Synthesis of thymol-based pyrazolines: An effort to perceive novel potent-antimalarials

Article abstract of DOI:10.1016/j.bioorg.2019.102933

A series of thymol based substituted pyrazolines and chalcones was synthesized and evaluated for antimalarial activity, using in-vitro and in-vivo malaria models. All the target compounds (5a-k and 6a-j)were found to be active against human malaria parasite strain Plasmodium falciparum NF54. Among all, compounds 5e and 5f of chalcone series and 6c and 6f of pyrazoline series exhibited prominent antimalarial activity with IC₅₀ less than 3 and 2 μM respectively, while other pyrazolines also significantly inhibited the P. falciparum with IC₅₀ less than 10 μM. The designed pharmacophores were found to be effective against P. falciparum. Compound 6f was found to be able to retard malaria progression in mice. This was evident through decreased parasitemia, increased mean survival time and hemoglobin content in the treated animals. Moreover, 6f was observed as an inhibitor of heme polymerization pathway of the malaria parasite. It also inhibited free heme degradation, which could be possibly responsible for higher reactive oxygen species (ROS)in parasite, thus inhibiting the rapid proliferation of the parasite. In addition to this, compound 6f was found to be non-toxic with a good selectivity index. Based on these observations, the compound 6f could be taken up for further antimalarial lead optimization studies.

Full text of DOI:10.1016/j.bioorg.2019.102933

Accepted Manuscript
Synthesis of thymol-based pyrazolines: An effort to perceive novel potent-an-
timalarials
Dushyant Singh Raghuvanshi, Narsingh Verma, Shiv Vardan Singh, Sonam
Khare, Anirban Pal, Arvind Singh Negi
PII:
S0045-2068(18)31050-2
https://doi.org/10.1016/j.bioorg.2019.102933
102933
DOI:
Article Number:
Reference:
YBIOO 102933
Bioorganic Chemistry
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21 September 2018
4 April 2019
15 April 2019
Please cite this article as: D. Singh Raghuvanshi, N. Verma, S. Vardan Singh, S. Khare, A. Pal, A. Singh Negi,
Synthesis of thymol-based pyrazolines: An effort to perceive novel potent-antimalarials, Bioorganic Chemistry
(2019), doi: https://doi.org/10.1016/j.bioorg.2019.102933
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Synthesis of thymol-based pyrazolines: An effort to perceive novel potent-
antimalarials
Dushyant Singh Raghuvanshia,*, Narsingh Vermaa, Shiv Vardan Singhb, Sonam Khareb, Anirban
Palb,*, Arvind Singh Negia
b
aDepartment of Medicinal Chemistry, Molecular Bioprospection Department, CSIR-Central
Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Kukrail Road, Lucknow 226015, India
dushyant.bhu@gmail.com
CIMAP Communication No. CIMAP/PUB/2017/27
Abstract
A series of thymol based substituted pyrazolines and chalcones was synthesized and evaluated for
antimalarial activity, using in-vitro and in-vivo malaria models. All the target compounds (5a-k
and 6a-j) were found to be active against human malaria parasite strain Plasmodium falciparum
NF54. Among all, compounds 5e and 5f of chalcone series and 6c and 6f of pyrazoline series
exhibited prominent antimalarial activity with IC50 less than 3 and 2 μM respectively, while other
pyrazolines also significantly inhibited the P. falciparum with IC50 less than 10 μM. The designed
pharmacophores were found to be effective against P. falciparum. Compound 6f was found to be
able to retard malaria progression. This was evident through decreased parasitemia, increased
mean survival time and hemoglobin content in the treated animals. Moreover, 6f was observed as
an inhibitor of heme polymerization pathway of the malaria parasite. It also inhibited free heme
degradation, which could be possibly responsible for higher reactive oxygen species (ROS) in
parasite, thus inhibiting the rapid proliferation of the parasite. In addition to this, compound 6f was
found to be non-toxic with a good selectivity index. Based on these observations, the compound 6f
could be taken up for further antimalarial lead optimization studies.
Keywords: Thymol; Pyrazolines; Huisgen; Zwitterion mechanism; Antiplasmodial studies;
Hemozoin, Toxicity.
1

1. Introduction
Malaria is one of the most important life-threatening diseases in the world apart from cancer,
tuberculosis and AIDS. Despite comprehensive global efforts for eradication of malaria, latest
WHO reports in 2016 revealed that there were 216 million cases of malaria and 4.29 million
deaths [1]. Surprisingly, malaria still remains a major killer of children especially under five years
old [1]. Recently the arsenal of antimalarial drugs viz. quinine, chloroquine, artemisinins along
with newer chemotherapeutic agents artesunate and arteether is at high risk due to bioavailability
issues and have also got trapped by the resistance problem [2-12]. Now a days, artemisinins are
used as most potent antimalarial drugs for curing chloroquine-resistant P. falciparum infections
[13-16]. The dearth of new affordable drugs has complicated the clinical management of malaria
in endemic areas resulting in an increased mortality rate necessitating the discovery of new natural
product inspired chemical entities. It is important to mention that most of the anti-malarial drugs
are from natural sources or natural product inspired viz, artemisinin, quinine, chloroquine,
atovaquone etc. (Fig. 1) [17].
Thymol is a major natural monoterpene present in the essential oil of various plants, such as
Trachyspermum ammi and Thymus vulgaris, etc [18,19] possessing broadspectrum antimicrobial,
anti-oxidant and anti-inflammatory activity along with antimalarial activity [19-21]. In earlier
reports, pyrazolines have been possess a broad range of biological activities such
as antibacterial [22,23], antidepressant [24], anticancer activities and antiviral activity etc [25,26].
Recent literature revealed that these classes of compounds are not very much explored for their
possible antimalarial activities [27-30]. To the best of our knowledge thymol based pyrazolines
have not been reported previously for antimalarial activity.
Stupendous role of natural products in antimalarial drug design and as a part of our endeavor to
develop highly efficient methods and designing of bioactive pharmacophore [31-34], we are
reporting a synthesis of a new series of thymol derived pyrazoline derivatives through Huisgen
zwitterion mechanism [35], their antimalarial activity and toxicity studies.
2

Fig. 1. Figure represents the structure of natural or natural product inspired antimalarials, thymol
and lead molecule.
2. Results and Discussion
2.1. Chemistry
The synthesis of pyrazoline analogues were outlined in scheme 1.
Scheme 1. Schematic representation of novel pyrazolines synthesis.
The chalcones were synthesised according to given method in Scheme 1 (supplementary
information Table 2). The studies were initiated to optimize the reaction conditions for a model
reaction of chalcone 5c with diisopropyl azodicarboxylate (DIAD) in the presence of a
stoichiometric amount of triphenylphosphine under microwave (MW) irradiation conditions and
the overall findings are given in Table 1. Interestingly, it was found that the reaction gave
promising results in the toluene as well as 1,2-dimethoxyethane at 100 °C (Table 1, entries 5 and
6) under microwave conditions. To screen the effect of solvent and temperature, the model
reaction was performed under microwave irradiation conditions at different temperatures in
3

various solvents. The optimum conversion was achieved in toluene or 1,2-dimethoxyethane at 100
°C. In order to optimize the microwave power, the above reaction was also studied by varying
microwave power (60 and 80 W) and it was found that 60 Watt (W) power at 100 °C was
necessary to achieve maximum conversion of product 6c.
Table 1 Optimization of reaction conditions for pyrazolines.a
S. No.
Solvent
Power
(W)
60
Temp(oC)
Time
(min.)
15
Yieldb
(%)
nrc
1.
2.
3.
4.
5.
Peg 400
Nitromethane
Nitobenzene
Benzene
100
100
100
100
100
60
15
nr
60
15
nr
60
15
55
1,2-
60
15
74
Dimethoxyethane
Toluene
6.
7.
60
80
60
60
60
60
100
100
120
80
100
100
15
15
15
15
20
15
75
73
70
66
68
23
Toluene
8.
Toluene
9.
Toluene
10.
11.
Toluene
Diglyme
aReaction conditions: 5c (1 mmol), DIAD (1.5 mmol), PPh3 (1.5 mmol), 15 min.
60 W, 100°C. bIsolated yield based on 5c. cnr = no reaction.
Under the optimized set of MW reaction conditions (60 W, 100 °C), a number of chalcones 5 were
subsequently allowed to react with DIAD and triphenylphosphine to afford various pyrazoline
derivatives (6a–6j) in reasonably excellent yields in 15 minutes (Table 2). Interestingly, different
functional groups are well tolerated in this reaction system.
4

