1-N-substituted thiocarbamoyl-3-phenyl-2-pyrazolines: Synthesis and in vitro antiamoebic activities

Article abstract of DOI:10.1016/j.ejmech.2005.03.015

The title compounds were prepared by reaction of Mannich bases with various N-4 substituted thiosemicarbazides. The chemical structures of the compounds were proved by means of their UV, IR, ¹H NMR, ¹³C NMR spectroscopic data and elemental analyses. The in vitro antiamoebic activities of these compounds were evaluated by microdilution method against HM1:IMSS strain of Entamoeba histolytica and compared with the standard drug, metronidazole. It was concluded that 3-chloro and 3-bromo substituents on the phenyl ring at position 3 of the pyrazoline ring enhanced the antiamoebic activity. Compounds 9, 17, 18, 20 and 21 showed less IC₅₀ value than metronidazole.

Full text of DOI:10.1016/j.ejmech.2005.03.015

European Journal of Medicinal Chemistry 40 (2005) 935–942
www.elsevier.com/locate/ejmech
Short Communication
1-N-Substituted Thiocarbamoyl-3-Phenyl-2-Pyrazolines:
Synthesis and In Vitro Antiamoebic Activities
Mohammad Abid, Amir Azam *
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, India
Received 10 December 2004; received in revised form 19 March 2005; accepted 23 March 2005
Available online 25 May 2005
Abstract
The title compounds were prepared by reaction of Mannich bases with various N-4 substituted thiosemicarbazides. The chemical structures
1
13
of the compounds were proved by means of their UV, IR, H NMR, C NMR spectroscopic data and elemental analyses. The in vitro
antiamoebic activities of these compounds were evaluated by microdilution method against HM1:IMSS strain of Entamoeba histolytica and
compared with the standard drug, metronidazole. It was concluded that 3-chloro and 3-bromo substituents on the phenyl ring at position 3 of
the pyrazoline ring enhanced the antiamoebic activity. Compounds 9, 17, 18, 20 and 21 showed less IC50 value than metronidazole.
©
2005 Elsevier SAS. All rights reserved.
Keywords: Pyrazoline; Mannich base; Thiocarbamoyl; Thiosemicarbazide; Entamoeba histolytica
1
. Introduction
various promising pharmacological properties [9]. As evi-
dent from the literature, it was noted that very little research
has been carried out on 1-N-substituted thiocarbamoyl-3-
phenyl-2-pyrazolines [10], but no work has been done on
screening of these compounds against E. histolytica. Earlier
we have reported different heterocyclic thiosemicarbazones,
their transition metal complexes and in vitro screening against
E. histolytica [11–13] and their in vivo and cytotoxicity stud-
ies are in progress. As literature survey reveals the pharma-
cological importance of pyrazolines and their derivatives, this
prompted us to synthesize new 1-N-substituted thiocarba-
moyl-3-phenyl-2-pyrazoline derivatives 1-21 (Table 1) and
their in vitro screening against HM1:IMSS strain of E. his-
tolytica. To the best of our knowledge this is the first report
against E. histolytica.
Parasitic protozoa continue to beleaguer and kill millions
of people in the subtropical and tropical regions of the world.
The amitochondrial protist E. histolytica is estimated to infect
up to 10% of the world’s population. Fifty million cases of
amoebic dysentery and liver abscess are reported each year
[
1]. These infections result in approximately 50,000 to
00,000 deaths annually [2]. Amoebiasis is primarily treated
with the drug metronidazole [1-(2-hydroxyethyl)-2-methyl-
-nitroimidazole], even though significant side effects, such
1
5
as neurological complications, and the possible selection of a
resistant E. histolytica strain have been reported [3–6]. In
addition, treatment failures among patients with amoebiasis
often raise the possibility of drug resistance. Therefore it is
desirable to search for new leads for amoebicidal drugs.
Pyrazoles and their reduced forms pyrazolines are well
known nitrogen containing heterocyclic compounds and vari-
ous procedures have been developed for their syntheses [7].
As a result, a wide variety of pyrazoles and pyrazolines have
hitherto been described in the literature [7,8]. The interest of
scientists in such compounds has been stimulated by their
2. Chemistry
The Mannich reaction of various ketones with formalde-
hyde and dimethylamine hydrochloride generates the Man-
nich base precursor [14]. The reaction is sensitive to both the
amount of hydrochloric acid and ethanol present. The reac-
tion works best when a minimum amount of ethanol and 2 µ
L of acid/mmol ketone is added. The methyl phenyl ketone
gave high yields above 80%, while the yields for 3-bromo
*
Corresponding author. Tel.: 0091 11 26981717 / 3253, fax: 0091 11
2
0
6980229 / 0091-11-26981232.
E-mail address: sumbul@yahoo.co.in (A. Azam).
223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.03.015

936
M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
Table 1
compounds [16,17]. All the compounds were purified by col-
umn chromatography to give crystalline solid compounds,
but in low yield (9-25%). The compounds are stable in the
solid as well as in the solution state. The structure of the com-
pounds was confirmed by IR, H NMR, C NMR, electronic
spectra and elemental analysis. The compounds are insoluble
in water but soluble in methanol, ethanol and DMSO.
Structure of 1-N-substituted thiocarbamoyl-3-phenyl-2-pyrazolines (1-21)
1
13
3
. Pharmacology
All the 1-N-substituted pyrazoline derivatives 1-21 were
screened in vitro for antiamoebic activity against HM1:IMSS
strain of E. histolytica by microdilution method [18]. E. his-
tolytica trophozoites were cultured in TYIS-33 growth
medium as described previously [19] in wells of 96-well
microtiter plate. Each compound tested was serially diluted
and added to the growing trophozoites in microtiter plate.
Effect on growth of trophozoites was monitored microscopi-
cally at regular interval and quantitative estimation of the drug
action was made by protein estimation. The % inhibition of
amoeba was calculated from the optical densities of the con-
trol and tested wells and was plotted against the logarithm of
concentration of the drug tested. Linear regression analysis
was used to determine the best fitting straight line from which
IC50 value was found.
