Benzofuran-2-Carboxamide Derivatives as Immunomodulatory Agents Blocking the CCL20-Induced Chemotaxis and Colon Cancer Growth.

The correlation between the CCL20/CCR6 axis and autoimmune and non-autoimmune disorders is widely recognized. Inhibition of the CCL20-dependent cell migration represents therefore a promising approach for the treatment of many diseases, such as inflammatory bowel diseases and colorectal cancer. We report herein our efforts to explore the biologically relevant chemical space around the benzofuran scaffold of MR120, a modulator of the CCL20/CCR6 axis previously discovered by our group. A functional screening allowed us to identify C4 and C5-substituted derivatives as the most effective inhibitors of the CCL20-induced chemotaxis of human peripheral blood mononuclear cells (PBMC). Moreover, selected compounds (16e and 24b) also proved to potently inhibit the growth of different colon cancer cell lines, with cytotoxic/cytostatic and antiproliferative activity.

Exploring the potential of human intestinal organoids: Applications, challenges, and future directions.

The complex and dynamic environment of the gastrointestinal tract shapes one of the fastest renewing tissues in the human body, the intestinal epithelium. Considering the lack of human preclinical studies, reliable models that mimic the intestinal environment are increasingly explored. Patient-derived intestinal organoids are powerful tools that recapitulate in vitro many pathophysiological features of the human intestine. In this review, the possible applications of human intestinal organoids in different research fields are highlighted. From physiologically relevant to intestinal disease modeling, regenerative medicine, and toxicology studies, the potential of intestinal organoids will be here presented and discussed. Despite the remarkable opportunities offered, limitations related to ethical concerns, tissue collection, reproducibility, and methodologies may hinder the full exploitation of this cell-based model into high throughput studies and clinical practice. Currently, distinct approaches can be used to overcome the numerous challenges found along the way and to allow the full implementation of this ground-breaking technology.

Development of inhalation powders containing lactic acid bacteria with antimicrobial activity against Pseudomonas aeruginosa.

OBJECTIVES: The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. METHODS: The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. RESULTS: Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 107 colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 106 colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. CONCLUSION: This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.

An Enhanced Dissolving Cyclosporin-A Inhalable Powder Efficiently Reduces SARS-CoV-2 Infection In Vitro.

This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.

Pharmacological characterization of second generation FXR agonists as effective EphA2 antagonists: A successful application of target hopping approach.

It is well demonstrated the key role of Eph-ephrin system, specifically of EphA2 receptor, in supporting tumor growth, invasion, metastasis and neovascularization. We previously identified FXR agonists as eligible antagonists of Eph-ephrin system. Herein we characterize new commercially available FXR (Farnesoid X Receptor) agonists as potential Eph ligands including Cilofexor, Nidufexor, Tropifexor, Turofexorate isopropyl and Vonafexor. Our exploration based on molecular modelling investigations and binding assays shows that Cilofexor binds specifically and reversibly to EphA2 receptor with a Ki value in the low micromolar range. Furthermore, Cilofexor interferes with the phosphorylation of EphA2 and the cell retraction and rounding in PC3 prostate cancer cells, both events depending on EphA2 activation. In conclusion, we can confirm that target hopping can be a successful approach to discover new moiety of protein-protein inhibitors.

Probing the effects of MR120 in preclinical chronic colitis: A first-in-class anti-IBD agent targeting the CCL20/CCR6 axis.

Concerning the growing interest in the role played by the CCL20/CCR6 axis in IBD pathogenesis and in the search for novel anti-IBD small molecules, we have recently discovered the first small-molecule (MR120) endowed with protective action against TNBS-induced colitis and zymosan-induced peritonitis. This protective action occurs through interference with the CCL20/CCR6 signaling. The aim of the present work is to expand the preclinical investigation of MR120, evaluating its beneficial anti-inflammatory effect on a model of chronic colitis obtained by cyclically exposing C57BL/6 mice to 3% DSS. Subcutaneous administration of MR120 at 1 mg/kg, the same dose effective against acute inflammation, helped attenuate several systemic and local inflammatory responses induced by DSS. Besides significantly improving murine health conditions, MR120 counteracted mucosal macroscopic injury, the increase of colonic edema and neutrophils oxidative activity, and mitigated spleen enlargement, while not significantly lowering intestinal IL-6 concentration. Overall, repeated daily treatment with MR120 for approximately 30 days was well tolerated and showed moderate protection in a relevant model of chronic colitis, in line with the beneficial effect previously observed in acute models of intestinal inflammation. Although more potent analogues of MR120 will be needed to more fully evaluate their clinical translatability, the present work provides a valuable example of in vivo efficacy of CCL20/CCR6 modulators in a chronic model of IBD.

