Tin(IV) compounds of tridentate thiosemicarbazone Schiff bases: Synthesis, characterization, in-silico analysis and in vitro cytotoxicity

Article abstract of DOI:10.1016/j.poly.2020.114729

Twelve tin(IV) compounds (5–16) derived from four tridentate thiosemicarbazone Schiff bases of 4-methyl-3-thiosemicarbazide with 2-hydroxy-3-methoxybenzaldehyde (1, 2) and 4-phenyl-3-thiosemicarbazide with 2,3-dihydroxybenzaldehyde (3, 4) of the general f

Full text of DOI:10.1016/j.poly.2020.114729

Journal Pre-proofs
Tin(IV) compounds of tridentate thiosemicarbazone Schiff bases: synthesis,
characterization, in-silico analysis and in vitro cytotoxicity
Enis Nadia Md Yusof, Alister J. Page, Jennette A. Sakoff, Michela I. Simone,
Abhi Veerakumarasivam, Edward R.T. Tiekink, Thahira B.S.A. Ravoof
PII:
S0277-5387(20)30386-7
DOI:
Reference:
https://doi.org/10.1016/j.poly.2020.114729
POLY 114729
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
2 July 2020
28 July 2020
30 July 2020
Please cite this article as: E.N.M. Yusof, A.J. Page, J.A. Sakoff, M.I. Simone, A. Veerakumarasivam, E.R.T.
Tiekink, T.B.S. Ravoof, Tin(IV) compounds of tridentate thiosemicarbazone Schiff bases: synthesis,
characterization, in-silico analysis and in vitro cytotoxicity, Polyhedron (2020), doi: https://doi.org/10.1016/
j.poly.2020.114729
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Tin(IV) compounds of tridentate thiosemicarbazone Schiff bases:
synthesis, characterization, in-silico analysis and in vitro
cytotoxicity
Enis Nadia Md Yusof 1,2,3, Alister J. Page 2,*, Jennette A. Sakoff 4, Michela I. Simone 2,5
,
Abhi Veerakumarasivam 6,7, Edward R. T. Tiekink 8 and Thahira B. S. A. Ravoof 1,9,
*
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia;
2
3
4
Discipline of Chemistry, School of Environmental and Life Sciences, University of
Newcastle, University Drive, Callaghan, NSW 2308, Australia;
Chemistry Section, School of Distance Education, Universiti Sains Malaysia, 11800,
Minden, Penang, Malaysia
Experimental Therapeutics Group, Department of Medical Oncology, Calvary Mater
Newcastle Hospital, Edith Street, Waratah NSW 2298, Australia;
Priority Research Centre for Drug Development, University of Newcastle, University
Drive, Callaghan, NSW 2308, Australia
5
6
Department of Biological Sciences, School of Science and Technology, Sunway
University, No. 5 Jalan Universiti, 47500 Bandar Sunway, Selangor Darul Ehsan,
Malaysia;
7
8
Medical Genetics Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
Research Centre for Crystalline Materials, School of Science and Technology, Sunway
University, No. 5 Jalan Universiti, 47500 Bandar Sunway, Selangor Darul Ehsan,
Malaysia;
Foundry of Reticular Materials for Sustainability (FORMS), Materials Synthesis and
Characterization Laboratory, Institute of Advanced Technology, Universiti Putra
Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia;
9
* Correspondence: thahira@upm.edu.my (T.B.S.A.R.), alister.page@newcastle.edu.au
(A.J.P.)
Abstract
Twelve tin(IV) compounds (5-16) derived from four tridentate thiosemicarbazone Schiff
bases of 4-methyl-3-thiosemicarbazide with 2-hydroxy-3-methoxybenzaldehyde (1, 2) and 4-
phenyl-3-thiosemicarbazide with 2,3-dihydroxybenzaldehyde (3, 4) of the general formulae
[R2Sn(Ln)] and [Sn(Ln)2] (where R = Ph or Me; Ln = 1, 2, 3 and 4) were synthesized and
characterized by elemental analysis, IR, UV-vis, mass spectrometry and multinuclear NMR
(1H, 13C and 119Sn) spectroscopy. X-ray crystallographic data was obtained for 11′, a 2:1 co-
crystal between Ph2Sn(L2) (11) and 3-methoxysalicylaldehyde azine, and Me2Sn(L2) (12)

where L2H2 is 2-(2-hydroxy-3-methoxybenzylidene)-N-phenylhydrazinecarbothioamide. The
analysis revealed distinct coordination geometries for 11 and 12 approaching trigonal-
bipyramidal. In the crystal of 11′, supramolecular dimers arising from amine-N–
H…S(thiolate) hydrogen bonding and {…HNCS}2 synthons are evident; π(chelate
ring)…π(oxidobenzylidene) stacking is also apparent. In the crystal of 12, supramolecular,
helical chains are generated by a combination of amine-N–H…O(phenoxide) hydrogen
bonding and Sn…S secondary bonding. The cytotoxic activity of the compounds against a
panel of ten cancer cell lines, [HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast),
A2780 (ovarian), H460 (lung), A431 (skin), DU145 (prostate), BE2-C (neuroblastoma) and
MIA (pancreas), and one normal cell line, MCF-10A (normal breast)] were investigated. The
thiosemicarbazone Schiff bases 1 and 4 as well as the diphenyltin(IV) compounds showed a
strong ability to inhibit the growth of cancer cells, with particular selectivity against HT29,
MCF-7, A2780, A431, BE2-C, SJ-G2 and MIA cell lines. The structure-activity relationship
of all these compounds were studied by evaluating the effect of alkyl and aryl groups
attached on the thiosemicarbazone backbone, the methoxy/hydroxyl groups present at the
meta-position of the phenyl ring and alkyl or aryl groups bound to the tin center.
1.
Introduction
Schiff bases that contain nitrogen, sulfur and oxygen as donor atoms, such as
thiosemicarbazones, semicarbazones and dithiocarbazates, and their metal complexes have
been of interest since 1946 [1] owing to their remarkable biological and pharmacological
properties, especially antitumor, antibacterial, antiviral, anti-tuberculosis, antifungal and
antimalarial activities [2], which are altered when small changes to the structures (e.g.,
changing of a functional group) are applied. Thiosemicarbazones are considered as privileged
ligands due to their potential donor atoms, π-delocalization and configurational flexibility,
which can produce various metal-ligand linkages [3]. Compounds having thiol groups have
also been proven to inhibit the ribonucleotide reductase (RR) enzyme, used in DNA
synthesis. Hence, by inhibiting or blocking the function of the RR enzyme, DNA replication
and synthesis of tumor cells can be controlled or prevented [4]. In many cases, complexation
with metal ions increases the bioactivity of the compounds, suggesting that coordination of
such ligands enhances their cytotoxicity.

Although much research has been devoted to the synthesis, characterization and biological
properties of ligands coordinated to transition metal ions, tin-based compounds have received
considerably less attention because of ecotoxicology effects at the biochemical, cellular and
organism levels [5]. In recent years, tin-based compounds have been of great interest because
of their ability to form stable bonds with hetero donor atoms, for instance nitrogen, sulfur and
oxygen atoms [6–15]. Tin(IV) compounds are now well-known for their applications as
cytotoxic, biocidal, antibacterial and antifungal agents [6,16–19]. Many studies have reported
the antimicrobial activities of tin(IV) compounds derived from thiosemicarbazone Schiff
bases. In particular, compounds containing the 3-methoxysalicylaldehyde thiosemicarbazone
Schiff base were tested for their in vitro cytotoxicity against human acute lymphoblastic
leukemia (Jurkat cells) [20]. The data indicated increasing potency in the order
dimethyltin(IV) < diphenyltin(IV) < dibutyltin(IV) compounds, with IC50 values of 260, 130
and 50 μM, respectively. This suggested that the cytotoxicity of dialkytin(IV) compounds
increased with the increase in the length of the organic chain. The cytotoxicity of diphenyl-
and dimethyltin(IV) compounds of pyruvic acid thiosemicarbazone have also been
investigated against human breast adenocarcinoma (MCF-7), bladder carcinoma (T24), non-
small cell lung carcinoma (A-549) and mouse fibroblast (L-929) cell lines, with IC50 values
in the range 0.43 to 19.73ꢀμΜ. The diphenyltin(IV) compound was most potent against T-24
cells with an IC50 value of 0.43ꢀμΜ, where it exhibited 96-fold better activity than cisplatin
[21].
Recently, tin(IV) compounds derived from the tridentate 2-hydroxy-5-methoxybenzaldehyde-
N(4)-methylthiosemicarbazone exhibited higher anticancer activity against the human
colorectal (HTC-116) cell line as compared to the reference drug 5-fluorouracil [8]. The
significant biological activity of the tin(IV) compounds were influenced by the types of
organo group attached to the tin center, diffusion, lipophilicity and steric effects [8,22–24].
As a continuation of our research on tridentate ONS Schiff bases and their tin(IV) compounds
[25,26], we report herein the preparation, spectroscopic characterization and bioactivity of
tin(IV) compounds (5-16) containing 2-hydroxy-3-methoxybenzyl- and 2,3-dihydroxybenzyl-
derived thiosemicarbazone Schiff bases (1-4). The diphenyl- (5, 8, 11, and 14) and
dimethyltin(IV) (6, 9, 12, and 15) compounds exhibited a penta-coordinated geometry,
whereas tin(IV) compounds were coordinated to two molecules of thiosemicarbazone Schiff

