Bifunctional [2′,6′-dimethyl-L-tyrosine1] endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed μ-agonist/δ-antagonist and dual μ-agonist/δ-agonist opioid ligands

Article abstract of DOI:10.1021/jm061238m

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH₂) and [Dmt¹]EM-2 (Dmt = 2′,6′-dimethyl-L-tyrosine) analogues, containing alkylated Phe³ derivatives, 2′-monomethyl (2, 2′), 3′,5′- and 2′,6′-dimethyl (3, 3′, and 4′, respectively), 2′,4′,6′-trimethyl (6, 6′), 2′-ethyl-6′-methyl (7, 7′), and 2′-isopropyl-6′- methyl (8, 8′) groups or Dmt (5, 5′), had the following characteristics: (i) [Xaa³]EM-2 analogues exhibited improved μ- and δ-opioid receptor affinities. The latter, however, were inconsequential (K_i^δ = 491-3451 nM). (ii) [Dmt ¹,-Xaa³]EM-2 analogues enhanced μ- and δ-opioid receptor affinities (K_i^μ = 0.069-0.32 nM; K _i^δ = 1.83-99.8 nM) without κ-opioid receptor interaction. (iii) There were elevated μ-bioactivity (IC₅₀ = 0.12-14.4 nM) and abolished δ-agonism (IC₅₀ > 10 μM in 2′, 3′, 4′, 5′, 6′), although 4′ and 6′ demonstrated a potent mixed μ-agonism/δ-antagonism (for 4′, IC₅₀^μ = 0.12 and pA₂ = 8.15; for 6′, IC₅₀^μ = 0.21 nM and pA₂ = 9.05) and 7′ was a dual μ-agonist/δ-agonist (IC₅₀ ^μ = 0.17 nM; IC₅₀^δ = 0.51 nM).

Full text of DOI:10.1021/jm061238m

© Copyright 2007 by the American Chemical Society
Volume 50, Number 12
June 14, 2007
Articles
Bifunctional [2′,6′-Dimethyl-L-tyrosine1]endomorphin-2 Analogues Substituted at Position 3 with
Alkylated Phenylalanine Derivatives Yield Potent Mixed µ-Agonist/δ-Antagonist and Dual
µ-Agonist/δ-Agonist Opioid Ligands
,†
Tingyou Li, Kimitaka Shiotani, Anna Miyazaki,† Yuko Tsuda, Akihiro Ambo,# Yusuke Sasaki,# Yunden Jinsmaa,‡
,†
Ewa Marczak,‡ Sharon D. Bryant,‡ Lawrence H. Lazarus,*,‡ and Yoshio Okada*,
The Graduate School of Food and Medicinal Sciences and Faculty of Pharmaceutical Sciences, Kobe Gakuin UniVersity,
Nishi-ku, Kobe 651-2180, Japan, Department of Biochemistry, Tohoku Pharmaceutical UniVersity, Aoba-ku, Sendai 981-8558,
Japan, and Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of EnVironmental Health Sciences,
Research Triangle Park, North Carolina 27709
ReceiVed October 20, 2006
Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt ) 2′,6′-dimethyl-L-tyrosine) analogues,
containing alkylated Phe3 derivatives, 2′-monomethyl (2, 2′), 3′,5′- and 2′,6′-dimethyl (3, 3′, and 4′,
respectively), 2′,4′,6′-trimethyl (6, 6′), 2′-ethyl-6′-methyl (7, 7′), and 2′-isopropyl-6′-methyl (8, 8′) groups
or Dmt (5, 5′), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved µ- and
δ-opioid receptor affinities. The latter, however, were inconsequential (Kiδ ) 491-3451 nM). (ii) [Dmt1,-
Xaa3]EM-2 analogues enhanced µ- and δ-opioid receptor affinities (Kiµ ) 0.069-0.32 nM; Kiδ ) 1.83-
99.8 nM) without κ-opioid receptor interaction. (iii) There were elevated µ-bioactivity (IC50 ) 0.12-14.4
nM) and abolished δ-agonism (IC50 > 10 µM in 2′, 3′, 4′, 5′, 6′), although 4′ and 6′ demonstrated a potent
µ
µ
mixed µ-agonism/δ-antagonism (for 4′, IC50 ) 0.12 and pA2 ) 8.15; for 6′, IC50 ) 0.21 nM and pA2 )
µ
δ
9.05) and 7′ was a dual µ-agonist/δ-agonist (IC50 ) 0.17 nM; IC50 ) 0.51 nM).
Introduction
to improve biological activity in vitro and in vivo by chemical
modification.15-25
Endomorphin-1 (EM-1 ) H-Tyr-Pro-Trp-Phe-NH2) and en-
domorphin-2 (EM-2 ) H-Tyr-Pro-Phe-Phe-NH2) are endog-
enous opioid peptides with remarkably high selectivity for
µ-opioid receptors.1 Since the endomorphins express pharma-
cological properties similar to those of morphine and are thought
to inhibit pain without its undesirable side effects,2,3 extensive
studies have been performed in order to clarify their pharma-
cological characteristics4-9 and bioactive conformation10-14 and
The aromatic amino acid residue in position 3 is the defining
structural determinant between EM-1 (Trp3) and EM-2 (Phe3).1,26
Moreover, extensive studies previously indicated that Dmta in
lieu of the N-terminal Tyr dramatically enhanced receptor
affinity and bioactivity of numerous opioid agonists and
antagonists,27,28 and data on endomorphin-2 analogues modified
at position 1 with tyrosine analogues alkylated on the tyramine
ring improved in vitro biological parameters.29 Similarly, an
alkylated Phe analogue, 2′,6′-dimethylphenylalanine (Dmp), was
an effective surrogate for phenylalanine in several opioid
* To whom correspondence should be addressed. For L.H.L.: phone,
+1-919-541-3238; fax, 1-919-541-5737; e-mail, lazarus@niehs.nih.gov.
For Y.O.: phone, +81-78-974-1551; fax, +81-78-974-5689; e-mail,
okada@pharm.kobegakuin.ac.jp.
The Graduate School of Food and Medicinal Sciences, Kobe Gakuin
University.
a Abbreviations: Dmp, 2′,6′-dimethyl-L-phenylalanine; 3,5Dmp, 3′,5′-
dimethyl-L-phenylalanine; Dmt, 2′,6′-dimethyl-L-tyrosine; Emp, 2′-ethyl-
6′-methyl-L-phenylalanine; Imp, 2′-isopropyl-6′-methyl-L-phenylalanine;
Mmp, 2′-methyl-L-phenylalanine; Tmp, 2′,4′,6′-trimethyl-L-phenylalanine;
Xaa, phenylalanine analogue.
† Faculty of Pharmaceutical Sciences, Kobe Gakuin University.
# Tohoku Pharmaceutical University.
‡ National Institute of Environmental Health Sciences.
10.1021/jm061238m CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/12/2007

2754 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Li et al.
rating the morphine tolerance and dependence due to µ-opioid
receptors,41 this study focused exclusively on these two impor-
tant and interrelated opioid systems. Furthermore, as our data
reveal (infra vide), these EM-2 analogues are essentially devoid
of activity toward κ-opioid receptors. In fact, the combination
of Dmt1 and an alkylated Phe3 substitution provides unique
evidence for the change in the bioactivities of several analogues,
including identification of potent bifunctional µ-opioid receptor
agonists containing δ-antagonist or δ-agonist properties.
Chemistry
Dmt was synthesized by following the method described by
Dygos et al.42 The Phe analogues (Figure 1) containing 2′-methyl
(Mmp) (R2), 3′,5′-dimethyl (3,5Dmp) (R3,R5), 2′,6′-dimethyl
(Dmp) (R2,R6), 2′,4′,6′-trimethyl (Tmp) (R2,R4,R6), 2′-ethyl-6′-
methyl (Emp) (R2,R6), and 2′-isopropyl-6′-methyl (Imp) (R2,R6)
were prepared as reported, through [Rh(1,5-COD)(R,R-DIPAM-
P)]BF4, [(R,R)-(-)-1,2-bis[(o-methoxyphenyl)(phenyl)phosphi-
no]ethane(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate] me-
diated asymmetric catalytic hydrogenation of the corresponding
acetamidoacrylate.43 Boc(tert-butyloxycarbonyl)-Tyr-Pro-OH44
and Boc-Dmt-Pro-OH40 were prepared as reported. Tyr/Dmt-
Pro-Xaa-Phe-NH2 (Xaa ) Mmp, 3,5Dmp, Dmp, Dmt, Tmp,
Emp, Imp) was formed by segment condensation method in
solution. Briefly, after deprotection of Boc-Xaa-Phe-NH2 with
HCl/dioxane, the resulting H-Xaa-Phe-NH2 was condensed with
Boc-Tyr/Dmt-Pro-OH using PyBop (benzotriazol-1-yloxytrispyr-
rolidinophosphonium hexafluorophosphate) as the coupling
reagent. The final Boc protecting group was removed with HCl/
dioxane in the presence of anisole, and the resulting peptide
was purified by semipreparative RP (reversed phase)-HPLC.
The identification and purity of the final compounds and their
intermediates were verified using MS, NMR, analytical HPLC,
and elemental analysis. Some analytical data of the final
compounds are summarized in Table 1. The final compounds
exhibiting greater than 98% purity were used for all biological
assays. Supporting Information provides detailed elemental
analysis results of all the compounds.
Figure 1. Schematic structure of endomorphin-2 with alkylation sites
(R1-R6) on Tyr1 and Phe3.
peptides such as dynorphin A,30 [Leu5]enkephalin,31 dermorphin,
deltorphin II,32 YrFB (H-Tyr-D-Arg-Phe-âAla-NH2),33 and en-
domorphin-2.34 Furthermore, the replacement of Tyr1 by Dmp
in deltorphin II and enkephalin also yielded analogues that were
surprisingly nearly as effective as the parental peptides,35
suggesting that alkylation of the aromatic ring enhances
hydrophobicity and stability and/or limits rotational freedom
in its solution conformation.
