Three C9 substituted N-phenethyl-5-phenylmorphans were found to be extremely G-protein biased potent mu opioid receptor partial agonists that did not recruit beta-arrestin at all in both the PathHunter assay and in the Tango assay.
Abstract
A series of compounds have been synthesized with a variety of substituents based on a three-carbon chain at the C9-position of 3-hydroxy-N-phenethyl-5-phenylmorphan (3-(2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol). Three of these were found to be μ-opioid receptor agonists in the inhibition of forskolin-induced cAMP accumulation assay and they did not recruit β-arrestin at all in the PathHunter assay and in the Tango assay. Compound 12 (3-((1S,5R,9R)-2-phenethyl-9-propyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), 13 (3-((1S,5R,9R)-9-((E)-3-hydroxyprop-1-en-1-yl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), and 15a (3-((1S,5R,9R)-9-(2-hydroxypropyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol) were partial μ-agonists. Two of them had moderate efficacies (EMAXca. 65%) and one had lower efficacy, and they were ca. 5, 3, and 4 times more potent, respectively, than morphine in vitro. Computer simulations were carried out to provide a molecular basis for the high bias ratios of the C9-substituted 5-phenylmorphans toward G-protein activation.
Introduction
The discovery of multiple intracellular signaling cascades beyond heterotrimeric G protein activation1 has increased our knowledge of the complexity of transduction pathways triggered by μ-opioid receptor (MOR) agonists. In addition to Gi-signaling, typical MOR agonists recruit the regulatory and signaling protein β-arrestin2, which contributes to receptor desensitization and activate distinct signaling pathways. A typical MOR agonist like morphine activates both pathways, and increasing evidence indicates β-arrestin signaling may be therapeutically relevant.2 In β-arrestin knockout mice, antinociceptive activity of morphine is increased and the development of tolerance eliminated.3,4 Other studies have suggested that many of the adverse effects elicited by classical opioids, such as respiratory depression, gastrointestinal effects, tolerance and dependence, were at least partially mediated through recruitment of β-arrestin.5,6 This has spurred the development of biased agonists which preferentially activate G-protein signaling in an effort to separate the side-effects long-considered inherent to the analgesic activity of MOR agonists.
The search for analgesics devoid of side-effects has been ongoing for more than a century, with perhaps tens of thousands of opioid analogs synthesized. A sound basis for their pharmacological action that would enable the synthesis of new analgesics with fewer side-effects remains elusive. In vivo hot-plate or tail-flick assays have been the most reliable tools for assessing analgesic activity of novel opioids and along with animal behavioral studies, divergence from the usual effects of opioids could be reliably assessed.7 The isolation and structural elucidation of opioid receptors now allows a more rational approach to the design and synthesis of new compounds, and have given new hope that improved analgesics may eventually become available. Decades of analog synthesis provided a structurally diverse array of MOR agonists, the vast majority of which appeared to have essentially the same side-effects as morphine, although a few interesting compounds were introduced. Hot-plate and tail-flick antinociceptive assays in mice led to the introduction of pentazocine,8 a 6,7-benzomorphan. pentazocine was found to have decreased tolerance and dependence compared to the older 4,5-epoxymorphinan and morphinan classical types of opioid and is now known to be a weak μ-antagonist, and a κ-agonist. While its clinical utility was limited due to its kappa receptor mediated dysphoric effects, it represented one of the first examples of a clinically useful opioid analgesic with decreased respiratory depression. Although the full pharmacological mechanism of action remains uncharacterized, pentazocine, along with buprenorphine, have been noted to display a “ceiling” effect on respiratory depression, wherein escalating doses beyond a certain level do not further decrease respiratory function.9,10 With the renewed interest in opioids over the past decade, mainly due to the severe side-effects of widely available heroin and fentanyl used illicitly, new methodology and approaches have been proposed that may help identify compounds with fewer or milder side effects than those of the well-known, clinically-used analgesics. One of these approaches is based on MOR biased agonism, and several agonists with varying degrees of biased activity have recently been reported (Fig. 1).11–13 These compounds are structurally distinct from the classical opioids based on morphine but they act primarily through the MOR and have been shown to have decreased liability for some or all of the usual opioid side-effects, e.g., tolerance, constipation, and respiratory depression. All of these G-protein biased compounds recruit β-arrestin-2 to some extent.
