Development of potent bifunctional endomorphin-2 analogues with mixed μ-/δ-opioid agonist and δ-opioid antagonist properties

Article abstract of DOI:10.1021/jm030649p

The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with aromatic, heteroaromatic, or aliphatic groups (X = phenethyl,benzyl, phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited μ-opioid receptor affinity in the nanomolar range (K_i = 2.41-6.59 nM), which, however, was 3-to 10-fold less than the parent peptide. Replacement of Tyr¹ by Dmt (2′,6′-dimethyl-L-tyrosine) (19-32) exerted profound effects: (i) acquisition of high μ-opioid receptor affinity (K_i = 0.11-0.52 nM) except 23 (Ph); (ii) presence of potent functional μ-opioid receptor agonism (IC₅₀ < 1 nM) for 19 ([Dmt¹]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association of weak δ-opioid antagonist activity (pA₂ = 5.41-7.18) except 19 ([Dmt¹]EM-2), 20 (H), 27 (1-naphthyl), and in particular 29 (5-quinolyl) with its potent δ-agonism (IC₅₀ = 0.62 nM, pA₂ = 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice, a bioactivity absent in the corresponding Tyr¹ analogue (14); and (v) preferential cis orientation (cis/trans = 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans orientation (cis/trans = 1:2 to 1:3). Thus, [Dmt¹]EM-2 analogues with hydrophobic C-terminal extensions provide model compounds with potent μ-opioid receptor bioactivity and dual functional agonism.

Full text of DOI:10.1021/jm030649p

J . Med. Chem. 2004, 47, 3591-3599
3591
Develop m en t of P oten t Bifu n ction a l En d om or p h in -2 An a logu es w ith Mixed
µ-/δ-Op ioid Agon ist a n d δ-Op ioid An ta gon ist P r op er ties
Yoshio Fujita,† Yuko Tsuda,†,‡ Tingyou Li,‡ Takashi Motoyama,† Motohiro Takahashi,† Yoshiro Shimizu,†
Toshio Yokoi,†,‡ Yusuke Sasaki,§ Akihiro Ambo,§ Atsuko Kita,# Yunden J insmaa,| Sharon D. Bryant,|
Lawrence H. Lazarus,| and Yoshio Okada*,†,‡
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences and High Technology Research Center,
Kobe Gakuin University, Nishi-ku, Kobe 651-2180, J apan, Department of Biochemistry, Tohoku Pharmaceutical University,
4-1, Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, J apan, Department of Pharmacology I, Discovery Research
Laboratories, Dainippon Pharmaceutical Co., Ltd., Enoki 33-94, Suita 564-0053, J apan, and Medicinal Chemistry Group,
LCBRA, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received December 30, 2003
The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with
aromatic, heteroaromatic, or aliphatic groups (X ) phenethyl,benzyl, phenyl, naphthyl, pyridyl,
quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid
activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited
µ-opioid receptor affinity in the nanomolar range (Ki ) 2.41-6.59 nM), which, however, was 3-
to 10-fold less than the parent peptide. Replacement of Tyr1 by Dmt (2′,6′-dimethyl-L-tyrosine)
(19-32) exerted profound effects: (i) acquisition of high µ-opioid receptor affinity (Ki ) 0.11-
0.52 nM) except 23 (Ph); (ii) presence of potent functional µ-opioid receptor agonism (IC50 < 1
nM) for 19 ([Dmt1]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association
of weak δ-opioid antagonist activity (pA2 ) 5.41-7.18) except 19 ([Dmt1]EM-2), 20 (H), 27
(1-naphthyl), and in particular 29 (5-quinolyl) with its potent δ-agonism (IC50 ) 0.62 nM, pA2
) 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice,
a bioactivity absent in the corresponding Tyr1 analogue (14); and (v) preferential cis orientation
(cis/trans ) 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans
orientation (cis/trans ) 1:2 to 1:3). Thus, [Dmt1]EM-2 analogues with hydrophobic C-terminal
extensions provide model compounds with potent µ-opioid receptor bioactivity and dual
functional agonism.
In tr od u ction 1
Pro-Leu-Gly-NH2),9,10 and Tyr-W-MIF-1 (Tyr-Pro-Trp-
Gly-NH2).11,12 The latter group of opioid peptides consists
of the enkephalins,13 endorphins,14,15 and dynorphins,16
which exhibit partial or weak selectivity for δ, µ, and κ
opioid receptor subtypes, respectively. Modification or
substitution at the second and fifth positions in en-
kephalin yielded DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-
ol)17 and DADLE ([D-Ala2,D-Leu5]enkephalin),18 which
acquired selectivity and agonism for µ- and δ-opioid
receptors, respectively. Similarly, the presence of a
D-isomer at position 2 of the natural occurring amphib-
ian skin opioid heptapeptides dermorphin19 and deltor-
phin20 suggests that the conformation around the Tyr1-
Xaa2 bond and residues C-terminal to Phe3 might affect
opioid activity. Although recent data revealed that the
peptide bond of Tyr-Pro of endomorphins and morphi-
ceptin occurs in a trans orientaion,21-23 other results
indicated that the cis orientation might be a character-
istic of the bioactive form in EM-223 and morphicep-
tin.23,24
Although the three major opioid receptor subtypes,
µ, δ, and κ, are important targets for developing
therapies to treat acute pain,2,3 only the µ-opioid recep-
tor appears to be involved in morphine tolerance and
addiction.4a However, opiate tolerance and physical
dependence could be blocked by δ-opioid receptor an-
tagonists without compromising the antinociception
produced by drug interaction at µ-opioid receptors.4b
Therefore, it might be ideal to produce opioidmimetics
with mixed µ-opioid agonist and δ-opioid antagonist
properties. The plethora of endogenous ligands that
interact with these receptors are structurally distinct
but can be clearly divided into two unique classes of
endogenous peptides: one contains the Tyr-Pro amide
bond and the other has the Tyr-Gly-Gly-Phe sequence.
