Rational Design, Chemical Syntheses, and Biological Evaluations of Peripherally Selective Mu Opioid Receptor Ligands as Potential Opioid Induced Constipation Treatment

Abstract

Opioid-induced constipation (OIC) is a common adverse effect of opioid analgesics. Peripherally acting μ opioid receptor antagonists (PAMORAs) can be applied in the treatment of OIC without compromising the analgesic effects. NAP, a 6β-N-4-pyridyl-substituted naltrexamine derivative, was previously identified as a potent and selective MOR antagonist mainly acting peripherally but with some CNS effects. Herein, we introduced a highly polar aromatic moiety, for example, a pyrazolyl or imidazolyl ring to decrease CNS MPO scores in order to reduce passive BBB permeability. Four compounds 2, 5, 17, and 19, when administered orally, were able to increase intestinal motility during morphine-induced constipation in the carmine red dye assays. Among them, compound 19 (p.o.) improved GI tract motility by 75% while orally administered NAP and methylnaltrexone showed no significant effects at the same dose. Thus, this compound seemed a promising agent to be further developed as an oral treatment for OIC.

Graphical Abstract

INTRODUCTION

Opioid analgesics play a critical role in cancer and noncancer pain management.^1,2 Opioids exert their pharmacological functions through interacting with one or more of the three classic opioid receptors, known as the μ opioid receptor (MOR), the κ opioid receptor (KOR), and the δ opioid receptor (DOR).³ Among them, the MOR is the primary target for most of the clinical opioid analgesics. Meanwhile, the activation of MOR accounts for various undesired side effects, such as tolerance, addiction, respiratory depression and constipation.⁴

The abuse liability of MOR agonists are widely known and have been intensively studied, and several treatments are available for opioid use disorders.⁵ At the same time, another unwanted side effect, that is, opioid-induced constipation (OIC), might be underestimated. This opioid-associated bowel dysfunction has occurred in 40–80% of MOR agonist-treated patients and contributes to a considerable percentages of treatment cessation, causing a substantial burden to the patients and health systems.^6,7 As neither dietary changes nor laxative products are effective enough in preventing or treating OIC, peripherally acting μ opioid receptor antagonists (PAMORAs) have become the major research of interest because of their targeting the underlying mechanisms of OIC, that is, MOR receptor activation in the gastrointestinal (GI) tract.⁸ As opioid pain relievers promote analgesia mainly in supraspinal sites and the spinal cord, the peripheral selectivity is essential for potential OIC therapeutic agents to avoid compromising the analgesic effects of opioid pain relievers.⁶

Currently, three PAMORAs have been approved by the U.S. FDA to treat OIC, namely, methylnaltrexone (MNTX),^9,10 naloxegol,^11,12 and naldemedine¹³ (Figure 1). A fourth PAMORA, alvimopan, however, is indicated mainly for postsurgery gastrointestinal recovery.¹⁴ Their mechanisms of peripheral nervous system (PNS) restriction are derived from the chemical structures: the N-methyl quaternary amine in MNTX, the PEGylation in naloxegol, and the high molecular weight and bulky side chain in naldemedine.¹⁴ Such structural features limit their blood-brain barrier (BBB) permeability and restrict their MOR-antagonizing effects to the periphery.^14,15 All these drugs have demonstrated sufficient efficacy and acceptable safety in a variety of clinical studies,^1,6,14 though some important clinical concerns and safety points have remained.

Figure 1.

Chemical structures of methylnaltrexone, naldemedine, and naloxegol.

Even though the positively charged quaternary amine dramatically lowers the lipophilicity of MNTX, indirect evidence has indicated that central effects can be elicited by MNTX.¹⁰ Abdominal pain has also been reported in 33–80% of the MNTX patients from two double-blind clinical trials,¹⁶ and cardiac-related adverse event occurred in less than 1% of patients in a long-term study.¹⁷ Moreover, although MNTX can be taken orally, three tablets of 150 mg are needed per day, and it costs at least two thousand dollars for a 30-day course.¹⁸ Naloxegol and naldemedine, on the other hand, are less pricey, but still require approximately four hundred dollars for one month.¹⁸ Moreover, naloxegol is only prescribed to noncancer pain patients. Also, the long-term use of naloxegol may induce many side effects, including abdominal pain, diarrhea, nausea, headache, and vomiting.¹² What is more concerning is that naloxegol has been implicated in the possible elevated risk for a life-threatening arrhythmia.¹⁹ The newest PAMORA, naldemedine, also has the reported side effects of diarrhea and tearing of the stomach or intestine wall.^6,20 Furthermore, both naloxegol and naldemedine are substrates of the CYP3A4 so that drug–drug interactions have been of concern.⁶ Therefore, it is still imperative to design and develop novel PAMORAs with more favorable tolerability profiles and to provide more choices for clinical applications.

In an effort to identify novel PAMORAs, a lead compound, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-pyridyl)acetamido]morphinan (NAP), discovered by our laboratories,²¹ was found to act as a peripherally selective MOR ligand based on in vitro and in vivo pharmacological and pharmacokinetic studies.^22–24 The ED₅₀ of NAP was 0.0088 mg/kg in a GI-tract motility assay, nearly 300-fold more potent than MNTX.²⁴ Nevertheless, NAP was able to moderately antagonize morphine’s antinociception with an AD₅₀ value of 4.51 mg/kg in a mouse pain model, which was significantly less potent than its gastric motility potency, but still suggesting its potential interference with opioid analgesic effects.²¹ Moreover, the observation of diarrhea associated with high doses of NAP also warrants further structure–activity relationships (SARs) studies on this class of compounds. Herein, a series of new opioid ligands have been rationally designed, synthesized, and biologically evaluated both in vitro and in vivo to identify potent peripherally selective MOR antagonists.

RESULTS AND DISCUSSION

Structure-Based and Physicochemical Property-Driven Drug Design.

Our laboratories have been engaged in the study of epoxymorphinan derivatives carrying diversified heterocyclic substituents at 14- or 6-position for over a decade. One of them is NAP, which possesses high MOR selectivity over the KOR and DOR. As NAP was observed to have therapeutic effects both systematically and peripherally, a series of SAR studies have been carried out to dissociate its PNS-favored characteristics from the central nervous system (CNS)-favored, and vice versa, to develop novel treatments for opioid use disorders and OIC.^2,25

From our previous studies, the heterocyclic ring in the C-6 side chain of NAP, that is, the pyridyl ring, was postulated to be critical in MOR binding and selectivity via a π–π stacking interaction with Trp318^7.35 and a potential cation–π interaction with Lys303^6.58.^26,27 Accordingly, an aromatic moiety in the side chain was proposed to be maintained in the lead optimization for CNS- or PNS-targeted MOR antagonists.

To further limit MOR antagonism by these ligands to the periphery, the focus of our studies has landed on design and syntheses of MOR antagonists impermeable through the BBB, which is a primary and critical physical barrier between the CNS and periphery.^28,29 One of the approaches established for assessing the potential of small molecules to penetrate the BBB is the in silico prediction and calculation based on physicochemical properties. To define the physicochemical properties space for CNS drug design, scientists at Pfizer developed a weighted scoring approach, called “CNS MPO (multiparameter optimization)” algorithm. In this scoring algorithm, six fundamental physicochemical properties, that is, ClogP (calculated partition coefficient), ClogD (calculated distribution coefficient at pH 7.4), TPSA (topological polar surface area), MW (molecular weight), HBD (number of hydrogen-bond donors), and pK_a (dissociation constant), are included.^30,31 Each property is weighted equally and defined as T0 with values between 0 and 1. Therefore, the collective CNS MPO score of a chemical entity may range from 0 to 6.0, with a desirable score greater than 4.0 as a widely used cutoff to select hits in CNS drug discovery programs.³⁰ Meanwhile, it should be noted that MPO score greater than 4 as a cutoff is solely based on the observation that 74% of already-marketed CNS-targeting drugs demonstrated a high CNS MPO (≥4), while a reasonable number of exceptions probably still exist.³⁰ Applying CNS MPO seems a practical approach for balancing multiple variables without the penalty of hard cut-offs and can be used prospectively in molecular design. In this context, we hypothesized that a CNS MPO score lower than 4.0 would suggest PNS-acting potential of designed small molecules.

