Planar Chiral Ferrocene Cyclopalladated Derivatives Induce Caspase

Article Cite This: Organometallics XXXX, XXX, XXX−XXX

Planar Chiral Ferrocene Cyclopalladated Derivatives Induce CaspaseDependent Apoptosis and Antimetastasis in Cancer Cells Guidong Gong,† Yuan Cao,† Fei Wang,*,‡ and Gang Zhao*,† †

College of Chemical Engineering, Sichuan University, Chengdu 610064, PR China Key Laboratory of Natural Medicine and Clinical Translation, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR Chian

‡

S Supporting Information *

ABSTRACT: A series of planar chiral ferrocene cyclopalladated compounds were synthesized and characterized. The absolute configurations of four compounds were determined by single-crystal X-ray analysis. The cytotoxic activities of the synthesized compounds and cisplatin exhibited different inhibition potencies on the viability of human liver, breast, and colon cancer cell lines. The dinuclear compound 7 was 16-fold more potent than cisplatin in hepatoma cells. Compound 7 was also more effective than cisplatin in the inhibition of hepatoma cell metastasis. Flow cytometry analysis and caspase activity assays indicated that compound 7 exerted antiproliferative activity in cancer cells through the induction of caspase-dependent apoptosis.

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INTRODUCTION Platinum-based antitumor drugs, such as cisplatin, carboplatin, and oxaliplatin, are used extensively as cancer therapeutics. However, the observed side effects and inefficiency against cisplatin-resistant tumors have severely limited the clinical application of those compounds.1,2 Recently, cyclopalladated compounds have attracted attention as potential agents with excellent antitumor activity.3,4 Structure−activity relationship studies indicated that the activity of cyclopalladated compounds was highly related to the N-group,5,6 the C−N cycle4,5 and the ligands of palladium.7 The anticancer mechanism of cyclopalladated compounds is still unclear. Some cyclopalladated compounds were found to interact with DNA in the same manner as high-dose cisplatin, although DNA was not considered to be their intended target.3,8 Other cyclopalladated compounds were found to induce cancer cell apoptosis through interactions with organelle membranethiol-groups, which led to organelle membrane permeabilization.9−13 Caspases are a family of cysteinyl-aspartate-specific proteases closely associated with apoptosis and it has been shown that certain cyclopalladated compounds can induce the caspase-dependent apoptosis of cancer cells.4,9,11 Cytosolic calcium mobilization and cathepsin B were also found to play important roles in palladacycle-induced cancer cell apoptosis. Ferrocene and its derivatives were used as alternative groups in some anticancer drugs.14−16 The active site of these compounds are the ferrocene groups, and they not only induce reactive oxygen species (ROS)-mediated apoptosis of cancer cell lines17−19 but also inhibit cathepsin B.16 Planar chirality is an important characteristic of ferrocene derivatives, and the optically pure ferrocene cyclopalladated compounds were isolated and used as catalyst in organic synthesis.20 Previously, © XXXX American Chemical Society

we found that cyclopalladated compounds with different planar chiral ferrocene showed different antitumor activity. However, only two compounds were studied and the antitumor mechanisms were not established.20e Therefore, it is of interest to synthesize more analogs and to identify their antitumor mechanism for the development of new anticancer drugs. Herein, we synthesized 24 optically pure planar chiral ferrocene cyclopalladated compounds (Scheme 1). The antitumor activities of the synthesized compounds were Scheme 1. Overview of Synthesized Compounds 3−26a

Reaction conditions: (i) C6H5CH3, 110 °C, 24 h. (ii) Pd(OAc)2, MeOH, r.t., 24 h. (iii) NaX (X = Cl, Br, or I) MeOH, r.t., 2 h. (iv) PPh3, CH3COCH3, r.t., 2 h. a

Received: December 16, 2017

A

DOI: 10.1021/acs.organomet.7b00897 Organometallics XXXX, XXX, XXX−XXX

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Organometallics evaluated in human liver, breast, and colon cancer cell lines. The interaction with plasmid DNA PET21a of all test compounds and cisplatin were examined. The most potent compound was selected for the examination of the antitumor mechanism.

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RESULT AND DISCUSSION Synthesis and Characterization of the Compounds. The structural formulas and the numbering of the compounds under study are presented in Scheme 1. Acetylferrocene hydrazones (+)-SC-2 and (−)-RC-2 were obtained by a condensation reaction between acetylferrocene and chiral hydrazines (−)-(S)-1-amino-2-(methoxymethyl)pyrrolidine [(−)-S-1] or (+)-(R)-1-amino-2-(methoxymethyl)pyrrolidine [(+)-R-1] in dry benzene.21,22 The acetylferrocene hydrazone were obtained as orange plates at a good yield. The compounds (+)-S-2 and (−)-R-2 have been characterized by 1H NMR 13C NMR (Figures S1−S4) melting point and optical rotation. (−)-R-2 or (+)-S-2 was mixed with Pd(OAc)2 and NaOAc in a 1:1:1 molar ratio in dry methanol at room temperature for 24 h, followed by treatment with excess amounts of NaCl for 2 h, producing the corresponding planar chiral cyclopalladated monomers compounds 3, 4 or 5, 6 in moderate yields (3/4 = 5/6 ≈ 9:1).21 Compounds 7−10 and 11−14 were produced by the same method with the substitution of NaBr or NaI for NaCl.21−24 They were fully characterized by 1H NMR, 13C NMR (Figures S15−S28), ESI-MS, elemental analysis (EA), melting point, and optical rotation. The monomers of cyclopalladated compounds 15−26 were obtained by bridge-splitting reactions of the dinuclear compounds 3−14 with PPh3 in a 1:5 molar ratio at room temperature for 2 h. Only compound 16 was acquired when preparing monomers of cyclopalladated compound, so we know the chiral molecular structure of compound 4 was (Spl,Sc,Sc,Spl). All compounds were fully characterized by 1H NMR, 13C NMR, 31P NMR (Figures S29−S64), ESI-MS, EA, and optical rotation. X-ray Diffraction Analysis. Single crystals of compounds 16 (CCDC 1546621), 18 (CCDC 1546620), 22 (CCDC 1546619), and 26 (CCDC 1546623) (Figure 1, thermal ellipsoids) were obtained by the slow evaporation of the solvents from CDCl 3 . The X-ray diffraction study of compounds 3, 5−8, and 10 have been reported previously.21,23,24 Crystal data, selected dihedral angles in compounds 16, 18, 22, and 26 are shown in Tables S1 and S2 separately. Due to the large triphenylphosphine group, the interplanar angle {CNPd1}/{Pd1 × 1X2} in compounds 16, 18, 22, and 26 was significantly folded (Table 1 and the Supporting Information). Cytotoxicity Assay. Liver cancer (Huh-7 and SK-hep-1), breast cancer (MCF-7), and colon cancer (HCT116) cells were used to examine the cytotoxicity of compounds 3−26 and cisplatin, respectively. As shown in Table 2, the compounds showed different cytotoxicity to different cancer lines. The planar chiral cyclopalladated ferrocene-derived ligands exerted more potent inhibition of the viability of SK-Hep-1 cells, usually with IC50 values less than 10 μM. All binuclear compounds except compounds 4, 9, and 11 showed lower IC50 values than did the monomeric cyclopalladated compounds. All dimeric cyclopalladated compounds 3−14 showed lower IC50 values in SK-Hep-1 cells, whereas for compounds 3, 5, and 11− 14, the dimers of the cyclopalladated compounds showed lower IC50 values in MCF-7. Finally, other dimeric cyclopalladated

Figure 1. X-ray crystal structures of compounds 16, 18, 22, and 26 presented as ellipsoid models; hydrogen atoms are omitted for clarity.

Table 1. Interplanar Angle in Compounds 16, 18, 22, and 26 compound

{CNPd1}/{Pd1 × 1X2} (deg)a

16 18 22 26

26.12 26.39 27.58 28.37

The interplanar angle of plane Pd1CN and plane Pd1 × 1X2, X1 = Cl1, X2 = P1 for 16; X1 = Cl1, X2 = P1 for 18; X1 = Br1, X2 = P1 for 16; X1 = I1, X2 = P1 for 18.

a

compounds except for compound 11 showed lower IC50 values in HCT116 cells. As for the dimeric cyclopalladated compounds mentioned above, the bridge atoms played a key role in cytotoxicity. The metal atoms (Pd) in dimeric planar chiral cyclopalladated ferrocene-derived ligands were connected by two halogen atoms. From Table 2, if decorated by the same planar chiral ferrocene and the same chiral N-group, then compounds with Br bridges showed lower IC50 values. The planar chirality of the C,N-palladacycle and the cental chirality of the N-substituent were secondary influences of these compounds on cytotoxicity, but the relationship requires further study. Compound 7 was shown to exhibit the strongest inhibition of viability in the four cancer cell lines; its IC50 value in SK-Hep-1 cells was 3.48 μM, approximately 16-fold more potent than cisplatin. We found that compound 7 potently inhibited the viability of SK-Hep-1 HCT116 cancer cells which suggested that compound 7 may warrant further development as a new antitumor agent. Electrochemistry. The redox behavior of compounds 3, 7, 11, 15, 19, and 23 was investigated as reported methed,25 and the half-wave potentials for oxidation of the ferrocene moiety were summarized in Table 3. It was found that compounds 3, 7, and 11 exhibited higher E1/2 and |Epa − Epc| than the corresponding monomers of cyclopalladated compounds 15, 19, and 23. However, the IC50 value of compounds 3 and 7 were more potent than compounds 15 and 19, though the IC50 value of compound 11 was less potent than compound 23. These results suggest the half-wave may have an impact on the B


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Organometallics

Table 2. IC50 Values (μM) of Compounds 3−26 and Cisplatin against Huh-7, SK-Hep-1, MCF-7, and HCT116 Cancer Cell Lines compound