4
. Results and Discussion
The synthesis of 1-N-substituted 3-phenyl-2-pyrazolines
(1-21) was done by cyclization of Mannich bases with vari-
ous N-4 substituted thiosemicarbazides in methanol under
basic conditions. The product mixture contained only unre-
acted Mannich base and the cyclization product, which was
purified by column chromatography using silica gel 60F254
eluted with dichloromethane: methanol (98:2). The yield of
cyclised product in unsubstituted thiosemicarbazide was in
the range of 50-70%; and 9-25% in the case of substituted
thiosemicarbazides. Analytical data of the compounds are in
good agreement with their composition. The structure of the
compounds were established by means of their spectral data
and 3-chloro acetophenones in the Mannich reaction were
lower in the range of 40-60%.All the thiosemicarbazides were
prepared by the method reported by O’Sullivan [15]. The con-
densation of Mannich reaction product with N-4 substituted
thiosemicarbazides by different cyclic amines lead to the for-
mation of 1-N-substituted thiocarbamoyl 3-phenyl-2-
pyrazolines 1-21 (Scheme 1). According to the currently
accepted mechanism, the formation of 1-N-substituted
thiocarbamoyl-3-phenyl-2-pyrazolines is favored via thi-
osemicarbazone formation, which undergo cyclization under
basic conditions to form desired pyrazoline ring in all the
1
13
(IR, UV, H NMR, C NMR) and elemental analyses and
are presented in Table 1.
Scheme 1. (i) Ethanol, hydrochloric acid, reflux; (ii) Methanol, NaOH, reflux.

M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
937
4
.1. IR and electronic spectral studies
zoline ring resonate at 43.8-49.6 and 73.5-78.5 ppm respec-
tively.All the compounds showed a signal at 154.5-159.3 ppm
due to the azomethine carbon of pyrazoline ring. Thiocarbam-
oyl carbon (C=S) displayed a signal at 174.1-182.5 ppm in
all the compounds. The signals from 136.5 to 120.1 ppm were
assumed due to the aromatic carbons. The carbons at N-1 sub-
stituted cyclic groups resonate at their usual positions and are
shown in the data given in the experimental section.
Selected diagnostic bands of the IR spectra of the 1-N-
substituted 3-phenyl-2-pyrazolines (1-21) showed useful
information about the structure of the compounds. All the
compounds showed intense bands in the region 1001-
–
1
1
113 cm due to the m(C=S) stretch of the thiocarbamoyl
group. The IR spectra of all the compounds showed m(C=N)
stretch at 1516-1599 cm because of the ring closure. In addi-
tion, the absorption bands at 1115-1261 cm were attributed
to the m(C-N) stretch vibrations, which also confirm the for-
mation of desired pyrazoline ring in all the compounds. The
compounds (1-12) showed additional sharp bands in the
region 3260-3375 cm due to the m(NH) stretch.
The electronic spectra of all the compounds studied in the
UV region in methanol, exhibited three absorption bands at
–1
–
1
4.3. Anti-amoebic activity
Two considerations governed the selection of compounds
to be prepared as thiosemicarbazone analogues for this study.
It was considered to have representatives of the three classes
of 1-N-substituted 3-phenyl-2-pyrazolines with 3-bromo and
–1
3
-chloro substituents on the phenyl ring at position 3 of pyra-
–
1
–1
–1
zoline ring and 1-N-substituted with different cyclic groups.
These include (1) 1-N- substituted derivatives, represented
by compounds 1-12; and (2) compounds where 1-N is con-
tained in a medium ring, which includes all remaining com-
pounds 13-21. The choices were made to correspond to thi-
osemicarbazones with these bulky groups that displayed a
high level of antiamoebic activity [11–13]. All the com-
pounds were evaluated for antiamoebic activity in vitro using
HM1:IMSS strain of E. histolytica to investigate the influ-
ence of the substitution. All the experiments were carried out
in triplicate at each concentration level and repeated thrice.
The IC50 values in µ M are shown in Table 2. The results
were estimated as the percentage of growth inhibition com-
pared with the untreated controls and plotted as probit values
as a function of the drug concentration. The IC50 and 95%
380.5-294 cm , 288.5-233 cm and 220-205.4 cm assign-
able to n→p*, p→p* and n→r* transitions respectively. The
band at 380.5-294 cm assigned to the n→p* transition
involving the thione portion (C=S) of thiocarbamoyl group.
The two other absorption bands at 288.5-233 cm and 220-
–
1
–1
–
1
205.4 cm were due to p→p* transition of phenyl ring and
n→r* transition of azomethine nitrogen, respectively.
4
.2. Nuclear Magnetic Resonance spectral studies
1
The H NMR spectra were recorded using CDCl as the
3
solvent clearly support the proposed structures of the com-
pounds [8a]. The pyrazoline protons at C and C carbons
4
5
appeared as broad triplets at 3.12-3.56 (J = 7.9-11.9 Hz) and
.03-4.67 (J = 7.9-11.8 Hz) ppm respectively. The strong
deshielding of the C protons compared with the C protons
4
5
4
Table 2
of the pyrazoline ring can be assumed due to its conforma-
tion A [14].
In vitro antiamoebic activities of 1-N-Substituted Thiocarbamoyl-3-Phenyl-
2-Pyrazolines against (HM1:IMSS) strain of E. histolytica
a
Compound
IC50(µM)
6.5
4.2
3.7
5.2
3.1
2.4
2.5
2.0
1.5
7.6
2.6
2.2
4.8
3.6
2.8
3.1
1.6
1.2
2.3
1.4
1.0
1.8
S.D.
0.5
0.3
0.3
0.1
0.1
0.2
0.3
0.1
0.2
0.1
0.2
0.2
0.1
0.1
0.3
0.2
0.1
0.3
0.3
0.1
0.1
0.1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
.
.
.
.
.
.
.
.
.
0.
1.
2.
3.
4.
5.
6.
7.
8.
9.
0.
The NH proton of thiocarbamoyl group of the compounds
1-12) showed an additional singlet at 7.89-8.15 ppm. The
(
protons belonging to the aromatic ring and the other cyclic
groups were observed with the expected chemical shift and
integral values.
1
3
The C NMR spectra of all the compounds were taken in
21.
CDCl and the signals obtained are in good agreement with
Metronidazole
3
a
the proposed structures. The C and C carbons of the pyra-
Standard deviation.
