Hypoxia-Activated O6-Benzylguanine Prodrugs

描述（申请人提供）：将DNA鸟嘌呤的O-6位氯乙基化的烷基化剂前药包括我们实验室设计合成的onrigin (cloretazine)，目前处于临床后期试验阶段；carmustine （BCNU）和temozolomide （TMZ）分别是FDA批准的临床使用的亚硝基脲和甲基化剂，KS119是一种选择性激活实体肿瘤缺氧细胞的磺酰肼前药。由胆酰化剂产生的DNA损伤，是DNA鸟嘌呤o6位的烷基化，导致G-C交联，容易被o6 -烷基鸟嘌呤-DNA烷基转移酶（AGT）修复，AGT是一种将鸟嘌呤o6位的烷基和甲基转移到AGT分子的蛋白质。这一作用代表了肿瘤和宿主组织对onigin、KS119、BCNU和TMZ耐药的主要机制。在癌症患者中，无毒性剂量的全身O6-BG已被证明可以消耗肿瘤中的AGT含量；这一作用也会消耗正常组织中的AGT，使两种肿瘤宿主组织对联合使用BCNU敏感。由于骨髓抑制，AGT的消耗需要减少80%的BCNU剂量，导致这种亚硝基脲的抗肿瘤水平无效。本申请的具体目的是：(a)设计和合成可处方的O6-BG前药，该前药含有对硝基苄基羰基触发物/连接物，通过还原实体肿瘤缺氧细胞中的酶选择性/优先激活，从而选择性地消耗肿瘤中的AGT，同时保留充氧正常组织；(b)体外评价O6-BG前药对缺氧和氧合肿瘤细胞的预处理效果，然后再用蛇原素、KS119、BCNU和TMZ；(c)在体内评估这些药物与合成的AGT抑制剂联合治疗多种含有不同水平AGT的移植人类和小鼠肿瘤的效果；(d)对合成前药作用机制的生化研究。预期实体肿瘤由于AGT的组成性高而对O6-DNA鸟嘌呤靶向药物的细胞毒性作用具有抗性，通过预处理O6-BG前药，可以选择性/优先地去除抗药诱导蛋白AGT，导致氯乙基化/甲基化药物耐药肿瘤转化为药物敏感肿瘤，从而增加可能受益于onrigin、KS119、BCNU和TMZ的恶性肿瘤的范围。

DESCRIPTION (provided by applicant): 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.