Synthesis, molecular modeling, and biological evaluation of novel chiral thiosemicarbazone derivatives as potent anticancer agents

Article abstract of DOI:10.1002/chir.22408

A series of new chiral thiosemicarbazones derived from homochiral amines in both enantiomeric forms were synthesized and evaluated for their in vitro antiproliferative activity against A549 (human alveolar adenocarcinoma), MCF-7 (human breast adenocarcinoma), HeLa (human cervical adenocarcinoma), and HGC-27 (human stomach carcinoma) cell lines. Some of compounds showed inhibitory activities on the growth of cancer cell lines. Especially, compound 17b exhibited the most potent activity (IC₅₀ 4.6 μM) against HGC-27 as compared with the reference compound, sindaxel (IC₅₀ 10.3 μM), and could be used as a lead compound to search new chiral thiosemicarbazone derivatives as antiproliferative agents.

Full text of DOI:10.1002/chir.22408

CHIRALITY 27:177–188 (2015)
Synthesis, Molecular Modeling, and Biological Evaluation of Novel
Chiral Thiosemicarbazone Derivatives as Potent Anticancer Agents
1
1
2
1
˙
˘
1
˘
*
DEMET TAS¸DEMIR, AYS¸EGÜL KARAKÜÇÜK-IYIDOGAN, MUSTAFA ULAS¸LI, TUGBA TAS¸KIN-TOK,
EMINE ELÇIN ORUÇ-EMRE, AND HASAN BAYRAM3
˙
˙
1Gaziantep University, Faculty of Science and Arts, Department of Chemistry, Gaziantep, Turkey
2Gaziantep University, Faculty of Medicine, Department of Medical Biology, Sehitkamil, Gaziantep, Turkey
3Gaziantep University, Faculty of Medicine, Department of Pulmonary Diseases, Sehitkamil, Gaziantep, Turkey
ABSTRACT
A series of new chiral thiosemicarbazones derived from homochiral amines in both
enantiomeric forms were synthesized and evaluated for their in vitro antiproliferative activity against
A549 (human alveolar adenocarcinoma), MCF-7 (human breast adenocarcinoma), HeLa (human cervi-
cal adenocarcinoma), and HGC-27 (human stomach carcinoma) cell lines. Some of compounds showed
inhibitory activities on the growth of cancer cell lines. Especially, compound 17b exhibited the most
potent activity (IC50 4.6 μM) against HGC-27 as compared with the reference compound, sindaxel
(IC50 10.3 μM), and could be used as a lead compound to search new chiral thiosemicarbazone
derivatives as antiproliferative agents. Chirality 27:177–188, 2015. © 2014 Wiley Periodicals, Inc.
KEY WORDS: chiral amine; hydrazinecarbothioamide; cytotoxicity; HipHop; pharmacophore model
INTRODUCTION
of chiral drugs in enantiopure form is very important in
synthetic organic chemistry, medicinal chemistry, natural
product chemistry, and the pharmaceutical industry.16,17
When a chiral compound is synthesized in several steps, it
can be important for practical and economic reasons to intro-
duce the proper stereochemistry at an early stage. This can
be achieved starting from chiral building blocks that are
enantiomerically pure and have functionalities that allow
them to be transformed into the desired product.18 According
to this approach, chiral amines can be considered convenient
starting materials in the synthesis of chiral thiosemicar-
bazones because they are commercially available, and gener-
ally accessible in two enantiomeric forms.
In continuation of our research on compounds endowed
with anticancer activity, we designed and synthesized
enantiopure thiosemicarbazones derived from homochiral
amines as chiral building blocks. Our previous results have
shown that the reaction in basic media between amines and
CS2 in CHCl3 is a very efficient procedure to synthesize the
chiral isothiocyanate derivatives. Furthermore, this process
retains the stereochemistry of the starting chiral amine. In
the present work, we prepared chiral thiosemicarbazones
with good yield and high enantiopurity and evaluated their
cytotoxic activity against human alveolar adenocarcinoma
(A549), human breast adenocarcinoma (MCF-7), human
cervical adenocarcinoma (HeLa), and human stomach carci-
noma (HGC-27) cell lines. In addition, a virtual screening of
the mentioned chiral thiosemicarbazone (17b) was per-
formed to illustrate its SAR (structure–activity relationships)
of active sites of protein–ligand interactions and possible
conformations by generating a pharmaphore hypothesis to
increase knowledge of the structure and mechanism.
Cancer is a significant cause of mortality and morbidity
worldwide and current chemotherapeutic agents are not suffi-
ciently effective. Delivery of anticancer drugs to specific tumor
tissues is a complex process involving various biochemical,
mechanical, and biophysical factors. Designing specific che-
motherapeutic drugs may be challenging for treatment but
using many advanced technologies such as next-generation
sequencing, microarray protein expression profiles, and signal-
ing pathways have supported the discovery of treatment
targets and personalized treatments.1 Recently, the majority
of drugs used for cancer treatment are not "cancer cell-
specific" and these drugs are cytotoxic for normal cells. There-
fore, pharmaceutical research has depended on the discovery
of new drugs for cancer treatment.2
Thiosemicarbazones are an important class of imine
derivatives that have very different kinds of pharmacological
activities, especially as anticancer agents.3 Their anticancer
activity is attributed to the ability to inhibit three target points
on which research is focused. First, thiosemicarbazone deriv-
atives inhibit the iron-containing ribonucleotide reductase,
which is involved in the biosynthesis of DNA by metal chela-
tion.4–6 Second, these compounds inhibit topoisomerase II by
stabilization of the cleavable complex between this enzyme
and DNA through a thiol alkylation.7–9 Third, recently
thiosemicarbazones were found to be inhibitors of ATP-
binding cassette (ABC) transporters, which are known to play
a critical role in the development of multidrug resistance.10,11
Biological systems (proteins, sugars, enzymes, etc.) recog-
nize an enantiomer pair as different substances and so two
enantiomers of a chiral drug molecule bind differently to tar-
get receptors. Consequently, an enantiomer may act as an ef-
ficient therapeutic drug while the other enantiomer is inactive
or highly toxic.12–14 Approximately 56% of the drugs currently
in use are chiral compounds, and about 88% of these chiral
synthetic drugs are used therapeutically as racemates. Unfor-
tunately, there are many racemic drugs where the stereospec-
ificity of the metabolism and/or the pharmacodynamic effects
of the enantiomers is not known.15 Therefore, the synthesis
*Correspondence to: Demet Taşdemir, Gaziantep University, Faculty of
Science and Arts, Department of Chemistry, 27310, Gaziantep, Turkey.
E-mail: demettasdemir@gmail.com
Received for publication 2 September 2014; Accepted 13 October 2014
DOI: 10.1002/chir.22408
Published online 14 November 2014 in Wiley Online Library
(wileyonlinelibrary.com).
© 2014 Wiley Periodicals, Inc.

