Bis-pyrazolines: Synthesis, characterization and antiamoebic activity as inhibitors of growth of Entamoeba histolytica

Article abstract of DOI:10.1016/j.ejmech.2007.11.005

The cyclization of chalcone with N-4 substituted thiosemicarbazides under basic condition led to the formation of new compounds, thiocarbamoyl bis-pyrazoline derivatives. The structure of the compounds were elucidated by UV, IR, ¹H NMR, ¹³C NMR and ESI-MS spectral data and thermogravimetric analysis, and their purities were confirmed by elemental analyses. The antiamoebic activity of these complexes was evaluated by microdilution method against HM1:IMSS strain of Entamoeba histolytica and the results were compared with the standard drug, metronidazole. Structure-activity relationship shows that the compound with aromatic substituents at the thiocarbamoyl group was more active than those with the cyclic groups. However, it was clear from the IC₅₀ values that the compounds 15 and 20 are more active and both showed a structural resemblance having an electron withdrawing groups attached to the phenyl ring. MTT assay showed that all the compounds are non-toxic to human kidney epithelial cell line.

Full text of DOI:10.1016/j.ejmech.2007.11.005

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 426e431
http://www.elsevier.com/locate/ejmech
Short communication
Bis-pyrazolines: Synthesis, characterization and antiamoebic activity as
inhibitors of growth of Entamoeba histolytica
1
Abdul R. Bhat, Fareeda Athar , Amir Azam
*
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 18 June 2007; received in revised form 30 October 2007; accepted 12 November 2007
Available online 19 November 2007
Abstract
The cyclization of chalcone with N-4 substituted thiosemicarbazides under basic condition led to the formation of new compounds, thiocar-
1
bamoyl bis-pyrazoline derivatives. The structure of the compounds were elucidated by UV, IR, H NMR, 13C NMR and ESI-MS spectral data
and thermogravimetric analysis, and their purities were confirmed by elemental analyses. The antiamoebic activity of these complexes was
evaluated by microdilution method against HM1:IMSS strain of Entamoeba histolytica and the results were compared with the standard drug,
metronidazole. Structureeactivity relationship shows that the compound with aromatic substituents at the thiocarbamoyl group was more active
than those with the cyclic groups. However, it was clear from the IC50 values that the compounds 15 and 20 are more active and both showed
a structural resemblance having an electron withdrawing groups attached to the phenyl ring. MTT assay showed that all the compounds are non-
toxic to human kidney epithelial cell line.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Bischalcone; Bis-Pyrazolines; Thiocarbamoyl; Antiamoebic activity
1. Introduction
derivatives (metronidazole, tinidazole, ornidazole). Metronida-
zole is the mainstay and used in combination with other drugs
such as iodoquinol, paromomycin and diloxanide furoate [6].
Tissue amoebicides such as metronidazole and tinidazole kill
amoeba in host tissue and organ whereas iodoquinol and paro-
momycin are active only in the intestinal lumen [7]. Despite
these, there is lack of ideal drug, and immunity acquired to
already available drugs and the side effects are a major hurdle
in eradicating these diseases [8e11].
Five-membered heterocyclic compounds natural as well as
synthetic are important for their biological activities. Com-
pounds with pyrazole ring are of interest due to their broad
spectrum of biological activities against monoamine oxidase
inhibitor [12], bacterial [13], depression [14], hypotensive
[15], pyretic and inflammatory diseases [16]. Some bis-
pyrazoline derivatives were also found with antimicrobial ac-
tivity [17]. The discovery of this class of drugs provides an
outstanding case of history of modern drug development
and also points out the unpredictability of biological activity
from structural modification. In view of the number of
Amoebiasis is the second leading cause of death from para-
sitic disease worldwide [1]. The causative amoeboid eukaryote
parasite, Entamoeba histolytica, is a potent pathogen. E. histo-
lytica causes approximately 50 million cases and approximately
100,000 deaths annually [2,3]. This parasite acts as a macro-
phage on steroids with pumped-up phagocytic, proteolytic,
and cytolytic capabilities, invading human colonic mucosa,
and occasionally penetrating through to the portal circulation,
reaching the liver and causing fatal liver abscesses [4]. Brain
abscess is the dreadful complication of this disease [5]. The cor-
nerstone of treatment for amoebiasis remains the nitroimidazole
* Corresponding author. Tel.: þ91 11 26981717/3253; fax: þ91 11
26980229/1232.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
1
Present address: Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.005

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 426e431
427
pharmacological significance of pyrazolines, we have synthe-
sized a new series of organic molecules containing two pyr-
azoline rings and have evaluated the same against the
HM1:IMSS strain of the E. histolytica, a protozoa responsible
for amoebiasis. The ongoing research in our laboratory
showed that pyrazoline derivatives [18e20] are good candi-
dates for these studies. On the basis of their activity and fa-
vorable therapeutic indexes, these compounds were identified
as viable leads for further studies.
methanol gave crystalline solid compounds in low yield.
