Syntheses and evaluation of 3-(3-bromo phenyl)-5-phenyl-1-(thiazolo [4,5-b] quinoxaline-2-yl)-2-pyrazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.10.026

A variety of 3-(3-bromo phenyl)-5-phenyl-1-(thiazolo [4,5-b] quinoxaline-2-yl)-2-pyrazoline were obtained by the refluxing of 1-N-thiocarbamoyl 3,5-diphenyl-2-pyrazoline with 2,3-dichloroquinoxaline. The chemical structures of the compounds were elucidated by UV, IR, ¹H NMR, and ¹³C NMR spectroscopy. The purity of the compounds was confirmed by their elemental analysis. The antiamoebic activity of these compounds was evaluated by microdilution method against HMI:IMSS strain of Entamoeba histolytica and the IC₅₀ values were compared with the standard drug metronidazole. Some of the quinoxaline derivatives showed less IC₅₀ values than metronidazole. To elucidate the toxic effect, MTT assay was performed using kidney epithelial cell line. The results showed that all the compounds are non-toxic.

Full text of DOI:10.1016/j.ejmech.2007.10.026

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European Journal of Medicinal Chemistry 43 (2008) 1749e1757
http://www.elsevier.com/locate/ejmech
Short communication
Syntheses and evaluation of 3-(3-bromo phenyl)-5-phenyl-1-
(thiazolo [4,5-b] quinoxaline-2-yl)-2-pyrazoline derivatives
*
Asha Budakoti, Abdul R. Bhat, Fareeda Athar, Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 7 July 2007; received in revised form 21 October 2007; accepted 22 October 2007
Available online 25 October 2007
Abstract
A variety of 3-(3-bromo phenyl)-5-phenyl-1-(thiazolo [4,5-b] quinoxaline-2-yl)-2-pyrazoline were obtained by the refluxing of 1-N-thiocar-
bamoyl 3,5-diphenyl-2-pyrazoline with 2,3-dichloroquinoxaline. The chemical structures of the compounds were elucidated by UV, IR, 1H
NMR, and 13C NMR spectroscopy. The purity of the compounds was confirmed by their elemental analysis. The antiamoebic activity of these
compounds was evaluated by microdilution method against HMI:IMSS strain of Entamoeba histolytica and the IC50 values were compared with
the standard drug metronidazole. Some of the quinoxaline derivatives showed less IC50 values than metronidazole. To elucidate the toxic effect,
MTT assay was performed using kidney epithelial cell line. The results showed that all the compounds are non-toxic.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Chalcone; Pyrazolines; Thiocarbamoyl; Quinoxaline: Antiamoebic activity
1. Introduction
Literature survey revealed that the quinoxaline compounds
showed better antiprotozoal, antimicrobial, and monamine ox-
idase inhibitors’ activity [8e11]. In our earlier report, we have
reported the synthesis of quinoxaline derivatives and it was
found that 3-bromo and 3-chloro substituents on the phenyl
ring and 4-methyl group in pyrazoline ring greatly effect anti-
amoebic activity [8]. These results increase as to synthesize
some new derivatives of quinoxalines. We report in this paper
the synthesis and antiamoebic activity of bromo substituted
derivatives of quinoxalines and compared the antiamoebic ac-
tivity of chalcones and pyrazolines.
Pathogenic intestinal protozoa are responsible for clinically
important infections in both the developed and the developing
world [1]. Amoebiasis is an infection of the mucous mem-
brane of the large intestine where Entamoeba histolytica is
the causative organism. This organism occurs in the intestine
in the form of trophozoites and cysts. It may be characterized
by abdominal pain, anorexia, fever, weight loss and hepato-
megaly [2]. Metronidazole is an orally active drug in the treat-
ment of several types of illness caused by protozoa, as well as
anaerobic bacteria [3e5]. Side effects are, however, common
and include a disulfiram-like reaction (predominantly nausea
and vomiting) when taken with alcohol, in addition to a dry
mouth and headache [6]. Entamoebas’ strain resistant to this
drug has also begun to appear [7]. It is therefore necessary
to search for new and effective amoebicidals.
2. Results and discussion
The Claisen-Schmidt reaction of various ketones and alde-
hyde generates different types of chalcones (1e8). The reac-
tion is sensitive to amount of NaOH. Different chalcones
with thiosemicarbazide lead to the formation of thiocarba-
moyl-3,5-diphenyl-2-pyrazolines (9e16) (Scheme 1) which
after refluxing with 2,3-dichloroquinoxaline in THF gave the
corresponding fused 3,5-diphenyl-2-(thiazolo [4,5-b] quinoxa-
line-2-yl)-2H-pyrazole (17e24). Analytical and spectral data
* Corresponding author. Tel.: þ91 11 26981717/3253; fax: þ91 11
26980229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.026

1750
A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
O
R
CO-CH3
+
NaOH
R - CHO
Br
Br
(1-8)
S
NH2-NH-C-NH2
R
N
N
N
S
N
R
NH2
N
THF
N
N
N
S
Cl
Cl
N
(9-16)
Br
(17-24)
CH3
CH3
R
=
N
H
CH3
CH3
CH3
CH2CH3
Cl
Cl
Cl
CH3
Scheme 1. Synthesis of quinoxaline derivatives.