178
TAS¸DEMIR ET AL.
EXPERIMENTAL
Measurement and Reagents
Anal. Calcd. for C16H17N3S: C, 67.81; H, 6.05; N, 14.83; S,
11.31%. Found: C, 67,90; H, 6.03; N, 14.78; S, 11.32%.
Melting points were determined in an EZ-Melt MPA120 Automated
Melting Point apparatus. The optical rotations were determined in a
PolAAr 3000 polarimeter at 20°C, using dimethyl sulfoxide (DMSO).
UV-vis spectra were recorded on a PG Instruments (UK) T80+ UV-visible
spectrophotometer in DMF. IR spectra were recorded on a Perkin Elmer
(Boston, MA) 1620 model FT-IR Spectrophotometer with a universal ATR
sampling accessory. 1H and 13C nuclear magnetic resonance (NMR)
spectra were obtained at room temperature with a Bruker (Billerica,
MA) AVANC-DPX NMR spectrophotometer in DMSO-d6 using
tetramethylsilane (TMS) as an internal standard at 400 MHz and
100 MHz, respectively. The chemical shifts are given in parts per million
(ppm) downfield from TMS. The splitting patterns of 1H NMR were de-
signed as follows: s: singlet, brs: broad singlet, d: doublet, dd: double dou-
blet, t: triplet, q: quartet, p: pentet, m: multiplet. The coupling constants
(J) are given in Hertz. The mass spectra were obtained using an ABSciex
3200 QTrap system liquid chromatography, tandem mass spectroscopy
(LC/MS/MS) in the electrospray mode. Elemental analyses (CHNS)
data were obtained by a Thermo Scientific (Pittsburgh, PA) Flash 2000
elemental analyzer.
(+)-(S)-2-benzylidene-N-(1-phenylethyl)hydrazinecarbothioamide
(3b). Yellow crystals; yield 75%; mp. 92–93°C; [α]D = +77.0
(c: 0.5, DMSO); UV (DMSO, λmax, nm): 334, 324, 256; IR
(νmax, cmÀ1): 3309 and 3183 (N-H), 1601 (C=N), 1203
(C=S); EI-MS (m/z): 283.1 [M]+; 1H NMR (400 MHz,
DMSO-d6): δ 1.58 (d, 3H, J = 6.8 Hz, H-6’), 5.75 (p, 1H,
H-1’), 7.25 (t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.35 (t, 2H, J1,
J2 = 7.6 Hz, H-4’), 7.42-7.45 (m, 5H, H-3’, H-4", H-5"), 7.83
(dd, 2H, J1, J2 = 7.2 Hz, H-3"), 8.11 (s, 1H, H-1"), 8.62 (d, 1H,
J = 8.8 Hz, H-4), 11.58 (s, 1H, H-2); 13C NMR (100 MHz,
DMSO-d6): δ 21.76 (C-6´), 52.87 (C-1’), 126.79 (C-5’), 126.19
(C-3’), 127.89 (C-4’), 128.68 (C-4"), 129.13 (C-3"), 130.37 (C-
5"), 134.53 (C-2"), 143.04 (C-2’), 144.30 (C=N, C-1"), 176.87
(C=S, C-3). Anal. Calcd. for C16H17N3S: C, 67.81; H, 6.05; N,
14.83; S, 11.31%. Found: C, 67,92; H, 6.01; N, 14.75; S, 11.36%.
(À)-(R)-2-(4-bromobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (4a). Yellow crystals; yield 87%; mp. 153–154°C;
[α]D = À72.4 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 340,
330, 260; IR (νmax, cmÀ1): 3356 and 3141 (N-H), 1593 (C=N)
1209; (C=S); EI-MS (m/z): 363.2 [M+H]+; 1H NMR
(400 MHz, DMSO-d6): δ 1.57 (d, 3H, J = 7.2 Hz, H-6’), 5.75 (p,
1H, H-1’), 7.25 (t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.36 (t, 2H, J1,
J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 8.8 Hz, H-3’), 7.62 (d, 2H,
J = 8.4 Hz, H-4"), 7.80 (d, 2H, J = 8.4 Hz, H-3"), 8.07 (s, 1H,
H-1"), 8.69 (d, 1H, J = 8.8 Hz, H-4), 11.61 (s, 1H, H-2); 13C
NMR (100 MHz, DMSO-d6): δ 21.71 (C-6’), 52.91 (C-1’),
123.57 (C-5’), 126.76 (C-5’), 127.18 (C-3’), 128.67 (C-4’), 129.78
(C-3"), 132.08 (C-4"), 133.85 (C-2"), 141.84 (C-2’), 144.22
(C=N, C-1"), 176.96 (C=S, C-3). Anal. Calcd. for C16H16BrN3S:
C, 53.04; H, 4.45; N, 11.60; S, 8.85%. Found: C, 53.06; H, 4.41;
N, 11.76; S, 8.81%.
All chemicals and solvents were purchased from Merck (Darmstadt,
Germany) and Aldrich Chemical (Milwaukee, WI) and used without fur-
ther purification. All reaction progress was monitored by thin layer chro-
matography (TLC). TLC was performed on silica gel plates (Merck Silica
Gel 60, F254, 0.2 mm) with visualization by exposure to iodine vapor and
UV-light using EtOAc/hexane (v/v 1:1 and 1:3) as the solvent system.
Synthesis of Thiosemicarbazones 3a-17a and 3b-17b: A
General Method
To a hot solution of chiral thiosemicarbazides 2a-2b (1.53 mmol) in
methanol (5 mL) was added dropwise a solution of the corresponding
p-substituted benzyaldehydes (1.53 mmol) in hot methanol (5 mL) over
15 min. The reaction mixture was refluxed and the progress of the reac-
tion was followed by TLC (10–12 h) and cooled. The product was filtered
and washed with petroleum ether. The crude product was recrystallized
from ethanol.
(+)-(S)-2-(4-bromobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (4b). Yellow crystals; yield 83%; mp. 154–155°C;
[α]D = +71.2 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 339,
330, 259; IR (νmax, cmÀ1): 3356 and 3145 (N-H), 1591
(C=N), 1208 (C=S); EI-MS (m/z): 363.1 [M+H]+; 1H NMR
(400 MHz, DMSO-d6): δ 1.57 (d, 3H, J = 7.2 Hz, H-6’), 5.76
(p, 1H, H-1’), 7.25 (t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.35 (t, 2H, J1,
J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 8.8 Hz, H-3’), 7.62 (d, 2H,
J = 8.4 Hz, H-4"), 7.81 (d, 2H, J = 8.4 Hz, H-3"), 8.07 (s, 1H,
H-1"), 8.70 (d, 1H, J = 9.2 Hz, H-4), 11.62 (s, 1H, H-2); 13C
NMR (100 MHz, DMSO-d6): δ 21.72 (C-6’), 52.92 (C-1’),
123.57 (C-5’), 126.76 (C-5’), 127.18 (C-3’), 128.66 (C-4’),
129.77 (C-3"), 132.08 (C-4"), 133.86 (C-2"), 141.84 (C-2’), 144.22
(C=N, C-1"), 176.99 (C=S, C-3). Anal. Calcd. for C16H16BrN3S: C,
53.04; H, 4.45; N, 11.60; S, 8.85%. Found: C, 53.08; H, 4.46; N,
11.72; S, 8.80%.
(À)-(R)-N-(1-phenylethyl)hydrazinecarbothioamide (2a). Yellow
viscose; yield 84%; IR (νmax, cmÀ1): 3272 and 3092 (N-H),
1514 (C-N), 1191 (C=S); H NMR (400 MHz, DMSO-d6): δ
1.44 (d, 3H, J = 6.8 Hz, H-6’), 4.62 (brs, 2H, H-1), 5.55 (p, 1H,
H-1’), 7.25 (t, 1H, J1, J2 = 6.8 Hz, H-5’), 7.30-7.35 (m, 4H, H-3’,
H-4’), 8.03 (d, 1H, J = 8.8 Hz, H-4), 8.74 (s, 1H, H-2).
1
(+)-(S)-N-(1-phenylethyl)hydrazinecarbothioamide (2b). Yellow
viscose; yield 77%; IR (νmax, cmÀ1): 3350 and 3157 (N-H),
1
1549 (C-N), 1128 (C=S); H NMR (400 MHz, DMSO-d6): δ
1.42 (d, 3H, J = 6.8 Hz, H-6’), 4.54 (brs, 2H, H-1), 5.51 (p, 1H,
H-1’), 7.23 (t, 1H, J1, J2 = 6.8 Hz, H-5’), 7.24-7.37 (m, 4H, H-3’,
H-4’), 8.00 (d, 1H, J = 8.8 Hz, H-4), 8.68 (s, 1H, H-2).
(À)-(R)-2-benzylidene-N-(1-phenylethyl)hydrazinecarbothioamide
(3a). Yellow crystals; yield 72%; mp. 88–89°C; [α]D = À76.6
(c: 0.5, DMSO); UV (DMSO, λmax, nm): 334, 320, 258; IR
(νmax, cmÀ1): 3309 and 3187 (N-H), 1601 (C=N), 1205
(C=S); EI-MS (m/z): 283.1 [M]+; 1H NMR (400 MHz,
DMSO-d6): δ 1.58 (d, 3H, J = 7.2 Hz, H-6’), 5.76 (p, 1H, H-1’),
7.25 (t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz,
H-4’), 7.43-7.45 (m, 5H, H-3’, H-4", H-5"), 7.84 (dd, 2H, J1,
J2 = 7.2 Hz, H-3"), 8.12 (s, 1H, H-1"), 8.62 (d, 1H, J = 9.2 Hz,
H-4), 11.58 (s, 1H, H-2); 13C NMR (100 MHz, DMSO-d6): δ
21.77 (C-6’), 52.88 (C-1’), 126.79 (C-5’), 126.19 (C-3’), 127.89
(C-4’), 128.68 (C-4"), 129.13 (C-3"), 130.37 (C-5"), 134.53
(C-2"), 143.04 (C-2’), 144.30 (C=N, C-1"), 176.90 (C=S, C-3).
(À)-(R)-2-(4-chlorobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (5a). White crystals; yield 75%; mp. 138–139°C;
[α]D = À82.7 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 339,
325, 259; IR (νmax, cmÀ1): 3359 and 3142 (N-H), 1596
(C=N), 1205 (C=S); EI-MS (negative ion mode, m/z): 316.0
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.57 (d, 3H,
J = 7.2 Hz, H-6’), 5.75 (p, 1H, H-1’), 7.25 (t, 1H, J1, J2 = 7.2 Hz,
H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 7.2 Hz,
H-3’), 7.49 (d, 2H, J = 8.8 Hz, H-4"), 7.87 (d, 2H, J = 8.4 Hz,
H-3"), 8.08 (s, 1H, H-1"), 8.69 (d, 1H, J = 8.8 Hz, H-4), 11.61
(s, 1H, H-2); 13C NMR 100 MHz, DMSO-d6): δ 21.72 (C-6’),
Chirality DOI 10.1002/chir

