Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold

Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents. Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling

Synthesis and structure-activity relationship studies of C(13)-desmethylene-(−)-zampanolide analogs

14 p.-4 fig.-2 tab.We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (−)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(−)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.3.1]heneicosane core by an intramolecular HWE reaction (67–95 % yield) and a stereoselective aza-aldol reaction with an (S)-BINOL-derived sorbamide transfer complex, to establish the C(20) stereocenter (24–71 % yield). As the sole exception, for the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclization product into the corresponding zampanolide analog was unsuccessful. All modifications led to reduced cellular activity and lowered microtubule-binding affinity compared to DM-ZMP, albeit to a different extent. For compounds incorporating the reactive enone moiety of ZMP, IC50 values for cancer cell growth inhibition varied between 5 and 133 nM, compared to 1–12 nM for DM-ZMP. Reduction of the enone double bond led to a several hundred-fold loss in growth inhibition. The cellular potency of 2,3-dihydro-13-desmethylene zampanolide, as the most potent analog identified, remained within a ninefold range of that of DM-ZMP.This workwas supported by the Swiss National Science Foundation (KHA,project200021_149253). Institutional support by the ETH Zurich is also gratefully acknowledged(KHA).Funding was also received from Ministerio de Ciencia e Innovación(Spain) (JFD,Project PID2019-104545RB-I00/AEI/10.13039/501100011033),the European Commission-NextGenerations EU(RegulationEU 2020/2094),through CSIC’s Global Health Platform(PTI Salud Global) and Proyecto de Investigación en Neurociencia Fundación Tatiana Pérez de Guzmán el Bueno 2020 (JFD).Peer reviewe

Polymeric glabrescione B nanocapsules for passive targeting of Hedgehog-dependent tumor therapy in vitro

Aim: With the purpose of delivering high doses of glabrescione B (GlaB) to solid tumors after systemic administration, long-circulating GlaB-loaded oil-cored polymeric nanocapsules (NC-GlaB) were formulated. Materials & methods: Synthesis of GlaB and its encapsulation in nanocapsules (NCs) was performed. Empty and GlaB- loaded NCs were assessed for their physico-chemical properties, in vitro cytotoxicity and in vivo biodistribution. Results: GlaB was ef ciently loaded into NCs (~90%), which were small (~160 nm), homogeneous and stable upon storage. Further, GlaB and NC-GlaB demonstrated speci c activities against the cancer stem cells. Preliminary studies in tumor-bearing mice supported the ability of NC to accumulate in pancreatic tumors. Conclusion: This study provides early evidence that NC-GlaB has the potential to be utilized in a preclinical setting and justi es the need to perform therapeutic experiments in mice

Natural products from higher plants and marine organisms as sources of new anticancer agents: synthesis and biological evaluation

