Synthesis and biological activities of cyclic lanthionine enkephalin analogues: δ-opioid receptor selective ligands

Article abstract of DOI:10.1021/jm020108k

The synthesis and biological test results of a series of enkephalin analogues incorporating the lanthionine modification are presented. The syntheses of four monosulfide-bridged analogues of enkephalins, Tyr-c[D-Ala_L-Gly-Phe-D-Ala_L]-OH (1a), Tyr-c[D-Val_L-Gly-Phe-D-Ala_L]-OH (1b), Tyr-c[D-Ala_L-Gly-Phe-Ala_L]-OH (1c), and Tyr-c[D-Val_L-Gly-Phe-Ala_L]-OH (1d), where Ala_L and Val_L denote the lanthionine amino acid ends linked by a monosulfide bridge to form the lanthionine structure, were successfully carried out via preparation of the linear peptide on solid support and cyclization in solution. In vitro binding assays against μ, δ-, and κ-opioid receptors and in vitro tests using GPI and MVD assays revealed that the dimethyl lanthionine analogues 1b and 1d, denoted as D-Val_L in position 2, show substantial selectivity toward the δ-opioid receptor, while the unsubstituted analogues 1a and 1c, denoted as D-Ala_L in position 2, bind to both μ- and δ-opioid receptors. The in vivo thermal escape assay by intrathecal administration showed that the analogues 1b and 1d are among the most potent ligands at producing antinociception through the δ-opioid receptor. The picomolar potencies of analogues 1a and 1c in the intrathecal (it.) assay strongly indicate that μ- and δ-opioid receptors interact synergistically to modulate the antinociceptive responses.

Full text of DOI:10.1021/jm020108k

3746
J . Med. Chem. 2002, 45, 3746-3754
Syn th esis a n d Biologica l Activities of Cyclic La n th ion in e En k ep h a lin
An a logu es: δ-Op ioid Recep tor Selective Liga n d s
Yosup Rew,† Shelle Malkmus,‡ Camilla Svensson,‡ Tony L. Yaksh,‡ Nga N. Chung,§ Peter W. Schiller,§
J oel A. Cassel,| Robert N. DeHaven,| and Murray Goodman*,†
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive,
La J olla, California 92093-0343, Department of Anesthesiology, University of California, San Diego, 9500 Gilman Drive,
La J olla, California 92093-0818, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal,
Quebec, H2W 1R7, Canada, and Adolor Corporation, 371 Phoenixville Pike, Malvern, Pennsylvania 19355
Received March 7, 2002
The synthesis and biological test results of a series of enkephalin analogues incorporating the
lanthionine modification are presented. The syntheses of four monosulfide-bridged analogues
of enkephalins, Tyr-c[D-AlaL-Gly-Phe-D-AlaL]-OH (1a ), Tyr-c[D-ValL-Gly-Phe-D-AlaL]-OH (1b),
Tyr-c[D-AlaL-Gly-Phe-AlaL]-OH (1c), and Tyr-c[D-ValL-Gly-Phe-AlaL]-OH (1d ), where AlaL and
ValL denote the lanthionine amino acid ends linked by a monosulfide bridge to form the
lanthionine structure, were successfully carried out via preparation of the linear peptide on
solid support and cyclization in solution. In vitro binding assays against µ-, δ-, and κ-opioid
receptors and in vitro tests using GPI and MVD assays revealed that the dimethyl lanthionine
analogues 1b and 1d , denoted as D-ValL in position 2, show substantial selectivity toward the
δ-opioid receptor, while the unsubstituted analogues 1a and 1c, denoted as D-AlaL in position
2, bind to both µ- and δ-opioid receptors. The in vivo thermal escape assay by intrathecal
administration showed that the analogues 1b and 1d are among the most potent ligands at
producing antinociception through the δ-opioid receptor. The picomolar potencies of analogues
1a and 1c in the intrathecal (it.) assay strongly indicate that µ- and δ-opioid receptors interact
synergistically to modulate the antinociceptive responses.
In tr od u ction
Methionine and leucine enkephalins, identified from
mammalian tissues in 1975, have been considered as
endogenous peptide ligands toward the δ-opioid recep-
tor.9 As with other bioactive endogenous peptides, there
are several major considerations that limit the clinical
applications of naturally occurring enkephalins includ-
ing rapid degradation under physiological conditions,
poor selectivity for receptor subtypes, modest absorp-
tion, and relatively poor central bioavailability.10 Since
their discovery, exhaustive synthetic modifications of
enkephalins have been conducted in an attempt to
improve their biological profiles. Studies to obtain
structure-activity relationships of enkephalins have
revealed that two aromatic groups of Tyr and Phe and
the protonated nitrogen at the N terminus act as
pharmacophores when enkephalins interact with the
receptors.11 Changes in the amino acid composition of
these endogenous peptides can lead to compounds with
high potency and selectivity for the δ-receptor, such as
DADLE ([D-Ala2, D-Leu5]-enkephalin),12 BUBU [Tyr-D-
Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)],13 and the cyclic
peptide DPDPE ([D-Pen2, D-Pen5]-enkephalin).14 These
and other δ-receptor selective peptides have been used
for in vitro studies, but their metabolic instability and/
or their relatively poor distribution properties and low
in vivo potency have limited their usefulness.
The pharmacological effects of opioids are due to
specific interactions of the drugs with opioid receptors.
To date, three major opioid receptors (µ, δ, and κ) have
been cloned and characterized.1 Morphine and the other
alkaloid opioids, currently in use for the relief of pain,
are µ-opioid receptor selective agonists. Many of the
serious side effects associated with morphine adminis-
tration such as respiratory depression, physiological
dependence, tolerance, and a reduction in gut motility
are mediated by the µ-opioid receptor,2 while κ-agonists
show some promise as nonaddictive analgesics.3 The
κ-agonists, however, are generally less efficacious as
analgesics4,5 and have been shown to cause dysphoric
side effects.6 The δ-opioid receptor is an attractive target
that may possess potential clinical benefits including
sufficient analgesic efficacy, negligible cross tolerance
to morphine, reduced respiratory depression, reduced
inhibition of gut motility, and minimal physical depen-
dence.7 No δ-opioid receptor selective agonist that
exhibits potent systemic in vivo activity has been found
to date.8 Therefore, our goal was to develop opioid
ligands with a high degree of selectivity and potency
for the δ-receptor subtype.
