2112 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA J. Med. Chem. 1991,34, zyxwv BA 2112-2120 Fisher reagent grade. All nonaqueous reactions were carried out under an inert atmosphere unless otherwise noted. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA [ 1 zyxwvutsr JIHGFEDCB S -[ 1R *,2R *,4R *( 1R *,2R *)]I-N-[ [ 54 Dimet hylamho)- l-naphthylenyl]sulfonyl]-l-phenylalanyl-N-[2-hydroxy-5- methyl-l-(2-methylpropyl)-4-[[[2-methyl-l-[[ (2-pyridinyl- met hyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]- zyxwvu EDCBA Nim-[ (4-methylphenyl)sulfonyl]-~-histidinamide. To a well-stirred solution of Boc-His(Tos)-LVA-Ile-Amp' (270 mg, 0.32 "01) in dichloromethane (5 zyxwvutsr CBA mL) was added trifluoroacetic acid (5 mL). The reaction mixture was stirred at room temperature for 0.5 h, the solvents were removed in vacuo, and the residue was redissolved in dichloromethane. The solution was washed with saturated sodium bicarbonate solution, water, and brine, dried over anhydrous sodium sulfate, and concentrated. The solid was diasolved in dry dimethylformamide (5 mL), dansyl-PheOSue (247 mg, 0.50 mmol) and triethylamine (101 mg, 1.0 mmol) were added, and the reaction mixture was stirred at mom temperature for 72 h. The dimethylformamide was removed in vacuo, and the residue was dissolved in dichloromethane and poured into brine. The aqueous phase was extracted with 5% methanol/dichloro- methane, and the extracts were combined, dried, (anhydrous sodium sulfate), and concentrated. The residue was chromato- graphed on silica gel (elution with 5% methanol/dichloromethane) to yield dansyl-Phe-His(Tos)-LVA-Ile-Amp (172 mg, 48%) as an amorphous light green solid: IR (mull, cm-') 3291, 1648, 1619, 1545,1173, 1093; MS (FAB) m / z 1106 [M + HI+; UV (MeOH, nm) 236 (ah, 23630), 252 (ah, 19260), 259 (ah, 17500), 266 (sh, 128700,338 (4120). [ 18 -[ 1R *,2R *,4R * ( 1R *,2R *)]I-N-[ [ 5- (Dimethylamino)- l-naphthylenyl]sulfonyl]-l-phenylalanyl-~-[2-hydroxy-5- methyl- 1-(2-methylpropyl)-4-[ [ [2-methyl- 1-[ [ (2-pyridinyl- methyl)amino]carbonyl] butyl]amino]carbonyl]hexyl]-L- histidinamide. A solution of daneyl-Phe-His(Tos)-LVA-neAmp (42 mg, 0.037 mmol) and 1-hydroxybenzotriazole (15 mg, 0.11 mmol) in methanol (2 mL) was stirred at room temperature for 24 h. The reaction mixture was concentrated and the residue was chromatographed on silica gel (elution with 5% methanol/di- chloromethane followed by 5% methanol (saturated with gaseous ammonia)/dichloromethane) to provide dansyl-Phe-His-LVA- Ile-Amp" (28 mg, 80%) as an amorphous solid: IR (mull, cm-') 3288,1640,1571,1545,1145,1061; MS (FAB) m / z 952 [M + HI+; UV (MeOH, nm) 255 (15950), 260 (ah, 15370), 266 (ah, 11560), 338 (4080); exact mass calcd for CB1H&07S 952.5119, found 952.5119. [ 1s -[ 1R *,2R *,4R *( 1R *,2R *)]I-N-[ [ zy UT 5 4 Dimethylamino)- l-naphthylenyl]sulfonyl]-l-phenylalanyl-N-[2-hydrox~S- methyl-l-(2-methylpropyl)-4-[ [ [2-methyl-l-[ [ (2-pyridinyl- methy1)aminolcarbon yl]butyl]amino]carbonyl] hexyl]-^- histidinamide 2-Hydroxy- 1,2,3-~ropanetricarboxy~~ (1:3). in methanol (2 mL) and citric acid (0.037 g, 0.176 mmol) was added. Upon complete solution, the methanol was removed in vacuo and the residue was dissolved in water (5 mL). The salt (88 mg, 95%) was obtained as a fluffy white solid after lyophi- lization. Dissociation Constants. The dissociation constants, Kd)s, of the renin inhibitors were determined by the fluorescenc displacement away! with the following modifications: 3 (23 nM, in duplicate) was added to recombinant human renin (33 nM) in 0.01 M Tris, pH 7.4, 5 mM 8-octyl glucoside, at 37 OC and incubated for 1.5 h. The fluorescence intensities were recorded prior to (F,,) and after (F-) the addition of dansylated inhibitor. Unlabeled inhibitors (6.6 and 13 nM) were then added, and the incubation was continued an additional 2.5 h, after which the final fluorescence intensity, F,, was recorded. The fluorescence in- tensities were normalized to a volume of 2.5 mL. Inhibition Constants? Recombinant human renin (0.010 77 or 0.0586 pM) was preincubated at 37 OC for 2 h with, for example, 2 (0-0.27 pM) or 3 (0-1.5 pM), respectively. Twenty-five mi- croliters of the renin/inhibitor preincubation mixture was added to 100 pL of RSP, 856 and 1344 pM, respectively in 0.01 M phosphate, 10 mM n-octyl 8-D-glucopyranoside, pH 6.5, to begin hydrolysis. Cleavage of RSP by renin was followed by quantitation of the products by HPLC. The data was plotted and fitted to an integrated form of the Michaelis-Menton equation (see tex Dansyl-PheHis-LVA-IleAmp (0.055 g, 0.058 "01) was dissolved (14) Amino acid analysis: Ile, 0.994; His, 1.002. The sulfonamide linkage of dansyl-Phe was not hydrolyzed under standard conditions. Synthesis of a Series of Nitrothiophenes with Basic or Electrophilic Substitu and Evaluation as Radiosensitizers and as Bioreductively Activated Cy Michael D. Threadgill,* Paul Webb, Peter O'Neill, Matthew A. Naylor, Miriam A. Stephens, Ian J. Stratford, Shirley Cole, Gerald E. Adams, and E. Martin Fielden Medical Research Council Radiobiology Unit, Chilton, Didcot, Oxfordshire OX11 ORD, U.K. Received November 14, zyx 1990 A series of 2- and 3-nitrothiophene-5-carboxamides bearing N-(w-aminoalkyl) side chains has been prepared by treatment of the thiophenecarbonyl chloride with the appropriate (protected) w-aminoalkylamine. Analogous N-(oxiranylmethyl)nitrothiophene-5-carboxamides have been synthesized by epoxidation of the corresponding N-allylamide. Compounds in both classes were evaluated in vitro both as radiosensitizers of hypoxic mammalian cells and as selective bioreductively activated cytotoxins. The most potent radiosensitizers were those agents strong tertiary amine bases or oxiranes in the side chain. Studies in vivo showed that 2-methyl-N-[2-(dimethyl- amino)ethyl]-3-nitrothiophene-5-carboxamide caused slight radiosensitization of the KHT sarcoma in mice given 0.34 "01 kg'. However, administration of this and related tertiary amines at higher dosee waa precluded by systemic toxicity. The relative resistance of cells in hypoxic regions of solid tumors to killing by ionizing radiation remains an im- portant reason for failure of local control of cancer by radiotherapy since molecular oxygen is required as an electron acceptor for the manifestation of damage to DNA. Electron-affinic nitroheterocycles can, however, act as mimics of molecular oxygen in this process and thus can be effective as radiosensitizers of hypoxic cells.lt2 Indeed, a correlation between the one-electron reduction potenti (E1,) of such compounds and their efficiency as sensitizers of hypoxic cells in vitro to ionizing radiation has been * Author to whom correspondence should be addressed at School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, U.K. OO22-2623/91/ 1834-21 12$02.50/0 (1) Adams, G. E. Br. Med. Bull. 1973,29,48. (2) Chapman, J. D.; Greenstock, C. L.; Reuvere, A. P.; McDonald, E.; Dunlop, I. Radiat. Res. 1973,53, 190. 0 1991 American Chemical Society