Nuevos inhibidores de chok no simétricos y simétricos más polares con actividad antitumoral y antimalárica

Resumo

Background Cancer is a group of diseases characterized by the excessive and uncontrolled growth of cells that invade and damage tissues and organs, eventually causing the death of the individual(1). In industrialized countries, is considered the leading cause of death after cardiovascular disease, but it is intended to be the first(2). Cancer treatment is in the need of selective drugs that can interfere specifically with signalling pathways affected during the carcinogenic process. Identification of new potential molecular targets is the key event in the design of new anticancer strategies. The relevance of deregulation of choline kinase (ChoK) in oncogene-driven cell transformation has been previously demonstrated(3-7). Choline kinase (ChoK) is a cytosolic enzyme present in various tissues, which catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is important for the generation of two major membrane phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM) and subsequently for the cell division. ChoK plays a vital role in cell signaling pathways and regulation of cell growth along with PCho involved in malignant transformation through ras oncogenes in different cancers such as breast, lung, colon, prostate, neuroblastoma, hepatic lymphomas, meningiomas and diverse murine tumours. The Ras effectors serine/threonine kinase (Raf-1), the Ral-GDP dissociation stimulator (Ral-GDS) and the phosphatidylinositol 3-kinase (PI3K) are involved in the activation of ChoK during tumorigenesis(8). It is well reported that in mammalian cells, the three known isoforms of ChoK (ChoK-¿1, ChoK-¿2, and ChoK-ß) are encoded by two separate genes: ChoK-¿ and ChoK-ß. The two functional isoforms of ChoK-¿, ChoK¿1 and ChK-¿2, are the result of alternative splicing of the Chk- ¿ transcript. None of the isoforms are active as monomers and the active enzyme consists of homo- or heterodimers9. ChoK¿1 differs from ChoK¿2 in only an extra stretch of 18 amino acids, while ChoKß differs from ChoK¿1 and ChoK¿2 in approximately 40% of amino acids. ChoK¿ and its product (PCho) have been implicated in malignant transformation. This hypothesis is supported by a number of observations. First, ras-, raf-, src- and increased PCho levels compared to the parental cells from which they were originated as a consequence of an increase in ChoK activity(10). An increase in both ChoK¿ protein levels and activity was also confirmed in different human tumour-derived cell lines and human tumour biopsies(11). Furthermore, a clear association between ChoK¿ expression and activity, and a more malignant phenotype and worse prognosis, has been established in human breast and lung tumours(12). In addition, ChoK activity has been related to the regulation of cell proliferation in both normal breast cells, and tumor-derived cell lines from breast cancers(13). On the other hand, recently has been reported that Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells(14). All these evidences indicate that ChoK¿ participates in the regulation of cell proliferation and its function is deregulated during carcinogenesis . The consideration of ChoK as a novel target for the development of new anticancer drugs is justified. The obtained ChoK inhibitors it was based in structural modifications of hemicholinium-3 (HC-3). Moreover It was be demonstrated that the inhibition of this enzyme by a selective inhibitor is rather specific to this enzyme with no effect on a variety of oncogene-activated signalling pathways involved in the regulation of cell proliferation. Nontransformed cells were able to resume cell proliferation after removal of the drug, while transformed cells were irreversibly affected(15). In normal cells, blockage of de novo phosphorylcholine (PCho) synthesis by inhibition of ChoK promotes the dephosphorylation of pRb, resulting in a reversible cell cycle arrest at G0/G1phase. In contrast, ChoK inhibition in tumor cells renders cells unable to arrest in G0/G1as manifested by a lack of pRb dephosphorylation. Furthermore, tumor cells specifically suffer a drastic wobble in the metabolism of main membrane lipids PC and sphingomyelin (SM). This lipid disruption results in the enlargement of the intracellular levels of ceramides. As a consequence, normal