Novel thiosemicarbazone derivatives as potential antitumor agents: Synthesis, physicochemical and structural properties, DNA interactions and antiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2008.03.006

The paper describes synthesis of several novel thiosemicarbazone derivatives. Furthermore, crystal and molecular structure of 4-diethylamino-salicylaldehyde 4-phenylthiosemicarbazone revealed planarity of conjugated aromatic system, which suggested the po

Full text of DOI:10.1016/j.bmc.2008.03.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5189–5198
Novel thiosemicarbazone derivatives as potential antitumor
agents: Synthesis, physicochemical and structural properties,
DNA interactions and antiproliferative activity
a
a
a
c
c
-
´
ˇ
´
´
Ivica Dilovic, Mirta Rubcic, Visnja Vrdoljak, Sandra Kraljevic Pavelic, Marijeta Kralj,
ˇ ´
Ivo Piantanidab, and Marina Cindric
a,
*
*
´
aFaculty of Science, Chemistry Department, Horvatovac 102a, 10000 Zagreb, Croatia
b
-
Division of Organic Chemistry and Biochemistry, Ruder Bosˇkovic´ Institute Zagreb, Croatia
c
Division of Molecular Medicine, Ruder Bosˇkovic´ Institute, Zagreb, Croatia
-
Received 17 January 2008; revised 27 February 2008; accepted 4 March 2008
Available online 6 March 2008
Abstract—The paper describes synthesis of several novel thiosemicarbazone derivatives. Furthermore, crystal and molecular struc-
ture of 4-diethylamino-salicylaldehyde 4-phenylthiosemicarbazone revealed planarity of conjugated aromatic system, which sug-
gested the possibility of DNA binding by intercalation, especially for here studied naphthalene derivatives. However, here
presented DNA binding studies excluded this mode of action. Physicochemical and structural properties of novel derivatives were
compared with previously studied analogues, taken as reference compounds, revealing distinctive differences. In addition, novel thi-
osemicarbazone derivatives (1, 2 and 5–8) clearly display stronger antiproliferative activity on five tumor cell lines than the reference
compounds 3 and 4, which supports their further investigation as potential antitumor agents.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Several mechanisms of antitumor action of thiosemicar-
bazones were proposed. For example, they could stabi-
lize cleavable complexes formed by topoisomerase II
(topoII) and DNA leading to apoptosis. The stabilizing
effect is mainly due to the alkylation of thiol residues on
the topo II–DNA complex.5 Besides, thiosemicarba-
zones could inhibit ribonucleotide reductase (RR) activ-
ity. RR catalyzes the synthesis of deoxyribonucleotides
required for DNA synthesis. Since deoxyribonucleotides
are present in extremely low levels in mammalian cells, it
is a crucial and rate-controlling step in the pathway
leading to the biosynthesis of DNA. Mammalian ribo-
nucleotide reductase (RR) is composed of two dissimilar
proteins, (R1), which contains polythiols and (R2),
which contains non-heme iron and a free tyrosyl radical.
Both the R1 and R2 subunits contribute to the active
site of the enzyme.6 Since thiosemicarbazones are
known iron chelators and as such can destabilize or
damage the non-heme iron-stabilized tyrosyl free radical
and thus inhibit the catalytical function of RR.
Thiosemicarbazones exhibit various biological activities
and have therefore attracted considerable pharmaceuti-
cal interest.1 They have been evaluated over the last 50
years as antiviral, antibacterial and anticancer therapeu-
tics. Thiosemicarbazones belong to a large group of
thiourea derivatives, whose biological activities are a
function of parent aldehyde or ketone moiety.2,3 Conju-
gated N–N–S tridentate ligand system of thiosemicarba-
zide (NH2–CS–NH–NH2) seems essential for anticancer
activity, possibly due to the observation that structural
alterations that hinder a thiosemicarbazone’s ability to
function as a chelating agent tend to destroy or reduce
its medicinal activity.4 Furthermore, the most active
thiosemicarbazones are those which possess the trans
isomer (i.e., hydrogen bonding involving Oꢀ ꢀ ꢀHN).
Keywords: Thiosemicarbazone; Antitumor evaluation; UV/vis spec-
troscopy; DNA binding.
Very few studies were done on the non-covalent interac-
tions of thiosemicarbazones with double stranded (ds-)
DNA, although DNA damage was often proposed as
possible mechanism of action. For example, biological
*
Corresponding authors. Tel.: +385 1 4571 210; fax: +385 1 46 80 195
(I.P.); tel.: +385 1 4606350 (M.C.); e-mail addresses: pianta@irb.hr;
marina@chem.pmf.hr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.006

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Table 1. General and crystal data, summary of intensity data
collection and structure refinement for the compound (5)
(5)
CHO
OH
H2N
NH
C
S
NH
R2
+
Empirical formula
Formula weight
C18H22N4OS
342.47
R1
Habit and color
Crystal dimension (mm3)
Crystal system, space group
Prismatic, colorless
0.62 · 0.21 · 0.20
Monoclinic, P21/c
CH3OH
NH
˚
Unit cell dimensions (A, ꢁ)
a
b
c
8.8570(3)
18.1129(6)
11.4739(3)
94.974(3)
1833.78(10)
4
CH
N
C
NH
R2
b
3
˚
S
Volume (A )
Z
Dcalc (g cmꢁ3
)
1.240
0.188
728
OH
R1
l (mmꢁ1
F(000)
)
2h range for data collection (ꢁ) 3.74–27.99
h, k, l range
Reflections observed/
ꢁ11 to 11, ꢁ23 to 23, ꢁ15 to 15
30,150/4410 (0.0324)
2576
-OCH
H
;
;
-N(CH CH )
3 2
;
3
R
=
=
2
1
independent (Rint
)
Observed reflections
[I > 2r(I)]
Data/restraints/parameters
Goodness-of-fit on F2
R/wR [I > 2r(I)]a
R/wR (all data)b
R
H ;
Ph
4410/0/305
0.951
2
Scheme 1. The general synthetic procedure and the structures of all
studied compounds.
