Design, Synthesis, and Biological Evaluation of NAP Isosteres: A Switch from Peripheral to Central Nervous System Acting Mu-Opioid Receptor Antagonists

Piyusha P Pagare; Mengchu Li; Yi Zheng; Abhishek S Kulkarni; Samuel Obeng; Boshi Huang; Christian Ruiz; James C Gillespie; Rolando E Mendez; David L Stevens; Justin L Poklis; Matthew S Halquist; William L Dewey; Dana E Selley; Yan Zhang

doi:10.1021/acs.jmedchem.2c00087

. Author manuscript; available in PMC: 2023 Apr 30.

Published in final edited form as: J Med Chem. 2022 Mar 7;65(6):5095–5112. doi: 10.1021/acs.jmedchem.2c00087

Design, Synthesis, and Biological Evaluation of NAP Isosteres: A Switch from Peripheral to Central Nervous System Acting Mu-Opioid Receptor Antagonists

Piyusha P Pagare ^1,^#, Mengchu Li ^2,^#, Yi Zheng ³, Abhishek S Kulkarni ⁴, Samuel Obeng ⁵, Boshi Huang ⁶, Christian Ruiz ⁷, James C Gillespie ⁸, Rolando E Mendez ⁹, David L Stevens ¹⁰, Justin L Poklis ¹¹, Matthew S Halquist ¹², William L Dewey ¹³, Dana E Selley ¹⁴, Yan Zhang ¹⁵

¹Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

²Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

³Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

⁴Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

⁵Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

⁶Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

⁷Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

⁸Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

⁹Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

¹⁰Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

¹¹Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

¹²Department of Pharmaceutics, Virginia Commonwealth University, Richmond, Virginia 23298, United States

¹³Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

¹⁴Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

¹⁵Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

P.P.P. and M.L. contributed equally.

Author Contributions

Y.Z. conceived and oversaw the project and finalized the manuscript with editorial input from D.E.S. P.P.P. and M.L. drafted the manuscript. Y.Z., A.S.K., B.H., C.R., and P.P.P. conducted the chemical syntheses. S.O., J.C.G., and R.E.M. conducted in vitro assays under the supervision of D.E.S. M.L. and D.L.S. conducted warm water tail immersion and withdrawal studies under the supervision of W.L.D. M.L., P.P.P., and J.L.P. performed BBB penetration and analysis under the supervision of W.L.D. and M.S.H.

^✉

Corresponding Author: Yan Zhang – Phone: +1(804) 828-0021; yzhang2@vcu.edu; Fax: +1(804) 825-7625

PMCID: PMC10149103 NIHMSID: NIHMS1881169 PMID: 35255685

Abstract

The μ opioid receptor (MOR) has been an intrinsic target to develop treatment of opioid use disorders (OUD). Herein, we report our efforts on developing centrally acting MOR antagonists by structural modifications of 17-cyclopropylmethyl-3,14-dihydroxy-4,5α-epoxy-6β-[(4′-pyridyl) carboxamido] morphinan (NAP), a peripherally acting MOR-selective antagonist. An isosteric replacement concept was applied and incorporated with physiochemical property predictions in the molecular design. Three analogs, namely, 25, 26, and 31, were identified as potent MOR antagonists in vivo with significantly fewer withdrawal symptoms than naloxone observed at similar doses. Furthermore, brain and plasma drug distribution studies supported the outcomes of our design strategy on these compounds. Taken together, our isosteric replacement of pyridine with pyrrole, furan, and thiophene provided insights into the structure–activity relationships of NAP and aided the understanding of physicochemical requirements of potential CNS acting opioids. These efforts resulted in potent, centrally efficacious MOR antagonists that may be pursued as leads to treat OUD.

Graphical Abstract

graphic file with name nihms-1881169-f0006.jpg

INTRODUCTION

Opioid use disorders (OUD) pose an imminent threat to human health worldwide with approximately 2.1 million Americans suffering from this epidemic.¹ According to the Centers of Disease Control and Prevention (CDC), in 2018, 69.5% of the annual overdose deaths in the US were caused by opioids.² This misuse of and addiction to opioids, including prescription pain medications as well as synthetic opioids, not only affects public health but also puts a burden on the economy and the healthcare system. Clinical use of opioids has been associated with several other side effects such as constipation (acute and prolonged uses) and respiratory depression (acute and prolonged uses).^3,4 Currently, detoxification and maintenance therapy are the two most commonly used approaches to treat opioid use disorders.^5–7 Methadone, buprenorphine, and naltrexone (Figure 1) are first-line opioid medicines approved by the US Food and Drug Administration (FDA) for opioid use disorders.⁵ Methadone and buprenorphine, μ opioid receptor (MOR) full and partial agonists, respectively, show good efficacy for opioid addiction maintenance therapy.^8–10 However, about 50% of patients relapse after being treated for opioid use disorders with methadone and buprenorphine.^11,12 While opioid antagonists naltrexone and naloxone have displayed the ability to manage opioid misuse and overdose and to reduce relapse, they carry some side effects such as dysphoria, depression, and even suicide.^13–15 One of the most concerning side effects are the withdrawal symptoms precipitated by naltrexone and naloxone, including abdominal cramps, nausea/vomiting, diarrhea, muscle aches, anxiety, confusion, and extreme sleepiness.^16,17 High doses of these drugs are also reported to show hepatotoxicity, cardiovascular, and pulmonary problems.^16,18 Some of these side effects of the opioid antagonists are related to their low selectivity to the MOR over the δ opioid receptor (DOR) and κ opioid receptor (KOR).¹³

Figure 1. — Chemical structures of FDA-approved opioid ligands as opioid use disorder treatments.

Opioid receptors belong to the G-protein coupled receptor (GPCR) super family. There are four opioid receptors, viz., MOR, KOR, DOR, and the nociception/orphanin FQ receptor (NOP).^19–21 MOR is the primary pharmacological target of most known opioids.^22,23 Activation of the MOR by its agonists, leads to G_i/o-mediated adenylyl cyclase inhibition, which in turn results in the reduction of intracellular cAMP levels, closing of voltage-gated Ca⁺² channels, and activation of inwardly rectifying K⁺ channels. The overall effect of MOR activation results in lowering of postsynaptic neuronal excitability or inhibition of presynaptic neurotransmitter release. The vast array of pharmacological effects as a result of MOR activation includes analgesia, euphoria, sedation, respiratory depression, cough suppression, and constipation.^24–28 Along with the analgesia produced by opioids, their ability to cause euphoria, due to activation of the MOR in several regions of the brain, often leads to opioid misuse.²⁹ Thus, there is an urgent need to develop highly potent, efficacious, and selective MOR ligands with minimum side effects as OUD medications.

Our laboratories have been engaged in identifying novel chemical entities toward developing selective opioid receptor ligands by employing the “message-address concept”.³⁰ Our drug design efforts have led to the development of a small molecule library encompassing different substituents on the epoxymorphinan skeleton.^31–38 Previously, we reported a highly selective MOR antagonist 17-cyclopropylmethyl-3,14-dihydroxy-4,5α-epoxy-6β-[(4′-pyridyl) carboxamido]-morphinan (NAP), which showed high binding affinity for the MOR and at least 500-fold and 150-fold selectivity over the DOR and KOR in vitro, respectively.^31,39 However, further in vivo pharmacological characterization of NAP revealed that it showed limited potency with an AD₅₀ of 4.51 (95% CL, 2.45–8.20) mg/kg, in part because of its constrained ability to penetrate the blood–brain barrier (BBB), making NAP a peripherally acting selective MOR antagonist.^39,40 Collectively, this data encouraged us to explore NAP as a lead for centrally acting MOR-selective antagonists with potential to treat OUD. Herein, we report our efforts toward developing centrally acting MOR antagonists by applying an isosteric replacement as a lead optimization strategy.

Lead compounds, though potent and efficacious, may possess undesirable side effects, poor bioavailability, metabolic instability, and/or lack of selectivity. It has been shown that isosteric replacements may enhance the biological properties of the lead compound as well as improve its safety and clinical utility.^41–43 A number of examples in the literature have demonstrated the importance of classical ring equivalents as well as their use in the discovery of many known FDA-approved drugs.^44–47

The physicochemical properties needed for BBB permeability have been extensively studied in an attempt to define the characteristics of successful CNS drug candidates using various approaches.^48,49 Several of them have consistently been found to be important in the context of designing molecules for optimal brain exposure, including lipophilicity (cLogP or cLogD), the number of hydrogen-bond donors (HBD) and acceptors (HBA), polar surface area (PSA), ionization state (pK_a), rotatable bond (RB) count, and molecular weight (MW), and a useful guidance for a desirable CNS candidate profile has been reported (e.g., cLogP = 2.8, cLogD = 1.7, pK_a = 8.4).^49–55

RESULTS AND DISCUSSION

Molecular Design.

Based on molecular modeling studies and site-directed mutagenesis experiments, we previously demonstrated that the C-6 side chain of NAP bearing the pyridine ring may be engaged in a π-π stacking interaction with Trp318^7.35 in conjugation with a plausible hydrogen bonding interaction between its pyridinyl nitrogen and a positively charged Lys303^6.58 residue that is absent in the KOR and DOR. We postulate that these residues may be involved in potentially imparting selectivity to NAP and its derivatives.^56,57

Hence, to apply NAP as a lead, we employed the concept of isosteric replacement to replace the pyridine ring in the address moiety of NAP with its isosteric counterparts: pyrrole, furan, and thiophene. Although some of these heterocycles may be structural alerts due to their potential for bio-activation and metabolic toxicity, it is known that inclusion of the heterocyclic moiety in drugs alone does not necessarily result in toxic effects.^58,59 Factors like drug dose and reactivity of oxidative metabolites are responsible for the fate of drugs bearing these heterocycles.^59,60 Thus, we reasoned that the presence of a structural alert itself is insufficient to exclude them from consideration. We hypothesized that this isosteric replacement would maintain the critical π-π stacking interaction between the aromatic pyridine ring of NAP and Trp318,^38,56 and would lead to development of compounds with comparable pharmacological properties (e.g., similar affinity, selectivity, and/or efficacy) as well as improved pharmacokinetic profiles. While there was a possibility of loss of hydrogen bonding interaction with the Lys303^6.58 residue resulting in lowered selectivity over the KOR and DOR, we speculated that owing to the high electron density of the heteroatoms in the heteroaromatic pyrrole, furan, and thiophene rings, they would form cation-dipole or cation-pi interaction with Lys303 effectively to compensate such a potential drawback. Additionally, compounds possessing an acetamido or n-propanamido linker between the “message” and “address” moieties were designed to help probe the influence of distance and flexibility of the aromatic ring on parameters such as affinity and selectivity. The linker substitution position was also varied to study the role of orientation of the aromatic ring on biological activities. Finally, although NAP has a defined stereocenter (β) at C6, the structure–activity relationship of the C6 stereochemistry has not been conclusive based on our previous studies. For example, while NAP³¹ and NBF³⁵ carry a β configuration, two other leads, NAQ³¹ and NAN³⁷ actually carry an α configuration at C6. Since NAP, NBF, NAQ, and NAN were identified as MOR-selective ligands, it seemed that the C6 stereochemistry had little effect on the interaction of the address portion of the molecule with Lys303. Therefore, we decided to synthesize compounds with both configurations at C6 in this study. Thus, a total of 36 compounds were designed with the following characteristics: (1) the stereochemistry at C(6) may be either α or β, (2) the linker between the epoxymorphinan skeleton and C(6) side chain may either be carboxamido, acetamido, or n-propanamido, and (3) the linker could be attached to either the 2′ or 3′ position of the aromatic ring (Figure 2). Of these, two compounds, 27 and 28 have been previously studied as potential alcohol-cessation agents.⁶¹ Thus a total of 34 new compounds were synthesized. Additionally, the physicochemical parameters were predicted for all the newly designed ligands by using ACD/Percepta (v2020.2.0),⁶² and it was found that the different parameters fell in the following range for all derivatives designed: cLogP = 1.71–3.04, cLogD = 0.94–2.40, and pK_a = 7–7.5 (see the Supporting Information). These values cover a range around the ideally expected values for reasonable BBB permeability as compared to NAP (cLogP = 1.18, cLogD = 0.98, and pKa = 7.19).

Figure 2. — Molecular design of NAP derivatives.

Chemical Synthesis.

All newly designed compounds were synthesized according to previously reported procedures.^31,35,37,38 Briefly, 6α- and 6β-naltrexamine (NTA) were synthesized by stereoselective reduction amination of naltrexone with benzylamine and dibenzylamine, respectively, followed by catalytic hydrogenation under acidic conditions. Various commercially available five-membered heterocyclic carboxylic acids were coupled with 6α- and 6β-naltrexamine utilizing the EDCI/HOBt coupling reaction under mild basic conditions. 6-Position monosubstituted free bases were then obtained in reasonable yields by treating them with K₂CO₃ in methanol (Scheme 1). These final compounds, obtained in yields ranging from 30 to 95%, were converted to their hydrochloric acid salt forms, fully characterized, and applied for in vitro and in vivo pharmacological characterization.

Scheme 1. — Synthetic Route for Target Compounds

In Vitro Pharmacological Studies.

Binding affinity and selectivity of all synthesized ligands on the three opioid receptors were determined using the competitive radioligand binding assay. Following the previously reported protocol,^31,35,38 opioid receptor-expressing CHO cell membranes were used where the MOR was labeled with [³H]naloxone, and the KOR and DOR with [³H]diprenorphine. The [³⁵S]GTPγS functional assay was then carried out to determine the agonist potency and efficacy of each ligand at the MOR by measuring its efficacy relative to the full agonist DAMGO for MOR activation. The binding, selectivity, potency, and efficacy results for the 6α-analogs are summarized in Table 1, and the results for 6β-analogs are summarized in Table 2.

Table 1.

Opioid Receptor Binding Affinity and MOR [³⁵S]GTPγS Functional Assay Results for 6α-Analogs



Compds.	R	K_i (nM)			Selectivity		MOR [³⁵S]GTPγS Binding
Compds.	R	MOR	KOR	DOR	δ/μ	κ/μ	EC₅₀ (nM)	% E_max of DAMGO

NLX^a	-	0.79 ± 0.02	1.1 ± 0.03	76 ± 2	69	1.0	NA	13 ± 1

NAP^b		0.37 ± 0.07	60.7 ± 5.6	277.5 ± 8.0	747	163	1.14 ± 0.38	22.72 ± 0.84

1		0.32 ± 0.03	1.58 ± 0.16	24.66 ± 2.22	76	5	3.39 ± 1.29	12.11 ± 2.64

3		0.46 ± 0.04	2.88 ± 0.38	3.85 ± 1.18	8	6	4.18 ± 1.21	25.01 ± 2.61

5		0.58 ± 0.03	0.54 ± 0.09	7.58 ± 0.37	13	1	3.79 ± 1.01	23.97 ± 3.59

7		1.31 ± 0.09	2.78 ± 0.18	40.38 ± 5.80	31	2	2.21 ± 0.68	16.65 ± 1.25

9		3.62 ± 0.43	19.0 ± 1.28	14.45 ± 1.81	4	5	51.36 ± 23.29	31.56 ± 2.87

11		0.70 ± 0.12	0.59 ± 0.07	8.62 ± 1.37	12	1	3.86 ± 1.39	25.24 ± 3.26

13		0.53 ± 0.33	1.97 ± 0.26	33.27 ± 3.51	63	4	9.49 ± 6.29	21.80 ± 5.19

15		0.59 ± 0.03	6.67 ± 1.09	4.61 ± 0.42	8	12	2.74 ± 0.57	22.65 ± 2.77

17		0.72 ± 0.10	1.52 ± 0.27	2.94 ± 0.26	4	2	2.77 ± 0.73	21.77 ± 2.76

19		0.50 ± 0.05	2.14 ± 0.36	28.63 ± 3.22	57	4	7.63 ± 4.22	18.14 ± 2.95

21		0.42 ± 0.04	6.45 ± 0.59	3.74 ± 0.49	9	15	8.32 ± 3.56	14.95 ± 1.46

23		0.44 ± 0.10	0.78 ± 0.04	3.54 ± 0.50	9	2	2.31 ± 0.87	27.62 ± 1.96

25		0.37 ± 0.01	1.59 ± 0.17	24.28 ± 5.08	65	4	0.44 ± 0.09	24.00 ± 2.89

27^c		0.46 ± 0.05	6.04 ± 0.83	6.92 ± 1.63	15	13	5.95 ± 1.67	25.14 ± 1.07

29		0.48 ± 0.03	1.76 ± 0.21	2.25 ± 0.43	7	4	3.06 ± 0.48	43.73 ± 1.71

31		0.38 ± 0.04	2.80 ± 0.12	10.71 ± 3.56	29	7	1.07 ± 0.22	30.66 ± 4.44

33		0.23 ± 0.01	0.65 ± 0.08	39.41 ± 11.35	173	3	1.21 ± 0.40	28.59 ± 1.15

35		0.23 ± 0.02	3.66 ± 0.44	18.55 ± 3.19	81	16	4.30 ± 2.29	33.40 ± 4.46

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The in vitro data for NLX adopted from ref 63.

The in vitro data for NAP adopted from ref 31.

Compound first published in ref 61.

Table 2.

Opioid Receptor Binding Affinity and MOR [³⁵S]GTPγS Functional Assay Results for 6β-Analogs^c



Compds.	R	K_i (nM)			Selectivity		MOR [³⁵S]GTPγS Binding
Compds.	R	MOR	KOR	DOR	δ/μ	κ/μ	EC₅₀ (nM)	% E_max of DAMGO

NLX^a	-	0.79 ± 0.02	1.1 ± 0.03	76 ± 2	69	1.0	NA	13 ± 1

NAP^b		0.37 ± 0.07	277.5 ± 8.0	60.7 ± 5.6	74	16	1.14 ± 0.38	22.72 ± 0.84

2		14.05 ± 0.95	40.70± 2.64	1639 ± 349	117	3	3.39 ± 1.29	12.43 ± 1.05

4		0.16 ± 0.02	1.35 ± 0.12	24.27 ± 4.51	152	8	0.80 ± 0.03	44.17 ± 1.52

6		0.52 ± 0.08	1.25 ± 0.13	36.71 ± 4.24	70	2	1.22 ± 0.12	39.34 ± 3.16

8		0.36 ± 0.04	4.51 ± 0.44	107.45 ± 16.44	298	12	2.03 ± 0.23	17.36 ± 2.04

10		0.28 ± 0.05	4.19 ± 0.46	37.78 ± 5.61	135	15	1.18 ± 0.06	53.26 ± 0.95

12		0.41 ± 0.08	2.72 ± 0.39	67.47 ± 11.18	164	7	1.60 ± 0.17	37.72 ± 0.87

14		0.30 ± 0.05	1.29 ± 0.12	84.68 ± 4.75	282	4	0.98 ± 0.09	24.2 ± 0.9

16		0.23 ± 0.03	3.38 ± 0.27	45.60 ± 5.12	198	15	0.97 ± 0.09	40.00 ± 0.9

18		0.28 ± 0.02	0.61 ± 0.07	33.00 ± 3.58	118	2	1.33 ± 0.15	44.53 ± 2.72

20		8.47 ± 0.4	29.36 ± 2.80	1980 ± 271	234	3	34.99 ± 5.02	24.99 ± 1.74

22		0.22 ± 0.03	3.68 ± 0.13	73.02 ± 7.08	332	17	0.91 ± 0.22	30.83 ± 2.07

24		0.21 ± 0.01	0.47 ± 0.03	32.70 ± 3.47	156	2	0.63 ± 0.12	42.61 ± 2.61

26		0.24 ± 0.01	0.79 ± 0.07	48.67 ± 1.62	206	3	0.54 ± 0.14	24.42 ± 2.04

28^b		0.24 ± 0.02	1.61 ± 0.2	64.93 ± 2.38	270	7	1.16 ± 0.35	25.24 ± 2.33

30		0.26 ± 0.01	0.23 ± 0.02	4.87 ± 1.19	19	1	1.52 ± 0.37	37.79 ± 4.30

32		0.25 ± 0.03	0.37 ± 0.06	152.9 ± 34.3	612	1	0.95 ± 0.3	26.49 ± 1.9

34		0.20 ± 0.02	1.31 ± 0.18	36.11 ± 10.17	176	6	3.24 ± 0.11	26.49 ± 3.17

36		0.21 ± 0.02	0.22 ± 0.04	51.01 ± 19.27	245	1	0.81 ± 0.08	64.66 ± 4.31

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The in vitro data for NLX adopted from ref 63.

The in vitro data for NAP adopted from ref 31.

Compound first published in ref 61.

Overall, the 6α-analogs (except compounds 7 and 9) showed sub nanomolar affinity for the MOR, similar to that seen for NAP. Interestingly, the isosteric replacement of the address moiety of NAP, with pyrrole, furan, and thiophene, in fact, resulted in improvement of KOR affinity leading to reduced selectivity between the MOR and KOR (Table 1). All analogs showed relatively higher affinity (single-to-double digit nanomolar K_i) at the DOR compared to NAP. It was observed that the change between three isosteres, pyrrole, furan, and thiophene, resulted in no significant change in the binding affinity and selectivity for the MOR over KOR and DOR. For example, compounds 1 (K_i κ/μ 4.85 and δ/μ 75.87), 13 (K_i κ/ μ 3.83 and δ/μ 62.83), and 25 (K_i κ/μ 4.25 and δ/μ 65.00) that differ only in the aromatic moiety in their address portion showed similar affinity and selectivity profiles. A similar trend was observed for all 6α-analogs. Compared to NAP, the compounds with the n-propamido linker showed increased KOR affinity, thereby lowering the κ/μ selectivity, making these analogs less selective between the KOR and MOR than the ones with a carboxamido or acetamido spacer. In general, the compounds with an acetamido linker exhibited the highest MOR-over-KOR selectivity.

Lastly, the attachment position of the linker, 2′ or 3′ of the aromatic ring, seemed to have little effect on either binding affinity or selectivity.

Among the 6β-analogs, it was observed that all compounds (except compounds 2 and 20) showed sub-nanomolar affinity for the MOR, nanomolar affinity for the KOR, and much lower affinity for the DOR (Table 2). Similar to their 6α-counterparts, these compounds also showed improved affinity for the KOR compared to NAP. The 6β-analogs, overall, showed much higher selectivity for the MOR over the DOR compared to their 6α-counterparts. Compound 32, with 3-thiophene in the address region showed the highest selectivity for the MOR over the DOR (K_i δ/μ 612.0), while compound 22 with 2-furanylmethyl in the address region showed the highest selectivity for the MOR over the KOR (K_i κ/μ 16.7). The 6β-analogs with an acetamido linker presented higher MOR-over-KOR selectivity than the ones with a carboxamido or n-propamido linker.

As seen in Table 1, all 6α-analogs exhibited partial agonism with similar potency and efficacy. In detail, compounds 9, 29, 31, and 35 show higher efficacy than NAP, ranging between 30 and 50%, whereas compounds 1, 7, 19, and 21 showed lower efficacy than NAP (<20%). The remaining 6α-analogs (3, 5, 11, 13, 15, 17, 19, 23, 25, 27, and 33) showed efficacies similar to NAP (20–30%) for G-protein activation in MOR-expressing CHO cells.^35,39,56 There appeared to be no significant effect of chain length or substitution position on the heterocyclic rings on their efficacies.

Among the 6β-analogs, all compounds except compounds 2 and 8 exhibited partial agonism with efficacies ranging between 25 and 65%. Compounds 2 and 8 showed low efficacies (2, E_max = 12.43 ± 1.05; 8, E_max = 17.36 ± 2.04) and were identified as antagonists. Interestingly, both compounds 2 and 8 are pyrrole derivatives with a methyl linker and differ only in the substitution position of the linker on the pyrrole ring.

Overall, except compounds 2 and 20, substituting the pyridine ring in the address region of NAP with its isosteres pyrrole, furan, and thiophene rings retained the high binding affinity as well as similar efficacy at the MOR.

In Vivo Warm-Water Tail Immersion Assay.

The warm-water tail immersion assay was conducted on all compounds to assess their antinociception potency and antagonism against morphine’s antinociception. In this assay, the duration for which mice kept their tails in the warm water was recorded. The longer the duration, giving higher percent maximum possible effects (%MPE), the higher the antinociceptive effects the studied compound possesses.

All 36 compounds’ antinociception were examined in tail immersion assay first to preclude any opioid receptor agonists. In this study, the test was conducted 20 min after each compound (10 mg/kg) was injected subcutaneously. As shown in Figure 3A,B, among the 36 compounds, most compounds showed no significant antinociceptive effects compared to the vehicle, which corresponded to their low efficacy at the MOR (Tables 1 and 2). Compounds 4, 6, 8, 19, 20, 30, and 36 exhibited antinociception that is reflected in their increased % MPE (Figure 3A,B). Among them, compounds 4, 6, 30, and 36 showed moderate efficacies at the MOR (Table 2), suggesting that their antinociceptive effects were most likely due to the activation at the MOR. On the other hand, compounds 8 and 19 acted as low-efficacy MOR agonists in the [³⁵S]GTPγS binding assay while showing high KOR affinity (Tables 1 and 2), suggesting that their antinociception may come from interacting with the KOR. Compound 20, however, demonstrated not only low efficacy and low potency at the MOR but also low to moderate binding affinity toward the DOR and KOR (Table 2). Hence, there might be mechanisms other than opioid receptor agonism responsible for the antinociception for 20. All other analogs showed no significant antinociception when compared to morphine and could potentially act as opioid antagonists.

Figure 3. — Water–water tail immersion assay results of (A) 6α-analogs (n = 6) as agonists at a single dose of 10 mg/kg s.c., (B) 6β-analogs (n = 6) as agonists at a single dose of 10 mg/kg s.c., and (C) compounds as antagonists at a single dose of 10 mg/kg s.c. in the presence of morphine (10 mg/kg). Saline and morphine were used as the negative and positive controls, respectively. Data are presented as mean values ± SD. *P < 0.05, **P < 0.01, and ***P < 0.0005, ****P < 0.0001, compared to 10 mg/kg morphine (s.c.).

The analogs defined as potential antagonists at the MOR were then studied for their ability to antagonize morphine’s antinociceptive effect. As seen from Figure 3C, compounds 1, 11, 14, 15, 16, 25, 26, 31, and 32 significantly antagonized morphine’s antinociceptive effect thereby showing pronounced antagonism of CNS antinociception. Interestingly, of the nine compounds identified as antagonists, six of them (compounds 1, 14, 25, 26, 31, and 32) possessed no linker methylene group between the amide bond and the address region.

All remaining compounds (2, 3, 5, 7, 9, 10, 12, 13, 17, 18, 21, 22–24, 27–29, 33–35) did not produce any significant antinociception nor were they able to antagonize morphine’s antinociceptive effect. It was observed that these compounds, except compounds 29 and 35, showed predicted clogP < 2.5. Taking their high in vitro binding affinity to the MOR into account, these compounds most likely lack CNS permeability, which resulted in their lack of in vivo activity.

Following this single-dose assessment, in vivo dose–response studies with eight identified antagonists were conducted. Compound 11 exhibited poor solubility in pyrogen-free isotonic saline as well as sterile-filtered distilled/deionized water at higher doses, while addition of 10% DMSO or 2% Tween80 did not improve its solubility significantly. Hence, 11 was excluded from the following studies. The antiantinociception potencies of the remaining eight compounds were determined where their AD₅₀ values ranged from 0.42 to 23.54 mg/kg. Six out of eight identified antagonists, except 15 and 16, possessed AD₅₀ values comparable to NAP (Table 3).

Table 3.

AD₅₀ Values of Compounds to Antagonize Morphine-Mediated Antinociception

compound	AD₅₀ (mg/kg) (95% CL)
NLX^a	0.05 (0.03–0.09)
NAP^b	4.51 (2.45–8.26)
1	4.71 (1.61–13.81)
14	6.04 (3.47–10.50)
15	12.31 (6.92–21.90)
16	23.54 (11.34–48.83)
25	0.42 (0.21–0.82)
26	1.62 (1.12–2.36)
31	1.51 (1.08–2.09)
32	6.81 (2.46–18.91)

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The in vitro data for NLX adopted from ref 63.

The in vitro data for NAP adopted from ref 31.

