4, 5-Dihydrooxazole-pyrazoline hybrids: Synthesis and their evaluation as potential antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2016.07.055

A new series of oxazoline-pyrazoline hybrids (4a-p) were synthesized by condensation reaction of substituted oxazoline based chalcones (3a-m) and substituted hydrazines in methanol. Some of the compounds exhibited promising in?vitro antimalarial activity for chloroquine sensitive CQ^S(3D7) strain and chloroquine resistant CQ^R(RKL9) strain. The most potent analogue 4i (IC₅₀0.322?μg/ml) exhibited significant in vivo antimalarial potential against Plasmodium berghei mouse model. The stable complex of 4i with hematin (1:1 stoichiometry) suggests that heme may be one possible target for these hybrid compounds. The study has revealed potential of title compounds as lead for the development of antimalarial agents.

Full text of DOI:10.1016/j.ejmech.2016.07.055

Accepted Manuscript
4, 5-dihydrooxazole-pyrazoline hybrids: Synthesis and their evaluation as potential
antimalarial agents
Ashutosh Kumar Pandey, Supriya Sharma, Minakshi Pandey, M. Mumtaz Alam, M.
Shaquiquzzaman, Mymoona Akhter
PII:
S0223-5234(16)30618-3
10.1016/j.ejmech.2016.07.055
EJMECH 8773
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 April 2016
Revised Date: 11 July 2016
Accepted Date: 23 July 2016
Please cite this article as: A.K. Pandey, S. Sharma, M. Pandey, M.M. Alam, M. Shaquiquzzaman, M.
Akhter, 4, 5-dihydrooxazole-pyrazoline hybrids: Synthesis and their evaluation as potential antimalarial
agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.055.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
4, 5-Dihydrooxazole-Pyrazoline hybrids: Synthesis and their evaluation as
potential antimalarial agents
Ashutosh Kumar Pandeya, Supriya Sharmab, Minakshi Pandeyc, M. Mumtaz Alam, M.
Shaquiquzzamana, Mymoona Akhtera*
Synthesis of Oxazoline- pyrazoline hybrid analogues from chalcone intermediates having significant
antimalarial activity