4
5

938
M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
confidence limits were interpolated in the corresponding dose-
response curve. Metronidazole had a 50% inhibitory concen-
tration (IC 1.7-1.8 µ M) in our experiments.All the 3-bromo
and 3- chloro substituted pyrazoline derivatives were found
to be more active than their respective unsubstituted ana-
logues indicated that the substitution of bromine or chlorine
on the phenyl ring at position 3 of pyrazoline ring enhances
the antiamoebic activity. Among all the bromo and chloro
derivatives, the most active compounds in this class were those
plates.All the chemicals were purchased fromAldrich chemi-
cal company (USA). Elemental analyses (C,H,N) were car-
ried out by Central Drug Research Institute, Lucknow, India,
and the results were within 0.4% of the theoretical values.
Melting points were recorded on a KSW melting point appa-
ratus and were uncorrected. Electronic spectra were recorded
in methanol on a Shimadzu UV- 1601 PC UV-Visible spec-
trophotometer. IR spectra on KBr disks were recorded on a
50
1
Perkin Elmer model 1620 FT-IR spectrophotometer. H and
13
1
-N-substituted 3-phenyl-2-pyrazoline derivatives, which have
cyclooctyl amine (9, IC50 = 1.5 µ M), N-phenyl piperizine
17, IC50 = 1.6 µ M), (18, IC50 = 1.2 µ M), N-benzyl piperi-
C NMR spectra were obtained at ambient temperature using
a Bruker Spectrospin DPX-300 MHz spectrophotometer in
(
CDCl using tetramethylsilane as an internal standard. Split-
3
dine (20, IC50 = 1.4 µ M), (21, IC50 = 1.0 µ M) as 1-N-
substitution. The results were statistically evaluated by analy-
sis of variance. The null hypothesis was tested using t-test.
The significativity of the difference between the IC50 values
of metronidazole and the compounds 9, 17, 18, 20 and 21
was evaluated by t-test. The values of the calculated T were
found higher than the Table value of T at 5% level, thus con-
cluding that the character under study is said to be signifi-
cantly influenced by the treatment. It is concluded that the
presence of these bulky groups at position 1-N of thiocarbam-
oyl group and substitution on the phenyl ring at position 3 of
pyrazoline ring with chlorine or bromine greatly enhanced
antiamoebic activity. Also important to note that the substi-
tution of chlorine at 1-N thiocarbamoyl group does not effect
the antiamoebic activity. Detailed studies of the toxicity of
these compounds, mechanism of action as well as in vivo
studies are in progress.
ting patterns are designated as follows; s, singlet; d, doublet;
t, triplet; m, multiplet. Chemical shift values are given in
(ppm) and coupling constants (J) in hertz. The ESI mass spec-
tra of a few representative compounds were recorded on a
MICROMASS QUATTRO II triple quadrupole mass spec-
trometer.
6.2. Synthesis of Mannich bases: A general method
A suspension of ketone (0.2 mol), dimethyl amine hydro-
chloride (0.26 mol) and paraformaldehyde (0.26 mol) in a
mixture of 35 mL of ethanol and 0.5 mL of conc. HCl was
refluxed for 2 hrs.After cooling, 200 mL of acetone was added
.The crystals formed were collected, washed with acetone and
dried in vacuo.
6.2.1. Acetophenone Mannich base (1)
White solid (chloroform). Yield: 87%; m.p.: 153 °C; IR:
–
1
mmax (cm ) 2952 (aliph. C-H), 1679 (C=O), 1468 (arom.
5. Conclusion
1
C=C), 1224 (C-N); H NMR (CDCl ): d 7.21-7.86 (m, 5H,
3
Aryl), 3.29 (t, 2H, -CH ), 2.97 (t, 2H, -CH ), 2.53 (s, 6H,
2
2
Although this class of compound is known since many
-CH3).
years, and even the synthetic procedure for obtaining them
has been recently published [10], very little is known about
their anti-amoebic activity. This research examined the anti-
amoebic activity of the new 1-N-substituted 3-phenyl-2-
pyrazolines (1-21) prepared by reaction of Mannich bases with
thiosemicarbazides substituted by different cyclic amines. In
vitro antiamoebic activity of the compounds was carried out
against HM1:IMSS strain of E. histolytica. The biological
behavior revealed that 3-chloro and 3-bromo substituents on
the phenyl ring at position 3 of the pyrazoline ring increases
the antiamoebic activity. All the bromo and chloro substi-
tuted pyrazoline derivatives show better antiamoebic activity
than their respective unsubstituted analogues. Moreover, the
compounds 9, 17, 18, 20 and 21 were found to be more active
than the reference drug, metronidazole.
6
.2.2. 3-Bromo acetophenone Mannich base (2)
White solid (chloroform). Yield: 55%; m.p.: 176 °C; IR:
–
1
mmax (cm ) 2968 (aliph. C-H), 1689 (C=O), 1470 (arom.
C=C), 1214 (C-N); H NMR (CDCl ): d 7.12-7.95 (m, 4H,
Aryl), 3.31 (t, 2H, -CH ), 2.95 (t, 2H, -CH ), 2.65 (s, 6H,
1
3
2
2
-CH3).
6
.2.3. 3-Chloro acetophenone Mannich base (3)
White solid (chloroform). Yield: 42%; m.p.: 189 °C; IR:
–
1
mmax (cm ) 2967 (aliph. C-H), 1689 (C=O), 1480 (arom.
C=C), 1218 (C-N); H NMR (CDCl ): d 7.17-7.76 (m, 4H,
Aryl), 3.33 (t, 2H, -CH ), 2.92 (t, 2H, -CH ), 2.71 (s, 6H,
1
3
2
2
-CH3).
