Enantiopure bicyclic lactams: synthesis and biological evaluation

N-Methyl-D-Aspartate (NMDA) receptors are fundamental for the normal function of the central nervous system and have an important role in memory and learning. An overactivation of these receptors results into an influx of excess of calcium cation (Ca2+) leading to neuronal loss associated with major degenerative disorders including Parkinson’s and Alzheimer’s diseases. Since the 80s, a big interest emerged of both academia and industry to develop drugs targeting the NMDA subtype of glutamate receptors with potential application for the treatment of some degenerative disorders. The main successes were amantadine (39) and memantine (40), two compounds which belong to adamantanes’s family, approved by FDA and used currently in the clinic. Another important area of therapeutic interest is cancer, which is considered the first and second causes of deaths on developed and developing countries, respectively. Statistical studies estimate that in 2030 the number of deaths caused by this disease will be 13.2 million. For this reason, it is essential the discovery of new methodologies and therapeutic agents for the treatment of cancer. The main goal of this thesis is the enantioselective synthesis of small molecules, more precisely phenylalaninol- and tryptophanol-derived bicyclic lactams, with potential application as NMDA receptor antagonists and antitumor agents, respectively. From the library of phenylalaninol derivatives, two new were promising NMDA receptor antagonists were identified. In particular, compounds 1c and 1d revealed to be more active than the hit compound, 1a, with IC50 values of 39 μM and 36 μM, respectively. Derivatives of another amino alcohol, (1S, 2R)-(−)-cis-1-amino-2-indanol, were also synthesized and a new antagonist, compound 6b, was identified with IC50 value of 51 μM. A hit-to-lead process of these three compounds was performed and the determination of respective IC50 values is in progress. For the series of tryptophanol derivatives, a hit compound (4c) was identified which revealed an IC50 value of 60 μM in MCF-7 cell lines (breast adenocarcinoma). A structural derivatization was performed, leading to five more active compounds (8b-f) with IC50 values between 6.7 and 9.0 μM, for the same cancer cell line. Furthermore, these five compounds were also evaluated in other cancer cell lines, and revealed to be selective for MCF-7 cell line, as well as non-toxic for normal cells (HEK 293)

Indolo[2,3-a]quinolizidines and derivatives: Bioactivity and asymmetric Synthesis

Corynantheine alkaloids with a tetracyclic indole[2,3-a]-quinolizidine motif are an important issue in academia and in the life science industries due to their broad bioactivity profile. In particular, the main biological effects described for indoloquinolizidines include analgesic, anti-inflammatory, antihypertensive, and antiarrhythmic activities, as well as inhibition of multiple ion channels, affinity for opioid receptors, and activity against Leishmania. For that reason, in the last decades, numerous efforts have been invested in the development of novel synthetic strategies to obtain the indole[2,3-a]-quinolizidine system. This review focuses on the synthetic methodologies developed to target the most important alkaloids of this family, and highlights the potential use of these alkaloids or analogs to treat several diseases, ranging from cancer to neurodegenerative disorders

Enhancing anticancer activity of spiropyrazoline oxindoles by disrupting p53-MDMs PPIs

Cancer is a major public health problem worldwide with 18.1 million new cases of cancer and 9.6 million deaths worldwide in 20181. The protein p53 is involved in many biological processes that are important to maintain the normal function of the cells (e.g. apoptosis, cell arrest, and DNA repair). It is an attractive target in oncology because it can modulate several additional cellular processes that are relevant for the suppression of tumour development, such as opposing oncogenic metabolic reprogramming, activating autophagy, and restraining invasion and metastasis. In all types of human cancers, the p53 tumour suppressor function is inactivated by mutation or gene deletion or by negative regulators such as MDM2 and MDMX. In the last years, the most popular approach among medicinal chemists to activate the wild-type p53 was the inhibition of p53-MDM2 protein-protein interaction (PPI) using small molecules. However, it is currently known that the full reactivation of p53 is only achieved when the interactions of p53 with both negative regulators are inhibited. Due to the lack of dual p53-MDM2/X PPIs inhibitors in clinical trials, it is urgent to develop small molecules that inhibit p53-MDMs PPIs2. Our research team has been working on the development and optimization of spiropyrazoline oxindoles to obtain dual p53-MDM2/X PPIs inhibitors. Hence, we have already developed derivatives with good antiproliferative activities in HCT-116 p53(+/+) human colorectal carcinoma cell line, which induce apoptosis and cell cycle arrest at G0/G1 phase, upregulate p53 steady-state levels, and lead to a decrease of MDM2 levels3. In this communication, we report the structure-based computational optimization of this chemical family for the development of novel p53-MDM2/X interactions inhibitors. Our studies will shed light on the possible binding mode of spirooxindole derivatives to MDM2 and MDMX and will drive the hit-to-lead optimization strategy. Furthermore, we report our most recent optimization of the synthesis of these new spiropyrazoline oxindoles derivatives and the first preliminary biological results. Acknowledgements: This work was supported by National Funds (FCT/MEC, Fundacao para a Ciencia e Tecnologia and Ministerio da Educacao e Ciencia) through UID/DTP/04138/2019 (iMed.ULisboa), project PTDC/QUI-QOR/29664/2017, Principal Investigator grant CEECIND/01772/2017 (M. M. M. Santos) and PhD fellowships SFRH/BD/137544/2018 (E.A. Lopes) and SFRH/BD/117931/2016 (M. Espadinha). 1Ferlay, J., Colombet, M., Soerjomataram, I., Mathers, C., Parkin, D., Pineros, M., Znaor, A. and Bray, F., Int. J. Cancer, 2019, 144, 1941-1953. 2Espadinha M., Barcherini V., Lopes E. A., Santos M. M. M., Curr. Top. Med. Chem. 2018, 18, 647-660. 3a) Nunes R., Ribeiro C. J. A., Monteiro Â., Rodrigues C. M. P., Amaral J. D., Santos M. M. M., Eur. J. Med. Chem., 2017, 139, 168-179. b) Amaral J. D., Silva D., Rodrigues C. M. P., Sola S., Santos M. M. M., Front. Chem., 2019, 7, article

Hybrid molecules of protoflavones and spirooxindole derivatives with selective cytotoxicity against triple-negative breast cancer cells

The combination of compounds with complementary bioactivities into hybrid molecules is an emerging concept in drug discovery. In this study, we aimed to synthesize new hybrid compounds based on p53-MDM2/X protein-protein interaction spiropyrazoline oxindole-based inhibitors and ataxia telangiectasia and Rad3-related (ATR) protoflavone-based inhibitors through copper(i) catalysed azide-alkyne cycloaddition. Five new hybrids were prepared along with three representative reference fragments. The compounds were tested against human breast cancer cell lines MCF-7 (hormone-dependent, wild-type p53) and MDA-MB-231 (triple-negative, mutant p53). Most of the new hybrids were more cytotoxic than their reference fragments and several showed 2-4 times selective toxicity against MDA-MB-231 cells. Relevant pharmacological benefit gained from the hybrid coupling was further confirmed by virtual combination index calculations using the Chou method. Compound 13 modulated doxorubicin-induced DNA damage response through inhibiting the ATR-dependent activation of Chk-1, while increasing the activation of Chk-2. Our results suggest that the new hybrids may serve as new leads against triple negative breast cancer

Design, synthesis and biological evaluation of novel p53 activators by targeting p53 protein-protein interactions

The p53 protein, also known as the “guardian of the genome”, has an important role in the tumor suppression and regulation of cell processes. The majority of human cancers show inactivation of the p53 pathway. This perturbation can occur either by negative regulation, either by mutation or deletion of its gene. In tumors harboring wt p53, the MDM2 and MDMX homologous proteins are the main contributors for suppressing the p53 functions. In the last years, the development of p53-MDM2 PPI small molecule inhibitors has been one of the most popular approaches to reactivate wt p53, with eight clinical candidates under evaluation. However, it is now considered that, to achieve a full p53 reactivation, a dual inhibition of MDM2 and MDMX is required. Until today, there is no dual small molecule inhibitors of p53-MDM2/X PPIs in clinical trials. In the last years, our group has been working on the design of five-membered spirooxindoles to develop novel anticancer agents. This work explores the design of the spiropyrazoline oxindole family to act as MDM2/X dual inhibitors. Here, we report an in silico-guided design, synthetic optimization, and biological evaluation of two libraries of spiropyrazoline oxindoles. p53 also interacts with CREBBP. The inhibition of the p53-CREBBP PPI in certain biological circumstances can result in the p53 stabilization. For this purpose, PROTAC technology that allows to degrade the CREBBP protein was implemented, based on a lead CREBBP ligand developed in the Conway group. Also, a yeast target-based screening of enantiopure tryptophanol derivatives led to the identification of dual p53-MDM2/X inhibitors, which were further optimized to compounds DIMP53-1, SYNAP and SLMP53-1, the last being also a mut p53 reactivator. To better understand the mechanism of action of this chemical family, in particular of SLMP53-1, two types of chemical probes were prepared, and preliminary in vitro cell assays were performed to evaluate their potential in future applications. Also, the preliminary biological and photocrosslinking results for SLMP53-1 photoaffinity-based probe showed its potential for the biological target profile of this compound. Overall, this PhD thesis has provided valuable insights in the development of p53 activators

