GC-Targeted C8-Linked Pyrrolobenzodiazepine−Biaryl Conjugates with Femtomolar in Vitro Cytotoxicity and in Vivo Antitumor Activity in Mouse Models Khondaker M

Article pubs.acs.org/jmc GC-Targeted C8-Linked Pyrrolobenzodiazepine−Biaryl Conjugates with Femtomolar in Vitro Cytotoxicity and in Vivo Antitumor Activity in Mouse Models Khondaker M. Rahman,† Paul J. M. Jackson,† Colin H. James,‡ B. Piku Basu,‡ John A. Hartley,§ Maria de la Fuente,‡ Andreas Schatzlein,‡ Mathew Robson,∥ R. Barbara Pedley,∥ Chris Pepper,⊥ Keith R. Fox,# Philip W. Howard,∇ and David E. Thurston*,† † Department of Pharmacy, Institute of Pharmaceutical Sciences, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom ‡ UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom § Cancer Research UK Drug−DNA Interactions Research Group, UCL Cancer Institute, Paul O'Gorman Building, 72 Huntley Street, London, WC1E 6BT, United Kingdom ∥ UCL Cancer Institute, University College London, Paul O’Gorman Building, 72 Huntley Street, London WC1E 6BT, United Kingdom ⊥ Institute of Cancer & Genetics, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom # Centre for Biological Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom ∇ Spirogen Ltd., QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom *S Supporting Information ABSTRACT: DNA binding 4-(1-methyl-1H-pyrrol-3-yl)- benzenamine (MPB) building blocks have been developed that span two DNA base pairs with a strong preference for GC-rich DNA. They have been conjugated to a pyrrolo[2,1- c][1,4]benzodiazepine (PBD) molecule to produce C8-linked PBD−MPB hybrids that can stabilize GC-rich DNA by up to 13-fold compared to AT-rich DNA. Some have subpicomolar IC50 values in human tumor cell lines and in primary chronic lymphocytic leukemia cells, while being up to 6 orders less cytotoxic in the non-tumor cell line WI38, suggesting that key DNA sequences may be relevant targets in these ultrasensitive cancer cell lines. One conjugate, 7h (KMR-28-39), which has femtomolar activity in the breast cancer cell line MDA-MB-231, has significant dose-dependent antitumor activity in MDA-MB- 231 (breast) and MIA PaCa-2 (pancreatic) human tumor xenograft mouse models with insignificant toxicity at therapeutic doses. Preliminary studies suggest that 7h may sterically inhibit interaction of the transcription factor NF-κB with its cognate DNA binding sequence. ■ INTRODUCTION known DNA-targeting agents with a view to designing novel fi DNA is a well-known target for chemotherapeutic intervention, molecules targeted to speci c sequences within genes to allow selective modulation of expression for both research and and the mechanism of action of many currently used anticancer 1,7 drugs involves the inhibition of DNA-related processes such as therapeutic purposes. This has led to substantial progress by 1 a number of groups, the best known example being the hairpin replication and transcription. Many small molecules are known 3,8 to interact in either the major or minor grooves of DNA, or polyamides of Dervan and co-workers. The PBDs are sequence-selective DNA minor-groove insert between base pairs in an intercalative mode. Some bind 9−13 noncovalently, whereas others form monocovalent bonds or binding agents. The naturally occurring PBDs produced inter- or intrastrand cross-links.2 Some molecules such as the by Streptomyces and Micrococcus species are monomeric (e.g., minor-groove noncovalently binding natural products netropsin anthramycin, 3; Figure 1) and form singly alkylated DNA 6,12,19 (1) and distamycin (2) (Figure 1) are selective for AT-rich adducts, whereas the synthetic PBD dimers consist of two sequences of DNA, whereas other families of agents such as the PBD units joined through a C8/C8′-linker and can form minor-groove covalently binding pyrrolo[2,1-c][1,4]- interstrand or intrastrand DNA cross-links in addition to benzodiazepines (PBDs) (e.g., anthramycin, 3) are selective − for GC-rich sequences.3 6 There has been significant interest in Received: December 21, 2012 trying to understand the molecular basis of the selectivity of Published: March 21, 2013 © 2013 American Chemical Society 2911 dx.doi.org/10.1021/jm301882a | J. Med. Chem. 2013, 56, 2911−2935 Journal of Medicinal Chemistry Article Figure 1. Structures of the AT-targeting minor-groove noncovalently binding agents netropsin (1) and distamycin (2), the GC-targeting covalently binding anthramycin (3), and the C8-linked PBD−polyamide conjugate GWL-78 (4). − monoadducts.14 17 One PBD dimer, SJG-136, has successfully the sequence selectivity of the two components. These − completed phase I clinical trials18 20 and is presently conjugates were also shown to have excellent cellular/nuclear undergoing phase II evaluation in ovarian and hematological penetration properties, and a degree of correlation was cancers. PBD molecules have a chiral center at their C11a(S)- observed between cytotoxicity and DNA-binding affinity. position, which provides them with an appropriate 3-dimen- Furthermore, a robust