Growth Inhibition by Induction Cell Apoptosis a - ACS Publications

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Effective Antitumor Candidates Based upon FerrocenylselenoDopamine Derivatives: Growth Inhibition by Induction Cell Apoptosis and Antivascular Effects Hai-Yan Zhou,† Meng Li,‡ Jian Qu,†,∥ Su Jing,*,† Heng Xu,*,§ Juan-Zhi Zhao,⊥ Jian Zhang,⊥ and Ming-Fang He‡ †

School of Chemistry and Molecular Engineering, ‡School of Pharmaceutical Sciences, and §Jiangsu Province Institute of Materia Medica, Nanjing Tech University, Nanjing 211816, People’s Republic of China ∥ Institute of Advanced Materials, Nanjing Tech University, Nanjing 210009, People’s Republic of China ⊥ Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, People’s Republic of China S Supporting Information *

ABSTRACT: One ferrocenyl-dopamine (L1) and four ferrocenylseleno-dopamine derivatives (L2−L5) were designed and prepared with different structural parameters, such as chalcogen atoms, cycle, and semirigidity. The best in vitro anticancer activity occurred with 1,5-diselena[8]ferrocenophane L5, which inhibited cancer cell growth at the lowest micromolar concentrations. The biological studies showed that L5 arrested the cell cycle in G1 phase and induced apoptosis by activating caspase 3/9, also inhibiting endothelial cell (HUVECs) formation. It exhibited better in vivo antitumor activity in mice bearing HepG2 tumor xenograft in comparison to free dopamine. The results suggest that the excellent biological activities can be attributed to the synergetic effect of the ferrocenophane, chalcogen atom, and catechol group.

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INTRODUCTION Since the discovery of ferrocene in the early 1950s,1 ferrocenyl derivatives have attracted tremendous interest in the field of catalysis, materials science, and pharmaceutical chemistry.2 Owing to the advantages of kinetic stability, hypotoxicity, ease of modification, and good redox properties, a number of bioferrocene species have been found to be promising anticancer candidates, such as ferrocifen type compounds and ferrocenyl nucleobases.3 Two main anticancer mechanisms were suggested for these ferrocenyl derivatives. The first is the ferrocene unit undergoing oxidation to form the ferrocenium cation in the presence of glutathione, this oxidation process catalyzes the production of reactive oxygen species (•OH etc.), thus generating cytotoxic effects.4 The second is ferrocenyl compounds forming linkages with the ATP binding site and inhibiting the catalytic activity of topoisomerase II to rapidly proliferate cancerous cells.5 In this endeavor, the best known example is ferrocifen type compounds; cytotoxic effects occur by senescence induction, formation of reactive oxygen species, and/ or antihormone effects.6 In addition, ferrocenyl phenols exhibit effects through in situ oxidation to form cytotoxic quinone methides.7 It has been found that the cyclic ferrocifens based upon [3]ferrocenophane show significant activity enhancement possibly due to the steric strain.8 In the same context, selenium has been found to play a role in anticarcinogenesis in two ways: as an essential nutrient providing © XXXX American Chemical Society

the catalytic centers of antioxidant enzymes or as a source that directly affects tumorigenesis.9 The Jacob group reported a series of peptidomimetic compounds containing redox-active chalcogens and quinones as potential anticancer agents.10 The LópezCortés group designed a series of ferrocenyl selenoamides, which show good anticancer potency superior to that of tamoxifen and cisplatin.11 Our continued interest in the study of ferrocenyl selenoethers led us to investigate their potential biomedical properties.12 Dopamine (DA) as a kind of neurotransmitter, has been reported to modulate tumor angiogenesis and immunity by inhibiting VEGF.13 Meanwhile, DA contains a catechol group that can be easily oxidized to reactive quinone, which then reacts with functional groups (−NH2 or −SH) of proteins in human blood and effectively helps deliver the drug to the blood.14 This quinone is then selected to incorporate into the skeleton of ferrocenyl selenoether compounds for the purpose of improving the antitumor activity, biocompatibility, and drug transport capacity. In this study, we designed and synthesized one ferrocenyl-dopamine derivative (L1) and four ferrocenylseleno-dopamine species (L2−L5) with different structural parameters. Their anticancer activities were evaluated, and 1,5diselena[8]ferrocenophane L5 was found to give the best Received: March 22, 2016

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DOI: 10.1021/acs.organomet.6b00237 Organometallics XXXX, XXX, XXX−XXX

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Organometallics Scheme 1. Synthetic Scheme of Ferrocenyl-Dopamine Derivatives L1−L5

more negative than those of B1−B5, which means the ferrocene moiety is easier to oxidize after the incorporation of the DA unit. It is not surprising to find that the ferrocene moiety in the ferrocenophane L5 is easier to oxidize than that of the acyclic analogues, which is in agreement with our previous studies.12 Studies have shown that the lower half -wave potential for oxidation of the ferrocene moiety is related to better anticancer activity.7b,16 The oxidation of the ferrocene moiety can induce the catechol group to form reactive o-quinone products,16 which have much greater advantages in inhibiting the thioredoxin reductases, the key targets in anticancer drug action mechanisms.5c L5 is then expected to have the best anticancer activity among the compounds tested. In Vitro Anticancer Activities. The growth inhibitory effects of the ferrocenylseleno-dopamine derivatives L2−L5 were evaluated on six human cancerous cell lines by cytotoxicity assays, using AGS (gastric adeno carcinoma), A2780 (ovarian carcinoma), A549 (nonsmall-cell lung carcinoma), BxPC-3 (pancreatic cancer), HepG2 (hepatocellular carcinoma), and MGC-803 (human gastric cancer). L1, B3 (as a representative of B1−B5), and DA were chosen as controls in order to clarify the roles played by the ferrocene part and selenium and catechol groups in anticancer properties. The IC50 values of the tested compounds against cancer cells are summarized in Table 2. From data in Table 2, we drew the conclusion that almost all cell lines were sensitive to L3−L5 with a low IC50 value of 100 μM) and compound DA without a ferrocene part

anticancer activity. Studies were then performed to elucidate the mode of action of L5 in HepG2. The biological studies both in vitro and in vivo revealed that ferrocenylseleno-dopamine derivatives could not only induce carcinoma cell apoptosis but also inhibit angiogenesis of vessels in hepatocellular carcinoma in vitro or in vivo.

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RESULTS AND DISCUSSION Synthesis. The five ferrocenyl-dopamine derivatives L1−L5 were synthesized as shown in Scheme 1. Solution-phase peptide coupling of ferrocenylcarboxylic acid (B1−B5) with free Nterminal dopamine gives the target products. These species were characterized by conventional spectroscopic techniques; all data are given in the Supporting Information and are in agreement with the proposed structures. Electrochemistry. The redox behavior was investigated by cyclic voltammetry (CV) in acetonitrile/methanol (v/v 1/1) with [NBu4][PF6] (0.1 M) as a supporting electrolyte. The halfwave potentials for oxidation of the ferrocene moiety are summarized in Table 1. The cyclic voltammograms of B1−B5 and L1−L5 show one quasi-reversible redox wave in the potential range −0.5 to +1.0 V (Figures S1−S5 in the Supporting Information), which is assigned to oxidation of the ferrocene moiety.15 The half-wave potentials for oxidation of the ferrocene moiety of L1−L5 are all Table 1. Cyclic Voltammetric Data of Compounds B1−B5 and L1−L5a E1/2 (|Epa − Epc|) (mV) B L ΔE1/2

