Syntheses, Crystal Structures and in vitro Anticancer Activities of Dibenzyltin

JIANG Wu-Jiu MO Tian-Zi ZHANG Fu-Xing KUANG Dai-Zhi TAN Yu-Xing

Syntheses, Crystal Structures and in vitro Anticancer Activities of Dibenzyltin Compounds Based on the-(2-Phenylacetic acid)-aroyl Hydrazone①

JIANG Wu-Jiu MO Tian-Zi ZHANG Fu-Xing KUANG Dai-Zhi TAN Yu-Xing②

(421008)

Twodibenzyltin compounds, {[C6H5O(O)C=N–N=C(Ph)COO] (-Cl–C6H4CH2)2- Sn(CH3OH)}2(1) and {[-Me–C6H4O(O)C=N–N=C(Ph)COO] (-Cl–C6H4CH2)2Sn(CH3OH)}2(2), have been synthesized by microwave “one pot” reaction with benzoylhydrazine (or-methyl benzhydrazine), phenylglyoxylic acid and di--chlorobenzyltin dichloride. Compound 2 contains twocrystals2a and 2b. The compoundshave been characterized with IR, elemental analysis,1H,13C and119Sn NMR spectra, H RMS. Three crystals are all binuclear molecules with a Sn2O2four-membered ring plane, and each Sn atom center is seven-coordinated with [SnO4C2N] to form a distorted pentagonal bipyramidal configuration.antitumor activities of all compounds and carboplatin were evaluated by MTT against three human cancer cells such as NCI-H460 (lung cancer cells), HepG2 (liver cancer cells) and MCF7 (breast cancer cells), and compound 2 exhibited better antitumor activity.

organotin complex, hydrazine, synthesis, crystal structure, biological activity;

1 INTRODUCTION

Cancer is a serious threat to human life and health as one of the important causes of human disease death. In the process of clinical use, cisplatin[1], a metal chemotherapy drug for early cancer treatment, has exposed some problems such as high toxicity and drug resistance. With further understanding of pharmacological action, many new metal compounds with anti-cancer activities have been synthesized[2-4]. Dialkyltin compounds have been concerned due to their similar structures to cisplatin and better anticancer activities in vitro than cisplatin and other platinum drugs[5, 6]. Researches show the biological activity of organotin compounds has been deter- mined with the organic groups connected to tin atoms and the ligands coordinated with them[7, 8]. Therefore, we can obtain metal anticancer com- pounds with different activities with different organic groups or ligands.

Hydrazone is obtained by dehydration after nucleophilic addition reaction between hydrazine and aldehyde or ketone, and is a special class of Schiff base compounds with –CONHN=CH–. In addition, the groups are found such acyl, sub-amino and imide in the hydrazone compounds. The stability of these compounds is good because the unshared electron of nitrogen atom on sub-amino group can formconjugation system with C=N groups of acyl group and imide. Furthermore, hydrazone compounds contain imino-nitrogen and carbonyl-oxygen atoms, as well as other coordination atoms with strong coordination ability, which can form stable com- pounds with transition metals, rare earth metals, major metals and nonmetals. Many experiments showed that the biological activity of acylhydrazone compounds was enhanced significantly compared with the corresponding ligands, and the compounds had extensive biological and drug activities[9-11].

Based on the previous researches, in the work, two new dibenzyltin compounds have been synthesized by the microwave “one pot” with benzoylhydrazine (or-methyl benzhydrazine), phenylglyoxylic acid and di--chlorobenzyltin dichloride. The groups of aroyl hydrazoneligands to inhibit cancer cell activity have been investigated preliminarily, which lay a foundation for screening new organic-metallic com- pounds with high anticancer activity.

2 EXPERIMENTAL

2.1 Instruments and reagents

Italian MILESTONE microwave synthesizer was employed for the compounds. IR spectra were recorded using the Shimadzu Prestige 21 infrared spectrometer in the range of 4000～400 cm–1(KBr pellets).1H,13C and119Sn NMR were measured by Bruker AVANCE-500 NMR. Elemental analysis was performed by the PE-2400(II) element analyzer. H RMS was determined by the Thermo Scientific LTQ Orbitrap XL (ESI source). Melting point was measured by Beijing Tektronix X-4 binocular photo- micrograph (thermometer not corrected). Thermogra- vimetric analyses were executed on a NETZSCH TG 209 F3 thermogravimetric analyzer.

The di--chlorobenzyltin dichloride was obtained according the literature[12]. Carboplatin was pur- chased from Balinway technologies Co. LTD. NCI- H460, HepG2 and MCF7 cells were obtained from the U.S. tissue culture library (ATCC). RPMI 1640 medium with 10% fetal bovine serum was purchased from GIBICO.