Table 2 Synthesis of functionalized pyrazolines.a,b
.
5

aMicrowave reaction conditions: 5 (1 mmol), DIAD (1.5 mmol), PPh3 (1.5 mmol), 15 min.,
60 W, 100°C. bIsolated yield based on 5.
2.2.
Biology
2.2.1. Antiplasmodial activity and cytotoxicity
Thymol based chalcones
In our preliminary in-vitro testing, thymol isolated from Trachyspermum ammi, exhibited a
promising IC50 (85.87±12.70 µM; Table 3) against human malaria parasite strain P. falciparum
NF54 (chloroquine sensitive strain-CQ sensitive). This initial finding prompted us for further
chemical modification of the thymol. Furthermore, thymol based chalcones (intermediates) were
also tested in-vitro, and found to be as promising antiplasmodial agents (Table 3), hence we
selected these intermediates for further chemical modification to perceive more active
antiplasmodial agents.
Table 3 Antiplasmodial activity of thymol and its chalcone analogues.
S. NO.
1.
Compounds
IC50 (µM)
85.87±12.70
24.49±5.70
17.60±3.25
21.93±4.61
23.71±8.95
2.80±0.22
Thymol
5a
2.
3.
5b
4.
5c
5.
5d
6.
5e
7.
5f
2.25±0.17
8.
5g
40.96±10.92
35.58±10.12
9.
5h
6

10.
11.
12.
5i
5j
20.41±3.31
27.51±5.56
15.78±6.20
5k
Data are the mean ± SD of three experiments in triplicate; values are in µM.
Thymol based pyrazolines
Interestingly upon chemical alteration and compared to thymol based chalcones, all the pyrazoline
derivatives were proved to be potential growth inhibitors (IC50 value <10 μM) of human malaria
parasite P. falciparum (NF54) (Table 4). Amongst all the derivatives, compound 6f was found to
be most active, with an IC50 value of 1.05±0.11µM. We have also evaluated the antiplasmodial
activity of most active lead 6f, against resistant strain of human malaria parasite (P. falciparum-
K1) and was recorded to be 3.07±0.65 µM. Additionally, all the synthesized derivatives were
found to have good therapeutic index against murine peritoneal macrophages, exhibited SI values
being higher than 10 (Table 4). Chloroquine used as a reference antimalarial exhibited an IC50
value of 0.0174±0.005 µM against P. falciparum NF54 strain (CQ-sensitive). Compound 6f was
found as the most active among the all pyrazolines, therefore selected as a lead for further studies
(in-vivo and mechanism based studies).
Table 4 Antiplasmodial activity of the pyrazoline derivatives of thymol and their selectivity index.
Inhibitory concentration
(IC50)
Cytotoxic concentration
(CC50)
Selectivity index
(SI)
Compounds
6a
6b
6c
6d
6e
6f
6g
6h
2.32±0.22
2.86±0.27
1.75±0.18
9.84±0.42
3.61±0.26
1.05±0.11
3.61±0.31
5.75±1.17
3.11±0.16
ND
101.18±3.95
113.61±8.14
147.46±4.67
177.48±4.44
137.64±3.69
103.19±2.54
120.85±8.47
140.91±7.63
123.59±5.61
ND
43.61
39.72
84.26
14.98
38.12
98.27
33.47
24.50
6i
6j
39.73
ND
Chloroquine
0.0174±0.005
148.17±23.52
8515.51
Data are the mean ± SD of three experiments in triplicate; values are in µM.
7

2.2.2. Selectivity towards host erythrocytes (RBCs)
The synthesized pyrazolines were also tested for their hemolytic potential to examine their
selectivity against human B+ve erythrocytes (separated using standard protocol). Upon 48 hrs
incubation, none of the derivative exhibited lytic potential upto 50 µM concentration. These results
are indicative for the selective antiplasmodial activity of the synthesized pyrazolines against
human malaria parasite (P. falciparum) in the host erythrocytes.
2.2.3. Compound 6f inhibited the hemozoin formation in vitro.
Hemozoin formation is a key metabolism of malaria parasite and a well known chemotherapeutic
target for many antimalarials, especially the quinoline derivatives viz., chloroquine [36]. As a lead
antiplasmodial agent of the present work, we have also investigated the effect of 6f on hemozoin
formation through in-vitro β-hematin (synthetic analog of hemozoin) formation assay. In our
results, 6f significantly inhibited this polymerization in a concentration dependent manner (Fig. 2)
with an IC50 value of 38.79±0.50 µM. Chloroquine as standard inhibitor of hemozoin formation
exhibited an IC50 value of 17.25±1.60 µM.
Fig. 2. Effect of compound 6f on in vitro β-hematin (hemozoin) formation. Results are represented
as mean percentage ± SD.
2.2.4. Compound 6f inhibited the free heme degradation.
The effect of compound 6f on H2O2 mediated free heme degradation was also monitored since it
has shown to have inhibitory effect of heme polymerization (hemozoin formation). As compared
8

to negative control, H2O2 degraded free hemin at 20 mM concentration in positive controls (Fig.
3). Furthermore, at the highest tested concentration of 100 µM, compound 6f significantly
(p<0.001) inhibited H2O2 mediated free heme degradation (Depicted in figure).
Fig. 3. Effect of compound 6f on peroxidative hemin degradation. Results are represented as mean
percentage ± SD. *p<0.05, **p<0.01 and ***p<0.001, ns- non significant.
2.2.5. Effect on total ROS
Compound 6f treatment caused generation of free radicals in the malaria parasite. The mean
fluorescence intensity values were recorded higher in the treated (in multiple of its IC50) parasite as
compared to untreated parasite (Fig. 4). Since compound 6f was found to be an inhibitor of free
heme polymerisation and degradation, so it might be responsible for free heme auto-oxidation
which consequently caused ROS generation in parasite, as also observed augmented here.
9

Fig. 4. Effect of compound 6f on total ROS generation in the treated parasites. 1X, 2X, and 3X are
the times concentration of IC50 value of compound 6f (1.05±0.11 µM). Results are represented as
mean percentage ± SD. *p<0.05, **p<0.01 and ***p<0.001.
2.2.6. Compound 6f inhibited malaria progression in mice
Under in vivo conditions, the compound 6f significantly inhibited P. berghei growth, exhibiting a
chemo-suppression of 25.29±5.14% and 55.60±9.71% at 50 and 100 mg/kg body weight,
respectively (Table 5). Additionally, 6f treated mice were found to exhibit a better lifespan (MST,
16.40 days), as compared to vehicle control group (MST, 8.80 days) (Table 4). These results are
also corroborated with the hemoglobin level of 6f treated animals estimated at peak parasitemia on
day 6, where 6f significantly (p<0.001) restored the hemoglobin levels deprivation in mice (Fig.
5).
Table 5 In-vivo antimalarial activity of compound 6f against rodent malaria parasite P. berghei in
Swiss mice.
Percent parasitemia
Mean survival
Percent
Doses
time (Days)
Day 4
2.14±0.36
2.06±0.55
0.59±0.17
0.0
Day 6
10.41±0.78
8.03±1.09
4.62±1.01
0.0
chemosupression
Vehicle
50 mg/kg
100 mg/kg
-
8.80
11.60
16.40
>28
25.29±5.14*
55.60±9.71**
100***
Chloroquine
(10 mg/kg)
*p < 0.05, **p < 0.01, ***p<0.001 vehicle vs treatment.
10