To validate our hypothesis, the CNS MPO scores were first calculated for NAP, MNTX, naldemedine, and naloxegol by adopting the CNS MPO desirability tool (Table S1). It is shown that both naldemedine and naloxegol showed CNS MPO scores lower than 4.0, which is in line with their PNS dominated properties. Meanwhile, NAP and MNTX showed CNS MPO scores of 4.38 and 4.64, respectively, suggesting their potential CNS-targeting characteristics, which is in agreement with the fact that both of them carried centrally mediated effects while somehow conferred peripheral selectivity.^10,21

Combined with the isosterism concept, we decided to introduce a highly polar moiety, that is, a pyrazolyl or imidazolyl ring, to replace the pyridyl ring in NAP in order to increase TPSA and HBD, as well as to decrease ClogP and ClogD of the whole molecule, in the hope to further lower CNS MPO scores. Moreover, as heteroaromatic ring systems, pyrazolyl and imidazolyl are also expected to maintain the essential interactions with Trp318^7.35 and Lys303^6.58. In the newly designed agents (1–24, Scheme 1), we also altered the stereochemistry at C(6) (α or β), the distance between the aromatic ring and the morphinan skeleton, and the linker substitution position on the aromatic rings to further explore the preferred physiochemical features of NAP analogs as PNS agents. Compounds 1–24 were calculated to possess CNS MPO scores ranging from 3.56 to 3.76, all lower than 4.0. It should be noted that all designed compounds have increased TPSA and HBD as well as decreased ClogP and ClogD values as expected (Table S1). In summary, we attempted to generate a focused library enriched with possibly active and selective molecules for the MOR, which should be more efficient to discover new lead compounds and drug candidates with PNS-selective potency.

Scheme 1. Syntheses of the Target Compounds 1–24^a.

^aReaction conditions: (a) BnNH₂, benzene, p-TsOH, reflux; (b) NaBH₄, EtOH, 4 Å MS, r.t.; (c) H₂, MeOH, HCl, Pd/C, r.t.; (d) Bn₂NH, PhCOOH, toluene, p-TsOH, reflux; (e) NaCNBH₃, EtOH, 4 Å MS, r.t.; (f) RCOOH, EDCI, HOBt, TEA, 4 Å MS, DMF, r.t.; (g) K₂CO₃, MeOH, r.t.; (h) 1.25 M HCl/MeOH, 0 °C to r.t.

Chemistry.

The chemical syntheses of target compounds 1–24 were performed following the synthetic route outlined in Scheme 1. Briefly, 6α- or 6β-naltrexamine was prepared by stereoselective reduction amination of naltrexone with benzyl-amine or dibenzylamine, respectively, followed by debenzylation under catalytic hydrogenation condition.²¹ A variety of commercially available pyrazole- or imidazole-bearing carboxylic acids, were coupled with 6α- or 6β-naltrexamine employing the EDCI/HOBt method. After treatment with potassium carbonate in methanol, the 6-position monosubstituted free bases were furnished. These free bases were then converted to their hydrochloric acid salt forms, fully characterized and submitted for in vitro and in vivo pharmacological studies.

In Vitro Radioligand Binding and MOR [³⁵S]-GTPγS Functional Assays.

To characterize the binding affinity and selectivity profiles of all newly synthesized compounds on the three opioid receptors, in vitro competitive radioligand binding assays were performed as previously described.²⁵ The results are summarized in Tables 1 and 2.

Table 1.

Binding Affinity, Selectivity, and MOR [³⁵S]-GTPγS Functional Assay Results of Compounds 1–12 (6α-Configuration)^a

^aThe values are the mean ± SEM of at least three independent experiments.

^bData have been reported in ref 32 and are presented, here, for comparison purposes. Human recombinant opioid receptors were used in the assay.

^cNA: Not applicable.

^dData have been reported in ref 21 and are presented here for comparison purposes.

Table 2.

Binding Affinity, Selectivity, and MOR [³⁵S]-GTPγS Functional Assay Results of Compounds 13–24 (6β-Configuration)^a

^aThe values are the mean ± SEM of at least three independent experiments.

^bData have been reported in refs 21 and 32 and are presented here for comparison purposes. Human recombinant opioid receptors were used in the assay.

^cNA: Not applicable.

^dData have been reported in ref 21 and are presented here for comparison purposes.

As shown in Table 1, all 6α-compounds 1–12 retained high binding affinity, subnanomolar to one-digit nanomolar, for the MOR. All compounds exhibited higher binding affinities than that of MNTX (K_i,MOR = 5.50 ± 1.11 nM), and most compounds possessed comparable K_i values to NAP (K_i,MOR = 0.37 ± 0.07 nM). It was also observed that compounds with a carboxamido linker showed higher MOR binding affinity than compounds with an acetamido linker (1 vs 2, 4 vs 5, 7 vs 8, 10 vs 11), which demonstrated the same trend as their corresponding CNS MPO scores (3.76 vs 3.66), while they may not show higher affinity than the compounds with an n-propanamido linker. In addition, nine out of 12 (1, 3, 4, 7–12) also exhibited one-digit nanomolar binding affinity for either the KOR or DOR, but these compounds still preserved reasonable δ/μ and κ/μ selectivity. More particularly, compounds 2 and 5 demonstrated at least a hundred-fold selectivity for the MOR over both the KOR and DOR. Also, in general, compounds bearing a pyrazolyl ring (1–6) presented higher selectivity toward the MOR than the ones bearing an imidazolyl ring (7–12).

Among the 6β-configuration compounds (Table 2), it was observed that all 6β-compounds also maintained high binding affinity for the MOR. Moreover, all 6β-compounds possessed subnanomolar K_i values and showed higher binding affinity for the MOR than MNTX and their 6α-counterparts except for 21. Meanwhile, among the six compounds (13–18) bearing a pyrazolyl ring, the ones with an acetamido linker showed the highest MOR affinity (13 vs 14 vs 15, 16 vs 17 vs 18), while for the compounds bearing an imidazolyl ring, the ones with a carboxamido linker did the same (19 vs 20 vs 21, 22 vs 23 vs 24). Unlike 6α-compounds, the binding affinities of 13–24, for the DOR were all at most double-digit nanomolar, thereby increasing the δ/μ selectivity. Particularly, compounds 14, 15, 17–19, 22, and 23 presented hundreds-fold δ/μ selectivity. The κ/μ selectivity was also preserved for 13–23. Though the newly designed compounds showed lower selectivity for the MOR than NAP, most compounds were still MOR-selective and some compounds (14, 17–19) continued to exhibit high selectivity over both the KOR and DOR.

The [³⁵S]-GTPγS binding assay was carried out as well to determine the functionality on the MOR of each compound. As presented in Tables 1 and 2, all compounds showed low efficacy with % E_max values ranging from 7.50 ± 1.37 to 23.5 ± 1.29, which indicated these compounds may behave as MOR antagonists similarly to NAP (% E_max = 22.7 ± 0.84). The EC₅₀ value of MNTX was >10 000 nM for MOR, indicating an insignificant agonist activity. Interestingly, the 6β-analogues, which shared the same C6 configuration with NAP, demonstrated lower efficacies than their 6α-counterparts (except for 23).

Taken together, the replacement of the pyridyl ring in NAP with its isosteric pyrazolyl and imidazolyl rings maintained high binding affinity, selectivity, and low-efficacy functionality at the MOR.

In Vivo Warm-Water Tail Immersion Assay.

The warm-water tail immersion assay is a well-established assay for evaluating opioid analgesics, especially MOR agonists. The antinociceptive effects in tail withdrawal test have been found to involve both spinal and supraspinal level of the CNS^33–35 and MOR antagonists that can effectively block the antinociception produced by MOR agonists are very possible central-acting agents. Hence, such practice has been employed to distinguish and preclude CNS-acting MOR ligands, either agonists or antagonists. More specifically, warm-water tail immersion pain model, which measures changes in latency of response to thermal stimulation at varying doses of tested compounds, has been widely used in our practice^25,36 and others^37–40 to test the in vivo acute agonistic or antagonistic effects of opioids and nonopioids. In this context, all 24 compounds were first evaluated for their acute antinociception tail-withdrawal assay (Figure 2A). Two compounds, 11 and 14, showed moderate antinociceptive effects with 36.9 ± 20.2% and 39.3 ± 17.2% maximum possible effects (MPE) indicating their potential CNS activity. Subsequently, the other 22 compounds exhibiting no apparent CNS antinociception were tested for blockade of morphine’s antinociception effect. As shown in Figure 2B, only two compounds, 10 and 22, significantly antagonized morphine’s antinociception effects to 9.2 ± 5.4% MPE and 32.7 ± 14.6% MPE, respectively. In the follow-up dose–response study (Figure S1), compound 10 was observed to possess an AD₅₀ of 5.3 mg/kg. The rest of the 20 compounds (1–9, 12–13, 15–21, 23–24) showed no evident antagonism against morphine in the tail immersion studies.

Figure 2.

