Hypoxia-selective antitumor agents. 10. bis(nitroimidazoles) and related Bis(nitroheterocycles): development of derivatives with higher rates of metabolic activation under hypoxia and improved aqueous solubility.

A series of analogues of the previously described compound N-[2-(2-methyl-5-nitroimidazol-1H-yl)ethyl]-4-(2-nitroimidazol- 1H-yl)butanamide (4), a novel hypoxic cell cytotoxin and radiosensitizer, have been prepared and evaluated for hypoxia-selective cytotoxicity and hypoxic cell radiosensitization in vitro. The new derivatives were designed to overcome the low aqueous solubility of 4 and its slow kinetics of killing under hypoxia. The nitroheterocycle unit had a significant effect on solubility, with 3-nitrotriazoles being about 6-fold more soluble than the corresponding 2-nitroimidazoles. Analogues with a range of neutral linker chains (polyhydroxy, alkanesulfonamide, and bisamide) showed only slightly improved solubility and were unable to be fully evaluated. However, a series of analogues with cationic amine linkers had adequate aqueous solubility (up to 280 mM). The amine analogues could not be prepared by direct reduction of precursor amides such as 4 and were most conveniently synthesized by aza-Wittig condensation of the appropriate azide and aldehyde components. The amine-linked compounds were more cytotoxic than 4, with the symmetrical bis(2-nitroimidazole) derivatives (13 and 14) up to 9-fold more potent. They showed hypoxic selectivities comparable to that of 4 (ca. 200-fold) but had much more rapid kinetics of killing under hypoxia, resulting in high hypoxic selectivity at early times in culture. The nature of the mechanism of cytotoxicity of these compounds remains unclear but appears not to be DNA cross-linking, with the compounds showing a lack of hypersensitivity toward repair-deficient UV4 cells. The enhanced solubility and hypoxia-selective cytotoxicity (at early times) of 13 compared with 4 represent significant potential advantages.

Hypoxia-selective antitumor agents. 6. 4-(Alkylamino)nitroquinolines: a new class of hypoxia-selective cytotoxins.

A series of isomeric 4-[[3-(dimethylamino)propyl]amino]nitroquinolines has been synthesized and evaluated as hypoxia-selective cytotoxins and as radiosensitizers of hypoxic cells. The compounds showed widely-differing hypersensitivity factors (ratios of cytotoxicity against wild-type and repair-deficient mammalian cells). Many compounds showed oxygen-sensitive bioreduction resulting in DNA alkylation, while others show oxygen-insensitive modes of action. Of the nitro isomers studied, the 5-nitro showed the greatest hypoxic selectivity. A series of ring-substituted analogues were then prepared, in an effort to lower its reduction potential of -286 mV. Structure-activity studies showed that the effects of substitution on reduction potential were complex, being mediated by electronic and steric effects on the nitro group, as well as by effects on quinoline pKa. Two compounds of lower reduction potential, the 3- and 8-methyl analogues, showed improved selectivity (47- and 60-fold in a clonogenic assay). These two compounds also showed the highest "in vitro therapeutic indices" of the series as hypoxic cell radiosensitizers. Despite these favorable in vitro properties, neither compound had activity against hypoxic cells in SCCVII tumors when administered at 60% of the MTD.

5-Nitro-4-(N,N-dimethylaminopropylamino)quinoline (5-nitraquine), a new DNA-affinic hypoxic cell radiosensitizer and bioreductive agent: comparison with nitracrine.

Targeting of electron-affinic radiosensitizers to DNA via noncovalent binding (e.g., intercalation) may offer the potential for increasing sensitizing efficiency. However, it has been suggested that high-affinity DNA binding may compromise sensitization by restricting the mobility of sensitizers along the DNA, and by decreasing rates of extravascular diffusion in tumors. The weak DNA intercalator nitracrine (1-NC) is a more efficient radiosensitizer than related nitroacridines with higher DNA-binding affinities (Roberts et al., Radiat. Res. 123, 153-164, 1990). The present study investigates whether electron-affinic agents of even lower DNA-binding affinity may be superior to nitroacridines. The quinoline analog of 1-NC, 5-nitraquine (5-NO), was shown to have an intrinsic association constant for calf thymus DNA in 20 mM phosphate buffer which was 12-fold lower than that of 1-NC. 5-Nitraquine was not accumulated as efficiently as 1-NC by AA8 cells, but, despite a similar one-electron reduction potential, was 2- to 3-fold more potent than 1-NC as a hypoxia-selective radiosensitizer in vitro when compared on the basis of average intracellular concentration. Thus the radiosensitizing potency of 5-NQ appears not to be compromised by its low DNA-binding affinity. The cytotoxic mechanisms of 5-NQ and 1-NC appear to be similar (hypoxia-selective formation of DNA monoadducts), but 5-NQ is 1200-fold less potent than 1-NC as a cytotoxin. Despite this advantage, 5-NQ was not active in vivo as a radiosensitizer in SCCVII tumors. This lack of activity appears to be due to its relatively high toxicity in vivo (intraperitoneal LD50 of 105 mumol kg-1 in C3H/HeN mice), high one-electron reduction potential (-286 mV), and rapid metabolism to the corresponding amine in mice. The in vitro therapeutic index (hypoxic radiosensitizing potency/aerobic cytotoxic potency) of this weak DNA binder was lower than that of the non-DNA targeted radiosensitizer misonidazole, suggesting that DNA targeting enhances cytotoxicity more than radiosensitization. Development of useful DNA-targeted radiosensitizers may require the exploitation of DNA binding modes different from those of the nitroacridines and nitroquinolines.

Correlation of the radiosensitization potency afforded by nitroacridine intercalators with their electron scavenging efficiency in DNA.

Nitracrine (1-NC) and its nitro-positional analogues are electron-affinic DNA intercalating compounds that act as hypoxic cell radiosensitizers in cell culture, but are too rapidly metabolized to provide radiosensitization in vivo. We have explored the electron-trapping efficiencies of nitroacridines to see if such compounds can scavenge radicals formed in irradiated DNA and if the efficiency of such trapping is related to their radiosensitization properties. We have shown that a correlation does indeed exist as 1-NC, the most potent radiosensitizer, scavenges approximately 45% of the migrating electrons (formed by attachment of eaq- to the DNA) compared with approximately 4% for 4-NC, the least efficient radiosensitizer. The quenching of the delayed fluorescence from acridine orange bound to DNA was also used to probe intracellular uptake and association with DNA. The results are in accord with earlier measurements of average uptake and a suggestion that intercalators which form DNA complexes with short residence times will be preferable to highly bound agents as radiosensitizers. Since 1-NC possesses the smallest association constant with DNA of the four compounds, it is suggested that its high radiosensitization potency may well be related to it being able to interact with more radical sites on the DNA than the other analogues, in addition to its capture of migrating electrons in DNA.

NLA-1: a 2-nitroimidazole radiosensitizer targeted to DNA by intercalation.

Targeting of electron affinic radiosensitizers to DNA via reversible non-covalent intercalative binding has potential for increasing sensitizer concentrations locally at the DNA target while decreasing accessibility to reductases responsible for bioactivation and cytotoxicity. We have prepared an DNA-targeted acridine-linked 2-nitroimidazole (NLA-1) as an example of such a compound. NLA-1 binds reversibly to DNA with an affinity similar to 9-aminoacridine, and is approximately 1000 times more potent than MISO as a cytotoxin, despite a similar reduction potential. It shows less enhancement of cytotoxicity under hypoxia (5- to 6-fold) than does MISO (approximately 11-fold), but is a potent hypoxia-selective radiosensitizer in AA8 cells with a concentration for an enhancement ratio of 1.6 (C1.6) of 9 microM. The mean intracellular concentration at the C1.6 is 400 microM, on which basis its potency is about twice that of MISO. The in vitro therapeutic index (aerobic cytotoxic potency/hypoxic C1.6) of NLA-1 is approximately 6-fold lower than that for MISO. NLA-1 lacks radiosensitizing activity against SCCVII or EMT6 tumors in vivo at the maximum tolerated dose (MTD) of 100 mumol.kg-1.

Radiosensitization of mammalian cells in vitro by nitroacridines.

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.

Hypoxia-selective radiosensitization of mammalian cells by nitracrine, an electron-affinic DNA intercalator.

The radiosensitizing ability of the 1-nitroacridine nitracrine (NC) is of interest since it is an example of a DNA intercalating agent with an electron-affinic nitro group. NC radiosensitization was evaluated in Chinese hamster ovary cell (AA8) cultures at 4 degrees C in order to suppress the rapid metabolism and potent cytotoxicity of the drug. Under hypoxic conditions, submicromolar concentrations of NC resulted in sensitization (SER = 1.6 at 1 mumol dm-3). Sensitization was also seen under aerobic conditions but a concentration more than 10-fold higher was required. In aerobic cultures NC radiosensitization was independent of whether cells were exposed before and during, or after, irradiation. Postirradiation sensitization was not observed under hypoxic conditions. The time dependence of NC uptake and the development of radiosensitization were similar (maximal at 30 min at 4 degrees C under hypoxia) suggesting that sensitization, unlike cytotoxicity, is due to unmetabolized drug. NC is about 1700 times more potent as a radiosensitizer than misonidazole. This high potency is adequately accounted for by the electron affinity of NC (E(1) value at pH7 of -275 mV versus NHE) and by its accumulation in cells to give intracellular concentrations approximately 30 times greater than in the medium. However, concentrations of free NC appear to be low in AA8 cells, presumably because of DNA binding. If radiosensitization by NC is due to bound rather than free drug, it suggests that intercalated NC can interact very efficiently with DNA target radicals. This is despite a binding ratio in the cell estimated as less than 1 NC molecule/400 base pairs under conditions providing efficient sensitization. This work suggests a new approach in the search for more effective clinical radiosensitizers, and poses questions on the means by which intercalated drugs can interact with DNA damage.

