Potent MOR Agonists from 2′-Hydroxy-5,9-dimethyl-N-phenethyl Substituted-6,7-benzomorphans and from C8-Hydroxy, Methylene and Methyl Derivatives of N-Phenethylnormetazocine

(−)-5,9-Dimethyl-6,7-benzomorphan (normetazocine) derivatives with a para-OH or ortho-F substituent in the aromatic ring of the N-phenethyl moiety were synthesized and found to have subnanomolar potency at MOR, and both were fully efficacious in vitro. These new compounds, (1R,5R,9R)-6,11-dimethyl-3-(2-fluorophenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol and (1R,5R,9R)-6,11-dimethyl-3-(4-hydroxyphenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol, were more potent than the unsubstituted compound N-phenethylnormetazocine and about 30 or 40 times more potent than morphine, respectively. A variety of substituents in the ortho, meta, or para position in the aromatic ring of the N-phenethyl moiety were synthesized, 25 of these compounds, and found to have varying effects on potency and efficacy as determined by the forskolin-induced cAMP accumulation assay. The N-phenethyl moiety was also modified by increasing chain length to form a N-phenylpropyl side chain with and without a para-nitro moiety, and by an N-cinnamyl side chain. Also, an indole ethylamine normetazocine was synthesized to replace the N-phenethylamine side chain in normetazocine. The phenylpropylamine, propenylamine (cinnamyl) and the para-nitropropylamine had little or no MOR potency. The indole-ethylamine on the normetazocine nucleus, however, had moderate potency (MOR EC50 = 12 nM), and was fully efficacious (%Emax = 102%) in the cAMP assay. Retention of the N-phenethyl moiety and the addition of alkyl and alkenyl moieties on C8 in (−)-N-phenethylnormetazocine gave a C8-methylene derivative that had subnanomolar potency at MOR and a C8-methyl analog that had nanomolar potency. Five C8-substituted compounds were synthesized.


