Synthesis, Characterization and Antitumor Activity of a Series of Polypyridyl Complexes

A series of polypyridyl complexes have been synthesized. All polypyridyl complexes and some of the soluble ligands have been assayed for antitumor activity in vitro against the HL-60 (the human leucocytoma) cells, BEL-7402 (the human liver carcinoma) cells, KB (the human nasopharyngeal carcinoma) cells and HELA (the human adenocarcinoma of cervix) cells. The results indicate that several complexes have relative activity against different cell lines. Especially, the complexes [Co(bpy)2(pip)]3+, [Co(phen)2(pip)]3+, [Ru(bpy)2(pztp)]2+ and [Ru(pztp)2(bpy)]2+ show relative high activity against four tumor cell lines. Moreover, they are slightly more effective than cisplatin. At the concentration of 100 μg/mL, the complexes show inhibitory rate of 72∼86% for the cancer cells and have no toxicity for MDCK and Vero cells. It is indicated that these complexes can inhibit cancer cells selectively.


Introduction
Currently, cis-diaminedichlorplatinum(II) (cisplatin) is one of the three most widely used anlttumor drugs in the world. It is highly effective in treating testicular and ovarian cancers'. Despite its success, cisplatin has several disadvantages that include severe toxicity such as nephrotoxicity, neurotoxicity, and emetogensis. The toxic side effects of cisplatin limit the dose that can be given to patients. Another platinum-based antitumor_ drug, diamine[1,1-cyclobutanedicaboxylato(2-)]-platinum(II) (carboplatin), received worldwide approval and achieved routine clinical use. Carboplatin is less toxic than cisplatin and can be given at a much higher dose than cisplatin. Unfortunately, carboplatin is still only }ctive n the same range of tumors as cisplatin and is still administered intravenously. Thus, it is necessary to develop a new class of anticancer agents.
Some metal comple3s containing polypyridyl ligands are known to be bound with DNA by intercalation. However, httle attention has.been paid to the anticancer activity of polypyridyl metal complexes and a systematic investigation on the anticancer activity and cytotoxcity of polypyridyl complexes is rare. In fact s,oe metal polypyridyl complexes display remarkable antibacterial and antitumor activity' In the present paper, we report for the fist time the cytotoxic activity of a series of Ru(II) / Co(III) based polypyridyl complexes. It was expected to find a new type of potential antitumor agent from these complexes. For experiments the complexes were dissolved in freshly prepared in 10 % DMSO, and diluted to the required concentration with culture when used. The stock solutions in distilled water were stored at +4C. The solutions in growth medium used in the experiments were prepared extempore. Cisp,atin was used as a reference compound and its solutions were prepared as described above-.

Cells and tumor cells
The four tumor cells strains HL-60 (the human leucocytoma) cell line, BEL-7402 (the human liver carcinoma) cell line, KB (the human nasopharyngeal carcinoma) cell line, and HELA (the human adenocarcinoma of cervix) cell line were provided by the National Key Laboratory of Natural and Bionic Drugs, Beijing Medical University.
Madin-Darby Canine Kidney MDCK cells and Vero cells were obtained from the Chinese Academy of Military Medical Sciences.
The cells and tumor cells were cultured at 37C as monolayers in RPMI-1640 medium (Flow Laboratories, USA) supplemented with antibiotics (penicillin and streptomycin) and 10% bovine serum. Serum concen'ation was reduced to 5% for growth of cells and tumor cells and for testing the complexes ".
Cytotoxicity assay of the compounds The compounds were dissolved in RPMI-1640 containing 2% Calf serum, filtered to remove germs, and diluted with cell maintenance medium to obtain the solution with different concentrations. Having formed a single layer, MDCK and Vero cells were digested b' pancreatin and counted, then made into a 2x10 cells/ml suspension and inoculated n the wells of flat-bottomed 96-well plastic culture tray (0.1 ml/well), thereafter incubated at 37C in a thermotank containing 5% CO2 for 24 h. After the cells had formed single layers, the original culture medium was removed and replaced with maintenance medium containing various concentrations of the compounds. After further incubatj)n for 5-7 days, the number of the living cells was calculated by MTT staining method The maximum nontoxic concentration (TC0) and the 50% toxic concentration (TC0) were also calculated.
Antitumor activity assay of the compounds The complexes and cisplatin were assayed for cytotoxicity in vitro against The four tumor cells strains HL-60 (the human leucocytoma) cell line, BEL-7402 (the human liver carcinoma) cell line, KB (the human nasopharyngeal carcinoma) cell line, and HELA (the human adenocarcinoma of cervix) cell line. The procedure for antitumor activity studies was similar to that reported earlier2'24. Briefly, in order to calculate the concentration of each rug that produces a 50% inhibition of cell growth (IC0), 100 1 of cell suspension (4x10 cell/ml) were exposed to various concentrations of complexes dissolved in sterile water. After incubation periods of 72 h for all cell lines, the cell concentrations were determined both in control and in drug-treated cultures. All experiments were made in quadruplicate.
The results show that several complexes have relative activity against different cell lines.

Results
All complexes are characterized by elemental analyses, UV-Vis spectroscopy and NMR spectroscopy and are in accordance with their proposed formula.
Cytotoxicity and antitumor activity of the complexes Microscopically according to the survival rates, the maximum nontoxic concentration (TCo) and the 50% toxic concentration (TC0) of the complexes can be calculated by Reed-Muench method, and the results are listed in Table 1.
Microscopically the MNC for complexes was determined as 300 490 tmol / L for MDCK cells and 340 470 lamol / L for Vero cells (Table 1). Additional data were found when the viability of cells was studied. The number of viable MDCK cells and Vero cultured in complexes medium was increased during the whole period of investigation as compared to that of untreated control. Moreover, the viability was significantly increased when the concentration of ACV was decreased. This is well manifested after 48 and 72 h.
In contrast, all complexes tested were toxic low for MDCK and Vero cell.
Polypyridyl complexes have been tested against four tumor cell lines. They were exposed to cells for 72 h and growth inhibition was assessed usinu the sulforhodamine B protein staining assay2. The corresponding 50% inhibitor dose (Iff0) values are shown in For instance,. [Ru(bpy)z(pp]zt ) s. hows much more hig.her activity gaainst all of the four cell lines than [Ru(phen)z(pztp)] 2 an spte of the smlarty of their structures. The results indicate clearly that the antitumor mechanisms of these complexes should be different and the central metal ions, the ligands and the shape of the complexes should play a role in the mechanism. Moreover, since all of these polypyridyl complexes bind to DNA noncovalently, the antitumor mechanisms should also be different from that of cisplatin. Recently published data also show that the inhibition of antitumor activity by polypyridyl ruthenium or cobalt was enhanced by reducing agents and that the mechanism of the activation is similar to that for polypyridyl complexes mediated   54.6* 32.5* 92.6* >100" >100" 92* >100" >100" 6.7** 12.6"* 6.9** 12.3"* >100" >100" 9.8** 6.5** 12.3"* 24.5** 65.4* 9.6** 14.6"* 6.8** 98.6* >100' >100" >100"