Design, syntheses, and pharmacological characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan analogues as opioid receptor ligands

Article abstract of DOI:10.1016/j.bmc.2015.02.055

A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1′- and/or 4′-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6′- and/or 7′-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca²⁺ increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6′-nitro group varied significantly in the different 'address' domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands.

Full text of DOI:10.1016/j.bmc.2015.02.055

Bioorganic & Medicinal Chemistry 23 (2015) 1701–1715
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, syntheses, and pharmacological characterization of 17-
cyclopropylmethyl-3,14b-dihydroxy-4,5
carboxamido)morphinan analogues as opioid receptor ligands
a-epoxy-6a
-(isoquinoline-30-
a
b
b
a
Yunyun Yuan a, , Saheem A. Zaidi , David L. Stevens , Krista L. Scoggins , Philip D. Mosier ,
⇑
Glen E. Kellogg a, William L. Dewey b, Dana E. Selley b, Yan Zhang a,
⇑
a Department of Medicinal Chemistry, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23298, USA
b Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North 12th Street, Richmond, VA 23298, USA
a r t i c l e i n f o
a b s t r a c t
A series of 17-cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a
-(isoquinoline-30-carboxamido)mor-
Article history:
Received 12 January 2015
Revised 11 February 2015
Accepted 26 February 2015
Available online 6 March 2015
phinan (NAQ) analogues were synthesized and pharmacologically characterized to study their struc-
ture–activity relationship at the mu opioid receptor (MOR). The competition binding assay showed
two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions
at the 10- and/or 40-position of the isoquinoline ring retained or improved MOR selectivity over the kappa
opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In
contrast, substitutions at the 60- and/or 70-position of the isoquinoline ring reduced MOR selectivity as
well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged
with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms
compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca2+
increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indi-
cated orientation of the 60-nitro group varied significantly in the different ‘address’ domains of the recep-
tors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current
findings provide valuable insights for future development of NAQ-based MOR selective ligands.
Published by Elsevier Ltd.
Keywords:
MOR
Antagonist
SAR
Selectivity
2
1. Introduction
The MOR interacts mostly with Gai/o
.
Following MOR activation,
G protein-gated inwardly rectifying K+ (GIRK) channels3 open up
while voltage-gated Ca2+ channels (VGCC)4 and intracellular
adenylate cyclase-mediated cyclic adenosine monophosphate
(cAMP)5 production are inhibited. All of these cascades lead to
membrane potential decrease, neuronal excitability, neurotrans-
mitter release, and downstream signaling through their second
messenger systems which ultimately affect gene expression.6
Behavioral effects manifested through MOR activation include
antinociception as well as reward-related behaviors such as sub-
stance (e.g., opioid and alcohol) abuse and addiction.6–8
There are three main types of opioid receptors: mu (MOR),
kappa (KOR) and delta (DOR), all of which belong to the Class A
rhodopsin-like G protein-coupled receptor (GPCR) superfamily.1
Abbreviations: cAMP, cyclic adenosine monophosphate; CHO, Chinese hamster
ovary; DAMGO, [D
-Ala2-MePhe4-Gly(ol)5]enkephalin; DFT, density functional the-
ory; DOR, delta opioid receptor; DPN, diprenorphine; EDCI, 1-ethyl-3-(3-dimethy-
laminopropyl)carbodiimide; GIRK, G protein-gated inwardly rectifying K+; GPCR, G
protein-coupled receptor; HOBt, hydrobenzotriazole; KOR, kappa opioid receptor;
MD, molecular dynamics; MOR, mu opioid receptor; NTX, naltrexone; NTI,
As highlighted in the 2013 World Drug Report, ‘Opioids
remained the most commonly reported group of substances
involved in drug-related deaths’.9 Opioid overdose was a major
cause of mortality. Currently, the available treatment for opioid
overdose is administration of opioid antagonists, such as naloxone
(short action) and naltrexone (long action).10 Opioid abuse and
addiction is a risk factor for opioid overdose. The MOR full agonist
methadone, partial agonist buprenorphine, and antagonist
naltrindole; NAQ, 17-cyclopropylmethyl-3,14b-dihydroxy-4,5
quinoline-30-carboxamido)morphinan; NAMD, NAnoscale Molecular Dynamics
program; NCQ, 17-cyclopropylmethyl-3,14b-dihydroxy-4,5 -epoxy-6 -(1-chloro-
4-methoxyisoquinoline-3-carboxamido)morphinan; NNQ, 17-cyclopropylmethyl-
3,14b-dihydroxy-4,5 -epoxy-6 -(6-nitroisoquinoline-3-carboxamido)morphinan;
PME, Particle Mesh Ewald; OPM, Orientations of Proteins in Membranes; SAR,
structure–activity relationship; TFF, tripos force field; VGCC, voltage-gated Ca2+
channels.
a-epoxy-6a-(iso-
a
a
a
a
⇑
Corresponding authors. Tel.: +1 804 828 0021; fax: +1 804 828 7625.
E-mail addresses: yyuan@vcu.edu (Y. Yuan), yzhang2@vcu.edu (Y. Zhang).
http://dx.doi.org/10.1016/j.bmc.2015.02.055
0968-0896/Published by Elsevier Ltd.

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Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
naltrexone are used to treat opioid addiction presently.11–13 These
three medications serve as ‘proof-of-concept’ that targeting MOR
would deliver therapeutic effect for opioid addiction while
their associated shortcomings, such as overdose risk or
hepatotoxicity,14–16 call for new molecules with improved
pharmacological profiles. Under such a context, scientists and
researchers have identified a number of MOR selective ligands.
Some of the representative antagonists are illustrated in
Figure 1.17–22 Among them, several newly developed ligands
in chronic morphine-dependent mice. This new ligand was also
docked into the recently determined crystal structures of three
opioid receptors followed by molecular dynamics simulations to
gain insights into its selectivity profile to guide future molecular
design.
2. Results and discussion
2.1. Molecular design
showed promising therapeutic benefit for alcohol (CP-86608720
)
and food (GSK152149821) addiction, while limited success has
been achieved for opioid addiction.
Previous modeling studies in which NAQ was docked into the
recently determined crystal structures of the MOR,26 KOR27 and
DOR28 yielded two different binding sites as ‘address’ domains that
interacted with the side chain of NAQ (i.e., the spacer plus the iso-
quinoline ring): one located at the top of transmembrane helices 6
and 7, and the other at the interface of helix 5 and extracellular
loop 2 (ECL2).29 While several residues (such as Trp318 and
Lys303) within these MOR ‘address’ domains were proposed to
form favorable hydrogen bonding or aromatic stacking interactions
with the side chain of NAQ, others (such as Glu229) may also pro-
vide favorable interactions upon modification of the NAQ side
chain. To further enrich our understanding of the postulated
MOR ‘address’ domains and more importantly, to study the SAR
of NAQ, a series of NAQ analogues were designed based on our
modeling studies and the Craig plots.30 The following features were
varied: the distance between the isoquinoline ring and the epoxy-
morphinan skeleton, the aromatic character of the C(6) side chain,
and the substitutions on the isoquinoline ring. To have a wide
Our research interest in developing MOR selective antagonists
for treatment of neurological disorders led to the identification of
a potent and highly selective MOR ligand NAQ, a C(6)-isoquinoline
substituted naltrexone derivative based on a MOR homology
model and the ‘message–address’ concept (Fig. 1).22 NAQ acted
as a low-efficacy MOR partial agonist in the [35S]GTP
assay, but antagonized the effects of MOR full agonist [
MePhe4-Gly(ol)5]enkephalin (DAMGO) in the [35S]GTP
S binding
c
S binding
D
-Ala2-
c
assay and MOR agonist morphine in the warm-water tail immer-
sion assay.22,23 NAQ produced weak intracranial self-stimulation
(ICSS) facilitation in opioid-naïve rats but more robust ICSS facil-
itation in opioid-experienced rats. It also significantly reversed
morphine withdrawal-associated depression of ICSS. These effects
of NAQ were similar to that of nalbuphine.24 The ICSS results thus
agree with the in vitro characterization of NAQ as a low-efficacy
MOR partial agonist and indicate that NAQ may serve as a
relatively safe option for treatment of opioid withdrawal or
dependence.
range of the partition constant
p, the Hammett substituent con-
stant , and the resonance constant ER values for appropriate SAR
r
NAQ, however, also showed relatively high efficacy and moder-
studies, different functional groups, such as –Cl and –NO2, –CN,
–OMe and –OH, –NMe2 and –Me were chosen as the substituents
on the isoquinoline ring to cover all the quadrants of the Craig
plots.
ate potency in the DOR [35S]GTP S binding assay.23 It has been pro-
c
posed that DOR activation is also involved in the development of
morphine dependence.25 Thus a NAQ analogue showing less DOR
activation while retaining the selective MOR low-efficacy partial
agonistic and antagonistic properties would possess more promis-
ing therapeutic potentials. We herein report the structure–activity
relationship (SAR) studies of the first generation of NAQ analogues
during such a pursuit. All the synthesized compounds were first
evaluated for binding affinities in opioid receptor radioligand com-
2.2. Chemistry
As outlined in the previous section, the proposed SAR study of
NAQ would mainly focus on its side chain while keeping the
epoxymorphinan core intact. Thus, the syntheses of substituted
isoquinoline-3-carboxylic acids and their saturated analogues
became the first major task. Interestingly, there are very limited
reports on the synthesis of isoquinoline-3-carboxylic acids or their
esters. Based on these literature reports and others describing
similar functional group transformation as well as commercial
petition binding assays, MOR functions in the MOR [35S]GTP
cS
binding assay, and acute antinociceptive agonistic and antagonistic
effects in the warm-water tail immersion assay. Compound 11
(NNQ), which demonstrated in vivo opioid antagonism activity,
was further tested for its potential opioid-withdrawal symptoms
N
N
N
N
OH
OH
HO
O
HN
CO2Me
O
O
O
O
HO
HO
N
N
OH HO
OH
7-aminomethyl oripavine
nalmefene
NHSO2Me
naloxonazine
N
N
NH
17
N
OH
H
H
H
N
N
O
F
H
6
N
OH
O
F
HN
OH
NAQ
GSK1521498
CP-866,087
Figure 1. Representative MOR antagonists used as therapeutic agents or under clinical/preclinical investigation.

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1703
availability of starting materials and synthetic challenges, five
groups of desirable substituted isoquinoline-3-carboxylic acids or
saturated analogues, including several new compounds, were syn-
thesized via multistep synthesis (Schemes 1–5, see details in
Supplementary data). The majority of the reactions went smoothly
with good to excellent yields. To be noted, the reductive amination
of intermediate 56 with NaBH3CN also reduced the aromatic ring
containing the nitrogen atom, which further yielded two products,
57 and 63, after being treated with 10% Pd/C in boiling xylene
(Scheme 4). (S)-2-Methyl-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid hydrochloride (61) was prepared by Eschweiler–
Clarke reaction adopting the reported procedure31 (Scheme 5).
To prepare the epoxymorphinan core part in the designed NAQ
analogues, naltrexone was reacted with benzylamine and sodium
borohydride, followed by catalytic hydrogenation in the presence
compare with previous results as well as for consistency purpose,
[3H]naloxone (NLX), [3H]naltrindole (NTI), and [3H]diprenorphine
(DPN) were used to label the MOR, DOR and KOR, respectively.
The MOR [35S]GTP
cS binding assay was conducted to determine
the efficacy of each ligand at the MOR as reported earlier.23 The
results were interpreted as the potency (EC50) and the relative effi-
cacy (% Emax, the maximum response of each compound to the
response of the MOR full agonist DAMGO at 3 lM) for MOR activa-
tion. A portion of the results have been briefly reported earlier.35
As seen in Table 1, all sixteen NAQ analogues retained sub-
nanomolar to single digit nanomolar binding affinities at the
MOR except the one carrying methoxy groups at the 60- and
70-positions of the isoquinoline ring (14). Therefore these two posi-
tions may not be suitable to introduce electron-donating groups
simultaneously when a potent MOR ligand is desirable.
of concentrated hydrochloric acid to yield
6
a
-naltrexamine
Comparing the binding data at both MOR and KOR, 10,40-disub-
stitution (4 and 5), 10-substitution (8–10), or an electron-donating
group at the 40- or 70-position (6 and 13) yielded comparable or
improved MOR/KOR selectivity relative to NAQ, while the other
structural modifications moderately decreased MOR/KOR selectiv-
ity. Among this series of NAQ analogues, 4 showed the highest
MOR/KOR selectivity, nearly 10-fold higher than that of naltrexone.
With respect to the MOR and the DOR binding affinities, ligands
containing different electronic characteristics substituents at the
10- and/or 40-position of the isoquinoline ring (4–10) showed
>50-fold MOR/DOR selectivity, except for 5. The remaining ana-
logues showed little to medium MOR/DOR selectivity. Compound
9 with a 10-cyano group was the most MOR/DOR selective ligand
in this series of NAQ analogues and displayed similar MOR/DOR
selectivity as NAQ.
dihydrocholoride (6
a
-NTAꢀ2HCl)32 in a total yield of 79%. Then
the prepared substituted isoquinoline-3-carboxylic acids or their
saturated counterparts were coupled to 6
a-NTAꢀ2HCl using EDCI/
HOBT method.33–35 After treating the coupling mixture with
K2CO3 in methanol, the products were then purified by silica gel
column chromatography with reasonable yields (Scheme 6). A total
of sixteen compounds were successfully synthesized as the first
generation of NAQ analogues.
2.3. In vitro and in vivo pharmacological studies
Both in vitro and in vivo assays were carried out to characterize
the first generation of NAQ analogues. The known MOR antagonist
naltrexone (NTX) was tested along as a control in all the studies.
A majority of the first generation of NAQ analogues showed less
2.3.1. In vitro radioligand binding assay and MOR [35S]GTP
functional assay
cS
than 30% of the maximal stimulation observed with 3
lM DAMGO
in the MOR [35S]GTP
cS binding assay, except for 4 and 9 (Table 1).
The synthesized NAQ analogues were first evaluated in radioli-
Among them, 3 with a glycine-unit spacer and 11–14 carrying sub-
stitutions with different electronic characteristics at the 60- and/or
70-position of the isoquinoline ring had less than 15% MOR stim-
ulation relative to DAMGO. Interestingly, NAQ and each of its ana-
logues examined here were 1–2 orders of magnitude less potent
gand competition binding assays and MOR [35S]GTP
cS functional
assay on mouse opioid receptor transfected Chinese hamster ovar-
ian (CHO) cell membranes for their binding affinity, selectivity and
MOR agonism/antagonism in vitro. The competitive radioligand
binding assays were performed as described previously.36,37 To
than naltrexone in the MOR [35S]GTP
c
S binding assay, especially
O
(A)
NBS, AIBN, CCl4
reflux, 1 h, 32%
Na CN, Na I, E tOH
EtOH, 2 N NaOH
CN
Br
re flux, 1 h
quantitative
100 °C, 1 h, 80%
N
N
N
N
OH
21
22
20
Ph
Ph
H
N
H
N
N
1 N NaOH
N
MeOH, H2SO4
NaH, benzene
(B)
(C)
O
CO2H
O
CO2Me
O
CO2Me
O
COOH
BnBr, reflux 24 h, 36%
100 °C, 1.5 h, 90%
reflux, 20 h, 59%
24
25
26
O
O
O
N
N
i) SOCl2, reflux, 4 h
Na BH4, MeOH
0 °C, 1 h, 70%
HCl (conc.)
OH
OH
ii) -10 °C, AlCl3, 2.5 h
80% two steps
reflux, 115 h, quantitative
N .HCl
OH
28
O
27
23
O
O
O
H2N
COOH
O
1 N NaOH, MeOH
3 h, quantitative
O
N
H
N
H
N
N
O
N
O
EDCI, HOBt, TEA
DMF, 12 h, 96%
30
29
Scheme 1. Synthetic routes of isoquinoline-3-carboxylic acids 20, 23, and 29 with different spacer lengths.

