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Mol Cancer Ther. 2006;5:2886-2894
© 2006 American Association for Cancer Research

Research Articles: Therapeutics, Targets, and Development

Synthesis and biological properties of bioreductively targeted nitrothienyl prodrugs of combretastatin A-4

Peter Thomson1, Matthew A. Naylor1, Steven A. Everett1, Michael R.L. Stratford1, Gemma Lewis1, Sally Hill1, Kantilal B. Patel1, Peter Wardman1 and Peter D. Davis2

1 University of Oxford, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom and 2 Angiogene Pharmaceuticals Ltd., The Magdalen Centre, Oxford Science Park, Oxon, United Kingdom

Requests for reprints: Peter Thomson, Gray Cancer Institute, University of Oxford, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom HA6 2JR. Phone: 441923828611. E-mail: thomson{at}gci.ac.uk

Abstract

Nitrothienylprop-2-yl ether formation on the 3'-phenolic position of combretastatin A-4 (1) abolishes the cytotoxicity and tubulin polymerization-inhibitory effects of the drug. 5-Nitrothiophene derivatives of 1 were synthesized following model kinetic studies with analogous coumarin derivatives, and of these, compound 13 represents a promising new lead in bioreductively targeted cytotoxic anticancer therapies. In this compound, optimized gem-dimethyl {alpha}-carbon substitution enhances both the aerobic metabolic stability and the efficiency of hypoxia-mediated drug release. Only the gem-substituted derivative 13 released 1 under anoxia in either in vitro whole-cell experiments or supersomal suspensions. The rate of release of 1 from the radical anions of these prodrugs is enhanced by greater methyl substitution on the {alpha}-carbon. Cellular and supersomal studies showed that this {alpha}-substitution pattern controls the useful range of oxygen concentrations over which 1 can be effectively released by the prodrug. [Mol Cancer Ther 2006;5(11):2886–94]

Introduction

Combretastatin A-4 (CA4; 1; Fig. 1 ) is an antineoplastic and vascular targeting stilbene that was isolated from the South African bush willow tree Combretum caffrum (1–4). This new class of therapeutic compounds are known primarily as vascular targeting agents, which have potential use in disease conditions or pathologies, such as cancer, where an abnormal growth of blood vessels is an essential component to the disease and its progression (5). The mechanism of action is through microtubule disruption, affecting the cytoskeleton of the endothelial cells lining the tumor vasculature (6–8). When this tubulin structure is disrupted, the endothelial cells change shape from flat to round, impeding blood flow through the capillary, starving the tumor of nutrients, and causing tumor cell death (8–10). Preclinical animal model studies and subsequent clinical trials have shown that the drug drastically reduces blood flow in tumors (11). Phase I human cancer clinical trials of the sodium CA4 phosphate prodrug (2; Fig. 1) have been successfully completed (12, 13) and the drug is currently undergoing phase II trials.


Figure 1
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  		Figure 1. Structures of CA4 (1) and its phosphate prodrug (2).

 
The discovery of 1 prompted the synthesis of many structural variations as improved vascular targeting agents (14–17). However, the combretastatins also have powerful antiproliferative activity against cancer cell lines in vitro (3, 14, 18–25), and it is thought that this is due to an antimitotic action brought about by inhibition of tubulin polymerization. In contrast to the antivascular activity, this antimitotic activity requires prolonged exposure of the cells to the compounds. Therefore, it is probable that the latter activity is generally not expressed in vivo because rapid elimination of the compounds preclude this exposure at nontoxic doses. We have sought to exploit tumor hypoxia and examine whether combretastatin analogues delivered by a hypoxia-driven fragmentation strategy may offer the potential to deliver prolonged tumor exposure that may be antimitotic while minimizing host toxicity. This strategy may also, via a bystander effect, retain vascular targeting activity. It is thus the object of this study to synthesize and evaluate prodrugs that on bioreductive activation break down to release an antimitotic stilbene compound (i.e., CA4; 1; Fig. 2 ).


Figure 2
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  		Figure 2. One-electron reduction of prodrug and elimination of CA4 (1).

 
Although the phenolic 3'-hydroxyl group of 1 is not essential for tubulin binding, large bulky groups in this region inhibit binding (9). This position was therefore considered a good candidate for prodrug derivatization. Bioreductive targeting of phenolic compounds by indolequinones has been shown, and much work has been documented on the factors that control this process (26–28). Nitroaromatic compounds have also long been known to exhibit similar redox properties and have been 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 down selectively under low oxygen tensions to release an anticancer agent, no such compound is yet in clinical use. Several problems have been encountered, including a lack of stability of the prodrugs toward nonbioreductive processes. Thus, carbonate-linked Taxol prodrugs were reported to be unstable toward enzymatic hydrolysis in cellular assays, thereby releasing Taxol by a nonreductive process (34). Hypoxia-activated nitroheterocyclic phosphoramidates have also been reported, which were unstable in vivo, displaying rapid metabolism and consequent elimination half-lives of only a few minutes (35, 36). Similarly, nitroheteroaryl quaternary salts have been synthesized as bioreductive prodrugs of mechlorethamine, but the compounds were too unstable with regard to nonspecific release of mechlorethamine to be of use as bioreductive agents (29, 30). Thus, prodrugs showing improved stability toward nonreductive processes are desirable. A further consideration is the rate of release of the active drug under hypoxic conditions. To be effective, the bioreductively activated prodrug needs to deliver the drug at a rate that competes with clearance of the prodrug and diffusion of the drug out of the solid tumor. Prodrugs that fragment faster or that fragment more efficiently at oxygen tensions commonly found in solid tumors are desirable.

