Structural and cytotoxic studies of cationic thiosemicarbazones

Article abstract of DOI:10.1016/j.molstruc.2017.02.031

Schiff bases from the thiosemicarbazones family with variable N4 substituents are known to show enhanced growth inhibitory properties. In view of these facts and as a part of our continuous interest in cationic Schiff bases, we have developed several Schiff base ligands from (3-formyl-4-hydroxyphenyl)methyltriphenylphosphonium (T) in present study. The compounds were characterized by various spectroscopic methods (infrared spectra, ¹H NMR, ¹³C NMR, HRESIMS and X-ray crystallography). Three of the N4 substituents, namely P(tsc)T, FP(tsc)T and EP(tsc)T exerted strong growth inhibitory properties by inhibiting the highly metastasis prostate cancer growth (PC-3). The thiosemicarbazone with ethylphenyl (EP) moiety displayed most potent activity against all cell lines tested. The MTT data obtained from analysis establishes that phenyl substituent enhances the growth inhibitory properties of the compound. The result affirms that EP(tsc)T would serve as a lead scaffold for rational anticancer agent development.

Full text of DOI:10.1016/j.molstruc.2017.02.031

Accepted Manuscript
Structural and cytotoxic studies of cationic thiosemicarbazones
Saravana Kumar Sinniah, Kae Shin Sim, Seik Weng Ng, Kong Wai Tan
PII:
S0022-2860(17)30177-1
10.1016/j.molstruc.2017.02.031
MOLSTR 23425
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 5 October 2016
Revised Date: 8 February 2017
Accepted Date: 9 February 2017
Please cite this article as: S.K. Sinniah, K.S. Sim, S.W. Ng, K.W. Tan, Structural and cytotoxic
studies of cationic thiosemicarbazones, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.02.031.
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ACCEPTED MANUSCRIPT
Structural and cytotoxic studies of cationic thiosemicarbazones
a
a
b,c
b*
Saravana Kumar Sinniah , Kae Shin Sim , Seik Weng Ng and Kong Wai Tan
a
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Malaysia
b
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia
c
Chemistry Department, Faculty of Science, King AbdulAziz University, P. O. box 80203,
Jeddah, Saudi Arabia.
Abstract
Schiff bases from the thiosemicarbazones family with variable N4 substituents are known to
show enhanced growth inhibitory properties. In view of these facts and as a part of our
continuous interest in cationic Schiff bases, we have developed several Schiff base ligands
from (3-formyl-4-hydroxyphenyl)methyltriphenylphosphonium (T) in present study. The
1
compounds were characterized by various spectroscopic methods (infrared spectra, H NMR,
1
3
C NMR, HRESIMS and X-ray crystallography). Three of the N4 substituents, namely
P(tsc)T, FP(tsc)T and EP(tsc)T exerted strong growth inhibitory properties by inhibiting the
highly metastasis prostate cancer growth (PC-3). The thiosemicarbazone with ethylphenyl
(EP) moiety displayed most potent activity against all cell lines tested. The MTT data
obtained from analysis establishes that phenyl substituent enhances the growth inhibitory
properties of the compound. The result affirms that EP(tsc)T would serve as a lead scaffold
for rational anticancer agent development.
Keywords: Thiosemicarbazone; Schiff base; MTT; growth inhibitory; antitumor.
1
. Introduction
The anticancer drug, 3-aminopyridine-2-caboxaldehyde thiosemicarbazone, known
commercially as Triapine, is a Schiff base that can be synthesized by reaction of an aromatic
*
Corresponding author- E-mail: kongwai@um.edu.my; Tel.: +603-79672144
1

