Synthesis, characterization, and evaluation of silver(I) complexes with mixed-ligands of thiosemicarbazones and diphenyl(p-tolyl)phosphine as biological agents

Article abstract of DOI:10.1080/00958972.2019.1577400

Five new silver(I) complexes were synthesized with mixed ligands of thiosemicarbazone derivatives and diphenyl(p-tolyl)phosphine in search of new biologically active compounds. A CHN elemental analysis, powder X-ray diffraction (PXRD) data and several spectroscopic techniques such as Fourier-transform infrared spectroscopy, energy-dispersive X-ray, ¹H, ¹³C, and ³¹P{¹H} NMR were performed to elucidate the structure of these complexes. Elemental analysis suggested that the stoichiometry of the complexes formed by the reaction of silver nitrate with thiosemicarbazone in the presence of (p-tolyl)PPh₂ was indeed 1:2:1 molar ratio. The silver ions were discovered to be coordinated to the sulfur of thiosemicarbazone and phosphorus of (p-tolyl)PPh₂, having a tetrahedral geometry based on the spectroscopic data obtained. The PXRD patterns were studied to see the degree of crystallinity of the complexes. The in vitro antiproliferative activity of these complexes was investigated toward the MDA-MB-231 and MCF-7 breast cancer cell lines, as well as the HT-29 colon cancer cell line, which yielded IC₅₀ values in low micromolar range. The antiplasmodial activity of these complexes was also examined against chloroquine-resistant Plasmodium falciparum parasite which demonstrated good activity and further tested for their selectivity index.

Full text of DOI:10.1080/00958972.2019.1577400

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Synthesis, characterization, and evaluation
of silver(I) complexes with mixed-
ligands of thiosemicarbazones and
diphenyl(p-tolyl)phosphine as biological agents
Syahrina Nur 'Ain Abdul Halim, Fariza Juliana Nordin, Mohd Ridzuan Mohd
Abd Razak, Nur Rahimah Fitrah Mohd Sofyan, Siti Nadiah Abdul Halim, Nor
Fadilah Rajab & Rozie Sarip
To cite this article: Syahrina Nur 'Ain Abdul Halim, Fariza Juliana Nordin, Mohd Ridzuan Mohd
Abd Razak, Nur Rahimah Fitrah Mohd Sofyan, Siti Nadiah Abdul Halim, Nor Fadilah Rajab & Rozie
Sarip (2019): Synthesis, characterization, and evaluation of silver(I) complexes with mixed-ligands
of thiosemicarbazones and diphenyl(p-tolyl)phosphine as biological agents, Journal of Coordination
Chemistry, DOI: 10.1080/00958972.2019.1577400
To link to this article: https://doi.org/10.1080/00958972.2019.1577400
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2019.1577400
Synthesis, characterization, and evaluation of silver(I)
complexes with mixed-ligands of thiosemicarbazones and
diphenyl(p-tolyl)phosphine as biological agents
a
b
Syahrina Nur ’Ain Abdul Halim , Fariza Juliana Nordin , Mohd Ridzuan Mohd
c
a
a
Abd Razak , Nur Rahimah Fitrah Mohd Sofyan , Siti Nadiah Abdul Halim ,
Nor Fadilah Rajab and Rozie Sarip
b
a
a
b
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; Faculty
c
of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; Bioassay Unit, Herbal
Medicine Research Centre, Institute for Medical Research, Kuala Lumpur, Malaysia
ABSTRACT
ARTICLE HISTORY
Five new silver(I) complexes were synthesized with mixed ligands of
thiosemicarbazone derivatives and diphenyl(p-tolyl)phosphine in
search of new biologically active compounds. A CHN elemental ana-
lysis, powder X-ray diffraction (PXRD) data and several spectroscopic
techniques such as Fourier-transform infrared spectroscopy, energy-
dispersive X-ray, H, C, and P{ H} NMR were performed to elucidate
the structure of these complexes. Elemental analysis suggested that
the stoichiometry of the complexes formed by the reaction of silver
Received 9 November 2018
Accepted 14 January 2019
KEYWORDS
Thiosemicarbazone; silver
complex; anticancer;
antimalarial; phosphine
1
13
31
1
2
nitrate with thiosemicarbazone in the presence of (p-tolyl)PPh was
indeed 1:2:1 molar ratio. The silver ions were discovered to be coordi-
nated to the sulfur of thiosemicarbazone and phosphorus of (p-tolyl)
2
PPh , having a tetrahedral geometry based on the spectroscopic data
obtained. The PXRD patterns were studied to see the degree of crys-
tallinity of the complexes. The in vitro antiproliferative activity of these
complexes was investigated toward the MDA-MB-231 and MCF-7
breast cancer cell lines, as well as the HT-29 colon cancer cell line,
which yielded IC50 values in low micromolar range. The antiplasmodial
activity of these complexes was also examined against chloroquine-
resistant Plasmodium falciparum parasite which demonstrated good
activity and further tested for their selectivity index.
CONTACT Rozie Sarip
Lembah Pantai, 50603 Kuala Lumpur, Malaysia
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
rozie@um.edu.my
Department of Chemistry, Faculty of Science, University of Malaya,