0.0415/0.1014
0.0749/0.1211
0.375 and ꢁ0.219
Largest diff. peak
and hole (eꢁ A )
3
˚
activity of some thiosemicarbazones substituted by con-
densed aromatic moieties was based on prior formation
of non-covalent complexes with DNA.7 Furthermore, 5-
nitroimidazole-thiosemicarbazone damaged both pyrim-
idine and purine bases8 but it was not clear by which
mode of interaction the active compound approached
DNA. However, a huge number of thiosemicarbazone
derivatives with the potential of direct DNA binding,
like, for example, many derivatives containing con-
densed aromatic moieties,5,9 were never tested for inter-
actions with ds-DNA.
P
P
2
a R ¼ kF o j ꢁ j F ck= F o, w ¼ 1=½r2ðF o2Þ þ ðg1PÞ þ g2Pꢂ where
2
1=2
P ¼ ðF 2o þ 2F 2cÞ=3, S ¼ R½wðF o2 ꢁ F c2Þ =ðNobs ꢁ NparamÞꢂ
.
2
2 1=2
b wR ¼ ½RðF o2 ꢁ F 2c Þ =RðF o2Þ ꢂ
.
2. Results and discussion
2.1. Chemistry
All thiosemicarbazones (1–8) were prepared according
to the modified procedure described in the literature10
starting from corresponding aldehyde and thiosemicar-
bazide or 4-phenylthiosemicarbazide (Scheme 1). Com-
pounds are white or yellow powders easily soluble in
warm DMF, DMSO, acetone, dichloromethane or
Therefore, condensation of various naphthyl- and sali-
cyl- or 3-methoxy-salicylaldehyde analogues with thio-
semicarbazide to form thiosemicarbazone derivatives
(Scheme 1) may result in highly biologically active com-
pounds because of all afore-mentioned reasons.
Figure 1. ORTEP plot of the (5) compound, showing atom-labeling scheme and displacement ellipsoids are drawn at 50% probability level.
Hydrogen atoms are shown as spheres of arbitrary small radii.

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
´
5191
˚
Table 2. Selected bond lengths (A)
chloroform. The IR spectra of powders are in accor-
dance to the literature data for the same type of com-
pounds,11 namely the characteristic intense bands in
the range of 1652–1610 cmꢁ1 are associated with the
m(C@N) and d(N–H) frequencies. The m(OH) band of
phenolic oxygen occurs almost at the same frequency
for all thiosemicarbazones at 3337 or 3339 cmꢁ1. The in-
C1–S1
1.6792(15)
1.342(2)
C1–N1
C2–N3
C9–N1
C17–N4
N2–N3
1.346(2)
1.286(2)
1.412(2)
1.457(2)
1.3866(17)
C1–N2
C6–N4
C16–N4
C4–O1
1.3734(19)
1.456(3)
1.3507(19)
Figure 2. (Top) Centrosymmetric dimers of (5) are held together by N–Hꢀ ꢀ ꢀS hydrogen bonds (R22ð8Þ). Intramolecular O–Hꢀ ꢀ ꢀN hydrogen bonds are
also shown (S11ð6Þ). View is projected down the crystallographic a axis. (Bottom) T-shaped C–Hꢀ ꢀ ꢀp interactions are added to the above-mentioned
interactions.

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
5192
tense m(C–S) band is observed in the range of 760–
775 cmꢁ1
2.2. Study of compounds 1–8 in aqueous solutions
.
2.2.1. Electronic absorption spectra of 1–8. All studied
compounds (1–8) were scarcely soluble in water, form-
ing colloids after agitation in the ultrasonic bath. Only
upon heating some compounds dissolved in water at
about 10ꢁ5 mol dmꢁ3 concentrations, which were not
suitable for the preparation of stock solutions. There-
fore, for easier manipulation in further experiments,
stock solutions of 1–8 were prepared in DMSO at
c = 5 · 10ꢁ3–1 · 10ꢁ2 mol dmꢁ3. For all experiments
small aliquots of DMSO stock solutions were added into
the aqueous medium to give homogeneous solutions
with DMSO content of less than 5%. The UV/vis spectra
of 1–8 are linearly dependent on the concentration of
The yellow prismatic crystals of 4-diethylamino-salicyl-
aldehyde 4-phenylthiosemicarbazone (5) suitable for
X-ray crystallography were obtained from a dichloro-
methane solution by slow evaporation.
General and crystal data for the compound (5) are given
in Table 1.
Like in many thiosemicarbazones, especially ones de-
rived from salicylaldehyde, the thione form (Fig. 1)
dominates in the solid state (C–S bond distance of
1.6792(15) is comparable to the one found in thio-
urea12). The trans configuration of the S1 atom with re-
spect to N3 is present (and with N1 in the cis
configuration with respect to N3). It has been seen that
the above-mentioned configuration of similar thiosemi-
carbazones changes upon complexation.13
compounds up to 8 · 10ꢁ6 mol dmꢁ3
.
Comparison of the UV/vis spectra (Fig. 3) of 1–4 with
those of 5–8 revealed that introduction of phenyl-substi-
tuent on the amino group of thiosemicarbazone influ-
enced only the intensity of the electronic absorption
properties of studied compounds, in most cases increasing
the molar absorptivity. However, electronic properties of
the substituent attached to the salicylic part of studied
thiosemicarbazones had much more pronounced influ-
ence on the UV/vis spectra of 1–8 (Fig. 3). If we consider
compound 4 with unsubstituted salicylic moiety as a refer-
ence, introduction of electron-donating substituents such
as diethylamino- (1) and methoxy- (2) caused merging of
two absorption maxima into one. However, only diethyl-
amino- (1) substituent-induced additionally strong bato-
chromic shift of maximum (DAbsmax(1–2) = 51 nm).
