Initiation of DNA Strand Cleavage by 1,2,4-Benzotriazine 1,4-Dioxide Antitumor Agents: Mechanistic Insight from Studies of 3-Methyl-1,2,4-benzotriazine 1,4-Dioxide

Abstract

The antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, TPZ, 1) gains medicinal activity through its ability to selectively damage DNA in the hypoxic cells found inside solid tumors. This occurs via one-electron enzymatic reduction of TPZ to yield an oxygen-sensitive drug radical (2) that leads to oxidatively generated DNA damage under hypoxic conditions. Two possible mechanisms have been considered to account for oxidatively generated DNA damage by TPZ. First, homolysis of the N-OH bond in 2 may yield the well known DNA-damaging agent, hydroxyl radical. Alternatively, it has been suggested that elimination of water from 2 generates a benzotriazinyl radical (4) as the ultimate DNA-damaging species. In the studies described here, the TPZ analogue 3-methyl-1,2,4-benzotriazine 1,4-dioxide (5) was employed as a tool to probe the mechanism of DNA damage within this new class of antitumor drugs. Initially, it was demonstrated that 5 causes redoxactivated, hypoxia-selective oxidation of DNA and small organic substrates in a manner that is completely analogous to TPZ. This suggests that 5 and TPZ damage DNA by the same chemical mechanism. Importantly, the methyl substituent in 5 provides a means for assessing whether the putative benzotriazinyl intermediate 7 is generated following one-electron reduction. Two complementary isotopic labeling experiments provide evidence against the formation of the benzotriazinyl radical intermediate. Rather, a mechanism involving the release of hydroxyl radical from the activated drug radical intermediates can explain the DNA-cleaving properties of this class of antitumor drug candidates.The compound 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, TPZ, 1, Scheme 1) is currently undergoing a variety of phase I, II, and III clinical trials for the treatment of human cancers. 1 TPZ gains medicinal activity from its ability to selectively damage DNA in the oxygen-poor (hypoxic) cells found inside solid tumors. [2][3][4][5][6][7][8] This DNA-damage process begins with intracellular enzymatic reduction of TPZ to yield the drug radical intermediate (2 , Scheme 1). [9][10][11][12] In normally-oxygenated cells, 2 undergoes relatively harmless oxidation back to the parent drug (Scheme 1), 9,10,13 while, under hypoxic conditions, the drug radical intermediate 2 leads to oxidatively generated DNA damage including hydroxylation of the nucleobases 22,23 and strand breaks initiated by the abstraction of hydrogen atoms from the sugar-phosphate backbone of DNA. [7][8][9][24][25][26][27][28] In the recent literature, two mechanisms have been considered to explain TPZ-mediated DNA damage. We have presented evidence [22][23][24]26,29 supporting a mechanism involving homolysis of the N-OH bond in the neutral drug radical (2) to yield the mono-N-oxide metabolite 3 and the well known DNA-damaging agent hydroxyl radical (Scheme 1, upper branch). 30 This *To whom correspondence should be addressed: gatesk@missouri.edu; phone: (573) FAX: (573) NIH-PA Author ManuscriptNIH-PA ...

Isotopic Labeling Experiments That Elucidate the Mechanism of DNA Strand Cleavage by the Hypoxia-Selective Antitumor Agent 1,2,4-Benzotriazine 1,4-Di-N-oxide

“…Overall, the reductively-activated, hypoxia-selective DNA-cleaving properties of 1b closely resemble those of tirapazamine and other 1,2,4-benzotriazine 1,4-dioxides. 13,21,25,43 …”

The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery

“…10 [Evans 1998] These free radicals cause DNA strand breaks. [11][12][13] [Junnotula 2009, Brown 2004, Yin 2012. The DNA damaging mechanism of TPZ and its analogues has been shown to be consistent with the release of the HO• radicals from the enzymatically reduced TPZ• radical.…”

Strategies on the Development of Small Molecule Anticancer Drugs for Targeted Therapy

“…(Fig. 1) [45] Platinum-based chemotherapy drugs are among the most investigated drug resistance examples. Ohmichi et al concluded that the reduced platinum accumulation plays a key role in drug resistance rather than the increased drug efflux because the main multidrug resistance efflux pump, Pglycoprotein, is not usually overexpressed in cisplatinresistant tumors [46].…”