Synthesis and Pharmacological Evaluation of Enantiopure N-Substituted Ortho-c Oxide-Bridged 5-Phenylmorphans

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).


Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay ( Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay ( Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay ( Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay ( Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay ( Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay (Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay (Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay (Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay (Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay (Table 2).

Introduction
The search for improved analgesics has been ongoing for a century [1][2][3][4]. Those that are now clinically used have side effects that cause concern. These side effects, respiratory depression, gastrointestinal effects, tolerance, dependence, etc., make the use of these analgesics problematic. We have sought a new scaffold that differed from that of the morphine-like compounds, with the hope that it might provide antinociceptives with fewer opioid-like side effects. One of these scaffolds was the ortho-C oxide-bridged phenylmorphan [5][6][7][8][9][10][11][12][13][14]. We have modified the N-substituent with the hope of influencing both opioid receptor affinity (Table 1) and efficacy and potency as determined in the forskolin-induced cAMP accumulation assay ( Table 2).    69.9 ± 9.5 135 ± 10 a Binding assays were typically conducted in at least three independent experiments, each performed with triplicate observations using whole rat brains excluding cerebellum; Ki ± SEM (nM); NT = not tested-inactive (<50% activity at 100 nM concentration in exploratory binding assays (displaced less than half of radioligand). Compounds with low binding affinity (>50 nM) were not further examined in functional assays).
We have synthesized and pharmacologically evaluated the 12 possible structurally rigid ortho-a and para-a through -f oxide-bridged phenylmorphans in racemic or enantiomeric form [5][6][7][8][9][10][11][12][13][14]. Of all of the a-through f-oxide-bridged phenylmorphans, the racemic N-phenethyl ortho-c oxide-bridged phenylmorphan (±-1, Figure 1) was found to have the highest mu-opioid receptor affinity (K i = 1.1 nM) [14]. The N-substituents in opioids play a major role in affinity and efficacy. We previously investigated the effects of N-substituents on a 5-phenylmorphan scaffold [14][15][16]. The 1R,5S-N-phenylcyclopropylmethyl and 1S,5R-N-phenylcyclopropylmethyl)-5-(3-hydroxyphenyl)morphans were found to have varying affinities at the mu-opioid receptor (MOR) (K i = 2-450 nM). Interestingly, compounds acted unusually as inverse agonists in the [ 35 S]GTPγS functional assay using nondependent cells that stably express the cloned human mu-opioid receptor [15,16]. Two of the N-substituted 5-phenylmorphan compounds with trans-2-phenyl-cyclopropylmethyl groups ((+)-2 and (-)-3, Figure 1) showed the highest affinity at MOR (K i = 3 and 4 nM, respectively), and possessed very potent mu-opioid antagonist activity (K e = 0.17 and 0.3 nM, respectively). We were interested in determining whether the combination of the most potent (ortho-c) oxide-bridged phenylmorphan scaffold and the conformationally restrained phenylcyclopropylmethyl moieties would have modifed potency and efficacy in functional assays. Herein, we report the synthesis of enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans, their binding affinities at opioid receptors, and their functional activity as agonists or antagonists. The chiral atoms in the ortho-c oxide-bridged phenylmorphans were either 3S,6aR,11aR or a 3R,6aS,11aS (see Figure 1 for atom numbering). For each of those ortho-c oxide-bridged phenylmorphan enantiomers, four N-2-phenylcyclopropylmethyl diastereomers needed to be synthesized. These eight stereoisomers and other N-substituted enantiomeric 5-phenylmorphans (N-E and Zcinnamyl compounds, and N-propyl compounds) were synthesized. The opioid binding affinity and functional activity of these compounds were compared with the resolved (-)-N-phenethylortho-c oxide-bridged 5-phenylmorphan (-)-1.  6.28 ± 0.43 (102.1 ± 0.2%) a Inhibition of forskolin-induced cAMP accumulation; cAMP Hunter TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRK1), and human δ-opioid receptor (OPRD1) were used for the forskolin-induced cAMP accumulation assay to determine potency and efficacy of the compounds following the previously established methods [17]. To determine % efficacy in forskolin-induced cAMP assays, data were blank subtracted with the vehicle control, followed by normalization to the forskolin control. Data were then analyzed in GraphPad Prism 8 (GraphPad, LaJolla, CA, USA) using nonlinear regression; values are expressed as the mean ± SEM of at least three independent experiments; b MOR antagonist potency (IC50) determined versus EC90 of fentanyl; degree of antagonism (Imax) normalized to naltrexone. c DOR antagonist potency (IC50) determined versus EC50 of SNC80; degree of antagonism (Imax) normalized to naltrexone. d KOR antagonist potency (IC50) determined versus EC90 of U50488H; degree of antagonism (Imax) normalized to nor-BNI.  6.28 ± 0.43 (102.1 ± 0.2%) a Inhibition of forskolin-induced cAMP accumulation; cAMP Hunter TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRK1), and human δ-opioid receptor (OPRD1) were used for the forskolin-induced cAMP accumulation assay to determine potency and efficacy of the compounds following the previously established methods [17]. To determine % efficacy in forskolin-induced cAMP assays, data were blank subtracted with the vehicle control, followed by normalization to the forskolin control. Data were then analyzed in GraphPad Prism 8 (GraphPad, LaJolla, CA, USA) using nonlinear regression; values are expressed as the mean ± SEM of at least three independent experiments; b MOR antagonist potency (IC50) determined versus EC90 of fentanyl; degree of antagonism (Imax) normalized to naltrexone. c DOR antagonist potency (IC50) determined versus EC50 of SNC80; degree of antagonism (Imax) normalized to naltrexone. d KOR antagonist potency (IC50) determined versus EC90 of U50488H; degree of antagonism (Imax) normalized to nor-BNI.  6.28 ± 0.43 (102.1 ± 0.2%) a Inhibition of forskolin-induced cAMP accumulation; cAMP Hunter TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRK1), and human δ-opioid receptor (OPRD1) were used for the forskolin-induced cAMP accumulation assay to determine potency and efficacy of the compounds following the previously established methods [17]. To determine % efficacy in forskolin-induced cAMP assays, data were blank subtracted with the vehicle control, followed by normalization to the forskolin control. Data were then analyzed in GraphPad Prism 8 (GraphPad, LaJolla, CA, USA) using nonlinear regression; values are expressed as the mean ± SEM of at least three independent experiments; b MOR antagonist potency (IC50) determined versus EC90 of fentanyl; degree of antagonism (Imax) normalized to naltrexone. c DOR antagonist potency (IC50) determined versus EC50 of SNC80; degree of antagonism (Imax) normalized to naltrexone. d KOR antagonist potency (IC50) determined versus EC90 of U50488H; degree of antagonism (Imax) normalized to nor-BNI. 6.28 ± 0.43 (102.1 ± 0.2%) a Inhibition of forskolin-induced cAMP accumulation; cAMP Hunter TM Chinese hamster ovary cells (CHO-K1) that express human µ-opioid receptor (OPRM1), human κ-opioid receptor (OPRK1), and human δ-opioid receptor (OPRD1) were used for the forskolin-induced cAMP accumulation assay to determine potency and efficacy of the compounds following the previously established methods [17]. To determine % efficacy in forskolin-induced cAMP assays, data were blank subtracted with the vehicle control, followed by normalization to the forskolin control. Data were then analyzed in GraphPad Prism 8 (GraphPad, LaJolla, CA, USA) using nonlinear regression; values are expressed as the mean ± SEM of at least three independent experiments; b MOR antagonist potency (IC50) determined versus EC90 of fentanyl; degree of antagonism (Imax) normalized to naltrexone. c DOR antagonist potency (IC50) determined versus EC50 of SNC80; degree of antagonism (Imax) normalized to naltrexone. d KOR antagonist potency (IC50) determined versus EC90 of U50488H; degree of antagonism (Imax) normalized to nor-BNI. Molecules 2022, 27, x FOR PEER REVIEW 5 of 23 Figure 1. Structures of N-substituted ortho-and para-a through f oxide-bridged phenylmorphans and selected ortho-c oxide-bridged phenylmorphan and 5-phenylmorphan ligands.

