Binding of norbinaltorphimine (norBNI) congeners to wild-type and mutant mu and kappa opioid receptors: Molecular recognition loci for the pharmacophore and address components of kappa antagonists

Article abstract of DOI:10.1021/jm000059g

Molecular modifications of both the kappa opioid antagonist norbinaltorphimine (norBNI, 1) and the kappa receptor have provided evidence that the selectivity of this ligand is conferred through ionic interaction if its N17' protonated amine group (an 'address') with a nonconserved acidic residue (Glu297) on the kappa receptor. In the present study, we have examined the effect of structural modifications on the affinity of norBNI analogues for wild-type and mutant kappa and mu opioid receptors expressed in COS-7 cells. Compounds 2, 3, and 7, which have an antagonist pharmacophore and basic N17' group in common with norBNI, retained high affinity for the wild-type kappa but exhibited greatly reduced affinity for mutant kappa receptors (E297K and E297A). Modification of the phenolic or N-substituent groups of the antagonist pharmacophore (4 and 5) or removal of basicity at the address N17' center (6) led to greatly reduced affinity for the wild-type and mutant receptors. The reduced affinity upon modification of the kappa receptor is consistent with the ionic interaction of the protonated N17' group of kappa antagonists (1-3, 7) with the carboxylate group of E297 at the top of TM6. This was supported by the greatly enhanced affinity of compounds 1-3 for the mutant mu receptor (K303E), as compared to the wild-type mu receptor, given that residue K303 occupies a position equivalent to that of E297 in the kappa receptor. In view of the high degree of homology of the seven TM domains of the kappa and mu opioid receptors, it is suggested that the antagonist pharmacophore is bound within this highly conserved region of the kappa or mutant mu receptor and that an anionic residue at the top of TM6 (E297 or K303E, respectively) provides additional binding affinity.

Full text of DOI:10.1021/jm000059g

J . Med. Chem. 2000, 43, 1573-1576
1573
Bin d in g of Nor bin a ltor p h im in e (n or BNI) Con gen er s to Wild -Typ e a n d Mu ta n t
Mu a n d Ka p p a Op ioid Recep tor s: Molecu la r Recogn ition Loci for th e
P h a r m a cop h or e a n d Ad d r ess Com p on en ts of Ka p p a An ta gon ists
†
†
‡
‡
,†
Dennis L. Larson, Robert M. J ones, Siv A. Hjorth, Thue W. Schwartz, and Philip S. Portoghese*
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, and
Laboratory for Molecular Pharmacology, Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark
Received February 15, 2000
Molecular modifications of both the kappa opioid antagonist norbinaltorphimine (norBNI, 1)
and the kappa receptor have provided evidence that the selectivity of this ligand is conferred
through ionic interaction if its N17′ protonated amine group (an “address”) with a nonconserved
acidic residue (Glu297) on the kappa receptor. In the present study, we have examined the
effect of structural modifications on the affinity of norBNI analogues for wild-type and mutant
kappa and mu opioid receptors expressed in COS-7 cells. Compounds 2, 3, and 7, which have
an antagonist pharmacophore and basic N17′ group in common with norBNI, retained high
affinity for the wild-type kappa but exhibited greatly reduced affinity for mutant kappa receptors
(E297K and E297A). Modification of the phenolic or N-substituent groups of the antagonist
pharmacophore (4 and 5) or removal of basicity at the address N17′ center (6) led to greatly
reduced affinity for the wild-type and mutant receptors. The reduced affinity upon modification
of the kappa receptor is consistent with the ionic interaction of the protonated N17′ group of
kappa antagonists (1-3, 7) with the carboxylate group of E297 at the top of TM6. This was
supported by the greatly enhanced affinity of compounds 1-3 for the mutant mu receptor
(
K303E), as compared to the wild-type mu receptor, given that residue K303 occupies a position
equivalent to that of E297 in the kappa receptor. In view of the high degree of homology of the
seven TM domains of the kappa and mu opioid receptors, it is suggested that the antagonist
pharmacophore is bound within this highly conserved region of the kappa or mutant mu receptor
and that an anionic residue at the top of TM6 (E297 or K303E, respectively) provides additional
binding affinity.