1

2

3

4

5

272 (80) 192 (59) −80

101 (94) 53 (75) −48

88 (91) 49 (78) −39

112 (72) 59 (72) −53

45 (76) −12 (54) −57

a

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


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Organometallics

Table 2. Anticancer Activity of Compounds L1−L5, B3, and DA: IC50 Measured against Cancer Cell Lines after 72 h of Treatment IC50 (μM)a L1 L2 L3 L4 L5 B3 DA

AGS

A2780

A549

BxPC-3

HepG2

MGC-803

>100.0 12.1 ± 0.6 12.8 ± 2.3 3.7 ± 0.1 2.4 ± 0.4 >100.0 >50.0

27.7 ± 1.7 18.5 ± 2.6 12.5 ± 0.3 8.4 ± 0.7 2.3 ± 0.3 >100.0 >100.0

>100.0 >50.0 48.4 ± 2.3 48.2 ± 3.7 4.8 ± 1.2 >100.0 28.2 ± 4.7

35.4 ± 0.7 >50.0 10.1 ± 0.9 6.6 ± 0.5 5.4 ± 0.7 >100.0 21.3 ± 1.9

16.6 ± 0.8 14.4 ± 1.8 10.6 ± 1.2 5.2 ± 0.8 2.2 ± 0.5 >100.0 >50.0

28.4 ± 3.1 24.8 ± 4.9 14.0 ± 0.9 12.7 ± 1.0 4.5 ± 0.1 >100.0 28.8 ± 6.1

IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are presented as the mean ± SD from the dose−response curves at least three independent experiments.

a

Figure 1. Anticancer activities of (a) L5 in different cancer cell lines and (b) L1−L5, B3, B5, and DA in the HepG2 cell line.

(IC50 > 50 μM) had no significant effect on inhibition of cancer cells. The compounds L2−L5 containing selenium showed greater cytotoxicity than L1 (Table 2 and Figure 2b), which may be due to selective biological activities of the chalcogen atom.10 L3, with

one more chalcogen atom (sulfur) and an extending peptide chain, showed cytotoxicity greater than that of L2. L4, containing a benzyl unit, exhibited higher cytotoxicity than L2. The IC50 values of L4 against HepG2, AGS, A2780, and BxPC-3 cells were significantly lower than those of L2. The semirigidity and the rich π-electron presence of the benzene ring between the ferrocene part and the catechol group in L4 may be the reason for the potent performance in anticancer activity during the interaction with DNA, which affected the replication of DNA.17 The results of cytotoxicity assays suggested that the catechol group, the ferrocene part, and the chalcogen atom played an important synergetic role in the anticancer properties. Cyclic species L5 had the most potent anticancer activity in vitro, which is in agreement with the electrochemical study. In addition, L5 was chosen to verify biological activity using HepG2 cells. Effects on Cell Cycle Progression and Apoptosis or Necrosis. The cell cycle distribution of HepG2 cells was analyzed after treatment with L5 (10, 20 μM) for 24 h. Indeed, marked G1 phase arrest was observed in L5-treated HepG2 cells (Figure 2a−c). As shown in Figure 2d, the percentage of cell population in the G1 phase was increased in comparison to that of the control, in a dose-dependent manner (p < 0.01), while the S population was dramatically decreased at 20 μM L5 treatment (p < 0.01), suggesting that the cell cycle G1 phase was arrested in response to L5 treatment. Meanwhile, significant decline from 11.82 to 6.07% was also observed in the G2/M phase of L5treated cancer cells in comparison to that of control groups, in a dose-dependent manner (p < 0.01). Because cell cycle arrest plays an important role in apoptosis of tumor cells,18 the abilities of L5 to induce cell apoptosis or cell death in HepG2 cells by flow cytometry were further investigated. The effects of different concentrations of L5 on HepG2 cells were compared by Annexin V/PI stain. The Annexin V positive cells were regarded as apoptotic cells, including early apoptotic cells (AV+/PI−) and late apoptotic cells (AV+/PI+). First, DA failed to significantly induce the early-

Figure 2. Flow cytometry of cell cycle phase distribution after 24 h (a− c) and the percentage of cell cycle population (d) shown after the treatment of DA 20 μM (as an isotype control) and L5 at 10 and 20 μM. Data shown are the mean values of three independent experiments ± SD. ***, **, and * represent p < 0.001, p < 0.01, and p < 0.05, respectively, in comparison to the control. p < 0.05 was considered as statistically significant. The percentage of DMSO in the medium was 0.25%. C


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Organometallics and late-stage apoptosis under the same molar conditions. As shown in Figure 3, in comparison to the isotype control, the late

Figure 4. Immunoblot analysis. (a) Time-dependent effects of L5 on the expressions of caspase-3 and caspase-9 in HepG2 cells. (b, c) Analysis of the effect of L5 on the expression of caspase-3 and caspase-9. Each value represents the mean ± SD of three experiments. ***, **, and * represent p < 0.001, p < 0.01, and p < 0.05, respectively, in comparison to control. p < 0.05 was considered as statistically significant.

Figure 3. L5 induces cell death in HepG2 cells. Cells were treated with DA 20 μM (as an isotype control) and L5 at 10 and 20 μM for 24 h. (a− c) Apoptosis was monitored by Annexin V/PI double staining. (d) Percentage of early apoptotic cells, late apoptotic cells, and dead cells in HepG2 treated with DA 20 μM (as an isotype control) and L5 at 10 and 20 μM. The values are means ± SD of at least three independent experiments. *** represents p < 0.001 in comparison to control. p < 0.05 was considered as statistically significant. The percentage of DMSO in the medium was 0.25%.

activation in the current study are consistent with the increase in the necrotic and apoptotic ratios of HepG2 cells treated with L5 in PI staining (Figure 3d), indicating that the activation of caspase-3 or caspase-9 is a crucial pathway in L5-induced apoptosis in HepG2 cells. Furthermore, L5 treatment altered the protein expression of one of the antiapoptotic members of the Bcl family (Bcl-2). The expression level of Bcl-2 on HepG2 cells was decreased from 12 to 48 h after treatment with L5, but Bax was increased (p < 0.01) (Figure 5). The proapoptotic family protein (Bax) promotes the release of cytochrome c, whereas an antiapoptotic member (Bcl2) is capable of antagonizing the proapoptotic proteins and preventing the loss of mitochondrial membrane potential.

apoptotic ratios of HepG2 cells treated with 10 and 20 μM L5 obviously increased in a dose-dependent manner, to 13.09% and 33.62%, respectively (p < 0.01). Moreover, the necrotic ratios of HepG2 cells also increased in a dose-dependent manner after 10 and 20 μM L5 treatments (p < 0.01). However, there were few differences in the early apoptotic ratios of HepG2 cells treated with 10 and 20 μM L5. In comparison to the control, the early apoptotic ratios of HepG2 cells were increased but not changed in a dose-dependent manner (p > 0.05). Thus, it is concluded that L5 induces HepG2 cell apoptosis and necrosis effectively in a dose-dependent manner, resulting in directly killing cells or inhibiting cell growth. Compound L5 Induces Activation of Caspases and down-Regulation of the Antiapoptotic Protein Bcl-2. Caspases are proteolytic enzymes that play critical roles in necrosis, inflammation, and apoptosis.19 The activations of caspases lead to irreversible biochemical and morphological changes in cells. Caspase-3 is the first of all effector caspases activated for amplifying downstream apoptotic process. The activation of caspase-3 is a very rapid process in the cell death process.20 Therefore, therapeutic strategies aimed at inducing apoptosis by activating caspase-3 and caspase-9 may evaluate the compounds’ cytotoxicity to cancer cells. It was investigated whether the effectors (caspase-3 or caspase-9) were activated on treatment with L5 at 12, 24, 36, and 48 h (Figure 4a). Indeed, caspase-9 was highly activated in a time-dependent manner, suggesting that the mitochondria apoptosis pathway was activated by L5 treatment (p < 0.01) (Figure 4c). Furthermore, there was a gradual increase in caspase-3 activity in a timedependent manner, suggesting that the apoptosis was irreversible (p < 0.01) (Figure 4b). Results of the caspase-3 and caspase-9