Abstract part A: The dysregulation of the Hedgehog (Hh) signaling pathway plays a pivotal role in the generation and cell-manteinance of many human cancers. Gli transcription factors, the final effectors of the pathway, represent the most promising target for the development of new drugs targeting the Hh pathway in tumors. In a previous work, a natural isoflavone, glabrescione B (GlaB) (I), was identified as the first small molecule binding Gli1. It is able to inhibit the transcriptional activity of Gli1, by interfering with its interaction with DNA. In order to perform further studies on the mechanism of action of GlaB (I) we developed a total synthesis, while NMR studies demonstrated the interaction of GlaB with Gli1 Biological studies have demonstrated its ability to interfere with the activity of Gli1 by inhibiting the growth of Gli-dependent-Hh-dependent tumor cells such as medulloblastoma (MB) and basal cell carcinoma (BCC) both in vitro and in allograft mouse models. In addition, our new synthetic route, which encompasses just three steps with an overall yield of 15%, provided an efficient synthetic means to enable the investigation of the role of GlaB ring-B in the interaction Gli1-GlaB. In fact, our synthetic strategy allowed the preparation of several GlaB derivativesin order to elucidate the structure-activity relationships (SARs) and to clarify the molecular mechanism underlying its Hedgehog signalling modulation.Abstract part B: The second part of this PhD thesis describes the work I have carried out during my research stay abroad at Swiss Federal Institute of Technology (ETH) in Zürich (Switzerland) in Prof. Dr. Karl-Heinz Altmann's laboratory. Marine natural products show higher incidence of bioactivity compared to terrestrial natural products. This is due to a high degree of chemical novelty and their high dilution in ocean water. (+)-Dactylolide (I) was isolated by Riccio and co-workers from a sponge of the genus Dactylospongia, collected off the coast of Vanuatu islands, in the South Pacific Ocean. The absolute and the relative configuration at C19 of the compound remained unassigned. The assignment of the relative and absolute configuration of (+)-dactylolide (I) is based on its first total synthesis by Smith and co-workers. As had been described for the natural product, synthetic dactylolide has been found to be dextrorotatory, but the magnitude of the specific rotation reported for the natural product and synthetic I were significantly different from each other. In addition, the discrepancies between the 13C-NMR spectra of synthetic and natural (+)-dactylolide (if ever so slight), also leave open the possibility that the configuration of C19 in natural (+)-dactylolide is R and not S (i. e. natural dactylolide could have the structure II instead of I). In order to demonstrate that, a total synthesis of both compounds was established. (+)-Dactylolide (I) is related to (-)-zampanolide (III), another marine macrolide. The latter shows low nanomolar cytotoxicity against both drug-sensitive and multi-drug resistant cancer cell lines, and induces microtubule bundle formation. While (-)-zampanolide (III) is a nM inhibitor of cancer cell growth in vitro, not many data about the activity of (+)-dactylolide (I) have been published. On the other hand, the biological activity of synthetic (-)-dactylolide (ent-I) is well known. This compound exhibits sub-μM IC50 values against a multitude of cancer cell lines, although (–)-zampanolide (III) is still 100- to 300-fold more potent. At the same time, not even synthetic (+)-zampanolide (ent-III) has ever been tested and the importance of the configuration of the macrocycle for the potency of dextrorotatory compounds remains unclear. Our goal was to synthesize (+)-dactylolide (I) and (+)-zampanolide (ent-III), in order to investigate how the macrocycle configuration would affect the biological activity of the compounds

The plant-derived triterpenoid tingenin B is a potent anticancer agent due to its cytotoxic activity on cancer stem cells of breast cancer in vitro

Despite the rapid advances in chemotherapy regimens, the outcome of patients with breast cancer is not satisfactory. One of the reasons of this dissatisfaction is that subsets of cells in tumors which referred as cancer stem cells (CSCs) show and/or gain resistance to therapies. Thus, compounds that target CSCs are urgently needed. Since some are already used in the clinic, natural products have great potential for further development as anti cancer drugs. The aim of this study is to investigate the cytotoxic activity of tingenin b (or 22 beta-hydroxytingenone) which is a quinone-methide triterpenoid structurally related to tingenone, against breast CSCs (stem-cell enriched population from MCF-7 cell line, MCF-7s). It has been found that tingenin b was cytotoxic against MCF-7s (IC50 value for 48 h was found to be 2.38 mu M) by inducing apoptosis. It was evident by Annexin V staining positivity, decreased mitochondria( membrane potential and Bcl-2 dephosphorylation with a concomitant increase in Bax protein expression. In addition, endoplasmic reticulum stress was also found to be involved in tingenin b-induced cell death. In conclusion, the results warrant further studies aimed at elucidating and corroborating its possible use in the treatment of breast cancer

One hundred faces of cyclopamine

The natural steroidal alkaloid cyclopamine has been identified as the first inhibitor of the Hedgehog (Hh) signaling pathway, which is implicated in embryonic development and tumorigenesis, as well as is hyperactivated in cancer stem cells (CSCs). The list of Hh-dependent tumors is steadily growing, and it has been estimated that about 25% of all cancer deaths show signs of aberrant Hh pathway activation. Notably, cyclopamine has been found to exert anticancer activity against several types of human cancer and to inhibit CSCs proliferation, thus highlighting the druggability of the Hh pathway and paving new opportunities in anticancer drug discovery. The aim of the present work is to review the main synthetic strategies to cyclopamine and its derivatives, with particular emphasis on the challenging chemical modifications aimed at improving the biological activity of the molecule

The plant-derived triterpenoid tingenin B is a potent anticancer agent due to its cytotoxic activity on cancer stem cells of breast cancer in vitro

