Novel Pd(II) complexes of 1-N-substituted 3-phenyl-2-pyrazoline derivatives and evaluation of antiamoebic activity

Article abstract of DOI:10.1016/j.ejmech.2007.03.021

Cyclization of Mannich base with N⁴-substituted thiosemicarbazides by different aliphatic, aromatic and cyclic amines afforded a series of new 1-N-substituted cyclised pyrazoline analogues of thiosemicarbazones (PYZ-TSC) 1-10. Reaction of [Pd(DMSO)₂Cl₂] with pyrazoline derivatives led to new palladium(II) complexes [Pd(PYZ-TSC)Cl₂] 1a-10a. The structures of all the compounds were characterized by spectroscopic methods. It was concluded that the pyrazoline thiosemicarbazone derivatives have two chelating arms, one attached at the 2-position of the pyrazole ring (that is, N donor) and other (S donor) linked to the thiosemicarbazone branch. The determination of antiamoebic activity of all the compounds was done using HM1:IMSS strain of Entamoeba histolytica, among all the complexes, 8a showed the most promising IC₅₀ = 0.37 μM vs. IC₅₀ = 1.81 μM of metronidazole, the reference drug. MTT assay showed that the compounds are non-toxic to human kidney epithelial cell line.

Full text of DOI:10.1016/j.ejmech.2007.03.021

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 393e403
http://www.elsevier.com/locate/ejmech
Short communication
Novel Pd(II) complexes of 1-N-substituted 3-phenyl-2-pyrazoline
derivatives and evaluation of antiamoebic activity
*
Kakul Husain, Mohammad Abid, Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 6 March 2006; received in revised form 22 January 2007; accepted 15 March 2007
Available online 5 April 2007
Abstract
Cyclization of Mannich base with N4-substituted thiosemicarbazides by different aliphatic, aromatic and cyclic amines afforded a series of
new 1-N-substituted cyclised pyrazoline analogues of thiosemicarbazones (PYZ-TSC) 1e10. Reaction of [Pd(DMSO)2Cl2] with pyrazoline
derivatives led to new palladium(II) complexes [Pd(PYZ-TSC)Cl2] 1ae10a. The structures of all the compounds were characterized by spec-
troscopic methods. It was concluded that the pyrazoline thiosemicarbazone derivatives have two chelating arms, one attached at the 2-position of
the pyrazole ring (that is, N donor) and other (S donor) linked to the thiosemicarbazone branch. The determination of antiamoebic activity of all
the compounds was done using HM1:IMSS strain of Entamoeba histolytica, among all the complexes, 8a showed the most promising
IC50 ¼ 0.37 mM vs. IC50 ¼ 1.81 mM of metronidazole, the reference drug. MTT assay showed that the compounds are non-toxic to human kidney
epithelial cell line.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Palladium(II) complexes; Cyclised pyrazoline analogues; Thiosemicarbazones; Entamoeba histolytica
1. Introduction
lengthy treatment or high doses often cause side effects such
as headache, nausea, vomiting, dry mouth, metallic taste, diz-
ziness, vertigo, stomatitis, glossitis and neurological complica-
tions [7e9]. Therefore, it is necessary to search for new
alternative medications that can eliminate the parasite without
generating carcinogenic effects.
The chemistry of cyclised heterocyclic systems especially
containing pyrazole moiety has been largely investigated due
to their effective use in pharmacological areas [10e21]. In
particular, the pyrazolate coordination chemistry has recently
received a major appraisal of structurally unprecedented and
exciting compounds of great pharmacological importance
and thus has attracted considerable attention in recent years
as versatile ligands in coordination chemistry [22e26]. In
the recent years, pyrazole-containing complexes have been re-
ported to possess antitumor activity comparable to that of cis-
platin [27e29].
Amoebiasis, caused by Entamoeba histolytica, an early
branching eukaryote, is one of the major threats to public
health in much of the developing world. More than 50 million
people worldwide are infected and up to 110,000 die every
year due to amoebiasis [1]. It is the third most common para-
sitic disease in humans after malaria and schistosomiasis [2,3].
The highest incidence is observed in children below the age of
5 years [4]. Trophozoites of the extracellular protozoan para-
site E. histolytica colonize the human gut, occasionally invade
the intestinal mucosa, and sometimes metastasize to other
organs [5]. Recently it is found that amoebic abscesses of
the brain are a dreadful complication of this disease [6]. The
effective treatment for amoebiasis is metronidazole; however,
Pioneering studies in our laboratory [30e32] have led to
synthesize a new series of 1-N-substituted cyclised pyrazoline
thiosemicarbazones and their palladium(II) complexes. The
* 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.2007.03.021

394
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
antiamoebic activity and toxicity study of these compounds
were performed against E. histolytica and KB cell lines,
respectively.
and sparingly soluble in methanol, ethanol and insoluble in wa-
ter. The complexes do not undergo any weight loss up to 230 ꢀC,
which suggests their fair thermal stability. The structures of all
the ligands and Pd(II) complexes were established by means
of elemental analysis, IR, UV, 1H NMR, 13C NMR, thermogra-
vimetric analysis and FAB MS (Scheme 2).
2. Results and discussion
2.1. Chemistry
2.1.1. Electronic spectral studies
The electronic spectra of the cyclised pyrazoline analogues
1e10 exhibited three absorption bands at 260e278, 218e230
and 202e212 nm assignable to n / p*, p / p* and n / s*
transitions, respectively. The band at 260e278 nm assigned to
the n / p* transition was due to the thione portion (C]S) of
thiocarboxamide group. The two other absorption bands at
218e230 and 202e212 nm were due to p / p* transition
of phenyl ring and n / s* transition of azomethine nitrogen,
respectively. The UV/vis spectrum of all the palladium(II)
complexes exhibits similar pattern, representing three bands
in the region 289e225, 237e245 and 212e221 nm. A careful
study of electronic spectral bands of complexes showed that
there was change in the energy of these bands due to extended
conjugation of ligands. Complexes 1ae10a show a single
band in the region 212e221 nm due to charge-transfer transi-
tion, which is in a higher-energy region with respect to li-
gands. Bands at higher energies 289e225 nm are attributed
to the p / p* transitions of the aromatic ring. In complexes,
the p / p* transition of azomethine chromophore shifted to
237e245 nm indicating that the imino N-atom is involved in
the coordination. An intense charge-transfer band is observed
for the complexes in the 371e373 nm region which is reason-
ably assignable to a combination of ligand to metal charge
transfer and metal ded band transitions. Such observations
have also been noticed earlier in other palladium(II) com-
plexes of similar ligand systems [36].