Figure 5. Immunoblot analysis. (a) Time-dependent effects of L5 on the expressions of P53, Bax, and Bcl-2 in HepG2 cells. Analysis of the effect of L5 on the expression of P53 (b), Bax (c), and Bcl-2 (d). Each value represents the mean ± SD of three experiments. ***, **, and * represent p < 0.001, p < 0.01, and p < 0.05, respectively, in comparison to control. p < 0.05 was considered as statistically significant. D


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Organometallics Altogether, the results indicate that L5 is able to activate caspase-3 and caspase-9 and downregulate the expression of antiapoptotic protein Bcl-2, demonstrating that cell apoptosis may be an important pathway for cell growth inhibition in L5treated HepG2 cells in vitro. As an important tumor suppressor, p53 plays a protective role in many cellular processes that are capable of activating multiple target genes, leading to cell cycle arrest. The expression levels of p53 on the L5-treated HepG2 cells were analyzed by immunoblotting (Figure 5). As expected, the expression level of p53 on HepG2 cells was significantly increased from 12 to 48 h after treatment with L5 (p < 0.01). Consistent with the previous result, the tumor suppressor protein p53 mediates cell cycle G1 phase arrest in response to a variety of stress stimuli.21 Therefore, both the distribution and expression of proteins results suggest that arresting the cell cycle may be an important mechanism of L5’s underlying anticancer activity. Evaluation of Antitumor Activity of Compound L5 in Vivo. The compound L5 was then performed in vivo antitumor evaluation in male BALB/c nude mice and HepG2 tumor xenograft model. HepG2 cells in PBS with matrigel/mouse were inoculated subcutaneously in the right leg flank. When the implanted tumor grew up to 65−75 mm3, 24 nude mice were separated randomly into four groups (the vehicle control group, the L5-treated group, and the DA-treated group) and moved into an isotype laboratory. Groups of mice were treated with L5 (50 mg/kg). The control group was treated with the same concentration of DMSO. The same molar mass of DA (17 mg/kg) as the concentration of L5 (50 mg/kg) was used as isotype control. The body weight and the tumor volume were measured twice per week. The results are shown in Figure 6a,b; in comparison to the mice in the vehicle group and the DA-treated group, the L5-treated group experienced a significant reduction in tumor volume (p < 0.01).

There have been some studies concentrating on the antitumor effect of free DA.22 Sood et al. reported that doses of 75 mg/kg significantly reduced tumor growth, with the greatest decrease being among SKOV3 tumor-bearing nude mice treated with 50, 75, and 100 mg/kg of free DA.23 To keep in accordance with the same molar concentration of DA to the dose of 50 mg/kg of L5, the dose of 17 mg/kg of DA was chosen as a control. In the present study, it is found that 17 mg/kg of DA treatment could not inhibit tumor growth in the HepG2 tumor xenograft model (p > 0.05). The low concentration of DA failed to inhibit tumor growth, suggesting that compound L5 has available inhibition in HepG2 tumor xenograft. Tumor weights were recorded after slaughter on treatment for 21 days, and the inhibitory rates on tumor growth are shown in Figure 6c. The compound L5 possessed significant antitumor activity in the HepG2 tumor xenograft model with an inhibitory rate of 53.8% in groups treated with L5 at 50 mg/kg (p < 0.01), and free DA showed no effect (p > 0.05). There were no significant changes in relative body weight (from 5.19% to 6.52%), although treatment of mice with L5 for the same periods of time led to slight weight loss (p > 0.05) (Figure 6d). No other adverse effects were observed among the mice in the vehicle control group and L5-treated groups, suggesting that L5 exhibits more potent antitumor activity and lower dose than does dopamine in vivo, without significant toxicity at these doses. The effect of L5 on tissues in the xenograft model was also observed. After the nude mice were slaughtered, the tumor was excised from subcutaneous tissue and the blood vessels were stained with an antibody against the endothelial marker CD31 and counted by IHC. As shown in Figure 7, the size and the number of the vessels were reduced in both the high-dose L5 group and low-dose L5 group, in comparison to the control or DA group. The reduction in number was about 20% after treatment with L5 and was statistically significant (p < 0.05) (Figure 8), indicating that L5 possessed inhibition properties of angiogenesis in vivo.24 There were no significant differences in HE slices among the four groups. During apoptosis, the insideout signaling of CD31 is somehow disabled so that the apoptotic cell no longer actively rejects the phagocyte. Therefore, the reduced expression of CD31 was shown in tissues from groups treated with L5. The formation of new blood vessels from preexisting ones is a crucial step for tumor growth, invasion, and metastasis. Solid tumors cannot grow without access to and recruitment of blood vessels.25 In the present study, we presumed that the compound L5 partially affect blood vessels, resulting in growth inhibition in the xenograft model. The effect of L5 on angiogenesis in vitro was further evaluated using HUVEC cells as a model. As shown in Figure 9, after 6 h of incubation, compound L5 clearly disrupted the network of HUVECs, in comparison with the control or DA treatment. After L5 treatment at 6 h, all of the tested concentrations were effective in altering the tubulelike structures, especially at a 6.0 μM concentration of L5 (Figure 9a). Image analysis26 was performed to obtain a quantitative measurement of the total length of the tubules covered by HUVECs and the number of branching points after DA or L5 treatments. It is important to stress that L5 exerted significant antivascular effects in vitro, and in a dosedependent manner (p < 0.01), but DA did only slightly (p < 0.05) (Figure 9b). The data of the total length of the tubules covered by HUVECs treated with different concentrations of L5 or DA are summarized in Table S1 in the Supporting Information, suggesting that L5 exhibits more potent antivascular activity than DA under the same testing concentrations that did not

Figure 6. In vivo anticancer activity of L5 in mice bearing HepG2 tumor xenograft. (a) Effect of L5 (50 mg/kg/p2d), DA (17 mg/kg/p2d), or vehicle (5% DMSO in saline, v/v) on growth of tumor xenograft. Tumor growth was tracked by the mean tumor volume (mm3) ± SD (n = 6). (b) Photographs of tumors from the treatment group and the control group. (c) Tumor weight recorded after the mice were killed. ** represents p < 0.05, p versus the vehicle control. (d) Body weight change (presented as percent change from initial weight). E


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Figure 9. Antivascular activity of compound L5 in vitro. (a) Inhibition of endothelial cell capillary-like tubule formation by L5. Representative pictures are selected fields of each group visualized and imaged under an inverted microscope (Nikon, Japan, at 100× magnification) of preformed capillary-like tubules treated with increasing concentrations of L5 for 6 h. (b) Relative level covered by HUVECs. * represents p < 0.05 versus control, and *** represents p < 0.001 versus control. Figure 7. In vivo effects of L5 on angiogenesis. HepG2 cells were injected into the right flank of BABL/c nude mice as described in the Experimental Section. Tumor tissues were embedded in paraffins for immunohistochemistry: CD31 immunohistochemistry and hematoxylin−eosin (HE) staining of tumor tissues from xenograft model (100× magnification).

