Sigma receptor and aquaporin modulators: chiral resolution, configurational assignment, and preliminary biological profile of RC752 enantiomers.

The key role of chiral small molecules in drug discovery programs has been deeply investigated throughout last decades. In this context, our previous studies highlighted the influence of the absolute configuration of different stereocenters on the pharmacokinetic, pharmacodynamic and functional properties of promising Sigma receptor (SR) modulators. Thus, starting from the racemic SR ligand RC752, we report herein the isolation of the enantiomers via enantioselective separation with both HPLC and SFC. After optimization of the eco-sustainable chiral SFC method, both enantiomers were obtained in sufficient amount (tens of mg) and purity (ee up to 95%) to allow their characterization and initial biological investigation. Both enantiomers a) displayed a high affinity for the S1R subtype (Ki = 15.0 ± 1.7 and 6.0 ± 1.2 nM for the (S)- and (R)-enantiomer, respectively), but only negligible affinity toward the S2R (> 350 nM), and b) were rapidly metabolized when incubated with mouse and human hepatic microsomes. Furthermore, the activity on AQP-mediated water permeability indicated a different functional profile for the enantiomers in terms of modulatory effect on the peroxiporins gating.

Peptides as Therapeutic Agents: Challenges and Opportunities in the Green Transition Era.

Peptides are at the cutting edge of contemporary research for new potent, selective, and safe therapeutical agents. Their rise has reshaped the pharmaceutical landscape, providing solutions to challenges that traditional small molecules often cannot address. A wide variety of natural and modified peptides have been obtained and studied, and many others are advancing in clinical trials, covering multiple therapeutic areas. As the demand for peptide-based therapies grows, so does the need for sustainable and environmentally friendly synthesis methods. Traditional peptide synthesis, while effective, often involves environmentally draining processes, generating significant waste and consuming vast resources. The integration of green chemistry offers sustainable alternatives, prioritizing eco-friendly processes, waste reduction, and energy conservation. This review delves into the transformative potential of applying green chemistry principles to peptide synthesis by discussing relevant examples of the application of such approaches to the production of active pharmaceutical ingredients (APIs) with a peptide structure and how these efforts are critical for an effective green transition era in the pharmaceutical field.

Discovery of RC-752, a Novel Sigma-1 Receptor Antagonist with Antinociceptive Activity: A Promising Tool for Fighting Neuropathic Pain.

Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It is a debilitating disorder that affects up to 10% of the world's population. Although opioid analgesics are effective in reducing pain, they present severe risks; so, there is a pressing need for non-opioid pain-relieving drugs. One potential alternative is represented by sigma-1 receptor (S1R) antagonists due to their promising analgesic effects. Here, we report the synthesis and biological evaluation of a series of S1R antagonists based on a 2-aryl-4-aminobutanol scaffold. After assessing affinity toward the S1R and selectivity over the sigma-2 receptor (S2R), we evaluated the agonist/antagonist profile of the compounds by investigating their effects on nerve growth factor-induced neurite outgrowth and aquaporin-mediated water permeability in the presence and absence of oxidative stress. (R/S)-RC-752 emerged as the most interesting compound for S1R affinity (Ki S1R = 6.2 ± 0.9) and functional antagonist activity. Furthermore, it showed no cytotoxic effect in two normal human cell lines or in an in vivo zebrafish model and was stable after incubation in mouse plasma. (R/S)-RC-752 was then evaluated in two animal models of NP: the formalin test and the spinal nerve ligation model. The results clearly demonstrated that compound (R/S)-RC-752 effectively alleviated pain in both animal models, thus providing the proof of concept of its efficacy as an antinociceptive agent.

Photo-Flow Technology for Chemical Rearrangements: A Powerful Tool to Generate Pharmaceutically Relevant Compounds.

In recent years, photochemistry has increasingly emerged as an enabling methodology in both academia and the pharmaceutical industry. Long photolysis times and the gradual reduction of light penetration remained for many years unsolved issues for photochemical rearrangements, triggering the generation of highly reactive species in an uncontrolled fashion and causing the formation of multiple side products. The emergence of continuous-flow chemistry significantly helped to overcome these issues, thus prompting the implementation of photo-flow-based approaches for the generation of pharmaceutically relevant substructures. This Technology Note highlights the benefits of flow chemistry for photochemical rearrangements, including Wolff, Favorskii, Beckmann, Fries, and Claisen rearrangements. We showcase recent advances for photo-rearrangements in continuous flow applied to the synthesis of privileged scaffolds and active pharmaceutical ingredients.

