Synthesis, spectral studies and antiamoebic activity of new 1-N-substituted thiocarbamoyl-3-phenyl-2-pyrazolines

Article abstract of DOI:10.1016/j.ejmech.2007.10.032

Thirty new pyrazoline derivatives were synthesized by cyclization of Mannich bases with thiosemicarbazides being substituted by different cyclic and aromatic amines. The structures of the compounds were elucidated by elemental analyses, UV, IR, ¹H and ¹³C NMR and ESI-MS spectral data. The in vitro antiamoebic activity was evaluated against Entamoeba histolytica in comparison with metronidazole used as reference substance. Out of the 30 compounds screened for antiamoebic activity, 10 (5, 6, 15, 18, 25-30) were found to be better inhibitors of E. histolytica since they showed lesser IC₅₀ values than metronidazole. The preliminary results indicated that the presence of 3-chloro or 3-bromo substituent on the phenyl ring at position 3 of the pyrazoline ring enhanced the antiamoebic activity as compared to unsubstituted phenyl ring. The study suggests that the preliminary activity of these compounds may further be explored for the development of new targets for amoebiasis.

Full text of DOI:10.1016/j.ejmech.2007.10.032

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European Journal of Medicinal Chemistry 44 (2009) 417e425
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, spectral studies and antiamoebic activity of new
1-N-substituted thiocarbamoyl-3-phenyl-2-pyrazolines
*
Mohammad Abid, Abdul Roouf Bhat, Fareeda Athar, Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, India
Received 13 April 2007; received in revised form 20 October 2007; accepted 29 October 2007
Available online 4 November 2007
Abstract
Thirty new pyrazoline derivatives were synthesized by cyclization of Mannich bases with thiosemicarbazides being substituted by different
1
cyclic and aromatic amines. The structures of the compounds were elucidated by elemental analyses, UV, IR, H and 13C NMR and ESI-MS
spectral data. The in vitro antiamoebic activity was evaluated against Entamoeba histolytica in comparison with metronidazole used as reference
substance. Out of the 30 compounds screened for antiamoebic activity, 10 (5, 6, 15, 18, 25e30) were found to be better inhibitors of
E. histolytica since they showed lesser IC50 values than metronidazole. The preliminary results indicated that the presence of 3-chloro or 3-bromo
substituent on the phenyl ring at position 3 of the pyrazoline ring enhanced the antiamoebic activity as compared to unsubstituted phenyl ring. The
study suggests that the preliminary activity of these compounds may further be explored for the development of new targets for amoebiasis.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrazolines; Mannich bases; Thiocarbamoyl; Entamoeba histolytica
1. Introduction
[5e8]. Therefore it is desirable to search for new lead mole-
cules, which can be effectively used against amoebiasis.
In drug designing programs an essential component of the
search for new leads is the synthesis of molecules, which is
novel yet resembles known biologically active molecules by vir-
tue of the presence of critical structural features. Certain small
heterocyclic molecules act as highly functionalized scaffolds
and are known pharmacophores of a number of biologically
active and medicinally useful molecules [9,10]. Pyrazoles and
their reduced forms, pyrazolines, are well known nitrogen con-
taining heterocyclic compounds, and various procedures have
been developed for their syntheses [10]. The interest of scien-
tists in such compounds has been stimulated by their various
promising pharmacological properties [11]. 2-Pyrazoline deriv-
atives have been reported to exhibit various pharmacological
activities such as antibacterial, antifungal, antimicrobial and an-
tidepressant [12e16]. In continuation to our ongoing research
work on pyrazoline derivatives [17], we describe herein the syn-
thesis, spectral studies and in vitro antiamoebic activity of new
1-N-substituted-2-pyrazoline derivatives against HM1:IMSS
strain of E. histolytica.
Amoebiasis is a protozoan infection caused by intestinal
parasite Entamoeba histolytica. The prevalence of amoebic co-
litis and liver abscess is greater in developing regions such as
Central and South America than in the industrialized world. It
is the third most common cause of death from parasitic dis-
eases after malaria and schistosomiasis. It is estimated that
40e50 million cases of amoebic colitis and liver abscess
due to E. histolytica occur worldwide and result in 100 000
deaths [1e3]. Amoebic abscesses of the brain are a dreadful
complication of E. histolytica infection [4]. The cornerstones
of amoebic liver abscess treatment are nitro imidazoles such
as metronidazole. However, metronidazole is mutagenic and
has been associated with serious side effects and some E. his-
tolytica strains resistant to this drug have also begun to appear
* 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 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.032

418
M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
2. Chemistry
The compounds were stable in the solid as well as in the solu-
tion state. Elemental analysis and spectral data were in agree-
ment with the structure of the synthesized compounds. The
compounds were insoluble in water but soluble in chloroform,
methanol and most of the organic solvents.
The aforementioned compounds were prepared according
to the synthetic sequences illustrated in Scheme 1. The Man-
nich bases were synthesized by the reaction of respective
ketones with paraformaldehyde and dimethylamine hydro-
chloride in a mixture of ethanol and conc. HCl [18]. High
yields were obtained when a minimum amount of ethanol
and 2 mL of acid/mmol of ketone are added in the Mannich
reaction. The methyl phenyl ketone gave high yields of above
80%, while the yields for substituted acetophenone in the
Mannich reaction were lower in the range of 40e60%. The
synthesis of 1-N-substituted thiocarbamoyl-3-phenyl-2-
pyrazolines (1e30) was carried out by reacting Mannich bases
with N4-substituted thiosemicarbazides [19]. The formation of
pyrazoline derivatives is favored via thiosemicarbazone
formation, which undergoes cyclization under basic conditions
to form the desired pyrazoline ring in all the compounds
[20,21]. The purity of the compounds was established by
thin layer chromatography (TLC) and elemental analyses.
The interest in the IR spectra of the compounds lies mainly
in the bands due to NHeC]S, C]N and CeN functional
groups. The compounds 16e30 may exist in thioneethiol
tautomerism since they contain a thioamide (eNHeC]S)
functional group. The IR spectra of these compounds indicate
that they retain their thione form in the solid state, as they
showed a strong band at 1023e1100 cmꢀ1 due to n(C]S)
stretching vibrations. A strong band appeared at 1516e
1605 cmꢀ1 was assigned to n(C]N) because of ring closure
[22]. In addition, the absorption bands at 1112e1262 cmꢀ1
were attributed to the n(CeN) stretching vibrations, which
also confirm the formation of desired pyrazoline ring in all
the compounds. The compounds 16e30 showed an additional
sharp band in the region 3214e3446 cmꢀ1 due to the n(NH)
stretch. The electronic spectra of all the compounds studied
S
KOH
HR
S
+
C
+
(HCHO)n
CH3
+
C
O
X
ClCH2COONa
S
NaOOCCH2
HOOCCH2
C
S
R
Ethanol, HCl
(CH3)2NH2Cl
HCl
S
C
S
R
HCl
N
CH3
CH3
C
X
NH2NH2.H2O
S
O
NH2-NH
C
R
Methanol, NaOH
N
R
N
S
X
Compounds 1-30
X=H, Br, Cl
R= Aromatic and Cyclic amines
Scheme 1.

M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
419
in the UV region, exhibited three absorption bands at 382e
284 cmꢀ1, 267e243 cmꢀ1 and 237e203 cmꢀ1 assignable to
n / p*, p / p* and n / s* transitions, respectively. The
band at 382e284 cmꢀ1 is assigned to the n / p* transition
involving the thione portion (C]S) of thiocarbamoyl group.
The two other absorption bands at 267e243 cmꢀ1 and
237e203 cmꢀ1 were due to p / p* transition of phenyl
ring and n / s* transition of azomethine nitrogen, respec-
tively. The 1H NMR spectra showed two broad triplets at
3.13e3.93 ppm (J ¼ 6.66e10.71 Hz) and 4.29e4.61 ppm
(J ¼ 6.23e12.00 Hz) due to pyrazoline protons at C4 and C5
carbons, respectively. The strong deshielding of the C5 protons
compared with the C4 protons of the pyrazoline ring can be
assumed due to its structure (Fig. 1).
The compounds 16e30 showed an additional singlet at
8.04e9.12 ppm due to NH proton of thiocarbamoyl group.
The protons belonging to the aromatic ring and the other
cyclic groups were observed with the expected chemical shift
and integral values. The C4 and C5 carbons of the pyrazoline
ring in the 13C NMR spectra showed two signals at 44.9e
52.1 ppm and 72.1e78.7 ppm. All the compounds showed
a signal at 154.2e159.7 ppm, which was assigned to the azo-
methine carbon of pyrazoline ring. Thiocarbamoyl carbon
(C]S) displayed a signal at 174.9e183.6 ppm in all the
compounds. The signals from 138.6 ppm to 120.1 ppm were
assumed to be due to the aromatic carbons. The carbons at
1-N-substituted cyclic groups resonate at their usual positions
and are shown in the data given in Section 4. The characteris-
tic peaks were observed in the mass spectra of pyrazoline de-
rivatives. Their mass spectra exhibit molecular ion peaks and
contain fragments that confirm the pyrazoline ring structure
in all the compounds. The spectra of all the compounds
suggest that the pyrazoline derivatives remain in thioneethiol
tautomerization. The fragmentation of compound 17 showed
an M ꢀ SH ion at m/z 342 due to the removal of thiol (SH)
functionality of the compound. This is followed by elimination
of CH3 radical to give substituted pyrazoline ion at m/z 329 as
the base peak. The fragmentation of the molecular ion may
also occur by elimination of CH3 radical followed by removal
of SH radical to give ion peak at m/z 329. The removal of CN
radical produced disubstituted pyrazoline ion at m/z 303. The
removal of phenyl radical produces ion peak at m/z 226. The
peak at m/z 182 was also observed due to m-bromo phenyl
cyanide ion after the removal of azirinium radical (m/z 42).
The fragmentation of compound 18 also shows the same
fragmentation pattern. It shows an M ꢀ SH ion at m/z 299
due to the removal of thiol (SH) functionality of the
compound. This is followed by elimination of CH3 radical
to give substituted pyrazoline ion at m/z 284 as the base
peak. The fragmentation of the molecular ion may also occur
by elimination of CH3 radical followed by removal of SH
radical to give ion peak at m/z 284. The removal of CN
radical produced disubstituted pyrazoline ion at m/z 261.
The removal of phenyl radical produces ion peak at m/z
185. The peak at m/z 141 was also observed due to m-chloro
phenyl cyanide ion.
3. Antiamoebic activity
All new pyrazoline derivatives were screened for antia-
moebic activity against HM1:IMSS strain of E. histolytica
using a microdilution method [23,24]. Three classes of
1-N-substituted thiocarbamoyl-3-phenyl-2-pyrazolines were
synthesized, the compounds having unsubstituted phenyl
ring, 3-bromo or 3-chloro substituents on the phenyl ring at
position 3 of pyrazoline ring and 1-N-thiocarbamoyl group
substituted with different cyclic and aromatic amines to estab-
lish the structureeactivity relationship (SAR). The IC50 values
in micromolar are shown in Table 1. The results were esti-
mated as the percentage of 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 interpolated in the corresponding doseeresponse curve.
Metronidazole had a 50% inhibitory concentration (IC50
1.68e1.81 mM) in our experiments. The pyrazoline derivatives
with unsubstituted phenyl ring showed IC50 in the range of
9.56e1.76 mM. The compounds 25 and 28 in this series
showed better antiamoebic activity (IC50 ¼ 1.76 mM for 25,
IC50 ¼ 1.79 mM for 28, versus IC50 ¼ 1.81 mM for metronida-
zole). The cyclized pyrazolines with bromo substituted phenyl
ring showed IC50 in the range of 5.33e0.58 mM. The com-
pounds 5, 26 and 29 in this series were found to be better
inhibitors of E. histolytica (IC50 ¼ 1.09 mM for 5, IC50
¼
0.67 mM for 26, IC50 ¼ 0.58 mM for 29 versus IC50 ¼ 1.68 mM
for metronidazole). Among all the chloro derivatives, the most
active compounds in this class were those pyrazoline deriva-
tives, which have hexamethyline imine (6, IC50 ¼ 0.89 mM),
tetrahydro quinoline (15, IC50 ¼ 1.77 mM), o-toluidine (18,
IC50 ¼ 1.68 mM), 2,4-difluoro aniline (27, IC50 ¼ 0.51 mM)
and adamantyl amine (30, IC50 ¼ 0.47 mM) as 1-N-substitution.
All the 3-bromo and 3-chloro substituted cyclized pyrazoline
derivatives were found to be more active than their respective
unsubstituted analogues. It was concluded that the presence of
bulky groups at position 1-N of thiocarbamoyl group and 3-
bromo or 3-chloro substituents on the phenyl ring at position
3 of pyrazoline ring greatly enhanced the antiamoebic activ-
ity. The results were statistically evaluated by analysis of
variance. The null hypothesis was tested using t-test. The
significance of the difference between the IC50 values of met-
ronidazole and the most active compounds was evaluated by
t-test. The values of the calculated t were found to be higher
than the table value of t at 5% level, thus concluding that the
character under study is said to be significantly influenced by
the treatment.
3
4
X
2 N
5
N1
R
S
Fig. 1.

420
M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
Table 1
In vitro antiamoebic activities of 1-N-substituted thiocarbamoyl-3-phenyl-2-
pyrazolines (1e30) against (HM1:IMSS ) strain of E. histolytica
apparatus and are uncorrected. The ESI mass spectra of a few
representative compounds were recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer. Electronic
spectra were recorded in methanol on a Shimadzu UV-1601
PC UVeVisible Spectrophotometer. IR spectra on KBr disks
were recorded on a Perkin Elmer model 1620 FT-IR spectro-
N
R
N
S
1
photometer. H and 13C NMR (300 MHz) spectra were ob-
X
tained at ambient temperature using a Bruker spectrospin
DPX-300 MHz spectrophotometer in CDCl3 using tetrame-
thylsilane as an internal standard. Chemical shifts are ex-
pressed as parts per million from tetramethylsilane. Splitting
patterns are designated as follows: s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet). Coupling constants (J
values) are given in hertz (Hz). The reactions were monitored
on Merck pre-coated aluminium TLC plates 60F254 and the
products were visualized by exposure to UV light. Merck sil-
ica gel (230e400 mesh) was used for column chromatography.
Elemental analysis (C, H, N) was carried out by Central Drug
Research Institute, Lucknow, India, and the results were within
0.4% of the theoretical values.
Compound
X
R
IC50 (mM)
S.D.a
1
2
3
H
3.73
2.82
2.31
0.15
0.25
0.10
Br
Cl
N
N
4
5
6
H
4.39
1.09
0.89
0.55
0.23
0.17
Br
Cl
7
8
9
H
5.94
5.25
3.71
0.31
0.25
0.19
Br
Cl
N
CH3
10
11
12
H
7.31
4.44
2.91
0.89
0.57
0.15
CH2
CH3
Br
Cl
N
4.1. Synthesis of Mannich base: a general method
13
14
15
H
6.19
2.78
1.77
0.90
0.33
0.53
A suspension of ketone (0.2 mol), dimethylamine hydro-
chloride (0.26 mol) and paraformaldehyde (0.26 mol) in a mix-
ture of 35 mL of ethanol and 0.5 mL of conc. HCl was
refluxed for 2 h. After cooling, 200 mL of acetone was added.
The crystals formed were collected, washed with acetone and
dried in vacuo.
Br
Cl
N
CH3
HN
16
17
18
H
4.78
3.82
1.68
0.53
0.76
0.27
Br
Cl
4.2. Synthesis of 1-N-substituted thiocarbamoyl-3-
phenyl-2-pyrazolines (1e30): a general method
19
20
21
H
5.01
3.35
2.81
0.35
0.23
0.56
Br
Cl
HN
CH3
CH3
Thiosemicarbazide (0.5 mmol) was dissolved in methanol
(5 mL) by refluxing under nitrogen. NaOH/H2O (0.18 mL,
1:2 w/v) was added to the reaction mixture. The Mannich
base (0.5 mmol) in methanol (5 mL) was added drop wise to
the reaction mixture and refluxed for 48e72 h. The reflux
time was dependent upon the thiosemicarbazide taken. Metha-
nol was removed in vacuo. The residue was dissolved in
dichloromethane, washed with water and dried over anhydrous
Na2SO4. The residual oil was purified via column chro-
matography on silica gel 60F254 eluted with dichloromethane:-
methanol (98:2) and crystallized using appropriate solvent
(chloroform or methanol).
22
23
24
H
9.56
5.33
2.40
1.10
0.75
0.45
Br
Cl
HN
F
25
26
27
H
1.76
0.67
0.51
0.24
0.11
0.10
Br
Cl
HN-
F
28
29
30
H
1.79
0.58
0.47
0.57
0.12
0.11
Br
Cl
N
H
Metronidazole
1.80
0.27
4.2.1. (3-Phenyl-4,5-dihydro-1H-pyrazol-1-yl)
(pyrrolidin-1-yl)methanethione (1)
a
Standard deviation.
Cream solid. Yield: 22%; mp: 105 ꢁC. Anal. Calcd. for
C14H17N3S: C, 64.86; H, 6.56; N, 16.22; found: C, 64.89; H,
6.54; N, 16.24%; lmax (MeOH)/(nm): 364, 321, 236, 204;
4. Experimental
1
nmax (cmꢀ1): 1541 (C]N), 1193 (CeN), 1033 (C]S); H
The Mannich base and thiosemicarbazides substituted by
different cyclic and aromatic amines were prepared using
a two-step synthetic route as reported earlier [18,19]. All the
chemicals were purchased from Aldrich Chemical Company
(USA). Melting points were recorded on a KSW melting point
NMR (CDCl3): (d, ppm) 7.18e7.63 (5H, m, Ph), 4.47
(2H, t, J ¼ 8.70 Hz, CH2), 3.34 (2H, t, J ¼ 9.32 Hz, CH2),
2.67e3.02 (4H, m, CH2), 1.53e2.05 (4H, m, CH2); 13C
NMR (CDCl3): (d, ppm) 178.2 (C]S), 156.1 (C]N),
133.7, 131.1, 128.4, 126.1, 123.3, 122.4 (Aryl-C), 78.4

M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
421
(CH2), 48.9 (CH2), 42.2 (2CH2), 31.3 (2CH2); m/z (ESI):
4.2.6. Azepan-1-yl(3-(3-chlorophenyl)-4,5-
260.5 (M þ 1).
dihydro-1H-pyrazol-1-yl)methanethione (6)
Dark brown solid. Yield: 16%; mp: 110 ꢁC. Anal. Calcd.
for C16H20N3SCl: C, 59.72; H, 6.22; N, 13.06; found: C,
59.79; H, 6.24; N, 13.1%; lmax (MeOH)/(cmꢀ1): 328.6,
246.1, 206.4; nmax (cmꢀ1): 1572 (C]N), 1169 (CeN), 1065
4.2.2. (3-(3-Bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
(pyrrolidin-1-yl)methanethione (2)
Yellow solid. Yield: 19%; mp: 119 ꢁC. Anal. Calcd. for
C14H16N3SBr: C, 49.70; H, 4.73; N, 12.43; found: C, 49.68;
H, 4.71; N, 12.46%; lmax (MeOH)/(nm): 371, 292, 243, 233,
221, 214; nmax (cmꢀ1): 1561 (C]N), 1189 (CeN), 1069
1
(C]S); H NMR (CDCl3): (d, ppm) 7.19e7.87 (4H, m, Ar),
4.43 (2H, t, J ¼ 6.23 Hz, CH2), 3.33 (2H, t, 2H, J ¼ 8.57 Hz,
CH2), 1.49e2.97 (12H, m, CH2); 13C NMR (CDCl3):
(d, ppm) 182.4 (C]S), 155.2 (C]N), 133.1, 131.4, 129.7,
127.3, 125.5, 121.8 (Aryl-C), 77.2 (CH2), 51.4 (CH2), 44.8
(2CH2), 33.3 (2CH2), 25.4 (2CH2).
1
(C]S); H NMR (CDCl3): (d, ppm) 7.18e7.97 (4H, m, Ar),
4.45 (2H, t, J ¼ 10.4 Hz, CH2), 3.37 (2H, t, J ¼ 7.5 Hz,
CH2), 2.49e2.88 (4H, m, CH2), 1.58e2.17 (4H, m, CH2);
13C NMR (CDCl3): (d, ppm) 179.2 (C]S), 155.7 (C]N),
135.1, 133.3, 129.2, 126.1, 124.8, 121.1 (Aryl-C), 78.3
(CH2), 48.4 (CH2), 39.7 (2CH2), 29.5 (2CH2); m/z (ESI):
339.5 (M þ 1).
