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Published in final edited form as: Med Chem Res. 2020 Jul 8;29(9):1697–1706. doi: 10.1007/s00044-020-02597-2

Exploring 1-adamantanamine as an alternative amine moiety for metabolically labile azepane ring in newly synthesized benzo[d]thiazol-2(3H)one σ receptor ligands

Sebastiano Intagliata 1,2, Hebaalla Agha 1, Theresa A Kopajtic 3, Jonathan L Katz 3, Shyam H Kamble 4, Abhisheak Sharma 4, Bonnie A Avery 4, Christopher R McCurdy 1,2
PMCID: PMC7880169  NIHMSID: NIHMS1610592  PMID: 33584084

Abstract

In this work we report the structure-activity relationships, binding properties, and metabolic stability studies of a series of benzo[d]thiazol-2(3H)one as sigma receptors (σRs) ligands. Specifically, to improve the metabolic stability of the cyclic amine fragment of our lead compound (SN56), the metabolically unstable azepane ring was replaced with a 1-adatamantamine moiety. Within the synthesized analogs, compound 12 had low nanomolar affinity for the σ1R (Ki = 7.2 nM) and moderate preference (61-fold) over the σ2R. In vitro metabolic stability studies showed a slight improvement of the metabolic stability for 7-12, even though an extensive metabolism in rat liver microsomes is being observed. Furthermore, metabolic soft spot identification of 12 suggested that the N-methyl group of the adamantyl moiety is a major site of metabolism.

Keywords: Sigma receptors, Sigma receptors ligands, 1-Adamantamine, Benzo[d]thiazol-2(3H)one, Synthesis, Binding properties, Metabolic stability, Soft spot identification

Introduction

The sigma receptors (σRs) were discovered in 1976 by Martin and coworkers (Martin et al. 1976). Initially they were classified as “sigma opioid receptors” based on the actions of (±)-SKF-10,047, a κ-opioid receptor partial agonist (Hayashi and Su 2005). Subsequently, further studies using enatiomerically pure ligands gave the first evidence that the binding protein was not a subtype of opioid receptors (Hayashi and Su 2005; Su 1982). In contrast with the past, σRs are now confirmed to be a unique class of receptors that modulate different proteins, including N-methyl-D-aspartate (NMDA) receptor, and Ca2+ ion channels (Monnet et al. 1990; Cheng et al. 2008). σRs are classified in two receptor subtypes named σ1 and σ2, which differ in molecular size, amino acid sequence, tissue distribution, binding properties, and pharmacological profile (Walker et al. 1990). Both subtypes can be found in the central and peripheral nervous systems (Hellewell and Bowen 1990; Quirion et al. 1992). In particular, σ1Rs are widely distributed in the brain, spinal cord and peripheral nerve (Bangaru et al. 2013; Mavlyutov et al. 2016). Many preclinical in vivo studies suggested that σ1R could be useful in different therapeutic fields such as depression, anxiety, schizophrenia, neuronal protection, analgesia, and treatment for drug abuse (Cobos et al. 2008; Cirino et al. 2019; Obeng et al. 2020; Patel et al. 2020; Xiao et al. 2020). The σ1R has been cloned from different tissues and species (Guitart et al. 2004), and its crystal structure has been released (Schmidt et al. 2016). The σ2R seems to be involved in different physiological functions including inflammation, cellular metabolism, and modulation of the intracellular Ca2+ concentrations (Vilner and Bowen 2000; Cassano et al. 2009). Moreover, σ2R has been associated with tumor cell proliferation and apoptosis driving the attention to this protein as “druggable” target for cancer treatment (Georgiadis et al. 2017; Sun et al. 2018; Nicholson et al. 2018). Most recently, a series of benzimidazolone-based selective σ2R ligands have been reported to attenuate cocaine-induced convulsions in rodents, suggesting new potential pharmacological tools to treat cocaine toxicity (Intagliata et al. 2019). Finally, the σ2R was cloned and identified as TMEM97, a transmembrane protein involved in cholesterol trafficking and homeostasis (Alon et al. 2017).

