Synthesis and binding affinity of novel mono- and bivalent morphinan ligands for κ, μ, and δ opioid receptors

Article abstract of DOI:10.1016/j.bmc.2011.03.052

A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors, and their functional activities were determined at κ and μ receptors in [³⁵S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (K_i values less than 1 nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity K_i values of 0.089 nM at the μ receptor and 0.073 nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists.

Full text of DOI:10.1016/j.bmc.2011.03.052

Bioorganic & Medicinal Chemistry 19 (2011) 2808–2816
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and binding affinity of novel mono- and bivalent morphinan ligands
for j, l, and d opioid receptors
Bin Zhang a, Tangzhi Zhang a, Anna W. Sromek a, Thomas Scrimale b, Jean M. Bidlack b, John L. Neumeyer a,
⇑
a Alcohol & Drug Abuse Research Center, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478-9106, USA
b Department of Pharmacology and Physiology, School of Medicinal and Dentistry, University of Rochester, Rochester, NY 14642, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of homo- and heterodimeric ligands containing
j
/
l
agonist and
l
agonist/antagonist phar-
Received 21 December 2010
Revised 16 March 2011
Accepted 22 March 2011
Available online 26 March 2011
macophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro
binding affinity at , and d opioid receptors, and their functional activities were determined at
and S functional assays. Most of these compounds had high binding affinity at
receptors in [35S]GTP
and receptors (Ki values less than 1 nM). Compound 15b, which contains butorphan (1) at one end
of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with bind-
j
,
l
j
l
c
l
j
Keywords:
ing affinity Ki values of 0.089 nM at the
l
receptor and 0.073 nM at the
j receptor. All of the morphinan-
Homo- and heterodimeric ligands
Kappa, delta and mu receptors
Opioids
derived ligands were found to be partial
j
and agonists; ATPM-derived ligands 12 and 11 were found to
l
be full
j agonists and partial l agonists.
Ó 2011 Elsevier Ltd. All rights reserved.
Morphinans
Butorphan
Butorphanol
Functional assays
1. Introduction
suggested that j opioid agonists with varying activity at the l
receptor effectively reduced cocaine self-administration in nonhu-
Bivalent ligands have the potential for bridging vicinal recep-
tors. Such bridging should be manifested by a substantial increase
in potency due to the high local concentration of the free pharma-
cophore in the vicinity of the proximal recognition site when the
bivalent ligand is bound in a monovalent mode.1,2 Due to homo-
man primates with fewer undesirable side effects than the highly
selective
develop novel bivalent ligands targeting both
Hence, we investigated a new series of homo- and hetero- bivalent
j
agonists.14–16 These results encouraged us to further
j
and l receptors.
ligands which contain the
j agonist, l partial agonist butorphanol
and heterooligomerization among the opioid receptors,3–5
a
(5)26 and its analogue 14-methoxybutorphanol (8). The synthesis
number of bivalent ligands containing opiate or peptide pharmaco-
phores have been designed and synthesized, which proved to
possess enhanced binding affinity and selectivity. For example,
bivalent ligands containing oxymorphone or naltrexamine phar-
macophores with specific spacer lengths have been reported to
have enhanced opioid agonist or antagonist potency and selectiv-
ity. Portoghese et al.2,6–10 has also reported a range of homo- and
heterodimeric ligands with varying linker lengths designed to
investigate pharmacodynamic and organizational features of opi-
oid receptors. These reported heterodimeric ligands were demon-
strated to possess significantly greater potency and selectivity
compared to their monomer congeners, providing further evidence
for the opioid receptor heterooligomerization phenomena.10–12
The development of bivalent ligands that bridge the gap
between binding sites on dimerized receptors could lead to a
new generation of analgesic drugs that may not cause physical
dependence or tolerance with chronic use.13 Behavioral studies
of a bivalent ligand (11) containing ATPM (10),27 which is a full
j
and partial
were evaluated in vitro for their binding affinity at
oid receptors, and for their pharmacological properties in the
35S]GTP
S binding assay.
We have previously reported that bivalent ligands incorporat-
l
agonist, has also been achieved. These ligands
j, l
, and d opi-
[
c
ing two molecules of butorphan (1) (‘homobivalent’) or two dis-
tinct (‘heterobivalent’) morphinan ligands, when connected by a
molecular spacer, can lead to compounds with improved binding
affinities and selectivities.17–21 Bivalent ligands 3 and 4, which con-
tain butorphan (1), a mixed
ing ester chain (Fig. 1), are the most potent ligands in this series,
with agonist and partial
agonist activity.17,18 Ligand 2 (Fig. 1)
derived from the linkage of a d-selective peptide antagonist Dmt-
Tic -tyrosine-1,2,3,4-tetrahydroisoquinoline-3-
(20,60-dimethyl-
carboxylic acid) and a agonist butorphan (1) through a
j/l
agonist24 at both ends of the link-
j
l
L
j/l
repeated 3-aminopropionyl spacer was found to maintain the
same characteristics as the two parent compounds.22 Thus, the
binding profile of such bivalent ligands is dependent on the
⇑
Corresponding author. Tel.: +1 617 855 3388.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.052

B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
2809
HO
N
N
N
H2N
O
O
O
HO
O
N
H
N
H
O
1, Butorphan
2
N
N
N
OH
O
O
HO
O
X
O
5, Butorphanol
3,
4,
X = (CH2)8
X =
Figure 1. Structure of homo- and heterobivalent ligands.
molecular structure of the opioid pharmacophore, the character
and length of the connecting spacer, and the connecting site of
the two pharmacophores.2,8,17–23,25
We reasoned that by varying the particular combination of mor-
phinans in each dimer, we would be able to modulate the combi-
morphinan 5 or 8 to afford 13, deprotecting the benzyl ether of
the latter, and condensing the resulting free acid 14 with 5, 8, or
naloxone in the presence of DCC and DMAP, as previously reported
(Scheme 3).18
nation of selectivity, activity, and potency at
j, l, and d opioid
3. Results and discussion
receptors for a given ligand. Such dimers, with specifically chosen
activity for each receptor subtype, could prove highly useful in
studying the biological effects of receptor oligomerization and
could also lead to a superior therapeutic agent for the treatment
of cocaine abuse. We thus investigated a new series of homo-
All the novel opioid ligands were evaluated for their binding
affinities and selectivity for l, j, and d opioid receptors with Chi-
nese hamster ovary (CHO) membranes stably expressing one of
the human opioid receptors. The data are summarized in Table 1.