Huh-7 15.67 19.76 34.6 22.96 13.92 15.83 16.34 18.02 57.43 31.62 46.49 50.22 55.74 52.87 48.66 46.92 80.4 52.08 53.1 71.14 39.89 94.18 58.5 56.7 23.14

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 cisplatin

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.54 1.16 3.89 1.45 1.18 5.5 1.33 0.46 3.18 3.39 7.21 2.46 10.4 8.09 6.09 5.11 5.72 4.98 6.32 4.75 4.38 6.4 9.92 1.97 0.34

SK-Hep-1

MCF-7

HCT116

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17.19 ± 3.12 16.47 ± 0.961 24.36 ± 1.59 28.74 ± 2.518 14.19 ± 0.698 14.57 ± 4.84 14.76 ± 1.38 24.21 ± 1.38 56.98 ± 8.03 27.92 ± 0.954 43.2 ± 4.27 60.9 ± 6.80 94.58 ± 32.61 66.86 ± 3.06 >100 141.2 ± 4.25 62.87 ± 3.41 87.6 ± 4.29 111.4 ± 9.74 >100 44.04 ± 1.42 >100 171.4 ± 9.35 >100 28.88 ± 3.84

35.09 ± 0.62 22.91 ± 0.72 20.04 ± 1.94 40.88 ± 6.86 17.9 ± 3.81 20.14 ± 4.89 18.32 ± 0.775 20.57 ± 2.02 46.46 ± 6.71 35.25 ± 1.14 42.12 ± 6.15 51.07 ± 3.58 69.56 ± 0.97 94.62 ± 26.2 46.11 ± 2.96 23.34 ± 3.11 50.07 ± 4.14 82.00 ± 4.595 64.55 ± 2.55 >100 38.24 ± 1.396 >100 58.78 ± 0.188 >100 50.05 ± 1.13

4.829 4.819 4.892 5.463 3.48 4.522 4.12 4.692 6.345 4.789 4.59 6.733 5.544 4.786 4.822 5.32 5.273 4.972 5.912 15.7 4.84 11.57 11.71 7.151 58.61

0.588 0.224 1.00 1.05 0.236 0.59 0.195 0.245 0.432 0.221 0.359 0.652 0.521 0.533 0.508 0.372 0.267 0.0976 1.22 1.09 0.242 1.29 2.10 2.28 1.63

Table 3. Cyclic Voltammetric Data of Compounds 3, 7, 11, 15, 19, and 23a |Epa − Epc| (mv) E1/2 (mV)

3

7

11

15

19

23

33 102

35 104

44 113

4 73

4 72

7 81

a

E1/2 values are quoted relative to FcH/[FcH]+. Epa is the oxidation peak potential, and Epc is the teduction peak potential

cytotoxicity of those cyclopalladated candidates, but it was not a major factor.25 DNA Interaction. The binding of palladium compounds 3− 26 to DNA was studied by their ability to modify the electrophoretic mobility of the supercoiled closed circular form of PET21a plasmid DNA. In this investigation, only the supercoiled closed circular (ccc) form in PET21a plasmid DNA was studied. As shown in Figure 2, cisplatin altered the electrophoretic mobility of PET21a at low concentrations (2.5−5 μM) and fragmented the DNA at slightly higher concentrations (10−25 μM), which was in accordance with previous reports.5 In contrast, dimeric cyclopalladated compounds showed no interaction with PET21a plasmid DNA, even at a high concentration (200 μM). Monomeric cyclopalladated compounds showed weak interactions with PET21a plasmid DNA at high concentrations (25−200 μM) with a stronger ability to destroy DNA than dimeric cyclopalladated compounds at the same concentrations. These results were not consistent with the IC50 values shown in Table 2 and suggested that DNA may not be the primary target for the planar chair ferrocene cyclopalladium compounds. Cell Cycle and Cell Apoptosis. As compound 7 showed the most potent inhibition of SK-Hep-1 cells, it was selected for further mechanistic studies. To examine the effect of com

Figure 2. Increasing concentrations of compounds 3−26 and cisplatin interact with PET21a DNA (0.8 μg). Line 1: DNA only. Line 2: 2.5 μM. Line 3: 5 μM. Line 4: 10 μM. Line 5: 25 μM. Line 6: 50 μM. Line 7: 100 μM. Line 8:200 μM.

pound 7 on the cancer cell cycle, SK- Hep-1 cells were incubated with different concentrations of compound 7 for 24 h and then analyzed by flow cytometry. As shown in Figure 3A, the incubation with 0, 5.0, and 7.5 μM compound 7 for 24 h had no effect on the cell cycle. Next, we examined the effect of compound 7 on the apoptosis of cancer cells. As shown in Figure 3B, the incubation of SK-Hep-1 with compound 7 at 5 or 7.5 μM significantly decreased the percentage of living cells with a consistent increase in apoptosis cells. An early apoptosis rate of 34.6% and a late apoptosis and necrosis rate of 13.2% were observed after C


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Organometallics

Figure 3. (A) Histograms from representative experiments and plots that depict the variation of the percentage of SK-Hep-1 in various phases of the cell cycle. Cell cycle was analyzed for SK-Hep-1 cells untreated (control) or treated with compound 7 at 5 μM and 7.5 μM. (B) Histograms from representative experiments and plots that depict the variation of the percentage of SK-Hep-1 cells which were alive (quadrant 4), in early apoptosis (quadrant 3), or in late apoptosis/ necrosis (quadrants 1 and 2). Apoptosis was analyzed for SK-Hep-1 cells untreated (control) or treated with compound 7 at 5 μM or 7.5 μM. (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 compared with the DMSO control.

Figure 4. (A) Compound 7 inhibited SK-Hep-1 cell migration in wound healing assay. Cells were wounded by the pipet and then treated with various concentrations of compounds for 24 h. Scale bar 50 μm. (B) compound 7 inhibited SK-Hep-1 cells migration in transwell invasion assay. The top chambers were seeded with 1 × 105 SK-Hep-1 cells in dulbecco’s modified eagle medium (DMEM) and treated with Cisplatin or compound7 for 24 h. Scale bare 50 μm.

potential agent for the treatment of metastatic HCC may be warranted. The effects of compound 7 on apoptotic pathways were examined. As shown in Figure 5A, caspase-3 activity in SK-Hep1 cells was markedly enhanced after treatment with 2.5 or 5 μM compound 7 for 12 h. Caspase-9 activity in SK-Hep-1 cells was

24 h treatment with 5 μM compound 7. Furthermore, when the concentration of compound 7 was increased to 7.5 μM, the percentage of cells in early apoptosis, late apoptosis, and necrosis increased from 21.3 to 48.1%, respectively. These results suggested that compound 7 exhibited cytotoxicity in cancer cells through the induction of apoptosis triggered by the mitochondrial- or lysosomal-dependent pathways.3,10,13 Compounds that contained the ferrocene group induced the ROSmediated apoptosis of certain cyclopalladated compounds were found to exercise their antiproliferative activity in cancer cells through the in cancer lines.26,27 ROS mediates apoptosis through mitochondrial and lysosomal membrane permeabilization in cells.9,10 Therefore, it is of interest to further identify whether compound 7 triggers ROS generation to induce apoptosis in cancer cells. Effects of Compound 7 on Tumor Cell Metastasis. The effect of compound 7 on the metastasis of cancer cells was compared with that of cisplatin by a wound healing assay and a transwell assay. As shown in Fiugre 4A, treatment with compound 7 at 5−10 μM for 24 h potently inhibited the migration of SK-Hep-1 cells, whereas treatment with cisplatin at the same concentrations slightly inhibited the migration of SK-Hep-1 cells. Compound 7 also potently inhibited the migration of SK-Hep-1 cells, as shown by the trans-well assay, at a much lower concentration than that of cisplatin (Figure 4B). Cathepsin B plays a key role in the cell migration and cyclopalladium compounds with ferrocene can inhibit cathepsin B.13,16 Therefore, it was possible that compound 7 inhibited the metastasis of cancer cells through the suppression of cathepsin B; however, this required further study. Unresectable or metastatic hepatocellular carcinoma (HCC) carries a poor prognosis. Systemic therapy with cytotoxic agents provides only marginal benefit. In this study, we found that cisplatin did not effectively inhibit the migration of SK-Hep-1 cells, which was consistent with the findings that cisplatin alone failed to demonstrate meaningful activity in metastatic HCC patients.2,28 We also found that compound 7 potently inhibited metastasis of HCC cells; thus, the development of compound 7 into a new

Figure 5. (A) Caspase-3, caspase-8, and caspase-9 activation in SKHep-1 cells after treatment with different concentrations of compound 7 for 12 h. (B) Cell lysates were immunoblotted with an anti-PARP antibody. GAPDH staining is shown as a loading control. All values are presented as the mean ± SD (n = 3). (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 compared with the DMSO control; n.s., not significant. D


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Organometallics also enhanced after treatment with 5 μM compound 7 for 12 h, but caspase-8 activity was unaffected. Consistent with its effect on caspase-3 activation, cleaved polyADP-ribose polymerase (cleaved-PARP) was detected after treatment with compound 7 (Figure 5B). Caspase-3 belongs to the “executioners” category, and caspase-8 and -9 are “initiators”.29,30 Furthermore, caspase8 mediates the exogenous apoptotic pathway and caspase-9 mediates endogenous apoptotic pathway.30,31 The activation of caspase-3 was necessary for cyclopalladated compounds to induce cancer cell apoptosis.9−13 Here, we found that compound 7 had no effect on caspase-8 activation but activated capase-3 and caspase-9, which suggested that compound 7 may exert its pro-apoptotic effect through the activation of the endogenous apoptotic pathway in cancer cells.

