Potent DNA-directed alkylating agents: Synthesis and biological activity of phenyl N-mustard–quinoline conjugates having a urea or hydrazinecarboxamide linker Rajesh Kakadiya a,d , Huajin Dong b , Amit Kumar a , Dodia Narsinh a , Xiuguo Zhang b , Ting-Chao Chou b , Te-Chang Lee a , Anamik Shah d , Tsann-Long Su a,c, * a Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan b Preclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA c Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan d Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India article info Article history: Received 15 December 2009 Revised 25 January 2010 Accepted 28 January 2010 Available online 4 February 2010 Keywords: DNA-directed alkylating agents Anticancer agents Quinolines Phenyl nitrogen mustards DNA interstrand cross-linking agents abstract A series of N-mustard–quinoline conjugates bearing a urea or hydrazinecarboxamide linker was synthe- sized for antitumor evaluation. The in vitro cytotoxicity studies revealed that compounds with hydrazin- ecarboxamide linkers were generally more cytotoxic than the corresponding urea counterparts in inhibiting human lymphoblastic leukemia and various solid tumor cell growths in culture. The therapeu- tic efficacy against human tumor xenografts in animal model was studied. It was shown that complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 17a, i and 18c, d was achieved. In the present study, it was revealed that both linkers are able to lower the chemically reactive N-mustard pharmacophore and thus the newly synthesized conjugates possess a long half-life in rat plasma. Moreover, the new N-mustard derivatives are able to induce DNA cross-linking either by modified comet assay or by alkaline agarose gel shift assay. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction DNA alkylating agents have been widely used in chemother- apy. 1–3 However, the progress of developing new alkylating agents is sluggish since these derivatives have several drawbacks including a lack of drug-specific affinity to tumor cells, a high chemical reactiv- ity and induced bone marrow toxicity. 4–6 Recently, there is renewed interest in the discovery of new alkylating agents for cancer chemo- therapy via the designing of DNA-directed alkylating agents or pro- drug to overcome the general drawbacks of alkylating agents. DNA-directed alkylating agents are generally synthesized by linking alkylating pharmacophores (such as N-mustard residue) to DNA-affinic molecules (such as DNA intercalating agents or DNA minor groove binder). 7–10 It has been demonstrated that these agents have higher cytotoxicity and better therapeutic efficacy than the corresponding untargeted alkylating agents. Studies on the structure–activity relationships of these conjugates suggest that selection of DNA-affinic molecule (carrier), N-mustard residue (alkyl or phenyl N-mustard) and the spacer (type and length) greatly affect their antitumor activity. For example, Tallimustine (1, FCE 24517, Chart 1) 11 was selected as an anticancer drug candi- date of this new class of cytotoxic compounds. It was evaluated in Phase I and II clinical trials and expressed a promising anticancer activity both in vivo and in vitro. 11 As for designing prodrugs, Springer et al. and other laboratories synthesized various N-mustard prodrugs by masking phenyl N- mustards with glutamic acid or tyramine via a urea (2, X = NH, 12 3, 13 and 4, 14 Chart 1), carbamate (2, X = O; 12 and 5, 15 ), and carbox- amide [6 (CMDA) 16,17 and 7 (CJS 1050) 18 ] spacer for antibody-di- rected enzyme prodrug therapy (ADEPT), 19 gene-directed enzyme prodrug therapy (GDEPT) 20 or melanocyte-directed enzyme pro- drug therapy (MDEPT). 13–15 These studies suggest that urea, carba- mate, and carboxamide linker are able to lower the reactivity of the reactive N-mustard pharmacophore. In our research on developing DNA-directed alkylating agents, we previously exemplified that alkyl N-mustard residue linked to the anilino ring or/and acridine chromophore of 9-anilinoacridines (alkyl N-mustard–9-anilinoacridine conjugates) or acridine via a methylene or alkoxy (O–C 1–4 ) linker possesses significant cytotox- icity in inhibiting various human leukemia and solid tumor cell growth in vitro as well as potent therapeutic effects against human tumor xenografts in animal model. 21–24 We have also shown that the 9-anilinoacridine conjugates were more cytotoxic than the cor- responding acridine derivatives. Moreover, we found that the length of the spacer also influences their potency. Among these 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.01.061 * Corresponding author. Tel.: +886 2 2789 9045; fax: +886 2 2782 5573. E-mail address: tlsu@ibms.sinica.edu.tw (T.-L. Su). Bioorganic & Medicinal Chemistry 18 (2010) 2285–2299 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc