (compound 5f,
5h, 5i, 5j, and5l) as well as three carbons aliphatic chain (compound 5n) enhances the potency
while branching of alkyl (compound 5m, 5o) and step down of one carbon (compound 5a)
decreases the activity against both strains.
Table 1. In vitro antiplasmodial activity, cell cytotoxicity and selectivity Index of the 1,3,5-
trisubstituted pyrazoline derivatives
S.
IC50 ((μM))a+ SD
Cytotoxicity
IC50 (μM)d
Compound
SIe
MRC-2b
RKL-9c
HO
0.081 + 0.051
0.471 + 0.27
35.39
75.14
5a
N
N
H
O
N
6

HO
0.110 + 0.014
1.650 + 0.114
49.68
30.12
5b
N
N
H
O
N
HO
0.094 + 0.022
0.544 + 0.334
54.54
100.26
5c
N
N
H
O
N
HO
H3CO
OCH3
0.040 + 0.034
0.330 + 0.063
56.76
172
5d
N
N
H
O
N
HO
H3CO
OCH3
0.030 + 0.055
0.251 + 0.054
53.57
213.43
5e
N
N
H
O
N
7

HO
O
0.214 + 0.121
2.550 + 0.051
11.42
4.48
5f
N
N
H
O
N
HO
OCH3
0.063 + 0.019
0.779 + 0.153
44.78
57.48
5g
N
N
H
O
N
HO
Br
0.141 + 0.213
0.970 + 0.166
58.71
60.5
5h
N
N
H
O
N
HO
F
2.500 + 0.220
2.902 + 0.091
52.43
18.07
5i
N
N
H
O
N
HO
NO2
1.566 + 0.051
2.441 + 0.027
18.62
7.63
5j
N
N
H
O
N
8

HO
OCH3
0.059 + 0.010
0.556 + 0.035
42.44
76.33
5k
N
N
H
O
N
HO
Cl
0.240 + 0.043
2.332 + 0.048
47.74
20.47
5l
N
N
H
O
N
HO
0.034 + 0.033
0.022 + 0.015
0.038 + 0.045
0.465 + 0.021
0.192 + 0.024
0.337 + 0.021
38.74
41.56
45.43
83.31
5m
5n
5o
N
N
H
O
N
HO
216.46
N
N
H
O
N
HO
O
138.93
N
N
H
O
N
9

Ref.
Chloroquine
0.050 + 0.031
0.401 + 0.102
27.00
67.33
a Concentration corresponding to 50% growth inhibition of the parasite.
bChloroquine sensitive strain of P. falciparum, IC50, μM+SD, n=2
cChloroquine resistant strain of P. falciparum, IC50, μM+SD, n=2
dCytotoxicity against HepG2 cell line. Values are the mean of one experiment in duplicate
eSI: Selectivity Index (IC50 value of Cytotoxicity activity / IC50 values of antiplasmodial activity
against RKL-9)
Ref. – Reference compound i.e. chloroquine
2.2.2 Cytotoxicity and selectivity index
Cytotoxicity experiments performed on HepG2 cell line and determine their selectivity
indexes so as to validate their real potential as selective antiplasmodial (Table 1). Among the series,
only two compounds 5f (IC50 = 11.42 μM) and 5j (IC50 = 18.62 μM) were found to be more toxic
than chloroquine (IC50 = 27.0 μM) rest thirteen compounds did not displayed cytotoxicity properties
in comparison with the standard chloroquine. Selectivity indexes for CQ-resistant (RKL-9) P.
falciparum strains ranging between 4 and 217. Selectivity ratios of two most potent hit compounds
5e and 5n were 213.43 and 216.46 indicates their selectivity towards antiplasmodial activity.
2.2.3 In vitro Heme Crystallization Inhibition Assay
Compounds were studied for their ability to inhibit the crystallization of hematin to β-
hematin (the synthetic equivalent of hemozoin) in vitro using a colorimetric β-hematin inhibition
assay. The results are given in Table 2. Compound 5a, 5b, 5c, 5m and 5n having alkyl substitutions,
inhibited the formation of β-hematin in the range of 48-81%. Among these compounds 5n inhibited
81% formation of β-hematin which is quite near to CQ (87%) Interestingly, compounds 5f, 5i and
5j which displayed least active in vitro antiplasmodial results also showed very less (<5%)
inhibition of β-hematin formation, this might be due to their inability to accumulate significantly
within the parasite’s digestive vacuole, which is the definitive site of hemoglobin catabolism and
therefore the site of action of hemozoin formation inhibitors. Compounds having alkoxy
substitution, e.g. 5d, 5e, 5g and 5k and 5o significantly inhibited β-hematin formation in the range
of 54-89%. Among these compounds 5e inhibited 89% formation of β-hematin which is more than
CQ (87%). Except a few, most of the synthesized compounds in this study, especially having alkyl
and alkoxy substitutions accumulated significantly within the parasite’s digestive vacuole which
10

indicates, chloroquine and 1,3,5- trisubstituted pyrazolines (5a–o) may have a similar
antiplasmodial mode of action. This finding further strengthens the hypothesis that heme
polymerization inhibition may be a possible mode of action for this series of compounds (5a–o).
Table 2. In vitro heme crystallization inhibition
assay of synthesized compounds 5a-5o
% Inhibition of β-hematin
Comp.
Formation*
53 + 0.24
57 + 0.39
67 + 0.29
71 + 0.67
89 + 0.71
< 5
5a
5b
5c
5d
5e
5f
66 + 0.24
76 + 1.00
< 5
5g
5h
5i
< 5
5j
54 + 0.03
31 + 0.06
48 + 0.54
81 + 0.62
78 + 0.71
87 + 2.65
5k
5l
5m
5n
5o
CQ
*Average of duplicate determinations and equivalents
of compounds (relative to heamatin)
2.5. In vitro antimicrobial activity
All the target compounds were evaluated for their in vitro antimicrobial activity against
Staphylococcus aureus (MTCC 3160) and Bacillus subtilis (MTCC 121) representing Gram-
positive bacteria, and Pseudomonas aeruginosa (MTCC 741) and Escherichia coli (MTCC 51)
representing Gram-negative bacteria. Compounds were also evaluated for their in vitro antifungal
activity against Candida albicans (MTCC 227) and Aspergillus niger (MTCC 8189). The results of
in vitro antibacterial as well as antifungal activities of compounds (5a–5o) are summarized in Table
3.
11

