DOI: 10.1002/cmdc.201300171 Antiproliferative Activity, Cell Cycle, and Apoptosis Studies of a Series of 6-Substituted 9H-Purin-9-yl-pyridinium Derivatives on a Human Cervical Carcinoma Cell Line BelØn Rubio-Ruiz, [a] Javier Ramos-Torrecillas, [b] Fermín Capitµn-CaÇadas, [c] Rosario Sµnchez- Martín, [d] Miguel ngel Gallo, [a] Antonio Espinosa, [a] Concepción Ruiz, [b] Ana Conejo-García,* [a] and Antonio Entrena* [a] Physicochemical parameters are among the most relevant properties to be evaluated in the early phase of drug develop- ment. Lipophilicity is one physicochemical parameter for which early prescreening is available. [1] Correlation analysis between lipophilicity and such biological activities as cytotoxicity, anti- proliferative effects, and apoptosis has a significant impact on drug discovery, as it can be used to predict and estimate bio- logical efficacy. [2–7] Choline kinase (ChoK) is overexpressed in human tumors. Small-molecule inhibitors that can interfere with ChoK activity have proven to be effective antitumor drugs in vitro and in vivo. [8–10] We recently reported the rational development of a series of nonsymmetrical ChoK inhibitors bearing a 4-substi- tuted pyridinium ring and an adenine moiety connected by linkers of various lengths (Figure 1, compounds 1–8). [11, 12] These structures were biologically evaluated and showed mod- erate potency as antiproliferative agents against the human hepatoma HepG2 cell line. [11] Considering that lipophilicity can be easily modified and determined by characterizing molecules prepared with different moieties, herein we present a new series of compounds in which the polar amino group at posi- tion 6 of the adenine (Figure 1, compounds 1–8,X = NH 2 ) was replaced with a benzylthio substituent (Figure 1, compounds 9–16,X = SCH 2 Ph) to increase their lipophilicity. The antiproli- ferative and cytotoxic effects of compounds 1–16 were evalu- ated against a human cervical carcinoma (HeLa) cell line to study how lipophilicity influences biological activity. Oncogenic transformations lead to cell-cycle aberration and dysregulation of apoptosis. The targeting of cell-cycle and apoptotic pathways has emerged as an attractive approach for the treatment of cancer. [13] In this study we evaluated the effect of compounds 9–16 on cell-cycle progression and apop- tosis in HeLa cells. Finally, the most apoptotic compound, 12, was selected for further investigations of downstream apoptot- ic signaling by analyzing the activation of caspase-3. Synthesis of the target compounds 9–16 was carried out as shown in Scheme 1. 6-Benzylthiopurine (17) was synthesized by a nucleophilic substitution reaction between benzyl bro- mide and 6-mercaptopurine. [14] The strategy for synthesizing monocationic intermediates 18–25 was based on our previous reported method. [11] Finally, the purine alkylation at N9 was car- ried out by microwave-assisted synthesis with DMF as a solvent, irradiating at 130 8C for 30 min. Carcinoma of the cervix is considered to be relatively resist- ant to chemotherapy. Few cytotoxic agents have shown signifi- cant activity in advanced and recurrent cervical cancer. [15] Thus, the effects on cell proliferation were investigated in HeLa cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay. [16] Table 1 lists the IC 50 values of Figure 1. Structures of monoquaternary pyridinium salts 1–16. [a] Dr. B. Rubio-Ruiz, Prof. M. . Gallo, Prof. A. Espinosa, Dr. A. Conejo-García, Prof. A. Entrena Departamento de Química FarmacØutica y Orgµnica Facultad de Farmacia, Universidad de Granada Campus de Cartuja, 18071 Granada (Spain) E-mail: aconejo@ugr.es aentrena@ugr.es [b] J. Ramos-Torrecillas, Prof. C. Ruiz Departamento de Enfermería, Facultad de Ciencias de la Salud Universidad de Granada, 18071 Granada (Spain) [c] F. Capitµn-CaÇadas Departamento de Bioquímica y Biología Molecular II Facultad de Farmacia, Universidad de Granada Campus de Cartuja, 18071 Granada (Spain) [d] Dr. R. Sµnchez-Martín Chemical Biology Section, School of Chemistry King’s Buildings, West Mains Road, EH9 3JJ Edinburgh (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201300171.  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 4 &1& These are not the final page numbers! ÞÞ CHEMMEDCHEM COMMUNICATIONS