Hypoxic Cytotoxicity Research Articles

Hypoxia-Activated Prodrugs: Substituent Effects on the Properties of Nitro seco-1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]indol-4-one (nitroCBI) Prodrugs of DNA Minor Groove Alkylating Agents

Nitrochloromethylbenzindolines (nitroCBIs) are a new class of hypoxia-activated prodrugs for antitumor therapy. The recently reported prototypes undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents and are selectively toxic to some but not all hypoxic tumor cell lines. Here we report a series of 31 analogues that bear an extra electron-withdrawing substituent that serves to raise the one-electron reduction potential of the nitroCBI. We identify a subset of compounds, those with a basic side chain and sulfonamide or carboxamide substituent, that have consistently high hypoxic selectivity. The best of these, with a 7-sulfonamide substituent, displays hypoxic cytotoxicity ratios of 275 and 330 in Skov3 and HT29 human tumor cell lines, respectively. This compound (28) is efficiently and selectively metabolized to the corresponding aminoCBI, is selectively cytotoxic under hypoxia in all 11 cell lines examined, and demonstrates activity against hypoxic tumor cells in a human tumor xenograft in vivo.

Cytotoxic palladium complexes of bioreductive quinoxaline N1, N4-dioxide prodrugs

Four new palladium(II) complexes with the formula Pd(L) 2, where L are quinoxaline-2-carbonitrile N 1, N 4-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and 1H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd( L1) 2 and Pd( L2) 2, where L1 is 3-aminoquinoxaline-2-carbonitrile N 1, N 4-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N 1, N 4-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd( L3) 2, where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N 1, N 4-dioxide, resulted in vitro more potent cytotoxin in hypoxia ( P = 5.0 μM) than the corresponding free ligand ( P = 9.0 μM) and tirapazamine ( P = 30.0 μM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd( L2) 2 introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd( L1) 2 and Pd( L3) 2 produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd( L3) 2 showed a more marked effect than Pd( L1) 2. Indeed, for the highest doses assayed, Pd( L3) 2 was even able to introduce positive supercoils on the plasmid DNA.

Design of antiangiogenic hypoxic cell radiosensitizers: 2-Nitroimidazoles containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety

We designed chiral 2-nitroimidazole derivatives containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety as antiangiogenic hypoxic cell radiosensitizers. Based on results of molecular orbital calculations, the 2-aminomethylene-4-cyclopentene-1,3-dione moiety is expected to show high electrophilicity comparable to that of the 2-methylene-4-cyclopentene-1,3-dione moiety included in TX-1123 and tyrphostin AG17. We evaluated the antiangiogenic and radiosensitizing effects of the new compounds, along with other biological properties including their activities as hypoxic cytotoxicities and protein tyrosine kinase (PTK) inhibitory activities. Among the compounds tested, 5 (TX-2036) proved to be the strongest antiangiogenic hypoxic cell radiosensitizer. All the other chiral 2-nitroimidazole derivatives having 2-aminomethylene-4-cyclopentene-1,3-dione moiety tested were also antiangiogenic hypoxic cell radiosensitizers. The PTK inhibitory activity of 5 (TX-2036) showed this to be a promising and potent EGFR kinase inhibitor, having an IC 50 value of lower than 2 μM. This compound also was an Flt-1 kinase inhibitor having an IC 50 value of lower than 20 μM. Our results show that these chiral 2-nitroimidazole derivatives that contain the 2-aminomethylene-4-cyclopentene-1,3-dione moiety as a potent antiangiogenic pharmacophoric descriptor are promising lead candidates for the development of antiangiogenic hypoxic cell radiosensitizers.