2
S. N. A. ABDUL HALIM ET AL.
1. Introduction
Thiosemicarbazone is classified as Schiff-base compound which functions as chelating
agent for numerous metal ions. Owing to the presence of sulfur and imine nitrogen
atoms, the various binding modes reaffirm its versatility [1–3]. Therefore, these ligands
exhibit diverse biological properties such as antimalarial [4–6], anticancer [7, 8] and
antiprotozoan through the inhibition of cysteine proteases and other targets [9–12],
which have been of great interest.
Although as free ligands, the thiosemicarbazones revealed good antimalarial activ-
ities, certain studies found that the introduction of transition metals would enhance
the properties of these ligands. For instance, Khanye et al. [13] found that the coordin-
ation of gold to thiosemicarbazone, compared to its free ligands, significantly
improved the antiplasmodial activity. Meanwhile, instead of employing gold, Adams
et al. [14] also achieved similar trend of antiplasmodial activity despite the utilization
of various thiosemicarbazone derivatives with the adoption of palladium. Nevertheless,
it was proved that the combination of metal and thiosemicarbazone derivatives in
complexes enhanced the antiproliferative ability [15, 16].
Hence, a thorough biological study on thiosemicarbazone metal complexes is of
increasing importance given that such metal coordination may positively affect the
antimalarial and antiproliferative activities. Other than thiosemicarbazone, transition
metal complexes with phosphine also revealed fascinating biological activities; for
example, coinage metals encompassing tertiary phosphine and diphosphines were
scrutinized for their anticancer properties [17–21] and displayed good activities.
Following that, the antimalarial activity of phosphine complexes against murine mal-
aria parasite, specifically Plasmodium berghei, was also examined, which demonstrated
the potential development of transmission-blocking antimalarial compound.
Furthermore, Molter et al. [22] discovered that the synthesized gold complex with
mixed ligands of thiosemicarbazone and phosphine revealed rather similar IC50 value
to that of chloroquine. Hence, this study synthesized mixed-ligands system of silver(I)
complexes and evaluated their anticancer and antimalarial properties. To date, several
structural studies of silver(I) thiosemicarbazone complexes were reported [23–26] but
studies on biological activities mainly focus on antibacterial properties [27, 28]. A thor-
ough search of relevant literatures revealed that there were no empirical findings on
anticancer and antimalarial activities of silver(I) complexes with both thiosemicarba-
zone and phosphine ligands. Herein, this study evaluated the synthesis of silver(I) com-
plexes with mixed-ligands of thiosemicarbazone derivatives and diphenyl(p-
tolyl)phosphine and their in vitro antiproliferative and antiplasmodial activities.
2. Experimental
2.1. Materials and instrumentation
All solvents and reagents in this study were of analytical grade and commercially pur-
chased from Sigma Aldrich Ltd., unless stated otherwise. The thiosemicarbazide, aro-
matic aldehydes, silver nitrate, diphenyl(p-tolyl) phosphine, acetic acid, diethyl ether,

JOURNAL OF COORDINATION CHEMISTRY
3
ethanol, methanol and acetonitrile were used without purification. The CHN analyses
were performed by Perkin Elmer CHNS/O 2400 Series II. The infra-red (IR) spectra were
determined using a Perkin Elmer Spectrum One FT-IR spectrophotometer (ATR) at a
ꢀ
1
frequency range of 450–4000 cm . JEOL FT-NMR ECX 400 (ECX 400) was employed to
1
13
31
1
measure the NMR spectra of H, C and P{ H} at 400 MHz in deuterated solvents
without internal reference. The presence of metals and other elements were detected
by energy-dispersive X-ray spectroscopy (EDX). Powder X-ray diffraction (PXRD) was
recorded on an X-ray diffractometer (PANalytical, Netherlands) with Cu Ka characteris-
tic radiation (k ¼ 0.154 nm) at the voltage of 40 kV and current of 40 mA and the scan-
ꢁ
ꢁ
ꢁ
ning rate was 4.25 /min and the scanning of 2h was from 0 to 90 at room
temperature (25 C).
ꢁ
2.2. Synthesis of thiosemicarbazone ligands
As described in the literature [29], the ligands were synthesized with minor modifica-
tions. In general, thiosemicarbazide (6.0 mmol, 0.55 g) was dissolved in ethanol (30 mL)
with the addition of an appropriate amount of aromatic benzaldehyde (6.3 mmol) and
a few drops of acetic acid. Subsequently, the mixture was refluxed for ca. 6 h. The
formed precipitates were filtered and dried at room temperature.
2
.2.1. 4-Hydroxy-benzaldehyde thiosemicarbazone [L1]
ꢁ
Yield 47%; m.p. 230 C. Anal. Calcd for C H N OS: C, 49.21; H, 4.65; N, 21.52. Found: C,
8
9 3
ꢀ
1
49.11; H, 4.63; N, 20.83. IR data (cm ): m(N–H) 3471; m(N–H) 3359; m(S–H) 2751;
1
m(C ¼ N) 1578. H NMR (400 MHz, DMSO), d (ppm): 11.20 (1H, s, N–H); 9.82 (1H, s, N–H);
13
8
.01 (1H, s, S–H); 7.91 (1H, s, N ¼ CH); 6.73–7.78 (4H, m, Ar–H); 7.78 (1H, s, Ar–OH).
NMR (400 MHz, DMSO), d (ppm): 177.93 (C–S); 159.76 (C–OH); 143.20 (N ¼ C–Ar);
16.06–129.58 (C ).
C
1
Ar
2
.2.2. 2,4-Dihydroxy-benzaldehyde thiosemicarbazone [L2]
ꢁ
Yield 56%; m.p. 238 C. Anal. Calcd for C H N O S: C, 45.49; H, 4.29; N, 19.89. Found: C,
8
9 3 2
ꢀ
1
4
5.38; H, 4.22; N, 19.81. IR data (cm ): m(N–H ) 3348, 3254; m(N–H) 3479; m(C ¼ S) 876;
2
1
m(C ¼ N) 1584. H NMR (400 MHz, DMSO), d (ppm): 11.13 (1H, s, N–H); 9.70 (2H, s,
N–H ); 8.20 (1H, s, N ¼ CH); 6.21–7.64 (3H, m, Ar–H); 7.91 (1H, s, Ar–OH); 7.70 (1H, s,
2
1
3
Ar–OH). C NMR (400 MHz, DMSO), d (ppm): 177.50 (C ¼ S); 160.09, 158.50 (C–OH);
1
41.17 (C ¼ N); 102.77–128.83 (C ).
Ar
2
.2.3. 5-Bromo-2-hydroxy-benzaldehyde thiosemicarbazone [L3]
ꢁ
Yield 85%; m.p. 250 C. Anal. Calcd for C H BrN OS: C, 35.05; H, 2.94; N, 15.33. Found:
C, 35.01; H, 2.49; N, 14.91. IR data (cm ): m(N–H) 3356; m(N–H) 3469; m(S–H) 2833;
8
ꢀ
8
3
1
1
m(C ¼ N) 1595. H NMR (400 MHz, DMSO), d (ppm): 11.374 (1H, s, N–H); 10.19 (1H, s,
N–H); 8.24 (1H, s, N ¼ CH); 6.76–8.16 (2H, m, Ar–H); 8.16–8.11 (3H, overlap peaks of
13
Ar–OH, Ar–H and S–H). C NMR (400 MHz, DMSO), d (ppm): 178.35 (C–S); 156.06
(C–OH); 137.79 (C ¼ N); 133.73–111.64 (CAr).

