Synthesis, hydroxyl radical production and cytotoxicity of analogues of bleomycin.

Two pyridine analogues of the metal complexing region of the anticancer drug bleomycin and two related but deactivated prodrugs have been linked to a 2,6-diphenylpyridine derivative as a DNA binding unit. The 2,6-diphenylpyridine system is structurally related to known amplifiers of the cytotoxicity of bleomycin. The conjugates were found to bind to DNA more strongly than bleomycin-A2 and were more cytotoxic than the corresponding compounds lacking the DNA binding unit. On exposure of a mixture of cells and prodrugs to hypoxia and then air, the prodrug containing the nitrohistidine unit was not bioreductively activated but the prodrug having an N-oxide group was bioreductively activated. This result represents a novel approach to the improvement of the therapeutic ratio of bleomycin analogues.

Phthalimide analogs of CB 1954: synthesis and bioactivation.

Four novel 4-substituted 5-nitrophthalimides (5-substituted-6-nitro-1,3-dihydro-isoindol-1,3-diones), 6, 7, 10 and 11, and the known 5 are prepared as analogs of the dinitrobenzamide prodrug CB 1954, 1, and considered as potential candidates for gene-directed enzyme prodrug therapy. All the phthalimides are poor substrates for Escherichia coli nitroreductase compared to CB 1954. However, 6, 7, 10 and 11 are reduced by both the human and rat forms of DT-diaphorase; 10 is a particularly good substrate but 7 decomposes in phosphate buffer. A cell-line panel consisting of V79 cells that have been engineered to express various levels of either the human or rat forms of DT-diaphorase in an identical cellular background was used to evaluate these compounds as prodrugs activated by this enzyme. The cytotoxic effect of CB 1954 is proportional to the activity of either the rat or human enzyme but cells expressing the rat enzyme were much more sensitive (10000-fold at higher levels of DT-diaphorase activity) than cells expressing comparable levels of the human enzyme. These results demonstrate that the resistance of human tumors to CB 1954 can be accounted for solely by the kinetic properties of the enzyme for this prodrug. The nitrophthalimide analogs overcome this kinetic failing of CB 1954. However, these compounds are not activated to produce cytotoxicity in these DT-diaphorase-expressing cell lines. It is postulated their reduction products fail to undergo an acylation reaction in a manner analogous to CB 1954. Thus, reduction by DT-diaphorase is not predictive of cytotoxicity in this class of prodrugs.

Synthesis and properties of prodrugs activated in hypoxia to give bleomycin analogues.

Prodrugs bioreductively activated to bleomycin analogues are reported. The production of hydroxyl radicals in the presence of FE(II) and dioxygen by both the prodrugs and the activated products are determined and their in vitro cytotoxicity measured.

Rates of reaction of indoleacetic acids with horseradish peroxidase compound I and their dependence on the redox potentials.

The rates of reaction of seven indole-3-acetic acid derivatives with horseradish peroxidase compound 1 at pH 5 were measured by stopped flow, and the reduction potentials and pKa of their radical cations were determined by pulse radiolysis. Reasonable correlation of these properties with Hammett substituent parameters was found, but not with Brown-Okamoto (theta +) parameters. The rates of reaction with compound I correlate well with the reduction potentials under the same conditions, with rates of reaction that increase by ca. 2.5 orders of magnitude with a 100 mV decrease in the reduction potential. This relationship is in agreement with that previously estimated for the reaction of compound I with phenols and anilines, suggesting that the rate of reaction depends solely on the reduction potential of the substrate radical, even for compounds of dissimilar structure.

Enhancement of lipid peroxidation by indole-3-acetic acid and derivatives: substituent effects.

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the N1 position. Rates of decarboxylation, pka values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.

Potential bioreductively activated hypoxia probes and post-irradiation radiosensitizers related to NITP.