6
.3. Synthesis of 1-N-substituted
thiocarbamoyl-3-phenyl-2-pyrazoline: A general method
6. Experimental protocols
6.1. Chemistry
Thiosemicarbazide (0.5 mmol) was dissolved in methanol
(
5 mL) upon refluxing under nitrogen. NaOH/H O (0.18 mL,
2
Reactions were monitored by thin-layer chromatography
TLC) using Merck silica gel 60 F254 precoated thin layer
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
(

M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
939
the reaction mixture and refluxed for 48-72 hrs. The metha-
nol was removed in vacuo. The residue was dissolved in
dichloromethane, washed with water and dried over anhy-
drous Na SO . The residual oil was purified via column chro-
3.47-3.63 (m, 1H, -CH), 3.25 (t, 2H, -CH , J = 10.7 Hz), 1.16-
2
1
3
2.26 (m, 10H, -CH2); C NMR(CDCl ): (d, ppm) 179.1
3
(C=S), 155.8 (C=N), 132.7, 130.5, 128.7, 126.2, 123.7, 121.4
(Aryl-C), 77.4 (CH ), 54.2 (CH), 48.2 (CH ), 33.2 (2CH ),
2
4
2
2
2
matography on silica gel 60F254 eluted with 98:2 dichloro-
methane/methanol and crystallized using appropriate solvent.
25.5 (2CH ), 23.8 (CH ); ESI-MS: 288 (M+1).
2 2
6
.3.5. 3-(3-Bromophenyl)-N-cyclohexyl-4,5-dihydro-1H-
6.3.1. N-cyclopentyl-3-phenyl-4,5-dihydro-1H-pyrazole-1-
pyrazole-1-thiocarboxamide (5)
thiocarboxamide (1)
White solid (chloroform).Yield: 16%; m.p.: 179 °C; Anal.
Brown solid (methanol). Yield: 24%; m.p.: 160 °C; Anal.
calc. (C H N S): C, 65.93; H, 6.96; N, 15.38; found: C,
calc. (C H N SBr): C, 52.46; H, 5.46; N, 11.48; found: C,
1
6 20 3
52.49; H, 5.40; N, 11.45; UV: k (nm): 377.4, 240, 220;
1
5
19
3
max
–
1
6
2
1
7
5.89; H, 6.91; N, 15.41%; UV: k (nm): 367, 324.1, 242.5,
IR: m
(cm ) 3297 (NH), 1553 (C=N), 1227 (C-N), 1111
max
(C=S); H NMR (CDCl ): d 7.23-7.88 (m, 4H, Aryl), 8.15 (s,
max
–
1
1
06.2; IR: m
(cm ) 3309 (NH), 1560 (C=N), 1261 (C-N),
max
1
3
098 (C=S); H NMR (CDCl ): d 7.19-7.64 (m, 5H, Aryl),
1H, -NH), 4.35 (t, 2H, -CH , J = 9.0 Hz), 3.36-3.68 (m, 1H,
3
2
.89 (s, 1H, -NH), 4.64 (t, 2H, -CH , J = 9.8 Hz), 3.29-3.60
-CH), 3.24 (t, 2H, -CH J = 9.1 Hz), 1.22-2.01 (m, 10H,
2
2,
1
3
(
8
m, 1H, -CH), 3.17 (t, 2H, -CH J = 9.0 Hz), 1.18-2.53 (m,
-CH2); C NMR (CDCl ): (d, ppm) 181.3 (C=S), 157.3
2,
3
1
3
H, -CH2); C NMR (CDCl ): (d, ppm) 181.2 (C=S), 157.4
(C=N), 136.3, 132.7, 129.7, 126.2, 124.4, 122.2 (Aryl-C), 78.1
(CH ), 54.5 (CH), 47.4 (CH ), 32.8 (2CH ), 25.5 (2CH ),
3
(
(
C=N), 131.7, 130.4, 129.1, 127.9, 125.3, 122.5 (Aryl-C), 74.2
CH ), 52.9 (CH), 45.9 (CH ), 31.9 (2CH ), 26.2 (2CH ).
2
2
2
2
23.8 (CH ); ESI-MS: 367 (M+1).
2
2
2
2
2
6
.3.2. 3-(3-Bromophenyl)-N-cyclopentyl-4,5-dihydro-1H-
6.3.6. 3-(3-Chlorophenyl)-N-cyclohexyl-4,5-dihydro-1H-
pyrazole-1-thiocarboxamide (6)
pyrazole-1-thiocarboxamide(2)
Yellow solid (chloroform: methanol). Yield: 18%; m.p.:
Light yellow solid (chloroform).Yield: 14%; m.p.: 161 °C;
1
1
3
72 °C; Anal. calc. (C H N SBr): C, 51.14; H, 5.11; N,
Anal. calc. for (C H N SCl): C, 59.72; H, 6.22; N, 13.06;
15
18
3
16 20 3
1.93; found: C, 51.07; H, 5.15; N, 11.91%; UV: k
(nm):
found: C, 59.69; H, 6.31; N, 13.17; UV: k
(nm): 370.5,
max
max
–1
–1
68, 241, 220; IR: m (cm ) 3375 (NH), 1522 (C=N), 1192
349.5, 288.5, 249; IR: m
(cm ) 3350 (NH), 1521 (C=N),
max
1
max
1
(C-N), 1070 (C=S); H NMR (CDCl ): d 7.21-7.88 (m, 4H,
1135 (C-N), 1060 (C=S); H NMR (CDCl ): d 7.21-7.97 (m,
3
3
Aryl), 8.10 (s, 1H, -NH), 4.43 (t, 2H, -CH J = 8.7 Hz), 3.63-
4H, Aryl), 8.09 (s, 1H, -NH), 4.37 (t, 2H, -CH , J = 8.1 Hz),
2,
2
3
.98 (m, 1H, -CH), 3.53 (t, 2H, -CH J = 9.1 Hz), 1.27-2.17
3.60-3.82 (m, 1H, -CH), 3.29 (t, 2H, -CH , J = 8.7 Hz), 1.37-
2,
2
1
3
13
(m, 8H, -CH2); C NMR (CDCl ) : (d, ppm) 180.3 (C=S),
2.11 (m, 10H, -CH2); C NMR(CDCl ): (d, ppm) 179.5
3
3
1
58.7 (C=N), 133.2, 131.9, 129.4, 127.1, 125.8, 122.4 (Aryl-
C), 73.8 (CH ), 55.5 (CH), 46.3 (CH ), 31.3 (2CH ), 25.7
(C=S), 156.4 (C=N), 135.7, 133.7, 131.4, 129.7, 125.8, 120.8
(Aryl), 77.5 (CH ), 54.5 (CH), 46.9 (CH ), 31.6 (2CH ), 24.1
2
2
2
2
2
2
(2CH2).