4
S. N. A. ABDUL HALIM ET AL.
2
.2.4. 2-Hydroxy-4-methoxy-benzaldehyde thiosemicarbazone [L4]
ꢁ
Yield 69%; m.p. 212 C. Anal. Calcd for C H N O S: C, 47.99; H, 4.92; N, 18.65. Found:
C, 47.51; H, 5.24; N, 18.76. IR data (cm ): m(N–H) 3245; m(N–H) 3455; m(S–H) 2815;
9
ꢀ
11 3 2
1
1
m(C ¼ N) 1544. H NMR (400 MHz, DMSO), d (ppm):11.21 (1H, s, N–H); 9.90 (1H, s, N–H);
8
.23 (1H, s, N ¼ CH); 6.36–6.40 (2H, m, Ar–H); 7.75–7.78 (2H, overlap peaks of Ar–OH
13
and Ar–H); 7.97 (1H, s, S–H); 3.69 (3H, s, O–CH ). C NMR (400 MHz, DMSO), d (ppm):
3
1
5
77.69 (C–S); 162.38 (C–OCH ); 158.39 (C–OH); 140.68 (C ¼ N); 128.75–101.29 (C );
3
Ar
5.66 (O–CH ).
3
2
.2.5. 5-Bromo-2-hydroxy-3-methoxy benzaldehyde thiosemicarbazone [L5]
ꢁ
Yield 95%; m.p. > 250 C. Anal. Calcd for C H BrN O S: C, 35.54; H, 3.31; N, 13.81.
Found: C, 35.54; H, 3.11; N, 12.81. IR data (cm ): m(N–H) 3352; m(N–H) 3450; m(S–H)
9
10
3 2
ꢀ
1
1
2
813; m(C ¼ N) 1529. H NMR (400 MHz, DMSO), d (ppm): 11.39 (1H, s, N–H); 9.429 (1H,
s, N–H); 8.29 (1H, s, N ¼ CH); 7.03–7.79 (2H, Ar–H); 8.09 (2H, overlap peaks of Ar–OH
13
and S–H); 3.78 (3H, s, O–CH ). C NMR (400 MHz, DMSO), d (ppm): 178.30 (C–S);
3
1
49.46 (C–OCH ); 145.78 (C–OH); 137.82 (C ¼ N); 145.78–111.30 (C ); 56.80 (O–CH ).
3
Ar
3
2.3. Synthesis of silver complexes
AgNO (0.1 mmol, 0.02 g) was dissolved in a solvent mixture (5 mL) of acetonitrile and
3
methanol (ratio of 2:3). Then separately, the synthesized thiosemicarbazone (0.2 mmol)
was dissolved in the same solvent mixture and was added dropwise to the silver solu-
ꢁ
tion. Following that, the reaction mixture was refluxed for 3 h at 50 C. The reaction
mixture was further treated with diphenyl(p-tolyl)phosphine (0.1 mmol, 0.03 g) in the
mixture of acetonitrile and methanol (5 mL) and the refluxing continued for another
2
h. The solution was then filtered to remove any impurities and then dried in vacuo.
The outline of the reaction as shown in Figure 1 and the structure of the product was
proposed referring to the previous study [30] and spectroscopic data attained as no
crystal data could be obtained to date.
2
.3.1. [Ag (PPh (p-tolyl)) (4-hydroxy-benzaldehyde thiosemicarbazone) ](NO ) [1]
2
2
2
ꢁ
4
3 2
Yield 26%; m.p. 130–134 C. Anal. Calcd for C H Ag N O P S : C, 54.27; H, 4.55; N,
70
70
2 12 4 2 4
ꢀ
1
10.85. Found: C, 53.64; H, 4.14; N, 11.37. IR data (cm ): m(N–H) 3407; m(N–H) 3278;
ꢀ
m(S–H) overlap with broad N–H and O–H peak; m(C ¼ N) 1591; m(P–C ) 1095; m(NO
)
Ar
3
1
1305. H NMR (400 MHz, DMSO), d (ppm): 11.64 (4H, s, N–H); 9.97 (4H, s, N–H); 7.93
(
4H, s, N ¼ CH); 6.75–7.62 (44H, m, Ar–H); 8.55 (4H, s, S–H); 8.32 (4H, s, Ar–OH); 2.24
13
(6H, s, Ar–CH ). C NMR (400 MHz, DMSO), d (ppm): 175.20 (C–S); 160.27 (C–OH);
3
31
1
1
45.76 (C ¼ N); 116.11–141.12 (C ); 21.41 (Ar–CH ). P{ H} NMR (400 MHz, DMSO), d
Ar
3
(ppm): 8.84.
2
.3.2. [Ag (PPh (p-tolyl)) (2,4-dihydroxy-benzaldehyde thiosemicarbazone) ](NO ) [2]
2 2 2 4 3 2
ꢁ
Yield 40%; m.p. 136–142 C. Anal. Calcd for C H Ag N O P S : C, 52.11; H, 4.37; N,
70
70
2
ꢀ
12 8 2 4
1
10.42. Found: C, 51.85; H, 4.32; N, 10.29. IR data (cm ): m(N–H ) 3278, 3171; m(N–H)
2
ꢀ
1
3
414; m(C ¼ S) 849; m(C ¼ N) 1600; m(P–C ) 1096; m(NO ) 1372. H NMR (400 MHz,
Ar
3
DMSO), d (ppm): 11.51 (4H, s, N–H); 9.86 (8H, d, J ¼ 13.3 Hz, N–H ); 8.27 (4H, s, N ¼ CH);
2