Continuous-Flow Technology for Chemical Rearrangements: A Powerful Tool to Generate Pharmaceutically Relevant Compounds.

The efficacy, safety, and scale-up of several chemical rearrangements remain unsolved problems due to the associated handling of hazardous, toxic, and pollutant chemicals and high-risk intermediates. For many years batch processes have been considered the only possibility to drive these reactions, but continuous-flow technology has emerged, for both academic laboratories and pharmaceutical companies, as a powerful tool for easy, controlled, and safer chemistry protocols, helping to minimize the formation of side products and increase reaction yields. This Technology Note summarizes recently reported chemical rearrangements using continuous-flow approaches, with a focus on Curtius, Hofmann, and Schmidt reactions. Flow protocols, general advantages and safety aspects, and reaction scope for the generation of both privileged scaffolds and active pharmaceutical ingredients will be showcased.

Novel S1R agonists counteracting NMDA excitotoxicity and oxidative stress: A step forward in the discovery of neuroprotective agents.

Sigma-1 receptor (S1R) has been considered a promising therapeutic target for several neurodegenerative diseases and S1R agonists have shown neuroprotective activity against glutamate excitotoxicity and oxidative stress. Starting from a previously identified low nanomolar S1R agonist, in this work we prepared and tested novel benzylpiperidine/benzylpiperazine-based compounds designed by applying a ring opening strategy. Among them, 4-benzyl-1-(2-phenoxyethyl)piperidine 6b (S1R Ki = 0.93 nM) and 4-benzyl-1-(3-phenoxypropyl)piperidine 8b (S1R Ki = 1.1 nM) emerged as high affinity S1R ligands and showed selectivity over S2R and N-methyl-d-aspartate receptor (NMDAR). Candidate compounds behaved as potent S1R agonists being able to enhance the neurite outgrowth induced by nerve growth factor (NGF) in PC12 cell lines. In SH-SY5Y neuroblastoma cell lines they exhibited a neuroprotective effect against rotenone- and NMDA-mediated toxic insults. The neuroprotective activity of 6b and 8b was reverted by co-treatment with an S1R antagonist, PB212. Compounds 6b and 8b were tested for cytotoxicity in-vitro against three human cancer cell lines (A549, LoVo and Panc-1) and in-vivo zebrafish model, resulting in a good efficacy/safety profile, comparable or superior to the reference drug memantine. Overall, these results encourage further preclinical investigations of 6b and 8b on in-vivo models of neurodegenerative diseases.

From Nature to Synthetic Compounds: Novel 1(N),2,3 Trisubstituted-5-oxopyrrolidines Targeting Multiple Myeloma Cells.

The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.

Urea-based anticancer agents. Exploring 100-years of research with an eye to the future.

Suramin was the first urea-based drug to be approved in clinic, and in the following century a number of milestone drugs based on this scaffold were developed. Indeed, urea soon became a privileged scaffold in medicinal chemistry for its capability to establish a peculiar network of drug-target interactions, for its physicochemical properties that are useful for tuning the druggability of the new chemical entities, and for its structural and synthetic versatility that opened the door to numerous drug design possibilities. In this review, we highlight the relevance of the urea moiety in the medicinal chemistry scenario of anticancer drugs with a special focus on the kinase inhibitors for which this scaffold represented and still represents a pivotal pharmacophoric feature. A general outlook on the approved drugs, recent patents, and current research in this field is herein provided, and the role of the urea moiety in the drug discovery process is discussed form a medicinal chemistry standpoint. We believe that the present review can benefit both academia and pharmaceutical companies' medicinal chemists to prompt research towards new urea derivatives as anticancer agents.

Biocatalysis: A smart and green tool for the preparation of chiral drugs.