ACCEPTED MANUSCRIPT
aldehyde and thiosemicarbazide. The potency of Triapine has propelled it to phase II in
clinical trials and inspires the development of new thiosemicarbazones derivatives [1-4].
Literature on cationic Schiff bases, particularly in the context of their medicinal uses, is
sparse, with references being limited to research carried out on their interactions with DNA
and bacteria [5-9].
Interestingly, Schiff bases from the thiosemicarbazones family having a substituent at the N4
atom, are known to show enhanced growth inhibitory properties [10]. In one of the structure
and activity study it showed that substitution of phenolic groups at terminal thioamide
nitrogen enhances the biological significance of thiosemicarbazone [11]. Similarly in another
study showed that 2-hydroxy-5-methoxyacetophenone thiosemicarbazone with the presence
of phenyl group at N(4) position enhances the antiproliferative potential [12].
Drugs for treating prostate cancer typically show chemoresistance and radioresistance [13,
1
4]. Among prostate tumor cell lines, PC-3 which lacks of androgen receptors is a useful
model in developing drugs for androgen independent cancer cell line [15-18]. Some
phosphonium salts have been reported to show antiproliferative activity in some cancer cell
lines [19]. Copper complexes with phosphonium containing hydrazone ligand for instance
have shown to exhibit cytotoxicity activity against PC-3 cells [20].
Based on these investigations, we intended to synthesize and characterize some new cationic
Schiff base ligands with cytotoxic properties. In addition, this research aimed to determine
structure activity relationship of cationic thiosemicarbazones by evaluating the growth
inhibitory properties of the compounds. In this study, a new series of triphenylphosphonium
1
13
containing thiosemicarbazones were synthesized and characterized by IR, H NMR,
C
NMR, CHN, HRESIMS and X-ray analysis. The growth inhibitory effect of
triphenylphosphonium containing thiosemicarbazone derivatives were evaluated on various
cancer cell lines namely PC3, A549, MCF7, MDA-MB-231, HCT116 and RWPE-1. This
study provides a good start in understanding the basis of structure activity relationship of
cationic thiosemicarbazones towards prostate cancer cell lines.
2
2
. Experimental
.1 Chemistry: Synthesis of compounds
2

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2
.1.1 Materials and instruments
All reagents and solvents used for chemical synthesis were purchased from Merck unless
stated otherwise. Sodium hydrogen carbonate (NaHCO ) was purchased from R&M
3
chemicals. 4-phenyl-3-thiosemicarbazide, 4-(4-fluorophenyl)-3-thiosemicarbazide, 4-ethyl-3-
thiosemicarbazide and 4-ethylphenyl-3-thiosemicarbazide were purchase from Alfa Aesar.
2
.1.2 Physical measurements
Infrared spectra were recorded from KBr discs on Perkin-Elmer Spectrum RX-1
1
13
spectrometer. H NMR and C NMR spectra were recorded in deuterated DMSO-d6 on a
JEOL JNM-LA400 or ECA 400 MHz instrument. Chemical shifts (δ) are given in ppm (parts
per million). Elemental analyses were carried out on a Thermo Finnigan Eager 300 - CHNS
elemental analyzer. HRMS were obtained on a JEOLAccuTOF mass spectrometer.
2
.1.3 Synthesis of 5-(chloromethyl)-2-hydroxybenzaldehyde
All the reagents and solvents were obtained from commercial sources and used as supplied.
2
0
-hydroxybenzaldehyde (10.64 ml, 0.076 mol) was stirred with paraformaldehyde (5.05 g,
.168 mol) and 50 ml of hydrochloric acid (fuming 37 %). The reaction mixture was stirred at
room temperature for 20 hours. After 20 hours, clumps of pinkish solid formed and it was
filtered off. The solid was washed with 0.5 % NaHCO solution and then with distilled water
3
until the pH was close to seven. It was crystallized using petroleum ether to give white needle
crystals. The crystals were dried under vacuum and kept for further synthesis [49, 50].
2
.1.4 Synthesis of (3-formyl-4-hydroxyphenyl) methyltriphenylphosphonium (T)
Briefly, 5-chloromethylsalicylaldehyde (0.170 g, 1 mmol) and triphenylphosphine (0.262 g, 1
mmol) were dissolved in 50 ml of toluene separately. Both solutions were mixed together and
refluxed for three hours and cooled. The cooled whitish-yellow precipitated phosphonium salt
was then filtered off and washed with diethyl ether. Slow evaporation of the compound in
ethanol yielded white crystals. The crystals were filtered, washed with water, dried in the air
and kept in desiccator over silica gel.
(
Yield: 41 %). Anal. Calc. for C H O P.Cl: (432.88): C, 72.22 %; H, 5.09 %. Found: C,
2
6
22
2
-1
7
1.79 %; H, 5.21 %. IR: (KBr, cm ): v(O-H): 3422 (w), v (C-H): 3021 (m), v(C=O): 1676 (s), v(C-
H): 1438 (s), v(C-O): 1112 (s), v(C-H): 687 (s). s = strong, m = medium and w = weak
3