JOURNAL OF COORDINATION CHEMISTRY
5
Figure 1. Synthetic route for mixed-ligands silver complexes.
13
6
.23–7.71 (40H, m, Ar–H); 8.32, 8.20 (4H, s, Ar–OH); 2.24 (6H, s, Ar–CH3). C NMR
(
1
400 MHz, DMSO), d (ppm): 175.20 (C ¼ S); 161.67, 158.98 (C–OH); 143.87 (C ¼ N);
31
1
02.75–141.29 (C ); 21.40 (Ar–CH ). P{ H} NMR (400 MHz, DMSO), d (ppm): 8.30.
Ar 3
2
.3.3. [Ag (PPh (p-tolyl)) (5-bromo-2-hydroxy-benzaldehyde thiosemicarbazone) ]
2
2
2
4
(
NO ) [3]
3 2
ꢁ
Yield 29%; m.p. 120–124 C. Anal. Calcd for C H Ag Br N O P S : C, 45.08; H, 3.57;
N, 9.01. Found: C, 44.88; H, 3.56; N, 8.61. IR data (cm ): m(N–H) 3322; m(N–H) 3409;
70
66
2
4 12 4 2 4
1
ꢀ
m(S–H) overlap with broad N–H and O–H peak; m(C ¼ N) 1602; m(P–C ) 1094.06;
Ar
ꢀ
1
m(NO ) 1316.04. H NMR (400 MHz, DMSO), d (ppm): 11.71 (4H, s, N–H); 10.34 (4H, s,
3
N–H); 8.29 (4H, s, N ¼ CH); 6.79–8.16 (41H, m, Ar–H); 8.57 (4H, s, S–H); 8.51 (4H, s,
13
Ar–OH); 2.26 (5.95H, s, Ar–CH ). C NMR (400 MHz, DMSO), d (ppm): 175.69 (C–S);
3
31
1
1
56.44 (C–OH); 141.29 (C ¼ N); 111.63–140.42 (C ); 21.42 (Ar–CH ). P{ H} NMR
Ar
3
(400 MHz, DMSO), d (ppm): 8.30.