In the former group, endomorphin-1 (EM-1, Tyr-Pro-
Trp-Phe-NH2) and endomorphin-2 (EM-2, Tyr-Pro-Phe-
Phe-NH2) from bovine5 and human brain6 are highly
µ-opioid receptor selective and share a common struc-
tural feature with morphiceptin (Tyr-Pro-Phe-Pro-
NH2),7 hemorphin (Tyr-Pro-Phe-Thr),8 Tyr-MIF-1 (Tyr-
Ra tion a le
The replacement of 2′,6′-dimethyl-L-tyrosine25 (Dmt)
for Tyr in opioid ligands dramatically enhanced their
binding affinities and functional potency.26-29 The struc-
ture-activity relationships in a series of [Dmt1]EM-2
analogues30 indicated that Dmt enhanced the binding
affinities to and bioactivity of δ- and µ-opioid receptors
* To whom correspondence should be addressed. Phone: 81-78-974-
1551. Fax: 81-78-974-5689. E-mail: okada@pharm.kobegakuin.ac.jp.
† Faculty of Pharmaceutical Sciences, Kobe Gakuin University.
‡ High Technology Research Center, Kobe Gakuin University.
§ Tohoku Pharmaceutical University.
#
Dainippon Pharmaceutical Co., Ltd.
| LCBRA, National Institute of Environmental Health Sciences.
10.1021/jm030649p CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/28/2004

3592 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Fujita et al.
F igu r e 1. Design of EM-2 analogues and [Dmt1]EM-2 analogues.
Sch em e 1. Synthetic Scheme for Dmt-Pro-Phe-NH-Bzl
(22)a
between Phe and 2-aminopyridine was formed by a
phosphazo method35 because of poor nucleophilicity of
the amino group. Other peptide bonds were formed by
a mixed-anhydride method using isobutyl chloroformate
(for activation of amino acids without functional side
chain),36 an azide method (for activation of Tyr or
Dmt),37 or PyBop reagent (for activation of Dmt).38
R-Amino protected peptide intermediates were charac-
terized by using TLC, 1H NMR, 13C NMR, and elemental
analysis. Deprotection of Boc was performed using HCl/
dioxane or TFA, and for deprotection of the Z group,
catalytic hydrogenation over Pd/C was employed. All
final products were purified by semipreparative reversed-
phase HPLC. The analytical data for compounds 4-18
and 22-32 are in Supporting Information (Tables 1 and
2). Each compound exhibited a single peak on analytical
HPLC with a unique retention time. Analysis by MALDI-
TOF mass spectrometry (MS) and by HPLC revealed
that all compounds were the desired peptides with
greater than 98% purity.
a
Reagents: (a) IBCF, NMM; (b) HCl/dioxane; (c) PyBop, DIPEA.
and that the Dmt1-Pro2 amide bond preferred a cis
orientation. It is recognized that the presence of ad-
ditional aromatic residues enhances the affinity and
selectivity of various opioid analogues, such as observed
with the TIPP family of δ-opioid receptor antagonists31
and the Dmt-Tic-NH-CH(R)R′ analogues with mixed µ-
and δ-opioid receptor agonist activity or with µ-agonist
and δ-antagonist profiles.32,33 In light of these observa-
tions and the effect that substitution with an ethylphen-
yl group at the C terminus of [Dmt1]EM-2 elicited
δ-antagonism,30 we sought to further investigate the
effect of an increase of the hydrophobicity of EM-2
analogues. This study details the alteration of affinity
and functional activity caused by replacement of the
C-terminal Phe4-NH2 with aromatic amines (benzyl,
phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl) or
aliphatic amines (tert-butyl, cyclohexyl, adamantyl)
(Figure 1). The alteration in receptor binding and
bioactivity profiles suggests that substitution of Phe-
NH2 at the fourth position by more hydrophobic amides
of [Dmt1]EM-2 appears to play a role also in the
acquisition of both µ- and δ-opioid receptor agonism,
comparable to that found in some Dmt-Tic-NH-CH(R)-
1H-benzimidazol-2-yl analogues.32,33
Resu lts
En d om or p h in -2 An a logu es. The binding affinities
for µ- and δ-opioid receptors for 1-18 are summarized
in Table 1. All the analogues exhibited negligible
δ-opioid receptor affinities (micromolar range). In the
substitution of the C-terminal Phe-NH2 by a monocyclic
aromatic residue, the µ-opioid receptor binding affinities
of 3 (C2H4-Ph), 4 (Bzl), and 5 (Ph) decreased as a
function of the loss in the number of interposing
methylene units (Ki ) 13.3, 21.6, and 33.5 nM, respec-
tively). Furthermore, the heteromonocyclic aromatic
residue analogues 6 (4-Pyr), 7 (3-Pyr), and 8 (2-Pyr)
exhibited similarly weak µ-opioid receptor affinities
(26.5-48.5 nM). On the other hand, the bicyclic aro-
matic residue 9 (Nph) exhibited a 5.5-fold higher affinity
(Ki ) 2.41 nM) than 3 (phenethyl). In the heterocyclic
group, only 11 (5-Qln) had moderate µ-opioid receptor
affinity (Ki ) 4.07 nM); analogues 10 (3-Qln), 12 (6-Qln),
13 (8-Qln), and 14 (5-Isq) had generally low µ-opioid
receptor affinities. Analogues containing bulky aliphatic
groups (15-18) also displayed comparable weak µ-opioid
receptor affinities (Table 1).