(A) Tail-withdrawal assay. Ten milligrams per kilogram of each compound, vehicle, or morphine was administered (s.c.) to a group of 6 mice. Compared with vehicle: # p < 0.1, F (25, 130) = 5.31. (B) Single-dose antagonism tail-withdrawal assay. Ten milligrams per kilogram of each compound, or vehicle, or 1 mg/kg naloxone (NLX) was given (s.c.) to a group of 6 mice 5 min prior to morphine injection (s.c.). Compared with vehicle + morphine group: **** p < 0.0001, *** p < 0.001, F (23, 119) = 5.47. Mean %MPE value of each group is presented and error bar represents SE.

Preliminary GI Tract Motility Study.

The carmine red dye study, charcoal meal test, and colonic bead expulsion assay (in vivo or ex vivo) are three commonly applied protocols to evaluate GI tract motility.^25,32,41,42 The charcoal meal test and colonic bead expulsion measures small and large intestinal transit, respectively, while the carmine red dye method measures the whole GI tract mobility. The carmine red dye assay, thus, was employed as our preliminary OIC animal model to examine the in vivo effects of potential peripherally restricted MOR ligands.

All of the compounds 1–9, 12–13, 15–21, and 23–24 showed very similar MOR binding affinity (Table 1 and 2), so based on their relatively higher MOR selectivity over the DOR and KOR and low MOR efficacy, compounds 2 and 5 (6α-configuration) and 17, 18, and 19 (6β-configuration) from two subseries were first considered to be evaluated in the red dye studies. Meanwhile, since compounds 2, 5, 17, and 18 all contain a pyrazolyl ring while 19 contains an imidazolyl ring, and compound 17 possessed higher MOR selectivity than 18, therefore, compounds 2, 5, 17, and 19, two from each subseries with structural diversity, were finally chosen to undergo the test first.

The carmine red dye study recorded the time required to defecate a red fecal pellet after oral administration. As shown in Figure 3A, compared to the naïve group, 10 mg/kg morphine elongated the pellet defecation time by 124 min. Then the selected four compounds were administered subcutaneously (s.c.) to the mice to alleviate the constipation. However, none of the compounds, though potent MOR antagonists/low efficacy partial agonists in vitro, were able to reduce the lengthened GI tract transit time (Figure 3A). It was speculated that, because of the high TPSA, their passive permeability may be too low to allow them to distribute from the injection site to the GI tract. Therefore, the administration route was changed from systematically s.c. to oral gavage for shortening the distribution path and increasing the compound concentration at the action site. To exclude the water interference, vehicle was also given orally in the naïve group and morphine group (Figure 3B). As presented in Figure 3B, all four compounds, 2, 5, 17, and 19, successfully reversed morphine-induced constipation via oral administration by 50%, 39%, 40%, and 38%, respectively. No statistical significance was observed from the results, which might be partially due to their possessing similar CNS MPO scores. The oral administration route appeared to be feasible and favorable for these new compounds to elicit in vivo GI effects. Furthermore, these collective results, ineffectiveness via s.c. and effectiveness via p.o., not only indicated their applicability as potentially orally available agents, but also further demonstrated their PNS selectivity.

Figure 3.

(A) Carmine red dye assay for compounds 2, 5, 17 and 19 administered subcutaneously; (B) Carmine red dye assay for compounds 2, 5, 17 and 19 administered orally. All testing compounds were given s.c. or via oral gavage at a dose of 10 mg/kg. Morphine was given subcutaneously at a dose of 10 mg/kg in all groups depicted above. Each group had at least 5 mice. The increased time requirement, compared with the respective vehicle group, was annotated above the bar for each group.

KOR and DOR [³⁵S]-GTPγS Functional Assays.

As noted, KOR activation may cause some adverse effects including sedation and dysphoria⁴³ and DOR agonism may induce convulsion and also cause constipation.⁴⁴ Because compound 2 showed the best anticonstipation effect, and compound 19 exhibited almost the same efficacy in reversing morphine-induced constipation as those of 5 and 17 (Figure 3B), while lower MOR efficacy, 2 (% E_max = 21.5 ± 0.63) and 19 (% E_max = 10.0 ± 1.81) were further characterized for their functionality on KOR and DOR in the [³⁵S]GTPγS binding assays, respectively. The results are shown in Table 3. MNTX was reported to possess EC₅₀ values greater than 10 000 nM for both KOR and DOR, suggesting that it did not behave as a potent agonist for these two receptors.³² Compared to NAP, compound 2 exhibited similar moderate efficacy at the KOR but with a much lower potency, and relatively high efficacy at the DOR with a comparable potency. Meanwhile, compound 19 demonstrated much lower efficacy at the KOR than NAP, and presented reasonably low efficacy at the DOR. Compared to compound 2 that could act as a KOR and DOR dual partial agonist, compound 19 might act as a KOR antagonist and DOR partial agonist with lower efficacy. Therefore, 19 might display fewer adverse effects, and was therefore selected for further carmine red dye studies.

Table 3.

KOR and DOR [³⁵S]-GTPγS Functional Assay Results of Compounds 2 and 19^a

	KOR [35S]-GTPγS binding		DOR [35S]-GTPγS binding
compounds	EC50 (nM)	% Emax of U50,488H	EC50 (nM)	% Emax of SNC80
2	99.0 ± 18.3	49.9 ± 1.22	20.5 ± 4.02	69.3 ± 4.78
19	11.7 ± 2.01	9.03 ± 0.85	19.1 ± 5.91	22.1 ± 0.70
NAPb	28.8 ± 14.4	45.5 ± 4.4	15.2 ± 15.2	10.2 ± 3.1
MNTXc	>10000	NAd	>10000	NA

^aAll values are the mean ± SEM of at least three independent experiments.

^bData have been reported in ref 22 and are presented here for comparison purposes.

^cData have been reported in refs 21 and 32 and are presented here for comparison purposes. Human recombinant opioid receptors were used in the assay.

^dNA: not applicable.

Further Carmine Red Dye Study.

Characterized as an orally active MOR-selective antagonist with no obvious CNS activity, compound 19 was then further examined in the OIC model to compare to NAP and MNTX. MNTX, when injected intraperitoneally, was reported⁴⁵ to be effective to block the antitransit effects of morphine (5 mg/kg, s.c.). In our studies, MNTX was instead evaluated by oral gavage, a more clinically relevant administration route. At the same time, all compounds were also evaluated against the constipation caused by 5 mg/kg morphine. First, 5 mg/kg morphine was injected to validate the significant induction of OIC under our testing conditions. Then compound 19, NAP, and MNTX were administrated by oral gavage. As expected, compound 19 dramatically reversed the inhibitory effects of morphine on GI motility by 75% with reasonable significance (Figure 4). Interestingly, no blockage was observed for NAP and MNTX, when given orally, of the GI motility inhibition by morphine. It is worth mentioning that we observed softened stool and increased pellets of poops in the MNTX group compared to the morphine group, though neither was quantitative. Another observation is that all the tested mice in the MNTX group possessed much less time to defecate the red fecal pellet than two tested mice in the morphine group (Table S2) though the average time of the MNTX group was similar to that of the morphine group. Both observations indicated that MNTX might exhibit somehow insignificant efficacy at a dose of 10 mg/kg in mice, while individual difference of mice may affect the test results. Although NAP was shown with high potency in alleviating OIC in previous studies,²² we postulated that maybe metabolites of NAP played major roles in its OIC reversal activity since the administration route was subcutaneous. However, it might not be the same case with MNTX because the active form of MNTX was MNTX itself and no metabolites were observed.⁴⁶ The absolute oral bioavailability of MNTX in human subjects has not been determined while its bioavailability in male rats was very low (<1%) after oral administration.⁴⁷ Indeed, there have been two studies reported to improve the oral bioavailability of MNTX in rats.^48,49 We think that the very low oral bioavailability of MNTX in rodents, resulting in insufficient concentration of pharmacologically active molecules at the action sites, could be a very possible reason that explains its insignificant in vivo efficacy in the present study.

Figure 4.

Carmine red dye assay for compound 19, NAP, and MNTX using 5 mg/kg morphine. All compounds were given via oral gavage at a dose of 10 mg/kg. Each group had at least 5 mice. The increased time requirement, compared with the vehicle group, was annotated above the bar for each group. Compared with 5 mg/kg morphine group: *p < 0.1, F (14,70) = 5.88).

CONCLUSIONS

By employing a structure-based and physicochemical property-driven drug design strategy, we designed and synthesized a set of NAP derivatives containing pyrazolyl and imidazolyl rings with decreased CNS MPO scores to improve PNS-selectivity. All the newly synthesized compounds maintained high binding affinity for the MOR and reasonable selectivity over the KOR and DOR. In the in vivo studies, most compounds showed marginal CNS effects. Among them, four selected compounds demonstrated efficaciousness in reversing OIC caused by morphine via oral administration but not subcutaneously. Taken together, these newly designed compounds seemed to possess reasonable PNS selectivity as designed. Most importantly, MPO score calculation worked well in our present practice, helping identify PNS-targeting lead compounds in an efficient way (with increased probability of success). As we can see from the in vivo studies, compound 19 was demonstrated to have dramatic improvement over NAP and MNTX in the carmine red dye assay to reverse OIC induced by morphine via p.o. route of administration. As the oral administration route is always clinically preferred, compared to MNTX, orally effective compound 19 may serve as a promising new lead for further development as an OIC treatment option.