Chemistry. Compounds (+)-(RC)-2, (−)-(SC)-2. Acetylferrocene (10 mmol) and chiral hydrazone (+)-(RC)-1 or (−)-(SC)-1 (10 mmol) were dissolved in dry benzene (100 mL). The flask containing the reaction mixture was connected to a condenser equipped with a Dean−Stark apparatus. The red solution was refluxed over an oil bath for about 6 h and then the carefully transferred into a Schlenk tube, into which 5 Å molecular sieve (3.0 g) were introduced. The mixture was further refluxed for 6 h and then washed with n-hexane. Characterization data were as follows: (−)-(RC)-2: Yield: 1.88 g (75%). Mp 67.7−68.2 °C. [α]20 D − 430.2 (c 1.0 in CHCl3). 1H NMR(400 MHz, CDCl3, 25 °C, TMS): δ = 4.67 [d, J = 1.3 Hz, 1H; H2 (C5H4)], 4.59 [d, J = 1.4, 1H; H5 (C5H4)], 4.33−4.25 [m, 2H; H3, H4 (C5H4)], 4.12 (s, 5H; C5H5), 3.50 (q, J = 7.2 Hz, 1H; CH) 3.38(s, 3H; OCH3), 3.34−3.20 (m, 2H; OCH2), 2.48 (dd, J = 17.1, 8.6 Hz, 1H; NCH2), 2.19 (s, 3H;CH3CN), 2.04 (dt, J = 6.72 Hz, 1H; NCH2), 1.93−1.81 (m, 2H; CHCH2CH2), 1.75− 1.65 (m, 2H; CH2CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 160.60 (CN), 84.36 [C1 (C5H3)], 75.67 [C2 (C5H3)], 69.55 [C5 (C5H3)], 69.14 (C5H5), 68.93 [C3 (C5H3)], 67.37 [C4 (C5H3)], 66.18 (OCH2), 66.42 (NCH), 59.25 (OCH2), 54.11(NCH2), 26.67 (CH2CH2), 22.31 (CH2CH2), 16.85(CNCH3). MS (ESI): calcd for [M + H]+: 341.13; found: 340.95. Anal. Calcd for C18H24FeN2O: C, 63.54; H, 7.11; N, 8.23. Found: C, 63.52; H, 7.09; N, 8. 24. (+)-(SC)-2: yield: 2.25 g (86%). Mp 75−76 °C. [α]20 D + 11.67 (c 1.0 in CHCl3). 1H NMR(400 MHz, CDCl3, 25 °C, TMS): δ = 4.67 [dd, J = 3.3,1.6 Hz, 1H; H2 (C5H4)], 4.59 [dd, J = 3.3, 1.7 Hz, 1H; H5 (C5H4)], 4.28 [t, J = 1.9 Hz, 2H; H3,H4 (C5H4)], 4.12 (s, 5H; C5H5), 3.50 (q, J = 7.2, 1H; CH), 3.38 (s, 3H, OCH3), 3.34−3.21 (m, 2H; OCH2), 2.47 (q, J = 8.48, 1H; NCH2), 2.19 (s, 3H; CH3CN), 2.08−1.99 (m, 1H; NCH2), 1.91−1.83 (m, 2H; CH2CH2), 1.78−1.65 (m, 2H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 160.63 (CN), 84.36 [C1 (C5H3)], 75.68 [C2 (C5H3)], 69.59 [C5 (C5H3)], 69.15 (C5H5), 68.94 [C3 (C5H3)], 67.37 [C4 (C5H3)], 66.82 (OCH2), 66.43 (NCH), 59.26 (OCH2), 54.12(NCH2), 26.68 (CH2CH2), 22.31 (CH2CH2), 16.85 (CNCH3). MS (ESI): calcd for [M + H]+: 341.13; found: 340.95. Anal. Calcd for C18H24FeN2O: C, 63.54; H, 7.11; N, 8.23. Found: C, 63.50; H, 7.12; N, 8. 20. Compounds (−)-(Spl,RC,RC,Spl)-3 and (+)-(Rpl,RC,RC,Rpl)-4. Hydrazone (−)-(RC)-2 (0.33 g, 1.0 mmol) was added to a methanolic (30 mL) solution containing Pd(OAc)2 (0.22 g, 1.0 mmol) and NaOAc· 3H2O (0.14 g, 1.0 mmol) and stirred at room temperature for 24 h, followed by treatment with excess NaCl and stirring at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into chloroform and passed through a SiO2-column with 10:1 chloroform/n-hexane. Concentration of the eluted solution of two successive red bands produced compounds (−)-(Spl,RC,RC,Spl)-3 and (+)-(Rpl,RC,RC,Rpl)-4 which were recrystallized from dichloromethane/n-hexane (1:3) as red plates [product ratio 9:1, total yield 0.24 g (49.17%)]. Characterization data were as follows: (−)-(Spl,RC,RC,Spl)-3: Mp > 210 °C. [α]20 D − 2738.6 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.82 [s, 1H; H5 (C5H3)], 4.74 [s, 1H; H5 (C5H3)], 4.38 [d, J = 10.6 Hz, 12H; C5H5 + H3 (C5H3)], 4.24 [s, 2H; H4 (C5H3)], 3.75 (s, 2H; CH), 3.43 (s, 4H; OCH2), 3.41 (s, 6H; OCH3), 3.26 (dd, J = 17.4, 8.6 Hz, 2H; NCH2), 2.82 (t, J = 7.6 Hz, 2H; NCH2), 2.27 (s, 6H; CH3CN), 2.04−1.54 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.01 (CN), 98.81 [C1 (C5H3)], 85.47 [C2 (C5H3)], 75.77 [C5 (C5H3)], 73.76[C3 (C5H3)], 70.62(C5H5), 67.40 [C4 (C5H3)], 65.61 (OCH2), 59.06 (NCH), 56.62 (OCH2), 54.82(NCH2), 26.66 (CH2CH2), 22.25 (CH2CH2), 15.20 (CNCH3). MS (ESI): m/z calcd for [3 − Cl]+: 927.01; found: 927.33. Anal. Calcd for C36H46Cl2Fe2N4O2Pd2: C, 44.94; H, 4.82; N, 5.82. Found: C, 44.91; H, 4.75; N, 5.87. (+)-(Rpl,RC,RC,Rpl)-4: Mp 195.6−196.2 °C. [α]20 D −876.6 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.89 [s, 1H; H5 (C5H3)], 4.80 [s, 1H; H5 (C5H3)], 4.54 [d, J = 17.3 Hz, 12H; H3 (C5H3) + C5H5], 4.27 [t, J = 2.4 Hz, 2H; H4 (C5H3)], 3.63 (d, J = 29.0 Hz, 2H; CH), 3.55−3.46 (m, 2H; NCH2), 3.32−3.11 (m, 10H; CH2OCH3),

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CONCLUSION In this study, optically pure planar chiral ferrocene derived cyclopalladated compounds were synthesized and characterized by NMR, optical rotation, and ESI-MS. The molecular structures of compounds 16, 18, 22, and 26 were confirmed by single-crystal X-ray diffraction analysis. The cytotoxicity assays showed that these compounds were cytotoxic to four cancer cell lines with different potencies. Compound 7 exhibited the most potent inhibitory effect, approximately 16 times greater than cisplatin against SK-Hep-1 liver cancer cells. However, unlike cisplatin, DNA was not the primary target of planar chiral ferrocene derived cyclopalladated compounds. Compound 7 had no effect on the cell cycle, but significantly induced apoptosis through the activation of the caspase-3/-9dependent pathway. Furthermore, compound 7 more potently inhibited the metastasis of SK-Hep-1 cells than cisplatin, which warranted the development of compound 7 as a novel therapeutic means for the treatment of HCC.

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EXPERIMENTAL SECTION

General. Melting points were measured on a Meltemp melting point apparatus. Optical rotation was measured with Perkin elmer model 341 polarimeter. 1H NMR spectra were recorded on Bruker AM400 NMR spectrometer. Chemical shifts were reported in parts per million (ppm) from tetramethylsilane with the solvent resonance as the internal standard (CDCl3, δ = 7.26 ppm). Spectra were reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants (Hz), integration and assignment. 13C NMR spectra were collected on commercial instruments (101 MHz) with complete proton decoupling. Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane with the solvent resonance as internal standard (CDCl3, δ = 77.0 ppm). Mass spectra were recorded on UPLC-Xevo TQMS system equipped with an ESI source. C, H, and N elemental determination were performed on a Euro EA 3000 elemental analyzer (Leeman). Cyclic voltammograms were measured with CH Instruments 660E electrochemical analyzer (Shanghai Chenhua). Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. The cisplatin was purchased from TCI Tokyo Chemical Industry (Shanghai) Co., Ltd. (Shanghai, China) Crystallography data of compounds 16, 18, 22, and 26 were measured on a MSC/Rigaku RAXIS IIC imaging-plate diffractometer. Intensities were collected at 294 K using graphite-monochromatized Mo Kα radiation (λ = 0.71073) from arotating-anode generator operating at 50 kV and 90 mA (2θmin = 3°, 2θmax = 55°, 2−5° oscillation frames in the range of 0−180°, exposure 8 min per frame). The X-ray diffraction data were collected on Xcalibur Eos diffractometer. The structure of the molecule was elucidated using Olex2.32 Besides, structural refinement were achieved via the ShelXL refinement package through least-squares minimization.33 E


