Diversity of complexes based on p-nitrobenzoylhydrazide, benzoylformic acid and diorganotin halides or oxides self-assemble: Cytotoxicity, the induction of apoptosis in cancer cells and DNA-binding properties

Article abstract of DOI:10.1016/j.bioorg.2019.103402

Eight organotin(IV) complexes (C1-C8) have been synthesized and characterized by elemental analysis, fourier transform infrared spectroscopy (FT-IR), multinuclear nuclear magnetic resonance (¹H, ¹³C and ¹¹⁹Sn NMR), high resolution mass spectroscopy (HRMS) and single crystal X-ray structural analysis. Crystallographic data show that C1 was a tetranuclear 16-membered macrocycle complex, C2-C4 and C7 were centrosymmetric dimer distannoxane and there was a Sn₂O₂ four-membered ring in the middle of the molecule, respectively, C5 and C6 are monoorganotin complexes due to the dehydroalkylation effect during the reaction, while C8 forms a one-dimensional chain structure. The cytotoxicity of all complexes were tested by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays against three human tumor cell lines NCI-H460, MCF-7 and HepG2. The dibutyltin complex C2 has been shown to be more potent antitumor agents than other complexes and carboplatin. Cell apoptosis study of C2 with the high activity on HepG2 and MCF-7 cancer cell lines was investigated by flow cytometry, it was shown that the antitumor activity of C2 was related to apoptosis, but it has different cell cycle arrest characteristics from platinum compounds, and the proliferation was inhibited by blocking cells in S phase. The DNA binding activity of the C2 was studied by UV–visible absorption spectrometry, fluorescence competitive, viscosity measurements and gel electrophoresis, results shown C2 can be well embedded in the double helix of DNA and cleave DNA.

Full text of DOI:10.1016/j.bioorg.2019.103402

Journal Pre-proofs
Diversity of complexes based on p-nitrobenzoylhydrazide, benzoylformic acid
and diorganotin halides or oxides self-assemble: cytotoxicity, the induction of
apoptosis in cancer cells and DNA-binding properties
Wujiu Jiang, Shanji Fan, Qian Zhou, Fuxing Zhang, Daizhi Kuang, Yuxing
Tan
PII:
S0045-2068(19)31049-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.103402
YBIOO 103402
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
3 July 2019
22 October 2019
24 October 2019
Please cite this article as: W. Jiang, S. Fan, Q. Zhou, F. Zhang, D. Kuang, Y. Tan, Diversity of complexes based
on p-nitrobenzoylhydrazide, benzoylformic acid and diorganotin halides or oxides self-assemble: cytotoxicity,
the induction of apoptosis in cancer cells and DNA-binding properties, Bioorganic Chemistry (2019), doi: https://
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Diversity of complexes based on p-nitrobenzoylhydrazide,
benzoylformic acid and diorganotin halides or oxides
self-assemble: cytotoxicity, the induction of apoptosis in cancer
cells and DNA-binding properties
Wujiu Jianga, *, Shanji Fan,b Qian Zhou,c Fuxing Zhang,a Daizhi Kuanga and Yuxing Tana, *
a. Key Laboratory of Functional Metal-Organic Compounds of Hunan Province, Key Laboratory of
Functional Organometallic Materials, University of Hunan Province, College of Chemistry and
Materials Science, Hengyang Normal University, Hengyang 421008, PR China.
b. Department of Oncologysurgery, The First Affiliated Hospital of University South China, Hengyang
421001, PR China.
c. School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, PR
China.
* Email: jwj_china@163.com, tanyuxing@hynu.edu.cn
Abstract: Eight organotin(IV) complexes (C1-C8) have been synthesized and characterized by
elemental analysis, fourier transform infrared spectroscopy (FT-IR), multinuclear nuclear magnetic
resonance (1H, 13C and 119Sn NMR), high resolution mass spectroscopy (HRMS) and single crystal
X-ray structural analysis. Crystallographic data show that C1 was a tetranuclear 16-membered
macrocycle complex, C2-C4 and C7 were centrosymmetric dimer distannoxane and there was a
Sn2O2 four-membered ring in the middle of the molecule, respectively, C5 and C6 are
monoorganotin complexes due to the dehydroalkylation effect during the reaction, while C8
forms a one-dimensional chain structure. The cytotoxicity of all complexes were tested by
3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays against three human
tumor cell lines NCI-H460, MCF-7 and HepG2. The dibutyltin complex C2 has been shown to be
more potent antitumor agents than other complexes and carboplatin. Cell apoptosis study of C2 with

the high activity on HepG2 and MCF-7 cancer cell lines was investigated by flow cytometry, it was
shown that the antitumor activity of C2 was related to apoptosis, but it has different cell cycle arrest
characteristics from platinum compounds, and the proliferation was inhibited by blocking cells in S
phase. The DNA binding activity of the C2 was studied by UV-visible absorption spectrometry,
fluorescence competitive, viscosity measurements and gel electrophoresis, results shown C2 can be
well embedded in the double helix of DNA and cleave DNA.
Keywords: organotin complex; self-assemble; synthesis; crystal structure; apoptosis;
1 Introduction
Cisplatin as the first organometallic anticancer drug [1] was still used as a front-line
wide-spectrum anticancer drug [2]. However, unbearable chemotherapy side-effects [3-8]
and the emergence resistance [9-11] have limited the clinical effectiveness of cisplatin and
related drugs. Screening new organometallic complexes with high anticancer activity, it
was expected to overcome the defect of platinum-based chemotherapy agent and open a
new way in organometallic anticancer drug treatment. Some complexes with high
anticancer activity in vitro have been discovered, such as organotin complexes [12-15],
ruthenium complexes [16, 17], gallium complexes [18, 19], metallocene complexes
[20-23], etc. Research shows the structure of the diorganotin complexes were highly
similar to cisplatin in the coordination mode, and most of them have better anticancer
activity in vitro than cisplatin [24-26], so it was considered to be the promising alternative
to platinum-based complexes as a new organometallic anticancer drug. Since Crowe [27]
reported that diorganotin complexes have anticancer activity in vitro, many organotin
complexes have been prepared, some of which have far superior anticancer activity to
platinum-based complexes in breast cancer [28, 29], ovarian cancer [30], nasopharyngeal
cancer [31, 32], colon cancer [33] and bladder cancer [34].
In addition, the activity of the diorganotin complex depends on multiple factors such
as the type of hydrocarbon group, ligand type, molecular structure, etc. Because the
diorganotin has a large lipid-water partition coefficient, we have designed a biocompatible
nitrogen-containing hydrophilic multidentate ligand to regulate the lipid-water partition