The shape of the UV/vis spectrum of the naphthalene ana-
logue (3) is quite similar to the spectrum of 4 but it is
shifted toward longer wavelengths by about 25 nm. Such
a shift is quite common between most benzene and
naphthalene analogues. The same substituent-induced
effects are observed for 5–7 in comparison to 8.
The molecule (Fig. 1) can be separated in two planar
fragments: salicylthiosemicarbazone (consisting of S1,
C1–C8, N2, N3, N4 and O1) and N-phenyl part (consist-
ing of N1, C9–C14) which are inclined by 58.21(4)ꢁ.
Planarity of the salicylthiosemicarbazone part of mole-
cule enhances delocalization of p electrons, which also
can be seen easily from the bond length distribution
(Table 2). It is interesting to see how p-system spreads
on the diethylamino moiety by achieving additional pla-
narity which is accompanied by shortening of C6–N4
distance. Because of steric hindrance, the methyl groups
(C15 and C18 with attached hydrogen atoms) of the
diethylamino moiety are found on the opposite sides
of the plane defined by C16, N4 and C17 atoms. The
endocyclic bond distances and angles found in the phe-
nyl rings are similar to those in normal sp2 hybridized
moieties.
2.2.2. Thermal dependence of the UV/vis spectra of 1–8.
The aqueous solutions of 1–8 were successively heated up
to 85 ꢁC and the changes were monitored by UV/vis spec-
trophotometry. Upon heating from 25 to 85 ꢁC the UV/
vis spectra of 1, 2, 4 changed less than 5% and upon cool-
ing back to 25 ꢁC the starting spectra are fully recovered.
Heating of aqueous solutions of naphthalene derivatives
3 and 7 caused partial precipitation. In contrast to 1, 2, 4,
the UV/vis spectra of their phenyl-substituted analogues
5, 6, 8 changed considerably upon heating (Fig. 4). Upon
cooling back to 25 ꢁC only starting spectrum of 6 was
fully recovered, while those of 5 and 8 remained signifi-
cantly changed, most likely due to the chemical changes
related to the phenyl-substituent.
As in many non-protonated thiosemicarbazones derived
from salicylaldehide derivatives,14 the salicylaldimine
part of molecule is characterized by an intramolecular
six-membered heteronuclear resonance-assisted hydro-
gen bond (Fig. 2). The thiosemicarbazone part is charac-
terized by an intramolecular five-membered (non-planar)
weak hydrogen.
Molecules are arranged in sheets parallel to the crystallo-
graphic (021) plane and connected by the T-shaped p inter-
actions and relatively close HꢀꢀꢀH contacts (Table 3).
Mentioned sheets are interconnected by N–HꢀꢀꢀS hydrogen
bonds (R22ð8Þ) forming centrosymmetric dimers (Fig. 2).
2.2.3. Fluorimetric properties of 1–8. Solutions of 1–8
exhibited strong fluorescence (Fig. 5), emission inten-
sity being linearly dependent on their concentration
up to 5 · 10ꢁ6 mol dmꢁ3. Similar to that observed in
the UV/vis spectra, introduction of phenyl-substituent
on the amino group of thiosemicarbazone (5–8) influ-
enced only the emission intensity but not the shape of
the fluorescence spectra of 1–4. The only exception is
significant difference between fluorimetric spectra of 3
and 7, where introduction of phenyl- (7) induced
˚
Table 3. Intermolecular contacts (A)
Type
Distance
C–Hꢀ ꢀ ꢀp
C9ꢀ ꢀ ꢀH17Aiii
C11ꢀ ꢀ ꢀH15Biii
C14ꢀ ꢀ ꢀH17Aiii
H10ꢀ ꢀ ꢀH7iii
2.892(23)
2.861(36)
2.885(23)
2.393(23)
Hꢀ ꢀ ꢀH
iii = ꢁx, ꢁy + 1, ꢁz.

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
´
5193
extraordinary strong batochromic shift of emission
maxima (DAbsmax(7–3) = 71 nm). The linear depen-
dence of the UV/vis and fluorimetric spectrum of 7
on the concentration does not support intermolecular
aggregates. Therefore, such an effect could be attrib-
uted to the strong intramolecular interaction between
naphthalene- and phenyl-moiety of 7.
4 and 5). The low solubility of naphthalene derivatives 3
and 7 hampered UV/vis titrations and thermal denatur-
ation experiments with calf thymus (ct-) DNA. Thermal
instability of 5 and 8 did not allow accurate thermal
denaturation experiments with ct-DNA. Thus, as the
representatives of salicylic derivatives (excluding naph-
thalene analogues 3 and 7), compounds 2 and 4 were
tested for binding to the double stranded ct-DNA.
If we consider compound 4 with the unsubstituted
salicylic moiety as a reference, the introduction of
diethylamino-substituent (1) results in hypsocromic
shift of emission maximum, while methoxy-substituent
(2) yields batochromic shift. Obviously, the electron-
donating nature of both substituents is not controlling
fluorescence properties of 1 and 2. It is interesting to
note that replacement of benzene (4) with naphtha-
lene (3) caused batochromic shift of emission
maximum.
