## 4.2. Synthesis of Indeno[1,2-b]indoles: Challenges and Insights from Literature to Experiment

Jean-Jacques Hélesbeux, Florence O Mccarthy, Maria Manuel Silva, Alcino J Leitão, Anthony Burke, Jorge a R Salvador, Rui Moreira, Emília Sousa, Vânia M Moreira, Pascal Marchand

► To cite this version:

Jean-Jacques Hélesbeux, Florence O Mccarthy, Maria Manuel Silva, Alcino J Leitão, Anthony Burke, et al.. 4.2. Synthesis of Indeno[1,2-b]indoles: Challenges and Insights from Literature to Experiment. 32nd Annual GP2A Medicinal Chemistry Conference, GP2A, Aug 2024, Coimbra, Portugal. pp.18-19, 10.3390/ddc4010002 . hal-05062204

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Conference Report

32nd Annual GP₂A Medicinal Chemistry Conference

Jean-Jacques Hélesbeux ¹ ID, Florence O. McCarthy ² ID, Maria Manuel Silva ³,⁴ ID, Alcino J. Leitão ³,⁴ ID,
Anthony Burke ³,⁵ ID, Jorge A. R. Salvador ³,⁴ ID, Rui Moreira ⁶ ID, Emília Sousa ⁷,⁸ ID, Vânia M. Moreira ³,⁴,* ID
and Pascal Marchand ⁹,* ID

1 Université Angers, SONAS, SFR QUASAV, F-49000 Angers, France; jj.helesbeux@univ-angers.fr

2 School of Chemistry and Analytical Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, T12K8AF Cork, Ireland; f.mccarthy@ucc.ie

3 Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; msilva@ff.uc.pt (M.M.S.); ajleitao@ff.uc.pt (A.J.L.); ajburke@ff.uc.pt (A.B.); salvador@ff.uc.pt (J.A.R.S.)

4 Centre for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, 3004-531 Coimbra, Portugal

5 Coimbra Chemistry Centre, Institute of Molecular Sciences, Chemistry Department, Faculty of Science and Technology, University of Coimbra, 3004-535 Coimbra, Portugal

6 Research Institute for Medicines (iMed), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; rmoreira@ff.ulisboa.pt

7 Faculdade de Farmácia, Universidade do Porto, Rua Jorge de Viterbo Ferreira 228, 4050-313 Porto, Portugal; esousa@ff.up.pt

8 Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Av. General Norton de Matos, s/n, 4450-208 Matosinhos, Portugal

9 Nantes Université, Cibles et Médicaments des Infections et de l'Immunité, IICiMed, UR 1155, F-44000 Nantes, France

* Correspondence: vmoreira@ff.uc.pt (V.M.M.); pascal.marchand@univ-nantes.fr (P.M.)

**Abstract:** The Group for the Promotion of Pharmaceutical Chemistry in Academia (GP2A) held its 32nd annual conference in August 2024 at the University of Coimbra, Portugal. There were 8 keynote presentations, 12 early career researcher oral presentations, and 34 poster presentations. Four awards were delivered, two for the best oral communications and two for the best poster presentations.

**Keywords:** medicinal chemistry; drug design; chemical biology; chemical tools; pharmaceutical chemistry; conference report

<figure><img src="image_2.png" /></figure>

check for
updates

Academic Editor: Philippe Loiseau

Received: 4 December 2024

Accepted: 11 December 2024

Published: 9 January 2025

**Citation:** Hélesbeux, J.-J.; McCarthy, F.O.; Silva, M.M.; Leitão, A.J.; Burke, A.; Salvador, J.A.R.; Moreira, R.; Sousa, E.; Moreira, V.M.; Marchand, P. 32nd Annual GP₂A Medicinal Chemistry Conference. *Drugs Drug Candidates* **2025**, *4, 2*. https://doi.org/10.3390/ddc4010002

**Copyright:** © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0/).

# 1. Introduction

After 32 years, the annual meeting of the Group for the Promotion of Pharmaceutical chemistry in Academia (GP₂A) was held, for the first time in its history, in Portugal, at the beautiful and historical University of Coimbra, a UNESCO World Heritage site. As before, our event brought together participants from across Europe and beyond, connecting university and industrial researchers working in the fields of medicinal chemistry and chemical biology. The conference was in line with its high scientific standing, providing a friendly atmosphere, and a plethora of exchanges of experience between senior and young researchers (doctoral or post-doctoral students). The conference was held at the “Auditório da Reitoria”, right in the heart of the old University buildings, and organized with the help of members of the Faculties of Pharmacy of the universities of Coimbra, Porto, and Lisbon. The meeting benefited from the outstanding support of our sponsors, some having joined us on site.

Altogether, there were 107 attendees from different countries, including Brazil, India, Ukraine, Poland, Sweden, Finland, France, the UK, Ireland, Spain, Germany, Portugal, and
Italy. We hosted eight plenary lectures (keynote lectures 2.1–2.7 below) from internationally renowned scientists, namely, Prof. Michael Decker (University of Würzburg, Würzburg, Germany), Dr. Mikael Altun (Karolisnka Institute, Stockholm, Sweden), Prof. Line Bourel (University of Strasbourg, Strasbourg, France), Prof. Stefania Butini (Università degli Studi di Siena, Siena, Italy), Prof. Paola Costi (University of Modena and Reggio Emilia, Modena and Reggio Emilia, Italy), Prof. Antimo Gioello (Università degli Studi di Perugia, Perugia, Italy), Prof. Diego Muñoz-Torrero (Universitat de Barcelona, Barcelona, Spain), and Prof. Tiago Rodriges (University of Lisbon, Lisbon, Portugal), covering topics ranging from infectious and neurodegenerative diseases, chemoprevention of cancer, protein degradation, approaches to target identification, hit identification and optimization for drug candidates, natural products, inflammatory diseases/pain, and the application of structural cheminformatics, machine learning, artificial intelligence, and new synthetic concepts. There were a further record number of 12 presentations from early career researchers (Section 3 below), and 34 poster communications (Section 4 below).

Four prizes were awarded, and the prize winners were as follows:

- Best Oral Presentation: Constance Dalton, University of Nottingham, sponsored by MDPI;

- Runner-up Oral Presentation: Lucia Gonzales, University of Santiago de Compostela, sponsored by Wiley;

- Best Poster Presentation: Johana Charles, Université Claude Bernard Lyon 1, sponsored by Wiley;

- Runner-up Poster Presentation: Sara Moura, University of Coimbra, sponsored by RSC Medicinal Chemistry.

The local organizing committee put together two social programs for the attendees, including a guided tour to the Palace of Schools, featuring the Baroque Library, Saint Michael's Chapel, and the Royal Palace of the University of Coimbra, and also a guided tour to the Old Cathedral of Coimbra plus a "Fado" show. The attendees also had the opportunity to listen to a typical "tuna" show, by the students comprising the Imperial TAFFUC. The conference banquet dinner was held in the fabulous premises of the Quinta das Lágrimas.

## 2. Keynote Lectures

### 2.1. *Chemical Neuroscience in Alzheimer's Disease: Hybrid Molecules and Photopharmacology*

Michael Decker¹

1. Pharmaceutical/Medicinal Chemistry, Julius-Maximilian-University Würzburg (JMU), 97074 Würzburg, Germany; michael.decker@uni-wuerzburg.de

Alzheimer's disease (AD) is still uncurable, and next to its pathophysiological hallmarks of β-amyloid plaques and τ-protein hyperphosphorylation, neuroinflammation is observed. It is observed that counteracting neuroinflammation is effectively reversing cognitive decline in animal models of AD. Such anti-neuroinflammatory action can be effectively mediated by inhibiting the enzyme human butyrylcholinesterase (BChE) [1], by activating the human muscarinic acetylcholinesterase receptor subtype 1 (*hACh*₁R or *hM*₁R), and/or by activating the human cannabinoid receptor subtype 2 (*hCB*₂R) [2].

We report on novel ways to selectively inhibit BChE by carbamates and thereby improve both short- and long-term memory using pseudo-irreversible inhibitors with tunable duration of action, which directly correlate with dosage necessary in vivo [3]. Following this approach, BChE inhibition was combined with concomitant agonist activity at the *hCB*₂R. Despite lower activity (at both targets) in the micromolar range, compounds
represent "small molecules" and were very potent in vivo, proving a synergistic mode of action [2].

"Photopharmacology" uses molecular photoswitchable moieties that are incorporated into bioactive molecules to control pharmacological activity by light, achieving an unprecedented spatio-temporal control of bioactivity. The challenges of this approach are illustrated for photoswitchable BChE inhibitors enabling the light control of each step of carbamoylation BChE and yielding a photoswitchable inhibitor, that was inactive as its *trans*-photoisomer in vivo, but active as its *cis*-photoisomer [4].

Furthermore, photoswitchable hCB2R agonists were developed representing "cis-on" affinity switches, as well as a compound that is a "trans-on" efficacy switchable photo-agonist [5]. This compound used novel benzimidazole-based photoswitches, which require visible, but not UV-light for switching [6]. Furthermore, the latter compound represents the first-in-class light-controllable class-A GPCR-biased agonist [5].

Finally, the most recent developments regarding photoswitchable *hM*₁*R* agonists are reported, which includes red-shifted orthosteric agonists as well as the first photoswitchable class-A allosteric modulator ("Photo-BQCA") enabling the light-control of *hM*₁*R* action [7].

*Financial support by the Deutsche Forschungsgemeinschaft (DFG DE 1546/10-1 and 12-1) and by the Elite Network of Bavaria for the Doctoral Network "Receptor Dynamics" is gratefully acknowledged.*

## 2.2. *The One Health Concept in Antiparasitic Drug Discovery, What Matters?*

Maria Paola Costi¹

1. Department of Life Science, University of Modena and Reggio Emilia, 41125 Modena, Italy; mariapaola costi@unimore.it

Nearly 75% of emerging human infections worldwide originate from animals. Current drugs for both human and animal (H&A) vector-borne diseases (VBD) are in short supply and have limited efficacy, potential toxicity, and limited resources. Emerging environmental issues in pharmaceutical use/manufacturing are increasing attention in this area. Collaboration and research between different expertise involving human and animal medicines are essential to define how new drugs can be developed using a more sustainable approach [8]. All the drug discovery processes can include greener principles [9]. OneHealthdrugs is a COST Action dedicated to the development of innovative strategies for the supply of drugs with an environmentally friendly profile. The new drugs should combat H&A VBD while maintaining the principles of an optimal profile for both organisms, improving their quality by also implementing delivery technologies. With these aims, we have developed different concepts. We highlight the scarcity of ecotoxicological data for commonly used antiparasitic drugs and stress the urgent need for considering the One Health concept; then, we advocate for employing predictive tools and nonanimal methodologies such as New Approach Methodologies at early stages of antiparasitic drug research and in the subsequent development [10].

In one representative case, we have investigated the biochemical mechanisms of resistance to antimonials, paromomycin, and miltefosine in three drug-resistant parasitic *Leishmania infantum* strains from human clinical isolates, using a whole-cell mass spectrometry proteomics approach (Figure 1). We identified 14 differentially expressed proteins that were validated with their transcripts. Next, we employed functional association networks to identify parasite-specific proteins as potential targets for novel drug discovery studies [11]. We used SeqAPASS analysis (Sequence Alignment to Predict Across Species Susceptibility, EPA tool) to predict the susceptibility based on the evolutionary conservation of protein drug targets across species. Five proteins identified at the end of the process emerged as top candidates. Among them, calpain-like cysteine peptidase was the best
selected protein. Overall, this work identifies new biological targets for designing drugs to prevent the development of Leishmania drug resistance, while aligning with One Health principles that emphasize the interconnected health of people, animals, and ecosystems. Other cases will be presented showing the interconnection between One Health approaches and the drug discovery process.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Whole-cell mass spectrometry proteomics approach.</figcaption></figure>

*Acknowledgment: COST Action CA21111. One Health Drugs against Parasitic Vector-Borne Diseases in Europe and Beyond*

## 2.3. Fighting Against Multiresistant Fungal Infections—A State-of-the-Art Update and the Beginning of a Chemical Biology Study of Synthetic Antifungal Substances

Line Bourel ¹,² and Mylène Lang ¹,²

1. Chemobiology and Pharmacognosy for Health Team, Laboratory of Therapeutic Innovation, UMR 7200 CNRS/Unistra, Faculty of Pharmacy, 74, Route du Rhin, F-67400 Illkirch, France

2. USIAS, University of Strasbourg, F-67000 Strasbourg, France; line.bourel@unistra.fr

Among the microbial causes for nosocomial infections, multiresistant fungi are frequently incriminated, but research thereon is somehow left behind [12]. Yet, worldwide, >1.6 million immunocompromised hospitalized patients die annually of invasive fungal diseases (IFDs), notably due to *Candida* spp., against which none of the four approved antifungal (AF) drug families is active [13]. The AF arsenal is composed of very toxic Amphotericin B, weakly selective 5-Fluorocytosine, and of the azole class, which are increasingly inefficient against IFDs. In the 2000s, Echinocandins (ECs) were introduced as semisynthetic lipopeptides (LPs) inhibiting β-(1,3)-D-glucan synthase (GS), an enzyme involved in the biosynthesis of the fungal cell wall. ECs trigger its disruption, which is lethal for yeasts like *Candida* spp. As this enzyme is absent in mammal cells, ECs were initially developed as safe AF drugs against IFDs. Unfortunately, nowadays, more and more *Candida* strains encountered in intensive care units exhibit a low EC susceptibility [14]. So, for this class and its target, mechanistic investigations are really urged. In the near future, the identification of new AF scaffolds active on this relevant target would be of great importance for public health [15].

The University of Strasbourg Institute for Advanced Study (USIAS) funds original research in all scientific domains and serves as a place of intellectual brainstorming and innovation. USIAS is a flagship project of the Excellence Initiative (IdEx) of the University of Strasbourg and supports researchers from any disciplines by kicking off their new scientific projects. Thus, the present research program has just begun.

First, a state-of-the-art update regarding fungal pathologies and antifungal substances will be presented. Second, we propose to implement the antifungal arsenal with original ECs-based synthetic LPs that can be more easily sourced and purposely chemically modified. In this project, their activity will be continuously assessed against fungal strains originating from patients hospitalized at the Hopitaux Universitaires de Strasbourg in
low-throughput screening EUCAST-grade conditions. In parallel, a medium-throughput
screening is planned and libraries of alternative original substances will be tested on whole
*Candida albicans*. Finally, we will expose the beginning of a chemical biology study. To
this purpose, fluorescent ECs have been synthesized and the bioproduction of a traceable
β-(1,3)-D-glucan synthase has just started, to permit the imaging of their interaction and
the structural analysis of the target. If successful, such bioproduction could be extended
to the GS of other strains that are also responsible for life-threatening IFDs. By all these
pathways, we hope to understand the intimate mechanism of action of synthetic ECs on GS
and, in parallel, to contribute to the identification of original AF substances.

## 2.4. *Machine Learning to Accelerate the Chemical Sciences*

Tiago Rodrigues¹

1. iMed.Ulisboa, Faculty of Pharmacy, University of Lisbon, 1649–003 Lisbon, Portugal; tiago.rodrigues@ff.ulisboa.pt

Machine learning and artificial intelligence (ML/AI) are reshaping the chemical sciences by changing research paradigms and enabling new discoveries. Provided there are enough quality data, ML/AI algorithms can identify hidden patterns that would remain unnoticed otherwise [16,17]. A central dogma in ML/AI is that the performance is tightly connected to the amount of training data. However, recent reports challenge this concept [18,19].

In several chemistry use cases, data are typically scarce, heterogeneous, and/or unstructured, which poses important challenges to the implementation of discriminative ML/AI tools [16]. Herein, we show that small amounts of high-quality data are viable starting points for ML/AI, in particular when active learning approaches are pursued. We discuss how smart experiment prioritization can efficiently unravel meaningful patterns in chemical reaction data, in the identification of false-positive hits in screening assays, and in the design of nanomaterials [20–23]. We discuss the impact that low-data ML/AI might have in next generation discovery sciences.