For comparison purposes, the data for naloxone, butorphan (1),
butorphanol (5), ligands 3, 4, and 10 are also included in Table 1.
Monovalent compound 13b containing a 10-carbon linking chain,
and hetero- bivalent ligands containing the
j agonist, l partial
agonist butorphanol (5)26 and its analogue 14-methoxybutorpha-
nol (8). The synthesis of the homobivalent ligand (11) which con-
tains ATPM (10),27 a full
j
but partial
achieved. These ligands were evaluated in vitro for their binding
affinity at , and d opioid receptors. These ligands retained or
l agonist, has also been
exhibits the same binding affinity at
ent ligand 8 but shows slightly lower affinity at d receptor. Simi-
larly, 13b shows slightly higher binding affinity at and
j and l receptors as the par-
j, l
j
l
displayed better affinity at
j and l receptors compared to their
receptors, and shows the same affinity at d receptor compared to
homobivalent ligand 9b. Heterobivalent ligands 15c and 15a exhi-
parent compounds.
bit comparable binding affinity at
binding affinity at both and d receptors when compared to nalox-
one. In contrast, ligand 15a shows a lower affinity at , and d
receptors than butorphanol. Ligand 15c also shows lower affinity
at and d receptors, but a slightly increased affinity at the recep-
tor than monomeric parent ligand 8. It is interesting to note that
15b has enhanced binding affinity both at and receptors when
compared to its parent compounds butorphan (1) and butorphanol
(5). Since it has been previously demonstrated that ATPM, a full
agonist with activity, effectively reduced cocaine self-adminis-
l receptor, but have increased
2. Chemistry
j
l, j
Butorphanol (1) free base was treated with BnBr/K2CO3 to give
the corresponding benzyl ether. After treatment with NaH/Me2SO4,
the benzyl group was removed to give 14-methoxybutorphanol (8)
(Scheme 1) and were pharmacologically characterized at the
oid receptor and the S] assay.
Treatment of sebacic acid (1 equiv) with oxalyl chloride gave seba-
coyl chloride, which was then coupled to the morphinans 8 and 10
(2 equiv) to afford the homobivalent compounds 9 and 11, respec-
tively (Scheme 2).18 The heterodimeric ligands were prepared step-
wise, by esterifying sebacoyl chloride monobenzyl ester with
l
j
j
opi-
l
j
l
opioid receptor using the [35GTP
c
j
l
tration in nonhuman primates with fewer undesirable side effects
than the highly selective kappa agonists,14–16 we analyzed homodi-
mers of ATPM. It was found that ATPM-derived homodimeric 11
N
OH
N
N
N
OH
OMe
OMe
Me2SO4
H2
BnBr
Pd-C
NaH
THF
K2CO3
DMF
HO
BnO
BnO
HO
8
6
7
5
Scheme 1.

2810
B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
N
N
OR
N
OR
RO
Sebacoyl chloride
Toluene/Et3N
95oC
O
O
HO
O
8, R = Me
9
O
N
N
N
Sebacoyl chloride
S
O
O
S
S
Toluene/Et3N
95oC
N
H2N
N
N
N
H
N
H
8
10, ATPM
11
N
N
LiAlH4
THF
S
S
N
N
N
H
N
8
H
12
Scheme 2.
N
OR1
N
1) (COCl)2, Cat. DMF
OR1
BnO2C
CO2H
Pd-C, H2
8
O
2) Et3N, DCM
CO2Bn
HO
5, R1 = H
O
R
1 = H, 13a
8, R1= Me
R1 = Me, 13b
N
N
OR1
OR1
DCC, DMAP
ROH Et3N
O
O
CO2R2
CO2H
O
O
R1 = H, 14a
R
1 = H, R2 = Naloxone, 15a
R1 = H, R2 = Butorphan, 15b
R1 = Me, R2 = Naloxone, 15c
R1 = Me, 14b
Scheme 3.
and 12 maintain high binding affinity at
1 nM), although they have lower affinity at all three receptors than
j
receptor (less than
All compounds were found to be partial
35S]GTP
S binding assay (Table 3).
l agonists in the
[
c
the parent compound ATPM.
The activities of 14-methoxybutorphanol (8), univalent ligand
13b, homobivalent ligands 9b, 12, 11, and heterobivalent ligands
4. Conclusions
Homo- and heterobivalent ligands, which contain
15c, 15b, and 15a at both
j
and
l receptors were assessed using
j/l agonist
the [35S]GTP
respectively.
cS binding assay, and reported in Tables 2 and 3,
butorphanol (5), its analogue 14-methoxybutorphanol (8) and
the aminothiazolomorphinan 1027 (ATPM), have been synthesized
The results indicate that all 14-methoxybutorphanol (8) and
butorphanol-derived univalent and bivalent ligands 8, 9b, 13b,
and their binding affinities evaluated at
tors. All of these ligands have been found to have high binding
affinity at and/or opioid receptors. The heterobivalent ligand
15b is more potent than either of its parent compounds butorphan
(1) or butorphanol (5) at and receptors, and was found to be
l, j, and d opioid recep-
15c, 15b, and 15a acted as partial
nist activity (Emax range between 74% and 130% maximal stimula-
tion) and varying degrees of antagonist activity (Imax ranged from
j agonists, all having high j ago-
l
j
j
l
j
23% to 43% maximal inhibition). In contrast, the ATPM-derived
homobivalent ligands 11 and 12 were found to be full agonists.
