New Pd(II) complexes of the synthesized 1-N-substituted thiosemicarbazones of 3-indole carboxaldehyde: Characterization and antiamoebic assessment against E. histolytica

Article abstract of DOI:10.1016/j.ejmech.2007.12.002

Reaction of 3-indole carboxaldehyde with aminothiocarbonyl hydrazines resulted in the formation of 3-indole carboxaldehyde thiosemicarbazones (TSCs) 1-13. The synthesized thiosemicarbazones were used as ligands in the formation of [Pd(TSC)Cl₂]

Full text of DOI:10.1016/j.ejmech.2007.12.002

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2016e2028
http://www.elsevier.com/locate/ejmech
Short communication
New Pd(II) complexes of the synthesized 1-N-substituted
thiosemicarbazones of 3-indole carboxaldehyde: Characterization
and antiamoebic assessment against E. histolytica
*
Kakul Husain, Abdul Roouf Bhat, Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 10 July 2007; received in revised form 3 December 2007; accepted 4 December 2007
Available online 14 December 2007
Abstract
Reaction of 3-indole carboxaldehyde with aminothiocarbonyl hydrazines resulted in the formation of 3-indole carboxaldehyde thiosemicar-
bazones (TSCs) 1e13. The synthesized thiosemicarbazones were used as ligands in the formation of [Pd(TSC)Cl2] complexes with palladium(II)
metal ion precursor, [Pd(DMSO)2Cl2]. The chemical structures of all the compounds were established by electronic, IR, 1H NMR and 13C NMR
spectral data. The structure of the complexes was further established by FABMS and DTA. It is concluded that the thione sulphur and the azo-
methine nitrogen atom of the ligands are bonded to the metal ion. The testing of the antiamoebic activity of these compounds against the pro-
tozoan parasite Entamoeba histolytica suggests that compounds 5, 3a, 5a and 8ae13a might be endowed with important antiamoebic properties
since they showed less IC50 values than metronidazole. Moreover, compound 12a displays remarkable antiamoebic activity than metronidazole
(IC50 values of 0.29 vs 1.81 mM, respectively). 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; Indole-3-carboxaldehyde; Thiosemicarbazones; Entamoeba histolytica
1. Introduction
with metronidazole has several side effects that include nau-
sea, vomiting, dry mouth, metallic taste, abdominal pain and
headache. In some cases additional side effects viz. dizziness,
vertigo, paresthesias and rarely encephalopathy or convulsions
have been reported leading to even discontinuation of the drug
[3]. Due to long-term treatment toxicity and clinical resistance
to drugs commonly used, new effective agents are urgently
needed. On the positive side, a great deal of flexibility is
offered by the replacement of the imidazole ring structure
by the heterocyclic moiety.
Of the various heterocyclic systems, the indole nucleus has
been reported as a common dominator of psychotropism and is
of great value in the field of medicine and biochemistry [4,5].
This nitrogen heterocycle and its derivatives have occupied
a unique place in the chemistry because of its varied biody-
namic properties such as anticancer [6e9], antidepressant
[10], antihypertensive [11], psychomimetic [12], antimicrobial
[13,14], antidiabetic [15], antimalarial [16] and anti-inflamma-
tory [17,18]. Moreover, thiosemicarbazones are a class of
Amoebiasis is the second leading cause of death among
parasitic diseases worldwide. The causative protozoan para-
site, Entamoeba histolytica, is a potent pathogen. Secreting
proteinases that dissolve host tissues, killing host cells on con-
tact, and engulfing red blood cells, E. histolytica trophozoites
invade the intestinal mucosa, causing amoebic colitis [1]. Met-
ronidazole (MTZ, 1-[2-hydroxyethyl]-2-methyl-5-nitroimida-
zole) is an antibacterial and antiprotozoal drug that has been
in use for over 35 years. In an event of overt clinical resistance
to metronidazole in the anaerobic protozoa, there is no alter-
native treatment for invasive amoebiasis, keeping in mind
the documented cross-resistance between currently used nitro-
imidazole drugs [2] and worldwide availability. Treatment
* 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.2007.12.002

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
2017
compounds very promising in the treatment of many diseases
and their development is still in progress [19e21]. In addition,
metal ions are known to accelerate drug action, and the efficacy
of a therapeutic agent may be enhanced upon coordination with
a metal ion [22]. In particular, neutral palladium(II) and (IV)
complexes exhibit potential antitumour activity and apoptosis
[23,24]. The literature reports that palladium(II) and platinu-
m(II) complexes of phenylacetaldehyde thiosemicarbazone
are able to in vitro bind to DNA, and present enhanced capacity
to form inter-strand crosslinks by comparison with cisplatinum
[22,25].
We have previously reported the structural and spectral
studies of several transition metal complexes of N4-substituted
thiosemicarbazones with the aim to correlate the structural
features and chelating ability to the antiprotozoal properties
[26,27]. Here the synthesis of 3-indole carboxaldehyde thiose-
micarbazones 1e13 and their subsequent bidentate Pd(II)
complexes 1ae13a were screened for their antiamoebic activ-
ities against HM1:IMSS strain of E. histolytica in vitro exper-
iments and it was found that the coordination of palladium to
thiosemicarbazone enhances the activity. The toxicity study of
these compounds was performed against KB cell lines.
presence of n (NeH) stretch at 3100e3300 cmꢁ1 in the
spectra of the ligands suggest that all the ligands remain in
the thione form in the solid state. The band appearing at
790e820 cmꢁ1 ascribed to n (C]S) [31] of ligands is shifted
to lower wave number by ca. 15e30 cmꢁ1, indicating that the
thione sulphur participates as a coordinating site. This coordi-
nation is confirmed by the presence of two new bands at
around 550 and 440 n (PdeN, PdeS) [32]. Thione form re-
veals preferential coordination of thionic sulphur over nitrogen
of indole is due to more nucleophilic character of the former.
The band due to n (CeNeC) (ring) of indole moiety remains
unaltered in 1ae13a, indicating non-participation of ring
nitrogen in coordination. The negative shift of 25e57 cmꢁ1
of n (C]N) stretch in the complexes indicates the involvement
of azomethine nitrogen in complexation [33]. The broad band
observed in region 3300 cmꢁ1 due to n (NeH) stretch is
slightly shifted in complex probably due to the adjustment
of current arising due to coordination of thionic sulphur,
thus, confirming the fact that ligands behave as neutral NS
donor bidentate in these complexes.
The electronic spectra of these complexes exhibited bands
at expected position as cited in the literature for the similar
system [34]. A perusal of these spectra revealed that they
were dominated by intense intra-ligand and charge transfer
bands. Thus a band appearing at 47 500e49 000 cmꢁ1 in the
spectra of complexes was assigned to pep* transition of in-
dole ring. Another band at 38 000e40 000 cmꢁ1 was attributed
to nep* transitions of indole ring. This band, followed by
a shoulder band at 29 000e31 000 cmꢁ1, appears due to the
thiosemicarbazones’ moiety. A comparison of these three
bands with free ligands revealed that there is increase in inten-
sity and decrease in the frequency that is attributed to extended
conjugation in the ligand moieties after complexation. The
complexes show absorption peaks in the visible region due
to the ded transition of the single d electron of the palladiu-
m(II) ion.
2. Results and discussion
The synthesis of indole-3-carboxaldehyde thiosemicarba-
zones and their Pd(II) complexes is represented in Scheme 1.
All the thioglycolic acids were prepared by the reported
O’Sullivan method [28]. Thiocarbonylhydrazines were pre-
pared by refluxing the alkaline solution of thioglycolic acid
with hydrazine hydrate and their thiosemicarbazones were
synthesized by refluxing aqueous solution of thiocarbonylhy-
drazines and ethanolic solution of 3-indole-carboxaldehyde
in equimolar ratio at 8 ꢀC for 3 h with continuous stirring.
After cooling at ca. 10 ꢀC for 24 h, the precipitated compound
was filtered and recrystallized from appropriate solvent. The
precursor [Pd(DMSO)2Cl2] used for the synthesis of Pd(II)
complexes was synthesized by the literature procedure [29,
30]. The complexes were prepared by mixing 1:1 ratio of
the appropriate ligand and [Pd(DMSO)2Cl2] and the reaction
mixture was heated under reflux for 1e3 h. After keeping
the solution at 0 ꢀC overnight the colored solid separated
out. This was filtered off and washed with hot water followed
by small quantity of methanol, and dried in vacuo over silica
gel to give amorphous solids. The complexes gave high yields
around 80%, while the yields for thiosemicarbazones showed
variable results. The chemical structures of all the compounds
were confirmed by means of elemental analysis and electronic,
IR, 1H NMR and 13C NMR spectral studies. The structures of
complexes were further established by thermogravimetric
analysis and FABMS. The analytical data of these compounds
are in good agreement with their composition (Table 1).
Selected significant IR bands revealed the structural charac-
teristics of the compounds. All the thiosemicarbazones 1e13
may exhibit in thioneethiol tautomerization since they contain
thione group (C]S) and a proton adjacent to thione group.
The absence of n (SeH) stretch at 2500e2600 cmꢁ1 and the
Further evidence for the formation of compounds was ob-
1
tained from the H NMR spectra, which provide diagnostic
tools for the positional elucidation of the protons. Assignments
of the signals are based on the chemical shifts and intensity
patterns. The ligands 1e13 do not show any resonance at ca.
4.0 ppm, attributed to eSH proton resonance, while the
appearance of a broad peak at 11.51e11.69 ppm due to the
eNH proton of thioamide group indicates that even in a polar
solvent such as DMSO they remain in the thione form. The
eNH proton signal of the thiosemicarbazones usually shifts
0.86e2.83 ppm upfield in their respective complexes. How-
ever, in complexes, we are unable to calculate the coupling
constant values for aromatic region. This might be assumed
due to the merging of peaks upon coordination. This informa-
tion suggests the adjustment of electronic current upon coordi-
nation of pC]S group to the metal ion. The protons belonging
to the aromatic ring and the other cyclic groups were observed
with the expected chemical shift and integral values in the
same region as those of free ligands.
Moreover, the 13C NMR spectra of all the ligands taken in
DMSO gave the spectral signals in good agreement with the