T.R. acknowledges all collaborators who contributed to the execution of the presented work.

## 2.5. *Accelerating Medicinal Chemistry by Flow-Enhanced Synthesis*

Antimo Gioiello¹

1. Laboratory of Medicinal and Advanced Synthetic Chemistry (@LabMASC), Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy; antimo.gioiello@unipg.it

In the target-rich post-genomics era, the competitive arena of the pharmaceutical industry requires constant innovation and accelerated timelines to foster drug discovery pipelines [24,25]. Despite decades of ground-breaking research, compound synthesis is still considered the rate-limiting factor in the pace of drug discovery.

In medicinal chemistry, the preparation of pure compound collections represents the bottleneck of the 'design-make-test-analyze' cycle, as the conventional bench synthesis is a labor-intensive and time-consuming work. Moreover, there exists a persisting need to develop enabling syntheses that can facilitate compound development addressing the process scalability and optimization challenges for large-scale production.

The goal of this lecture is to introduce the concepts and potential of continuous flow technology in medicinal chemistry, and to showcase the efforts made by our group with the aim to improve synthetic capabilities and expedite the medicinal chemistry learning cycle [26]. Particular attention will be devoted to case studies that have shown the utility of flow chemistry to simplify usual batch operations and, in turn, the synthesis of com-
pound libraries readily available for screenings, as well as to realize sustainable synthetic approaches for lead discovery and development.

## 2.6. Novel Inhibitors of the Endocannabinoids' Catabolic Enzymes as Potential Therapeutics for Epilepsy and Neuroinflammatory Conditions

Stephania Butini¹

1. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 2, 53100 Siena, Italy; butini3@unisi.it

The endocannabinoids (ECs) anandamide (AEA) and 2-arachidonoylglycerol (2-AG), by stimulating cannabinoid CB1 and CB2 receptors (CB1R and CB2R), regulate relevant signaling pathways. The key enzymes involved in ECs catabolism are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Their unique role in terminating ECs signaling and regulating the intracellular levels of AEA, 2-AG, and other ECs supports their potential as therapeutic targets. The selective or dual inactivation of ECs degrading enzymes represents an attractive approach for eliciting the desirable effects of CBR activation, while avoiding the negative (psychotropic, among others) effects of CB1R stimulation.

Epilepsy is the second most common neurological disorder with an incidence rate of 0.3–0.5% worldwide. Most of the conventional anti-epileptic drugs have a narrow therapeutic margin and require therapeutic drug monitoring. Despite the introduction of many II-generation anti-epileptic drugs, pharmacoresistant epilepsy has not been significantly reduced. A reduction in AEA in patients affected by temporal lobe epilepsy (TLE) has been clearly documented. Similarly, the neuroprotective role of AEA was confirmed by the kainic acid-induced increase of AEA in the hippocampus, which, without affecting 2-AG, provides “on demand” protection against acute excitotoxicity. Seizure activity at the cellular level initiates a significant influx of calcium. Over time, impaired neuronal calcium homeostasis increases the activity of pro-oxidant cellular systems. The enocannabinoid system can delay or prevent excitotoxic damage by re-balancing the main excitatory/inhibitory systems and directly modulating mitochondrial function, thus preventing oxidative stress–related epileptogenesis. This rationale led to the proposal of FAAH inhibitors as a therapeutic option for the prevention of epileptic disorders. To strengthen this hypothesis, we demonstrated that our potent and selective FAAH inhibitor (NF1245, Ki = 160 pM [27]) ameliorated the acute epileptic behavior and prevented hippocampal oxidative damage in a rat model of pilocarpine-induced epilepsy, when tested at 10 mg/kg [28]. The same dose was also effective in TLE generated by electric kindling. Based on our recent results [29], the rational structural modifications produced improved the analogues that were able to reduce the oxinflammation state by decreasing the DNA-binding activity of NF-kB p65, devoid of a cytotoxic effect and unwanted cardiac effects, while being able to reduce the severity of the pilocarpine-induced status epilepticus [28].

Neuro-inflammation, mainly regulated by microglia and astrocytes, is a common feature of several neurodegenerative diseases, amyotrophic lateral sclerosis, and multiple sclerosis. In fact, neurological disorders can be triggered by chronic neuro-inflammatory conditions that, through different mechanisms in which oxidative stress is involved, may lead to neuronal cell death. Oxidative stress is caused by increased levels of reactive oxygen species (ROS) deriving from mitochondrial dysfunctions. Elevating endogenous levels of the ECs through FAAH and MAGL inhibition is a valuable approach to enhance the anti-inflammatory, anti-nociceptive [30,31], and neuroprotective effects mediated by the ECs. On these bases, we have recently designed a new library of FAAH inhibitors of nanomolar potency embedding ionizable functions, for improving the water solubility and chemical stability [32]. For these compounds, effective in reducing the ROS production in 1321N1 astrocytes, the anti-inflammatory activity was evaluated ex vivo in hippocampal slice cul-
tures. New and exceptionally potent irreversible MAGL inhibitors were recently developed for attaining brain permeability [33]. The exploration of different polypharmacological profiles will be also discussed [34].

## 2.7. *Serendipity and Drug-Unlikeness in the Discovery of a New Antimalarial Agent*

Diego Muñoz-Torrero ¹,²

1. Laboratory of Medicinal Chemistry (CSIC Associated Unit), Faculty of Pharmacy and Food Sciences, Av. Joan XXIII 27-31, 08028-Barcelona, Spain; dmunoztorrero@ub.edu

2. Institute of Biomedicine (IBUB), University of Barcelona, Av. Diagonal 643, 08028-Barcelona, Spain

All of us, as medicinal chemists, have interiorized the notion that our hits, leads, and candidates must feature a number of general attributes or properties, which directly derive from their chemical structure and would give them more chances to become a drug. Thus, we expect that our compounds align with a sort of "structural beauty pattern" that we call drug-likeness. Starting with the working hypothesis by Xavier Fernàndez-Busquets (Head of the Nanomalaria group, at Institute for Bioengineering of Catalonia (IBEC) and ISGlobal Barcelona) that protein aggregation, which seems to be abundant in *Plasmodium falciparum*, has a functional role, and, hence, that the inhibition of protein aggregation might be a new way to treat malaria, he serendipitously found that a dye used to monitor protein aggregation was indeed able to kill the malaria parasite with nanomolar potency. This compound, YAT2150, is everything but a "beautiful" drug-like molecule. Notwithstanding this, we initiated a joint project to go deeper into the physicochemical and biological properties of YAT2150 and to perform a lead optimization campaign to establish a first-in-class family of antimalarial agents based on the inhibition of protein aggregation.

This has resulted in very interesting findings on the profile of YAT2150: (i) it has a nanomolar potency on chloroquine- and artemisinin-resistant *P. falciparum* lines; (ii) it arrests asexual blood parasites at the trophozoite stage; (iii) it interferes with sexual and hepatic forms of the parasite; (iv) it reduces the amount of aggregated proteins in *P. falciparum* cultures; (v) it fluoresces when it accumulates in its main localization in the parasite cytosol, thereby being useful as a theranostic agent; and (vi) it is active against the leishmaniasis parasite *Leishmania infantum* [35,36].

Moreover, after modifications at different points of the chemical structure of YAT2150, we have developed new analogs with improved antiplasmodial potencies, selectivity indexes, and DMPK properties. Overall, these compounds, featuring a novel chemical scaffold and mechanism of action, might be the spearhead of a new generation of antimalarial drugs in the post-artemisinin era and pave the way for the single-drug treatment of the geographically overlapped malaria and leishmaniasis, the two most deadly parasitic diseases.

*Funding from MICIU/AEI/10.13039/501100011033 (grants PID2020-118127RB-I00 and PDC2022-133085-I00 (with European Union NextGenerationEU/PRTR funds)) and AGAUR (grant 2021SGR00357) is gratefully acknowledged.*

# 3. Oral Communications

## 3.1. *A MTDL's Approach Against Alzheimer's Disease: Deconstruction of a Tricycle*

Thomas Guiselin¹, Cédric Lecoutey¹, Christophe Rochais¹ and Patrick Dallemagne¹

1. Normandie Univ, UNICAEN, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), 14000 Caen, France; thomas.guiselin@unicaen.fr

Alzheimer's disease (AD) has been described for the first time more than a century ago, and its molecular causes are now the subject of a relative consensus around the
hyperphosphorylation of the TAU protein and the aggregation of the β-amyloid (Aβ) peptide into neurotoxic oligomers.

5-HT6R is a valuable target of therapeutic interest in AD. The inactivation leads, in particular, by the inhibition of the mTOR pathway, to procognitive effects observed in rodents. 5-HT6R antagonists are also able to promote the 5-HT concentration in the brain. As a result, 5-HT6R antagonists have shown positive effects against the memory disorders affecting these animals. The serotonin transporter (SERT), on the other hand, has also demonstrated its interest as a target in the treatment of AD. Indeed, its antagonists, which selectively inhibit serotonin reuptake (SSRI) at the pre-synaptic level, have also demonstrated their ability to reduce amyloid deposition and senile plaques in transgenic AD mice and in humans.

The objective of this project is to design a multi-target directed ligand, able to both inhibit 5-HT reuptake and selectively antagonize 5-HT6R (Figure 2).

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Hypothetical mechanisms of a dual SSRI/5-HT6R antagonist.</figcaption></figure>

Starting from a hit, selected from the CERMN's chemical library, a first set of derivatives has been synthesized in order to establish the structure-activity relationships and improve the expected activities (Figure 3).

<figure><img src="image_5.png" /><figcaption>**Figure 3.** MTDL's scaffolds.</figcaption></figure>
3.2. *Advances in the Development of Synthetic Allosteric Modulators of Protein Kinase C*

Katia Sirna¹, Riccardo Provenzani¹, Saara Lautala², Artturi Koivuniemi², Alex Bunker², Evgeni Grazhdankin¹, Henri Xhaard¹, Raimo K. Tuominen³, Virpi Talman³ and Jari Yli-Kauhaluoma¹

1. Drug Research Programme and Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland

2. Drug Research Programme and Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland

3. Drug Research Programme and Division of Pharmacology and Pharmacology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland; katia.sirna@helsinki.fi

Protein kinases C (PKCs) are a family of serine/threonine kinases implicated in several cellular processes, such as the modulation of gene expression, cell division, migration, proliferation and differentiation, cell survival, and apoptosis, and, therefore, they are involved in different pathologies. Thus, the development of PKC-targeted compounds represents a highly promising strategy to identify new drug candidates [37]. We have previously developed 5-(hydroxymethyl)isophthalate derivatives (HMIs) (Figure 4), which demonstrated binding to the C1 domain of PKCs and exhibited anticancer, antifibrotic, and neuroprotective effects [38–40]. However, their high lipophilicity negatively affects their drug-like properties.

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Pros and cons of previous (HMIs and PRDs) and current (bicyclic derivatives) allosteric modulators of PKC.</figcaption></figure>

To improve the solubility of HMIs while retaining the activity, several nitrogen-
containing cores had already been considered. The last generation of compounds, pyridines
(PRDs), showed a comparable affinity to the C1 domain while reducing the overall
lipophilicity compared to the HMIs [41]. Using the PRDs as a starting point and considering
the molecular structure of some of the natural and synthetic ligands of the C1 domain [42],
we designed bicyclic derivatives, bearing a hydroxymethyl group and alkyl/phenyl sub-
stituents, as new potential allosteric modulators of PKC (Figure 4). The new small molecules
were tested for binding to the C1 domain, revealing a higher affinity than the HMIs. More-
over, a preliminary biological evaluation suggested that the new compounds are able to
activate PKC.

3.3. *Synthesis and Evaluation of Thio-Linked Pyrimidine-Based Compounds Against Leishmania Donovani: In Silico and In Vitro Assessments*

Inês Costa ¹,², André C. Augusto ¹, Margarida M. Alves ³, Rita C. Guedes ³, Philippe M. Loiseau ⁴, Sandrine Cojean ⁵,⁶ and Maria L. S. Cristiano ¹,²

1. Center of Marine Sciences, CCMAR, Gambelas Campus, University of Algarve, UAlg, 8005-139 Faro, Portugal; a52917@ualg.pt
2. Department of Chemistry and Pharmacy, Faculty of Sciences and Technology, FCT, Gambelas Campus, University of Algarve, UAlg, 8005-139 Faro, Portugal

3. Research Institute for Medicines (iMed.Ulisboa), Computational Medicinal Chemistry Lab, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal

4. BioCIS Biomolécules: conception, Isolement, synthèse, chimiothérapie antiparasitaire, Université Paris-Saclay, 91400 Orsay, France

5. Université Paris-Saclay, 91400 Orsay, France

6. UMR BIPAR, Laboratory of Animal Health, Anses, INRAe, EnvA, 94700 Maisons-Alfort, France

Leishmaniases are a cluster of syndromes recognized by the WHO as neglected tropi-
cal diseases, caused by Leishmania parasites. Available chemotherapy for treating these
infections is unsatisfactory, mainly due to emerging resistance. There is thus a huge need
for novel drugs, with a better efficacy, safety, and reduced tendency to resistance devel-
opment [43]. In the search for novel biomolecular targets, trypanothione reductase (TR)
becomes attractive due to its vital role in maintaining the parasite's redox machinery.
TR reduces the disulfide bridge of trypanothione disulfide (TS₂) into reduced trypanoth-
ione T(SH)₂, a di-thiol responsible for eliminating reactive species for Leishmania [44,45].
Moreover, TR owns several advantages, namely its structural similarity among all *Try-
panosomatidae* parasites and its absence in the mammalian host [46]. Among the search
for better antileishmanial options, a pilot study identified the potential of aryl sulfides in
inactivating TR [45,47]. Considering these results, we disclose the synthesis of a library of
novel thio-linked pyrimidine-based compounds based on the Buchwald–Hartwig method-
ology. Different products were formed by changing the reaction time, some of them being
confirmed by X-ray crystallographic studies. The results of our computational studies
indicate that all compounds from our library interact with residue Arg222, thus restricting
the interaction between the enzyme and its cofactor NADPH. Finally, we also disclose the
results of the in vitro antileishmanial activity of all compounds against *L. donovani* axenic
and intramacrophage amastigotes, as well as their cytotoxicity against Raw 264.7 cells.
Compounds **IC69** and **IC90** proved to be the most active against both forms of *Leishmania*,
exhibiting IC₅₀ values on a nanomolar scale (Figure 5).

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Representation of the research approach followed, with indication of best-performing thio-linked pyrimidine-based conjugates among those synthesized and studied within the presented work.</figcaption></figure>

3.4. *Development of New Antivirals Using a High-Throughput Fragment-Merging Strategy*

Hadia Almahli ¹, Jasmin Aschenbrenner ², Max Winokan ², Daren Fearon ², Kate Fieseler ², Mathew Golding ², Stephanie Wills ², Warren Thompson ², Rahman Shah Zaib Saleem ¹ and Matthew Todd ¹

1. Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University College London (UCL), London WC1N 1AX, UK; h.almahli@ucl.ac.uk

2. Diamond light source Ltd., Diamond House, Harwell Science and Innovation Campus, Didact OX11 0DE, UK
Viral infections continue to pose health risks coupled to major healthcare costs. A broad range of viruses including SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and CHIKV (CHIKungunya Virus) [48] continues to evolve and pose a pandemic threat. The dawn of this decade witnessed how SARS-CoV-2, responsible for the COVID-19 pandemic, caused more than 774 million infections and led to more than 7 million deaths [49]. Developing effective antivirals with new mechanisms of action is essential for pandemic preparedness. In the READDI-AViDD consortium, we focus on the development of new small-molecule antivirals based on hits derived from in silico, fragment, and high-throughput screening. For hits validated through orthogonal assays, hit to lead campaigns are conducted. All data and ideas are placed in the public domain for others to use. In the READDI-AViDD project, we carry out the design, synthesis, and biological evaluation of broad-spectrum antivirals targeting different viral proteins: SARS-CoV-2-RdRp, SARS-CoV-2-nsp14/10, SARS-CoV-2-nsp14, CHIKV-nsp2-protease, and CHIKV-nsp3-MD.

