European Journal of Medicinal Chemistry 89 (2015) 42e50

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Original article

Synthesis, crystal structure and antitumor effect of a novel copper(II) complex bearing zoledronic acid derivative Ling Qiu, Gaochao Lv, Liubin Guo, Liping Chen, Shineng Luo, Meifen Zou, Jianguo Lin* Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 April 2014 Received in revised form 8 October 2014 Accepted 12 October 2014 Available online 13 October 2014

A great majority of Cu(II) complexes currently studied in the anticancer research field exert their antiproliferative activities through ligand exchange. In this work, we present the synthesis and structural characterization of two novel Cu(II) complexes, {[Cu3(ZL)2(H2O)6]$6H2O}n (1) (ZL ¼ 1-hydroxy-2-(1Himidazol-1-yl)ethane-1,1-diyldiphosphonic acid) and [Cu(IPrDP)2]$3H2O (2) (IPrDP ¼ 1-hydroxy-3-(1Himidazol-1-yl)propane-1,1-diyldiphosphonic acid). Due to the insolubility of polymer 1 in common solvents, only the biological activities of complex 2 were investigated. The antitumor activity of complex 2 was evaluated against a panel of human cancer cell lines, including U2OS, A549, HCT116, MDA-MB-231 and HepG2. Complex 2 exhibited comparable cytotoxic effect to cisplatin (CDDP) against the human colon carcinoma cells HCT116, and superior selectivity for inhibiting human hepatocarcinoma cells rather than normal liver cells. The cell cycle distribution analysis indicates that complex 2 inhibits human carcinoma cells by inducing the cell cycle arrest at the G2/M phase, showing a similar mechanism of action to that of CDDP. The binding interaction of complex 2 with calf thymus DNA (CT-DNA) has been explored by UVevis absorption and circular dichroism (CD), demonstrating complex 2 has a moderate binding affinity for DNA through intercalation. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Copper(II) complex Zoledronic acid Crystal structure Antitumor activity Hepatotoxicity DNA binding

1. Introduction In the last decades, extensive researches have proposed a growing number of antineoplastic agents [1e3]. Among them, transition metal-based complexes represent an important class of chemotherapeutics that has been used extensively for clinical treatments. Platinum complexes as typical chemotherapeutic agents have received remarkable attention since first being introduced to treat the disease almost four decades ago, such as cisplatin, carboplatin and oxaliplatin [4,5]. Though many of these drugs are quite effective in treating primary cancers, some drugs fail to remain effective once the tumor has become metastatic [6]. Moreover, due to lack of specificity, platinum-based anticancer chemotherapies are often associated with various severe side effects including nephrotoxicity, hepatotoxicity, ototoxicity, neurotoxicity, gastrointestinal toxicity, etc. The intrinsic and acquired resistances possessed by various cancers also limit the clinical efficacy of these drugs [5,7]. Therefore, it is essential to design novel

* Corresponding author. E-mail address: [email protected] (J. Lin). http://dx.doi.org/10.1016/j.ejmech.2014.10.028 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

metal complexes with less toxicity and better specificity to overcome these adverse effects [8e10]. Up to now, a plenty of new compounds featuring alternative metals in preclinical studies have been proved to be promising antitumor pharmaceuticals, such as copper [11], which is an essential metal ion involved in several highly conserved biochemical processes [12]. Copper is taken up by the cell through human copper transporter 1 (hCTR1) and delivered by chaperones to its intracellular destinations [13e15]. The ability of iterating between two oxidation states (þ1 and þ2) is one of the key features, which has been exploited by organisms throughout evolution [16]. In recent years, copper complexes were designed and preliminarily screened to show promise as both antiproliferative and antimetastatic agents [16e21]. For instance, Raja et al. demonstrated the antioxidative and cytotoxic activity of a series of copper complexes with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde derivatives, which showed significant radical scavenging activity and effective cytotoxicity against cancerous cell lines such as human cervical cancer cells (HeLa), human laryngeal epithelial carcinoma cells (HEp-2), human liver carcinoma cells (HepG2), and human skin cancer cells (A431) [17,18]. Castle et al. reported a series of copper complexes of semicarbazones, which possessed fascinating