4.2.7. N-cyclohexyl-N-methyl-3-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (7)
Bright yellow solid. Yield: 15%; mp: 127 ꢁC. Anal. Calcd.
for C17H23N3S: C, 67.77; H, 7.64; N, 13.95; found: C, 67.79;
H, 7.62; N, 13.97%; lmax (MeOH)/(nm): 374, 321, 206, 204;
1
4.2.3. (3-(3-Chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
(pyrrolidin-1-yl)methanethione (3)
nmax (cmꢀ1): 1568 (C]N), 1128 (CeN), 1069 (C]S); H
NMR (CDCl3): (d, ppm) 7.21e7.68 (5H, m, Ph), 4.41 (2H,
t, J ¼ 10.0 Hz, CH2), 3.13 (2H, t, J ¼ 8.33 Hz, CH2),
2.90e3.07 (1H, m, CH ), 1.62e2.04 (10H, m, CH2), 1.56
(3H, s, CH3); 13C NMR (CDCl3): (d, ppm) 179.3 (C]S),
156.2 (C]N), 133.2, 131.9, 128.9, 126.5, 124.4, 121.1
(Aryl-C), 74.5 (CH2), 54.2 (CH), 47.9 (CH2), 34.5 (2CH2),
26.3 (2CH2), 22.6 (CH2), 20.7 (CH3); m/z (ESI): 301.6
(M þ 1).
Brown solid. Yield: 15%; mp: 129 ꢁC. Anal. Calcd. for
C14H16N3SBr: C, 57.24; H, 5.45; N, 14.31; found: C, 57.29;
H, 5.39; N, 14.35%; lmax (MeOH)/(nm): 371, 291.5, 249,
234, 224; nmax (cmꢀ1): 1569 (C]N), 1191 (CeN), 1077
1
(C]S); H NMR (CDCl3): (d, ppm) 7.22e7.91 (4H, m, Ar),
4.41 (2H, t, J ¼ 8.13 Hz, CH2), 3.33 (2H, t, J ¼ 9.50 Hz,
CH2), 2.41e2.97 (4H, m, CH2), 1.61e2.25 (4H, m, CH2);
13C NMR (CDCl3): (d, ppm) 177.4 (C]S), 154.5 (C]N),
134.2, 133.8, 131.7, 129.4, 126.2, 122.2 (Aryl-C), 76.1
(CH2), 48.1 (CH2), 40.2 (2CH2), 32.4 (2CH2).
4.2.8. 3-(3-Bromophenyl)-N-cyclohexyl-N-
methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (8)
Dark brown solid. Yield: 13%; mp: 151 ꢁC. Anal. Calcd.
for C17H22N3SBr: C, 53.68; H, 5.79; N, 11.05; found: C,
53.45; H, 5.81; N, 11.11%; lmax (MeOH)/(nm): 382, 371,
351, 290, 251; nmax (cmꢀ1): 1560 (C]N), 1190 (CeN),
1037 (C]S); 1H NMR (CDCl3): (d, ppm) 7.19e7.97
(4H, m, Ar), 4.33 (2H, t, J ¼ 8.57 Hz, CH2), 3.16 (2H, t,
J ¼ 8.57 Hz, CH2), 3.36e3.56 (1H, m, CH ), 1.68e2.12
(10H, m, CH2), 1.57 (3H, s, CH3); 13C NMR (CDCl3):
(d, ppm) 181.2 (C]S), 159.4 (C]N), 136.7, 133.2, 129.7,
127.4, 125.2, 122.4 (Aryl-C), 76.3 (CH2), 55.3 (CH), 47.3
(CH2), 33.2 (2CH2), 27.3 (2CH2), 23.2 (CH2), 20.9 (CH3);
m/z (ESI): 380.7 (Mþ).
4.2.4. Azepan-1-yl(3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)
methanethione (4)
Brown solid. Yield: 18%; mp: 106 ꢁC. Anal. Calcd. for
C16H21N3S: C, 66.89; H, 7.32; N, 14.63; found: C, 66.95; H,
7.31; N, 14.59%; lmax (MeOH)/(nm): 382, 377, 322, 243,
203; nmax (cmꢀ1): 1540 (C]N), 1112 (CeN), 1076 (C]S);
1H NMR (CDCl3): (d, ppm) 7.21e7.78 (5H, m, Ph), 4.43
(2H, t, J ¼ 8.75 Hz, CH2), 3.93 (2H, t, J ¼ 7.50 Hz, CH2),
1.57e3.15 (12H, m, CH2); 13C NMR (CDCl3): (d, ppm)
179.7 (C]S), 156.9 (C]N), 133.3, 130.5, 127.1, 125.3,
123.5, 121.2 (Aryl-C), 76.6 (CH2), 48.3 (CH2), 42.7 (2CH2),
33.1 (2CH2), 24.8 (2CH2); m/z (ESI): 288 (M þ 1).
4.2.9. 3-(3-Chlorophenyl)-N-cyclohexyl-N-
methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (9)
Brown solid. Yield: 12%; mp: 131 ꢁC. Anal. Calcd. for
C17H22N3SCl: C, 60.80; H, 6.56; N, 12.52; found: C, 60.75;
H, 6.49; N, 12.61%; lmax (MeOH)/(cmꢀ1): 372.5, 318.2,
245.6, 213.3; nmax (cmꢀ1): 1584 (C]N), 1179 (CeN), 1067
4.2.5. Azepan-1-yl(3-(3-bromophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)methanethione (5)
Yellow solid. Yield: 13%; mp: 169 ꢁC. Anal. Calcd. for
C16H20N3SBr: C, 52.46; H, 5.46; N, 11.48; found: C, 52.44;
H, 5.49; N, 11.45%; lmax (MeOH)/(nm): 371, 288, 247, 234,
224, 214; nmax (cmꢀ1): 1562 (C]N), 1140 (CeN), 1067
1
(C]S); H NMR (CDCl3): (d, ppm) 7.19e7.91 (4H, m, Ar),
4.47 (2H, t, J ¼ 8.07 Hz, CH2), 3.31 (2H, t, J ¼ 9.23 Hz,
CH2), 3.47e3.54 (1H, m, CH ), 1.43e2.09 (10H, m, CH2),
1.63 (3H, s, CH3); 13C NMR (CDCl3): (d, ppm) 182.3
(C]S), 158.4 (C]N), 136.3, 133.8, 131.2, 128.9, 125.2,
121.4 (Aryl-C), 77.2 (CH2), 52.4 (CH), 48.3 (CH2), 32.2
(2CH2), 26.6 (2CH2), 22.4 (CH2), 19.3 (CH3); m/z (ESI):
336.5 (M þ 1).