The Mannich reaction of propiophenone with formaldehyde
and dimethylamine hydrochloride gives the Mannich base pre-
cursor. The reaction works best when a minimum amount of
ethanol and 2 mL of acid/mmol ketone is added. Propiophe-
none gave satisfying yield around 90% in the Mannich reac-
tion. The condensation of this Mannich reaction product
with N4-substituted thiosemicarbazides by different amines
leads to the formation of 1-N-substituted cyclised pyrazoline
analogues of thiosemicarbazones (Scheme 1). The product
was generally crystalline in case of aliphatic substituted thio-
semicarbazides and oil in rest of the compounds. According to
the currently accepted mechanism [33,34] the formation of the
cyclised pyrazoline analogues is favored via thiosemicarba-
zones formation, which undergoes cyclization under basic con-
ditions to form the desired pyrazoline ring. The product mixture
contained only unreacted starting material and the cyclization
product, which was purified by column chromatography using
silica gel 60F254 eluted with chloroform:methanol (98:2).
The yield of cyclised products was assorted from low to high.
The compounds obtained are stable. [Pd(DMSO)2Cl2] serves
as a good starting material to prepare complexes reported
here thereby giving novel palladium(II) complexes of 1-N-
substituted cyclised pyrazoline analogues of thiosemicarba-
zones (1ae10a). The precursor [Pd(DMSO)2Cl2] used for the
synthesis of palladium complexes was synthesized by the liter-
ature procedure [35]. Reaction of 1-N-substituted cyclised pyr-
azoline analogues of thiosemicarbazones and [Pd(DMSO)2Cl2]
in equimolar ratio in refluxing methanol gave amorphous com-
plexes with satisfactory yields. Crystallization was done in suit-
able solvents. All the complexesare soluble in DMFand DMSO,
2.1.2. IR spectral analysis
Assignments of selected characteristic IR band positions
provide significant indication for the formation of the cyclised
pyrazoline analogues of thiosemicarbazones 1e10. All the
compounds showed intense bands in the region 1021e
1109 cmꢁ1 due to the n(C]S) stretch of the thiocarboxamide
group along with a weak band appeared in the region 2500e
2600 cmꢁ1 due to n(CeSH) [36, 37] suggesting that all the
cyclised pyrazoline analogues exhibit thione 4 thiole tautom-
erism as depicted in Fig. 1. The IR spectra of all the com-
pounds showed n(C]N) stretch at 1525e1598 cmꢁ1
because of the ring closure.
HCl
.
i
CH3
CH3
CH3
(HCHO)n
+
N
CH3
CH3
(CH3)2NH2Cl
O
O
H
N
R
ii
H2N
S
In addition, the absorption bands at 1102e1198 cmꢁ1 were
attributed to the n(CeN) stretch vibrations, which also confirm
the formation of the desired pyrazoline ring in all the com-
pounds. The mono-substituted amines showed additional sharp
bands in the region 3275e3389 cmꢁ1 due to the n(NH) stretch.
The IR spectra in the range 40,000e400 cmꢁ1 show that the
ligands are coordinated to the Pd(II) metal ion giving 1ae
10a. The most striking bands of the ligands (n(C]N), n(Ne
H), and n(CeN)) increase their frequency when they are part
of the complexes. Moreover, the band appearing in the region
H3C
R
S
iii
N
N
N
Pd(DMSO)2Cl2
R
N
Pd
S
Cl
Cl
Complexes (1a-10a)
Ligands (1-10)
R= Acyclic, Aromatic and Cyclic amine
Scheme 1. Reagents and conditions: (i) methanol, NaOH (11%), 15e28 ꢀC;
(ii) ethanol, reflux, NaOH; (iii) dry MeOH, 70 ꢀC.

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
395
CH3
N
N
N
S
Pd
Cl
+
Cl
[Pd-H]+
M = m/z 454
m/z = 107
-PdCl2
H3C
H3C
+.
+.
+.
N
N
.
N
N
N
N
S
S
+
m/z 276
[M- PdCl2]
m/z 276
.
.
-CH3
-SH
H3C
H
N
N
N
N
N
N
+
S
m/z 243
m/z 261
.
-SH
.
+.
-CH3
N
.
C
N
N
N
m/z 104
m/z 228
.
-CN
N
+
+.
N
+.
N
m/z 202
N
N
m/z 146
.
Scheme 2. Proposed mass fragmentation pattern for complex.
1001e1050 cmꢁ1 n(C]S) in the spectrum is shifted to lower
wave number by ca. 15e30 cmꢁ1 after complexation, indicat-
ing that the thione sulphur participates as a coordinating site.
The far-IR spectra of the complexes exhibit bands in the
region 443e489 cmꢁ1 tentatively attributed to n(PdeN) and
n(PdeS) stretches, further indicating the involvement of
(eC]N) nitrogen and (eC]S) sulphur in these complexes.
This confirms the fact that in this case cyclised pyrazolyl thi-
osemicarbazone ligands behave as neutral NS donor bidentate
upon complexation.
and the other as a doublet of doublets ranging from 3.96 to
4.96 ppm (J ¼ 10.3e14.3 Hz) which is due to vicinal coupling
with the two magnetically non-equivalent protons of the meth-
ylene group at position 4 and the geminal proton at position 5
of the pyrazoline ring which may be due to its conformation A
given below.
Hc
Hb
H3C
Ha
S
4
5
N
1
R
3
N
2
A
2.1.3. Nuclear magnetic resonance spectral studies
Further evidence for the formation of pyrazoline com-
The CH3 protons attached to the pyrazoline nucleus at C4
appear as a doublet in d 1.30e1.36 ppm region. The NH pro-
ton of different substituted thiocarboxamide group of the
compounds 1e10 showed a singlet at 8.37e8.96 ppm. The
protons belonging to the aromatic ring and the other aliphatic
groups were observed with the expected chemical shift and
1
pounds was obtained by the H NMR spectra. The pyrazoline
proton Hc, at C4 carbon appears downfield in the region 3.16e
3.87 as multiplets in all compounds and the geminal protons
Ha and Hb, at C5 carbon exhibit two signals, one as triplet
in the range 3.60e4.76 ppm (J ¼ 3.6e5.1, 10.3e14.3 Hz)

396
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
H
N
2.1.5. FAB MS analysis
H3C
H3C
The structure of the pyrazolyl palladium complexes 1ae10a
was additionally confirmed from its mass spectral fragments
and gave the molecular ion peak as (M þ 1)þ ion. It is assumed
that the fragmentation pathway is initiated by loss of the two
chlorine ions simultaneously, giving (M ꢁ Cl2) ion peaks at im-
plicit positions. Elimination of Pd metal from cyclised pyrazo-
line thiosemicarbazone complexes and its further fragmentation
follows a similar pathway in all the complexes which occurs via
cleavage of [SH] and [CH3] moieties giving ideal peaks in all
the compounds which correspond to the [M ꢁ SH]þ or
[M ꢁ CH3]þ ion. This is followed by elimination of CN to pro-
duce a disubstituted pyrazoline ion. Further elimination of alkyl
radical of the substituted amines leads to the formation of
mono-substituted pyrazoline ion at m/z 146 as a base peak in
all the complexes. The major fragmentation pathway involved
the split of pyrazoline ring giving fragment at m/z 104. The
azirinium ions are barely detected and the ions at m/z 13, 91,
77 arise from further decomposition in the usual way. The
FAB MS fragmentation pattern of compound 3 is depicted in
Scheme 2.