Enantiopure bicyclic lactams: synthesis and biological evaluation

N-Methyl-D-Aspartate (NMDA) receptors are fundamental for the normal function of the central nervous system and have an important role in memory and learning. An overactivation of these receptors results into an influx of excess of calcium cation (Ca2+) leading to neuronal loss associated with major degenerative disorders including Parkinson’s and Alzheimer’s diseases. Since the 80s, a big interest emerged of both academia and industry to develop drugs targeting the NMDA subtype of glutamate receptors with potential application for the treatment of some degenerative disorders. The main successes were amantadine (39) and memantine (40), two compounds which belong to adamantanes’s family, approved by FDA and used currently in the clinic. Another important area of therapeutic interest is cancer, which is considered the first and second causes of deaths on developed and developing countries, respectively. Statistical studies estimate that in 2030 the number of deaths caused by this disease will be 13.2 million. For this reason, it is essential the discovery of new methodologies and therapeutic agents for the treatment of cancer. The main goal of this thesis is the enantioselective synthesis of small molecules, more precisely phenylalaninol- and tryptophanol-derived bicyclic lactams, with potential application as NMDA receptor antagonists and antitumor agents, respectively. From the library of phenylalaninol derivatives, two new were promising NMDA receptor antagonists were identified. In particular, compounds 1c and 1d revealed to be more active than the hit compound, 1a, with IC50 values of 39 μM and 36 μM, respectively. Derivatives of another amino alcohol, (1S, 2R)-(−)-cis-1-amino-2-indanol, were also synthesized and a new antagonist, compound 6b, was identified with IC50 value of 51 μM. A hit-to-lead process of these three compounds was performed and the determination of respective IC50 values is in progress. For the series of tryptophanol derivatives, a hit compound (4c) was identified which revealed an IC50 value of 60 μM in MCF-7 cell lines (breast adenocarcinoma). A structural derivatization was performed, leading to five more active compounds (8b-f) with IC50 values between 6.7 and 9.0 μM, for the same cancer cell line. Furthermore, these five compounds were also evaluated in other cancer cell lines, and revealed to be selective for MCF-7 cell line, as well as non-toxic for normal cells (HEK 293)

Indolo[2,3-a]quinolizidines and derivatives: Bioactivity and asymmetric Synthesis

Corynantheine alkaloids with a tetracyclic indole[2,3-a]-quinolizidine motif are an important issue in academia and in the life science industries due to their broad bioactivity profile. In particular, the main biological effects described for indoloquinolizidines include analgesic, anti-inflammatory, antihypertensive, and antiarrhythmic activities, as well as inhibition of multiple ion channels, affinity for opioid receptors, and activity against Leishmania. For that reason, in the last decades, numerous efforts have been invested in the development of novel synthetic strategies to obtain the indole[2,3-a]-quinolizidine system. This review focuses on the synthetic methodologies developed to target the most important alkaloids of this family, and highlights the potential use of these alkaloids or analogs to treat several diseases, ranging from cancer to neurodegenerative disorders

Tryptophanol-Derived Oxazolopyrrolidone Lactams as Potential Anticancer Agents against Gastric Adenocarcinoma

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Gastric cancer is one of the deadliest cancers in modern societies, so there is a high level of interest in discovering new drugs for this malignancy. Previously, we demonstrated the ability of tryptophanol-derived polycyclic compounds to activate the tumor suppressor protein p53, a relevant therapeutic target in cancer. In this work, we developed a novel series of enantiomerically pure tryptophanol-derived small molecules to target human gastric adenocarcinoma (AGS) cells. From an initial screening of fourteen compounds in AGS cell line, a hit compound was selected for optimization, leading to two derivatives selective for AGS gastric cells over other types of cancer cells (MDA-MB-231, A-549, DU-145, and MG-63). More importantly, the compounds were non-toxic in normal cells (HEK 293T). Additionally, we show that the growth inhibition of AGS cells induced by these compounds is mediated by apoptosis. Stability studies in human plasma and human liver microsomes indicate that the compounds are stable, and that the major metabolic transformations of these molecules are mono- and di-hydroxylation of the indole ring.This research was funded by FCT (Fundação para a Ciência e a Tecnologia) through iMed.ULisboa (UIDB/04138/2020), CQE (UID/QUI/00100/2020), project PTDC/QUI-QOR/29664/2017, principal investigator grants CEECIND/02001/2017 (A. M. M. Antunes), and CEECIND/03143/2017 (L. M. Gonçalves), and PhD fellowships SFRH/BD/117931/2016 (M. Espadinha) and PD/BD/143126/2019 (V. Barcherini), and funded by Severo Ochoa FunFuture CEX2019-917-S.info:eu-repo/semantics/publishedVersio