A bivalent ligand (KMN-21) antagonist for μ/κ heterodimeric opioid receptors

Article abstract of DOI:10.1016/j.bmcl.2009.10.045

In an effort to develop antagonists for κ-μ opioid receptor heterodimers, a series of bivalent ligands 3-6 containing κ- and μ-antagonist pharmacophores were designed and synthesized. Evaluation of the series in HEK-293 cells revealed 4 (KMN-21) to select

Full text of DOI:10.1016/j.bmcl.2009.10.045

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6978–6980
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A bivalent ligand (KMN-21) antagonist for l/j heterodimeric opioid receptors
*
Shijun Zhang, Ajay Yekkirala, Ye Tang, Philip S. Portoghese
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to develop antagonists for j–l opioid receptor heterodimers, a series of bivalent ligands 3–6
Received 7 September 2009
Revised 8 October 2009
Accepted 9 October 2009
Available online 4 November 2009
containing
in HEK-293 cells revealed 4 (KMN-21) to selectively antagonize the activation of
gesting possible bridging of receptors when the bivalent ligand spacer contains 21 atoms.
Published by Elsevier Ltd.
j
- and
l
-antagonist pharmacophores were designed and synthesized. Evaluation of the series
j–l
heterodimers, sug-
Keywords:
Opioid receptors
Dimerization
Bivalent ligand
Heterodimers
The opioid receptors belong to the superfamily of G-protein
coupled receptors (GPCRs) and play important roles in pain percep-
tion and regulation. Three major types of opioid receptors includ-
j
–d bivalent ligands15,16 and our desire to maintain a favorable
hydrophilic and lipophilic balance coupled with flexibility. This in-
cluded a spacer that contains (1) glycine units that maintain a
favorable hydrophilic-lipophilic balance, (2) a succinyl unit that
contributes to the flexibility for favorable interaction with hetero-
dimers, and (3) an alkylamine moiety attached to pharmacophore
1 which permits variation of the spacer length by one atom incre-
ments (Fig. 1).
ing
j-, d-, and l-opioid receptors have been well characterized
and cloned.1 Numerous reports have demonstrated the dimeriza-
tion/oligomerization of GPCRs,2 and the existence of dimers for
opioid receptors in cultured cells.3–8 In addition, pharmacological
evidence had suggested that multiple phenotypic
j-opioid recep-
tor subtypes are related to heterodimeric opioid receptors. For
The synthetic protocol for 3–6 is shown in Schemes 1 and 2.
Briefly, coupling reaction between carboxylic acid 7 and naltrexam-
ine 8, which were synthesized as reported15,18, gave intermediate 9,
which on hydrolysis with TFA in dichloromethane afforded carbox-
ylic acid intermediate 10 (Scheme 1). The reactions of monoprotect-
ed alkyldiamines 11–14 with KSCN followed by Cbz-protection
yielded 15–18, which upon condensation with 19 in the presence
of HgCl2 and Et3N followed by deprotection, provided 20–23 in good
example,
j–d and j–l heterodimers have been linked to the j2
subtype.7,9 Therefore it would be of value to develop chemical tools
to assist in the pharmacological characterization of opioid receptor
heterodimers.
In this regard, bivalent ligands have been developed as pharma-
cological tools to study the dimerization of opioid receptors.10–14
Recently, we had reported two selective bivalent ligands KDN-21
and KDAN-18, that have suggested the association of d1 and j2
phenotypes with
j
–d heterodimers.15,16 In view of the promising
results from this bivalent ligand strategy and evidence showing
the association of opioid receptors via homo- and hetero-dimeriza-
tion,4–8 we have extended the bivalent strategy to design tools for
N
N
H
N
OH
O
HO
NH2
NH
N
H
j
–
l
heterodimers.
Here we report on the synthesis and in vitro biological charac-
terization of a series of bivalent ligands 3–6 containing a -opioid
antagonist pharmacophore 50-guanidinonaltrindole (50-GNTI) (1)17
O
2
H2N
OH
O
OH
1
j
N
OH
and a l-opioid antagonist pharmacophore b-naltrexamine (b-NTX)
N
O
O
H
H
N
NH
H
H
N
H
N
OH
O
(2)18 linked through a spacer of varying length. The design ratio-
N
N
N
H
N
H
N
H
n
nale of these bivalent ligands is based on our previous studies of
O
O
O
O
N
H
OH
3-6: n=2-5
Figure 1. Designed bivalent ligands.
OH
* Corresponding author. Tel.: +1 612 624 9174; fax: +1 202 513 8609.
E-mail address: porto001@umn.edu (P.S. Portoghese).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.10.045

S. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6978–6980
6979
N
O
O
H
N
DCC, HOBt
O
HO
N
+
2
2
NH2
N
H
O
O
DMF
O
O
7
OH
8
N
TFA/CH2Cl2
O
O
O
H
N
H
N
O
2
OH
2
N
H
N
N
N
H
2
2
H
H
O
O
O
O
O
O
9
10
OH
OH
Scheme 1. The synthetic route for the carboxylic acid intermediate 10.
NCbz
HN
O
S
1) 19, HgCl2, Et3N
HN
N
Cbz-Cl
KSCN
OH
O
n
NH2
H2N NHBoc
n
11-14: n=2-5
Ph
O
N
H
N
H
nNHBoc
2) HCl in ether
N
H
15-18: n=2-5
20-23: n=2-5
OH
NH2
N
OH
1) 10, DCC, HOBt, DMF
2) Cyclohexadiene/Pd-C
N
H
O
19
OH
Bivalent ligands 3-6
Scheme 2. The synthetic route for the designed bivalent ligands 3–6.
yield. Finally, standard amide coupling reactions of 10 with 20–23
followed by catalytic hydrogenation afforded the desired bivalent li-
gands 3–6 (Scheme 2). The chemical structures of bivalent ligands
were characterized and confirmed by 1H NMR and Fast-atom bom-
bardmentmassspectra(FABMS)(ESI). Thepurityofdesignedligands
was determined by reverse HPLC (Acetonitrile/H2O/TFA: 50/50/0.1
and MeOH/H2O/TFA: 30/70/0.1) to be >98%.
After synthesis, the antagonist activities of 3–6 were evaluated
by measuring inhibition of Ca2+ release in HEK-293 cells that stably
express j- and l-opioid receptors singly or together (Fig. 2). A chi-
meric G-protein was transiently transfected in this assay for Ca2+
release.19 The selective agonists used to evaluate the antagonism
selectivity of the target compounds were U69, 59320
(j
), and DAM-
GO (l lM concentra-
).21 While all of the bivalent ligands 3–6 (1
tion) antagonized Ca2+ release in cells containing singly
expressed receptors, only bivalent ligand 4 (KMN-21) significantly
antagonized both U69593- and DAMGO-induced Ca2+ release in
cells containing coexpressed
suggest that bivalent ligand 4 with a spacer length of 21 atoms en-
gages opioid heterodimers more efficiently than homodimers.
In this regard, it is noteworthy that the –d bivalent ligand antag-
onist, KDN-21, also contains a 21-atom spacer, suggesting common
bridging modes to and
–d heterodimeric receptors.16 To
j- and l-opioid receptors. These data
j–l
j
j–l
j
further understand the interactions of bivalent ligand 4 with j–l
heterodimers, additional studies would be required.
In summary, a series of bivalent ligands containing
j- and l-
opioid antagonist pharmacophores attached to variable length
spacers were designed and synthesized as chemical tools to inves-
Figure 2. Compound 4 (KMN-21) is an antagonist in HEK-293 cells coexpressing
l-
and -opioid receptors. Intracellular calcium release experiments were performed
j
tigate
revealed that bivalent ligand 4 with 21 atoms in its spacer signifi-
cantly antagonized Ca2+ release of activated
- and -opioid recep-
j–l heterodimers. Biological evaluation in HEK-293 cells
using appropriate agonist ligands and compounds 3–6 in HEK-293 cells stably
expressing (a) mu, (b) kappa, (c and d) mu/kappa opioid receptors. Cells were
transiently transfected with chimeric G-protein,
40,000 cells) and pre-incubated with antagonist compounds 3–6 (1
D
6-Gqi4-myr (200 ng for every
M) and dye
j
l
l
tors. Together with our previous results of different bivalent
ligands,16,17 the results further exemplifies the power of bivalent
ligands as pharmacological tools in investigating the dimerization
of opioid receptors in particular and GPCRs in general.
from calcium release kit (Molecular Devices). Intracellular calcium release was
measured in a Flexstation apparatus (Molecular Devices) using DAMGO or U69593
(1
l
M). Experiments were performed in triplicate (n = 4) and significance was
**
measured using ANOVA ( = p < 0.01).

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S. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6978–6980
10. Portoghese, P. S.; Ronsisvalle, G.; Larson, D. L.; Yim, C. B.; Sayre, L. M.; Takemori,
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We thank Mike Powers for capable technical assistance. This re-
search is supported by grant DA01533 from National Institute on
Drug Abuse.
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