Verifying the role of 3-hydroxy of 17-cyclopropylmethyl-4,5α-epoxy-3,14β-dihydroxy-6β-[(4′-pyridyl) carboxamido]morphinan derivatives via their binding affinity and selectivity profiles on opioid receptors

Article abstract of DOI:10.1016/j.bioorg.2021.104702

In the present study, the role of 3-hydroxy group of a series of epoxymorphinan derivatives in their binding affinity and selectivity profiles toward the opioid receptors (ORs) has been investigated. It was found that the 3-hydroxy group was crucial for the binding affinity of these derivatives for all three ORs due to the fact that all the analogues 1a-e exhibited significantly higher binding affinities compared to their counterpart 3-dehydroxy ones 6a-e. Meanwhile most compounds carrying the 3-hydroxy group possessed similar selectivity profiles for the kappa opioid receptor over the mu opioid receptor as their corresponding 3-dehydroxy derivatives. [³⁵S]-GTPγS functional assay results indicated that the 3-hydroxy group of these epoxymorphinan derivatives was important for maintaining their potency on the ORs with various effects. Further molecular modeling studies helped comprehend the remarkably different binding affinity and functional profiles between compound 1c (NCP) and its 3-dehydroxy analogue 6c.

Full text of DOI:10.1016/j.bioorg.2021.104702

Bioorganic Chemistry 109 (2021) 104702
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Verifying the role of 3-hydroxy of
17-cyclopropylmethyl-4,5α
-epoxy-3,14β-dihydroxy-6β-[(4′-pyridyl)
carboxamido]morphinan derivatives via their binding affinity and
selectivity profiles on opioid receptors
,
Boshi Huanga, Rama Guntaa, Huiqun Wanga, Mengchu Lia, Danni Caob d, Rolando E. Mendezc,
James C. Gillespiec, Chongguang Chenb, Lan-Hsuan M. Huangb, Lee-Yuan Liu-Chenb,
,
*
Dana E. Selleyc, Yan Zhanga
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 800 E Leigh Street, Richmond, VA 23298, United States
b Center for Substance Abuse Research and Department of Pharmacology, Lewis Katz School of Medicine, Temple University, 3500 North Broad Street, MERB 851,
Philadelphia, PA 19140, United States
c Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North 12th Street, Richmond, VA 23298, United States
d Beijing Key Laboratory of Neuropsychopharmacology, State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, the role of 3-hydroxy group of a series of epoxymorphinan derivatives in their binding
affinity and selectivity profiles toward the opioid receptors (ORs) has been investigated. It was found that the 3-
hydroxy group was crucial for the binding affinity of these derivatives for all three ORs due to the fact that all the
analogues 1a-e exhibited significantly higher binding affinities compared to their counterpart 3-dehydroxy ones
6a-e. Meanwhile most compounds carrying the 3-hydroxy group possessed similar selectivity profiles for the
kappa opioid receptor over the mu opioid receptor as their corresponding 3-dehydroxy derivatives. [35S]-GTPγS
functional assay results indicated that the 3-hydroxy group of these epoxymorphinan derivatives was important
for maintaining their potency on the ORs with various effects. Further molecular modeling studies helped
comprehend the remarkably different binding affinity and functional profiles between compound 1c (NCP) and
its 3-dehydroxy analogue 6c.
3-Hydroxy group
Opioid receptors
Binding affinity
Selectivity
Molecular docking
1. Introduction
MOR agonists, including morphine have been the frontline medication
in the management of moderate to severe pain for a long time [5].
The opioid receptors (ORs) belong to G-protein-coupled receptors
(GPCRs) and generally fall into four subtypes: opioid receptor (MOR),
Naloxone and naltrexone, two primary MOR antagonists, have been
utilized in the treatment of opioid use disorders [6]. Nalbuphine, a KOR
agonist and MOR partial agonist, is the only approved non-scheduled
opioid analgesic in the U.S [7]. Norbinaltorphimine (nor-BNI),
behaved as a KOR antagonist in animal models, and has demonstrated
antidepressant and antipanic-like activities [8]. As a highly selective
DOR antagonist, naltrindole (NTI) was found to significantly attenuate
the discriminative stimulus properties of cocaine in rats [9], and showed
promise in the treatment of stress disorders [10]. However, these OR
ligands are also accompanied by a battery of potential side effects which
have compromised their applications [11]. Therefore, novel small
μ
κ opioid receptor (KOR), δ opioid receptor (DOR), and the nociceptin/
orphanin FQ (N/OFQ) peptide (NOP) receptor [1,2]. ORs couple to Gi/
Go G-proteins, generating the efflux of K+, closing voltage-gated Ca2+
channels and inhibiting adenylyl cyclase to reduce formation of cyclic
adenosine monophosphate (cAMP) [3]. It has been extensively reported
that activation of different types of ORs may yield various pharmaco-
logical behaviors [4]. As a result, a large number of full agonists, partial
agonists and antagonists targeting ORs have been applied to treat
different diseases or used in pharmacological research. For example, the
* Corresponding author.
E-mail address: yzhang2@vcu.edu (Y. Zhang).
https://doi.org/10.1016/j.bioorg.2021.104702
Received 16 July 2020; Received in revised form 10 September 2020; Accepted 28 January 2021
Available online 9 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
molecules targeting ORs with minimal side effects are still highly
desirable for various clinical applications.
In the past decade, we have been constructing an opioid ligands li-
brary, with small molecules featuring diversified heterocyclic sub-
stituents introduced onto the 4,5-epoxymorphinan skeleton [4,12–21],
and have explored the potential therapeutic applications of these new
opioid ligands in preclinical studies [6,22–28]. In an attempt to develop
peripheral selective MOR ligands as therapeutic agents toward opioid-
induced constipation (OIC) [12,14,15,21–23,25,28], we obtained com-
pounds 1a-e which were preliminarily identified as KOR/MOR dual
selective ligands with subnanomolar binding affinities through the
competitive radioligand binding assays, among which 1b and 1c
exhibited moderate KOR selectivity over the MOR (Ki ratio,
9.62, respectively) [15] (Fig. 1).
μ/κ: 3.5 and
Fig. 2. General structure of 4,5-epoxymorphinan containing the para-
hydroxyphenylethylamine moiety.
It has been well-accepted that the 4,5-epoxymorphinan skeleton
embeds a prevalent para-hydroxyphenylethylamine moiety that corre-
sponds to the N-terminal tyrosine residue in the endogenous opioid
peptides, e.g. enkephalins, endorphins and dynorphins (Fig. 2) [29] and
such a chemical structural feature should be critical for opioid receptor
recognition in general, which was derived from previous studies of
opioid ligands that were mainly from medicinal chemists’ perspective
[30–37].
in the field. In 2018, the crystal structure of human KOR in complex with
a potent 4,5-epoxymorphinan opioid agonist and an active-state-
stabilizing nanobody was reported [37]. In this study, binding affin-
ities of nalfurafine for the KOR and MOR were determined respectively,
with KOR Ki = 0.32 nM and MOR Ki = 4.20 nM, and μ/κ Ki ratio at 13.
Meanwhile, removal of the 3-hydroxy group in nalfurafine yielded
compound CT-18 (Fig. 3). It was found that binding affinities of CT-18
for both the KOR and MOR (for KOR, Ki = 1.50 nM; for MOR, Ki = 533
It is noteworthy that nalfurafine (TRK-820), approved for the treat-
ment of uremic pruritus in Japan, also bears the 4,5-epoxymorphinan
skeleton while acting as a selective KOR agonist [38]. It has been re-
ported that the 3-hydroxy, an essential functional group in the critical
para-hydroxyphenylethylamine moiety, of nalfurafine derivatives,
bearing the 4,5-epoxymorphinan skeleton or the decahydro(iminoe-
thano)phenanthrene skeleton, has been proven indispensable for
maintaining binding affinities for the opioid receptors [33,39,40].