Table 3. In vitro antimicrobial activity of synthesized compounds 5a–5o (MIC in μg/ml)
Compound
S. aureus B. subtilis E. coli P. aeruginosa A. niger C. albicans
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
50
25
12.5
25
50
50
50
12.5
12.5
25
50
50
12.5
3.25
6.5
25
50
25
12.5
25
6.5
6.5
25
25
12.5
6.5
12.5
25
12.5
6.5
12.5
12.5
25
50
25
25
12.5
6.5
12.5
25
25
25
25
12.5
3.25
25
25
12.5
-
100
50
50
75
75
3.25
50
50
12.5
12.5
50
6.5
12.5
25
25
25
6.5
12.5
6.5
50
12.5
3.25
12.5
75
12.5
50
50
75
25
-
25
75
50
3.25
12.5
12.5
50
12.5
-
12.5
12.5
50
25
-
3.25
12.5
6.5
12.5
-
6.5
-
12.5
Ciprofloxacina
Fluconazoleb
25
* Values are mean ± SD of three replicates
aciprofloxacin was used as a positive control against bacteria species
bfluconazole was used as a positive control against fungi species
Compounds 5c, 5h, 5i, 5j, 5m and 5n showed better antimicrobial effects with a MIC value
of 12.5, 6.5, 12.5, 6.5, 12.5 and 12.5 μg/ml respectively, than ciproafloxacin (MIC = 25 μg/ml)
against S. aureus. Similarly, 5b, 5d and 5g showed activity comparable to the standard against S.
aureus. In case of B. subtilis, compounds 5b, 5c, 5h, 5i, 5j and 5l with MIC values in the range
3.25–12.5 μg/ml exhibited better antibacterial activity than all other compounds and standard drug
ciproafloxacin (MIC = 25 μg/ml).
In general, it has been observed that synthesized compounds displayed better antibacterial
performance against the Gram-negative bacteria than the Gram-positive bacteria. Compounds 5b,
5c, 5i, 5j, 5m and 5o showed superior activity with MIC value of 3.25, 6.5, 6.5, 6.5, 3.25 and 6.5
μg/ml than ciproafloxacin (MIC = 12.5 μg/ml) against E. coli. Likewise, 5a, 5g, 5n showed
antibacterial activity equipotent with the standard drug against E. coli. In case of P. aeruginosa, the
compounds 5c, 5g and 5l with MIC values in the range 3.25–12.5 μg/ml exhibited better activity
than all other compounds and the standard drug. Compounds 5b, 5c, 5h, 5i and 5j showed excellent
12

broad spectrum activity in the range 3.25-25 μg/ml concentration, against all the tested Gram-
positive and Gram-negative bacterial strains.
The results of the antifungal activities of synthesized compounds (5a–o) were summarized
in Table 3. In comparison with standard antifungal drug, fluconazole, compounds 5b, 5c, 5g, 5h, 5i,
5j, 5m and 5n showed equivalent inhibitory activity against A. niger and the compounds 5d, 5e, 5f,
5k, 5l and 5o exhibited greater antifungal activity than standard drug against A. niger. Compounds
5e and 5l with MICs 6.5 and 3.25 μg/ml, respectively were most active against A. niger. Similarly,
compounds 5f, 5l and 5o with MICs 3.25, 6.5 and 6.5 μg/ml respectively, showed greater activity
than standard drug against C. albicans. Compounds 5i, 5j and 5m displayed a similar level of
activity than standard drug against C. albicans.
3. Conclusion
A new series of 1,3,5-trisubstituted pyrazolines was prepared in two steps through a simple
and efficient synthesis pathway. Among these fifteen compounds four antiplasmodial hit
compounds were identified from an in vitro screening. Compound 5n was identified as most potent
antiplasmodial hit, displaying an IC50=0.022 μM against CQ-sensitive (MRC-2) and IC50=0.192 μM
against CQ-resistant (RKL-9) P. falciparum strains. The structure activity relationships clearly
show that the substitution at para position with electron releasing groups has great influence on
antiplasmodial activity as compared to electron withdrawing groups. Most of the compounds have
shown safe toxicological profile toward HepG2 cell line and high selectivity indexes compared to
chloroquine indicating as selective antiplasmodial agents. In order to assess the mechanism of
antiplasmodial effect, compounds were also evaluated for in vitro heme crystallization inhibition
assay. Compound 5e (89%) and 5n (81%) significantly inhibit the β-hematin formation.
Additionally, to investigate potential of pyrazoline scaffold in other pharmacological profile,
synthesized compounds have been screened for antimicrobial against a panel of S. aureus, B.
subtilis. E.coli, P. aeruginosa and antifungal activity against a panel of A. niger, C. albicans. These
observations indicated that the 1,3,5-trisubstituted pyrazolines constitute attractive chemical
scaffold for the establishment of new chemical entities with antimalarial potential.
4. Experimental
4.1. General
Melting points were determined in one end open capillary tubes on a Buchi 530 melting
point apparatus and are uncorrected. Infrared (IR) spectra were recorded for the compounds on
13
1
Agilent Cary 630 FTIR Spectrometer in KBr. C and H NMR spectra were recorded on Bruker
13

DRX - 300 instruments in DMSO-d6 at 27C. Chemical shifts are reported in parts per million
(ppm) using tetramethylsilane (TMS) as an internal standard. The coupling constants (J) are given
in Hertz. Spin multiples are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet) and
m (multiplet). Mass spectra were recorded on a JEOL Sx 102/DA-6000 mass spectrometer using
fast atomic bombardment (FAB). Elemental analysis was undertaken with Elemental vario EL III
Carlo Erba 1108 analyzer. The results of elemental analysis (C, H, and N) were within ± 0.4% of
the calculated values. The progress of the reaction and purity of the obtained compounds was
monitored by thin layer chromatography (TLC) on pre-coated silica gel 60F254 (mesh) and a solvent
system by using a mixture of ethyl acetate and hexane (3:7 v/v) for chalcones and using a mixture
of acetone and petroleum ether (40:60 v/v) as mobile phase for 1, 3, 5- trisubstituted pyrazolines.
The spots were detected by exposure to the UV lamp at 254 nm. Overall synthesis of titled 1, 3, 5-
trisubstituted pyrazolines (5a-5o) were performed as outlined in fig.1.
4.2. General procedure for the synthesis of chalcones (3a to 3o)
To the solution of (0.05 mole, 6.8 g) of 1-(4-Hydroxy-phenyl) -ethanone (1) in 30 ml of methanol
on an ice bath, freshly prepared 2 N methanolic NaOH solution (50 ml) was added and stirred for
20 min. To this 0.05 mole of appropriate aldehyde (2a–l) was added and the reaction mixture was
stirred at room temperature for 20-24 h. The reaction mixture was cooled in an ice bath and
neutralized with dilute hydrochloric acid. The obtained precipitate was separated by filtration and
washed with distilled water to give the crude product. The product (3a-o) so obtained was
recrystallized from 75% methanol. The purity of the products was checked on TLC by using a
mixture of ethyl acetate and hexane as mobile phase.
4.2.1. 3-(2-Ethyl-phenyl)-1-(4-hydroxy-phenyl)-propenone (3a)
Prepared by the above method from 2a (0.05 mole, 6.6 ml) and 1 (0.05 mole, 6.8 g); yield: 72% as
pale yellow crystalline solid; Rf = 0.64 in EtOAc/hexane, 3:7; mp: 202–204 oC. FTIR (neat) νmax
3085 (CH, aromatic), 767 (CH, substituted phenyl ring), 1686 (C=O), 1640 (C=C, conjugated to
C=O), 1590 (
, aromatic), 2961–2835 (CH), 1443 (CH); 1H NMR (300 MHz, d): 5.1(s, 1H,
:
C
C str
Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J = 7.3 Hz), 8.25 (d, 1H, 1-
ethylene, J = 6.4 Hz), 7.1 (m, 3H, ArH), 7.25 (d, 1H, ArH, J = 6.9 Hz), 2.55 (m, 2H, methylene),
1.25 (t, 3H, methyl, J = 7.4 Hz).
4.2.2. 1-(4-Hydroxy-phenyl) -3-(2-isopropyl-phenyl) -propenone (3b)
14