1
(IR, UV, H NMR, 13C NMR) of all the synthesized com-
pounds were in full agreement with the proposed structures.
All the compounds were obtained in good yields and are stable
in the solid as well as in the solution state.
the H NMR and 13C NMR spectra, which provide diagnostic
tools for the positional elucidation of the protons. Assignments
of the signals are based on the chemical shifts and intensity
patterns. The pyrazoline protons HA and HB (Fig. 1) are gem-
inal 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 doublets in the region
of 6.32e5.40 ppm due to vicinal coupling with two non-equiv-
alent geminal protons of C4 carbon. These protons were
slightly shifted in case of quinoxaline compounds. HA and
HB protons at C4 carbon, appears in the region 3.99e3.11
and 3.80e2.78 ppm as doublet of doublets in all quinoxaline
compounds (17e24). The CH proton also appeared as doublet
of doublets in the region of 6.58e5.11 ppm due to vicinal cou-
pling with two non-equivalent geminal protons of C4 carbon.
1
2.1. IR and electronic spectral studies
The electronic spectra of the cyclised pyrazoline analogues
(9e16) studied in the UV region in methanol exhibited three
absorption bands at 371e290, 270e236 and 232e205 nm as-
signable to n / p*, p / p* and n / s* transitions, respec-
tively. The band at 371e290 nm was assigned to the transition
involving the thione portion (C]S) of thiocarbamoyl group.
The two other absorption bands at 270e236 and 232e
205 nm were due to p / p* transition of phenyl ring and
n / s* transition of azomethine nitrogen, respectively. The
UV spectral data of quinoxaline derivatives (17e24) were
also studied which showed the same type of transitions as ob-
served in compounds 9e16. It showed three spectral bands at
388.3e299, 287e248 and 239e204 nm assigned to n / p*,
p / p* and n / s* transitions of thiocarbamoyl group
(CeSeC), aromatic ring and azomethine nitrogen, respectively.
Br
HA
4
3
HB
HX
N
R
Br
N
N
S
N
2N
S
5
N
1N
C
R'
NH2
2.2. Nuclear Magnetic Resonance spectral studies
17-24
9-16
Further evidence for the formation of pyrazoline com-
pounds and their quinoxaline derivatives was obtained from
Fig. 1.

A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
1751
The NH proton of different substituted thiocarbamoyl pyrazo-
line compounds showed a doublet at 9.24e7.17 ppm. In the
13C NMR spectra, the C4 and C5 carbons of the pyrazoline
ring in compounds 9e16 resonate at 37.43e37.06 and
62.98e60.49 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 204.23e169.42 ppm. The qui-
noxaline compounds 17e24 showed two signals at 151.66e
152.36 and 143.35e140.11 ppm due to azomethine carbon
of the pyrazoline ring and quinoxaline ring, respectively. Thio-
carbamoyl carbon (CeSeC) displayed a signal at 143.07e
139.31 ppm in all the compounds.
than their corresponding chalcone and pyrazoline analogues.
The better antiamoebic quinoxaline derivatives of pyrazolines
(17e24) were subjected to MTT assay. At a concentration of
60 mM (Table 2), these compounds did not show any inhibitory
activity. To investigate the selectivity of the compounds, the
‘‘safety index’’ (SI) was calculated and defined as: Toxicity
IC50/Protozoal IC50; where toxicity IC50 is defined as the con-
centration of compound that kills 50% of the human (kidney ep-
ithelial) cell line and protozoal IC50 is the concentration that
kills 50% of amoeba protozoa. This allows an estimate of which
compounds might be efficacious or toxic against human cells
and potentially in vivo. The numerical results for each com-
pound are given in Table 2. It is interesting to note that the com-
pound 18 has lowest toxicity and highest antiamoebic activity
and overall compounds show more favorable safety profile
along with the most promising antiamoebic activity.
2.3. In vitro antiamoebic activity
The antiamoebic activity of all the compounds was deter-
mined against HM1:IMSS strain of E. histolytica and the re-
sults are summarized in Tables 1 and 2. A comparative
study of IC50 values of chalcones (1e8), pyrazolines (9e
16), quinoxaline analogues (17e24) and reference compound
metronidazole is done. The results were estimated as the per-
centage 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 doseeresponse curve. The differ-
ent degree of inhibition of the growth of E. histolytica by three
series may reflect sound differences in the ability of the activ-
ities of the three types of compounds. Chalcone (1e8) ex-
hibited antiamoebic activity with IC50 of 3.48e1.41 mM. All
the pyrazoline derivatives (9e16) showed IC50 ¼ 0.23e
4.37 mM. In case of quinoxaline derivatives activities varying
in the range IC50 ¼ 0.08e2.49 mM. All the quinoxaline com-
pounds were more potent than their corresponding chalcone
and pyrazoline analogues. The introduction of quinoxaline
moiety enhanced activity by many folds in all the compounds.