Over the last decades, biocatalysis has achieved growing interest thanks to its potential to enable high efficiency, high yield, and eco-friendly processes aimed at the production of pharmacologically relevant compounds. Particularly, biocatalysis proved an effective and potent tool in the preparation of chiral molecules, and the recent innovations of biotechnologies and nanotechnologies open up a new era of further developments in this field. Different strategies are now available for the synthesis of chiral drugs and their intermediates. Enzymes are green tools that offer several advantages, associated both to catalysis and environmentally friendly reactants. Specifically, the use of enzymes isolated from biological sources or of whole-cell represents a valuable approach to obtain pharmaceutical products. The sustainability, the higher efficiency, and cost-effectiveness of biocatalytic reactions result in improved performance and properties that can be translated from academia to industry. In this review, we focus on biocatalytic approaches for synthesizing chiral drugs or their intermediates. Aiming to unveil the potentialities of biocatalysis systems, we discuss different examples of innovative biocatalytic approaches and their applications in the pharmaceutical industry.

Repurposing the Antiplatelet Agent Ticlopidine to Counteract the Acute Phase of ER Stress Condition: An Opportunity for Fighting Coronavirus Infections and Cancer.

Different pathological conditions, including viral infections and cancer, can have a massive impact on the endoplasmic reticulum (ER), causing severe damage to the cell and exacerbating the disease. In particular, coronavirus infections, including SARS coronavirus-2 (SARS-CoV-2), responsible for COVID-19, cause ER stress as a consequence of the enormous amounts of viral glycoproteins synthesized, the perturbation of ER homeostasis and the modification of ER membranes. Therefore, ER has a central role in the viral life cycle, thus representing one of the Achilles' heels on which to focus therapeutic intervention. On the other hand, prolonged ER stress has been demonstrated to promote many pro-tumoral attributes in cancer cells, having a key role in tumor growth, metastasis and response to therapies. In this report, adopting a repurposing approach of approved drugs, we identified the antiplatelet agent ticlopidine as an interferent of the unfolded protein response (UPR) via sigma receptors (SRs) modulation. The promising results obtained suggest the potential use of ticlopidine to counteract ER stress induced by viral infections, such as COVID-19, and cancer.

Bitopic Sigma 1 Receptor Modulators to Shed Light on Molecular Mechanisms Underpinning Ligand Binding and Receptor Oligomerization.

The sigma 1 receptor (S1R) is an enigmatic ligand-operated chaperone involved in many important biological processes, and its functions are not fully understood yet. Herein, we developed a novel series of bitopic S1R ligands as versatile tools to investigate binding processes, allosteric modulation, and the oligomerization mechanism. These molecules have been prepared in the enantiopure form and subjected to a preliminary biological evaluation, while in silico investigations helped to rationalize the results. Compound 7 emerged as the first bitopic S1R ligand endowed with low nanomolar affinity (Ki = 2.6 nM) reported thus far. Computational analyses suggested that 7 may stabilize the open conformation of the S1R by simultaneously binding the occluded primary binding site and a peripheral site on the cytosol-exposed surface. These findings pave the way to new S1R ligands with enhanced activity and/or selectivity, which could also be used as probes for the identification of a potential allosteric site.

Sigma-1 Receptor Agonists Acting on Aquaporin-Mediated H2O2 Permeability: New Tools for Counteracting Oxidative Stress.

Sigma1 Receptor (S1R) is involved in oxidative stress, since its activation is triggered by oxidative or endoplasmic reticulum stress. Since specific aquaporins (AQP), called peroxiporins, play a relevant role in controlling H2O2 permeability and ensure reactive oxygen species wasted during oxidative stress, we studied the effect of S1R modulators on AQP-dependent water and hydrogen peroxide permeability in the presence and in the absence of oxidative stress. Applying stopped-flow light scattering and fluorescent probe methods, water and hydrogen peroxide permeability in HeLa cells have been studied. Results evidenced that S1R agonists can restore water permeability in heat-stressed cells and the co-administration with a S1R antagonist totally counteracted the ability to restore the water permeability. Moreover, compounds were able to counteract the oxidative stress of HeLa cells specifically knocked down for S1R. Taken together these results support the hypothesis that the antioxidant mechanism is mediated by both S1R and AQP-mediated H2O2 permeability. The finding that small molecules can act on both S1R and AQP-mediated H2O2 permeability opens a new direction toward the identification of innovative drugs able to regulate cell survival during oxidative stress in pathologic conditions, such as cancer and degenerative diseases.