coefficient of diorganotin and it can be used to regulate molecular structure types. In order
to simplify the synthesis step, we have synthesized a series of organotin complexes by
microwave-assisted one-pot self-assembly using the coordination ability of the ligand and
organotin.
NO2
O2N
Ph
O
N
O
R
N
Sn
N
R
R
O
O
N
Sn
R
R
O
O
Ph
Ph
N
O
O
R
Sn
N
O
O
O
R
Sn
R
N
O
N
Ph
NO2
O2N
(C1)
O2N
OH
Sn
H
N
O
O
Ph
R
R
NH2
O
N
N
O
O
O2N
NO2
N
N
O
Sn
+
O
R
R
Ph
O
HO
Ph
MeOH
OH
Microwave
O
(C2, C3, C4, C7)
O2N
+
Ph
N
O
N
R2SnCl2 / R2SnO
O
O
Sn
Cl
R
OH
C1: R= Ph-
C2: R= n-Bu-
(C5, C6)
C3: R= PhCH2-
NO2
C4: R= p-Me-Ph-CH2-
C5: R= o-Me-Ph-CH2-
C6: R= p-Cl-Ph-CH2-
C7: R= o-Cl-Ph-CH2-
C8: R= 2,4-Cl2-Ph-CH2-
N
O
R
R
Ph
O
N
Sn
O
HO
n
(C8)
Scheme 1. The reactions of C1-C8.
In this paper, we report four types of organotin complexes, the cytotoxicity of these
complexes against three human cancer cell lines (NCI-H460, MCF-7 and HepG2) was
initially tested. HepG2 and MCF-7 cancer cell apoptosisinduced by C2 was examined by flow

cytometry analysis, and the interaction mode of C2 and DNA was studied by UV,
fluorescence and viscosity, etc.
2 Experimental
2.1 Materials and methods
The microwave synthesis reaction was completed using Sineo Microwave MDS-10
High-throughput Microwave Sample Preparation Workstation. Elemental analyses for C,
H, and N were determined on a PE-2400(II) analyzer. IR spectrum was obtained for KBr
pellets on Shimadzu Prestige-21 spectrophotometer in the 4000-400 cm-1. 1H, 13C and 119Sn
NMR analysis were performed on a Bruker AVANCE NMR spectrometer. HRMS was
obtained by Thermo Scientific LTQ Orbitrap XL with ESI. Crystal structure was
determined on a CCD area detector X-ray diffractometer. Thermogravimetric analyes
(TGA) were recorded on a NETZSCH TG 209 F3 instrument at a heating rate of 20°C /min
from room temperature to 900°C under air. Melting point measurement was executed on an
X-4 binocular micromelting point apparatus with the temperature unadjusted. Cytotoxicity
studies was test by Thermo Scientific Multiskan GO. UV-Vis absorption spectra was
measured by UV-2550 spectrometer. Fluorescence spectra was obtained with a Hitachi
F-7000 spectrophotometer with quartz cuvette (path length=1cm). Viscosity experiments
were conducted on an Ubbelodhe viscometer. Gel electrophoresis was measured by
DYY-6C Electrophoresis power supply. Cell apoptosis and cell cycle were measured by
BD FACSCalibur CellSorting System.
Calf thymus DNA (Type XV, Activated, lyophilized powder) were from
Sigma-Aldrich LLC., pBR322 DNA used in this study was synthesized by Sangon Biotech
Co., Ltd. (Shanghai, China). 4-Nitrobenzhydrazide were from J&K Scientific Ltd.
Dibutyltin oxide and benzoylformic acid were from TCI (Shanghai) Development Co., Ltd.
Diphenyltin dichloride were from Alfa Aesar (China) Chemical Co., Ltd. Other chemicals
were from Sinopharm Chemical Reagent Co., Ltd. All reagents were of analytical grade
obtained from commercial sources and used without further purification. Ultrapure water
(18.2 MΩ·cm) obtained from a Milli-Q water purification system (Millipore Co., USA)
was used in all experiments. Tris-HCl (0.01mol·L-1) buffer solution was prepared by a
certain amount of Tris dissolved in super pure water before using, the pH of the solution

was adjusted to 7.40 with Hydrochloric acid solution (0.1 mol·L-1). The purity of CT-DNA
was determined by comparing the absorbance at 260 and 280 nm (A260/A280=1.8 ~ 1.9/1).
The concentration of CT-DNA was calculated by measuring the absorbance at 260nm
(ε260=6600 L·mol-1·cm-1). The reserve solution is stored at 4°C. The ethidium bromide
solution was prepared by a certain amount of ethidium bromide solid dissolved in Tris-HCl
(0.01 mol·L-1) buffer solution.
2.2 Synthesis
To a mixture of p-nitrobenzoylhydrazide (1 mmol), benzoylformic acid (1 mmol), the
corresponding diorganotin halides or oxides (1 mmol) and methanol (30 mL) were added
to the microware reaction kettle, and subjected to microwave treatment with 30 min at 100
°C. When the reaction kettle had cooled to room temperature, the solution in the kettle was
filtered. The complexes crystals were obtained by controlling solvent evaporation.
{[p-NO2-C6H4(O)C=N-N=C(Ph)COO](C6H5)2Sn}4 (C1): Yellow crystals, Yield 81%.
m.p.: 221-223°C. FT-IR (KBr, cm-1): 3421, 3072, 3055, 2991, 1695, 1616, 1600, 1587,
1568, 1525, 1479, 1384, 1340, 1321, 1294, 1263, 1251, 1089, 1172, 1153, 1024, 1012, 871,
1
852, 815, 736, 712, 690, 592, 547, 449. H NMR (500 MHz, CDCl3, δ/ppm): 8.44 (d, J =
8.9 Hz, 2H), 8.34 (d, J = 8.9 Hz, 2H), 8.15-8.17 (m, 2H), 7.86-7.88 (m, 4H), 7.26-7.59 (m,
9H). 13C NMR (126 MHz, CDCl3, δ/ppm): 173.24, 162.53, 152.49, 150.28, 137.91, 136.11,
135.35, 132.47, 132.44, 131.71, 129.75, 129.60 (1JSn-C = 89.5 Hz), 127.99, 127.86, 123.65.
119Sn NMR (187 MHz, CDCl3, δ/ppm): -294.09. HRMS (ESI) m/z calcd for C27H20N3O5Sn+
[M+H]+ 586.0419 , found 586.0411.
{[p-NO2-C6H4(O)C=N-N=C(Ph)COO](n-Bu2)Sn(CH3OH)}2 (C2): Yellow crystals,
Yield 76%. m.p.: 98-100°C (dec.). FT-IR (KBr, cm-1): 3419, 3076, 3055, 2956, 2927, 2870,
2858, 1631, 1598, 1587, 1529, 1496, 1400, 1382, 1342, 1315, 1305, 1292, 1255, 1153,
1168, 1085, 1024, 1012, 869, 852, 821, 804, 731, 719, 707, 690, 630, 594, 574, 542, 410.
1H NMR (500 MHz, CDCl3, δ/ppm): 8.27-8.32 (m, 4H), 8.12-8.14 (m, 2H), 7.58-7.60 (m,
3H), 3.49 (s, 3H), 1.72-1.81 (m, 4H), 1.50-1.66 (m, 4H), 1.33-1.40 (m, 4H), 0.88 (t, J = 7.2
Hz, 6H). 13C NMR (126 MHz, CDCl3, δ/ppm): 173.51, 164.30, 151.89, 150.04, 138.38,
132.08, 131.90, 129.73, 128.61, 127.83, 123.47, 50.85, 26.86, 26.42, 23.35 (1JSn-C = 618.0