Rationale
It is well-known that a subtle change in both the hydropho-
bicity and spatial conformation of an opioid ligand contributes
not only to a modification of its overall activity profile (receptor
affinity, bioactivity)30,32,34,35 and physicochemical properties
(stability, conformation)14,18,21,31 but also to its biological
efficacy (in vitro, in vivo) as an antinociceptive agent27,28,36 with
an enhanced ability to transit through the blood-brain bar-
rier.36,37 In this regard, N,N-alkylation converted a potent
δ-opioid antagonist into an inverse agonist38 and led to the
appearance of elevated µ-opioid receptor affinity and increased
δ-antagonism among numerous analogues of the Dmt-Tic
(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) pharmacoph-
oric compounds.39 On the other hand, the acquisition of a greater
degree of hydrophobicity through alkylation of the tyramine ring
of Tyr1 permitted further differentiation of the biological
properties between the structurally related endomorphin-1 and
-2 molecules.29,36 In light of the ability of the dialkylated
derivative of phenylalanine (Dmp) to substitute for Tyr1 in
deltorphin II35 and for Phe in other opioids,30-35 the importance
of the residue in the third position of the endomorphins, which
is known to affect their receptor selectivity and bioactivity,1-4,6,7
should be amenable to alterations in the hydrophobic milieu of
the aromatic moiety of Phe. An overall change in conformation
or reduced freedom of rotation imparted by the alkyl substituents
should provide a gauge of the potential effect of the substitution
on receptor interaction and functional bioactivity parameters
(Figure 1). Furthermore, in light of the importance of µ-opioid
receptors in modulating pain, appetite, and alcohol-induced
elevation of spontaneous inhibition of postsynaptic currents
(IPSC)40 and the involvement of δ-opioid receptors in amelio-
Results and Discussion
Opioid Receptor Affinity. Improved affinities for µ-opioid
receptors were observed in the [Xaa3]EM-2 compounds (2, 4,
6-8) compared to the parent peptide (1), except 3 and 5 which
exhibited weaker affinities (Table 2). Introduction of a single
methyl group at the 2′-position of Phe (2) enhanced µ-opioid
receptor affinity 6-fold; a second methyl group at the 6′-position
(4) induced a further 5-fold increase. Sterically bulky groups,
such as a third methyl group at the 4′-position (6) or an ethyl
(7) or isopropyl (8) at the 6′-position (Figure 1), reduced receptor
affinity by a factor of 10 relative to [Dmp3]EM-2 (4), which
exhibits the highest µ-selectivity of any known µ-opioid
agonist;1,34 nonetheless, the µ-affinities of 6-8 were still 3-fold
greater than that of the naturally occurring EM-2 (1) (Table 2).
Another dimethylated Phe derivative, [3,5Dmp3]EM-2 (3),
displayed the lowest µ-opioid receptor affinity of all the
analogues (Table 2), being 141-fold lower than [Dmp3]EM-2
(4) and about 5-fold less than 1, suggesting that the positions
of the alkyl groups on the Phe3 aromatic ring were a critical
factor for receptor affinity.
Among the [Dmt1,Xaa3]EM-2 ligands (1′-8′) (Table 2),
alkylated Phe3 analogues essentially enhanced the affinities for
both µ- and δ-opioid receptors, although minor discrepancies
in µ-opioid receptor affinity were noted for 6′, 7′, and 8′ in
comparison to the [Xaa3]EM-2 substances. Although they

Bifunctional Endomorphin-2 Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2755
Table 1. Analytical Data of H-Tyr/Dmt-Pro-Xaa-Phe-NH2 Endomorphin-2 Analogues
TOF mass [M + 1]
HPLC (min)
b
c
compd
peptide
[R]D (deg)
c in H2O
TLCa Rf
calcd
found
tR
tR
2
2′
3
3′
4′
5
5′
6
6′
7
7′
8
H-Tyr-Pro-Mmp-Phe-NH2
H-Dmt-Pro-Mmp-Phe-NH2
H-Tyr-Pro-3,5Dmp-Phe-NH2
H-Dmt-Pro-3,5Dmp-Phe-NH2
H-Dmt-Pro-Dmp-Phe-NH2
H-Tyr-Pro-Dmt-Phe-NH2
H-Dmt-Pro-Dmt-Phe-NH2
H-Tyr-Pro-Tmp-Phe-NH2
H-Dmt-Pro-Tmp-Phe-NH2
H-Tyr-Pro-Emp-Phe-NH2
H-Dmt-Pro-Emp-Phe-NH2
H-Tyr-Pro-Imp-Phe-NH2
H-Dmt-Pro-Imp-Phe-NH2
-38.63
-4.18
-36.0
-1.51
-2.71
-34.22
0.88
0.26
0.23
0.24
0.22
0.42
0.34
0.45
0.23
0.21
0.24
0.24
0.21
0.25
0.76
0.76
0.76
0.78
0.72
0.75
0.71
0.77
0.80
0.79
0.80
0.76
0.77
586.7
614.8
600.7
628.8
628.8
616.7
644.8
614.8
642.8
614.8
642.8
628.8
656.8
586.8
615.0
600.7
628.9
628.8
616.8
644.8
615.0
642.7
614.8
642.9
628.9
656.7
15.27
16.08
16.44
17.33
16.69
13.67
14.47
16.63
17.35
16.39
17.19
17.26
18.09
27.53
29.50
30.57
32.59
30.25
23.26
25.29
31.10
32.59
30.25
32.70
32.81
34.51
-42.67
0.98
-36.53
-1.97
-33.25
1.75
8′
a Solvent: n-BuOH/H2O/AcOH/pyridine ) 4:1:1:2. b The column was eluted at a flow rate of 1 mL/min with a linear gradient of 90% solvent A to 10%
solvent A in 30 min. c The column was eluted at a flow rate of 1 mL/min with a linear gradient of 90% solvent A to 50% solvent A in 40 min.
Table 2. Receptor Affinities of H-Tyr/Dmt-Pro-Xaa-Phe-NH2 Endomorphin-2 Analoguesa
compd
peptide sequence
Kiµ (nM)b
n
Kiδ (nM)c
n
Kiδ/Kiµ
Kiκ (nM)d
n
Kiκ/Kiµ
1
1′
2
2′
3
3′
4
4′
5
5′
6
6′
7
7′
8
Tyr-Pro-Phe-Phe-NH2
Dmt-Pro-Phe-Phe-NH2
Tyr-Pro-Mmp-Phe-NH2
Dmt-Pro-Mmp-Phe-NH2
Tyr-Pro-3,5Dmp-Phe-NH2
Dmt-Pro-3,5Dmp-Phe-NH2
1.33 ( 0.15
0.26 ( 0.01
0.22 ( 0.05
0.18 ( 0.015
6.19 ( 0.24
0.11 ( 0.005
0.044 ( 0.003
0.069 ( 0.008
2.63 ( 0.25
0.092 ( 0.015
0.44 ( 0.04
0.18 ( 0.004
0.46 ( 0.071
0.21 ( 0.025
0.45 ( 0.02
0.32 ( 0.027
3
4
3
3
5
3
3
3
3
3
4
3
3
3
4
3
6085 ( 1215
99.2 ( 7.9
1741 ( 177
4.61 ( 0.3
3451 ( 624
11.6 ( 1.6
1440 ( 94
2.27 ( 0.44
3020 ( 161
80.8 ( 6.2
1358 ( 128
1.83 ( 0.22
491 ( 14
3
3
5
3
5
5
3
4
5
4
4
3
4
3
4
3
4575
382
7878
26
558
105
32730
33
1148
878
3093
10
1074
14
>4000
489 ( 135
ND
176 ( 64
ND
830 ( 148
>4000
94.2 ( 10.5
ND
998 ( 363
ND
>3000
e
3
3
3
3
3
3
3
3
1880
994
7480
>91000
1365
e
Tyr-Pro-Dmp-Phe-NH2
Dmt-Pro-Dmp-Phe-NH2
Tyr-Pro-Dmt-Phe-NH2
Dmt-Pro-Dmt-Phe-NH2
Tyr-Pro-Tmp-Phe-NH2
Dmt-Pro-Tmp-Phe-NH2
Tyr-Pro-Emp-Phe-NH2
Dmt-Pro-Emp-Phe-NH2
Tyr-Pro-Imp-Phe-NH2
Dmt-Pro-Imp-Phe-NH2
10850
945
172 ( 35
ND
278 ( 66
ND
3.03 ( 0.09
543 ( 42
1320
641
1221
14
8′
4.61 ( 0.3
205 ( 73
a The italicized compounds represent parental opioid peptides. b Versus [3H]DAMGO. c Versus [3H]deltorphin-II. d Versus [3H]U-69,593. n is the number
of independent repetitions conducted for each analogue using five to eight doses of peptide. ND: not determined. e These data are nearly identical to the µ-
and δ-opioid affinites and selectivities previously published for 1′ and 4 (refs 29 and 34, respectively).
displayed improved δ-opioid receptor affinity, µ-opioid selectiv-
ity remained. As published previously, the presence of Dmt1 in
opioids enhanced δ-opioid receptor affinity by orders of
magnitude;27-29,45 however, with µ-opioid selective agonists,
an N-terminal Dmt residue enhanced affinity to δ-sites to a
greater extent than found with the prototype δ-opioid antagonists
containing the Dmt-Tic pharmacophore,45 as seen by the 634-
fold increase in δ-opioid affinity in 4′ relative to 4 with the
concomitant loss of µ-selectivity by 3 orders of magnitude
(Table 2). In these Dmt derivatives, the highest µ-opioid
selectivity occurred with [Dmt1,3]EM-2 (5′) (Kiδ/Kiµ ) 878),
which was less (37-fold) than the most selective [Xaa3]EM-2
compound (4) but nearly 3-fold greater than 1′ (Table 2). One
analogue of considerable interest is Tmp3 (6′); with a 44-fold
greater interaction than Dmt3 (5′) toward δ-opioid receptors,
the data suggest that the hydrogen donor capability of the
hydroxyl group of Dmt is apparently less effective in affecting
receptor interaction when situated within the peptide than the
hydrophobicity of the 4′ methyl group; i.e., the hydroxyl group
may contribute a negative influence as an internal residue
supporting the original study on the endomorphins.1
Functional Bioactivity. The functional bioactivities (Table
3) of the [Xaa3]EM-2 analogues exhibited considerable variation
in µ-agonism relative to EM-2 (1); analogues 2 and 6-8 were
2- to 5-fold more active, while 3 and 5 were inactive. The
increase in hydrophobicity and positioning of the methyl groups
on Phe3 changed µ-agonism relative to EM-2 (1); i.e., while
alkylation at the 3′ and 5′ positions of Phe3 inactivated the
molecule (3), substitutions at positions 2′ and 6′ were very well
tolerated (4) producing dual µ-agonism/δ-agonism.34 Moreover,
trimethylation (6) yielded a highly selective µ-agonist.
The [Dmt1,Xaa3]EM-2 analogues (Table 3) generally doubled
µ-opioid bioactivities (2′, 4′), remained essentially unchanged
(5′, 6′, 7′, 8′), or lost bioactivity (3′) relative to 1′.29 Compound
3′ was an anomaly; the biopotency of this analogue correlated
with µ-opioid receptor affinity, suggesting that the methyl groups
at positions 3′ and 5′ (3, 3′) presented an unfavorable conforma-
tion for activation of the µ-opioid receptor, whereas the dimethyl
alkylation at positions 2′ and 6′ in Phe3 (4′ and 434) was
permitted. This was equally true for the trimethyl- (6′),
ethylmethyl- (7′), and isopropylmethyl-Phe3 (8′) derivatives.
This observation was repeated in the [Xaa3]EM-2 series (6-
8), reflecting a change that presumably enabled the ligands to
adopt a more compatible ligand-receptor conformation to
trigger a biological response. Substitution of Phe3 by Dmt3 only
yielded a bioactive compound in the presence of Dmt1 (compare
5 and 5′). Moreover, replacement of the 4′ OH of Dmt3 (5′) by
a methyl group to yield Tmp3 (6′) displayed slightly enhanced
µ-agonism and significantly improved δ-antagonism. Tmp3 (6′)
As seen in Table 2, κ-opioid receptor affinities for the Dmt
derivatives (1′-8′) and parental compounds (1, 4) were quite
weak relative to the interaction of these peptides to both µ- and
δ-opioid receptors, although the control U-69593 demonstrated
good affinity (1.04 ( 0.45 nM). Owing to the lack of high
κ-opioid affinity, none of the analogues were investigated for
their κ-functional bioactivity.