Meanwhile, apparently such an observation mainly came from the
structure–activity relationship (SAR) studies of nalfurafine derivatives
with very limited information from structural biology due to technical
limitation previously.
nM; μ/κ: 355) decreased compared to those of nalfurafine while its MOR
binding affinity appeared to be affected much more significantly than
for the KOR [37]. Thus removing 3-hydroxy group seemed more
favorable for opioid ligand selectivity for the KOR over the MOR. Such
observation motivated us to reconsider the role of 3-hydroxy group for
its influence on binding affinity and particularly on the selectivity pro-
files to the three ORs.
Moreover, another example from a previous study demonstrated that
chemical modification on the 3-hydroxy group of levorphanol with the
aminothiazole moiety was well tolerant, and a highly potent KOR se-
lective ligand ATPM was generated which the selectivity for the KOR
over the MOR was reversed [46] (Fig. 4). These results further suggested
that 3-hydroxy group may not be as critical as a required pharmaco-
phore in at least some epoxymorphinan derivatives.
With vast progresses achieved in modern technology, a large number
of GPCR crystal structures have been resolved in the last two decades.
More recently, the crystal complexes of all three major ORs (i.e. MOR,
KOR, and DOR) at both active and inactive states have been obtained
[37,41–45], which allow us to re-examine the roles of some “indis-
pensable” moieties in opioid ligands from the angle of structural biology
and possibly to revisit many taken-for-granted concepts and conclusions
Take together, it seems that certain modifications at the 3-position
may be acceptable from both perspectives of structural biology and
medicinal chemistry. Therefore, we believe that it is imperative to re-
investigate the influence of 3-hydroxy group of epoxymorphinan de-
rivatives on especially KOR selectivity over the MOR. More specifically,
we implemented the current study in our epoxymorphinan derivatives
1a-e as KOR/MOR dual selective ligands by removing their 3-hydroxy
group and verifying such an operation on ligand selectivity and func-
tional profiles toward opioid receptors. Herein, we report the synthesis
of the newly designed compounds 6a-e, their SAR and molecular
modeling study results.
2. Results and discussion
2.1. Chemistry
The chemical synthesis of compounds 1a-e has been reported [15].
The synthetic route of compounds 6a-e is outlined in Scheme 1. The
Fig. 1. Chemical structures of compounds 1a-e.
Fig. 3. Chemical structures of nalfurafine and compound CT-18.
2

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
stereoselective reductive amination to give the 6β-dibenzyl protected
compound 4. Subsequent deprotection of the dibenzyl group of com-
pound 4 under catalytic hydrogenation condition afforded the key in-
termediate 5. Finally, the desired compounds 6a-e were prepared
through the amide coupling reaction of 5 with substituted pyr-
idylcarboxylic acids and subsequent salt formation with reasonable
yields. All the target compounds were fully characterized.
2.2. In vitro radioligand binding assays and [35S]-GTPγS functional
assays
Fig. 4. Chemical structures of levorphanol and compound ATPM.
To determine the binding affinity and selectivity profiles of the new
compounds 6a-e on the three opioid receptors, the in vitro competitive
radioligand binding assays were conducted using Chinese hamster ovary
(CHO) cells overexpressing monoclonal opioid receptors. The KOR and
DOR were labeled with [3H]diprenorphine, while the MOR was labeled
with [3H]naloxone, respectively.
syntheses of compounds 6a-e was performed in a similar way to that of a
previous report [47] and to that of 1a-e with the removal of 3-hydroxy
group first: the tetrazolyl ether 2 was obtained by reacting the starting
material naltrexone with 5-chloro-1-phenyl-1H-tetrazole. Catalytic hy-
drogenation of compound 2 in glacial acetic acid provided the 3-desoxy-
naltrexone 3 with a yield of 82%. The intermediate 3 was undergone a
As shown in Table 1, all the compounds carrying 3-hydroxy group, i.
e. 1a-e, possessed subnanomolar binding affinity for the KOR and
Scheme 1. Synthesis of the target compounds 6a-e.
Table 1
The radioligand binding affinity to the KOR, MOR, and DOR and selectivity profiles of compounds 1a-e and 6a-e.a
Ki (nM)
Selectivity
/κ
Compounds
R1
R2
KOR
MOR
DOR
μ
δ/κ
1a
OH
H
Cl
0.15 ± 0.04b
10.46 ± 1.38
0.18 ± 0.03b
7.44 ± 0.7c
0.10 ± 0.04b
18.84 ± 3.01
0.63 ± 0.18b
24.51 ± 3.23c
1.25 ± 0.55b
58.27 ± 5.16c
0.39 ± 0.20b
39.03 ± 2.56
0.60 ± 0.23b
45.63 ± 4.59
602.20 ± 22.30b
2123.66 ± 101.18
173.70 ± 134.90b
2086.15 ± 71.59
75.30 ± 7.00b
0.67
1.8
4015
203
965
280
579
551
155
38.0
349
67.9
6a
Cl
1b
OH
H
Br
3.5
6b
Br
3.3
1c (NCP)
6c
OH
H
CN
0.13 ± 0.01b
6.13 ± 0.74c
0.58 ± 0.12b
96.07 ± 14.84
0.46 ± 0.04b
86.91 ± 9.22
9.6
CN
3375.73 ± 743.96
9.5
1d
OH
H
CH3
CH3
OCH3
OCH3
90.10 ± 17.10b
0.67
0.41
1.3
6d
3649.72 ± 158.17
160.40 ± 6.60b
1e
OH
H
6e
5900.42 ± 300.78
0.53
a
The values are the mean ± SEM of at least three independent experiments.
b
c
Data have been reported in Reference [15], and are presented here for comparison purposes.
Data have been reported in Reference [48], and are presented here for comparison purposes.
3

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
subnanomolar to one-digit nanomolar binding affinity for the MOR,
while they bound to the DOR with relatively low affinity of Ki values at
two- digit nanomolar to submicromolar level. Moreover, the 3-dehy-
droxy compounds 6a-e exhibited lower binding affinity for the KOR,
with Ki values at the one- to two-digit nanomolar level. They also bound
to the MOR and DOR with Ki values at the two-digit nanomolar level and
the micromolar level, respectively, which were substantially lower in
affinity compared to their 3-hydroxylated counterparts. These results
clearly demonstrated that the 3-hydroxy was crucial for the affinities of
these epoxymorphinan ligands in binding to all three opioid receptors.
In addition, the effects of different substituents varying in size and
electronic natures at the 2′-position of the pyridine ring on binding af-
finity for the KOR and MOR were examined. It seemed that electron-
withdrawing groups were more favorable than electron-donating
groups for KOR binding affinity though not significantly. Among these
compounds, compound 1c (NCP) was found to display the highest
binding affinity for the KOR, and displayed the highest selectivity for the
Table 2
[
35S]-GTPγS Functional Assay Results of Compounds 1b, 1c, 6b, and 6c.a
KOR [35S]-GTPγS
MOR [35S]-GTPγS
DOR [35S]-GTPγS
binding
binding
binding
Compounds
EC50
(nM)
% Emax of
EC50
(nM)
% Emax
of
EC50
(nM)
% Emax
of
U50,488H
DAMGO
DPDPE
1b
0.34
98.76 ±
1.52
0.42 ±
0.05
61.29 ±
1.11
15.79
± 2.96
2675
57.34
± 0.03
81.88
± 9.09
0.28
± 2.48
145.10
± 12.31
55.68
6b
99.27 ±
2.79
290.90
± 41.99
1.82 ±
0.28
33.62 ±
3.92
± 689
29.88
± 3.99
1850
1c (NCP)
6c
97.14 ±
1.50
58.14 ±
1.48
± 0.03
92.00
± 8.47
± 1.71
113.90
± 3.10
100.20 ±
2.37
338.43
± 31.10
26.48 ±
3.73
± 152
a
The values are the mean ± SEM of at least three independent experiments.
much lower potency. Combined together, these results indicated that the
3-hydroxy group of these epoxymorphinan derivatives appeared to have
different influences on activation profiles of the three ORs.
