European Journal of Medicinal Chemistry 79 (2014) 391e398

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Original article

Synthesis, characterization and anti-proliferative activity of heterocyclic hypervalent organoantimony compounds Yi Chen a, b, c, Kun Yu c, Nian-Yuan Tan c, Ren-Hua Qiu c, Wei Liu a, Ning-Lin Luo c, Le Tong b, Chak-Tong Au c, d, Zi-Qiang Luo a, *, Shuang-Feng Yin c, * a

School of Basic Medicine, Central South University, Changsha 410013, PR China Medical College, Hunan University of Chinese Medicine, Changsha 410208, PR China State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China d Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 August 2013 Received in revised form 6 April 2014 Accepted 7 April 2014 Available online 12 April 2014

Three heterocyclic hypervalent organoantimony chlorides RN(CH2C6H4)2SbCl (2a R ¼ t-Bu, 2b R ¼ Cy, 2c R ¼ Ph) and their chalcogenide derivatives [RN(CH2C6H4)2Sb]2O (3a R ¼ t-Bu, 3b R ¼ Cy, 3c R ¼ Ph) were synthesized and characterized by techniques such as 1H NMR, 13C NMR, X-ray diffraction, and elemental analysis. It is found that the anti-proliferative activity detected over these compounds can be attributed to the coordination bond between the antimony and nitrogen atoms of these compounds. Moreover, a preliminary study on mechanistic action suggests that the inhibition effect is ascribable to cell cycle arrest and cell apoptosis. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Hypervalent organoantimony Synthesis Characterization Anti-proliferative activity Cell cycle arrest Apoptosis

1. Introduction Cancer is a major health problem. Many types of cancer are incurable, and mortality decline is mainly based on early detection and appropriate treatments [1]. In the fighting against cancers, new metal compounds are synthesized as antitumor agents [2,3]. Antimony is a group-15 element. It shows remarkable therapeutic efficacy on patients who suffer from leishmaniasis and acute promyelocytic leukemia [4e7]. Despite antimony compounds are used clinically for quite a number of diseases, they are rarely used as antitumor agents. In the 1990’s, Silvestru and coworkers reported for the first time the antitumor activity of organoantimony(III) compounds [4,8,9]. Fifteen years later Wang et al. [10] and Ludmila et al. [11] reported the relatively high antitumor activity of organoantimony(V) compounds. So far the most studied antimony compounds in the context of antitumor activity are

organometallic, and they are compounds with antimony-carbon bonds having ligands such as arylhydroxamates [10], lapachol [11], thioamides [12], and hydrazones [13]. The antitumor performance of these compounds, however, is unsatisfactory. It is hence meaningful to explore the chemical and pharmacological properties of new organoantimony compounds for the purpose of developing anticancer drugs. In the present study, we investigated the anti-proliferative activity of organoantimony compounds that are heterocyclic and hypervalent in nature. Through the use of different nitrogen substituent groups, we controlled the steric and substitution patterns of the organoantimony compounds. It is demonstrated that this kind of compounds can be used for the fabrication of anticancer drugs.

2. Results and discussion Abbreviations: t-Bu, tertiary butyl; Cy, cyclohexyl; Ph, phenyl; PI, propidium iodide; mM, mmol/L; mM, mmol/L; RT, room temperature. * Corresponding authors. E-mail addresses: [email protected] (Z.-Q. Luo), [email protected] (S.-F. Yin). http://dx.doi.org/10.1016/j.ejmech.2014.04.026 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

2.1. Chemistry Dilithiation of tertiary amines (2-Br-C6H4CH2)2NR (1a R ¼ t-Bu, 1b R ¼ Cy, 1c R ¼ Ph) with n-BuLi, followed by subsequent reaction