(2CH ), 22.2 (CH ).
2
2
6
.3.3. 3-(3-Chlorophenyl)-N-cyclopentyl-4,5-dihydro-1H-
6.3.7. N-cyclooctyl-3-phenyl-4,5-dihydro-1H-pyrazole-1-
thiocarboxamide (7)
pyrazole-1-thiocarboxamide (3)
Light yellow solid (chloroform).Yield: 15%; m.p.: 149 °C;
Anal. calc. (C H N SCl): C, 58.54; H, 5.85; N, 13.66;
Yellow solid (chloroform).Yield: 17%; m.p.: 128 °C; Anal.
calc. (C H N S): C 68.57, H 7.94, N 13.33; found: C 68.45,
1
5
18
3
18 25 3
found: C, 58.47; H, 5.78; N, 13.69; UV: k (nm): 371, 351,
H, 7.92, N, 13.41; k
(nm): 377.4, 324.7, 245, 207; IR:
max
max
–
1
–1
2
88, 249; IR: mmax (cm ) 3375 (NH), 1516 (C=N), 1196
mmax (cm ) 3375 (NH), 1523 (C=N), 1259 (C-N), 1113
1
1
(
C-N), 1080 (C=S); H NMR (CDCl ): d 7.22-7.99 (m, 4H,
(C=S); H NMR (CDCl ): d 7.16-7.67 (m, 5H, Aryl), 7.92 (s,
3
3
Aryl), 8.10 (s, 1H, -NH), 4.19 (t, 2H, -CH , J = 8.0 Hz), 4.55-
1H, -NH), 4.04 (t, 2H, -CH J = 11.8 Hz), 3.39-3.60 (m, 1H,
2
2,
4
.67 (m, 1H, -CH), 3.29 (t, 2H, -CH , J = 8.2 Hz), 1.57-2.30
-CH), 3.24 (t, 2H, -CH J = 11.8 Hz), 1.25-2.16 (m, 14H,
2
2,
1
3
13
(m, 8H, -CH2); C NMR(CDCl ): (d, ppm) 180.8 (C=S),
-CH2); C NMR (CDCl ): (d, ppm) 180.4 (C=S), 155.5
3
3
1
59.2 (C=N), 135.2, 133.9, 128.2, 126.8, 123.7, 121.5
(C=N), 132.1, 129.4, 127.4, 126.1, 124.5, 122.4 (Aryl-C), 76.6
(CH ), 52.9 (CH), 47.7 (CH ), 33.9 (2CH ), 30.3 (2CH ),
(Aryl-C) 78.5 (CH ), 53.9 (CH), 46.5 (CH ), 29.7 (2CH ),
2
2
2
2
2
2
2
25.3 (2CH2).
24.8 (2CH ), 22.4 (CH ).
2
2
6
.3.4. N-cyclohexyl-3-phenyl-4,5-dihydro-1H-pyrazole-1-
6.3.8. 3-(3-Bromophenyl)-N-cyclooctyl-4,5-dihydro-1H-
pyrazole-1-thiocarboxamide (8)
thiocarboxamide (4)
Cream solid (chloroform: methanol). Yield: 20%; m.p.:
Yellow solid (chloroform: methanol). Yield: 13%; m.p.:
1
58 °C; Anal. calc. (C H N S): C, 66.90; H, 7.32; N, 14.63;
177 °C; Anal. calc. for (C H N SBr): C, 54.82, H, 6.09, N,
16
21
3
18 24 3
found: C, 66.81; H, 7.19; N, 14.65; UV: k (nm): 367, 325.5,
10.66; found: C, 54.79, H, 6.12, N, 10.55; k
(nm): 377.4,
max
max
–1
–1
219.1, 205.5; IR: m
(cm ) 3296 (NH), 1526 (C=N), 1137
328.4, 241.2, 210.6; IR: m (cm ) 3349 (NH), 1526 (C=N),
max
1
max
1
(
C-N), 1089 (C=S); H NMR (CDCl ): d 7.20-7.72 (m, 5H,
1155 (C-N), 1068 (C=S); H NMR(CDCl ): d 7.17-7.86 (m,
3
3
Aryl), 8.06 (s, 1H, -NH), 4.40 (t, 2H, -CH , J = 10.1 Hz),
4H, Aryl), 8.06 (s, 1H, -NH), 4.41 (t, 2H, -CH J = 8.5 Hz),
2
2,

940
M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
4
2
.55-4.68 (m, 1H, -CH), 3.24 (t, 2H, -CH , J = 7.9 Hz), 1.26-
134.5, 131.9, 127.9, 125.7-120.9 (Aryl-C), 76.1 (CH ), 51.8
2
(CH ), 47.2 (CH ).
2 2
2
1
3
.20 (m, 14H, -CH2); C NMR(CDCl ): (d, ppm) 182.5
3
(C=S), 158.1 (C=N), 133.5, 131.2, 129.5, 126.1, 124.5, 122.8
(
3
Aryl-C), 76.1 (CH ), 58.7 (CH), 45.3 (CH ), 33.2 (2CH ),
6.3.13. 4-Methyl-1-[(3-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)thiocarbamoyl]piperidine (13)
2
2
2
0.7 (2CH ), 26.3 (2CH ), 24.1 (CH ).