Prepared by the above method from 2b (0.05 mole, 7.6 ml) and 1 (0.05 mole, 6.8 g); yield: 63% as
off white crystalline solid; Rf = 0.66 in EtOAc/hexane, 3:7; mp: 199–201 oC. FTIR (neat) νmax: 3094
(CH, aromatic), 765(CH, substituted phenyl ring), 1689 (C=O), 1642 (C=C, conjugated to C=O),
1
1584 (
, aromatic ring), 2956–2835 (C-H), 1444(C-H def); H NMR (300 MHz, d): 5.1(s,
C
C str
1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J = 7 Hz), 8.25 (d, 1H,
1-ethylene, J = 6.5 Hz), 7.08 (dd, 2H, ArH, J = 5.5 Hz, J = 6.2 Hz), 7.03 (s, 1H, ArH), 7.22(d, 1H,
ArH, J = 6.7 Hz), 3.10 (m, 1H, methine), 1.3 (dd, 6H, methyl, J = 7.1 Hz, J = 8 Hz).
4.2.3. 1-(4-Hydroxy-phenyl) -3-(2,4,6-trimethyl-phenyl) -propenone (3c)
Prepared by the above method from 2c (0.05 mole, 7.4 ml) and 1 (0.05 mole, 6.8 g); yield: 65% as
yellow crystalline solid; Rf = 0.62 in EtOAc/hexane, 3:7; mp: 246–248 oC. FTIR (neat) νmax: 3083
(C-H, aromatic ring), 776 (C-H, substituted phenyl ring), 1688 (C=O), 1641 (C=C, conjugated to
1
C=O), 1578 (
aromatic ring), 2960–2848 (C-H) 1437 (C-H def); H NMR (300 MHz, d):
C
C str
5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J = 6.6 Hz), 8.25
(d, 1H, 1-ethylene, J = 7.1 Hz), 2.35 (s, 9H, methyl), 6.65 (d, 2H, ArH, J = 6.4 Hz).
4.2.4. 3-(2,5-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propenone (3d)
Prepared by the above method from 2d (0.05 mole, 8.3 g) and 1 (0.05 mole, 6.8 g); yield: 69% as
light brown crystalline solid; Rf = 0.55 in EtOAc/hexane, 3:7; mp: 211-213 oC. FTIR (neat) νmax
:
3085 (C-H, aromatic ring), 767 (C-H, substituted phenyl ring), 1689 (C=O), 1638 (C=C, conjugated
1
to C=O), 1579 (
aromatic ring), 2962–2843 (C-H), 1447 (C-H def); H NMR (300 MHz,
C
C str
d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J = 6.4 Hz),
8.25 (d, 1H, 1-ethylene, J = 7.2 Hz), 3.70 (s, 6H, methoxy), 6.65 (m, 3H, ArH).
4.2.5. 3-(3,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propenone (3e)
Prepared by the above method from 2e (0.05 mole, 8.3 g) and 1 (0.05 mole, 6.8 g); yield: 70% as
pale yellow crystalline solid; Rf = 0.52 in EtOAc/hexane, 3:7; mp: 219-221 oC. FTIR (neat) νmax
:
3095 (C-H, aromatic ring), 764 (C-H, substituted phenyl ring), 1690 (C=O), 1642 (C=C, conjugated
1
to C=O), 1580 (
C
C str of aromatic ring), 2970–2834 (C-H), 1433 (C-H def); H NMR (300
MHz, d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J = 6.5
Hz), 8.25 (d, 1H, 1-ethylene, J = 7.2 Hz), 3.75 (s, 6H, methoxy), 6.65 (m, 3H, ArH).
4.2.6. 3-(4-Benzyloxy-phenyl)-1-(4-hydroxy-phenyl)-propenone (3f)
15

Prepared by the above method from 2f (0.05 mole, 10.6 g) and 1 (0.05 mole, 6.8 g); yield: 66% as
yellow crystalline solid; Rf = 0.47 in EtOAc/hexane, 3:7; mp: Decomposed at 232 oC. FTIR (neat)
νmax: 3094 (C-H, aromatic ring), 771 (C-H, substituted phenyl ring), 1692 (C=O), 1639 (C=C,
1
conjugated to C=O), 1589 (
aromatic ring), 2964–2825 (C-H), 1446 (C-H def); H NMR
C
C str
(300 MHz, d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J
= 6.3 Hz), 8.25 (d, 1H, 1-ethylene, J = 7.2 Hz), 5.20 (s, 2H, methylene), 7.25 (m, 5H, ArH).
4.2.7. 1-(4-Hydroxy-phenyl)-3-(4-methoxy-3-methyl-phenyl)-propenone (3g)
Prepared by the above method from 2g (0.05 mole, 7.3 g) and 1 (0.05 mole, 6.8 g); yield: 76% as
yellow amorphous solid; Rf = 0.52 in EtOAc/hexane, 3:7; mp: 219-221 oC. FTIR (neat) νmax: 3095
(C-H, aromatic ring), 764 (C-H, substituted phenyl ring), 1690 (C=O), 1642 (C=C, conjugated to
C=O), 1580 (
, aromatic ring), 2970–2834 (C-H), 1433 (C-H def); 1H NMR (300 MHz, d):
C C str
5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J = 6.5 Hz), 8.25
(d, 1H, 1-ethylene, J = 7.3 Hz), 3.7 (s, 3H, methoxy), 2.4 (s, 3H, methyl), 6.6 (s, 1H, ArH), 7.0 (m,
2H, ArH).
4.2.8. 3-(4-Bromo-phenyl)-1-(4-hydroxy-phenyl)-propenone (3h)
Prepared by the above method from 2h (0.05 mole, 9.3 g) and 1 (0.05 mole, 6.8 g); yield: 62% as
off white crystalline solid; Rf = 0.48 in EtOAc/hexane, 3:7; mp: 174–175 oC. FTIR (neat) νmax: 3095
(C-H, aromatic ring), 765 (C-H, substituted phenyl ring), 1693 (C=O), 1640 (C=C, conjugated to
1
C=O), 1584 (
aromatic ring), 2957–2828 (C-H), 1447 (C-H def); H NMR (300 MHz, d):
C
C str
5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J = 6.4 Hz), 8.25
(d, 1H, 1-ethylene, J = 7.3 Hz), 7.20 (m, 2H, ArH), 7.4 (m, 2H, ArH).
4.2.9. 3-(4-Fluoro-phenyl)-1-(4-hydroxy-phenyl)-propenone (3i)
Prepared by the above method from 2i (0.05 mole, 6.6 mL) and 1 (0.05 mole, 6.8 g); yield: 68% as
pale yellow crystalline solid; Rf = 0.45 in EtOAc/hexane, 3:7; mp: 181-183 oC. FTIR (neat) νmax
3094 (C-H, aromatic ring), 759 (C-H, substituted phenyl ring), 1690 (C=O), 1641 (C=C, conjugated
to C=O), 1592 (
C str, aromatic ring), 2960–2810 (C-H), 1444 (C-H def); 1H NMR (300 MHz,
:
C
d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J = 6.5 Hz),
8.25 (d, 1H, 1-ethylene, J = 7.5 Hz), 7.30 (m, 2H, ArH), 7.0 (m, 2H, ArH).
4.2.10. 1-(4-Hydroxy-phenyl)-3-(4-nitro-phenyl)-propenone (3j)
16