* To whom correspondence should be addressed. Phone: (858) 534-
4466. Fax: (858) 534-0202. E-mail: mgoodman@ucsd.edu.
† Department of Chemistry and Biochemistry, University of Cali-
fornia, San Diego.
Lanthionines are monosulfide analogues of cystine
and are key constituents of bioactive peptide lantibiotics.
They can act as food preservatives, antibacterials,
immunostimulants, and antitumor agents.15-18 For
‡ Department of Anesthesiology, University of California, San Diego.
§ Clinical Research Institute of Montreal.
| Adolor Corporation.
10.1021/jm020108k CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/04/2002

Lanthionine Enkephalin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3747
F igu r e 1. Enkephalins, DPDPE, and lanthionine enkephalin analogues.
Sch em e 1. Synthesis of Lanthionine Building Blocks
some time, our group has pursued the introduction of
the lanthionine structure as novel peptidomimetics into
various drug families, i.e., sandostatin,19 cell adhesion
modulator,20 and somatostatin.21-23 The monosulfide
bridge of lanthionine provides more constrained peptide
structures with greater stability toward enzymatic
degradation compared to the labile disulfide bridge of
cystine in natural or unnatural cyclic peptide sequences.
Molecular modeling and X-ray diffraction studies of
DPDPE indicated that the δ-selectivity of DPDPE is due
to the presence of the gem-dimethyl group in the 2
position, which causes more severe steric interference
at the µ-receptor than at the δ-receptor.24,25 On the basis
of this finding, we designed and synthesized a series of
enkephalin analogues incorporating the lanthionine
modification (1a -d , Figure 1).26-28
disulfide with a thioether side chain linkage and the
consequent reduced ring size will lead to more stable
and selective opioids.
Previously, we reported the synthesis of similar
lanthionine enkephalin analogues by several methods
that have been developed to prepare synthetic lanthio-
nines or natural lantibiotics including sulfur extrusion
and biomimetic Michael addition of cysteine to dehy-
droalanine.29,30 Unfortunately, inherent problems such
as lack of regioselectivity and epimerization made these
methods impractical for the development of a general-
ized synthetic route for the target lanthionine ana-
logues. To overcome the lack of stereoselectivity, we
developed a facile synthesis of orthogonally protected
lanthionine using the â-lactone31 developed by Vederas
et al.32 Because this route did not provide a building
block in a suitably protected form for Fmoc solid-phase
peptide synthesis,33 the target lanthionine enkephalins
were synthesized in solution.34 Although a preliminary
report of the synthesis of lanthionine opioids has ap-
peared, the method of synthesis in solution led to serious
problems of yield and optical purity of the target
structures.
In this paper we present novel routes for the synthesis
of lanthionine enkephalin analogues and the results of
biological assays (in vitro opioid binding assays, in vitro
GPI and MVD assays, and in vivo thermal escape assay
by intrathecal (it.) administration).
Resu lts a n d Discu ssion
Design a n d Syn th esis of Ta r get P ep tid es. To
compare the biological activities and stabilities of the
lanthionine analogues with DPDPE, we synthesized
lanthionine enkephalin analogues with a minimal change
of the spatial arrangement of the pharmacophoric
groups (Figure 1). We believe that the replacement of a
Recently, the synthesis of Fmoc-protected lanthio-
nines for solid-phase peptide synthesis was reported by
Me´nez (Scheme 1, route A).35 The N-trityl-3-iodoalanine
derivatives were used to avoid epimerization at the
R-carbon or â-elimination. However, when the reaction
to form the lanthionine was carried out in solution, a

3748 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Sch em e 2. Synthesis of Lanthionine Enkephalin Analoguesa
Rew et al.
a
(a) (i) 20% piperidine in NMP, (ii) Trt-Gly-OH, PyBOP, HOBt, DIEA, DMF, (iii) DCM/TFA/TIS (97/2/1), (iv) preactivated FMOC-D-
Cys(Trt)-OH (for 3a ) or Fmoc-D-Pen(Trt)-OH (for 3b) with DIC, DIEA, DMF/DCM (1/1), (v) 20% piperidine in NMP, (vi) Cbz-Tyr(Bzl)-OH,
PyBOP, HOBt, DIEA, DMF, (vii) DCM/TFA/TIS (94/3/3); (b) (i) 4a or 4b, Cs2CO3, DMF, (ii) TFA/H2O/TIS (93/5/2), (iii) 4 N HCl/dioxane;
(c) DPPA, NaHCO3, DMF, 0 °C, 72 h; (d) (i) HBr/AcOH, room temp, 4 h, (ii) purification by RP-HPLC.
significant amount of undesired aziridine was obtained
(up to 35%). As a result, we applied this method to solid-
phase reactions in which any aziridine formed was
removed by washing the solid support (Scheme 1, route
B). The target lanthionine enkephalin analogues (1a -
d ) were successfully synthesized via preparation of the
linear peptide on the solid support and cyclization in
solution (Scheme 2).26-28 Synthesis of analogue 1a
commenced with commercially available Fmoc-Phe Wang
resin 2. The tetrapeptide chain 3 was initially assembled
on the Wang resin. The Fmoc-Phe Wang resin 2 was
allowed to react with Trt-Gly-OH using PyBOP/HOBt,
after deprotection of the Fmoc group with 20% piperi-
dine in NMP. The Trt-Gly-Phe Wang resin was then
deprotected with CH2Cl2/TFA/TIS (97/2/1, v/v/v) and
coupled with Fmoc-D-Cys(Trt)-OH, which was preacti-
vated with DIC and HOBt under neutral conditions to
prevent diketopiperazine formation and epimeriza-
tion.36,37 The Fmoc protecting group of the tripeptide
resin was removed by 20% piperidine in DMF, and Cbz-
Tyr(Bzl)-OH was then coupled to the resulting peptide
resin using PyBOP/HOBt. After removal of the trityl
protecting group of cysteine by CH2Cl2/TFA/TIS (94/3/
3, v/v/v), the lanthionine linkage was introduced by
reaction of N-trityl-D-3-iodoalanine benzyl ester 4a with
the tetrapeptide resin 3a in the presence of Cs2CO3 in
DMF. The cleavage of the pentapeptide 5a from the
resin and the removal of the Trt protecting group were
achieved simultaneously by TFA/H2O/TIS (93/5/3,
v/v/v). The resulting crude peptide was cyclized in dilute
DMF solution in the presence of DPPA and NaHCO3.
