Novel delta opioid receptor agonists exhibit differential stimulation of signaling pathways

Article abstract of DOI:10.1016/j.bmc.2009.07.007

A novel family of 1,3,5-trisubstituted 1,2,4-triazoles was discovered as potent and selective ligands for the δ opioid receptor by rational design. Compound 5b exhibited low-nanomolar in vitro binding affinity (IC₅₀ = 5.8 nM), excellent selecti

Full text of DOI:10.1016/j.bmc.2009.07.007

Bioorganic & Medicinal Chemistry 17 (2009) 6442–6450
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel delta opioid receptor agonists exhibit differential stimulation
of signaling pathways
Youyi Peng a, , Qiang Zhang a, , Sonia Arora a, Susan M. Keenan a,à, Sandhya Kortagere a,§
,
Kenneth M. Wannemacher b,–, Richard D. Howells b, William J. Welsh a,
*
a Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (UMDNJ-RWJMS) and the Informatics Institute
of UMDNJ, Piscataway, NJ 08854, United States
b Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School (UMDNJ-NJMS), Newark, NJ 07101, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel family of 1,3,5-trisubstituted 1,2,4-triazoles was discovered as potent and selective ligands for
Received 11 May 2009
Revised 26 June 2009
Accepted 3 July 2009
Available online 9 July 2009
the d opioid receptor by rational design. Compound 5b exhibited low-nanomolar in vitro binding affinity
(IC50 = 5.8 nM), excellent selectivity for the d opioid receptor over the alternative
tors, full agonist efficacy in receptor down-regulation and MAP kinase activation assays, and low-efficacy
partial agonist activity in stimulation of GTP S binding. The apparent discrepancy observed in these func-
l and j opioid recep-
c
tional assays may stem from different signaling pathways involved in each case, as found previously for
other G-protein coupled receptors. More biological studies are underway to better understand the differ-
ential stimulation of signaling pathways by these novel compounds.
Keywords:
Delta opioid receptor
Agonists
Differential stimulation
Selectivity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
have been shown to modulate the development of tolerance16,17
and dependence on
l
agonists such as morphine,18 to act as anti-
Opioid receptors belong to class A within the superfamily of G-
protein coupled receptors (GPCRs). It has been well documented
tussive agents19 and to elicit anti-alcoholism20 and anti-obesity20
effects. The d selective opioid ligands thus represent attractive can-
didates for a broad range of pharmaceutical applications.
that at least three distinct opioid receptors (d,
l, and j) exist in
the central nervous system and periphery.1–4 Opioid agonists are
very effective analgesics for acute and postoperative pain through
The interactions between opioid receptors and their ligands have
been described in terms of the ‘message–address’ concept,21,22
illustrated here using the d opioid receptor selective antagonist nal-
trindole,23 agonists SNC80,24 TAN67,25 and SB21982526 (Fig. 1). The
‘message’ (red in Fig. 1) represents structural features involved in
activation of the
been reported with opioid analgesics, including respiratory depres-
sion, physical dependence, constipation, nausea, and vomiting.5
l opioid receptor. Many serious side effects have
A
significant body of evidence has suggested that d selective opioid
binding to all opioid receptors (d, l, and j), while the ‘address’
receptor agonists and antagonists offer great potential as therapeu-
(blue in Fig. 1) refers to specific structural features that confer high
selectivity for a particular opioid receptor subtype (e.g., d).23,26
Lipophilic, sterically bulky d ‘address’ groups, such as diethylcarba-
moyl in SNC80 and SB219825 and phenyl in naltrindole and
TAN67, have been reported to confer d receptor selectivity (Fig. 1).
More recently, Bernard et al.27 developed quantitative pharmaco-
phore models for d opioid ligands that reinforce the importance of
the hydrophobic ‘address’ moiety in conferring selective d receptor
activity.
tic agents devoid of the adverse side effects associated with
l
agonists (e.g., morphine).6–9 d selective agonists have been reported
to produce many pharmacological effects including analgesia,9 car-
dioprotection,10 neuroprotection,11,12 antidepression,13,14 and anti-
Parkinson activities.15 On the other hand, d selective antagonists
* Corresponding author. Tel.: +1 732 235 3234; fax: +1 732 235 3475.
E-mail address: welshwj@umdnj.edu (W.J. Welsh).
In an earlier study,28 we reported a novel family of 3,4,5-trisub-
stituted 1,2,4-triazoles as d selective opioid receptor antagonists
that were discovered by ligand-based drug design approaches in
view of the ‘message–address’ concept (Fig. 1).28–30 In this series,
the 4-tert-butyl was chosen as the hydrophobic ‘address’ to confer
d selectivity. When published, these compounds represented the
first nonpeptidic d receptor selective ligands lacking a highly basic
Present address: Intra-Cellular Therapies, Inc., 3960 Broadway, New York, NY
10032, United States.
à
Present address: School of Biological Sciences, University of Northern Colorado,
Greeley, CO 80639, United States.
§
Present address: Department of Microbiology and Immunology, Drexel University
College of Medicine, Philadelphia, PA 19129, United States.
–
Present address: Department of Medicine, University of Pennsylvania, Philadel-
phia, PA 19104, United States.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.007

Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
6443
O
N
OH
N
N
N
NH
O
O
OH
Naltrindole
SNC80
O
N
N
N
N
N
H
OH
OH
TAN-67
SB219825
R3
N1
R3
N2
N4
N2
N1
N4
R2
R1
R2
R1
3,4,5-trisubstituted 1,2,4-triazole
1,3,5-trisubstituted 1,2,4-triazole
8 R1 = 3-OH, R2 = 4-tert-butyl, R3 = N(Me)2
16 R1 = 3-OH, R2 = 3-tert-butyl, R3 = CH2N(Me)2
17 R1 = 3-OH, R2 = 4-tert-butyl, R3 = CH2N(Me)2
5a R1 = 3-OH, R2 = 3-tert-butyl, R3 = CH2N(Me)2
5b R1 = 3-OH, R2 = 4-tert-butyl, R3 = CH2N(Me)2
5c R1 = 3-OH, R2 = 2,4-(Me)2, R3 = CH2N(Me)2
5d R1 = 3-tert-butyl, R2 = 3-OH, R3 = CH2N(Me)2
5e R1 = 4-tert-butyl, R2 = 3-OH, R3 = CH2N(Me)2
Figure 1. Structures of d selective opioid receptor ligands. Structures of our previous 3,4,5-trisubstituted triazoles are exemplified by compounds (8, 16, 17) mentioned in this
article.