Fig. 1. G-Protein biased MOR agonists SR-17018,12 TRV130,5 and PZM21.11.
Increased bias factor (the degree to which G-protein activation is favored over β-arrestin recruitment) has been shown to be directly correlated to enhanced analgesia without a concomitant increase in respiratory depression, widening the potential therapeutic window for these biased agonists.12 It is still uncertain whether recruitment of β-arrestin-2 does influence respiratory depression. Recent experiments using β-arrestin-2 knockout and knockin mice suggest that β-arrestin-2 signaling does not play a role in respiratory depression.14,15 In this study, we introduce a new series of biased compounds, namely, C9-substituted 5-phenylmorphan MOR agonists that display the highest bias factors possible for potent antinociceptive compounds. These new analogs are unable to recruit β-arrestin-2 through MOR activation and retain morphine-like or better potency.16
The 5-(3-hydroxy)phenylmorphans were first synthesized as structurally simpler relatives of morphine over 50 years ago by May and Murphy,17 and remain of considerable interest for their diverse functional activity at opioid receptors. Expanding our previous study of potent analgesics bearing this template,18 here we explore the effect of three-carbon chains at the C9 position of 5-phenylmorphans. This study includes compounds containing a three-carbon spacer between C9 and the terminal oxygen, and another with n-propyl at C9; some of these compounds showed equivalent or higher MOR agonist potency than morphine in vitro while failing to recruit β-arrestin at detectable levels. Computer simulations provided a rationale for the biased property of this series of compounds.
Results and discussion
Chemistry
Our strategy for exploring the structure–activity relationship of the C9-position on the 5-phenylmorphan scaffold relied on the previously published intermediate (±)-1, separable into enantiopure (–)-1 and (+)-1 through optical resolution with l-(+) and d-(–)-tartaric acid, respectively (Scheme 1).18,19
Scheme 1. Optical resolution of (±)-1.
A small library of 5-phenymorphan compounds bearing a C9-propyl chain with and without terminal oxygen atoms were examined to probe their effect on β-arrestin recruitment and potency in vitro. Intermediate 4 (Scheme 2) was obtained through Von Braun demethylation of 1, followed by alkylation and subsequent O-demethylation of 3 using previously described procedures (Scheme 2).18,20
Scheme 2. Representative synthesis of intermediate 4. Reagents and conditions: (a) CNBr, K2CO3, acetonitrile, rt; then 3N HCl, 100 °C, 65%; (b) 2-phenylethyl bromide, K2CO3, acetonitrile, 80 °C, 84%; (c) BBr3, DCM, –78–0 °C, 94%.
Homologation of the C9 position was achieved in two steps through Wittig olefination and subsequent hydrolysis of the methyl vinyl ether to an epimeric mixture of aldehydes (Scheme 3). The reactivity and isomeric selectivity of both reactions were highly sensitive to the N-substituent, aryl functionality and hydrolysis conditions. The E stereochemistry of methyl vinyl ether intermediates 5–7 was assigned based on diagnostic NOE interaction between the C9-alkenyl and C1-methine protons.
Scheme 3. Wittig olefination of C9 carbonyl. Reagents and conditions: a. LiHMDS, (methoxymethyl)triphenylphosphonium chloride, THF, 0 °C.
Wittig olefination of (–)-1 gave a 5 : 1 ratio of E/Z isomers (Scheme 3), but with the larger N-substituent in 3 this ratio was reversed in favor of the Z-isomer 1 : 4 E/Z, and reversed yet again by reaction with phenolic 4 to give a 20 : 1 ratio of E/Z isomers. The E/Z selectivity observed in this reaction is also likely to be sensitive to the conditions employed during the workup. Hydrolysis of the resultant mixtures of E/Z isomers gave varying levels of selectivity depending on the isomeric ratio and conditions (Table 1).