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4, 5-Dihydrooxazole-Pyrazoline hybrids: Synthesis and their evaluation as
potential antimalarial agents
Ashutosh Kumar Pandeya, Supriya Sharmab, Minakshi Pandeyc, M. Mumtaz Alama, M.
Shaquiquzzamana, Mymoona Akhtera*
aDrug Design and Medicinal Chemistry Lab, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy and Bioinformatics Infrastructure Facility(BIF), Jamia Hamdard, New Delhi-110062, India
bNational Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi-110077, India
cBBS Institute of Pharmaceutical & Allied Sciences, Greater Noida-201310, U.P., India
Corresponding author Email: *mymoonaakhter@gmail.com, Tel: +91 9891369482. Fax: 011- 26059663.
ABSTRACT
A new series of oxazoline-pyrazoline hybrids (4a-p) were synthesized by condensation reaction
of substituted oxazoline based chalcones (3a-m) and substituted hydrazines in methanol. Some
of the compounds exhibited promising in vitro antimalarial activity for chloroquine sensitive
CQS (3D7) strain and chloroquine resistant CQR (RKL9) strain. The most potent analogue 4i
(IC50 0.322 µg/ml) exhibited significant in vivo antimalarial potential against P. berghei mouse
model. The stable complex of 4i with hematin (1:1 stoichiometry) suggests that heme may be
one possible target for these hybrid compounds. The study has revealed potential of title
compounds as lead for the development of antimalarial agents.
Keywords: Plasmodium falciparum, Oxazoline, Pyrazoline, Antimalarial, IC50.
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Introduction:
The worldwide impact of malaria still remains significant with annual deaths of millions of
people. Out of reported cases of malaria, falciparum malaria is most fatal which is due to
infection of Plasmodium falciparum that accumulates in brain capillary (cerebral malaria), leads
to coma and death. Other species of plasmodium which cause malaria are P. vivax, P. malariae,
P. ovale and the simian P. knowlesi. Due to unavailability of a licensed malaria vaccine, the
approved drugs present in the market are only option for prophylaxis and treatment of the disease
[1-2]. One of the common and concerning problem for majority of available drugs are drug
resistance (single drug/ multiple drug resistance). Thus, need of new chemical entities
encourages for further research in order to prevent issue of drug resistance. Recently, WHO
recommended the use of two or more therapeutic agents to minimize the drug resistance and
failures during treatment of malaria. Example-Artemisinin-based combination therapy-
Artemether-Lumefantrine and Artesunate+Sulfadoxine-Pyrimethamine.
The concept of hybridization has been employed to develop some heterocyclic nucleuses
with interesting biological activity. There may be enhanced biological activity of one single
molecule having more than one structurally active moiety. The research based on design of
hybrid molecules bearing potential scaffolds like chalcones, oxazolines and pyrazolines has been
extensively reported. Wanare et al. [3] synthesized and reported α-pyranochalcones hybrid
analogues (i) as P. falciparum growth inhibitors. Smit et al. [4] highlighted 4-aminoquinolinyl-
chalcone amides hybrids (ii) for its antimalarial and cytotoxic activity. Similarily, Tadigoppula et
al. [5] developed chalcone analogues and focused its antimalarial potential (iii). Some
researchers have reported oxazolines derivatives (iv-vi) with antitubercular, antioxidant and
antimicrobial activity [6-7]. Pyrazoline analogues (vii-ix) are well known in the area of
pharmaceutical research for their wide range of biological potential like cytotoxic [8], CNS
depressant [9], antimicrobial [10] and antimalarial activity [11-13] (Fig. 1). We here in report the
synthesis of oxazoline based chalcones and their conversion to pyrazoline hybrids through
Claisen- Schimdt condensation method and cyclocondensation mechanism respectively. We have
focused on in vitro and in vivo antimalarial potential of the synthesized hybrids.
Figure 1
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2. Results and discussion
2.1 Chemistry
Starting material compound 2 was obtained from 1 by treatment with ethanolamine. Compound 2
was converted to different chalcones (3a-m) through Claisen Schimdt condensation which on
cyclization yielded the title compounds (4a-p). The structure of all the compounds was
confirmed by IR, NMR and mass spectra. IR spectra showed the peaks of carbonyl function,
ether function and azomethine stretching at appropriate frequencies. The details are provided in
1
experimental section. In H NMR spectra the presence of etheric methylene moiety was
confirmed by peak at 4.56 ppm and cyclic protons (OCH2 and NCH2) could be picked at 3.77
and 3.55 ppm in compound 2. Successful synthesis of ethylenic protons of chalcone were
confirmed by observing peaks one deshielded proton between 7.68-8.36 ppm and other less
deshielded proton 7.59-7.86 ppm. The coupling constant between ethylenic protons was found to
be 15.5 Hz indicating trans isomer. In NMR of title compounds the spectra showed three
different protons of pyrazoline in double-doublets at specified positions. The stereogenic proton
appears between 4.55-5.45 ppm with coupling constant value 12.0 Hz. The other two methylenic
protons of pyrazoline was found between 3.33-3.97 ppm with coupling constant 17.2 Hz and
13
between 3.02-3.33 ppm with coupling constant 17.2 Hz. In C NMR, peaks were observed at
167-170 (C=N) and 159-162 (Car -O) further confirming the desired structures.
Scheme 1, Scheme 2
2.2 Biological Activity
All the synthesized hybrids (4a-p) and their intermediates (2 and 3a-m) were tested for their in
vitro antimalarial activity against CQS (3D7) strain and some selected compounds were tested
for CQR (RKL9) strain using chloroquine as reference drug [14-17]. Active compounds were
also tested for cytotoxicity [18]. Compounds (4a-p) showed excellent antimalarial activity (IC50
0.322- 2.154 µg/ml) compared to their precursor chalcones (0.515-4.233 µg/ml) indicating the
cyclization affects positively on the antimalarial activity. The SAR of chalcones revealed that
substitution by electron withdrawing groups (Cl, F, NO2) is favorable for activity (3a-c, 3g) and
substitution by electron donating group reduces the activity (3d-f, 3i). When phenyl group were
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replaced with benzfuzed moieties like dioxane, the activity diminishes probably due to dioxane
group not suitably fitted into the receptor cavity e.g. compound (3m). The SAR of pyrazoline
series (4a-p) revealed that substitution at nitrogen of pyrazoline highly affects the activity. An
increase in activity could be observed when R1 was substituted from hydrogen to unsubstituted
phenyl ring to benzenesulfonamide. The benzenesulfonamide substituted compounds (4c-e, 4i-j,
and 4l) exhibits excellent activity. The results are presented in Table 1 and 2.
Table 1, Table 2
Most potent compound 4i was selected for in vivo antimalarial studies [19-23] against P. berghei
mouse model. The results obtained were encouraging as the parasitaemia suppression of 79.33%
and 63.89% was observed at a dose 50 and 25 mg/kg body weight respectively on day 4 (Table
3). The mean survival days for treated mice (50mg/kg body weight) were 15.6 days that is
comparable to 7.2 days for negative control mice (vehicle treated) and 30 days for chloroquine
treated mice. Compound 4i was also tested for acute oral toxicity as per OECD guidelines 423
for testing of chemicals [24-25]. The histopathological study of liver taken from survived rat on
14th day post treatment reveals presence of minimal inflammatory cells infiltration in periportal
area which is not significant (Fig. 2).
Figure 2, Figure 3, Table 3
To study the mechanism of action of synthesized compounds heme binding studies were carried
out. The standard drug chloroquine is reported to exhibit its antimalarial activity by inhibiting the
formation of hemozoin inside food vacuole of parasites by accumulating in food vacuole and
binding with parasite heme/hematin [26-27]. It was hypothesized earlier that the polymerization
of heme results the formation of hemozoin but hemozoin is now considered as a crystalline
cyclic dimer of ferriprotoporphyrin IX. Hematin monomer, heme released as a result of digestion
of parasite hemoglobin and transformed into hemozoin. In aqueous solution, chloroquine-
ferriprotoporphyrin IX (CQ-FPIX) complex was formed which was evident by altering in the
UV-spectrum of aqueous hematin in presence of drug. The present study has conducted to
determine the binding of most potent compound (4i) with heme. A hematin solution in 40%
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DMSO exhibited a Soret band at 402 nm signifies the availability of monomeric heme under
conditions used 0.02 M HEPES buffer (pH 7.4) and 0.02 M MES buffer (pH 5.6). A substantial
reduction in intensity of the ferriprotoporphyrin IX Soret band without shifting of absorption
maximum was seen with stepwise addition (0-100 µM) of 4i into a fixed concentration of
monomeric heme (Fig. 4 and 5). It depicts association of compound with hematin.
Figure 4, Figure 5
Job’s plot was obtained by molar fraction method for expressing 1:1 stoichiometry of the most
stable complexes of 4i with monomeric heme at pH 7.4 and 5.6. Chloroquine also reported for
binding with heme dimer (µ-oxo-heme), so we have conducted the binding study of 4i with µ-
oxodimeric heme at pH 5.8. In this study, there was decrease in absorbance intensity at 362 nm
when different concentration of 4i (0-100 µM) was added to 10 µM µ-oxodimeric solution in 20
mM phosphate buffer at pH 5.8. The job’s plot gives 1:1 stoichiometry for the complex (Fig. 6).
Figure 6
The association constant for the complexes was determined by titration method (Table 4). The
value of association constant for the 4i complexed with monomeric heme was calculated as log
K 5.87 which is comparable to standard drug chloroquine (log K 5.20) at pH 7.4. On decreasing
the pH from 7.4 to 5.6 (food vacuole pH) the log K value reduced (log K 4.98) which depicts
binding is less strong as compare to pH 7.4. Similarly, the binding with µ-oxodimeric heme (log
K 4.34) at pH 5.8 is lesser than monomeric binding at pH 7.4. All the above results were
comparable with binding results of standard drug chloroquine. Thus the possible mechanism of
action of synthesized compounds may be similar to that of chloroquine i.e. inhibiting the
formation of β-hematin.
Table 4
3. Conclusion:
In present research work, we have reported synthesis of different analogues of oxazoline based
chalcones and their efficacious conversion into pyrazoline based hybrid compounds. Most of the
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compounds showed significant antimalarial potential. Compound 4i showed excellent in vitro as
well as in vivo activity i.e. comparable with standard drug (chloroquine). This compound
exhibited insignificant oral toxicity characterized by minimal inflammatory cell infiltrations
resulted from liver histopathology study of treated rat. The probable mode of action of the 4i
may be inhibition of hematin monomer, heme as the binding capability of this compound with
monomeric heme was comparable with chloroquine. These results indicate that the synthesized
hybrids are promising lead and provide significant model for further structural and biological
optimization.
4. Experimental:
4.1 General:
All Chemicals were purchased from authorized commercial suppliers. Melting point was
determined by open capillary method by digital melting point apparatus by VEEGO and are
uncorrected. 1H NMR and 13C NMR spectra were obtained on Bruker Avance II 400 and Bruker
Avance II 500 spectrometer using DMSO-d6 as solvent and TMS as internal standard. The
Chemical shift in NMR was represented in delta (δ) values. Infra-red (IR) spectra were recorded
on Bruker (Alpha-T) spectrophotometer and mass data were recorded in Waters Acquity UPLC-
MS spectrometer. Reactions were monitored by TLC using pre-coated silica gel aluminium
plates (0.25mm Merck silica-gel plates (60, F254). TLC spots were visualized under UV light
and by exposing them to iodine vapors. Swiss albino mice (20-25 g) and Wistar rats (180-200 g)
were procured from central animal house facility of Jamia Hamdard (Project No. 1138 and 1249)
to carry out in vivo antimalarial activity and toxicity study respectively using guidelines for
ethical conduct in the care and use of animals of the university.
4.2 Synthesis of 1-(4-((4, 5-dihydrooxazol-2-yl) methoxy) phenyl) ethanone (2):
To an equimolar mixture of p-hydroxyacetophenone and ethyl bromoacetate in dry acetone (25
ml), fused potassium carbonate (3-5g) was added. The reaction mixture was refluxed at 60ºC
with continuous stirring for specified time by monitoring of reaction by TLC. The reaction
mixture was allowed to cool at room temperature. The solvent were evaporated completely and
transferred the concentrate into mixture of methanol and hydrochloric acid (10:1). The reaction
mixture was stirred continuously at room temperature with TLC monitoring. After completion of
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reaction, the reaction mixture was poured into crushed ice and stirred properly. The precipitate
obtained was filtered, dried and crystallized to get acid intermediate. This intermediate was
further refluxed with equimolar quantity of ethanolamine in methanol to obtain oxazoline based
acetophenone (2).
Colourless solid, Yield 69%, Rf = 0.33 (CHCl3:MeOH/9:1), mp 140-142ºC, 1H NMR (400 MHz,
CDCl3): δ 2.47 (s, 3H, CH3), 3.53 (dd, J = 10.4, 5.6 Hz, 2H, oxazoline-NCH2), 3.77 (t, J =
4.8Hz, 2H, oxazoline-OCH2), 4.56 (s, 2H, OCH2), 6.96 (d, J = 8.8 Hz, 2H, Ar-H), 7.95 (d, J =
8.8 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1664 and 1549 (CO), 1251 and 1054
(OCH2); anal. calcd. for C12H13NO3 (219.24): C, 65.74; H, 5.98; N, 6.39; Found C, 65.70; H,
5.94; N, 6.41.
4.3 General Procedure for synthesis of Chalcones (3a-m):
To an equimolar mixture of compound (2) and substituted aldehydes in methanol (25 ml),
crushed sodium hydroxide pellets were added. The reaction mixture was refluxed at 75ºC for
specified time with monitoring of reaction by TLC. The reaction mixture was allowed to cool at
room temperature and quenched by pouring the mixture into crushed ice. The solution was
neutralized to by cold dilute hydrochloric acid solution drop wise manner. The precipitate
obtained was filtered, washed with water, dried and crystallized.
4.3.1. 3-(4-chlorophenyl)-1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)prop-2-en-1-one (3a):
1
Yellow solid, Yield 64%, Rf = 0.8 (CHCl3:MeOH/9:1), mp 290-292ºC, H NMR (500 MHz,
DMSO-d6): δ 3.38 (s, 2H, oxazoline-NCH2), 4.40 (s, 4H, OCH2, oxazoline-OCH2), 6.95 (d, J =
7.0 Hz, 2H, Ar-H), 7.52 (d, J = 7.0 Hz, 2H, Ar-H), 7.68 (d, J = 15.5 Hz, 1H, =CH), 7.93 (d, J =
7.0 Hz, 2H, Ar-H), 7.97 (d, J = 16.0 Hz, 1H, COCH=), 8.11 (d, J = 7.5 Hz, 2H, Ar-H), FT-IR
(Alpha ATR Crystal) cm-1: 1656 and 1601 (CO), 1243 and 1055 (OCH2); anal. calcd. for
C19H16ClNO3 (341.79): C, 66.77; H, 4.72; N, 4.10; Found C, 66.74; H, 4.74; N, 4.11.
4.3.2. 3-(3-chlorophenyl)-1-(4-((4, 5-dihydrooxazol-2-yl)methoxy)phenyl)prop-2-en-1-one (3b):
1
Yellow solid, Yield 77%, Rf = 0.68 (CHCl3:MeOH/9:1), mp 280-282ºC, H NMR (500 MHz,
DMSO-d6): δ 3.21 (s, 2H, oxazoline-NCH2), 3.58 (s, 2H, oxazoline-OCH2), 4.31 (s, 2H, OCH2),
6.94 (d, J = 8 Hz, 2H, Ar-H), 7.48 (d, J = 8.0 Hz, 2H, Ar-H), 7.67 (d, J = 15.5 Hz, 1H, =CH),
7.82 (d, J = 15.5 Hz, 1H, COCH=), 7.99-8.08 (m, 2H, Ar-H), 8.13 (d, J = 8.0 Hz, 2H, Ar-H),
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FT-IR (Alpha ATR Crystal) cm-1: 1656 and 1602 (CO), 1224 and 1055 (OCH2); anal. calcd. for
C19H16ClNO3 (341.79): C, 66.77; H, 4.72; N, 4.10; Found C, 66.78; H, 4.74; N, 4.11.
4.3.3. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3c):
Colourless solid, Yield 75%, Rf = 0.67 (CHCl3:MeOH/9:1), mp 265-267ºC, 1H NMR (500 MHz,
DMSO-d6): δ 3.82 (s, 2H, oxazoline-NCH2), 4.61 (s, 4H, OCH2, oxazoline-OCH2), 7.02 (d, J =
6.0 Hz, 2H, Ar-H ), 7.31 (t, J = 7.5 Hz, 2H, Ar-H), 7.71 (d, J = 15.0 Hz , 1H, =CH), 7.84 (d, J =
8.0 Hz, 1H, COCH=), 7.91-7.97 (m, 2H, Ar-H), 8.13 (t, J = 8.0 Hz, 2H, Ar-H), FT-IR (Alpha
ATR Crystal) cm-1: 1656 and 1592 (CO), 1229 and 1168 (OCH2); anal. calcd. for C19H16FNO3
(325.33): C, 70.14; H, 4.96; N, 4.31; Found C, 70.18; H, 4.98; N, 4.31.
4.3.4. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3d):
1
Yellow solid, Yield 69%, Rf = 0.48 (CHCl3:MeOH/9:1), mp 280-282ºC, H NMR (500 MHz,
DMSO-d6): δ 3.40 (s, 3H, OCH3), 3.82 (s, 4H, oxazoline-OCH2, NCH2), 4.34 (s, 2H, OCH2),
6.93 (d, J = 7.0 Hz, 2H, Ar-H), 7.01 (d, J = 7.5 Hz, 2H, Ar-H), 7.67(d, J = 15.5 Hz, 1H, =CH),
7.79 (s, 1H, COCH=), 7.84 (d, J = 7.0 Hz, 2H, Ar-H), 8.09 (d, J = 7.0 Hz, 2H, Ar-H), FT-IR
(Alpha ATR Crystal) cm-1: 1596 and 1506 (CO), 1237 and 1173 (OCH2); anal. calcd. for
C20H19FNO4 (337.37): C, 71.20; H, 5.68; N, 4.15; Found C, 71.21; H, 5.68; N, 4.14.
4.3.5. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(3-methoxyphenyl)prop-2-en-1-one
(3e): Yellow solid, Yield 65%, Rf = 0.64 (CHCl3:MeOH/9:1), mp 90-92ºC, 1H NMR (500 MHz,
DMSO-d6): δ 3.21 (s, 2H, oxazoline-NCH2), 3.58 (s, 2H, oxazoline-OCH2), 3.83 (s, 3H, OCH3),
4.51 (s, 2H, OCH2), 6.66 (d, J = 8.0 Hz, 1H, Ar-H), 6.99-7.02 (m, 2H, Ar-H), 7.37 (d, J = 8.0
Hz, 1H, Ar-H), 7.38 (t, J = 9.5 Hz , 1H, Ar-H), 7.47 (s, 1H, =CH), 7.67(d, J = 15.5 Hz, 1H,
COCH=), 7.88-7.99 (m, 2H, Ar-H), 8.13 (d, J = 8.0 Hz, 1H, Ar-H), FT-IR (Alpha ATR Crystal)
cm-1: 1651 and 1590 (CO), 1248 and 1166 (OCH2); anal. calcd. for C20H19NO4 (337.37): C,
71.20; H, 5.68; N, 4.15; Found C, 71.18; H, 5.66; N, 4.12.
4.3.6. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(p-tolyl)prop-2-en-1-one (3f): Yellow
1
solid, Yield 68%, Rf = 0.61 (CHCl3:MeOH/9:1), mp 300-302ºC, H NMR (500 MHz, DMSO-
d6): δ 2.34(s, 3H, CH3), 3.21 (s, 2H, oxazoline-NCH2), 3.51 (s, 2H, oxazoline-OCH2), 4.25 (s,
2H, OCH2), 6.92 (d, J = 8 Hz, 2H, Ar-H), 7.26 (d, J = 6.5 Hz, 2H, Ar-H), 7.66 (d, J = 15.5 Hz,
1H, =CH), 7.75(d, J = 7.0 Hz, 2H, Ar-H), 7.87 (d, J = 15.5 Hz, 1H, COCH=), 8.08 (d, J = 8.0
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Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1 : 1657 and 1600 (CO), 1237 and 1176 (OCH2);
anal. calcd. for C10H19NO3 (321.37): C, 74.75; H, 5.96; N, 4.36; Found C, 74.74; H, 5.94; N,
4.38.
4.3.7. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one (3g):
1
Yellow solid, Yield 70%, Rf = 0.37 (CHCl3:MeOH/9:1), mp 172-174ºC, H NMR (500 MHz,
DMSO-d6): δ 3.22 (s, 2H, oxazoline-NCH2), 3.40 (s, 2H, oxazoline-OCH2), 4.64 (s, 2H, OCH2),
7.13 (d, J = 8.0 Hz, 2H, Ar-H), 7.79 (d, J = 15.5 Hz, 1H, =CH), 8.15 (s, 1H, COCH=), 8.17-
8.22 (m, 4H, Ar-H), 8.29 (d, J = 7.5 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1651 and
1598 (CO), 1257 and 1174 (OCH2); anal. calcd. for C19H16N2O5 (352.34): C 64.77, H 4.58, N
7.95, Found C, 64.76; H, 4.54; N, 7.94.
4.3.8. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(3-nitrophenyl)prop-2-en-1-one (3h):
1
Yellow solid, Yield 71%, Rf = 0.39 (CHCl3:MeOH/9:1), mp 168-170ºC, H NMR (500 MHz,
DMSO-d6): δ 3.40 (s, 4H, oxazoline-OCH2, NCH2), 4.64 (s, 2H, OCH2), 7.12 (d, J = 8.0 Hz, 2H,
Ar-H), 7.79 (d, J = 15.5 Hz, 1H, =CH), 8.15 (s, 1H, COCH=), 8.17 (d, J = 10.5 Hz, 2H, Ar-H),
8.21 (d, J = 8.0 Hz, 2H, Ar-H), 8.28 (d, J = 7.5 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1:
1655 and 1598 (CO), 1256 and 1175 (OCH2); anal. calcd. for C19H16N2O5 (352.34): C, 64.77; H,
4.58; N, 7.95; Found C, 64.75; H, 4.58, N, 7.94.
4.3.9. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(3i): Yellow solid, Yield 75%, Rf = 0.2 (CHCl3:MeOH/9:1), mp 160-162ºC, 1H NMR (500 MHz,
DMSO-d6): δ 3.22 (s, 2H, oxazoline-NCH2), 3.58 (s, 2H, oxazoline-OCH2), 4.83 (s, 2H, OCH2),
6.86 (d, J = 8.0 Hz, 1H, Ar-H), 7.06 (d, J = 8.0 Hz, 2H, Ar-H), 7.22-7.27 (m, 2H, Ar-H), 7.31 (d,
J = 7.0 Hz, 1H, Ar-H), 7.62 (d, J = 15.0 Hz, 1H, =CH), 7.84 (d, J = 15.5 Hz, 1H, COCH=), 8.15
(d, J = 8.0 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1635 and 1584 (CO), 1208 and
1161(OCH2); anal. calcd. for C19H17NO4 (323.12): C, 70.58; H, 5.30; N, 4.33; Found C, 70.56;
H, 5.31; N, 4.31.
4.3.10. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(3-hydroxyphenyl)prop-2-en-1-one (3j)
1
: Yellow solid, Yield 65%, Rf = 0.34 (CHCl3:MeOH/9:1), mp 142-144ºC, H NMR (500 MHz,
DMSO-d6): δ 3.21 (s, 2H, oxazoline-NCH2 ), 3.57 (s, 2H, oxazoline-OCH2), 4.83 (s, 2H, OCH2),
6.86 (d, J = 8.0 Hz, 1H, Ar-H), 7.05 (d, J = 8.0Hz, 2H, Ar-H), 7.22-7.27 (m, 2H, Ar-H), 7.31 (d,
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J = 7.0 Hz, 1H, Ar-H), 7.61 (d, J = 15.0, 1H, CH= ), 7.85 (d, J = 15.5 Hz, 1H, COCH=), 8.14 (d,
J = 8.0 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1652 and 1581 (CO), 1168 and 1060
(OCH2); anal. calcd. for C19H17NO4 (323.12): C, 70.58; H, 5.30; N, 4.33; Found C, 70.59; H,
5.34; N, 4.31.
4.3.11. (1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-phenylprop-2-en-1-one (3k): Yellow
solid, Yield 70%, Rf = 0.64 (CHCl3:MeOH/9:1), mp 85-87ºC, 1H NMR (500 MHz, DMSO-d6): δ
3.17 (s, 2H, oxazoline-NCH2), 3.57 (s, 2H, oxazoline-OCH2), 4.79 (s, 2H, OCH2), 6.66 (d, J =
8.0 Hz, Ar-H), 6.99 (t, J = 9.0 Hz, 1H, Ar-H), 7.06 (d, J = 8.5 Hz, 2H, Ar-H), 7.43-7.46 (m, 2H,
Ar-H), 7.72 (d, J = 15.5 Hz, 1H, COCH=), 7.84-7.94 (m, 2H, Ar-H), 7.96 (d, J = 15.0 Hz, 1H,
CH=), 8.16 (d, J = 8.0 Hz, 1H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1656 and 1589 (CO),
1221 and 1166 (OCH2); anal. calcd. for C19H17NO3 (307.34): C, 74.25; H, 5.58; N, 4.56; Found
C, 74.23; H, 5.54; N, 4.53.
4.3.12. 1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-3-(1H-indol-3-yl)prop-2-en-1-one (3l):
1
Yellow solid, Yield 63%, Rf = 0.54 (CHCl3: MeOH/9:1), mp 175-177ºC, H NMR (400 MHz,
DMSO-d6): δ 3.55 (s, 4H, oxazoline-OCH2, NCH2), 4.63 (s, 2H, OCH2), 6.96 (d, J = 7.6 Hz, 1H,
=CH ), 7.18-7.22 (m, 5H, Ar-H), 7.48 (d, J = 7.6 Hz, 1H, COCH=), 7.61(d, J = 8.8 Hz, 2H, Ar-
H), 8.06 (d, J = 6.0 Hz, 2H, Ar-H), 12.14 (s, 1H, indole-NH), FT-IR (Alpha ATR Crystal) cm-1:
3153 and 2974 (NH), 1628 and 1514 (CO), 1236 and 1125 (OCH2); anal. calcd. for C21H18N2O3
(346.38): C, 72.82; H, 5.24; N, 8.09; Found C, 72.80; H, 5.24; N, 8.09.
4.3.13. 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)
prop-2-en-1-one (3m): Yellow solid, Yield 65%, Rf = 0.61 (CHCl3:MeOH/9:1), mp 85-90ºC, 1H
NMR (500 MHz, DMSO-d6): δ 3.54 (s, 2H, oxazoline-NCH2), 4.29 (s, 8H, oxazoline-OCH2,
dioxane OCH2), 6.92 (t, J = 8.5 Hz, 3H, Ar-H), 7.33 (d, J = 7.5 Hz, 1H, Ar-H), 7.46 (s, 1H, Ar-
H), 7.58 (d, J = 15.5 Hz, 1H, =CH), 7.77 (d, J = 15.5 Hz, 1H, COCH=), 8.08 (d, J = 7.5 Hz, 2H,
aromatic-H), FT-IR (Alpha ATR Crystal) cm-1: 1649 and 1585 (CO), 1246 and 1056 (OCH2);
anal. calcd. for C21H19NO5 (365.38): C, 69.03; H, 5.24; N, 3.83; Found C, 68.99; H, 5.24; N,
3.81.
4.4 General Procedure for synthesis of Oxazoline-Pyrazoline hybrids (4a-p):
10