2756 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Li et al.
Table 3. Functional Bioactivities of H-Tyr/Dmt-Pro-Xaa-Phe-NH2 Endomorphin-2 Analoguesa
MVD assayb
IC50 (nM)c
GPI assayb
IC50 (nM)
d
compd
peptide
pA2
1
1′
2
2′
3
3′
4
4′
5
5′
6
6′
7
7′
8
Tyr-Pro-Phe-Phe-NH2
Dmt-Pro-Phe-Phe-NH2
Tyr-Pro-Mmp-Phe-NH2
Dmt-Pro-Mmp-Phe-NH2
Tyr-Pro-3,5Dmp-Phe-NH2
Dmt-Pro-3,5Dmp-Phe-NH2
6.9 ( 0.9
344 ( 93
e
0.26 ( 0.038
1.33 ( 0.27
0.16 ( 0.041
389 ( 166
0.59 ( 0.18
15.7 ( 5.1
>10000 (0.00%)
>10000 (8.0%)
>10000 (7.1%)
1.39 ( 0.17
>10000 (15.6%)
978 ( 113
>10000 (<1%)
>10000 (36.2%)
>10000 (22.2%)
277 ( 29
6.59
6.77
8.15
7.06
9.05
14.4 ( 5.4
f
Tyr-Pro-Dmp-Phe-NH2
0.38 ( 0.104
0.12 ( 0.020
541 ( 178
Dmt-Pro-Dmp-Phe-NH2
Tyr-Pro-Dmt-Phe-NH2
Dmt-Pro-Dmt-Phe-NH2
Tyr-Pro-Tmp-Phe-NH2
Dmt-Pro-Tmp-Phe-NH2
Tyr-Pro-Emp-Phe-NH2
Dmt-Pro-Emp-Phe-NH2
Tyr-Pro-Imp-Phe-NH2
Dmt-Pro-Imp-Phe-NH2
1.94 ( 0.21
1.90 ( 0.49
0.21 ( 0.077
2.35 ( 0.53
0.17 ( 0.072
2.74 ( 1.20
0.20 ( 0.050
0.51 ( 0.24
>10000 (17.7%)
5.56 ( 2.47
8′
a The italicized compounds represent parental opioid peptides. b The data are the mean of over five independent repetitions used in different isolated
tissue preparations. IC50 is the concentration required for 50% inhibition of the electrically induced contraction in muscle derived from a dose-response
curve. c Values in parentheses indicate maximal inhibition of the tissue contraction at a concentration of 10 000 nM. d Negative log of the molar concentration
required to double the deltorphin II concentration to achieve the original response. e Data cited from ref 29. f Data cited from ref 34.
became a mixed µ-agonist/δ-antagonist with δ-antagonism being
2 orders of magnitude greater than that for Dmt3 (5′).
Conclusion
This study demonstrated that alkylated Phe3 residues incor-
porated into the third position of EM-2 act as effective amino
acid surrogates, in particular the [Dmt1,Xaa3]EM-2 analogues,
which exhibited significantly higher affinity for both µ- and
δ-opioid receptors and yet retained µ-opioid bioactivity. The
lack of interaction of the analogues with κ-opioid receptors
revealed that these compounds maintained the selectivity of
endomorphin for µ-opioid receptors. The most intriguing
observation was the unique appearance of δ-antagonism in the
endomorphins, which normally lack interaction with δ-opioid
receptors and have high µ-opioid receptor selectivity and
prominent µ-opioid agonism.1 These bifunctional molecules are
targets in the design of new antinociceptive opioids that could
potentially alleviate acute or chronic pain with low physical
dependence and tolerance,41 especially due to the acquisition
of δ-opioid antagonism. Pharmacological and biochemical
evidence further reveal that µ- and δ-opioid receptors readily
form heterodimers48 and that the δ-opioid receptors modulate
the function of µ-opioid receptors in this complex.49 Thus, our
opioid derivatives displaying dual µ-agonism/δ-agonism (7′) and
mixed µ-agonism/δ-antagonism (6′, 4′) characteristics that are
shown to exhibit a low tendency to develop dependence and
tolerance50 and verified using δ-receptor knockout mice51 and
a δ-receptor antagonist52 might be applicable for antinoci-
ception with a low degree of dependence and tolerance.
Furthermore, structural clarification of the subtle differences
among these interrelated opioid ligands14,23 should provide
insight on the molecular mechanism of action between agonists
and antagonists.53
Recently, Dmt-Pro-Trp-Phe-NH2 ([Dmt1]EM-1) was reported
to be a mixed µ-opioid agonist/δ-antagonist (GPI IC50 ) 0.272
nM; MVD pA2 ) 8.60).36b On the other hand, [Dmt1]EM-2 (1′)
is a dual µ-opioid agonist/δ-opioid agonist.29 The only difference
between [Dmt1]EM-1 and 1′ is Trp3 or Phe3. This indicates that
the bulky side chain of Trp in combination with Dmt1 causes
either a steric hindrance in the conformation of the peptide or
a shift in hydrophobicity to potentiate the induction of δ-opioid
receptor antagonism.
In terms of the δ-functional bioactivities, the majority of the
ligands failed to elicit substantial δ-agonism except 7′ and to a
lesser extent both 8′ (10-fold loss) and 2 (30-fold decrease), in
which the bioactivity profile of 7′ resembled the bioactivity
profiles of the reference compounds (1′ (cf. ref 24) and 434).
Nonetheless, the [Dmt1,Xaa3]EM-2 compounds established
bioactive profiles distinctly different from the profiles of the
[Xaa3]EM-2 analogues: (i) the appearance of δ-antagonism
(2′, 3′, 4′, 5′, 6′) ranging from pA2 ) 6.59 (2′) to a potent pA2
) 9.05 (6′) and (ii) the appearance of δ-agonism with 7′ and
8′. The δ-opioid antagonism of [Dmt1,Tmp3]EM-2 (6′) is about
8-fold more potent than the prototype δ-antagonist H-Dmt-Tic-
OH (pA2 ) 8.48).45 Furthermore, [Xaa3]EM-2 analogues
remained µ-agonists except for the inactive ligands 3 and 5 and
the dual µ-agonism/δ-agonism of 2. An N-terminus Dmt in 4′
eliminated δ-agonism of 4 with the appearance of δ-antagonism
(4′). In fact, the loss of δ-agonism in the [Dmt1,Xaa3]EM-2
analogues was associated with the δ-antagonism (6′ > 4′ > 5′,
3′, 2′).
Substitution of Phe3 in EM-2 by other phenylalanine ana-
logues, such as Hfe (homophenylalanine), Phg (phenylglycine),
D-Phg,46 N-methyl-Phe,22 D-2-Nal [D-3-(2-naphthyl)-alanine],23
L- and D-R-aminoxy-Phe,21 â-methyl-Phe,18 4′-NH2-Phe, and 4′-
NCS-Phe,47 failed to improve the affinity toward the µ-opioid
receptor or provide unique functional bioactivities in the
resultant ligands. Our results clearly indicated that modification
of Phe3 with alkyl groups not only enhnaced receptor affinity
but also transformed the functional bioactivity to elicit phar-
macological properties as bifunctional opioid ligands (dual
µ-agonists/δ-agonists and mixed µ-agonists/δ-antagonists).
Experimental Section
Melting points were determined on a Yanagimoto micromelting
point apparatus and are uncorrected. TLC was performed on
precoated plates of silica gel F254 (Merk, Darmstadt, Germany).
Rf1, Rf2, Rf3, and Rf4 values refer to CHCl3/H2O/AcOH (acetic acid)
(90:8:2), AcOEt (ethyl acetate), AcOEt/MeOH (methanol) (20:1),
and n-BuOH (butanol)/H2O/AcOH/pyridine (4:1:1:2), respectively.
Optical rotations were measured with a DIP-1000 automatic
polarimeter (Japan Spectroscopic Co.). A Waters Delta 600 with
COSMOSIL C18 column (4.6 mm × 250 mm) was used for
analytic RP-HPLC. The solvents for analytical HPLC were the

Bifunctional Endomorphin-2 Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2757
following: solvent A, 0.05% TFA (trifluoroacetic acid) in water;
solvent B, 0.05% TFA in CH3CN. Elution was at a flow rate of 1
mL/min with a linear gradient of 90% A to 10% A in 30 min and
with a linear gradient of 90% A to 50% A in 40 min. The retention
time was reported as tR (min). 1H and 13C NMR spectra were
measured on a Bruker DPX-400 spectrometer at 25 °C. Chemical
shift values are expressed as ppm downfield from tetramethylsilane.
General Procedure for Boc Protected Phe Analogues Boc-
Xaa-OH (Xaa ) Mmp, 3,5Dmp, Dmp, Dmt, Tmp, Emp, Imp).
(Boc)2O (di-tert-butyl dicarbonate, l5.5 mmol) in dioxane (15 mL)
was added to a solution of amino acid (5.0 mmol) in H2O (15 mL)
containing TEA (triethylamine, 15.0 mmol). The reaction mixture
was stirred at 0 °C for 15 min and then at room temperature for 2
h, and the solvent was removed under vacuum. The residue was
adjusted to pH 2-3 by using cooled 10% citric acid; the precipitated
product was extracted with AcOEt. The combined extract was
washed with saturated NaCl solution, dried over Na2SO4, filtered,
and concentrated. The residue was diluted with hexane, and the
solid was collected by filtration and dried under vacuum. Elemental
analysis results of Boc-Xaa-OH are summarized in Supporting
Information (Table 1).
by filtration, and dried under vacuum. Elemental analysis results
of Boc-Xaa-Phe-NH2 are summarized in Supporting Information
(Table 2).
Nr-tert-Butyloxycarbonyl-2′-methyl-L-phenylalanylphenyla-
lanylamide. Yield 470 mg (97.0%); mp 164-166 °C; Rf2 ) 0.73;
[R]2D5 -21.04° (c 0.34, MeOH). 1H NMR (400.1 MHz, DMSO-d6)
δ: 7.89 (d, 0.15H, J ) 6.80 Hz, Phe NH), 7.79 (d, 0.84H, J )
8.25 Hz, Phe NH), 7.45, 7.36 (2s, 1.0H, CONH2), 7.30-6.96 (m,
10.9H, CONH2, Mmp NH, Phe Ar-H, Mmp Ar-H), 6.52 (d, 0.1H,
J ) 6.8 Hz, Mmp NH), 4.60-4.43 (m, 1.0H, Phe RH), 4.11 (dt,
1.0H, J ) 4.45, 9.80 Hz, Mmp RH), 3.03 (dd, 1.0H, J ) 4.87,
13.72 Hz, Phe âH), 2.90-2.70 (m, 2H, Phe âH, Mmp âH), 2.65
(dd, 1.0H, J ) 10.15, 14.12 Hz, Mmp âH), 2.24 (s, 3H, Mmp CH3),
1.29, 1.14 (2s, 9.0H, Boc). 13C NMR (100.6 MHz, DMSO-d6) δ:
172.45, 171.11, 155.01, 137.58, 136.01 (5q), 129.73, 129.25,
127.89, 126.13, 125.39 (5t, Mmp and Phe Ar-C), 78.16 (q, Boc),
4.71 (t, Mmp RC), 53.29 (t, Phe RC), 37.67 (s, Phe âC), 34.78 (s,
Mmp âC), 28.00, 27.57 (2p, Boc), 18.90 (p, Mmp CH3).