Discovery of small-molecules targeting the CCL20/CCR6 axis as first-in-class inhibitors for inflammatory bowel diseases.

The CCL20/CCR6 axis is implicated in the migration of CCR6+ immune cells towards CCL20, its sole ligand, whose expression is increased during inflammatory processes and is known to play a pivotal role in triggering different autoimmune-mediated inflammatory diseases. Herein, we report a drug discovery effort focused on the development of a new pharmacological approach for the treatment of inflammatory bowel diseases (IBDs) based on small-molecule CCR6 antagonists. The most promising compound 1b was identified by combining in silico studies, sustainable chemistry and in vitro functional/targeted assays, and its efficacy was finally validated in a classic murine model of colitis (TNBS-induced) and in a model of peritonitis (zymosan-induced). These data provide the proof of principle that a pharmacological modulation of the CCL20/CCR6 axis may indeed represent the first step for the development of an orally bioavailable drug candidate for the treatment of IBD and, potentially, other diseases regulated by the CCL20/CCR6 axis.

Bithiazole Inhibitors of Phosphatidylinositol 4-Kinase (PI4KIIIß) as Broad-Spectrum Antivirals Blocking the Replication of SARS-CoV-2, Zika Virus, and Human Rhinoviruses.

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIß, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIß block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIß inhibition, the role of PI4KIIIß in SARS-CoV-2 entry/replication is debated.

UniPR1331: Small Eph/Ephrin Antagonist Beneficial in Intestinal Inflammation by Interfering with Type-B Signaling.

Eph receptors, comprising A and B classes, interact with cell-bound ephrins generating bidirectional signaling. Although mainly related to carcinogenesis and organogenesis, the role of Eph/ephrin system in inflammation is growingly acknowledged. Recently, we showed that EphA/ephrin-A proteins can modulate the acute inflammatory responses induced by mesenteric ischemia/reperfusion, while beneficial effects were granted by EphB4, acting as EphB/ephrin-B antagonist, in a murine model of Crohn's disease (CD). Accordingly, we now aim to evaluate the effects of UniPR1331, a pan-Eph/ephrin antagonist, in TNBS-induced colitis and to ascertain whether UniPR1331 effects can be attributed to A- or B-type signaling interference. The potential anti-inflammatory action of UniPR1331 was compared to those of the recombinant proteins EphA2, a purported EphA/ephrin-A antagonist, and of ephrin-A1-Fc and EphA2-Fc, supposedly activating forward and reverse EphA/ephrin-A signaling, in murine TNBS-induced colitis and in stimulated cultured mononuclear splenocytes. UniPR1331 antagonized the inflammatory responses both in vivo, mimicking EphB4 protection, and in vitro; EphA/ephrin-A proteins were inactive or only weakly effective. Our findings represent a further proof-of-concept that blockade of EphB/ephrin-B signaling is a promising pharmacological strategy for CD management and highlight UniPR1331 as a novel drug candidate, seemingly working through the modulation of immune responses.

Impact of S1P Mimetics on Mesenteric Ischemia/Reperfusion Injury.

Mesenteric ischemia/reperfusion (I/R), following the transient deprivation of blood flow to the gut, triggers an acute flogistic process involving the disruption of endothelial and epithelial barriers integrity, the activation of immune cells, and the abundant release of inflammatory mediators. Among them, the lipid mediator sphingosine-1-phosphate (S1P) is involved in maintaining epithelial and endothelial barrier integrity and in governing the migration of immune cells through the interaction with S1P1-5 receptors. Therefore, the present work aims to investigate the involvement of S1P signaling in intestinal I/R-induced injury by studying the effects of FTY720, the non-selective S1P1,3-5 agonist, and comparing them with the responses to ozanimod, selective S1P1,5 agonist, in a murine model of gut I/R. Intestinal edema, gut and lung neutrophil infiltration, and oxidative stress were evaluated through biochemical and morphological assays. The collected results highlight the protective action of FTY720 against the inflammatory cascade elicited by mesenteric I/R injury, mainly through the control of vascular barrier integrity. While these beneficial effects were mimicked by ozanimod and can be therefore attributed largely to the effects exerted by FTY720 on S1P1, the recruitment of myeloid cells to the injured areas, limited by FTY720 but not by ozanimod, rather suggests the involvement of other receptor subtypes.