In fact, compounds 25, 26, and 31 were significantly more potent than NAP with 25 (AD₅₀ = 0.42 mg/kg) showing 10-fold higher potency (Table 3). Also, compound 25 showed much higher potency than other NAP derivatives, i.e., NFP (AD₅₀ = 2.82 mg/kg) and NYP (AD₅₀ = 1.75 mg/kg), which were identified in previous studies.³⁶ With the exception of compound 32, the predicted CNS-relevant physicochemical properties also correlated well with the potency wherein compounds 11, 14, 15, and 16 showed clogP < 2.5 and clogD < 1.5, whereas compounds 25, 26, and 31 showed identical physicochemical properties (cLogP = 2.77, cLogD = 2.11) that predict higher CNS permeability.

KOR and DOR [³⁵S]GTPγS Binding Assays.

As predicted and observed from the binding assay results (Table 2), many compounds resulted in decreased selectivity over the KOR and DOR compared to NAP, which we wondered that could potentially lead to some undesired off-target effects. Hence, before investigating their pharmacology further, the functionalities of the eight selected antagonists (1, 14–16, 25, 26, 31, and 32) on the KOR and DOR were investigated. In the KOR [³⁵S]GTPγS binding assays, all compounds showed moderateto-high efficacy with single- or double-digit nanomolar potencies (Table 4). As KOR agonists may help treat morphine or oxycodone addiction and opioid-induced pruritus,^64,65 the partial agonism exhibited by these compounds on the KOR may in fact be beneficial in OUD treatments. On the other hand, the high potency and efficacy of compound 32 toward the KOR, which were not observed in the in vivo antinociception study, could be concerning clinically as a full KOR agonist could also elicit dysphoria and sedation.⁶⁶ Except for compound 15, all other compounds remained highly selective over the DOR with none displaying high potency or high efficacy in the DOR functional study (Table 4). Thus, although compound 15 showed relatively low δ/μ selectivity and a nanomolar level EC₅₀, we speculated that its partial agonism displayed at the DOR would not result in severe side effects such as convulsion. Therefore, the comparatively low κ/μ and δ/μ selectivity seemed acceptable for further pursuing these MOR ligands as potential therapeutic agents for OUD except compound 32.

Table 4.

Potencies and Efficacies at the KOR and DOR of Compounds 1, 14–16, 25, 26, 31, and 32

	selectivity		KOR [³⁵S]GTPγS Binding		DOR [³⁵S]GTPγS Binding
compd	κ/μ	δ/μ	EC₅₀ (nM)	% E_max of U50,488H	EC₅₀ (nM)	% E_max of SNC80
NAP^a	163	747	28.8 ± 14.4	45.5 ± 4.4	15.2 ± 15.2	10.2 ± 3.1
1	5	76	14.8 ± 0.99	43.4 ± 1.36	26.9 ± 3.23	50.9 ± 5.25
14	4	282	6.3 ± 0.85	67.2 ± 2.53	35.5 ± 8.71	23.1 ± 0.97
15	12	8	85.4 ± 6.29	63.3 ± 2.50	6.2 ± 0.83	66.8 ± 4.24
16	15	198	13.3 ± 0.59	49.3 ± 1.07	42.3 ± 6.03	31.2 ± 1.49
25	4	65	1.7 ± 0.24	50.6 ± 0.95	59.4 ± 6.71	48.6 ± 3.41
26	3	206	7.0 ± 0.31	67.4 ± 3.00	70.4 ± 38.51	43.6 ± 7.31
31	7	29	16.3 ± 1.73	47.8 ± 0.85	38.8 ± 5.83	58.5 ± 3.13
32	1	612	1.0 ± 0.44	90.8 ± 2.34	31.4 ± 4.89	47.8 ± 0.95

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The in vitro data for NAP adopted from ref 31.

In Vivo Opioid Withdrawal Studies.

Opioid antagonists such as naloxone (NLX) and naltrexone are associated with significant withdrawal symptoms when administered to opioiddependent patients.^67,68 Such drawbacks have limited the clinical application of naloxone and naltrexone. Since compounds 25, 26, and 31 appeared to be the most potent antagonists in vivo among others, they were selected to be studied for their potency to produce withdrawal symptoms. Somatic symptoms of opioid withdrawal including wet dog shakes, jumps, and paw tremors were monitored and recorded over a period of 20 min, starting 3 min after each injection with the tested compounds given to morphine-pelleted mice. As shown in Figure 3, NLX precipitated withdrawal symptoms at 1 mg/kg similar to that of previously reported.^35,36

At 1 mg/kg dose, compounds 25, 26, and 31 produced significantly fewer wet dog shakes, escape jumps, and paw tremors than 1 mg/kg NLX (Figure 4A–C) in morphine-pelleted mice. Both 6α-analogs 25 and 31 started showing wet dog shakes and jumps at a 5 mg/kg dose. While paw tremoring was noteworthy for compound 25 at 5 mg/kg, it remained mild through all doses for compound 31. Interestingly, the 6β-analog 26 did not precipitate significant withdrawal symptoms at a dose as high as 10 times that of NLX. Interestingly, compound 25 showed fewer withdrawal symptoms at 10 mg/kg compared to 5 mg/kg as well as observed fewer wet dog shakes than that seen for compound 31. We have observed some similar cases previously in our studies with other analogs, which could be due to the nature of the behavioral in vivo study and the individual conditions of mice. Meanwhile, the symptoms shown at both doses 5 and 10 mg/kg were not significantly different statistically. Additionally, wet dog shakes and jumps were seen at 20 and 33.8 mg/kg equivalent to those seen for 1 mg/kg of naloxone (data not shown). Although the compounds exhibit lower potency than NLX, the partial agonism shown by the new analogs compared to NLX’s neutral antagonism could be a reason of their reduced withdrawal symptoms. Overall, the results suggest that these compounds, especially compound 26, precipitate much less withdrawal effects than NLX, thus making them promising candidates to develop for the treatment for opioid use disorders.

BBB-Penetration Studies.

As our goal is to develop centrally acting MOR antagonists, we designed NAP analogs by applying the isosteric replacement strategy and physicochemical parameter predictions. Additionally, CNS penetrance of selected compounds was further estimated using other models including Swiss ADME,⁶⁹ Pfizer’s central nervous system multiparameter optimization (CNS-MPO) index,⁷⁰ and ligand-lipophilic efficiency indices (LLE).⁷¹ Swiss ADME predicted compounds 25, 26, and 31 to be CNS-non-permeant. CNS-MPO estimates a score higher than 4 as a criterion for CNS hit selection.⁷⁰ Determination these scores for these compounds revealed naltrexone (MPO score of 5.5) and NAP (MPO score of 4.4) to be CNS-permeable, while compounds 25, 26, and 31 (MPO score of 3.8) to be CNS-non permeable (Table S2). Similarly, determination of the LLE indicated that NAP (PNS-acting) has a higher index of 8.3, while NLX (LLE of 7.0) and compounds 25 (LLE of 6.7), 26 (LLE of 6.85), and 31 (LLE of 6.65) showed slightly lower indices (Table S2). While compounds 25, 26, and 31 seemed significantly more potent than NAP in vivo, MPO and LLE scores appeared to have their limitations to predict the CNS permeability of NAP analogs.

Several factors govern in vivo efficacy including intrinsic clearance and efflux transport in addition to CNS permeability. The low in vivo potentcy of NAP mainly is due to its poor CNS permeability. Although NAP was determined to be a P-gp substrate, efflux by P-gp was not the most critical issue of impeded CNS permeability for the NAP’s already very low diffusional permeability (P_app,A-B = 0.6 ± 0.17 and P_app,B-A = 7.8 ± 1.0 in units of 10⁻⁶ cm/s). The permeability of NAP was not lower than naltrexone but was similar to mannitol, a paracellular permeability marker.⁴⁰ Therefore, we postulated that the major concern was physicochemical properties of NAP (cLogP = 1.18, cLogD = 0.98, pK_a = 7.17, and TPSA = 94.39) that hampered its passive permeability.

Subsequently, passive permeability of the most potent compound (25) was assessed. Compound 25 showed a P_app,A-B = 11.9 ± 0.91 10⁻⁶ cm/s and a P_app,B-A = 26.5 ± 0.86 10⁻⁶ cm/s, thus suggesting that it was highly permeable as compared to NAP. Further, in vivo time-dependent BBB-penetration studies were carried out. Compound 25 was administered s.c. at the tested dose of 10 mg/kg following which mice were sacrificed at 5, 10, and 30 min, and their plasma and blood samples were collected. After the blood samples were centrifuged to obtain plasma, the plasma and brain homogenate samples were then analyzed to determine the amount of compound 25 using liquid chromatography–tandem mass spectrometry (LC–MS/MS), and the brain-to-plasma ratios were calculated (Table 5). Compound 25 appeared in plasma with the highest concentration (4.13 μg/mL) as early as 5 min after s.c. administration. Brain concentrations of compound 25 after 5, 10, and 30 min were 0.283, 0.366, and 0.459 μg/g, respectively, indicating that compound 25 penetrated into the CNS after subcutaneous administration. Additionally, the brain-to-plasma concentration ratio of compound 25 increased over time indicating its progressive BBB-penetration.

Table 5.

BBB Penetration of Compound 25 (10 mg/kg, s.c) in Mice (n = 3, Mean ± SD) at Various Time Points

time (min)	5	10	30
brain (μg/g)	0.283 ± 0.09	0.366 ± 0.08	0.459 ± 0.08
plasma (μg/mL)	4.130 ± 1.31	3.150 ± 1.87	1.547 ± 0.96
brain-to-plasma ratio	0.068	0.116	0.297

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CONCLUSIONS

In summary, an isosteric ring replacement strategy was utilized to design a novel series of NAP derivatives as potential CNS-permeable and MOR-selective antagonists. This isosteric replacement aimed to improve CNS-permeability by substituting the pyridine ring in NAP with pyrrole, furan, and thiophene systems. In general, all compounds retained high MOR binding affinity, though in general, their selectivity over the KOR and DOR was lower as compared to NAP. It was observed that the heteroaromatic ring and position of substitution had no significant influence on the binding affinity and selectivity. However, the linker length and the configuration of C(6) seemed to affect their MOR selectivity over KOR and DOR. Moreover, from the in vivo studies, it was observed that at least 16 compounds (seven agonists and nine antagonists) showed improved CNS permeability, indicating the success of our isostere replacement as a lead modification strategy. Furthermore, out of the nine CNS-active MOR antagonists identified in the in vivo study, compounds 25, 26, and 31 demonstrated remarkable CNS antagonism against morphine and precipitated fewer withdrawal symptoms than NLX. Interestingly, all three compounds contain a thiophene moiety with no linker carbon (n = 0) between the amide bond and the address moieties. These compounds also showed identical CNS-relevant physicochemical properties (cLogP = 2.77, cLogD = 2.11, and pK_a = 7.34) predicting them to be BBB permeable as compared to NAP (cLogP = 1.18, cLogD = 0.98, and pKa = 7.19), which was further confirmed by in vivo BBB-penetration studies for the most potent compound 25. Thus, these novel thiophene isosteres of NAP showed promising potential for their utility in the treatment of opioid use disorders and may serve as lead compounds in the design of future small molecule MOR-selective antagonists. Furthermore, the presented structure- and physiochemical property-based drug design practice may help design not only CNS but also PNS agents.

EXPERIMENTAL SECTION

Chemistry.

All nonaqueous reactions were carried out under a pre-dried nitrogen gas atmosphere. Naloxone-d₅ was purchased from Cerilliant Corp. All other solvents and reagents were purchased from Sigma-Aldrich, Alfa Aesar, and Fisher Scientific and were used as received without further purification. Melting points were measured on an MPA100 OptiMelt automated melting point apparatus without correction. The IR spectra were recorded on a Thermo Scientific Nicolet iS10 FT-IR spectrometer. Analytical thin-layer chromatography (TLC) analyses were carried out on Analtech Uniplate F254 plates, and flash column chromatography (FCC) was performed over silica gel (230–400 mesh, Merck). The ¹H (400 MHz) and ¹³C (100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield 400 Plus spectrometer, and chemical shifts were expressed in parts per million. The high-resolution mass spectra were obtained on an Applied BioSystems 3200 Q trap with a turbo V source for TurbolonSpray. Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed on a Varian ProStar 210 system using an Agilent Microsorb-MV 100–5 C18 column (250 × 4.6 mm). All analyses were conducted at ambient temperature with a flow rate of 0.8 mL/min. The mobile phase is acetonitrile/water (90:10) with 0.1% trifluoroacetic acid (TFA). The UV detector was set up at 210 nm. Compound purities were calculated as the percentage peak area of the analyzed compound, and retention times (Rt) were presented in minutes. The purity of all newly synthesized compounds was identified as ≥95%.

General Procedure for the Amide Coupling/Hydrolysis Reaction.

A solution of carboxylic acid (2.5 equiv) in dry DMF (1.5 mL) was added with hydrobenzotriazole (HOBt, 3 equiv), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 3 equiv), 4 Å molecular sieves, and triethylamine (5 equiv) on an icewater bath. After 1 h, a solution of 6α-naltrexamine or 6β-naltrexamine (1 equiv) in pre-dried DMF (1.5 mL) was added dropwise. The resulting mixture was stirred at room temperature. Once TLC indicated complete consumption of the starting material, the reaction mixture was filtered through Celite. The filtrate was concentrated to dryness and dissolved in anhydrous methanol (3 mL), and then K₂CO₃ (2.5 equiv) was added. The resulting mixture was stirred overnight at room temperature and filtered again over Celite. After being concentrated, the residue was purified by flash column chromatography with CH₂Cl₂/MeOH (1% NH₃·H₂O) as the eluent to give the free base.³⁸ After structural confirmation by ¹H NMR, the corresponding free base was then converted into a hydrochloride salt, which was fully characterized by ¹H NMR, ¹³C NMR, IR, HRMS, and HPLC.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6a-[(2′-pyrrolyl)carboxamido]morphinan Hydrochloride (1).

Compound 1 was synthesized as shown in the general procedure with 37% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 11.53 (s, 1H), 10.15 (s, 1H), 9.21 (s, 1H), 8.86 (s, 1H), 7.57 (d, J = 8 Hz, 1H), 6.89 (m, 1H), 6.85 (m, 1H), 6.72 (d, J = 8 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 6.33 (s, 1H), 6.10 (m, 1H), 4.72 (d, J = 3.8 Hz, 1H), 4.57 (m, 1H), 3.93 (d, J = 6.8 Hz, 1H), 3.25 (m, 2H), 2.96 (m, 1H), 2.73 (m, 1H), 1.92 (m, 1H), 1.64 (m, 1H), 1.46 (m, 1H), 1.12 (m, 2H), 0.69 (m, 1H), 0.62 (m, 1H), 0.49 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.9, 146.0, 138.7, 128.7, 125.9, 122.0, 121.4, 119.0, 118.3, 110.9, 108.4, 87.5, 69.3, 61.0, 57.0, 45.3, 45.1, 45.1, 30.2, 29.2, 23.4, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3259, 1749, 1621, 1453, 1116, 1067, 1035, 748. HRMS m/z: calc. 436.2158 [M + H]⁺; obs., 436.2232 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.56 min) and was found to be 97.72% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′pyrrolyl)carboxamido]morphinan Hydrochloride (2).