Table 1. Hydrolysis of enol ethers 5–7.
| |||
| Cmpd | Acid | Time | α : β ratio |
| 5 (E/Z = 5 : 1) | 90% formic acid | 48 h | 1 : 1.5 |
| 5 | aq TFA | 24 h | 1 : 1 |
| 5 | aq HClO4 | 24 h | 1 : 1 |
| 5 | 3N HCl | 12 h | 1 : 9 |
| 5 | 3N HCl | 24 h | 1 : 2 |
| 5 | 6N HCl | 1.5 h | 1 : 3 |
| 6 (E/Z = 1 : 4) | 3N HCl | 36 h | 1 : 3 |
| 7 (E/Z = 20 : 1) | 3N HCl | 12 h | 1 : 7 |
Hydrolysis of 7 with 3N HCl over 12 h was found to be optimal for obtaining the 1S,5R,9R-epimer, with longer reaction times leading to epimerization and a degradation of C9R-epimer selectivity. Conversion of the Z- to the E-isomer was observed during the hydrolysis of 6; in view of the longer reaction times, this suggests that the E-isomer was hydrolyzed significantly faster. All attempts at chromatographic separation of the epimers generated an intensely blue colored impurity and a significant loss in yield. Therefore, the epimeric mixtures were used without any attempt at purification. Additionally, initial synthetic studies showed that late-stage O-demethylation of the aryl ether with either Lewis or Brønsted acids resulted in poor yields or decomposition, and thus utilizing phenol 4 as a common intermediate largely avoided these difficulties.
Using hydrolysis conditions optimized for the C9R-epimer, the epimeric mixture of aldehydes 10 was subjected to Horner–Wadsworth–Emmons olefination which resulted in a chromatographically separable diastereomeric mixture of enones, giving the C9R-epimer 11 as the major product (Scheme 4). Alternatively, Wittig olefination of 10 and immediate hydrogenation afforded propyl substituted 12. Reduction of 11 with LiAlH4 resulted in allyl alcohol 13.
Scheme 4. Representative synthesis of analogs 11–13. Reagents and conditions: (a) methyl diethylphosphonoacetate, NaH, THF, 57%; (b) ethyltriphenylphosphonium iodide, LiHMDS, THF; then 5% Pd/C, 50 psi H2, MeOH, 28%; (c) LiAlH4, THF, 0 °C, 59%.
Hydrogenation of enone 11 afforded common intermediate 14, which was reduced to propyl alcohol 15, transesterified to ethyl ester 16, and hydrolyzed to carboxylic acid 17 (Scheme 5). The relative configuration of the C9 position was determined by X-ray crystallographic analysis of 14b (Scheme 5 and Fig. 2).
Scheme 5. Synthesis of analogs 14–17. Reagents and conditions: (a) 5% Pd/C, 50 psi H2, AcOH, MeOH, 75%; (b) LiAlH4, THF, 0 °C, 88%; (c) EtOH, cat. H2SO4, 78 °C, 63%; (d) 1N aq LiOH, MeOH, 60 °C, 25%.
Fig. 2. X-ray crystallographic structure of methyl 3-((1R,5S,9S)-5-(3-hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-9-yl)propanoate hydrobromide (14b). The ellipsoids are shown at the 50% probability level.
The C9S-epimer (22) of 14a was synthesized in 5 steps from aldehyde 8 (Scheme 6). Horner–Wadsworth–Emmons olefination of the aldehyde gave enone 18, N-demethylation to 19 resulted in a mixture of methyl and ethyl esters, which were carried forward as a mixture through the rest of the synthesis. N-Alkylation with phenylethyl bromide resulted in 20 and hydrogenation of the enone gave 21. O-Demethylation of 21 in refluxing HBr resulted in concomitant hydrolysis of the mixture of esters; immediate esterification with MeOH and trimethyl orthoformate resulted in the methyl ester 22.
Scheme 6. Synthesis of 9S epimer 22. Reagents and conditions: (a) triethylphosphonoacetate, NaH, THF, 73%; (b) 1-chloroethyl chloroformate, DCE, 80 °C; then MeOH, 60 °C, 91%; (c) 2-phenylethyl bromide, K2CO3, ACN, 80 °C, 68%; (d) 5% Pd/C, 50 psi H2, MeOH, 98%; (e) 48% HBr, toluene, 120 °C; then MeOH, HC(OMe)3, H2SO4, 60 °C, 66%.