L. Qiu et al. / European Journal of Medicinal Chemistry 89 (2015) 42e50

biological activities and potential applications as anticancer agents as well as diagnostic and therapeutic radiopharmaceuticals [19e21]. These results stimulated wide interests in the search of effective antitumor copper complexes. The chemical feature of the ligands has been recognized to be the main determinant of biological activities of complexes [22], which can (i) modulate the permeability through the cell membranes by tuning their lipophilic character, (ii) direct the toxicities of the metals toward specific intracellular targets, and (iii) exhibit an intrinsic cytotoxic activity when they dissociate from the center metals. Diphosphonates (DPs) are a class of drugs with PeCeP backbone structure, showing high affinity for bone mineral and other calcified tissues, and used as therapeutic agents against hypercalcemia of malignancy (caused by several tumors), skeletal metastases, Paget's disease, and postmenopausal osteoporosis [23]. In addition to being prescribed as drugs, DPs can serve as drug targeting and delivery vehicles for therapeutic and diagnostic applications [24e26]. Compared with other molecules, DPs have more advantages for targeted delivery of therapeutic agents to bone because their binding affinity and biological activity can be tuned by changing their R1 and R2 substituents [27]. Therefore, combination of the bifunctional diphosphonate and the copper moiety can ideally promote the specific accumulation of the drug in the bone-related tumors or metastases with significant improvement in the chemotherapy efficacy and reduction in the systemic toxicity. Zoledronic acid (ZL), a typical third-generation diphosphonate, is the most potent and widely used in the clinical treatment of skeletal diseases [28]. Due to the multidentate chelating ability, ZL is known to form stable chelates with many metals and envisaged as a possible carrier moiety to develop metal-based radiopharmaceuticals with great potential applications in the nuclear medicine [29e34]. Also it exhibits multiply bridging properties to combine with diverse transition metal ions to form versatile architectures with promising applications in the material science [35e38]. In a continuing effort to find metal-based compounds with high anticancer activity and low toxicity, we herein report two novel copper complexes based on ZL and its derivative IPrDP (Scheme 1), {[Cu3(ZL)2(H2O)6]$6H2O}n (1) (ZL ¼ 1-hydroxy-2-(1H-imidazol-1yl)ethane-1,1-diyldiphosphonic acid) and [Cu(IPrDP)2]$3H2O (2) (IPrDP ¼ 1-hydroxy-3-(1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid). This is the first case of copper complex containing the diphosphonate ligand reported as an anticancer agent. The present work includes the synthesis, structural characterization, antitumor activity, hepatotoxicity, action mechanism, and DNA binding studies.

2. Experimental 2.1. Materials and methods All chemicals were purchased as reagent grade and used without further purification. CDDP was purchased from Shandong BoYuan pharmaceutical Co., Ltd. The diphosphonic acid ligand ZL and IPrDP were prepared according to the method as described in our previous work [32]. The human cancer cell lines U2OS (human osteosarcoma cell line), A549 (human lung cancer cell line), HCT116

(human colon carcinoma cell line), MDA-MB-231 (human breast cancer cell line) and HepG2 (human liver carcinoma cell line) as well as the human normal liver cell lines (LO2) were obtained from the Cell Bank of Chinese Academy of Sciences, Shanghai, China. The reagent 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) used for cell lysis was purchased from Sigma (St. Louis, MO, USA). Dulbecco's modified eagle medium (DMEM) and RPMI 1640 were purchased from Gibco Company (USA). Dimethyl sulfoxide (DMSO) and propidium iodide (PI) were purchased from Beyotime Institute of Biotechnology. Cell culture plates were products of Corning. Calf thymus DNA (CT-DNA) were purchased from SigmaeAldrich and used as received. Elemental analysis (C, H, N) was carried out using a Vario EL III Elementar analyzer. IR spectra (4000e400 cm1) were obtained with KBr disks on a Nicolet Nexus470 Fourier transform IR spectrophotometer. Single crystal structural determination was performed on a Bruker SMART APEX CCD diffractometer using Cu-Ka (l ¼ 1.5418 Å) at room temperature, in which the X-ray tube was operated at 40 kV and 40 mA. The UVevis absorption spectra were recorded on a UV-3600 spectrophotometer (Shimadzu, Japan), and circular dichroism (CD) spectra were recorded on a MOS 450 spectropolarimeter (BioLogic, France). 2.2. Synthesis of copper complexes 2.2.1. Synthesis of {[Cu3(ZL)2(H2O)6]·6H2O}n (1) ZL (0.05 mmol, 13.6 mg) and CuSO4$5H2O (0.10 mmol, 25.0 mg) were mixed in 5 mL distilled water. The mixture was refluxed for 20 min. Then, the resulting blue solution was filtered and left unperturbed to slowly concentrate. After several days, pale blue blocky crystals suitable for X-ray diffraction were obtained. Yield: 16.4 mg (47.8%). Anal. Calcd for C10H38Cu3N4O26P4: C, 12.71; H, 4.05; N, 5.93%. Found: C, 12.70; H, 4.09; N, 5.88%. IR (KBr pellet, cm1): 3444(w), 3167(w), 1627(s), 1400(s), 1279(m), 1151(s), 1077(s), 1031(m), 990(m), 965(w), 842(w), 638(w), 603(w). 2.2.2. Synthesis of [Cu(IPrDP)2]·3H2O (2) IPrDP (0.05 mmol, 14.3 mg) and CuSO4$5H2O (0.10 mmol, 25.0 mg) were mixed in 5 mL distilled water. The mixture was refluxed for 20 min. Then, the resulting blue solution was filtered Table 1 Crystallographic data for complexes 1 and 2. Complex