The yield of cyclized compounds was in the range of 12e
32%. All the compounds are insoluble in water but soluble
in most of the organic solvents. The structures of the com-
pounds were established by means of their IR, UV, 1H
NMR, 13C NMR and mass spectral studies.
Assignment of selected characteristic IR bands provides
significant indications for the formation of the cyclized pyr-
azoline analogues of the thiosemicarbazones (12e21). The
absence of the band at/or around 1658 cmꢀ1 showed the ab-
sence of the carbonyl group in all the compounds (12e21).
The appearance of an intense band between 1357 and
1393 cmꢀ1 regions showed n (C]S) stretch of the thiocarba-
moyl group. The cyclization of the product was confirmed
by the appearance of the absorption bands in the region
1583e1618 cmꢀ1 and 1055e1096 cmꢀ1. The former band
is attributed to n (C]N) stretch and the latter band to n
(CeN) stretch vibrations. The compounds 12 and 16e21
showed additional bands in the region 3322e3396 cmꢀ1
due to n (NeH) stretch.
2. Results and discussion
Bischalcone was prepared according to ClaiseneSchmidt
reaction, i.e., by treating acetophenone with NaOH (60%)
and terephthalaldehyde gave above 80% yield. All the thiose-
micarbazides were prepared by reported method [21]. The cy-
clization of chalcone with N-4 substituted thiosemicarbazides
under basic condition led to the formation of new compounds
thiocarbamoyl bis-pyrazoline derivatives, Scheme 1. Accord-
ing to the currently accepted mechanism, formation of the cy-
clized pyrazoline analogues is favored via thiosemicarbazone
formation, which undergo cyclization under basic conditions
to form desired pyrazoline ring in all the compounds (12e
21), (Table 1) [22,23]. The compounds recrystallized by
The electronic spectra of the cyclized pyrazoline analogue
studies in the UV region in methanol, exhibited three absorp-
tion bands at 371e338 nm, 269e231 nm and 229e210 nm as-
signed to n / p*, p / p* and n / s* transitions,
respectively. The band at 371e338 nm was assigned to the
O
KOH + S=C=S + HR
O
O
CH3
ClCH2COONa
2
H
H
+
S
NaOOCCH2SC-R
S
HOOCCH2SC-R
O
O
NH2NH2.H2O
S
1
NH2NHC-R
2-11
N
N
N
N
S=C
C=S
R
R
12-21
Scheme 1. General representation for the synthesis of bis-pyrazoline derivatives.

428
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 426e431
Table 1
Different substituent groups ‘R’
protons belonging to the aromatic ring were observed within
the expected chemical shift region along with integral values.
The 13C NMR spectra of all the compounds were taken in
CDCl3. The C4 and C5 carbons of the pyrazoline ring resonate
at 40.3e48.5 ppm and 62.3e67.9 ppm, respectively. All the
compounds showed a signal at 153.2e158.2 ppm assigned to
azomethine carbon of pyrazoline ring. Thiocarboxamide car-
bon (C]S) displayed a signal at 175.0e179.1 ppm in all the
compounds. The signals due to the aromatic carbons and the
carbons at 1-N-substituted aliphatic groups resonate at their
usual positions shown in the experimental section.
HN
NO2
N
H
N
NH2
N
CH3
12
13
16
14
15
CH3
F
HN
CH3
N
H
N
H
HN
HN
F
17
21
18
19
20
2.1. In vitro antiamoebic activity
Preliminary experiments were carried out to determine the
antiamoebic activity in vitro of all the compounds (12e21) by
microdilution method using HM1:IMSS strain of E. histoly-
tica. The results of the bioassays are summarized in Table 2.
The data are present in terms of percent growth inhibition
relative to untreated controls, and plotted as probit values as
a function of drug concentration. The IC50 values were inter-
polated in the corresponding dose response curves. The IC50
values for compounds 15 (0.42 mM), 20 (0.62 mM), and 18
(0.920 mM), correspond to 1.95e4.28 fold increase in activity,
than the standard drug metronidazole (1.8 mM), thus proved to
be better inhibitors of E. histolytica growth. The compounds
17 (1.16 mM) and 12 (1.57 mM) showed moderate activity
than metronidazole. The results were statistically evaluated
by analyses of variance. The null hypothesis was tested using
T-Test and the significance of the differences between the IC50
value(s) of metronidazole vs. 12e21 was evaluated. The cal-
culated T-values were higher than the table values at the 4%
level. Hence, the character under study was significantly influ-
enced by the treatment. Structureeactivity relationship shows
that the compound with aromatic substituents at the thiocarba-
moyl group was more active than those with the cyclic groups.
