Aroylhydrazone Diorganotin Complexes Causes DNA Damage and Apoptotic Cell Death: From Chemical Synthesis to Biochemical Effects

Under microwave irradiation, eighteen new aroylhydrazone diorganotin complexes (1a–9b) were produced through the reaction of aroylhydrazine, 2-ketobutyric acid, and the corresponding diorganotin. Fourier transform infrared spectroscopy, 1H, 13C, and 119Sn nuclear magnetic resonance spectroscopies, high-resolution mass spectroscopy, X-ray crystallography, and thermogravimetric analysis (TGA) were performed to characterize the complexes. The in vitro anticancer activity for complexes were assessed using a CCK-8 assay on human cancer cells of HepG2, NCI-H460, and MCF-7. Complex 4b revealed more intensive anticancer activity against MCF-7 cells than the other complexes and cisplatin. Flow cytometry analysis and transmission electron microscope observation demonstrated that complex 4b mediated cell apoptosis of MCF-7 cells and arrested cell cycle in S phase. Western blotting analysis showed that 4b induced DNA damage in MCF-7 cells and led to apoptosis by the ATM-CHK2-p53 pathway. The single cell gel electrophoreses assay results showed that 4b induced DNA damage. The DNA binding activity of 4b was studied by UV–Visible absorption spectrometry, fluorescence competitive, viscosity measurements, gel electrophoresis, and molecular docking, and the results show that 4b can be well embedded in the groove and cleave DNA.

During the last few years, it has been noticeable that organotin compounds occupy an important place in cancer chemotherapy reports [15][16][17] because of their cytotoxic effects, ability to bind with DNA, anti-proliferating nature, and apoptotic-inducing nature. Thus, organotins have emerged as impending biologically active metallopharmaceuticals [18,19]. During the research and development of anticancer drugs, the growth inhibition experiment of drugs in vitro cancer cells is the first step in verifying the drug anticancer effects. It is significant in clarifying drug anticancer spectrums and screening efficient anticancer

Crystal Structure
The molecular structures of 1a-9b are shown in Figure 1. The selected bond lengths and angles for the complexes are listed in Table S5 (Supplementary Materials). The structures of complexes 1a-9b can be divided into four different coordinate types. First, in complexes 1a, 2a, 2b, 3a, 4a, 5a, 8a, and 9a, one-dimensional infinite chain structure was formed by a Sn-O bond between two adjacent asymmetric units (one-dimensional chain structure of complex 1a is shown in Figure 2a, the other in the Supplementary Materials), it was found that the length of the Sn-O bond in each complex was different, but which were much less than the sum of the Van der Waals radii for Sn and O (0.36 nm), which proves that there is a strong interaction between Sn1 and O3 i [30]. The central tin atoms were all six coordinations of the distorted octahedral configuration. Second, in complexes 1b, 4b, 5b, 6b, 7a, 7b, 8b, and 9b, they are centrosymmetric dimer distannoxanes, that is, they are a dinuclear molecule containing a central Sn 2 O 2 four-membered ring, each carboxyl oxygen atom bridges the two tin atoms in an antisymmetric unit. The Sn1-O2-Sn1 i -O2 i system exhibits a 0.0 • torsional angle in every complex, indicating that the four-membered Sn 2 O 2 ring is a planar structure. The central tin atoms are a seven-coordinate structure exhibiting distorted pentagonal pyramidal geometry. These structures are similar to those reported in the literature [31][32][33]. Third, for complex 3b, a two-dimensional network structure is formed by a Sn-O bond between two adjacent asymmetric units (in Figure 2b

