SYNTHESIS, CHARACTERIZATION, ANTICANCER ACTIVITY AND REACTIVITY TOWARD BIOLOGIC

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。本提案的主要目的是寻找与目前使用的金属基抗癌药物（如顺铂、卡铂等）相比性能更好的新型铂类药物。，并通过研究它们对一些有希望发现的药物的重要生物分子（DNA、寡核苷酸）的反应性，发现它们细胞毒性的分子基础。为了实现这些目标，我们计划确定两个系列铂（II）配合物的合成和表征的较好条件。1.-以苯基苯并杂唑或重要的生物活性羧酸的酰胺衍生物为配体的铂（II）阴离子三halo配合物。这些配合物是基于先前合成的苯基苯并杂唑三溴阴离子配合物的抗癌活性而设计的，该配合物与顺铂相比具有不同的交联模式。虽然没有对这些配合物进行毒性研究，但我们希望通过在其他系列化合物上用与铂（II）中心结合的氯化物取代溴化物配体来减少任何可能的毒性。为了研究配体对这些配合物的影响，将使用的新的苯基苯并杂唑配体通过在苯并唑和/或苯乙烯基上放置其他取代基（电子给性和/或电子吸性）以及改变配体中苯并唑部分的杂原子（NH， S， O）来进行修饰。用于阴离子三halo配合物的其他配体包括从报道的生物活性羧酸衍生的酰胺，如2-苯基乙酸、苯基丁酸、支链和非支链饱和脂肪酸以及不饱和和多不饱和脂肪酸。2.-中性铂（II）配合物，其中母体药物中的一个或两个氯基被上述生物活性羧酸取代。母药包括顺铂、顺- pt (DACH)CI2和顺- pt (en)CI2，其中DACH代表1,2-二氨基环己烷，en代表乙二胺。这些配体的使用有望增加新复合物的脂性和膜通透性，并可能诱导与生物活性主要靶点相互作用的新途径。此外，羧酸基团的存在预计会产生一种多功能化合物，这种化合物的离开基团在与DNA结合之前由铂配合物实现。因此，释放的羧酸应该对细胞发挥自己的生物活性。所有这些化合物将被纯化并通过元素分析、紫外可见光谱、1H、3C、195Pt和二维核磁共振实验进行表征。它们也将被结晶，以获得足够好的晶体用于x射线衍射研究。针对癌细胞系的生物测试将在UPR，里约热内卢Piedras校区的生物测试实验室进行。该项目的长期目标是研究有前景的配合物与重要生物分子（如DNA）的相互作用，这是已知铂配合物的主要目标。这些相互作用将遵循标准程序，通过核磁共振波谱和高效液相色谱，并对其产物进行表征。为了获得新复合物的首选结合位点信息，将铂化DNA进行酶解，并将其加合物进行色谱分离和1H NMR鉴定。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The major objective of this proposal is to search for new platinum drugs with improved properties compared to currently used metal based anticancer agents such as cisplatin, carboplatin, etc..., and to discover the molecular basis of their cytotoxicity by studying their reactivity toward biological important molecules (DNA, oligonucleotides) of some of the promising discovered drugs. In order to achieve these goals we plan to determine the better conditions for the synthesis and characterization of two series of platinum(II) complexes. 1.- anionic trihalo complexes of platinum(II) in which either a styrylbenzoheterazole or amide derivatives of important biological active carboxylic acids will be used as ligands. These complexes were designed based on the reported anticancer activity of previously synthesized tribromo anionic complexes of styrylbenzoheterazole which demonstrated a different pattern of cross-linking compared to cisplatin. Although no toxicity studies have been performed on these complexes, we expect to decrease any possible unwanted toxicity by substitution of the bromide ligands by chloride bound to the platinum(II) center on other series of compounds. To study the effect of the ligand on these complexes, the new styrylbenzoheterazole ligands to be used will be modified by placing other substituents (electrondonating and/or electronwithdrawing) on either the benzazolo and/or the styryl moieties, and also by changing the heteroatom (NH, S, O) on the benzazolo part of the ligands. Other ligands to be used for the anionic trihalo complexes include amides derived from reported bioactive carboxylic acids, such as 2-phenylacetic acid, phenylbutyric acid, branched and unbranched saturated fatty acids and unsaturated and polyunsaturated fatty acids. 2.- Neutral platinum(II) complexes in which the one or both of the chloride leaving groups in the parent drugs are substituted by the biological active carboxylic acids mentioned above. The parent drugs involves the use of cisplatin, cis-Pt(DACH)CI2, and cis-Pt(en)CI2, where DACH stands for 1,2-diaminocyclohexane and en for ethylenediamine. The use of these ligands are expected to increase the lipophyllicity and membrane permeability of the new complexes and probably to induce new ways of interactions with the main targets for bioactivity. Also the presence of the carboxylate groups are expected to produce a multifunctional compound in such a way that is known that the leaving groups are realized by the platinum complexes before it is bound to DNA. So, the freed carboxylates should exert their own bioactivity on the cells. All of these compounds will be purified and characterized by elemental analysis, UV-Vis spectroscopy, 1H, 3C, 195Pt and two dimension NMR experiments. They will be also crystallized to obtain crystal good enough for X-Ray Diffraction studies. The biological testing against cancer cell lines will be performed at the Biotesting Laboratory at UPR, Rio Piedras Campus. The long term objectives of the proposed projects is to study the interaction of promising complexes with important biological molecules, such as DNA, which is the main target of known platinum complexes. These interactions will be followed by standard procedures andby NMR spectroscopy and HPLC, and their products characterized. In order to obtain information on the preferred sites of binding of the new complexes, the platinated DNA will be enzymatically digested and their adducts separated chromatographically and identified by 1H NMR spectroscopy.