1704
Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
OH
OH
CO2Me
COOH
1 N NaOH, EtOH
reflux, 3 h, 98%
(A)
N
N
Cl
Cl
N
F
5
a
33
M
O
D
H
A
,
c
2
,
1
3
,
O
1
%
5
C
0
s
p
%
9
s
C
,
i
,
,
P
d
I
h
4
e
2
7
/
C
M
1
h
,
,
t
6
r
6
%
(B)
OMe
OMe
(C)
OH
OH
COOH
CO2Me
CO2Me
1 N NaOH, EtOH
reflux, 1.5 h, 91%
COOH
CO OH
1 N NaOH, EtOH
N
N
reflux, 1 h
N
N
N
quantitative
Cl
Cl
35
34
N
31
32
(D)
N
O
CO
2
Et
N
CO2Et
N
CO2Et
SOCl2, CHCl3, DMF
reflux, 4.5 h, 97%
TEA, 10% Pd/C
1 N NaOH, MeOH
rt , 1 h, 93%
NH
N
EtOH, H2, 15 psi
4h, 14%
N
Cl
37
38
36
Scheme 2. Synthetic routes of isoquinoline-3-carboxylic acids 31, 33, 34, and 36 with 40 or 10,40-substitutions.
CO 2Me
O
COOH
CHO
CO2Me
CHO
Ph
N
H
COOH
i) KOH, MeOH
reflux, 2 h
MeI, K2CO3, DMF
acetone, 12 h, 95%
(A)
Ac2O, NaOAc
N
ii) 1 N HCl, 100 °C
87%, two steps
100 °C, 3 h, 50%
O
40
Ph
O
41
CO2Me
CO2Me
N
CO2H
POCl3, DIPEA
toluene, reflux, 5 h, 87%
1 N NaOH, EtOH
reflux, 0.5 h, 92%
NH
N
O
Cl
Cl
42
43
39
CO2Me
N
CO 2H
Pd2(d ba)3, dppf, ZnCN2, Zn
CH3CO N(CH3)2, 120 °C, 2 h, 59%
1 N NaOH, EtOH
rt, overnight, 53%
(B)
(C)
43
N
CN
CN
44
45
CO2Me
CO 2H
SnMe4, Pd(PPh3)2Cl2
1 N NaOH, EtOH
reflux, 0.5 h, 97%
43
N
N
DMF, 130 ° C, 30 min, 87%
CH3
CH3
46
47
Scheme 3. Synthetic routes of isoquinoline-3-carboxylic acids 39, 44, and 46 with 10-substitution.
for 11, 12 and 14 (P200-fold). The EC50 and Emax for morphine (M)
and buprenorphine (B) in the mMOR [35S]GTP
S binding assay
Collectively, the in vitro competition binding studies of NAQ
revealed that a two-atom spacer and an aromatic ring system is
favorable for MOR selectivity whereas different positions and elec-
tronic characteristics of substituents on the isoquinoline ring affect
MOR selectivity in different patterns. Nonetheless, the in vitro
radioligand binding studies of the first generation of NAQ ana-
logues identified five ligands (4, 5, 9, 10, and 13) with over 10-fold
c
were previously tested in our labs: M, 120 24 nM and
106 3.0%; B, 3.6 1.2 nM and 43 2.4%.38 It thus seems that 4,
the most efficacious MOR ligand in this series of NAQ analogues,
is more potent but less efficacious than morphine, whereas it pos-
sesses similar potency and efficacy as buprenorphine.

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1705
(A)
MeO2C
HO2C
HO2C
HNO3 (conc.)
MeOH
NO2
NO2
H2SO4 (conc.), -10 °C
2 h, 97%
HN
HN
C
HN
reflux, 48 h
50, 6-NO2, 18%
51, 7-NO2, 51%
49
MeO
2
Me O2C
HO2C
10% Pd/ C
1N NaOH
NO2
NO2
NO2
xylene, reflux, 48 h
THF, MeOH
overnight
HN
N
N
50, 51
52, 6-NO2, 13%
54, 7-NO2, 53%
48, 6-NO2, 99%
53, 7-NO2, quantitative
MeO2C
N
10% Pd/C
MeO2C
HO2C
1) HCHO, NaBH3CN
ACN, 5 h
H2, 60psi, 5h
1N NaOH
(B)
·2HCl
NH2
54
N
N
HCl (conc.)
MeOH, 97%
2) 10% Pd/ C, xylene
reflux, 48 h
THF, MeOH, 73%
overnight
NMe
2
NMe2
56
55
57, 21%
HO2C
MeO2C
Me
1N NaOH
N
N
THF, MeOH, 48 h
93%
NMe2
Me
NMe2
63, 54%
62
HO2C
MeO2C
OMe
OMe
OMe
OMe
OMe
OMe
Me O2C
OMe
OMe
(C)
H2SO4 (conc.)
10% Pd/C
1N NaOH
MeOH, reflux, 87%
overnight
HN
N
HN
N
xylene, reflux, 72 h
64%
THF, MeOH, 74%
overnight
59
60
HO2C
58
Scheme 4. Synthetic routes of isoquinoline-3-carboxylic acids 48, 53, 55, 58, and 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 62 with 60 and/or 70-substitutions.
O
O
2.3.2. Warm-water tail immersion assay
1) HCO2H, HCHO
reflux, 4 h
The warm-water tail immersion assay is one of the tests used to
measure the efficacy of analgesics.40 The advantage of this assay is
its relative reproducibility41 and was thus employed in the current
study. Each NAQ analogue was tested for its ability to produce
antinociception and/or to antagonize the antinociceptive effects
of morphine in mice. The results are interpreted as the percentage
of maximum possible effect (% MPE). A higher % MPE indicates a
stronger antinociception effect by the ligand. Figure 2A depicts
the intrinsic antinociceptive effects of the sixteen NAQ analogues
at 10 mg/kg (this dose was chosen so as to compare with 10 mg/
kg morphine, which provides 90–100% MPE in the tail immersion
test). Not surprisingly, 4 (NCQ), which showed the highest MOR
OH
OH
• HCl
N
NH
2) HCl (conc.)
93%
CH3
61
Scheme 5. Synthetic route of (S)-2-methyl-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid hydrochloride 61.
MOR/KOR and 15-fold MOR/DOR selectivity, which is comparable
to that of the reported MOR antagonist cyprodime.39 As for the
MOR efficacy study, the most significant observation was that sub-
stitutions at the 60- and/or 70-position of the isoquinoline ring and
the glycine-unit spacer reduced MOR efficacy. The studies also pro-
vide us valuable information for future molecular design. For
example, combining the encouraging MOR selectivity feature of
10,40-disubstitutions with the low MOR efficacy property of
60/70-substitution might yield more selective MOR ligands with
relative low efficacy.
maximal stimulation in the in vitro [35S]GTP
cS binding assay, acted
as a full agonist in the in vivo warm-water tail immersion assay,
resembling the MOR agonist morphine. The ED50 values of 4 and
morphine were calculated to be 0.73 (0.55–0.97), and 3.24 (2.44–
4.31) mg/kg (95% CL), respectively. The much smaller difference
between the potencies of4 and morphine in the tail immersion
N
N
N
OH
O
OH
O
OH
O
R
1) RCOOH, EDCI, HOBt,
TEA, DMF
1) NH2Bn, TsOH,
Toluene
NH2
H
NH
2) K2CO3, MeOH
2) NaBH4, EtOH
O
H
3) Pd/C, MeOH,
O
HCl (conc.)
•2HCl
OH
OH
OH
19
1–16
Scheme 6. Synthetic route used to generate the first generation of NAQ analogues 1–16.

1706
Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
Table 1
Binding affinity, selectivity and stimulation of MOR [35S]GTP
c
S binding for NAQ analoguesa
Compd
R
Ki (nM)
Selectivity
d/
MOR [35S]GTP
c
S binding
ClogP
l
j
d
j
/
l
l
EC50 (nM)
0.16 0.04
% Emax of DAMGO
NTX
NAQ
NA
0.33 0.02
1.44 0.11
143.5 13.7
4.4
12
435
146
5.4 0.8
1.11 0.07
1.20 0.04
0.68 0.05
13.3 1.1
1.10 0.15
1.61 0.04
161.9 15.0
12.5 0.7
8.4 0.7
3.3 0.4
4.6 0.6
20.8 1.2
18.6 1.1
N
1
2
0.9
2.4
10
12
N
1.14 0.11
27.38 0.35
N
O
3
2.7 1.4
0.61 0.04
22.2 2.1
9.2 0.4
0.2
40
3.4
62
14.1 4.1
13.9 1.9
51.0 0.4
N
H
N
OMe
4 (NCQ)
0.55 0.01
33.9 0.5
1.74 0.13
N
Cl
OH
5
0.73 0.07
18.3 1.9
17.4 1.8
25
24
1.23 0.09
19.8 0.8
N
Cl
OH
6
7
0.45 0.02
1.11 0.06
4.0 0.4
5.1 0.3
32.8 1.5
78.8 0.7
8.9
4.6
73
71
1.06 0.03
6.0 1.5
20.5 0.9
21.6 0.8
N
N
N
N
8
1.26 0.04
0.99 0.07
2.1 0.2
10.8 1.2
10.1 0.5
29.1 0.7
79.8 2.4
129.9 9.6
117.5 7.3
8.6
10
14
63
2.62 0.38
3.32 0.24
7.2 0.5
26.4 0.9
37.5 0.7
22.2 0.4
Cl
N
N
9
131
56
CN
Me
10
NO2
NO2
11 (NNQ)
5.7 1.7
3.6 1.1
27.9 2.0
16.0 1.4
94.7 1.1
55.3 1.8
4.9
4.5
16
16
31.5 18.7
42.7 23.7
12.5 1.4
13.4 0.6
3.10
3.10
N
N
12
13
14
15
16
4.4 0.4
13.4 0.4
2.9 1.3
88.0 6.9
61.0 2.8
6.76 0.54
4.91 0.23
68.6 2.5
69.2 4.8
9.3 0.3
4.5 0.1
20
16
4.70 0.62
85.2 8.6
19.4 10.3
11.4 0.9
6.32 0.30
14.0 0.4
24.8 1.3
26.2 0.6
3.83
N
N
NMe2
OMe
4.6
2.3
2.7
5.2
3.2
2.5
OMe
N
Me
1.79 0.06
N
Me
NMe2
a
The values are the means SEM of four independent experiments. Membranes were prepared from mMOR-, mKOR-, and mDOR-CHO cells. The Bmax values for the MOR,
KOR, and DOR are 3.20 0.41, 1.500 0.139, and 3.667 0.002 pmol/mg. [3H]naloxone, [3H]naltrindole and [3H]diprenorphine were used to label the MOR, DOR and KOR,
respectively. The percentage maximal stimulation to DAMGO (% Emax of DAMGO) is the maximal response of the compound compared to that of 3 lM DAMGO (normalized to
100%).
assay (ꢁ9-fold) compared to that in the MOR [35S]GTP
c
S binding
Figure 2B portrays the antinociceptive effect of morphine at
10 mg/kg in the presence of each NAQ analogue at 1 mg/kg. Due
to the full agonism of 4 as seen in Figure 2A, it was not tested in
this antagonism study. Compound 11 (NNQ) effectively blocked
morphine-mediated antinociception. Its calculated AD50 value is
0.92 (0.51–1.67) mg/kg (95% CL), which is less potent than naltrex-
one (Table 2). This may be partially due to their different binding
affinities at the MOR: naltrexone is nearly 20-fold more potent
than 11 (Table 1). The other fourteen NAQ analogues had marginal
impact on the antinociceptic effect of morphine. For example, com-
pound 13 showed similar affinity to the MOR compared to 11 with
assay (ꢁ70 fold) suggests that pharmacokinetics may play signifi-
cant roles in the in vivo study. Compound 9 produced a % MPE of
56.9 12.7, which was also consistent with its second highest
MOR maximal stimulation in the [35S]GTP
cS binding assay. The %
MPEs of the remaining NAQ analogues, except for 6 and 10, were
less than 20%, indicating either weak opioid receptor agonism (as
shown in the in vitro MOR [35S]GTP
cS binding assay) or unfavor-
able pharmacokinetic properties (such as low CNS permeability)
since tail immersion response is mainly mediated at the spinal
levels of CNS.42