Introduction
The 6,7-benzomorphans (Figure 1) have been of interest for the past several decades and analgesics based on them have been found to be clinically useful [1,2].The 6,7benzomorphan structure is derived from the morphinans.It lacks the morphinan C-ring, making the 6,7-benzomorphans less complex (Figure 1) than the morphinan or epoxymorphinan opioid structures.This structural modification did not necessarily lead to improved analgesics: e.g., a prototypical 6,7-benzomorphan, metazocine, N-methyl-5, 9-dimethyl-6,7benzomorphan, not only acts at the mu opioid receptor (MOR) essentially like morphine, but also has dysphoric and hallucinogenic side-effects attributed to interaction with the kappa opioid receptor (KOR).Several of the derivatives of the 6,7-benzomorphans have been found to act as MOR agonists or antagonists, depending on the N-substituent.The racemic phenazocine (N-phenethyl relative of metazocine, Figure 1) was found to have milder side-effects in vivo (e.g., physical dependence, sedation, depression) than equivalent doses of morphine.Pentazocine, the N-dimethylallyl relative of metazocine, acts at both MOR and KOR and has been found to be a clinically useful analgesic with milder opioid-like side-effects.It is in clinical use and has been combined with naloxone to lessen its illicit use.The (+)-N-allylnormetazocine (SKF 10.047, Alazocine) is the prototypical sigma-1 receptor agonist [3] and does not interact at opioid receptors.Its (−)-enantiomer was found to be a MOR antagonist [4].
Molecules 2023, 28, x FOR PEER REVIEW 2 of 31 benzomorphan structure is derived from the morphinans.It lacks the morphinan C-ring, making the 6,7-benzomorphans less complex (Figure 1) than the morphinan or epoxymorphinan opioid structures.This structural modification did not necessarily lead to improved analgesics: e.g., a prototypical 6,7-benzomorphan, metazocine, N-methyl-5, 9-dimethyl-6,7-benzomorphan, not only acts at the mu opioid receptor (MOR) essentially like morphine, but also has dysphoric and hallucinogenic side-effects attributed to interaction with the kappa opioid receptor (KOR).Several of the derivatives of the 6,7-benzomorphans have been found to act as MOR agonists or antagonists, depending on the N-substituent.The racemic phenazocine (N-phenethyl relative of metazocine, Figure 1) was found to have milder side-effects in vivo (e.g., physical dependence, sedation, depression) than equivalent doses of morphine.Pentazocine, the N-dimethylallyl relative of metazocine, acts at both MOR and KOR and has been found to be a clinically useful analgesic with milder opioid-like side-effects.It is in clinical use and has been combined with naloxone to lessen its illicit use.The (+)-N-allylnormetazocine (SKF 10.047, Alazocine) is the prototypical sigma-1 receptor agonist [3] and does not interact at opioid receptors.Its (−)enantiomer was found to be a MOR antagonist [4].A considerable amount of effort has been expended on the synthesis of derivatives of the classical opioids [5], as well as the 6,7-benzomorphans [6][7][8][9][10][11][12], in an attempt to ameliorate their undesirable side-effects, with relatively little success [13].
Various alkyl moieties have been added to the 5,9-positions in the benzomorphan, cis and trans, and this has influenced the potency of the compound, but the side-effects were those noted for the classical opioids.When the phenolic hydroxyl was converted to a carbonamide function, it was found to have increased potency and, most interestingly, some of those benzomorphans were partial agonists in the [ 35 S]GTPgS (GTP) assay [14,15].Depending on the alkyl substituents at C5 and C9 and the stereochemistry of the molecule, changing the N-substituent has been shown to have a considerable effect on the benzomorphan [12], sometimes improving its potency and, in a few instances, changing its receptor interaction to that of a bifunctional ligand (MOR-σ1) [16].
We have found that substituents on the aromatic ring of the N-phenethyl substituent can modify the potency and efficacy of a different type of compound, norhydromorphone, as measured in the inhibition of forskolin-induced cAMP accumulation (cAMP) and [ 35 S]GTPgS (GTP) functional assays [17].The p-chlorophenethyl moiety was found to transform norhydromorphone into a bifunctional ligand that had subnanomolar potency at MOR and DOR in the cAMP assay, and that compound did not depress respiration in a rodent assay.We posed the question of whether we could obtain similar results with substituents on the aromatic ring in the N-phenethyl moiety of the 6,7-benzomorphans.
Many N-substituents have been synthesized in the 6,7-benzomorphans, including N-methyl through decyl [10], heterocyclic derivatives of the N-methyl substituent [18], cyanoalkyl, N-allyl and N-alkynyl [13], N-napthyl and quinolyl substituents, N-phenylpro panamide [12] and N-propyl amides [19].N-Phenethyl-6,7-benzomorphans are very wellknown [17,20], but a study of the in vitro effect of substituents on the aromatic ring in the N-phenethyl substituent is novel.In this work, a series of (−)-N-phenethylnormetazocines were synthesized with electron-withdrawing or -donating substituents on the aromatic ring of the N-phenethyl moiety to determine whether they could alter the functional effects of the 6,7-benzomorphans as measured in the cAMP assay and, possibly, convert them to partial agonists, compounds that are not as efficacious as morphine, with the hope that this would lessen their side-effects.Some partial agonists have been noted to have less effect than fully efficacious agonists like heroin or fentanyl on respiratory depression [21,22], a major cause of death due to opioid overdose.
The effect of substituents at C9 in the 5-(3-hydroxy)phenylmorphans, a position comparable to the C8 in normetazocine (Figure 1), has been extensively explored [20][21][22].Substituents at C9 in the phenylmorphans and C8 in the benzomorphans are relatively close to the tertiary nitrogen atom.Substituents at C8 in 6,7-benzomorphan enantiomers are not well-known, and C8-alkyl and alkenyl-substituted N-phenethylnormetazocine enantiomers have not been previously examined.The (−)-N-phenethylnormetazocine enantiomers with a C8-methyl or C8 methylene substituent were found to be very potent MOR agonists and were fully efficacious.