Ch em istr y
Optically pure (98%) Dmt was prepared as de-
scribed,25 and the purity was ascertained by both HPLC
using a chiral column [CROWNPAK CR(+)] and reac-
tion with D- and L-amino acid oxidases34 followed by
amino acid analysis. Boc-Dmt-OH was prepared as
published.29 EM-2 and [Dmt1]EM-2 analogues were
synthesized by a solution method (Scheme 1) and
detailed in Figure 1. The Boc group and the Z group
were used for R-amino protection. The peptide bond
[Dm t1]en d om or p h in -2 An a logu es. Table 2 sum-
marizes the opioid receptor affinities of the [Dmt1]EM-
2 analogues (19-32). With the exception of Dmt-Pro-

Endomorphin-2 Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3593
Ta ble 1. Receptor Binding Profile of EM-2 Analoguesa
µ-opioid receptor
Ki (nM)
δ-opioid receptor
Ki (nM)
binding selectivity
compd
peptide
Ki(δ)/Ki(µ)
1
EM-2
Tyr-Pro-Phe-NH-X
X ) H
X ) C2H4-Ph
X ) Bzl
X ) Ph
X ) 4-Pyr
X ) 3-Pyr
X ) 2-Pyr
X ) 1-Nph
X ) 3-Qln
X ) 5-Qln
X ) 6-Qln
X ) 8-Qln
X ) 5-Isq
X ) But
0.69 ( 0.16
9230 ( 200
13400
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
46.3 ( 3.8
13.3 ( 2.6
21.6 ( 3.8
33.5 ( 6.1
48.2 ( 2.1
48.5 ( 5.0
26.5 ( 3.9
2.41 ( 0.5
55.8 ( 5.03
4.07 ( 0.45
72.9 ( 6.6
249 ( 20.6
61.1 ( 14.2
19.6 ( 4.5
5.79 ( 1.7
23.6 ( 1.7
6.59 ( 0.06
15900 ( 2300
4310 ( 635
1730 ( 292
343
324
80
(3)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(5)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(4)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
23170 ( 11500
25070 ( 2710
19360 ( 3330
19660 ( 633
3640 ( 1260
18600 ( 1740
12700 ( 2470
9880 ( 2640
17100 ( 3590
3480 ( 640
13300 ( 1940
8110 ( 1760
6260 ( 1310
5560 ( 1770
692
521
400
742
1510
333
3110
136
68
57
680
1400
265
844
X ) cyclo-HX
X ) 1-AD
X ) 2-AD
a
Displacement of [3H]DAMGO (µ-selective) and [3H]DPDPE (δ-selective) from rat brain membrane synaptosomes. The Ki values are
the mean ( SE.
Ta ble 2. Receptor Binding Profile of [Dmt1]EM-2 Analoguesa
µ-opioid receptor
δ-opioid receptor
Ki (nM)
binding selectivity
compd
peptide
Ki (nM)
Ki(δ)/Ki(µ)
1
19
EM-2
0.69 ( 0.16
9230 ( 200
28.2 ( 8.1
13400
188
[Dmt1]EM-2
Dmt-Pro-Phe-NH-X
X ) H
0.15 ( 0.035
(3)
(3)
20
21
22
23
24
25
26
27
28
29
30
31
32
0.12 ( 0.09
0.51 ( 0.15
0.52 ( 0.055
1.11 ( 0.18
0.36 ( 0.063
0.17 ( 0.02
0.13 ( 0.01
0.29 ( 0.037
0.33 ( 0.017
0.11 ( 0.014
0.22 ( 0.038
0.49 ( 0.057
0.19 ( 0.018
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(4)
(4)
(3)
(4)
(5)
(5)
53.2 ( 6.1
18.0 ( 2.5
13.8 ( 1.3
20.6 ( 2.5
52.7 ( 9.9
287.5 ( 55
157.2 ( 25
19.9 ( 3.0
190.4 ( 22.3
30.0 ( 3.8
46.6 ( 2.4
33.1 ( 1.71
98.3 ( 8.83
(3)
(3)
(3)
(3)
(3)
(4)
(4)
(4)
(3)
(3)
(3)
(4)
(4)
443
35
27
X ) C2H4-Ph
X ) Bzl
X ) Ph
19
X ) 4-Pyr
X ) 3-Pyr
X ) 2-Pyr
X ) 1-Nph
X ) 3-Qln
X ) 5-Qln
X ) 6-Qln
X ) 8-Qln
X ) 5-Isq
424
1732
1191
68
577
283
212
68
517
a
Displacement of [3H]DAMGO (µ-selective) and [3H]DPDPE (δ-selective) from rat brain membrane synaptosomes. The Ki values are
the mean ( SE.
Phe-NH-Ph (23) (Ki ) 1.11 nM), all of the [Dmt1]EM-2
analogues demonstrated µ-opioid receptor affinity higher
than EM-2 (Ki ) 0.69 nM) with Ki ranging from 0.11 to
0.52 nM. Concomitantly, the δ-opioid receptor affinity
increased, thereby decreasing µ-opioid receptor selectiv-
ity. However, 24 (4-Pyr), 25 (3-Pyr), 26 (2-Pyr), 28 (3-
Qln), and 32 (5-Isq) indicated moderate µ-opioid receptor
selectivities (424, 1732, 1191, 577, and 517, respec-
tively).
Ta ble 3. In Vitro Bioactivity Profile of [Dmt1]EM-2 Analogues
GPI assaya MVD assayb pA2 value vs
compd
peptide
IC50 (nM)
IC50 (nM) dertorphin II
1
19
EM-2
5.79 ( 0.4
344 ( 93
[Dmt1]EM-2
Dmt-Pro-Phe-NH-X
X ) H
0.07 ( 0.016 1.87 ( 0.61
20
21
22
23
24
25
26
27
28
29
30
31
32
2.33 ( 0.49 113 ( 35
5.03 ( 0.99 >10000
22.0 ( 6.1
37.7 ( 7.0
11.8 ( 2.7
72.8 ( 13.0 >10000
15.0 ( 3.2 >10000
0.49 ( 0.183 5.47 ( 1.01
9.14 ( 0.81 >10000
0.26 ( 0.04 0.616 ( 0.17
6.21 ( 3.1
X ) C2H4-Ph
X ) Bzl
X ) Ph
7.05
7.18
6.94
6.52
6.33
6.70
>10000
>10000
>10000
X ) 4-Pyr
X ) 3-Pyr
X ) 2-Pyr
X ) 1-Nph
X ) 3-Qln
X ) 5-Qln
X ) 6-Qln
X ) 8-Qln
X ) 5-Isq
P h a r m a cologica l Activity. All [Dmt1]EM-2 ana-
logues acted as µ-opioid agonists in a guinea pig ileum
(GPI) bioassay (Table 3). In particular, [Dmt1]EM-2 (19)
and analogues 27 (1-Nph), 29 (5-Qln), and 32 (5-Isq)
exhibited potent µ-opioid receptor agonism (IC50 < 1
nM). In the mouse vas deferens (MVD) bioassay, all
analogues were weak δ-opioid receptor antagonists (pA2
) 5.41-7.18) except 27 (1-Nph) and 29 (5-Qln), which
were also δ-opioid receptor agonists (IC50 ) 5.47 and
0.62 nM, respectively). Interestingly, 29 also exhibited
weak δ-opioid antagonism (pA2 ) 5.88) but only at high
concentrations.