N atom. The most potent compound (8 in Fig. 1) described in this
previous study28 exhibited d binding affinity of 50 nM and moder-
in 2.0 M HCl at 5 °C gave diazonium salts, which were reacted with
ethyl-2-chloro-3-oxobutanoate to yield intermediates 2. Amina-
tion of 2 with ammonia produced amidrazones in 100% yield typ-
ically within 2 h. The 1,2,4-triazole core ring was then closed by
refluxing of amidrazones with acyl chlorides in toluene to give
the key intermediates 3.33 The ethyl esters in 3 were converted
to alcohols using LiAlH4 as reducing reagent, which were further
converted to iodide by Mitsonobu reaction.34 The iodides, without
isolation, were substituted by N,N-dimethylamine in refluxing
chloroform to produce 4. Yields of 4 from 3 were excellent (80–
90%). Compounds 4a–4e were then converted to phenols 5a–5e
by BBr3 with yields greater than 95%.
ate selectivity for d over the
ratio: d/ = 80; d/ >200).
l and j opioid receptors (selectivity
l
j
Our next aim was to improve the binding affinity and selectivity
for the d opioid receptor. Guided by computational molecular mod-
eling, we reconfigured the substitution pattern around the triazole
ring to yield the 1,3,5-trisubstituted 1,2,4-triazole series (Fig. 1).
The majority of known potent opioids contain a basic N atom
which would be expected to exist predominantly in its protonated
form at physiological conditions (pH 7.4); this protonated N atom
has been suggested to form a salt bridge with a highly conserved
Asp residue in TM3 of opioid receptors.31,32 We incorporated this
feature by judiciously adding a basic amino group (–CH2N(Me)2)
on the core triazole ring of the 1,2,4-triazole compounds. A limited
series of 10 compounds (4a–4e and 5a–5e in Scheme 1) was then
synthesized to establish proof of concept. Here we report
the chemical synthesis and initial biological testing of this series
of 1,3,5-substituted 1,2,4-triazoles as d selective opioid receptor
ligands.
3. Results and discussion
Structural alignment between our triazole-based compounds
with known d opioid ligands SNC80 and naltrindole was performed
to explore their degree of overlap in three-dimensional space
(Fig. 2). For clear comparison, compounds 1,3,5-substituted 5b
and 3,4,5-substituted 17, which have identical substitutions at
R1, R2, and R3, are both shown in Fig. 2. In general, compound 5b
aligns very well with both SNC80 and naltrindole in terms of the
‘message’ (the basic N atom and the phenolic moiety) and the ‘ad-
dress’ (respectively, tert-butyl vs diethylcarbamoyl; and tert-butyl
vs phenyl). In contrast, the ‘address’ group (tert-butyl) of
2. Chemistry
The synthesis of 1,3,5-substituted triazoles 4a–4e and 5a–5e
(Table 1) is depicted in Scheme 1. Reaction of amines 1 with NaNO2

6444
Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
O
CO2C2H5
3. NH3(gas)
1. NaNO2, HCl
0-5oC
H
NH2
R2
N N
OC2H5
N
Cl
N
N
2. CH3COCHClCO2C2H5
rt
4. R2C6H4COCl
toluene,reflux
R1
R1
3a R1 = 3-OCH3; R2 = 3-tert-butyl
3b R1 = 3-OCH3; R2 = 4-tert-butyl
3c R1 = 3-OCH3; R2 = 2,4-(Me)2
3d R1 = 3-tert-butyl; R2 = 3-OCH3
3e R1 = 4-tert-butyl; R2 = 3-OCH3
R1
1
2
3a-3e
R2
R2
N
N
N
N
5. LiAlH4, THF
N
N
8. BBr3
N
N
5a R1 = 3-OH; R2 = 3-tert-butyl
5b R1 = 3-OH; R2 = 4-tert-butyl
5c R1 = 3-OH; R2 = 2,4-(Me)2
5d R1 = 3-tert-butyl; R2 = 3-OH
5e R1 = 4-tert-butyl; R2 = 3-OH
6. Ph3P, C H 3I, DEAD
R1
R1
7. N(C2H5)2
reflux
4a-4e
5a-5e
R1 and R2 are the same as in 3a-3e
Scheme 1.
velop lined by the side-chains of residues of Phe202 (EL2); Val217
(TM5); and Phe218 (TM5); Val281 (TM6), Trp284 (TM6) and
Thr285 (TM6); and Leu300 (TM7). The N4 atom (Fig. 3) in the tri-
azole ring is located near the side-chain hydroxyl group of Tyr129
(dNꢀ ꢀ ꢀO = 4.52 Å) in TM3, suggesting a potential hydrogen bond or
an electrostatic interaction.
These same ligand–receptor interactions of 5b, viz., the salt
bridge, the hydrogen bonds and the hydrophobic interaction, are
well conserved in the binding of known d receptor selective opioid
agonists. Ala221 but not His278 has been reported to form a hydro-
gen bond with the tyramine hydroxyl of nonpeptidic opioid ago-
nists,35 which is the case for our compounds. Some of the
residues (Tyr129, Phe218, Val281, Trp284, and Leu300) in contact
with 5b have been reported using mutagenesis studies to play
important roles in the binding of d selective opioid agonists
(BW373U86, DPDPE, SNC80, and TAN67).37–39 The similarity in li-
gand–receptor interactions between the subject compounds and
known d opioids offers compelling structural evidence of their po-
tential binding affinity to the d opioid receptor.
Table 1
Binding affinity of the subject compounds to the opioid receptors
Compd
IC50 (nM) SEM
Selectivity
da
lb
jc
l
/d
j/d
4a
5a
4b
5b
4c
5c
4d
5d
2195 354
48.9 8.3
2307 393
5.8 1.2
>10,000
273 82
1139 311
28.8 3.3
>10,000
121 6.1
2600
>10,000
2447 661
>10,000
>10,000
>10,000
2435 372
>10,000
245 20
>10,000
>10,000
>10,000
>10,000
6066 478
>10,000
341 20
>10,000
3851 311
>10,000
351 95
>10,000
46.3 8.2
>10,000
760 127
>10,000
>10,000
3601 523
>4.6
50
>4.6
7.0
>4.3
>1724
N/A
8.9
>4.3
664
N/A
1.3
>8.8
8.5
>8.8
1.6
4e
5e
N/A
>80
N/A
6.1
16d
17d
SNC80
>3.9
>21.6
7583
>3.9
>21.6
4501
460
0.8 0.1
a
IC50 for inhibition of [3H]DPDPE binding to HEK 293 cells expressing the murine
d receptor.
b
IC50 for inhibition of [3H]DAMGO binding to HEK 293 cells expressing the
murine receptor.