2
2
2
Dark Brown solid (methanol). Yield: 18%; m.p.: 104 °C;
Anal. calc. for (C H N S): C, 66.90, H, 7.32, N, 14.63;
6.3.9. 3-(3-Chlorophenyl)-N-cyclooctyl-4,5-dihydro-1H-
1
6 21 3
pyrazole-1-thiocarboxamide (9)
found: C, 66.92, H, 7.29, N, 14.65; k
(nm): 360.5, 321.1,
max
–
1
creamish yellow solid (chloroform). Yield: 15%; m.p.:
234, 205.4; IR: m
(C=S); H NMR (CDCl ): d 7.18-7.69 (m, 5H, Aryl), 4.67 (t,
(cm ) 1596 (C=N), 1145 (C-N), 1101
max
1
1
1
3
1
4
3
1
24 °C; Anal. calc. for (C H N SCl) : C, 61.80, H, 6.87, N,
18
24
3
3
2.02; found: C, 61.84, H, 6.79, N, 12.12; k
(nm): 378,
2H, -CH , J = 11.3 Hz), 3.13 (t, 2H, -CH , J = 11.1 Hz), 3.23-
max
2
2
–1
20, 247.4, 207; IR: mmax (cm ) 3346 (NH), 1539 (C=N),
3.57 (m, 4H, -NCH ), 1.89-2.21 (m, 1H, -CH), 1.19-1.69 (m,
2
1
13
115 (C-N), 1059 (C=S); H NMR (CDCl ): d 7.18-7.92 (m,
4H, -CH ), 1.01 (d, 3H, -CH , J = 5.6 Hz); C NMR (CDCl ):
3
2
3
3
H, Aryl), 8.14 (s, 1H, -NH), 4.56 (t, 2H, -CH , J = 8.7 Hz),
(d,ppm) 178.5 (C=S), 157.5 (C=N), 132.3, 129.4, 127.6,
125.1, 122.4, 121.5 (Aryl-C), 77.0 (CH ), 52.7 (2CH ), 46.9
2
.67-3.73 (m, 1H, -CH), 3.33 (t, 2H, -CH , J = 9.1 Hz), 1.57-
2
2
2
1
3
.99 (m, 14H, -CH2); C NMR(CDCl ): (d, ppm) 181.6
(CH ), 36.2 (CH), 32.8 (2CH ), 13.3 (CH ).
3
2
2
3
(
(
C=S), 156.9 (C=N), 133.4, 131.7, 129.4, 126.1, 124.5, 121.2
Aryl-C), 75.2 (CH ), 52.9 (CH), 47.7 (CH ), 33.9 (2CH ),
6.3.14. 1-{[3-(3-Bromophenyl)-4,5-dihydro-1H-pyrazol-1-
yl]thiocarbamoyl}-4-methyl piperidine (14)
2
2
2
3
0.3 (2CH ), 24.8 (2CH ), 22.4 (CH ).
2
2
2
Brown solid (methanol). Yield: 12%; m.p.: 118 °C; Anal.
calc. for (C H N SBr): C, 52.46, H, 5.46, N, 11.48; found:
6
1
.3.10. N-[(2-chlorophenyl)methyl]-3-phenyl-4,5-dihydro-
H-pyrazole-1-thiocarboxam-ide (10)
White solid (chloroform).Yield: 14%; m.p.: 189 °C; Anal.
1
6 20 3
C, 52.47, H, 5.49, N, 11.46; k
(nm): 324.1, 295.5, 244,
max
–1
217.4; IR: m (cm ) 1564 (C=N), 1248 (C-N), 1069 (C=S);
max
1
calc. for (C H N SCl): C, 61.91, H, 4.85, N, 12.75; found:
H-NMR (CDCl ): d 7.22-7.88 (m, 4H, Aryl), 4.43 (t, 2H,
1
7
16
3
3
C, 61.89, H, 4.84, N, 12.77; k
(nm): 313, 244, 211.4; IR:
-CH , J = 9.2 Hz), 3.16 (t, 2H, -CH , J = 9.2 Hz), 3.36-3.77
max
2
2
–
1
mmax (cm ) 3262 (NH), 1587 (C=N), 1130 (C-N), 1038
(m, 4H, -NCH ), 2.21-2.59 (m, 1H, -CH), 1.75-2.16 (m, 4H,
2
1
13
(
C=S); H NMR (CDCl ): d 7.18-7.85 (m, 9H, Aryl), 8.14 (s,
-CH ), 1.02 (d, 3H, -CH , J = 4.6 Hz); C NMR(CDCl ): (d,
3
2
3
3
1
H, -NH), 4.47 (t, 2H, -CH , J = 8.0 Hz), 4.98 (d, 2H, -CH ,
ppm) 177.2 (C=S), 156.4 (C=N), 135.4, 133.8, 131.2, 128.9,
125.2, 122.7 (Aryl-C), 77.2 (CH ), 52.4 (2CH ), 48.3 (CH ),
2
2
1
3
J = 5.4 Hz), 3.27 (t, 2H, -CH , J = 7.9 Hz); C NMR
2
2
2
2
(
CDCl ): (d, ppm) 181.4 (C=S), 158.1 (C=N), 134.5, 133.3,
40.2 (CH), 33.7 (2CH ), 13.1 (CH ).
3
2
3
1
31.4, 130.5, 127.9, 125.4 – 121.6 (Aryl-C), 77.4 (CH ), 52.6
2
(
CH ), 49.6 (CH ); ESI-MS: 331 (M+1).
6.3.15. 1-{[3-(3-Chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl]thiocarbamoyl}-4-methyl piperidine (15)
2
2
6
4
.3.11. 3-(3-Bromophenyl)-N-[(2-chlorophenyl)methyl]-
,5-dihydro-1H-pyrazole-1-thiocarboxamide (11)
Yellow solid (Chloroform: methanol). Yield: 15%; m.p.:
147 °C; Anal. calc. for (C H N SCl): C, 59.72, H, 6.22, N,
1
6 20 3
Yellow solid (chloroform: methanol). Yield: 11%; m.p.:
77 °C; Anal. calc. for (C H N SBrCl): C, 49.94, H, 3.67,
13.06; found: C, 59.89, H, 6.27, N, 13.03; k
(nm): 380.5,
max
–
1
1
347.5, 293.5, 287, 246; IR: m
(C-N), 1060 (C=S); H NMR (CDCl ): d 7.21-7.87 (m, 4H,
(cm ) 1549 (C=N), 1189
17
15
3
max
1
N, 10.28; found: C, 49.95, H, 3.89, N, 10.31; k (nm): 377.4,
max
3
–1
2
1
8
4
96, 243.5, 210.2; IR: m
129 (C-N), 1036 (C=S); H NMR (CDCl ): d 7.24-7.94 (m,
(cm ) 3260 (NH), 1568 (C=N),
Aryl), 4.52 (t, 2H, -CH , J = 8.2 Hz), 3.36 (t, 2H, -CH ,
max
1
2
2
J = 8.7 Hz), 3.47-3.69 (m, 4H, -NCH ), 2.13-2.46 (m, 1H,
3
2
H, Aryl), 8.14 (s, 1H, -NH), 4.46 (t, 2H, -CH , J = 7.9 Hz),
-CH), 1.79-2.08 (m, 4H, -CH ), 1.11 (d, 3H, -CH ,
2
2
3
1
3
.78 (d, 2H, -CH , J = 5.1 Hz), 3.27 (t, 2H, -CH , J = 8.1 Hz);
J = 5.0 Hz); C NMR (CDCl ): (d, ppm) 177.9 (C=S), 158.9
2
2
3
1
3
C NMR(CDCl ): (d, ppm) 179.8 (C=S), 159.2 (C=N), 135.3,
(C=N), 135.2, 131.8, 129.7, 126.2, 124.1, 121.3 (Aryl-C), 75.3
(CH ), 53.7 (2CH ), 47.9 (CH ), 38.4 (CH), 32.8 (2CH ),
3
1
32.6, 130.3, 128.4, 125.7-122.8 (Aryl-C), 78.3 (CH ), 56.3
2
2
2
2
2
(
CH ), 48.9 (CH ).