NITP (1) is an effective marker of hypoxia in tumours for both microscopy and cell sorting studies and, additionally, the compound shows post-irradiation sensitization, probably by inhibition of repair of radiation damage to DNA. However, NITP does not have the substitution pattern which the immunochemical reagents are raised to recognize and the compound has very low solubility in water. We report the synthesis of an isomer (13) of NITP which has the desirable substitution pattern and is also soluble in very weak aqueous base. The successful synthesis of 13 uses a nitrosation and cyclization of a substituted uracil (16), but earlier approaches from 5 and 12 yielded the pyridoxanthine derivative 6. The preparative use of nitro group displacement reactions from 8-nitrocaffeine is shown to be a useful entry to a range of 8-substituted caffeines and is utilized to obtain two derivatives of NITP which carry aliphatic amine chains, i.e. 34 and 35.

Bioreductive markers for hypoxic cells: 2-nitroimidazoles with biotinylated 1-substituents.

The interference by oxygen with the bioreductive metabolism and binding within cells of 2-nitroimidazoles has been used to identify hypoxic cells. Three novel compounds were synthesized with a 1-substituent containing a biotin moiety. Bound adducts of these compounds could be identified in hypoxic cells in vitro by the biotin binding proteins, avidin or streptavidin, labeled with fluorescein. The metabolism and discrimination of these compounds between well-oxygenated and hypoxic cells was evaluated by flow cytometry. Ester or amide links between the 2-nitroimidazole and the biotin were degraded in the presence of mouse serum, but a compound with a C5 hydrocarbon link was stable, and this compound was suitable for evaluation in an in vivo tumor model.

DNA-binding properties of nitroarene oligopeptides designed as hypoxia-selective agents.

The DNA-binding properties of six candidate nitroarene-functionalized oligopeptides 8-13 (i.e. 5-nitrofuran, 2-nitroimidazole and 4-nitrobenzene derivatives) with potential hypoxia-selective activity, developed as analogues of the archetypal minor groove-binding ligands, netropsin 1 and distamycin 2, have been examined using an extended molecular mechanics and dynamics (MM/MD) modelling approach. The energies calculated for interaction with the d(CGCGAATTCGCG) duplex, or d(CGCAAATTTGCG)2 in the case of the elongated nitrobenzene 13, correlate with fluorimetric data obtained by indirect competitive displacement of ethidium bromide from double-stranded calf thymus DNA. The results suggest that the mode of interaction for these agents resembles the behavior of structurally unrelated bis(amidine) derivatives that also bind to AT-rich stretches of duplex B-DNA via the minor groove. Analysis of the binding behaviour indicates that the interactions are determined by enthalpic rather than entropic energy terms, suggesting that specific or differential hydration effects for the DNA and ligand may be unimportant. A rational structure-activity relationship is established for the interaction of this family of oligopeptides with DNA that also encompass the bis(amidine) class of ligands.

Hypoxia-specific inhibition of recovery from radiation damage by a novel 2-nitroimidazole with a theophylline side chain.

A novel 2-nitroimidazole with a theophylline side-chain, 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline, (NITP) was as efficient a hypoxic-cell radiosensitizer as misonidazole. However, if cells were irradiated with NITP under hypoxic conditions and then exposed to the drug under aerobic conditions, a much larger radiosensitizing effect was observed, partly because of a reduction in the size of the shoulder of the survival curve. There was little effect of NITP on the radiosensitivity of well oxygenated cells, even with post-irradiation drug contact. Split-dose survival curves showed that the drug inhibited recovery from radiation damage only when the cells were irradiated under hypoxia but not when irradiations were under oxic conditions. A reduction in the size of the shoulder of the survival curve should allow the hypoxic-cell radiosensitizing efficiency of NITP to be maintained with low doses of radiation used in multifraction cancer radiotherapy. Bifunctional drugs containing both electron-affinic and repair inhibiting groups may represent a new approach to the synthesis of hypoxic-cell targeted adjuncts to radiotherapy.

Bioreductive fluorescent markers for hypoxic cells: a study of 2-nitroimidazoles with 1-substituents containing fluorescent, bridgehead-nitrogen, bicyclic systems.