N
N
R
N
N
R
N
SH
S
Thione form
Thiole form
Fig. 1. Structure of pyrazoline thiosemicarbazones exhibiting thione 4 thiole
tautomerism.
1
integral values. Comparing the H NMR spectra of pyrazolic
ligands and complexes, a slight downfield shift of thioamide
(NHeC]S) proton signal in the region 8.54e9.18 ppm for
complexes was observed which indicates the involvement
of thione sulphur in complex formation. The complexes
1ae10a do not show any resonance at ca. 4.0 ppm, attributed
to eSH proton resonance, indicating that even in a polar
solvent such as DMSO they remain in the thione form.
This information suggests the adjustment of electronic cur-
rent upon coordination of pC]S group to the metal ion.
As a result, establishing the direct involvement of ]Ne
NeC]S group in coordination. Other protons, namely viz.
CH3 protons, CH2 protons and aryl protons in complexes
1ae10a resonate nearly at the same region as that of free
ligands.
The 13C NMR spectra of all the cyclised pyrazoline an-
alogues of thiosemicarbazones 1e10 were taken in CDCl3
and the signals obtained further confirm the proposed struc-
tures. The C4 and C5 carbons of the pyrazoline ring reso-
nate at 46.3e51.1 and 50.8e54.1 ppm, respectively. All
the compounds showed a signal at 150.6e166.3 ppm which
was assigned to the azomethine carbon of pyrazoline ring.
Thiocarboxamide carbon (C]S) displayed a signal at
173.9e184.3 ppm in all the compounds. The signals in the
range 139.1e125.0 ppm were due to the aromatic carbons.
The carbons at 1-N-substituted amino groups resonate at
their usual positions as is shown in the data given in
Section 4.
2.1.6. In vitro antiamoebic activity
All the 1-N-substituted thiocarboxamide-4-methyl-3-phe-
nyl 2-pyrazolines with thiosemicarbazides of variegated nature
and their Pd(II) complexes were screened for antiamoebic
activity against HM1:IMSS strain of E. histolytica. The com-
pounds under study are 1-N-substituted with different
aliphatic, alicyclic and aromatic groups to investigate the in-
fluence of the substitution on the antiamoebic activity. The
IC50 values in mM are indexed in Table 1. The results were es-
timated 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 was used as the reference drug and had
a 50% inhibitory concentration (IC50 1.6e1.8 mM) in our
experiments. All the compounds showed IC50 values in the
range 0.37e15.25 mM. Among all the cyclised ligands, the
N-cyclo-octyl amine 7 in this series showed a close value for
antiamoebic activity with the reference drug, metronidazole
(IC50 ¼ 2.3 mM vs. IC50 ¼ 1.8 mM of metronidazole). The
complexation of cyclised pyrazoline thiosemicarbazones 1e
10 with palladium(II) results in complexes 1ae10a, which
showed IC50 ¼ 0.37e5.65 mM. All the metal complexes were
found more active than their respective ligands indicating
that the complexation to metal enhances the activity of the li-
gand. Fig. 2 represents the graph of pyrazoline thiosemicarba-
zones and Pd(II) complexes exhibiting comparison of IC50
values in mM. This may be explained by Tweedy’s theory
[38], according to which chelation reduces the polarity of
the central metal atom because of partial sharing of its positive
charge with the ligand, which favors permeation of the com-
plexes through the lipid layer of cell membrane. Moreover,
the complexes 2a and 4ae10a displayed the most promising
in vitro amoebicidal activity and were found better inhibitors
of the parasite than metronidazole. The Pd-complex precursor
2.1.4. TGA analysis
Thermograms of Pd(II) complexes of 1-N-substituted cy-
clised pyrazoline analogues of thiosemicarbazones 1ae10a
were recorded under nitrogen with a heating rate of 10 ꢀC/
min between room temperature and 800 ꢀC. These complexes
did not lose weight up to 230 ꢀC. Further increments of tem-
perature caused decomposition of the complexes in two steps,
the temperature range for the first step being 230e290 ꢀC for
the palladium(II) complexes where losses of mixed fragments
were observed. The second step starts immediately after the
first one and continues until complete decomposition of the
ligand and formation of PdS as the end product were observed.
Although decomposed fragments of the ligands could not be
approximated due to continuous weight loss, the total percent-
age weight loss of the complexes corresponds to the loss of the
respective ligand after considering the transfer of one sulphur
atom to the metal ion and residues correspond to the palladium
sulphide.

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
397
Table 1
In vitro antiamoebic activity of compounds against E. histolytica and cytotoxic profile
CH3
N
R
CH3
N
S
N
N
Pd
Cl
Cl
S
R
(1-10)
(1a-10a)
Compound
R
Antiamoebic activity IC50 (mM)
S.D.a
Toxicity profile IC50 (mM)
Safety index (SI)
1
1a
eNH2
10.32
5.65
0.11
0.01
>100
>100
>9.68
>17.69
HN-CH-CH3
2
2a
15.25
1.68
0.20
0.19
>100
>100
>6.55
>59.52
CH3
3
3a
eHNeCH2eCH2eCH2eCH3
4.32
3.57
0.09
0.03
>100
>100
>23.14
>28.01
HN-CH2-CH--CH3
CH3
4
4a
9.13
1.76
0.06
0.02
>100
>100
>10.95
>56.81
5
5a
8.75
1.81
0.24
0.02
>100
>100
>11.42
>55.24
N
H
6
6a.
6.63
1.49
0.10
0.18
>100
>100
>15.08
>67.11
HN
7
7a
2.32
1.39
0.08
0.05
>100
>100
>43.10
>71.94
HN
8
8a
8.69
0.37
0.07
0.03
>100
>100
>11.50
>270.27
N
N
H3C
HN
9
9a
4.57
0.58
0.24
0.01
>100
>100
>21.88
>172.40
Cl
10
10a
4.62
0.82
0.17
0.02
>100
>100
>21.64
>121.95
N-CH2-
H
[Pd(DMSO)2Cl2]
METRONIDAZOLE (MNZ)
8.00
1.81
0.34
0.03
¼100
>100
>12.50
>55.24
a
Standard deviation.
[Pd(DMSO)2Cl2] was also evaluated for antiameobic activity
and compared with Pd(II) complexes and metronidazole,
which showed no activity against E. histolytica. It was con-
cluded that the presence of bulky groups at position N4 of
the thiosemicarbazone moiety greatly enhanced the antiamoe-
bic activity. The IC50 values indicate that all the Pd(II) com-
plexes cause a marked inhibition, while the parent ligands
are less active than complexes. 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 metronidazole and the compounds was eval-
uated by T-test. The values of the calculated T were found
higher than the tabulated T at 5% level, thus concluding that
the character under study is significantly influenced by the
treatment. Detailed studies of the toxicity, in vivo and mecha-
nism of action of these complexes are in progress.
2.1.7. Toxicity profile
The pyrazoline ligands and their palladium complexes were
studied against human kidney epithelial cell line to ensure that
compounds were not toxic to human cells. None of the com-
pounds inhibited cell growth at a concentration of 100 mM.