Compound 2 was synthesized as shown in the general procedure with 38% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.56 (s, 1H), 9.33 (s, 1H), 8.82 (s, 1H), 8.17 (d, J = 8 Hz, 1H), 6.71 (d, J = 8 Hz, 1H), 6.63 (d, J = 8 Hz, 1H), 6.60 (m, 1H), 6.16 (s, 1H), 5.90 (m, 1H), 5.81 (m, 1H), 4.58 (d, J = 8 Hz, 1H), 3.83 (d, J = 4 Hz, 1H), 3.43 (s, 1H), 3.34 (m, 2H), 3.05 (m, 2H), 2.84 (m, 1H), 2.43 (m, 2H), 1.73 (m, 2H), 1.51 (m, 2H), 1.32 (m, 1H), 1.05 (m, 1H), 0.67 (m, 1H), 0.58 (m, 1H), 0.50 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.1, 157.5, 146.6, 133.2, 130.4, 128.2, 126.0, 120.1, 116.9, 109.9, 108.5, 91.2, 69.6, 61.5, 56.7, 50.6, 46.1, 45.5, 29.2, 27.4, 23.7, 23.3, 5.6, 5.0, 2.6. IR (diamond, cm⁻¹) ν_max: 3160, 1639, 1298, 1124, 748. HRMS m/z: calc. 436.2158 [M + H]⁺; obs., 436.2246 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.45 min) and was found to be 96.98% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6a-[2′-(2′-pyrrolyl)acetamido]morphinan Hydrochloride (3).

Compound 3 was synthesized as shown in the general procedure with 53% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.21 (s, 1H), 9.26 (s, 1H), 8.84 (s, 1H), 7.97 (d, J = 8 Hz, 1H), 7.54 (m, 1H), 6.73 (d, J = 8 Hz, 1H), 6.56 (d, J = 8 Hz, 1H), 6.38 (m, 1H), 6.29 (s, 1H), 6.22 (m, 1H), 4.60 (d, J = 4 Hz, 1H), 4.40 (m, 1H), 3.91 (d, J = 8 Hz, 1H), 3.59 (s, 2H), 3.27 (m, 2H), 2.95 (m, 1H), 2.70 (m, 1H), 2.43 (m, 1H), 1.87 (m, 1H), 1.61 (m, 1H), 1.40 (m, 2H), 1.06 (m, 1H), 1.05 (m, 1H), 0.68 (m, 1H), 0.60 (m, 1H), 0.48 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.3, 150.0, 145.9, 141.7, 138.8, 128.7, 122.0, 119.0, 118.2, 110.4, 107.1, 87.3, 69.3, 60.9, 56.9, 45.3, 45.1, 34.9, 30.1, 29.1, 23.4, 19.6, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3236, 1634, 1455, 1116, 1066, 1032, 720. HRMS m/z: calc. 450.2315 [M + H]⁺; obs., 450.2397 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.51 min) and was found to be 99.84% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[2′-(2′-pyrrolyl)acetamido]morphinan Hydrochloride (4).

Compound 4 was synthesized as shown in the general procedure with 72% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.56 (s, 1H), 9.33 (s, 1H), 8.83 (s, 1H), 8.16 (d, J = 4 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.64 (d, J = 8 Hz, 1H), 6.61 (m, 1H), 6.16 (s, 1H), 5.91 (m, 1H), 5.83 (m, 1H), 4.60 (d, J = 8 Hz, 1H), 3.85 (d, J = 4 Hz, 1H), 3.05 (m, 2H), 2.85 (m, 1H), 2.43 (m, 1H), 1.72 (m, 2H), 1.53 (m, 1H), 1.43 (m, 1H), 1.34 (m, 1H), 1.07 (m, 1H), 0.68 (m, 1H), 0.59 (m, 1H), 0.52 (m, 1H), 0.43 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.2, 142.0, 141.2, 129.5, 125.2, 120.5, 119.2, 117.8, 116.7, 107.1, 105.8, 89.8, 69.6, 61.6, 56.6, 50.8, 46.4, 45.5, 35.0, 29.2, 27.3, 23.5, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3076, 1655, 1502, 1316, 1127, 1033, 726. HRMS m/z: calc. 450.2315 [M + H]⁺; obs., 450.2372 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.34 min) and was found to be 97.71% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α[3′-(2′-pyrrolyl)propanamido]morphinan Hydrochloride (5).

Compound 5 was synthesized as shown in the general procedure with 72% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.50 (s, 1H), 9.21 (s, 1H), 8.84 (s, 1H), 7.68 (d, J = 8 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.56 (m, 2H), 6.26 (s, 1H), 5.81 (m, 1H), 5.73 (m, 1H), 4.59 (m, 1H), 4.41 (m, 1H), 3.90 (m, 1H), 3.35 (m, 2H), 3.25 (m, 1H), 3.00 (m, 3H), 2.73 (m, 3H), 2.44 (m, 2H), 1.85 (m, 1H), 1.60 (m, 1H), 1.39 (m, 2H), 1.07 (m, 1H), 0.92 (m, 1H), 0.69 (m, 1H), 0.60 (m, 1H), 0.48 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.0, 145.9, 138.7, 130.8, 128.7, 122.0, 119.0, 118.1, 115.9, 107.0, 104.1, 87.5, 69.3, 60.9, 56.9, 45.1, 45.1, 44.8, 35.3, 30.1, 29.1, 23.4, 23.2, 19.6, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3223, 1635, 1455, 1116, 1033, 729. HRMS m/z: calc. 464.2471 [M + H]⁺; obs., 464.2532 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.58 min) and was found to be 99.65% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[3′-(2′-pyrrolyl)propanamido]morphinan Hydrochloride (6).

Compound 6 was synthesized as shown in the general procedure with 71% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.50 (s, 1H), 9.20 (s, 1H), 8.84 (s, 1H), 7.68 (d, J = 8 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.56 (m, 2H), 6.25 (s, 1H), 5.86 (m, 1H), 5.73 (m, 1H), 4.59 (d, J = 4 Hz, 1H), 4.42 (m, 1H), 3.91 (m, 1H), 3.27 (m, 2H), 3.04 (m, 2H), 2.94 (m, 1H), 2.73 (m, 2H), 2.44 (m, 2H), 1.85 (m, 1H), 1.61 (m, 1H), 1.38 (m, 1H), 1.07 (m, 1H), 0.93 (m, 1H), 0.69 (m, 1H), 0.60 (m, 1H), 0.48 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.1, 142.1, 141.2, 130.7, 129.6, 120.5, 119.1, 117.8, 115.9, 107.0, 104.2, 89.9, 69.7, 61.6, 56.6, 50.5, 46.4, 45.5, 35.8, 29.3, 27.2, 23.6, 23.1, 8.4, 5.6, 5.0, 2.6. IR (diamond, cm⁻¹) ν_max: 3162, 1644, 1407, 1125, 1032, 746. HRMS m/z: calc. 464.2471 [M + H]⁺; obs., 464.2554 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.45 min) and was found to be 98.97% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3′-pyrrolyl)carboxamido]morphinan Hydrochloride (7).

Compound 7 was synthesized as shown in the general procedure with 95% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 11.52 (s, 1H), 9.21 (s, 1H), 8.84 (s, 1H), 7.56 (m, 1H), 6.89 (s, 1H), 6.84 (s, 1H), 6.71 (d, J = 8 Hz, 1H), 6.58 (d, J = 8 Hz, 1H), 6.31 (s, 1H), 6.11 (m, 1H), 4.73 (m, 1H), 4.55 (m, 1H), 3.91 (s, 1H), 3.29 (m, 2H), 2.95 (m, 1H), 2.72 (m, 1H), 1.91 (m, 1H), 1.64 (m, 1H), 1.45 (m, 2H), 1.25 (m, 1H), 1.10 (m, 2H), 0.70 (m, 1H), 0.61 (m, 1H), 0.49 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 161.1, 146.0, 145.3, 143.8, 138.7, 128.6, 122.5, 122.0, 119.0, 118.2, 109.2, 87.1, 69.3, 56.9, 45.4, 45.1, 30.3, 30.2, 29.1, 23.4, 19.2, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3240, 1654, 1540, 1115, 1033, 917, 746. HRMS m/z: calc. 436.2158 [M + H]⁺; obs., 436.2231 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.44 min) and was found to be 99.20% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-pyrrolyl)carboxamido]morphinan Hydrochloride (8).

Compound 8 was synthesized as shown in the general procedure with 74% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.16 (s, 1H), 9.26 (s, 1H), 8.84 (s, 1H), 7.97 (d, J = 8 Hz, 1H), 7.54 (m, 1H), 6.73 (d, J = 8 Hz, 1H), 6.56 (d, J = 8 Hz, 1H), 6.38 (m, 1H), 6.28 (s, 1H), 6.22 (m, 1H), 4.60 (d, J = 4 Hz, 1H), 4.40 (m, 1H), 3.90 (d, J = 8 Hz, 1H), 3.59 (m, 1H), 2.94 (m, 1H), 2.71 (m, 1H), 2.48–2.41 (m, 1H), 1.86 (m, 1H), 1.61 (m, 1H), 1.40 (m, 2H), 1.06 (m, 1H), 0.94 (m, 1H), 0.69 (m, 1H), 0.60 (m, 1H), 0.48 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.3, 150.0, 145.9, 141.7, 138.8, 128.7, 122.0, 119.0, 118.2, 110.4, 107.1, 87.3, 69.3, 60.9, 56.9, 45.3, 45.1, 34.9, 30.1, 29.1, 23.4, 19.6, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3046, 1634, 1500, 1125, 1032, 748. HRMS m/z: calc. 436.2158 [M + H]⁺; obs., 436.2231 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.24 min) and was found to be 96.13% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-pyrrolyl)acetamido]morphinan Hydrochloride (9).

Compound 9 was synthesized as shown in the general procedure with 76% yield. ¹H NMR (400 MHz DMSO-d₆) δ: 10.51 (s, 1H), 9.23 (s, 1H), 8.81 (s, 1H), 7.72 (d, J = 8 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.60 (m, 1H), 6.56 (d, J = 8 Hz, 1H), 6.22 (s, 1H), 5.90 (m, 1H), 5.82 (m, 1H), 4.59 (d, J = 3.88 Hz, 1H), 4.39 (m, 1H), 3.87 (d, J = 8 Hz, 1H), 3.44 (s, 2H), 3.40 (m, 1H), 3.29 (m, 1H), 3.05 (m, 2H), 2.93 (m, 1H), 2.70 (m, 1H), 2.43 (m, 1H), 1.84 (m, 1H), 1.62 (m, 1H), 1.40 (m, 2H), 1.04 (m, 1H), 0.93 (m, 1H), 0.68 (m, 1H), 0.60 (m, 1H), 0.47 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.6, 169.8, 149.5, 131.2, 129.9, 123.3, 119.4, 117.8, 117.6, 116.5, 116.1, 115.8, 113.5, 108.1, 107.9, 89.0, 69.0, 60.7, 57.0, 45.0, 34.6, 32.1, 29.7, 29.1, 23.8, 19.5, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3222, 1749, 1644, 1494, 1116, 1068, 748. HRMS m/z: calc. 450.2315 [M + H]⁺; obs., 450.2390 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.50 min) and was found to be 95.14% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-pyrrolyl)acetamido]morphinan Hydrochloride (10).

Compound 10 was synthesized as shown in the general procedure with 74% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.55 (s, 1H), 9.34 (s, 1H), 8.82 (s, 1H), 8.16 (d, J = 8 Hz, 1H), 6.71 (d, J = 8 Hz, 1H), 6.63 (d, J = 8 Hz, 1H), 6.61 (m, 1H), 6.15 (s, 1H), 5.90 (m, 1H), 5.82 (m, 1H), 4.58 (d, J = 8 Hz, 1H), 3.83 (m, 1H), 3.39 (s, 2H), 3.3 (m, 1H), 3.04 (m, 3H), 2.84 (m, 1H), 2.42 (m, 2H), 1.72 (m, 2H), 1.51 (m, 1H), 1.43 (m, 1H), 1.33 (m, 2H), 1.07 (m, 1H), 0.67 (m, 1H), 0.58 (m, 1H), 0.50 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.8, 142.1, 141.2, 129.6, 120.5, 119.1, 117.8, 117.3, 115.8, 108.0, 89.9, 69.6, 61.6, 56.6, 50.6, 46.4, 45.5, 34.8, 29.2, 27.3, 23.6, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3062, 1656, 1315, 1127, 1033, 726. HRMS m/z: calc. 450.2315 [M + H]⁺; obs., 450.2369 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.47 min) and was found to be 99.86% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3-′pyrrolyl)propanamido]morphinan Hydrochloride (11).

Compound 11 was synthesized as shown in the general procedure with 41% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 11.53 (s, 1H), 10.11 (s, 1H), 9.23 (s, 1H), 8.86 (s, 1H), 7.58 (d, J = 8 Hz, 1H), 6.89 (m, 1H), 6.85 (m, 1H), 6.71 (d, J = 8 Hz, 1H), 6.58 (d, J = 8 Hz, 1H), 6.31 (s, 1H), 6.10 (m, 1H), 4.72 (d, J = 4 Hz, 1H), 4.56 (m, 1H), 3.93 (d, J = 4 Hz, 1H), 3.41 (s, 1H), 3.28 (m, 3H), 2.96 (m, 1H), 2.73 (m, 1H), 1.91 (m, 1H), 1.64 (m, 1H), 1.46 (m, 2H), 1.11 (m, 2H), 0.70 (m, 1H), 0.62 (m, 1H), 0.50 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.9, 146.0, 138.7, 128.7, 125.9, 122.0, 121.4, 119.1, 118.3, 110.8, 108.5, 87.5, 69.3, 61.0, 56.9, 45.3, 45.1, 45.1, 45.1, 30.2, 29.2, 23.4, 19.5, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3224, 1635, 1456, 1117, 1032, 944, 746. HRMS m/z: calc. 464.2471 [M + H]⁺; obs., 464.2522 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.41 min) and was found to be 96.13% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-pyrrolyl)propanamido]morphinan Hydrochloride (12).