Addition of ct-DNA did not yield any measurable
change in the UV/vis spectra of 2 and 4, while fluorimet-
ric titrations revealed only very weak quenching of 2 and
4 fluorescence (<10%) upon titration with ct-DNA. Pro-
cessing of the fluorimetric titration data by Scatchard
equation15 gave us the only possible result values of ra-
tio n[bound compd]/[ct-DNA] ꢃ 1 and Ks ꢄ 105 M. In ther-
mal denaturation experiments addition of 2 or 4 at
ratios r[compd]/[ct-DNA] = 0.3 did not alter thermal dena-
turation properties of DNA, therefore intercalation of
2 and 4 within DNA double helix can be excluded. Fur-
thermore, compounds 2 and 4 do not possess any posi-
tive charge, which would allow significant interaction
with the DNA phosphates. On the other hand, neutral,
aromatic molecules of 2 and 4 are strongly hydrophobic
and therefore should tend to accumulate within the lyp-
2.3. Study of interactions of 1–8 with calf thymus (ct-)
DNA in aqueous solutions
The spectroscopic properties of all studied compounds
in buffered aqueous solutions (Na cacodylate, pH 7,
I = 0.05 M) are comparable to those in pure water (Figs.
1
2
H
2
C
OCH3
OH
N
CH
3
N
H C
2
CH
3
OH
N
S
S
N
H
NH2
N
H
4.5x104
4.0x104
3.5x104
3.0x104
NH
2
1
2
3
4
6
OH
N
4
5
S
6
OH
N
7
N
H
8
NH2
S
3
5
2.5x104
N
H
2.0x104
1.5x104
1.0x104
5.0x103
NH2
H
C
2
OCH
CH
3
3
N
3
H
C
OH
2
CH
OH
N
S
N
N
H
0.0
S
N
H
260 280 300 320 340 360 380 400 420 440
N
H
N
H
λ / nm
7
8
OH
N
OH
N
S
S
N
H
N
H
N
H
N
H
Figure 3. The UV/vis spectra of 1–8 in water.

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
5194
A
B
C
25
25
0.30
40
40
H
L
T
U8
V8
W8
X8
50
50
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
60
0.6
0.5
0.4
0.3
0.2
0.1
0.0
60
70
0.25
0.20
0.15
0.10
0.05
80
70
90
85
95
25 ponovo
25 pon
isosbestic point at
λ = 347 nm
280
300
320
340
360
380
400
420
320
340
360
380
400
420
440
280
300
320
340
360
λ / nm
λ / nm
λ / nm
Figure 4. The dependence of the UV/vis spectra of aqueous solutions of 5 (A), 6 (B) and 8 (C) on the temperature.
320
binding and therefore calculated Scatchard ratio
n[bound compd]/[ct-DNA] is significantly higher than 1. Con-
sequently, calculated binding constants (Ks) are mainly
related to the interactions between molecules of 2 or 4,
respectively.
300
280
260
240
220
200
180
160
140
120
100
80
1*
2
3
4
5
6
7**
8
In conclusion, compounds 2 and 4 as representatives of
other salicylic derivatives (1, 5, 6, and 8), do not show
noteworthy interactions with double-stranded DNA.
The naphthalene derivatives 3 and 7 could not be tested
due to their low solubility.
60
40
20
0
400
2.4. The effect of thiosemicarbazones on the proliferation
of tumor and normal cells
420
440
460
480
500
520
540
560
580
λ / nm
Figure 5. Fluorescence emission spectra of 1–8 in water, 25 ꢁC, c(1–
8) = 1.4 · 10ꢁ6 mol dmꢁ3; excitation wavelength see longer kmax in
Supplementary Material. *1 and **7: due to the very low emission
intensities wider slits had to be used, thus intensity of the shown
spectra is not comparable with the spectra of 2, 3, 4, 5, 6, and 8.
Compounds 1–8 were tested for their potential anti-
proliferative effects using MTT test (as described in
Section 4) on a panel of six human cell lines, five of
which were derived from different cancer types includ-
ing HeLa (cervical carcinoma), MCF-7 (breast carci-
noma), SW620 (colon carcinoma), MiaPaCa-2
(pancreatic carcinoma), Hep-2 (laryngeal carcinoma)
and one from normal diploid fibroblasts, WI 38 (Ta-
ble 4). All tested compounds showed noticeable anti-
proliferative effect having IC50 values in the low
micromolar, or submicromolar range (Table 4).
Regarding the structure–activity relationship, the low-
est activity exhibited previously published compounds
4 and 3,3 which can be considered as reference
ophilic DNA grooves. Therefore, changes in fluores-
cence spectra of 2 and 4 upon addition of ct-DNA most
likely originate from aromatic interactions between mol-
ecules of 2 (or 4) within the DNA grooves and do not
represent interaction of 2 (or 4) with the ct-DNA. Such
agglomerates of 2 (or 4) would allow presence of more
small molecules per one DNA base pair than it is
theoretically possible for intercalation or minor groove
Table 4. In vitro inhibition of thiosemicarbazones 1–8 on the growth of tumor cells and normal human fibroblasts (WI 38)
Compound
IC50 (lM)a
Hep-2
HeLa
MiaPaCa-2
SW620
MCF-7
WI 38
1
2
3
4
5
6
7
8
0.9 0.1
1.4 0.3
0.2 0.2
P5
0.3 0.3
1.6 0.9
0.7 0.5
2.4 0.4
2
2
>5
3
1
0.4 0.2
0.3 0.1
>5
0.2 0.1
0.3 0.2
P5
P5
0.2 0.06
0.4 0.1
>5
>5
0.5 0.5
4.7 0.3
>5
P5
0.7 0.08
1.7 0.2
P5
1.2
P5
0.2 0.3
2.5 0.7
0.6 0.1
2.4 0.8
0.6 0.5
1.6 0.8
0.3 0.2
1
2
0.3 0.1
1.4 1.2
0.6 0.08
1.3 1.1
3
2 0.7
1.5 0.3
3.3 1.3
1
2
0.08
4
a IC50, the concentration that causes a 50% reduction of the cell growth.

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´
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compounds due to unsubstituted salicylic and amino-
parts. The introduction of phenyl-substituent on the
amino group of 3 and 4 increased the activity (com-
pounds 7 and 8, respectively). The exception is a very
strong activity of 3 toward HeLa and Hep-2 cells.
