Cyclooctadiene Ru(II) complexes of thiophene-2-carboxaldehyde-derived thiosemicarbazones: synthesis, characterization and antiamoebic activity

Article abstract of DOI:10.1016/j.ejmech.2006.01.014

Thiosemicarbazones (TSC) 1-10 were synthesized by condensing substituted thiosemicarbazide with thiophene-2-carboxaldehyde. These thiosemicarbazones were further reacted with [Ru(η⁴ -C₈H₁₂)(CH₃CN)₂Cl<

Full text of DOI:10.1016/j.ejmech.2006.01.014

European Journal of Medicinal Chemistry 41 (2006) 592–598
http://france.elsevier.com/direct/ejmech
Original article
Cyclooctadiene Ru(II) complexes of thiophene-2-
carboxaldehyde-derived thiosemicarbazones: synthesis,
characterization and antiamoebic activity
Shailendra Singh a, Fareeda Athar a, Mannar R. Maurya b, Amir Azam a,*
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
b Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India
Received 13 July 2005; revised and accepted 12 January 2006
Available online 20 March 2006
Abstract
Thiosemicarbazones (TSC) 1–10 were synthesized by condensing substituted thiosemicarbazide with thiophene-2-carboxaldehyde. These
thiosemicarbazones were further reacted with [Ru(η4 –C8H12)(CH3CN)2Cl2] to form complexes of the type [Ru(η4 –C8H12)(TSC)Cl2] 1a–10a.
Thiosemicarbazones exhibited antiamoebic activity in the range IC50 = 1.09–5.42 μM. In vitro assessment of antiamoebic activity indicated that
the thiosemicarbazones 3, IC50 = 1.67 μM, 4, IC50 = 1.11 μM and 6, IC50 = 1.09 μM showed substantially less IC50 value than metronidazole
(IC50 = 1.87 μM), a commonly used drug against amoebiasis. Cyclooctadiene Ru(II) complexes of thiosemicarbazones showed significant im-
provement in antiamoebic activity (IC50 = 0.30–1.39 μM). All the complexes possess noteworthy potencies and showed less IC50 values than
metronidazole against HK-9 strain of Entamoeba histolytica. Among all the complexes, the most promising antiamoebic activities was shown by
the complexes 4a and 6a (IC50 = 0.31 μM of 4a and IC50 = 0.30 μM of 6a versus metronidazole).
© 2006 Elsevier SAS. All rights reserved.
Keywords: Thiophene-2-carboxaldehyde; Thiosemicarbazones; Ru(II) Complexes; Antiamoebic activity; Entamoeba histolytica.
1. Introduction
apeutic demand. Thiosemicarbazones, a class of compounds,
has been tested for their potential antitumor [5], antimalarial
[6], antiviral activity [7], radio protector [8], anticonvulsant
[9], trypanocidal agents [10–12], ulcer inhibitor and anticancer
agents [13–15]. The thiosemicarbazones of thiophene-2-car-
boxaldehyde and their antileukemic activity are reported pre-
viously [16,17]. The significant leishmanicidal, anticancer and
anti-HIV activity [18,19] of hydrazones of thiophene-2-carbox-
aldehyde led us to study the screening of thiosemicarbazones
against E. histolytica.
(ImH)[trans-Ru(Im)2Cl4] has been used as anticancer agent
[20]. Earlier studies on Ru complexes such as, cis-
RuCl2(DMSO)4, as antineoplastic agents, have suggested a
DNA binding mechanism [21]. The complex RuCl2(CTZ)2
(CTZ = clotrimazole) displays good activity against Trypano-
soma cruzi, [22] while RuCl2(CQ)2 (CQ = chloroquine) is effi-
cient against Plasmodium falciparum [23]. In our earlier stu-
dies, some thiophene-2-carboxaldehyde thiosemicarbazones
and Ru(II) and Pd(II) complexes showed promising results
against E. histolytica [24,25]. In this paper we report the synth-
esis and characterization of new thiosemicarbazones of thio-
Amoebiasis, a protozoan infection, is the second leading
cause of death. It is associated with liver and brain abscess
and afflicts over 50 million people per annum leading to
50,000–100,000 deaths annually [1]. Recently, a case of inva-
sive lung amoebiasis in which patient showed superior vena
cava (SVC) syndrome and a brain abscess without liver invol-
vement is reported. [2] The drugs currently used in the clinical
treatment, nitroimidazole/metronidazole are considered to be
the most effective against this parasitic disease but do not war-
rant complete cure. Entamoeba histolytica, the causative agent
of amoebiasis, is able to adapt to therapeutically relevant levels
of the drug and may develop resistance to nitroimidazoles/me-
tronidazole [3]. The long-term use of medications produces un-
desirable side effects in patients [4]. Therefore, research on
new drugs for the treatment still constitutes an important ther-
* Corresponding author. Tel.: +91 11 2698 1717x3253;
fax: +91 11 2698 0229, +91 11 2698 1232.