Purification of the less polar desired cyclic peptide from
polar impurities was effected by silica gel column
chromatography, followed by removal of benzyloxycar-
bonyl, benzyl ether, and benzyl ester protecting groups
by 30% HBr/AcOH. Final purification by RP-HPLC
provided the target peptide 1a . The other target pep-
tides (1b-d ) were synthesized by this synthetic route.
Biologica l Resu lts. The synthesized lanthionine
enkephalin analogues were evaluated for their in vitro
binding affinities at µ-, δ-, and κ-opioid receptors by
measuring the inhibition of binding of [3H]-diprenor-
phine to cloned human opioid receptors expressed in
CHO cell membranes (Table 1). Morphine and DPDPE
were used as reference compounds. As shown in Table
1, analogues 1b and 1d , which have a gem-dimethyl
group in the â-position of D-AlaL2 at the second residue,
are δ-opioid receptor selective peptides, while analogues
1a and 1c, lacking the gem-dimethyl group in position
2, bind with high affinity to both µ- and δ-receptors. The
lanthionine enkephalin analogues bind to the δ-receptor
with affinities ranging from 0.63 to 2.0 nM and thus
are slightly more potent than DPDPE. The binding
affinity for the µ-receptor of analogue 1b is reduced 320-
fold relative to that of the parent compound 1a , whereas
analogue 1d has a 60-fold-reduced µ-affinity compared
with analogue 1c. Analogue 1b is the most δ-selective
ligand among these analogues.
The functional in vitro activities of the analogues were
determined by measuring the inhibition of electrically
evoked contractions of isolated muscle preparations. The
guinea pig ileum (GPI) contains µ-and κ-opioid recep-
tors, of which 70% are µ-receptors, and the mouse vas
deferens (MVD) contains µ- and δ-opioid receptors, of
which 80% are δ-receptors. The resulting IC50 values
determined in the GPI and MVD assays delineate the

Lanthionine Enkephalin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3749
Ta ble 1. In Vitro Binding Affinities of Lanthionine Enkephalin Analogues at Cloned Human µ-, δ-, and κ-Opioid Receptors in CHO
Cell Membranesa
Ki (nM)
Ki ratio
analogues
µ
δ
κ
µ/δ
1a
2.0
2.0
1600
1
Tyr-c[D-AlaL-Gly-Phe-D-AlaL]-OH
(1.8-2.2)
n ) 11
(1.7-2.3)
n ) 5
(1400-1900)
n ) 7
1b
630
(580-680)
n ) 3
0.93
(0.35-2.5)
n ) 3
>10 000
677
Tyr-c[D-ValL-Gly-Phe-D-AlaL]-OH
1c
2.3
0.63
6400
3.7
Tyr-c[D-AlaL-Gly-Phe-AlaL]-OH
(1.3-3.7)
n ) 5
(0.62-0.65)
n ) 4
(4700-8500)
n ) 4
1d
130
(110-150)
n ) 4
0.79
(0.66-0.95)
n ) 4
>1000
165
Tyr-c[D-ValL-Gly-Phe-AlaL]-OH
DPDPE
>10 000
2.2
(1.0-3.9)
n ) 5
>10 000
>4550
morphine
17
150
260
0.11
(6.8-43)
n ) 4
(63-360)
n ) 4
(100-690)
n ) 3
a
The Ki values are presented with 95% confidence intervals and the number (n) of determinations. Reference ligand is [3H]-diprenorphine.
Ta ble 2. In Vitro and In Vivo Bioactivities of Lanthionine Enkephalin Analogues
a
IC50 [nM]
d
IC50 ratio
GPI/MVD
ED50
[nM]
analogues
GPIb
MVDc
1a
0.56 ( 0.037
n )3
1.58 ( 0.13
n ) 3
0.35
0.0015
(0.0010-0.0024)
n ) 24
Tyr-c[D-AlaL-Gly-Phe-D-AlaL]-OH
1b
730 ( 136
n )3
2.33 ( 0.02
n ) 3
313
0.26
(0.17-0.41)
n ) 24
Tyr-c[D-ValL-Gly-Phe-D-AlaL]-OH
1c
1.06 ( 0.15
n ) 5
0.35 ( 0.04
n ) 5
3.02
0.0018
(0.0010-0.0034)
n ) 20
Tyr-c[D-AlaL-Gly-Phe-AlaL]-OH
1d
82.0 ( 11.5
n ) 5
0.26 ( 0.03
n ) 5
315
0.12
(0.04-0.33)
n ) 13
Tyr-c[D-ValL-Gly-Phe-AlaL]-OH
DPDPE
7300 ( 1700
n ) 3
4.1 ( 0.5
n ) 3
1780
0.09e
130
(98-163)
n ) 24
morphine
58.8e
644e
15
(10-24)
n ) 30
a
d
IC50 values are given (SEM with number (n) of determinations. b,c GPI for µ receptor and MVD for δ receptor. ED50 values are
presented with 95% confidence intervals and the number (n) of determinations. e Data taken from reference 60.
opioid activities of the synthesized analogues at the µ-
and δ-receptors, respectively. The ratio of the IC50
values in the GPI versus the MVD therefore provides a
functional index of the µ- or δ-selectivity (Table 2). The
compounds displayed a wide range of potencies in the
GPI assay, from IC50 values of 0.56 and 1.06 nM for
analogues 1a and 1c, respectively, to IC50 values of 730
and 82 nM for analogues 1b and 1d , respectively. In
contrast, the IC50 values of these lanthionine analogues
displayed a narrow range of potencies in the MVD assay
from 0.26 to 2.33 nM. These data indicated that
analogues 1a and 1c had low nanomolar potencies at
both µ- and δ-receptors. On the other hand, the dimeth-
ylated analogues 1b and 1d maintained the high
potency of analogues 1a and 1c toward the δ-receptor
while showing much decreased µ-activities. These re-
sults are consistent with the binding assay data. Ana-
logue 1b, which has a D residue at the fifth position,
showed reduced µ-potency with the same δ-potency
(greater selectivity for the δ-receptor) compared to
analogue 1d , which possesses an L residue at the fifth
position.
