Development of potent μ-opioid receptor ligands using unique tyrosine analogues of endomorphin-2

Article abstract of DOI:10.1021/jm049384k

Six analogues of tyrosine, which contained alkyl groups at positions 2′, 3′, and 6′, either singly or in combination on the tyramine ring, were investigated for their effect on the opioid activity of [Xaa¹]endomorphin-2 (EM-2). The opioid analo

Full text of DOI:10.1021/jm049384k

586
J. Med. Chem. 2005, 48, 586-592
Development of Potent µ-Opioid Receptor Ligands Using Unique Tyrosine
Analogues of Endomorphin-2
Tingyou Li,† Yoshio Fujita,† Yuko Tsuda,†,‡,§ Anna Miyazaki,‡ Akihiro Ambo,| Yusuke Sasaki,| Yunden Jinsmaa,
Sharon D. Bryant, Lawrence H. Lazarus,*, and Yoshio Okada*,†,‡,§
The Graduate School of Food and Medicinal Sciences, Faculty of Pharmaceutical Sciences, and High Technology Research
Center, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan, Tohoku Pharmaceutical University,
Aoba-ku, Sendai 981-8558, Japan, and Medicinal Chemistry Group, LPC, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina 27709
Received July 31, 2004
Six analogues of tyrosine, which contained alkyl groups at positions 2′, 3′, and 6′, either singly
or in combination on the tyramine ring, were investigated for their effect on the opioid activity
of [Xaa1]endomorphin-2 (EM-2). The opioid analogues displayed the following characteristics:
(i) high µ-opioid receptor affinity [Ki(µ) ) 0.063-2.29 nM] with selectivity [Ki(δ)/Ki(µ)] ranging
from 46 to 5347; (ii) potent functional µ-opioid agonism [GPI assay (IC50 ) 0.623-0.924 nM)]
and with a correlation between δ-opioid receptor affinities and functional bioactivity using
MVD; (iii) intracerebroventricular administration of [Dmt1]- (14) and [Det1]EM-2 (10) produced
a dose-response antinociception in mice, with the former analogue more active than the latter;
and (iv) a marked shift occurred from the trans-orientation at the Tyr1-Pro2 bond to a cis-
conformer compared to that observed previously with [Dmt1]EM-2 (14) (Okada et al. Bioorg.
Med. Chem. 2003, 11, 1983-1984) except [Mmt1]EM-2 (7). The active profile of the [Xaa1]EM-
2 analogues indicated that significant modifications on the tyramine ring are possible while
high biological activity is maintained.
Introduction1
tion of affinity, modification of receptor selectivity, and
change in the spectrum of their bioactivity profile.11,12
Recent studies detailed the effects of [Dmt1]EM-2 (14)
and its C-terminally modified analogues, which were
characterized with potent µ-opioid receptor binding
affinity, µ-receptor agonism, or mixed µ-/δ-opioid ago-
nism and δ-opioid antagonism properties in some of the
C-terminally modified analogues.13 Interestingly, the
formation of opioid mimetic compounds containing two
N-termini of Dmt separated by either an unbranched
alkyl diamine14 or 3,6-diaminoalkylpyrazinone15 exhib-
ited unique µ-opioid receptor agonism in vitro and
potent morphine-like analgesia in vivo.
Despite the structural diversity among endogenous
mammalian opioid ligands, the enkephalins,2 endor-
phins,3 dynorphins,4 Tyr-W-MIF-1,5 and endomorphins,6
as well as the exogenous amphibian skin opioid pep-
tides,7,8 an N-terminal Tyr residue is a common struc-
tural element. According to the concept of the message
and address domains,9 the Tyr residue acts as part of
the message domain to anchor the opioid peptide within
the receptor. Since µ-opioid receptors mediate the
pharmacological effects associated with morphine, the
high selectivity for these receptor sites by the endog-
enous opioid peptides endomorphin-1 (EM-1, H-Tyr-Pro-
Trp-Phe-NH2) and endomorphin-2 (EM-2, H-Tyr-Pro-
Phe-Phe-NH2)6 may be important model peptides in the
search for new analgesics.
The introduction of 2′,6′-dimethyl-L-tyrosine (Dmt)
into various opioid ligands provided evidence that a
single residue can elicit substantial changes in the
activity profile of an opioid peptide.10 The most dramatic
representation of this effect involved the development
of derivatives containing the Dmt-Tic pharmacophore
that displayed extremely potent δ-opioid receptor an-
tagonism.10 Similarly, structure-activity studies on a
variety of opioid ligands containing Dmt revealed a
dramatic alteration in their activity through the eleva-
Rationale
Determination of the structure-activity profile of
opioid peptides has been approached largely through the
modification of side chains or alterations on various
functional groups of many residues including Tyr. For
example, N-alkylation of Tyr with bulky groups reduced
the affinity of the Dmt-Tic pharmacophore for the
δ-opioid receptor,10c although methylation exhibited
minimal effects on δ-opioid receptor affinity and bio-
activity.10a The increase in functional activity may be
due to increased resistance to enzymatic degradation16
as well as providential hydrophobic interactions within
the receptor.10f On the other hand, O-methylation of Tyr
eliminated activity17 as well as the introduction of
various types of functional groups (CO2H, CONH2, CH2-
OH).18 Further, introduction of I, OCH3, NO2, NH2,19
* Corresponding authors. Y.O.: Tel, +81-78-974-1551; fax, +81-78-
974-5689; e-mail, okada@pharm.kobegakuin.ac.jp. L.H.L.: Tel, +1-919-
541-3238; fax, +1-919-541-0696. E-mail: lazarus@niehs.nih.gov.
† The Graduate School of Food and Medicinal Sciences, Kobe Gakuin
University.
20
or CH3 in the 3′-position of Tyr induced negative
‡ Faculty of Pharmaceutical Sciences, Kobe Gakuin University.
§ High Technology Research Center, Kobe Gakuin University.
| Tohoku Pharmaceutical University.
consequences. Substitution of the hydroxyl group with
NO2,20 its deletion,21 or merely shifting its position in
the ring from the 4′- to the 3′-position20 completely
National Institute of Environmental Health Sciences.
10.1021/jm049384k CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/23/2004

Analogues of Endomorphin-2 as µ-Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 587
Scheme 1. Synthetic Scheme for Six Tyr Analoguesa
Figure 1. Structure of tyrosine analogues and substituents.
abolished activity. On the other hand, the bisubstituted
Dmt-containing peptides11-15 and those having a single
methyl group at the 2′-position of the Tyr residue in
DALDA22 and DPDPE19 maintained or increased recep-
tor affinity and functional bioactivity. Furthermore, the
replacement of Tyr with Dmp in endomorphin ([Dmp1]-
EM-2)23 and dermorphin24 demonstrated that high
biological activity was surprisingly maintained without
the important hydroxyl group on the tyramine ring.
The enhancement of opioid activity upon inclusion of
Dmt11b and Dmp23,24 in the sequence of opioid peptides
provided the impetus to develop analogues with sys-
tematic modifications at the 2′- and 6′-positions of the
Tyr aromatic ring and investigate the impact on the
activity of EM-2. In the present study, six tyrosine
analogues containing different alkyl groups were pre-
pared, namely, 2′-monomethyltyrosine (Mmt), 2′,3′,6′-
trimethyltyrosine (Tmt), 2′-ethyl-6′-methyltyrosine (Emt),
2′-isopropyl-6′-methyltyrosine (Imt), 2′,6′-diethyltyrosine
(Det), and 2′,6′-diisopropyltyrosine (Dit) (Figure 1) in
order to produce [Xaa1]EM-2 analogues and compare
them against [Dmt1]EM-2. The EM-2 analogues were
analyzed for their interactions with µ- and δ-opioid
receptors in vitro and in vivo, and the conformation
around the Xaa1-Pro2 bond was analyzed using 1H NMR
to investigate the effect of alkyl groups on the cis/trans
isomerization of the EM-2 analogues.
a Reagent and conditions: (i) KI/KIO3, HCl/MeOH; (ii) Ac2O/
pyridine, 50 °C; (iii) 2-acetamidoacrylate, Pd(OAc)2, (2-MeC6H4)3P,
Et3N/MeCN, reflux, 12 h. For 3e: 2-acetamidoacrylate, Pd(OAc)2,
(C6H4)3P, Et3N, 130 °C, 20 h; (iv) H2 (60 psig), [Rh(1,5-COD)(R,R-
DIPAMP)]BF4, 60 °C; (v) 12 mol/L HCl, reflux, 6 h; (vi) (Boc)2O,
Et3N.
Table 1. Analytical Data of the Tyrosine Analogues
Mmt Emt Imt
Det
Dit
Tmt
tR (C18)a
tR (WH)b
9.8 11.65 12.47 12.55 13.88
11.92
Chemistry
Although two procedures exist for the stereoselective
synthesis of L-Dmt, developed independently by Dygos
et al.25 and Hruby et al.,26 preparation of the Tyr
analogues in this investigation followed the method of
Dygos et al. (Scheme 1).25 The starting materials,
3-ethyl-5-methylphenol,27 3,5-diethylphenol,28 and 3,5-
diisopropylphenol,29 were prepared in our laboratory
according to procedures in the literature, and the other
phenol derivatives were commercially available. Briefly,
the alkylphenols were first iodinated in the 4-position
(1a-f) and the phenolic hydroxyl group was protected
with an acetyl group (2a-f). It was followed by a Heck
reaction30 to construct the dehydroamino acids (3a-f).
Compound 2a-d and 2f proceeded well by the Heck
reaction with exception of the 3,5-diisopropyl-4-iodophe-
nyl acetate (2e), because of the bulky steric hindrance
under the conditions described by Dygos et al.25 The
desired compound 3e was obtained by using a relatively
small ligand, (C6H5)3P, and reaction at higher temper-
ature, 130 °C. In the case of 3c, we could not remove
the starting material 3-isopropyl-5-methylphenol from
1c at the iodination step, due to the identical Rf values
of these two compounds. Thus, a sample of 1c containing
a small amount of starting material was used directly
for acetylation and the impurity was removed after Heck
reaction. The obtained dehydroamino acids (3a-f) were
reduced enantioselectively in the presence of the chiral
catalyst, [Rh(1,5-COD)(R,R-DIPAMP)BF4] to give 4a-
D analogue
16.9c 22.4c 22.5c 26.2c 30.4c
32.2 29.1 32.3 35.4 49.7
49.0d
51.6
L analogue
ee (recrystallization) 96.3 96
a Retention time with COSMOSIL 5C18-AR-II, 95:5 to 10:90 (30
min). b Retention time with CROWNPAK WH. c 50 °C, 1.5 mL/
min, 1 mM CuSO4:MeOH ) 90:10. d 50 °C, 1.5 mL/min, 0.5 mM
CuSO4:MeOH ) 95:5.
96.2 98
94.5 >99.0
f; the protecting groups were removed with 12 mol/L
HCl to yield 5a-f. The synthetic amino acids had ee
values ranging from 75% to 96%. To obtain optically
pure amino acids, the crude products were recrystallized
from 5 mol/L HCl and analyzed by chromatography on
a chiral column to yield enantiomeric purity > 95% ee
(Table 1). The amino group of the Tyr analogues was
protected with a Boc group (6a-f) before use in peptide
synthesis.
As shown in Scheme 2, the EM-2 analogues were
synthesized in solution using Boc-protection methods.
After deprotection of Boc-Pro-Phe-Phe-NH213a with HCl/
dioxane, the resulting amino component was condensed
with Boc-protected Tyr analogues (6a-f) using PyBop
as the coupling reagent. The final Boc protecting group
was removed with TFA in the presence of anisole, and
the resulting peptides were precipitated and purified
when necessary by semipreparative RP-HPLC. The
identification and purity of the final compounds were
verified using MS, NMR, analytical HPLC, and elemen-
tal analysis. The compounds exhibiting greater than

588 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Li et al.
Table 2. Physicochemical Properties of the [Xaa1]EM-2 Analogues
[R]D
c in
H2O
TLCa
Rf
tof-mass
[M + 1]
HPLC
compd
peptide
(deg)
tR (min)b
7
8
H-Mmt-Pro-Phe-Phe -NH2
H-Emt-Pro-Phe-Phe-NH2
H-Imt-Pro-Phe-Phe-NH2
H-Det-Pro-Phe-Phe-NH2
H-Dit-Pro-Phe-Phe-NH2
H-Tmt-Pro-Phe-Phe-NH2
-14.2
-4.57
-7.92
-2.70
-15.26
-2.05
0.44
0.41
0.36
0.36
0.43
0.51
0.56
0.68
0.74
0.72
0.78
0.70
586. 7
614. 8
628. 8
628. 8
656. 8
614. 8
14.98
15.89
16.6
16.55
17.85
16.12
9
10
11
12
a Solvent: n-BnOH:HOAc:H2O ) 4:1:5 (upper layer). b A:B ) 90:10 to A:B ) 10:90 for 30 min.