IC50 for inhibition of [3H]U69593 binding to HEK 293 cells expressing the rat
receptor.
l
c
Competitive binding assays were conducted against the three
j
opioid receptors d,
l, and j (Table 1). The results revealed that
d
Ki values using [3H]bremazocine as the radiolabeled ligand, Ref. 28.
structural modification from 3,4,5-substituted 1,2,4-triazoles to
1,3,5-substituted 1,2,4-triazoles significantly improves the binding
affinity and selectivity for the d opioid receptor, which supports the
molecular modeling studies and our intuitive deductions. For
example, 5a and 5b in the present 1,3,5-substituted series demon-
strated binding affinities of 48.9 and 5.8 nM for the d receptor. In
comparison, their corresponding compounds (16 and 17 in Fig. 1)
from our previously reported 3,4,5-substituted series exhibited
weak d binding affinity (Ki = 2600 and 460 nM, respectively, Table
1).28 For all compounds in this series, the presence of a hydroxyl
(e.g., 5a–5c) rather than a methoxyl (e.g., 4a–4c) at R1 dramatically
enhanced the binding affinity for the d opioid receptor, which is
consistent with the structure–activity relationship (SAR) of mor-
phine-like opioids40–43 and SNC80 derivatives.44 Likewise, the
presence of the sterically bulky tert-butyl group (5a and 5b) rather
than methyl (5c) at R2 greatly improved the binding affinity and
selectivity for the d opioid receptor, in agreement with the
‘address’ requirement for lipophilic, sterically bulky groups at the
position. Shifting the tert-butyl group at R2 from the meta position
to para position on the phenyl ring improved both the binding
affinity and selectivity for the d opioid receptor. Interchanging
the points of attachment of the phenolic and 4-tert-butylphenyl
compound 17 deviates somewhat from the orientation of the
diethylcarbamoyl in SNC80 and the phenyl in naltrindole. Clearly,
reconfiguring the substitution pattern on the core triazole ring im-
proves the pharmacophore alignment with known opioids of the
1,3,5-substituted compounds compared with the 3,4,5-substituted
compounds. In addition, one of the triazole N atoms (N4 in Fig. 2)
appears to correspond to the C-14 hydroxyl of naltrindole, which
has been proposed to hydrogen bond with the side chain of
Tyr129.35 This high degree of structural alignment of the subject
compounds with naltrindole and SNC80 strengthened our confi-
dence in their likelihood as d opioid receptor ligands.
Docking of 5b to a homology model of the d opioid receptor
based on the X-ray crystal structure of bovine rhodopsin36 revealed
that the basic N atom is well positioned to form a salt bridge with
the side-chain carboxylate group of Asp128 in TM3 (Fig. 3). Like-
wise, the 3-hydroxyl at R1 of 5b is situated within hydrogen bond-
0
ing distance (dO–O = 3.31 ÅA) of the backbone carbonyl O atom of
Ala221 in TM5. The 4-tert-butyl phenyl moiety, that is, the delta
‘address’ in this series, is appropriately buried in a hydrophobic en-

Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
6445
Figure 2. Structural alignment of 5b (green) and 17 (brown) with SNC80 (purple) and naltrindole (cyan). The compounds are rendered in ball-and-stick mode. Nitrogen: blue;
oxygen: red; carbon: green (5b), purple (SNC80), cyan (naltrindole) and brown (17). The N4 atom of 5b is labeled in purple text. The phenolic moiety and ‘address’ are
indicated with red and purple ovals, respectively.
Compound 5b was submitted to the NIMH Psychoactive Drug
Screening Program (PDSP)45 to screen against a panel of G-protein
coupled receptors and transporters. The screening results (Supple-
mentary data, Table 1) revealed that compound 5b is highly
selective for the d opioid receptor over 5HT receptors, adrenergic
receptors, dopamine receptors, histamine receptors, muscarinic
subtype receptors, and transporters of DAT, NET and SERT.
The functional activities of these novel compounds at the opioid
receptors were determined by ligand-induced receptor down-reg-
ulation (Table 2), stimulation of GTPcS binding (Table 2), and by
phospho-MAP kinase assays (for SNC80 and 5b, Fig. 5). In the
receptor down-regulation assays, incubation of HEK293 cells
expressing the d opioid receptor with 5a–5e produced a decrease
in receptor binding activity, due to proteolysis of the receptor.46
The level of down-regulation for 5b, 5d, and 5e was nearly equal
to SNC80, suggesting that the subject compounds at 10
lM act as
full d agonists in this assay. In the GTP S binding assay, however,
c
the level of stimulation was much less for 5b, 5d, and 5e than for
SNC80. Even 5b, the most potent compound in this series, was at
least threefold less stimulatory than SNC80 when both were as-
sayed at 10 lM. Consequently, results from the GTPcS binding as-
say indicate that the subject compounds act as low-efficacy partial
opioid agonists at this concentration. To further explore the func-
tional efficacy of 5b, its activity was compared with SNC80 for ago-
nist-induced phosphorylation and activation of MAP kinase.47 In
this assay, like the agonist-induced down-regulation assay, 5b
was equally efficacious as SNC80 for activation of MAP kinase
(Fig. 5).
Figure 3. Docked conformation of compound 5b in the active site of the d opioid
receptor structural model. Compound 5b is rendered in ball-and-stick mode and
colored by atom-type (C: green; N: blue; O: red; H: pale blue). The opioid receptor
is rendered as a line ribbon and colored cyan. Residues in contact with 5b are
rendered in stick mode and colored by Sybyl v7.2 atom-type. Hydrogen bonds are
depicted as yellow dotted lines. The N4 atom of 5b is labeled in purple text. To
obtain further experimental confirmation of the structures of these ligands, X-ray
crystallographic analysis was conducted on compounds 5b and 5e in this series
(Fig. 4). (CCDC 728550 (5b) and CCDC 728551 (5e) contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif.)