the most potent ligand in this series. With the exception of the
two ATPM-derived ligands 11 and 12, all other ligands which were

B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
2811
Table 1
Ki values for the inhibition of
l
,
j
, and d opioid binding to CHO membranesa
Compound
Structure
Kia (nM)
Selectivity
:d
[3H]DAMGO
l
[3H]U69,593
j
[3H]Naltrindoled
l:j
N
Naloxoneb
0.23 0.05
0.25 0.02
38
3
1:1:152
3:1:75
2:1:100
1:1:58
O
HO
HO
HO
HO
O
N
N
N
Butorphanc
1
0.23 0.01
0.079 0.003
0.12 0.0068
0.20 0.020
5.9 0.6
OH
Butorphanol 5
0.22 0.012
0.14 0.014
0.090 0.004
12 1.1
OMe
MCL-691
8
8.1 0.071
N
N
MCL-144
0.049 0.001
4.2 0.4
2:1:90
3d
O
O
O
O
8
N
N
MCL-145
0.2 0.03
0.08 0.01
9.4 0.5
3:1:120
O
4d
O
O
O
O
N
N
N
OMe
MeO
MCL-692
9b
0.26 0.037
0.15 0.014
0.55 0.10
0.37 0.040
0.23 0.011
0.14 0.019
13 1.0
14 3.8
39 6.1
1:1:50
1:1:93
4:1:280
O
O
O
O
8
MeO
MCL-693
13b
O
O
OBn
8
N
N
N
OH
MeO
MCL-694
15c
O
O
O
O
O
O
8
N
HO
MCL-695
15b
0.089 0.012
0.073 0.001
8.0 2.4
1:1:110
3:1:130
O
O
O
O
8
N
N
OH
HO
MCL-696
15a
0.82 0.11
0.28 0.014
35 11
O
O
O
O
O
O
8
(continued on next page)

2812
B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
Table 1 (continued)
Compound
Structure
Kia (nM)
Selectivity
:d
[3H]DAMGO
l
[3H]U69,593
j
[3H]Naltrindoled
l:j
N
10e
MCL-147
ATPM
1.5 0.21
0.049 0.0046
29
2
31:1:592
24:1:460
S
N
N
H2N
N
N
MCL-715
11
8.8 0.66
0.37 0.024
170 7.1
O
O
S
S
S
S
N
N
N
N
H
N
H
8
N
MCL-714
12
2.5 0.19
0.27 0.0023
39 4.8
9:1:140
N
N
H
N
H
8
a
Membranes from Chinese hamster ovary (CHO) cells, stably expressing either the human j, l, or d opioid receptors, were incubated with 12 different concentrations of
the compounds in the presence of receptor-specific radioligands at 25 °C, in a final volume of 1 mL of 50 mM Tris–HCl, pH 7.5. Nonspecific binding was determined using
10 M naloxone. Data are the mean values SEM from three experiments, performed in triplicate.
l
b
Ref. 19.
Ref. 23.
Ref. 17.
Ref. 27.
c
d
e
evaluated in this study were partial agonists at the
(Table 2) and partial agonists at the receptor (Table 3). In con-
trast, the two ATPM-derived ligands 11 and 12 were full agonists
at the receptor, but like the other ligands in this study, were also
partial agonists at the receptor. These results suggest that
appropriate design of bivalent ligands may enhance the binding
affinity and selectivity of opioid ligands, and also provide evidence
for opioid receptor oligomerization phenomena. These bivalent li-
gands may be used as chemical and pharmacological tools to elu-
cidate the pharmacodynamic and organizational features of
opioid receptors.
j
receptor
J = 8.4, 1H), 6.93–6.62 (m, 2H), 5.02 (s, 2H), 4.82–3.80 (br, 1H),
3.04 (d, J = 18.2, 1H), 2.75 (dd, J = 6.2, 18.2, 1H), 2.63 (d, J = 6.0,
2H), 2.54–1.37 (m, 19H), 0.99 (d, J = 12.4, 1H); 13C NMR (75 MHz,
CDCl3) d 157.4, 142.8, 137.1, 128.7, 128.5, 128.2, 127.9, 127.6,
112.1, 111.9, 70.0, 69.5, 61.3, 60.5, 44.7, 41.5, 37.0, 33.9, 31.7,
30.2, 27.0, 26.8, 24.9, 21.7, 21.6, 18.7.
l
j
l
5.1.2. (ꢀ)-3-Benzyloxy-14-methoxybutorphanol (7)
To a solution of 3-benzyloxybutorphanol (6, 3.8 g, 9.11 mmol)
in anhydrous DMF (50 mL) was added NaH (60% in mineral oil,
3.64 g, 91.1 mmol) at room temperature and stirred for 15 min.
Next, Me2SO4 (2.6 mL, 27 mmol) was added to the suspension,
and the reaction mixture was stirred at room temperature for
1 h. The reaction was quenched carefully by addition of water at
0 °C. Next, EtOAc (200 mL) was added, and the organic layer was
washed with water (50 mL ꢁ 2) and brine, and dried over Na2SO4.
After the solvent was removed under reduced pressure, the crude
product was purified on silica gel (gradient: EtOAc/hexane 1:20
to EtOAc/hexane 1:3) to give a colorless oil (3.88 g, 98%); 1H
NMR (300 MHz, CDCl3) d 7.51–7.29 (m, 5H), 7.01 (d, J = 8.4, 1H),
6.87 (d, J = 2.5, 1H), 6.77 (dd, J = 2.4, 8.3, 1H), 5.02 (s, 2H), 3.22 (s,
3H), 3.13 (d, J = 18.2, 1H), 3.00 (d, J = 4.8, 1H), 2.62–2.35 (m, 5H),
2.27–1.16 (m, 16H), 0.89 (d, J = 12.0, 1H); 13C NMR (75 MHz, CDCl3)
d 157.3, 143.3, 137.1, 129.5, 128.5, 128.0, 127.8, 127.6, 111.9,
111.7, 74.2, 70.0, 61.3, 52.8, 46.9, 45.8, 42.1, 35.8, 34.4, 29.2,
28.1, 27.2, 25.5, 24.7, 21.5, 20.8, 18.8. Anal. Calcd for
5. Experimental section
5.1. General synthetic methods
1H and 13C NMR spectra were recorded at 300 MHz (75 MHz) on
a Varian Mercury 300 spectrometer. Chemical shifts are given as d
value (ppm) downfield from tetramethylsilane as an internal refer-
ence. Melting points were determined on a Thomas-Hoover capil-
lary tube apparatus and are reported uncorrected. Elemental
analyses, performed by Atlantic Microlabs, Atlanta, GA, were with-
in 0.4% of theoretical values. Analytical thin-layer chromatography
(TLC) was carried out on 0.2 lm Kieselgel 60F-254 silica gel alumi-
num sheets (EM Science, Newark, NJ). Flash chromatography was
used for the routine purification of reaction products. Eluent sys-
tems are described for the individual compounds.