Substituents on the Aromatic Ring in the N-Phenethyl Side Chain
With the optically resolved (−)-normetazocine in hand, a series of N-substituted analogs were prepared using procedures in the literature (Scheme 2) [17].The resolved (−)-2 was used as the bromocamphorsulfonate salt since it was found that the use of the isolated free base resulted in poorer yields.NaHCO3 (3 equivalents) was added to the reaction to generate the free base in situ.Heating the reaction at 100 °C overnight in DMF gave the desired N-substituted analogs in good yields.Various substituted N-phenethyl bromides and other miscellaneous alkyl bromides were used as alkylating agents.

Substituents on the Aromatic Ring in the N-Phenethyl Side Chain
With the optically resolved (−)-normetazocine in hand, a series of N-substituted analogs were prepared using procedures in the literature (Scheme 2) [17].The resolved (−)-2 was used as the bromocamphorsulfonate salt since it was found that the use of the isolated free base resulted in poorer yields.NaHCO 3 (3 equivalents) was added to the reaction to generate the free base in situ.Heating the reaction at 100 • C overnight in DMF gave the desired N-substituted analogs in good yields.Various substituted N-phenethyl bromides and other miscellaneous alkyl bromides were used as alkylating agents.

Substituents on the Aromatic Ring in the N-Phenethyl Side Chain
With the optically resolved (−)-normetazocine in hand, a series of N-substituted analogs were prepared using procedures in the literature (Scheme 2) [17].The resolved (−)-2 was used as the bromocamphorsulfonate salt since it was found that the use of the isolated free base resulted in poorer yields.NaHCO3 (3 equivalents) was added to the reaction to generate the free base in situ.Heating the reaction at 100 °C overnight in DMF gave the desired N-substituted analogs in good yields.Various substituted N-phenethyl bromides and other miscellaneous alkyl bromides were used as alkylating agents.(22), and the unsubstituted compound ( 23)), were converted to their HCl salts by dissolving the free base in hot alcohol (isopropanol, ethanol, or methanol) and then adding concentrated hydrochloric acid.The HCl salt crystallized after stirring the solution for a few hours, employing a slow diffusion method with diethyl ether.The HCl salts were recrystallized from hot isopropanol.

Modification of the N-Phenethyl Side Chain
Several other types of (−)-N-substituted compounds were synthesized (Scheme 3) and evaluated for their potency and efficacy (Table 1): an N-phenylpropyl substituent (−)-24, an N-cinnamyl substituent (−)-25, and an indole ((−)-26.(22), and the unsubstituted compound ( 23)), were converted to their HCl salts by dissolving the free base in hot alcohol (isopropanol, ethanol, or methanol) and then adding concentrated hydrochloric acid.The HCl salt crystallized after stirring the solution for a few hours, employing a slow diffusion method with diethyl ether.The HCl salts were recrystallized from hot isopropanol.

Synthesis of C8-Oxo-Normetazocine
A large-scale synthesis and subsequent optical resolution of a C8-oxo key intermediate (rac-2′-methoxy-8-oxo-N-phenethylnormetazocine, rac-34) was undertaken to obtain the starting material for the synthesis of alkenyl and alkyl substituents at that C8 position.The synthesis of the 2′-methoxy racemate rac-34 proceeded according to the literature [25,26] (for the comparable phenol), in good overall yield via a three-step process from 29 Scheme 4. Synthesis of the 1-methyl piperidine analog (−)-28.

Synthesis of C8-Oxo-Normetazocine
A large-scale synthesis and subsequent optical resolution of a C8-oxo key intermediate (rac-2′-methoxy-8-oxo-N-phenethylnormetazocine, rac-34) was undertaken to obtain the starting material for the synthesis of alkenyl and alkyl substituents at that C8 position.The synthesis of the 2′-methoxy racemate rac-34 proceeded according to the literature [25,26] (for the comparable phenol), in good overall yield via a three-step process from 29 Scheme 5. Synthesis of the 4-nitrophenylpropyl substituent (−)-31.