EXPERIMENTAL SECTION

Chemistry.

All nonaqueous reactions were carried out under a predried nitrogen gas atmosphere. All solvents and reagents were purchased from either Comi-blocks, Sigma-Aldrich, or Enamine LLC, and were used as received without further purification. Melting points (mp) were measured on an MPA100 OptiMelt automated melting point apparatus without correction. Analytical thin-layer chromatography (TLC) analyses were carried out on Analtech Uniplate F254 plates and flash column chromatography (FCC) was performed using silica gel (230–400 mesh, Merck). ¹H (400 MHz) and ¹³C (100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield 400 Plus spectrometer. Chemical shifts were expressed in δ units (ppm), using TMS as an internal standard, and J values were reported in hertz (Hz). Mass spectra were obtained on an Applied BioSystems 3200 Q trap with a turbo V source for Turbolon Spray. Analytical reversed-phase high performance liquid chromatography (HPLC) was performed on a Varian ProStar 210 system using Agilent Microsorb-MV 100–5 C18 column (250 × 4.6 mm). All analyses were conducted at an ambient temperature with a flow rate of 0.5 mL/min. HPLC eluent condition: acetonitrile/water (with 0.1% trifluoroacetic acid), acetonitrile increased from 40% to 100% in gradient within 20 min of test. The UV detector was set up at 210 nm. The injection volume was 5 μL. The purities of final compounds were calculated as the percentage peak area of the analyzed compound, and retention time (Rt) was presented in minutes. The purity of all newly synthesized compounds was identified as ≥95%.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(3′-pyrazolylcarboxamido)morphinan (1).

¹H NMR (400 MHz, DMSO-d₆) δ 9.36 (brs, 1H, exchangeable), 8.84 (brs, 1H, exchangeable), 7.82 (d, J = 1.4 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H, exchangeable), 6.73 (d, J = 8.1 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 6.59 (d, J = 8.1 Hz, 1H), 6.29 (brs, 1H, exchangeable), 4.73 (d, J = 3.8 Hz, 1H), 4.63–4.56 (m, 1H), 3.90 (d, J = 6.7 Hz, 1H), 3.27–3.23 (m, 1H), 3.11–3.04 (m, 3H), 2.97–2.92 (m, 1H), 2.78–2.66 (m, 1H), 2.47–2.45 (m, 1H), 1.94–1.86 (m, 1H), 1.66 (dd, J = 13.2, 2.6 Hz, 1H), 1.58–1.51 (m, 1H), 1.48–1.42 (m, 1H), 1.09–1.03 ( m, 1H), 1.01–0.92 (m, 1H), 0.73–0.66 (m, 1H), 0.65–0.58 (m, 1H), 0.51–0.45 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.30, 143.13, 142.57, 136.22, 128.57, 126.24, 119.60, 116.98, 115.67, 102.73, 85.19, 66.70, 58.35, 54.47, 45.97, 42.72, 42.58, 42.15, 27.60, 26.66, 20.93, 17.42, 3.13, 2.71. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2180. mp 269.7–271.7 °C dec % Purity: 97.78. Rt: 6.709 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(3′-pyrazolylacetamido)morphinan (2).

¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (brs, 1H, exchangeable), 8.04 (d, J = 7.9 Hz, 1H, exchangeable), 7.75 (m, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 6.30 (m, 1H), 4.59 (d, J = 3.9 Hz, 1H), 4.44–4.37 (m, 1H), 3.92 (d, J = 6.8 Hz, 1H), 3.62 (s, 2H), 3.31–3.22 (m, 2H), 3.08–2.93 (m, 3H), 2.75–2.65 (m, 1H), 2.49–2.41 (m, 2H), 1.92–1.84 (m, 1H), 1.62–1.58 (m, 1H), 1.43–1.37 (m, 2H), 0.99–0.92 (m, 1H), 0.70–0.65 (m, 1H), 0.63–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.42–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.87, 146.00, 142.90, 138.85, 132.77, 128.73, 122.08, 119.09, 118.32, 104.88, 87.40, 69.32, 60.92, 56.97, 45.15, 33.66, 30.11, 29.16, 23.47, 19.66, 5.67, 5.14, 2.54. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2355, [M + Na]⁺ (m/z): 473.2178. mp 253.7–254.5 °C dec % Purity: 100. Rt: 6.443 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3′-(pyrazolyl-3″-yl)propanamido]morphinan (3).

¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (brs, 2H, exchangeable), 8.87 (brs, 1H, exchangeable), 7.84–7.82 (m, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 6.32 (d, J = 2.2 Hz, 1H 4.57 (d, J = 3.9 Hz, 1H), 4.43–4.36 (m, 1H), 3.92 (d, J = 6.8 Hz, 1H), 3.30–3.21 (m, 2H), 3.07–2.93 (m, 3H), 2.89 (t, J = 7.4 Hz, 2H), 2.75–2.65 (m, 1H), 2.56 (t, J = 7.3 Hz, 2H), 2.48–2.41 (m, 1H), 1.91–1.83 (m, 1H), 1.62–1.58 (m, 1H), 1.42–1.32 (m, 2H), 1.11–1.04 (m, 1H), 0.98–0.87 (m, 1H), 0.71–0.62 (m, 1H), 0.61–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.42–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.37, 147.14, 146.02, 138.81, 133.66, 128.73, 122.08, 119.04, 118.32, 104.16, 87.47, 69.32, 60.93, 56.97, 45.19, 45.13, 44.90, 33.98, 30.11, 29.15, 23.47, 21.73, 19.71, 5.67, 5.15, 2.54. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2477, [M + Na]⁺ (m/z): 487.2296. mp 255.3–256.4 °C dec % Purity: 100. Rt: 6.708 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(4′-pyrazolylcarboxamido)morphinan (4).

¹H NMR (400 MHz, DMSO-d₆) δ 8.85 (brs, 1H, exchangeable), 8.13 (s, 2H), 7.72 (d, J = 7.7 Hz, 1H, exchangeable), 6.71 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 6.32 (brs, 1H, exchangeable), 4.71 (d, J = 3.8 Hz, 1H), 4.60–4.52 (m, 2H), 3.92 (d, J = 6.8 Hz, 1H), 3.32–3.24 (m, 2H), 3.10–3.03 (m, 2H), 2.98–2.92 (m, 1H), 2.77–2.67 (m, 1H), 2.47–2.43 (m, 1H), 1.95–1.87 (m, 1H), 1.61 (dd, J = 12.8, 1.9 Hz, 1H), 1.49–1.41 (m, 2H), 1.18–1.13 (m, 1H), 0.72–0.66 (m, 1H), 0.64–0.58 (m, 1H), 0.52–0.46 (m, 1H), 0.42–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 159.25, 146.88, 143.43, 136.01, 131.73, 126.20, 119.63, 116.65, 115.64, 114.95, 84.97, 66.76, 62.38, 58.43, 54.43, 42.62, 27.70, 26.60, 20.92, 16.79, 12.58, 3.12, 2.68. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2181. mp 259.1–261.2 °C dec % Purity: 98.45. Rt: 6.261 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(4′-pyrazolylacetamido)morphinan (5).

¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (brs, 1H, exchangeable), 7.94 (d, J = 8.0 Hz, 1H, exchangeable), 7.72 (s, 2H), 6.73 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 6.33 (brs, 2H, exchangeable), 4.58 (d, J = 3.9 Hz, 1H), 4.42–4.35 (m, 1H), 3.92 (d, J = 7.0 Hz, 1H), 3.39 (s, 2H), 3.31–3.22 (m, 2H), 3.08–3.01 (m, 2H), 2.98–2.92 (m, 1H), 2.75–2.65 (m, 1H), 2.47–2.41 (m, 1H), 1.91–1.83 (m, 1H), 1.62–1.58 (m, 1H), 1.42–1.36 (m, 2H), 1.06–1.03 (m, 1H), 0.98–0.89 (m, 1H), 0.71–0.65 (m, 1H), 0.63–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.41–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.61, 146.02, 138.83, 132.82, 128.74, 122.07, 119.05, 118.29, 87.39, 69.31, 60.93, 56.96, 48.55, 45.18, 45.17, 45.12, 45.07, 30.94, 30.12, 29.17, 23.46, 19.65, 5.67, 5.14, 2.53. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2351, [M + Na]⁺ (m/z): 473.2165. mp 252.5–253.6 °C dec % Purity: 100. Rt: 6.337 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3′-(pyrazolyl-4″-yl)propanamido]morphinan (6).