The oxygen-sensitive bioreductive binding of 2-nitroimidazoles labeled with fluorescent side chains has been used to stain hypoxic mammalian cells selectively. Several novel compounds were synthesized with a 1-substituent containing a fluorescent, bicyclic system having a bridgehead-nitrogen atom. Additional amine and secondary alcohol substituents were also included in the link between the fluorophor and the nitroimidazole to improve water solubility. Their ability to discriminate between hypoxic and oxic cells was compared by flow cytometric analysis. A wide range of cellular fluorescence and hypoxic-oxic differentials in fluorescence was observed when compounds with indolizine fluorophors were incubated with cells, and one such compound was considered suitable for further evaluation in vivo. Two compounds with bimane fluorophors gave very little cellular fluorescence when incubated with hypoxic cells.

Fluorescent markers for hypoxic cells: a study of nitroaromatic compounds, with fluorescent heterocyclic side chains, that undergo bioreductive binding.

Several novel compounds having both a 2-nitroimidazole nucleus and a fluorescent ring system in their molecular structure were prepared and evaluated as potential fluorescent probes for hypoxia. Bioreduction of nitroimidazoles, which is inhibited by oxygen, is known to lead to binding of bioreductive metabolites to cellular macromolecules and this provides a mechanism for binding the fluorescent moiety to hypoxic cells. These compounds can incorporate a wide range of fluorophors and can therefore be designed to suit the laser-line wavelengths available for excitation of fluorescence in the flow cytometer. Several nitroimidazoles with naphthalimide side chains were rapidly taken up into cells and became concentrated in the cells, thus reducing their concentration in the extracellular medium. This suggests a potential microscopic bioavailability problem with probes of this type when used in vivo as they would become progressively depleted in the extracellular fluid as they diffused from blood vessels, through layers of packed cells in tumors, to the hypoxic cells where they could undergo hypoxia-specific metabolism. Synthesis of nitroimidazoles with coumarin fluorophors led to several potentially useful probes for hypoxia; substituents on the coumarin fluorophor had a marked effect on the cellular fluorescence of these compounds.

Fluorescent markers for hypoxic cells. A study of novel heterocyclic compounds that undergo bio-reductive binding.

The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells. Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc. as a diagnostic method. In this paper, the synthesis of some heteroaromatic nitro-compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours. Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry. Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic:oxic differential fluorescence was observed. The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo.

Flow cytometric evaluation of hypoxic cells in solid experimental tumours using fluorescence immunodetection.

Numerous methods have been proposed for the detection of hypoxic cells using nitroimidazoles labelled with both radioactive and stable isotopes where the isotopic label becomes bound as a result of reductive metabolism of the nitro group. A new probe for hypoxia, 7-(4'-(2-nitroimidazol-l-yl)-butyl)-theophylline, is described where an immunologically recognisable hapten (theophylline) is covalently linked to a 2-nitroimidazole. Bioreduction of the nitroimidazole leads to binding of bioreductive metabolites, and hence the theophylline side-chain, to intracellular molecules. Immunochemical procedures are then used to stain cells containing the bound theophylline using an FITC-conjugated anti-serum. Flow cytometric analysis of stained cells is facilitated by co-staining cellular DNA, which allows discrimination of single cells in the sample and rejection of cell clumps and debris. The alternative use of an immunoperoxidase-conjugated anti-serum has been used to demonstrate the localisation of hypoxic cells in frozen tumour sections.

Nitroaryl compounds as potential fluorescent probes for hypoxia. I. Chemical criteria and constraints.

Cellular reduction of nitroaryl compounds is efficiently inhibited by oxygen, and detection of products characteristic of reduction could form the basis for diagnostic tests for the presence of hypoxic cells in tumors. The criteria for suitable compounds include a high sensitivity and selectivity of detection response between oxic and hypoxic cells, which can be provided using fluorescence detection and suitable nitroaryl compounds which have very low fluorescence until reduced. Examples described include a nitroacridine and nitronaphthalimides. Although the intercalating ability of these ring systems lead to high sensitivity for detection of reduced metabolites in vitro by flow cytometry, poor bioavailability is an unwanted consequence of intercalation. The application of several model reducing systems for reduction of potential fluorescent probes for hypoxia is described, and the absorption and fluorescence spectral characteristics of other examples of structures which could form the basis for useful probes are outlined.