13.7 (CH3).
2
2
6
4
.3.12. 3-(3-Chlorophenyl)-N-[(2-chlorophenyl)methyl]-
,5-dihydro-1H-pyrazole-1-thiocarboxamide (12)
6.3.16. 4-Phenyl-1-[(3-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)thiocarbamoyl]piperazine (16)
Dark yellow solid (Chloroform: methanol). Yield: 9%;
Golden yellow solid (chloroform: methanol). Yield: 15%;
m.p.: 167 °C; Anal. calc. for (C H N S): C, 68.57, H, 6.29,
m.p.: 189 °C; Anal. calc. for (C H N SCl ): C, 56.04, H,
17
15
3
2
20 22 4
4
3
1
8
4
.12, N, 11.54; found: C, 56.11, H, 4.15, N, 11.58; k (nm):
N, 16.00; found: C, 68.59, H, 6.27, N, 15.98; k (nm): 371,
max
max
–1
–1
23.5, 245, 216; IR: mma x1 (cm ) 3295 (NH), 1561 (C=N),
295.4, 233, 206; IR: m
1001 (C=S); H NMR (CDCl ): d 7.20-7.71 (m, 10H, Aryl),
(cm ) 1597 (C=N), 1156 (C-N),
max
1
185 (C-N), 1070 (C=S); H NMR (CDCl ): d 7.19-7.83 (m,
3
3
H, Aryl), 8.11 (s, 1H, -NH), 4.36 (t, 2H, -CH , J = 9.1 Hz),
4.03 (t, 2H, -CH2, J = 11.1 Hz), 3.36 (t, 2H, -CH ,
2
2
1
3
.82 (d, 2H, -CH , J = 5.2 Hz), 3.29 (t, 2H, -CH , J = 9.6 Hz);
J = 11.8 Hz), 3.65 (t, 4H, -NCH ), 3.19 (t, 4H, -NCH );
C
2
2
2
2
1
3
C NMR(CDCl ): (d,ppm) 177.4 (C=S), 155.7 (C=N), 136.2,
NMR (CDCl ): (d, ppm) 174.1 (C=S), 156.3 (C=N), 131.4,
3
3

M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
941
1
30.8, 127.6, 125.1-121.2 (Aryl-C), 73.5 (CH ), 56.2 (2CH ),
6.3.21. 1-{[3-(3-Chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
2
2
4
6.7 (CH ), 42.9 (2CH ).
yl]thiocarbamoyl}-4-(phenyl methyl)piperazine (21)
Cream solid (Chloroform),Yield: 10%; m.p.: 154 °C; Anal.
calc. for (C H N SCl): C, 66.42, H, 6.04, N, 10.57; found:
2
2
6
.3.17. 1-{[3-(3-Bromophenyl)-4,5-dihydro-1H-pyrazol-1-
2
2 24 3
yl]thiocarbamoyl}-4-phenyl piperazine (17)
C, 66.49, H, 6.11, N, 10.54; k
(nm): 325.3, 233, 216; IR:
max
–
1
1
Yellow solid (chloroform: methanol). Yield: 12%; m.p.:
mmax (cm ) 1585 (C=N), 1169 (C-N), 1065 (C=S); H NMR
(CDCl ): d 7.21-7.85 (m, 9H, Aryl), 4.45 (t, 2H, -CH ,
8
1
2
1
4
3
8 °C; Anal. calc. for (C H N SBr): C, 55.94, H, 4.89, N,
20 21 4
3
2
3.05; found: C, 56.01, H, 4.91, N, 13.06; k
(nm): 377.4,
J = 9.2 Hz), 2.63 (d, 2H, -CH , J = 5.8 Hz), 3.32 (t, 2H, -CH ,
max
2
2
–1
89.2, 245.3, 216.3; IR: m (cm ) 1564 (C=N), 1188 (C-N),
J = 8.9 Hz), 2.19-2.46 (m, 1H, -CH), 3.39-3.76 (m, 4H,
max
1
13
068 (C=S); H NMR (CDCl ): d 7.18-7.85 (m, 9H, Aryl),
-NCH ), 1.32-1.97 (m, 4H, -CH ); C NMR(CDCl ):(d,ppm)
3
2
2
3
.25 (t, 2H, -CH , J = 9.8 Hz), 3.56 (t, 2H, -CH , J = 10.0 Hz),
175.3 (C=S), 159.3 (C=N), 136.5, 133.8, 130.2, 128.1, 125.7-
120.7 (Aryl-C), 75.9 (CH ), 58.1 (2CH ), 43.8 (CH ), 36.9
2
2
1
3
.63-3.76 (m, 4H, -NCH ), 3.19-3.47 (m, 4H, -NCH );
C
2
2
2
2
2
NMR(CDCl ): (d, ppm) 176.3 (C=S), 157.1 (C=N), 134.1,
(CH ), 30.5 (2CH ), 33.9 (CH).
3
2
2
1
5
32.2, 130.9, 129.4, 127.5, 126.3-122.9 (Aryl-C), 74.7 (CH2),
6.7 (2CH ), 45.8 (2CH ), 48.9 (CH ).