2018
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
H
N
H
EtOH
NH2
N
+
H
HN
C
O
H
C
C
S
R
N
HN
C
H
N
S
R
1-13
H
C
N
DMSO
DMSO
Cl
Cl
Pd
Cl
HN
C
Pd
dry MeOH, reflux
Cl
S
R
1a-13a
CH2
CH3
CH2
NH2
HN
HN
1/1a
2/2a
CH CH3
CH3
HN
CH2 CH2 CH2
CH3
3/3a
4/4a
CH3
CH2 CH
CH3
HN
CH2 CH3
CH2 CH3
N
5/5a
7/7a
9/9a
6/6a
CH3
CH2 CH3
CH2
CH2
CH3
HN
HN
HN
8/8a
H3C
CH3
Cl
HN
10/10a
CH2
CH3
H2C
HN
N
11/11a
12/12a
F
HN
F
13/13a
Scheme 1.

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
2019
Table 1
Analytical and physicochemical data of thiosemicarbazones and their Pd(II) complexes
Compound/stoichiometry
Yield (%)
Mp/dec temp (ꢀC)
Found (calcd)
C
H
N
Cl
1/3-ICA-TSC C10H10N4S
1a/[Pd(3-ICA-TSC)2Cl2] C10H10N4S
2/3-ICA-PrTSC C13H16N4S
76
64
13
50
62
45
61
83
67
84
61
27
62
62
20
87
21
86
62
43
19
41
57
65
20
49
220
160
226
267
200
160
170
160
145
295
140
285
140
160
200
235
198
344
192
354
180
267
130
257
160
215
54.71 (55.04)
30.10 (30.35)
59.55 (60.00)
32.81 (32.95)
59.87 (60.00)
35.95 (35.64)
61.02 (61.31)
37.20 (37.22)
61.29 (61.31)
37.70 (37.90)
61.70 (61.31)
37.37 (37.22)
62.39 (62.50)
38.83 (38.84)
66.31 (66.23)
42.12 (42.03)
66.00 (66.23)
42.52 (42.03)
66.46 (66.23)
42.10 (42.03)
67.00 (67.08)
43.37 (43.25)
59.21 (59.56)
39.12 (39.24)
58.28 (58.18)
37.80 (37.84)
4.35 (4.58)
2.50 (2.52)
6.01 (6.15)
3.97 (3.65)
6.23 (6.15)
3.31 (3.65)
6.42 (6.56)
3.98 (3.98)
6.54 (6.56)
3.83 (3.98)
6.54 (6.56)
3.87 (3.98)
6.85 (6.94)
4.43 (4.29)
4.99 (5.19)
3.43 (3.29)
5.60 (5.19)
3.08 (3.29)
5.03 (5.19)
3.42 (3.29)
5.24 (5.59)
3.35 (3.60)
4.47 (4.37)
2.93 (2.88)
3.96 (3.63)
2.38 (2.36)
25.66 (25.68)
14.25 (14.16)
22.00 (21.53)
12.76 (12.80)
21.54 (21.53)
12.76 (12.75)
20.77 (20.43)
12.65 (12.40)
20.41 (20.43)
12.72 (12.39)
20.39 (20.43)
12.47 (12.40)
19.90 (19.44)
12.31 (12.02)
17.97 (18.18)
11.31 (11.52)
18.05 (18.18)
11.68 (11.52)
18.15 (18.18)
11.67 (11.52)
17.27 (17.39)
11.22 (11.21)
16.39 (16.35)
10.31 (10.77)
16.97 (16.96)
11.15 (11.03)
e
17.92 (17.96)
e
16.21 (16.23)
2a/[Pd(3-ICA-PTSC)2Cl2] C13H16N4SCl2Pd
3/3-ICA-iso-PrTSC C13H16N4S
e
16.07 (16.23)
3a/[Pd(3-ICA-IPTSC)2Cl2] C13H16N4SCl2Pd
4/3-ICA-BuTSC C14H18N4S
e
15.70 (15.73)
4a/[Pd(3-ICA-BTSC)2Cl2] C14H18N4SCl2Pd
5/3-ICA-iso-BuTSC C14H18N4S
e
15.54 (15.71)
5a/[Pd(3-ICA-IBTSC)2Cl2] C14H18N4SCl2Pd
6/3-ICA-diETSC C14H18N4S
e
15.39 (15.73)
6a/[Pd(3-ICA-DETSC)2Cl2] C14H18N4SCl2Pd
7/3-ICA-NMButTSC C15H20N4S
e
12.04 (12.06)
7a/[Pd(3-ICA-NMButTSC)2Cl2] C15H20N4SCl2Pd
8/3-ICA-p-TolTSC C17H16N4S
e
14.04 (14.62)
8a/[Pd(3-ICA-p-TolTSC)2Cl2] C17H16N4SCl2Pd
9/3-ICA-o-TolTSC C17H16N4S
e
14.64 (14.64)
9a/[Pd(3-ICA-o-TolTSC)2Cl2] C17H16N4SCl2Pd
10/3-ICA-m-TolTSC C17H16N4S
e
14.59 (14.62)
10a/[Pd(3-ICA-m-TolTSC)2Cl2] C17H16N4SCl2Pd
11/3-ICA-NMBzTSC C18H18N4S
e
11a/[Pd(3-ICA-NMBzlTSC)2Cl2] C18H18N4SCl2Pd
12/3-ICA-2-CBzTSC (C17H15N4SCl)
12a/[Pd(3-ICA-2-CBTSC)2Cl2] C17H15N4SCl3Pd
13/3-ICA-2,4-diFATSC C16H12N4SF
13a/[Pd(3-ICA-2,4-DFATSC)2Cl2] C16H12N4SF2Cl2Pd
14.39 (14.21)
e
20.64 (20.48)
e
14.01 (13.99)
probable structures. All the ligands showed two signals at
172.49e179.12 and 134.11e141.73 ppm assigned due to the
thioamide (C]S) and azomethine carbon (C]N), respec-
tively. The signals from 111.6 to 138.8 ppm were due to the
indole ring carbons. The signal at around 140 ppm is attributed
to the CeH part of azomethine group. The carbons at 1-N-
substituted cyclic groups resonate at their usual positions
and are shown in the data given in Section 3. 13C NMR spectra
also provide diagnostic tools for the elucidation of the coordi-
nation mode of the ligands in complexes. Assignments of the
signals are based on the chemical shifts and intensity patterns
and coordination induced shift (CIS), Dd [Dd ¼ d (com-
plex) ꢁ d (free ligand)], of the signals for carbon atom in the
vicinity of the coordinating functions. Thus the C]S carbon
in ligands experiences CIS value of 5e7 ppm in complexes
1ae13a, indicating the coordination of thione sulphur. As
a result of variation of electron density on coordination, azo-
methine carbon signal is shifted downfield by 2e4 ppm in
their respective complexes, which indicates coordination of ni-
trogen lone pair to metal. Other carbons (CH3, CH2 and aryl
carbons) in these complexes resonate nearly at the same region
as those of free ligands.
180e240 ꢀC. DTA plots for complexes suggest their exother-
mic nature of decomposition in the subsequent stages. Third
stage of decomposition starts immediately after second one
and continues until complete decomposition of the ligands
and formation of PdS as the end product. The final temperature
at which PdS obtained varied from complex to complex but in
any case the formation of final stable product does not go be-
yond 900 ꢀC. Although decomposed fragments of the ligands
could not be approximated due to continuous weight loss, the
total % weight loss of the complexes corresponds to the loss of
the respective ligands after considering the transfer of one
sulphur atom to the metal ion and residues corresponds to
the palladium sulphide.
Additionally, a general splitting pathway with the charac-
teristic peaks was observed by the thiosemicarbazone Pd(II)
complexes 1ae13a. The positive ion FAB mass spectrum of
all the complexes was recorded using m-nitro benzyl alcohol
(NBA) as the matrix. The spectra of Pd(II) complexes showed
a number of informative fragment ions of different intensities
confirming their molecular weights. The result presented here
is interpreted in terms of simple bond cleavages and ligand
losses. The molecular ion peaks were observed as [M]ꢂþ or
[M þ 1]ꢂþ and the major fragmentation pathway involved the
cleavage of thioamide group (CSeNH) giving fragment at
m/z 157 which is the highest mass ion. A peak at m/z 117
corresponds to the indole ring fragment. The azirinium ions
are barely detected and the ions at m/z 13, 91, 77 arise from
further decomposition in the usual way. The FABMS fragmen-
tation pattern of compound 9a is depicted in Scheme 2.
Thermal behavior of all complexes (1ae13a) was studied
in the temperature range 25e900 ꢀC. Fig. 1 provides the
TGA and DTA plots of a representative complex 8a. All the
dehydrated complexes are stable up to ca.110 ꢀC and then de-
compose in three major steps. The temperature range for the
first endothermic step being 110e180 ꢀC which is followed
by exothermic second step covering the temperature range