Complex IC 50 /µM
Therefore, the structure-activity analysis of the anticancer activity of the complex shows that: (i) for most complexes, the drug sensitivity of MCF-7 cells is greater than that of HepG2 and NCI-H460 cells, the dependence of anticancer activity (IC 50 value) both on the type of alkyl as well as the type of aromatic ring on the ligand; (ii) when the alkyl is stronger, the substituent in the ligand aromatic ring has less effect on the activity, and the inhibitory activity of the butyl group is greater than that of the phenyl group, which proves that the alkyl group attached to the tin atom is the main pharmacophore; (iii) analyzing the same type of alkyl substitution of the central tin atom, with the butyl substituent, the order of influence of the aromatic ring in the ligand is as follows: p-Me-Ph > p-MeO-Ph > p-NO 2 -Ph > 2-furyl > p-OH-Ph > o-OH-Ph > 2-thienyl > Ph > p-t-Bu-Ph, when the phenyl group is the substituent, the order is as follows: p-NO 2 -Ph > p-Me-Ph > p-OH-Ph > Ph > o-OH-Ph > p-MeO-Ph > p-t-Bu-Ph > 2-thienyl > 2-furyl; (iv) the anticancer activity of aroylhydrazone diorganotin complexes may be related to the synergistic effect of alkyl group and ligand; (v) comparing the toxicity of 4b to normal cells HL-7702, 4b shows lower cytotoxic activity in the normal human liver cell line (IC 50 = 7.31 ± 0.61 µM) than in the MCF-7 cell line (IC 50 = 0.47 ± 0.04 µM), which indicated the obvious specificity for this type of tumour cell and high selectivity index factors of more than 3.0. In order to further clarify the anticancer mechanism of aroylhydrazone diorganotin complexes, we selected complex 4b, which had the best inhibitory activity on MCF-7, for subsequent experiments.

Cell Apoptosis Analysis
Because of the superior inhibitory activity of 4b for MCF-7 cells, a flow cytometry test was conducted to detect the apoptosis level when MCF-7 cells were exposed to 4b. Treatment of MCF-7 cells with 4b for 24 h led to the concentration-dependent apoptosis in Figure 3a,c. The apoptosis percentage is expressed as regions Q2 (early apoptotic cells) and Q3 (late apoptotic cells) and total percentages of apoptosis. The 4b exhibited a negligible apoptosis induction effect on MCF-7 cells at low concentrations (0.2 µM). The 4b concentration increased with induction of apoptosis. Total apoptosis percentages were 7.32% and 27.25 % when the concentrations were 0.4 and 0.8 µM, respectively, which were higher than those of the control. As indicated by the data, 4b can mediate MCF-7 cancer cell apoptosis, which is consistent with the strong anticancer activity in 4b.
gible apoptosis induction effect on MCF-7 cells at low concentrations (0.2 μM). The concentration increased with induction of apoptosis. Total apoptosis percentages w 7.32% and 27.25 % when the concentrations were 0.4 and 0.8 μM, respectively, which w higher than those of the control. As indicated by the data, 4b can mediate MCF-7 can cell apoptosis, which is consistent with the strong anticancer activity in 4b.

Cell Cycle Distribution Analysis
Cell cycle analysis was performed to investigate the anti-proliferative effects of 4b. Difference between phases of the cell cycle is based on the content of genetic material. The S phase of the cell cycle is the DNA replication phase, during which a large amount of genetic material is synthesized, so this phase contains more DNA than quiescent cells. As shown from the cell cycle detection (Figure 3b,d), after treatment with 4b, there was significant upregulation in the S phase and significant downregulation in the G2/M phase, blocking the progression of cancer cells from the S phase to the G2/M phase, thus blocking the mitosis of the cells. 4b induces apoptosis by inhibiting DNA replication and transcription, and achieves an anti-tumor effect. These results indicated that 4b can induce cell cycle arrest. Significantly, the cell cycle arrest at S phase of 4b is similar to the reported metal complexes [34][35][36] and is different from cisplatin.

TEM Observation of Cell Morphology
In order to further clarify the cell death mode induced by 4b, we used transmission electron microscopy to observe the effect of 4b on cell ultrastructure. As shown in Figure 4a,b, the cell membrane, nucleus, and other organelles of MCF-7 cells in the control group did not show abnormal changes. After 4b treatment, the ultrastructure of MCF-7 cancer cells were obviously destroyed. According to Figure 4c,d, we observed that the cytoplasm was concentrated and the cell volume shrank, so the microvillus on the cell membrane surface disappeared, and the structure of the other organelles were blurred. The nuclear envelope still existed, but its shape was irregular. The chromatin in the nucleus condensed and fragmented into several pieces, which were scattered in the nucleus. It is very obvious that the above appearance belonged to the specific characteristics of apoptosis. cycle arrest. Significantly, the cell cycle arrest at S phase of 4b is similar to the re metal complexes [34][35][36] and is different from cisplatin.