In tr od u ction
K303E, in the latter. The results support the proposal
that a glutamate residue (E297) in the wild-type kappa
receptor or in the mutant (K303E) mu receptor interacts
with the protonated N17′ group of norBNI and its potent
analogues. Moreover, the data suggest that the kappa
and mutant mu receptor environments surrounding the
bound antagonist pharmacophore of norBNI are simi-
The prototypical kappa opioid receptor antagonist,
norbinaltorphimine, 1 (norBNI), contains two basic
nitrogens, N17 and N17′.1,2 The N17 basic nitrogen is
associated with the antagonist pharmacophore, whereas
N17′ was proposed to function as an “address” in
conferring kappa selectivity. It was proposed that the
3
8
lar.
“address” recognition locus on the kappa receptor con-
tains a uniquely positioned acidic residue that ion-pairs
with N17′. Support for this was obtained from structure-
activity relationship (SAR) studies which demonstrated
that the second pharmacophore is not necessary for the
kappa antagonist activity of norBNI and that the N17′
basic nitrogen (the “address”) is a key requirement for
selectivity.4 The finding that amidation of N17′ de-
stroys kappa antagonist activity was consistent with
Ch em istr y
The synthesis and biological activities of compounds
3
,6
1-4 and 6 have been reported earlier. Compound 5
was prepared by first forming the 3-O-methyl ether 9
by treatment of naltrexone (8) with diazomethane
(Scheme 1) and then reacting 9 with excess oxymor-
phone (10) in the presence of hydrazine under acidic
conditions. Excess oxymorphone helped to minimize
formation of the 3-O-methyl ether dimer.
,5
5
,6
this proposal.
Subsequent studies on mutant and
chimeric kappa-mu opioid receptors revealed this
residue to be Glu297 (E297) which is located at the top
of transmembrane helix 6 (TM6).7
Compound 7 was obtained by reaction of the corre-
sponding 17′-desalkyl analogue 11 (Scheme 2) with
5
Here we present data on the binding of norBNI-
related ligands (2-7) to cloned kappa and mu receptors
that have been mutated at position 297 (E297X, X ) K
or A) in the former and at an equivalent position,
bisBOC-thiopseudourea and mercury(II) chloride using
Kim’s guanidylation procedure, followed by removal of
the BOC groups with trifluoroacetic acid.
9
Op ioid Recep tor Bin d in g Affin ity
*
To whom correspondence should be addressed. Tel: 612-624-6184.
Receptor binding was carried out on whole transfected
COS-7 cells. The effect of exchanging key amino acid
residues in cloned kappa (E297X) and mu (K303E)
Fax: 612-626-6891. E-mail: porto001@tc.umn.edu.
†
University of Minnesota.
Rigshospitalet.
‡
1
0.1021/jm000059g CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/30/2000

1
574 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Larson et al.
Sch em e 1
with 17′-cyclopropylmethyl, -methyl, and -amidine groups
(2, 3, 7) all retain high affinity for the wild-type kappa
receptor. On the other hand, since the acetyl group
eliminates the basicity of N17′, it greatly reduces the
affinity of 6, presumably because such a modification
eliminates counterionic attraction for E297.
The finding that the mutant kappa receptors (E297K
and E297A) display greatly reduced affinity for norBNI
(1) and its analogues (2, 3, 7) that contain a basic N17′
group is consistent with the participation of counterions
for high-affinity binding. This is due to the fact that
lysine and alanine are unable to interact with N17′ as
counterions. Thus, the cationic “address” (protonated
N17′) of these ligands and the anionic carboxylate group
(E297) of the kappa receptor possibly may confer affinity
for the kappa receptor through the formation of a salt
bridge.
The fact that E297 is not conserved among mu and
delta receptors appears to be the basis for the kappa
selectivity of norBNI, inasmuch as the equivalent posi-
tion in mu and delta opioid receptors contains lysine
opioid receptors on the binding affinity of norBNI
analogues is presented in Table 1. Among these com-
pounds, norBNI (1) showed the highest binding affinity
for the wild-type kappa receptor and exhibited a 100-
fold loss of binding affinity for both mutant receptors
compared to that for the wild type. Compounds 2, 3, and
(
K303) and tryptophan (W284) residues, respectively.10
In this regard, it is likely that the protonated amino
group of K303 inhibits binding of norBNI through ionic
repulsion of the protonated N17′ group in the active
kappa antagonists. The bulky, hydrophobic tryptophan
residue (W284) in the delta receptor may inhibit binding
through steric repulsion.