ACCEPTED MANUSCRIPT
To an equimolar mixture of compound (3a-m) and different hydrazine derivatives in methanol
(25ml), 2-3 drops of hydrochloric acid were added. The reaction mixture was refluxed with
stirring at 70ºC for specified time. The reaction progress was monitored by TLC. After
completion of reaction, the mixture was allowed to cool at room temperature and quenched by
pouring the mixture into crushed ice. The precipitate obtained was filtered, dried and
crystallized.
4.4.1. 2-((4-(5-(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-
1
dihydrooxazole (4a): Brown solid, Yield 67%, Rf = 0.41 (CHCl3:MeOH/9:1), mp 98-100ºC, H
NMR (400 MHz, DMSO-d6): δ 3.03 (dd, J = 11.2, 6.0 Hz, 1H, pyrazoline-CH2), 3.66-3.68 (m,
4H, oxazoline-OCH2, NCH2), 3.84 (dd, J = 17.6, 12.0 Hz, 1H, pyrazoline-CH2), 4.82 (s, 2H,
OCH2), 5.42 (dd, J = 12.0, 6.0 Hz, 1H, pyrazoline-CH), 6.66 (t, J = 7.2 Hz, 1H, Ar-H), 6.91-
6.96 (m, 4H, Ar-H), 7.05-7.13 (m, 2H, Ar-H), 7.26 (d, J = 8.4 Hz, 2H, Ar-H), 7.36 (d, J = 8.4
Hz, 2H, Ar-H), 7.64 (d, J = 8.8 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1594 and
1491 (C=N), 1211 and 1080 (OCH2); HRMS m/z = 433.1418 (M+2)+; anal. calcd. for
C25H22ClN3O2 (431.91): C, 69.52; H, 5.13; N, 9.73; Found C, 69.50; H, 5.14; N, 9.71.
4.4.2. 1-(5-(4-chlorophenyl)-3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (4b): Reddish-brown solid, Yield 64%, Rf = 0.62 (CHCl3:MeOH/9:1), mp
120-122ºC, 1H NMR (400 MHz, DMSO-d6): δ 2.22 (s, 3H), 3.08 (dd, J = 18.0, 4.8 Hz, 1H,
pyrazoline-CH2), 3.29 (s, 4H, oxazoline-OCH2, NCH2), 3.78 (dd, J = 18.0, 12.0 Hz, 1H,
pyrazoline-CH2), 4.72 (s, 2H, OCH2), 5.48 (dd, J = 12.0, 4.8 Hz, 1H, pyrazoline-CH), 6.96 (d, J
= 8.8 Hz, 2H, Ar-H), 7.17 (d, J = 8.4 Hz, 2H, Ar-H), 7.34 (d, J = 8.4 Hz, 2H, Ar-H), 7.68 (d, J =
13
8.8 Hz, 2H, Ar-H), C NMR (δ ppm, CDCl3, 100 MHz): 22.15, 59.19, 65.00, 115.22, 124.45,
127.99, 128.74, 129.05, 132.14, 141.91, 154.37, 159.92, 167.70, 170.34, FT-IR (Alpha ATR
Crystal) cm-1: 1595 (C=N), 1230 and 1066 (OCH2); HRMS m/z = 399.1203 (M+2)+; anal. calcd.
for C21H20ClN3O3 (397.85): C, 63.40; H, 5.07; N, 10.56; Found C, 63.40; H, 5.04; N, 10.53.
4.4.3. 4-(5-(4-chlorophenyl)-3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4c): Brown solid, Yield 72%, Rf = 0.56 (CHCl3:MeOH/9:1),
mp 50-52ºC,1H NMR (500 MHz, DMSO-d6): δ 3.13 (dd, J = 18.5, 4.8 Hz, 1H, pyrazoline-CH2),
3.55 (s, 2H, oxazoline-NCH2), 3.70 (s, 2H, oxazoline-OCH2), 3.93 (dd, J = 17.6, 15.0 Hz, 1H,
pyrazoline-CH2), 4.88 (s, 2H, OCH2), 5.61 (dd, J = 17.6, 11.5 Hz, 1H, pyrazoline-CH), 6.99 -
11