Synthesis of C8-Oxo-Normetazocine
A large-scale synthesis and subsequent optical resolution of a C8-oxo key intermediate (rac-2 -methoxy-8-oxo-N-phenethylnormetazocine, rac-34) was undertaken to obtain the starting material for the synthesis of alkenyl and alkyl substituents at that C8 position.The synthesis of the 2 -methoxy racemate rac-34 proceeded according to the literature [25,26] (for the comparable phenol), in good overall yield via a three-step process from normetazocine (1) (Scheme 6).Boc protection of the secondary amine in normetazocine, followed by methylation of the phenol and subsequent removal of the Boc group, provided 32 in 98% yield.The C8 position was oxidized using chromium trioxide to form the ketone 33 (63%).Finally, the key intermediate, rac-34, was synthesized via N-alkylation of the 8-oxo-2methoxy-normetazocine (34) with phenethyl bromide in high yield (80%).The ketone in 34 was found to be resistant to olefination under Wittig or HWE conditions as well as with oxo-nucleophiles.It did, however, react with organometallics such as alkyl lithiates and Grignard reagents.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 31 normetazocine (1) (Scheme 6).Boc protection of the secondary amine in normetazocine, followed by methylation of the phenol and subsequent removal of the Boc group, provided 32 in 98% yield.The C8 position was oxidized using chromium trioxide to form the ketone 33 (63%).Finally, the key intermediate, rac-34, was synthesized via N-alkylation of the 8-oxo-2′-methoxy-normetazocine (34) with phenethyl bromide in high yield (80%).The ketone in 34 was found to be resistant to olefination under Wittig or HWE conditions as well as with oxo-nucleophiles.It did, however, react with organometallics such as alkyl lithiates and Grignard reagents.

Synthesis of the Optical Isomers of C8-oxo m-Methoxy-N-phenethyl-6,7-benzomorphan
While we had successfully resolved normetazocine based on methodology in the literature [27], we preferred to carry the synthesis of the C8-substituted compounds up to rac-34 (Scheme 6) and resolve that compound because that would enable us to run initial exploratory reactions on the available racemic compound.After screening conditions for crystallization, a combination of DMF and acetone was found to be ideal for the optical resolution of rac-34 with (+)-tartaric acid as the chiral acid (Scheme 7).Crystallization occurred after cooling to 0 °C.Conversion to the free base gave, after crystallization, 1S,5R,9R (−)-34.Its (+)-enantiomer (1R,5S,9S-(+)-34) was obtained after treatment with (−)tartaric acid of the rac-34 base obtained from the residual (+)-tartrate salt of (−)-34.These enantiomers had optical rotations of −41.3° and +44.4°, respectively.The resolution of rac-34 was scaled up to provide the needed quantity, and both enantiomers were collected at greater than 90% yield (Scheme 7).2.5.Synthesis of the Optical Isomers of C8-oxo m-Methoxy-N-phenethyl-6,7-benzomorphan While we had successfully resolved normetazocine based on methodology in the literature [27], we preferred to carry the synthesis of the C8-substituted compounds up to rac-34 (Scheme 6) and resolve that compound because that would enable us to run initial exploratory reactions on the available racemic compound.After screening conditions for crystallization, a combination of DMF and acetone was found to be ideal for the optical resolution of rac-34 with (+)-tartaric acid as the chiral acid (Scheme 7).Crystallization occurred after cooling to 0 • C. Conversion to the free base gave, after crystallization, 1S,5R,9R (−)-34.Its (+)-enantiomer (1R,5S,9S-(+)-34) was obtained after treatment with (−)tartaric acid of the rac-34 base obtained from the residual (+)-tartrate salt of (−)-34.These enantiomers had optical rotations of −41.3 • and +44.4 • , respectively.The resolution of rac-34 was scaled up to provide the needed quantity, and both enantiomers were collected at greater than 90% yield (Scheme 7).
Molecules 2023, 28, x FOR PEER REVIEW 6 of 31 normetazocine (1) (Scheme 6).Boc protection of the secondary amine in normetazocine, followed by methylation of the phenol and subsequent removal of the Boc group, provided 32 in 98% yield.The C8 position was oxidized using chromium trioxide to form the ketone 33 (63%).Finally, the key intermediate, rac-34, was synthesized via N-alkylation of the 8-oxo-2′-methoxy-normetazocine (34) with phenethyl bromide in high yield (80%).The ketone in 34 was found to be resistant to olefination under Wittig or HWE conditions as well as with oxo-nucleophiles.It did, however, react with organometallics such as alkyl lithiates and Grignard reagents.

