New heterocyclic hybrids of pyrazole and its bioisosteres: Design, synthesis and biological evaluation as dual acting antimalarial-antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2015.02.038

A new series of pyrazole derivatives were synthesized by hybridization with five-membered heterocyclic moieties such as thiazoles, thiazolidinones, 1,3,4-thiadiazoles and pyrazolines. The compounds were evaluated for their in vivo antimalarial activity ag

Full text of DOI:10.1016/j.ejmech.2015.02.038

Accepted Manuscript
New heterocyclic hybrids of pyrazole and its bioisosteres: Design, synthesis and
biological evaluation as dual acting antimalarial-antileishmanial agents
Adnan A. Bekhit, Ahmed M.M. Hassan, Heba A. Abd El Razik, Mostafa M.M. El-
Miligy, Eman J. El-Agroudy, Alaa El-Din A. Bekhit
PII:
S0223-5234(15)00134-8
10.1016/j.ejmech.2015.02.038
EJMECH 7720
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2014
Revised Date: 1 November 2014
Accepted Date: 19 February 2015
Please cite this article as: A.A. Bekhit, A.M.M. Hassan, H.A. Abd El Razik, M.M.M. El-Miligy, E.J. El-
Agroudy, A.E.-D.A. Bekhit, New heterocyclic hybrids of pyrazole and its bioisosteres: Design, synthesis
and biological evaluation as dual acting antimalarial-antileishmanial agents, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.038.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Series of new pyrazole derivatives hybridized with five-membered heterocyclic
moieties were evaluated for dual activity as antimalarial-antileishmanial agents. Six
compounds displayed dual activity against malaria and leishmaniasis.

ACCEPTED MANUSCRIPT
New heterocyclic hybrids of pyrazole and its bioisosteres: Design, synthesis
and biological evaluation as dual acting antimalarial-antileishmanial agents
a
a
a
a
Adnan A. Bekhit , Ahmed M. M. Hassan , Heba A. Abd El Razik , Mostafa M. M. El-Miligy ,
a
b
Eman J. El-Agroudy , Alaa El-Din A. Bekhit
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21521, Egypt
b
Food Science, University of Otago, Dunedin, New Zealand
ABSTRACT
A new series of pyrazole derivatives were synthesized by hybridization with five-membered
heterocyclic moieties such as thiazoles, thiazolidinones, 1,3,4-thiadiazoles and pyrazolines.
The compounds were evaluated for their in vivo antimalarial activity against Plasmodium
berghei infected mice and the most active derivatives were further examined for their in vitro
antimalarial activity against chloroquine resistant (RKL9) strain of P. falciparum. Compounds
2
c, 2d, 4b, 4c, 4d, 5a, 6c, 8c and 9b had more than 90% parasite suppression activity of that
found with the antimalarial reference standard drug, chloroquine phosphate and had lower IC50
values than chloroquine. Compounds 4b and 9b were the most active derivatives, and their
activities were 5-fold higher than chloroquine. All the newly synthesized compounds were
evaluated for their in vitro antileishmanial activity against Leishmania aethiopica
promastigotes and antiamastigote. The results showed that compounds 2c, 2d, 3d, 4b, 4c, 4d
and 5a had lower or similar IC50 values than the reference standard drugs, amphotericin B and
miltefosine. Compound 3d had the highest antileishmanial activity. Collectively, compounds
2
c, 2d, 4b, 4c, 4d and 5a exhibited dual activity against malaria and leishmaniasis and were
safe and well tolerated by the experimental animals orally up to 300 mg/ kg and parenterally
up to 100 mg/ kg.
Keywords
Pyrazole, thiazole, thiazolidinone, 1,3,4-thiadiazole, antimalarial, antileishmanial,.
Correspondence: Adnan A. Bekhit,
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, AlexandriaUniversity,
Alexandria21521,
Egypt.
E-mail: adnbekhit@pharmacy.alexu.edu.eg
Fax: 20-3-4873273
1

ACCEPTED MANUSCRIPT
1
. Introduction
Malaria and leishmaniasis are dangerous tropical protozoal diseases that continue to
increase in prevalence and cause morbidity all over the world. Despite attempts to control
malaria over the last few decades, its prevalence has increased in developing countries [1].
Leishmaniasis is very common disease in Africa. Globally, it has been estimated that there are
about 12-15 million people currently infected with leishmaniasis and it is predicted that this
number will increase by about 2 million annually as a result of new infected cases [2]. Current
drugs used for the treatment of both diseases are effective, but resistance and side effects have
been reported almost for all the current therapeutic agents [2].
Pyrazole derivatives are very interesting compounds that have been reported to possess
versatile biological activities, including; anticancer [3, 4], anti-inflammatory [5, 6], antiviral
[7, 8] and antimicrobial [9, 10] activities. Furthermore, several reports showed that pyrazole
derivatives A[11], B[12], and C[13] had antimalarial activity, whereas compounds D[14],
E[15], and F[16] exhibited antileishmanial activity (Figure 1).
There is high interest in utilizing different heterocyclic skeletons for the development
of dual therapeutic drugs [12, 17]. It has been reported that some five-membered heterocyclic
ring systems, such asthiazolidin-4-one G [18], thiazole H [19], 1,3,4-thiadiazole I [20] and
pyrazoline J [21] (Figure 2), have beneficial biological activities against malaria and
leishmaniasis. As a continuation of our research toward the discovery of new antimalarial
and/or antileishmanial agents [12, 22-24]; we designed, synthesized some pyrazole hybrids
with these five-membered bioisosteres I and II (Figure 3) and evaluated the compounds for
their dual activity as antimalarial-antileishmanial agents. The target compounds were designed
to be attached to different moieties to impart variable electronic and lipophilic properties with
the aim of understanding the effects of these properties on the antimalarial and/or
antileishmanial activities. Nitro-heterocyclic compounds were found to be effective against
various bacterial and parasitic infections due to nitro-reductases mediated activation within the
pathogen that produce toxic effects on bacteria and parasites [25]. Therefore, a nitro group was
added as one of the substituents to increase the antiprotozoal activities of the targeted
compounds.
2
. Chemistry
The target compounds were synthesized as outlined in Schemes 1 and 2. In scheme 1,
the starting compounds 1,3-diaryl-1H-pyrazole-4-carboxaldehydes (1a-d) were synthesized by
reacting their hydrazones with Vilsmeir-Haack reagent, as described earlier [26-28]. The 1,3-
diaryl-1H-pyrazole-4-carboxaldehydes (1a-d) were then reacted with phenylthiosemicarbazide
to obtain the key intermediate N-phenylhydrazinecarbothioamide derivatives (2a-d) [29-31],
which were cyclized with acetic anhydride to give diacetyl-1,3,4-thiadiazole derivatives (3a-
d)[30, 32], or with ethylbromoacetate to afford 3-aryl-4-thiazolidinone derivatives (4a-d) [30,
3
2, 33], or with 4-bromophenacyl bromide to yield the corresponding 3,4-diarylthiazole
derivatives (5a-d)[7, 34].
2

ACCEPTED MANUSCRIPT
In scheme 2, the key intermediates 3-(1,3-diaryl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-ones
(6a-c) were afforded from the condensation of the starting pyrazole aldehydes 1a, 1b and 1c
with acetophenonein alcoholic solution of potassium hydroxide [35, 36]. The obtained
pyrazoline derivatives intermediates (6a-c) were then reacted with phenylhydrazine
hydrochloride in acidic medium to afford 1,3-diaryl-4-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-
5
-yl)pyrazoles 7a-c [35, 37] or with hydrazine hydrate in ethanol, acetic acid and propanoic
acid to give 1,3-diaryl-4-(3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)pyrazoles (8a-c) [35, 38, 39],
1
1
,3-diaryl-4-(1-acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)pyrazoles (9a-c) [35, 40] and
,3-diaryl-4-(3-phenyl-1-propanoyl-4,5-dihydro-1H-pyrazol-5-yl)pyrazoles (10a-c) [41],
respectively.
3
. Results and discussion
3
.1. Analytical results
The IR spectra of the starting pyrazole 4-carboxaldehydes (1a-d) showed a
-1
characteristic peak at 1669-1688 cm , which was attributed to the aldehydic C=O group. Their
1
H-NMR spectra displayed two singlet that were attributed to pyrazole-C -H and the aldehydic
5
1
proton. The H-NMR spectra of carbothioamide derivatives (2a-d) showed the disappearance
of the aldehydic proton and the existence of two deuterium exchangeable singlet, which were
1
attributed to CH=N-NH-CS-NH-phenyl. The H-NMR spectra of thiadiazole, thiazolidinone
and thiazoline derivatives (3a-d), (4a-d) and (5a-d) lacked deuterium exchangeable singlet,
which is characteristic for the two NH of thiosemicarbazone moiety. Compounds (3a-d)
showed a singlet of thiadiazole-C proton at 7.08-7.17 ppm, while compounds (4a-d) and (5a-
5
d) showed one singlet at 4.11-4.15 ppm and a singlet at 6.70-6.77 ppm, which were assigned
1
for thiazolidine-C proton and thiazoline-C proton, respectively. The H-NMR spectra of
5
5
chalcones (6a-c) showed two doublets that appeared in the range of 7.59-7.98 ppm; each was
integrated for one proton and was assigned to the vinylic protons. The E configuration was
obtained as the coupling constant of the vinylic protons and was equal to 15.3 Hz [42].
The IR spectra of all pyrazoline derivatives (7-10 a-c) lacked the carbonyl peak in the
1
corresponding chalcones, which verified their synthesis. H-NMR spectra of target pyrazoline
derivatives showed the characteristic peaks for pyrazoline-C and C protons, at their expected
4
5
1
chemical shifts. H-NMR spectra of compounds (8a, b) showed broad singlet at 7.51-7.55
ppm, which is deuterium exchangeable and was assigned for pyrazoline NH proton, whereas
NH proton in compound 8c appeared as doublet, due to coupling with pyrazoline-C proton.
5
1
The H-NMR spectra of compounds (9a-c) showed singlet representing the COCH
3
group. In
compounds (10a-c), characteristic triplet and quartet peaks of propanoyl protons were found.
3
3
.2. Biological screening
.2.1. In vivo antimalarial activity testing against P. berghei
Results for the in vivo anti-malarial activity of the synthesized compounds are shown
in Table 1. All the compounds showed parasitaemia level lower than the negative control (<
5
0% of that found in the control), indicating that they have significant suppressive effect
3

ACCEPTED MANUSCRIPT
against P. berghei. Compounds 2c, 2d, 4b, 4c, 4d, 5a, 6c, 8c and 9b exhibited the highest
activity with percent suppression > 90%.
Pyrazolethiosemicarbazone derivatives (2c and 2d) had high antimalarial activity, and
the activity was increased upon cyclization into 3-aryl-4-thiazolidinone (4b, 4c and 4d). Also,
the antimalarial activity of pyrazolethiosemicarbazone derivatives (2a and 2b) was
significantly increased upon cyclization into 4,5-diarylthiazole (5a and 5b). The low
dissolution rate of some derivatives of 3-phenyl-2,3-dihydrothiazole (5c and 5d) may explain
their lower percent suppression compared to their similar analogs. The presence of three atoms
spacer between the pyrazole ring and its bioisosteres in the cyclized derivatives exhibited
better antimalarial activity than the directly attached ones. For instance, compounds obtained
from the hybridization between pyrazole and 4-thiazolidinone (4b, 4c and 4d) or thiazole
moiety (5a) showed better antimalarial activity than the hybridization with 1,3,4-thiadiazole
(3a-d) or pyrazoline moieties except in (8c and 9b).
It was clear that most of the highly active antimalarial compounds were bearing a nitro
group (2c, 2d, 4c, 4d, 6c, 8c), which highlight the importance of nitro compounds in treatment
of some infectious diseases. Compound 8c was almost as active as its chalcone 6c. This
probably can be attributed to the presence of nitro groups in both compounds that might be
responsible for activity and masked any minor difference due to synthesis. Hybridization
between pyrazole and pyrazoline ring possessed a significant antimalarial activity and this
activity varied according to the substitution at position 1. The 1H-pyrazoline derivatives (8a-c)
showed good activity, which was slightly increased when H was replaced by phenyl (7a-c),
with the exception of 8c, which was more active than 7c. The 1- acetylpyrazoline derivatives
(9a and 9b) showed higher activity than unsubstituted pyrazoline (8a and 8b) and
phenylpyrazoline (7a-c) compounds. Compound 9c was less active compared to 9a and 9b,
which might be due to the low solubility of this compound and likely subsequent low
absorption. The 1-propanoylpyrazoline derivatives (10a-c) showed low antimalarial activity
compared with their parent unsubstituted compounds. Generally, hybridization of pyrazoles
with other bioisosteres increased the antimalarial activity more than compounds lacking
hybridization (2a-d) and (6a-c).
3
.2.2. In vitro antimalarial activity testing against P. falciparum
The most active compounds 2c, 2d, 4b, 4c, 4d, 5a, 6c, 8c and 9b that showed the
highest in vivo percent suppression against P. berghei, were then evaluated for their in vitro
anti-plasmodial activity against chloroquine resistant (RKL9) strains of P. falciparum using
the standard method of Trager and Jensen [43]. Results revealed that all selected compounds
had greater activity than chloroquine phosphate (IC50 = 0.1920 ꢀM) against chloroquine
resistant (RKL9) strain of P. falciparum. Compounds 4b and 9b were the most potent against
RKL9 strains (Table 2). This finding was rationalized by performing molecular docking for
these compounds on P. falciparum dihydrofolate reductase (Pf-DHFR) active site.
3
.2.3. In vitro antileishmanial activity on L. aethiopica promastigotes
®
A quantitative colorimetric assay using the oxidation-reduction indicator Alamar blue
was developed to measure cytotoxicity of the synthesized compounds against the protozoan
parasite L. aethiopica. Alamar blue assay was used to determine the viability of promastigotes
and evaluate the antileishmanial activity of the synthesized compounds. Compounds 2c, 2d,
4