To investigate the selectivity of the compounds, the ‘‘safety in-
dex’’ (SI) was calculated and defined as: toxicity IC50/proto-
zoal IC50, where toxicity IC50 is defined as the concentration
of compound that kills 50% of the human (kidney epithelial)

398
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
DOSE RESPONSE GRAPH
among all the complexes, 2a and 4ae10a were found to possess
remarkable amoebistatic to amoebicidal values ranging from
0.37 to 1.81 mM i.e., equal to or less than metronidazole. It
was found that upon complexation, the activity of ligand was
considerably increased. The complexes of ligands having aro-
matic moieties showed most promising antiamoebic activity
as compared to complexes of ligands having aliphatic and ali-
cyclic moieties that can be attributed to the presence of bulky
aryl groups such as N-phenyl piperizine, N-o-toluidine, N-2-
chlorobenzyl. MTT assay showed that the compounds are
non-toxic to human kidney epithelial cell line.
LIGANDS
Pd PRECURSOR
Pd PRECURSOR
METRONIDAZOLE
16
14
12
10
8
6
4
2
0
1
2
3
4
5
6
TREATMENT
7
8
9
10 11 12
4. Experimental
All the chemicals were purchased from Aldrich chemical
company (USA). Elemental analyses (C, H, N) were per-
formed by Central Drug Research Institute, Lucknow, India,
and the results were within 0.4% of the theoretical values.
Chlorine was estimated by standard method. Melting points
were recorded on a KSW melting point apparatus and were
uncorrected. Electronic spectra were recorded in methanol
on a Shimadzu UV-1601 PC UV/vis spectrophotometer. IR
spectra on KBr disks were recorded on a PerkineElmer model
Fig. 2. Pyrazoline thiosemicarbazones and Pd(II) complexes exhibiting com-
parison of IC50 values (mM).
cell line and protozoal IC50 is the concentration that kills 50%
of amoeba protozoa. This allows to estimate which compounds
might be efficacious or toxic against human cells and poten-
tially in vivo. The numerical results for each compound are
given in Table 1. These safety indices were then plotted
against antiamoebic IC50 values, obtaining the results depicted
in Fig. 3. These results show that none of the pyrazoline li-
gands show significant values of SI. In contrast, complex 8a
has lowest cytotoxicity and highest antiamoebic activity and
in an overall result, complexes 2a and 4ae10a show the
more favorable safety profile along with the most promising
antiamoebic activity.
1
1620 FT-IR spectrophotometer. H NMR and 13C NMR spec-
tra were obtained at ambient temperature using a Bruker Spec-
trospin DPX-300 MHz spectrophotometer in CDCl3 using
tetramethylsilane as an internal standard. Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. Chemical shift values are given in (parts per mil-
lion) and coupling constants (J ) in hertz. The FAB mass
spectra of all the complexes were recorded on a JEOL SX
102/DA-6000 Mass Spectrometer/Data System using argon/
xenon 6 kV, 10 mA as the FAB gas and m-nitro benzyl alcohol
(NBA) was used as the matrix. Thermogravimetric analysis of
the complexes was performed on a TG 51 thermogravimetric
analyzer under nitrogen atmosphere with the heating rate of
10 ꢀC/min. Reactions were monitored by thin-layer chroma-
tography (TLC) using Merck silica gel 60 F254 precoated
thin-layer plates.
3. Conclusion
This research examined the antiamoebic activities of novel
palladium(II) complexes of 1-N-substituted cyclised pyrazoline
analogues of thiosemicarbazones. It was found that the pyrazo-
line thiosemicarbazone derivatives have two chelating arms and
act as neutral NS bidentate chelators. Moreover, the substitu-
ents did not have any influence on the coordination pattern of
compounds. In vitro antiamoebic evaluation of the ligands
and metal complexes was carried out against E. histolytica us-
ing HM1:IMSS strain. The biological behavior revealed that
4.1. Synthesis of Mannich base
A suspension of propiophenone (0.2 mol), dimethylamine
hydrochloride (0.26 mol) and paraformaldehyde (0.26 mol) in
a mixture of 35 mL of ethanol and 0.5 mL of conc. HCl was re-
fluxed for 2 h. After cooling, the solvent was removed in vacuo.
A few drops of HCl were added, and the mixture was worked up
with dichloromethane and water. The aqueous layer was ad-
justed to basic and extracted with dichloromethane (3ꢂ). The
dichloromethane layer was combined and dried. The product
was obtained as colorless oil. The compound has been reported
earlier [39], we have characterized this compound by IR and 1H
NMR spectra. Yield: 91%; colorless oil; IR: nmax (cmꢁ1) 3025
(arom. CeH), 2950 (aliph. CeH), 1677 (C]O), 1221 (CeN);
1H NMR (CDCl3): (d, ppm) 7.33e7.49 (5H, m, aryl), 3.68 (H,
m, eCH ), 2.71e2.55 (2H, dd, eCH2, J ¼ 5.7, 10.7 Hz), 2.17
(6H, s, eCH3), 1.12 (3H, d, eCH3, J ¼ 7.09 Hz).
Fig. 3.

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
399
4.2. Cyclised pyrazoline analogue of
thiosemicarbazones: a general method
(CDCl3): (d, ppm) 8.69 (s, 1H, NH ), 7.20e7.74 (m, 5H,
AreH ), 4.41 (t, 1Ha, CH, J ¼ 11.5 Hz), 4.20 (dd, 1Hb, CH,
J ¼ 4.3, 11.5 Hz), 3.71 (m, 1Hc, CH ), 3.54 (m, 2H, CH2),
1.76 (m, 2H, CH2), 1.49 (m, 2H, CH2), 1.03 (t, 3H, CH3,
J ¼ 7.2 Hz), 1.32 (d, 3H, CH3, J ¼ 6.39 Hz); 13C NMR
(CDCl3): (d, ppm) 174.5 (C]S), 157.1 (C]N), 135.8,
129.5, 129.3, 128.6, 128.3, 126.7 (aryl-C ), 52.5 (CH2), 51.5
(CH2), 51.0 (CH), 44.9 (CH2), 30.6 (CH2), 19.9 (CH2), 14.6
(CH3), 13.5 (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 dropwise to
the reaction mixture and refluxed for 48e72 h. The reflux
time was dependent upon the thiosemicarbazides taken. N4-
Substituted thiosemicarbazides were cyclised with Mannich
base within 48 h while for N4,N4-disubstituted thiosemicarba-
zides the refluxing time was 48e72 h. The methanol was
removed in vacuo. The residue was dissolved in dichlorome-
thane, washed with water and dried over anhydrous Na2SO4.
In some cases precipitate was formed and the product was
obtained by filtration in ice-cold ethanol. If no precipitate
was formed then 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).
4.2.4. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-isobutyl)thiocarboxamide (4)
Yield 36%. Rf 0.69; pale yellow crystals (CHCl3); m.p.
