Accepted Manuscript Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with norcantharidin and benzimidazole derivatives Wen-Ji Song, Qiu-Yue Lin, Wen-Jiao Jiang, Fang-Yuan Du, Qing-Yuan Qi, Qiong Wei PII: DOI: Reference:
S1386-1425(14)01260-8 http://dx.doi.org/10.1016/j.saa.2014.08.069 SAA 12590
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
23 June 2014 17 August 2014 23 August 2014
Please cite this article as: W-J. Song, Q-Y. Lin, W-J. Jiang, F-Y. Du, Q-Y. Qi, Q. Wei, Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with norcantharidin and benzimidazole derivatives, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/ 10.1016/j.saa.2014.08.069
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Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with norcantharidin and benzimidazole derivatives
Wen-Ji Songa,b, Qiu-Yue Lin a,b,*, Wen-Jiao Jiangb, Fang-Yuan Dub, Qing-Yuan Qi b,*, Qiong Weib a
Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces, Zhejiang Normal
University, 321004, P. R. China b
College of Chemical and Life Science, Zhejiang Normal University, 321004, P. R. China
Corresponding authors: Qiu-Yue Lin; Qing-Yuan Qi Tel.: +86-579-82283353; Fax: +86-579-82282269 Email: [email protected]; [email protected]
Abstract: Two novel complexes [Cu(L)2(Ac)2]·3H2O (1) (L= N-2-methyl benzimidazole demethylcantharate imide, C16H15N3O3, Ac = acetate, C2H3O2) and [Cu(bimz)2(DCA)] (2) (bimz = benzimidazole, C7H6N2; DCA = demethylcantharate, C8H8O5) were synthesized and characterized by elemental analysis, infrared spectra and X-ray diffraction techniques. Cu(II) ion was four-coordinated in complex 1, Cu(II) ion was five-coordinated in complex 2. A large amount of intermolecular hydrogen-bonding and π-π stacking interactions were observed in these complex structures. The DNA-binding properties of these complexes were investigated using electronic absorption spectra, fluorescence spectra, viscosity measurements and agarose gel electrophoresis. The interactions between the complexes and bovine serum albumin (BSA) were investigated by fluorescence spectra. The antiproliferative activities of the complexes against human hepatoma cells (SMMC7721) were tested in vitro. And the results showed that these complexes could bind to DNA in moderate intensity via partial intercalation, and complexes 1 and 2 could cleave plasmid DNA through hydroxyl radical mechanism. Title complexes could effectively quench the fluorescence of BSA through static quenching. Meanwhile, title complexes had stronger antiproliferative effect compared to L and Na2(DCA) within the tested concentration range. And complex 1 possessed more antiproliferative active than complex 2. Keywords: Norcantharidin; Benzimidazole Derivatives; Copper complex; DNA Binding; Antiproliferative Activity.
1 Introduction 2
In recent years, the interactions of Cu(II) complexes with DNA and protein molecules drew more and more scholars’ attention [1, 2]. Copper(II) complexes are well suited for DNA hydrolysis due to the strong Lewis acid properties of the cupric ion. Several copper(II) complexes have been developed as artificial nucleases, and showed versatile DNA cleavage properties in the absence or presence of a redox agent [3, 4]. Planar heterocyclic based complexes have received considerable interest in nucleic-acid chemistry because of their diverse chemical reactivity, unusual electronic properties, and peculiar structure, which results in non-covalent interactions with DNA [5]. Benzimidazole derivatives possess a variety of biological activities and pharmacological effects. Several compounds containing benzimidazole group, have been reported to exhibit antimicrobial, anticancer, antifungal and anti-inflammatory activities [6]. Especially, the combinations of the pharmaceutical agents with some metal ions can further improve their biological activity. Demethylcantharidin (NCTD, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylc acid anhydride) and disodium demethylcantharate (Na2(DCA)), as the derivatives of cantharidin, have been applied in clinical use [7]. Meanwhile, demethylcantharate (DCA) could inhibit the activities of protein phosphatases 1 (PP1) and 2A( PP2A) effectively [8, 9]. A range of amines were applied to react with norcantharidin, and results showed high level of cytotoxicity [10, 11]. Based on our previous investigations and as a continuation of our research program on complexes containing demethylcantharidin [12, 13], we synthesized two novel Cu(II)
3
complexes containing demethylcantharidin. The interactions of these complexes with DNA and bovine serum albumin (BSA) were investigated. In addition, antiproliferative activities against human hepatoma cells (SMMC-7721) were tested in vitro. 2 Experimental Sections 2.1 Materials and instruments All reagents and chemicals were obtained from commercial sources. Demethylcantharidin (NCTD, C8H8O4) was obtained from Nanjing Zelang Medical Technology Co. Ltd.; Na2(DCA) was prepared in accordance with the literature described technique[14]; 2-Aminomethylbenzimidazole dihydrochloride (ambi·2HCl) was prepared using the literature technique [15]; Benzimidazole (bimz, C7H6N2) and ct-DNA were obtained from Sinopharm Chemical Reagent Co. Ltd.; ct-DNA (ρ = 200 µg·mL-1, c = 3.72×10 -4 mol·L-1), with A260 / A280 = 1.8 - 2.0, was prepared using 50 mmol·L-1 NaCl; Plasmid DNA (pDsRed2-C1) was purchased from Clontech Co. Ltd. America; Bovine Serum Albumin (BSA) was purchased from Beijing BioDee BioTech Co. Ltd. and was stored at 4 °C; BSA (ρ = 500 µg·mL-1, c = 7.47×10-6 mol·L-1) was prepared using 5 mmol·L-1 NaCl solution; MTT(methyl thiazolyl tetrazolium) was purchased from the Sigma Company; Human hepatoma cells (SMMC-7721) was purchased from Shanghai Institute of Cell Bank. Other chemical reagents in analytical reagent grade were used without further purification. Elemental analyses of C, H and N were carried out in Vario EL III elemental analyzer. Infrared spectra were obtained using the KBr disc method by NEXUS-670
4
FT-IR spectrometer in the spectral range of 4000-400 cm-1. Diffraction intensities of the complexes were collected at 293 K on Bruker SMART APEX II CCD diffractometer. Electronic absorption spectra were obtained using UV-2501 PC spectrophotometer. Viscosity experiments were carried on Ubbelodhe viscometer. Fluorescence
emission
spectra
were
obtained
by
Perkin-Elmer
LS-55
spectrofluorometer. Agarose gel electrophoresis was performed on PowerPac Basic electrophoresis apparatus (BIO-RAD). Gel image formation were obtained on UNIVERSAL HOOD 11-S.N. (BIO-RAD Laboratories).
2.2 Synthesis of L N-2-methyl benzimidazole demethylcantharate imide (L= C16H15N3O3) was prepared in accordance to the literature techniques [16]. Mixture of 1 mmol norcantharidin (NCTD), 1 mmol 2-Aminomethylbenzimidazole dihydrochloride, 1 mmol cadmium acetate, and 10 mL distilled water was sealed in a 25 mL teflon-lined stainless vessel and heated at 433 K for 3 d, then cooled slowly to room temperature. The solution was then filtered and was allowed to stand still for 3 weeks until forming colorless crystals. Anal. Calcd. (%) for C16H15N3O3: C, 64.65; H, 5.05; N, 14.14. Found (%): C, 64.62; H, 5.03; N, 14.16. IR (KBr pellet, cm-1): 1617, 1392 (υ (C=O)); 1446(υ(C=N)); 1258, 1033, 1001(υ(C-O-C)).
2.3 Synthesis of the complexes 2.3.1 Synthesis of the complex 1 In a 20mL weighing bottle, Cu(Ac)2·H2O ( 0.06g, 0.3 mmol) was dissolved in water (2 mL). The L (0.089 g, 0.3 mmol) solution in mixed solvents of water and
5
ethanol (2:1, v/v) (10 mL) was then added dropwisely with stirring under room temperature. The mixture solution was filtered after two hours. One week after, blue crystals with suitable size for single-crystal X-ray diffraction were obtained. Anal. Calcd. (%) for C36H42N6O13Cu (1): C, 52.05; H, 5.06; N, 10.12. Found (%): C, 52.01; H, 5.03; N, 10.29. IR (KBr pellet, cm-1): 3445(υ(OH)); 1572, 1395 (υ (C=O)); 1464(υ(C=N)); 1254, 1057, 984(υ(C-O-C)). 2.3.2 Synthesis of the complex 2 A mixture of Cu(Ac)2·H2O (0.5mmol) and Na2DCA (0.5mmol) was dissolved in water. And 1.0mmol benzimidazole (bimz) in ethanol was added dropwisely into the mixed solution and stirring at room temperature. The solution was filtered after two hours. One week later, blue crystals with suitable size for single-crystal X-ray diffraction were obtained. Anal. Calcd. (%) for Cu(C22 H20 N4 O5) (2): C, 54.55; H, 4.13; N, 11.57. Found (%): C, 54.25; H, 4.01; N, 11.78. IR (KBr pellet, cm-1): 3432(υ(OH)); 1635, 1396 (υ(C=O)); 1432(υ(C=N)); 1251, 1032, 981(υ(C-O-C)).
