Synthesis, Biological Evaluation and Ligand Based Pharmacophore Modeling of New Aromatic Thiosemicarbazones as Potential Anticancer Agents

Article abstract of DOI:10.1007/s11094-019-01968-3

Two series of new aromatic thiosemicarbazone derivatives were synthesized by condensation of N-(4-cyanophenyl)hydrazine carbothioamide (I) and N-(4-methylsulfanylphenyl)hydrazine carbothioamide (II) with appropriate aromatic aldehydes in order to investig

Full text of DOI:10.1007/s11094-019-01968-3

DOI 10.1007/s11094-019-01968-3
Pharmaceutical Chemistry Journal, Vol. 53, No. 2, May, 2019 (Russian Original Vol. 53, No. 2, February, 2019)
SYNTHESIS, BIOLOGICAL EVALUATION AND LIGAND BASED
PHARMACOPHORE MODELING OF NEW AROMATIC
THIOSEMICARBAZONES AS POTENTIAL ANTICANCER AGENTS
A. Karaküçük-Ýyidoðan,1,* B. Aydýnöz,1 T. Taþkýn-Tok,1
E. E. Oruç-Emre,1 and J. Balzarini2
Original article submitted March 27, 2019.
Two series of new aromatic thiosemicarbazone derivatives were synthesized by condensation of
N-(4-cyanophenyl)hydrazine carbothioamide (I) and N-(4-methylsulfanylphenyl)hydrazine carbothioamide
(II) with appropriate aromatic aldehydes in order to investigate their antiviral and cytostatic potency. The
chemical structures of all compounds were fully characterized by elemental analysis and spectroscopic tech-
niques. The results of the bioassays indicated that compounds Id, Ie, If and IIf proved inhibitory against influ-
enza virus A (EC50 = 13 – 27 mg/mL for strain H1N1 and 9.3 – 18 mg/mL for strain H3N2). Compounds Ig
and IIg were the most cytostatic compounds with inhibition of HeLa cell proliferation at an IC50 = 0.3 mg/mL
for Ig and 1.9 mg/mL for IIg. Especially, compound Ig showed the highest cytostatic activity with IC50 of 0.30,
0.70 and 2.50 mg/mL against HeLa, CEM and L1210 cell lines, respectively. This inhibition range was within
the same order of magnitude as that for cisplatin. Furthermore, molecular modeling was carried out to exam-
ine the cytostatic activity and determine the best pharmacophore model as a guide for the design and develop-
ment of potential prodrugs in future studies.
Keywords: aromatic thiosemicarbazone; antiviral activity; cytostatic activity; anti-influenza activity;
pharmacophore modeling.
1. INTRODUCTION
Triapine) inhibited the biosynthesis of DNA in murine leuke-
mia L1210 cells by blocking the activity of ribonucleotide
reductase [5].
Thiosemicarbazones are important class of synthetic
products and potential biologically active compounds.
Domagk, et al. [1] first reported that thiosemicarbazone
pharmacophore had antituberculosis activity. Then, antiviral
activity of benzaldehyde thiosemicarbazone derivatives
against vaccina virus in mice was found by Hamre, et al. [2].
The antiviral activity of isatin-b-thiosemicarbazone (IBT)
and N-methyl-isatin-b-thiosemicarbazone (commercially
known as Methisazone or Marboran) was widely investi-
gated against orthopoxviruses in the 1960s [3]. After these
discoveries, thiosemicarbazones draw considerable interest
due to their application in the pharmaceutical chemistry and
proved to be chemotherapeutic agents potentially useful for
inhibiting cancer cells [4]. For example, 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (commercially known as
In recent years, it has been commonly accepted that
agents containing more than one pharmacophore can have
superior efficacy as compared to single-pharmacophore
drugs [6]. Pharmacophore hybridization is a method of ratio-
nal drug design, and a single molecule containing different
modes of action can be beneficial for the treatment of di-
seases. As an important pharmacophore, bis-alkylating nitro-
gen mustard such as chlormethine, melphalan, chlorambucil
and many more have anticancer activities against hematolo-
gic tumors, myeloma, ovarian cancer and solid tumors [7].
Alkylating agents are electrophilic entities that interact with
nucleophilic moieties of DNA resulting in the covalent trans-
fer of an alkyl group. Another pharmacophore para-substi-
tuted aryl nitrile, which is bioisostere of a ketone group, has
a variety of anticancer activities, e.g., against hormonally-re-
sponsive breast cancer (letrozole), breast cancer (neratinib),
prostate cancer (bicalutamide), pancreatic cancer, non-small
cell lung cancer, head and neck cancer (L-778,123, phase I),
1
Department of Chemistry, Gaziantep University, 27310 Gaziantep, Tur-
key.
2
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Re-
search, KU Leuven, 3000 Leuven, Belgium.
*
e-mail: iyidogan@gantep.edu.tr
139
0091-150X/19/5302-0139 © 2019 Springer Science+Business Media, LLC

140
A. Karaküçük-Ýyidoðan et al.
Fig. 1. Design of compound Ig.
and chronic myelogenous leukemia (bosutinib, SKI-606,
phase III) [8].
tives. All thiosemicarbazone derivatives (Ia-g and IIa-g) ex-
hibited two intense bands in the region of 3396 – 3266 cm-1
and 3162 – 3120 cm-1 due to n(N-H) stretching. The strong
band at 839 – 818 cm-1 was present due to n(C=S) stretching.
It was suggested that thiosemicarbazones in the solid phase
remain in the thione form. In addition, the characteristic
azomethine stretching vibrations n(C=N) at 1587 –
1543 cm-1 were observed, which also confirmed the forma-
Previously, we reported in vitro cytostatic and broad-
spectrum antiviral activity of the thiosemicarbazones derived
from 5-substitutedthiophene-2-carboxaldehydes and their
platinum(II) and palladium(II) complexes [9]. Encouraged
by these results and aimed at developing effective anticancer
agents, we designed two series of thiosemicarbazones by the
pharmacophore hybridization method using two or more dif-
ferent pharmacophores in view of prospecting their cyto-
static and antiviral activity (Fig. 1). In order to gain detailed
information about this issue and to develop effective
anticancer agents for future studies, we have performed vir-
tual screening of the aforementioned compounds by using
Gaussian09 and Discovery Studio 3.5 software [10, 11].
The intermediates N-(4-cyanophenyl)hydrazinecarbo-
thioamide (I) and N-(4-methylthiophenyl)hydrazinecarbo-
thioamide (II) were prepared from 4-cyanophenyl isothio-
cyanate and 4-methylthiophenyl isothiocyanate by reaction
with hydrazine monohydrate, respectively [12]. N-(4-cy-
anophenyl)hydrazinecarbothioamide (I) was reported previ-
ously [13] and N-(4-methylthiophenyl)hydrazinecarbothio-
amide (II) was commercially available. The novel thiosemi-
carbazones (Ia – Ig and IIa – IIg) as well as the known
analog of Ib [14] were synthesized in high yield (71 – 92%)
from the corresponding 4-substitutedbenzaldehydes by treat-
ment with thiosemicarbazides in methanol, according to the
general procedure of a previously described method [15].