Figure 8. Percent of microvessel number in tumor tissues. Quantitative analysis of tumor section stained with CD31 for blood vessel number. Data are represented as the mean ± SD of five mice per group. * represents p < 0.05 versus control, and *** represents p < 0.001 versus control. Figure 10. Effect of L5 on migration abilities in HUVEC cells. (A) Images of migrating cells after treatment with L5 and DA for 8 h. The images were visualized and imaged under an inverted microscope (Nikon, Japan, at 100 × magnification). (B) Mean relative migrating cells shown in the histogram. *** represents p < 0.001 versus the control.

affect HUVEC proliferation, though the IC50 values of L5 and DA against HUVEC cells are 7.6 and >50 μM, respectively (Figure S24 in the Supporting Information). Endothelial cell migration to the tumor site is an important mechanism of angiogenesis,27 and the suppression of this process is a significant strategy to arrest the development of tumor vasculature. Endothelial cell migration was then evaluated by a Boyden chamber migration assay. As shown in Figure 10, the migratory ability of HUVEC cells with L5 or DA treatment was assessed using 8 μm pore transwell chambers. The percentages of

migrating HUVEC cells decreased in the L5 treatment group, in a dose-dependent manner, reducing to less than 40% of the control at 1.5 and 2.5 μM concentrations of L5 (p < 0.01). However, in comparison to the control, DA treatment could not F


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The synthetic procedure and characterization data of B2−B5 are available in the Supporting Information. Synthesis and Characterization Data of L1. Ferrocenecarboxylic acid (0.230 g, 1 mmol), DA (0.190 g, 2 mmol), HOBT (0.135 g, 1 mmol), and WSC (0.25 mL, 1 mmol) were placed in a 250 mL threenecked, round-bottomed flask containing dry ethanol (150 mL) under a nitrogen atmosphere; the resulting mixture was stirred at room temperature overnight. Ethanol was removed from the reaction mixture under reduced pressure, the residue was dissolved in dichloromethane (50 mL), and this solution was washed with water three times. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using dichloromethane/ethyl acetate (3/1 v/v) to obtain a yellow solid. Yield: 0.146 g (40%), mp 197−198 °C. νmax/cm−1: 3434, 3390, 2923, 2595, 23360, 1595, 1556, 1304, 1279, 1124, 1004, 972, 818, 781, 499. 1H NMR (400 MHz, DMSO-d6): 8.75, 8.66 (2H, s, −OH), 7.78−7.80 (1H, t, J = 5.6 Hz, −CONH−), 6.64−6.66, 6.49−6.52 (3H, m, ArH), 4.75− 4.76, 4.32−4.33 (4H, m, η5-C5H4), 4.11 (5H, s, η5-C5H5), 3.30−3.33 (2H, d, J = 7.3 Hz, −NHCH2), 2.62−2.66 (2H, t, J = 7.4 Hz, −CH2Ph). 13 C NMR (δ, 100.6 MHz, CDCl3): 172.3, 144.2, 143.0, 130.7, 120.7, 117.6, 115.8, 115.5, 75.2, 70.5, 36.7, 34.8, 34.5, 32.5, 32.3, 32.0, 31.8, 30.3,30.1, 28.0, 27.9, 27.7. ESIMS: calcd for C19H19NO3Fe [M+] 366.07, found 366.25. Anal. Calcd for C19H19NO3Fe: C, 62.49; H, 5.24; N, 3.84. Found: C, 62.46; H, 5.26; N, 3.87. Synthesis and Characterization Data of L2. The synthesis was similar to that of L1, except that B2 (0.338 g, 1 mmol) was used instead of ferrocenecarboxylic acid. The crude product was purified by column chromatography using dichloromethane/ethyl acetate (3/1 v/v). The target product was obtained as a yellow solid. Yield: 0.220 g (45%), mp 113−114 °C. νmax/cm−1: 3345, 2913, 2860, 2359, 1557, 1523, 1464, 1359, 1262, 1022, 794, 499. 1H NMR (δ, 400 Hz, DMSO-d6): 8.73, 8.64 (2H, s, −OH), 7.88 (1H, t, J = 5.5 Hz, −CONH−), 6.62, 6.56, 6.44 (d, J = 1.8 Hz, 1H, ArH), 6.43 (1H, s, ArH), 6.41 (dd, J = 7.9, 1.8 Hz, 1H, ArH), 4.32, 4.25 (4H, d, η5-C5H4), 4.18 (5H, s, η5-C5H5), 3.14 (2H, dd, J = 13.8, 6.9 Hz, −NHCH2), 2.70 (4H, m, SeCH2CH2, PhCH2), 2.37 (2H, t, J = 7.4 Hz, SeCH2). 13C NMR (δ, 100.6 MHz, DMSO-d6): 170.4, 145.0, 143.5, 130.5, 119.2, 115.9, 115.4, 74.4, 69.3, 69.0, 36.5, 34.6, 24.0. ESIMS: calcd for C21H23NO3SeFe [M+] 474.02, found 474.08. Anal. Calcd for C21H23NO3SeFe: C, 53.41; H, 4.91; N, 2.97. Found: C, 53.44; H, 4.90; N, 2.95. Synthesis and Characterization Data of L3. The synthesis was similar to that of L1, except that B3 (0.412 g, 1 mmol) was used instead of ferrocenecarboxylic acid. The crude product was purified by column chromatography using dichloromethane/ethyl acetate (3/1 v/v), and the target product was obtained as a yellow oil. Yield: 0.230 g (42%). νmax/cm−1: 3422, 3128, 2923, 2360, 1520, 1279, 816, 493. 1H NMR (δ, 400 MHz, CDCl3): 8.73, 8.63 (2H, s, −OH), 7.89−7.91 (1H, t, J = 5.5 Hz, −CONH-−, 6.61−6.63, 6.56−6.57, 6.41−6.44 (3H, m, ArH), 4.31− 4.32, 4.23−4.24 (4H, m, η5-C5H4), 4.18 (5H, s, η5-C5H5), 3.13−3.18 (2H, dd, J = 14.3, 6.3 Hz, −NHCH2), 2.59−2.65, 2.53−2.55 (8H, m, SCH2CH2, CH2S, PhCH2), 2.27−2.31 (2H, t, J = 7.3 Hz, SeCH2CH2), 1.71−1.79 (2H, m, SeCH2). 13C NMR (δ, 100.6 MHz, CDCl3): 170.2, 145, 143.5, 130.2, 119.2, 115.9, 115.4, 74.3, 70.6, 69.3, 69.0, 35.8, 34.6, 30.6, 29.6, 27.4, 27.0. ESIMS: calcd for C24H29NO3SSeFe [M+] 547.36, found 547.50. Anal. Calcd for C24H29NO3SSeFe: C, 52.76; H, 5.35; N, 2.56. Found: C, 52.75; H, 5.36; N, 2.54. Synthesis and Characterization Data of L4. The synthesis was similar to that of L1, except that B4 (0.400 g, 1 mmol) was used instead of B1. The crude product was purified by column chromatography using dichloromethane/ethyl acetate (3/1 v/v), and the target product was obtained as a yellow solid. Yield: 0.213 g (41%), mp 51−52 °C. νmax/ cm−1: 3399, 3083, 2928, 2359, 1608, 1528, 1502, 1443, 1285, 1189, 1105, 1018, 857, 818, 704, 589, 498. 1H NMR (δ, 400 Hz,DMSO-d6): 8.83, 8.72 (2H, s, −OH), 8.50 (1H, t, J = 5.5 Hz, −CONH−), 7.74, 7.22, 6.75−6.64, 6.52 (7H, m, ArH), 4.38−4.27, 4.27−4.15 (9H, br, η5-C5H4, η5-C5H5), 3.93 (2H, s, SeCH2Ph), 3.40 (2H, s, −NHCH2), 2.69 (2H, t, J = 7.6 Hz, CH2CH2Ph). 13C NMR (δ, 100.6 MHz, DMSO-d6): 165.7, 145.0, 143.5, 143.0, 130.3, 128.4, 127.0, 119.2, 116.0, 115.5, 74.6, 70.5, 69.51, 69.0, 41.3, 34.7, 32.0. ESIMS: calcd for C26H25NO3SeFe [M+]

significantly inhibit endothelial cell migration, such as HUVEC cells, at 0.65, 1.5, or 2.5 μM. It seems that L5 could potently suppress the migration of endothelial cells, in a dose-dependent manner, resulting in destroying the development of tumor vasculature.