2.4 DNA binding 2.4.1 Electronic absorption spectra Electronic absorption spectra were collected at 25 °C by fixing the concentrations of the complexes, with DNA concentration ranging from 0 to 7.44×10-5 mol·L-1. Absorption spectra measurements were carried out at 200 nm - 400 nm, and DNA in Tris-HCl buffer solution (pH = 7.4) was used as reference. 2.4.2 Fluorescence spectra Fluorescence quenching experiments were carried out by adding DNA solutions
6
(0-7.44×10-4 mol·L-1) to the samples containing 2.00 × 10-5 mol·L-1 complexes. The mixture were diluted by Tris-HCl buffer solution (pH = 7.4). Fluorescence for 1 was recorded at excitation wavelength (λex) of 248 nm and emission wavelength (λem) between 250 nm and 500 nm. Fluorescence for 2 was recorded at 244 nm excitation wavelength (λex) and emission wavelength (λem) between 255 nm and 450 nm (λem). 2.4.3 Viscosity measurement Viscosity measurements were performed. Compounds were added to DNA solution (3.72×10 -4 mol·L-1) with microsyringes. The concentration of the compounds were controlled within the range of 0 - 3.33×10 -6 mol·L-1. The relative viscosities η were calculated using equation [17]: η = (t - t0) / t0, where t0 and t represent the flow time of DNA solution through the capillary in the absence and presence of complex. The average values of three replicated measurements were used to evaluate the viscosity of the samples. Data were presented as (η/η0)1/3 versus the ratio of the concentration of compounds to DNA, where η was the viscosity of DNA in the presence of compound and η0 was the viscosity of DNA. 2.4.4 Interaction with pDsRed2-C1 plasmid DNA Interactions between the complexes and pDsRed2-C1 plasmid DNA were studied using agarose gel electrophoresis. The samples were incubated at 37°C for 3h, followed by addition of 0.25% bromo-phenol blue and 1 mmolL-1 EDTA. The DNA cleavage products were submitted to electrophoresis in 1.0% agarose gel containing 0.5μgmL-1 ethidium bromides. The bands were photographed.
2.5 Interaction with BSA 7
2.5.1 Fluorescence spectra The complexes (0-26.7×10-9 mol·L-1) were added to solution containing 4.98×10 -7 mol·L-1 BSA and Tris-HCl buffer (pH = 7.4). Fluorescence spectra were obtained by recording the emission spectra (285nm-480nm) at excitation wavelength of 280 nm.
2.6 Antiproliferative activity evaluation The antiproliferative activities of the compounds (1, 2, L and Na2(DCA)) were evaluated by human hepatoma cells (SMMC-7721). The MTT assay was applied to measure the antiproliferative activities [18]. The compounds were dissolved in DMSO as 100 mmol·L-1 stock solutions, and diluted in culture medium before using. The target concentration of DMSO in the medium was less than 0.1%, and it did not interfere with the tested bioactivity results[19]. Cells were seeded for 24 h before adding compounds, and incubated for 72 h. Then 100 µL MTT (1 mg·mL-1, dissolved in DMEM nutrient solution) was added into each well and incubated for 4 h (37 °C). The absorbance was measured by microplate reader at 570 nm. The inhibition rate was calculated accordingly. The errors quoted were standard deviations, which three replicates were involved in the calculation [20].
2.7 Crystal structure determination Single crystals, sized 0.345 mm×0.279 mm×0.214 mm (1) and 0.345 mm×0.287 mm×0.156 mm (2), were used for X-ray diffraction analysis. The structures were solved by direct methods and refined by full-matrix least-squares techniques using the SHELXTL-97 program package [21, 22]. All non-hydrogen atoms were refined
8
anisotropically. Besides the hydrogen atoms on oxygen atoms, which were located from the difference Fourier maps, other hydrogen atoms were generated geometrically. Crystal data and experimental details for structural analyses are listed in Table 1.
PLAESE INSTERT TABLE 1
3 Results and discussion 3.1 Structural description of complexes Two novel complexes have been characterized by X-ray single crystal diffraction. The spectral results indicated that the space groups of the complexes were C2/C (1) and Pna2 1 (2). Selected bond lengths and angles of complexes 1, 2 were listed in Table 2 and 3. Hydrogen bond lengths and angles of complex 1, 2 were listed in Table S1 and S2. Molecular structures of the title complexes were shown in Fig. 1. The packing diagram was shown in Fig. S1. In complex 1, the Cu(II) ion was four-coordinated. Each Cu(II) coordinated with two imine nitrogen N(2) (or N(2A)) from ligand (L), and two oxygen atoms of different carboxyl groups from acetate ions, forming electrically neutral complex. This molecule was centrally symmetric, with the symcenter at the centre of CuN2O2. The bond angles of O(1)-Cu(1)-O(1)#1、O(1)-Cu(1)-N(2)、O(1)#1-Cu(1)-N(2)#1 and N(2)-Cu(1)-N(2)#1 are 88.38(14)° 、 90.23(10)° 、 90.23(10)° and 97.25(14)° ， respectively, all of which are close 90°. Thus, a slightly distorted quadrangle was formed around Cu(1) by N(1), N(2), O(1), and O(3). The composition of the complex
9
was [Cu(L)2(Ac)2]·3H2O(1). Fig. S1 showed that the hydrogen-bonding formed due to the presence of the nitrogen atoms and the oxygen atoms from the imide(L) and acetate ligands, and crystallization water molecules. The complex is rich in intramolecular and intermolecular hydrogen bonds, such as N(1)-H(1A)...O(1W)； O(3W)-H(3WA)...O(4)；O(2W)-H(2WA)...O(2). These hydrogen-bonding stabilized this crystal structure. In complex 2, Cu(II) ion was five-coordinated. Each Cu(II) coordinated with two azomethine nitrogen N(1) (or N(3)) from two bimz, two carboxylate oxygen atoms O2 and O3 in two different carboxylate groups, and one bridge oxygen atoms O1 from demethylcantharate, forming a distorted tetragonal pyramid structure. The composition of the complex was [Cu(bimz)2(DCA)](2). Fig. S1 showed that the hydrogen-bonding formed due to the presence of the nitrogen atom from the bimz and the oxygen atoms from demethylcantharate, such as N(2)-H(2A)...O(5)#1, N(4)-H(4A)...O(3)#2. Meanwhile, complexes 1 and 2 contain the benzimidazole group, resulting л-л stacking effects among the complexes. Therefore, we concluded that the synergistic effect, including π-π stacking and hydrogen-bonding interactions, existed between the complexes and biomacromolecule, which could be the fundamental cause of the biological activity change found in macromolecules [23].
PLEASE INSERT FIGURE 1 PLAESE INSTERT TABLE 2-3
10
3.2. DNA binding studies 3.2.1. Electronic absorption spectra The application of electronic absorption spectroscopy is one of the most useful techniques in DNA-binding studies [24]. Changes observed in the UV spectra upon titration can provide evidence for the intercalative interaction mode pattern, since hypochromism would occur from π-π stacking interactions [25]. To further investigate the possible binding modes and to obtain the binding constants (Kb) of complex to DNA, we also studied the effect of DNA titration to the title complexes by electronic absorption spectra at 298 K. Results are shown in Fig. 2 ((a): 1; (b): 2). The intrinsic binding constant (Kb) was determined by the equation: [DNA] / (εA εF) = [DNA] / (εB - εF) + 1 / [Kb (εB - εF)], where [DNA] was the concentration of DNA, εA, εF and εB corresponded to the apparent extinction coefficient, the extinction coefficient for the free compounds and its fully DNA-bound combination, respectively
[26]. Kb/(L·mol-1) values of the compounds were 5.52×103 (1), and 1.93×10 3 (2). The values suggested that the binding ability of complex 1 was more intense than complex 2, which agrees with the observation that complex 1 has a better planar aromatic structure result in more intermolecular interactions, including π-π stacking and hydrogen-bonding interactions. The complexes overlap the DNA base pairs via benzimidazole ring insertion, which leads to the hypochromicity of complexes absorption [27].