2020
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
100
90
80
70
60
50
40
30
13.65
10
DeltaY = 9.208%
5
0
DeltaY=17.383%
–5
–10
–15
–20
DeltaY = 26.691%
–25
–27.45
4050
100
150
200 250
Temperature(°C)
300
350
400
Fig. 1. DTA plot for thermal behavior of Pd(II) complex 8a.
2.1. In vitro antiamoebic activity
antiamoebic activity and compared with Pd(II) complexes
and metronidazole, which showed no activity against E. histo-
lytica. It was concluded that the presence of bulky groups at
position N4 of the thiosemicarbazone moiety greatly enhanced
antiamoebic activity. Moreover, it was found that aliphatic
compounds and their complexes were less active than aromatic
compounds and their complexes. The IC50 values indicate that
all the Pd(II) complexes cause a marked inhibition, while the
parent ligands are less active than complexes. The results were
estimated 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. 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 5, 3a, 5a and
8ae13a was evaluated by T-test. The values of the calculated
T were found higher than the table value of T at 5% level, thus
concluding that the character under study is thus significantly
influenced by the treatment. The results indicate that the com-
plexation to Pd increases the activity of parent ligands.
The thiosemicarbazone ligands and their palladium com-
plexes were studied against human kidney epithelial cell line
to ensure that compounds were not toxic to human cells.
None of the compounds inhibited cell growth at a concentra-
tion of 100 mM. To investigate the selectivity of the com-
pounds, the ‘‘safety index’’ (SI) was calculated and defined
as: toxicity IC50/protozoal IC50, where toxicity IC50 is defined
as the concentration of compound that kills 50% of the human
(kidney epithelial) cell line and protozoal IC50 is the concen-
tration that kills 50% of amoeba protozoa. It gives an idea
about the efficacy or toxicity of compounds against human
cells and potentially active 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
Two considerations governed the selection of compounds to
be prepared as thiosemicarbazone analogues for this study. It
was considered to have representatives of the two classes of
1-N-substituted 3-indole carboxaldehyde thiosemicarbazones,
the compounds having 1-N-substituted with different aliphatic
groups. These include: (1) 1-N-substituted derivatives with dif-
ferent aliphatic groups represented by compounds 1e7; and
(2) with different aromatic groups represented by compounds
8e13. In our previous communications it was found that the
activity of the resulting thiosemicarbazones enhanced due to
the substitution of bulky group at N4 position of thiosemicar-
bazides [22,26]. All the compounds were evaluated for their
antiamoebic activity in vitro using HM1:IMSS strain of E. his-
tolytica to investigate the influence of the substitution. The
IC50 values in mM are shown in Table 2 and a relative graph
has been plotted in Fig. 2. Metronidazole had a 50% inhibitory
concentration (IC50 1.6e1.8 mM) in our experiments. 3-Indole
carboxaldehyde thiosemicarbazones taken as ligands showed
IC50 in the range of 0.29e11.52 mM. Compound 12 in this
series showed notable antiamoebic activity in comparison
with the reference drug, metronidazole (IC50 ¼ 0.29 mM vs
IC50 ¼ 1.8 mM of metronidazole). The complexation of thiose-
micarbazones 1e13 with palladium(II) results in complexes
1ae13a, which showed IC50 ¼ 0.29e11.52 mM. All the metal
complexes were found more active than their respective li-
gands indicating that the complexation to metal enhances the
activity of the ligands. This may be explained by Tweedy’s
theory [35], 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 complexes through the lipid layer of cell membrane. More-
over, the complexes 3a, 5a and 8ae13a displayed the most
promising in vitro antiamoebic activity and were found to be
better inhibitors of the parasite than metronidazole. The Pd-
complex precursor [Pd(DMSO)2Cl2] was also evaluated for