POTENTIAL OF CHIRAL CANCER DRUGS
179
52.90 (C-1’), 126.77 (C-5’), 127.17 (C-3’), 128.54 (C-4’), 130.50
(C-4"), 133.53 (C-3"), 134.75 (C-2"), 141.71 (C-5"), 143.99
(C-2’), 144.24 (C=N, C-1"), 176.97 (C=S, C-3). Anal. Calcd.
for C16H16ClN3S: C, 60.46; H, 5.07; N, 13.22; S, 10.09%.
Found: C, 60.49; H, 5.06; N, 13.21; S, 10.10%.
J = 7.6Hz, H-3’), 7.87 (d, 2H, J = 8.4 Hz, H-4’), 8.04 (d, 2H,
J =8.0Hz, H-3"), 8.14 (s, 1H, H-4"), 8.75 (d, 1H, J=8.8Hz,
H-4), 11.73 (s, 1H, H-2). 13C NMR (100MHz, DMSO-d6): δ
21.66 (C-6’), 53.00 (C-1’), 112.01 (C-5"), 119.26 (C≡N), 126.76
(C-3"), 127.19 (C-5’), 128.67 (C-3’), 130.34 (C-4’), 133.66 (C-4"),
139.13 (C-2"), 140.91 (C-2’), 144.13 (C=N, C-1"), 177.19 (C=S,
C-3). Anal. Calcd. for C17H16N4S: C, 66.21; H, 5.23; N, 18.17;
S, 10.40%. Found: C, 66.29; H, 5.17; N, 18.19; S, 10.52%.
(+)-(S)-2-(4-chlorobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (5b). White crystals; yield 81%; mp. 138–139°C;
[α]D = +81.2 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 340,
330, 260; IR (νmax, cmÀ1): 3359 and 3139 (N-H), 1595
(C=N), 1205 (C=S); EI-MS (negative ion mode, m/z): 316.1
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.57 (d, 3H,
J = 7.2 Hz, H-6’), 5.76 (p, 1H, H-1’), 7.26 (t, 1H, J1, J2 = 7.2 Hz,
H-5’), 7.36 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 7.2 Hz,
H-3’), 7.48 (d, 2H, J = 8.8 Hz, H-4"), 7.87 (d, 2H, J = 8.4 Hz,
H-3"), 8.09 (s, 1H, H-1"), 8.71 (d, 1H, J = 8.8 Hz, H-4), 11.63
(s, 1H, H-2); 13C NMR (100 MHz, DMSO-d6): δ 21.71 (C-6’),
52.89 (C-1’), 126.77 (C-5’), 127.17 (C-3’), 128.53 (C-4’),
130.51 (C-4"), 133.53 (C-3"), 134.76 (C-2"), 141.69 (C-5"),
143.96 (C-2’), 144.24 (C=N, C-1"), 176.96 (C=S, C-3). Anal.
Calcd. for C16H16ClN3S: C, 60.46; H, 5.07; N, 13.22; S,
10.09%. Found: C, 60.42; H, 5.09; N, 13.19; S, 10.05%.
(+)-(S)-2-(4-cyanobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (7b). Yellow crystals; yield 72%; mp. 166–167°C;
[α]D = +81.2 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 360,
348; 260; IR (νmax, cmÀ1): 3370 and 3139 (N-H), 2224
(C≡N), 1595 (C=N), 1206 (C=S); EI-MS (negative ion mode,
m/z): 307.0 [M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.58 (d,
3H, J = 7.2 Hz, H-6’), 5.77 (p, 1H, H-1), 7.25 (t, 1H, J1,
J2 = 7.2 Hz H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43
(d, 2H, J = 7.6 Hz, H-3’), 7.88 (d, 2H, J = 8.0 Hz, H-3"), 8.05
(d, 2H, J = 7.6 Hz, H-4"), 8.14 (s, 1H, H-1"), 8.76 (d, 1H,
J = 8.8 Hz, H-4), 11.73 (s, 1H, H-2); 13C NMR (100 MHz,
DMSO-d6): δ 21.65 (C-6’), 53.00 (C-1’), 112.01 (C-5"),
119.28 (C≡N), 126.76 (C-3"), 127.19 (C-5’), 128.68 (C-3’),
130.34 (C-4’), 133.67 (C-4"), 139.13 (C-2"), 140.89 (C-2’),
144.13 (C=N, C-1"), 177.16 (C=S, C-3). Anal. Calcd. for
C17H16N4S: C, 66.21; H, 5.23; N, 18.17; S, 10.40%. Found: C,
66.31; H, 5.19; N, 18.22; S, 10.44%.
(À)-(R)-2-(4-fluorobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (6a). White crystals; yield 71%; mp. 131–132°C;
[α]D = À84.0 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 335,
320, 260; IR (νmax, cmÀ1): 3339 and 3172 (N-H), 1600 (C=N),
1204 (C=S); EI-MS (m/z): 301.2 [M]+; 1H NMR (400MHz,
DMSO-d6): δ 1.57 (d, 3H, J = 6.8 Hz, H-6’), 5.75 (p, 1H, H-1),
7.25 (t, 3H, J1, J2 = 6.8 Hz, H-5’, H-3"), 7.35 (t, 2H, J1,
J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 7.2Hz, H-3’), 7.91 (dd, 2H, J1,
J2 =8.8Hz, H-4"), 8.09 (s, 1H, H-1"), 8.67 (d, 1H, J =9.2Hz,
H-4), 11.58 (s, 1H, H-2); 13C NMR (100MHz, DMSO-d6): δ
22.77 (C-6’), 52.89 (C-1’), 116.06 (C-4"), 126.75 (C-5’), 127.19
(C-3’), 128.68 (C-4’), 130.15 (C-2"), 131.14 (C-3"), 141.97
(C-2’), 144.26 (C=N, C-1"), 164.73 (C-5"), 176.89 (C=S, C-3).
Anal. Calcd. for C16H16FN3S: C, 63.43; H, 5.65; N, 13.93; S,
2.34%. Found: C, 63.56; H, 5.45; N, 13.84; S, 2.50%.
(À)-(R)-2-(4-hydroxybenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (8a). Yellow crystals; yield 87%; mp. 171–172°C; [α]
D = À69.5 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 344, 330,
304; IR (νmax, cmÀ1): 3359 and 3151 (N-H), 1605 (C=N), 1200
(C=S); EI-MS (m/z): 300.1 [M+H]+; 1H NMR (400 MHz,
DMSO-d6): δ 1.56 (d, 3H, J =6.8Hz, H-6’), 5.73 (p, 1H, H-1),
6.81 (d, 2H, J = 8.8 Hz, H-4"), 7.26 (t, 1H, J1, J2 = 7.2HzH-5’),
7.35 (t, 2H, J1, J2 =7.6Hz, H-4’), 7.42 (d, 2H, J =8.4Hz, H-3’),
7.65 (d, 2H, J =8.8Hz, H-3"), 8.01 (s, 1H, H-1"), 8.47 (d, 1H,
J =9.2Hz, H-4), 9.94 (s, 1H, OH), 11.39 (s, 1H, H-2); 13C
NMR (100 MHz, DMSO-d6): δ 21.15 (C-6’), 52.03 (C-1’),
115.33 (C-4"), 124.75 (C-2"), 126.05 (C-5’), 126.46 (C-3’),
127.97 (C-4’), 128.95 (C-3"), 142.81 (C-2’), 143.69 (C=N, C-1"),
159.09 (C-5"); 175.74 (C=S, C-3). Anal. Calcd. for C16H17N3OS:
64.19; H, 5.72; N, 14.04; S, 10.71%. Found: C, 64.28; H, 5.68; N,
14.19; S, 10.72%.
(+)-(S)-2-(4-fluorobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (6b). White crystals; yield 77%; mp. 134–135°C;
[α]D = +83.2 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 335,
320, 260; IR (νmax, cmÀ1): 3339 and 3167 (N-H), 1601
(C=N), 1203 (C=S); EI-MS (m/z): 301.0 [M]+; 1H NMR
(400 MHz, DMSO-d6): δ 1.57 (d, 3H, J = 6.8 Hz, H-6’), 5.75
(p, 1H, H-1), 7.25 (m, 3H, H-5’, H-3"), 7.35 (t, 2H, J1,
J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 7.6 Hz, H-3’), 7.91 (dd, 2H,
J1, J2 = 8.4 Hz, H-4"), 8.10 (s, 1H, H-1"), 8.64 (d, 1H,
J = 8.8 Hz, H-4), 11.56 (s, 1H, H-2); 13C NMR (100 MHz,
DMSO-d6): δ 22.77 (C-6’), 52.89 (C-1’), 116.07 (C-4"),
126.74 (C-5’), 127.19 (C-3’), 128.68 (C-4’), 130.16 (C-2"),
131.11 (C-3"), 141.96 (C-2’), 144.26 (C=N, C-1"), 164.72
(C-5"), 176.90 (C=S, C-3). Anal. Calcd. for C16H16FN3S: C,
63.43; H, 5.65; N, 13.93; S, 2.34%. Found: C, 63.52; H, 5.49;
N, 13.86; S, 2.42%.
(+)-(S)-2-(4-hydroxybenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (8b). Yellow crystals; yield 87%; mp. 173–174°C;
[α]D =+70.6 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 344,
330, 305; IR (νmax, cmÀ1): 3360 and 3151 (N-H), 1605 (C=N),
1201 (C=S); EI-MS (m/z): 299.0 [M]+; 1H NMR (400 MHz,
DMSO-d6): δ 1.56 (d, 3H, J=7.2Hz, H-6’), 5.73 (p, 1H, H-1), 6.82
(d, 2H, J =8.8Hz, H-4"), 7.26 (t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.35
(t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J =8.4 Hz, H-3’), 7.65
(d, 2H, J =8.4Hz, H-3"), 8.01 (s, 1H, H-1"), 8.47 (d, 1H,
J =9.2Hz, H-4), 9.96 (s, 1H, OH), 11.40 (s, 1H, H-2); 13C NMR
(100MHz, DMSO-d6): δ 21.19 (C-6’), 52.02 (C-1’), 115.32
(C-4"), 124.76 (C-2"), 126.08 (C-5’), 126.48 (C-3’), 127.96 (C-4’),
128.97 (C-3"), 142.83 (C-2’), 143.68 (C=N, C-1"), 159.03 (C-5");
175.71 (C=S, C-3). Anal. Calcd. for C16H17N3OS: 64.19; H, 5.72;
N, 14.04; S, 10.71%. Found: C, 64.16; H, 5.75; N, 14.11; S, 10.76%.
(À)-(R)-2-(4-cyanobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (7a). Yellow crystals; yield 85%; mp. 167–168°C;
[α]D = À79.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 359,
345, 259; IR (νmax, cmÀ1): 3369 and 3139 (N-H), 2224
(C≡N), 1596 (C=N), 1214 (C=S); EI-MS (negative ion mode,
m/z): 307.0 [M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.59
(d, 3H, J = 7.2 Hz, H-6’), 5.76 (p, 1H, H-1), 7.25 (t, 1H, J1,
J2 = 7.2 Hz, H-5’), 7.35 (t, 2H, J1, J2 = 7.6Hz , H-4’), 7.43 (d, 2H,
(À)-(R)-2-(4-methoxybenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (9a). White crystals; yield 75%; mp. 143–144°C; [α]
D = À65.4 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 345, 330,
260; IR (νmax, cmÀ1): 3361 and 3149 (N-H), 1605 (C=N), 1201
Chirality DOI 10.1002/chir