6
S. N. A. ABDUL HALIM ET AL.
2
.3.4. [Ag (PPh (p-tolyl)) (2-hydroxy-4-methoxy-benzaldehyde thiosemicarbazone) ]
2
2
2
4
(
NO ) [4]
3 2
ꢁ
Yield 39%; m.p. 122–126 C. Anal. Calcd for C H Ag N O P S : C, 53.24; H, 4.71; N,
1
74
78
2 12 8 2 4
ꢀ
1
0.07. Found: C, 52.97; H, 4.26; N, 9.73. IR data (cm ): m(N–H) 3414; m(N–H) 3281;
ꢀ
m(S–H) overlap with broad N–H and O–H peak; m(C ¼ N) 1627; m(P–C ) 1095; m(NO
)
Ar
3
1
1
324. H NMR (400 MHz, DMSO), d (ppm): 11.51 (4H, s, N–H); 10.02 (4H, s, N–H); 8.27
(
4H, s, N ¼ CH); 6.38–7.81 (41H, m, Ar–H); 8.39 (4H, s, S–H); 8.18 (4H, s, Ar–OH); 3.70
13
(12H, s, O–CH ); 2.26 (6H, s, Ar–CH ). C NMR (400 MHz, DMSO), d (ppm): 175.10 (C–S);
3 3
1
2
58.73 (C–OH); 162.84 (C–OCH ); 142.36 (C ¼ N); 101.24–141.22 (C ); 55.71 (O–CH );
3
Ar
3
31
1
1.42 (Ar–CH ). P { H} NMR (400 MHz, DMSO), d (ppm): 8.07.
3
2
.3.5. [Ag (PPh (p-tolyl)) (5-bromo-2-hydroxy-3-methoxy-benzaldehyde
2
2
2
thiosemicarbazone) ](NO ) [5]
4
3 2
ꢁ
Yield 46%; m.p. 126–130 C. Anal. Calcd for C H Ag Br N O P S : C, 44.78; H, 3.76;
N, 8.47. Found: C, 45.06; H, 3.42; N, 7.84. IR data (cm ): m(N–H) 3424; m(N–H) 3288;
74
74
2
4 12 8 2 4
1
ꢀ
ꢀ
m(S–H) overlap with broad N–H and O–H peak; m(C ¼ N) 1597; m(P–C ) 1093; m(NO
)
Ar
3
1
1
311. H NMR (400 MHz, DMSO), d (ppm): 11.71 (4H, s, N–H); 10.34 (4H,s, N–H); 8.35
(
4H, s, N ¼ CH); 7.09–7.79 (36H, m, Ar–H); 8.58 (4H, s, S–H); 8.51 (4H, s, Ar–OH); 3.80
13
(12H, s, O–CH ); 2.26 (6H, s, Ar–CH ). C NMR (400 MHz, DMSO), d (ppm): 175.67 (C–S);
3 3
1
46.22 (C–OH); 149.54 (C–OCH ); 141.34 (C ¼ N); 111.35–140.56 (C ); 21.49 (Ar–CH ).
3
Ar
3
3
1
1
P { H} NMR (400 MHz, DMSO), d (ppm): 8.84.
2.4. Biological assays
2.4.1. Antiproliferative assay
A sulforhodamine B (SRB) assay was performed to determine the inhibition concentra-
tion (IC ) of all compounds, as described in previous studies [31]. Each cell was
5
0
treated at varied concentrations: 5.053–161.686 mM (for 1), 4.843–154.960 mM (for 2),
.189–134.055 mM (for 3), 4.680–149.752 mM (for 4) and 3.936–125.944 mM (for 5).
4
After 48 h, each cell was fixed in the plate with 50 mL of 50% (w/v) trichloroacetic acid
ꢁ
(
TCA) solution and incubated for 1 h at 4 C. Following that, these plates were washed
with tap water for five times then air dried. Then, the cells were stained with 100 mL
of 0.4% (w/v) SRB staining solution and incubated for 10 min at room temperature.
Subsequently, the plates were washed with 1% (v/v) acetic acid for three times (to
remove unbound stains) and air dried. Then, 200 mL of 10 mM Trizma base was added
into each well then were shaken well for 10 min. The absorbance was read at 490 nm
using a microplate reader. Meanwhile, the IC50 values were calculated based on the
following formula: IC50 = (OD sample/OD control)ꢂ100. It should be noted that these
experiments were triplicated.
2
.4.2. In vitro P. falciparum HRPII assay
All compounds were evaluated in vitro for antiplasmodial activity using HRPII assay
32, 33] as described elsewhere, with certain modifications [34]. Briefly, ring-infected
[
red blood cells (RBCs) with 0.05% parasitemia and 1.5% haematocrit were exposed
ꢁ
with serially diluted compounds in a candle jar for 72 h at 37 C. The final tested