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1707
(A)
(B)
100
80
60
40
20
0
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
3
1
2
3
5
6
7
8
9
10 11 12
1
14 15 16
10 11 12 13 14 15 16
NTX
Figure 2. Warm-water tail immersion assay in mice (n P 6) at 56 0.1 °C. All tested compounds were administered subcutaneously (s.c.). (A) Antinociceptive effects of NAQ
analogues 1–16. Compounds (10 mg/kg) were injected at Time 0. Twenty minutes after injection, tail flick was assessed with hot water. (B) Blockage of the antinociceptive
effect of morphine by NAQ analogues 1–3, 5–16, and naltrexone (NTX). Tested compounds (1 mg/kg) were injected at Time 0. Five minutes later, morphine (10 mg/kg) were
administered. Twenty minutes after morphine injection, tail flick was tested using hot water.
60-guanidinium and Glu297 led to KOR activation. Further
investigation is certainly warranted to see if similar scenario might
happen to compound 12.
Table 2
AD50 values of 11 to antagonize morphine-mediated (10 mg/kg) antinociceptive effect
in warm-water tail immersion assay in mice (n P 6)a
Compd
AD50 values (mg/kg (95% CL))
To be noticed, the binding affinity of morphine at the mMOR-
CHO membrane under the same assay conditions is
1.00 0.03 nM ([3H]DAMGO as the radioligand), which is com-
parable to most of the NAQ analogues, except for 14 (Table 1).
Because the majority of these NAQ analogues (1 mg/kg) did
not antagonize the antinociceptive effects of 10 mg/kg morphine,
we decided not to pursue further studies on them except 4
and 11.
Naltrexone
NAQb
11 (NNQ)
0.006 (0.003–0.014)
0.45 (0.27–0.78)
0.92 (0.51–1.67)
a
All drugs and test compounds were administered subcutaneously (s.c.) to a
group of at least six mice.
Data taken from Li et al.22
b
2.3.3. The KOR and DOR [35S]GTP
cS binding assays for 4 and 11
lower MOR efficacy than 11 in the in vitro [35S]GTP
cS binding
To further understand the underlying mechanisms of 4 (a full
agonist) and 11 (low-efficacy partial agonist and moderate-potency
antagonist) in the warm-water tail immersion assay, KOR and DOR
assay but showed no significant antinociception effect. This could
possibly due to more rapid metabolic excretion rate of the
dimethylamino moiety43 compared to a nitro group44 at the
in vivo level so that 13 could not block the antinoceiception of
morphine in the in vivo warm-water tail withdrawal assay.
Of note, 11 and 12 are regioisomers. They shared a similar
pharmacological profile including MOR binding affinity, selectivity,
[
35S]GTP
cS functional assays were performed for these two
compounds to see if KOR and DOR play any roles for the observed
activities. The KOR full agonist ( )-trans-2-(3,4-dichlorophenyl)-
N-methyl-N-(-2-pyrrolidin-1-ylcyclohexyl) acetamide (U50,488)46
and DOR full agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpi-
perazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide
(SNC80)47 were included in the assays as reference compounds for a
maximal effect at the KOR and the DOR, respectively.
[
35S]GTP
cS binding, and intrinsic agonism in the tail immersion
test. On the other hand, they acted differently when challenged
with morphine in the tail immersion assay. Compound 11 was able
to antagonize morphine while compound 12 showed no effect.
Distinct pharmacological profiles of regioisomers have been
reported for other opioid ligands, such as 50-guanidinonaltrindole
(50-GNTI) and 60-GNTI. In that case, both ligands have comparable
binding affinities at the MOR and the DOR, with 50-GNTI is 8-fold
more potent than 60-GNTI at the KOR. However, 50-GNTI acts as a
KOR antagonist while 60-GNTI behaves like a KOR full agonist when
tested on the electrically stimulated guinea pig ileal longitudinal
muscle.45 It was suggested that a conformational change of trans-
membrane helix 6 that was induced by the interaction between
As seen in Table 3, compared to naltrexone and NAQ, 4 acted as
a low-potency KOR partial agonist with low efficacy (7.1 0.5%).
Compound 11 displayed a comparable KOR efficacy to naltrexone,
while nearly 80-fold less potent than naltrexone. The potency of 11
was nearly 6-fold lower than that of NAQ though it had a relatively
higher KOR efficacy than NAQ. The reduced potency of 11 at KOR
[
35S]GTP
cS binding compared to both naltrexone and NAQ is par-
ticularly encouraging since KOR activation has been reported to
be considerably involved in dysphoria and anhedonia associated
Table 3
Stimulation of KOR and DOR [35S]GTP
c
S binding for 4 and 11a
Compd
KOR [35S]GTP
c
S binding
DOR [35S]GTP
c
S binding
Receptor function
selectivityb
EC50 (nM)
% Emax of U50,488
EC50 (nM)
% Emax of SNC80
j
/
l
d/l
NTX
0.81 0.08
10.9 7.9
26.4 3.9
62.6 10.4
20.8 0.9
13.1 2.0
7.1 0.5
4.4 1.6
98.6 23.7
32.6 5.1
108.1 0.8
5.6 0.6
53.5 5.4
54.8 0.1
67.7 2.0
5.1
3.3
15
28
30
19
3.4
NAQc
4 (NCQ)
11 (NNQ)
21.1 0.4
2.0
a
The values are the means SEM of three independent experiments. The percentage maximal stimulation (% Emax) to ( )-trans-U50,488 or SNC80 is the
maximal response of the compound compared to that of ( )-trans-U50,488 (3 lm) or SNC80 (5 lm).
b
Calculated as the ratio of the EC50 values of a ligand at given two opioid receptors in the [35S]GTP
Data taken from Yuan et al.23
cS binding assay.
c

1708
Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
with drug withdrawal, stress-induced aversion states, and stress-
induced relapse-like behavior.48 NAQ,
and 11 all induced
ꢁ10-fold higher maximal stimulation than naltrexone at the
DOR. However, intriguingly, they were 7- to 25-fold less potent
compared to naltrexone.
agonism to augment intracellular Ca2+ level. Interestingly, 4 did
not show any significant agonist activity either when compared
to the MOR full agonist DAMGO. As reported in the literature
recently, a number of GPCR ligands have been identified to display
‘biased signaling’, that is, the ability of a ligand to activate only
some subset(s) of its receptor’s signaling repertoire. Different
4
With respect to their relative potencies in the opioid receptor
[
35S]GTP
cS binding assays, 4 retained moderate MOR/DOR func-
ligand-induced receptor conformational states, diversified G
tion selectivity compared to naltrexone and NAQ, while displayed
a significantly higher MOR/KOR function selectivity than both nal-
trexone and NAQ. Compound 11, however, exhibited less MOR/
DOR function selectivity than either naltrexone or NAQ and
showed similar MOR/KOR function selectivity to naltrexone and
NAQ. Thus the full agonism of 4 in the warm-water tail immersion
assay could be primarily mediated by the MOR. Hough and
coworkers have shown that the KOR full agonist U50,488 (s.c) pro-
duced 25% MPE at 55 °C in the tail immersion assay while the DOR
proteins and scaffolding/signaling partners, as well as different
receptor oligomerization states can all lead to distinctive function
and signaling.51 In light of this phenomenon, we hypothesize that
the characteristic agonism of 4 in [35S]GTP
cS binding and warm-
water tail immersion assays compared to its insignificant agonism
in Ca2+ flux assay might be due to its biased signaling towards
different signaling pathways, which would be interesting to fur-
ther verify. The determined EC50 of DAMGO is 7.5 1.6 nM
(Fig. 3A), which is comparable to the data obtained from
Cytosensor micrometer (9.61 2.78 nM).50 Both 4 and 11 dose-
dependently blocked the Ca2+ increase trigged by DAMGO
(Fig. 3B). The calculated IC50 values for 4 and 11 are 23.7
3.9 nM and 2728 452 nM, respectively. Compound4 was about
10-fold more potent than 11 in the cell membrane-based radioli-
gand competition binding assay (Table 1). Therefore, the observed
100-fold higher potency of 4 to 11 in Ca2+ flux antagonism study
indicates intrinsic signal amplification occurs in the whole
cell-based assay. The IC50 value for naltrexone under same testing
conditions is 15.5 0.1 nM.
full agonist [D D
-Pen2, -Pen5]enkephalin (intracerebroventricular)
exerted a DOR-independent antinociceptive effect at this tempera-
ture.49 Based on their report and the function selectivity profile of
11, the weak partial agonism of 11 (8.9 3.4% MPE) as seen in the
tail immersion agonism study (Fig. 2A) might be mediated through
both the MOR and the KOR. Since the analgesic effect of morphine
has been mainly attributed to MOR activation, the antagonism of
11 as seen in the tail immersion antagonism study (Fig. 2B) would
be mainly due to its action at the MOR.
2.3.4. Ca2+ flux assays for 4 and 11
Cytosolic Ca2+ level undergoes rapid and transient increase
upon activation of Gq-signaling pathways in CHO cells.50 The intra-
cellular Ca2+ measurement has thus become one of the primary
assays to assess GPCR function. To further profile 4 and 11 at the
MOR, Ca2+ flux functional assays were carried out in Gaqi5 trans-
fected hMOR-CHO cells. Compound 11 displayed no apparent
2.3.5. Opioid withdrawal assays
Since 11 antagonized morphine-mediated antinociception in
the warm-water tail immersion assay, it was further tested in
the chronic opioid withdrawal assays in morphine-pelleted mice.
First, 11 itself did not induce any significant withdrawal symptoms
in placebo-pelleted mice at 30 mg/kg (Fig. 4A and B, first columns)
(A)
(B)
110
80
DAMGO
90
60
40
20
0
70
4
11
50
30
10
-10
-10
-9
-8
-7
-6
-11 -10
-9
-8
-7
-6
-5
-4
Log[M]
Log[M]
Figure 3. Ca2+ flux assays in Gqi5 transfected hMOR-CHO cells. (A) DAMGO dose-dependently increased intracellular Ca2+ levels. (B) Compounds 4 and 11 antagonized the
intracellular Ca2+ increase triggered by activation of the MOR with DAMGO. The results shown are representative of at least three independent experiments.
(A)
(B)
20
Morphine-pelleted
Morphine-pelleted
50
40
30
20
10
0
15
10
5
*
*
*
*
0
)
)
)
)
)
g
g
g)
g)
g
g
g)
g)
g)
k
k
kg
/k
/k
/k
/k
/k
/
/k
/k
g
g
g/
mg/
m
mg
mg
mg
m
mg
mg
m
0
1 mg
0
0
3
0
0
0
0
0
3
00
1
(
(1
(
(3
(1
(3
1
X (1
NT
X (
NT
1
1
(
1
(1
1
1
1
11
1
11
1
11
1
Figure 4. Compound 11 (s.c.) in opioid-withdrawal assays in chronic morphine-exposed mice (n P 6): (A) escape jumps; (B) wet-dog shakes. The first column in each figure
represents placebo-pelleted mice while the second to the fifth represent morphine-pelleted mice. ⁄Indicates P < 0.05, compared to naltrexone (NTX, s.c.).