The apparent discrepancy between the receptor down-regula-
tion, GTPcS binding, and phospho-MAP kinase assays for 5b may
stem from different signaling pathways involved in each case. This
phenomenon has also been observed for other GPCRs, for example,
serotonin 5HT receptors,48 adrenergic receptors,49 and angiotensin
receptors.50 It has been reported that activation of GPCRs can in-
duce differential cellular signaling patterns by both G-protein
dependent and G-protein independent mechanisms, such as
through arrestin.49,51–54 Thus, a molecule classified as an agonist
based on one functional assay may behave differently in another
assay. For example, a well-established b2 adrenergic receptor in-
verse agonist (ICI118551) for adenylyl cyclase stimulated the acti-
vation of ERK that depends on b-arrestin-2 expression.49 Similarly,
groups on the triazole ring (5a vs 5d; 5b vs 5e) altered both the
binding affinity and selectivity for the d receptor. Although the
small number of examples in this study precludes us from discern-
ing a clear SAR, these encouraging results have motivated the syn-
thesis and biological evaluation of an expanded series of analogs
for further structural optimization of these triazole-based d selec-
tive opioids.

6446
Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
Figure 4. X-ray crystal structures of compounds 5b and 5e. Atoms are depicted as 50% thermal probability ellipsoids.
Table 2
by rational design. Among the compounds tested thus far, 5b
exhibited potent binding affinity of 5.8 nM and excellent selectiv-
Functional activity ( SEM) of the subject compounds at 10
receptor
lM for the d opioid
ity for the d opioid receptor (selectivity ratio: d/
l >1712; d/
Compd
Down-regulation control%
GTPcS control%
j
= 664). Screening assays performed by PDSP reveal that 5b
exhibits high selectivity for the d opioid receptor over a panel of
GPCRs and transporters. The subject compounds exhibited differ-
ential agonistic efficacy in different functional assays: receptor
Control
SNC80
Naloxone
5a
5b
5c
100.0 2.9
16.0 2.3
103.3 1.8
70.0 5.8
15.0 2.9
82.0 5.8
20.0 2.9
19.2 2.6
101.7 2.2
824.3 31.3
101.0 2.6
120.0 7.5
251.0 27.7
125.7 8.8
210.7 24.3
183.7 10.9
down-regulation, stimulation of GTPcS binding, and MAP kinase
activation. This discrepancy between different functional assays
has been observed for the d receptor and other GPCRs like seroto-
nin, angiotensin, and adrenergic receptors. It has been suggested
that different signaling pathways are involved in each functional
assay. Currently, more biological studies are underway to better
understand the mechanism of differential stimulation by these no-
vel compounds. A broader range of analogs will be synthesized to
more fully explore the structure–activity relationships of this ser-
ies of compounds. This task will be facilitated by an integrated ap-
proach that encompasses both ligand-based and structure-based
drug design methods. Structural models for the d opioid receptor
will be constructed using homology modeling approaches based
on the recently published X-ray crystal structures of several
GPCRs.49,56–60
5d
5e
Wei et al.50 have shown that a modified angiotensin peptide could
stimulate arrestin-dependent activation of ERK1/2 but not activa-
tion of G-protein. More recently, it was reported that the d receptor
agonists DPDPE and TIPP inhibited adenylyl cyclase activity and
activated MAP kinase; however, only DPDPE, but not TIPP or mor-
phine, induced desensitization and internalization of the d recep-
tor.55 These preliminary results for our novel compounds provide
another example that agonist efficacy is dependent on the cellular
signaling pathway. However, the underlying mechanism for the
differential stimulation remains to be determined although this
phenomenon has been observed for a variety of GPCRs. Currently,
more biological studies are underway in our laboratory to better
understand signaling mechanisms of the subject compounds.
5. Experimental
5.1. General information
4. Conclusion
1H NMR spectra were recorded at 400 MHz on Varian Gemini-
400 spectrometer, in deuterated chloroform (CDCl3) or DMSO
(DMSO-d6) solution at room temperature, using TMS (0.00 ppm)
We report here a novel family of 1,3,5-trisubstituted 1,2,4-tria-
zoles as potent and selective d opioid receptor ligands discovered
Figure 5. HEK293 cells expressing the d receptor were treated with saline (control), SNC80 (10 lM)) or 5b (10 lM) for 10 min. Whole cell lysates were prepared and analyzed
by western blot analysis using antibodies selective for phosphorylated MAP kinase or total (phosphorylated and nonphosphorylated) MAP kinase. Statistical analysis was
performed using one-way ANOVA (**, p<0.01; ***, p<0.001) Newman–Keuls multiple comparison test (n = 5).

Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
6447
as internal standards and were reported in parts per million (ppm).
5.5. Ethyl 5-(3-tert-butylphenyl)-1-(3-methoxyphenyl)-1H-
13C NMR spectra were recorded on a Varian Gemini 100 MHz NMR
spectrometer at room temperature in CDCl3 and were internally
referenced to CDCl3 (77.23 ppm). Abbreviations for signal coupling
are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; w, wide. Coupling constants, J, are reported (Hz). Analytical
thin layer chromatography (TLC) was performed on pre-coated
1,2,4-trazole-3-carboxylate (3a)
The crystal of 2a 668 mg (2.6 mmol) was dissolved in 10 mL
THF, and ammonia gas was bubbled through the solution at room
temperature. Two hours later, TLC indicated all 2a had been con-
verted to amidrazone. The solvent and excess ammonia were evap-
orated, the residue was dissolved in 15 mL chloroform, and the
clear solution was cooled to 0–5 °C. 3-tert-Butylbenzoyl chloride
510 mg (2.6 mmol) was dissolved in 5 mL chloroform and added
dropwise carefully into the amidrazone solution. The mixture
was refluxed overnight. After cooling to room temperature, 5 mL
triethylamine was added and the solution was extracted by water
(3 ꢁ 10 mL). The organic layer was dried over MgSO4, and the
product was purified by chromatography using EtOAc and hexane
as the eluent to give 886 mg (90%) of 3a as colorless liquid. 1H NMR
(400 MHz, CDCl3): d 1.16 (s, 9H), 1.44(t, J = 1.9 Hz, 3H), 3.78 (s, 3H),
4.52 (q, J = 2.9 Hz, 2H), 6.91 (d, J = 6.9 Hz, 2H), 6.96 (d, J = 7.3 Hz,
1H),7.24 (s, 1H), 7.27 (d, J = 7.2 Hz, 2H), 7.40–7.44 (m, 2H).
plastic backed plates purchased from Aldrich (Silica Gel 60 F254
;
0.25 mm thickness). Flash column chromatography was conducted
with Silica Gel 60 (230–400 mesh) from Natland Co. HPLC was per-
formed using the Waters ACQUITYTM HPLC system, which was
equipped with a Synergi 4u Fusion-RP80A column (2.0 mm ꢁ
50 mm) and a PDA detector. All samples were run under gradient
conditions with CH3CN/H2O modified with 0.1% formic acid. All
samples had purity >95% under 254 nm. MS and MS–MS were con-
ducted on a Finnigan LCQ DUO Mass Spectrum (Thermo Quest Co.)
