Unpredictable stereochemical preferences for mu opioid receptor activity in an exhaustively stereodiversified library of 1,4-enediols.

Article abstract of DOI:10.1021/ol027237f

Using olefin cross-metathesis, we synthesized a novel stereodiversified library of compounds 3 containing a trans-1,4-enediol. Screening this library for mu opioid receptor (MOR) affinity identified multiple high-affinity ligands and revealed that the ste

Full text of DOI:10.1021/ol027237f

ORGANIC
LETTERS
2003
Vol. 5, No. 5
633-636
Unpredictable Stereochemical
Preferences for Mu Opioid Receptor
Activity in an Exhaustively
Stereodiversified Library of 1,4-Enediols
Zhangjie Shi,† Bryce A. Harrison,† and Gregory L. Verdine*
Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
Verdine@chemistry.harVard.edu
Received November 5, 2002
ABSTRACT
Using olefin cross-metathesis, we synthesized a novel stereodiversified library of compounds 3 containing a trans-1,4-enediol. Screening this
library for mu opioid receptor (MOR) affinity identified multiple high-affinity ligands and revealed that the stereochemical configuration varied
widely among those ligands having the highest affinity. It was not possible to predict the configurations of the most active compounds 3 on
the basis of the configuration of endomorphin-2, a known MOR peptide ligand, validating the diversity-based approach to ligand discovery.
Diversity-based approaches for discovering bioactive small
molecules are most attractive when the biologic properties
of the molecules are least predictable.1 Approaches to
molecular diversity have mostly focused on constitutional
variation, often starting with a fixed, cyclic scaffold.2 We
are investigating a complementary approach wherein stereo-
chemical diversity of densely branched acyclic molecules
generates geometric variation among library members.3
We recently reported the synthesis of an exhaustively
stereodiversified library of 1,5 enediols of structure 2,4 based
on biasing elements from endomorphin-2 (1),5 a highly potent
and selective mu opioid receptor (MOR)6 peptide agonist.
Screening of this library for MOR affinity identified several
active stereoisomers. The most potent, (S,S,S,R)-2,7 had a
Ki of 8.8 nM in a radioligand competitive binding assay,
with 57- and 150-fold selectivity for MOR over the delta
opioid receptor (DOR) and kappa opioid receptor (KOR),
respectively. This compound acted as a partial agonist for
MOR in functional assays. Interestingly, the stereochemical
configuration of 2 strongly impacted the affinity and
selectivity. The five stereoisomers exhibiting the highest
affinity had an 18-fold range in MOR affinity with a 17-
* To whom correspondence should be addressed.
† These authors contributed equally to this work.
(1) (a) Schreiber, S. L. Science 2000, 287, 1964-1969. (b) Stockwell,
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(4) Harrison, B. A.; Gierasch, T. M.; Neilan, C.; Pasternak, G. W.;
Verdine, G. L. J. Am. Chem. Soc. 2002, 124, 13352-13353.
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Y. H.; Loh, H. H. Biopolymers 1999, 51, 440-455.
(7) All stereochemical labels are given in the C2 f C8 direction as shown
in compounds 2 and 3.
(2) For examples, see: (a) Pelish, H. E.; Westwood, N. J.; Feng, Y.;
Kirchhausen, T.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 6740-6741.
(b) Nicolaou, K. C.; Pfefferkorn, J. A.; Mitchell, H. J.; Roecker, A. J.;
Barluenga, S.; Cao, G.-Q.; Affleck, R. L.; Lillig, J. E. J. Am. Chem. Soc.
2000, 122, 9954-9967. (c) Tan, D. S.; Foley, M. A.; Shair, M. D.; Schreiber,
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(3) (a) Harrison, B. A.; Verdine, G. L. Org. Lett. 2001, 3, 2157-2159.
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Nolan, S. P.; Verdine, G. L. Org. Lett. 2000, 2, 3999-4002. (c) Michielin,
O.; Zoete, V.; Gierasch, T. M.; Eckstein, J.; Napper, A.; Verdine, G.;
Karplus, M. J. Am. Chem. Soc. 2002, 124, 11131-11141.
10.1021/ol027237f CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/11/2003