Radiosensitization by non-nitro compounds.

The effects of 23 non-nitro compounds on the radiosensitivity of hypoxic Chinese hamster V79-379A or E. coli AB 1157 cells in vitro are outlined. Imidazole derivatives substituted with several alternative electron-withdrawing groups are described; the dicyanovinyl function conferred considerable radiosensitizing activity. 2,4,5-Tribromoimidazole and 2,4-dinitrophenol may show unusual radiosensitizing activity because of interference with oxidative phosphorylation. Attempts to influence radiosensitivity by compounds potentially capable of depleting intracellular sulphydryls are also described.

Structure-activity relationships in the development of hypoxic cell radiosensitizers. III. Effects of basic substituents in nitroimidazole sidechains.

The effects of substituting 2-nitroimidazoles with groups carrying basic functions were studied. Prototropic, redox, lipophilicity and protein-binding properties were compared with the efficiency in radiosensitizing hypoxic Chinese hamster V79-379A cells in vitro and the cytotoxicities of the compounds after chronic aerobic exposure. Seventeen compounds were (2-nitro-1-imidazolyl)alkylamines in which the effects of changes in the terminating base and of alkyl chain length were investigated. About an order of magnitude increase in sensitization efficiency could be observed in some compounds without any increase in the aerobic cytotoxicity compared to simple, uncharged 2-nitroimidazoles such as misonidazole. The behaviour of five hydrazones was similar to that of uncharged analogues. The methiodide quaternary salts of two of the (2-nitro-1-imidazolyl)alkylamines showed that quaternization considerably reduced sensitization efficiency. (Nitro-1-imidazolyl)alkylamines appear worthy of further investigation as hypoxic cell radiosensitizers in vivo.

Structure-activity relationships in the development of hypoxic cell radiosensitizers. I. Sensitization efficiency.

The efficiency of 35 nitroaromatic and nitroheterocyclic compounds in radiosensitizing hypoxic Chinese Hamster cells in vitro was determined. The concentration C of the compound required to achieve an enhancement ratio of 1.6 was measured, and the redox and partition properties were quantified as the one-electron reduction potential at pH 7, E, and the octanol: water partition coefficient, P, respectively. Most of the compounds studied were 2-nitroimidazoles, but some 4- and 5-nitromidazoles, 5-nitrofurans and nitrobenzenes were investigated for comparison. Together with data for nine nitroimidazoles previously reported, the results were fitted to a structure-activity relationship of the form -log C = b0 + b1E + b2 log P + b3 (log P)2 using multiple linear regression analysis. Statistical tests showed that the coefficients b2 and b3 were not significantly different from zero and the simpler equation, obtained by omitting the terms in log P, explained 85 per cent of the variance in log C. Earlier reports that the radiosensitization efficiency of nitro compounds in vitro largely depends on the reduction potential were confirmed. The conclusive demonstration that P is unimportant in vitro is valuable in interpreting the results of experiments in vivo, where P is expected to have a much greater influence on biological response.

Structure-activity relationships in the development of hypoxic cell radiosensitizers. II. Cytotoxicity and therapeutic ratio.

This paper describes measurements of the aerobic cytotoxicity of 42 nitroaromatic and nitroheterocyclic compounds towards Chinese Hamster cells in vitro. The results of acute and chronic exposure were quantified, and the concentration C required to achieve a standard response estimated. Fitting the data to an equation of the form - log C = b0 + b1E, where E is the one-electron reduction potential, explained 47 and 71 per cent of the variance in the acute and chronic aerobic cytotoxicity respectively. The addition of further terms to the equation, quantifying partition properties, was not statistically significant. The coefficient b1 was similar for both acute and chronic exposure; the dependence of both cytotoxicity and radiosensitization efficiency on reduction potential was also similar. A therapeutic ratio derived from these in vitro measurements showed no dependence on redox or partition properties. The insensitivity of cytotoxicity and radiosensitization properties to variations in molecular structure, other than those which influence redox behaviour, offers exceptional flexibility in developing compounds of improved therapeutic ratio.