Furthermore, the introduction of polar, electron-
donating substituents as diethylamino- (1) and meth-
oxy- (2) to the salicylic moiety of 4 also increased
the activity. Moreover, the introduction of phenyl-
substituents (5) and (6) abolished the selectivity
among different cell lines observed for unsubstituted
analogues 1 and 2.
on five tumor cell lines than the reference compounds
3 and 4, which supports their further investigation as
potential antitumor agents.
4. Experimental
4.1. Materials
All aldehydes, 4-phenylthiosemicarbazide and thiosemi-
carbazide, were of reagent grade and used as purchased.
C, H, N analyses were provided by the Analytical Ser-
The antiproliferative activity of compounds 3 and 4
has already been described by Lovejoy and Richard-
son,3 whereby compound 4 had lowest activity, which
is in perfect accordance with our results. Moreover,
in our experiments, as well as in the above-mentioned
study, MCF-7 cells were least affected by the treatment
with 3 among tumor cells tested. However, although 3
is in our experiments less active on normal human
fibroblast (WI 38) than on HeLa and Hep-2 tumor
cells, it is equally active as on other tumor cells. There-
fore, significantly lower rate of proliferation of WI 38
cells did not cause the resistance of these cells to 3,
pointing to cytotoxic mode of action. Therefore, the
hypothesis that thiosemicarbazone 3 shows selectivity
in regard to normal cells, as discussed by Lovejoy
and Richardson,3 could not be generalized. Neverthe-
less, here described novel thiosemicarbazone derivatives
(1, 2 and 5–8) clearly display stronger antiproliferative
activity than the parent compounds 3 and 4, which
supports their further investigation as potential antitu-
mor agents.
-
ˇ
´
vices Laboratory of Ruder Boskovic Institute, Zagreb.
1H and 13C NMR spectra were recorded on either a Var-
ian Gemini 300 or a Bruker Avance DPX 300 spectrom-
eter using TMS as an internal standard in DMSO-d6.
IR spectra were recorded as KBr pellets using Perkin-El-
mer Fourier-Transform Spectrum RX1 Spectrophotom-
eter in the region 4500–450 cmꢁ1
.
Diffracted intensities were collected on the Oxford Dif-
fraction Xcalibur 3 diffractometer using graphite mono-
chromated Mo Ka radiation at 293 K. The programs
CrysAlis CCD17 and CrysAlis RED17 were used for data
collection, cell refinement and data reduction. The struc-
ture was solved by direct methods (SHELXS18). Refine-
ment procedure (SHELXL-9719) by full-matrix least
squares methods based on F2 values against all reflec-
tions included anisotropic displacement parameters for
all non-H atoms. The two last-mentioned programs
operated under the WinGX20 program package. The
hydrogen atom positions were obtained from a differ-
ence Fourier map and they were included in the refine-
ment process with isotropic displacement parameters.
A summary of crystal data is presented in Table 1. Pro-
grams PLATON,21 PARST,22 ORTEP23 and Mercury24
were used for analysis of the structure and drawings
preparation.
Another perspective of these novel compounds is
their potential as promising chelators for a number
of biologically relevant cations. Namely, metal chela-
tion is a very important process, useful to afford new
chemical features to metal complexes in order to
make them suitable for practical purposes (e.g., phar-
macological applications). For example, copper(II)
complexes, containing aromatic thiosemicarbazones
were determined to induce apoptosis in human leuke-
mia cell lines.16 Therefore, further studies of these
novel thiosemicarbazones with various metal cations
are underway.
4.2. Synthesis
4.2.1. Synthesis of 4-diethylamino-salicylaldehyde thio-
semicarbazone (1). Thiosemicarbazide (0.92 g, 10 mmol)
was dissolved in 40 mL of dry methanol with stirring
and warming over a period of 30 min. To the warm
thiosemicarbazide solution, 4-diethylamino-salicylalde-
hyde (1.94 g, 10 mmol) in 20 mL of dry methanol
was added and the mixture was stirred and slowly re-
fluxed for 2 h. The mixture was then cooled down to
room temperature when the yellow crystalline com-
pound precipitated. The compound was collected by
filtration, washed well with cold methanol and dried
in vacuum.
3. Conclusions
The crystal and molecular structure of one of the no-
vel thiosemicarbazone derivatives (5) revealed the pla-
narity of the conjugated aromatic system, which
suggested the possibility of DNA binding by intercala-
tion. However, the presented DNA binding studies ex-
cluded this mode of action. Physicochemical and
structural properties of novel derivatives (1, 2 and
5–8) were compared with previously studied analogues
(3 and 4), taken as reference compounds, revealing
distinctive differences. Most likely as a consequence
of substituent effects, the novel thiosemicarbazone
derivatives displayed stronger antiproliferative activity
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 1.15 g (43%). Anal. Calcd for C12H18N4OS: C,
54.11; H, 6.81; N, 21.03; S, 12.04. Found: C, 54.00;
H, 6.90; N, 21.11; S, 11.98%. IR (cmꢁ1) in KBr:
3337 (m), 3150 (s), 2996 (m), 1622 (m), 1610 (m),

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
5196
1596 (s), 760 (m); 1H NMR (d, DMSO-d6, 25 ꢁC,
ppm): 11.07 (s, 1H, NHCS), 9.61 (s, 1H, OH), 8.19
(s, 1H, CH@N), 7.66 and 7.87 (2br s, 1H each,
NH2), 3.33 (q, 4H, CH2N), 1.10 (t, 6H, CH3), 6.09–
7.52 (m, 3H, aromatic); 13C NMR (d, DMSO-d6,
25 ꢁC, ppm): 176.75 (C@S), 142.45 (CH@N), 43.94
(CH2), 12.96 (CH3), 158.26, 150.20, 129.12, 107.51,
104.08, 97.40 (C-aromatic).
4.2.4. Synthesis of salicylaldehyde thiosemicarbazone (4).
Thiosemicarbazide (0.92 g, 10 mmol) was dissolved in
40 mL of dry methanol with stirring and warming over
a period of 30 min. To the warm thiosemicarbazide solu-
tion, salicylaldehyde (1.21 g, 10 mmol) in 10 mL of dry
methanol was added and the mixture was stirred and
slowly refluxed for 2 h. The mixture was then cooled
down to room temperature when the white crystalline
compound precipitated. The compound was collected
by filtration, washed well with cold methanol and dried
in vacuum.
4.2.2. Synthesis of 3-methoxy-salicylaldehyde thiosemi-
carbazone (2). Thiosemicarbazide (0.92 g, 10 mmol)
was dissolved in 40 mL of dry methanol with stirring
and warming over a period of 30 min. To the warm
thiosemicarbazide solution, 3-methoxy-salicylaldehyde
(1.53 g, 10 mmol) in 10 mL of dry methanol was
added and the mixture was stirred and slowly refluxed
for 2 h. The mixture was then cooled down to room
temperature when the white crystalline compound
precipitated. The compound was collected by filtra-
tion, washed well with cold methanol and dried in
vacuum.
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 0.97 g (50%). Anal. Calcd for C8H9N3OS: C,
49.23; H, 4.61; N, 21.54; S, 16.42. Found: C, 49.09; H,
4.50; N, 21.41; S, 16.39%. IR (cmꢁ1) in KBr: 3337
(m), 3133 (s), 2886 (m), 1622 (s), 1610 (s), 1576 (s),
1
775 (m); H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.39
(s, 1H, NHCS), 9.88 (s, 1H, OH), 8.39 (s, 1H, CH@N),
8.12 and 7.94 (2br s, 1H each, NH2), 6.82–7.91 (m, 4H,
aromatic); 13C NMR (d, DMSO-d6, 25 ꢁC, ppm): 177.56
(C@S), 139.55 (CH@N), 156.28, 130.97, 126.65, 120.22,
119.15, 115.92 (C-aromatic).
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 0.85 g (38%). Anal. Calcd for C9H11N3O2S: C,
47.98; H, 4.92; N, 18.65; S, 14.23. Found: C, 47.76; H,
5.00; N, 18.67; S, 14.20%. IR (cmꢁ1) in KBr: 3337
(m), 3130 (s), 2986 (m), 1632 (s), 1613 (s), 1576 (s),
4.2.5. Synthesis of 4-diethylamino-salicylaldehyde 4-phe-
nylthiosemicarbazone (5).
4.2.5.1. Method A. 4-Phenylthiosemicarbazide
(1.67 g, 10 mmol) was dissolved in 40 mL of dry metha-
nol with stirring and warming over a period of 30 min.
To the cold thiosemicarbazide solution, 4-diethyl-
amino-salicylaldehyde (1.94 g, 10 mmol) in 20 mL of
dry methanol was added. A yellow product began to
immediately separate from the red solution. The mixture
was stirred additionally at room temperature for 3 h.
The solution was evaporated and product was filtered
and dried in vacuum without washing.
1
765 (m); H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.39
(s, 1H, NHCS), 9.17 (s, 1H, OH), 8.40 (s, 1H, CH@N),
7.88 and 8.10 (2br s, 1H each, NH2), 3.81(s, 3H, OCH3),
6.77–7.53 (m, 3H, aromatic); 13C NMR (d, DMSO-d6,
25 ꢁC, ppm): 178.05 (C@S), 139.83 (CH@N), 56.25
(CH3O), 148.26, 146.34, 121.14, 119.33, 118.52, 113.19
(C-aromatic).
4.2.3. Synthesis of 2-hydroxy-naphthaldehyde thiosemi-
carbazone (3). Thiosemicarbazide (0.92 g, 10 mmol)
was dissolved in 40 mL of dry methanol with stirring
and warming over a period of 30 min. To the warm
thiosemicarbazide solution, 2-hydroxy-naphthaldehyde
(1.73 g, 10 mmol) in 20 mL of dry methanol was
added and the mixture was stirred at room tempera-
ture for 2 h. The yellow crystalline compound ob-
tained after standing at room temperature over the
night was filtered, washed well with cold methanol
and dried in vacuum.
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 0.97 g (28%). Anal. Calcd for C18H22N4OS: C,
63.13; H, 6.48; N, 16.36; S, 9.36. Found: C, 63.09; H,
6.32; N, 16.30; S, 9.34. IR (cmꢁ1) in KBr: 3339 (m),
3131 (s), 2980 (m), 1652 (s), 1623 (s), 1577 (s), 765 (s);
1H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.49 (s, 1H,
NHCS), 9.85 (s, 1H, NHPh), 9.58 (s, 1H, OH), 8.33 (s,
1H, CH@N), 3.33 (q, 4H, CH2N), 1.10 (t, 6H, CH3),
7.16–7.61 (m, 3H, aromatic), 6.12–7.70 (m, 5H, C6H5);
13C NMR (d, DMSO-d6, 25 ꢁC, ppm): 174.52 (C@S),
142.53 (CH@N), 43.95 (CH2), 12.65 (CH3), 139.43,
128.06, 125.24, 124.86 (C6H5), 158.43, 150.36, 129.20,
107.49, 104.07, 97.32 (C-aromatic).
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 1.27 g (52%). Anal. Calcd for C12H11N3OS: C,
58.76; H, 4.52; N, 17.13; S, 13.07. Found: C, 58.68; H,
4.50; N, 17.07; S, 13.11%. IR (cmꢁ1) in KBr: 3337
(m), 3118 (s), 2986 (m), 1615 (s), 1623 (m), 1586 (s),
1
4.2.5.2. Method B. 4-Diethylamino-salicylaldehyde
(0.24 g, 1.24 mmol) was dissolved in 5 mL of dichloro-
methane and the solution of 4-phenylthiosemicarbazide
(0.21 g, 1.25 mmol) in 20 mL of dichloromethane was
added. The reaction mixture was then stirred at room
temperature for 3 h. The resulting yellow prismatic crys-
tals were collected by filtration and dried over KOH.
