Synthesis of new 2-(5-substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-b]quinoxaline derivatives and evaluation of their antiamoebic activity

Article abstract of DOI:10.1016/j.ejmech.2008.02.002

In an effort to develop potent antiamoebic agents, we have synthesized chalcones (1-8), amino-5-substituted-(3-phenyl(2-pyrazolinyl))methane-1-thione derivatives (1a-8a) and 2-(5-substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-b]quinoxaline derivat

Full text of DOI:10.1016/j.ejmech.2008.02.002

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1317e1325
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of new 2-(5-substituted-3-phenyl-2-pyrazolinyl)-1,
3-thiazolino[5,4-b]quinoxaline derivatives and evaluation
of their antiamoebic activity
*
Asha Budakoti, Abdul Roouf Bhat, Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 21 March 2007; received in revised form 25 January 2008; accepted 8 February 2008
Available online 4 March 2008
Abstract
In an effort to develop potent antiamoebic agents, we have synthesized chalcones (1e8), amino-5-substituted-(3-phenyl(2-pyrazolinyl))me-
thane-1-thione derivatives (1ae8a) and 2-(5-substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-b]quinoxaline derivatives (1be8b) and eval-
uated for their in vitro antiamoebic activity against HM1:IMSS strain of E. histolytica. All the compounds were characterized by electronic, IR,
1H NMR and mass spectroscopic data. It was observed that the antiamoebic activity enhances on modifying the structure of chalcones to the
pyrazolines and further to quinoxalines. The MTT assay was performed on human kidney epithelial cell line to check the cytotoxicity of the
compounds and the results were compared with metronidazole. Compound 6b showed better antiamoebic activity and less toxicity than met-
ronidazole.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Chalcone; Pyrazolines; Thiocarbamoyl; Quinoxaline; Antiamoebic activity
1. Introduction
complication and possible selection of a resistant E. histoly-
tica strain have been reported [3]. Therefore, the develop-
ment of new alternative antiamoebic drugs devoid of side
effects is still needed.
Ameobiasis is the infection of the human gastrointestinal
tract by Entamoeba histolytica [1]. E. histolytica causes ap-
proximately 50 million cases and approximately 100,000
deaths annually. Amoebic liver abscesses are the most fre-
quent and severe clinical presentations of amoebiasis. Symp-
tomatic patients, typically present abdominal pain,
tenderness, diarrhea and bloody stools. The drugs used to
eradicate E. histolytica, such as nitroimidazoles, have been
shown to pose several important problems as mutagenic
and toxic for the host when they are used at high doses,
amoeba strains are able to develop resistance to these drugs
[2]. Amoebiasis is treated with the drug metronidazole, even
though significant side effects, such as neurological
Among the various classes of nitrogen containing hetero-
cyclic compounds, quinoxaline derivatives are the important
components of several pharmacologically active compounds
[4e9]. Although rarely described in nature, synthetic qui-
noxaline ring is part of number of antibiotics such as echi-
nomycin and actinomycin, which are known to inhibit the
growth of Gram-positive bacteria and are also active against
various transplantable tumors [10e12]. There are many
examples of biologically active quinoxalines, which showed
very interesting pharmacological properties such as antibac-
terial, antiviral, anticancer, antifungal, anthelmintic, and in-
secticidal [13e18]. All literature survey reveals the
pharmacological importance of quinoxalines. The new
quinoxaline derivatives, 1be8b, were synthesized and
screened in vitro for their ability to inhibit the growth of
E. histolytica.
* Corresponding author. Tel.: þ91 11 26981717/3253; fax: þ91 11
26980229/1232.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.002

1318
A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
2. Results and discussion
band at 371e292 nm was assigned to the transition involving
the thione portion (C]S) of thiocarbamoyl group. The two
other absorption bands at 288e236 and 232e205 nm were
due to p / p* transition of phenyl ring and n / s* transi-
tion of azomethine nitrogen, respectively. The UV spectral
data of 1be8b were also studied which showed the same
type of transitions as observed in compounds 1ae8a. It
showed three spectral bands at 388.3e299, 287e248 and
239e204 nm assigned to n / p*, p / p* and n / s* tran-
sitions of thiocarbamoyl group (CeSeC), aromatic ring and
azomethine nitrogen, respectively.
The synthetic routes of the proposed compounds 1e8, 1ae8a
and 1be8b are outlined in Scheme 1. ClaiseneSchmidt conden-
sation between acetophenone and aromatic aldehydes in the
presence of methanolic solution of sodium hydroxide resulted
in the formation of chalcones (1e8) in excellent yields (49e
1
93%). They were characterized by UVevis, IR and H NMR
spectroscopic techniques. Cyclisation of different chalcones
with thiosemicarbazide under basic condition in refluxing etha-
nol leads to the formation of new amino-5-substituted-(3-phe-
nyl(2-pyrazolinyl))methane-1-thione (1ae8a) in modest
yields (11e24%). They were also fully characterized using var-
ious spectroscopic techniques. The 2-(5-substituted-3-phenyl-
2.2. Nuclear magnetic resonance spectral studies
2-pyrazolinyl)-1,3-thiazolino[5,4-b]quinoxaline
derivatives
Further evidence for the formation of 1e8, 1ae8a and 1be
1
8b was obtained by H NMR spectroscopy, which provide
diagnostic tools for the positional elucidation of the protons.