Scheme 2. Synthetic Scheme for [Xaa1]EM-2 analogues
(7-12)a
binding affinity by one-third relative to that of EM-2
(13), suggesting that the bulky isopropyl group inter-
fered with the binding to receptor. On the other hand,
Imt1 (9), which bears one bulky isopropyl and a methyl
group, exhibited a binding affinity similar to that of
Dmt1 (14). While the affinity of Mmt1 (7) was analogous
to that of Dmt1 (14), it had a 20-fold higher µ-opioid
receptor selectivity.
The results indicated that all of the Tyr1-substituted
EM-2 analogues had higher µ- and δ-opioid receptor
affinities than EM-2 (13), except for [Dit1]EM-2 (11), and
[Dmt1]EM-2 (14) exhibited the highest δ-opioid receptor
affinity (Table 3). Of these analogues, [Mmt1]EM-2 (7)
and [Tmt1]EM-2 (12) had the weakest δ-affinities and
subsequently the highest µ-receptor selectivities [Ki(δ)/
Ki(µ) ) 4005 and 5347, respectively], suggesting that
either the reduced lipophilicity of the aromatic ring (7)
or steric hindrance of the 3′-methyl group (12) was
unfavorable for ligand-receptor interaction at the
δ-opioid receptor. This also demonstrated a subtle
difference in the composition of the binding sites be-
tween µ- and δ-receptors.
One model representing the possible receptor-ligand
interaction of [Imt1]EM-2 (9) implies that one side of
the aromatic side chain could be inserted into a binding
pocket, which could accommodate a relatively small
alkyl group on the aromatic ring, such as methyl or
ethyl, but is insufficient for a bulky isopropyl group. In
other words, the portion of the aromatic ring containing
the isopropyl group might protrude outside the binding
pocket and therefore does not substantially interfere
with ligand-receptor interaction. While the binding
pocket accommodates either a methyl or an ethyl group,
the higher affinity of [Det1]EM-2 (10) and [Emt1]EM-2
(8) (0.06 nM and 0.08 nM, respectively) suggested a
preference for the ethyl derivative relative to the smaller
methyl group. Furthermore, the [Xaa1]EM-2 analogues
containing Dmt (14), Tmt (12), Mmt (7), and Imt (9)
exhibited comparable affinities (0.13-0.15 nM), but Dit
(11), which has two bulky isopropyl groups, exhibited
the weakest µ-opioid receptor affinity (2.29 nM).
a (i) HCl/dioxane; (ii) IBCF, Et3N, THF/DMF; (iii)PyBop, DIPEA,
DMF; (iv) TFA, anisole. bXaa: 7, Mmt; 8, Emt; 9, Imt; 10, Det;
11, Dit; 12, Tmt.
Table 3. Opioid Receptor Binding Affinity of [Xaa1]EM-2
Analogues
Ki(δ)/
compd
peptide
Ki(µ) (nM)a nc Ki(δ) (nM)b nc Ki(µ)
7
8
[Mmt1]EM-2 0.132 ( 0.008
3
5
5
3
4
3
528.6 ( 47
55.7 ( 6.2
190 ( 10
6
3
3
3
3
5
4005
884
1266
830
46
5347
13400
188
[Emt1]EM-2 0.063 ( 0.006
9
[Imt1]EM-2
[Det1]EM-2
[Dit1]EM-2
0.15 ( 0.013
0.084 ( 0.006
2.29 ( 0.37
10
11
12
13
14
69.7 ( 5.3
105 ( 16
[Tmt1]EM-2 0.111 ( 0.002
593.5 ( 80
9230 ( 200
28.2 ( 8.1
EM-2d
[Dmt1]EM-2d 0.15 ( 0.04
0.69 ( 0.16
a Versus [3H]DAMGO. b Versus [3H]DPDPE. c The number of
independent repetitions used different synaptosomal preparations.
d Data cited from ref 13a.
98% purity were used for all biological assays. The
physicochemical constants are shown in Table 2 and
given in more detail in Supporting Information.
Results and Discussion
Opioid Receptor Affinity. The opioid receptor bind-
ing affinities of the EM-2 analogues are summarized in
Table 3. The µ-opioid receptor affinity increased 4.6-fold
after replacement of the Tyr1 by Dmt (14) relative to
the parental peptide (13), as seen previously.13a Intro-
duction of a third methyl group at the 3′-position in the
Dmt aromatic ring (Tmt1) (12) further increased the
µ-opioid receptor affinity by nearly 50%, being 6.4-fold
higher than that of EM-2 (13). Substitution of one or
two methyl groups in Dmt by ethyl groups substantially
enhanced the binding affinity of the peptide; namely,
[Emt1]EM-2 (8) and [Det1]EM-2 (10) were about 11- and
8-fold more potent, respectively, than EM-2 (13), and
about twice as active as [Dmt1]EM-2 (14). These results
suggested that the side chain of Emt and Det might be
more accessible to residues within the binding pocket
than even Dmt. On one hand, introduction of two
isopropyl groups on the Tyr ring (Dit1) (11) decreased
In general, affinity of opioid ligands to the receptors
increased when Tyr was substituted by Dmt,11 suggest-
ing that the side chain of Dmt fits quite tightly and
precisely into a hydrophobic cavity situated within the
receptor. In addition, we conclude, on the basis of the
data from the [Xaa1]EM-2 analogues (7-12, 14) and
other studies,13b including those dealing with C-termi-
nally extended analogues of Dmt-Tic,10a,c,e,f that the
µ-opioid receptor apparently accommodates a bulkier
ligand than the δ-opioid receptor, which has different
requirements for ligand interaction.31
Functional Bioactivity in Vitro. The in vitro
biological activities were evaluated using isolated guinea

Analogues of Endomorphin-2 as µ-Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 589
Table 4. Functional Bioactivity of [Xaa1]EM-2 Analogues
GPI assay
MVD assay
compd
peptide
IC50 ( SE (nM)
na
IC50 ( SE (nM)
na
antagonismb
7
8
[Mmt1]EM-2
[Emt1]EM-2
[Imt1]EM-2
[Det1]EM-2
[Dit1]EM-2
[Tmt1]EM-2
EM-2
0.924 ( 0.431
0.623 ( 0.113
10.6 ( 2.7
6
5
11
5
8
12
33
10
28.7 ( 12.7
1.08 ( 0.31
601 ( 168
17
7
13
13
-
16
14
7
++
+
9
+
10
11
12
13
14
0.903 ( 0.326
299 ( 62
47.1 ( 17.9
>10000
+
nd
++
+
2.31 ( 0.74
7.96 ( 0.76
0.261 ( 0.038
46.4 ( 11.1
344 ( 93
0.59 ( 0.182
[Dmt1]EM-2
+
a The number of independent repetitions used different isolated tissue preparations. b Antagonism by CTAP (200 nM) with the percent
recovery of electrically evoked contraction: ++, >50%; +, <50%; nd, none detected.