Hz), 13.51. 119Sn NMR (187 MHz, CDCl3, δ/ppm): -509.34. HRMS (ESI) m/z calcd for
C23H28N3O5Sn+ [M-CH3OH+H]+ 546.1045, found 546.1035.
{[p-NO2-C6H4(O)C=N-N=C(Ph)COO](C6H5CH2)2Sn(CH3OH)}2 (C3): Yellow crystals,
Yield 72%. m.p.: 102-104°C (dec.). FT-IR (KBr, cm-1): 3568, 3080, 3057, 3024, 2943,
1643, 1632, 1597, 1582, 1524, 1493, 1383, 1346, 1302, 1244, 1171, 1155, 1088, 868, 854,
802, 760, 739, 718, 696, 631, 594, 552. 459. 1H NMR (500 MHz, CDCl3, δ/ppm): 8.25 (d,
J = 8.5 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.47-7.55 (m, 3H), 7.37 (d, J = 7.5 Hz, 2H), 7.04
2
(d, J = 7.5 Hz, 4H), 6.93-6.97 (m, 6H), 3.53 (d, J = 11.6 Hz, JSn-H = 128.4 Hz, 2H), 3.49
(s, 3H), 3.45 (d, J = 11.6 Hz, 2JSn-H = 128.4 Hz, 2H). 13C NMR (126 MHz, CDCl3, δ/ppm):
173.48, 168.76, 149.88, 149.40, 138.75, 136.49, 131.13, 129.78, 128.97, 128.60, 128.49,
128.31, 127.46, 125.70, 123.34, 50.88, 36.91 (1JSn-C = 748.4 Hz). 119Sn NMR (187 MHz,
CDCl3, δ/ppm): -637.87. HRMS (ESI) m/z calcd for C29H24N3O5Sn+ [M-CH3OH+H]+ 614.0732,
found 614.0725.
{[p-NO2-C6H4(O)C=N-N=C(Ph)COO](p-Me-C6H4CH2)2Sn(CH3OH)}2 (C4): Yellow
crystals, Yield 61%. m.p.:116-118°C (dec.). FT-IR (KBr, cm-1): 3482, 3082, 3049, 2999,
2920, 1678, 1624, 1605, 1591, 1528, 1510, 1499, 1383, 1344, 1319, 1306, 1290, 1254,
1
1169, 1086, 1024, 870, 853, 814, 733, 710, 691, 631, 592, 546, 501, 461. H NMR (500
MHz, CDCl3, δ/ppm): 8.26 (d, J = 8.5 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.53 (t, J = 7.3
Hz, 1H), 7.47 (t, J = 7.3 Hz, 2H), 7.32-7.33 (m, 2H), 6.92 (d, J = 7.9 Hz, 4H), 6.77 (d, J =
2
7.9 Hz, 4H), 3.50 (s, 3H), 3.45 (d, J = 11.6 Hz, JSn-H = 126.2 Hz, 2H), 3.39 (d, J = 11.6
2
Hz, JSn-H = 126.2 Hz, 2H), 2.12 (s, 6H). 13C NMR (126 MHz, CDCl3, δ/ppm): 173.41,
168.31, 149.82, 149.57, 138.96, 135.24, 133.30, 131.10, 130.97, 129.76, 129.14, 128.45,
128.22, 127.34, 123.27, 50.88, 36.22 (1JSn-C = 748.9 Hz), 20.83. 119Sn NMR (187 MHz,
CDCl3, δ/ppm): -638.91. HRMS (ESI) m/z calcd for C31H28N3O5Sn+ [M-CH3OH+H]+ 642.1045,
found 642.1036.
[p-NO2-C6H4(O)C=N-N=C(Ph)COO](CH3OH)(o-Me-C6H4CH2)SnCl
(C5):
Light
yellow crystals, Yield 41%. m.p.: 145-147°C (dec.). FT-IR (KBr, cm-1): 3107, 3053, 3022,
2970, 1667, 1593, 1526, 1479, 1445, 1385, 1346, 1321, 1304, 1290, 1258, 1175, 1094,
1001, 853, 741, 718, 689, 637, 596, 557, 436. 1H NMR (500 MHz, CDCl3, δ/ppm): 8.31 (d,
J = 8.6 Hz, 2H), 8.18 (d, J = 7.9 Hz, 2H), 7.53 (s, 1H), 7.30 (s, 2H), 7.22 (s, 2H), 6.84 (s,

2
4H), 3.95 (s, JSn-H = 153.3 Hz, 2H), 3.49 (s, 3H), 2.03 (s, 3H). 13C NMR (126 MHz,
d6-DMSO, δ/ppm): 170.64, 161.06, 149.85, 146.58, 137.56, 135.91, 135.00, 131.56,
131.45, 131.25, 129.94, 129.78, 129.39, 127.78, 125.84, 125.73, 123.87, 48.52, 34.50
(1JSn-C = 1038.5 Hz), 19.71. 119Sn NMR (187 MHz, d6-DMSO, δ/ppm): -452.74. HRMS
(ESI) m/z calcd for C23H19ClN3O5Sn+ [M-CH3OH+H]+ 572.0030, found 572.0038.
[p-NO2-C6H4(O)C=N-N=C(Ph)COO](CH3OH)(p-Cl-C6H4CH2)SnCl (C6): Light yellow
crystals, Yield 45%. m.p.: 144-146°C (dec.). FT-IR (KBr, cm-1): 3113, 3082, 3059, 2940,
2778, 1665, 1609, 1593, 1528, 1479, 1445, 1387, 1344, 1323, 1290, 1258, 1171, 1155,
1
1090, 1013, 993, 870, 853, 833, 718, 691, 637, 596, 556, 476, 420, 401. H NMR (500
MHz, CDCl3, δ/ppm): 8.62 (s, 2H), 8.32 (d, J = 8.5Hz, 2H), 7.79 (d, J = 8.5Hz, 3H), 7.44
2
(d, J = 8.4Hz, 4H), 6.86 (s, 2H), 3.73 (s, JSn-H = 153.6 Hz, 2H), 3.49 (s, 3H). 13C NMR
(126 MHz, d6-DMSO, δ/ppm): 170.45, 161.32, 149.61, 146.71, 144.52, 137.97, 135.98,
131.32, 130.60, 129.69, 129.29, 128.99, 127.92, 127.58, 123.77, 48.50 (1JSn-C = 1041.2
Hz). 119Sn NMR (187 MHz, d6-DMSO, δ/ppm): -454.17. HRMS (ESI) m/z calcd for
C22H16Cl2N3O5Sn+ (M-CH3OH+H)+ 591.9483, found 591.9484.
{[p-NO2-C6H4(O)C=N-N=C(Ph)COO](o-Cl-C6H4CH2)2Sn(CH3OH)}2
(C7):
Red
crystals, Yield 73%. m.p.: 102-104°C (dec.). FT-IR (KBr, cm-1): 3451, 3057, 3019, 2936,
2826, 1636, 1607, 1591, 1528, 1501, 1474, 1439, 1387, 1344, 1288, 1256, 1169, 1157,
1
1086, 1015, 868, 851, 812, 750, 733, 719, 687, 633, 592, 573, 544, 440. H NMR (500
MHz, CDCl3, δ/ppm): 8.16 (d, J = 8.8Hz, 2H), 8.05 (s, 1H), 7.85-7.87 (m, 3H), 7.47-7.53
(m, 3H), 7.27-7.29 (m, 1H), 7.13-7.16 (m, 3H), 6.90-6.96 (m, 4H), 3.48 (s, 3H), 3.44 (d, J
= 12.0 Hz, 2JSn-H = 226.6 Hz, 2H), 3.33 (d, J = 12.0 Hz, 2JSn-H = 226.6 Hz, 2H). 13C NMR
(126 MHz, CDCl3, δ/ppm): 172.96, 163.54, 151.38, 149.87, 138.12, 124.40, 132.88,
131.73, 129.73, 130.62, 129.73, 129.04, 127.74, 127.35, 126.97, 126.83, 123.19, 50.88,
30.14 (1JSn-C = 573.6 Hz). 119Sn NMR (187 MHz, CDCl3, δ/ppm): -644.87. HRMS (ESI)
m/z calcd for C29H22Cl2N3O5Sn+ [M-CH3OH+H]+ 681.9953, found 681.9953.
{[p-NO2-C6H4(O)C=N-N=C(Ph)COO](2,4-Cl2-C6H3CH2)2Sn(CH3OH)}n (C8): Yellow
crystals, Yield 77%. m.p.: 122-124°C (dec.). FT-IR (KBr, cm-1): 3618, 3231, 3055, 2945,
1622, 1578, 1526, 1499, 1472, 1385, 1342, 1292, 1256, 1173, 1155, 1109, 1088, 1045,
1
1013, 870, 853, 820, 762, 719, 691, 656, 592, 540, 442. H NMR (500 MHz, CDCl3,