cells remain unaffected, but tumor cells are promoted to apoptosis(16). By the other hand, Malaria is a major global threat that results in more than 2 million deaths each year caused by parasites of the genus Plasmodium. The treatment of malaria is becoming extremely difficult due to the emergence of drug-resistant parasites, the absence of an effective vaccine, and the spread of insecticide-resistant vectors. Thus, malarial therapy needs new chemotherapeutic approaches leading to the search for new drug targets(17). High levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in Plasmodium-infected cells have been observed. Genetic and pharmacological data confirm that the biosynthesis pathways of novo both phospholipids are essential for the survival of the parasite(18). The intracellular Kennedy pathway is the route of synthesis of new PC in the Plasmodium and it has been proposed as a therapeutic target for the development of new antimalarial drugs, in order to reduce and combat the drug-resistance. In particular, the ChoK enzyme has been proposed as therapeutic target since intervenes in the Kennedy pathway of the Plasmodium with a regulatory activity in the biosynthesis of PC. The PfCK gene, unlike the human choline kinase, exists as a single copy in the Plasmodium genome and therefore leads to the absence of the isoforms present in the ChoK(18). Various mono- and biscuaternary compounds with aliphatic chains are able to inhibit the growth of Plasmodium in vitro and in vivo(19). Although the mechanism of action of these is not yet entirely clear, recent studies have suggested the capacity, by these molecules, attack at Kennedy pathway via the enzymatic passages(20). These results indicate that inhibition of ChoK is a rather specific strategy for the cytotoxic treatment of transformed cells and open the doors for the development of new drugs against the Plasmodium infected cells, justifying the research of new and better rational antitumoral and antimalarial drug design. Aims The previously mentioned antecedents indicate that the selective inhibition of choline kinase (ChoK) could lead to the development of new antitumor drugs with minimized adverse effects that are present in other chemotherapy agents. Our research group have developed several series of compounds that can act as choline kinase inhibitors and as antiproliferative agents, justifying the correlation between both types of biological activities. These series of compounds show a biscationic, cyclophanic or bicyclophanic structures, which include different linkers and cationic heads. Docking studies performed with some of these compounds in a homology model of human choline kinase and the recent crystallization of this enzyme indicate that the inhibitors could be inserted simultaneously in both ATP and Cho binding sites. Since the nature of both binding sites is different, the design, synthesis and analysis of new monocationic non-simmetrical inhibitors were considered. However, new biscationic structures have also been synthetized to study the influence of new linkers on the biological activity. The objetives of this Thesis are: Objective 1.- The design and synthesis of new monocationic non-simmetric inhibitors (SOS-1 to SOS-29) with the following structure: Objective 1a.- Study of the influence of some functional groups present in the fragment that can be inserted into the ATP binding site on the biological activity of these new inhibitors. Objective 1b.- Study of the influence of various cationic heads on the inhibition of ChoK. Objective 1c.- Study of the influence of different aryl alkyl linkers. Objective 2.- Continuing the line of research developed by our research group, and in order to further deep in the influence of both the cationic head and the polarity of the molecule, the desing and synthesis of new compounds with biscationic structure have been performed. Objective 2a.- Introduction of a quinuclidine moiety as a cationic head and various aryl alkyl linkers (SOS-30 to 33). Objective 2b.- Introduction of two ether bonds in the linker, in order to see the influence of these groups on the antiproliferative and inhibitory activity of ChoK, a various cationic heads previously synthesized by our group in other compounds (SOS- 34 to 44). Objective 3.- Unambiguous identification of all synthesized compounds by means of nuclear magnetic resonance techniques (1H-RMN, 13C, HMBC y HSQC) and mass spectrometry. Objective 4.