Here, we describe the Fragment-Based Drug Discovery (FBDD) approach that we are using in the development of new inhibitors targeting CHIKV-nsp3-Macrodomain and the most promising in vitro methodologies that can expedite this process (Figure 6).

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Fragment-merged compound designed by Fragmenstein showing binding interactions with CHIKV-nsp3-MD at the adenine-binding site.</figcaption></figure>

Fragmestein [50] is used in this project to develop fragment-merged compounds, and we also used Fragalysis [51], which allowed us to study the binding interactions of the merged compounds with the target protein (CHIKV-nsp3-Macrodomain) based on their original fragments, and then to follow up with the synthesis of these compounds aiming to explore new antivirals which could also assist with the elimination of other viral pathogens [52].

### 3.5. *Development of Novel Sirtuin-1 Activators: Promising Therapeutic Tools Against Neurodegenerative Diseases*

Rodrigo F.N. Ribeiro<sup>1,2,3,4</sup>, Andrea Magni<sup>5</sup>, Luís Pereira de Almeida<sup>1,2,3,4</sup>, Cláudia Cavadas<sup>1,2,3</sup>, Giorgio Colomboe<sup>5,*</sup> and Maria Manuel Silva<sup>1,2,3,*</sup>

1. Center for Neuroscience and Cell Biology (CNC-UC), University of Coimbra, 3004-504 Coimbra, Portugal; rribeiro@cnc.uc.pt

2. Center for Innovation in Biomedicine and Biotechnology (CIBB), University of Coimbra, 3004-531 Coimbra, Portugal

3. Faculty of Pharmacy, University of Coimbra (FFUC), 3000-548 Coimbra, Portugal

4. Gene Therapy Center of Excellence (GeneT), 3004-504 Coimbra, Portugal
5. Department of Chemistry, University of Pavia, 27100 Pavia, Italy

Machado-Joseph disease is a fatal neurodegenerative disorder characterized by the expression of the toxic mutant ataxin-3 protein. Importantly, our group found that resveratrol, through sirtuin-1 (SIRT1) activation, is effective in alleviating the phenotype of transgenic MJD mice [53]. Moreover, similar positive effects have been described in other neurodegenerative disorders [54]. To overcome the low potency, low bioavailability, and lack of SIRT1 specificity of resveratrol, diverse synthetic activators were developed [53]. Despite high expectations, clinical trials with novel sirtuin-activating compounds (STACs) were disappointing, mainly due to pharmacokinetic (PK) issues. Therefore, the present study aimed to take advantage of state-of-the-art computer-aided drug design to discover an improved potent and safe STAC.

All the available 3D structures of SIRT1 were analyzed and prepared (Pymol; Jalview; AlphaFold). The two models containing the STAC-binding domain (PDB ID: 4ZZJ/5BTR) were further optimized and subjected to 1-microsecond molecular dynamics (MD) simulations to explore the behavior of SIRT1-ligand complexes (Desmond, Schrödinger). After extensive examination, trajectory frame clustering was performed, according to the protein backbone or loops deviations, and the top five clusters for each analysis were retrieved. This resulted in 20 representative structures of SIRT1. A comprehensive compound database comprising nearly 5 million structures, including our own virtual library, was created, and virtual screening simulations were performed using Glide (Schrödinger). The compounds were ranked based on their docking score, and, more importantly, the number of times each was present in the top molecules per cluster, allowing us to predict not only the possibility of a strong binding but also the consistency across SIRT1 dynamics. Ligand optimization and redocking were performed with Glide (Schrödinger) and SeeSAR (Bio-SolveIT). Hits were defined as molecules with good docking scores (Glide) and binding affinities (SeeSAR), when present in the top compounds of most of the 20 clusters. Specialized ADME and toxicity prediction modules were used to filter our compounds to ensure drug-likeness properties and surpass the limitations of existing STACs (QikProp; SwissADME; pkCSM; Optibrium). Moreover, MD are being performed providing crucial insights into ligand-protein interactions and stability profiles.

We obtained a set of virtual STACs with a promising activity and PK profile.
This study has the potential to bridge the gap between our previous work and fu-
ture pharmacotherapy, also providing proof-of-principle for treatment of high-burden
neurodegenerative disorders.

Funded by the ERDF (COMPETE 2020) and FCT (UIDB/04539/2020, UIDP/04539/2020, and LA/P/0058/2020). R.F.N.R. is funded by FCT (2020.04850.BD) and EMBO (Scientific Exchange Grant 10569).

3.6. *From Inhibitors to Degraders: Application to the Design of Innovative Compounds for the Treatment of Chemoresistant Ovarian Cancer*

Thomas Lemaitre ¹, Charline Kieffer ¹, Marc Since ¹ and Anne-Sophie Voisin-Chiret ¹

1. Normandie Univ, UNICAEN, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), 14000 Caen, France; thomas.lemaitre@unicaen.fr

Ovarian cancer is one of the most common gynecologic cancers that has the highest mortality rate. The diagnosis is often late, and the cancers are thus at a too-advanced stage, making therapeutic strategies ineffective. The standard treatment consists of cytoreduction surgery combined with chemotherapy, but resistance to platinum salts constitutes the primary cause of therapeutic failure. Escaping apoptosis is a key feature of the tumor process, partly due to the overexpression of anti-apoptotic proteins from the Bcl-2 family, including Bcl-2, Bcl-xL, and Mcl-1. These proteins are privileged targets to be inhibited to
overcome resistance, and their simultaneous inhibition restores apoptosis [55]. However, the inhibition of both proteins leads to cardiotoxicity and thrombocytopenia due to on-target/off-tumor effects.

In order to restore apoptosis, achieve tissue selectivity, and avoid toxicity, we chose to develop compounds that simultaneously degrade these proteins by hijacking the UPS (Ubiquitin Proteasome System) pathway. The UPS is one of the main pathways responsible for protein degradation in vivo and is involved in almost 80% of protein degradation in the cell. It is a quality control process that uses E3 ligases to identify damaged or misfolded proteins so that they can be degraded via the proteasome (Figure 7), also known as the “cell’s trash-disposal machinery” [56].

<figure><img src="image_9.png" /><figcaption>**Figure 7.** Schematic representation of a degrader ternary complex with the POIs and E3 Ligases (CRBN and VHL).</figcaption></figure>

Our work is therefore to design and synthesize new degraders, and more precisely PROTACs and Molecular Glues [57], directed against two proteins of interest (POI): Mcl-1 and Bcl-xL. The molecular scaffolds used to start this work have been studied in previous work of the research team and have shown interesting inhibition activities [58]. The synthesis of 25 degraders will be presented in this study, including in silico, in vitro, and in vivo results for some of them.

### 3.7. A Versatile Conjugation Platform for the Development of PROTACS and Molecular Probes for Their Characterization

Lucía González ¹,², Maria Majellaro ², Thomas Webb ³, Olliver Hsia ³, Alessio Ciulli ³ and Eddy Sotelo ¹

1. Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) University of Santiago de Compostela, 15706 Santiago de Compostela, Spain; luciagonzalez.pico@usc.es or lucia.gonzalez@celtarys.com

2. Celtarys Research, spin-off of the University of Santiago de Compostela, 15706 Santiago de Compostela, Spain

3. Centre For Protein Degradation (CeTPD), University of Dundee, DD14HN Dundee, UK

PROTACs (PROteolyisis TArgetting Chimeras) are heterobifunctional molecules capable of hijacking E3 ligases in order to degrade a specific protein of interest (PoI). This unique mechanism of action has led to the development of a vast number of PROTACs targeted towards different PoIs, always using a limited pool of E3 ligases. Indeed, most PROTACs developed so far hijack CRLCRBN and CRLVHL, using thalidomide derivatives and VH 032 variants [59,60].

The limited expansion of the E3 ligase toolbox has led to a need for pharmacological tools dedicated to screening E3 ligase ligands. In this context, Celtarys Research's expertise in developing fluorescent tools using an efficient and convergent proprietary conjugation
technology is a perfect fit for designing and synthesizing new fluorescent probes for different ligases [61].

Herein, we report the data obtained during a proof-of-concept using Celtarys' technology (Figure 8) to obtain a total of 10 PROTACs, as well as the process behind the development and synthesis of novel VHL and CRBN fluorescent probes and the preliminary affinity data of eight VHL fluorescent ligands, developing a TR-FRET assay for the best ligand.

<figure><img src="image_10.png" /><figcaption>**Figure 8.** (A) PROTAC and probe design using Celtarys' proprietary technology. (B) TR-FRET assay concentration optimization.</figcaption></figure>

3.8. Benchmarking Traditional and Deep Learning Models for Protein Target Activity Prediction

Ana Laura Dias ¹, Carlos M. M. Afonso ², Gonçalo J. L. Bernardes ³,⁴ and Tiago Rodrigues ¹

1. Research Institute for Medicines (iMed), University of Lisbon, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal; ana.laura.fernandes@edu.ulisboa.pt

2. Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), University of Porto, Av. General Norton de Matos, 4450-208 Matosinhos, Portugal

3. Instituto de Medicina Molecular João Lobo Antunes, University of Lisbon, Avenida Professor Egas Moniz, 1649-028 Lisbon, Portugal

4. Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

Machine learning (ML) algorithms can learn subtle, hidden patterns in sparse chemical data and use this knowledge to make predictions on unseen data. Small molecule target discovery, a key step in the drug discovery process, has been accelerated by ML models [62,63]. ML methods, ranging from traditional approaches (e.g., Random Forest) to deep learning (DL), such as Transformers, can be exploited for activity prediction and target discovery. Although the success of DL in a wide range of applications suggests its advantage over traditional methods, its performance has shown to be only marginally better or even inferior [64].

Therefore, we assessed the performance of several ML methods for the activity prediction task. This benchmarking study was conducted on the ChEMBL database (version 31), with 1167 protein targets (50–8000 entries). The algorithms were designed to predict normalized activity values, using CATS2 pharmacophore descriptors for fixed representation and SMILES for representation learning, and were optimized through hyperparameter tuning. The model performance was evaluated using regression metrics such as the coefficient of determination (R²), Mean Absolute Error (MAE), and Mean Squared Error (MSE). Metrics were obtained from a 10-fold cross-validation with different random state values.

We found that activity predictions for the ChEMBL database were significantly better with traditional methods, specifically tree-based methods which outperformed all DL methods, including representation learning. This difference could be attributed to the data-intensive nature of DL models, which may struggle with smaller, more specific datasets
typically associated with biological research. Consequently, our findings emphasize the importance of selecting the appropriate algorithm for activity prediction, with traditional ML methods proving more suitable for activity predictions.

*This work was supported by the Fragment-Screen (101094131) project. A.L Dias acknowledges Fundação para a Ciência e Tecnologia (FCT) for the financial support from the PhD studentship 2022.09827.BD.*

### 3.9. *Development of New Nature-Inspired Synthetic Flavonoids for Marine Biofouling Prevention*

Daniela Pereira ¹,², Vitor Vasconcelos ²,³, Madalena Pinto¹,², Joana R. Almeida ², Marta Correia-da-Silva ¹,² and Honorina Cidade ¹,²

1. Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira n° 228, 4050-313 Porto, Portugal; danielamvpereira@gmail.com

2. CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal

3. Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4069-007 Porto, Portugal

Marine biofouling, caused by the settlement and accumulation of marine micro and macroorganisms on submerged surfaces, namely on boats, is responsible for several economic, environmental, and health concerns [65]. Antifouling coatings containing biocides have been used over the past decades to prevent this phenomenon. However, these biocides presented a high toxicity for the environment and human health, so more environmentally safe antifoulants are urgently needed [66]. Aiming to discover new sustainable antifouling compounds with a low ecotoxicity and considering the potential of some marine flavonoids as antifoulants, our research group has been focused on the synthesis and evaluation of antifouling activity of synthetic flavonoid derivatives [67,68]. Among them, several flavonoids showed promising antifouling activity, namely the prenylated chalcone C1P (Figure 8) [68]. Based on these results, and in an effort to develop more effective antifoulants, a series of chemically related flavonoid analogues was prepared using lead optimization strategies of Medicinal Chemistry (Figure 9). The antifouling activity was investigated using the in vivo anti-settlement bioassay with *Mytilus galloprovincialis* larvae and marine biofouling microorganisms. Complementary assays with *Artemia salina* were also performed to evaluate their toxicity against non-target marine organisms. These studies resulted in the identification of several flavonoids with antifouling activity and low toxicity against macro- and microfouling species.

<figure><img src="image_11.png" /><figcaption>**Figure 9.** Structure of C1P and synthetic research plan.</figcaption></figure>

*This research was supported by national funds through FCT (Foundation for Science and Technology) within the scope of UIDB/04423/2020, UIDP/04423/2020, and the project PTDC/CTA-AMB/0853/2021. D.P. acknowledges FCT for the PhD scholarship (grant SFRH/BD/147207/2019).*
3.10. Design and Synthesis of Novel Allosteric Modulators for the Prostaglandin EP2 G Protein-Coupled Receptor

Constance Dalton ¹, Charles Laughton ¹, Nicholas Holliday ² and Shailesh N. Mistry ¹

1. Division of Biomolecular Science and Medicinal Chemistry, School of Pharmacy, The University of Nottingham Biodiscovery Institute, University Park, Nottingham NG7 2RD, UK; pcycd4@nottingham.ac.uk

2. School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK

The prostagladin EP₂ receptor (EP₂) is a widely expressed G protein-coupled receptor activated endogenously by prostaglandin E₂ (PGE₂), which contributes to the development of chronic inflammation in cancer and has roles in diseases such as Parkinson's, endometriosis, arthritis, intercranial aneurysms, glioblastoma, and epilepticus (Figure 10) [69,70]. EP₂ antagonism is therefore considered a possible therapeutic approach to treat these diseases. Previously, numerous orthosteric antagonists (i.e., those that bind to the PGE₂ binding site) have been synthesized [69,71]. In 2020, “**Compound 1**” (Figure 10) was reported as the first allosteric EP₂ antagonist that demonstrates a reversible, agonist-dependent mode of action.

<figure><img src="image_12.png" /><figcaption>**Figure 10.** (A) Structure of “**Compound 1**” highlighting three regions of interest for structural modification; dioxane (blue—where most structural changes occurred in the literature), tetrahydrofuran (pink), and quinoxaline (green). (B) PGE₂ binds and activates EP₂, Gαs-mediated induction of adeny late cyclase to increase cytoplasmic cAMP levels. Downstream events are then mediated through protein kinase A. EP₂ activation also induces β-arrestin2, which is known to promote tumor cell growth and migration [69,70].</figcaption></figure>

As part of this communication, we will report, for the first time, the synthetic route to “**Compound 1**”, as well as our exploration of an expanded structure-activity relationship dataset focusing on modifications at the tetrahydrofuran (pink) moiety. Initial work has identified novel analogues of “**Compound 1**”, displaying an improved affinity and potency for EP₂ compared to the literature compound, whilst demonstrating an insurmountable mode of action indicative of a negative allosteric modulator. Pharmacological characterization was conducted using both a NanoBRET competition binding study employing the G protein mimetic peptide TMR-Gαs19cha18, and a NanoBiT complementation assay demonstrating selectivity at the hEP2 receptor compared to the closely related hEP4 receptor [71,72].
### 3.11. 4-Anilinoquinazoline Derivatives as New Potent and Selective NOD1-RIPK2 Inhibitors for the Treatment of Inflammatory Diseases

Morgane Rivoal¹, Amélie Barzyck¹, Perrine Six¹, Claire Devos¹, Xavier Dezitter¹, Min-Jeong Cornu-Choi¹, Amaury Farce¹, Mathilde Body-Malapel¹, Madjid Djouina¹, Karine Huard², Erika Pellegrini²,³, Stephen Cusack², Laurent Dubuquoy¹, Régis Millet¹ and Natascha Leleu-Chavain ¹,*

1. Université de Lille, Inserm, U1286-INFINITE-Lille Inflammation Research International Center, F-59000 Lille, France; morgane.rivoal.etu@univ-lille.fr

2. European Molecular Biology Laboratory, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France

3. IBS, 38000 Grenoble, France

Inflammation is a defense mechanism with the function to restore tissue damages and to eliminate pathogens. Inflammation can be triggered by danger signals (DAMP: Danger-Associated Molecular Pattern) or pathogenic signals (PAMP: Pathogen-Associated Molecular Pattern) recognized by immune cells present in tissues, through pattern-recognition receptors (PRR). Among these PRR, Nucleotide Oligomerization Domain 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses. It has been reviewed that the inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 vs. NOD2 inhibitors at the nanomolar range, based on a 4-anilinoquinazoline scaffold [73,74]. We demonstrated that NOD1 inhibition occurs through the inhibition of Receptor-Interacting Protein Kinase 2 (RIPK2), a serine/threonine kinase, involved in its downstream signaling pathways (Figure 11) [74]. Our best inhibitors demonstrate no cytotoxicity, a selectivity for RIPK2 over EGFR and VEGFR, and a capacity to reduce pro-inflammatory cytokine IL-8 secretion in HEK-Blue™-hNOD1. Moreover, two of our compounds showed promising in vivo activity on a DSS-induced colitis murine model [74]. The 4-anilinoquinazoline scaffold offers novel perspectives to design NOD1-RIPK2 inhibitors, potentially useful to treat inflammatory diseases.

