Hypoxia-Activated O6-Benzylguanine Prodrugs

描述（申请人提供）：将DNA鸟嘌呤O-6位氯乙基化的烷化剂前药包括onrigin氯雷他嗪（cloretazine）是本实验室自行设计合成的药物，目前处于临床试验后期;卡莫司汀（BCNU）和替莫唑胺（TMZ），分别是FDA批准的临床使用的亚硝基脲和甲基化剂，以及KS 119，将使用在实体瘤的低氧细胞中选择性活化的磺酰肼前药。由氯乙基化剂产生的DNA损伤，即DNA鸟嘌呤O-6位的烷基化，导致G-C交联，易于被O-6-烷基鸟嘌呤-DNA烷基转移酶（AGT）修复，AGT是一种将烷基和甲基从鸟嘌呤O-6位转移到AGT分子的蛋白质。这种作用代表了肿瘤和宿主组织对onrigin、KS 119、BCNU和TMZ的抗性的主要机制。在癌症患者中，无毒剂量的全身性O 6-BG已被证明可以消耗肿瘤中的AGT含量;该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的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无效剂量水平。本申请的具体目的是（a）设计和合成可配制的O 6-BG前药，其含有通过实体瘤的缺氧细胞中的还原酶选择性/优先活化的对硝基苄基羰基触发剂/接头，从而选择性地消耗AGT的肿瘤，同时保留含氧正常组织;（B）体外评价用O 6-BG前药预处理缺氧和充氧的肿瘤细胞，然后用奥瑞金、KS 119、BCNU和TMZ预处理;（c）在体内评价这些药物与合成的AGT抑制剂的组合对含有不同水平AGT的各种移植的人和鼠肿瘤的作用，和（d）合成的前药的作用机制的生物化学研究。由于组成性高水平的AGT而对O 6-DNA鸟嘌呤靶向剂的细胞毒性作用具有抗性，通过用O 6-BG前药预处理，将选择性/优先地耗尽抗性诱导蛋白AGT，导致氯乙基化/甲基化剂抗性肿瘤转化为药物敏感性肿瘤，从而增加可从Onrigin、KS 119、BCNU和TMZ获益的恶性肿瘤谱。 公共卫生相关性：缺氧肿瘤组织中AGT抑制性前药的选择性活化增强了使DNA鸟嘌呤的O 6-位氯乙基化和甲基化的烷基化和甲基化药物的抗肿瘤潜力，以及逆转了这类药物的主要耐药机制（即，靶向DNA鸟嘌呤的O 6-位的烷化剂）。新AGT抑制剂的设计、合成和表征与特定已知DNA O 6-鸟嘌呤靶向剂的持续开发相结合使用，可以很好地证明产生用于多种目前无应答肿瘤的独特的新治疗。

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.