E-mail address: amir_sumbal@yahoo.co.in (A. Azam).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.01.014

S. Singh et al. / European Journal of Medicinal Chemistry 41 (2006) 592–598
593
1
phene-2-carboxaldehyde and their ruthenium (II) complexes, in
which the N-4 thiosemicarbazone moiety is replaced by cyclic
and arylic amines. These compounds were tested in vitro for
their ability to inhibit the growth of E. histolytica using micro-
dilution method.
1493 (C = C), 1141 (C–N), 1067 (C = S); H NMR (CDCl3):
(δ, ppm) 9.07 (1H, s, –NH), 7.89 (1H, s, –CH = N), 7.20–7.59
(3H, m, aryl), 4.91 (1H, m, –CH), 4.79 (8H, m, –CH2), 1.78
(3H, d, J = 5.3 Hz, –CH3),.
2.1.3. 4-(2-Ethylpiperidine)-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-2EPTSC] (3)
2. Experimental
Yellow solid (methanol/chloroform). Yield: 59%; m.p.
210 °C. Anal. calc. for C13H19N3S2: C 55.48, H 6.80, N
14.93; found: C 55.46, H 6.84, N 15.08%; UV–vis: νmax
(cm–1) 28985, 37037, 48780; IR: νmax (cm–1) 3147 (NH),
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 chemi-
cal company (USA). Elemental analyses (C, H, N) were carried
out by Central Drug Research Institute, Lucknow, India, and
the results were within 0.4% of the theoretical values. Chlorine
was estimated by decomposing the complexes with
Na2O2/NaOH and precipitated as AgCl with AgNO3 after dis-
solving in dil. HNO3. Melting points were recorded on a KSW
melting point apparatus and were uncorrected. Electronic spec-
tra were recorded in methanol on a Shimadzu UV-1601 PC
UV-Visible spectrophotometer. IR spectra on KBr disks were
1
1531 (C = N), 1492 (C = C), 1130 (C–N), 1045 (C = S); H
NMR (CDCl3): (δ, ppm) 8.81 (1H, s, –NH), 8.24 (1H, s, –
CH = N), 7.09–7.98 (3H, m, aryl), 3.61 (1H, m, –CH), 3.27
(10H, m, –CH2), 1.75 (3H, d, J = 5.1 Hz, –CH3).
2.1.4. 4-(4-Benzylpiperidine)-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-4BPTSC] (4)
Yellow solid (methanol). Yield: 62%; m.p. 200 °C. Anal.
calc. for C18H21N3S2: C 62.97, H 6.12, N 12.24; found: C
63.04, H 6.18, N 12.20%; UV–vis: νmax (cm–1) 29940,
37453, 47846; IR: νmax (cm–1) 3212 (NH), 1542 (C = N),
recorded on
a
Perkin Elmer model 1620 FT-IR
spectrophotometer. 1H NMR data were obtained at ambient
temperature using a Bruker Spectrospin DPX-300 MHz spec-
trophotometer in CDCl3 for ligands and DMSO for complexes
using tetramethylsilane as an internal standard. Splitting pat-
terns are designated as follows; s, singlet; d, doublet; t, triplet;
m, multiplet. Chemical shift values are given in (ppm). Ther-
mograms of the complexes were recorded under nitrogen on a
TG 51 thermogravimetric analyzer with increasing the tem-
perature at 10 °C min–1.
1
1499 (C = C), 1145 (C–N), 1072 (C = S); H NMR (CDCl3):
(δ, ppm) 8.99 (1H, s, –NH), 8.24 (1H, s, –CH = N), 7.09–7.49
(8H, m, aryl), 4.80 (1H, m, –CH), 4.75 (10H, m, –CH2).
2.1.5. 4-(4-Phenylpiperazine)-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-NPPTSC] (5)
Light brown solid (methanol). Yield: 63%; m.p. 115 °C.
Anal. calc. for C16H18N4S2: C 58.15, H 5.49, N 16.95; found:
C 58.13, H 5.52, N 17.05%; UV–vis: νmax (cm–1) 29154,
37843, 48309; IR: νmax (cm–1) 3120 (NH), 1600 (C = N),
2.1. Synthesis of thiosemicarbazones: general method
1
1545 (C = C), 1132 (C–N), 1018 (C = S); H NMR (CDCl3):
All thiosemicarbazones were synthesized by mixing an aqu-
eous solution of thiocarbonylhydrazines (0.003 mole in 10 ml)
and ethanolic solution of thiophene-2-carboxaldehyde (0.003
mole in 10 ml) at 25 °C for 3 h with continuous stirring. After
cooling, the precipitated compound was filtered and recrystal-
lized from appropriate solvent.