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Organometallics 3.02 (d, J = 37.7 Hz, 2H; NCH2), 2.29 (s, 6H; CH3CN), 2.23−1.60 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.32 (CN), 110.49 [C1 (C5H3)], 86.29 [C2 (C5H3)], 75.32 [C5 (C5H3)], 69.32[C3 (C5H3)], 70.80(C5H5), 66.25 [C4 (C5H3)], 63.82 (OCH2), 59.09 (NCH), 57.73 (OCH2), 53.73(NCH2), 26.58 (CH2CH2), 21.94 (CH2CH2), 15.37 (CNCH3). MS (ESI): m/z calcd for [4 − Cl]+: 927.01; found: 927.27. Anal. Calcd for C36H46Cl2Fe2N4O2Pd2: C, 44.94; H, 4.82; N, 5.82. Found: C, 44.89; H, 4.88; N, 5.81. Compounds (+)-(Rpl,SC,SC,Rpl)-5 and (−)-(Spl,SC,SC,Spl)-6. The hydrazone (+)-(S)-2 (0.33 g, 1.0 mmol) was added to a methanolic (30 mL) solution containing Pd(OAc)2 (0.22 g, 1.0 mmol) and NaOAc·3H2O (0.14 g, 1.0 mmol) and stirred at room temperature for 24 h, followed by treatment with excess NaCl and stirring at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into chloroform and passed through a SiO2-column with 10:1 chloroform/n-hexane. Concentration of the eluted solution of two successive red bands produced compounds (+)-(Rpl,SC,SC,Rpl)-5 and (−)-(Spl,SC,SC,Spl)-6 which were recrystallized from dichloromethane/n-hexane (1:3) as red plates [product ratio 9:1, total yield 0.26 g (54.00%)]. Characterization data were as follows: (+)-(Rpl,SC,SC,Rpl)-5: Mp 228.1 −229.6 °C. [α]20 D + 2677 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.82 [s, 1H; H5 (C5H3)], 4.73 [s, 1H; H5 (C5H3)], 4.38 [d, J = 10.7 Hz, 12H; C5H5], 4.24 [s, 2H; H4 (C5H3)], 3.74 (s, 2H; CH), 3.43 (s, 4H; OCH2), 3.41 (s, 6H; OCH3), 3.26 (dd, J = 17.6, 8.6 Hz, 2H; NCH2), 2.82 (t, J = 7.9 Hz, 2H; NCH2), 2.27 (s, 6H; CH3CN), 2.23−1.60 (m, 8H; CH2CH2). 13 C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.03 (CN), 101.41 [C1 (C5H3)], 85.47 [C2 (C5H3)], 75.77 [C5 (C5H3)], 73.76[C3 (C5H3)], 70.62(C5H5), 67.39 [C4 (C5H3)], 65.62 (OCH2), 63.31 (NCH), 59.06 (OCH2), 54.83(NCH2), 26.70 (CH2CH2), 22.25 (CH2CH2), 15.20 (CNCH3). MS (ESI): calcd for [5 − Cl]+: 925.01; found: 925.55. Anal. Calcd for C36H46Cl2Fe2N4O2Pd2: C, 44.94; H, 4.82; N, 5.82. Found: C, 44.77; H, 4.74; N, 5.90. (−)-(Spl,SC,SC,Spl)-6: Mp >210 °C. [α]20 D − 898.1 (c 1.0 in CHCl3). 1 H NMR (400 MHz, CDCl3) δ = 4.89 [s, 1H; H5 (C5H3)], 4.80 [s, 1H; H5 (C5H3)], 4.38 [s, 2H; H3 (C5H3)]4.34 [s, 10H; C5H5], 4.27 [t, J = 2.4 Hz, 2H; H4 (C5H3)], 3.79−3.41 (m, 4H; CH + NCH2), 3.43− 3.16 (m, 10H; CH2OCH3), 3.01 (d, J = 37.7 Hz, 2H; NCH2), 2.29 (s, 6H; CH3CN), 2.15−1.60 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 186.85 (CN), 88.68 [C1 (C5H3)], 85.07 [C2 (C5H3)], 74.20 [C5 (C5H3)], 71.00[C3 (C5H3)], 70.78(C5H5), 67.70 [C4 (C5H3)], 65.74 (OCH2), 62.31 (NCH), 58.89 (OCH2), 54.34(NCH2), 26.08 (CH2CH2), 22.09 (CH2CH2), 15.08 (C NCH3). MS (ESI): m/z calcd for [3-Cl]+: 925.01; found: 927.27. MS (ESI): m/z calcd for [6 − Cl]+: 927.01; found: 927.15. Anal. Calcd for C36H46Cl2Fe2N4O2Pd2: C, 44.94; H, 4.82; N, 5.82. Found: C, 44.91; H, 4.84; N, 5.79. Compounds (−)-(Spl,RC,RC,Spl)-7 and (+)-(Rpl,RC,RC,Rpl)-8. Hydrazone (−)-(R)-2 (0.33 g, 1.0 mmol) was added to a methanolic (30 mL) solution containing Pd(OAc)2 (0.22 g, 1.0 mmol) and NaOAc· 3H2O (0.14 g, 1.0 mmol) and stirred at room temperature for 24 h, followed by treatment with excess NaBr and stirring at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into chloroform and passed through a SiO2-column with chloroform. Concentration of the eluted solution of two successive red bands produced compounds (−)-(Spl,RC,RC,Spl)-7 and (+)-(Rpl,RC,RC,Rp;)-8 which were recrystallized from dichloromethane/n-hexane (1:3) as red plates [product ratio 9:1, total yield 0.33 g (62.3%)]. Characterization data were as follows: (−)-(Spl,RC,RC,Spl)-7: Mp 238.1−238.7 °C. [α]20 D − 3516.0 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.86 [s, 2H; H5 (C5H3)], 4.37 [s, 12H; H5 (C5H3) + C5H5], 4.24 [s, 2H; H4 (C5H3)], 3.78 (s, 2H; CH), 3.41 (s, 10H; CH2OCH3), 3.30 (dd, J = 17.4, 8.7 Hz, 2H; NCH2), 2.82 (t, J = 7.6 Hz, 2H; NCH2), 2.28 (s, 6H; CH3CN), 2.04−1.61 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.15 (CN), 103.39 [C1 (C5H3)], 85.67 [C2 (C5H3)], 75.54 [C5 (C5H3)], 75.16[C3 (C5H3)], 70.74(C5H5), 67.92 [C4

(C5H3)], 65.76 (OCH2), 63.35 (NCH), 59.07 (OCH2), 54.90(NCH2), 26.60 (CH2CH2), 22.10 (CH2CH2), 15.28 (CNCH3). MS (ESI): m/z calcd for [7 − Br]+: 970.96; found: 971.29. Calcd for [7+H]+: 1048.88; found: 1048.73. Anal. Calcd for C36H46Br2Fe2N4O2Pd2: C, 41.13; H, 4.41; N, 5.33. Found: C, 41.10; H, 4.39; N, 5.32. (+)-(Rpl,RC,RC,Rpl)-8: Mp > 220 °C. [α]20 D + 2300.7 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.48 [(d, J = 1.7 Hz, 2H; H5 (C5H3)], 4.39 [s, 12H; H3 (C5H3) + C5H5], 4.32−4.29 [m, 2H; H4 (C5H3)], 3.52−3.44 (m, 2H; CH), 3.12 (t, J = 12.5 Hz, 2H; NCH2), 3.04−2.91 (m, 10H; CH2OCH3), 2.82 (dd, J = 10.5, 3.8 Hz, 2H; NCH2), 2.27 (s, 6H; CH3CN), 2.89−1.43 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.34 (CN), 102.00 [C1 (C5H3)], 86.34 [C2 (C5H3)], 75.30 [C5 (C5H3)], 74.63[C3 (C5H3)], 70.86(C5H5), 69.35 [C4 (C5H3)], 66.30 (OCH2), 63.76 (NCH), 59.07 (OCH2), 55.30(NCH2), 26.54 (CH2CH2), 21.89 (CH2CH2), 15.34 (CNCH3). MS (ESI): m/z calcd for [8 − Br]+: 970.96; found: 970.74. Anal. Calcd for C36H46Br2Fe2N4O2Pd2: C, 41.13; H, 4.41; N, 5.33. Found: C, 41.15; H, 4.44; N, 5.32. Compounds (+)-(Rpl,SC,SC,Rpl)-9 and (−)-(Spl,SC,SC,Spl)-10. Hydrazone (+)-(S)-2 (0.33 g, 1.0 mmol) was added to a methanolic (30 mL) solution containing Pd(OAc)2 (0.22 g, 1.0 mmol) and NaOAc·3H2O (0.14 g, 1.0 mmol) and stirred at room temperature for 24 h, followed by treatment with excess NaBr and stirring at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into chloroform and passed through a SiO2column with 10:1 chloroform/n-hexane. Concentration of the eluted solution of two successive red bands produced compounds (+)-(Rpl,SC,SC,Rpl)-9 and (−)-(Spl,SC,SC,Spl)-10 which were recrystallized from dichloromethane/n-hexane (1:3) as red plates [product ratio 9:1, total yield 0.30 g (57.42%)]. Characterization data were as follows: (+)-(Rpl,SC,SC,Rpl)-9: Mp 217.7−218.9°C. [α]20 D + 3500.9 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.93−4.78 [m, 2H; H5 (C5H3)], 4.37 [s, 12H; H3 (C5H3) + C5H5], 4.25 [s, 2H; H4 (C5H3)], 3.78 (s, 2H; CH), 3.64−3.22 (s, 14H; CH3OCH2 + NCH2), 2.82 (t, J = 7.3 Hz, 2H; NCH2), 2.28 (s, 6H; CH3CN), 2.21−1.59 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.15 (CN), 103.39 [C1 (C5H3)], 85.67 [C2 (C5H3)], 75.54 [C5 (C5H3)], 75.16[C3 (C5H3)], 70.74(C5H5), 67.92 [C4 (C5H3)], 65.76 (OCH2), 63.35 (NCH), 59.07 (OCH2), 54.90(NCH2), 26.60 (CH2CH2), 22.10 (CH2CH2), 15.28 (CNCH3). MS (ESI): m/z calcd for [9 − Br]+: 970.96; found: 969.55. Anal. Calcd for C36H46Br2Fe2N4O2Pd2: C, 41.13; H, 4.41; N, 5.33. Found: C, 41.10; H, 4.44; N, 5.37. (−)-(Spl,SC,SC,Spl)-10: Mp 241.0−241.7 °C. [α]20 D − 2359.0 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.95 [d, J = 23.8 Hz 2H; H5 (C5H3)], 4.37 [s, 2H; H3 (C5H3)], 4.33 [s, 10H; C5H5], 4.27 [s, 2H; H4 (C5H3)], 3.65 (s, 2H; CH), 3.58−3.45 (m, 2H; NCH2), 3.45− 3.34 (m, 10H; NCH2), 3.33−3.13 (m, 2H; CH3OCH2), 2.28 (s, 6H; CH3CN), 2.18−1.62 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 186.84 (CN), 97.92 [C1 (C5H3)], 86.79 [C2 (C5H3)], 75.83 [C5 (C5H3)], 72.79[C3 (C5H3)], 70.53(C5H5), 68.61 [C4 (C5H3)], 66.68 (OCH2), 63.57 (NCH), 59.09 (OCH2), 54.99(NCH2), 26.85 (CH2CH2), 22.29 (CH2CH2), 15.11 (C NCH3). MS (ESI): m/z calcd for [10 − Br]+: 970.96; found: 971.11. Anal. Calcd for C36H46Br2Fe2N4O2Pd2: C, 41.13; H, 4.41; N, 5.33. Found: C, 41.19; H, 4.42; N, 5.29. Compounds (−)-(Spl,RC,RC,Spl)-11 and (+)-(Rpl,RC,RC,Rpl)-12. Hydrazone (−)-(R)-2 (0.33 g, 1.0 mmol) was added to a methanolic (30 mL) solution containing Pd(OAc)2 (0.22 g, 1.0 mmol) and NaOAc· 3H2O (0.14 g, 1.0 mmol) and stirred at room temperature for 24 h, followed by treatment with excess NaI and stirring at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into chloroform and passed through a SiO2-column with chloroform. Concentration of the eluted solution of two successive red bands produced compounds (−)-(Spl,RC,RC,Spl)-11 and (+)-(Rpl,RC,RC,Rpl)-12 which were recrystallized from dichloromethane/n-hexane (1:3) as red plates [product ratio 9:1, total yield 0.38 g (65.9%)]. Characterization data were as follows: F


