Matthew A. Naylor

The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions

Analogues of EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole-4-7-dione]prop-2-e n-1-ol) which lack functionality at either the C-2 or C-3 position were synthesised. The aim was to establish the importance of each group towards toxicity and to give an indication as to whether substitution at either position altered activation and toxicity after metabolism by cellular NADPH: cytochrome c (P450) reductase (P450R). MDA231 breast cancer cells were transfected with the cDNA for human P450R and stable clones were isolated. These high P450R-expressing clones were used to determine the aerobic and hypoxic toxicity of EO9 and the two analogues that lacked functionality at either C-2 or C-3. The results showed that P450R was strongly implicated in the bioactivation of EO9 and its analogues under both of these conditions. This data also showed that the C-3 functionality was primarily implicated in hypoxic toxicity.

Bioreductively-activated prodrugs for targeting hypoxic tissues: Elimination of aspirin from 2-nitroimidazole derivatives

2-Nitroimidazoles were synthesised substituted with aspirin or salicylic acid, as leaving groups linked through the (imidazol-5-yl)methyl position. Activation of aqueous solutions by CO2*- (a model one-electron reductant) resulted in release of aspirin or salicylate, probably via the 2-hydroxyaminoimidazole. The analogous 2-nitroimidazole with bromide as leaving group eliminated bromide in < 1 ms via the radical-anion.

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

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 α-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 α-carbon. Cellular and supersomal studies showed that this α-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]

Enhancement of vascular targeting by inhibitors of nitric oxide synthase

PURPOSE This study investigates the enhancement of the vascular targeting activity of the tubulin-binding agent combretastatin A4 phosphate (CA4P) by various inhibitors of nitric oxide synthases. METHODS AND MATERIALS The syngeneic tumors CaNT and SaS growing in CBA mice were used for this study. Reduction in perfused vascular volume was measured by injection of Hoechst 33342 24 h after drug administration. Necrosis (hematoxylin and eosin stain) was assessed also at 24 h after treatment. Combretastatin A4 phosphate was synthesized by a modification of the published procedure and the nitric oxide synthase inhibitors L-NNA, L-NMMA, L-NIO, L-NIL, S-MTC, S-EIT, AMP, AMT, and L-TC, obtained from commercial sources. RESULTS A statistically significant augmentation of the reduction in perfused vascular volume by CA4P in the CaNT tumor was observed with L-NNA, AMP, and AMT. An increase in CA4P-induced necrosis in the same tumor achieved significance with L-NNA, L-NMMA, L-NIL, and AMT. CA4P induced little necrosis in the SaS tumor, but combination with the inhibitors L-NNA, L-NMMA, L-NIO, S-EIT, and L-TC was effective. CONCLUSIONS Augmentation of CA4P activity by nitric oxide synthase inhibitors of different structural classes supports a nitric oxide-related mechanism for this effect. L-NNA was the most effective inhibitor studied.

S-2-AMINO-5-AZOLYLPENTANOIC ACIDS RELATED TO L-ORNITHINE AS INHIBITORS OF THE ISOFORMS OF NITRIC OXIDE SYNTHASE (NOS)

S-2-Amino-5-(2-aminoimidazol-1-yl)pentanoic acid and S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid have been used as weakly inhibitory lead compounds in the design of 2-amino-5-azolylpentanoic acids which are more potent in their inhibition of nitric oxide synthases. Treatment of 2-(Boc-amino)-5-bromopentanoic acid t-butyl ester with appropriate imidazoles and 1,2,4-triazoles and with tetrazole under basic conditions, followed by acidolytic deprotection, gave many of the required 2-amino-5-azolylpentanoic acids. Tetrazole was alkylated at 1-N and at 2-N in approximately equal amounts whereas the 1,2,4-triazoles reacted principally at 1-N. A nitrile was introduced at the 2-position of the imidazole by reaction of the 2-unsubstituted precursor with 1-cyano-4-dimethylaminopyridine. Of this series of compounds, 2-amino-5-(imidazol-1-yl)pentanoic acid was identified as the most potent member against rat iNOS, rat nNOS and a human-derived cNOS. Examination of the structure-activity relationships for the identity and substitution of the azoles has led to the proposal of a model for the binding of the inhibitors to the binding site for the natural substrate.

Comparing the anti-tumor effect of several bioreductive drugs when used in combination with photodynamic therapy (PDT)

PURPOSE To compare the effect on the RIF-1 murine sarcoma of nine bioreductive agents from five different classes when used in combination with either photodynamic therapy or clamping. METHODS AND MATERIALS RIF-1 tumors implanted intradermally in C3H mice were treated with either 50J photodynamic therapy or with 120 min clamping in combination with either misonidazole, pimonidazole, metronidazole, nimorazole, RB6145, RSU1069, SR4233, mitomycin-C, or RB90740. The tumors were measured 3 times-per-week until reaching 4 x their initial treatment volume. RESULTS RSU1069 produced the greatest anti-tumor activity in combination with both photodynamic therapy and clamping. RB6145 also substantially enhanced the effect of photodynamic therapy and clamping whereas misonidazole induced a smaller, but significant increase. Mitomycin-C had no effect under clamped conditions, but greatly increased the tumorcidal effect of photodynamic therapy. Mitomycin-C also induced an effect when given with light alone. None of the other agents showed any augmentation of the tumor cell killing induced by photodynamic therapy. CONCLUSION Of the bioreductive agents studied RSU1069, RB6145 and mitomycin-C showed the greatest anti-tumor response in combination with photodynamic therapy.