(δ, ppm) 10.37 (1H, s, –NH), 8.54 (1H, s, –CH = N), 6.87-7.85
(8H, m, aryl), 4.21 (8H, m, –CH2).
2.1.6. 4-(2-Adamantyl)-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-ADMTSC] (6)
White solid (ethanol/acetone). Yield: 57%; m.p. 115 °C.
Anal. calc. for C16H22N3S2: C 59.96, H 6.92, N 13.11; found:
C 60.09, H 7.06, N 13.09%; UV–vis: νmax (cm–1) 29154,
38167, 48076; IR: νmax (cm–1) 3200 (NH), 1650 (C = N),
2.1.1. 4-Cyclohexyl-4-methyl-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-NMCHTSC] (1)
Light brown solid (chloroform). Yield: 65%; m.p. 192 °C.
Anal. calc. for C13H19N3S2: C 55.48, H 6.80, N 14.93; found:
C 55.50, H 6.85, N 14.90%; UV–vis: νmax (cm–1) 29154,
38023, 47846; IR: νmax (cm–1) 3154 (NH), 1585 (C = N),
1
1535 (C = C), 1135 (C–N), 1057 (C = S); H NMR (CDCl3):
(δ, ppm) 9.67 (1H, s, –NH), 8.45 (1H, d, –NH), 7.69 (1H, s, –
CH = N), 7.02–7.45 (3H, m, aryl), 4.05–4.13 (18H, m, ada-
mantyl ring).
1
1497 (C = C), 1143 (C–N), 1076 (C = S); H NMR (CDCl3):
(δ, ppm) 9.32 (1H, s, –NH), 8.71 (1H, s, –CH = N), 7.26–7.98
(3H, m, aryl), 3.69 (1H, m, –CH), 3.57 (10H, m, –CH2), 1.60
(3H, s, –CH3).
2.1.7. 4-o-Toluidine-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-o-TolTSC] (7)
White solid (acetone). Yield: 47%; m.p. 205 °C. Anal. calc.
for C13H13N3S2: C 56.70, H 4.76, N 15.26; found: C 56.75, H
4.80, N 15.30%; UV–vis: νmax (cm–1) 29850, 37526, 47393;
IR: νmax (cm–1) 3215 (NH), 1548 (C = N), 1490 (C = C), 1107
2.1.2. 4-Methylpiperidine-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-4MPTSC] (2)
Yellow solid (methanol). Yield: 68%; m.p. 140 °C. Anal.
calc. for C12H17N3S2: C 53.90, H 6.41, N 15.71; found: C
53.81, H 6.45, N 15.79%; UV–vis: νmax (cm–1) 30120,
37174, 49019; IR: νmax (cm–1) 3167 (NH), 1663 (C = N),
1
(C–N), 1040 (C = S); H NMR (CDCl3): (δ, ppm) 9.97 (2H, s,
–NH), 8.87 (1H, s, –CH = N), 7.65–8.11 (7H, m, aryl), 2.30
(3H, s, –CH3).

594
S. Singh et al. / European Journal of Medicinal Chemistry 41 (2006) 592–598
2.1.8. 4-m-Toluidine-1-(thiophene-2-ylmethylene)
2.2.2. Dichloro (4-methylpiperidine-1-(thiophene-2-
thiosemicarbazide, [2-TCA-m-TolTSC] (8)
ylmethylene)thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-4MPTSC)Cl2] (2a)
White solid (acetone). Yield: 45%; m.p. 196 °C. Anal. calc.
for C13H13N3S2: C 56.70, H 4.76, N 15.26; found: C 56.75, H
4.81, N 15.30%; UV–vis: νmax (cm–1) 29585, 38759, 47393;
IR: νmax (cm–1) 3168 (NH), 1556 (C = N), 1511 (C = C), 1119
Dark brown solid (methanol/DMSO); yield: 71%; dec. temp.
271 °C. Anal. calc. for C20H29N3S2Cl2Ru: C 43.87, H 5.34, N
7.67, Cl 12.95; found: C 43.90, H 5.39, N 7.70, Cl 12.90%;
UV–vis: νmax (cm–1) 22331, 27548, 36995, 49816; IR: νmax
(cm–1) 3284 (NH), 1591 (C = N), 1497 (C = C), 1039 (C = S),
1
(C–N), 1041 (C = S); H NMR (CDCl3): (δ, ppm) 9.71 (2H, s,
–NH), 8.24 (1H, s, –CH = N), 7.18–7.49 (7H, m, aryl), 2.35
(3H, s, –CH3).
1
508, 470, 438 (Ru-N, Ru-S); H NMR (DMSO-d6): (δ, ppm)
7.89 (1H, s, –CH = N), 7.13–7.69 (3H, m, aryl), 4.70 (1H, m,
–CH), 4.39 (8H, m, –CH2), 3.49 (1H, s, –NH), 2.45 (3H, d,
–CH3), 2.39 (4H, m, exo CH2), 2.01 (4H, m, endo CH2).
2.1.9. 4-p-Toluidine-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-p-TolTSC] (9)
Light yellow solid (methanol). Yield: 54%; m.p. 192 °C.
Anal. calc. for C13H13N3S2: C 56.70, H 4.76, N 15.26; found:
C 56.71, H 4.82, N 15.31%; UV–vis: νmax (cm–1) 29761,
38167, 48309; IR: νmax (cm–1) 3260 (NH), 1537 (C = N),
1492 (C = C), 1163 (C–N), 1087 (C = S); H NMR (CDCl3):
(δ, ppm) 9.53 (2H, s, –NH), 8.80 (1H, s, –CH = N), 6.96–7.33
(7H, m, aryl), 2.29 (3H, s, –CH3).
2.2.3. Dichloro (4-(2-ethylpiperidine)-1-(thiophene-2-
ylmethylene)thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-2EPTSC)Cl2] (3a)
Dark brown solid (methanol/DMSO); yield: 65%; dec. temp.
286 °C. Anal. calc. for C21H31N3S2Cl2Ru: C 44.91, H 5.56, N
7.48, Cl 12.63; found: C 44.95, H 5.60, N 7.50, Cl 12.57%;
UV–vis: νmax (cm–1) 21945, 26316, 37878, 49160; IR: νmax
(cm–1) 3435 (NH), 1561 (C = N), 1509 (C = C), 1018 (C = S),
1
1
2.1.10. 4-(4-Methylbenzyl)-1-(thiophene-2-ylmethylene)
thiosemicarbazide, [2-TCA-NMBzlTSC] (10)
489, 456 (Ru-N, Ru-S); H NMR (DMSO-d6): (δ, ppm) 8.54
(1H, s, –CH = N), 7.07–7.39 (3H, m, aryl), 4.95 (1H, s, –
NH), 3.58 (1H, m, –CH), 3.31 (10H, m, –CH2), 2.65 (4H, m,
exo CH2), 2.39 (4H, m, endo CH2), 1.95 (3H, t, –CH3).
Yellow solid (methanol/chloroform). Yield: 56%; m.p.
195 °C. Anal. calc. for C14H15N3S2: C 58.10, H 5.22, N
14.52; found: C 58.05, H 5.24, N 14.61%; UV–vis: νmax
(cm–1) 29498, 37598, 47619; IR: νmax (cm–1) 3170 (NH),
2.2.4. Dichloro (4-(4-benzylpiperidine)-1-(thiophene-2-
ylmethylene)thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-4BPTSC)Cl2] (4a)
1
1598 (C = N), 1515 (C = C), 1147 (C–N), 1088 (C = S); H
NMR (CDCl3): (δ, ppm) 9.17 (1H, s, –NH), 7.89 (1H, s, –
CH = N), 6.96–7.61 (8H, m, aryl), 3.42 (2H, s, –CH2), 1.79
(3H, s, –CH3).
Brown solid (methanol/DMSO); yield: 69%; dec. temp.
279 °C. Anal. calc. for C26H33N3S2Cl2Ru: C 50.07, H 5.33,
N 6.74, Cl 11.37; found: C 49.95, H 5.39, N 6.80, Cl
11.40%; UV–vis: νmax (cm–1) 21890, 36736, 49508; IR: νmax
(cm–1) 3437 (NH), 1553 (C = N), 1485 (C = C), 1056 (C = S),
2.2. Preparation of complexes: general method
1
519, 487, 442 (Ru-N, Ru-S); H NMR (DMSO-d6): (δ, ppm)
A solution of appropriate thiosemicarbazone (0.001 mole)
in dry methanol (10 ml) was added to a stirred suspension of
[Ru(η4-C8H12) (CH3CN)2Cl2] (0.001 mole) in hot methanol
(10 ml). The obtained mixture was heated at reflux on a water
bath for 4 h during which period starting material dissolved
and complex started to separate. After keeping the reaction
flask at room temperature for 2 h, the brown solid was filtered,
washed with methanol and dried in vacuo over silica gel.