Prepared by the above method from 2j (0.05 mole, 7.6 g) and 1 (0.05 mole, 6.8 g); yield: 74% as
yellow crystalline solid; Rf = 0.49 in EtOAc/hexane, 3:7; mp: 222–223 oC. FTIR (neat) νmax: 3094
(C-H, aromatic ring), 765 (C-H, substituted phenyl ring), 1688 (C=O), 1643 (C=C, conjugated to
1
C=O), 1585 (
aromatic ring), 2945–2823 (C-H), 1456 (C-H def); H NMR (300 MHz, d):
C
C str
5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J = 6.6 Hz), 8.25
(d, 1H, 1-ethylene, J = 7.6 Hz), 7.55 (m, 2H, ArH), 8.2 (m, 2H, ArH).
4.2.11. 1-(4-Hydroxy-phenyl)-3-(4-methoxy-phenyl)-propenone (3k)
Prepared by the above method from 2k (0.05 mole, 6.1 ml) and 1 (0.05 mole, 6.8 g); yield: 70% as
straw yellow amorphous solid; Rf = 0.53 in EtOAc/hexane, 3:7; mp: 217–218oC. FTIR (neat) νmax
3103 (C-H, aromatic ring), 766 (C-H, substituted phenyl ring), 1695 (C=O), 1640 (C=C, conjugated
to C=O), 1589 (
, aromatic ring), 2972–2848 (C-H), 1432 (C-H def); 1H NMR (300 MHz,
:
C
C str
d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J = 6.5 Hz),
8.25 (d, 1H, 1-ethylene, J = 7.8 Hz), 7.2 (m, 2H, ArH), 6.7 (m, 2H, ArH), 3.7 (s, 3H, methoxy).
4.2.12. 3-(2-Chloro-phenyl)-1-(4-hydroxy-phenyl)-propenone (3l)
Prepared by the above method from 2l (0.05 mole, 5.6 ml) and 1 (0.05 mole, 6.8 g); yield: 79% as
pale yellow amorphous solid; Rf = 0.56 in EtOAc/hexane, 3:7; mp: 229–231 oC. FTIR (neat) νmax
3097 (C-H, aromatic ring), 775 (C-H, substituted phenyl ring), 1691 (C=O), 1642 (C=C, conjugated
to C=O), 1586 (
, aromatic ring), 2961–2834 (C-H), 1448 (C-H def); 1H NMR (300 MHz,
:
C
C str
d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.40 (d, 1H, 1-ethylene, J = 6.9 Hz),
8.25 (d, 1H, 1-ethylene, J = 7.5 Hz), 7.20 (m, 2H, ArH), 7.0 (m, 2H, ArH).
4.2.13. 1-(4-Hydroxy-phenyl)-3-(4-isopropyl-phenyl)-propenone (3m)
Prepared by the above method from 2m (0.05 mole, 7.5 g) and 1 (0.05 mole, 6.8 g); yield: 62% as
yellow amorphous solid; Rf = 0.66 in EtOAc/hexane, 3:7; mp: decomposed at 248 oC. FTIR (neat)
νmax: 3095 (C-H, aromatic ring), 764 (C-H, substituted phenyl ring), 1690 (C=O), 1642 (C=C,
conjugated to C=O), 1580 (
, aromatic ring), 2970–2834 (C-H), 1433 (C-H def); 1H NMR
C C str
(300 MHz, d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.45 (d, 1H, 1-ethylene, J
= 6 Hz), 8.25 (d, 1H, 1-ethylene, J = 7.2 Hz), 3.1 (dd, 6H, methyl, J = 5.4 Hz, J = 7.4 Hz), 1.3 (m,
1H, methine), 7.2 (m, 2H, ArH), 7.0 (m, 2H, ArH).
4.2.14. 1-(4-Hydroxy-phenyl)-3-(4-propyl-phenyl)-propenone (3n)
Prepared by the above method from 2n (0.05 mole, 7.3 g) and 1 (0.05 mole, 6.8 g); yield: 73% as
yellow amorphous crystalline solid; Rf = 0.59 in EtOAc/hexane, 3:7; mp: 206-208 oC. FTIR (neat)
17

νmax: 3095 (C-H, aromatic ring), 764 (C-H, substituted phenyl ring), 1690 (C=O), 1642 (C=C,
conjugated to C=O), 1580 (
, aromatic ring), 2970–2834 (C-H), 1433 (C-H def); 1H NMR
C C str
(300 MHz, d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J
= 6.5 Hz), 8.25 (d, 1H, 1-ethylene, J = 7.5 Hz), 2.6 (t, 2H, methylene, J = 6.1 Hz), 1.7 (m, 2H,
methylene), 1.1 (t, 3H, methyl, J = 8.2 Hz), 7.2 (m, 2H, ArH), 7.0 (m, 2H, ArH).
4.2.15. 1-(4-Hydroxy-phenyl)-3-(4-isopropoxy-phenyl)-propenone (3o)
Prepared by the above method from 2o (0.05 mole, 7.9 g) and 1 (0.05 mole, 6.8 g); yield: 70% as
pale yellow amorphous solid; Rf = 0.49 in EtOAc/hexane, 3:7; mp: 233-234 oC. FTIR (neat) νmax
3095 (C-H, aromatic ring), 764 (C-H, substituted phenyl ring), 1690 (C=O), 1642 (C=C, conjugated
to C=O), 1580 (
, aromatic ring), 2970–2834 (C-H), 1433 (C-H def); 1H NMR (300 MHz,
:
C C str
d): 5.1(s, 1H, Ar-OH), 6.9 (m, 2H, ArH), 7.55 (m, 2H, ArH), 7.44 (d, 1H, 1-ethylene, J = 6.6 Hz),
8.25 (d, 1H, 1-ethylene, J = 7.4 Hz), 4.0 (m, 1H, methine), 1.4 (dd, 6H, methyl, J = 5.6 Hz, J = 6.1
Hz), 7.2 (m, 2H, ArH), 6.7 (m, 2H, ArH).
4.3. General procedure for the synthesis of 1, 3, 5- trisubstituted pyrazolines (5a-5o)
To the solution of (4 mmole) of appropriate chalcone (3a–o) in 10 ml of n-butanol, nicotinic
acid hydrazide (4) (4 mmole, 0.55 g) was added and the reaction mixture was refluxed for 8–22 h.
Progress of the reaction was monitored in every 60 min interval on silica coated TLC plates using a
mixture of acetone and petroleum ether (40:60 V/V) as mobile phase. The excess of solvent was
removed under reduced pressure and the reaction mixture was cooled in an ice bath. The product
precipitated out at low temperature was washed with distilled water, reconstituted in a minimum
amount of methanol and dried under reduced pressure. All the 1, 3, 5 – trisubstituted pyrazolines
(compounds 5a-5o) were synthesized by this method. Corresponding reflux time is given to individual
products.
4.3.1.[5-(2-Ethyl-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-methanone
(5a)
Prepared by the above method from 3a (4 mmole, 1.01 g) and 4 (4 mmole, 0.55 g) after 17h reflux;
yield: 87% as shiny white amorphous solid; mp: 188-190 oC. FTIR (neat) νmax: 3087 (C-H, aromatic
ring), 765 (C-H, substituted phenyl ring), 1667 (C=O), 1591 (
C
C str, aromatic ring), 2960–2836
(C-H), 1440 (C-H def), 1609 (C=N), 3334 (C-N); 1H NMR (300 MHz, d): 7.0-7.1(m, 4H, ArH), 2.7
(m, 2H, methylene), 1.2 (m, 3H, methyl CH), 5.1(s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H,
18