Biological results
Given recent reports on the potential utility of G-protein biased MOR agonists, this series of compounds were evaluated for functional activity at the MOR utilizing two assays, one of which measured G-protein signaling, and in a separate assay, β-arrestin recruitment. Chinese hamster ovary cells (CHO-K1) that express human MOR (OPRM1) were used in forskolin-induced cAMP assays as previously described21 to measure functional activity through G-protein signaling (Table 2). Many of these compounds, 12, 13, 14a, 15a, were found to be potent partial agonists at MOR, while 16 was found to have low nanomolar potency at MOR as a full agonist. These compounds were also evaluated for their potency and efficacy at the human KOR (OPRK1) and DOR (OPRD1) to determine selectivity using the aforementioned forskolin-induced cAMP assay. The majority of these compounds displayed weak to no functional activity at the DOR in comparison with their EC50 values at MOR, and even less activity was observed at KOR. Taken together, these data suggest that these compounds are selective for activity at the MOR.
Table 2. Inhibition of forskolin-induced cAMP accumulation: functional activity for cAMP and MOR-mediated β-arrestin2 recruitment.
| # |
|
MOR cAMP |
KOR cAMP |
DOR cAMP |
β-Arrestin2 recruitment
a
(PathHunter assay) |
β-Arrestin2 recruitment
b
(Tango assay) |
||||||||
| R1 | R2 | EC50 c , d (nM) | E MAX % | EC50 (nM) | E MAX % | EC50 (nM) | E MAX % | EC50 (nM) | E MAX % | Bias factor | EC50 (nM) | E MAX % | Bias factor | |
| 11a | CH CH–CO2Me | H | 132 ± 62 | 52.6 ± 6.5% | >10 000 | — | 143.0 ± 18.0 | 27.2 ± 3.7% | >25 000 e | — | NC f | NT g | NT g | NT g |
| 12 | CH2CH2CH3 | H | 0.95 ± 0.35 | 63.3 ± 3.9% | >10 000 | — | >10 000 | — | >25 000 e | — | NC f | >25 000 e | — | NC f |
| 13 | CH CH–CH2OH | H | 2.31 ± 0.78 | 33.5 ± 5.9% | >10 000 | — | >10 000 | — | >25 000 e | — | NC f | >25 000 e | — | NC f |
| 14a | CH2CH2–CO2Me | H | 0.46 ± 0.09 | 94 ± 2.3% | >10 000 | — | >10 000 | — | 1.72 ± 0.17 | 4.5 ± 0.38% | 0.55 | 12.5 | 123.4 | 0.15 |
| 15a | CH2CH2CH2OH | H | 1.2 ± 0.37 | 65 ± 6% | >10 000 | — | 80.9 ± 33.9 | 22.62 ± 6% | >25 000 e | — | NC f | >25 000 e | — | NC f |
| 16 | CH2CH2–CO2Et | H | 0.24 ± 0.09 | 100.1 ± 0.7% | 35.3 ± 19.9 | 36.5 ± 3.8% | 49.1 ± 10.1 | 74.2 ± 4.9% | 11.57 ± 3.1 | 18.26 ± 1.7% | 1.89 | >25 000 e | — | NC f |
| 17 | CH2CH2–CO2H | H | 31.7 ± 11.4 | 49 ± 2.5% | >10 000 | — | >10 000 | — | >25 000 e | — | NC f | >25 000 e | — | NC f |
| 22 | H | CH2CH2–CO2Me | 854 ± 114 | 63 ± 2.4% | 322.8 ± 110.5 | 55.01 ± 5.0% | >10 000 | — | >25 000 e | — | NC f | NT g | NT g | NT g |
|
MOR cAMP |
KOR cAMP |
DOR cAMP |
β-Arrestin2 recruitment
a
(PathHunter assay) |
β-Arrestin2 recruitment
b
(Tango assay) |
|||||||||
| R1 | R2 | EC50 c , d (nM) | E MAX % | EC50 (nM) | E MAX % | EC50 (nM) | E MAX % | EC50 (nM) | E MAX % | Bias factor | EC50 (nM) | E MAX % | Bias factor | |
| 14b | H | CH2CH2–CO2Me | 31.3 ± 4.6 | 42 ± 4.8% | >10 000 | — | >10 000 | — | >25 000 | — | NC f | >25 000 e | — | NC f |