5.93
5.88
5.41
6.14
6.12
>10000
445 ( 11.0 2981 ( 685
0.94 ( 0.112 >10000
a
b
Values are the mean of seven experiments ( SE. Values are
the mean of six experiments ( SE.
(Figure 2) and was antagonized completely by naltrex-
one, a µ-opiate antagonist (Figure 3). This indicates that
the antinociceptive effect of 32 acts through µ-opioid
receptors similar to that of morphine. This antinocice-
ptive effect of 32 was weaker and had a duration shorter
In Vivo An tin ocicep tion . Dmt-Pro-Phe-NH-5-Isq
(32) produced a dose-dependent antinociceptive effect
after ic administration, which subsided within 60 min

3594 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Fujita et al.
F igu r e 2. Antinociceptive effect of Dmt-Pro-Phe-NH-5-Isq
(32) after intracisternal (ic) administration in the tail pressure
test in mice. (A) Time course of the antinociceptive effect
induced by Dmt-Pro-Phe-NH-5-Isq (32). Each symbol repre-
sents the mean ( SE of five mice: (O) saline; (2) Dmt-Pro-
Phe-NH-5-Isq 32 (1 µg/mouse); (0) Dmt-Pro-Phe-NH-5-Isq 32
(3 µg/mouse); (b) Dmt-Pro-Phe-NH-5-Isq 32 (10 µg/mouse); (4)
Dmt-Pro-Phe-NH-5-Isq 32 (30 µg/mouse). (B) The area under
the curve (AUC) was calculated from the time course from 0
to 30 min after administration. Each column represents the
mean ( SE of 5 mice: (//) P < 0.01, significantly different
from the saline-treated group (Dunnett’s munltiple comparison
test). (C) Time course of the antinociceptive effect induced by
morphine HCl. Each symbol represents the mean ( SE of four
or five mice: (O) saline; (2) morphine HCl (0.3 µg/mouse); (0)
morphine HCl (1 µg/mouse); (b) morphine HCl (3 µg/mouse).
(D) The area under the curve (AUC) was calculated from the
time course from 0 to 30 min after administration. Each
column represents the mean ( SE of four or five mice: (/) P
< 0.05, (//) P < 0.01, significantly different from the saline-
treated group (Dunnett’s multiple comparison test).
F igu r e 3. Antagonism by naltrexone of the antinociceptive
effect induced by Dmt-Pro-Phe-NH-5-Isq (32) and morphine
HCl in the tail pressure test in mice. (A) Time course of the
antinociceptive effect induced by Dmt-Pro-Phe-NH-5-Isq (32),
showing the dose-response relation in the antagonism by
naltrexone. Each symbol represents the mean ( SE of five
mice. Naltrexone was administration sc 15 min before intra-
cisternal (ic) administration of Dmt-Pro-Phe-NH-5-Isq 32: (O)
saline (sc) + saline (sc); (4) saline (sc) + Dmt-Pro-Phe-NH-5-
Isq 32 (10 µg/mouse, ic); (b) naltrexone HCl (1 mg/kg, sc) +
Dmt-Pro-Phe-NH-5-Isq 32 (10 µg/mouse, ic); (0) naltrexone
HCl (3 mg/kg, sc) + Dmt-Pro-Phe-NH-5-Isq 32 (10 µg/mouse,
ic); (2) naltrexone HCl (5 mg/kg, sc) + Dmt-Pro-Phe-NH-5-
Isq 32 (10 µg/mouse, ic). (B) The area under the curve (AUC)
was calculated from the time course from 0 to 30 min after
administration. Each column represents the mean ( SE of five
mice: (//) P < 0.01, significantly different from the saline-
treated group (Student’s t-test); (#) P < 0.05, (##) P < 0.01,
significantly different from the Dmt-Pro-Phe-NH-5-Isq (32)
alone-treated goup (Dunnett’s multiple comparison test). (C)
Time course of the antinociceptive effect induced by morphine
HCl, showing the dose-response relation in the antagonism
by naltrexone. Each symbol represents the mean ( SEM of
five mice. Naltrexone was administered sc 15 min before
intracisternal (ic) administration of morphihe HCl: (O) saline
(sc) + saline (sc); (4) saline (sc) + morphine HCl 1 µg/mouse
(ic); (b) naltrexone HCl 1 mg/kg (sc) + morphine HCl 1 µg/
mouse (ic). (D) The area under the curve (AUC) was calculated
from the time course from 0 to 30 min after administration of
morphine HCl. Each column represent the mean ( SEM of
five mice: (//) P < 0.01, significantly different from the saline-
treated group (Student’s t-test); (#) P < 0.05, (##) P < 0.01,
significantly different from the morphine alone-treated group
(Student’s t-test).
than that of morphine (Figure 2). The maximum effect
of analogue 32 (30 µg/mouse) was about one tenth of
that with morphine.
Solu tion Con for m a tion a l Stu d y. NMR studies of
[Dmt1]EM-2 analogues were conducted in DMSO-d6.
Despite the molecular differences in the compounds, the
chemical shift of the Pro â and γ positions of the cis and
trans conformations in 13C NMR remained constant;
chemical shifts of cis γ, trans γ, trans â, and cis â were
observed at 21, 24, 29, and 31 ppm, respectively. The
1
cis and trans isomers were identified by H NMR, 13C
NMR, H-H COSY, C-H COSY, and NOESY. Figure 4
shows the characteristic NOE pairs of the cis and trans
conformations at the Dmt-Pro peptide bonds. In the
cis isomer, the methyl groups and the CR proton of Dmt
are spatially close to the CR proton of Pro. As a result,
the chemical shift of Pro CR H of the cis isomer was
observed at a higher field than that of trans isomer.
While Dmt exists as a trans isomer, the methyl groups
and the CR proton of Dmt are spatially close to the Cδ
proton of Pro; consequently, the chemical shift of Pro
Cδ H of the trans isomer was observed at higher field
than that of the cis isomer. The cis/trans ratios of the
[Dmt1]EM-2 analogues were determined from relative
peak intensities obtained by 1H NMR spectroscopy
(Table 4).
Discu ssion
In flu en ce of Dm t a n d th e Su bstitu tion a t th e
F ou r th P osition on P ep tid e Activity. The receptor
binding profiles of EM-2 analogues and [Dmt1]EM-2
analogues summarized in Tables 1 and 2 demonstrated

Endomorphin-2 Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3595
F igu r e 4. Characteristic NOE pairs of cis and trans confor-
mations at the Dmt-Pro peptide bond.