- Study of the inhibition capacity of ChoK by these new inhibitors. Objective 5.- Study of the antiproliferative capacity of the designed and synthesized ChoK inhibitors. Objective 6.- Study of the antimalarial activity of the designed and synthesized compounds. Objective 7.- Molecular modelling techniques will be use in order to perform theoretical study of the viability or impracticality of various synthesized inhibitors. Designed and Synthesized compounds In this Thesis have been designed, synthesized and characterized forty-four compounds. Of wich twenty-nine are non-symmetrical monocationics salts of bromide (Family A) and fifteen are symmetrical biscationics salts of bromide (Family B), with different semirigid spacers. Chemistry- Family A The general procedure used in the syntheses of the final monocationics non-symmetric compounds (A Family) are performed by means of three different steps. The first step is the bromomethylation of the four different linkers used in the synthesis (benzene, biphenyl, 1,2-diphenylethane or 1,4-diphenylbutane), in order to obtain the intermediates 1-4. 1,4-Bis(bromomethyl)benzene 1 is commercially available, and was not synthetized. Reaction of biphenyl or 1,2-diphenylethane with formaldehyde and hydrogen bromide in the presence of H3PO4 yields 4,4¿-bis(bromomethyl)biphenyl 221 and 1,2-bis[(bromomethyl)phenyl]ethane 3 (22), respectively. Finally, 1,4-diphenylbutane was prepared by electrolysis hydrogenation H-Cube Full-H2 of trans,trans-1,4-diphenyl-1,3-butadiene, in presence of MeOH, and was bisbromomethylated following the previous mentioned conditions to yield the intermediated 1,4-bis[4-(bromomethyl)phenyl]butane 4 (22). In the second step different conditions have been used depending on the intermediate compounds that will be obtained. The intermediates derivatives of pyridine is the reaction of the bisbromomethylated linkers 1-4 with pyrrolidinopyridine or dimethylaminepyridine (2:1 equivalent relationships) to obtain the 1a-4a and the 1b-4b. This reaction was performed in butanone at room temperature during 3 days, and intermediates 1a-4a and 1b-4b were isolated as a white precipitate which was filtered and washed with butanone, ethyl acetate and diethyl ether to obtain the product as white solid. The intermediates derivatives of quinuclidine 1c-3c was obtained using different solvents depending on the intermediates compounds that will be obtained. Compound 1c was obtained using toluene, compound 2c using butanone and compound 3c using dioxane as solvent. The third step allows the insertion of the m-aminophenol fragment in the other extreme of the bisbromomethylated linker. Different conditions have been used depending on the final compound that would be obtained. In the synthesis of compounds of Subfamily A1, the 3-aminophenol hydroxyl group is firstly protected, in order to avoid the reaction through the OH group, by treatment with TBDMSCl, TEA and DMAP, in CH2Cl2 at room temperature during 6h. The obtained 3-(tert-butyldimethylsilyloxy)aniline was treated with compounds 1a-4a, 1b-4b and 1c-3c in DMF in the presence of K2CO3 during 20-24 h at 110 °C. In this reaction the OH protecting group is liberated, probably due to the acid pH generated by the HBr formed during the nuclephilic substitution, being compounds SOS-1 to SOS-11 obtained the final products of the reaction. In the synthesis of compounds of Subfamily A2, the compounds 1b-4b and 1c-3c was treated with 3-nitrophenol and NaH, in DMF at reflux during 20h, yielding final compounds SOS-12 to SOS-18 In the synthesis of compounds of Subfamily A3, two different routes have been used. The first one is the treatment of the intermediates 1a-4a with 3-aminophenol and HNa, in DMF at a temperature of 110 °C during 22 h. This route allows the isolation of compounds SOS-19 to SOS-22. In the second route has the reduction of the nitro group of the compounds of the Subfamily A2 by treatment with Fe/ SO4Fe in water at reflux temperature during 3 h, yielding the final compounds SOS-23 to SOS-29. Chemistry- Family B The general procedure used in the synthesis of the final biscationic symmetric compounds of the Subfamily B1 are performed by means of two different steps. The first step is the bromomethylation of the four different linkers used in the synthesis of the A Family to obtain 1-4. The second step is the reaction of the bisbromomethylated linkers 1-4 with quinuclidine (1:2 equivalent relationships) to obtain the SOS-30 to SOS-33 final compounds. This reaction was performed in butanone at room temperature during 6 days, and compounds SOS-30 to SOS-33 were isolated as a white precipitate, filtered and washed with butanone to obtain the product as white solid. Scheme 8.