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
H3C
2021
analyses (C, H, N) were performed 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 re-
corded 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 spectrophotometer. 1H
NMR and 13C NMR spectra were obtained at ambient temper-
ature using a Bruker Spectrospin DPX-300 MHz spectropho-
tometer 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 million 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 us-
ing argon/xenon (6 kV, 10 mA) as the FAB gas and m-nitro
benzyl alcohol (NBA) as the matrix. Thermogravimetric anal-
ysis of the complexes was performed on a TG 51 thermogravi-
metric analyzer under nitrogen atmosphere with the heating
rate of 10 ꢀC/min.
NH
HN
C
H
S
C
N
Pd
N
H
+
Cl
Cl
[Pd-H]
.
+
m/z = 486
m/z = 107
H
N
PdCl2
C
HN
H
CH3
S
C
N
CH3
N
H
m/z = 309
H
N
HN
C
H
S
C
N
C10H9N4S
m/z = 411
3.1. Synthesis of indole-3-carboxaldehyde
thiosemicarbazones: a general method
N
H
+
H
C
+
N
.
N
N
N
All thiosemicarbazones were synthesized by refluxing an
ethanolic solution of cycloalkylaminothiocarbonylhydrazines
(3 mmol) and indole-3-carboxaldehyde (3 mmol) at 80 ꢀC
for 3 h with continuous stirring. After cooling at ca. 10 ꢀC
for 24 h, the precipitated compound was filtered and recrystal-
lized from appropriate solvent.
H
N
N
H
C9H6N3
m/z = 157
.
+
.
+
CH
C
3.1.1. Indole-3-carboxaldehyde-N(4) thiosemicarbazone,
3-ICA-TSC (1)
N
NH2
H
Light brown solid (methanolechloroform); UV/vis: n
(cmꢁ1) 30 303, 37 313, 48 780; IR: nmax (cmꢁ1) 3378, 3232
(NH), 1614 (C]N), 1538 (C]C), 1251 (CeN), 752 (C]S);
1H NMR (DMSO): (d, ppm) 12.16 (1H, s, eNH), 11.60 (1H,
s, eNH), 11.18 (2H, s, eNH2), 8.29 (1H, s, eCH]N), 8.23
(1H, d, indole C4eH, J ¼ 7.14 Hz), 8.03 (1H, s, indole C2e
H), 7.43 (1H, d, indole C7eH, J ¼ 7.14 Hz), 7.10e7.22 (2H,
m, indole C5eH and C6eH); 13C NMR (CDCl3): (d, ppm)
173.10 (C]S), 139.96 (C]N), 136.70, 128.35, 122.6, 121.8,
120.29, 116.51, 111.63 (aryleC).
C8H7N
m/z =117
Scheme 2. Proposed mass fragmentation pattern for compound 9a.
of the thiosemicarbazone ligands show significant values of SI.
In contrast, compound 12a has lowest cytotoxicity and highest
antiamoebic activity and in an overall result, complexes 3a, 5a
and 8ae13a show the more favorable safety profile along with
the most promising antiamoebic activity. The result indicates
that MTT assay showed that the compounds are non-toxic to
human kidney epithelial cell line. Detailed studies on the
mechanism of action of these compounds as well as further
modifications of these and other related thiosemicarbazones
are in progress.
3.1.2. Indole-3-carboxaldehyde-N(4)propyl
thiosemicarbazone, 3-ICA-PrTSC (2)
Greenish black solid (methanolechloroform); UV/vis: n
(cmꢁ1) 28 818, 38 610, 48 309; IR: nmax (cmꢁ1) 3265, 3120
(NH), 1614 (C]N), 1550 (C]C), 1243 (CeN), 758
1
3. Experimental
(C]S); H NMR (DMSO): (d, ppm) 12.14 (1H, s, NeNH),
11.68 (1H, s, NeNH), 11.18 (1H, t, CSeNH), 8.90 (1H, s,
eCH]N), 8.29 (1H, d, indole C4eH, J ¼ 8 Hz), 7.81 (1H,
s, indole C2eH), 7.43 (1H, d, indole C7eH, J ¼ 8 Hz),
7.12e7.24 (2H, m, indole C5eH and C6eH), 1.69 (2H, m,
eCH2), 3.6 (2H, t, eCH2, J ¼ 6.6 Hz), 0.94 (3H, t, eCH3,
J ¼ 6.97 Hz); 13C NMR (CDCl3): (d, ppm) 175.86 (C]S),
Reactions were monitored by thin-layer chromatography
(TLC) using Merck silica gel 60 F254 precoated thin-layer
plates. All the chemicals were purchased from Aldrich Chem-
ical Company (USA). All the cycloalkylaminothiocarbonyl-
hydrazines were prepared as reported earlier [28]. Elemental

2022
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
Table 2
In vitro antiamoebic activity of compounds against E. histolytica and cytotoxic profile
H
HN
HN
H
C
N
C
N
Cl
Cl
HN
HN
Pd
C
C
S
R
R
S
(1-13)
(1a-13a)
Compound no.
R
Antiamoebic activity
Toxicity profile
IC50 (mM)
Safety
index S.I.
IC50 (mM)
S.D.a
1
1a
eNH2
11.52
3.57
0.01
0.02
>100
>100
>8.68
>28.01
HN-CH-CH3
CH3
2
6.84
0.11
>100
>14.61
2a
3
4.06
7.64
1.44
4.09
2.78
0.02
0.01
0.11
0.03
0.08
>100
>100
>100
>100
>100
>24.63
>13.08
>69.44
>24.44
>35.97
eNHeCH2eCH2eCH2eCH3
eNHeCH2eCH2eCH3
3a
4
4a
HN-CH2-CH-CH3
CH3
5
5.76
0.02
0.13
>100
>100
>17.36
>80.64
5a
1.24
CH2-CH3
N
6
2.58
2.22
0.04
0.08
>100
>100
>38.75
>45.04
CH2-CH3
6a
CH2-CH2-CH2-CH3
N
7
5.50
2.14
0.03
0.08
>100
>100
>18.18
>46.72
CH3
7a
CH3
8
3.25
0.01
0.09
>100
>100
>30.76
>76.92
HN
8a
1.30
H3C
HN
9
2.05
0.02
0.02
>100
>100
>48.78
9a
0.58
>172.41
10
1.29
0.52
0.09
0.07
>100
>100
>77.51
HN
H3C
10a
>192.30
N-CH2-
CH3
11
2.46
0.10
0.02
>100
>100
>40.65
11a
0.74
>135.13
Cl
12
3.28
0.04
0.03
>100
>100
>30.48
N-CH2-
H
12a
0.29
>344.82