The benzo[d]thiazol-2(3H)one heterocycle is a widely used scaffold in drug discovery, and several examples of benzothiazolone-based ligands have been reported in the literature (Poupaert et al. 2005; Romeo et al. 2019). These ligands are able to bind several CNS targets, such as dopaminergic, serotoninergic, and σRs (Wu et al. 2005; Salerno et al. 2014; Ucar et al. 1997). The benzo[d]thiazol-2(3H)one derivative, compound SN56 (Fig. 1), was reported by our research group to have high affinity and selectivity for the σ1Rs (Yous et al. 2005). Moreover, the fluorine-18 radiolabelled analog of SN56 ([18F]FTC-146) is currently under phase I clinical trials as a diagnostic agent to use in positron emission tomography (PET) (James et al. 2012; Avery et al. 2017; Shen et al. 2017a; Shen et al. 2017b; Hjornevik et al. 2017).

Fig. 1.

Fig. 1

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Structure of 3-[2-(azepan-1-yl)ethyl]-6-propylbenzo[d]thiazol-2(3H)-one (SN56), (+)-cis-(2-{[1-adamantyl(methyl)amino]-methyl}−1-phenylcyclopropyl)methanol (A), and general structure of new benzo[d]thiazol-2(3H)one derivatives

Unfortunately, SN56 suffers from a poor metabolic stability profile which includes short in vitro half-life (t1/2 < 1 min) in rat liver microsomes (RLMs). Despite the exact metabolic soft spots of SN56 are unknown, it is likely that the metabolically unprotected azepane moiety significantly contribute to the poor metabolic stability. Cytochromes P450 (CYP) are commonly involved in the metabolism of cyclic tertiary amines by catalyzing the oxidation of the carbon atom in α-position to the nitrogen and resulting in the formation of chemically unstable intermediates that lead to ring cleavage (Fig. 2) (Kalgutkar et al. 2002). Therefore, in an attempt to improve the metabolic stability of SN56, in this work we described the synthesis, structure-activity relationship (SAR), and in vitro metabolic stability studies of a new series of benzo[d]thiazol-2(3H)one derivatives (7-12, Fig. 1) bearing the 1-adamantamine moiety as cycloalkanamine portion (Fig 1). The 1-adamantamine group was selected due to its multifaceted value in drug design. In particular, the steric hindrance of the adamantyl group might result in limited access to the metabolic enzymes at the site of metabolism, thus improving the metabolic stability of the new compounds (Liu et al. 2011; Lamoureux and Artavia 2010). Moreover, the 1-adamantamine moiety has already been used for the development of the high affinity σRs ligand A (Fig. 2), providing the evidence that 1-adamantanamine is able to bind effectively with the σRs binding site (Marrazzo et al. 2002).

Fig. 2.

Fig. 2

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Proposed metabolic pathway of the cycloalkanamine moiety for compound SN56

Materials and methods

Chemistry

Reagents and starting materials were obtained from commercial suppliers and were used without purification. Precoated silica gel 60 F254 aluminum backed plates from Merck were used for thin-layer chromatography (TLC). Column chromatography was performed on silica gel 60 (Sorbent Technologies). 1H and 13C NMR spectra were obtained on a Bruker APX400 at 400 and 100 MHz, respectively, in deuterated chloroform (CDCl3) solution. Chemical shift (δ) values are given in parts per million (ppm) relative to tetramethylsilane (TMS, δ = 0.00 ppm) as the internal standard; coupling constants (J values) are given in hertz (Hz). For signal multiplicities, the following abbreviations are used as follows: s (singlet), d (doublet), dd (doublets of doublet), t (triplet), q (quartet), br s (broad singlet) and m (multiplet). The mass spectra (MS) were recorded on a Waters Aquity Ultra Performance LC with ZQ detector in ESI positive ionization mode. The high-resolution mass spectra (HRMS) were recorded on Agilent 6540 QToF instrument in ESI positive ionization mode. The purity of final compounds (>95%) was verified by HPLC on a Waters 2695 system equipped with a Waters X-Bridge C18 column and a Waters 996 UV detector set at 254 nm. The following conditions were used; method 1: a flow rate of 1 mL/min, a gradient run from 55% eluent A (H2O/triethylamine, 99.8 : 0.2), 45% eluent B (acetonitrile) to 0% eluent A, 100% eluent B for 10 min; method 2: a flow rate of 1 mL/min, a isocratic run of 90% eluent A (acetonitrile) (H2O), 10% eluent B (H2O) for 10 min.