ACCEPTED MANUSCRIPT
3
d, 4b, 4c, 4d and 5a showed higher activity than the reference standard drugs amphotericin B
and miltefosine, whereas compounds 1a, 1c, 1d, 2b, 5b, 6a and 7c showed lower
antileishmanial activity compared to both standard drugs. Other compounds were found to be
less active than amphotericin B but more potent than miltefosine (Table 3). The
antileishmanial activity of the most active compounds was rationalized through molecular
docking of these compounds in the active site of Leishmania major pteridine reductase crystal
structure Lm-PTR1.
The hybridization between pyrazole and 3-aryl-4-thiazolidinone (4b, 4c, 4d) or 3,4-
diarylthiazole (5a), through three atoms spacers, showed high inhibitory effect on the
promastigotes, with IC50 values ranging from 0.0142- 0.0341 ꢀg/ml. Some carbothioamide
derivatives (2c and 2d) also showed high inhibitory effect. The nitrated carbothioamide
derivatives (2c and 2d) and their cyclized 4-thiazolidinone derivatives (4c and 4d) showed
higher antileishmanial activity than the non-nitrated compounds. This may be explained by the
presence of nitro group that may contribute the activity of some trypanosomes, especially in
Leishmania.
The hybridization with 1,3,4-thiadiazole, compounds 3a, 3b and 3c, resulted in lower
inhibitory effect on the promastigotes compared to amphotericin B. This may be due to the
direct attachment of the heterocyclic ring to the parent pyrazole ring. An exceptionally high
antileishmanial activity was found for compound 3d. This high activity was further explored
using molecular docking to PTR1 active site.
The attachment of pyrazoline ring directly to the parent pyrazole resulted in
pronounced antileishmanial activity. For example, unsubstituted pyrazoline derivatives 8a-c
possessed better antileishmanial activity than their phenyl-substituted derivatives 7a-c.
Similarly, 1-propanoylpyrazoline derivatives 10a-c had better antileishmanial activity
compared to their phenyl substituted ones 7a-c and the 1-acetylpyrazoline derivatives (9a and
9
b). The higher antileishmanial activity of 1-acetylpyrazoline derivative 9c (IC50 = 1.0882 ꢀg
/
ml) relative to all other pyrazoline derivatives may be explained by its strong binding
affinities with receptor active site, good solubility and the presence of nitro group in its
structure. Most of the 1,3-diarylpyrazole-4-carboxaldehyde derivatives (1a, 1c and 1d)
showed pronounced antileishmanial activity but their activities were lower than the standard
drug miltefosine. However, the condensation of these aldehydes into carbothioamides 2a-d
and chalcones 6a-c resulted in increased antileishmanial activity.
2
.2.4. In vitro antileishmanial activity on L. aethiopica amastigotes
The nitrophenylpyrazolylthiadiazole 3d and the nitrophenylpyrazolylthiazolidinone 4d
had lower IC values than amphotericin B and miltefosine against L. aethiopica amastigotes.
5
0
The
nitrophenylpyrazolylthiosemicarbazones
2c
and
2d,
as
well
as
nitrophenylpyrazolylthiazolidinone 4c were less active than amphotericin B but were more
potent than miltefosine (Table 4). The thiazoline derivative 5a was found to be equipotent to
miltefosine against L. aethiopica amastigotes.
3
.2.5. In vivo acute toxicity testing
The most active antimalarial and antileishmanial compounds, 2c, 2d, 3d, 4b, 4c, 4d,
a, 6c, 8c and 9b, were tested for their toxicity in mice. The experimental mice did not have
5
5

ACCEPTED MANUSCRIPT
any toxicity signs after treatment with test compounds. There was no significant difference in
the weight of the mice and no death cases was recorded during 3 days of observation post
administration of the test compounds (data not shown). The test compounds were well
tolerated by the experimental animals orally up to 300 mg/kg. Moreover, these compounds
were tested for their toxicity through the parenteral route and the results revealed that all the
selected test compounds were nontoxic up to 100 mg/ kg.
3
.3. Molecular docking
In the current study, molecular docking was performed in order to rationalize the
obtained biological results. The interactions of the synthesized compounds with the active site
of target macromolecules were investigated to study the mode of binding and their orientations
and related to their antimalarial and/ or antileishmanial activity. Two characteristic proteins
were used: Plasmodium falciparum dihydrofolate reductase Pf-DHFR (PDB ID: 1J3I) and
Leishmania major pteridine reductase Lm-PTR (PDB ID: 2BFM).
3
.3.1. Antimalarial docking study
The binding site of P. falciparum wild-type dihydrofolate reductase Pf-DHFR (PDB
ID: 1J3I) was explored computationally, which provides some insights in the discovery of
novel antimalarial drugs and necessary structural requirement for antifolates in antimalarial
chemotherapy [44]. The interaction of Pf-DHFR with its co-crystallized ligand 6,6-dimethyl-
1
-[3-(2,4,5-trichlorophenoxy)propoxy]-1,6-dihydro-1,3,5-triazine-2,4-diamine
(WR99210)
showed hydrogen bond interaction with Ile 14, Asp 54 and Ile 164, in addition to hydrophobic
interactions with other amino acid residues such as Cys 15, Ala 16, Met 55 and Phe 58 (Figure
4
). Docking results for compound 4b showed that thiazolidinone carbonyl moiety exhibited
hydrogen bond interaction with Arg 122 (Figure 5). On the other hand, compound 9b revealed
hydrogen bondg interaction between the pyrazoline moiety and Ser 108 (Figure 6).
3
.3.2. Antileishmanial docking study
The binding site of Leishmania major pteridine reductase Lm-PTR1 (PDB ID: 2BFM)
was explored computationally, which provides discernment of the novel antileishmanial drugs.
PTR1 represents a target for the development of improved therapies for infections caused by
this protozoan [45]. The determination of three dimensional co-crystal structure of Lm-PTR1
complex with 5-[(3,4,5-trimethoxyphenyl)methyl]pyrimidine-2,4-diamine (trimethoprim,
TOP) showed hydrogen bond interaction with Tyr 194, a water molecule is trapped between
the diaminopyrimidine, which led to a hydrogen bond with TOP N , and Ser 111, as well as
2
hydrophobic interactions with Phe 113 and other amino acid residues (Figure 7). Leishmanial
docking results for compound 3d showed hydrogen bond interaction between acetamide
carbonyl moiety and Arg 17, with scoring of interactions equal to 54% which was the best
scoring result among all tested compounds (Figure 8). Compound 4c formed hydrogen
bonding interaction between side chain nitrogen and Tyr 194, in addition to other hydrophobic
interactions with Phe 113 and Arg 17 (Figure 9).
4
. Conclusion
6

ACCEPTED MANUSCRIPT
The objectives of the present study were to design, synthesize and investigate the
antimalarial and antileishmanial activities of some pyrazole hybrids with five-membered
bioisosteres to synthesize new structure leads serving as dual antimalarial-antileishmanial
agents. Results of the in vivo antimalarial screening in mice infected with chloroquine
sensitive P. berghei revealed that compounds 2c, 2d, 4b, 4c,4d, 5a, 6c, 8c and 9b exhibited
parasitemia suppression > 90%. These compounds were further tested for their in vitro
antimalarial activity against chloroquine resistant (RKL9) strain of P. falciparum. These
compounds showed IC50 values better than the standard drug chloroquine phosphate.
Compounds 4b and 9b were the most active and were 5-fold more active than chloroquine.
These findings were supported by the docking results obtained for these molecules, which
demonstrated that these compounds established hydrogen bond interactions with some amino
acid residues in the pocket of Pf-DHFR, in addition to some hydrophobic interactions with
good scoring results.
The in vitro antipromastigote activity demonstrated that compounds 2c, 2d, 3d, 4b, 4c,
4
d and 5a had IC50 better than both standard drugs miltefosine and amphotericin B
deoxycholate, which indicates their high antileishmanial activity against L. aethiopica.
Compound 3d was 3-fold more active than amphotericin and 225 folds more active than
miltefosine. Furthermore, compounds 3d and 4d showed lower IC values than amphotericin
5
0
B and miltefosine against L. aethiopica amastigotes. These results were rationalized through
molecular docking. These compounds showed hydrogen bonding with some amino acid
residues in Lm-PTR1 active site, which showed good binding profile. Toxicity studies for the
most active compounds indicated their safety orally and parenterally up to 300 and 100 mg/
kg, respectively. In conclusion, compounds 2c, 2d, 4b, 4c, 4d and 5a demonstrated dual
activity against both malaria and leishmania and represent fruitful scaffolds for the
development of dual acting antileishmanial-antimalarial agents.
5
. Experimental:
5
.1. Chemistry
Melting points were determined in open-glass capillaries using a Griffin melting point
apparatus and are all uncorrected. Infrared spectra (IR) were recorded on Perkin-Elmer 1430
1
13
infrared spectrophotometer. H-NMR and C-NMR were scanned on Jeol-400 MHz NMR-
spectrometer (DMSO-d6) and chemical shifts are given in δ (ppm) downfield from
tetramethylsilane (TMS) as internal standard. Microanalyses were performed on Vario El Fab-
Nr elemental analyzer. Following up of the reactions as performed by thin-layer
chromatography (TLC) on silica gel (60 GF254) coated glass plates and the spots were
visualized by exposure to iodine vapors or UV-lamp at λ 254 nm for few seconds.
5
.1.1. General method for preparation of 1,3-diaryl-1H-pyrazole-4-carboxaldehydes 1a-d
Few drops of glacial acetic acid, p-substituted phenylhydrazine hydrochloride (25
mmole) and anhydrous sodium acetate (2.05 g, 25 mmole) were added to a solution of p-
substituted acetophenone (25 mmole) in ethanol (10 ml). The reaction mixture was heated
under reflux for 2-5 h, and then allowed to cool to room temperature. The separated solid
3
product was filtered, washed with ethanol and dried. POCl (7.20 g, 4.3 ml, 25 mmole) was
7