| 15b | H | CH2CH2CH2–OH | 35.2 ± 12.59 | 48.7 ± 3.7% | >10 000 | — | >10 000 | — | >25 000 e | — | NC f | >25 000 e | — | NC f |
| 23 (ref. 16) | H | OH g | 0.017 ± 0.004 | 101% | 322.8 ± 110.5 | 55.01 ± 5.0% | 2.6 ± 0.5 | 74.0 ± 20.8% | 6.08 ± 0.74 | 116 ± 5.4% | 2.16 | NT h | NT h | NT h |
| Standards | ||||||||||||||
| Morphine | 4.7 ± 0.6 | 103 ± 0.6% | NT h | NT h | NT h | NT h | 379.6 ± 40.7 | 25.7 ± 0.6% | 2.3 | NT h | NT h | NT h | ||
| SR 17018 i (ref. 12) | 15.6 ± 5.1 | 101.2 ± 1.8% | NT h | NT h | NT h | NT h | 2975 ± 1623 | 89.2 ± 3.4% | 1.96 | NT h | NT h | NT h | ||
| PZM21 (ref. 11) | 2.5 ± 0.4 | 101 ± 0.6% | NT f | NT f | NT f | NT f | 57.1 ± 8.5 | 4.3 ± 0.4% | 2.98 | NT h | NT h | NT h | ||
| Salvinorin A | NT h | NT h | 0.03 ± 0.007 | 99.8 ± 0.3% | NT h | NT h | NT h | NT h | NT h | NT h | NT h | NT h | ||
| DADLE | NT h | NT h | NT h | NT h | 0.02 ± 0.004 | 92.1 ± 0.7% | NT h | NT h | NT h | NT h | NT h | NT h | ||
| DAMGO | 0.3 ± 0.04 | 101.5 ± 0.5 | NT h | NT h | NT h | NT h | 44.1 ± 3.9 | 103 ± 0.4 | 1.0 | NT h | NT h | NT h |
aHitHunter 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. PathHunter CHO cells expressing human μ-opioid receptor β-arrestin-2 EFC cells were used for the β-arrestin-2 EFC recruitment assay. All cell lines were purchased from Eurofins DiscoverX (Fremont, CA). Cell culture was performed as previously described.21
bThermo Fisher Scientific's SelectScreen™ Profiling Service: 10 point titration agonist results.25
cEfficacy values were determined by normalization to DAMGO.
dMean ± standard error of the mean; n ≥ 2.
e E MAX = 0% at the concentrations tested.
fNC = not calculable. Bias factor could not be calculated because recruitment of β-arrestin2 was not observed under any of the conditions tested.
gThe C9–OH compound 23 has 1R,5R,9S stereochemistry.18
hNT = not tested.
iPurchased from Cayman Chemical, Ann Arbor, MI (CAS 2134602-45-0; labelled purity ≥98%), HRMS-ESI (m/z): [M + H]+ calcd. for C19H19 Cl3N3O 410.0594, found 410.0592.
The ability of these compounds to modulate the interaction of arrestin with the MOR was also investigated in OPRM1 β-arrestin-2 cells as described previously (Table 2).21 Bias factors were calculated in comparison to the reference ligand [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) using previously reported methods.22,23 A bias factor of ca. 1 indicates no preference for either signaling pathway and a bias factor greater than 1 indicates a bias toward G-protein signaling. The new compounds in Table 2 were compared with data obtained with these assays for the standards, morphine, SR17018 (ref. 12) and PZM21 (ref. 11) (Fig. 1), and the structurally simplest relative of the new compounds, 5-(3,9-dihydroxy)phenylmorphan (5-(3-hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-9-ol) 23.18,20,24 Further investigation of signaling bias was conducted commercially through the use of a Tango GPCR assay system (Thermo Fisher Scientific).