Compound 12 was synthesized as shown in the general procedure with 72% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 11.52 (s, 1H), 10.01 (s, 1H), 9.23 (s, 1H), 8.84 (s, 1H), 7.75 (d, J = 8 Hz, 1H), 6.89 (m, 1H), 6.85 (m, 1H), 6.72 (d, J = 8 Hz, 1H), 6.58 (d, J = 8 Hz, 1H), 6.32 (s, 1H), 6.10 (m, 1H), 4.73 (m, 1H), 4.52 (d, J = 4 Hz, 1H), 3.92 (d, J = 8 Hz, 1H), 3.29 (m, 2H), 2.96 (m, 1H), 2.72 (m, 1H), 2.46 (m, 1H), 1.92 (m, 1H), 1.64 (m, 1H), 1.46 (m, 2H), 1.08 (m, 2H), 0.70 (m, 1H), 0.62 (m, 1H), 0.49 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.6, 142.1, 141.2, 129.6, 121.5, 120.5, 119.1, 117.8, 117.2, 114.5, 107.3, 89.9, 69.7, 61.6, 56.6, 50.5, 46.4, 45.5, 37.5, 29.2, 27.3, 23.6, 22.9, 22.7, 5.7, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3067, 1656, 1315, 1127, 748. HRMS m/z: calc. 464.2471 [M + H]⁺; obs., 464.2523 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.33 min) and was found to be 96.93% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(2′furanyl)carboxamido]morphinan Hydrochloride (13).

Compound 13 was synthesized as shown in the general procedure with 87% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.25 (s, 1H), 8.87 (s, 1H), 7.86 (m, 1H), 7.74 (d, J = 8 Hz, 1H), 7.20 (m, 1H), 6.73 (d, J = 8 Hz, 1H), 6.65 (m, 1H), 6.58 (d, J = 8 Hz, 1H), 6.35 (s, 1H), 4.72 (d, J = 4 Hz, 1H), 4.57 (m, 1H), 3.93 (m, 1H), 3.32 (s, 1H), 3.26 (m, 1H), 3.08 (m, 3H), 2.97 (m, 1H), 2.72 (m, 1H), 1.93 (m, 1H), 1.64 (m, 1H), 1.47 (m, 1H), 1.20 (t, J = 8 Hz, 1H), 0.69 (m, 1H), 0.61 (m, 1H), 0.51 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 157.1, 147.4, 145.8, 145.1, 138.8, 128.6, 122.0, 119.2, 118.3, 113.9, 111.9, 87.2, 69.2, 64.8, 60.9, 57.0, 45.2, 30.1, 29.1, 23.4, 19.5, 15.1, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3245, 1634, 1505, 1117, 1032, 727. HRMS m/z: calc. 437.1998 [M + H]⁺; obs., 437.2079 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.72 min) and was found to be 100% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′furanyl)carboxamido]morphinan Hydrochloride (14).

Compound 14 was synthesized as shown in the general procedure with 64% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.30 (s, 1H), 8.86 (s, 1H), 8.57 (d, J = 8 Hz, 1H), 7.85 (m, 1H), 7.13 (m, 1H), 6.73 (d, J = 8 Hz, 1H), 6.66 (d, J = 8 Hz, 1H), 6.64 (m, 1H), 6.18 (m, 1H), 4.84 (d, J = 8 Hz, 1H), 3.38 (s, 1H), 3.64 (m, 1H), 3.08 (m, 3H), 2.87 (m, 1H), 2.71 (m, 1H), 2.45 (m, 1H), 1.91 (m, 1H), 1.77 (m, 1H), 1.54 (m, 1H), 1.42 (m, 2H), 1.20 (t, J = 8 Hz, 1H), 1.08 (m, 1H), 0.69 (m, 1H), 0.60 (m, 1H), 0.52 (m, 1H), 0.42 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 157.3, 147.8, 144.9, 142.0, 141.3, 129.6, 120.5, 119.2, 117.8, 113.3, 111.8, 89.6, 69.6, 61.7, 56.6, 50.4, 45.5, 29.4, 27.2, 23.7, 23.0, 8.4, 5.7, 5.0, 2.6. IR (diamond, cm⁻¹) ν_max: 3010, 1644, 1503, 1126, 748. HRMS m/z: calc. 437.1998 [M + H]⁺; obs., 437.2087 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 6.31 min) and was found to be 98.05% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(2′-furanyl)acetamido]morphinan Hydrochloride (15).

Compound 15 was synthesized as shown in the general procedure with 74% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.54 (s, 1H), 9.25 (s, 1H), 8.87 (s, 1H), 7.78 (d, J = 8 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.60 (m, 1H), 6.55 (d, J = 8 Hz, 1H), 6.30 (s, 1H), 5.89 (m, 1H), 5.82 (m, 1H), 4.58 (d, J = 4 Hz, 1H), 4.38 (m, 1H), 3.93 (m, 1H), 3.44 (s, 1H), 3.38 (m, 1H), 3.27 (m, 2H), 3.04 (m, 2H), 2.94 (m, 1H), 2.70 (m, 1H), 2.43 (m, 1H), 1.87 (m, 1H), 1.59 (m, 1H), 1.39 (m, 2H), 1.07 (m, 1H), 0.93 (m, 1H), 0.67 (m, 1H), 0.60 (m, 1H), 0.48 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.0, 145.9, 138.8, 128.7, 125.4, 122.0, 119.0, 118.2, 116.6, 107.1, 105.8, 87.4, 69.3, 64.8, 60.9, 56.9, 48.5, 45.1, 34.8, 30.1, 29.1, 23.4, 19.7, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3055, 1660, 1563, 1119, 745. HRMS m/z: calc. 451.2155 [M + H]⁺; obs., 451.2207 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 4.84 min) and was found to be 96.72% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′-furanyl)acetamido]morphinan Hydrochloride (16).

Compound 16 was synthesized as shown in the general procedure with 53% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.34 (s, 1H), 8.83 (s, 1H), 8.34 (d, J = 8 Hz, 1H), 7.55 (s, 1H), 6.72 (d, J = 8 Hz, 1H), 6.64 (d, J = 8 Hz, 1H), 6.39 (m, 1H), 6.21 (m, 2H), 4.59 (d, J = 8 Hz, 1H), 3.85 (d, J = 4 Hz, 1H), 3.51 (s, 2H), 3.05 (m, 3H), 2.85 (m, 1H), 2.73 (m, 1H), 2.42 (m, 2H), 1.71 (m, 2H), 1.54 (m, 1H), 1.44 (m, 1H), 1.34 (m, 1H), 1.13 (m, 2H), 0.69 (m, 1H), 0.59 (m, 1H), 0.51 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.3, 149.7, 142.0, 141.8, 141.2, 129.5, 120.5, 119.2, 117.9, 110.4, 107.3, 89.7, 69.6, 61.6, 56.6, 50.9, 46.4, 45.5, 35.3, 29.2, 27.3, 23.5, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3005, 1658, 1466, 1128, 747. HRMS m/z: calc. 451.2155 [M + H]⁺; obs., 451.2225 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.45 min) and was found to be 97.44% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(2′-furanyl)propanamido]morphinan Hydrochloride (17).

Compound 17 was synthesized as shown in the general procedure with yield 65%. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.87 (s, 1H), 9.17 (s, 1H), 8.82 (s, 1H), 7.74 (d, J = 8 Hz, 1H), 7.49 (s, 1H), 6.71 (d, J = 8 Hz, 1H), 6.56 (d, J = 8 Hz, 1H), 6.34 (m, 1H), 6.22 (s, 1H), 6.11 (m, 1H), 4.59 (d, J = 4 Hz, 1H), 4.41 (m, 1H), 3.88 (d, J = 7 Hz, 1H), 3.34 (d, J = 16 Hz, 1H), 3.27 (m, 1H), 3.06 (m, 6H), 2.93 (m, 1H), 2.84 (m, 2H), 2.70 (m, 1H), 2.46 (m, 1H), 1.84 (m, 1H), 1.60 (m, 1H), 1.37 (m, 2H), 1.07 (m, 1H), 0.93 (m, 1H), 0.69 (m, 1H), 0.61 (m, 1H), 0.46 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.3, 154.7, 145.9, 141.2, 138.7, 128.7, 122.0, 119.0, 118.1, 110.3, 104.9, 87.4, 69.3, 61.0, 56.9, 45.4, 45.1, 45.1, 44.9, 33.2, 29.1, 23.4, 19.6, 8.4, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3188, 2981, 2947, 1635, 1506, 1455, 1116, 1033, 744. HRMS m/z: calc. 465.2311 [M + H]⁺, 487.2209 [M + Na]⁺; obs., 465.2404 [M + H]⁺, 487.2220 [M + Na]⁺. The purity of the compound was checked by HPLC (Rt = 6.25 min) and was found to be 97.59% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′-furanyl)propanamido]morphinan Hydrochloride (18).

Compound 18 was synthesized as shown in the general procedure with 72% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.31 (s, 1H), 8.82 (s, 1H), 8.19 (m, 1H), 7.50 (m, 1H), 6.72 (d, J = 8 Hz, 1H), 6.64 (d, J = 8 Hz, 1H), 6.35 (m, 1H), 6.16 (m, 1H), 6.10 (m, 1H), 4.55 (d, J = 8 Hz, 1H), 3.83 (s, 1H), 3.40 (m, 2H), 3.06 (m, 2H), 2.83 (m, 3H), 2.41 (m, 4H), 1.70 (m, 2H), 1.47 (m, 2H), 1.33 (m, 1H), 1.12 (m, 1H), 0.70 (m, 1H), 0.59 (m, 1H), 0.51 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.4, 154.6, 142.0, 141.2, 141.2, 129.5, 120.5, 119.2, 117.8, 110.3, 105.0, 89.8, 69.6, 61.6, 56.6, 55.9, 50.6, 46.4, 45.5, 33.7, 29.2, 27.3, 23.4, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3161, 1639, 1538, 1298, 1124, 1032, 748. HRMS m/z: calc. 465.2311 [M + H]⁺; obs., 465.2374 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.70 min) and was found to be 99.80% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-furanyl)carboxamido]morphinan Hydrochloride (19).

Compound 19 was synthesized as shown in the general procedure with 34% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.22 (s, 1H), 8.85 (s, 1H), 8.28 (s, 1H), 7.88–7.89 (m, 1H), 7.74 (m, 1H), 6.93 (s, 1H), 6.71 (d, J = 8 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 6.31 (s, 1H), 4.72 (d, J = 4 Hz, 1H), 4.56 (m, 1H), 3.91 (m, 1H), 3.28 (m, 2H), 3.27 (m, 3H), 2.95 (m, 1H), 2.71 (m, 2H), 1.92 (m, 1H), 1.63 (m, 1H), 1.45 (m, 2H), 1.04 (m, 1H), 0.70 (m, 1H), 0.61 (m, 1H), 0.49 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 161.1, 146.0, 145.3, 143.8, 138.7, 128.6, 122.5, 122.0, 119.0, 109.2, 87.1, 69.3, 56.9, 45.4, 45.1, 30.2, 29.1, 23.4, 19.2, 8.4, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3034, 1640, 1504, 1116, 1030, 946. HRMS m/z: calc. 437.1998 [M + H]⁺; obs., 437.2053 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 4.76 min) and was found to be 97.49% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-furanyl)carboxamido]morphinan Hydrochloride (20).

Compound 20 was synthesized as shown in the general procedure with 67% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.54 (s, 1H), 9.25 (s, 1H), 8.86 (s, 1H), 7.77 (d, J = 8 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.60 (m, 1H), 6.56 (d, J = 8 Hz, 1H), 6.29 (s, 1H), 5.90 (m, 1H), 5.82 (m, 1H), 4.58 (d, J = 3.84 Hz, 1H), 4.39 (m, 1H), 3.91 (d, J = 4 Hz, 1H), 3.31 (s, 1H), 3.24 (m, 1H), 3.03 (m, 2H), 2.95 (m, 1H), 2.70 (m, 1H), 2.44 (m, 1H), 1.86 (m, 1H), 1.61 (m, 1H), 1.39 (m, 2H), 1.07 (m, 1H), 0.94 (m, 1H), 0.69 (m, 1H), 0.60 (m, 1H), 0.49 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.0, 145.9, 138.8, 128.7, 125.3, 122.0, 119.0, 118.2, 116.6, 107.1, 105.8, 87.4, 69.3, 60.9, 57.5, 56.9, 45.1, 34.8, 30.1, 29.1, 23.4, 19.6, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3333, 1647, 1456, 1119, 870, 748. HRMS m/z: calc. 437.1998 [M + H]⁺, 459.1896 [M + Na]⁺; obs., 437.2091 [M + H]⁺, 459.1911 [M + Na]⁺. The purity of the compound was checked by HPLC (Rt = 6.41 min) and was found to be 97.12% pure.

17-Cyclopropylmetyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-furanyl)acetamido]morphinan Hydrochloride (21).

Compound 21 was synthesized as shown in the general procedure with 73% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.55 (s, 1H), 9.26 (s, 1H), 8.85 (s, 1H), 7.78 (d, J = 8 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.60 (m, 1H), 6.56 (d, J = 8 Hz, 1H), 6.29 (s, 1H), 5.91–5.89 (m, 1H), 5.82 (m, 1H), 4.59 (d, J = 4 Hz, 1H), 4.39 (m, 1H), 3.91 (d, J = 8 Hz, 1H), 3.34 (d, J = 20 Hz, 1H), 3.24 (m, 1H), 3.03 (m, 2H), 2.94 (m, 1H), 2.70 (m, 1H), 2.43 (m, 1H), 1.86 (m, 1H), 1.59 (m, 1H), 1.41 (m, 2H), 1.07 (m, 1H), 0.98 (m, 1H), 0.70 (m, 1H), 0.60 (m, 1H), 0.49 (m, 1H), 0.38 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.0, 145.9, 138.8, 128.7, 125.3, 122.0, 119.0, 118.2, 116.6, 107.1, 105.8, 87.4, 69.3, 60.9, 56.9, 48.5, 45.1, 35.1, 30.0, 29.1, 23.4, 19.6, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3231, 1640, 1319, 1117, 1032, 725. HRMS m/z: calc. 451.2155 [M + H]⁺; obs., 451.2209 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 4.80 min) and was found to be 96.01% pure.