¹H NMR (400 MHz, DMSO-d₆) δ 9.16 (brs, 2H, exchangeable), 8.87 (brs, 1H, exchangeable), 7.75 (d, J = 8.0 Hz, 1H, exchangeable), 7.71 (s, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 4.56 (d, J = 3.8 Hz, 1H), 4.44–4.36 (m, 1H), 3.92 (d, J = 6.8 Hz, 1H), 3.35–3.22 (m, 2H), 3.07–2.93 (m, 3H), 2.74–2.68 (m, 3H), 2.46–2.42 (m, 3H), 1.91–1.82 (m, 1H), 1.62–1.58 (m, 1H), 1.41–1.30 (m, 2H), 1.11–1.04 (m, 1H), 0.97–0.86 (m, 1H), 0.71–0.65 (m, 1H), 0.64–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.42–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.88, 146.03, 138.79, 132.06, 128.73, 122.08, 119.83, 119.04, 118.32, 87.53, 69.31, 60.95, 56.97, 45.19, 45.13, 44.80, 36.04, 30.11, 29.17, 23.46, 19.74, 19.65, 5.67, 5.14, 2.54. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2511, [M + Na]+ (m/z): 487.2326. mp 260.8–261.5 °C dec % Purity: 95.42. Rt: 6.658 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(5′-imidazolylcarboxamido)morphinan (7).

¹H NMR (400 MHz, DMSO-d₆) δ 9.24 (brs, 1H, exchangeable), 8.91 (brs, 2H, including 1H exchangeable), 8.49 (brs, 1H, exchangeable), 8.37 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 6.43 (brs, 1H, exchangeable), 4.70 (d, J = 3.8 Hz, 1H), 4.65–4.58 (m, 1H), 3.97 (d, J = 6.8 Hz, 1H), 3.41–3.35 (m, 1H), 3.33–3.23 (m, 2H), 3.10–3.03 (m, 2H), 3.01–2.96 (m, 1H), 2.76–2.67 (m, 1H), 2.55–2.45 (m, 1H), 1.98–1.90 (m, 1H), 1.65–1.62 (m, 1H), 1.55–1.42 (m, 2H), 1.18–1.12 (m, 1H), 0.72–0.66 (m, 1H), 0.64–0.58 (m, 1H), 0.52–0.46 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.84, 157.24, 145.86, 138.62, 135.61, 128.60, 122.17, 120.47, 119.31, 118.31, 87.04, 69.22, 60.79, 56.91, 45.50, 45.19, 45.09, 30.10, 28.96, 23.43, 19.31, 5.64, 5.19, 2.51. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]+ (m/z): 437.2167. mp 263.7–264.9 °C dec % Purity: 99.36. Rt: 6. 970 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(5′-imidazolylacetamido)morphinan (8).

¹H NMR (400 MHz, DMSO-d₆) δ 14.37 (brs, 2H, exchangeable), 9.28 (s, 1H, exchangeable), 9.01 (d, J = 1.2 Hz, 1H), 8.85 (brs, 1H, exchangeable), 8.24 (d, J = 8.0 Hz, 1H, exchangeable), 7.48 (s, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 6.33 (s, 1H, exchangeable), 4.59 (d, J = 3.9 Hz, 1H), 4.45–4.38 (m, 1H), 3.92 (d, J = 6.8 Hz, 1H), 3.71 (s, 2H), 3.26–3.22 (m, 2H), 3.08–3.01 (m, 2H), 2.98–2.93 (m, 1H), 2.72–2.66 (m, 1H), 2.45–2.40 (m, 1H), 1.92–1.84 (m, 1H), 1.60 (dd, J = 12.8, 2.1 Hz, 1H), 1.46–1.37 (m, 2H), 1.11–1.03 (m, 1H), 1.01–0.92 (m, 1H), 0.71–0.65 (m, 1H), 0.63–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.42–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.82, 145.73, 138.60, 133.49, 128.65, 127.60, 122.09, 119.25, 118.04, 116.83, 87.22, 69.20, 60.82, 56.91, 48.44, 45.27, 45.07, 30.74, 30.07, 28.93, 23.37, 19.40, 5.60, 5.17, 2.45. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2360, [M + Na]⁺ (m/z): 473.2178. mp 288.2–290.1 °C dec % Purity: 96.87. Rt: 6.859 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3′-(imidazol-5″-yl)propanamido]morphinan (9).

¹H NMR (400 MHz, DMSO-d₆) δ 14.33 (brs, 1H, exchangeable), 9.24 (brs, 1H, exchangeable), 8.88 (d, J = 1.2 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H, exchangeable), 7.36 (d, J = 0.7 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.32 (s, 1H, exchangeable), 4.55 (d, J = 4.0 Hz, 1H), 4.43–4.35 (m, 1H), 3.91 (d, J = 6.8 Hz, 1H), 3.35–3.30 (m, 2H), 3.26–3.21 (m, 1H), 3.06–3.00 (m, 2H), 2.98–2.93 (m, 1H), 2.88 (t, J = 7.3 Hz, 2H), 2.72–2.65 (m, 1H), 2.57 (t, J = 7.3 Hz, 2H), 2.45–2.40 (m, 1H), 1.91–1.82 (m, 1H), 1.59 (dd, J = 12.8, 2.0 Hz, 1H), 1.41–1.32 (m, 2H), 0.98–0.86 (m, 1H), 0.71–0.64 (m, 1H), 0.63–0.57 (m, 1H), 0.50–0.44 (m, 1H), 0.41–0.35 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.09, 145.91, 138.60, 133.20, 132.65, 128.71, 122.16, 119.13, 118.23, 115.34, 87.41, 69.21, 60.83, 56.93, 48.43, 45.07, 44.86, 33.37, 30.07, 29.03, 23.43, 20.15, 19.59, 5.64, 5.16, 2.52. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2519. mp 286.9–288.4 °C dec % Purity: 97.03. Rt: 6.692 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(2′-imidazolylcarboxamido)morphinan (10).

¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (brs, 1H, exchangeable), 8.59–8.46 (m, 1H, exchangeable), 7.62 (s, 1H), 7.56 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 8.2 Hz, 1H), 6.42 (brs, 1H, exchangeable), 4.75 (d, J = 3.9 Hz, 1H), 4.64–4.59 (m, 1H), 3.98–3.97 (m, 1H), 3.38–3.23 (m, 2H), 3.11–3.04 (m, 2H), 3.01–2.97 (m, 1H), 2.76–2.67 (m, 1H), 2.54–2.44 (m, 1H), 1.98–1.89 (m, 1H), 1.66–1.58 (m, 2H), 1.52–1.45 (m, 1H), 1.11–1.07 (m, 2H), 0.72–0.66 (m, 1H), 0.65–0.58 (m, 1H), 0.52–0.46 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.76, 145.60, 138.65, 137.81, 128.55, 122.25, 122.17, 119.52, 118.27, 86.84, 69.20, 60.76, 56.92, 48.43, 45.84, 45.23, 45.12, 29.95, 28.83, 23.42, 19.49, 5.63, 5.19, 2.50. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2196. mp 268.4–270.2 °C dec % Purity: 98.60. Rt: 6.675 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(2′-imidazolylacetamido)morphinan (11).

¹H NMR (400 MHz, DMSO-d₆) δ 14.31 (s, 2H, exchangeable), 9.30 (s, 1H, exchangeable), 8.87 (brs, 1H, exchangeable), 8.50 (d, J = 8.0 Hz, 1H, exchangeable), 7.58 (s, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 6.38 (brs, 1H, exchangeable), 4.59 (d, J = 3.9 Hz, 1H), 4.47–4.39 (m, 1H), 4.08 (s, 2H), 3.93 (d, J = 6.8 Hz, 1H), 3.31–3.22 (m, 2H), 3.08–3.01 (m, 2H), 2.98–2.93 (m, 1H), 2.75–2.66 (m, 1H), 2.45–2.40 (m, 1H), 1.93–1.85 (m, 1H), 1.63–1.60 (m, 1H), 1.48–1.37 (m, 2H), 1.10–1.05 (m, 1H), 1.02–0.93 (m, 1H), 0.71–0.65 (m, 1H), 0.63–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.42–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.08, 143.28, 138.88, 136.15, 126.15, 119.64, 116.79, 116.34, 115.63, 84.72, 66.72, 58.27, 54.41, 43.00, 42.62, 29.53, 29.11, 27.59, 26.43, 20.90, 16.90, 3.13, 2.70. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2361. mp 264.1–265.8 °C dec % Purity: 96.56. Rt: 6.849 min.