Gas chromatographic analyses were performed on a Hewlett–
Packard 6890 GC–MS instrument with a FID detector using
25 m ꢁ 0.20 mm capillary column with cross-linked methylsilox-
ane as a stationary phase. Melting points were taken on a Mel-
temp melting point apparatus in open capillary tubes without
calibration.
5.6. Ethyl 5-(4-tert-butylphenyl)-1-(3-methoxyphenyl)-1H-
1,2,4-trazole-3-carboxylate (3b)
All reactions were carried out with anhydrous solvents in oven-
dried and argon-charged glassware. All anhydrous solvents except
as mentioned were freshly distilled and stored in 4 Å molecular
sieves. All solvents used in workup, extraction procedures, re-crys-
tallization process, and chromatography were used as received
from commercial supplier without further purification. All re-
agents were purchased from Aldrich Chemical Company.
Prepared as 3a, yield was 93%. 1H NMR (400 MHz, CDCl3): d 1.17
(s, 9H), 1.43(t, J = 2.3 Hz, 3H), 3.74 (s, 3H), 4.48 (q, J1 = 2.7 Hz,
J2 = 2.0 Hz, 2H), 6.86 (m, 3H), 7.22 (s, 1H), 7.32 (d, J = 6.9 Hz, 2H),
7.96 (d, J = 7.6 Hz, 2H).
5.7. Ethyl 5-(2,4-dimethylphenyl)-1-(3-methoxyphenyl)-1H-
1,2,4-trazole-3-carboxylate (3c)
5.2. Ethyl 2-chloro-2-(2-(3-methoxyphenyl)hydrazono)acetate
(2a)
Prepared as 3a, yield was 87%. 1H NMR (400 MHz, CDCl3): d 1.22
(s, 9H), 1.56 (t, J = 6.4 Hz, 3H), 2.01 (s, 3H), 2.32 (s, 3H), 3.67 (s, 3H),
4.48 (q, J1 = 5.9 Hz, 2H), 6.82 (d, J = 5.9 Hz, 2H), 7.01 (s, 1H), 7.16–
7.20 (m, 2H), 7.51 (d, J = 5.1 Hz, 1H), 7.68 (d, J = 3.7 Hz, 1H).
The 3-methoxylaniline 2.46 g (20.0 mmol) was dissolved in
50 mL 2.0 N hydrochloride to give a clear solution. The solution
was cooled to 5 °C and NaNO2 1.38 g (20.0 mmol) which was
dissolved in 5 mL water was added dropwise. The temperature
was kept <5 °C. Thirty minutes later, the solution was cooled
to <1 °C and ethyl 2-chloro-3-oxobutanoate 3.28 g (20.0 mmol)
was added dropwise multiple portions. The solution was stirred
overnight at room temperature. Chloroform 30 mL was added
and the organic layer was dried over MgSO4. After concentrating,
the product was purified by chromatography using EtOAc and
hexane as eluent to obtain 3.53 g (69%) 2a as colorless liquid
which became a solid while standing. 1H NMR (400 MHz, CDCl3):
d 1.39 (t, J = 5.5 Hz, 3H), 3.82 (s, 3H), 4.37 (dd, J = 5.5 Hz, 2H),
6.59 (d, J = 5.7 Hz, 1H), 6.76 (d, J = 6.3 Hz, 1H), 6.83 (s, 1H),
7.19–7.25 (m, 1H), 8.32 (s, 1H). 13C NMR (100 MHz, CDCl3): d
14.2, 55.3, 62.8, 100.4, 107.0, 108.7, 116.1, 130.3, 142.8, 160.8,
197.9.
5.8. Ethyl 1-(3-tert-butylphenyl)-5-(3-methoxyphenyl)-1H-
1,2,4-trazole-3-carboxylate (3d)
Prepared as 3a, yield was 89%. 1H NMR (400 MHz, CDCl3): d 1.32
(s, 9H), 1.46 (t, J = 7.2 Hz, 3H), 3.67 (s, 3H), 4.53 (q, J1 = 7.1 Hz, 2H),
6.92 (d, J = 4.7 Hz, 1H), 6.99–7.04 (m, 3H), 7.20–7.22 (m, 2H), 7.32
(s, 1H), 7.45 (d, J = 4.1 Hz, 1H).
5.9. Ethyl 1-(4-tert-butylphenyl)-5-(3-methoxyphenyl)-1H-
1,2,4-trazole-3-carboxylate (3e)
Prepared as 3a, yield was 92%. 1H NMR (400 MHz, CDCl3): d 1.31
(s, 9H), 1.44 (t, J = 6.9 Hz, 3H), 3.71 (s, 3H), 4.49 (q, J1 = 6.9 Hz, 2H),
6.92 (d, J = 4.7 Hz, 1H), 7.10 (d, J = 7.8 Hz, 2H),7.22 (d, J = 7.4 Hz,
1H), 7.32 (d, J = 4.6 Hz, 2H),7.45 (d, J = 4.6 Hz, 2H).
5.3. Ethyl 2-(2-(3-tert-butylphenyl)hydrazono)-2-chloroacetate
(2b)
5.10. (5-(3-tert-Butylphenyl)-1-(3-methoxyphenyl)-1H-1,2,4-
triazol-3-yl)-N,N-dimethylmethanamine (4a)
Prepared as 2a to get 3.61 g (72%) as yellow solid. 1H NMR
(400 MHz, CDCl3): d 1.31 (s, 9H), 1.41(t, J = 4.9 Hz, 3H), 4.39 (dd,
J = 7.2 Hz, 2H), 7.15 (d, J = 5.9 Hz, 2H), 7.20 (s, 1H), 7.24 (t,
J = 6.1 Hz, 1H), 8.35 (s, 1H).