δ/ppm): 8.19-8.22 (m, 2H), 7.85-7.88 (m, 4H), 7.55-7.80 (m, 3H), 7.19 (d, J = 2.1Hz, 2H),
2
7.10 (d, J = 8.4Hz, 2H), 6.93-6.95 (m, 2H), 3.50 (s, 3H), 3.43 (d, J = 12.0 Hz, JSn-H
=
114.4 Hz, 2H), 3.33 (d, J = 12.0 Hz, 2JSn-H = 114.4 Hz, 2H). 13C NMR (126 MHz, CDCl3,
δ/ppm):173.09, 163.42, 151.63, 150.02, 137.82, 133.40, 133.11, 132.39, 132.11, 131.72,
131.27, 129.35, 128.85, 128.16, 127.91, 127.33, 123.37, 50.90, 30.11 (1JSn-C = 607.2 Hz).
119Sn NMR (187 MHz, CDCl3, δ/ppm): -279.95. HRMS (ESI) m/z calcd for
C29H20Cl4N3O5Sn+ [M-CH3OH+H]+ 749.9174, found 749.9151.
2.3 Single Crystal Structure Determination
X-ray diffraction data for crystals were performed with graphite monochromated
Mo-Kα radiation (λ= 0.71073 Å ) on a Bruker Smart Apex II CCD diffractometer, and
collected by the φ-ω scan technique at ambient temperature. Multi-scan absorption
correction was applied to the data. The crystal structures were solved by direct methods
and refined by full-matrix least-squares on F2. All the non-hydrogen atoms were located in
successive difference Fourier syntheses and then refined anisotropically. Hydrogen atoms
were placed in calculated positions or located from the Fourier maps, and refined
isotropically with the isotropic vibration parameters related to the non-hydrogen atom to
which they are bonded. All calculations were performed with SHELXL [35] programs
within WINGX [36]. A summary of the crystallographic data and selected experimental
information are given in Table S1. Crystallographic data for the structures of compounds
reported in this paper have been deposited with the Cambridge Crystallographic Data
Center as supplementary publication Nos. CCDC 1479715 (C1); 1479712 (C2); 1479716
(C3); 1479713 (C4); 1897744 (C5); 1897745 (C6); 1897746 (C7) and 1479714 (C8).
2.4 In vitro cytotoxic activity
NCI-H460(ATCC No: HTB-177), HepG2(ATCC No: HB-8065) and MCF-7(ATCC
No: HTB-22) cells were obtained from American Tissue Culture Collection (ATCC).
Normal human liver cell line (HL-7702) were obtained from the Shanghai Institute of Cell
Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained at
37°C in a 5% CO2 incubator in RPMI 1640 (GIBICO, Invitrogen) containing 10% fetal
bovine serum (GIBICO, Invitrogen). 5mM stock solutions of complexes were prepared in
DMSO and diluted in fresh medium for use. The final concentration of DMSO never

exceeded 0.1% (v/v). Cell proliferation was assessed by MTT assay. 100 μL of cells
(5×107 cells·L-1) were seeded into 96 well plates. The cells incubated for 24 h at 37 °C, 5%
CO2. Then the medium was replaced with the respective medium containing complexes at
different concentrations and incubated for 48 h. 10 μL of MTT was added and the medium
was removed after 4 h of incubation. Finally, crystal violet was solubilized in 100 μL
DMSO and the absorbance was measured at 570 nm. Six concentrations (10 nM-10 μM)
were set for the compounds and at least 3 parallels of every concentration were used. All
experiments were repeated at least three times. The data was calculated using Graph Pad
Prism version 7.0. The IC50 were fitted using a non-linear regression model with a
sigmoidal dose response [13].
2.5 Cell apoptosis assay
HepG2 and MCF-7 cells were seeded in sterile six-well plates at density of 1
×
108 cell·L-1
and 1×
109 cell·L-1 (1mL per well), respectively. After the cells were attached for 24 h, the
supernatant was discard. Then cells were exposed to C2 solution at concentrations of 0.1, 0.3 and
0.5 uM. After 48 h incubation, the drugs were washed two times by cold PBS. Re-suspended cells
in 100 ml binding buffer, Annexin V-PE (5 ml) and 7AAD (5 ml) were added to cells. And then
cells were incubated for 15 min at room temperature in the dark and then analyzed by flow
cytometry. Each group of experiments was measured in parallel 3 times [31].
2.6 Cell cycle analysis
HepG2 and MCF-7 cells were seeded in sterile six-well plates at density of 1
×
108 cell·L-1
and 1
×
109 cell·L-1 (1mL per well), respectively. After the cells were attached for 24 h, the
supernatant was discard. Then cells were exposed to C2 solution at concentrations of 0.1, 0.2 and
0.4 M. After 48 h incubation, the drugs were washed two times by cold PBS. 70% ethanol was
added. After the cells were fixed at 20°C overnight, RNaseA (10 L) and PI (10 L) were added
μ
μ
μ
to cells. And then cells were incubated for 15 min at room temperature in the dark and then
detected by flow cytometry. Each group of experiments was measured in parallel 3 times [32].
2.7 DNA-binding studies
2.7.1 UV-visible absorption spectrometry
The investigation of the possible binding modes of complex to DNA and the
calculation of the corresponding DNA-binding constants (Kb) were carried out by UV-vis

spectroscopy. UV-visible absorption spectrometry experiments were carried out with a
constant concentration of C2 (50 μM), and varying the concentration of CT-DNA (0-50
μM) in tris-HCl (0.01mol·L-1) buffer solution. The intrinsic binding constants (Kb) was
calculated according the following the Wolfe-Shimmer equation [37]:
cDNA / (εA - εF) = cDNA / (εB - εF) + 1 / Kb (εB - εF)
where cDNA is the concentration of CT-DNA, εA is the observed extinction coefficient at
arbitrary DNA concentration, εF is the extinction coefficient of the free complex, εB is the
extinction coefficient of the complex when fully combinate to CT-DNA. The
DNA-binding constants Kb were determined by the Wolfe-Shimmer equation and the plots
cDNA / (εA - εF) versus cDNA, Kb is given by the ratio of the slope to the intercept.
2.7.2 Fluorescence competitive study
In this fluorescence study, a mixture of the CT-DNA (30 μM), EB (30 μM) and
different concentration complex C2 solution (0-80 μM) was placed in a 5 mL volumetric
flask in tris-HCl (0.01mol·L-1) buffer solution. After 3 h, the fluorescence spectra were
acquired at 25 °C. The excitation wavelength was 258 nm, and the emission wavelength is shown
in spectrum. The slit scanning width of emission and excitation is 5.0 nm. Finally
quenching constant (Ksv) values of C2 were determined by using Stern-Volmer equation
[13].
2.7.3 Viscosity measurements
The viscosity measurements were carried out in the Ubbelohde viscometer, and it was
keep in the water bath with 25.00 ± 0.02 °C. Viscosity experiments were performed by
fixed DNA concentration (50 μM) and increasing the concentration of C2 (0-50 μM) in
tris-HCl (0.01mol·L-1) buffer solution. The flow time of sample was measured with a
digital stopwatch. The values of viscosity were calculated from the flow times of DNA
containing solutions corrected for the flow time of buffer alone (t0), η = (t − t0) [38]. Data
was presented as (η/η0)1/3 versus (ccomplex/cDNA), where η is the viscosity of DNA in the
presence of the complex, η0 is the viscosity of DNA alone, ccomplex is the concentration of
the complex and cDNA is the concentration of DNA.
2.7.3 Gel electrophoresis studies