Ta ble 4. Cis/ Trans Ratio of [Dmt1]EM-2 Analogues in DMSOa
compd
peptide
cis/trans
Tyr-Pro-Trp-Phe-NH2 (EM-1)
Tyr-Pro-Phe-Phe-NH2 (EM-2)
Tyr-Pro-Phe-NH2
[Dmt1]EM-2
Dmt-Pro-Phe-NH-X
X ) H
1/3
1/2
1/2
70/30
1
2
19
F igu r e 5. Superimposition of the endomorphin-2 analogues,
Dmt-Pro-Phe-NH-1-Nph (27) (green), Dmt-Pro-Phe-NH-5-Qln
(29) (red), and Dmt-Pro-Phe-NH-6-Qln (30) (yellow). The
1
models were developed on the basis of data from H NMR and
20
21
22
23
24
25
26
27
28
29
30
32
65/35
65/35
65/35
60/40
60/40
65/35
65/35
60/40
65/35
60/40
60/40
60/40
X ) C2H4-Ph
X ) Bzl
molecular modeling. Carbon atoms are displayed in green,
oxygen atoms in red, and nitrogen atoms in blue with the
exception the C-terminal heteroaromatics, which are colored
according to the substitution. Hydrogens are not displayed.
X ) Ph
X ) 4-Pyr
X ) 3-Pyr
X ) 2-Pyr
porting a hypothesis that the orientation of the hetero-
cyclic ring may be important for the appearance of
δ-agonism.
X ) 1-Nph
X ) 3-Qln
X ) 5-Qln
The µ-opioid receptor affinities of EM-2 analogues
containing a third aromatic residue, such as 3 (C2H4-
Ph, 13.3 nM), 4 (Bzl, 21.6 nM), and 5 (Ph, 33.5 nM),
decreased as a function of the loss in the number of
methylene units. Therefore, the distance between the
phenol moiety of the Tyr residue and the other aromatic
residue might be an important factor in a proper fit
within an opioid receptor active site.43 On this point,
Dmt-Tic-NH-CH(R)-R′ analogues containing Bid (1H-
benzimidazol-2-yl) as the R′ functional group acquired
high affinity and moderate agonist activity for µ-opioid
receptors, whereas the change in spacer (R) influenced
the acquisition of agonist or antagonist activity.32,33
While all the analogues studied were µ-opioid receptor
agonists, the functional bioactivity of [Dmt1]EM-2 ana-
logues for the δ-opioid receptor indicated considerable
differences, the analogues behaving as agonists (19, 20,
27) or antagonists (21-26, 28, 30, 32) or having mixed
agonist/antagonist properties (29, 31). Interestingly,
with respect to the δ-receptor, those compounds con-
taining monocyclic aromatic residues (21-26) exhibited
antagonist activity but in the presence of a bicyclic
aromatic residue attached through the R-position (27,
29, 31) tended to act as agonists, while those covalently
bound through the â position (28, 30) exhibited antago-
nist activity.
X ) 6-Qln
X ) 5-Isq
Determined from relative peak intensities obtained by 1H
a
NMR.
that Dmt enhanced interaction with the receptors and
altered their bioactivity profiles. Similarly, the replace-
ment of Tyr with Dmt in the Tyr-Tic pharmacophore39
resulted in a potent and highly selective δ-opioid recep-
tor antagonist,40-42 and our previous studies indicated
that the replacement of Tyr with Dmt in EM-2 enhanced
both δ- and µ-opioid receptor affinities.30 These data and
other observations substantiate the fact that Dmt plays
a dominant role in determining the efficacy of the
interaction of an opioid ligand with receptors.
In addition to a hypothesis that the dimethyl groups
on the tyramine ring interact with hydrophobic binding
sites within the receptor pocket,43 experimental evidence
provided herein demonstrates that Dmt also shifts the
conformation at the Dmt-Pro amide bond in endomor-
phin-2 from trans to cis. A common structural feature
of Dmt-Pro and Dmt-Tic is the cyclic imino acid at the
second position; however, the size of these rings (five-
membered ring and six-membered ring, respectively)
and the existence of a second aromatic residue in the
latter compound represent major differences between
Pro and Tic. The cis/trans equilibrium at the Dmt-Pro
peptide bond in DMSO favored the cis orientation in
contrast to the Tyr-Pro amide bond, which indicated a
higher ratio for the trans orientation.30 The preferred
cis orientation of the Dmt-Pro amide bond may be
explained by the presence of methyl groups at the 2′
and 6′ positions of Dmt that could make the trans
orientation less favorable (Table 4). Molecular modeling
and energy minimization of 1-Nph (27) (µ/δ-agonist),
5-Qln (29) (µ/δ-agonist and δ-antagonist), and 6-Qln (30)
(µ-agonist, weak δ-antagonist) revealed a different
orientation of the heteroaromatic ring (Figure 5), sup-
In Vivo An tin ocicep tive Activity of a [Dm t1]EM-
2 An a logu e. Peptides are easily hydrolyzed by pro-
teolytic enzymes such as aminopeptidase, carboxypep-
tidase, and enkephalinase.44 While the Pro2-Phe3 amide
bond of endomorphin-2 was cleaved by dipeptidyl pep-
tidase IV,45a [D-Pro2]EM-2 is totally resistant.45b Cardillo
et al.46 reported that endomorphin-1 analogues contain-
ing homo-Pro acquired resistance to enzymatic degrada-
tion and the replacement of Pro by homo-Pro effected a
significant change in peptide-enzyme recognition. En-
zymatic stability of opioid peptides containing Dmt at
position 1 was examined by use of aminopeptidase and
rat brain homogenates, which revealed that they were

3596 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Fujita et al.
(IBCF),36 the phosphazo method,35 the azide method37 or using
the PyBop reagent.38 The Boc and Z groups were used for
N-terminal protection. Deprotection of Boc was performed
using hydrochloric acid in dioxane (HCl/dioxane) or TFA.