Assignment of the signals are based on the chemical shifts
1
and intensity patterns. Two doublets in the H NMR spectra
(1be8b) were obtained in (16e49%) yields by heating at reflux
amino-5-substituted-(3-phenyl(2-pyrazolinyl))methane-1-thione
(1ae8a) with 2,3-dichloroquinoxaline. The progress of each
reaction was monitored by thin layer chromatography. Com-
1
pounds 1be8b were characterized by IR, UVevis, H NMR,
of chalcones (1e8) in the region (6.86e7.74) and (5.82e
7.66) ppm appears due to (eCOeCH]) and (]CHeAr) pro-
tons favor their formation. In the 1H NMR spectra of the
cyclised product of chalcone, 1ae8a, pyrazoline protons HA
and HB are geminal protons at C4 carbon, appears in the region
3.90e3.09 and 3.32e3.98 ppm as doublet of doublets in all
compounds. The CH proton also appeared as doublet of dou-
blets in the region of 6.32e5.40 ppm due to vicinal coupling
with two non-equivalent geminal protons of C4 carbon. These
protons HA and HB protons at C4 carbon were slightly shifted
in case of quinoxaline compounds 1be8b and appears in the
region 3.99e3.11 and 3.80e2.78 ppm as doublet of doublets
in all quinoxaline compounds. The CH proton also appeared
as doublet of doublets in the region of 6.58e5.11 ppm due
to vicinal coupling with two non-equivalent geminal protons
of C4 carbon. The NH proton of different substituted thiocar-
bamoyl pyrazoline compounds showed a doublet at 9.24e
7.17 ppm. Compounds 1ae8a and 1be8b were additionally
characterized by 13C NMR spectroscopy. In the 13C NMR
spectra, the C4 and C5 carbons of the pyrazoline ring in com-
pounds 1ae8a resonate at 37.43e37.06 and 62.98e
60.49 ppm, respectively. The phenyleC resonates at
149.93e132.71 ppm. All the pyrazoline compounds showed
a signal at 189.23e170.16 ppm, which was assigned to azo-
methine carbon of pyrazoline ring. Thiocarbamoyl carbon
(C]S) displayed a signal at 169.42e204.23 ppm. The qui-
noxaline compounds 1be8b showed two signals at 151.66e
152.36 and 140.11e143.35 ppm due to azomethine carbon
of the pyrazoline ring and quinoxaline ring, respectively.
Thiocarbamoyl carbon (CeSeC) displayed a signal at
143.07e139.31 ppm in all the compounds.
13C NMR and mass spectroscopy. The purity of all the com-
pounds was checked by elemental analysis.
2.1. IR and electronic spectral studies
The positions of IR band provide significant indication for
the formation of 1e8, 1ae8a and 1be8b. The bands due to
n(C]O) and n(C]C) stretch at (1669e1750) cmꢀ1 and
(1523e1580) cmꢀ1, respectively, favors the formation of chal-
cone derivatives (1e8). The absence of n(C]O) and n(C]C)
bands in the IR spectra of 1ae8a obtained by cyclisation of
chalcone 1e8 shows the formation of 1ae8a. Compounds
1ae8a showed intense bands in the region 1333e1370 cmꢀ1
due to the n(C]S) stretch of the thiocarbamoyl group. The
IR spectra of all the compounds showed n(C]N) stretch at
1542e1590 cmꢀ1 due to the ring closure. In addition, the ab-
sorption bands at 1024e1122 cmꢀ1 were attributed to the
n(CeN) stretch vibrations, which also confirm the formation
of desired pyrazoline ring in all the compounds. The com-
pounds showed sharp bands in the region 3228e3454 cmꢀ1
due to the n(NH) stretch. Compounds 1ae8a were further
reacted with 2,3-dichloroquinoxaline to get compounds 1be
8b. The bands due to n(NH) stretch and n(C]S) were absent
in these compounds. Selected diagnostic bands of the IR spec-
tra of 2-(5-substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazo-
lino[5,4-b]quinoxaline derivatives (1be8b) showed intense
bands at 839e945 cmꢀ1 due to the n(CeS) stretch. The
compounds show two strong bands at 1486e1563 and
1517e1510 cmꢀ1 due to n(C]N) stretch of azomethine nitro-
gen of pyrazoline ring and quinoxaline ring, respectively. In
addition, the absorption band at 1078e1192 cmꢀ1 attributed
to the n(CeN) stretch vibrations, which also confirm the for-
mation of desired pyrazoline ring in all the compounds [19].
The electronic spectra of the cyclised pyrazoline analogues
studied in the UV region in methanol, exhibited three absorp-
tion bands at 371e290, 270e236 and 223e205 nm assignable
to n / p*, p / p* and n / s* transitions, respectively. The
2.3. ESI-MS analysis
The characteristic peaks observed in the mass spectra of qui-
noxaline derivative (8b) are summarized in Scheme 2. The mass
spectrum exhibits molecular ion peaks and contains fragments
that confirm the quinoxaline structure in all the compounds.

A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
1319
O
CO-CH3
R
NaOH/MeOH
5-28°C, 18 h
+
R - CHO
(1-8)
S
NaOH/ethanol
reflux, 8h
NH2-NH-C-NH2
R
N
reflux, 8h
ethanol
N
N
S
N
R
N
N
N
N
N
Cl
Cl
NH2
S
(1b-8b)
(1a-8a)
CH3
1.
6.
R =
2.
CH2CH3
3.
N
H
4.
CH3
5.
CH3
CH3
CH3
CH3
Cl
Cl
Cl
8.
7.
Scheme 1. Synthesis of 2-(5-substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-b]quinoxaline derivatives (1be8b).
The fragmentation of compound 8b showed the molecular ion
2.4. In vitro antiamoebic activity
peak (M þ 1). The fragmentation of the compound occurs via
removal of [eCl2] moiety giving desired peaks in all the
compounds which correspond to the [M ꢀ Cl2]þ ion. This is
followed by elimination of eC6H5 [20].
All the compounds were evaluated for antiamoebic activity in
vitro using HM1:IMSS strain of E. histolytica. The IC50 values
in mM are shown in Table 1. The results were estimated as the
+.
N
N
N
N
N
N
N
Cl2
N
N
S
N
S
Cl
Cl
m/z=477
m/z=406
_
.
+.
N
N
N
S
N
N
m/z=328
Scheme 2. Mass fragmentation pattern of compound 8b.