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
2023
Table 2 (continued)
Compound no.
R
Antiamoebic activity
Toxicity profile
IC50 (mM)
Safety
index S.I.
IC50 (mM)
S.D.a
0.04
F
F
13
NH
6.05
>100
>16.52
13a
Pd(DMSO)2Cl2
Metronidazole (MNZ)
0.62
8.00
1.81
0.01
0.34
0.03
>100
¼100
>100
>161.29
>12.50
>55.24
a
Standard deviation.
140.55 (C]N), 136.50, 128.38, 122.4, 121.7, 120.25, 116.51,
111.63 (aryleC), 21.94 (CH2), 11.41 (CH3).
s, eNH), 11.16 (1H, s, eNH), 8.29 (1H, s, eCH]N), 8.19
(1H, d, indole C4eH, J ¼ 8.1 Hz), 7.82 (1H, s, indole C2eH),
7.44 (1H, d, indole C7eH, J ¼ 8.1 Hz), 7.12e7.23 (2H, m,
indole C5eH and C6eH), 1.63 (2H, m, eCH2), 1.42 (2H,
m, eCH2), 1.05 (3H, t, eCH3, J ¼ 7.1 Hz); 13C NMR
(CDCl3): (d, ppm) 173.22 (C]S), 140.55 (C]N), 136.74,
128.38, 122.67, 121.98, 121.45, 117.89, 112.03 (aryleC),
44.61 (CH2), 30.64 (CH2), 19.90 (CH2), 13.59 (CH3).
3.1.3. Indole-3-carboxaldehyde-N(4)isopropyl
thiosemicarbazone, 3-ICA-iso-PrTSC (3)
Light brown solid (methanolechloroform); UV/vis: n
(cmꢁ1) 29 940, 37 453, 47 846; IR: nmax (cmꢁ1) 3360, 3131
(NH), 1615 (C]N), 1540 (C]C), 1232 (CeN), 760
1
(C]S); H NMR (DMSO): (d, ppm) 12.53 (1H, s, eNH),
11.63 (1H, s, eNH), 11.22 (1H, s, eNH), 8.31 (1H, s, e
CH]N), 8.15 (1H, d, indole C4eH, J ¼ 7.2 Hz), 7.83 (1H,
s, indole C2eH), 7.45 (1H, d, indole C7eH, J ¼ 7.2 Hz),
7.11e7.23 (2H, m, indole C5eH and C6eH), 3.58 (1H, m,
eCH), 1.14 (6H, d, eCH3, J ¼ 6 Hz); 13C NMR (CDCl3):
(d, ppm) 173.80 (C]S), 141.26 (C]N), 136.58, 128.13,
122.12 121.74, 120.48, 117.11, 111.74 (aryleC), 41.41
(CH), 21.30 (2CH3).
3.1.5. Indole-3-carboxaldehyde-N(4)isobutyl
thiosemicarbazone, 3-ICA-iso-BuTSC (5)
Brown solid (methanolechloroform); UV/vis: n (cmꢁ1
)
29 411, 37 878, 48 780; IR: nmax (cmꢁ1) 3360, 3131 (NH),
1
1614 (C]N), 1545 (C]C), 1232 (CeN), 761 (C]S); H
NMR (CDCl3): (d, ppm) 11.63 (1H, s, eNH), 12.15 (1H,
s, eNH), 11.22 (1H, s, eNH), 8.31 (1H, s, eCH]N),
8.28 (1H, d, indole C4eH, J ¼ 8.5 Hz), 7.81 (1H, s, indole
C2eH), 7.42 (1H, d, indole C7eH, J ¼ 8.5 Hz), 7.10e7.21
(2H, m, indole C5eH and C6eH), 3.49 (2H, t, eCH2,
J ¼ 6.4 Hz), 2.02 (1H, m, eCH), 0.94 (6H, d, eCH3,
J ¼ 6.6 Hz); 13C NMR (CDCl3): (d, ppm) 173.90 (C]S),
141.73 (C]N), 137.84, 127.08, 123.36, 122.08, 121.45,
117.89, 111.78 (aryleC), 51.78 (CH2), 28.10 (CH), 20.42
(2CH3).
3.1.4. Indole-3-carboxaldehyde-N(4)butyl
thiosemicarbazone, 3-ICA-BuTSC (4)
Light brown solid (methanolechloroform); UV/vis: n
(cmꢁ1) 28 985, 37 037, 48 780; IR: nmax (cmꢁ1) 3365, 3139
(NH), 1612 (C]N), 1540 (C]C), 1233 (CeN), 762 (C]S);
1H NMR (CDCl3): (d, ppm) 12.14 (1H, s, eNH), 11.61 (1H,
DOSE RESPONSE GRAPH
12
10
8
Ligands
Pd(II) Complex
Pd(II) Precursor
Metronidazole
6
4
2
0
1
2
3
4
5
6
7
8
TREATMENT
9
10
11
12
13
14
Fig. 2. In vitro activity of indole thiosemicarbazones and their Pd(II) complexes exhibiting comparison of IC50 values (mM).

2024
K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
Fig. 3. Correlation of safety index and antiamoebic activity.
3.1.6. Indole-3-carboxaldehyde-N(4,4)diethyl
thiosemicarbazone, 3-ICA-diETSC (6)
7.07 (2H, d, phenyl-H, J ¼ 7.8 Hz), 2.32 (3H, s, eCH3); 13C
NMR (CDCl3): (d, ppm) 173.08 (C]S), 134.11 (C]N),
137.19, 136.01, 134.1, 132.71, 130.74, 127.99, 126.7, 122.6,
121.8, 120.96, 120.28, 111.45 (aryleC), 21.00 (CH3).
Yellow solid (methanolechloroform); UV/vis: n (cmꢁ1
)
30 395, 37 037, 47 846; IR: nmax (cmꢁ1) 3363, 3170 (NH),
1
1616 (C]N), 1518 (C]C), 1243 (CeN), 759 (C]S); H
NMR (DMSO): (d, ppm) 12.15 (1H, s, eNH), 11.51 (1H, s,
eNH), 8.90 (1H, s, eCH]N), 8.32 (1H, d, indole C4eH,
J ¼ 7.9 Hz), 7.85 (1H, s, indole C2eH), 7.43 (1H, d, indole
C7eH, J ¼ 7.9 Hz), 7.11e7.31 (2H, m, indole C5eH and
C6eH), 3.82 (6H, m, eCH3), 1.21 (4H, t, eCH2, J ¼ 6 Hz);
13C NMR (CDCl3): (d, ppm) 177.90 (C]S), 140.63 (C]N),
138.87, 128.08, 123.67, 122.56, 121.74, 117.43, 111.33
(aryleC), 41.21 (2CH2), 11.80 (2CH3).
3.1.9. Indole-3-carboxaldehyde-N(4)o-toluidine
thiosemicarbazone, 3-ICA-o-TolTSC (9)
Light brown solid (DMSO); UV/vis: n (cmꢁ1) 29 498,
38 759, 47 393; IR: nmax (cmꢁ1) 3272, 3149 (NH), 1607
1
(C]N), 1547 (C]C), 1249 (CeN), 760 (C]S); H NMR
(DMSO): (d, ppm) 12.17 (1H, s, eNH), 11.65 (2H, s,
eNH), 11.52 (2H, s, eNH), 8.40 (1H, s, eCH]N), 8.30
(1H, d, indole C4eH, J ¼ 7.8 Hz), 7.87 (1H, s, indole C2eH),
7.50 (2H, d, phenyl-H, J ¼ 7.8 Hz), 7.42 (1H, d, indole
C7eH, J ¼ 7.8 Hz), 7.15e7.28 (2H, m, indole C5eH and
C6eH), 7.07e7.13 (2H, m, phenyl-H), 2.28 (3H, s, eCH3);
13C NMR (CDCl3): (d, ppm) 172.82 (C]S), 134.17 (C]N),
144.9, 136.02, 133.02, 127.99, 126.90, 124.68, 123.74,
122.6, 121.8, 120.80, 120.30, 116.05, 111.67 (aryleC),
17.90 (CH3).
3.1.7. Indole-3-carboxaldehyde-N(4,4)methyl butyl
thiosemicarbazone, 3-ICA-NMButTSC (7)
Light brown solid (chloroform); UV/vis: n (cmꢁ1) 29 239,
38 167, 48 076; IR: nmax (cmꢁ1) 3215, 3112 (NH), 1621
1
(C]N), 1578 (C]C), 1244 (CeN), 765 (C]S); H NMR
(DMSO): (d, ppm) 12.14 (1H, s, eNH), 11.71 (1H, s, eNH),
8.90 (1H, s, eCH]N), 0.91 (3H, t, eCH3, J ¼ 6.7 Hz), 8.34
(1H, d, indole C4eH, J ¼ 7.1 Hz), 7.91 (1H, s, indole
C2eH), 7.48 (1H, d, indole C7eH, J ¼ 7.1 Hz), 7.15e7.29
(2H, m, indole C5eH and C6eH), 3.96 (2H, t, eCH2,
J ¼ 6.7 Hz), 3.67 (3H, s, eCH3), 2.27 (2H, m, eCH2), 1.59
(2H, m, eCH2); 13C NMR (CDCl3): (d, ppm) 177.76
(C]S), 140.73 (C]N), 137.00, 128.07, 123.37, 122.45,
121.67, 117.34, 112.56 (aryleC), 37.40 (CH3), 28.71 (CH2),
14.03 (CH3).
3.1.10. Indole-3-carboxaldehyde-N(4)m-toluidine
thiosemicarbazone, 3-ICA-m-TolTSC (10)
Green solid (DMSO); UV/vis: n (cmꢁ1) 29 585, 37 735,
47 393; IR: nmax (cmꢁ1) 3267, 3182 (NH), 1609 (C]N),
1
1550 (C]C), 1244 (CeN), 758 (C]S); H NMR (DMSO):
(d, ppm) 12.18 (1H, s, eNH), 11.69 (1H, s, eNH), 11.58
(1H, s, eNH), 8.40 (1H, s, eCH]N), 8.41 (1H, s, phenyl-
H), 8.35e8.82 (3H, m, phenyl-H), 8.33 (1H, d, indole C4eH,
J ¼ 7.8 Hz), 7.91 (1H, s, indole C2eH), 7.45 (1H, d, indole
C7eH, J ¼ 7.8 Hz), 7.00e7.20 (2H, m, indole C5eH and
C6eH), 2.33 (3H, s, eCH3); 13C NMR (CDCl3): (d, ppm)
174.23 (C]S), 134.16 (C]N), 137.20, 136.21, 136.01,
134.10, 132.56, 130.78, 128.01, 126.45, 122.76, 121.89,
120.96, 120.28, 111.67 (aryleC), 21.13 (CH3).
3.1.8. Indole-3-carboxaldehyde-N(4)p-toluidine
thiosemicarbazone, 3-ICA-p-TolTSC (8)
Light yellow solid (DMSO); UV/vis: n (cmꢁ1) 29 411,
38 759, 47 393; IR: nmax (cmꢁ1) 3256, 3154 (NH), 1610
1
(C]N), 1544 (C]C), 1213 (CeN), 763 (C]S); H NMR
(DMSO): (d, ppm) 12.17 (1H, s, eNH), 11.69 (1H, s, eNH),
11.55 (1H, s, eNH), 8.40 (1H, s, eCH]N), 8.23 (1H, d, in-
dole C4eH, J ¼ 7.6 Hz), 7.91 (1H, s, indole C2eH), 7.48
(1H, d, indole C7eH, J ¼ 7.6 Hz), 7.50 (2H, d, phenyl-H,
J ¼ 7.8 Hz), 7.12e7.23 (2H, m, indole C5eH and C6eH),
3.1.11. Indole-3-carboxaldehyde-N(4,4)methylbenzyl
thiosemicarbazone, 3-ICA-NMBzTSC (11)
Dark yellow solid (DMSO); UV/vis: n (cmꢁ1) 29 498,
37 735, 47 846; IR: nmax (cmꢁ1) 3388, 3161 (NH), 1613