bases (7, 10, 13, and 16) suggesting a hexa-coordinated geometry, according to 119Sn NMR
analysis.
The experimental data (FTIR, electronic and single crystal X-ray diffraction analysis) of the
synthesized compounds were in excellent agreement with the computed data, as evidenced by
density functional theory (DFT) calculations using the B3LYP/LanL2DZ/6-311G(d,p) level
of theory. The cytotoxicity of all the compounds against a panel of ten cancer cell lines and
one normal cell line was investigated. The results indicated that small differences in the
structures of the compounds (Figure 1) had significant effects on their activity. These studies
provide fundamental data for future drug design development in cancer treatment.
Figure 1. The structure of the thiosemicarbazone Schiff bases 1-4.
2.
Experimental
2.1. Physical measurements
Melting points were determined using an Electrothermal digital melting point apparatus. IR
spectra were recorded using the Perkin Elmer Spectrum 100 with Universal ATR Polarization
in the range 4000–280 cm−1. C, H and N elemental analyses were carried out using a LECO
CHNS-932 instrument. Molar conductivities of 10−3 M solutions of the organotin(IV)
compounds in DMSO were measured at 27 °C using a Jenway 4310 conductivity meter fitted
with a dip-type cell with a platinized electrode. Electronic spectra were recorded on a
1
Shimadzu UV-1650 PC recording spectrophotometer (1000–200 nm). H and 13C NMR
spectra were recorded using an NMR JNM ECA400 spectrometer. 119Sn NMR were
measured using a Bruker BioSpin Avance III (600MHz) spectrometer. The mass spectra were
recorded using a Shimadzu GC-MS QP2010Plus mass spectrometer.

2.2. Materials
All solvents and reagents were of analytical reagent grade and used without further
purification.
Chemicals:
hydroxide,
4-methyl-3-thiosemicarbazide,
4-phenyl-3-thiosemicarbazide, potassium
2,3-dihydroxybenzaldehyde,
2-hydroxy-3-methoxybenzaldehyde,
dichlorodiphenyltin(IV), dichlorodimethyltin(IV), tin(II) chloride. Solvents: absolute ethanol,
99.8%, ethanol, 95%, methanol, dimethylsulfoxide.
2.3. Syntheses
2.3.1. Syntheses of 2-(2-hydroxy-3-methoxybenzylidene)-N-methylhydrazinecarbothioamide
(1) and 2-(2-hydroxy-3-methoxybenzylidene)-N-phenylhydrazinecarbothioamide (2)
Compounds 1 and 2 were prepared according to the procedure described in the literature
[27,28] with some modifications.
4-Methylthiosemicarbazide (1.05 g, 10 mmol)/4-phenylthiosemicarbazide (1.67 g, 10 mmol)
was dissolved in methanol (40 cm3) with stirring and heating (40 °C) over a period of 30
minutes. 3-Methoxysalicylaldehyde (1.52 g, 10 mmol) in 10 cm3 of methanol was added to
the thiosemicarbazide solution and stirred at room temperature for 4 h. Upon cooling, a
crystalline product began to form which was filtered, washed with cold methanol and dried in
a desiccator over anhydrous silica gel.
2-(2-Hydroxy-3-methoxybenzylidene)-N-methylhydrazinecarbothioamide (1)
Colorless crystalline solid. Yield: 92 %. Melting point: 242-243 °C. Analysis calculated for
C10H13N3O2S: C, 50.19; H, 5.48; N, 17.56. Found: C, 49.93; H, 5.38; N, 17.22 %. FT-IR
1
(ATR, cm-1): 3337 v(OH), 3304 v(NH), 1610 v(C=N), 1109 v(N-N), 1037 v(C=S). H NMR
(DMSO-d6) δ (ppm.): 2.99 (d, 3H, CH3), 3.79 (s, 3H, O-CH3), 6.93-7.54 (m, 3H, Ar-H), 8.37
(s, 1H, CH), 8.39 (q, 1H, C(=S)-NH), 9.18 (s, 1H, OH), 11.42 (s, 1H, NH-N). 13C NMR
(DMSO-d6) δ (ppm.): 30.8 (N-CH3), 56.4 (O-CH3), 113.1, 118.2, 119.2, 121.2, 139.6, 146.7
(Ar-C), 148.4 (C=N), 177.6 (C=S).
2-(2-Hydroxy-3-methoxybenzylidene)-N-phenylhydrazinecarbothioamide (2)
White crystalline solid. Yield: 90 %. Melting point: 209-210 °C. Analysis calculated for
C15H15N3O2S: C, 59.78; H, 5.02; N, 13.94. Found: C, 59.99; H, 5.15; N, 13.80 %. FT-IR
1
(ATR, cm-1): 3300 v(NH), 1609 v(C=N), 1103 v(N-N), 908 v(C=S). H NMR (DMSO-d6) δ

(ppm.): 3.80 (s, 3H, O-CH3), 6.77-7.69 (m, 8H, Ar-H), 9.26 (s, 1H, CH), 10.02 (s, 1H, OH),
11.78 (s, 1H, NH-N). 13C NMR (DMSO-d6) δ (ppm.): 56.3 (O-CH3), 113.4, 118.8, 119.5,
121.2, 125.4, 126.4, 128.6, 139.7, 140.4, 146.6 (aromatic-C), 148.6 (C=N), 176.1 (C=S).
2.3.2. Syntheses of 2-(2,3-dihydroxybenzylidene)-N-methylhydrazinecarbothioamide (3) and
2-(2,3-dihydroxybenzylidene)-N-phenylhydrazinecarbothioamide (4)
Compounds 3 and 4 were prepared according to the procedure described in the literature
[29,30]. A 25 cm3 ethanolic solution of 2,3-dihydroxybenzaldehyde (1.38 g, 10 mmol) was
added to an equimolar ethanolic solution (10 cm3) of 4-methyl-3-thiosemicarbazide (1.05 g,
10 mmol)/4-phenyl-3-thiosemicarbazide (1.67 g, 10 mmol). The mixture was stirred for 3
hours at room temperature and the title compound was filtered. Each of the title compounds
was then recrystallized from methanol to remove all the impurities and kept in desiccator
over anhydrous silica gel.
2-(2,3-Dihydroxybenzylidene)-N-methylhydrazinecarbothioamide (3)
Pale yellow solid. Yield: 83 %. Melting point: 231-232 °C. Analysis calculated for
C9H11N3O2S: C, 47.99; H, 4.92; N, 18.65. Found: C, 46.88; H, 4.85; N, 18.38 %. FT-IR
1
(ATR, cm-1): 3418 v(OH), 3140 v(NH), 1601 v(C=N), 1112 v(N-N), 1035 v(C=S). H NMR
(DMSO-d6) δ (ppm.): 3.00 (d, 3H, CH3), 6.67-7.38 (m, 3H, Ar-H), 9.02 (s, 1H, CH), 8.37,
8.38 (2 x s, 2H, OH), 9.49 (s, 1H, C(=S)-NH), 11.40 (s, 1H, NH-N). 13C NMR (DMSO-d6) δ
(ppm.): 31.3 (N-CH3), 116.7, 117.4, 119.4, 121.5, 140.1, 145.6 (Ar-C), 146.0 (C=N), 178.0
(C=S).
2-(2,3-Dihydroxybenzylidene)-N-phenylhydrazinecarbothioamide (4)
Pale yellow solid. Yield: 70 %. Melting point: 215-216 °C. Analysis calculated for
C14H13N3O2S: C, 58.52; H, 4.56; N, 14.62. Found: C, 57.61; H, 4.69; N, 14.67 %. FT-IR
1
(ATR, cm-1): 3443 v(OH), 3129 v(NH), 1597 v(C=N), 1047 v(N-N), 1029 v(C=S). H NMR
(DMSO-d6) δ (ppm.): 6.67-7.57 (m, 8H, Ar-H), 8.49 (s, 1H, CH), 8.96, 9.52 (2 x s, 2H, OH),
10.01 (s, 1H, C(=S)-NH), 11.75 (s, 1H, NH-N). 13C NMR (DMSO-d6) δ (ppm.): 117.1, 117.9,
119.5, 121.3, 125.6, 126.0, 128.5, 139.6, 141.3, 145.9 (Ar-C), 146.0 (C=N), 176.0 (C=S).

2.3.3. General procedure for the synthesis of tin(IV) compounds derived from 1 and 3
To a solution of 1 (0.24 g, 1 mmol)/ 3 (0.23 g, 1 mmol) in 100 cm3 of methanol, KOH (0.11
g, 2 mmol) was added and the mixture was stirred and heated for 30 minutes in methanol.
Then, 1 mmol of the tin precursor (Ph2SnCl2 (0.34 g)/ Me2SnCl2 (0.22 g)/ SnCl2 (0.19 g)) was
added to the mixture and refluxed for 2 hours under nitrogen. The mixture was filtered while
hot and then the filtrate was placed in a freezer until a bright yellow solid formed. The solid
residue obtained was recrystallized from methanol.
Diphenyltin(IV) compound of 2-(2-hydroxy-3-methoxybenzylidene)-N-methylhydrazine
carbothioamide (5)
Bright yellow solid. Yield: 74 %. Melting point: 138-139 °C. Analysis calculated for
C22H21N3O2SSn: C, 51.79; H, 4.15; N, 8.24. Found: C, 51.03; H, 4.23; N, 8.16 %. FT-IR
1
(ATR, cm-1): 3299 v(N-H), 1596 v(C=N), 1066 v(N-N), 973 v(C=S). H NMR (CDCl3) δ
(ppm.): 3.01 (d, 3H, N-CH3), 3.96 (s, 3H, O-CH3), 7.96 (s, 1H, CH), 7.36-8.12 (m, 13H, Ar-
H), 8.59 (s, 1H, NH). 13C NMR (CDCl3) δ (ppm.): 29.8 (NH-CH3), 56.5 (O-CH3), 115.7,
116.7, 117.2, 125.1, 128.6, 129.9, 135.9, 142.5, 151.6 (Ar-C), 157.1 (C=N), 160.3 (S-C-S).
119Sn NMR (CDCl3) δ (ppm.): -236.
Dimethyltin(IV) compound of 2-(2-hydroxy-3-methoxybenzylidene)-N-methylhydrazine
carbothioamide (6)
Bright yellow solid. Yield: 42 %. Melting point: 164-168 °C. Analysis calculated for
C12H17N3O2SSn: C, 37.33; H, 4.44; N, 10.88. Found: C, 39.00; H, 4.76; N, 11.00 %. FT-IR
1
(ATR, cm-1): 3222 v(N-H), 1590 v(C=N), 1066 v(N-N), 973 v(C=S). H NMR (CDCl3) δ
(ppm.): 0.91 (s, 6H, Sn-CH3), 2.97 (d, 3H, N-CH3), 3.94 (s, 3H, O-CH3), 7.26 (s, 1H, CH),
13
6.66-6.87 (m, 3H, Ar-H), 8.55 (s, 1H, NH). C NMR (CDCl3) δ (ppm.): 8.7 (Sn-CH3), 31.3
(NH-CH3), 56.4 (O-CH3), 115.7, 115.8, 117.8, 118.5, 125.8, 151.3 (Ar-C), 156.5 (C=N),
178.0 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): -154.
Tin(IV)
compound
of
2-(2-hydroxy-3-methoxybenzylidene)-N-methylhydrazine
carbothioamide (7)
Compound 7 was prepared following the same procedure as described for 5, using 1 (0.48 g,
2 mmol). Bright yellow solid. Yield: 31 %. Melting point: 118-119 °C. Analysis calculated
for C20H22N6O4S2Sn: C, 40.49; H, 3.74; N, 14.17. Found: C, 40.50; H, 3.33; N, 14.17 %. FT-

IR (ATR, cm-1): 3308 v(N-H), 1590 v(C=N), 1066 v(N-N), 973 v(C=S). 1H NMR (DMSO-d6)
δ (ppm.): 2.36 (d, 6H, N-CH3), 3.82 (s, 6H, O-CH3), 7.60 (s, 2H, CH), 6.67-7.31 (m, 6H, Ar-
13
H), 8.88 (s, 2H, NH). C NMR (DMSO-d6) δ (ppm.): 19.3 (NH-CH3), 56.7 (O-CH3), 105.4,
116.7, 117.4, 126.5, 128.6, 129.0, 130.7, 130.8, 134.9, 149.7, 151.8, 158.3 (Ar-C), 163.7
(C=N), 170.6 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): -354.
Diphenyltin(IV)
compound
of
2-(2,3-dihydroxybenzylidene)-N-methylhydrazine
carbothioamide (8)
Yellow solid. Yield: 49 %. Melting point: 186-192 °C. Analysis calculated for
C21H19N3O2SSn: C, 50.83; H, 3.86; N, 8.47. Found: C, 48.06; H, 4.27; N, 8.02 %. FT-IR
1
(ATR, cm-1): 1593 v(C=N), 1006 v(N-N), 953 v(C=S). H NMR (DMSO-d6) δ (ppm.): 3.00
13
(s, 3H, NH-CH3), 8.57 (s, 1H, CH), 6.37-8.20 (m, 13H, Ar-H), 11.27 (s, 1H, NH). C NMR
(DMSO-d6) δ (ppm.): 30.9 (NH-CH3), 112.8, 113.7, 115.7, 118.4, 128.3, 129.2, 136.0, 136.1,
141.1, 145.4, 153.3 (aromatic-C), 154.3 (C=N), 177.0 (S-C-S). 119Sn NMR (DMSO-d6) δ
(ppm.): -227.
Dimethyltin(IV)
compound
of
2-(2,3-dihydroxybenzylidene)-N-methylhydrazine
carbothioamide (9)
Yellow solid. Yield: 32 %. Melting point: 223-225 °C. Analysis calculated for
C11H15N3O2SSn: C, 35.51; H, 4.06; N, 11.29. Found: C, 35.87; H, 3.86; N, 11.35 %. FT-IR
1
(ATR, cm-1): 1596 v(C=N), 1006 v(N-N), 951 v(C=S). H NMR (DMSO-d6) δ (ppm.): 0.62
(s, 6H, Sn-CH3), 3.00 (d, 3H, N-CH3), 6.32-7.10 (m, 3H, Ar-H), 8.38 (s, 1H, CH), 11.30 (s,
1H, NH). 13C NMR (DMSO-d6) δ (ppm.): 6.8 (Sn-CH3), 30.7 (NH-CH3), 112.6, 113.7, 115.6,
118.6, 140.7, 153.9 (Ar-C), 155.0 (C=N), 177.2 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): -
123.
Tin(IV) compound of 2-(2,3-dihydroxybenzylidene)-N-methylhydrazinecarbothioamide (10)
Compound 10 was prepared following the same procedure as described for 5, using 1 (0.46 g,
2 mmol). Orange solid. Yield: 79 %. Melting point: >300 °C. Analysis calculated for
C18H18N6O4S2Sn: C, 38.25; H, 3.21; N, 14.87. Found: C, 36.62; H, 2.92; N, 14.21 %. FT-IR
(ATR, cm-1): 1585 v(C=N), 993 v(N-N), 951 v(C=S). 1H NMR (DMSO-d6) δ (ppm.): 2.83 (s,
3H, N-CH3), 6.65-7.52 (m, 6H, Ar-H), 8.34 (s, 1H, OH), 8.77 (s, 1H, CH), 11.27 (s, 1H, NH).

13C NMR (DMSO-d6) δ (ppm.): 30.6 (NH-CH3), 113.8, 114.3, 116.7, 118.9, 140.0. 150.1
(Ar-C), 150.6 (C=N), 177.5 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): 519.
2.3.4. General procedure for the syntheses of tin(IV) compounds derived from 2
Compound 2 (0.30 g, 1 mmol) was dissolved in methanol (100 cm3) and Et3N (0.28 cm3, 2
mmol) was added dropwise to the solution of 2. The mixture was heated (40 °C) for about 2
hours until the solution was reduced by half. Next 1 mmol of the tin precursor (Ph2SnCl2
(0.34 g)/Me2SnCl2 (0.22 g)/ SnCl2 (0.19 g)) was added to the mixture. The mixture was
refluxed under nitrogen for about 2 hours and filtered while hot to remove the triethylamine
salt and the filtrate was kept at room temperature until a bright yellow product formed.
Diphenyltin(IV) compound of 2-(2-hydroxy-3-methoxybenzylidene)-N-phenylhydrazine
carbothioamide (11)
Yellow crystals. Yield: 73 %. Melting point: 205-207 °C. Analysis calculated for
C27H23N3O2SSn: C, 56.67; H, 4.05; N, 7.34. Found: C, 57.53; H, 4.26; N, 7.87%. FT-IR
1
(ATR, cm-1): 3331 v(N-H), 1586 v(C=N), 1075 v(N-N), 832 v(C=S). H NMR (CDCl3) δ
(ppm.): 3.96 (s, 3H, O-CH3), 7.99 (s, 1H, CH), 6.69-7.56 (m, 18H, Ar-H), 8.70 (s, 1H, NH).
13C NMR (CDCl3) δ (ppm.): 56.7 (O-CH3), 115.2, 116.2, 116.9, 119.4, 120.7, 123.4, 124.1,
125.4, 128.7, 128.9, 130.0, 135.9, 139.3, 142.1, 148.3, 149.7, 151.7 (Ar-C), 162.5 (C=N),
164.8 (S-C-S). 119Sn NMR (CDCl3) δ (ppm.): -242.
Dimethyltin(IV) compound of 2-(2-hydroxy-3-methoxybenzylidene)-N-phenylhydrazine
carbothioamide (12)
Yellow crystals. Yield: 64 %. Melting point: 176-179 °C. Analysis calculated for
C17H19N3O2SSn: C, 45.56; H, 4.27; N, 9.38. Found: C, 45.86; H, 4.40; N, 7.08%. FT-IR
1
(ATR, cm-1): 3294 v(N-H), 1577 v(C=N), 1059 v(N-N), 824 v(C=S). H NMR (CDCl3) δ
(ppm.): 3.85 (s, 3H, O-CH3), 7.53 (s, 1H, CH), 6.69-7.32 (m, 8H, Ar-H), 8.65 (s, 1H, NH).
13C NMR (CDCl3) δ (ppm.): 6.5 (Sn-CH3), 56.2 (O-CH3), 115.3, 116.6, 116.7, 120.5, 123.3,
125.4, 128.9, 139.4, 151.3, 156.8 (Ar-C), 162.5 (C=N), 163.9 (S-C-S). 119Sn NMR (CDCl3) δ
(ppm.): -115.
Tin(IV)
compound
of
2-(2-hydroxy-3-methoxybenzylidene)-N-phenylhydrazine
carbothioamide (13)