However, it was clear from the IC50 values that the compounds
15 and 20 are more active and both showed a structural resem-
blance having an electron withdrawing groups attached to the
phenyl ring. Out of the cyclic substituent, compound 18 with
cyclooctyl ring was found to be more active than cyclohexyl
and/or cyclopentyl rings.
transition involving the thione portions (C]S) of thiocarba-
moyl group. The two other absorption bands at 269e231 nm
and 229e210 nm were due to p / p* transition of phenyl
ring and n / p* transition of azomethine nitrogen.
The structure of the compounds was further confirmed by
1H NMR and 13C NMR. The pyrazoline protons Ha and Hb
(A) are geminal protons, appear in the region of 3.05e
3.82 ppm and 2.91e2.16 ppm as doublet of doublets in all
the compounds.
HA
HA
4
3
HB
HB
HX
HX
2N
N
5
1N
N
C=S
R
S=C
R
(A)
The CH proton appeared as doublet of doublets in the
region 5.73e6.52 ppm due to vicinal coupling with two non-
equivalent geminal protons of adjacent carbon atom. The
NH proton of different substituted thiocarbamoyl compounds
12, and 16e21 showed singlet at 8.36e10.89 ppm. The
Table 2
In vitro antiamoebic activity against E. histolytica HM1:IMSS strain and cyto-
toxicity profile of 12e21
2.2. Toxicity profile
Sample
code
Compound
Antiamoebic
activity
Toxicity
profile
(IC50 (mM))
Safety
Index
(S.I.)
To ensure that the bis-pyrazolines were not toxic to human
cells these compounds were tested against a human kidney ep-
ithelial cell line. None of the compounds inhibited cell growth
at a concentration of 90 mM (Table 2). To investigate the selec-
tivity of the compounds, the ‘‘safety index’’ (S.I.) was calcu-
lated 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 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 compound are given in
IC50 (mM)
S.D.
12
13
C26H24N6S2
C34H36N6S2
C42H40N6S2
C38H30N8S2O4
C36H40N6S2
C38H44N6S2
C42H52N6S2
C40H36N6S2
C38H28N6S2F4
C40H36N6S2
C6H9N3O3
1.57
>1.8
>1.8
0.42
>1.8
1.16
0.92
>1.8
0.62
>1.8
1.8
0.51
0.61
0.47
0.15
0.61
0.39
0.41
0.73
0.24
0.62
0.39
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>100
>57.3
>50.0
>50.0
>214
14
15
16
17
>50.0
>77.6
>97.8
>50.0
>145
18
19
20
21
>50.0
>55.55
MNZ
S.D. ¼ standard deviation. S.I. ¼ toxicity IC50/protozoal IC50
.

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 426e431
429
Table 2. These results show that the compound 15 has lowest
3.1.3. Synthesis of thiocarbamoyl bis-pyrazoline derivatives
(12e21)
A mixture of bischalcone 1 (1 equiv), thiosemicarbazides 2e
11 (4 equiv) and NaOH (2.5 equiv) was refluxed in ethanol for 3
days. The solution was poured into ice water. The precipitate
was filtered and recrystallized from methanol to yield 12e21.
cytotoxicity and highest antiamoebic activity and overall com-
pounds show more favorable safety profile along with the most
promising antiamoebic activity. Thus the accumulation of the
bis-pyrazoline derivatives will remain toxic to the parasite,
whilst in the human host there will be a decreased likelihood
of toxicity.
3.1.3.1. Amino[5-(4-{1-(aminothioxomethyl)-3-phenyl(2-pyra-
zoline-5-yl-phenyl)}-3-phenyl(2-pyrazolinyl)]methane-1-thione
(12). Brownish yellow crystal (chloroform); yield 16%; m.p.
231 ꢁC; Anal. Calc. for C26H24N6S2: C 64.43, H 4.99, N
17.34, found C 64.71, H 5.21, N 17.61%; UV/vis lmax (nm)
356, 247, 223; IR n (cmꢀ1) 3318 (NH), 3096 (AreH), 2925
3. Experimental
Reactions were conducted in oven dried glass wear. All the
chemicals were purchased from Aldrich chemical company
(USA). Analytical thin-layer chromatography was performed
on precoated silica gel 60 F254 plates and column chromatogra-
1
(CH), 1590 (CH]N), 1373 (C]S), 1096 (CeN); H NMR
(CDCl3): d/ppm 8.36 (s, 4H, NH2), 7.86 (s, 4H, AreH),
7.10e7.28 (m, 10H, AreH), 5.91 (dd, 2H, Hx, JxA 8.93 Hz,
JxB 16.81 Hz), 3.82 (dd, 2H, HA, JAB 6.41 Hz, JAx 8.93 Hz),
2.9 (dd, 2H, HB, JBA 6.41 Hz, JBx 16.81 Hz); 13C NMR
(DMSO): d/ppm 176.4 (C]S), 154.9 (C]N), 141.1, 130.5,
130.1, 128.2, 126.6, 125.2 (AreC), 62.3 (CH), 42.0 (CH2).