ACCEPTED MANUSCRIPT
added to dry dimethylformamide (19.38 g, 20.4 ml, 265 mmole) in ice bath drop wise over a
period of 30 minutes, stirred for 45 minutes, then the previously prepared hydrazone was
added drop wise at 0 °C, stirred and left to reach room temperature The reaction mixture was
then heated at 70–80 °C for 3-5 h. The reaction mixture was poured onto crushed ice and
boiled. The precipitate obtained was then filtered, washed with water, dried and crystallized
from ethanol.
1
,3-Di(4-methylphenyl)-1H-pyrazole-4-carboxaldehyde1a
The product was obtained as a yellow solid. Yield 85%; m.p. 130°C; IR (cm ): 1670
-1
1
(
C=O), 1519 (C=N); H-NMR (DMSO-d
6
, δ ppm): 2.36, 2.38 (2s, each 3H, 2CH
-C3,5-H), 7.36 (d, J = 7.8 Hz, 2H, N-C -CH -C3,5-H), 7.82 (d, J =
-C2,6-H), 7.86 (d, J = 8 Hz, 2H, C-C -CH -C2,6-H), 9.21 (s, 1H,
pyrazole C -H); 9.96 (s, 1H, CHO). C-NMR (DMSO-d , δ ppm):21.02, 21.42 (2 CH ),
3
), 7.31 (d, J
=
7
8 Hz, 2H, C-C
.8 Hz, 2H, N-C
6
H
4
-CH
-CH
3
6
H
4
3
6
H
4
3
6
H
4
3
1
3
5
6
3
1
19.61 (N-methylphenyl-C , ), 122.45 (pyrazole-C ), 129.02 (C-methylphenyl-C ), 129.08 (C-
2 6 4 1
methylphenyl-C , ), 129.62 (C-methylphenyl-C , ), 130.56 (N-methylphenyl-C , ), 134.91
2
6
3 5
3 5
(
pyrazole-C ), 136.91 (C-methylphenyl-C ), 137.70 (N-methylphenyl-C ), 139.18 (N-
5 4 4
methylphenyl-C ), 153.08 (pyrazole-C ), 185.08 (C=O); Elemental analysis: Calcd for
1
3
C H N O: C 78.24, H 5.84; N 10.14, found C78.51, H 5.64, N 10.32.
1
8
16
2
1
-(4-Bromophenyl)-3-(4-methylphenyl)-1H-pyrazole-4-carboxaldehyde 1b
-1
The compound was obtained as a yellow solid. Yield 91%; m.p. 138-140°C; IR (cm ):
1
1
669 (C=O), 1520 (C=N), 725 (C-Br); H-NMR (DMSO-d , δ ppm): 2.37 (s, 3H, CH ), 7.31
6
3
(d, J = 7.65 Hz, 2H, C-C
6
H
4
-CH
3
-C3,5-H), 7.75 (d, J = 8 Hz, 2H, C
6
H
4
-Br-C2,6-H), 7.81 (d, J =
8
Hz, 2H, C
6 4 6 4 3
H -Br-C3,5-H), 7.95 (d, J = 7.65 Hz, 2H, C-C H -CH -C2,6-H), 9.30 (s, 1H,
+
•
pyrazole C
3
1
5
-H), 9.97 (s, 1H, CHO); EIMS m/z (% relative abundance): 342 (100) (M +2),
+
•
41 (55), 340 (100) (M ), 399 (34), 325 (42), 232 (11), 157 (12), 155 (13), 130 (14), 116 (15),
15 (15), 91 (26), 90 (11), 89 (16), 80 (34), 77 (11), 76 (22), 75 (19), 65 (17), 64 (17), 63 (13);
Elemental analysis Calcd for C H BrN O: C 59.84, H 3.84, N 8.21, found C 60.02, H 3.58,
1
7
13
2
N 8.44.
1
-(4-Methylphenyl)-3-(4-nitrophenyl)-1H-pyrazole-4-carboxaldehyde 1c
-
1
The compound was obtained as a yellow solid. Yield 80%; m.p. 150-152°C; IR (cm ):
1
1
7
679 (C=O), 1601 (C=N), 1516, 1345 (NO ); H-NMR (DMSO-d , δ ppm): 2.38 (s, 3H, CH ),
2
6
3
.40 (d, J = 8.6 Hz, 2H, C H -CH -C -H), 7.88 (d, J = 8.6 Hz, 2H, C H -CH -C -H), 8.27
6
4
3
3,5
6
4
3
2,6
(
d, J = 7.8 Hz, 2H, C H -NO -C -H), 8.37 (d, J= 7.8 Hz, 2H, C H -NO -C -H), 9.37 (s, 1H,
6 4 2 2,6 6 4 2 3,5
pyrazole C -H), 10.01 (s, 1H, CHO); Elemental analysis Calcd for C H N O : C 66.44, H
5
17 13
3
3
4
1
1
.26, N 13.67, found C 66.40, H 4.38, N 13.39.
-(4-Bromophenyl)-3-(4-nitrophenyl)-1H-pyrazole-4-carboxaldehyde 1d
The compound was obtained as a yellow solid. Yield 40%; m.p. 198-200°C; IR (cm ):
-
1
1
688(C=O), 1602 (C=N), 1525, 1344 (NO
-Br-C2,6-H), 7.99 (d, J = 8.3Hz, 2H, C
Hz, 2H, C H -NO -C -H), 8.36 (d, J = 8.6 Hz, 2H, C H -NO -C -H), 9.46 (s, 1H,
2
), 713(C-Br); H-NMR (DMSO-d
6
, δ ppm): 7.80
-Br-C3,5-H), 8.27 (d, J = 8.6
(d, J = 8.3 Hz, 2H, C
6
H
4
6 4
H
6
4
2
2,6
6
4
2
3,5
pyrazoleC -H), 10.03 (s, 1H, CHO); Elemental analysis Calcd for C H BrN O : C 51.63, H
5
16 10
3
3
2
.71, N 11.29; found C 51.82, H 2.93, N 11.32.
8

ACCEPTED MANUSCRIPT
5
.1.2. General method for preparation of2-[(1,3-diaryl-1H-pyrazol-4-yl)methylene]-N-
phenylhydrazinecarbothioamides 2a-d
An equivalent amount of phenylthiosemicarbazide (0.67 g, 4 mmole) was added to a
suspension of the appropriate 1,3-diaryl-1H-pyrazole-4-carboxaldehyde 1a-d (4 mmole) in
absolute ethanol (15 ml). The reaction mixture was heated under reflux for 2-4 h and allowed
to cool to room temperature. The separated solid product was filtered, washed with ethanol
and crystallized from proper solvent(s).
2
-[(1,3-Di(4-methylphenyl)-1H-pyrazol-4-yl)methylene]-N-phenylhydrazine-
carbothioamide 2a
The product was crystallized from DMF:ethanol (1:5) as a white solid. Yield 99%; m.p.
-
1
1
94-196°C; IR (cm ): 3330 (NH), 1596 (C=N), 1542, 1268, 1204, 957 (NCS amide I, II, III
1
and IV bands, respectively); H-NMR (DMSO-d , δ ppm): 2.41, 2.42 (2s, each 3H, 2CH ),
6
3
7
.27 (t, J = 9 Hz, 1H, phenyl-C -H), 7.36-7.47 (m, 6H, phenyl-C2,3,5,6-H and C-C H -CH -C -
4 6 4 3 3,5
H), 7.62- 7.66 (m, 4H, N-C H -CH -H), 7.83 (d, J = 8.25 Hz, 2H, C-C H -CH -C -H), 8.37
6
4
3
6
4
3
2,6
(
s, 1H, N=CH), 9.20 (s, 1H, pyrazole-C -H), 9.81, 11.75 (2s, each 1H, N-NH and CS-NH,
5
D
2
O exchangeable); Elemental analysis Calcd for C25
23 5
H N S: C 70.56, H 5.45, N 16.46, found
C 69.98, H 5.63; N 16.12.
2
-[(1-(4-Bromophenyl)-3-(4-methylphenyl)-1H-pyrazol-4-yl)methylene]-N-phenylhydrazine-
carbothioamide 2b
The product was crystallized from DMF:ethanol (1:5) as an off white solid. Yield 98%;
-1
m.p. 258-260°C; IR (cm ): 3433 (NH), 1623 (C=N), 1535, 1296, 1214, 954 (NCS amide I, II,
III and IV bands, respectively), 729 (C-Br); H-NMR (DMSO-d , δ ppm): 2.38 (s, 3H, CH ),
1
6
3
7
7
.22 (t, J = 7.35 Hz, 1H, phenyl-C -H), 7.34 (d, J = 7.8 Hz, 2H, C H -CH -C3,5-H), 7.42 (t, J =
4 6 4 3
Hz, 2H, phenyl-C -H), 7.59 (d, J = 7.95Hz, 2H, C H -Br-C -H), 7.60 (d, J = 7.95 Hz, 2H,
3
,5
6
4
2,6
C H -Br-C -H), 7.76 (d, J = 8.7 Hz, 2H, C H -CH -C , -H), 7.87 (d, J = 8.4 Hz, 2H, phenyl-
6
4
3,5
6
4
3
2 6
C , -H), 8.32 (s, 1H, N=CH), 9.23 (s, 1H, pyrazole-C -H), 9.75, 11.74 (2s, each 1H, N-NH and
2
6
5
+
•
CS-NH, D O exchangeable); EIMS m/z (% relative abundance): 491 (72) (M +2), 489 (58)
M ), 129 (100); Elemental analysis Calcd for C H BrN S: C 58.78, H 4.11, N 14.28, found
2
+
•
(
24 20 5
C 59.12, H 3.98, N 13.95.
2
-[(1-(4-Methylphenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)methylene]-N-phenyl-
hydrazinecarbothioamide 2c
The product was crystallized from DMF:H
2
O (9:1) as a yellow solid. Yield 85%; m.p.
70-272°C; IR (cm ): 3322 (NH), 1596 (C=N), 1550, 1333, 1200, 960 (NCS amide I, II, III
-1
2
1
and IV bands, respectively), 1550, 1333 (NO ); H-NMR (DMSO-d , δ ppm): 2.38 (s, 3H,
2
6
CH ), 7.22 (t, J = 7.95 Hz, 1H, phenyl-C -H), 7.36-7.41 (m, 4H, phenyl-C -H and C H -CH -
3
4
3,5
6
4
3
C -H), 7.58 (d, J = 7.5 Hz, 2H, phenyl-C -H), 7.82 (d, J = 8.4 Hz, 2H, C H -CH -C -H),
3
,5
2,6
6
4
3
2,6
9

ACCEPTED MANUSCRIPT
8
.02 (d, J = 8.85 Hz, 2H, C H -NO -C -H), 8.36 (d, J = 8.85 Hz, 2H, C H -NO -C -H), 8.38
6
4
2
2,6
6
4
2
3,5
(
s, 1H, N=CH), 9.23 (s, 1H, pyrazole-C -H), 9.78, 11.75 (2s, each 1H, N-NH and CS-NH,
5
1
3
D O exchangeable); C-NMR (DMSO-d , δ ppm): 20.48 (CH ), 117.46 (pyrazole-C ), 118.74
2
6
3
4
(
N-methylphenyl-C ), 123.90 (NO -phenyl-C ), 125.32 (NO -phenyl-C ), 128.18 (phenyl-
2,6 2 3,5 2 2,6
C ), 128.51 (phenyl-C ), 129.15 (phenyl-C ), 130.07 (N-methylphenyl-C ), 135.69
2
,6
4
3,5
3,5
(
pyrazole-C ), 135.15 (methylphenyl-C ), 136.97 (phenyl-C ), 138.61 (NO -phenyl-C ),
5 4 1 2 1
1
38.75 (HC=N), 147.14 (NO -phenyl-C ), 148.94 (pyrazole-C ), 175.31 (C=S); Elemental
2 4 3
20 6 2
analysis Calcd for C24H N O S: C 63.14, H 4.42, N 18.41, found C 63.22, H 4.63, N 18.68.
2
-[(1-(4-Bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)methylene]-N-phenyl-
hydrazinecarbothioamide 2d
2
The product was crystallized from dioxane:H O (9:1) as a yellow solid. Yield 98%; m.p.
-
1
>
300°C; IR (cm ): 3439 (NH), 1598 (C=N), 1549, 1254, 1108, 960 (NCS amide I, II, III and
1
IV bands, respectively), 1549, 1337 (NO ), 705 (C-Br); H-NMR (DMSO-d , δ ppm): 7.22 (t,
2
6
J = 6.6 Hz, 1H, phenyl-C -H), 7.38 (t, J = 7.8 Hz, 2H, phenyl-C -H), 7.58 (d, J = 8.55 Hz,
4
3,5
2
H, C H -Br-C -H), 7.79 (d, J = 8.55 Hz, 2H, C H -Br-C -H), 7.91 (d, J = 8.4 Hz, 2H,
6 4 2,6 6 4 3,5
phenyl-C -H), 8.02 (d, J = 8.25 Hz, 2H, C H -NO -C -H), 8.36 (d, J = 8.25 Hz, 2H, C H -
2
,6
6
4
2
2,6
6
4
NO -C -H), 8.37 (s, 1H, N=CH), 9.30 (s, 1H, pyrazole-C -H), 9.76, 11.78 (2s, each 1H, N-
2
3,5
5
NH and CS-NH, D O exchangeable); Elemental analysis Calcd for C H BrN O S: C 52.98,
2
23 17
6
2
H 3.29, N 16.12, found C 53.22, H 3.02, N 15.88.
5
.1.3. General method for preparation ofN-[4-acetyl-5-(1,3-diaryl-1H-pyrazol-4-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]-N-phenylacetamides 3a-d
A solution of the selected thiosemicarbazone 2a-d (1 mmole) in acetic anhydride (5 ml)
was heated under reflux for 3-5 h. The reaction mixture was left to attain room temperature,
cold water (10 ml) was then added and the mixture was stirred for 30 min to decompose
excess acetic anhydride. The separated solid product was filtered, washed with water, dried
and crystallized from the proper solvent.
N-{4-Acetyl-5-[1,3-di(4-methylphenyl)-1H-pyrazol-4-yl]-4,5-dihydro-1,3,4-
thiadiazol-2-yl}-N-phenylacetamide 3a
The product was crystallized from ethanol:H O (3:1) as a yellow solid. Yield 80%; m.p.
2
-1
1
of 152-154°C; IR (cm ): 1678, 1657 (C=O), 1593 (C=N), 1298, 1064 (C-S-C); H-NMR
(
DMSO-d , δ ppm): 1.87, 1.88 (2s, each 3H, 2COCH ), 2.35, 2.38 (2s, each 3H, 2CH ), 7.08
6
3
3
(
s, 1H, thiadiazole C -H), 7.31 (d, J = 8.1 Hz, 2H, C-C H -CH -C -H), 7.32 (d, J = 8.4 Hz,
5 6 4 3 3,5
2
H, N-C H -CH -C -H), 7.50- 7.54 (m, 5H, phenyl-H), 7.62 (d, J = 8.4 Hz, 2H, N-C H -
6 4 3 3,5 6 4
CH -C -H), 7.76 (d, J = 8.4 Hz, 2H, C-C H -CH -C -H), 8.22 (s, 1H, pyrazole C -H);
3
2,6
6
4
3
2,6
5
Elemental analysis Calcd for C29
.64, N 13.98.
27 5 2
H N O S: C 68.35, H 5.34, N 13.74, found C 68.52, H
5
N-{4-Acetyl-5-[1-(4-bromophenyl)-3-(4-methylphenyl)-1H-pyrazol-4-yl]-4,5-
dihydro-1,3,4-thiadiazol-2-yl}-N-phenylacetamide3b
The product was crystallized from ethanol:H O (3:1) obtained as a yellow solid. Yield
2
-1
7
(
8%; m.p. 190-192°C; IR (cm ): 1688, 1662 (C=O), 1592 (C=N), 1233, 1068 (C-S-C), 703
1
C-Br); H-NMR (DMSO-d , δ ppm): 1.85, 1.88 (2s, each 3H, 2COCH ), 2.38 (s, 3H, CH ),
6
3
3
10