For the gel electrophoresis experiments, the gel electrophoresis experiments were
performed by fixed pBR322 plasmid DNA concentration and increasing the concentration
of C2 (0, 20, 40, 60, 80, 100 μM), and the contents were incubated for 1 h at 25 °C. and
they were electrophoresed for 1 h at 150 V on 1.0% agarose gel in TAE buffer (pH=8.0).
Goldview was used for staining, the image was photographed by using gel imaging
documentation system under UV light mode.
2.7.4 Molecular Docking.
Molecular docking was performed using MOE (Molecular Operating Environment)
software. The crystal structure of DNA molecule was downloaded from Protein Data Bank
(code: 453D)[39], and the embedded small molecule and water was removed by MOE
software, hydrogenation was added to the DNA as it is required for the electrostatics, and
point charge was calculated and the energy was minimized[40, 41]. The CIF file of
complex is converted into a PDB file by Mercury 3.8 and imported into the MOE software,
then removed the symmetrical unit and the coordinating solvent molecule, the energy
minimized to obtain the complex for docking. Molecules docking results were obtained by
Discovery Studio 3.5.
3 Results and discussion
3.1 Synthesis
The syntheses procedures are shown in Scheme 1. The organotin complexes (C1-C8) were
synthesized by equimolar amounts of p-nitrobenzoylhydrazide, benzoylformic acid and the
corresponding diorganotin halides or oxides under the microwave radiation, all reactant and
solvent are added to the microware reaction kettle at one time, and after the reaction is finished, it
is cooled to room temperature and then purified. The reaction time is 30 min. Obviously, the
reaction operation is simple and efficient.
Through comparative experiments, we found that the yield of the complexes synthesized by
traditional heating reflux method is not as good as that of microwave-assisted synthesis, and the
yield of the stepwise synthesis is also not as high as that of the “one-pot”. So microware reaction
has the characteristics of short reaction time, simple reaction step and good reproducibility.
In addition, it is easy to see that C1-C4, C7 and C8 are diorganotin complex in these
complexes, C5 and C6 are monoorganotin compounds. The structures of the C5 and C6 are

different from the initial experimental design. It is speculated that the dehydrocarbyl reaction
occurs during the reaction [42]. Initially, we suspected that it was related to microwave radiation,
but the guess is not true after the comparison experiment, it found that C5 and C6 still obtained
the dehydroalkylation product by the traditional heating reflux method. Therefore, it is
suspected that the following factors may lead to dehydroalkylation: stronger nucleophiles
than hydrocarbons attack the tin atoms, steric hindrance, charge density, etc. In summary, a
more stable dehydrocarbyl compound is formed after removal of a alkyl group.
3.2 Spectroscopic Data Discussion
The wavenumbers (cm-1) of different vibrational modes of of ligand (H2L) and C1-C8 was
shown in the table 1. In the IR spectra of the complexes, a broad band in the range
3419-3618 cm-1 appearing in the C2-C8, as ν(OH) vibrations, indicating that methanol was
involved in coordination. The gap between νasym(COO-) and νsym(COO-) is important to
find out the binding mode of COO- moiety with Sn atom. The literature [43] reveals the
Δ[νasym(COO-)-νsym(COO-)] value in the range of 150-200 cm-1 indicates a bridging
behaviour while a value >200 cm-1 exhibits monodentate coordination, other is considered
to be practically bidentate. It was found that the Δ[νasym(COO-)-νsym(COO-)] value of
C2-C7 was 204-216 cm-1 by calculation. C1 and C8 are less than 200 cm-1. Therefore, the
coordination mode of COO- and Sn atom in complexes C2-C7 are monodentate
coordination, the C1 and C8 are bridging coordination. The results of FT-IR study are
consistent with the X-ray structure. Besides, the typical absorptions for Sn-C, Sn-N, Sn-O,
Sn-O-Sn vibrations in the complexes are located in the normal range of similar organotin
complexes [44, 45].
Table 1 The wavenumbers (cm-1) of different vibrational modes of of ligand (H2L) and C1-C8
Comp. ν(OH) νasym(COO- νsym(COO- ν(Sn-O ν(Sn-
ν(Sn-
N)
ν(Sn-
O)
)
)
-Sn)
~
C)
H2L
C1
3271
3421
3419
3568
~
~
~
~
~
1568
1598
1597
1384
1382
1383
690
592
594
594
547
542
552
449
429
459
C2
690
696
C3

C4
C5
C6
C7
C8
3482
3107
3113
3451
3618
1591
1593
1593
1591
1578
1383
1385
1387
1387
1385
691
689
691
687
691
592
596
596
592
592
546
557
556
554
540
461
436
476
440
442
1
The H NMR spectrum show the ratio of the integral area of each group is consistent
with the expected number of protons in each group [46]. It is observed that the substituted
benzylic methylene protons in C5 and C6 were a normal single peak and a pair of small
satellite peaks, this is the results of Sn-H coupling [47]. However, the substituted benzylic
methylene protons in C3, C4, C7 and C8 were presenting two double peaks. From the
analysis of single-crystal X-ray diffraction results, it is known the complexes C5 and C6
are a monobenzyltin compound and the complexes C3, C4, C7 and C8 are dibenzyltin, so
it is speculated that the reason is that C3, C4, C7 and C8 cannot be freely rotated due to its
large steric hindrance, resulting in the geminal coupling. Other hydrogen protons have
similar chemical shifts in those complexes. The important 1H NMR peak and their
assignment of ligand (H2L) and C1-C8 was shown in the table 2.
Table 2 The important 1H NMR peak and their assignment of ligand (H2L) and C1-C8
Comp.
H2L
C1
R
-
The important 1H NMR peak (δ/ppm)
12.92 (s, 1H, -COOH)
Ph-
-
1.72-1.81 (m, 4H, -CH2CH2CH2CH3), 1.50-1.66 (m, 4H,
-CH2CH2CH2CH3), 1.33-1.40 (m, 4H, -CH2CH2CH2CH3), 0.88
(t, J = 7.2 Hz, 6H, -CH2CH2CH2CH3)
C2
n-Bu-
3.53 (d, J = 11.6 Hz, 2H, -CH2Ph), 3.45 (d, J = 11.6 Hz, 2H,
-CH2Ph).
C3
C4
PhCH2-
3.45 (d, J = 11.6 Hz, 2H, -CH2Ph), 3.39 (d, J = 11.6 Hz, 2H,
-CH2Ph), 2.12 (s, 6H, -CH2Ph-CH3)
p-Me-Ph-CH2-
C5
C6
o-Me-Ph-CH2- 3.95 (s, 2H, -CH2Ph), 2.03 (s, 3H, -CH2Ph-CH3).
p-Cl-Ph-CH2- 3.73 (s, 2H, -CH2Ph).

