Synthesis and cytotoxicity of 2,6,9-trisubstituted purines derivatives

ISBN 978-85-85905-21-7

Área

Química Orgânica

Autores

Espinosa-bustos, C. (PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE) ; Salas, C. (PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE) ; Faundez, M. (PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE) ; Gallardo, C. (PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE)

Resumo

A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method on three cancer cells lines and Vero cells. Nine out of ten compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide on HeLa (Human cervix adenocarcinoma) and HL-60 (Human promyelocytic leukaemia) cells.

Palavras chaves

purines; cytotoxicity; MTT assay

Introdução

Purine scaffold is often found in several molecules with biological properties, receiving the title of privileged scaffold. Therefore, the purine core is an interesting structural fragment that has been incorporated into new drugs. Some examples of major purine-based drugs that are currently being used are: anticancer agents (6-mercaptopurine, thioguanine), antiviral agents to overcome infections such as herpes or AIDS (acyclovir, ganciclovir, carbovir, abavavir), among others. In recent years, several di- and tri-substituted purine derivatives have been synthesized and tested on cancer cells. Furthermore, purine’s 2,6,9-substitution pattern is found in several compounds with anti-cancer properties such as myoseverin and roscovitine. Considering the importance of having new anticancer therapies, in this work we show the synthesis and cytotoxic evaluation of tri- substituted purine derivatives.

Material e métodos

2,6-Dichloropurine, benzyl amines and the alkyl halides, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture medium, fetal bovine serum and penicillin-streptomycin were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Etoposide, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) and all other chemicals were obtained from Sigma- Aldrich. Nuclear magnetic resonance spectra were recorded on a Bruker AM-400 apparatus using CDCl3 solutions containing TMS as internal standard. Thin layer chromatography (TLC) was performed using Merck GF-254 type 60 silica gel. Column chromatography was carried out using Merck silica gel 60 (70–230 mesh).

Resultado e discussão

Chemistry To carry out the synthesis of compounds 4a-l, 6-chloro-2-fluoropurine 1 was used as starting material, according to Scheme 1. First, through a reaction of N-alkylation using benzyl chloride, the derivative substituted at position 9 of the purine 1, with 48 % yield it was achieved. Additionally, was obtained the regioisomer alkylated at the 7 position with a 13 % (not shown). Later, position 6 of purine 1 core was substituted with trifluoromethoxyphenyl group by Suzuki-Miyaura C-C coupling using the respective boronic acid and Pd(PPh3)2Cl2 as palladium source under dioxane reflux, furnishing the compound 3 with a 67 % yield. Finally, derivatives 4a-l were obtained in good yields (43 – 94 %) using 12 different benzylamines, n-butanol as solvent and DIPEA. All compounds (finals and intermediates) were characterized by NMR spectroscopy of 1H, 13C and 19F, IR spectra and high-resolution mass spectres. The final compounds 4a-l shown as characteristic signals the proton of NH group in the zone of 5.50 – 5.70 ppm as a broad signal, and the hydrogens of the benzylamine methylene group at 4.60 – 4.80 ppm as a doublet signal. Biology The potential in vitro antitumor activity of compounds 4a–l was initially tested for their cytotoxic effects on HL-60, HCT116 and HeLa cancer cell lines and Vero cells. A conventional colorimetric assay was set up to estimate the IC50 values, which represent the concentration of a drug that is required for 50 % inhibition in vitro after 72 h of continuous exposure to compounds. Four serial dilutions (from 12.5 to 100 μM) for each sample were evaluated in triplicate and etoposide was used as a positive control and reference drug. In general, all compounds showed high cytotoxicity in cancerous cells and low toxicity in Vero cells.

scheme 1

Scheme 1. Reagents and conditions: i) benzyl bromide, K2CO3, DMF, 6 h., r.t. ii) trifluoromethoxy phenyl boronic acid, Pd(PPh3)2Cl2, K2CO3 2M, Dioxane, 2h, reflux. Iii) benzylamines, DIPEA, n-BuOH, 12 h, reflux.

Table 1

Table 1. In vitro cytotoxicity of compounds 4a–4l on cancer cell lines and Vero cells.

Conclusões

New 2,6,9-trisubstituted purines was synthesized and evaluated their cytotoxicity on three cancer cell lines and Vero cells. The vast majority of these compounds were more potent than the reference drug etoposide and exhibited selectivity against three cancer cell lines in comparison with VERO cells. These results showed that these compounds are promising leads for the development of new antitumor drugs.