ACCEPTED MANUSCRIPT
7
.09 (s, 1H, thiadiazole C -H), 7.33 (d, J = 7.95 Hz, 2H, C H -CH -C -H), 7.67 (d, J = 8.85
5
6
4
3
3,5
Hz, 2H, C H -Br-C -H), 7.46-7.57 (m, 5H, phenyl-H), 7.70 (d, J = 8.85 Hz, 2H, C H -Br-
6
4
2,6
6
4
C -H), 7.88 (d, J = 7.95 Hz, 2H, C H -CH -C -H), 8.33 (s, 1H, pyrazole C -H); EIMS m/z
3
,5
6
4
3
2,6
5
+
•
+•
(
% relative abundance): 576 (2) (M +3), 575 (4) (M +2); Elemental analysis Calcd for
C H BrN O S: C 58.54, H 4.21, N 12.19, found C 58.81, H 4.50, N 11.84.
2
8
24
5
2
N-{4-Acetyl-5-[1-(4-methylphenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl]-4,5-
dihydro-1,3,4-thiadiazol-2-yl}-N-phenylacetamide 3c
The product was crystallized from dioxane:H
2
O (9:1) as a yellow solid. Yield 60%; m.p.
98-200°C; IR (cm ): 1690, 1664 (C=O), 1597 (C=N), 1545, 1339 (NO ), 1230, 1075 (C-S-
), 2.36 (s, 3H, CH ), 7.17
-C3,5-H), 7.51-7.54 (m, 5H,
phenyl-H), 7.80 (d, J = 8.65 Hz, 2H, C H -CH -C -H), 8.06 (d, J = 8.85 Hz, 2H, C H -NO -
-1
1
2
1
C); H-NMR (DMSO-d
6
, δ ppm): 1.85, 1.87 (2s, each 3H, 2COCH
-CH
3
3
(
s, 1H, thiadiazole C -H), 7.34 (d, J = 8.65 Hz, 2H, C
5
6
H
4
3
6
4
3
2,6
6
4
2
1
3
C -H), 8.34 (d, J = 8.85 Hz, 2H, C H -NO -C -H), 8.40 (s, 1H, pyrazole C -H); C-NMR
2
,6
6
4
2
3,5
5
(
DMSO-d , δ ppm): 21.01 (methylphenyl-CH ), 21.88 (thiadiazole-CO-CH ), 24.00
6
3
3
(thiadiazole-N-CO-CH3), 66.89 (thiadiazole-C5), 119.18 (methylphenyl-C2,6), 123.76
(
pyrazole-C ), 124.44 (NO -phenyl-C ), 128.22 (pyrazole-C ), 129.23 (NO -phenyl-C ),
4
2
3,5
5
2
2,6
1
1
29.46 (phenyl-C ), 129.70 (phenyl-C ),130.14 (phenyl-C ), 130.48 (methylphenyl-C ),
2,6 4 3,5 3,5
37.04 (methylphenyl-C ), 137.28 (methylphenyl-C ), 139.58 (phenyl-C ), 139.91 (NO -
4
1
1
2
phenyl-C ), 146.80 (NO -phenyl-C ), 147.45 (thiadiazole-C ), 149.17 (pyrazole-C ), 168.47
1
2
4
2
3
(
thiadiazole-C=O), 171.07 (thiadiazole-N-C=O); Elemental analysis Calcd for C H N O S:
28 24 6 4
C 62.21, H 4.47, N 15.55, found C 61.92, H 4.22, N 15.84.
N-{4-Acetyl-5-[1-(4-bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl]-4,5-
dihydro-1,3,4-thiadiazol-2-yl}-N-phenylacetamide 3d
The product was crystallized from dioxane:H
2
O (9:1) as a yellow solid. Yield 94%; m.p.
22-224°C; IR (cm ): 1686, 1660 (C=O), 1599 (C=N), 1540, 1343 (NO ), 1230, 1111 (C-S-
C), 705 (C-Br); H-NMR (DMSO-d , δ ppm):1.85, 1.87 (2s, each 3H, 2COCH ), 7.17 (s, 1H,
-1
2
2
1
6
3
thiadiazole C -H), 7.45-7.55 (m, 5H, phenyl-H), 7.74 (d, J = 8.85 Hz, 2H, C H -Br-C -H),
5
6
4
2,6
7
.92 (d, J = 8.85 Hz, 2H, C H -Br-C -H), 8.07 (d, J = 8.85 Hz, 2H, C H -NO -C -H), 8.34
6 4 2,6 6 4 2 2,6
(
d, J = 8.85 Hz, 2H, C H -NO -C -H), 8.50 (s, 1H, pyrazole C -H); Elemental analysis Calcd
6 4 2 3,5 5
for C H BrN O S: C 53.56, H 3.50, N 13.88, found C 53.28, H 3.81, N 14.20.
2
7
21
6
4
5
.1.4. General method for preparation of 2-{[(1,3-diaryl-1H-pyrazol-4-yl)methylene]-
hydrazono}-3-phenylthiazolidin-4-ones 4a-d
A mixture of the selected thiosemicarbazone 2a-d (1 mmole), ethyl bromoacetate (0.09
ml, 0.14 g, 1 mmole), anhydrous sodium acetate (0.08 g, 1 mmole) in absolute ethanol (12 ml)
was refluxed for 3-8 h. The obtained precipitate was collected by filtration, washed with
water, dried, and crystallized from a proper solvent(s).
2
-{[(1,3-Di(4-methylphenyl)-1H-pyrazol-4-yl)methylene]hydrazono}-3-phenylthiazolidin-4-
one 4a
The product was crystallized from dioxane:H O (9:1) as a white solid. Yield 84%; m.p.
2
-1
1
2
22-224°C; IR (cm ): 1721 (C=O), 1615(C=N), 1245, 1116 (C-S-C); H-NMR (DMSO-d , δ
6
11

ACCEPTED MANUSCRIPT
ppm): 2.36 (s, 6H, 2CH ), 4.11 (s, 2H, thiazole-C -H), 7.29 (d, J = 7.95 Hz, 2H, C-C H -CH -
3
5
6
4
3
C -H), 7.32- 7.47 (m, 5H, N-phenyl-H); 7.50 (d, J = 7.65 Hz, 2H, N-C H -CH -C -H); 7.73
3
,5
6
4
3
3,5
(
d, J = 7.65 Hz, 2H, N-C H -CH -C -H); 7.82 (d, J = 7.95 Hz, 2H, C-C H -CH -C -H); 8.25
6 4 3 2,6 6 4 3 2,6
(
s, 1H, N=C-H); 8.82 (s, 1H, pyrazole-C -H); Elemental analysis Calcd for C H N OS: C
5
27 23
5
6
9.65, H 4.98, N 15.04, found C 69.48, H 5.16, N 14.84.
2
-{[(1-(4-Bromophenyl)-3-(4-methylphenyl)-1H-pyrazol-4-yl)methylene]-
hydrazono}-3-phenylthiazolidin-4-one 4b
The product was crystallized from DMF:H
22-224°C; IR (cm ): 1728 (C=O), 1607 (C=N), 1237, 1069 (C-S-C), 825 (C-Br); H-NMR
2
O (9:1) as a white solid. Yield 86%; m.p.
-1
1
2
6 3 5
(DMSO-d , δ ppm): 2.35, 2.37 (2s, 3H, CH , E and Z isomers), 4.11, 4.15 (2s, 2H, thiazole-C -
H, E and Z isomers), 7.29-7.95 (m, 13H, aromatic H), 8.21, 8.24 (2s, 1H, HC=N, E and Z
isomers), 7.76, 8.93 (2s, 1H, pyrazole-C -H, E and Z isomers); EIMS m/z (% relative
5
+
•
+•
abundance): 531 (100) (M +2), 529 (97) (M ); Elemental analysis Calcd for C H BrN OS:
2
6
20
5
C 58.87, H 3.80, N13.20, found C 59.16, H 4.08, N 13.54.
2
-{[(1-(4-Methylphenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)methylene]-
hydrazono}-3-phenylthiazolidin-4-one 4c
The product was crystallized from dioxane:H O (9:1) as a white solid. Yield of 81%;
2
-1
m.p. 266-268°C; IR (cm ): 1720 (C=O), 1617 (C=N), 1525, 1338 (NO ), 1239, 1114 (C-S-C);
H-NMR (DMSO-d , δ ppm): 2.37 (s, 3H, CH ), 4.12 (s, 2H, thiazole-C -H), 7.38 (d, J = 8.7
2
1
6
3
5
Hz, 2H,C H -CH -C -H), 7.50-7.52 (m, 5H, N-phenyl-H), 7.82 (d, J = 8.7 Hz, 2H, C H -
6
4
3
3,5
6
4
CH
3
-C2,6-H), 8.27 (d, J = 9 Hz, 2H, C
6
H
4
-NO
2
-C2,6-H), 8.34 (d, J = 9 Hz, 2H, C
6
H
4
-NO
, δ ppm): 20.50
), 118.76 (methylphenyl-C2,6), 123.93
-phenyl-C2,6), 128.16 (phenyl-C2,6), 128.50 (phenyl-C ),
), 129.16 (phenyl-C3,5), 130.05 (methylphenyl-C3,5), 135.13
methylphenyl-C ), 136.57 (methylphenyl-C ), 136.98 (phenyl-C ), 138.76 (NO -phenyl-C ),
2
-C3,5-
1
3
H), 8.37 (s, 1H, HC=N), 8.91 (s, 1H, pyrazole-C
5
-H); C-NMR (DMSO-d
), 117.46 (pyrazole-C
-phenyl-C3,5), 125.31 (NO
28.94 (pyrazole-C
6
(
(
1
CH
NO
3
), 34.30 (thiazolidinone-C
5
4
2
2
4
5
(
4
1
1
2
1
1
1
1
47.17 (NO -phenyl-C ), 148.96 (CH=N), 154.73 (pyrazole-C ), 155.68 (thiazolidinone-C ),
2 4 3 2
75.33 (thiazolidinone-C ); Elemental analysis Calcd for C H N O S: C 62.89, H 4.06, N
4
26 20
6
3
6.93, found C 62.66, H 3.84, N 17.12.
2
-{[(1-(4-Bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)methylene]-
hydrazono}-3-phenylthiazolidin-4-one 4d
The product was crystallized from dioxane:H O (9:1) as a white solid. Yield 93%; m.p.
2
-1
2
(
42-244°C; IR (cm ): 1725 (C=O), 1624 (C=N), 1528, 1338 (NO ), 1241, 1109 (C-S-C), 756
C-Br); H-NMR (DMSO-d , δ ppm): 4.12 (s, 2H, thiazole-C -H), 7.36-7.59 (m, 5H, N-
2
1
6
5
phenyl-H), 7.76 (d, J = 9 Hz, 2H, C
6
H
4
-Br-C2,6-H), 7.92 (d, J = 9 Hz, 2H, C
6
H
4
-Br-C3,5-H),
-C3,5-H), 8.37 (s,
-H); Elemental analysis Calcd for C25 17BrN S: C
3.48, H 3.05, N 14.97, found C 53.68, H 3.35, N 14.72.
8
1
5
.02 (d, J = 9 Hz, 2H, C
H, N=C-H), 9.00 (s, 1H, pyrazole-C
6 4 2 6 4 2
H -NO -C2,6-H), 8.25 (d, J = 9 Hz, 2H, C H -NO
5
H
6 3
O
5
.1.5. General method for preparation 2-{[(1,3-diaryl-1H-pyrazol-4-yl)methylene]-
hydrazono}-4-(4-bromophenyl)-3-phenyl-2,3-dihydrothiazoles 5a-d
12