JOURNAL OF COORDINATION CHEMISTRY
7
concentrations ranged between 0.25 and 15.7 mg/mL. The negative control was the
infected RBC without compounds or replaced with sterile H O. After 72 h of incuba-
2
ꢁ
tion, the test plates were kept overnight at ꢀ80 C. The test plates were thawed to
lyse the infected RBCs at room temperature. The parasite-compound exposure activity
(end point) was measured using HRPII assay, as described by Noedl et al. [33, 35]. The
collected data were then transferred to HN-NonLin software (malaria.farch.net) to get
5
0% effective concentration (EC ) values directly from the graph.
50
2
.4.3. In vitro cytotoxicity assay
The MDBK cells were maintained in complete DMEM culture medium containing
5 mM HEPES, 0.4% sodium bicarbonate (NaHCO ) and 100 U of Penstrep (100 U peni-
2
3
cillin and 100 U streptomycin) supplemented with 10% fetal bovine serum (FBS). The
cytotoxicity of the compounds were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-
3
diphenyltetrazolium bromide (MTT) assay [36]. Briefly, the MDBK cells (1 ꢂ 10 cells per
well) were exposed with serially diluted (two-fold serial dilutions) compounds with the
range of final concentration between 0.78 and 25 mg/mL in triplicates. The positive
control for cell growth was the cell suspension without test substance, whereas the
negative control was the cell suspension with 0.05% of Triton X 100. The culture was
ꢁ
incubated for 72 h in a CO2 incubator (CO2 concentration of 5% at 37 C).
Subsequently, 50 mL of MTT solution (5 mg MTT in 1 mL PBS and 2.5 mL DMEM media)
was added into each well. Likewise, the plates were further incubated for 4 h in a CO2
ꢁ
incubator (CO concentration of 5% at 37 C). The medium was then removed and
2
replaced with 200 mL of DMSO to dissolve the MTT formazan product. The solution
was subsequently mixed for 15 min and once for 30 s before the measurement of
absorbance at 540 nm was taken with microplate reader (FLUOstar Omega, Germany).
Both growth inhibition and 50% cytotoxic concentration (CC ) values were estimated
50
in percentage based on dose–response curve. A selectivity index (SI), which corre-
sponded to the ratio between the antiplasmodial and cytotoxic activities, was calcu-
lated according to the following formula:
CC5
0 normal cell lines
SIPlasmodium
¼
EC5
0 Plasmodium
3. Results and discussion
During the synthesis, 1 and 2 produced clear solution throughout the experiment.
However, 3, 4 and 5 produced precipitates after reacting with thio-ligand for few
minutes. As the reaction progressed, the precipitates were reduced upon the addition
of diphenyl(p-tolyl)phosphine ligand. However, those three complexes did not produce
clear solution. Hence, this is presumably contributed by the presence of another sub-
stituent in the aromatic ring, besides the hydroxyl group as in complexes 1 and 2.
Thiosemicarbazone ligands exhibit thione–thiol tautomerism, as shown in Figure 2.
Depending on the reaction condition, this reversible reaction occurs in numerous thio-
amide-type derivatives as majority give rise to S-alkyl and N-alkyl derivatives [37]. The
spectroscopic data suggested that all synthesized thiosemicarbazone ligands and their
corresponding complexes exist in the thiol formation except for L2 and its

8
S. N. A. ABDUL HALIM ET AL.
S
SH
H N
N
H
HN
N
H
2
thione
thiol
Figure 2. Thione–thiol tautomerism.
corresponding complex 2. It is postulated that the occurrence of tautomerism is con-
tributed by the presence of various functional groups in the aromatic ring, which sub-
sequently promotes conjugation and electron delocalization in these compounds.
Up to this point, it was revealed that the synthesized silver(I) complexes were non-
hygroscopic which it would not decompose after being stored for an extended dur-
ation. It was reaffirmed by periodic monitoring of its NMR spectrum.
3.1. Spectroscopic data analysis
1
13
31
1
Several spectroscopic techniques such as FT-IR, H NMR, C NMR and P{ H} NMR
were employed to characterize the synthesized thiosemicarbazone ligands and their
complexes. The information of complex coordination through the shape and intensity
of d–d bands was acquired using ultraviolet–visible (UV–vis) spectroscopy. However,
this is not applicable for silver(I) complexes as the d–d transition of Ag(I) is principally
forbidden [27]. Therefore, EDX spectroscopy was utilized to detect the presence of
Ag(I) instead.
3.1.1. FT-IR spectroscopy
The IR spectra of the complexes validated the coordination of silver to thiosemicarba-
zone ligand, which revealed the presence of m(C ¼ N) vibrational modes in the com-
ꢀ
1
plexes 1–5. Nevertheless, a very weak band around 2800–2500 cm of m(S–H) was
observed in L1, L3, L4 and L5 shows that the ligands exist as thiols. However, this
band was not present in the spectrum of their corresponding complexes. A study by
Lobana et al. (2009) [26] shows the presence of intramolecular and intermolecular
hydrogen-bonding of hydroxyl and amino groups in silver thiosemicarbazone complex
ꢀ
1
contributes to band broadening in the 3600–3000 cm region, which causes overlap-
ping of the bands around this region; thus, this may be the plausible rationalization
for the disappearance of m(S–H) in the complex. Meanwhile, m(S–H) was not present in
L2 or its corresponding complex (2), which suggested that it appears in thione form.
ꢀ
1
Thioamide band, m(C ¼ S), was located at 848.87 cm for 2, which demonstrated the
ꢀ
1
shift to lower energy region (875.60 cm ) compared to that of L2. This could be due
to the weakening of C ¼ S bond with the coordination of silver via sulfur atom. The
coordination of phosphorus to silver center was confirmed by the presence of charac-
ꢀ
1
teristic peak m(P–C ) in the region of 1091–1095 cm . The sharp band around
ph
ꢀ
1
ꢀ
1
300 cm , attributed to NO3 bend peak, demonstrated the presence of non-coordi-
nated NO3 (outside the coordination sphere) in all five complexes in this study.
ꢀ