Nr-tert-Butyloxycarbonyl-3′,5′-dimethyl-L-phenylalanylphe-
nylalanylamide. Yield 493 mg (98.6%); mp 193-195 °C; Rf2
)
0.74; [R] 2D5 -16.02° (c 0.39, MeOH). 1H NMR (400.1 MHz,
DMSO-d6) δ: 8.0-7.92 (br, 0.14H, Phe NH), 7.86 (d, 0.80H, J )
8.23 Hz, Phe NH), 7.45, 7.35 (2s, 1H, CONH2), 7.30-7.14 (m,
5H, Phe Ar-H), 7.14-6.96 (m, 1H, CONH2), 6.91-6.71 (m,
3.75H, 3,5Dmp Ar-H, 3,5Dmp NH), 6.40-6.32 (br, 0.12H, 3,5Dmp
NH), 4.36-4.39 (m, 1H, Phe RH), 4.12-3.98 (m, 1H, 3,5Dmp RH),
Nr-tert-Butyloxycarbonyl-2′-methyl-L-phenylalanine. Yield
1.25 g (89.2%); mp 118-119 °C; Rf1 ) 0.56; [R] 2D5 -15.54°
1
(c 1.03, MeOH). H NMR (400.1 MHz, CDCl3) δ: 7.20-7.05
(m, 4H), 6.63 (br, 0.38H), 4.93 (br, 0.62H), 4.60-4.37 (m, 1H),
3.33-3.20 (m, 1H), 3.07-2.80 (m, 1H), 2.36 (s, 3H), 1.40, 1.17
(2s, 9H).
3.01 (dd, 1.0H, J ) 5.04, 13.75 Hz, Phe âH), 2.84 (dd, 1.0H, J )
Nr-tert-Butyloxycarbonyl-3′,5′-dimethyl-L-phenylalanine. Yield
3,5
8.65, 13.75 Hz, Phe âH), 2.77 (dd, 1.0H, J ) 4.26, 13.68 Hz,
-
0.71 g (96.5%); mp 115-116 °C; Rf1 ) 0.56; [R] 2D5 +16.09°
Dmp âH), 2.59 (dd, 1.0H, J ) 10.09, 13.68 Hz, 3,5Dmp âH), 2.21
(s, 6H, 3,5Dmp CH3), 1.31, 1.16 (2s, 9H, Boc). 13C NMR (100.6
MHz, DMSO-d6) δ: 172.54, 171.14, 155.02, 137.77, 137.62, 136.65
(6q), 129.20, 127.91 (2t, Phe Ar-C), 127.49, 126.77 (2t, 3,5Dmp
Ar-C), 126.13 (t, Phe Ar-C), 78.04 (q, Boc), 55.88 (t, 3,5Dmp
RC), 53.36 (t, Phe RC), 37.79 (s, Phe âC), 37.16 (s, 3,5Dmp âC),
28.01 (p, Boc), 20.81 (p, 3,5Dmp CH3).
1
(c 0.67, MeOH). H NMR (400.1 MHz, CDCl3) δ: 6.89 (s, 1H),
6.79 (s, 2H), 4.89 (br, 1H), 4.52 (br, 1H), 3.12 (dd, 1H, J ) 5.34,
13.84 Hz), 3.05-2.91 (m, 1H9, 2.28 (s, 3H), 1.42 (s, 9H).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-phenylalanine. Yield
1.3 g (93.0%); mp 135-136 °C; Rf1 ) 0.40; [R] 2D5 -17.85°
1
(c 0.41, MeOH). H NMR (400.1 MHz, CDCl3) δ: 7.13-6.95
(m, 3.50H), 5.03-4.92 (br, 0.4H), 4.58-4.46 (m, 1H), 3.25-3.02
(m, 2H), 2.40, 2.37 (2s, 6H), 1.36, 1.06 (2s, 9H).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-phenylalanylphe-
nylalanylamide. Yield 491 mg (98.0%); mp 177-179 °C; Rf2
)
0.73; [R] 2D5 -15.28° (c 0.58, MeOH). 1H NMR (400.1 MHz,
DMSO-d6) δ: 7.95 (d, 0.2H, J ) 7.51 Hz, Phe NH), 7.83 (d, 0.75H,
J ) 8.48 Hz, Phe NH), 7.45 (br, 0.23H, CONH2), 7.33-7.21 (m,
4.68H, CONH2 and Ar-H), 7.21-7.13 (m, 1H, Ar-H), 7.08 (s,
1.0H, CONH2), 7.0-6.86 (m, 3.73H, Ar-H and Dmp NH), 6.54
(d, 0.14H, J ) 9.54 Hz, Dmp NH), 4.58-4.45 (m, 1.0H, Phe RH),
4.19-4.03 (m, 1.0H, Dmp RH), 3.02 (dd, 1.0H, J ) 5.05, 13.62
Hz, Phe âH), 2.88-2.55 (m, 3.0H, Phe âH and Dmp âH), 2.07 (s,
6.0H, Dmp CH3), 1.26, 1.04 (2s, 9.0H, Boc). 13C NMR (100.6 MHz,
DMSO-d6) δ: 172.48, 170.91, 154.64, 137.66, 136.61, 134.68 (6q),
129.28, 127.86, 127.78, 126.12, 125.85 (5t, Ar-C), 78.13 (q, Boc),
54.91 (t, Dmp RC), 53.35 (t, Phe RC), 37.84 (s, Phe âC), 32.30 (s,
Dmp âC), 27.97, 27.34 (2p, Boc), 19.80 (p, Dmp CH3).
Nr-tert-Butyloxycarbonyl-2′,4′,6′-trimethyl-L-phenylala-
nine. Yield 1.37 g (88.2%); mp 153-154 °C; Rf1 ) 0.56; [R] D25
1
-13.26° (c 0.94, MeOH). H NMR (400.1 MHz, CDCl3) δ: 7.03
(br, 0.60H), 6.84 (s, 2H), 4.97 (br, 0.40H), 4.53-4.40 (m, 1H),
3.23-3.13 (m, 1H), 3.05 (dd, 1H, J ) 9.95, 14.10 Hz), 2.35 (s,
6H), 2.24 (s, 3H), 1.37, 1.08 (2s, 9H).
Nr-tert-Butyloxycarbonyl-2′-ethyl-6′methyl-L-phenylala-
nine. Yield 1.32 g (85.7%); mp 117-118 °C; Rf1 ) 0.56; [R] D25
1
-14.33° (c 1.0, MeOH). H NMR (400.1 MHz, CDCl3) δ: 7.26
(br, 0.77H), 7.14-7.00 (m, 3H), 4.97 (br, 0.23H), 4.60-4.50 (m,
1H), 3.28-3.17 (m, 1H), 3.17-3.06 (m, 1H), 2.86-2.65 (m, 2H),
2.42, 2.39 (2s, 3H), 1.36 (s, 2.2H), 1.22 (t, 3H, J ) 7.51 Hz), 1.05
(s, 6.8H).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylphenylala-
nylamide. Yield 483 mg (93.2%); mp 184-186 °C; Rf2 ) 0.93;
[R] 2D5 -19.08° (c 0.33, MeOH). 1H NMR (400.1 MHz, DMSO-d6)
δ: 8.66 (s, 1H, Dmt OH), 7.91 (d, 0.20H, J ) 8.18 Hz, Phe NH),
7.79 (d, 0.78H, J ) 8.44 Hz, Phe NH), 7.47, 7.32 (2s, 1.0H,
CONH2), 7.29-7.02 (m, 6H, Phe Ar-H, CONH2), 6.86 (d, 0.74H,
J ) 9.30 Hz, Dmt NH), 6.43 (d, 0.17H, J ) 9.69 Hz, Dmt NH),
6.34 (s, 2H, Dmt Ar-H), 4.58-4.43 (m, 1.0H, Phe RH), 4.10-
3.93 (m, 1.0H, Dmt RH), 3.02 (dd, 1.0H, J ) 5.09, 13.60 Hz, Phe
âH), 2.81 (dd, 1.0H, J ) 9.01, 13.60 Hz, Phe âH), 2.65 (dd, 1.0H,
J ) 5.14, 14.40 Hz, Dmt âH), 2.55 (dd, 1.0H, J ) 9.16, 14.40 Hz,
Nr-tert-Butyloxycarbonyl-2′-isopropyl-6′-methyl-L-phenyla-
lanine. Yield 1.45 g (90.3%); mp 121-122 °C; Rf1 ) 0.56; [R]25
D
1
-12.78° (c 0.93, MeOH). H NMR (400.1 MHz, CDCl3) δ: 7.26
(br, 0.76H), 7.17-7.07 (m, 2H), 7.05-6.95 (m, 1H), 4.95 (br,
0.24H), 4.56-4.45 (m, 1H), 3.47-3.30 (m, 0.76H), 3.30-3.05 (m,
2.24H), 2.43, 2.40 (2s, 3H), 1.35 (s, 2.3H), 1.25 (d, 3H, J ) 6.73
Hz), 1.18 (d, 3H, J ) 6.42 Hz), 1.05 (s, 6H).
General Procedure for Synthesis of Boc-Xaa-Phe-NH2 (Xaa
) Mmp, 3,5Dmp, Dmp, Dmt, Tmp, Emp, Imp). The H-Phe-NH2
hydrochloride salt (0.25 g, 1.25 mmol) was dissolved in DMF (N,N-
dimethylformamide, 10 mL) containing DIPEA (diisopropylethy-
lamine, 0.50 mL, 2.87 mmol), and to this solution Boc-Xaa-OH
(1.14 mmol) and PyBop (0.65 g, 1.25 mmol) were added. The
reaction mixture was stirred at 0 °C for 10 min, then at room
temperature for 4 h. After removal of solvent, the residue was
diluted with AcOEt (80 mL). The diluted mixture was washed with
ice-cooled 10% citric acid (3 × 15 mL), 5% Na2CO3 (3 × 15 mL),
and saturated NaCl (3 × 20 mL), dried over Na2SO4, and evaporated
to dryness. The residue was purified by flash chromatography (SiO2,
AcOEt). The compound was precipitated with hexane, collected
Dmt âH), 2.13 (s, 6.0H, Dmt CH3), 1.28, 1.09 (2s, 9.0H, Boc). 13
C
NMR (100.6 MHz, DMSO-d6) δ: 172.49, 171.13, 154.90, 154.69,
137.65 (5q), 129.26, 127.83, 126.09 (3t, Phe Ar-C), 124.96 (q),
114.61 (t, Dmt Ar-C), 78.05 (q, Boc), 55.38 (t, Dmt RC), 53.30
(t, Dmt RC), 37.82 (s, Phe âC), 31.58 (s, Dmt âC), 27.99, 27.39
(2p, Boc), 19.95 (p, Dmt CH3).