ArH), 1.8 (m, 2H, CH2 pyrazoline), 4.9 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-
13
H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 124, 130.4, 115.8, 159.6, 116
and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.6, 40 and 39.2 (C3, C4, and C5), 168 (1C, C=O),
129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 142, 138.7, 127.7, 126.4, 125.5 and
127.1 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’), 22.4 (1C, methylene), 16.6 (1C, methyl); ESI-MS (m/z):
371.16 (M+); Anal. Calcd for C23H21N3O2: C, 74.37; H, 5.70; N, 11.31. Found: C, 74.31; H, 5.65;
N, 11.27
4.3.2.[3-(4-Hydroxy-phenyl)-5-(2-isopropyl-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5b)
Prepared by the above method from 3b (4 mmole, 1.06 g) and 4 (4 mmole, 0.55 g) after 18h reflux;
yield: 81% as white crystalline solid; mp: 154-155 oC. FTIR (neat) νmax: 3092 (C-H, aromatic ring),
761(C-H, substituted phenyl ring), 1670 (C=O), 1580 (
, aromatic ring), 2960–2830 (C-H),
C str
C
1442 (C-H def), 1605 (C=N), 3335 (C-N); 1H NMR (300 MHz, d): 7.05 (m, 4H, ArH), 3.1 (m, 1H,
methine), 1.3 (dd, 6H, methyl CH, J = 6.5 Hz, J = 8 Hz), 5.0 (s, 1H, Ar-OH), 7.4 (m, 2H, ArH), 6.8
(m, 2H, ArH), 2.0 (m, 2H, CH2 pyrazoline), 4.8 (m, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85
13
(s, 1H, Cd-H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 125, 130.4, 115.8,
159.6, 115.8 and 129.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.6, 40.2 and 40 (C3, C4, and C5), 168.2
(1C, C=O), 129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 140.1, 147.3, 126.1, 126.2,
126 and 126.8 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’), 25.5 (1C, methine), 24.7 (2C, methyl); ESI-MS
(m/z): 385.18 (M+); Anal. Calcd for C24H23N3O2: C, 74.78; H, 6.01; N, 10.90. Found: C, 74.72; H,
5.99; N, 10.87
4.3.3. [3-(4-Hydroxy-phenyl)-5-(2,4,6-trimethyl-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5c)
Prepared by the above method from 3c (4 mmole, 1.06 g) and 4 (4 mmole, 0.55 g) after 16h reflux;
o
yield: 90% as off white amorphous solid; mp: 166-168 C. FTIR (neat) νmax: 3081 (C-H, aromatic
ring), 778 (C-H, substituted phenyl ring), 1667 (C=O), 1580 (
C
C str, aromatic ring), 2965–2846
(C-H) 1437 (C-H def) 1620 (C=N) 3343 (C-N); 1H NMR (300 MHz, d): 2.45 (m, 9H, C-H methyl),
6.6 (m, 2H, ArH), 5.2 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.6 (m, 2H, ArH), 1.9 (m, 2H, CH2
pyrazoline), 4.9 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-H), 7.60 (s, 1H, Ce-H),
13
8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 123.8, 130.4, 115.8, 158.6, 115.8 and 130.4 (C1’, C2’,
C3’, C4’, C5’ and C6’), 155.6, 40.5 and 32.4 (C3, C4, and C5), 168 (1C of C=O), 129.6, 152.5, 148.3,
124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 17.9 (2C, methyl on C2’’ and C6’’), 21.0 (1C, methyl on C4’’),
19

141, 136, 127.4, 135.5, 127 and 136 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’); ESI-MS (m/z): 385.18 (M+);
Anal. Calcd for C24H23N3O2: C, 74.78; H, 6.01; N, 10.90. Found: C, 74.7; H, 6.0; N, 10.87.
4.3.4.
[5-(2,5-Dimethoxy-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5d)
Prepared by the above method from 3d (4 mmole, 1.14 g) and 4 (4 mmole, 0.55 g) after 21h reflux;
yield: 78% as dark brown solid; mp: 212-214 oC. FTIR (neat) νmax: 3090 (C-H, aromatic ring), 763
(C-H, substituted phenyl ring), 1664 (C=O), 1577 (
, aromatic ring), 2960–2840 (C-H),
C str
C
1
1446 (C-H def), 1615 (C=N), 3330 (C-N); H NMR (300 MHz, d): 3.7 (s, 6H, C-H methoxy), 6.6
(s, 1H, ArH), 6.3 (s, 1H, ArH), 6.5 (s, 1H, ArH), 5.0 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H,
ArH), 1.7 (m, 2H, CH2 pyrazoline), 4.9 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-
13
H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 124, 130.4, 115.8, 159.6, 116
and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.6, 40 and 35.8 (C3, C4, and C5), 168 (1C of C=O),
129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 129, 152.8, 114.6, 113.1, 154 and 113.6
(C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’), 56.3 (1C, methoxy on C2’’), 56.0 (1C, methoxy on C5’’); ESI-MS
(m/z): 403.15 (M+); Anal. Calcd for C23H21N3O4: C, 68.47; H, 5.25; N, 10.42. Found: C, 68.44; H,
5.21; N, 10.4
4.3.5.
[5-(3,4-Dimethoxy-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5e)
Prepared by the above method from 3e (4 mmole, 1.14 g) and 4 (4 mmole, 0.55 g) after 22h reflux;
yield: 91% as mud brown solid; mp: 202-204 oC. FTIR (neat) νmax: 3098 (C-H, aromatic ring), 761
(C-H, substituted phenyl ring), 1672 (C=O), 1581 (
, aromatic ring), 2970–2833 (C-H),
C str
C
1432 (C-H def), 1622 (C=N), 3341 (C-N); 1H NMR (300 MHz, d): 3.73 (s, 6H, C-H methoxy), 6.5
(s, 1H, ArH), 6.66 (s, 1H, ArH), 6.57 (s, 1H, ArH), 5.0 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.9 (m,
2H, ArH), 1.9 (m, 2H, CH2 pyrazoline), 4.8 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H,
13
Cd-H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 123.8, 130.4, 116, 161.6,
115.8 and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.6, 39.9 and 45.7 (C3, C4, and C5), 168.3 (1C of
C=O), 129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 135.8, 113.6, 147.1, 145.6, 114.6
and 120.3 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’), 56.9 (2C, methoxy on C3’’ and C4’’); ESI-MS (m/z):
403.15 (M+); Anal. Calcd for C23H21N3O4: C, 68.47; H, 5.25; N, 10.42. Found: C, 68.43; H, 5.22;
N, 10.4
20

4.3.6.
[5-(4-Benzyloxy-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5f)
Prepared by the above method from 3f (4 mmole, 1.32 g) and 4 (4 mmole, 0.55 g) after 17h reflux;
o
yield: 93% as brownish yellow amorphous solid; mp: 178-180 C. FTIR (neat) νmax: 3090 (C-H,
aromatic ring), 767 (C-H, substituted phenyl ring), 1671 (C=O), 1587 (
, aromatic ring),
C str
C
2960–2820 (C-H), 1440 (C-H def), 1612 (C=N), 3344 (C-N); 1H NMR (300 MHz, d): 7.0 (m, 2H,
ArH), 6.8 (m, 2H, ArH), 5.2 (s, 2H, methylene), 7.6 (m, 5H, Cb’’-H, Cc’’-H, Cd’’-H, Ce’’-H and Cf’’-
H), 5.0 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H, ArH), 1.7 (m, 2H, CH2 pyrazoline), 4.9 (s,
1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H);
13C NMR (300 MHz, d): 122.8, 130.4, 115.8, 159.6, 116.6 and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’),
155.6, 38.5 and 45.4 (C3, C4, and C5), 167.6 (1C of C=O), 129.6, 152.5, 148.3, 124.9 and 137 (Ca,
Cb, Cc, Ce and Cf), 134.8, 128.0, 113.6, 160, 113.6 and 128 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’), 78
(1C of –OCH2– ), 141, 127.3, 128.7, 127.4, 129 and 127.3 (Ca’, Cb’, Cc’, Cd’, Ce’ and Cf’); ESI-MS
(m/z): 449.17 (M+); Anal. Calcd for C28H23N3O3: C, 74.82; H, 5.16; N, 9.35. Found: C, 74.8; H,
5.13; N, 9.34.
4.3.7. [3-(4-Hydroxy-phenyl)-5-(4-methoxy-3-methyl-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-
3-yl-methanone (5g)
Prepared by the above method from 3g (4 mmole, 1.07 g) and 4 (4 mmole, 0.55 g) after 18h reflux;
o
yield: 81% as pale yellow solid; mp: 203-205 C. FTIR (neat) νmax: 3099 (C-H, aromatic ring), 768
(C-H, substituted phenyl ring), 1666 (C=O), 1574 (
, aromatic ring), 2950–2810 (C-H),
C str
C
1445 (C-H def), 1605 (C=N), 3335 (C-N); 1H NMR (300 MHz, d): 2.35 (s, 3H, methyl), 3.7 (s, 3H,
methoxy), 6.6-6.8 (m, 3H, ArH), 5.0 (s, 1H, aromatic C-OH), 6.79 (m, 2H, ArH), 7.36 (m, 2H,
ArH), 1.85 (m, 2H, CH2 pyrazoline), 4.8 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H,
13
Cd-H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 123.8, 130.4, 115, 159.6,
115.8 and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.7, 37.8 and 39.6 (C3, C4, and C5), 168.9 (1C of
C=O), 129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 12.3 (1C, methyl), 56.5 (1C,
methoxy), 134.9, 126, 128.4, 127.7, 128.7 and 126 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’); ESI-MS
(m/z): 387.16 (M+); Anal. Calcd for C23H21N3O3: C, 71.30; H, 5.46; N, 10.85. Found: C, 71.24; H,
5.40; N, 10.81.
4.3.8.
[5-(4-Bromo-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5h)
21