The ethyl ester 3a 177 mg (0.46 mmol) was dissolved in 4 mL
anhydrous ether and LiAlH4 41 mg (1.05 mmol) was added in small
portions. The mixture was stirred for 1 h and ethyl acetate was
added dropwise to quench reaction. Two milliliters of water and
10 mL chloroform were added and organic layer was dried and
concentrated to yield the alcohol with 100% yield. The alcohol
146 mg (0.43 mmol) was dissolved in 4 mL THF and 143 mg CH3I
(1.0 mmol), and 174 mg PPh3 (0.64 mmol) were added in sequence.
The solution was then cooled to 0 °C. Diethyl diazocarboxylate
(DEAD) 113 mg (0.64 mmol) was slowly added dropwise into the
solution above, which was then stirred at room temperature. TLC
5.4. Ethyl 2-(2-(4-tert-butylphenyl)hydrazono)-2-chloroacetate
(2c)
Prepared as 2a to get 3.97 g (76%) as yellow solid. 1H NMR
(400 MHz, CDCl3): d 1.29 (s, 9H), 1.41(t, J = 7.2 Hz, 3H), 4.38 (dd,
J = 7.2 Hz, 2H), 7.16 (d, J = 5.9 Hz, 2H), 7.36 (d, J = 4.5 Hz, 2H),
8.31 (s, 1H).

6448
Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
indicated that the reaction finished within 4 h. Diethylamine
(2.0 M in THF) 6.0 mL was introduced to above solution, and the
solution was heated to reflux overnight. Solvent and excess dieth-
ylamine were removed and residual was purified by chromatogra-
phy using chloroform and methanol as eluent to give 167 mg 4a as
white solid. 1H NMR (400 MHz, CDCl3): d 1.12 (s, 9H), 2.43 (s, 6H),
3.57 (s, 2H), 3.72 (s, 3H), 6.85–7.18 (m, 3H), 7.21 (d, J = 5.1 Hz, 1H),
7.26 (m, 2H), 7.35 (d, J = 4.9 Hz, 1H), 7.49 (m, 1H). MS (ESI): 365.5
(M++1).
3.60 (s, 2H), 6.27 (s, 1H), 6.82 (d, J = 5.9 Hz, 1H), 6.87 (d,
J = 5.0 Hz, 1H), 7.21–7.25 (m, 6H). 13C NMR (100 MHz, CDCl3): d
31.1, 34.8, 44.9, 55.6, 112.8, 116.0, 116.9, 124.4, 125.5, 128.5,
130.3, 138.5, 153.3, 154.4, 158.0, 159.4. MS (ESI): 351.3 (M++1).
5.17. 3-(3-((Dimethylamino)methyl)-5-(2,4-dimethylphenyl)-
1H-1,2,4-triazol-1-yl)phenol (5c)
Prepared as 5a, white solid, yield was 71% from 4c. Mp: 167–
169 °C. 1H NMR (400 MHz, CDCl3): d 1.96 (s, 3H), 2.28 (s, 3H),
2.40 (s, 6H), 3.72 (s, 2H), 6.54 (s, 1H), 6.63 (d, J = 7.9 Hz, 1H), 6.72
(d, J = 6.7 Hz, 1H), 7.01 (t, J = 7.1 Hz, 2H) 7.03–7.24 (m, 3H). 13C
NMR (100 MHz, CDCl3): d 19.6, 21.3, 44.6, 55.2, 111.1, 114.8,
115.9, 125.2, 126.7, 129.9, 130.0, 131.4, 137.0, 138.3, 140.2,
157.3158.5, 161.9. MS (ESI): 323.4 (M++1).
5.11. (5-(4-tert-Butylphenyl)-1-(3-methoxyphenyl)-1H-1,2,4-
triazol-3-yl)-N,N-dimethylmethanamine (4b)
Prepared as 4a, white solid, yield was 87% from 3b. 1H NMR
(400 MHz, CDCl3): d 1.28 (s, 9H), 2.41 (s, 6H), 3.67 (s, 2H), 3.74
(s, 3H), 6.92 (d, J = 5.9, 1H), 6.95 (s, 1H), 7.25–7.30 (m, 2H), 7.31
(d, J = 8.9 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H). MS (ESI): 365.5 (M++1).
5.18. 3-(2-(3-tert-Butylphenyl)-5-((dimethylamino)methyl)-
2H-1,2,4-triazol-3-yl)phenol (5d)
5.12. (5-(2,4-Dimethylphenyl)-1-(3-methoxyphenyl)-1H-1,2,4-
triazol-3-yl)-N,N-dimethylmethanamine (4c)
Prepared as 5d, white solid, yield was 78% from 4d. Mp: 177–
178 °C. 1H NMR (400 MHz, CDCl3): d 1.19 (s, 9H), 2.45 (s, 6H),
3.79 (s, 2H), 6.82 (s, 1H), 6.85 (d, J = 6.0 Hz, 2H), 7.10–1.15 (m,
2H), 7.20 (s, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.37 (d, J = 6.7 Hz, 1H).
13C NMR (100 MHz, CDCl3): d 31.0, 34.7, 44.8, 55.3, 95.4, 116.0,
117.9, 120.3, 121.9, 122.3, 125.6, 128.5, 128.9, 129.7, 137.4,
152.7, 153.5, 156.9. MS (ESI): 351.3 (M++1).
Prepared as 4a, white solid, yield was 89% from 3c. 1H NMR
(400 MHz, CDCl3): d 2.01 (s, 3H), 2.31 (s, 3H), 2.42 (s, 6H), 3.64
(s, 3H), 3.72 (s, 2H), 6.77–6.82 (m, 2H), 6.98 (d, J = 5.6 Hz, 1H),
7.12–7.17 (m, 3H). MS (ESI): 337.3 (M++1).