Article

Organometallics (−)-(Spl,RC,RC,Spl)-11: Mp 205.0−206.1 °C; [α]D20 − 2676.1 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.98 [d, J = 16.2 Hz 2H; H5 (C5H3)], 4.38 [s, 1H; H3 (C5H3)], 4.35 (s, 10H; C5H5), 4.27 [s, 2H; H4 (C5H3)], 3.80 (td, J = 10.5, 5.3 Hz, 2H; CH), 3.42 (s, 10H; CH2OCH3), 3.33 (s, 2H; NCH2), 2.82 (t, J = 7.7 Hz, 2H; NCH2), 2.28 (s, 6H; CH3CN), 2.07−1.62 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.32 (CN), 106.54 [C1 (C5H3)], 86.40 [C2 (C5H3)], 76.30 [C5 (C5H3)], 75.34[C3 (C5H3)], 70.81(C5H5), 69.30 [C4 (C5H3)], 66.23 (OCH2), 63.83 (NCH), 59.09 (OCH2), 55.29(NCH2), 26.58 (CH2CH2), 21.94 (CH2CH2), 15.37 (CNCH3). MS (ESI): m/z calcd for [11 − I]+: 1016.94; found: 1018.22. Calcd for [11 − I]+: 1018.94; found: 1018.22. Anal. Calcd for C36H46Fe2I2N4O2Pd2: C, 37.76; H, 4.05; N, 4.89. Found: C, 37.69; H, 4.37; N, 4.59. (+)-(Rpl,RC,RC,Rpl)-12: Mp 204.3−205.4 °C; [α]20 D + 2262.2 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 5.06 [d, J = 19.5 Hz, 2H; H5 (C5H3)], 4.38 [s, 2H; H3 (C5H3)], 4.32 (s, 10H; C5H5) 4.29 [s, 2H; H4 (C5H3)], 3.72 (d, J = 19.2 Hz; CH), 3.43 (s, 2H; NCH2), 3.34−3.14 (m, 10H; CH2OCH3), 2.99 (d, J = 29.7 Hz, 2H; NCH2), 2.28 (s, 6H; CH3CN), 2.21−1.67 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.15 (CN), 107.82 [C1 (C5H3)], 85.90 [C2 (C5H3)], 77.22 [C5 (C5H3)], 74.23 [C3 (C5H3)], 70.37 (C5H5), 69.65 [C4 (C5H3)], 66.26 (OCH2), 63.00 (NCH), 58.93 (OCH2), 55.10(NCH2), 26.16 (CH2CH2), 21.86 (CH2CH2), 15.27 (CNCH3). MS (ESI): m/z calcd for [12 − I]+: 1018.94; found: 1019.23. Anal. Calcd for C36H46Fe2I2N4O2Pd2: C, 37.76; H, 4.05; N, 4.89. Found: C, 37.72; H, 4.24; N, 4.62. Compound (+)-(Rpl,SC,SC,Rpl)-13 and (−)-(Spl,SC,SC,Spl)-14. Hydrazone (+)-(S)-2 (0.33 g, 1.0 mmol) was added to a methanolic (30 mL) solution containing Pd(OAc)2 (0.22 g, 1.0 mmol) and NaOAc·3H2O (0.14 g, 1.0 mmol) and stirred at room temperature for 24 h, followed by treatment with excess NaI and stirring at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into chloroform and passed through a SiO2column with 10:1 chloroform/n-hexane. Concentration of the eluted solution of two successive red bands produced compounds (+)-(Rpl,SC,SC,Rpl)-13 and (−)-(Spl,SC,SC,Spl)-14 which were recrystallized from dichloromethane/n-hexane (1:3) as red plates [product ratio 9:1, total yield 0.30 g (52.23%)]. Characterization data were as follows: (+)-(Rpl,SC,SC,Rpl)-13: Mp 199.7−200.0 °C. [α]20 D + 2690.0 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 4.98 [d, J = 15.5 Hz, 2H; H5 (C5H3)], 4.38 [s, 2H; H3 (C5H3)],4.35 (s, 10H; C5H5), 4.27 [s, 2H; H4 (C5H3)], 3.80 (td, J = 10.2, 5.0 Hz, 2H; CH), 3.42 (s, 10H; CH3OCH2), 3.33 (d, J = 8.3 Hz, 2H; NCH2), 2.82 (t, J = 7.5 Hz, 2H; NCH2), 2.28 (s, 6H; CH3CN), 2.06−1.65 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.31 (CN), 106.50 [C1 (C5H3)], 86.37 [C2 (C5H3)], 78.05 [C5 (C5H3)], 75.33 [C3 (C5H3)], 70.80 (C5H5), 69.30 [C4 (C5H3)], 66.23 (OCH2), 63.78 (NCH), 59.08 (OCH2), 55.28(NCH2), 26.56 (CH2CH2), 21.90 (CH2CH2), 15.37 (CNCH3). MS (ESI): m/z calcd for [13 − I]+: 1018.94; found: 1018.41. Anal. Calcd for C36H46Fe2I2N4O2Pd2: C, 37.76; H, 4.05; N, 4.89. Found: C, 37.89; H, 4.21; N, 4.54. (−)-(Spl,SC,SC,Spl)-14: Mp 204.3−204.7 °C. [α]20 D − 2273.1 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 5.06 [d, J = 17.8 Hz, 2H; H5 (C5H3)], 4.38 [s, 2H; H3 (C5H3)], 4.32 [s, 12H; H4 (C5H3) + C5H5], 3.73 (d, J = 21.4 Hz, 2H; NCH), 3.53−3.39 (m, 2H; NCH2), 3.34−3.12 (m, 10H; CH2OCH3), 3.00 (d, J = 26.4 Hz, 2H; NCH2), 2.28 (s, 6H; CH3CN), 2.15−8 1.65 (m, 8H; CH2CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ = 187.20 (CN), 104.29 [C1 (C5H3)], 85.91 [C2 (C5H3)], 77.72 [C5 (C5H3)], 74.42 [C3 (C5H3)], 71.10 (C5H5), 69.69 [C4 (C5H3)], 66.27 (OCH2), 63.00 (NCH), 58.94 (OCH3), 55.10 (NCH2), 26.18 (CH2CH2), 21.86 (CH2CH2), 15.27 (CNCH3). MS (ESI): m/z calcd for [14 + H]+: 1146.86; found: 1147.15. Anal. Calcd for C36H46Fe2I2N4O2Pd2: C, 37.76; H, 4.05; N, 4.89. Found: C, 37.92; H, 4.27; N, 4.96 Compound 15−18. The corresponding binuclear cyclopalladated compounds 3−6 (0.19 g, 0.2 mmol) were added to a acetone (10 mL) solution containing PPh3 (0.26 g 10 mmol), stirred at room temperature for 2 h. The resultant reaction mixture was dried under