Pharmacokinetic Assay of MOR Antagonist (+)-17
The in vitro data from the forskolin-induced cAMP accumulation assay (Table 2) indicated that (+)-17 was a little more potent than naltrexone and had a higher degree of antagonism than naloxone. To determine whether its enzymatic effect was similar to or different than naltrexone, a pharmacokinetic study was undertaken.
Compound (+)-17, naltrexone, and naloxone were analyzed for their inhibitory effects on the activity of selected human liver microsomal cytochrome P450 isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) ( Table 3). At 10 µM, (+)-17 showed significant (>50%) inhibitory activity on the 2D6 isozyme. It also displayed inhibitory effects on 1A2, 2C9, 2C19, and 3A to various extents (ranging from 6.8% to 46.6%) indicating this molecule may cause potential drug-drug interactions. Unfortunately, the level of inhibition caused by (+)-17 was greater than that seen with naltrexone and naloxone. Microsomal metabolic stability of compound (+)-17, naltrexone, and naloxone were also evaluated using both human and mouse liver microsomes (Table 4). In human liver microsomes, naltrexone and naloxone showed a much longer half-life than (+)-17. A similar pattern was also observed in mouse liver microsome but to a lesser extent (57.4 and 14.4 min vs. 6.9 min). The metabolism of (+)-17 in the human and mouse microsomes appears to be mostly through NADPH-dependent mechanisms.

General Information
TLC analyses were carried out on Analtech silica gel GHLF 0.25 mm plates with UV and I 2 detection. Melting points were determined in open glass capillaries on a Thomas Hoover melting point apparatus or MP70 melting point system (manufactured by Mettler Toledo) and were uncorrected. Elemental analyses (C, H, N) were performed by Micro-Analysis, Inc, Wilmington, DE, and were within ±0.4% of the theoretical values. 1 H NMR and 13 C NMR spectra were recorded on a Bruker DMX wide-bore spectrometer in CDCl 3 (unless otherwise noted) at 400 or 500 MHz and 100 or 125 MHz, respectively, with the values given in ppm and J (Hz) assignments of 1 H resonance coupling. For 1 H NMR spectra (CDCl 3 ), the residual solvent peak was used as the reference (7.26 ppm) while the central solvent peak was used as the 13 C NMR reference (77.0 ppm in CDCl 3 ). The high-resolution electrospray ionization (ESI) mass spectra were obtained on a Waters LCT Premier timeoff light (TOF) mass spectrometer. Flash column chromatography was performed with Bodman silica gel LC 60 A. The chiral HPLC was performed on an Agilent 1100 series analytical instrument equipped with UV detector G1315-DAD using (R, R)-WHELK-O1 column (manufactured by Regis Technologies Int.), 250 × 4.6 mm. The samples for HPLC analyses were dissolved in CH 2 Cl 2 . A mixture of hexane, CH 2 Cl 2 and 2-propanol (80/15/5), and 0.1% v/v TFA was used as eluent and the flow rate was 1.5 mL/min. The optical rotation was measured with a PerkinElmer 341 polarimeter.