1320
A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
Table 1
In vitro antiamoebic activity against HM1:IMSS strain of E. histolytica and toxicity studies
R
N
N
N
S
N
O
N
N
R
N
R
S
NH2
1-8
1a-8a
1b-8b
Compound
R
Antiamoebic activity
Toxicity profile
IC50 (mM)
S.D.a
IC50 (mM)
Safety index
N
1
1.70
1.61
1.49
0.23
0.34
0.18
>50
>50
>50
>29.41
>31.05
>33.55
5
5
5
1a
1b
2
1.80
1.77
1.29
0.48
0.23
0.17
>44
>54
>18
>24.43
>30.51
>13.96
3
5
5
2a
2b
N
H
3
2.12
1.58
0.30
0.34
0.18
0.17
>50
>50
>14
>23.58
>31.68
>46.40
3
3
3
3a
3b
CH3
CH3
4
1.41
4.37
3.20
0.23
0.34
0.18
>50
>50
>35
>35.46
>11.44
>10.78
5
5
5
CH3
4a
4b
CH3
5
1.79
1.61
1.49
0.05
0.04
0.09
>50
>50
>50
>27.93
>31.05
>33.55
3
3
3
CH2CH3
5a
5b
CH3
CH3
6
3.48
0.23
0.17
0.48
0.23
0.17
>46
>50
>50
>13.25
>217.39
>294.11
3
3
4
6a
6b
7
2.60
2.23
1.90
0.98
0.02
0.33
>50
>50
>50
>19.23
>22.42
>26.31
3
5
5
Cl
7a
7b
8
2.01
1.09
0.34
0.18
0.17
>50
>45
>50
>24.87
>41.18
>56.17
5
5
5
Cl
8a
8b
0 .89
Cl
Metronidazole
1.80
0.05
>100
>55.56
5
N ¼ number of times the experiment has been repeated.
a
Standard deviation.
percentage of the growth inhibition compared with the untreated
controls and plotted as probit values as a function of the drug
concentration. The IC50 and 95% confidence limits were inter-
polated in the corresponding dose-response curve. We have syn-
thesized chalcone derivatives (1e8) having various aromatic
substituents. Chalcones have already been known for their ex-
cellent biological activity against number of diseases [21e23].
They exhibited antiamoebic activity with IC50 of 1.41e
3.48 mM. Out of eight chalcone derivatives only two were
found with more inhibition activity for E. histolytica than

A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
1321
metronidazole, the standard drug. From these chalcones we
4. Experimental
have prepared amino-5-substituted-(3-phenyl(2-pyrazolinyl))-
methane-1-thione derivatives (1ae8a) and their activities
were compared. It was observed that due to their modification
as pyrazolines, their potencies were increased with IC50 0.23e
4.37 mM. Out of eight compounds three were found with better
inhibitory tendency than metronidazole. The compound
amino{5-[2-(methylethylphenyl)-3-phenyl(2-pyrazolinyl)}me-
thane-1-thione (6a) having 2-methylethylphenyl substituents,
IC50 ¼ 0.23 mM was w1.6 times more active than metronida-
zole (IC50 ¼ 1.8 mM). From these very effective compounds,
we have synthesized 2-(5-substituted-3-phenyl-2-pyrazolinyl)-
1,3-thiazolino[5,4-b]quinoxaline derivatives (1be8b) and their
potencies were further enhanced by the introduction of quinoxa-
line moiety (IC50 ¼ 0.17e1.90 mM). Out of eight compounds
six had better inhibitory tendency than metronidazole as well
as their parent compounds. Compound 6b with IC50 ¼ 0.17 mM
was found to be the most active among all the compounds.
All the chemicals were purchased from Aldrich Chemical
Company (U.S.A) and were used without further purifica-
tion. The reactions were monitored by pre-coated aluminium
silica gel 60F254 thin layer plates procured from Merck
(Germany). The purity of compounds was confirmed by C,
H and N analysis carried out at Central Drug Research Insti-
tute, Lucknow, India. Melting points were recorded on KSW
melting point apparatus and are uncorrected. Electronic
spectra were recorded in methanol on a Shimadzu UV-
1601 PC UVevis spectrophotometer. IR spectra on KBr
disks were recorded on a Perkin Elmer model 1620 FT-IR
1
spectrophotometer. H and 13C NMR spectra were obtained
at ambient temperature using a BRUCKER SPECTROSCO-
PIN DPX-300 MHz spectrophotometer in CDCl3 using tetra-
methylsilane as an internal standard. The ESI mass spectra
of a few representative compounds were recorded on a
MICRO MASS QUATTRO II triple quadrupole mass
spectrometer.
2.5. Toxicity profile
The toxic activities of compounds 1e8, 1ae8a and 1be8b
were evaluated in vitro on human kidney epithelial cell line.
The numerical results for each compound are given in Table 1.
These activities were expressed as micromolar concentrations
of the compounds that inhibited 50% of cell proliferation
(human kidney epithelial). All the compounds inhibited cell
growth at varied concentration (IC50 > 14e50 mM) while for
metronidazole it is (IC50 > 100 mM). Except a few (2b, 3b
and 4b) all the compounds inhibited cell growth at a concentra-
tion of >50 mM. To investigate the selectivity of the com-
pounds, the ‘‘safety index’’ (SI) was calculated and defined
as: toxicity IC50/protozoal IC50; where toxicity IC50 is defined
as the concentration of compound that kills 50% of the human
(kidney epithelial) cell line and protozoal IC50 is the concentra-
tion that kills 50% of amoeba protozoa. This allows an estimate
of which compounds might be efficacious or toxic against hu-
man cells and potentially in vivo. Safety index profile showed
that compounds 6a, 6b and 8b were less toxic than metronida-
zole. The results implicate that compound 6b has less toxicity
and better antiamoebic activity than metronidazole.
4.1. Synthesis of chalcones: general procedure
A solution of acetophenones (50 mmol) and appropriate al-
dehyde (50 mmol) in methanolic NaOH was stirred for 18 h at
5e28 ꢁC. Solid obtained was washed with ice-cold water and
then ethanol, dried and recrystallized by the adequate solvent
as indicated for each chalcone [24e26].