Novel analgesic agents obtained by molecular hybridization of orthosteric and allosteric ligands.

Despite the high incidence of acute and chronic pain in the general population, the efficacy of currently available medications is unsatisfactory. Insufficient management of pain has a profound impact on the quality of life and can have serious physical, psychological, social, and economic consequences. This unmet need reflects a failure to develop novel classes of analgesic drugs with superior clinical properties and lower risk of abuse. Nevertheless, recent advances in our understanding of the neurobiology of pain are offering new opportunities for developing different therapeutic approaches. Among those, the activation of M2 muscarinic acetylcholine receptors, which play a key role in the cholinergic regulation of the nociceptive transmission, constitutes one of the most promising strategies. We have recently developed a small library of novel pharmacological agents by merging the structures of known orthosteric and allosteric muscarinic ligands through their molecular hybridization, an emerging approach in medicinal chemistry based on the combination of pharmacophoric moieties of different bioactive substances to produce a new compound with improved pharmacological properties. Herein we report the functional characterization of the new ligands in vitro and the assessment of their efficacy as analgesic agents and tolerability in mice. This work provides new insights for the development and optimization of novel muscarinic hybrid compounds for the management of pain.

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach †.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIß and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.

Targeting the Eph/Ephrin System as Anti-Inflammatory Strategy in IBD.

Besides their long-known critical role in embryonic growth and in cancer development and progression, erythropoietin-producing hepatocellular carcinoma type B (EphB) receptor tyrosine kinases and their ephrin-B ligands are involved in the modulation of immune responses and in remodeling and maintaining the integrity of the intestinal epithelial layer. These processes are critically involved in the pathogenesis of inflammatory-based disorders of the gut, like inflammatory bowel diseases (IBDs). Accordingly, our aim was to investigate the role of the EphB/ephrin-B system in intestinal inflammation by assessing the local and systemic effects produced by its pharmacological manipulation in 2,4,6-trinitrobenzenesulfonic acid (TNBS)- (Th1-dependent model) and dextran sulphate sodium (DSS)- (innate response model) induced colitis in mice. To this purpose, we administered chimeric Fc-conjugated proteins, allegedly able to uni-directionally activate either forward (ephrin-B1-Fc) or reverse (EphB1-Fc) signaling, and the soluble monomeric EphB4 extracellular domain protein, that, simultaneously interfering with both signaling pathways, acts as EphB/ephrin-B antagonist.The blockade of the EphB/ephrin-B forward signaling by EphB4 and EphB1-Fc was ineffective against DSS-induced colitis while it evoked remarkable beneficial effects against TNBS colitis: it counteracted all the evaluated inflammatory responses and the changes elicited on splenic T lymphocytes subpopulations, without preventing the appearance of a splice variant of ephrin-B2 gene elicited by the haptenating agent in the colon. Interestingly, EphB4, preferentially displacing EphB4/ephrin-B2 interaction over EphB1/ephrin-B1 binding, was able to promote Tumor Necrosis Factor alpha (TNFα) release by splenic mononuclear cells in vitro. On the whole, the collected results point to a potential role of the EphB/ephrin-B system as a pharmacological target in intestinal inflammatory disorders and suggest that the therapeutic efficacy of its blockade seemingly works through the modulation of immune responses, independent of the changes at the transcriptional and translational level of EphB4 and ephrin-B2 genes.

Bifidobacterium bifidum PRL2010 alleviates intestinal ischemia/reperfusion injury.

Mesenteric ischemia/reperfusion is a clinical emergency with high morbidity and mortality due to the transient reduction of blood supply to the bowel. In recent years, the critical contribution of gut microbiome to human health and proper gastrointestinal functions has gradually emerged. In the current study, we investigated the protective effects of five days supplementation with Bifidobacterium bifidum PRL2010 in a murine model of gut ischemia/reperfusion. Our findings indicate that animals pretreated with B. bifidum PRL2010 showed lower neutrophil recruitment in the lungs, remarkably reduced bacterial translocation and decreased transcription levels of TNFalpha and IL-10 both in liver and kidneys, at the same time increasing those of IL-12 in kidneys. Inhibiting the adhesion of pathogenic bacteria and boosting host innate immunity responses are among the possible protective mechanisms enacted by the probiotic. These results demonstrate that short-period treatment with B. bifidum PRL2010 is a potential strategy to dampen remote organ injury due to mesenteric ischemia/reperfusion.