7
, which also possess an antagonist pharmacophore unit
in their structures, exhibited binding affinity compa-
rable to that of 1 for the kappa wild-type receptor and
also showed a similar degree of loss in mutant kappa
receptor binding affinity. On the other hand, compound
6
, which possesses the same antagonist pharmacophore
moiety as 1 but differs by an amide group at N17′
versus the basic character at N17′ for the other
The role of E297 as an anionic residue that interacts
with the cationic “address” of kappa antagonists has
received added support from studies with the mu
receptor mutant whose lysine residue was replaced by
(
compounds), exhibited a >100-fold less binding affinity
for the wild-type kappa receptor.
7
glutamate (K303E). In this mutant, the glutamate
The importance of the N-cyclopropyl and phenolic
functions to the antagonist activity of bimorphinans, as
reported earlier in functional assays, is complemented
residue is located in a position equivalent to that of E297
in the kappa receptor. The mutant mu receptor dis-
played greatly enhanced affinity for 1-3 relative to the
wild-type mu receptor, again highlighting the critical
role of E297 in the kappa receptor. What is more, the
data suggest that similar binding domains of the kappa
and mutant mu receptors recognize the antagonist
3
in the present study. Compound 5, which contains a
methoxy function in its antagonist pharmacophore,
exhibited a 100-fold reduction in binding affinity for the
kappa wild-type receptor.
The binding of 1-6 on the wild-type mu receptor was
uniformly low. However, the affinity of compounds 1-3
for the mutant mu receptor (K303E) was increased by
a factor of 100-200-fold over that for the wild-type mu
receptor. The difference was similar to that observed
between the wild-type and mutant κ opioid receptors.
7
pharmacophore of the high-affinity ligands (1-3). This
supports the idea that the recognition locus for the
pharmacophore is in the central cavity created by the
seven TM helices, as they have the greatest homology
compared to potential extracellular binding sites on the
8
receptor.
It can be noted that ligands (4, 5) with an “address”,
but without an accompanying antagonist pharmaco-
phore, possess low affinity for the kappa receptor. Since
the phenolic OH and the N17-cyclopropylmethyl group
are required for recognition of the pharmacophore, their
absence in 4 and 5 leads to the >200-fold loss of affinity
Discu ssion
The binding data from the present study complements
published functional SAR studies of norBNI analogues.
The substituent attached to N17′ does not appear to be
critical as long as basicity is maintained. Thus, ligands
3
-6
Sch em e 2

Binding of norBNI Congeners to Opioid Receptors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1575
Ta ble 1. Effect of Site-Directed Mutagenesis on Binding to Kappa and Mu Opioid Receptors
a
i
K (nM)
compd
R1
R2
R3
R4
R5
wtκ
(E297K)κ
(E297A)κ
wtµ
(K303E)µ
1b
CH
CH
CH
CH
CH
CH
CH
CH(CH
CH(CH
CH(CH
)
)
)
H
H
H
H
CH
H
H
CH
CH
CH
CH
CH
CH(CH
)
)
OH
H
OH CH
3
OH
OH
OH
OH
H
H
0.13 ( 0.04 (10) 12.9 ( 1.7 (9)
0.32 ( 0.04 (12) 39.9 ( 6.2 (4)
0.39 ( 0.03 (11) 28 ( 2.6 (4)
21.7 ( 4.8 (10) >1000 (4)
12.8 ( 1.7 (9)
39.6 ( 6.2 (4)
17 ( 10.6 (4)
>1000 (4)
70.0 ( 17.9 (4) 0.94 ( 0.09 (10)
2
2
2
3
2
2
2
2
2
2
2
2
2
2
3
2
3
3
2
2
2
2
b
b
b
2
3
4
5
6
7
40.0 ( 5.5 (4)
276 ( 32.3 (2)
>1000 (4)
0.16 ( 0.07 (3)
2.7 ( 0.3 (3)
41.7 ( 10.4 (4)
52 ( 19 (4)
CH(CH
H
H
H
H
CH(CH
CH(CH
CH(CH
2
2
2
)
)
)
2
2
2
3
36.3 ( 3.0 (8)
23.6 ( 4.6 (9)
0.57 (1)
>1000 (3)
104 ( 8.4 (4)
189 (2)
>1000 (2)
165 ( 2.3 (4)
102 ( 27.5 (4)
c
COCH
(CdNH)NH
3
104 ( 8.4 (4)
42 ( 4.8 (3)
2
diprenor-
phine
0.28 ( 0.05 (11) 0.37 ( 0.05 (16) 0.38 ( 0.05 (16) 0.27 ( 0.05 (10) 0.33 ( 0.10 (12)
a
3
Ki values were determined from competition binding curves using [ H]diprenorphine as the radioligand and analyzed on intact
transfected COS-7 cells in the same manner as described previously.7 Column headings, such as “wtκ”, refer to the wild-type kappa
receptor preparation, and “(E297K)κ” relates to the preparation resulting from mutagenic exchange of glutamic acid residue 297 for
lysine of the kappa receptor. Data are presented as mean ( standard error with the number of replicate experiments indicated in
parentheses. Preparation and smooth muscle pharmacology described in ref 3. c Preparation and smooth muscle pharmacology described
in ref 5.