2.2 Syntheses of the compounds

The methanol solution (20.0 mL) of benzoylhydra- zine (or-methyl benzoylhydrazine, 1.0 mmol), phenylglyoxylic acid (1.0 mmol) and di--chloro- benzyltin dichloride (1.0 mmol) was added into 100.0 mL microwave reaction tank with microwave reaction for 30 min at 100 °C, then the mixture was cooled to room temperature and filtered into a conical flask. The solvent was controlled for volatile- zation into crystals1, 2a and 2b.

Crystal1: The product was yellow crystal. Yield: 68%. m. p.: 108～110 °C. Anal. Calcd. (C30H26Cl2N2O4Sn): C, 53.93; H, 3.92; N, 4.19%. Found: C, 53.94; H, 3.92; N, 4.20%. FT-IR (KBr, cm–1): 3489, 3057, 2961, 2941, 1618, 1593, 1566, 1493, 1479, 1435, 1425, 1391, 1317, 1287, 1252, 1173, 1155, 1088, 1070, 1020, 999, 866, 804, 781, 731, 712, 687, 631, 594, 577, 498, 476, 432.1H NMR (500 MHz, CDCl3,/ppm): 7.97 (d,= 7.3 Hz, 2H), 7.52～7.55 (m, 6H), 7.40～7.43 (m, 2H), 7.09 (s, 2H), 6.88 (s, 6H), 3.39 (s, 4H).13C NMR (126 MHz, CDCl3,/ppm): 176.06, 168.79, 147.38, 138.77, 134.35, 132.47, 132.31, 131.40, 130.98, 129.70, 129.19, 128.38, 128.31, 128.18, 127.47, 126.43, 125.72, 35.81.119Sn NMR (Me4Sn, 187 MHz, CDCl3,/ppm): –638.95. H RMS (ESI) m/z calcd. for C29H23Cl2N2O3Sn+[M-CH3OH+H]+637.01022, found 637.01093.

Crystal 2a: The product was yellow crystal in the yield of 37%. m. p.: 144～146 °C. Anal. Calcd. (C31H28Cl2N2O4Sn): C, 54.58; H, 4.14; N, 4.11%. Found: C, 54.60; H, 4.16; N, 4.10%. FT-IR (KBr, cm–1): 3462, 3049, 3011, 2938, 2828, 1624, 1609, 1595, 1584, 1570, 1476, 1427, 1393, 1306, 1281, 1250, 1177, 1155, 1090, 1078, 1063, 1018, 870, 806, 781, 745, 729, 691, 619, 594, 575, 471, 432.1H NMR (500 MHz, CDCl3,/ppm): 7.86 (d,= 7.9 Hz, 2H), 7.51～7.52 (m, 5H), 7.21 (d,= 7.6 Hz, 2H), 7.09 (s, 2H), 6.84～6.89 (m, 6H), 3.40 (s, 4H), 2.43 (s, 3H).13C NMR (126 MHz, CDCl3,/ppm): 176.20, 169.51, 146.39, 143.09, 138.96, 134.23, 131.33, 130.75, 129.62, 129.29, 128.91, 128.57, 128.37, 128.17, 127.40, 126.45, 125.59, 36.57, 21.82.119Sn NMR (Me4Sn, 187 MHz, CDCl3,/ppm): –637.74. HRMS (ESI) m/z calcd. for C30H25Cl2N2O3Sn+[M-CH3OH+H]+651.02587, found 651.02594.

Crystal 2b: The product was red-brown crystal in 35% yield. m.p.: 144～146 °C. Anal. Calcd. (C31H28Cl2N2O4Sn): C, 54.58; H, 4.14; N, 4.11%. Found: C, 54.55; H, 4.13; N, 4.12%. FT-IR (KBr, cm–1): 3433, 3059, 3013, 2951, 1624, 1595, 1570, 1493, 1476, 1443, 1425, 1391, 1310, 1290, 1254, 1175, 1155, 1088, 1078, 1016, 866, 837, 804, 785, 746, 729, 692, 621, 594, 573, 488, 473, 434.1H NMR (500 MHz, CDCl3,/ppm): 7.86 (d,= 7.9 Hz, 2H), 7.50～7.54 (m, 5H), 7.21 (d,= 7.6 Hz, 2H), 7.09 (s, 2H), 6.84～6.89 (m, 6H), 3.38 (s, 4H), 2.43 (s, 3H).H RMS (ESI) m/z calcd. for C30H25Cl2N2O3Sn+[M-CH3OH+H]+651.02587, found 651.02661.