5.13. (1-(3-tert-Butylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-
triazol-3-yl)-N,N-dimethylmethanamine (4d)
5.19. 3-(2-(4-tert-Butylphenyl)-5-((dimethylamino)methyl)-
2H-1,2,4-triazol-3-yl)phenol (5e)
Prepared as 4a, white solid, yield was 81% from 3d. 1H NMR
(400 MHz, CDCl3): d 1.22 (s, 9H), 2.43 (s, 6H), 3.66 (s, 3H), 3.70
(s, 2H), 6.90 (d, J = 6.5 Hz, 1H), 7.02 (s, 1H), 7.16–7.22 (m, 2H),
7.32 (s, 1H), 7.32–7.42 (m, 3H). MS (ESI): 365.3 (M++1).
Prepared as 5a, white solid, yield was 72% from 4e. Mp: 153–
155 °C. 1H NMR (400 MHz, CDCl3): d 1.29 (s, 9H), 2.37 (s, 6H),
3.70 (s, 2H), 6.72 (s, 1H), 6.84 (d, J = 4.7 Hz, 1H), 6.90 (d,
J = 5.1 Hz, 1H), 7.09–7.16 (m, 3H).7.34 (d, J = 4.5 Hz, 2H). 13C NMR
(100 MHz, CDCl3): d 31.2, 34.7, 44.9, 55.4, 115.9, 118.1, 124.5,
126.2, 128.3, 129.8, 135.2, 152.0, 154.4, 157.1, 159.3. MS (ESI):
351.3 (M++1).
5.14. (1-(4-tert-Butylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-
triazol-3-yl)-N,N-dimethylmethanamine (4e)
Prepared as 4a, white solid, yield was 81% from 3e. 1H NMR
(400 MHz, CDCl3): d 1.32 (s, 9H), 2.43 (s, 6H), 3.66 (s, 3H), 3.69
(s, 2H), 6.91 (d, J = 6.5 Hz, 1H), 7.04–7.09 (m, 1H), 7.09 (s, 1H),
7.19 (d, J = 8.9 Hz, 1H), 7.27 (d, J = 10.2 Hz, 2H), 7.41 (d, J = 9.8 Hz,
2H). MS (ESI): 365.3 (M++1).
5.20. Receptor binding assay
Cell membrane preparations were obtained from HEK293-sta-
ble transfectants expressing either the murine d receptor,47
l
receptor,46 or the rat
j
receptor61 by ultracentrifugation. Opioid
5.15. 3-(5-(3-tert-Butylphenyl)-3-((dimethylamino)methyl)-
1H-1,2,4-triazol-1-yl)phenol (5a)
receptor binding assays were conducted in duplicate at least three
times on membrane preparations that had been re-suspended in
50 mM Tris–HCl, pH 7.5, utilizing [3H]DPDPE, [3H]DAMGO and
36.5 mg 4a (0.1 mmol) was dissolved in 5 mL dichloromethane
and 5.0 equiv of BBr3 was added dropwise at ꢂ78 °C. The solution
was stirred at room temperature; TLC indicated the reaction was
finished within 5 h. Ten milliliters of chloroform and 10 mL water
were added in sequence and mixture was extracted by water
(3 ꢁ 5 mL). The organic layer was dried over MgSO4 and concen-
trated. The final product 5a was purified by chromatography using
chloroform and methanol as the eluent to obtain 130 mg 5a (78%)
as white solid. Mp: 192–194 °C. 1H NMR (400 MHz, CDCl3): d 1.11
(s, 9H), 2.13 (s, 6H), 3.57 (s, 2H), 6.16 (s, 1H), 6.85 (d, J = 6.9 Hz, 1H),
6.92 (d, J = 7.1 Hz, 1H), 7.14(d, J = 5.6 Hz, 1H), 7.16 (d, J = 4.9 Hz,
1H), 7.25–7.30 (m, 4H). 13C NMR (100 MHz, CDCl3): d 30.9, 34.5,
44.7, 55.5, 112.6, 116.0, 116.8, 125.9, 126.0, 126.8, 127.1, 128.2,
130.3, 138.5, 151.5, 154.8, 158.0, 159.2. MS (ESI): 351.3 (M++1).
[3H]U69593 as radioligand for d,
tively. Cyclazocine (10
l
, and
j opioid receptors, respec-
lM) was used to define nonspecific binding.
Following 1-h incubation on ice, binding assays were terminated
by filtration through Whatman GF/B filters. Filters were soaked
in Ecoscint liquid scintillation solution (National Diagnostics, Man-
ville, NJ) and filter-bound radioactivity was measured using a Pack-
ard Tri-Carb 2100 TR liquid scintillation analyzer. Receptor binding
data were analyzed by nonlinear regression of saturation and com-
petition curves using Prism 3.0 software (GraphPad Software, San
Diego, CA). Protein concentrations were determined with the Bio-
Rad Bradford protein assay using bovine serum albumin as the
standard.
5.21. Receptor down-regulation assays
5.16. 3-(5-(4-tert-butylphenyl)-3-((dimethylamino)methyl)-
1H-1,2,4-triazol-1-yl)phenol (5b)
HEK293 cells expressing the FLAG-tagged d receptor were
grown to near confluence in 100 mm dishes in DMEM media sup-
plemented with 10% fetal calf serum and 0.25 mg/ml G418. Media
was replaced with serum-free media prior to overnight incubation
Prepared as 5a, white solid, yield was 82% from 4b. Mp: 191–
193 °C. 1H NMR (400 MHz, CDCl3): d 1.25 (s, 9H), 2.23 (s, 6H),
with 10 lM of 5a–5e or control compounds. Media was aspirated

Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
6449
and cell washed twice with cold PBS. Cell homogenates were col-
lected and further washed thrice with 50 mM Tris–HCl, pH 7.5.
Protein concentration was measured using the Bio-Rad Bradford
to optimize the geometries of all compounds with protonated N
atoms. Three atoms (basic nitrogen atom N, centroid of phenolic
group, and centroid of pyrrole at naltrindole, or phenyl at SNC80,
5b and 17) were selected for the structural alignment using the
Atom Fit scheme within Sybyl7.2.
reagent, and an equal amount of protein (50 lg) was used for the
binding assays. Binding assays were performed using [3H]dipre-
norphine as the radioactive ligand. Binding assays were conducted
as described above. The assays were run in duplicate at least three
times.
