Design, Synthesis and In Vitro Cytotoxic Activity ... Design, Synthesis and In Vitro Cytotoxic Activity of New 6,9-Disubstituted Purine Analogues

A series of new 6,9-disubstituted purine analogs with 4-substituted piperazine at C-6 and 4-substituted benzyl at N-9 were designed and synthesized in four steps. All synthesized compounds ( 7 – 26 ) were screened initially for their in vitro anticancer activity on Huh7 liver, HCT116 colon and MCF7 breast carcinoma cell lines. Cytotoxic bioactivity studies revealed that all compounds screened, with compound 19 being the exception, were found to have promising cytotoxic activities at IC 50 range of 0.05–21.8 μM against cancer cells Huh7, HCT116 and MCF7. Among the prepared purine analogs, two of them ( 12 and 22 ) exhibited excellent cytotoxic activities, with IC 50 0.08–0.13 μM, on Huh7 cells comparable to camptothecin (CPT) and better than cladribine, fludarabine and 5-FU. Afterwards, the evaluation of cytotoxicity of the most potent purine analogs was screened against further hepatocellular cancer (HCC) cell lines. The 6-(4-(4-trif-luoromethylphenyl)piperazine ( 12 ) and 6-(4-(3,4-dichlorophenyl)piperazine analogs ( 25 ) displayed a significant IC 50 values (IC 50 < 0.1–0.13 μM) comparable to CPT and better cytotoxic bioactivity when compared with 5-FU, cladribine and fludarabine on HCC cells (Huh7 and HepG2). (100%) + ), 271.24 (55%) (M+2). Anal. Calcd for C 11 H 10 Cl 2 N 4 : C, 49.09; H, 3.75; N, 20.82. Found: C, 49.14; H, 3.66; N, 20.42.


Introduction
The purine nucleus is involved in the biological molecules that play a key role in the signaling pathways of all living organisms. 1For this reason, the purine structure is an interesting organic moiety included in new drugs.Purine and purine nucleoside analogs exhibit a variety of biological activities.These analogs have been extensively studied as enzyme inhibitors, 2-5 cytotoxic, [6][7][8][9][10] antiviral, [11][12][13][14] antihyperglycemic, 15 immunostimulatory, 16 antifungal, and antibacterial [17][18][19][20][21][22] agents due to their potential activities, even though their antiviral and anticancer effects are more commonly known.5-Fluorouracil (5-FU), a well known fluorinated nucleobase analogue, is highly preferred for the treatment of various cancers in clinics. 235][26] Purine nucleobase analogs such as 6-mercaptopurine and 6-thioguanine, are specifically used in pediatric acute lymphoblastic leukemia as an inhibitor of nucleic acid metabolism. 27,284][35] Also, various purine scaffolds and their analogs have been studied as potential anticancer agents that contain cyclin-dependent kinase and heat shock protein inhibition.Olomoucine, 36 roscovitine, 37 purvalanol A, B, amino-purvalanol 38 (Figure 1) have been synthesized and screened as cyclin-dependent kinase inhibitors.Especially, R-roscovitine is under investigation as a chemotherapeutic agent against non-small cell lung cancer and other malignancies.Also, olomoucine with its 2,6,9-trisubstitued purine structure is another purine derivative with antiproliferative effects on cancer cell lines.
The heat shock protein 90 (Hsp90) has been an existing target in cancer since its inhibition may lead to the breakdown of many cancer-associated proteins.Furthermore, inhibitors of Hsp90 kill cancer cells at lower concentrations than is required to harm normal human cells.In recent years, purine analogues, which are Hsp90 inhibitors, have entered clinical trials as drugs in the therapy of solid tumors and hematologic malignancies. 50n our previous studies, 51,52 we have reported important cytotoxic activities of 9-(cyclopentyl/β-D-ribofuranosyl/para-toluenesulfonyl)-6-(4-substituted piperazino)purine analogs A, B, C (Figure 2).In this work, we report the synthesis of new analogs of purines A, B, C as 9-(4-substituted benzyl)purines and evaluate their cytotoxic activities against liver (Huh7), colon (HCT116) and and breast (T47D) carcinoma cell lines.We further investigate the most active compounds (7-18, 20, 22-26) on a panel of liver cancer cells.

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 MHz for 1 H, 100.6 MHz for 13 C).TMS was used as internal standard for the 1 H NMR 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 the ESI+ 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 (0.040-0.063 mm particle size).The chemical reagents used in the synthesis were purchased from Merck, Fluka, Sigma and Aldrich.5-Amino-4,6-dichloropyrimidine (2) was synthesized according to the reported method.ed benzylamines (2 mmol) and Et 3 N (3 mmol) were added.The mixture was refluxed for 15 h.The reaction mixture was concentrated in vacuo, and the residue was purified by column chromatography (EtOAC-hexane, 1:4 to 1:2).

