In Vitro and in Vivo Anticancer Activity of Copper Bis

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In vitro and In vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes Duraippandi Palanimuthu, Sridevi Shinde, Kumaravel Somasundaram, and Ashoka Gnanadoss Samuelson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300938r • Publication Date (Web): 15 Jan 2013 Downloaded from http://pubs.acs.org on January 17, 2013

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Journal of Medicinal Chemistry

In vitro and In vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes Duraippandi Palanimuthu,† Sridevi Vijay Shinde,‡ Kumaravel Somasundaram,*‡ and Ashoka G. Samuelson*†

†

Duraippandi Palanimuthu and Prof. A. G. Samuelson, Department of Inorganic and Physical

Chemistry, Indian Institute of Science, Bangalore, India 560012, Fax: (+91) 80-2360-1552. e-mail: [email protected]

‡

Sridevi Vijay Shinde and Prof. Kumaravel Somasundaram, Department of Microbiology and

Cell Biology, Indian Institute of Science, Bangalore, India 560012, Fax: (+91) 80-23602697. e-mail: [email protected] ABSTRACT Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl3-thiosemicarbazone) (GTSCH2) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle 1 ACS Paragon Plus Environment


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analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. Complex Cu(GTSCHCl) caused distinct DNA cleavage and Topo IIα inhibition unlike Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice. INTRODUCTION The discovery of cisplatin ushered in an era of cancer therapy that included inorganic metal complexes extensively.1 Though cisplatin is widely used as an anticancer drug to cure many cancers, severe side effects and acquired resistance due to prolonged treatment have spurred investigators to find alternatives for circumventing drug resistance.2 With this aim, several classes of metal complexes have been synthesized using various ligands and metal ions, and their anticancer activity has been successfully evaluated both in vitro and in vivo.3–8 Transition metal based thiosemicarbazone complexes are one such class of complexes that have been investigated intensely over five decades.9–13 Among them, copper bis(thiosemicarbazone) complexes have attracted more attention, because many of them displayed promising anticancer activity.14–20 The mono(thiosemicarbazone) complexes of copper containing one or two equivalents of the ligand are well understood, and they manifest cytotoxicity mainly through ROS generation.21–23 However, the mechanism of action of copper bis(thiosemicarbazone) complexes is less studied though significant progress has been achieved in elucidating the mechanistic pathway of copper biacetyl-bis(4-methyl-3-thiosemicarbazone) [Cu(ATSM)], copper 2-ethoxy2-ketobutyraldehyde-bis(thiosemicarbazone) [Cu(KTS)] and copper glyoxal-bis(4-methyl-3thiosemicarbazone) [Cu(GTSM)].24–26 These complexes exhibit their anticancer activity by

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activating multiple pathways such as inhibition of DNA and RNA synthesis and disruption of ATP production and surprisingly none of them function through ROS generation. More recently, some copper bis(thiosemicarbazone) complexes have shown selectivity to cells having low levels of oxygen compared to normal cells.27–29 A good example is Cu(ATSM) which selectively accumulates in hypoxic cancer cells.29 This might be attributed at least partly, to irreversible reduction of Cu(II) to Cu(I) and subsequent trapping of copper within the cells. Whereas in normal cells, Cu(I) is likely to be re-oxidized to Cu(II), because of the high oxygen tension present in these cells, and suffer expulsion from the cell. Short synthesis times have enabled synthesis of radiolabeled copper complexes that are theranostic, permitting diagnosis and therapy simultaneously.25 Thus radiolabeled

64

Cu(ATSM) has been used as a radiotherapy

agent against hamsters bearing human GW39 colon cancer tumors, significantly increasing their survival time (6 fold compared to controls).30 Besides its therapeutic utility, it has also shown promise as a positron emission tomography-computed tomography (PET/CT) agent to diagnose the progress of cervical carcinoma, and has successfully cleared clinical human trial Phase II recently.31,32 Following the successful story of

64

Cu(ATSM), a number of copper

bis(thiosemicarbazone) complexes with different radiolabeled 60Cu, 61Cu, 62Cu and 64Cu nuclides have been tested as PET/CT imaging agents.33–37 Recently, we have shown that the ligand bis(thiosemicarbazone) of glyoxal (GTSCH2) can be used for live cell imaging in conjunction with zinc.38 We have now directed our attention towards the corresponding copper complexes and their anticancer activity. A series of new copper

bis(thiosemicarbazone)

complexes

derived

from

biacetyl-bis(4-pyrrolidinyl-3-

thiosemicarbazone) (ATSCH2), benzil-bis(4-pyrrolidinyl-3-thiosemicarbazone) (BTSCH2) and glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH2) ligands have been synthesized