The amino acid sequences of the human d opioid receptor
(hDOR accession number: P02699) and bovine rhodopsin were re-
trieved from the Swiss-Prot database.65 The sequence alignment
was performed by using ClustalW.66 The default matrix BLOSUM
and a gap open penalty 25 were chosen for the sequence align-
ment. The residues indicated in red in Fig. 1 (Supplementary data)
include Asn67, Asp95, D/ERY motif in TM3, Trp173, Pro225,
Pro276, NPXXY motif in TM7, Cys121 and Cys198. The conserved
two Cys residues are involved in formation of the conserved disul-
fide bridge between TM3 and extracellular loop 2 (EL2). The same
boundaries of the TM domains were applied to the opioid receptors
as identified in the X-ray crystal structure for the corresponding
sequence of bovine rhodopsin.
5.22. GTPcS assays
Cell membranes were prepared from HEK293-stable transfec-
tants expressing murine d opioid receptors using ultracentrifuga-
tion. Protein concentration was measured using the Bio-Rad
Bradford reagent. Ten micrograms of membrane protein expressing
the delta opioid receptor was incubated at room temperature for
90 min with assay buffer containing [S-35]GTP
ence or absence of 10 M of each compound except for compound
5c which was used at 50 M concentration. Following incubation,
cS and GDP in pres-
l
l
Aftertheoptimalalignmentwasachieved, the3Dstructuralmod-
els of the d opioid receptor were constructed using the HOMOLOGY
module in the Insight package67 based on the X-ray crystal structure
of bovine rhodopsin (pdb ID: 1F88) as the template.36 The coordi-
nates of TMs were copied from the corresponding TMs in the tem-
plate structure. Loop searches in the PDB were performed on all
extracellular and intracellular loops. The loop hits showing the high-
est sequence identity and lowest rms deviation were selected for the
corresponding regions. The N and C termini, located, respectively, in
theperiplasmand cytosol, aredistant fromtheligandbindingpocket
and therefore were excluded in the final models. Energy minimiza-
tion was performed with AMBER8.0 using AMBER99 force field on
initial models for about 500 steps to relax close interactions.68 The
d receptor selective agonist (+)SNC80 and antagonist naltrindole
were manually docked to the putative active site based on the pub-
lished mutation data and molecular modeling studies. The resulting
ligand–receptor complexes were submitted to energy minimization
and 1.5 ns molecular dynamics simulations at constant NTP
(T = 25 °C; P = 1 atm) using AMBER8.0. The averaged structure was
energy-minimized. Removing the ligands from the minimized
complexes yielded one final set of coordinates for the agonist-bound
and antagonist-bound d opioid receptors, which were used for
docking of compound 5b with the GOLD docking program (version
3.1).69 The number of poses for each compound was set to 100. After
docking, all binding poses of each ligand were visually checked.
Only the poses that exhibited a high GOLDscore and formed a salt
bridge with Asp128 and hydrogen bond to TM5 or TM6 were
selected as realistic docked conformations. The resulting recep-
tor–ligand complexes were submitted to energy minimization with
AMBER8.0. The binding interactions were analyzed with Sybyl v7.2.
The binding pose of 5b shown in Fig. 2 is from the agonist-
bound structure of the d opioid receptor. The key contact residues
with 5b are the same for agonist-bound and antagonist-bound
structures.
samples were filtered through Whatman GFB filters and washed
three times with ice cold 50 mM Tris–HCl pH 7.4. Filters were soaked
in Ecoscint liquid scintillation solution (National Diagnostics, Man-
ville, NJ) and filter-bound radioactivity was measured using a Pack-
ard Tri-Carb 2100 TR liquid scintillation analyzer for S-35 isotope.
The assays were performed in duplicate at least three times.
5.23. MAP kinase assays
HEK293 cells expressing the d receptor were placed in serum-
free media overnight to reduce basal MAP kinase signaling. The fol-
lowing day, the media was replaced with fresh serum-free media
with or without SNC80 or 5b at 10 lM and cells were incubated
at 37 °C for 10 min. Reactions were terminated by aspiration of
the media and solubilization of the cells on the dish with 1% dode-
cyl-b-D-maltoside, 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM
CaCl2, 1 mM MgCl2, 10% glycerol, protease inhibitor cocktail (Sig-
ma, 1:100) and phosphatase inhibitor cocktail 1 and 2 (Sigma, each
1:100). The detergent lysate was centrifuged at 16,000g for 20 min,
and the supernatant was recovered. The protein concentration in
the supernatant was determined using the Dc protein assay (Bio-
Rad). Protein (60 lg per lane) was resolved using 12% SDS/PAGE
at 120 V for approximately 1.5 h and transferred to Immobilon P
polyvinylidene difluoride membranes (Millipore, Bedford, MA) at
100 V for 1 h. Immunoblots were incubated in blocking buffer fol-
lowed by overnight incubation with either mouse monoclonal
anti-phospho-MAP kinase antibody (1:1000, Santa Cruz Biotech-
nology, Santa Cruz, CA) or rabbit anti-MAP kinase antibody that
recognizes total (phosphorylated and nonphosphorylated) MAP ki-
nase (1:3000, Upstate Biotechnology, Charlottesville, VA). Goat
anti-mouse IgG conjugated with horseradish peroxidase (Santa
Cruz Biotechnology) or goat anti-rabbit IgG conjugated with horse-
radish peroxidase (Santa Cruz Biotechnology) were used as second-
ary antibodies. Membranes were developed using ECL Western
blotting substrate (Pierce Biotechnology, Rockford, IL) and HyBlot
CL autoradiography film (Denville Scientific, Metuchen, NJ). Blots
were quantified using SynGene Software (Synoptics Ltd, Cam-
bridge, England).
Acknowledgments
Access to the computing facilities of the Informatics Institute at
University of Medicine and Dentistry of New Jersey is sincerely
appreciated. We also want to acknowledge Dr. Nigam Rath of the
University of Missouri-St. Louis for his X-ray crystallographic anal-
ysis of compounds 5b and 5e.
5.24. Molecular modeling
All molecular modeling operations were performed on
Linux workstations and Solaris server. The crystal structures of
(ꢂ)SNC8062 and morphine63 were used as the starting conforma-
tions to build the structures of (+)SNC80 and naltrindole using Syb-
yl v7.2.64 Compounds 5b and 17 were constructed with Sybyl7.2 as
well. The MMFF94 force field and partial atomic charges were used
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.007.

6450
Y. Peng et al. / Bioorg. Med. Chem. 17 (2009) 6442–6450
K.; Hruby, V. J.; Roeske, W. R.; Yamamura, H. I. Mol. Pharmacol. 1996, 50,
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