pig ileum (GPI) for µ-opioid receptors and mouse vas
deferens (MVD) for δ-opioid receptors (Table 4). The GPI
tissue contains predominantly µ-opioid receptors, while
MVD includes δ-opioid receptors. The analogues [Mmt1]-
(7), [Emt1]- (8), [Det1]- (10), and [Tmt1]EM-2 (12)
exhibited high GPI potencies (IC50 ) 0.924, 0.623, 0.903,
and 2.31 nM, respectively), although their activities
were less than that of [Dmt1]EM-2 (14). In the MVD
assay, most analogues except for [Imt1]EM-2 (9) and
[Dit1]EM-2 (11) exhibited an unexpectedly higher po-
Figure 2. Effect of intracerebroventricularly injected EM-2
(13), [Det1]EM-2 (10), and [Dmt1]EM-2 (14) in the tail-flick
(A) and hot-plate (B) tests. Each compound was administered
at a dose of 3 µg/mouse and each point represents the mean (
SE determined with five or six mice. The asterisk denotes
values that were significantly different than the control mice
by the Student test (***, p < 0.001; **, p < 0.01).
tency than that based on the δ-opioid receptor affinity.
To confirm receptor preferences, the MVD activity of
these analogues was examined using a specific µ-opioid
receptor antagonist, CTAP.32 As shown in Table 4, the
MVD activity of the analogues and even that of parent
peptide EM-2 was effectively inhibited by CTAP; the
most effective antagonism was observed with [Tmt1]EM-
2 (7) and [Mmt1]EM-2 (12), which have relatively low
δ-opioid receptor affinity, suggesting that the MVD
activity of these two analogues was, for the most part,
elicited via a µ-opioid receptor which coexists in the
MVD tissues. The activity of the other analogues [Emt1]-
(8), [Det1]- (10), [Dmt1]- (14), and EM-2 (13) was also
inhibited by CTAP, suggesting that their MVD potencies
were mediated in part by the µ-opioid receptor in MVD
Table 5. The Cis/Trans Conformer Ratio of [Xaa1]EM-2
cis/trans (DMSO-d6)a
compd
peptide
cis
trans
7
8
[Mmt1]EM-2
[Emt1]EM-2
[Imt1]EM-2
[Det1]EM-2
[Dit1]EM-2
[Tmt1]EM-2
EM-2b
35
65
75
75
90
65
1
65
35
25
25
10
35
2
9
10
11
12
13
14
tissues. A similar phenomenon was also observed with
[Dmt1]EM-2b
70
30
23,24
other high-affinity µ-opioid receptor ligands.
The
a Determined by relative peak intensities from 1H NMR analy-
ses. b Data cited from ref 13a.
very high MVD potency of [Dmt1]EM-2 (14) and [Emt1]-
EM-2 (8) may then be attributable to the concomitant
µ-opioid receptor in MVD tissues as well as a relatively
high δ-opioid receptor affinity of their own (Table 1). It
should be noted that [Dit1]EM-2 (11) has unexpectedly
low GPI and MVD potencies while it still retained high
receptor affinity for both µ- and δ-opioid receptors,
implying that this analogue may have a mixed agonist/
antagonist property for both receptor types. As a
consequence, the antagonism experiments using the
MVD tissues strongly suggest that this series of ana-
logues are potent µ-opioid receptor agonists.
the 2′ and 6′-positions of Tyr represent the optimal alkyl
groups for interaction with and activation of µ- and
δ-receptors. Although [Dmt1]EM-2 (14) was the most
potent analogue in terms of the duration of the analgesic
response [60 min in contrast to 20 min with EM-2 (13)],
it was approximately 16% as effective as morphine (data
not shown). Naloxone (10 mg/kg sc) blocked antinoci-
ception (data not shown), which supports the observa-
tion that opioid receptors are involved in producing
analgesia.
In Vivo Biological Activity. The analgesic effect of
[Dmt1]EM-2 (14) and [Det1]EM-2 (10) were determined
by the tail-flick test (spinally mediated mechanism) and
the hot-plate test (supraspinal effects) in comparison to
the effect produced by EM-2 (13). The results in Figure
2 showed that all three compounds produced analgesia
with qualitatively similar results in the tail-flick test;
however, a stronger activity was observed with [Dmt1]-
EM-2 (14) based on the analysis of hot-plate tests. In
summary, [Dmt1]EM-2 (14) > [Det1]EM-2 (10) > EM-2
(13), yielding activity ratios of 1.00:0.86:0.65 in the tail-
flick test and 1.00:0.47:0.30 in the hot-plate tests. These
results indicated that the methyl side chains located at
Cis/Trans Conformation. With the presence of a
proline residue in the second position of the peptide
sequence, cis/trans isomerization readily occurs at the
Xaa1-Pro2 peptide bond. The cis/trans ratio can be easily
determined by 1H NMR in DMSO as described by Okada
et al.13 The analysis revealed that [Xaa1]EM-2 analogues
8, 9, 10, and 12 potentially adopt the cis conformer with
a cis/trans ratio of approximately 70:30 (Table 5),
equivalent to that of [Dmt1]EM-2 (14). Interestingly,
[Dit]1EM-2 (11), with two bulky alkyl groups in the
aromatic ring, had a cis/trans ratio of 90:10, while
[Mmt1]EM-2 (7) was trans rich, similar to EM-2 (13).
These observations suggest that alkyl groups at the 2′-

590 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Li et al.
was stirred at 0 °C for 10 min and then room temperature for
4 h. After removal of the solvent, the residue was diluted with
AcOEt. The dilution was washed with ice cold 10% citric acid
(3 × 10 mL), 5% Na2CO3 (3 × 10 mL), and saturated NaCl (3
× 10 mL); dried over Na2SO4; and evaporated to dryness. The
residue was purified by flash chromatograph. The compound
was precipitated with hexane, filtered, and dried under
vacuum. Elemental analysis data are summarized in the
Supporting Information (Table 2) along with the data for yield,
and 6′-positions of the tyramine moiety affected the cis/
trans ratio of the EM-2 analogues and the bulkier
groups produced greater cis isomers.