11
PLEASE INSERT FIGURE 2
3.2.2 Fluorescence quenching studies Complexes 1 and 2 showed an intense fluorescence emission around 248 nm (1) and 244 nm (2). The fluorescence emission of the complexes 1 and 2 are quenched in presence of DNA. The complexes showed strong emission bands at around 297 nm(1) and 294 nm (2), as shown in Fig. 3. According to the Stern-Volmer equation: F0/F = 1+ Ksv [Q], F0 and F represent the fluorescence intensities in the absence and presence of quencher, respectively [28], [Q] is the quencher concentration and Ksv is the Stern-Volmer constant, Ksv were calculated as 4.26× 103 mol·L-1(1) and 2.68 × 10 3 mol·L-1(2). The binding intensity of complex 1 was stronger than complex 2, this is consistent to the results found in the electronic absorption spectra. PLEASE INSERT FIGURE 3
3.2.3. Viscosity measurements To further study the binding mode of the compounds interacting with DNA, DNA viscosity at 25°C was investigated (Fig. 4). The experimental data showed that the relative viscosity of DNA steadily decreased after adding complexes and L, and it could increase after adding benzimidazole. But there was no significant viscosity change occurred after adding Na2DCA. The possible explanation is that the complexes and L were partially inserted to the DNA base pairs and resulting in a kink in the DNA helix, therefore decreased the DNA effective length [29]. Because of the
12
planar benzimidazole ring could also insert to the DNA base pair, and the steric hindrances of complexes were enhanced due to the non-planar structure of demethylcantharate (DCA). From fig. 4, the interactions of complex(1) with DNA is significantly stronger than complex(2). The result agrees with the electronic absorption spectra and fluorescence spectra conclusion. PLEASE INSERT FIGURE 4 3.2.4. Interaction with pDsRed2-C1 plasmid DNA The cleavage reaction on pDsRed2-C1 plasmid DNA can be monitored by agarose gel electrophoresis. When pDsRed2-C1 plasmid DNA is subjected to electrophoresis, different migration speeds were observed [30]. Relatively fast migrations were observed at the intact supercoil form (Form I). If scission occurs on one strand (nicking), the supercoil will partially relax to generate a slow moving open-circular form (Form II) [31]. Fig. 5 gave the electrophoretograms of the interaction of pDsRed2-C1 plasmid DNA with increasing concentrations of complexes. Complexes are capable of cleaving plasmid DNA when the concentration of complexes was greater than 500µM. When the concentration of complexes increased, the amount of Form I diminished gradually, and Form II increased. Comparing channel 4 to 6, the cleavage ability of complexes was enhanced by adding ascorbic acid. In order to investigate the reaction mechanism, dimethylsulfoxide (DMSO) was introduced to the experimental design. DMSO as a radical scavenger could inhibit the cleavage ability of complexes significantly in channel 5 and 7. With increasing
13
amount of ascorbic acid (Vc), Cu(II) complex was reduced to Cu(I) complex. The Cu(I) complex then reacts with dissolved oxygen generating ROS: superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH). Finally, the ROS attacks the plasmid DNA leading to the single and double DNA strand breaks. So the cleavage process was via hydroxyl radical mechanism [32]. PLEASE INSERT FIGURE 5
3.3 Interaction with BSA 3.3.1 Fluorescence spectra and quenching mechanism The results of title complexes quenching the BSA fluorescence were shown in Fig. 6. Complex 1 and 2 showed similar quenching patterns. It showed that the intensity of the fluorescence peak of BSA decreased with increasing concentration of complexes, which inferred that strong interactions existed between complexes and BSA [33].
PLEASE INSERT FIGURE 6
Fluorescence quenching can undergo two different mechanisms: static quenching and dynamic quenching. For dynamic quenching, the mechanism can be described by the Stern-Volmer equation: F0 / F = 1 + Kq τ0 [Q], where F0 and F are the fluorescence intensities of BSA in the absence and presence of the complexes, respectively [34], [Q] is the concentration of the complexes. For many proteins, τ0 is known to be approximately equal to 10-8s [35]. The calculated quenching rate constants Kq were
14
1.47×1015 L·mol-1·s-1(1) and 1.15×1015 L·mol-1·s-1(2). These values were much greater than the maximum possible value of diffusion-limited quenching in water. The result also suggested that the quenching mechanism of complexes to BSA was static quenching, which generated via intense interaction [36]. 3.3.2 Binding constants and binding sites Assuming there were n identical and independent binding sites in protein, the binding constant KA can be calculated using equation [37]: lg (F0 - F) / F = lg KA + n lg [Q]. The values of KA were 1.59×106L·mol-1 (1), 5.4×104 L·mol-1 (2), and 2.78×10 4 L·mol-1 (Na2DCA). The values of n were 0.88(1), 0.68(2) and 0.66 (Na2DCA). The results indicated that strong binding interaction existed between the complexes and BSA. The binding intensity of complexes was stronger than Na2DCA, and the binding site of complexes was one.
3.4 Antiproliferative activity evaluation As shown in Fig. 7, the antiproliferative activity of complex 1, complex 2, L and Na2DCA at the given concentration showed a dose-dependent manner against human hepatoma cells (SMMC-7721) in vitro. The inhibition ratios tested revealed that complex 1 and 2 had strong antiproliferative activities against human hepatoma cells (SMMC-7721) lines in vitro
compare to L and Na2DCA. The inhibition rates of complex(1) against SMMC-7721
lines (IC50=24.55±0.48 µmol·L-1) is much higher than that of L (IC50=116.63±2.66
µmol·L-1) [38]. The inhibition rates of complex(1) against SMMC-7721 lines is much
higher than complex(2) (IC50=41.82±3.90 µmol·L-1). The inhibition rates of two novel
15
complexes were higher than that of the transition metal complexes of demethylcantharate and thiazole derivatives [12, 13] against SMMC-7721 cells.
which suggests that various compositions and structures of complexes would lead to different antiproliferative activities, and this can be important in designing and
synthesizing novel anti-cancer drugs [39]. It is clear that the strong interaction found
between complexes and biomacromolecules (DNA or BSA ) is directly correlated to
the antiprolififerative activity of complexes. PLEASE INSERT FIGURE 7
4. Conclusions Two
novel
Cu(II)
complexes
[Cu(L)2(Ac)2]·3H2O(1)(
L=
N-2-methyl
benzimidazole demethylcantharate imide, C16H15N3O3, Ac = acetate, C2H3O2 ) and [Cu(bimz)2(DCA)](2)( bimz = benzimidazole, C7H6N2; DCA = demethylcantharate, C8H8O5) were synthesized and characterized. The crystal structure of complex 1 and 2 were determined by X-ray diffraction. These complexes had strong DNA and BSA
binding intensity and high inhibition rates against human hepatoma cells (SMMC-7721) in vitro. Complex(1) had intense antiproliferative activities against the human hepatoma cells(SMMC-7721) in vitro, which had the potential to develop as an anti-cancer drug in the future.
5. Acknowledgment We thank Institute of Zhejiang Academy of Medical Science for helping with
16
antiproliferative activity test.
Supplementary materials Crystallographic data for the structure reported in this article has been deposited with the Cambridge Crystallographic Data Center CCDC 909444(1), 918105(2). Copies of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB21EZ, UK ([email protected]). The packing diagrams of complexes were shown in Fig. S1. Hydrogen bond lengths and angles of complex 1, 2 were listed in Table S1, S2.
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[20] X. L. Zheng, H. X. Sun, X. L. Liu, Y. X. Chen, B. C. Qian, Acta Pharmacol. Sin. 25 (2004) 1090-1095. [21] G. M. Sheldrick, SHELXS-97, Program for the Solution of Crystal Structures, University of Göttingen, Germany, 1997. [22] G. M. Sheldrick, SHELXL-97, Program for the Refinement of Crystal Structures, University of Göttingen, Germany, 1997. [23] Z. Rehman, N. Muhammad, S. Shuja, S. Ali, I. S. Butler, A. Meetsma, M. Khan. Polyhedron , 28 (2009) 3439-3448. [24] Z. C. Liu, B. D. Wang, Z. Y. Yang, Y. Li, D. D. Qin, T. R. Li, Eur. J. Med. Chem. 44 (2009) 4477-4484. [25] C. S. Kalliopi, P. Franc, T. Iztok, P. K. Dimitris, P. George, J. Inorg. Biochem. 104 (2010) 740-745. [26] K. Paramasivam, S. Palanisamy, R. B. Rachel, H. C. Alan, S. P. B. Nattamai, D. Nallasamy. Dalton Trans. 41 (2012) 4423-4436. [27] S. Roy, S. Saha, R. Majumdar, R. R. Dighe, A. R. Chakravarty, Polyhedron 29 (2010) 2787-2794. [28] I. M. Khan, A. Ahmad, M. F. Ullah, Spectrochim. Acta A 102 (2013) 82-87. [29] J. Liu, T. X. Zhang, T. B. Lu, L. H. Qu, H. Zhou, Q. L. Zhang, L. N. Ji, J. Inorg. Biochem. 91 (2002) 269-276. [30] N. Wang, Y. Y. Wang, X. X. Wang, X. L. Zheng, D. M. Yan, Q. Y. Lin, Chinese J. Chem. 29 (2011) 473-477. [31] X. Q. He, Q. Y. Lin, R. D. Hu, X. H. Lu, Spectrochim. Acta A 68 (2007) 184-190.
