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Purpose: To compare oxygen dependence and tissue transport properties of a new hypoxia-activated prodrug, PR-104A, with tirapazamine, and to evaluate the implications for antitumor activity when combined with radiotherapy. Methods and Materials: Oxygen dependence of cytotoxicity was measured by clonogenic assay in SiHa cell suspensions. Tissue transport parameters were determined using SiHa multicellular layers. Spatially resolved pharmacokinetic (PK) and pharmacodynamic (PD) models were developed to predict cell killing in SiHa tumors and tested by clonogenic assay 18 h after treatment with the corresponding phosphate ester, PR-104. Results: The K-value (oxygen concentration to halve cytotoxic potency) of PR-104A was 0.126 ± 0.021 μM (10-fold lower than tirapazamine at 1.30 ± 0.28 μM). The diffusion coefficient of PR-104A in multicellular layers (4.42 ± 0.15 × 10−7 cm2 s−1) was lower than that of tirapazamine (1.30 ± 0.05 × 10−6 cm2 s−1) but PK modeling predicted better penetration to hypoxic cells in tumors because of its slower metabolism. The tirapazamine PK/PD model successfully predicted the measured activity in combination with single-dose radiation against SiHa tumors, and the PR-104A model underpredicted the activity, which was greater for PR-104 than for tirapazamine (at equivalent host toxicity) both with radiation and as a single agent. Conclusion: PR-104/PR-104A has different PK/PD properties from tirapazamine and superior activity with single-dose radiotherapy against SiHa xenografts. We have inferred that PR-104A is better able to kill cells at intermediate partial pressure of oxygen in tumors than implied by the PK/PD model, most likely because of a bystander effect resulting from diffusion of its activated metabolites from severely hypoxic zones. [Copyright &y& Elsevier]

Graphical abstract Abstract Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs. [ABSTRACT FROM AUTHOR]

The article presents spectral and kinetic evidence that protonated form of tirapazamine (TPZ) alters dehydration to the benzotriazinyl radical causing oxidative damage to DNA and oxidizing TPZ. It proposes that the form of TPZ goes through a bond homolysis to discharge the oxidizing radical and the 1-oxide of TPZ. Moreover, it reveals that the metabolism of 1,2,4-benzotriazine 1,4-dioxide class of hypoxia-selective prodrugs (HSPs) leads to the formation of carbon- and nitrogen-centered radicals.

SN30000 is a second-generation benzotriazine-N-oxide hypoxia-activated prodrug scheduled for clinical trial. Previously we showed that covalent binding of the hypoxia probe EF5 predicts metabolic activation of SN30000 in a panel of cancer cell lines under anoxia, suggesting that they are activated by the same reductases. However the identity of these reductases is unknown. Here, we test whether forced expression of nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R3, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) enhances oxic or anoxic reduction of SN30000 and EF5 by HCT116 cells. Covalent binding of 14C-EF5 and reduction of SN30000 to its 1-oxide and nor-oxide metabolites was highly selective for anoxia in all lines, with significantly elevated anoxic metabolism of both compounds in lines over-expressing POR, MTRR, NOS2A or NDOR1. There was a strong correlation between EF5 binding and SN30000 metabolism under anoxia across the cell lines (R2 = 0.84, p = 0.0001). Antiproliferative potency of SN30000 under anoxia was increased most strongly by overexpression of MTRR and POR. Transcript abundance in human tumours, evaluated using public domain mRNA expression data, was highest for MTRR, followed by POR, NOS2A and NDOR1, with little variation between tumour types. Immunostaining of tissue microarrays demonstrated variable MTRR protein expression across 517 human cancers with most displaying low expression. In conclusion, we have identified four diflavin reductases (POR, MTRR, NOS2A and NDOR1) capable of reducing both SN30000 and EF5, further supporting use of 2-nitroimidazole probes to predict the ability of hypoxic cells to activate SN30000. [ABSTRACT FROM AUTHOR]

Abstract: 5-Bromomethyl-4-nitroimidazoles have utility as bioreductive trigger precursors for the preparation of hypoxia-selective prodrugs. Here we describe an efficient two-step synthesis of 5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole, a preferred precursor, employing an N-bromosuccinimide mediated radical bromination. Use of this precursor to prepare SN29966, a promising hypoxia-selective irreversible pan-ErbB inhibitor is reported along with the preparation of four other prodrug candidates. 5-Bromomethyl-4-nitroimidazole analogues bearing electron-donating and electron-withdrawing substituents at the N-1 and C-2 positions are also described. [Copyright &y& Elsevier]

Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure–activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation. Single-electron metabolism was achieved by exposing the human carcinoma cell line T47D to each agent under hypoxic conditions, whilst concerted two-electron metabolism was assessed by stably expressing the cDNA for human NQO1 in a cloned cell line of T47D. The C-3 and C-5 positions of the indolequinone nucleus were modified to manipulate reactivity of the reduction products and the four prodrugs were identified as NQO1 substrates of varying specificity. Two of the four prodrugs, in which both C-3 and C-5 groups remained functional, proved to be NQO1-directed cytotoxins with selectivity ratios of 60- to 80-fold in the T47D (WT) versus the NQO1 overexpressing T47D cells. They also retained selectivity as hypoxic cytotoxins with oxic/hypoxic ratios of 20- to 22-fold. Replacement of the C-3 hydroxymethyl leaving group with an aldehyde group ablated all selectivity in air and hypoxia in both cell lines. Addition of a 2-methyl group on the C-5 aziridinyl group to introduce steric hinderance reduced but did not abolish NQO1-dependent metabolism. However, it enhanced single-electron metabolism-dependent DNA cross-linking in a manner that was independent of cytotoxicity. These data demonstrate that subtle structure–activity relationship exists for different cellular reductases and under certain circumstances distinct forms of DNA damage can arise, the cytotoxic consequences of which can vary. This study identifies a candidate indolequinone analogue for further development as a dual hypoxia and NQO1-directed prodrug. [Copyright &y& Elsevier]

Various synthetic methods leading to 5-(bromomethyl)-4-nitroimidazoles are described. [ABSTRACT FROM AUTHOR]