KOR over the MOR (μ/κ = 9.62) as well as very high selectivity for the
KOR over the DOR (δ/κ = 579). Overall, these compounds exhibited
higher binding affinities for the KOR and MOR than for the DOR. Based
on our previous studies [5,12,15,21], it has been demonstrated that the
binding pocket in both the KOR and MOR can form aromatic stacking,
hydrophobic and hydrogen bonding interaction with these ligands
containing a 4′-pyridyl moiety.
2.3. Molecular modeling study
From the binding and functional assays results, it was found that
NCP (Fig. 5a) possessed about 50-fold higher binding affinities for both
the KOR and MOR than those of compound 6c (Fig. 5b). In addition,
both NCP and 6c acted as KOR full agonists and MOR partial agonists,
with NCP showing higher efficacy than compound 6c on the MOR.
Notably, both of NCP and 6c contained a ‘message’ moiety (epox-
ymorphinan moiety) and an ‘address’ moiety (2-cyanopyridyl moiety),
which were similar to other epoxymorphinan derivatives [4,20,21]. The
only difference between the chemical structures of NCP and compound
6c was the 3-hydroxy group on their phenyl ring.
The selectivity of these compounds for the KOR over the MOR and
DOR was also compared. In general, compounds carrying electron-
withdrawing groups, e.g. CN and Br, exhibited higher selectivity for
the KOR over the MOR and DOR than compounds with electron-
donating groups (CH3 and OCH3). As discussed above, compounds 1c
and 6c with the cyano group at 2′-position of the 4′-pyridyl ring
possessed the highest selectivity for the KOR over the MOR and DOR
overall. Interestingly, the selectivity profile for the KOR over the MOR
was relatively uninfluenced by the bearing of 3-hydroxy group (e.g. 1b
vs 6b, 1c vs 6c, and 1d vs 6d), suggesting that the impact of removing 3-
hydroxy group on the binding affinities was similar for the KOR and
MOR because of the nearly same degree of selectivity profile shift be-
tween these two sets of compounds. This observation was somewhat
different from the previous report [37]. On the other hand, the selec-
tivity profiles for the KOR over the DOR of all the 3-hydroxy group
bearing analogues were relatively higher than those of the correspond-
ing 3-dehydroxy analogues, revealing that the 3-hydroxy group may
exert noteworthy influence on the selectivity profiles for the KOR over
the DOR.
To investigate why NCP and compound 6c with such similar chem-
ical structures exhibited significantly different binding affinity and
functional profiles at the KOR and MOR, NCP was docked into the active
KOR and MOR crystal structures, and compound 6c was docked into the
active KOR and the inactive MOR ones. The docking solution with the
highest CHEM-PLP score was merged into the respective receptor to
afford the ligand-receptor complex. Subsequently, energy minimization
was further conducted on the four ligand-receptor complexes
(NCP_KORactive, NCP_MORactive, compound 6c_KORactive, and compound
6c_MORinactive, Fig. S1). Furthermore, 100 ns molecular dynamics (MD)
simulations were conducted on the four ligand-receptor complexes and
their binding modes from the MD simulations of each complex evaluated
by the root-mean-square deviation (RMSD, Fig. S2) were shown in
Fig. 6.
Compounds 1b and 1c (NCP) together with their 3-dehydroxy ana-
logues 6b and 6c were chosen to further determine their potencies and
relative efficacies at the KOR, MOR, and DOR. [35S]-GTPγS functional
assays were conducted on KOR-, MOR- and DOR-expressed CHO cells.
EC50 and Emax values are measures of potency and efficacy, respectively.
As shown in Fig. 6, NCP and compound 6c exhibited similar in-
teractions with the orthosteric sites in the NCP_KORactive, NCP_MOR-
active, compound 6c_KORactive, and compound 6c_MORinactive complexes,
which was the same to the molecular docking results (Fig. S1). The
orthosteric sites of the KOR and MOR accommodated the rigid and bulky
4, 5-epoxymorphinan moiety of NCP and compound 6c. Three types of
interactions between the key residues located at the orthosteric site and
the two ligands were established. First, residues M3.36, W6.48, I6.51, and
H6.52 formed hydrophobic interactions with the 4, 5-epoxymorphinan
moiety. Second, D3.32 formed ionic interaction with the piperidine
quaternary ammonium nitrogen atom of the ligands’ 4, 5-epoxymor-
phinan nucleus. Third, a hydrogen bonding interaction was observed
between Y3.33 and the dihydrofuran oxygen atom of the ligands. How-
ever, the interactions between compound 6c and the allosteric site in
compound 6c_MORinactive complex were different from those of NCP in
NCP_KORactive and NCP_MORactive complexes and compound 6c in
compound 6c_KORactive complex. The binding subdomain of the 2-cya-
nopyridyl moiety of compound 6c in compound 6c_MORinactive com-
plex showed a significant change after 100 ns MD simulations (Fig. 6)
E
max values are expressed as % of the maximal response produced by the
full agonists U50,488H, DAMGO, and DPDPE at the KOR, MOR, and
DOR, respectively (Table 2). As shown in Table 2, compounds 1b and 1c
(NCP) acted as full agonists of the KOR with % Emax values of 98.76%
and 97.14%, and exhibited subnanomolar potency with EC50 values of
0.34 nM and 0.28 nM, respectively. Their counterparts without a 3-hy-
droxy group (6b and 6c) also demonstrated full agonism of KOR (%
Emax = 99.27% and 100.20%, respectively) but with much lower po-
tency (EC50 = 81.88 nM and 92.00 nM, respectively). Apparently, the 3-
hydroxy group did not significantly affect the efficacy at the KOR
whereas its removal led to a dramatic decrease in KOR potency. Addi-
tionally, compound 1b and 1c (NCP) behaved as MOR partial agonists
with moderate efficacy (1b, % Emax = 61.29%; 1c, % Emax = 58.14%),
while 6b and 6c acted as MOR partial agonists with low efficacy (6b, %
Emax = 33.62%; 6c, % Emax = 26.48%). Moreover, compounds 1b and 1c
(NCP) acted as DOR partial agonists with moderate efficacy (1b, % Emax
= 57.34%; 1c, % Emax = 55.68%), while 6b and 6c exhibited full ago-
nism to DOR (6b, % Emax = 145.10%; 6c, % Emax = 113.90%) but with
4

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
Fig. 5. The chemical structures of NCP (a) and compound 6c (b). The structures with notions were derived from the optimized NCP_KORactive and compound
6c_KORactive complexes.
