We have previously designed and synthesized several series of bifunctional alkylating agents that were found to have potent activity against a variety of cancer xenograft

models [28], [29] and [30]. Among these agents, the compound BO-1012 (Figure W1A), which is a bis(methylcarbamate) derivative of 3a-aza-cyclopenta[α]indene, was shown to have potent therapeutic efficacy against inherited resistance H460 cells and bladder cancer cells with acquired cisplatin resistance (NTUB1/P) in nude mice when used in combination with arsenic trioxide [31]. Another derivative, BO-1090, was found to be effective PARP inhibitor against a variety of oral cancer cells both in vitro and in vivo [30]. Compound BO-1012 displays potent therapeutic efficacy and was selected as a lead compound for further development as an antitumor agent. However, this agent was not suitable for large-scale preparation because of the explosive and severely hazardous properties of methyl isocyanate, which was used to introduce the bis(methylcarbamate) functional group into the final product. For lead optimization, we synthesized compound 3-(4-methoxyphenyl)-9H-pyrrolo[1,2-a]indole-1,2-diyl)bis(methylene)

bis(ethylcarbamate) (BO-1509) ( Figure W1), which bears a bis(ethylcarbamate) group and can be prepared in large amounts. Notably, we found that BO-1509 possessed the ability to kill various cancer cell lines. ICLs formed by bifunctional alkylating agents are usually repaired by a complex pathway [32]. The combination of a PI3K inhibitor MEK inhibitor with an anticancer agent is therefore believed to increase the efficacy of the drug or to decrease drug resistance [33] and [34]. In this study, we investigated the anticancer activity of BO-1509 in combination with LY294002 against non–small cell lung cancer (NSCLC), which accounts for approximately 80% of lung cancer cases [35]. Protirelin More than half of patients with NSCLC have epidermal growth factor receptor (EGFR) mutations and are promisingly treated with tyrosine kinase inhibitors (TKIs), such as erlotinib or gefitinib [36], [37], [38] and [39]. Unfortunately, the emergence of resistance to targeted therapeutics

occurs nearly in all patients in a short period [40]. Therefore, in this study, we demonstrated that the combination of BO-1509 with LY294002 significantly suppressed the growth of several lung cancer cell lines, including EGFR-mutant NSCLC lines, PC9 and PC9/gef B4 cells, both in vitro and in vivo. H460 and A549 cells were obtained from the American Type Culture Collection (Manassas, VA). Gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/gef B4) cells were kindly provided by Dr Chih-Hsin Yang (Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan) [41]. CL1-5, CL83, and CL25 cells were provided by Dr Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan) [42]. A549 cells were maintained in Dulbecco’s modified Eagle’s medium.