Synthesis and Characterization of a "Clickable" PBR28 TSPO-Selective Ligand Derivative Suitable for the Functionalization of Biodegradable Polymer Nanoparticles.

Reactive microgliosis is a pathological hallmark that accompanies neuronal demise in many neurodegenerative diseases, ranging from acute brain/spinal cord injuries to chronic diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and age-related dementia. One strategy to assess and monitor microgliosis is to use positron emission tomography (PET) by exploiting radioligands selective for the 18 kDa translocator protein (TSPO) which is highly upregulated in the brain in pathological conditions. Several TSPO ligands have been developed and validated, so far. Among these, PBR28 has been widely adopted for PET imaging at both preclinical and clinical levels, thanks to its high brain penetration and high selectivity. For this reason, PBR28 represents a good candidate for functionalization strategies, where this ligand could be exploited to drive selective targeting of TSPO-expressing cells. Since the PBR28 structure lacks functional moieties that could be exploited for derivatization, in this work we explored a synthetic pathway for the synthesis of a PBR28 derivative carrying an alkyne group (PBR-alkyne), enabling the fast conjugation of the ligand through azide-alkyne cycloaddition, also known as click-chemistry. As a proof of concept, we demonstrated in silico that the derivatized PBR28 ligand maintains the capability to fit into the TSPO binding pocked, and we successfully exploited PBR-alkyne to decorate zwitterionic biodegradable polymer nanoparticles (NPs) resulting in efficient internalization in cultured microglia-like cell lines.

Enantiomeric Resolution and Absolute Configuration of a Chiral δ-Lactam, Useful Intermediate for the Synthesis of Bioactive Compounds.

During the past several years, the frequency of discovery of new molecular entities based on γ- or δ-lactam scaffolds has increased continuously. Most of them are characterized by the presence of at least one chiral center. Herein, we present the preparation, isolation and the absolute configuration assignment of enantiomeric 2-(4-bromophenyl)-1-isobutyl-6-oxopiperidin-3-carboxylic acid (trans-1). For the preparation of racemic trans-1, the Castagnoli-Cushman reaction was employed. (Semi)-preparative enantioselective HPLC allowed to obtain enantiomerically pure trans-1 whose absolute configuration was assigned by X-ray diffractometry. Compound (+)-(2R,3R)-1 represents a reference compound for the configurational study of structurally related lactams.

Setup and Validation of a Reliable Docking Protocol for the Development of Neuroprotective Agents by Targeting the Sigma-1 Receptor (S1R).

Sigma-1 receptor (S1R) is a promising molecular target for the development of novel effective therapies against neurodegenerative diseases. To speed up the discovery of new S1R modulators, herein we report the development of a reliable in silico protocol suitable to predict the affinity of small molecules against S1R. The docking method was validated by comparing the computational calculated Ki values of a test set of new aryl-aminoalkyl-ketone with experimental determined binding affinity. The druggability profile of the new compounds, with particular reference to the ability to cross the blood-brain barrier (BBB) was further predicted in silico. Moreover, the selectivity over Sigma-2 receptor (S2R) and N-methyl-D-aspartate (NMDA) receptor, another protein involved in neurodegeneration, was evaluated. 1-([1,1'-biphenyl]-4-yl)-4-(piperidin-1-yl)butan-1-one (12) performed as the best compound and was further investigated for acetylcholinesterase (AchE) inhibitor activity and determination of antioxidant activity mediated by aquaporins (AQPs). With a good affinity against both S1R and NMDA receptor, good selectivity over S2R and favorable BBB penetration potential together with its AChE inhibitory activity and its ability to exert antioxidant effects through modulation of AQPs, 12 represents a viable candidate for further development as a neuroprotective agent.

Exploring the RC-106 Chemical Space: Design and Synthesis of Novel (E)-1-(3-Arylbut-2-en-1-yl)-4-(Substituted) Piperazine Derivatives as Potential Anticancer Agents.