The synthetic route of target compounds Ia – Ig and IIa – IIg
is shown in Scheme 1.
1
tion of thiosemicarbazones [16]. Results of the H NMR in-
tegrations and signal multiplicities were in line with the pro-
posed structures and other spectral data. The 1H NMR spec-
tra of Ia – Ig and IIa – IIg showed a singlet peak attributable
to the =N-NH proton in arange of d = 12.60 – 11.62 ppm as
well as a singlet peak attributable to the PhNH proton in the
range d = 10.80 – 9.92 ppm. The signal of the azomethine
proton (HC=N) appeared as a singlet at d = 8.63 – 8.04 ppm
[16]. All aromatic protons were observed with the expected
chemical shift (d = 8.48 – 6.80 ppm) and coupling constant
in the NMR spectra of all thiosemicarbazone derivatives.
The 13C NMR spectra of the thiosemicarbazones exhibited
two important signals at d = 176.92 – 174.23 and at
d = 143.89 – 142.57 ppm assigned to thioamide (C=S) and
imine (C=N) carbon atoms, respectively. The signals at
d = 164.01 – 112.06 ppm in the spectra were assumed to be
due to the aromatic carbons. The MS spectra of all
thiosemicarbazones were in line with the proposed struc-
tures.
2. EXPERIMENTAL CHEMICAL PART
The chemical structures of new compounds were
throughly eludicated by elemental analysis and spectral data
1
The progress of all reactions were monitored by thin
layer chromatography (TLC). TLC was performed on silica
(UV-Vis, IR, H NMR, 13C NMR, and ESI-MS). Assign-
ments of selected diagnostic IR bands provided significant
indication for the formation of the thiosemicarbazone deriva-
gel plates (Merck Silica Gel 60, F , 0.2 mm) with visual-
254
ization by exposure to iodine vapor and UV light using

Synthesis, Biological Evaluation and Ligand
141
Scheme 1. Synthetic pathway of thiosemicarbazones Ia – Ig and IIa – IIg. Reagents and conditions: (a) NH NH .H O (98%), Et O, rt, 24 h;
2
2
2
2
(b) appropriate aldeyhde in MeOH, reflux.
EtOAc/hexane (v/v 1:1 and 1:3) as solvent system. Melting
points were determined on a EZ-Melt MPA120 Automated
Melting Point apparatus and were uncorrected. The IR spec-
tra were recorded on a Perkin Elmer 100 FT-IR spectrometer
N-(4-cyanophenyl)hydrazinecarbothioamide
Light yellow solid (methanol). Yield: 1.14 g (95%); m.p.:
185 – 186°C. Anal. calc. for C H N S: C, 49.98; H, 4.19; N,
(I).
8
8
4
29.14; S, 16.68%. Found: C, 49.86; H, 4.29; N, 29.16; S,
1
with universal ATR sampling accessory. The H NMR and
13C NMR spectra were obtained at room temperature with a
16.84%. UV (DMSO, lmax, nm): 325; 293; 236. IR (nmax
cm-1): 3338, 3196 (N-H); 2229 (CºN); 1208 (C-N); 1083
,
Bruker Avance-DPX-400 NMR spectrometer in DMSO-d
1
(N-N); 831 (C=S). H NMR (DMSO-d ; d, ppm): 9.53 (s,
6
6
using TMS as the internal standard. The mass spectra were
obtained using an LC/MS Agilent 1100 MSD series spec-
trometer in the electrospray mode. Elemental (CHNS) analy-
ses were performed using a VarioMICRO elemental ana-
lyzer.
1H, NHNH ); 9.40 (s, 1H, PhNH); 8.10 (d, 2H, ArH,
2
J = 8.23 Hz, orto protons to -CN group); 7.76 (d, 2H, ArH,
J = 8.26 Hz, meta protons to -CN group); 4.99 (brs, 2H,
NH ).
2
N-(4-methylsulfanylphenyl)hydrazinecarbothioamide
(II). White solid (methanol). Yield: 1.17 g (88%); m.p.:
181 – 182°C. Anal. calc. for C H N S : C, 45.04; H, 5.20;
2.1. Synthesis of Thiosemicarbazides I and II
8
11
3 2
N, 19.70; S, 30.06%. Found: C, 45.24; H, 5.43; N, 19.63; S,
General procedure. A solution of hydrazine monohyd-
rate (7.50 mmol) in diethylether (20 mL) was added drop-
wise to a solution of 4-substitutedphenyl isothiocyanate
(6.25 mmol) in diethyl ether (20 mL) at room temperature
with vigorous stirring. The mixture was allowed to stand
overnight. The precipitated solid was filtered and washed
two times with water. The crude product was recrystallized
from methanol.
30.07%. UV (DMSO, lmax, nm): 330; 293; 237. IR (nmax
,
cm-1): 3346, 3274, 3175 (N-H); 1203 (C-N); 1064 (N-N);
828 (C=S). 1H NMR (DMSO-d ; d, ppm): 9.60 (s, 1H,
6
NHNH ); 9.12 (s, 1H, PhNH); 7.59 (d, 2H, ArH,
2
J = 8.57 Hz, meta protons to -SCH group); 7.21 (d, 2H,
3
ArH, J = 8.61 Hz, orto protons to -SCH group); 4.80 (brs,
3
2H, NH ); 2.46 (s, 3H, SCH ).
2
3

142
A. Karaküçük-Ýyidoðan et al.
2.2. Synthesis of Thiosemicarbazones Ia – Ig and IIa – IIg
J = 8.62 Hz, orto protons to -CN group); 7.91 (dd, 2H, ArH,
J = 3.58 Hz, J = 2.40 Hz, orto protons at phenyl ring); 7.83
1
2
General procedure: To a hot solution of thiosemicarba-
zide (1.04 mmol) in methanol (25 mL) was added dropwise a
solution of the appropriate aldehyde (1.04 mmol) in metha-
nol (10 mL) with continuous stirring. After the addition of a
catalytic amount of glacial acetic acid, the reaction mixture
was refluxed for 6 – 24 h. The progress of the reaction was
monitored by TLC. The reaction mixture was cooled and the
precipitate was filtered. The crude product was washed with
cold diethylether or ethanol and recrystallized from appropri-
ate solvent. Table 1 presents data of mass spectroscopy and
elemental analysis for aromatic thiosemicarbazones Ia – Ig
and Iýa – IIg.
(d, 2H, ArH, J = 8.62 Hz, meta protons to -CN group); 7.46
(t, 3H, ArH, J = 3.42 Hz, J = 2.93 Hz, para and meta pro-
1
2
tons at phenyl ring). 13C NMR (DMSO-d ; d, ppm): 176.49
6
(C=S), 143.78 (C=N), 141.99, 133.83, 132.34, 131.75,
130.16, 129.13, 128.55, 118.67 (ArC), 108.62 (CºN).