17-Cyclopropylmethyl-3,14β-d/hydroxy-4,5α-epoxy-6α-[(3′-(imidazol-2″-yl)propanamldo]morphinan (12).

¹H NMR (400 MHz, DMSO-d₆) δ 14.28 (brs, 2H, exchangeable), 9.23 (brs, 1H, exchangeable), 8.86 (brs, 1H, exchangeable), 7.97 (d, J = 8.0 Hz, 1H, exchangeable), 7.52 (s, 2H), 6.73 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 6.33 (brs, 1H, exchangeable), 4.55 (d, J = 3.9 Hz, 1H), 4.41–4.33 (m, 1H), 3.93 (d, J = 6.6 Hz, 1H), 3.28–3.21 (m, 1H), 3.12 (t, J = 7.1 Hz, 2H), 3.06–2.95 (m, 4H), 2.77 (t, J = 7.0 Hz, 2H), 2.72–2.65 (m, 1H), 2.47–2.40 (m, 1H), 1.91–1.83 (m, 1H), 1.60–1.57 (m, 1H), 1.41–1.35 (m, 2H), 1.08–1.04 (m, 1H), 0.96–0.87 (m, 1H), 0.71–0.64 (m, 1H), 0.63–0.57 (m, 1H), 0.51–0.45 (m, 1H), 0.41–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.10, 144.34, 143.29, 136.05, 126.22, 119.70, 116.80, 116.02, 115.87, 115.60, 84.87, 66.72, 58.31, 54.45, 45.99, 42.59, 42.52, 29.09, 27.58, 26.50, 20.92, 18.68, 18.16, 16.96, 3.15, 2.75. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2510, [M + Na]⁺ (m/z): 487.2321. mp 279.0–281.4 °C dec % Purity: 98.12. Rt: 7.021 min.

17-Cyclopropylmethyl-3,14β-d/hydroxy-4,5α-epoxy-6β-(3′-pyrazolylcarboxamldo)morphinan (13).

¹H NMR (400 MHz, DMSO-d₆) δ 9.33 (brs, 1H, exchangeable), 8.86 (s, 1H, exchangeable), 8.39 (d, J = 8.3 Hz, 1H, exchangeable), 7.76 (d, J = 1.8 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.68 (d, J = 1.9 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.19 (brs, 1H, exchangeable), 4.90 (d, J = 7.7 Hz, 1H), 3.87 (d, J = 5.1 Hz, 1H), 3.67–3.62 (m, 2H), 3.36–3.31 (m, 2H), 3.11 (d, J = 5.9 Hz, 1H), 3.06–3.03 (m, 1H), 2.89–2.85 (m, 1H), 2.47–2.44 (m, 1H), 1.97–1.88 (m, 1H), 1.77–1.74 (m, 1H), 1.57–1.53 (m, 1H), 1.46–1.38 (m, 2H), 1.12–1.04 (m, 1H), 0.70–0.66 (m, 1H), 0.63–0.59 (m, 1H), 0.54–0.49 (m, 1H), 0.45–0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.66, 139.53, 138.57, 128.71, 127.14, 118.12, 116.86, 115.32, 102.64, 87.24, 67.12, 59.06, 54.11, 47.77, 45.95, 43.84, 43.06, 39.69, 26.93, 24.73, 21.14, 20.36, 3.11, 2.63. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2193, [M + Na]⁺ (m/z): 459.2004. mp 268.7–270.2 °C dec % Purity: 99.99. Rt: 6.197 min.

17-Cyclopropylmethyl-3,14β-d/hydroxy-4,5α-epoxy-6β-(3′-pyrazolylacetamldo)morphinan (14).

¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (brs, 1H, exchangeable), 8.44 (d, J = 7.4 Hz, 1H, exchangeable), 7.75 (s, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 6.28 (s, 1H), 4.60 (d, J = 7.8 Hz, 1H), 3.86 (d, J = 5.1 Hz, 1H), 3.59–3.54 (m, 2H), 3.44–3.36 (m, 1H), 3.34–3.27 (m, 2H), 3.08–3.01 (m, 2H), 2.89–2.83 (m, 1H), 2.46–2.37 (m, 2H), 1.81–1.70 (m, 2H), 1.53–1.49 (m, 1H), 1.43–1.41 (m, 1H), 1.36–1.29 (m, 1H), 1.11–1.04 (m, 1H), 0.70–0.66 (m, 1H), 0.62–0.55 (m, 1H), 0.54–0.48 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.28, 142.73, 141.95, 141.04, 132.49, 129.53, 120.60, 119.37, 117.82, 104.86, 89.76, 69.56, 61.50, 56.61, 50.78, 46.33, 45.52, 34.02, 29.20, 27.22, 23.44, 22.86, 5.60, 5.10, 2.51. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2339, [M + Na]⁺ (m/z): 473.2152. mp 250.2–251.4 °C dec % Purity: 99.20. Rt: 6.401 min.

17-Cyclopropylmethyl-3,14β-d/hydroxy-4,5α-epoxy-6β-[(3′-(pyrazolyl-3′-yl)propanamldo]morphinan (15).

¹H NMR (400 MHz, DMSO-d₆) δ 8.85 (brs, 1H, exchangeable), 8.22 (brs, 1H, exchangeable), 7.74 (d, J = 2.0 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 6.22 (d, J = 2.0 Hz, 1H), 4.55 (d, J = 7.8 Hz, 1H), 3.85 (brs, 1H), 3.44–3.38 (m, 1H), 3.34–3.27 (m, 2H), 3.08–3.01 (m, 2H), 2.90–2.84 (m, 3H), 2.47–2.32 (m, 4H), 1.72–1.65 (m, 2H), 1.51–1.41 (m, 2H), 1.36–1.28 (m, 1H), 1.10–1.02 (m, 1H), 0.70–0.64 (m, 1H), 0.62–0.55 (m, 1H), 0.53–0.47 (m, 1H), 0.43–0.36 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.95, 147.52, 142.59, 141.78, 134.15, 130.14, 121.05, 119.68, 118.38, 104.69, 90.37, 70.17, 62.03, 57.13, 51.15, 46.96, 46.03, 34.97, 29.78, 27.78, 24.13, 23.48, 22.13, 6.21, 5.59, 3.10. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2503, [M + Na]⁺ (m/z): 487.2322. mp 216.8–217.7 °C dec % Purity: 100. Rt: 6.812 min.

17-Cyclopropylmethyl-3,14β-d/hydroxy-4,5α-epoxy-6β-(4′-pyrazolylcarboxamldo)morphinan (16).

¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (brs, 1H, exchangeable), 8.26 (d, J = 8.1 Hz, 1H, exchangeable), 8.04 (s, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.18 (brs, 1H, exchangeable), 4.73 (d, J = 7.9 Hz, 1H), 3.86 (d, J = 5.0 Hz, 1H), 3.66–3.57 (m, 1H), 3.35–3.31 (m, 2H), 3.11–3.03 (m, 2H), 2.87–2.83 (m, 1H), 2.45–2.40 (m, 2H), 1.85–1.73 (m, 2H), 1.59–1.55 (m, 1H), 1.48–1.36 (m, 2H), 1.10–1.04 (m, 1H), 0.69–0.64 (m, 1H), 0.62–0.55 (m, 1H), 0.54–0.48 (m, 1H), 0.44–0.38 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.78, 142.03, 141.10, 133.90, 129.60, 120.61, 119.32, 117.80, 117.72, 89.87, 69.60, 61.58, 56.61, 50.25, 48.44, 46.38, 45.53, 29.28, 27.25, 23.89, 22.93, 5.63, 5.12, 2.53. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2172, [M + Na]⁺ (m/z): 459.1988. mp 275.9–277.1 °C dec % Purity: 98.78. Rt: 6.332 min.

17-Cyclopropylmethyl-3,14β-dlhydroxy-4,5α-epoxy-6β-(4′-pyrazolylacetamldo)morphinan (17).

¹H NMR (400 MHz, DMSO-d₆) δ 8.84 (brs, 1H, exchangeable), 8.21 (d, J = 8.0 Hz, 1H, exchangeable), 7.59 (s, 2H), 6.71 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.26 (brs, 1H, exchangeable), 4.58 (d, J = 7.8 Hz, 1H), 3.84 (d, J = 5.3 Hz, 1H), 3.44–3.32 (m, 2H), 3.30–3.25 (m, 3H), 3.08–2.97 (m, 2H), 2.88–2.82 (m, 1H), 2.46–2.36 (m, 2H), 1.78–1.68 (m, 2H), 1.52–1.46 (m, 1H), 1.44–1.41 (m, 1H), 1.36–1.24 (m, 1H), 1.09–1.01 (m, 1H), 0.70–0.64 (m, 1H), 0.61–0.55 (m, 1H), 0.52–0.48 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.87, 165.97, 142.00, 141.07, 132.75, 129.56, 120.58, 119.30, 117.81, 114.26, 89.81, 69.57, 61.50, 56.60, 50.65, 46.35, 45.50, 31.23, 29.22, 27.24, 23.51, 22.89, 5.63, 5.10, 2.54. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2339, [M + Na]⁺ (m/z): 473.2159. mp 231.8–233.2 °C dec % Purity: 96.02. Rt: 6.338 min.