Synthesis of the Optical Isomers of C8-oxo m-Methoxy-N-phenethyl-6,7-benzomorphan
While we had successfully resolved normetazocine based on methodology in the literature [27], we preferred to carry the synthesis of the C8-substituted compounds up to rac-34 (Scheme 6) and resolve that compound because that would enable us to run initial exploratory reactions on the available racemic compound.After screening conditions for crystallization, a combination of DMF and acetone was found to be ideal for the optical resolution of rac-34 with (+)-tartaric acid as the chiral acid (Scheme 7).Crystallization occurred after cooling to 0 °C.Conversion to the free base gave, after crystallization, 1S,5R,9R (−)-34.Its (+)-enantiomer (1R,5S,9S-(+)-34) was obtained after treatment with (−)tartaric acid of the rac-34 base obtained from the residual (+)-tartrate salt of (−)-34.These enantiomers had optical rotations of −41.3° and +44.4°, respectively.The resolution of rac-34 was scaled up to provide the needed quantity, and both enantiomers were collected at greater than 90% yield (Scheme 7).With the free bases in hand, a way to quantitatively confirm the enantiomeric excess was needed, and several NMR chiral solvating reagents were studied for that purpose (Figure 2).With the free bases in hand, a way to quantitatively confirm the enantiomeric excess was needed, and several NMR chiral solvating reagents were studied for that purpose (Figure 2).The only reagent that was found to be successful for the separation of the enantiomers of 34 so that they could be clearly observed in their NMR spectra was 1-(anthracen-9-yl)-2,2,2-trifluoroethan-1-ol.The two peaks that were most resolved in the 1 H-NMR spectra and that could be used for determining enantiomeric excess were the methyl doublet and the farthest down-field aryl signal.After a single recrystallization, an enantiomeric excess of >98% was achieved for both enantiomers (see Supplementary Materials for NMR spectra).With a successful resolution method, we needed to determine the absolute configuration of the two enantiomers.Although tartaric acid resolved rac-34 well, it did not provide a proper crystalline form for X-ray spectroscopic determination.A small amount of each enantiomer base was dissolved in acetone and seven drops of concentrated hydrobromic acid added.As the acetone evaporated under ambient pressure, colorless needles formed.The crystals of the HBr salt of (+)-34 were used for the X-ray crystallographic analysis (Figure 3).The crystal structure analysis confirmed both the overall structure and the absolute configuration for the enantiomeric compounds.The only reagent that was found to be successful for the separation of the enantiomers of 34 so that they could be clearly observed in their NMR spectra was 1-(anthracen-9-yl)-2,2,2-trifluoroethan-1-ol.The two peaks that were most resolved in the 1 H-NMR spectra and that could be used for determining enantiomeric excess were the methyl doublet and the farthest down-field aryl signal.After a single recrystallization, an enantiomeric excess of >98% was achieved for both enantiomers (see Supplementary Materials for NMR spectra).With a successful resolution method, we needed to determine the absolute configuration of the two enantiomers.Although tartaric acid resolved rac-34 well, it did not provide a proper crystalline form for X-ray spectroscopic determination.A small amount of each enantiomer base was dissolved in acetone and seven drops of concentrated hydrobromic acid added.As the acetone evaporated under ambient pressure, colorless needles formed.The crystals of the HBr salt of (+)-34 were used for the X-ray crystallographic analysis (Figure 3).The crystal structure analysis confirmed both the overall structure and the absolute configuration for the enantiomeric compounds.With the free bases in hand, a way to quantitatively confirm the enantiomeric excess was needed, and several NMR chiral solvating reagents were studied for that purpose (Figure 2).The only reagent that was found to be successful for the separation of the enantiomers of 34 so that they could be clearly observed in their NMR spectra was 1-(anthracen-9-yl)-2,2,2-trifluoroethan-1-ol.The two peaks that were most resolved in the 1 H-NMR spectra and that could be used for determining enantiomeric excess were the methyl doublet and the farthest down-field aryl signal.After a single recrystallization, an enantiomeric excess of >98% was achieved for both enantiomers (see Supplementary Materials for NMR spectra).With a successful resolution method, we needed to determine the absolute configuration of the two enantiomers.Although tartaric acid resolved rac-34 well, it did not provide a proper crystalline form for X-ray spectroscopic determination.A small amount of each enantiomer base was dissolved in acetone and seven drops of concentrated hydrobromic acid added.As the acetone evaporated under ambient pressure, colorless needles formed.The crystals of the HBr salt of (+)-34 were used for the X-ray crystallographic analysis (Figure 3).The crystal structure analysis confirmed both the overall structure and the absolute configuration for the enantiomeric compounds.With the enantiopure (−)-and (+)-34 in hand, we pursued the synthesis of the C8hydroxy, methylene and methyl compounds.

