High-affinity mu opioid receptor ligands discovered by the screening of an exhaustively stereodiversified library of 1,5-enediols

Article abstract of DOI:10.1021/ja027150p

In this communication, we report the synthesis of an exhaustively stereodiversified library of 16 1,5-enediols (2) and the screening of these compounds for mu opioid receptor (MOR) binding. The stereochemical configuration of 2 strongly impacted the bindi

Full text of DOI:10.1021/ja027150p

Published on Web 10/23/2002
High-Affinity Mu Opioid Receptor Ligands Discovered by the Screening of an
Exhaustively Stereodiversified Library of 1,5-Enediols
Bryce A. Harrison,† Tiffany Malinky Gierasch,† Claire Neilan,‡ Gavril W. Pasternak,‡ and
Gregory L. Verdine*,†
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138, and
Laboratory of Molecular Neuropharmacology, Memorial Sloan Kettering Cancer Center,
New York, New York 10021
Received June 3, 2002
Scheme 1 a
The discovery of small organic molecules with potent biologic
activity is a key goal of life science research.1 Success in this arena
is frequently dependent on access to libraries of diverse chemical
compounds.2 Diversity may be incorporated into these libraries
through the attachment of diverse functional groups to a fixed, cyclic
scaffold.3 A complementary approach is to use stereochemical
variation and acyclic stereocontrol to generate geometric diversity
among the members of the library.4
To illustrate the latter concept, we have focused our efforts on
the discovery of nonpeptidic ligands for peptide receptors. A fertile
ground for such efforts lies in the discovery of ligands for the opioid
class of G-protein coupled receptors,5 which are found in the
nervous system and mediate the sensation of pain.6 An endogenous
ligand for the mu opioid receptor (MOR) is the tetrapeptide
endomorphin-2 (1), a potent agonist of MOR with high (104-fold)
selectivity for MOR over the delta opioid receptor (DOR) and the
kappa opioid receptor (KOR).7
a Reagents and conditions: (a) Cl2(PCy3)(IMesH2)RuCHPh, CH2Cl2, 25
°C, 25%; (b) 95% TFA; (c) TPSH, piperidine, NMP, 100%.
showed significant binding under these conditions; Ki values for
Stereodiverse, nonpeptidic compounds 2 are designed to target
MOR. In compounds 2, the N-terminal tripeptide unit of 1 has been
replaced by a nonpeptidic, stereodiverse unit incorporating a 1,5-
enediol moiety. The dense array of stereocenters combined with
the rigidifying olefin in 2 are intended to generate geometric
diversity. An explicit goal of this study was to investigate how
such geometric diversification impacts binding to MOR. Here we
report the synthesis of an exhaustively stereodiversified library
comprising 16 stereoisomers of 2 and the screening of these
compounds for binding to MOR. Furthermore, we report diversi-
fication of the central hydrocarbon linkage and of the residual
C-terminal amino acid.
Ligands 2 were synthesized in parallel using a solid-phase cross
metathesis approach (Scheme 1).8 Resin-bound 4 was treated with
Cl2(PCy3)(IMesH2)RuCHPh9 and an excess of 3 to give 5, which
was then deprotected and cleaved from the resin with 95% TFA.
Although the yield for this process was modest (24-38%), the
products were obtained in good purity.
the five most active compounds were then determined in competi-
3
tive binding assays with H-labeled DAMGO (Table 1).10
The stereoisomer with the highest affinity, (S,S,S,R)-2,11 exhibited
a Ki of 8.8 nM, within an order of magnitude of the Ki measured
for 1 (1.2 nM) under the same conditions. Variation of configuration
at even a single stereocenter of (S,S,S,R)-2 had a strong impact on
binding affinity. For example, inversion of only the C-7 hydroxyl
[(S,S,S,R)-2 f (S,S,R,R)-2] resulted in a 3-fold loss in affinity.
Inversion of only the C-3 hydroxyl [(S,S,S,R)-2 f (S,R,S,R)-2] gave
an 18-fold reduction in binding affinity. Inversion of either the C-2
or the C-8 aryl groups resulted in significant reduction in affinity
at 10 µM (not shown in Table 1). Most of the high-affinity ligands