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
2025
1
(C]N), 1540 (C]C), 1243 (CeN), 756 (C]S); H NMR
(DMSO): (d, ppm) 12.13 (1H, s, eNH), 11.53 (1H, s, eNH),
8.46 (1H, s, eCH]N), 8.26 (1H, d, indole C4eH,
J ¼ 7.5 Hz), 7.88 (1H, s, indole C2eH), 7.74 (1H, d, indole
C7eH, J ¼ 7.5 Hz), 7.26e7.46 (2H, m, indole C5eH and
C6eH), 7.04e7.24 (5H, m, phenyl-H), 5.21 (2H, s, eCH2),
3.18 (3H, s, eCH3); 13C NMR (CDCl3): (d, ppm) 179.12
(C]S), 140.73 (C]N), 139.75, 136.71, 132.71, 129.62,
129.59, 127.94, 127.73, 122.6, 121.80, 120.96, 120.28,
111.98 (aryleC), 53.91 (CH3), 38.90 (CH3).
(NH), 1587 (C]N), 1218 (CeN), 723 (C]S), 514, 457
1
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.16 (1H,
s, eNH), 8.68 (2H, s, eNH), 8.56 (1H, s, eNH), 8.13 (1H,
s, eCH]N), 7.09e7.64 (5H, m, aryleH); 13C NMR
(CDCl3): (d, ppm) 178.21 (C]S), 142.92 (C]N), 136.70,
128.35, 122.6, 121.8, 120.29, 116.51, 111.63 (aryleC);
FABMS: m/z 396 [M], 367, 323, 157, 117, 107.
3.2.2. Dichloro(indole-3-carboxaldehyde-N(4)propyl
thiosemicarbazone) palladium(II), [Pd(3-ICA-PTSC)2Cl2] (2a)
Dark brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
3.1.12. Indole-3-carboxaldehyde-N(4)2-chlorobenzyl
thiosemicarbazone, 3-ICA-2-CBzTSC (12)
21 097, 36 954, 34 013, 45 248; IR: nmax (cmꢁ1) 3263, 3118
(NH), 1590 (C]N), 1221 (CeN), 725 (C]S), 514, 459
Dark yellow solid (DMSO); UV/vis: n (cmꢁ1) 29 411,
38 759, 48 076; IR: nmax (cmꢁ1) 3289, 3166 (NH), 1611
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.14 (1H,
1
s, eNH), 10.53 (1H, s, eNH), 8.47 (1H, s, eNH), 8.02 (1H,
s, eCH]N), 1.63 (2H, m, eCH2), 3.61 (2H, t, eCH2,
J ¼ 6.6 Hz), 0.96 (3H, t, eCH3, J ¼ 6.98 Hz), 7.09e8.25 (5H,
m, aryleH); 13C NMR (CDCl3): (d, ppm) 183.01 (C]S),
143.03 (C]N), 136.50, 128.38, 122.4, 121.7, 120.25, 116.51,
111.63 (aryleC), 21.94 (CH2), 11.41 (CH3); FABMS: m/z 437
[M], 383, 209, 157, 117, 107.
1
(C]N), 1547 (C]C), 1243 (CeN), 757 (C]S); H NMR
(DMSO): (d, ppm) 12.15 (1H, s, eNH), 11.65 (1H, s, eNH),
11.46 (1H, t, eNH), 8.44 (1H, s, eCH]N), 8.28 (1H, d, indole
C4eH, J ¼ 8 Hz), 7.85 (1H, s, indole C2eH), 8.08 (1H, d, in-
dole C7eH, J ¼ 8 Hz), 7.42e7.52 (2H, m, indole C5eH and
C6eH), 7.08e7.33 (4H, m, phenyl-H), 4.75 (2H, d, eCH2,
J ¼ 6.5 Hz); 13C NMR (CDCl3): (d, ppm) 175.02 (C]S),
140.55 (C]N), 139.63, 136.70, 134.57, 132.71, 130.87,
130.21, 128.99, 127.91, 127.68, 122.60, 121.80, 120.96,
120.28, 111.63 (aryleC), 49.31 (CH2).
3.2.3. Dichloro(indole-3-carboxaldehyde-N(4)isopropyl
thiosemicarbazone) palladium(II),
[Pd(3-ICA-IPTSC)2Cl2] (3a)
Dark brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
3.1.13. Indole-3-carboxaldehyde-N(4)2,4-difluoroaniline
thiosemicarbazone, 3-ICA-2,4-diFATSC (13)
23 584, 26 666, 34 246, 45 248; IR: nmax (cmꢁ1) 3357, 3128
(NH), 1590 (C]N), 1233 (CeN), 728 (C]S), 512, 457
Grayish brown solid (DMSO); UV/vis: n (cmꢁ1) 29 239,
38 461, 48 076; IR: nmax (cmꢁ1) 3291, 3184 (NH), 1610
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.52 (1H,
1
s, eNH), 10.23 (1H, s, eNH), 8.15 (1H, s, eCH]N), 3.56
(1H, m, eCH), 1.15 (6H, d, eCH3, J ¼ 6 Hz), 6.95e8.46
(5H, m, aryleH); 13C NMR (CDCl3): (d, ppm) 179.08
(C]S), 144.08 (C]N), 136.58, 128.13, 122.12, 121.74,
120.48, 117.11, 111.74 (aryleC), 41.41 (CH), 21.30 (2CH3);
FABMS: m/z 437 [M], 366, 214, 157, 117, 107.
1
(C]N), 1540 (C]C), 1244 (CeN), 758 (C]S); H NMR
(DMSO): (d, ppm) 12.14 (1H, s, eNH), 11.62 (1H, s, eNH),
11.18 (1H, s, eNH), 9.07 (1H, br s, eCH]N), 8.60 (1H, s,
phenyl-H), 8.27 (2H, d, phenyl-H, J ¼ 8.8 Hz), 8.15 (1H, d, in-
dole C4eH, J ¼ 9.5 Hz), 7.81 (1H, s, indole C2eH), 7.40 (1H,
d, indole C7eH, J ¼ 9.5 Hz), 7.11e7.23 (2H, m, indole C5eH
and C6eH); 13C NMR (CDCl3): (d, ppm) 172.49 (C]S),
134.34 (C]N), 166.82 (CeF), 158.38 (CeF), 136.87,
132.67, 129.16, 127.99, 125.86, 122.6, 121.8, 120.96, 120.42,
120.28, 112.09 (aryleC).
3.2.4. Dichloro(indole-3-carboxaldehyde-N(4)butyl
thiosemicarbazone) palladium(II),
[Pd(3-ICA-BTSC)2Cl2] (4a)
Deep red solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
23 584, 26 954, 34 364, 46 512; IR: nmax (cmꢁ1) 3363, 3135
(NH), 1595 (C]N), 1233 (CeN), 1099 (C]S), 528, 453
3.2. Synthesis of Pd(II) complexes of
thiosemicarbazones: a general method
1
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.14 (1H,
s, eNH), 8.65 (1H, s, eNH), 8.15 (1H, s, eCH]N), 1.63
(2H, m, eCH2), 1.42 (2H, m, eCH2), 1.04 (3H, t, eCH3,
J ¼ 7.1 Hz), 7.22e7.73 (5H, m, aryleH); 13C NMR
(CDCl3): (d, ppm) 180.56 (C]S), 142.76 (C]N), 136.74,
128.38, 122.67, 121.98, 121.45, 117.89, 112.03 (aryleC),
44.61 (CH2), 30.64 (CH2), 19.90 (CH2), 13.59 (CH3);
FABMS: m/z 452 [M], 273, 323, 209, 157, 117, 107.
To a hot solution of the appropriate ligand (2 mmol) in
methanol (10 mL) was added a solution of palladium chloride
(2 mmol) dissolved in minimum quantity of methanol and the
reaction mixture was heated under reflux for 1e3 h. After
keeping the solution at 0 ꢀC overnight the colored solid sepa-
rated out. This was filtered off and washed with hot water
followed by small quantity of methanol and dried to give
amorphous solids.
3.2.5. Dichloro(indole-3-carboxaldehyde-N(4)isobutyl
thiosemicarbazone) palladium(II),
3.2.1. Dichloro(indole-3-carboxaldehyde-N(4)
[Pd(3-ICA-IBTSC)2Cl2] (5a)
thiosemicarbazone) palladium(II), [Pd(3-ICA-TSC)2Cl2] (1a)
Deep red solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
Reddish brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
23 697, 26 954, 34 247, 45 872; IR: nmax (cmꢁ1) 3357, 3129
(NH), 1586 (C]N), 1232 (CeN), 731 (C]S), 517, 423
24 390, 26 954, 34 602, 45 248; IR: nmax (cmꢁ1) 3376, 3229