compared with that from molecular docking operation (Fig. S1). As
shown in Fig. 6, the 2-cyanopyridyl moiety of NCP and compound 6c
was adopted in a subdomain of the allosteric site of the KOR and MOR,
formed by residues Q2.60, WECL1, and L(KOR)/I(MOR)3.29. These resi-
dues seemed to establish hydrophobic interactions with the pyridyl ring
of NCP. Similar interactions were also formed in compound 6c_KORactive
complex. While in compound 6c_MORinactive complex, the 2-cyanopyr-
idyl moiety of compound 6c bound with another subdomain of the
allosteric site of the MOR, formed by residues V6.55 and W7.35. To be
noted, the binding modes of NCP in the KOR and MOR and compound 6c
in the KOR, were almost identical to those of the KOR agonist MP1104 in
the active KOR and the MOR agonist BU72 in the active MOR (Fig. S3)
[37,42]. Additionally, our previous molecular modeling studies revealed
that the ‘address’ moiety of an epoxymorphinan derivative interacted
with the subdomain formed by residues Q2.60, WECL1, and I3.29 of the
allosteric site of the MOR may exhibit positive allosteric modulation to
the ‘message’ moiety and induce the ligand to display agonism profile to
the MOR. On the contrary, the ‘address’ moiety of an epoxymorphinan
derivative bound with the subdomain formed by residues V6.55 and
W7.35 of the allosteric site of the MOR may demonstrate negative allo-
steric modulation to the ‘message’ moiety and leads to the ligand acting
as an antagonist to the MOR [20,49]. Therefore, we speculated that
these findings might explain why NCP and compound 6c exhibited
similar agonism activities at the KOR as that of MP1104 and why NCP
possessed a different activation profile from that of compound 6c at the
MOR.
shown in the binding mode of NCP_KORactive complex after 100 ns MD
simulations (Fig. 6), these water-mediated hydrogen bonds were also
established between the 3-hydroxy group of NCP and residues Y3.33
,
K
5.39, and H6.52. However, in the compound 6c_KORactive complex, due
to the lack of the 3-hydroxy group, compound 6c did not form any
water-mediated hydrogen bonds with residues Y3.33, K5.39, and H6.52
.
These similar observations could also be found in the NCP_MORactive and
compound 6c_MORinactive complexes, in which NCP formed the water-
mediated hydrogen-bonding interactions with residues Y3.33, K5.39
,
and H6.52 through its 3-hydroxy group while compound 6c did not. More
importantly, it can be concluded from Table S1 that the water-mediated
hydrogen-bonding interactions could strengthen the interactions be-
tween NCP and residues Y3.33, K5.39, and H6.52 compared with that in the
compound 6c_KORactive and compound 6c_MORinactive complexes.
Therefore, the lack of water-mediated hydrogen bonding interactions
due to removal of the 3-hydroxy group may lead to decrease in binding
affinities for the KOR and MOR, which provided a computational
explanation to the lower binding affinities of compound 6c to the KOR
and MOR compared to those of NCP.
3. Conclusion
In summary, we have investigated the role of the 3-hydroxy group of
the epoxymorphinan derivatives in the binding affinity and selectivity
profiles toward the opioid receptors for a series of compounds from our
library. All the 3-dehydroxy analogues 6a-e demonstrated reduced
binding affinities for the ORs compared to compounds 1a-e containing
the 3-hydroxy group, providing further evidence that the 3-hydroxy is
critical for the affinity of epoxymorphinan derivatives binding to all
three ORs. Moreover, most compounds carrying the 3-hydroxy group
In addition, as observed in the crystal structures of the active KOR
and MOR, the 3-hydroxy group of the phenolic moiety of the original
ligands, MP1104 and BU72, may interact with residues Y3.33, K5.39, and
H6.52 through water-mediated hydrogen bonds [37,42]. Similarly, as
5

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
Fig. 6. The binding modes of NCP_KORactive
,
NCP_MORactive
,
compound 6c_KORactive
, and
compound 6c_MORinactive complexes after 100 ns
MD simulations. The receptors were shown as
cartoon models, the active KOR in light-pink, the
active MOR in light-blue, and the inactive MOR
in light-green. NCP, compound 6c, and key
amino acid residues were shown as stick models.
Carbon atoms: NCP in cyan; compound 6c in
yellow; key amino acid residues of the KOR in
magenta, and the MOR in orange. The dashed
lines in yellow represented potential water-
mediated hydrogen bonds. The key residues at
the allosteric site of the MOR were shown as stick
and ball models in magentas. The key residues at
the allosteric site of the KOR were shown as stick
and ball models in orange. The water molecules
were shown as sphere models in red. (For inter-
pretation of the references to colour in this figure
legend, the reader is referred to the web version
of this article.)
possessed similar selectivity profiles for the KOR over the MOR as those
of their corresponding 3-dehydroxy analogues, suggesting that the 3-hy-
droxy group may not be as influential to ligand selectivity profiles be-
tween the two receptors as expected. Further [35S]-GTPγS functional
assay results indicated that the 3-hydroxy group of these epox-
ymorphinan derivatives played a vital role in the potency to activate the
opioid receptors, and demonstrated different degrees of influence on
maximal activation profiles at the three ORs. Binding modes from the
MD simulations showed that the 3-dehydroxy compound 6c and NCP
exhibited similar interactions with the orthosteric sites, while the in-
teractions between compound 6c and the allosteric site in compound
6c_MORinactive complex were different from those of NCP in
NCP_KORactive and NCP_MORactive complexes and compound 6c in
compound 6c_KORactive complex. Moreover, NCP formed water-
mediated hydrogen bonds with residues Y3.33, K5.39, and H6.52 in both
complexes through its 3-hydroxy group, while the 3-dehydroxy com-
pound 6c did not. These findings furnished possible explanations for the
different in vitro binding affinity profiles and functional behaviors be-
tween compounds 1c (NCP) and 6c in the KOR and MOR.
melting point apparatus without correction. IR spectra were recorded on
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). 1H (400 MHz) and 13C (100
MHz) nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Ultrashield 400 Plus spectrometer. Chemical shifts were
expressed in δ units (ppm), using TMS as an internal standard, and J
values were reported in hertz (Hz). Mass spectra were obtained on an
Applied BioSystems 3200 Q trap with a turbo V source for Turbolon
Spray. Analytical reversed-phase high performance liquid chromatog-
raphy (HPLC) was performed on a Varian ProStar 210 system using
Agilent Microsorb-MV 100–5 C18 column (250 × 4.6 mm). All analyses
were conducted at an ambient temperature with a flow rate of 0.5 mL/
min. HPLC eluent condition: acetonitrile/water (with 0.1% trifluoro-
acetic acid), acetonitrile increased from 40% to 100% in gradient within
20 min of test. The UV detector was set up at 210 nm. The injection
volume was 5 µL. The purities of final compounds were calculated as the
percentage peak area of the analyzed compound, and retention time (Rt)
was presented in minutes. The purity of all newly synthesized com-
pounds was identified as ≥ 95%.