C
29H37NO2ꢂHCl 0.5H2O: C, 73.01; H, 8.24; N, 2.94. Found: C,
72.78; H, 7.97; N, 2.86.
5.1.1. (ꢀ)-3-Benzyloxybutorphanol (6)
5.1.3. (ꢀ)-14-Methoxybutorphanol (8)
To a stirring suspension of butorphanol free base (5, 7.52 g,
23.0 mmol) and K2CO3 (9.52 g, 69.0 mmol) in DMF (100 mL) was
added BnBr (4.13 g, 24.15 mmol), and the reaction mixture was
stirred overnight at room temperature. The next day, EtOAc
(200 mL) was added, and the organic layer was washed with water
(100 mL ꢁ 3) and brine (50 mL), dried over Na2SO4, and concen-
trated under reduced pressure. The crude product was then puri-
fied over silica gel (EtOAc/hexane 1:3) to afford a colorless oil
(9.04 g, 94%); 1H NMR (300 MHz, CDCl3) d 7.37 (m, 5H), 7.01 (d,
Pd/C (20 mg) was added to a stirring solution of 3-benzyloxy-
11-methoxybutorphanol (230 mg, 0.533 mmol) in MeOH (5 mL),
and the suspension was hydrogenated at room temperature over
night. The next day, the suspension was filtered off and solvent
was removed under reduced pressure to give a colorless oil
(170 mg, 93%) which was used directly without purification; 1H
NMR (300 MHz, CDCl3) d 6.56 (d, J = 8.2, 1H), 6.42 (s, 1H), 6.30 (d,
J = 8.1, 1H), 6.23–5.85 (br, 1H), 2.85 (s, 3H), 2.83–2.60 (m, 2H),

B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
2813
Table 2
Pharmacological characterization of morphinan monovalent and bivalent ligands at the kappa opioid receptor using the [35S]GTP
cS binding assay
Compound
Structure
Kappa (AgonMCL-692, MCL-693, ist)
Kappa (antagonist)
IC50 (nM) Imax (% maximal
EC50 (nM)
Emax (% maximal
stimulation over basal)
inhibition)
N
OMe
MCL-691
2.8 0.90
90 3.6
22 3.7
17 2.9
6.4 2.7
7.7 3.3
37 3.0
8
HO
N
N
MeO
OMe
MCL-692
9b
2.4 0.57
2.1 0.076
18 4.5
83 0.95
43 4.3
30 4.7
33 3.8
23 2.9
O
O
O
O
O
O
O
8
N
MeO
MCL-693
13b
90 6.2
OBn
8
N
N
OH
MeO
MCL-694
15c
80 4.8
O
O
O
O
O
O
8
N
N
HO
MCL-695
15b
0.84 0.089
100 0.95
3.7 1.2
O
O
O
O
8
N
OH
N
HO
MCL-696
12 4.9
74 16
49 13
35 2.2
O
O
15a
O
O
O
N
O
8
N
N
N
MCL-715
11
5.5 0.31
130 3.3
NIa
NIa
O
O
S
S
N
N
H
N
H
8
N
MCL-714
12
7.3 0.81
130 12
NIa
NIa
S
S
N
N
N
H
N
H
8
Membranes from CHO cells that expressed the human
60 min at 30 °C. Nonspecific binding was measured by the inclusion of 10
j
opioid receptor were incubated with 12 concentrations of the compound in the presence of 0.08 nM [35S]GTP
M GTP S binding was stimulated by the addition of
S. For the inhibition experiments, [35S]GTP
cS for
l
c
c
100 nM U50,488. Data are the mean SEM from three experiments performed in triplicate.
a
NI = no inhibition.
2.30–0.83 (m, 21H), 0.50 (d, J = 11.9, 1H); 13C NMR (75 MHz, CDCl3)
d 154.8, 143.1, 128.2, 128.1, 113.3, 112.0, 74.5, 61.0, 52.8, 47.0,
45.9, 41.9, 35.5, 34.2, 29.2, 28.2, 27.3, 25.7, 24.8, 21.6, 20.9, 18.8.
Mp (HCl salt): 195 °C (dec). Anal. Calcd for C22H31NO2ꢂHCl H2O:
C, 66.73; H, 8.65; N, 3.54. Found: C, 66.40, H, 8.27; N, 3.54.
the resulting solid was filtered, washed with two portions of ether,
and dried under vacuum to give the corresponding HCl salt.
5.2.1. Bis((ꢀ)-14-methoxy-17-N-cyclobutylmethyl)morphinan-
3-yl) decanedioate (9)
Representative procedure: To
a solution of sebacic acid
5.2. General procedure for converting morphinan ligands to the
HCl salts
(0.0981 mmol, 30.2 mg; 1 equiv) in anhydrous CH2Cl2 (2 mL) was
added oxalyl chloride (1.962 mmol, 0.173 mL; 20 equiv) and two
drops of DMF. Gas evolution was observed and the solution was
stirred overnight. The next day, the reaction mixture was concen-
trated under reduced pressure. The resulting yellow oil was
To a solution of the free base in a minimal amount of ethyl ace-
tate was added excess ethereal 1 N HCl. A precipitate formed, and

2814
B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
Table 3
Pharmacological characterization of morphinan monovalent and bivalent at the mu opioid receptor using the [35S]GTP
cS binding assay
Compound
Structure
Mu (agonist)
Mu (antagonist)
Imax (% maximal
EC50 (nM)
3.6 1.8
Emax (% maximal
stimulation over basal)
IC50 (nM)
inhibition)
N
OMe
MCL-691
8
50 2.7
42 2.1
54 7.0
62 3.4
9.3 3.1
50 3.4
HO
N
N
OMe
MeO
MeO
MCL-692
9b
1.8 0.36
1.7 0.39
10 3.0
260 92
7.6 2.3
19 3.0
44 11
28 2.2
45 2.8
O
O
O
O
O
O
O
8
8
N
MCL-693
13b
OBn
N
N
N
OH
MeO
HO
MCL-694
15c
O
O
O
O
O
O
8
8
N
MCL-695
15b
1.2 0.23
46 4.1
3.4 0.63
50 5.7
O
O
O
O
O
N
N
N
OH
HO
MCL-696
15a
2.0 0.42
19 3.5
34 4.5
73 7.7
O
O
O
O
O
8
N
N
N
MCL-715
11
130 42
25 1.8
NAa
74 at 10 lM
O
O
S
S
N
N
H
N
H
8
N
MCL-714
12
22 5.3
38 3.7
390 100
71 5.3
S
S
N
N
N
N
8
H
H
Membranes from CHO cells that expressed the human
60 min at 30 °C. Nonspecific binding was measured by the inclusion of 10
l
opioid receptor were incubated with 12 concentrations of the compound in the presence of 0.08 nM [35S]GTP
M GTP S binding was stimulated by the addition of
S. For the inhibition experiments, [35S]GTP
cS for
l
c
c
200 nM DAMGO. Data are the mean SEM from three experiments performed in triplicate.