In several instances, as shown in Table 2, bias factors were not calculable due to a lack of detectable β-arrestin2 recruitment at 25 000 nM. Compounds 11a–13 were all found to lack β-arrestin2 recruitment, with 12 and 13 showing potent partial agonist activity for G-protein mediated signaling as measured by adenylyl cyclase inhibition. Methyl and ethyl esters 14a and 16, respectively, showed full agonist efficacy for G-protein signaling and potent partial-agonist β-arrestin2 signaling, with bias factors lower than 2. Given that several of our potent compounds (12, 13, 14b, 15a, 15b, 16, and 17) displayed a very strong bias for the G-protein pathway utilizing an enzyme fragment complementation assay, we chose to re-evaluate β-arrestin signaling using the Tango GPCR assay,25 an assay that does not depend on the knowledge of the G-protein signaling specificity of the MOR. The transcription-mediated response following β-arrestin recruitment relies on very specific intracellular interactions that are not altered by other signaling pathways.26 Generally, the result on all of the compounds evaluated in the Tango GPCR assay were in agreement with the PathHunter assay results. One exception was compound 16. While 16 had a relatively strong EC50 value (11.57 ± 3.1 nM), it was not very efficacious (Emax = 18.26 ± 1.7%). It should be noted that this level of efficacy is lower than morphine (Emax = 25.7 ± 0.6%) and SR 17018 (Emax = 89.2 ± 3.4%). Overall, extension of the carbon chain at the C9 position not only enhances the selectivity for the MOR, but also significantly reduces or completely eliminates β-arrestin recruitment.
To understand the receptor–ligand interactions responsible for the biased property, we conducted molecular dynamics (MD) simulations of compounds 12, 14 and 15 (both 1S,5R and 1R,5S configurations) bound to the receptor. The MOR structure was taken from our previous study,20 which contains a modeled intracellular loop 3 (ICL3, cf. Experimental section, ESI†). The receptor was embedded in a zwitterionic POPC membrane and solvated in a water solution with physiological concentrations of ions (see simulation setup in the Experimental section, ESI†). For comparative analysis, a simulation of the empty receptor, i.e., containing only a crystallographic Na+ ion,27 was taken as a reference; a structure at the end of this simulation was used as the starting point for all the other simulations.
The analysis shows that the biased property of the 1S,5R series stems from strong interactions, mainly hydrophobic, between the C9 substituent and transmembrane helix 3 (TMH3) at the mid-membrane level (Fig. 3). In 12 and 15a, the spacer and its OH substituent are critical to adopt conformations suitable for developing these interactions. Longer chains, such as methyl ester (14a), bend upward, interacting with the upper residues of the receptor, including extracellular loop 2 (ECL2); the resulting weaker interactions with TMH3 appears to be ineffective in preventing β-arrestin recruitment. However, in compound 22, the C9S-epimer of the methyl ester (14a), the chain is reoriented and regains the favorable interactions with TMH3, resulting in bias ratio comparable to 12 and 15a. Reorienting the chain in this fashion also resulted in a dramatic loss of agonist potency at the MOR. This stereospecificity could be further exploited to design novel, highly-potent biased agonists. On the other hand, a very potent μ-agonist phenylmorphan 23 (1R,5R,9S) lacks a carbon spacer at the C9 position and has a bias ratio (2.2) comparable to morphine. Previous simulations20 showed that the C9S–OH in 23 has little effect on the movement of TMH3, consistent with its non-biased activity; this is also the case for morphine20 despite its different scaffold.
Fig. 3. From left to right, compounds 12, 14a, and 15a showing the interactions of the C9 substituents with the receptor, mainly with TMH3 (helix numbers in parentheses). Hydrophobic interactions are more prevalent, but the polar groups in the chain can have important effects in its orientations, with Y84 playing a role to stabilize either the ester or hydroxy groups. Opposite to the direction of the spacer are TMH6 and TMH7 (Fig. S1†), which help stabilize the body of the ligands also through hydrophobic interactions; the head and tail of the ligands are stabilized by similar interactions in all the compounds (not shown). Not all the interactions are present simultaneously, especially in 14a, where the H-bonding with Y84 is less frequent than in 15a. Dashed straight lines indicate H-bonding interactions (H colored in white; C, green; N, blue; and O, red).