3-{2-[(Adamantan-1-yl)amino]ethyl}benzo[d]thiazol-2(3H)-one (7)

The bromo derivative 4 (2.32 mmol) was added to a suspension of 1-adamantanamine (4.17 mmol) and sodium bicarbonate (7.02 mmol) in dimethylformamide (13 mL). The reaction mixture was stirred at 60 °C for 24 hours. After being cooled, the mixture was poured into water and then extracted in chloroform. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by flash column chromatography over silica gel using a mixture of ethyl acetate/hexanes (6:4, v/v) as eluent, and then triturated with petroleum ether to give compound 7 as a pure white solid (66%). 1H NMR (400 MHz, CDCl3) δ 7.35 (dd, J = 7.8, 1.3 Hz, 1H, aromatic), 7.25 (td, J = 7.8, 1.3 Hz, 1H, aromatic), 7.13 – 7.03 (m, 2H, aromatic), 3.94 (t, J = 7.2 Hz, 2H, CONCH2), 2.86 (t, J = 7.3 Hz, 2H, CH2NH), 1.97 (br s, 3H, adamantane), 1.63 – 1.46 (m, 13H, adamantane). 13C NMR (101 MHz, CDCl3) δ 170.6 (NCOS, benzothiazolone), 137.7 (benzothiazolone), 126.8 (benzothiazolone), 123.5 (benzothiazolone), 123.2 (benzothiazolone), 123.1 (benzothiazolone), 111.2 (benzothiazolone), 51.0 (Cq, adamantane), 44.7 (CONCH2), 43.2 (3 CH2, adamantane), 38.6 (CH2NH), 37.1 (3 CH2, adamantane), 30.0 (3 CH, adamantane). MS (ESI) m/z 330 [M + H]+. HPLC (method 1): tR = 5.03 min. HRMS (ESI) m/z: [M + H]+ calcd for C19H25N2OS 329.1682; found 329.1689.

3-{2-[(Adamantan-1-yl)amino]ethyl}−6-propionylbenzo[d]thiazol-2(3H)-one (8)

The title compound was obtained from bromo derivative 5 (4.96 mmol) and 1-adamantanamine (8.94 mmol) following the same procedure for the preparation of 7. The crude product was purified by flash chromatography using a mixture of ethyl acetate/hexanes (7:3, v/v) as eluent, and then triturated with petroleum ether to give compound 8 as a pure white solid (57%). 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 1.8 Hz, 1H, aromatic), 7.89 (dd, J = 8.5, 1.8 Hz, 1H, aromatic), 7.13 (d, J = 8.5 Hz, 1H, aromatic), 3.95 (t, J = 7.1 Hz, 2H, CONCH2), 2.93 (q, J = 7.2 Hz, 2H, CH3CH2CO), 2.87 (t, J = 7.1 Hz, 2H, CH2NH), 1.96 (br s, 3H, adamantane), 1.64 – 1.40 (m, 12H, adamantane), 1.17 (t, J = 7.2 Hz, 3H + 1H, CH3CH2 + NH adamantane). 13C NMR (101 MHz, CDCl3) δ 199.3 (C=O), 170.7 (NCOS, benzothiazolone), 141.3 (benzothiazolone), 132.5 (benzothiazolone), 127.3 (benzothiazolone), 123.4 (benzothiazolone), 123.2 (benzothiazolone), 110.9 (benzothiazolone), 51.0 (Cq, adamantane), 45.1 (CONCH2), 43.2 (3 CH2, adamantane), 38.6 (CH2NH), 37.0 (3 CH2, adamantane), 32.0 (CH3CH2CO), 29.9 (3 CH, adamantane), 8.7 (CH3CH2). MS (ESI) m/z 385 [M + H]+. HPLC (method 1): tR = 7.64 min. HRMS (ESI) m/z: [M + H]+ calcd for C22H29N2O2S 385.1944; found 385.1858.