Fig. 5. At peak parasitemia on day 6, level of hemoglobin in the experimental animals treated with
6f at the doses of 50 and 100 mg/kg body weight. Results are represented as mean percentage ±
SD. *p<0.05, **p<0.01 and ***p<0.001, Infected vs. treatment, n=6.
2.2.7. Acute oral toxicity
The most active compound 6f was also studied for its safety profile for its further selection for a
lead optimization particular to malaria. No significant changes in the serum and biochemical
parameters in the treated animals as compared to vehicle received control animals (Table 6).
Animals on gross pathological study showed no changes in any of the major organs studied
including their absolute weight (Fig. 6). These results of acute oral toxicity clearly revealed that
the compound 6f has been found to be safe upto the dose of 2000 mg/kg body weight.
Table 6 Effect of 6f as a single acute oral dose at 2000 mg/kg body weight on hematological and
serum biochemical parameters in swiss albino mice.
Effect of 6f in various parameters as a single oral dose in Swiss
albino mice
Parameters
Treatment @ 2000
Vehicle
mg/kg
20.49±2.35
11.58±1.34
8.62±0.91
12.10±1.54
21.25±2.32
11.08±0.28
7.99±1.19
12.07±0.51
Body weight (gm)
Body weight (gm)
Hemoglobin (gm/dl)
RBC count (cells x 106/µL)
WBC count (cells x 104/µL)
Hematological (Unit)
Serum biochemical (Unit)
11

109.65±14.85
201.32±61.75
0.10±0.06
108.51±29.59
204.70±39.85
0.07±0.04
Total cholesterol (mg/dL)
Triglycerides (mg/dL)
Serum creatinine (mg/dL)
Albumin (gm/dL)
Cholesterol profile
Kideny
Test
Function
2.33±0.13
2.31±0.09
25.35±9.54
16.07±6.05
30.34±9.43
33.56±8.54
21.28±5.42
28.82±4.77
Alkaline phosphatase (U/dL)
SGPT (U/L)
Liver Function Test
SGOT (U/L)
Data are mean ±SE of six animals in each group
Fig. 6. Acute effect of the compound 6f at the dose of 2000 mg/kg bd. wt. on the absolute
organ weight of the experimental animals.
Discussion
Medicinal plants are a rich source of diverse pharmacologically active secondary metabolites
especially in case of antimalarial drug development. This has already been confirmed by the
developments of the new natural product derived antimalarials. Thymol is
a
natural monoterpenoid phenol widely distributed in nature and isolated from the Trachyspermum
ammi (ajwain seeds) and others. Earlier thymol showed significant antimalarial activity against P.
falciparum (IC50 ~5 µg/mL) [19]. In recent years, pyrazoline nucleus has received significant
attention in the area of malaria. Acharya et al., reported the in-vivo antimalarial activity of tri-
substituted pyrazolines against P. berghei strain and showed 60% chemosuppression at 50 mg/kg
12

at 4th day of experiment [29]. This novel finding provoked us to designed thymol based
pyrazolines, and herein our report showed significant in-vitro antimalarial activity against P.
falciparum (IC50 ~ 1.05 and 1.75 µM) and in-vivo against P. berghei showed significant
chemosuppression 72% at 100 mg/kg at 4th day. In our case dose is higher but chemosuppression
and mean survival time is significantly higher than previous report. Pyrazoline analogues resulted
in potent antimalarial agents against both chloroquine sensitive (NF54) and chloroquine resistant
strains (K1) of P. falciparum. The antimalarial activity of designed pharmacophore reveals that the
phenyl ring with methoxy and bromo substituent was found to be very much favourable. (Table 3
& 4). This SAR is full agreement with published antimalarial report [37]. The compound 6f is
found to be safe up to 2000 mg/kg under in-vivo condition [19,29].
Our study was initiated to achieve a natural product derived novel antimalarial agent. To
accomplish the objective, thymol was isolated from the seeds of Trachyspermum ammi
(supplementary data: Table 1) and was further modified. Thymol (1) and 2-O-alkylthymol (2) was
converted into corresponding aldehydes (3) by Vilsmeier-Haack or MgCl2/HCHO reaction.
Compound 3 was further converted into the desired chalcones 5 (supplementary data: Table 2)
Finally, substituted pyrazolines (6) were synthesised by the reaction of chalcones and Diisopropyl
azodicarboxylate through Huisgen Zwitterion mechanism (Scheme 1). All the studied compounds
were characterized using 1H and 13C NMR and mass spectrometry (Supplementary data).
All the thymol derived molecules including chalcone and pyrazoline scaffolds in one frame (5 &
6) were evaluated for their antiplasmodial activity against CQ sensitive (NF54) strain of P.
falciparum. Among the tested thymol based chalcones (5), compounds 5e and 5f exhibited
promising antiplasmodial activity with IC50 values of 2.80±0.22 and 2.25±0.17 μM respectively.
Interestingly, thymol based pyrazolines 6, which were synthesised from chalcones (5), exhibited
an IC50 value of < 10 µM. In pyrazoline series (6a-j), compound 6f exhibited better antiplasmodial
activity against human malaria parasite P. falciparum (IC50; 1.05±0.11 μM). The difference in the
IC50 values can be attributed to the factors such as number of carbon atoms, nitrogenous ring, and
the substitutions on the pyrazoline ring. The significant activity of the above mentioned
compounds might be due to the presence of nitrogenous ring and trimethoxy system, since both
trimethoxy containing molecule 5f and 6f exhibited potential antiplasmodial activity against P.
falciparum. To elucidate the role of thymol based chalcones and pyrazolines in antimalarial
activity, we derivatized the compounds (5a-k & 6a-j) as outlined in Fig. 2. Additionally, all the
13

synthesized derivatives were found to have good selective index against murine peritoneal
macrophages, thus exhibited an acceptable selectivity index. In order to find out the possible mode
of action of lead compound 6f, we have studied its effect on heme polymerization and degradation
pathway of malaria parasite. The compound 6f exhibited promising inhibitory activity, hence
could be responsible for the generation of heme mediated toxicity towards malaria parasite, and
therefore for the delayed progression of parasite, as observed in our result also. As a lead
compound, 6f was selected for further detailed study under in-vivo conditions against rodent
malaria parasite P. berghei in a dose dependent manner. Compound 6f significantly inhibited the
growth of malaria parasite in vivo, whereas the level of hemoglobin in the treated animals was also
found to be higher, as compared the vehicle received animals. The increased level of hemoglobin
in the treated parasite is indicative for the growth inhibition of malaria parasite, since hemoglobin
is used as nutrient source by the parasite. We have also assessed the antiplasmodial activity of
compound 6f against the resistant strain of malaria parasite, where it exhibited significant
inhibition of resistant parasite, which makes it a dual action molecule for antimalarial therapy.
Overall, the above observations are indicative for further detailed prospection of compound 6f in
antimalarial combination therapy.
3. Conclusion
In conclusion, a series of thymol based chalcones and novel pyrazolines were synthesized with
highly efficient methods and evaluated for their antimalarial efficacy. Compound 6f, a pyrazoline
derivative of thymol was found to be the most active antimalarial lead, being biologically active
against P. falciparum (in vitro) and P. berghei (in vivo). Furthermore, compound 6f was found as
an inhibitor of heme polymerization and degradation pathway of the malaria parasite, hence
augmented total ROS in the treated parasite, which might be responsible for inhibition of parasite
growth. It was also found to be non-toxic with good selectivity index. Currently, we are focusing
towards the lead optimisation of these thymol derivatives towards the other aspects of
pharmacology for a better in vivo antimalarial activity.
14