Compound 13 was prepared following the same procedure as described for 11, using 2 (0.60
g, 2 mmol). Yellow solid. Yield: 50 %. Melting point: 293-294 °C. Analysis calculated for
C30H26N6O4S2Sn: C, 50.23; H, 3.65; N, 11.71. Found: C, 49.85; H, 3.73; N, 11.60%. FT-IR
(ATR, cm-1): 3303 v(N-H), 1581 v(C=N), 1063 v(N-N), 824 v(C=S). 1H NMR (DMSO-d6) δ
(ppm.): 3.58 (s, 6H, O-CH3), 9.08 (s, 2H, CH), 6.80-7.73 (m, 16H, Ar-H), 9.70 (s, 2H, NH).
13C NMR (DMSO-d6) δ (ppm.): 56.4 (O-CH3), 117.9, 118.2, 121.0, 123.4, 126.8, 129.2,
140.4, 151.4, 154.8 (Ar-C), 160.3 (C=N), 162.2 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): -
451.
2.3.5. General procedure for the synthesis of tin(IV) compounds derived from 4
Compound 4 (0.29 g, 0.001 mol) was dissolved in methanol (100 cm3) and KOH (0.11 g, 2
mmol) was added dropwise to the solution of 4. The mixture was refluxed for about 30
minutes, whereupon the color changed from light yellow to orange. Next 1 mmol of the tin
precursor (Ph2SnCl2 (0.34 g)/Me2SnCl2 (0.22 g)/SnCl2 (0.19 g)) was added to the mixture.
The mixture was refluxed for 6 hours and filtered while hot to remove the triethylamine salt
and the filtrate was kept at room temperature until the product, an orange precipitate, formed.
Diphenyltin(IV)
compound
of
2-(2,3-dihydroxybenzylidene)-N-phenylhydrazine
carbothioamide (14)
Orange solid. Yield: 71 %. Melting point: 133-137 °C. Analysis calculated for
C26H21N3O2SSn: C, 55.94; H, 3.79; N, 7.53. Found: C, 56.30; H, 3.99; N, 7.42%. FT-IR
(ATR, cm-1): 1587 v(C=N), 998 v(N-N), 957 v(S-C-S). 1H NMR (DMSO-d6) δ (ppm.): 6.57-
13
9.52 (m, 18H, Ar-H), 9.87 (s, 1H, CH), 11.69 (s, 1H, NH). C NMR (DMSO-d6) δ (ppm.):
120.5, 120.7, 125.4, 125.5, 125.6, 125.7, 128.5, 128.6, 128.7, 128.8, 128.9, 129.0, 129.1,
129.5, 134.6, 135.2, 135.5, 136.3, 136.4, 136.7, 139.7 (Ar-C), 148.8 (C=N), 175.2 (S-C-S).
119Sn NMR (DMSO-d6) δ (ppm.): -328.
Dimethyltin(IV)
compound
of
2-(2,3-dihydroxybenzylidene)-N-phenylhydrazine
carbothioamide (15)
Orange solid. Yield: 42 %. Melting point: 153-156 °C. Analysis calculated for
C16H17N3O2SSn: C, 44.27; H, 3.95; N, 9.68. Found: C, 44.32; H, 3.72; N, 9.90%. FT-IR
(ATR, cm-1): 1575 v(C=N), 1001 v(N-N), 917 v(S-C-S). 1H NMR (DMSO-d6) δ (ppm.): 0.63
(s, 6H, CH3), 8.51 (s, 1H, CH), 6.33-7.75 (m, 8H, Ar-H), 9.84 (s, 1H, OH). 13C NMR

(DMSO-d6) δ (ppm.): 7.1 (Sn-CH3), 110.2, 114.7, 128.3, 128.4, 128.7, 128.8, 135.3, 152.1,
152.7, 153.6 (Ar-C), 153.8 (C=N), 167.5 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): -103.
Tin(IV) compound of 2-(2,3-dihydroxybenzylidene)-N-phenylhydrazinecarbothioamide (16)
Compound 16 was prepared following the same procedure as described for 14, using 4 (0.58
g, 2 mmol). Orange solid. Yield: 48 %. Melting point: >300 °C. Analysis calculated for
C28H22N6O4S2Sn: C, 48.78; H, 3.22; N, 12.19. Found: C, 48.30; H, 2.97; N, 12.52 %. FT-IR
(ATR, cm-1): 1588 v(C=N), 1001 v(N-N), 939 v(S-C-S). 1H NMR (DMSO-d6) δ (ppm.): 6.66-
13
7.55 (m, 16H, Ar-H), 8.48 (s, 2H), 10.00 (s, 2H, CH), 11.74 (s, 2H, NH). C NMR (DMSO-
d6) δ (ppm.): 117.1, 119.5, 121.2, 125.6, 125.7, 125.9, 126.0, 128.4, 128.5, 128.5, 139.6 (Ar-
C), 145.9, 146.0 (C=N), 175.7, 176.1 (S-C-S). 119Sn NMR (DMSO-d6) δ (ppm.): -541.
2.4. X-ray structure determination
Intensity data for light-yellow crystals of 11′ (0.05 × 0.08 × 0.12 mm) and 12 (0.07 × 0.13 ×
0.18 mm) a were measured at 150 K on an Oxford Diffraction Gemini Eos CCD
diffractometer (Rigaku Oxford Diffraction, United Kingdom) fitted with Mo Kα radiation (λ
= 0.71073 Å). Data reduction and empirical absorption corrections, based on a multi-scan
technique, were applied [31]. The structures were solved by direct methods [32] and refined
on F2 with anisotropic displacement parameters, with C-bound H atoms in the riding model
approximation [33]. The oxygen- and nitrogen-bound H atoms were refined with distance
restraints of O‒H = 0.84 ± 0.01 Å and N‒H = 0.88 ± 0.01 Å, respectively. A weighting
2
2
scheme of the form w = 1/[σ2(Fo ) + (aP)2], where P = (Fo + 2Fc2)/3, was introduced in each
case; for 11′ a = 0.033 and for 12 a = 0.022. The absolute structure of 12 was determined
based on differences in Friedel pairs included in the data set. The molecular structure
diagrams were generated at the 70% probability level by ORTEP for Windows [34] and the
packing diagrams were prepared with DIAMOND [35]. Additional analysis was conducted
with PLATON [36].
Crystal data for 11′: C27H23N3O2SSn, 0.5(C16H16N2O4), M = 722.43, monoclinic, P21/c, a =
14.7977(5), b = 13.0726(4), c = 17.0997(6) Å, β = 105.421(3)°, V = 3188.75(19) Å3, Z = 4,
Dx = 1.505 g cm−3, F(000) = 1468 and μ = 0.912 mm−1. No. reflections measured = 14861
(θmax = 29.4°), no. independent reflections = 7337, no. reflections with I ≥ 2σ(I) = 5378, R
(obs. data) = 0.041 and wR2 (all data) = 0.087. CCDC deposition number: 1975499.

Crystal data for 12: C17H19N3O2SSn, M = 448.10, orthorhombic, P212121, a = 7.7253(2), b =
12.4692(3), c = 18.2692(5) Å, V = 1759.84(8) Å3, Z = 4, Dx = 1.691 g cm−3, F(000) = 896
and μ = 1.585 mm−1. No. reflections measured = 11201 (θmax = 29.3°), no. independent
reflections = 4191, no. reflections I ≥ 2σ(I) = 3900, R (obs. data) = 0.030 and wR2 (all data) =
0.096. CCDC deposition number: 1975500.
2.5. Density Functional Theory (DFT) calculations
DFT calculations were performed using Gaussian09 (Gaussian Inc., Wallingford, CT, USA)
[37] and Gaussview5 (Semichem, Inc., Shawnee Mission, KS, USA) [38] software. The
molecular structures and geometries of the Schiff bases and tin(IV) compounds were fully
optimized using the DFT method with the B3LYP [39,40] hybrid exchange correlation
functional, the LanL2DZ pseudopotential on Sn [41–43] and 6-311G(d,p) Pople basis set for
all other atoms. The initial single crystal X-ray molecular structures and geometries for the
tin compounds in 11 and 12 were used for the DFT calculations, using the same functional
and basis set. Vibrational frequencies were scaled using a scaling factor of 0.9682 [44]. The
electronic stabilities of the optimized geometries were computed using the time-dependent
density functional theory (TD-DFT) formalism [45,46] and included solvation effects
(DMSO) via the polarizable continuum method (PCM) [47–49], using the same basis set.
These DFT calculations were performed in the same way as reported in a previous
publication [25].
2.6. In vitro cytotoxic assay
The cytotoxicity of the tin(IV) compounds against HT29 (colon), U87 and SJ-G2
(glioblastoma), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin), DU145
(prostate), BE2-C (neuroblastoma) and MIA (pancreas) cell lines, and one normal breast cell
line, MCF-10A, were measured using an MTT assay, by the same method as previously
reported [25,50,51].
Cell culture and stock solutions. Stock solutions were prepared as follows and stored at −20
°C: The synthesised compounds and cisplatin were stored as 10 mM solutions in DMSO and
saline solution respectively. All cell lines were cultured in a humidified atmosphere 5% CO2
at 37 °C. The cancer cell lines were maintained in Dulbecco’s modified Eagle’s medium