1
(C]S); H NMR (CDCl3): (d, ppm) 7.26e7.95 (4H, m, Ar),
4.40 (2H, t, J ¼ 8.03 Hz, CH2), 3.27 (2H, t, J ¼ 10.71 Hz,
CH2), 1.56e2.92 (12H, m, CH2); 13C NMR (CDCl3): (d,
ppm) 180.2 (C]S), 157.7 (C]N), 137.2, 135.7, 131.8,
129.4, 126.1, 124.7 (Aryl-C), 75.4 (CH2), 52.1 (CH2), 45.5
(2CH2), 35.7 (2CH2), 27.8 (2CH2); m/z (ESI): 366.3 (Mþ).

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M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
4.2.10. N-Benzyl-N-methyl-3-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (10)
249, 243, 237, 223; nmax (cmꢀ1): 1582 (C]N), 1172 (Ce
N), 1045 (C]S); 1H NMR (CDCl3): (d, ppm) 7.22e7.99
(8H, m, Aryl), 4.33 (2H, t, J ¼ 7.79 Hz, CH2), 3.16 (2H, t,
J ¼ 8.17 Hz, CH2), 1.68e3.19 (6H, m, CH2); 13C NMR
(CDCl3): (d, ppm) 181.2 (C]S), 159.4 (C]N), 135.2,
133.8, 131.2, 129.7, 125.2e122.4 (Aryl-C), 76.3 (CH2), 55.3
(CH2), 47.3 (CH2), 33.2 (CH2), 27.3 (CH2).
Creamish yellow solid. Yield: 17%; mp: 96 ꢁC. Anal.
Calcd. for C18H19N3S: C, 69.90; H, 6.15; N, 13.59; found:
C, 69.85; H, 6.19; N, 13.54%; lmax (MeOH)/(nm): 374, 231,
208; nmax (cmꢀ1): 1560 (C]N), 1146 (CeN), 1074 (C]S);
1H NMR (CDCl3): (d, ppm) 7.21e7.79 (10H, m, Ph), 4.47
(2H, t, J ¼ 6.8 Hz, CH2), 3.13 (2H, t, J ¼ 6.8 Hz, CH2), 2.61
(2H, s, CH2), 1.58 (3H, s, CH3); 13C NMR (CDCl3): (d,
ppm) 178.4 (C]S), 155.3 (C]N), 133.3, 131.7, 130.4,
129.6, 125.2e120.8 (Aryl-C), 76.9 (CH2), 46.7 (CH2), 38.4
(CH2), 19.6 (CH3).
4.2.15. (3-(3-Chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
(3,4-dihydroquinolin-1(2H )-yl)methanethione (15)
Yellow solid. Yield: 12%; mp: 184 ꢁC. Anal. Calcd. for
C19H18N3SCl: C, 64.13; H, 5.06; N, 11.81; found: C, 64.21;
H, 5.01; N, 11.85%; lmax (MeOH)/(nm): 371, 293.5, 246,
232, 215; nmax (cmꢀ1): 1541 (C]N), 1123 (CeN), 1079
(C]S); 1H NMR (CDCl3): (d, ppm) 7.19e7.91 (8H, m,
Aryl), 4.47 (2H, t, J ¼ 9.14 Hz, CH2), 3.31 (2H, t,
J ¼ 8.79 Hz, CH2), 1.89e3.23 (6H, m, CH2); 13C NMR
(CDCl3): (d, ppm) 178.3 (C]S), 156.4 (C]N), 136.3, 133.8,
128.9, 125.2e121.4 (Aryl-C), 77.2 (CH2), 52.4 (CH2), 48.3
(CH2), 33.7 (CH2), 29.1 (CH2); m/z (ESI): 355.2 (Mþ).
4.2.11. N-Benzyl-3-(3-bromophenyl)-N-methyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (11)
Dark brown solid. Yield: 15%; mp: 179 ꢁC. Anal. Calcd.
for C18H18N3SBr: C, 55.67; H, 4.64; N, 10.82; found: C,
55.59; H, 4.61; N, 10.85%; lmax (MeOH)/(nm): 371, 291,
250, 230, 221; nmax (cmꢀ1): 1549 (C]N), 1169 (CeN),
1
1078 (C]S); H NMR (CDCl3): (d, ppm) 7.19e8.01 (9H,
m, Ar), 4.37 (2H, t, J ¼ 7.79 Hz, CH2), 3.25 (2H, t,
J ¼ 8.57 Hz, CH2), 2.93 (2H, s, CH2), 1.96 (3H, s, CH3);
13C NMR (CDCl3): (d, ppm) 181.6 (C]S), 159.3 (C]N),
135.4, 133.9, 131.2, 127.6, 125.8e121.1(Aryl-C), 72.1
(CH2), 49.7 (CH2), 32.6 (CH2), 18.5 (CH3).
4.2.16. 3-Phenyl-N-o-tolyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide (16)
Reddish brown solid. Yield: 18%; mp: 124 ꢁC. Anal. Calcd.
for C17H17N3S: C, 69.15; H, 5.76; N, 14.24; found: C, 69.11;
H, 5.79; N, 14.21%; lmax (MeOH)/(nm): 374, 325, 246, 213.5;
nmax (cmꢀ1): 3322 (NH), 1574 (C]N), 1169 (CeN), 1078
(C]S); 1H NMR (CDCl3): (d, ppm) 8.84 (1H, s, NH ),
7.16e7.99 (9H, m, Ph), 4.50 (2H, t, J ¼ 6.66 Hz, CH2), 3.39
(2H, t, J ¼ 6.66 Hz, CH2), 2.36 (3H, s, CH3); 13C NMR
(CDCl3): (d, ppm) 179.1 (C]S), 155.9 (C]N), 133.3,
130.5, 128.7, 126.2e122.6 (Aryl-C), 77.4 (CH2), 48.2
(CH2), 24.9 (CH3).
4.2.12. N-Benzyl-3-(3-chlorophenyl)-N-methyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (12)
Light brown solid. Yield: 14%; mp: 148 ꢁC. Anal. Calcd.
for C18H18N3SCl: C, 62.88; H, 5.24; N, 12.23; found: C,
62.85; H, 5.22; N, 12.25%; lmax (MeOH)/(nm): 371, 290,
249.5, 230, 221, 207; nmax (cmꢀ1) 1541 (C]N), 1151
(CeN), 1077 (C]S); 1H NMR (CDCl3): (d, ppm)
7.24e7.97 (9H, m, Ar), 4.29 (2H, t, J ¼ 8.04 Hz, CH2), 3.24
(2H, t, J ¼ 8.17 Hz, CH2), 2.98 (2H, s, CH2), 1.91 (3H, s,
CH3); 13C NMR (CDCl3): (d, ppm) 176.2 (C]S), 157.8
(C]N), 136.9, 134.1, 132.2, 129.7, 126.8e120.6 (Aryl-C),
74.3 (CH2), 48.6 (CH2), 33.6 (CH2), 19.1 (CH3).