and 9-fold range in selectivity for MOR over DOR and KOR,
respectively. These ligands all conserved the (S)-configura-
tion at C2, and four of the five conserved the (R)-
configuration at C8, corresponding to the configuration of 1
at the respective positions. In an effort to discover MOR
ligands with improved agonist activity, we sought to
synthesize a new class of stereodiverse compounds 3
containing a 1,4-enediol moiety.
bromide to give (S,R)-6 and (S,S)-6, separable by flash
chromatography, in a 1.6:1 ratio in 75% combined yield.
To determine the relative configurations of 6, these com-
pounds were cyclized to 7 by treatment with KHMDS in
THF. In (S,R)-7, a 1.4% NOE was observed between H4 and
H1/H1′ and a 0% NOE between H4 and H2, while in (S,S)-7,
a 2.3% NOE was observed between H4 and H2 and a 0%
NOE between H4 and H1/H1′. Starting with Boc-D-Tyr(tBu)-
OH, (R,S)-6 and (R,R)-6 were synthesized by the same
procedure.
Compounds 6 were coupled with compounds 84 by olefin
cross-metathesis10 to give 9 (Scheme 2). Our previously
Scheme 2. Synthesis of 3a
a Reagents and conditions: (a) Cl2(PCy3)(IMesH2)RuCHPh,
CH2Cl2, 40 °C, 53%; (b) LiOH, H2O2, THF, H2O, 79%; (c) HBTU,
HOBt, DIPEA, NMP, Phe-NH-Rink amide AM resin; (d) 95%
TFA.
To investigate fully the impact of stereochemical diversity,
we prepared 16 stereoisomers of 3 using a modular approach,
beginning with the synthesis of the four stereoisomers 6
(Scheme 1).8 Boc-L-Tyr(t-Bu)-OH was subjected to Arndt-
Eistert homologation to give methyl ester (S)-5 by way of
diazoketone (S)-4.9 Compound (S)-5 was reduced to the
aldehyde with DIBAL-H and reacted with vinylmagnesium
reported synthesis of 2 featured a olefin cross-metathesis
between an allylic and a homoallylic alcohol;4 however, the
synthesis of 9 represented our first efforts at the cross-
metathesis of two allylic alcohols in a stereodiverse manner.
In this system, both metathesis partners have a similar
substitution pattern around the olefin, which could reduce
the selectivity of the coupling. Moreover, the increased steric
congestion around the olefin could hinder the metathesis
reaction. The coupling was attempted first with different
ratios of 6 and 8 using second generation Grubbs catalyst11
in refluxing CH2Cl2. A 1:1 or 5:1 ratio of 6 to 8 gave low
yields of 9. However, a 1:5 ratio of 6 to 8 gave 9 in 30-
56% yield for eight diastereomers. In an optimized procedure,
6 was added slowly by syringe pump to excess 8 in the
presence of second generation Grubbs catalyst in refluxing
CH2Cl2. Using only a 1:2 ratio of 6 to 8, this procedure gave
9 in 51-81% yield for the eight diastereomers. By a
combination of these methods, we completed the synthesis
of all 16 stereoisomers of 9.
Scheme 1. Synthesis of 6a
Compounds 9 were hydrolyzed to the free acids and
coupled with phenylalanine supported on Rink Amide AM
(8) Gierasch, T. M.; Shi, Z.; Verdine, G. L. Manuscript submitted.
(9) Gordon, E. M.; Godfrey, J. D.; Delaney, N. G.; Asaad, M. M.; Von
Langen, D.; Cushman, D. W. J. Med. Chem. 1988, 31, 2199-2211.
(10) (a) Blackwell, H. E.; O’Leary, D. J.; Chatterjee, A. K.; Washenfelder,
R. A.; Bussmann, D. A.; Grubbs, R. H. J. Am. Chem. Soc. 2000, 122, 58-
71. (b) Fu¨rstner, A. Angew. Chem., Int. Ed. 2000, 39, 3012-3043.
(11) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1,
953-956.
a Reagents and conditions: (a) ClCO2iBu, N-methylmorpholine,
THF; (b) CH2N2, THF, 85% for two steps; (c) PhCO2Ag, NEt3,
MeOH; (d) DIBAL-H, THF, 43% for two steps; (e) vinyl-MgBr,
THF, 1.6:1 ratio of (S,R)-6 to (S,S)-6, 75%; (f) KHMDS, THF,
92-96%.
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Org. Lett., Vol. 5, No. 5, 2003