4.
Experimental section
4.1. Chemistry
4.1.1. General Experimental
The reactions were performed in round bottom flask and microwave vial using Teflon-coated
magnetic stirring bar with constant stirring. Column chromatography was performed on silica gel
(Merck 100-200 mesh) column. Merck GF254 plates (thickness 0.25 mm) were used for thin layer
chromatography (TLC). TLC plate spots visualization was accomplished with UV light and
1
13
staining in I2 chamber. H and C NMR were recorded on 300/500 MHz and 125 MHz Bruker
FTNMR spectrometer. Chemical shifts in NMR are expressed in δ values and tetramethylsilane
(TMS) used as internal standard.
4.1.2. Reagents
All the chemicals including triphenylphosphine, DIAD, and other solvents and reagents were
procured from Sigma-Aldrich and Alfa-aesar and were used as received. Thymol was isolated
from trachyspermum ammi and used after purification.
4.1.3. Conversion of thymol to thymol methyl ether (2).
A 250 ml round bottom flask was charged with potassium carbonate (6 g) and dry acetone (30 ml),
thymol 1a (2 g) and dimethyl sulphate (3 ml) were added to this mixture and the reaction mixture
was stirred for 5 h at room temperature. On completion, the reaction mixture was filtered and
washed with acetone and distilled off to get thymol ether 2 (2.1g).
4.1.4. Synthesis of 5-isopropyl-4-methoxy-2-methylbenzaldehyde (3a).
In a round bottom flask, thymol methyl ether 2 (4 g) was stirred in dimethylformamide (20 ml) in
ice bath having temperature range between 0 to -10°C, subsequently POCl3 (12 ml) was added to
the mixture and temperature of the reaction was maintained below 0°C. After complete addition of
POCl3, reaction was heated at 80 °C for 2-3 h. Upon completion, the reaction mixture was poured
into cold water, and extracted with ethyl acetate. The organic layer was washed with water, dried
over anhydrous sodium sulphate, evaporated and purified by column chromatography to get the
desired compound of formula 3a (3.6 g).
15

4.1.5. Synthesis of 2-hydroxy-3-isopropyl-6-methylbenzaldehyde (3b).
In a100-ml three-necked round-bottomed flask anhydrous magnesium dichloride (4.76 g, 50
mmol) and solid paraformaldehyde (2.25 g, 75 mmol) were added in dry CH3CN (125 ml) in an
inert atmosphere. Triethylamine (5.06 g, 50 mmol) was added drop wise to the above mixture and
stirred for 10 min, subsequently, thymol (3.75 g, 25 mmol) was added and refluxed for 4 hr. After
completion of the reaction, flask was cooled to room temperature and quenched with 10% HCl
(aq) solution. The biphasic reaction was extracted with 3 x 50 ml ethyl acetate. The combined
extracts were washed with saturated sodium chloride. The organic phase was dried over anhydrous
Na2SO4, and was purified by column chromatography to get the desired compound of formula 3b
(70%).
4.1.6. General procedure for the preparation of chalcones (5).
To a solution of 3a/3b (1.5 mmol) in methanol (5 ml), acetophenones 4a-j (1 mmol) and KOH (10
mmol) were added. The reaction mixture was allowed to stirrer for 4 h at 40°C. Upon completion
of reaction (monitored through TLC), the mixture was cooled to room temperature by treating with
cold water (15 ml). The precipitate was filtered and purified by recrystallization or column
chromatography using a mixture of ethyl acetate/n-hexane as eluent to afford the desired thymol
based chalcones 5.
4.1.6.1. (E)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1-p-tolylprop-2-en-1-one 5a.
1H NMR (300 MHz, CDCl3, δ ppm): 1.25 (d, J=7.2 Hz, 6H, 2×CH3), 2.45 (s, 3H, CH3), 2.48 (s,
3H, CH3), 3.27-3.34 (m, 1H, CH), 3.87 (s, 3H, OCH3), 6.69 (s, 1H, ArH), 7.27-7.40 (m, 3H, CH,
ArH), 7.57 (s, 1H, ArH), 7.94 (d, J=8.4 Hz, 2H, ArH), 8.07 (d, J=15.6 Hz, 1H, CH).
13C NMR (75 MHz, CDCl3, δ ppm): 20.28, 22.05, 23.03, 27.18, 55.81, 113.00, 120.83, 124.86,
126.38, 129.00, 129.37, 129.64, 135.60, 136.53, 138.30, 142.69, 143.61, 159.15, 190.71.
ESI-MS (C21H24O2): 309 [M+H]+.
4.1.6.2. (E)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1-(naphthalen-2-yl)prop-2-en-1-one
5b.
1H NMR (300 MHz, CDCl3, δ ppm): 1.26 (d, J=6.3 Hz, 6H, 2×CH3), 2.48 (s, 3H, CH3), 3.28-3.32
(m, 1H, CH), 3.87 (s, 3H, OCH3), 6.69 (s, 1H, ArH), 7.47-7.62 (m, 4H, CH, ArH), 7.89-8.02 (m,
3H, CH, ArH), 8.09-8.18 (m, 2H, ArH), 8.54 (s, 1H, ArH).
16

13C NMR (75 MHz, CDCl3, δ ppm): 20.33, 23.07, 27.25, 55.83, 113.04, 120.87, 124.95, 125.07,
126.35, 127.09, 128.22, 128.50, 128.59, 128.85, 129.92, 130.12, 133.04, 135.67, 135.78, 136.47,
138.48, 143.14, 159.29, 191.07.
ESI-MS (C24H24O2): 345 [M+H]+.
4.1.6.3. (E)-1-(3,4-dimethoxyphenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)prop-2-en-
1-one 5c.
1H NMR (300 MHz, CDCl3, δ ppm): 1.25 (d, J=6.9 Hz, 6H, 2×CH3), 2.48, (s, 3H, CH3), 3.25-3.34
(m, 1H, CH), 3.87 (s, 3H, OCH3), 3.98 (s, 6H, 2×OCH3), 6.69 (s, 1H, ArH), 6.94 (d, J=8.4 Hz, 1H,
ArH), 7.35 (d, J=15.6 Hz, 1H, CH), 7.56 (s, 1H, ArH), 7.64-7.72 (m, 2H, ArH), 8.06 (d, J=15.6
Hz, 1H, CH).
13C NMR (75 MHz, CDCl3, δ ppm): 20.28, 23.02, 27.18, 55.80, 56.44, 110.41, 111.35, 113.00,
120.55, 123.24, 124.83, 126.47, 132.19, 135.57, 138.19, 142.30, 149.61, 153.43, 159.09, 189.38.
ESI-MS (C22H26O4): 355 [M+H]+.
4.1.6.4. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)prop-2-
en-1-one 5d.
1H NMR (300 MHz, CDCl3, δ ppm): 1.24 (d, J=6.9 Hz, 6H, 2×CH3), 2.47 (s, 3H, CH3), 3.23-3.32
(m, 1H, CH), 3.86 (s, 3H, OCH3), 6.06 (s, 2H, CH2), 6.68 (s, 1H, ArH), 6.89 (d, J=8.4 Hz, 1H,
ArH), 7.29 (d, J=15.6 Hz, 1H, CH), 7.54 (s, 2H, ArH), 7.64-7.67 (m, 1H, ArH), 8.05 (d, J=15.3
Hz, 1H, CH).
13C NMR (75 MHz, CDCl3, δ ppm): 20.28, 23.03, 27.18, 55.81, 102.18, 108.27, 108.88, 113.00,
120.34, 124.81, 124.87, 126.36, 133.86, 135.60, 138.31, 142.56, 148.60, 151.82, 159.14, 188.90.
ESI-MS (C21H22O4): 339 [M+H]+.
4.1.6.5. (E)-1-(4-bromophenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)prop-2-en-1-one
5e.
1H NMR (500 MHz, CDCl3, δ ppm): 1.24 (d, J=7.0 Hz, 6H, 2×CH3), 2.46, (s, 3H CH3), 3.26-3.29
(m, 1H, CH), 3.86 (s, 3H, OCH3), 6.68 (s, 1H, ArH), 7.28 (d, J=15.5 Hz, 1H, CH), 7.55 (s, 1H,
ArH), 7.62-7.65 (m, 2H, ArH), 7.87-7.90 (m, 2H, ArH), 8.08 (d, J=15.5 Hz, 1H, CH).
13C NMR (75 MHz, CDCl3, δ ppm): 19.88, 22.61, 26.78, 55.43, 112.64, 119.66, 124.49, 125.65,
127.47, 130.00, 131.83, 135.32, 137.47, 138.25, 143.28, 159.05, 189.62.
ESI-MS (C20H21BrO2): 371 [M-H]+.
4.1.6.6. (E)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one 5f.
17