105 ꢀC. Anal. calc. for C15H21N3S: C 65.45, H 7.63, N
15.27; found: C 65.37, H 7.21, N 15.25; lmax (cmꢁ1): 268,
230, 204; IR: nmax (cmꢁ1) 3361 (NH), 2956 (CH), 1541
1
(C]N), 1457 (C]C), 1102 (CeN), 1057 (C]S); H NMR
(CDCl3): (d, ppm) 8.68 (s, 1H, NH ), 6.93e8.08 (m, 5H,
AreH ), 4.43 (t, 1Ha, CH, J ¼ 11.1 Hz), 4.22 (dd, 1Hb, CH,
J ¼ 4.2, 11.1 Hz), 3.75 (m, 1Hc, CH ), 3.56 (t, 2H, CH2,
J ¼ 6.4 Hz), 1.35 (d, 6H, CH3, J ¼ 6.6 Hz), 1.32 (d, 3H,
CH3, J ¼ 7.2 Hz); 13C NMR (CDCl3): (d, ppm) 174.5
(C]S), 157.0 (C]N), 136.0, 129.0, 129.0, 128.7, 128.3,
126.9 (aryl-C ), 52.5 (CH2), 51.5 (CH2), 51.0 (CH), 30.3
(CH), 20.4 (2CH3), 14.6 (CH3).
4.2.1. 3-Phenyl-4-methyl-2-pyrazoline-1-
thiocarboxamide (1)
Yield 51%. Rf 0.72; pale yellow solid; m.p. 97 ꢀC. Anal.
calc. (C11H13N3S): C 60.27, H 5.93, N 19.20; found: C
60.54, H 5.88, N 18.97; lmax (nm): 262, 222, 202; IR: nmax
/
cmꢁ1 3289 (NH), 2921 (CeH), 1530 (C]N), 1457 (C]C),
1
1125 (CeN), 1021 (C]S); H NMR (CDCl3): (d, ppm) 8.54
4.2.5. 3-Phenyl-4-methyl-2-pyrazoline-1-
(s, 2H, NH2), 7.26e7.74 (m, 5H, AreH ), 4.43 (t, 1Ha, CH,
J ¼ 11.4 Hz), 4.19 (dd, 1Hb, CH, J ¼ 4.5, 11.4 Hz), 3.82 (m,
1Hc, CH ), 1.36 (d, 3H, CH3, J ¼ 6.4 Hz); 13C NMR
(CDCl3): (d, ppm) 175.3 (C]S), 156.0 (C]N), 135.9,
129.3, 129.2, 128.7, 126.4 (aryl-C ), 50.8 (CH2), 48.8 (CH2),
14.6 (CH3).
(N-cyclopentyl)thiocarboxamide (5)
Yield 27%. Rf 0.74; yellow crystals (CHCl3); m.p. 158 ꢀC.
Anal. calc. for C16H21N3S: C 66.89, H 7.31, N 14.63; found: C
66.56, H 6.92, N 14.15; lmax (cmꢁ1): 272, 230, 208; IR: nmax
(cmꢁ1) 3301 (NH), 2925 (CH), 1525 (C]N), 1457 (C]C),
1
1171 (CeN), 1054 (C]S); H NMR (CDCl3): (d, ppm) 8.68
(s, 1H, NH ), 7.21e7.38 (m, 5H, AreH ), 4.41 (t, 1Ha, CH,
J ¼ 11.3 Hz), 4.19 (dd, 1Hb, CH, J ¼ 4.3, 11.3 Hz), 3.71 (m,
1Hc, CH ), 1.35 (d, 3H, CH3, J ¼ 7.1 Hz), 1.30e1.74 (m,
8H, CH2); 13C NMR (CDCl3): (d, ppm) 177.3 (C]S), 158.0
(C]N), 138.8, 129.5, 129.3, 128.7, 128.3, 126.9 (aryl-C ),
51.5 (CH2), 51.1 (CH2), 51.0 (CH2), 32.3 (2CH2), 22.9
(2CH2), 14.6 (CH3).
4.2.2. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-isopropyl)thiocarboxamide (2)
Yield 57%. Rf 0.63; cream white crystals (CHCl3); m.p.
120 ꢀC. Anal. calc. for C14H19N3S: C 64.36, H 7.27, N
16.09; found: C 63.70, H 7.21, N 16.04; lmax (cmꢁ1): 267,
224, 205; IR: nmax (cmꢁ1) 3344 (NH), 2924 (CH), 1536
1
(C]N), 1459 (C]C), 1108 (CeN), 1064 (C]S); H NMR
(CDCl3): (d, ppm) 8.69 (s, 1H, NH ), 7.13e7.74 (m, 5H,
AreH ), 4.61 (m, 1Hc, CH), 4.41 (t, 1Ha, CH, J ¼ 11.3 Hz),
4.19 (dd, 1Hb, CH, J ¼ 5.1, 10.3 Hz), 3.71 (m, 1Hc, CH ),
1.30 (d, 6H, CH3, J ¼ 6.6 Hz), 1.32 (d, 3H, CH3,
J ¼ 6.39 Hz); 13C NMR (CDCl3): (d, ppm) 175.4 (C]S),
157.1 (C]N), 135.8, 129.5, 129.3, 128.7, 128.6, 126.4
(aryl-C ), 51.5 (CH2), 51.1 (CH), 41.8 (CH2), 14.6 (CH3).
4.2.6. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-cyclohexyl)thiocarboxamide (6)
Yield 41%. Rf 0.76; pale yellow crystals (CHCl3); m.p.
140 ꢀC. Anal. calc. for C17H23N3S: C 67.77, H 7.64, N
13.95; found: C 67.04, H 7.11, N 14.05; lmax (cmꢁ1): 272,
226, 210; IR: nmax (cmꢁ1) 3303 (NH), 2925 (CH), 1527
1
(C]N), 1457 (C]C), 1140 (CeN), 1056 (C]S); H NMR
4.2.3. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-butyl)thiocarboxamide (3)
Yield 67%. Rf 0.71; pale yellow crystals (CHCl3); m.p.
180 ꢀC. Anal. calc. for C15H21N3S: C 65.45, H 7.63, N
15.27; found: C 65.41, H 7.29, N 15.45; lmax (cmꢁ1): 270,
228, 208; IR: nmax (cmꢁ1) 3338 (NH), 2962 (CH), 1545
(CDCl3): (d, ppm) 8.67 (s, 1H, NH ), 7.24e7.35 (m, 5H,
AreH ), 4.40 (t, 1Ha, CH, J ¼ 11.5 Hz), 4.19 (dd, 1Hb, CH,
J ¼ 4.2, 11.5 Hz), 3.71 (m, 1Hc, CH ), 1.34 (d, 3H, CH3,
J ¼ 7.1 Hz), 1.27e1.85 (m, 10H, CH2); 13C NMR (CDCl3):
(d, ppm) 178.9 (C]S), 159.1 (C]N), 135.9, 129.7, 129.5,
128.9, 128.5, 125.0 (aryl-C ), 51.5 (CH2), 51.0 (CH2), 50.4
(CH2), 32.2 (2CH2), 25.6 (2CH2), 25.1 (CH2), 14.6 (CH3).
1
(C]N), 1456 (C]C), 1142 (CeN), 1081 (C]S); H NMR

400
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
4.2.7. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-cyclooctyl)thiocarboxamide (7)
129.3, 129.2, 129.1, 128.9, 127.2, 126.4 (aryl-C ), 51.5
(CH2), 51.1 (CH), 50.0 (CH2), 14.6 (CH3).
Yield 77%, Rf 0.72; pale yellow crystals (CHCl3); m.p.
160 ꢀC. Anal. calc. for C19H27N3S: C 69.72, H 7.64, N 12.84;
found: C 69.70, H 7.12, N 12.67; lmax (cmꢁ1): 278, 224, 212;
IR: nmax (cmꢁ1) 3299 (NH), 2926 (CH), 1526 (C]N), 1454
4.3. Pd(II) complexes of cyclised pyrazoline
analogues of thiosemicarbazones
1
(C]C), 1129 (CeN), 1023 (C]S); H NMR (CDCl3): (d,
General procedure: to a hot solution of the appropriate li-
gand (2 mmol) in dry methanol (10 mL) was added a solution
of [Pd(DMSO)2Cl2] (2 mmol) dissolved in minimum quantity
of dry methanol and the reaction mixture was heated under re-
flux for 1e3 h. After keeping the solution at 0 ꢀC overnight the
colored solid separated out. This was filtered off and washed
with ice-cold ethanol followed by small quantity of methanol
and dried to give amorphous solids.
ppm) 8.67 (s, 1H, NH ), 7.26e7.73 (m, 5H, AreH ), 4.40 (t,
1Ha, CH, J ¼ 11.1 Hz), 4.19 (dd, 1Hb, CH, J ¼ 4.1, 11.4 Hz),
3.71 (m, 1Hc, CH ), 1.32 (d, 3H, CH3, J ¼ 7.1 Hz), 1.27e2.00
(m, 14H, CH2); 13C NMR (CDCl3): (d, ppm) 177.3 (C]S),
157.0 (C]N), 135.8, 129.3, 128.7, 128.5, 126.4 (aryl-C ),
51.5 (CH2), 51.1 (CH), 51.0 (CH2), 31.7 (CH2), 26.6 (2CH2),
25.2 (CH2), 21.9 (2CH2), 14.6 (CH3).
4.2.8. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-phenylpiperizenyl)thiocarboxamide (8)
4.3.1. 3-Phenyl-4-methyl-2-pyrazoline-1-thiocarboxamide
palladium(II) chloride [Pd{3-Ph-4-Me-2-Pz-1-N-
TC}Cl2] (1a)
Yield 13%, Rf 0.78; creamish yellow solid (CHCl3); m.p.
160 ꢀC. Anal. calc. for C21H24N4S: C 69.23, H 6.59, N
15.38; found: C 68.99, H 6.59, N 15.06; lmax (cmꢁ1): 269,
228, 208; IR: nmax (cmꢁ1) 2923 (CH), 1598 (C]N), 1457
Yield: 48%; dark brown solid (DMSO). Anal. calc. for
C11H13N3SCl2Pd: C 33.30, H 3.28, N 10.59, Cl 17.91; found:
C 33.52, H 3.19, N 10.50, Cl 17.80; UV/vis: lmax (nm) 371,
295, 242, 216; IR: nmax (cmꢁ1) 3255 (NH), 2925 (CH), 1547
(C]N), 1134 (CeN), 1001 (C]S), 487, 446 (PdeN, Pde
1
(C]C), 1146 (CeN), 1024 (C]S); H NMR (CDCl3): (d,
ppm) 6.85e7.97 (m, 10H, AreH ), 4.81 (t, 1Ha, CH,
J ¼ 11.4 Hz), 4.17 (dd, 1Hb, CH, J ¼ 4.3, 11.4 Hz), 3.43 (m,
1Hc, CH ), 3.12e3.41 (m, 8H, CH2), 1.34 (d, 3H, CH3,
J ¼ 7.1 Hz); 13C NMR (CDCl3): (d, ppm) 186.6 (C]S),
157.2 (C]N), 151.1, 136.1, 129.7, 129.3, 129.2, 129.0,
128.8, 126.3, 120.3, 117.2 (aryl-C ), 53.8 (2CH2), 52.1
(CH2), 47.6 (2CH2), 46.7 (CH), 14.6 (CH3).
1
S); H NMR (CDCl3): (d, ppm) 8.95 (s, 2H, NH2), 7.14e
7.79 (m, 5H, AreH ), 4.41 (t, 1H, CH, J ¼ 11.5 Hz), 4.20
(dd, 1H, CH, J ¼ 4.5, 11.5 Hz), 3.81 (m, 1H, CH ), 1.36 (d,
3H, CH3, J ¼ 6.3 Hz); FAB MS: m/z 398 [M þ 1], 362.5,
327, 220, 205, 187, 172, 145, 103.
4.3.2. 3-Phenyl-4-methyl-2-pyrazoline-1-
4.2.9. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-o-toluidine)thiocarboxamide (9)
(N-isopropyl)thiocarboxamide palladium(II) chloride
[Pd{3-Ph-4-Me-2-Pz-1-N-isoPrTC}Cl2] (2a)
Yield 62%; Rf 0.62; yellow crystals (CHCl3); m.p. 157 ꢀC.
Anal. calc. for C18H19N3S: C 69.90, H 6.14, N 13.59; found: C
69.48, H 6.13, N 13.60 %; UV/vis: lmax (nm) 270, 220, 204;
IR: nmax (cmꢁ1) 3339 (NH), 2962 (CH), 1545 (C]N), 1458
Yield 94%; orangish brown solid (DMSO). Anal. calc. for
C14H19N3SCl2Pd: C 38.32, H 4.33, N 9.58, Cl 16.19; found:
C 38.55, H 4.35, N 9.04, Cl 15.20; UV/vis: lmax (nm) 371,
289, 244, 221; IR: nmax (cmꢁ1) 3245 (NH), 2977 (CH), 1580
(C]N), 1134 (CeN), 1012 (C]S), 487, 445 (PdeN, Pde
1
(C]C), 1142 (CeN), 1081 (C]S); H NMR (DMSO): (d,
1
ppm) 8.86 (1H, s, eNH ), 7.17e8.08 (m, 9H, AreH ), 4.52
(t, 1Ha, CH, J ¼ 14.3 Hz), 4.28 (dd, 1Hb, CH, J ¼ 3.6,
14.3 Hz), 3.75 (m, 1Hc, CH ), 1.59 (s, 3H, CH3), 1.33 (d,
3H, CH3, J ¼ 7.8 Hz); 13C NMR (CDCl3): (d, ppm) 174.5
(C]S), 150.6 (C]N), 144.0, 139.1, 136.4, 133.9, 129.4,
129.3, 128.9, 128.7, 126.4, 126.3, 123.2, 116.9, 116.4 (aryl-
C ), 51.8 (CH2), 46.9 (CH), 17.9 (CH3), 14.6 (CH3).