Nr-tert-Butyloxycarbonyl-2′,4′,6′-trimethyl-L-phenylalanylphe-
nylalanylamide. Yield 483 mg (93.5%); mp 224-225 °C; Rf2
)
0.76; [R] 2D5 -16.38° (c 0.41, MeOH). 1H NMR (400.1 MHz,

2758 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Li et al.
DMSO-d6) δ: 7.94 (d, 0.18H, J ) 7.62 Hz, Phe NH), 7.83 (d,
0.79H, J ) 8.46 Hz, Phe NH), 7.43 (br, 0.20H, CONH2), 7.30-
7.13 (m, 5.80H, CONH2 and Tyr Ar-H), 6.92 (d, 0.77H, J ) 9.29
Hz, Tmp NH), 6.73 (s, 2H, Tmp Ar-H), 6.48 (d, 0.15H, J ) 9.46
Hz, Tmp NH), 4.60-4.40 (m, 1H, Phe RH), 4.15-3.96 (m, 1H,
Tmp RH), 3.02 (dd, 1H, J ) 5.03, 13.54 Hz, Phe âH), 2.81 (dd,
1H, J ) 9.05, 13.54 Hz, Phe âH), 2.72 (dd, 1H, J ) 5.31, 14.15
Hz, Tmp âH), 2.63 (dd, 1H, J ) 9.22, 14.10 Hz, Tmp âH), 2.18,
2.15 (2s, 9H, Tmp CH3), 1.27, 1.06 (2s, 9H, Boc). 13C NMR (100.6
MHz, DMSO-d6) δ: 172.49, 171.00, 154.67, 153.62, 137.68,
136.40, 134.50, 131.56 (8q), 129.26 (t, Phe Ar-C), 128.48 (t, Tmp
Ar-C), 127.84, 126.09 (2t, Phe Ar-C), 78.11 (q, Boc), 55.04 (t,
Tmp RC), 53.34 (t, Phe RC), 37.79 (s, Phe âC), 31.91 (s, Tmp
âC), 27.96, 27.31 (2p, Boc), 20.33, 19.71 (2p, Tmp CH3).
MHz, DMSO-d6) δ: 9.21 (s, 0.19H, Tyr cis-OH), 9,15 (s, 0.76H,
Tyr trans-OH), 8.46 (d, 0.16H, J ) 8.39 Hz, Phe cis-NH), 8.01 (d,
0.76H, J ) 7.57 Hz, Phe trans-NH), 7.86 (d, 0.78H, J ) 8.20 Hz,
Mmp trans-NH), 7.76 (d, 0.18H, J ) 8.12 Hz, Mmp cis-NH), 7.28-
7.14 (m, 5.0H, Phe Ar-H), 7.14-6.93 (m, 8.9H, Tyr Ar-H, Mmp
Ar-H, CONH2, Tyr NH), 6.68-6.57 (m, 2.1H, Tyr Ar-H, Tyr
NH), 4.48-4.34 (m, 2.0H, Phe RH, Tyr RH), 4.34-4.10 (m, 1.77H,
Pro trans-RH, Mmp RH), 3.82 (d, 0.17H, J ) 7.67 Hz, Pro cis-
RH), 3.64-3.53 (m, 0.65H, Pro trans-δH), 3.53-3.37 (m, 0.96H,
Pro trans-δH), 3.24-2.54 (m, 6.39H, Pro cis-δH, Phe âH, Tyr âH,
Mmp âH), 2.25 (s, 3.0H, Mmp CH3), 1.98-1.87 (m, 0.70H, Pro
trans-âH), 1.87-1.66 (m, 2.66H, Pro cis-âH, Pro trans-âH, Pro
trans-γH), 1.48-1.38 (m, 0.27H, Pro cis-γH), 1.38-1.14 (m, 9.2H,
Boc, Pro cis-âH), 1.0-0.86 (m, 0.15H, Pro cis-γH). 13C NMR
(100.6 MHz, DMSO-d6) δ: 172.39, 171.56, 170.86, 170.65, 170.35,
170.25, 155.96, 155.67, 155.36, 155.24, 137.84, 137.65, 136.04,
135.79 (14q), 130.30, 130.09, 129.77, 129.05, 128.94, 127.94,
126.44, 126.20, 126.12, 125.53, 114.89, 114.77 (12t, Ar-C), 78.32,
77.87 (2q, Boc), 59.52 (t, Pro trans-RC), 59.39 (t, Pro cis-RC), 54.10
(t, Tyr trans-RC), 53.88 (t, Tyr cis-RC), 53.71 (t, Phe RC), 53.49
(t, Mmp cis-RC), 53.16 (t, Mmp trans-RC), 46.62 (s, Pro trans-
δC), 46.04 (s, Pro cis-δC), 37.60 (s, Phe cis-âC), 37.17 (s, Phe
trans-âC), 36.85 (s, Tyr cis-âC), 35.45 (s, Tyr trans-âC), 34.23 (s,
Mmp trans-âC), 33.77 (s, Mmp cis-âC), 29.99 (s, Pro cis-âC), 28.54
(s, Pro trans-âC), 28.06 (p, trans-Boc), 27.75 (p, cis-Boc), 24.39
(s, Pro trans-γC), 21.01 (s, Pro cis-γC), 19.01 (p, Mmp trans-CH3),
18.91 (p, Mmp cis-CH3).
Nr-tert-Butyloxycarbonyl-2′-ethyl-6′-methyl-L-phenylalanylphe-
nylalanylamide. Yield 481 mg (93.2%); mp 208-210 °C; Rf2
)
0.78; [R] 2D5 -15.16° (c 0.35, MeOH). 1H NMR (400.1 MHz,
DMSO-d6) δ: 7.93 (d, 0.16H, J ) 7.63 Hz, Phe NH), 7.81 (d,
0.77H, J ) 8.46 Hz, Phe NH), 7.50-6.88 (m, 10.87H, CONH2,
Emp NH, Phe Ar-H, Emp Ar-H), 6.55 (d, 0.16H, J ) 9.29 Hz,
Emp NH), 4.57-4.40 (m, 1H, Phe RH), 4.16-3.97 (m, 1H, Emp
RH), 3.03 (dd, 1H, J ) 5.02, 13.62 Hz, Phe âH), 2.85-2.53 (m,
5H, Phe âH, Emp âH, Emp CH2CH3), 2.24 (s, 3H, Emp Ar-CH3),
1.26 (s, 7.38H, Boc), 1.10 (t, 3H, J ) 7.42 Hz, Emp CH2CH3),
1.04 (s, 1.62H, Boc). 13C NMR (100.6 MHz, DMSO-d6) δ: 172.46,
170.89, 154.65, 142.64, 137.62, 136.65, 133.90 (7q), 129.29, 127.83
(2t, Phe Ar-C), 127.67 (t, Emp Ar-C), 126.10 (t, Phe Ar-C and
Emp Ar-C), 125.94 (t, Emp Ar-C), 78.12 (q, Boc), 55.56 (t, Emp
RC), 53.28 (t, Phe RC), 37.88 (s, Phe âC), 31.44 (s, Emp âC),
27.95, 27.35 (2p, Boc), 25.03 (s, Emp, CH2CH3), 19.90 (p, Emp
Ar-CH3) 15.03 (p, Emp CH2CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-2′-
methyl-L-phenylalanylphenylalanylamide. Yield 266 mg (84.5%);
mp 222-228 °C; Rf3 ) 0.71; [R] 25 -45.81° (c 0.36, MeOH). H
1
NMR (400.1 MHz, DMSO-d6) δ:D 9.05 (s, 0.45H, Dmt cis-OH),
8.86 (s, 0.43H, Dmt trans-OH), 8.33 (d, 0.44H, J ) 8.22 Hz, Phe
NH), 8.10 (d, 0.15H, J ) 7.22 Hz, Phe NH), 8.03 (d, 0.33H, J )
7.52 Hz, Phe NH), 7.83 (d, 0.48H, J ) 8.14 Hz, Mmp NH), 7.65
(d, 0.43H, J ) 8.06 Hz, Mmp NH), 7.29-7.14 (m, 5.5H, Phe Ar-
H, CONH2), 7.14-6.95 (m, 6.0H, Mmp Ar-H, CONH2, Dmt NH),
6.75 (d, 0.33H, J ) 8.54 Hz, Dmt NH), 6.43-6.28 (m, 2.1H, Dmt
Ar-H, Dmt NH), 4.48-4.23 (m, 3.0H, Pro trans-RH, Mmp RH,
Phe RH, Dmt trans-RH), 4.17-4.08 (m, 0.46H, Dmt cis-RH), 3.52-
3.40 (m, 0.49H, Pro trans-δH), 3.20-2.65 (m, 8.0H, Pro cis-RH,
Pro cis-δH, Pro trans-δH, Phe âH, Mmp âH, Dmt âH), 2.25 (s,
3.0H, Mmp CH3), 2.16, 2.09 (2s, 6.0H, Dmt CH3), 1.96-1.62 (m,
2.53H, Pro cis-âH, Pro trans âH, Pro trans-γH), 1.45-1.07 (m,
9.50H, Boc, Pro cis-γH), 1.05-0.91 (m, 0.51H, Pro cis-âH), 0.86-
0.70 (m, 0.47H, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6)
δ: 172.36, 172.19, 171.42, 171.06, 170.63, 170.26, 170.14, 155.51,
155.41, 154.92, 154.65, 138.08, 137.85, 137.81, 137.53, 136.05,
136.01, 135.85 (18q), 129.79, 129.75, 129.07, 129.01, 128.89,
127.94, 126.13, 125.53, 125.50 (9t, Ar-C), 124.24, 122.95 (2q),
114.83, 114.62 (3t, Ar-C), 78.54, 77.88 (2q, Boc), 59.78 (t, Pro
trans-RC), 59.14 (t, Pro cis-RC), 53.74 (t, Mmp cis-RC), 53.45 (t,
Phe RC), 53.18 (t, Mmp trans-RC), 51.47 (t, Dmt RC), 46.55 (s,
Pro trans-δC), 46.18 (s, Pro cis-δC), 37.89 (s, Phe cis-âC), 37.20
(s, Phe trans-âC), 34.12 (s, Mmp cis-âC), 33.48 (s, Mmp trans-
âC), 29.93 (s, Pro cis-âC), 28.61 (s, Pro trans-âC), 28.18, 27.98,
27.45 (3p, Boc), 24.34 (s, Pro trans-γC), 20.99 (s, Pro cis-γC),
20.11 (p, Dmt trans-CH3), 19.43 (p, Dmt cis-CH3), 18.99, 18.94
(2p, Mmp CH3).