1H NMR (500 MHz, CDCl3, δ ppm): 1.24 (d, J=7.0 Hz, 6H, 2×CH3), 2.47 (s, 3H CH3), 3.26-3.29
(m, 1H, CH), 3.86 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.94 (s, 6H, 2×OCH3), 6.69 (s, 1H, ArH),
7.26-7.29 (m, 3H, CH, ArH), 7.54 (s, 1H, ArH), 8.05 (d, J=15.5 Hz, 1H, CH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.91, 22.60, 26.83, 55.44, 56.42, 60.98, 106.18, 112.62,
120.54, 124.53, 125.91, 134.06, 135.26, 137.92, 142.26, 142.73, 153.12, 158.88, 189.91.
ESI-MS (C23H28O5): 385 [M+H]+.
4.1.6.7. (E)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
5g.
1H NMR (500 MHz, CDCl3, δ ppm): 1.25 (d, J=7.0 Hz, 6H, 2×CH3), 2.47 (s, 3H CH3), 3.27-3.30
(m, 1H, CH), 3.86 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.68 (s,1H, ArH), 6.97-7.00 (m, 2H, ArH),
7.36 (d, J=15.5 Hz, 1H, CH), 7.56 (s, 1H, ArH), 8.03-8.10 (m, 3H, CH, ArH).
13C NMR (75 MHz, CDCl3, δ ppm): 20.30, 23.04, 27.16, 55.81, 55.87, 112.98, 114.17, 120.57,
124.79, 126.44, 131.12, 131.75, 131.95, 135.55, 138.24, 142.27, 159.06, 163.60, 189.41.
ESI-MS (C21H24O3): 325 [M+H]+.
4.1.6.8. (E)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1-(thiophen-2-yl)prop-2-en-1-one 5h.
1H NMR (300 MHz, CDCl3, δ ppm): 1.24 (d, J=6.9 Hz, 6H, 2×CH3), 2.47 (s, 3H, CH3), 3.23-3.33
(m, 1H, CH), 3.86 (s, 3H, OCH3), 6.68 (s, 1H, ArH), 7.16-7.28 (m, 2H, CH, ArH), 7.55 (s, 1H,
ArH), 7.65 (d, J=4.8 Hz, 1H, ArH), 7.86 (d, J=3.6 Hz, 1H, ArH), 8.10 (d, J=15.3 Hz, 1H, CH).
13C NMR (75 MHz, CDCl3, δ ppm): 20.30, 23.02, 27.20, 55.82, 113.02, 120.25, 124.88, 126.08,
128.53, 131.81, 133.76, 135.62, 138.56, 142.31, 146.37, 159.31, 182.70.
ESI-MS (C18H20O2S): 301 [M+H]+.
4.1.6.9. (E)-1-(3,5-dimethoxyphenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)prop-2-en-
1-one 5i.
1H NMR (500 MHz, CDCl3, δ ppm): 1.24 (d, J=7.0 Hz, 6H, 2×CH3), 2.46 (s, 3H CH3), 3.25-3.30
(m, 1H, CH), 3.86 (s, 3H, OCH3), 3.86 (s, 6H, 2×OCH3), 6.66-6.68 (m, 2H, ArH), 7.15 (d, J=2.5
Hz, 2H, ArH), 7.27 (d, J=15.5 Hz, 1H, CH), 7.54 (s, 1H, ArH), 8.07 (d, J=15.5 Hz, 1H, CH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.90, 22.63, 26.78, 55.42, 55.63, 104.62, 106.39, 112.60,
120.40, 124.53, 125.83, 135.29, 138.05, 140.79, 142.88, 158.89, 160.86, 190.45
ESI-MS (C22H26O4): 355 [M+H]+.
18

4.1.6.10. (E)-1-(4-flurophenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)prop-2-en-1-one
5j.
1H NMR (500 MHz, CDCl3, δ ppm): 1.25 (d, J=6.5 Hz, 6H, 2×CH3), 2.47 (s, 3H CH3), 3.26-3.31
(m, 1H, CH), 3.86 (s, 3H, OCH3), 6.69 (s, 1H, ArH), 7.15-7.19 (m, 2H, ArH), 7.32 (d, J=15.5 Hz,
1H, CH), 7.56 (s, 1H, ArH), 8.04-8.12 (m, 3H, ArH, CH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.90, 22.64, 26.77, 55.43, 112.61, 115.55 (d, JC-C-F = 21.25
Hz, CH), 119.72, 124.43, 125.71, 130.97 (d, JC-C-C-F = 8.75 Hz, CH), 135.01 (d, JC-C-C-C-F = 2.50
Hz, C), 135.27, 138.17, 142.91, 158.95, 164.43 (d, JC-F = 252.50 Hz, C), 189.06.
ESI-MS (C20H21FO2): 313 [M+H]+.
4.1.6.11. (E)-3-(2-hydroxy-3-isopropyl-6-methylphenyl)-1-(4-methoxyphenyl)prop-2-en-1-
one 5k.
1H NMR (500 MHz, CDCl3, δ ppm): 1.26 (d, J= 7.0 Hz, 6H, 2×CH3), 2.36, (s, 3H CH3), 3.19-3.22
(m, 1H, CH), 3.89 (s, 3H, OCH3), 5.54 (s, 1H, OH), 6.79 (d, J=8.0 Hz, 1H, ArH), 6.96-6.98 (m,
2H, CH, ArH), 7.10 (d, J=7.5 Hz 1H, ArH), 7.59 (d, J=16 Hz, 1H, CH), 7.94-7.97 (m, 1H, ArH),
8.01-8.03 (m, 2H, CH, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.51, 22.37, 26.63, 55.23, 112.51, 115.27, 119.89, 124.25,
125.76, 130.08, 130.97, 135.08, 137.88, 142.06, 158.71, 161.71, 189.99.
ESI-MS (C20H22O3): 311 [M+H]+.
4.2. Synthesis of functionalized pyrazolines 6.
Diisopropyl azodicarboxylate (1.5 mmol), thymol based chalcones 5 (1.0 mmol), PPh3 (1.5 mmol)
and toluene (2 ml) were put in a pressure regulation 10 ml pressurized vial with ‘snap-on’ cap and
flushed with nitrogen before closing the vial. The reaction mixture was irradiated in a single-mode
microwave system at 60W power and 100 °C for 15 min. After completion of the reaction
(checked by TLC), reaction mixture was cooled at room temperature and the solvent was removed
under reduced pressure. The residue was subjected to column chromatography embedded with
silica gel. Hexane/ethyl acetate was used as eluent to afford the functionalized pyrazolines 6.
4.2.1. diisopropyl 3-(5-isopropyl-4-methoxy-2-methylphenyl)-5-p-tolyl-1H-pyrazole-1,2(3H)
dicarboxylate 6a.
1H NMR (500 MHz, CDCl3, δ ppm): 0.99 (d, J=6.0 Hz, 3H, CH3), 1.07 (d, J=6.5 Hz, 3H, CH3),
1.12-1.16 (m, 6H, 2×CH3), 1.30-1.34 (m, 6H, 2×CH3), 2.36 (s, 3H, CH3), 2.50 (s, 3H, CH3), 3.17-
3.22 (m, 1H, CH), 3.81 (s, 3H, OCH3), 4.81-4.86 (m, 1H, CH), 5.03-5.08 (m, 1H, CH), 5.60 (d,
19