2-(4-Hydroxybenzylidene)-N-(4-cyanophenyl)hydra-
zinecarbothioamide (Ib). Yellow solid (acetone). Yield:
0.22 g (71%); m.p.: 224 – 225°C. IR (nmax, cm-1): 3396,
3153 (O-H and N-H); 2229 (CºN); 1579 (C=N); 1207
1
(C-N); 1060 (N-N); 833 (C=S). H NMR (DMSO-d ; d,
6
ppm): 11.93 (s, 1H, CSNH); 10.20 (s, 1H, PhNH); 9.98 (s,
1H, OH); 8.10 (s, 1H, HC=N); 7.98 (d, 2H, ArH,
J = 8.69 Hz, orto protons to -CN group); 7.82 (d, 2H, ArH,
J = 8.67 Hz, meta protons to -CN group); 7.74 (d, 2H, ArH,
J = 8.64 Hz, meta protons to -OH group); 6.82 (d, 2H, ArH,
J = 8.64 Hz orto protons to -OH group). 13C NMR
2-Benzylidene-N-(4-cyanophenyl)hydrazinecarbothio-
amide (Ia). Yellow solid (acetone). Yield: 0.26 g (90%);
m.p.: 193 – 194°C. IR (nmax, cm-1): 3275, 3140 (N-H); 2220
(CºN); 1580 (C=N); 1200 (C-N); 1063 (N-N); 834 (C=S).
1H NMR (DMSO-d ; d, ppm): 12.10 (s, 1H, CSNH); 10.33
6
(s, 1H, PhNH); 8.22 (s, 1H, HC=N); 7.97 (d, 2H, ArH,
(DMSO-d ; d, ppm): 175.56 (C=S), 154.42 (ArC, ipso car-
6
TABLE 1. Molecular Formulas, Mass Spectroscopy Data and Elemental Analysis of Aromatic Thiosemicarbazones Ia-Ig and IIa-IIg
% Elemental analysis of C, H, N, S: Found (Calcd.)
Molecular
MS (m/z)
Compd
Ia
R
R¢
formula
C
H
N
S
-CN
-H
C15H12N4S
279.1 [M-H]-
295.1 [M-H]-
309.1 [M-H]-
64.47
4.48
19.76
11.39
(64.26)
(4.31)
(19.98)
(11.44)
Ib
-CN
-OH
-OCH3
-OCF3
-F
C15H12N4OS
C16H14N4OS
60.45
4.18
18.65
10.41
(60.79)
(4.08)
(18.91)
(10.52)
Ic
-CN
61.99
4.98
18.35
10.14
(61.92)
(4.75)
(18.05)
(10.33)
Id
-CN
C16H11F3N4OS 363.1 [M-H]-
298.1 [M]+
C15H11N5O2S 324.1 [M-H]-
52.68
3.04
15.78
8.95
(52.74)
(3.21)
(15.58)
(8.80)
Ie
-CN
C15H11FN4S
60.04
3.42
18.53
10.92
(60.29)
(3.52)
(18.78)
(10.75)
If
-CN
-NO2
55.23
3.92
21.85
9.97
(55.38)
(3.81)
(21.63)
(9.86)
Ig
-CN
-N(CH2CH2Cl)2 C19H19Cl2N5S 419.0 [M-H]-
54.24
4.36
16.82
7.34
(54.29)
(4.56)
(16.66)
(7.63)
IIa
IIb
IIc
IId
IIe
IIf
IIg
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-H
-OH
C15H15N3S2
300.1 [M-H]-
59.65
5.27
13.73
21.23
(59.77)
(5.02)
(13.84)
(21.28)
C15H15N3OS2 316.0 [M-H]-
C16H17N3OS2 330.1 [M-H]-
C16H14F3N3OS2 384.0 [M-H]-
56.90
4.98
13.52
20.44
(56.76)
(4.76)
(13.34)
(20.20)
-OCH3
-OCF3
-F
57.62
5.34
12.80
19.26
(57.78)
(5.27)
(12.68)
(19.35)
49.68
3.87
10.84
16.64
(49.86)
(3.66)
(10.90)
(16.64)
C15H14FN3S2
319.1 [M]+
56.12
4.71
13.58
20.37
(56.30)
(4.62)
(13.28)
(20.08)
-NO2
C15H14N4O2S2 345.0 [M-H]-
52.68
4.49
16.72
18.93
(52.41)
(4.27)
(16.47)
(18.72)
-N(CH2CH2Cl)2 C19H22Cl2N4S2 440.1 [M-H]-
51.69
5.28
12.39
14.47
(51.70)
(5.02)
(12.49)
(14.53)

Synthesis, Biological Evaluation and Ligand
143
bon to -OH group), 143.48 (C=N), 141.98, 132.64, 131.73,
130.21, 127.47, 119.44, 116.72 (ArC), 107.52 (CºN).
2-(4-Methoxybenzylidene)-N-(4-cyanophenyl)hydra-
zinecarbothioamide (Ic). Beige solid (ethanol). Yield: 0.29
g (92%); m.p.: 217 – 218°C. IR (nmax, cm-1): 3273, 3158
(N-H); 2227 (CºN); 1543 (C=N); 1201 (C-N); 1022 (N-N);
NMR (DMSO-d ; d, ppm): 176.39 (C=S); 148.23 (ArC, ipso
6
carbon to -NO group); 143.64 (C=N); 141.62, 140.55,
2
132.71, 128.97, 125.88, 124.17, 119.27 (ArC), 107.53
(CºN).
2-{4-[Bis(2-chloroethyl)amino]benzylidene}-N-(4-cya-
nophenyl)hydrazinecarbo thioamide (Ig). Light brown
solid (acetone). Yield: 0.33 g (76%); m.p.: 174 – 175°C. IR
(nmax, cm-1): 3290, 3148 (N-H); 2228 (CºN); 1582 (C=N);
1
835 (C=S). H NMR (DMSO-d ; d, ppm): 12.03 (s, 1H,
6
CSNH); 10.25 (s, 1H, PhNH); 8.15 (s, 1H, HC=N); 7.97 (d,
2H, ArH, J = 8.73 Hz, orto protons to -CN group); 7.85 (d,
1
1199 (C-N); 1076 (N-N); 829 (C=S); 719 (C-Cl). H NMR
2H, ArH, J = 8.83 Hz, meta protons to -OCH group); 7.82
(DMSO-d ; d, ppm): 11.90 (s, 1H, CSNH); 10.18 (s, 1H,
3
6
(d, 2H, ArH, J = 8.70 Hz, meta protons to -CN group); 7.01
PhNH); 8.08 (s, 1H, HC=N); 7.99 (d, 2H, ArH, J = 8.50 Hz,
orto protons to -CN group); 7.81 (d, 2H, ArH, J = 8.53 Hz,
meta protons to -CN group); 7.73 (d, 2H, ArH, J = 8.70 Hz,
meta protons to -N(CH CH Cl) group); 6.81 (d, 2H, ArH,
(d, 2H, ArH, J = 8.81 Hz, orto protons to -OCH group);
3
3.81 (s, 3H, OCH ). 13C NMR (DMSO-d ; d, ppm): 176.38
3
6
(C=S), 158.32 (ArC, ipso carbon to -OCH group), 143.76
3
2
2
2
(C=N), 142.58, 140.35, 133.09, 129.17, 126.76, 124.86,
J = 8.75 Hz, orto protons to -N(CH CH Cl) group);
2
2
2
3.76 – 3.81 (m, 8H, -N(CH CH Cl) ). 13C NMR (DMSO-d ;
119.49 (ArC), 106.98 (CºN), 56.12 (OCH ).
