Synthesis, Cytotoxicity Evaluation, and Computational Insights of Novel 1,4-Diazepane-Based Sigma Ligands.

Letter Cite This: ACS Med. Chem. Lett. 2020, 11, 651−656 pubs.acs.org/acsmedchemlett Synthesis, Cytotoxicity Evaluation, and Computational Insights of Novel 1,4-Diazepane-Based Sigma Ligands Daniele Zampieri,*,† Sara Fortuna,† Antonella Calabretti,† Maurizio Romano,‡ Renzo Menegazzi,‡ Dirk Schepmann,§ Bernhard Wünsch,§ and Maria Grazia Mamolo† † Department of Chemical and Pharmaceutical Sciences, P.le Europa 1-Via Giorgieri 1, University of Trieste, 34127 Trieste, Italy Department of Life Sciences, Via Valerio 28/1, University of Trieste, 34127 Trieste, Italy § Institute of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149 Münster, Germany ‡ S Supporting Information * ABSTRACT: Among several potential applications, sigma receptor ligands can be used as antipsychotics, antiamnesics, and against other neurodegenerative disorders as well as neuroprotective agents. We present herein a new series of diazepane-containing derivatives as σR ligands obtained by a conformational expansion approach of our previously synthesized piperidine-based compounds. The best results were reached by benzofurane 2c, 3c and quinoline 2d, 3dsubstituted diazepane derivatives, which showed the highest σR affinity. The cytotoxic activities of synthesized compounds were evaluated against two cancer cell lines, and the results indicated that none of the compounds induced significant toxicity in these cells. We also evaluated the antioxidant activity by radical scavenging capacity of our best compounds on ABTS and H2O2. The results obtained reveal that our new derivatives possess an excellent antioxidant profile and could be protective for the cells. Overall, the benzofurane derivative 2c due to its strong interaction with the active site of the receptor, as confirmed by molecular dynamic simulations, emerged as the optimum compound with high σ1R affinity, low cytotoxicity, and a potent antioxidant activity. KEYWORDS: Sigma receptor, molecular dynamics, binding studies, cytotoxicity, antioxidant activity T protein-97 (TMEM97),19 an endoplasmic-reticulum-resident transmembrane molecule implicated in cholesterol homeostasis due to its association with the lysosomal transporter NPC1.20,21 The σ2R crystal structure is still elusive, but several pharmacophore models have been proposed.22−25 The σ2Rs are overexpressed in many cancer cell lines including lung cancer,26,27 breast cancer,28 ovarian cancer,29 glioma cancer,30 and gastric cancer.31 In this context, since σ2R agonists can induce tumor cell death, they have been proposed as potential antitumor drugs. On the other hand, the σ2Rs are widely expressed in cerebellum, red nucleus, and substantia nigra and are a potential target for the treatment of movement disorders and of neuroleptic-induced acute dystonia.32 In addition, σ1R antagonists as well as σ2R agonists can modulate neuropathic pain.33,34 In the past decade, our group has synthesized and biologically evaluated an extensive series of compounds both he sigma receptors (σR) are a class of proteins initially classified, by Martin and co-workers,1 as a subtype of the opiate receptors. Further studies revealed them to be a different receptor class comprising two distinct subtypes: σ1 and σ2.2−5 The σ1R is a chaperone protein, cloned in 1996 from several tissues including human, consisting of 223 amino acids6,7 with a MW of 25.3 kDa.8 Crystallized 20 years later, it revealed a trimeric protein organization.9 The σ1R subtype is primarily localized to mitochondria-associated ER membranes (MAM) of neuronal and peripheral cells, such as cardiac myocytes and hepatocytes. This receptor can also translocate to the plasma membrane or ER-membrane and regulate the activity of other proteins by modulating different ionic channels via an IP3-indipendent mechanism.10,11 The σ1Rs have neuroprotective and antiamnesic activity12,13 and modulate opioid analgesia14 as well as drug addiction,15 and their antagonists seem to be effective against the negative manifestations of schizophrenia without producing extrapyramidal side effects.16,17 In addition, several studies suggest a role for σ1R in tumor biology, since its expression increased in some cancers.18 After 40 years from the discovery of σRs,1 in 2017, the σ2R subtype has been purified and identified as transmembrane © 2019 American Chemical Society Special Issue: In Memory of Maurizio Botta: His Vision of Medicinal Chemistry Received: November 13, 2019 Accepted: December 16, 2019 Published: December 16, 2019 651 DOI: 10.1021/acsmedchemlett.9b00524 ACS Med. Chem. Lett. 2020, 11, 651−656 ACS Medicinal Chemistry Letters Letter of preferential affinity for σ1R and σ2R subtypes. Following up our studies in this field, we report herein the development of a new class of sigma ligands designed through the expansion of the conformational selection paradigm applied to our previously synthesized piperidine-based σ1R ligands 1 (Figure 1).35 and NaCNBH3, to give the final subseries 3a−g. These compounds were purified by DCVC technique. The σ1R and σ2R affinities of the test compounds were determined in competition experiments by radiometric assays, using [3H]-(+)-Pentazocine as radioligand for the σ1R assay and [3H]-DTG (di-o-tolylguanidine) as radioligand in the σ2R assay. Compounds 2a−g and 3a−g were tested against σ1R and σ2R of animal origin, prepared from guinea pig brain and rat liver membranes by homogenization, centrifugation, and washing of the respective tissues. We also performed a competition experiment toward GluN2b subunit containing NMDA receptors in a radioligand binding assay. This receptor subtype plays important roles in synaptic transmission and plasticity, learning, memory, and other physiological and pathological processes.37,38 Hence, antagonists of the GluN2b subunit are of interest as neuroprotective drugs for various CNS disorders. The radioligand used in the competition assay was [3H]-labeled Ifenprodil, a prototypical allosteric inhibitor of the GluN2b subunit (Supporting Information). For compounds with affinity value higher than 100 nM, only one measure was performed. The σ1R, σ2R, and GluN2b affinities of compounds 2a−g and 3a−g are presented in Table 1. From the obtained data we can summarize the following: (i) the bulky diazepane spacer retained, or even improved, the σR affinity to both σ1 and σ2, with respect to the piperidine ring; (ii) only bicycle derivatives displayed moderate to high affinity toward both σR subtypes, while the corresponding monocycle analogues were weak inhibitors or avoiding of σR affinity; (iii) the best results against σ1R were reached by benzofurane derivative 2c, while its 2,4-dimethyl substituted analogue 3c gave the best pan-affinity with Ki values of 8.0 and 28 nM toward both σR subtypes and also the best GluN2b inhibition value of 59 nM; (iv) the 2,4-dimethyl substitution on benzyl moiety derivatives improves the σ2 over σ1 affinity of the bicycle derivatives, as well as the affinity toward the GluN2b subunit rec (...truncated)