In general, the putative binding modes of the 1S,5R series, where the tertiary nitrogen remains H-bonded to D83 in TMH3, force the C9-chain to point in a direction with close packing of side chains and limited access to water (Fig. S1†). By contrast, in the 1R,5S series, the chain points in a direction that is less constrained sterically and more hydrated (Fig. S1†) to make multiple, potentially favorable interactions with the receptor, either directly or through water bridges (as in 23).20 These differences in the solvation environment of the chain may have consequences in the binding affinities since electrostatic/H-bonding interactions can be compensated differently by the desolvation penalty, especially if the chain contains a polar substituent (e.g., 14 and 15). At the same time, hydrophobic groups in the chains of the 1R,5S series yield unstable structures due to the limited availability of nonpolar residues. This is the case of the enantiomer of 12, which failed to stabilize in any of the simulations, frequently losing the H-bonding interaction with TMH3 and finding alternate modes of binding. One exception is 14b; due to the chain length, it bends and interacts with TMH3, targeting the helix in regions also targeted by 12 and 15a (Fig. S1†), hence its biased effect. In particular, the ester oxygens of 14b are stabilized by direct H-bonds with Q60 (a residue found to be critical in imparting stereospecificity of 23), whereas C76, V79, I80 and the nonpolar moiety of D83 stabilizes the chain through hydrophobic interactions (Fig. S1†). These observations are consistent with the fact that, although the 1S,5R series of compounds were generally more potent than their 1R,5S analogs, there were compounds in both series that did not recruit β-arrestin.
Conclusions
We formerly noted that H-bonding interactions provided by the C9S–OH of the phenylmorphan strongly influenced the interaction with MOR.18,24 While we initially thought that the extreme G-protein bias for 13 and 15 could be due to such H-bonding interactions, a very similar bias profile for the C9-propyl-substituted 12 suggests this may not be the case. Interestingly, while the methyl ester 14a has a relatively small bias factor reflecting its ability to recruit β-arrestin, its enantiomer 14b shows a complete lack of β-arrestin recruitment. Although the 1S,5R series of compounds were generally more potent than their 1R,5S analogs, there were compounds in both series that did not recruit β-arrestin. Also, the stereochemistry at the C9 position was noted to be an important factor affecting the interaction of the 5-phenylmorphans with the MOR, and that was clearly indicated by an C9S-epimer 22 that was far less potent as a MOR agonist than its C9R-epimer 14a.
In this series of 3-carbon chain analogs at C9, we have identified three compounds, all in the 1S,5R,9R series, that will be of interest for further study in vivo. These were 12 (3-((1S,5R,9R)-2-phenethyl-9-propyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), 13 (3-((1S,5R,9R)-9-((E)-3-hydroxyprop-1-en-1-yl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), and 15a (3-((1S,5R,9R)-9-(3-hydroxypropyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), which were found to be ca. 5, 3, and 4-fold more potent than morphine in vitro, respectively, have no detectable activity at KOR, and weak activity at DOR. Compounds 12, 13, and 15a failed to recruit β-arrestin in two different assays for β-arrestin recruitment. Additionally, 12, 13, and 15a display selectivity for the MOR, which would mitigate potential side effects that may be elicited by activating the other opioid receptors. Molecular dynamic simulations show that more direct engagement of TMH3 is essential to impart biased activity; the strong non-polar interactions between this helix and the 3-carbon spacer at C9 likely confers the extreme G-protein bias to 12 and 15a.
Author contributions
The manuscript was written with contributions from all the authors, and all authors have given approval of the final version of the manuscript.
Abbreviations
- EC
Extracellular
- IC
Intracellular
- ICL
Intracellular loop
- ECL
Extracellular loop
- TMH
Transmembrane helix
- MOR
μ-Opioid receptor
- cAMP
Cyclic adenosine monophosphate
- DAMGO
[d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin
- PDB
Protein Data Base
Conflicts of interest
The authors declare no competing financial interest. ESG, EB, FL, AEJ, and KCR are inventors on a patent assigned to NIH covering biased potent opioid-like agonists.
Supplementary Material
Acknowledgments
The work of ESG, EB, FL, AS, AEJ, and KCR was supported by the NIH Intramural Research Program (IRP) of the National Institute on Drug Abuse and the National Institute of Alcohol Abuse and Alcoholism. The work of SK, RC, and TEP was supported by DA018151 (to TEP) and GM008545 (to RC). The work of Y-SL and SAH was supported by the NIH IRP through the CIT and utilized the high-performance computer capabilities of the Biowulf HPC cluster at the NIH. The X-ray crystallographic work was supported by NIDA through an Interagency Agreement #Y1-DA1101 with the Naval Research Laboratory (NRL). NIH, DHHS. We thank Dr. John Lloyd (Mass Spectrometry Facility, NIDDK) for the mass spectral data.
Footnotes
†Electronic supplementary information (ESI) available. CCDC 1914554. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0md00104j
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