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1
Characteristics H NMR (400MHz, DMSO-d , TMS, s=singlet; d= duplet; m=multiplet) δ
6
(
ppm): 5.07-5.11 (2H, d, CH ); 6.90 (1H, s, aromatic CH); 7.03-7.07 (1H, s, aromatic CH);
2
7
.19-7.20 (1H, s, aromatic CH); 7.62-7.90 (15H, m, aromatic CH); 10.13 (1H, s, CHO); 11.03
1
3
(
1H, s, OH). C NMR (DMSO-d , TMS, ppm 100MHz): 190.13 (C=O); 161.21 (C-OH);
6
117.67-138.62 (Ar); 27.92, 27.46 (CH ). HRESIMS m/z 397.1356 (calcd for C H O P,
2 26 22 2
3
97.1352).
2
.1.5 Synthesis of [4-hydroxy-3-[(E)-
(phenylcarbamothioylhydrazono)methyl]phenyl]methyl-triphenyl-phosphonium
chloride P(tsc)T
The (3-formyl-4-hydroxy-phenyl) methyl-triphenyl-phosphonium chloride (0.382g, 1mmol)
and 4-phenyl-3-thiosemicarbazide (0.168g, 1mmol) were dissolved in 30 ml and 20 ml of
ethanol, respectively. Both solutions were then mixed together and refluxed for four hours
and cooled. The yellowish precipitate formed was filtered off using filter paper and washed
again with ethanol to give pure Schiff base. The product was air dried at room temperature.
(
Yield: 59 %). Anal. Calc. for C H N OPS.Cl.CH O: (614.14): C, 66.56 %; H, 5.38 %; N,
33 29 3 4
-1
6
.85 %. Found: C, 66.51 %; H, 5.15 %; N, 6.88 %. IR: (KBr, cm ): v(O-H): 3303 (s), v(C=N)
:
1
594 (m), v(C-H): 1438 (s), v(C=S): 1111 (s), v(C=S): 830 (m), v(C-H): 691 (s). s = strong, m =
medium and w = weak.
1
Characteristic H NMR (400MHz, DMSO-d , TMS, s=singlet; d= duplet and m=multiplet) δ
6
(
ppm): 5.00-4.97 (2H, d, CH ); 6.83-6.75 (1H, s, aromatic CH); 7.41-7.39 (2H, s, aromatic
2
CH ); 7.52-7.44 (2H, m, CH ); 7.70-7.59 (15H, m, aromatic CH); 7.81-7.77 (3H, m, CH );
2
2
3
1
3
8
.30 (1H, s, aromatic CH); 9.45 (1H, s, CH=N); 10.41 (1H, s, OH); 11.87 (1H, s, NH). C
NMR (DMSO-d , TMS, ppm 100MHz): 176.06 (C=S); 157.10 (C-OH); 139.21 (C=N);
6
117.12-135.49 (Ar); 31.23 (CH ). HRESIMS m/z 546.1747 (calcd for C H N OPS,
2 33 29 3
5
46.1769).
2
.1.6 Synthesis of [3-[(E)-[(4-fluorophenyl)carbamothioylhydrazono]methyl]-4-hydroxy-
phenyl]methyl-triphenyl-phosphonium chloride FP(tsc)T
4