J=3.0 Hz, 1H, CH), 6.01 (d, J=3.0 Hz, 1H, CH), 6.66 (s, 1H, ArH), 7.13-7.17 (m, 3H, ArH), 7.40
(d, J=8.5 Hz, 2H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.25, 21.35, 21.62, 21.75, 22.08, 22.14, 22.56, 22.70,
26.94, 55.44, 63.87, 70.32, 70.44, 111.32, 112.78, 124.92, 127.11, 128.64, 128.78, 129.17, 133.48,
134.92, 138.49, 142.93, 155.29, 156.32.
ESI-MS (C29H38N2O5): 495 [M+H]+.
4.2.2. diisopropyl3-(5-isopropyl-4-methoxy-2-methylphenyl)-5-(naphthalen-2-yl)-1H
pyrazole-1,2(3H)-dicarboxylate 6b.
1H NMR (500 MHz, CDCl3, δ ppm): 1H NMR (500 MHz, CDCl3, δ ppm): 0.95 (d, J=6.0 Hz, 3H,
CH3), 1.02 (d, J=6.5 Hz, 3H, CH3), 1.16-1.21(m, 6H, 2×CH3), 1.35-1.39 (m, 6H, 3×CH3), 2.54 (s,
3H, CH3), 3.22-3.25 (m, 1H, CH), 3.84 (s, 3H, OCH3), 4.83-4.88 (m, 1H, CH), 5.09-5.14 (m, 1H,
CH), 5.79 (d, J=3.0 Hz, 1H, CH), 6.08 (d, J=3.0 Hz, 1H, CH), 6.70 (s, 1H, ArH), 7.22 (s, 1H,
ArH), 7.46-7.49 (m, 2H, ArH), 7.64-7.66 (m, 1H, ArH), 7.82-7.85 (m, 3H, ArH), 7.99 (s, 1H,
ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.26, 21.58, 21.72, 22.15, 22.60, 22.71, 27.00, 29.70,
30.87, 55.45, 64.13, 70.44, 70.61, 112.52, 112.84, 124.87, 125.27, 126.16, 126.26, 126.33, 127.43,
127.69, 128.22, 128.76, 129.53, 132.95, 133.34, 133.40, 135.05, 142.87, 155.27, 156.39.
ESI-MS (C32H38N2O5): 531 [M+H]+.
4.2.3. diisopropyl 5-(3,4-dimethoxyphenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1H
pyrazole-1,2(3H)-dicarboxylate 6c.
1H NMR (500 MHz, CDCl3, δ ppm): 1.01 (d, J=6.5 Hz 3H, CH3), 1.07 (d, J=6.5 Hz, 3H, CH3),
1.13-1.16 (m, 6H, 2×CH3), 1.26-1.34 (m, 6H, 2×CH3), 2.49 (s, 3H, CH3), 3.18-3.23 (m, 1H, CH),
3.81 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.82-4.87 (m, 1H, CH), 5.03-5.08
(m, 1H, CH), 5.57 (d, J=3.0 Hz, 1H, CH), 6.00 (d, J=2.5 Hz, 1H, CH), 6.66 (s, 1H, ArH), 6.85 (d,
J=8.0 Hz, 1H, ArH), 7.03 (d, J=1.5 Hz, 1H, ArH), 7.09 -7.11 (m, 1H, ArH), 7.15 (s, 1H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.24, 21.69, 21.76, 21.95, 22.10, 22.12, 22.56, 22.71,
26.95, 55.43, 55.90, 55.94, 63.96, 70.31, 70.47, 110.50, 110.59, 110.88, 112.72, 120.05, 124.80,
124.92, 128.85, 133.31, 134.97, 142.59, 148.44, 149.47, 155.26, 156.31.
ESI-MS (C30H40N2O7): 541 [M+H]+.
20

4.2.4. diisopropyl 5-(benzo[d][1,3]dioxol-5-yl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1H
pyrazole-1,2(3H)-dicarboxylate 6d.
1H NMR (500 MHz, CDCl3, δ ppm): 1.04 (d, J=6.0, Hz, 3H, CH3), 1.13-1.18 (m, 9H, 3×CH3),
1.31-1.36 (m, 6H, 2×CH3), 2.52 (s, 3H, CH3), 3.19-3.24 (m, 1H, CH), 3.83 (s, 3H, OCH3), 4.84-
4.89 (m, 1H, CH), 5.05-5.10 (m, 1H, CH), 5.59 (d, J=3.0 Hz, 1H, CH), 5.96 (s, 2H, CH2) 6.00-
6.03 (m, 1H, CH), 6.68 (s, 1H, ArH), 6.81 (d, J=8.0 Hz, 1H, ArH), 7.01 (d, J=2.0 Hz, 1H, ArH),
7.05-7.07 (m, 1H, ArH), 7.12 (s, 1H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.25, 21.64, 21.67, 21.80, 22.07, 22.14, 22.57, 22.69,
26.94, 55.44, 63.81, 70.37, 70.52, 101.20, 107.81, 107.96, 111.21, 112.80, 121.14, 124.87, 126.13,
128.64, 133.54, 134.91, 142.58, 147.31, 147.93, 155.32, 156.35.
ESI-MS (C29H36N2O7): 525 [M+H]+.
4.2.5. diisopropyl
5-(4-bromophenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1H-
pyrazole-1,2(3H)-dicarboxylate 6e.
1H NMR (500 MHz, CDCl3, δ ppm): 1.01 (d, J=6.5 Hz, 3H, CH3), 1.08 (d, J=6.0 Hz, 3H, CH3),
1.12-1.15 (m, 6H, 2×CH3), 1.30-1.34 (m, 6H, 2×CH3), 2.49 (s, 3H, CH3), 3.13-3.25 (m, 1H, CH),
3.81 (s, 3H, OCH3), 4.82-4.87 (m, 1H, CH), 5.03-5.08 (m, 1H, CH), 5.67 (d, J=3.0 Hz, 1H, CH),
6.01 (d, J=3.0 Hz, 1H, CH), 6.66 (s, 1H, ArH), 7.10 (s, 1H, ArH), 7.37 (d, J=8.5 Hz, 2H, ArH),
7.48 (d, J=8.5 Hz, 2H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.23, 21.46, 21.75, 22.07, 22.13, 22.56, 22.68, 26.93,
55.44, 64.03, 70.51, 70.82, 112.80, 122.56, 124.75, 128.37, 128.72, 131.00, 131.16, 133.40,
135.03, 141.86, 155.24, 156.41.
ESI-MS (C28H35BrN2O5): 559 [M+H]+.
4.2.6. diisopropyl 3-(5-isopropyl-4-methoxy-2-methylphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazole-1,2(3H)-dicarboxylate 6f.
1H NMR (500 MHz, CDCl3, δ ppm): 1.02 (d, J=6.5 Hz, 3H, CH3), 1.08 (d, J=6.0 Hz, 3H, CH3),
1.14-1.17 (m, 6H, 2×CH3), 1.30-1.35 (m, 6H, 2×CH3), 2.49 (s, 3H, CH3), 3.19-3.25 (m, 1H, CH),
3.81 (s, 3H, OCH3), 3.85 (m, 3H, OCH3), 3.86 (s, 6H, 2×OCH3), 4.83-4.88 (m, 1H, CH), 5.04-5.09
(m, 1H, CH), 5.63 (d, J=3.0 Hz, 1H, CH), 6.01 (d, J=3.0 Hz, 1H, CH), 6.67 (s, 1H, ArH), 6.75 (s,
2H, ArH), 7.18 (s, 1H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.23, 21.67, 21.75, 22.09, 22.55, 22.73, 26.92, 55.39,
56.13, 60.93, 64.15, 70.37, 70.57, 104.59, 111.81, 112.68, 124.64, 127.70, 128.68, 133.18, 135.01,
138.54, 142.64, 152.86, 155.19, 156.34.
GC-MS (C31H42N2O8): 571 [M+H]+.
21