3-{2-[(Adamantan-1-yl)amino]ethyl}−6-propylbenzo[d]thiazol-2(3H)-one (9)

The title compound was obtained from bromo derivative 6 (6.60 mmol) and 1-adamantanamine (11.83 mmol) following the same procedure for the preparation of 7. The crude product was purified by flash chromatography using a mixture of ethyl acetate/hexanes (7:3, v/v) as eluent, to give compound 9 as a pure pale yellow oil (66%). 1H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 1.5 Hz, 1H, aromatic), 7.04 (dd, J = 8.2, 1.8 Hz, 1H, aromatic), 6.96 (d, J = 8.3 Hz, 1H, aromatic), 3.91 (t, J = 7.3 Hz, 2H, CONCH2), 2.85 (t, J = 7.3 Hz, 2H, CH2NH), 2.52 (t, J = 7.6 Hz, CH2CH2Ar), 1.97 (s, 3H, adamantane), 1.61 – 1.46 (m, 2H + 12H, CH3CH2CH2 + adamantane), 1.31 (s, 1H, adamantane), 0.87 (t, J = 7.3 Hz, 3H, CH3CH2). 13C NMR (101 MHz, CDCl3) δ 170.2 (NCOS, benzothiazolone), 137.9 (benzothiazolone), 135.4 (benzothiazolone), 126.7 (benzothiazolone), 122.7 (benzothiazolone), 122.4 (benzothiazolone), 110.6 (benzothiazolone), 50.7 (Cq, adamantane), 44.4 (CONCH2), 42.8 (3 CH2, adamantane), 38.3 (CH2NH), 37.7 (CH2CH2Ar), 36.7 (3 CH2, adamantane), 29.6 (3 CH, adamantane), 24.8 (CH3CH2CH2), 13.8 (CH3CH2). MS (ESI) m/z 371 [M + H]+. HPLC (method 1): tR = 5.30 min. HRMS (ESI) m/z: [M + H]+ calcd for C22H31N2OS 371.2152; found 371.2156.

3-{2-[(Adamantan-1-yl)(methyl)amino]ethyl}benzo[d]thiazol-2(3H)-one (10)

Iodomethane (1.62 mmol) was added to a suspension of sodium bicarbonate (2.05 mmol) and amine derivative 7 (1.37 mmol) in dimethylformamide (18 mL). The reaction mixture was stirred at 80 °C for 3 hours. After completion of the reaction, the mixture was partitioned between ethyl acetate and water. The extracted organic layer was washed with brine, dried over anhydrous sodium sulfate, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography using a mixture of hexanes/ethyl acetate (6:4, v/v), to give 10 as a pure pale yellow solid (51%). 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 7.7 Hz, 1H, aromatic), 7.23 (d, J = 7.8 Hz, 1H, aromatic), 7.14 – 6.98 (m, 2H, aromatic), 3.91 (t, J = 7.3 Hz, 2H, CONCH2), 2.67 (t, J = 7.3 Hz, 2H, CH2N), 2.30 (s, 3H, CH3N), 1.97 (br s, 3H, adamantane), 1.60 – 1.41 (m, 12H adamantane). 13C NMR (101 MHz, CDCl3) δ 170.0 (benzothiazolone), 137.6 (benzothiazolone), 126.3 (benzothiazolone), 123.0 (benzothiazolone), 122.7 (benzothiazolone), 122.6 (benzothiazolone), 110.9 (benzothiazolone), 54.7 (Cq, adamantane), 46.7 (CH2N), 42.9 (CONCH2), 38.6 (3 CH2, adamantane), 36.7 (3 CH2, adamantane), 34.5 (CH3N), 29.6 (3 CH, adamantane). MS (ESI) m/z 344 [M + H]+. HPLC (method 1): tR = 8.18 min. HRMS (ESI) m/z: [M + H]+ calcd for C20H27N2OS 343.1839; found 343.1843.