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This compound was synthesized according to our previously published procedure [51]. The
(
3-formyl-4-hydroxyphenyl) methyltriphenylphosphonium chloride (0.432 g, 1 mmol) and 4-
4-fluorophenyl)-3-thiosemicarbazide (0.186 g, 1 mmol) were dissolved in 30 ml and 20 ml
(
of ethanol, respectively. Both solutions were mixed together and refluxed for four hours and
cooled. The yellowish precipitate formed was filtered off using filter paper and washed again
with ethanol to give pure Schiff base. The product was air dried at room temperature. Slow
evaporation of the compound in methanol yielded yellow crystals. The crystals were kept in a
desiccator over silica gel.
(
Yield: 55 %). Anal. Calc. for C H FN OPS.Cl.CH O: (632.13): C, 64.66 %; H, 5.07 %; N,
33 28 3 4
-1
6
.66 %. Found: C, 64.83 %; H, 4.71 %; N, 6.86 %. IR: (KBr, cm ): v(O-H): 3328 (s), v(C=N)
:
1
560 (m), v(C-H): 1438 (s), v(C=S): 1112 (s), v(C=S): 839 (s), v(C-H): 691 (s). s = strong, m =
medium and w = weak.
1
Characteristic H NMR (400MHz, DMSO-d , TMS, s=singlet; d= duplet and m=multiplet) δ
6
(
ppm): 5.05-5.01 (2H, d, CH ); 6.82-6.80 (1H, s, aromatic CH); 7.27-7.24 (1H, s, aromatic
2
CH); 7.28-7.27 (2H, m, CH ); 7.80-7.60 (15H, m, aromatic CH); 7.85-7.80 (3H, m, CH );
2
3
1
3
8
.32 (1H, s, aromatic CH); 9.51 (1H, s, CH=N); 10.50 (1H, s, OH); 11.91 (1H, s, NH). C
NMR (DMSO-d , TMS, ppm 100MHz): 175.97 (C=S); 156.59 (C-OH); 135.05 (C=N);
6
114.85-134.92 (Ar); 27.80 (CH ). HRESIMS m/z 564.1677 (calcd for C H N OFPS,
2 33 28 3
5
64.1675).
2
.1.7 Synthesis of [3-[(E)-(ethylcarbamothioylhydrazono)methyl]-4-hydroxyphenyl]
methyltriphenylphosphonium chloride E(tsc)T
The (3-formyl-4-hydroxyphenyl) methyltriphenylphosphonium chloride (0.432 g, 1 mmol)
and 4-ethyl-3-thiosemicarbazide (0.119 g, 1 mmol) were dissolved in 30 ml and 20 ml of
ethanol, respectively. Both solutions were mixed together and refluxed for four hours and
cooled. The yellowish precipitate formed was filtered off using filter paper. The precipitate
was washed again with ethanol to give pure Schiff base. The product was air dried at room
temperature.
(
Yield: 50 %). Anal. Calc. for C H N OPS.Cl.C H O: (580.12): C, 64.25 %; H, 6.04 %; N,
29 29 3 2 6
-1
7
.25 %. Found: C, 64.04 %; H, 5.60 %; N, 7.35 %. IR: (KBr, cm ): v(O-H): 3290 (w), v(C=N):
5

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1
560 (m), v(C-H): 1439 (s), v(C=S): 1109 (s), v(C=S): 822 (w), v(C-H): 687 (m). s = strong, m =
medium and w = weak.
1
Characteristic H NMR (400MHz, DMSO-d , TMS, s=singlet; d= duplet and m=multiplet) δ
6
(
ppm): 5.02-4.99 (2H, d, CH ); 6.80-6.77 (1H, s, aromatic CH); 6.86-6.83 (1H, s, aromatic
2
CH); 7.27-7.22 (1H, s, aromatic CH); 7.45-7.40 (2H, d, CH ); 7.69-7.54 (15H, m, aromatic
2
CH); 7.83-7.78 (3H, m, CH ); 8.32 (1H, s, NH); 9.48 (1H, s, CH=N); 10.50 (1H, s, OH);
3
13
1
1.87 (1H, s, NH). C NMR (DMSO-d , TMS, ppm 100MHz): 175.57 (C=S); 156.57 (C-
6
OH); 138.63 (C=N); 116.57-134.96 (Ar); 27.79 (CH ). HRESIMS m/z 498.1781 (calcd for
2
C H N OPS, 498.1769).
29
29
3
2
.1.8 Synthesis of [3-[(E)-[(4-ethylphenyl)carbamothioylhydrazono]methyl]-4-
hydroxy-phenyl]methyl-triphenyl-phosphonium chloride EP(tsc)T
The (3-formyl-4-hydroxyphenyl) methyltriphenylphosphonium chloride (0.432 g, 1 mmol)
and 4-ethylphenyl-3-thiosemicarbazide (0.195 g, 1 mmol) were dissolved in 30 ml and 20 ml
of ethanol, respectively. Both solutions were mixed together and refluxed for four hours and
cooled. The yellowish precipitate formed was filtered off using filter paper and washed again
with ethanol to give pure Schiff base. The product was air dried at room temperature.
(
Yield: 57 %). Anal. Calc. for C H N OPS.Cl: (610.15): C, 68.97 %; H, 5.42 %; N, 6.90 %.
35 33 3
-1
Found: C, 68.45 %; H, 5.43 %; N, 6.83 %. IR: (KBr, cm ): v(O-H): 3330 (s), v(C=N): 1600 (m),
v(C-H): 1438 (s), v(C=S): 1108 (s), v(C=S): 845 (m), v(C-H): 687 (s). s = strong, m = medium and w
=
weak.
1
Characteristic H NMR (400MHz, DMSO-d , TMS, s=singlet; d= duplet and m=multiplet) δ
6
(ppm): 2.49 (3H, m, CH ); 4.98-4.94 (2H, d, CH ); 6.75-6.73 (1H, s, aromatic CH); 6.82-6.80
3 2
(
1H, s, aromatic CH); 7.25-7.24 (2H, s, aromatic CH ); 7.46 (2H, m, CH ); 7.67-7.48 (15H,
2
2
m, aromatic CH); 7.81-7.77 (3H, m, CH ); 8.28 (1H, s, aromatic CH); 9.37 (1H, s, CH=N);
3
1
3
1
0.41 (1H, s, OH); 11.80 (1H, s, NH). C NMR (DMSO-d , TMS, ppm 100MHz): 176.11
6
(
C=S); 157.05 (C-OH); 138.82 (C=N); 117.12-135.52 (Ar); 31.23 (CH ). HRESIMS m/z
2
5
74.2105 (calcd for C H N OPS, 574.2082).
35 33 3
2
.2 Biological studies
6