Our data revealed interesting observations concerning
the cis/trans equilibrium: first, [Mmt1]EM-2 (7) with a
cis/trans ratio of 35:65 contrasts remarkably with [Dmt1]-
EM-2 (14), with a ratio of 70:30, yet both compounds
had the same µ-opioid receptor affinities (0.13 and 0.15
nM, respectively) (Table 3); second, while [Dit1]EM-2
(11) had the highest cis/trans ratio (90:10), it exhibited
the lowest µ-opioid receptor affinity (Table 3) and
weakest functional bioactivity (Table 4). Thus, the cis/
trans ratio about the Xaa1-Pro2 bond may not be a
predictor of biological activity.
1
melting point, optical rotation, Rf, H NMR, and 13C NMR.
General Procedure for Synthesis of TFA-H-Xaa-Pro-
Phe-Phe-NH2 (7-12; Xaa ) Mmt, Emt, Imt, Det, Dit, Tmt).
Boc-Xaa-Pro-Phe-Phe-NH2 (0.21 mmol) was treated with TFA
(7 mmol) and anisole (0.46 mmol) for 1 h at room temperature.
The reaction solution was diluted with hexane, and the solid
was collected by filter, dried over KOH pellets, and purified
by semipreparative RP-HPLC. The purified peptide was ly-
ophilized to give amorphous powder. Elemental analysis data
are summarized in the Supporting Information (Table 3) along
Conclusions
Six L-tyrosine analogues were incorporated at the
N-terminus of the EM-2 sequence. EM-2 containing Det
(10), Emt (8), and Tmt (12) were the most potent
analogues in the binding assays (Table 3), which signi-
fies that the overall hydrophobicity of the peptides might
be an important factor for ligand-receptor interaction;
however, bulkier moieties, such as the isopropyl groups
of Dit (11), substantially interfere with ligand-receptor
interaction. Furthermore, the data suggest that the
µ-opioid receptor can readily accommodate a bulkier
N-terminal Tyr derivative than the δ-opioid receptor.
The affinities of the EM-2 analogues, which are char-
acteristic of µ-receptor selective ligands, were verified
and supported by the functional bioactivity in vitro. In
conclusion, the present study demonstrated that the Tyr
analogues used in this research are useful as surrogate
residues in the potential design of new opioid mimetics
with unique biological properties.
1
with the data for yield, H NMR, and 13C NMR.
Opioid Receptor Binding Assays. Opioid receptor affini-
ties were determined under equilibrium conditions [2.5 h at
room temperature (23 °C)] in a competition assay using brain
P2 synaptosomal membranes prepared from Sprague-Dawley
rats. Synaptosomes were preincubated to remove endogenous
opioids, washed in excess ice-cold buffer containing protease
inhibitor, and stored in a glycerol-containing buffer with
protease inhibitor at -80 °C as described previously.33 The δ-
and µ-opioid receptors were radiolabeled with [3H]DPDPE and
[3H]DAMGO, respectively,13-15,33 and excess unlabeled peptide
(2 µM) established the level of nonspecific binding. After
incubation, the radiolabeled membranes were rapidly filtered
on Whatman GF/C glass fiber filters presoaked in 0.1%
polyethylenimine in order to optimize the signal-to-noise ratio,
washed with ice-cold BSA buffer, and dried at 75 °C. Radio-
activity was determined using EcoLume (ICN, Costa Mesa,
CA). All analogues are analyzed in duplicate using five to seven
peptide dosages and several synaptosomal preparations in
independent repetitions (n values noted in Table 3) to ensure
statistical significance. The affinity constants (Ki) were cal-
culated according to Cheng and Prusoff.34
Experimental Section
Biological Activity in Isolated Tissue Preparation. The
myenteric plexus longitudinal muscle preparations (2-3 cm
segments) from the small intestine of male Hartley strain
guinea pigs (GPI) measured µ-opioid receptor agonism, and a
single mouse vas deferens (MVD) was used to determine
δ-opioid receptor agonism as described previously.23 The
isolated tissues were suspended in organ baths containing
balanced salt solutions in a physiological buffer, pH 7.5.
Agonists were tested for the inhibition of electrically evoked
contraction, expressed as IC50 (nM) obtained from the dose-
response curves. The IC50 values represent the mean ( SE of
5-33 separate assays. To measure whether µ-opioid receptor-
mediated antagonism occurred in the MVD, the test compound
was added to the tissue preparation at the IC50 dose value;
after 5 min incubation, CTAP (200 nM) was added and the
percent recovery (reversal rate) of electrically evoked contrac-
tion was then calculated.
Determination of Analgesia in Vivo. Tail-flick and hot-
plate latencies in Swiss-Webster male mice measured the effect
of icv administration of EM-2 (13), [Det1]EM-2 (10), and [Dmt1]-
EM-2 (14) as follows: compounds were dissolved in physi-
ological saline and injected into mice icv.15 Tail-flick tests
(spinal analgesia) were performed by applying radiant heat
to the dorsal surface of the tail (Columbus Instruments,
Columbus, OH). The latency period for removal of the tail,
defined as the tail-flick latency (TFL), was adjusted between
2 and 3 s (preresponse time), and a cut-off time was set at 8 s
to avoid external heat-related damage. The analgesic response
was measured beginning at 10 min following icv administra-
tion of EM-2 (13), [Det1]EM-2 (10), or [Dmt1]EM-2 (14),
respectively, and testing was terminated when TFL ap-
proached the preresponse time. The AUC (area under the
curve) was obtained by plotting the response time (s) versus
Mass spectra were measured with a KRATOS MALDI-TOF
analyses (matrix-assisted laser desorption ionization time-of-
flight mass spectrometry). Melting points were determined on
a Yanagimoto micromelting point apparatus and are uncor-
rected. TLC was performed on precoated plates of silica gel
F254 (Merk, Darmstadt, Germany). Optical rotations were
determined with a DIP-1000 automatic polarimeter (Japan
Spectroscopic Co.). Semipreparative RP-HPLC and analytic
RP-HPLC used a Waters Delta 600 with COSMOSIL C18
column (20 mm × 250 mm) and COSMOSIL C18 column (4.6
mm × 250 mm), respectively. The solvents for analytical HPLC
were as follows: A, 0.05% TFA in water; B, 0.05% TFA in CH3-
CN. The column was eluted at a flow rate of 1 mL/min with a
linear gradient of 90% A to 10% A in 30 min; the retention
1
time is reported as tR (min). H NMR spectra were measured
on a Bruker DPX-400 spectrometer at 25 °C. Chemical shift
values are expressed as ppm downfield from tetramethyl-
siliane.