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and characterized. Their cytotoxicity was estimated in different human cancer cell lines, where the copper complexes derived from GTSCH2 were found to be most cytotoxic in all the cell lines tested. Further, we have studied their cellular accumulation and distribution in HCT116 cells and observed significant accumulation of copper in cytoplasm when compared to nucleus in 1 h. Tritiated thymidine incorporation assay in HCT116 cells with Cu(GTSC) and Cu(GTSCHCl) showed reduction in DNA synthesis. Cell cycle analyses results further suggest that these complexes induce significant apoptosis. Biophysical studies like DNA interaction, DNA cleavage and Topo IIα inhibition throw light on the mechanistic aspects of their anticancer activity. In vivo cytotoxicity studies in mice bearing colon cancer (HCT116) showed impaired tumor formation in response to Cu(GTSC) treatment. RESULTS AND DISCUSSION Synthesis and Characterization The bis(thiosemicarbazone) ligands and their copper complexes were synthesized using the previously reported procedure with minor modifications (Figure 1).39–42 The ligands were synthesized by refluxing thiosemicarbazide and a 1,2-diketone in 2:1 mole ratio with a few drops of concentrated sulphuric acid (Scheme S1, Supporting Information). The ligands were characterized by multinuclear (1H and

13

C) NMR spectroscopy. The copper complexes, in both

neutral and cationic forms, were prepared by refluxing one equivalent of the ligand with the appropriate copper salts (copper(II) perchlorate salt was used for cationic complex preparation and copper(II) chloride or copper(II) acetate was used for the preparation of the neutral complexes) in ethanol or in a mixture of ethanol-chloroform (1:1) for 4 h. The resulting copper complexes were characterized by spectroscopic techniques like UV–visible and IR spectroscopy, ESI-MS and elemental analysis. In addition, the complexes were subjected to cyclic 4 ACS Paragon Plus Environment

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voltammetry, molar conductivity and when single crystals were available, through X-ray crystallography.

Figure 1. Proposed chemical structures of bis(thiosemicarbazone) ligands and their copper complexes.



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Figure 2. ORTEP representations of A) Cu(ATSC), B) Cu(BTSC) and C) Cu(GTSC) complexes. Thermal ellipsoids are shown at the 40% probability level and solvent molecules, if any, are omitted for clarity.


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The electronic absorption and infrared spectra of these compounds are consistent with the N2S2 coordination proposed with a possible coordination from a chloride or the counter ion as in the case of perchlorate. The presence of the perchlorate counter ion could be deduced from the molar conductance in DMF (1 mM). Thus, complexes Cu(ATSCH)ClO4 and Cu(BTSCH)ClO4 showed molar conductances of 62 and 66 S m2 M-1 respectively, indicating that both complexes were

1:1

electrolytes.

For

comparison,

the

well

known

complex

bis(1,10-

phenanthroline)copper(II) nitrate {[Cu(phen)2](NO3)2}, a 1:2 electrolyte, was shown to have a molar conductance of 115 S m2 M-1 under the same conditions. In contrast, Cu(ATSC), Cu(BTSC), Cu(GTSC) and Cu(GTSCHCl) show molar conductances of 3, 18, 6 and 13 S m2 M-1 respectively, confirming the neutral nature of the complexes. The molecular structures of the three complexes Cu(ATSC), Cu(BTSC) and Cu(GTSC) are depicted in Figure 2, and their crystallographic data given as supplementary information in Table S1. In all structures, the Cu(II) centre adopted a distorted square planar geometry by coordinating to two sulphur and two nitrogen atoms. The ligand is present in a syn-conformation at the diimine core and wraps itself around the metal center. In the case of Cu(GTSC), the structure is quite different from the zinc complex characterized earlier which exhibited a trimeric structure, [Zn(GTSC)]3, and the ligand has an anti-conformation at the diimine.34 The C-S bond length in all neutral complexes is 1.75 Å which clearly indicates thiolate coordination.43 The reduction potential of Cu(II) in copper bis(thiosemicarbazone) complexes is an important parameter that modulates their cellular retention and hypoxia selectivity.40 Recently, Dearling and Fujibayashi illustrated through in vitro and in vivo experiments that complexes which are difficult to reduce, with an E1/2 that is lower than –0.50 V can show hypoxia selectivity whereas the easily reducible complexes do not show such selectivity.28,35 We have 7 ACS Paragon Plus Environment