17-Cyclopropylmethyl-3,14β-dlhydroxy-4,5α-epoxy-6β-[(3′-(pyrazolyl-4″-yl)propanamldo]morphinan (18).

¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (brs, 1H, exchangeable), 8.18 (d, J = 7.9 Hz, 1H, exchangeable), 7.70 (s, 2H), 6.73 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.26 (brs, 2H, exchangeable), 4.54 (d, J = 7.9 Hz, 1H), 3.87 (d, J = 4.9 Hz, 1H), 3.45–3.37 (m, 1H), 3.33–3.26 (m, 2H), 3.08–3.01 (m, 2H), 2.90–2.84 (m, 1H), 2.71–2.67 (m, 2H), 2.46–2.40 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.74–1.64 (m, 2H), 1.48–1.40 (m, 2H), 1.35–1.28 (m, 1H), 1.11–1.04 (m, 1H), 0.70–0.64 (m, 1H), 0.62–0.54 (m, 1H), 0.53–0.48 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.36, 141.99, 141.02, 132.13, 129.57, 120.63, 119.58, 119.34, 117.84, 89.89, 69.57, 61.48, 56.60, 50.38, 46.31, 45.48, 36.55, 29.24, 27.20, 23.52, 22.86, 19.51, 5.60, 5.10, 2.51. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2490. mp 229.8–231.0 °C dec % Purity: 99.85. Rt: 6.526 min.

17-Cyclopropylmethyl-3,14β-dlhydroxy-4,5α-epoxy-6β-(5′-imldazolylcarboxamldo)morphinan (19).

¹H NMR (400 MHz, DMSO-d₆) δ 9.36 (brs, 1H, exchangeable), 9.14 (brs, 1H, exchangeable), 8.88 (brs, 1H, exchangeable), 8.80 (brs, 1H), 8.20 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.26 (s, 1H, exchangeable), 4.84 (d, J = 7.8 Hz, 1H), 3.89 (d, J = 5.4 Hz, 1H), 3.70–3.61 (m, 2H), 3.36–3.33 (m, 1H), 3.10 (d, J = 5.9 Hz, 1H), 3.06–3.03 (m, 1H), 2.89–2.85 (m, 1H), 2.46–2.40 (m, 2H), 1.95–1.85 (m, 1H), 1.80–1.77 (m, 1H), 1.60–1.56 (m, 1H), 1.46–1.36 (m, 2H), 1.11–1.05 (m, 1H), 0.71–0.65 (m, 1H), 0.62–0.56 (m, 1H), 0.54–0.48 (m, 1H), 0.44–0.38 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 157.07, 141.89, 141.22, 135.61, 129.52, 128.36, 120.65, 120.19, 119.45, 117.86, 89.50, 69.56, 61.45, 56.61, 50.79, 46.36, 45.60, 29.33, 27.19, 23.58, 22.93, 5.66, 5.12, 2.57. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2175, [M + Na]⁺ (m/z): 459.1992. mp 270.8–272.5 °C dec % Purity: 99.62. Rt: 6.885 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(5′-imidazolylacetamido)morphinan (20).

¹H NMR (400 MHz, DMSO-d₆) δ 14.37 (brs, 2H, exchangeable), 9.36 (s, 1H, exchangeable), 9.00 (d, J = 1.2 Hz, 1H), 8.84 (brs, 1H, exchangeable), 8.59 (d, J = 7.9 Hz, 1H, exchangeable), 7.47 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.26 (s, 1H, exchangeable), 4.60 (d, J = 7.8 Hz, 1H), 3.86 (d, J = 5.3 Hz, 1H), 3.66 (s, 2H), 3.46–3.37 (m, 2H), 3.27–3.22 (m, 1H), 3.08–3.02 (m, 2H), 2.89–2.84 (m, 1H), 2.44–2.38 (m, 2H), 1.83–1.71 (m, 2H), 1.54–1.51 (m, 1H), 1.44–1.42 (m, 1H), 1.36–1.24 (m, 1H), 1.09–1.05 (m, 1H), 0.69–0.64 (m, 1H), 0.62–0.57 (m, 1H), 0.53–0.48 (m, 1H), 0.43–0.38 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.88, 142.03, 141.30, 133.64, 129.59, 127.51, 120.58, 119.26, 117.88, 117.04, 89.75, 69.64, 61.55, 56.66, 51.09, 46.46, 45.58, 31.10, 29.26, 27.30, 23.57, 22.98, 5.71, 5.09, 2.61. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2345, [M + Na]⁺ (m/z): 473.2158. mp 292.4–294.3 °C dec % Purity: 98.23. Rt: 6.525 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-(imidazol-5″-yl) propanamido]morphinan (21).

¹H NMR (400 MHz, DMSO-d₆) δ 14.50 (brs, 1H, exchangeable), 14.32 (brs, 1H, exchangeable), 9.35 (brs, 1H, exchangeable), 8.98 (d, J = 1.0 Hz, 1H), 8.85 (brs, 1H, exchangeable), 8.32 (d, J = 7.9 Hz, 1H, exchangeable), 7.38 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.26 (brs, 1H, exchangeable), 4.55 (d, J = 7.8 Hz, 1H), 3.87 (d, J = 5.0 Hz, 1H), 3.33–3.26 (m, 4H), 3.07–3.02 (m, 2H), 2.87 (t, J = 7.2 Hz, 3H), 2.46–2.38 (m, 3H), 1.76–1.67 (m, 2H), 1.48–1.40 (m, 2H), 1.34–1.27 (m, 1H), 1.11–1.04 (m, 1H), 0.68–0.64 (m, 1H), 0.62–0.57 (m, 1H), 0.54–0.49 (m, 1H), 0.43–0.38 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.11, 142.09, 141.28, 133.27, 132.54, 129.64, 120.59, 119.20, 117.89, 115.43, 89.87, 69.66, 61.48, 56.62, 50.64, 46.45, 45.53, 33.90, 29.30, 27.26, 23.64, 22.98, 20.04, 5.72, 5.10, 2.61. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2516, [M + Na]⁺ (m/z): 487.2334. mp 278.7–280.9 °C dec % Purity: 99.23. Rt: 6.977 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(2′-imidazolylcarboxamido)morphinan (22).

¹H NMR (400 MHz, DMSO-d₆) δ 9.53 (brs, 1H, exchangeable), 9.33 (brs, 1H, exchangeable), 8.85 (brs, 1H, exchangeable), 7.56 (s, 2H), 6.73 (d, J = 8.1 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 6.22 (brs, 1H, exchangeable), 4.87 (d, J = 7.8 Hz, 1H), 3.86 (d, J = 5.3 Hz, 1H), 3.72–3.63 (m, 1H), 3.36–3.29 (m, 2H), 3.13–3.03 (m, 2H), 2.88–2.84 (m, 1H), 2.46–2.40 (m, 1H), 1.98–1.87 (m, 1H), 1.79–1.76 (m, 1H), 1.63–1.59 (m, 1H), 1.50–1.39 (m, 2H), 1.06–1.02 (m, 1H), 0.71–0.65 (m, 1H), 0.63–0.56 (m, 1H), 0.54–0.48 (m, 1H), 0.44–0.38 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.82, 141.95, 141.14, 139.86, 129.58, 123.69, 120.63, 119.41, 117.85, 89.61, 69.59, 61.50, 56.61, 50.58, 46.35, 45.60, 29.45, 27.22, 23.52, 22.89, 5.63, 5.11, 2.54. HRMS calculated for C₂₄H₂₈N₄O₄ m/z: 436.2111. Found [M + H]⁺ (m/z): 437.2177. mp 270.7–272.4 °C dec % Purity: 96.44. Rt: 6.439 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(2′-imidazolylacetamido)morphinan (23).