TEM Observation of Cell Morphology
In order to further clarify the cell death mode induced by 4b, we used transm electron microscopy to observe the effect of 4b on cell ultrastructure. As shown in 4a,b, the cell membrane, nucleus, and other organelles of MCF-7 cells in the contro did not show abnormal changes. After 4b treatment, the ultrastructure of MCF-7 cells were obviously destroyed. According to Figure 4c,d, we observed that the cyt was concentrated and the cell volume shrank, so the microvillus on the cell mem surface disappeared, and the structure of the other organelles were blurred. The envelope still existed, but its shape was irregular. The chromatin in the nucleus con and fragmented into several pieces, which were scattered in the nucleus. It is very o that the above appearance belonged to the specific characteristics of apoptosis.

Western Blotting
After the MCF-7 cell line was treated with different concentrations of 4b, compared with the control group, ATM and Chk2 protein had no obvious change trend, and the difference was not statistically significant. With the increase in 4b concentration, Bax, cleaved caspase-3, Cytochrome c, p53, p-ATM, and p-Chk2 had a significant upward-regulation trend, Bcl-2 expression was significantly reduced, and the difference was statistically significant ( Figure 5).
with the control group, ATM and Chk2 protein had no obvious change trend, and difference was not statistically significant. With the increase in 4b concentration, B cleaved caspase-3, Cytochrome c, p53, p-ATM, and p-Chk2 had a significant upward-r ulation trend, Bcl-2 expression was significantly reduced, and the difference was stati cally significant ( Figure 5). ATM is the direct receptor of DNA double-strand breaks (DSBs). After damage curs, ATM automatically undergoes phosphorylation and is activated. Among them, autophosphorylation of p-ATM (Ser1981) is a recognized marker of ATM activation. AT is very sensitive to DNA damage, and only a small amount of DSBs can cause rapid ph phorylation of most molecules at the Ser1981 site. The results of western blotting show that the protein level of total ATM did not change, while the phosphorylated ATM w significantly upregulated with the increase in drug concentration, indicating that ph phorylated ATM was its activated form. Among the many downstream effectors, Ch plays a very important role. Chk2 is a serine/threonine kinase that interferes with damage repair mechanisms. When there is no DNA damage, Chk2 is mostly uniform dispersed in the cell nucleus in the state of inactive monomers. Once the external envir mental conditions cause DSBs, they will gather at the damaged DNA site. The activa ATM interacts with Chk2 to phosphorylate a series of sites. The DNA damage repair pa way is to complete the repair of damaged DNA through a series of phosphorylated tar proteins that bind to the DNA damage site together. If the DNA damage cannot be eff tively repaired, the damaged cell will initiate the apoptosis mechanism. One pathway, DNA damage activates ATM kinase, and ATM directly activates the p53ser15 site to m ATM is the direct receptor of DNA double-strand breaks (DSBs). After damage occurs, ATM automatically undergoes phosphorylation and is activated. Among them, the autophosphorylation of p-ATM (Ser1981) is a recognized marker of ATM activation. ATM is very sensitive to DNA damage, and only a small amount of DSBs can cause rapid phosphorylation of most molecules at the Ser1981 site. The results of western blotting showed that the protein level of total ATM did not change, while the phosphorylated ATM was significantly upregulated with the increase in drug concentration, indicating that phosphorylated ATM was its activated form. Among the many downstream effectors, Chk2 plays a very important role. Chk2 is a serine/threonine kinase that interferes with cell damage repair mechanisms. When there is no DNA damage, Chk2 is mostly uniformly dispersed in the cell nucleus in the state of inactive monomers. Once the external environmental conditions cause DSBs, they will gather at the damaged DNA site. The activated ATM interacts with Chk2 to phosphorylate a series of sites. The DNA damage repair pathway is to complete the repair of damaged DNA through a series of phosphorylated target proteins that bind to the DNA damage site together. If the DNA damage cannot be effectively repaired, the damaged cell will initiate the apoptosis mechanism. One pathway, the DNA damage activates ATM kinase, and ATM directly activates the p53ser15 site to mediate MCF-7 cell apoptosis. Another pathway is that when DNA damage occurs, it is detected by ATM and other related kinases, and then the damage signal is transmitted to downstream Chk2 and p53. The increase in p53 expression leads to the downregulation of the apoptosis inhibitor Bcl-2 expression, activates the expression of pro-apoptotic factor Bax, and opens the mitochondrial membrane channel [37]. The Cytochrome c is released into the cytoplasm, and finally activates the apoptosis executive protein caspase-3 to trigger cell apoptosis.