FABMS (M þ 1): m/z 485.32, calc. 484.20.
˚
phy was accomplished using Silica gel, 60 A (200e400 mesh)
and basic alumina. Elemental analyses were performed by Cen-
tral Drug Research Institute, Lucknow, using Heraeus Vario EL
III analyzer, and the results were within 0.3% of the theoretical
values. Electronic spectra were recorded in methanol on a Shi-
madzu UV-1601 PC UVeVisible spectrophotometer. IR spec-
tra on KBr disks were recorded on a PerkineElmer model 1620
FT-IR spectrophotometer. 1H NMR spectra were taken in
CDCl3 at ambient temperature using a Bruker Spectrospin
DPX-300 MHz spectrophotometer with TMS as internal stan-
dard. Splitting patterns are designated as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. Chemical shift values are
given in ppm.
3.1.3.2. (3-Phenyl-5-{4-3-phenyl-1-(pyrrolidinylthioxomethyl)
(2-pyrazoline-5-yl-phenyl}(2-pyrazolinyl))pyrrolidinylmethane-
1-thione (13). Brownish yellow crystal (chloroform); yield
12%; m.p. 271 ꢁC; Anal. Calc. for C34H36N6S2: C 68.88, H
6.12, N 14.18, found: C 69.05, H 6.42, N 14.41%; UV/vis
lmax (nm) 342, 297, 228; IR n (cmꢀ1) 3041 (AreH), 2961
1
(CH), 1615 (CH]N), 1386 (C]S), 1064 (CeN); H NMR
(CDCl3): d/ppm 7.53 (s, 4H, AreH), 7.18e7.46 (m, 10H, Are
H), 6.52 (dd, 2H, Hx, JxA 8.72 Hz, JxB 16.21 Hz), 3.94e4.19
(16H, m, eCH2), 3.58 (dd, 2H, HA, JAB 6.32 Hz, JAx 8.72 Hz),
2.16 (dd, 2H, HB, JBA 6.32 Hz, JBx 16.21 Hz); 13C NMR
(DMSO): d/ppm 175.8 (C]S), 153.4 (C]N), 142.4, 130.1,
129.9, 128.6, 125.7, 124.2 (AreC), 65.3 (CH), 63.1, 61.4,
60.9, 43.1 (CH2); FABMS (M þ 1): m/z 593.69, calc. 592.29.
3.1. Chemistry
3.1.1. General procedure for the synthesis
of bischalcone (1)
A solution of acetophenone (2 equiv) and terapthalaldehyde
(1 equiv) in methanolic solution of NaOH (60%) was stirred
for 20 h at room temperature. The solution was poured into
ice cold water of pH w 2 (pH adjusted by HCl). The solid
was separated and dissolved in CH2Cl2, washed with saturated
solution of NaHCO3 and evaporated to dryness. The residual
was purified by column chromatography using CH2Cl2 as elu-
ent. The compound was recrystallized in ethanol.
Pale yellow crystal (chloroform); yield 83%; m.p. 158;
Anal. Calc. for C24H18O2: C 85.17, H 5.36, found: C 85.5,
H 5.73%; UV/vis lmax (nm) 374, 288, 239, 219; IR n
(cmꢀ1) 3060 (AreH), 2924 (CH), 1653 (C]O), 1610
3.1.3.3. (Methylphenylamino)[5-(4-{1[(methylphenylamino)-
thioxomethyl]-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-phenyl
(2-pyrazolinyl)]methane-1-thione (14). Brownish yellow crys-
tal (chloroform); yield 26%; m.p. 235 ꢁC; Anal. calc. for
C42H40N6S2: C 72.8, H 5.82, N 12.13, found: C 72.97, H
5.61, N 12.38%; UV/vis lmax (nm), 366, 247, 216; IR n
(cmꢀ1) 3071 (AreH), 2914 (CH), 1588 (CH]N), 1357
1
(C]S), 1083 (CeN); H NMR (CDCl3): d/ppm 7.25e7.61
(m, 22H, AreH), 7.82 (s, 4H, AreH), 6.13 (dd, 2H, Hx, JxA
9.71 Hz, JxB 17.31 Hz), 5.02 (2H, s, eNCH2e), 3.38 (3H, s,
eNeCH3), 3.15 (dd, 2H, HA, JAB 5.91 Hz, JAx 9.71 Hz), 2.46
(dd, 2H, HB, JBA 5.91 Hz, JBx 17.31 Hz); 13C NMR (DMSO):
d/ppm 177.2 (C]S), 155.0 (C]N), 132.2, 131.2, 128.4,
128.1, 126.9, 125.3 (AreC), 65.8 (CH2), 62.3 (CH), 43.8
(CH2), 40.6 (CH3). FABMS (M þ 1): m/z 693.52, calc. 692.32.