4.1.1. 1,3-Diphenyl prop-2-en-1-one (1)
Pale yellow crystals (chloroform); yield: 93%; m.p. 55 ꢁC;
Anal. calc. for C15H12O: C 86.21, H 4.86; found: C 86.50, H
4.90; UVevis lmax (nm): 371, 291, 236, 217; IR nmax (cmꢀ1):
1664 (C]O), 1620 (CH]CH), 1580 (C]C); 1H NMR
(CDCl3) (d, ppm): 7.80 (m, 10H, Ar), 7.74 (d, H, J ¼ 15 Hz,
1H), 7.63 (d, H, J ¼ 15 Hz, 1H).
4.1.2. 3-Indol-3-yl-1-phenylprop-2-en-1-one (2)
Dark yellow solid (chloroform); yield: 56%; m.p. 182 ꢁC;
Anal. calc. for C17H12NO: C 77.86, H 4.58, N 5.34; found:
C 77.83, H 4.52, N 5.33; IR (KBr) (cmꢀ1): 3160 (NH),
3. Conclusion
1
We report herein the synthesis and in vitro antiamoebic
activity of chalcone (1e8), amino-5-substituted-(3-phenyl
(2-pyrazolinyl))methane-1-thione derivatives (1ae8a) and 2-
(5-substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-
b]quinoxaline derivatives (1be8b) against HM1:IMSS strain
of E. histolytica. Modification of the compounds from chal-
cones, 1e8 to pyrazolines, 1ae8a and further to quinoxa-
lines, 1be8b results in an increase in antiamoebic activity.
The toxic studies on human kidney epithelial cell lines
showed that albeit all the compounds were not toxic but
more toxic than metronidazole except 6a, 6b and 8b and
compound 6b has most promising toxicity profile with potent
antiamoebic activity.
3020 (aromatic, CeH), 1710 (C]O), 1630 (eCH]CH); H
NMR (CDCl3) (d, ppm): 8.60 (1H, s, NH of indole), 7.11e
7.89 (9H, m, AreH), 6.86 (1H, d, J ¼ 14.8 Hz, ]CHeAr),
5.82 (1H, d, J ¼ 14.8 Hz, COeCH]).
4.1.3. 3-(4-Methylphenyl)-1-phenyl prop-2-en-1-one (3)
Pale yellow crystals (chloroform); yield: 54%; m.p. 175 ꢁC;
Anal. calc. for C16H14O: C 86.48, H 6.30; found: C 86.51, H
6.26; UVevis lmax (nm): 291, 236, 217; IR nmax (cmꢀ1): 1710
(C]O), 1643 (CH]CH), 1550 (C]C); 1H NMR (CDCl3) (d,
ppm): 7.90 (m, 10H, Ar), 7.71 (d, H, J ¼ 15 Hz, 1H), 7.66 (d,
H, J ¼ 15 Hz, 1H), 2.35 (s, 3H, CH3).

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A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
4.1.4. 1-Phenyl 3-(2,4,6-trimethylphenyl)
prop-2-en-1-one (4)
217; IR nmax (cmꢀ1): 3435 (NeH), 2924 (CeH), 1558
1
(C]N), 1357 (C]S), 1122 (CeN); H NMR (CDCl3) (d,
Pale yellow crystals (chloroform); yield: 49%; m.p. 128 ꢁC;
Anal. calc. for C18H18O: C 86.40, H 7.20; found: C 86.43, H
7.16; UVevis lmax (nm): 371, 291, 236, 217; IR nmax (cmꢀ1):
1721 (C]O), 1630 (CH]CH), 1552 (C]C); 1H NMR
(CDCl3) (d, ppm): 7.81 (m, 7H, Ar), 7.70 (d, H, J ¼ 15 Hz,
1H), 7.16 (d, H, J ¼ 15 Hz, 1H), 2.50 (s, 9H, CH3).
ppm): 6.51e7.50 (m, 10H, Ar), 3.48 (dd, 1H, HA, JAB:
18.5 Hz, JAX: 9.25), 3.49 (dd, 1H, HB, JAB: 18.5, JBX: 9.25),
6.32 (dd, 1H, HX, JAX: 11.10, JBX: 9.25), 2.13 (s, 3H, CH3),
9.24 (d, 2H, NH2); 13C NMR (CDCl3) (d, ppm): 176.19
(C]S), 149.57 (C]N), 144.52e125.72 (PhenyleC), 62.86
(CeH), 37.06 (CH2).
4.1.5. 3-(2-Ethylphenyl)-1-phenylprop-2-en-1-one (5)
4.2.2. Amino(5-indol-3-yl-3-phenyl(2-pyrazolinyl))methane-
1-thione (2a)
Pale yellow crystals (chloroform); yield: 76%; m.p. 108 ꢁC;
Anal. calc. for C17H18O: C 86.44, H 6.77; found: C 86.48, H
6.80; UVevis lmax (nm): 271, 236, 217; IR nmax (cmꢀ1): 1750
Pale yellow crystal (chloroform); yield: 21%; m.p. 185 ꢁC;
Anal. calcd. for C17H15N4S: C, 66.44, H, 4.88, N, 18.24;
found: C, 66.46, H, 4.85, N, 18.39; UVevis lmax (nm): 371,
270, 236; IR: nmax (cmꢀ1): 3233 (NeH), 2928 (CeH), 1590
1
(C]O), 1622 (CH]CH), 1523 (C]C); H NMR (CDCl3)
(d, ppm): 7.71 (d, H, J ¼ 15 Hz, 1H), 7.53 (d, H, J ¼ 15 Hz,
1H), 7.11 (m, 10H, Ar), 1.20e2.10 (m, 2H, CH2), 3.20
(t, 3H, CH3).
1
(C]N), 1336 (C]S), 1052 (CeN); H NMR (CDCl3) (d,
ppm): 6.67e7.75 (m, 15H, Ar), 3.31 (dd, 1H, HA, JAB:
17.33, JAX: 9.33), 3.32 (dd, 1H, HB, JAB: 16.6, JAX: 9.52),
6.02 (dd, 1H, HX, JAX: 9.33, JBX: 8.75), 7.89 (d, 2H, NH2),
13C NMR (CDCl3) (d, ppm): 170.16 (C]S), 149.57 (C]N),
149. 93e126.07 (PhenyleC), 38.28 (CH), 33.86 (CH2).