b
relative to norBNI. Consequently, it is the combined
affinity of the pharmacophore and the “address” that
contributes to the high affinity of norBNI and other
potent kappa antagonists in the series.
Con clu sion
Molecular modification of both norBNI and opioid
receptors has led to greater insight into the molecular
recognition of norBNI and its analogues by kappa opioid
receptors. The “address” (N17′) of the ligands must be
sufficiently basic to be in the protonated state at
physiologic pH in order for it to associate with the
carboxylate group of E297 in the kappa receptor. The
protonated N17′ address is rigidly held in a specific
orientation by a scaffold which directs it to E297 when
F igu r e 1. Model of norBNI (1) docked to its recognition site
on the wild-type kappa receptor. Note the association of N17
and N17′ with residues D138 in TM3 and E297 in TM6,
respectively. Transmembrane helices TM1 and TM4 have been
deleted to aid visualization.
the ligand is bound to the kappa receptor.1
1,12
In this
regard, the geometry and length of the scaffold are
important for bringing the counterions together, as it
has been reported that a nonlinear scaffold leads to
1
1
greatly reduced kappa antagonist potency. Using
norBNI as a template, the distance between the N17
and N17′ basic groups is ∼11 Å, and this approximately
matches the distance between the conserved aspartate
residue in TM3 (D138) and the nonconserved glutamate
4
00 mesh; Aldrich Chemical Co.) as the stationary phase and
nitrogen pressure. Thin-layer chromatography was performed
on silica gel 60 F254 0.25-nm aluminum-backed sheets (E.
Merck) and visualized with UV light, phosphomolybdic acid,
or iodine vapor. Chromatographic elution solvent systems are
reported as volume/volume ratios. IR spectra were recorded
on a Perkin-Elmer PE-281 spectrophotometer or a Nicolet
1
3
(
E297) at the top of TM6 (Figure 1). This correspon-
dence suggests that the cationic groups of norBNI and
its potent analogues are proximal to D138 and E297
when bound in the central cavity of the kappa receptor.
Given that the seven-TM domain of kappa and mu
receptors possesses a high degree of homology,1 and the
finding that the mutant mu receptor (K303E) binds
kappa antagonists with high affinity, it appears likely
that the antagonist pharmacophore is bound within this
highly conserved region of the kappa receptor.
5
DXC FT-IR spectrometer using KBr disks. NMR spectra were
obtained using a Varian Unity 300 MHz or Varian Inova 300
MHz instrument at room temperature and CDCl , CD OD, or
3 2
CD ) SdO as solvents. The δ scale (ppm) was in reference to
3
3
(
4
the deuterated solvent, and coupling constants (J ) are reported
in hertz (Hz). Mass spectra (FAB) were obtained on a VG-
7
07EHF spectrometer using a m-nitrobenzyl alcohol (MNOBA)
matrix or (EI) on a Finnigan MAT 95 instrument. The purity
of compounds was assessed by analytical HPLC on an Alltech
C18, 5-µm, 4.6-mm × 250-mm column using 10-15% aqueous
3
acetonitrile + 0.1% CF COOH run isocratically. Melting points
Exp er im en ta l Section
were determined in open capillary tubes with a Thomas-
Hoover melting point apparatus and are uncorrected.