Despite the fact that significant advances in treatment of common cancers have been achieved over the years, orphan tumors still represent an important unmet medical need. Due to their complex multifactorial origin and limited number of cases, such pathologies often have very limited treatment options and poor prognosis. In the search for new anticancer agents, our group recently identified RC-106, a Sigma receptor modulator endowed with proteasome inhibition activity. This compound showed antiproliferative activity toward different cancer cell lines, among them glioblastoma (GB) and multiple myeloma (MM), two currently unmet medical conditions. In this work, we directed our efforts toward the exploration of chemical space around RC-106 to identify new active compounds potentially useful in cancer treatment. Thanks to a combinatorial approach, we prepared 41 derivatives of the compound and evaluated their cytotoxic potential against MM and GB. Three novel potential anticancer agents have been identified.

Sigma-1 receptor antagonists: promising players in fighting neuropathic pain.

Sigma-1 receptors (S1Rs) are strongly correlated to neuropathic pain (NP), since their inactivation may decrease allodynia or dysesthesia, promoting analgesic effects. In the recent patent landscape, S1R antagonists endowed with nanomolar S1Rs affinity emerged as potent antinociceptive agents. So far, three patented compounds have been proposed for counteracting NP. Particularly PV-752 and AV1066, disclosed by the University of Pavia (Italy) and Anavex, respectively, showed good analgesic activity in preclinical studies. Moreover, E-52862 developed by Esteve (Spain) has been proved to be effective, both in preclinical and Phase II clinical trials, against several symptoms of NP. These patents ascertain S1R antagonists as potential drugs, alone or in combination with other analgesic drugs, for managing NP in humans.

New Insights into the Opening of the Occluded Ligand-Binding Pocket of Sigma1 Receptor: Binding of a Novel Bivalent RC-33 Derivative.

Significant progresses have been made to understand the molecular basis of the Sigma1 receptor (S1R) operating in normal and pathological conditions. S1R is a transmembrane protein that participates in a wide variety of processes at the central nervous system; hence, its function has been associated with mental and neurological disorders. Several ligands have been proposed to regulate the function of S1R revealing a high plasticity of the ligand-binding pocket. Previous drug-design studies have been mainly based on pharmacophore models; however, the recently revealed crystal structure of S1R provides an excellent opportunity for verifying previous predictions and for evaluating the binding of novel compounds. Interestingly, the crystal structure shows that the binding pocket of S1R is highly occluded from solvent; therefore, it is not clear how ligands access this site. In the present work, we applied steered molecular dynamics (SMD) simulations to open the occluded ligand-binding pocket in the S1R crystal structure and to determine the preferred ligand pathway to enter and exit the binding site. The intracellular surface of the ß-barrel ligand-binding region was found the most favorable route to accommodate ligands. This route supports the binding of RC-33 (our in-house-developed S1R modulator) and a new bivalent derivative that constitutes the first divalent structure shown to interact with S1R. Free energy calculations of these compounds associated with S1R agree with experimental Ki values and provide molecular insights of the binding mode of modulators that could access the S1R ligand-binding pocket through the cytoplasmic region.

Docking, Interaction Fingerprint, and Three-Dimensional Quantitative Structure-Activity Relationship (3D-QSAR) of Sigma1 Receptor Ligands, Analogs of the Neuroprotective Agent RC-33.

The human Sigma1 receptor (S1R), which has been identified as a target with an important role in neuropsychological disorders, was first crystallized 3 years ago. Since S1R structure has no relation with another previous crystallized structures, the presence of the new crystal is an important hallmark for the design of agonists and antagonists against this important target. Some years ago, our group identified RC-33, a potent and selective S1R agonist, endowed with neuroprotective properties. In this work, drawing on new structural information, we studied the interactions of RC-33 and its analogs with the S1R binding site by using computational methods such as docking, interaction fingerprints, and receptor-guided alignment three dimensional quantitative structure-activity relationship (3D-QSAR). We found that RC-33 and its analogs adopted similar orientations within S1R binding site, with high similitude with orientations of the crystallized ligands; such information was used for identifying the residues involved in chemical interactions with ligands. Furthermore, the structure-activity relationship of the studied ligands was adequately described considering classical QSAR tests. All relevant aspects of the interactions between the studied compounds and S1R were covered here, through descriptions of orientations, binding interactions, and features that influence differential affinities. In this sense, the present results could be useful in the future design of novel S1R modulators.