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1709
Table 4
NNQ–opioid receptor interaction energies (kcal/mol)
Radiusa (Å)
MOR–NNQ
VDWc
KOR–NNQ
DOR–NNQ
VDWc
Eb
Total
Eb
VDWc
Total
Eb
Total
10
8
6
ꢂ13.76
ꢂ14.69
ꢂ17.44
ꢂ16.45
ꢂ71.64
ꢂ69.89
ꢂ67.83
ꢂ63.89
ꢂ85.40
ꢂ84.58
ꢂ85.27
ꢂ80.34
ꢂ22.52
ꢂ21.43
ꢂ23.00
ꢂ19.27
ꢂ66.35
ꢂ66.04
ꢂ62.90
ꢂ58.52
ꢂ88.87
ꢂ87.47
ꢂ85.90
ꢂ77.79
ꢂ8.37
ꢂ63.49
ꢂ63.24
ꢂ63.82
ꢂ60.49
ꢂ71.86
ꢂ71.42
ꢂ70.53
ꢂ72.19
ꢂ8.18
ꢂ6.71
5
ꢂ11.70
a
b
c
Distance from the docked ligand NNQ.
E: electrostatic interaction.
VDW: Van der Waals’ interaction.
Figure 5. Lowest interaction energy-associated NNQ poses after a 15-ns molecular dynamic simulation with three opioid receptors: (A) MOR; (B) KOR; (C) DOR. NNQ is
represented by balls and sticks (green carbon atoms) and the interacting opioid receptor residues are shown as capped sticks (MOR = yellow; KOR = white; DOR = cyan). Ionic/
dipole–dipole interactions and hydrogen bonds are shown with black dashed lines. Opioid receptor amino acid residues are labeled with their sequence number and
Ballesteros–Weinstein index.53
while naltrexone (1 mg/kg) induced significant withdrawal signs
almost immediately after injection in morphine-pelleted mice
and gave 30.0 12.1 times escape jumps and 14.8 2.2 times
wet-dog shakes (Fig. 4A and B, second columns). Second, 11 did
not precipitate significant jumps in morphine-pelleted mice until
30 mg/kg and reached a plateau at 100 mg/kg (Fig. 4A, columns
3–5). Compared to naltrexone, 11 also produced significantly less
wet-dog shakes, even at dose as high as 100 mg/kg (Fig. 4B,
columns 3–5). Thus, 11 had marginal opioid withdrawal potential
at a dose 10-fold of its AD50 value and caused less significant with-
drawal symptoms than naltrexone overall. This might be due to its
weak partial agonism as seen in the tail immersion agonism study
(Fig. 2A). Such observations render 11, an opioid low-efficacy par-
tial agonist/antagonist lacking severe opioid withdrawal capacity,
as a promising lead compound towards developing novel ligands
for substance addiction treatment.

1710
Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
2.4. Molecular modeling studies
structure–activity relationship (SAR) at three opioid receptors, par-
ticularly the mu opioid receptor (MOR). All ligands bound to the
MOR with sub-nanomolar to nanomolar affinity. Substitutions at
the 10- and/or 40-position of the isoquinoline ring retained or
increased MOR selectivity over the KOR while still possessing more
than 20-fold MOR selectivity over the DOR. In contrast, substitution
at the 60- and/or 70-position of the isoquinoline ring decreased MOR
selectivity. A two-atom spacer and an aromatic side chain were pre-
ferred for optimal MOR selectivity. Meanwhile, substitutions at the
60- and/or 70-position of the isoquinoline ring also reduced MOR
efficacy while other structural modifications either retained or
To provide insights into the opioid receptor selectivity profile of
11 (NNQ) compared with NAQ and guide future molecular design,
molecular modeling studies of NNQ in three opioid receptors were
conducted. NNQ was first docked into the optimized crystal struc-
tures of three opioid receptors (see details in Section 4). Molecular
dynamics (MD) simulations on the best scored solutions were then
carried out for all three receptors embedded in a lipid bilayer and
solvated with water to allow the receptor–ligand complexes to
equilibrate in this simulated biological environment. Energy analy-
ses were then performed on the resulting NNQ–receptor com-
plexes. The most favorable non-bonded interaction energy,
calculated using NAMD (a molecular dynamic program) Energy,
between NNQ and its surrounding environment (including protein
and water molecules) within different cutoff distances of the
ligand were determined and summarized in Table 4.
As seen in Table 4, the choice of cutoff distance from the ligand
in general did not significantly affect the non-bonded interaction
energy between the receptor and the ligand NNQ, except for those
of MOR and KOR at 5 Å, in which moderate increases of total
energy were observed compared to the corresponding values at
larger cutoff radii. The total interaction energies at 5 Å for the
NNQ–receptor complexes followed the trend: MOR < KOR < DOR,
that is NNQ bound most favorably to the MOR followed by the
KOR and then to the DOR, which is supported by a similar trend
observed in the experimental binding affinities of NNQ at the three
receptors (Table 1).
The binding poses associated with the lowest total energy for
NNQ in the three opioid receptors are shown in Figure 5. The
epoxymorphinan core remained in its crystallographically-deter-
mined binding site and maintained the same interactions as pre-
viously observed.26–28 However, orientation of the 60-nitro group,
located in the extracellular loop ‘address’ regions of the opioid
receptors,52 was more varied. For the MOR–NNQ complex, the 60-
nitro group was directed toward the top of helix 5, interacting with
Glu2295.35 through two bridging water molecules (Fig. 5A, 5.35 is
the Ballesteros–Weinstein index53). In contrast, the isoquinoline
ring was placed between helixes 2 and 3 in the KOR–NNQ complex,
with the 60-nitro group interacting with Gln1152.70 and the back-
bone NH group of Ser210ECL2 via a water molecule (Fig. 5B).
However, in the DOR–NNQ complex (Fig. 5C), the isoquinoline ring
was oriented toward helix 7 with the nitro group interacting with
Arg291ECL3 and His3017.36 via hydrogen bonds. This resulted in the
loss of the ionic interaction between the carboxylate group of
Asp128 and the protonated N(17) atom of the epoxymorphinan
skeleton, leaving only one hydrogen bond formed between
Asp128 residue and the 14-hydroxyl group. This loss of favorable
hydrogen bonding interaction was also reflected by the relatively
high electrostatic interaction energy for the DOR–NNQ complex,
compared to the MOR–NNQ and KOR–NNQ complexes (Table 4).
Taken together, the molecular modeling study of the interac-
tions between 11 (NNQ) and three opioid receptors well matched
with the experimental binding data. Thus it could be utilized as a
reliable tool to guide future molecular design. The modeling study
also implicated that the nitrogen atom in the nitro group was
involved in a dipole–dipole interaction which only occurred in
the KOR–NNQ complex. Thus replacing the nitro group with
another hydrogen bond acceptor lacking dipole–dipole interaction
potential might improve its MOR selectivity over the KOR.
increased MOR efficacy in the [35S]GTP
cS binding assay. Among this
series of NAQ analogues, 4 behaved as a full agonist in the warm-
water tail immersion assay, while 11 acted as a low-efficacy partial
agonist or a moderate-potency antagonist in the absence or pres-
ence of morphine, respectively. The full agonism of 4 in the tail
immersion study was mediated primarily by the MOR whereas
the weak partial agonism of 11 may be mediated by both the
MOR and the KOR. Nevertheless, 11 exerted its antagonism mainly
through the MOR. Both 4 and 11 antagonized the intracellular Ca2+
increase induced by DAMGO in Gqi5 transfected hMOR-CHO cells.
Furthermore, 11 produced less significant withdrawal symptoms
than naltrexone in the chronic opioid withdrawal assay. In addition,
molecular dynamics simulation studies of 11 in the crystal struc-
tures of three opioid receptors revealed that orientation of the 60-ni-
tro group varied considerably in the structurally different ‘address’
regions of the receptors and played an important role in the
observed binding affinities. All together, the current study identi-
fied a new compound (11, NNQ) carrying interesting pharmacologi-
cal profiles which warrants further investigation on the SAR of NAQ,
particularly at the 60-position of the isoquinoline ring. Meanwhile,
the potential application of 11 in treatment of different neurologi-
cal disorders, such as substance addiction remains to be explored.
4. Experimental
4.1. Chemical synthesis
4.1.1. General methods
Reagents were purchased from either Sigma–Aldrich or Alfa
Aesar. TLC analyses were carried out on the Analtech Uniplate
F254 plates. Chromatographic purification was carried out on silica
gel (230–400 mesh, Merck) columns. Melting points were obtained
with a Fisher scientific micro melting point apparatus without cor-
rection. All IR spectra were acquired on
a Nicolet iS10
instrument.1H (400 MHz) and 13C (100 MHz)nuclear magneticreso-
nance (NMR) spectra were recorded at ambient temperature with
tetramethylsilane as the internal standard on Varian Mercury
400 MHz NMR spectrometer. LC–MS analysis was performed on a
Waters Micromass high resolution (HR) QTOF-II instrument (ESI
source), or an Applied Bio Systems 3200 Q trap with a turbo V source
for TurbolonSpray. HPLC analysis was done with a Varian ProStar
210 system on Microsorb-MV 100-5 C8/C18 column
(250 mm ꢃ 4.6 mm) eluting with acetonitrile (0.1% TFA)/water at
1 mL/min over 15–45 min. Elemental analysis was conducted by
Atlantic Microlab, Inc. All the analytical methods listed above were
used to determine the purity of the newly synthesized compounds
and analyses indicated by the symbols of the elements or HRMS
were within 0.4% of the theoretical values. Yields were not
maximized.
3. Conclusion
4.1.2. General procedure for amide coupling/saponification
reaction
In conclusion, the first generation of NAQ analogues were syn-
thesized and pharmacologically characterized to study their
On an ice-water bath, to the solution of acid (3 equiv, prepared
in house, see Supplementary data for details) in anhydrous