6.4. In vitro testing against E. histolytica
2
2
2
6
.3.18. 1-{[3-(3-Chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
All the 1-N-substituted 3-phenyl-2-pyrazoline derivatives
(1-21) were screened in vitro for antiamoebic activity against
(HM1:IMSS) strain of E. histolytica by using a microplate
method [18]. DMSO (40 µ L) [20,21] was added to all the
samples (~1 mg) followed by enough culture medium to
obtain concentration of 1 mg/mL. Samples were dissolved or
suspended by mild sonication in a sonicleaner bath for a few
minutes and then further diluted with medium to concentra-
tions of 0.1 mg/mL. Two-fold serial dilutions were made in
the wells of 96-well microtiter plate (Costar) in 170 µ L of
medium. Each test included metronidazole as a standard
amoebicidal drug, control wells (culture medium plus amoe-
bae) was prepared from a confluent culture by pouring off the
medium, adding 2 m L of medium and chilling the culture on
ice to detach the organisms from the side of the flask. The
number of amoeba per m L was estimated with a heamocy-
tometer and trypan blue exclusion was used to confirm viabil-
yl]thiocarbamoyl}-4-phenyl piperazine (18)
Brown solid (methanol), Yield: 10%; m.p.: 117 °C; Anal.
calc. for (C H N SCl): C, 62.42, H, 5.46, N, 14.56; found:
C, 62.45, H, 5.41, N, 14.59; k
IR: mmax (cm ) 1599 (C=N), 1148 (C-N), 1015 (C=S); H
NMR (CDCl ): d 7.23-7.87 (m, 9H, Aryl), 4.41 (t, 2H, -CH ,
2
0 21 4
(nm): 370.5, 294, 249.5;
max
–
1
1
3
2
J = 8.7 Hz), 3.35 (t, 2H, -CH , J = 8.2 Hz), 3.59-3.79 (m, 4H,
2
13
-NCH ), 3.17-3.30 (m, 4H, -NCH ); C NMR(CDCl ): (d,
2 2 3
ppm) 178.5 (C=S), 156.8 (C=N), 136.2, 134.9, 132.7, 130.4,
125.5-120.1 (Aryl-C), 77.1 (CH ), 54.3 (2CH ), 44.2 (2CH ),
2 2 2
4
6.8 (CH2).
.3.19. 1-[(3-Phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiocar-
6
bamoyl]-4-(phenylmethyl)- piperazine (19)
Yellow solid (chloroform: methanol). Yield: 12%; m.p.:
1
1
2
1
4
2
43 °C; Anal. calc. for (C H N S): C, 72.73, H, 6.89, N,
22 25 3
5
1.57; found: C, 72.74, H, 6.87, N, 11.54; k
(nm): 319.2,
ity. The cell suspension used was diluted to 10 organism/mL
max
–1
80.1, 246.4, 210.5; IR: m (cm ) 1597 (C=N), 1216 (C-N),
by adding fresh medium and 170 µ L of this suspension was
added to the test and control wells in the plate so that the
wells were completely filled (total volume, 340 µ L). An
max
1
056 (C=S); H NMR (CDCl ): d 7.16-7.75 (m, 10H, Aryl),
3
.42 (t, 2H, -CH , J = 9.2 Hz), 2.59 (d, 2H, -CH ), 3.36 (t,
H, -CH , J = 8.9 Hz), 2.26-2.43 (m, 1H, -CH), 3.62-3.97
2
2
4
inoculum of 1.7 X 10 organisms/well was chosen so that
2
1
3
(
(
m, 4H, -NCH ), 1.50-1.86 (m, 4H, -CH ); C NMR
CDCl ): (d, ppm) 179.3 (C=S), 154.5 (C=N), 131.0, 129.3,
confluent, but not excessive growth took place in control wells.
Plates were sealed with expanded polystyrene (0.5 thick).
Secured with tape, placed in a modular incubating chamber
(flow laboratories, High wycombe, UK), and gassed for
2
2
3
128.7, 125.3-122.4 (Aryl-C), 76.7 (CH ), 56.4 (2CH ), 45.1
2 2
(CH ), 38.6 (CH ), 29.4 (2CH ), 34.5 (CH).
2 2 2
1
0 min with nitrogen before incubation at 37 °C for 72 h.
6.3.20. 1-{[3-(3-Bromophenyl)-4,5-dihydro-1H-pyrazol-1-
yl]thiocarbamoyl}-4-(phenyl methyl)piperazine (20)
Dark brown solid (methanol). Yield: 9%; m.p.: 133 °C;
Anal. calc. for (C H N SBr): C, 59.73, H, 5.43, N, 9.50;
6.5. Assessment of antiamoebic activity
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. Plate
was then immediately washed once in sodium chloride solu-
tion (0.9%) at 37 °C. This procedure was completed quickly,
and the plate was not allowed to cool in order to prevent the
detachment of amoebae. The plate was allowed to dry at room
temperature, and the amoebae were fixed with methanol, when
dry, stained with (0.5%) aqueous eosin for 15 min. Stained
plate was washed once with tape water and then twice with
2
2 24 3
found: C, 59.81, H, 5.47, N, 9.49; k
(nm): 377.4, 247.4,
max
–1
2
09.2; IR: mmax (cm ) 1559 (C=N), 1187 (C-N), 1097 (C=S);
1
H NMR (CDCl ): d 7.22-7.90 (m, 9H, Aryl), 4.41 (t, 2H,
3
-
CH , J = 11.2 Hz), 2.64 (d, 2H, -CH , J = 7.1 Hz), 3.12 (t,
2
2
2
3
H, -CH , J = 11.9 Hz), 2.29-2.57 (m, 1H, -CH, J = 4.8 Hz),
2
1
3
.37-3.69 (m, 4H, -NCH ), 1.59-2.13 (m, 4H, -CH );
C
2
2
NMR (CDCl ): (d, ppm) 181.4 (C=S), 157.2 (C=N), 134.3,
3
1
33.7, 131.4, 129.8, 125.1-121.6 (Aryl-C), 76.3 (CH ), 56.6
2
(
2CH ), 48.4 (CH ), 35.7 (CH ), 28.9 (2CH ), 33.6 (CH).
2 2 2 2

942
M. Abid, A. Azam / European Journal of Medicinal Chemistry 40 (2005) 935–942
distilled water and allowed to dry. A 200 µ L portion of 0.1 N
sodium hydroxide solution was added to each well to dis-
solve 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 determine
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