Prepared by the above method from 3h (4 mmole, 1.21 g) and 4 (4 mmole, 0.55 g) after 9h reflux;
yield: 84% as brownish yellow solid; mp: 146-148 oC. FTIR (neat) νmax: 3092 (C-H, aromatic ring),
761 (C-H, substituted phenyl ring), 1670 (C=O), 1582 (
, aromatic ring), 2955–2825 (C-H),
C str
C
1440 (C-H def), 1612 (C=N), 3344 (C-N); 1H NMR (300 MHz, d): 7.07 (m, 2H, ArH), 7.5 (m, 2H,
ArH), 5.0 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H, ArH), 1.8 (m, 2H, CH2 pyrazoline), 4.8 (s,
1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H);
13C NMR (300 MHz, d): 123.8, 130.4, 115.8, 159.6, 115.8 and 131 (C1’, C2’, C3’, C4’, C5’ and C6’),
155.6, 41 and 45.4 (C3, C4, and C5), 168 (1C of C=O), 129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb,
Cc, Ce and Cf), 141.5, 129.2, 131.6, 121, 131.6 and 129.2 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’); ESI-MS
(m/z): 421.04 (M+); Anal. Calcd for C21H16BrN3O2: C, 59.73; H, 3.82; N, 9.95. Found: C, 59.7; H,
3.81; N, 9.92.
4.3.9.
[5-(4-Fluoro-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5i)
Prepared by the above method from 3i (4 mmole, 0.97 g) and 4 (4 mmole, 0.55 g) after 8h reflux;
o
yield: 82% as off white amorphous solid; mp: 230-232 C. FTIR (neat) νmax: 3090 (C-H, aromatic
ring), 765 (C-H, substituted phenyl ring), 1675 (C=O), 1588 (
, aromatic ring), 2955–2810
C str
C
1
(C-H), 1441 (C-H def), 1615 (C=N), 3336 (C-N); H NMR (300 MHz, d): 7.4 (m, 2H, ArH), 6.7
(m, 2H, ArH), 5.5 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H, ArH), 2.0 (m, 2H, CH2
pyrazoline), 4.9 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-H), 7.60 (s, 1H, Ce-H),
13
8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 123.8, 133.4, 115.8, 159.6, 120.1 and 130.4 (C1’, C2’,
C3’, C4’, C5’ and C6’), 155.6, 39.9 and 45.4 (C3, C4, and C5), 167.9 (1C of C=O), 129.6, 152.5, 148.3,
124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 138, 128.6, 115, 160, 115.3 and 128.6 (C1’’, C2’’, C3’’, C4’’,
C5’’ and C6’’); ESI-MS (m/z): 361.12 (M+); Anal. Calcd for C21H16FN3O2: C, 69.80; H, 4.46; N,
11.63. Found: C, 69.76; H, 4.42; N, 11.6.
4.3.10.
[3-(4-Hydroxy-phenyl)-5-(4-nitro-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5j)
Prepared by the above method from 3j (4 mmole, 1.08 g) and 4 (4 mmole, 0.55 g) after 14h reflux;
o
yield: 80% as brownish yellow amorphous solid; mp: 224-226 C. FTIR (neat) νmax: 3094 (C-H,
aromatic ring), 767 (C-H, substituted phenyl ring), 1679 (C=O), 1587 (
C
C str, aromatic ring),
2945–2823 (C-H), 1450 (C-H def), 1615 (C=N), 3335 (C-N); 1H NMR (300 MHz, d): 7.38 (m, 2H,
ArH), 8.14 (m, 2H, ArH), 5.0 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H, ArH), 1.7 (m, 2H, CH2
pyrazoline), 4.7 (s, 1H, CH of pyrazoline ring), 9.10 (s, 1H, Cb-H of 4-pyridine), 8.85 (s, 1H, Cd-H),
22

13
7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 123.8, 130.4, 115.8, 159.6, 115.8
and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.6, 39.9 and 45.4 (C3, C4, and C5), 168 (1C of C=O),
129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 148.6, 127.9, 123.1, 146.4, 123.1 and
127.9 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’); ESI-MS (m/z): 388.12 (M+); Anal. Calcd for C21H16N4O4:
C, 64.94; H, 4.15; N, 14.43. Found: C, 64.90; H, 4.11; N, 14.39.
4.3.11.
[3-(4-Hydroxy-phenyl)-5-(4-methoxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5k)
Prepared by the above method from 3k (4 mmole, 1.02 g) and 4 (4 mmole, 0.55 g) after 19h reflux;
yield: 84% as white amorphous solid; mp: 206-207 oC. FTIR (neat) νmax: 3099 (C-H, aromatic ring),
764 (C-H, substituted phenyl ring), 1675 (C=O), 1586 (
, aromatic ring), 2974–2838 (C-H),
C str
C
1437 (C-H def), 1627 (C=N), 3338 (C-N); 1H NMR (300 MHz, d): 3.73 (s, 3H, methoxy), 6.95 (m,
2H, ArH), 6.7 (m, 2H, ArH), 5.0 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.9 (m, 2H, ArH), 1.9 (m, 2H,
CH2 pyrazoline), 4.8 (s, 1H, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H, Cd-H), 7.60 (s, 1H, Ce-
13
H), 8.35 (s, 1H, Cf-H); C NMR (300 MHz, d): 123.6, 130.4, 115.8, 159.7, 115.8 and 130.4 (C1’,
C2’, C3’, C4’, C5’ and C6’), 155.6, 39.9 and 45.7 (C3, C4, and C5), 168.3 (1C of C=O), 129.6, 152.5,
148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 134.7, 128.8, 113.4, 161, 113.4 and 128.8 (C1’’, C2’’,
C3’’, C4’’, C5’’ and C6’’), 56.9 (2C, methoxy on C3’’ and C4’’); ESI-MS (m/z): 373.14 (M+); Anal.
Calcd for C22H19N3O3: C, 70.76; H, 5.13; N, 11.25. Found: C, 70.72; H, 5.10; N, 11.24.
4.3.12.
[5-(2-Chloro-phenyl)-3-(4-hydroxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5l)
Prepared by the above method from 3l (4 mmole, 1.03 g) and 4 (4 mmole, 0.55 g) after 9.5 h reflux;
o
yield: 76% as brown amorphous solid; mp: 162-164 C. FTIR (neat) νmax: 3097 (C-H, aromatic
ring), 775 (C-H, substituted phenyl ring), 1677 (C=O), 1587 (
C
C str, aromatic ring), 2959–2830
(C-H), 1448 (C-H def), 1611 (C=N), 3338 (C-N); 1H NMR (300 MHz, d): 7.02 (s, 1H, ArH), 7.07
(s, 1H, ArH), 7.0 (s, 1H, ArH), 7.2 (s, 1H, ArH), 5.5 (s, 1H, Ar-OH), 6.8 (m, 2H, ArH), 7.4 (m, 2H,
ArH), 2.0 (m, 2H, CH2 pyrazoline), 4.9 (s, 1H, m, CH pyrazoline), 9.10 (s, 1H, Cb-H), 8.85 (s, 1H,
Cd-H), 7.60 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); 13C NMR (300 MHz, d): 123.8, 133.4, 115.8, 159.6,
120.1 and 130.4 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.6, 39.9 and 45.4 (C3, C4, and C5), 167.9 (1C of
C=O), 129.6, 152.5, 148.3, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf), 142.8, 128.5, 126.4, 127.9, 128.7
and 132.4 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’); ESI-MS (m/z): 377.09 (M+); Anal. Calcd for
C21H16ClN3O2: C, 66.76; H, 4.27; N, 11.12. Found: C, 66.72; H, 4.23; N, 11.10.
23