a
An IC50 value could not be determined because the inhibition curve had not reached a plateau at 10 lM of the compound.
redissolved in anhydrous CH2Cl2 (5 mL), 14-methoxybutorphanol
(8) (100 mg, 0.294 mmol; 3 equiv) and Et3N (40 mg, 0.392 mmol;
4 equiv) was added to the solution, and the mixture was stirred
at room temperature overnight. The organic layer was washed
with NaHCO3 solution, dried over Na2SO4. The solvent was re-
moved and the result crude product was purified on silica gel
(EtOAc/Et3N/MeOH 200:1:1) to give the pure compound as color-
less oil (70 mg, 84%); 1H NMR (300 MHz, CDCl3) d 7.08 (d, J = 8.3,
2H), 6.92 (d, J = 2.2, 2H), 6.84 (dd, J = 2.3, 8.2, 2H), 3.21 (s, 6H),
3.16 (d, J = 18.8, 2H), 3.01 (d, J = 5.4, 2H), 2.62–1.17 (m, 54H),
0.88 (d, J = 10.9, 2H); 13C NMR (75 MHz, CDCl3) d 172.4, 149.3,
143.4, 134.4, 128.1, 118.6, 118.1, 74.1, 61.3, 52.8, 46.9, 45.6, 42.1,
35.8, 34.4, 29.2, 29.05, 29.02, 28.1, 27.2, 25.5, 25.1, 24.8, 21.4,
20.8, 18.8; mp (HCl salt): 154 °C (dec). Anal. Calcd for
C
54H76N2O6ꢂ2HCl H2O: C, 68.99; H, 8.58; N, 2.98. Found: C, 69.03,
H, 8.58; N, 2.96.
5.2.2. Bis((ꢀ)-3-aminothiazolo-N-cyclopropyl-
methylmorphinan)sebacamide hydrochloride (11)
A mixture of compound 10 (88 mg, 0.25 mmol), sebacoyl chlo-
ride (30 lL, 0.14 mmol) and triethyl amine (45 lL, 0.30 mmol) in
2 mL toluene was refluxed overnight. After cooling to room tem-
perature, the reaction mixture was directly purified on silica gel
(EtOAc/MeOH/Et3N = 60:1:1). A slightly yellow foam (52 mg, 54%)

B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
2815
as product was obtained; 1H NMR (300 MHz, CDCl3) d 7.66 (s, 2H),
7.55 (s, 2H), 3.10 (m, 4H), 2.77 (m, 4H), 2.43 (m, 10H), 1.92 (m, 4H),
1.37 (m, 30H), 0.88 (m, 2H), 0.51 (d, J = 8.0, 4H), 0.11 (m, 4H); 13C
NMR (75 MHz, CDCl3) d 171.98, 159.00, 146.92, 139.88, 134.44,
129.24, 119.91, 116.88, 59.96, 55.71, 45.59, 45.02, 42.57, 38.06,
36.87, 36.33, 28.80, 28.74, 26.90, 26.58, 24.95, 24.86, 22.18, 9.40,
4.10, 3.63. Mp (HCl salt): 270 °C (dec). Anal. Calcd for
and brine, and dried over Na2SO4. The crude product was purified
on silica gel (EtOAc/hexanes 1:1) to a give yellow oil (110 mg, 31%).
1H NMR (300 MHz, CDCl3) d 7.35 (m, 5H), 7.09 (d, J = 8.3, 1H),
6.92 (s, 1H), 6.85 (dd, J = 2.3, 8.2, 1H), 5.12 (s, 2H), 3.22
(s, 3H), 3.16 (d, J = 18.5, 1H), 3.02 (d, J = 5.4, 1H), 2.60–1.20 (m,
37H), 0.89 (d, J = 11.1, 1H); 13C NMR (75 MHz, CDCl3) d 173.5,
172.3, 149.3, 143.3, 136.0, 134.2, 128.4, 128.1, 118.5, 118.1, 74.0,
66.0, 61.2, 52.8, 46.9, 45.6, 42.0, 35.6, 34.3, 34.24, 34.19, 29.1,
29.0, 28.9, 28.0, 27.1, 25.4, 25.0, 24.81, 24.78, 21.4, 20.8, 18.7. Mp
(HCl salt): 165–168 °C. Anal. Calcd for C39H53NO5ꢂHCl 2.5H2O: C,
67.17; H, 8.53; N, 2.01. Found: C, 67.21; H, 8.35; N, 2.22.
C
52H68N6O2S2ꢂ2HCl 3.5H2O: C, 61.88; H, 7.69; N, 8.33. Found: C,
61.75; H, 7.51; N, 7.94. HPLC analysis indicates a single compound
(100%).
5.2.3. (ꢀ)-3,30-(Decane-1,10-
diaminothiazolo)bis(cyclopropylmethyl)morphinan
hydrochloride (12)
5.3. General procedure for the preparation of morphinans 14a
and 14b
To the solution of amide (52 mg, 0.06 mmol) in 3 mL anhydrous
THF was added LiAlH4 (10 mg, 0.24 mmol) at room temperature.
