As aforementioned this study aimed to

As aforementioned, this study aimed to design, synthesize and investigate aromatase inhibitory and anti-breast cancer activities of N,N′-disubstituted thiourea derivatives. Based on the attractive phenotypic molecule 1, the synthetic N,N′-disubstituted thioureas have been designed including monothiourea type I (4), para-bisthiourea type II (5) and meta-bisthiourea type III (6) (Fig. 1). In addition, a molecular docking was carried out to reveal possible binding modes and the crucial protein-ligand interactions toward the aromatase.
Results and discussion
Conclusions Twenty-three thiourea derivatives (4–6) have been achieved by a simple one step synthesis using commercially available and inexpensive starting materials. Eight thiourea derivatives displayed aromatase inhibitory activity. In particular, the meta-bisthiourea bearing NO2 (6f) and 3,5-diCF3 (6h) were shown to be the promising potent agents with IC50 values of 0.8 and 0.6 μM, respectively. The molecular docking revealed that one of the thiourea moieties of these two compounds (6f and 6h) could form hydrophobic interactions with the enzyme residues (Val370, Leu477, Thr310, and Phe221 for 6f, Val370, Leu477, Ser478, and Ile133 for 6h) to mimic steroidal backbone of the ASD substrate, whereas another substituted thiourea moiety interacted with the enzyme via the formation of π-π interaction with Phe221 (6h) and cation-π interaction with Fe3+ (6f). H-bonding interactions with the enzyme residues (Gln218, Thr310, and Ser478) were only observed for the nitro derivative 6f. The present work provides bisthiourea as a novel skeleton with potentially useful for the development of aromatase inhibitors.
Experimental section
Introduction Aromatase is a limiting enzyme responsible for a key step in the biosynthesis of endogenous estrogen (Lephart, 1996) and thus is involved in various reproductive functions, including reproductive behaviors and development (Britt et al., 2000, Vosges et al., 2010). While the role of aromatase in reproductive functions is well established, recent studies have shown that this enzyme is involved in a number of CNS non-reproductive functions, including nociceptive processing. The suppression of aromatase activity has been shown to reduce nociceptive behaviors in tgx (Evrard and Balthazart, 2004), rats (Ghorbanpoor et al., 2014) and mice (O'Brien et al., 2015), suggesting that aromatase plays a role in nociceptive processing. Moreover, the expression of aromatase has been shown to be increased within astrocytes in a variety of animal pain models, including a model of cancer-induced pain (O'Brien et al., 2015) and spinal cord injury-induced pain (Ghorbanpoor et al., 2014). While mounting evidences suggest that aromatase is involved in modulating nociception, the mechanisms involved in this regulation in both acute and chronic pain conditions are currently poorly understood. The activity of aromatase is reported to be modulated through rapid post-translational modifications, including phosphorylation (Charlier et al., 2013). Thus, brain aromatase activity is rapidly decreased by PKC-, PKA-, and CAMK-controlled Ca2+-dependent phosphorylation in the presence of ATP/Mg2+/Ca2+ (Balthazart et al., 2003, Balthazart et al., 2005). However, in the presence of acid protein phosphatase or a protein kinase inhibitor, aromatase activity is increased (Balthazart et al., 2005). Further studies have revealed that mutation of specific aromatase amino acids, particularly serine residues, blocked the phosphorylation of aromatase and led to increased aromatase activity (Evrard, 2006). Collectively, these studies demonstrate that a reduction in aromatase phosphorylation represents a critical mechanism in the rapid regulation of aromatase activation, and raises the possibility that dephosphorylation of aromatase may contribute to its role in acute nociception. The sigma-1 receptor is a unique ligand-operated molecular receptor which implicated in a myriad of biological processes. As a protein which resides in the mitochondria-associated endoplasmic reticulum membrane (MAM), sigma-1 receptors serve as molecular chaperones and correct the conformation of the IP3 receptor type 3 in order to sustain proper Ca2+ signaling (Hayashi and Su, 2007). Moreover, under stimulation by agonists or stressors, the sigma-1 receptor is translocated from MAM to plasma membrane and interacts with various ion channel, receptors, and kinases (Su et al., 2010, Chu and Ruoho, 2016). In the CNS, sigma-1 receptors have been reported to be predominantly localized within spinal astrocytes in mice (Moon et al., 2014) and activated sigma-1 receptors potentiate the intracellular Ca2+ concentration through NMDA receptor-mediated Ca2+ influx (Monnet et al., 2003). The sigma-1 receptors further activate Ca2+-dependent kinases and phosphatases, resulting in the modulation of the phosphorylation status of various intracellular enzymes and signaling molecules (Roh et al., 2011, Xu et al., 2015). Our laboratories have previously demonstrated that sigma-1 receptors act as important trigger receptors in initiating chronic neuropathic pain via calcineurin-dependent dephosphorylation of nNOS (Roh et al., 2011). Since the change in the phosphorylation status of aromatase has been reported to be affected by an intracellular Ca2+-dependent pathway (Balthazart et al., 2003), these observations suggest that activation of sigma-1 receptors may also induce the dephosphorylation of aromatase, resulting in the rapid activation of aromatase.