ACCEPTED MANUSCRIPT
To a suspension of the selected thiosemicarbazone2a-d (1 mmole) in absolute ethanol (8
ml), 4-bromophenacyl bromide (0.28 g, 1 mmole) and anhydrous sodium acetate (0.08 g, 1
mmole) were added. The reaction mixture was heated under reflux for 2–4 h then allowed to
cool. The obtained precipitate was filtered, washed with ethanol, dried, and crystallized from
DMF/ H O (9:1).
2
4
-(4-Bromophenyl)-2-{[(1,3-di(4-methylphenyl)-1H-pyrazol-4-yl)methylene]-
hydrazono}-3-phenyl-2,3-dihydrothiazole 5a
-1
The product was obtained as a white solid. Yield 98%; m.p. 208-210°C; IR (cm ):
1
1
2
=
594(C=N), 1217, 1068 (C-S-C), 730 (C-Br); H-NMR (DMSO-d , δ ppm): 2.34-2.39 (m, 6H,
6
CH , E and Z isomers), 6.70, 6.77 (2s, 1H, thiazole-C -H, E and Z isomers), 7.10, 7.15 (2d, J
3
5
8.7 Hz , 2H, C
6
H
4
6 4 6 4
-Br-C2,6-H), 7.24-7.54 (m, 13.5H, phenyl-H, C H -Br-C3,5-H, C-C H -
CH
H, C-C
pyrazole-C
N 11.58, found C 65.81, H 4.02, N 11.83.
3
-C3,5-H, N-C
6
H
4
-CH
-C2,6-H), 8.13, 8.47 (2s, 1H, HC=N, E and Z isomers), 8.75 (s, 0.5H,
-H, E and Z isomers); Elemental analysis Calcd for C33 S: C 65.56, H 4.33,
3 5
-H and pyrazole-C -H, E and Z isomers), 7.70, 7.83 (2d, J = 8.4 Hz ,
2
6 4 3
H -CH
5
H26BrN
5
4
-(4-Bromophenyl)-2-{[(1-(4-bromophenyl)-3-(4-methylphenyl)-1H-pyrazol-4-
yl)methylene]hydrazono}-3-phenyl-2,3-dihydrothiazole 5b
-1
The product was obtained as a white solid. Yield 87%; m.p. 200-202°C; IR (cm ): 1589
1
(
C=N), 1214, 1070 (C-S-C), 729 (C-Br).; H-NMR (DMSO-d , δ ppm): 2.36, 2.38 (2s, 3H,
6
CH , E and Z isomers), 6.70, 6.77 (2s, 1H, thiazole-C -H, E and Z isomers), 7.06, 7.16 (2d, J =
3
5
8
7
.4 Hz, 2H, thiazole-C H -Br-C -H), 7.24- 7.46 (m, 7H, phenyl-H and C H -CH -C -H),
6 4 2,6 6 4 3 3,5
.51-7.57 (m, 4H, pyrazole-C H -Br-H), 7.70, 7.72 (2d, J = 8.55 Hz, 2H, thiazole-C H -Br-
6
4
6
4
C -H), 7.77, 7.93 (2d, J = 9 Hz, 2H, C H -CH -C -H), 8.12, 8.49 (2s, 1H, N=C-H, E and Z
3
,5
6
4
3
2,6
isomers), 7.48, 8.49 (2s, 1H, pyrazole-C -H, E and Z isomers); Elemental analysis Calcd for
5
32 2 5
C H23Br N S: C 57.41, H 3.46, N 10.46, found C 57.74, H 3.72, N 10.64.
4
-(4-Bromophenyl)-2-{[(1-(4-methylphenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-
yl)methylene]hydrazono}-3-phenyl-2,3-dihydrothiazole 5c
-1
The product was obtained as a white solid. Yield 60%; m.p. 236-238°C; IR (cm ):
1
1
2
597(C=N), 1553, 1336 (NO ), 1215, 1070 (C-S-C), 719(C-Br); H-NMR (DMSO-d , δ ppm):
2
6
.37, 2.40 (2s, 3H, CH , E and Z isomers), 6.71, 6.79 (2s, 1H, thiazole-C -H, E and Z
3
5
isomers), 7.11, 7.16 (2d, J = 8.4 Hz, 2H, thiazole-C H -Br-C -H), 7.26-7.39 and 7.45-7.52
6
4
2,6
(
2m, 9H, phenyl-H and C H -CH -H), 7.83, 7.96 (2d, J = 8.4 Hz, 2H, thiazole-C H -Br-C -
6 4 3 6 4 3,5
H), 8.26 (d, J = 8.8 Hz, 2H, C H -NO -C -H), 8.34 (d, J = 8.8 Hz, 2H, C H -NO -C -H),
6
5
2
2,6
6
5
2
3,5
7
.43, 8.55 (2s, 1H, HC=N, E and Z isomers), 7.63, 8.83 (2s, 1H, pyrazole-C -H, E and Z
5
1
3
isomers); C-NMR (DMSO-d , δ ppm): 20.46, 20.47 (CH ), 102.34, 102.70 (thiazole-C ),
6
3
5
1
14.56, 117.27 (pyrazole-C ), 118.12, 118.15 (methylphenyl-C ), 118.56, 118.71 (Br-phenyl-
4 2,6
C ), 121.73, 121.89 (phenyl-C ), 123.46, 123.88 (phenyl-C ), 127.98, 128.46 (NO -phenyl-
4
4
2,6
2
C ), 128.65, 128.96 (NO -phenyl-C ), 129.51, 129.63 (Br-phenyl-C ), 129.96, 130.12 (Br-
3
.5
2
2,6
2,6
phenyl-C
1
), 130.18, 130.30 (phenyl-C3,5), 131.04, 131.22 (methylphenyl-C3,5), 136.18, 136.50
pyrazole-C ), 136.71, 136.86 (methylphenyl-C ), 137.32, 137.68 (methylphenyl-C ), 138.38,
39.02 (Br-phenyl-C3,5), 144.13, 144.44 (NO -phenyl-C ), 145.67, 145.93 (phenyl-C ),
46.85, 147.11 (thiazole-C ), 148.79, 149.78 (NO -phenyl-C ), 152.03, 152.07 (CH=N),
(
1
1
5
4
1
2
1
1
4
2
4
13

ACCEPTED MANUSCRIPT
1
53.52, 153.64 (pyrazole-C ), 168.87, 171.19 (thiazole-C ); Elemental analysis Calcd for
3
2
C H BrN O S: C 60.48, H 3.65, N 13.22, found C 60.81, H 3.38, N 13.58.
3
2
23
6
2
4
-(4-Bromophenyl)-2-{[(1-(4-bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-
yl)methylene]hydrazono}-3-phenyl-2,3-dihydrothiazole 5d
-1
The product was obtained as a white solid. Yield 77%; m.p. 270-272°C; IR (cm ): 1617
1
(
C=N), 1550, 1335 (NO ), 1210, 1068 (C-S-C), 717 (C-Br); H-NMR (DMSO-d , δ ppm):
2
6
6
.71 (s, 1H, thiazole-C
5
-H), 7.11 (d, J = 8.4 Hz, 2H, thiazole-C
4
-C
6
H
4
-Br-C2,6-H), 7.26-7.39
(
2
m, 5H, phenyl-H), 7.44 (d, J = 8.4 Hz, 2H, thiazole-C
4
-C
-Br-C2,6-H), 7.94 (d, J = 8.7 Hz, 2H, pyrazole-C
-NO -C2,6-H), 8.35 (d, J = 7.95 Hz, 2H, C
-H); EIMS m/z (% relative abundance): 700 (35)
M +2), 80 (100); Elemental analysis Calcd for C H Br N O S: C 53.16, H 2.88, N 12.00,
6
H
4
-Br-C3,5-H), 7.76 (d, J = 8.7 Hz,
H, pyrazole-C
1
-C
6
H
4
1
-C -Br-C3,5-H), 8.23
6 4
H
(s, 1H, HC=N), 8.24 (d, J = 7.95 Hz, 2H, C
6
H
4
2
6 4
H -
NO -C3,5-H), 8.92 (s, 1H, pyrazole-C
(
2
5
+
•
3
1
20
2
6
2
found C 52.82, H 3.22, N 11.80.
5
.1.6. Synthesis of 3-(1,3-diaryl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-ones 6a-c
A suspension of the appropriate 1,3-diaryl-1H-pyrazole-4-carboxaldehyde 1a,b,d (10
mmole) in dioxane:ethanol (3 ml:7 ml) was added portion wise to a mixture of acetophenone
1.23 g, 1.2 ml, 10 mmole) in 3% alcoholic KOH (20 ml). The reaction mixture was stirred for
4 h at RT. The yellow precipitate formed was then filtered, washed with ethanol, dried and
crystallized from proper solvent.
(
2
3
-[1,3-Di(4-methylphenyl)-1H-pyrazol-4-yl]-1-phenylprop-2-en-1-one 6a
The product was crystallized from ethanol as a yellow solid. Yield 72%; m.p. 174°C;
-1
1
IR (cm ): 1645 (C=O), 1581 (C=N); H-NMR (DMSO-d
CH ), 7.36 (d, J = 8.4 Hz, 2H, C-C -CH -C3,5-H), 7.37 (d, J = 7.95 Hz, 2H, N-C
3,5-H), 7.75 (d, J = 7.95 Hz, 2H, N-C -CH -C2,6-H), 7.54-7.65 (m, 3H, phenyl-C3,4,5-H),
.69 (d, J = 15.3 Hz, 1H, CH=CH-CO), 7.82 (d, J = 8.4 Hz, 2H, C-C H -CH -C -H), 7.84 (d,
6
, δ ppm): 2.37, 2.39 (2s, each 3H,
2
C
7
3
6
H
4
3
6
H
4
-CH -
3
6
H
4
3
6
4
3
2,6
J = 15.3 Hz, 1H, CH=CH-CO), 8.08 (d, J = 8.7 Hz, 2H, phenyl-C -H), 9.37 (s, 1H, pyrazole-
2
,6
1
3
C -H); C-NMR (DMSO-d , δ ppm): 21.01, 21.40 (2 CH ), 118.07 (pyrazole-C ), 119.01 (N-
5
6
3
4
methylphenyl-C ), 121.57 (CH=CH-CO), 128.75 (C-methylphenyl-C ), 128.84 (phenyl-
2
,6
2,6
C ), 128.95 (C-methylphenyl-C ), 129.26 (phenyl-C ), 129.72 (pyrazole-C ), 129.92 (C-
2
,6
1
3,5
5
methylphenyl-C ), 130.54 (N-methylphenyl-C ), 133.47 (C-methylphenyl-C ), 135.02
3
,5
3,5
4
(
phenyl-C ), 137.01 (N-methylphenyl-C ), 137.33 (N-methylphenyl-C ), 138.29 (phenyl-C ),
4 4 1 1
1
38.64 (CH=CH-CO), 153.31 (pyrazole-C ), 189.25 (C=O); Elemental analysis Calcd for
3
C H N O: C 82.51, H 5.86, N 7.40, found C 82.18, H 5.50, N 7.08.
2
6
22
2
3
-[1-(4-Bromophenyl)-3-(4-methylphenyl)-1H-pyrazol-4-yl]-1-phenylprop-2-en-1-
one 6b
The product was crystallized from ethanol as a white solid. Yield 78%; m.p. 176°C; IR
-1
1
(
cm ): 1663 (C=O), 1603 (C=N), 698 (C-Br); H-NMR (DMSO-d
CH ), 7.37 (d, J = 7.8 Hz, 2H, C -CH -C3,5-H), 7.56 (d, J = 8.4 Hz, 2H, C
.59-7.73 (m, 4H, phenyl-C3,4,5-H and CH=CH-CO), 7.76-7.87 (m, 3H, phenyl-C -H and
6
, δ ppm): 2.40 (s, 3H,
3
6
H
4
3
6 4
H
-Br-C2,6-H),
7
2,6
CH=CH-CO), 7.91 (d, J = 8.4 Hz, 2H, C H -Br-C -H), 8.07 (d, J = 7.8 Hz, 2H, C H -CH -
6
4
3,5
6
4
3
+
•
C -H), 9.43 (s, 1H, pyrazole-C -H); EIMS m/z (% relative abundance): 444(20) (M +2), 442
2
,6
5
14