1
(CH]CH), 1541 (C]C); H NMR (CDCl3): d/ppm 6.9 (d,
2H J ¼ 15 Hz, C]CH), 7.25e7.49 (m, 10H, AreH), 7.6 (d,
4H, AreH), 7.92 (d, 2H J ¼ 15 Hz, COeCH); 13C NMR
(DMSO): d/ppm 190.2 (C]O), 143.5 (C]C), 138.0
(C]C), 136.8, 132.9, 128.9, 128.6, 128.5, 123.0 (AreC).
FABMS (M þ 1): m/z 339.1, calc. 338.
3.1.3.4. (4-Nitrophenylamino)[5-(4-{1-[(4-nitrophenylamino)
thioxomethyl]-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-phenyl(2-
pyrazolinyl)]methane-1-thione (15). Brownish yellow crystal
(chloroform); yield 19%; m.p. 274 ꢁC; Anal. Calc. for
3.1.2. Synthesis of thiosemicarbazides (2e11)
Substituted thiosemicarbazides (2e11) were prepared by
a reported method [21].

430
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 426e431
C38H30N8O4S2: C 62.78, H 4.16, N 15.42, found: C 62.39, H
4.32, N 15.69%; UV/vis lmax (nm) 338, 239, 215; IR n (cmꢀ1
3034 (AreH), 2989 (CH), 1583 (CH]N), 1393 (C]S), 1055
m, eNeCHe (cyclooctyl ring)), 4.31e4.43 (28H, m, cyclooc-
tyl ring), 3.32 (dd, 2H, HA, JAB 5.52 Hz, JAx 9.92 Hz), 2.71
(dd, 2H, HB, JBA 5.52 Hz, JBx 17.35 Hz); 13C NMR
(DMSO): d/ppm 175.0 (C]S), 153.2 (C]N), 143.2, 133.1,
132.3, 130.6, 130.2, 128.4 (AreC), 67.8 (CH), 66.3, 65.9,
62.9, 61.3, 61.1, 60.2, 40.3 (CH2). FABMS (M þ 1): m/z
705.93, calc. 704.41.
)
1
(CeN); H NMR (CDCl3): d/ppm 9.62 (s, 2H, NH), 7.82 (s,
4H, AreH), 7.41e7.79 (m, 18H, AreH), 5.85 (dd, 2H, Hx,
JxA 9.68 Hz, JxB 17.39 Hz), 3.49 (dd, 2H, HA, JAB 6.71 Hz,
JAx 9.68 Hz), 2.36 (dd, 2H, HB, JBA 6.71 Hz, JBx 17.39 Hz);
13C NMR (DMSO): d/ppm 176.7 (C]S), 157.8 (C]N),
143.4, 130.5, 130.1, 128.2, 127.5, 126.6, 125.9, 125.2 (Are
C), 67.9 (CH), 46.1 (CH2); FABMS (M þ 1): m/z 727.50,
calc. 726.24.
3.1.3.8. [(3-Methylphenyl)amino][5-(4-{1-{[(3-methylphenyl)
amino]thioxomethyl}-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-
phenyl(2-pyrazolinyl)]methane-1-thione (19). Brownish yel-
low crystal (chloroform); yield 26%; m.p. 249 ꢁC; Anal.
calc. for C40H36N6S2: C 72.25, H 5.46, N 12.64, found: C
72.68, H 5.76, N 12.91%; UV/vis lmax (nm) 371, 246, 210;
IR n (cmꢀ1) 3376 (NH), 3073 (AreH), 2921 (CH), 1593
3.1.3.5. (Cyclopentylamino)[5-(4-{1-[(cyclopentylamino)thiox-
omethyl]-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-phenyl(2-pyra-
zolinyl)]methane-1-thione (16). Brownish yellow crystal
(chloroform); yield 32%; m.p. 246 ꢁC; Anal. calc. for
C36H40N6S2: C 69.64, H 6.49, N 13.54, found: C 69.79, H
1
(CH]N), 1377 (C]S), 1064 (CeN); H NMR (CDCl3): d/
ppm 10.83 (s, 2H, NH), 7.62 (s, 4H, AreH), 7.19e7.53 (m,
18H, AreH), 5.93 (dd, 2H, Hx, JxA 9.54 Hz, JxB 16.29 Hz),
3.47 (dd, 2H, HA, JAB 5.97 Hz, JAx 9.54 Hz), 2.56 (dd, 2H,
HB, JBA 5.97 Hz, JBx 16.29 Hz), 2.15 (3H, s, eCH3); 13C
NMR (DMSO): d/ppm 179.1 (C]S), 154.8 (C]N), 141.6,
132.5, 131.6, 131.1, 130.8, 128.3, 128.1, 126.4, 126.0, 125.9
(AreC), 63.1 (CH), 47.1 (CH2), 46.9 (CH3). FABMS
(M þ 1): m/z 665.86 calc. 664.28.