The mixture was stirred at room temperature for 4 h, and then
three drops water was added to quench the reaction, which was
then directly purified on silica gel (EtOAc/MeOH/Et3N = 60:1:1) to
give slightly yellow foam (33 mg product, 66%); 1H NMR
(300 MHz, CDCl3) d 7.46 (s, 2H), 7.32 (s, 2H), 5.92 (s, 2H), 3.74–
3.65 (m, 6H), 3.00 (m, 6H), 2.66 (m, 4H), 2.45 (m, 2H), 2.22 (m,
4H), 1.40 (m, 36H), 0.63 (d, J = 7.9, 4H), 0.29 (d, J = 10.8, 4H). 13C
NMR (75 MHz, CDCl3) d 167.10, 152.11, 137.55, 128.38, 119.46,
115.23, 62.56, 58.99, 56.16, 45.90, 45.40, 37.32, 36.15, 30.03,
29.10, 26.47, 26.03, 24.89, 21.87, 4.30, 4.27. Mp (HCl salt): 252 °C
(dec). Anal. Calcd for C52H72N6S2ꢂ4HCl 2.2H2O: C, 60.17; H, 7.88;
N, 8.10. Found: C, 60.27; H, 7.91; N, 7.92.
Pd/C (42 mg) was added to a solution of benzyl ester 4 (418 mg)
in MeOH (20 mL). The reaction mixture was hydrogenated at room
temperature overnight. The next day, the suspension was filtered
and MeOH was removed under reduced pressure to give the prod-
uct, which was used directly without purification.
5.3.1. Decanedioic acid (ꢀ)-butorphanol-3-yl ester (14a)
Colorless oil (205 mg, 97%); 1H NMR (300 MHz, CD3OD) d 6.09
(d, J = 8.1, 1H), 5.86 (d, J = 2.1, 1H), 5.77 (dd, J = 2.1, 8.3, 1H),
2.87–0.14 (m, 41H); 13C NMR (75 MHz, CD3OD) d 174.6, 171.1,
148.8, 138.6, 128.8, 127.4, 118.5, 117.0, 67.1, 59.8, 55.8, 45.5,
38.3, 31.9, 31.8, 29.2, 29.1, 27.24, 27.20, 27.14, 27.07, 27.00, 25.3,
23.6, 23.0, 22.8, 19.1, 18.5, 16.4.
5.3.2. Decanedioic acid (ꢀ)-14-methoxyl-butorphanol-3-yl ester
(14b)
5.2.4. Benzyl ((ꢀ)-14-hydroxy-17-N-cyclobutylmethyl)
morphinan-3-yl) decanedioate (13a)
Yellow solid (342 mg, 96%); 1H NMR (300 MHz, CD3OD) d 7.31
(d, J = 8.4, 1H), 7.08 (d, J = 2.1, 1H), 6.99 (dd, J = 2.1, 8.3, 1H), 3.83
(d, J = 5.8, 1H), 3.55–3.09 (m, 4H), 3.37 (s, 3H), 3.04 (d, J = 8.6,
1H), 2.87–1.14 (m, 34H); 13C NMR (75 MHz, CDCl3) d 178.5,
172.3, 149.6, 142.5, 132.6, 128.2, 119.2, 118.2, 77.2, 73.8, 60.0,
52.9, 47.5, 45.3, 41.6, 35.8, 34.3, 34.2, 32.9, 29.05, 29.00, 28.2,
27.4, 25.5, 24.8, 21.1, 20.6, 18.7.
Method B: See the synthesis of 13b.
1H NMR (300 MHz, CDCl3) d 7.34 (s, 5H), 7.08 (d, J = 8.2, 1H),
6.91 (s, 1H), 6.84 (d, J = 8.2, 1H), 5.10 (s, 2H), 3.07 (d, J = 18.5,
1H), 2.78 (m, 1H), 2.62 (d, J = 5.9, 1H), 2.44 (m, 8H), 1.77 (m,
29H), 0.99 (d, J = 11.9, 1H); 13C NMR (75 MHz, CDCl3) d 173.6,
172.4, 149.3, 142.9, 136.0, 133.7, 128.5, 128.2, 128.1, 118.7,
118.3, 69.3, 66.0, 61.1, 60.5, 44.5, 41.5, 36.9, 34.3, 34.2, 33.7,
31.5, 30.1, 28.99, 28.96, 26.9, 26.7, 25.2, 24.83, 24.78, 21.6, 18.7.
Mp (HCl salt): 87–92 °C. Anal. Calcd for C38H51NO5ꢂHCl 0.8H2O: C,
69.93; H, 8.28; N, 2.15. Found: C, 70.04; H, 8.21; N, 2.19.
5.4. General procedure for the preparation of morphinans 15a,
15b, and 15c
Representative procedure: To
a solution of morphinan
5.2.5. Benzyl (ꢀ)-14-methoxy-butorphanol-3-yl decanedioate
(13b)
(0.162 mmol; 1 equiv) and sebacic acid monomorphinan ester
(0.162 mol; 1 equiv) in anhydrous CH2Cl2 (2 mL) was added DCC
(0195 mmol; 1.2 equiv) and DMAP (0.0162 mmol; 0.1 equiv) and
the reaction mixture was stirred overnight at room temperature.
CH2Cl2 was removed and an equal volume of ethyl acetate was
added. The resulting white solid was removed by filtration, and
the organic layer was washed with saturated NaHCO3 solution
and brine, and dried over Na2SO4. Ethyl acetate was removed to
give the crude product, which was purified on silica gel (EtOAc/
Et3N 100:1) to give pure product.
Method A: To the solution of sebacic acid monobenzyl ester
(1.08 g, 3.53 mmol) in anhydrous CH2Cl2 (15 mL) was added oxalyl
chloride (0.62 mL, 7.04 mmol) and two drops of DMF. Gas evolu-
tion could be observed and the solution was stirred overnight.
Next, CH2Cl2 and excess oxalyl chloride was removed under re-
duced pressure. The yellow oil was redissolved in anhydrous
CH2Cl2. 14-Methoxy-butorphanol (8) (1.00 g, 2.93 mmol) and
Et3N (2.0 mL, 14.1 mmol) was added to the solution and the mix-
ture was stirred overnight. The organic layer was washed with
NaHCO3 solution and dried over Na2SO4. After solvent was re-
moved under reduced pressure, the crude product was purified
on silica gel (EtOAc/hexanes 1:4 and EtOAc/hexanes 1:1) to give
a yellow oil (460 mg, 25%).