ACCEPTED MANUSCRIPT
7.04 (m, 6H, Ar-H), 7.17 (t, J = 8.0 Hz, 2H, Ar-H), 7.29 (bs, 2H, SO2NH2), 7.57 (d, J = 8 Hz,
2H, Ar-H), 7.72 (d, J = 7.5 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1635 and 1584
(C=N), 1208 and 1161(OCH2); HRMS m/z = 512.1150 (M+2)+; anal. calcd. for C25H23ClN4O4S
(510.99): C, 58.76; H, 4.54; N, 6.94; Found C, 58.79; H, 4.54; N, 6.93.
4.4.4. 4-(5-(3-chlorophenyl)-3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4d): Yellow solid, Yield 69%, Rf = 0.56 (CHCl3:MeOH/9:1),
1
mp 70-72ºC, H NMR (500 MHz, DMSO-d6): δ 3.18 (dd, J = 21.0, 12.5 Hz, 1H, pyrazoline-
CH2), 3.57 (s, 2H, oxazoline- NCH2), 3.70 (s, 2H, , oxazoline-OCH2), 3.92 (dd, J = 15.0, 12.5
Hz, 1H, pyrazoline-CH2), 4.97 (s, 2H, OCH2), 5.61 (dd, J = 11.5, 4.8 Hz, 1H, pyrazoline-CH),
7.01-7.06 (m, 4H, Ar-H), 7.17 (bs, 2H, SO2NH2), 7.34 (t, J = 8.5 Hz, 3H, Ar-H), 7.59 (bs, 3H,
Ar-H), 7.72 (d, J = 6.0 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1587 and 1498 (C=N),
1217 and 1145 (OCH2); HRMS m/z = 512.1102 (M+2)+; anal. calcd. for C25H23ClN4O4S
(510.99): C, 58.76; H, 4.54; N, 6.94; Found C, 58.75; H, 4.54; N, 6.93.
4.4.5. 4-(3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4e): Creamish solid, Yield 70%,
Rf =0.61
(CHCl3:MeOH/9:1), mp 100-103ºC, 1H NMR (400 MHz, DMSO-d6): δ 3.14 (dd, J = 17.6, 4.8
Hz, 1H, pyrazoline-CH2), 3.67 (s, 4H, oxazoline-OCH2, NCH2), 3.90 (dd, J = 17.6, 12 Hz, 1H,
pyrazoline-CH2), 4.84 (s, 2H, OCH2), 5.60 (dd, J = 12.0, 4.8 Hz, 1H, pyrazoline-CH), 6.97-7.02
(m, 6H, Ar-H), 7.23 (d, J = 8.4 Hz, 2H, SO2NH2), 7.37 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.8 Hz,
2H), 7.69 (d, J = 8.8 Hz, 2H), 13C NMR (δ ppm, DMSO, 100 MHz): 43.38, 52.33, 61.99, 65.06,
112.29, 115.25, 125.40, 127.65, 128.19, 128.25, 129.54, 132.56, 133.35, 141.13, 146.31, 150.03,
159.05, 169.50, FT-IR (Alpha ATR Crystal) cm-1: 1639 and 1594 (C=N), 1218 and 1152
(OCH2); HRMS m/z = 495.1585 (M+H)+; anal. calcd. for C25H23FN4O4S (494.54): C, 60.72; H,
4.69; N, 11.33; Found C, 60.70; H, 4.64; N, 11.33.
4.4.6. 2-((4-(5-(4-fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-
1
dihydrooxazole (4f): Yellow solid, Yield 68%, Rf = 0.61 (CHCl3:MeOH/9:1), mp 95-97ºC, H
NMR (400 MHz, DMSO-d6): δ 3.04 (dd, J = 7.2, 6.0 Hz, 1H, pyrazoline-CH2), 3.67 (s, 4H,
oxazoline-OCH2, NCH2), 3.84 (dd, J = 7.2, 12.0 Hz, 1H, pyrazoline-CH2), 4.82 (s, 2H, OCH2),
5.42(dd, J = 12.0, 6.4 Hz, 1H, pyrazoline-CH2), 6.67 (t, J = 7.2 Hz, 1H, Ar-H), 6.95 (t, J = 8.8
Hz, 4H, Ar-H), 7.09-7.15 (m, 4H, Ar-H), 7.29 (d, J = 2.0 Hz, 2H, Ar-H), 7.65 (d, J = 8.8 Hz,
12