Previous work demonstrated the presence of µ- and
δ-receptors in the spinal dorsal horn,38 and the activa-
tion of each results in antinociception.39 The thermal
escape latency assay with it. drug administration mea-
sures the magnitude of the spinal antinociceptive effect.
The results are reported as the dosage necessary to
produce a half-maximum effect (ED50) (Table 2). All
agents examined produced a dose-dependent increase
in the thermal escape latency with a maximum eleva-
tion produced by the highest doses of all drugs. At doses
where a maximum thermal escape latency was ob-
served, there was no concomitant change in motor
function or general behavior. All of the lanthionine
analogues had much lower ED50 values than DPDPE

3750 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Rew et al.
or morphine. The ED50 values for 1a and 1c were 0.0015
and 0.0018 nM. Dimethylated analogues were less
potent, and the ED50 values were 0.26 nM for 1b and
0.12 nM for 1d . The ED50 values of analogues 1a and
1c indicate that these compounds were almost 106 times
more potent than DPDPE and 104 times more potent
than morphine. The dimethylated lanthionine analogues
1b and 1d , which are δ-selective agonists, exhibited
ED50 values that reflect potencies 60-120 times greater
than morphine as well as nearly 103 times greater than
DPDPE.
account for the potency of analogues 1a and 1c relative
to µ-agonists such as morphine and DAMGO. Because
the it. potency for the three δ-preferring ligands (1b,
1d , and DPDPE) in this study and other µ/δ-peptido-
mimetics previously studied covary with the in vitro
affinity and the activity for both the µ- and δ-receptors,
we cannot exclude the possibility that some of their
apparent potency also reflects a minimal µ-contribution
to their apparent antinociceptive potency.
Con clu sion
The enzymatic degradation studies of 1b and DPDPE
have been carried out by incubating the peptides in the
rat brain homogenates solution for 24 h.40 The results
indicate that both 1b and DPDPE are too stable to
establish a quantitative difference between them. The
extraordinary resistances is presumably caused by the
presence of a â,â-dimethyl group in the position 2 or
position 5, which stablizes the compound toward enzy-
matic degradation. A previous study of enzymatic
degradation of a similar lanthionine enkephalin amide,
which does not have a â,â-dimethyl group in the position
2, has shown that the lanthionine enkephalinamide Tyr-
c[D-AlaL-Gly-Phe-AlaL]-NH2 is approximately 5 times
more stable than the corresponding disulfide analogue
Tyr-c[D-Cys-Gly-Phe-Cys]-OH toward degradation by
rat brain homogenates.41 The enhanced potencies of
the δ-selective lanthionine analogues 1b and 1d over
DPDPE can be explaind in part by improved biostablilty
of the lanthionine structure compared with the disulfide
bond.
Synthesis of the target lanthionine analogues (1a -
d ) was accomplished by the reaction of thiol with
N-trityl-D- or N-trityl-L-3-iodoalanine benzyl ester on a
solid support. As shown by the data for in vitro binding
affinity, the â,â-dimethylation at the second residue led
to a dramatic decrease in binding affinity at the
µ-receptor for both 1b and 1d analogues without alter-
ing δ binding affinity. Furthermore, the Ki value at the
5
µ-receptor for 1b, which has a D-AlaL substitution, is
5-fold higher at the µ-receptor and decreases 5 times
5
more than that of 1d , which has an AlaL substitution.
The functional assay results from GPI and MVD assays
parallel the results from the binding assay. All the
lanthionine analogues had much lower antinociceptive
ED50 values than DPDPE and were also more potent
than morphine after spinal delivery. The subnanomolar
analgesic potencies of δ-selective lanthionine analogues
may be due to their potent δ-activity and improved
stability. The picomolar potencies of nonselective
lanthionine analogues is consistent with the existence
of synergistic interactions between spinal µ- and δ-re-
ceptors. The previous work systemically examining
spinal µ- and δ-interactions have indeed demonstrated
such synergy. Thus, the lanthionine-bridged enkepha-
lins represent a new family of interesting δ-receptor
selective opioid agonists.
It is important to stress that spinal δ-receptors can
mediate significant antinociception. Thus, spinal deliv-
ery of agents with high δ-selectivity as defined by
binding and in vitro bioassays (e.g., analogues 1b and
1d ) can yield potent analgesia. To determine the role
of the δ-opioid receptor in mediating the spinal action
of the analogue 1b, we examined the effects of the
δ-receptor-preferring antagonist naltrindole, given spi-
nally (30 µg/10 µL) on the antinociception produced by
a just maximally effective dose of intrathecal 1b (30 µg).
Alone, this agonist produced a maximum possible effect
(MPE) of 92 ( 9%. Following pretreatment with nal-
trindole, the MPE was significantly reduced to 23 ( 4%;
P < 0.05; n ) 5. In contrast, this antagonist treatment
had no effect on the antinociception produced by in-
trathecal morphine (data not shown).40
Further biological studies (ip administration, agonist
and antagonist tests) and acid stabilities of these
lanthionine enkephalin analogues for systemic avail-
ability will be reported elsewhere soon,40 and the
conformational analyses of these lanthionine analogues
by NMR are underway.