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CONCLUSION In this work, four ferrocencylseleno-dopamine derivatives were designed and synthesized. Anticancer assay results indicated that their structures have a relationship with anticancer activity, in which 1,5-diselena[8]ferrocenophane L5 exhibits superior anticancer activity according to the IC50 values. An electrochemical study showed that the ferrocene moiety in L5 is easily oxidized, which may change the catechol group into a reactive oquinone product and then inhibit the thioredoxin reductases. It is found that dose-increased L5 induced HepG2 cell apoptosis and necrosis and induced accumulation of cells in the G1 phase. Further evidence indicated that L5 activated the expression levels of caspase-3 and caspase-9, the enzymes relating cells undergoing apoptosis. Both the distribution and expression of proteins suggest that arresting the cell cycle may be important mechanism of L5’s underlying anticancer activity. Additionally, the activation of the tumor suppressor p53 suggested that L5 has a potential effect in antitumor activity. An in vivo experiment demonstrated that L5 inhibited the tumor growth effectively in nude mice bearing HepG2 tumor xenografts. In an antivascular activity assay, L5 not only inhibited tubule formation but also depressed endothelial cell (HUVECs) migration. The results summarized here support the idea that ferrocenylseleno-dopamine derivatives could be potential anticancer drugs.

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EXPERIMENTAL SECTION

Chemistry. All manipulations involving air-free syntheses were performed using standard Schlenk-line techniques under a nitrogen atmosphere. NMR spectra were recorded at ambient temperature on a Bruker AV400 spectrometer (1H NMR at 400 MHz, 13C NMR at 100.6 MHz). Chemical shifts (δ) are given in ppm using CDCl3 or DMSO-d6 as solvent unless otherwise stated. 1H and 13C chemical shifts were measured relative to solvent peaks considering TMS at 0 ppm. HR-ESI mass spectra were recorded on a Micromass Quattro II triplequadrupole mass spectrometer using electrospray ionization and analyzed using the MassLynx software suite. Melting points (mp) were determined with an X-5 Microscopic melting point meter. IR spectra were recorded in the range 4000−400 cm−1 using KBr pellets on a Bruker Vector 22 FT-IR spectrophotometer. THF and acetonitrile were distilled from K alloy and calcium hydride, respectively. Ethanol was degassed before use. 3-Bromopropylselenoferrocene, diferrocenyl diselenide (Fc2Se2; Fc = [Fe(η5-C5H5)(η5-C5H4)]), and 1,2,3triselena[3]ferrocenophane (fcSe3; fc = [Fe(η5-C5H4)(η5-C5H4)]) were prepared according to literature methods.28 Ferrocenecarboxylic acid (98%), 3-bromopropionic acid (98%), thiohydracrylic acid (98%), 4-bromomethylbenzoic acid (98%), 3-bromo-2-(bromomethyl)propionic acid (98%), dopamine (DA; 98%), 1-hydroxybenzotriazole (HOBT; 99%), and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (WSC; 95%) were purchased from Energy Chemical. Cyclic voltammograms were recorded in acetonitrile/methanol (v/v 1/1), with 0.1 M tetra-n-butylammonium hexafluorophosphate ([NBu4][PF6]) as a supporting electrolyte, using a CH Instruments 660D electrochemical analyzer (Shanghai Chenhua). The experiments were carried out at room temperature. A three-electrode cell consisting of a 0.5 mm Pt working electrode, a platinum-wire counter electrode, and an Ag|Ag+ (0.01 M AgNO3 in CH3CN) reference electrode was used. The scan rate was 0.1 V s−1. The concentration range of the ferrocene compounds was 1.0 mM in acetonitrile. The E1/2 values obtained for the test samples were referenced relative to the ferrocene/ ferrocenium redox couple. G


Article

Organometallics 536.03, found 536.08. Anal. Calcd for C26H25NO3SeFe: C, 58.45; H, 4.71; N, 2.62. Found: C, 58.47; H, 4.73; N, 2.60. Synthesis and Characterization Data of L5. The synthesis was similar to that of L1, except B5 (0.430 g, 1 mmol) was used instead of B1. The crude product was purified by column chromatography using dichloromethane/ethyl acetate (3/1 v/v), and the target product was obtained as a yellow solid. Yield: 0.226 g (40%), mp 79−80 °C. νmax/ cm−1: 3435, 3075, 2936, 2359, 1644, 1602, 1520, 1443, 1410, 1359, 1282, 1193, 1113, 1026, 878, 814, 479. 1H NMR (δ, 400 Hz, DMSO-d6): 8.77, 8.67 (2H, s, −OH), 8.02−8.04 (1H, br, −CONH−), 6.63−6.65, 6.58−6.59, 6.44−6.46 (3H, br, ArH), 4.33−4.34, 4.29−4.31, 4.27−4.28 (8H, m, η5-C5H4), 3.41−3.52 (4H, m, −SeCH2), 3.16−3.21 (2H, m, −CH2Ph), 2.93−2.96 (1H, m, −CH−), 2.53−2.55 (2H, d, J = 7.6 Hz, −NHCH2). 13C NMR (δ, 100.6 MHz, CD3CN): 120.2, 115.5, 114.9, 102.0, 71.2, 70.8, 68.4, 68.1, 47.6, 40.3, 34.2, 25.1. ESIMS: calcd for C22H23NO3Se2Fe [M+] 565.94, found 566.08. Anal. Calcd for C22H23NO3Se2Fe: C, 46.92; H, 4.12; N, 2.49. Found: C, 46.96; H, 4.10; N, 2.47. Cells and Materials. The cell lines A549 (nonsmall-cell lung carcinoma), A2780 (ovarian carcinoma), BxPC-3 (pancreatic cancer), HepG2 (hepatocellular carcinoma), and AGS (gastric adeno carcinoma) were obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC protocols. MGC-803 was a human gastric cancer cell obtained from Dr. Wan-Zhou Zhao (Sino-EU Biomedical Innovation Center, Nanjing, People’s Republic of China). All cells were grown in media supplemented with 0.5 μL/mL of streptomycin (Invitrogen, USA) at 37 °C with 5% CO2. Stock solutions (100 mM) of the different compounds were obtained by dissolving them in dimethyl sulfoxide (DMSO). HUVECs were obtained from Lonza (USA) and cultured on endothelial growth medium-2 (EGM-2, Walkersville, MD, USA). HUVECs at early passages (passages 2−7) were used in the experiments and incubated at 37 °C in 5% CO2 (v/v). Cytotoxicity Assay. The effects of the compounds on cancer cell proliferation were performed. Cell viability was determined using cell counting kit-8 (CCK-8, Dojindo Laboratories, Japan). In brief, cancer cells were seeded into 96-well plates at a density of 3 × 103 (50 μL) per well, then incubated for 24 h at 37 °C. After incubation, according to compound concentration, the compounds were serially diluted in the medium from 200 μM, respectively. Then, 50 μL suspensions were mixed with cells at an equal volume. After incubation for 72 h at 37 °C, cells were incubated with 10 μL of CCK-8 for 4 h at 37 °C. Then, the absorbance was measured at 450 nm by use of a microplate reader (Thermo, USA). Wells with untreated cells or DMSO were used as controls. Cell survival rate curves were plotted as a percentage of untreated control cells according to standard curves, and IC50 values were calculated. The percentage of DMSO in the medium never exceeded 0.25%. This was also the maximum DMSO concentration in all cell-based assays described below. Cell Cycle Analysis. HepG2 cells were cultured in the presence of DA (20 μM) and two different concentrations of the L5 (10 and 20 μM) for 24 h. The same concentrations of DMSO were used as isotype control. HepG2 cells were harvested, fixed with 70% ethanol at 4 °C overnight, and then stained with 50 μg/mL propidium iodide containing 0.25 mg/mL of RNase A at room temperature for 30 min. Analysis was performed by flow cytometry (Becton-Dickinson FACSCalibur). Cell Death Analysis. The extent of apoptosis was measured with an Annexin V FITC/PI apoptosis detection kit (Invitrogen, CA, USA) according to the manufacturer’s instructions. HepG2 cells were treated with DA (20 μM) and L5 (10 and 20 μM) for 24 h. The same concentrations of DMSO were used as isotype control. HepG2 cells were then washed with ice-cold PBS and incubated with a combination of 5 μL of FITC Annexin V and 5 μL of PI at room temperature in the dark for 15 min. Analysis was performed by flow cytometry (BD, USA). Each determination was repeated three times. Western Blotting. A portion of 1 × 106 HepG2 cells was treated with L5 (20 μM) and collected at 0, 12, 24, 36, and 72 h after the compound incubation. After centrifugation at 500 rpm for 5 min, the pellet was lysed with P-MER cell lyse buffer (1 mL) (Thermo Scientific, USA) containing 1% Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, USA) for 30 min on ice. The supernatant was

collected after 10 min of centrifugation, equaled by spectrophotometer, denatured with sample loading buffer for 10 min at 95 °C, and stored at 4 °C for future use. Cell lysate proteins (50 μg per lane) were loaded on an SDS-PAGE instrument, transferred to a PVDF instrument (Bio-Rad, USA), immune-blotted with mAb AC10364 at 4 °C overnight, and probed with specific Abs as indicated, such as β-Actin and anti-P53 (Santa Cruz) and anticaspase-3, caspase-9, anti-Bcl-2, and anti-Bax (Cell Signaling). Tumor Xenograft. To study the efficacy of L5 as an anticancer drug, tumor-bearing nude mouse experiments were performed through a HepG2 tumor xenograft. The nude mice of BALB/C strain (male, 19− 20 g body weight) were purchased from the Vitalriver Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were raised at the Center for Laboratory Animals, Nanjing Medical University. All protocols were approved by the Institutional Animal Care and Use Committee at Nanjing Medical University. A portion of 2 × 106 HepG2 cells in 100 μL of PBS with matrigel (BD Biosciences, USA) (1/1) per mouse were inoculated subcutaneously in the right leg flank. When the implanted tumor grew to 65−75 mm3, 24 nude mice were separated randomly into four groups (to be described later) and moved into the isotope laboratory. The body weight and the tumor volume were measured twice per week. A group of mice was treated with L5 (50 mg/ kg). The control group was treated with same concentration of DMSO. The same molar mass of DA (17 mg/kg) as the concentration of L5 (50 mg/kg) was used as isotype control. The compounds (i.p.) were managed once every 2 days for 3 weeks. Tumor volume was measured by calipers every 2 days post-treatment and calculated as 0.5 × length × (width)2. Tumors were weighed and collected after slaughter. Gross Pathology and Histopathology. Pathological changes in tumor xenograft excised from nude mice were evaluated by a professional clinician. Samples of these tissues were fixed in 10% neutral buffered formalin for 24 h, dehydrated, embedded in paraffin wax, and sectioned (3−5 μm). The sections were mounted on conventional glass slides for histochemistry studies. All sections were stained by hematoxylin and eosin (HE). Immunohistochemistry (IHC) detection of the blood vessels was performed on tumor xenografts using anti-CD31 antibody (BD bioscience). Quantification was performed by counting the number of CD31 positive vessels in five fields per section (40× objective). Evaluation of Antivascular Activity in Vitro. HUVECs were obtained from Lonza (USA) and cultured on endothelial growth medium-2 (EGM-2, Walkersville, MD, USA). HUVECs at early passages (passages 2−7) were used in the experiments and incubated at 37 °C in 5% CO2 (v/v). In Vitro Proliferation Assay. HUVEC (3000 per well) were seeded in a 96-well plate in the appropriate growth medium for 12 h for attachment. Then cells were treated for 72 h with growth medium containing various concentrations of L5 and DA. Cells receiving 0.1% DMSO only served as a control. Cell growth was assessed using a cellcounting kit-8 (CCK-8, Dojindo, Japan) according to the protocol provided. The spectrophotometric absorbance of each well was measured by a multidetection microplate reader (Synergy HT, BioTeks, USA) at a wavelength of 450 nm. Each treatment was performed in triplicate. The IC50 value was calculated by GraphPad Prism 6 statistical software (San Diego, CA, USA). In Vitro Migration Assay. Boyden chamber migration assay: HUVECs (8000 per well) were seeded on a 48-well chamber (8 μm pore size, AP48, Neuro Probe). The top chamber contained the vehicle or various concentrations of L5 and DA. Cells were allowed to migrate for 8 h. Nonmigrated cells were scraped with a cotton swab, and migrated cells were fixed with 100% methanol and stained with 0.05% crystal violet. In Vitro Tube Formation Assay. Matrigel (growth factor reduced; BD Biosciences, USA) was thawed at 4 °C overnight. Each well of prechilled 96-well plates was coated with 100 μL of Matrigel, incubated, and solidified at 37 °C for 120 min. HUVECs at a density of 2 × 104 per well in EGM-2 containing different concentrations of test compounds (DA and L5) or 2 μM SU5416 were placed onto the Matrigel layer and incubated for 12 h. The network formation of four randomly selected fields in each group was visualized and imaged under an inverted H


