[en] ¹H resonance assignments in the NMR spectra of the self-complementary hexadeoxyribonucleoside pentaphosphate d(5'-GCATGC)₂ and its complex with the antibiotic nogalamycin, together with interproton distance constraints obtained from two-dimensional nuclear Overhauser effect (NOE) spectra, have enabled the authors to characterize the three-dimensional structure of these species in solution. In the complex described, two drug molecules are bound per duplex, in each of two equivalent binding sites, with full retention of the dyad symmetry. Twenty-eight NOE distance constraints between antibiotic and nucleotide protons define the position and orientation of the bound drug molecule. Nogalamycin intercalates at the 5'-CA and 5'-TG steps with the major axis of the anthracycline chromophore aligned approximately at right angles to the major axes of the base pairs. The nogalose sugar occupies the minor groove of the helix and makes many contacts with the deoxyribose moieties of three nucleotides along one strand of the duplex in the 5'-TGC segment. The charged dimethylamino group and hydroxyl functions of the bicyclic sugar lie in the major groove juxtaposed to the guanine base, the bridging atoms of the bicyclic sugar making contacts with the methyl group of the thymine. Thus the antibiotic is not symmetrically disposed in the intercalation site but is in close contact in both grooves with atoms comprising the 5'-TGC strand. The intercalation cavity is wedge-shaped, the major axes of the base pairs forming the site being tilted with respect to one another

[en] New approaches to killing chemoresistant and radioresistant hypoxic cells of solid tumours include the selective release of potent cytotoxins from relatively non-toxic prodrugs through reductive metabolism and/or radiolytic reduction. Central to these studies, is an understanding of the mechanism of cytotoxin release and the basis of hypoxia-selectivity, since such information can be used to design compounds of high potency against solid tumours. Pulse radiolysis studies can offer unique insights into these underlying mechanisms in aqueous solution through the determination of thermodynamic one-electron reduction potentials of the prodrugs, rate constants for the formation and spectral charaterization of one-electron reduced prodrugs, the kinetics release of the cytotoxins from one-electron reduced prodrugs and the influence of molecular oxygen on the obligate radical intermediates. A series of different triggers, which are found to vary greatly in the rate constant for release of the effectors upon one-electron reduction of the prodrugs, will be discussed. Release of effector from a prodrug does not solely depend upon the type of trigger but can also be dependent on the type of linker and released effector. For example, whereas fast quantitative release of the mustard effector mechlorethamine is seen from the quaternary nitroimidazole upon one electron reduction, release of N-[2-(dimethylamino)ethyl] acridine-4-carboxamide (DACA), requires a higher level of reduction of the same trigger. Release of cytotoxic ligands from metal complexes requires that the metal centre is reduced. When the metal centre is lower than DACA bound as a ligand, reduction is seen to occur solely on the ligand without release from the metal centre

[en] The nitroacridine nitracrine (1-NC) is a DNA intercalator and a hypoxia-selective, electron-affinic radiosensitizer. Sensitization of Chinese hamster fibroblast cultures at 0 degrees C by the nitro positional isomers of 1-NC has now been compared to help establish structure-activity relationships. The des-nitro analog (E(1) at pH 7 = -899 mV) did not sensitize, suggesting that an electron-affinic chromophore is required. All the nitroacridines (E(1) range -376 to -257 mV) sensitized hypoxic cells with a maximum sensitizer enhancement ratio of about 1.7, but with a 200-fold range in potency. When mean intracellular drug concentrations were compared, 2-, 3-, and 4-NC had potencies which were similar, independent of E(1), and no greater than predicted for non-DNA binding nitroheterocycles. Sensitization by these three isomers occurred at intracellular concentrations likely to saturate the potential intercalation sites on DNA. A large fraction of the radical sites sensitized by O2 are apparently inaccessible to these drugs. It is suggested that sensitization results from electron transfer from migrating transient charge carriers of low reduction potential to immobile bound intercalators. An additional sensitizing mechanism may be available to 1-NC, which was 20 times more potent, a potency not accounted for by E(1), cell uptake, or DNA binding affinity. The dissociation kinetics of the DNA-drug complex was faster for 1-NC than for the other isomers. The higher potency of 1-NC may reflect a short mean residence time (less than 1 ms) in its intercalation site, allowing significant mobility on the DNA within the lifetime of relatively stable radiation-induced target radicals