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Notes. NMR spectra were
obtained on Varian HG-400 (400 MHz) and Bruker AMX 500
(500 MHz) spectrometers. Chemical shifts (δ) are reported in
parts per million (ppm) relative to residual undeuterated
solvent as an internal reference. The following abbreviations
were used to explain the multiplicities: s, single; d, doublet;
t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of
triplets; m, multiplet; b, broad. Optical rotations were recorded
on a Perkin-Elmer 241 polarimeter. IR spectra were recorded
on a Nicolet Magna-IR 550 series II spectrometer. Mass
spectroscopic analyses were carried out by the facility at The
Scripps Research Institute, La J olla, CA. The final products
were purified and analyzed by RP-HPLC using Vydac protein
peptide C18 columns. Column dimensions were 4.5 mm × 250
mm (90 Å silica, 5 µm) for analytical HPLC and were 22 mm
× 250 mm (90 Å silica, 10 µm) for preparative HPLC. UV
absorbance was monitored at 220 nm. A binary system of
water and acetonitrile, both containing 0.1% TFA, was used
throughout. Two analytical HPLC profiles were obtained on a
Waters Millennium PDA system using both a linear gradient
of 10-50% acetonitrile over 30 min (condition A, tRa) and an
isocratic elution of 16% acetonitrile at 1 mL/min flow rate
Studies in recombinant mice, where expression of the
µ-opioid receptor was disrupted, demonstrated that
δ-opioid receptor selective agonists do not require
functional µ-receptors to mediate antinociception.42
While spinal δ-agonists are clearly distinct in their
pharmacology from µ-opioid agonists,43 the role played
by the δ-opioid receptor is clearly complicated. For
instance, while the effects of δ-selective agonists are
antagonized by naltrindole and those of µ-agonists are
not, CTAP, a µ-selective antagonist, antagonizes the
effects of intrathecally administrated µ-agonists and
DPDPE, a δ-selective agonist, with the same potency.44
It has also been reported that δ-receptor-mediated
analgesia is partially decreased in µ-opioid receptor
deficient mice.45
These observations are consistent with synergistic
interaction between µ- and δ-opioid receptors and could

Lanthionine Enkephalin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3751
(condition B, tRb). Preparative HPLC was carried out at 10 mL/
min flow rate using condition A, and the materials so obtained
were further purified by condition B in the aforementioned
system.
DMF to yield the tetrapeptide resin, which contained a free
thiol group 3a .
To an argon-agitated suspension of resin 3a and CsCO3 (4
equiv) in DMF was added N-Trt-D-3-iodoalanine benzyl ester
4a (3 equiv), and this was agitated for 8 h. After successive
washings with DMF, H2O, MeOH, benzene, and CH2Cl2, the
resin was treated with TFA/H2O/TIS (93/5/2, v/v/v, 15 mL) for
2 h. The filtrate from the cleavage reaction was collected and
combined with TFA washes (2 × 10 mL) of the cleaved peptide
resin. After addition of 4 N HCl in dioxane (1 mL), concentra-
tion of the combined filtrates under reduced pressure, pre-
cipitation in n-hexane/IPE (1/1, v/v, 10 mL), and centrifugation
yielded a crude linear intermediate 5a (310 mg, 0.33 mmol)
[MS(ESI) 890 [M + H]+, 912 [M + Na]+] as a yellow solid,
which was used without further purification in the next step.
Crude intermediate 5a (310 mg, 0.33 mmol) was dissolved
in DMF (130 mL), and NaHCO3 (280 mg, 3.3 mmol) was added.
The solution was cooled to -20 °C under Ar, and DPPA (0.280
mL, 1.3 mmol) was added. The reaction mixture was then
slowly allowed to reach 0 °C, and stirring was continued for
72 h. The solvent was removed under reduced pressure, and
flash chromatography (CH2Cl2/MeOH, 100/1 to 60/1, v/v)
afforded a crude cyclic intermediate (88 mg) [Rf ) 0.20 (CH2-
Cl2/MeOH, 15/1, v/v); MS(ESI) 894 [M + Na]+, 906 [M - Cl]-]
as an oily solid, which was used without further purification
in the next step.
All reagents were purchased from Aldrich Chemical unless
otherwise indicated. DMF was purchased from Fisher Scien-
tific and treated with sodium aluminosilicate molecular sieves
(4 Å nominal pore diameter) obtained from Sigma and with
Amberlite IR 120(plus) cation-exchange resin. CH2Cl2 was
distilled from calcium hydride. NMP was purchased from
Applied Biosystems and used without further purification.
Protected amino acids, Fmoc-Phe Wang resin, and PyBOP
were purchased from Novabiochem. Fmoc-D/L-Pen(Trt)-OH
were purchased from BACHEM Americas. Trt-D/L-Ser-OBzl
were prepared according to the method of Nakajima et al.46
The reactions were monitored by thin-layer chromatography
carried out on 0.25 mm E. Merck silica gel plates (60F-254)
using UV light as the visualizing agent and 7% ethanolic
phosphomolybdic acid and heat as the developing agent. The
Kaiser test47 was used as the qualitative test for the presence
or absence of free amino groups, and the Ellman test48,49 was
employed for the detection of a free thiol group during
reactions on solid support.
N-Tr t-D/L-3-iod oa la n in e Ben zyl Ester (4a /4b). To a solu-
tion of Trt-D-Ser-OBzl (8.16 g, 18.7 mmol), Ph3P (6.87 g, 26.2
mmol), and imidazole (1.79 g, 26.2 mmol) in acetonitrile/ether
(2/1, v/v, 90 mL) was added I2 (6.63 g, 52.5 mmol) slowly at 0
