2886 Synthesis and biological properties of bioreductively targeted nitrothienyl prodrugs of combretastatin A-4 Peter Thomson,1 Matthew A. Naylor,1 cytoskeleton of the endothelial cells lining the tumor Steven A. Everett,1 Michael R.L. Stratford,1 vasculature (6–8). When this tubulin structure is disrupted, Gemma Lewis,1 Sally Hill,1 Kantilal B. Patel,1 the endothelial cells change shape from flat to round, Peter Wardman,1 and Peter D. Davis2 impeding blood flow through the capillary, starving the tumor of nutrients, and causing tumor cell death (8–10). 1University of Oxford, Gray Cancer Institute, Mount Vernon Preclinical animal model studies and subsequent clinical Hospital, Northwood, Middlesex, United Kingdom and 2 trials have shown that the drug drastically reduces blood Angiogene Pharmaceuticals Ltd., The Magdalen Centre, Oxford flow in tumors (11). Phase I human cancer clinical trials of Science Park, Oxon, United Kingdom the sodium CA4 phosphate prodrug (2; Fig. 1) have been successfully completed (12, 13) and the drug is currently Abstract undergoing phase II trials. 1 Nitrothienylprop-2-yl ether formation on the 3¶-phenolic The discovery of prompted the synthesis of many position of combretastatin A-4 (1) abolishes the cytotox- structural variations as improved vascular targeting agents icity and tubulin polymerization-inhibitory effects of the (14–17). However, the combretastatins also have powerful drug. 5-Nitrothiophene derivatives of 1 were synthesized antiproliferative activity against cancer cell lines in vitro followingmodel kinetic studies with analogouscoumarin (3, 14, 18–25), and it is thought that this is due to an derivatives, and of these, compound 13 represents a antimitotic action brought about by inhibition of tubulin promisingnew lead in bioreductively targetedcytotoxic polymerization. In contrast to the antivascular activity, this anticancer therapies. In this compound, optimized gem- antimitotic activity requires prolonged exposure of the cells dimethyl A-carbon substitution enhances both the aerobic to the compounds. Therefore, it is probable that the latter metabolic stability and the efficiency of hypoxia-mediated activity is generally not expressed in vivo because rapid drugrelease. Only the gem-substituted derivative 13 elimination of the compounds preclude this exposure at released 1 under anoxia in either in vitro whole-cell nontoxic doses. We have sought to exploit tumor hypoxia experiments or supersomal suspensions. The rate of and examine whether combretastatin analogues delivered release of 1 from the radical anions of these prodrugs is by a hypoxia-driven fragmentation strategy may offer the enhanced by greater methyl substitution on the A-carbon. potential to deliver prolonged tumor exposure that may be Cellular and supersomal studies showed that this antimitotic while minimizing host toxicity. This strategy A-substitution pattern controls the useful range of oxygen may also, via a bystander effect, retain vascular targeting concentrations over which 1 can be effectively released activity. It is thus the object of this study to synthesize by the prodrug. [Mol Cancer Ther 2006;5(11):2886–94] and evaluate prodrugs that on bioreductive activation break down to release an antimitotic stilbene compound 1 Introduction (i.e., CA4; ; Fig. 2). Although the phenolic 3¶-hydroxyl group of 1 is not Combretastatin A-4 (CA4; 1; Fig. 1) is an antineoplastic and essential for tubulin binding, large bulky groups in this vascular targeting stilbene that was isolated from the South region inhibit binding (9). This position was therefore Combretum caffrum African bush willow tree (1–4). This considered a good candidate for prodrug derivatization. new class of therapeutic compounds are known primarily Bioreductive targeting of phenolic compounds by indole- as vascular targeting agents, which have potential use in quinones has been shown, and much work has been disease conditions or pathologies, such as cancer, where an documented on the factors that control this process abnormal growth of blood vessels is an essential compo- (26–28). Nitroaromatic compounds have also long been nent to the disease and its progression (5). The mechanism known to exhibit similar redox properties and have been of action is through microtubule disruption, affecting the studied as potential bioreductively activated prodrug delivery systems for a variety of drugs (29–33). However, despite the body of work about compounds that break Received 7/21/06; revised 9/1/06; accepted 9/25/06. down selectively under low oxygen tensions to release an The costs of publication of this article were defrayed in part by the anticancer agent, no such compound is yet in clinical use. payment of page charges. This article must therefore be hereby marked Several problems have been encountered, including a lack advertisement in accordance with 18 U.S.C. Section 1734 solely to of stability of the prodrugs toward nonbioreductive indicate this fact. processes. Thus, carbonate-linked Taxol