2.3 Crystal structuredetermination

The suitable samples (1: 0.23 × 0.21 × 0.21 mm3, 2a: 0.22 × 0.21 × 0.21 mm3and 2b: 0.22 × 0.21 × 0.21 mm3) were chosen for crystallographic study and then mounted at the Bruker SMART APEX II CCD single crystal diffractometer with graphite-mo- nochromated Mo-radiation (= 0.071073 nm) with ascan mode. All the data were corrected byfactors and empirical absorbance. The structure was solved by direct methods. All non-hydrogen atoms were determined in successive difference Fourier synthesis, and all hydrogen atoms were added according to theoretical models. All hydrogen and non-hydrogen atoms were refined by their isotropic and anisotropic thermal parameters through full-matrix least-squares techniques. All calculations were completed by the SHELXTL-97 program[13]. The selected bond lengths and bond angles for 1, 2a, 2b are listed in Tables 2, respectively.

2.4 Thermogravimetry studies

Thermogravimetric analyses were executed on a NETZSCH TG 209 F3 thermogravimetric analyzer with the heating speed at 20 °C·min–1and gas flow rate of 20 mL·min–1in the air.

2.5 Anticancer activity studies

The drug was dissolved in a small amount of DMSO. The mixture was diluted with water to the required concentration, and maintain the final con- centration of DMSO < 0.1%. NCI-H460, HepG2 and MCF7 cells were cultured in vitro using RPMI 1640 (GIBICO) culture medium containing 10% fetal bovine serum in a 5% (volume fraction) CO2, 37 °C saturated humidity incubator. In vitro anti- cancer drug sensitivity test was determined by MTT assay. Graph Pad Prism version 7.0 program was used for data processing, and IC50was obtained by fitting the nonlinear regression model with S-shaped dose response in the program.

3 RESULTS AND DISCUSSION

3.1 Syntheses

The synthetic routes of compounds are depicted in Fig. 1. As we know, the conventional synthesis methods of organotin compound include the reflux method, solvent-thermal and interfacial diffusion one[14], while the first one is the most common. However, we adopted the microwave “one pot”, which has the characteristics of short reaction time, simple reaction steps, easy purification and good reproducibility compared with traditional heating reflux method. Moreover, the yields of dibenzyltin compounds are around 70%, suggesting the yield of microwave methods is improved greatly[15].

In addition, in the syntheses of 2, two kinds of crystals with different colors and shapes were pre- cipitated at the same time. One is a yellow fine-block crystal (2a), and the other a red-brown rod-shaped crystal (2b), with their crystal morphology shown in Fig. 2. Two crystals were characterized by elemental analysis, IR, NMR, and X-ray single-crystal dif- fraction, showing their chemical compositions and molecular structures are the same, but the cell parameters were different, indicating 2a and 2b are homogeneous polycrystalline.

Fig. 1. Synthesis routes of the compounds

Fig. 2. Crystal appearance of 2a and 2b

3.2 Spectraanalyses

In the infrared spectra, the absorption bands at 3489 (1), 3462 (2a) and 3433 (2b) cm–1indicate the O–H stretching of coordinated methanol molecules. The peaks of 1566, 1570 and 1570 cm–1can be attributed to the C=N–N=C of acyl hydrazone[16, 17]. The carboxylate group appeared at 1618 (1), 1624 (2a) and 1624 (2b) cm–1for the antisymmetric stretching vibration and 1391 (1), 1393 (2a) and 1391 (2b) cm–1for symmetric stretching vibration. Furthermore, the Δvalues (Δ=asym(COO–)–sym(COO–)) are 227 (1), 231 (2a), 233 (2b) cm–1(2b), respectively, suggesting all Sn atoms of compounds coordinated the O atoms of carboxylic with a monodentate chelating modes[18], which are consistent with their crystal structures. The charac- teristic peaks of Sn–O–Sn，Sn–O，Sn–N and Sn–C are located at 619~631, 573~577, 471~476 and 432~434 cm–1, respectively, which are consistent with the peak locations of similar compounds re- ported in the literature[19, 20], indicating preliminarily that 1, 2a and 2b have similar structures.

In1H NMR spectrum, the ratio of the integral area of each group of the three crystals is consistent with the number of protons in each group of the expected structure[21]. Furthermore, it can be found the chemical displacements and peak shapes of all hydrogen protons in 2a and 2b remain the same, which further suggests 2a and 2b have the same asymmetric units. While the peak situation of hydrogen protonsin 1 is similar to that of 2a and 2b, which speculated 1 has similar asymmetric unit structure to 2a and 2b.

In the13C NMR spectrum, the peak positions of carboxyl carbon, hydrazine carbon and imino carbon in compounds 1 and 2a are consistent, and the peaks of the remaining groups are consistent with the number of carbon atoms in the theoretical struc- ture[21]. Based on the1H NMR and13C NMR spectra, it can be further inferred that the structures of 1 and 2a are similar, which is consistent with the X-ray single-crystal diffraction results.