S); H NMR (CDCl3): (d, ppm) 8.97 (s, 1H, NH ), 7.47e
7.90 (m, 5H, AreH ), 4.53 (m, 1H, CH ), 4.34 (t, 1H, CH,
J ¼ 11.2 Hz), 4.18 (dd, 1H, CH, J ¼ 5.1, 10.5 Hz), 3.71 (m,
1H, CH ), 1.28 (d, 6H, CH3, J ¼ 6.7 Hz), 1.32 (d, 3H, CH3,
J ¼ 6.39 Hz); FAB MS: m/z 440 [M þ 1], 404.5, 369, 262,
247, 229, 214, 188, 145, 103.
4.3.3. 3-Phenyl-4-methyl-2-pyrazoline-1-
4.2.10. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-butyl)thiocarboxamide palladium(II)
(N-2-chlorobenzyl)thiocarboxamide (10)
chloride[Pd{3-Ph-4-Me-2-Pz-1-N-BuTC}Cl2] (3a)
Yield 83%; mud brown solid (DMSO). Anal. calc. for
C15H21N3SCl2Pd: C 39.80, H 4.45, N 9.28, Cl 15.69; found:
C 39.79, H 4.64, N 9.28, Cl 15.70; UV/vis: lmax (nm) 373,
289, 241, 221; IR: nmax (cmꢁ1) 3202 (NH), 2967 (CH), 1584
(C]N), 1142 (CeN), 1021 (C]S), 488, 446 (PdeN, Pde
Yield 18%, Rf 0.65; yellow crystals (CHCl3); m.p. 120 ꢀC.
Anal. calc. for C18H18N3SCl: C 62.88, H 5.24, N 12.22; found:
C 63.01, H 5.89, N 12.21; lmax (cmꢁ1): 264, 228, 202; IR: nmax
(cmꢁ1) 3316 (NH), 2920 (CH), 1560 (C]N), 1457 (C]C),
1
1154 (CeN), 1037 (C]S); H NMR (CDCl3): (d, ppm) 8.90
1
(s, 1H, NH ), 6.27e8.05 (m, 9H, AreH ), 4.96 (t, 1Ha, CH,
J ¼ 11.6 Hz), 4.76 (dd, 1Hb, CH, J ¼ 4.6, 11.6 Hz), 3.62 (m,
1Hc, CH ), 3.46 (d, 2H, CH2), 1.33 (d, 3H, CH3,
J ¼ 7.1 Hz); 13C NMR (CDCl3): (d, ppm) 176.5 (C]S),
163.7 (C]N), 139.1, 134.4, 133.5, 130.7, 129.5, 129.4,
S); H NMR (CDCl3): (d, ppm) 9.16 (s, 1H, NH ), 7.47e
7.96 (m, 5H, AreH ), 4.51 (t, 1H, CH, J ¼ 11.5 Hz), 4.19
(dd, 1H, CH, J ¼ 4.3, 11.5 Hz), 3.81 (m, 1H, CH ), 3.66 (m,
2H, CH2),1.68 (m, 2H, CH2), 1.38 (m, 2H, CH2), 1.03 (t,
3H, CH3, J ¼ 7.3 Hz), 1.34 (d, 3H, CH3, J ¼ 6.39 Hz); FAB

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
401
MS: m/z 454 [M þ 1], 418.5, 383, 276, 261, 243, 228, 202,
11.4 Hz), 3.71 (m, 1H, CH ), 1.32 (d, 3H, CH3, J ¼ 7.1 Hz),
1.27e2.00 (m, 14H, CH2); FAB MS: m/z 508 [M þ 1], 472.5,
437, 330, 315, 297, 282, 256, 145, 103.
145, 103.
4.3.4. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-isobutyl)thiocarboxamide palladium(II)
4.3.8. 3-Phenyl-4-methyl-2-pyrazoline-1-(N-phenylpi-
perizenyl)thiocarboxamide palladium(II) chloride
[Pd{3-Ph-4-Me-2-Pz-1-N-PPTC}Cl2] (8a)
chloride [Pd{3-Ph-4-Me-2-Pz-1-N-isoBuTC}Cl2] (4a)
Yield 36%; brown solid (DMSO). Anal. calc. for
C15H21N3SCl2Pd: C 39.79, H 4.64, N 9.28, Cl 15.69; found:
C 39.25, H 4.46, N 9.24, Cl 15.29; UV/vis: lmax (nm) 371,
291, 244, 220; IR: nmax (cmꢁ1) 3211 (NH), 2962 (CH), 1582
(C]N), 1156 (CeN), 1045 (C]S), 487, 446 (PdeN, Pde
Yield 24%; brown solid (DMSO). Anal. calc. for
C21H24N4SCl2Pd: C 46.55, H 4.43, N 10.34, Cl 13.11; found:
C 46.27, H 4.52, N 10.27, Cl 13.23; UV/vis: lmax (nm) 371,
290, 237, 213; IR: nmax (cmꢁ1) 2920 (CH), 1599 (C]N),
1157 (CeN), 1017 (C]S), 481, 446 (PdeN, PdeS); 1H
NMR (CDCl3): (d, ppm) 6.84e7.97 (m, 10H, AreH ), 4.79
(t, 1H, CH, J ¼ 11.3 Hz), 4.17 (dd, 1H, CH, J ¼ 4.3,
11.4 Hz), 3.42 (m, 1H, CH ), 3.12e3.42 (m, 8H, CH2), 1.34
(d, 3H, CH3, J ¼ 6.7 Hz); FAB MS: m/z 543 [M þ 1], 507.5,
472, 365, 350, 332, 317, 291, 145, 103.
1
S); H NMR (CDCl3): (d, ppm) 9.18 (s, 1H, NH ), 7.41e
8.32 (m, 5H, AreH ), 4.40 (t, 1H, CH, J ¼ 11.3 Hz), 4.29
(dd, 1H, CH, J ¼ 4.2, 11.1 Hz), 3.67 (m, 1H, CH ), 3.54 (t,
2H, CH2, J ¼ 6.3 Hz), 1.34 (d, 6H, CH3, J ¼ 6.5 Hz), 1.32
(d, 3H, CH3, J ¼ 7.2 Hz); FAB MS; m/z 454 [M þ 1], 418.5,
383, 276, 261, 243, 228, 202, 145, 103.
4.3.5. 3-Phenyl-4-methyl-2-pyrazoline-1-
4.3.9. [3-Phenyl-4-methyl-2-pyrazoline-1-
(N-cyclopentyl)thiocarboxamide palladium(II) chloride
[Pd{3-Ph-4-Me-2-Pz-1-N-CPTC}Cl2] (5a)
(N-o-toluidine)thiocarboxamide] palladium(II) chloride
[Pd{3-Ph-4-Me-2-Pz-1-N-o-tolTC}Cl2] (9a)
Yield 27%; brownish yellow solid (DMSO). Anal. calc. for
C16H21N3SCl2Pd: C 34.12, H 4.52, N 9.04, Cl 15.29; found:
C 41.07, H 4.69, N 9.00, Cl 15.23; UV/vis: lmax (nm): 371,
290, 242, 212; IR: nmax (cmꢁ1) 3231 (NH), 2936 (CH), 1569
(C]N), 1184 (CeN), 1047 (C]S), 487, 443 (PdeN, PdeS);
1H NMR (CDCl3): (d, ppm) 8.86 (s, 1H, NH ), 7.47e7.99 (m,
5H, AreH ), 4.57 (t,1H, CH, J ¼ 11.1 Hz), 4.17 (dd, 1H, CH,
J ¼ 4.3, 11.1 Hz), 3.71 (m, 1H, CH ), 1.36 (d, 3H, CH3,
J ¼ 7.3 Hz), 1.31e1.77 (m, 8H, CH2); FAB MS: m/z 466
[M þ 1], 430.5, 395, 288, 273, 255, 240, 214, 145, 103.