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1711
DMF (7 mL), was added 1-ethyl-3-(3-dimethylaminopropyl)
4.1.5. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
carbodiimide hydrochloride (EDCI, 3 equiv), hydrobenzotriazole
(HOBt, 3 equiv), 4 Å molecular sieves, and TEA (8.0 equiv) under
[2-(isoquinoline-3-carboxamido)acetamido}morphinan (3)
The title compound was prepared in 72% yield. 1H NMR
(400 MHz, CD3OD) d 9.24 (s, 1H), 8.49 (s, 1H), 8.12 (d, J = 8.12 Hz,
1H), 8.02 (d, J = 8.12 Hz, 1H), 7.81 (dt, J = 1.28 Hz, 7.54 Hz, 1H),
7.75 (dt, J = 1.16 Hz, 7.50 Hz, 1H), 6.63 (d, J = 8.08 Hz, 1H), 6.49
(d, J = 8.12 Hz, 1H), 4.58 (d, J = 3.36 Hz, 1H), 4.51 (dt, J = 4.19 Hz,
12.95 Hz, 1H), 4.20 (s, 2H), 3.12 (d, J = 6.68 Hz, 1H), 3.03 (d,
J = 18.6 Hz, 1H), 2.63 (d, J = 7.00 Hz, 1H), 2.58 (dd, J = 6.90 Hz,
18.70 Hz, 1H), 2.35 (m, 1H), 2.33 (m, 1H), 2.26 (d, J = 11.72 Hz,
2H), 1.71 (dt, J = 9.24 Hz, 14.80 Hz, 1H), 1.55 (m, 1H), 1.48 (d,
J = 9.60 Hz, 1H), 1.42 (dd, J = 8.80 Hz, 14.80 Hz, 1H), 1.03 (m, 1H),
0.85 (m, 1H), 0.51 (m, 2H), 0.13 (m, 2H); 13C NMR (100 MHz,
CD3OD) d 170.44, 167.48, 152.91, 147.11, 144.33, 139.39, 137.12,
132.46, 132.13, 131.24, 130.40, 129.08, 128.89, 126.78, 121.35,
120.21, 118.40, 90.52, 71.10, 63.33, 60.63, 48.36, 47.73, 44.42,
43.69, 34.83, 30.43, 23.73, 21.72, 10.15, 4.59, 4.07. HRMS m/z found
N2 protection. Fifteen minutes later,
a solution of 6a-nal-
trexamine hydrochloride (19, 1.0 equiv) in DMF (5 mL) was added
dropwise. The resulted mixture was allowed to warm up to ambi-
ent temperature gradually. Upon completion of the reaction, the
mixture was then filtered through celite. The filtrate was concen-
trated to remove DMF. Methanol (10 mL), and K2CO3 (3 equiv)
were then added to the residue and stirred at ambient tempera-
ture overnight. The mixture was then filtered through celite
again. The filtrate was concentrated to remove methanol. The
residue was partitioned between CH2Cl2 (50 mL) and brine
(50 mL). The organic layer was separated and dried over anhy-
drous MgSO4, concentrated under reduced pressure. The residue
was then purified by column chromatography, eluting with
CH2Cl2/MeOH (1% NH3ꢀH2O) to afford the corresponding com-
pound as free base. Upon confirmation of the structure by 1H
NMR and 13C NMR, the compound was then transformed into
hydrochloride salt by dissolving the free base in MeOH (0.1 mL)
and DCM (2 mL), adding HCl methanol solution (1.25 M, 4 equiv)
under an ice-water bath, and stirred for 5 min. Diethyl ether
(10 mL) was then added. Two hours later, the precipitate was col-
lected by filtration, dried in vacuum to give the target compound
as hydrochloride salt, which was then used in the HPLC, LC–MS,
elemental analysis, and biological screenings.
555.2635, calculated 555.2602 (M+H)+. IR (Diamond, cmꢂ1
) mmax
3295.8, 2928.6, 1651.7, 1505.9, 1231.8. Mp 138–140 °C. Anal.
(C32H34N4O5ꢀ2HClꢀH2O) C, H, N (see details in Supplementary data).
4.1.6. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(1-chloro-4-methoxyisoquinoline-3-carboxamido)morphinan
(4)
The title compound was prepared in 70% yield. 1H NMR
(400 MHz, CDCl3) d 8.33–8.30 (m, 2H), 8.10 (d, J = 8.44 Hz, 1H,
exchangeable), 7.86–7.76 (m, 2H), 6.71 (d, J = 8.08 Hz, 1H), 6.56
(d, J = 8.12 Hz, 1H), 4.84–4.78 (m, 2H), 4.15 (s, 3H), 3.14 (d,
J = 6.04 Hz, 1H), 3.06 (d, J = 18.48 Hz, 1H), 2.69–2.60 (m, 2H),
2.42–2.23 (m, 4H), 1.90–1.79 (m, 2H), 1.58 (d, J = 10.44 Hz, 1H),
1.47 (m, 1H), 1.18 (m, 1H), 0.87 (m, 1H), 0.54 (m, 2H), 0.14 (m,
2H); 13C NMR (100 MHz, CDCl3) d 162.45, 152.84, 145.24, 143.86,
137.28, 134.72, 133.27, 131.48, 130.96, 130.13, 129.15, 126.66,
126.08, 123.52, 119.27, 117.06, 90.50, 69.59, 63.81, 62.28, 59.73,
47.37, 46.20, 43.23, 33.57, 29.24, 22.92, 21.09, 9.40, 3.94, 3.87.
HRMS m/z found 562.2128, calculated 562.2103 (M+H)+. IR
4.1.3. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
[2-(isoquinolin-3-yl)acetamido)morphinan (1)
The title compound was prepared in 70% yield. Hydrochloride
salt: 1H NMR (400 MHz, DMSO-d6) d 9.67 (s, 1H), 9.31 (br s, 1H,
exchangeable), 8.84 (br s, 1H, exchangeable), 8.37 (d, J = 8.56 Hz,
1H), 8.33 (d, J = 8.04 Hz, 1H), 8.17–8.15 (m, 2H), 8.04 (t,
J = 7.58 Hz, 1H), 7.87 (t, J = 7.46 Hz, 1H), 6.75 (d, J = 8.12 Hz,
1H), 6.58 (d, J = 8.16 Hz, 1H), 6.29 (br s, 1H), 4.64 (d,
J = 3.88 Hz, 1H), 4.45 (m, 1H), 4.06 (s, 2H), 3.90 (d, J = 6.64 Hz,
1H), 3.34 (d, J = 19.96 Hz, 1H), 3.26 (m, 1H), 3.09–2.97 (m, 2H),
2.94 (m, 1H), 2.70 (m, 1H), 2.43 (m, 1H), 1.87 (m, 1H), 1.62
(m, 1H), 1.50–1.35 (m, 2H), 1.08–0.95 (m, 2H), 0.67 (m, 1H),
0.61 (m, 1H), 0.47 (m, 1H), 0.39 (m, 1H); 13C NMR (100 MHz,
(Diamond, cmꢂ1
) mmax 3256.8, 1652.1, 1503.9, 1313.2, 1117.0,
945.5, 768.8, 726.8. Mp 202–204 °C.
4.1.7. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
DMSO-d6)
d
169.19, 148.48, 147.37, 141.37, 140.74, 140.50,
(1-chloro-4-hydroxyisoquinoline-3-carboxamido)morphinan
138.04, 131.87, 131.35, 130.11, 128.49, 127.87, 126.71, 123.43,
121.08, 119.65, 89.12, 71.00, 63.70, 59.21, 47.52, 47.17, 47.11,
40.01, 31.67, 30.62, 25.02, 21.02, 6.86, 6.24, 3.42. HRMS m/z
found 512.2699, calculated 512.2544 (M+H)+. IR (Diamond,
(5)
The title compound was prepared in 44% yield. Hydrochloride
salt: 1H NMR (400 MHz, DMSO-d6) d 13.61 (s, 1H, exchangeable),
9.42 (s, 1H, exchangeable), 8.88 (br s, 1H, exchangeable), 8.37 (m,
1H), 8.32 (m, 1H), 8.25 (d, J = 8.72 Hz, 1H, exchangeable), 8.03–
8.01 (m, 2H), 6.75 (d, J = 8.08 Hz, 1H), 6.63 (d, J = 8.12 Hz, 1H),
6.36 (s, 1H, exchangeable), 4.85 (d, J = 3.96 Hz, 1H), 4.71 (m,
1H), 3.93 (d, J = 6.96 Hz, 1H), 3.41–3.27 (m, 2H), 3.15–3.05 (m,
2H), 2.96 (m, 1H), 2.72 (m, 1H), 2.50 (m, 1H), 1.94 (dt,
J = 8.97 Hz, 15.36 Hz, 1H), 1.69–1.66 (m, 2H), 1.50 (dd,
J = 8.92 Hz, 15.32 Hz, 1H), 1.19–1.08 (m, 2H), 0.70 (m, 1H), 0.63
(m, 1H), 0.49 (m, 1H), 0.42 (m, 1H); 13C NMR (100 MHz,
CD3OD) d 169.81, 155.29, 147.22, 140.78, 140.56, 132.36 (ꢃ2),
131.39, 130.59, 129.87, 127.42, 124.28, 122.98, 121.92, 121.12,
120.04, 89.14, 71.05, 63.73, 59.10, 47.23, 46.83, 31.62, 30.32,
24.92, 21.19, 6.85, 6.28, 3.34 (ꢃ2). HRMS m/z found 548.1970, cal-
cmꢂ1
) mmax 3232.8, 1651.9, 1615.6, 1236.4, 1117.6, 765.6, 747.5.
Mp 203 °C dec.
4.1.4. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
[3-(isoquinolin-3-yl)propanamido)morphinan (2)
The title compound was prepared in 89% yield. 1H NMR
(400 MHz, CDCl3) d 9.19 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.74 (d,
J = 8.2 Hz, 1H), 7.66 (dt, J = 1.04 Hz, 6.95 Hz, 1H), 7.54 (m, 2H),
6.73 (d, J = 8.04 Hz, 1H), 6.49 (d, J = 8.12 Hz, 1H), 6.41 (d,
J = 8.44 Hz, 1H, exchangeable), 4.49 (m, 2H), 3.27 (m, 2H), 3.06
(d, J = 5.12 Hz, 1H), 2.99 (d, J = 18.52 Hz, 1H), 2.69 (dt, J = 3.28 Hz,
7.32 Hz, 2H), 2.63 (m, 1H), 2.54 (dd, J = 6.48 Hz, 18.56 Hz, 1H),
2.33 (m, 2H), 2.18 (m, 2H), 1.65 (m, 1H), 1.55–1.43 (m, 2H), 1.28
(m, 1H), 0.83 (m, 2H), 0.52 (m, 2H), 0.11 (m, 2H); 13C NMR
culated 548.1947 (M+H)+. IR (Diamond, cmꢂ1
2952.6, 1620.6, 1532.1, 1319.0, 1118.3, 1032.3, 948.3, 768.0. Mp
) mmax 3076.8,
(100 MHz, CDCl3)
d
171.63, 153.19, 151.90, 145.73, 137.74,
207–210 °C.
136.68, 131.10, 130.81, 127.63, 127.22, 126.86, 126.29, 125.74,
119.39, 119.13, 117.66, 89.77, 69.45, 62.25, 59.64, 47.00, 45.89,
43.28, 36.56, 33.44, 33.15, 28.84, 22.84, 21.25, 9.33, 3.92, 3.84.
HRMS m/z found 526.2710, calculated 526.2700 (M+H)+. IR
4.1.8. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(4-hydroxyisoquinoline-3-carboxamido)morphinan (6)
The title compound was prepared in 60% yield. 1H NMR
(400 MHz, CDCl3) d 13.36 (s, 1H, exchangeable), 8.58 (s, 1H),
8.34 (d, J = 8.04 Hz, 1H), 8.29 (d, J = 8.44 Hz, 1H, exchangeable),
(Diamond, cmꢂ1
) mmax 1628.3, 1502.9, 1117.4, 859.0, 748.0. Mp
106–108 °C.