JOURNAL OF COORDINATION CHEMISTRY
9
1
Figure 3. Comparison of H NMR thione–thiol tautomerism between L1 and L2.
3.1.2. NMR spectroscopy
The absence of NH signal for L1, L3, L4 and L5 and their corresponding complexes
2
of 1, 3, 4 and 5 indicated that they exist as thiols. Furthermore, the presence of S–H
signal around 7.97–8.11 ppm for these ligands and 8.39–8.59 ppm for their correspond-
ing complexes reaffirmed the formation of thiol tautomer. However, the NH signal for
2
L2 was present at 9.70 ppm, whereas the NH signal for its complex (2) was present at
2
9
.86 ppm. Accordingly, the absence of S–H peak suggested that they appear as thio-
1
nes instead. Figure 3 reveals the comparison of H NMR thione–thiol tautomerism
between L1 and L2.
The spectra of silver(I) complexes closely resembled their respective free ligands
but the major shift of S–H signal downfield in these complexes reaffirmed the occur-
rence of coordination of ligands to silver ions through sulfur atom of thiol without
being deprotonated. Minor downfield shift of protons attached to N , N of thiosemi-
1
2
carbazone and hydroxyl group in these complexes resulted upon their participation in
hydrogen bonding, namely, intermolecular, intramolecular or both. The bonding of
phosphine was proved by the presence of extra aromatic protons in the region of
7.15–7.50 ppm in these complexes, which were absent in the ligands of thiosemicarba-
zone. The latter conformation of the complex formation is in agreement with Altaf
et al. [30].
13
Compared to the free ligands, there was an upfield shift in C spectra of these
complexes for thiocarbonyl carbon signal with the coordination of metal to the ligand
through sulfur atom. The presence of p-tolyl methyl resonance in the region of
2
1.26–21.42 ppm distinguished the coordination of phosphine ligand to these com-
1
3
plexes. The C NMR signal for aromatic group of diphenyl(p-tolyl)phosphine was dis-
covered in the form of doublet, specifically for C(-P), C-2 and C-3. Meanwhile, C-4
appeared as singlet. These results are indeed proven to be observed in other previous
related study [38].
The chemical shifts in P{ H} NMR for all silver(I) complexes displayed one sharp
singlet peak, which were attributed to diphenyl(p-tolyl)phosphine. This one peak con-
firmed the chemical environment of phosphorus atoms is chemically equivalent, hence
31
1

1
0
S. N. A. ABDUL HALIM ET AL.
Figure 4. EDX spectra of complexes 1–5.
the geometry around silver(I) ion should be tetrahedral and not square planar [39].
Compared to its free phosphine ligand, the resonance of phosphorus in these com-
plexes shifted downfield, which confirmed the coordination of metal to ligand.
Considering the limited studies on silver(I) diphenyl(p-tolyl)phosphine complex, the
31
1
obtained chemical shifts of P{ H} for these complexes were compared to the
observed PPh values [30]. It should be noted that the observed values were rather
3
similar and conclusively, the obtained results are in agreement with one another.
3.1.3. EDX analysis
The obtained complexes were subjected to EDX analysis to validate the presence of
metal in the compound, as shown in Figure 4. The EDX analysis showed the presence
of silver metal and other components that are present in all these synthesized com-
plexes. Hence, this indicated successful metal–ligand complexation.
3.1.4. X-ray diffraction analysis
Several techniques were applied to crystallize these complexes to obtain suitable sin-
gle-crystal for analysis including the previous successful mentioned methods [30, 38,
40]. Regrettably, to date, none of these methods were able to generate crystals that
are suitable for single-crystal X-ray diffraction.
Therefore, PXRD has been carried out to study the degree of crystallinity of the
ꢁ
metal complexes by observing the diffraction pattern in the range of 5–40 (2h).
Figure 5 shows the broad peaks in low intensities for the synthesized silver complexes
2
, 3, 4 and 5 indicating their amorphous nature. Meanwhile, silver complex 1 displays
crystalline nature based on the diffraction pattern as shown in Figure 6. Although it

JOURNAL OF COORDINATION CHEMISTRY
11
Figure 5. PXRD pattern of silver complexes 2–5.
1
L1
5
10
15
20
25
30
35
40
Figure 6. PXRD pattern of silver complex 1.
was crystalline, unfortunately, the data were not sufficient to be analyzed by the sin-
gle-crystal X-ray diffraction.
3.2. Antiproliferative activity
Transition metals such as silver have been long regarded as antimicrobial agents but
their potential in cancer therapeutics has been underexplored [41, 42]. The in vitro
anticancer activity of silver was previously reported [43]. Essentially, the advantage of
silver is that it has lower toxicity compared to other metals such as platinum (e.g. cis-
platin) [44]. Meanwhile, extensive antiproliferative activity of thiosemicarbazones was
found on different tumor cell lines and display common features of other compounds