Synthesis of Tyr Analogues (Mmt, Emt, Imt, Det, Dit,
Tmt). The six unique L-Tyr analogues were synthesized
according to Scheme 1. The detailed synthetic procedure for
each Tyr analogue and the elemental analysis data are
summarized in Table 1 and presented in the Supporting
Information.
General Procedure for Synthesis of Boc-Xaa-Pro-Phe-
Phe-NH2 (Xaa ) Mmt, Emt, Imt, Det, Dit, Tmt). Boc-Pro-
Phe-Phe-NH213a (0.492 mmol) was treated with 8.8 mol/L HCl/
dioxane (9.84 mmol) to remove the Boc group at room
temperature for 30 min. The product was precipitated with
ether, filtered, and dried at vacuum. The resulted hydrochlo-
ride salt was dissolved in DMF (10 mL) containing DIPEA
(1.19 mmol), and to this solution were added Boc-Xaa-OH
(0.541 mmol) and PyBop (0.595 mmol). The reaction mixture

Analogues of Endomorphin-2 as µ-Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 591
(10) (a) Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.; Bianchi,
C.; Bryant, S. D.; Copper, P. S.; Lazarus, L. H. Evolution of the
Dmt-Tic pharmacophore: N-terminal methylated derivatives
with extraordinary δ opioid antagonist activity. J. Med. Chem.
1997, 40, 3100-3108. (b) Schiller, P. W.; Fundytus, M. E.;
Merovitz, L.; Weltrowska, G.; Nguyen, T. M.-D.; Lemieux, C.;
Chung N. N.; Coderre, T. J. The opioid µ agonist/δ antagonist
DIPP-NH2[Ψ] produces a potent analgesic effect, no physical
dependence, and less tolerance that morphine in rats. J. Med.
Chem. 1999, 42, 3520-3526. (c) Salvadori, S.; Guerrini, R.;
Balboni, G.; Bianchi, C.; Bryant, S. D.; Copper, P. S.; Lazarus,
L. H. Further studies on the Dmt-Tic pharmacophore: Hydro-
phobic substituents at the C-terminus endow δ antagonists to
manifest µ agonism or µ antagonism. J. Med. Chem. 1999, 42,
5010-5019. (d) Page, D.; Naismith, A.; Schmidt, R.; Coupal, M.;
Labarre, M.; Gosselin, M.; Bellemare, D.; Payza, K.; Brown, W.
Novel C-terminus modifications of the Dmt-Tic motif: A new
class of dipeptide analogues showing altered pharmacological
profiles toward the opioid receptors. J. Med. Chem. 2001, 44,
2387-2390. (e) Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi,
C.; Rizzi, D.; Bryant, S. D.; Lazarus, L. H. Evaluation of the Dmt-
Tic pharmacophore: Conversion of a potent δ opioid antagonist
into a potent δ opioid agonist and ligands with mixed properties.
J. Med. Chem. 2002, 45, 713-720. (f) Balboni, G.; Salvadori, S.;
Guerrini, R.; Negri, L.; Giannini, E.; Bryant, S. D.; Jinsmaa, J.;
Lazarus, L. H. Synthesis and opioid activity of N,N-dimethyl-
Dmt-Tic-NH-CH(R)-R′ analogues: Acquisition of potent δ an-
tagonism. Bioorg. Med. Chem. 2003, 11, 5435-5441.
(min) after administration of the compound. Morphine was
used as a positive control and naloxone was used as a general
opioid antagonist.
For the hot-plate test (supraspinal analgesia), mice were
set on an electrically heated plate at 55 ( 0.1 °C (IITC INC.,
Woodland Hills, CA) following the same drug injection para-
digm as above. Hot-plate latency (HPL) was measured as the
interval between the placement of the mice on the hot plate
and observing movements consisting of either jumping, licking,
or shaking their hind paws with a baseline latency of 15 s and
maximal cut-off time of 30 s. The area under the curve (AUC)
was derived from data based on the response (mean ( SE) of
five to seven mice per time point. The analgesic effect of EM-2
(13), [Det1]EM-2 (10), and [Dmt1]EM-2 (14) are relative to the
saline control.
Acknowledgment. This work was supported in part
by a Grant-in-Aid for Japan Society for the Promotion
of the Science (JSPS) Fellows (1503306) to T.L.
Supporting Information Available: Experimental pro-
cedures and analytical data for the synthesis of Tyr analogues
and their derivatives (1a-f to 6a-f) are presented herein.
Yield, melting point, [R]25D, Rf, and NMR values of [Boc-Xaa1]-
EM-2 as well as the yield, melting point, and NMR values of
[Xaa1]EM-2 (7-12) are given, including the elemental analy-
sis data of [Boc-Xaa1]EM-2 and [Xaa1]EM-2 (7-12). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(11) (a) Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.;
Balboni, G.; Salvadori, S. Design of δ-opioid peptide antagonists
for emerging drug application. Drug Discovery Today 1998, 3,
284-294. (b) Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada,
Y.; Lazarus, L. H. Dmt and opioid peptides: A potent alliance.
Biopolymers (Pept. Sci.) 2003, 71, 86-102.
(12) (a) Chandrakumar, N. S.; Yonan, P. K.; Stapelfeld, A.; Savage,
M.; Rorbacher, E.; Contreras, P. C.; Hammond, D. Preparation
and opioid activity of analogues of the analgesic dipeptide 2,
6-dimethyl-l-tyrosyl-N-(3-phenylpropyl)-d-alaninamide. J. Med.
Chem. 1992, 35, 223-233. (b) Hansen, J. D. W.; Stapelfeld, A.;
Savage, M. A.; Reichman, M.; Hammond, D. L.; Haaseth, R. C.;
Mosberg, H. I. Systemic analgesic activity and δ-opioid selectivity
in [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin. J. Med. Chem.
1992, 35, 684-687. (c) Sasaki, Y.; Suto, T.; Ambo, A.; Ouchi,
H.; Yamamoto Y. Biological properties of opioid peptides replac-
ing Tyr at position 1 by 2,6-dimethyl-Tyr. Chem. Pharm. Bull.