- Synthesis of compounds of Subfamily B1. The general procedure used in the synthesis of the final biscationics symmetric compounds of Sufamily B2 and B3 are performed by means of three different steps. The first step is the formation of the linker used in the synthesis (1,2-bis(4-bromomethylphenoy)ethane 7), (I) Reaction of 4-methoxyphenol 1 with NaOH in presence of EtOH at room Tª for 30 min, (II) afterward 1,2-dibromoethane was added and microwave irradiation at 140°C for 28 min to obtain 1,2-bis(p-methylphenoxy)ethane 6. (III) Finally in order to yield the linker 1,2-bis(4-bromomethylphenoy)ethane 7 the bromomethylation of 6 was carried out in presence of benzoyl peroxide, NBS and CCl4 by microwave irradiation at 120°C, 21 min. The second step is the synthesis of the cationics head. The cationic head 4-pirrolidinopiridine (9), 4-dimethylaminopiridine (10), quinuclidine (11) and 3-hidroxyquinuclidine (12) are commercially available while that the cationic heads derivatives of quinoline (13-19) were obtained following the procedure previously described23, starting of 4-quinoline and 7,4-dichloroquinoline which were treated with N-methylaniline, 4-chloro-N-methylaniline, perhydroazepine or pyrrolidine to obtain 13 (4-perhydroazepinoquinoline), 14 (4-N-methylanilinoquinoline), 15 (4-(4-chloro-N-methylanilino)quinoline), 16 (7-chloro-4-(N-methylanilino)quinoline), 17 (7-chloro-4-(4-clhoro-N-methylanilino)quinoline), 18(7-chloro-4 (pyrrolidino)quinoline) and/or 19 (7-chloro-4-perhidroazepinoquinoline). The third step is the synthesis of the final biscationics compounds of Subfamily B3 and B3. A solution of 1eq 1,2-bis(4-bromomethylphenoy)ethane 7 in dry CH3CN was added drop to drop to a solution of 9-19 (2 eq) in dry CH3CN under argon conditions. The mixture was heated under reflux for a further 3 days and, after cooling down to room temperature, washed with diethyl ether and hexane, filtered and dry to vacuom to afford SOS-34 to SOS-44 as a solid product. All compounds have been unequivocally established through the combined use of various spectroscopic techniques (1H-RMN, 13C-RMN, HMBC y HSQC) and calculation of the mass. Results and discussion Molecular modelling and drug design Human choline kinase ChoK¿1 (PDB id: 3G15) was chosen for the docking studies due to the presence in the same crystal structure of both the HC-3 and ADP. Molecular modeling studies were performed using Sybyl-X program(23). Initial docking studies, preformed on ChoK¿1 with the Family A (Subfamily A1 and A3) of non-symmetrical monocationic inhibitors described in this Thesis, show that the m-aminophenol moiety can mimics the adenine moiety that is present in the first family of previously published compounds, being inserted into de ATP binding site being stabilized by hydrogen bonds with Arg146, Asp206, Gln207 or Ile209(24). Docking studies also indicate that there are different modes for the binding of the inhibitors inside the enzyme. The length of the linker in compounds SOS-1, SOS-5, SOS-19 and SOS-23 is too short to allow these molecules to be allocated in both ATP and Cho binding sites simultaneously. The obtained poses for these compounds indicate that they can be inserted into the Cho binding site or into the ATP binding site. Finally, in the other compounds of these Subfamilies, the length of the linker is enough to allow these molecules to be inserted simultaneously in both ATP and choline binding sites. Consequently, it would be expected higher activity for those compounds that has the longer linker, like in the first series of non-symmetrical monocationic inhibitors previously described(23). Biological results The data indicate that the best inhibitors of this new family of non-symmetrical monocationic choline kinase inhibitors (Family A) are characterized by the presence of both a biphenyl linker and a dimethylaminepyridinium fragment, and that the nature of the m-aminophenol does not significantly affect