4.2.7. diisopropyl 5-(4-methoxyphenyl)-3-(5-isopropyl-4-methoxy-2 methylphenyl)-1H-
pyrazole-1,2(3H)-dicarboxylate 6g.
1H NMR (500 MHz, CDCl3, δ ppm): 1.00 (d, J=6.5 Hz, 3H, CH3), 1.08 (d, J=6.5 Hz, 3H, CH3),
1.12-1.16 (m, 6H, 2×CH3), 1.30-1.34 (m, 6H, 2×CH3), 2.50 (s, 3H, CH3), 3.17-3.22 (m, 1H, CH),
3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.81-4.86 (m, 1H, CH), 5.03-5.08 (m, 1H, CH), 5.56 (d,
J=3.5 Hz, 1H, CH), 6.00 (d, J=2.0 Hz, 1H, CH), 6.66 (s, 1H, ArH), 6.88 (d, J=9.0 Hz, 2H, ArH),
7.13 (s, 1H, ArH), 7.44 (d, J=9.0 Hz, 2H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.26, 21.66, 21.77, 22.09, 22.15, 22.58, 22.71, 26.94,
55.32, 55.43, 63.82, 70.33, 70.43, 110.53, 112.74, 113.39, 124.61, 124.88, 128.55, 128.85, 133.46,
134.89, 142.55, 155.32, 156.29, 159.91.
ESI-MS (C29H38N2O6): 511 [M+H]+.
4.2.8. diisopropyl 3-(5-isopropyl-4-methoxy-2-methylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-
1,2(3H)-dicarboxylate 6h.
1H NMR (500 MHz, CDCl3, δ ppm): 0.97 (d, J=6.5 Hz, 3H, CH3), 1.04-1.08 (m, 9H, 3×CH3),
1.22-1.26 (m, 6H, 2×CH3), 2.42 (s, 3H, CH3), 3.09-3.15 (m, 1H, CH), 3.74 (s, 3H, OCH3), 4.78-
4.83 (m, 1H, CH), 4.95-5.00 (m, 1H, CH), 5.62 (d, J=3.0 Hz, 1H, CH), 5.95 (s, 1H, CH), 6.59 (s,
1H, ArH), 6.93-6.95 (m, 1H, ArH), 7.04 (s, 1H, ArH), 7.17-7.19 (m, 1H, ArH), 7.22-7.23 (m, 1H,
ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.32, 21.64, 21.78, 22.10, 22.57, 22.70, 26.92, 55.44,
63.69, 70.52, 70.79, 112.42, 112.73, 124.81, 125.86, 126.94, 127.17, 128.32, 133.53, 133.82,
134.92, 136.51, 155.34, 156.35.
ESI-MS (C26H34N2O5S): 487 [M+H]+.
4.2.9. diisopropyl 5-(3,5-dimethoxyphenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1H-
pyrazole-1,2(3H)-dicarboxylate 6i.
1H NMR (500 MHz, CDCl3, δ ppm): 1.00 (d, J=6.0 Hz, 3H, CH3), 1.07 (d, J=6.5 Hz, 3H, CH3),
1.13-1.16 (m, 6H, 2×CH3), 1.24-1.34 (m, 6H, 2×CH3), 2.49 (s, 3H, CH3), 3.17-3.23 (m, 1H, CH),
3.79 (s, 3H, OCH3), 3.81 (s, 6H, 2×OCH3), 4.82-4.87 (m, 1H, CH), 5.03-5.08 (m, 1H, CH), 5.65
(d, J=3.5 Hz, 1H, CH), 6.01 (d, J=3.0 Hz, 1H, CH), 6.44-6.45 (m, 1H, ArH), 6.66-6.68 (m, 3H,
ArH), 7.13 (s, 1H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.23, 21.61, 21.72, 21.77, 22.07, 22.12, 22.56, 22.70,
26.93, 29.70, 55.39, 55.43, 64.02, 70.38, 70.56, 100.86, 105.44, 112.50, 112.77, 124.81, 128.57,
133.99, 135.01, 142.75, 155.20, 156.36, 160.37.
ESI-MS (C30H40N2O7): 541 [M+H]+.
22

4.2.10. diisopropyl 5-(4-hydroxyphenyl)-3-(5-isopropyl-4-methoxy-2-methylphenyl)-1H-
pyrazole-1,2(3H)-dicarboxylate 6j.
1H NMR (500 MHz, CDCl3, δ ppm): 1.00 (d, J=6.0 Hz, 3H, CH3), 1.07 (d, J=6.5 Hz, 3H, CH3),
1.13-1.16 (m, 6H, 2×CH3),1.30-1.34 (m, 6H, 2×CH3), 2.50 (s, 3H, CH3), 3.18-3.23 (m, 1H, CH),
3.81 (s, 3H, OCH3), 4.81-4.86 (m, 1H, CH), 5.04-5.09 (m, 1H, CH), 5.62 (d, J=3.0 Hz, 1H, CH),
6.02 (d, J=3Hz, 1H, CH), 6.66 (s, 1H, ArH), 7.03-7.07 (m, 2H, ArH), 7.12 (s, 1H, ArH), 7.48-7.51
(m, 2H, ArH).
13C NMR (125 MHz, CDCl3, δ ppm): 19.25, 21.63, 21.75, 22.09, 22.14, 22.58, 22.70, 26.92,
55.43, 63.96, 70.48, 70.69, 112.01, 112.77, 114.92, 115.09, 124.76, 128.21, 128.24, 128.53,
128.94, 129.01, 133.41, 134.99, 141.84, 155.23, 156.37, 161.87, 163.85.
ESI-MS (C28H35FN2O5): 499 [M+H]+.
5.
Biology
5.1. In vitro antiplasmodial activity
The in vitro antiplasmodial activity of synthesized compounds was evaluated against chloroquine
sensitive (NF54) and resistant (K1) strains of human malaria parasite P. falciparum, by using the
protocol described earlier in our report [33]. Compounds were dissolved in DMSO (Dimethyl
sulfoxide) at stock concentration of 10 µM, and the two-fold serial dilutions of test samples were
made in 96 well plates and incubated with 1.0% parasitized cell suspension containing 1%
parasitemia (more than 90% ring stages). The plates were incubated at 37 ºC in CO2 incubator in
an atmosphere containing 5% CO2 and air mixture. Onwards 72 h incubation, culture supernatant
was discarded gently and thin blood smears were prepared from each well. Slides were fixed in
methanol and stained with Giemsa dye (10%) upto 30 min, the percent parasitemia in each smear
was evaluated by counting a total 1000 erythrocytes from each smear. Antiplasmodial activity of
the tested samples was expressed in term of concentration inhibiting 50% of parasite growth
(IC50). IC50 values were calculated using pre-programmed excel spreadsheet by Logit regression
analysis. In order to assess the antiplasmodial activity three replicates were carried out, and
chloroquine was used as reference antimalarial drugs.
5.2. Cytotoxicity and selectivity index
In order to estimate the selectivity (therapeutic) index of the synthesized molecules, cell
cytotoxicity of samples was also evaluated against murine intraperitoneal primary macrophages.
23