1999, 47, 1506-1509. (d) Schiller, P. W.; Berezowska, I.; Nguyen,
T. M.-D.; Schimidt, R.; Lemieux, C.; Chung, N. N.; Falcone-
Hindley, M. L.; Yao, W.; Liu, J.; Iwama, S.; Smith, A. B.;
Hirschmann, R. Novel ligands lacking a positive charge for the
δ-and µ-opioid receptors. J. Med. Chem. 2000, 43, 551-559. (e)
Lu, Y.; Nguyen, T. M.-D.; Weltrowska, G.; Berezowska, I.;
Lemiex, C.; Chung, N. N.; Schiller, P. W. [2′,6′-Dimethyltyrosine]-
dynorphin A (1-11)-NH2 analogues lacking an N-terminal amino
group: Potent and selective κ opioid antagonists. J. Med. Chem.
2001, 44, 3048-3053. (f) Sasaki, Y.; Hirabuki, M.; Ambo, A.;
Ouchi, H.; Yamamoto, Y. Enkephalin analogues with 2′,6′-
dimethylphenylalanine replacing phenylalanine in position 4.
Bioorg. Med. Chem. Lett. 2001, 11, 327-329. (g) Harrison, B.
A.; Pasternak, G. W.; Verdine, G. L. 2,6-Dimethyltyrosine
analogues of a stereodiversified ligand library: Highly potent,
selective, nonpeptidic µ opioid receptor agonists. J. Med. Chem.
2003, 46, 677-680. (h) Weltrowska, G.; Lemieux, C.; Chung, N.
N.; Schiller, P. W. A chimeric opioid peptide with mixed µ
agonist/δ antagonist properties. J. Pept. Res. 2004, 63, 63-68.
(13) (a) Okada, Y.; Fujita, Y.; Motoyama, T.; Tsuda, Y.; Yokoi, T.; Li
T.; Sasaki, Y.; Ambo, A.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L.
H. Structural studies of [2′,6′-dimethyl-L-tyrosine1]endomorphin-
2 analogues: Enhanced activity and cis orientation of the Dmt-
Pro amide bond. Bioorg. Med. Chem. 2003, 11, 1983-1984. (b)
Fujita, Y.; Tsuda, Y.; Li, T.; Motoyama, T.; Takahashi, M.;
Shimizu, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Kita, A.; Jinsmaa,
Y.; Bryant, S. D.; Lazarus, L. H.; Okada, Y. Development of
potent bifunctional endomorphin-2 analogues with mixed µ-/δ-
opioid agonist and δ-opioid antagonist properties. J. Med. Chem.
2004, 47, 3591-3599.
References
(1) Abbreviations. In additional to the IUPAC-IUB Commission on
Biochemical Nomenclature (J. Biol. Chem. 1985, 260, 14-42),
this paper uses the following symbols and abbreviations: Ac2O,
acetic anhydride; AcOEt, ethyl acetate; Boc, tert-butyloxycarbo-
nyl; BSA, bovine serum albumin; CTAP, D-Phe-cyclo-(Cys-Tyr-
D-Trp-Arg-Thr-Pen)-Thr-NH2; DALDE, [D-Ala,2D-Leu5]enkephalin;
DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; DIPEA, diiso-
propylethylamine; DMF, N,N-dimethylformamide; Dmp, 2′,6′-
dimethyl-L-phenylalanine; DMSO, dimethyl sulfoxide; DPDPE,
cyclo-[D-Pen2,5]enkephalin; GPI, guinea-pig ileum; IBCF, isobutyl
chloroformate; IC50, concentration required for 50% inhibition
of the electrically induced contraction in muscle derived from a
dose-response curve; MVD, mouse vas deferens; NMR, nuclear
magnetic resonance; Pd(OAc)2, palladium(II) acetate; PyBOP,
benzoltriazol-1-yloxytrispyrrolidinophosphonium hexafluorphos-
phate; [Rh(1,5-COD) (R,R-DIPAMP)]BF4, (R,R)-(-)1,2-bis[(O-
methoxyphenyl)(phenyl) phosphino]ethane(1,5-cyclooctadiene)-
rhodium(I) tetrafluoroborate; RP-HPLC, reverse-phase high
performance liquid chromatography; TFA, trifluoroacetic acid;
TLC, thin-layer chromatography.
(2) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Forthergill, L. A.;
Morga, B, A.; Morris, H. R. Identification of two related pen-
tapeptides from the brain with potent opiate agonist activity.
Nature 1975, 258, 577-579.
(3) (a) Ling, N; Guillemin, R. Morphinomimetics activity of synthetic
fragments of â-lipotropin and analogues. Proc. Natl. Acad. Sci.
U.S.A. 1976, 7, 3308-3310. (b) Cox, B. M.; Goldstein, A.; Li, C.
H. Opioid activity of a peptide â-lipotropin-(61-91), derived form
â-lipotropin. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 1821-1823.
(4) Goldstein, A.; Fischli, W.; Lowney, L. I.; Hunkapilier, M.; Hood,
L. Porcine pituitary dynorphin: Complete amino acid sequence
of the biologically active heptadecapeptide. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 7219-7223.
(5) Echergyi, J.; Kastin, A. J.; Zadina, J. E. Isolation of a novel
tetrapeptide with opiate and anti-opiate activity form human
brain cortex: Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1). Peptides
1992, 13, 623-631.
(6) (a) Zadina, J. E.; Hackler, L.; Ge, L.-J.; Kastin, A. J. A potent
and selective endogenous agonist for the µ-opiate receptor.
Nature 1997, 386, 499-502. (b) Hackler, L.; Zadina, J. E.; Ge,
L.-J.; Kastin, A. J. Isolation of relatively large amounts of
endomorphin-1 and endomorphin-2 form human brain cortex.
Peptides 1997, 18, 1635-1639.
(7) Melchiorri, P.; Negri, L. The dermorphin peptide family. Gen.
Pharmacol. 1996, 27, 1099-1107.
(8) Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Salvadori, S. What
peptides these deltorphins be. Prog. Neurobiol. 1999, 57, 377-
420.
(9) Portoghese, P. S. Bivalent ligands and the message-address
concept in the design of selective opioid receptor antagonists.
Trends Pharmacol. Sci. 1989, 10, 230-235.
(14) Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.;
Konishi, R.; Nagata, M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S.
D.; Lazarus, L. H. Unique high-affinity synthetic µ-opioid
receptor agonists with central-and systemic-mediated analgesia.