1

2

Empirical formula Formula weight Crystal system Space group T (K) a (Å) b (Å) c (Å) a ( ) b ( ) g ( ) V (Å3) Z Dcalcd (g cm3) m (mm1) F(000) Goodness-of-fit on F2 R1 [I > 2s (I)] wR2 [I > 2s (I)] R1 (all data)a wR2 (all data)b

C10H38N4O26P4Cu3 944.94 Triclinic P-1 (2) 296(2) 8.6975(12) 9.9815(13) 10.1160(13) 93.095(2) 111.272(2) 105.188(2) 778.87(18) 1 2.01 2.342 481 1.106 0.0303 0.0841 0.0308 0.0844

C12H28N4O17P4Cu 687.8 Monoclinic P2/c 153(2) 8.229(3) 9.071(3) 16.762(7) 90.00 102.561(4) 90.00 1221.26(79) 4 1.87 1.246 706 0.998 0.0336 0.0974 0.0420 0.1025

a

R1 ¼ SjjFoj  jFcjj/SjFoj. wR2 ¼ jSw(jFoj2  jFcj2)j/Sjw(Fo)2j1/2, where w ¼ 1/[s2(F2o) þ (aP)2 þ bP]. P ¼ (F2o þ 2F2c )/3. b

Scheme 1. Chemical structures of the ligands ZL and IPrDP.

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and left unperturbed to concentrate slowly. After several days, blue blocky crystals suitable for X-ray diffraction were obtained. Yield: 11.0 mg (46.9%). Anal. Calcd for C12H28N4O17P4Cu: C, 20.93; H, 4.07; N, 8.14. Found: C, 21.05; H, 4.11; N: 8.10%. IR (KBr pellet, cm1): 3419(w), 3179(w), 1691(m), 1582(s), 1282(m), 1145(s), 1090(s), 1040(s), 976(w), 958(m), 890(w), 629(w), 579(m).

(concentration of drug that inhibits cell growth by 50%) was determined accordingly. Triplicate experiments were performed and the medium without the drug served as a control, as well as the cytotoxicity of CDDP evaluated by means of the same method served as a positive control. 2.6. Morphology study

2.3. X-ray crystallography Suitable single crystals of complexes 1 and 2 were selected and mounted in air onto thin glass fibers. X-ray intensity data for 1 and 2 were collected on a Bruker Smart Apex CCD-based diffractometer with graphite-monochromatic Mo-Ka radiation (l ¼ 0.71073 Å) at 293(2) K using the u-scan technique. Data reductions and absorption corrections were performed with the SAINT [39] and SADABS [40] software packages, respectively. The crystal structure was solved by direct method using the SHELXS-97 software package [41] and all the non-hydrogen atoms were refined anisotropically on F2 by the full-matrix least-squares technique using the SHELXL-97 software package [42]. All hydrogen atoms were generated geometrically and refined isotropically using the riding model. The details of the crystal parameters, data collection and refinements for the complexes were summarized in Table 1. Selected bond lengths and angles were listed in Table S1. Crystallographic data for the structures reported in this article have been deposited with the Cambridge Crystallographic Data Centre (CCDC) with CCDC reference numbers of 901874 and 979656 for 1 and 2. Copies of the data can be obtained free of charge from the CCDC via http://www.ccdc.cam.ac.uk/data_request/cif. 2.4. Cell culture and drug treatment Except that the human normal liver cell lines LO2 and human lung cancer cell lines A549 were cultured in the RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin, other four human cancer cell lines (U2OS, HCT116, MDA-MB-231 and HepG2) were cultured in the DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. All these cells were plated ( 1 104 cells/mL) in 100 mL per well in 96-well plates respectively and incubated in a humidified atmosphere of 5% CO2 and 95% air at 37  C for 24 h, which allowed for the cell attachment. After 24 h, these cells were treated with the test drug. Subsequently, these cells were cultured in a humidified incubator of 5% CO2 and 95% air at 37  C for another 48 h and 72 h, respectively. 2.5. Cytotoxicity assay The cytotoxicity of novel copper(II) complexes was evaluated by means of conventional MTT assay, respectively. Briefly, cells were seeded at a density of 7e10  103 cells per well in 96-well flatbottom plates and incubated at 37  C overnight to allow cells attachment. Then the aqueous solution of complex diluted with the medium DMEM or RPMI 1640 was added to each well to give the indicated final concentration, and the plate was incubated for additional 48 h or 72 h. After drug treatment, 20 mL MTT (5 mg/mL in PBS) was added directly to each well and the cells were incubated at 37  C for 4 h. After that, the medium was removed from each well and 100 mL DMSO was added to each well to lyse the precipitate. Finally, the plate was shaken and the absorbance of each well was measured at 490 nm using a microplate reader (BioRad Model 3550, CA, USA) to evaluate the inhibition of cell growth induced by the drug. A graph of the percentage of cell inhibition versus concentration can be plotted and the IC50 value