Forskolin-Induced cAMP Accumulation Assay for In Vitro Determination of the Potency and Efficacy of the Benzomorphans
Both electron-withdrawing (e.g., NO 2 , (−)-3) and -donating (e.g., OH, (−)-20) substituents at the para position led to highly MOR potent compounds in vitro (Table 1) with EC 50 values in the subnanomolar range EC 50 = 0.3 nM and 0.13 nM, respectively.Some of the ortho-substituents (e.g., 2-F (−)-8) were also extremely potent MOR agonists (EC 50 = 0.2 nM).The para-OH (−)-20 and the ortho-F ((−)-8) compounds were among the few synthesized compounds that were more potent than the unsubstituted benzomorphan (−)-23 (EC 50 = 0.27 nM).The meta-substituted compounds were in general much less potent than the comparable orthoand para-analogs, except for the meta-fluoro analog (−)-7 (EC 50 = 0.6 nM) that was more potent than the para-fluoro analog (−)-6 (EC 50 = 1.8 nM).Addition of a second chloro substituent 2,4-dichloro, 21 (EC 50 = 5 nM) or 2,6-dichloro, 22 (EC 50 = 4 nM) on the aromatic ring reduced the potency of the ortho (17, EC 50 = 1.4 nM) or para (15, EC 50 = 1.3 nM) monochloro-compound.No clear pattern between agonist potency and electron donating or withdrawal effects was observed.A steric effect due to bulky substituents was also not observed (e.g., the bulky para-Br (12, EC 50 = 0.7 nM) and the less bulky para-fluoro substituent (6, EC 50 = 1.8 nM)).A second example of that could be seen with the bulky para-nitro (3) substituted compound having subnanomolar MOR affinity (EC 50 = 0.3 nM).However, the bulky trifluoromethyl at the para position was less potent (9, EC 50 = 2 nM) than compounds with bromo, chloro, or nitro substituents in the para position on the aromatic ring, and the dichloro compounds (21 and 22) were also less potent than the para-monochloro compound 15.A steric effect that might influence potency at MOR was not clearly observed.Three compounds showed an increase in DOR potency, compared with the unsubstituted N-phenethylnormetazocine (23).The para-nitro, para-trifluoromethyl, para-bromo, and para-chloro substituted compounds (3, 9, 12, and 15, respectively) were several-fold more potent than the unsubstituted compound 23, and these substituents did not modify efficacy-they were all partial DOR agonists.All of the examined compounds were less potent at KOR than the unsubstituted compound 23.However, 23 had very low efficacy at KOR (EC 50 = 3.4 nM, %E max = 25%), and most of the compounds with substituents in the aromatic ring in the N-phenethyl side chain had greater efficacy and would, theoretically, be more likely to show the dysphoric and hallucinogenic side-effects known to occur with KOR agonists than the unsubstituted compound 23.
All the C8 compounds were fully efficacious.As expected, the (+)-C8-compounds (+)-36, (+)-38, and (+)-39 were much less potent than the levorotatory compounds (−)-38 and (−)-39 at MOR.This was expected, since dextrorotatory benzomorphans are usually much less active than their levorotatory relatives.Two (+)-compounds, the C8-hydroxy and methyl derivatives (+)-36 and (+)-39, had moderate MOR potency (EC 50 = 25.2 and 21.7 nM, respectively) and the (+)-methylene compound (+)-38 was considerably less potent (EC 50 = 70 nM).The methylene compound (−)-38 was found to have MOR subnanomolar potency (EC 50 = 0.23 nM); it was about 25 times more potent than morphine.The C8-methyl compound (−)-39 was also quite potent, with an EC 50 = 1.2 nM.Both the C8-methylene ((-)-38) and methyl ((-)-39) compounds had moderate potency at DOR, and neither was as potent as the unsubstituted compound 23 at DOR or KOR.Both compounds had nearly full efficacy at DOR, and both had little efficacy at KOR.  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 [28]; 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; N/D = not determined (MOR antagonist activity was not determined when a compound had potent, fully efficacious MOR agonist activity; DOR and KOR agonist activity were not determined if the MOR agonist activity was less than 20 nM).b MOR antagonist potency (IC 50 ) determined versus EC 90 of fentanyl; degree of antagonism (I max ) normalized to naltrexone.c DOR antagonist potency (IC 50 ) determined versus EC 50 of SNC80; degree of antagonism (I max ) normalized to naltrexone.d KOR antagonist potency (IC 50 ) determined versus EC 90 of U50488H; degree of antagonism (Imax) normalized to nor-BNI.

