Synthesis and Evaluation of Folate-Based Chlorambucil Delivery Systems for Tumor-Targeted Chemotherapy Annalisa Guaragna,* ,†,‡ Angela Chiaviello, §,‡ Concetta Paolella, † Daniele D’Alonzo, † Giuseppe Palumbo, § and Giovanni Palumbo † † Dipartimento di Chimica Organica e Biochimica, Universita ̀ di Napoli Federico II, via Cinthia, 4 I-80126 Napoli, Italy § Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Universita ̀ di Napoli Federico II, via S. Pansini, 5 I-80131 Napoli, Italy ABSTRACT: The development of tumor-targeting drug delivery systems, able to selectively transport cytotoxic agents into the tumor site by exploiting subtle morphological and physiological differences between healthy and malignant cells, currently stands as one of the most attractive anticancer strategies used to overcome the selectivity problems of conventional chemotherapy. Owing to frequent overexpression of folate receptors (FRs) on the surface of malignant cells, conjugation of cytotoxic agents to folic acid (FA) via suitable linkers have demonstrated to enhance selective drug delivery to the tumor site. Herein, the chemical synthesis and biological evaluation of two novel folate-conjugates bearing the anticancer agent chlorambucil (CLB) tethered to either an aminoether (4,7,10-trioxa-1,13-tridecanediamine) or a pseudo-β-dipeptide (β-Ala-ED-β-Ala) linker is reported. The two drug delivery systems have been prepared in high overall yields (54% and 34%) through straightforward and versatile synthetic routes. Evaluation of cell specificity was examined using three leukemic cell lines, undifferentiated U937 (not overexpressing FRs, FR - ), TPA-differentiated U937 (overexpressing FRs, FR + ), and TK6 (FR + ) cells. Both conjugates exhibited high specificity only to FR + cells (particularly TK6), demonstrating comparable antitumor activity to CLB in its free form. These data confirm the reliability of folate-based drug delivery systems for targeted antitumor therapy; likewise, they lay the foundations for the development of other folate-conjugates with antitumor potential. ■ INTRODUCTION With the exception of coronary and heart diseases, cancer remains the major cause of death in the Western world. Despite significant progress in the development of anticancer technologies (involving tumor detection, prevention, surgery, and chemotherapeutic treatments), there is still no efficient cure for patients with malignant diseases. One of the major drawbacks in anticancer chemotherapy is the lack of selectivity of cytotoxic drugs, which usually do not accurately discriminate between healthy and malignant cells, leading to systemic toxicity. To limit the onset of severe side effects, anticancer chemotherapeutics are often given at suboptimal doses, thus hampering their ability to reach tumor-destroying drug concentrations while prompting, after prolonged treatments, the development of resistance phenomena. Therefore, the construction of innovative tumor-specific drug delivery systems is urgently needed. Several approaches 1 aimed at improving selectivity of anticancer agents are currently being pursued: they are mostly based on subtle biochemical differences discriminating healthy from malignant cells. Among such approaches, those involving conjugation of the drug to tumor-specific ligands (the so-called “guided molecular missiles” or “molecular Trojan horses”) have been deeply investigated over the last years. 2 A tumor-targeting drug delivery system typically consists of a tumor recognition moiety and a cytotoxic warhead connected directly, or through a suitable linker, to form a conjugate which is inactive and sufficiently stable while in circulation; then, once selectively internalized into the cancer cell, cleavage of the conjugate restores the active cytotoxic warhead. The devised mechanism enables the conjugate to exhibit a strongly selective antitumor effect; however, to efficiently deliver the drug to the tumor site, it is important to have a detailed knowledge of the specific morphological and physiological differences distin- guishing malignant from healthy tissues. Rapidly dividing cancer cells require various nutrients and vitamins to maintain their rapid growth. Therefore, those receptors involved in the uptake of the essential vitamins for cell growth are often overexpressed on the surface of malignant cells. This key finding has allowed identification of useful biomarkers either for the imaging and detection of tumor cells or for the delivery of Received: August 12, 2011 Revised: October 31, 2011 Published: November 28, 2011 Article pubs.acs.org/bc © 2011 American Chemical Society 84 dx.doi.org/10.1021/bc200410d | Bioconjugate Chem. 2012, 23, 84-96