Alkylating agent prodrugs that chloroethylate the O-6 position of DNA guanine include onrigin (cloretazine), an agent designed and synthesized in our laboratory, currently in late stage clinical trial;carmustine (BCNU) and temozolomide (TMZ), an FDA approved clinically used nitrosourea and methylating agent, respectively, and KS119, a sulfonylhydrazine prodrug selectively activated in hypoxic cells of solid tumors will be employed. The DNA lesion, produced by the cholorethylating agents, an alkylation of the O6-position of DNA guanine which leads to a G-C crosslink, is susceptible to repair by O6-alkylguanine-DNA alkyltransferase (AGT), a protein that transfers alkyl and methyl groups from the O-6 position of guanine to the AGT molecule. This action represents the primary mechanism of tumor and host tissue resistance to onrigin, KS119, BCNU, and TMZ. Non-toxic doses of systemic O6-BG have been shown in cancer patients to deplete the AGT content of tumors;this action also depletes AGT in normal tissue, sensitizing both tumor host tissues to BCNU used in combination. The depletion of AGT necessitates an 80% decrease in dosage of BCNU because of myelosuppression, leading to an ineffective antineoplastic level of this nitrosourea.
The Specific Aims of this application are (a) the design and synthesis of formulatable O6-BG prodrugs containing a p-nitrobenzylcarbonyl trigger/linker activated selectively/preferentially by reducing enzymes in oxygen-deficient cells of solid tumors, thereby selectively depleting tumors of AGT while sparing oxygenated normal tissue;(b) evaluation of pretreatment of hypoxic and oxygenated tumor cells with O6-BG prodrugs in vitro followed by onrigin, KS119, BCNU, and TMZ;(c) evaluation in vivo of combinations of these agents with synthesized AGT inhibitors against a variety of transplanted human and murine tumors containing varied levels of AGT and (d) biochemical studies of the mechanism of action of the synthesized prodrugs The expectation is that solid tumors, resistant to the cytotoxic actions of O6-DNA guanine targeting agents because of constitutively high levels of AGT, will be selectively/preferentially depleted of the resistance inducing protein AGT by pretreatment with the O6-BG prodrug, leading to the conversion of chloroethylating/methylating agent resistant neoplasms to drug sensitive ones, thereby increasing the spectrum of malignancies that may derive benefit from onrigin, KS119, BCNU and TMZ.

Public Health Relevance

The selective activation of AGT inhibitory prodrugs in oxygen-deficient tumor tissue enhance the antitumor potential of alkylating and methylating drugs that chloroethylate and methylate the O6-position of DNA guanine, as well as reverse the primary mechanism of resistance of this class of agents (i.e., alkylating agents targeting the O6-position of DNA guanine). The design, synthesis and characterization of new AGT inhibitory agents in concert with the continued development of specific known DNA O6-guanine targeting agents to use in combination may well prove to yield unique new treatments for a variety of currently non-responsive tumors.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Lees, Robert G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Yale University
Schools of Medicine
New Haven
United States
Zip Code
Penketh, Philip G; Patridge, Eric; Shyam, Krishnamurthy et al. (2014) Influence of glutathione and glutathione S-transferases on DNA interstrand cross-link formation by 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the active anticancer moiety generated by laromustine. Chem Res Toxicol 27:1440-9
Penketh, Philip G; Shyam, Krishnamurthy; Zhu, Rui et al. (2014) Influence of phosphate and phosphoesters on the decomposition pathway of 1,2-bis(methylsulfonyl)-1-(2-chloroethyhydrazine (90CE), the active anticancer moiety generated by Laromustine, KS119, and KS119W. Chem Res Toxicol 27:818-33
Lin, Z Ping; Ratner, Elena S; Whicker, Margaret E et al. (2014) Triapine disrupts CtIP-mediated homologous recombination repair and sensitizes ovarian cancer cells to PARP and topoisomerase inhibitors. Mol Cancer Res 12:381-393
Zhu, Rui; Baumann, Raymond P; Penketh, Philip G et al. (2013) Hypoxia-selective O6-alkylguanine-DNA alkyltransferase inhibitors: design, synthesis, and evaluation of 6-(benzyloxy)-2-(aryldiazenyl)-9H-purines as prodrugs of O6-benzylguanine. J Med Chem 56:1355-9
Zhu, Rui; Baumann, Raymond P; Patridge, Eric et al. (2013) Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells. Bioorg Med Chem Lett 23:1853-9
Ishiguro, Kimiko; Shyam, Krishnamurthy; Penketh, Philip G et al. (2013) Expression of O(6)-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue. J Cancer Ther 4:919-931
Zhu, Rui; Seow, Helen A; Baumann, Raymond P et al. (2012) Design of a hypoxia-activated prodrug inhibitor of O6-alkylguanine-DNA alkyltransferase. Bioorg Med Chem Lett 22:6242-7
Patridge, Eric V; Eriksson, Emma S E; Penketh, Philip G et al. (2012) 7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide. Arch Toxicol 86:1613-25
Penketh, Philip G; Shyam, Krishnamurthy; Baumann, Raymond P et al. (2012) A strategy for selective O(6)-alkylguanine-DNA alkyltransferase depletion under hypoxic conditions. Chem Biol Drug Des 80:279-90
Penketh, Philip G; Baumann, Raymond P; Shyam, Krishnamurthy et al. (2011) 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119): a cytotoxic prodrug with two stable conformations differing in biological and physical properties. Chem Biol Drug Des 78:513-26

Showing the most recent 10 out of 18 publications