4.1.6. 3-(2-Methylethylphenyl)-1-phenylprop-2-en-1-one (6)
Pale yellow crystals (chloroform); yield: 54%; m.p. 144 ꢁC;
Anal. calc. for C18H17O: C 86.74, H 6.82; found: C 86.70, H
6.79; UVevis lmax (nm): 291, 236, 217; IR nmax (cmꢀ1): 1710
(C]O), 1613 (CH]CH), 1530 (C]C); 1H NMR (CDCl3) (d,
ppm): 7.80 (m, 9H, Ar), 7.74 (d, H, J ¼ 15 Hz, 1H), 7.63 (d, H,
J ¼ 15 Hz, 1H), 2.10 (m, 1H, CH), 1.80 (m, 6H, CH3).
4.2.3. Amino[5-(4-methylphenyl)-3-phenyl(2-pyrazolinyl)]
methane-1-thione (3a)
Pale yellow crystal (chloroform); yield: 11%; m.p. 160 ꢁC;
Anal calcd. for C17H17N3S: C 69.15, H 5.76, N 14.23; found:
C 69.21, H 5.70, N 14.19; UVevis lmax (nm): 371, 291, 245;
IR nmax (cmꢀ1): 3448 (NeH), 2963 (CeH), 1542 (C]N),
1364 (C]S), 1024 (CeN); 1H NMR (CDCl3) (d, ppm):
4.1.7. 3-(2-Chlorophenyl)-1-phenylprop-2-en-1-one (7)
Pale yellow crystals (chloroform); yield: 63%; m.p. 135 ꢁC;
Anal. calc. for C17H13O: C 86.21, H 4.86; found: C 86.50, H
4.90; UVevis lmax (nm): 371, 291, 236, 217; IR nmax (cmꢀ1):
1743 (C]O), 1630 (CH]CH), 1523 (C]C); 1H NMR
(CDCl3) (d, ppm): 7.78 (m, 10H, Ar), 7.67 (d, H, J ¼ 15 Hz,
1H), 7.20 (d, H J ¼ 15 Hz, 1H).
6.70e7.60 (m, 9H, Ar), 3.80 (dd, 1H, HA JAB: 18.5, JAX:
9.25 Hz), 3.60 (dd, 1H, HB, JAB: 18.5, JBX: 9.5), 5.90 (dd,
1H, HX, JAX: 9.25, JBX: 9.5), 2.32 (s, 3H, CH3), 7.17 (d,
2H, NH2).13C NMR (CDCl3) (d, ppm): 172.19 (C]S),
146.57 (C]N), 144.52e125.72 (PhenyleC), 62.86 (CH),
37.02 (CH2), 21.09 (CH3).
4.1.8. 3-(3,4-Dichlorophenyl)-1-phenylprop-2-en-1-one (8)
Pale yellow crystals (chloroform); yield: 85%; m.p. 118 ꢁC;
Anal. calc. for C17H13NO2: C 86.21, H 4.86; found: C 86.24,
H 5.16; UVevis lmax (nm): 371, 291, 236, 217; IR nmax
(cmꢀ1): 1710 (C]O), 1630 (CH]CH), 1550 (C]C); 1H
NMR (CDCl3) (d, ppm): 7.80 (m, 10H, Ar), 7.74 (d, 1H,
J ¼ 15 Hz,), 7.63 (d, 1H, J ¼ 15 Hz).
4.2.4. Amino[3-phenyl-5-(2,4,6-methylphenyl)(2-pyrazolinyl)]
methane-1-thione (4a)
Pale yellow powder (chloroform); yield: 24%; m.p. 175 ꢁC;
Anal. Calc. for C19H21N3S: C 70.58, H 6.50, N 13.00; found:
C 70.54, H 6.49, N 13.03; UVevis lmax (nm): 371, 290, 236,
217; IR nmax (cmꢀ1): 3435 (NeH), 2924 (CeH), 1558
1
4.2. Synthesis of amino-5-substituted-(3-phenyl(2-
pyrazolinyl))methane-1-thione (1ae8a):
general procedure
(C]N), 1357 (C]S), 1122 (CeN); H NMR (CDCl3) (d,
ppm): 6.72e7.50 (m, 7H, Ar), 3.48 (dd, 1H, HA, JAB:
18.5 Hz, JAX: 9.25), 3.49 (dd, 1H, HB, JAB: 18.5, JBX: 9.25),
6.32 (dd, 1H, HX, JAX: 11.10, JBX: 9.25) 2.13 (s, 3H, CH3),
7.79 (d, 1H, NH2); 13C NMR (CDCl3) (d, ppm): 189.23
(C]S), 146.57 (C]N), 132.78e125.32 (PhenyleC),
20.98e20.58 (CH3), 37.43 (CH2), 62.86 (CH).
A mixture of chalcone 1e8 (10 mmol), thiosemicarbazide
(10 mmol) and NaOH (25 mmol) was refluxed in ethanol
(25 ml) for 8 h. The solution was poured into ice water. The
precipitate was filtered and recrystallized from the suitable
solvent [20].
4.2.5. Amino[5-(2-ethylphenyl)-3-phenyl
(2-pyrazolinyl)]methane-1-thione (5a)
4.2.1. Amino[3,5-diphenyl(2-pyrazolinyl)]methane-
1-thione (1a)
Pale yellow powder (chloroform); yield: 24%; m.p. 175 ꢁC;
Anal. calc. for C16H15N3S: C 68.32, H 5.33, N 14.94; found: C
68.30, H 5.31, N 14.89; UVevis lmax (nm): 371, 290, 236,
Bright orange crystal (chloroform); yield: 18%; m.p.