3.44 (d, J = 12.0 Hz, 2H, -CH2Ph), 3.33 (d, J = 12.0 Hz, 2H,
C7
C8
o-Cl-Ph-CH2-
-CH2Ph).
3.43 (d, J = 12.0 Hz, 2H, -CH2Ph), 3.33 (d, J = 12.0 Hz, 2H,
-CH2Ph)
2,4-Cl2-Ph-CH2-
13
In the C NMR spectrum, the peaks of each group are consistent with the theoretical
prediction of the number of carbon atoms in the structure [46]. The carboxyl carbon,
hydrazide carbon and imino carbon of complexes can be clearly identified, they were
located at the low field position. The peak positions of the all carbon atoms in the similar
complexes are basically consistent.
In the 119Sn spectrum, 119Sn chemical shifts of the all complexes are observed. It
presents only a sharp singlet, this indicates that the new complex has been generated and
the R2SnCl2 or R2SnO as a reactant is disappear.
3.3 Thermal stability
The thermal stabilities of both complexes were carried out from 40 to 800 °C at a rate of 20
°C min-1, under an air atmosphere with a flowing rate of 20.0 mL·min-1. As shown in Fig.
S33~S40, the weight loss curves of C2~C8 are relatively similar, three stages of weight loss can
be observed, the mass loss in the first stage corresponds to the loss of the methanol molecules.
However, only two weight loss stages were observed in C1, indicating that methanol was not
involved in coordination, the results are consistent with the X-ray structure. The remaining
weight indicates the final products are SnO2 by calculation. In addition, as can be seen
from the figure, all complex are stable up to about 100°C.
3.4 Crystal structure
The molecular structures of C1-C8 are shown in Fig. 1. The geometric parameters of
hydrogen bond in C2, C3, C5, C6 and C7 are in the Table 3. Selected bond lengths and bond
angles of complexes are listed in Tables S2 (Supporting Information).
C1 shows a 16-membered macrocycle where four adjacent diphenyl tin centers are
bridged to each other by the bridging 2-oxo-2-phenylacetic acid p-nitrobenzoyl hydrazide
ligands to afford a tetranuclear. As can be seen from Fig. 1, all the tin atoms are distorted
octahedral configuration, it is surrounded by two equatorial C atoms from phenyl groups
and four axial O, N atoms from hydrazide ligands. The distances between the

symmetrically opposite tin centers in the macrocycle are 7.515 Å, it is worth mentioning
that although the size of the cavity in the composite is large enough to capture some guest
molecules, there are no small molecules in the cavity, and it is speculated that the cavity is
almost completely occupied by the phenyl group on the tin atom.
Fig.1 Molecular structure of C1~ C8 (The phenyl group on the tin atom is omitted for
clarity in C1.)
C2, C3, C4 and C7 are centrosymmetric dimer distannoxane, there is a Sn2O2
four-membered ring in the middle of the molecule, respectively. The center of the ring is
the symmetry center of the molecule. In the four-membered Sn2O2 ring, the sum of the
interior angles is 360°, indicating the four-membered Sn2O2 ring is planar. Each carboxyl
oxygen atom bridges the two tin atoms in an antisymmetric unit, the Sn1-O2 bond belongs
to the normal Sn-O covalent bond, The Sn1-O2i bond distances (2.8395(22) Å C2;
2.8593(15) Å C3; 2.7595(12) Å C4; 2.6414(24) Å C7) are greater than covalent radii of Sn
and O 2.13 Å, while are much less than the sum of the van der Waals radii 3.69 Å. Besides,
C2, C3 and C7 form two-dimensional network structure and a one-dimensional band
structure by weak action of C-H…O, respectively (Fig.2 (a), (b), (c)).

Fig.2 1D or 2D structures form by hydrogen bonds or Sn-O interaction in complexes (the
2,4-dichlorobenzyl group on the tin atom is omitted for clarity in (f))

C5 and C6 are monomer structure and dehydroxylation products. The molecule
structures of the C5 and C6 are almost the same, with only small differences in bond
lengths and bond angles. C5 and C6 are monobenzyl tin compounds, and their central tin
atoms are all six-coordinate in a distorted octahedral configuration and they form t a
one-dimensional band structure by weak action of C-H…O and O-H…O, respectively
(Fig.2 (d), (e)). The bond lengths of C-H…O and O-H…O are not much different from the
literature report [48-50].
In the complex C8, the central tin atoms are all seven coordination of the distorted
pentagonal bipyramidal configuration. It is worth noting that the bond length of Sn1-O3i is
2.9415(21) Å, which is much less than the sum of the Van der Waals radii for Sn and O, it
proves that there is a strong interaction between Sn1 and O3i. So the one-dimensional
infinite chain structure is formed by Sn1-O3i (Fig.2 (f)). In the one-dimensional infinite
chain
of
C8,
each
asymmetric
unit
contains
two
p-NO2-C6H4-(O)C=N-N=C(Ph)COO](CH3OH)(2,4-Cl2-C6H3CH2)2Sn with a pitch of
10.2876(7) Å. The distance between two adjacent Sn atoms is 6.4394(4) Å and the Sn
atoms are arrange in a relationship with the Sn…Sn…Sn angle of 106.032(4)°.
Table 3 Geometric parameters of hydrogen bond in C2, C3, C5, C6 and C7
Distance, Å
Comp.
D-H…A
Angle DHA, °
D-H
H…A
2.579
2.526
1.863
2.656
1.963
2.639
2.705
2.715
D…A
3.482
3.239
2.589
3.150
2.635
3.240
3.310
3.319
C2
C3
C5
C17-H17B…O4i
C29-H29…O5i
O4-H4A…O3i
C24-H24A…O6i
O4-H4A…O3i
C23-H23B…O6i
C28-H28…O5i
C29-H29…O5i
0.970
0.930
0.738
0.960
0.673
0.969
0.931
0.931
155.08
166.91
168.26
112.48
177.30
121.10
123.46
123.34
C6
C7
3.5 Cell-cytotoxicity assays
Complexes C1-C8 and carboplatin were evaluated for cytotoxicity in vitro by MTT
assay against human non-small cell lung cancer (NCI-H460), human breast