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with SbCl3, produces the organoantimony chlorides RN(CH2C6H4)2SbCl (2a R ¼ t-Bu, 2b R ¼ Cy, 2c R ¼ Ph) (Scheme 1). Treatment of these chlorides with KOH gives the organoantimony chalcogenide derivatives [RN(CH2C6H4)2Sb]2O (3a R ¼ t-Bu, 3b R ¼ Cy, 3c R ¼ Ph). The molecular structures of 2aec and 3aec are confirmed by elemental analyses and NMR techniques (1H and 13C NMR). Due to stronger electron-attracting ability of Sb, the protons linked with the carbon atom adjacent to Sb atom shift downfield (8.25 ppm, 2a; 8.26 ppm, 2b; 8.24 ppm, 2c) in comparison with those of the corresponding starting materials (7.55 ppm, 1a; 7.59 ppm, 1b; 7.60 ppm, 1c). Meanwhile, the 13C NMR data of the carbon atoms adjacent to Sb shift downfield (145.11 ppm, 2a; 144.01 ppm, 2b; 147.96 ppm, 2c) in comparison with those of the corresponding starting materials (139.10 ppm, 1a; 139.68 ppm, 1b; 136.27 ppm, 1c). On the contrary, compared with the 1H NMR spectra of the corresponding starting materials (8.25 ppm, 2a; 8.26 ppm, 2b; 8.24 ppm, 2c), stronger electron-donating ability of O counteracts the electron-attracting ability of Sb, leading to upfield shift of the protons linked with the carbon atoms adjacent to Sb atom (8.12 ppm, 3a; 8.21 ppm, 3b; 8.12 ppm, 3c). Crystal structures of 2a and 3a (Fig. 1) were determined by singlecrystal X-ray diffraction analysis, and selected bond lengths and angles are listed in Table 1. One can see that the coordination polyhedron around the centre Sb of hypervalent compounds 2a and 3a can be best described as a strongly distorted pseudo-trigonal bipyramid. The N(1), Cl(1) and O(1) atoms are located at the apical positions, while the C(1), C(10) and C(14) atoms are situated at the equatorial positions along with an electron lone pair of Sb. The Sb(1)N(1) distance (2.4638(14)  A) in 2a and that (2.6546(17)  A) in 3a is longer than that (2.397(3)  A) in 12-chloro-6-cyclohexyl-5,6,7,12tetrahydrodibenzo[c,f] [1,5]-azastibocine [14]. The results suggest that the N / Sb coordination in 2a and 3a is weaker than that of the latter. Furthermore, the two NeSb distances in 2a and 3a are slightly longer than the sum of the covalent radii (2.11  A) [15] but much shorter than the sum of the van der Waals radii (3.74  A) [16], indicating that there is coordination bonding between the antimony and the nitrogen atoms. According to Musher’s idea of hypervalent molecules [17], compounds 2aec and 3aec with high Sb valences can be considered as hypervalent. The SbeO bond length (2.0055(14)  A) in 3a is similar to those of organoantimony oxides such as [{2(Me2NCH2)C6H4}2Sb]2O (1.986(3)  A), (Ph2Sb)2O (1.978(3)  A), and (Me2Sb)2O (2.099(6)  A) [18e20]. Due to constrain imposed by intramolecular N / Sb coordination, the NeSbeCl angle in compound 2a (161.36(3) ) and NeSbeO angle in compound 3a (159.029(43) ) are significantly deviated from the ideal case of 180 . 2.2. Biological activity 2.2.1. Anti-proliferative activity Using CCK-8 assay, the anti-proliferative effect of compounds 1aec, 2aec and 3aec on A549 cells was examined. The concentrations of compounds required to inhibit 50% of cell growth (i.e.

Scheme 1. Synthesis of compounds 2aec and 3aec.