Table 1. MOR Affinity, Selectivity, and Efficacy of Compounds 1 and 3a
compound
Ki MORb (nM)
Ki DORc (µM) (Ki DOR/Ki MOR)
Ki KORd (µM) (Ki KOR/Ki MOR)
% GTP-γ-S bounde
endomorphin-2 (1)
(S,S,R,S)-3
(S,R,R,S)-3
(S,R,S,S)-3
(S,R,S,R)-3
(R,R,S,S)-3
(R,S,R,R)-3
1.0 ( 0.1
14 ( 1
28 ( 6
42 ( 5
32 ( 6
38 ( 13
20 ( 3
25 ( 1 (25 000)
1.2 ( 0.1 (82)
2.5 ( 3 (89)
2.5 ( 0.3 (60)
0.74 ( 0.04 (23)
1.2 ( 0.1 (30)
2.5 ( 0.3 (125)
10.4 ( 8.6 (10 000)
0.45 ( 0.06 (32)
1.2 ( 0.6 (42)
3.1 ( 2.6 (74)
0.29 ( 0.1 (9)
100 ( 6
25 ( 5
48 ( 8
57 ( 12
37 ( 7
30 ( 6
68 ( 10
0.88 ( 0.55 (23)
0.27 ( 0.03 (14)
a Errors on measurements represent two standard deviations from the mean (95% confidence interval). b Competitive binding assay with 3H-DAMGO for
hMOR-1 stably transfected into CHO cells. c Competitive binding assay with 3H-DPN for hDOR-1 stably transfected into HEK-293 cells. d Competitive
binding assay with 3H-U-69 593 for KOR in guinea pig cerebellum preparation. e Specific binding of GTP-γ-35S by G-proteins in CHO membrane preparations
stably transfected with hMOR-1, in the presence of GDP and 1 or 3 (10 µM), expressed as a percentage of DAMGO-induced GTP-γ-35S-specific binding.
resin. The product was cleaved from the resin, deprotected
with 95% TFA, and purified by reverse-phase HPLC to give
16 stereoisomers of 3.
Compounds 3 were screened for MOR affinity at 1 µM
concentration in a competitive binding assay with 3H-
DAMGO (Figure 1).12 The stereoisomers of 3 showed
for MOR affinity was not predictable from the configuration
of 1, as only one of the six ligands had both the (S)-
configuration at C2 and the (R)-configuration at C8. In fact,
the (S)-configuration at C8 was present in four of the six
ligands. Moreover, the effects of stereochemical changes to
the ligands were unpredictable as well. For example, invert-
ing C2, C7, or C8 of (S,S,R,S)-3, the highest affinity ligand,
greatly reduced affinity. However, inverting C4 [(S,S,R,S)-
3 f (S,R,R,S)-3] resulted in only a 2-fold reduction in
affinity. Compound (S,R,S,S)-3 was the least sensitive to
stereochemical changes; inverting C2, C7, or C8 actually
improved MOR affinity. Compound (R,S,R,R)-3 was the most
sensitive to stereochemical changes; inverting any one
stereocenter significantly reduced the affinity. Overall, no
stereochemical configuration at any of the stereocenters was
conserved among all six of the highest affinity ligands. This
result suggests that binding affinity resulted from the
combined impact of multiple stereocenters on the ligand-
receptor interaction.
To determine MOR selectivity, the six most potent ligands
were screened for affinity for DOR and KOR (Table 1). The
ligands showed 23-125-fold selectivity for MOR over DOR
Figure 1. Competitive binding assay with 3H-DAMGO (1.3 nM)
for hMOR-1 stably transfected into CHO cells. Specific binding
was defined by the difference in radioligand bound between assays
with no ligand (blank) and with 10 µM naloxone. Error bars
represent two standard deviations from the mean (95% confidence
interval) on triplicate measurements.
varying degrees of affinity for MOR, displacing 28-90%
of 3H-DAMGO from MOR. The six most potent ligands from
this screen were assayed at multiple concentrations to obtain
MOR binding curves for Ki determination (Table 1). These
experiments identified (S,S,R,S)-3 as the highest affinity
ligand with a Ki of 14 nM, about 14-fold less active than 1,
and the six highest affinity ligands showed a 3-fold range in
affinity. Most interestingly, the stereochemical preference
Figure 2. Specific binding of GTP-γ-35S by G-proteins in CHO
membrane preparations stably transfected with hMOR-1, in the
presence of GDP and 1 or 3 at multiple concentrations, expressed
as a percentage of DAMGO-induced GTP-γ-35S specific binding.
Error bars represent two standard deviations from the mean (95%
confidence interval) on triplicate measurements.
(12) (a) Pasternak, G. W. Mod. Methods Pharmacol. 1990, 6, 1-17. (b)
Bylund, D. B.; Yamamura, H. I. In Methods in Neurotransmitter Receptor
Analysis; Yamamura, H. I., Ed.; Raven: New York, 1990; pp 1-35.
Org. Lett., Vol. 5, No. 5, 2003
635