°C. After the reaction was stirred for 1 h (monitored by TLC)
at room temperature, the reaction was suspended in water
(300 mL) and extracted with EtOAC (3 × 200 mL). The
combined organic layers were rinsed with saturated aqueous
Na2S2O3 solution (300 mL), saturated aqueous CuSO4 solution
(300 mL), and brine (300 mL), dried over MgSO4, and
concentrated under reduced pressure. The resulting reaction
mixture was triturated with n-hexane/ether (2/1, v/v) and
filtered to remove the triphenylphospine oxide. The filtrate was
then concentrated under reduced pressure. Flash chromatog-
raphy (n-hexane/EtOAc, 15/1 to 9/1, v/v) afforded 4a as a pale-
yellow paste (9.52 g, 17.4 mmol, 93%): Rf ) 0.70 (n-hexane/
To the protected cyclic peptide 6a (88 mg) was added 30%
HBr/AcOH (5 mL) at 0 °C under Ar. After the reaction mixture
was stirred for 2 h at room temperature, precipitation in
n-hexane/IPE (1/1, v/v, 10 mL) and centrifugation yielded the
target compound 1a (45 mg, 0.07 mmol; overall yield based
on the Fmoc Wang resin is 7%). Compound 1a was further
purified by RP-HPLC as described in General Procedures
and Notes: 1H NMR (DMSO-d6) δ 9.33 (s, 1H, OH), 9.00 (dd,
J ) 7.2 and 4.0 Hz, 1H, Gly3NH), 8.84 (d, J ) 7.6 Hz, 1H,
D-Ala2NH), 8.30 (d, J ) 8.4 Hz, 1H, Phe4NH), 8.06 (bs, 3H,
Tyr1NH3), 7.35 (d, J ) 5.2 Hz, 1H, D-Ala5NH), 7.25-7.18 (m,
5H, Phe4HAr), 7.01 (d, J ) 8.4 Hz, 2H, Tyr1H2,6), 6.71 (d, J )
8.4 Hz, 2H, Tyr1H3,5), 4.61 (m, 1H, d-Ala2HR), 4.39 (m, 1H,
Phe4HR), 4.00 (m, 2H, Gly3HR, Tyr1HR), 3.46-3.20 (m, 4H,
D-Ala5HR, d-Ala5Hâ2, Gly3HR), 3.12 (dd, J ) 14.0 and 5.2 Hz,
1H, Phe4Hâ), 2.93 (dd, J ) 14.0 and 6.0 Hz, 1H, Tyr1Hâ), 2.85-
2.71 (m, 4H, Phe4Hâ, D-Ala2Hâ2, Tyr1Hâ); MS(ESI) 558 [M +
H]+, 580 [M + Na]+, 556 [M - H]-; HRMS (MALDI) [M + H]+
calcd for C26H31N5O7S 558.2017, found 558.2015; RP-HPLC
EtOAc, 4/1, v/v); [R]25 -14° (c 1.06, CHCl3); 1H NMR (CDCl3)
D
δ 7.50 (bd, J ) 7.2 Hz, 6H), 7.40-7.17 (m, 14 H), 4.79 (d, J )
12.0 Hz, 1H), 4.60 (d, J ) 12.0 Hz, 1H), 3.54 (m, 1H), 3.32
(dd, J ) 9.6 and 3.2 Hz, 1H), 3.30 (dd, J ) 9.6 and 7.2 Hz,
1H), 2.91 (d, J ) 10.0 Hz, 1H); 13C NMR (CDCl3) δ 171.8, 145.4,
134.9, 128.5-126.4 (complex), 71.1, 67.2, 56.2, 9.9; MS (ESI)
570 [M + Na]+; HRMS(MALDI) [M - I]+ calcd for C29H26NO2
420.1963, found 420.1962; IR (cm-1) 3318, 2059, 3032, 1733,
1490, 1455, 1210, 1167, 746, 698. 4b was prepared as described
above for 4a but starting with Trt-Ser-OBzl to provide 4b (95%
yield) as a pale-yellow paste: [R]25D +14.7° (c 0.37, chloroform);
a
b
tR ) 14.39, tR ) 9.34.
Tyr -c[D-Va lL-Gly-P h e-D-Ala L]-OH (1b). The target com-
pound (1b) was obtained using the described procedure for the
synthesis of compound 1a with Fmoc-D-Pen(Trt)-OH and 4a .
During the synthesis, intermediates 5b and 6b were confirmed
by mass spectroscopy and used without further purification
in the next steps as described above. 5b: MS(ESI) 918 [M +
H]+, 916 [M - H]-. 6b: Rf ) 0.25 (CH2Cl2/MeOH, 15/1, v/v);
MS(ESI) 900 [M + H]+, 922 [M + Na]+, 934 [M + Cl]-, 1012
[M + TFA]-.
[R]25 from the reference is +5°.50
D
Tyr -c[D-Ala L-Gly-P h e-D-Ala L]-OH (1a ). The Fmoc-Phe
Wang resin 2 (1.0 g, 1.0 mmol/g) was treated with 20%
piperidine in NMP (10 mL) for 10 min and was washed
sequentially with CH2Cl2, MeOH, CH2Cl2, and DMF. The resin
in DMF (10 mL) was allowed to react with Trt-Gly-OH (2.5
equiv), PyBOP (2.5 equiv), HOBt (1.0 equiv), and DIEA (4
equiv) for 12 h. After being washed with DMF, MeOH, and
CH2Cl2, the resin product was treated twice with CH2Cl2/TFA/
TIS (97/2/1, v/v/v, 10 mL) for 5 min and then was washed again
with CH2Cl2, MeOH, CH2Cl2, and DMF. The washed resin was
suspended in DMF/CH2Cl2 (1/1, v/v, 15 mL) and was treated
with a solution of Fmoc-D-Cys(Trt)-OH (2.5 equiv), which was
preactivated with DIC (2.5 equiv) and HOBt (2.5 equiv) in CH2-
Cl2 (5 mL) and 2,6-lutidine (1 equiv) for 12 h. After being
washed with DMF, MeOH, CH2Cl2, and NMP, the resin was
treated with 20% piperidine in NMP (10 mL) for 20 min and
was washed (CH2Cl2, MeOH, CH2Cl2, and DMF). The washed
resin was suspended once more in DMF (10 mL), treated with
Cbz-Tyr(Bzl)-OH (2.5 equiv), PyBOP (2.5 equiv), HOBt (1.0
equiv), and DIEA (4.5 equiv) for 12 h, and then washed
successively with DMF, MeOH, and CH2Cl2. The resin was
treated twice with CH2Cl2/TFA/TIS (94/3/3, v/v/v, 10 mL) for
5 min and then was washed with CH2Cl2, MeOH, CH2Cl2, and
The analytical results for compound 1b are as follows: 1H
NMR(DMSO-d6) δ 9.32 (s, 1H, OH), 8.84 (d, J ) 7.2 Hz, 1H,
Phe4NH), 8.81 (dd, J ) 8.0 and 2.4 Hz, 1H, Gly3NH), 8.71 (d,
J ) 8.8 Hz, 1H, D-Val2NH), 8.00 (b, 3H, Tyr1NH3), 7.43 (d,
J ) 7.2 Hz, 1H, D-Ala5NH), 7.29-7.17 (m, 5H, Phe4HAr), 7.09
(d, J ) 8.0 Hz, 2H, Tyr1H2,6), 6.70 (d, J ) 8.4 Hz, 2H, Tyr1H3,5),
4.71 (d, J ) 9.2 Hz, 1H, D-Ala2HR), 4.31-4.13 (m, 3H, D-Ala5HR,
Phe4HR, Gly3HR), 3.25-3.06 (m, 4H, d-Ala5Hâ2, Gly3HR, Phe4Hâ),
2.94 (dd, J ) 14.0 and 6.0 Hz, 1H, Tyr1Hâ), 2.84 (dd, J ) 14.0
and 11.6 Hz, 1H, Phe4Hâ), 2.78 (dd, J ) 13.6 and 8.4 Hz, 1H,
Tyr1Hâ), 1.16 (s, 3H, D-Val2Hγ3), 1.07 (s, 3H, D-Val2Hγ3);
MS(ESI) 586 [M + H]+, 608 [M + Na]+, 630 [M +2Na]+, 584
[M - H]-; HRMS (MALDI) [M + Na]+ calcd for C28H35N5O7S
a
b
608.2149, found 608.2141; RP-HPLC tR ) 15.05, tR ) 10.36.
Tyr -c[D-Ala L-Gly-P h e-Ala L]-OH (1c). The target com-
pound was obtained using the described procedure for the
synthesis of compound 1a with Fmoc-D-Cys(Trt)-OH and 4b.
During the synthesis, intermediates 5c and 6c were confirmed
by mass spectroscopy and used without further purification

3752 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Rew et al.
in the next steps as described above. 5c: MS(ESI) 890 [M +
H]+, 912 [M + Na]+. 6c: Rf ) 0.21 (CH2Cl2/MeOH, 15/1, v/v);
MS(ESI) 894 [M + Na]+, 906 [M + Cl]-.
added to each filter, and radioactivity on the filters was
determined by scintillation spectrometry in a Packard Top-
Count.
[3H]-Diprenorphine was purchased from Amersham Life
Science, Inc. (Arlington Heights, IL) and had a specific activity
of 66 Ci/mmol. Preliminary experiments were performed to
show that no specific binding was lost during the wash of the
filters, that binding achieved equilibrium within the incubation
time and remained at equilibrium for at least an additional
60 min, and that binding was linear with regard to protein
concentration. Nonspecific binding, determined in the presence
of 10 µM unlabeled naloxone, was less than 10% of total
binding.
The analytical results for compound 1c are as follows: 1H
NMR (DMSO-d6) δ 9.37 (s, 1H, OH), 8.90 (dd, J ) 7.6 and 3.6
Hz, 1H, Gly3NH), 8.72 (d, J ) 8.4 Hz, 1H, D-Ala2NH), 8.40 (d,
J ) 9.2 Hz, 1H, Phe4NH), 8.09 (bs, 3H, Tyr1NH3), 7.39 (d, J )
8.0 Hz, 1H, Ala5NH), 7.26-7.15 (m, 5H, Phe4HAr), 7.00 (d,
J ) 8.8 Hz, 2H, Tyr1H2,6), 6.71 (d, J ) 8.4 Hz, 2H, Tyr1H3,5),
4.50 (m, 2H, d-Ala2HR, Phe4HR), 4.35 (m, 1H, Ala5HR), 4.02 (dd,
J ) 14.0 and 8.0 Hz, 1H, Gly3HR), 3.93 (m, 1H, Tyr1HR,), 3.24
(dd, J ) 13.6 and 3.6 Hz, 1H, Gly3HR), 3.14 (dd, J ) 14.0 and
4.8 Hz, 1H, Phe4Hâ), 3.02 (dd, J ) 12.0 and 9.2 Hz, 1H, Ala5HR),
2.92 (dd, J ) 12.0 and 3.6 Hz, 1H, Ala5HR), 2.88-2.77 (m, 3H,
Tyr1Hâ,Phe4Hâ), 2.65 (dd, J ) 12.8 and 4.4 Hz, 1H, D-Ala2Hâ),
2.35 (dd, J ) 12.8 and 10.0 Hz, 1H, D-Ala2Hâ); MS(ESI) 580
The data from competition experiments were fit by nonlin-
ear regression analysis by Prism (GraphPad Software, Inc.,
San Diego, CA) using the four-parameter equation for one-
site competition and subsequently calculating the Ki from the
EC50 value by the Cheng-Prusoff equation.
[M + Na]+, 556 [M - H]-; HRMS (MALDI) [M + Na]+ calcd
a
for C26H31N5O7S 580.1842, found 580.1846; RP-HPLC tR
13.81, tR ) 8.38.
)
b
In Vitr o Biologica l Assa y. The in vitro bioactivities of the
compounds were tested in the GPI52 and MVD53 assays as
reported elsewhere.54,55 A log dose-response curve was deter-
mined with [Leu5]-enkephalin as the standard for each ileum
and vas preparation, and the IC50 values of the compounds
were normalized according to a published procedure.56
In Vivo Th er m a l Esca p e Assa y. 1. Ra t P r ep a r a tion .
Animal surgeries and tests were approved by the institutional
animal care committee of the University of California, San
Diego. Male Sprague-Dawley rats (Harlan Industries, India-
napolis, IN), weighing 250-350 g, were housed in separate
plastic cages and maintained on a 12 h cycle (on, 6:00 a.m.;
off, 6:00 p.m.) with food and water given ad libitum.
Chronic intrathecal (it.) catheters were implanted un-
der 2-3% isoflurane (50% O2/air) anesthesia.57 Briefly, for
intrathecal injection, an 8.5 cm polyethylene catheter (PE-10)
was inserted through a slit in the cisternal atlanto-occipital
membrane and was passed to the rostral edge of the lumbar
(L2) subarachnoid space. The external portion was tunneled
subcutaneously to exit at the top of the skull. Prior to insertion,
the catheter was flushed with saline and, after insertion,
plugged with stainless steel wire to prevent leakage of cere-
brospinal fluid.
2. Assa y of Th er m a l Nocicep tion . This test was described
previously in detail.58 Briefly, the animal was placed on a glass
surface that was maintained at 30 °C by a feedback-controlled,
under-glass, forced-air heating system.59 A projection bulb
under the glass was focused on the foot pad of the animal,
which induced an abrupt withdrawal. A cutoff time was set
at 20 s to prevent tissue damage. The test was started
approximately 1 h after the animal was placed in the chamber
for acclimation. After the baseline latency was measured, the
drugs were injected intrathecally and the test was performed.
3. Dr u gs a n d In jection s. All agonists were administrated
it. in a total volume of 10 µL followed by 10 µL of saline to
flush the catheter. Lanthionine enkephalin analogues (1a -
d ) and DPDPE ([D-Pen2,D-Pen5]-enkephalin; RBI, MA) were
dissolved in 20% 2-hydroxypropyl-â-cyclodextrin (Research
Biochemicals, Inc., Natick, MA). Morphine sulfate (Merck,
Sharp and Dohme, West Point, PA) was dissolved in physio-
logical saline (0.9% NaCl w/v).
Tyr -c[D-Va lL-Gly-P h e-Ala L]-OH (1d ). The target com-
pound was obtained using the described procedure for the
synthesis of compound 1a with Fmoc-D-Pen(Trt)-OH and 4b.
During the synthesis, intermediates 5d and 6d were confirmed
by mass spectroscopy and used without further purification
in the next steps as described above. 5d : MS(ESI) 918 [M +
H]+, 916 [M - H]-. 6d : Rf ) 0.28 (CH2Cl2/MeOH, 15/1, v/v);
MS(ESI) 900 [M + H]+, 922 [M + Na]+, 898 [M - H]-, 934
[M + Cl]-.
The analytical results for compound 1d are as follows: 1H
NMR (DMSO-d6) δ 9.34 (s, 1H, OH), 8.75 (d, J ) 9.2 Hz, 1H,
D-Val2NH), 8.69 (dd, J ) 8.8 and 1.6 Hz, 1H, Gly3NH), 8.29
(d, J ) 8.0 Hz, 1H, Phe4NH), 8.05 (s, 3H, Tyr1NH3), 7.39 (d,
J ) 9.2 Hz, 1H, Ala5NH), 7.26-7.15 (m, 5H, Phe4HAr), 7.09
(d, J ) 8.4 Hz, 2H, Tyr1H2,6), 6.69 (d, J ) 8.4 Hz, 2H, Tyr1H3,5),
4.59 (d, J ) 9.6 Hz, 1H, D-Val2HR), 4.42-4.30 (m, 2H, Phe4HR,
D-Ala5HR), 4.22 (dd, J ) 13.6 and 9.6 Hz, 1H, d-Ala5Hâ), 4.12
(dd, J ) 6.8 and 6.0 Hz, 1H, Tyr1HR), 3.15-3.06 (m, 3H,
Gly3HR, Phe4Hâ, Ala5Hâ), 2.98-2.83 (m, 3H, Ala5Hâ, Tyr1Hâ,
Phe4Hâ), 2.76 (dd, J ) 13.6 and 8.8 Hz, 1H, Tyr1Hâ), 1.16 (s,
3H, D-Val2Hγ3), 0.97 (s, 3H, D-Val2Hγ3); MS(ESI) 586 [M + H]+,
584 [M - H]-; HRMS (MALDI) [M + Na]+ calcd for C28H35
-
)
a
b
N5O7S 608.2155, found 608.2138; RP-HPLC tR ) 17.66, tR
10.73.
In Vitr o Ra d ioliga n d Bin d in g Assa y. 1. P r ep a r a tion
of Cell Mem br a n es Exp r essin g Op ia te Recep tor s. This
method is a modification of the method of Raynor et al.51 The
cloned human µ-, δ-, and κ-receptors were expressed in CHO
cells, and these CHO cells were harvested from the culture
flasks. The cells were centrifuged at 1000g for 10 min,
resuspended in assay buffer (50 mM tris(hydroxymethyl)-
aminomethane HCl, pH 7.8, 1.0 mM ethylene glycol bis(â-
aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA free acid),
5.0 mM MgCl2, 10 mg/L leupeptin, 10 mg/L pepstatin A, 200
mg/L bacitracin, 0.5 mg/L aprotinin), and centrifuged again.
The resulting pellet was resuspended in assay buffer homog-
enized with a Polytron homogenizer (Brinkmann, Westbury,
NY) for 30 s at a setting of 1. The homogenate was centrifuged
at 48000g for 10 min at 4 °C, and the pellet was resuspended
at 1 mg of protein/mL of assay buffer and stored at -80 °C
until use.
2. [3H]-Dip r en or p h in e Bin d in g to µ-, δ-, a n d K-Op ioid
Recep tor s. After dilution in assay buffer and homogenization
as before, membrane proteins (50-100 µg) in 250 µL of assay
buffer were added to mixtures containing test compound and
[3H]-diprenorphine (final concentration of 0.4 nM, 30 000 dpm)
in 250 µL of assay buffer in 96-well deep-well polystyrene titer
plates (Beckman) and were incubated at room temperature
for 60 min. Reactions were terminated by vacuum filtration
with a Brandel MPXR-96T harvester through GF/B filters in
filter bottom 96-well plates that had been pretreated with a
solution of 0.5% polyethylenimine and 0.1% bovine serum
albumin for at least 1 h. The filters were washed four times
with 1.0 mL of ice-cold 50 mM Tris-HCl, pH 7.8, 30 µL of
Microscint-20 (Packard Instrument Co., Meriden, CT) was
4. Da ta An a lysis. Response latency data from the thermal
escape test are presented as the mean ( SE at 0, 15, 30, 60,
90, and 120 min after drug injection and were converted to
percent maximum possible effect (% MPE) according to the
formula
postdrug latency - predrug latency
cutoff latency* - predrug latency
% MPE )
× 100%
where the cutoff latency was 20 s in this study.
Abbr evia tion s. All optically active amino acids are of the
L variety unless otherwise stated. Other abbreviations are the
following: Bzl, benzyl; Cbz, benzyloxycarbonyl; CDCl3, deu-
terated chloroform; CHO, Chinese hamster ovary; DIC, N,N′-
diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMF,
dimethylformamide; DMSO-d6, fully deuterated dimethyl sul-

Lanthionine Enkephalin Analogues
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Ack n ow led gm en t. This work was supported by
NIH Grant R01-DA 05539 (M.G.). We thank Dr. Li
Zhang for the interpretation of NMR spectra and
Sandra Blaj Moore for her helpful assistance in the
preparation of this manuscript.
Su p p or tin g In for m a tion Ava ila ble: HRMS spectra and
HPLC profiles of the final products 1a -d . This material is
available free of charge via the Internet at http://pubs.acs.org.
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