IC50) are shown in Table 2. It is observed that 2aec and 3aec show much higher anti-proliferative activity than their starting materials 1aec. The compounds with the same nitrogen substituent show anti-proliferative effects that follow the order: 3a (IC50 ¼ 3.5 mM) > 2a (IC50 ¼ 6.6 mM) > 1a (IC50 > 30 mM), 3b (IC50 ¼ 5.5 mM) > 2b (IC50 ¼ 31.4 mM) > 1b (IC50 > 30 mM, inhibition ratio is 9.39% at 30 mM). With IC50 above 30 mM, compounds 1c, 2c and 3c are all weak in anti-proliferative activity. In other words, the order of anti-proliferative effect of these compounds can also be arranged according to the nitrogen substituents: t-Bu > Cy > Ph. Since compared to Cy and Ph groups, t-Bu group is stronger in electron-donating ability but weaker in steric effect, it is hence deduced that the anti-proliferative activity towards A549 cells can be related to the coordination bonding between the antimony and nitrogen atoms of these compounds. In view that compounds 2a and 3a (both with same nitrogen substituent) showed stronger anti-proliferative activity, they were adopted to examine the time course at various concentrations (Fig. 2) as well as to examine the dose effect on anti-proliferative activity (Fig. 3). The results suggest that the anti-proliferative activity is concentration as well as time dependence. Based on the results of optimization, we adopted compound concentration of 10 mM and incubation period of 48 h for further investigation. To assess the possible side effects of administrating these compounds, the anti-proliferation activity of them on normal human bronchial epithelial cells (HBEC) was evaluated. After 48 h incubation, the IC50 values of compounds 2a and 3a on HBEC are 18.7 and 11.2 mM, giving IC50 (HBEC)/IC50(A549) ratio of 2.83 and 3.20, respectively. When the commercial anticancer drug cisplatin was adopted, the IC50 value on A549 cells under the same experimental conditions is above 30 mM. With anti-proliferation activity towards A549 cells stronger than that of cisplatin, the heterocyclic hypervalent compounds 2a and 3a can be further studied as antitumor drugs. 2.2.2. Effect of the compounds on the cell cycle There are five stages of the cell cycle: (1) the G1 phase that follows mitosis, a period for the synthesis of enzymes needed for DNA replication; (2) the S phase, a period of DNA replication; (3) the G2 phase where the cell continues to grow and produce new proteins; (4) the M phase where the cell divides into two daughter cells; and (5) the quiescent G0 phase where the cell remains stable until it begins the cell cycle again. To determine the possible effect of the heterocyclic hypervalent compounds on the progression of cell cycle, we performed flow cytometric analysis to quantify the percentage of A549 cells after cell permeabilization and propidium iodide (PI) labeling. In the analysis, the amount of bound dye is correlated with the DNA content of a given cell. In other words, DNA fragmentation in apoptotic cells is translated to fluorescence intensity, giving fluorescence peak that is lower in intensity than that of G0/G1 cells, i.e. a sub-G0/G1 peak. In A549 cells incubated with compounds 2a and 3a (10 mM for 48 h), the proportion of cells in the sub-G0/G1 phase increased to 4.86% and 8.01%, respectively, a large increase compared to 0.74% of the untreated control (Fig. 4). This increase in sub-G0/G1 phase was accompanied by an increase in cell number of the G2/M phase (compared with that of untreated cells), showing values of 28.76 versus 6.06% and 20.06 versus 6.06% (Fig. 4), indicating inhibiting effect of compounds 2a and 3a on cell mitosis. In other words, the increased proportion of cells in the sub-G0/G1 phase confirms that the apoptosis of A549 cells is a result of DNA degradation induced by compounds 2a and 3a. On the other hand, after treatment with compounds 2a and 3a, the cells in S phase decreased from the control value of 23.38% to 15.02% and 14.92%, individually, indicating the inhibiting effect of compounds 2a and 3a on DNA replication.

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Fig. 1. Molecular structure of compounds 2a and 3a (Hydrogen atoms are omitted).

Therefore, it is reasonable to deduce that the heterocyclic hypervalent organoantimony compounds have an inhibitory effect on cell proliferation, and the observed growth retardation was caused by cell cycle arrest mainly at the S and G2/M phases. 2.2.3. Effect of compounds on the cell apoptosis The ability of compounds 2a and 3a to induce apoptosis in the A549 cells was evaluated by means of Annexin-V and PI double staining using a FACSCalibur flow cytometer. As shown in Fig. 5, treatment with 3 mM of compound 3a for 48 h results in 6.29% of the A549 cells becoming apoptotic (early þ late), higher than the 3.98% value of apoptotic cells in an untreated control. Moreover, with the increase of 3a concentration to 10 mM, there is enhancement of apoptotic cells (early þ late) to 16.17%. The results demonstrate that there is significant cell apoptosis induced by 3a at a concentration of 10 mM. On the other hand, when the concentration of 2a is increased from 3 to 10 mM, the change of apoptotic cells (early þ late) is from 5.41% to 9.08%, relatively slight compared to that induced by 3a. 2.3. Stability of compounds 2a and 3a In order to check the stability of compounds 2a (IC50 ¼ 6.6 mM) and 3a (IC50 ¼ 3.5 mM), they were subject to NMR analysis with the employment of deuterated dimethyl sulfoxide (DMSO-d6) and DMSO-d6 þ H2O as solvent (see SI-Fig. 1).