the inhibitory effect. IC50 ChoK inhibition value has been calculated only for the most active compound SOS-6 (IC50 ChoK = 6.37 ± 0.53 µM) and SOS-24 (IC50= 7.89 ¿ 0.05 µM), since the inhibition percentage of the other molecules are, in general, very small. No clear structure-activity relationships can be established in the antiproliferative activity, since most of these molecules originate toxicity on the HepG2 line cells when they are tested at concentrations of 10 or 50 ¿M. In relation to the linker, compounds with bibenzyl (SOS-7, SOS-14) or 1,4-diphenylbutane (SOS-8, SOS-15) show better antiproliferative activity. Compounds with a pyrrolidinopyridinium cationic head show higher antiproliferative activity than their analogues with dimethylaminopyridinium moiety. Finally, Subfamily A3 molecules also shows in general higher antiproliferative percentage than their analogues from Subfamily A1. Unfortunately, biological results do not confirm the initial docking studies performed for the design of these inhibitors, neither the previously published results related to the influence of the linker of the first family of non-symmetrical inhibitors on the ChoK inhibitory activity23. The fact that all molecules with long linkers are inactive or show low inhibition percentage indicate that the conclusions obtained from the initial docking studies are not correct. There is only one reason for this behaviour: even the docking studies have indicated that the m-aminophenol fragment could bind inside the ATP binding site, the affinity of the ATP adenine fragment for its binding site in the choline kinase should be higher than that of the m-aminophenol. Consequently, in the biological assays, all these non-symmetrical compounds could not compete with the ATP adenine moiety, and they could not bind simultaneously in both binding sites. With the object of justify the obtained biological results, new docking studies have been performed using only the Cho binding site as the possible zone for the insertion of the ligands. With regard to the Family B, in general, these compounds behave as good ChoK inhibitors, however its antiproliferative activity against HepG2 are very low. Compunds of Subfamily B1 and B2 ( SOS-32, SOS-33, SOS-41 and SOS-42) presents good IC50 ex vivo values against ChoK, however they do not have antiproliferative activity against HepG2, which suggest that they are too polar structures to pass through the plasma membrane Structures of Subfamily B2 containing a quinoline substituted at the 4 position by a rest of a cycloalkilamine presents good results of ChoK inhibition (SOS-38, SOS-39 y SOS-40), as well as a relatively good antiproliferative activity , stablishing a clear structure-activity relationships. All compounds have submitted a good activity against Plasmodium falciparum but its necessary to make inhibition assays against ChoK of P. falciparum, to verify that these compounds really act by the inhibition of this enzyme or if they had another mechanism of action. Bibliografía 1. Muñoz, A. Cáncer. Genes y Nuevas terapias. Eds. Feduchi, E.; Irunzun, A.: Hélice: Madrid, 1997. 2. Gibbs, Jackson B.; Mechanism-Based Target Identification and Drug Discovery in Cancer Research. Science, 2000, 287, 1969. 3. Elsayed, Y. A.; Sausville, E. A. Selected novel anticancer treatments targeting cell signalling proteins. Oncologist, 2001, 6, 517-537. 4. Campos, J. et al.; Choline kinase inhibitory effect and antiproliferative activity of new 1,1',1''-(benzene-1,3,5-trylmethylene)tris{4-[(disubstituted)amino]pyridinium} tribromides. Chem. Eur. J., 2003, Vol. 38, 109-116. 5. Campos Joaquin M. et al.; QSAR-derived choline kinase inhibitors: How rational can antiproliferative drug design be? Curr. Med. Chem., 2003, 10, 1095-1112. 6. Ramírez de Molina, A. et al.; Overexpression of choline kinase is a frequent feature in human tumor-derived cell lines and in lung, prostate, and colorectal human cancers. Biochemical and Biophysical Research Communications, 2002c, 296, 580-583. 7. Ramírez de Molina et al.; Choline Kinase Activation Is a Critical Requirement for the Proliferation of Primary Human Mammary Epithelial Cells and Breast Tumor Progression. Cancer Research, 2004, 64, 6732-6739. 8. a) C. Kent. Regulation of phosphatidylcholine biosynthesis. Prog. Lipid. Res 29 (1990) 87¿105. b) A. Yalcin, B Clem, S. Makoni, A. Clem, K. Nelson, J. Thornburg, D. Siow, A.N. Lane, S.E. Brock, U. Goswami, J.W. Eaton, S. Telang, J. Chesney. Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signalling. Oncogene , 2010, 29, 139¿149. 9. C. Aoyama, H. Liao, K. Ishidate. Structure and function of choline kinase isoforms in mammalian cells. Prog. Lipid. Res, 2004, 43, 266¿281. 10. R. Hernández-Alcoceba, L. Saniger, J. Campos, M. C. Nunez, F. Khaless, M. A. Gallo, A. Espinosa, J. C. Lacal. Choline kinase inhibitors as a novel approach for antiproliferative drug design. Oncogene, 1997, 15, 2289-2301. 11. (a) A. Ramírez de Molina, A. Rodríguez-González, R. Gutiérrez, L. Martínez-Pineiro, J. Sánchez, F. Bonilla, R. Rosell, J. C. Lacal. Overexpression of choline kinase is a frequent feature in human tumour-derived cell lines and in lung, prostate, and colorectal human cancers. Biochem. Biophys. Res. Commun., 2002, 296, 580-583. b) G. Eliyahu, T. Kreizman, H. Degani. Phosphocholine as a biomarker of breast cancer: molecular and biochemical studies. Int. J. Cancer., 2007, 120, 1721-1730. c) K. Nakagami, T. Uchida, S. Ohwada, Y. Koibuchi, Y. Suda, T. Sekine, Y. Morishita, Increased choline kinase activity and elevated phosphocholine levels in human colon cancer. Jpn. J. Cancer Res., 1999, 90, 419-424. 12. a) A. Ramírez de Molina, R. Gutiérrez, M. A. Ramos, J. M. Silva, J. Silva, F. Bonilla, J. J. Sánchez, J. C. Lacal. Increased choline kinase activity in human breast carcinomas: clinical evidence for a potential novel antitumor strategy. Oncogene, 2002, 21, 4317-4322. b) A. Ramirez de Molina, J. Sarmentero-Estrada, C. Belda-Iniesta, M. Taron, V. Ramirez de Molina, P. M. Cejas, Skrzypski, D. J. Gallego- Ortega, de Castro, E. Casado, M. A. Garcia-Cabezas, J. J. Sanchez, M. Nistal, R. Rosell, M. Gonzalez-Baron, J. C. Lacal, Expression of choline kinase alpha to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study. Lancet Oncol., 2007, 8, 889-897. 13. Ramírez de Molina et al.; Choline Kinase Activation Is a Critical Requirement for the Proliferation of Primary Human Mammary Epithelial Cells and Breast Tumor Progression. Cancer Research, 2004, 64, 6732-6739. 14. T. Shah, F. Wildes, M.F. Penet, P.T. Winnard Jr, K. Glunde, D. Artemov, E. Ackerstaff, B. Gimi, S. Kakkad, V. Raman, Z. M. Bhujwalla. Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells NMR Biomed., 2010, 23, 633¿642. 15. Lacal, J. C. et al.; Inhibition of Choline kinase as a specific cytotoxic strategy in oncogene-transformed cells. Oncogene, 2003, 22, 8803-8812. 16. Rodríguez-Gonzalez, A., Ramírez de Molina, A., Fernéndez, F., Lcal, J.C. Choline kinase inhibition induces the increase in ceramides resulting in a highly specific and selective cytotoxic antitumoral strategy as a potential mechanism of action. Oncogene, 2004, 23, 8247-8259. 17. Athar Alam, Manish Goyal, Mohd Shameel, Iqbal, Chinmay Pal, Sumanta Dey, Samik Bindu, Pallab Maity and Uday Bandyopadhyay. Novel antimalarial drugs targets: hope for new antimalarial drugs. Expert Rev. Clin. Pharmacol., 2009, 2(5), 469-489. 18. Alberge, B. et al. Comparison of the cellular and biochemical properties of plasmodium falciparum choline and ethanolamine kinanses. Biochem J., 2009, 425(1), 149-58. 19. Ancelin, ML.; Calas, M.; Vidal-Sailhan, V.; Herbute, S.; Ringwald, P.; Vial, HJ. Potent inhibitors of Plasmodium phospholipid metabolism with a broad spectrum of in vitro antimalarial activities. Antimicrob. Agents Chemother., 2003, 47(8), 2590¿2597. 20. Choubey, V. et al. Molecular characterization and localization of Plasmodium falciparum choline kinase. Biochimica et Biophysica, 2006, 1027-1038. 21. H. A. Staab, M. Haenel, Transanulare wechselwirkungen bei [2.2]Phanen, II. Synthesen won [2.2](4,4')biphenylophan, [2.2](2,7)phenanthrenophan und [2](4,4')biphenylo[2](2,7)phenanthrenophan. Chem. Ber., 1973, 106, 2190-2202. 22. D. J. Cram, H. Steinberg, Macro rings. I. Preparation and spectra of the paracyclophanes, J. Am. Chem. Soc., 1951, 73, 5691-5704. 23. SYBYL-X 1.2, Tripos International, 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA. http://www.tripos.com 24. B. Rubio-Ruíz, A. Conejo-García, P. Ríos-Marco, M. P. Carrasco-Jiménez, J. Segovia, C. Marco, M. A. Gallo, A. Espinosa, A. Entrena Guadix. Design, synthesis, theoretical calculations and biological evaluation of new non-symmetrical choline kinase inhibitors. Eur. J. Med. Chem. (2012), 50, 154-162.