19
[32] C. A. Detmer III, F. V. Pamatong, J. R. Bocarsly, Inorg. Chem. 35 (1996) 6292-6298. [33] P. Sathyadevi, P. Krishnamoorthy, E. Jayanthi, R. R. Butorac, A. H. Cowley, N. Dharmaraj, Inorg. Chim. Acta 384 (2012) 83-96. [34] Q. Guo, L. Z. Li, J. F. Dong, H. Y. Liu, T. Xu, J. H. Li, Spectrochim. Acta A 106 (2013) 155-162. [35] X. W. Li, Y. T. Li, Z. Y. Wu, C. H. Yan, Inorg. Chim. Acta 390 (2012) 190-198. [36] Y. J. Wang, R. D. Hu, D. H. Jiang, P. H. Zhang, Q. Y. Lin, Y. Y. Wang, J. Fluoresc. 21 (2011) 813-823. [37] P. Sathyadevi, P. Krishnamoorthy, M. Alagesan, K. Thanigaimani, P. T. Muthiah, N. Dharmaraj, Polyhedron 31 (2012) 294-306. [38] Song W. J., Cheng J. P., Jiang D. H., Guo L., Cai M. F., Yang H. B., Lin Q. Y., Spectrochim. Acta A, 121(2014) 70-76. [39] F. Zhang, Q. Y. Lin, W. L. Hu, W. J. Song, S. T. Shen, P. Gui, Spectrochim. Acta A 110 (2013) 100–107.
Table captions Table 1. Crystal data of complex 1 and 2 Table 2. Selected bond lengths(Å) and angles(°) for complex 1 Table 3. Selected bond lengths(Å) and angles(°) for complex 2
Figure captions Fig. 1 Labeled ORTEP diagrams of complex 1(a) and 2(b) with 30% thermal probability ellipsoids shown. 20
Fig. 2 Absorption spectra of the complex 1(a) and 2(b) in the presence of increasing amount of DNA. [complex] = 3.00×10-6 mol· L-1 , from (1) to (5): (a). [DNA]×105 = 0, 0.74, 1.48, 2.24 and 2.98 mol· L-1, respectively. (b). [DNA]×105 = 0, 1.86, 3.72, 5.58 and 7.44 mol· L-1, respectively Fig. 3 Fluorescence spectra of the complex 1(a) and 2(b) in the absence and presence of increasing the amount of DNA; Insert in Fig. 3-5: fluorescence quenching curve of the complex by DNA. λex=248 nm (1), λex=244 nm (2), [complex]=2×10-5 mol· L-1; [DNA]/(10-4 mol· L-1), from 1 to 5: 0, 1.86, 3.72, 5.58, and 7.44, respectively. Fig. 4 Effect of increasing amounts of the compounds on the relative viscosity of DNA. [DNA] = 3.72×10-4 mol·L-1; [complex]/10-6 = 0, 0.67, 1.33, 2.00, 2.67 and 3.33 mol·L-1, respectively. Fig. 5 Electrophoretic separation of pDsRed2-C1 DNA induced by complexes 1(a) and 2(b) Lane 1: DNA alone; lane 2: DNA + complex (250 µ M); Lane 3: DNA + complex (500 µ M); lane 4: DNA + complex (750 µM); lane 5: DNA + complex (750 µ M) + DMSO (750µ M); lane 6: DNA + complex (750 µM) + Vc (750 µ M), lane 7: DNA + complex (750 µM) + Vc (750 µ M) + DMSO (750µM). [DNA] =3.0 µ g·mL-1. Fig. 6 Fluorescence spectra of BSA in the absence and the presence of complex 1(a) and 2(b) Inset: Stern-Volmer plots of the fluorescence titration data of the complexes. [BSA] = 4.98×10-7 mol· L-1; [complex]×109 = 0, 6.67, 13.3, 20.0, and 26.7 mol· L-1, from (1) to (5), respectively (a): complex 1, (b): complex 2. Fig. 7 Inhibition effects of compounds on SMMC-7721 cell growth. Data represent mean + S.D. and all assays were performed in triplicate for three independent experiments.
21
Fig. 1
22
Fig. 2
23
Fig. 3
24
Fig. 4
25
Fig. 5
26
Fig. 6
27
Fig. 7
28
Table 1 Crystal data of complex 1 and 2 Complex
1
2
CuC36H42N6O13
CuC22H20N4 O5
Formula weight
794.28
483.97
Crystal system
Monoclinic
orthorhombic
Space group
C2/C
Pna21
a(Å)
21.572 (4)
15.8667(3)
b(Å)
11.3687 (19)
9.9975(2)
c(Å)
17.371 (4)
12.5228(2)
α(°)
90.00
90.00
β(°)
111.528 (18)
90.00
γ(°)
90.00
90.00
Volume(Å3)
3963.0 (13)
1986.46(6)
Z
4
4
Crystal size (mm)
0.345×0.279×0.214
0.345×0.287×0.156
Shape
block
block
Colour
blue
blue
Dc (g/cm-3)
1.331
1.618
θ Rang for data collection(°)
2.03 to 27.86
2.41to 27.57
Reflections collected/Unique
6486 / 3106
19898 / 4472
0.1288
0.0844
0.632
1.145
F(000)
1672
996
R/wR [I>2σ(I)]
0.0514 / 0.1291
0.0791 / 0.1953
Restraints/parameters
9 / 285
1/ 289
Goodness-of-fit on F2
0.939
1.160
Largest diff. Peak and hole(e/Å-3)
0.459, -0.663
1.131, -1.887
Chemical formula
R(int) Absorption coefficient(mm-1)
29
Table 2. Selected bond lengths(Å) and angles(°) for complex 1 Bond
(Å)
Bond
(Å)
Cu(1)-O(1)
1.977(2)
Cu(1)-N(2)
1.991(2)
Cu(1)-O(1)#1
1.977(2)
Cu(1)-N(2)#1
1.991(2)
Angle
(°)
Angle
(°)
O(1)-Cu(1)- O(1)#1
88.38(14)
O(1)#1-Cu(1)-N(2)
160.71(8)
O(1)-Cu(1)-N(2)
90.23(10)
O(1)#1-Cu(1)-N(2)#1
90.23(10)
O(1)-Cu(1)-N(2)#1
160.71(8)
N(2)-Cu(1)-N(2)#1
97.25(14)
Symmetry transformations used to generate equivalent atoms:
30
#1 -x, y, -z+1/2
Table 3. Selected bond lengths(Å) and angles(°) for complex 2 Bond
(Å)
Bond
(Å)
Cu(1)-O(3)
1.983(7)
Cu(1)-N(3)
1.990(7)
Cu(1)-O(2)
1.942(6)
Cu(1)-N(1)
2.023(8)
Cu(1)-O(1)
2.256(6)
Angle
(°)
Angle
(°)
O(3) -Cu(1)-O (2)
88.1(3)
O(2)-Cu(1)-N(3)
167.0(3)
O(3)-Cu(1)-O(1)
91.7(2)
O(2)-Cu(1)-N(1)
88.1(3)
O(3)-Cu(1)-N(3)
90.3(3)
O(1)-Cu(1)- N(3)
98.3(3)
O(3)-Cu(1)-N(1)
174.2(3)
O(1)-Cu(1)-N(1)
93.1(3)
O(2)-Cu(1)-O(1)
94.7(2)
N(3)-Cu(1)-N(1)
92.3(3)
31
Two novel complexes demethylcantharate
[Cu(L)2(Ac)2]·3H2O(1) (L= N-2-methyl benzimidazole
imide,
C15H13N2O3,
Ac
=
acetate,
C2H3O2)
and
[Cu(bimz)2(DCA)](2) (bimz = benzimidazole, C7H6N2; DCA = demethylcantharate, C8H8O5) were synthesized and characterized. The DNA-binding properties of complexes were investigated by electronic absorption spectra, fluorescence spectra, viscosity measurements and agarose gel electrophoresis. The interaction between the complexes and bovine serum albumin (BSA) was investigated by fluorescence spectra. The antiproliferative activities of the complexes against human hepatoma cells (SMMC7721) were tested in vitro.
32
►two novel Cu(II) complexes with norcantharidin derivatives have been synthesized.
►Complexes structure was determined by X-ray diffraction. ►The complexes and ligands bound DNA moderately via partial intercalation modes. ►Complexes could cleave plasmid DNA via hydroxyl radical mechanism.