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K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
1
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.14 (1H, s,
eNH), 8.45 (1H, s, eCH]N), 3.26 (2H, t, eCH2,
J ¼ 6.4 Hz), 2.02 (1H, m, eCH), 0.94 (6H, d, eCH3,
J ¼ 6.6 Hz), 7.23e8.64 (5H, m, aryleH); 13C NMR
(CDCl3): (d, ppm) 178.80 (C]S), 143.84 (C]N), 137.84,
127.08, 123.36, 122.08, 121.45, 117.89, 111.78 (aryleC),
51.78 (CH2), 28.10 (CH), 20.42 (2CH3); FABMS: m/z 452
[M], 273, 241, 157, 117, 107.
(NH), 1594 (C]N), 1231 (CeN), 1108 (C]S), 549, 447
1
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.17 (1H, s,
eNH), 9.09 (1H, s, eNH), 8.64 (1H, s, eNH), 8.59 (1H, s,
eCH]N), 2.23 (3H, s, eCH3), 7.19e7.59 (5H, m, aryleH);
13C NMR (CDCl3): (d, ppm) 176.17 (C]S), 137.96 (C]N),
144.9, 136.02, 133.02, 127.99, 126.90, 124.68, 123.74, 122.6,
121.8, 120.80, 120.30, 116.05, 111.67 (aryleC), 17.90
(CH3); FABMS: m/z 486 [M], 451, 411, 397, 309, 157, 117,
107, 89.
3.2.6. Dichloro(indole-3-carboxaldehyde-N(4,4)diethyl
thiosemicarbazone) palladium(II),
[Pd(3-ICA-DETSC)2Cl2] (6a)
3.2.10. Dichloro(indole-3-carboxaldehyde-N(4)m-toluidine
thiosemicarbazone) palladium(II), [Pd(3-ICA-m-
TolTSC)2Cl2] (10a)
Brown solid (methanoleDMSO); UV/vis: n (cmꢁ1) 24 155,
31 546, 34 364, 44 643; IR: nmax (cmꢁ1) 3361, 3167 (NH), 1587
(C]N), 1243 (CeN), 735 (C]S), 548, 437 (PdeN, PdeS);
1H NMR (DMSO-d6): (d, ppm) 12.15 (1H, s, eNH), 10.37
(1H, s, eNH), 8.64 (1H, s, eCH]N), 3.71 (6H, m, eCH3),
1.21 (4H, t, eCH2, J ¼ 6 Hz), 7.00e8.75 (5H, m, aryleH);
13C NMR (CDCl3): (d, ppm) 182.11 (C]S), 144.37 (C]N),
138.87, 128.08, 123.67, 122.56, 121.74, 117.43, 111.33 (ar-
yleC), 41.21 (2CH2), 11.80 (2CH3); FABMS: m/z 465 [M],
379, 273, 157, 117, 107.
Brown solid (methanoleDMSO); UV/vis: n (cmꢁ1) 20 747,
26 954, 34 843, 45 872; IR: nmax (cmꢁ1) 3265, 3181 (NH),
1595 (C]N), 1231 (CeN), 739 (C]S), 535, 447 (PdeN,
1
PdeS); H NMR (DMSO-d6): (d, ppm) 12.49 (1H, s, eNH),
9.25 (1H, s, eNH), 8.79 (1H, s, eNH), 8.48 (1H, s, e
CH]N), 2.34 (3H, s, eCH3), 6.77e8.19 (5H, m, aryleH);
13C NMR (CDCl3): (d, ppm) 179.04 (C]S), 138.21 (C]N),
137.20, 136.21, 136.01, 134.10, 132.56, 130.78, 128.01,
126.45, 122.76, 121.89, 120.96, 120.28, 111.67 (aryleC),
21.13 (CH3). FABMS: m/z 486 [M], 470, 411, 309, 279,
209, 157, 117, 107, 88.
3.2.7. Dichloro(indole-3-carboxaldehyde-N(4,4)methyl
butyl thiosemicarbazone) palladium(II), [Pd(3-ICA-
NMButTSC)2Cl2] (7a)
Light brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
3.2.11. Dichloro(indole-3-carboxaldehyde-
N(4,4)methylbenzyl thiosemicarbazone) palladium(II),
[Pd(3-ICA-NMBzlTSC)2Cl2] (11a)
20 619, 26 810, 34 130, 45 249; IR: nmax (cmꢁ1) 3213, 3110
(NH), 1550 (C]N), 1231 (CeN), 737 (C]S), 537, 446
1
Dark brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.14 (1H,
21 978, 26 810, 34 130, 46 512; IR: nmax (cmꢁ1) 3386, 3159
(NH), 1588 (C]N), 1230 (CeN), 732 (C]S), 512, 437
s, eNH), 9.51 (1H, s, eNH), 8.86 (1H, s, eNH), 8.74 (1H,
s, eCH]N), 3.91 (2H, t, eCH2, J ¼ 6.7 Hz), 3.65 (3H, s, e
CH3), 2.27 (2H, m, eCH2), 1.57 (2H, m, eCH2), 7.02e7.69
(5H, m, aryleH); 13C NMR (CDCl3): (d, ppm) 181.96
(C]S), 145.01 (C]N), 137.00, 128.07, 123.37, 122.45,
121.67, 117.34, 112.56 (aryleC), 37.40 (CH3), 28.71 (CH2),
14.03 (CH3); FABMS: m/z 465 [M], 258, 157, 117, 107.
1
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.12 (1H,
s, eNH), 11.83 (1H, s, eNH), 8.72 (1H, s, eCH]N), 4.83
(2H, s, eCH2), 3.18 (3H, s, eCH3), 7.08e8.79 (10H, m, ar-
yleH); 13C NMR (CDCl3): (d, ppm) 185.56 (C]S), 143.76
(C]N), 139.75, 136.71, 132.71, 129.62, 129.59, 127.94,
127.73, 122.6, 121.80, 120.96, 120.28, 111.98 (aryleC),
53.91 (CH3), 38.90 (CH3); FABMS: m/z 499 [M], 289, 214,
209, 157, 117, 107, 105.
3.2.8. Dichloro(indole-3-carboxaldehyde-N(4)p-toluidine
thiosemicarbazone) palladium(II), [Pd(3-ICA-
p-TolTSC)2Cl2] (8a)
Orange solid (methanoleDMSO); UV/vis: n (cmꢁ1) 20 747,
26 954, 34 602, 47 393; IR: nmax (cmꢁ1) 3254, 3150 (NH), 1586
(C]N), 1231 (CeN), 734 (C]S), 548, 457 (PdeN, PdeS);
1H NMR (DMSO-d6): (d, ppm) 12.17 (1H, s, eNH), 9.25
(1H, s, eNH), 8.79 (1H, s, eNH), 8.48 (1H, s, eCH]N),
2.32 (3H, s, eCH3), 7.16e7.69 (5H, m, aryleH); 13C NMR
(CDCl3): (d, ppm) 179.01 (C]S), 138.09 (C]N), 137.19,
136.01, 134.1, 132.71, 130.74, 127.99, 126.7, 122.6, 121.8,
120.96, 120.28, 111.45 (aryleC), 21.00 (CH3); FABMS: m/z
486 [M], 391, 379, 157, 117, 107, 77.
3.2.12. Dichloro(indole-3-carboxaldehyde-
N(4)2-chlorobenzyl thiosemicarbazone) palladium(II),
[Pd(3-ICA-2-CBTSC)2Cl2] (12a)
Dark brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
22 831, 26 954, 34 364, 45 872; IR: nmax (cmꢁ1) 3289, 3164
(NH), 1596 (C]N), 1232 (CeN), 736 (C]S), 544, 446
(PdeN, PdeS); 1H NMR (DMSO-d6): (d, ppm) 12.07
(1H, s, eNH), 11.59 (1H, s, eNH), 8.71 (1H, s, eNH),
8.62 (1H, s, eCH]N), 4.74 (2H, d, eCH2, J ¼ 6.6 Hz),
7.12e8.13 (9H, m, aryleH); 13C NMR (CDCl3): (d,
ppm) 183.72 (C]S), 144.07 (C]N), 139.63, 136.70,
134.57, 132.71, 130.87, 130.21, 128.99, 127.91, 127.68,
122.60, 121.80, 120.96, 120.28, 111.63 (aryleC), 49.31
(CH2); FABMS: m/z 520 [M], 504, 368, 260, 214, 157,
117, 107.
3.2.9. Dichloro(indole-3-carboxaldehyde-N(4)o-toluidine
thiosemicarbazone) palladium(II), [Pd(3-ICA-o-
TolTSC)2Cl2] (9a)
Maroon solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
20 161, 26 954, 34 483, 46 512; IR: nmax (cmꢁ1) 3265, 3181