Mesenteric ischemia-reperfusion: an overview of preclinical drug strategies.

Mesenteric ischemia is a surgical emergency caused by a transient reduction in blood perfusion to the bowel. Despite accounting for only 0.1% of hospital admissions and 1-2% of gastrointestinal diseases, its elusive symptoms often lead to dramatically high morbidity and mortality rates. The complex cascade of inflammatory events and mediators triggered by mesenteric ischemia-reperfusion (I/R) accounts for the plethora of proposed pharmacological targets and for the current lack of an efficacious drug strategy for its management. It is hoped that a deeper understanding of its pathogenesis and the preclinical therapeutic strategies identified to date and described herein will improve the translation into the clinical setting of the pharmacological armamentarium against a life-threatening disorder that is currently mainly managed surgically.

α7 Nicotinic Agonist AR-R17779 Protects Mice against 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis in a Spleen-Dependent Way.

The existence of a cholinergic anti-inflammatory pathway negatively modulating the inflammatory and immune responses in various clinical conditions and experimental models has long been postulated. In particular, the protective involvement of the vagus nerve and of nicotinic Ach receptors (nAChRs) has been proposed in intestinal inflammation and repeatedly investigated in DSS- and TNBS-induced colitis. However, the role of α7 nAChRs stimulation is still controversial and the potential contribution of α4ß2 nAChRs has never been explored in this experimental condition. Our aims were therefore to pharmacologically investigate the role played by both α7 and α4ß2 nAChRs in the modulation of the local and systemic inflammatory responses activated in TNBS-induced colitis in mice and to assess the involvement of the spleen in nicotinic responses. To this end, TNBS-exposed mice were sub-acutely treated with various subcutaneous doses of highly selective agonists (AR-R17779 and TC-2403) and antagonists (methyllycaconitine and dihydro-ß-erythroidine) of α7 and α4ß2 nAChRs, respectively, or with sulfasalazine 50 mg/kg per os and clinical and inflammatory responses were evaluated by means of biochemical, histological and flow cytometry assays. α4ß2 ligands evoked weak and contradictory effects, while α7 nAChR agonist AR-R17779 emerged as the most beneficial treatment, able to attenuate several local markers of colitis severity and to revert the rise in splenic T-cells and in colonic inflammatory cytokines levels induced by haptenization. After splenectomy, AR-R17779 lost its protective effects, demonstrating for the first time that, in TNBS-model of experimental colitis, the anti-inflammatory effect of exogenous α7 nAChR stimulation is strictly spleen-dependent. Our findings showed that the selective α7 nAChRs agonist AR-R17779 exerted beneficial effects in a model of intestinal inflammation characterized by activation of the adaptive immune system and that the spleen is essential to mediate this cholinergic protection.

Protection by the Eph-Ephrin System Against Mesenteric Ischemia-Reperfusion Injury.

Mesenteric ischemia-reperfusion (I/R)-induced injury targets primarily endothelial and epithelial cells, leading to a cascade of inflammatory events, eventually culminating in life-threatening syndromes. Hitherto, the role of Eph, the largest family of tyrosine kinase receptors, and of their cell-bound ephrin ligands, whose interaction generates a bidirectional signaling, is still debated in I/R injury. The aim of the present work was therefore to investigate the effects produced by unidirectional activation of forward signaling (administration of chimeric protein ephrinA1-Fc), of reverse signaling (EphA2-Fc), or inhibition of both signals (monomeric EphA2 and the protein-protein interaction inhibitor UniPR1331) on the local and systemic inflammatory responses triggered by mesenteric I/R in mice.When administered at 200 µg/kg i.v., ephrin-A1-Fc prevented intestinal and lung I/R-induced injury, decreasing in the pulmonary district leukocytes recruitment, IL-1ß and TNFα levels, and EphA2 overexpression by mesenteric I/R. Blockade of Eph-ephrin signaling by equimolar EphA2 efficiently antagonized I/R-induced gut edema formation, an effect shared also by UniPR1331, mitigated lung mucosal injury, and counteracted the increase in pro-inflammatory cytokines levels. EphA2-Fc 180 µg/kg or equimolar Fc alone did not significantly modify the inflammatory responses to I/R.Our data suggest that the Eph-ephrin system is directly involved in the development of the acute inflammatory process activated in the gut by hypoxia-reoxygenation and in its amplification to distant organs, revealing that a fine pharmacological tuning of this signaling pathway may represent an attractive strategy to contain the I/R-induced inflammatory cascade.