Nr-tert-Butyloxycarbonyl-2′-isopropyl-6′-methyl-L-phenyla-
lanylphenylalanylamide. Yield 495 mg (93.0%); mp 109-110 °C;
Rf2 ) 0.72; [R] 2D5 -22.11° (c 0.33, MeOH). 1H NMR (400.1
MHz, DMSO-d6) δ: 7.93 (d, 0.17H, J ) 7.29 Hz, Phe NH), 7.83
(d, 0.76H, J ) 8.56 Hz, Phe NH), 7.51, 7.43 (2s, 0.87H, CONH2),
7.30-6.86 (m, 10H, CONH2, Ar-H, Imp NH), 6.60 (d, 0.17H, J
) 9.47 Hz, Imp NH), 4.60-4.48 (m, 1H, Phe RH), 4.10-3.97 (m,
1H, Imp RH), 3.22 (hept, 1H, J ) 6.68 Hz, CH(CH3)2), 3.04 (dd,
1H, J ) 4.98, 13.52 Hz, Phe âH), 2.83 (dd, 1H, J ) 9.14, 13.52
Hz, Phe âH), 2.78-2.58 (m, 2H, Imp âH), 2.25 (s, 3H, Imp Ar-
CH3), 1.23 (s, 7H, Boc), 1.17 (d, 3H, J ) 6.68 Hz, CH(CH3)2),
1.10 (d, 3H, J ) 6.68 Hz, CH(CH3)2), 1.02 (s, 2H, Boc). 13C NMR
(100.6 MHz, DMSO-d6) δ: 172.53, 170.85, 154.60, 147.38, 137.59,
136.48, 133.01 (7q), 129.34, 127.81, 127.39, 126.22, 126.11, 122.74
(6t, Ar-C), 78.10 (q, Boc), 55.74 (t, Imp RC), 53.19 (t, Phe RC),
38.03 (s, Phe âC), 30.97 (s, Imp âC), 27.92 (t, CH(CH3)2; p, Boc),
27.36 (p, Boc), 24.37, 23.73 (2p, CH(CH3)2), 20.13 (p, Imp Ar-
CH3).
General Procedure for Synthesis of Boc-Tyr/Dmt-Pro-Xaa-
Phe-NH2 (Xaa ) Mmp, 3,5Dmp, Dmp, Dmt, Tmp, Emp, Imp).
Boc-Xaa-Phe-NH2 (0.44 mmol) was treated with 7.7 M HCl/dioxane
(1.15 mL, 8.8 mmol) to remove the Boc group at room temperature
for 30 min. The product was precipitated with ether, filtered, and
dried under vacuum. The resulting hydrochloride salt was dissolved
in DMF (10 mL) containing DIPEA (184 µL, 1.06 mmol), and to
this solution Boc-Xaa-Pro-OH (0.44 mmol) and PyBop (252 mg,
0.49 mmol) were added. The reaction mixture was stirred at 0 °C
for 10 min, then at room temperature for 4 h. After removal of
solvent, the residue was diluted with AcOEt. The dilution was
washed with ice-cooled 10% citric acid (3 × 15 mL), 5% Na2CO3
(3 × 15 mL), and saturated NaCl (3 × 20 mL), dried over Na2-
SO4, and evaporated to dryness. The residue was purified by flash
chromatography (SiO2, AcOEt/MeOH ) 10:1). The compound was
precipitated with hexane, filtered, and dried under vacuum.
Elemental analysis results of Boc-Tyr/Dmt-Pro-Xaa-Phe-NH2 are
summarized in Supporting Information (Table 3).
Nr-tert-Butyloxycarbonyltyrosylprolyl-3′,5′-dimethyl-L-phe-
nylalanylphenylalanylamide. Yield 282 mg (91.4%); mp 126-
128 °C; Rf3 ) 0.67; [R] 2D5 -44.94° (c 0.48, MeOH). H NMR
1
(400.1 MHz, DMSO-d6) δ: 9.21 (s, 0.19H, Tyr cis-OH), 9.13 (s,
0.80H, Tyr trans-OH), 8.28 (d, 0.17H, J ) 7.94 Hz, Phe cis-NH),
7.94 (d, 0.81H, J ) 8.23 Hz, Phe trans-NH), 7.82-7.72 (m, 0.96H,
3,5Dmp NH), 7.30-6.88 (m, 9.90H, Phe Ar-H, Tyr Ar-H, Tyr
NH, CONH2), 6.77 (s, 3.0H, 3,5Dmp Ar-H), 6.71-6.54 (m, 2.1H,
Tyr Ar-H, Dmt NH), 4.47-4.07 (m, 3.83H, Pro trans-RH, 3,5Dmp
RH, Phe RH, Tyr RH), 3.76 (d, 0.17H, J ) 7.72 Hz, Pro cis-RH),
3.64-3.53 (m, 0.78H, Pro trans-δH), 3.53-3.47 (m, 1.0H, Pro
trans-δH), 3.27-3.16 (m, 0.22H, Pro cis-δH), 3.10-2.52 (m, 6.0H,
Nr-tert-Butyloxycarbonyltyrosylprolyl-2′-methyl-L-phenyla-
lanylphenylalanylamide. Yield 289 mg (95.6%); mp 138-140 °C;
Rf3 ) 0.64; [R] 2D5 -50.30° (c 0.64, MeOH). 1H NMR (400.1

Bifunctional Endomorphin-2 Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2759
Phe âH, 3,5Dmp âH, Tyr âH), 2.20, 2.18 (2s, 6.0H, 3,5Dmp CH3),
2.0-1.65 (m, 3.47H, Pro cis-âH, Pro trans-âH, Pro trans-γH),
1.53-1.42 (m, 0.33H, Pro cis-γH), 1.35, 1.29, 1.23 (3s, 9.0H, Boc),
1.11-0.94 (m, 0.1H, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-
d6) δ: 172.51, 171.31, 170.89, 170.56, 170.39, 170.27, 170.19,
155.97, 155.68, 155.34, 155.25, 137.81, 137.61, 137.25, 136.76
(15q), 130.25, 130.08, 129.03, 127.95, 127.91, 127.60, 126.80,
126.64, 126.13, 114.92, 114.78 (11t, Ar-C), 78.28, 77.85 (2q, Boc),
59.56 (t, Pro RC), 54.79, 54.10, 54.06, 53.68, 53.42 (5t, 3,5Dmp
RC, Phe RC, Tyr RC), 46.60 (s, Pro δC), 37.33 (s, Phe âC), 36.81
(s, 3,5Dmp âC), 35.47 (s, Tyr âC), 28.56 (s, Pro âC), 28.07 (p,
trans-Boc), 27.75 (p, cis-Boc), 24.28 (s, Pro γC), 20.79 (p, 3,5Dmp
trans-CH3), 20.75 (p, 3,5Dmp cis-CH3).
âC), 30.93 (s, Dmt trans-âC), 30.65 (s, Dmt cis-âC), 29.66 (s, Pro
cis-âC), 28.66 (s, Pro trans-âC), 28.17, 28.00, 27.44 (3p, Boc),
24.35 (s, Pro trans-γC), 20.90 (s, Pro cis-γC), 20.11, 19.84, 19.53
(3p, Dmt CH3, Dmp CH3).
Nr-tert-Butyloxycarbonyltyrosylprolyl-2′,6′-dimethyl-L-ty-
rosylphenylalanylamide. Yield 307 mg (97.2%); mp 154-156 °C;
Rf3 ) 0.60; [R] 2D5 -43.85° (c 0.33, MeOH). 1H NMR (400.1
MHz, DMSO-d6) δ: 9.21, 9.15 (2s, 0.96H, Tyr OH), 8.89, 8.84
(2s, 0.95H, Dmt OH), 8.44 (d, 0.21H, J ) 8.88 Hz, Dmt cis-NH),
7.91 (d, 0.76H, J ) 7.83 Hz, Dmt trans-NH), 7.79 (d, 0.77H, J )
8.10 Hz, Phe trans-NH), 7.69 (d, 0.22H, J ) 8.07 Hz, Phe cis-
NH), 7.57-7.12 (m, 2.15H, Tyr Ar-H, Tyr NH), 7.10-6.83 (m,
4.84H, Tyr Ar-H, Tyr NH, CONH2), 6.79-6.53 (m, 2.15H, Tyr
Ar-H, Tyr NH), 6.35-6.33 (m, 2.0H, Dmt Ar-H), 4.52-4.12 (m,
3.79H, Pro trans-RH, Tyr RH, Phe RH, Dmt RH), 3.88 (d, 0.21H,
J ) 7.66 Hz, Pro cis-RH), 3.66-3.55 (m, 0.65H, Pro trans-δ),
3.55-3.38 (m, 0.85H, Pro trans-δH), 3.24-3.10 (m, 0.50H, Pro
cis-δH), 3.10-2.94 (m, 1.24H, Phe trans-âH, Dmt trans-âH), 2.94-
2.54 (m, 4.73H, Phe trans-âH, Phe cis-âH, Tyr âH, Dmt trans-
âH), 2.14 (s, 6.0H, Dmt CH3), 2.01-1.88 (m, 0.76H, Pro trans-
âH), 1.88-1.77 (m, 1.55H, Pro trans-âH), 1.77-1.64 (m, 0.97H,
Pro trans-âH, Pro cis-âH), 1.50-1.40 (m, 0.29H, Pro cis-γH), 1.31,
1.24 (2s, 9.0H, Boc), 1.20-1.06 (m, 0.37H, Pro cis-âH), 0.98-
1.83 (m, 0.16H, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6)
δ: 172.34, 172.26, 171.13, 170.93, 170.79, 170.42, 170.18, 155.97,
155.68, 155.46, 155.24, 154.99, 154.90, 137.85, 137.66 (15q),
130.39, 130.11, 129.02, 128.97, 127.91, 126.36, 126.07 (7t, Ar-
C), 124.82, 124.72 (2q), 114.86, 114.77, 114.69 (3t, Ar-C), 78.41,
77.87 (2q, Boc), 59.45 (t, Pro RC), 54.12 (t, Tyr RC), 53.81 (t,
Phe RC), 53.54 (t, Dmt trans-RC), 52.62 (t, Dmt cis-RC), 46.62 (s,
Pro trans-δC), 46.20 (s, Pro cis-δC), 37.15 (s, Phe trans-âC), 36.82
(s, Phe cis-âC), 35.56 (s, Tyr âC), 31.24 (s, Dmt trans-âC), 30.24