180
TAS¸DEMIR ET AL.
(C=S); EI-MS (m/z): 313.1 [M]+; 1H NMR (400MHz,
DMSO-d6): δ 1.57 (d, 3H, J = 6.8 Hz, H-6’), 3.80 (s, 3H, OCH3,
H-6"), 5.74 (p, 1H, H-1), 6.99 (d, 2H, J = 8.8 Hz, H-4"), 7.25
(t, 1H, J1, J2 = 7.2Hz, H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’),
7.43 (d, 2H, J =8.0 Hz, H-3’), 7.77 (d, 2H, J = 8.8 Hz, H-3"), 8.05
(s, 1H, H-1"), 8.54 (d, 1H, J= 9.2 Hz, H-4), 11.46 (s, 1H, H-2);
13C NMR (100MHz, DMSO-d6): δ 21.83 (C-6’), 52.79 (C-1’),
55.74 (ÀOCH3, C-6"), 114.62 (C-4"), 126.77 (C-2"), 127.07
(C-5’), 127.17 (C-3’), 128.67 (C-4’), 129.53 (C-3"), 143.03
(C-2’), 144.37 (C=N, C-1"), 161.20 (C-5"), 176.58 (C=S, C-3).
Anal. Calcd. for C17H19N3OS: 65.15; H, 6.11; N, 13.41; S,
10.23%. Found: C, 65.22; H, 6.16; N, 13.45; S, 10.27%.
64.46; H, 4.92; N, 10.25; S, 7.82%. Found: C, 64.52; H, 4.87; N,
10.20; S, 7.88%.
(R)-(À)-2-[4-(4-fluorophenoxy)benzylidene]-N-(1-phenylethyl)hydra-
zine carbothioamide (11a). Yellow crystals; yield 74%; mp. 99–
100°C; [α]D = À71.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
344, 330, 259; IR (νmax, cmÀ1): 3356 and 3158 (N-H), 1601
(C=N), 1237 (C=S); EI-MS (m/z): 393.2 [M]+; 1H NMR
(400MHz, DMSO-d6): δ 1.57 (d, 3H, J = 7.2Hz, H-6’), 5.75
(p, 1H, H-1), 7.11 (dd, 4H, J1, J2 =8.8Hz, H-8", H-9"), 7.25
(t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’),
7.43 (d, 2H, J = 7.6 Hz, H-3’), 7.46 (d, 2H, J =8.8Hz, H-3"), 7.86
(d, 2H, J =8.8Hz, H-4"), 8.11 (s, 1H, H-1"), 8.55 (d, 1H,
J =8.8Hz, H-4), 11.51 (s, 1H, H-2); 13C NMR (100MHz,
DMSO-d6): δ 21.82 (C-6’), 52.83 (C-1’), 118.23 (C-9"), 119.02
(C-4"), 126,77 (C-2"), 127.18 (C-5’), 128.22 (C-3’), 129.86
(C-4’), 132.50 (C-3"), 142.29 (C-2’), 144.31 (C=N, C-1"), 153.26
(C-7"), 155.44 (C-10"), 158.23 (C-5"), 176.80 (C=S, C-3). Anal.
Calcd. for C22H20FN3OS: 67.15; H, 5.12; N, 10.68; S, 8.15%.
Found: C, 67.19; H, 5.12; N, 10.76; S, 8.10%.
(+)-(S)-2-(4-methoxybenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (9b). White crystals; yield 77%; mp. 145–146°C; [α]
D =+64.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 345, 330,
262; IR (νmax, cmÀ1): 3361 and 3149 (N-H), 1609 (C=N) 1200
(C=S); EI-MS (m/z): 313.0 [M]+; 1H NMR (400MHz,
DMSO-d6): δ 1.57 (d, 3H, J = 6.8 Hz, H-6’), 3.81 (s, 3H, OCH3,
H-6"), 5.74 (p, 1H, H-1), 6.99 (d, 2H, J = 8.8 Hz, H-4"), 7.25
(t, 1H, J1, J2 = 7.2Hz, H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’),
7.43 (d, 2H, J =8.0 Hz, H-3’), 7.77 (d, 2H, J = 8.8 Hz, H-3"), 8.06
(s, 1H, H-1"), 8.54 (d, 1H, J= 8.8 Hz, H-4), 11.46 (s, 1H, H-2);
13C NMR (100MHz, DMSO-d6): δ 21.84 (C-6’), 52.75 (C-1’),
55.80 (ÀOCH3, C-6"), 114.63 (C-4"), 126.77 (C-2"), 127.08
(C-5’), 127.17 (C-3’), 128.67 (C-4’), 129.52 (C-3"), 143.08
(C-2’), 144.36 (C=N, C-1"), 161.21 (C-5"), 176.61 (C=S, C-3).
Anal. Calcd. for C17H19N3OS: 65.15; H, 6.11; N, 13.41; S,
10.23%. Found: C, 65.12; H, 6.08; N, 13.51; S, 10.19%.
(+)-(S)-2-[4-(4-fluorophenoxy)benzylidene]-N-(1-phenylethyl)hydra-
zinecarbothioamide (11b). Yellow crystals; yield 87%; mp. 101–
102°C; [α]D =+72.6 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
344, 330, 259; IR (νmax, cmÀ1): 3350 and 3156 (N-H), 1604
(C=N), 1236 (C=S); EI-MS (m/z): 393.2 [M]+; 1H NMR
(400 MHz, DMSO-d6): δ 1.57 (d, 3H, J =7.2Hz, H-6’), 5.75
(p, 1H, H-1), 7.11 (dd, 4H, J1, J2 =8.8Hz, H-8", H-9"), 7.25
(t, 1H, J1, J2 = 7.2Hz, H-5’), 7.35 (t, 2H, J1, J2 = 7.6Hz, H-4’),
7.43 (d, 2H, J = 7.6Hz, H-3’), 7.46 (d, 2H, J =8.8Hz, H-3"), 7.87
(d, 2H, J = 8.8Hz, H-4"), 8.10 (s, 1H, H-1"), 8.58 (d, 1H,
J =8.8Hz, H-4), 11.53 (s, 1H, H-2); 13C NMR (100MHz,
DMSO-d6): δ 21.81 (C-6’), 52.84 (C-1’), 118.23 (C-9"), 119.02
(C-4"), 126,77 (C-2"), 127.18 (C-5’), 128.25 (C-3’), 129.87
(C-4’), 132.51 (C-3"), 142.29 (C-2’), 144.31 (C=N, C-1"), 153.26
(C-7"), 155.44 (C-10"), 158.23 (C-5"), 176.79 (C=S, C-3). Anal.
Calcd. for C22H20FN3OS: 67.15; H, 5.12; N, 10.68; S, 8.15%.
Found: C, 67.20; H, 5.16; N, 10.61; S, 8.22%.
(À)-(R)-2-[4-(4-chlorophenoxy)benzylidene]-N-(1-phenylethyl)hydra-
zinecarbothioamide (10a). Yellow crystals; yield 70%; mp. 136–
137°C; [α]D = À53.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
344, 330, 259; IR (νmax, cmÀ1): 3350 and 3155 (N-H), 1602
(C=N), 1207 (C=S); EI-MS (m/z): 409.1 [M]+; 1H NMR
(400MHz, DMSO-d6): δ 1.57 (d, 3H, J= 6.8Hz, H-6’), 5.75
(p, 1H, H-1), 7.01 (d, 2H, J = 8.8Hz, H-4"), 7.19 (m, 2H, H-9"),
7.24 (t, 3H, J1, J2 = 7.2 Hz, H-5’, H-8"), 7.34 (t, 2H, J1, J2 =7.6Hz,
H-4’), 7.43 (d, 2H, J = 8.0Hz, H-3’), 7.84 (d, 2H, J = 8.4Hz,
H-3"), 8.10 (s, 1H, H-1"), 8.52 (d, 1H, J = 8.8 Hz, H-4), 11.50
(s, 1H, H-2); 13C NMR (100MHz, DMSO-d6): δ 21.81 (C-6’),
52.84 (C-1’), 118.23 (C-4"), 122.13 (C-8"), 126,77 (C-2"), 127.18
(C-5’), 127.58 (C-10"), 128.68 (C-3’), 129.87 (C-4’), 130.81
(C-9"), 132.48 (C-3"), 142.29 (C-2’), 144.31 (C=N, C-1"), 157.82
(C-7"), 161.01 (C-5"), 176.78 (C=S, C-3). Anal. Calcd. for
C22H20ClN3OS: 64.46; H, 4.92; N, 10.25; S, 7.82%. Found: C,
64.49; H, 4.98; N, 10.21; S, 7.76%.
(À)-(R)-2-(4-methylthiobenzylidene)-N-(1-phenylethyl)hydrazine-
carbothioamide (12a). Yellow crystals; yield 77%; mp. 106–
107°C; [α]D = À84.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
360, 345, 259; IR (νmax, cmÀ1): 3350 and 3153 (N-H), 1593
(C=N), (C=S) 1238; EI-MS (m/z): 329.6 [M]+; 1H NMR
(400MHz, DMSO-d6): δ 1.57 (d, 3H, J = 6.8Hz, H-6’), 2.53
(s, 3H, SCH3, H-6"), 5.76 (p, 1H, H-1), 7.25 (t, 1H, J1 =8.0Hz,
J2 =7.2Hz, H-5’), 7.28 (d, 2H, J =8.0Hz, H-4"), 7.35 (t, 2H,
J1 =7.2Hz, J2 =7.6Hz, H-4’), 7.43 (d, 2H, J = 8.0 Hz, H-3’), 7.81
(d, 2H, J =8.4Hz, H-3"), 8.06 (s, 1H, H-1"), 8.62 (d, 1H,
J =8.8Hz, H-4), 11.53 (s, 1H, H-4); 13C NMR (100MHz,
DMSO-d6): δ 14.76 (SCH3, C-6"), 21.79 (C-6’), 52.85 (C-1’),
125.95 (C-5’), 126.78 (C-3’), 127.18 (C-4"), 128.48 (C-4’),
129.16 (C-3"), 130.99 (C-2"), 141.21 (C-2’), 142.68 (C-5"),
144.32 (C=N, C-1"), 176.73 (C=S, C-3). Anal. Calcd. for
C17H19N3S2: C, 61.97; H, 5.81; N, 12.75; S, 19.46%. Found: C,
61.85; H, 5.83; N, 12.61; S, 19.36%.
(+)-(S)-2-[4-(4-chlorophenoxy)benzylidene]-N-(1-phenylethyl)hydra-
zinecarbothioamide (10b). Yellow crystals; yield 85%; mp. 138–
139°C; [α]D =+52.7 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
344, 330, 259; IR (νmax, cmÀ1): 3350 and 3157 (N-H), 1605
(C=N), 1207 (C=S); EI-MS (m/z): 409.1 [M]+; 1H NMR
(400MHz, DMSO-d6): δ 1.57 (d, 3H, J =6.8 Hz, H-6’), 5.75
(p, 1H, H-1), 7.01 (d, 2H, J = 8.8Hz, H-4"), 7.15 (m, 2H, H-9"),
7.24 (t, 3H, J1, J2 = 7.2 Hz, H-5’, H-8"), 7.35 (t, 2H, J1, J2 =7.6Hz,
H-4’), 7.43 (d, 2H, J= 8.4 Hz, H-3’), 7.84 (d, 2H, J = 8.4Hz, H-
3"), 8.09 (s, 1H, H-1"), 8.53 (d, 1H, J = 8.8Hz, H-4), 11.50
(s, 1H, H-2); 13C NMR (100MHz, DMSO-d6): δ 21.81 (C-6’),
52.84 (C-1’), 118.23 (C-4"), 122.13 (C-8"), 126,77 (C-2"), 127.18
(C-5’), 128.67 (C-3’), 127.58 (C-10"), 129.86 (C-4’), 130.81 (C-9"),
132.48 (C-3"), 142.29 (C-2’), 144.31 (C=N, C-1"), 157.82 (C-7"),
161.03 (C-5"), 176.80 (C=S, C-3). Anal. Calcd. for C22H20ClN3OS:
(+)-(S)-2-(4-methylthiobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (12b). Yellow crystals; yield 81%; mp. 109–110°C; [α]
D = +83.2 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 360, 345, 259;
IR (νmax, cmÀ1): 3350 and 3151 (N-H), 1593 (C=N), 1238 (C=S);
EI-MS (m/z): 330. 1 [M+H]+; 1H NMR (400 MHz, DMSO-d6): δ
1.57 (d, 3H, J=6.8Hz, H-6’), 2.52 (s, 3H, H-6"), 5.76 (p, 1H, H-1),
7.24 (t,1H, J1 =8.0Hz, J2 =7.2Hz, H-5’), 7.29 (d, 2H, J=8.0Hz,
Chirality DOI 10.1002/chir