J. Med. Chem. 2003, 46, 3201-3209.
(15) Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.; Li, T.; Fujisawa, Y.;
Shiotani, K.; Tsuda, Y.; Yokoi, T.; Ambo, A.; Sasaki, Y.; Bryant,
S. D.; Lazarus. L. H.; Okada Y. Oral bioavailability of a new
class of µ-opioid receptor agonists containing 3,6-bis[Dmt-NH-
(CH2)n]-2(1H)-pyrazinone with central-mediated analgesia. J.
Med. Chem. 2004, 47, 2599-2610.

592 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Li et al.
(16) Bradbury, A. F.; Smyth, D. G.; Snell, C. R.; Deakin, J. E. W.;
Wendlandt, S. Comparison of the analgesic properties of lipo-
tropin C-fragment and stabilized enkephalins in the rat. Bio-
chem. Biophy. Res. Commun. 1977, 74, 748-754.
glycine and alanine. Tetrahedron 2001, 57, 6375-6328. (b) Tang,
X.; Soloshonok, V. A.; Hruby, V. J. Convenient, asymmetric
synthesis of enantiomerically pure 2′,6′-dimethyltyrosine (DMT)
via alkylation of chiral equivalents of nucleophilic glycine.
Tetrahedron: Asymmetry 2000, 11, 2917-2925.
(17) Schiller, P. W.; Yam, C. H.; Lis, M. Evidence for topographical
analogy between methionine-enkephalin and morphine deriva-
tives. Biochemistry 1977, 16, 1831-1838.
(27) Balaban, A. T.; Nenitzescu, C. D. Pyrylium salts obtained by
diacylation of olefins. Part II. The two pyrylium salts formed in
diacetylation of 2-methylbut-2-ene. J. Chem. Soc. 1961, 3553-
3561.
(28) Tessier, D. M.; Clark, J. M. Hapten design in the development
of competitive enzyme-linked immunosorbent assays for geno-
toxic metabolites of alachlor. J. Agric. Food Chem. 1999, 47,
3925-3933.
(29) Seidel, F.; Schulze, M.; Baltz, H. Autoxidation of 1,3,5-triiso-
propylbenzene, a new synthesis of phloroglucinol. J. Prakt.
Chem. 1956, 3, 278-298.
(30) Heck, R. F. Palladium-catalyzed vinylation of organic halides.
Org. React. 1982, 25, 345-390.
(31) Bryant, S. D.; Salvadori, S.; Okada, Y.; Takahshi, M.; Sasaki,
Y.; Lazarus, L. H. Computational chemistry and opioidmimet-
ics: Receptor ligand interaction of three classes of biologically
potent peptides. In Peptides 2000, Martinez, J., Fehrentz, J.-A.,
Eds.; EDK: Paris, 2000; pp 407-408.
(32) Pelton, J. T.; Kazmierski, W.; Gulya, K.; Yamamura, H. I.;
Hruby, V. J. Design and synthesis of conformationally con-
strained somatostatin analogues with high potency and specific-
ity for µ opioid receptors. J. Med. Chem. 1986, 29, 2370-2375.
(33) (a) Lazarus, L. H.; Salvadori, S.; Santagaga, V.; Tomatis, R.;
Wilson, W. E. Function of negative charge in the “address
domain” of deltorphins. J. Med. Chem. 1991, 34, 1350-1355.
(b) Lazarus, L. H.; Salvadori, S.; Attila, M.; Grieco, P.; Bundy,
D. M.; Wilson, W. E.; Tomatis, R. Interaction of deltorphin with
opioid receptors: Molecular determinants for affinity and se-
lectivity. Peptides 1993, 14, 21-28.
(18) Arnold, Z. S.; Schiller P. T. Optically active aromatic amino acid.
Part VI. Synthesis and properties of (leu5)-enkephalin analogues
containing O-methyl-l-tyrosine1 with ring substitution at position
3′. J. Pept. Sci. 2000, 6, 280-289.
(19) Toth, G.; Russell, K. C.; Landis, G.; Kramer, T. H.; Fang, L.;
Knapp, R.; Davis, P.; Burks, T. F.; Yamamura, H. I.; Hruby, V.
Ring substituted and other conformationally constrained
tyrosine analogues of [D-Pen2,D-Pen5]enkephalin with δ opioid
receptor selectivity. J. Med. Chem. 1992, 35, 2384-2391.
(20) Coy, D. H.; Kastin, A. J. Tyrosine-modified analogues of me-
thionine-enkephalin and their effects on the mouse vas deferens.
Peptides 1980, 1, 175-177.
(21) Darlak, K.; Grzonka, Z.; Janicki, P.; Czlonkowski, A.; Gumulka,
W. Structure-activity studies of dermorphin. Synthesis and
some pharmacological data of dermorphin and its 1-substituted
analogues. J. Med. Chem. 1983, 26, 1445-1457.
(22) Schiller, P. W.; Nguyen, T. M.-D.; Berezowska, I.; Dupuis, S.;
Weltrowska, G.; Chang, N. N.; Lemieux, C. Synthesis and in
vitro opioid activity profiles of DALDA analogues. Eur. J. Med.
Chem. 2000, 35, 895-901.
(23) Sasaki, Y.; Sasaki, A.; Niizuma, H.; Goto, H.; Ambo, A. Endo-
morphin 2 analogues containing Dmp residue as an aromatic
amino acid surrogate with high µ-opioid receptor affinity and
selectivity. Bioorg. Med. Chem. 2003, 11, 675-678.
(24) Ambo, A.; Niizuma, H.; Sasaki, A.; Kohara, H.; Sasaki, Y.
Dermorphin tetrapeptide analogues with 2′,6′-dimethylpheny-
lalanine (Dmp) substituted for aromatic amino acids have high
µ opioid receptor binding and biological activities. Bioorg. Med.
Chem. Lett. 2003, 13, 1269-1272.
(25) Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson, O. J.;
Getman, D. P.; Periana, R. A.; Beck, G. R. A convenient
asymmetric synthesis of the unnatural amino acid 2,6-dimethyl-
L-tyrosine. Synthesis 1992, 741-743.
(26) (a) Soloshonok, V. A.; Tang, X.; Hruby, V. J. Large-scale
asymmetric synthesis of novel sterically constrained 2′,6′-
dimethyl- and R-2′,6′-trimethyltyrosine and -phenylalanine de-
rivatives via alkylation of chiral equivalents of nucleophilic
(34) Cheng, Y.-C.; Prusoff, W. H. Relationships between the inhibition
constant (Ki) and the concentration of inhibition which cause
50% inhibition (I50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
JM049384K