6.73, N 13.81%; UV/vis lmax (nm) 363, 245, 211; IR n (cmꢀ1
)
3349 (NH), 3012 (AreH), 2947 (CH), 1595 (CH]N), 1371
1
(C]S), 1076 (CeN); H NMR (CDCl3): d/ppm 9.15 (s, 2H,
NH), 7.61 (s, 4H, AreH), 7.03e7.41 (m, 10H, AreH), 5.73
(dd, 2H, Hx, JxA 8.83 Hz, JxB 17.46 Hz), 4.55e4.63 (2H, m, e
NeCHe (cyclopentyl ring)), 3.64 (dd, 2H, HA, JAB 6.23 Hz,
JAx 8.83 Hz), 2.13 (dd, 2H, HB, JBA 6.23 Hz, JBx 17.46 Hz),
1.43e1.69 (16 H, m, cyclopentyl ring); 13C NMR (DMSO): d/
ppm 178.0 (C]S), 155.3 (C]N), 142.2, 132.1, 131.9, 128.4,
127.3, 124.2 (AreC), 63.8 (CH), 62.8, 61.2, 59.0, 46.3 (CH2);
FABMS (M þ 1) m/z 621.81, calc. 620.32.
3.1.3.9. [(2,4-Difluorophenyl)amino[5-(4-{1-{[(2,4-difluoro-
phenyl)amino]thioxomethyl}-3-phenyl(2-pyrazoline-5-yl-phenyl)}-
3-phenyl(2-pyrazolinyl)]methane-1-thione (20). Brownish yellow
crystal (chloroform); yield 16%; m.p. 253 ꢁC; Anal. Calc. for
C38H28N6S2F4: C 64.38, H 3.98, N 11.86, found: C 64.73, H
3.1.3.6. (Cyclohexylamino)[5-(4-{1-[(cyclohexylamino)thioxo-
methyl]-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-phenyl(2-pyra-
zolinyl)]methane-1-thione (17). Brownish yellow crystal
(chloroform); yield 23%; m.p. 261 ꢁC; Anal. Calc. for
C38H44N6S2: C 70.33, H 6.84, N 12.27, found: C 70.94, H
6.43, N 12.56%; UV/vis lmax (nm) 363, 251, 229; IR n
3.86, N 11.93%; UV/vis lmax (nm) 358, 269, 218; IR n (cmꢀ1
)
3365 (NH), 3065 (AreH), 2892 (CH), 1587 (CH]N), 1358
1
(C]S), 1073 (CeN); H NMR (CDCl3): d/ppm 10.05 (s, 2H,
NH), 7.69 (s, 4H, AreH), 7.12e7.44 (m, 16H, AreH), 6.41
(dd, 2H, Hx, JxA 9.32 Hz, JxB 17.45 Hz), 3.05 (dd, 2H, HA,
JAB 5.63 Hz, JAx 9.32 Hz), 2.37 (dd, 2H, HB, JBA 5.63 Hz, JBx
17.45 Hz); 13C NMR (DMSO): d/ppm 176.2 (C]S), 155.1
(C]N), 133.4, 133.1, 131.8, 130.3, 128.4, 128.1, 126.9, 126.4,
125.1 (AreC), 64.2 (CH), 42.3 (CH2). FABMS (M þ 1): m/z
709.49, calc. 708.22.
(cmꢀ1
) 3316 (NH), 3019 (AreH), 2947 (CH), 1609
1
(CH]N), 1369 (C]S), 1086 (CeN); H NMR (CDCl3): d/
ppm 8.81 (s, 2H, NH), 7.42e7.81 (m, 10H, AreH), 7.31 (s,
4H, AreH), 6.03 (dd, 2H, Hx, JxA 9.61 Hz, JxB 16.81 Hz),
4.14e4.29 (2H, m, eNeCH (cyclohexyl ring)), 3.28 (dd,
2H, HA, JAB 5.81 Hz, JAx 9.61 Hz), 2.95 (dd, 2H, HB, JBA
5.81 Hz, JBx 16.81 Hz), 1.23e1.97 (20H, m, cyclohexyl
ring); 13C NMR (DMSO): d/ppm 177.3 (C]S), 154.5
(C]N), 134.2, 132.6, 130.4, 128.3, 126.2 (AreC), 65.9
(CH), 63.3, 62.1, 61.6, 61.0, 59.9, 41.8 (CH2). FABMS
(M ꢀ 1): m/z 647.2, calc. 648.35.
3.1.3.10. [(4-Methylphenyl)amino][5-(4-{1-{[(4-methylphenyl)
amino]thioxomethyl}-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-
phenyl(2-pyrazolinyl)]methane-1-thione (21). Brownish yel-
low crystal (chloroform); yield 18%; m.p. 236 ꢁC; Anal.
Calc. for C40H36N6S2: C 72.25, H 5.46, N 12.64, found: C
72.61, H 5.71, N 12.87%; UV/vis lmax (nm) 368, 251, 317;
IR n (cmꢀ1) 3396 (NH), 3058 (AreH), 2929 (CH), 1605
3.1.3.7. (Cyclooctylamino)[5-(4-{1-[(cyclooctylamino)thioxo-
methyl]-3-phenyl(2-pyrazoline-5-yl-phenyl)}-3-phenyl(2-pyrazo-
1
(CH]N), 1368 (C]S), 1072 (CeN); H NMR (CDCl3): d/
linyl)]methane-1-thione
(18). Brownish
yellow
crystal
ppm 10.65 (s, 2H, NH), 7.81 (s, 4H, AreH), 7.31e7.57 (m,
18H, AreH), 5.96 (dd, 2H, Hx, JxA 8.68 Hz, JxB 16.52 Hz),
3.18 (dd, 2H, HA, JAB 5.99 Hz, JAx 8.68 Hz), 2.91 (dd, 2H,
HB, JBA 5.99 Hz, JBx 16.52 Hz), 2.33 (3H, s, eCH3); 13C
NMR (DMSO): d/ppm 178.3 (C]S), 158.2 (C]N), 132.8,
131.8, 131.4, 130.5, 128.2, 128.0, 126.9, 126.2, 125.3 (Are
C), 65.2 (CH), 48.5 (CH2), 47.2 (CH3). FABMS (M þ 1): m/
z 665.50, calc. 664.28.
(chloroform); yield 17%; m.p. 268 ꢁC Anal. Calc. for
C42H52N6S2: C 71.54, H 7.44, N 11.92, found: C 71.76, H
7.13, N 12.14%; UV/vis lmax (nm) 369, 231, 220; IR n
(cmꢀ1) 3312 (NH), 3059 (AreH), 2893 (CH), 1618 (CH]N),
1
1368 (C]S), 1063 (CeN); H NMR (CDCl3): d/ppm 8.56 (s,
2H, NH), 7.56 (s, 4H, AreH), 7.21e7.39 (m, 10H, AreH),
6.42 (dd, 2H, Hx, JxA 9.92 Hz, JxB 17.35 Hz), 4.65e5.01 (2H,

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 426e431
431
4. Antiamoebic activity
compounds. 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 formazan crys-
tals. The absorbance was read at the test wavelength of 540 nm
and reference wavelength of 690 nm and the percentage cellu-
lar viability calculated with appropriate controls taken into ac-
count. The mean ꢃ S.D. values of IC50 values in Table 2 are
from three independent experiments.
The compounds 12e21 were screened in vitro for antia-
moebic activity against the HM1:IMSS strain of E. histolytica
by the microdilution method [24]. E. histolytica trophozoites
were cultured in TYIS-33 growth medium as described previ-
ously, in wells of 96 microtiter plate (Costar) [25]. The test
compounds were dissolved in DMSO (40 ml), at which level
no inhibition of the amoeba occurs [26,27]. Then, culture me-
dium was added to obtain a concentration of 1 mg/ml. Two
fold serial dilutions were then made. Each test includes metro-
nidazole as a standard amoebic drug, control wells (culture
medium plus amoeba), and a blank (culture medium only).
The number of amoeba per millimeter was estimated with a he-
mocytometer, and trypan blue exclusion was used to confirm
viability. The cell suspension used was diluted to 105 organ-
isms/ml by adding fresh medium, and 170 ml of this suspen-
sion 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. The plates
were sealed, and gassed for 10 min with N2, and then incu-
bated at 37 ꢁC for 72 h. After incubation the growth of the
amoeba was checked with a low power microscope. The cul-
ture medium was removed by inverting the plate and shaking
gently. The plates were immediately washed with 0.9% aq.