3-{2-[(Adamantan-1-yl)(methyl)amino]ethyl}−6-propionylbenzo[d]thiazol-2(3H)-one (11)

The title compound was obtained from amine derivative 8 (1.91 mmol) and iodomethane (2.30 mmol) following the same procedure for the preparation of 10. The pure product was obtained by recrystallization from acetonitrile, to give 11 as pure pale yellow crystals (47%). 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 1.9 Hz, 1H, aromatic), 7.88 (dt, J = 8.5, 2.0 Hz, 1H, aromatic), 7.09 (d, J = 8.5 Hz, 1H, aromatic), 3.92 (t, J = 6.8 Hz, 2H, CONCH2), 2.93 (q, J = 7.3, 2H, CH3CH2CO), 2.67 (t, J = 6.8 Hz, 2H, CH2N), 2.27 (s, 3H, adamantane), 1.94 (br s, 3H, CH3N), 1.57 – 1.39 (m, 12H, adamantane), 1.17 (td, J = 7.2, 2.2 Hz, 3H, CH3CH2). 13C NMR (101 MHz, CDCl3) δ 198.7 (C=O), 169.9 (NCOS, benzothiazolone), 141.1 (benzothiazolone), 131.7 (benzothiazolone), 126.5 (benzothiazolone), 122.6 (benzothiazolone), 122.5 (benzothiazolone), 110.4 (benzothiazolone), 46.6 (CH2N), 43.0 (CONCH2), 38.3 (3 CH2, adamantane), 36.4 (3 CH2, adamantane), 34.2 (CH3N), 31.4 (CH3CH2CO), 30.7 (Cq, adamantane), 29.2 (3 CH, adamantane), 8.1 (CH3CH2). MS (ESI) m/z 400 [M + H]+. HPLC (method 1): tR = 7.82 min. HRMS (ESI) m/z: [M + H]+ calcd for C23H31N2O2S 399.2101; found 399.2109.

3-{2-[(Adamantan-1-yl)(methyl)amino]ethyl}−6-propylbenzo[d]thiazol-2(3H)-one (12)

The title compound was obtained from amine derivative 9 (2.38 mmol) and iodomethane (2.86 mmol) following the same procedure for the preparation of 10. The crude product was purified by flash chromatography using a mixture of ethyl acetate/hexanes (6:4, v/v) as eluent, to give compound 12 as a pure pale yellow oil (59%). 1H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 1.7 Hz, 1H, aromatic), 7.04 (dd, J = 8.2, 1.8 Hz, 1H, aromatic), 6.93 (d, J = 8.2 Hz, 1H, aromatic), 3.88 (t, J = 7.3 Hz, 2H, CONCH2), 2.65 (t, J = 7.4 Hz, 2H, CH2N), 2.53 (t, J = 7.6 Hz, 2H, CH2CH2Ar), 2.30 (s, 3H, CH3N), 1.97 (s, 3H, adamantane), 1.64–1.43 (m, 2H + 12H, CH3CH2CH2 + adamantane), 0.87 (t, J = 7.4 Hz, 3H, CH3CH2). 13C NMR (101 MHz, CDCl3) δ 169.7 (benzothiazolone), 137.4 (benzothiazolone), 135.4 (benzothiazolone), 126.2 (benzothiazolone), 122.4 (benzothiazolone), 122.0 (benzothiazolone), 110.3 (benzothiazolone), 54.1 (Cq, adamantane), 46.5 (CH2N), 42.8 (CONCH2), 38.4 (3 CH2, adamantane), 37.4 (CH2CH2Ar), 36.5 (3 CH2, adamantane), 34.2 (CH3N), 29.4 (3 CH, adamantane), 24.4 (CH3CH2CH2), 13.4 (CH3CH2). MS (ESI) m/z 386 [M + H]+. HPLC (method 2): tR = 5.63 min. HRMS (ESI) m/z: [M + H]+ calcd for C23H33N2OS 385.2308; found 385.2323.