Yield 64%; black solid (DMSO). Anal. calc. for
C18H18N3SCl2Pd: C 44.50, H 3.70, N 8.65, Cl 21.94; found:
C 44.37, H 3.70, N 8.66, Cl 21.90; UV/vis: lmax (nm) 371,
292, 242, 221; IR: nmax (cmꢁ1) 3257 (NH), 2918 (CH),
1587 (C]N), 1167 (CeN), 1025 (C]S), 478, 443 (PdeN,
PdeS); 1H NMR (CDCl3): (d, ppm) 8.98 (s, 1H, NH ),
7.21e7.78 (m, 9H, AreH ), 4.78 (t, 1H, CH, J ¼ 11.4 Hz),
4.18 (dd, 1H, CH, J ¼ 4.4, 11.4 Hz), 3.62 (m, 1H, CH ),
3.46 (d, 2H, CH2), 1.33 (d, 3H, CH3, J ¼ 7.1 Hz); FAB MS:
m/z 489 [M þ 1], 453.5, 418, 310, 295, 277, 262, 236, 145,
103.
4.3.6. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-cyclohexyl)thiocarboxamide palladium(II) chloride
[Pd{3-Ph-4-Me-2-Pz-1-N-CHTC}Cl2] (6a)
4.3.10. 3-Phenyl-4-methyl-2-pyrazoline-1-(N-2-chloroben-
zyl)thiocarboxamide palladium(II) chloride [Pd{3-
Yield 50%; orangish yellow crystals (DMSO). Anal. calc.
for C17H23N3SCl2Pd: C 43.00, H 4.85, N 8.85, Cl 14.96; found:
C 42.87, H 4.52, N 8.27, Cl 14.23; UV/vis: lmax (nm) 373, 293,
239, 217; IR: nmax (cmꢁ1) 3230 (NH), 2936 (CH), 1569 (C]N),
1157 (CeN), 1050 (C]S), 483, 444 (PdeN, PdeS); 1H NMR
(CDCl3): (d, ppm) 8.86 (s, 1H, NH ), 7.47e7.99 (m, 5H, Are
H ), 4.31 (t, 1H, CH, J ¼ 11.5 Hz), 4.17 (dd, 1H, CH, J ¼ 4.1,
11.4 Hz), 3.68 (m, 1H, CH ), 1.35 (d, 3H, CH3, J ¼ 7.3 Hz),
1.27e1.87 (m, 10H, CH2); FAB MS: m/z 479 [M þ 1], 443.5,
409, 302, 287, 269, 254, 228, 145, 103.
Ph-4-Me-2-Pz-1-N-2-ClBzTC}Cl2] (10a)
Yield 70%; mud brown solid (DMSO). Anal. calc. for
C18H19N3SCl3Pd: C 41.40, H 3.45, N 8.06, Cl 20.41; found:
C 41.27, H 3.52, N 8.09, Cl 20.43; UV/vis: lmax (nm) 371,
291, 242, 216; IR: nmax (cmꢁ1) 3209 (NH), 2961 (CH), 1567
(C]N), 1157 (CeN), 1023 (C]S), 489, 447 (PdeN, Pde
1
S); H NMR (DMSO): (d, ppm) 8.97 (1H, s, eNH ), 7.33e
8.09 (m, 9H, AreH ), 4.49 (t, 1H, CH, J ¼ 14.1 Hz), 4.27
(dd, 1H, CH, J ¼ 3.5, 14.1 Hz), 3.79 (m, 1H, CH ), 1.58 (s,
3H, CH3), 1.35 (d, 3H, CH3, J ¼ 7.8 Hz); FAB MS: m/z
533.5 [M þ 1], 488, 452.5, 345.5, 330.5, 312.5, 297.5, 271.5,
145, 103.
4.3.7. 3-Phenyl-4-methyl-2-pyrazoline-1-
(N-cyclooctyl)thiocarboxamide palladium(II) chloride
[Pd{3-Ph-4-Me-2-Pz-1-N-COTC}Cl2] (7a)
4.4. In vitro testing against E. histolytica
Yield 88%; orangish yellow crystals (DMSO). Anal. calc.
for C19H27N3SCl2Pd: C 45.03, H 5.33, N 8.29, Cl 14.02; found:
C 44.87, H 5.07, N 8.27, Cl 14.01; UV/vis: lmax (nm): 371, 292,
245, 217; IR: nmax (cmꢁ1) 3300 (NH), 2923 (CH), 1565 (C]N),
1134 (CeN), 1016 (C]S), 482, 445 (PdeN, PdeS); 1H NMR
(CDCl3): (d, ppm) 8.91 (s, 1H, NH ), 7.47e7.99 (m, 5H, Are
H ), 4.38 (t, 1H, CH, J ¼ 11.4 Hz), 4.17 (dd, 1H, CH, J ¼ 4.3,
All the 1-N-substituted cyclised pyrazoline analogues of
thiosemicarbazones 1e10 and their palladium complexes
1ae10a were screened in vitro for antiamoebic activity against
HM1:IMSS strain of E. histolytica by using a microplate
method [40]. DMSO (40 mL) was added to all the samples
(w1 mg) followed by enough culture medium to obtain

402
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 393e403
concentration of 1 mg/mL [41,42]. 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 mL of me-
dium. Each test included metronidazole as a standard amoebi-
cidal drug, control wells (culture medium plus amoebae) was
prepared from a confluent culture by pouring off the medium,
adding 2 mL of medium and chilling the culture on ice to de-
tach the organisms from the side of the flask. The number of
amoeba per mL was estimated with a heamocytometer and try-
pan blue exclusion was used to confirm viability. The cell sus-
pension was diluted to 105 organism/mL by adding fresh
medium and 170 mL of this suspension was added to the test
and control wells in the plate so that the wells were completely
filled (total volume, 340 mL). An inoculum of 1.7 ꢂ 104 organ-
isms/well was chosen so that 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 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. 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 metha-
nol, 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 optical density of the resulting solution in each
well was determined at 490 nm with a microplate reader
(Labsystem Multiskane Bichromatic, UK). The percentage in-
hibition 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.
of 540 nm and reference wavelength of 690 nm and the per-
centage cellular viability calculated with appropriate con-
trols was taken into account. All assays were performed
in triplicate.
Acknowledgements
The authors are thankful to Prof. Alok Bhattacharya and
Prof. Sudha Bhattacharya, School of Life Sciences and School
of Environmental Sciences, Jawaharlal Nehru University, New
Delhi, respectively, for providing Laboratory facilities for anti-
amoebic activities.
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