Fused pyrazine mono-N-oxides as bioreductive drugs. III. Characterization of RB 90740 in vitro and in vivo.

RB 90740 is the lead compound in a series of aromatic mono-N-oxide bioreductive drugs. The compound shows considerably greater toxicity towards hypoxic verses aerobic mammalian cells in vitro. The differential in concentration required to give the same level of cell killing under these conditions ranges from 3.5 in a human bronchio-alveolar tumor cell line up to 120 in a rodent cell line defective in the repair of DNA strand breaks. The ability of RB 90740 to cause DNA strand breaks under hypoxic conditions was confirmed by alkaline sucrose gradient and pulsed field gel electrophoresis techniques. Despite these properties demonstrated in vitro, RB 90740 was shown not to be cytotoxic to hypoxic cells in experimental murine tumors in vivo. This may be due, in part, to the level of hypoxia (< 0.02% O2) necessary to produce toxicity in vitro..

Assessment of a range of novel nitro-aromatic radiosensitizers and bioreductive drugs.

In a directed search for the best compounds for clinical evaluation, some 150 selected nitroaromatic compounds, representing 6 distinct types, namely, furans, thiophenes, imidazoles, pyrazoles, pyrroles, and triazoles, have been synthesized and tested as hypoxic cell radiosensitisers and bioreductive drugs. These compounds have a wide range of one-electron redox potentials, ranging from -700 mV for 3-nitropyrroles to -250 mV for 5-nitrofurans. Within each series, those agents bearing alkylating moieties on the side chain are generally the more effective radiosensitisers in vitro. Studies in vivo demonstrated that the bifunctional nitroimidazoles were superior to the other nitroarenes tested. In terms of bioreductive cell killing, the best differential between oxic and hypoxic cell toxicity was shown for the bifunctional 2-nitroimidazoles, which had values greater than 20. In contrast, the other classes of nitroarines generally showed differential toxicities of less than 10.

Iminoxyl radicals and stable products from the one-electron oxidation of 1-methylindole-3-carbaldehyde oximes

The radical intermediates and the stable products formed on one-electron oxidation of 1-methylindole-3-carbaldehyde oxime 2a were compared with those of 1-methylindole-3-carboxamidine oxime 4a in aqueous solution. The dibromide radical anion generated radiolytically by pulse radiolysis reacted with both 2a and 4a CNOH to yield the radical cations [CNOH]˙+, which exist in prototropic equilibria with the neutral iminoxyl radicals [CNO]˙ (pKa = 3.53 ± 0.03 and 5.01 ± 0.01 at ionic strength 0.05 M, respectively). This was confirmed by the observed primary salt-effect which accelerated the decay of the radical cations but not the iminoxyl radicals. Methylation of the N-hydroxyimino function in both 2a and 4a precluded deprotonation of the corresponding radical cations [CNOCH3]˙+. At low concentrations of 2a and high dose rates the 2a radicals [CNO]˙ decayed bimolecularly via unstable dimers to the aldehyde CO, with higher concentrations and lower dose rates favouring the chain-catalysed isomerisation of the N-hydroxyimino moiety. Radicals from 4a decay bimolecularly to form unstable dimers which degrade to produce an amide, nitrile and carboxylic acid. The observed differences in the oxidation chemistry of 2a and 4a probably reflect the enhanced stabilisation of iminoxyl radicals through α-amino substitution.

Fused pyrazine mono-n-oxides as bioreductive drugs. II cytotoxicity in human cells and oncogenicity in a rodent transformation assay

PURPOSE To determine what structural moieties of the fused pyrazine mono-N-oxides are determining factors in their in vitro cytotoxicity and oncogenicity. METHODS AND MATERIALS A new series of experimental bioreductive drugs, fused pyrazine mono-N-oxides, was evaluated in vitro for aerobic and hypoxic cytotoxicity in the HT29 human colon adenocarcinoma cell line by using clonogenic assays. The relative oncogenicities of these compounds were also determined in aerobic cultures of C3H 10T1/2 mouse embryo fibroblasts by using a standard transformation assay. RESULTS Removal of the 4-methyl piperazine side chain from the parent compound, RB 90740, reduced the potency of the hypoxic cytotoxin. Reduction of the N-oxide function increased the aerobic cytotoxicity and eliminated most of the hypoxic/aerobic cytotoxic differential. The reduced N-oxide also had significant oncogenicity, consistent with a mechanism of genotoxicity following bioreduction of RB 90740. CONCLUSION This new series of bioreductive compounds may be effective in cancer therapy, particularly the lead compound RB 90740. The oncogenic potential of these compounds is similar to that for other cancer therapies. Further studies should include evaluation of these compounds in vivo and the development of analogs with reduced oncogenic potential and retention of the hypoxic/aerobic cytotoxicity differential.