4. Experimental
4.1. Chemistry
4.1.1. Preparation of target compounds 6a-e
All nonaqueous reactions were carried out under a pre-dried nitrogen
gas atmosphere. All solvents and reagents were purchased from com-
mercial suppliers, and were used as received without further purifica-
tion. Melting points were measured on a MPA100 OptiMelt automated
4.1.1.1. 17-Cyclopropylmethyl-4,5α-epoxy-14β-hydroxy-3-[(1-phenyl-1H-
tetrazol-5-yl)oxy]-morphinan-6-one (2). To a mixture of naltrexone
(1.30 g, 3.81 mmol) and potassium carbonate (1.16 g, 8.38 mmol) in dry
DMF (10 mL, dried over 4 Å molecular sieves) was added 5-chloro-1-
6

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
phenyl-1H-tetrazole (756 mg, 4.19 mmol) in portions. The resulting
mixture was stirred at room temperature for 6 h. After completion of the
reaction (monitored by TLC), DMF was removed under reduced pres-
sure. The crude residue was extracted with DCM, and the combined
organic layers were washed with water and brine, dried over Na2SO4,
and filtered. The filtrate was concentrated to give a brown solid. The
crude product was washed with methanol to afford the pure interme-
diate 2 as a white solid. Yield: 97%. Mp: 151.1–151.8 ◦C. 1H NMR (400
MHz, CDCl3): δ 7.89–7.87 (m, 2H), 7.61–7.57 (m, 2H), 7.52–7.47 (m,
2H), 7.18 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 4.76 (brs, 1H),
3.14 (d, J = 19.0 Hz, 1H), 3.12–3.03 (m, 1H), 2.95–2.87 (m, 1H),
2.73–2.40 (m, 4H), 2.32 (dt, J = 14.6, 3.0 Hz, 1H), 2.22 (brs, 1H), 1.99
(brs, 1H), 1.66–1.58 (m, 3H), 0.95 (brs, 1H), 0.61 (d, J = 6.3 Hz, 2H),
0.22 (brs, 2H). 13C NMR (100 MHz, CDCl3): 207.40, 159.44, 146.61,
135.77, 133.12, 131.44, 131.22, 129.71 (Ph-C × 2), 129.40, 122.45 (Ph-
C × 2), 121.19, 119.87, 91.24, 69.96, 61.81, 59.20, 50.91, 43.40, 36.10,
31.30, 30.70, 23.00, 9.37, 4.06, 3.81. HRMS (ESI) m/z: 486.2049 [M +
H]+, 508.1864 [M + Na]+, C27H27N5O4 (485.2063).
Hz, 4H), 7.27–7.24 (m, 4H), 7.16 (t, J = 7.3 Hz, 2H), 6.92 (t, J = 7.7,
1H), 6.53 (d, J = 7.6, 1H), 6.48 (d, J = 7.8, 1H), 4.69 (d, J = 7.8 Hz, 1H),
3.89 (d, J = 14.2 Hz, 2H), 3.65 (d, J = 14.2 Hz, 2H), 3.03–2.97 (m, 2H),
2.61 (dd, J = 11.8, 5.5 Hz, 1H), 2.57–2.51 (m, 2H), 2.38–2.30 (m, 2H),
2.21 (td, J = 12.4, 5.0 Hz, 1H), 2.10–1.94 (m, 2H), 1.65–1.54 (m, 2H),
1.42 (dd, J = 12.4, 2.2 Hz, 1H), 1.20 (td, J = 13.0, 2.8 Hz, 1H),
0.90–0.78 (m, 1H), 0.55–0.47 (m, 2H), 0.14–0.07 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ 156.55, 140.53, 133.19, 130.74, 128.62, 128.43,
128.16, 126.69, 117.59,108.39, 91.00, 70.47, 62.64, 59.53, 59.37,
54.59, 47.09, 44.07, 30.96, 30.73, 23.46, 19.00, 9.61, 4.02, 3.98. IR
(Diamond, cmꢀ 1
) νmax: 3390, 3025, 2910, 2835, 1629, 1493, 1454,
1374, 1245, 1136, 1049, 925, 731, 697. HRMS (ESI) m/z: 507.3032 [M
+ H]+, C34H38 N2O2 (506.2933).
4.1.1.4. 17-Cyclopropylmethyl-4,5α-epoxy-6β-amino-14β-hydrox-
ymorphinan hydrochloride (5⋅2HCl). To a suspension of intermediate 4
(750 mg, 1.48 mmol) in 12 mL of anhydrous methanol was added
concentrated HCl (0.3 mL, pH = 2). Then, 10% Pd/C (262 mg) was
added and the resulting mixture was hydrogenated at room temperature
under 65 psi pressure for 4 days. After completion of the reaction (by
TLC monitoring), the reaction mixture was filtered through Celite and
the Celite was washed with methanol. The combined filtrate was
concentrated to afford the key intermediate 5 as an off-white solid.
Yield: 81%. Mp: > 250 ◦C. This compound could be used in the next step
without any further purification. A small quantity of 5 was recrystallized
using methanol/ether to get a pure product for characterization. 1H
NMR (400 MHz, DMSO‑d6): δ 9.01 (brs, 1H, exchangeable), 8.58 (brs,
3H, exchangeable), 7.21 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.7, 1H), 6.80
(d, J = 7.9, 1H), 6.56 (brs, 1H), 4.77 (d, J = 7.3 Hz, 1H), 4.13 (brs, 1H,
exchangeable), 3.45 (d, J = 19.8 Hz, 1H), 3.39–3.34 (m, 1H), 3.19–3.12
(m, 1H), 2.05 (d, J = 10.3 Hz, 1H), 2.92–2.88 (m, 1H), 2.73 (brs, 1H),
2.48–2.44 (m, 2H), 2.02 (q, J = 12.8 Hz, 1H), 1.88 (d, J = 13.9 Hz, 1H),
1.77 (d, J = 9.8 Hz, 1H), 1.43 (d, J = 10.8 Hz, 1H), 1.28 (t, J = 13.4 Hz,
1H), 1.10–1.07 (m, 1H), 0.69–0.65 (m, 1H), 0.62–0.58 (m, 1H),
0.56–0.50 (m, 1H), 0.43–0.39 (m, 1H). 13C NMR (100 MHz, DMSO‑d6):
155.13, 131.44, 129.71, 127.69, 119.33, 109.01, 88.22, 69.30, 61.10,
56.66, 52.34, 46.03, 44.96, 28.67, 27.07, 23.62, 21.31, 5.68, 5.09, 2.63.
4.1.1.2. 17-Cyclopropylmethyl-4,5α-epoxy-14β-hydroxymorphinan-6-one
(3). A mixture of compound 2 (1.87 g, 3.85 mmol), 10% Pd/C (675 mg),
and 7 mL of glacial acetic acid was hydrogenated (H2, 52 psi) at 40 ◦C for
10 h. The reaction mixture was filtered through Celite and the Celite was
washed with DCM (3 times). The filtrate was concentrated to give a light
brown oil. The oil was treated with 4 N NaOH (6 mL) and extracted with
DCM. The combined organic layers were washed with brine, dried over
Na2SO4, filtered and concentrated under reduced pressure. The residue
was further purified by column chromatography (MeOH/DCM = 1/100)
to provide the pure compound 3 as a white solid. Yield: 82%. Mp:
143.2–143.9 ◦C. 1H NMR (400 MHz, CDCl3): δ 7.05 (td, J = 7.8, 1.1 Hz,
1H), 6.73 (d, J = 7.9, 1H), 6.67 (d, J = 7.6, 1H), 4.63 (s,1H), 3.19 (d, J =
5.9 Hz, 1H), 3.10 (d, J = 18.8 Hz, 1H), 3.03 (td, J = 14.5, 5.0 Hz, 1H),
2.69 (dd, J = 12.0, 4.8 Hz, 1H), 2.62 (dd, J = 18.8, 6.0 Hz, 1H),
2.46–2.38 (m, 3H), 2.29 (d, J = 14.5 Hz, 1H), 2.12 (td, J = 12.0, 3.5 Hz,
1H), 1.87 (dt, J = 13.2, 4.0 Hz, 1H), 1.66–1.58 (m, 1H), 1.54–1.51 (m,
1H), 0.92–0.82 (m, 1H), 0.57–0.53 (m, 2H), 0.16–0.12 (m, 2H). 13C
NMR (100 MHz, CDCl3): δ 209.08, 157.40, 133.16, 129.18, 127.88,
118.66, 108.05, 89.85, 70.17, 62.08, 59.25, 50.11, 43.53, 36.20, 31.39,
30.82, 23.33, 9.43, 3.99, 3.84. HRMS (ESI) m/z: 326.1728 [M + H]+,
IR (Diamond, cmꢀ 1
) νmax: 3342, 3207, 2967, 2832, 1630, 1610, 1514,
1455, 1389, 1338, 1225, 1161, 1056, 909, 784. HRMS (ESI) m/z:
C
20H23NO3 (325.1678).