ACCEPTED MANUSCRIPT
+
•
(
20) (M ), 337 (100); Elemental analysis Calcd for C H BrN O: C 67.73, H 4.32, N 6.32,
25 19 2
found C 67.50, H 4.54, N 6.68.
3
-(1-(4-Bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-
one 6c
The product was crystallized fromethanol:H O (9:1) as a yellow solid. Yield 98%; m.p.
2
-1
1
2
36-238°C; IR (cm ): 1665 (C=O), 1603(C=N), 1537, 1340 (NO ), 691 (C-Br); H-NMR
2
(
DMSO-d
CH=CH-CO), 7.94-7.98 (m, 3H, C
-NO -C2,6-H), 8.12 (d, J = 9.3 Hz, 2H, phenyl-C2,6-H), 8.44 (d, J = 9.3 Hz, 2H, C
-C3,5-H), 9.50 (s, 1H, pyrazole-C -H); Elemental analysis Calcd for C24
0.77, H 3.40, N 8.86, found C 60.98, H 3.62, N 9.04.
6
, δ ppm): 7.63- 7.74 (m, 3H, phenyl-C3,4,5-H),7.82-7.89 (m, 3H, C
6 4
H -Br-C2,6-H and
6
H
4
-Br-C3,5-H and CH=CH-CO), 8.01 (d, J = 9 Hz, 2H,
C
NO
6
6
H
2
4
2
6 4
H -
H16BrN O : C
3 3
5
5
5
.1.7. General method for preparation of 1,3-diaryl-4-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-
-yl)pyrazoles 7a-c
A mixture of the appropriate 3-(1,3-diaryl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-one
a-c (1 mmole), phenylhydrazineHCl (0.14 g, 1 mmole) and anhydrous sodium acetate (0.08
6
g, 1 mmole) in ethanol: glacial acetic acid (12 ml: 1 ml) was heated under reflux for 18- 48 h
then allowed to cool. The obtained precipitate was filtered, washed with ethanol then with
H O, dried and crystallized from proper solvent.
2
1
,3-Di(4-methylphenyl)-4-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)pyrazole 7a
The product was crystallized from ethanol:H O (3:1) as a white solid. Yield 43%; m.p.
20-122°C; IR (cm ): 1592 (C=N); H-NMR (DMSO-d , δ ppm): 2.50, 2.51 (2s, each 3H,
CH ), 3.42-3.48 (m, 2H, pyrazoline C -H), 4.35 (t, J= 5.06 Hz, pyrazoline-C -H), 6.98 (t, J =
.02 Hz, 1H, N-phenyl-C -H), 7.07-7.28 (m, 6H, N-phenyl-H and C-methylphenyl-C3,5-H),
.32-7.49 (m, 7H, C-methylphenyl-H and C-phenyl-C3,4,5-H), 7.68-7.77 (m, 4H, C-
2
-1
1
1
2
7
7
6
3
4
5
4
methylphenyl-C -H and C-phenyl-C -H), 8.27 (s, 1H, pyrazole-C -H); Elemental analysis
2
,6
2,6
5
Calcd for C H N : C 82.38, H 5.62, N 12.01; found C 82.12, H 5.80, N 11.78.
3
2
26
4
1
-(4-Bromophenyl)-3-(4-methylphenyl)-4-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)pyrazole
7
b
The product was crystallized from ethanol:H O (3:1) as a white solid. Yield 33%; m.p.
2
-1
1
1
94-196°C; IR (cm ): 1632 (C=N), 689 (C-Br); H-NMR (DMSO-d , δ ppm): 2.38 (s, 1H,
6
CH ),3.29 (distorted dd, pyrazoline C -H), 4.06 (dd, J = 16, 12 Hz, pyrazoline-C -H), 5.50
3
4
4
(
dd, J = 12, 7.6 Hz, pyrazoline-C -H), 6.73 (t, J = 7.4 Hz, 1H, pyrazoline-N-phenyl-C -H),
5
4
6
.98 (d, J = 7.4 Hz, 2H, pyrazoline-N-phenyl-C2,6-H), 7.15 (t, J = 7.4 Hz, 2H, pyrazoline-N-
-CH -C3,5-H), 7.38 (t, J = 7.4 Hz, 1H, pyrazoline-
-H), 7.44 (t, J = 7.4 Hz, 2H, pyrazoline-C -phenyl-C3,5-H), 7.62 (d, J = 8.9 Hz,
-Br-C2,6-H), 7.69 (d, J = 8 Hz, 2H, C -CH -C2,6-H), 7.76 (d, J = 7.4 Hz, 2H,
pyrazoline-C -phenyl-C2,6-H), 7.83 (d, J = 8.9 Hz, 2H, C -Br-C3,5-H), 8.39 (s, 1H, pyrazole-
C -H); C-NMR (DMSO-d , δ ppm): 18.99 (CH ), 40.00 (pyrazoline-C , under DMSO),
phenyl-C3,5-H), 7.33 (d, J = 8 Hz, 2H, C
-phenyl-C
H, C
6
H
4
3
C
2
3
4
3
6
H
4
6
H
4
3
3
6 4
H
1
3
5
6
3
4
5
1
6.50 (pyrazoline-C ), 113.73 (N-phenyl-C ), 118.98 (pyrazole-C ), 119.45 (N-phenyl-C ),
5 2,6 4 4
20.54 (bromophenyl-C ), 123.70 (bromophenyl-C ), 126.25 (methylphenyl-C ), 127.65
2
,6
4
2,6
15

ACCEPTED MANUSCRIPT
(
pyrazole-C ), 128.33 (C-phenyl-C ), 129.06 (C-phenyl-C ), 129.20 (methylphenyl- C ),
5
2,6
3,5
1
1
29.32 (N-phenyl-C ), 129.83 (methylphenyl-C3,5), 130.06 (C-phenyl-C4), 132.73
3,5
(
bromophenyl-C ), 132.77 (methylphenyl-C ), 138.22 (C-phenyl-C ), 138.90 (bromophenyl-
3,5 4 1
C ), 145.07 (N-phenyl-C ), 148.12 (pyrazole-C ), 150.56 (pyrazoline-C ); Elemental analysis
1
1
3
3
Calcd for C H BrN : C 70.06, H 4.36, N 10.54; found C 69.78, H 4.48, N 10.22.
3
1
23
4
1
-(4-Bromophenyl)-3-(4-nitrophenyl)-4-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazole 7c
The product was crystallized from dioxane:H
2
O (9:1) as a white solid. Yield 50%; m.p.
-1
1
2
18-220°C; IR (cm ): 1599 (C=N), 1539, 1340 (NO
ppm): 3.43-3.46 (m, 2H, pyrazoline-C -H), 4.35 (t, J = 4.5 Hz, pyrazoline-C
.2 Hz, 1H, N-phenyl-C -H), 7.44 (t, J = 7.2 Hz, 2H, N-phenyl-C2,6-H), 7.61-7.99 (m, 11H, C-
phenyl-H, C H -Br-H and N-phenyl-C -H), 8.33 (d, J = 8.04 Hz, 2H, C H -NO -C -H),
2
), 691 (C-Br); H-NMR (DMSO-d
6
, δ
4
5
-H), 7.17 (t, J =
7
4
6
4
3,5
6
4
2
2,6
8
.42 (d, J = 8.04 Hz, 2H, C H -NO -C -H), 8.48 (s, 1H, pyrazole-C -H); Elemental analysis
6 4 2 3.5 5
Calcd for C H BrN O : C 64.07, H 3.58, N 12.45, found C 64.30, H 3.50, N 12.72.
3
0
20
5
2
5
.1.8. General method for preparation of 1,3-diaryl-4-(3-phenyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazoles 8a-c
A mixture of the appropriate α, β-unsaturated ketone 6a-c (1 mmole) and NH NH .H O
2
2
2
9
9% (0.2 g, 0.19 ml, 4 mmole) in ethanol (10 ml) was heated under reflux for 15-30 minutes
then allowed to cool down. The white precipitate obtained after cooling was filtered, washed
with ethanol, dried and crystallized from ethanol.
1
,3-Di(4-methylphenyl)-4-(3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)pyrazole 8a
The product was obtained as a white solid. Yield 89%; m.p. 146-148°C; IR (cm ):
-1
1
3
322 (NH), 1609 (C=N); H-NMR (DMSO-d
-H), 3.46 (dd, J=16.2, 10.8 Hz, pyrazoline C
J = 10.8 Hz, pyrazoline C -H), 7.28- 7.34 (m, 4H, C-C H -CH -C -H and N-C H -CH -C -
6
, δ ppm): 2.33, 2.35 (2s, each 3H, 2CH
3
), 2.97
(dd, J =16.2, 10.8 Hz, pyrazoline C
4
4
-H), 4.96 (t,
5
6
4
3
3,5
6
4
3
3,5
H) 7.36- 7.41 (m, 3H, phenyl-C3,4,5-H), 7.55 (br d, 1H, pyrazoline-NH, D O exchangeable),
2
7
.62- 7.66 (m, 4H, N-C H -CH -C -H and phenyl-C -H), 7.76 (d, J = 8.4 Hz, 2H, N-C H -
6 4 3 2,6 2,6 6 4
1
3
CH -C -H), 8.49 (s, 1H, pyrazole C -H); C-NMR (DMSO-d , δ ppm):20.96, 21.38 (2CH ),
3
2,6
5
6
3
4
1
0.00 (pyrazoline-C , under DMSO), 55.80 (pyrazoline-C ), 118.59 (N-methylphenyl-C ),
4 5 2,6
23.29 (pyrazole-C ), 126.02 (C-methylphenyl-C ), 127.56 (pyrazole-C ), 128.37 (phenyl-
4
2,6
5
C ), 128.65 (C-methylphenyl-C ),129.01 (phenyl-C ), 129.70 (C-methylphenyl-C ),
2
,6
1
3,5
3,5
1
30.45 (N-methylphenyl-C ), 130.74 (phenyl-C ), 133.78 (C-methylphenyl-C ), 135.96 (N-
3,5 4 4
methylphenyl-C ), 137.74 (phenyl-C ), 137.86 (N-methylphenyl-C ), 149.83 (pyrazole-C ),
4
1
1
3
1
50.58 (pyrazoline-C ); Elemental analysis Calcd for C H N : C 79.56, H 6.16, N 14.27;
3 26 24 4
found C 79.30, H 5.92, N 13.84.
1
-(4-Bromophenyl)-3-(4-methylphenyl)-4-(3-phenyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazole 8b
-1
The product was obtained as a white solid. Yield 87%; m.p. 156-158°C; IR (cm ): 3317
1
(NH), 1589, (C=N), 727 (C-Br); H-NMR (DMSO-d , δ ppm): 2.36 (s, 3H, CH ), 2.97 (dd, J =
6
3
1
1
6.2, 10.8 Hz, pyrazoline C -H), 3.47 (dd, J = 16.2, 10.8 Hz, pyrazoline C -H), 4.97 (t, J =
4
4
0.8 Hz, pyrazoline C -H), 7.28- 7.41 (m, 5H, C H -CH -C -H and phenyl-C3,4,5-H), 7.51 (br
5
6
4
3
3,5
16