ACCEPTED MANUSCRIPT
2H, Ar-H), FT-IR (Alpha ATR Crystal) cm-1: 1595 and 1497 (C=N), 1216 and 1086 (OCH2);
HRMS m/z = 416.1797 (M+H)+; anal. calcd. for C25H22FN3O2 (415.46): C, 72.27; H, 5.34; N,
10.11; Found C, 72.28; H, 5.34; N, 10.11.
4.4.7. 2-((4-(5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-dihydro-
1
oxazole (4g): Brown solid, Yield 62%, Rf = 0.43 (CHCl3:MeOH/9:1), mp 80-82ºC, H NMR
(400 MHz, DMSO-d6): δ 2.70 (dd, J = 16.0, 10.8 Hz, 1H, pyrazoline-CH2), 3.29 (dd, J = 16.0 ,
11.2 Hz, 1H, pyrazoline-CH2), 3.69 (s, 4H, oxazoline-OCH2, NCH2), 4.29 (s, 2H, OCH2), 4.68 (t,
J = 12.0 Hz, 1H, pyrazoline-CH2), 6.80 (d, J = 8.0 Hz, 2H, Ar-H), 6.85 (d, J = 8.4 Hz, 2H, Ar-
H), 7.24 (d, J = 8.4 Hz, 2H, Ar-H), 7.46 (d, J = 8.0 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal)
cm-1: 1588 and 1510 (C=N), 1244 and 1174 (OCH2), HRMS m/z = 352.1637 (M+H)+, anal.
calcd. for C20H21N3O3 (351.40): C, 68.36; H, 6.02; N, 11.96; Found C, 68.34; H, 6.04; N, 11.93.
4.4.8. 2-((4-(5-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-
1
dihydrooxazole (4h): Brown solid, Yield 65%, Rf = 0.91 (CHCl3:MeOH/9:1), mp 70-72ºC, H
NMR (400 MHz, DMSO-d6): δ 3.05 (dd, J = 16.8, 6.8 Hz, 1H, pyrazoline-CH2), 3.75 (s, 3H,
OCH3), 3.86-3.83 (m, 3H, pyrazoline-CH2,oxazoline-NCH2), 4.70-4.64 (m, 4H, OCH2,
oxazoline-OCH2), 5.17 (dd, J = 12.0, 7.2 Hz, 1H, pyrazoline-CH), 6.75 (t, J = 7.2 Hz, 1H, Ar-H),
6.80-6.84 (m, 3H, Ar-H), 6.87-6.98 (m, 4H, Ar-H), 7.04 (d, J = 8 Hz, 2H, Ar-H), 7.32 (d, J = 8.4
Hz, 2H, Ar-H), 7.64 (d, J = 8.8 Hz, 2H, Ar-H), 7.83 (d, J = 8.8 Hz, 1H, Ar-H), 13C NMR (δ ppm,
CDCl3, 100 MHz): 43.78, 52.33, 55.28, 64.04, 65.30, 104.39, 113.34, 114.48, 118.88, 119.47,
125.31, 126.77, 127.26, 128.89, 130.76, 140.23, 144.25, 145.11, 146.42, 151.45, 157.71, 158.14,
158.96, 159.60, 169.19, 169.41, FT-IR (Alpha ATR Crystal) cm-1: 1589 and 1493 (C=N), 1232
and 1167 (OCH2); HRMS m/z = 428.1943 (M+H)+; anal. calcd. for C26H25N3O3 (427.50): C,
73.05; H, 5.89; N, 9.83; Found C, 73.06; H, 5.90; N, 9.81.
4.4.9. 4-(3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4i): Brown solid, Yield 66%, Rf = 0.38 (CHCl3:MeOH/9:1),
mp 60-62ºC,1H NMR (400 MHz, DMSO-d6): δ 3.13 (dd, J = 17.6, 5.2 Hz, 1H, pyrazoline-CH2),
3.67 (s, 7H, oxazoline-OCH2, NCH2,OCH3), 3.89 (dd, J = 17.6, 12.0 Hz, 1H, pyrazoline-CH2),
4.83 (s, 2H, OCH2), 5.52 (dd, J = 1.6, 4.8 Hz, 1H, pyrazoline-CH), 6.74 (d, J = 7.6, 1H, Ar-H),
6.79 (d, J = 6.0 Hz, 2H, Ar-H), 6.97-7.00 (m, 4H, Ar-H), 7.02 (s, 2H, SO2NH2), 7.19 (t, J = 8.4
Hz, 1H, Ar-H), 7.54 (d, J = 8.8 Hz, 2H, Ar-H), 7.69 (d, J = 8.8 Hz, 2H, Ar-H), 13C NMR (δ ppm,
13