(s, Dmt cis-âC), 29.79 (s, Pro cis-âC), 28.58 (s, Pro trans-âC),
28.08 (p, trans-Boc), 27.77 (p, cis-Boc), 24.42 (s, Pro trans-γC),
21.05 (s, Pro cis-γC), 20.00 (p, Dmt, trans-CH3), 19.96 (p, Dmt
cis-CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-2′,6′-
dimethyl-L-tyrosylphenylalanylamide. Yield 257 mg (80.3%); mp
160-162 °C; Rf3 ) 0.63; [R] 2D5 -34.23° (c 0.53, MeOH). 1H
NMR (400.1 MHz, DMSO-d6) δ: 9.06, 8.87 (2br, 2H, Dmt OH),
8.24 (d, 0.48H, J ) 8.25 Hz, Dmt3 NH), 8.03-7.87 (m, 0.52H,
Dmt3 NH), 7.82-7.70 (m, 0.49H, Phe NH), 7.53 (d, 0.51H, J )
7.66 Hz, Phe NH), 7.34-6.81 (m, 7.7H, Phe Ar-H, CONH2, Dmt1
NH), 6.73 (d, 0.30H, J ) 8.13 Hz, Dmt1 NH), 6.48-6.30 (m, 4.0H,
Dmt Ar-H), 4.53-4.03 (m, 3.40H, Pro trans-RH, Dmt3 RH, Phe
RH, Dmt1 RH), 3.56-3.43 (m, 0.40H, Pro trans-δH), 3.28-3.17
(m, 0.60H, Pro cis-RH), 3.17-2.62 (m, 7.60H, Pro trans-δH, Pro
cis-δH, Phe âH, Dmt3 âH, Dmt1 âH), 2.30-1.98 (m, 12.0H, Dmt
CH3), 1.95-1.83 (m, 0.40H, Pro trans-âH), 1.83-1.57 (m, 2.0H,
Pro cis-âH, Pro trans-γH), 1.47-1.04 (m, 9.65H, Boc, Pro cis-
γH), 1.04-0.88 (m, 0.51H, Pro cis-âH), 0.84-0.65 (m, 0.45H, Pro
cis-γH). 13C NMR (100.6 MHz, DMSO-d6) δ: 172.26, 172.07,
171.25, 170.96, 170.91, 170.58, 170.29, 170.00, 155.64, 155.44,
154.98, 154.95, 154.65, 138.12, 137.87, 137.81, 137.53 (19q),
129.01, 128.96, 127.95, 127.90, 126.12, 126.05 (6t, Phe Ar-C),
124.80, 122.93 (2q), 114.84, 114.66 (2t, Dmt Ar-C), 78.63, 77.89
(2q, Boc), 59.70 (t, Pro trans-RC), 59.26 (t, Pro cis-RC), 53.84 (t,
Dmt3 cis-RC), 53.56 (t, Dmt3 trans-RC), 53.48 (t, Phe cis-RC), 53.14
(t, Phe trans-RC), 51.50 (t, Dmt1 cis-RC), 51.41 (t, Dmt1 trans-
RC), 46.48 (s, Pro trans-δC), 46.39 (t, Pro cis-δC), 38.01 (s, Phe
cis-âC), 37.17 (s, Phe trans-âC), 31.31 (s, Dmt3 trans-âC), 31.19
(s, Dmt3 cis-âC), 30.95 (s, Dmt1 trans-âC), 29.98 (s, Dmt1 cis-
âC), 29.80 (s, Pro cis-âC), 28.65 (s, Pro trans-âC), 28.19, 27.99,
27.47 (3p, Boc), 24.35 (s, Pro trans-γC), 20.95 (s, Pro cis-γC),
20.11, 19.99, 19.51 (3p, Dmt CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-3′,5′-
dimethyl-L-phenylalanylphenylalanylamide. Yield 281 mg (87.6%);
mp 208-210 °C; Rf3 ) 0.73; [R] 25 -35.02° (c 0.31, MeOH). H
1
NMR (400.1 MHz, DMSO-d6) δ:D9.06 (br, 0.47H, Dmt cis-OH),
8.89 (br, 0.39H, Dmt trans-OH), 8.17 (d, 0.47H, J ) 7.83 Hz, 3,5
-
Dmp cis-NH), 7.93-7.83 (m, 0.67H, 3,5Dmp trans-NH, Phe trans-
NH), 7.74 (d, 0.81H, J ) 7.98 Hz, Phe cis-NH), 7.30-6.95 (m,
3,5
7.68 H, Phe Ar-H, CONH2, Dmt NH), 6.85-6.72 (m, 3.0H,
-
Dmp Ar-H), 6.69 (d, 0.34H, J ) 8.49 Hz, Dmt NH), 6.39, 6.40
(2s, 2.0H, Dmt Ar-H), 6.24 (d, 0.10H, J ) 8.52 Hz, Dmt NH),
4.48-4.19 (m, 3.0H, Pro trans-RH, 3,5Dmp RH, Phe RH, Dmt trans-
RH), 4.19-4.10 (m, 0.50H, Dmt cis-RH), 3.55-3.44 (m, 0.50H,
Pro trans-δH), 3.20-2.64 (m, 8.0H, Pro cis-RH, Pro trans-δH, Pro
cis-δH, Phe âH, 3,5Dmp âH, Dmt âH), 2.21, 2.19 (2s, 3,5Dmp CH3),
2.17, 2.09 (2s, Dmt CH3), 1.96-1.83 (m, 0.50H, Pro trans-âH),
1.83-1.60 (m, 2.0H, Pro cis-âH, Pro trans-âH, Pro trans-γH),
1.46-1.06 (m, 9.54H, Boc, Pro cis-γH), 1.06-0.92 (m, 0.47H, Pro
cis-âH), 0.92-0.77 (m, 0.49H, Pro cis-γH). 13C NMR (100.6 MHz,
DMSO-d6) δ: 172.46, 172.25, 171.22, 171.08, 170.71, 170.40,
170.22, 170.08, 155.51, 155.43, 154.96, 154.62, 138.06, 137.85,
137.76, 137.51, 137.39, 136.79, 136.78 (19q), 129.04, 127.95,
127.92, 127.57, 127.48, 126.70, 126.61, 126.13 (8t, Ar-C), 124.25,
122.99 (2q), 114.84, 114.66 (2t, Ar-C), 78.46, 77.89 (2q, Boc),
59.78 (t, Pro trans-RC), 59.11 (t, Pro cis-RC), 55.02 (t, 3,5Dmp trans-
RC), 54.22 (t, 3,5Dmp cis-RC), 53.72 (t, Phe cis-RC), 53.43 (t, Phe
trans-RC), 51.44 (t, Dmt cis-RC), 51.35 (t, Dmt trans-RC), 46.51
(s, Pro trans-δC), 45.94 (s, Pro cis-δC), 37.94 (s, Phe cis-âC), 37.56
(s, Phe trans-âC), 36.61 (s, 3,5Dmp cis-âC), 36.00 (s, 3,5Dmp trans-
âC), 31.26 (s, Dmt cis-âC), 30.82 (s, Dmt trans-âC), 30.03 (s, Pro
cis-âC), 28.63 (s, Pro trans-âC), 28.19, 27.97, 27.42 (3p, Boc),
24.18 (s, Pro trans-γC), 20.94 (s, Pro cis-γC), 20.78 (p, 3,5Dmp,
cis-CH3), 20.76 (p, 3,5Dmp trans-CH3), 20.13 (p, Dmt trans-CH3),
19.39 (p, Dmt cis-CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-2′,6′-
dimethyl-L-phenylalanylphenylalanylamide. Yield 284 mg (84.9%);
mp 145-146 °C; Rf3 ) 0.88; [R] 25 -39.32° (c 0.52, MeOH). H
1
NMR (400.1 MHz, DMSO-d6) δ:D 9.05, 8.86 (2s, 1H, Dmt OH),
8.35 (d, 0.45H, Dmp NH), 8.10-7.94 (m, 0.55H, Dmp NH), 7.88-
7.55 (m, 0.40H, Phe NH), 7.70-7.54 (m, 0.60H, Phe NH), 7.40-
6.61 (m, 10.9H, Phe Ar-H, Dmp Ar-H, CONH2, Dmt NH), 6.50-
6.22 (m, 2.1H, Dmt Ar-H, Dmt NH), 4.65-4.28 (m, 2.80H, Pro
trans-RH, Phe RH, Dmp RH, Dmt trans-RH), 4.28-3.98 (m, 0.53H,
Dmt cis-RH), 3.57-3.44 (m, 0.47H, Pro trans-δH), 3.28-2.63 (m,
8.20H, Pro cis-RH, Pro trans-δH, Pro cis-δH, Phe âH, Dmp âH,
Dmt âH), 2.42-1.98 (m, 12.0H, Dmt CH3, Dmp CH3), 1.98-1.54
(m, 2.54H, Pro cis-âH, Pro trans-âH, Pro trans-γH), 1.54-1.00
(m, 9.75H, Boc, Pro cis-γH), 1.00-0.82 (m, 0.41H, Pro cis-âH),
0.73-0.53 (m, 0.32H, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-
d6) δ: 172.21, 172.09, 171.16, 170.97, 170.70, 170.12, 169.98,
155.60, 155.42, 154.92, 138.11, 137.88, 137.82, 137.56, 136.78,
136.54, 134.57, 134.11 (18q), 129.12, 129.00, 128.95, 127.96,
127.91, 127.82, 126.13, 126.07, 125.98, 125.90, 114.83, 114.63 (12t,
Ar-C), 78.64, 77.90 (2q, Boc), 59.65 (t, Pro trans-RC), 59.30 (t,
Pro cis-RC), 53.82 (t, Phe trans-RC), 53.53 (t, Phe cis-RC), 53.05
(t, Dmp trans-RC), 52.43 (t, Dmp cis-RC), 51.47 (t, Dmt RC), 46.53
(s, Pro trans-δC), 46.34 (s, Pro cis-δC), 37.93 (s, Phe trans-âC),
37.17 (s, Phe cis-âC), 31.89 (s, Dmp cis-âC), 31.40 (s, Dmp trans-
Nr-tert-Butyloxycarbonyltyrosylprolyl-2′,4′,6′-trimethyl-L-
phenylalanylphenylalanylamide. Yield 237 mg (75.3%); mp 215-
217 °C; Rf3 ) 0.76; [R] 2D5 -46.69° (c 0.35, MeOH). H NMR
1
(400.1 MHz, DMSO-d6) δ: 9.20 (s, 0.23H, Dmt cis-OH), 9.15 (s,
0.72H, Dmt trans-OH), 8.50 (s, 0.20H, J ) 8.93 Hz, Phe cis-NH),

2760 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Li et al.