In the quinoxaline series, out of eight compounds six had bet-
ter activity than metronidazole. If we further take into account
the substituent effect, in general all the compounds substituted
with methyl phenyl groups had shown less potency than the
phenyl, indolyl and chlorophenyl substituents. The compound
18 with IC50 ¼ 0.17 mM was found most active among all the
compounds which has indolyl group along with quinoxaline.
The results were statistically evaluated by analysis of variance.
The null hypothesis was tested using the t-test and the signif-
icance of the differences between the IC50 value(s) of metro-
nidazole versus 1, 2, 8, 9, 12, 16e20, 23 and 24 was
evaluated. The calculated t-values were higher than the table
values at the 4% level. Hence, the character under study was
influenced by the treatment.
3. Conclusion
This research involved the syntheses of chalcones (1e8),
the cyclization of these chalcones by thiosemicarbazide and
synthesis of pyrazoline derivatives (9e16) and their quinoxa-
line derivatives (17e24). We have further examined the antia-
moebic activities of all the compounds. The biological
behavior revealed that the most of the compounds showed bet-
ter activity. The toxicity studies of quinoxaline derivatives
(17e24) showed that these compounds will remain toxic to
the parasite, whilst in the human host there will be a decreased
likelihood of toxicity.
4. Experimental
4.1. Materials and methods
All the chemicals were purchased from Aldrich Chemical
Company (U.S.A.) and were used without further purification.
The reactions were monitored by pre-coated aluminium silica
gel 60F254 thin layer plates procured from Merck (Germany).
All thiosemicarbazides were prepared by a reported method
[12] and their purity was confirmed by C, H and N analyses car-
ried out at Central Drug Research Institute Lucknow, India.
Chlorine was estimated by decomposing the complexes with
Na2O2/NaOH and precipitated as AgCl with AgNO3 after dis-
solving in dil. HNO3. Melting points were recorded on KSW
melting point apparatus and are uncorrected. Electronic spectra
were recorded in methanol on a Shimadzu UV-1601 PC UVe
Visible spectrophotometer. IR spectra on KBr disks were re-
corded on a Perkin Elmer model 1620 FT-IR spectrophotometer.
1H and 13C NMR spectra were obtained at ambient temperature
using a brucker spectroscopin DPX-300 MHZ spectrophotome-
ter in CDCl3 using tetramethylsilane as an internal standard.
2.4. Toxicity profile
4.2. Synthesis of bromo acetophenone chalcones:
general procedure
The in vitro toxic activity of newly synthesized compounds
was studied on human kidney epithelial cell line. All the qui-
noxaline compounds showed significant antiamoebic activity
A solution of bromo acetophenones (50 mmol) and appro-
priate aldehyde (50 mmol) in methanolic NaOH was stirred

1752
A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
Table 1
In vitro antiamoebic activity of substituted chalcones (1e8) and pyrazolines (9e16)
O
Br
HA
R
4
3
HB
HX
Br
2N
1-8
5
N
C
R'
1
S
NH2
9-16
R group
Compound
Antiamoebic activity
IC50 (mM)
Compound
Antiamoebic activity
IC50 (mM)
S.D.
0.05
S.D.
0.04
1
1.39
9
0.19
2
1.05
0.48
10
2.28
0.23
N
H
3
4
2.49
1.80
0.98
0.34
11
12
4.38
1.71
0.02
0.18
CH3
CH3
CH3
CH3
5
6
3.63
1.92
0.48
0.23
13
14
2.61
2.34
0.54
0.34
CH2CH3
CH3
CH3
7
8
2.23
1.11
0.18
0.03
15
16
1.90
0.56
0.09
0.03
Cl
Cl
Cl
Metronidazole
1.8
0.05

A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
1753
Table 2
In vitro antiamoebic activity and toxicity of substituted quinoxaline derivatives of pyrazolines
N
Br
N
N
S
N
N
R
17-24
Compound
R group
Antiamoebic activity
IC50 (mM)
Toxicity profile,
IC50 (mM)
Safety Index
(SI)
S.D.