<figure><img src="image_13.png" /><figcaption>**Figure 11.** Representation of our compounds' mode of action in inhibiting NOD1 inflammatory signaling pathways.</figcaption></figure>

## 4. Poster Presentations

### 4.1. Use of Machine Learning Methods and Pharmacophore Screening to Search for Compounds with Dual Antiparasitic and Anticancer Activity Among Different Thiazolidinone and Pyrazoline Derivatives

Anna Kryshchyshyn-Dylevych¹ and Roman Lesyk¹

1. Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv, Ukraine; kryshchyshyn.a@gmail.com
Thiazolidinone ring derivatives, such as 2,4-thiazolidinedione, rhodanine (2-thioxo-4-thiazolidinone), 2-alkyl(aryl)-substituted, and 2-amino(imino)-substituted 4-thiazolidinones, are widely used as privileged structures and sub-structures for the design of "drug-like" molecules, including those with anticancer and antiparasitic properties [75]. The analysis of sub-libraries of compounds based on the synthesized various thiazolidinones from the "in-house" library of the Department of Pharmaceutical, Organic and Bioorganic Chemistry with the anticancer activity on Leukemic cell line HL-60 (NCI protocol) and antiparasitic activity against *Trypanosoma brucei brucei* (in vitro studies) were chosen for the ML studies. To classify the compounds with the antitrypanosomal activity as an independent variable an IC₅₀ calculated in the growth inhibition assay towards *Tb brucei* was selected. The classes were divided as follows: (i) class 1: IC₅₀ < 5 µM or inhibition > 90% at 10 mg/mL; (ii) class 2: IC₅₀ 5–10 µM or inhibition < 90% > 50% at 10 mg/mL; and (iii) class 3: IC₅₀ > 10 µM or inhibition < 50% at 10 mg/mL.

Compounds from class 1 (with the highest inhibition rates towards *Tb brucei*) were selected in one cluster when applying both the agglomerative hierarchical clustering and k-means clustering algorithms. Selecting compounds from the same classes into the same clusters when applying the above-mentioned ML techniques testifies the robustness of the algorithms. The agglomerative hierarchal clustering is based on dendogram creation. An unsupervised ML algorithm k-means clustering aims to minimize the sum of distances between the points and their respective cluster centroid.

Pharmacophore screening was carried out for five compounds (from the 1st class) identified in one cluster: 3,5-arylidene-1H-pyrazol-thiazol-2-one and 3,5-arylidene-1H-pyrazol-isatine hybrid molecules, 5-enamine-rhodanine-3-phenylpropionate, and 5-ene-2-amino-4-thiazolidone derivatives to a pharmacophore merged from a series of BCL-2 complexes with antagonist venetoclax (PDB codes: 6O0K, 6O0M, 6O0P), which is used for chronic lymphocytic leukemia and acts via promoting apoptosis. The results of the pharmacophore modeling (LigandScout 4.4 Software) characterized all studied molecules as active (pharmacophore-fit scores 38–58.7). The obtained pharmacophore model was then used for the screening of a set of hybrid molecules bearing combination of 4-thiazolidinone or thiazole core and phenyl-indole or 6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole fragment with the in vitro studied submicromolar levels of IC₅₀ on *Tb brucei* and *Tb gambiense* [76]. All 19 tested compounds fit the pharmacophore model with pharmacophore-fit scores within the range of 36.01–55.55.

The conclusions are as follows: A set of thiazolidine derivatives with the established dual antitrypanosomal and anticancer activity was analyzed by clustering methods resulting in selecting a group of highly active antitrypanosomals with good anticancer properties. The latter underwent pharmacophore screening (BCL-2-venetoclax complexes). The same pharmacophore model, when applied to a set of highly active antitrypanosomals—indole and imidazothiadiazole thiazolidinone hybrids, also showed high pharmacophore-fit scores, suggesting they might possess antiproliferative properties as well.

*Acknowledgment.* We are grateful to the LigandScout developers for providing the free academic license.

## 4.2. Synthesis of Indeno[1,2-b]indoles: Challenges and Insights from Literature to Experiment

Johana Charles ¹, Christelle Marminon ¹, Noël Pinaud ², Jean Guillon ³, Karsten Niefind ⁴, Joachim Jose ⁵ and Marc Le Borgne ¹

1. University Claude Bernard Lyon 1, Centre de Recherche en Cancérologie de Lyon, UMR CNRS 5286, INSERM 1052, 69373 Lyon, France; johana.charles@univ-lyon1.fr

2. University of. Bordeaux, Institut des Sciences Moléculaires, UMR CNRS 5255, 33405 Talence, France
3. University of Bordeaux, Laboratoire Acides Nucléiques: Régulations Naturelles et Artificielles, UMR CNRS 5320, INSERM U1212, 33000 Bordeaux, France

4. Department of Chemistry, Universität zu Köln, Institut für Biochemie, 50674 Köln, Germany

5. Westfälische Wilhelms-Universität Münster, Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, 48149 Münster, Germany

Indeno[1,2-**b**]indole has been demonstrated to be a key scaffold for the design of new bioactive molecules in oncology (e.g., inhibitors of protein kinase CK2 and inhibitors of the breast cancer resistance protein ABCG2). Depending on the synthetic route chosen, a large possibility of specific functionalizations can be achieved [77]. Initially, we focused our SAR study on substituents (from R1 to R5) in order to develop new indeno[1,2-**b**]indoles as CK2 inhibitors. Four functionalized indeno[1,2-**b**]indoles were synthesized using the two-step reaction described by Hemmerling et al. [78] and Gozzi et al. [79] (Figure 12).

<figure><img src="image_14.png" /><figcaption>**Figure 12.** Comparison of two-step synthesis from Gozzi et al. [79] and one-pot synthesis.</figcaption></figure>

It has been observed that the reaction conditions described become inappropriate depending on the substituents used. Indeed, the following unexpected results have been observed: an incomplete reaction progress, very low yields, and a significant formation of by-products.

In this presentation, we will discuss the optimization work concerning the quantities of reagents and the selection of solvents used in the synthesis process. Based on the optimal reaction conditions determined, a one-pot synthesis method has been developed to improve yields and make the synthesis more convenient and straightforward.

*This work was supported by ANR-DFG (XPLOR_CK2, ANR-22-CE92-0081).*

### 4.3. Fragment-Based Drug Design: An Effective Approach for Aiming at Challenging Targets to Discover New Anti-Inflammatory Agents

Florian Schwalen¹, Charline Kieffer¹ and Anne-Sophie Voisin-Chiret¹

1. Centre d'Etudes et de Recherche sur le Médicament de Normandie, Normandie University, 14000 Caen, France; florian.schwalen@unicaen.fr

Various methods can be used to discover new drug candidates. These include the fragment-based drug design method. We report the successful use of this method in the development of both selective and affine compounds for the BIR2 domain of the XIAP protein. The X-chromosome-linked inhibitor of the apoptosis protein (XIAP) is a protein involved in various signaling pathways, in particular thanks to its different baculovirus IAP repeat (BIR) domains, which are involved in different protein-protein interactions. XIAP is implicated in a pro-inflammatory pathway, via its BIR2 domain. XIAP interacts with the receptor-interacting protein 2 (RIP2), ultimately leading to pro-inflammatory signaling [80]. Therefore, selectively blocking the XIAP-BIR2 domain could reduce this inflammatory signaling. The main problem in developing compounds that disrupt this protein-protein interaction is the high homology between the BIR2 and BIR3 domains of XIAP and other members of the IAP family (in particular cIAPs) leading to off-target effects [81]. In order to avoid them, it is crucial to develop compounds that are selective for XIAP-BIR2. The aim
of the "XIAP project", currently developed in CERMN, is to design molecules that interact specifically with XIAP-BIR2, in order to disrupt the XIAP-RIPK2 interaction and obtain potentially anti-inflammatory molecules.

To achieve this, we embarked on a chemoinformatics-assisted "de novo drug design" process, using a fragment-based drug design method. This first stage enabled us to explore the domains of interaction between XIAP-BIR2 and RIPK2 and, among other things, to determine the differences between XIAP-BIR2 and XIAP-BIR3. Thanks to these data, we synthesized an initial library of 50 fragments. An in vitro affinity evaluation of fragments for both XIAP-BIR2 and XIAP-BIR3 was carried out using AlphaScreen® and Fluorescent Polarization Assay (FPA) technologies. The modulation of the best fragments led to the obtention of a hit molecule, affine, and selective for XIAP-BIR2. The validation of the inhibitory activity of XIAP in cellular models was carried out using 'HEK-Blue NOD' cell lines, which specifically control the expression of the NOD1 and NOD2 signaling pathways (Figure 13A). Thanks to this proof of concept, we are now continuing our pharmacomodulation work to improve the affinity of the hit while maintaining selectivity for XIAP-BIR2, such as shown in Figure 13B.

<figure><img src="image_15.png" /><figcaption>**Figure 13.** Current results (A) and objectives (B) of the "XIAP project".</figcaption></figure>

4.4. Semisynthetic Anticancer Halimane Derivatives from Plectranthus Ornatus Codd

Gabrielle Bangay ¹,², Florencia Z. Brauning ¹, Tomasz Kowalczyk ³,⁴, Anna Merecz-Sadowska ⁵, Ewelina Synowiec ⁶, Tomasz Śliwinski ⁶, Nuno Candeias ⁷, Monica S. Estevão ⁸, Carlos A.M. Afonso ⁸, Vânia André ⁹, Przemysław Sitarek ³ and Patrícia Rijo ¹,⁸,*

1. Center for Research in Biosciences & Health Technologies (CBIOS), Universidade Lusófona, 1749-024 Lisbon, Portugal; gabrielle.bangay@ulusofona.pt

2. Universidad de Alcalá de Henares. Facultad de Farmacia, Departamento de Ciencias Biomédicas (Área de Farmacología; Nuevos Agentes Antitumorales, Acción Tóxica Sobre Células Leucémicas. Ctra. Madrid-Barcelona km. 33,600, 28805 Alcalá de Henares, Madrid, España

3. Department of Medical Biology, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland

4. Department of Molecular Biotechnology and Genetics, Faculty of Biology and Environmental Protection, University of Lodz, 90-136 Lodz, Poland

5. Department of Allergology and Respiratory Rehabilitation, Medical University of Lodz, 90-725 Lodz, Poland

6. Department of Molecular Genetics, Faculty of Biology and Environmental Protection. University of Lodz, 90-136 Lodz, Poland

7. LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
8. Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisbon, Portugal

9. Centro de Quimica Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisbon, 1649-004 Lisbon, Portugal

Natural products have long been a cornerstone in drug discovery, providing a vast array of bioactive compounds with therapeutic potential. The *Plectranthus* genus is widely recognized for its medicinal benefits. *Plectranthus ornatus* Codd., in particular, is traditionally used in Africa to treat gastric and liver ailments, with its leaves known for their antibiotic properties [82]. The primary component of *P. ornatus* is the halimane compound, 11 *R*\*-acetoxyhalima–5,13*E*–dien–15–oic acid (**Hal**), which is noted for its anticancer effect [83]. This study aimed to enhance the efficacy of the halimane lead compound, and an additional physiochemical characterization of **Hal** was conducted. To our knowledge, this research is the first to publish data on the absolute configurations using SCXRD and the thermal stability of **Hal**. Reactions of **Hal** with various amines were performed to create new semisynthetic derivatives, and their structures were elucidated. The cytotoxicity of these derivatives was tested against three leukemia cell lines (CCRF-CEM, K562, and HL-60). Their antioxidant activity was examined using H₂O₂-induced HGF-1 cells, and their anti-inflammatory activity was studied using RT-PCR and ELISA. The findings revealed that the amide derivatives of **Hal** exhibited a moderate cytotoxicity and more potent activity compared to the parent molecule, providing insights into the structure-activity relationship (SAR) of **Hal**. These derivatives also showed protection against DNA oxidative damage. Furthermore, they demonstrated anti-inflammatory properties by modulating gene and protein expression of cytokines IL-1β, TNF-α, and IL-6, induced by LPS in normal HGF-1 cells. Overall, this study offers valuable insights into the improved biological activities of semisynthetic **Hal** derivatives, laying the groundwork for novel cancer therapy drug formulations.

*The authors acknowledge and thank Fundação para a Ciência e a Tecnologia (FCT, Portugal), for funding this project through DOI 10.54499/UIDP/04567/2020, DOI 10.54499/UIDB/04567/2020, UI/BD/151422/2021, DOI 10.54499/UIDP/00100/2020, DOI 10.54499/UIDB/00100/2020, DOI 10.54499/LA/P/0056/2020, and contract DOI 10.54499/CEECIND/00283/2018/CP1572/CT0004. The authors also thank Fundação Calouste Gulbenkian for the support for this work through grant N° 275123. Dr. Auguste Fernandes is acknowledged for the TGA/DSC data.*

4.5. Modifying Positions 2 or 8 of the Imidazo[1,2-a]Pyrazine Lead Compound CTN1122 Affects Its Antileishmanial Activity, L-CK1.2 Kinase Inhibition, and Safety Profile

Lhana Tisseur ¹, Marc-Antoine Bazin ¹, Sandrine Cojean ²,³, Fabrice Pagniez ¹, Cédric Logé ¹, Guillaume Bernadat ³, Christian Cavé ³, Isabelle Ourliac-Garnier ¹, Carine Picot ¹, Olivier Leclercq ⁴, Blandine Baratte ⁵, Najma Rachidi ⁴, Stéphane Bach ⁵, Philippe Loiseau ³, Patrice Le Pape ¹ and Pascal Marchand ¹

1. Nantes Université, Cibles et médicaments des infections et de l'immunité, IICiMed, UR 1155, F-44000 Nantes, France; lhana.tisseur@etu.univ-nantes.fr

2. UMR BIPAR, Laboratory of Animal Health, Anses, INRAe, EnvA, F-94700 Maisons-Alfort, France

3. Université Paris-Saclay, Chimiothérapie Antiparasitaire, BIOmolécules: Conception, Isolement et Synthèse BioCIS UMR 8076 CNRS F-91400 Orsay, France

4. Institut Pasteur and INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, F-75015 Paris, France

5. Sorbonne Université, UPMC Paris 06, CNRS USR3151 "Protein Phosphorylation and Human Disease" group, Station Biologique, F-29680 Roscoff, France
Leishmaniasis is a parasitic disease classified as a neglected tropical disease by the WHO. It manifests in various forms—cutaneous, mucocutaneous, and visceral—and poses a serious public health issue, with 12 million people infected globally and over 40,000 deaths annually. The disease is endemic in many regions worldwide, with its emergence in Europe attributed to global warming. Current treatments are far from ideal due to their high toxicity, significant costs, and administration methods that limit the accessibility for disadvantaged populations. Additionally, increasing parasite resistance to these treatments is diminishing their effectiveness in some areas. Consequently, there is an urgent need to develop new treatments that are safer, more effective, and target novel proteins to overcome these resistances. A promising recent discovery is **CTN1122** [84], which targets a specific *Leishmania* Casein Kinase 1 protein (L-CK1.2) [85,86] and demonstrates strong antileishmanial properties. In this context, we have decided to synthesize **CTN1122** analogues to enhance the pharmacological activity profile (Figure 14).