high vacuum. The product was extracted into dichloromethane and passed through a SiO2-column with 4:1 petroleum ethe/ethyl acetate. Concentration of the eluted solution of one successive red band produced compound (−)-(Spl,RC)-15, (+)-(Rpl,RC)-16, (+)-(Rpl,SC)17, or (−)-(Spl,SC)-18 which was recrystallized from dichloromethane/ n-hexane (1:5) as reddish yellow plates in a high yield [0.27 g (92.45%), 0.27 g (92.27%), 0.24 g (83.97%), and 0.25 g (91.65%) respectively] Characterization data were as follows: (−)-(Spl,RC)-15: Mp 201.0−202.0 °C. [α]20 D − 917.6 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.88−7.70 [m, 6H; H2,H6 (C6H5)], 7.40 [d, J = 8.5 Hz, 9H; H3, H4, H5 (C6H5)], 4.40 [s, 1H; H5 (C5H3)], 4.25 [s, 1H; H3 (C5H3)], 3.99 [s, 1H; H4 (C5H3)], 3.90 (s, 5H; C5H5), 3.73 (d, J = 7.8 Hz, 1H; CH), 3.44−3.34 (m, 5H; CH2OCH3), 3.27 (s, 1H; NCH2), 2.94 (t, J = 7.1 Hz, 1H; NCH2), 2.48−2.24 (m, 4H; CH3CN + CH2), 1.97 (d, J = 9.6 Hz, 1H; CH2), 1.77 (dd, J = 18.9, 9.7 Hz, 1H; CH2), 1.65−1.59 (m, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 186.15 (CN), 135.06 [C1 (C6H5)], 132.03 [C2, C6 (C6H5)], 130.29 [C3, C5 (C6H5)], 127.93 [C4 (C6H5)], 100.91 [C1 (C5H3)], 86.68 [C5 (C5H3)], 76.53 [C3 (C5H3)], 76.23 [C4 (C5H3)], 70.20 (C5H5), 68.63 (OCH2), 66.49 (NCH), 63.07 (OCH2), 58.99 (OCH3), 55.00 (NCH2), 26.99 (CH2), 22.79 (CH2), 15.18 (CNCH3). MS (ESI): m/z calcd for [15 − Cl]+: 707.11; found: 707.28. Anal. Calcd for C36H38ClFeN2OPPd: C, 58.16; H, 5.15; N, 3.77. Found: C, 58.45; H, 5.52; N, 3.86. (+)-(Spl,RC)-16: Mp 221.0−221.6 °C. [α]20 D + 1122.2 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.78 [dd, J = 11.2, 6.8 Hz 6H; H2,H6 (C6H5)], 7.41 [t, J = 7.1 Hz, 9H; H3, H4, H5 (C6H5)], 4.39 [d, J = 2.0 Hz, 1H; H5 (C5H3)], 4.29 [s, 1H; H3 (C5H3)], 4.02 [s, 1H; H4 (C5H3)], 3.80 (s, 6H; C5H5 + NCH), 3.41 (s, 1H; NCH2),3.39 (s, 3H; OCH3) 3.31 (ddd, J = 24.6, 9.9, 4.9 Hz, 2H; OCH2), 3.02 (t, J = 7.2 Hz, 1H; NCH2), 2.36 (s, 3H; CNCH3), 2.33−2.22 (m, 1H; CH2), 2.13−2.05 (m, 1H; CH2), 1.87−1.75 (m, 1H; CH2), 1.69−1.59 (s, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 185.75 (CN), 134.87 [C1 (C6H5)], 132.18 [C2, C6 (C6H5)], 130.25 [C3, C5 (C6H5)], 127.99 [C4 (C6H5)], 99.98 [C1 (C5H3)], 86.16 [C5 (C5H3)], 76.96 [C3 (C5H3)], 75.44 [C4 (C5H3)], 70.54 (C5H5), 68.95 (OCH2), 66.42 (NCH), 62.06 (OCH2), 58.82 (OCH3), 54.91 (NCH2), 26.67 (CH2), 22.44 (CH2), 15.15 (CNCH3). MS (ESI): m/z calcd for [16 − Cl]+: 707.11; found: 706.92. Anal. Calcd for C36H38ClFeN2OPPd: C, 58.16; H, 5.15; N, 3.77. Found: C, 58.19; H, 5.30; N, 3.42. (+)-(Spl,RC)-17: Mp 159.0−159.7 °C. [α]20 D + 1308.9 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.88−7.70 [m, 6H; H2,H6 (C6H5)], 7.41 [t, J = 8.3 Hz, 9H; H3, H4, H5 (C6H5)], 4.39 [s, 1H; H5 (C5H3)], 4.25 [s, 1H; H3 (C5H3)], 3.90 [s, 1H; H4 (C5H3)], 3.90 (s, 5H; C5H5), 3.73 (dd, J = 15.1, 7.2 Hz, 1H; CH), 3.40 (s, 5H; CH2OCH3), 3.27 (s, 1H; NCH2), 2.94 (t, J = 7.5 Hz, 1H; NCH2), 2.48−2.38 (m, 1H; CH2), 2.36 (s, 3H; CNCH3), 1.97 (dd, J = 17.3, 10.4 Hz, 1H; CH2), 1.85−1.71 (m, 1H; CH2), 1.67−1.58 (s, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 186.15 (CN), 135.06 [C1 (C6H5)], 132.02 [C2, C6 (C6H5)], 130.28 [C3, C5 (C6H5)], 127.93 [C4 (C6H5)], 100.91 [C1 (C5H3)], 86.68 [C5 (C5H3)], 76.58 [C3 (C5H3)], 76.22 [C4 (C5H3)], 70.20 (C5H5), 68.62 (OCH2), 66.49 (NCH), 63.06 (OCH2), 58.99 (OCH3), 55.01 (NCH2), 26.98 (CH2), 22.78 (CH2), 15.17 (CNCH3). MS (ESI): m/z calcd for [17 − Cl]+: 707.11; found: 707.46. Anal. Calcd for C36H38ClFeN2OPPd: C, 58.16; H, 5.15; N, 3.77. Found: C,58.14; H,5.32; N,3.54. (−)-(Spl,RC)-18: Mp 181.2−182.0 °C. [α]20 D − 1010.6 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.85−7.72 [m, 6H; H2,H6 (C6H5)], 7.48−7.33 [m, 9H; H3, H4, H5 (C6H5)], 4.38 [d, J = 2.2 Hz, 1H; H5 (C5H3)], 4.30 [s, 1H; H3 (C5H3)], 4.01 [s, 1H; H4 (C5H3)], 3.80 (s, 6H; C5H5 + NCH), 3.40 (s, 1H; NCH2),3.39−3.24 (m, 5H; CH2OCH3), 3.02 (t, J = 7.4 Hz, 1H; NCH2), 2.36 (s, 3H; CNCH3), 2.33−2.23 (m, 1H; CH2), 2.13−2.04 (m, 1H; CH2), 1.88−1.76 (m, 1H; CH2), 1.69−1.60 (s, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 185.76 (CN), 134.88 [C1 (C6H5)], 132.18 [C2, C6 (C6H5)], 130.27 [C3, C5 (C6H5)], 128.00 [C4 (C6H5)], 103.10 [C1 (C5H3)], 86.17 [C5 (C5H3)], 76.98 [C3 (C5H3)], 75.45 [C4 (C5H3)], 70.55 (C5H5), 68.94 (OCH2), 66.43 (NCH), 62.07 (OCH2), 58.82 (OCH3), 54.92 (NCH2), 26.68 (CH2), 22.44 (CH2), 15.16 (CNCH3). MS (ESI): m/z calcd for [18 − Cl]+: 707.11; found: 707.46. Anal. Calcd for G