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2
.2.1 Materials
Cisplatin was purchased from Merck (Darmstadt, Germany), MTT (3-(4,5-Dimethylthiazol-
-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich (Missouri, USA).
2
Penicillin-Streptomycin as an antibacterial and amphotericin B solution as an antifungal were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Accutase solution as a cell detacher
and fetal bovine serum (FBS) used as undefined supplement in cell culture, were purchased
from Sigma-Aldrich (Missouri, USA). Phosphate-buffered saline (PBS) from Sigma-Aldrich
(St. Louis, MO, USA) was purchased in tablet form, and each tablet was dissolved in 200 ml
of distilled water. Dimethyl sulfoxide (DMSO) and trypan blue were purchased from Sigma-
Aldrich (St. Louis, MO, USA) as well. Medium used for cell culture are RPMI 1640
purchased from Sigma-Aldrich (St. Louis, MO, USA) and keratinocyte serum free medium
purchased from Invitrogen (Carlsbad, CA, USA).
2
.2.2 Cell culture
The human cell lines used in this study were PC-3 (prostate adenocarcinoma), A549 (non-
small cell lung cancer), MCF7 (breast carcinoma), MDA-MB-231 (breast adenocarcinoma),
HCT116 (colorectal carcinoma) and RWPE-1 (prostate normal). The cell lines were
purchased from American Type Culture Collection (Chicago, IL, USA). PC-3, A549, MCF7
and HCT116 were cultured in RPMI 1640 medium, supplemented with 10 % heat-inactivated
fetal bovine serum, 2 % penicillin-streptomycin and 1 % of amphotericin b solution. DMEM
medium supplemented with 10 % heat-inactivated fetal bovine serum, 2 % penicillin-
streptomycin and 1 % of amphotericin b solution was used to culture MDA-MB-231 cells.
RWPE-1 cells were maintained in keratinocyte serum free medium supplemented with 2 %
penicillin-streptomycin and 1 % of amphotericin. The medium were added with two additives
required to grow this cell line which are the bovine pituitary extract (BPE) and human
recombinant epidermal growth factor (EGF). Cells were cultured as monolayers and
maintained in exponential growth at 37 °C in a humidified atmosphere containing 5 % CO2.
2
o
Cells were cultured in filtered 25 cm culture flasks in a humidified incubator at 37 C with 5
%
CO . When the cells reach 70 % confluence, they were harvested using accutase by
2
o
incubating at 37 C for 10 minutes to obtain complete cell detachment. The cells were
counted using trypan blue exclusion dye method with appropriate cell number for 72 hours in
1
00 µl dilution per well. Cells were seeded in triplicate into 96-well tissue culture plates. The
cells were incubated in CO incubator under conditions appropriate for the cell line for 18
2
7

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hours. After 18 hours the medium was removed carefully without causing cell detachment.
Next anticancer drugs ranging from 0.1 to 30 µM dissolved in fresh medium were added into
designated wells, with the final volume of 200 µl each well. The plates were further
incubated for 72 hours. Wells with media alone omitting the anticancer drugs were used as
negative control and empty wells were used as the blank. Cisplatin were used as positive
control for comparison.
2
.2.3 MTT growth inhibitory assay
MTT solution was prepared by weighing out 100 mg of MTT powder into 20 ml of PBS.
MTT solution was filter-sterilize through a 0.2 ꢀM syringe filter into a sterile, light protected
container. The solution was stored at -20 °C protected from light. Exactly three hours after
each of the desired time points, 20 µl of the solution was added using multichannel pipette
o
into each well and cells were incubated at 37 C for further three hours. After three hours the
medium was gently removed completely and 200 ml of DMSO was added into each well. The
plate was stirred on a shaker for 15 minutes to completely dissolve the precipitants. The
absorbance was detected at 570 nm with a reference wavelength at 690 nm on a Multiskan
GO UV/Vis microplate spectrophotometer from Thermo Scientific (Waltham, MA, USA)
[
52].
3
3
. Results and discussion
.1 Synthesis and characterization
The thiosemicarbazones were prepared by condensation reaction of (T) with
thiosemicarbazide and its N-(4)-substituted derivatives in accordance to the reaction scheme
shown in Scheme 1. Compound 1 is white in color and compound 2-5 are yellow in color. All
compounds are non-hygroscopic, stable both in solid and solution phases, and highly soluble
1
in MeOH, DMSO and DMF. The compounds were characterized by infrared spectra (IR), H
1
3
NMR, C NMR and HRESIMS which were in conformity with the structure of targeted
compounds.
8