As shown in SI-Figs. 2 and 3, there is no obvious difference between the RT 1H NMR spectrum of a fresh 2a sample and that of a 2a sample that was kept in open air at RT for 4 days. It is clear that compound 2a is stable. Meanwhile, compound 3a in DMSO-d6 was found to remain intact over a period of 4 days (see SI-Fig. 4). With the introduction of H2O, compound 3a in DMSO-d6 undergoes transformation to give the corresponding organoantimony hydroxide (see SI-Fig. 1 and SI-Fig. 5). It is noted that there is the establishment of equilibrium between compound 3a and its hydroxide, and the hydroxide can be converted back to compound 3a once H2O is removed (See SI-Fig. 1). Moreover, the organoantimony hydroxide is found to be stable in DMSO-d6 þ H2O (See SI-Fig. 5). Based on these evidences, we deduce that the active specie for antiproliferative activity in the biological evaluation with compound 3a is in fact the organoantimony hydroxide derivative. 3. Conclusion In this study, we synthesized heterocyclic hypervalent organoantimony compounds 2aec and 3aec, and characterized their structures. The results of structure determination by means of single-crystal X-ray diffraction reveal that there is coordination bond between the antimony and the nitrogen atoms in compounds 2a and 3a. The organoantimony compounds 2a and 3a show good Table 2 Anti-proliferation activities against A549 and HBEC. No.

Table 1 Selected bond distances ( A) and angles ( ) for compounds 2a and 3a. 2a Sb(1)eC(10) Sb(1)eC(1) Sb(1)eCl(1) Sb(1)eN(1) C(10)eSb(1)eC(1) C(10)eSb(1)eN(1) C(1)eSb(1)eN(1) C(10)eSb(1)eCl(1) C(1)eSb(1)eCl(1) N(1)eSb(1)eCl(1)

3a 2.1474(17) 2.1520(16) 2.5505(4) 2.4638(14) 95.70(6) 76.99(5) 74.96(5) 91.05(5) 92.35(5) 161.36(3)

Sb(1)eC(1) Sb(1)eC(14) Sb(1)eO(1) Sb(1)eN(1) C(1)eSb(1)eC(14) C(1)eSb(1)eN(1) C(14)eSb(1)eN(1) C(1)eSb(1)eO(1) C(14)eSb(1)eO(1) N(1)eSb(1)eO(1) Sb(1)eO(1)eSb(1a)

2.146(2) 2.158(2) 2.0055(14) 2.6546(17) 92.59(9) 72.622(79) 73.808(81) 92.33(9) 92.88(7) 159.029(43) 115.96(12)

1a 1b 1c 2a 2b 2c 3a 3b 3c Cisplatin a

IC50 (mM)a A549

HBEC

>30 >30 >30 6.6  1.85c 31.4  8.19 >30 3.5  0.71 5.5  0.40 >30 >30

ntb Nt Nt 18.7  2.1 Nt Nt 11.2  1.8 Nt Nt Nt

IC50 was obtained through the Probit regression model. Not tested. Each point is the mean  S.D. for three different experiments performed in triplicate. b c

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Fig. 3. Dose effect on anti-proliferation activity of compounds 2a and 3a. A549 cells were treated for 48 h. Cell viability was examined using CCK-8 assay. Each point is the mean  S.D. for three different experiments performed in triplicate. #P < 0.05, ## P < 0.01 versus 10 mM.

recorded at 25  C over an INOVA-400M (Varian) instrument calibrated using tetramethylsilane (TMS) as internal standard. Elemental analysis was performed over a VARIO EL III (Elementar) instrument. Melting points were determined over a XT-4 micro melting point apparatus (Beijing Tech Instrument Co., Ltd.).

Fig. 2. Time course of anti-proliferative effect of compound 2a (A) and compound 3a (B). A549 cells were treated at various concentrations of the compounds (1, 3, 10, and 30 mM) for periods as indicated. Cell viability was examined using CCK-8 assay. Each point is the mean  S.D. for three different experiments performed in triplicate. # P < 0.05, ##P < 0.01 versus 24 h; &P < 0.05, &&P < 0.01 versus 48 h.