►Complex(1) has strongest activity against human hepatoma cells.
33
S1386-1425(14)01260-8 http://dx.doi.org/10.1016/j.saa.2014.08.069 SAA 12590
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
23 June 2014 17 August 2014 23 August 2014
Please cite this article as: W-J. Song, Q-Y. Lin, W-J. Jiang, F-Y. Du, Q-Y. Qi, Q. Wei, Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with norcantharidin and benzimidazole derivatives, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/ 10.1016/j.saa.2014.08.069
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Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with norcantharidin and benzimidazole derivatives
Wen-Ji Songa,b, Qiu-Yue Lin a,b,*, Wen-Jiao Jiangb, Fang-Yuan Dub, Qing-Yuan Qi b,*, Qiong Weib a
Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces, Zhejiang Normal
University, 321004, P. R. China b
College of Chemical and Life Science, Zhejiang Normal University, 321004, P. R. China
Corresponding authors: Qiu-Yue Lin; Qing-Yuan Qi Tel.: +86-579-82283353; Fax: +86-579-82282269 Email: [email protected]; [email protected]
Abstract: Two novel complexes [Cu(L)2(Ac)2]·3H2O (1) (L= N-2-methyl benzimidazole demethylcantharate imide, C16H15N3O3, Ac = acetate, C2H3O2) and [Cu(bimz)2(DCA)] (2) (bimz = benzimidazole, C7H6N2; DCA = demethylcantharate, C8H8O5) were synthesized and characterized by elemental analysis, infrared spectra and X-ray diffraction techniques. Cu(II) ion was four-coordinated in complex 1, Cu(II) ion was five-coordinated in complex 2. A large amount of intermolecular hydrogen-bonding and π-π stacking interactions were observed in these complex structures. The DNA-binding properties of these complexes were investigated using electronic absorption spectra, fluorescence spectra, viscosity measurements and agarose gel electrophoresis. The interactions between the complexes and bovine serum albumin (BSA) were investigated by fluorescence spectra. The antiproliferative activities of the complexes against human hepatoma cells (SMMC7721) were tested in vitro. And the results showed that these complexes could bind to DNA in moderate intensity via partial intercalation, and complexes 1 and 2 could cleave plasmid DNA through hydroxyl radical mechanism. Title complexes could effectively quench the fluorescence of BSA through static quenching. Meanwhile, title complexes had stronger antiproliferative effect compared to L and Na2(DCA) within the tested concentration range. And complex 1 possessed more antiproliferative active than complex 2. Keywords: Norcantharidin; Benzimidazole Derivatives; Copper complex; DNA Binding; Antiproliferative Activity.
1 Introduction 2
In recent years, the interactions of Cu(II) complexes with DNA and protein molecules drew more and more scholars’ attention [1, 2]. Copper(II) complexes are well suited for DNA hydrolysis due to the strong Lewis acid properties of the cupric ion. Several copper(II) complexes have been developed as artificial nucleases, and showed versatile DNA cleavage properties in the absence or presence of a redox agent [3, 4]. Planar heterocyclic based complexes have received considerable interest in nucleic-acid chemistry because of their diverse chemical reactivity, unusual electronic properties, and peculiar structure, which results in non-covalent interactions with DNA [5]. Benzimidazole derivatives possess a variety of biological activities and pharmacological effects. Several compounds containing benzimidazole group, have been reported to exhibit antimicrobial, anticancer, antifungal and anti-inflammatory activities [6]. Especially, the combinations of the pharmaceutical agents with some metal ions can further improve their biological activity. Demethylcantharidin (NCTD, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylc acid anhydride) and disodium demethylcantharate (Na2(DCA)), as the derivatives of cantharidin, have been applied in clinical use [7]. Meanwhile, demethylcantharate (DCA) could inhibit the activities of protein phosphatases 1 (PP1) and 2A( PP2A) effectively [8, 9]. A range of amines were applied to react with norcantharidin, and results showed high level of cytotoxicity [10, 11]. Based on our previous investigations and as a continuation of our research program on complexes containing demethylcantharidin [12, 13], we synthesized two novel Cu(II)
3
complexes containing demethylcantharidin. The interactions of these complexes with DNA and bovine serum albumin (BSA) were investigated. In addition, antiproliferative activities against human hepatoma cells (SMMC-7721) were tested in vitro. 2 Experimental Sections 2.1 Materials and instruments All reagents and chemicals were obtained from commercial sources. Demethylcantharidin (NCTD, C8H8O4) was obtained from Nanjing Zelang Medical Technology Co. Ltd.; Na2(DCA) was prepared in accordance with the literature described technique[14]; 2-Aminomethylbenzimidazole dihydrochloride (ambi·2HCl) was prepared using the literature technique [15]; Benzimidazole (bimz, C7H6N2) and ct-DNA were obtained from Sinopharm Chemical Reagent Co. Ltd.; ct-DNA (ρ = 200 µg·mL-1, c = 3.72×10 -4 mol·L-1), with A260 / A280 = 1.8 - 2.0, was prepared using 50 mmol·L-1 NaCl; Plasmid DNA (pDsRed2-C1) was purchased from Clontech Co. Ltd. America; Bovine Serum Albumin (BSA) was purchased from Beijing BioDee BioTech Co. Ltd. and was stored at 4 °C; BSA (ρ = 500 µg·mL-1, c = 7.47×10-6 mol·L-1) was prepared using 5 mmol·L-1 NaCl solution; MTT(methyl thiazolyl tetrazolium) was purchased from the Sigma Company; Human hepatoma cells (SMMC-7721) was purchased from Shanghai Institute of Cell Bank. Other chemical reagents in analytical reagent grade were used without further purification. Elemental analyses of C, H and N were carried out in Vario EL III elemental analyzer. Infrared spectra were obtained using the KBr disc method by NEXUS-670
4
FT-IR spectrometer in the spectral range of 4000-400 cm-1. Diffraction intensities of the complexes were collected at 293 K on Bruker SMART APEX II CCD diffractometer. Electronic absorption spectra were obtained using UV-2501 PC spectrophotometer. Viscosity experiments were carried on Ubbelodhe viscometer. Fluorescence
emission
spectra
were
obtained
by
Perkin-Elmer
LS-55
spectrofluorometer. Agarose gel electrophoresis was performed on PowerPac Basic electrophoresis apparatus (BIO-RAD). Gel image formation were obtained on UNIVERSAL HOOD 11-S.N. (BIO-RAD Laboratories).
2.2 Synthesis of L N-2-methyl benzimidazole demethylcantharate imide (L= C16H15N3O3) was prepared in accordance to the literature techniques [16]. Mixture of 1 mmol norcantharidin (NCTD), 1 mmol 2-Aminomethylbenzimidazole dihydrochloride, 1 mmol cadmium acetate, and 10 mL distilled water was sealed in a 25 mL teflon-lined stainless vessel and heated at 433 K for 3 d, then cooled slowly to room temperature. The solution was then filtered and was allowed to stand still for 3 weeks until forming colorless crystals. Anal. Calcd. (%) for C16H15N3O3: C, 64.65; H, 5.05; N, 14.14. Found (%): C, 64.62; H, 5.03; N, 14.16. IR (KBr pellet, cm-1): 1617, 1392 (υ (C=O)); 1446(υ(C=N)); 1258, 1033, 1001(υ(C-O-C)).
2.3 Synthesis of the complexes 2.3.1 Synthesis of the complex 1 In a 20mL weighing bottle, Cu(Ac)2·H2O ( 0.06g, 0.3 mmol) was dissolved in water (2 mL). The L (0.089 g, 0.3 mmol) solution in mixed solvents of water and
5
ethanol (2:1, v/v) (10 mL) was then added dropwisely with stirring under room temperature. The mixture solution was filtered after two hours. One week after, blue crystals with suitable size for single-crystal X-ray diffraction were obtained. Anal. Calcd. (%) for C36H42N6O13Cu (1): C, 52.05; H, 5.06; N, 10.12. Found (%): C, 52.01; H, 5.03; N, 10.29. IR (KBr pellet, cm-1): 3445(υ(OH)); 1572, 1395 (υ (C=O)); 1464(υ(C=N)); 1254, 1057, 984(υ(C-O-C)). 2.3.2 Synthesis of the complex 2 A mixture of Cu(Ac)2·H2O (0.5mmol) and Na2DCA (0.5mmol) was dissolved in water. And 1.0mmol benzimidazole (bimz) in ethanol was added dropwisely into the mixed solution and stirring at room temperature. The solution was filtered after two hours. One week later, blue crystals with suitable size for single-crystal X-ray diffraction were obtained. Anal. Calcd. (%) for Cu(C22 H20 N4 O5) (2): C, 54.55; H, 4.13; N, 11.57. Found (%): C, 54.25; H, 4.01; N, 11.78. IR (KBr pellet, cm-1): 3432(υ(OH)); 1635, 1396 (υ(C=O)); 1432(υ(C=N)); 1251, 1032, 981(υ(C-O-C)).