adenocarcinoma (MCF-7) and human liver hepatocellular carcinoma (HepG2) cell lines.
Table 4 lists the cytotoxicity screening results for all organotin complexes. The complexes
C1 and C2 exerted strong cytotoxic effects against tested cancer cell lines with a lower
IC50 value (<2.0
C3, C4, C7 and C8 showed moderate cytotoxicity. The complexes C5 and C6 did not
show 50% inhibition at a concentration of 10 M. Among these cell lines, the HepG2 cell
μM), and better than the positive control drug Carboplatin. The complexes
μ
line was the most sensitive towards C2 with IC50 values of 0.28 ± 0.06 μM, and the MCF-7
cell is second (IC50=0.67 ± 0.07). They have a lower IC50 value than the anticancer activity
of other organotin compounds [51-54].
Table 4 The cytotoxicity of complexes(C1-C8) in vitro.
IC50 / μM
Comp.
NCI-H460
1.91 ± 0.12
1.18 ± 0.03
5.74 ± 0.14
4.50 ± 0.09
>10a
MCF-7
1.51 ± 0.09
0.67 ± 0.07
3.92 ± 0.23
3.06 ± 0.04
>10
HepG2
1.14 ± 0.08
0.28 ± 0.06
3.65 ± 0.11
2.74 ± 0.17
>10
HL-7702
C1
C2
4.25 ± 0.06 (15.17) b
C3
C4
C5
C6
>10
>10
>10
C7
7.26 ± 0.15
8.73 ± 0.21
7.26 ± 0.32
3.24 ± 0.09
4.90 ± 0.17
8.22 ± 0.41
4.36 ± 0.09
4.52 ± 0.07
7.70 ± 0.25
C8
Carboplatin
a Because of the limited solubility of complexes, the maximum concentration of complexes
can only reach 10 μM.
b
The selectivity index factor, defined as IC50 (normal HL-7702 cells)/IC50 (HepG2 cancer
cells), is given in parentheses.
Based on this observation, possible structure-activity relationship can be recognized as
follows: dibutyltin complex
> diphenyltin complex > dibenzyltin complex >
monobenzyltin complex. It can be presumed that the number of alkyl substituted on tin
played a key role in the killing of cancer cells. C5 and C6 are monobenzyltin complex,

their tumor cytotoxicity are inferior to other diorganotin complexes. However, in
diorganotin complexes, the difference in cytotoxicity might be attributed to the small steric
hindrance or small molecular weight. This trend is in accordance with the results reported
in the literature [55, 56].
To gain more insights into the possible in vitro cytoselectivity of C2 with the high
cytotoxicity for HepG2 and MCF-7 cell line, MTT assays were also performed in normal
human liver cells (HL-7702) (Table 1). C2 shows the lower cytotoxic activity in the
normal human liver cells line (IC50 = 4.25 ± 0.06 μM) than in HepG2 cell line (IC50 = 0.28 ±
0.06 μM), which demonstrates the obvious specificity for this type of tumor cell and high
selectivity index factors of more than 3.0 [57-59]. So the complex C2 may be used as a
candidate compound for the anticancer drug after further chemical optimization.
3.6 Cell apoptosis analysis by flow cytometry
Table 5 The percentages of apoptosis of C2 in different concentrations.
Q2 (late
Q4
Concentration
Total
Cell
Comp.
apoptosis and
necrotic cell)
(early
(μM)
percentage
apoptosis)
Control
0.64
2.40
0.31
0.82
5.76
11.00
0.82
0.43
1.77
1.89
0.95
3.22
C2
0.1
0.3
0.5
HepG2
21.75
40.94
4.11
27.51
51.94
4.93
Control
C2
0.1
0.3
0.5
5.53
5.96
MCF-7
19.82
36.97
21.59
37.86
On the basis of the cytotoxicity effect study, C2 with good activity was selected for
further experiments. Since C2 has a better inhibitory activity against HepG2 and MCF-7
cells, we performed a flow cytometry assay to determine the level of apoptosis of HepG2
and MCF-7 cells exposed to C2. Early stages of apoptosis are characterized by
perturbations in the cellular membrane. It leads to a redistribution of phosphatidylserine
(PS) to the external side of the cell membrane, which causes a Ca flux. Annexin V is a
Ca-dependent phospholipidbinding protein with high affinity for PS. Therefore,

fluorescently labeled annexin V can be used to identify early apoptosis cells32. The
percentage of apoptosis was presented in Table and Fig. 3 by the region Q4 (early
5
apoptosis) and Q2 (late apoptosis and necrotic cell) and total percentages of apoptosis. We
can see that C2 did not significantly induce apoptosis of HepG2 and MCF-7 cells at low
concentrations (0.1 μM). With the increase of the concentration of C2, the apoptosis
induction obviously increased. The total apoptosis percentages are 27.51%, 51.94%
(HepG2) and 21.59%, 38.86% (MCF-7) at the concentration of 0.3 and 0.5 μM, respectively,
which are also higher than the control. These data show C2 is able to induce HepG2 or
MCF-7 cancer cell apoptosis at some extent, which might partially related to the strong
antitumor activity of C2.
Fig. 3 Effect of C2 on apoptosis of HepG2 (a~d) and MCF-7 cells (e~h). Apoptosis detection in
HepG2 and MCF-7 cells using the Annexin V assay after 24 h. The total percentage of apoptotic
cells was considered as Q2 + Q4. Plot presents the fluorescence data of 7-aminoactinomycin D
(7-AAD) and Annexin V fluorescein corresponding to (a) 0.95% (control) apoptotic cells, (b)
3.22% (0.1 μM), (c) 27.51% (0.3 μM), (d) 51.94% (0.5 μM), (e) 4.93% (control) apoptotic cells,
(f) 5.96% (0.1 μM), (g) 21.59% (0.3 μM), (h) 38.86% (0.5 μM).
3.7 Cell cycle distribution analysis by flow cytometry
Table 6 Cell populations of HepG2 and MCF-7 cells after treating by C2
Concentration
Cell
Comp.
G0/G1 (%)
G2/M (%)
S (%)
(μM)
HepG2
Control
67.69
10.43
21.88

C2
0.1
0.2
0.4
64.58
61.51
55.42
65.80
63.31
57.72
59.20
10.98
10.55
8.55
24.44
27.94
36.03
21.57
25.67
28.27
30.85
Control
12.62
11.03
14.01
9.95
C2
0.1
0.2
0.4
MCF-7
Fig. 4 Effects of C2 on cycle of HepG2 and MCF-7. HepG2 and MCF-7 cells were exposed
various concentrations (0.1-0.4 μM) of C2 in 24 h, and the cells were harvested and flow
cytometry analyzed for cell cycle distribution by propidium iodide (PI) staining.
Cell cycle analysis was performed to investigate the anti-proliferative effects of the C2.
Difference between phases of the cell cycle is based on the content of genetic material. The
cell population in the S phase (DNA replication phase) is synthesizing genetic material,
and thus contains more DNA than quiescent cells. The subsequent G2/M phase
(interphase/mitosis) is characterized by the presence of two copies of DNA [31]. Therefore,
changes in these stages are used as a basis for the treatment of cancer. In order to study
DNA cell content changes in cell cycle, cell cycle analysis was performed by PI single
labeling (Fig. 4). This experiment found that the C2 can change the cell cycle distribution
of HepG2 and MCF-7 (Table 6). Compared with the control group, as the concentration
increases, the proportion of cells in the G2/M phase is reduced, the proportion of cells in
the S phase significantly increased. Apoptosis rate is also significantly improved. It is

suggested that the C2 can arrest the cell cycle in the S phase, and block the progression of
cancer cells from the S phase to the G2/M phase, so that block the mitosis of the cells. The
C2 induces apoptosis by inhibiting DNA replication and transcription, and achieves an
anti-tumor effect. Significantly, the cell cycle arrest at S phase of C2 is entirely different
from reported dibutyltin complexes [24, 31] or platinum-based chemotherapy drug [60,
61]
.
3.8 DNA-binding studies
3.8.1 UV-visible absorption spectrometry
Fig. 5 Electronic spectra of C2 in Tris-HCl buffer upon addition of CT-DNA, ccomplex = 50
μmol·L-1; from a to f, cDNA = 0, 10, 20, 30, 40, 50 μmol·L-1, respectively. The arrow shows the
absorbance changes upon increasing DNA concentrations. Inset: plot of cDNA/(εA − εF) vs ccomplex
.
The electronic absorption spectra of C2 in the absence and presence of CT-DNA are
shown in Fig.5. It can be seen from the Fig.5 that the decrease in absorbance occurs with
the increase of DNA concentration. Hypochromism occurs own to the breakage of the
secondary structure of DNA, whereas hypochromism is due to the stabilization of DNA
duplex through a strong interaction such as intercalation.
From the absorption spectroscopy tests, Kb values of C2 were calculated as 2.35×104
L·mol-1 (r2 = 0.993), as can be seen that it is similar to the reported complexes in the
literature [62, 63], and it explained the intercalation of complexes and DNA are relatively
stronger. It preliminary indicated there are intercalation in the interaction of between the
C2 and the DNA.
3.8.2 Fluorescence competitive study