1712
Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
7.90 (d, J = 7.64 Hz, 1H), 7.75–7.67 (m, 2H), 6.74 (d, J = 8.08 Hz,
1H), 6.56 (d, J = 8.12 Hz, 1H), 4.79 (m, 2H), 3.14 (d, J = 6.52 Hz,
1H), 3.06 (d, J = 18.48 Hz, 1H), 2.69–2.61 (m, 2H), 2.42–2.24 (m,
4H), 1.87 (dt, J = 9.01 Hz, 14.54 Hz, 1H), 1.77 (m, 1H), 1.60 (m,
1H), 1.47 (dd, J = 9.22 Hz, 14.14 Hz, 1H), 1.19 (m, 1H), 0.87 (m,
1H), 0.57–0.53 (m, 2H), 0.14 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 169.20, 155.12, 145.36, 141.77, 137.42, 131.39, 130.87,
129.94, 129.58, 128.31, 127.14, 126.04, 122.79, 122.13, 119.42,
117.29, 90.41, 69.62, 62.25, 59.76, 47.45, 45.93, 43.17, 33.72,
29.33, 22.99, 21.04, 9.40, 3.95, 3.87. HRMS m/z found 514.2514,
4.1.12. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
(1-methylisoquinolin-3-carboxamido)morphinan (10)
a-epoxy-6a-
The title compound was prepared in 65% yield. 1H NMR
(400 MHz, CDCl3) d 8.54 (d, J = 8.44 Hz, 1H, exchangeable), 8.45
(s, 1H), 8.11 (d, J = 8.20 Hz, 1H), 7.93 (d, J = 7.92 Hz, 1H), 7.73–
7.63 (m, 2H), 6.72 (d, J = 8.08 Hz, 1H), 6.56 (d, J = 8.08 Hz, 1H),
4.87–4.84 (m, 2H), 3.13 (d, J = 6.40 Hz, 1H), 3.06 (d, J = 18.48 Hz,
1H), 2.93 (s, 3H), 2.68–2.60 (m, 2H), 2.41–2.22 (m, 4H), 1.88–1.80
(m, 2H), 1.58 (m, 1H), 1.48 (dd, J = 8.94 Hz, 11.70 Hz, 1H), 1.23
(m, 1H), 0.87 (m, 1H), 0.55 (m, 2H), 0.14 (m, 2H); 13C NMR
calculated 514.2339 (M+H)+. IR (Diamond, cmꢂ1
)
mmax 3211.9,
(100 MHz, CDCl3) d 164.29, 157.57, 145.29, 142.41, 137.30,
1624.9, 1529.8, 1456.4, 1118.2, 952.0, 764.1, 745.5. Mp 128–
131 °C.
136.10, 131.04, 130.41, 128.86, 128.61, 128.47, 126.12, 125.67,
119.15, 119.08, 116.96, 90.57, 69.63, 62.34, 59.71, 47.38, 46.17,
43.27, 33.53, 29.11, 22.94, 22.48, 21.31, 9.41, 3.92, 3.83. HRMS
m/z found 512.2569, calculated 512.2544 (M+H)+. Salt: IR (dia-
4.1.9. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(4-quinazoline-2-carboxamido)morphinan (7)
mond, cmꢂ1
) mmax 3207.9, 1660.7, 1506.7, 1457.6, 1320.3, 1117.4,
The title compound was prepared in 63% yield. 1H NMR
(400 MHz, CDCl3) d 9.45 (s, 1H), 8.45 (d, J = 8.96 Hz, 1H), 8.17 (d,
J = 8.44 Hz, 1H), 7.99–7.93 (m, 2H), 7.72 (dt, J = 0.76 Hz, 7.52 Hz,
1H), 6.73 (d, J = 8.08 Hz, 1H), 6.56 (d, J = 8.12 Hz, 1H), 4.88 (m,
1H), 4.82 (d, J = 4.4 Hz, 1H), 3.14 (d, J = 6.16 Hz, 1H), 3.06 (d,
J = 18.48 Hz, 1H), 2.69–2.60 (m, 2H), 2.42–2.22 (m, 4H), 1.89–
1.80 (m, 2H), 1.58 (m, 1H), 1.48 (m, 1H), 1.25 (m, 1H), 0.88 (m,
1H), 0.55 (m, 2H), 0.14 (m, 2H); 13C NMR (100 MHz, CDCl3) d
162.07, 160.68, 153.97, 149.87, 145.26, 137.59, 134.77, 130.92,
129.38, 129.22, 127.15, 125.79, 124.87, 119.25, 117.33, 90.08,
69.65, 62.32, 59.68, 47.32, 46.70, 43.28, 33.44, 28.95, 22.96,
21.13, 9.35, 3.91, 3.84. HRMS m/z found 499.2515, calculated
1032.6, 782.8. Mp 224 °C dec.
4.1.13. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(6-nitroisoquinoline-3-carboxamido)morphinan (11)
26
The title compound was prepared in 62% yield. [
a
]
ꢂ308.89° (c
D
0.09, MeOH). 1H NMR (400 MHz, DMSO-d6) d 9.63 (s, 1H), 9.27 (d,
J = 1.96 Hz, 1H), 9.16 (br s, 1H), 8.94 (s, 1H), 8.53 (d, J = 8.96 Hz,
1H), 8.51 (d, J = 8.32 Hz, 1H), 8.49 (dd, J = 2.18 Hz, 9.02 Hz, 1H),
6.63 (d, J = 8.08 Hz, 1H), 6.51 (d, J = 8.08 Hz, 1H), 4.67–4.62 (m,
2H), 3.12 (d, J = 6.4 Hz, 1H), 3.01 (d, J = 18.56 Hz, 1H), 2.51–2.50
(m, 2H), 2.38–2.32 (m, 2H), 2.29–2.14 (m, 2H), 1.71 (dt,
J = 9.22 Hz, 14.47 Hz, 1H), 1.60 (t, J = 11.24 Hz, 1H), 1.46–1.37 (m,
2H), 1.05 (m, 1H), 0.90 (m, 1H), 0.50 (m, 2H), 0.14 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 162.58, 152.04, 148.56, 145.39,
144.61, 138.01, 135.04, 130.76, 130.64, 130.36, 124.81, 124.45,
122.33, 121.31, 118.89, 117.22, 88.73, 69.12, 61.23, 58.69, 46.56,
45.86, 42.80, 33.49, 29.23, 22.36, 20.64, 8.98, 3.84, 3.37. HRMS
m/z found 543.2243, calculated 543.2238 (M+H)+. IR (Diamond,
499.2340 (M+H)+. IR (Diamond, cmꢂ1
) mmax 3369.5, 1675.0,
1506.4, 1117.3, 777.8. Mp 151–153 °C.
4.1.10. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
a-epoxy-6a-
(1-chloroisoquinoline-3-carboxamido)morphinan (8)
The title compound was prepared in 29% yield. 1H NMR
(400 MHz, CDCl3) d 8.55 (s, 1H), 8.36 (d, J = 8.24 Hz, 1H), 8.18
(d, J = 8.76 Hz, 1H, exchangeable), 7.99 (d, J = 7.52 Hz, 1H), 7.83–
7.75 (m, 2H), 6.74 (d, J = 8.12 Hz, 1H), 6.57 (d, J = 8.12 Hz, 1H),
4.86–4.81 (m, 2H), 3.13 (d, J = 6.52 Hz, 1H), 3.07 (d, J = 18.48 Hz,
1H), 2.69–2.61 (m, 2H), 2.42–2.24 (m, 4H), 1.89–1.73 (m, 2H),
1.62 (d, J = 8.96 Hz, 1H), 1.48 (m, 1H), 1.21 (m, 1H), 0.88 (m,
1H), 0.57–0.53 (m, 2H), 0.15–0.12 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 162.82, 150.33, 145.21, 142.79, 137.88, 137.42, 131.80,
130.86, 130.00, 128.68, 127.93, 126.58, 125.94, 120.68, 119.33,
117.22, 90.35, 69.63, 62.31, 59.72, 47.39, 46.46, 43.24, 33.59,
29.15, 22.95, 21.13, 9.40, 3.90, 3.82. HRMS m/z found 532.2002,
cmꢂ1
) mmax 3368.3, 1663.2, 1630.6, 1531.8, 1507.1, 1457.3,
1341.8, 1116.3. Mp 141–143 °C.
4.1.14. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(7-nitroisoquinoline-3-carboxamido)morphinan (12)
26
The title compound was prepared in 37% yield. [
a
]
ꢂ320.00° (c
D
0.1, MeOH). 1H NMR (400 MHz, CDCl3) d 9.37 (s, 1H), 9.00 (m, 1H),
8.73 (s, 1H), 8.53 (dd, J = 2.06 Hz, 8.94 Hz, 1H), 8.46 (d, J = 8.64 Hz,
1H), 8.16 (d, J = 8.96 Hz, 1H), 6.73 (d, J = 8.04 Hz, 1H), 6.58 (d,
J = 8.04 Hz, 1H), 4.89–4.82 (m, 2H), 3.15 (d, J = 6.36 Hz, 1H), 3.08
(d, J = 18.4 Hz, 1H), 2.70–2.61 (m, 2H), 2.42–2.24 (m, 4H), 1.89–
1.82 (m, 2H), 1.62–1.59 (m, 1H), 1.49 (dd, J = 9.28 Hz, 12.4 Hz,
1H), 1.23–1.19 (m, 1H), 0.88 (m, 1H), 0.56 (m, 2H), 0.14 (m, 2H);
13C NMR (100 MHz, CDCl3) d 163.03, 152.80, 147.13, 146.82,
145.21, 138.68, 137.28, 130.91, 130.14, 128.33, 126.12, 124.42,
124.22, 119.98, 119.41, 117.08, 90.45, 69.61, 62.21, 59.71, 47.41,
46.46, 43.19, 33.62, 29.16, 22.94, 21.09, 9.39, 3.96, 3.86. HRMS
m/z found 543.2218, calculated 543.2238 (M+H)+. IR (Diamond,
calculated 532.1998 (M+H)+. IR (diamond, cmꢂ1
) mmax 3380.5,
2926.0, 1656.5, 1506.2, 1259.8, 1116.5, 1033.8, 988.7, 786.4. Mp
210–212 °C.
4.1.11. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
a-epoxy-6a-
(1-cyanoisoquinoline-3-carboxamido)morphinan (9)
The title compound was prepared in 40% yield. 1H NMR
(400 MHz, CDCl3) d 8.82 (s, 1H), 8.39 (m, 1H), 8.16 (d, J = 8.68 Hz,
1H, exchangeable), 8.09 (m, 1H), 7.92–7.87 (m, 2H), 6.75 (d,
J = 8.08 Hz, 1H), 6.61 (d, J = 8.08 Hz, 1H), 4.84 (m, 2H), 3.15 (d,
J = 6.36 Hz, 1H), 3.08 (d, J = 18.48 Hz, 1H), 2.69–2.63 (m, 2H),
2.42–2.27 (m, 4H), 1.89–1.79 (m, 2H), 1.60 (m, 1H), 1.50 (m, 1H),
1.30 (m, 1H), 0.87 (m, 1H), 0.55 (m, 2H), 0.15 (m, 2H); 13C NMR
cmꢂ1
) mmax 3367.7, 1664.1, 1631.3, 1522.7, 1508.7, 1487.5,
1459.2, 1342.9, 1117.0. Mp 230 °C dec.
4.1.15. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(7-dimethylaminoisoquinoline-3-carboxamido)morphinan (13)
26
The title compound was prepared in 75% yield. [
a]
ꢂ224.62° (c
D
(100 MHz, CDCl3)
d
162.39, 144.98, 144.18, 137.62, 136.51,
0.1, MeOH). 1H NMR (400 MHz, CDCl3) d 8.91 (s, 1H), 8.45 (s, 1H),
8.32 (d, J = 8.72 Hz, 1H, exchangeable), 7.80 (d, J = 9.08 Hz, 1H),
7.34 (dd, J = 9.12 Hz, 2.48 Hz, 1H), 6.94 (d, J = 2.16 Hz, 1H), 6.73
(d, J = 8.08 Hz, 1H), 6.55 (d, J = 8.12 Hz, 1H), 4.86–4.79 (m, 2H),
3.20–3.00 (m, 8H), 2.68–2.59 (m, 2H), 2.38–2.27 (m, 4H), 1.90–
1.76 (m, 2H), 1.59 (m, 1H), 1.46 (m, 1H), 1.16 (m, 1H), 0.87 (m,
1H), 0.57–0.52 (m, 2H), 0.15–0.11 (m, 2H); 13C NMR (100 MHz,
133.16, 132.48, 131.38, 130.70, 130.08, 128.97, 125.63, 125.52,
124.35, 119.56, 117.44, 115.46, 89.90, 69.72, 62.31, 59.69, 47.35,
46.61, 43.36, 33.42, 28.78, 22.96, 21.21, 9.43, 3.95, 3.87. HRMS
m/z found 523.2339, calculated 523.23398 (M+H)+. Salt: IR (dia-
mond, cmꢂ1
793.8, 748.9. Mp 251 °C dec.
) mmax 3076.2, 2161.7, 1507.3, 1318.1, 1116.8, 1033.1,

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1713
CDCl3) d 164.63, 150.28, 149.12, 145.46, 140.09, 137.49, 131.73,
130.99, 129.02, 128.27, 125.88, 120.54, 120.22, 119.19, 117.21,
104.52, 90.72, 69.62, 62.31, 59.75, 47.33, 46.07, 43.22, 40.42
(ꢃ2), 33.62, 29.38, 22.95, 21.21, 9.39, 3.94, 3.86. HRMS m/z found
NMR (400 MHz, DMSO-d6) d 10.75 (br s, 1H, exchangeable), 9.27
(br s, 1H, exchangeable), 8.97–8.95 (m, 2H, exchangeable), 7.09
(d, J = 7.32 Hz, 1H), 6.86 (m, 1H), 6.75 (d, J = 8.08 Hz, 1H), 6.64
(m, 1H), 6.58 (d, J = 8.08 Hz, 1H), 6.45 (br s, 1H, exchangeable),
4.70 (d, J = 3.8 Hz, 1H), 4.60–4.25 (m, 4H), 3.98 (m, 1H), 3.35 (d,
J = 20.16 Hz, 1H), 3.30–2.80 (m, 15H), 2.71 (m, 1H), 2.45 (m, 1H),
1.93 (m, 1H), 1.70–1.35 (m, 3H), 1.09 (m, 1H), 1.01 (m, 1H), 0.69
(m, 1H), 0.62 (m, 1H), 0.48 (m, 1H), 0.40 (m, 1H); 13C NMR
(100 MHz, DMSO-d6) d 166.35, 147.40, 145.93, 138.65, 128.81,
128.66 (ꢃ2), 122.25, 121.21, 119.36, 118.59, 114.76, 111.47,
86.73, 69.23, 64.85, 62.91, 60.80, 57.00, 54.80, 45.67, 45.22,
41.51, 40.87, 29.98, 29.67, 28.92, 23.51, 19.73, 19.54, 5.65, 5.17,
2.57. Hydrochloride salts of the mixture: HRMS m/z found
541.3001, calculated 541.2809 (M+H)+. IR (Diamond, cmꢂ1
) mmax
3375.0, 1616.2, 1518.4, 1504.5, 1156.4, 810.0, 726.1. Mp 268 °C
dec.
4.1.16. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
a-epoxy-6a-
(6,7-dimethoxyisoquinoline-3-carboxamido)morphinan (14)
26
The title compound was prepared in 72% yield. [
a]
ꢂ262.59° (c
D
0.25, MeOH). 1H NMR (400 MHz, DMSO-d6)
d
9.16 (s, 1H,
exchangeable), 9.13 (s, 1H), 8.42 (m, 2H), 7.625 (s, 1H), 7.619 (s,
1H), 6.63 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 8.04 Hz, 1H), 4.92 (br s,
1H, exchangeable), 4.61 (m, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.09
(d, J = 6.48 Hz, 1H), 3.00 (d, J = 18.52 Hz, 1H), 2.65–2.56 (m, 2H),
2.40–2.27 (m, 2H), 2.26–2.13 (m, 2H), 1.69 (dt, J = 9.32 Hz,
J = 14.49 Hz, 1H), 1.56 (m, 1H), 1.45–1.36 (m, 2H), 0.99 (m, 1H),
0.88 (m, 1H), 0.50 (m, 2H), 0.13 (m, 2H); 13C NMR (100 MHz,
559.3279, calculated 559.32788 (M+H)+; IR (Diamond, cmꢂ1
) mmax
3348, 3233, 1671, 1618, 1459, 1236, 1118, 1034, 811; mp >250 °C.
4.2. Biological evaluation
4.2.1. Drugs
DMSO-d6)
d
163.46, 153.21, 151.30, 148.62, 145.38, 141.84,
Morphine sulfate was purchased from Mallinckrodt (St. Louis,
MO). Naloxone and naltrexone were purchased from Sigma–
Aldrich (St. Louis, MO). All drugs and test compounds were dis-
solved in pyrogen-free isotonic saline (Baxter Healthcare,
Deerfield, IL).
137.89, 132.08, 130.70, 125.69, 124.88, 118.75, 118.45, 117.09,
106.20, 105.80, 89.00, 69.06, 61.19, 58.71, 55.86, 55.74, 46.55,
45.52, 42.65, 33.62, 29.27, 22.25, 20.69, 9.06, 3.74, 3.36. HRMS
m/z found 558.2615, calculated 558.2599 (M+H)+. IR (Diamond,
cmꢂ1
) mmax 3486.3, 3382.4, 1669.1, 1506.4, 1457.0, 1250.1,
1220.7, 1177.2, 1004.9, 830.7. Mp 250 °C dec.
4.2.2. Animals
Male Swiss–Webster mice (Harlan, Indianapolis, IN) weighing
25–30 g were housed six per cage in animal care quarters at
22 2 °C on a 12 h light/dark cycle. Food and water were available
ad libitum. The mice were brought to a test room (22 2 °C, 12 h
light/dark cycle), marked for identification, and allowed 18 h to
recover from transport and handling. Protocols and procedures
were approved by the Institutional Animal Care and Use
Committee at Virginia Commonwealth University Medical Center
and complied with the recommendations of the International
Association for the Study of Pain.
4.1.17. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
[(S)-(2-methyl-1,2,3,4-tetrahydroisoquinoline-3-
carboxamido)]morphinan (15)
a-epoxy-6a-
The title compound was prepared in 87% yield. 1H NMR
(400 MHz, CDCl3) d 7.15–7.09 (m, 3H), 7.01 (m, 1H), 6.69 (d,
J = 8.08 Hz, 1H), 6.53 (d, J = 8.16 Hz, 1H), 4.93 (br s, 1H), 4.66 (d,
J = 4.64 Hz, 1H), 4.52 (m, 1H), 3.78 (d, J = 14.96 Hz, 1H), 3.64 (d,
J = 14.92 Hz, 1H), 3.29 (t, J = 6.92 Hz, 1H), 2.99–3.05 (m, 4H), 2.64
(d, J = 5.84 Hz, 1H), 2.55 (dd, J = 6.50 Hz, 18.46 Hz, 1H), 2.43 (s,
3H), 2.33 (dd, J = 3.6 Hz, 6.40 Hz, 2H), 2.22 (m, 2H), 1.48–1.66 (m,
3H), 1.19 (m, 1H), 0.81–0.95 (m, 2H), 0.53 (m, 2H), 0.11 (m, 2H);
13C NMR (100 MHz, CDCl3) d 172.03, 145.06, 137.57, 133.94,
133.17, 130.81, 128.23, 126.91, 126.26, 126.18, 125.61, 119.29,
117.43, 89.73, 69.51, 64.56, 62.25, 59.67, 55.88, 47.13, 45.74,
43.29, 42.07, 33.28, 29.49, 28.62, 22.86, 21.29, 9.40, 3.90, 3.85.
HRMS m/z found 516.2854, calculated 516.2857 (M+H)+. IR
4.2.3. In vitro competitive radioligand binding and functional
assay
The radioligand binding assay and the [35S]GTP
cS binding assay
were conducted using monocloned mice opioid receptor-expressed
Chinese hamster ovarian (CHO) cell lines as described
previously.36,37 Briefly, for the competition binding assay,
[3H]NLX, [3H]DPN, and [3H]NTI were used to label the MOR, the
KOR, and the DOR, respectively. Aliquots of a membrane protein
(Diamond, cmꢂ1
) mmax 3200.0, 2930.5, 1641.3, 1504.4, 1253.9,
1118.8. Mp 137–139 °C. Anal. (C31H37N3O4ꢀ2HClꢀH2O) C, H, N (see
details in Supplementary data).
(30 lg) were incubated with the corresponding radioligand in
the presence of different concentrations of the ligand under
investigation in TME buffer (50 mM Tris, 3 mM MgCl2, 0.2 mM
EGTA, pH 7.7) at 30 °C for 1.5 h. The bound radioactive ligand
was separated from the free radioligand by filtration using the
4.1.18. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6a-
(7-dimethylamino-2-methyl-1,2,3,4-tetrahydroisoquinoline-3-
carboxamido)morphinan (16)
The title compound was prepared in 76% yield as a mixture of
Brandel harvester (Biomedical Research
&
Development
diastereoisomers with a ratio of 3:2 based on the proton NMR
Laboratories, MD). Specific (i.e., opioid receptor-related) binding
was determined as the difference in binding obtained in the
26
spectrum analysis. [
a
]
D
ꢂ308.96° (c 0.25, MeOH). Hydrochloride
salt of one isomer: 1H NMR (400 MHz, DMSO-d6) d 10.75 (br s,
1H, exchangeable), 9.27 (br s, 1H, exchangeable), 8.97–8.95 (m,
2H, exchangeable), 7.15 (d, J = 7.60 Hz, 1H), 6.86 (m, 1H), 6.75 (d,
J = 8.08 Hz, 1H), 6.64 (m, 1H), 6.58 (d, J = 8.08 Hz, 1H), 6.45 (br s,
1H, exchangeable), 4.65 (d, J = 3.8 Hz, 1H), 4.60–4.25 (m, 4H),
3.98 (m, 1H), 3.35 (d, J = 20.16 Hz, 1H), 3.30–2.80 (m, 15H), 2.71
(m, 1H), 2.45 (m, 1H), 1.93 (m, 1H), 1.70–1.35 (m, 3H), 1.09 (m,
1H), 1.01 (m, 1H), 0.69 (m, 1H), 0.62 (m, 1H), 0.48 (m, 1H), 0.40
(m, 1H); 13C NMR (100 MHz, DMSO-d6) d 166.32, 147.40, 145.83,
138.65, 129.04, 128.66 (ꢃ2), 122.25, 121.21, 119.36, 118.59,
114.76, 111.47, 86.88, 69.23, 64.85, 62.91, 60.80, 57.00, 54.80,
45.59, 45.18, 41.51, 40.71, 29.92, 29.45, 28.96, 23.51, 19.71,
19.54, 5.65, 5.17, 2.57. Hydrochloride salt of another isomer: 1H
absence and presence of
5 lM naltrexone, 5 lM ( )-trans-
U50,488, and 5 M SNC80 for the MOR, the KOR, and the DOR,
l
respectively. The potency of the drugs in displacing the specific
binding of the radioligand was determined by linear regression
analysis of Hill plots. The IC50 values were determined and con-
verted to Ki values using the Cheng–Prusoff equation. The
[
35S]GTP
c
S functional assays were conducted in the same cell
membranes used for the receptor binding assays. Membrane pro-
teins (10 g) were incubated with varying concentrations of com-
pounds, GDP (10 S in assay buffer
M) and 0.1 nM [35S]GTP
l
l
c
(50 mM Tris, 3 mM MgCl2, 100 mM NaCl, 0.2 mM EGTA, pH 7.7)
for 1.5 h at 30 °C. Nonspecific binding was determined with
20 lM unlabeled GTPcS while basal binding was assessed in the

1714
Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
presence of GDP and absence of drug. DAMGO (3
l
M), ( )-trans-
at 72 h from pellet implantation with naltrexone (1.0 mg/kg, s.c.),
and the testing compound (s.c.) at indicated doses. Withdrawal
commenced within 3 min after antagonistadministration. Escape
jumps and wet dog shakes were quantified by counting their
occurrences over 20 min for each mouse using at least six mice
per drug.
U50,488 17 (5 M), and SNC80 18 (5 M) were included as the
l
l
internal standard in the assay for a maximal effect of a full agonist
for the MOR, KOR, and DOR, respectively. The bound radioactive
ligand was separated from the free radioligand by rapid filtration
and three washes and then subjected to liquid scintillation
spectrophotometry. Percent maximal stimulation is defined as
(net stimulated binding by test compound/by internal stan-
dard) ꢃ 100%. The normalized data were then subjected to nonlin-
ear regression analysis to determine EC50 and efficacy (Emax) values
with Prism 3.0 software (GraphPad Software, San Diego, CA).
4.2.7. Statistical analysis
One-way ANOVA followed by the post hoc Dunnett test were
performed to assess significance using the Prism 3.0 software
(GraphPad Software, San Diego, CA).
4.2.4. Ca2+ flux assay
4.3. Molecular modeling studies
The Ca2+ flux assay was performed similarly to the procedure
described earlier29,54 except as indicated below. The hMOR-CHO
cells were maintained in DMEM/F12 supplemented with 10% fetal
The molecular structure of the ligand (11, NNQ) was sketched in
SYBYL-X 2.0, and its Gasteiger–Hückel charges were assigned
before energy minimization (10,000 iterations) with the Tripos
bovine serum, 100 units/mL penicillin, 100 lg/mL streptomycin,
and 0.25 mg/mL hygromycin B at 37 °C and 5% CO2. Four hours
after transfection, cells were plated at 30,000 cells per well into a
clear bottom, black 96-well plate (Greiner Bio-one) and incubated
for 24 h. The growth media was then decanted and the wells were
force field (TFF). The X-ray crystal structures for MOR (4DKL)26
,
KOR (4DJH)27 and DOR (4EJ4)28 were retrieved from PDB Data
Bank (http://www.pdb.org). SYBYL-X 2.0 was also used to prepare
the obtained protein coordinates for ligand docking by extracting
the crystallized ligand and the fusion protein at intracellular loop
3, followed by addition of hydrogen atoms and subsequent energy
minimization of only the added hydrogen atoms. Automated dock-
ing on these ‘cleaned’ receptor structures was performed utilizing a
genetic algorithm-based docking program GOLD 5.2.57 The binding
washed with 100
then incubated with either 80
(antagonism study) of Fluo4 loading buffer (40
AM (Invitrogen), 84 L 2.5 mM probenacid, in 8 mL or 5.5 mL assay
buffer) for 30 min. For antagonism study, 25 L of varying concen-
l
L of 50:1 HBSS/HEPES assay buffer. Cells were
(agonism study) or 55
L 2 M Fluo4-
l
L
lL
l
l
l
l
trations of testing compounds were added in triplicates and the
plate was incubated for an additional 15 min. Plates were then
read on a FlexStation3 microplate reader (Molecular Devices) at
site was defined to include all atoms within 10 Å of the c-carbon
atom of Asp3.32 for the three opioid crystal structures along with
a hydrogen bond constraint between the N(17) nitrogen atom
and the carboxylate group oxygen atoms of Asp3.32. The best
CHEM-PLP-scored solutions were chosen for molecular dynamics
(MD) studies. Gaps in the protein sequence including those due
to extraction of the fusion proteins were modeled and refined
employing MODELLER9v10.58,59 Force field parameter and topol-
ogy files for NNQ were generated utilizing SwissParam.60 Density
functional theory (DFT) calculations at the 6-31G⁄ level were
employed to calculate partial atomic charges of the NNQ atoms
using NWChem 6.0.61 Coordinates for the spatial arrangement of
the receptors within the lipid bilayer were retrieved from the
Orientations of Proteins in Membranes (OPM) database.62 The sim-
ulation system, consisting of the receptor–ligand complex embed-
ded in a lipid (POPC) bilayer surrounded with saline solution
(0.15 M NaCl) was created in VMD 1.9.163 using the CHARMM force
field topology file.64,65 All simulations were performed under
hybrid CHARMM force field parameters that included protein,
494/516 ex/em for a total of 120 s. After 15 s of reading, 20 lL of
varying concentrations of testing compounds in triplicates (ago-
nism study) or 500 nM of DAMGO (NIDA, antagonism study) in
assay buffer, or assay buffer alone, was added to the wells to bring
the total volume up to 100 l
L. The changes in Ca2+ flux were moni-
tored and peak height values were recorded. The obtained values
were then subjected to nonlinear regression analysis to determine
EC50 or IC50 values using Prism 3.0 (GraphPad Software, San Diego,
CA). All experiments were repeated at least three times.
4.2.5. Tail immersion test
The warm-water tail immersion assay was performed according
to Coderre and Rollman55 using a water bath with the temperature
maintained at 56 0.1 °C. Before injecting, the baseline latency
(control) of the mice was determined. Only mice with a reaction
time from 2 to 4 s were used. The average baseline latency for
the experiment was 3.0 0.1 s. For the agonism study, the test
latency after drug treatment was assessed at 20 min (time that
morphine effect starting to peak) following drug administration.
For the antagonism study, NAQ analogues were administered
5 min before morphine injection, and 20 min after morphine treat-
ment the test latency was assessed. A 10 s maximum cutoff time
was imposed to prevent tissue damage. Antinociception was quan-
tified according to the method of Harris and Pierson56 as the per-
centage of maximum possible effect (% MPE), which was
calculated as: % MPE = [(test latency ꢂ control latency)/(10 ꢂ con-
trol latency)] ꢃ 100. Percent MPE was calculated for each mouse
using at least six mice per drug.
lipids and ligand with
a time-step of 2 femtoseconds (fs).
Periodic boundary conditions were employed, and Particle Mesh
Ewald (PME) summation was used to calculate long-range electro-
static interactions. Non-bonded interactions were calculated with
a smooth cutoff between 10 and 12 Å with a frequency of 1 fs.
The temperature was maintained at 310 K via Langevin dynamics.
All molecular modeling simulations were performed using NAMD
2.8.66 MD simulations were carried out in four stages. In the first
stage, equilibration of the fluid-like lipid bilayer was performed
via minimization (1000 iterations) followed by NPT equilibration
(pressure equilibration, 0.5 fs) of the lipid tails only. In the second
stage, an NPT equilibration of the system was run for a period of
1 ns with harmonic constraints placed on protein and NNQ atoms
(5 kcal/(mol-Å)). The harmonic restraint was released in stage 3
and the entire system was equilibrated using the NVT canonical
ensemble for a further 1 ns. The final production run was con-
ducted for 15 ns using an NVT ensemble. Energy landscape analysis
was performed using the NAMD Energy 1.4 plug-in; non-bonded
interaction analyses were performed at various distances with a
dielectric constant of 6.5.67 The best-scored poses based on the
NAMD non-bonded interactions were selected for further analysis.
4.2.6. Opioid withdrawal assays
A 75 mg morphine pellet was implanted into the base of the
neck of male Swiss–Webster mice following the reported
procedure.23 The animals were allowed to recover in their home
cages before testing. Mice were then allowed for 30 min habitua-
tion to an open-topped, square, clear Plexiglas observation cham-
ber (26 ꢃ 26 ꢃ 26 cm3) with lines partitioning the bottom into
quadrants before given antagonist. Withdrawal was precipitated