Article

Organometallics

106.64 [C1 (C5H3)], 86.02 [C5 (C5H3)], 77.26 [C3 (C5H3)], 75.58 [C4 (C5H3)], 70.61 (C5H5), 68.73 (OCH2), 66.17 (NCH), 62.22 (OCH2), 58.81 (OCH3), 55.10 (NCH2), 26.68 (CH2), 22.30 (CH2), 15.22 (CNCH3). MS (ESI): m/z calcd for [22 − Br]+: 707.11; found: 706.92 . Anal. Calcd for C36H38BrFeN2OPPd: C, 54.88; H, 4.86; N, 3.56. Found: C, 54.50; H, 4.65; N, 3.42. Compound 23−26. Corresponding binuclear cyclopalladium compounds 11−14 (0.23 g, 0.2 mmol) were added to an acetone (10 mL) solution containing PPh3 (0.26 g 10 mmol), stirred at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into dichloromethane and passed through a SiO2-column with 4:1 petroleum ether/ethyl acetate. Concentration of the eluted solution of one successive red band produced compound (−)-(Spl,RC)-23, (+)-(Rpl,RC)-24, (+)-(Rpl,SC)25, or (−)-(Spl,SC)-26 which were recrystallized from dichloromethane/n-hexane (1:5) as reddish yellow plates in a high yield [0.30 g (90.80%), 0.30 g(89.69%), 0.25 g (84.73%), and 0.28 g (86.32%) respectively] Characterization data are as follows: (−)-(Spl,RC)-23: Mp 184.1−184.6 °C. [α]20 D − 1262.6 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.78 [dd, J = 11.2, 7.7 Hz, 6H; H2,H6 (C6H5)], 7.40 [t, J = 10.3 Hz, 9H; H3, H4, H5 (C6H5)], 4.38 [s, 2H; H5 (C5H3) + H3 (C5H3)], 3.96 [s, 1H; H4 (C5H3)], 3.88 (s, 5H; C5H5), 3.79 (td, J = 10.7, 2.6 Hz, 1H; CH), 3.40 (s, 6H; CH2OCH3 + NCH2), 2.90 (t, J = 7.6 Hz, 1H; NCH2), 2.51 (dq, J = 18.4, 9.3 Hz, 1H; CH2), 2.38 (s, 3H; CNCH3), 2.08 (d, J = 9.5 Hz, 1H; CH2), 1.93−1.79 (m, 1H; CH2), 1.69−1.59 (m, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 187.54 (CN), 135.07 [C1 (C6H5)], 133.98 [C2, C6 (C6H5)], 130.18 [C3, C5 (C6H5)], 127.86 [C4 (C6H5)], 108.43 [C1 (C5H3)], 86.84 [C5 (C5H3)], 76.29 [C3 (C5H3)], 75.85 [C4 (C5H3)], 70.33 (C5H5), 68.30 (OCH2), 66.19 (NCH), 64.32 (OCH2), 58.97 (OCH3), 56.00 (NCH2), 26.90 (CH2), 22.41 (CH2), 15.61 (CNCH3). MS (ESI): m/z calcd for [23 − I]+: 707.11; found: 707.28. Anal. Calcd for C36H38FeIN2OPPd: C, 51.79; H, 4.59; N, 3.36. Found: C, 51.77; H, 4.74; N, 3.32. (+)-(Spl,RC)-24: Mp 188.9−190.0 °C. [α]20 D + 1115.7 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.80 [ddd, J = 11.4, 7.8, 1.4 Hz, 6H; H2, H6 (C6H5)], 7.52−7.32 [m; 9H; H3, H4, H5 (C6H5)], 4.46−4.38 [m, 1H; H5 (C5H3)], 4.36 [d, J = 2.1 Hz; 1H; H3 (C5H3)], 3.97 [t, J = 2.1 Hz, 1H; H4 (C5H3)], 3.79 (s, 5H; C5H5), 3.58 (s, 1H; NCH),3.49−3.25 (m, 5H; CH2OCH3) 2.98 (t, J = 7.8 Hz, 1H; NCH2), 2.46−2.32 (m, 4H; CNCH3 + NCH2), 2.26−2.13 (m, 1H; CH2), 1.95−1.80 (m, 1H; CH2), 1.71−1.59 (s, 2H; CH2). 13C NMR (100 MHz, CDCl3) δ = 186.15 (CN), 134.95 [C1 (C6H5)], 133.97 [C2, C6 (C6H5)], 130.18 [C3, C5 (C6H5)], 127.97 [C4 (C6H5)], 112.12 [C1 (C5H3)], 85.94 [C5 (C5H3)], 76.98 [C3 (C5H3)], 75.73 [C4 (C5H3)], 70.72 (C5H5), 68.40 (OCH2), 65.76 (NCH), 62.67 (OCH2), 58.84 (OCH3), 55.64 (NCH2), 26.57 (CH2), 22.10 (CH2), 15.36 (CNCH3). MS (ESI): m/z calcd for [24 − Cl]+: 707.11; found: 707.28. Anal. Calcd for C36H38FeIN2OPPd: C, 51.79; H, 4.59; N, 3.36. Found: C, 51.71; H, 4.48; N, 3.38. (+)-(Spl,RC)-25: Mp 190.1−190.9 °C. [α]20 D + 1065.7 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.78 [dd, J = 11.3, 6.7 Hz, 6H; H2, H6 (C6H5)], 7.47−7.30 [m, 9H; H3, H4, H5 (C6H5)], 4.38 [s, 1H; H5 (C5H3)], 4.37−4.31 [m, 1H; H3 (C5H3)], 3.96 [s, 1H; H4 (C5H3)], 3.88 (s, 5H; C5H5), 3.83−3.73 (m, 1H; CH), 3.40 (s, 6H; CH2OCH3 + NCH2), 2.90 (t, J = 7.8 Hz, 1H; NCH2), 2.51 (dq, J = 18.2, 9.0 Hz, 1H; CH2), 2.38 (s, 3H; CNCH3), 2.08 (dt, J = 11.7, 8.4 Hz, 1H; CH2), 1.94−1.78 (m, 1H; CH2), 1.69−1.59 (s, 1H; CH2). 13 C NMR (100 MHz, CDCl3) δ = 187.57 (CN), 135.06 [C1 (C6H5)], 134.01 [C2, C6 (C6H5)], 130.19 [C3, C5 (C6H5)], 127.89 [C4 (C6H5)], 108.47 [C1 (C5H3)], 86.87 [C5 (C5H3)], 76.32 [C3 (C5H3)], 75.88 [C4 (C5H3)], 70.36 (C5H5), 66.22 (OCH2), 64.35 (NCH), 64.35 (OCH2), 59.00 (OCH3), 56.03 (NCH2), 26.93 (CH2), 22.44 (CH2), 15.65 (CNCH3). MS (ESI): m/z calcd for [25 − I]+: 707.11; found: 707.44. Anal. Calcd for C36H38FeIN2OPPd: C, 51.79; H, 4.59; N, 3.36. Found: C, 52.16; H, 4.66; N, 3.22. (−)-(Spl,RC)-26: Mp 199.7−202.0 °C. [α]20 D − 970.0 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.80 [dd, J = 10.9, 7.6 Hz, 6H; H2, H6 (C6H5)], 7.39 [t, J = 7.6 Hz, 9H; H3, H4, H5 (C6H5)], 4.39 [s, 1H; H5 (C5H3)], 4.36 [s, 1H; H3 (C5H3)], 3.97 [s, 1H; H4

C36H38ClFeN2OPPd: C, 58.16; H, 5.15; N, 3.77. Found: C, 58.38; H, 5.33; N, 3.79. Compound 19−22. Corresponding binuclear cyclopalladium compounds 7−10 (0.21 g, 0.2 mmol) were added to an acetone (10 mL) solution containing PPh3 (0.26 g 10 mmol), stirred at room temperature for 2 h. The resultant reaction mixture was dried under high vacuum. The product was extracted into dichloromethane and passed through a SiO2-column with 4:1 petroleum ether/ethyl acetate. Concentration of the eluted solution of one successive red band produced compound (−)-(Spl,RC)-19, (+)-(Rpl,RC)-20, (+)-(Rpl, SC)21, or (−)-(Spl,SC)-22 which was recrystallized from dichloromethane/ n-hexane (1:5) as reddish yellow plates in a high yield [0.26 g (81.66%), 0.30 g (95.72%), 0.25 g (80.91%), and 0.28 g (90.13%), respectively] Characterization data are as follows: (−)-(Spl,RC)-19: Mp 171.1−172.0 °C. [α]20 D − 1323.4 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.85−7.72 [m, 6H; H2,H6 (C6H5)], 7.40 [d, J = 8.5 Hz, 9H; H3, H4, H5 (C6H5)], 4.39 [s, 1H; H5 (C5H3)], 4.30 [s, 1H; H3 (C5H3)], 3.98 [s, 1H; H4 (C5H3)], 3.88 (s, 5H; C5H5), 3.78 (dd, J = 15.7, 7.8 Hz, 1H; CH), 3.39 (t, J = 3.6 Hz, 5H; CH2OCH3), 3.33 (s, 1H; NCH2), 2.93 (t, J = 7.1 Hz, 1H; NCH2), 2.52−2.39 (m, 1H; CH2), 2.37 (s, 3H; CNCH3), 2.07−1.94 (m, 1H; CH2), 1.88−173 (m, 1H; CH2), 1.66−1.59 (m, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 186.75 (CN), 135.04 [C1 (C6H5)], 132.77 [C2, C6 (C6H5)], 130.22 [C3, C5 (C6H5)], 127.89 [C4 (C6H5)], 103.86 [C1 (C5H3)], 86.68 [C5 (C5H3)], 76.43 [C3 (C5H3)], 76.15 [C4 (C5H3)], 70.26 (C5H5), 68.49 (OCH2), 66.41 (NCH), 63.74 (OCH2), 58.98 (OCH3), 55.36 (NCH2), 26.99 (CH2), 22.67 (CH2), 15.34 (CNCH3). MS (ESI): m/z calcd for [19 − Br]+: 707.11; found: 707.29. Anal. Calcd for C36H38BrFeN2OPPd: C, 54.88; H, 4.86; N, 3.56. Found: C, 55.01; H, 4.75; N, 3.33 (+)-(Spl,RC)-20: Mp 182.5−183.5 °C. [α]20 D + 1043.2 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.77 [dd, J = 18.4, 6.8 Hz 6H; H2,H6 (C6H5)], 7.46−7.35 [m, 9H; H3, H4, H5 (C6H5)], 4.39 [d, J = 2.0 Hz, 1H; H5 (C5H3)], 4.30 [s, 1H; H3 (C5H3)], 4.00 [d, J = 11.3 Hz, 1H; H4 (C5H3)], 3.90 (s, 1H; NCH) 3.80 (s, 5H; C5H5), 3.44− 3.24 (m, 6H; CH2OCH3 + NCH2), 3.06−2.97 (m, 1H; NCH2), 2.36 (s, 3H; CNCH3), 2.33−2.05 (m, 2H; CH2), 2.04−1.72 (m, 2H; CH2). 13C NMR (100 MHz, CDCl3) δ = 185.87 (CN), 135.10 [C1 (C6H5)], 132.33 [C2, C6 (C6H5)], 130.38 [C3, C5 (C6H5)], 128.12 [C4 (C6H5)], 102.33 [C1 (C5H3)], 86.29 [C5 (C5H3)], 77.36 [C3 (C5H3)], 75.60 [C4 (C5H3)], 70.50 (C5H5), 69.40 (OCH2), 66.53 (NCH), 62.19 (OCH2), 58.93 (OCH3), 55.04 (NCH2), 26.82 (CH2), 22.56(CH2), 15.26 (CNCH3). MS (ESI): m/z calcd for [20 − Br]+: 707.11; found: 707.27. Anal. Calcd for C36H38BrFeN2OPPd: C, 54.88; H, 4.86; N, 3.56. Found: C, 54.87; H, 4.85; N, 3.59; (+)-(Spl,RC)-21: Mp 193.0−194.0 °C. [α]20 D + 1262.7 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.78 [dd, J = 10.6, 7.2 Hz, 6H; H2,H6 (C6H5)], 7.51−7.30 [m, 9H; H3, H4, H5 (C6H5)], 4.39 [s, 1H; H5 (C5H3)], 4.35−4.25 [m, 1H; H3 (C5H3)], 3.97 [t, J = 2.2 Hz, 1H; H4 (C5H3)], 3.88 (s, 5H; C5H5), 3.83−3.71 (m, 1H; CH), 3.39 (s, 5H; CH2OCH3), 3.22 (s, 1H; NCH2), 2.92 (t, J = 7.5 Hz, 1H; NCH2), 2.54−2.39 (m, 1H; CH2), 2.36 (s, 3H; CNCH3), 2.01 (dd, J = 18.2, 9.5 Hz, 1H; CH2), 1.88−1.74 (m, 1H; CH2), 1.66−1.58 (s, 1H; CH2). 13 C NMR (100 MHz, CDCl3) δ = 185.98 (CN), 134.85 [C1 (C6H5)], 132.90 [C2, C6 (C6H5)], 130.18 [C3, C5 (C6H5)], 127.97 [C4 (C6H5)], 106.64 [C1 (C5H3)], 86.02 [C5 (C5H3)], 75.58 [C3 (C5H3)], 77.26 [C4 (C5H3)], 70.61 (C5H5), 68.73 (OCH2), 66.17 (NCH), 62.22 (OCH2), 58.81 (OCH3), 55.10 (NCH2), 26.68 (CH2), 22.30 (CH2), 15.22 (CNCH3). MS (ESI): m/z calcd for [21 − Br]+: 707.11; found: 707.38. Anal. Calcd for C36H38BrFeN2OPPd: C, 54.88; H, 4.86; N, 3.56. Found: C, 54.87; H, 4.89; N, 3.53. (−)-(Spl,RC)-22: Mp 185.1−186.0 °C. [α]20 D − 650.7 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3) δ = 7.80 [dd, J = 11.3, 6.7 Hz, 6H; H2,H6 (C6H5)], 7.39 [d, J = 7.0 Hz, 9H; H3, H4, H5 (C6H5)], 4.37 [d, J = 2.0 Hz, 2H; H5 (C5H3) + H3 (C5H3)], 4.00 [s, 1H; H4 (C5H3)], 3.79 (s, 6H; C5H5 + NCH), 3.49 (s, 1H; NCH2),3.43−3.25 (m, 5H; CH2OCH3), 3.00 (t, J = 7.7 Hz, 1H; NCH2), 2.36 (s, 3H; CNCH3), 2.30−2.08 (m, 2H; CH2), 2.05−1.78 (m, 2H; CH2). 13C NMR (100 MHz, CDCl3) δ = 185.98 (CN), 134.85 [C1 (C6H5)], 132.90 [C2, C6 (C6H5)], 130.18 [C3, C5 (C6H5)], 127.97 [C4 (C6H5)], H