ACCEPTED MANUSCRIPT
DMSO, 100 MHz): 52.33, 55.48, 62.66, 65.05, 79.11, 79.44, 79.77, 112.24, 113.01, 115.24,
118.03, 125.50, 127.60, 128.13, 130.80, 133.17, 143.83, 146.53, 150.02, 159.00, 160.11, 169.51,
FT-IR (Alpha ATR Crystal)cm-1: 1590 and 1500 (C=N), 1146 and 1053 (OCH2); HRMS m/z =
507.1657 (M+H)+, anal. calcd. for C26H26N4O5S (506.57): C, 61.65; H, 5.17; N, 11.06; Found C,
61.70; H, 5.14; N, 11.08.
4.4.10. 4-(3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-5-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4j): Cream color solid, Yield 62%,
Rf = 0.39
(CHCl3:MeOH/9:1), mp 60-63ºC, 1H NMR (400 MHz, DMSO-d6): δ3.09 (dd, J = 17.6, 4.8 Hz,
1H, pyrazoline-CH2), 3.67 (s, 7H, oxazoline-OCH2, NCH2, OCH3), 3.86 (dd, J = 17.6, 12.0 Hz,
1H, pyrazoline-CH2), 4.83 (s, 2H, OCH2), 5.50 (dd, J = 11.6, 4.8 Hz, 1 H, pyrazoline-CH), 6.86
(d, J = 8.8 Hz, 2H, Ar-H), 6.97-7.01 (m, 4H, Ar-H), 7.03 (s, 2H, SO2NH2), 7.13 (d, J = 8.4 Hz,
2H, Ar-H), 7.54 (d, J = 8.8 Hz, 2H, Ar-H), 7.69 (d, J = 8.4 Hz, 2H, Ar-H), FT-IR (Alpha ATR
Crystal) cm-1: 1589 and 1500 (C=N), 1148 and 1054 (OCH2); HRMS m/z = 507.1659 (M+1)+;
anal. calcd. for C26H26N4O5S (506.57): C, 61.65; H, 5.17; N, 11.06; Found C, 61.68; H, 5.14; N,
11.03.
4.4.11. 2-((4-(1-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-dihydro
oxazole (4k): Brown solid, Yield 60%, Rf = 0.46 (CHCl3:MeOH/9:1), mp 95-96ºC,1H NMR (400
MHz, DMSO-d6): δ 2.22 (s, 3H), 3.00 (dd, J = 17.2, 6.4 Hz, 1H, pyrazoline-CH2), 3.67 (s, 4H,
oxazoline-OCH2, NCH2), 3.82 (dd, J = 17.2, 12.0 Hz, 1H, pyrazoline-CH2), 4.82 (s, 2H, OCH2),
5.33 (dd, J = 12.0, 6.4 Hz, 1H, pyrazoline-CH), 6.65 (t, J = 7.2 Hz, 1H, Ar-H), 6.95 (t, J = 7.6
Hz, 4H, Ar-H), 7.07-7.15 (m, 6H, Ar-H), 7.64 (d, J = 8.4 Hz, 2H), 13C NMR (δ ppm, DMSO-d6,
125 MHz): 21.11, 31.15, 52.32, 63.38, 65.02, 113.29, 115.17, 118.73, 126.12, 126.25, 127.65,
129.27, 129.99, 136.97, 140.15, 144.97, 147.49, 158.53, 169.55, FT-IR (Alpha ATR Crystal) cm-
1: 1596 and 1494 (C=N), 1210 and 1056 (OCH2); HRMS m/z = 412.1992 (M+H)+; anal. calcd.
for C26H25N3O2 (411.50): C, 75.89; H, 6.12; N, 10.21; Found C, 75.90; H, 6.14; N, 10.22.
4.4.12. 4-(3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4l): Reddish brown solid, Yield 64%, Rf = 0.30 (CHCl3:
1
MeOH/9:1), mp 110-112ºC, H NMR (500 MHz, DMSO-d6): δ3.16 (dd, J = 17.6, 5.0 Hz, 1H,
pyrazoline-CH2), 3.70 (s, 4H, oxazoline-OCH2, NCH2), 3.93 (dd, J = 17.6, 12 Hz, 1H,
pyrazoline-CH2), 4.86 (s, 2H, OCH2), 5.63 (dd, J = 12.0, 5.0 Hz, 1H, pyrazoline-CH), 7.00 -
14

