Design, synthesis, and evaluation of opioid analogues with non-peptidic β-turn scaffold: Enkephalin and endomorphin mimetics

Article abstract of DOI:10.1021/jm0155897

We have identified a μ-selective opioid receptor agonist without a cationic amino group in the molecule from libraries of bicyclic β-turn peptidomimetics. The biologically active conformation of the lead is proposed to mimic an endomorphin type III 4 → 1

Full text of DOI:10.1021/jm0155897

© Copyright 2002 by the American Chemical Society
Volume 45, Number 7
March 28, 2002
Letters
conformational analyses of peptides in membrane-
mimicking environments.5 Several studies suggested
reverse turn conformations as the potential biologically
active conformation of the opioid peptides based upon
computational models,6 X-ray crystallography,7 and
spectroscopic analyses.5b,8
Design , Syn th esis, a n d Eva lu a tion of
Op ioid An a logu es w ith Non -P ep tid ic
â-Tu r n Sca ffold : En k ep h a lin a n d
En d om or p h in Mim etics
Masakatsu Eguchi,*,‡,§ Richard Y. W. Shen,†
We have developed a â-turn peptidomimetic with four
J . Paul Shea,† Min S. Lee,† and Michael Kahn*,‡,§
Molecumetics Ltd., 2023 120th Avenue NE, Suite 400,
Bellevue, Washington 98005, Department of Pathobiology
University of Washington, Seattle, Washington 98195,
and Pacific Northwest Research Institute, 720 Broadway,
Seattle, Washington 98122
Received November 20, 2001
Abstr a ct: We have identified a µ-selective opioid receptor
agonist without a cationic amino group in the molecule from
libraries of bicyclic â-turn peptidomimetics. The biologically
active conformation of the lead is proposed to mimic an
endomorphin type III 4 f 1 â-turn conformation.
sites of diversity readily accessible through solid-phase
synthesis from commercially available diversity com-
ponents. This privileged template was shown by X-ray
crystal structural analysis9a and solution-phase 2D
NMR spectroscopy9 to mimic a type I â-turn conforma-
tion accurately on a rigid bicyclic structure. This highly
constrained 6,6-bicyclic system incorporating function-
ality at the i to i + 3 positions affords us an opportunity
to probe the biologically active conformation of peptides
that potentially adopt a reverse turn conformation.
Therefore, we applied this scaffold to the preparation
of mimetics of Leu-enkephalin (YGGFL)1 to identify
potent and selective ligand(s) for opioid receptors (µ, δ,
and κ)10 and to elucidate their structure-activity rela-
tionship (SAR). In addition to the conformational stabil-
ity induced by this template, its non-peptidic nature can
potentially improve the undesirable therapeutic char-
acteristics of peptides, which include poor bioavailabil-
ity, short duration of action, and lack of oral activity.11
During the course of our SAR studies, we found a
striking similarity between the three-dimensional struc-
ture and the receptor selectivity profile of our lead
compounds and those of the recently isolated opioid
peptides, endomorphin-1 (YPWF) and endomorphin-2
(YPFF).12 Herein, we report the design, synthesis, and
evaluation of opioid peptide analogues utilizing our
privileged bicyclic â-turn scaffold to discover potent
The isolation and identification of the endogenous
opioid peptides, methionine enkephalin and leucine
enkephalin,1 initiated an intensive effort to discern the
biologically active conformation of enkephalin through
comparison with the rigid morphine skeleton, which is
believed to share a common binding motif.2 It has been
shown that the proper spatial orientation of a cationic
amine, a phenol group, and an additional hydrophobic
group is necessary to manifest biological activity through
interaction with opioid receptors.2a,c However, the in-
herent conformational flexibility of the opioid peptides
has hampered numerous attempts to accurately assess
the bioactive conformation(s). Significant effort has been
devoted to the elucidation of the receptor-bound con-
formation through systematic studies of conformation-
ally constrained peptides3 or peptidomimetics4 or by
* To whom correspondence should be addressed. Address: Pacific
Northwest Research Institute, 720 Broadway, Seattle, WA 98122.
Phone: 206-726-1200. Fax: 206-726-1217. E-mail (for M.E.): meguchi@
pnri.org. E-mail (for M.K.): mkahn@pnri.org.
‡ Pacific Northwest Research Institute.
§ University of Washington.
† Molecumetics Ltd.
10.1021/jm0155897 CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/03/2002