Exponentially growing cells were seeded into 6-well plates at a concentration of 2  105 cells per well. After 24 h incubation at 37  C for cell attachment, the culture medium was removed and replaced with fresh medium containing the complex 2 at the concentration of 60 mM. The cells were incubated for another 48 h. Thereafter, the cells were observed under the inverted light microscopy and photographed by a Panasonic Lumix DMC-FH2. 2.7. Cell cycle analysis Cells were seeded at a density of 1e2  105 cells per well in 6well flat-bottom plates and incubated at 37  C overnight to allow cells attachment. Then the drugs (complex and CDDP) were added to each well to give the indicated final concentration. After 72 h exposure to the drug, cells were collected by trypsinization, washed with ice-cold PBS, suspended with 70% ethanol (stored at 20  C) and fixed at 4  C overnight. Then the cells were centrifuged to remove ethanol, washed with PBS, and then resuspended in 1 mL of DNA staining reagent containing 50 mg/ml RNase, 50 mg/ml PI, 0.1% Triton X-100, and 0.1 mM EDTA (pH 7.4) for 30 min at 4  C. Finally, the cell cycle profile in each sample was analyzed using a FACS Calibur flow cytometer equipped with a 488-nm argon ion laser and a Cell Quest 3.1 software. DNA histograms obtained from Cell Quest 3.1 were further analyzed under Window system using WinMDI 2.8 software (Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA). 2.8. DNA binding study CT-DNA was dissolved in buffer solution (5 mM TriseHCl, 50 mM NaCl, pH 7.4) as a stock solution, which was stored at 4  C for 24 h to reach homogeneous phase and used within three days. The solution of CT-DNA in the buffer gives a UV absorbance ratio (A260/A280) of about 1.8, indicating that CT-DNA is sufficiently free of protein. The concentration of CT-DNA was determined by measuring the UV absorption at 260 nm, taking 6600 M1 cm1 as its molar absorption coefficient. A solution of complex 2 (1.0  104 M) in the buffer (5 mM TriseHCl, 50 mM NaCl, pH 7.4) was incubated for 2 h at 25  C before the CT-DNA solution was added. Then an increasing amount of CTDNA (104 M, 100 mL per time) was gradually added to the above solution. And the absorption spectrum was recorded after each successive addition of CT-DNA solution. The binding constant (Kb) can be determined using the following equation: [DNA]/(εa  εf) ¼ [DNA]/(εb  εf) þ 1/Kb(εb  εf)

(1)

here, εa, εf, and εb correspond to Aobsd/[Cu], the extinction coefficient for the free copper complex, and the extinction coefficient for the copper complex in the fully bound form, respectively. A plot of [DNA]/(εa  εf) versus [DNA] gives the binding constant Kb as the ratio of the slope to the intercept. The circular dichroism (CD) spectra for CT-DNA were recorded as follows. Each sample of CT-DNA (1.0  104 M) was incubated with different concentrations of complex 2 at 37  C for 24 h in the dark, where the concentration ratio of the complex to CT-DNA was 0, 0.2, 0.4, 0.8 and 1.0, respectively. Each sample was scanned in the

L. Qiu et al. / European Journal of Medicinal Chemistry 89 (2015) 42e50

wavelength range of 220e320 nm at a speed of 10 nm/min and the buffer background was subtracted. 3. Results and discussion 3.1. Synthesis, IR spectroscopy and crystal structure Scheme 2 shows the formation of complexes 1 and 2 with ZL and IPrDP as ligands respectively. They were fully characterized by the elemental analysis, IR spectroscopy and X-ray crystallography. The IR spectra exhibit both complicated and important peaks in the range of 900e1300 cm1 for the complexes 1e2 and the corresponding ligands ZL and IPrDP (Fig. S1). For the ligand ZL, the strong absorptions at 1178 and 1094 cm1 are ascribed to the P]O stretching vibrations, and the absorptions at 1056 and 1016 cm1 are assigned to the asymmetric stretching vibrations of PeO. And the symmetric stretching vibrations of PeO locate at 969 and 904 cm1. For complex 1, the corresponding peaks shift to 1151 and 1078 cm1 (ns,P]O, Dn is 27 and 16 cm1), 1031 and 990 cm1 (nas,PeO, Dn is 25 and 26 cm1), 939 and 897 cm1 (ns,PeO, Dn is 30 and 7 cm1). Similarly, the strong absorptions of the ligand IPrDP at 1196 and 1097 cm1 are ascribed to the P]O stretching vibrations and the absorptions at 1053 and 998 cm1 are assigned to the asymmetric stretching vibrations of PeO. And the symmetric stretching vibrations of PeO locate at 968 and 941 cm1. For complex 2, the corresponding peaks shift to 1145 and 1091 cm1 (ns,P]O, Dn is 51 and 6 cm1), 1040 and 976 cm1 (nas,PeO, Dn is 13 and 22 cm1), 958 and 932 cm1 (ns,PeO, Dn is 10 and 9 cm1). All these reveal that complexes 1 and 2 both exhibit blue shift compared with their ligands, due to the coordination of the ligand to copper atoms. Furthermore, the broad adsorption band at 3444 and 3418 cm1 in the IR spectra of complexes 1 and 2 clearly show the presence of water molecules, and the peaks around 3000e3200 cm1 are attributed to the stretching vibrations of NeH and CeH in the imidazole ring. This demonstrates that the imidazole nitrogen atom is protonated, which is not only consistent with the crystal structures of ZL and ZL derivatives [43], but also consistent with those of other metal complexes of ZL [38,44]. X-ray diffraction study displays that complex 1 is a neutral coordination polymer and crystallizes in triclinic space group P-1. As shown in Fig. 1, there are two crystallographically independent copper atoms in the asymmetric unit with Cu1 atom sitting in an inversion center and Cu2 atom at a general position. Both of them have a distorted octahedral geometry. For the Cu1 atom, the equatorial sites are occupied by two pairs of phosphonate oxygen atoms (O4, O6 and O4B, O6B) from two equivalent ZL ligands, while the remaining two axial sites are occupied by two equivalent water