POTENTIAL OF CHIRAL CANCER DRUGS
181
H-4"), 7.35 (t, 2H, J1 =7.2Hz, J2 =7.6Hz, H-4’), 7.43 (d, 2H,
J=8.0Hz, H-3’), 7.80 (d, 2H, J= 8.0 Hz, H-3"), 8.05 (s, 1H,
H-1"), 8.60 (d, 1H, J= 8.8 Hz, H-4), 11.54 (s, 1H, H-2); 13C
NMR (100 MHz, DMSO-d6): δ 14.76 (SCH3, C-6"), 21.79
(C-6’), 52.85 (C-1’), 125.97 (C-5’), 126.78 (C-3’), 127.18 (C-4"),
128.41 (C-4’), 129.16 (C-3"), 130.99 (C-2"), 141.20 (C-2’), 142.68
(C-5"), 144.31 (C=N, C-1"), 176.74 (C=S, C-3). Anal. Calcd. for
C17H19N3S2: C, 61.97; H, 5.81; N, 12.75; S, 19.46%. Found: C,
61.92; H, 5.76; N, 12.80; S, 19.51%.
265, 255; IR (νmax, cmÀ1): 3339 and 3167 (N-H), 1600 (C=N),
1205 (C=S); EI-MS (negative ion mode, m/z): 327.1 [M-H]+;
1H NMR (400 MHz, DMSO-d6): δ 1.59 (d, 3H, J = 6.8 Hz, H-
6’), 5.77 (p, 1H, H-1), 7.25 (t, 1H, J1, J2 = 7.2 Hz, H-5’), 7.35 (t,
2H, J1, J2 = 7.6 Hz, H-4’), 7.42 (d, 2H, J = 7.6 Hz, H-3’), 8.12 (d,
2H, J = 8.0 Hz, H-4"), 8.18 (s, 1H, H-1"), 8.26 (d, 2H,
J = 8.4 Hz, H-3"), 8.88 (d, 1H, J = 8.8 Hz, H-4), 11.84 (s, 1H, H-
2); 13C NMR (100 MHz, DMSO-d6): δ 21.64 (C-6’), 53.04 (C-
1’), 124.25 (C-4"), 126.76 (C-3"), 127.21 (C-5’), 128.68 (C-3’),
129.18 (C-4’), 140.44 (C-2"), 141.04 (C-2’), 144.09 (C=N, C-
1"), 148.06 (C-5"), 177.21 (C=S, C-3). Anal. Calcd. for
C16H16N4O2S2: C, 58.52; H, 4.91; N, 17.06; S, 9.76%. Found:
C, 58.56; H, 4.87; N, 17.04; S, 9.69%.
(À)-(R)-2-(4-trifluoromethylthiobenzylidene)-N-(1-phenylethyl)hydra-
zinecarbothioamide (13a). White crystals; yield 76%; mp. 146–
147°C; [α]D =À72.4 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
335, 260; IR (νmax, cmÀ1): 3360 and 3141 (N-H), 1600 (C=N),
1235 (C=S); EI-MS (negative ion mode, m/z): 382.0 [M-H]+;
1H NMR (400MHz, DMSO-d6): δ 1.58 (d, 3H, J=6.8Hz, H-6’),
5.75 (p, 1H, H-1), 7.25 (t, 1H, J1, J2 =7.6Hz, H-5’), 7.35 (t, 2H,
J1, J2 =7.6Hz, H-4’), 7.43 (d, 2H, J=7.2 Hz, H-3’), 7.75 (d, 2H,
J=8.0Hz, H-4"), 7.99 (d, 2H, J=8.4Hz, H-3"), 8.14 (s, 1H, H-
1"), 8.71 (d, 1H, J=8.8Hz, H-4), 11.69 (s, 1H, H-2); 13C NMR
(100MHz, DMSO-d6): δ 21.71 (C-6’), 52.95 (C-1’), 126.76 (C-
5’), 127.19 (C-4"), 128.46 (C-3’), 129.04 (C-4’), 131.52 (C-3"),
134.62 (C-2"), 136.73 (SCF3, C-6"), 137.56 (C-5"), 141.23 (C-
2’), 144.18 (C=N, C-1"), 177.08 (C=S, C-3). Anal. Calcd. for
C17H16F3N3S2: C, 53.25; H, 4.21; N, 10.96; S, 16.72%. Found:
C, 53.16; H, 4.31; N, 11.09; S, 16.68%.
(À)-(R)-2-[(4-pyrrolidin-1-yl)benzylidene])-N-(1-phenylethyl)hydra-
zinecarbothioamide (15a). Brown crystals; yield 77%; mp. 142–
143°C; [α]D = À105.4 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
365, 339, 260; IR (νmax, cmÀ1): 3361 and 3124 (N-H), 1597
(C=N) 1200 (C=S); EI-MS (m/z): 352.8 [M]+; 1H NMR
(400 MHz, DMSO-d6): δ 1.57 (d, 3H, J = 6.8 Hz, H-6’), 1.96
(s, 4H, CH2, H-7"), 3.28 (s, 4H, NCH2, H-6"), 5.72 (p, 1H,
H-1), 6.56 (d, 2H, J = 8.8 Hz, H-4"), 7.25 (t, 1H, J1, J2 = 7.2 Hz,
H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43 (d, 2H, J = 7.6 Hz,
H-3’), 7.58 (d, 2H, J = 8.4 Hz, H-3"), 7.99 (s, 1H, H-1"), 8.34
(d, 1H, J = 8.8 Hz, H-4), 11.27 (s, 1H, H-2) ; 13C NMR
(100 MHz, DMSO-d6): δ 22.60 (C-6’), 26.05 (NCH2, C-6"),
48.34 (CH2, C-7"), 53.31 (C-1’), 112.55 (C-4"), 121.68 (C-2"),
126.81 (C-5’), 127.81 (C-3’), 129.33 (C-4’), 130.00 (C-3"),
144.50 (C=N, C-1"), 145.12 (C-2’), 149.93 (C-5"), 176.64 (C=S,
C-3). Anal. Calcd. for C20H24N4S: C, 68.15; H, 6.86; N, 15.89;
S, 9.10%. Found: C, 68.16; H, 6.95; N, 15.77; S, 9.19%.
(+)-(S)-2-(4-trifluoromethylthiobenzylidene)-N-(1-phenylethyl)hydra-
zinecarbothioamide (13b). Yellow crystals; yield 80%; mp. 150–
151°C; [α]D =+71.9 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
340, 260, IR (νmax, cmÀ1): 3360 and 3141 (N-H), 1600 (C=N),
1235 (C=S); EI-MS (negative ion mode, m/z): 382.1 [M-H]+;
1H NMR (400MHz, DMSO-d6): δ 1.58 (d, 3H, J = 7.2 Hz, H-
6’), 5.75 (p, 1H, H-1), 7.25 (t, 1H, J1, J2 = 7.6 Hz, H-5’), 7.35
(t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43 (d, 2H, J = 7.6Hz, H-3’), 7.75
(d, 2H, J= 8.4 Hz, H-4"), 7.99 (d, 2H, J = 8.4 Hz, H-3"), 8.14
(s, 1H, H-1"), 8.74 (d, 1H, J= 8.8 Hz, H-4), 11.70 (s, 1H, H-2);
13C NMR (100MHz, DMSO-d6): δ 21.75 (C-6’), 52.95 (C-1’),
126.75 (C-5’), 127.19 (C-4"), 128.45 (C-3’), 129.03 (C-4’),
131.52 (C-3"), 134.61 (C-2"), 136.72 (SCF3, C-6"), 137.56
(C-5"), 141.21 (C-2’), 144.18 (C=N, C-1"), 177.09 (C=S, C-3).
Anal. Calcd. for C17H16F3N3S2: C, 53.25; H, 4.21; N, 10.96; S,
16.72%. Found: C, 53.32; H, 4.18; N, 10.89; S, 16.74%.
(+)-(S)-2-[(4-pyrrolidin-1-yl)benzylidene])-N-(1-phenylethyl)hydra-
zinecarbothioamide (15b). Brown crystals; yield 78%; mp.
140–141°C; [α]D = +104.4 (c: 0.5, DMSO); UV (DMSO, λmax
,
nm): 365, 339, 259; IR (νmax, cmÀ1): 3357 and 3131 (N-H)
1
1599 (C=N), 1200 (C=S); EI-MS (m/z): 352.8 [M+]; H NMR
(400 MHz, DMSO-d6): δ 1.56 (d, 3H, J=6.8Hz, H-6’), 1.96 (s, 4H,
CH2, H-7"), 3.26 (s, 4H, NCH2, H-6"), 5.71 (p, 1H, H-1), 6.55
(d, 2H, J=8.8Hz, H-4"), 7.25 (t, 1H, J1, J2 =7.2Hz, H-5’), 7.35
(t, 2H, J1, J2 =7.6Hz, H-4’), 7.42 (d, 2H, J=7.6Hz, H-3’), 7.58
(d, 2H, J=8.4Hz, H-3"), 7.98 (s, 1H, H-1"), 8.33 (d, 1H, J=8.8Hz,
H-4), 11.28 (s, 1H, H-2); 13C NMR (100 MHz, DMSO-d6): δ 22.61
(C-6’), 26.06 (NCH2, C-6"), 48.33 (CH2, C-7"), 53.30 (C-1’), 112.53
(C-4"), 121.63 (C-2"), 126.80 (C-5’), 127.82 (C-3’), 129.37 (C-4’),
130.01 (C-3"), 144.49 (C=N, C-1"), 145.12 (C-2’), 149.92 (C-5"),
176.62 (C=S, C-3). Anal. Calcd. for C20H24N4S: C, 68.15; H, 6.86;
N, 15.89; S, 9.10%. Found: C, 68.21; H, 6.87; N, 15.82; S, 9.12%.
(À)-(R)-2-(4-nitrobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (14a). Yellow crystals; yield 84%; mp. 170–171°C;
[α]D = À42.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 380,
265, 254; IR (νmax, cmÀ1): 3355 and 3107 (N-H), 1599
(C=N), 1205 (C=S); EI-MS (negative ion mode, m/z): 327.0
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.59 (d, 3H,
J = 7.2 Hz, H-6’), 5.77 (p, 1H, H-1), 7.25 (t, 1H, J1, J2 = 7.2 Hz,
H-5’), 7.36 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43 (d, 2H, J = 8.0 Hz,
H-3’), 8.12 (d, 2H, J = 8.8 Hz, H-4"), 8.19 (s, 1H, H-1"), 8.25
(À)-(R)-2-[(4-morpholin-4-yl)benzylidene])-N-(1-phenylethyl)hydra-
zinecarbothioamide (16a). Yellow crystals; yield 69%; mp. 98–
99°C; [α]D = À94.0 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
360, 349, 259; IR (νmax, cmÀ1): 3348 and 3143 (N-H), 1599
(C=N), 1225 (C=S); EI-MS (negative ion mode, m/z): 367.2
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.57 (d, 3H,
J =7.2Hz, H-6’), 3.21 (t, 4H, J1, J2 =4.8Hz, NCH2, H-7"), 3.74
(t, 4H, J1, = 4.4Hz, J2 = 5.2Hz, OCH2, H-6"), 5.74 (p, 1H, H-1),
6.96 (d, 2H, J =8.8Hz, H-4"), 7.25 (t, 1H, J1, J2 =7.2HzH-5’),
7.35 (t, 2H, J1, J2 = 7.6Hz, H-4’), 7.43 (d, 2H, J =7.6Hz, H-3’),
7.67 (d, 2H, J =8.8Hz, H-3"), 8.01 (s, 1H, H-1"), 8.46 (d, 1H,
J =9.2Hz, H-4), 11.38 (s, 1H, H-2); 13C NMR (100MHz,
DMSO-d6): δ 21.88 (C-6’), 47.95 (NCH2, C-6"), 52.75 (C-1’),
66.41 (OCH2, C-7"), 114.72 (C-4"), 124.73 (C-2"), 126.78
(C-5’), 127.17 (C-3’), 128.68 (C-4’), 129.09 (C-3"), 143.50
(d, 2H, J = 8.4 Hz, H-3"), 8.89 (d, 1H, J = 8.8 Hz, H-4), 11.86
13
(s, 1H, H-2);
C NMR (100 MHz, DMSO-d6): δ 21.64
(C-6’), 53.04 (C-1’), 124.25 (C-4"), 126.76 (C-3"), 127.21
(C-5’), 128.68 (C-3’), 129.16 (C-4’), 140.44 (C-2"), 141.03
(C-2’), 144.07 (C=N, C-1"), 148.05 (C-5"), 177.22 (C=S, C-3).
Anal. Calcd. for C16H16N4O2S2: C, 58.52; H, 4.91; N, 17.06;
S, 9.76%. Found: C, 58.49; H, 4.86; N, 17.11; S, 9.66%.
(+)-(S)-2-(4-nitrobenzylidene)-N-(1-phenylethyl)hydrazinecarbo-
thioamide (14b). Yellow crystals; yield 74%; mp. 167–168°C;
[α]D = +43.7 (c: 0.5, DMSO); UV (DMSO, λmax, nm): 370,
Chirality DOI 10.1002/chir