Hypoxia-Selective 3-Alkyl 1,2,4-Benzotriazine 1,4-Dioxides: The Influence of Hydrogen Bond Donors on Extravascular Transport and Antitumor Activity

Tirapazamine (TPZ) and related 1,2,4-benzotriazine 1,4 dioxides (BTOs) are selectively toxic under hypoxia, but their ability to kill hypoxic cells in tumors is generally limited by their poor extravascular transport. Here we show that removing hydrogen bond donors by replacing the 3-NH2 group of TPZ with simple alkyl groups increased their tissue diffusion coefficients as measured in multicellular layer cultures. This advantage was largely retained using solubilizing 3-alkylaminoalkyl substituents provided these were sufficiently lipophilic at pH 7.4. The high reduction potentials of such compounds resulted in rates of metabolism too high for optimal penetration into hypoxic tissue, but electron-donating 6- and 7-substituents moderated metabolism. Pharmacokinetic/pharmacodynamic model-guided screening was used to select BTOs with optimal extravascular transport and hypoxic cytotoxicity properties for evaluation against HT29 human tumor xenografts in combination with radiation. This identified four novel 3-alkyl BTOs providing greater clonogenic killing of hypoxic cells than TPZ at equivalent host toxicity, with the 6-morpholinopropyloxy-BTO 22 being 3-fold more active.

Chemical synthesis and biological evaluation of a NAD(P)H:quinone oxidoreductase-1–targeted tripartite quinone drug delivery system

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V(max) and K(m) values of 11.86 +/- 3.09 micromol/min/mg and 2.70 +/- 1.14 micromol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.

Synthesis, Structure and Hypoxic Cytotoxicity of 3‐Amino‐1,2,4‐benzotriazine‐1,4‐dioxide Derivatives.

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Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia

Hypoxia is a common trait found in many solid tumours and thus represents a therapeutic target with considerable potential. PR-104, a hypoxia-activated prodrug currently in clinical trial, is a water-soluble phosphate ester which is converted in vivo to the corresponding alcohol, PR-104A. This 3,5-dinitrobenzamide-2-nitrogen mustard is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and 5-amine (PR-104M) in hypoxic cells. The clinical effectiveness of PR-104 will depend in part on the expression of reductases within tumours that can effect this reduction. Here, we evaluate the roles of NADPH:cytochrome P450 oxidoreductase (CYPOR; E.C.1.6.2.4) and NAD(P)H:quinone oxidoreductase (NQO1; E.C.1.6.99.2) as candidate PR-104A reductases. A weak correlation was observed between NQO1 activity and aerobic cytotoxicity in a panel of eight tumour cell lines. However, overexpression of human NQO1 did not increase cytotoxicity of PR-104A or the formation of PR-104H/M, showing that PR-104A is not a substrate for NQO1. Overexpression of human CYPOR did, however, increase the hypoxic cytotoxicity of PR-104A, and its metabolism to PR-104H and PR-104M, demonstrating it to be a PR-104A reductase. To assess the contribution of CYPOR to overall activation of PR-104A in hypoxic SiHa cells, a combination of siRNA transfection and antisense expression were used to suppress CYPOR protein by 91% (±3%), a phenotype which conferred 45% (±7%) decrease in cytotoxic potency of PR-104A. Regression analysis of all CYPOR depletion data was found to correlate with cytoprotection and metabolism ( p < 0.001). Residual PR-104A reductase activity could be inhibited by the flavoprotein inhibitor diphenyliodonium. We conclude that CYPOR is an important PR-104A reductase, but that other flavoenzymes also contribute to its activation in hypoxic SiHa cells.

Synthesis, Structure and Hypoxic Cytotoxicity of 3‐Amino‐1,2,4‐benzotriazine‐1,4‐dioxide Derivatives

A series of novel 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives were synthesized and screened for their in vitro cytotoxicity against promyelocytic leukemia HL-60, androgen-independent prostate tumor PC3, hepatocellular carcinoma Bel-7402, human esophagus tumor ECA-109, and human breast tumor MCF-7 cell lines in hypoxia and in normoxia. Most compounds showed higher cytotoxic activity both in hypoxia and in normoxia. Among them, compounds 61 and 62 showed more potent cytotoxic activity and hypoxic selectivity when compared to tirapazamine.