1396 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Letters
F igu r e 1. Diversity components for enkephalin and endo-
morphin mimetics.
µ-receptor ligands that bear no cationic amine and we
evaluate the relevance of the conformational and the
functional requirements for biological activity.
The inhibitory activity of each mimetic at 1 µM and/
or 0.1 µM was evaluated through competition of specific
binding of radioligand ([3H]naloxone) for relatively
nonselective opioid receptors in rat cerebral cortex.14
The specific radioligand binding to the receptors is
defined as the difference between total binding and
nonspecific binding determined in the presence of an
excess of unlabeled naloxone (1 µM). Results are ex-
pressed as a percent inhibition of the specific binding
obtained in the presence of the test compounds (see
Supporting Information for the preliminary results).
Among the 12 hydrophobic residues introduced at the i
+ 3 position, the benzyl and 2-pyridylmethyl groups
showed the highest inhibitory activity and the other
alkyl groups provided almost the same level of activity
when the i + 2 position is benzyl. Diversification of the
R-amino acids at the i + 2 position varies the inhibitory
activity from 99% to 11% while holding the functionality
at the i + 3 position as n-butyl. The phenethyl group at
this position afforded the best result. The activities
decrease in the order of phenethyl > p-aminobenzyl >
benzyl > other alkyls, hydrogen, and phenyl groups >
p-hydroxybenzyl group. Extension of the alkyl chain
between the phenol group and the template at the i
position (from ethyl to propyl) causes significant loss of
inhibitory activity.
To validate this preliminary SAR result, we resyn-
thesized four of the most active compounds. â-Turn
mimetics with a benzyl group or phenethyl group at the
i + 2 position and a benzyl group or n-butyl group at
the i + 3 position were prepared, and the IC50 values of
these four compounds versus the relatively nonselective
opioid receptors in rat cerebral cortex were determined
as shown in Table 1. These compounds represent an
enkephalin 5 f 2 â-turn model with one methylene
length shorter at the i position and an endomorphin type
III 4 f 1 â-turn model with one methylene length longer
at the i position.15 The IC50 value of 6a was measured
as 149 nM. The replacement of the n-butyl group of 6a
at the i + 3 position with a benzyl group, 6b, improved
the inhibitory activity almost 2-fold to 80 nM, and the
replacement of the benzyl group of 6a at the i + 2 with
a phenethyl group, 6c, improved the inhibitory activity
almost 5-fold to 27 nM. The inhibitory activity of 6c was
further enhanced 3-fold to 9 nM by replacing the n-butyl
group at the i + 3 position with a benzyl group, 6d . All
four compounds are lipophilic and bear no cationic
Enkephalin can adopt any type of â-turn conformation
in solution because of the presence of two glycine
residues at the second and third positions. Indeed,
various types of â-turn conformations (types I, I′, II′,
and III)5a-c,7,8a,b of enkephalin have been observed in
various media. By employing our â-turn scaffold, we can
investigate the following hypothetical bioactive â-turn
conformation models; enkephalin 4 f 1 â-turn model
(A), enkephalin 5 f 2 â-turn model (B), and endomor-
phin 4 f 1 â-turn model (C). To examine these models,
we incorporate the designed diversity elements into the
scaffold at the i to i + 3 positions. Twelve primary
amines at the i + 3 position, 13 R-amino acids at the i
+ 2 position, one â-alanine at the i + 1 position, and a
phenol component with three different lengths of spacer
at the i position were selected as shown in Figure 1.
A focused library of opioid peptide mimetics with
these components was prepared according to our previ-
ously published solid-phase synthetic protocol.9 Nucleo-
philic displacement of the bromide, 1, with a number
of primary amines gave the corresponding secondary
amine, 2, which was then coupled with the appropriate
Fmoc-R-amino acids with HOAt/DIC in NMP. Treat-
ment of 3 with 25% piperidine in DMF followed by
coupling with Fmoc-â-alanine afforded 4. After depro-
tection of the Fmoc group of 4 with 25% piperidine, the
resin was treated with the corresponding alkyl p-
nitrophenyl carbonates in the presence of DIEA to
produce 5. Cleavage from the acetal resin followed by
stereoselective tandem acyliminium cyclization was
accomplished by treatment with formic acid at room
temperature to give the 6,6-bicyclic â-turn mimetic, 6.
All products were purified by preparative TLC (silica
gel) to afford the pure product in good overall yields
(11-37%; see Supporting Information). The tryptophan
derivatives at the i + 2 position and p-hydroxylbenzyl-
carbamate derivatives at the i position failed to afford
the desired bicyclic scaffold.13