Growth in cadmium-containing medium induces resistance to heat in E. coli.

We have shown that 10 microM Cd2+ in the growth medium can induce resistance to subsequent heat treatment in E. coli B/r. Resistance was shown by cells during an extended lag phase and, especially, during log phase. The results contrast with the effect of Cd2+ exposure on radiation lethality, for which sensitization was previously reported in cells from lag and stationary phase cultures.

Interactions between cadmium and radiation in the killing of Escherichia coli.

CdCl2 (10 microM) added to the growth medium caused an extension to the lag phase in the growth of Escherichia coli. Radiation sensitivity in the extended lag phase was increased, particularly at low radiation doses. However, exposure to Cd2+ did not affect log-phase cultures which are normally more sensitive than lag-phase cultures. The increase in radiation resistance normally seen in stationary phase was delayed and reduced. When the cells had adapted to growth in the Cd2+-containing medium, further Cd2+ exposure was ineffective unless a Cd2+-free medium was provided for several hours. Prior growth in 25 microM Zn2+ partially protected against the effects of Cd2+ on growth and radiation sensitivity.

Combination of misonidazole and m-AMSA: effects on radiation sensitivity and toxicity in E. coli.

Simultaneous administration of misonidazole and m-AMSA resulted in additive radio-sensitization and toxicity in E. coli B/r. At low drug doses the toxicities may be super-additive. However, experiments in N2 and O2 indicated that a therapeutic benefit from the combination is unlikely. Depending upon the actual sequence and the presence of O2, pretreatment by one drug could lead to enhancement or reduction of the toxicity of a subsequent exposure to the other drug. In particular, an m-AMSA pretreatment made the toxicity of misonidazole independent of the oxygen concentration. Possible implications of the interaction of the two chemicals are discussed.

Enhanced killing of mammalian cells by radiation combined with m-AMSA.

m-AMSA is an intercalating agent at present on Phase II trial as a chemotherapeutic drug. A 30 min exposure of Chinese hamster (Line V79-753B) cells to submicromolar concentrations of m-AMSA killed 50% of the cells. The survivors had an enhanced sensitivity to radiation-induced cell killing. Depending upon the conditions, m-AMSA enhanced the radiation effect by either a decrease in the survival-curve shoulder or by an increase in slope. m-AMSA may act partly by suppressing the accumulation of sublethal damage but, if so, recovery from damage as measured in split-dose experiments with cells pretreated with the drug is not affected to its radiation effect from selective toxicity to cells in a radioresistant phase of the cell cycle cannot be excluded. Radiation and the drug interacted to give increased cell killing, even when the exposures to each agent were separated in time. It is concluded that m-ASMA may behave like actinomycin D and adriamycin, and enhance clinical radiation responses. In vivo testing to determine the effect of m-AMSA on the therapeutic index is recommended.

Radiosensitization of E. coli B/r by 9-anilinoacridines.

Six anilinoacridine derivatives have been tested for the ability to act as radiosensitizers. Two gave good sensitization at concentrations of 100 microM or less. Both of these are known to possess significant activity against experimental tumours, and one (m-AMSA) is in Phase II clinical trial as a chemotherapeutic drug. Anilinoacridines may have potential as drugs with both a chemotherapeutic and radiosensitizing role. In spite of their structural similarity, the 2 derivatives which sensitize do so by different mechanisms. Compound VI behaves like a typical hypoxic cell sensitizer but Compound I (m-AMSA) interferes with the accumulation of sublethal damage in either the presence or absence of O2. The latter also displays a post-irradiation sensitizing effect. Differences in mechanism may be related to the relative DNA-binding abilities and electronic differences between the 2 drugs.

Radiosensitization of E. coli B/r by the cytotoxic agent procarbazine: a hypoxic cell sensitizer preferentially toxic to aerobic cells and easily oxidized.

Procarbazine has been shown to be a hypoxic cell sensitizer of moderate ability in E. coli B/r, with an achievable enhancement ratio of 1.4 at subtoxic concentrations. The drug appears to act in a manner similar to the expected with the electron-affinic radiosensitizers. However, procarbazine and the electron-affinic sensitizers differ in two important respects. Unlike the electron-affinic sensitizers, procarbazine is not easily reduced, but is easily oxidized. It is more toxic to aerobic than to hypoxic cells. At the drug dosages in present clinical use, procarbazine is likely to be only a weak radiosensitizer. The possible implications of the data for the further development of a new class of sensitizers and combination therapy are discussed.