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Scheme 1: Reaction scheme and proposed structures
9

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3
.2 Infrared spectra
-1
The IR spectra of the ligands were analyzed in the region of 4000 – 400 cm . The IR
-1
spectrum of T showed the presence of band at 3021 cm which is ascribed to aromatic v(C-
-1
H) stretch. The bands from 3290 to 3422 cm are assigned to v(O-H) for all the compounds
[
21]. Meanwhile, the bands from 3124 to 3176 are assigned to N-H stretching frequency [22-
-1
2
4]. In the IR spectrum of T, a strong band at 1676 cm appeared but disappeared in all
thiosemicarbazones is assigned to the stretching vibration of carbonyl v(C=O) [20, 25-27].
The IR spectra of (tsc)T showed IR absorption of 1598, meanwhile the rest of the compounds
-1
showed IR absorption of 1560 cm which are ascribed to the v(C=N) azomethine stretching
frequencies [28-30]. Appearance of strong band in thiosemicarbazones for the region of 1108
-1
-1
to 1112 cm and 822 to 839 cm are ascribed to v(C-S) stretching vibration [31]. The above
assignments are in good agreement with the literature values.
1
3
.3 H NMR
1
The H NMR spectra of the compounds are recorded in DMSO-d . In the spectrum of T, a
6
complex multiplet that appeared in the region of 7.91 to 7.62 ppm is assigned to aromatic
protons of triphenylphosphine [20, 32, 33]. Upon formation of Schiff base, the multiplet
shifted upfield as presented in the experimental section. The aldehydic proton (-CHO) of T
appeared in the spectrum at 10.13 ppm but upon Schiff base formation there was no
resonance attributable to (-CHO). Singlet observed at 8.11 to 9.48 ppm is ascribed to the
imine proton(-CH=N) [34]. The prominent doublet signal that appears in all compounds in
the range of 5.00 ppm is assigned to the methylene CH proton, whereas the multiplets that
2
appear at the region of 7.60 to 7.89 ppm are ascribed to the aromatic proton of
1
triphenylphosphine. The H-NMR spectral data are in good agreement with the literatures of
the similar compounds therefore the results strongly affirms that the proposed compounds
have been formed.
1
3
3
.4 C NMR
1
3
13
The C NMR spectra of the compounds are recorded in DMSO-d . The C NMR spectrum
6
of compound T, showed chemical shift at 190.12 ppm and 161.21 ppm which is assigned to
C=O and C-OH respectively. Upon the formation of Schiff base ligand, the chemical shift of
(C=O) for compound T is replaced by azomethine carbon (C=N) for all thiosemicarbazone
which appeared in the region of 135.05 to 144.80 ppm. The prominent doublet that falls
1
0

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around 27.00 ppm is assigned to the methylene carbon for the compounds. Additionally the
signal at 175.61 to 179.06 ppm is ascribed to (C=S) bond of thiosemicarbazones.
3
.5 X-ray crystal structures
Single-crystal X-ray analysis reveals that E(tsc)T crystallized in the monoclinic system
meanwhile P(tsc)T in the triclinic form. P(tsc)T is having space group of P-1 and E(tsc)T
having a space group of P2 /n. Selected crystallographic data are given in Table 1. Selected
1
bond lengths and angles are given in Table S1. Figure 1 and 2 shows ORTEP plots of
P(tsc)T and E(tsc)T respectively. The crystallized compound P(tsc)T and E(tsc)T adopt an
E configuration about the C=N bond with bond distance of 1.285(2) and 1.281(4)
respectively. Presence of C=S bond is detected in both P(tsc)T and E(tsc)T with bond length
of 1.6813(16) and 1.675(3) Å respectively. The O-H and N-H hydrogen atoms are syn for
both compounds [35, 36]. With respect to the central hydroxybenzene ring in the cation, the
o
o
phosphonium-P atom lies to the ring with torsion angle of 75.11 and 115.4 for P(tsc)T and
E(tsc)T respectively via P -C -C -C . The hydrazone residue of E(tsc)T showed planar
1
19 20
25
o
o
torsion angle of 0.4 via N - N -C -C -C . P(tsc)T have a torsion angle of 15.7 via N -
1
1
27 26 25
1
C -C -C for similar position.
26
24 25
11