Sulforhodamine B (SRB) Assay for Cytotoxicity Screening
Huh7, HCT116, MCF7, HepG2, Mahlavu, and FO-CUS cells were inoculated (2000−10000 cells/well in 200 μL) in 96-well plates.The next day, the media was 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 7-26 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 dark for 1 h.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 tempera-ture.The protein-bound and dried SRB dye was then solubilized with 10 mMTris-Base pH 8.The absorbance values were obtained at 515 nm in a microplate reader.The data 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. Biological Evaluation
The antitumor activities of newly synthesized purine analogues were first analyzed on three human cancer cell lines including Huh7 (liver), HCT116 (colon) and MCF7 (breast) cancer cells by using the sulforhodamine B (SRB) method.The IC 50 values of the purine compounds were calculated in comparison with DNA topoisomerase inhibitor camptothecin (CPT) and the known cell growth inhibitors fludarabine, cladribine, 5-fluorouracil (5-FU).The data are summarized in Table 1.
All synthesized purine derivatives in this study, except for compound 19, exhibited important cytotoxic activity against cancer cells Huh7, HCT116, MCF7 with IC 50 from 0.05 to 21.7 μM.
As seen from the data in Table 1, all the 6-(trifluoromethylphenyl)piperazine purines, 12 and 22 exhibited excellent cytotoxic activities with IC 50 0.08-0.13μM on Huh7 cells comparable to CPT and better than cladribine, fludarabine and 5-FU.In addition, compounds 15 and 25 bearing a 3,4-dichlorophenyl group at the piperazine of the purine, presented a higher cytotoxic activity than known nucleoside drugs cladribine, fludarabine and nucleobase drug 5-FU on Huh7 cells.For the 4-fluorophenyl substituted derivatives 13 and 23, their best activity is observed for the 9-(4-chlorobenzyl) purine derivative 23 with IC 50 value of 0.57 μM on Huh7.Cytotoxic activity differences were not observed in the nonsubstituted phe-   We then screened the anticancer activity of the most potent purine analogs against further hepatocellular cancer (HCC) cells lines (Table 2, Figure 3).We observed that the most important cell growth inhibition in the presence of 6-(4-(4-trifluoromethylphenyl)piperazine)-9-(4-trifluoromethylbenzyl)purine derivative 12 and its 9-(4-chlorobenzyl) analogue 22, with IC 50 values of < 0.1-0.23 μM against all the HCC cell lines.Compounds 12 and 22 also showed comparable cytotoxic effects with CPT and cladribine on these cell lines.Furthermore, 12 and 22 showed a better biological activity than the standard anticancer agents 5-FU and fludarabine in HCC cell lines (Table 2).The 6-(4-(2,4-dichlorophenyl)piperazine analogs 15, 25 were also very active (IC 50 < 0.1-0.24μM) against Huh7 and HepG2 cell lines.

Conclusion
We designed and synthesized twenty novel purine analogs 7-26 bearing substituted piperazine at the C-6, substituted benzyl group at the N-9, by the multistep reactions, starting from 4,6-dichloro-5-nitropyrimidine.The cytotoxic activities of the compounds were evaluated first in human liver (Huh7), breast (MCF7), colon (HCT116) and then in hepatocellular carcinoma cells (HCC): Huh7, HepG2, Mahlavu and FOCUS.Our results demonstrated that the 6-(trifluoromethylphenyl)piperazine analogs 12, 22 with IC 50 values less than 0.5 μM were promising molecules as cytotoxic agents on Huh7, MCF7 and HCT116 cancer cells.In order to investigate the use of potential cytotoxic agents on HCC, the bioactivity of the purine analogs was also tested in a panel of liver cancer cells.Molecules 12 and 22, that were synthesized as putative cytotoxic compounds, displayed the best anticancer bioactivities (IC 50 < 0.1-0.23 μM) against HCC cell lines (Figure 4).These results indicate that these compounds can be considered as promising lead molecules for the development of potential anticancer agents.

Figure 3 .
Figure 3. Percent cell death in the presence of 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.

Figure 4 .
Figure 4. a) Chemical structures of the most active purine analogs 12, 22, 15 and 25 b) Percent cell death in the presence of most active compounds (12, 22, 15 and 25).Huh7 cells were inoculated in 96-well plates.All molecules and their DMSO controls were administered to the cells in triplicate with corresponding different concentrations: 0.625, 1.25, 2.5, 5, 10, 20, and 40μM.After 72 h of incubation, SRB assays were generated, and the cell death percentages were calculated in comparison with DMSO-treated wells.

Table 1 .
In vitro cytotoxicity of compounds 7-26 on different human cancer cell lines

Cancer cell lines, IC 50 (μM) 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. a