<figure><img src="image_16.png" /><figcaption>**Figure 14.** Modulations from the hit compound CTN1122.</figcaption></figure>

Two types of pharmacomodulation on the imidazo[1,2-a]pyrazine ring of the lead compound CTN1122 will be presented and compared. One by modifying the substituent in position 8 with various amines and amides and the other by modifying the substituent in position 2 with variously substituted aryls. The study of these analogues will enable us to discuss the structure-activity relationship concerning their antileishmanial properties and their ability to inhibit the target protein L-CK1.2, and to consider their toxicity profile.

## 4.6. *In Vitro* Evaluation of Potential Substance P Antagonists Designed for Dermatological Applications

Márcia S. Martins ¹,², Sara Cravo ¹,², Artur M. S. Silva ³, Madalena M. M. Pinto ¹,², Isabel F. Almeida ⁴,⁵, Maria T. Cruz ⁶,⁷ and Emília Sousa ¹,²

1. Laboratory of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; esousa@ff.up.pt

2. CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, 4450-208 Matosinhos, Portugal

3. LAQV-REQUIMTE & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

4. UCIBIO—Applied Molecular Biosciences Unit, MedTech, Laboratory of Pharmaceutical Technology, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

5. Associate Laboratory i4HB, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

6. Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal

7. Center for Neuroscience and Cell Biology, 3000-548 Coimbra, Portugal

Chronic pruritus has been linked with the activation of the neurokinin 1 receptor (NK1R) by substance P (SP), making the design of SP antagonists an upcoming strategy to alleviate this dermatological condition [87]. The identification of a natural SP antagonist, along with the disclosure of a NK1R crystallographic structure, facilitated the design of
new SP antagonists. Given the common association between pruritus and inflammation, our objective was to utilize structure-based drug design to develop novel SP antagonists inspired by a promising marine natural product and evaluate them in vitro in representative skin cell lines. This approach seeks to create innovative compounds suitable for the topical treatment of inflammatory skin diseases associated with pruritus.

Seventy designed ligands were evaluated for their binding affinity to NK1R (PDB 6E59, AutoDock Vina) together with their pharmacokinetic properties, using in silico methods. Eighteen compounds were selected and evaluated in vitro (keratinocytes, macrophages, and fibroblasts) to assess both the cytotoxicity and potential anti-inflammatory activity. A significant reduction in the pro-inflammatory mediator nitric oxide (NO), together with a significant decrease in inducible nitric oxide synthase (iNOS) protein levels, and the absence of NO-scavenging potential, suggest the inhibition of inflammatory pathways upstream of the synthesis of iNOS. The presence of an aromatic moiety appears crucial for the observed activity, prompting further design studies and new synthetic approaches. A total of ten new potential substance P antagonists were obtained and structurally characterized for further studies.

*Acknowledgments: FCT-UIDP/04378/2020, UIDB/04378/2020, LA/P/0140/2020, UIDB/04423/2020, UIDP/04423/2020, UIDB/04539/2020, UIDP/04539/2020, LA/P/0058/2020, and 10.54499/2021.05964.B.*

### 4.7. Synthesis and Anti-Tumor Evaluation of Thieno[2,3-b]Pyrazine Derivatives

Maria F. Martins¹, Ana Borges ²,³, Ricardo C. Calhelha ²,³ and Maria-João R. P. Queiroz ¹,*

1. Centro de Química da Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal; mjrpq@quimica.uminho.pt

2. Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal

3. Laboratório Associado para Sustentabilidade e Tecnologia em Regiões de Montanha (SusTEC), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal

The thieno[2,3-b]pyrazine skeleton has been found in natural products such as urothion and its derivatives, and in biologically active synthetic compounds useful in the prevention or treatment of inflammation, cell proliferation, and immune-related conditions and disease [88,89].

Herein, we present the synthesis and preliminary antitumor evaluation of thieno[2,3-b]pyrazine derivatives obtained by Pd-catalysed C-N Buchwald-Hartwig (compounds 1), [90] and Pd/Cu-catalyzed Sonogashira (compounds 2) couplings, starting from the methyl 3-amino or 3-bromothieno[2,3-b]pyrazine-2-carboxylate. Furthermore, in the Sonogashira coupling reaction, compounds 3, resulting from 6-endo-dig cyclization, were also obtained as minor products (Figure 15).

The series of compounds 1, 2, and 3 were evaluated for their cytotoxic activity in several human tumor cell lines (Caco-2, MCF-7, AGS, HeLa, and NCI-H460) and, in Vero cells or in a porcine liver primary cell culture (PLP2) as toxicity models. For the most promising compounds, characterized by low GI₅₀ values and no toxicity in non-tumor cells, further studies on apoptosis induction and cell cycle profile were conducted to gain insights into their mechanisms of action.
<figure><img src="image_17.png" /><figcaption>**Figure 15.** Synthesis of thieno[2,3-b]pyrazine derivatives 1, 2, and 3. Reagents and conditions: A: X= NH₂, Pd(OAc)₂ (10 mol%), Xantphos (12 mol%), Cs₂CO₃ (2.8 equiv.), 1,4-dioxane, 120 °C, 3–5 h. B: X=Br, Pd(OAc)₂ (6 mol%), rac-BINAP (8 mol%), Cs₂CO₃ (2 equiv.), toluene, 100 °C, 2–6 h.</figcaption></figure>

Acknowledgments are due to FCT-Portugal through the financial support of CQUM (UID/QUI/0686/2020), CIMO (UIDB/00690/2020 and UIDP/00690/2020), and SusTEC (LA/P/0007/2020), and national funding by FCT through the Institutional Scientific Employment Program-Contract for R. Calhelha contract.

### 4.8. Targeting Tuberculosis: Exploring Dual-Action Terpenoid Derivatives as Promising Agents

Mariia Nesterkina ¹, Vidhisha Sonawane ², Lorenz Latta ¹, Andreas Kany ¹, Tobias Neu ¹, Norbert Reiling ²,³, Olga Vashchenko ⁴, Longin Lisetski ⁴, Iryna Kravchenko ¹, Claus-Michael Lehr ¹,⁵ and Anna K. H. Hirsch ¹,⁵

1. Helmholtz Institute for Pharmaceutical Research Saarland (HIPS)-Helmholtz Centre for Infection Research (HZI), Campus Building E 8.1, 66123 Saarbrücken, Germany; mariia.nesterkina@helmholtz-hips.de

2. Microbial Interface Biology, Research Center Borstel, Leibniz Lung Center, Parkallee 22, D-23845 Borstel, Germany

3. German Center for Infection Research (DZIF), Site Hamburg-Lübeck-Borstel-Riems, 23845 Borstel, Germany

4. Institute for Scintillation Materials of National Academy of Sciences of Ukraine, Nauky Ave. 60, 61072 Kharkiv, Ukraine

5. Department of Pharmacy, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany

Tuberculosis (TB), caused by *Mycobacterium tuberculosis* (*Mtb*), remains a significant global health threat with substantial morbidity and mortality rates. The efficacy of the first-line anti-TB drugs, isoniazid (INH) or rifampicin (RIF), was compromised by the development of chromosomal mutations in *Mtb* over recent decades. However, the amount of new anti-TB drugs entering clinical trials is insufficient. Alternatively, the semi-synthesis or structural modifications of well-known first-line antitubercular drugs offer a versatile approach to yield effective medications. Thus, the aim of the present study is to employ the prodrug/codrug strategy to optimize the INH structure in order to enhance its efficacy against *Mtb* (Figure 16).
<figure><img src="image_18.png" /><figcaption>**Figure 16.** Investigation of terpenoid derivatives with dual action.</figcaption></figure>

Terpenoid derivatives exhibited significant activity against the virulent *Mtb* strain H37Rv, with MIC₉₀ values ranging from 0.5 to 8 µM. Given the intracellular nature of *Mtb* growth, the synthesized compounds attenuated the expression of pro-inflammatory cytokines TNF-α and IL-6 in LPS-induced THP-1-derived macrophages. Additionally, a qRT-PCR analysis revealed the downregulation of IL-6, IL-10, TNF, CCL3, and CCL4 gene expression in macrophages upon treatment with INH derivatives. The most promising candidate exhibits activity against *Mtb*-infected human monocyte-derived macrophages at a concentration of 0.5 µM. Furthermore, we conducted in vitro ADME profiling, and assessed the cytotoxicity against HepG2 cells and THP-1 macrophages for the most active terpenoid derivatives.

Thus, terpenoid-based INH derivatives are active against the virulent *Mtb* strain H37Rv, modulate pro-inflammatory cytokine expression, downregulate specific gene expression, and exhibit a promising efficacy against *Mtb*-infected human macrophages, suggesting them as anti-TB agents with favorable in vivo ADMET.

## 4.9. Translational Approach to Combat RNA Viral Infections in Horses: A Pathway Towards Equine and Human Health Protection

Mirjana Antonijevic ¹,², Anne-Lise Pocheron ¹, Peggy Suzanne ², Cécile Schimmich ¹, Christophe Rochais ², Patrick Dallemagne ² and José-Carlos Valle-Casuso ¹

1. ANSES Animal Health Laboratory, PhEED Unit, 14430 Goustranville, France; mirjana.antonijevic@unicaen.fr

2. Normandie Univ., UNICAEN, CERMN, 14000 Caen, France

RNA viruses present significant challenges to animal health, particularly in equine populations, where infections like the Equine Arteritis Virus (EAV) and Equine Infectious Anemia Virus (EIAV) have become increasingly prevalent [91]. Despite advancements in veterinary medicine, the arsenal against these viruses remains limited, urging innovative strategies for effective disease management. Our interdisciplinary research initiative is aimed at integrating veterinary virology with medicinal chemistry to address the pressing need for novel antiviral therapies against EAV and EIAV. After some successful preliminary tests exploring this hypothesis [92], we have employed cutting-edge technologies, including in silico screening of CERMN's library of compounds and structure-based drug design, to identify and develop new antiviral molecules. Also, our approach encompasses drug repurposing strategies, leveraging the diverse chemical space of existing compounds to expedite the discovery of potential therapeutics, that would enable us to rationally design and optimize drug candidates, targeting essential viral proteins and host cell factors critical for viral replication and pathogenesis (Figure 17).
<figure><img src="image_19.png" /><figcaption>**Figure 17.** Representation of the research process.</figcaption></figure>

Finally, we want to explore the translational potential of our research by investigating structural and functional similarities between equine and human viral infections, offering insights into the development of therapeutics against human pathogens such as HIV and coronaviruses like SARS-CoV-2 [93].

4.10. Covalent Modification of Wild-Type p53 with a Tryptophanol-Derived Oxazoloisoindolinone

Ricardo J. F. Ferreira ¹, Ana P. Leandro ¹, Alexandra M. M. Antunes ² and Maria M. M. Santos ¹,*

1. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1600-277 Lisboa, Portugal; ricardojferreira@edu.ulisboa.pt

2. Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal; alexandra.antunes@tecnico.ulisboa.pt

The protein p53 has a crucial role in cells, for preventing genome mutations. Mutations in the TP53 gene are present in about 50% of human tumors, and in these cases, p53 is unable to carry out its tumor-suppressing functions. The presence of mutated forms of p53 is linked to the development of more aggressive tumors with an increased metastatic potential [94,95]. Consequently, there is growing interest in developing small molecules able to modify mutant p53 to the wild-type (wt) p53 functional form. Until now, few mut-p53 activators have been developed and entered clinical trials, and none have yet reached clinical use. Over the last few years, our research group has been involved in the development of tryptophanol-derived oxazoloisoindolinones as p53 activators [96,97]. The optimization of one hit compound led to the development of a more active and more selective derivative for colon cancer cells expressing p53. In this communication, we will disclose our latest results on the study of the mechanism of action of this new derivative. The lead compound was tested against wt-p53, R273H, and R280K mutant p53 using a differential scanning fluorimetry (DSF) assay, showing that it stabilizes the proteins. A mass spectrometry-based covalent binding assay with the wt-p53 protein showed that the compound can react and covalently bind to cysteines in the wt p53 DNA binding domain (Figure 18).
<figure><img src="image_20.png" /><figcaption>**Figure 18.** Overview of the assays.</figcaption></figure>

*This work was supported by national funds (Fundação para a Ciência e a Tecnologia) through iMed.ULisboa (UIDB/04138/2020), project PTDC/QUI-QOR/1304/2020, and PhD fellowship 2022.11539.BD (R. Ferreira).*

4.11. *Synthesis of Glycosyl-Triazole-Phthalimides as Antiviral Agents*

Ronaldo N. de Oliveira ¹, Willyenne M. Dantas ¹,², Valentina N. M. de Oliveira ³, Poliana G. da Silva ², Renata P. G. Mendes ², Bruna M. Guimarães ¹, Sumit Kumar ⁴, Brijesh Rathi ⁵ and Lindomar J. Pena ²

1. Department of Chemistry, Federal Rural University of Pernambuco, Recife 52171-900, Brazil; ronaldo.noliveira@ufrpe.br

2. Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), Recife 50670-420, Brazil

3. Federal Institute of Education, Science and Technology of Pernambuco, Campus Ipojuca, Ipojuca 55590-000, Brazil

4. Infectious Diseases Division, HeteroChem InnoTech, Hansraj College Premises, Delhi 110007, India

5. Laboratory for Translational Chemistry and Drug Discovery, Department of Chemistry, Hansraj College, University of Delhi, Delhi 110007, India

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for causing coronavirus disease 2019 (COVID-19) [98]. Since its discovery, research has rapidly advanced to develop treatments for patients and combat the virus's spread. However, the need for updated drugs persists, due to the emergence of new variants and severe cases that are more difficult to treat. Using a click reaction [99], 12 molecular hybrids were synthesized by combining azido-phthalimide derivatives and 2,3-unsaturated alkynyl-O-glycosides, resulting in the formation of 1H-1,2,3-triazole-based compounds with excellent yields (54-99%) (Figure 19). The cytotoxic activity on Vero cells and activity against the SARS-CoV-2 virus were investigated in vitro. The compounds exhibited 20% cytotoxic concentrations (CC₂₀) ranging from 208.5 µM to over 1000 µM. Following screening, all compounds showed significant differences in statistical analyses against the SARS-CoV-2 virus. Compound 3e had a selectivity index (SI) value of 195.8, and compound 3f had an IC₅₀ value of 11.2 µM. In silico analyses, including docking and molecular dynamics, were also performed to elucidate the mechanism of action and interactions between the synthesized compounds and the target proteins of SARS-CoV-2 [100]. The computational results indicated that 3CLpro could be the plausible target for these analogs, with compound 3f emerging as the best candidate.
<figure><img src="image_21.png" /><figcaption>**Figure 19.** Results of the post-infection assay of compounds **3e** and **3f** against the SARS-CoV-2 virus.</figcaption></figure>

Acknowledgments: CAPES, FACEPE, and CNPq.

4.12. *Effect of LUSO's Thermal Water on LPS-Induced Macrophage Inflammation*

Cláudia Cid ¹,²,*, Beatriz Rodrigues ¹,*, Rosa Resende ¹, Mylène Carrascal ³, Maria do Céu Sousa ¹,², Conceição Egas ¹,⁴, Cláudia F. Pereira ¹,⁵, Anabela Marisa Azul ¹, Maria Teresa Cruz ¹,², Rui Soares ¹,⁵,⁶,⁷,§ and Ana Silva ¹,²,§

1. CIBB-Center for Innovative Biomedicine and Biotechnology, University of Coimbra, 3004-504 Coimbra, Portugal

2. Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, 3000-348; anacrs@cnc.uc.pt

3. Tecnimede Group, 2710-089 Sintra, Portugal

4. Biocant-Transfer Technology Association, Biocant Park, 3060-197 Cantanhede, Portugal

5. Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal

6. Clinical Pathology Service, Portuguese Oncology Institute of Coimbra, 3000-075 Coimbra, Portugal

7. Termas de Luso, 3050-230 Luso, Portugal

* Equally contributing authors;

§ Equally contributing senior authors.