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Table 1
Crystallographic data for P(tsc)T and E(tsc)T
P(tsc)T
E(tsc)T
Empirical Formula
Formula Weight
Crystal system
a (Å)
b (Å)
c (Å)
C H N OPS.Cl.CH O
C H N OPS.ClC H OH
33
29
3
4
29 29
3
2
5
614.14
580.12
Triclinic, P-1
8.7119 (4)
11.3037 (5)
16.2991 (7)
102.0448
99.8760
99.6329
1511.72 (12)
2
Monoclinic, P2 /n
1
9.9634 (5)
17.6847 (10)
16.0134 (7)
90
107.899
90
2685.0 (2)
4
α (º)
β (º)
γ (º)
3
V (Å )
Z
F (000)
634
1.329
0.28
1120
1.321
0.31
-3
Dx (Mg m )
-1
µ (mm )
T (K)
293(2)
100(2)
0.15 x 0.10 x 0.05
Yellow, Block
4655
Crystal size (mm)
Crystal color, habit
Observed data [I>2σ(I)]
Rint
θmax ( )
θmin ( )
0.50 x 0.40 x 0.30
Yellow, Block
6096
0.025
27.5
1.9
0.040
27.6
2.4
o
o
h
k
l
-11→11
-14→14
-21→21
0.037
-12→12
-14→23
-20→15
0.050
2
2
R[F >2σ(F )]
wR(F )
2
0.103
0.124
S
ꢁ
ꢁ
1.05
0.53
0.58
1.02
0.66
0.66
-
3
ρmax (e Å )
-
3
ρmin (e Å )
CCDC no
1443549
1024896
1
2

ACCEPTED MANUSCRIPT
Figure 1: ORTEP diagram of C H N OPS.Cl [P(tsc)T]. Thermal ellipsoids are shown at 50%
3
3
29
3
probability
Figure 2: ORTEP diagram of C H N OPS.Cl [E(tsc)T]. Thermal ellipsoids are shown at 50%
2
9
29
3
probability
1
3

ACCEPTED MANUSCRIPT
3
.6 Growth inhibitory effect on PC-3 human prostate cancer cells as determined by
MTT
The results summarized in Table 2 were expressed as IC values averaged from three
5
0
independent experiments. In this study, we used hormone independent PC-3 cells, whereby
their growth is not affected by the absence of androgen [13, 37]. Globally prostate cancer is
the second-most frequently diagnosed malignancy [38]. Furthermore, PC-3 cell line is also
known for being the more aggressive forms of prostatic adenocarcinoma [39]. A normal
human prostate cell line (RWPE-1) was used to determine the specificity of cancerous cells.
Four human cancer cell lines, namely MCF7, A549, MDA-MB-231 and HCT 116 were
included in this study for growth inhibitory activity comparison. As shown in Table 2, out of
four compounds tested, E(tsc)T showed poor inhibitory effect with IC value more than 30
5
0
µM against PC-3, MCF7 and A549 cells. Compounds that showed poor inhibitory effects
were not further tested against MDA-MB-231 and HCT 116 cells. The thiosemicarbazone
with ethylphenyl moiety displayed most potent activity against PC-3 and HCT 116 cells with
IC value of 1.07 ± 0.38 µM and 0.9 ± 0.10 µM respectively, surpassing that of the positive
5
0
control cisplatin (IC value of 5.47 ± 0.06 and 4.8 ± 0.35 µM, respectively). The results in
5
0
Table 2 showed that EP(tsc)T exerted least damage to RWPE-1 cells (IC value of 6.60 ±
50
0
.10 µM; giving selectivity index of 6.2 and 7.3 against PC-3 and HCT 116 respectively). It
is highly desirable to have a compound that is actively killing cancer cells but exerted least
damage to the normal cells. This result suggests thiosemicarabazone with ethylphenyl
substituent at N4 atom exerted the highest growth inhibitory properties. The value is closely
followed by two other N4 substituents namely P(tsc)T and FP(tsc)T with IC value of 3.73
5
0
±
0.42 and 2.47 ± 0.12 µM respectively against PC-3. The same N4 substituents P(tsc)T and
FP(tsc)T showed good properties towards HCT 116 cells with IC value of 3.0 ± 0.40 µM
5
0
and 6.73 ± 0.12 µM respectively. In this study, the results showed that phenyl compound
further enhances the growth inhibitory properties of the compound. Phenyl group were also
proven to have growth inhibitory activity towards other cancer cell line such as MRC5
fibroblasts and A375 melanoma cells [40]. The advent of fluorouracil has exemplified the
role of organofluorine compounds in therapeutic chemistry with approximately 20% of all
currently approved drugs contain at least one fluorine atom [41]. Fluorouracil unique
structure is known to interfere with DNA and RNA replication in cells, and concurrently
stops cancer cells growth [42-47]. Fluorouracil is still a widely used antineoplastic agent
which played a major role in treatment of various cancer types such as colorectal, breast,
stomach, head and neck cancer [48]. Similarly in this study, the introduction of a fluorine
1
4