Elucidating the gut microbiome of ulcerative colitis: bifidobacteria as novel microbial biomarkers.

Ulcerative colitis (UC) is associated with a substantial alteration of specific gut commensals, some of which may be involved in microbiota-mediated protection. In this study, microbiota cataloging of UC patients by 16S rRNA microbial profiling revealed a marked reduction of bifidobacteria, in particular the Bifidobacterium bifidum species, thus suggesting that this taxon plays a biological role in the aetiology of UC. We investigated this further through an in vivo trial by testing the effects of oral treatment with B. bifidum PRL2010 in a wild-type murine colitis model. TNBS-treated mice receiving 10(9) cells of B. bifidum PRL2010 showed a marked reduction of all colitis-associated histological indices as well as maintenance of mucosal integrity as it was shown by the increase in the expression of many tight junction-encoding genes. The protective role of B. bifidum PRL2010, as well as its sortase-dependent pili, appears to be established through the induction of an innate immune response of the host. These results highlight the importance of B. bifidum as a microbial biomarker for UC, revealing its role in protection against experimentally induced colitis.

Dual Role of Endogenous Serotonin in 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis.

BACKGROUND AND AIMS: Changes in gut serotonin (5-HT) content have been described in Inflammatory Bowel Disease (IBD) and in different experimental models of colitis: the critical role of this monoamine in the pathogenesis of chronic gastrointestinal inflammation is gradually emerging. Aim of the present study was to evaluate the contribution of endogenous 5-HT through the activation of its specific receptor subtypes to the local and systemic inflammatory responses in an experimental model of IBD. MATERIALS AND METHODS: Colitis was induced by intrarectal 2,4,6-TriNitroBenzene Sulfonic acid in mice subacutely treated with selective antagonists of 5-HT1A (WAY100135), 5-HT2A (Ketanserin), 5-HT3 (Ondansetron), 5-HT4 (GR125487), 5-HT7 (SB269970) receptors and with 5-HT1A agonist 8-Hydroxy-2-(di-n-propylamino)tetralin. RESULTS: Blockade of 5-HT1A receptors worsened TNBS-induced local and systemic neutrophil recruitment while 5-HT1A agonist delayed and mitigated the severity of colitis, counteracting the increase in colonic 5-HT content. On the contrary, blockade of 5-HT2A receptors improved global health conditions, reduced colonic morphological alterations, down-regulated neutrophil recruitment, inflammatory cytokines levels and colonic apoptosis. Antagonism of 5-HT3, 5-HT4, and 5-HT7 receptor sites did not remarkably affect the progression and outcome of the pathology or only slightly improved it. CONCLUSION: The prevailing deleterious contribution given by endogenous 5-HT to inflammation in TNBS-induced colitis is seemingly mediated by 5-HT2A and, to a lesser extent, by 5-HT4 receptors and coexists with the weak beneficial effect elicited by 5-HT1A stimulation. These findings suggest how only a selective interference with 5-HT pro-inflammatory actions may represent an additional potential therapeutic option for intestinal inflammatory disorders.

Novel Potent N-Methyl-d-aspartate (NMDA) Receptor Antagonists or σ1 Receptor Ligands Based on Properly Substituted 1,4-Dioxane Ring.

Two series of 1,4-dioxanes (4-11 and 12-19) were rationally designed and prepared to interact either with the phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptor or with σ1 receptors, respectively. The biological profiles of the novel compounds were assessed using radioligand binding assays, and the compounds with the highest affinities were investigated for their functional activity. The results were in line with the available pharmacophore models and highlighted that the 1,4-dioxane scaffold is compatible with potent antagonist activity at NMDA receptor or high affinity for σ1 receptors. The primary amines 6b and 7 bearing a cyclohexyl and a phenyl ring or two phenyl rings in position 6, respectively, were the most potent noncompetitive antagonists at the NMDA receptor with IC50 values similar to those of the dissociative anesthetic (S)-(+)-ketamine. The 5,5-diphenyl substitution associated with a benzylaminomethyl moiety in position 2, as in 18, favored the interaction with σ1 receptors.