0.09
17
0.14
>60
>428
18
19
0.08
0.10
0.03
0.06
>60
>750
N
H
>60
>600
CH3
20
1.29
0.56
>60
>46.51
CH3
CH3
CH3
21
22
23
24
2.49
1.87
0.68
0.70
1.90
0.05
0.30
0.09
>60
>60
>60
>60
>24.09
>32.08
>88.23
>85.71
CH2CH3
CH3
CH3
Cl
Cl
Cl
Metronidazole
1.8
0.05
>100
>55.55

1754
A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
for 18 h at 5e28 ꢀC. Solid obtained, washed with ice-cold wa-
ter and then rectified spirit, dried and recrystallized by ethanol
[12,13].
10.9; found: C 56.51, H 3.12, Cl 1.93%. UV/vis lmax (nm):
371, 291, 236, 217; IR nmax (cmꢁ1): 1710 (C]O), 1630
1
(CH]CH), 1550 (C]C); H NMR (CDCl3) (d, ppm): 7.66
(m, 10H, Ar), 7.52 (d, HA, J ¼ 15 Hz, 1H), 7.63 (d, HB,
4.2.1. 1-3-Br-ph-3-ph-prop-2-ene-1-one (1)
J ¼ 15 Hz, 1H).
Pale yellow crystals (chloroform); yield: 93%; m.p.:
121 ꢀC. Anal. calc. for (C15H11OBr): C 62.93, H 3.84; found:
C 62.82, H 3.81%. UV/vis lmax (nm): 371, 291, 236, 217; IR
nmax (cmꢁ1): 1656 (C]O), 1622 (CH]CH), 1580 (C]C); 1H
NMR (CDCl3) (d, ppm): 7.8 (m, 10H, Ar), 7.74 (d, 1H, 1H,
J ¼ 15 Hz), 7.63 (d, 1H, HB, J ¼ 15 Hz).
4.2.8. 1-(3-Br-ph)-3-(2,4 diCl-ph) prop-2-ene-1-one (8)
Pale yellow crystals (chloroform); yield: 54%; m.p.:
175 ꢀC. Anal. calc. for (C15H9OBrCl2): C 50.70, H 2.53, Cl
20; found: C 50.65, H 2.57%. UV/vis lmax (nm): 371, 291,
236, 217; IR nmax (cmꢁ1): 1710 (C]O), 1630 (CH]CH),
1550 (C]C); 1H NMR (CDCl3) (d, ppm): 7.89 (m, 10H,
Ar), 7.2 (d, HA, J ¼ 15 Hz, 1H), 7.51 (d, HB, J ¼ 15 Hz, 1H).
4.2.2. 1-(3-Br-ph)-3-indolyl prop-2-ene-1-one (2)
Pale yellow crystals (chloroform); yield: 55%; m.p.:
123 ꢀC. Anal. calc. for (C17H12NOBr): C 62.76, H 3.69, N
4.30; found: C 60.05, H 3.11, N 4.34%. UV/vis lmax (nm):
371, 291, 236, 217; IR nmax (cmꢁ1): 1664 (C]O), 1620
4.3. Synthesis of pyrazoline derivatives
A mixture of chalcone (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 methanol [14].
1
(CH]CH), 1568 (C]C); H NMR (CDCl3) (d, ppm): 5.82
(d, 1H, CH), 6.86 (1H, d, CHeAr), 7.11e7.70 (9H, m, Are
H), 9.01 (1H, s, NH of indole).
4.2.3. 1-(3-Br-ph)-3-(4-me-ph)-prop-2-ene-1-one (3)
4.3.1. 1-N-tc-3-(3-br-ph)-5-ph-2-pz (9)
Pale yellow crystals (chloroform); yield: 73%; m.p.:
144 ꢀC. Anal. calc. for (C16H13OBr): C 64, H 4.33; found: C
64.05, H 4.16%. UV/vis lmax (nm): 371, 291, 236, 217; IR
nmax (cmꢁ1): 1710 (C]O), 1630 (CH]CH), 1550 (C]C);
1H NMR (CDCl3) (d, ppm): 7.8 (m, 10H, Ar), 7.74 (d, HA,
J ¼ 15 Hz, 1H), 7.63 (d, HB, J ¼ 15 Hz, 1H), 2.5 (s, 3H, CH3).
Pale yellow powder (chloroform); yield: 24%; m.p.:
175 ꢀC. Anal. calc. (C16H14N3SBr): C 53.48, H 3.89, N
11.69; found: C 53.45, H 3.84, N 11.63%. UV/vis lmax
(nm): 371, 290, 236, 217; IR nmax (cmꢁ1): 3435 (NeH),
1
2924 (CeH), 1558 (C]N), 1357 (C]S), 1122 (CeN); H
NMR (CDCl3) (d, ppm): 6.5e7.5 (m, 10H, Ar), 3.48 (dd,
1H, HA, JAB 18.5, JAx 9.25), 3.49 (dd, 1H, HB, JAB 18.5,
JBx 9.25), 6.3 (dd, 1H, Hx, JAx 11.10, JBx 9.25), 9.2 (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).