(DMEM) (Trace Biosciences) supplemented with 10% fetal bovine serum, 10 mM sodium
bicarbonate, penicillin (100 IU/mL), streptomycin (100 μg/mL) and glutamine (4 mM). The
normal cell line, MCF-10A, was cultured in DMEM:F12 (1:1) cell culture media, 5% heat
inactivated horse serum, supplemented with penicillin (50 IU/mL), streptomycin (50 μg/mL),
20mM Hepes, L-glutamine (2 mM), epidermal growth factor (20 ng/mL), hydrocortisone
(500 ng/mL), cholera toxin (100 ng/mL) and insulin (10 μg/mL).
In vitro growth inhibition assay. Cells in logarithmic growth were transferred to 96-well
plates. The cytotoxicity was determined by plating cells in duplicate in 100 μL medium at a
density of 2500–4000 cells/well. On day 0 (24 h after plating), when the cells were in
logarithmic growth, 100 μL medium, with or without the test agent, were added to each well.
After 72 h drug exposure, the growth inhibitory effects were evaluated using an MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and the absorbance was read at
540 nm. Percentage growth inhibition was determined at a fixed drug concentration of 25
μM. A value of 100% was indicative of complete cell growth inhibition. Those analogs
showing appreciable percentage growth inhibition underwent further dose response analysis,
allowing for the calculation of a GI50 value. This value is the drug concentration at which cell
growth is 50% inhibited based on the difference between the optical density values on day 0
and those at the end of the drug exposure [52,53].
3.
Results and discussion
3.1. Synthesis
The synthetic pathways for the Schiff bases (1-4) and their tin(IV) compounds (5-16) are
indicated in Schemes 1 and 2. The Schiff bases were synthesized by the condensation
reaction between 2-hydroxy-3-methoxybenzaldehyde/2,3-dihydroxybenzaldehyde and the
corresponding
thiosemicarbazide
(4-methyl-3-thiosemicarbazide
and
4-phenyl-3-
thiosemicarbazide) in alcoholic solution, which was as previously reported [27,54,55]. The
Schiff bases were then reacted with Ph2SnCl2, Me2SnCl2 and SnCl2 separately, in the
presence of potassium hydroxide (KOH)/triethyamine (Et3N) by conventional methods or
under reflux. The isolated yellow or orange colored tin(IV) compounds were obtained in
acceptable yields (31-79%), however, some were produced in low yields due to their
instability at room temperature. The tin(IV) compounds were soluble in most organic

solvents, especially dimethylsulfoxide (DMSO) and dimethylformamide (DMF). The molar
conductance values of the compounds were in the range 0.88-7.85 Ω-1 cm2 mol-1, which was
well below 25 Ω-1 cm2 mol-1, indicating that all the compounds were non-electrolytic in
nature. This means that no counter ions were present in the outer coordination sphere [56].
R2
HO
R2
S
S
MeOH/EtOH
HO
R1
N
R1
NH2
N
N
N
N
heat and stir
(3-4 hours)
H
H
H
H
O
Scheme 1.
Synthetic pathway to the thiosemicarbazone Schiff bases 1-4.
R2
R1 = CH3, R2 = OCH3 (5)
R1 = CH3, R2 = OH (8)
R1 = C6H5, R2 = OCH3 (11)
R1 = C6H5, R2 = OH (14)
Sn
O
SnPh2Cl2
methanol
S
R1
N
N
N
H
CH3
Sn
R2
R2
R1 = CH3, R2 = OCH3 (6)
R1 = CH3, R2 = OH (9)
R1 = C6H5, R2 = OCH3 (12)
R1 = C6H5, R2 = OH (15)
H3C
S
O
SnMe2Cl2
methanol
HO
S
R1
N
R1
N
N
N
N
N
H
H
H
H
N
N
N
R1
R1 = CH3, R2 = OCH3 (7)
SnCl2
R1 = CH3, R2 = OH
R1 = C6H5, R2 = OCH3 (13)
R1 = C6H5, R2 = OH
(10)
S
R2
O
Sn
methanol
O
(16)
R2
S
R1
N
N
N
H
Scheme 2.
Synthetic pathway of the thiosemicarbazone Schiff bases to the tin(IV)
compounds (5-16)
3.2. Spectroscopic and spectrometric data
3.2.1. FTIR analysis

The experimental and calculated frequencies in the infrared spectra of the thiosemicarbazone
Schiff bases (1-4) and their tin(IV) compounds (5-16) were determined in the range 4000-280
cm-1 and 4000-0 cm-1, respectively. Important infrared vibrations and their assignments are
summarized in Table S1 for both experimental and calculated frequencies. The calculated
frequencies were employed to assign prominent peaks with maximum accuracy, which
resulted in excellent correlation with the experimental data (Figure S1). In the spectra of 1
and 2, the v(OH) band was not observed, which suggested that the v(OH) band overlapped
with the v(N-H) band due to hydrogen bonding (NH...OH) between the two groups [57,58].
The v(OH) band was observed in the calculated spectra because they were generated from gas
phase structures, while the experimental spectra were analyzed in the solid state where the
compounds are in a more concentrated form, resulting in either intermolecular and/or
intramolecular hydrogen bonding, similar to that observed in structurally-related Schiff bases
[23,59,60]. Conversely, the v(OH) band was observed in the spectra of 3 and 4, which were
comparable to previous literature [23]. As a result, the loss of v(OH) upon complexation was
difficult to assign by FTIR alone, due to the intra- and intermolecular hydrogen bonding
between the molecules. The v(N-H) band of the thiosemicarbazone Schiff bases disappeared
upon complexation due to the deprotonation of the NH group and the involvement of the
resulting nitrogen atom in coordination to the Sn center. Furthermore, the IR spectra of 1, 2, 3
and 4 exhibited a strong intensity band due to the presence of v(C=N)azomethine at 1610, 1609,
1601 and 1597 cm-1, respectively. This band shifted to lower frequencies in the spectra of the
tin(IV) compounds, suggesting coordination to the tin center via the azomethine nitrogen
atom. Other than that, the v(C=S) and v(N-N) absorptions shifted to lower frequencies upon
complexation, indicating coordination via the thiolate sulfur and azomethine nitrogen atoms
forming five-membered chelate rings. A small deviation was observed in the vibrational
frequencies which can be explained by the fact that the experimental spectra were obtained in
the solid state, while the DFT calculations were run in the gas phase.
3.2.2. Multinuclear (1H, 13C and 119Sn) NMR spectral analysis
1
The H and 13C NMR spectra of 1-4 were recorded in DMSO-d6 solution and 5-16 were
recorded in DMSO-d6/CDCl3 solution at room temperature. The assignments of the relevant
1
signals are compiled in Tables S2 and S3. The H NMR spectra of 1-4 showed signals at δ
11.42, 11.78, 11.40 and 11.75 ppm, respectively, which indicated the presence of -NH-
protons. These -NH- proton signals were not present in the spectra of the tin(IV) compounds,
indicating that the Schiff bases were coordinated to the tin atoms via the nitrogen donor atom.

Proton signals appeared at δ 9.18 and 10.02 ppm for 1 and 2 corresponding to the hydroxyl
1
atom, which disappeared in the H NMR spectra of the tin(IV) compounds, indicating the
coordination of the hydroxyl proton to the tin center [61]. Contrastingly, the two signals for
hydroxyl groups at δ 8.37, 8.38 ppm (3) and δ 8.96, 9.52 ppm (4) disappeared upon
complexation, which indicated the presence of intra- and intermolecular hydrogen bonding in
the compounds [23,62].
13
The C NMR spectra of 1, 2, 3 and 4 showed carbon signals at δ 177.6, 176.1, 178.0 and
176.0 ppm, respectively in the downfield region attributed to -S-C(=S)N. The position of
these carbon signals proved that the compounds are predominately thione tautomers, even in
DMSO-d6 solution. The signals shifted to the upfield region in the spectra of the tin(IV)
compounds, indicative of the involvement of the -S-C(-S)N moiety in the complexation and
decreasing electron density at the carbon atom when the sulfur atom is chelated to the tin
atom. The C=N signal was assigned at δ 148.4, 148.6, 146.0 and 146.0 ppm for 1-4,
respectively, and appeared downfield as the carbon atom is bonded to electronegative atoms.
However, the C=N signals shifted downfield in the spectra of the tin(IV) compounds due to
the increasing electron density around the atom upon complexation. The -CH3- unit of the
methoxy group appeared in the upfield region at δ 56.4 (1) and 56.3 ppm (2). A similar
carbon signal was observed for the -CH3- unit of the methoxy group in the spectra of the
tin(IV) compounds, indicating that the methoxy group does not coordinate to the tin atom.
The 119Sn NMR data was used to predict the geometry of the tin-containing compounds. The
119Sn NMR spectra of compounds 5-16 were evaluated in DMSO/CDCl3 solutions at room
temperature using SnCl4 (δ = -150 ppm) as an external standard. The 119Sn chemical shift
strongly depends on the alkyl/aryl group attached to the tin atom and the electronegativity of
the ligand coordinated to the tin atom, as well as the temperature employed in the
experiments. Theoretically, as the coordination number increases, the 119Sn chemical shift
moves towards the shielding region [63]. The spectra showed one sharp signal which
indicated that the tin(IV) compounds had only a single tin atom species. The 119Sn NMR
values of the penta-coordinated diphenyl- (5, 8, 11 and 14) and dimethyltin(IV) (6, 9, 12 and
15) compounds fell in the ranges δ -227 to -328 and -103 to -154 ppm, respectively, similar to
that reported previously for diphenyl- and dimethyltin(IV) compounds [64–67]. The 119Sn
NMR values of the hexa-coordinated tin(IV) compounds (7, 10, 13 and 16) were observed in
the range δ -354 to -541 ppm. The 119Sn NMR values of compounds 8, 10, 14, and 16 were