[en] The conformation at the dA-dT junction in k-(AAAAATTTTT)₂ was investigated by using a variety of phase-sensitive two-dimensional nuclear magnetic resonance experiments at 500 MHz for detailed studies of the deoxyribose ring puckers. Conformational constraints were collected from two-dimensional nuclear Overhauser enhancement spectra recorded with short mixing times and from quantitative simulations of the cross-peaks in two-dimensional correlated spectra. Overall, the decamer duplex adopts a conformation of the B-DNA type, and for dA₄ and dA₅ the pseudorotation phase angle P is in the standard range 150-180 degree. The deoxyribose puckers for the other nucleotides deviate significantly from the standard B-DNA structure. Spectrum simulations assuming either static deviations from standard B-DNA or a simple two-state dynamic equilibrium between the C2'-endo and C3'-endo forms of the deoxyribose were used to analyze the experimental data. In agreement with recent papers on related duplexes containing (dA)_n tracts, the authors observed prominent nuclear Overhauser effects between adenine-2H and deoxyribose-1'H, which could be largely due to pronounced propeller twisting as observed in the crystal structures of (dA)_n-containing compounds

[en] The reactions of nitroquinolines with radical sites on DNA, formed by OH^. radical attack in aqueous solution, have been studied. It is found that the small concentrations of compound, unbound to DNA, oxidise the essentially immobile DNA radicals with diffusion-controlled rate constants of ca. 5 x 10⁹ dm³ mol^-1 s^-1 to form absorbing radical anions. Oxidation of the radical sites by the proportion of the compounds bound to DNA does not occur, except to a small extent in the case of the 6-nitro regioisomer. Calculations based on the measured intrinsic association constant and migration rate along DNA of the 6-nitro regioisomer yield a jump time between neighbouring sites on the DNA of 2.76 ns. Competition studies between such compounds which blind to DNA and xenobiotica for OH^. radical-damaged sites on DNA is illustrated by studies with three thiol compounds which yield second-order rate constants for repair of the DNA radicals, through H-atom donation; glutathione, 1.2 x 10⁶ dm³ mol^-1 S^-1, cysteine, 6 x 10⁶ dm³ mol^-1 s^-1 and cysteamine, 1.6 x 10⁸ dm³ mol^-1 s^-1. (author)

[en] Resistance of noncycling cells to amsacrine (m-AMSA) has been widely reported and may limit the activity of this drug against solid tumors. The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblasts (AA8 cells, a subline of Chinese hamster ovary-cells) in log- and plateau-phase spinner cultures. In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity induced by a 1-h exposure to m-AMSA or to its solid tumor-active analogue, CI-921. Studies with radiolabeled m-AMSA established that similar levels of drug were accumulated by log- and plateau-phase cells and that there was no significant drug metabolism in either of these cultures after 1 h. However, marked differences in sensitivity to m-AMSA-induced DNA breakage were observed using a fluorescence assay for DNA unwinding. Changes in sensitivity to DNA breakage occurred in parallel with changes in sensitivity to m-AMSA-induced cell killing. DNA breaks disappeared rapidly after drug removal (half-time approximately 4 min), suggesting that these lesions were probably mediated by DNA topoisomerase II. Resistance to m-AMSA may therefore be associated with changes in topoisomerase II activity in noncycling cells

[en] The clinically-used antitumour drug nitracrine (9-[3-dimethylaminopropyl]amino-l-nitroacridine) shows increased toxicity towards mammalian tumour cells under hypoxic conditions, but several closely-related nitroacridines do not show this selectivity. To aid studies of the mode of action of this class of compound four different tritium-labelled nitroacridines [G-³H]9-amino-l-nitroacridine, [G-³H]9-methylamino-l-nitroacridine, 9-(3-(dimethylamino)propylamino) [G-³H]-l-nitroacridine, and 9-(3-(dimethylamino)propylamino)[G-³H]-3-nitroacridine were prepared from tritiated sodium N-(3'-nitrophenyl)anthranilate. Optimum conditions for addition of the amine sidechain to the intermediate chloronitroacridines varied markedly for each derivative. The tritiated compounds proved stable as solid salts, but dilute solutions rapidly hydrolysed to the nitroacridones, in a reaction which was assisted by light. (author)