Comet Assay
Slight DNA damage usually leads to cell cycle arrest, while severe DNA damage can lead to cell apoptosis and necrosis. Comet assay is a versatile and sensitive method for measuring DNA damage based on DNA single and double-strand breaks. The comet assay was performed using MCF-7 cells to research the effect of complex 4b on the DNA of the cells. As shown in Figure 6, in the control (a), no DNA damage was seen. However, MCF-7 cells incubated with the 0.4 µM 4b for 24 h showed a well-shaped comet (b), and the length of the comet's tail represents the extent of DNA damage. The results confirm that 4b can induce DNA damage, which is further evidence of apoptosis.

Comet Assay
Slight DNA damage usually leads to cell cycle lead to cell apoptosis and necrosis. Comet assay i measuring DNA damage based on DNA single an say was performed using MCF-7 cells to research t the cells. As shown in Figure 6, in the control (a), MCF-7 cells incubated with the 0.4 μM 4b for 24 h the length of the comet's tail represents the extent that 4b can induce DNA damage, which is further  illustrates the absorption spectra of complex 4b in the presence of increasing amounts of CT-DNA. A strong absorption peak around 380 nm was observed. After interaction with the increasing concentration of CT-DNA, it could be seen that hypochromism was observed along with a red shift of 18 nm. The reason for hypochromism is that the interaction between the complex and the DNA causes the change in the molecule conformation. The change in the spectrum is related to the binding force, where the stronger the effect, the more obvious the hypochromism. From the absorption spectroscopy tests, K b was calculated as 0.82 × 10 4 L·mol −1 (r 2 = 0.998), and the observed values of K b were in good agreement with the reported values (the order of 10 4 L·mol −1 ) of similar intercalated metal-complexes [38][39][40]. The relatively large amount of K b and red shift suggested that there was strong interaction between these complexes and the CT-DNA double helix. measured as 2.3 × 10 4 L•mol −1 , which was similar to the reported value [41][42][43]. This resul implies that 4b can intercalate into DNA. In case the tin atom in 4b interacts with the bas pairs of DNA, the insertion of terminal ligands into such pairs can be achieved to realiz EB competition. Thus, squeezing of EB from the double helix of DNA can be achieved through 4b.

Viscosity Measurements
The viscosity experiments clearly showed that the relative viscosity of CT-DNA steadily increased with a concentration of 4b, however, this trend was not obvious fo Bu2SnO and the ligand. As can be seen from Figure 7c, the order of increase in viscosity was 4b > Ligand > Bu2SnO. This can be explained that the 4b and DNA employed a clas sical intercalation model, which demanded that the DNA helix must be extended, causing an increase in DNA viscosity [44,45]. The results further suggest an intercalating binding mode of the complex with DNA and is also consistent with the spectroscopic results and molecular docking.

Gel Electrophoresis Studies
As can be seen from Figure 7d, complex 4b can effectively cut the pBR322, and it cutting activity is related to the concentration of the complex. As the concentration of th complex is gradually increased, Form II was also observed. However, when the concen tration reached 80 μmol•L −1 , there was still no Form III. Thus, complex 4b can effectively cut pBR322 from Form I to Form II.  Figure 7b illustrates the fluorescence quenching curve of complex 4b at varying concentrations in the EB-DNA system. Fluorescence in the system reduced when 4b was added, which revealed that 4b could quench the fluorescence in the EB-DNA system. The complex could compete with EB for binding to the site on CT-DNA. The more 4b is added, the more EB originally bound to CT-DNA will be replaced by the 4b, which will reduce the fluorescence intensity of the EB-DNA system. Relevant illustrations are displayed to confirm that the EB-DNA fluorescence quenching by 4b can be attributed to 4b rivalry against EB. By using the Stern-Volmer correction formula: I 0 /I = 1 + K SV c complex , the K SV was measured as 2.3 × 10 4 L·mol −1 , which was similar to the reported value [41][42][43]. This result implies that 4b can intercalate into DNA. In case the tin atom in 4b interacts with the base pairs of DNA, the insertion of terminal ligands into such pairs can be achieved to realize EB competition. Thus, squeezing of EB from the double helix of DNA can be achieved through 4b.

Viscosity Measurements
The viscosity experiments clearly showed that the relative viscosity of CT-DNA steadily increased with a concentration of 4b, however, this trend was not obvious for Bu 2 SnO and the ligand. As can be seen from Figure 7c, the order of increase in viscosity was 4b > Ligand > Bu 2 SnO. This can be explained that the 4b and DNA employed a classical intercalation model, which demanded that the DNA helix must be extended, causing an increase in DNA viscosity [44,45]. The results further suggest an intercalating binding mode of the complex with DNA and is also consistent with the spectroscopic results and molecular docking.

Gel Electrophoresis Studies
As can be seen from Figure 7d, complex 4b can effectively cut the pBR322, and its cutting activity is related to the concentration of the complex. As the concentration of the complex is gradually increased, Form II was also observed. However, when the concentration reached 80 µmol·L −1 , there was still no Form III. Thus, complex 4b can effectively cut pBR322 from Form I to Form II.

Molecular Docking
Docking results of Figure 8 prove that the binding of 4b to DNA relies on intercalation. The ligand part of 4b was completely inserted into the double helix of DNA. The atoms are substantially planar on the ligand, and the steric hindrance is relatively small. The deoxyribonucleotide ring formed a coordinate bond with the central tin atom, with a bond length of 2.605 Å (see Figure S119). The results indicate that 4b can bind to DNA via intercalation, which was determined through spectroscopic investigations. Docking results of Figure 8 prove that the binding of 4b to DNA relies on intercalation. The ligand part of 4b was completely inserted into the double helix of DNA. The atoms are substantially planar on the ligand, and the steric hindrance is relatively small. The deoxyribonucleotide ring formed a coordinate bond with the central tin atom, with a bond length of 2.605 Å (see Figure S119). The results indicate that 4b can bind to DNA via intercalation, which was determined through spectroscopic investigations.
( The microwave synthesis reaction was completed using the Sineo Microwave MDS-10 High-throughput Microwave Sample Preparation Workstation (Shanghai, China). Elemental analyses for C, H, and N were determined on a PE-2400(II) analyzer (Waltham, MA, USA). The IR spectrum was obtained for KBr pellets on a Shimadzu Prestige-21 spectrophotometer in the 4000-400 cm −1 (Kyoto, Japan). 1 H, 13 C, and 119 Sn NMR analysis were performed on a Bruker AVANCE NMR spectrometer (Karlsruhe, Germany). HRMS was obtained by Thermo Scientific LTQ Orbitrap XL (Waltham, MA, USA) or Waters UPLC I-Class Xevo G2 XS-Q Tof (Milford, MA, USA) with ESI. Crystal structure was determined on a Bruker SMART APEX II X-ray diffractometer (Karlsruhe, Germany). Thermogravimetric analyses (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 (Selb, Germany). Melting point measurement was executed on an X-4 binocular micromelting point apparatus with the temperature unadjusted (Beijing, China). UV-Vis absorption spectra was measured by a Shimadzu UV-2550 spectrometer (Kyoto, Japan). Fluorescence spectra were obtained with a Hitachi F-7000 spectrophotometer (Tokyo, Japan) with a quartz cuvette (path length = 1 cm). Viscosity experiments were conducted on an Ubbelodhe viscometer (Shanghai, China). Gel electrophoresis was measured by DYY-6C electrophoresis power supply (Beijing, China). Cell apoptosis was measured by the BD FACSCalibur CellSorting System (Franklin Lakes, NJ, USA).