more negative due to the presence of hydroxyl groups at the meta position, which are more
electronegative than the methoxy groups [68].
3.2.3. Mass spectrometric analysis
Mass spectral data for 1-4 were recorded in DMSO and were found to be consistent with the
proposed formulation of the Schiff bases. The mass spectra displayed prominent peaks at m/z
239, 301, 225, and 287 for Schiff bases 1-4, respectively, which correspond to the
[C10H13N3O2S]+, [C15H15N3O2S]+, [C9H11N3O2S]+ and [C14H13N3O2S]+ ions; the mass spectra
for 1-4 are supplied in Figure S2.
3.2.4 Electronic spectral analysis
The experimental and calculated electronic data of compounds 1-16 in DMSO are tabulated
in Table S4. The prominent experimental electronic absorptions for 1-4 were observed in the
range 328-334 nm, which were best correlated with the calculated absorptions by B3LYP in
the range 317-330 nm. The frontier molecular orbitals of compounds 1-16 are shown in
Figure S3, where the figure illustrates the excitation of electrons from the HOMO of non-
bonding electrons at sulfur and nitrogen atoms that were excited to the LUMO, which is
largely centered on the thiosemicarbazone backbone, 2,3-dihydroxy phenyl ring and as well
as the oxygen atom attached to the phenyl ring. Thus, this supported the transition of
electrons from n→π* and π→π* of the Schiff bases. For the tin(IV) compounds (5-16), the
HOMOs are largely centered on the thiosemicarbazone Schiff base, whereas the LUMOs are
centered on the entire thiosemicarbazone Schiff base except the methyl or phenyl groups
attached to the nitrogen atom, thiolate sulfur and oxygen atoms attached to the phenyl ring.
3.3. X-ray structure crystallography of 11’ and 12
3.3.1. Molecular structures
The crystallographic asymmetric unit of 11′ comprises a molecule of Ph2Sn(L2) (11) and half
a molecule of 3-methoxysalicylaldehyde azine, with the molecular structures of each shown
in Figure 2. The presence of the azine molecule in 11′ presumably arises from the prolonged
standing of the acetonitrile:methanol (1:1) solution during the crystallization of an
authenticated sample of 11, which resulted in partial decomposition of 11 and subsequent
condensation of hydrazine and o-vanillin to form the azine. The tin center in 11′ is
coordinated by two ipso-carbon atoms of the phenyl substituents as well as the imine-N,

phenoxide-O and thiolate-S atoms derived from the di-negative, tridentate Schiff base ligand.
The resulting coordination geometry defined by the C2NOS donor set is highly distorted from
an ideal trigonal-bipyramidal geometry. This is quantified by the value of τ = 0.60, which
lies between the extreme values of 1.0 and 0.0 for the ideal geometries of trigonal-
bipyramidal and square pyramidal [69]. The angle closest to being trans is the S1–Sn–O1
angle of 161.81(7)°, with the next widest angle of 125.85(10)° being for N2–Sn–C16.
Selected geometric parameters are collated in the caption to Figure 2. While the crystal
structure of L2H2 is not available for comparison, that of the 4-methoxy analogue, L5H2, is
available [70]. In L2H2 the formally C1=S1 thione bond is 1.6769(14) Å, which is
considerably shorter than the C1–S1 bond length of 1.748(3) Å in 11′. The other parameters
of interest relate to the shortening of the C1–N1 bond in 11′ to 1.290(4) Å compared with
1.3441(17) Å in L5H2 and the small increase in the formally imine-C2=N2 bond to 1.309(4)
compared with 1.2798(18) Å in L5H2 [70].
Figure 2. The molecular structure of the constituents of 11′ showing the atom-labelling
scheme and 70% probability displacement ellipsoids. The 3-methoxysalicylaldehyde azine
molecule in (b) is disposed about a crystallographic center of inversion with unlabelled atoms
related by the symmetry operation -x, 3-y, -z. Selected geometric parameters: Sn–S1 =

2.5475(8), Sn–O1 = 2.0853(19), Sn–N2 = 2.176(3), S1–C1 = 1.748(3), N1–N2 = 1.394(3),
N1–C1 = 1.290(4), and N2–C2 = 1.309(4) Å. Details of the intramolecular hydroxy-O–
H…N(azine) hydrogen bond: H3o...N4 = 1.84(3) Å, O3…N4 = 2.598(4) Å with the angle at
H3o = 150(3)°.
The major distortions in the coordination geometry about the tin atom in 11′ can be traced to
the formation of five- (Sn,S1,N1,N2,C1) and six-membered (Sn,O1,N2,C2-C4) chelate rings
by the tridentate ligand, resulting in tight S1–Sn–N2 [77.76(6)°] and O1–Sn–N2 [84.62(9)°]
chelate angles. Each chelate ring is essentially planar, as seen in the values of the root mean
square (r.m.s.) deviations of 0.047 Å [maximum deviation = 0.035(3) Å for the C1 atom] and
0.025 Å [0.026(3) Å for C3] for the five- and six-membered rings, respectively. The dihedral
angle formed between the chelate rings is 2.81(9)°, indicating these rings are coplanar, and
the dihedral angle between the terminal rings is 5.36(15)°, indicating the Schiff base di-anion
is essentially planar.
The second constituent of 11′ is a half a molecule of 3-methoxysalicylaldehyde azine, with
the full molecule being generated by the application of crystallographic inversion symmetry.
The molecule is constructed about a central azine-N4–N4i bond [1.401(5) Å for symmetry
operation (i) -x, 3-y, -z] and features intramolecular hydroxy-O–H…N(azine) hydrogen bonds
which close S(6) loops; see Figure 2 for details. The crystal structure determination of this
molecule has been reported several times and in two polymorphs. A form is known [71]
where the molecule is disposed about a center of inversion [N–N = 1.4025(14) Å], as in 11′,
as well as a non-symmetric version [N–N = 1.402(5) Å], which approximates a
centrosymmetric conformation [72].
To a first approximation, the molecular structure of 12, Figure 3, mirrors that found for the
diphenyltin compound in 11′. Thus, a similar coordination mode is adopted by the L1 di-
anion, but in this case, based on a value of τ = 0.0 [69], the coordination geometry is distorted
square pyramidal. In this description, the Sn atom lies 0.6358(18) Å out of the basal plane
defined by the S1, O1, N2 and C16 atoms [r.m.s. deviation = 0.0078 Å] in the direction of the
axially-bound C17 atom. This arises as the two widest angles, that is S1–Sn–O1 [145.67(9)°]
and N2–Sn–C16 [145.45(15)°] are virtually identical. The Sn-S1 [2.5475(8)] and Sn-N2
[2.257(3)] bond lengths are, respectively, approximately 0.05 Å shorter and 0.08 Å longer in
12 than the equivalent bonds in 11′, while the Sn-O1 bond lengths remain the same.

Figure 3. The molecular structure of the constituents of 12 showing atom-labelling scheme
and 70% probability displacement ellipsoids. Selected interatomic parameters: Sn–S1 =
2.4982(12), Sn–O1 2.085(3), Sn–N2 = 2.257(3), S1–C1 = 1.751(4), N1–N2 = 1.396(5), N1–
C1 = 1.306(5) and N2–C2 = 1.288(5) Å.
The five- and six-membered chelate rings in 12 exhibit r.m.s. deviations of 0.080 Å
[maximum deviation = 0.071(3) Å for the N2 atom] and 0.200 Å [0.189(1) Å for Sn],
suggesting deviations from planarity. Indeed, the five- and six-membered rings may each be
described as having an envelope conformation where, for the smaller ring, the Sn atom lies
0.248(6) Å out of the plane defined by the remaining four atoms [r.m.s. deviation = 0.0056
Å]. The envelope is more pronounced for the larger ring, with the Sn atom 0.612(5) Å above
the plane [r.m.s. deviation = 0.0229 Å]. The dihedral angle between the chelate rings is
17.88(12)°, but this reduces to 12.1(2)° when the angle between the planar regions is
computed. The dihedral angle between the outer rings is 6.2(2)°.
Thus far, no specific mention of the tin-bound substituents in 11′ and 12 has been made. The
Sn–C bond lengths in 11′ are equivalent at 2 × 2.134(3) and, in turn, these are experimentally
equivalent to those in 12, that is, 2.134(4) Å [Sn–C16] and 2.128(4) Å [Sn–C17]. A
difference is seen in the C–Sn–C angles, however. Thus, in 11′, this angle is 121.46(12)°
which is significantly wider than the equivalent angle of 114.82(18)° in 12. This disparity is