The ingestion of natural mineral water (NMW) contributes to important mineral intake, which might explain its extensive use in human nutrition with acknowledged health benefits, particularly related to an anti-inflammatory effect [101,102]. NMW's therapeutic properties are empirically recognized but lack scientific validation, particularly in Portugal. In this study, we focused on LUSO NMW, a hyposaline water, mainly composed of chlorine, sodium, and potassium, with relevant traces of magnesium, calcium, and bicarbonate. LUSO NMW-based therapies (indicated primarily for circulatory, nephro-urinary, and respiratory system diseases, and rheumatic and musculoskeletal disorders), are often directed to inflammatory-associated pathologies. Hence, we investigated the effect of LUSO NMW in an in vitro model of inflammation, specifically the murine macrophage cell line (RAW 264.7) cultured in normal culture medium or LUSO NMW-containing medium, for 48 h, in the absence or presence of Lipopolysaccharide (LPS; 100 ng/mL). In these cells, we evaluated the (1) cellular metabolism (Resazurin assay); (2) M1 (pro-inflammatory) and M2 (anti-inflammatory) profile (Flow cytometry); (3) phagocytic capacity (Flow cytometry); (4) NF-κB nuclear translocation (immunofluorescence); (5) gene expression (real-time RT-PCR); (6) protein levels (Western blotting); and (7) nitric oxide (NO) (Griess assay) cytokines secretion levels (ELISA). The results showed that LUSO NMW increased the macrophage metabolism and phagocytosis. Moreover, the expression of the M2 marker, CD163, was significantly higher in cells exposed to LUSO NMW. In an inflammatory context (with LPS), LUSO NMW decreased the Il6 and Tnfa gene expression, pro-IL-1β protein levels, and
IL-1β, IL-6, and TNF-α secretion levels. In contrast, cells exposed to LUSO NMW exhibited higher mRNA and protein levels of the antioxidant enzyme HMOX1. Overall, these results validate the anti-inflammatory effect of LUSO NMW and support its antioxidant potential. In conclusion, LUSO NMW's therapeutic effect should be deeply explored, including as an adjuvant in immune- and anti-inflammatory-based therapies.

*Acknowledgments: ee want to acknowledge 'Sociedade Central de Cervejas' for funding.*

### 4.13. Discovery and SAR Exploration of Novel $a_{2a}$ Antagonists as Anticancer Agents

Asier Selas ¹,², Hugo Fojo-Carballo ¹,², Jhonny Azuaje ¹, Arielle Pacha-Amores ², Xerardo García-Mera ¹, M. Rita Paleo ¹, José M. Brea ³, María I. Loza ³ and Eddy Sotelo ¹,²,*

1. Center for Research in Chemical Biology and Molecular Materials (CIQUS), University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; asier.selas@usc.es

2. Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

3. Center for Research in Metabolic Diseases (CIMUS). University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

Adenosine is a key immunosuppressive metabolite that regulates one of the major mechanisms supporting immune tolerance in tumors [103]. In normal cells, $A_{2A}$ and $A_{2B}$ receptors are engaged in the regulatory mechanisms that protect tissues against excessive immune response [103,104]. However, in the tumor microenvironment, an elevated adenosine concentration hijacks this protective pathway and obstructs anti-tumor immunity [104].

Adenosine inhibits the biological functions of T lymphocytes, infiltrating the cancer tissue by binding to the $A_{2A}$ receptor. Similarly, the activation of the $A_{2A}$ receptor on NK cells leads to a loss of their effector capacity, and therefore hinders the elimination of cancerous cells [104]. In this regard, $A_{2A}$ and $A_{2B}$ receptor antagonists constitute an emerging family of immunotherapeutic agents for cancer treatment. Accordingly, we herein document the design, synthesis, and SAR studies of a large collection of $A_{2A}$ receptor antagonists.

Inspired by the Lundbeck 28 ligand [105], we carried out a pharmacomodulation process with the aim to improve the potency and selectivity of the series by introducing new diversities that potentiate non-orthosteric interactions (Figure 20). The classical UGICR and several of its variants were employed for library synthesis, thus enabling us to identify new ligands with excellent $A_{2A}$ affinity/selectivity profiles. The antiproliferative and antimetastatic effect observed for selected $A_{2A}$ antagonists will be presented.

<figure><img src="image_22.png" /><figcaption>**Figure 20.** Selected compounds obtained from the MCR-assisted pharmacomodulation of the *Lundbeck28* ligand.</figcaption></figure>
4.14. Marine-Derived Cyclodepsipeptides: Synthesis Though an Emerging Alternative to Conventional Peptide Cyclization

Ricardo Ribeiro ¹,², Eugénia Pinto ²,³, Emília Sousa ¹,² and Carla Fernandes ¹,²

1. Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; cfernandes@ff.up.pt

2. CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, 4450-208 Matosinhos, Portugal.

3. Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal.

Unnarmicins A and C are cyclodepsipeptides produced by marine bacteria *Photobacterium* sp. [106], that can inhibit the fungal ABC transporters whose overexpression is the cause of the highest levels of multidrug resistance of pathogenic fungi [107]. Structurally, they comprise a tetrapeptide and a hydroxy aliphatic chain linked by an ester bond, differing only in the length of the aliphatic chain (Figure 21). Current methods for the chemical synthesis of cyclodepsipeptides in solution is a great challenge, particularly the head-to-tail cyclization step due to the formation of dimers or oligomers that reduces the overall yields and restricts the chemical diversity of cyclic molecules [108]. Therefore, the trend is to explore other synthetic approaches [109].

<figure><img src="image_23.png" /><figcaption>**Figure 21.** Structures of cyclodepsipeptides, unnarmicin A and C.</figcaption></figure>

Herein, we report the synthesis of new cyclodepsipeptides, analogues of unnarmicins A and C, though an emerging alternative to conventional peptide cyclization. First, the peptide building blocks were synthetized through a solution-phase synthetic strategy by using coupling agents and protecting agents such as *tert*-butyloxycarbonyl (Boc), benzyl ester, and methyl groups. Deprotections were planned according to the labile acid and base groups. In addition, chiral liquid chromatography was used to enantioseparate and determine the enantiomeric purity of chiral building blocks comprising a hydroxy fatty acid chain. The structures of all the synthetized compounds were established by spectroscopic methods. To our knowledge, it is the first report of the synthesis of cyclodepsipeptides by this peptide cyclization approach. Further studies will include the evaluation of their antimicrobial activities.

*This research was supported by FCT within the scope of UIDB/04423/2020 and UIDP/04423/2020. Ricardo Ribeiro also thanks the BYTPhD program by CIIMAR, grant provided by FCT UI/BD/150912/2021.*

4.15. Introducing Chirality into Nature-Inspired Flavones as Promising Eco-Friendly Antitumor Agents

Cláudia Pinto ¹,², João Machado ¹, Flávia Barbosa ³, Patrícia Silva ³, Hassan Bousbaa ³, Odília Queirós ³, Madalena Pinto ¹,², Honorina Cidade ¹,² and Maria Elizabeth Tiritan ¹,³,⁴
1. Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; up201604041@up.pt

2. CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, 4450-208 Matosinhos, Portugal

3. UNIPRO-Oral Pathology and Rehabilitation Research Unit, University Institute of Health Sciences (IUCS), CESPU, 4585-116 Gandra, Portugal

4. TOXRUN-Toxicology Research Unit, University Institute of Health Sciences (IUCS), CESPU, 4585-116 Gandra, Portugal

Searching for more environmentally friendly drug production and development practices leading to compounds with greater biodegradability and less ecotoxicity is an emerging issue given the negative impact of their improper disposal in the environment [110]. Flavones are an important class of natural products with great interest in Medicinal Chemistry due to their multiple biological activities, particularly antitumor [111]. Frequently, their combination with a chiral moiety, such as amino acids, results in highly biodegradable compounds with greater selectivity for the target [112]. Over the last decades, several reports have focused on the synthesis of chiral flavonoids to evaluate the influence of chirality on their pharmacological profile [113].

Following the concept of "benign by design" [114] and according to the research group experience, a library of chiral derivatives of flavones (CDF) was prepared by the combination of four different flavone skeletons with enantiomerically pure esters of tryptophan and tyrosine, to assess the influence of chirality on their antitumor activity. Liquid chromatography using a polysaccharide-based column confirmed the success of the enantioselective reactions, with enantiomeric ratios above 89%. Moreover, biological assays performed on three human tumor cell lines showed that CDF displayed better antiproliferative activity compared to their precursors. Particularly, Trice derivatives conjugated with methyl ester of tryptophan stood out for their lower GI₅₀ values. Interestingly, enantioselectivity was proven in most enantiomeric pairs, where usually, one enantiomer demonstrated prominent antiproliferative activity towards the other enantiomer. Additional studies to predict the toxicity and evaluate the mechanisms underlying the most promising compounds are being carried out.

*Acknowledgments: Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 (Group of Marine Natural Products and Medicinal Chemistry), through national funds provided by the FCT- (ERDF) within the framework of the program PT2020, and Flav4Tumor-GI2-CESPU-2022 funded by CESPU, Crl. DP and CP acknowledge for their FCT grants (SFRH/BD/147207/2019 and 2022.12419.BD, respectively).*

4.16. *Conjugation of Siderophore Mimetics with Antibiotics/Antimicrobial Adjuvants as a New Approach to Overcome Antibacterial Resistance*

Mariana C. Almeida ¹,² Paulo M. da Costa ²,³ Emília Sousa ¹,² and Diana I.S.P Resende ¹,²,³

1. Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; esousa@ff.up.pt

2. CIIMAR-Centro Interdisciplinar de Investigação Marinha e Ambiental, 4450-208 Matosinhos, Portugal

3. ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal

Antibacterial resistance is already a global health emergency [115]. In consequence, the development of innovative strategies to fight antibacterial resistance is now a top priority. Siderophores are metal-chelating compounds with a high affinity for iron which
are produced by numerous microorganisms as well as some plants [116]. The conjugation of siderophores or their synthetic mimetics with antibiotics constitutes a promising approach to the development of target-directed antibacterial agents, with encouraging results already reported in the literature [117]. Inspired by the vast potential of this strategy, in this work, we aimed to synthesize siderophore mimetics and conjugate them with an antibiotic or with an antimicrobial adjuvant, such as an efflux pump inhibitor.

Herein, several siderophore mimetics were synthesized and conjugated with an efflux pump inhibitor, to obtain five novel conjugates (Figure 22). The efflux pump inhibitor was first attached to a linker and then combined with the siderophore mimetic through sequential coupling reactions. Efforts in the coupling of the siderophore mimetics with a known antibiotic are currently underway. Additionally, the effect of siderophore mimetics in the growth of a reference strain of Escherichia coli and the antibacterial activity of the obtained conjugates against a panel of human pathogenic bacteria was investigated. Future work includes the assessment of possible synergism with antibiotics and investigation of the conjugates' capacity to inhibit bacterial efflux pumps.

<figure><img src="image_24.png" /><figcaption>**Figure 22.** Schematic representation of the main goal of this work.</figcaption></figure>

FCT-UIDB/04423/2020, UUID/04423/2020, 2021.05224.BD doi:10.54499/2021.05224.BD, 2022.00379.CEECIND/CP1728/CT0001 doi:10.54499/2022.00379.CEECIND/CP1728/CT0001.

## 4.17. Drug Repurposing to Discover Novel Antibacterial Agents: Our Experience

Matej Zore ¹, Jari Yli-Kauhaluoma ¹ and Jayendra Z. Patel ¹

1. Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, FI-00014 Helsinki, Finland; jayendra.patel@helsinki.fi

Drug repurposing offers significant advantages in the field of drug discovery over traditional approaches. It features shorter development times, reduced costs, and improved safety profiles by utilizing existing drugs or drug candidates for new indications or targets [118]. In early 2022, our colleagues screened an FDA-approved drug library and identified the S1PR modulator fingolimod as a promising antibacterial agent against *S. aureus* and *A. baumannii* [119]. Inspired by this discovery, we screened a selected set of commercially available S1PR modulators, leading to the identification of etrasimod as a promising antibacterial agent against several Gram-positive bacteria, including multidrug-resistant (MDR) strains [120]. In addition to repurposing S1PR modulators, we have structurally optimized them to enhance their antibacterial activity [121]. Concurrently, by combining our experience with S1PR modulators and available in-house data, we have identified the main structural features necessary to achieve antibacterial activity against Gram-positive bacteria. Additionally, as S1PRs are part of the GPCR superfamily, we screened a small set of other GPCR-targeting drugs and clinical candidates. This effort resulted in the identification of AMG837, a well-known GPR40 agonist albeit a previously failed clinical candidate, as a possible starting point for developing antibacterial agent against Gram-positive bacteria (unpublished data).
In summary, our recent success in identifying S1PR-targeting drugs as promising antibacterial agents against Gram-positive bacteria underscores the importance of the drug repurposing approach (Figure 23). Our work also opens new avenues for exploring other GPCR-targeting drugs and clinical candidates as potential starting points for discovering effective antibacterial agents to combat MDR bacteria.

## Biological screening against *S. aureus*

<figure><img src="image_25.png" /><figcaption>**Figure 23.** Overview of our research work in the antibacterial field using a drug repurposing approach.</figcaption></figure>

### 4.18. QN6 as a New Ligand for the Therapy of Diseases of Aging and Stroke

Francisco López-Muñoz¹, Ricardo Martínez-Murillo² and José Marco-Contelles³

1. Faculty of Health, Camilo José Cela University of Madrid, 28692 Madrid, Spain

2. Neurovascular Research Group, Cajal Institute (CSIC), 28006 Madrid, Spain

3. Laboratory of Medicinal Chemistry, Institute of General Organic Chemistry (CSIC), 28006 Madrid, Spain; iqoc21@iqog.csic.es

We have recently identified (Z)-*N*-benzyl-1-(8-hydroxyquinolin-2-yl)methanimine oxide (**QN6**) as a potent hBuChE (IC₅₀ = 1.06 ± 0.31 nM) and hMAO-B (IC₅₀ = 4.46 ± 0.18 µM) inhibitor, showing antioxidant and biometal chelator properties, able to cross the blood–brain barrier, non-cytotoxic, acting as a neuroprotector agent in a 6-hydroxydopamine cell model of Parkinson’s disease (PD), and as an anti-amnesic ligand in the scopolamine-induced mouse model of Alzheimer’s disease (AD), and that chronic treatment of double transgenic APPswe-PS1δE9 mice with **QN6** reduced the amyloid plaque load in the hippocampus and cortex of female mice [122].

Here, we report the neuroprotective properties and the antioxidant power of **QN6** for their potential application in treating stroke. In vitro neuroprotection studies of **QN6** in an oxygen-glucose deprivation model of cerebral ischemia, in human neuroblastoma cell cultures, indicate that **QN6** is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC₅₀ = 3.97 ± 0.78 µM), as well as necrotic (EC₅₀ = 3.79 ± 0.83 µM) and apoptotic cell death (EC₅₀ = 3.99 ± 0.21 µM) [123], showing a high capacity to decrease superoxide production (EC₅₀ = 3.94 ± 0.76 µM). Furthermore, in an experimental permanent focal ischemia model, **QN6**-treated animals exhibited a very significant reduction (75.21 ± 5.31%) in the brain lesion volume size.

Overall, **QN6** as a unique, single and multivalent agent for the combined therapy of diseases of aging such as PD/AD, and stroke [122,123].