NaCl solution at 37 ꢁC. This procedure was performed
quickly, and the plate was not allowed to cool, to prevent
the detachment of amoebae. The plate was allowed to dry at
room temperature, and the amoebas were fixed with chilled
MeOH by keeping it in an ice bath for 15 min, dried, and
stained with 0.5% aq. eosin for 15 min. The stained plate
was washed once with tap H2O, and then twice with distilled
H2O, and allowed to dry. Then 0.1 N aq. NaOH solution
(200 ml) was added to each well to dissolve the protein and
to release the dye (eosin). The optical density of the resulting
solution in each well was determined at 490 nm with a micro-
plate reader. The inhibition (in %) 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 determine
the best-fitting straight line, from which IC50 values were
determined.
References
[1] S.L. Stanley Jr., Lancet 361 (2003) 1025e1034.
[2] WHO, Wkly. Epidemiol. Rec. 14 (1997) 97e100.
[3] C.D. Huston, R. Haque, W.A. Petri Jr., Expert Rev. Mol. Med. (1999)
1e11.
[4] J.L. Ravdin, Clin. Infect. Dis. 20 (1995) 1453e1466.
[5] F.D. Rocco, G. Sabatino, G. Tamburrini, O. Ranno, P. Valentini,
M. Caldarlli, Acta Neurochir. 146 (2004) 1271e1272.
[6] D.C.K. Ng, S.Y. Kwok, Y. Cheng, C.C. Chung, M.K.W. Li, Hong Kong
Med. J. 12 (2006) 71e73.
[7] A. Azam, S.M. Agarwal, Curr. Bioact. Compd. 3 (2007) 121e123.
[8] F. Roe, Surgery 93 (1983) 158e164.
[9] J.C. Samuelson, A. Burke, J.M. Courval, Antimicrob. Agents Chemother.
36 (1992) 2392e2397.
[10] I.S. Adagu, D. Nolder, D.C. Warhurst, J.F. Rossignol, J. Antimicrob.
Chemother. 49 (2002) 103e111.
[11] G. Turan-Zitouni, A. Ozdemir, K. Guven, Arch. Pharm. (Weinheim) 338
(2005) 96e104.
[12] N. Gokhan, A. Yesilada, G. Ucar, K. Erol, A.A. Bilgin, Arch. Pharm.
Med. Chem. 336 (2003) 362e371.
[13] B.S. Holla, P.M. Akbarali, M.K. Shivanada, IL Farmaco 55 (2000)
256e263.
[14] Y.R. Prassad, A.L. Rao, L. Prasoona, K. Murali, P.R. Kumar, Bioorg.
Med. Chem. Lett. 15 (2005) 5030e5034.
[15] J.T. Zitouni, P. Chevallet, F.S. Kilic, K. Erol, Eur. J. Med. Chem. 35
(2000) 635e641.
[16] O. Bruno, A. Ranise, F. Bondavalli, F. Schenone, M. D’ Amico,
A. Filipelli, W. Filipelli, R. Francesco, IL Farmaco 48 (1993) 949e966.
[17] D.B. Reddy, B. Seenaiah, S. Eswaraiah, T. Seshamma, M.V.R. Reddy, J.
Indian Chem. Soc. 66 (1989) 893e896.
[18] M. Abid, A. Azam, Bioorg. Med. Chem. 13 (2005) 2213e2220.
[19] M. Abid, A. Azam, Bioorg. Med. Chem. Lett. 16 (2006) 2812e2816.
[20] M. Abid, A. Azam, Eur. J. Med. Chem. 40 (2005) 935e942.
[21] D.G. O’ Sullivan, P.W. Sadler, C. Webley, Chem. Commun. 7 (1) (1963)
17e26.
[22] H. Ferres, W.R. Jackson, J. Chem. Soc., D. Chem. Commun. 6 (1969)
261e262.
[23] R.H. Wiley, C.H. Jarboe, F.N. Hayes, E. Hansbury, J.T. Nielson,
P.X. Callahan, M.C. Sellars, J. Org. Chem. 23 (1958) 732e738.
[24] W. Wright, M.J. O’Neill, J.D. Phillipson, D.C. Warhurst, Antimicrob.
Agents Chemother. 32 (1988) 1725e1729.
4.1. MTT toxicity assay
For the toxicity assay, transformed human kidney epithe-
lium (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 as-
say was used to determine the toxicity profile of the com-
pounds [28]. The trypsinized cell suspension was adjusted to
0.5 million cells/ml and plated out with the various
[25] L.S. Diamond, D.R. Harlow, C.C. Cunnick, Trans. R. Soc. Trop. Med.
Hyg. 72 (1978) 431e432.
[26] F.D. Gillin, D.S. Reiner, M. Suffness, Antimicrob. Agents Chemother. 22
(1982) 342e345.
[27] A.T. Keen, A. Harris, J.D. Phillipson, D.C. Warhurst, Planta Med. (1986)
278e284.
[28] T. Mosmann, J. Immunol. Methods 65 (1e2) (1983) 55e63.