anti-proliferation activity towards cancer cells as a result of apoptosis and cell cycle arrest. Compared to cisplatin, compounds 2a and 3a show stronger anti-proliferation activity towards A549 cells. This is the first example that hypervalent organoantimony compounds show ability to inhibit the growth of human tumor cell lines. It is expected that compounds 2a and 3a will find broad medicinal applications, especially for the combat of cancers. 4. Experimental 4.1. Chemistry The manipulations of air-sensitive materials were performed under an atmosphere of nitrogen using the standard Schlenk techniques. Most of the chemicals were purchased from Aldrich. Co., Ltd. and used as received unless otherwise indicated. Et2O was dried and distilled from a purple solution of sodium/benzophenone ketyl. The N,N-bis(2-bromobenzyl)- 2-methylpropan-2-amine (1a), N,N-bis(2-bromobenzyl)cyclohexanamine (1b), and N,N-bis(2bromobenzyl)aniline (1c) compounds were prepared according to procedures reported by Kakusawa et al. [21]. The NMR spectra were

4.1.1. General procedure for the synthesis of compounds 2aec To a solution of N,N-bis(2-bromobenzyl)-amine (1 equiv) in Et2O (1.0 mol/L), n-BuLi (2.0e2.2 equiv, 2.5 M in hexane) was added dropwise at 50  C. The mixture was gradually warmed to room temperature (RT) over a period of 3 h, and then a solution of SbCl3 (1 equiv) in Et2O (0.8e1.0 mol/L) was added at 78  C. The obtained mixture was gradually warmed to RT over a period of 12 h. After removal of solvent under vacuum, the residue was subject to toluene extraction, and the insoluble material was filtered out. The organic layer was washed with deionized water and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure and the residue was recrystallized from CH2Cl2/hexane to give heterocyclic hypervalent organoantimony chlorides 2aec in the form of colorless crystals. 4.1.1.1. Synthesis of compound 2a. The reaction was performed according to procedure for the synthesis of compounds 2aec using 1a (4.11 g, 10.0 mmol) in Et2O (100 mL), n-BuLi (8.4 mL, 21 mmol, 2.5 M in hexane), and SbCl3 (2.28 g, 10 mmol) in Et2O (100 mL). Yield: 3.52 g (85.7%). Mp: 213e215  C; 1H NMR (CDCl3, 400 MHz, TMS): d 1.34 (9H, s), 3.96 (2H, d, J ¼ 15.2 Hz), 4.36 (2H, d, J ¼ 15.6 Hz), 7.09 (2H, d, J ¼ 7.2 Hz), 7.25 (2H, t, J ¼ 8.0 Hz), 7.32 (2H, t, J ¼ 7.2 Hz), 8.25 (2H, d, J ¼ 7.6 Hz); 13C NMR (CDCl3, 100 MHz, TMS): d 27.11, 57.31, 60.50, 124.57, 128.57, 129.00, 135.07, 140.00, 145.11. Anal. Calc. for C18H21ClNSb (408.6): C, 52.91; H, 5.18; N, 3.43. Found: C, 52.78; H, 5.26; N, 3.52. 4.1.1.2. Synthesis of compound 2b. The reaction was performed according to procedure for the synthesis of compounds 2aec using 1b (2.19 g, 5.0 mmol) in Et2O (50 mL), n-BuLi (4.4 mL, 11 mmol, 2.5 M in hexane), and SbCl3 (1.14 g, 5.0 mmol) in Et2O (60 mL). Yield: 2.02 g (83.4%). Mp: 254e256  C; 1H NMR (400 MHz, CDCl3, TMS): d 1.07e1.13 (1H, m), 1.22e1.41 (4H, m), 1.66 (1H, d,

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Fig. 4. Cell cycle of A549 cells as examined in 48 h treatment of compounds 2a and 3a at 10 mM concentration. Cell cycle was assessed by FACS analysis. Each point is the mean  S.D. for three different experiments performed in triplicate. *P < 0.05, **P < 0.01 versus control.

J ¼ 13.2 Hz), 1.83 (2H, d, J ¼ 13.2 Hz), 2.03 (2H, d, J ¼ 12 Hz), 3.01e 3.08 (1H, m), 4.05 (2H, d, J ¼ 14.8 Hz), 4.20 (2H, d, J ¼ 15.2 Hz), 7.10 (2H, d, J ¼ 7.2 Hz), 7.27 (2H, td, J ¼ 0.8 Hz, 7.2 Hz), 7.34 (2H, t, J ¼ 7.2 Hz), 8.26 (2H, d, J ¼ 7.2 Hz); 13C NMR (100 MHz, CDCl3, TMS): d 25.44, 25.64, 29.54, 57.80, 65.43, 124.70, 128.72, 128.87, 134.99, 140.13. 144.01. Anal. Calc. for C20H23ClNSb (434.6): C, 55.27; H, 5.33; N, 3.22. Found: C, 55.39; H, 5.25; N, 3.14.

chlorides RN(CH2C6H4)2SbCl (1.0 mmol) in 20 mL toluene. The mixture was stirred at RT for 12 h, then the organic layer was separated and the water phase was washed with toluene (3  10 mL). The toluene solution was dried over anhydrous Na2SO4. Heterocyclic hypervalent organoantimony chalcogenide derivatives [RN(CH2C6H4)2Sb]2O in the form of white solid was obtained after solvent removal under vacuum.