4.2.17. 3-(3-Bromophenyl)-N-o-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (17)
Brown solid. Yield: 14%; mp: 160 ꢁC. Anal. Calcd. for
C17H16N3SBr: C, 54.55; H, 4.28; N, 11.23; found: C, 54.61;
H, 4.29; N, 11.21%; lmax (MeOH)/(nm): 374, 362, 213, 220;
nmax (cmꢀ1): 3214 (NH), 1538 (C]N), 1132 (CeN), 1072
(C]S); 1H NMR (CDCl3): (d, ppm) 8.79 (1H, s, NH ),
7.19e7.95 (8H, m, Ar), 4.51 (2H, t, J ¼ 10.71 Hz, CH2),
3.33 (2H, t, J ¼ 10.71 Hz, CH2), 2.46 (3H, s, CH3); 13C
NMR (CDCl3): (d, ppm) 181.4 (C]S), 156.7 (C]N),
136.6, 133.9, 131.2, 130.8, 129.6, 125.4e121.7 (Aryl-C),
78.2 (CH2), 46.8 (CH2), 22.6 (CH3); m/z (ESI): 373, 359,
342, 329, 303, 225, 182.
4.2.13. (3,4-Dihydroquinolin-1(2H )-yl)
(3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanethione (13)
Brown solid. Yield: 12%; mp: 181 ꢁC. Anal. Calcd. for
C19H19N3S: C, 71.03; H, 5.92; N, 13.08; found: C, 71.11; H,
5.97; N, 13.06%; lmax (MeOH)/(nm): 372, 367, 267, 293,
254, 214.5; nmax (cmꢀ1): 1605 (C]N), 1181 (CeN), 1095
1
(C]S); H NMR (CDCl3): (d, ppm) 7.16e7.83 (9H, m, Ph),
4.34 (2H, t, J ¼ 8.17 Hz, CH2), 3.32 (2H, t, J ¼ 8.97 Hz,
CH2), 1.76e3.12 (6H, m, CH2); 13C NMR (CDCl3): (d,
ppm) 178.1 (C]S), 154.2 (C]N), 133.5, 131.9, 129.5,
127.6, 125.5e120.7 (Aryl-C), 78.7 (CH2), 53.2 (CH2), 44.9
(CH2), 34.5 (CH2), 31.7 (CH2).
4.2.18. 3-(3-Chlorophenyl)-N-o-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (18)
White solid. Yield: 11%; mp: 124 ꢁC. Anal. Calcd. for
C17H16N3SCl: C, 61.91; H, 4.85; N, 12.75; found: C, 61.89;
H, 4.84; N, 12.77%; lmax (MeOH)/(nm): 329, 235.4, 209;
nmax (cmꢀ1): 3446 (NH), 1541 (C]N), 1169 (CeN), 1025
(C]S); 1H NMR (CDCl3): (d, ppm) 8.80 (1H, s, NH ),
7.21e7.98 (8H, m, Ar), 4.52 (2H, t, J ¼ 6.87 Hz, CH2), 3.36
(2H, t, J ¼ 7.04 Hz, CH2), 2.36 (3H, s, CH3); 13C NMR
4.2.14. (3-(3-Bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
(3,4-dihydroquinolin-1(2H )-yl)methanethione (14)
Light yellow solid. Yield: 10%; mp: 191 ꢁC. Anal. Calcd.
for C19H18N3SBr: C, 57.00; H, 4.50; N, 10.50; found: C,
56.95; H, 4.49; N, 10.52%; lmax (MeOH)/(nm): 371, 284,

M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
423
(CDCl3): (d, ppm) 177.8 (C]S), 156.1 (C]N), 134.6, 132.1,
130.8, 127.6, 125.4e120.1 (Aryl-C), 76.5 (CH2), 47.6 (CH2),
21.9 (CH3); m/z (ESI): 332, 317, 299, 284, 261, 185, 141.
4.2.23. 3-(3-Bromophenyl)-N-p-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (23)
Light yellow solid. Yield: 12%; mp: 134 ꢁC. Anal. Calcd.
for C17H16N3SBr: C, 54.55; H, 4.28; N, 11.23; found: C,
54.48; H, 4.24; N, 11.25%; lmaxþ(MeOH)/(nm): 371, 294,
250, 229.5, 220; nmax (cmꢀ1): 3365 (NH), 1521 (C]N),
1221 (CeN), 1070 (C]S); 1H NMR (CDCl3): (d, ppm)
8.97 (1H, s, NH ), 7.20e8.06 (8H, m, Ar), 4.40 (2H, t,
J ¼ 11.04 Hz, CH2), 3.33 (2H, t, J ¼ 9.78 Hz, CH2), 2.45
(3H, s, CH3); 13C NMR (CDCl3): (d, ppm) 181.4 (C]S),
156.3 (C]N), 135.4, 133.3, 131.7, 129.6, 126.2e122.5
(Aryl-C), 78.4 (CH2), 46.1 (CH2), 28.6 (CH3); m/z (ESI):
374.3 (Mþ).
4.2.19. 3-Phenyl-N-m-tolyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide (19)
Brown solid. Yield: 20%; mp: 112 ꢁC. Anal. Calcd. for
C17H17N3S: C, 69.15; H, 5.76; N, 14.24; found: C, 69.09; H,
5.71; N, 14.22%; lmax (MeOH)/(nm): 373, 293, 289, 249,
209; nmax (cmꢀ1): 3320 (NH), 1541 (C]N), 1179 (CeN),
1
1075 (C]S); H NMR (CDCl3): (d, ppm) 8.97 (1H, s, NH ),
7.19e7.87 (9H, m, Ph), 4.43 (2H, t, J ¼ 7.07 Hz, CH2), 3.23
(2H, t, J ¼ 9.82 Hz, CH2), 2.38 (3H, s, CH3); 13C NMR
(CDCl3): (d, ppm) 178.3 (C]S), 155.2 (C]N), 133.7,
129.2, 127.6, 125.8e120.5 (Aryl-C), 76.4 (CH2), 48.1
(CH2), 27.3 (CH3).