[en] Published in summary form only

[en] Complete text of publication follows. The bicyclic nitroimidazoles PA-824 and OPC-67683 are new candidate drugs for treating TB, and are in clinical trial. The drugs appear to act in an atypical manner compared to related nitroimidazoles, which are generally reduced by cellular reductases to the nitro radical anion, which either undergoes redox cycling to form superoxide (oxic conditions), or leads to the formation of nitroso- and on to hydroxylamine cytotoxins (under hypoxia). The major metabolites of PA-824 and OPC-67683 determined under aerobic and hypoxic conditions respectively, are their desnitro compounds. Recently it has been reported that these drugs release the reactive nitrogen species (RNS) NO when metabolized in the bacterium. To explore possible mechanisms by which RNS can be released, we have studied a series of nitroimidazoles related to PA-824, using pulse and steady-state radiolysis. Propan-2-ol medium (neat and with addition of 5% vol. water) has been chosen as a model for a solvent-restricting active site. In contrast to results obtained in aqueous solution, where reduction of the imidazole ring preceeds the reduction of the nitro group, we observed the release of nitrite upon the steady-state radiolytic reduction of PA-824 and analogs, with yields up to approximately one-electron equivalent. The yield of nitrite release was dependent on the electronic properties of the 3-substituent attached to the imidazole ring which forms part of the 6-membered saturated ring of PA-824. The analogs with SO and SO₂ as substituents did not yield nitrite upon one-electron reduction, and CH₂, S substituents produced low yields of nitrite. Changing the saturated 6-member ring to a 7-membered ring, also containing O as the substituent, inhibited the production of nitrite. Parallel experiments for PA-824 in 95/5% propan- 2-ol/water, did not change the nitrite yield. MS analysis of the des-nitro product, formed on radiolytic reduction, gave an m/e mass increase over that of the parent compound of +13. This product is assigned as the adduct between 2-propanol and the desnitro compound and could result from radical-radical reaction. Pulse radiolysis was used to produce the transient spectrum arising from one-electron reduction in 95/5% vol. propan-2-ol/water. The spectrum is similar to that of related nitroimidazoles in aqueous solutions. Spectra in deaerated and N₂O saturated solutions exhibit differences in 280 nm region; in deaerated solution dose independent formation of the des-nitro radical of the parent compound at 6000 s^-1 was observed. The absorption band centred at 450 nm, typical of one-electron reduced nitroimidazoles, was observed to decay with 1^st-order kinetics at 700 s^-1, independent of both the absorbed dose and nitroimidazole concentration.

[en] Tirapazamine (TPZ; 3-amino-1,2,4-benzotriazine 1,4-dioxide) is a hypoxic cytotoxin currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin based chemotherapy. To develop improved TPZ analogs we have synthesized thirty-nine 3-amino-1,2,4-benzotriazine 1,4-dioxides (BTOs) to examine structure-activity relationships (SAR) for ring substitution. The electronic, hydrophobic and steric parameters of substituents at the 5, 6, 7, and 8 positions were systematically varied, and the aqueous solubility and one-electron reduction potentials [E(1)] of the analogs were determined. For each compound we determined cell killing of mouse SCCVII tumor cells in vitro under aerobic and hypoxic conditions by clonogenic survival and determined their relative hypoxic toxicity (RHT; relative to TPZ) and hypoxic cytotoxicity ratio (HCR). A subset of compounds was independently evaluated using a 96-well SRB proliferation assay, the data from which correlated well with that derived by the clonogenic endpoint. Most substituents, except 5- and 8-dimethylamino and 8-diethylamino, gave analogs less soluble than TPZ. E(1) values ranged from -240 mV through -670 mV (with TPZ having a value of -456 mV), and correlated well with the electronic parameter sigma for substituents at the 5-, 6-, 7-, and 8-positions. The cell killing potency of the analogs under aerobic conditions showed a relatively weak positive correlation with hydrophobicity (logP) but a strong positive correlation with E(1) (i.e. electron-withdrawing substituents increased aerobic toxicity). Hypoxic cytotoxicity also generally increased with increasing E(1), with a maximum (RHT up to 3.9-fold) seen in halo- and trifluoromethyl-substituted BTO derivatives having E(1) between ca. -370 and -400 mV. The results suggest that substituents in the benzene ring of BTO analogs can be used to predictably vary one-electron reduction potentials and also provide a much better definition than previously of the optimum range of these reduction potentials for a desirable biological activity profile (high HCR, RHT and solubility)