CHEMICAL STUDIES OF TAXOL AND MICROTUBULES

紫杉醇是从太平洋红豆杉中分离出来的一种二萜化合物，它具有 在I期临床试验中显示出抗肿瘤活性 黑色素瘤、腺癌、难治性卵巢癌、非小细胞肺癌 癌症、胃癌、结肠癌、头颈部癌和淋巴母细胞癌 白血病。与顺铂联合使用时，观察到了活性 抗晚期大细胞肺癌，非小细胞肺癌， 乳腺癌，头颈部，结肠癌，晚期卵巢癌， 还有黑色素瘤。紫杉醇在第二阶段试验中也显示出活性。 联合化疗对晚期卵巢上皮性肿瘤的治疗作用 放射和化疗，其中一些被认为对 顺铂。 与紫杉醇作为抗肿瘤药物的发展相关的三个领域， 并以其独特的行动模式的开发为根本 管理肿瘤疾病的新的总体战略是 已在此应用程序中寻址。 目前，可供使用的紫杉醇数量有限 不足以在癌症化疗中广泛应用。部分 通过将关键的A环侧链连接到更多 容易获得但没有活性的天然物质可以缓解这种情况 有问题。我们将开发三个新的A形环侧链附件 方法学，其中两个涉及5，6-二氢-6-酮-1，3恶嗪化学 (包括将不对称侧链合成与 依附于二萜核共分五步)，一步 这涉及到烯酮的方法学。前一个战略的成功之处在于 结合侧链的初步结果提示 可变功能。 紫杉醇在微管上的结合部位(S)保持完好 没有特征的。这种情况妨碍了在 紫杉醇独特的作用模式的分子水平，这反过来， 通过设计新药来破坏它的开发。三个领域 调查的目标是化学相关的表征 紫杉醇结合部位将被追查。首先，我们将进行实验评估 (进一步的结构-活性研究和构象分析 通过NOE和13C T1研究的紫杉醇类似物)和计算 (分子力学建模和动力学计算)假设 紫杉醇及其生物活性类似物是预先组织起来的 结合到微管上。这一观点得到了初步的有力支持 结构-活性和建模结果。如果事实证明这是真的， 关于结合部位的性质的重要信息 药物设计的优化将在短期内实现。第二, 我们将对结合部位进行光亲和标记。这些 研究将揭示微管的结构域和氨基酸残基 负责紫杉醇的识别。第三，我们将把 来自前两项研究的信息和关于 紫杉醇结合计算模型中微管蛋白的结构 地点。即将出现的画面将揭示三维 紫杉醇与重要化学物质相互作用的化学特征 微管蛋白结合部位的结构元件。 最后，我们将继续制备紫杉醇衍生物，这将是 在细胞和亚细胞水平上对研究 紫杉醇细胞毒性的基础生物学。例如，由于该站点位于 紫杉醇结合的微管不可能为此目的而进化，它 紫杉醇有可能模仿一种内源性物质 生物活性。针对紫杉醇产生的抗体将用于 寻找这样的内源性物质。相似的抗体和荧光 用于紫杉醇与微管结合的成像的紫杉醇类似物 准备好了。

Taxol, a diterpene isolated from the Pacific yew, Taxus brevifolia, has exhibited antineoplastic activity in phase I clinical trials against melanoma, adenocarcinoma, refractory ovarian carcinoma, non-small cell lung carcinoma, gastric, colon, and head and neck carcinomas, and lymphoblastic leukemias. In combination with cisplatin, activity has been observed against advanced large cell lung carcinoma, non-small cell lung carcinoma, breast, head and neck, and colon carcinomas, advanced ovarian carcinoma, and melanoma. Activity also has been displayed by taxol in phase II trials against advanced ovarian epithelial tumors in patients pretreated with radiation and chemotherapy, some of whom were considered resistant to cisplatin. Three areas associated with the development of taxol as an antitumor agent, and with the exploitation of its unique mode of action as a fundamentally new and general strategy for the management of neoplastic disease are addressed in this application. At the present time, the limited quantities of taxol available are insufficient for its broad application in cancer chemotherapy. Partial synthesis through the attachment of the critical A-ring side chain to more readily available but inactive natural substances can alleviate this problem. We will develop three new A-ring side chain attachment methodologies, two that involve 5,6-dihydro-6-keto-1,3 oxazine chemistry (including one that coalesces asymmetric side chain synthesis with attachment to the diterpenoid nucleus into a total of five steps), and one that involves ketene methodology. The success of the former strategy is suggested by preliminary results on the attachment of side chains with variable functionality. The binding site(s) of taxol on microtubules remain completely uncharacterized. This state of affairs prevents the understanding at the molecular level of taxol's unique mode of action, which, in turn, undermines its exploitation through the design of new drugs. Three areas of investigation aimed at the chemically relevant characterization of the taxol binding site will be pursued. First, we will evaluate experimentally (further structure-activity studies and the conformational analysis of taxol analogues through NOE and 13C T1 studies) and computationally (molecular mechanics modeling and dynamical calculations) the hypothesis that taxol and its biologically active analogues are preorganized for tight binding to microtubules. This notion is supported strongly by preliminary structure-activity and modeling results. Should this prove to be true, significant information on the nature of the binding site and for the optimization of drug design will be available in the short term. Second, we will engage in photoaffinity labeling of the binding site. These studies will reveal the domains and amino acid residues of microtubules responsible for the recognition of taxol. Third, we will combine the information from the first two studies with the available data on the structure of the tubulins in a computational model of the taxol binding site. The picture that will emerge will reveal the three-dimensional chemical features of the interaction between taxol and the important structural elements of tubulin at the binding site. Finally, we will pursue the preparation of taxol derivatives that will be useful at the cellular and subcellular levels for the investigation of the biology that underlies taxol's cytotoxicity. For example since the site on microtubules to which taxol binds cannot have evolved for that purpose, it is possible that taxol mimics an endogenous substance with similar biological activity. Antibodies generated to taxol will be employed in a search for such endogenous substances. Similar antibodies and fluorescent taxol analogues for the imaging of taxol bound to microtubules will be prepared.