Binding studies

Frozen whole guinea pig brains were used and processed as previously reported (Garces-Ramirez et al. 2011; Bowen et al. 1993). Ligand binding experiments were conducted for σ1R studies with 3 nmol/L [3H](+)-pentazocine (specific activity 28 Ci/mmol) and 8.0 mg tissue. Nonspecific binding was determined using 10 μmol/L haloperidol. For σ2R studies, each tube contained 3 nmol/L [3H]1,3-di(2-tolyl)guanidine ([3H]DTG) (specific activity 48 Ci/mmol), 200 nmol/L (+)-pentazocine, and 8.0 mg tissue. Nonspecific binding was determined using 100 μmol/L haloperidol.

In vitro microsomal stability

In vitro metabolic stability of 7-12 was conducted by incubating each compound at 1 μM concentration with RLMs in a bench-top shaker for 30 min at 37 ± 0.5 °C. An aliquot (1 μL) of stock solution (200 μM) was spiked in 0.2 mL of metabolic reaction mixture [50 mM phosphate buffer (pH 7.4), 1 mg protein/mL RLMs and 3 mM magnesium chloride]. The reaction mixture was pre-incubated for 5 min at 37 ± 0.5 °C and then reaction was initiated by addition of 1 mM NADPH to obtain 1 mM final concentration. Verapamil was used as positive control. The metabolic reaction mixture without NADPH was used as negative control. Aliquots (25 μl) were withdrawn at 0, 5, 10, 15, 20 and 30 min and quenched with of ice-cold acetonitrile (200 μL) with internal standard The samples were filtered through 0.45 μm membrane filtration 96 well plate by centrifugation at 1,000 g for 10 min. The filtrate was injected onto LC-MS/MS system.

Metabolic soft spot identification of 12 in RLMs

The metabolic soft spot identification of 12 was performed by incubating the compound at 10 μM concentration with 1 mg/ml RLMs supplemented with NADPH (1 mM final concentration). Initially, the compound was spiked in 0.4 mL reaction mixture [50 mM phosphate buffer (pH 7.4), 1 mg protein/mL RLMs] and the reaction mixture was then pre-incubated for 5 min in a bench-top shaker at 37 ± 0.5 °C. After preincubation reaction was initiated by addition of 1 mM NADPH. An aliquot (150 μl) was taken at 0 (control sample) and 10 min and mixed with equal volume of ice-cold acetonitrile. The samples were then vortex mixed for 2 min and centrifuged at 10,000 g for 5 min. The supernatant was then injected onto LC-HRMS (Agilent QToF instrument).

Results and Discussion

Chemistry

The starting materials 2-6 were prepared following known procedures (Bhat et al. 2013; Intagliata et al. 2017). The new target compounds 7-9, were synthesized through a nucleophilic substitution of the corresponding 3-(2-bromoethyl)benzo[d]thiazol-2(3H)-one intermediates 4-6 with 1-adamantanamine, according to Scheme 1. Subsequently, N-methylated derivatives 10-12 were obtained by treatment of the respective amine with iodomethane in DMF under base catalyzed conditions (Scheme 1). Physicochemical data for the newly synthesized compounds were consistent with their structure.

Scheme 1.

Scheme 1

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Reagents and conditions: (a) 1,2-dibromoethane, K2CO3, DMF, 60 °C, 3 h, 70–75%; (b) 1-adamantanamine, NaHCO3, DMF, 70 °C, 24 h, 57–66%; (c) iodomethane, NaHCO3, DMF, 80 °C, 3 h, 47–59%.

Binding studies

New derivatives 7-12 were tested on both σRs subtypes in guinea pig brain. Briefly, the σ1Rs were labeled with [3H](+)-pentazocine, and the σ2Rs were labeled with [3H]DTG in the presence of (+)-pentazocine to block σ1Rs. Nonspecific binding was determined in the presence of haloperidol. Results are summarized in Table 1 and are expressed as Ki (nM).

Table 1.

Binding affinities data of the investigated benzo[d]thiazol-2(3H)one derivatives 7-12, SN56, and haloperidolat σ1R and σ2R

Compd. Ki (nM ± SEM)a Ki σ2R /Ki σ1R
σ1R σ2R
7 131 ± 9.94 64.1 ± 3.25 0.5
8 11.2 ± 1.26 71.2 ± 3.11 6.3
9 11.2 ± 0.68 69.2 ± 5.56 6.2
10 12.9 ± 1.80 85.8 ± 8.02 6.6
11 12.9 ± 1.84 113 ± 16 8.7
12 7.2 ± 0.88 440 ± 40 61
SN56 b 0.56 >1000 >1000
Haloperidol c 1.9 ± 0.30 79.8 ± 20.6 42
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a

The values represent the mean ± SEM from triplicate assays

b

Data from (Yous et al. 2005)

c

Data from (Bowen et al. 1993)

One of the aims of this research was to evaluate the effect of the replacement of the azepane ring with the 1-adamantamine moiety on the σRs binding affinity and selectivity. In particular, this modification led to substantial structural changes, such as enlargement and conversion of the cycloalkanamine ring to an exocyclic amine moiety. Replacement of the azepane ring by 1-adamantanamine group was well tolerated in terms of affinity for the σ1R. Furthermore, shifting from a cycloalkanamine to an exocyclic amine did not abolish the σRs affinity, despite a reduction in σ1Rs selectivity has been observed. In general, all final derivatives possess good σRs affinity for both subtypes with slight σ1R preference, with the exception of compound 7 (Table 1). Indeed, 6-substituted analogs, bearing an acyl or alkyl group, were preferred rather than unsubstituted derivatives (9 and 8 vs 7). Finally, methylation of the primary amine group of 7-8 did not improve the affinity or selectivity at the σ1R (10–11), whereas introduction of the N-methyl group on the 6-propyl substituted compound 9 led to the optimal substitution for affinity and selectivity for σ1R among the series synthesized (12, Table1).

In vitro microsomal stability and metabolic soft spot identification

The second goal of this work was to evaluate whether the steric hindrance of a bulky amine moiety, like the 1-adamantamine group, led to improving the metabolic stability. To do so, in vitro metabolic stability studies of 7-12 were performed. Results indicated that the new derivatives exhibited fast metabolism in RLMs (Table 2). Importantly, the adamantine derivatives 7-12 showed slightly improved metabolic stability (t1/2 = 1.23–4.32 min) compared to compound SN56 (t1/2 = 0.98 min). Similarly, the in vitro metabolic intrinsic clearance (Clint) of compound 7-12 improved (1.1 to 4.4-fold) compared to Clint of compound SN56 (Table 2). These results suggest that, although to a smaller extent, the steric hindrance of the cyclic group did contribute towards improving the metabolic stability. One potential reason that antagonized the metabolic stability improvement could be the significant increase in lipophilicity (e.g., clogP) as a consequence of the introduction of the adamantyl group (Tsopelas et al. 2017; Nassar et al. 2004).

Table 2.

In vitro pharmacokinetic parameters in RLMs of 7-12, and SN56

Compd. Parametersa
t1/2 (min) C1int (mL/min/mg protein)
7 1.50 0.46
8 4.32 0.16
9 2.46 0.28
10 1.06 0.65
11 1.23 0.56
12 1.89 0.37
SN56 0.98 0.71
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a

t1/2 = in vitro hepatic microsomal half-life, Clint = intrinsic clearance

To further investigate the site of metabolism of the most active analog, the metabolic soft spot identification was performed. It can be seen that a total of 14 metabolites of compound 12 were detected after incubation for 10 min with RLMs supplemented with NADPH (Fig 3). The percentage of UV relative abundance of each metabolite is shown in Table 3.