17-Meth yl-17′-cyclop r op ylm eth yl-3′-m eth oxy-6,6′,7,7′-
tetr a d eh yd r o-4,5r:4′,5′r-d iep oxy-6,6′-(im in o)[7,7′-bim or -
All reagents were obtained from the Aldrich Chemical Co.
unless stated otherwise. Naltrexone was obtained from
Mallinckrodt. All reactions described were conducted in an
inert atmosphere of argon or nitrogen unless otherwise stated.
Column chromatography was performed using silica gel (200-
1
5
p h in a n ]-3,14,14′-tr iol (5). Naltrexone 3-O-methyl ether (9)
was prepared by reacting naltrexone (315 mg, 1.0 mmol) with

1
576 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Larson et al.
excess diazomethane (generated from 1-methyl-3-nitro-1-ni-
trosoguanidine and sodium hydroxide in biphasic Et O/water)
in a mixture of tetrahydrofuran (5 mL) and diethyl ether (5
mL) at ambient temperature. Upon completion of the reaction,
the solvent was removed under reduced pressure to afford an
isolated by filtration and purified by RP-HPLC (Alltech C18,
2
10 µm, 22 mm × 250 mm; water-acetonitrile-TFA, 90:10:
0.25, isocratic) to afford 7‚2CF COOH as an amorphous solid:
3
1
yield 15 mg (34%); mp >230 °C; H NMR (CD OD, 300 MHz)
3
δ 6.49 (m, H1, H2, H1′, H2′), 5.45 (s, 1H, H5), 5.42 (s, 1H,
H5′), 4.17 (d, 1H, J ) 7.0 Hz, H9′), 3.71 (dd, 1H, H9, J ) 4.7
Hz, J ) 14.5 Hz), 3.11-3.42 (m, 5H), 2.61-3.11 (m, 5H), 2.20-
oil, which was chromatographed on silica using CHCl
3
-NH
0.52
) δ 6.68 & 6.60 (2d,
);
4
-
OH (99:1) to afford 9 as an oil: 270 mg, 76%; TLC R
f
1
(
2
CHCl
3
4 3
-NH OH, 99:1); H NMR (CDCl
2
1
.61 (m, 6H), 1.73 (m, 1H, H15), 1.60 (m, 1H, H15′), 0.96 (m,
H, J ) 8.15 Hz, C1 & C2), 4.66 (s, 1H, C5), 3.88 (s, 3H, OCH
EI MS m/z 355 (M , 100%); mp 236 °C (hydrochloride salt).
3
13
H, H19), 0.60 (m, H
2
O & H21), 0.29 (m, H20 & H21); C NMR
+
(CD
3
OD, 75.4 MHz) δ 159.44, 145.04, 144.76, 141.56, 141.12,
Oxymorphone (10) (344 mg, 1.14 mmol) was dissolved in
glacial acetic acid (5 mL) and mixed with a solution of 9 (135
mg, 0.38 mmol) in glacial acetic acid (5 mL). Hydrazine
dihydrochloride (80 mg, 0.76 mmol) was added, and the
reaction mixture was stirred at 90 °C for 24 h and for an
additional 24 h at 95 °C under a stream of nitrogen. The
reaction mixture was cooled, and the volatile components were
removed under reduced pressure with the aid of a heptane
azeotrope. The oily residue was subjected to column chroma-
132.27, 131.81, 131.63, 126.66, 126.40, 126.24, 126.01, 125.04,
124.83, 119.91, 119.77, 119.50, 118.44, 117.94, 116.70, 116.17,
86.18, 85.91, 74.67, 74.63, 74.48, 63.52, 61.01, 60.46, 55.18,
44.95, 41.11, 35.94, 33.64, 33.41, 32.72, 30.84, 30.45, 29.94,
2
C
4.05, 21.85, 10.19, 4.62, 4.14; HRMS (FAB) m/z (%) calcd for
+
37
H
39
N
5
O
6
(M + H) 650.2978, obsd 650.2849.
Ack n ow led gm en t. This work was supported by the
National Institute on Drug Abuse. We thank Dr. M.