Article

Organometallics

Labib, A. A. Appl. Organomet. Chem. 2007, 21, 613−625. (g) Gasser, G.; Ott, I.; Metzler-Nolte, N. J. Med. Chem. 2011, 54, 3−25. (h) Kowalski, K.; Skiba, J.; Oehninger, L.; Ott, I.; Solecka, J.; Rajnisz, A.; Therrien, B. Organometallics 2013, 32, 5766−5773. (i) Kowalski, K. Coord. Chem. Rev. 2016, 317, 132−156. (j) Nguyen, H. V.; Sallustrau, A.; Balzarini, J.; Bedford, M. R.; Eden, J. C.; Georgousi, N.; Hodges, N. J.; Kedge, J.; Mehellou, Y.; Tselepis, C.; Tucker, J. H. R. J. Med. Chem. 2014, 57, 5817−5822. (4) (a) Yeary, R. A. Toxicol. Appl. Pharmacol. 1969, 15, 666−676. (b) Neuse, E. W. J. Inorg. Organomet. Poly. Mater. 2005, 15, 3−32. (c) Tabbi, G.; Cassino, C.; Cavigiolio, G.; Colangelo, D.; Ghiglia, A.; Viano, I.; Osella, D. J. Med. Chem. 2002, 45, 5786−5796. (5) (a) Sai Krishna, A. D.; Panda, G.; Kondapi, A. K. Arch. Biochem. Biophys. 2005, 438, 206−216. (b) Turley, H.; Comley, M.; Houlbrook, S.; Nozaki, N.; Kikuchi, A.; Hickson, I. D.; Gatter, K.; Harris, A. L. Br. J. Cancer 1997, 75, 1340−1346. (c) Kondapi, A. K.; Mulpuri, N.; Mandraju, R. K.; Sasikaran, B.; Subba Rao, K. Int. J. Dev. Neurosci. 2004, 22, 19−30. (d) Larsen, A. K.; Escargueil, A. E.; Skladanowski, A. Pharmacol. Ther. 2003, 99, 167−181. (6) (a) Vessières, A.; Corbet, C.; Heldt, J. M.; Lories, N.; Jouy, N.; Laïos, I.; Leclercq, G.; Jaouen, G.; Toillon, R.-A. J. Inorg. Biochem. 2010, 104, 503−511. (b) Bruyère, C.; Mathieu, V.; Vessières, A.; Pigeon, P.; Top, S.; Jaouen, G.; Kiss, R. J. Inorg. Biochem. 2014, 141, 144−151. (c) Citta, A.; Folda, A.; Bindoli, A.; Pigeon, P.; Top, S.; Vessières, A.; Salmain, M.; Jaouen, G.; Rigobello, M. P. J. Med. Chem. 2014, 57, 8849− 8859. (d) Pigeon, P.; Görmen, M.; Kowalski, K.; Müller-Bunz, H.; McGlinchey, M. J.; Top, S.; Jaouen, G. Molecules 2014, 19, 10350− 10369. (7) (a) Payen, O.; Top, S.; Vessières, A.; Brulé, E.; Plamont, M.-A.; McGlinchey, M. J.; Müller-Bunz, H.; Jaouen, G. J. Med. Chem. 2008, 51, 1791−1799. (b) Hillard, E.; Vessières, A.; Thouin, L.; Jaouen, G.; Amatore, C. Angew. Chem., Int. Ed. 2006, 45, 285−290. (8) (a) Görmen, M.; Pigeon, P.; Top, S.; Vessières, A.; Plamont, M.-A.; Hillard, E. A.; Jaouen, G. MedChemComm 2010, 1, 149−151. (b) Cázares-Marinero, J.; de, J.; Buriez, O.; Labbé, E.; Top, S.; Amatore, C.; Jaouen, G. Organometallics 2013, 32, 5926−5934. (c) Gormen, M.; Plażuk, D.; Pigeon, P.; Hillard, E. A.; Plamont, M.A.; Top, S.; Vessières, A.; Jaouen, G. Tetrahedron Lett. 2010, 51, 118− 120. (d) Plażuk, D.; Vessières, A.; Hillard, E. A.; Buriez, O.; Labbé, E.; Pigeon, P.; Plamont, M.-A.; Amatore, C.; Zarkzewski, J.; Jaouen, G. J. Med. Chem. 2009, 52, 4964−4967. (9) (a) Combs, G. F., Jr; Gray, W. P. Pharmacol. Ther. 1998, 79, 179− 192. (b) Combs, G. F., Jr. Med. Klin. 1999, 94, 18−24. (c) Zeng, H. W.; Combs, G. F., Jr. J. Nutr. Biochem. 2008, 19, 1−7. (d) Combs, G. F., Jr. Br. J. Cancer 2004, 91, 195−199. (e) Zeng, H. W.; Cao, J. J.; Combs, G. F., Jr. Nutrients 2013, 5, 97−110. (10) (a) Shaaban, S.; Diestel, R.; Hinkelmann, B.; Muthukumar, Y.; Verma, R. P.; Sasse, F.; Jacob, C. Eur. J. Med. Chem. 2012, 58, 192−205. (b) Shaaban, S.; Sasse, F.; Burkholz, T.; Jacob, C. Bioorg. Med. Chem. 2014, 22, 3610−3619. (11) Gutiérrez-Hermández, A. I.; López-Cortés, J. G.; Ortega-Alfaro, M. C.; Ramírez-Apan, M. T.; Cázares-Marinero, J. J.; Toscano, R. A. J. Med. Chem. 2012, 55, 4652−4663. (12) (a) Jing, S.; Morley, C. P.; Webster, C. A.; Vaira, M. D. Dalton Trans. 2006, 4335−4342. (b) Ji, W.; Jing, S.; Liu, Z. Y.; Shen, J.; Ma, J.; Zhu, D. R.; Cao, D. K.; Zheng, L. M.; Yao, M. X. Inorg. Chem. 2013, 52, 5786−5793. (c) Xiao, F.; Shen, J.; Qu, J.; Jing, S.; Zhu, D. R. Inorg. Chem. Commun. 2013, 35, 69−71. (d) Shen, J.; Liu, T.; Li, Y.; Ji, W.; Jing, S.; Zhu, D. R.; Guan, G. F. Inorg. Chem. Commun. 2014, 44, 6−9. (e) Liu, Y. Q.; Ji, W.; Zhou, H. Y.; Li, Y.; Jing, S.; Zhu, D. R.; Zhang, J. RSC Adv. 2015, 5, 42689−42697. (13) (a) Basu, S.; Nagy, J. A.; Pal, S.; Vasile, E.; Eckelhoefer, I. A.; Bliss, V. S.; Manseau, E. J.; Dasgupta, P. S.; Dvorak, H. F.; Mukhopadhyay, D. Nat. Med. 2001, 7, 569−574. (b) Sarkar, C.; Chakroborty, D.; Mitra, R. B.; Banerjee, S.; Dasgupta, P. S.; Basu, S. Am. J. Physiol-Heart. C 2004, 287, H1554−H1560. (c) O’Brien, P. J. Chem.-Biol. Interact. 1991, 80, 1− 41. (d) Bisaglia, M.; Mammi, S.; Bubacco, L. J. Biol. Chem. 2007, 282, 15597−15605.

microscope (Nikon, Japan) at 100× magnification. The tube length was quantified by Image Pro Plus 6.0 software. The values were expressed as percent change from control cultures grown with complete medium. Statistical Analysis. All experiments were performed in triplicate; statistical analysis was performed with the aid of commercially available software (GraphPad Prism 6, GraphPad Software Inc., San Diego, CA). Results are expressed as the mean ± SD. One-way ANOVA was used to compare groups. p < 0.05 was considered as statistically significant.

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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.6b00237. Synthesis procedure and characterization data of B2−B5, the total length of the tubules covered by HUVECs, the effect of L5 or DA on HUVEC cells, cyclic voltammograms, and 1H NMR and 13C NMR spectra for compounds L1−L5. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*S.J.: tel, 86-25-58139150; e-mail, [email protected]. *H.X.: tel, 86-25-58849764; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation (Grant No. 21171092) and National Basic Research Program of China (973 Program) (No. 2013CB733504).