182
TAS¸DEMIR ET AL.
(C-2’), 144.42 (C=N, C-1"), 152.60 (C-5"), 176.36 (C=S, C-3).
Anal. Calcd. for C20H24N4OS: C, 65.19; H, 6.56; N, 15.20; S,
8.70%. Found: C, 65.09; H, 6.50; N, 15.33; S, 8.76%.
Cell Culture
Human breast adenocarcinoma (MCF-7), human alveolar adenocarci-
noma (A549), human cervix adenocarcinoma (HELA), and human stomach
adenocarcinoma (HGC-27) cells were maintained in Dulbecco’s Modified
Eagle Medium (DMEM; Sigma, St. Louis, MO) containing 10% fetal calf
serum (Thermo, Waltham, MA), 100 IU of penicillin/mL, and 100 μg/mL
of streptomycin (both from Life Technologies, Rochester, NY).
(+)-(S)-2-[(4-morpholin-4-yl)benzylidene])-N-(1-phenylethyl)hydra-
zine carbothioamide (16b). Yellow crystals; yield 80%; mp. 96–
97°C; [α]D = +95.6 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
360, 349, 259; IR (νmax, cmÀ1): 3348 and 3136 (N-H), 1600
(C=N), 1226 (C=S); EI-MS (negative ion mode, m/z): 367.0
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.56 (d, 3H,
J = 6.8 Hz, H-6’), 3.21 (t, 4H, J1, J2 = 4.8 Hz, NCH2, H-7"), 3.74
(t, 4H, J1, = 4.4 Hz, J2 = 5.2 Hz, OCH2, H-6"), 5.74 (p, 1H, H-1),
6.97 (d, 2H, J = 8.8 Hz, H-4"), 7.24 (t, 1H, J1, J2 = 7.2 Hz, H-5’),
7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43 (d, 2H, J = 7.6 Hz, H-3’),
7.67 (d, 2H, J = 8.8 Hz, H-3"), 8.01 (s, 1H, H-1"), 8.46 (d, 1H,
J = 8.8 Hz, H-4), 11.38 (s, 1H, H-2); 13C NMR (100 MHz,
DMSO-d6): δ 21.88 (C-6’), 47.94 (NCH2, C-6"), 52.75 (C-1’),
66.40 (OCH2, C-7"), 114.72 (C-4"), 124.73 (C-2"), 126.78
(C-5’), 127.16 (C-3’), 128.67 (C-4’), 129.08 (C-3"), 143.50
(C-2’), 144.42 (C=N, C-1"), 152.59 (C-5"), 176.34 (C=S, C-3).
Anal. Calcd. for C20H24N4OS: C, 65.19; H, 6.56; N, 15.20; S,
8.70%. Found: C, 65.23; H, 6.51; N, 15.28; S, 8.79%.
Cell Viability Assay
Cells were seeded at 5 × 104 cells/mL of a 24-well cell culture plate and
incubated for 48 h. After 48 h incubation, the cells were treated with a dif-
ferent concentration (2.5, 10, 50, and 100 μM) of compounds for 24 h. Cell
viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazoliumbromide) assay. For the MTT assay, MTT solution (mg/mL)
was added onto the cells (500 μl/well) and incubated for 1 h at 37°C. Absor-
bance was measured at 570 nm using an Epoch microplate spectrophotom-
eter (BioTek Instruments, Winooska, VT).
Statistical Analyses
All of data were analyzed with an analysis of variance (ANOVA) test
(prism 5 software). P < 0.05 was considered statistically significant.
Molecular Modeling Study
In an effort to explain the possible mechanism by which the new chiral
thiosemicarbazone derivative (17b) displayed antiproliferative activity to-
ward HGC-27 and guide further SAR studies, the preferential pharma-
cophore modeling was carried out via Discovery Studio (DS; Accelyrs
Software, San Diego, CA) 3.5. Compound 17b was designed and minimized
using the conjugate gradient algorithm with the help of DS 3.5.19 After-
wards, to generate conformations a subprotocol of DS 3.5 was applied to
determine the stable conformations of the mentioned compound. We deter-
mined the stable two forms of the compound as listed in Table 1.
(À)-(R)-2-[(4-piperidin-1-yl)benzylidene])-N-(1-phenylethyl)hydra-
zinecarbothioamide (17a). Yellow crystals; yield 69%; mp. 80–
81°C; [α]D = À67.3 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
365, 355, 259; IR (νmax, cmÀ1): 3371 and 3196 (N-H), 1599
(C=N), 1221 (C=S); EI-MS (negative ion mode, m/z): 365.0
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.56 (d, 3H,
J = 7.2 Hz, H-6’), 1.57 (brs, 6H, CH2CH2, H-7", H-8") 3.25
(t, 4H, J1, J2 = 5.2 Hz, NCH2, H-6"), 5.73 (p, 1H, H-1), 6.93
(d, 2H, J = 8.8 Hz, H-4"), 7.25 (t, 1H, J1, = 6.8 Hz, J2 = 7.6 Hz,
H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43 (d, 2H, J = 7.6 Hz,
H-3’), 7.62 (d, 2H, J = 8.8 Hz, H-3"), 7.99 (s, 1H, H-1"), 8.42
(d, 1H, J = 9.2 Hz, H-4), 11.35 (s, 1H, H-2); 13C NMR
(100 MHz, DMSO-d6): δ 21.96 (C-6’), 24.39 (CH2, C-8"),
25.02 (CH2, C-7"), 48.17 (NCH2, C-6") 52.72 (C-1’), 113.60
(C-4"), 123.53 (C-2"), 126.76 (C-5’), 127.15 (C-3’), 128.70
(C-4’), 129.18 (C-3"), 143.68 (C-2’), 144.48 (C=N, C-1"),
152.72 (C-5"), 176.20 (C=S, C-3). Anal. Calcd. for C21H26N4S:
C, 68.82; H, 7.15; N, 15.29; S, 8.75%. Found: C, 68.79; H,
7.12; N, 15.33; S, 8.88%.
Pharmacophore Generation
The results of the SAR study reported that compound 17b was more
active than the others and also the reference compound against HGC-27
cells. Hence, we attempted to determine its pharmacophore model. In
this study, HipHop,20,21 which is one method of a pharmacophore model,
was used for the mentioned reasons. HipHop provides a feature-based
alignment of an active compound. Furthermore, the chemical features
of the molecule were superimposed against the reference compound
using the HipHop method. Conformational models of the compound
17b were investigated in this application.
RESULTS AND DISCUSSION
Chemistry
The synthesis of the target chiral thiosemicarbazones was
accomplished using the reaction sequence in Scheme 1. The
chiral isothiocyanates (1a-b) as key intermediates were
synthesized by treatment of (R)-(+)-α-methylbenzylamine and
(S)-(À)-α-methylbenzylamine with CSCl2 in the presence of
NaOH in a water–chloroform medium at 0°C for 24h, as
described in previously reported methods.22 Thus, the chiral
center from chiral amines was retained. Subsequently, chiral
thiosemicarbazides (2a-b) were prepared by reacting
hydrazine monohydrate with chiral isothiocyanates (1a-b) in
diethylether at room temperature.23 A series of chiral
thiosemicarbazones (3a-17a and 3b-17b) as an enantiomeric
pair were synthesized by refluxing an equimolar ratio of the
p-substituted benzaldehydes and chiral thiosemicarbazides in
methanol medium without using a catalyst. The reactions were
followed until complete consumption of thiosemicarbazides.
The novel chiral thiosemicarbazones (3a-17a and 3b-17b)
were obtained in good yields (69–87%) and high enantiopurity
after recrystallization from ethanol.
(+)-(S)-2-[(4-piperidin-1-yl)benzylidene])-N-(1-phenylethyl)hydra-
zinecarbothioamide (17b). Yellow crystals; yield 72%; mp. 79–
80°C; [α]D = +66.8 (c: 0.5, DMSO); UV (DMSO, λmax, nm):
364, 355, 260; IR (νmax, cmÀ1): 3372 and 3195 (N-H), 1599
(C=N), 1222 (C=S); EI-MS (negative ion mode, m/z): 364.8
[M-H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.56 (d, 3H,
J = 7.2 Hz, H-6’), 1.57 (brs, 6H, CH2CH2, H-7", H-8") 3.25
(t, 4H, J1, J2 = 5.2 Hz, NCH2, H-6"), 5.73 (p, 1H, H-1), 6.93
(d, 2H, J = 8.8 Hz, H-4"), 7.25 (t, 1H, J1, = 6.8 Hz, J2 = 7.6,
H-5’), 7.35 (t, 2H, J1, J2 = 7.6 Hz, H-4’), 7.43 (d, 2H, J = 7.6 Hz,
H-3’), 7.62 (d, 2H, J = 8.8 Hz, H-3"), 7.99 (s, 1H, H-1"), 8.42
(d, 1H, J = 9.2 Hz, H-4), 11.36 (s, 1H, H-2); 13C NMR
(100 MHz, DMSO-d6): δ 21.90 (C-6’), 24.36 (CH2, C-8"),
25.04 (CH2, C-7"), 48.18 (NCH2, C-6") 52.71 (C-1’), 113.65
(C-4"), 123.57 (C-2"), 126.78 (C-5’), 127.16 (C-3’), 128.67
(C-4’), 129.16 (C-3"), 143.68 (C-2’), 144.44 (C=N, C-1"),
152.79 (C-5"), 176.21 (C=S, C-3). Anal. Calcd. for
C21H26N4S: C, 68.82; H, 7.15; N, 15.29; S, 8.75%. Found: C,
68.88; H, 7.07; N, 15.36; S, 8.67%.
Chirality DOI 10.1002/chir