4.3.13.
[3-(4-Hydroxy-phenyl)-5-(4-isopropyl-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5m)
Prepared by the above method from 3m (4 mmole, 1.06 g) and 4 (4 mmole, 0.55 g) after 17h reflux;
yield: 81% as off white amorphous solid; mp: 184-185 oC. FTIR (neat) νmax: 3096 (C-H, aromatic
ring), 760 (C-H, substituted phenyl ring), 1670 (C=O), 1585 (
, aromatic ring), 2975–2840
C C str
(C-H), 1435 (C-H def), 1620 (C=N), 3340 (C-N); 1H NMR (300 MHz, d): 1.25 (dd, 6H, methyl, J =
9 Hz, J = 7.6 Hz), 3.1 (s, 1H, methine), 7.05 (m, 2H, ArH), 7.1 (m, 2H, ArH), 5.0 (s, 1H, Ar-OH),
6.7 (m, 2H, ArH), 7.9 (m, 2H, ArH), 1.9 (m, 2H, CH2 pyrazoline), 4.9 (s, 1H, CH pyrazoline), 9.15
(s, 1H, Cb-H), 8.85 (s, 1H, Cd-H), 7.65 (s, 1H, Ce-H), 8.35 (s, 1H, Cf-H); 13C NMR (300 MHz, d):
123.4, 130.5, 115.8, 159.5, 115.8 and 130.5 (C1’, C2’, C3’, C4’, C5’ and C6’), 155.5, 39.6 and 45.0
(C3, C4, and C5), 168.6 (1C of C=O), 129.6, 152.6, 148.5, 124.9 and 137 (Ca, Cb, Cc, Ce and Cf),
139.7, 127.0, 126.4, 146.5, 126.4 and 127.0 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’), 31.9 (1C, methine),
24.5 (2C, methyl); ESI-MS (m/z): 385.18 (M+); Anal. Calcd for C24H23N3O2: C, 74.78; H, 6.01; N,
10.90. Found: C, 74.72; H, 6.0; N, 10.71.
4.3.14.
[3-(4-Hydroxy-phenyl)-5-(4-propyl-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5n)
Prepared by the above method from 3n (4 mmole, 1.06 g) and 4 (4 mmole, 0.55 g) after 17.5h
o
reflux; yield: 78% as off white amorphous solid; mp: 196-197 C. FTIR (neat) νmax: 3090 (C-H,
aromatic ring), 765 (C-H, substituted phenyl ring), 1678 (C=O), 1584 (
, aromatic ring),
C C str
1
2970–2830 (C-H), 1435 (C-H def), 1625 (C=N), 3330 (C-N); H NMR (300 MHz, d): 1.1 (t, 3H,
methyl), 1.7 (m, 2H, methylene), 2.5 (m, 2H, C-H methylene), 6.95 (m, 2H, ArH), 7.0 (m, 2H,
ArH), 5.1 (s, 1H, Ar-OH), 6.75 (m, 2H, ArH), 7.85 (m, 2H, ArH), 1.95 (m, 2H, CH2 pyrazoline),
4.85 (s, 1H, CH pyrazoline), 9.15 (s, 1H, Cb-H), 8.8 (s, 1H, Cd-H), 7.65 (s, 1H, Ce-H), 8.3 (s, 1H,
13
Cf-H); C NMR (300 MHz, d): 123.5, 130.5, 116.0, 159.6, 116.0 and 130.5 (C1’, C2’, C3’, C4’, C5’
and C6’), 155.7, 39.8 and 45.2 (C3, C4, and C5), 168.4 (1C of C=O), 129.7, 152.5, 148.4, 124.9 and
137.2 (Ca, Cb, Cc, Ce and Cf), 138.9, 127.6, 128.4, 137, 128.4 and 127.6 (C1’’, C2’’, C3’’, C4’’, C5’’ and
C6’’), 37.9 (1C, methylene), 25.4 (1C, methylene), 13.9 (1C, methyl); ESI-MS (m/z): 385.18 (M+);
Anal. Calcd for C24H23N3O2: C, 74.78; H, 6.01; N, 10.90. Found: C, 74.69; H, 6.03; N, 10.84.
4.3.15.
[3-(4-Hydroxy-phenyl)-5-(4-isopropoxy-phenyl)-4,5-dihydro-pyrazol-1-yl]-pyridin-3-yl-
methanone (5o)
Prepared by the above method from 3o (4 mmole, 1.13 g) and 4 (4 mmole, 0.55 g) after 22h reflux;
o
yield: 76% as reddish brown amorphous solid; mp: 215-217 C. FTIR (neat) νmax: 3095 (C-H,
24