and 9-74-fold selectivity for MOR over KOR. Selectivity
did not correlate highly with MOR affinity, with the most
potent ligand (S,S,R,S)-3 exhibiting only average selectivity
(82- and 32-fold selective for MOR over DOR and KOR).
The six highest affinity ligands 3 also were tested for the
ability to induce MOR-mediated GTP-γ-35S binding by
G-proteins in CHO membrane preparations (Table 1). At 10
µM, compounds 3 induced 25-68% of the GTP-γ-35S
binding induced by the full MOR agonist DAMGO, indicat-
ing that compounds 3 are partial agonists for MOR. Of the
ligands tested, (R,S,R,R)-3 (68%) and (S,R,S,S)-3 (57%) had
the highest activity in the GTP-γ-35S assay. Activation curves
were measured for these two compounds (Figure 2), which
gave EC50 values of 390 ( 110 and 390 ( 40 nM for
(R,S,R,R)-3 and (S,R,S,S)-3, respectively.
as in 2, the effects of stereochemical variation in 3 on MOR
affinity, selectivity, and efficacy were not predictable on the
basis of either the configuration of 1 or the properties of
any given stereoisomer of 3. These results illustrate the need
to sample stereochemistry broadly when attempting to
identify bioactive small molecules having multiple stereo-
centers.
Acknowledgment. We are grateful to Loriann Mahurter,
Claire Neilan, and Gavril Pasternak for providing expertise
and KOR guinea pig brain preparations. B.A.H. is the
recipient of a predoctoral fellowship from the National
Science Foundation. This work was supported by a gift from
Enanta Pharmaceuticals.
In conclusion, we developed an efficient olefin cross-
metathesis procedure for accessing a new class of stereodi-
verse 1,4-enediol compounds, 3. Screening of these com-
pounds for MOR activity identified multiple potent and
selective partial agonists. Although stereochemical diversity
did not impact the properties of these compounds as greatly
Supporting Information Available: Experimental pro-
cedures and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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Org. Lett., Vol. 5, No. 5, 2003