1
2
S. N. A. ABDUL HALIM ET AL.
TABLE 1. The antiproliferative activities of silver complexes 1–5 (IC50 in mM).
Silver complex
MDA-MB-231
HT-29
MCF-7
1
2
3
4
5
4.45 ± 1.88
3.26 ± 1.37
4.66 ± 1.53
2.55 ± 0.67
3.68 ± 1.63
2.84 ± 0.27
2.83 ± 0.32
3.11 ± 0.56
2.20 ± 0.60
2.52 ± 0.68
3.89 ± 2.11
4.53 ± 1.86
3.74 ± 1.29
3.80 ± 1.88
3.20 ± 1.55
TABLE 2. The antimalarial, cytotoxicity (EC50 in mM) and selectivity index (SI) of silver com-
plexes 1–5.
Silver complex
HRPII
Normal MDBK
Selectivity index (SI)
1
2
3
4
5
1.45 ± 0.31
1.16 ± 0.32
2.02 ± 1.07
1.69 ± 1.54
0.75 ± 0.42
0.64 ± 0.06
0.58 ± 0.08
0.51 ± 0.02
0.54 ± 0.02
0.77 ± 0.16
0.44
0.50
0.25
0.32
1.03
with carcinogenic potency [45, 46]. On the other hand, it was demonstrated that silver
phosphine compounds exerted in vitro antiproliferative effect [47, 48]. Based on IC50
values, all the synthesized compounds in this study displayed significantly high poten-
tial as antiproliferative agents for breast, MDA-MB-231, MCF-7 and colorectal HT-29
cell lines (Table 1). Complex 4 which consists of methoxy and hydroxyl group was
found to have the best activity for colon cancer, HT-29 with IC =2.20 ± 0.60 lM and
50
one of the breast cancer cell lines which is MDA-MB-231 with IC =2.55 ± 0.67 lM as
50
compared to the other complexes. Meanwhile, breast cancer MCF-7 shows the best
result with complex 5 with IC =3.20 ± 1.55 lM in the presence of bromine, hydroxyl
50
and methoxy moieties. In comparison with clinically used metal compounds such as
cisplatin, these compounds showed greater antiproliferative effect toward MDA-MB-
2
31 (IC =25.28 mM) [49], MCF-7 (IC =35 mM) [50] and HT-29 (IC =5.28 mM) [51].
50 50 50
Thus, in-depth studies on these compounds as metallotherapeutic agents for cancer
diseases are required.
3
.3. Antiplasmodial activity
The obtained EC50 values of the complexes are shown in Table 2. All the complexes
–5 showed significantly promising antiplasmodial activity with the obtained EC50 val-
1
ues ranged between 0.75 and 2.02 lM. Among these silver(I) complexes, 5 was found
to be the most potent with the presence of hydroxyl, methoxy and bromine moieties
in the aromatic ring of thiosemicarbazone. On the contrary, the absence of bromine
(in 3) and methoxy (in 4) in the aromatic ring demonstrated almost two-fold reduction
in the antiplasmodial activity. However, the antiplasmodial activity was maintained for
complexes 1 and 2 with the presence of either one or two hydroxyl groups in the aro-
matic ring of thiosemicarbazone. In comparison with the commercialized antimalarials,
the EC50 values of the silver complexes 1–5 were higher as most of the antimalarials
such as pyrimethamine (antifolate), chloroquine (4-aminoquinoline) and artemisinin
derivatives (endoperoxides) scored EC50 values lower than 0.1 mM against the drug-
sensitive P. falciparum and more than 0.1 mM against the drug-resistant P. falciparum
[52]. Nevertheless, the importance of each functional group in enhancing the

JOURNAL OF COORDINATION CHEMISTRY
13
selectivity of silver complexes was unclear, which should be promptly addressed in
future studies.
To know the safety of these complexes, the cytotoxicity against MDBK cells was fur-
ther examined. The selectivity indices were calculated (Table 2) for all silver(I) com-
plexes which revealed low SI values (<1). In other words, lack of selectivity was
demonstrated as the inhibitory activity against the P. falciparum parasite was lower
than the normal mammalian cell line, MDBK cells. Studies on silver compounds against
malaria have been limited. A related study conducted in 1975 discovered that the
splenectomized mice with the infection of P. berghei were cured when silver sulfadia-
zine was administered orally and subcutaneously in doses not exceeding 1050 mg/kg/
day for 5 days to these CF-1 mice [53]. Following in 2013, another study reported that
silver(I) complexes with N-heterocyclic carbine ligands showed promising in vitro anti-
plasmodial activity but with further testing, these compounds exhibited strong hemo-
lytic properties on parasite culture even with the weakest doses, which indicated toxic
activity [54]. In short, this study revealed that the toxicity of silver was debatable des-
pite the recognition of silver as one of the least toxic metals [55].
4. Conclusion
A series of silver(I) complexes with mixed-ligands of thiosemicarbazone derivatives and
diphenyl(p-tolyl)phosphine were synthesized. The silver atom was coordinated to the
sulfur atom of thiosemicarbazone and phosphorus atom of diphenyl(p-tolyl) phos-
phine. Moreover, the obtained spectroscopic data agreed with suggested structures.
Evaluation of these complexes against three cancer cell lines revealed potential in the
antiproliferative activity as these complexes yielded low IC50 values (2.2–4.6 lM) in all
cell lines. The antiplasmodial activity of these complexes against chloroquine-resistant
P. falciparum parasite were also examined, which demonstrated good activity, but
unfortunately they were not selective. Further modification on the structural frame of
thiosemicarbazone or phosphine may improve the SI value.
Acknowledgement
This work was supported by the Research Grant University of Malaya (UMRG) - AFR (Frontier
Science), Grant No. RG357-15AFR and Fundamental Research Grant Scheme (FRGS), Grant No.
FP047-2014A.
Disclosure statement
No potential conflict of interest was reported by the author.
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