327.2082 [M + H]+, C20H26N2O2 (326.1994).
4.1.1.3. 17-Cyclopropylmethyl-4,5
α
-epoxy-6β-dibenzylamino-14β-hydrox-
4.1.1.5. General procedure for the preparation of target compounds 6a-e.
To a solution of substituted pyridylcarboxylic acids (2 equiv) in anhy-
drous DMF (2 mL) were added 4 Å molecular sieves, EDCI (1.5 equiv),
HOBt (1.5 equiv), and Et3N (5.0 equiv) at 0 ◦C (ice-water bath) under N2
atmosphere. After stirring for 1 h, a solution of 5 (hydrochloride salt, 1.0
equiv) in DMF (1 mL) was added dropwise. The resultant mixture was
allowed to warm to room temperature and was stirred for 4–5 days.
Upon completion of the reaction, the mixture was then filtered through
Celite. The filtrate was concentrated under reduced pressure to remove
DMF. The crude residue was purified by column chromatography
(MeOH/DCM) to provide the desired free bases of 6a-e. After confir-
mation by 1H NMR, the free bases were converted into their hydro-
chloride salts 6a-e. In this regard, the free base (1 equiv) was dissolved
in MeOH (0.5–1 mL) and cooled in an ice-water bath. Next, HCl in
methanol solution (1.25 M, 4 equiv) was added dropwise and the
resultant mixture was stirred for 1 h. Finally, ether (25 mL) was added
and the mixture was stirred at room temperature overnight. Next day,
the precipitate was collected by filtration and dried to obtain the desired
hydrochloride salt (6a-e), which was then used for further analysis and
biological assays.
ymorphinan (4). To a mixture of 3 (400 mg, 1.23 mmol), benzoic acid
(165 mg, 1.35 mmol), and a trace amount of p-toluenesulfonic acid (p-
TsOH, 20 mg) in anhydrous toluene (120 mL) and ethanol (30 mL) was
added dibenzylamine (255 mg, 1.29 mmol) under N2 atmosphere. The
reaction mixture was heated to reflux, employing a Dean-Stark trap for
removal of generated water. The solvent was removed from the trap
until the volume of the reaction mixture reduced to ca. 30 mL over 8 h.
After cooling down to room temperature, additional anhydrous toluene
(120 mL) and ethanol (30 mL) was added and the mixture was refluxed
overnight. Next day, the solvent was removed from the trap to ca. 30 mL
left again and the mixture was cooled to ambient temperature. Molec-
ular sieves (3 g, 4 Å) and anhydrous ethanol (30 mL) were added fol-
lowed by the addition of sodium cyanoborohydride (62 mg, 0.99 mmol).
The reaction mixture was stirred at room temperature for 24 h under N2
atmosphere. After completion of the reaction (monitored by TLC), the
reaction mixture was filtered through Celite and the filtrate was
concentrated under reduced pressure. The residue was treated with 3%
ammonia water and extracted with ethyl acetate (three times). The
combined organic layers were washed with brine and dried over
Na2SO4. Removal of ethyl acetate under reduced pressure gave a brown
oil which was further purified by column chromatography (16%-18%
ethyl acetate/hexane) to deliver compound 4 as a white solid. Yield:
72%. Mp: 119.8–120.4 ◦C. 1H NMR (400 MHz, CDCl3): δ 7.45 (d, J = 7.4
4.1.1.6. 17-Cyclopropylmethyl-4,5α
-epoxy-14β-hydroxy-6β-{[4′-(2′-
chloropyridyl)]carboxamido}morphinan hydrochloride (6a). Free base of
6a was synthesized following the general procedure by reacting 2-
7

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
chloroisonicotinic acid (79 mg, 0.50 mmol) with 5 (100 mg, 0.25 mmol).
4.1.1.9. 17-Cyclopropylmethyl-4,5
α
-epoxy-14β-hydroxy-6β-{[4′-(2′-
Yellow sticky solid. Yield: 56%. The free base was then converted into its
methylpyridyl)]carboxamido}morphinan (6d). Free base of 6d was syn-
thesized following the general procedure by reacting 2-methylisonico-
tinic acid (55 mg, 0.40 mmol) with 5 (80 mg, 0.20 mmol). Yellow
sticky solid. Yield: 79%. The free base was then converted into its hy-
drochloride salt 6d. Off-white solid. Yield: 71%. Mp: decomposed at
234.4 ◦C. 1H NMR (400 MHz, DMSO‑d6): δ 9.42 (d, J = 7.6 Hz, 1H), 8.97
(brs, 1H, exchangeable), 8.83 (d, J = 5.7 Hz, 1H), 8.15 (s, 1H), 8.04 (d, J
= 5.2 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.7, 1H), 6.76 (d, J =
7.9, 1H), 6.37 (brs, 1H, exchangeable), 4.85 (d, J = 7.8 Hz, 1H), 3.97 (d,
J = 4.8 Hz, 1H), 3.74–3.66 (m, 1H), 3.46 (d, J = 19.9 Hz, 1H), 3.41–3.31
(m, 1H), 3.20 (dd, J = 19.7, 5.6 Hz, 1H), 3.06 (d, J = 8.3 Hz, 1H),
2.92–2.88 (m, 1H), 2.74 (s, 3H), 2.45–2.42 (m, 2H), 2.02–1.92 (m, 1H),
1.85 (d, J = 13.5 Hz, 1H), 1.58–1.55 (m, 1H), 1.45 (d, J = 9.8 Hz, 1H),
1.43–1.37 (m, 1H), 1.11–1.07 (m, 1H), 0.70–0.66 (m, 1H), 0.64–0.59
(m, 1H), 0.56–0.51 (m, 1H), 0.44–0.42 (m, 1H). 13C NMR (100 MHz,
DMSO‑d6): δ 162.63, 156.05, 155.81, 131.38, 131.25, 129.59, 128.32,
124.24, 120.90, 118.96 (2 × C), 109.05, 90.00, 69.64, 61.49, 56.74,
51.57, 46.28, 45.00, 29.38, 27.24, 23.78, 23.44, 20.88, 5.79, 5.19, 2.71.
1
◦
hydrochloride salt 6a. Off-white solid. Yield: 79%. Mp: > 250 C. H
NMR (400 MHz, DMSO‑d6): δ 9.13 (d, J = 8.2 Hz, 1H), 8.94 (brs, 1H,
exchangeable), 8.58 (d, J = 5.1 Hz, 1H), 7.93 (s, 1H), 7.82 (dd, J = 5.1,
1.3 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.7, 1H), 6.76 (d, J =
7.8, 1H), 6.29 (s, 1H, exchangeable), 4.80 (d, J = 7.7 Hz, 1H), 3.94 (d, J
= 5.1 Hz, 1H), 3.72–3.64 (m, 1H), 3.46 (d, J = 19.9 Hz, 1H), 3.39–3.32
(m, 1H), 3.20 (dd, J = 19.7, 5.6 Hz, 1H), 3.05 (d, J = 7.4 Hz, 1H),
2.90–2.86 (m, 1H), 2.46 (d, J = 8.6 Hz, 2H), 1.99–1.89 (m, 1H), 1.82 (d,
J = 13.8 Hz, 1H), 1.60–1.54 (m, 1H), 1.46 (d, J = 8.8 Hz, 1H), 1.43–1.36
(m, 1H), 1.10–1.07 (m, 1H), 0.70–0.66 (m, 1H), 0.63–0.57 (m, 1H),
0.55–0.51 (m, 1H), 0.45–0.41 (m, 1H). 13C NMR (100 MHz, DMSO‑d6): δ
162.63, 155.82, 151.00, 150.83, 144.56, 131.33, 129.59, 128.38,
121.94, 120.93, 118.93, 109.07, 90.06, 69.64, 61.55, 56.74, 51.40,
46.27, 44.98, 29.37, 27.25, 23.77, 23.44, 5.76, 5.18, 2.70. IR (Diamond,
cmꢀ 1
) νmax: 3083, 1652, 1540, 1459, 1363, 1333, 1117, 1031, 909, 751.
HRMS (ESI) m/z: 466.1901 [M + H]+, C26H28ClN3O3 (465.1819). HPLC
purity: 99.80%. Rt: 7.888 min.
IR (Diamond, cmꢀ 1
) νmax: 3363, 3118, 1660, 1624, 1552, 1454, 1425,
4.1.1.7. 17-Cyclopropylmethyl-4,5α
-epoxy-14β-hydroxy-6β-{[4′-(2′-bro-
1333, 1278, 1128, 1029, 920, 749. HRMS (ESI) m/z: 446.2452 [M +
H]+, C27H31N3O3 (445.2365). HPLC purity: 96.99%. Rt: 7.058 min.
mopyridyl)]carboxamido}morphinan (6b). Free base of 6b was synthe-
sized following the general procedure by reacting 2-bromoisonicotinic
acid (81 mg, 0.40 mmol) with 5 (80 mg, 0.20 mmol). Light brown solid.
Yield: 62%. The free base was then converted into its hydrochloride salt
6b. Off-white solid. Yield: 81%. Mp: > 250 ◦C. 1H NMR (400 MHz,
DMSO‑d6): δ 9.13 (d, J = 8.2 Hz, 1H), 8.94 (brs, 1H, exchangeable), 8.56
(d, J = 5.0 Hz, 1H), 8.07 (d, J = 0.6 Hz, 1H), 7.84 (dd, J = 5.1, 1.4 Hz,
1H), 7.19 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.8
Hz, 1H), 6.30 (s, 1H, exchangeable), 4.80 (d, J = 7.8 Hz, 1H), 3.94 (d, J
= 5.2 Hz, 1H), 3.71–3.63 (m, 1H), 3.46 (d, J = 19.8 Hz, 1H), 3.41–3.32
(m, 1H), 3.20 (dd, J = 19.8, 5.8 Hz, 1H), 3.09–3.02 (m, 1H), 2.90–2.86
(m, 1H), 2.46 (d, J = 8.8 Hz, 2H), 1.98–1.89 (m, 1H), 1.82 (d, J = 13.9
Hz, 1H), 1.58–1.54 (m, 1H), 1.46 (d, J = 9.0 Hz, 1H), 1.42–1.36 (m, 1H),
1.11–1.07 (m, 1H), 0.72–0.66 (m, 1H), 0.63–0.57 (m, 1H), 0.55–0.49
(m, 1H), 0.45–0.41 (m, 1H). 13C NMR (100 MHz, DMSO‑d6): δ 162.50,
155.82, 151.34, 144.15, 141.95, 131.33, 129.60, 128.30, 125.55,
121.24, 118.94, 109.08, 90.06, 69.64, 61.55, 56.74, 51.40, 46.27,
4.1.1.10. 17-Cyclopropylmethyl-4,5α
-epoxy-14β-hydroxy-6β-{[4′-(2′-
methoxypyridyl)]carboxamido}morphinan (6e). Free base of 6e was
synthesized following the general procedure by reacting 2-methoxyiso-
nicotinic acid (77 mg, 0.50 mmol) with 5 (100 mg, 0.25 mmol). Yellow-
orange solid. Yield: 67%. The free base was then converted into its hy-
drochloride salt 6e. White solid. Yield: 91%. Mp: decomposed at
235.0 ◦C. 1H NMR (400 MHz, DMSO‑d6): δ 8.95 (d, J = 7.9 Hz, 2H,
exchangeable), 8.30 (dd, J = 5.3, 0.4 Hz, 1H), 7.39 (dd, J = 5.3, 1.1 Hz,
1H), 7.23 (s, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 7.7, 1H), 6.76 (d,
J = 7.9, 1H), 4.81 (d, J = 7.8 Hz, 1H), 3.94 (d, J = 4.5 Hz, 1H), 3.90 (s,
3H), 3.71–3.63 (m, 1H), 3.46 (d, J = 19.9 Hz, 1H), 3.39–3.32 (m, 1H),
3.20 (dd, J = 19.7, 5.9 Hz, 1H), 3.05 (d, J = 6.6 Hz, 1H), 2.90–2.85 (m,
1H), 2.45 (d, J = 8.7 Hz, 2H), 1.98–1.88 (m, 1H), 1.81 (d, J = 13.5 Hz,
1H), 1.57–1.52 (m, 1H), 1.46 (d, J = 9.0 Hz, 1H), 1.42–1.36 (m, 1H),
1.11–1.07 (m, 1H), 0.72–0.66 (m, 1H), 0.63–0.56 (m, 1H), 0.55–0.49
(m, 1H), 0.45–0.39 (m, 1H). 13C NMR (100 MHz, DMSO‑d6): δ 164.23,
163.74, 155.88, 147.65, 144.49, 131.37, 129.57, 128.41, 118.90,
114.75, 109.07, 108.44, 90.14, 69.69, 61.49, 56.74, 53.71, 51.23,
46.30, 45.00, 29.41, 27.27, 23.80, 23.58, 5.82, 5.22, 2.73. IR (Diamond,
44.98, 29.37, 27.25, 23.76, 23.43, 5.76, 5.18, 2.70. IR (Diamond, cmꢀ 1
)
νmax: 3082, 2937, 1650, 1533, 1493, 1456, 1332, 1252, 1133, 1029,
907, 733. HRMS (ESI) m/z: 510.1388 [M + H]+, C26H28BrN3O3
(509.1314). HPLC purity: 99.21%. Rt: 8.038 min.
cmꢀ 1
) νmax: 3407, 3114, 1640, 1504, 1457, 1425, 1386, 1299, 1126,
1030, 916, 751. HRMS (ESI) m/z: 462.2382 [M + H]+, C27H31N3O4
4.1.1.8. 17-Cyclopropylmethyl-4,5α
-epoxy-14β-hydroxy-6β-{[4′-(2′-cya-
(461.2315). HPLC purity: 97.10%. Rt: 7.694 min.
nopyridyl)]carboxamido}morphinan (6c). Free base of 6c was synthe-
sized following the general procedure by reacting 2-cyanoisonicotinic
acid (59 mg, 0.40 mmol) with 5 (80 mg, 0.20 mmol). Off-white solid.