Single Crystal Structure Determination
X-ray diffraction spectral collection for crystals was achieved using a Smart Apex II CCD diffractometer from Bruker through Mo-K α (λ = 0.71073 Å) radiation using a graphite monochromator. The ϕ-ω scan technique was used for diffraction spectral gathering at an ambient temperature. The acquired data were subjected to multiscan correction of absorption. The crystal structures were determined through direct manipulation, and full-matrix least-squares was performed to refine the crystal structures on F 2 . Before anisotropical refinement, successive difference Fourier syntheses yielded all the remaining atoms (nonhydrogen). The hydrogen atoms were positioned in measured locations or placed onto the Fourier maps, followed by isotropical refinement using the atom (nonhydrogen)-associated variables of isotropic vibration they were bound to. Measurements were performed using SHELXL [46] on WINGX [47]. Complex-related crystal information and structure refinement parameters are presented in  1a, 1b, 2a, 2b, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b, 9a, and 9b).

In Vitro Anticancer 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). HL-7702 cell was obtained from the Chinese Academy of Sciences. The cells were maintained at 37 • C in a 5% CO 2 incubator in RPMI 1640 (GIBICO, Invitrogen) containing 10% fetal bovine serum (GIBICO, Invitrogen). The 5 mM 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 the CCK8 assay. A total of 100 µL of cells (40 cells·µL −1 ) incubated at 37 • C, 5% CO 2 was seeded into 96-well plates. Then, the medium was replaced with the respective medium containing complexes at different concentrations and incubated for 24 h. Ten µL CCK8 solution was added to each well and incubated at 37 • C for an additional 4 h. The optical density was detected with a microplate reader at 450 nm. Eleven concentrations (0.03 µM-32 µM) were set for the compounds and at least three parallels of every concentration were used. All experiments were repeated at least three times. The data were calculated using Graph Pad Prism version 7.0. The IC 50 was fitted using a non-linear regression model with a sigmoidal dose response.

Cell Apoptosis Assay
Approximately 1 × 10 5 cells were inoculated on each well of a six-well plate and subjected to 24 h cultivation. Next, cells were processed using candidate complexes at varying concentrations for 24 h, and were then subjected to two rounds of cold PBS rinsing, followed by assaying with an Annexin-V-FITC/PI Apoptosis Kit (MultiSciences Biotech, China) according to the manufacturer's instructions. Cell apoptosis was tested using a flow cytometer (BD TM FACS Calibur, Franklin Lakes, NJ, USA).

Cell Cycle Analysis
Approximately 1 × 10 5 cells were inoculated on each well of a six-well plate and subjected to 24 h cultivation. The cell cycle progression after treatment with 4b was performed by using the Cell Cycle and Apoptosis Analysis Kit (Beyotime Biotechnology, China), according to the manufacturer's instructions and reference methods. MCF-7 cells were treated with indicated concentration of 4b in 2 mL total medium for 24 h before harvest. The cells were fixed with ice-cold 70% ethanol overnight at 4 • C. After centrifugation, the cell pellets were washed with cold PBS once, and RNaseA (10 µL) and PI (10 µL) were added to the cells. Cells were then incubated for 15 min at room temperature in the dark and then detected by flow cytometry. Parallel determination was conducted three times.

Transmission Electron Microscope (TEM)
MCF-7 cell aggregates were fixed with 2.5% glutaraldehyde-PBS buffer for 2 h at 4 • C. After rinsing three times with PBS buffer for 30 min, the samples were post-fixed with 1% OsO 4 for another 2 h at 4 • C. Then, the samples were dehydrated in a graded series of ethanol-acetone with gradient ascent (50-70-90-100%), and embedded in epoxy resins. The sample was sliced through with an ultramicrotome, stained with uranyl acetate, and examined under electron microscope (Talos F200C, FEI, Hillsboro, OR, USA).