4.1.1.3. Synthesis of compound 2c. The reaction was performed according to procedure for the synthesis of compounds 2aec using 1c (2.16 g, 5.0 mmol) in Et2O (50 mL), n-BuLi (4.0 mL, 10 mmol, 2.5 M in hexane), and SbCl3 (1.14 g, 5.0 mmol) in Et2O (60 mL). Yield: 3.7 g (86.3%). Mp: 222e224  C; 1H NMR (CDCl3, 400 MHz, TMS): d 4.52 (2H, d, J ¼ 14.8 Hz), 4.70 (2H, d, J ¼ 15.6 Hz), 7.22 (4H, m), 7.26 (1H, d, J ¼ 7.6 Hz), 7.30e7.35 (4H, m), 7.42 (2H, t, J ¼ 7.2 Hz), 8.24 (2H, d, J ¼ 7.6 Hz); 13C NMR (CDCl3, 100 MHz, TMS): d 61.19, 119.74, 125.28, 125.43, 129.17, 129.24, 129.58, 135.25, 140.56, 143.06, 147.96. Anal. Calc. for C20H17ClNSb (428.6): C, 56.05; H, 4.00; N, 3.27. Found: C, 55.73; H, 4.12; N, 3.35.

4.1.2.1. Synthesis of compound 3a. The reaction was performed according to procedure for the synthesis of compounds 3aec using 2a (0.409 g, 1.0 mmol). Yield: 0.768 g (99%). Mp: 235e237  C; 1H NMR(400 Hz, acetone-d6, TMS): d ¼ 1.35 (18H, s), 3.83 (4H, d, J ¼ 15.5 Hz), 4.37 (4H, d, J ¼ 15.5 Hz), 7.13 (4H, d, J ¼ 0.5 Hz), 7.14e 7.12 (6H, m), 7.24 (4H, t, J ¼ 7.5 Hz), 7.29 (2H, dt, J ¼ 7.2 Hz), 8.12 (2H, d, J ¼ 7.2 Hz); 13C NMR (125 Hz, acetone-d6, TMS): d ¼ 27.06, 29.40, 29.56, 29.71, 29.87, 30.02, 30.12, 30.18, 30.27, 30.33, 56.55, 59.39, 125.96, 127.73, 128.00, 133.65, 143.85, 147.05. Anal. Calc. for C36H42N2OSb2: C, 56.72; H, 5.55; N, 3.68; Found: C, 56.65; H, 5.58; N, 3.70.

4.1.2. General procedure for the synthesis of compounds 3aec A solution of KOH (0.4 g, 10 mol) in 20 mL deionized water was added to a solution of heterocyclic hypervalent organoantimony

4.1.2.2. Synthesis of compound 3b. The reaction was performed according to procedure for the synthesis of compounds 3aec using 2b (0.435 g, 1.0 mmol). Yield: 0.750 g (92%). Mp: 227e229  C; 1H

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NMR (400 Hz, CDCl3, TMS): d ¼ 3.87 (1H, s), 3.91 (1H, s), 4.06 (1H, s), 4.10 (1H, s), 6.99 (1H, t, J ¼ 3.2 Hz), 7.08 (2H,d, J ¼ 7.6 Hz), 7.16 (3H, dt, J ¼ 7.6 Hz), 7.20e7.28 (4H, m), 8.21 (2H, d, J ¼ 7.2 Hz); 13C NMR (100 Hz, CDCl3, TMS): d ¼ 15.28, 21.46, 25.78, 25.84, 25.94, 29.19, 29.24, 56.40, 56.58, 63.87, 64.11, 125.04, 125.20, 125.30, 127.62, 127.66, 128.07, 128.23, 129.04, 132.85, 134.16, 144.44, 144.97, 145.42. Anal. Calc. for C40H46N2OSb2: C, 59.00; H, 5.69; N, 3.44; Found: C, 59.10; H, 5.65; N, 3.42. 4.1.2.3. Synthesis of compound 3c. The reaction was performed according to procedure for the synthesis of compounds 3aec using 2c (0.429 g, 1.0 mmol). Yield: 0.722 g (90%). Mp: 295e297  C; 1H