2
2
2
6
3
2-[(4-Trifluoromethoxy)benzylidene]-N-(4-cyanophen-
yl)hydrazinecarbothioamide (Id). White solid (acetone).
Yield: 0.28 g (72%); m.p.: 219 – 220°C. IR (nmax, cm-1):
3291, 3149 (N-H); 2223 (CºN); 1549 (C=N); 1201 (C-N);
d, ppm): 176.49 (C=S), 149.25 (ArC, ipso carbon to
-N(CH CH Cl) group), 143.33 (C=N), 136.64, 134.12,
2
2
2
130.45, 127.68, 125.96, 123.87, 112.65 (ArC), 107.78
(CºN), 53.18 (N(CH CH Cl) ), 42.54 (N(CH CH Cl) ).
2
2
2
2
2
2
1079 (N-N); 839 (C=S). 1H NMR (DMSO-d ; d, ppm): 12.60
2-Benzylidene-N-[(4-methylthio)phenyl]hydrazinecar-
bothioamide (IIa). White solid (acetone). Yield: 0.27 g
(85%); m.p.: 211 – 212°C. IR (nmax, cm-1): 3299, 3149
(N-H); 1573 (C=N); 1082 (C-N); 1062 (N-N); 823 (C=S). 1H
6
(s, 1H, CSNH); 10.80 (s, 1H, PhNH); 8.63 (s, 1H, HC=N);
8.48 (d, 2H, ArH, J = 8.27 Hz, meta protons to -OCF
3
group); 8.37 (d, 2H, ArH, J = 8.65 Hz, orto protons to -CN
group); 8.27 (d, 2H, ArH, J = 8.66 Hz, meta protons to -CN
NMR (DMSO-d ; d, ppm): 11.84 (s, 1H, CSNH); 10.09 (s,
6
group); 7.86 (d, 2H, ArH, J = 8.22 Hz, orto protons to -OCF
1H, PhNH); 8.17 (s, 1H, HC=N); 7.91 (dd, 2H, ArH,
J = 3.80 Hz, J =2.05 Hz, orto protons at phenyl ring); 7.53
3
group). 13C NMR (DMSO-d ; d, ppm): 176.24 (C=S), 146.45
6
1
2
(ArC, ipso carbon to OCF ), 142.76 (C=N), 140.37, 139.63,
(d, 2H, ArH, J = 8.58 Hz, meta protons to -SCH group);
3
3
131.85, 128.79 (ArC), 126.41 (OCF ), 124.19, 123.07,
7.44 (t, 3H, ArH, J = 3.47 Hz, J =2.27 Hz, para and meta
3
1
2
protons at phenyl ring); 7.28 (d, 2H, ArH, J = 8.59 Hz, orto
protons to -SCH group), 2.49 (s, 3H, SCH ). 13C NMR
115.54 (ArC), 107.05 (CºN).
2-(4-Fluorobenzylidene)-N-(4-cyanophenyl)hydrazine-
carbothioamide (Ie). White solid (acetone). Yield: 0.23 g
(73%); m.p.: 206 – 207°C. IR (nmax, cm-1): 3284, 3146
(N-H); 2226 (CºN); 1581 (C=N); 1201 (C-N); 1076 (N-N);
3
3
(DMSO-d ; d, ppm): 174.23 (C=S), 143.85 (C=N), 135.47,
6
134.92, 133.76, 131.67, 131.01, 129.28, 128.85, 125.71
(ArC), 15.43 (SCH ).
3
1
827 (C=S). H NMR (DMSO-d ; d, ppm): 12.51 (s, 1H,
2-(4-Hydroxybenzylidene)-N-[(4-methylthio)phenyl]-
hydrazinecarbothioamide (IIb). White solid (methanol).
Yield: 0.26 g (81%); m.p.: 202 – 203°C. IR (nmax, cm-1):
3580 (O-H); 3266, 3153 (N-H); 1583 (C=N); 1164 (C-N);
6
CSNH); 10.73 (s, 1H, PhNH); 8.61 (s, 1H, HC=N); 8.42 (dd,
2H, ArH, J = 8.79 Hz, meta protons to -F atom); 8.37 (d, 2H,
ArH, J = 8.74 Hz, orto protons to -CN group); 8.26 (d, 2H,
ArH, J = 8.73 Hz, meta protons to -CN group); 7.72 (t, 2H,
ArH, J = 8.86 Hz, orto protons to -F atom). 13C NMR
1069 (N-N); 818 (C=S). 1H NMR (DMSO-d ; d, ppm): 11.65
6
(s, 1H, CSNH); 9.94 (s, 1H, PhNH); 9.92 (s, 1H, OH); 8.07
(s, 1H, HC=N); 7.73 (d, 2H, ArH, J = 8.59 Hz, meta protons
to -OH group); 7.54 (d, 2H, ArH, J = 8.57 Hz, meta protons
(DMSO-d ; d, ppm): 176.71 (C=S), 163.99 (ArC, ipso car-
6
bon to -F atom), 142.77 (C=N), 139.78, 134.14, 132.42,
130.23, 126.47, 118.53, 116.77 (ArC), 106.96 (CºN).
2-(4-Nitrobenzylidene)-N-(4-cyanophenyl)hydrazine-
carbothioamide (If). Dark yellow solid (ethanol). Yield:
0.27 g (81%); m.p.: 256 – 257°C. IR (nmax, cm-1): 3280,
to -SCH group); 7.26 (d, 2H, ArH, J = 8.59 Hz, orto protons
3
to -OH group); 6.81 (d, 2H, ArH, J = 8.57 Hz orto protons to
-SCH group), 2.49 (s, 3H, SCH ). 13C NMR (DMSO-d ; d,
3
3
6
ppm): 176.92 (C=S), 153.29 (ArC, ipso carbon to -OH
group), 142.57 (C=N), 131.27, 129.62, 128.51, 126.13,
3140 (N-H); 2224 (CºN); 1579 (C=N); 1499 (NO , asym-
2
125.48, 120.03, 115.68 (ArC), 16.67 (SCH ).
metric); 1337 (NO , symmetric); 1200 (C-N); 1088 (N-N);
3
2
1
839 (C=S). H NMR (DMSO-d ; d, ppm): 12.35 (s, 1H,
2-(4-Methoxyybenzylidene)-N-[(4-methylthio)phenyl]-
hydrazinecarbothioamide (IIc). White solid (acetone).
Yield: 0.25 g (73%); m.p.: 194 – 195°C. IR (nmax, cm-1):
3309, 3126 (N-H); 1570 (C=N); 1164 (C-N); 1068 (N-N);
6
CSNH); 10.51 (s, 1H, PhNH); 8.31 (d, 2H, ArH, J = 8.86 Hz,
orto protons to -NO group); 8.28 (s, 1H, HC=N); 8.21 (d,
2
2H, ArH, J = 8.87 Hz, meta protons to -NO group); 7.95 (d,
2
1
823 (C=S). H NMR (DMSO-d ; d, ppm): 11.72 (s, 1H,
2H, ArH, J = 8.62 Hz, orto protons to -CN group); 7.82 (d,
2H, ArH, J = 8.65 Hz, meta protons to -CN group). 13C
6
CSNH); 10.01 (s, 1H, PhNH); 8.11 (s, 1H, HC=N); 7.85 (d,

144
A. Karaküçük-Ýyidoðan et al.
2H, ArH, J = 8.74 Hz, meta protons to -SCH group); 7.53
yellow solid (chloroform). Yield: 0.40 g (88%); m.p.:
192 – 193°C. IR (nmax, cm-1): 3299, 3162 (N-H); 2228 1545
(C=N); 1177 (C-N); 1061 (N-N); 818 (C=S); 712 (C-Cl). 1H
3
(d, 2H, ArH, J = 8.57 Hz, meta protons to -OCH group);
3
7.27 (d, 2H, ArH, J = 8.57 Hz, orto protons to -OCH
3
group); 6.99 (d, 2H, ArH, J = 8.74 Hz, orto protons to -SCH
NMR (DMSO-d ; d, ppm): 11.62 (s, 1H, CSNH); 9.92 (s,
3
6
group); 3.81 (s, 3H, OCH ), 2.49 (s, 3H, SCH ). 13C NMR
3
3
1H, PhNH); 8.04 (s, 1H, HC=N); 7.72 (d, 2H, ArH,
(DMSO-d ; d, ppm): 175.93 (C=S), 161.25 (ArC, ipso car-
J = 8.82 Hz, meta protons to -SCH group); 7.55 (d, 2H,
6
3
bon to -OCH group), 143.36 (C=N), 136.67, 134.83, 129.66,
ArH, J = 8.58 Hz, meta protons to -N(CH CH Cl) group);
3
2
2
2
126.83, 126.73, 126.10, 114.51 (ArC), 55.70 (OCH ), 15.44
7.26 (d, 2H, ArH, J = 8.60 Hz, orto protons to
-N(CH CH Cl) group); 6.80 (d, 2H, ArH, J = 8.90 Hz, orto
3
(SCH ).
3
2
2
2
2-[(4-Trifluoromethoxy)benzylidene]-N-[(4-methyl-
thio)phenyl]hydrazinecarbo thioamide (IId). White solid
(chloroform). Yield: 0.25 g (74%); m.p.: 198 – 199°C. IR
(nmax, cm-1): 3324, 3121 (N-H); 1577 (C=N); 1160 (C-N);
protons to -SCH
group); 3.75 – 3.82 (m, 8H,
3
-N(CH CH Cl) protons); 2.50 (s, 3H, -SCH ). 13C NMR
2
2
2
3
(DMSO-d ; d, ppm): 175.96 (C=S), 148.41 (ArC, ipso car-
6
bon to -N(CH CH Cl) group), 143.89 (C=N), 136.70,
2
2
2
1077 (N-N); 822 (C=S). 1H NMR (DMSO-d ; d, ppm): 11.91
6
134.56, 129.72, 126.44, 126.12, 122.63, 112.06 (ArC), 52.32
(N(CH CH Cl) ), 41.87 (N(CH CH Cl) ), 15.57 (SCH ).
(s, 1H, CSNH); 10.15 (s, 1H, PhNH); 8.17 (s, 1H, HC=N);
2
2
2
2
2
2
3
8.06 (d, 2H, ArH, J = 8.57 Hz, meta protons to -SCH
3
group); 7.51 (d, 2H, ArH, J = 8.55 Hz, meta protons to
3. EXPERIMENTAL BIOLOGICAL PART
-OCF group); 7.42 (d, 2H, ArH, J = 8.52 Hz, orto protons to
3
-OCF group); 7.28 (d, 2H, ArH, J = 8.56 Hz, orto protons to
3
-SCH group); 2.49 (s, 3H, -SCH ). 13C NMR (DMSO-d ; d,
3.1. Antiviral Activity Assay
3
3
6
ppm): 176.40 (C=S), 147.36 (ArC, ipso carbon to -OCF
3
The antiviral assays were based on the inhibition of vi-
rus-induced cytopathicity in confluent cell cultures, and the
cytostatic assays on inhibition of tumor cell proliferation in
exponentially growing tumor cell cultures according to pre-
viously described methods [17]. The synthesized compounds
were evaluated against the following viruses: herpes simplex
virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient
(TK–) HSV-1 KOS strain resistant to ACV (ACVr), herpes
simplex virus type 2 (HSV-2) strain G, cytomegalovirus
strains AD-169 and David, varicella zoster virus (VZV)
strains OKA and YS, vaccinia virus Lederle strain, respira-
tory syncytial virus (RSV) strain Long, vesicular stomatitis
virus (VSV), Coxsackie B4 virus, parainfluenza 3 virus, in-
fluenza virus A (subtypes H1N1, H3N2), influenza virus B,
reovirus-1, Sindbis and Punta Toro virus. The antiviral as-
says were based on inhibition of virus-induced cytopathicity
or plaque formation (for VZV) in human embryonic lung
(HEL) fibroblasts, African green monkey cells (Vero), hu-
man epithelial cervix carcinoma cells (HeLa) or
Madin-Darby canine kidney cells (MDCK). Confluent cell
cultures in microtiter 96-well plates were inoculated with
100 CCID of virus (1 CCID being the virus dose to infect
group), 143.71 (C=N), 141.11, 139.78, 136.33, 129.81
(ArC), 127.04 (OCF ), 125.92, 122.49, 116.12 (ArC), 15.84
3
(SCH ).
3
2-(4-Fluorobenzylidene)-N-[(4-methylthio)phenyl]hyd-
razinecarbothioamide (IIe). White solid (chloroform).