2.4 DNA binding 2.4.1 Electronic absorption spectra Electronic absorption spectra were collected at 25 °C by fixing the concentrations of the complexes, with DNA concentration ranging from 0 to 7.44×10-5 mol·L-1. Absorption spectra measurements were carried out at 200 nm - 400 nm, and DNA in Tris-HCl buffer solution (pH = 7.4) was used as reference. 2.4.2 Fluorescence spectra Fluorescence quenching experiments were carried out by adding DNA solutions
6
(0-7.44×10-4 mol·L-1) to the samples containing 2.00 × 10-5 mol·L-1 complexes. The mixture were diluted by Tris-HCl buffer solution (pH = 7.4). Fluorescence for 1 was recorded at excitation wavelength (λex) of 248 nm and emission wavelength (λem) between 250 nm and 500 nm. Fluorescence for 2 was recorded at 244 nm excitation wavelength (λex) and emission wavelength (λem) between 255 nm and 450 nm (λem). 2.4.3 Viscosity measurement Viscosity measurements were performed. Compounds were added to DNA solution (3.72×10 -4 mol·L-1) with microsyringes. The concentration of the compounds were controlled within the range of 0 - 3.33×10 -6 mol·L-1. The relative viscosities η were calculated using equation [17]: η = (t - t0) / t0, where t0 and t represent the flow time of DNA solution through the capillary in the absence and presence of complex. The average values of three replicated measurements were used to evaluate the viscosity of the samples. Data were presented as (η/η0)1/3 versus the ratio of the concentration of compounds to DNA, where η was the viscosity of DNA in the presence of compound and η0 was the viscosity of DNA. 2.4.4 Interaction with pDsRed2-C1 plasmid DNA Interactions between the complexes and pDsRed2-C1 plasmid DNA were studied using agarose gel electrophoresis. The samples were incubated at 37°C for 3h, followed by addition of 0.25% bromo-phenol blue and 1 mmolL-1 EDTA. The DNA cleavage products were submitted to electrophoresis in 1.0% agarose gel containing 0.5μgmL-1 ethidium bromides. The bands were photographed.
2.5 Interaction with BSA 7
2.5.1 Fluorescence spectra The complexes (0-26.7×10-9 mol·L-1) were added to solution containing 4.98×10 -7 mol·L-1 BSA and Tris-HCl buffer (pH = 7.4). Fluorescence spectra were obtained by recording the emission spectra (285nm-480nm) at excitation wavelength of 280 nm.
2.6 Antiproliferative activity evaluation The antiproliferative activities of the compounds (1, 2, L and Na2(DCA)) were evaluated by human hepatoma cells (SMMC-7721). The MTT assay was applied to measure the antiproliferative activities [18]. The compounds were dissolved in DMSO as 100 mmol·L-1 stock solutions, and diluted in culture medium before using. The target concentration of DMSO in the medium was less than 0.1%, and it did not interfere with the tested bioactivity results[19]. Cells were seeded for 24 h before adding compounds, and incubated for 72 h. Then 100 µL MTT (1 mg·mL-1, dissolved in DMEM nutrient solution) was added into each well and incubated for 4 h (37 °C). The absorbance was measured by microplate reader at 570 nm. The inhibition rate was calculated accordingly. The errors quoted were standard deviations, which three replicates were involved in the calculation [20].
2.7 Crystal structure determination Single crystals, sized 0.345 mm×0.279 mm×0.214 mm (1) and 0.345 mm×0.287 mm×0.156 mm (2), were used for X-ray diffraction analysis. The structures were solved by direct methods and refined by full-matrix least-squares techniques using the SHELXTL-97 program package [21, 22]. All non-hydrogen atoms were refined
8
anisotropically. Besides the hydrogen atoms on oxygen atoms, which were located from the difference Fourier maps, other hydrogen atoms were generated geometrically. Crystal data and experimental details for structural analyses are listed in Table 1.
PLAESE INSTERT TABLE 1
3 Results and discussion 3.1 Structural description of complexes Two novel complexes have been characterized by X-ray single crystal diffraction. The spectral results indicated that the space groups of the complexes were C2/C (1) and Pna2 1 (2). Selected bond lengths and angles of complexes 1, 2 were listed in Table 2 and 3. Hydrogen bond lengths and angles of complex 1, 2 were listed in Table S1 and S2. Molecular structures of the title complexes were shown in Fig. 1. The packing diagram was shown in Fig. S1. In complex 1, the Cu(II) ion was four-coordinated. Each Cu(II) coordinated with two imine nitrogen N(2) (or N(2A)) from ligand (L), and two oxygen atoms of different carboxyl groups from acetate ions, forming electrically neutral complex. This molecule was centrally symmetric, with the symcenter at the centre of CuN2O2. The bond angles of O(1)-Cu(1)-O(1)#1、O(1)-Cu(1)-N(2)、O(1)#1-Cu(1)-N(2)#1 and N(2)-Cu(1)-N(2)#1 are 88.38(14)° 、 90.23(10)° 、 90.23(10)° and 97.25(14)° ， respectively, all of which are close 90°. Thus, a slightly distorted quadrangle was formed around Cu(1) by N(1), N(2), O(1), and O(3). The composition of the complex
9
was [Cu(L)2(Ac)2]·3H2O(1). Fig. S1 showed that the hydrogen-bonding formed due to the presence of the nitrogen atoms and the oxygen atoms from the imide(L) and acetate ligands, and crystallization water molecules. The complex is rich in intramolecular and intermolecular hydrogen bonds, such as N(1)-H(1A)...O(1W)； O(3W)-H(3WA)...O(4)；O(2W)-H(2WA)...O(2). These hydrogen-bonding stabilized this crystal structure. In complex 2, Cu(II) ion was five-coordinated. Each Cu(II) coordinated with two azomethine nitrogen N(1) (or N(3)) from two bimz, two carboxylate oxygen atoms O2 and O3 in two different carboxylate groups, and one bridge oxygen atoms O1 from demethylcantharate, forming a distorted tetragonal pyramid structure. The composition of the complex was [Cu(bimz)2(DCA)](2). Fig. S1 showed that the hydrogen-bonding formed due to the presence of the nitrogen atom from the bimz and the oxygen atoms from demethylcantharate, such as N(2)-H(2A)...O(5)#1, N(4)-H(4A)...O(3)#2. Meanwhile, complexes 1 and 2 contain the benzimidazole group, resulting л-л stacking effects among the complexes. Therefore, we concluded that the synergistic effect, including π-π stacking and hydrogen-bonding interactions, existed between the complexes and biomacromolecule, which could be the fundamental cause of the biological activity change found in macromolecules [23].
PLEASE INSERT FIGURE 1 PLAESE INSTERT TABLE 2-3
10
3.2. DNA binding studies 3.2.1. Electronic absorption spectra The application of electronic absorption spectroscopy is one of the most useful techniques in DNA-binding studies [24]. Changes observed in the UV spectra upon titration can provide evidence for the intercalative interaction mode pattern, since hypochromism would occur from π-π stacking interactions [25]. To further investigate the possible binding modes and to obtain the binding constants (Kb) of complex to DNA, we also studied the effect of DNA titration to the title complexes by electronic absorption spectra at 298 K. Results are shown in Fig. 2 ((a): 1; (b): 2). The intrinsic binding constant (Kb) was determined by the equation: [DNA] / (εA εF) = [DNA] / (εB - εF) + 1 / [Kb (εB - εF)], where [DNA] was the concentration of DNA, εA, εF and εB corresponded to the apparent extinction coefficient, the extinction coefficient for the free compounds and its fully DNA-bound combination, respectively
[26]. Kb/(L·mol-1) values of the compounds were 5.52×103 (1), and 1.93×10 3 (2). The values suggested that the binding ability of complex 1 was more intense than complex 2, which agrees with the observation that complex 1 has a better planar aromatic structure result in more intermolecular interactions, including π-π stacking and hydrogen-bonding interactions. The complexes overlap the DNA base pairs via benzimidazole ring insertion, which leads to the hypochromicity of complexes absorption [27].