aromatic ring), 770 (C-H, substituted phenyl ring), 1660 (C=O), 1580 (
, aromatic ring),
C C str
2965–2830 (C-H), 1440 (C-H def), 1620 (C=N), 3345 (C-N); 1H NMR (300 MHz, d): 1.4 (dd, 6H,
C-H methyl), 4.0 (m, 1H, C-H methine), 6.95 (m, 2H, ArH), 6.65 (m, 2H, ArH), 5.0 (s, 1H, Ar-
OH), 6.9 (m, 2H, ArH), 7.6 (m, 2H, ArH), 1.95 (m, 2H, CH2 pyrazoline), 4.85 (s, 1H, CH
13
pyrazoline), 9.10 (s, 1H, Cb-H), 8.80 (s, 1H, Cd-H), 7.60 (s, 1H, Ce-H), 8.30 (s, 1H, Cf-H); C
NMR (300 MHz, d): 123.6, 130.5, 116.2, 159.4, 116.2 and 130.5 (C1’, C2’, C3’, C4’, C5’ and C6’),
155.5, 39.6 and 45.5 (C3, C4, and C5), 168.4 (1C of C=O), 129.3, 152.5, 148.3, 125.1 and 137.2 (Ca,
Cb, Cc, Ce and Cf), 134.2, 127.8, 113.9, 157.2, 113.9 and 127.8 (C1’’, C2’’, C3’’, C4’’, C5’’ and C6’’),
70.9 (1C, methine), 22.9 (2C, methyl); ESI-MS (m/z): 401.17 (M+); Anal. Calcd for C24H23N3O3: C,
71.80; H, 5.77; N, 10.47. Found: C, 71.76; H, 5.67; N, 10.39.
4.4. In vitro antimalarial assay30,31
The in vitro antimalarial activities of the compounds were assessed against CQ sensitive and
resistant isolates of P. falciparum and compared with clinically used antimalarial drug chloroquine.
The half maximal inhibitory concentrations (IC50) were obtained. In brief, the cultures of
asynchronous parasites of P. falciparum (MRC-2 and RKL-9) were synchronized using 5%
aqueous solution of sorbitol. All other stages except rings were degenerated. Degenerated stages
had been removed by centrifuge for 5 minutes at 1500 rpm. Parasitemia was adjusted to about 1%
for assay by diluting with fresh washed RBCs. The synthesized compounds were dissolved in 100
μL of dimethylsulfoxide (DMSO) and required dilutions were made with a RPMI-1640 medium.
The tests were done in 96 well plate using CQ sensitive and resistant isolates. Different
concentrations of synthesized compounds were dispensed in 96 well plate in triplicate. The first
well in all the rows was without any drug and considered as control. The synchronized parasites
were inoculated to all the wells, including control wells. The plates were incubated in a CO2
incubator at 37C for 24-30 h depending on the maturation of the schizont, thereafter; smears were
prepared from all the wells, fixed with methanol, stained with Giemsa’s stain and examined under
light microscope, 100 x oil immersion. Growth of parasites in the test wells was compared to that of
negative controls and the inhibition of parasite growth was expressed as a percentage. The half-
maximal inhibitory concentration (IC50) responses were estimated by the probit method.
4.5. Cell cytotoxicity assay
Toxicity is an important consideration in any drug development program; therefore we
studied cytotoxicity of these compounds against (Table 1) HepG2 cell lines using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay32 at ACTREC, Tata Memorial
25

Centre, Mumbai. Briefly, cell cultures were routinely maintained in RPMI 1640 medium
supplemented with 10% bovine foetal calf serum (FCS). For cytotoxicity evaluations, 5 x 102 cells
in 180 ml medium were seeded in each well of a 96-well plate and incubated at 37C under a 5%
CO2 atmosphere for 1 h. Aliquots of 20 μL of serial dilutions of the test compounds (stocks in
DMSO) were added to the wells. Untreated wells received 20 μL of culture medium while
additional solvent controls were prepared with medium containing DMSO to account for any
possible effect of DMSO on cell viability. The plates were then incubated at 37C under an
atmosphere of 5% CO2 for seven days and 20 μL MTT (5 mg/ml) was added to each well. The
plates were further incubated for an additional 4 h at 37C under an atmosphere of 5% CO2 and
then centrifuged for 10 minutes at 800 G. The supernatant was carefully aspirated from each well
without disturbing the pellet and the cells were washed with 150 μl of phosphate buffered saline
(PBS) followed by centrifugation for 10 minutes at 800 G. The supernatant was again carefully
aspirated and the plates were dried at 37C for an hour. The 100 μL ethanol was added to each well
to solubilise the resultant formazan crystals, aided by a gentle mechanical shacking for 1-2 h.
Absorbance were measured on a Universal Microplate Reader (ELx800 UV, Bio-tek Instrument) at
a wavelength of 570 nm and the percentage cell growth in drug treated well were calculated and
plotted against log drug concentration to determine the corresponding IC50 values by non-linear
regression analysis.
4.6. In vitro heme crystallization inhibition assay
The compounds after Schizont Maturation Inhibition Assay, were further screened for the
inhibition of the hemozoin formation by the method reported by Ncokazi and Egan (2004)33. Heme
solution is prepared by dissolving 5.2 mg of haemin chloride (Sigma Chemical Co., USA) in 1 ml
of DMSO. Chloroquine and synthesized compounds with different molar equivalents to haemin
were prepared by dissolving in DMSO.
The test compounds dissolved in DMSO in doses ranging from 0.12 to 5 M equivalents to
haemin chloride in 50 μL of 8 mM and of haemin chloride solution in DMSO, 50 μL DMSO as
control were taken for the assay. By adding 100 μL of 8M acetate buffer (pH 5.0) the hemozoin
formation was initiated. Culture plates were incubated at 37 C for 18 h, centrifuged and the soluble
fraction of unprecipitated material was collected. Thereafter, 200 μL of DMSO was added to re-
suspend the remaining pellet in order to remove unreacted haematin and the plates were centrifuged
26

again, the DMSO soluble fraction was collected and the residual pellet (which consists of pure
precipitate of haematin) was dissolved in 200 μL of 0.1M NaOH. Thereafter 75 μL of it was
transferred to new tubes and diluted four times by adding 0.1M NaOH. The amount of haematin
was determined spectrophotometrically at 405 nm wavelength. The % inhibition of hemozoin
formation was calculated by the below given formula:
The percentage inhibition of hemozoin of the standard drug was compared with the control.
4.7. In vitro antimicrobial activity
Minimal Inhibitory Concentrations (MICs, μg/ml) were determined on different microbes
using Broth Micro Dilution procedure according to the recommendations of National Committees
for Clinical Laboratory Standards (NCCLS) 34-36. MIC was defined as the lowest concentration of
compound that inhibited visible growth of microbes after incubation at 35C for 24 h for bacteria
and 48 h for fungi. Strains of gram negative bacteria Pseudomonas aeruginosa (MTCC 741),
Escherichia coli (MTCC 51) and gram positive bacteria Staphylococcus aureus (MTCC 3160),
Bacillus subtilis (MTCC 121) and fungal strains Candida albicans (MTCC 227), Aspergillus niger
(MTCC 8189) were used for testing antibacterial and antifungal activities. Bacterial strains were
grown in Mueller–Hinton Broth and fungal strains were grown in Sabouraud Liquid medium. The
inoculum densities of 5 x 105 CFU/ml for bacteria and 0.5–2.5 x 103 CFU/ml for fungi were
prepared. Ciproafloxacin and fluconazole were used as standard antibiotic powder. The synthesized
compounds were dissolved in DMSO and further dilutions were prepared in sterile distilled water of
various concentrations by twofold serial dilution method to obtain the required concentration.
Ciproafloxacin and fluconazole were diluted in sterile distilled water. After dilution was completed,
microbe suspensions were inoculated into each well of row. MIC values were given as μg/ml. The
turbidity was monitored by the unaided eye and the lowest concentration, at which no growth was
seen, recorded and considered as MIC of that particular compound. The MICs of the synthesized
compounds and standard drug has been summarized in Table 3.
Acknowledgments
Authors are thankful to The Director, National Institute of Malaria Research, New Delhi, India for
providing facilities and assistance for in-vitro antimalarial bioassay. One of the authors, Vikash K.
Mishra, is grateful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for
Senior Research Fellowship.
27

References
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Malaria
Report
2016
http://www.who.int/malaria/publications/world_malaria_report_2016/report/en/.
2. Altıntopa MD, Özdemira A, Turan-Zitounia G, Ilgınb S, Atlıb O, Demirelc R, Kaplancıklıa
ZA. A novel series of thiazolyl–pyrazoline derivatives: Synthesis and evaluation of
antifungal activity, cytotoxicity and genotoxicity. Eur J Med Chem. 2015;92: 342–352.
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