Potentiation of the Cytotoxicity of the Anticancer Agent Tirapazamine by BenzotriazineN-oxides: The Role of Redox Equilibria

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide), the lead bioreductive drug with selective toxicity for hypoxic cells in tumors, is thought to act by forming an active oxidizing radical of high one-electron reduction potential, E(1), when reduced by reductases. It has a dual mechanism of action, both generating DNA radicals, following its one-electron reduction and subsequently oxidizing these DNA radicals to form labile cations or hydrolyzable lactones through transferring an O atom, resulting in DNA strand breaks. These parallel secondary reactions have been proposed to be also initiated by its two-electron reduced metabolite, the 1-oxide. We have used pulse radiolysis to show that the benzotriazinyl radical of a highly soluble analogue of tirapazamine, the 3-(N,N-dimethyl-1,2-ethanediamine) analogue, is able to oxidize tirapazamine itself. We have found that both tirapazamine and the 1-oxides are in equilibrium with their respective benzotriazinyl radicals, with high concentrations of the more soluble 1-oxide maintaining a high concentration of the more reactive oxidizing radical of tirapazamine. The one-electron reduction potentials, E(1), of the 1-oxides and related compounds have been measured and, together with the E(1) values of tirapazamine and the 2-nitroimidazole radiosensitizer, misonidazole, are shown to predict the published percentages of electron transfer. This radical chemistry study gives an insight into the mechanisms of the potentiation of radical damage, reported for DNA, that underlies the hypoxic cytotoxicity of electron affinic compounds. The E(1) values of the benzotriazinyl radicals of the benzotriazine compounds govern the position of the redox equilibria, which determine the amount of initial radical damage. The E(1) values of the 1,4-dioxides and 1-oxide compounds govern the degree of potentiation of the initial radical damage once formed.

Radical properties governing the hypoxia-selective cytotoxicity of antitumor 3-amino-1,2,4-benzotriazine 1,4-dioxides

Revealing the free radical mechanism by which the anticancer drug tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) induces hypoxia-selective cytotoxicity, is seen as a way forward to develop clinically useful bioreductive drugs against chemo- and radiation-resistant hypoxic tumor cells. Our previous studies point to the formation of an active benzotriazinyl radical following the one-electron reduction of tirapazamine and its elimination of water from the initial reduction intermediate, and have suggested that this species is a cytotoxin. In this paper we have used pulse radiolysis to measure the one-electron reduction potentials of the benzotriazinyl radicals E(B*,H(+)/B) of 30 analogues of tirapazamine as well as the one-electron reduction potentials of their two-electron reduced metabolites, benzotriazine 1-oxides E(B/B*-). The redox dependencies of the back-oxidation of the one-electron reduced benzotriazine 1,4-dioxides by oxygen, their radical prototropic properties and water elimination reactions were found to be tracked in the main by the one-electron reduction potentials of the benzotriazine 1,4-dioxides E(A/A*-). Multiple regression analysis of published aerobic and hypoxic clonogenic cytotoxicity data for the SCCVII murine tumor cell line with the physical chemistry parameters measured in this study, revealed that hypoxic cytotoxicity is dependent on E(B*, H(+)/B) thus providing strong evidence that the benzotriazinyl radicals are the active cytotoxic species in hypoxia, while aerobic cytotoxicity is dependent on E(B/B*-). It is concluded that maximizing the differential ratio between these two controlling parameters, in combination with necessary pharmacological aspects, will lead to more efficacious anticancer bioreductive drugs.

498 Selective potentiation of the hypoxic cytotoxicity of the bioreductive drug tirapazamine

A series of 10 novel nitro-analogues of cryptolepine (1) has been synthesised and these compounds were evaluated for their in-vitro cytotoxic properties as well as their potential for reductive activation by the cytosolic reductase enzymes NQO1 and NQO2. Molecular modelling studies suggest that cryptolepine is able to fit into the active site of NQO2 and thus raising the possibility that nitro-analogues of 1 could act as bioreductive prodrugs and be selectively reduced by NQO1 and NQO2 to more toxic species in cancer cells in which these enzymes are over-expressed. Analogues were screened against the RT112 cell line (high in NQO2), in the presence and absence of the essential cofactor dihydronicotinamide riboside (NRH), whereby all analogues were shown to be cytotoxic (IC50 < 2 μM) in the absence of NRH. With the addition of NRH, one analogue, 2-fluoro-7,9-dinitrocryptolepine (7), exhibited a 2.4-fold increase in cytotoxic activity. Several nitro-derivatives were also evaluated as substrates for purified human NQO1 and analogues that were found to be substrates were subsequently tested against the H460 (high NQO1) and BE (low NQO1) cell lines to detect in-vitro activation by NQO1. The analogue 8-chloro-9-nitrocryptolepine (9) was found to be the best substrate for NQO1 but it was not more toxic to H460 than to BE cells. Fluorescence laser confocal microscopy of 1 and several analogues showed that in contrast to 1 the analogues were not localised into the nucleus suggesting that their cytotoxic mode(s) of action are different. This study has identified novel substrates for both NQO1 and NQO2 and further work on nitrocryptolepine derivatives as a lead towards novel anticancer agents would be worthwhile.