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1397
Ta ble 1. Effects of the Mimetics on the Specific Radioligand
([3H]Naloxone) Binding to the Relatively Nonselective Opioid
Receptor
Ta ble 2. Effects of the 6c and 6d on the Specific Radioligand
Binding to the Human Opioid Receptors and IC50 Values for
the Reference Compoundsa
F igu r e 2. Intravenous mouse hot plate analgesia assay of 6c.
reference compounds
6c
6d
receptors
0.1 µM
0.1 µM
IC50 (nM)
δ (h)
κ (h)
µ (h)
<10%
<10%
91%
<10%
<10%
101%
DPDPE
U 50488
DAMGO
3.5
0.69
1.6
a
Radioligands [3H]DPDPE, [3H]U 69593, and [3H]DAMGO were
used for assays for human δ-, κ-, and µ-opioid receptors, respec-
tively. IC50 values are within accepted limits of historic averages
obtained ( 0.5 log unit.
amino group in the molecule.16 Compounds 6c and 6d
were further studied using cloned human µ-, δ-, and
κ-receptors to examine the receptor selectivity as shown
in Table 2. Both of the compounds, 6c and 6d , showed
excellent µ-selectivity for the human opioid receptors.
For comparison, a linear peptide 7 corresponding to
6c was prepared and tested for its affinity for the rat
cerebral cortex relatively nonselective opioid receptors.
Although the same functional groups, such as p-hydroxy-
phenethyl, (S)-homophenylalanyl, and n-butyl groups,
are displayed on the surface of the linear amide back-
bone structure, no binding activity was observed at the
1 µM level. This result suggests that the correct spatial
orientations and conformational restrictions of these
functional groups by the rigid template are critical for
high receptor affinity.
We carried out an in vivo evaluation of the analgesic
effect of 6c. Three groups of 10 male CD-1 mice were
administered 0.1, 1.0, or 10 mg/kg of 6c intravenously
(iv) at 5.0 mL/kg. One group of 10 male CD-1 mice was
administered vehicle (propylene glycol and saline, 1:1)
iv at 5.0 mL/kg. Another group of 10 male CD-1 mice
received the positive control, morphine sulfate at 10 mg/
kg, iv, at 5.0 mL/kg. The mice were sequentially placed
on a hot plate analgesia meter (set at 55 °C) and
observed for a reaction to the heat stimulus. The
reaction times were obtained at 5, 15, 30, 60, and 120
min following iv administration. The administration of
morphine sulfate at 10 mg/kg, iv, produced statistically
significant (p e 0.05) increases in mean reaction times
of 279%, 234%, 164%, 113%, and 22% analgesia respec-
tively at 5, 15, 30, 60, and 120 min postadministration.
The administration of 6c at 1.0 mg/kg, iv, produced a
statistically significant (p e 0.05) increase in the mean
reaction time of analgesia 104% at 5 min postdose. The
administration of 6c at 10 mg/kg, iv, showed statistically
significant (p e 0.05) increases in the mean reaction
times of 337%, 189%, and 75% analgesia respectively
at 5, 15, and 30 min postadministration. The results of
the intravenous mouse hot plate analgesia assay are
F igu r e 3. Overlay of the dominant low-energy conformer of
6d (azure) and type III â-turn conformation of endomorphin
(rose, ref 18).
summarized in Figure 2. It appears that the initial level
of analgesic activity of 6c is the same as the initial
activity level of morphine, although the in vivo half-life
of 6c was almost 2-fold less than that of morphine.
Our 2D NMR experiments in CDCl3 at -20 °C showed
that the bicyclic scaffold of 6c and 6d adopts the same
ring conformation as observed by X-ray crystallography
for the corresponding sulfonamide derivative9a and that
6c and 6d exist as a mixture of two rotamers about the
urethane linkage. A Monte Carlo conformational search
for 6d using the MMFF force field in vacuo as imple-
mented in MacroModel 7.117 generated the bicyclic ring
structure of the lowest energy conformer that matched
the core ring structure deduced from the 2D NMR
experiments. The overlaid structure of 6d with a trans-
endomorphin-1 â-turn type III like structure18 revealed
the structural similarity of these molecules with an
rmsd value of 0.39 Å at six atom positions19 as shown
in Figure 3. J udging from the similarity of the three-
dimensional structure and the µ-selectivity profile of 6d ,
it would be reasonable to deduce that the opioid
analogue 6d mimics the endomorphin type III 4 f 1
â-turn model.
In conclusion, we have identified a nanomolar affinity
µ-opioid receptor agonist lacking a positive charge in
the molecule by employing a privileged â-turn scaffold.
The stereochemistries of 6c and 6d in solution were
analyzed by 2D NMR in combination with molecular
mechanics, which suggest that the biologically active
conformation of endomorphin is a type III 4 f 1 â-turn
conformation and that the µ-active conformation of
enkephalin is a 5 f 2 â-turn conformation. Finally, we
have demonstrated the application of our â-turn pepti-

1398 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Letters
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J M0155897