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measured the E1/2 using cyclic voltammetry for the newly synthesized complexes in dry DMF vs SCE and the results are given in Table 1. Representative cyclic voltammogram for Cu(GTSC) complex (5 mM) is shown in the supplementary data Figure S1. These half-wave potentials (E1/2) were assignable to the Cu(II)/Cu(I) redox couple. It is known that small modifications on the diketone backbone changes the redox potential to a large extent.29 We also find that the substitution of pyrrolidine moiety on the N-terminus of thiosemicarbazide, as in Cu(ATSC) or Cu(ATSCH)ClO4 results in a reduction potential close to that of Cu(ATSM). On the other hand, the replacement of electron donating methyl groups in Cu(ATSM) with phenyl groups in Cu(BTSC) and Cu(BTSCH)ClO4 or hydrogens in Cu(GTSC) on the 1,2-diketone backbone of bis(thiosemicarbazone), markedly increases the reduction potential to –0.34, –0.36 and –0.18 V, respectively. Thus complexes Cu(ATSC) and Cu(ATSCH)ClO4 are likely to be hypoxia selective as they have a large negative redox potential, but they do not exhibit significant cytotoxicity like Cu(GTSC) vide infra making them less interesting for therapeutic applications. The five coordinated complex Cu(GTSCHCl) is unique as it is cytotoxic vide infra and at the same time, showed a completely irreversible reduction potential at –0.66 V which is conducive to hypoxia selectivity. Table 1. Cyclic voltammetry data for copper bis(thiosemicarbazone) complexes. S. No Complex E1/2 (V)a ∆Ep (mV)b  ipa / ipc c 1 Cu(ATSM) –0.58 91 0.99 2 Cu(ATSC) –0.53 90 1.03 3 Cu(ATSCH)ClO4 –0.54 94 1.29 4 Cu(BTSC) –0.34 73 1.02 5 Cu(BTSCH)ClO4 –0.36 95 1.06 6 Cu(GTSC) –0.18 93 0.99 d 7 Cu(GTSCHCl) – – – All reduction potentials are w.r.t SCE and 10 mVs-1 scan rate was used for all the measurements. a

Half-wave reduction potential is given by E1/2 = (Epa+Epc)/2. 8 ACS Paragon Plus Environment

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b

The difference in potential is given by ∆Ep = Epa–Epc, where Epa and Epc are the anodic and

cathodic peak potentials, respectively. c

Ratio of anodic peak current over the cathodic peak current.

d

It showed an irreversible reduction peak at –0.66 V with a ∆Ep of 330 mV and  ipa / ipc of

0.30. In vitro Cytotoxicity The cytotoxic activity of the bis(thiosemicarbazone) ligands and their copper complexes against various human cancer cell lines such as SiHa (cervical cancer), MCF-7 (breast cancer), PC-3 (prostate cancer), A-2780 (ovarian cancer) and HepG2 (hepatocellular liver cancer) were evaluated. For comparison, the corresponding zinc complexes were synthesized according to the literature procedures and their cytotoxicity evaluated.38,44 Cultured cancer cells were treated with compounds with different concentrations for 48 h, and the percentage of cell growth inhibition was determined by sulforhodamine-B (SRB) assay.45 Adriamycin was used as a positive control. The GI50 values are given in Table 2. The copper complexes of BTSCH2 were found to be inactive (> 100 µM) in almost all the cell lines tested, except the complex Cu(BTSCH)ClO4 which is highly toxic (2.36 µM) to HepG2 cells. Complexes of ATSCH2 having a low redox potential (E1/2 ~ – 0.54 V) showed anticancer activity against certain cell lines: Cu(ATSC) in MCF-7 (2.60 µM) and Cu(ATSCH)ClO4 in MCF-7 (2.50 µM), A-2780 (0.12 µM) and HepG2 (100 >100 >100 >100 Cu(BTSC) >100 >100 >100 >100 Cu(BTSCH)ClO4 >100 >100 >100 >100 Zn(BTSC) >100 >100 >100 >100 ATSCH2 >100 –b >100 >100 Cu(ATSC) >100 2.60 >100 >100 Cu(ATSCH)ClO4 >100 2.50 >100 0.12 Zn(ATSCHCl) >100 >100 >100 0.13 GTSCH2 51.30 >100 80.40 100 2.27 >100 >100 Adriamycind 0.15

In Vitro and in Vivo Anticancer Activity of Copper Bis

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