4.2.4. 1-(3-Br-ph)-3-(2,4,6 tri-me-ph)-prop-2-ene-1-one (4)
Pale yellow crystals (chloroform); yield: 62%; m.p.: 175 ꢀC.
Anal. calc. for (C18H17OBr): C 65.85, H 5.18; found: C 65.78,
H 5.16%. UV/vis lmax (nm): 299, 236, 217; IR nmax (cmꢁ1):
1710 (C]O), 1630 (CH]CH), 1550 (C]C); 1H NMR
(CDCl3) (d, ppm): 7.8 (m, 10H, Ar), 7.01 (d, HA, J ¼ 15 Hz,
1H), 7.23 (d, HB, J ¼ 15 Hz, 1H), 3.01 (s, 3H, CH3).
4.3.2. 1-N-tc-3-(3-br-ph)-5-(1H-3-indolyl)-2-pz (10)
Pale yellow crystal (chloroform); yield: 21%; m.p.: 185 ꢀC.
Anal. calc. for (C17H15N4SBr): C 52.85, H 3.88, N 14.50;
found: C 52.76, H 3.82, N 14.50%. UV/vis lmax (nm); 371,
270, 236; IR nmax (cmꢁ1): 3233 (NeH), 2928 (CeH), 1590
4.2.5. 1-(3-Br-ph)-3-(3-isopropyl ph)-prop-2-ene-1-one (5)
Pale yellow crystals (chloroform); yield: 44%; m.p.:
113 ꢀC. Anal. calc. for (C17H15OBr): C 64.96, H 4.77; found:
C 63.98, H 4.68%. UV/vis lmax (nm): 291, 258, 236; IR nmax
(cmꢁ1): 1722 (C]O), 1623 (CH]CH), 1554 (C]C); 1H
NMR (CDCl3) (d, ppm): 7.7 (m, 10H, Ar), 7.4 (d, HA,
J ¼ 13.6 Hz, 1H), 7.6 (d, HB, J ¼ 15 Hz, 1H), 1.2e2.1 (m,
2H, CH2).
1
(C]N), 1336 (C]S), 1052 (CeN); H NMR (CDCl3) (d,
ppm): 6.61e7.17 (m, 15H, Ar), 3.31 (dd, 1H, HA, JAB
17.33, JAx 9.33), 3.35 (dd, 1H, HB, JAB 16.6, JAx 9.52), 6.0
(dd, 1H, Hx, JAx 9.33, JBx 8.75), 7.81 (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).
4.2.6. 1-(3-Br-ph)-3-[3-(me-ethyl)-ph] prop-2-ene-1-one (6)
Pale yellow crystals (chloroform); yield: 56%; m.p.:
129 ꢀC. Anal. calc. for (C18H17OBr): C 65.85, H 5.18; found:
C 65.85, H 5.12%. UV/vis lmax (nm): 371, 291, 236, 217; IR
nmax (cmꢁ1): 1698 (C]O), 1643 (CH]CH), 1555 (C]C); 1H
NMR (CDCl3) (d, ppm): 7.8 (m, 10H, Ar), 7.5 (d, HA,
J ¼ 14.8 Hz, 1H), 7.2 (d, HB, J ¼ 15 Hz, 1H), 2.5 (s, 3H, CH3).
4.3.3. 1-N-tc-3-(3-br-ph)-5-(4-me-ph)-2-pz (11)
Pale yellow crystal (chloroform); yield: 21%; m.p.: 185 ꢀC.
Anal. calc. for (C17H16N3SBr): C 54.69, H 4.28, N 11.26;
found: C 54.69, H 4.22, N 11.34%. UV/vis lmax (nm); 371,
270, 236; IR nmax (cmꢁ1): 3242 (NeH) 2911 (CeH), 1567
1
(C]N), 1354 (C]S), 1078 (CeN); H NMR (CDCl3) (d,
ppm): 6.43e7.10 (m, 15H, Ar), 3.31 (dd, 1H, HA; JAB
17.33, JAx 9.33), 3.20 (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).
4.2.7. 1-(3-Br-ph)-3-(2-Cl-ph) prop-2-ene-1-one (7)
Pale yellow crystals (chloroform); yield: 54%; m.p.:
175 ꢀC. Anal. calc. for (C17H10OBrCl): C 56.25, H 3.12, Cl

A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
1755
4.3.4. 1-N-tc-3-(3-br-ph)-5-(2,4,6 tri-me-ph)-2-pz (12)
(NeH), 2924 (CeH), 1498 (C]N), 1357 (C]S), 1152
(CeN); 1H NMR (CDCl3) (d, ppm): 6.51e7.50 (m, 10H,
Ar), 3.48 (dd, 1H, HA, JAB 18.5, 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), 9.33 (d, 2H, NH2); 13C NMR
(CDCl3) (d, ppm): 176.19 (C]S), 149.57 (C]N), 144.52e
125.72 (PhenyleC), 62.86 (CeH), 37.06 (CH2).
