pubs.acs.org/jmc Published on Web 03/10/2010 r 2010 American Chemical Society J. Med. Chem. 2010, 53, 2927–2941 2927 DOI: 10.1021/jm901722v Structure-Activity Relationships of Monomeric C2-Aryl Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) Antitumor Agents Dyeison Antonow, †,‡ Maciej Kaliszczak, § Gyoung-Dong Kang, ‡ Marissa Coffils, ‡ Arnaud C. Tiberghien, ‡ Nectaroula Cooper, † Teresa Barata, † Sibylle Heidelberger, † Colin H. James, † Mire Zloh, † Terence C. Jenkins, ) Anthony P. Reszka, ^ Stephen Neidle, ^ Sylvie M. Guichard, § Duncan I. Jodrell, §,3 John A. Hartley, # Philip W. Howard,* ,†,‡ and David E. Thurston* ,†,‡ † Cancer Research UK Gene Targeted Drug Design Research Group, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, U.K., ‡ Spirogen Ltd, 29/39 Brunswick Square, London WC1N 1AX, U.K., § Cancer Research UK Pharmacology and Drug Development Group, University of Edinburgh Cancer Research Centre, Crewe Road, Edinburgh EH4 2XR, U.K., ) Morvus Technology Limited, Ty ˆ Myddfai, Llanarthne, Carmarthen SA32 8HZ, U.K., ^ Cancer Research UK Biomolecular Structure Research Group, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, U.K., and # Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, 72 Huntley Street, London WC1E 6BT, U.K. 3 Current address: Cancer Research UK Cambridge Research Institute, University of Cambridge. Received November 21, 2009 A comprehensive SAR investigation of the C2-position of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) mono- mer antitumor agents is reported, establishing the molecular requirements for optimal in vitro cytotoxicity and DNA-binding affinity. Both carbocyclic and heterocyclic C2-aryl substituents have been studied ranging from single aryl rings to fused ring systems, and also styryl substituents, establishing across a library of 80 analogues that C2-aryl and styryl substituents significantly enhance both DNA-binding affinity and in vitro cytotoxicity, with a correlation between the two. The optimal C2-grouping for both DNA-binding affinity and cytotoxicity was found to be the C2-quinolinyl moiety which, according to molecular modeling, is due to the overall fit of the molecule in the DNA minor groove, and potential specific contacts with functional groups in the floor and walls of the groove. This analogue (14l) was shown to delay tumor growth in a HCT-116 (bowel) human tumor xenograft model. Introduction Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepine (PBD) a compounds are isolated from the fermentation broth of Streptomyces species and are known for their antibiotic and antitumor properties. 1,2 They can exist as N10-C11 carbino- lamine methyl ethers (e.g., anthramycin, 1), carbinolamines (e.g., sibiromycin, 2), or imines (e.g., the synthetic C2-aryl PBD-imines, 3, 4, and 5), although these different forms can in- terconvert at the N10-C11 position depending on solvent and isolation conditions (Figure 1). However, in all cases, the C11- position is electrophilic and enables the molecules to alkylate the NH 2 group of a guanine in the minor groove of DNA. 1 This reaction is sequence-selective and preferentially targets 5 0 -Pu- G-Pu sequences. The PBDs have a right-handed twist due to the “S”-configuration at their C11a-position, which allows them to fit perfectly within the DNA minor groove. These properties differentiate PBDs from other families of DNA- alkylating agents, and both the chemical and biological aspects of these compounds have been explored by several research groups since the discovery of anthramycin in the 1960s. 3-20 Structure-activity relationship (SAR) information for the PBDs was initially established based on biological data obtained from natural compounds. 21-23 The development of an understanding of their mode of action prompted the rational design of synthetic analogues which provided addi- tional chemical and biological information. 1,2 The recent growth of interest in PBDs is partly due to the progress of SJG-136, which is the first synthetic PBD-based compound to enter phase II clinical trials with success. 24-27 Joining two PBD monomeric units together to make PBD dimers such as SJG-136 results in greater in vitro cytotoxicity and in vivo antitumor efficacy due to the ability of these molecules to cross-link DNA. 25,28 However, PBD dimer syntheses are generally lengthier than those for PBD monomers, and this has limited the scope for structural diversification. Since the success of SJG-136, there has been interest in progressing a PBD monomer to clinical trials due to the potential advan- tages including lower molecular weight, ease of synthesis, and greater potential for structural diversification. In addition, compared to PBD dimers, the monomers are poor substrates for ABC transporters 29 which can lead to multidrug resis- tance in a clinical setting. For example, SJG-136 is a substrate for the ABC transporter P-glycoprotein (ABCB1/P-gp). 30,31 Also, other preclinical data 32 indicate that, as a class, C2- modified PBD monomers have in vivo antitumor activity in *To whom correspondence should be addressed. Phone: þ44 (0) 207 753 5932. Fax: þ44 (0) 207 753 5965. E-mail: philip.howard@ spirogen.com (P.W.H.); david.thurston@pharmacy.ac.uk (D.E.T.). a Abbreviations: BAIB, [bis(acetoxy)iodo]benzene; CT-DNA, calf thymus DNA; ΔT m , enhancement in thermal denaturation temperature of calf-thymus DNA; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbo- diimide hydrochloride salt; HCT-116, human colorectal tumor cell line; NaHMDS, sodium bis(trimethylsilyl)amide; 2D NOESY two-dimen- sional nuclear Overhauser enhancement spectroscopy; PBD, pyrrolo- [2,1-c][1,4]benzodiazepine; Pu, purine nucleotides; SAR, structure- activity relationship; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; TLC, thin layer chromatography.