Yield: 0.24 g (72%); m.p.: 175 – 176°C. IR (nmax, cm-1):
3323, 3133 (N-H); 1581 (C=N); 1228 (C-F); 1150 (C-N);
1069 (N-N); 826 (C=S). 1H NMR (DMSO-d ; d, ppm): 11.83
6
(s, 1H, CSNH); 10.11 (s, 1H, PhNH); 8.15 (s, 1H, HC=N);
7.99 (dd, 2H, ArH, J = 8.70 Hz, meta protons to -F atom);
7.51 (d, 2H, ArH, J = 8.57 Hz, meta protons to -SCH
3
group); 7.28 (t, 2H, ArH, J = 8.77 Hz, orto protons to -F
atom); 7.27 (d, 2H, ArH, J = 8.55 Hz, orto protons to -SCH
3
group); 2.49 (s, 3H, -SCH ). 13C NMR (DMSO-d ; d, ppm):
3
6
176.89 (C=S), 164.01 (ArC, ipso carbon to -F atom), 143.18
(C=N), 140.04, 133.95, 131.56, 130.19, 127.76, 119.31,
115.83 (ArC), 16.24 (SCH ).
3
2-(4-Nitrobenzylidene)-N-[(4-methylthio)phenyl]hyd-
razinecarbothioamide (IIf).
Orange solid (acetone). Yield: 0.30 g (82%); m.p.:
225 – 226°C. IR (nmax, cm-1): 3296, 3120 (N-H); 1587
(C=N); 1435 (NO , asymmetric); 1336 (NO , symmetric);
50
50
2
2
1185 (C-N); 1083 (N-N); 836 (C=S). 1H NMR (DMSO-d ; d,
50% of the cell cultures) or 20 or 100 plaque forming units
(PFU) (for VZV and CMV, respectively) in the presence of
varying concentrations of the test compounds. Viral
cytopathicity or plaque formation was recorded as soon as it
reached completion in the control virus-infected cell cultures
that were not treated with the test compounds. Antiviral ac-
6
ppm): 12.10 (s, 1H, CSNH); 10.30 (s, 1H, PhNH); 8.27 (d,
2H, ArH, J = 8.96 Hz, orto protons to -NO group); 8.24 (s,
2
1H, HC=N); 8.21 (d, 2H, ArH, J = 8.95 Hz, meta protons to
-NO group); 7.50 (d, 2H, ArH, J = 8.58 Hz, meta protons to
2
-SCH group); 7.29 (d, 2H, ArH, J = 8.59 Hz, orto protons to
3
-SCH group); 2.50 (s, 3H, -SCH ). 13C NMR (DMSO-d ; d,
tivity was expressed as EC (50% effective concentration)
50
3
3
6
defined as the compound concentration required to reduce
virus-induced cytopathogenicity or viral plaque formation by
ppm): 176.57 (C=S), 149.15 (ArC, ipso carbon to -NO
2
group), 142.86 (C=N), 140.54, 139.27, 133.66, 130.83,
50%). Inhibition of HIV-1(III )- and HIV-2(ROD)-induced
129.11, 128.77, 126.35 (ArC), 15.88 (SCH ).
B
3
cytopathicity in CEM cell cultures was measured in
microtiter 96-well plates containing ~3 ´ 105 CEM cells/mL
2-{4-[Bis(2-chloroethyl)amino]benzylidene}-N-[(4-me-
thylthio)phenyl]hydrazine carbothioamide (IIg). Light

Synthesis, Biological Evaluation and Ligand
145
infected with 100 CCID of HIV per milliliter and contain-
(cyan), two ring aromatic (orange), one hydrogen bond do-
nor (pink) and one hydrogen acceptor (green).
50
ing appropriate dilutions of the test compounds. After 4 – 5
days of incubation at 37°C in a CO -controlled humidified
2
atmosphere, CEM giant (syncytium) cell formation was ex-
5. RESULTS AND DISCUSSION
amined microscopically and characterized by the EC value
50
(50% effective concentration inhibiting HIV-induced giant
cell formation by 50%).
All newly synthesized compounds were evaluated for
their antiviral, cytostatic, and cytotoxic properties. None of
the compounds effectively inhibited DNA virus replication
(i.e. HSV-1, HSV-2, VV), except I and II that showed slight
activity against vaccinia virus replication in HEL cell cul-
3.2. Cytostatic/Toxic Activity Assay
All cytostatic/toxic activity assays were performed in
96-well microliter plates. To each well were added
(5 – 7.5) ´ 104 tumor cells and a given amount of the test
compound. The cells were allowed to proliferate for 48 h
tures (EC = 45 – 50 mg/mL) and compounds Ib, Ic and IIc
50
bearing an electron-donating group (–OH and –OCH )
3
against feline herpes virus (EC = 17.8 – 54.3 mg/mL). All
50
compounds were also not significantly inhibitory against a
wide variety of RNA viruses, including vesicular stomatitis
virus (VSV), respiratory syncytial virus and Coxsackie virus
in HeLa cell cultures at subtoxic concentrations (Table 2).
However, in Vero cell cultures compound Ig consistently
+
(murine leukemia L1210 cells) or 72 h (human CD
4
T-lymphocytic CEM and human cervix carcinoma HeLa
cells) at 37°C in a humidified CO -controlled atmosphere. At
2
the end of the incubation period, the cells were counted in a
Coulter counter. The IC (50% inhibitory concentration)
50
showed an antiviral activity of 3 mg/mL (EC ) against
50
was defined as the concentration of the compound that inhib-
ited cell proliferation by 50%. Cisplatin was purchased from
SIGMA and used as a reference drug. Compound concentra-
tions of 100, 20, 4, 0.8, 0.16 and 0.032 mg/mL have been
tested (5-fold dilutions) and the data represent the mean
SD (standard deviation) of at least two to three independent
experiments. All values are significantly different from
cisplatin (less active), except for Ig (no statistical difference,
p > 0.05).
Coxsackie virus B4 but there was no inhibitory activity ob-
served for parainfluenza virus-3, reovirus-1, Sindbis virus
and Punta Toro virus (Table 2). The fact that the activity of
Ig against Coxsackie virus B4 was only observed in one cell
line (monkey kidney Vero), but not in another cell line (hu-
man cervix carcinoma HeLa) led us to conclude that this
compound should not be considered as a consistent new anti-
viral lead agent.
A marginal activity was observed for Ib, Ic and IIc
against feline herpes virus (EC = 17.8 – 54.3 mg/mL) and
50
4. EXPERIMENTAL MOLECULAR MODELING
PART
for Ib, Ic and IIa against feline corona (FIPV) virus
(EC = 7.0 – 31.5 mg/mL) in CRFK cell cultures (Table 3).