17-Cyclopropylmetyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-furanyl)acetamido]morphinan Hydrochloride (22).

Compound 22 was synthesized as shown in the general procedure with 46% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.31 (s, 1H), 8.81 (s, 1H), 8.24 (d, J = 8 Hz, 1H), 7.58 (m, 1H), 7.51 (s, 1H), 6.72 (d, J = 8 Hz, 1H), 6.64 (d, J = 8 Hz, 1H), 6.42 (m, 1H), 6.13 (m, 1H), 4.58 (d, J = 8 Hz, 1H), 3.83 (m, 1H), 3.25 (m, 2H), 3.05 (m, 2H), 2.84 (m, 1H), 2.42 (m, 2H), 1.71 (m, 2H), 1.52 (m, 1H), 1.44 (m, 1H), 1.34 (m, 1H), 1.07 (m, 1H), 0.68 (m, 1H), 0.60 (m, 1H), 0.51 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.4, 142.8, 141.9, 141.0, 139.9, 129.8, 120.7, 119.2, 119.1, 117.9, 111.5, 89.6, 69.6, 61.8, 56.4, 50.7, 46.4, 45.5, 32.0, 29.3, 27.2, 23.5, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3057, 1659, 1500, 1129, 1011, 771. HRMS m/z: calc. 451.2155 [M + H]⁺; obs., 451.2241 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 7.50 min) and was found to be 98.73% pure.

17-Cyclopropylmetyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-furanyl)propanamido]morphinan Hydrochloride (23).

Compound 23 was synthesized as shown in the general procedure with 51% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.15 (s, 1H), 8.84 (s, 1H), 7.69 (d, J = 8 Hz, 1H), 7.54 (m, 1H), 7.44 (m, 1H), 6.72 (d, J = 8 Hz, 1H), 6.56 (d, J = 8 Hz, 1H), 6.39 (m, 1H), 6.24 (s, 1H), 4.58 (d, J = 4 Hz, 1H), 4.43–4.37 (m, 1H), 3.90 (d, J = 4 Hz, 1H), 3.26 (m, 1H), 3.04 (m, 2H), 2.95 (m, 1H), 2.71 (m, 1H), 2.63 (t, 2H), 2.45 (d, J = 4 Hz, 1 H), 2.40 (t, 2H), 1.85 (m, 1H), 1.61 (m, 1H), 1.37 (m, 2H), 1.05 (m, 1H), 0.91 (m, 1H), 0.68 (m, 1H), 0.61 (m, 1H), 0.48 (m, 1H), 0.39 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.9, 145.9, 142.9, 138.8, 138.7, 128.7, 124.0, 122.0, 119.0, 118.2, 111.1, 87.5, 69.3, 64.8, 61.0, 56.9, 45.1, 44.8, 35.3, 30.1, 29.1, 23.4, 20.4, 19.6, 15.1, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3229, 1651, 1452, 1117, 1068, 749. HRMS m/z: calc. 465.2311 [M + H]⁺; obs., 465.2403 [M + H]⁺. The purity of the compound was checked by HPLC (Rt = 4.88 min) and was found to be 100% pure.

17-Cyclopropylmetyl-3, 14β-dihydroxy-4,5α-epoxy-6β-[(3′-furanyl)propanamido]morphinan Hydrochloride (24).

Compound 24 was synthesized as shown in the general procedure with 57% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.22 (s, 1H), 8.84 (s, 1H), 8.28 (s, 1H), 7.89–7.87 (m, 1H), 7.74 (t, J = 1.64 Hz, 1H), 6.93 (s, 1H), 6.72 (d, J = 8 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 6.30 (m, 1H), 4.72 (d, J = 3.84 Hz, 1H), 4.55 (m, 1H), 3.90 (m, 1H), 2.94 (m, 1H), 2.72 (m, 2H), 1.91 (m, 1H), 1.63 (m, 1H), 1.46 (m, 2H), 1.11 (m, 3H), 0.69 (m, 1H), 0.61 (m, 1H), 0.49 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.0, 142.8, 142.1, 141.2, 138.9, 129.6, 123.9, 120.5, 119.1, 117.8, 111.2, 89.8, 69.6, 61.6, 56.6, 50.5, 46.4, 45.5, 35.9, 29.3, 27.3, 23.6, 22.9, 20.3, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3059, 1634, 1500, 1124, 873, 748. HRMS m/z: calc. 465.2311 [M + H]⁺, 487.2209 [M + Na]⁺; obs., 465.2400 [M + H]⁺, 487.2225 [M + Na]⁺. The purity of the compound was checked by HPLC (Rt = 6.43 min) and was found to be 97.61% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-(2′-thienylcarboxamido)morphinan Hydrochloride (25).

Compound 25 was synthesized as shown in the general procedure with 82.57% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.30 (s, 1H, exchangeable), 8.82 (s, 1H, exchangeable), 8.17 (d, J = 7.7 Hz, 1H, exchangeable), 7.88 (d, J = 3.6 Hz, 1H), 7.77 (d, J = 5.0 Hz, 1H), 7.17 (t, J = 4.3 Hz, 1H), 6.71 (d, J = 8.2 Hz, 1H), 6.59 (d, J = 8.1 Hz, 1H), 6.25 (s, 1H, exchangeable), 4.76 (d, J = 3.7 Hz, 1H), 4.55 (s, 1H), 3.07 (m, 3H), 2.93 (s, 2H), 2.71 (d, J = 21.5 Hz, 1H), 1.89 (d, J = 15.3 Hz, 1H), 1.64 (d, J = 13.5 Hz, 1H), 1.48 (m, 2H), 1.19 (m, 2H), 1.04 (d, J = 6.1 Hz, 1H), 0.70 (m, 1H), 0.62 (m, 1H), 0.47 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.8, 146.4, 145.9, 145.4, 139.5, 138.7, 131.0, 128.5, 127.8, 119.1, 87.1, 69.2, 65.6, 61.1, 57.0, 47.5, 46.0, 45.2, 29.2, 23.3, 20.9, 15.1, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3069, 1621, 1537, 1456, 1316, 1031, 745. HRMS m/z: calc. 453.1803 [M + H]⁺; obs., 453.1854 [M + H]⁺. Mp 233.5–235.8 °C. The purity of the compound was checked by HPLC (Rt = 6.49 min) and was found to be 97.47% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(2′thienylcarboxamido)morphinan Hydrochloride (26).

Compound 26 was synthesized as shown in the general procedure with 30.55% yield. ¹H NMR (400 MHz, DMSO-d₆) δ:. 9.36 (s, 1H, exchangeable), 8.86 (s, 1H, exchangeable), 8.72 (d, J = 8.3 Hz, 1H, exchangeable), 7.83 (dd, J = 3.7, 1.1 Hz, 1H), 7.78 (dd, J = 5.1, 1.1 Hz, 1H), 7.18 (dd, J = 5.0, 3.7 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 6.18 (s, 1H, exchangeable), 4.80 (d, J = 7.8 Hz, 1H), 3.87 (d, J = 5.6 Hz, 1H), 3.69–3.57 (m, 1H), 3.12 (d, J = 6.0 Hz, 1H), 3.10–3.01 (m, 1H), 2.85 (t, J = 9.8 Hz, 1H), 2.45 (dd, J = 10.4, 3.3 Hz, 2H), 1.89 (m, 1H), 1.77 (m, 1H), 1.59 (m, 1H), 1.44 (m, 2H), 1.09 (m, 1H), 0.69 (m, 1H), 0.60 (m, 1H), 0.52 (m, 1H), 0.42 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.7, 141.9, 141.1, 139.9, 139.8, 130.9, 129.5, 128.0, 127.9, 120.5, 119.3, 117.8, 89.6, 69.5, 61.6, 56.6, 50.9, 46.4, 45.5, 29.2, 27.2, 23.7, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3074, 1646, 1543, 1462, 1319, 1021, 746. HRMS m/z: calc. 453.1803 [M + H]⁺, 475.1701 [M + Na]⁺, 927.3588 [2 M + Na]⁺; obs., 453.1845 [M + H]⁺, 475.1646 [M + Na]⁺, 927.3049 [2 M + Na]⁺. Mp 289–292.6 °C. The purity of the compound was checked by HPLC (Rt = 6.42 min) and was found to be 99.82% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(2′-thienylacetamido)morphinan Hydrochloride (27).

Compound 27 was synthesized as shown in the general procedure with 40.17% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.22 (s, 1H, exchangeable), 8.81 (s, 1H, exchangeable), 8.01 (d, J = 7.9 Hz, 1H, exchangeable), 7.36 (dd, J = 5.1, 1.4 Hz, 1H), 6.70 (m, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 6.19 (s, 1H, exchangeable), 4.60 (s, 1H), 4.40 (s, 1H), 3.87 (s, 1H), 3.73 (d, J = 1.0 Hz, 2H), 3.18 (d, J = 4.5 Hz, 1H), 3.03 (s, 2H), 2.92 (d, J = 13.6 Hz, 1H), 2.71 (d, J = 24.2 Hz, 1H), 2.45 (d, J = 18.2 Hz, 1H), 1.83 (s, 1H), 1.62 (d, J = 13.6 Hz, 1H), 1.42 (dd, J = 14.8, 9.8 Hz, 2H), 1.25 (s, 1H), 1.00 (m, 2H), 0.68 (m, 1H), 0.61 (m, 1H), 0.47 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.6, 145.9, 138.7, 137.6, 128.6, 126.4, 125.88, 124.7, 119.0, 118.1, 87.3, 69.2, 60.9, 56.9, 48.5, 45.1, 36.2, 30.1, 29.1, 23.4, 19.5, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3134, 1634, 1543, 1457, 1321, 1033, 746. HRMS m/z: calc. 467.1960 [M + H]⁺, 489.1858 [M + Na]⁺; obs., 467.2001 [M + H]⁺, 489.1813 [M + Na]⁺. Mp 198.5–200.1 °C. The purity of the compound was checked by HPLC (Rt = 6.68 min) and was found to be 97.70% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6β-(2′-thienylacetamido)morphinan Hydrochloride (28).

Compound 28 was synthesized as shown in the general procedure with 86.61% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.34 (s, 1H, exchangeable), 8.81 (s, 1H, exchangeable), 8.41 (d, J = 7.8 Hz, 1H, exchangeable), 7.36 (dd, J = 5.1, 1.3 Hz, 1H), 6.96 (dd, J = 5.2, 3.4 Hz, 1H), 6.92 (dd, J = 3.4, 1.3 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.13 (s, 1H, exchangeable), 4.58 (d, J = 7.8 Hz, 1H), 3.82 (m, 1H), 3.66 (m, 2H), 3.08 (dq, J = 7.3, 2.6 Hz, 1H), 3.03 (dt, J = 10.4, 4.5 Hz, 1H), 2.85 (m, 1H), 2.41 (m, 1H), 1.71 (td, J = 14.9, 14.2, 3.0 Hz, 2H), 1.54 (m, 1H), 1.45 (d, J = 10.3 Hz, 1H), 1.34 (m, 1H), 1.07 (m, 1H), 0.68 (m, 1H), 0.59 (m, 1H), 0.50 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.7, 142.0, 141.2, 137.4, 129.5, 126.5, 125.9, 124.8, 120.5, 119.2, 117.9, 89.7, 69.6, 61.6, 56.6, 50.9, 46.4, 45.5, 36.7, 29.2, 27.3, 23.4, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3003, 1658, 1553, 1466, 1314, 1031, 749. HRMS m/z: calc. 467.1960 [M + H]⁺, 489.1858 [M + Na]⁺; obs., 467.2032 [M + H]⁺, 489.1842 [M + Na]⁺. Mp 210–212 °C. The purity of the compound was checked by HPLC (Rt = 6.53 min) and was found to be 96.39% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[3′-(thiophen-2″-yl)propanamido]morphinan Hydrochloride (29).

Compound 29 was synthesized as shown in the general procedure with 72.17% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.20 (s, 1H, exchangeable), 8.84 (s, 1H, exchangeable), 7.76 (d, J = 8.1 Hz, 1H, exchangeable), 7.30 (d, J = 5.1 Hz, 1H), 6.93 (dd, J = 5.1, 3.4 Hz, 1H), 6.88 (d, J = 3.4 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 6.25 (s, 1H, exchangeable), 4.59 (d, J = 4.1 Hz, 1H), 4.43 (tt, J = 8.4, 4.1 Hz, 1H), 3.89 (d, J = 7.0 Hz, 1H), 3.28 (m, 1H), 3.05 (m, 4H), 2.94 (m, 1H), 2.71 (m, 1H), 2.44 (dd, J = 13.5, 4.9 Hz, 1H), 1.85 (dt, J = 15.4, 9.4 Hz, 1H), 1.62 (dd, J = 13.0, 3.5 Hz, 1H), 1.41 (dd, J = 14.9, 9.5 Hz, 2H), 1.27 (t, J = 7.2 Hz, 1H), 1.04 (m, 1H), 0.93 (tt, J = 13.4, 8.2 Hz, 1H), 0.69 (m, 1H), 0.62 (m, 1H), 0.48 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.3, 145.9, 143.7, 138.7, 128.6, 126.7, 124.4, 123.6, 122.0, 119.0, 118.1, 87.4, 69.3, 64.8, 61.0, 56.9, 45.1, 44.8, 36.8, 30.1, 29.1, 25.2, 23.4, 19.6, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3065, 1644, 1587, 1456, 1319, 1032, 746. HRMS m/z: calc. 481.2116 [M + H]⁺, 503.2014 [M + Na]⁺; obs., 481.2159 [M + H]⁺, 503.1978 [M + Na]⁺. Mp 173.4–175.6 °C. The purity of the compound was checked by HPLC (Rt = 6.71 min) and was found to be 98.46% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6β-[3′-(thiophen-2″-yl)propanamido]morphinan Hydrochloride (30).

Compound 30 was synthesized as shown in the general procedure with 53.44% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.34 (s, 1H, exchangeable), 8.80 (s, 1H, exchangeable), 8.18 (d, J = 7.9 Hz, 1H, exchangeable), 7.30 (dd, J = 5.1, 1.3 Hz, 1H), 6.93 (dd, J = 5.1, 3.4 Hz, 1H), 6.85 (m, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.10 (s, 1H, exchangeable), 4.54 (d, J = 7.9 Hz, 1H), 3.81 (d, J = 5.6 Hz, 1H), 3.03 (td, J = 7.3, 3.3 Hz, 4H), 2.82 (m, 2H), 2.75 (dd, J = 5.1, 2.8 Hz, 1H), 2.66 (m, 1H), 2.43 (m, 2H), 1.68 (d, J = 12.7 Hz, 2H), 1.53 (d, J = 8.4 Hz, 1H), 1.44 (d, J = 11.0 Hz, 1H), 1.35 (m, 1H), 1.26 (t, J = 5.9 Hz, 1H), 1.07 (m, 1H), 0.69 (d, J = 5.6 Hz, 1H), 0.59 (m, 1H), 0.55 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.5, 143.5, 142.0, 141.2, 129.5, 126.8, 124.5, 123.6, 120.5, 119.2, 117.8, 89.8, 69.6, 61.6, 56.6, 50.6, 46.4, 45.5, 37.3, 29.2, 27.2, 25.1, 23.5, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3053, 1644, 1532, 1455, 1298, 1061, 749. HRMS m/z: calc. 481.2116 [M + H]⁺; obs., 481.2166 [M + H]⁺. Mp 207.4–209.8 °C. The purity of the compound was checked by HPLC (Rt = 6.74 min) and was found to be 97.48% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(3′thienylcarboxamido)morphinan Hydrochloride (31).

Compound 31 was synthesized as shown in the general procedure with 32.40% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.20 (s, 1H, exchangeable), 8.81 (s, 1H, exchangeable), 8.22 (dd, J = 2.9, 1.3 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H, exchangeable), 7.61 (dd, J = 5.0, 2.9 Hz, 1H), 7.55 (dd, J = 5.0, 1.3 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 8.1 Hz, 1H), 6.22 (s, 1H, exchangeable), 4.77 (d, J = 3.9 Hz, 1H), 4.61–4.51 (m, 1H), 3.88 (d, J = 7.1 Hz, 2H), 3.11 (d, J = 6.6 Hz, 1H), 3.05 (m, 1H), 2.94 (m, 1H), 2.68 (d, J = 2.3 Hz, 1H), 2.34 (d, J = 2.2 Hz, 1H), 1.89 (m, 1H), 1.66 (d, J = 13.3 Hz, 1H), 1.48 (m, 2H), 1.18 (d, J = 8.0 Hz, 1H), 1.05 (m, 2H), 0.70 (m, 1H), 0.63 (m, 1H), 0.48 (m, 1H), 0.42 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 161.6, 146.0, 138.7, 137.5, 128.9, 128.6, 127.1, 126.5, 122.0, 119.0, 118.2, 87.1, 69.3, 64.8, 61.0, 56.9, 46.4, 45.6, 45.1, 29.2, 23.4, 19.2, 13.6, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3069, 1621, 1537, 1456, 1316, 1031, 745. HRMS m/z: calc. 453.1803 [M + H]⁺; obs., 453.1858 [M + H]⁺. Mp 198.3–200.3 °C. The purity of the compound was checked by HPLC (Rt = 5.65 min) and was found to be 96.98% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6β-(3′-thienylcarboxamido)morphinan Hydrochloride (32).

Compound 32 was synthesized as shown in the general procedure with 67.60% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.31 (s, 1H, exchangeable), 8.83 (s, 1H, exchangeable), 8.49 (d, J = 8.0 Hz, 1H, exchangeable), 8.16 (dd, J = 3.0, 1.3 Hz, 1H), 7.61 (dd, J = 5.0, 2.9 Hz, 1H), 7.53 (dd, J = 5.0, 1.3 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 6.12 (s, 1H, exchangeable), 4.79 (d, J = 7.8 Hz, 1H), 3.85 (d, J = 5.6 Hz, 1H), 3.66 (dt, J = 12.9, 6.3 Hz, 1H), 3.12 (d, J = 5.9 Hz, 1H), 3.05 (m, 1H), 2.99 (d, J = 8.9 Hz, 1H), 2.85 (t, J = 9.4 Hz, 1H), 2.45 (m, 2H), 1.87 (q, J = 13.0 Hz, 1H), 1.76 (d, J = 13.5 Hz, 1H), 1.62 (t, J = 15.4 Hz, 1H), 1.45 (m, 2H), 1.26 (t, J = 7.0 Hz, 1H), 1.09 (d, J = 8.9 Hz, 1H), 0.70 (m, 1H), 0.61 (m, 1H), 0.52 (m, 1H), 0.43 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 161.5, 142.1, 141.3, 137.7, 129.6, 128.7, 126.7, 120.5, 119.2, 117.9, 89.8, 69.7, 61.8, 56.7, 50.8, 46.4, 45.5, 29.3, 27.3, 23.0, 5.6, 5.0, 2.6. IR (diamond, cm⁻¹) ν_max: 3025, 1636, 1557, 1462, 1304, 1032, 747. HRMS m/z: calc. 453.1803 [M + H]⁺; obs., 453.1852 [M + H]⁺. Mp 289.5–291.6 °C. The purity of the compound was checked by HPLC (Rt = 6.22 min) and was found to be 98.85% pure.

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-(3′-thienylacetamido)morphinan Hydrochloride (33).

Compound 33 was synthesized as shown in the general procedure with 44.64% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.22 (s, 1H, exchangeable), 8.81 (s, 1H, exchangeable), 7.93 (d, J = 7.9 Hz, 1H, exchangeable), 7.46 (dd, J = 5.0, 3.0 Hz, 1H), 7.28 (t, J = 2.0 Hz, 1H), 7.06 (dd, J = 4.9, 1.3 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 6.19 (s, 1H, exchangeable), 4.62 (d, J = 4.0 Hz, 1H), 4.39 (s, 1H), 3.87 (d, J = 6.9 Hz, 1H), 3.51 (s, 2H), 3.06 (dd, J = 19.7, 7.7 Hz, 2H), 2.95 (d, J = 14.2 Hz, 1H), 2.72 (m, 1H), 2.42 (m, 1H), 1.83 (dd, J = 15.9, 8.7 Hz, 1H), 1.62 (d, J = 13.5 Hz, 1H), 1.42 (dd, J = 15.3, 9.5 Hz, 2H), 1.02 (m, 2H), 0.69 (m, 1H), 0.61 (m, 1H), 0.47 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.2, 145.9, 138.8, 136.1, 128.7, 128.6, 125.5, 122.0, 122.0, 119.0, 118.1, 87.3, 69.2, 61.0, 56.9, 45.1, 36.8, 30.1, 29.1, 23.4, 19.5, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 3133, 1629, 1538, 1457, 1320, 1034, 746. HRMS m/z: calc. 467.1960 [M + H]⁺, 489.1858 [M + Na]⁺; obs., 467.2010 [M + H]⁺, 489.1820 [M + Na]⁺. Mp 196–198.2 °C. The purity of the compound was checked by HPLC (Rt = 6.47 min) and was found to be 97.35% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(3′thienylacetamido)morphinan Hydrochloride (34).

Compound 34 was synthesized as shown in the general procedure with 62.31% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.34 (s, 1H, exchangeable), 8.83 (s, 1H, exchangeable), 8.34 (d, J = 7.9 Hz, 1H), 7.46 (dd, J = 4.9, 2.9 Hz, 1H), 7.25 (dd, J = 2.9, 1.3 Hz, 1H), 7.04 (dd, J = 4.9, 1.3 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 8.2 Hz, 1H), 6.17 (s, 1H, exchangeable), 4.59 (d, J = 7.8 Hz, 1H), 3.84 (d, J = 5.6 Hz, 1H), 3.05 (m, 2H), 2.86 (ddt, J = 11.0, 7.7, 3.5 Hz, 1H), 2.73 (dd, J = 4.9, 1.4 Hz, 1H), 2.42 (m, 2H), 1.73 (m, 2H), 1.51 (dt, J = 12.8, 4.4 Hz, 1H), 1.44 (d, J = 9.8 Hz, 1H), 1.33 (td, J = 13.8, 3.1 Hz, 1H), 1.07 (m, 1H), 0.68 (m, 1H), 0.60 (m, 1H), 0.51 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.4, 142.0, 141.2, 135.9, 129.5, 128.5, 125.6, 122.0, 120.5, 119.2, 117.9, 89.8, 69.6, 61.6, 56.6, 50.8, 46.4, 45.5, 37.2, 29.2, 27.3, 23.5, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3061, 1644, 1540, 1457, 1325, 1034, 745. HRMS m/z: calc. 467.1960 [M + H]⁺; obs., 467.2019 [M + H]⁺. Mp 249.6–252.2 °C. The purity of the compound was checked by HPLC (Rt = 6.29 min) and was found to be 98.08% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[3′-(thiophen-3″-yl)propanamido]morphinan Hydrochloride (35).

Compound 35 was synthesized as shown in the general procedure with 42.61% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.20 (s, 1H, exchangeable), 8.83 (s, 1H, exchangeable), 7.70 (d, J = 8.0 Hz, 1H, exchangeable), 7.44 (dd, J = 4.9, 2.9 Hz, 1H), 7.18 (d, J = 2.9 Hz, 1H), 7.01 (dd, J = 4.9, 1.3 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 6.22 (s, 1H, exchangeable), 4.59 (d, J = 4.1 Hz, 1H), 4.41 (ddd, J = 13.1, 8.3, 4.0 Hz, 1H), 3.87 (d, J = 6.9 Hz, 1H), 3.05 (dd, J = 19.6, 7.0 Hz, 2H), 2.95 (m, 2H), 2.89–2.77 (m, 3H), 2.72 (m, 1H), 1.84 (dt, J = 15.1, 9.3 Hz, 1H), 1.63 (m, 1H), 1.41 (dd, J = 15.9, 10.4 Hz, 2H), 0.95 (m, 2H), 0.69 (m, 1H), 0.62 (m, 1H), 0.48 (m, 1H), 0.40 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.9, 145.9, 141.5, 138.7, 128.7, 128.3, 125.7, 122.0, 120.3, 87.4, 69.2, 61.0, 56.9, 45.1, 44.84, 35.8, 31.8, 30.1, 29.1, 25.6, 23.42, 15.1, 5.6, 5.1, 2.5. IR (diamond, cm⁻¹) ν_max: 2945, 1640, 1538, 1455, 1319, 1032, 747. HRMS m/z: calc. 481.2116 [M + H]⁺, 503.2014 [M + Na]⁺; obs., 481.2154 [M + H]⁺, 503.1965 [M + Na]⁺. Mp 175.4–178 °C. The purity of the compound was checked by HPLC (Rt = 6.85 min) and was found to be 96.47% pure.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[3′-(thiophen-3″-yl)propanamido]morphinan Hydrochloride (36).

Compound 36 was synthesized as shown in the general procedure with 78.26% yield. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.33 (s, 1H, exchangeable), 8.80 (s, 1H, exchangeable), 8.13 (d, J = 7.9 Hz, 1H,exchangeable), 7.44 (dd, J = 4.9, 2.9 Hz, 1H), 7.16 (dd, J = 2.9, 1.3 Hz, 1H), 7.00 (dd, J = 4.9, 1.3 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.11 (s, 1H, exchangeable), 4.54 (d, J = 7.9 Hz, 1H), 3.82 (d, J = 5.5 Hz, 1H), 3.08 (d, J = 5.8 Hz, 1H), 3.02 (dd, J = 11.7, 5.0 Hz, 2H), 2.82 (tt, J = 7.3, 4.2 Hz, 3H), 2.74 (m, 1H), 2.39 (q, J = 7.6 Hz, 3H), 1.68 (d, J = 12.6 Hz, 2H), 1.47 (m, 2H), 1.34 (m, 1H), 1.22 (m, 1H), 1.1 (m, 1H), 0.68 (m, 1H), 0.60 (m, 1H), 0.50 (m, 1H), 0.41 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.0, 142.1, 141.4, 141.2, 129.6, 128.3, 125.6, 120.5, 120.5, 119.2, 117.8, 89.8, 69.6, 61.7, 56.6, 50.5, 48.5, 45.5, 36.4, 29.3, 27.2, 25.5, 23.5, 22.9, 5.6, 5.0, 2.5. IR (diamond, cm⁻¹) ν_max: 3053, 1644, 1530, 1455, 1298, 1087, 750. HRMS m/z: calc. 481.2116 [M + H]⁺; obs., 481.2174 [M + H]⁺. Mp 209.8–211.6 °C. The purity of the compound was checked by HPLC (Rt = 6.66 min) and was found to be 99.50% pure.

Biological Evaluation of Drugs.

Morphine (morphine sulfate pentahydrate salt) was purchased from Mallinckrodt (St. Louis, MO) or provided by the National Institute of Drug Abuse (NIDA). Naltrexone and naloxone were purchased as their hydrochloride salts from Sigma-Aldrich (St. Louis, MO). All drugs and test compounds were dissolved in pyrogen-free isotonic saline (Baxter Healthcare, Deerfield, IL) or terile-filtered distilled/deionized water. All other reagents and radioligands were purchased from either Sigma-Aldrich or Thermo Fisher.

Animals.

Male Swiss Webster mice (25–35 g, 6–8 weeks, Harlan Laboratories, Indianapolis, IN) were housed in a temperaturecontrolled (20–22 °C) AAALAC-accredited facility in which they had ad libitum access to food and water. The mice were maintained on a 12 h/12 h light–dark cycle (0600–1800 lights on) for the duration of the experiment and were tested during the light segment of this cycle. Mice arrived at the vivarium housed 4/cage and, following 1 week habituation, were separated into individual cages. Mice were allowed to acclimate to individual caging for at least 24 h and then were randomly assigned to the various treatment conditions before the start of studies. Experimenters were blinded to these treatment conditions during the duration of the experiment and data analysis. No adverse events occurred during the experiment, and no mice were excluded from data analysis. Protocols and procedures (Animal Welfare Assurance Number D16–00180) were approved by the Institutional Animal Care and Use Committee (IACUC) at the Virginia Commonwealth University Medical Center and complied with the recommendations of the IASP (International Association for the Study of Pain).

In Vitro Competitive Radioligand Binding Assay.