Deprotection of Z was performed by catalytic hydrogenolysis
using Pd/C. On TLC (Kieselgel G/ Merck), Rf values refer to
the systems CHCl3/MeOH/AcOH (90:8:2), hexane/AcOEt (1:
1), and hexane/AcOEt (1:3). These peptides exhibited greater
than 98% purity by analytical HPLC (Waters model 600 E),
using a Cosmosil 5C18-AR column (NAKARAI TESQUE, Inc.,
25 cm × 4.6 mm) with the absorbance monitored at 220 nm,
and run (1 mL/min) in the following solvents: A, 0.05% TFA
in H2O; B, 0.05% TFA in CH3CN.
Gen er a l P r oced u r e for Syn th esis of Boc-P h e-NH-X (X
) Bzl, P h , 4-P yr , 3-P yr , Np h , Qln , Isq, Bu t, Cyclo-HX, AD).
A mixed anhydride [prepared from Boc-Phe-OH (3.8 mmol),
IBCF (3.8 mmol), and NMM (4.2 mmol)] in THF (30 mL) was
added to a solution of the corresponding amine component (4.2
mmol) in DMF (20 mL) at -15 °C. The reaction mixture was
stirred at 0 °C for 15 min and at 4 °C overnight. After removal
of the solvent, the residue was extracted with AcOEt. The
extract was washed with 10% citric acid, 5% Na2CO3, and
saturated NaCl, dried over Na2SO4, and evaporated. Petroleum
ether was added to the residue to form crystals, which were
collected by filtration. The yield, melting point, [R] 2D0, Rf, and
elemental analysis data are summarized in Table 3 in Sup-
porting Information along with the 1H NMR values for the
intermediates in the peptide synthesis.
Z-P h e-NH-2-P yr . PCl3 was added to distilled pyridine (50
mL) containing 2-aminopyridine (1.0 g, 10.6 mmol) at 0 °C.
After 1 h, Z-Phe-OH (3.17 g, 10.6 mmol) was added to the
mixture. The mixture was stirred at 80 °C for 5 h and at room
temperature for 2 days. After removal of the solvent, the
residue was extracted with AcOEt. The extract was washed
with 10% citric acid, 5% Na2CO3, and water, dried over Na2-
SO4, and evaporated to dryness. Petroleum ether was added
to the residue to form crystals, which were collected by
filtration: yield 2.93 g (73.6%), mp 137-139 °C, [R] 2D0 +8.9° (c
1.0, MeOH), Rf ) 0.77 (CHCl3/MeOH/AcOH ) 90:8:2). Anal.
Calcd for C22H21N3O3‚1/4H2O: C, 69.6; H, 5.66; N, 11.00.
Found: C, 69.4; H, 5.64; N, 10.6.
more stable than the original peptides.29 Therefore, the
[Dmt1]EM-2 analogue Dmt-Pro-Phe-NH-5-Isq (32) might
be similarly resistant to enzymatic degradation by
aminopeptidase or carboxypeptidase, although the Pro2-
Phe3 bond is metabolized.45a Therefore, modification of
the Pro2-Phe3 peptide bond might be necessary for
future research in order to develop a more potent and
long lasting opioid ligand.
Con clu sion s
The data indicated that Dmt markedly enhanced the
biological properties of EM-2 analogues in both the
competitive binding and in vitro biological assays.
Furthermore, the binding affinity of 19-32, except for
23 (Ph), is also higher than that of EM-2 and their in
vitro bioactivity profiles are also different from that of
EM-2. Although [Dmt1]EM-2 and its analogues are
µ-opioid agonists, Dmt-Pro-Phe-NH-1-Nph (27) and -5-
Qln (29) exhibited µ- and δ-opioid agonism. These
remarkable changes are comparable to those observed
in other, unrelated opioid peptides such as enkephalin26
and analogues of deltorphin,19 dynorphin A (1-11),20 as
well as the Dmt-Tic-R32 and Dmt-Tic-NH-(R)-R′ family
of peptides.33 The ic administration of Dmt-Pro-Phe-NH-
5-Isq (32) produced a significant dose-dependent anti-
nociceptive effect that was antagonized completely by
naltrexone, proving that the effect was mediated by the
µ-opioid receptors in a manner similar to that of
morphine. Thus, these data clearly indicate that N-
terminal Dmt has the potential to act in the develop-
ment of novel bioactive opioidomimetics for potential
therapeutic and clinical applications. The difference in
the conformation about the Xaa-Pro amide bond in
solution in peptides containing Tyr (trans-rich) and Dmt
(cis-rich) (Table 4) suggests that conformational features
of the peptide affect receptor recognition and biological
function. Finally, the methyl groups on the tyramine
ring of Dmt undoubtedly play a dominant role in the
interaction within the opioid binding domains by direct
interaction with hydrophobic side chains of receptor
residues or by stabilization of a favored cis conformer
in solution prior to and during binding.
Gen er a l P r oced u r e for Syn th esis of Boc-P r o-P h e-
NH-X (X ) Bzl, P h , 4-P yr , 3-P yr , Np h , Qln , Isq, Bu t, Cyclo-
HX, AD). A mixed anhydride [prepared from Boc-Pro-OH (11
mmol), IBCF (11 mmol), and NMM (11 mmol)] in THF (30 mL)
was added to a solution of the corresponding amine component
[prepared from Boc-Phe-NH-X (10.1 mmol) and 7.4 N HCl/
dioxane (30 mmol)] in DMF (30 mL) containing NMM (21
mmol) at -15 °C. The reaction mixture was stirred at 0 °C for
15 min and at 4 °C overnight. After removal of the solvent,
the residue was extracted with AcOEt. The extract was worked
up in a manner similar to that for Z-Phe-NH-2-Pyr to give
crystals. The yield, melting point, [R] 2D0, Rf, and elemental
analysis data are summarized in Supporting Information
Exp er im en ta l Section
P ep tid e Syn th esis. Melting points were determined on a
Yanagimoto micromelting point apparatus and are uncor-
rected. Optical rotations were measured with an automatic
polarimeter, model DIP-1000 (Japan Spectroscopic Co.). MALDI-
TOF mass spectrometry (MS) was carried out on a KOMPACT
MALDI IV instrument (Kratos Analytical Co.) using sinapinic
acid as the matrix. 1H (400.13 MHz) and 13C (100.62 MHz)
NMR spectra were recorded on a Bruker DPX-400 spectrom-
eter. Final compounds (30 mg) were dissolved in 1.0 mL of
DMSO-d6 (99.9% isotopic purity). Chemical shift values were
expressed as ppm downfield from tetramethylsilane, used as
an internal standard (δ value). The J values are given in Hz.
