Studies on Some New Ru(III) Complexes Using aryl-azo Pentane- 2,4-dione and 2,6-bis (2'-Benzimidazolyl) Pyridine as Ligands: Synthesis, Spectroscopic, Luminescent, Electrochemical and Biological Activities

Some ruthenium(III) complexes with aryl-azo 2,4-pentanedione as co-ligands (L1H - L3H2) have been synthesized and characterized spectroscopically IR, 1H NMR, UV/Vis, ESR, conductimetric) along with elemental analysis and FAB-mass data. Their luminescent and redox properties have been studied. The antibacterial, anti-HIV and antitmnour activities have also been reported.

Thus, in order to avoid isomeric complexities, we selected a terpyridyl type tridentate ligand vz.
2,6-bis(2'-benzimidazolyl)pyridine. Thes selectivity of the benzimidazolyl terpyridyl ligand was also based on its involvement in the strong intramoleeular stacking interactions between the DNA strands hence in bioactivities [7] ofthe resulting complexes. In this context it is also of worth to mention that azo/hydrazo compounds have also been subjected [8] to many biological reactions such as in protein synthesis inhibition, nitrogen fixation and antitumour properties which have been understood in their action as DNA erosslinking agents. Additionally, ruthenium(Ill) chloro complexes have been reported [9] to bind covalently to calfthymus DNA. In this context reports [10][11] by Keppler and Sava et al. on the tumour inhibiting properties of ruthenium(Ill) ehloro complexes have been found very encouraging.

Materials and Methods
All the solvents purchased from E. Merck were distilled using standard procedure prior to use.
Neutral alumina for column chromatography was supplied by E. Merck and used as such. All the reactions were carried out under N2 atmosphere. Ru(llI) Complexes of Aryl-Azo Pentane-2, 4-Dione and 2,6-Bis(2 '-Benzimidazolyl)-pyridine Microanalysis (C, H and N) performed on a Carlo Erba Elemental Analyzer 1105 and FAB-mass _data using a JEOL SX-102 mass spectrometer were carried out at the Central Drug Research Institute Lucknow, India. IR (KBr p_ellets) and LW/Vis data were obtained using a JASCO FT IR 5300 spectrometer and a Shimadzu UV-1601 speetrophotometer whereas ESR spectra in the solid state_ as well as in solution (DMSO) at room temperature and liquid N2 temperature were recorded at the Indian Institute of Technology, Mumbai, India. Eleetroefiemical measurements were made using an electrochemical interface SI1287 potentios.tate, using a graphite disc as working electrode, platinum wire as auxiliary electrode and Ag/Ag as reference electrode in a three electrode configuration. Luminescent spectra were reetrdd at the University of Tokyo, Japan using a Shimadzu RF 5300 speetrophotometer at 25C. The antibacterial study was carried out at the School of Biotechnology, B.H.U., Varanasi, India whereas antitumour and anti-HIV activities were evaluated at the School-bf Pharmacy, University ofNorth Carolina, USA.