Germana Paterlini for valuable assistance in construct-
ing colored graphics.
tography using an elution gradient of CHCl
0%)-NH OH (1%) to yield pure 5: 32 mg (18%); mp (dihy-
drochloride salt) >260 °C; TLC R 0.44 (EtOAc-MeOH-
, 300 MHz) δ 8.47 (s, 1H,
pyrrole NH), 6.52 (m, 4H, H2, H2′, H1, H1′), 5.41 (s, 1H, H5),
.37 (s, 1H, H5′), 3.70 (s, 3H, CH O), 2.26 (s, 3H, NCH ), 0.79
m, 1H, cyclopropane CH), 0.41 (m, 4H, cyclopropane CH R),
, 75.4 MHz)
3
-MeOH (0-
1
4
f
1
4 3
NH OH, 90:10:1); H NMR (CDCl
Refer en ces
5
3
3
(
0
2
(1) Portoghese, P. S.; Lipkowski, A. W.; Takemori, A. E. Bimorphi-
nans as highly selective potent kappa receptor antagonists. J .
Med. Chem. 1987, 30, 238-239.
1
3
.12 (m, 4H, cyclopropane CH
2
â); C NMR (CDCl
3
δ 144.7 (C3′), 143.3 & 143.0 (C4 & C4′), 139.0 (C3), 130.8 (C12),
30.5 (C12′), 125.6 (C11), 125.1 (C11′), 124.6 (C6 & C6′), 118.7
C1), 118.1 (C1′), 117.3 (C2), 116.2 (C7), 116.1 (C7′), 113.2 (C2′),
5.8 (C5), 85.2 (C5′), 73.1 (C14), 72.8 (C14′), 65.0 (C9), 62.4
C9′), 59.4 (C18 & C18′), 56.1 (CH O), 47.8 (C13), 47.4 (C13′),
), 31.6, 31.3 (C8 & C8′), 28.9,
(
2) Portoghese, P. S.; Lipkowski, A. W.; Takemori, A. E. Binaltor-
phimine and nor-binaltorphimine, potent and selective κ-opioid
receptor antagonists. Life Sci. 1987, 40, 1287-1292.
1
(
8
(3) Portoghese, P. S.; Nagase, H.; Lipkowski, A. W.; Larson, D. L.;
Takemori, A. E. Binaltorphimine-related bivalent ligands and
their κ opioid receptor antagonist selectivity. J . Med. Chem.
(
3
4
2
4
1
5.4 (C16), 43.7 (C16′), 42.9 (NCH
3
1
988, 31, 836-841.
8.8 (C10 & C10′), 23.1, 22.4 (C15 & C15′), 7.5 (C19 & C19′),
.0 (C20 & C20′), 3.8 (C21 & C21′); IR (KBr) cm-1 3416 (s),
(4) Portoghese, P. S.; Nagase, H.; Takemori, A. E. Only one
pharmacophore is required for the kappa opioid antagonist
selectivity of norbinaltorphimine. J . Med. Chem. 1988, 31, 1344-
636 (m), 1506 (m), 1457 (w), 1328 (w), 1121 (m), 1046 (m);
+
HRMS (FAB) m/z calcd for C38
obsd 636.3088.
H
41
N
3
O
6
(M + H) 636.3074,
1
347.
(5) Portoghese, P. S.; Lin, C.-E.; Farouz-Grant, F.; Takemori, A. E.
Structure-activity relationship of N17′-substituted norbinal-
torphinine congeners. Role of the N17′ basic group in the
interaction with a putative address subsite on the κ opioid
receptor. J . Med. Chem. 1994, 37, 1495-1500.
1
7-Cyclop r op ylm eth yl-17′-gu a n id in yl-6,6′,7,7′-tetr a d e-
h yd r o-4,5r:4′,5′r-d iep oxy-6,6′-(im in o)[7,7′-bim or p h in a n ]-
′,3,14,14′-tetr ol (7). 17-Cyclopropylmethyl-6,6′,7,7′-tetrade-
hydro-4,5R:4′,5′R-diepoxy-6,6′-(imino)[7,7′-bimorphinan]-
3
(
6) Lin, C. E.; Takemori, A. E.; Portoghese, P. S. Synthesis and
kappa-opioid antagonist selectivity of a norbinaltorphimine
congener. Identification of the address moiety required for
kappa-antagonist activity. J . Med. Chem. 1993, 36, 2412-2415.
7) Hjorth, S. A.; Thirstrup, K.; Grandy, D. K.; Schwartz, T. W.