¹H NMR (400 MHz, DMSO-d₆) δ 14.17 (brs, 2H, exchangeable), 9.35 (s, 1H, exchangeable), 8.81 (d, J = 7.7 Hz, 2H, exchangeable), 7.57 (s, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.20 (s, 1H, exchangeable), 4.59 (d, J = 7.8 Hz, 1H), 4.00 (s, 2H), 3.84 (d, J = 5.4 Hz, 1H), 3.45–3.37 (m, 3H), 3.08–3.02 (m, 2H), 2.88–2.82 (m, 1H), 2.46–2.42 (m, 2H), 1.82–1.70 (m, 2H), 1.58–1.54 (m, 1H), 1.45–1.43 (m, 1H), 1.38–1.30 (m, 1H), 1.11–1.04 (m, 1H), 0.71–0.65 (m, 1H), 0.62–0.55 (m, 1H), 0.53–0.47 (m, 1H), 0.43–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.61, 141.89, 141.17, 129.50, 120.95, 120.62, 119.41, 118.88, 117.85, 89.60, 69.52, 61.44, 56.62, 51.23, 46.35, 45.57, 32.15, 29.22, 27.25, 23.46, 22.92, 5.66, 5.12, 2.58. HRMS calculated for C₂₅H₃₀N₄O₄ m/z: 450.2267. Found [M + H]⁺ (m/z): 451.2320, [M + Na]⁺ (m/z): 473.2133. mp 275.6–277.0 °C dec % Purity: 97.80. Rt: 6.210 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-(imidazol-2″-yl)propanamido]morphinan (24).

¹H NMR (400 MHz, DMSO-d₆) δ 14.28 (s, 2H, exchangeable), 9.38 (brs, 1H, exchangeable), 8.86 (brs, 1H, exchangeable), 8.41 (d, J = 7.9 Hz, 1H, exchangeable), 7.51 (s, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 6.27 (s, 1H, exchangeable), 4.56 (d, J = 7.8 Hz, 1H), 3.87 (d, J = 5.2 Hz, 1H), 3.37–3.26 (m, 3H), 3.12–3.01 (m, 4H), 2.89–2.84 (m, 1H), 2.71 (t, J = 7.2 Hz, 2H), 2.45–2.37 (m, 2H), 1.77–1.67 (m, 2H), 1.48–1.40 (m, 2H), 1.32–1.26 (m, 1H), 1.10–1.04 (m, 1H), 0.70–0.63 (m, 1H), 0.61–0.56 (m, 1H), 0.54–0.48 (m, 1H), 0.42–0.37 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.54, 146.90, 142.03, 141.26, 129.58, 120.55, 119.23, 118.41, 117.89, 89.79, 69.64, 61.53, 56.63, 50.78, 46.44, 45.54, 31.99, 29.26, 27.27, 23.61, 22.96, 21.13, 5.71, 5.09, 2.60. HRMS calculated for C₂₆H₃₂N₄O₄ m/z: 464.2424. Found [M + H]⁺ (m/z): 465.2502, [M + Na]⁺ (m/z): 487.2324. mp 285.1–287.2 °C dec % Purity: 97.83. Rt: 6.984 min.

Biological Evaluation. Drugs.

The free base of naltrexone was provided through NIDA Drug Supply Program. All drugs and test compounds were dissolved in sterile-filtered distilled/deionized water. All other reagents and radioligands were purchased from either Sigma-Aldrich or PerkinElmer.

In Vitro Competitive Radioligand Binding Assay.

The competition binding assay was conducted using the monoclonal mouse opioid μ or κ receptor expressed in CHO cell lines (monoclonal human δ opioid receptor was used in the DOR assay). In this assay, 30 μg of membrane protein was incubated with the corresponding radioligand in the presence of different concentrations of test compounds in TME buffer (50 mM Tris, 3 mM MgCl₂, and 0.2 mM EGTA, pH 7.4) for 1.5 h at 30 °C. The bound radioligand was separated by filtration using the Brandel harvester. Specific (i.e., opioid receptor-related) binding to the MOR, KOR, and DOR was determined as the difference in binding obtained in the absence and presence of 5 μM of naltrexone, U50,488, and SNC80, respectively. Relative affinity values (IC₅₀) were determined by fitting displacement binding inhibition values by nonlinear regression using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA), where %inhibition value was calculated as follows: %inhibition = 100% – (binding in the presence of tested compound – nonspecific binding)/specific binding × 100%. The IC₅₀ values were converted to K_i values using the Cheng-Prusoff equation: K_i = IC₅₀/ [1 + ([L*]/K_D)], where [L*] is the concentration of the radioligand and K_D is the K_D of the radioligand.50

In Vitro [³⁵S]GTPγS Functional Assay.

The [³⁵S]GTPγS functional assay was conducted to determine the efficacy of the compounds at the MOR, KOR and DOR. In this assay, 10 μg of MOR-CHO/KOR-CHO/DOR-CHO membrane protein was incubated in a final volume of 500 μL containing TME with 100 mM NaCl, 20 μM GDP, 0.1 nM [³⁵S]GTPγS, and varying concentrations of the compound under investigation for 1.5 h in a 30 °C water bath. The Bradford protein assay was utilized to determine and adjust the concentration of protein required for the assay. Nonspecific binding was determined with 20 μM unlabeled GTPγS. Furthermore, 3 μM DAMGO/U50488H/SNC80 was included in the assay as the maximally effective concentration of a full agonist for the MOR/KOR/DOR. After incubation, the bound radioactive ligand was separated from the free radioligand by filtration through a GF/B glass fiber filter paper using a Brandel harvester. Bound radioactivity was determined by liquid scintillation counting. All assays were determined in duplicate and repeated at least three times. Net stimulated [³⁵S]GTPγS binding was defined as agonist-stimulated minus basal binding in the absence of agonist. Percent of DAMGO/U50488H/SNC80 stimulated [³⁵S]GTPγS binding was defined as (net-stimulated binding by ligand/net-stimulated binding by 3 μM DAMGO U50488H/SNC80) × 100%.

Animals.

5–8 week old 25–35 g male Swiss Webster mice were housed in cages (5 maximal per cage) in animal care quarters and maintained at 22 ± 2 °C on a 12 h light-dark cycle. Food (standard chow) and water were available ad libitum. The mice were brought to the lab (22 ± 2 °C, 12 h light-dark cycle) and allowed at least 18 h to recover from transport. Protocols and procedures were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University Medical Center and comply with the recommendations of the International Association for the Study of Pain. All mice were used only once.

Tail-Withdrawal Study.

The tail-withdrawal test was performed using a water bath with the temperature maintained at 56 ± 0.1 °C. Baseline latency was measured before any injections. Each mouse was gently wrapped in a cloth with only the tail exposed. The distal one-third of the tail was immersed perpendicularly in water, and the mouse rapidly flicked the tail from the bath was seen as the first sign of discomfort. The duration of time the tail remained in the water bath was counted as the baseline latency. Untreated mice with baseline latency reaction times ranging from 2 to 4 s were used. Test latency was obtained 20 min later after each injection. A 10-s maximum cutoff latency was used to prevent any tissue damage. Antinociception was quantified as the percentage of maximal possible effect (%MPE), which was calculated as %MPE = [(test latency – control latency)/(10 – control latency)] × 100. The %MPE value was calculated for each mouse using 6 mice per group. Testing compounds were given 10 mg/kg (s.c.) to each mouse. In the morphine challenge study, testing compounds or controls were administered 5 min prior to the subcutaneous injection of 10 mg/kg morphine.

Carmine Red Dye Study.

Each mouse was placed in an individual cage. Five minutes prior to morphine (or vehicle) injection, the testing compound or vehicle was given (s.c. or p.o.) to a group of 5–6 mice. After 20 min of the morphine administration, 0.2 mL red dye solution containing 0.5% carboxymethyl cellulose (CMC) and 6% carmine red dye in ddH₂O was given to each mouse (p.o.) and the time when mouse was fed was recorded as time 0. Then the time which costed each mouse to defecate the first red pellet was measured and recorded. Cut-off time was 6 h.

Statistical Analysis.

One-way ANOVA followed by the corrected Dunnett test were performed to assess significance using Prism 8.0 software (GraphPad Software, San Diego, CA).

ABBREVIATIONS

BBB

blood–brain barrier

CHO

Chinese hamster ovary

CMC

carboxymethyl cellulose

CNS

central nervous system

DAMGO

[d-Ala2-MePhe4-Gly(ol)5]enkephalin

DOR

δ opioid receptor

EDCI

1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide

FCC

flash column chromatography

GI

gastrointestinal

HBD

hydrogen bond donor

HOBt

hydroxybenzotriazole

HPLC

high-performance liquid chromatography

KOR

κ opioid receptor

MNTX

methylnaltrexone

MOR

μ opioid receptor

mp

melting points

MPE

maximal possible effect

MPO

multiparameter optimization

MW

molecular weight

NIDA

National Institute of Drug Abuse

NLX

naloxone

NMR

nuclear magnetic resonance

OIC

opioid-induced constipation

PAMORAs

peripherally acting μ-opioid receptor antagonists

PNS

peripheral nervous system

SARs

structure–activity relationships

s.c.

subcutaneously

SEM

standard error of mean

TLC

thin-layer chromatography

TPSA

topological polar surface area