11
PLEASE INSERT FIGURE 2
3.2.2 Fluorescence quenching studies Complexes 1 and 2 showed an intense fluorescence emission around 248 nm (1) and 244 nm (2). The fluorescence emission of the complexes 1 and 2 are quenched in presence of DNA. The complexes showed strong emission bands at around 297 nm(1) and 294 nm (2), as shown in Fig. 3. According to the Stern-Volmer equation: F0/F = 1+ Ksv [Q], F0 and F represent the fluorescence intensities in the absence and presence of quencher, respectively [28], [Q] is the quencher concentration and Ksv is the Stern-Volmer constant, Ksv were calculated as 4.26× 103 mol·L-1(1) and 2.68 × 10 3 mol·L-1(2). The binding intensity of complex 1 was stronger than complex 2, this is consistent to the results found in the electronic absorption spectra. PLEASE INSERT FIGURE 3
3.2.3. Viscosity measurements To further study the binding mode of the compounds interacting with DNA, DNA viscosity at 25°C was investigated (Fig. 4). The experimental data showed that the relative viscosity of DNA steadily decreased after adding complexes and L, and it could increase after adding benzimidazole. But there was no significant viscosity change occurred after adding Na2DCA. The possible explanation is that the complexes and L were partially inserted to the DNA base pairs and resulting in a kink in the DNA helix, therefore decreased the DNA effective length [29]. Because of the
12
planar benzimidazole ring could also insert to the DNA base pair, and the steric hindrances of complexes were enhanced due to the non-planar structure of demethylcantharate (DCA). From fig. 4, the interactions of complex(1) with DNA is significantly stronger than complex(2). The result agrees with the electronic absorption spectra and fluorescence spectra conclusion. PLEASE INSERT FIGURE 4 3.2.4. Interaction with pDsRed2-C1 plasmid DNA The cleavage reaction on pDsRed2-C1 plasmid DNA can be monitored by agarose gel electrophoresis. When pDsRed2-C1 plasmid DNA is subjected to electrophoresis, different migration speeds were observed [30]. Relatively fast migrations were observed at the intact supercoil form (Form I). If scission occurs on one strand (nicking), the supercoil will partially relax to generate a slow moving open-circular form (Form II) [31]. Fig. 5 gave the electrophoretograms of the interaction of pDsRed2-C1 plasmid DNA with increasing concentrations of complexes. Complexes are capable of cleaving plasmid DNA when the concentration of complexes was greater than 500µM. When the concentration of complexes increased, the amount of Form I diminished gradually, and Form II increased. Comparing channel 4 to 6, the cleavage ability of complexes was enhanced by adding ascorbic acid. In order to investigate the reaction mechanism, dimethylsulfoxide (DMSO) was introduced to the experimental design. DMSO as a radical scavenger could inhibit the cleavage ability of complexes significantly in channel 5 and 7. With increasing
13
amount of ascorbic acid (Vc), Cu(II) complex was reduced to Cu(I) complex. The Cu(I) complex then reacts with dissolved oxygen generating ROS: superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH). Finally, the ROS attacks the plasmid DNA leading to the single and double DNA strand breaks. So the cleavage process was via hydroxyl radical mechanism [32]. PLEASE INSERT FIGURE 5
3.3 Interaction with BSA 3.3.1 Fluorescence spectra and quenching mechanism The results of title complexes quenching the BSA fluorescence were shown in Fig. 6. Complex 1 and 2 showed similar quenching patterns. It showed that the intensity of the fluorescence peak of BSA decreased with increasing concentration of complexes, which inferred that strong interactions existed between complexes and BSA [33].
PLEASE INSERT FIGURE 6
Fluorescence quenching can undergo two different mechanisms: static quenching and dynamic quenching. For dynamic quenching, the mechanism can be described by the Stern-Volmer equation: F0 / F = 1 + Kq τ0 [Q], where F0 and F are the fluorescence intensities of BSA in the absence and presence of the complexes, respectively [34], [Q] is the concentration of the complexes. For many proteins, τ0 is known to be approximately equal to 10-8s [35]. The calculated quenching rate constants Kq were
14
1.47×1015 L·mol-1·s-1(1) and 1.15×1015 L·mol-1·s-1(2). These values were much greater than the maximum possible value of diffusion-limited quenching in water. The result also suggested that the quenching mechanism of complexes to BSA was static quenching, which generated via intense interaction [36]. 3.3.2 Binding constants and binding sites Assuming there were n identical and independent binding sites in protein, the binding constant KA can be calculated using equation [37]: lg (F0 - F) / F = lg KA + n lg [Q]. The values of KA were 1.59×106L·mol-1 (1), 5.4×104 L·mol-1 (2), and 2.78×10 4 L·mol-1 (Na2DCA). The values of n were 0.88(1), 0.68(2) and 0.66 (Na2DCA). The results indicated that strong binding interaction existed between the complexes and BSA. The binding intensity of complexes was stronger than Na2DCA, and the binding site of complexes was one.
3.4 Antiproliferative activity evaluation As shown in Fig. 7, the antiproliferative activity of complex 1, complex 2, L and Na2DCA at the given concentration showed a dose-dependent manner against human hepatoma cells (SMMC-7721) in vitro. The inhibition ratios tested revealed that complex 1 and 2 had strong antiproliferative activities against human hepatoma cells (SMMC-7721) lines in vitro
compare to L and Na2DCA. The inhibition rates of complex(1) against SMMC-7721
lines (IC50=24.55±0.48 µmol·L-1) is much higher than that of L (IC50=116.63±2.66
µmol·L-1) [38]. The inhibition rates of complex(1) against SMMC-7721 lines is much
higher than complex(2) (IC50=41.82±3.90 µmol·L-1). The inhibition rates of two novel
15
complexes were higher than that of the transition metal complexes of demethylcantharate and thiazole derivatives [12, 13] against SMMC-7721 cells.
which suggests that various compositions and structures of complexes would lead to different antiproliferative activities, and this can be important in designing and
synthesizing novel anti-cancer drugs [39]. It is clear that the strong interaction found
between complexes and biomacromolecules (DNA or BSA ) is directly correlated to
the antiprolififerative activity of complexes. PLEASE INSERT FIGURE 7
4. Conclusions Two
novel
Cu(II)
complexes
[Cu(L)2(Ac)2]·3H2O(1)(
L=
N-2-methyl
benzimidazole demethylcantharate imide, C16H15N3O3, Ac = acetate, C2H3O2 ) and [Cu(bimz)2(DCA)](2)( bimz = benzimidazole, C7H6N2; DCA = demethylcantharate, C8H8O5) were synthesized and characterized. The crystal structure of complex 1 and 2 were determined by X-ray diffraction. These complexes had strong DNA and BSA
binding intensity and high inhibition rates against human hepatoma cells (SMMC-7721) in vitro. Complex(1) had intense antiproliferative activities against the human hepatoma cells(SMMC-7721) in vitro, which had the potential to develop as an anti-cancer drug in the future.
5. Acknowledgment We thank Institute of Zhejiang Academy of Medical Science for helping with
16
antiproliferative activity test.
Supplementary materials Crystallographic data for the structure reported in this article has been deposited with the Cambridge Crystallographic Data Center CCDC 909444(1), 918105(2). Copies of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB21EZ, UK ([email protected]). The packing diagrams of complexes were shown in Fig. S1. Hydrogen bond lengths and angles of complex 1, 2 were listed in Table S1, S2.
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[20] X. L. Zheng, H. X. Sun, X. L. Liu, Y. X. Chen, B. C. Qian, Acta Pharmacol. Sin. 25 (2004) 1090-1095. [21] G. M. Sheldrick, SHELXS-97, Program for the Solution of Crystal Structures, University of Göttingen, Germany, 1997. [22] G. M. Sheldrick, SHELXL-97, Program for the Refinement of Crystal Structures, University of Göttingen, Germany, 1997. [23] Z. Rehman, N. Muhammad, S. Shuja, S. Ali, I. S. Butler, A. Meetsma, M. Khan. Polyhedron , 28 (2009) 3439-3448. [24] Z. C. Liu, B. D. Wang, Z. Y. Yang, Y. Li, D. D. Qin, T. R. Li, Eur. J. Med. Chem. 44 (2009) 4477-4484. [25] C. S. Kalliopi, P. Franc, T. Iztok, P. K. Dimitris, P. George, J. Inorg. Biochem. 104 (2010) 740-745. [26] K. Paramasivam, S. Palanisamy, R. B. Rachel, H. C. Alan, S. P. B. Nattamai, D. Nallasamy. Dalton Trans. 41 (2012) 4423-4436. [27] S. Roy, S. Saha, R. Majumdar, R. R. Dighe, A. R. Chakravarty, Polyhedron 29 (2010) 2787-2794. [28] I. M. Khan, A. Ahmad, M. F. Ullah, Spectrochim. Acta A 102 (2013) 82-87. [29] J. Liu, T. X. Zhang, T. B. Lu, L. H. Qu, H. Zhou, Q. L. Zhang, L. N. Ji, J. Inorg. Biochem. 91 (2002) 269-276. [30] N. Wang, Y. Y. Wang, X. X. Wang, X. L. Zheng, D. M. Yan, Q. Y. Lin, Chinese J. Chem. 29 (2011) 473-477. [31] X. Q. He, Q. Y. Lin, R. D. Hu, X. H. Lu, Spectrochim. Acta A 68 (2007) 184-190.