ACCEPTED MANUSCRIPT
7.04 (m, 7H, Ar-H), 7.17 (d, J = 8.0 Hz, 2H, SO2NH2), 7.29 (bs, 1H, Ar-H), 7.58 (d, J = 8.0 Hz,
2H, Ar-H), 7.72 (d, J = 7.5 Hz, 2H, Ar-H), FT-IR (Alpha ATR Crystal)cm-1: 1589 and 1506
(C=N), 1150 and 1090 (OCH2); HRMS m/z = 522.1472 (M+H)+;
anal. calcd. for
C25H23N5O6S(521.55): C, 57.57; H, 4.44; N, 13.43; Found C, 57.55; H, 4.42; N, 13.45.
4.4.13. 4-(3-(4-((4,5-dihydrooxazol-2-yl)methoxy)phenyl)-5-(3-nitrophenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4m): Reddish brown solid, Yield 65%, Rf = 0.42 (CHCl3:
MeOH/9:1), mp 105-107ºC, 1H NMR (500 MHz, DMSO-d6): δ 3.19 (dd, J = 17.6, 5.8 Hz, 1H,
pyrazoline-CH2), 3.71 (s, 4H, oxazoline-OCH2, NCH2), 3.92 (dd, J = 25.0, 12.5 Hz, 1H,
pyrazoline-CH2) 4.88 (s, 2H, OCH2), 5.62 (dd, J = 25.0, 11.5 Hz, 1H, pyrazoline-CH), 7.00-
7.06 (m, 4H, Ar-H), 7.17 (bs, 2H, Ar-H, SO2NH2), 7.34 (t, J = 15.5 Hz, 3H, Ar-H), 7.59 (bs, 3H,
Ar-H), 7.73 (d, J = 6.5 Hz, 2H), FT-IR (Alpha ATR Crystal)cm-1: 1599 and 1511(C=N), 1159
and 1098 (OCH2); HRMS m/z = 522.1427 (M+H)+; anal. calcd. for C25H23N5O6S (521.55): C,
57.57; H, 4.44; N,13.43; Found C, 57.55; H, 4.42; N, 13.45.
4.4.14. 2-((4-(5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-
dihydrooxazole (4n): Yellow solid, Yield 59%, Rf = 0.57 (CHCl3: MeOH/9:1), mp 175-177ºC,
1H NMR (400 MHz, DMSO-d6): δ 3.90 (dd, J = 19.2, 5.2 Hz, 1H, pyrazoline-CH2), 4.23 (dd, J =
18.4, 10.0 Hz, 1H, pyrazoline-CH2), 4.69 (s, 4H, oxazoline-OCH2, NCH2), 4.87 (s, 2H, OCH2),
6.28 (dd, J = 9.6, 5.2 Hz, 1H, pyrazoline-CH), 6.92-6.97 (m, 1H, Ar-H), 7.16-7.21 (m, 3H, Ar-
H), 7.45-7.48 (m, 2H, Ar-H), 7.75 (s, 2H, Ar-H), 8.31 (d, J = 7.2 Hz, 2H, Ar-H), 8.87 (s, 1H, Ar-
H), 11.67 (s, 1H, indole-NH), FT-IR (Alpha ATR Crystal)cm-1: 1609 and 1504 (C=N), 1230 and
1120 (OCH2); HRMS m/z = 361.1681 (M+H)+; anal. calcd. for C21H20N4O2 (360.41): C, 69.98;
H, 5.59; N,15.55; Found C, 69.95; H, 5.58; N, 15.55.
4.4.15. 2-((4-(5-(1H-indol-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)methyl)-4,5-
dihydrooxazole (4o): Brown solid, Yield 58%, Rf = 0.43(CHCl3: MeOH/9:1), mp 120-122ºC, 1H
NMR (400 MHz, DMSO-d6): δ 3.56 (dd, J = 20.0, 9.6Hz, 1H, pyrazoline-CH2), 4.62 (dd, J =
18.4, 10.0 Hz, 1H, pyrazoline-CH2), 4.78 (s, 4H, oxazoline-OCH2, NCH2), 4.92 (s, 2H, OCH2),
5.35 (dd, J = 10.0, 5.5 Hz, 1H, pyrazoline-CH), 7.00-7.04 (m, 4H, Ar-H), 7.13-7.21(m, 6H, Ar-
H), 7.60 (d, J = 8.0 Hz, 3H, Ar-H), 8.24 (d, J = 7.5 Hz, 2H, Ar-H), 9.8 (s, 1H, indole-CH), 11.3
(s, 1H, indole-NH), FT-IR (Alpha ATR Crystal) cm-1: 1589 and 1495 (C=N), 1229 and 1166
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(OCH2); HRMS m/z = 437.1991 (M+H)+; anal. calcd. for C27H24N4O2 (436.51): C, 74.29; H,
5.54; N, 12.84; Found C, 74.25; H, 5.53; N, 12.85.
4.4.16. 4-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-((4,5-dihydrooxazol-2-yl)methoxy)
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4p): Brown solid, Yield 62%, Rf =
0.43 (CHCl3:MeOH/9:1), mp 115-117ºC, 1H NMR (500 MHz, DMSO-d6): δ3.14 (dd, J = 17.6,
5.2 Hz, 1H, pyrazoline-CH2), 3.70 (s, 8H, oxazoline-OCH2, NCH2, dioxane-OCH2 ), 3.85 (dd, J
= 17.6, 12.0 Hz, 1H, pyrazoline-CH2), 4.87 (s, 2H, OCH2), 5.49 (dd, J = 11.8, 5.0 Hz, 1H,
pyrazoline-CH2), 6.61-6.70 (m, 4H, Ar-H), 6.80 (d, J = 8.0 Hz, 2H, SO2NH2), 7.50-7.55 (m, 4H,
Ar-H), 7.58 (d, J = 8.0 Hz, 1H, Ar-H), 7.64 (d, J = 8.5 Hz, 2H, Ar-H), 13C NMR (δ ppm, DMSO-
d6, 100 MHz): 31.17, 43.54, 52.35, 64.40, 64.52, 65.03, 112.25, 114.71, 115.23, 118.13, 118.86,
125.54, 127.62, 128.12, 133.06, 135.16, 143.21, 143.96, 146.44, 150.00, 158.96, 169.53, FT-IR
(Alpha ATR Crystal)cm-1: 1589 and 1501(C=N), 1287 and 1150 (OCH2); HRMS m/z =
535.1652 (M+H)+; anal. calcd. for C27H26N4O6S (534.58): C, 60.66; H, 4.90; N, 10.48; Found C,
60.65; H, 4.92; N, 10.45.
4.5 In-vitro antimalarial activity:
The activity is based on schizontocidal activity of test samples. In this assay, Parasitized blood
was made by infecting red blood cells with CQS (3D7) strain and CQR (RKL9) strain of P.
falciparum with 2-3% parasitemia and 8% hematocrit in RPMI 1640 medium supplemented with
human AB+ serum. Parasitized blood was added to the wells of 96 - well plate containing 100 µl
of test sample (1 mg/ml conc.) diluted in medium at different concentrations. The plates were
incubated at 37ºC in gas mixture of 90% N2, 5% CO2 and 5% O2 for 24 hours. Then blood smear
was prepared from all wells and stained with giemsa stain. Schizonts stage (3 or more merozoites
containing) were counted and result was analysed using dose-response curves by non-linear
regression analysis using HN-NonLin Regression analysis (malaria.farch.net).
4.8 In-vivo antimalarial activity:
The in vivo antimalarial efficacy was determined by Peter’s 4 day suppressive test [19-21] by
using P. berghei NK65 strain and female swiss albino mice (20-25 g of body weight). Groups of
five mice were innoculated intraperitonially with approximately 10-15% of P. berghei infected
erythrocytes from a donor mouse. Compound 4i was administered at a dose of 25 mg/kg body
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weight and 50 mg/kg body weight by oral route after four hours. Chloroquine (5 mg/kg body
weight) was administered positive (standard) control group. Negative control group (5 mice)
were administered the same amount of solvent used to suspend the test compound. The doses
were administered on day 0, day 1, day 2 and day 3. On day 4, blood was taken from tail vein,
smears were made, fix with methanol and stained with Giemsa and parasitemia was examined
microscopically (1000 magnification). The % parasitemia and average % suppression of
parasitemia were determined as compare to negative control group. Mean survival Days (MSD)
was determined by calculating the death of mice during 30 days observation period.
4.6 In-vitro cytotoxicity assay:
Cytotoxicity of the compounds was carried out with Vero cell line using Mosmann method with
certain modifications. The cells were incubated for 72h with different dilutions of selected
compounds using MTT as reagent for the detection of cytotoxicity. 50% cytotoxic concentration
(CC50) values represent the concentration of compound required to kill 50% of the fibroblast
cells. The selectivity index (SI) (Table 1 and 2) was calculated using the formula, SI = CC50/IC50
4.7 Acute oral toxicity study:
Acute oral toxicity study of selected compound 4i was carried out by using three female Wister
rats in treated group and three rats in control group as per OECD guidelines 423 for testing of
chemicals [24-25]. The safe dose for 4i was determined as approximately below 300 mg/kg body
weight after 14 days observation period. The survived rat was sacrificed on 14th day and liver
was taken out, fixed in 10% formalin and processed for histopathological study.
4.9 Heme binding studies:
4.9.1. Binding of compound 4i with monomeric heme:
Hemin stock solution (1.2 mM) was prepared by dissolving 7.8 mg hemin chloride in 10 ml
DMSO (analytical grade). The working solutions were prepared by mixing 20 µl stock solutions
with 4 ml DMSO and 1 ml 0.2 M HEPES buffer (pH 7.4) and finally making it up to 10 ml with
double distilled deionized water. Stock solution of compound 4i (2 mM) was prepared in DMSO
for titration. The increasing concentrations (0-100 µM) of 4i were used for titration of heme (2.4
mM) and absorbance was recorded at 402 nm. The exactly same procedure was adopted for
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conducting the experiment at pH 5.6, except that 2-[N-morpholino] ethanesulphonate (MES)
buffer (pH 5.4) was substituted for HEPES buffer.
4.9.2. Binding of 4i with µ- oxo dimeric heme:
Heme stock solution (10mM) was prepared by dissolving hemin chloride in 0.1 M NaOH and
sonicated for 30 minutes. Heme solution (60 µM) was prepared by diluting stock solution with
20mM phosphate buffer (pH 5.8). Stock solution (2mM) of 4i was prepared in DMSO. Titrations
were done by specified addition of of 4i stock solution to 60 µM heme solution and absorbance
was recorded at 362 nm.
Acknowledgements: The authors thank to Jamia Hamdard for providing research laboratories
(chemistry and microbiology), animal house and instrumentation facilities (NMR, mass and IR)
at the institute. One of the authors, Pandey, A.K. expresses thanks to National Institute of
Malaria Research, Dwarka, New Delhi for providing guidance and co-operation for conducting
antimalarial activity and to IIIM, (CSIR Lab), Jammu for their help in recording HRMS of
compounds.
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Table Captions
1. Table 1: In vitro antimalarial activity of compounds (3a-m) against CQS (3D7) and CQR
(RKL9) strains
2. Table 2 In vitro antimalarial activity of title compounds (4a-p) against CQS (3D7) and
CQR (RKL9) strains
3. Table 3 In vivo antimalarial activity of 4i in mice infected with P. berghei
4. Table 4 Association constants for 4i and chloroquine with heme
Figure Captions
5. Fig. 1. Design strategy for hybrid compounds bearing potential scaffolds
6. Fig. 2. Liver histopathogical study (10X): (a) Showing normal architecture of
hepatocytes in control group; (b) Showing minimal inflammatory cells infiltration in
treated group
7. Fig. 3. Microscopic view (1000 X) of (a) chloroquine (5mg/kg body weight) treated mice
RBC and (b) 4i (50 mg/kg body weight) treated mice RBC (day 4)
8. Fig. 4. (a) Titration of 4i with monomeric heme at pH 5.6 and (b) Job’s plot of
monomeric heme complex formation with 4i at pH 5.6
9. Fig. 5. (a) Titration of 4i with monomeric heme at pH 7.4 and (b) Job plot of monomeric
heme complex formation with 4i at pH 7.4
10. Fig. 6. (a) Titration of 4i with µ-oxodimeric heme at pH 5.8 and (b) Job’s plot of µ-
oxodimeric heme complex formation with 4i at pH 5.8
11. Scheme 1. Synthesis of intermediate compound (2)
12. Scheme 2. Synthesis of title compounds (4a-p) with chalcone intermediates (3a-m).
21