K. Husain et al. / European Journal of Medicinal Chemistry 43 (2008) 2016e2028
2027
3.2.13. Dichloro(indole-3-carboxaldehyde-N(4)2,4-
difluoroaniline thiosemicarbazone) palladium(II),
[Pd(3-ICA-2,4-DFATSC)2Cl2] (13a)
of the resulting solution in each well was determined at
490 nm with a microplate reader (Labsystem Multiskane
Bichromatic, UK). 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
the best-fitting straight line from which the IC50 value was
found.
Blackish brown solid (methanoleDMSO); UV/vis: n (cmꢁ1
)
21 277, 36 810, 34 483, 47 619; IR: nmax (cmꢁ1) 3289, 3183
(NH), 1590 (C]N), 1221 (CeN), 730 (C]S), 545, 461
1
(PdeN, PdeS); H NMR (DMSO-d6): (d, ppm) 12.14 (1H, s,
eNH), 11.05 (1H, s, eNH), 10.48 (1H, s, eNH), 8.96 (1H,
s, eCH]N), 7.08e8.89 (8H, m, aryleH); 13C NMR
(CDCl3): (d, ppm) 179.06 (C]S), 139.96 (C]N), 166.82
(CeF), 158.38 (CeF), 136.87, 132.67, 129.16, 127.99,
125.86, 122.6, 121.8, 120.96, 120.42, 120.28, 112.09 (aryle
C). FABMS: m/z 543 [M], 442, 184, 157, 117, 107, 91.
3.4. MTT toxicity assay
For the toxicity assay, transformed human kidney epithe-
lium (Graham) cells were continuously maintained in culture
at 37 ꢀC in 5% CO2. The MTT (3-[4,5-dimethylthiazol-2yl]-
2,5-diphenyltetrazolium bromide, USB) cellular viability
assay was used to determine the toxicity profile of the com-
pounds [40]. The trypsinized cell suspension was adjusted to
0.5 million cells/mL and plated out with the various com-
pounds. After 44 h of incubation, 2 mM MTT was added to
the plates and incubated for a further 4 h, DMSO was then
added to stop the reaction and dissolve the formazan crystals.
The absorbance was read at the test wavelength of 540 nm and
reference wavelength of 690 nm and the percentage cellular
viability calculated with appropriate controls taken into ac-
count. The mean ꢄ S.D. values of IC50 values in Table 2 are
from three independent experiments.
3.3. In vitro testing against E. histolytica
All the N(4)-substituted indole thiosemicarbazones (1e13)
and their palladium complexes (1ae13a) were screened in
vitro for antiamoebic activity against (HM1:IMSS ) strain of
E. histolytica by using a microplate method [36,37]. DMSO
(40 mL) [38,39] was added to all the samples (w1 mg) fol-
lowed by enough culture medium to obtain a 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 concentrations of 0.1 mg/mL.
Twofold serial dilutions were made in the wells of 96-well
microtiter plate (Costar) in 170 mL of medium. Each test in-
cluded metronidazole as a standard amoebicidal 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 detach the organ-
isms from the side of the flask. The number of amoeba per mL
was estimated with a heamocytometer and trypan blue exclu-
sion test was used to confirm viability. The cell suspension
used was diluted to 105 organisms/mL by adding fresh me-
dium 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 ꢃ 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 cm thick), Secured with tape,
placed in a modular incubating chamber (Flow Laboratories,
High Wycombe, UK), and gassed for 10 min with nitrogen be-
fore incubation at 37 ꢀC for 72 h.
Acknowledgements
This work was supported from a grant received from
Department of Science and Technology [Grant no. VII-
PRDSF/44/2004-05/TT], New Delhi, India.
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