Fig. 3.

Fig. 3

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Extracted ion chromatograms of metabolites of compound 12 upon incubation with RLMs, supplemented by NADPH (10 min incubation)

Table 3.

The percentage of UV relative abundance of metabolites of compound 12in RLMs supplemented with NADPH

Met/Parent Modification RT in MS (min) UV % relative abundancea M+H
12 10.54 6.2 385.2317
Met 1 N-dealkylation 5.72 MSb 166.1590
Met 2 P+2O 6.54 1.4 417.2206
Met 3 N-demethylation+2O 6.60 5.1 403.2050
Met 4 P+2O 6.78 9.4 417.2206
Met 5 N-demethylation+2O 7.03 7.1 403.2050
Met 6 P+2O-2H 7.30 28.6 415.2050
Met 7 N-demethylation+2O-2H 7.39 1.7 401.1893
Met 8 N-demethylation+2O-2H 7.61 4.3 401.1893
Met 9 N-demethylation+O 8.01 1.2 387.2101
Met 10 N-demethylation+O 8.25 27.2 387.2101
Met 11 P+O 8.35 2.5 401.2257
Met 12 N-demethylation+O 8.62 2.7 387.2101
Met 13 P+O 8.77 2.7 401.2257
Met 14 N-demethylation+O −2H 385.1944
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a

UV wavelength = 249 nm.

b

Peak detected in mass detector only.

Compound 12 was extensively metabolized in RLMs, with ca. 6% of it remaining unmodified at the end of 10 min incubation (Table 3). The major metabolites of compound 12 were found to be either the N-methylated metabolite Met 6 and the N-demethylated metabolite Met 10 (Table 3, and Fig. 4). In particular, Met 6 was formed by the monooxidation of the adamantine moiety and concurrent monooxidation+dehydrogenation of the ethyl linker or propyl side chain. Similarly, Met 10 was formed by monooxidation of either the propyl side chain or the ethyl linker of N-demethylated metabolite. Moreover, the predominant metabolic pathway, which includes Met 3, 5, 7–9, 12, and 14 (Table 3), was detected as the downstream metabolites of the N-demethylated metabolite after further multiple oxidations, overall contributing ~55% towards the total metabolism. Other important metabolic pathways include the oxidation of the adamantyl moiety, 6-propyl chain, or ethylamine linker resulting in the formation of Met 2, 4, 6, 11, or 13 (Table 3). These results suggest that the N-methyl group is a major metabolic soft spot. Conversely, the adamantyl moiety also underwent substantial metabolism, thus minimizing the metabolic stability improvement of compound 12.

Fig. 4.

Fig. 4

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Ion fragment analysis and probable structure of major metabolite Met 6 (top panel) and Met 10 (bottom panel)

Conclusion

In this work, we reported the design and synthesis of new benzo[d]thiazol-2(3H)one derivatives with nanomolar affinity for σRs. Among them, 12 showed good affinity for σ1Rs (Ki = 7.2 nM) and stronger preference for σ1Rs over σ2Rs (σ2R/σ1R = 61). Surprisingly, the steric hindrance of the adamantyl group had a marginal effect on improving the overall in vitro metabolic stability of the newly synthesized compounds in the RLMs assay. The metabolic soft spots of 12 were found to be both the N-ethyl-N-methyladamantan-1-amine moiety, and the 6-propyl chain. Nevertheless, the introduction of a bicyclic moiety such as 1-adamantamine was well tolerated maintaining the compounds affinities in the low nanomolar range at σ1R. Altogether, these results are useful to lead the synthesis of new benzothiazolone-based analogs with improved metabolic stability, and further studies are currently being developed.

Supplementary Material

44_2020_2597_MOESM1_ESM

Acknowledgments

This study was supported, in part, by grants from the National Institutes of Health NIDA R01 DA023205 (CRM) and from the Department of Defense CMRMP PR161310/P1 (CRM).

Footnotes

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

Conflict of interest

The authors declare that they have no conflict of interest regarding the publication of this paper.

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