159 ꢁC, Anal. calcd. for C18H19 N3S: C 69.90, H 6.14, N
13.59; found: C 69.95, H 6.18, N 13.50; UVevis lmax
(nm): 371, 291, 245, IR nmax (cmꢀ1): 3228 (NeH) 2925
1
(CeH), 1578 (C]N), 1333 (C]S), 1066 (CN); H NMR

A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
1323
(CDCl3) (d, ppm): 6.7e7.6 (m, 9H, Ar), 3.9 (dd, 1H, HA,
AB: 18.5, JAX: 9.25 Hz), 3.6 (dd, 1H, HB, JAB: 18.5, JBX
The solvent was evaporated under vacuo. The residue was re-
crystallized from acetone [27].
J
:
9.5), 5.4 (dd, 1H, HX, JAX: 9.25, JBX: 9.5), 2.32 (s, 3H,
CH3), 7.19 (d, 2H, NH2), 13C NMR (CDCl3): (d, ppm):
172.19 (C]S), 146.50 (C]N), 144.52e125.72 (Phenyle
C), 62.86 (CH), 37.06 (CH2), 27.90 (ethyleCH2), 15.70
(CH3).
4.3.1. 2-(3,5-Diphenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-b]
quinoxaline (1b)
Pale yellow crystal (chloroform); yield: 43%; m.p. 181 ꢁC;
Anal. calc. for C24H17N5S: C 70.76, H 4.18, N 17.20; found: C
70.80, H 4.16, N 17.21; UVevis lmax (nm): 371, 290, 236,
217; IR nmax (cmꢀ1): 2954 (CeH), 1558 (C]N), 1542
(C]N), 1122 (CeN), 920 (CeS); 1H NMR (CDCl3) (d,
4.2.6. Amino{5-[2-(methylethylphenyl)-3-phenyl
(2-pyrazolinyl)]}methane-1-thione (6a)
Orange crystal (chloroform); yield: 22%; m.p: 189 ꢁC;
Anal.calcd. for C20H23N3S: C 70.80, H 6.21, N 13.04; found:
C 70.83, H 6.22, N 13.10; UVevis lmax (nm): 366, 301, 223,
205, 246; IR nmax (cmꢀ1): 3255 (NH) 2923 (CeH), 1545
(C]N), 1349 (C]S), 1033 (CeN); 1H NMR (d, ppm):
7.10e7.72 (m, 15H, Ar), 3.09 (dd, 1H, HA), 3.5 (dd, 1H,
HB, JAB: 16.6, JAX: 9.52), 6.0e5.9 (dd, 1H, HX, JAX, 2.58,
ppm): 8.72e7.50 (14H, m, Ar), 3.48 (dd, 1H, HA, JAB:
18.5 Hz, JAX, 9.25), 3.80 (dd, 1H, HB, JAB: 18.5 Hz,), 6.32
(dd, 1H, HX, JAX: 11.10, JBX: 9.25). 13C NMR (CDCl3) (d,
ppm): 143.07 (CeS), 152.36 (C]N), 143.35 (C]N),
136.33e125.10 (PhenyleC), 35.82 (CH2), 61.60 (CH).
4.3.2. 2-(5-Indol-3-yl-3-phenyl-2-pyrazolinyl)-1,
3-thiazolino[5,4-b]quinoxaline (2b)
J
BX: 7.75), 2.51 (s, 3H, CH3)), 7.80 (d, 1H, NH2); 13C NMR
Yellow crystal (chloroform); yield: 49%; m.p. 175 ꢁC;
Anal. calc. for C26H17N6S: C 70.11, H 3.82, N 18.87; found:
C 70.12, H 3.80, N, 18.82; UVevis lmax (nm): 369, 290,
236, 215; IR nmax (cmꢀ1): 2924 (CeH), 1516 (C]N), 1510
(C]N), 1099 (CeN), 862 (CeS); 1H NMR (CDCl3) (d,
(CDCl3) (d, ppm): 175.49 (C]S), 143.85 (C]N), 148.52e
115 (PhenyleC), 24.08 (CH3), 62.86 (CH), 37.43 (CH2).
4.2.7. Amino[5-(2-chlorophenyl)-3-phenyl
(2-pyrazolinyl)]methane-1-thione (7a)
Yellow crystal (chloroform); yield: 18%; m.p. 210 ꢁC;
Anal. calc. for C16H14N3SCl: C 60.85, H 4.43, N 13.31;
found: C 60.80, H 4.40, N 13.29; UVevis lmax (nm): 371,
291, 245, 223, 211; IR nmax (cmꢀ1): 3421 (NH), 2922 (Ce
H), 1549 (C]N), 1370 (C]S), 1076 (CeN); 1H NMR
(CDCl3) (d, ppm): 7.58e7.51 (m, 9H, Ar), 3.59 (dd, 1H,
HA, JAB: 18.75 JAX: 12.5), 3.98 (dd, 1H, HB, JAB: 17.5,
ppm); 7.50e7.01 (14H, m, Ar), 3.11 (dd, 1H, HA, JAB:
18.5 Hz, JAX: 9.25), 2.80 (dd, 1H, HB, JAB: 18.5, JBX: 9.25),
5.11 (dd, 1H, HX, JAX: 11.10, JBX: 9.25), 9.24 (d, 1H, NH),
9.24 (d, 1H, NH). 13C NMR (CDCl3) (d, ppm): 140.31 (Ce
S), 150.23 (C]N), 140.11 (C]N), 136.33e125.10 (Phe-
nyleC), 35.82 (CH2), 61.60 (CH).
J
BX: 6.77), 6.32 (dd, 1H, HX, JAX: 10.96, JBX: 6.77), 7.77
4.3.3. 2-(5-(4-Methylphenyl)-3-phenyl-2-pyrazolinyl)-1,3-
thiazolino[5,4-b]quinoxaline (3b)
(d, 2H, NH2).13C NMR (CDCl3) (d, ppm): 175.49 (C]S),
143.85 (C]N), 148.52e115 (PhenyleC), 37.21 (CH2),
62.98 (CH).