*This research was supported by grants OPTICOMC903 and NACONT from the Camilo José Cela University (Madrid).*

### 4.19. New Azole Antifungals with a Fused Imidazopyrimidone Scaffold

Mathieu Scaviner¹, Rémi Guillon¹, Sophie Gazzola¹, Isabelle Ourliac-Garnier¹, Carine Picot¹, Marjorie Albassier¹, Aurélien Planchat², Fabrice Pagniez¹, Patrice Le Pape¹ and Cédric Logé¹
1. Nantes Université, Cibles et Médicaments des Infections et de l'Immunité, IICiMed UR1155, Institut de Recherche en Santé 2, F-44200 Nantes, France; mathieu.scaviner@etu.univ-nantes.fr

2. Nantes Université, CNRS, CEISAM UMR 6230, F-44000 Nantes, France

Currently, drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat invasive fungal infections (IFIs) [12]. The antifungal armamentarium includes the polyenes (amphotericin B formulations), the pyrimidine analogue (flucytosine), echinocandins, and the azoles (fluconazole, itraconazole (first generation), voriconazole, posaconazole, and isavuconazole (second generation)). Isavuconazole, and its structural analogue ravuconazole, share a similar 4-(1,3-thiazol-4-yl)benzonitrile moiety (Figure 24), and both are active against clinically important yeasts and molds including *Candida* spp. and *Aspergillus* spp. [124,125]. These azoles inhibit cytochrome P450-dependent lanosterol 14α-demethylation (CYP51), leading to a depletion of ergosterol (major component of the fungal cell membrane). As part of our research project, we replaced the thiazolyl substituent of ravuconazole by an imidazo[1,2-c]pyrimidin-5-one scaffold while retaining the benzonitrile moiety or substituting it by a more hydrophilic pyridine-2-carbonitrile (Figure 23).

<figure><img src="image_26.png" /><figcaption>**Figure 24.** Chemical structures of some antifungal agents and our synthesized compounds.</figcaption></figure>

These compounds were tested against pathogenic *Candida* spp. (fluconazole-susceptible and fluconazole-resistant) and against some filamentous fungi such as *Aspergillus fumigatus*, and the zygomycetes *Rhizopus oryzae*, and *Mucor circinelloides*. Compound I was also evaluated against two murine models of lethal systemic infections caused by *Candida albicans*.

## 4.20. Conformational Restriction of Designer Drugs Provides Potent and Selective CB2 Agonists with Neuroprotective Effect

Sandra Ortigueira-Noya ¹,², Claudia Gioé-Gallo ¹,², Jhonny Azuaje ¹,², Maria Majellaro ¹,², Xerardo García-Mera ², Rita Paleo ¹, José M. Brea ³, Maria I. Loza ³, Gemma Navarro ⁴, Rafael Franco ⁴, Marialessandra Contino ⁵, Angela Stefanachi ⁵ and Eddy Sotelo ¹,²,*

1. Center for Research in Chemical Biology and Molecular Materials, CiQUS, 15705 Santiago de Compostela, Spain; sandra.ortigueira.noya@usc.es

2. Faculty of Pharmacy, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain

3. Center for Research in Molecular Medicine and Chronic Diseases, CiMUS, 15705 Santiago de Compostela, Spain

4. Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain

5. Department of Pharmacy-Drug Sciences, Università degli studi di Bari Aldo Moro, 70121 Bari, Italy

Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances that mimic the psychotropic effects of THC. Introduced into the illicit drug market in the 2000s,
they are sold over the Internet under names like Spice, K2, or "synthetic marijuana" [126]. The consumption of SCRs is associated with serious adverse effects, including psychosis, seizures, cardiotoxicity, and renal injury, and has led to significant mortality. However, their effects in humans remain largely unknown, posing challenges for scientists, healthcare professionals, and regulatory authorities [126,127]. The first part of this study documents the synthesis and pharmacological characterization of a library of 64 enantiopure SCRs. Pharmacological data demonstrated that these compounds are highly potent at both CB1 and CB2 receptors [128]. The collection was developed to provide standard compounds for enforcement agencies to monitor SCRs in the illicit drug market. The second part of the study focuses on increasing selectivity for the CB2 receptor. Designing molecules with a 5- or 6-carbon ring at the amide terminus resulted in 64 new compounds with conformational restriction (Figure 25). This modification preserved their activity and made them highly selective for the CB2 receptor while maintaining their potency. Biased agonism studies were conducted to understand the specific signaling pathways activated by these compounds. Additionally, a small sample of six compounds was evaluated for neuroprotective effects, revealing promising data.

<figure><img src="image_27.png" /><figcaption>**Figure 25.** Graphical representation of the pharmacological data of SCRs and cyclic amides. It shows the transition of compounds from being very potent and non-selective to highly selective for the CB2 receptor.</figcaption></figure>

## 4.21. Development of Novel Small Molecules to Reactivate R273H Mutant p53

Tomás R. G. Monteiro¹ Ricardo J. F. Ferreira¹ Lídia M. D. Gonçalves¹ A. Paula Leandro¹ Maria M. M. Santos¹,*

1. Research Institute for Medicines (iMed. ULisboa), Faculdade de Farmácia Universi-dade de Lisboa, 1649-003 Lisboa, Portugal; tomasmonteiro1@edu.ulisboa.pt; mari-asantos@ff.ulisboa.pt

The tumor-suppressor protein p53 is often referred to in the scientific community as the "guardian of the genome". In almost 50% of tumors, the p53 gene, TP53, is mut-tated [129]. These mutations preferentially occur in the DNA-binding domain of the protein and can be classified as structural (e.g., R175H and Y220C) or DNA contact (e.g., R273H and R280K) [130]. Cancers that harbor mutant (mut) p53 R273H are currently one of the most difficult to treat, since this mutation enhances cell proliferative and metastatic properties [131]. Only a few small molecules have been developed to treat mut p53 tu-mors by restoring its wild-type (wt) conformation, and inhibiting cell proliferation and tumor growth. However, none of them act on the R273H mutation, and the molecular mechanisms underlying the action of these compounds remain obscure. Nevertheless,
some have entered clinical trials, i.e., APR246 against mut p53 R175H, reaching Phase I and/or II as monotherapy [129]. Enantiopure tryptophanol-derived isoindolinones were previously identified as direct reactivators of wt-p53 and mut p53, and some derivatives showed p53-dependent in vivo antitumor activity [97,132]. In this communication, we will disclose our results on the hit-to-lead optimization of this chemical scaffold with the goal of obtaining novel reactivators of mut p53 R273H able to restore the p53 wt-conformation. New substituents were inserted to explore the chemical space and to improve solubility (Figure 26). The synthesized compounds were evaluated against mut p53 R273H using a differential scanning fluorimetry (DSF) assay, and the most promising compounds were also tested against several cancer cell lines expressing different p53 status.

<figure><img src="image_28.png" /><figcaption>**Figure 26.** Hit-to-lead optimization on the tryptophanol-derived scaffold to target mut p53 R273H.</figcaption></figure>

*This work was supported by national funds (Fundação para a Ciência e a Tecnologia) through iMed.ULisboa (UIDB/04138/2020), project PTDC/QUI-QOR/1304/2020, and PhD fellowship 2022.11539.BD (R. Ferreira).*

## 4.22. Design, Synthesis, and Biological Evaluation of New MT5-MMP Inhibitor as Potential Therapeutic Interest in Alzheimer's Disease

Antoine Magniez ¹, C. Rémondin ¹, A. Davis, A. Lepailleur ¹, P. Dallemagne ¹ and C. Rochais ¹

1. Centre d'Etudes et de Recherche sur le Médicament de Normandie, Université de Caen Normandie, 14000 Caen, France, Boulevard Becquerel; antoine.magniez@unicaen.fr

Alzheimer's Disease (AD) is one of the most common senile dementias in the world and a global issue in health care. Though several efforts have been made to find therapeutics, most of them broke down in clinical trials, with 98% fails since 2003 [133]. This pushed scientists to a better understanding of the physiopathology, which is still not totally known nowadays. Even though AD is a multifactorial disease, the main hypothesis is the aggregation of the amyloid β peptide (Aβ) from the amyloid precursor protein (APP) cleavage which results in the formation of senile plaques in the brain cortex. Finding a way to prevent this aggregation has always been a goal since the discovery of Aβ. Recently, MT5-MMP was discovered as a new target playing a role in Aβ formation [134]. MT5-MMP is a metalloproteinase which acts as η-secretase and releases a neurotoxic Aη-α fragment from the APP cleavage and, in a second time, is also a proamyloidogenic that promotes the production of Aβ. The inhibition of this protein can therefore become an interesting new therapeutic strategy to treat AD. In this context, we have recently identified a new family of MT5-MMP inhibitors possessing an amide linker. In order to explore new analogs, several sulfonamides and triazoles linkers have been introduced and the structure of the ligand (Figure 27) has been compared using X-ray analysis with the goal to develop potent and selective inhibitors.
<figure><img src="image_29.png" /><figcaption>**Figure 27.** General structure of the designed inhibitors.</figcaption></figure>

4.23. Targeting IRE1 for an Adjuvant Therapy in Glioblastoma

Sébastien Sueron ¹,², Diana Pelizzari-Raymundo ², Leif A. Eriksson ³, Eric Chevet ², François-Hugues Porée ¹ and Xavier Guillory ²

1. ISCR UMR CNRS 6226 Faculty of Pharmacy, Rennes University, F-35000 Rennes, France; francois-hugues.poree@univ-rennes.fr

2. Inserm U1242 Centre de Lutte Contre le Cancer Eugène Marquis, Rennes University, F-35000 Rennes, France.

3. Department of Chemistry & Molecular Biology, University of Gothenburg, 40530 Göteborg, Sweden.

Glioblastoma (GB) is the most frequent and malignant primary brain tumor. Its highly infiltrative nature and heterogeneity leads to the systematic failure of current therapeutics with a median survival of 15 to 18 months post-diagnosis. Signaling by the inositol-requiring enzyme 1 (IRE1), a bifunctional serine/threonine kinase, has been identified as a pro-survival adaptative mechanism playing an instrumental role in several cancers [135]. This makes IRE1 inhibition an appealing therapeutic strategy in oncology, either as a monotherapy or as an adjuvant therapy alongside established treatments [136,137]. Our group established the relevance of targeting IRE1 in GB and showed in GB mouse models that IRE1 inhibition resulted in a reduced tumor aggressiveness and increased sensitivity to temozolomide (TMZ), the reference chemotherapy [138]. Using a structure-driven drug discovery pipeline, we identified Z4P, a blood brain barrier-permeable and kinase site-bound ligand showing inhibitory activities in GB cell models, the sensitization of tumor cells to TMZ, and, more strikingly, preventing tumor relapse in mice when used in combination with TMZ (Figure 28) [139]. We will disclose here our results concerning the structure-activity relationships (SAR) on the Z4P series.

<figure><img src="image_30.png" /><figcaption>**Figure 28.** (a) Quantitative analysis of bioluminescence imaging signal across 200 days of experiment with temolozomide alone (blue) and the combination of temolozomide/Z4P (red). (b) Structure-Activity Relationships (SAR) strategy around the Z4P scaffold.</figcaption></figure>
Financial support from La Ligue Contre le Cancer, Plan Cancer and Fondation pour la Recherche Médicale is acknowledged.

## 4.24. Approaches to the Synthesis of Isatin-Based Molecules as Inhibitors of BACE-1 and Cholinesterases

Catarina A. Montargil¹, Rosa Resende², Elisabete P. Carreiro³, Alfonso T. García-Sosa⁴, Armanda E. Santos²,⁵ and Anthony J. Burke ¹,²,³,⁶

1. Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; catarinamontargil@gmail.com

2. Center for Neuroscience and Cellular Biology (CNC), University of Coimbra, 3004-504 Coimbra, Portugal.

3. LAQV-REQUIMTE, University of Évora, Institute of Research and Advanced Training (IIFA), 7000-671 Évora, Portugal.

4. Institute of Chemistry, University of Tartu, Ravila 14 A, 50411 Tartu, Estonia.

5. Laboratory of Biochemistry and Biology, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal.

6. Coimbra Chemistry Centre–Institute of Molecular Sciences (CQC-IMS), Chemistry Department, University of Coimbra, 3004-535 Coimbra, Portugal.

Alzheimer's disease (AD) remains a significant neurodegenerative disease with limited therapeutic options. Some of the targets involved in AD are considered undruggable, meaning that these biomolecules do not respond to conventional small molecules [140]. Consequently, larger molecules have begun to be considered as potential therapeutics for these targets. Macrocycles for a long time have held a privileged status for the treatment of various diseases, particularly cancer (Dolastatin, Laulimalide A, Peloruside), and as an anti-microbial (Erythronolide B) and immunosuppressants (Rapamycin, FK-506), showing favorable pharmacological properties.

Our interest has been the development of novel oxindole-based macrocycles, which show a large spectrum of biological activities against neurodegenerative diseases, particularly Alzheimer's [141,142].

In this study, we report our efforts on synthesizing a series of isatin-based macrocycles and their precursors and evaluating these precursors against some key AD targets. The compounds were tested for inhibitory activity against beta-secretase 1 (BACE-1) and cholinesterases (ChE), both crucial enzymes implicated in AD pathology. Molecular docking studies have been carried out, providing further insights. Additionally, we assessed the cytotoxicity and neuroprotective effects of these compounds using the N2A-APPswe cell line, a well-established in vitro model for AD.

We thank FCT for funding through the strategic project UIDB/00313/2020 | UIDP/00313/2020 to CQC-IMS.

## 4.25. The Bucherer–Bergs Multicomponent Reaction for Assessing Chiral Spiro-Oxindole-Hydantoins: Screening Against Leishmania Donovani

Maria B. V. Moura ¹,², Anthony J. Burke ¹,²,³ and Maria C. Sousa ²,³

1. University of Coimbra, Coimbra Chemistry Centre-Institute of Molecular Sciences (CQC-IMS), Departamento de Química, 3004-535 Coimbra, Portugal; beatrizmoura14@hotmail.com

2. University of Coimbra, Faculty of Pharmacy, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.

3. University of Coimbra, Center for Neurosciences and Cellular Biology (CNC), Polo I, Rua Larga Faculdade de Medicina, 3004-504 Coimbra, Portugal.
Leishmaniasis is a neglected tropical disease (NTDs), being the second biggest cause of death for a parasitic disease, after malaria [143]. According to the World Health Organization, it is estimated that 700,000 to 1 million new cases are reported every year. Current treatments include antimony compounds, amphotericin B, pentamidine, and miltefosine, among others [144]. However, these pharmaceuticals show toxicity and require prolonged usage, as well as being expensive [145].

Interestingly, several spiro compounds have already demonstrated antileishmanial activity [146]. The oxindole unit is a well-known pharmacophore [147] and, in fact, compounds containing oxindole have been reported for their antileishmanial activity [148]. Hydantoins (imidazolidine-2,4-ones) are also biologically active [149]. In this communication, we will discuss our results on the multicomponent synthesis (Bucherer-Bergs reaction) of a library of spiro-oxindole-hydantoins. Our studies included the use of Natural Deep Eutectic Solvents (NADES) and organocatalysts (Figure 29).

<figure><img src="image_31.png" /><figcaption>**Figure 29.** Synthesis of chiral spiro-oxindoles-hydantoins (* stereogenic center*).</figcaption></figure>

We are currently evaluating these compounds for their antileishmanial activity in *Leishmania donovani*, and, gratifyingly, some of these compounds were found to be active.

We thank the FCT for funding through the strategic project UIDB/00313/2020 | UIDP/00313/2020 to Coimbra Chemistry Centre–Institute of Molecular Sciences (CQC-IMS).

## 4.26. Leveraging the Identification of Novel Ubiquitin Specific Protease 7 (USP7) Inhibitors Through a Structure-Based Virtual Screening Protocol

Rita Oliveira ¹/², Laura D. Carreira ¹/², Jorge A. R. Salvador ¹/²

1. Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; rita.oliveira@cnc.uc.pt

2. Centre for Innovative Biomedicine and Biotechnology & Center for Neuroscience and Cell Biology, 3000-504 Coimbra, Portugal.