50
Whereas no activity was found in MDCK cell cultures
against influenza B virus, compounds Id, Ie, If and IIf which
have an electron-withdrawing group (–CF , –F and –NO )
The biological activity depended on only on the chemical
structure of substituents, but also on the three dimensional
(3D) configuration of compound Ig. For this reason, we
again applied computational methods and ligand-based
pharmacophore modeling to evaluate this state. The new de-
rivatives of thiosemicarbazones (Ia – Ig and IIa – IIg) were
drawn and converted from 2D to 3D, and the PM3MM basis
set was assigned and then minimized using a semi-empirical
method with the aid of Gaussian09 program package. A
conformational search of the ligands was carried out using
BEST algorithm for 3D pharmacophore generation. The
Auto Pharmacophore Generation protocol in Discovery Stu-
dio 3.5 was used based on a bioactive conformation. In this
study, we used compound Ig as the bioactive conformation
3
2
proved inhibitory against influenza
A
virus (EC :
50
12.7 – 26.7 mg/mL for strain H1N1 and 9.3 – 18.5 mg/mL for
strain H3N2). These compounds were only 5- to 20-fold less
effective than the clinically used drug oseltamivir and were
neither cytotoxic (MCC) nor cytostatic (CC ) at 100 mg/mL
50
against MDCK cells (Table 3). Thus, a certain degree of selec-
tivity was present for these compounds against influenza A vi-
rus in cell culture and should be further considered for optimi-
zation as potential anti-influenza virus lead compounds.
The synthesized compounds were also evaluated for their
cytostatic activity against murine leukemia L1210, human
+
CD T-lymphocyte CEM and cervix carcinoma HeLa cells
4
with IC values between 0.30 and 2.50 mg/mL. The
50
(Table 4). Most compounds in the IIa – IIg series did not
pharmacophore models were generated based on the main
features of compound Ig. These features were Hb-Acceptor,
Hb-Donor, Hydrophobic and Ring Aromatic in the
pharmacophore models. Besides, the Genetic Function Ap-
proximation (GFA) model for the selectivity of
pharmacophores was used. The best model was determined
with the help of Clusters pharmacophores subprotocol. The
model contains six features, representing two hydrophobic
show pronounced cytostatic activity (IC ³ 100 mg/mL).
50
However, IIb was moderately cytostatic (IC = 13 –
50
15 mg/mL), whereas IIg seemed to be poorly inhibitory to
L1210 and CEM cell proliferation, but had a pronounced
antiproliferative activity against HeLa cells (IC
=
50
1.9 mg/mL). This unusual selectivity between tumor cell
lines should be further investigated. In fact, the I series of

146
A. Karaküçük-Ýyidoðan et al.
compounds were often somewhat more cytostatic than their
ring affected the cytostatic activity. Compounds Ia and IIa
without any substituent group on the benzene ring did not
show cytostatic activity. Then, we examined the effect of in-
troducing various substituent groups in the benzene ring of
compounds Ia and IIa. The -OH, -OCH , -CF , -F, -NO and
corresponding II series of compounds. It may not be a coin-
cidence that Ig (IC = 0.3 mg/mL against HeLa cells) was
50
also most cytostatic among series I and also showed the most
pronounced antiproliferative potency against HeLa cells.
The structure – activity relationship (SAR) studies were
performed to determine how the substituent on the benzene
3
3
2
-N(CH CH Cl) groups at the para position of the benzene
2
2
2
ring of compounds Ia and IIa were used to form compounds
TABLE 2. Cytotoxicity and Antiviral Activity of Thiosemicarbazides (I and II) and thiosemicarbazones (Ia – Ig and IIa – IIg) in HEL, HeLa
and Vero Cell Cultures
HEL cell culture
EC50b) (mg/mL)
HeLa cell culture
Vero cell culture
EC50b) (mg/mL)
EC50b)(mg/mL)
Herpes
simplex
virus-1
TK KOS
ACVr
MCCa)
MCCa)
MCCa)
Compound
Herpes Herpes
Vesicu-
lar
virus-1 virus-2 virus stomatiti
(KOS) (G) s virus
Vesicu-
lar
Respira-
Para in-
flu- Reo vi- Sindbis
Coxsack
ie virus
B4
Coxsack Punta
ie virus Toro vi-
B4 rus
simpex simplex Vaccina
tory
(mg/mL)
(mg/mL)
(mg/mL)
stomatiti
s virus
syncytic
al virus
enza-3 rus-1
virus
virus
I
>100 >100 >100 50
>100 >100 >100 >100 >100 >100
100 >20 >20 >20 >20 >20
>100 >100
>100 >100 >100 100
>20
>20
>20
>20
>20
³100
³100
100
Ia
>100 >100 >100 >100 >100 >100 >100 >100 >100
Ib
Ic
>20
>20
>20
>20
>20
>20
>20
>20
>20
100
100
100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>100 >100 >100 >100 >100 >100 100
>100 >100 >100 >100 >100 >100 100
Id
Ie
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 100
³100
³100
³20
If
>100 >100 >100 >100 >100 >100 >100 >100 >100
Ig
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
100
100
>4
>4
>4
3
>4
II
>100 >100 >100 45
100 >20 >20 >20
>100 100
>20 >20
100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
IIa
IIb
IIc
IId
IIe
IIf
IIg
100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 100
>20
>20
>20
100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
100
100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>100 >100 >100 >100 100
>100 >100 >100 >100 100
>100 >100 >100 >100 >100 >100 100
>20
>20
>20
100
100
>20
>20
100
>20
10
>20
>20
>100 >100 >100 100
³100
Brivudin >250 0.04
(mM)
>250 100
Cidofovir >250
(mM)
2
2
29
>250
2
Acyclovir >250 0.4
0.2
0.06
>250 >250 112
>100 >100 10
(mM)
Ganciclovi >100 0.03
r (mM)
DS-5000c)
>100
2
34
0.6
>100 >100 >100 100
9
100
(S)-DHPA
d)(mM)
>250 >250 >250 >250 >250 >250 >250 >250 >250 >250
Ribavirin
>250
5
112
10
>250 112
>250 >250 >250 146
(mM)
a) Required to cause a microscopically detectable alteration of normal cell morphology.
b) Required to reduce virus-induced cytopathogenicity by 50%.
c)DS-5000: dextran sulfate (molecular weight, 5.000).
d)(S)-DHPA: (S)-9-(2,3-dihydroxypropyl)adenine.

Synthesis, Biological Evaluation and Ligand
147
(Ib – Ig and IIb – IIg). Especially Ig with a bis(2-chloro-
cisplatin obtained against the three tumor cell lines
(p = 0.647). The results showed that compounds Ia – Ig con-
taining electron-withdrawing group, such as -CN located at
the para position of the benzene ring, increased the cytostatic
ethyl)amino moiety, the IC values (0.30 – 2.50 mg/mL)
50
were the lowest (most effective) among all compounds and
compared to cisplatin as a reference compound. A two-tailed,
unpaired t-test (GraphPad Prism) revealed no significant dif-
ference between the cytostatic inhibition values of Ig and
activity, according to the electron-donating group (-SCH ) at
3
the same position of the structure (like compounds
TABLE 3. Cytotoxicity and Antiviral Activity of Thiosemicarbazides (I and II) and Thiosemicarbazones (Ia – Ig and IIa – IIg) in CRFK and
MDCK Cell Cultures.
CRFK cell culture
MDCK cell culture
EC50b) (mg/mL)
EC50b) (mg/mL)
Influenza RH1N1
SUBTYPE
Influenza A
H3N2 subtype
Influenza B
a)
a)
Compound
CC50
CC50
MCCc)
(mg/mL)
(mg/mL)
(mg/mL)
Feline
Corona
Virus
Feline
Herpes
Virus
visual CPE
score
MTS
visual CPE
MTS
visual CPE
MTS
score
score
(FIPV)
I
84.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>20
>100
31.5
>20
>100
54.3
78.6
44.3
>100
>100
>100
>100
>100
0.4
100
³20
100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
Ia
Ib
Ic
>20
>20
>20
>20
>20
>20
23.1
25.0
100
>20
>20
>20
>20
>20
>20
Id
Ie
>100
>100
>100
>100
>100
7.0
>100
>100
>100
>100
>100
>100
>100
17.8
>100
>100
>100
0.8
27.0
14.5
10.5
>0.16
>20
26.3
10.9
17.1
>0.16
>20
9.5
13.9
14.0
17.5
>0.16
>20
>100
>100
>100
>0.16
>20
>100
>100
>100
>0.16
>20
23.0
15.0
>0.16
>20
If
Ig
II
90.2
>100
0.8
100
IIa
IIb
IIc
IId
100
>20
>20
>20
>20
>20
>20
>100
>100
>100
0.8
>0.16
>20
>0.16
>20
>0.16
>20
>0.16
>20
>0.16
>20
>0.16
>20
>100
100
>100
>100,
>100
>100
>100
>100
>100
>100
³20100
IIe
>100
>100
>100
>100,
>100
>100
>100
>100
>100
>100
³20100
IIf
>100
>100
>100
33.2
>100
>100
36.2
1.6
>100
>100
16.5
1.8
>100
>100
>100
>100
11.0
16.5
9.0
9.5
>100
>100
>100
>100
IIg
>100
>100
>100
>100
HHAd)
UDAe)
Ganciclovir
>100
>100
7.3
(mM)
Oseltamivir
carboxylate
>100
>100
2.0
2.7
1.0
0.8
34.0
29.1
Ribavirin
>100
>200
>200
>100
>200
>200
9.0
8.0
8.3
5.4
9.0
0.9
0.9
5.7
0.3
0.4
9.0
3.4
Amandatine
Rimandatine
>200
>200
>200
>200
18.0
25.7
a) 50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan- based MTS assay.
b) 50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by measuring the
cell viability with the colorimetric formazan-based MTS assay.
c) Required to cause a microscopically detectable alteration of normal cell morphology.
d)HHA: Hippeastrum hybrid Lectin (Amoryllis bulbs).
e)UDA: Urtica dioica agglutinin.

148
A. Karaküçük-Ýyidoðan et al.
IIa – IIg). Furthermore, the introduction of electron-donat-
modeling. Especially, 3D-conformation of the investigated
compounds showed the major effect in this study. Unfortu-
nately, compounds Ia – If and IIa – IIf did not show any ac-
tivity in comparison to compounds Ig and IIg. The proposed
methods were able to provide valuable information about
key features and interactions that are important for the bio-
logical activity of compound Ig. Thus, ligand-based
pharmacophore modeling was increasingly successful in de-
lineating why compound Ig displayed better cytostatic activ-
ity than compound IIg.
ing groups (-OH, -OCH ) at the para position of the benzene
3
ring weakened the cytostatic effects. While the same situa-
tion for electron-withdrawing groups (-F, -NO -CF ) was
2,
3
also observed, compound IIb exhibited moderate activity
against all tumor cell types. Besides, compound 1g displayed
the better cytostatic activity than its analog compound IIg,
which has an electron-donating group (-SCH ) at the para
3
position. These results indicated that the introduction of the
bulky alkylating group (bis(2-chloroethyl)amino) and a
cyano group reinforced the cytostatic activity.
The pharmacophore model (Fig. 2A) was hypothetically
superimposed with compounds Ig (fit value 4.6216) and IIg
(fit value 3.0032). Figures 2B and 2C summarize the results
of model fitting obtained for compounds Ig and IIg. The
other compounds were not superimposed to fit with the best
model. In addition to comparing the results of modeling with
experimental data, they show compatible and significant
ACKNOWLEDGEMENTS
The present work was supported by the Scientific Re-
search Projects Governing Unit Council of Gaziantep Uni-
versity (Grant No. FEF.08.10), Gaziantep, Turkey and by the
KU Leuven (GOA 10/14). The authors are grateful to Leentje
Persoons, Frieda De Meyer, Leen Ingels, Anita Camps, Ste-
ven Carmans, Lies Van den Heurck and Lizette van
Berckelaer for excellent technical assistance and Dr.
Annelies Stevaert for statistical analysis.
trends. It is established that Hb-acceptor (-CN; -SCH ), hyd-
3
rophobic groups (-N(CH CH Cl) ), and 3D-conformations
2
2
2
are responsible for the cytostatic activity of compounds such
as Ig and IIg, according to ligand-based pharmacophore
+
TABLE 4. Inhibitory Effect of Compounds I, Ia – Ig and II, IIa – IIg on the Proliferation of Murine Leukemia (L1210), Human CD T-Lym-
4
phocyte (CEM) and Human Cervix Carcinoma (HeLa) Cells
IC50a) (mg/mL)
Compound
L1210
CEM
HeLa
I
Ia
20 ± 3
79 ± 48
50 ± 2
>200
21 ± 1
83 ± 59
107 ± 13
>200
11 ± 0
67 ± 10
91 ± 18
31 ± 23
>200
Ib
Ic
Id
>200
>200
Ie
29 ± 12
>200
38 ± 27
>200
68 ± 9
If
142 ± 20
0.30 ± 0.17
18 ± 8
Ig
2.5 ± 0.2
83 ± 24
>200
0.70 ± 0.16
20 ± 0
II
IIa
IIb
IIc
IId
IIe
IIf
>200
185 ± 21
13 ± 2
14 ± 4
15 ± 14
121 ± 24
145 ± 68
³ 200
>200
112 ± 8
61 ± 5
³ 200
>200
>200
>200
>200
>200
IIg
Cisplatinb)
³ 200
50 ± 23
2.20 ± 0.1
1.9 ± 1.7
0.22 ± 0.03
2.53 ± 0.3
a)
50%-Inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%. Compound concentration of
100, 20, 4, 0.8, 0.16 and 0.032 mg/mL have been tested (5-fold dilutions) and data represent the mean ± SD (standard deviation) of at least two to
three independent experiments. All values were significantly different from cisplatin (less active), except for Ig (no statistical difference, p
higher than 0.05).
b)
Cisplatin was purchased from SIGMA.

Synthesis, Biological Evaluation and Ligand
149
Fig. 2. (A) Pharmacophore model of the docking pose of compound Ig; (B) the mapping of compound Ig (fit value 4.6216) to hypo10; (C) the
mapping of compound IIg (fit value 3.0032) to hypo10.
9. A. Karaküçük-Iyidoðan, D. Taþdemir, E. E. Oruç-Emre, et al.,
Eur. J. Med. Chem., 46, 5616 – 5624 (2011).
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