Selective potentiation of the hypoxic cytotoxicity of tirapazamine by its 1-N-oxide metabolite SR 4317.

Tirapazamine (TPZ), a bioreductive drug with selective toxicity for hypoxic cells in tumors, is currently in Phase III clinical trials. It has been suggested to have a dual mechanism of action, both generating DNA radicals and oxidizing these radicals to form DNA breaks; whether the second (radical oxidation) step is rate-limiting in cells is not known. In this study we exploit the DNA radical oxidizing ability of the 1-N-oxide metabolite of TPZ, SR 4317, to address this question. SR 4317 at high, but nontoxic, concentrations potentiated the hypoxic (but not aerobic) cytotoxicity of TPZ in all four of the human tumor cell lines tested (HT29, SiHa, FaDu, and A549), thus providing a 2-3-fold increase in the hypoxic cytotoxicity ratio. In potentiating TPZ, SR 4317 was 20-fold more potent than the hypoxic cell radiosensitizers misonidazole and metronidazole but was less potent than misonidazole as a radiosensitizer, suggesting that the initial DNA radicals from TPZ and radiation are different. SR 4317 had favorable pharmacokinetic properties in CD-1 nude mice; coadministration with TPZ provided a large increase in the SR 4317 plasma concentrations relative to that for endogenous SR 4317 from TPZ. It also showed excellent extravascular transport properties in oxic and anoxic HT29 multicellular layers (diffusion coefficient 3 x 10(-6) cm(2)s(-1), with no metabolic consumption). Coadministration of SR 4317 (1 mmol/kg) with TPZ at a subtherapeutic dose (0.133 mmol/kg) significantly enhanced hypoxic cell killing in HT29 tumor xenografts without causing oxic cell killing, and the combination at its maximum tolerated dose was less toxic to hypoxic cells in the retina than was TPZ alone at its maximum tolerated dose. This study demonstrates that benzotriazine mono-N-oxides have potential use for improving the therapeutic utility of TPZ as a hypoxic cytotoxin in cancer treatment.

DNA-targeted 1,2,4-benzotriazine 1,4-dioxides: potent analogues of the hypoxia-selective cytotoxin tirapazamine.

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.

Improved potency of the hypoxic cytotoxin tirapazamine by DNA-targeting

To improve the potency of the hypoxic cytotoxin tirapazamine (TPZ), we have constructed an analog, SN26955, with the TPZ moiety attached to an acridine chromophore to target the drug to DNA. The underlying reason for this is our previous finding that the hypoxic cytotoxicity of TPZ is a result of its ability to produce DNA double-strand breaks, whereas many of the toxicities of the drug in clinical use are likely the result of its metabolism in the cytoplasm and effects on mitochondria. We found that the DNA-targeted TPZ analog was more potent than TPZ in killing hypoxic cells by 1–2 orders of magnitude, yet it retained the hypoxic selectivity for cell killing of TPZ. We show that SN26955 is only active in producing DNA damage when it is enzymatically reduced while bound to, or in close association with, the DNA. We also show that it has a different cofactor dependence than TPZ for reduction leading to DNA double-strand breaks, suggesting the involvement of a different reductase for production of the lethal lesion than for TPZ. These results show the promise of DNA-targeting of TPZ to produce a DNA compound with greater clinical efficacy than TPZ itself.

Structure-activity relationships of 1,2,4-benzotriazine 1,4-dioxides as hypoxia-selective analogues of tirapazamine.

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxic cytotoxin currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. As part of a program to develop TPZ analogues with improved solubility/potency and therapeutic indices, we synthesized 34 1,2,4-benzotriazin-3-amine 1,4-dioxides (BTO) 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 analogues 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 analogues 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. Aerobic cytotoxic potency showed 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 to -400 mV. Analogues with high HCRs (>50) all had E(1)s in the range -450 to -510 mV (weakly electron-donating substituents) with the exception of the 8-CF(3) analogue, which had an HCR of 112 against SCCVII despite a high E(1) of -372 mV). The results suggest that ring-A substituents in BTO analogues 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).