Ubiquitin-specific protease 7 (USP7) is a member of one of the most largely studied families of deubiquitylating enzymes, which are proteases in control of the dynamics of ubiquitination. It plays a key role in modulating the levels of multiple proteins, including tumor suppressors, transcription factors, epigenetic modulators, DNA repair proteins, and regulators of the immune response [150–156]. The abnormal expression of USP7 is found in various malignant tumors, and a high expression signature generally indicates poor tumor prognosis. This suggests USP7 as a promising prognostic and druggable target that offers interesting new avenues for cancer therapy [150–156]. Therefore, the main goal of this study was the identification of promising small molecules that could potentially inhibit USP7 enzymatic activity. The work was conducted according to an integrated molecular
modelling protocol, including structure-based pharmacophore and molecular docking virtual screening. Such a protocol disclosed new USP7 hit compounds, highlighting the utility of computer-aided drug discovery in the early steps of the drug discovery process, and paved the way for the identification of promising USP7 inhibitors that might represent a steppingstone for cancer treatment.

Rita I. Oliveira and Laura D. Carreira thank the Portuguese Research Agency FCT—Fundação para a Ciência e a Tecnologia, I.P., for funding the individual research grants No. 2021.07538.BD and 2022.10811. BD, respectively.

4.27. *Design, Synthesis and Biological Evaluation of Novel Madecassic Acid Derivatives with Anticancer Potential*

Sara Moura ¹,², Pedro J. M. Sobral ¹,², Ana S. C. Valdeira ¹,²,³, Emad Darvishi ³, Girma M. Woldemichael ³,⁴, John A. Beutler ³, Kirk R. Gustafson ³ and Jorge A. R. Salvador ¹,²

1. Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; spmoura17@gmail.com

2. Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal

3. Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA

4. Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute, Frederick, MD 21702-1201, USA

Madecassic acid (1) is a pentacyclic triterpenoid found in the traditional medicinal plant *Centella asiatica* (L.) Urban that has various pharmacological activities [157]. In particular, this natural compound demonstrated anticancer activity by inducing apoptosis in an in vivo mouse model bearing the CT26 cancer cell line [158]. There are a limited number of 1 derivatives, and few of them have been explored for their anticancer activity [159,160]. Thus, a series of new derivatives of 1 was synthesized and screened for cytotoxicity against the NCI-60 panel of cancer cell lines. All the tested derivatives exhibited superior antiproliferative activity compared to that of 1. Among them, compound 29 showed $GI_{50}$ values ranging from 0.3 to 0.9 µM against 26 different tumor cell lines, demonstrating more selectivity for one colon (COLO 205) and two melanoma (SK-MEL-5 and UACC-257) cell lines at the TGI level. The mechanism of action of compound 29 was predicted using a CellMiner bioinformatics analysis and validated by biochemical assays. These studies demonstrated that treatment with 29 induced cell cycle arrest at the G1/S transition and disrupted the mitochondrial membrane potential in tumor cells. In conclusion, derivative 29 shows promise as a lead compound for the development of new anticancer drugs and merits further research (Figure 30) [161].

<figure><img src="image_32.png" /><figcaption>**Figure 30.** Structure of madecassic acid (1) and its derivative 29 with potential anticancer activity.</figcaption></figure>
## 4.28. Development of Emerging Strategies to Target the Ser/Thr Kinase RIPK2

Morgane Rivoal¹, Amélie Barzyck¹, Xavier Dezitter¹, Perrine Six¹, Min-Jeong Cornu-Choi¹, Nicolas Renault¹, Corentin Bedart¹, Amaury Farce¹, Laurent Dubuquoy¹, Régis Millet¹ and Natascha Leleu-Chavain ¹,*

1. Univ. Lille, Inserm, U1286-INFINITE-Lille Inflammation Research International Center, F-59000 Lille, France; natascha.leleu@univ-lille.fr

Inflammation is a defense mechanism designed to repair tissue damage and eliminate pathogens. Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is a crucial mediator of innate immune signaling pathways, and particularly those initiated by pattern-recognition receptors (PRRs) such as NOD-like receptors (NLRs) NOD1 and NOD2 [162]. RIPK2 plays a central role in the regulation of inflammatory and immune responses by activating signaling cascades leading to the production of pro-inflammatory cytokines. It functions by recruiting and activating downstream signaling molecules, including the IκB kinase (IKK) complex and mitogen-activated protein kinase (MAPK) pathways, resulting in the activation of transcription factors such as NF-κB and AP-1. Given its pivotal role in inflammatory signaling pathways, RIPK2 has garnered significant interest as a therapeutic target for various inflammatory diseases [163]. Several small molecule inhibitors targeting RIPK2 have been developed and are currently under investigation for their therapeutic potential in diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis, and other inflammatory disorders. In view of our team's expertise in the field of inflammatory diseases, we decided to focus on this promising target.

Our project has been divided into several tasks. The first led to the establishment of structure-activity relationships (SARs) of a series of quinazolines that identified the elements required for activity. In pursuit of strategies that have the potential to lead to highly selective compounds, two emerging strategies have been selected by our team and will be presented in this second poster. The strategy of degrading kinases is emerging and has shown potential for providing a greater selectivity compared to conventional inhibitors. These degraders have a tripartite structure consisting of a RIPK2 ligand selected from our series of quinazolines, a hydrophobic tag separated by a linker. The hydrophobic tag mimics the exposed parts of misfolded proteins, attracting chaperone proteins that lead our target to degradation by the proteasome. The aim of synthesizing these degraders is first to vary the linker and hydrophobic tags to establish the initial RSAs and proof of concept. A second strategy aims to identify the inhibitors of the XIAP-RIPK2 interaction through virtual screening. This approach is justified by the identification of XIAP as the primary ligase responsible for RIPK2 ubiquitination, which is essential for RIPK2 activation of inflammatory pathways. An electron microscopy model (PDB:8AZA) [164] was optimized using the AMBER14 method to identify the amino acids involved in the interaction. Then, a pharmacophore search using Discovery Studio software led to the identification of peptides due to the extent of the interaction. Virtual screening was performed by docking a peptide library obtained via the Protein Data Base (PDB) using AutoDock CrankPep software. These peptides have a defined three-dimensional structure, which reduces the risk of conformational changes during experimental testing. They will be the subject of further testing at our institute for their potential as new *hits*.

## 4.29. Carvone Derivatives: Synthesis and Screening of Anti-Inflammatory Activity

Lara Mingatos¹, Gabriela Moço¹, Cátia Sousa ², Ana Capitão ³, Alexandrina Ferreira Mendes ¹,³,⁴ and Alcino Leitão ¹,³,⁴

1. Faculty of Pharmacy, University of Coimbra, 3004-548 Coimbra, Portugal; laramingatos@gmail.com
2. NOVA Medical School | Faculdade de Ciências Médicas (NMS | FCM), NOVA University of Lisbon, 1649-004 Lisboa, Portugal

3. Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal

4. Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, 3004-504 Coimbra, Portugal

Natural products are increasingly used for their anti-inflammatory properties and as sources of new anti-inflammatory compounds [165]. Further chemical modifications are a useful strategy to improve their bioavailability and/or potency.

Previous studies elucidated the structure-activity relationship of monoterpene compounds, derived from *p*-menthane, as potential anti-inflammatory drugs. (*S*)-(*+*)-carvone (1) (Figure 31) was identified as the most potent of the compounds tested and may be efficient in halting inflammation-related diseases like osteoarthritis [166].

<figure><img src="image_33.png" /><figcaption>**Figure 31.** (*S*)-(*+*)-carvone (1) and 8-hydroxycarvotanacetone (2).</figcaption></figure>

The *α*,*β*-unsaturated ketone group of carvone seems to be critical for activity. The replacement of the isopropenyl group at C5 by a 2-hydroxyisopropanyl group, such as in 8-hydroxycarvotanacetone (2) (Figure 30), lowered the potency but provided a hydroxyl group, important to manage the lipophilic properties. Another relevant feature for activity must be the chirality, so both enantiomers of carvone must be studied [166].

Based on these premises, recently, our group reported the synthesis of some carvone derivatives and performed a screening in silico and in vitro of their anti-inflammatory activity and pharmacokinetic properties [167]. Although the tested compounds still have a low potency and specificity, they are presenting anti-inflammatory and some advantageous ADME properties, and the results encouraged us to design new structures that may overcome the detected drawbacks and yield more promising drugs. New carvone derivatives have been synthesized, namely 9-hydroxycarvone, using a three-step synthesis, and their esters-using acyl halides under basic catalysis. This strategy allowed us to maintain the functionalities and differences at positions 9 and 8 of the molecule, and either maintain or not maintain the double bond, respectively. After the purification and structural analysis, the cytotoxicity and anti-inflammatory activity of selected derivatives were evaluated in vitro.

*Funding from the European Regional Development Fund (ERDF), through the COMPETE 2020-Operational Programme for Competitiveness and Internationalization and Portuguese national funds via FCT-Fundação para a Ciência e a Tecnologia, under projects UIDB/04539/2020, UIDP/04539/2020, and LA/P/0058/2020. Acknowledgments to UCQFarma for FTIR/ATR facility and UC-NMR for NMR facility.*

### 4.30. Discovery of Potent Isoquinolinequinone N-Oxides to Overcome Cancer Multidrug Resistance

Ryan D. Kruschel ¹, Mélanie A. G. Barbosa ²,³,⁴, Maria João Almeida ²,³, Cristina P. R. Xavier ²,³, M. Helena Vasconcelos ²,³,⁴ and Florence O. McCarthy ¹,*

1. School of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork, T12 K8AF Cork, Ireland; f.mccarthy@ucc.ie
2. i3S–Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal;

3. Cancer Drug Resistance Group, IPATIMUP–Institute of Molecular Pathology and Immunology, University of Porto, 4200-135 Porto, Portugal;

4. FFUP–Faculty of Pharmacy of the University of Porto, 4050-313 Porto, Portugal

Multidrug resistance (MDR) of human tumors has resulted in an immediate need to de-
velop appropriate new drugs. Our research has previously identified isoquinolinequinones
(IQQ) as effective against cancer cell lines and uniquely against the NCI-ADR/RES MDR
cell line [168]. This work outlines the development of twenty potent IQQ N-oxide deriva-
tives in two isomeric families, both exhibiting nano-molar GI₅₀ values against human
tumor cell lines [169]. Preliminary NCI-60 tumor screening sees the C(6) isomers achieve
a mean GI₅₀ > 2 times lower than the corresponding C(7) isomers. The MDR evaluation
of nine selected compounds reveals that each presents lower GI₅₀ concentrations in two
MDR tumor cell lines. Four of the series display nanomolar GI₅₀ values against MDR cells,
having selectivity ratios up to 2.7 versus the sensitive (parental) cells. The most potent
compound 25 inhibits the activity of drug efflux pumps in MDR cells, causes significant
ROS accumulation, and potently inhibits cell proliferation, causing alterations in the cell
cycle profile. Our findings are confirmed by 3D spheroid models, providing new candidates
against MDR cancer (Figure 32).

<figure><img src="image_34.png" /><figcaption>**Figure 32.** IQQ N-oxides as a framework for the potent inhibition of MDR cancer cell lines.</figcaption></figure>

4.31. *Design, Synthesis, and Evaluation of Benzhydrylpiperazine-Based Novel Molecular Hybrids as Potent COX-2/5-LOX Inhibitors with Anti-Proliferative Activity*

Poorvi Saraf ¹ and Sushant Kumar Shrivastava ¹

1. Department of Pharmaceutical Engineering & Technology, IIT (BHU), Varanasi 221005, India; poorvisaraf.rs.phe19@itbhu.ac.in

The strategy of the development of safer anti-inflammatory drugs resulted in effective
and promising leads for the treatment of multifaceted inflammatory diseases. In the current
research, we have performed the virtual screening of piperazine-containing compounds
from the ChEMBL database and optimized the hit to design and synthesize molecular hy-
brids of benzhydrylpiperazine and 1,3,4-oxadiazole [170–173]. The envisioned compounds
have been successfully synthesized, characterized, and assessed biologically. The biological
activities revealed an appealing inhibitory potential of the synthesized derivatives against
the target enzymes. Surprisingly, the novel compounds also displayed an antiproliferative
activity in A549-Lung cancer cell lines and COLO-205-colon cancer cell lines. Amongst
the investigated compounds, 9d containing 4-Cl substitution at the terminal phenyl ring
appeared to be the most promising lead with an inhibition of COX-2 (IC₅₀ = 0.25 ± 0.03 µM), and 5-LOX (IC₅₀ = 7.87 ± 0.33 µM). The enzyme kinetics investigation of **9d** against COX-2 indicated a competitive inhibition (Kd = 0.22 µM). Compounds **9d** and **9g** were evaluated in paw edema models of inflammation, which indicated a significant anti-inflammatory response. These potential compounds demonstrated a reduction in PGE2, IL-6, and TNF-α, and an increase in IL-10 concentrations. Furthermore, the promising compounds displayed gastrointestinal (GI), liver, and kidney safety along with antioxidant properties. Moreover, treatment with compounds **9d** and **9g** limits platelet aggregation and suggests no indication of cardiotoxicity when examined on rats with myocardial infarction. The most promising compound **9d** also displayed anticancer potential in the *Drosophila flies* model. The results were also correlated with in silico docking and molecular dynamics simulation studies.

### 4.32. *Mapping the Protein Kinases' Cysteinome by a Kinase-Focused Covalent Fragment Library*

Nico J. Seidler¹, Guiqun Wang², Sandra Röhm², Stefan Knapp², Matthias Gehringer¹,²,³

1. Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, 72074 Tübingen, Germany; matthias.gehringer@uni-tuebingen.de

2. Goethe-University Frankfurt, Structural Genomics Consortium (SGC), 60323 Frankfurt am Main, Germany

3. Division for Medicinal Chemistry, Institute for Biomedical Engineering, Tübingen University Hospital, 72076 Tübingen, Germany

Selectivity remains a major challenge in the development of new protein kinase inhibitors. One of the prime strategies to gain selectivity within the “kinome” is to target poorly conserved, non-catalytic cysteine residues adjacent to the ATP-binding pocket via an electrophilic functional group, often referred to as the “warhead” [174]. Despite the ongoing development of such covalent kinase inhibitors in recent years, many of the accessible cysteine positions in the “cysteinome” of protein kinases have not yet been covalently targeted [175–177]. Our goal was to establish a library of kinase-focused covalent fragments which enables the identification of starting points for the future development of new inhibitors that can covalently bind these previously neglected cysteine residues.

Therefore, we synthesized a set of fragment-like electrophilic compounds that are designed to target different cysteine sites by combining a hinge-binding motif with different linker systems and a warhead. The synthesis of most fragments was carried out by a Suzuki-type cross-coupling reaction of a hinge-binding aryl halide with various acrylamide-derived boronic acid pinacol esters. A thermal shift assay was used to screen the library against a panel of more than 40 kinases, which all bear a cysteine in proximity to the ATP-binding pocket. Furthermore, covalent binding was assessed by mass spectrometry. This generated promising hits for different cysteine positions, also demonstrating the high selectivity of many compounds within our kinase panel, which indicates that the binding is not primarily driven by the reactivity of the warhead. Promising compounds were further profiled, and we successfully obtained several co-crystal structures of inhibitors bound to their respective protein targets, many of which belong to the understudied fraction of the kinome. This allowed us to gain a deeper understanding of the binding mode of these inhibitors, providing a strong basis for future medicinal chemistry programs.

## 5. Conclusions

The 32nd GP₂A conference was successfully held at the University of Coimbra, in Portugal, for the first time in its history. We acknowledge the outstanding support of our sponsors, namely CEM, Specanalítica, QLabo, Laborspirit, Solítica, MT Brandão, Unicam, Santander, the Portuguese Society of Chemistry, MDPI, Wiley, and the RSC Medicinal Chemistry. We also greatly acknowledge the work of the undergraduate and post-graduate
students of the Faculty of Pharmacy of the University of Coimbra, for their help with our
event on-site.

The 33rd edition of the GP₂A conference will be held as the “Joint Medicinal Chemistry congress GP₂A–Paul Ehrlich MedChem 2025”, at the University of Nantes, France, on 13–15 June 2025.

**Author Contributions:** All authors contributed to compiling and formatting the conference abstracts and all authors approved the submission of the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available on request from the respective authors.

**Conflicts of Interest:** The authors declare no conflicts of interest.

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