4.2.24. 3-(3-Chlorophenyl)-N-p-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (24)
Brown solid. Yield: 12%; mp: 161 ꢁC. Anal. Calcd. for
C17H16N3SCl: C, 61.91; H, 4.86; N, 12.75; found: C, 61.95,
H, 4.81; N, 12.71%; lmax (MeOH)/(nm): 371, 351, 291,
248.5; nmax (cmꢀ1): 3369 (NH), 1516 (C]N), 1196 (CeN),
4.2.20. 3-(3-Bromophenyl)-N-m-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (20)
Brown solid. Yield: 17%; mp: 135 ꢁC. Anal. Calcd. for
C17H16N3SBr: C, 54.55; H, 4.28; N, 11.23; found: C, 54.49;
H, 4.26; N, 11.25%; lmax (MeOH)/(nm): 371, 351, 292.5,
249; nmax (cmꢀ1): 3328 (NH), 1543 (C]N), 1179 (CeN),
1
1080 (C]S); H NMR (CDCl3): (d, ppm) 8.10 (1H, s, NH ),
7.22e7.99 (8H, m, Ar), 4.39 (2H, t, J ¼ 9.13 Hz, CH2), 3.29
(2H, t, J ¼ 10.07 Hz, CH2), 2.39 (3H, s, CH3); 13C NMR
(CDCl3): (d, ppm) 180.3 (C]S), 159.7 (C]N), 136.3,
135.2, 131.9, 127.8, 125.8e122.5 (Aryl-C), 78.6 (CH2), 46.5
(CH2), 29.7 (CH3); m/z (ESI): 331.4 (M þ 1).
1
1094 (C]S); H NMR (CDCl3): (d, ppm) 9.01 (1H, s, NH ),
7.19e7.96 (8H, m, Ar), 4.51 (2H, t, J ¼ 12.00 Hz, CH2),
3.36 (2H, t, J ¼ 10.5 Hz, CH2), 2.38 (3H, s, CH3); 13C NMR
(CDCl3): (d, ppm) 182.6 (C]S), 158.3 (C]N), 136.2,
134.8, 133.3, 129.1, 127.3, 125.6e122.7 (Aryl-C), 77.2
(CH2), 46.3 (CH2), 29.5 (CH3); m/z (ESI): 374.2 (Mþ).
4.2.25. N-(2,4-Difluorophenyl)-3-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (25)
Yellow solid. Yield: 14%; mp: 176 ꢁC. Anal. Calcd. for
C16H13N3SF2: C, 60.56; H, 4.10; N, 13.25; found: C, 60.62;
H, 4.14; N, 13.24%; lmax (MeOH)/(cmꢀ1): 345.6, 306.8,
234.6; nmax (cmꢀ1): 3336 (NH), 1554 (C]N), 1184 (CeN),
4.2.21. 3-(3-Chlorophenyl)-N-m-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (21)
Yellow solid. Yield: 13%; mp: 163 ꢁC. Anal. Calcd. for
C17H16N3SCl: C, 61.91; H, 4.85; N, 12.75; found: C, 61.87;
H, 4.79; N, 12.72%; lmax (MeOH)/(nm): 371, 352, 292,
249.5, 243.5; nmax (cmꢀ1): 3314 (NH), 1539 (C]N), 1261
1
1086 (C]S); H NMR (CDCl3): (d, ppm) 8.95 (1H, s, NH ),
7.17e8.20 (8H, m, Ar), 4.49 (2H, t, J ¼ 11.32 Hz, CH2),
3.40 (2H, t, J ¼ 9.90 Hz, CH2); 13C NMR (CDCl3): (d, ppm)
181.4 (C]S), 156.3 (C]N), 137.3, 136.9, 134.5, 130.7,
127.6, 125.4e123.8 (Aryl-C), 76.9 (CH2), 48.7 (CH2).
1
(CeN), 1100 (C]S); H NMR (CDCl3): (d, ppm) 8.04 (1H,
s, NH ), 7.19e7.72 (8H, m, Ar), 4.41 (2H, t, J ¼ 9.87 Hz,
CH2), 3.38 (2H, t, J ¼ 8.53 Hz, CH2), 2.13 (3H, s, CH3); 13C
NMR (CDCl3): (d, ppm) 176.2 (C]S), 157.9 (C]N),
136.2, 133.8, 130.5, 127.6, 125.4e122.7 (Aryl-C), 77.4
(CH2), 49.3 (CH2), 28.8 (CH3).
4.2.26. 3-(3-Bromophenyl)-N-(2,4-difluorophenyl)-
4,5-dihydro-1H-pyrazole-1-carbothioamide (26)
Yellow solid. Yield: 10%; mp: 192 ꢁC. Anal. Calcd. for
C16H12N3SF2Br: C, 48.48; H, 3.03; N, 10.61; found: C,
48.39; H, 3.02; N, 10.56%; lmax (MeOH)/(nm): 371, 352,
291, 246; nmax (cmꢀ1): 3345 (NH), 1560 (C]N), 1170
4.2.22. 3-Phenyl-N-p-tolyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (22)
1
(CeN), 1045 (C]S); H NMR (CDCl3): (d, ppm) 9.31 (1H,
Dark yellow solid. Yield: 24%; mp: 121 ꢁC. Anal. Calcd.
for C17H17N3S: C, 69.15; H, 5.76; N, 14.24; found: C,
69.04; H, 5.69; N, 14.22%; lmax (MeOH)/(nm): 367.5,
321.4, 229, 225, 220, 207; nmax (cmꢀ1): 3344 (NH), 1531
s, NH ), 7.27e8.27 (7H, m, Ar), 4.61 (2H, t, J ¼ 7.17 Hz,
CH2,), 3.38 (2H, t, J ¼ 8.63 Hz, CH2); 13C NMR (CDCl3):
(d, ppm) 182.6 (C]S), 158.2 (C]N), 138.6, 136.5, 133.9,
131.8, 129.7, 126.4e123.6 (Aryl-C), 77.2 (CH2), 46.3
(CH2); m/z (ESI): 396.5 (Mþ).
1
(C]N), 1126 (CeN), 1037 (C]S); H NMR (CDCl3): (d,
ppm) 9.00 (1H, s, NH ), 7.17e7.77 (8H, m, Ar), 4.49 (2H, t,
J ¼ 6.82 Hz, CH2), 3.50 (2H, t, J ¼ 10.23 Hz, CH2), 2.35
(3H, s, CH3); 13C NMR (CDCl3): (d, ppm) 179.6 (C]S),
156.9 (C]N), 136.4, 134.6, 132.9, 129.7, 126.3e123.8
(Aryl-C), 76.1 (CH2), 47.4 (CH2), 27.3 (CH3); m/z (ESI):
296.4 (M þ 1).