Quinoxaline 1,4-dioxides: hypoxia-selective therapeutic agents.

A problem that confronts clinicians in the treatment of cancer is the resistance of hypoxic tumors to chemotherapy and radiation therapy. Thus, the development of new drugs that are toxic to hypoxic cells found in solid tumors is an important objective for effective anticancer chemotherapy. We recently showed that the heterocyclic aromatic N-oxides, quinoxaline 1,4-dioxides (QdNOs), are cytotoxic to tumor cells cultured under hypoxia. In this study, we evaluated the hypoxia-selective toxicity of four diversely substituted QdNOs and determined their effect on the expression of hypoxia inducible factor (HIF) 1alpha in the human colon cancer cell line T-84. The various QdNOs were found to possess a 50- to 100-fold greater cytotoxicity to T-84 cells cultured under hypoxia compared with oxia. Interestingly, the hypoxia cytotoxicity ratio (HCR), the ratio of equitoxic concentrations of the drug under aerobic/anoxic conditions, was highly structure related and depended on the nature of the substituents on the QdNO heterocycle. The most cytotoxic 2-benzoyl-3-phenyl-6,7-dichloro derivative of QdNO (DCQ) was potent at a dose of 1 microM with an HCR of 100 and significantly reduced the levels of HIF-1alpha transcript and protein. The 2-benzoyl-3-phenyl derivative (BPQ) had a hypoxia potency of 20 microM and an HCR of 40. By contrast, the 2-aceto-3-methyl and the 2,3-tetramethylene (TMQ) derivatives of QdNO were much less cytotoxic under hypoxia (HCRs of 8.5 and 6.5, respectively) and reduced the expression of HIF-1alpha mRNA to a much lesser extent. Because the nonchlorinated analogue BPQ did not demonstrate behavior similar to that of DCQ, we hypothesize that the C-6, C-7-chlorine of DCQ might play a significant role in the selective hypoxic cytotoxicity of the drug.

Design, synthesis and biological activities of antiangiogenic hypoxic cytotoxin, triazine- N-oxide derivatives

For cancer therapy, hypoxia represents an important tumor specific target. Therefore we designed and synthesized antiangiogenic hypoxic cytotoxins as ‘hypoxia modifiers’. They can be activated bioreductively in hypoxic cells to kill the oxygen-deficient tumor cells selectively and prevent their re-growth. The aromatic heterocycle di- N-oxides, tirapazamine (TPZ), TX-1102, and TX-402 inhibited growth of EMT6/KU cells, SAS/neo cells, and SAS/Trp248 cells (mutant p53 gene transformant) under hypoxic condition. They also induced apoptosis selectively at a dose of 10 μM each under hypoxic condition for 5 h. Their hypoxic cytotoxicities and apoptosis inducing activities were p53-independent because the activities in SAS/neo cells were almost similar to that in SAS/Trp248 cells. In angiogenesis inhibition assay using chick embryo chorioallantoic membrane (CAM), TPZ, TX-1102, TX-402 and TX-1033 showed 40, 25, 60 and 60% inhibition of angiogenesis each at a dose of 10 μg/CAM. On the other hand, the nitrosopyrimidine, TX-1041 had neither antiangiogenic activity nor cytotoxicity. Therefore the di- N-oxide group is thought to be required for the biological activities. TX-1102 was a potent antiangiogenic hypoxic cytotoxin inducing apoptosis p53-independently.