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[32] C. A. Detmer III, F. V. Pamatong, J. R. Bocarsly, Inorg. Chem. 35 (1996) 6292-6298. [33] P. Sathyadevi, P. Krishnamoorthy, E. Jayanthi, R. R. Butorac, A. H. Cowley, N. Dharmaraj, Inorg. Chim. Acta 384 (2012) 83-96. [34] Q. Guo, L. Z. Li, J. F. Dong, H. Y. Liu, T. Xu, J. H. Li, Spectrochim. Acta A 106 (2013) 155-162. [35] X. W. Li, Y. T. Li, Z. Y. Wu, C. H. Yan, Inorg. Chim. Acta 390 (2012) 190-198. [36] Y. J. Wang, R. D. Hu, D. H. Jiang, P. H. Zhang, Q. Y. Lin, Y. Y. Wang, J. Fluoresc. 21 (2011) 813-823. [37] P. Sathyadevi, P. Krishnamoorthy, M. Alagesan, K. Thanigaimani, P. T. Muthiah, N. Dharmaraj, Polyhedron 31 (2012) 294-306. [38] Song W. J., Cheng J. P., Jiang D. H., Guo L., Cai M. F., Yang H. B., Lin Q. Y., Spectrochim. Acta A, 121(2014) 70-76. [39] F. Zhang, Q. Y. Lin, W. L. Hu, W. J. Song, S. T. Shen, P. Gui, Spectrochim. Acta A 110 (2013) 100–107.
Table captions Table 1. Crystal data of complex 1 and 2 Table 2. Selected bond lengths(Å) and angles(°) for complex 1 Table 3. Selected bond lengths(Å) and angles(°) for complex 2
Figure captions Fig. 1 Labeled ORTEP diagrams of complex 1(a) and 2(b) with 30% thermal probability ellipsoids shown. 20
Fig. 2 Absorption spectra of the complex 1(a) and 2(b) in the presence of increasing amount of DNA. [complex] = 3.00×10-6 mol· L-1 , from (1) to (5): (a). [DNA]×105 = 0, 0.74, 1.48, 2.24 and 2.98 mol· L-1, respectively. (b). [DNA]×105 = 0, 1.86, 3.72, 5.58 and 7.44 mol· L-1, respectively Fig. 3 Fluorescence spectra of the complex 1(a) and 2(b) in the absence and presence of increasing the amount of DNA; Insert in Fig. 3-5: fluorescence quenching curve of the complex by DNA. λex=248 nm (1), λex=244 nm (2), [complex]=2×10-5 mol· L-1; [DNA]/(10-4 mol· L-1), from 1 to 5: 0, 1.86, 3.72, 5.58, and 7.44, respectively. Fig. 4 Effect of increasing amounts of the compounds on the relative viscosity of DNA. [DNA] = 3.72×10-4 mol·L-1; [complex]/10-6 = 0, 0.67, 1.33, 2.00, 2.67 and 3.33 mol·L-1, respectively. Fig. 5 Electrophoretic separation of pDsRed2-C1 DNA induced by complexes 1(a) and 2(b) Lane 1: DNA alone; lane 2: DNA + complex (250 µ M); Lane 3: DNA + complex (500 µ M); lane 4: DNA + complex (750 µM); lane 5: DNA + complex (750 µ M) + DMSO (750µ M); lane 6: DNA + complex (750 µM) + Vc (750 µ M), lane 7: DNA + complex (750 µM) + Vc (750 µ M) + DMSO (750µM). [DNA] =3.0 µ g·mL-1. Fig. 6 Fluorescence spectra of BSA in the absence and the presence of complex 1(a) and 2(b) Inset: Stern-Volmer plots of the fluorescence titration data of the complexes. [BSA] = 4.98×10-7 mol· L-1; [complex]×109 = 0, 6.67, 13.3, 20.0, and 26.7 mol· L-1, from (1) to (5), respectively (a): complex 1, (b): complex 2. Fig. 7 Inhibition effects of compounds on SMMC-7721 cell growth. Data represent mean + S.D. and all assays were performed in triplicate for three independent experiments.
21
Fig. 1
22
Fig. 2
23
Fig. 3
24
Fig. 4
25
Fig. 5
26
Fig. 6
27
Fig. 7
28
Table 1 Crystal data of complex 1 and 2 Complex
1
2
CuC36H42N6O13
CuC22H20N4 O5
Formula weight
794.28
483.97
Crystal system
Monoclinic
orthorhombic
Space group
C2/C
Pna21
a(Å)
21.572 (4)
15.8667(3)
b(Å)
11.3687 (19)
9.9975(2)
c(Å)
17.371 (4)
12.5228(2)
α(°)
90.00
90.00
β(°)
111.528 (18)
90.00
γ(°)
90.00
90.00
Volume(Å3)
3963.0 (13)
1986.46(6)
Z
4
4
Crystal size (mm)
0.345×0.279×0.214
0.345×0.287×0.156
Shape
block
block
Colour
blue
blue
Dc (g/cm-3)
1.331
1.618
θ Rang for data collection(°)
2.03 to 27.86
2.41to 27.57
Reflections collected/Unique
6486 / 3106
19898 / 4472
0.1288
0.0844
0.632
1.145
F(000)
1672
996
R/wR [I>2σ(I)]
0.0514 / 0.1291
0.0791 / 0.1953
Restraints/parameters
9 / 285
1/ 289
Goodness-of-fit on F2
0.939
1.160
Largest diff. Peak and hole(e/Å-3)
0.459, -0.663
1.131, -1.887
Chemical formula
R(int) Absorption coefficient(mm-1)
29
Table 2. Selected bond lengths(Å) and angles(°) for complex 1 Bond
(Å)
Bond
(Å)
Cu(1)-O(1)
1.977(2)
Cu(1)-N(2)
1.991(2)
Cu(1)-O(1)#1
1.977(2)
Cu(1)-N(2)#1
1.991(2)
Angle
(°)
Angle
(°)
O(1)-Cu(1)- O(1)#1
88.38(14)
O(1)#1-Cu(1)-N(2)
160.71(8)
O(1)-Cu(1)-N(2)
90.23(10)
O(1)#1-Cu(1)-N(2)#1
90.23(10)
O(1)-Cu(1)-N(2)#1
160.71(8)
N(2)-Cu(1)-N(2)#1
97.25(14)
Symmetry transformations used to generate equivalent atoms:
30
#1 -x, y, -z+1/2
Table 3. Selected bond lengths(Å) and angles(°) for complex 2 Bond
(Å)
Bond
(Å)
Cu(1)-O(3)
1.983(7)
Cu(1)-N(3)
1.990(7)
Cu(1)-O(2)
1.942(6)
Cu(1)-N(1)
2.023(8)
Cu(1)-O(1)
2.256(6)
Angle
(°)
Angle
(°)
O(3) -Cu(1)-O (2)
88.1(3)
O(2)-Cu(1)-N(3)
167.0(3)
O(3)-Cu(1)-O(1)
91.7(2)
O(2)-Cu(1)-N(1)
88.1(3)
O(3)-Cu(1)-N(3)
90.3(3)
O(1)-Cu(1)- N(3)
98.3(3)
O(3)-Cu(1)-N(1)
174.2(3)
O(1)-Cu(1)-N(1)
93.1(3)
O(2)-Cu(1)-O(1)
94.7(2)
N(3)-Cu(1)-N(1)
92.3(3)
31
Two novel complexes demethylcantharate
[Cu(L)2(Ac)2]·3H2O(1) (L= N-2-methyl benzimidazole
imide,
C15H13N2O3,
Ac
=
acetate,
C2H3O2)
and
[Cu(bimz)2(DCA)](2) (bimz = benzimidazole, C7H6N2; DCA = demethylcantharate, C8H8O5) were synthesized and characterized. The DNA-binding properties of complexes were investigated by electronic absorption spectra, fluorescence spectra, viscosity measurements and agarose gel electrophoresis. The interaction between the complexes and bovine serum albumin (BSA) was investigated by fluorescence spectra. The antiproliferative activities of the complexes against human hepatoma cells (SMMC7721) were tested in vitro.
32
►two novel Cu(II) complexes with norcantharidin derivatives have been synthesized.
►Complexes structure was determined by X-ray diffraction. ►The complexes and ligands bound DNA moderately via partial intercalation modes. ►Complexes could cleave plasmid DNA via hydroxyl radical mechanism.
►Complex(1) has strongest activity against human hepatoma cells.
33