emphasized in the overlay diagram shown in Figure 4, as are the differences in the relative
orientations of the L2 di-anions.
Figure 4. Overlay diagram of the R2Sn(L2) molecules in 11′ (R = Ph; red image) and
inverted-12 (R = Me; blue image), whereby the five-membered rings are coincident.
3.3.2. Supramolecular structures
The most notable aspect of the molecular packing of 11′ is the formation of eight-membered
{…HNCS}2 synthons through the agency of amine-N–H…S(thiolate) hydrogen bonds between
centrosymmetrically related Ph2Sn(L2) molecules, Figure 5(a). Additional interactions
between molecules of note are of the type π(chelate ring)…π(oxidobenzylidene) stacking, as
illustrated in Figure 5(b). Such interactions are increasingly being recognized as being
important in providing points of contact in coordination chemistry [73] and computational
chemistry indicates these provide energies of stabilization greater than conventional π-
stacking interactions between organic residues [74]. The dimeric aggregates are connected
into a supramolecular layer in the ab-plane via L1-imine-C–H…O(hydroxy) and imine-C–
H…O(methoxy) interactions, as shown in Figure 5(c).
In essence, each 3-
methoxysalicylaldehyde azine molecule links four symmetry related Ph2Sn(L2) molecules.
The layers stack along the c-axis direction, being connected by tin-bound-phenyl-C-H…π(Sn-

phenyl, oxidobenzylidene) and azine-methoxy-C-H…π(N-phenyl) interactions, to consolidate
the three-dimensional architecture, Figure 5(d).
Figure 5. Molecular packing in the crystal of 11′: (a) a view of the supramolecular dimer
sustained by amine-N–H…S(thiolate) hydrogen bonds, shown as orange dashed lines [N3–
H3n…S1i: H3n…S1i = 2.60(3) Å, N3…S1i = 3.380(3) Å and angle at H3n= 150(3)° for
symmetry operation (i) 1-x, 2-y, -z], (b) a view of the dimer aggregate connected by π(chelate
ring)…π(oxidobenzylidene) stacking interactions [Cg(Sn,O1,N2,C1-C3)…Cg(C3-C8)ii
=
3.8613(15) Å and angle of inclination = 2.13(11)° for (ii) -x, 2-y, -z] shown as pink dashed
lines, (c) supramolecular layer whereby the aggregate shown in (a) is connected by L2-imine-
C–H…O(hydroxy) and imine-C–H…O(methoxy) interactions (blue dashed lines) [C2–
H2…O3: H2…O3 = 2.40 Å, C2…O3 = 3.336(4) Å and angle at H2 = 168°; C34–H34...O2iii:
C34–H34...O2iii = 2.40 Å, C34...O2iii = 3.230(4) Å and angle at H34 = 146° for (iii) x, 1+y, z],
and (d) a view of the unit-cell contents in projection down the b-axis with C–H…π

interactions shown as purple dashed lines [C18–H18…Cg(C22-C27)iv: H18…Cg(C22-C27)iv =
2.79 Å and angle at H18 = 148°; C19–H19…Cg(C3-C8)iv: H19…Cg(C3-C8)iv = 2.73 Å and
angle at H19 = 138°; C35–H35c…Cg(C10-C15)v: H35c…Cg(C10-C15)v = 2.85 Å and angle at
H35c = 133° for (iv) x, 3/2-y, -1/2+z and (v) x, 5/2-y, 1/2+z]. In (a)-(c), non-participating
hydrogen atoms have been omitted for clarity.
The most prominent aspect of the molecular packing of 12 is the formation of supramolecular
chains supported by amine-N–H…O(phenoxide) hydrogen bonding. The chains are aligned
along the a-axis and have a helical topology, being propagated by 21-screw symmetry, Figure
6(a). Further stability to the aforementioned chains is provided by secondary bonding [73,75]
of the type Sn…S, well known in organotin chemistry [76]. As detailed in Figure 6(b), the
sulfur atom approaches the tin atom from the basal plane to establish a 5+1 coordination
geometry; the C17–Sn–S1 angle = 157.48(11)°. When considered in conjunction with the
hydrogen bonding, six-membered, {…HNCS…SnO} heterosynthons are established. The
chains are assembled into a three-dimensional architecture by amine-N-phenyl-C–
H…π(oxidobenzylidene) interactions as each chain forms two donor and two acceptor
interactions, Figure 6(c).

Figure 6. Molecular packing in the crystal of 12: (a) a view of the supramolecular helical
chain sustained by amine-N–H…O(phenoxide) hydrogen bonds, shown as orange dashed lines
[N3–H3n…O1i: H3n…O1i = 2.26(4) Å, N3…O1i = 3.089(5) Å and angle at H3n = 160(3)° for
symmetry operation (i) 1/2+x, 1/2-y, 1-z], (b) detail of the Sn…S secondary bonding [Sn…S1ii
= 3.4928(12) Å for (ii) -1/2+x, 1/2-y, 1-z] within the chain shown in (a) and the resulting six-
membered, {…HNCS…SnO} heterosynthon, and (c) a view of the unit-cell contents in a
projection down the a-axis with one supramolecular chain highlighted in the space-filling

mode. Chains are connected by amine-N-phenyl-C–H…π(oxidobenzylidene) interactions
[C12–H12…Cg(C3-C8)iii: H12…Cg3iii = 2.98 Å and angle at H12 = 129° for (iii) 1/2-x, 1-y,
1/2+z] shown as purple dashed lines.
3.4. Cytotoxic activity
Compounds 1-16 were screened for their cytotoxicity against a panel of ten cancer cell lines,
HT29, U87, SJ-G2, MCF-7, A2780, H460, A431, DU145, BE2-C and MIA, and one normal
cell line, MCF-10A (Table 1). However, it was not possible to determine the cytotoxicity
values of 7 due to its insolubility in 100% DMSO at 1mM concentration. Cisplatin was used
as a positive control to induce cell death. The growth inhibition concentrations of the
compounds required to inhibit 50% cell proliferation (GI50) were recorded after 72 hours of
cell exposure to the compounds. The stability of the compounds in DMSO and in a mixture
of DMSO and H2O were studied by UV-vis spectroscopic analysis, where the spectra
remained unchanged after 72 hours, which indicated that the compounds are stable in both
solvent systems.
The cytotoxicity evaluation of the 2-hydroxy-3-methoxybenzyl thiosemicarbazone Schiff
base analogues (1 and 2) revealed an increase in potency when a methyl substituent was
attached to the α-nitrogen atom, where 1 exhibited 10 to 20 times higher anti-proliferative
activity as compared that of 2, 3 and 4 in the panel of cancer cell lines tested. Table 1 shows
the high level for the cytotoxic potency of 1 against HT-29, A2780, A431, BE2-C and MIA
cell lines. Compound 1 was approximately ~10-100 times more potent than similar
synthesized structures, 2-[(1E)-({[(benzylsulfanyl)methanethioyl]amino}-imino)methyl]-6-
methoxyphenol
(SBOVaH)
[25]
and
2-hydroxy-5-methoxybenzaldehyde-N(4)-
methylthiosemicarbazone (H2dmmt) [8] against all the cancer cell lines tested, except for the
DU145 cell line. 1 also showed excellent cytotoxicity against the panel of cancer cell lines
compared to the reference drug (cisplatin). No obvious cytotoxicity pattern was observed for
2, which was similar to that of a similar analogue, SBOVaH [25]. In contrast, the 2,3-
dihydroxybenzyl thiosemicarbazone Schiff bases (3 and 4) showed a different pattern of
cytotoxicity, which could be attributed to the phenyl group attached to the α-nitrogen atom,
where the phenyl group potentially facilitates binding to biological molecules by π
interactions [23]. Compounds 2 and 4 are similar in structure, with the difference being only
in the methoxy (2) and hydroxyl (4) group substituents at the meta position of the benzene