Comparison of aromatic and tertiary amine N-oxides of acridine DNA intercalators as bioreductive drugs : Cytotoxicity, DNA binding, cellular uptake, and metabolism

Some N-oxide derivatives of DNA intercalators are bioreductive prodrugs that are selectively toxic under hypoxic conditions. The hypoxic selectivity is considered to result from an increase in DNA binding affinity when the N-oxide moiety is reduced. This study investigated whether differences in DNA binding affinity between N-oxides and their corresponding amines, measured by equilibrium dialysis, can account for the hypoxic cytotoxicity ratios (HCR) of tertiary amine N-oxide (-tO) and aromatic N-oxide (-aO) derivatives of the 1-nitroacridine nitracrine (NC) and its non-nitro analogue 9-[3-( N,N-dimethylamino)propylamino]acridine (DAPA). Cytotoxicity was measured in aerobic and hypoxic suspensions of Chinese hamster ovary (CHO) AA8 cells by clonogenic assay. HCR were much greater for NC-tO (820-fold) than for NC (5-fold) or NC-aO (4-fold), whereas DAPA and its N-oxides lacked hypoxic selectivity (1-fold). DNA binding measurements demonstrated that binding affinity is lowered more by aromatic than tertiary amine (side-chain) N-oxides, an observation that does not correlate with HCR. Compounds were accumulated in cells to high concentrations (C i/C e ≈ 10–200), with the exception of the tertiary amine N-oxides, for which the ratio of intracellular to extracellular drug was less than unity. For NC-tO this probably resulted from low p K a values for both the acridine chromophore and the side-chain, whereas DAPA-tO may be too hydrophilic for efficient membrane permeation. Bioreductive drug metabolism, assessed by HPLC, was faster for the NC than the DAPA N-oxides. The high HCR of NC-tO relative to NC-aO is ascribed to the rapid and selective reduction of its N-oxide moiety, followed by activation of the NC intermediate by O 2-sensitive reduction of its 1-nitro group to the corresponding 1-amine. The metabolism studies suggest that unmasking of DNA binding affinity by reductive removal of the N-oxide moiety, although not the only determinant, is important and needs to occur before nitroreduction for optimal effect.

Synthesis and evaluation of nitroheterocyclic phosphoramidates as hypoxia-selective alkylating agents.

A series of novel nitroheterocyclic phosphoramidates has been prepared, and the cytotoxicity of these compounds has been evaluated in clonogenic assays against B16, wild-type and cyclophosphamide-resistant MCF-7, and HT-29 cells under aerobic conditions and HT-29 cells under hypoxic conditions. All compounds were comparable in toxicity to wild-type and resistant MCF-7 cells and were also selectively toxic to HT-29 cells under hypoxic conditions (selectivity ratios 1.7 to >20). Analogues lacking the nitro group were not cytotoxic. Electron-withdrawing substituents increased cytotoxicity under aerobic conditions and thereby decreased hypoxic selectivity. In contrast, an electron-donating substituent markedly decreased both aerobic and hypoxic cytotoxicity but enhanced hypoxic selectivity. Chemical reduction of the nitro group resulted in rapid expulsion of the cytotoxic phosphoramide mustard. The most potent of these compounds show significant cytotoxicity under both aerobic and hypoxic conditions.

Bioreductive activation of a series of indolequinones by human DT-diaphorase: structure-activity relationships.

A series of indolequinones including derivatives of EO9 bearing various functional groups and related indole-2-carboxamides have been studied with a view to identifying molecular features which confer substrate specificity for purified human NAD(P)H:quinone oxidoreductase (DT-diaphorase), bioreductive activation to DNA-damaging species, and selectivity for DT-diaphorase-rich cells in vitro. A broad spectrum of substrate specificity exists, but minor changes to the indolequinone nucleus have a significant effect upon substrate specificity. Modifications at the 2-position are favorable in terms of substrate specificity as these positions are located at the binding site entrance as determined by molecular modeling studies. In contrast, substitutions at the (indol-3-yl)methyl position with bulky leaving groups or a group containing a chlorine atom result in compounds which are poor substrates, some of which inactivate DT-diaphorase. Modeling studies demonstrate that these groups sit close to the mechanistically important amino acids Tyr 156 and His 162 possibly resulting in either alkylation within the active site or disruption of charge-relay mechanisms. An aziridinyl group at the 5-position is essential for potency and selectivity to DT-diaphorase-rich cells under aerobic conditions. The most efficient substrates induced qualitatively greater single-strand DNA breaks in cell-free assays via a redox mechanism involving the production of hydrogen peroxide (catalase inhibitable). This damage is unlikely to form a major part of their mechanism of action in cells since potency does not correlate with extent of DNA damage. In terms of hypoxia selectivity, modifications at the 3-position generate compounds which are poor substrates for DT-diaphorase but have high hypoxic cytotoxicity ratios.