The 13C signals were assigned with the aid of distortionless
enhancement by polarization transfer (DEPT) and two-
dimensional experiments (H-H COSY, C-H COSY), and
classification of carbon atoms are indicated by CH3 (primary),
CH2 (secondary), CH (tertiary), or Cq (quaternary). Dmt was
prepared according to the method of Dygos et al.,25 and its
chirality was assessed by both HPLC [column, CROWNPAK
CR(+) (Daisel Chemical, Ltd., 15 cm × 4.6 mm); flow rate, 0.4
mL/min; mobile phase, perchloric acid (pH 2); UV detection,
220 nm] and amino acid analysis. Peptide bonds were formed
by the mixed-anhydride method using isobutyl chloroformate
1
(Table 4) along with the H NMR and 13C NMR values for the
intermediates in peptide synthesis.
Boc-P r o-P h e-NH-2-P yr . The title compound was prepared
as for the synthesis of Boc-Pro-Phe-NH-X starting from Boc-
Pro-OH (0.50 g, 2.32 mmol), IBCF (0.30 mL, 2.32 mmol), NMM
(0.25 mL, 2.32 mmol), and H-Phe-NH-2-pyridine‚2AcOH [pre-
pared from hydrogenolysis of Z-Phe-NH-2-pyridine (1.0 g, 2.66
mmol) in MeOH (30 mL) in the presence of Pd/C and acetic
acid].
Gen er a l P r oced u r e for Syn th esis of Boc-Tyr -P r o-P h e-
NH-X (X ) P h , P yr , Np h , Qln , Isq, Bu t, Cyclo-HX, AD).
Boc-Tyr-NHNH2 (2.28 mmol) was dissolved in DMF (10 mL).
Under cooling with ice/NaCl, 7.2 N HCl/dioxane (4.56 mmol)
and isoamyl nitrite (2.5 mmol) were added. Stirring was
continued for 10 mim, when the hydrazine test became
negative. The solution, after neutralization with NMM (4.56
mmol), was combined with a solution of the corresponding
amine component [prepared from Boc-Pro-Phe-NH-X (2.28
mmol) and 7.2 N HCl/dioxane (22.8 mmol)] and NMM (6.84
mmol) in DMF (20 mL). After the reaction mixture was stirred

Endomorphin-2 Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3597
at 4 °C overnight, the solvent was removed. The residue was
extracted with AcOEt. The extract was worked up in a manner
similar to that for Z-Phe-NH-2-Pyr to give crystals. The yield,
melting point, [R] 2D0, Rf, and elemental analysis data are
summarized in Table 5 in Supporting Information along with
Ra d ioliga n d Bin d in g Assa ys. Synaptosomal brain mem-
brane P2 preparations from Sprague-Dawley rats were pre-
pared and used as the source for µ- and δ-opioid receptors after
the removal of endogenous opioids.47,48 The competitive dis-
placement assay used 5.57 nM [3H]DPDPE (NEN-DuPont) and
3.5 nM [3H]DAMGO (Amersham) for the δ and µ sites,
respectively, as published.47,48 Affinity constants (Ki) were
determined as given earlier.48
In Vitr o Bioa ssa y. For the GPI assay, the myenteric
plexus-longitudinal muscle was obtained from male Hartley
strain guinea pig (250-300 g) ileum as described by Rang,49
and the tissue was mounted in a 10 mL bath that contained
aerated (95% O2, 5% CO2) Krebs-Henseleit solution at 35 °C.
The tissue was stimulated transmurally between the platinum
1
the H NMR and 13C NMR values for the intermediates in the
peptide synthesis.
Boc-Tyr (Bu t)-P r o-P h e-NH-Bzl. A mixed anhydride [pre-
pared from Boc-Tyr(But)-OH (2.22 mmol), IBCF (2.22 mmol),
and NMM (2.22 mmol)] in THF (30 mL) was added to a
solution of H-Pro-Phe-NH-Bzl‚HCl [prepared from Boc-Pro-
Phe-NH-Bzl (2.22 mmol) and 7.4 N HCl/dioxane (7.0 mmol)]
in DMF (30 mL) containing NMM (2.44 mmol) at -15 °C. The
reaction mixture was stirred at 0 °C for 30 min and at 4 °C
overnight. After removal of the solvent, the residue was
extracted with AcOEt. The extract was worked up in a manner
similar to that for Z-Phe-NH-2-Pyr to give crystals. Yield 1.17
g (71.8%), mp 151-160 °C, [R] 2D0 -45.9° (c 1.0, MeOH), Rf )
wire electrodes using pulses of 0.5 ms duration with
a
frequency of 0.1 Hz at the supramaximal voltage. Longitudinal
contractions were recorded via an isometric transducer. For
the MVD assay, the vas deferens of male ddY strain mouse
(25-35 g) were prepared as described by Hughes et al.50
A
0.78 (CHCl3/MeOH/AcOH ) 90:8:2). Anal. Calcd for C39H50
-
pair of vasa was mounted in a 10 mL bath that contained
aerated (95% O2, 5% CO2), modified Mg2+-free Krebs solution
containing ascorbic acid (0.1 mM) and EDTA‚4Na (0.027 mM)
at 35 °C. The tissue was stimulated transmurally with trains
of rectilinear pulses of 1 ms. Stimulation trains were given at
intervals of 20 s and consisted of seven stimuli of 1 ms duration
with intervals of 10 ms.
N4O6: C, 69.8; H, 7.51; N, 8.35. Found: C, 69.6; H, 7.53; N,
8.31.
Gen er a l P r oced u r e for Syn th esis of HCl‚H-Tyr -P r o-
P h e-NH-X (4-18) (X ) Bzl, P h , P yr , Np h , Qln , Isq, Bu t,
Cyclo-HX, AD). Boc-Tyr-Pro-Phe-NH-X (0.068 mmol) was
treated with 6.9 N HCl/dioxane (0.68 mmol) for 1 h at room
temperature. Et2O was added to the solution until the product
precipitated. The precipitate was collected by centrifugation,
dried over KOH pellets, and purified by semipreparative
reversed-phase HPLC. The purified peptide was lyophilized
from 1 N HCl to give an amorphous powder. The yield, HPLC
retention time, and MS and elemental analysis data are
summarized in Table 1 of Supporting Information.