NMR (400 Hz, CDCl3, TMS): d ¼ 4.42 (2H, d, J ¼ 15.2 Hz), 4.75 (2H, d, J ¼ 14.8 Hz), 6.99 (1H, t, J ¼ 3.2 Hz), 7.16 (2H, d, J ¼ 8.0 Hz), 7.20 (2H, d, J ¼ 6.8 Hz), 7.22e7.27 (5H, m), 7.29 (2H, dt, J ¼ 7.2 Hz), 8.12 (2H, d, J ¼ 7.2 Hz); 13C NMR (100 Hz, CDCl3, TMS): d ¼ 59.06, 118.00, 122.55, 125.44, 128.14, 128.22, 129.12, 133.93, 143.64, 145.35, 149.16. Anal. Calc. for C40H34N2OSb2: C, 59.89; H, 4.27; N, 3.49; Found: C, 59.79; H, 4.29; N, 3.52. 4.1.3. X-ray crystal structural determination of hypervalent compounds 2a and 3a X-ray single crystal diffraction analysis of compounds 2a and 3a was performed on a Bruker SMART APEX diffractometer with a CCD

Fig. 5. Apoptosis of A549 cells induced by compounds 2a and 3a. Cells were treated with compounds 2a and 3a at 3, 10 mM for 48 h and apoptosis was analyzed using Annexin-V and propidium iodide (PI) double staining by FACS analysis. Percentage of early apoptotic (annexin-Vþ/PI), late apoptotic (annexin-Vþ/PIþ), and dead (annexin-V/PIþ) cells are shown, respectively.

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detector using graphite monochromated radiation (MoKa, l ¼ 0.71073  A). The collected frames were processed with SAINTþ [22] software; and the collected reflections were subject to absorption correction (SADABS) [23]. The structure was solved by the direct methods using SHELXS-97, and refined by full-matrix leastsquares analyses on F2 [24]. Hydrogen atoms were generated in their idealized positions and all non-hydrogen atoms were refined anisotropically (Table 3).

8 was added and the plates were incubated for an additional 2 h. The optical density of the formazane yellow formed in the cells was measured at 490 nm, using an iMark microplate absorbance reader (Bio-Rad, USA). The IC50 values were obtained through the Probit regression model between inhibition ratio and concentration. Except the case of 48 h treatment, the cell viability of organometallic compounds in human bronchial epithelial cell was conducted under the same experimental conditions.

4.2. Biological evaluation

4.2.3. Cell cycle by FACS analysis For cell cycle analysis, 3  105 cells per well were incubated with the addition of compounds 2a and 3a (10 mM) for 48 h. At the end of the incubation, the cells were trypsinized and washed twice with phosphate-buffered saline (PBS) and separated by centrifugation. With the addition of 1 mL of cold 70% ethanol, the cells were once more incubated for 1 h at 4  C. After centrifugation, the pellet was resuspended in 1 mL of DNAase-free RNase A (200 mg/mL) and stored at 37  C. After 1 h, the cells were stained with 40 mg/mL propidium iodide (PI) for 20 min at RT. The percentage of cells in each stage of the cell cycle was determined using a FACSCalibur flow cytometer by counting 1  105 cells and analyzed using Cellquest software (BectoneDickinson, France).

All compounds were dissolved in dimethyl sulfoxide (DMSO) at 20 mM concentration and stocked at 4  C prior to use. The Cell Counting kit-8 (CCK-8) from Dojindo Molecular Technologies, Inc. (Shanghai, China) was used to measure the optical density of the formazane yellow formed in the cells. All other reagents were from Sigma and of the highest quality commercially available. All data were obtained from at least three separate experiments and the results were expressed as mean  S.D. Data were analyzed for statistical significance by one-way ANOVA, and pb0.05 was considered statistically for the indication of significant difference. 4.2.1. Cell cultures Human cancer cells A549 (human alveolar adenocarcinoma cell line) and human normal cells HBEC (human bronchial epithelial cell) used in this study were from the Cancer Research Institute, Central South University (Changsha, Hunan, PR China). A549 cells and HBEC cells were cultured under standard culture conditions (37  C, 95% air: 5% CO2) in RPMI1640 medium (HyClone) supplemented with 10% newborn calf serum (HyClone) and 1% penicilline streptomycin (HyClone). 4.2.2. Cell viability The cell viability of the organometallic compounds in human alveolar adenocarcinoma cell line was determined by the CCK-8 assay using a modified method according to the manufacturer’s instructions. In all assays, the compounds were quickly dissolved in DMSO and diluted in sterile culture medium before being added to the cell culture (DMSO concentration < 0.1% (v/v)). The cells (5  103 cells per well) were seeded in each well of the 96-well plates. After 12 h, compounds of designated concentrations (1, 3, 10 and 30 mM) were added. After 24-, 48-, and 72-h treatment, CCKTable 3 Crystal data and structure refinement parameters for compounds 2a and 3a. Parameters