Pale yellow crystal (chloroform); yield: 22%; m.p. 169 ꢁC;
Anal. calc. for C25H19N5S: C 71.25, H 4.51, N 16.62; found: C
71.31, H 4.55, N 16.69; UVevis lmax (nm): 371, 290, 236,
217; IR nmax (cmꢀ1): 2933 (CeH), 1558 (C]N), 1576
(C]N), 1122 (CeN), 915 (CeS); 1H NMR (CDCl3) (d,
4.2.8. Amino[5-(2,3-dichlorophenyl)-3-phenyl
(2-pyrazolinyl)]methane-1-thione (8a)
Yellow crystal (chloroform); yield: 13%; m.p: 190 ꢁC;
Anal. calcd. for C16H13N3SCl2: C 54.85, H 3.71, N 12; found:
C 54.80, H 3.75, N 12.12%; UVevis lmax (nm): 371, 270,
236; IR nmax (cmꢀ1): 3454 (NH), 2925 (CeH), 1585
ppm): 6.72e7.50 (14H, m, Ar), 3.66 (dd, 1H, HA, JAB
:
18.5 Hz, JAX: 9.25), 3.49 (dd, 1H, HB, AB: 18.5, JBX: 9.25),
J
6.58 (dd, 1H, HX, JAX: 11.10, JBX: 9.25) 2.13 (s, 3H, CH3)
13C NMR (CDCl3) (d, ppm): 140.31 (CeS), 152.01 (C]N),
141.78 (C]N), 133.33e126.10 (PhenyleC), 33.58 (CH2),
59.56 (CH),17.85 (CH3).
1
(C]N), 1350 (C]S), 1052 (CeN); H NMR (CDCl3) (d,
ppm): 6.78e7.51, (m, 10H, Ar), 3.38 (dd, 1H, HA, JAB
:
17.33, JAX: 9.33), 3.48 (dd, 1H, HB, JAB: 16.7, JBX: 8.1),
6.21 (dd, 1H, HX, JAX: 11.6, JBX: 10.2), 7.7 (d, 2H, NH2),
1.2e2.1 (m, 10H, CH2): 13C NMR (CDCl3): (d, ppm):
177.23 (C]S), 140.66 (C]N), 136.33e125.10 (PhenyleC),
37.43 (CH2), 60.49 (CH).
4.3.4. 2-[3-Phenyl-5-(2,4,6-trimethylphenyl)-2-pyrazolinyl]-
1,3-thiazolino[5,4-b] quinoxaline (4b)
Yellow crystal (chloroform); yield: 24%; m.p. 182 ꢁC;
Anal. calc. for C27H23N5S: C 72.16, H 5.12, N 15.59; found:
C 72.20, H 5.10, N 15.50; UVevis lmax (nm): 377, 289,
256, 217; IR nmax (cmꢀ1): 2928 (CeH), 1509 (C]N), 1538
(C]N), 1103 (CeN), 839 (CeS); 1H NMR (CDCl3) (d,
4.3. Synthesis of 2-(5-substituted-3-phenyl-2-
pyrazolinyl)-1,3-thiazolino[5,4-b]quinoxaline derivatives
(1be8b): general procedure
ppm): 7.72e7.50 (14H, m, Ar), 3.48 (dd, 1H, HA, JAB
:
18.5 Hz, JAX: 9.25), 3.23 (dd, 1H, HB, JAB: 18.5, JBX: 9.25),
A mixture of 1-N-thiocarbamoyl-3,5-diphenyl pyrazoline
compounds 1ae8a (10 mmol) and 2,3-dichloroquinoxaline
(10 mmol) in absolute ethanol (15 ml) was refluxed for 8 h.
5.69 (dd, 1H, HX, JAX: 11.10, JBX: 9.25). 13C NMR (CDCl3)
(d, ppm): 140.22 (CeS), 150.23 (C]N), 140.11 (C]N),
136.33e125.10 (PhenyleC), 35.82 (CH2), 61.60 (CH).

1324
A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
4.3.5. 2-[5-(2-Ethylphenyl)-3-phenyl-2-pyrazolinyl]-1,
3-thiazolino[5,4-b]quinoxaline (5b)
4.4. In vitro testing against E. histolytica
Pale yellow crystal (chloroform); yield: 18%; m.p. 187 ꢁC;
Anal. calc. for C26H21N5S: C 71.72, H 4.83, N 16.09; found: C
71.75, H 4.77, N 16.10; UVevis lmax (nm): 373, 288, 236,
219; IR nmax (cmꢀ1): 2924 (CeH),1563 (C]N), 1558
(C]N), 1192 (CeN), 945 (CeS); 1H NMR (CDCl3) (d,
All the cyclised pyrazoline analogues were screened in vitro
for antiamoebic activity against HM1:1MSS strain of E. histo-
lytica by microdilution method [28]. E. histolytica trophozoites
were cultured in TYIS-33 growth medium as described previ-
ously in wells of 96 well microtiter plate [29]. All the com-
pounds were dissolved in DMSO (40 ml) at which level no
inhibition of amoeba occurs [30e31] and the stock solutions
of the compounds were prepared freshly before use at a concen-
tration of 1 mg/ml. Two-fold serial dilutions were made in the
wells of 96-well microtiter plate (Costar). Each test includes
metronidazole as a standard amoebicidal drug, control wells
(culture medium plus amoebae) and a blank (culture medium
only). The number of amoeba per ml was estimated with a hae-
mocytometer and trypan blue exclusion was used to confirm
viability. The cell suspension used was diluted to 105 organ-
ism/ml by adding fresh medium and 170 ml of this suspension
was added to the test and control wells in the plate.
ppm): 7.23e6.72 (14H, m, Ar), 3.48 (dd, 1H, HA, JAB
:
18.5 Hz, JAX: 9.25), 3.49 (dd, 1H, HB, AB: 18.5, JBX: 9.25),
J
6.32 (dd, 1H, HX, JAX: 11.10, JBX: 9.25) 2.13 (s, 3H, CH3),
9.24 (d, 1H, NH) 13C NMR (CDCl3) (d, ppm): 140.24 (Ce
S), 150.11 (C]N), 141.22 (C]N), 136.33e125.10 (Phe-
nyleC), 35.82 (CH2), 27.90 (CH2) 61.60 (CH).