ACCEPTED MANUSCRIPT
d, 1H, pyrazoline-NH, D O exchangeable), 7.62- 7.69 (m, 6H, C H -Br-H and phenyl-C -H),
2
6
4
2,6
7
.87 (d, J = 8.7 Hz, 2H, C H -CH -C -H), 8.58 (s, 1H, pyrazole C -H); EIMS m/z (% relative
6 4 3 2,6 5
+
•
+•
abundance): 458 (4) (M +2), 456 (100) (M ); Elemental analysis Calcd for C H BrN : C
2
5
21
4
6
5.65, H 4.63, N 12.25, found C 65.26, H 4.84, N 11.90.
1
-(4-Bromophenyl)-3-(4-nitrophenyl)-4-(3-phenyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazole 8c
The product was obtained as a white solid. Yield 60%; m.p. 204-206°C; IR (cm ): 3313
-1
1
(
1
1
NH), 1597 (C=N), 1543, 1342 (NO
2
), 719 (C-Br); H-NMR (DMSO-d
-H), 3.56 (dd, J = 16.5, 10.8 Hz, pyrazoline C
-H), 7.30- 7.42 (m, 3H, phenyl-C3,4,5-H), 7.60 (d, J = 3.6 Hz, 1H,
O exchangeable), 7.62- 7.65 (m, 2H, phenyl-C2,6-H), 7.71 (d, J = 9 Hz, 2H,
C H -Br-C -H), 7.92 (d, J = 9 Hz, 2H, C H -Br-C -H), 8.08 (d, J = 9 Hz, 2H, C H -NO -
6
, δ ppm): 2.98 (dd, J =
6.5, 10.8 Hz, pyrazoline C
0.8, 3.3 Hz, pyrazoline C
4
4
-H), 5.08 (td, J =
5
pyrazoline-NH, D
2
6
4
2,6
6
4
3,5
6
4
2
C -H), 8.32 (d, J = 9 Hz, 2H, C H -NO -C -H), 8.70 (s, 1H, pyrazole C -H); Elemental
2
,6
6
4
2
3,5
5
analysis Calcd for C H BrN O : C 59.03, H 3.72, N 14.34; found C 58.88, H 3.56, N 14.71.
2
4
18
5
2
5
1
.1.9. General method for preparation of 4-(1-acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-
,3-diarylpyrazoles9a-cand1,3-diaryl-4-(3-phenyl-1-propanoyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazoles 10a-c
A mixture of the appropriate α, β-unsaturated ketone 6a-c (1 mmole) and NH NH .H O
2
2
2
9
9% (0.2 g, 0.19 ml, 4 mmole) in the appropriate carboxylic acid (acetic acid or propionic
acid) (10 ml) was heated under reflux for 4-8 h then allowed to cool. The obtained precipitate
was filtered, washed with ethanol and crystallized from the proper solvent.
4
-(1-Acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1,3-di-(4-methylphenyl)-
pyrazole 9a
The product was crystallized from ethanol as a white solid. Yield 64 %; m.p. 196-198
-1
1
°
C; IR (cm ): 1661 (C=O), 1617 (C=N); H-NMR (DMSO-d , δ ppm): 2.32 (s, 6H, 2CH ),
6
3
2
.36 (s, 3H, COCH ), 3.20 (dd, J = 18, 5.2 Hz, pyrazoline C -H), 3.89 (dd, J = 18, 12 Hz,
3 4
pyrazoline C -H), 5.68 (dd, J = 12, 5.2 Hz, pyrazoline C -H), 7.26 (d, J = 6.9 Hz, 2H, C-C H -
4
5
6
4
CH -C -H), 7.28 (d, J = 6.3 Hz, 2H, N-C H -CH -C -H), 7.44-7.48 (m, 3H, phenyl-C3,4,5H),
3
3,5
6
4
3
3,5
7
.67 (d, J = 8.4 Hz, 2H, N-C H -CH -C -H), 7.74- 7.77 (m, 4H, phenyl-C -H and C-C H -
6 4 3 2,6 2,6 6 4
1
3
CH -C -H), 8.28 (s, 1H, pyrazole C -H); C-NMR (DMSO-d , δ ppm): 20.39, 20.82 (2CH ),
3
2,6
5
6
3
2
1
1.91 (COCH ), 42.18 (pyrazoline-C ); 51.84 (pyrazoline-C ), 118.02 (N-methylphenyl-C ),
3 4 5 2,6
23.06 (pyrazole-C ), 126.01 (pyrazole-C ), 126.12 (C-methylphenyl-C ), 126.56 (C-
4
5
1
methylphenyl-C ), 127.82 (phenyl-C
), 128.63 (C-methylphenyl-C ),129.05 (phenyl-
2
,6
2,3,5,6
3,5
C ), 129.71 (C-methylphenyl-C ), 130.11 (N-methylphenyl-C ), 131.25 (N-methylphenyl-
4
4
3,5
C
4
), 135.35 (phenyl-C
1
), 137. 16 (N-methylphenyl-C
1
3
), 148.98 (pyrazole-C ), 153.84
(pyrazoline-C
3
), 167.54 (C=O); Elemental analysis Calcd for C28
26 4
H N O: C 77.39, H 6.03, N
1
2.89, found C 77.02, H 6.36, N 12.58.
4
-(1-Acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1-(4-bromophenyl)-3-(4-
methylphenyl)pyrazole 9b
The product was crystallized from ethanol as a white solid. Yield 73%; m.p. 232-234°C;
-1
1
IR (cm ): 1665 (C=O), 1592 (C=N), 689 (C-Br); H-NMR (DMSO-d , δ ppm): 2.31 (s, 3H,
6
17

ACCEPTED MANUSCRIPT
COCH ), 2.36 (s, 3H, CH ), 3.20 (dd, J = 18, 5.2 Hz, pyrazoline C -H), 3.89 (dd, J = 18, 12
3
3
4
Hz, pyrazoline C -H), 5.68 (dd, J = 12, 5.2 Hz, pyrazoline C -H), 7.29 (d, J = 8.1 Hz, C H -
4
5
6
4
CH -C -H), 7.44- 7.48 (m, 3H, phenyl- C -H), 7.64 (d, J = 8.7 Hz, C H -Br-C -H), 7.68
3
3,5
3,4,5
6
4
2,6
(
d, J = 8.1 Hz, 2H, C H -Br- C -H), 7.74-7.77 (m, 2H, phenyl- C -H), 7.86 (d, J = 8.7 Hz,
6 4 3,5 2,6
C H -CH -C -H), 8.37 (s, 1H, pyrazole C -H); EIMS m/z (% relative abundance): 500 (6)
6
4
3
2,6
5
+
•
+•
(
M +2), 498 (5) (M ), 80 (100); Elemental analysis Calcd for C H BrN O: C 64.94, H 4.64,
27 23 4
N 11.22, found C 65.20, H 4.32, N 11.64.
4
-(1-Acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1-(4-Bromo-phenyl)-3-(4-
nitrophenyl)pyrazole 9c
The product was crystallized fromdioxane:ethanol (5:1) as a white solid. Yield 73%;
-1
1
m.p. > 300°C; IR (cm ): 1659 (C=O), 1696, 1499 (C=N), 1553, 1339 (NO
2
), 714 (C-Br); H-
NMR (DMSO-d , δ ppm): 2.37 (s, 3H, COCH ), 3.35 (distorted dd, pyrazoline C -H), 4.01
6
3
4
(
dd, J = 18, 11.85 Hz, pyrazoline C -H), 5.81 (dd, J = 11.85, 5.2 Hz, pyrazoline C -H), 7.47-
4
5
7
.54 (m, 3H, phenyl-C3,4,5-H), 7.74 (d, J = 9 Hz, 2H, C H -Br-C -H), 7.80- 7.84 (m, 2H,
6 4 2,6
phenyl-C -H), 7.95 (d, J = 9 Hz, 2H, C H -Br- C -H), 8.16 (d, J = 8.85 Hz, 2H, C H -NO -
2
,6
6
4
3,5
6
4
2
C -H), 8.38 (d, J = 8.85 Hz, 2H, C H -NO - C -H), 8.57 (s, 1H, pyrazole C -H); Elemental
2
,6
6
4
2
3,5
5
analysis Calcd for C H BrN O : C 58.88, H 3.80, N 13.20, found C 59.06, H 3.84, N 13.06.
2
6
20
5
3
1
,3-Di(4-methylphenyl)-4-(3-phenyl-1-prpanoyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazole 10a
The product was crystallized from ethanol as a white solid. Yield 75%; m.p. 202-
-1
1
2
04°C; IR (cm ): 1659 (C=O), 1596 (C=N); H-NMR (DMSO-d
Hz, 3H, CH -CH ), 2.31, 2.36 (2s, each 3H, 2CH ), 2.74 (q, J = 7.65 Hz, 2H, CH
dd, J = 18, 5.1 Hz, pyrazoline C -H), 3.87 (dd, J = 18, 12 Hz, pyrazoline C -H), 5.66 (dd, J =
-H), 7.26 (d, J = 7.8 Hz, 2H, C-C -CH -C3,5-H), 7.28 (d, J = 7.2
-C3,5-H), 7.44-7.46 (m, 3H, phenyl-C3,4,5-H), 7.67 (d, J = 7.2 Hz, 2H, N-
C H -CH -C -H), 7.74- 7.77 (m, 4H, phenyl- C -H and C-C H -CH -C -H), 8.26 (s, 1H,
6
, δ ppm): 1.06 (t, J = 7.65
2
3
3
2
-CH ), 3.19
3
(
1
4
4
2, 5.1 Hz, pyrazoline C
-CH
5
6
H
4
3
Hz, 2H, N-C
6
H
4
3
6
4
3
2,6
2,6
6
4
3
2,6
1
3
pyrazole C -H); C-NMR (DMSO-d , δ ppm): 8.85 (CO-CH -CH ), 20.34, 20.82 (2CH ),
5
6
2
3
3
2
6.90 (CO-CH -CH ), 42.00 (pyrazoline-C ), 51.95 (pyrazoline-C ), 118.02 (N-methylphenyl-
2 3 4 5
C ), 123.23 (pyrazole-C ), 125.97 (pyrazole-C ), 126.04 (C-methylphenyl-C ), 126.56 (C-
2
,6
4
5
1
methylphenyl-C ), 127.87 (phenyl-C ), 128.63 (phenyl-C ), 129.09 (C-methylphenyl-C ),
2
,6
2,6
3,5
3,5
1
29.76 (N-methylphenyl-C ), 130.06 (phenyl-C ), 130.11 (C-methylphenyl-C ), 131.34 (N-
3,5 4 4
methylphenyl-C ), 135.36 (phenyl-C ), 137.18 (N-methylphenyl-C ), 149.10 (pyrazole-C ),
4
1
1
3
1
6
53.71 (pyrazoline-C ), 170.82 (C=O); Elemental analysis Calcd for C H N O: C 77.65, H
.29, N 12.49, found C 77.28, H 6.02, N 12.78.
3
29 28
4
1
-(4-Bromophenyl)-3-(4-methylphenyl)-4-(3-phenyl-1-propanoyl-4,5-dihydro-1H-pyrazol-5-
yl)pyrazole 10b
The product crystallized fromdioxane:ethanol (5:1) as a white solid. Yield 87%; m.p.
-
1
1
2
1
38-240°C; IR (cm ): 1660 (C=O), 1591 (C=N), 691 (C-Br); H-NMR (DMSO-d
6
, δ ppm):
), 3.19
.07 (t, J = 7.5 Hz, 3H, CH -CH ), 2.36 (s, 3H, CH ), 2.74 (q, J = 7.5 Hz, 2H, CH -CH
2
3
3
2
3
(
dd, J = 18, 5.4 Hz, pyrazoline C -H), 3.88 (dd, J = 18, 12 Hz, pyrazoline C -H), 5.66 (dd, J =
4
4
1
2, 5.4 Hz, pyrazoline C -H), 7.29 (d, J = 7.5 Hz, C H -CH -C -H), 7.45-7.47 (m, 3H,
5 6 4 3 3,5
phenyl-C3,4,5-H), 7.64 (d, J = 9 Hz, 2H, C H -Br-C -H), 7.68 (d, J = 8.1 Hz, C H -CH -C -
6
4
2,6
6
4
3
2,6
18

ACCEPTED MANUSCRIPT
H), 7.74- 7.77 (m, 2H, phenyl-C -H), 7.85 (d, J = 9 Hz, 2H, C H -Br-C -H), 8.36 (s, 1H,
2
,6
6
4
3,5
+
•
pyrazole C -H); EIMS m/z (% relative abundance): 514 (2) (M +2), 64 (100); Elemental
5
analysis Calcd for C H BrN O: C 65.50, H 4.91, N 10.91, found C 65.44, H 5.21, N 11.26.
2
8
25
4
1
-(4-Bromophenyl)-3-(4-nitrophenyl)-4-(3-phenyl-1-propanoyl-4,5-dihydro-1H-
pyrazol-5-yl)pyrazole 10c
The product crystallized fromdioxane:ethanol (5:1) as a white solid. Yield 54%; m.p. >
-
1
1
3
d
(
1
00°C; IR (cm ):1656 (C=O), 1596 (C=N), 1552, 1339 (NO
, δ ppm): 1.11 (t, J = 7.65 Hz, 3H, CH -CH ), 2.79 (q, J = 7.65 Hz, 2H, CH
-H), 3.99 (dd, J = 17.7, 11.1 Hz, pyrazoline C -H), 5.79 (dd, J =
-H), 7.50-7.55 (m, 3H, phenyl-C3,4,5-H), 7.73 (d, J = 9.15 Hz, 2H,
-Br-C2,6-H), 7.79- 7.83 (m, 2H, phenyl-C2,6-H), 7.93 (d, J = 9.15 Hz, 2H, C
H), 8.16 (d, J = 9 Hz, 2H, C H -NO -C -H), 8.38 (d, J = 9 Hz, 2H, C H -NO - C -H ), 8.54
2
), 690 (C-Br); H-NMR (DMSO-
6
2
3
2
-CH ), 3.28
3
distorted dd, pyrazoline C
4
4
1.1, 4.8 Hz, pyrazoline C
5
C
6
H
4
6 4
H -Br- C3,5-
6
4
2
2,6
6
4
2
3,5
(
s, 1H, pyrazole C -H); Elemental analysis Calcd for C H BrN O : C 59.57, H 4.07, N
5
27 22
5
3
1
2.86, found: C 59.22, H 3.89, N 12.48.
5
.2. Biological Screening
5
.2.1. In vivo antimalarial activity testing against P. berghei
Swiss albino mice of both sexes, weighing 24-38 g and 4-6 weeks of age, were used in
the antimalarial activity test. The animals were acclimatized for a period of 7 days at room
o
temperature (23-25 C) and relative humidity of 60-65%. The animals were housed in standard
cages and maintained on standard pelleted diet and water. In vivo antimalarial activity test of
the synthesized compounds was performed using a 4-day standard suppressive test using P.
berghei ANKA strain infected mice as described by Fidock et al [46]. This is the most widely
used test in which the efficacy of a compound is assessed by comparison of blood parasitemia
level and mouse survival time in treated and untreated mice [47]. On day 0, the test mice were
7
injected intravenously with 0.2 ml of 2 x 10 parasitized erythrocytes infected with P. berghei
ANKA strain. These parasite erythrocytes were obtained from the blood of a donor mouse
with approximately 20-30% parasitemia and the blood was diluted with normal saline. After 2
h of injection, the infected mice were weighed and randomly divided into 37 groups of five
mice per cage. Groups 1-35 (35 cages) received the synthesized compounds orally, each at
0
.048 mmol/kg/day dose levels and served as treatment groups. Group 36 received the drug
vehicle (7% Tween, 3% ethanol in distilled water) and served as a negative control, while
Group 37 received chloroquine phosphate at dose level of 25 mg/kg/day (0.048 mmol/kg/day)
and served as a positive control. On days 1 to 3, the treatment groups were treated with the
same single dose of the synthesized compound at 24 h intervals. On day 4 (i.e. 24 h after the
last dose), blood smear from all test animals was prepared using Giemsa stain. Level of
parasitemia was determined microscopically by counting 5 fields of approximately 100
erythrocytes per field. The difference between the mean value for the negative control group
(taken as 100%) and those of the experimental groups was calculated and expressed as percent
suppression or activity. Chloroquine treated mice were completely cured of the parasite.The
survival time for each test mouse was recorded and the mean survival time was calculated in
19