Analysis of selective binding epitopes for the κ-opioid receptor
antagonist nor-binaltorphimine. Mol. Pharmacol. 1995, 47,
5
3
′,3,14,14′-tetrol (11) (73.4 mg, 0.12 mmol) was dissolved in
anhydrous dimethylformamide (3.0 mL) and cooled to 0 °C in
an ice bath. 1,3-Bis(tert-butoxycarbonyl)-2-methylthiopseudourea
(
(36 mg, 0.13 mmol) and triethylamine (0.052 mL, 0.37 mmol)
were added sequentially, and the reaction mixture was allowed
to stir for 10 min at 0 °C. Mercury(II) chloride (1.1 equiv, 35
mg) was added in one portion, and rapid stirring was main-
tained for 20 min, after which the ice bath was removed and
the reaction mixture was allowed to attain ambient temper-
ature over 1 h. The mixture was filtered through a Celite pad
under vacuum to remove mercuric sulfide, and the pad was
subsequently washed repeatedly with methanol. Removal of
all volatile components under reduced pressure produced an
oil, which was subjected to flash column chromatography using
1
089-1094.
(8) J ones, R. M.; Hjorth, S. A.; Schwartz, T. W.; Portoghese, P. S.
Mutational evidence for a common kappa antagonist binding
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(
9) Kim, K. Y.; Quian, L. Improved method for the preparation of
guanidines. Tetrahedran Lett. 1988, 29, 3183.
(
10) http://www.opioid.umn.edu.
(
11) Bolognesi, M. L.; Ojala, W. H.; Gleason, W. B.; Griffin, J . F.;
Farouz-Grant, F.; Larson, D. L.; Takemori, A. E.; Portoghese,
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CHCl
3
4
-MeOH-NH OH (80:10:1) to afford the N17′-bisBOC-
guanidinyl derivative 12 as a white crystalline solid: yield 74
mg (73%); H NMR (CD OD, 300 MHz) δ 6.49 (m, H1, H2, H1′,
1
3
H2′), 5.45 (s, 1H, H5), 5.42 (s, 1H, H5′), 4.17 (d, 1H, J ) 7.0
Hz, H9′), 3.71 (dd, 1H, J ) 4.7 Hz, J ) 14.5 Hz, H9), 3.11-
(
12) Portoghese, P. S.; Garzon-Aburbeh, A.; Nagase, H.; Lin, C. E.;
Takemori, A. E. Role of the spacer in conferring kappa opioid
receptor selectivity to bivalent ligands related to norbinaltor-
phimine. J . Med. Chem. 1991, 34, 1292-1296.
3
1
.42 (m, 5H), 2.61-3.11 (m, 5H), 2.20-2.61 (m, 6H), 1.73 (m,
H, H15), 1.60 (m, 1H, H15′), 1.43 (s, 9H, t-BOC), 1.41 (s, 9H,
t-BOC), 0.96 (m, 1H, H19), 0.60 (m, H20R & H21R), 0.29 (m,
(13) Metzger, T. G.; Paterlini, M. G.; Portoghese, P. S.; Ferguson, D.
M. Application of the message-address concept to the docking
of naltrexone and selective naltrexone-derived opioid antagonists
into opioid receptor models. Neurochem. Res. 1996, 21, 1287-
H20â & H21â); HRMS (FAB) m/z (%) calcd for C47
58 5 10
H N O
+
(
M + H) 853.4262, obsd 853.3829.
Removal of the BOC groups was accomplished by dissolving
1
294.
1
2 (50 mg, 0.05 mmol) in anhydrous dichloromethane (4 mL)
(14) Satoh, M.; Minami, M. Molecular pharmacology of the opioid
under nitrogen, cooling the solution to 0 °C, and then adding
trifluoroacetic acid (1 mL) dropwise over a 10-min period. The
reaction mixture was allowed to stir at ambient temperature
for a further 48 h, and then the volatile components were
removed under reduced pressure and the resultant oil was
washed with diethyl ether. The residual white precipitate was
receptors. Pharmacol. Ther. 1995, 68, 343-364.
(
15) Archer, S.; Seyed-Mozaffari, A.; Ward, S.; Kosterlitz, H. W.;
Paterson, S. J .; McKnight, A. T.; Corbett, A. D. 10-Ketonal-
trexone and 10-Ketooxymorphone. J . Med. Chem. 1985, 28,
9
74-976.
J M000059G