Fig. 6 Effects of complex C2 on the fluorescent spectra of EB-DNA system, cDNA = 30 μmol·L-1;
cEB = 3 μmol·L-1; from a to i, ccomplex = 0, 10, 20, 30, 40, 50, 60, 70, 80 μmol·L-1, respectively. The
arrow shows the emission intensity changes upon increasing DNA concentrations. Inset: plot of
I0/I vs ccomplex, λex = 258nm.
Fig.6 is fluorescence quenching curve of different concentration C2 on the EB-DNA
system. The fluorescence of EB-DNA system was obviously decreased after the addition
of C2, it is showed that the presence of C2 makes the fluorescence of EB-DNA system
quenched. The fluorescence quenching is up to 68.8% of the initial EB-DNA fluorescence
intensity indicating the competition of the complex with EB in the binding to DNA. The
results show C2 may displace EB from the DNA-EB, interacting with DNA by the
intercalative mode. According to Stern-Volmer correction equation [64]:
I0/I=1+KSVccomplex, the KSV was calculated as 2.54×104 L·mol-1, this value is larger than the
reported in the literature [65, 66]. It indicated that the C2 has a strong intercalation with
DNA, and can be assumed that the tin atom of C2 combined the base pairs of DNA, the
terminal ligands inserted into the DNA base pairs to compete with EB, so that EB is
squeeze out the DNA double helix by C2.
3.8.3 Viscosity measurements

Fig. 7 Effect of increasing amounts of the complex C2, Bu2SnO and Ligand on the relative
viscosity at 25.00 ± 0.02 °C
Viscosity experimental results clearly showed that the relative viscosity of CT-DNA
steadily increasing with concentration of C2, however, this trend is unconspicuous in
Bu2SnO and ligand. The literature [55, 67] reported that a significant increase in the
viscosity of DNA on the addition of a complex indicates the classical intercalative mode of
binding to DNA. In contrast, if the interaction of the complex with the DNA is caused by
partial or non-classical insertion, the viscosity change of the DNA solution is not obvious
or unchanged. As can be seen from the Fig.7, the order of increase in viscosity is C2 >
Ligand > Bu2SnO. This observation can be explained by the fact that the C2 and DNA
adopt a classical intercalation model, which requires that the DNA helix must be extended,
resulting in an increase in DNA viscosity. The result further suggests an intercalating
binding mode of the complexes with DNA and also parallels the above spectroscopic
results.
3.8.4 Gel electrophoresis studies
The effect of complex on DNA cleavage was studied by agarose gel electrophoresis.
The activity depends mainly on the properties, concentration and reaction time of the
complex itself. When the complex interacts with the supercoiled plasmid DNA, the
closed-loop supercoiled structure (Form I) becomes an open-loop notch type (Form II) if it
causes a gap in one strand of the closed-loop supercoil, and when two chains break at the
same position, they become linear structures (Form III) [68]. The migration rate of the
three kinds of plasmid DNA in agarose gel electrophoresis is different. The closed-loop

supercoiled (Form I) moves closer to the front through the gel due to its tight structure,
followed by the linear structures (Form III). The open-loop notch type (Form II) is
restrained due to its loose structure, and the movement to the positive electrode is
suppressed. As can be seen from the Fig.8, upon increasing concentrations of C2 from 0,
20, 40, 60, 80, 100μmol·L-1, the effect on DNA mobility was negligible and even at highest
concentration the complex was able to cleave the DNA, and it can convert pBR322 from
Form I to Form II, and the DNA fracture degree increased with increasing complex
concentration. It explain that C2 have higher cleavage activity and is well inserted into
DNA.
Fig. 8 Agarose gel electrophoresis of pBR322 treated with different concentrations C2, from a to
f, ccomplex = 0, 20, 40, 60, 80, 100 μmol·L-1, respectively.
3.8.5 Molecular Docking
Fig.9 Molecular modeling of the interaction between C2 and DNA
Molecular docking can obviously express the characteristic information of the
interaction between complex and DNA, and guide us to understand the binding mode of

DNA and complex from the molecular level. From Fig. 9, the docking results showed that
C2 bind to DNA by intercalation. It can be seen that the whole C2 molecule inserted into
the double helix of DNA. It suggested that C2 was capable of binding to DNA through
intercalation binding. So docking studies revealed that C2 interacted with DNA through
intercalation binding which was also in accordance with spectroscopic investigations.
Based on the anti-cancer activity data, it is speculated from the docking results that the
whole molecule structure of C2 can be embedded in the base pair of DNA and reversibly
bind to the double helix of DNA, resulting in DNA cleavage. And transcription and
synthesis of DNA is affected, so the anti-tumor effect is finally achieved.
4. Conclusions
Diorganotin complexes (C1, C2, C3, C4, C7, C8) have better in vitro anticancer activity than
monoorganotin complexes (C5, C6). Among these complexes, dibutyltin complex C2 has an
exceptionally high anticancer activity against HpeG2 and MCF-7 cells. In the past, dibutyltin
complexes were considered to be a cell cycle nonspecific agent, as were platinum-based
chemotherapy drugs. However, it was found that the dibutyltin complex of 2-oxo-2-phenylacetic
acid p-nitrobenzoyl hydrazide (C2) is a cell cycle specific agents through flow cytometry in this
paper. It can block HpeG2 and MCF-7 cells in S phase and eventually causes apoptosis. In further
experiments, it was found that C2 can be well embedded in the double helix of DNA and cleave
DNA. This indicates that C2 inhibits DNA synthesis of cancer cells by binding to DNA, so as to
inhibit the growth of cancer cells. In summary, the C2 may be a potential candidate for further
chemical optimization and cancer therapy.
Acknowledgements
This work was supported by the Science and Technology Development Plan Project of
Hengyang City (grant number: 2018KJ121); Hengyang Normal University Industrial Supported
Project (grant number: HXKJ201908, HXKJ201911, HXKJ201946, HXKJ201947); and the
Foundation of Key Laboratory of Functional Organometallic Materials, University of Hunan
Province (grant number: GN19K07).
Appendix A. Supplementary material

Supplementary Material Crystallographic data for the structural analysis have been deposited
with the Cambridge Crystallographic Data Centre, CCDC No. 1479715 (C1); 1479712 (C2);
1479716 (C3); 1479713 (C4); 1897744 (C5); 1897745 (C6); 1897746 (C7) and 1479714
(C8).
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