Synthesis , Characterization and In-Vitro cytotoxic Studies of 5 , 10 , 15 , 20 Tetra Pyridyl Porphyrin Coordinated to Four [ Ru ( bipy ) 2 Cl ] + Groups

Ruthenium possesses several favorable properties suited to rational anticancer drug design when conjugation with the porphyrin moiety was accomplished through peripheral pyridyl rings. The ruthenium porphyrin conjugates are soluble at least moderately in aqueous solution and are thus suitable for biological investigations in particular for cytotoxicity and photocyotoxicity tests. In present study the compound 5,10,15,20 tetra pyridyl porphyrin coordinated to four [Ru (bipy)2 Cl]+ groups (meso-5,10,15,20 tetrakis {4(chloro-bis-bipyridyl ruthenium(II)) pyridyl} porphyrin) is synthesized by modified Alder method. This compound is characterized by UV-Visible Spectroscopy, FT-IR Spectroscopy, 1H-NMR spectroscopy, Fluorescence Spectroscopy and Cyclic Voltametry. In-Vitro anticancer activity of the compounds have been evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay method. The results show that the compound is cytotoxic against human lymphoma cancer cells.

when coordinated to metal porphyrin complexes or to other metal complexes, unique supramolecular assemblies with multi electron redox and photochemical activities 8 .The transition metal centers are biologically active.It was reported that tetra ruthenated porphyrin can bind to DNA with high affinity .The organometallic fragments are modulate the biological properties of porphyrin complexes.The addition of the organometallic fragments increases the hydrophilicity of the highly hydrophobic porphyrin ligand [9][10][11][12][13][14]

Synthesis of 5,10,15,20 tetra pyridyl porphyrin (TPyP)
Synthesis of TPyP was done according to literature data 15 : A mixture of 9.41 ml (100 mmol) of 4-pyridinecarboxaldehyde and 13 ml (100 mmol) propionic anhydride was dissolved in 300 ml propionic acid and 6.93 ml (100 mmol) pyrrole dissolved in 100 ml of propionic acid, was added drop wise to the mixture heated to reflux, for 1/2 h.The whole reaction mixture was refluxed for over 2h.After cooling to room temperature, the solvent was evaporated to dryness, and the oily residue was repeatedly washed with hot water, neutralized by aqueous ammonia (25%), and washed again with hot water.The purple solids obtained by this procedure were filtered and dried.

Anticancer Activity Studies Cell culture
The U937 (human histiocytic lymphoma cell line) was procured from American Type Culture Collection (ATCC, USA).The cells were cultured in RPMI medium with 10% Fetal bovine serum (FBS), 100 U/mL penicillin and 100 ¼g/mL streptomycin in T25cm 2 , T75 cm 2 , and 96-well culture plates at 37°C in a humidified atmosphere containing 5% CO 2 .All experiments were performed using cells from passage 15 or less.

Cell viability assay
The MTT assay was carried out as previously described by Mossman (1983).Briefly, U937 cells were plated at a density of 1.5 x 10 4 cells per well in 200¼L of fresh culture medium.After overnight growth, cells were treated at different concentrations (25-125 ¼g/mL) of complexes 1-3 for 24 and 48 h.After incubation, 20 ¼L of MTT solution (5 mg/mL in phosphate-buffered saline (PBS) were added to each well.The plates were wrapped with aluminum foil and incubated for overnight at 37 °C.The plates were centrifuged and supernatant was carefully removed.Purple color formazan crystals were dissolved by the addition of 100¼L of DMSO to each well.The absorbance was monitored at 570 (measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, CA, USA).Data were collected for three replicates each and used to calculate the mean.The percentage inhibition was calculated, from this data, using the formula: Mean abasorbance of untreated cells (control)-Mean abasorbance of treated cells × 100 Mean absorbance of untreated cells (control)

NMR studies
The Proton NMR spectra of [µ-(H 2 TPyP) {Ru-(bpy) 2 Cl} 4 ] 4+ (Fig. 5) shows doublet at 9.975 ppm corresponds to 8 identical protons at pyrrole ring.Two doublets at 8.64 ppm and 8.48 ppm correspond to pyridyl protons  and  to N -atom respectively.The triplets at 8.07 ppm assigned to bipyridyl protons meta and triplet at 7.77 ppm assigned to bipyridyl protons para to N-atom attached to Ruthenium metal ion.The other peaks between 7.79 ppm to 7.09 ppm assigned to other bipyridyl protons.The diamagnetic ring current shields the N-H protons, therefore it shifts to upfield, even further than the tetramethylsilane (TMS) peak 17 .

Cyclic voltammetry studies
Tetra ruthenated tetra pyridyl porphyrin is cycled in the anodic direction there is a quasireversible redox couple with +0.88V associated Wavelength (nm) with Ru (III/II).In the cathodic direction two quasi reversible redox couple with -0.68V and -1.11V are porphyrin centered redox process (Fig. 3).An additional quasi reversible redox couple with -1.42V is present in the cyclic voltagram of the ruthenated porphyrin solution, but it's not present in the free base porphyrin cyclic voltagram.This redox couple is attributed to the bipyridine ligands coordinated to the Ru(II) centers.The area under the redox wave is associated with the Ru(II) center is approximately twice the area of the first reduction process.This agrees with the two to one ruthenium to porphyrin stoichiometry predicted for complex Scheme 1: