Novel ligands lacking a positive charge for the δ- and μ-opioid receptors

Article abstract of DOI:10.1021/jm990461z

Recently we reported using minilibraries to replace Lys [somatostatin (SRIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D- Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,-301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals, into a selective NK-1 receptor antagonist with an IC₅₀ of 2 nM in vitro. During the screening of the same libraries for ligands of the δ- opioid receptor, we identified four compounds (1-4) which represent a new class of δ-opioid antagonists, some of which were also NK-1 receptor antagonists. The most potent δ-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr- Thr-Phe-] (2), showed a K(e) value of 128 nM in the mouse vas deferens assay and a δ-receptor binding affinity constant of 152 nM in the rat brain membrane binding assay. These results are of interest because they represent a novel class of δ-opioid antagonists and, like two previously reported δ- opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the δ-opioid ligands, we prepared two analogues of the β-casomorphin-derived mixed μ-agonist/δ-antagonist, H- Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) or by the deletion of this N-terminal amino group (6). These latter compounds proved to be δ-opioid antagonists with K(e) values in the 16-120 nM range, as well as fairly potent μ-opioid antagonists (K(e) ? 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in μ- and δ-opioid receptor antagonists. In addition, cyclic, hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the prevailing assumptions about δ- and μ-opioid receptor binding need revision and that the receptors for these opioid ligands have much in common with the NK-1 and somatostatin receptors.

Full text of DOI:10.1021/jm990461z

J . Med. Chem. 2000, 43, 551-559
551
Expedited Articles
Novel Liga n d s La ck in g a P ositive Ch a r ge for th e δ- a n d µ-Op ioid Recep tor s
Peter W. Schiller,*,† Irena Berezowska,† Thi M.-D. Nguyen,† Ralf Schmidt,†,§ Carole Lemieux,† Nga N. Chung,†
Margaret L. Falcone-Hindley,‡ Wenqing Yao,‡, J osephine Liu,‡ Seiji Iwama,‡,| Amos B. Smith, III,*,‡ and
Ralph Hirschmann*,‡
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montre´al, 110 Pine Avenue West,
Montre´al, Quebec, Canada H2W 1R7, and Department of Chemistry, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received September 9, 1999
Recently we reported using minilibraries to replace Lys9 [somatostatin (SRIF) numbering] of
the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]) to generate the
potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel
cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,-
301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals,24
into a selective NK-1 receptor antagonist with an IC50 of 2 nM in vitro. During the screening
of the same libraries for ligands of the δ-opioid receptor, we identified four compounds (1-4)
which represent a new class of δ-opioid antagonists, some of which were also NK-1 receptor
antagonists. The most potent δ-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-] (2), showed
a Ke value of 128 nM in the mouse vas deferens assay and a δ-receptor binding affinity constant
of 152 nM in the rat brain membrane binding assay. These results are of interest because they
represent a novel class of δ-opioid antagonists and, like two previously reported δ-opioid
antagonists, they lack a positive charge. To examine further the requirement for a positive
charge in the δ-opioid ligands, we prepared two analogues of the â-casomorphin-derived mixed
µ-agonist/δ-antagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the
positive charge either through formylation of the primary amino group (5) or by the deletion
of this N-terminal amino group (6). These latter compounds proved to be δ-opioid antagonists
with Ke values in the 16-120 nM range, as well as fairly potent µ-opioid antagonists (Ke ≈ 200
nM). These six compounds provide the most convincing evidence to date that there is no
requirement for a positive charge in µ- and δ-opioid receptor antagonists. In addition, cyclic
hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the
prevailing assumptions about δ- and µ-opioid receptor binding need revision and that the
receptors for these opioid ligands have much in common with the NK-1 and somatostatin
receptors.
In tr od u ction
an ionic interaction between the amino group of agonist
and antagonist ligands and the â2-adrenergic receptor
via the Asp113 residue of the latter.5 This proposition
was based in part on the fact that a mutant protein, in
which Asp113 was replaced by the isosteric Asn (D113N),
required 8000-40000-fold increases in the amount of
agonist needed for adenylate cyclase stimulation, while
the â2-adrenergic antagonist propranolol displayed a Ki
value that was increased 10000-fold.6 In later work,
Strader and co-workers7 described another mutant of
the â2-adrenergic receptor in which the Asp113 was
replaced by Ser. This mutant similarly failed to bind
conventional agonists or antagonists efficiently. Subse-
quent screening of catechols, in which the amine sub-
stituents on the side chain were replaced by ketones or
esters, activated the D113S mutant but not the wild
type receptor. The authors proposed that the agonism
in the D113S mutant resulted from hydrogen bond
interactions which replaced the ionic binding interaction
with the wild type receptor.7 Taken together, these
The sequencing of diverse G-protein coupled receptors
has demonstrated the presence of a common structural
motif consisting of seven membrane spanning R-heli-
ces.2,3 These helices contain several conserved residues,
including two aspartates, which are believed to bind to
positively charged ligands such as biogenic amines.4
Peptide hormone and neurotransmitter receptors such
as tachykinin NK-1, neuropeptide Y, somatostatin, and
the enkephalins display sufficient diversity which makes
it more difficult to identify specific binding sites.4
In a seminal 1987 paper, Strader, Sigal et al. proposed
* Corresponding author: Ralph F. Hirschmann. Phone: 215-898-
7398. Fax: 215-573-9711. E-mail: rfh@sas.upenn.edu.
† Clinical Research Institute of Montre´al.
‡ University of Pennsylvania.
| Present address: Department of Material Science, Osaka City
University, Sugimoto, Sumiyoshi 558-8585, J apan.
§ Present address: AstraZeneca Research Centre Montreal, 7171
Frederick Banting, St. Laurent, Quebec, Canada H4S 1Z9.
Present address: DuPont Pharmaceuticals Co., P.O. Box 80500,
Experimental Station, E500/3402B, Wilmington, DE 19880-0500.
10.1021/jm990461z CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/05/2000

552 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schiller et al.
studies demonstrated convincingly that interactions via
salt bridges that were thought to be an absolute
requirement for the binding of catecholamines and their
receptors are replaceable, in this case via hydrogen
bonding.
Th e δ-Op ioid Recep tor
Similarly, it has been assumed that the binding of
an opioid ligand to its receptor requires an ionic
interaction, involving a positively charged atom, gener-
ally a basic amino nitrogen.8 The basis of this view
stems from the pioneering work of Kosterlitz9 and
Ro˜mer10 which demonstrated that elimination of the
positively charged nitrogen in the enkephalins, generat-
ing the corresponding desamino analogues, produces
loss of agonism and binding.9,10 Acetylation of the
N-terminal amino group also produced inactive com-
pounds.11 On the other hand, sulfonium analogues of
levorphanol and levallorphan, in which the nitrogen
atom of these alkaloids has been replaced by tertiary
S-methyl and S-allyl groups, were found to retain
significant potency. This result was interpreted to mean
that a positively charged heteroatom, other than nitro-
gen, can also interact with an anionic site on opioid
receptors and induce agonism.12 Ronai et al. reported
in 1992 that Boc-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu)-OH,
an opioid peptide lacking a positive charge, was a
moderately potent δ-opioid antagonist in the mouse vas
deferens (MVD) assay (Ke ≈ 30 nM).13 However, the
δ-receptor affinity in the rat brain membrane binding
assay was very weak (Kiδ ) 300-1000 nM), diminishing
the impact of this report.13,14 Conversely, the diketopip-
erazine c(-Dmt-Tic-), which also lacks a positive charge,
was reported by Balboni et al. to be a δ-antagonist.
However, the affinity in the MVD assay (Ke ) 3.8 µM)
was very low.15
F igu r e 1. Cyclic hexapeptides tested at the δ-opioid receptor.
Because two neutral δ-opioid antagonists have been
reported, albeit with only modest activity13,15 (see
above), we wished to examine further the requirement
of a positively charged heteroatom in ligands for the
opioid receptor. Believing cyclic hexapeptides to repre-
sent privileged platforms,21 similar to such scaffolds as
steroids, benzodiazepines, â-D-glucose derivatives,22 and
the so-called tricyclics, typified by amitriptyline,23 we
screened cyclic hexapeptides (Figure 1), available from
the NK-1 receptor program, which had already identi-
fied a potent NK-1 antagonist (IC50 ) 2.0 nM).24 Of a
total of 12 arbitrarily selected cyclic hexapeptides, all
lacking a positive charge, two compounds, 1 and 4, were
found to bind selectively to the δ-opioid receptor and to
produce δ-opioid antagonistic effects in the functional
MVD assay. Compound 4 lacks both a positive charge
and a phenolic hydroxyl group. Conversely, the struc-
turally related parent SRIF agonist, L-363,301 (c[-Pro-
Phe-D-Trp-Lys-Thr-Phe-]),25 containing the free ꢀ-amino
nitrogen of Lys does not bind to these receptors. Finally,
one of us (W.Y.) synthesized 2 and 3 as designed opioid
receptor ligands.
In 1992, two reports described the cloning of the
δ-opioid receptor16,17 which subsequently permitted site-
directed mutagenesis experiments. Kong et al. investi-
gated the role of Asp95, believed to be involved in the
binding of δ-opioid ligands.18 These authors reported
that the enkephalins and enkephalin-derived agonists
bind to the wild type receptor but not to the D95N
mutant, consistent with conventional thinking. In con-
trast, δ-receptor-selective antagonists such as naltrin-
dole, its benzofuran analogue, and 7-benzylidenaltrex-
one bound equally well to the wild type and mutant
receptors. Furthermore, the nonselective opioid agonist
bremazocine, which interacts with the wild type δ-, κ-,
and µ-receptors, also interacted well with the mutant
δ-receptor. The authors emphasized that “δ-selective
agonists bind differently to the cloned δ-opioid receptor
than do δ-selective antagonists or nonselective opioid
agonists”; they did not comment about specific struc-
tural requirements.
Our results, therefore, establish via the MVD and rat
brain membrane receptor binding assays that a positive
charge is neither a necessary nor a sufficient require-
ment for δ-selective opioid antagonistic binding. These
results encouraged us to eliminate the positive charge
contained in another class of cyclic peptides, the potent
â-casomorphin analogue H-Dmt-c[-D-Orn-2-Nal-D-Pro-
Gly-] (7), a mixed µ-opioid agonist/δ-opioid antagonist
(Figure 2).26 Formylation or deletion of the N-terminal
amino group resulted in compounds 5 and 6, respec-
tively, which retained δ-opioid antagonist activity in the
Believing the requirement for a positively charged
nitrogen atom to be the common property of opioid
ligands,8 Befort et al. investigated the possibility that
Asp128 of the mouse δ-opioid receptor provides the
anionic component of a salt bridge.19 However, replace-
ment of this amino acid with alanine gave an active
receptor, leading them to speculate that Asp128 is not
directly involved in the binding of the ligand but affects
binding site conformation.19,20
F igu r e 2. â-Casomorphin analogues tested.

Ligands Lacking Positive Charge for δ,µ-Opioid Receptors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 553
MVD assay. Interestingly, these transformations con-
verted agonists at the µ-receptor into moderately potent
µ-opioid antagonists.
Op ioid R ecep t or Bin d in g Assa ys a n d in Vit r o
Bioa ssa ys. Binding affinities of compounds for µ- and
δ-opioid receptors were determined by displacing [3H]-
DAMGO and [3H]DSLET, respectively, from rat brain
membrane binding sites; κ-opioid receptor affinities
were measured by displacement of [3H]U69,593 from
guinea pig brain membrane binding sites. For the
determination of their in vitro opioid activities, com-
pounds were tested in bioassays based on inhibition of
electrically evoked contractions of the guinea pig ileum
(GPI) and MVD. The GPI assay is usually considered
as being representative for µ-receptor interactions, even
though the ileum also contains κ-receptors. In the MVD
assay, opioid effects are primarily mediated by δ-recep-
tors; however µ- and κ-receptors also exist in this tissue.
Ke values for δ-antagonists were determined in the MVD
assay against the δ-agonist DPDPE, while the highly
selective µ-agonist TAPP (H-Tyr-D-Ala-Phe-Phe-NH2)30
was used for the determination of Ke values of µ-receptor
antagonists in the GPI assay.
Ch em istr y
Cyclic Hexa p ep tid e Syn th esis. Cyclic hexapeptides
1-4 were prepared from protected, linear precursors,
the latter prepared via solid-phase peptide synthesis.26,27
The peptides were removed from the resin with 0.4%
TFA in CH2Cl2 and cyclized with DPPA via the method
previously reported from these laboratories.28 The pro-
tecting groups were then removed from the cyclic
peptides using our standard conditions (TFA:DCM:EDT:
H2O, 50:45:3:2; 1 h at room temperature)24 and purified
by reversed-phase HPLC. The resultant cyclic peptides
1
1-4 were characterized by H and 13C NMR spectros-
copy, high-resolution mass spectrometry, and analytical
HPLC using two different solvent systems to confirm
structure and purity.
â-Ca som or p h in An a logu e Syn th esis. Synthesis of
the formyl analogue CHO-Dmt-c[-D-Orn-2-Nal-D-Pro-
Gly-] (5) proceeded with the addition of p-nitrophenyl
formate and DIPEA to 7. Purification was performed
by HPLC to furnish 5 in 92% yield.
For the synthesis of the desamino analogue Dhp-c[-
D-Orn-2-Nal-D-Pro-Gly-] (6), 3-(2′,6′-dimethyl-4′-hydrox-
yphenyl)propionic acid (Dhp, 13) was required (Scheme
1). To this end, 3,5-dimethylphenol was iodinated, using
the procedure of Hu¨nig and Schwarz29 to afford 9 in 24%
yield. Subsequent acetylation (95%) and Heck coupling
with methyl acrylate yielded 11 in 89% yield. Catalytic
hydrogenation (H2/Pd-C, 60 psi, 60 °C) followed by
hydrolysis with 12 N HCl then afforded 13 in 87% yield.
Resu lts
Replacement of the Lys9 residue of the potent soma-
tostatin receptor agonist L-363,301, c[-Pro-Phe-D-Trp-
Lys-Thr-Phe-], by tyrosine generated 1, c[-Pro-Phe-D-
Trp-Tyr-Thr-Phe-],24 which showed significant δ-antag-
onist activity (Ke ) 238 ( 26 nM) against the selective
δ-agonist DPDPE in the MVD assay (Table 1).
Further structural modification through replacement
of the Phe between Pro and D-Trp with 3-(1-naphthyl)-
alanine (1-Nal) led to compound 2, c[-Pro-1-Nal-D-Trp-
Tyr-Thr-Phe-], with a 2-fold increase in δ-antagonist
potency (Ke ) 128 ( 18 nM). Interestingly, the corre-
sponding analogue 3 with 2-Nal in place of 1-Nal was
20 times less potent as a δ-opioid antagonist (Table 1).
Finally, replacement of the Lys residue in L-363,301
with cyclohexylalanine (Cha)24 produced compound 4,
which also demonstrated δ-antagonist activity (Ke ) 202
( 38 nM). Unlike the compounds by Ro´nai14 and by
Balboni,15, the δ-opioid receptor affinities of compounds
1-4, measured in the rat brain membrane binding
assay (Table 2), were in good agreement with their
respective δ-antagonist potencies determined in the
MVD assay (Table 1). None of these compounds showed
antagonist or agonist activity in the µ-receptor-selective
GPI assay, and none of them bound to µ- or κ-receptors
at concentrations up to 10 µM. Various other amino acid
replacements at Phe2, D-Trp3, or Lys4 of c[-Pro-Phe-D-
Trp-Lys-Thr-Phe-] led to compounds that either had
weak δ-antagonist potency (Ke > 2 µM) or failed to bind
altogether and showed neither µ-agonist nor µ-antago-
nist activity in the GPI assay.
Sch em e 1
To examine the requirement for a positive charge in
opioid receptor ligands further, we prepared analogues
of the cyclic â-casomorphin peptide H-Dmt-c[-D-Orn-2-
Nal-D-Pro-Gly-] (7) in which the positive charge on the
N-terminal amino group was eliminated by either
formylation or deamination. Compound 7 is a potent
δ-antagonist (Ke ) 2.13 ( 0.51 nM, MVD assay) and a
potent µ-agonist (IC50 ) 7.88 ( 0.94 nM, GPI assay)
(Table 1).26 It has subnanomolar binding affinity for
both µ- and δ-receptors (Table 2). Formylated analogue
5 retained high δ-opioid antagonist potency in the MVD
The linear peptide precursor Dhp-D-Orn-2-Nal-D-Pro-
Gly-OH (15) was prepared by standard solid-phase
peptide synthesis techniques using Boc chemistry.30 The
protecting groups of Orn(Fmoc) and Dhp(Boc) were
removed with 20% (v/v) piperidine in DMF and 50%
(v/v) TFA in CH2Cl2, respectively. Cyclization between
the δ-amino group of D-Orn and the C-terminal carboxyl
group in dilute solution with DPPA, followed by pre-
parative HPLC, yielded Dhp-c[-D-Orn-2-Nal-2-Pro-
Gly-] (6).

554 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schiller et al.
Ta ble 1. Ke Values of Opioid Antagonists Determined in the MVD and GPI Assays Compared with NK-1 Results
compd
NK-1 IC50 (nM)a
MVD Ke (nM)b,c
GPI Ke (nM)b,d
1
2
3
4
5
6
7
c[-Pro-Phe-D-Trp-Tyr-Thr-Phe-]
c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-]
c[-Pro-2-Nal-D-Trp-Tyr-Thr-Phe-]
c[-Pro-Phe-D-Trp-Cha-Thr-Phe-]
CHO-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-]
Dhp-c[-D-Orn-2-Nal-D-Pro-Gly-]
H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-]31
1045 ( 230
165
N/A
216 ( 141
N/A
238 ( 26
128 ( 18
2460 ( 240
202 ( 38
16.3 ( 5.3
121 ( 16
2.13 ( 0.51
inactivee
inactivee
inactivee
inactivee
216 ( 28
N/A
N/A
237 ( 19, agonist
(IC50 ) 7.88 ( 0.94 nM)
a
b
d
As reported in ref 24. Mean of three to six determinations ( SEM. c Determined against DPDPE. Determined against TAPP.
e Inactive at concentrations up to 10 µM. N/A ) not available.
Ta ble 2. Binding Affinities of Peptide Analogues at µ- and δ-Receptors in Rat Brain Membrane Homogenates
compd
Kiδ (nM)a,b
Kiµ (nM)a,c
Kiµ/Kiδ
1
2
3
4
5
6
7
8
c[-Pro-Phe-D-Trp-Tyr-Thr-Phe-]
c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-]
c[-Pro-2-Nal-D-Trp-Tyr-Thr-Phe-]
c[-Pro-Phe-D-Trp-Cha-Thr-Phe-]
CHO-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-]
Dhp-c[-D-Orn-2-Nal-D-Pro-Gly-]
H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-]31
[Leu5]enkephalin
759 ( 140
152 ( 16
>10000
>13.2
>65.8
>9.35
>20.6
6.65
4.13
1.02
3.73
>10000
1070 ( 270
486 ( 65
>10000
>10000
218 ( 28
450 ( 77
0.476 ( 0.015
9.43 ( 2.07
32.8 ( 1.6
109 ( 12
0.467 ( 0.066
2.53 ( 0.35
a
b
Mean of three to six determinations ( SEM. Displacement of [3H]DSLET. c Displacement of [3H]DAMGO.
assay (Ke ) 16.3 ( 5.3 nM) although it was about 8
times less potent than the parent peptide (Table 1). In
agreement with this result, compound 5 also showed
marked δ-receptor affinity (Kiδ ) 32.8 ( 1.6 nM) in the
rat brain membrane binding assay. In the GPI assay, 5
was found to be a µ-antagonist (Ke ) 216 ( 28 nM)
against the selective µ-agonist TAPP in good agreement
with the relatively low µ-receptor affinity (Kiµ ) 218 (
28 nM). Desamino analogue 6 was a δ-opioid antagonist
in the MVD assay (Ke ) 121 ( 16 nM) with a 7-fold
lower potency than analogue 5 and was a µ-antagonist
about equipotent with analogue 5 in the GPI assay. The
µ- and δ-receptor affinities of 6 in the rat brain
membrane binding assays were in good agreement with
the Ke values obtained in the functional assays.
Neither 5 nor 6 bound to κ opioid receptors at
concentrations up to 10 µM. These results indicate that
compounds 5 and 6 retain significant δ-antagonist
potency. These analogues also show significant µ-opioid
receptor affinity and, in contrast to the µ-agonist peptide
7, are µ-opioid antagonists. They represent the first
compounds lacking a positive charge with significant
µ-opioid antagonist activity. Here again, our results
show good agreement between binding affinities and
potency in functional assays.
binding affinity (Kd ) 300-1000 nM) was too weak to
be consistent with the results of the MVD assay, this
report did not significantly change the view that a
positive charge is needed in the ligand.14 Similarly,
Balboni et al.15 stated that the neutral diketopiperazine
c(-Dmt-Tic-) is a δ-opioid antagonist. Again, this paper
did not significantly challenge the conventional thinking
because the diketopiperazine had very low affinity in
the MVD assay (Ke ) 3.8 µM).
The successful cloning of the δ-opioid receptor16,17
enabled Kong et al.18 to replace Asp95 by Asn via site-
directed mutagenesis. The enkephalins and related
compounds bound only to the wild type receptor, but
not to the D95N mutant, suggesting that Asp95 repre-
sents the anion that forms a salt bridge with the
positively charged ligand. Their results are consistent
with the conventional assumption that a salt bridge
between an aspartyl residue of the receptor and a
positive charge on the ligand is required for binding.
However, Kong et al. reported also that the nonselective
bremazocine binds to both the wild type and mutant
δ-receptors as an agonist. The authors concluded that
δ-selective agonists bind to the cloned δ-opioid receptor
differently than do δ-selective antagonists or nonselec-
tive agonists, but the requirement for a positive charge
in opioid ligands was not addressed.
Discu ssion
Different binding modes of chemically closely related
compounds with similar biological profiles are fre-
quently observed in medicinal chemistry.33,34 In the
steroid field, it is well-precedented that the oxygen atom
at C3, long thought to be an absolute requirement for
binding, can be replaced without loss of activity.35 Thus,
a wide array of experiments supported the view that a
C3-ketone, as in cortisol, is an absolute requirement for
antiinflammatory activity. This view was later shown
to be erroneous.36 The work of Strader and co-workers
is also relevant.5-7 Their work showed that while Asp113
of the â2-adrenergic receptor binds catecholamines via
a salt bridge, the mutant D113N binds catechols lacking
a basic nitrogen as agonists.6 Last, recent work by
Schwartz and co-workers demonstrated that by chang-
ing Asp113 of the â2-adrenergic receptor to a His residue,
Ionic bond formation is thought to play a key role in
the binding of G-protein coupled receptors to their
ligands.2,3 When the δ-opioid receptor was first cloned
in 1992, it became apparent that it was a member of
the G-protein coupled receptor family of membrane
proteins.16,17 Extensive literature reports have sup-
ported the widely accepted conclusion that all ligands
for opioid receptors carry a positively charged hetero-
atom (nitrogen or sulfur) which interacts with the
receptor, presumably via a salt bridge.8,32 This view
remained unchallenged until 1992 when Ronai and
collaborators13 reported that the linear hexapeptide Boc-
Tyr-Pro-Gly-Phe-Leu-Thr(OtBu)-OH, which lacks a posi-
tive charge, was a moderately potent δ-opioid antagonist
in the MVD assay (Ke ) 30 nM). Because the δ-receptor

Ligands Lacking Positive Charge for δ,µ-Opioid Receptors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 555
signal transduction can be induced with zinc or copper
ions in the absence of catecholamines.37 Taken together
such reports demonstrate, convincingly, that a given
binding mode (i.e., a salt bridge) can be modified by
substitutions, for example, hydrogen bonding, metal
chelation, or hydrophobic interactions.
tors have more features in common than had heretofore
been appreciated.
Con clu sion s
The cyclic hexapeptides described in this paper rep-
resent a new class of δ-opioid antagonists. The â-caso-
morphin analogues 5 and 6 displayed both δ- and
µ-antagonist properties and represent the first neutral
compounds with significiant µ-antagonist activity. The
results reported herein strengthen the evidence that an
electrostatic interaction is not an absolute requirement
for δ- and µ-opioid receptor-ligand interactions. Our
results also support the suggestion that neutral mol-
ecules can interact with δ- and µ-opioid receptors as
antagonists via aromatic and/or aliphatic side chains.
It is noteworthy that 4, in which Tyr has been replaced
by Cha, lacks a phenolic hydroxyl group but, neverthe-
less, retains the ability to act as an antagonist at the
δ-opioid receptor. Taken together, the results reported
herein show that δ-opioid receptors can bind ligands via
diverse binding modes and that cyclic hexapeptides are
privileged platforms.44 Further work is underway, both
in Montre´al and in Philadelphia, to explore these
findings more fully.
The experiments described herein do not involve
replacement of receptor aspartate residues by aspar-
agine but, like the studies of Ronai and of Balboni,
involve the generation of small molecules lacking a
positive charge. The one common feature of the peptides
reported herein (especially 1-4) is their large content
of aromatic amino acids. Balboni et al. pointed out that
aromaticity and hydrophobicity are important factors
in the binding of the δ-antagonist c(-Dmt-Tic-) to
δ-receptors.15 Such aromatic/aromatic interactions are
very common.38 We also show that in the cyclic â-caso-
morphin analogues 5 and 6 removal of the positive
charge through formylation or elimination of the N-
terminal amino group resulted in a 70-230-fold reduc-
tion in δ-receptor binding affinity which corresponds to
a 2.6-3.3 kcal/mol loss in binding energy (Table 2). For
the ligands described above, this loss in binding energy
may be replaced in part via energy gained from the
interaction with aromatic side chains of the δ-opioid
receptor.38
Exp er im en ta l Section
A recent report suggested that the high-affinity
binding site of δ-selective peptide agonists may be
located in the transmembrane (TM) region of helices
V-VII, as well as part of TM III.39 Site-directed mu-
tagenesis of several aromatic residues in TMs IV-VII
revealed varying degrees of importance of individual
residues.40 These aromatic residues, conserved across
the opioid receptor subtypes, are believed to form a
general binding domain for opioids and, possibly, signal
transduction.39,40
Gen er a l Meth od s. All solvents and reagents were obtained
from commercial sources and used without further purification.
Assembly of linear peptides was carried out on a model 431A
Applied Biosystems peptide synthesizer using L-Pro-2-chlo-
rotrityl polystyrene resin (Advanced Chem Tech or Anaspec).
N-R-FMOC amino acids (Bachem Bioscience or Advanced
Chem Tech) were employed throughout. Precoated plates
(silica gel F254, 250 µm; Merck, Darmstadt, FRG) were used
for TLC in the following solvent systems (all v/v): (I) CHCl3/
MeOH/AcOH (85:10:5), (II) hexane/EtOAc (4:1), (III) toluene/
ethyl acetate (2:3), (IV) n-BuOH/AcOH/H2O (4:1:5, organic
phase), and (V) n-BuOH/pyridine/AcOH/H2O (15:10:3:12).
Proton magnetic resonance spectra were recorded at 25 °C
on either a Varian VXR-400S spectrometer or a Bruker AM
500 MHz spectrometer using tetramethylsilane or residual
solvent as the internal standards. Molecular weights of
compounds were determined by either FAB mass spectrometry
on an MS-50 HMTCTA mass spectrometer interfaced to a DS-
90 data system (Dr. M. Evans, Department of Chemistry,
University of Montreal) or by ESI mass spectrometry on a VG
70/70H micromass spectrometer. Optical rotations were mea-
sured on a Perkin-Elmer model 241 polarimeter.
HP LC An a lysis. The HPLC system GOLD (Beckman)
consisting of a programmable solvent module 126 and a diode
array detector module 168 was used for the purification and
purity determination of the â-casomorphin peptides. Analytical
reversed-phase HPLC chromatography was carried out on a
Vydac 218-TP column (4.6 × 250 mm) under isocratic condi-
tions with 50% MeOH in 0.1% TFA at a flow rate of 1.0 mL/
min. Preparative reversed-phase HPLC was performed using
a Vydac 218-TP column (22 × 250 mm) with a linear gradient
of 20-45% acetonitrile in 0.1% TFA at a flow rate of 7 mL/
min, absorption being measured at 216 and 280 nm. Analytical
HPLC for the cyclic hexapeptides was performed on a Waters
The phenolic hydroxyl group of the N-terminal Tyr
residue in opioid peptides is an important binding
element in the interaction with opioid receptors.8 Re-
placement of the Tyr1 hydroxyl group in linear and cyclic
opioid peptides, by Phe, has been shown to result in a
potency decrease by 1 or 2 orders of magnitude.41 In
marked constrast, the replacement in Tyr of 1 by Cha
(affording 4) had little or no effect on potency. This
result suggests that the hydroxyl group of the Tyr
residue in the cyclic hexapeptide need not be required
for binding to the δ-receptor and that the resulting
compounds have a receptor binding mode that is dif-
ferent from opioid peptides which contain an N-terminal
Tyr residue.
Finally, the route which this work has taken is also
of interest. It was originally discovered by Terenius that
SRIF has weak affinity for the µ-opioid receptor.42
Utilizing this fact, Hruby designed and synthesized
somatostatin analogues that have high potency at the
µ-opioid receptor and low affinity at the SRIF receptor,
thus changing the biological profile.43 Our previous work
has demonstrated that through design and synthesis,
we converted a SRIF agonist into a NK-1 antagonist,
changing ligand-receptor affinity.24 This paper dem-
onstrates that an SRIF analogue can be modified also
to produce an opioid ligand; however, this time we
targeted the δ-opioid receptor. Finally this paper also
demonstrates that the SRIF, NK-1, and δ-opioid recep-
600E multisolvent delivery system equipped with
a 996
photodiode array detector. Reversed-phase HPLC chromatog-
raphy was performed with a Vydac 238TP54 column (4.6 ×
250 mm) with a 0-100% gradient of aqueous 0.1% TFA against
either acetonitrile or methanol at a flow rate of 1.0 mL/min.
Preparative HPLC was performed on a Rainin solvent delivery
system equipped with a Dynamax detector and utilizing a
Dynamax C18 (300 Å, 21.4 × 250 mm) column with a 30-
70% gradient of aqueous 0.1% TFA to MeCN.

556 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schiller et al.
Gen er a lized Cyclic Hexa p ep tid e Syn th esis. Assembly
of the peptides started with either 0.1 or 0.25 mmol of Fmoc-
L-Pro-2-chlorotrityl polystyrene resin and sequential addition
of the approriately protected N-R-Fmoc amino acids.27 Peptides
were removed from the resin using a solution of 0.40% TFA
in CH2Cl2. After 30 min, the slurry was filtered and washed
with additional TFA solution. The filtrate was concentrated,
azeotroped with benzene, and dried. Cyclization occurred by
suspending the peptide (1 equiv) in anhydrous DMF, along
with NaHCO3 (15 equiv) and DPPA (1.5 equiv).28 This solution
was stirred for at least 18 h at 0 °C and monitored by HPLC.
Once complete, the DMF was removed and the peptide was
subjected to silica gel chromatography. The protecting groups
were removed from the cyclic peptides using TFA:CH2Cl2:EDT:
H2O (50:45:3:2), followed by precipitation with ethyl ether:
hexane. The crude peptide was filtered, dried, subjected to
HPLC purification, and lyophilized as an amorphous solid.
dimethylphenol (24.4 g, 0.20 mol) in MeOH (406 mL) and concd
HCl (162 mL) was added a mixture of KI (22.4 g, 0.14 mol)
and KIO3 (13.8 g, 4.5 mmol). The mixture was initially cooled
in an ice bath and allowed to warm to room temperature
overnight. The precipitated product was collected by filtration
and washed with MeOH/H2O (1:1). The crude product was
dissolved in toluene, filtered through Celite, and precipitated
with pentane to give 9 (12.2 g, 24%): mp 133-135 °C (lit.29
131 °C); TLC Rf 0.85 (I).
3,5-Dim eth yl-4-iod op h en yl Aceta te (10).45 To a solution
of 9 (12.0 g, 48.8 mmol), in pyridine (12.0 mL) was added acetic
anhydride (6.9 mL, 72.6 mmol). The reaction was heated to
50 °C for 30 min, cooled to room temperature and diluted with
0.5 N HCl (200 mL) to effect crystallization. The product was
washed with 120 mL 0.5 N HCl followed by 60 mL portions of
H2O until the final wash reached a pH of 5. Vacuum drying
yielded 13.4 g (95%) of crystalline 10: mp 46-47 °C (lit.45 51
°C); TLC Rf 0.9 (II).
Meth yl 3-(2′,6′-Dim eth yl-4′-acetoxyph en yl)acr ylate (11).
To a solution of 10 (5.0 g, 17.24 mmol), methylacrylate (1.63
mL, 18.08 mmol), tri-o-tolylphosphine (0.27 g, 0.91 mmol), and
NEt3 (4.77 mL, 34.0 mmol) was added Pd(AcO)2 (71.6 mg, 0.32
mmol) in MeCN (24.3 mL). The solution was heated to reflux
for 28 h prior to cooling to room temperature. The catalyst
was filtered through Celite, and the solvent removed in vacuo.
The crude product was dissolved in H2O (30 mL) and extracted
with EtOAc (3 × 40 mL). The combined EtOAc extracts were
washed with brine (3 × 30-mL), treated with activated carbon
and dried over MgSO4. Solvent removal in vacuo yielded a red
oil which crystallized upon standing at 5 °C for 18 h. Recrys-
tallization from ethyl acetate/hexane afforded 3.80 g (89%) of
11: mp 59-60 °C; TLC Rf 0.48 (II); 1H NMR (500 MHz, CDCl3)
δ 2.26 (s, 3H, COCH3), 2.36 (s, 6H, Ar-CH3), 3.80 (s, COOCH3),
6.03 (d, 1H, J ) 16.4 Hz), 6.78 (s, 2H, Ar), 7.76 (d, 1H, J )
16.4 Hz).
Cyclo(1-Na l-D-Tr p -Tyr -Th r -P h e-P r o) (2): [R]25 -0.70°
D
1
(c 0.19, MeOH); H NMR (500 MHz, CD3OD) δ 0.78 (m, 2H),
0.86, (m, 1H), 1.15 (d, 3H, J ) 6.4 Hz), 1.37 (m, 1H), 1.73 (m,
1H), 2.49 (dd, 1H, J ) 5.4, 13.9 Hz), 2.56 (dd, 1H, J ) 5.0,
14.2 Hz), 2.78 (m, 2H), 2.88 (m, 1H), 3.03 (m, 2H), 3.18 (m,
1H), 3.41 (dd, 1H, J ) 7.7, 15.6 Hz), 3.52 (dd, 1H, J ) 7.4,
13.6 Hz), 3.61 (d, 1H), 4.11 (dd, 1H, J ) 4.7, 6.4 Hz), 4.15 (dd,
1H, J ) 5.1, 8.0 Hz), 4.30 (dd, 1H, J ) 5.6, 10.8 Hz), 4.38 (d,
1H, J ) 4.6 Hz), 4.44 (dd, 1H, J ) 5.5, 9.9 Hz), 4.82 (d, 1H, J
) 7.6 Hz), 6.56 (s, 4H), 6.80 (s, 1H), 7.06 (t, 1H, J ) 7.5), 7.14
(t, 1H, J ) 10.0 Hz), 7.18 (m, 2H), 7.25 (m, 6H), 7.37 (d, 1H,
J ) 8.2 Hz), 7.42 (d, 1H, J ) 7.9 Hz), 7.44 (m, 1H), 7.73 (d,
1H, J ) 8.1 Hz), 7.83 (d, 1H, J ) 7.8 Hz), 8.22 (d, 1H, J ) 8.4
Hz); 13C NMR (125 MHz, CD3OD) δ 18.9, 22.2, 28.4, 31.6, 36.4,
36.6, 38.6, 47.3, 55.3, 55.8, 56.2, 57.3, 57.9, 62.5, 68.6, 110.3,
112.6, 116.4, 119.3, 119.9, 122.6, 124.4, 124.9, 126.4, 126.8,
128.1, 128.4, 128.6, 128.7, 128.9, 129.8, 130.0, 130.6, 130.9,
133.6, 134.3, 135.4, 136.8, 138.0, 157.2, 171.4, 171.9, 172.6,
173.1, 173.7, 174.2; HRMS (ESI) m/z calcd for C51H53N7O8
+
Meth yl 3-(2′,6′-Dim eth yl-4′-a cetoxyp h en yl)p r op ion a te
(12). To a solution of 11 (3.72 g, 15 mmol) in MeOH/AcOH
(1:1) (50 mL) was added 10% Pd-C (2.42 g). The reaction
vessel was purged with argon and pressurized to 60 psi with
H2. The reaction mixture was stirred vigorously and the
temperature raised to 60 °C. When the pressure dropped to 0
psi, the mixture was cooled to 10 °C, vented with argon and
repressurized to 60 psi with H2. This procedure was repeated
10 times until TLC analysis (III) indicated complete reduction.
The mixture was filtered through Celite and the solvents
removed in vacuo to yield 3.05 g (81.3%) of 12 as an oil: TLC
Rf 0.36 (II), Rf 0.76 (III); 1H NMR (400 MHz, CDCl3) δ 2.28 (s
3H, OCH3), 2.32 (s, 6H, CH3 Ar), 2.44 (t, 2H, CH2), 2.94 (t,
2H, CH2), 3.71 (s, 3H, COOCH3), 6.74 (s, 2H, Ar).
Na 914.3853, found 914.3830 [(M + Na)+].
Cyclo(2-Na l-D-Tr p -Tyr -Th r -P h e-P r o) (3): [R]25 -2.01°
D
1
(c 0.17, MeOH); H NMR (500 MHz, CD3OD) δ 0.80 (m, 2H),
1.12 (d, 3H, J ) 6.4 Hz), 1.25 (m, 1H), 1.68 (m, 1H), 2.65 (dd,
1H, J ) 5.0, 14.2 Hz), 2.70 (dd, 1H, J ) 6.4, 14.0 Hz), 2.78
(dd, 1H, J ) 8.3, 14.2 Hz), 2.90 (m, 2H), 3.06 (m, 3H), 3.20 (m,
2H), 3.60 (d, 1H, J ) 7.6 Hz), 4.09 (dd, 1H, J ) 4.5, 6.4 Hz),
4.20 (dd, 1H, J ) 5.0, 8.3 Hz), 4.37 (m, 1H), 4.44 (dd, 1H, J )
6.5, 9.0 Hz), 4.76 (dd, 1H, J ) 6.7, 7.7 Hz), 6.57 (d, 2H, J )
6.0 Hz), 6.64 (d, 2H, J ) 6.5 Hz), 6.89 (s, 1H), 7.05 (t, 1H, J )
7.5 Hz), 7.13 (m, 1H), 7.25 (m, 6H), 7.37 (m, 4H), 7.44 (s, 1H),
7.64 (m, 2H), 7.74 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 17.5,
20.6, 26.9, 30.1, 35.3, 37.3, 38.3, 45.9, 53.9, 54.5, 54.6, 55.9,
56.4, 61.1, 67.1, 109.1, 111.2, 115.0, 117.9, 118.5, 121.2, 123.1,
125.3, 125.7, 126.9, 127.0, 127.1, 127.2, 127.3, 127.6, 127.7,
128.6, 129.2, 129.5, 132.5, 133.4, 134.1, 135.5, 136.7, 155.9,
170.0, 170.6, 171.1, 171.8, 172.3, 173.0; HRMS (ESI) m/z calcd
for C51H53N7O8 + Na 914.3853, found 914.3853 [(M + Na)+].
