Synthesis of Some Substituted 6-Phenyl Purine Analogues and Their Biological Evaluation as Cytotoxic Agents

A series of 6-(4-substituted phenyl)-9-(tetrahydropyran-2-yl)purines 3–9, 6-(4-substituted phenyl)purines 10–16, 9-((4substituted phenyl)sulfonyl)-6-(4-substituted phenyl)purines 17–32 were prepared and screened initially for their in vitro anticancer activity against selected human cancer cells (liver Huh7, colon HCT116, breast MCF7). 6-(4-Phenoxyphenyl)purine analogues 9, 16, 30–32, had potent cytotoxic activities. The most active purine derivatives 5–9, 14, 16, 18, 28–32 were further screened for their cytotoxic activity in hepatocellular cancer cells. 6-(4-Phenoxyphenyl)-9(tetrahydropyran-2-yl)-9H-purine (9) had better cytotoxic activity (IC50 5.4 μM) than the well-known nucleobase analogue 5-FU and known nucleoside drug fludarabine on Huh7 cells. The structure–activity relationship studies reported that the substitution at C-6 positions in purine nucleus with the 4-phenoxyphenyl group is responsible for the anti-cancer activity.

Furthermore, purine nucleosides such as fludarabine, cladribine, and pentostatine (Fig. 3) were approved in FDA for the therapy of hematologic disorders between 1991 and 1992. 18,19 Experimental

1. Chemistry
Melting points were recorded with a capillary melting point apparatus (Electrothermal 9100) and are uncorrected.NMR spectra were recorded on a VARIAN Mercury 400 FT-NMR spectrometer (400 for 1 H, 100.6 MHz for 13 C).TMS was used as internal standard for the 1 H and 13 C NMR spectra; values are given in δ (ppm) and J values are in Hz.Mass spectra were taken on Waters Micromass ZQ by using ESI+ ionization method.Elemental analyses (C, H, N) were determined on a Leco CHNS 932 instrument and gave values within ±0.4% of the theoretical values.Column chromatography was accomplished on silica gel 60 (40-63 mm particle size).The chemical reagents used in synthesis were purchased from Merck, Fluka, Sigma and Aldrich.

Fludarabine
Cladribine Pentostatine Hepatocellular carcinoma (HCC) is one of the deadly cancers and affects most of the world population.] Chronic liver damage is due to viral diseases, chemical exposure, environmental toxins or autoimmune diseases that are the risk factors for HCC.These conditions cause an acquired tolerance to genotoxic stress, but finally result in a cancerous case that does not respond to the mechanism of cell death. 23he diagnosis of HCC patients is usually very poor and HCC tumors are resistant to chemotherapeutic agents.Lately, a multikinase inhibitor Sorafenib, was approved by the FDA and the EU for the treatment of hepatocellular carcinoma. 246][27] Therefore, it is essential to discover new livercancer-specific drugs for hepatocellular carcinoma treatment.

1. Cells and Culture
The human primary liver cancer cell lines (Huh7, HepG2, Mahlavu and FOCUS) were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen GIBCO), with 10% fetal bovine serum (FBS) (Invitrogen GIBCO), nonessential amino acids, and 1% penicillin (Biochrome).It was incubated in 37 °C with 5% CO 2 .DMSO (Sigma) was used as the solvent for the compounds.The concentration of DMSO was always less than 1% in the cell culture medium.The cytotoxic drugs (5-FU, Fludarabine and Cladribine) used as positive controls were from Calbiochem.

Sulforhodamine B (SRB) Assay for Cytotoxicity Screening
Huh7, HCT116, MCF7, HepG2, Mahlavu, and FOCUS cells were inoculated (2000-10000 cells/well in 200 μL) in 96-well plates.The next day, the media were refreshed and the compounds dissolved in DMSO were applied in concentrations between 1 and 40 μM in parallel with DMSO-only treated cells as negative controls.At the 72nd hour of treatment with compounds 3-32 and the other drugs, the cancer cells were fixed with 100 μL of 10% (w/v) trichloroacetic acid (TCA) and kept at +4 °C in the dark for one hour.TCA fixation was terminated by washing the wells with ddH 2 O five times.Air-dried plates were stained with 0.4% sulphorhodamine B (SRB) dissolved in 1% acetic acid solution for 10 min in the dark and at room temperature.The protein-bound and dried SRB dye was then solubilized with 10 mM Tris-Base pH 8.The absorbance values were obtained at 515 nm in a microplate reader.The data were normalized against DMSO only treated wells, which were used as controls in serial dilutions.In all experiments, a linear response was observed, with serial dilutions of the compounds and the drugs.

2. Cytotoxic Activity and Structure-Activity Relationship (SAR)
The in vitro cytotoxicity of the compounds 3-32 were initially analyzed on human cancer cells (liver Huh7, colon HCT116, breast MCF7), using a sulforhodamine B (SRB) assay.The IC 50 values for each compound were also calculated in comparison with the known cell growth   1.
Analyzing the data presented in Table 1, highlights the 4-phenoxyphenyl substitution as the group at C-6 as the most responsible for the anti-cancer activity against Huh7.When we compared their IC 50 values with the nucleobase analogue 5-FU and nucleoside analogue Fludarabine, we observed that our compounds 9, 16, 30, 31 and 32 had showed lower values in micromolar concen- trations and these molecules had a better cytotoxic activity on Huh7 cells (5.4,16.0, 14.3, 13.6 and 11.0 vs 30.6 μM and 28.4 for 5-FU and Fludarabine).Compound 29, bearing a 4-tert-butylphenyl substituent at C-6 position of the purine, was active derivative with greater potency against Huh7 cell line than 5-FU and Fludarabine.The  structure-activity relationship (SAR) results are summarized in Scheme 2. Notably 6,9-disubstituted derivative 9 showed superior cytotoxic activity (IC 50 7.4 μM) compared with Fludarabine (IC 50 15.2μM) against MCF7 tumor cell line.Within the tested purine analogues on HCT116 cell, compounds 9 and 31 with 4-phenoxyphenyl group at N-9 position, showed good cytotoxic activity (IC 50 15.9 and 13.1 μM, respectively).

inhibitors 5 -
fluorouracil (5-FU), fludarabine and cladribine and the results are summarized in Table

aFigure 4 .
Figure 4. Percent cell death in the presence of the most active compounds.Huh7, HepG2, Mahlavu and FOCUS cells were inoculated in 96-well plates.All molecules and their DMSO controls were administered to the cells in triplicate with five different concentrations: 40, 20, 10, 5, and 2.5 μM.After 72 h of incubation, SRB assays were generated and the cell death percentages were calculated in comparison with DMSO-treated wells.

Cancer cell lines, IC 50 (μM) a
a IC 50 values were calculated from the cell growth inhibition percentages obtained with 5 different concentrations (40, 20, 10, 5, and 2.5 μM) of each molecule incubated for 72 h.NI: No inhibition