Yield: 41%. The free base was then converted into its hydrochloride salt
6c. Off-white solid. Yield: 86%. Mp: > 250 ◦C. 1H NMR (400 MHz,
DMSO‑d6): δ 9.24 (d, J = 8.2 Hz, 1H), 8.93 (dd, J = 5.0, 0.4 Hz, 2H,
exchangeable for 1H), 8.43 (d, J = 0.7 Hz, 1H), 8.13 (dd, J = 5.1, 1.6 Hz,
1H), 7.20 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.7, 1H), 6.77 (d, J = 7.8, 1H),
6.29 (s, 1H, exchangeable), 4.80 (d, J = 7.8 Hz, 1H), 3.93 (d, J = 5.2 Hz,
1H), 3.74–3.65 (m, 1H), 3.46 (d, J = 19.9 Hz, 1H), 3.40–3.33 (m, 1H),
3.21 (dd, J = 19.9, 5.7 Hz, 1H), 3.06 (d, J = 7.3 Hz, 1H), 2.90–2.85 (m,
1H), 2.46 (d, J = 8.8 Hz, 2H), 1.99–1.90 (m, 1H), 1.82 (d, J = 13.9 Hz,
1H), 1.60–1.55 (m, 1H), 1.47 (d, J = 9.0 Hz, 1H), 1.44–1.38 (m, 1H),
1.11–1.07 (m, 1H), 0.70–0.66 (m, 1H), 0.64–0.57 (m, 1H), 0.55–0.49
(m, 1H), 0.45–0.40 (m, 1H). 13C NMR (100 MHz, DMSO‑d6): δ 162.30,
155.80, 152.21, 142.45, 133.30, 131.33, 129.63, 128.26, 126.53,
125.35, 118.97, 117.26, 109.09, 90.05, 69.63, 61.58, 56.75, 51.49,
46.27, 44.98, 29.35, 27.25, 23.76, 23.41, 5.75, 5.17, 2.69. IR (Diamond,
4.2. Biological evaluation
The free base of naltrexone was provided through NIDA Drug Supply
Program. All drugs and test compounds were dissolved in sterile-filtered
distilled/deionized water. All other reagents and radioligands were
purchased from either Sigma-Aldrich or Perkin-Elmer.
4.2.1. In vitro competitive radioligand binding assay
The competition binding assay was conducted using monoclonal
mice opioid receptor expressed in CHO cell lines (monoclonal human δ
opioid receptor was used in the DOR assay). In this assay, 30 µg of
membrane protein was incubated with the corresponding radioligand in
the presence of different concentrations of test compounds in TME buffer
(50 mM Tris, 3 mM MgCl2, and 0.2 mM EGTA, pH 7.4) for 1.5 h at 30 ◦C.
The bound radioligand was separated by filtration using the Brandel
harvester. Specific (i.e., opioid receptor-related) binding to the KOR,
MOR, and DOR was determined as the difference in binding obtained in
the absence and presence of 5 µM of U50,488, DAMGO, and SNC80,
respectively. Relative affinity values (IC50) were determined by fitting
cmꢀ 1
) νmax: 3185, 3082, 1660, 1630, 1552, 1458, 1384, 1254, 1169,
1132, 1050, 917, 782. HRMS (ESI) m/z: 457.2245 [M + H]+,
C
27H28N4O3 (456.2161). HPLC purity: 100%. Rt: 7.404 min.
8

B. Huang et al.
Bioorganic Chemistry 109 (2021) 104702
displacement binding inhibition values by non-linear regression using
GraphPad Prism 8.0 (GraphPad Software, San Diego, CA), where %in-
hibition value was calculated as follows: %inhibition = 100% - (binding
in the presence of tested compound - nonspecific binding)/specific
binding × 100%. The IC50 values were converted to Ki values using the
Cheng-Prusoff equation: Ki = IC50/ [1 + ([L*]/KD)], where [L*] is the
concentration of the radioligand and KD is the KD of the radioligand
[50].
GUI website service [54] and then further conducted 100 ns molecular
dynamics (MD) simulations on each system by Amber14.0 [55] to obtain
reliable and stable ligand-receptor complexes. In the process of the MD
simulations, periodic boundary conditions were applied. The long-range
electrostatic interactions were calculated by Particle Mesh Ewald (PME)
method [56]. The cut-off of the non-bonded van der Waals interactions
was set at 10 Å. The constant temperatures at 310 K was controlled by
the Langevin thermostat. After 100 ns MD simulation, the root-mean-
square deviations (RMSD) of the backbone atoms of the amino acid
residues and ligand in each complex during the 100 ns MD simulations
were computed to evaluate the equilibrium of the system (Fig. S2).
4.2.2. In vitro [35S]-GTPγS functional assay
[
35S]-GTPγS functional assays were conducted in the rat MOR,
human KOR, mouse DOR cell membranes. Membrane proteins (10
μg)
were incubated with varying concentrations of drugs, GDP (15
μM), and
Declaration of Competing Interest
80 pM [35S]-GTPγS in assay buffer for 1 h at 30 ◦C. Nonspecific binding
was determined with 10
μM unlabeled GTPγS. U50,488H (5 μM),
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
DAMGO (5 μM), and DPDPE (5 μM) were included in the assay for a
maximally effective concentration of a full agonist for the KOR, MOR,
and DOR, respectively. The Emax values for receptors were calculated as
relative to net full agonist-stimulated [35S]-GTPγS binding, which is
defined as (net-stimulated binding by ligand/net-stimulated binding by
maximally effective concentration of a full agonist) × 100%. By using
the equation Ke = [Ant]/DR-1, where [Ant] is the concentration of
antagonist and DR is the ratio of the EC50 values of the full agonist in the
presence and absence of antagonist, Ke values in the competitive
antagonism studies were determined.
Acknowledgements
The authors are grateful to NIDA Drug Supply Program for providing
the free base of naltrexone. This work was partially supported by NIH/
NIDA Grants R01DA024022, R01DA044855 and UG3DA050311 (Y.Z.);
and R01DA041359, P30DA013429 and R21DA045274 (L.-Y.L.-C.) The
content is solely the responsibility of the authors and does not neces-
sarily represent the official views of the National Institute on Drug Abuse
or the National Institutes of Health.
4.3. Molecular modeling study
The PDB files of crystal complexes of the agonist MP1104 with KOR
(PDB ID: 6B73) [37], the agonist BU72 with MOR (PDB ID: 5C1M) [42],
and the antagonist β-FNA with MOR (PDB ID: 4DKL) [41] were down-
loaded from Protein Data Bank (http://www.rcsb.org). Prior to con-
ducting the molecular docking, the three receptors were firstly prepared.
The monomers of the KOR and MOR, and the three crystallographic
water molecules involved in the water-mediated hydrogen bonding in-
teractions between residues Y3.33, K5.39, and H6.52 and the original li-
gands (MP1104, BU72, and β-FNA) in the three crystal structures were
remained. While the three original ligands, the active-state-stabilizing
nanobody in 6B73, the G protein-mimetic camelid-antibody fragment
in 5C1M, the T4 lysozyme fragment in 4DKL, solvent molecules, and
other water molecules in the three crystal structures were removed.
Subsequently, hydrogen atoms were added to each receptor and the two
ligands, NCP and compound 6c, were sketched by applying SYBYL-X 2.1
(Tripos Inc., St Louis). Moreover, the two compounds were assigned
with Gasteiger-Hückel charges and energy minimized under the Tripos
force field (TFF) [51]. After preparing the structures of the ligands and
the receptors, GOLD 5.6 [52] was applied to dock the two ligands, NCP
and compound 6c, into the above three receptors. In the process of
docking study, the binding sites of the KOR and MOR were defined by
the atoms within 10 Å of the γ-carbon atom of D3.32 in the three re-
ceptors [53] and two distance constraints were applied. One is the dis-
tance between the piperidine quaternary ammonium nitrogen atom of
the ligands’ 4, 5-epoxymorphinan nucleus and D3.32. The other one is the
distance between the ligands’ dihydrofuran oxygen atom and the
phenolic oxygen atom of Y3.33. In addition, 50 docking solutions were
obtained after molecular docking study. Except for above setting pa-
rameters, default parameters were applied for the other docking pro-
cedure. The docking solution with the highest CHEM-PLP score was
selected as the optimal docking pose of each compound. Subsequently,
the optimal docking poses were merged into their respective receptors to
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104702.
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