3-(2′,6′-Dim eth yl-4′-h ydr oxyph en yl)pr opion ic Acid (Dh p)
(13). A mixture of 12 (3.05 g, 12.2 mmol) and 12 N HCl (14
mL) was heated to reflux for 7.5 h, followed by a cooling to 4
°C overnight. The solid product was collected and recrystallized
from boiling water to give 13 (2.06 g, 87%): mp 123-124 °C;
1
TLC Rf 0.18 (II), Rf 0.77 (III); H NMR (400 MHz, CDCl3) δ
CHO-Dm t-cyclo(D-Or n -2-Na l-D-P r o-Gly) (5). To a solu-
tion of peptide 7 (15 mg, 0.02 mmol) at 0 °C were added DIPEA
(0.005 mL, 0.029 mmol) and p-nitrophenyl formate (0.304 mL,
0.029 mmol). The reaction was stirred for 2 h and allowed to
warm to room temperature. Solvent was removed and the
2.32 (s, 6H, CH3 Ar), 2.44 (t, 2H, CH2), 2.94 (t, 2H, CH2), 6.81
(s, 2H, ar); FAB-MS m/e 195.
O-Boc Der iva tive of 3-(2′,6′-Dim eth yl-4′-h yd r oxyp h en -
yl)p r op ion ic Acid [Dh p (OBoc)] (14). To a solution of 13
(2.06 g, 10.61 mmol) in THF/H2O (1:1) (24 mL) at 0 °C were
added DMAP (0.288 g, 2.35 mmol), NEt3 (3.86 mL, 27.56 mmol)
and Boc2O (2.67 g, 11.67 mmol) in the THF/H2O (1:1) (10 mL).
The reaction stirred for 1.5 h before the THF was removed in
vacuo. Ethyl acetate (30 mL) was added, followed by addition
of a 5% aqueous solution of KHSO4 until a pH of 6 was reached.
The organic phase was separated, dried over MgSO4, and
evaporated to yield the crude product as an oil. Crystallization
from ethyl acetate/hexane afforded crystalline 14 (2.35 g,
peptide was directly purified by HPLC (12.2 mg, 92.4%): [R]25
D
-1.02° (c 0.26, MeOH); 1H NMR (500 MHz, CD3OD) δ 1.03
(m, 1H), 1.28 (m 3H), 1.54 (m, 3H), 1.67 (m, 1H), 2.26 (s, 6H),
2.30 (m, 1H), 2.82 (d, 1H, J ) 13.2 Hz), 2.96 (dd, 1H, J ) 4.8,
13.8 Hz), 3.10 (m, 2H), 3.43 (m, 3H), 3.53 (m, 1H), 4.10 (dd,
1H, J ) 4.5, 8.0 Hz), 4.28 (m, 2H), 4.32 (dd, 1H, J ) 5.4, 11.0
Hz), 4.43 (dd, 1H, 4.3, 10.9 Hz), 6.50 (s, 2H), 7.36 (dd, 1H, J )
1.7, 8.4 Hz), 7.48 (m, 2H), 7.69 (s, 1H), 7.83 (m, 3H), 8.09 (d,
1H, J ) 1.0 Hz); 13C NMR (125 MHz, CD3OD) δ 20.3, 23.5,
25.0, 27.2, 29.9, 31.9, 37.9, 38.8, 43.2, 48.3, 54.2, 54.7, 56.2,
62.9, 116.2, 125.2, 127.2, 127.6, 128.6, 128.7, 129.1, 129.4,
134.1, 134.4, 134.9, 139.9, 157.1, 163.6, 171.8, 174.0, 174.1,
174.3, 174.5; HRMS (ESI) m/z calcd for C37H44N6O7 + Na
707.3169, found 707.3169 [(M + Na)+].
1
80%): mp 108-110 °C; TLC Rf 0.87 (I); H NMR (400 MHz,
CDCl3): δ 1.55 (s, 9H, C(CH3)3), 2.31 (s, 6H, CH3 Ar), 2.45 (t,
2H, CH2), 2.95 (t, 2H, CH2), 6.82 (s, 2H, Ar); FAB-MS m/e 295.
Dh p -D-Or n -2-Na l-D-P r o-Gly-OH (15). Peptide synthesis
was performed by the manual solid-phase technique using a
Merrifield Boc-Gly-OH resin (1% cross-linked, 100-200 mesh,
3,5-Dim eth yl-4-iod op h en ol (9).29 To a solution of 3,5-

Ligands Lacking Positive Charge for δ,µ-Opioid Receptors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 557
0.76 mmol; Bachem Bioscience, King of Prussia, PA). The
peptide was assembled using Boc-protected amino acids
(Bachem) and DIC/HOBt coupling according to a published
protocol.26,31 The protecting groups of Orn (Fmoc) and Dhp
(Boc) were removed with 20% (v/v) piperidine in DMF and 50%
(v/v) TFA in CH2Cl2, respectively. The peptide was removed
from the resin by treatment with HF (20 mL) and anisole (1.0
mL) for 60 min at 0 °C. After evaporation of the HF, the resin
was extracted three times with Et2O, followed by extraction
with glacial AcOH. The linear peptide 15 was obtained in solid
form through lyophilization of the acetic acid extract and
purification by preparative HPLC: TLC Rf 0.51 (IV), Rf 0.59
(V); FAB-MS m/e 659 (M+).
nM. IC50 values were determined from log dose-displacement
curves, and Ki values were calculated from the obtained IC50
values by means of the equation of Cheng and Prusoff,51 using
values of 1.3, 2.6, and 2.9 nM for the dissociation constants of
3[H]DAMGO, [3H]DSLET, and [3H]U69,593, respectively.
Ack n ow led gm en t. This work was supported by the
Medical Research Council of Canada (Grant MT-5655
to P.W.S.) and the National Institute on Drug Abuse
(Grant DA-04443 to P.W.S.). We thank Dr. M. Evans
(Department of Chemistry, University of Montreal) and
Mr. J ohn Dykins (Department of Chemistry, University
of Pennsylvania) for performing the FAB and ESI mass
spectrometric determinations, respectively. Additional
support (R.H., A.B.S.) was provided by a grant (GM-
41821) from the National Institutes of Health (Institute
of General Medical Sciences), Merck Research Labora-
tories, and DuPont Pharmaceuticals Co. The authors
(R.H., A.B.S.) thank Dr. George Furst for assistance in
obtaining NMR spectra and Dr. J oseph Barbosa for
many fruitful discussions.