sequence-selective blockade of tran- sional shape to fit perfectly within the DNA minor groove.12 scription at sites approximately corresponding to their DNA They also possess an electrophilic N10−C11 moiety (i.e., footprints was observed. interconvertible imine, carbinolamine, or carbinolamine methyl To develop molecules capable of targeting longer GC-rich ether functionalities) that can form a reversible covalent aminal DNA sequences as potential transcription factor inhibitors, we linkage between their C11-position and the nucleophilic C2- decided to explore replacement of the polypyrrole units within 12,21 NH2 group of a guanine base. PBD monomers such as 3 these PBD conjugates with GC-recognizing fragments. The lack typically span three base pairs of DNA with a reported of GC-preference for polypyrrole molecules such as distamycin preference for 5′-Pu-G-Pu-3′ sequences,1,12 although more and netropsin is thought to relate to the wider minor-groove recent data suggest that they have a kinetic preference for 5′- width in GC-rich regions of DNA and the presence of the Py-G-Py-3′ sequences.22 It is known from the literature that the exocyclic 2-amino groups of guanine bases which protrude into DNA binding affinity of PBD molecules correlates well with the minor groove. This prevents these molecules, most of their in vitro cytotoxicity in tumor cell lines,6,15,23 and PBD which are naturally curved, from achieving multiple close van dimers that can cross-link DNA have a greater cytotoxic der Waals contacts with functional groups in the floor and walls potency compared to PBD monomers that can only of the minor groove.3,4 Several studies using empirical force monoalkylate.15 As a result of covalently binding to DNA, fields have concluded that van der Waals contacts are the PBDs can mediate a number of biological effects including the dominant factor in sequence recognition, and Dervan and co- inhibition of endonucleases,24 RNA polymerase,6,25 and workers have devised a sequence rule based on the pairing of transcription factor binding.26,27 For the PBD dimers, the different five-membered heterocycles in a hairpin polyamide most likely basis for their antitumor activity is thought to be DNA motif.8 However, these rules are only valid when preferential DNA repair in healthy cells compared to tumor polyamides are linked through a hairpin motif, and so do not cells, with the repair response depending on the cell type, and apply to linear polyamides. Furthermore, poor cellular uptake the extent and duration of exposure to the agent.28 Tumor cells and nuclear penetration of the relatively high molecular weight are often deficient in one or more relevant DNA repair hairpin polyamides have been major disadvantages for these pathways, thus leading to selective cytotoxicity and antitumor molecules, and this has limited their use as tools in cellular activity in vivo.29 Many PBD molecules also have significant experiments and as potential therapeutic agents. Therefore, in a − antimicrobial activity.30 35 different approach to this problem, we have focused on the In 2006 we reported a series of C8-linked PBD−poly(N- development of a series of biaryl building blocks designed to methylpyrrole) conjugates (e.g., GWL-78, 4; Figure 1) which possess a unique curvature in order to enhance van der Waals demonstrated the synergistic effect of joining a GC-specific contacts within the GC-tracts of the DNA minor groove. Such PBD unit to an AT-recognizing polypyrrole fragment, as building blocks, which span two DNA base pairs, offer a illustrated by a significant increase in DNA binding affinity (up synthetic advantage when designing longer more complex gene- to 50-fold) for 4 compared to its component PBD and targeting molecules, in that fewer building blocks are required tripyrrole fragments.6 DNA footprinting experiments showed to span a greater number of base pairs. that these molecules bind to DNA sequences, with the binding We started by designing a library of fragments to span two site size increasing with molecular length and with the majority DNA base pairs based on simple phenyl-substituted hetero- ′ of sites conforming to the consensus motif 5 -XGXWz (X = any cycles of appropriate length (e.g., Figure 2). With each base but preferably a purine, W = A or T, z =3± 1), reflecting containing one heterocyclic and one carbocyclic component, 2912 dx.doi.org/10.1021/jm301882a | J. Med. Chem. 2013, 56, 2911−2935 Journal of Medicinal Chemistry Article interaction at one or more of the NF-κB consensus recognition sites, thus inhibiting the NF-κB signaling pathway (Figure 8A). The novel biaryl building blocks developed through this study contribute to the pool of molecular fragments available for the synthesis of DNA-targeting agents, and differentiate themselves from other agents through their CG-recognition properties. The significant in vitro and in vivo activity of 7h, the large differential effect on tumor compared to non-tumor (i.e., Figure 2.

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