8.61 (1H, s, –CH = N), 7.11–7.38 (8H, m, aryl), 4.95 (1H, s, –
NH), 3.61 (1H, m, –CH), 3.10 (10H, m, –CH2), 2.60 (4H, m,
exo CH2), 2.38 (4H, m, endo CH2).
2.2.5. Dichloro (4-(4-phenylpiperazine)-1-(thiophene-2-
ylmethylene)thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-NPPTSC)Cl2] (5a)
Dark brown solid (methanol/DMSO); yield: 72%; dec.
temp. 285 °C. Anal. calc. for C24H30N4S2Cl2Ru: C 47.21, H
4.95, N 9.18, Cl 11.61; found: C 47.25, H 5.00, N 9.22, Cl
11.66%; UV–vis: νmax (cm–1) 21431, 24938, 36995, 49513;
IR: νmax (cm–1) 3256 (NH), 1629 (C = N), 1538 (C = C),
2.2.1. Dichloro (4-cyclohexyl-4-methyl-1-(thiophene-2-
ylmethylene)thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-NMCHTSC)Cl2] (1a)
1
Brown solid (methanol/DMSO); yield: 54%; dec. temp.
207 °C. Anal. calc. for C21H31N3S2Cl2Ru: C 44.91, H 5.56,
N 7.48, Cl 12.63; found: C 45.07, H 5.60, N 7.40, Cl
12.71%; UV–vis: ν (cm–1) 22010, 36736, 49419; IR: νmax
(cm–1) 3237 (NH), 1607 (C = N), 1484 (C = C), 1057
1012 (C = S), 497, 452 (Ru-N, Ru-S); H NMR (DMSO-d6):
(δ, ppm) 8.14 (1H, s, –CH = N), 7.13–7.61 (8H, m, aryl), 4.19
(1H, s, –NH), 3.92 (8H, s, –CH2), 2.65 (4H, m, exo CH2), 2.19
(4H, m, endo CH2).
1
(C = S), 514, 578, 541 (Ru-N, Ru-S); H NMR (DMSO-d6):
2.2.6. Dichloro (4-(2-adamantyl)-1-(thiophene-2-ylmethylene)
thiosemicarbazide) cyclooctadiene ruthenium(II)
(δ, ppm) 8.69 (1H, s, –CH = N), 6.94–7.18 (3H, m, aryl),
4.98 (1H, s, –NH), 3.57 (1H, m, –CH), 3.21 (3H, s, –CH3),
2.92 (10H, s, –CH2), 2.54 (4H, m, exo CH2), 2.18 (4H, m,
endo CH2).
[Ru(η4-C8H12)(2-TCA-ADMTSC)Cl2] (6a)
Brown solid (methanol/DMSO); yield: 54%; dec. temp.
292 °C. Anal. calc. for C24H33N3S2Cl2Ru: C 48.07, H 5.55,

S. Singh et al. / European Journal of Medicinal Chemistry 41 (2006) 592–598
595
N 7.01, Cl 11.82; found: C 47.97, H 5.59, N 7.10, Cl 11.79%;
UV–vis: νmax (cm–1) 21998, 36315, 49513; IR: νmax (cm–1)
3281 (NH), 1669 (C = N), 1528 (C = C), 1027 (C = S), 511,
4.21 (1H, s, –NH), 3.44 (2H, s, –CH2), 3.31 (3H, s, –CH3),
2.71 (4H, m, exo CH2), 2.22 (4H, m, endo CH2).
1
2.3. Organism culture and in vitro testing against E. histolytica
478, 437 (Ru-N, Ru-S); H NMR (DMSO-d6): (δ, ppm) 8.03
(1H, s, –CH = N), 7.13–7.49 (3H, m, aryl), 3.83 (1H, s, –NH),
3.69–3.78 (18H, m, adamantyl ring), 2.53 (4H, m, exo CH2),
2.34 (4H, m, endo CH2).
Preliminary experiments were carried out to determine the
antiamoebic activities of the in vitro culture against the HK-9
strain of E. histolytica as previously described [26]. The
E. histolytica strain HK-9 was cultured in tubes (15 cm3) by
using Diamond TYIS-33 medium [27]. All the compounds
were dissolved in DMSO (40 μl) [28,29] followed by adding
enough culture medium to obtain concentration of 1 mg ml–1.
Stock solutions of the compounds were prepared freshly before
use at a concentration of 0.1 mg ml–1. Twofold serial dilutions
were made in the wells of 96-well microtiter plate (Costar).