2a

3a

Formula Formula weight Temperature Crystal size (mm) Crystal system Space group a ( A) b ( A) c ( A) a ( ) b ( ) g ( ) V ( A3) Z Density (Mg m3) m (mm1) F (000) Reflections collected Independent reflections R (int) Goodness-of-fit on F2 R1, wR2 [I > 2s(I)]

C18H21ClNSb 408.56 120 K 0.25  0.15  0.10 Monoclinic P21/n 9.747 (2) 15.8808 (3) 11.388 (2) 90 110.030 (1) 90 1656.2 (5) 4 1.639 1.82 816 16,671 3735 0.017 1.09 0.017, 0.039

C36H42N2OSb2 762.22 133 0.35  0.30  0.15 Monoclinic C2/c 26.119 (3) 14.1149 (15) 18.997 (2) 90 111.245 (1) 90 6527.7 (12) 8 1.551 1.69 3056 22,860 7088 0.024 1.02 0.024, 0.067

4.2.4. Apoptosis assessment by FACS analysis For apoptosis assessment, 3  105 cells per well were incubated with the addition of compounds 2a and 3a (0, 3, 10 mM) for 48 h. At the end of the incubation, cells were trypsinized and subsequently collected by centrifugation. The cells were washed twice with PBS and resuspended in 500 mL of binding buffer and incubated with 5 mL of annexin V-FITC and 5 mL PI for 20 min at RT in the dark. Then the cells were analyzed with a FACSCalibur flow cytometer (BectoneDickinson, France). In each sample, 1  105 cells were analyzed. Data analysis was performed with Cellquest software (BectoneDickinson, France). The results were interpreted as follows: cells in the lower left quadrant (annexin-V/PI) were considered as living cells, in the lower right quadrant (annexin-Vþ/ PI) as early apoptotic cells, in the upper right quadrant (annexinVþ/PIþ) as late apoptotic cells, and in the upper left quadrant (annexin-V/PIþ) as dead cells. The total apoptotic rate was the rate of cells in the lower right quadrant (annexin-Vþ/PI) plus in the upper right quadrant (annexin-Vþ/PIþ). 4.3. Stability of the compounds Stability analysis of these compounds was performed on an INOVA-400M (Varian) instrument. Initially, compound 2a was dissolved in DMSO-d6 and divided into two samples. Then 55 mL double distilled water (volume ratio of DMSO-d6/H2O ¼ 10:1) was added to one sample. The 1H NMR spectrum was recorded respectively after the sample was prepared and kept at RT for 4 days. The same experiments were performed with compound 3a. It is noted that the sample portion of 3a contains certain amount of the corresponding organoantimony hydroxide (see SI-Fig. 4). Acknowledgments This work was supported by the Science Research Foundation for Higher Universities of Hunan Province (10A092), ResearchBased Learning and Innovation Experiment Project for Undergraduates of Hunan Province (2011-483), the National Natural Science Foundation of China (21003040), Program for Changjiang Scholars and Innovative Research Team in University (IRT1238), and the Program for New Century Excellent Talents in Universities (NCET-10-0371). CTA thanks HNU for an adjunct professorship. Y. Chen, K. Yun and N.Y. Tan contributed equally.

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Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.04.026.

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Synthesis, characterization and anti-proliferative activity of heterocyclic hypervalent organoantimony compounds.

Three heterocyclic hypervalent organoantimony chlorides RN(CH2C6H4)2SbCl (2a R = t-Bu, 2b R = Cy, 2c R = Ph) and their chalcogenide derivatives [RN(CH...
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