Síntesis y evaluación antimitótica de espiroacetales y análogos de pironetina

Resumen

1. INTRODUCTION Cancer continues to be one of the most feared diseases of the modern world. It is the disease, after cardiovascular illness, most affecting the population of developed countries. Cancer is not a disease but a broad unitary and complex group of diseases (over 200 types described) whose only common element is the uncontrolled proliferation and subsequent aberrant cell dispersion from a point source or body. Given the complexity of the process there is no single type of compound that can exert a curative action in all cases. It is understood, therefore, that the action against cancerous phenomenon with multiple approach requires a precise knowledge on the molecular level thereof,[1] which is associated with the problem of finding products which prevent or at least retard the uncontrolled proliferation mentioned.[2] 1.1. SPONGISTATIN 1 In 1993, the research groups of Pettit, Kitagawa/Kobayashi and Fusetani research groups independently reported a new class of highly potent bis-spiroacetal-containing marine macrolides with captivating structures. The research led to the identification and structure determination of spongistatins 1-9,[3] that were isolated from Spongia species in the East Indian Ocean. These marine macrolides are very strong inhibitors of the growth of many types of cancer cells.[4] As the most potent member, spongistatin 1 exhibited an in-vitro mean panel GI50 of 0.15 nM (e.g., HL-60, SR leukemias, NCI-H226, DMS 114, HT29, KM12 and OVCAR-3 ovarian cancer) and was also exceedingly potent against a subset of highly chemoresistant tumor types (typical GI50, 0.03 nM), while in-vivo human melanoma and ovarian carcinoma xenograft studies showed curative responses at extremely low doses. Despite these exceptional and highly promising biological profiles, further testing of the spongistatins was curtailed by the vanishingly small natural supply from the sponge sources. 1.2. PIRONETIN Pironetin was isolated almost simultaneously from two different microorganisms of the genus Streptomyces, in one of them as a vegetal growth regulator (Streptomyces sp. NK10958),[5] and in the other as a product with immunosuppressive activity (Streptomyces prunicolor PA48153), named PA-48153C.[6] Pironetin has a high pharmacological potential. The most important property of this compound is its ability to arrest the cell cycle in G2/M phase through inhibition of microtubule polymerization. The presence of the conjugated double bond between C-2 and C-3 is essential for activity. This result supports the hypothesis that this double bond is essential for the ability of pironetin to act as a Michael acceptor, whereby a covalent binding to residue Lys352 is formed.[7] In this model it can be observed that the pironetin is located near the Asn258 residue, generating three possible hydrogen bonds. 2. METHODOLOGY 2.1. SPIROACETAL DERIVATIVES. RESULTS AND DISCUSSION In this thesis we have designed a versatile synthetic way to obtain 8 diastereomeric spiroacetals. These compounds have been designed taking the cytotoxic macrolide of spongistatin 1 as the reference model. The asymmetric synthesis of these spiroacetal derivatives was achieved using 1,5-anti stereoinduction in aldol reactions, asymmetric allylations and spiroacetalization reactions as key steps. After its synthesis, biological evaluation of these compounds was carried out: the cytotoxicity of the aforementioned compounds on human ovary carcinoma cell lines A2780 and A2780AD (MDR overexpressing P-glycoprotein) was determined, studies concerning the effect of the compounds upon cytoplasmatic microtubule network, DNA and cell cycle of A549 lung carcinoma cells were performed, and the critical concentrations required for tubulin assembly were measured.[8] Spiroacetals 3.17 and 3.20 were found to retain a significant fraction of the cytotoxicity of the parent compound by means of interaction with tubulin. In vitro assembly studies showed that the compounds are able to bind to purified tubulin and to inhibit its assembly, although with different degrees of potency. However, the inmunofluorescence studies of cells indicated that the main cellular effect of spiroacetals 3.17 and 3.20 with tubulin is not the depolymerization of the cytoskeleton but a bundling effect: the microtubular network appears disorganized and the microtubules are forming bundles, a typical effect of microtubule-stabilizing agents and suggests that, either the compounds are modified inside the cell and/or, at the intracellular concentrations and perhaps in the presence of other cellular proteins, the interactions of the two compounds with tubulin produce additional effects not observed in experiments in vitro. Anyway, both compounds 3.17 and 3.20 were able to accumulate cells in the G2/M phase as shown in cell cycle experiments. 2.2. PIRONETIN ANALOGUES. RESULTS AND DISCUSSION In this thesis we have prepared six pironetin analogues, based on iterative ozonolysis/Brown allylation sequences as well as aldol reactions as key steps. In all these compounds, the configurations at the oxygenated carbons C-5, C-7 and C-9 are as in natural pironetin. Synthetic intermediates (Z)-5.114, (Z)-5.127 and (E)-5.127 were also included in the biological studies, since they possessed all functional groups that are required in the natural product pironetin for interaction with a-tubulin. As regards the biological evaluation of these compounds, the following studies were carried out: the cytotoxicity of the aforementioned compounds on human ovary carcinoma cell lines A2780 and A2780AD (MDR overexpressing P-glycoprotein) was determined, studies concerning the effect of the compounds upon cytoplasmatic microtubule network, DNA and cell cycle of A549 lung carcinoma cells were performed, and the critical concentrations required for tubulin assembly were measured.[9] Most compounds proved cytotoxic in the low micromolar range against both non-resistant and multidrug resistant P-glycoprotein overexpressing, ovarian carcinoma cell lines, similar IC50 values being found in both cell lines. Thus, most of the aforementioned compounds are able to inhibit microtubule assembly, both in vitro and in cell cultures, sharing the same general mechanism of action of tubulin assembly inhibition by the natural pyrone pironetin. 3. CONCLUSIONS In summary, we have been able to achieve the stereoselective synthesis of eight diastereoisomeric spiroacetals through a convergent synthetic route. The methodology that has been applied in the synthesis allows the formation of spiroacetals in a relatively easy and quick route. In the spiroacetalization step, two diastereomers can be generated, with each of them being able to adopt four different conformations. However, the spiroacetal obtained was in all cases the one having the maximum number of anomeric effects and, where possible, the minimum number of 1,3-diaxial interactions. Thus, the allyl group was located in all cases in the equatorial position in order to minimize these unfavourable 1,3-diaxial interactions. In summary, we have demonstrated that our synthetic spiroacetals display cytotoxicity and interact with tubulin having a measurable effect on microtubule polymerization. The precise mechanism of action and the potential binding site of these spiroacetal derivatives are still not known. Through the implementation of aldol additions using chiral auxiliaries such as Evans oxazolidinones and Crimmins thiazolidinthiones, asymmetric allylations, Z-selective olefinations and ring closing metathesis as key steps, we have been able to achieve the asymmetric synthesis of some a,b-unsaturated dihydropyrones related to pironetin. The obtained biological results suggest that all alkyl pendants are necessary for the full biological activity, perhaps with a certain emphasis on the role of ethyl group at C-4 (compound 5.102 is the most potent pironetin analogue that has been synthesized so far by our research group.). 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