4.2.27. 3-(3-Chlorophenyl)-N-(2,4-difluorophenyl)-
4,5-dihydro-1H-pyrazole-1-carbothioamide (27)
Dark brown solid. Yield: 9%; mp: 140 ꢁC. Anal. Calcd. for
C16 H12N3SF2Cl: C, 54.62; H, 3.41; N, 11.95; found: C, 54.67;
H, 3.44; N, 11.91%; lmax (MeOH)/(nm): 382, 371, 347, 292;

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M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
nmax (cmꢀ1): 3445 (NH), 1541 (C]N), 1262 (CeN), 1023
(C]S); 1H NMR (CDCl3): (d, ppm) 9.12 (1H, s, NH ),
7.24e8.14 (7H, m, Ar), 4.55 (2H, t, J ¼ 8.17 Hz, CH2), 3.41
(2H, t, J ¼ 9.89 Hz, CH2); 13C NMR (CDCl3): (d, ppm)
183.6 (C]S), 157.9 (C]N), 136.1, 135.4, 133.9, 130.6,
127.8, 126.1e124.9 (Aryl-C), 76.2 (CH2), 48.3 (CH2).
by microdilution method. E. histolytica trophozoites were
cultured in TYIS-33 growth medium as described previously
in wells of 96-well microtiter plate [23,24]. All the compounds
(ca. 1 mg) were dissolved in DMSO (40 mL) and the stock so-
lutions of the compounds were prepared freshly before use by
adding enough culture medium to obtain a concentration of
1 mg/mL. Dissolution was facilitated by mild sonication in
a sonicleaner bath for a few minutes. As evidenced by a com-
parison of UVevis spectra of the compounds dissolved in
DMSO on one hand, and DMSO þ TYIS-33 medium on the
other hand, the compounds preserve their integrity under bio-
logical conditions. Under these conditions, the compounds are
stable and no inhibition of amoeba occurs [25,26]. Two-fold
serial dilutions were made in the wells of 96-well microtiter
plate (Costar) in 170 mL of medium. Each test included metro-
nidazole as a standard amoebicidal drug, control wells (culture
medium plus amoebae) and a blank (culture medium only).
The control wells were prepared from a confluent culture by
pouring off the medium, adding 2 mL of fresh medium and
chilling the culture on ice to detach the organism from the flask
wall. The number of amoeba per millilitre was estimated with
a heamocytometer, and trypan blue exclusion was used to con-
firm viability. The cell suspension used was diluted to 105 or-
ganism/mL by adding fresh culture medium and 170 mL of this
suspension was added to the test and control wells so that the
wells are completely filled (total volume 340 mL). An inocu-
lum of 1.7 ꢂ 104 organisms/well was chosen so that confluent,
but not excessive growth took place in control wells. The
plates were sealed with expanded polystyrene, secured with
tape, placed in a modular incubation chamber and gassed for
10 min with nitrogen before incubation at 37 ꢁC for 72 h.
After incubation, the growth of amoebae in the plate was
checked with a low power microscope. The culture medium
was removed by inverting the plate and shaking gently. The
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 pre-
vent the detachment of amoebae. The plate was allowed to dry
at room temperature, and the amoebae were fixed with meth-
anol and, when dry, stained with (0.5%) aqueous eosin for
15 min. Stained plate was washed once with tap 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 op-
tical density of the resulting solution in each well was deter-
mined at 490 nm with a microplate reader. The percentage
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 determine the best-fitting straight line
from which the IC50 value was found.
4.2.28. N-(2-Adamantyl)-3-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (28)
Dark yellow solid. Yield: 11%; mp: 163 ꢁC. Anal. Calcd.
for C20H25N3S: C, 70.79; H, 7.37; N, 12.39; found: C,
70.81; H, 7.35; N, 12.41%; lmax (MeOH)/(nm): 374, 368,
248, 228, 207; nmax (cmꢀ1): 3446 (NH), 1540 (C]N), 111.4
1
(CeN), 1077 (C]S); H NMR (CDCl3): (d, ppm) 8.06 (1H,
s, NH ), 7.21e7.89 (5H, m, Ar), 4.67 (1H, t, J ¼ 6.45 Hz,
CH ), 4.39 (2H, t, J ¼ 9.12 Hz, CH2), 3.26 (2H, t,
J ¼ 10.13 Hz, CH2), 1.45e2.07 (14H, m, adaman. ring); 13C
NMR (CDCl3): (d, ppm) 177.8 (C]S), 156.2 (C]N),
133.2, 131.3, 129.8, 127.1, 125.7, 121.4 (Aryl-C), 74.4
(CH2), 44.3 (CH2), 55.30, 52.24, 38.55, 34.21, 32.98, 32.14,
30.86, 31.11, 31.02, 26.111 (adaman. ring).
4.2.29. N-(2-Adamantyl)-3-(3-bromophenyl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (29)
Creamish yellow solid. Yield: 13%; mp: 181 ꢁC. Anal.
Calcd. for C20H24N3SBr: C, 57.42; H, 5.74; N, 10.05; found:
C, 57.45; H, 5.77; N, 10.01%; lmax (MeOH)/(nm): 381, 368,
328, 288, 249, 237, 224, 216; nmax (cmꢀ1): 3369 (NH), 1569
1
(C]N), 1142 (CeN), 1076 (C]S); H NMR (CDCl3): (d,
ppm) 8.14 (1H, s, NH ), 7.23e7.89 (4H, m, Ar), 5.02 (1H, t,
J ¼ 5.16 Hz, CH ), 4.39 (2H, t, J ¼ 8.07 Hz, CH2), 3.21 (2H,
t, J ¼ 9.14 Hz, CH2), 1.36e2.17 (14H, m, adaman. ring);
13C NMR (CDCl3): (d, ppm) 181.3 (C]S), 156.3 (C]N),
136.5, 133.7, 129.2, 126.3, 124.7, 122.9 (Aryl-C), 73.3
(CH2), 49.4 (CH2), 54.13, 52.24, 39.22, 35.18, 32.32, 32.87,
32.22, 31.45, 31.02, 26.11 (adaman. ring).
4.2.30. N-(2-Adamantyl)-3-(3-chlorophenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (30)
Dark yellow solid. Yield: 9%; mp: 161 ꢁC. Anal. Calcd. for
C20H24N3SCl: C, 64.26; H, 6.43; N, 11.24; found: C, 64.24; H,
6.45; N, 11.27%; lmax (MeOH)/(nm): 366, 334, 306, 249, 230;
nmax (cmꢀ1): 3340 (NH), 1565 (C]N), 1179 (CeN), 1070
(C]S); 1H NMR (CDCl3): (d, ppm) 8.11 (1H, s, NH ),
7.19e7.87 (4H, m, Ar), 5.12 (1H, t, J ¼ 5.92 Hz, CH ), 4.33
(2H, t, J ¼ 9.23 Hz, CH2), 3.29 (2H, t, J ¼ 9.87 Hz, CH2),
1.42e2.34 (15H, m, adaman. ring); 13C NMR (CDCl3): (d,
ppm) 174.9 (C]S), 155.2 (C]N), 137.0, 134.9, 132.7,
127.4, 125.2, 123.8 (Aryl-C), 77.4 (CH2), 48.6 (CH2), 56.32,
51.40, 39.75, 35.80, 33.83, 32.57, 31.86, 31.07, 30.09, 27.19
(adaman. ring).
4.3. Organism culture and assessment
of antiamoebic activity
Acknowledgements
This work was supported by Department of Science and
Technology (Grant no. Vll-PRDSF/44/2004-05/TT). The
authors are thankful to Prof. Alok Bhattacharya and Prof.
All the pyrazoline derivatives were screened in vitro for
antiamoebic activity against HM1:IMSS strain of E. histolytica

M. Abid et al. / European Journal of Medicinal Chemistry 44 (2009) 417e425
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