5-Nitrothiophenes have the required reduction potential to be of use in this respect (37, 38), and we have sought to exploit this moiety for the reductive delivery of the phenolic stilbene 1 in this manner. We report herein the synthesis of combretastatin nitrothiophene ether-linked conjugates, optionally substituted on the {alpha}-carbon atom. Substitution at this position was carried out to facilitate manipulation of rates of reductive elimination and metabolic stability (39). Initial compounds incorporated 7-hydroxy-4-methylcoumarin (3) as a model because of facile detection of its release in bioreductive conjugates (28). The analogous CA4 derivatives were then synthesized for further chemical and biological evaluation.

Materials and Methods

Chemistry
Nuclear magnetic resonance (NMR) spectra were obtained at 500 MHz using a Bruker AVC500, at 250 MHz using a Bruker AVC250, and at 60 MHz using a Jeol MY60 spectrometer with tetramethylsilane as internal standard. Melting points were obtained using an Electrothermal IA9100. High-resolution mass spectra (HRMS) on chromatographically homogeneous compounds were recorded on a VG Trio 2000 mass spectrometer. Elemental analyses were carried out by Medac Ltd., Brunel Science Centre, Egham, Surrey, United Kingdom. Silica gel for flash chromatography was Merck Kieselgel 60H grade (230–400 mesh). High-performance liquid chromatography (HPLC)-mass spectrometry (MS) was done using a Waters Integrity System with electron effect ionization. Chromatography was carried out using a Hichrom (Theale, United Kingdom) RPB column (100 x 3.2 mm) with eluents A: 5% acetonitrile, 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 µm). Analytic TLC was done on precoated silica gel plates (60 F254, 0.2 mm thick, VWR). Visualization of the plates was accomplished using UV light and/or potassium permanganate staining. Solutions in organic solvents were dried by standard procedures, and dichloromethane, benzene, dimethylformamide, and tetrahydrofuran were anhydrous commercial grades. Solvents used for chromatography were HPLC grade and obtained from Sigma-Aldrich Chemical Co. (Dorset, United Kingdom) 7-Hydroxy-4-methylcoumarin (3), diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), and 1,1-(azodicarbonyl)dipiperidine (ADDP) were all obtained from Sigma-Aldrich Chemical Co. CA4 (1) was synthesized according to the procedure of Pettit and Rhodes (40). 2-Hydroxymethyl-5-nitrothiophene (4; ref. 41), 2-(1-hydroxyethyl)-5-nitrothiophene (6; ref. 42), and ethyl 2-hydroxy-2-(5-nitrothien-2-yl)propanoate (10; ref. 43) were prepared by literature methods.

2-Bromomethyl-5-Nitrothiophene (5) (44). Compound 4 (6.0 g, 38 mmol) was dissolved in dichloromethane (30 mL) and the solution was cooled to 0°C. Phosphorus tribromide (2.5 g, 50 mmol) in dichloromethane (30 mL) was then added dropwise and the solution was stirred for a further 0.5 hour. Dichloromethane (250 mL) was added and the solution was washed with water (2 x 250 mL) and brine (100 mL), dried, and evaporated in vacuo. The product was used without further purification (6.0 g, 72%). 1H NMR (60 MHz, CDCl3) {delta} 4.61 (s, 2H), 7.02 (d, J = 4.2 Hz, 1H), 7.74 (d, J = 4.2 Hz, 1H) ppm.

2-(1-Bromoethyl)-5-Nitrothiophene (7). Compound 6 (5.0 g, 29 mmol) was dissolved in dichloromethane (60 mL) and the solution was cooled to 0°C. Phosphorus tribromide (2.5 g, 50 mmol) in dichloromethane (5 mL) was then added dropwise and the solution was stirred for a further 2 hours. Dichloromethane (150 mL) was added and the solution was washed with water (2 x 250 mL) and brine (100 mL), dried, and evaporated in vacuo. The residue was purified on silica (25% ethyl acetate/hexane) to give a yellow oil (4.0 g, 58%). 1H NMR (60 MHz, CDCl3) {delta} 2.11 (d, J = 6.6 Hz, 3H), 5.32 (q, J = 6.6 Hz, 1H), 7.03 (d, J = 4.2 Hz, 1H), 7.74 (d, J = 4.2 Hz, 1H) ppm. Liquid chromatography-retention time, 6.42 minutes (TFA 50–100%); MS (m/z, %) 237 (M+, 1), 235 (M+, 1), 156 (100), 141 (6), 125 (8), 110 (12).

