Hypoxia-selective antitumor agents. 13. Effects of acridine substitution on the hypoxia-selective cytotoxicity and metabolic reduction of the bis-bioreductive agent nitracrine N-oxide.

A series of nuclear-substituted derivatives of nitracrine N-oxide (2; a bis-bioreductive hypoxia-selective cytotoxin) were prepared and evaluated, seeking analogues of lower nitroacridine reduction potential. Disubstitution with Me or OMe groups at the 4- and 5-positions did not provide analogues with one-electron reduction potentials significantly lower than those of the corresponding monosubstituted derivatives (E(1) ca. -350 mV for both the 4-OMe and 4,5-diOMe compounds). This appears not to be due to a concomitant raising of the acridine pKa but to a lack of direct electronic effect of substituents in the ring not bearing the nitro group. Conversely, placing two OMe groups in the nitro-bearing ring does result in a substantial further lowering of reduction potential (the 2,4-diOMe analogue has an E(1) of -401 mV). The mono- and disubstituted N-oxides have substantially lower cytotoxicities than the parent nitracrine N-oxide 2 but generally retain very high hypoxic selectivity. The OMe-substituted N-oxides all showed greater metabolic stability than 2 in hypoxic AA8 cell cultures, and the 4-OMe compound 6 had improved activity in EMT6 multicellular spheroids suggesting that this metabolic stabilization may allow more efficient diffusion in tumor tissue. The parent compound 2 was selectively toxic to hypoxic cells in KHT tumors in vivo and clearly superior to nitracrine itself (although only at doses which would eventually be lethal to the host). The analogues of lower E(1), including 6, were not superior to 2 in vivo, indicating that metabolic stabilization of the nitro group is not alone sufficient to improve therapeutic utility.

Bis-bioreductive agents as hypoxia-selective cytotoxins: nitracrine N-oxide.

Drugs with two reducible centers, both of which must be metabolized by oxygen-inhibitable processes for full activation ("bis-bioreductive agents"), offer potential for the development of hypoxia-selective cytotoxins with improved oxygen sensitivity. The sidechain N-oxide (1-NCO) of the (mono)bioreductive agent nitracrine (1-NC) has been synthesized and evaluated as a potential example of such an approach. The association constant for reversible DNA binding of 1-NCO was 15-fold lower than that of 1-NC, as measured by equilibrium dialysis in a low ionic strength buffer, indicating that the N-oxide has the potential to act as a less toxic pro-drug of 1-NC. Cell uptake and aerobic cytotoxicity of 1-NCO were much lower than for 1-NC whereas its hypoxic selectivity as a cytotoxin was greatly increased. In stirred suspension cultures of AA8 cells, pure (less than 0.02% 1-NC) 1-NCO was 1000-1500 times more potent under hypoxia than in 20% O2. For 1-NC the corresponding ratio was 10 +/- 1. 1-NCO had greater hypoxic selectivity in this system than misonidazole (ratio 11), RSU 1069 (ratio 25), 8Me-5NQ (ratio 60), or SR 4233 (ratio 80). Studies of 1-NCO metabolism indicate rapid, O2-inhibited reduction to 1-NC. The data are consistent with a two-step bioactivation mechanism, with reduction of the N-oxide generating a DNA intercalator of increased binding affinity, followed by reduction of the nitro group of this DNA-targeted cytotoxin to form reactive cytotoxic metabolites.

Hypoxia-selective antitumor agents. 1. Relationships between structure, redox properties and hypoxia-selective cytotoxicity for 4-substituted derivatives of nitracrine.

The nitroacridine derivative 9-[[3-(dimethylamino)propyl]amino]-1-nitroacridine (nitracrine) is selectively cytotoxic to hypoxic tumor cells in culture. However, the compound undergoes reductive metabolism too rapidly, with the reduction not being sufficiently inhibited by molecular oxygen in aerobic tissues, for it to demonstrate the same activity in vivo. In a search for derivatives with lower reduction potentials, we have synthesized and evaluated a series of derivatives bearing 4-substituents with a wide range of electronic properties. The one-electron reduction potentials (E(1] of these compounds, when compared under conditions of equivalent ionization, were highly correlated with sigma p values. However, at pH 7 the influence of substituent electronic properties was modified by prototrophic equilibria, with the basic nature of the acridine limiting the extent to which ring substituent electronic effects can be used to modulate reduction potential of the 1-nitro group. Nevertheless, comparison of the kinetics of the killing of AA8 cells under hypoxia suggests that some metabolic stabilization of the compounds can be achieved by the use of electron-donating substituents, with such compounds retaining the hypoxia-selective toxicity of nitracrine in cell culture. However, the 4-substituted nitracrines show no clear relationship between E(1) and cytotoxic potency, in distinct contrast to simpler nitroheterocycles such as nitroimidazoles.