Despite the rapid advances in chemotherapy regimens, the outcome of patients with breast cancer is not satisfactory. One of the reasons of this dissatisfaction is that subsets of cells in tumors which referred as cancer stem cells (CSCs) show and/or gain resistance to therapies. Thus, compounds that target CSCs are urgently needed. Since some are already used in the clinic, natural products have great potential for further development as anti cancer drugs. The aim of this study is to investigate the cytotoxic activity of tingenin b (or 22β-hydroxytingenone) which is a quinone-methide triterpenoid structurally related to tingenone, against breast CSCs (stem-cell enriched population from MCF-7 cell line, MCF-7s). It has been found that tingenin b was cytotoxic against MCF-7s (IC50 value for 48 h was found to be 2.38 μM) by inducing apoptosis. It was evident by Annexin V staining positivity, decreased mitochondrial membrane potential and Bcl-2 dephosphorylation with a concomitant increase in Bax protein expression. In addition, endoplasmic reticulum stress was also found to be involved in tingenin b-induced cell death. In conclusion, the results warrant further studies aimed at elucidating and corroborating its possible use in the treatment of breast cancer

Occurrence of Enantioselectivity in Nature: The Case of (S)-Norcoclaurine

This review article is aimed at providing a monographic overview on (S)-norcoclaurine (NC) alkaloid from three diverse points of view, collected all together for the first time: 1) the synthetic one, where the compound is seen as a target chiral molecule to be obtained in the highest optical purity and as a starting point for the development of biocatalytic asymmetric syntheses of tetrahydroisoquinoline alkaloids; 2) the chromatographic one, which addresses the HPLC separation of the two NC enantiomers; and 3) the biochemical one, for which a thorough understanding of the topology and mechanism of action of norcoclaurine synthase (NCS) enzyme is still a matter of debate. Special emphasis on the most recent studies in the field is given by discussing the results published by the main research groups who are working on NC and NCS

A method for the stereoselective construction of the hemiaminal center in zampanolides

4 p.-1 fig.-1 tab.-i graph. abst.-5 schem.We have developed a new method for the stereoselective establishment of the N-acyl hemiaminal moiety in zampanolide-type structures that involves the reaction of (Z,E)-sorbamide (3) with BINAL-H and subsequent amide transfer froma putative aluminum carboximidoate complex to the aldehyde moiety of a dactylolide precursor, such as 2 or 5. The method has enabled the efficient synthesis of 13-desmethylene-(−)-zampanolide (4), which was found to be an equipotent cell growth inhibitor as the natural product (−)-zampanolide (1).This work was supported by the Swiss National Science Foundation (Projects 200021_149253 and 200020_175744 (KHA)). Funding was also obtained from the Ministerio de Ciencia e Innovación PID2019-104545RB-I00 and H2020-MSCA-ITN-2019 860070 TUBINTRAIN (JFD).Peer reviewe

Synthesis of Analogs of Waltheriones S and T and Their Activity Against Trypanosoma cruzi

Several alkaloids of the waltherione family exhibit antitrypanosomal activity in the sub-micromolar or nanomolar range. While the overwhelming majority of waltheriones are based on a quinoline core structure, two structurally simpler pyridone-based congeners, waltheriones S and T, have recently been isolated and found to inhibit Trypanosoma cruzi with single-digit micromolar potency. Here, we report on the synthesis of a series of analogs of waltheriones S and T based on pyridone ring formation via cyclization of an appropriate triketone precursor with ammonia and the assessment of their activity against Trypanosoma cruzi. The data show that the methoxy group at the C(3)-position of the pyridone ring can be removed without significant loss in potency. Further modification of 3-desmethoxy waltherione T through methoxylation at the C(1\u27) position of the C(6)-side chain or double methoxylation at the C(1\u27)-position and the pyridone nitrogen had no significant impact on antitrypanosomal activity. These findings contrast with the activity differences between the corresponding quinoline-based natural waltheriones M, Q, and H, where the methoxy-bearing waltheriones Q and H are one order of magnitude more potent than the unsubstituted parent compound waltherione M. Our data indicate that the SAR for monocyclic waltheriones S and T does not simply parallel that of the quinoline-based congeners and they point to the importance of a rigid quinoline core for potent activity against T. cruzi