ACCEPTED MANUSCRIPT
comparison with that of the negative group. Percentage parasitemia and percentage
suppression were calculated using the following formulae:
5
.2.2. In vitro antimalarial activity against P. falciparum
The most active compounds, 2c, 2d, 4b, 4c, 4d, 5a, 6c, 8c and 9b, were further
examined for their antiplasmodial activities against chloroquine resistant (RKL9) P.
falciparum strain. The P. falciparum strain RKL9 was maintained in a continuous culture
using the standard method described by Trager and Jensen[43]. The assay was carried out in
o
9
6 well microtitre flat-bottomed tissue culture plates incubated at 37 C for 24 h. Two-fold
serial dilutions of test compounds and chloroquine diphosphate were examined for their effect
on schizont maturation. The initial culture was maintained in small vials with 10%
haematocrit, i.e. 10 ml erythrocytes containing 1.0% ring stage parasite in 100ml complete
media. The assay culture volume was 100 ml per well. Number of parasites for the assay was
adjusted to 1–1.5% by dilution with fresh human B (+) RBC. Compounds were dissolved in
ethanol and further diluted with RPMI 1640 medium (thefinal ethanol concentration did not
exceed 0.5%, which did not affect parasite growth). Chloroquine diphosphate was dissolved in
aqueous medium. Test was done in duplicate wellsfor each dose of the drugs. Solvent control
culture containing the same concentrations of thesolvent as present in the test wells was done
with RPMI-1640 containing 10% AB (+) serum. Parasite growth was found to be unaffected at
the solvent concentrations used. Growth of theparasites from duplicate wells of each
concentration was monitored in Giemsa stained blood smears by counting number of schizont
per 100 asexual parasites. Percent schizont maturation inhibition was calculated using the
formula:
Where, Nt and Nc represent the number of schizonts in the test and control well respectively.
5
.2.3. In vitro antileishmanial activity on L. aethiopica promastigotes
All the compounds, dissolved in DMSO, were evaluated for their antileishmanial
activity to a final concentration of 1 mg/ml. The final concentration of DMSO did not exceed
.1 % so that it did not have any effect on parasite. Both test and standard solutions were
0
serially diluted to appropriate concentrations using fresh complete media [20, 48]. The test
compounds were prepared by three-fold serial dilutions (starting from 10 to 0.04 ꢀg/ml).
Amphotericin B deoxycholate and miltefosine were used as positive controls for comparison
of the antileishmanial activity of the test compounds and were used in three-fold serial
dilutions. Promastigote forms of L. aethiopica were used for the assay. A 100 ꢀl of culture
20

ACCEPTED MANUSCRIPT
6
media containing 3 x 10 promastigotes of L. aethiopica were seeded in each well of a 96 well
flat bottom plate. Various dilutions of test compounds (10, 3.33, 1.11, 0.37, 0.12, 0.04 ꢀg/ml)
were added to the parasites. The assay was done in duplicates. Wells containing only the
parasites, media and DMSO were served as negative control. The plates were then kept at
room temperature (21 ± 1 °C). After 24 h, 10 ꢀl of Alamar blue (12.5 mg resazurin dissolved
in 100 ml of distilled water) [49] was added to each of the wells. Absorbance of the resulting
mixture was measured after 48 h at 540 and 630 nm using a plate reader. Alamar blue works
through the conversion of resazurin (7-hydroxy-3H-phenoxazine-3-one-10-oxide), the active
®
ingredient of Alamar blue (blue and non-fluorescent), to resorufin (pink and highly
fluorescent) through reduction reactions of metabolically active cells. The amount of
fluorescence produced is proportional to the number of living cells[50, 51].
5
.2.4. In vitro antileishmanial activity on L. aethiopica amastigotes
The test compounds were serially diluted in a 96-well microtitre plate to a final test
concentration of 0.04 to 10 ꢀg/ml in 50 ꢀl culture medium and 50 ꢀl suspensions containing 2
7
×
10 cells/ml axenic amastigotes were added to each well. The plate contents were then
incubated in humidified atmosphere at 31°C under a 5% CO for 72 h. After 68 h of
2
incubation, a 10 ꢀl of fluorochrome resazurin solution (12.5 ꢀg dissolved in 100 ml of PBS
(pH=7.2)) was added to each well and the fluorescence intensity was measured after a total
incubation time of 72 h using 37 Victor3 Multilabel Counter at excitation wavelength of 530
nm and emission wavelength of 590 nm. The EC50 value for each extract was evaluated from
sigmoidal dose–response curves using computer software Graph pad prism 3.0 and the results
were expressed as mean ± SD of triplicate experiments with each test concentration measured
in duplicate. Assays with standard anti-leishmanial drugs and negative controls (medium alone
and 1% DMSO) were also performed to have reference values. The background fluorescence
intensity of each extract and reference drug was measured [52].
5
.2.5. In vivo acute toxicity testing
The most active compounds, which showed promising dual antileishmanial and
antimalarial activities, were tested for their oral acute toxicity in mice. Eleven groups of mice,
each group consisting of six male mice (26-32 g) were used for testing acute toxicity [53].The
mice in each group were fasted overnight and weighed prior to test. The compounds were
prepared in suspension form in aqueous vehicle containing 1% gum acacia. Mice in group one
to ten were given 25, 50, 100, 200 and 300 mg/kg/day of the synthesized compounds and the
eleventh group was treated orally with the vehicle (control group) at a maximum dose of 1
ml/100 g of body weight. The mortality percentage in each group was recorded after 24 h [24,
3
7]. Additionally, the test compounds were investigated for their parenteral acute toxicity in
groups of six mice each as reported earlier [12]. The compounds, or their vehicle, propylene
glycol (control), were given by intraperitoneal injection in doses of 10, 25, 50, 75, 100 mg/kg.
The survival percentage was followed up to seven days [54].
5
.3. Molecular docking
All computer-aided docking studies were carried out using Molecular Operating
Environment (MOE Dock 2008) software [55]. The crystal structure of Pf-DHFR with the
bound (WR99210) (PDB ID: 1J3I) and Lm-PTR1 with the bound TOP (PDB ID: 2BFM) were
21

ACCEPTED MANUSCRIPT
downloaded from the protein data bank. Target compounds were constructed using the builder
interface of the MOE program and all hydrogens were added. Conformational analyses were
done through energy minimization using Force Field MMFF94x. The active sites of both
proteins were generated using the MOE- Alpha Site Finder, and then ligands were docked
within this active site using MOE Dock. MOE was also used to calculate the best score
between the ligands and the enzymes’ binding sites. Scoring is determined as a total of two
specific scoring functions: alpha hydrogen bonding and London forces. Thirty conformers of
the ligand were retained with highest and best score by default. The obtained conformers that
showed the best ligand-enzyme interactions were selected.
Acknowledgements
The authors would to thank Dr. Ashenafi Damtew, Addis Ababa University, for his expert
assistance with the biological screening of the compounds.
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24

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Figure captions
Figure 1: Structures of lead antimalarial or antileishmanial pyrazoles
Figure 2: Structures of lead antimalarial or antileishmanial agents containing five-
membered rings
Figure 3: General formulae of the target compounds
Figure 4: 3D View from a molecular modeling study, of the minimum-energy structure
of the complex of WR99210 docked in Pf-DHFR (PDB ID: 1J3I). Viewed using
Molecular Operating Environment (MOE) module.
Figure 5: 3D View from a molecular modeling study, of the minimum-energy structure
of compound 4b docked in Pf-DHFR (PDB ID: 1J3I). Viewed using Molecular Operating
Environment (MOE) module.
Figure 6: 3D View from a molecular modeling study, of the minimum-energy structure
of compound 9b docked in Pf-DHFR (PDB ID: 1J3I). Viewed using Molecular Operating
Environment (MOE) module.
Figure 7: 3D View from a molecular modeling study, of the minimum-energy structure
of the complex of TOP docked in Lm-PTR (PDB ID: 2BFM). Viewed using Molecular
Operating Environment (MOE) module.
Figure 8: 3D View from a molecular modeling study, of the minimum-energy structure
of compound 3d docked in Lm-PTR (PDB ID: 2BFM). Viewed using Molecular
Operating Environment (MOE) module.
Figure 9: 3D View from a molecular modeling study, of the minimum-energy structure
of compound 4c docked in Lm-PTR (PDB ID: 2BFM). Viewed using Molecular
Operating Environment (MOE) module.
25

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Table 1: In vivo antimalarial activity of test compounds against P. berghei at dose
4
8.4 μM/ kg/day
1
2
Comp.
No.
R
R
%
%
Mean
Parasitemia*
Suppression survival time
(days)
1
a
b
CH3
CH3
NO2
NO2
CH3
CH3
NO2
NO2
CH3
CH3
CH3
Br
33±0.4
30±0.8
32±0.6
35±0.8
34±1.0
30±1.6
5±0.8
61.62
65.11
62.79
59.30
60.47
65.12
94.19
94.19
83.72
86.05
8.62
1
8.40
7.28
8.71
9.23
8.84
1
c
CH3
Br
1d
2a
2b
CH3
Br
2
c
CH3
Br
14.66
14.84
12.22
12.68
2d
3a
3b
5±0.4
CH3
Br
14±1.2
12±0.4
3
c
NO2
NO2
CH3
CH3
CH3
Br
18±0.2
12±0.4
10±1.6
4±0.2
79.07
86.05
88.37
95.35
10.40
16.86
10.64
16.86
3d
4a
4b
CH3
Br

ACCEPTED MANUSCRIPT
4
c
NO2
NO2
CH3
CH3
NO2
NO2
CH3
CH3
3±0.4
96.51
97.67
97.67
83.72
74.42
67.44
69.77
16.62
16.44
16.84
11.22
12.12
12.26
11.60
4d
5a
5b
Br
2±0.6
CH3
Br
2±0.8
14±2.8
22±0.6
28±0.2
26±1.6
5
c
CH3
Br
5
d
a
6
CH3
6
b
CH3
NO2
CH3
CH3
NO2
Br
Br
22±1.4
3±0.2
74.42
96.51
69.77
74.42
62.79
11.80
16.22
10.82
10.64
9.84
6
c
7
a
b
CH3
Br
26±1.2
22±1.6
32±2.8
7
7
c
Br
8
a
b
CH3
CH3
NO2
CH3
CH3
NO2
CH3
Br
30±0.4
28±0.8
4±0.4
65.12
67.44
95.35
79.07
96.51
69.77
7.62
7.88
8
8
c
Br
17.60
10.08
16.40
6.62
9
a
b
CH3
Br
18±1.4
3±0.6
9
9
c
Br
26±1.2

ACCEPTED MANUSCRIPT
1
0a
CH3
CH3
NO2
…….
CH3
38±0.6
42±1.8
34±0.2
86±1.2
0.0
55.81
51.16
60.47
0.00
8.84
6.86
6.82
5.24
ND**
1
0b
0c
Br
1
Br
Control
…….
…….
Chloroquine …….
phosphate
100
*
Values are M± SD, P< 0.05
** ND: No death recorded during experimental period
Table 2: In vitro anti-plasmodial activity against
chloroquine-resistant (RKL9) strain of P. falciparum.
Comp. No.
IC , µM±SD*
50
2
c
0.0378±0.006
0.0384±0.002
0.0364±0.004
0.0402±0.002
0.0392±0.012
0.0418±0.008
0.0388±0.010
0.0420±0.006
0.0368±0.008
0.1920±0.003
2
4
d
b
4
c
4
d
a
5
6
8
c
c
9
b
Chloroquine
*
Results of two separate determinations.