45

molecules (O4W and O4WB). For the Cu2 atom, three of its six coordination sites are filled with O1, O3 and O5 from one ZL ligand, one site with O7a from the other ZL ligand, and the remaining two sites with two water molecules (O1w and O2w). Because of the JahneTeller effect for the d9 configuration of Cu2þ, there are weak interactions between Cu1 and coordination water molecules at the axial site. Hence, the octahedral structure was stretched to a distorted one and the distances of Cu1eO range from 1.9564(18) to 2.4425(28) Å. For the same reason, the distances of Cu2eO vary from 1.9211(18) to 2.7111(20) Å. Among them, Cu1eO4w and Cu2eO1 are slightly longer (2.4425 and 2.7111 Å, respectively), but they are still non-negligible interactions [45]. The bond distances of PeO are similar and the shorter one corresponds to P]O [P1]O2, 1.5083 Å; P2]O7, 1.5132 Å]. Due to the coordination of O7 with Cu(II) atom, the bond P2]O7 is slightly longer than P1]O2, which accords well with the studies of Cao et al. [37]. In the complex 1, ZL serves as a hexadentate ligand which bridges Cu1 and Cu2 by five phosphonate oxygen atoms (O3, O4, O5, O6 and O7) and one hydroxyl oxygen (O1) (Fig. 1A). As shown in Fig. 2, complex 1 contains a dimer unit of {Cu2eO7eP2eO5eCu2} and these dimers are further connected by the octahedral unit {Cu1O6}, forming a one-dimensional (1-D) polymeric chain running along the c axis. The shortest distance of Cu1/Cu2 is 4.8642(7) Å and the distance between adjacent chains is 14.8582(14) Å. Furthermore, there exists plentiful hydrogen bonding interactions within the chain. The neighbor chains are further connected by extensive intermolecular hydrogen bonds, including O3w/O3iii, O1w/O4wiii, O6w/O2viii, O5w/O6ii, O5w/O7ii, O1w/O5wi and O2w/O5w, forming a plane (Fig. S2 and Table S2). Numerous hydrogen bonds are also observed between adjacent planes [O6w/O2v, O6w/O2viii, O3w/O2vi, O2w/O6wv and N2/O4iv], featuring a three-dimensional (3-D) supramolecular structure (Fig. S2). The H-bond distances (D/A) range from 2.720(3) to 3.016(4) Å. Notably, the imidazole nitrogen atom is protonated and involved in the hydrogen bonds mentioned above (N2/O4iv), which is similar to other metal complexes of ZL and ZL derivatives [35e38,44]. X-ray diffraction study displays that complex 2 is mononuclear and crystallizes in the monoclinic space group P2/c.The asymmetric unit of complex 2 consists of one IPrDP ligand with a protonated imidazole ring and one Cu(II) metal ion locating at specific position with 1/2 site occupancy, which accounts for the charge neutrality of the whole structure. As shown in Fig. 1B, Cu1 is coordinated by four oxygen atoms (O1, O1A, O4, O4A) from two IPrDP ligands with square-planar coordination geometry, which is similar to the structures of other reported copper complexes [17e21]. Notably, although each phosphonate of the ligand remove one proton

Scheme 2. Synthetic route for complexes {[Cu3(ZL)2(H2O)6]$6H2O}n (1) and [Cu(IPrDP)2]$3H2O (2).

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L. Qiu et al. / European Journal of Medicinal Chemistry 89 (2015) 42e50

Fig. 1. ORTEP view of the molecular structures and atomic labeling of complexes 1 (left) and 2 (right). Thermal ellipsoids are drawn at the 30% probability level.