Pale yellow powder (chloroform); yield: 24%; m.p.:
175 ꢀC. Anal. calc. (C19H20N3S): C 56.85, H 4.98, N 10.47
found: C 56.85, H 4.96, N 10.43%. UV/vis lmax (nm): 371,
290, 236, 217; IR nmax (cmꢁ1): 3444 (NeH), 2971 (CeH),
1
1543 (C]N), 1321 (C]S), 1127 (CeN); H NMR (CDCl3)
(d, ppm): 6.51e7.50 (m, 10H, Ar), 3.48 (dd, 1H, HA, JAB
18.5, 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).
4.4. Synthesis of 3,5 substituted diphenyl-1-
(thiazolo [4,5-b] quinoxaline-2-yl)-2-pyrazole
derivatives; general procedure
A mixture of 1-N-thiocarbamoyl 3,5 substituted diphenyl
pyrazoline compound (3.47 g, 0.01 mol) and 2,3-dichloroqui-
noxaline (1.99 g, 0.01 mol) in THF (10 ml) was refluxed for
24 h. The solvent was evaporated under reduced pressure.
The residue was recrystallized from ethanol [15].
4.3.5. 1-N-tc-3-(3-br-ph)-5-(2-ethyl ph)-2-pz (13)
Pale yellow powder (chloroform); yield: 24%; m.p.:
175 ꢀC. Anal. calc. (C18H18N3SBr): C 55.81, H 4.65, N
10.85; found: C 55.78, H 4.61, N 10.85%. UV/vis lmax
(nm): 371, 290, 236, 217; IR nmax (cmꢁ1): 3226 (NeH),
1
2924 (CeH), 1505 (C]N), 1357 (C]S), 1105 (CeN); H
4.4.1. 3-(3-Br-ph)-5-ph-1-(tz [4,5-b] qx-2-yl)-2-pz (17)
Yellow solid (chloroform); yield: 18%; m.p.: 162 ꢀC. Anal.
calc. (C24H16N5SBr): C 59.38, H 3.29, N 14.43; found: C
59.89, H 3.89, N 14.97%. UV/vis lmax (nm): 371, 298, 266,
236, 212; IR nmax (cmꢁ1): 2892 (CeH), 1542 (C]N), 1539
(C]N), 1085 (CeN), 885 (CeS); 1H NMR (CDCl3) (d,
ppm): 6.72e7.50 (14H, m, Ar), 3.48 (dd, 1H, HA, JAB 18.5,
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).
NMR (CDCl3) (d, ppm): 6.4e7.5 (m, 10H, Ar), 3.48
(dd, 1H, HA, JAB 18.5, 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), 8.10 (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).
4.3.6. 1-N-tc-3-(3-br-ph)-5-[(2-isoprop)-ph]-2-pz (14)
Pale yellow powder (chloroform); yield: 24%; m.p.:
175 ꢀC. Anal. calc. (C19H20N3SBr): C 56.85, H 4.98, N
10.47; found: C 56.43, H 4.88, N 10.48%. UV/vis lmax
(nm): 371, 290, 236, 217; IR nmax (cmꢁ1): 3362 (NeH),
4.4.2. 3-(3-Br-ph) 5-(3-indolyl)-1-(tz [4,5-b] qx-2-yl)
2-pz (18)
Yellow solid (chloroform); yield: 34%; m.p.: 180 ꢀC. Anal.
calc. (C26H18N6S Br): C 59.38, H 3.29, N 14.43; found: C
59.89, H 3.89, N 14.97%. UV/vis lmax (nm): 371, 290, 236,
217; IR nmax (cmꢁ1): 2992 (CeH), 1489 (C]N), 1566
(C]N), 1108 (CeN), 881 (CeS); 1H NMR (CDCl3) (d,
ppm): 6.61e7.71 (14H, m, Ar), 3.32 (dd, 1H, HA, JAB 18.5,
JAx 9.25), 3.47 (dd, 1H, HB, JAB 18.5, JBx 9.25), 5.81 (dd,
1H, Hx, JAx 11.10, JBx 9.25).
1
2995 (CeH), 1489 (C]N), 1377 (C]S), 1038 (CeN); H
NMR (CDCl3) (d, ppm): 6.5e7.5 (m, 10H, Ar), 3.48 (dd,
1H, HA, JAB 18.5, 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).
4.3.7. 1-N-tc-3-(3-br-ph)-5-(4-Cl-ph)-2-pz (15)
4.4.3. 3-(3-Br-ph)-5-(4-me-ph)-1-(tz [4,5-b] qx-2-yl)-
2-pz (19)
Pale yellow powder (chloroform); yield: 24%; m.p.:
175 ꢀC. Anal. calc. (C16H15N3SClBr): C 48.79, H 3.30, N
10.68, Cl 9.03; found: C 48.56, H 3.33, N 10.81, Cl 9.12%.