ACCEPTED MANUSCRIPT
atom in FP(tsc)T resulted in good inhibitory activities towards PC-3, MCF7, MDA-MB-231
and HCT 116 cells.
1
5

ACCEPTED MANUSCRIPT
Table 2 Growth inhibitory effect (IC µM) of tested compounds
5
0
A
D
IC50 µM (SI)
Compounds
C
PC-3
MCF7
5.63 ± 0.93
10.4 ± 1.06
>30
A549
6.94 ± 0.98
>30
MDA-MB-231
28.47 ± 0.81
27.2 ± 0.20
NT
HCT 116
3.0 ± 0.40 (1.2)
6.73 ± 0.12
NT
RWPE-1
P(tsc)T
FP(tsc)T
E(tsc)T
3.73 ± 0.42
3.57 ± 0.25
4.83 ± 0.06
17.11 ± 3.32
6.60 ± 0.10
12.04 ± 1.63
2.47 ± 0.12 (2.0)
>30
>30
EP(tsc)T
1.07 ± 0.38 (6.2)
5.47 ± 0.06 (2.2)
9.13 ± 0.64
7.53 ± 1.21
4.2 ± 0.79 (2.9)
6.07 ± 0.92 (1.1)
0.9 ± 0.10 (7.3)
B
Cisplatin
5.1 ± 0.1 (2.4)
3.47 ± 0.23 (3.5)
4.8 ± 0.35 (2.5)
A
B
C
Data are presented as mean ± SD from of three independent experiments triplicate for each; Positive control; Non-cancerous cell line;
D
E
SI= Selectivity Index (normal cells/cancer cells); NT= not tested
16

ACCEPTED MANUSCRIPT
4
Conclusions
The new thiosemicarbazone derivatives have been synthesized and characterized by various
spectroscopic and analytical techniques; the results were in conformity with the structure of
targeted compounds. Three of the N4 substituent namely P(tsc)T, FP(tsc)T and EP(tsc)T
exerted good growth inhibitory properties by inhibiting the highly metastasis prostate cancer
growth. The MTT data obtained from the analysis proves that phenyl substituent enhances the
growth inhibitory properties of the compound and this data were supported by published
literatures. Taken together, these data suggest that the three compounds; warrants further
study as anticancer agents and it can serve as building block for further modifications.
Acknowledgements
The authors wish to acknowledge the Ministry of Higher Education of Malaysia (MOHE) for
scholarship and financial assistance. The author also would like to thank Toray Science
Foundation (55-02-03-1060 Grant) for their financial assistance. Financial assistance from
University of Malaya is deeply appreciated [UMRG-RG049/11BIO and PPP-PV015/2012A].
Authors Contribution
Saravana Kumar Sinniah performed analysis on all samples, interpreted data and wrote the
manuscript. Sim Kae Shin and Tan Kong Wai supervised development of work, helped in
data interpretation and manuscript evaluation. Ng Seik Weng performed X-ray
crystallography analysis and helped in critical revision of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest
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Highlights
1
13
•
•
•
•
•
Compounds were confirmed by IR, H/ C NMR, HRESIMS and X-ray crystallography.
P(tsc)T, FP(tsc)T and EP(tsc)T inhibits the highly metastasis PC-3 cells.
EP(tsc)T showed S.I of 6.2 and 7.3 against PC-3 and HCT 116 respectively.
The thiosemicarbazone with ethylphenyl (EP) moiety displayed most potent activity.
P(tsc)T, FP(tsc)T and EP(tsc)T warrants further study as anticancer agents.