Results and Discussion
The composition of the complexes assigned on the bases of their elemental (C, H and N) analysis and FAB-mass data is shown in Table I. The complexes were found thermally stable at room temperature. Complexes 1, 4 and 5 were soluble in acetone, acetomtrile, DMF and DMSO whereas 2 and 3 were soluble only in DMF and DMSO. The molar conductance of the complexes shown in Table I is in consistence with the number of counteranions present in the complexes 12,17]. IR Spectra: In the IR spectra (KBr) of the complexes, peaks observed at 1620 1640 cm due to vC=O were found to be lower as compared to free ligands values observed at 1674-1687 cm . This indicated the participation of both the >C=O groups in the bonding with ruthenium. However in complexes 3, 4 strong peak observed at 1102-1108 cmwas assigned to S-coordinated DMSO in view of an earlier report [18]. A strong and sharp peak observed at 839-840 cm in the spectra of thHe complexes 4 and 5 was assigned to v(PF6").
NMR spec,tra: To get further structurdl support from H NMR spectra, one representative complex [Ru(L'H)3] 3C1 was reduced into the Ru(II) form in the presence of N-ethyl morpholine using a reported procedure [19]. The H NMR spectnnn recorded in DMSO-de showed two peaks at 8 2.6 and 2.9 ppm due to methyl protons, a complex pattern at 7-8 ppm due to phenyl protons and a singlet at b 14.20 ppm due to the NH proton.   Table II. The data reported in Table II show mainly three transitions in the range 265-272; 334-423 and 540-604 nm. The former two transitions in the higher energy regions wcrc considered to be ligand centered (LC) and latter transition in the range 540-604 nm was assigned to ligand (*) to metal (t2sg) transition in view of earlier reports [4,5] on ruthcnium(III) systems. Substitution of chloro groups in the complex 2 by 2,6-bis(2'-bcnzimidazolyl) pyridinc lowered the LMCT transition wavelength by 10 nm which is also consistent with the earlier report [20]. Almost double intensity of the peak observed for complex 5 as compared to complex 4 could bc considered duc to the dinuclcar nature [21]  1.80-2.50 wcrc found to lic in the range as reported earlier [22,23]. All the peaks were observed with equal spacing and equal intensity. However for the complexes 1 and 3 a broad spccmnn was observed in the solid state at 298 K and 77 K temperatures but in solution (DMSO) they showed thrcc signals and g values, again calculated to bc in the range 1.9-2.5.
Thus on the basis of spectroscopic data (IR, 1H NMR, UV/Vis, ESR, conductimctric) alongwith elemental analysis and FAB mass data, the proposed structures for the complexes arc shown in  (10-M) in the potential range +2V using Ag/Ag / as reference and graphite disc as working and Ag/Ag* as rcfcrcncc clcctrodcs. Rcdoxpotential (E) data arc reported in Table III. Oxidation Cyclic-voltammogram of the mononuclear complex showed two irreversible oxidations at +0.82 and +1.15 V. Since free ligand also showed an oxidation peak at /1.00 V so distinction between ligand-based and metal-based oxidation was difficult. However in view of earlier report [24] the latter peak at 1.15 V could be considered to arise from Ru(III) --, Ru(IV) oxidation further more dinuclear complex [RuEL H2C16(DMSO)2] showed two oxidation peaks at +1.40 and 1.20 V which could arise due to subsequent oxidation oftwo ruthenium centres.
Other complexes showed only one broad oxidation peak indicating that ligand based oxidation is overlapping with the metal-based oxidation. Reductions The complexes 1 3 showed two reduction peaks in the range -0.72 to -0.86V and -1.37 to 1.51 V whereas complexes 4 and 5 showed three reduction peaks in the range -0.72 to -0.79, -1.44 to 1.46V and-1.80 to-1.79V which were consistent with earlier report [25]. Room temperature emission spectra Emission properties of the complexes have been studied in DMF solution (10 5 M) at room temperature and the data are presented in Table II. The complexes 1 3 after excitation at 330 nm emitted at 460, 436 and 555 nm respectively indicating that the dinuclear complex 3 emitted at lower energy than the complexes 1 and 2. The complexes 4 and 5 showed two emissions at 370, 719 and 375, 725 nm respectively. In view of an earlier report [26] two emissions in the range 370 Ru(III) Complexes of Aryl-Azo Pentane-2,4-Dione and 2,6-Bis(2 '-Benz midazolyl)-pyridine 375 and 719-720 rim were considered to arise probably from bridging ligand to metal and terminal ligand to metal charge-transfer. The better luminescence observed for the mononuclear complex 4 as compared to dinuclear complex 5 was also found to be consistent with the earlier report [27].

Antibacterial studies
The antibacterial activity of the ruthenium (II,I) complexes was evaluated against Pseudomonas aeruginosa and E. coli in DMSO solution (10"M) using the susceptibility testing method [28] as reported earlier [12]. Inhibitory effects by the free ligands (LH-L3H2) against Pseudomonas aerugmosa have been discussed earlier [12]  The antitumour and anti-HIV activities were evaluated using standard procedures [29,30]. The cytotoxicity data of free ligands (L,H L3H2) alongwith their ruthenium complexes are shown in Table V. Among the free ligands (L'H LaH2),_the activity trend was observed to be L3H2 > LiH > L2H2 indicating that the dinueleating ligand LH2 was most active as compared to L2H2 against both tumour cells viz. A549 and U87-MG. However, the antitumour activity of ruthenium (III) complexes 1-5 was found to be significant as compared to their free ligands. The highest activity was shown by complex 3 which contains the most active ligand (L3H2) against both the turnout cells. The mononuclear complex I showed better activity as compared to dinuclear complex 2. The better activity of complex 4 as compared to complex 1 against U87-MG was considered in terms of the presence of the bio-active benzimidazolyl group. The complex 5 did not show activity against any of both cells however it had a significant effect on the growth of only the glioblastoma cell line (clumping behaviour). A detailed mechanism ofthese activities is yet to be explored.
The anti-HIV activities (Table V) trend of the free ligands were again in the sequence L3H2 > LH > L2H2 indicating that the ligand L3H2 was the most active. However ruthenium (III)complexes showed a better activity as compared to the free ligands. The activity of these compounds was compared with the standard AZT (azido-thymidine) treated as control under the similar experimental conditions. NA Not active inhibition < or 5% at 20 tg/mL. *The sample was not active but had a significant unique impact on growth behaviour of U87-MG cells at 20 and 10 lag/mL. AZT Azido-thymidme