Method B: To the solution of sebacic acid monobenzyl ester
(206 mg, 0.676 mmol) and 14-methoxy-butorphanol (192 mg,
0.563 mmol) in anhydrous CH2Cl2 (6 mL) was added DCC
(139 mg, 0.676 mmol), and DMAP (7 mg, 0.0563 mmol), and the
reaction mixture was stirred overnight. The next day, after solvent
was removed under reduced pressure, the residue was redissolved
in an equal volume of ethyl acetate and the white solid was filtered
off. The organic layer was washed with saturated NaHCO3 solution
5.4.1. (5a-17-Fllyl-14-hydroxy-6-oxo-4,5-epoxymorphinan-3-
yl-17-((ꢀ)-N-cyclobutylmethyl-14-hydroxyl-mophinan-3-yl)
decanedioate (15a)
Yellow oil (68 mg, 51%); 1H NMR (300 MHz, CDCl3) d 7.09 (d,
J = 8.3, 1H), 6.87 (ddd, J = 3.3, 8.2, 11.9, 3H), 6.69 (d, J = 8.2, 1H),
5.82 (ddt, J = 6.4, 10.1, 16.5, 1H), 5.21 (m, 2H), 4.69 (s, 1H),
3.25–1.20 (m, 54H), 0.91 (m, 1H); 13C NMR (75 MHz, CDCl3) d
207.6, 172.4, 171.3, 149.3, 147.7, 142.8, 133.6, 132.5, 129.9,
128.2, 122.9, 119.3, 118.8, 118.2, 90.5, 69.3, 62.0, 61.1, 60.5, 57.6,
50.5, 41.4, 34.3, 29.6, 29.0, 26.7, 22.9, 21.6, 14.1. Mp (HCl salt):
170–174 °C. Anal. Calcd for C50H64N2O8ꢂ2HCl 2.4H2O: C, 64.08; H,
7.61; N, 2.99. Found: C, 63.85; H, 7.28; N, 2.98.

2816
B. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 2808–2816
5.4.2. ((ꢀ)-17-N-Cyclobutylmethyl)morphinan-3-yl-((ꢀ)-14-
hydroxyl-17-N-cyclobutylmethyl) morphinan-3-yl)
decanedioate (15b)
7.4, 3
35S]GTP
cific binding was measured by the inclusion of 10
l
M GDP, 3 mM MgCl2, 0.2 mM EGTA, and 100 mM NaCl.
S was added at a final concentration of 0.08 nM. Nonspe-
M GTPgS. After
[
c
l
Colorless oil (52 mg, 42%); 1H NMR (300 MHz, CDCl3) d 7.09 (d,
J = 8.3, 2H), 6.87 (m, 4H), 3.04 (dd, J = 18.4, 28.6, 1H), 2.82 (d, J = 5.8,
1H), 2.65–0.98 (m, 64H); 13C NMR (75 MHz, CDCl3) d 172.4, 149.3,
149.2, 142.9, 135.1, 133.7, 128.4, 128.2, 118.7, 118.4, 118.3, 118.0,
69.3, 61.4, 60.5, 55.7, 49.0, 45.6, 44.8, 44.5, 41.6, 41.5, 37.7, 36.9,
36.5, 34.8, 34.3, 33.9, 33.7, 31.5, 30.1, 29.02, 28.98, 27.8, 26.9,
26.7, 26.4, 25.6, 25.2, 24.9, 24.8, 24.3, 22.0, 21.6, 18.8, 18.7. Mp
(HCl salt): 142–145 °C. Anal. Calcd for C52H72N2O5ꢂ2HCl 1.8H2O:
C, 68.60; H, 8.59; N, 3.08. Found: C, 68.77; H, 8.42; N, 3.34.
a 60-min incubation at 30 °C, samples were filtered through GF/B
glass fiber filters, which were washed three times with 3-mL of
cold 50 mM Tris–HCl, pH 7.5. The filters were counted in 2 mL of
ScintSafe 30% scintillation fluid. The EC50 value and Emax values
were calculated using SIGMAPLOT software.
To determine if the compound had antagonistic properties at
the
above. To stimulate [35S]GTP
added. Twelve different concentrations of the compound were
added. To determine if a compound was an antagonist at the
receptor, membranes expressing the opioid receptor were incu-
bated with 12 concentrations of the compound and 200 nM DAM-
GO to stimulate [35S]GTP
S binding. IC50 and Imax values were
j
opioid receptor, membranes were incubated as described
cS binding, 100 nM U50,488 was
l
5.4.3. (5
a
)-17-Allyl-14-hydroxy-6-oxo-4,5-epoxymorphinan-3-
l
yl-((ꢀ)-14-methoxyl-17-N-cyclobutylmethyl)morphinan-3-yl)
decanedioate (15c)
c
Yellow oil (36 mg, 51%); 1H NMR (300 MHz, CDCl3) d 7.09 (d,
J = 8.4, 1H), 6.92 (s, 1H), 6.88–6.78 (m, 2H), 6.68 (d, J = 8.2, 1H),
5.91–5.70 (m, 1H), 5.33–5.10 (m, 3H), 4.69 (s, 1H), 3.29–2.88 (m,
12H), 2.66–1.16 (m, 42H), 0.89 (d, J = 12.3, 1H); 13C NMR
(75 MHz, CDCl3) d 207.7, 172.4, 171.3, 149.3, 147.7, 143.4, 135.0,
134.3, 132.5, 130.0, 129.9, 128.1, 122.9, 119.3, 118.6, 118.2,
118.1, 90.5, 74.1, 70.0, 62.0, 61.2, 57.6, 52.7, 50.5, 46.9, 45.6, 43.1,
42.0, 36.0, 35.7, 34.4, 33.9, 31.1, 30.5, 29.2, 29.0, 28.9, 28.1, 27.2,
25.4, 25.0, 24.8, 24.7, 22.9, 21.4, 20.8, 18.8. Mp (HCl salt): 175 °C
(dec). Anal. Calcd for C51H66N2O8ꢂ2HCl 2H2O: C, 64.89; H, 7.69; N,
2.97. Found: C, 65.07; H, 7.61; N, 3.06.
calculated with SIGMAPLOT software.
Acknowledgment
This work was supported by NIH Grants R01-DA14251 (J.L.N.)
and K05-DA00360 (J.M.B.)