General Information
All reactions were performed in oven-dried glassware under an argon atmosphere unless otherwise noted.Proton ( 1 H NMR) and carbon ( 13 C NMR) spectra were recorded on a Varian Gemini-400 spectrometer at 400 MHz for 1 H NMR and 101 MHz for 13 C NMR. Mass spectra (HRMS) were recorded on a Waters (Mitford, MA, USA) Xevo-G X5 QTof.The optical rotation data were obtained on a PerkinElmer polarimeter model 341, and melting points were obtained using a Thomas-Hoover melting point apparatus.Thin layer chromatography (TLC) was performed on a 250 mm Analtech GHLF.Visualization was accomplished under UV or by staining in an iodine chamber.Flash column chromatography was performed with Fluka silica gel 60 (mesh 220−400).Elemental analyses were performed by Robertson Microlit Laboratories, Ledgewood, NJ, USA.

X-ray Crystal Structure Experimental Data
Single-crystal X-ray diffraction data on compound (+)-34 were collected using Cu Kα radiation and a Bruker SMART APEX II CCD area detector.The crystal was prepared for data collection by coating with high-viscosity microscope oil.The oil-coated crystal was mounted on a micromesh mount (MiTeGen, Inc., Ithaca, NY, USA) and transferred to the diffractometer, and a data set collected at 293(2) K.The 0.097 × 0.074 × 0.060 mm 3

Table 1 .
Opioid Receptor Activity Measured in the Forskolin-induced cAMP Accumulation Assay a .

Table 1 .
Opioid Receptor Activity Measured in the Forskolin-induced cAMP Accumulation Assay a .