POTENTIAL OF CHIRAL CANCER DRUGS
183
TABLE 1. IC50 values (in μM) for chiral thiosemicarbazones
Cancer cell lines
Compound
R
MCF-7 (Breast)
A549 (Lung)
HeLa (Cervix)
HGC-27 (Stomach)
3a
-H
-H
-Br
-Br
-CI
-CI
-F
-F
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
7.65
20.1
16.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
42.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7.56
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
45.7
42.8
>100
>100
46.4
38.0
>100
48.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.6
12.5
13.4
>100
>100
>100
>100
>100
43.5
>100
>100
40.3
46.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.2
>100
4.6
3b
4a
4b
5a
5b
6a
6b
7a
-CN
-CN
-OH
-OH
-OCH3
-OCH3
4-chlorophenoxy
4-chlorophenoxy
4-fluorophenoxy
4-fluorophenoxy
-SCH3
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
Taxol (Sindaxel)
-SCH3
-SCF3
-SCF3
-NO2
-NO2
-prryolidine
-prryolidine
-morpholine
-morpholine
-piperidine
-piperidine
10.3
of chiral thiosemicarbazones (3a-17a and 3b-17b),
stretching vibrations attributed to the ν(N-H) at 3309–3372 cmÀ1
and 3124–3196 cm-1 were observed, while the characteristic
intense bands at 1591–1609 cmÀ1 and 1200–1238 cmÀ1 were
attributed to ν(C=N) and ν(C=S), respectively. It was suggested
that in the solid phase the thiosemicarbazones will be retained in
the thione form. The 1H NMR spectra of chiral thiosemicarbazones
(3a-17a and 3b-17b) showed a singlet peak attributable to the –
NH (H-2) proton in the range δ 11.27–11.87 ppm as well as a singlet
peak attributable to the –NH (H-4) proton in the range δ
8.33–8.89ppm. The signal of the azomethine proton (H-1")
appeared as a singlet at δ 7.98–8.19ppm. All aromatic protons
were observed with the expected chemical shifts and coupling
constants. The proton at the chiral center was observed at δ
5.72–5.77ppm as a pentet peak. The 13C NMR spectra of the
thiosemicarbazones exhibited two important signals at δ
175.71–177.22 and at δ 143.68–144.50ppm attributed to the
thioamide (C=S) and imine (C=N) carbon atoms, respectively.
The signals at δ 112.01–164.73ppm in the spectra were
Scheme 1. Synthesis of thiosemicarbazones in (R)-enantiomeric form. Re-
agents and conditions: (a) CSCl2, NaOH/water, CHCl3, rt, 24 h; (b) NH2NH2.
H2O (98%), Et2O, rt, 24 h; (c) 4-substitutedbenzyaldehydes, CH3OH, reflux,
10–12 h.
All thiosemicarbazones (3a-17a and 3b-17b) were
1
attributed to the aromatic carbons. The signal at
53.31–52.02ppm was attributed to the chiral carbon atom. MS
δ
characterized by the combined use of UV-vis, IR, H NMR,
13CNMR, 2D NMR, and MS spectral data. In the IR spectra
Chirality DOI 10.1002/chir

184
TAS¸DEMIR ET AL.
spectra of all chiral thiosemicarbazones were in line with the
proposed structures.
hand, the IC50 value of compound 8a (24.6 μM) was mea-
sured on MCF-7 cells as well (Fig. 2), whereas compound
8a has no potential effects on A549 cells. The IC50 values of
3a were evaluated on A549 and HGC-27 as 20.1 μM and
12.5 μM, respectively (Fig. 1). The activity of 3b against
HGC-27 and A549 cells were assessed as mean IC50 values
of 16.9 μM and 13.4 μM, respectively (Fig. 1). The greatest
reduction of the maximum dose shown by 17b was against
the HGC-27 cell line. Compound 17b showed a more promi-
nent inhibitory effect (IC50 4.6 μM) than the reference drug,
Sindaxel (IC50 10.3 μM) (Fig. 2). But interestingly, compound
17a had no significant cytotoxic effect on any of the cell lines.
Compound 17b will be selected as a promising candidate for
further development. IC50 (μM) values are listed in Table 1.
Antiproliferative Activity Assays
In order to assess any possible toxic effects of the com-
pounds they were evaluated according to the MTT assay.
The serial dilutions 2.5, 10, 50, and 100 μM of the compounds
were incubated with HeLa, A549, MCF-7, and HGC-27 cell
lines for 24 h. Sindaxel is an anticancer drug used as a control
group. The compounds of 8a (IC50 45.7 μM), 8b (IC50
42.8 μM), 10a (IC50 46.4 μM), 10b (IC50 38.0 μM), and 11b
(IC50 48.8 μM) showed weaker activity than the reference
drug (IC50 9.6 μM) on HELA cells. Similarly, the compounds
6b (IC50 43.5 μM), 8a (IC50 40.3 μM), and 8b (IC50 46.9 μM)
showed less cytotoxic effect by comparison with sindaxel
(IC50 10.3 μM) on the HGC-27 cell line. Sindaxel (IC50
7.56 μM and IC50 7.65 μM) showed a better cytotoxic effect
than compounds 8b (IC50 42.2 μM) and 13a (IC50 48.6 μM)
on A549 and MCF-7 cell lines, respectively. On the other
Structure–Activity Relationship Study
The SAR studies were applied to define how the substituent
on the benzene ring and chiral structures of thiosemi-
carbazone derivatives (3a-17a versus 3b-17b) involved the
Fig. 1. Cell viability of compounds: Effect of compound 3a and 3b against A549 and HGC-27 cell lines.
Chirality DOI 10.1002/chir

POTENTIAL OF CHIRAL CANCER DRUGS
185
anticancer activity. Compounds 3a and 3b have the same
backbone, but different stereochemistry. These compounds
without any substituent group on the benzene ring show anti-
proliferative activities against the A549 and HGC-27 cells. In
this part of the study, the effect of the different substituent
group(s) on the benzene ring of compounds 3a and 3b were
evaluated. First, we used electron-withdrawing groups such
as –Br, ÀCl, ÀF, ÀCN, ÀNO2 instead of a hydrogen atom
on the 3-position of the benzene ring. Compound 6b exhib-
ited activity against HGC-27 cells; however, the activity value
(IC50 43.5 μM) was less than compounds 3a-3b (IC50 12.5
and 13.4 μM) and also the reference compound, sindaxel
(IC50 10.3 μM). In addition, the electron-donor groups
(ÀOH, OCH3, ÀOPhCI, ÀOPhF, ÀSCH3, ÀSCF3) were used
under the same conditions. This time, compounds 10a, 10b,
and 11b exhibited poor activities, with IC50 values of 38.0–
48.8 μM against HeLa cells, but not others. Furthermore,
the pyrrolidine, morpholine, and piperidine groups were uti-
lized to observe the activity change according to compounds
3a-3b and the reference compound. Compound 17b was
the most potent of all the compounds and the reference com-
pound, which indicated that the exchange of the piperidine
group including the S form of the chiral thiosemicarbazone
improved the antiproliferative activity against HGC-27 cells.
Compared with 17b, compound 17a, with a bulky substitu-
ent which connected with the chiral carbon, had more potent
activity. The substituent in the benzene ring and R- and S- no-
menclature of the chiral carbon widely influenced the activity.
Moreover, all of the thiosemicarbazone derivatives showed
low activity toward MCF-7, A549, and HeLa. Thus, compound
17b exhibited HGC-27 selectivity.
17b, shown in Figure 3, was used to derive common feature-
based alignments. It is considered a reference compound
(Fig. 3) and 17b with a principal value of 2 and maximum
omitting features (MaxOmitFeat) value of 0. Then 10
pharmacophore hypotheses were generated from compound
17b using the Auto Pharmacophore Generation protocol in
Discovery Studio 3.5. Within the generated 10 hypotheses,
the hypothesis is Figure 4A was determined for future steps.
These features were one Hb-Acceptor, one Hb-Donor, two hy-
drophobic, and two ring aromatic in 10 pharmacophore
models. Figure 4A–D and Figure 5E were projected as the
common feature functions to elucidate the pharmacophore
site of compound 17b. In addition, Figure 4C lists the map-
ping of 17b on the anticipated pharmacophore model meant
for the most active molecule. The reference compound,
sindaxel, was also investigated to compare it with the men-
tioned compound 17b by using pharmacophore modeling
subprotocol of DS 3.5. We first defined the pharmacophore
model of sindaxel and then the possible conformations occur-
ring via the generate conformation subprotocol of DS 3.5. Fi-
nally, the best model was chosen to evaluate the differences
with compound 17b, which has six features containing four
Hb-Donors, one hydrophobic, and one ring Aromatic,
possessing the highest ranking score in Figure 5F,G.
The generated pharmacophore models showed that the
two hydrophobic features were found to be significant for an-
tiproliferative activity. Two hydrophobic features indicated
the appropriate active shape of the molecule. This molecule
showed the required placement of bulky ring moieties. The
two hydrophobic atoms or groups at the defined positions
Pharmacophore Generation
In the present study, the HipHop was used to form a
pharmacophore hypotheses to elucidate the specification of
the SARs of the pharmacophore sites of the novel chiral
thiosemicarbazone, 17b. The obtained hypotheses could be
correlated with the activity of the mentioned molecule. Com-
pound 17b exhibited 2-fold more antiproliferative activity
than the reference compound against HGC-27 cells (Table 1).
According to the results of the antiproliferative activities of
the thiosemicarbazone derivatives, the most active molecule,
Fig. 3. The 3D chemical structures of the compound 17b and reference
compound, sindaxel, respectively.
Fig. 2. Cell viability of compounds: Effect of compound 8a against MCF-7 cell line and effect of compound 17b against HGC-27 cell line.
Chirality DOI 10.1002/chir