764 (m); H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.38
(s, 1H, NHCS), 10.41 (s, 1H, OH), 9.05 (s, 1H, CH@N),
7.81 and 8.20 (2br s, 1H each, NH2), 7.19–8.51 (m, 6H,
aromatic); 13C NMR (d, DMSO-d6, 25 ꢁC, ppm): 177.41
(C@S), 142.92 (CH@N), 156.49, 132.35, 131.40, 128.56,
127.95, 127.75, 123.33, 122.70, 118.23, 109.61 (C-
aromatic).

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
´
5197
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
(m); 1H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.79 (s,
1H, NHCS), 10.68 (s, 1H, OH), 10.11 (s, 1H, NHPh),
9.20 (s, 1H, CH@N), 7.21–8.51 (m, 6H, aromatic),
7.21–7.62 (m, 5H, C6H5); 13C NMR (d, DMSO-d6,
25 ꢁC, ppm): 175.85 (C@S), 143.65 (CH@N), 139.32,
128.33, 125.23, 125.22 (C6H5), 156.78, 132.73, 131.76,
128.19, 128.89, 127.95, 123.63, 122.62, 118.63, 109.83
(C-aromatic).
Yield: 0.42 g (100%). Anal. Calcd for C18H22N4OS: C,
63.13; H, 6.48; N, 16.36; S, 9.36. Found: C, 63.19; H,
6.42; N, 16.35; S, 9.30. IR (cmꢁ1) in KBr: 3339 (m),
3131 (s), 2980 (m), 1652 (s), 1623 (s), 1577 (s), 765 (s);
1H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.49 (s, 1H,
NHCS), 9.85 (s, 1H, NHPh), 9.58 (s, 1H, OH), 8.33 (s,
1H, CH@N), 3.33 (q, 4H, CH2N), 1.10 (t, 6H, CH3),
7.16–7.61 (m, 3H, aromatic), 6.12–7.70 (m, 5H, C6H5);
13C NMR (d, DMSO-d6, 25 ꢁC, ppm): 174.52 (C@S),
142.53 (CH@N), 43.95 (CH2), 12.65 (CH3), 139.43,
128.06, 125.24, 124.86 (C6H5), 158.43, 150.36, 129.20,
107.49, 104.07, 97.32 (C-aromatic).
4.2.8. Synthesis of salicylaldehyde 4-phenylthiosemicar-
bazone (8). 4-Phenylthiosemicarbazide (1.67 g, 10 mmol)
was dissolved in 40 mL of dry methanol with stirring
and warming over a period of 30 min. To the cold thio-
semicarbazide
solution,
salicylaldehyde
(1.23 g,
10 mmol) in 10 mL of dry methanol was added and mix-
ture was stirred at room temperature for a 4 h. Within
10 min a white product began to separate out. After
standing at room temperature over the night, the prod-
uct was filtered off, washed with small volume of cold
methanol and dried in vacuum.
4.2.6. Synthesis of 3-methoxy-salicylaldehyde 4-phenyl-
thiosemicarbazone (6). 4-Phenylthiosemicarbazide (1.67 g,
10 mmol) was dissolved in 40 mL of dry methanol with
stirring and warming over a period of 30 min. To the cold
thiosemicarbazide solution, 3-methoxy-salicylaldehyde
(1.53 g, 10 mmol) in 10 mL of dry methanol was added
and mixture was stirred at room temperature for a 4 h.
Within 10 min a white product began to separate out.
After standing at room temperature over the night, the
product was filtered off, washed with small volume of
cold methanol and dried in vacuum.
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 1.65 g (60%). Anal. Calcd for C14H13N3OS: C,
61.99; H, 4.79; N, 15.49; S, 11.82. Found: C, 61.72; H,
4.68; N, 15.40; S, 11.78%. IR (cmꢁ1) in KBr: 3359
(m), 3259 (s), 2986 (m), 1641 (s), 1630 (s), 1566 (s),
1
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
758 (m); H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.79
(s, 1H, NHCS), 10.06 (s, 1H, NHPh), 9.99 (s, 1H,
OH), 8.53 (s, 1H, CH@N), 6.85–8.10 (m, 4H, aromatic),
7.20–7.60 (m, 5H, C6H5); 13C NMR (d, DMSO-d6,
25 ꢁC, ppm): 176.49 (C@S), 140.80 (CH@N), 139.91,
128.79, 126.41, 125.92 (C6H5), 157.36, 132.09, 127.86,
121.00, 119.99, 116.81 (C-aromatic).
Yield: 1.65 g (54%). Anal. Calcd for C15H15N3O2S: C,
59.81: H, 15.12; N, 13.95; S, 10.64. Found: C, 59.65;
H, 14.99; N, 13.78; S, 10.43%. IR (cmꢁ1) in KBr: 3339
(m), 3159 (s), 2976 (m), 1642 (s), 1633 (s), 1576 (s),
1
768 (m). H NMR (d, DMSO-d6, 25 ꢁC, ppm): 11.74
(s, 1H, NHCS), 10.03 (s, 1H, NHPh), 9.26 (s, 1H,
OH), 8.53 (s, 1H, CH@N), 3.82 (s, 3H, OCH3), 6.80–
7.71 (m, 3H, aromatic), 7.19–7.58 (m, 5H, C6H5); 13C
NMR (d, DMSO-d6, 25 ꢁC, ppm): 175.82 (C@S),
140.04 (CH@N), 55.99 (CH3O), 139.23, 128.10, 125.76,
125.25 (C6H5), 146.23, 147.99, 120.76, 119.02, 118.58,
113.11 (C-aromatic).