rings. Compound 4 was more active than 2 against all cancer cell lines tested. This was
possibly due to the formation of hydrogen bonding interactions of two hydroxyl groups with
the active site of the amino acids of various enzymes in the cancer cells [77]. Compound 3
showed poorer cytotoxicity at the 25 µM single point dose evaluation pre-screening and was
not selected for further GI50 determination as it was considered to be inactive. The
cytotoxicity of the Schiff bases was tested using the non-cancerous normal human breast cell
line (MCF10A), where 1 showed lower toxicity towards the normal cells, which was evident
from its higher GI50 value (less active) as compared to the GI50 values of most of the cancer
cells, except U87, H460 and DU145. Compound 4 also exhibited a higher GI50 value against
MCF10A than HT29, MCF-7 and A2780. This suggests that 1 and 4 exhibit notable
anticancer properties against certain cancer cells as compared to normal cells.
The cytotoxicities of the tin(IV) compounds are comparable to those of related compounds
[25] where the diphenyltin(IV) compounds exhibit higher activities against certain cell lines
as compared to their Schiff bases and other tin(IV) compounds. In particular, 5 showed
higher cytotoxicities than its Schiff base (1) towards MCF-7, A2780, H460 and DU145 cells.
It was also observed that 5 exhibited a 2.5-fold lower activity than 1 against MIA cells.
Compounds 8 and 11 exhibited higher activities than 3 and 2 respectively across all cancer
cell lines. In a similar vein, compound 14 was more active than 4 for all cells, except MCF-7,
A2780 and H460 cells. The dimethyltin(IV) (6, 9, 12, and 15) and tin(IV) (10, 13, and 16)
compounds exhibited no significant differences as compared to their Schiff bases. It can be
concluded that the presence of two phenyl groups attached to the tin atom at the center
improved the cytotoxicity against all the tested cancer cell lines. The planarity of the aromatic
π system makes it available for stacking and provides easier penetration into the double helix
of the DNA of cancer cells [78]. Overall, the cytotoxicity data indicate that HT29, MCF-7,
A2780, A431, BE2-C and MIA are more sensitive, whereas H460 and DU145 cells are more
resistant against the Schiff bases and tin(IV) compounds that were investigated in this study
than cisplatin. By comparing the toxicity of the compounds tested, all the tin(IV) compounds
(5-16) showed lower toxicity for MCF10A cells as the GI50 values of MCF10A were higher
(less active) than the GI50 values of certain cancer cells. However, MCF10A is positive for
telomerase reverse transcriptase [79] which is known to be up-regulated in many cancer cells
as well. The decrease in cell viability after treatment with the synthesised compounds may be
due to the inactivation of this enzyme. The use of MCF10A in this study is to act as a
benchmark for the cytotoxicity data obtained [80].

Table 1. In vitro cytotoxicity of the tin(IV) compounds (5-16) derived from the thiosemicarbazone Schiff bases (1-4) against several cell lines,
determined by an MTT assay and expressed as GI50 values with standard errors. GI50 is the concentration at which cell growth is inhibited by 50%
72 hours post-incubation.
Growth inhibition concentration, GI50 (µM)
Compounds
HT29
U87
MCF-7
A2780
H460
A431
DU145
nd
BE2-C
SJ-G2
MIA
MCF10A
0.037 ±
0.07
0.43 ± 0.29
1
0.09 ± 0.06 0.26 ± 0.11 0.12 ± 0.08
0.42 ± 0.19 0.09 ± 0.06
0.03 ± 0.02 0.15 ± 0.03 0.04 ± 0.02
2.0 ± 0.40
1.5 ± 0.27 2.4 ± 0.26 5.1 ± 0.58
2.5 ± 0.77
> 25
3.3 ± 0.26
> 25
2
3b
1.80 ± 0.22 3.5 ± 0.40 1.9 ± 0.46
1.0 ± 0.15
2.1 ± 0.49
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
0.041 ±
0.02
0.020 ±
0.00
1.0 ± 0.03
2.1 ± 0.09
0.8 ± 0.49
1.5 ± 0.40
4
5
0.29 ± 0.25 1.6 ± 0.29 0.03 ± 0.01
0.05 ± 0.02 0.14 ± 0.06 0.02 ± 0.00
1.4 ± 0.43
0.7 ± 0.51
2.5 ± 1.2
0.86 ± 0.42
0.15 ± 0.08
0.02 ± 0.00 0.09 ± 0.04 0.11 ± 0.09
0.22 ± 0.06 0.07 ± 0.04
6.9 ± 5.1
nd
1.3 ± 0.53
nd
5.0 ± 3.1
nd
0.48 ± 0.14
nd
1.0 ± 0.71
nd
6
0.30 ± 0.05
0.13 ± 0.03 0.20 ± 0.01
0.35 ± 0.12
0.15 ± 0.03
0.15 ± 0.03
nd
nd
nd
nd
nd
nd
7
0.42 ± 0.18
0.34 ± 0.15
3.2 ± 0.33
0.81 ± 0.60
1.2 ± 0.41
3.2 ± 0.07
4.4 ± 0.60
0.37 ± 0.08
0.30 ± 0.02
8
0.15 ± 0.07 0.38 ± 0.10 0.10 ± 0.05 0.15 ± 0.04 0.60 ± 0.10 0.49 ± 0.31 0.50 ± 0.18 0.15 ± 0.04
0.23 ± 0.07
0.79 ± 0.36
1.4 ± 0.67
>50
9.1 ± 3.4
25 ± 4.00
1.4 ± 0.20
1.5 ± 0.39 1.6 ± 1.2
9
0.36 ± 0.07 2.2 ± 0.33 0.22 ± 0.04 0.26 ± 0.01
3.5 ± 3.3
1.2 ± 0.81
nd
10
11
12
13
14
15
0.09 ± 0.06
0.09 ± 0.07 0.10 ± 0.08
0.15 ± 0.11 0.22 ± 0.04 0.95 ± 0.21
1.1 ± 0.03
0.44 ± 0.11
1.3 ± 0.54
1.0 ± 0.35 0.57 ± 0.13 0.31 ± 0.13 0.27 ± 0.01
1.1 ± 0.26
0.29 ± 0.06
1.1 ± 0.20
0.35 ± 0.08
2.0 ± 0.88
3.3 ± 0.72
4.8 ± 0.75
3.7 ± 1.3
2.1 ± 0.43
2.6 ± 0.37
2.0 ± 0.18 2.8 ± 0.46 4.8 ± 0.73
1.8 ± 0.38
1.7 ± 0.74
1.6 ± 0.75
2.6 ± 0.48
3.2 ± 0.15 2.6 ± 0.22 5.3 ± 0.67 1.8 ± 0.07 5.0 ± 1.97
0.19 ± 0.05 0.32 ± 0.04 0.12 ± 0.03 0.16 ± 0.04 0.53 ± 0.19 0.38 ± 0.19 0.20 ± 0.05 0.13 ± 0.02 0.20 ± 0.03 0.10 ± 0.02
0.12 ± 0.03 0.40 ± 0.07 0.10 ± 0.03 0.21 ± 0.05 0.83 ± 0.37 0.27 ± 0.11 0.56 ± 0.27 0.20 ± 0.05 0.23 ± 0.03 0.17 ± 0.05
0.27 ±
0.34 ± 0.02
nd
16
0.18 ± 0.02 0.44 ± 0.06 0.17 ± 0.08
0.47 ± 0.07 0.26 ± 0.03 0.42 ± 0.15 0.25 ± 0.05 0.26 ± 0.02 0.23 ± 0.07
0.023
Cisplatin
11.0 ± 2.0 4.0 ± 1.0
6.5 ± 0.8
1.0 ± 0.1
0.9 ± 0.2
2.4 ± 0.3
1.2 ± 0.1
1.9 ± 0.2
0.4 ± 0.1
8.0 ± 1.0
a ‘nd’ = not determined; b percentage growth inhibition at 25 μM compound concentration

4. Conclusions
A series of twelve tin(IV) compounds derived from four thiosemicarbazone Schiff bases have
been synthesized and characterized by physicochemical and spectroscopic techniques, as well
as X-ray crystallographic analysis. X-ray crystallography indicated a highly distorted
trigonal-bipyramidal coordination geometry for Ph2Sn(L1) in 11′ and a distorted square
pyramidal geometry for Me2Sn(L1) in 12. An interesting pattern of cytotoxicity was observed
where compound 1 was selectively active against HT29, A2780, A431, BE2-C and MIA,
while 3 was inactive against all cancer cells. Both were similar in structure, the difference
being the methoxy (1) and hydroxyl (3) substituent at the meta position of the phenyl ring. In
contrast, compound 4, having a hydroxyl group on the phenyl ring, demonstrated greater
activity than compound 2 which had a methoxy group. Diphenyltin(IV) compound 5
displayed excellent activity in the range 0.016-0.22 μM against all the cancer cells tested.
Overall, the diphenyltin(IV) compounds showed the most promising anticancer potential.
Based on findings in this study, the thiosemicarbazone Schiff bases and their tin(IV)
compounds have significant anticancer potential and further mechanism of action and in vivo
studies are required to determine the action of these compounds in vivo for a better
intracellular understanding.
Appendix A. Supplementary data CCDC 1975499 and 1975500 contains the supplementary
crystallographic data for 11’ and 12. These data can be obtained free of charge
via http://www.ccdc.cam.ac.uk/conts/retrieving.html,
or
from
the
Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-
336-033; or e-mail: deposit@ccdc.cam.ac.uk.
Funding: This research was funded by Universiti Putra Malaysia under the Geran Putra IPS
(9504600) and Geran Putra IPB (9581001) (UPM) and the Malaysian Fundamental Research
Grant Scheme (FRGS No. 01-01-16- 1833FR).
Acknowledgements: We thank the Department of Chemistry, the Molecular Genetics
Laboratory and the Department of Biomedical Sciences, Universiti Putra Malaysia, Malaysia
and the Discipline of Chemistry, University of Newcastle and the Calvary Mater Hospital,
Australia for their facilities. Sunway University Sdn Bhd is thanked for X-ray
crystallographic analysis and financial support (Grant. no. STR-RCTR-RCCM-001-2019).

E.N.M.Y. wishes to thank Ministry of Higher Education Malaysia for the award of MyPhD,
MyBrain15 and the University of Newcastle for the award of University of Newcastle
International Postgraduate Research Scholarship. We would also like to thank Karen A.
Crouse, Robert Burns and Adam McCluskey for helpful discussions.
Conflicts of Interest: The authors declare no conflict of interest.
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