2-Thien-2-yl-Propan-2-ol (8). 2-Acetylthiophene (8.8 g, 70 mmol) was dissolved in anhydrous diethyl ether (200 mL) and the solution was cooled to 0°C. MeMgBr (3.0 mol/L in diethyl ether, 30 mL, 90 mmol) was then added via syringe under nitrogen. The solution was warmed to 20°C and stirring was continued for 3 hours. The solution was poured onto ice/water (25 g) and 0.1 mol/L HCl (250 mL) was added. The solution was extracted with diethyl ether (3 x 70 mL), dried, and evaporated. The residue was purified on silica (20% ethyl acetate/hexane) and then a second silica column (dichloromethane) to give a colorless oil (3.5 g, 35%). 1H NMR (60 MHz, CDCl3) {delta} 1.65 (s, 6H), 2.16 (s, 1H), 7.0 (m, 3H) ppm. LC-RT 3.09 minutes (TFA 50–100%); MS (m/z, %) 142 (M+, 12), 124 (100), 109 (93).

2-(5-Nitrothien-2-yl)Propan-2-ol (9) (45). Compound 8 (3.0 g, 16 mmol) was dissolved in acetic anhydride (50 mL) and the solution was cooled to –70°C. Fuming nitric acid (1.2 mL, 20 mmol) was then added gradually with vigorous stirring. The solution was stirred for 1 hour at –70°C and then allowed to warm to –40°C and stirred for further 1 hour at this temperature. Ice/water (300 g) was then added and the slurry was stirred for 30 minutes before extracting with ethyl acetate (3 x 75 mL). The organic layer was washed with sodium bicarbonate (saturated, 100 mL) and brine (50 mL), dried, and evaporated. The residue was purified on silica (20% ethyl acetate/hexane) to give an orange wax (1.5 g, 50%). 1H NMR (60 MHz, CDCl3) {delta} 1.67 (s, 6H), 2.1 (br, 1H), 6.88 (d, J = 4 Hz, 1H), 7.8 (d, J = 4 Hz, 1H) ppm. LC-RT 3.26 minutes (TFA 50–100%); MS (m/z, %) 187 (M+, 8), 172 (100), 157 (9), 142 (11), 127 (13).

1-(4-Methoxy-3-(5-Nitrothien-2-yl)Methyloxy)Phenyl-2-(3,4,5-Trimethoxy)Phenyl-Z-Ethene (11). Compound 4 (500 mg, 3.14 mmol) was dissolved in tetrahydrofuran (5 mL) together with triphenylphosphine (1.68 g, 6.28 mmol) and 1 (1.98 g, 6.28 mmol). To this solution was added DEAD (1.09 g, 6.28 mmol) and the solution was heated at 50°C for 3 hours and evaporated to dryness and the residue was purified on silica (25% ethyl acetate/hexane) to give a pale yellow solid (mp 88–90°C, 810 mg, 57%) after recrystallization from ethyl acetate/hexane. 1H NMR (60 MHz, CDCl3) {delta} 3.69 (s, 6H), 3.85 (s, 3H), 3.94 (s, 3H), 5.05 (s, 2H), 6.47 (bs, 4H), 6.88 (bs, 3H), 7.24 (bs, 1H), 7.79 (bs, 1H) ppm. LC-RT 9.98 minutes (TFA, 50–100%); MS (m/z, %) 457 (M+, 85), 316 (61), 301 (47), 252 (100). Anal. C; 60.47, H; 5.11, N; 2.88% C23H23NO7S requires C; 60.38, H; 5.07, N; 3.06%.

1-(4-Methoxy-3-(1-(1-(5-Nitrothien-2-yl))Ethoxy))Phenyl-2-(3,4,5-Trimethoxy)Phenyl-Z-Ethene (12). DEAD (357 mg, 2.05 mmol) was added dropwise to a solution of alcohol 6 (55 mg, 0.32 mmol), 1 (648 mg, 2.05 mmol), triphenylphosphine (288 mg, 1.10 mmol), and tetrahydrofuran (3 mL). The reaction was stirred for 16 hours at ambient temperature and then partitioned (ethyl acetate and brine), aqueous phase extracted (ethyl acetate), organic phase washed (H2O and brine), dried (MgSO4), and concentrated in vacuo. The residue was purified on silica (33% then 50% ethyl acetate/hexane and finally 100% ethyl acetate) to give a yellow oil (15 mg, 10%). 1H NMR (500 MHz, CDCl3) {delta} 1.82 (d, J = 5.0 Hz, 3H), 3.75 (s, 6H), 3.91 (s, 3H), 3.95 (s, 3H), 5.28 (q, J = 5.0 Hz, 1H), 6.50 (d, J = 5.0 Hz, 4H), 6.85 (m, 3H), 6.91 (d, J = 5.0 Hz, 1H), 7.80 (d, J = 5 Hz, 1H) ppm. LC-RT 3.85 minutes (100% CH3CN); MS (m/z, %) 471 (M+, 34), 425 (14), 316 (79), 301 (71), 252 (93), 141 (76), 125 (100); HRMS found 472.1426 C24H25NO7S requires 472.1424 (M+H).

1-(4-Methoxy-3-(2-(5-Nitrothien-2-yl)Prop-2-Yloxy))Phenyl-2-(3,4,5-Trimethoxy)Phenyl-Z-Ethene (13). 9 (200 mg, 1.07 mmol) was dissolved in benzene (2.5 mL) together with 1 (320 mg, 1 mmol) and ADDP (250 mg, 1 mmol) and the solution was maintained under argon with stirring. Tributylphosphine [200 mg, 1 mmol, dissolved in benzene (0.5 mL)] was then added via syringe and under argon. The solution was stirred for 24 hours at 20°C and then partitioned with ethyl acetate/water (100 mL) and the organic layer was washed with brine (50 mL), dried, and evaporated. The residue was purified on silica (33% ethyl acetate/hexane) and then on a second silica column (dichloromethane) to give a pale yellow oil (150 mg, 31%). 1H NMR (500 MHz, CDCl3) {delta} 1.60 (s, 3H), 1.63 (s, 3H), 3.75 (s, 3H), 3.76 (s, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 6.475 (d, J = 5 Hz, 4H), 6.74 (s, 1H), 6.81 (s, 1H), 6.86 (d, J = 5 Hz, 1H), 7.05 (d, J = 5 Hz, 1H), 7.775 (d, J = 5 Hz, 1H) ppm. LC-RT 4.34 minutes (100% CH3CN); MS (m/z, %) 485 (M+, 43), 316 (100), 301 (56); HRMS found 486.1582 C25H27NO9S requires 486.1581 (M+H).

Ethyl 2-(2-Methoxy-5Z-[2-(3,4,5-Trimethoxyphenyl)Vinyl]-Phenoxy-2-(5-Nitrothien-2-yl)Propanoate (14). DIAD (128 mg, 0.63 mmol) was added dropwise to a solution of 10 (54 mg, 0.22 mmol), 1 (100 mg, 0.32 mmol), triphenylphosphine (166 mg, 0.63 mmol), and tetrahydrofuran (1 mL). The reaction was stirred for 16 hours then adsorbed onto flash silica in vacuo. The residue was purified on silica (25% ethyl acetate/hexane) then on a second silica column (3% ethyl acetate/dichloromethane) to give a yellow oil (50 mg, 42%). TLC Rf = 0.2, 30% ethyl acetate/hexane; 1H NMR (250 MHz, CDCl3) {delta} 1.27 (t, J = 7.2 Hz, 3H), 1.78 (s, 3H), 3.76 (s, 6H), 3.85 (s, 3H), 3.89 (s, 3H), 4.26 (q, J = 7.3 Hz, 2H), 6.49 (s, 4H), 6.85 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 4.3 Hz, 1H), 7.06 (m, J = 8.4 Hz, 2H), 7.82 (d, J = 4.3 Hz, 1H) ppm; LC-RT 5.75 minutes (TFA, 50–100%); MS (m/z, %) 543 (M+, 9), 497 (8), 470(2), 316 (100), 301 (90), 283 (59), 252 (90), 241 (27), 226 (12), 213 (15), 197 (15), 183 (16), 168 (11), 154 (16), 139 (11); HRMS found 544.1632 C27H29NO9S requires 544.1636 (M+H).

(4-Methylcoumarin-7-yl)Oxymethyl-5-Nitrothiophene (15). 3 (1.0 g, 5.68 mmol) was dissolved in chloroform (15 mL) together with silver (I) oxide (1.0 g, 4.24 mmol). 5 (1.0 g, 4.5 mmol) was then added in five portions over 6 hours, and the solution was stirred for a further 48 hours. The mixture was filtered, evaporated and then dissolved in a minimum amount of chloroform, and purified by column chromatography on silica (dichloromethane followed by a second column with 10% then 50% ethyl acetate/hexane) to give a yellow oil (13 mg, 4%). 1H NMR (60 MHz, CDCl3) {delta} 1.84 (s, 6H), 2.37 (s, 3H), 6.14 (s, 1H), 6.8 (m, 2H), 6.96 (d, J = 4.2 Hz, 1H), 7.35 (m, 3H), 7.79 (d, J = 4.2 Hz, 1H). LC-RT 6.214 minutes (TFA, 50–100%); MS (m/z, %) 317 (M+, 4), 271 (16), 176 (42), 142 (100). Anal. C; 56.82, H; 3.54, N; 4.40% C17H15NO5S requires C; 56.78, H; 3.49, N; 4.41%.

2-(1-(4-Methylcoumarin-7-yl)Oxy)Ethyl-5-Nitrothiophene (16). 3 (37 mg, 0.212 mmol) was dissolved in chloroform (1 mL) together with silver (I) oxide (50 mg, 0.212 mmol). 7 (50 mg, 0.212 mmol) was then added, and the solution was stirred for a further 48 hours. The mixture was filtered, evaporated, and then dissolved in a minimum amount of chloroform and purified by column chromatography on silica (dichloromethane followed by a second a column with dichloromethane) to give a pale yellow solid recrystallized from diethyl ether (15 mg, 21%, mp 146–148°C). 1H NMR (60 MHz, CDCl3) {delta} 1.84 (s, 6H), 2.37 (s, 3H), 6.14 (s, 1H), 6.8 (m, 2H), 6.96 (d, J = 4.2 Hz, 1H), 7.35 (m, 3H), 7.79 (d, J = 4.2 Hz, 1H). LC-RT 6.214 minutes (TFA, 50–100%); MS (m/z, %) 331 (M+, 2), 176 (100), 156 (42). Anal. C; 57.76, H; 4.04, N; 4.15% C17H15NO5S requires C; 58.00, H; 3.95, N; 4.23%.

2-(1-Methyl-1-(4-Methylcoumarin-7-yl)Oxy)Ethyl-5-Nitrothiophene (17). 9 (187 mg, 1 mmol) was dissolved in tetrahydrofuran (2 mL) together with triphenylphosphine (472 mg, 1.8 mmol) and 3 (580 mg, 3.3 mmol). DEAD (574 mg, 3.3 mmol) was then added and the solution was heated at 105°C for 3.5 hours, cooled, and evaporated. The residue was dissolved in a minimum amount of acetone and purified by column chromatography on silica (dichloromethane followed by a second column with 25% ethyl acetate/hexane) to give a yellow oil. This material was purified by preparative HPLC (10% H2O/CH3CN) to give a white solid [13 mg, 4%, mp 156–158°C (dec.)] 1H NMR (60 MHz, CDCl3) {delta} 1.84 (s, 6H), 2.37 (s, 3H), 6.14 (s, 1H), 6.8 (m, 2H), 6.96 (d, J = 4.2 Hz, 1H), 7.35 (m, 3H), 7.79 (d, J = 4.2 Hz, 1H). LC-RT 2.29 minutes (100% CH3CN); MS (m/z, %) 176 (100), 170 (88), 148 (36). Anal. C; 58.99, H; 4.48, N; 4.07% C17H15NO5S requires C; 59.12, H; 4.38, N; 4.06%.

Ethyl 2-(4-Methylcoumarin-7-yl)Oxy-2-(5-Nitrothien-2-yl)Propanoate (18). DEAD (100 mg, 0.57 mmol) was added dropwise to a solution of 10 (50 mg, 0.22 mmol), 3 (120 mg, 0.68 mmol), triphenylphosphine (100 mg, 0.38 mmol), and tetrahydrofuran (2 mL). The reaction was stirred for 72 hours and then concentrated in vacuo. The residue was dissolved in a minimum amount of acetone and purified by column chromatography on silica (dichloromethane followed by a second column with 25% ethyl acetate/hexane) to give a yellow oil (10 mg, 11%). 1H NMR (60 MHz, CDCl3) {delta} 1.33 (t, J = 7.2 Hz, 3H), 2.02 (s, 3H), 2.38 (s, 3H), 4.22 (q, J = 7.3 Hz, 2H), 6.15–7.80 (m, 7H) ppm; LC-RT 2.724 minutes (100% MeCN); MS (m/z, %) 404 (M+, 5), 357 (10), 330 (22), 283 (16), 228 (90), 213 (12), 200 (45), 176 (100), 154 (38); Anal. C; 56.48, H; 4.37, N; 3.44% C19H17NO7S requires C; 56.57, H; 4.25, N; 3.47%.

Pulse Radiolysis
Prodrug radicals were formed by reduction of the parent nitro compounds (typically 50 µmol/L) by the 2-propanol radical generated radiolytically in a N2O-saturated 2-propanol/water mixture (50% v/v) with 4 mmol/L potassium phosphate buffer (pH 7.4–9). Experiments were done using a 6 MeV linear accelerator to generate an electron pulse (typically ~500 ns) as described previously (46). The absorbed radiation dose per electron pulse (typically 5–35 Gy, equivalent to 3–23 µmol/L reducing radicals) was determined by the thiocyanate dosimeter. Changes in absorbance were measured using a tungsten lamp and photodiode detector preceded by a single-pass monochromator.

Steady State {gamma}-Radiolysis
Prodrug solutions in 50% IPA/buffer (typically 40 µmol/L or below depending on solubility) were saturated with N2O in gas-tight syringes and then irradiated in a 60Co source. An absorbed dose of 1 Gy = 0.62 µmol/L 2-propanol radicals in N2O-saturated 2-propanol/water mixture (50% v/v) as determined by ferricyanide reduction. A dose rate of ~3.9 Gy min–1 was used, as determined by Fricke dosimetry.

HPLC
HPLC analysis was done using a Waters 2695 separations module, Waters 2996 photodiode array detector, and Waters 474 fluorescence detector. Data were collected using Waters Millennium software. The column was a C18 Hichrom RPB (100 x 3.2 mm) and the eluents were A: 10% acetonitrile, 90% water; B: 75% acetonitrile, 25% water. A gradient from 65% to 100% B was used over 3 minutes, with a 1-minute hold at 100%, and the flow rate was 1 mL/min. Detection was by absorbance at 292 nm (prodrugs) and by fluorescence (320 nm excitation and 390 nm emission; CA4).

Supersome Experiments
Prodrugs were dissolved in DMSO to a concentration of 625 µmol/L, and 20 µL of this stock solution were added to a mixture of 60 µL Supersomal P450 reductase (P450R; Gentest, BD Biosciences, Oxford, United Kingdom), 20 µL NADPH (10 mmol/L), and 2.4 mL potassium phosphate buffer [250 mmol/L (pH 7.4)] to give a final prodrug concentration of 5 µmol/L and incubated at 37°C. For anoxic experiments, the mixture was degassed with N2 for 20 minutes before prodrug addition and then overgassed with N2 during incubation. Samples (100 µL) were added to acetonitrile (100 µL), mixed, and then centrifuged at 14,300 rpm for 2 minutes before HPLC analysis.

Later experiments, showing comparative release of CA4 (1) over a range of oxygen tensions, were carried out by dissolving prodrugs in DMSO to a concentration of 52 µmol/L, and 60 µL were added to a mixture of 10 µL Supersomal P450R, 10 µL NADPH (10 mmol/L), and 1.17 mL potassium phosphate buffer [250 mmol/L (pH 7.4)] to give a final prodrug concentration of 2.5 µmol/L and incubated at 37°C. Anoxic and hypoxic experimental mixtures were degassed with either N2, 0.02%, 0.04%, 0.06%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, or 5% O2, or air for 20 minutes before prodrug addition and then overgassed with the appropriate gas during incubation. Samples (100 µL) were added to acetonitrile (80 µL), mixed, and then centrifuged at 14,300 rpm for 2 minutes before HPLC analysis. The oxygen content in the samples was measured using an Oxy Lab fitted with an oxygen-monitoring probe (Oxford Optronix, Oxford, United Kingdom).

A549 Whole-Cell Experiments
Prodrugs were dissolved in DMSO to a concentration of 625 µmol/L, and 80 µL were added to 10 mL A549 cells in Eagle's MEM (~106 cells/mL) to give a final prodrug concentration of 5 µmol/L and incubated at 37°C. For anoxic experiments, the mixture was degassed with N2 + 5% CO2 for 30 minutes before prodrug addition and then overgassed with N2 during incubation. Hypoxic experimental mixtures were degassed with either 0.1% or 0.3% O2 (5% CO2, balance N2) for 30 minutes before prodrug addition and then overgassed with the appropriate gas during incubation. Samples (1 mL) were added to 100 mmol/L hydrochloric acid (0.2 mL), mixed, and then extracted (via solid-phase extraction) before HPLC analysis.

Liver Metabolism
Metabolism of 5 µmol prodrug in air was done with 0.5 mL mouse liver homogenate [4 mg protein (Bradford assay)] with 100 µmol/L NADPH in 50 µmol/L potassium phosphate buffer at pH 7.4 incubated at 37°C. Samples were taken at regular intervals, added to an equivalent volume of acetonitrile, mixed, and centrifuged at 14,300 rpm for 2 minutes before HPLC analysis.

Tubulin Binding
A tubulin binding assay kit (Totam Biologicals Ltd., Peterborough, United Kingdom) was used to determine the relative binding efficiencies of the prodrugs. Composition A (100 µL) was added to a vial containing 250 µg bovine tubulin protein and either 2 µL DMSO for control or 2 µL of the appropriate concentration of drug in DMSO. The sample was mixed well and transferred to a spectrophotometer cuvette in a cell holder previously equilibrated at 37°C and the absorbance was read immediately at 340 nm. Absorbance readings were continued at 2-minute intervals for 1 hour.

Results and Discussion

The primary and secondary 5-nitrothienyl alcohols, 4 and 6, were prepared by sodium borohydride reduction of commercially available 5-nitrothiophene-2-carboxaldehyde and 2-acetyl-5-nitrothiophene, respectively (Fig. 3 ). Preparation of the gem-dimethylalcohol 9 involved Grignard addition of methylmagnesium bromide to 2-acetylthiophene and then careful nitration of the intermediate tertiary alcohol 8 with fuming nitric acid and acetic anhydride. {alpha}-Hydroxy ester 10 was synthesized via a vicarious nucleophilic substitution reaction as described by Lawrence et al. (43).


Figure 3
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  		Figure 3. Synthesis of nitrothiophene triggers. Reagents: (i) NaBH4, MeOH, 0°C; (ii) PBr3, DCM, 0°C; (iii) MeMgBr, Et2O, 0°C; (iv) Ac2O, f.HNO3, –40°C; (v) ref. 43.

 
Two different sets of Mitsunobu conditions (47) were used to couple the alcohols to stilbene 1 (Fig. 4 ). The more commonly used reagent mixture of DEAD (or DIAD)/triphenylphosphine was used to synthesize primary and secondary alkyl-aryl ethers, 11 and 12. Surprisingly, etherification of the hindered {alpha}-hydroxy ester 14 was effected using the same conditions, whereas a combination of ADDP/tributylphosphine was necessary for successful preparation of the hindered tertiary alkyl-aryl ether, 13. Synthesis of the analogous coumarins (Fig. 4) was carried out in the same manner or by treating the bromides 5 and 7, synthesized by phosphorus tribromide bromination of the alcohols, with silver (I) oxide and 7-hydroxy-4-methylcoumarin (3).


Figure 4
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  		Figure 4. Synthesis of combretastatin prodrugs and 7-hydroxy-4-methyl-coumarin analogues. Reagents: for 11, 12, 14–16, and 18: either (i) DEAD (or DIAD), triphenylphosphine, THF, or (ii) Ag2O, CHCl3 (with 5 or 7; for 13 and 17. ADDP, tributylphosphine, benzene.

 
Bioreductive activation of prodrugs may involve obligate one-electron donors, typically flavoproteins, such as NADPH:cytochrome P450R, or two-electron reduction, typically diaphorases, such as NQ01. In the present work, we have emulated reduction by one-electron processes in the hypoxic environments of solid tumors, and the inhibition of this process in the normoxic environments of normal tissues, by generating prodrug radical anions radiolytically. This enables us to evaluate the relative abilities of bioreductively activated prodrugs to release the active drug after one-electron reduction. Pulse radiolysis with spectrophotometric detection allows the generation and monitoring of the prodrug radical (Eqs. 1–4) in microseconds and subsequent radical fragmentation or reaction with O2, usually in milliseconds.

Formula 1(1)

Formula 2(2)

Formula 3(3)

Formula 4(4)
where ArNO2– is the prodrug radical anion.

Table 1 shows radical anion half-lives of conjugates of nitrothiophenes with both CA4 (1) and coumarin derivatives. The data clearly show that geminal {alpha}-dimethyl substitution increases both the rate and the efficiency of reductive elimination from reduced nitrothiophenes, with a 10-fold decrease in radical half-life for the {alpha}-gem-substituted CA4 conjugate compared with its {alpha}-monosubstituted analogue. The related coumarin derivatives exhibited a similar, although more pronounced trend, with relative half-lives of 850:45:1 for the series, {alpha}-unsubstituted, {alpha}-methyl, and {alpha}-gem-dimethyl. The efficiency of reductive elimination (fragmentation efficiency) was also increased by geminal substitution within this series.


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  		Table 1. Characterization of radical fragmentation and effector release efficiency using radiolytic production of radicals

 
The selection of 13 over the related lesser-substituted prodrugs was supported following studies of reduction by cytochrome P450R supersomes (Fig. 5 ). These data show that 1 is selectively produced by 13 in P450R supersomes under anoxia, whereas 11 and 12 were ineffective in this respect.


Figure 5
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  		Figure 5. CA4 (1) production in P450R supersomes under air (A) and anoxia (B). Prodrugs were dissolved in DMSO to a concentration of 625 µmol/L, and 20 µL were added to a mixture of 60 µL Supersomal P450R (Gentest), 20 µL NADPH (10 mmol/L), and 2.4 mL potassium phosphate buffer (pH 7.4) to give a final prodrug concentration of 5 µmol/L and incubated at 37°C. For anoxic experiments, the mixture was degassed with N2 for 20 min before prodrug addition and then overgassed with N2 during incubation. Samples (100 µL) were added to acetonitrile (100 µL), mixed, and then centrifuged at 14,300 rpm for 2 min before HPLC analysis. Points, single observation at each time.

 
In oxic liver homogenates, 13 was metabolically stable over an incubation period of 16 hours, whereas the other, lesser-substituted analogues 11 and 12 were not. These data are presented in Fig. 6 and suggest that such substitution has also successfully inhibited undesired aerobic metabolism, which was suffered by 11 and 12 during this incubation period. Benzylic ethers, with at least one hydrogen atom on the {alpha}-carbon, are prone to oxidation in oxic liver metabolites to give the corresponding carbonyl compound (i.e., an ester), which can then be cleaved by cellular esterases thus producing 1. Compound 13 was also stable when incubated in aerobic A549 whole-cell suspensions but released 1 efficiently under anoxic conditions (Fig. 7 ). The efficiency of this drug release in supersomal suspensions over a range of clinically relevant oxygen concentrations was determined and the results are presented in Fig. 8 . The radiolysis studies with the coumarin model prodrugs 15 to 18 showed the fragmentation rate-enhancing effect of {alpha}-substitution. Fragmentation is expected to be in competition with the reversal of reduction by oxygen (Eq. 5).

Formula 5(5)


Figure 6
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  		Figure 6. Prodrug metabolism in oxic liver homogenates. Metabolism of 5 µmol prodrug in air was done with 0.5 mL mouse liver homogenate (~4 mg protein by the Bradford assay) with 100 µmol/L NADPH in 50 mmol/L potassium phosphate buffer at pH 7.4 incubated at 37°C. Samples were taken at regular intervals, added to an equivalent volume of acetonitrile, mixed, and then centrifuged at 14,300 rpm for 2 min before HPLC analysis. Points, single observation at each time.

 

Figure 7
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  		Figure 7. CA4 (1) production from 13 in A549 whole cells. ANG704 (13) was dissolved in DMSO to a concentration of 625 µmol/L, and 80 µL were added to 10 mL A549 cells in Eagle's MEM (~106 cells/mL) to give a final prodrug concentration of 5 µmol/L and incubated at 37°C. For anoxic experiments, the mixture was degassed with N2 for 30 min before prodrug addition and then overgassed with N2 during incubation. Samples (1 mL) were added to 100 mmol/L hydrochloric acid (0.2 mL), mixed, and then extracted (via solid-phase extraction) before HPLC analysis. Points, single observation at each time.

 

Figure 8
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  		Figure 8. Release of CA4 (1) from 13 under different oxygen concentrations in A549 cells. ANG704 (13) was dissolved in DMSO to a concentration of 625 µmol/L, and 80 µL were added to 10 mL A549 cells in Eagle's MEM (~106 cells/mL) to give a final prodrug concentration of 5 µmol/L and incubated at 37°C. Anoxic and hypoxic experimental mixtures were degassed with either N2 or 0.1% or 0.3% O2 for 30 min before prodrug addition and then overgassed with the appropriate gas during incubation. Samples (1 mL) were added to 100 mmol/L HCl (0.2 mL), mixed, and then extracted (via solid-phase extraction) before HPLC analysis. Columns, mean of three replicates of one experiment; bars, SD.

 
This suggests that such substitution might extend the range of oxygenation status within the cells that could be targeted. It was thus predicted, based on these model studies, that the geminally substituted 13 would exhibit the broadest range of oxygen tensions that could be targeted, and Fig. 8 does indeed indicate that significant CA4 release was achieved up to a 0.3% oxygen concentration in A549 cells with this compound. The range of oxygen concentrations over which CA4 was released in supersomal suspensions by compounds 11 to 13 is shown in Fig. 9 . The unsubstituted analogue 11 only released CA4 over a narrow range of low oxygen concentrations (<0.01% O2) with complete inhibition above this level. The monosubstituted (12) and geminal (13)-substituted compounds, on the other hand, efficiently released the drug over a much broader range of oxygen concentrations, with 13 being >50% inhibited (compared with N2) only above 0.5% O2.


Figure 9
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  		Figure 9. Oxygen concentration dependence of CA4 reductive release from prodrugs 11 to 13. Prodrugs were dissolved in DMSO to a concentration of 52 µmol/L, and 60 µL were added to a mixture of 10 µL Supersomal P450R, 10 µL NADPH (10 mmol/L), and 1.17 mL potassium phosphate buffer [250 mmol/L (pH 7.4)] to give a final prodrug concentration of 2.5 µmol/L and incubated at 37°C. Anoxic and hypoxic experimental mixtures were degassed with either N2, 0.02 %, 0.04 %, 0.06 %, 0.1 %, 0.2 %, 0.3 %, 0.5 %, 1 %, 2%, or 5% O2, or air for 20 min before prodrug addition and then overgassed with the appropriate gas during incubation. Samples (100 µL) were added to acetonitrile (80 µL), mixed, and then centrifuged at 14,300 rpm for 2 min before HPLC analysis. Points, mean of three replicates of one experiment; bars, SD.

 
The prodrug 13 did not inhibit the growth of A549 cells up to concentrations of 2 µmol/L (Fig. 10 ) and similarly had no effect on tubulin polymerization (Fig. 11 ), further showing the prodrug status of the compound and the successful masking of the activity of 1 through ether linkage to a nitrothiophene moiety.


Figure 10
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  		Figure 10. Cytotoxicity studies of 1 and 13 in A549 cells. A549 cells were seeded in Eagle's MEM supplemented with 10% FCS and nonessential amino acids at 103 per well on a 96-well plate and allowed to attach for 24 h. Compounds were dissolved in DMSO and diluted with cell culture medium before addition. The cells were exposed to test compound (0–2 µmol/L) for 6 h and then incubated for a further 72 h. Viable cells were determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega Corp., Southampton, United Kingdom). Points, mean of eight wells of one experiment.

 

Figure 11
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  		Figure 11. Inhibition of tubulin polymerization by 1 and 13. Done with 250 µg pure bovine brain tubulin (Totam Biologicals) in G-PEM buffer comprising 80 mmol/L piperazine-N,N'-bis(2-ethanesulfonic acid) sesquisodium salt; 0.5 mmol/L magnesium chloride; 0.5 mmol/L ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N',-tetraacetic acid; 1 mmol/L GTP (pH 6.8). Tubulin polymerization was observed by measuring the absorbance (340 nm) of the solution in a quartz cuvette at 37°C over time. Points, single observation.

 
In conclusion, the presence of two {alpha}-substituents bestows beneficial properties on the compounds by inhibiting aerobic metabolism. The increased steric bulk provided by these substituents may thus stabilize the compounds against release of the cytotoxic or cytostatic drug moiety by chemical or enzymatic processes other than the desired bioreductive processes. Furthermore, the absence of a hydrogen atom {alpha} to the aromatic group is likely to prevent metabolic oxidation at this position, which can lead to release of the effector outside hypoxic regions. The electronic effects of substituents R1 and R2 clearly enhance the rate of reductive elimination and leads to an extended range of hypoxic oxygen tensions at which the cytotoxic or cytostatic moiety is released (up to 1% oxygen for compound 13). This may ultimately provide effective and selective delivery of the cytotoxic or cytostatic compound to a solid tumor.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/21/06; revised 9/ 1/06; accepted 9/25/06.

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