186
TAS¸DEMIR ET AL.
Fig. 4. A. Anticipated pharmacophore model generated for HGC-27cell inhibitor activity of tested thiosemicarbazone derivatives. B. Distance and torsional angles
between the generated common features calculated in the participated pharmacophore model. Alignment of the 3D generated conformer structures of compound 17b
as the reference AdeABC efflux pump inhibitors. C. Pharmacophore mapping of the compound 17b. D. Pharmacophore mapping of the compound 17a.
Fig. 5. E. Pharmacophore mapping of reference compound, sindaxel less active than compound 17b. F. The generated slab of the anticipated pharmacophore
model with the compound 17b. G. Anticipated pharmacophore model generated for HGC-27 cell inhibitor activity of the reference compound, sindaxel. H. The
generated slab of the anticipated pharmacophore model with the reference compound, sindaxel.
were necessary in the molecule to show antiproliferative ac-
tivity against HGC-27 cells and bind to the target protein.
All novel chiral thiosemicarbazone derivatives with their
conformational models were mapped onto the generated
HipHop pharmacophore model using a "rigid" fitting method
and "best mapping only" option to obtain the bioactive confor-
mation of each molecule in the Ligand Pharmacophore
Mapping protocol in Discovery Studio 3.5. The results of map-
ping of the features onto the compounds are given in Table 2,
and compound 17b, which was experimentally found as the
most active chiral thiosemicarbazone derivative, showed the
best fit value of 6.000 for the generated pharmacophore
Chirality DOI 10.1002/chir
hypothesis, fitting all the mapped common features in the
anticipated model with a specified "Pharmprint" value of
11111.
Additionally, the novel chiral thiosemicarbazone deriva-
tives were substituted with a piperidine group instead of the
hydrogen atom at 3-position of the benzene ring, and com-
pound 17b possessed a better match with all the features in
the anticipated model in Figure 3C. When compound 17b
was also compared with compound 17a, we observed that
the (S)-enantiomer (17b) was better matched than the
(R)-enantiomer (17a) (see Fig. 3D). This shows that the
(R)- and (S)-enantiomer greatly influenced the activity.

POTENTIAL OF CHIRAL CANCER DRUGS
187
TABLE 2. Alignment of common-feature
pharmacophore model with test set
used to define how the substituent on the benzene ring
and chiral structures of thiosemicarbazone derivatives (3a-
17a versus 3b-17b) involved the anticancer activity. In con-
clusion, the generated 3D common feature pharmacophore
hypothesis showed that the conformational properties and
substituent groups of the compounds were significant for
antiproliferative activity against any cells. In addition, the
compound possessing the (S)-(+)-2-[(4-piperidin-1-yl)ben-
zylidene])-N-(1-phenylethyl)hydrazinecarbothioamide (17b)
structure was important for improving HGC-27 inhibitor
potency, rather than (2α,5β,7β,10β,13α)-4,10-Diacetoxy-13-
[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy-1,7-
dihydroxy-9-oxo-5,20-epoxytax-11-en-2-yl benzoate (sindaxel) (see
Figs. 4C, 5E) in these attempted thiosemicarbazone derivatives.
Compound
Fit Value
3a
No map found
No map found
2.05092
5.25537
1.93746
3b
4a
4b
5a
5b
5.19604
6a
No map found
No map found
No map found
No map found
No map found
No map found
2.2815
6b
7a
7b
8a
8b
9a
ACKNOWLEDGMENTS
9b
5.15034
2.37954
4.88218
2.10885
5.30328
3.20682
5.25216
2.07216
This work was supported by the Scientific Research Projects
Governing Unit Council of Scientific Research Projects (Grant
no. FEF 12.15), Gaziantep, Turkey. The authors thank Esin
Akı Yalcin and the research group for technical assistance.
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
Sindaxel
LITERATURE CITED
1. Cho JY. Molecular diagnosis for personalized target therapy in gastric
cancer. J Gastric Cancer 2013;13:129–135.
2. Boulikas T, Vougiouka M. Cisplatin and platinum drugs at the molecular
level. (review). Oncol Rep 2003;10:1663–1682.
3. da Silva AP, Martini MV, de Oliveira CM, Cunha S, de Carvalho JE, Ruiz AL, da
Silva CC. Antitumor activity of (À)-alpha-bisabolol-based thiosemicarbazones
against human tumor cell lines. Eur J Med Chem 2010;45:2987–2993.
4. Tian J, Peehl DM, Zheng W, Knox SJ. Anti-tumor and radiosensitization
activities of the iron chelator HDp44mT are mediated by effects on intra-
cellular redox status. Cancer Lett 2010;298:231–237.
5. Richardson DR, Kalinowski DS, Lau S, Jansson PJ, Lovejoy DB. Cancer
cell iron metabolism and the development of potent iron chelators as
anti-tumour agents. Biochim Biophys Acta 2009;1790:702–717.
5.31915
No map found
No map found
3.82272
5.35914
2.26292
5.32714
2.78772
6.00000
No map found
As mentioned, compound 17b exhibited 2-fold more anti-
proliferative activity than the reference compound against
HGC-27 cells (Table 1). Hence, pharmacophore model was
also generated for the reference compound, sindaxel, to eluci-
date this state. The obtained pharmacophore model was
better matched with compound 17b; however, the reference
compound was not able to show any match with the deter-
mined pharmacophore model, as given in Figure 3E. This
observation explains why the (S)-(+)-2-[(4-piperidin-1-yl)
benzylidene])-N-(1-phenylethyl)hydrazine carbothioamide (17b)
structure is more favorable than sindaxel for increasing the
potency in this set of compounds.
6. Richardson DR. Iron chelators as therapeutic agents for the treatment of
cancer. Crit Rev Oncol Hematol 2002;42:267–281.
7. Kashyap M, Kandekar S, Baviskar AT, Das D, Preet R, Mohapatra P,
Satapathy SR, Siddharth S, Guchhait SK, Kundu CN, Banerjee UC.
Indenoindolone derivatives as topoisomerase II-inhibiting anticancer
agents. Bioorg Med Chem Lett 2013;23:934–938.
8. Dilovic I, Rubcic M, Vrdoljak V, Kraljevic Pavelic S, Kralj M, Piantanida
I, Cindric M. Novel thiosemicarbazone derivatives as potential antitu-
mor agents: Synthesis, physicochemical and structural properties,
DNA interactions and antiproliferative activity. Bioorg Med Chem
2008;16:5189–5198.
9. Yalowich JC, Wu X, Zhang R, Kanagasabai R, Hornbaker M, Hasinoff BB.
The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory
effects as catalytic inhibitors or poisons of DNA topoisomerase IIalpha.
Biochem Pharmacol 2012;84:52–58.
10. Wu CP, Shukla S, Calcagno AM, Hall MD, Gottesman MM, Ambudkar
CONCLUSION
SV. Evidence for dual mode of action of
a thiosemicarbazone,
NSC73306: a potent substrate of the multidrug resistance linked ABCG2
Here we synthesized a series of novel chiral thiosemi-
carbazones derived from homochiral amines in both enantio-
meric forms and evaluated their in vitro antiproliferative
activity against the A549, MCF-7, HeLa, and HGC-27 cell
lines. Sindaxel showed much stronger activity against the
MCF-7, A549, HeLa cancer cells, but did not show good activ-
ity against HGC-27 cells, according to compound 17b in this
study. If we can investigate the 3D chemical structures of
these compounds, we can see that they many differences,
such as atom, molecular weight, conformation, etc. It is
known that these properties are very effective in determining
and evaluating the activity of any compound against any cell.
Because of this, we examined the SAR study of the com-
pounds in this study. In other words, the SAR studies were
transporter. Mol Cancer Ther 2007;6Pt 1):3287–3296.
11. Moitra K, Lou H, Dean M. Multidrug efflux pumps and cancer stem cells:
insights into multidrug resistance and therapeutic development. Clin
Pharmacol Ther 2011;89:491–502.
12. Smith SW. Chiral toxicology: it’s the same thing…only different. Toxicol
Sci 2009;110:4–30.
13. McConathy J, Owens MJ. Stereochemistry in drug action. prim care com-
panion. J Clin Psychiatry 2003;5:70–73.
14. Caner H, Groner E, Levy L, Agranat I. Trends in the development of chiral
drugs. Drug Discov Today 2004;9:105–110.
15. Nguyen LA, He H, Pham-Huy C. Chiral drugs: an overview. Int J Biomed
Sci 2006;2:85–100.
16. Nunez MC, Garcia-Rubino ME, Conejo-Garcia A, Cruz-Lopez O, Kimatrai M,
Gallo MA, Espinosa A, Campos JM. Homochiral drugs: a demanding ten-
dency of the pharmaceutical industry. Curr Med Chem 2009;16:2064–2074.
Chirality DOI 10.1002/chir

188
TAS¸DEMIR ET AL.
17. Hutt AJ. Chirality and pharmacokinetics: an area of neglected dimension-
21. Yildiz I, Ertan T, Bolelli K, Temiz-Arpaci O, Yalcin I, Aki E. QSAR and
pharmacophore analysis on amides against drug-resistant S. aureus.
SAR QSAR Environ Res 2008;19:101–113.
22. Karakucuk-Iyidogan A, Tasdemir D, Oruc-Emre EE, Balzarini J. Novel
platinum(II) and palladium(II) complexes of thiosemicarbazones derived
from 5-substitutedthiophene-2-carboxaldehydes and their antiviral and cy-
totoxic activities. Eur J Med Chem 2011;46:5616–5624.
ality? Drug Metabol Drug Interact 2007;22:79–112.
18. Vandresen F, Falzirolli H, Almeida Batista SA, da Silva-Giardini AP, de
Oliveira DN, Catharino RR, Ruiz AL, de Carvalho JE, Foglio MA, da
Silva CC. Novel R-(+)-limonene-based thiosemicarbazones and their
antitumor activity against human tumor cell lines. Eur J Med Chem
2014;79:110–116.
˙
˙
˙
19. Accelrys Software Inc AS. Discovery Studio 3.5. San Diego, CA; 2013.
23. Karaküçük-Iyidog˘an A, Mercan Z, Oruç-Emre EE, Tas¸demir D, Is¸ler I,
˙
Kılıç IH, Özaslan M. Synthesis, characterization, and biological evaluation
of some novel thiosemicarbazones as possible antibacterial and antioxi-
dant agents. Phosphorus Sulfur Silicon Relat Elem 2014;189:661–673.
20. Krovat EM, Fruhwirth KH, Langer T. Pharmacophore identification, in
silico screening, and virtual library design for inhibitors of the human
factor Xa. J Chem Inf Model 2005;45:146–159.
Chirality DOI 10.1002/chir