In both assays, three to four washings were done with
intervals of 15 min between each dose. Dose-response curves
were constructed, and IC50 values (concentration causing a 50%
reduction of the electrically induced twitches) were calculated
graphically. For antagonism experiments, the test sample was
added to the bath 15 min prior to addition of deltorphin II as
agonist. The concentration-response curves of the agonist
were performed in the absence and in the presence of increas-
ing concentrations of the test compound, and the pA2 values
were calculated according to the method of Arunlakshana and
Schild.51
Deter m in a tion of An tin ocicep tion . Antinociception was
evaluated by the tail-pressure test in male ddY mice. Incre-
mental pressure was applied onto the tail of mice at a constant
rate with an analgeseometer (Ugo Basile), and the pressure
required to elicit a struggle behavior was measured as the
pressure threshold before and at various times after admin-
istration of test compound. A cutoff value of 500 g was used.
The test compound was dissolved in sterile saline or water,
and intracisternal administration (ic) was done. Intracisternal
administration was performed according to the method of Ueda
et al.52 at a volume of 10 µL/mouse.
Molecu la r Mod elin g. Molecular models of Dmt-Pro-Phe-
NH-1-Nph (27), Dmt-Pro-Phe-NH-5-Qln (29), and Dmt-Pro-
Phe-NH-6-Qln (30) were developed on the basis of 2D (NOESY)
1H NMR analyses and computational modeling strategies.
Molecular modeling was performed on a SGI Octane2 com-
puter system using Insight98 software (Accelrys). The starting
structures were derived on the basis of torsion angles and
approximate H-H distances from J coupling constants and
NOEs from 2D 1H NMR analyses. Constrained energy mini-
mizations were performed on each molecule using the CFF91
force field and the distant-dependent dielectric constant. The
steepest descent algorithm was used for 100 cycles followed
by another 1000 cycles of minimization using the conjugate
gradient algorithm in the same force field, stopping with a root-
mean-square gradient of 0.001 kcal mol-1 Å-1. Internal rota-
tion coordinates and energies for the three compounds, Dmt-
Pro-Phe-NH-1-Nph (27), Dmt-Pro-Phe-NH-5-Qln (29), and
Dmt-Pro-Phe-NH-6-Qln (30), are listed in Table 7 in Support-
ing Information.
Gen er a l P r oced u r e for Syn th esis of Boc-Dm t-P r o-P h e-
NH-X (X ) Bzl, P h , P yr , 5-Qln , 6-Qln ). To a solution of the
corresponding amine component [prepared from Boc-Pro-Phe-
NH-X (1.1 mmol) and 6.9 N HCl/dioxane (4.3 mmol)] in DMF
(30 mL) containing DIPEA (3.3 mmol), Boc-Dmt-OH (1.1
mmol) and PyBop (1.1 mmol) were added. The reaction
mixture was stirred at 4 °C overnight. After removal of the
solvent, the residue was extracted with AcOEt. The extract
was worked up in a manner similar to that for Z-Phe-NH-2-
Pyr to give crystals. The yield, melting point, [R] 2D0, Rf, and
elemental analysis data are summarized in Table 6 in Sup-
porting Information along with the 1H NMR and 13C NMR
values for the intermediates in the peptide synthesis.
Gen er a l P r oced u r e for Syn th esis of Boc-Dm t-P r o-P h e-
NH-X (X ) Np h , 3-Qln , 8-Qln , 5-Isq). Boc-Dmt-NHNH2 (2.35
mmol) was dissolved in DMF (10 mL). Under cooling with ice/
NaCl, 5.0 N HCl/dioxane (2.82 mmol) and isoamyl nitrite (2.82
mmol) were added. Stirring was continued for 10 mim, when
the hydrazine test became negative. The solution, after
neutralization with NMM (5.88 mmol), was combined with a
solution of the corresponding amine component [prepared from
Boc-Pro-Phe-NH-X (2.17 mmol) and 5.0 N HCl/dioxane (7.0
mmol)] and NMM (4.7 mmol) in DMF (20 mL). After the
reaction mixture was stirred at 4 °C overnight, the solvent
was removed and the residue was extracted with AcOEt. The
extract was worked up in a manner similar to that for Z-Phe-
NH-2-Pyr to give crystals. The yield, melting point, [R] 2D0, Rf,
and elemental analysis data are summarized in Table 6 in
Supporting Information along with the 1H NMR and 13C NMR
values for the intermediates in the peptide synthesis.
Gen er a l P r oced u r e for Syn th esis of HCl‚H-Dm t-P r o-
P h e-NH-X (22-32) (X ) Bzl, P h , P yr , Np h , Qln , Isq). Boc-
Dmt-Pro-Phe-NH-X (0.068 mmol) was treated with 6.9 N HCl/
dioxane (0.68 mmol) for 1 h at room temperature. Et2O was
added to the solution until the product precipitated. The
precipitate was collected by centrifugation, dried over KOH
pellets, and purified by semipreparative reversed-phase HPLC.
The purified peptide was lyophilized from 1 N HCl to give an
amorphous powder. The yield, HPLC retention time, and MS
and elemental analysis data are summarized in Table 2 in
Supporting Information along with the 1H NMR and 13C NMR
values.
Su p p or tin g In for m a tion Ava ila ble: The yield, analytical
data, 1H NMR and 13C NMR values of intermediates; the yield
and analytical data of EM-2 analogues; a list of internal
rotation coordinates and energies for the three compounds 27,
29, and 30. This material is available free of charge via the
Internet at http://pubs.acs.org.

3598 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Fujita et al.
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Refer en ces
(1) Abbreviations used in this report for amino acids, peptide, and
their derivatives are those recommended by IUPAC-IUB Com-
mission on Biochemical Nomenclature: Biochemistry 1966, 5,
2485-2489; 1966, 6, 362-364; 1972, 11, 1726-1732. The
customary L-configuration for amino acid residues is omitted.
The following additional abbreviations are used: AcOEt, ethyl
acetate; AcOH, acetic acid; AD, adamantine; Boc, tert-butyloxy-
carbonyl; Bu, butyl; Bzl, benzyl; DADLE, [D-Ala2,D-Leu5]-
enkephain; DAMGO, Tyr-D-Ala-Gly-N-MePhe-Gly-ol; DIPEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
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