Fig. 2. A fragment of 1-D chain in the complex 1 along the c axis. Some H atoms are omitted for clarity.

respectively to coordinate to Cu(II) atom, the imidazole group is protonated to balance the charge of the complex. This is consistent with the crystal structure of complex 1 and the IR spectroscopy analysis. The bond length of Cu1eO4 (1.919 Å) is slightly longer than that of Cu1eO1 (1.909 Å), while the bond length of P1eO1 (1.516 Å) is shorter than P1eO3 (1.558 Å) that may be also due to the coordination of the oxygen atom (O1) to Cu(II) atom. On the whole, the mononuclear structure of 2 is similar to that of the reported analog Cu(ImhedpH3)2 [37], which was obtained under the elaborate hydrothermal condition. In the mononuclear complex 2, extensive hydrogen-bonding interactions are also observed among the phosphonate oxygens, the protonated imidazole groups, the hydroxyl groups and the water molecules (see Fig. S3 and Table S2). The hydrogen bonds (D/A) range from 2.7686 to 3.3443 Å. A 3-D supramolecular structure is formed through the hydrogen bonds between adjacent planes [O8w/O2, O8w/O7i and O9w/O8wii]. Moreover, the imidazole groups in different molecules form stacks in the crystal via CeH/p interactions with the interplanar separation of about 3.7 Å and the dihedral angle of 24.22 , as shown in Fig. S4. This implies that the copper(II) complex 2 with square-planar geometry can be considered as a potential DNA intercalator, which is similar to other copper complexes with square-planar or square-pyramidal geometry that showing a remarkable intercalative binding affinity for DNA [17e21]. Comparison between complexes 1 and 2 shows that besides their crystal structures are different, their physicochemical properties are also completely different. The biggest difference between two complexes is the solubility. The complex 2 is water-soluble, but the complex 1 is quite insoluble in common solvents (such as DMSO, methanol, acetonitrile and water) due to the fact that it is a coordination polymer. 3.2. Cytotoxicity assay Due to the insolubility of complex 1, only the biological activities of complex 2 were studied here. The cytotoxic effect of complex 2 against U2OS, A549, HCT116, MDA-MB-231 and HepG2 cell lines was evaluated using MTT assay after 48 h and 72 h of treatment,

respectively. The results were also compared with those of CDDP. As shown in Fig. 3 and Fig. S5, both complex 2 and CDDP produce dose- and time-dependent cytotoxic effects on these human cancer cell lines. The cytotoxicity became more evident after exposure to a higher concentration of complex 2. Remarkably, complex 2 shows a moderate cytotoxic potency in U2OS, A549, MDA-MB-231 and HepG2 cells after treatment for 72 h with IC50 values of 44.65, 38.30, 36.09 and 37.66 mM, respectively (Table 2). Excitingly, the cytotoxicity of complex 2 against HCT116 cell lines is comparable to that of CDDP with the IC50 of 16.05 and 11.16 mM vs 16.28 and 11.80 mM at 48 and 72 h, respectively. The findings of in vitro cytotoxic activities of the copper(II) complex will prompt us to further explore its mechanism of action. It is known that platinum-based drugs in current clinical use usually have significant hepatotoxicity side effect, which affects their practical clinical efficacy and limits their wide use. In order to test whether our compound has the hepatotoxicity side effect, the cytotoxic effect of complex 2 against the human normal liver cell lines LO2 was investigated [46], using CDDP as a control. It is clear that the inhibition effects of complex 2 on the proliferation of LO2 and HepG2 cell lines at the concentration less than 20 mM are both unconspicuous, while those of CDDP at the concentration less than 5 mM are already remarkable (Fig. S5). At the same concentration of drug treated (5, 10, and 20 mM), the cytotoxicities of complex 2 against LO2 and HepG2 cell lines are both far lower than those of CDDP after treatment of 48 and 72 h. That is, the cell viabilities of human normal liver cell lines and liver cancer cell lines treated by complex 2 are much higher than those treated by CDDP. As the concentration of complex 2 increasing up to 50 mM, its cytotoxicities against LO2 and HepG2 cell lines are both comparable to those of CDDP at a lower concentration of 20 mM, with the cell viability of LO2 (58.58% vs 52.01%) and HepG2 (28.58% vs 28.75%) after treatment of 48 h. As the time of treatment prolonging to 72 h, however, the cell viability of LO2 treated by complex 2 (55.03%) is much higher than that treated by CDDP (25.02%) and the cell viability of HepG2 treated by complex 2 (18.76%) is slightly larger than that treated by CDDP (5.50%), as shown in Fig. 4. All these indicate that the cytotoxicity of complex 2 against the human normal liver cells is far lower than that of CDDP at the same concentration, although

L. Qiu et al. / European Journal of Medicinal Chemistry 89 (2015) 42e50

47

Fig. 3. Sensitivity of five human cancer cell lines to complex 2 treated for 48 and 72 h.