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Organometallics (C5H3)], 3.79 (s, 6H; C5H5 + NCH), 3.58 (s, 1H; NCH2),3.46−3.25 (m, 5H; CH2OCH3), 2.98 (t, J = 7.6 Hz, 1H; NCH2), 2.46−2.31 (m, 4H; CNCH3 + CH2), 2.24−2.14 (m, 1H; CH2), 1.87 (td, J = 16.9, 8.1 Hz, 1H; CH2), 1.69−1.60 (s, 1H; CH2). 13C NMR (100 MHz, CDCl3) δ = 186.12 (CN), 134.93 [C1 (C6H5)], 133.95 [C2, C6 (C6H5)], 130.16 [C3, C5 (C6H5)], 127.95 [C4 (C6H5)], 112.08 [C1 (C5H3)], 85.93 [C5 (C5H3)], 76.96 [C3 (C5H3)], 75.71 [C4 (C5H3)], 70.70 (C5H5), 68.37 (OCH2), 65.73 (NCH), 62.65 (OCH2), 58.82 (OCH3), 55.63 (NCH2), 26.54 (CH2), 22.08 (CH2), 15.34 (C NCH3). MS (ESI): m/z calcd for [26 − I]+: 707.11; found: 707.27. Anal. Calcd for C36H38FeIN2OPPd: C, 51.79; H, 4.59; N, 3.36. Found: C, 51.64; H, 4.38; N, 3.56. Electrochemistry. The cyclic voltammograms (CVs) were recorded as a reported method.25 In brief, the methanol/acetonitrile (v/v 1/1) solution was used as the electrolyte with 0.1 M [NBu4][PF6] as the supporting electrolyte. The CVs were recorded at room temperature and platinum electrode used as working electrode, glassy carbon electrode used as counter electrode, and calomel electrode used as reference electrode. The scan rate was 100 mV/s and the ferrocene/ferrocenium redox couple (1.0 mM) were taken as reference when the E1/2 values were measured. Biological Studies. Human Huh-7 cells, SK-Hep-1 cells, MCF-7 cells, and HCT116 cells were maintained in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. Cell Viability Assay. Cells were seeded in 96-well plates at a density of 5 × 103 cells/well with 100 μL of medium. Cultured cells were treated with compounds 3−26 or cisplatin at the indicated concentrations. After 24 h, 10 μL of Alamar Blue reagent (Solarbio, Beijing, China) was added to the medium and the cells were incubated for 2−4 h until the color turned from blue to pink. The relative fluorescence intensity was measured using a Thermo Scientific Varioskan Flash multimode reader. DNA Interaction Studies. A stock solution (10 mL) of compounds 3−26 was prepared in high-purity DMSO. Then, serial dilutions were made in Milli-Q water (1:1). Plasmid DNA PET21a (Novagen, Madison, WI) was obtained using PureYieldTM Plasmid Maxiperp System as described by the manufacturer (Promega, Beijing, China). Interaction of drugs with PET21a plasmid DNA was analyzed by agarose gel electrophoresis following a modification of the method previously described.5 Plasmid DNA aliquots (40 μg/mL) were incubated in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) with different concentrations of test compounds ranging from 0 to 200 μM at 37 °C for 24 h. Final DMSO concentration in the reactions was always lower than 1%. Cisplatin was dissolved in normal saline and used as positive control. Aliquots of 20 μL of incubated solutions of compounds containing 0.8 μg of DNA were subjected to 1% agarose gel electrophoresis in TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0). The gel was stained in TAE buffer containing goldview I DNA coloring agent (Solarbio), visualized and photographed under UV light. Cell Cycle and Apoptosis Assay. A total of 2 × 105 SK-Hep-1 cells were plated in a six-well plate for 24 h and then treated with compound 7 for 24 h at 37 °C. After incubation, the cells were harvested and washed with ice-cold PBS. The cell cycle progression was analyzed using a cell cycle and apoptosis analysis kit (Beyotime, Haimen, China), and the apoptosis ratio was performed with an annexin V-FITC Apoptosis Detection Kit (Beyotime). Wound Healing Assay and Transwell Invasion Assay. SKHep-1 cells were cultured to confluence in 24-well plates and wounded using a sterilized pipet tip to make a straight scratch. Cells were rinsed with physiological saline gently, and then PBS was replaced with DMEM medium containing compound 7 or cisplatin. Pictures were taken by a 600D Nikon Camera under microscope. For evaluation of wound closure, four randomly selected points along each wound area were marked, and the horizontal distance of migrating cells were from the initial wound was measured.34 The cells invasion assay was performed as described.34b The 2 × 105 cells in 200 μL of DMEM medium were added in the upper chamber and incubating 15 min at 37 °C, then replaced with basal DMEM medium (containing 10% FBS

and 1% penicillin/streptomycin) containing compound 7 or cisplatin. The plate was transferred to incubator, after 24 h, invasion terminated by removing the cells on the top with a cotton swab, and the filters fixed with 3% paraformaldehyde and stained with crystal violet (Selleck, Shanghai, China). Pictures were taken by a 600D Nikon Camera under microscope at five different places. Caspase-3, -8, and -9 Activity Assays. The activities of caspase3, -8, and -9 in SK-Hep-1 cells were assessed based on the specific protease-peptide substrate chromogenic reaction.35,36 In brief, SKHep-1 cells cultured in 6-well plates at 4 × 105 cells/well were treated for 12 h with PBS (as control) and different concentrations of compound 7. After that, the cells were harvested, lysed, and centrifugated. Then, aliquots of supernatants were collected and incubated with the peptide substrates of caspase-3 (Ac-DEVD-pNA), -8 (Ac-IETD-pNA), and -9 (Ac-LEHD-pNA) (Enzo Life Sciences, Inc., Farmingdale, NY), respectively. The activities of caspase-3, -8, and -9 were determined based on the absorbance at 405 nm by Thermo Scientific Varioskan Flash multimode reader. The total protein content of each sample was determined to normalize the obtained values by a BCA protein assay kit (Bestbio, Shanghai, China), and the activity ratio was calculated as compared to the blank control. Western Blot Analysis. Cells were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Sigma, Shanghai, China). The protein concentration was determined using a BCA protein assay kit (Bestbio). Aliquots of total cell lysates (40 μg protein) were mixed with loading buffer, boiled for 5 min, and subjected to 10% SDS-PAGE. Proteins were blotted onto nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin and then incubated at 4 °C overnight with anti-PARP (Beyotime) and anti-GAPDH (Huabio, Hangzhou, China) antibodies. Next, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotech, CA) and developed using an enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ). The intensity of each signal was determined by a computer imaging analysis system (Quantity One, Bio-Rad, Hercules, CA).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.7b00897. NMR Spectra and X-ray diffraction analysis of compounds 16, 18, 22, and 26 (PDF) Accession Codes

CCDC 1546619−1546621 and 1546623 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/ cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gang Zhao: 0000-0002-7272-3270 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Sichuan University High Level Talent Project, Sichuan Province 1,000 Talents Plan Project, the National Natural Science Foundation of China (Nos. I


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Organometallics

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21561142003 and 21672207), and Science & Technology Department of Sichuan Province (No. 2016JZ0022).

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Planar Chiral Ferrocene Cyclopalladated Derivatives Induce Caspase

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