Each test includes metronidazole as a standard amoebicidal
drug, with control wells (culture medium plus amoebae) and
a blank (culture medium only). All the experiments were car-
ried out in triplicate at each concentration level and repeated
thrice. The amoebae suspension was prepared from a confluent
culture by pouring off the medium at 37 °C and adding 5 ml of
fresh medium, chilling the culture tube on ice to detach the
organisms from the side of the flask. The number of amoeba
per ml was estimated with a haemocytometer, using trypan
blue exclusion to confirm the viability. The suspension was
diluted to 105 organism per ml by adding fresh medium and
170 μl of this suspension was added to the test and control
wells in the plate so that the wells were completely filled (total
volume, 340 μl). An inoculum of 1. 7 × 104 organisms per well
was chosen so that confluent, but not excessive growth, took
place in control wells. Plates were sealed 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. By inverting the plate, the cul-
ture media was removed with gentle shaking and then imme-
diately washed with sodium chloride solution (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.
It was allowed to dry at room temperature. After drying amoe-
ba were fixed with chilled methanol by keeping it in ice bath
for 15 min. dried and stained with (0.5%) aqueous eosin for
15 min. The stained plate was washed once with tap water,
then twice with distilled water and allowed to dry. A 200 μl
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. The % inhibition of amoe-
bal growth was calculated from the optical densities of the con-
trol and test wells and plotted against the logarithm of the dose
of the drug tested. Linear regression analysis was used to de-
termine the best fitting straight line from which the IC50 value
was found.
2.2.7. Dichloro (4-o-toluidine-1-(thiophene-2-ylmethylene)
thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-o-TolTSC)Cl2] (7a)
Dark brown solid (methanol/DMSO); yield: 74%; dec.
temp. 241 °C. Anal. calc. for C21H25N3S2Cl2Ru: C 45.40, H
4.54, N 7.56, Cl 12.76; found: C 45.45, H 4.60, N 7.60, Cl
12.77%; UV–vis: νmax (cm–1) 21946, 24630, 37736, 49915;
IR: νmax (cm–1) 3355 (NH), 1557 (C = N), 1489 (C = C),
1
1070 (C = S), 509, 478, 439 (Ru-N, Ru-S); H NMR (DMSO-
d6): (δ, ppm) 8.10 (1H, s, –CH = N), 7.23–7.69 (7H, m, aryl),
3.93 (1H, s, –NH), 2.59 (4H, m, exo CH2), 2.19 (4H, m, endo
CH2), 2.04 (3H, s, –CH3).
2.2.8. Dichloro (4-m-toluidine-1-(thiophene-2-ylmethylene)
thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-m-TolTSC)Cl2] (8a)
Brown solid (methanol/DMSO); yield: 68%; dec. temp.
235 °C. Anal. calc. for C21H25N3S2Cl2Ru: C 45.40, H 4.54,
N 7.56, Cl 12.79; found: C 45.32, H 4.61, N 7.60, Cl
12.80%; UV–vis: νmax (cm–1) 22127, 25915, 36418, 49423;
IR: νmax (cm–1) 3295 (NH), 1581 (C = N), 1502 (C = C),
1
1054 (C = S), 484, 445 (Ru-N, Ru-S); H NMR (DMSO-d6):
(δ, ppm) 7.93 (1H, s, –CH = N), 7.10–7.38 (7H, m, aryl), 4.20
(1H, s, –NH), 2.61 (4H, m, exo CH2), 2.24 (4H, m, endo CH2),
2.01 (3H, s, –CH3).
2.2.9. Dichloro (4-p-toluidine-1-(thiophene-2-ylmethylene)
thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-p-TolTSC)Cl2] (9a)
Dark brown solid (methanol/DMSO); yield: 65%; dec.
temp. 250 °C. Anal. calc. for C21H25N3S2Cl2Ru: C 45.40, H
4.54, N 7.56, Cl 12.79; found: C 45.45, H 4.59, N 7.50, Cl
12.80%; UV–vis: νmax (cm–1) 21929, 28985, 37593, 49216;
IR: νmax (cm–1) 3305 (NH), 1594 (C=N), 1515 (C=C), 1019
(C = S), 496, 458 (Ru-N, Ru-S); 1H NMR (DMSO-d6): (δ,
ppm) 7.89 (1H, s, –CH = N), 7.01–7.25 (7H, m, aryl), 3.99
(1H, s, –NH), 2.43 (4H, m, exo CH2), 2.13 (4H, m, endo
CH2), 1.98 (3H, s, –CH3).
2.2.10. Dichloro (4-(4-methylbenzyl)-1-(thiophene-2-
ylmethylene)thiosemicarbazide) cyclooctadiene ruthenium(II)
[Ru(η4-C8H12)(2-TCA-NMBzlTSC)Cl2] (10a)
Dark brown solid (methanol/DMSO); yield: 66%; dec.
temp. 298 °C. Anal. calc. for C22H27N3S2Cl2Ru: C 46.39, H
4.78, N 7.38, Cl 12.45; found: C 46.41, H 4.79, N 7.40, Cl
12.50%; UV–vis: νmax (cm–1) 21445, 25773, 37154, 49152;
IR: νmax (cm–1) 3254 (NH), 1621 (C = N), 1509 (C = C),
3. Results and discussion
1
1057 (C = S), 516, 479, 438 (Ru-N, Ru-S); H NMR (DMSO-
The thiosemicarbazone derivatives were synthesized using
the method as reported earlier [30]. These thiosemicarbazones
d6): (δ, ppm) 8.72 (1H, s, –CH = N), 7.03–7.18 (8H, m, aryl),

596
S. Singh et al. / European Journal of Medicinal Chemistry 41 (2006) 592–598
are insoluble in water and highly soluble in chloroform, metha-
nol, DMF and DMSO. The precursor
[Ru(η4
–C8H12)(CH3CN)2Cl2] used for the synthesis of Ru(II) com-
ent amines. All the thiosemicarbazones 1–10 may exhibit
thione–thiol tautomerism, since they have a thione group
(C = S) and proton adjacent to the thione group [32]. It has
been stated that the thione group (C = S) is relatively unstable
in the monomeric form and tends to turn into a stable C–S
single bond by tautomerism, if there is at least one hydrogen
atom adjacent to the C = S bond. However, all the compounds
showed intense, strong bands in the region 1018–1092 cm–1
due to ν(C = S) stretch and no band near 2570 cm–1 due to
ν(C–SH), suggesting that these ligands remain in the thione
form in the solid-state. The highest frequency band observed
in the 3147–3343 cm–1 region, due to the amine group stretch-
ing vibration in ligands also remains unaffected in the com-
plexes. The downward (C = S) band shift (16–37 cm–1) in the
complexes suggested the coordination of thiocarbonyl sulfur.
The spectra of all the thiosemicarbazones exhibit a strong band
at 1531–1653 cm–1 region due to the ν(C = N) stretch of the
azomethine linkage. In the complexes this band shifted
(14–46 cm–1) to lower frequency. This lowering of the
ν(C = N) stretch on complexation may be attributed to lower-
ing of the C = N bond order as a result of the M-N band for-
mation. Bands in the region ~ 450 cm–1 are assigned to the
ν(M-N) band which further supports the coordination of the
azomethine nitrogen. The two strong bands observed at 716–
680 and 605–597 cm–1 regions are attributed to the thiophene
ring deformation modes in the ligands. These thiophene ring
deformation vibrations are not affected in the complexes.
The electronic spectra of the ligands exhibit three bands in
the region 28,985–30,395, 37,037–38,759 and 47,393–48,
880 cm–1. The probable assignment for the first two bands is
due to the n π* and while last one is due to π π* transitions.
The thiosemicarbazones and their complexes exhibits π π*
band at ca. 48,000 cm–1 and n π* band at ca. 37,000 cm–1,
with little change in the energy of these bands. In complexes
n π* band was located at ca. 25,000 cm–1. In addition, a charge
transfer band was located in the lower energy region. For some
complexes the two bands, which are intraligand, merge to form
one band while in others the lower energy intraligand bands
merge with charge transfer band.
plexes was synthesized by the literature procedure [31]. All
1
compounds were characterized by elemental analysis, IR, H
NMR and electronic spectra and their purity was established
by TLC and melting point. Analytical and spectral data are in
good agreement with the composition of thiosemicarbazones 1-
10 (Fig. 1) and their 1,5-cyclooctadiene Ru(II) complexes 1a–
10a (Fig. 2).
3.1. IR and electronic spectral studies
The IR spectra of the compounds are quite similar despite
the presence of different substituent, which shows that the
charge delocalization was not influenced by substituting differ-
1
3.2. H NMR spectral studies
All the thiosemicarbazones exhibit signal due to the –NH
proton in the region 8.81–11.39 ppm. This signal usually shifts
to upfield and appears at 3.05–4.95 ppm after complexation.
However, in some complexes, –NH proton signal could not
be located in the 0–15 ppm range. This either merges with
aromatic protons or resonate beyond 15 ppm. All other protons
and aromatic protons resonate nearly at the same region with
slight shielding to that of the corresponding free thiosemicar-
bazone.
Fig. 1. Structure of the thiophene-2-carboxaldehyde thiosemicarbazones (1–
10).
3.3. Thermal studies (TGA)
The thermograms of the complexes along with the % loss at
different temperatures were recorded under nitrogen. The T.G.
profiles showed the complexes to be non-solvated and stable
Fig. 2. General structure of the thiophene-2-carboxaldehyde thiosemicarbazone
Ru(II) complexes (1a–10a).