The competition binding assay was conducted using the monoclonal mouse opioid receptors expressed in Chinese hamster ovary (CHO) cell lines (the monoclonal human δ opioid receptor was used in the DOR assay). In this assay, 20–30 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 7.7) 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 at the MOR, KOR, and DOR was determined as the difference in binding obtained in the absence and presence of 5 μM naltrexone, U50,488, and SNC80, respectively. All competition binding data were transformed to % bound = specific binding in the presence of competing ligand/specific binding in the absence of competing ligand × 100%.

In Vitro [³⁵S]GTPγS Functional Assay.

The [³⁵S]GTPγS functional assay was conducted to determine the efficacy of the compounds at the MOR.⁷² In this assay, 10 μg of MOR-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 was included in the assay as the maximally effective concentration of a full agonist for the MOR. After incubation, the bound radioactive ligand was separated from the free radioligand by filtration through a GF/B glass fiber filter paper and rinsed three times with ice-cold wash buffer (50 mM Tris–HCl, pH 7.2) using the 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 the agonist. Percent of DAMGO-stimulated [³⁵S]GTPγS binding was defined as (net-stimulated binding by ligand/net-stimulated binding by 3 μM DAMGO) × 100%.

Data Analysis of Receptor Binding and [³⁵S]GTPγS Functional Assay.

The assays of all samples were conducted in duplicate and repeated at least three times for a total of ≥3 independent determinations. Results were reported as mean values ± SEM. Concentration–effect curves were fitted by nonlinear regression to a four parameter model with the minimum constrained to 0, using GraphPad Prism software, to determine Hill deficient, EC₅₀, and E_max values. IC₅₀ values were obtained from nonlinear regression fitting to the four parameter model with the maximum (absence of competitor) constrained 100% and the minimum constrained to 0 using GraphPad Prism software. By using the Cheng–Prusoff equation K_i = IC₅₀/[1 + ([L]/K_D)], where [L] is the concentration of the competitor and K_D is the K_D of the radioligand; binding K_i values were determined from IC₅₀ values.⁷³

Warm-Water Tail Immersion Assay.

The antinociceptive effect of synthesized compounds was determined using the warm-water tail immersion assay.⁷⁴ Swiss Webster mice (six male mice for each group, 25–35 g, 6–8 weeks old) were used in this assay. Antinociception for all compounds was examined in male Swiss Webster mice. The water bath temperature was set as 56 ± 0.1 °C. The baseline latency (control) was determined before administration of the compounds to the mice, and only mice with a baseline latency of 2 to 4 s were used. In the agonism study, the tail immersion was done 20 min (time that the morphine effect starts to peak) after injecting the test compounds subcutaneously (s.c.). To prevent tissue damage, a 10 s maximum cutoff time was imposed. Antinociceptive response was calculated as the percentage of the maximum possible effect (%MPE), where % MPE = [(test − control latency)/(10 − control latency)] × 100. When being studied for their antagonist effects to morphine, the test compounds were given (s.c.) 5 min before morphine. The tail immersion test was then conducted 20 min after giving morphine (s.c.). %MPE was calculated for each mouse. AD₅₀ values were calculated using the least-squares linear regression analysis followed by calculation of 95% confidence interval by the Bliss method.

Opioid-Withdrawal Studies.

Swiss Webster mice (six male mice for each group, 25–35 g, 6–8 weeks old) were used for opioid-withdrawal studies. Following a previously reported protocol,^35,36 a 75 mg morphine pellet was implanted into the back of the mice, and the mice were allowed to recover in their home cages. Before being tested, a 30 min period was allowed for habituation to an open-topped, square, clear Plexiglas observation chamber (26 × 26 × 26 cm³) with lines partitioning the bottom into quadrants. All drugs and test compounds were administered (s.c.). The withdrawal was precipitated 72 h from pellet implantation with naloxone (1 mg/kg, s.c.) or the test compounds at varying doses. Withdrawal commenced within 3 min after antagonist administration. Escape jumps, paw tremors, and wet dog shakes were quantified by counting their occurrences over 20 min for each mouse. The data are presented as the mean ± SEM.

Caco-2 Permeability Studies.

Human epithelial colorectal adenocarcinoma (Caco-2) cells (HTB-37) were cultured in T75 flasks using complete Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS), 1% glutamine, 1% penicillin, and 1% streptomycin, at 37 °C in a 5% CO₂ atmosphere. Cells were passaged at 80–90% confluency using 0.05% trypsin–EDTA, and the medium was changed every other day. Following this, the cells were trypsinized, suspended in the medium, and applied to a Millipore 96-well plate where they were cultured as monolayers at a density of 25,000 cells/well. The cells were incubated in a 37 °C/5% CO₂ incubator to allow cell attachment and proliferation. Media was changed every 2–3 days for 21 days when cells reached 100% confluency. For apical → basolateral (A → B) permeability, 10 μM compound 25 was added to the apical (A) side and the amount of permeation as determined on the basolateral (B) side; for basolateral → apical (B → A) permeability, 10 μM compound 25 was added to the B-side and the amount of permeation was determined on the A side. The A-side buffer contained 100 μM lucifer yellow dye, in transport buffer (1.98 g/L glucose in 10 mM HEPES, 1x Hank’s balanced salt solution) pH 7.4, and the B-side buffer used was the transport buffer at pH 7.4. Caco-2 cells were incubated with 10 μM compound 25 in these buffers for 1 h. Ranitidine and Colchicine (low permeability), Labetalol and Propranolol (high permeability) were used as controls. At the end of the assay, donor and receiver side solution samples were collected, quenched by 100% methanol containing an internal standard, and centrifuged at 5000 rpm for 10 min at 4 °C. Following centrifugation, the supernatant for donor and receiver side samples was analyzed by HPLC-MS/MS to determine peak area ratios.

Data was expressed as Papp (cm/s):

Papp = \frac{(VR \times CR, end)}{dt} \times \frac{1}{A \times (CD, mid - CR, mid)}

(1)

where VR is the volume of the receiver chamber. CR,end is the concentration of the test compound in the receiver chamber at the end time point, dt is the incubation time, and A is the surface area of the cell monolayer. CD,mid is the calculated mid-point concentration of the test compound on the donor side, which is the mean value of the donor concentration at time 0 min and the donor concentration at the end time point. CR,mid is the mid-point concentration of the test compound on the receiver side, which is one-half of the receiver concentration at the end time point. Concentrations of the test compound were expressed as peak areas of the test compound.

In Vivo BBB Penetration Studies.

Swiss Webster mice (three mice each time point) were given compound 25 (10 mg/kg, s.c.) or the vehicle. At 5, 10, and 30 min time points post administration, the mice were decapitated, and brain samples and blood samples were collected. Blood samples were centrifuged for 10 min at 15,000g at 4 °C following which plasma was collected. Brain and plasma samples were stored at −80 °C until further analysis.

LCMS/MS Analysis.

The identification and quantification of compound 25 in mouse plasma and brain were performed using a modification of a previously described method with naloxone-d₅ as the internal standard.⁷⁵ Chromatographic separation of compound 25 and naltrexone-d₅ was achieved using a Shimadzu Nexera X2 liquid chromatography system with a Zorbax XDB-C18 4.6 × 75 mm, 3.5 μm column (Agilent Technologies, Santa Clara, CA). Mobile phase A consisted of water with 1 g/L ammonium formate and 0.1% formic acid, and mobile Phase B consisted of methanol. The flow rate was 1 mL/min. The systems’ detector was a Sciex 6500 QTRAP system with an IonDrive Turbo V source for TurbolonSpray (Sciex, Ontario, Canada). The following quantification and qualifying transition ions were monitored in a positive multiple reaction monitoring mode with collisions energies in parentheses: compound 25, 453 > 435 (27), 453 > 308 (35), and 455 > 267(43); naloxone-d₅, 333 > 212 (45), 333 > 315 (25), and 333 > 273. Concentrations were determined by a linear regression plot based on peak area ratios of the calibrators.

Statistical Analysis.

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

Supplementary Material

jm2c00087-si001

NIHMS1881169-supplement-jm2c00087-si001.pdf^{(9.9MB, pdf)}

jm2c00087-si002

NIHMS1881169-supplement-jm2c00087-si002.csv^{(3.4KB, csv)}

ACKNOWLEDGMENTS

The authors are grateful to the NIDA Drug Supply Program for providing the free base of naltrexone. This work was partially supported by NIH/NIDA DA024022, DA044855, DA050311 (Y.Z.), and NIH P30DA033934 (W.L.D. and M.S.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Drug Abuse or the National Institutes of Health.

ABBREVIATIONS USED

BBB: brain–blood barrier
cAMP: cyclic adenosine monophosphate
CHO: Chinese hamster ovary
CL: confidence level
CNS: central nervous system
DAMGO: D-Ala2-MePhe4-Gly(ol)5]enkephalin
DOR: δ opioid receptor
EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
GPCR: G protein-coupled receptor
HOBt: hydroxybenzotriazole
KOR: κ opioid receptor
MOR: μ opioid receptor
NAP: 17-cyclopropylmethyl-3,14-dihydroxy-4,5a-epoxy-6β-[(4′-pyridyl)carboxamido]morphinan
NIDA: National Institute of Drug Abuse
NLX: naloxone
NOP: nociception/orphanin FQ receptor
NTA: naltrexamine
OUD: opioid use disorder; % MPE, percentage maximum possible effect

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00087.

Spectra data for target compounds (¹H NMR, ¹³C NMR, and HPLC graphs); in silico physicochemical property prediction of target compounds; CNS-MPO and LLE calculations of the most potent compounds (PDF)

Molecular formula strings and some data (CSV)

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.2c00087

Contributor Information

Piyusha P. Pagare, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

Mengchu Li, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

Yi Zheng, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

Abhishek S. Kulkarni, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States

Samuel Obeng, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

Boshi Huang, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

Christian Ruiz, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

James C. Gillespie, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

Rolando E. Mendez, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

David L. Stevens, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

Justin L. Poklis, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

Matthew S. Halquist, Department of Pharmaceutics, Virginia Commonwealth University, Richmond, Virginia 23298, United States

William L. Dewey, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

Dana E. Selley, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

Yan Zhang, Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, United States.

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PERMALINK

Design, Synthesis, and Biological Evaluation of NAP Isosteres: A Switch from Peripheral to Central Nervous System Acting Mu-Opioid Receptor Antagonists

Piyusha P Pagare

Mengchu Li

Yi Zheng

Abhishek S Kulkarni

Samuel Obeng

Boshi Huang

Christian Ruiz

James C Gillespie

Rolando E Mendez

David L Stevens

Justin L Poklis

Matthew S Halquist

William L Dewey

Dana E Selley

Yan Zhang

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

RESULTS AND DISCUSSION

Molecular Design.

Figure 2.

Chemical Synthesis.

Scheme 1.

In Vitro Pharmacological Studies.

Table 1.

Table 2.

In Vivo Warm-Water Tail Immersion Assay.

Figure 3.

Table 3.

KOR and DOR [35S]GTPγS Binding Assays.

Table 4.

In Vivo Opioid Withdrawal Studies.

Figure 4.

BBB-Penetration Studies.

Table 5.

CONCLUSIONS

EXPERIMENTAL SECTION

Chemistry.

General Procedure for the Amide Coupling/Hydrolysis Reaction.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6a-[(2′-pyrrolyl)carboxamido]morphinan Hydrochloride (1).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′pyrrolyl)carboxamido]morphinan Hydrochloride (2).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6a-[2′-(2′-pyrrolyl)acetamido]morphinan Hydrochloride (3).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[2′-(2′-pyrrolyl)acetamido]morphinan Hydrochloride (4).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α[3′-(2′-pyrrolyl)propanamido]morphinan Hydrochloride (5).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[3′-(2′-pyrrolyl)propanamido]morphinan Hydrochloride (6).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3′-pyrrolyl)carboxamido]morphinan Hydrochloride (7).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-pyrrolyl)carboxamido]morphinan Hydrochloride (8).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-pyrrolyl)acetamido]morphinan Hydrochloride (9).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-pyrrolyl)acetamido]morphinan Hydrochloride (10).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3-′pyrrolyl)propanamido]morphinan Hydrochloride (11).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-pyrrolyl)propanamido]morphinan Hydrochloride (12).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(2′furanyl)carboxamido]morphinan Hydrochloride (13).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′furanyl)carboxamido]morphinan Hydrochloride (14).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(2′-furanyl)acetamido]morphinan Hydrochloride (15).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′-furanyl)acetamido]morphinan Hydrochloride (16).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(2′-furanyl)propanamido]morphinan Hydrochloride (17).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′-furanyl)propanamido]morphinan Hydrochloride (18).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-furanyl)carboxamido]morphinan Hydrochloride (19).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-furanyl)carboxamido]morphinan Hydrochloride (20).

17-Cyclopropylmetyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-furanyl)acetamido]morphinan Hydrochloride (21).

17-Cyclopropylmetyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-furanyl)acetamido]morphinan Hydrochloride (22).

17-Cyclopropylmetyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[(3′-furanyl)propanamido]morphinan Hydrochloride (23).

17-Cyclopropylmetyl-3, 14β-dihydroxy-4,5α-epoxy-6β-[(3′-furanyl)propanamido]morphinan Hydrochloride (24).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-(2′-thienylcarboxamido)morphinan Hydrochloride (25).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(2′thienylcarboxamido)morphinan Hydrochloride (26).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(2′-thienylacetamido)morphinan Hydrochloride (27).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6β-(2′-thienylacetamido)morphinan Hydrochloride (28).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-[3′-(thiophen-2″-yl)propanamido]morphinan Hydrochloride (29).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6β-[3′-(thiophen-2″-yl)propanamido]morphinan Hydrochloride (30).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(3′thienylcarboxamido)morphinan Hydrochloride (31).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6β-(3′-thienylcarboxamido)morphinan Hydrochloride (32).

17-Cyclopropylmethyl-3, 14β-dihydroxy-4,5α-epoxy-6α-(3′-thienylacetamido)morphinan Hydrochloride (33).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(3′thienylacetamido)morphinan Hydrochloride (34).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[3′-(thiophen-3″-yl)propanamido]morphinan Hydrochloride (35).

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[3′-(thiophen-3″-yl)propanamido]morphinan Hydrochloride (36).

Biological Evaluation of Drugs.

Animals.

KOR and DOR [³⁵S]GTPγS Binding Assays.

In Vitro [³⁵S]GTPγS Functional Assay.

Data Analysis of Receptor Binding and [³⁵S]GTPγS Functional Assay.