Comet Assay
MCF-7 cells (2 × 10 5 cells/mL) were performed to plant in 6-well plates and incubated for 24 h. Cells were dealt with 4b (0.4 uM) for 24 h. Moreover, cells were collected and washed with PBS, then cell suspension and 1% low melting point agarose were mixed to drop onto slides and placed with glass coverslips, which was allowed to settle for 10 min at 4 • C. Coverslips were removed and cells were lysed for 1 h at 4 • C. The slides were then placed in freshly prepared alkaline electrophoresis solution to unwind DNA for 20 min. Electrophoresis was performed under alkaline conditions for 20 min at 25 V and 300 mA. After electrophoresis the cells were stained with ethidium bromide (EB) solution for 20 min in darkness at 4 • C. Comet images were obtained by fluorescence microscope (Nikon ECLIPSE Ti, Japan).

UV-Visible Absorption Spectrometry
The potential modes of the complex binding with DNA were assessed with a UV-Vis spectroscope, which was also used for the determination of K b , the relevant intrinsic DNA-binding constants. The following Wolfe-Shimmer equation was used for K b value determination [48]: where c DNA refers to the CT-DNA concentration; ε A represents the extinction coefficient detected at a random concentration of DNA; ε F denotes the free complex's extinction coefficient; and ε B denotes the extinction coefficient during full complex-CT-DNA binding. We recorded the UV-Vis data of DNA under a 25 • C condition over 0-80 µM concentrations at a complex concentration of 50 µM. We collected the spectra in a 270-800 nm range. Corresponding slit width was set to 5 nm. The K b values were determined using the Wolfe-Shimmer formula plus c DNA /(ε A − ε F ) vs. c DNA plots .

Fluorescence Competitive Study
For the fluorescence investigation, a volumetric flask (5 mL) was used to prepare a mixture comprising 30 µM CT-DNA, 3 µM EB, and 0-80 µM complex. Fluorescence data collection was performed for 3 h under 25 • C. The results revealed emission wavelengths ranging from 540 to 700 nm and an excitation wavelength of 258 nm. Slit width setting was 5 nm for both emission and excitation. Finally, the Stern-Volmer equation was used to determine the quenching constant (Ksv) of the complex.

Viscosity Measurements
Viscosity tests were performed using the DNA concentration of 0-50 µM. The test was performed on the Ubbelohde viscometer. The obtained samples were stored at 25 (±0.02) • C in a water bath. For the determination of the flow time in samples, the digital stopwatch was used. Viscosity values were computed based on the flow time of DNA including solutions to correct the flow time in buffer (t 0 ), η = (t − t 0 ) [49]. The data are presented by (η/η 0 ) 1/3 vs. (c complex /c DNA ), in which η indicates the DNA viscosity of the complex; η 0 represents the DNA viscosity; and c complex refers to the complex concentration. Here, c DNA denotes the DNA concentration.

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 4b (0, 20, 40, 60, 80 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 a gel imaging documentation system under UV light mode.

Molecular Docking
The molecular operating environment (MOE) program was used to perform docking between molecules. We obtained the crystal architecture information of the DNA molecules from the Protein Data Bank (code: 453D) and eliminated water and embedded small molecules through MOE. Hydrogen atoms were introduced into DNA according to the electrostatic requirements. Next, point charges were computed, with minimal corresponding energy. The CIF data for the complex were transformed into its PDB counterpart by using Mercury 3.8 before it was imported into MOE. Next, symmetrical units and coordinated solvent molecules were erased, along with minimization of corresponding energies, thus obtaining the complex for subsequent docking. Discovery Studio 3.5 could be used to derive the molecular docking outcomes.

Conclusions
Eighteen diorganotin (IV) complexes were synthesized through one-pot microwave irradiation. NMR, HRMS, and X-ray single-crystal diffraction were performed to characterize complex structure. Biological assay results indicated that all the complexes exhibited anticancer activity against test cancer cells (HepG2, NCI-H460, MCF-7), and 4b exhibited superior inhibitory activity in MCF-7 cell lines to other complexes. More importantly, we studied the anti-tumor mechanism of complex 4b, where the results demonstrated that 4b causes cell apoptosis by damaging DNA. DNA binding studies have shown that complex 4b can interact with DNA through insertion mode.