UV/vis lmax (nm): 371, 290, 236, 217; IR nmax (cmꢁ1): 3367
(NeH), 2939 (CeH), 1533 (C]N), 1357 (C]S), 1079
(CeN); 1H NMR (CDCl3) (d, ppm): 6.51e7.50 (m, 10H,
Ar), 3.48 (dd, 1H, HA, JAB 18.5, 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), 9.43 (d, 2H, NH2); 13C NMR
(CDCl3): (d, ppm): 176.19 (C]S), 149.57 (C]N), 144.52e
125.72 (PhenyleC), 62.86 (CeH), 37.06 (CH2).
Pale yellow crystal (chloroform); yield: 16%; m.p.: 155 ꢀC.
Anal. calc. (C25H18N5SBr): C 60.12, H 3.60, N 14.02; found:
C 60.45, H 3.89, N 14.21%. UV/vis lmax (nm): 367, 299, 256,
236, 217; IR nmax (cmꢁ1): 2924 (CeH), 1558 (C]N), 1516
(C]N), 1142 (CeN), 912 (CeS); 1H NMR (CDCl3) (d,
ppm): 7.12e7.14 (14H, m, Ar), 3.11 (dd, 1H, HA, JAB 18.5,
JAx 9.25), 3.39 (dd, 1H, HB, JAB 18.5, JBx 9.25), 6.18 (dd,
1H, Hx, JAx 11.10, JBx 9.25).
4.4.4. 3-(3-Br-ph) 5-(4-ethyl ph)-1-(tz [4,5-b] qx-2-yl)-
2-pz (20)
4.3.8. 1-N-tc-3-(3-br-5-(2,3 di-Cl-ph)-2-pz (16)
Pale yellow crystal (chloroform); yield: 26%; m.p.: 188 ꢀC.
Anal. calc. (C26H20N5SBrCl): C 60.81, H 3.89, N 13.64;
found: C 60.78, H 3.89, N 13.56%. UV/vis lmax (nm): 373,
288, 236, 217; IR nmax (cmꢁ1): 2910 (CeH), 1498 (C]N),
Pale yellow powder (chloroform); yield: 24%; m.p.:
175 ꢀC. Anal. calc. (C16H14N3SCl2Br): C 44.65, H 3.25, N
9.76, Cl 16.51; found: C 44.78, H 3.22, N 9.66, Cl 16.40%.
UV/vis lmax (nm): 371, 290, 236, 217; IR nmax (cmꢁ1): 3283
1
1558 (C]N), 1116 (CeN), 869 (CeS); H NMR (CDCl3)

1756
A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
(d, ppm): 6.72e7.11 (14H, m, Ar), 3.44 (dd, 1H, HA, JAB 18.5,
JAx 9.25), 3.49 (dd, 1H, HB, JAB 18.5, JBx 9.25).
to make a concentration of 1 mg/ml. Twofold serial dilutions
were made in the wells of 96-well microtiter plate (Costar)
in 170 ml of culture medium. Each test includes metronidazole
as a standard amoebicidal drug, control wells (culture medium
plus amoebae) and a blank (culture medium only). The num-
ber of amoeba per ml was estimated with a haemocytometer
and trypan blue exclusion was used to confirm viability. The
cell suspension used was diluted to 105 organism/ml by adding
fresh medium and 170 ml of this suspension was added to the
test and control wells in the plate. 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.
4.4.5. 3-(3-Br-ph) 5-[2-(isopropyl) ph]-1-(tz [4,5-b]
qx-2-yl)-2-pz (21)
Pale yellow crystal (chloroform); yield: 18%; m.p.: 158 ꢀC.
Anal. calc. (C27H19N5SBr): C 61.83, H 3.62, N 13.35; found:
C 61.34, H 3.89, N 13.29%. UV/vis lmax (nm): 371, 298, 266,
236, 209; IR nmax (cmꢁ1): 2969 (CeH), 1538 (C]N), 1161
1
(CeN), 876 (CeS); H NMR (CDCl3) (d, ppm): 7.23e7.44
(14H, m, Ar), 3.11 (dd, 1H, HA, JAB 18.5, JAx 9.25), 3.76
(dd, 1H, HB, JAB 18.5, JBx 9.25), 5.89 (dd, 1H, Hx, JAx
11.10, JBx 9.20), 6.28 (dd, 1H, Hx, JAx 11.10, JBx 9.25).
4.4.6. 3-(3-Br-ph) 5-(2,4,6 tri-me-ph)-1-(tz [4,5-b] qx-2-yl)-
2-pz (22)
4.6. Assessment of antiamoebic activity
Orange crystal (chloroform); yield: 22%; m.p.: 192 ꢀC.