Dh p -c[-D-Or n -2-Na l-D-P r o-Gly-] (6). To a solution of 15
(100 mg, 0.151 mmol) in DMF (20 mL) were added DPPA (65
µL, 0.302 mmol) and NMM (16.5 µL, 0.151 mmol) in DMF (138
mL). The reaction was cooled to -25 °C and the cyclization
was monitored by HPLC analysis. Additional DPPA (32 µL,
0.151 mmol) and NMM (16.5 µL, 0.151 mmol) were added to
the reaction mixture until HPLC analysis indicated a complete
reaction (48 h). Evaporation of the DMF and trituration of the
residue with petroleum ether resulted in a crude oil. The
product was dissolved in EtOH, precipitated with 5% aq
KHSO4, filtered, and washed with H2O. The product was
redissolved in EtOAc, precipitated with diisopropyl ether, and
filtered. The crude product was purified by preparative HPLC
and was >98% pure by analytical HPLC: TLC Rf 0.65 (IV),
Rf 0.89 (V); [R]25D +7.33 ° (c 0.15, MeOH); 1H NMR (500 MHz,
CD3OD) δ 1.26 (m, 1H), 1.68 (m, 2H), 1.70 (t, 2H, J ) 7.3 Hz),
1.75 (d, 2H, J ) 8.0 Hz), 2.22 (s, 6 H), 2.25 (dd, 1H, J ) 7.6,
14.3 Hz), 2.34 (dd, 1H, J ) 6.0, 14.3 Hz), 2.66 (m, 1H), 2.78 (t,
2H, J ) 7.8 Hz), 2.89 (d, 1H, J ) 12.9 Hz), 3.22 (m, 2H), 3.48
(dd, 1H, J ) 3.4, 14.0 Hz), 3.55 (dd, 1H, J ) 11.5, 11.5 Hz),
3.70 (m, 1H), 4.16 (dd, 1H, J ) 5.6, 6.9 Hz), 4.28 (d, 1H, J )
9.0 Hz), 4.32 (br s, 1H), 4.62 (m, 1H), 6.45 (s, 2H), 6.80 (d, 1H,
J ) 9.6 Hz), 7.39 (d, 1H, J ) 8.4 Hz), 7.44 (m, 2H), 7.57 (d,
1H, J ) 5.4 Hz), 7.71 (br s, 2H), 7.80 (m, 5H), 7.94 (br s, 1H);
13C NMR (125 MHz, CD3OD) δ 20.1, 23.6, 25.3, 25.6, 27.5, 30.0,
36.6, 37.4, 38.9, 43.2, 55.6, 55.8, 63.1, 116.0, 127.2, 127.6, 128.4,
128.6, 128.7, 129.0, 129.5, 129.7, 134.1, 134.4, 134.9, 138.6,
156.2, 171.9, 174.3, 174.7, 175.0, 176.2; FAB-MS m/e 641 (M+);
HRMS (ESI) m/z calcd for C36H44N5O6 + Na 665.3189, found
665.3218 [(M + Na)+].
In Vitr o Bioa ssa ys a n d Recep tor Bin d in g Assa ys. The
GPI46 and MVD47 bioassays were carried out as reported in
detail elsewhere.47,48 Ke values for antagonists were determined
from the ratio (DR) of IC50 values obtained with an agonist in
the presence and absence of a fixed antagonist concentration
(a), using the equation: Ke ) a/(DR - 1).49 δ-Antagonist Ke
values of all compounds were determined in the MVD assay
against the δ-agonist DPDPE using antagonist concentrations
ranging from 50 to 4000 nM. The µ-antagonist Ke values of
the cyclic â-casomorphin analogues were determined in the
GPI assay against the µ-agonist TAPP with an antagonist
concentration of 500 nM.
Opioid receptor binding studies were performed as described
in detail elsewhere.47 Due to their poor water solubility, the
cyclic hexapeptides (∼2 mg) were first dissolved in 150 µL of
DMSO and then 9.85 mL of buffer was added. Since some
precipitation occurred after addition of the buffer, the precipi-
tated peptide was removed by filtration and the actual peptide
concentration of the clear solution was determined by optical
density measurement. To prevent peptide degradation, an
enzyme inhibitor cocktail50 consisting of bestatin (100 µM),
captopril (1.0 µM), thiorphan (1.0 µM), and L-leucyl-L-leucine
(100 µM) was added to the buffer. Binding affinities for µ- and
δ-opioid receptors were determined by displacing, respectively,
[3H]DAMGO (Multiple Peptide Systems, San Diego, CA) and
[3H]DSLET (Multiple Peptide Systems) from rat brain mem-
brane binding sites, and κ opioid receptor affinities were
measured by displacement of [3H]U69,593 (Amersham) from
guinea pig brain membrane binding sites. Incubations were
performed for 2 h at 25 °C with [3H]DAMGO, [3H]DSLET, and
[3H]U69,593 at respective concentrations of 0.72, 0.78, and 0.80
Su p p or t in g In for m a t ion Ava ila b le: Analytical HPLC
traces of compounds 1-4 and 6 and Table 3 of low-activity
compounds tested in the MVD assay. This information is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Symbols and abbreviations are in accordance with recommenda-
tions of the IUPAC-IUB J oint Commission on Biochemical
Nomenclature: Nomenclature and Symbolism for Amino Acids
and Peptides. Biochem J . 1984, 219, 345-373. The other
abbreviations are as follows: Boc, tert-butoxycarbonyl; Cha,
cyclohexylalanine; CTOP, H-D-Phe-c[-Cys-Tyr-D-Trp-Orn-Thr-
Pen-]-Thr-NH2; DAMGO, H-Tyr-D-Ala-Gly-NRMePhe-Gly-ol; Dhp,
3-(2′,6′-dimethyl-4′-hydroxyphenyl)propionic acid; DIC, 1,3-di-
isopropylcarbodiimide; Dmt, 2′,6′-dimethyltyrosine; DPDPE, H-
Tyr-c[-D-Pen-Gly-Phe-D-Pen-]; DIPEA, diisopropylethylamine;
DPPA, diphenyl phosphorazidate; DSLET, H-Tyr-D-Ser-Gly-Phe-
Leu-Thr-OH; FAB-MS, fast atom bombardment mass spectrom-
etry; Fmoc, 9-fluorenylmethoxycarbonyl; GPI, guinea pig ileum;
HOBt, 1-hydroxybenzotriazole; DIC, 1,3-diisopropylcarbodiimide;
MVD, mouse vas deferens; 1-Nal, 3-(1-naphthyl)alanine; 2-Nal,
3-(2-naphthyl)alanine; NMM, N-methylmorpholine; DMAP,
4-(dimethylamino)pyridine, TAPP, H-Tyr-D-Ala-Phe-Phe-NH2;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; U69,593,
(5R,7R,8â)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-
8-yl]benzeneacetamide.
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