Published: March 10, 2011 r2011 American Chemical Society 2378 dx.doi.org/10.1021/jm101530j | J. Med. Chem. 2011, 54, 2378–2390 ARTICLE pubs.acs.org/jmc Structure-Activity Study To Develop Cationic Lipid-Conjugated Haloperidol Derivatives as a New Class of Anticancer Therapeutics Krishnendu Pal, †,|| Subrata Kumar Pore, † Sutapa Sinha, ‡ Rajiv Janardhanan, § Debabrata Mukhopadhyay, ‡ and Rajkumar Banerjee* ,† † Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh 500007, India ‡ Department of Biochemistry and Molecular Biology and § Department of Radiology, Mayo Clinic Foundation, Rochester, Minnesota 55902, United States b S Supporting Information ’ INTRODUCTION σ receptors, a unique family of membrane-bound orphan receptor proteins, exist mainly in the central nervous system (CNS) but are expressed in various peripheral tissues at basal levels as well. 1,2 Moreover, tumor cell lines from diverse origins including neuroblastoma, glioma, melanoma, and carcinomas of breast, prostate, and lung overexpress σ receptors. 3-9 Interestingly, σ receptors are more highly expressed in rapidly proliferating cells and are down-regulated when cells become quiescent. 10,11 Their high density in various tumor cell types and particularly in rapidly proliferating cells makes σ receptors a potential target for diagnostic imaging and therapeutic agents. 8,9 Several studies suggest that σ receptor agonists induce cell death in various tumor cell lines including prostate and breast carcinoma and melanoma, with features consistent with apoptosis. 12,13 Haloperidol (Scheme 1) is one of the σ receptor agonists that has been shown to inhibit cell proliferation and induce apoptosis in neuroblastomas, melanomas, and mammary and colon carcinomas in moderate concentrations. 12-15 Some recent studies attributed this cytotoxicity to haloperidol-induced Akt-sensitive mitochondrial translocation of proapoptotic Bcl- XS 14,15 and disruption of PI3K/PDK1/Akt pathway through differential subcellular compartmentalization of PI3K and PDK1. 16 However, in spite of the fact that haloperidol shows significant antiproliferative activity toward cancer cells, its full potential has never been realized through proper chemical modifications. Recently we have shown that the targeting ability of haloper- idol toward σ receptor remains potentially unchanged when the tertiary OH group of haloperidol is chemically conjugated to phospholipids with a PEG spacer between. 17 This observation prompted us to investigate whether the apoptosis inducing ability of haloperidol can be enhanced without hampering the targeting ability. To this end, we synthesized various haloperidol derivatives where the tertiary OH group of haloperidol was conjugated to cationic lipids of varying chain lengths. Twin carbon chain containing cationic lipids with its natural affinity toward predominantly negatively charged cell membrane bind and interact to transfer genes into cells and hence constitute a distinct class of gene delivery agents. 18 We hypothesized that upon conjugation of twin carbon chain cationic lipid to haloper- idol, the resultant molecule will maintain its affinity for σ receptor and thus will not compromise its cellular entry in σ receptor overexpressing cancer cells. Moreover, the cellular entry may be further assisted by twin chain cationic lipids conjugated with it. This way the molecules’ local concentration and availability inside the cell will be enhanced in comparison to unconjugated Received: November 30, 2010 ABSTRACT: Haloperidol (HP), a neuroleptic drug, shows high affinity toward σ receptors (SR). HP and reduced-HP at higher concentration were known to induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we report the development of cationic lipid-conjugated haloperidol as a new class of anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue (HP-C8) showed significantly high, SR-assisted antiproliferative activity against cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt. Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was signifi- cantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt- protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby indicating a possible protein kinase down-regulatory and apoptosis inducing role in tumor-associated vascular cells. In conclusion, we developed σ receptor- targeting cationic lipid-modified HP derivatives as a promising class of anticancer therapeutic that concurrently affects cancer and tumor environment associated angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.