4.3. Spectroscopic measurements
The electronic absorption spectra were recorded on Var-
ian Cary 100 Bio spectrometer, and fluorescence emis-
sion spectra were recorded on Varian Eclipse
fluorimeter, in all cases using quartz cuvettes (1 cm).
The measurements were performed in the aqueous buffer
solution (pH 7.0; sodium cacodylate buffer,
I = 0.05 mol dmꢁ3). Under the experimental the condi-
tions used absorbance and fluorescence intensities of
studied compounds were proportional to their
concentrations.
4.2.7. Synthesis of 2-hydroxy-naphthaldehyde 4-phenyl-
thiosemicarbazone (7). 4-Phenylthiosemicarbazide (1.67 g,
10 mmol) was dissolved in 40 mL of dry methanol with
stirring and warming over a period of 30 min. To the cold
thiosemicarbazide solution, 2-hydroxynaphtaldehyde
(1.73 g, 10 mmol) in 20 mL of dry methanol was
added and the mixture was stirred at room temperature
for 4 h. After 30 min the yellow crystalline product
began to separate out. The product was filtered,
washed with small volume of cold methanol and dried
in vacuum.
4.3.1. Interactions with DNA. The calf thymus DNA (ct-
DNA) was purchased from Aldrich, dissolved in the so-
dium cacodylate buffer, I = 0.05 mol dmꢁ3, pH 7.0,
additionally sonicated and filtered through a 0.45 lm fil-
ter and the concentration of corresponding solution
determined spectroscopically as the concentration of
phosphates. In fluorimetric titrations, excitation wave-
length of k > 300 nm was used to avoid inner filter ef-
fects caused by absorption of excitation light by added
polynucleotide. Spectroscopic titrations were performed
by adding portions of polynucleotide solution into the
solution of the studied compound. The stability con-
stant (Ks) and [bound compound]/[polynucleotide phos-
phate] ratio (n) were calculated according to the
The product is soluble in warm acetone, methanol, eth-
anol, dichloromethane, chloroform, DMF and DMSO.
Yield: 0.88 g (27%). Anal. Calcd for C18H15N3OS: C,
67.27; H, 4.70; N, 13.07; S, 9.98. Found: C, 67.11; H,
4.58; N, 16.97; S, 9.78%. IR (cmꢁ1) in KBr: 3349 (m),
3157 (s), 2876 (m), 1641 (s), 1635 (s), 1577 (s), 766

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I. Dilovic et al. / Bioorg. Med. Chem. 16 (2008) 5189–5198
5198
Scatchard equation by non-linear least-square fitting,15
giving excellent correlation coefficients (>0.999) for ob-
tained values for Ks and n.
Croatia (Grant Nos. 119-1101342-1082, 119-1193079-
1084, 098-0982914-2918, and 098-0982464-2514).
Thermal melting curves for ct-DNA and its complexes
with studied compounds were determined as previously
described by following the absorption change at 260 nm
as a function of temperature.25 The absorbance of the li-
gand was subtracted from every curve, and the absor-
bance scale was normalized. Obtained Tm values are
the midpoints of the transition curves, determined from
the maximum of the first derivative or graphically by a
tangent method. Given DTm values were calculated sub-
tracting Tm of the free nucleic acid from Tm of complex.
Every DTm value here reported was the average of at
least two measurements, the error in DTm is 0.5 ꢁC.
Supplementary data
Additional crystallographic and spectroscopic data.
Crystallographic data have been deposited with the
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
(fax: +44 1223 366033; e-mail: deposit@ccdc.cam.ac.uk
or www://www.ccdc.cam.ac.uk) and are available on re-
quest, quoting the deposition number 674277 for the
compound 5. Supplementary data associated with this
article can be found, in the online version, at
doi:10.1016/j.bmc.2008.03.006.
4.3.2. Antiproliferative activity assay. The HeLa (cervical
carcinoma), Hep-2 (laryngeal carcinoma), MCF-7
(breast carcinoma), SW620 (colon carcinoma), Mia-
PaCa-2 (pancreatic carcinoma) and Hep-2 (laringeal
carcinoma) and WI 38 (normal diploid fibroblasts) cells
(obtained from American Type Culture Collection
(ATCC, Rockville, MD, USA)) were cultured as mono-
layers and maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin
and 100 lg/mL streptomycin in a humidified atmo-
sphere with 5% CO2 at 37 ꢁC. The growth inhibition
activity was assessed as described previously, according
to the slightly modified procedure of the National Can-
cer Institute, Developmental Therapeutics Program.25
The cells were inoculated onto standard 96-well microti-
ter plates on day 0. The cell concentrations were ad-
justed according to the cell population doubling time
(PDT): 1 · 104/mL for HeLa, Hep-2, MiaPaCa-2 and
SW620 cell lines (PDT = 20–24 h), 2 · 104/mL for
MCF-7 cell line (PDT = 33 h) and 3 · 104/mL for WI
38 (PDT = 47 h). Test agents were then added in five
dilutions (10ꢁ8 to 5 · 10ꢁ6 mol/l) and incubated for a
further 72 h. Working dilutions were freshly prepared
on the day of testing. After 72 h of incubation, the cell
growth rate was evaluated by performing the MTT as-
say, which detects dehydrogenase activity in viable cells.
Each test was performed in quadruplicate in three indi-
vidual experiments. The results are expressed as IC50,
which is the concentration necessary for 50% of inhibi-
tion. The IC50 values for each compound are calculated
from concentration–response curves using linear regres-
sion analysis by fitting the test concentrations that give
PG values above and below the reference value (i.e.,
50%). If however, for a given cell line all of the tested
concentrations produce PGs exceeding the respective
reference level of effect (e.g., PG value of 50), then the
highest tested concentration is assigned as the default
value, which is preceded by a ‘>’ sign. Each result is a
mean value from three separate experiments.
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Acknowledgments
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