Y. Yuan et al. / Bioorg. Med. Chem. 23 (2015) 1701–1715
1715
25. Abdelhamid, E. E.; Sultana, M.; Portoghese, P. S.; Takemori, A. E. J. Pharmacol.
Exp. Ther. 1991, 258, 299.
Figures were generated using the PyMOL Molecular Graphics
System, Version 1.5.0.4.
26. Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara,
R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Nature 2012, 485, 321.
27. Wu, H.; Wacker, D.; Mileni, M.; Katritch, V.; Han, G. W.; Vardy, E.; Liu, W.;
Thompson, A. A.; Huang, X.-P.; Carroll, F. I.; Mascarella, S. W.; Westkaemper, R.
B.; Mosier, P. D.; Roth, B. L.; Cherezov, V.; Stevens, R. C. Nature 2012, 485, 327.
28. Granier, S.; Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Weis, W. I.;
Kobilka, B. K. Nature 2012, 485, 400.
29. Zaidi, S. A.; Arnatt, C. K.; He, H.; Selley, D. E.; Mosier, P. D.; Kellogg, G. E.; Zhang,
Y. Bioorg. Med. Chem. 2013, 21, 6405.
30. Craig, P. N. J. Med. Chem. 1971, 14, 680.
31. Gray, A. P.; Platz, R. D.; Henderson, T. R.; Chang, T. C.; Takahashi, K.; Dretchen,
K. L. J. Med. Chem. 1988, 31, 807.
32. Sayre, L. M.; Portoghese, P. S. J. Org. Chem. 1980, 45, 3366.
33. Yuan, Y.; Elbegdorj, O.; Chen, J.; Akubathini, S. K.; Beletskaya, I. O.; Selley, D. E.;
Zhang, Y. Bioorg. Med. Chem. Lett. 2011, 21, 5625.
34. Zhang, Y.; Elbegdorj, O.; Yuan, Y.; Beletskaya, I. O.; Selley, D. E. Bioorg. Med.
Chem. Lett. 2013, 23, 3719.
35. Yuan, Y.; Elbegdorj, O.; Beletskaya, I. O.; Selley, D. E.; Zhang, Y. Bioorg. Med.
Chem. Lett. 2013, 23, 5045.
Acknowledgements
We are grateful to Drs. Lee-Yuan Liu-Chen (Temple University)
and Ping-Yee Law (University of Minnesota) for the generous gift of
mice opioid receptor-expressed CHO cell lines. Y.Y. thanks Orgil
Elbegdorj, Irma B. Adams, Joanna C. Jacob, Christopher K. Arnatt,
and Thomas Raborg for their technical guidance on the radioligand
binding and Ca2+ flux assays. Y.Y. would also like to thank Yunfei
Mao and Brianna Mackie for providing HRMS data. The work was
funded by PHS grants from NIH/NIDA DA024022 (Y.Z.), NIH
Grant R01 DA017204 (P.D.M.), and National Center for Research
Resources Award S10RR027411 (V.C.U.). 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.
36. Yuan, Y.; Elbegdorj, O.; Chen, J.; Akubathini, S. K.; Zhang, F.; Stevens, D. L.;
Beletskaya, I. O.; Scoggins, K. L.; Zhang, Z.; Gerk, P. M.; Selley, D. E.; Akbarali, H.
I.; Dewey, W. L.; Zhang, Y. J. Med. Chem. 2012, 55, 10118.
37. Yuan, Y.; Zaidi, S. A.; Elbegdorj, O.; Aschenbach, L. C. K.; Li, G.; Stevens, D. L.;
Scoggins, K. L.; Dewey, W. L.; Selley, D. E.; Zhang, Y. J. Med. Chem. 2013, 56,
9156.
Supplementary data
38. Selley, D. E.; Sim, L. J.; Xiao, R.; Liu, Q.; Childers, S. R. Mol. Pharmacol. 1997, 51,
87.
39. Spetea, M.; Schuellner, F.; Moisa, R. C.; Berzetei-Gurske, I. P.; Schraml, B.;
Doerfler, C.; Aceto, M. D.; Harris, L. S.; Coop, A.; Schmidhammer, H. J. Med.
Chem. 2004, 47, 3242.
40. Janssen, P. A. J.; Niemegeers, C. J. E.; Dony, J. G. H. Arzneim. Forsch. 1963, 13, 502.
41. Wilson, S. G.; Mogil, J. S. Behav. Brain Res. 2001, 125, 65.
42. Caggiula, A. R.; Epstein, L. H.; Perkins, K. A.; Saylor, S. Psychopharmacology 1995,
122, 301.
Supplementary data (acid side chain synthesis and character-
ization; HPLC, elemental analysis, and NMR spectra of the final
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2015.02.055.
References and notes
43. Logan, C. J.; Hutson, D. H.; Hesk, D. Xenobiotica 1985, 15, 391.
44. Kestell, P.; Pruijn, F. B.; Siim, B. G.; Palmer, B. D.; Wilson, W. R. Cancer
Chemother. Pharmacol. 2000, 46, 365.
45. Sharma, S. K.; Jones, R. M.; Metzger, T. G.; Ferguson, D. M.; Portoghese, P. S. J.
Med. Chem. 2001, 44, 2073.
1. Dhawan, B. N.; Cesselin, F.; Raghubir, R.; Reisine, T.; Bradley, P. B.; Portoghese,
P. S.; Hamon, M. Pharmacol. Rev. 1996, 48, 567.
2. Chakrabarti, S.; Prather, P. L.; Yu, L.; Law, P. Y.; Loh, H. H. J. Neurochem. 1995, 64,
2534.
3. Miyake, M.; Christie, M. J.; North, R. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3419.
4. Ortiz-Miranda, S. I.; Dayanithi, G.; Coccia, V.; Custer, E. E.; Alphandery, S.;
Mazuc, E.; Treistman, S.; Lemos, J. R. J. Neuroendocrinol. 2003, 15, 888.
5. Barchfeld, C. C.; Maasen, Z. F.; Medzihradsky, F. Life Sci. 1982, 31, 1661.
6. Thorsell, A. Alcohol Alcohol 2013, 48, 402.
7. Herz, A. J. Neural Transm. Suppl. 1983, 18, 227.
8. Sadée, W.; Wang, Z. Adv. Exp. Med. Biol. 1995, 373, 85.
9. World Drug Report 2013. Report from the United Nations Office on Drugs and
46. Von Voightlander, P. F.; Lewis, R. A. Prog. Neuro-Psychopharmacol. Biol.
Psychiatry 1982, 6, 467.
47. Calderon, S. N.; Rothman, R. B.; Porreca, F.; Flippen-Anderson, J. L.; McNutt, R.
W.; Xu, H.; Smith, L. E.; Bilsky, E. J.; Davis, P.; Rice, K. C. J. Med. Chem. 1994, 37,
2125.
48. Butelman, E. R.; Yuferov, V.; Kreek, M. J. Trends Neurosci. 2012, 35, 587.
49. Hough, L. B.; Nalwalk, J. W.; Chen, Y.; Schuller, A.; Zhu, Y.; Zhang, J.; Menge, W.
M.; Leurs, R.; Timmerman, H.; Pintar, J. E. Brain Res. 2000, 880, 102.
50. Coward, P.; Chan, S. D.; Wada, H. G.; Humphries, G. M.; Conklin, B. R. Anal.
Biochem. 1999, 270, 242.
51. Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.; Kobilka, B.;
Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos, A.; Sexton, P. M.; Miller,
K. J.; Spedding, M.; Mailman, R. B. J. Pharmacol. Exp. Ther. 2007, 320, 1.
52. Filizola, M.; Devi, L. A. Nature 2012, 485, 314.
53. Ballesteros, J. A.; Weinstein, H. Methods Neurosci. 1995, 25, 366.
54. Yuan, Y.; Arnatt, C. K.; El-Hage, N.; Dever, S. M.; Jacob, J. C.; Selley, D. E.; Hauser,
K. F.; Zhang, Y. Medchemcomm 2013, 4, 847.
55. Coderre, T. J.; Rollman, G. B. Life Sci. 1983, 32, 2139.
56. Harris, L. S.; Pierson, A. K. J. Pharmacol. Exp. Ther. 1964, 143, 141.
57. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
58. Eswar, N.; Eramian, D.; Webb, B.; Shen, M.-Y.; Sali, A. Methods Mol. Biol. 2008,
426, 145.
59. Fiser, A.; Do, R. K. G.; Šali, A. Protein Sci. 2000, 9, 1753.
60. Zoete, V.; Cuendet, M. A.; Grosdidier, A.; Michielin, O. J. Comput. Chem. 2011, 32,
2359.
Crime,
http://www.unodc.org/unodc/secured/wdr/wdr2013/World_Drug_
Report_2013.pdf.
10. White, J. M.; Irvine, R. J. Addiction 1999, 94, 961.
11. Bart, G. J. Addict. Dis. 2012, 31, 207.
12. Stotts, A. L.; Dodrill, C. L.; Kosten, T. R. Expert Opin. Pharmacother. 2009, 10,
1727.
13. Veilleux, J. C.; Colvin, P. J.; Anderson, J.; York, C.; Heinz, A. J. Clin. Psychol. Rev.
2010, 30, 155.
14. Isbell, H.; Vogel, V. H. Am. J. Psychiatry 1949, 105, 909.
15. Minozzi, S.; Amato, L.; Vecci, S.; Davoli, M.; Kirchmayer, U.; Verster, A. Cochrane
Database Syst. Rev. 2011, CD001333.
16. Walsh, S. L.; Preston, K. L.; Stitzer, M. L.; Cone, E. J.; Bigelow, G. E. Clin.
Pharmacol. Ther. 1994, 55, 569.
17. Niciu, M. J.; Arias, A. J. CNS Drugs 2013, 27, 777.
18. Chien, C.-C.; Lee, Y.-J.; Fan, L.-W.; Ho, I.-K.; Tien, L.-T. Toxicol. Lett. 2012, 212, 61.
19. Moynihan, H. A.; Derrick, I.; Broadbear, J. H.; Greedy, B. M.; Aceto, M. D.; Harris,
L. S.; Purington, L. C. S.; Thomas, M. P.; Woods, J. H.; Traynor, J. R.; Husbands, S.
M.; Lewis, J. W. J. Med. Chem. 2012, 55, 9868.
20. McHardy, S. F.; Heck, S. D.; Guediche, S.; Kalman, M.; Allen, M. P.; Tu, M.; Bryce,
D. K.; Schmidt, A. W.; Vanase-Frawley, M.; Callegari, E.; Doran, S.; Grahame, N.
J.; McLean, S.; Liras, S. MedChemComm 2011, 2, 1001.
61. Valiev, M.; Bylaska, E. J.; Govind, N.; Kowalski, K.; Straatsma, T. P.; Van Dam, H.
J. J.; Wang, D.; Nieplocha, J.; Apra, E.; Windus, T. L.; de Jong, W. A. Comput. Phys.
Commun. 2010, 181, 1477.
21. Ziauddeen, H.; Chamberlain, S. R.; Nathan, P. J.; Koch, A.; Maltby, K.; Bush, M.;
Tao, W. X.; Napolitano, A.; Skeggs, A. L.; Brooke, A. C.; Cheke, L.; Clayton, N. S.;
Sadaf Farooqi, I.; O’Rahilly, S.; Waterworth, D.; Song, K.; Hosking, L.; Richards,
D. B.; Fletcher, P. C.; Bullmore, E. T. Mol. Psychiatry 2013, 18, 1287.
22. Li, G.; Aschenbach, L. C.; Chen, J.; Cassidy, M. P.; Stevens, D. L.; Gabra, B. H.;
Selley, D. E.; Dewey, W. L.; Westkaemper, R. B.; Zhang, Y. J. Med. Chem. 2009, 52,
1416.
23. Yuan, Y.; Li, G.; He, H.; Stevens, D. L.; Kozak, P.; Scoggins, K. L.; Mitra, P.; Gerk, P.
M.; Selley, D. E.; Dewey, W. L.; Zhang, Y. ACS Chem. Neurosci. 2011, 2, 346.
24. Altarifi, A. A.; Yuan, Y.; Zhang, Y.; Selley, D. E.; Negus, S. S. Psychopharmacology
2015, 232, 815.
62. Lomize, M. A.; Lomize, A. L.; Pogozheva, I. D.; Mosberg, H. I. Bioinformatics 2006,
22, 623.
63. Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33.
64. Feller, S. E.; Gawrisch, K.; MacKerell, A. D., Jr. J. Am. Chem. Soc. 2002, 124, 318.
65. MacKerell, A. D., Jr.; Feig, M.; Brooks, C. L. III J. Comput. Chem. 2004, 25, 1400.
66. Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorsh id, E.; Villa, E.; Chipot,
C.; Skeel, R. D.; Kalé, L.; Schulten, K. J. Comput. Chem. 2005, 26, 1781.
67. Kukic, P.; Farrell, D.; McIntosh, L. P.; García-Moreno, E. B.; Jensen, K. S.; Toleikis,
Z.; Teilum, K.; Nielsen, J. E. J. Am. Chem. Soc. 2013, 135, 16968.