preserved the stereochemical configuration corresponding to that
of a natural L-amino acid for the aryl side chains; however,
(S,S,R,S)-2, with the nonnatural configuration at the C-8 benzyl
side chain, exhibited the fourth-highest affinity among the stere-
oisomers of 2.
Ligands 2 were assayed against DOR and KOR, to determine
MOR selectivity (Table 2). Ligands 2 exhibited stereochemically
dependent selectivity for MOR, although none was as selective as
1. (S,S,S,R)-2, the highest affinity MOR ligand, was 57- and 150-
fold selective for MOR over DOR and KOR, respectively, while
(S,R,S,R)-2 was only 5- and 18-fold selective. Interestingly,
Sixteen stereoisomers of 2 were screened at a concentration of
10 µM for competitive binding to MOR. Several stereoisomers
* To whom correspondence should be addressed. E-mail: verdine@chemistry.
harvard.edu.
† Harvard University.
‡ Memorial Sloan Kettering Cancer Center.
9
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J. AM. CHEM. SOC. 2002, 124, 13352-13353
10.1021/ja027150p CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Binding Affinity for MOR
reduced binding affinity and selectivity for MOR relative to 2
having the same configuration, presumably owing to their reduced
conformational preorganization. Isomerization of the central CdC
bond from trans (2) to cis (9) resulted in a significant loss of binding
affinity.
Ki MOR (nM)a,b
1.2
± 0.1
1
Configuration:
S,S,S,R
S,S,R,R
S,R,R,R
S,R,S,R
S,S,R,S
To investigate the role of the C-terminal Phe in MOR binding
of 2, three stereoisomers of 10-12 and one stereoisomer of 13
were synthesized and screened for MOR affinity and selectivity
(Scheme 2). Monomers 3 and 7 were coupled by solution phase
cross metathesis,8 followed by hydrolysis of the thioester, amidation,
and deprotection. Analogues 10 and 11 contain only one amide
bond, yet had similar or higher affinity and selectivity for MOR
than 2. For example, (S,S,S,R)-11 had a Ki value for MOR of 10
nM, and 110- and 600-fold selectivity for MOR over DOR and
KOR, respectively. Compounds 12 and 13 also retained significant
MOR affinity and selectivity, indicating that C-terminal modifica-
tions are well tolerated.
2
6
8.8 ( 0.7
98 ( 31
370 ( 150
21 ( 1
25 ( 5
95 ( 61
74 ( 12
29 ( 8
16 ( 1
22 ( 4
67 ( 38
120 ( 30
400 ( 110
53 ( 6
160 ( 40
190 ( 20
260 ( 70
79 ( 23
9
10
11
12
13
10 ( 2
28 ( 5
20 ( 1
37 ( 8
37 ( 1
a Competitive binding assay with 3H-DAMGO for hMOR-1 stably
transfected into CHO cells. b Errors represent 95% confidence interval.
Table 2. Selectivity of Ligands for MOR versus DOR and KOR
Ki DORa/Ki MOR, Ki KORb/Ki MOR
10 000, 9000
Configuration:
1
In conclusion, screening of an exhaustively stereodiversified
library has resulted in the identification of novel, nonpeptidic ligands
for the MOR. The best of these ligands, (S,S,S,R)-2, -10, and -11,
bind MOR with low nanomolar affinity and 57-600-fold selectivity
for MOR over other opioid receptors. Functional assays show that
these compounds are partial agonists for MOR (data not shown),
and we are currently searching for derivatives showing full agonist
activity. The results provide encouraging signs that stereochemical
diversity will be a valuable strategy for the discovery of nonpeptidic
ligands for peptide receptors.
S,S,S,R
S,S,R,R
S,R,R,R
S,R,S,R
S,S,R,S
2
6
57, 150
22, 40
6, 16
45, 48
31, 8
58, 22
42, 120
39, 180
28, 35
21, 18
26, 13
23, 15
55, 53
34, 120
15, 26
5, 18
21, 35
39, 7
86, 16
9
10
11
12
13
170, 86
110, 600
34, 53
24, 160
a Competitive binding assay with H-DPDPE for hDOR-1 stably trans-
fected into HEK-293 cells. b Competitive binding assay with 3H-U-69 593
for KOR in guinea pig cerebellum preparation.
3
Scheme 2 a
Acknowledgment. We thank G. Warner and Enanta Pharma-
ceuticals for running the initial screens, A. Marcus for help in
preparing 3 and 4, and L. Mahurter for technical assistance.
Supporting Information Available: Experimental procedures and
spectral data for all new compounds (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
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