S. Singh et al. / European Journal of Medicinal Chemistry 41 (2006) 592–598
597
up to 200 °C. Further increment of temperature caused decom-
position of all the complexes. In all the decomposition steps
loss of mixed fragments occurred which made it difficult to
predict the loss of any particular group at any step. Although
decomposed fragments of the ligands could not be approxi-
mated due to continuous weight loss of the respective ligand.
The residue corresponds to the ruthenium sulfide, i.e. after con-
sidering the transfer of one sulfur atom to the metal ion.
to the 2- position, rather than 3- or 4- position of the hetero-
cyclic ring and also to those in which a thiocarbonyl, rather
than a carbonyl group, is present [34]. All the complexes 1a–
10a (0.30–1.39 μM) showed less IC50 value than metronida-
zole. The cyclic analogues of thiosemicarbazones were found
to be more active than the arylic analogues. The most promis-
ing among them are Ru(II) complexes 4a and 6a having 4-
benzylpiperidine and 2-adamantyl as N4 substitution. The re-
sults were statistically evaluated by analysis of variance. The
null hypothesis was tested using t-test. The significativity of
the difference between the IC50 values of metronidazole and
the complexes 1a–10a 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 said
to be significantly influenced by the treatment. The Ru-com-
plex precursor [RuCl2(η4-C8H12)(MeCN)2] was also evaluated
for antiamoebic activity. The IC50 value of the metal precursor
establishes that it does not have any effect on growth of
E. histolytica. The results indicate that the complexation to
Ru increases the activity of parent ligands. Detailed studies of
the toxicity of these compounds, mechanism of action as well
as in vivo studies are in progress.
3.4. Biological activity
The new thiosemicarbazones and their ruthenium(II) com-
plexes were screened in vitro against HK-9 strain of
E. histolytica for antiamoebic activity and the results are listed
in Table 1. Metronidazole was used as a reference amoebicidal
drug. The biological test was carried out using DMSO as the
solvent in which all the compounds are stable. Metronidazole
had an inhibitory concentration (IC50) of 1.87 μM in our ex-
periment, which is close to the previously reported IC50 of 1.81
μM obtained against same strain of E. histolytica [33]. The
results were estimated as the percentage of growth inhibition
compared with the untreated controls and plotted as probit va-
lues as a function of the drug concentration. The IC50 and 95%
confidence limits were interpolated in the corresponding dose-
response curve. The thiosemicarbazones 1–10 exhibit IC50 va-
lues in the range (1.09–5.42 μM). The ligands (3, IC50 = 1.67
μM, 4, IC50 = 1.11 μM and 6, IC50 = 1.09 μM) showed better
amoebicidal activity as compared to metronidazole (IC50
= 1.87 μM). It was noted that antiparasitic activity was limited
to those compounds in which the alkylidene group is attached
4. Conclusion
In conclusion, thiosemicarbazones derivatives and their new
Ruthenium(II) complexes derived from thiophene-2-carboxal-
dehyde represent an interesting new family of compounds to
control growth of E. histolytica. The biological behavior
revealed that the thiosemicarbazones 3, 4 and 6 containing
2-ethylpiperidine, 4-benzylpiperidine and adamantylamino as
N4 substitution showed better IC50 value than metronidazole
against E. histolytica. The chelation induced significant
changes in the biological activity of the ligands. Though all
the ruthenium complexes 1a–10a have shown less IC50 value
than metronidazole, the better antiamoebic activity was shown
by cyclic analogues than arylic analogues. The most promising
among them are the Ru(II) complexes 4a and 6a having 4-ben-
zylpiperidine and adamantylamino as N4 substitution.
Table 1
In vitro antiamoebic activity of thiophene-2-carboxaldehyde thiosemicarba-
zones and their Ru(II) complexes against (HK-9) strain of E. histolytica
Compound
1
IC50 (μM)
4.41
1.23
2.58
0.75
1.67
0.52
1.11
0.31
5.42
1.39
1.09
0.30
2.87
0.81
3.13
0.69
3.02
0.74
2.56
0.76
14.36
1.87
S.D.
0.71
1a
0.13
0.18
0.05
0.13
0.03
0.07
0.02
0.77
0.15
0.13
0.06
0.18
0.12
0.35
0.11
0.27
0.09
0.31
0.13
3.47
0.47
2
2a
3
3a
Acknowledgements
4
4a
This work was supported from Council of Scientific and
Industrial Research (Grant no. 27(0117)/02/EMR-ll), New Del-
hi, India.
5
5a
6
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