References and notes
1. Dwyer, J. D.; Bloomfield, V. A. Biopolymers 1981, 20, 2323.
2. Erez, M.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem. 1982, 25, 847.
3. Jordan, B. A.; Cvejic, S.; Devi, L. A. Neuropsychopharmacology 2000, 23, S5.
4. Levac, B. A.; O’Dowd, B. F.; George, S. R. Curr. Opin. Pharmacol. 2002, 2, 76.
5. Rios, C. D.; Jordan, B. A.; Gomes, I.; Devi, L. A. Pharmacol. Ther. 2001, 92, 71.
6. Portoghese, P. S.; Ronsisvalle, G.; Larson, D. L.; Takemori, A. E. J. Med. Chem.
1986, 29, 1650.
7. Portoghese, P. S.; Larson, D. L.; Sayre, L. M.; Yim, C. B.; Ronsisvalle, G.; Tam, S.
W.; Takemori, A. E. J. Med. Chem. 1986, 29, 1855.
8. Portoghese, P. S.; Larson, D. L.; Yim, C. B.; Sayre, L. M.; Ronsisvalle, G.;
Lipkowski, A. W.; Takemori, A. E.; Rice, K. C.; Tam, S. W. J. Med. Chem. 1985, 28,
1140.
5.5. Opioid binding to the human l, j, and d opioid receptors
Chinese hamster ovary (CHO) cells stably transfected with the
human opioid receptor, d opioid receptor were obtained from
Dr. L.-Y. Liu-Chen (Temple University, Philadelphia, PA) and Dr. L.
Toll (SRI International, Palo Alto, CA), respectively. The opioid
j
l
receptors were obtained from Dr. G. Uhl (NIDA Intramural Pro-
gram, Baltimore, MD). The cells were grown in 100-mm dishes in
Dulbecco’s modified Eagle’s media supplemented with 10% fetal
bovine serum and penicillin–streptomycin (10,000 U/mL) at 37 °C
in a 5% CO2 atmosphere. The affinity and selectivity of the com-
pounds for the multiple opioid receptors were determined by incu-
bating the membranes with radiolabeled ligands and 12 different
concentrations of the compounds at 25 °C in a final volume of
1 mL of 50 mM Tris–HCl, pH 7.5. Incubation times of 60 min were
9. Portoghese, P. S.; Nagase, H.; Takemori, A. E. J. Med. Chem. 1988, 31, 1344.
10. Portoghese, P. S. J. Med. Chem. 2001, 44, 2259.
11. Daniels, J. D.; Kulkarni, A.; Xie, Z.; Bhushan, R. G.; Portoghese, P. S. J. Med. Chem.
2005, 48, 1713.
12. Bhushan, R. J.; Sharma, S. K.; Xie, Z.; Daniels, D. J.; Portoghese, P. S. J. Med. Chem.
2004, 47, 2969.
13. Owens, J. Nat. Rev. Drug Disc. 2006, 5, 105.
14. Negus, S. S.; Mello, N. K.; Portoghese, P. S.; Lin, C.-E. J. Pharmacol. Exp. Ther.
1997, 282, 44.
15. Mello, N. K.; Negus, S. S. J. Pharmacol. Exp. Ther. 1998, 286, 812.
16. Bowen, C. A.; Negus, S. S.; Zong, R.; Neumeyer, J. L.; Bidlack, J. M.; Mello, N. K.
Neuropsychopharmacology 2003, 28, 1125.
17. Neumeyer, J. L.; Zhang, A.; Xiong, W.; Gu, X.; Hilbert, J. E.; Knapp, B. I.; Negus, S.
S.; Mello, N. K.; Bidlack, J. M. J. Med. Chem. 2003, 46, 5162.
18. Peng, X.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. J. Med. Chem. 2006, 49, 256.
19. Peng, X.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. J. Med. Chem. 2007, 50, 2254.
20. Decker, M.; Fulton, B. F.; Zhang, B.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. J.
Med. Chem. 2009, 52, 7389.
used for the l j-selective li-
-selective peptide [3H]DAMGO and the
gand [3H]U69,593. A 3-h incubation was used with the d-selective
antagonist [3H]naltrindole. Nonspecific binding was measured by
the inclusion of 10 lM naloxone. Samples were filtered through
GF/B glass fiber filters, which were washed three times with 3-
mL cold 50 mM Tris–HCl, pH 7.5. The filters were counted in
2 mL of ScintiSafe 30% scintillation fluid. IC50 values were deter-
mined by log-probit analysis and were converted to Ki values by
the equation of Cheng and Prusoff.28
21. Mathews, J. L.; Peng, X.; Xiong, W.; Zhang, A.; Negus, S. S.; Neumeyer, J. L.;
Bidlack, J. M. J. Pharmacol. Exp. Ther. 2005, 315, 821.
22. Neumeyer, J. L.; Peng, X. M.; Knapp, B. I.; Bidlack, J. M.; Lazarus, L. H.; Salvadori,
S.; Trapella, C.; Bolboni, G. J. Med. Chem. 2006, 49, 5640.
23. Neumeyer, J. L.; Bidlack, J. M.; Zong, R.; Bakthavachalam, V.; Gao, P.; Cohen, D.
J.; Negus, S. S.; Mello, N. K. J. Med. Chem. 2000, 43, 114.
24. Gates, M.; Montzka, T. A. J. Med. Chem. 1964, 7, 127.
25. Portoghese, P. S. J. Med. Chem. 2000, 44, 2259.
5.6. [35S]GTP
cS binding assay to measure pharmacological
properties mediated by the kappa and mu opioid receptors
26. Garner, H. R.; Burke, T. F.; Lawhorn, C. D.; Stoner, J. M.; Wessinger, W. J.
Pharmacol. Exp. Ther. 1997, 282, 1253.
27. Zhang, A.; Xiong, W.; Hilbert, J. E.; DeVita, E. K.; Bidlack, J. M.; Neumeyer, J. L. J.
Med. Chem. 2004, 47, 1886.
28. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
To determine the agonist properties of the compounds at the
j
l
and
l opioid receptors, membranes expressing either the j or
receptor were incubated with 12 different concentrations of the
compound in 0.5 mL of buffer containing 50 mM Tris–HCl, pH