prodrugs were Requests for reprints: Peter Thomson, Gray Cancer Institute, University of Oxford, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom reported to be unstable toward enzymatic hydrolysis in HA6 2JR. Phone: 441923828611. E-mail: [email protected] cellular assays, thereby releasing Taxol by a nonreductive Copyright C 2006 American Association for Cancer Research. process (34). Hypoxia-activated nitroheterocyclic phos- doi:10.1158/1535-7163.MCT-06-0429 phoramidates have also been reported, which were Mol Cancer Ther 2006;5(11). November 2006 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Molecular Cancer Therapeutics 2887 0.1% trifluoroacetic acid (TFA); B: 100% acetonitrile; gradient 20% to 50% or 50% to 100% B, 4 minutes, or isocratically at 100% acetonitrile, at a flow rate of 0.5 mL/ min. Reverse-phase chromatography was conducted on Varian C18 Bond Elut straight barrel columns (sorbent mass, 1 g; volume, 6 mL; and particle size, 40 Am). Analytic TLC was done on precoated silica gel plates (60 F254, 0.2 mm Figure 1. Structures of CA4 (1) and its phosphate prodrug (2). thick, VWR). Visualization of the plates was accomplished using UV light and/or potassium permanganate staining. Solutions in organic solvents were dried by standard unstable in vivo, displaying rapid metabolism and conse- procedures, and dichloromethane, benzene, dimethylfor- quent elimination half-lives of only a few minutes (35, 36). mamide, and tetrahydrofuran were anhydrous commercial Similarly, nitroheteroaryl quaternary salts have been grades. Solvents used for chromatography were HPLC synthesized as bioreductive prodrugs of mechlorethamine, grade and obtained from Sigma-Aldrich Chemical Co. but the compounds were too unstable with regard to (Dorset, United Kingdom) 7-Hydroxy-4-methylcoumarin nonspecific release of mechlorethamine to be of use as (3), diethyl azodicarboxylate (DEAD), diisopropyl azodicar- bioreductive agents (29, 30). Thus, prodrugs showing boxylate (DIAD), and 1,1-(azodicarbonyl)dipiperidine improved stability toward nonreductive processes are (ADDP) were all obtained from Sigma-Aldrich Chemical desirable. A further consideration is the rate of release of Co. CA4 (1) was synthesized according to the procedure of the active drug under hypoxic conditions. To be effective, Pettit and Rhodes (40). 2-Hydroxymethyl-5-nitrothiophene the bioreductively activated prodrug needs to deliver the (4; ref. 41), 2-(1-hydroxyethyl)-5-nitrothiophene (6; ref. 42), drug at a rate that competes with clearance of the prodrug and ethyl 2-hydroxy-2-(5-nitrothien-2-yl)propanoate (10; and diffusion of the drug out of the solid tumor. Prodrugs ref. 43) were prepared by literature methods. that fragment faster or that fragment more efficiently at 2-Bromomethyl-5-Nitrothiophene (5) (44). Compound 4 oxygen tensions commonly found in solid tumors are (6.0 g, 38 mmol) was dissolved in dichloromethane (30 mL) desirable. and the solution was cooled to 0jC. Phosphorus tribromide 5-Nitrothiophenes have the required reduction potential (2.5 g, 50 mmol) in dichloromethane (30 mL) was then to be of use in this respect (37, 38), and we have sought to added dropwise and the solution was stirred for a further exploit this moiety for the reductive delivery of the 0.5 hour. Dichloromethane (250 mL) was added and the 1 phenolic stilbene in this manner. We report herein the solution was washed with water (2 Â 250 mL) and brine synthesis of combretastatin nitrothiophene ether-linked (100 mL), dried, and evaporated in vacuo. The product a 1 conjugates, optionally substituted on the -carbon atom. was used without further purification (6.0 g, 72%). HNMR Substitution at this position was carried out to facilitate y (60 MHz, CDCl3) 4.61 (s, 2H), 7.02 (d, J = 4.2 Hz, 1H), 7.74 manipulation of rates of reductive elimination and meta- (d, J = 4.2 Hz, 1H) ppm. bolic stability (39). Initial compounds incorporated 7- 2-(1-Bromoethyl)-5-Nitrothiophene (7). Compound 6 (5.0 3 hydroxy-4-methylcoumarin ( ) as a model because of facile g, 29 mmol) was dissolved in dichloromethane (60 mL) detection of its release in bioreductive conjugates (28). The and the solution was cooled to 0jC. Phosphorus tribromide analogous CA4 derivatives were then synthesized for (2.5 g, 50 mmol) in dichloromethane (5 mL) was then added further chemical and biological evaluation. dropwise and the solution was stirred for a further 2 hours. Dichloromethane (150 mL) was added and the solution was Materials and Methods washed with water (2 Â 250 mL) and brine (100 mL), dried, Chemistry and evaporated in vacuo. The residue was purified on silica Nuclear magnetic resonance (NMR) spectra were (25% ethyl acetate/hexane) to give a yellow oil (4.0 g, 58%). 1 y obtained at 500 MHz using a Bruker AVC500, at 250 H NMR (60 MHz, CDCl3) 2.11 (d, J = 6.6 Hz, 3H), 5.32 MHz using a Bruker AVC250, and at 60 MHz using a (q, J = 6.6 Hz, 1H), 7.03 (d, J = 4.2 Hz, 1H), 7.74 (d, J =4.2 Jeol MY60 spectrometer with tetramethylsilane as internal Hz, 1H) ppm.