3.3. Morphological study To determine the characteristics of human cancer cell lines' death induced by the complex 2, morphologic changes in the cancer cell lines U2OS, A549, HCT116, and HepG2 were examined. As well known, apoptotic cells are identified by their characteristic nuclei condensation, fragmentation and bright staining, whereas nuclei from normal cells demonstrate a normal uniform chromatin pattern. After treatment with complex 2 at the indicated concentrations for 48 h, all these five kinds of cells exhibit apoptosisrelated morphologies, such as cell shrinkage, rounding up, and cell membrane blebbing, compared with the control groups (Fig. 5). It is also clear that the density of cells decreased significantly after treated by complex 2. This indicates that complex 2 can not only inhibit the proliferation of the cancer cell lines, but also induce the apoptosis of the cancer cell lines. 3.4. Cell cycle analysis The cell cycle distribution of five different human cancer cell lines induced by complex 2 was analyzed using a FACS Calibur flow cytometer. The percentage of cell population at different phases were obtained from the Cell Quest software package and presented Table 2 Cytotoxicity of complex 2 against five human cancer cell lines in comparison with CDDP (IC50, mM).a Complex Cell line U2OS 2 (48 h) 2 (72 h) CDDP (48 h) CDDP (72 h)

A549

HCT116

MDA-MB-231 HepG2

54.33 ± 3.84 48.49 ± 2.99 16.05 ± 1.16 48.96 ± 2.01 44.65 ± 1.47 38.30 ± 1.00 11.16 ± 0.52 36.09 ± 1.39 11.04 ± 0.34 19.85 ± 1.49 16.28 ± 0.44 29.94 ± 1.65

41.38 ± 0.57 37.66 ± 0.91 6.78 ± 0.54

10.82 ± 0.37 15.38 ± 0.58 11.80 ± 0.26 14.05 ± 1.01

5.63 ± 0.34

a IC50 values determined by MTT assay were presented as the mean ± SD of three independent experiments.

in Fig. 6. It is evident that the cell populations of five human cancer cell lines at the G0/G1 phase all decrease with the increasing concentration, while the cell populations at S and G2/M phases both increase. This indicates that the complex 2 can cause tumor cell cycle to arrest at the G2/M phase, so that no more cells can go from G2/M to G0/G1. Furthermore, some cell apoptosis induced by the complex 2 can be observed since the cell populations at the subG1 phase increase, especially in the inhibition of HCT116 and HepG2 cell growth. Investigating the cell cycles of these human cancer cell lines treated by CDDP after 72 h, the same conclusion can be drawn that tumor cell cycle was arrested at the G2/M phase (Table S3). Therefore, it is inferred that the action mechanism of complex 2 is similar to that of CDDP which inhibits human cancer cell lines by inducing the cell cycle to arrest at the G2/M phase [47]. 3.5. DNA binding study Electronic absorption spectroscopy and circular dichroism spectroscopy are usually utilized to determine the binding ability of complexes with the DNA helix. The latter is also known as a sensitive technique to trace conformational changes of DNA or proteins. A complex bound to DNA through intercalation is characterized by the change in absorbance or intensity (hypochromism) and red shift in wavelength, due to the intercalative mode involving a strong stacking interaction between the aromatic chromophore and the DNA base pairs. The extent of hypochromism

100

Cell viability (%)

its inhibition effect on the human liver carcinoma cells is slightly lower than that of CDDP. In other words, complex 2 can be used as a potential anticancer drug with good selectivity for inhibiting human hepatocarcinoma cells rather than normal liver cells and hence low hepatotoxicity, which is worthy of further investigation on the animal model to elucidate it more thoroughly.

HepG2-48 h HepG2-72 h LO2-48 h LO2-72 h

80 60 40 20 0

20 M CDDP

20 M complex 2

50 M complex 2

Fig. 4. Cytotoxicity of complex 2 and CDDP against HepG2 and LO2.

48

L. Qiu et al. / European Journal of Medicinal Chemistry 89 (2015) 42e50

Fig. 5. Morphological changes in five human cancer cells treated with complex 2 at the indicated concentration. After exposure to complex 2 for 48 h, cells were photographed using inverted microscope (magnification 200).

Fig. 6. Cell cycle perturbation of five cancer cell lines treated by complex 2 after 72 h.

Fig. 7. Absorption spectra of complex 2 in the absence and presence of increasing concentration of CT-DNA in the buffer (5 mM TriseHCl, 50 mM NaCl, pH 7.4). The inset shows the fit of [DNA]/(εa  εf) vs [DNA] for the complex.