4.3.6. 2-{5-[2-(Methylethyl)phenyl]-3-phenyl-
2-pyrazolinyl}-1,3-thiazolino[5,4-b]quinoxaline (6b)
Pale yellow crystal (chloroform); yield: 16%; m.p: 175 ꢁC;
Anal. calc. for C27H23N5S: C 72.16, H 5.12, N 15.59; found: C
72.13, H 5.10, N 15.49; UVevis lmax (nm): 373, 288, 236,
217; IR nmax cmꢀ1: 2955 (CeH), 1545 (C]N), 1516
(C]N), 1078 (CeN), 892 (CeS); 1H NMR (CDCl3) (d,
An inoculum of 1.7 ꢂ 104 organisms/well was chosen so
that confluent, but not excessive growth took place in control
wells. Plates were sealed and gassed for 10 min with nitrogen
before incubation at 37 ꢁC for 72 h.
ppm): 6.72e7.50 (14H, m, Ar), 3.48 (dd, 1H, HA, JAB
:
18.5 Hz, JAX: 9.25), 3.49 (dd, 1H, HB, AB: 18.5, JBX: 9.25),
J
6.32 (dd,1H, HX, JAX: 11.10, JBx: 9.25) 2.13 (s, 3H, CH3),
13C NMR (CDCl3) (d, ppm): 140.31 (CeS), 150.23 (C]N),
140.11 (C]N), 136.33e125.10 (PhenyleC), 35.82 (CH2),
60.56 (CH),17.85 (CH3), 32.90 (CH).
4.5. Assessment of antiamoebic activity
After incubation, the growth of amoebae in the plate was
checked with a low power microscope. Inverting the plate
and shaking gently removed the culture medium. Plate was
then immediately washed once in sodium chloride solution
(0.9%) at 37 ꢁC. This procedure was completed quickly, and
the plate was not allowed to cool in order to prevent the de-
tachment of amoebae. The plate was allowed to dry at room
temperature and the amoebae were fixed with methanol and,
when dry, stained with 0.5% aqueous eosin for 15 min. Stained
plate was washed once with tape water and then twice with
distilled water and allowed to dry. A 200 mL portion of
0.1 N sodium hydroxide solution was added to each well to
dissolve the protein and release the dye. The optical density
of the resulting solution in each well was determined at
490 nm with a microplate reader. The % inhibition of amoebal
growth was calculated from the optical densities of the control
and test wells and plotted against the logarithm of the dose of
the drug tested. Linear regression analysis was used to deter-
mine the best-fitting straight line from which the IC50 value
was found. The results are reported in Table 1.
4.3.7. 2-[5-(2-Chlorophenyl)-3-phenyl-2-pyrazolinyl]-1,
3-thiazolino[5,4-b]quinoxaline (7b)
Pale yellow crystal (chloroform); yield: 34%; m.p: 177 ꢁC;
Anal. calc. for C24H16N5SCl: C 65.23, H 3.62, N 15.85; found:
C 65.25, H 3.60; N 15.85; UVevis lmax (nm): 371, 290, 227,
216; IR nmax (cmꢀ1): 2933 (CeH), 1558 (C]N), 1545
(C]N), 1162 (CeN), 920 (CeS); 1H NMR (CDCl3) (d,
ppm): 7.20e7.01(14H, m, Ar), 3.21 (dd, 1H, HA, JAB
:
18.5 Hz, JAX: 9.25), 3.49 (dd, 1H, HB, JAB: 18.5, JBX: 9.25),
5.82 (dd, 1H, HX, JAX: 11.10, JBX: 9.25); 13C NMR (CDCl3)
(d, ppm): 139.90 (CeS), 151.66 (C]N), 141.13 (C]N),
136.33e125.10 (PhenyleC), 32.78 (CH2), 62.33 (CH).
4.3.8. 2-[5-(2,3-Dichlorophenyl)-3-phenyl-2-
pyrazolinyl]-1,3-thiazolino[5,4-b]quinoxaline (8b)
Pale yellow crystal (chloroform); yield: 22%; m.p. 170 ꢁC;
Anal. calc. for C24H15N5SCl2: C 60.50, H 3.15, N 14.70;
found: C 60.53, H 3.15, N 14.76; UVevis lmax (nm): 371,
290, 258, 236, 217; IR nmax (cmꢀ1): 2924 (CeH), 1486
4.6. Toxicity screening
1
(C]N), 1511 (C]N), 1122 (CeN), 945 (CeS); H NMR
(CDCl3) (d, ppm): 6.72e7.50 (14H, m, Ar), 3.99 (dd, 1H,
HA, JAB: 18.5 Hz, JAX: 9.25), 2.78 (dd, 1H, HB, JAB: 18.5,
The toxic activities of compounds 1e8, 1ae8a and 1be8b
were evaluated in vitro on human kidney epithelial cell line.
For the toxicity assay, transformed human kidney epithelium
(Graham) cells was continuously maintained in culture at
37 ꢁC in 5% CO2. The MTT (3-[4,5-dimethylthiazol-2yl]-
2,5-diphenyltetrazolium bromide, USB) cellular viability
assay was used to determine the toxicity profile of these com-
pounds [32]. The trypsinized cell suspension was adjusted to
J
BX: 9.25), 6.21 (dd, 1H, HX, JAX: 11.10, JBX: 9.25) 2.13 (s,
3H, CH3); 13C NMR (CDCl3) (d, ppm): 139.31 (CeS),
150.23 (C]N), 140.11 (C]N), 136.33e125.10 (PhenyleC),
35.82 (CH2), 61.60 (CH),17.85 (CH3), 32.90 (CH), ESI-MS:
477 (M þ 1), 428, 306.

A. Budakoti et al. / European Journal of Medicinal Chemistry 44 (2009) 1317e1325
1325
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Acknowledgement
This work was supported by Department of Science and
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