Anal. calc. (C27H22N5SBr): C 61.48, H 4.17, N 13.28; found:
C 61.04, H 4.89, N 13.98%. UV/vis lmax (nm): 367, 290, 236,
221; IR nmax (cmꢁ1): 2924 (CeH), 1485 (C]N), 1522
(C]N), 1122 (CeN), 892 (CeS); 1H NMR (CDCl3) (d,
ppm): 6.7e7.4 (14H, m, Ar), 3.48 (dd, 1H, HA, JAB 18.5,
JAx 9.25), 3.51 (dd, 1H, HB, JAB 18.5, JBx 9.25), 6.32 (dd,
1H, Hx, JAx 11.10, JBx 9.25).
After incubation the growth of the amoeba was checked
with a low power microscope. The culture medium was re-
moved by inverting the plate and shaking gently. 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 Tables 1 and 2.
4.4.7. 3 (3-Br-ph) 5-(2-Cl-ph)-1-(tz [4,5-b] qx-2-yl)-
2-pz (23)
Pale yellow crystal (chloroform); yield: 18%; m.p.: 177 ꢀC.
Anal. calc. (C24H15N5SClBr): C 55.49, H 3.09, N 13.48;
found: C 55.64, H 3.89, N 13.61%. UV/vis lmax (nm): 371,
290, 231, 217; IR nmax (cmꢁ1): 2916 (CeH), 1576 (C]N),
1
1558 (C]N), 1051 (CeN), 947 (CeS); H NMR (CDCl3)
(d, ppm): 6.72e7.50 (14H, m, Ar), 3.38 (dd, 1H, HA, JAB
18.5, JAx 9.25), 3.56 (dd, 1H, HB, JAB 18.5, JBx 9.25), 5.44
(dd, 1H, Hx, JAx 11.10, JBx 9.25).
4.4.8. 3 (3-Br-ph) 5-(2,3diCl-ph)-1-(tz [4,5-b] qx-2-yl)-
2-pz (24)
Pale yellow crystal (chloroform); yield: 34%; m.p.: 175 ꢀC.
Anal. calc. (C24H14N5SCl2Br): C 62.07, H 2.53, N 12.65;
found: C 62.33, H 2.22, N 12.34%. UV/vis lmax (nm): 371,
290, 236, 217; IR nmax (cmꢁ1): 2924 (CeH), 1558 (C]N),
4.7. MTT toxicity assay
The quinoxaline derivatives of pyrazoline (17e24) which
showed the better antiamoebic results than their parent com-
pounds i.e.; chalcones (1e8) and pyrazolines (9e16) were as-
sessed for toxic profile. For the toxicity assay, transformed
human kidney epithelium (Graham) cells were continuously
maintained in culture at 37 ꢀC in 5% CO2. The MTT (3-[4,5-di-
methylthiazol-2yl]-2,5-diphenyltetrazolium bromide, USB)
cellular viability assay was used to determine the toxicity pro-
file of these compounds [20]. The trypsinized cell suspension
was adjusted to 0.5 million cells/mL and plated out with the
compounds (17e24). After 44 h of incubation, 2 mM MTT
was added to the plates and incubated for a further 4 h.
DMSO was then added to stop the reaction and dissolve the for-
mazan crystals. The absorbance was read at the test wavelength
of 540 nm and reference wavelength of 690 nm and the per-
centage cellular viability calculated with appropriate controls
1
1593 (C]N), 1122 (CeN), 920 (CeS); H NMR (CDCl3)
(d, ppm): 6.72e7.50 (14H, m, Ar), 3.71 (dd, 1H, HA, JAB
18.5, JAx 9.25), 3.86 (dd, 1H, HB, JAB 18.5, JBx 9.25), 5.79
(dd, 1H, Hx, JAx 11.10, JBx 9.25).
4.5. In vitro testing against E. histolytica
All the cyclised pyrazoline analogues were screened in vi-
tro for antiamoebic activity against HM1:1MSS strain of E.
histolytica by microdilution method [16]. E. histolytica tropho-
zoites were cultured in TYIS-33 growth medium as described
previously in wells of 96 well microtiter plate [17]. All the
compounds were dissolved in DMSO (40 ml) at which level
no inhibition of amoeba occurs [18,19] and the solution was
further diluted to 1 ml by adding freshly prepared medium

A. Budakoti et al. / European Journal of Medicinal Chemistry 43 (2008) 1749e1757
1757
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Acknowledgements
This work was supported by Department of Science and
Technology (Grant No. Vll-PRDSF/44/2004-05/TT). The au-
thors are thankful to Prof. Alok Bhattacharya and Prof. Sudha
Bhattacharya, School of Life Sciences and School of Environ-
mental Sciences, Jawaharlal Nehru University, New Delhi, re-
spectively for providing laboratory facilities.
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