Cells were cultivated in Minimum Essential Medium (Sigma, USA) supplemented with 10% heat-
inactivated fetal bovine serum (Gibco, USA). The evaluation of cell viability was performed
through MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay [38]. The
data was expressed as percent cell viability in term of 50% cytotoxic concentration (CC50), where
DMSO was taken as negative control. The selectivity index of the samples was expressed as a ratio
of CC50 and IC50 value and considered safe above than 5 as per WHO guidelines [39].
5.3. Hemolytic assay
The synthesized compounds were also evaluated for their hemolytic (RBC damage) potential.
Briefly, B+ve blood group human erythrocytes (2% hematocrit in RPMI media) were incubated for
48 hr in a 96-well plate, in the presence of varying concentrations (1-50 µM) of compounds. In
each experiment 0.5% saponin (positive control) and DMSO (negative control) were used. The
RBC damage was calculated by following the absorbance at 540 nm and expressed in term of
hemolysis [40].
5.4. Effect on in vitro heme polymerization
In an effort to eliminate the detrimental destructions experienced during hemoglobin degradation
and utilization, malaria parasite exploited its survival through heme polymerization. In the present
work, the inhibitory effect of synthesized compounds on heme polymerization pathway of the
malaria parasite was evaluated using the method reported earlier [41]. Briefly, male Swiss mice,
weighing 15-20 g were inoculated (i.p.) with 1×106 P. yoelii infected red blood cells. Blood from
infected animals at approx. 50% parasitemia was collected by cardiac puncture in 2.0% citrate
saline buffer and centrifuged at 2500 rpm for 15 minutes at 4 °C for the isolation plasma. The
plasma was used for β-hematin formation inhibitory (BHIA) assay. The assay mixture contained
100 mM sodium acetate buffer pH (5.1), 100 μL plasma, 100 μM hemin as the substrate and 1-50
μM compound in a total volume of 1.0 mL. The control tubes contained all the reagents except
compounds. The triplicate reaction mixture was incubated at 37 °C for 16 hrs in a rotary shaker.
The reaction was stopped by centrifugation at 10,000 rpm for 10 minutes at 30 °C. The pellet was
suspended in 100 mM tris-HCl buffer pH (7.4) containing 2.5% SDS for washing the pellet (three
times). The pellet obtained after centrifugation was again washed with distilled water (TDW) to
remove free hemin attached to polymerized β-hematin. The pellet was finally solubilized in 0.1N
24

NaOH. Absorbance was measured at 400 nm and the IC50 value was determined using non-linear
regression analysis dose response curves.
5.5. Inhibition of hydrogen peroxide-mediated hemin degradation
As a part of parasite rescue mechanism(s), free heme released upon hemoglobin degradation in
malaria parasite is degraded by intra-parasitic H2O2. The ability of synthesized compounds to
inhibit this degradation was also estimated using in vitro experiments. The effect of lead
compounds on peroxidative decomposition of hemin was estimated by using the protocol
established earlier by Loria et al., 1999 [42]. Briefly, a hemin stock solution (1 mM in 0.1 N
NaOH) was freshly prepared and 20 µL of this was distributed in each well of 96-well flat bottom
microplate. Onwards this, the lead compounds were added from 10-100 µM final concentrations,
chloroquine was used as reference inhibitor of heme degradation. The plates were left to
equilibrate (pre-incubated) for 30 min at room temperature after addition of 150 µL bovine serum
albumin (stock 1 mg/mL) in sodium acetate 0.1 M (pH 5.1), as similar to the pH range of P.
falciparum food vacuole. The peroxidative reaction was initiated by the addition of 20 µL H2O2 (2
M) and followed by measuring the decrease in absorption at the Soret band (404 nm) after 30 of
incubation, using a multiplate reader (SpectraMax). Negative control with addition of TDW
instead of H2O2, was also included in each experiment. Results were expressed as the percentage
of remained hemin in the reaction mixture.
5.6. Measurement of total ROS (oxidative stress)
The Intracellular oxidative stress (total ROS) in the treated parasite produced during the
chemotherapeutic treatment of compound 6f in malaria parasite was monitored by measuring the
alteration of fluorescence resulted from the oxidation of 2’,7’-dichlorofluorescein diacetate (DCF-
DA) [43]. Briefly, as mentioned above, P. falciparum culture at trophozoite stage (~85%
synchronized) and ~5% parasitemia was treated with varying concentrations (1X, 2X and 3X of
IC50 value) of test compounds for 16 hr (overnight). Upon treatment, 10 μM of DCFDA (stock in
1X PBS) was added in each well with subsequent incubation for 1hr. After incubation, pre-labeled
cells were treated with saponin (0.075% final concentration) to isolate parasite which were lysed
by mild sonication. The relative fluorescence intensity in parasite lysate (200 μl) was estimated by
measuring the using fluorescence plate reader (FLx 800, BIO-Tek, India) and determined with an
25

excitation and emission wavelengths of 485±10 and 535±10 nm respectively. The ROS level was
calculated as the mean fluorescence intensity (MFI) and expressed as percentage of the control
value.
5.7. In vivo antimalarial activity
The chloroquine-sensitive strain maintained in laboratory of rodent malaria parasite (P. berghei K-
173) was used for the evaluation of antimalarial activity of lead derivative, using Peter’s 4 day
chemo-suppressive test [44]. Briefly, on day 0, malarial infection in experimental mice was
induced with an intra-peritoneal injection of the parasitized RBCs (1 X 106 cells, in 0.2 mL ACD)
and six mice were randomly assigned in each group. Onwards day 4, microscopic examination of
the stained (Giemsa) blood smears was also performed to quantify the parasitic burden (percent
parasitemia) on host. Chemotherapeutic doses were prepared in 1% Tween 80 (v/v in water) and
given through oral administration (each 0.5 mL at 24 h interval), for a period of 4 days.
Chloroquine was used as standard antimalarial at the dose of 10 mg/kg body weight. Control
untreated group animals received equal volumes of vehicle (1% Tween 80). The treatment was
followed upto 28 days with regular examination of the percentage parasitemia (number of infected
erythrocytes per 100 erythrocytes). Clinical sign and mortality in each group was observed during
the experiment for evaluation of percent survival and mean survival time (MST). Hemoglobin in
the experimental animals was also estimated by using the Drabkin’s reagent [45].
5.8. Acute oral toxicity
In order to explore the therapeutic value of the most active antimalarial derivative of the present
work, acute oral toxicity study of the compound 6f was also performed in Swiss albino mice.
Experiment was performed in accordance with the Organization for Economic Co-operation and
Development (OECD) test guideline No 423 (1987). Briefly, 12 mice (6 male and 6 female) were
randomly divided into two groups comprising 3 male and 3 female mice in each group (20±2 g).
The animals were maintained at 22±5 °C with humidity control and dark and light cycle of 12
hours each. The animals were fed with standard mice feed and provided ad libitum drinking water.
Group 1 was considered as control group (received vehicle only) while group 2 as experimental
group where the received the compound 6f at a single dose of 2000 mg/kg body weight. The
experimental mice were fasted overnight before the dosing of the test material and upon dosing the
26

animals were monitored for mortality and clinical signs of illness (skin irritation, lachrymation,
piloerection, gait, posture and diarrhea) at hourly interval on day of test sample administration and
there after once in a day. In addition to observational study, body weight and major organ weight
were also recorded from the experimental animals. Blood and serum samples were collected from
all the animals on 7th day of the experiment in acute oral toxicity. The blood was used to analyze
the total RBC, WBC, differential leucocytes count, hemoglobin percentage. Serum was used for
biochemical parameters like ALKP, SGPT, SGOT, total cholesterol, triglycerides, creatinine,
bilirubin, serum protein and high density lipoprotein (HDL).
Supporting information.
Supplementary data related to this article can be found at online.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgment
DSR is thankful to Department of Science & Technology, New Delhi, India for the DST-INSPIRE
faculty award. The authors are also thankful to Director CSIR-CIMAP, Dr CS Chanotiya for
GC/MS analysis and Curator National Gene bank of CSIR-CIMAP, Lucknow for providing
authenticated plant material.
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