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ABBREVIATIONS USED DA, dopamine; HOBT, 1-hydroxybenzotriazole; WSC, 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide; [NBu4][PF6], tetra-n-butylammonium hexafluorophosphate; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DMSO, dimethyl sulfoxide; A549, nonsmall-cell lung carcinoma; A2780, ovarian carcinoma; BxPC-3, pancreatic cancer; HepG2, hepatocellular carcinoma; AGS, gastric adeno carcinoma; HUVEC, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; PBS, phosphate buffered saline; HE, hematoxylin and eosin; IHC, immunohistochemistry; EGM-2, endothelial growth medium-2

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REFERENCES

(1) (a) Kealy, T. J.; Pauson, P. L. Nature 1951, 168, 1039−1040. (b) Miller, S. A.; Tebboth, J. A.; Tremaine, J. F. J. Chem. Soc. 1952, 632− 635. (c) Wilkinson, G.; Rosenblum, M.; Whiting, M. C.; Woodward, R. B. J. Am. Chem. Soc. 1952, 74, 2125−2126. (d) Woodward, R. B.; Rosenblum, M.; Whiting, M. C. J. Am. Chem. Soc. 1952, 74, 3458−3459. (e) Fischer, E. O.; Pfab, W. Z. Naturforsch., B: J. Chem. Sci. 1952, 7, 377− 379. (f) Eiland, P. F.; Pepinsky, R. J. Am. Chem. Soc. 1952, 74, 4971− 4971. (g) Dunitz, J. D.; Orgel, L. E. Nature 1953, 171, 121−122. (2) (a) Ferrocenes: ligands, materials and biomolecules; Štěpnička, P., Ed.; Wiley: Hoboken, NJ, 2008. (b) Ferrocenes: Homogeneous catalysis-organic synthesis-materials science; Togni, A., Hayashi, T., Eds.; VCH: Weinheim, Germany, 1995. (3) (a) Braga, S. S.; Silva, A. M. S. Organometallics 2013, 32, 5626− 5639. (b) Jaouen, G.; Vessières, A.; Top, S. Chem. Soc. Rev. 2015, 44, 8802−8817. (c) Neuse, E. W. J. Inorg. Organomet. Poly. Mater. 2005, 15, 3−32. (d) Ornelas, C. New J. Chem. 2011, 35, 1973−1985. (e) Hartinger, C. G.; Metzler-Nolte, N.; Dyson, P. J. Organometallics 2012, 31, 5677− 5685. (f) Fouda, M. F. R.; Abd-Elzaher, M. M.; Abdelsamaia, R. A.; I


Article

Organometallics (14) (a) Burzio, L. A.; Waite, J. H. Biochemistry 2000, 39, 11147− 11153. (b) Liu, Y.; Meng, H.; Konst, S.; Sarmiento, R.; Rajachar, R.; Lee, B. P. ACS Appl. Mater. Interfaces 2014, 6, 16982−16992. (15) Chen, W. S.; Ou, W. Z.; Wang, L. Q.; Hao, Y. Q.; Cheng, J. S.; Li, J.; Liu, Y. N. Dalton. Trans. 2013, 42, 15678−15686. (16) (a) Savage, D.; Alley, S. R.; Gallagher, J. F.; Goel, A.; Kelly, P. N.; Kenny, P. T. M. Inorg. Chem. Commun. 2006, 9, 152−155. (b) Goel, A.; Savage, D.; Alley, S. R.; Hogan, T.; Kelly, P. N.; Draper, S. M.; Fitchett, C. M.; Kenny, P. T. M. J. Organomet. Chem. 2006, 691, 4686−4693. (c) Goel, A.; Savage, D.; Alley, S. R.; Kelly, P. N.; O’Sullivan, D.; Mueller-Bunz, H.; Kenny, P. T. M. J. Organomet. Chem. 2007, 692, 1292−1299. (d) Corry, A. J.; Goel, A.; Alley, S. R.; Kelly, P. N.; O’Sullivan, D.; Savage, D.; Kenny, P. T. M. J. Organomet. Chem. 2007, 692, 1405−1410. (e) Corry, A. J.; O’Donovan, N.; Mooney, Á .; O’Sullivan, D.; Rai, D. K.; Kenny, P. T. M. J. Organomet. Chem. 2009, 694, 880−885. (f) Mooney, Á .; Corry, A. J.; O’Sullivan, D.; Rai, D. K.; Kenny, P. T. M. J. Organomet. Chem. 2009, 694, 886−894. (g) Corry, A. J.; Mooney, Á .; O’Sullivan, D.; Kenny, P. T. M. Inorg. Chim. Acta 2009, 362, 2957−2961. (h) Mooney, Á .; Corry, A. J.; Ruairc, C. N.; Mahgoub, T.; O’Sullivan, D.; O’Donovan, N.; Crown, J.; Varughese, S.; Draper, S. M.; Rai, D. K.; Kenny, P. T. M. Dalton Trans. 2010, 39, 8228−8239. (i) Harry, A. G.; Butler, W. E.; Manton, J. C.; Pryce, M. T.; O’Donovan, N.; Crown, J.; Rai, D. K.; Kenny, P. T. M. J. Organomet. Chem. 2014, 766, 1−12. (17) (a) Ihmels, H.; Otto, D. Top Curr. Chem. 2005, 258, 161−204. (b) Martínez, R.; Chacón-García, L. Curr. Med. Chem. 2005, 12, 127− 151. (c) Graves, D. E.; Velea, L. M. Curr. Org. Chem. 2000, 4, 915−929. (d) Spencer, J.; Amin, J.; Wang, M. H.; Packham, G.; Alwi, S. S. S.; Tizzard, G. J.; Coles, S. J.; Paranal, R. M.; Bradner, J. E.; Heightman, T. D. ACS Med. Chem. Lett. 2011, 2, 358−362. (18) Kowalski, K.; Hikisz, P.; Szczupak, Ł.; Therrien, B.; Koceva-Chyła, A. Eur. J. Med. Chem. 2014, 81, 289−300. (19) Salvesen, G. S.; Riedl, S. J. Adv. Exp. Med. Biol. 2008, 615, 13−23. (20) Tyas, L.; Brophy, V. A.; Pope, A.; Rivett, A. J.; Tavaré, J. M. EMBO Rep. 2000, 1, 266−270. (21) Sun, X. X.; Dai, M. S.; Lu, H. J. Biol. Chem. 2008, 283, 12387− 12392. (22) (a) Peters, M. A. M.; Walenkamp, A. M. E.; Kema, I. P.; Meijer, C.; de Vries, E. G. E.; Oosting, S. F. Drug Resist. Updates 2014, 17, 96−104. (b) Sarkar, C.; Chakroborty, D.; Basu, S. J. Neuroimmune. Pharm. 2013, 8, 7−14. (c) Chakroborty, D.; Sarkar, C.; Basu, B.; Dasgupta, P. S.; Basu, S. Cancer Res. 2009, 69, 3727−3730. (d) Lu, K.; Basu, S. Sci. Rep. 2015, 5, 9642. (23) Moreno-Smith, M.; Lu, C. H.; Shahzad, M. M. K.; Pena, G. N. A.; Allen, J. K.; Stone, R. L.; Mangala, L. S.; Han, H. D.; Kim, H. S.; Farley, D.; Berestein, G. L.; Cole, S. W.; Lutgendorf, S. K.; Sood, A. K. Clin. Cancer Res. 2011, 17, 3649−3659. (24) Wu, J. B.; Lin, T. P.; Gallagher, J. D.; Kushal, S.; Chung, L. W. K.; Zhau, H. E.; Olenyuk, B. Z.; Shih, J. C. J. Am. Chem. Soc. 2015, 137, 2366−2374. (25) (a) Sherwood, L. M.; Parris, E. E.; Folkman, J. N. Engl. J. Med. 1971, 285, 1182−1186. (b) Rak, J.; Yu, J. L.; Kerbel, R. S.; Coomber, B. L. Cancer Res. 2002, 62, 1931−1934. (26) Guidolin, D.; Vacca, A.; Nussdorfer, G. G.; Ribatti, D. Microvasc. Res. 2004, 67, 117−124. (27) Bergers, G.; Benjamin, L. E. Nat. Rev. Cancer 2003, 3, 401−410. (28) Burgess, M. R.; Jing, S.; Morley, C. P. J. Organomet. Chem. 2006, 691, 3484−3489.

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