is commonly consistent with the strength of the intercalative interaction [48]. The intrinsic binding constant (Kb) of complex 2 to CT-DNA was first determined by UVevis absorption titration and the subsequent regression analysis. In Fig. 7, the absorption spectra of complex 2 in the absence and presence of increasing concentration of CT-DNA were shown. Upon addition of an increasing amount of DNA to a solution of the complex, an apparent hypochromism and a slight red shift (~4 nm) were observed near 260 nm, which is assigned to the intercalation involving stacking interactions between the aromatic chromophore (imidazole ring in complex 2) and the base pairs of DNA. The Kb value obtained from the regression analysis is 1.23  104 M1, suggesting that the new copper(II) complex 2 has a moderate binding affinity for DNA [49]. The CD spectra of CT-DNA complexed with the copper(II) complex 2 were shown in Fig. 8. In general, the characteristic CD spectrum of DNA exhibits a positive band at 275 nm due to the base stacking, a negative band at 245 nm due to the helicity of B-type DNA, and crossover point near 258 nm, respectively [50]. As shown in Fig. 8, the spectra exhibit characteristic features of the canonical

L. Qiu et al. / European Journal of Medicinal Chemistry 89 (2015) 42e50

B-type DNA conformation, although the intensities of both 245 and 275 nm peaks were altered in the presence of complex 2. So, it is possible to conclude that the fixation of complex 2 on DNA does not greatly affect its secondary structure. From Fig. 8, a slight increase in the intensity of both positive and negative bands was observed upon addition of increasing amount of the test complex 2, which is different from those of the classical DNA$platinum complexes that usually accompanied by an increase in the positive band and a decrease in the negative band [51]. An increase in the positive band indicates that the stacking of DNA base pairs increases, while the increase in the negative band suggests that the stability of DNA double helix structure enhances. These spectral features suggest that the square-planar copper(II) complex 2 interacts with the CT-DNA through the partial intercalation binding mode, which is one of the most important DNA binding modes because it invariably leads to cellular degradation [18]. As well known, the planarity, coordination geometry, and type of donor atom in ligands play key roles in determining the intercalating ability of complexes with DNA [17,18]. When the squareplanar copper(II) complex 2 intercalates the base pairs of CT-DNA, the p*-orbital of the aromatic imidazole ring in the complex will couple with the p-orbital of the base pairs of CT-DNA. With the concentration of the test compound increasing, the intercalation would increase. Therefore, the stability of the base-pair stacking and the tightness of the DNA double helix structure increase accordingly. On the whole, the complex 2 has an efficient binding affinity for DNA, which may be beneficial to the anticancer efficacy. This is also consistent with the conclusion drawn from the UVevis absorption study. 4. Conclusion In summary, two novel copper(II) complexes based on ZL and its derivative IPrDP were designed and synthesized, {[Cu3(ZL)2(H2O)6]$6H2O}n (1) and [Cu(IPrDP)2]$3H2O (2). Due to different coordination modes in the homologous diphosphonates, 1-D polymer of 1 and mononuclear complex of 2 were formed respectively. In both complexes, extensive hydrogen-bonding interactions are found among the phosphonate oxygen atoms, hydroxy groups, protonated imidazole groups and water molecules. Complex 2 shows dose- and time-dependent cytotoxic effects on the human cancer cell lines U2OS, A549, HCT116, MDA-MB-231 and HepG2. Although the cytotoxicities of complex 2 against U2OS, A549, MDA-MB-231 and HepG2 cells are moderate, its cytotoxic

Fig. 8. CD spectra of CT-DNA (5  103 M) in the presence of increasing concentration ratios of complex 2 (r ¼ 0, 0.20, 0.40, 0.80 and 1.00) in buffer (5 mM TriseHCl, 50 mM NaCl, pH 7.4) at 37  C.

49

effect on HCT116 cells is comparable to that of CDDP, and it has a better selectivity than CDDP for inhibiting hepatocarcinoma cells rather than normal liver cells. The complex 2 inhibits the proliferation of human cancer cell lines by inducing the cell cycle arrest at the G2/M phase, showing a similar action mechanism to that of CDDP. Through the absorption and circular dichroism spectra studies, complex 2 demonstrates a moderate binding affinity for DNA through intercalation. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21371082, 21001055), Natural Science Foundation of Jiangsu Province (BK20141102), Key Medical Talent Project of Jiangsu Province (RC2011097) and Public Service Platform for Science and Technology Infrastructure Construction Project of Jiangsu Province (BM2012066). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.10.028. References [1] M.J. Clarke, F. Zhu, D.R. Frasca, Non-platinum chemotherapeutic metallopharmaceuticals, Chem. Rev. 99 (1999) 2511e2534. [2] P.J. Dyson, G. Sava, Metal-based antitumour drugs in the post genomic era, Dalton Trans. (2006) 1929e1933. [3] W. Liu, R. Gust, Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs, Chem. Soc. Rev. 42 (2013) 755e773. [4] P.J. Loehrer, L.H. Einhorn, Drugs five years later. Cisplatin, Ann. Intern. Med. 100 (1984) 704e713. [5] E. Wong, C.M. Giandomenico, Current status of platinum-based antitumor drugs, Chem. Rev. 99 (1999) 2451e2466. [6] E.W. Thompson, J.T. Price, Mechanisms of tumour invasion and metastasis: emerging targets for therapy, Expert Opin. Ther. Targets 6 (2002) 217e233.
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