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Scheme 1. Synthesis of intermediate compound (2)
22

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Scheme 2. Synthesis of title compounds (4a-p) with chalcone intermediates (3a-m)
23

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Table 1 In vitro antimalarial activity of compounds (3a-m) against CQS (3D7) and CQR (RKL9)
strains
Compd.
R
Pf3D7
IC50 (µg/ml)a
PfRKL9
IC50 (µg/ml)a
RId
cLogPd
SIb
SIb
0.737
0.557
0.577
0.813
1.571
1.822
2.68
ND
ND
1.822
ND
ND
ND
3.27
ND
ND
ND
ND
ND
ND
ND
ND
3.05
ND
1.5891
2.6291
2.6291
2.0591
1.8351
1.8351
2.4151
1.6591
1.6591
1.2491
1.2491
1.9161
2
-
4-Cl
25.5
ND
ND
ND
ND
ND
ND
ND
ND
23.6
ND
7.8
ND
ND
ND
ND
ND
ND
ND
ND
7.7
3a
3b
3c
3d
3e
3f
3-Cl
4-F
ND
4-OMe
3-OMe
4-Me
4-NO2
3-NO2
4-OH
3-OH
Ph
ND
ND
ND
3g
3h
3i
0.723
1.571
2.437
0.515
1.773
ND
ND
ND
1.571
ND
3j
3k
ND
-
-
0.936
4.233
ND
ND
ND
ND
ND
ND
ND
ND
3.6221
1.8401
3l
3m
aIC50: Concentration corresponding to 50% growth inhibition of the parasite.
bSI: IC50value of cytotoxic activity/ IC50 value of antimalarial activity
cLogPc: calculated by Chem Draw Ultra Software, Version 12.0
dRI (Resistance Index) = IC50:PfRKL9 / IC50: Pf3D7

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Table 2 In vitro antimalarial activity of title compounds (4a-p) against CQS (3D7) and CQR
(RKL9) strains
Compd.
R1
R
Pf3D7
PfRKL9
IC50
RId
cLogPc
IC50
bSI
bSI
(µg/ml)a
0.477
(µg/ml)a
1.571
Ph
COMe
PhSO2NH2
PhSO2NH2
PhSO2NH2
Ph
4-Cl
4-Cl
23.3
29.5
22.2
34.2
19.8
ND
ND
30.4
12.5
18.4
ND
ND
ND
ND
21.3
ND
-
7.1
9.6
5.7
8.7
4.6
ND
ND
3.29
3.06
3.87
3.90
4.23
ND
6.5122
3.5772
4.6752
4.6752
4.1052
5.9422
3.2232
5.7182
3.8812
3.8812
6.2982
3.7052
3.7052
1.6092
4.1042
3.2282
-
4a
4b
4c
4d
4e
4f
0.513
0.471
0.552
0.419
0.645
1.032
0.545
0.322
0.413
1.009
0.627
1.02
1.571
1.822
2.154
1.773
ND
4-Cl
3-Cl
4-F
4-F
H
4-OMe
4-OMe
ND
ND
4g
4h
4i
Ph
1.571
0.737
0.936
ND
10.5 2.88
PhSO2NH2 4-OMe
PhSO2NH2 3-OMe
5.5
8.1
ND
ND
ND
ND
2.3
ND
-
2.29
2.27
ND
ND
ND
ND
9.22
ND
-
4j
Ph
4-Me
4k
4l
PhSO2NH2
4-NO2
ND
PhSO2NH2
3-NO2
ND
4m
4n
4o
4p
CQ
-
-
-
-
H
0.657
0.459
2.154
0.405
ND
Ph
4.233
ND
PhSO2NH2
-
2.154

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Table 3 In vivo antimalarial activity of 4i in mice infected with P. berghei
Compound
dose
Route
% reduction Mean survival
(mg/kg/day X 4)
parasitaemia
(day 4)
days (MSD)
4i
4i
25
50
5
po
po
po
-
63.89
79.33
97.10
-
11.4
15.6
30
Chloroquine
Negative Control
-
7.2

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Table 4 Association constants for 4i and chloroquine with heme
Compound
Monomeric heme
µ-oxo heme
Log K± σ
pH 5.8
Log K± σ
pH 5.6
pH 7.4
4.98
4.82
5.87
5.20
4.34
5.60
4i
Chloroquine

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Fig. 1. Design strategy for hybrid compounds bearing potential scaffolds