In119Sn NMR spectrum, the single peak of tin atoms present at 638.95 and 637.74 ppm in 1 and 2a, respectively indicate 1 and 2a show only a class of tin, and their chemical shifts of the tin atoms are close. Therefore, tin atoms have similar chemical environments with similar structures.

In HRMS spectrum, the mass peaks at m/z 637.01093 (1), 651.02594 (2a) and 651.02661 (2b) are attributed to the absorption peaks of [M– CH3OH+H]+, indicating the weak Sn–O bonds will be broken in the solution state and present monomer structure in three crystals, which also further sug- gests 2a and 2b show similar structures in solution state.

3.3 Description of the structure

Fig. 3. Molecular structures of compounds (Symmetry codes: 1,i–, –, –; 2a,i1–, –, –; 2b,i1–, 1–, 1–)

Table 1. Crystallographic Data of the Compounds

Compounds 1 and 2 have similar structural units, showing a distorted seven-coordinated pentagonal bipyramidal configuration with only little difference in bond parameters, which is consistent with the conclusion obtained by spectral characterization.

3.4 Thermal stability

Thermal analyses were performed for the synthesi- zed compounds under an air atmosphere, as shown in Fig. 4. Three crystals exhibit similar weight loss processes with three stages. At the initial stage of 40～180 °C, the weight loss of 1 was 5.1% (calcd. 4.8%), 2a was 4.9% (calcd. 4.7%) and 2b was 4.9% (calcd. 4.7%), which are attributed to the removal of methanol molecules. And further weight loss begins at 180 °C, corresponding to the elimination of organic molecules by assuming a SnO221.0% (1, calcd. 22.4%), 21.1% (2a, calcd. 21.9%) and 21.8% (2b, calcd. 21.9%) phase as the final residue. As a result, three crystals are stable until 100 °C, and 2a and 2b have the same weight loss processes and ratios, indicating they are homogeneous polycrys- talline.

3.5 Anticancer activity

Table 3 lists the inhibitory activities of 1, 2a and 2b and carboplatin on the cultured cancer cells NCI-H460, HepG2 and MCF7 in vitro. It can be seen that the approximation IC50of 2a and 2b to three cancer cells indicates that different crystal systems have little influence on the activity of compounds. In addition, compared with IC50, a conclusion can be drawn that the inhibitory activities of 1, 2a and 2b on NCI-H460 are similar with no significant difference, and are weaker than one of carboplatin. However, for HepG2 and MCF-7, the inhibitory activitiesof 2a and 2b were significantly better than that of 1, and even the one on MCF7 was better than that of carboplatin. Therefore, three crystals had certain inhibitory effects on the three cancer cells, and 2a and 2b are stronger than 1. Furthermore, combined with the structures of 1, 2a and 2b, they have similar structures, but only the aromatic ring of the hydrazone ligand is different. The aromatic ring of 1 is phenyl, while the aromatic ring of 2a and 2b is-methyl phenyl, which indicates the different aromatic ring of ligand has also certain influence on the anticancer activity of the compound.

Table 2. Parts of the Bond Lengths (Å) and Bond Angles (°) of (1), (2a) and (2b)

Fig. 4. TG-DTG curves of 1, 2a and 2b

Table 3. Inhibition Action of Compounds to Cancer Cell in Vitro

4 CONCLUSION

Twodibenzyltin compounds have been synthesi- zed by the microwave “one pot” method. Compound 2 has two crystals of 2a and 2b with different colors and shapes. Three crystals containfour-membered ring Sn2O2plane as the center of symmetry to construct a distorted pentagonal bipyramidal con- figuration. Three crystals can exist stably until 100 °C. The in vitro inhibitory activity on cancer cells NCI-H460, HepG2 and MCF7 showed the inhibitory activity of 2a and 2b is the same, and three crystals have obvious inhibitory effect on the three cancer cells, and the inhibitory effects of 2a and 2b are better than that of 1.

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8 July 2019;

25 November 2019 (CCDC 1938462, 1938463 and 1938464)

① The project was supported by the Hengyang Normal University College Students Innovation and Entrepreneurship Training Program (No. cxcy1944), Scientific Research Fund of Hunan Provincial Education Department of China (No. 19C0279), Hengyang Normal University Industrial Supported Project (Nos.HXKJ201907,HXKJ201908, HXKJ201911, HXKJ201946, HXKJ201947),and Foundation of Key Laboratory of Functional Organometallic Materials, University of Hunan Province (No. GN19K07)

. Tan Yu-Xing, E-mail: tanyuxing@hynu.edu.cn

10.14102/j.cnki.0254–5861.2011–2522