7.96 (d, 0.75H, J ) 7.87 Hz, Phe trans-NH), 8.22 (d, 0.74H, J )
8.22 Hz, Tmp trans-NH), 7.75 (d, 0.22H, J ) 8.16 Hz, Tmp cis-
NH), 7.27-7.12 (m, 5.0H, Phe Ar-H), 7.10-6.97 (m, 3.2H, Tyr
Ar-H, Tyr NH), 6.94 (d, 0.70H, J ) 8.17 Hz, Tyr NH), 6.80 (br,
0.24H, cis-CONH2), 6.76-6.55 (m, 4.8H, Tmp Ar-H, Tyr Ar-
H, CONH2, Tyr NH), 4.58-4.49 (m, 0.23H, Phe cis-RH), 4.43-
4.20 (m, 3.38H, Pro trans-RH, Tyr trans-RH, Tmp RH, Phe trans-
RH), 4.38-4.10 (m, 0.13H, Tyr cis-RH), 3.92 (d, 0.21H, J ) 7.67
Hz, Pro cis-RH), 3.65-3.54 (m, 0.70H, Pro trans-δH), 3.54-3.37
(m, 0.80H, Pro trans-δH), 3.20-3.10 (m, 0.50H, Pro cis-δH), 3.10-
2.85 (m, 2.3H, Phe âH, Tmp âH), 2.85-2.53 (m, 3.7H, Phe âH,
Tyr âH, Tmp âH), 2.28-2.08 (m, 9.0H, Tmp CH3), 2.0-1.87 (m,
0.71H, Pro trans-âH), 1.87-1.76 (m, 1.56H, Pro trans-γH), 1.76-
1.63 (m, 1.0H, Pro cis-âH, Pro trans-âH), 1.48-1.37 (m, 0.23H,
Pro cis-γH), 1.30, 1.24 (2s, 9.0H, Boc), 1.20-1.10 (m, 0.29H, Pro
cis-âH), 0.97-0.82 (m, 0.17H, Pro cis-γH). 13C NMR (100.6 MHz,
DMSO-d6) δ: 172.33, 172.24, 171.12, 170.77, 170.23, 170.32,
170.16, 155.95, 155.68, 155.36, 155.23, 137.88, 137.73, 136.59,
136.44, 134.69, 134.54, 131.21 (18q), 130.39, 130.11, 128.99,
128.94, 128.57, 128.48, 127.90, 126.37, 126.05, 114.77 (10t, Ar-
C), 78.34, 77.86 (2q, Boc), 59.37 (t, Pro RC), 54.10 (t, Tyr RC),
53.74 (t, Tmp trans-RC), 53.53 (t, Tmp cis-RC), 43.14 (t, Phe trans-
RC), 52.16 (t, Phe cis-RC), 46.61 (s, Pro trans-δC), 46.21 (s, Pro
cis-δC), 37.42 (s, Phe cis-âC), 37.10 (s, Phe trans-âC), 36.88 (s,
Tyr cis-âC), 35.55 (s, Tyr trans-âC), 31.61 (s, Tmp trans-âC), 30.72
(s, Tmp cis-âC), 29.87 (s, Pro cis-âC), 28.58 (s, Pro trans-âC),
28.08 (p, trans-Boc), 27.78 (p, cis-Boc), 24.42 (s, Pro trans-γC),
20.95 (s, Pro cis-γC), 20.36, 20.32, 19.75, 19.71 (4p, Tmp CH3).
Emp RH), 3.90 (d, 0.24H, J ) 7.62 Hz, Pro cis-RH), 3.66-3.60
(m, 0.62H, Pro trans-δH), 3.40-3.38 (m, 0.77H, Pro trans-δH),
3.23-3.08 (m, 0.62H, Pro cis-δH), 3.08-2.53 (m, 8.0H, Phe âH,
Tyr âH, Emp âH, Emp CH2CH3), 2.26 (s, 3.0H, Emp Ar-CH3),
2.02-1.88 (m, 0.75H, Pro trans-âH), 1.88-1.77 (m, 1.5H, Pro
trans-γH), 1.77-1.63 (m, 1.0H, Pro trans-âH, Pro cis-âH), 1.47-
1.20 (m, 9.35H, Pro cis-γH, Boc), 1.20-1.02 (m, 3.25H, Pro cis-
âH, Emp CH2CH3), 0.9-0.75 (m, 0.15H, Pro cis-γH). 13C NMR
(100.6 MHz, DMSO-d6) δ: 172.33, 172.18, 171.21, 170.79, 170.61,
170.21, 170.14, 155.96, 155.69, 155.46, 155.24, 142.80, 142.69,
137.84, 137.67, 136.89, 136.75, 133.76, 133.59 (19q), 130.40,
130.10, 129.01, 127.91, 127.79, 126.37, 126.27, 126.07, 126.00,
114.83, 114.78 (11t, Ar-C), 78.39, 77.86 (2q, Boc), 59.43 (t, Pro
RC), 54.13 (t, Tyr RC), 53.77 (t, Emp RC), 53.62 (t, Phe trans-
RC), 52.67 (t, Phe cis-RC), 46.62 (s, Pro trans-δC), 46.14 (s, Pro
cis-δC), 37.54 (s, Phe cis-âC), 37.14 (s, Phe trans-âC), 36.80 (s,
Tyr cis-âC), 35.56 (s, Tyr trans-âC), 31.06 (s, Emp trans-âC), 30.13
(s, Emp cis-âC), 29.76 (s, Pro cis-âC), 28.58 (s, Pro trans-âC),
28.06, 27.76 (2p, Boc), 25.18 (s, Emp CH2CH3), 24.43 (s, Pro trans-
γC), 21.00 (s, Pro cis-γC), 19.95 (p, Emp trans-Ar-CH3), 19.91
(p, Emp cis-Ar-CH3), 15.56 (p, Emp CH2CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-2′-
ethyl-6′-methyl-L-phenylalanylphenylalanylamide. Yield 280 mg
(85.6%); mp 135-137 °C; Rf3 ) 0.75; [R] 2D5 -38.20° (c 0.39,
1
MeOH). H NMR (400.1 MHz, DMSO-d6) δ: 9.06 (br, 0.44H,
Dmt cis-OH), 8.87 (br, 0.36H, Dmt trans-OH), 8.30 (d, 0.50H, J
) 8.77 Hz, Phe NH), 8.09-7.97 (m, 0.50H, Phe NH), 7.86-7.74
(m, 0.50H, Emp NH), 7.56 (d, 0.50H, J ) 8.10 Hz, Emp NH),
7.30-7.10 (m, 5.70H, Phe Ar-H, CONH2, Dmt NH), 7.10-6.87
(m, 4.41H, Emp Ar-H, CONH2), 6.84-6.68 (m, 0.73H, CONH2,
Dmt NH), 6.47-6.27 (m, 2.1H, Dmt Ar-H, Dmt NH), 4.50-4.23
(m, 3.0H, Pro trans-RH, Tmp RH, Phe RH, Dmt cis-RH), 4.16-
4.04 (m, 0.50H, Dmt cis-RH), 3.23 (d, 0.50H, J ) 7.37 Hz, Pro
cis-RH), 3.17-2.52 (m, 10.0H, Pro δH, Phe âH, Emp âH, Dmt
âH, Emp CH2CH3), 2.25 (s, 3.0H, Emp Ar-CH3), 2.15, 2.11 (2s,
6.0H, Dmt CH3), 1.97-1.84 (m, 0.49H, Pro trans-âH), 1.84-1.58
(m, 2.1H, Pro cis-âH, Pro trans-âH, Pro trans-γH), 1.47-1.10 (m,
9.67H, Dmt CH3, Emp Ar-CH3, Pro cis-γH), 1.10-1.00 (m, 3.0H,
Emp CH2CH3), 1.10-0.84 (m, 0.42H, Pro cis-âH), 0.70-0.53 (m,
0.38H, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6) δ: 172.17,
172.05, 171.13, 171.02, 170.59, 170.02, 155.63, 155.43, 154.90,
154.65, 142.77, 142.58, 138.11, 137.86, 137.81, 137.51, 136.86,
136.59, 133.74, 133.62 (20q), 128.90, 128.96, 127.96, 127.89,
127.76, 126.24, 126.14, 126.07, 125.96 (9t, Ar-C), 124.21, 122.93
(2q), 114.83, 114.61 (2t, Ar-C), 78.65, 77.89 (2q, Boc), 59.68 (t,
Pro trans-RC), 59.26 (t, Pro cis-RC), 53.80 (t, Tmp cis-RC), 53.66
(t, Tmp trans-RC), 53.50 (t, Phe cis-RC), 53.09 (s, Phe trans-RC),
51.48 (t, Dmt RC), 46.54 (s, Pro trans-δC), 46.31 (s, Pro cis-δC),
38.00 (s, Phe cis-âC), 37.16 (s, Phe trans-âC), 31.32 (s, Emp cis-
âC), 31.11 (s, Emp trans-âC), 30.09 (s, Dmt trans-âC), 29.77 (s,
Dmt cis-âC), 29.71 (s, Pro cis-âC), 28.68 (s, Pro trans-âC), 28.16,
27.99, 27.46 (3p, Boc), 25.20 (s, Emp cis-CH2CH3), 25.14 (s, Emp
trans-CH2CH3), 24.37 (s, Pro trans-γC), 20.88 (s, Pro cis-γC), 20.10
(p, Emp trans-Ar-CH3), 19.96 (p, Emp cis-Ar-CH3), 19.95 (p, Dmt
trans-CH3), 19.50 (p, Dmt cis-CH3), 15.58 (p, Emp CH2CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-2′,4′,6′-
trimethyl-L-phenylalanylphenylalanylamide. Yield 250 mg
(76.4%); mp 148-150 °C; Rf3 ) 0.80; [R] 2D5 -35.74° (c 0.35,
1
MeOH). H NMR (400.1 MHz, DMSO-d6) δ: 9.05 (br, 0.51H,
Dmt cis-OH), 8.86 (br, 0.43H, Dmt trans-OH), 8.31 (d, 0.48H, J
) 8.76 Hz, Phe NH), 8.06-7.93 (m, 0.51H, Phe NH), 7.86-7.76
(m, 0.49H, Tmp NH), 7.60 (d, 0.49H, J ) 8.14 Hz, Tmp NH),
7.28-7.10 (m, 5.59H, Phe Ar-H, Dmt NH, CONH2), 7.06, 6.98
(2s, 1.41H, CONH2), 6.80-6.65 (m, 2.84H, Tmp Ar-H, Dmt NH,
CONH2), 6.45-6.28 (m, 2.15H, Dmt Ar-H, Dmt NH), 4.50-4.22
(m, 3.0H, Pro trans-RH, Tmp RH, Phe RH, Dmt trans-RH), 4.16-
4.06 (m, 0.49H, Phe cis-RH), 3.55-3.43 (m, 0.56H, Pro trans-δH),
3.27-2.66 (m, 8.55H, Pro cis-RH, Pro cis-δH, Phe âH, Tmp âH,
Dmt âH), 2.28-2.05 (m, 15.0H, Dmt CH3, Tmp CH3), 1.95-1.58
(m, 2.74H, Pro cis-âH, Pro trans-âH, Pro trans-γH), 1.14-1.08
(m, 9.47H, Boc, Pro cis-γH), 1.05-0.9 (m, 0.42H, Pro cis-âH),
0.78-0.63 (m, 0.42H, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-
d6) δ: 172.25, 172.10, 171.16, 170.95, 170.76, 170.54, 170.20,
169.98, 155.53, 155.41, 154.92, 154.65, 138.11, 137.86, 137.57,
136.57, 136.32, 134.67, 134.60, 131.42, 131.27 (21q), 128.99,
128.95, 128.55, 128.50, 127.95, 127.90, 126.11, 126.06 (8t, Ar-
C), 124.22, 122.95 (2q), 114.83, 114.63 (2t, Ar-C), 78.60, 77.90
(2q, Boc), 59.64 (t, Pro trans-RC), 59.23 (t, Pro cis-RC), 53.78,
53.49 (2t, Tmp RC), 53.20, 52.68 (2t, Phe RC), 51.45 (t, Dmt RC),
46.53 (t, Pro trans-δC), 46.40 (t, Pro cis-δC), 37.90 (s, Phe cis-
âC), 37.12 (s, Phe trans-âC), 31.59 (s, Tmp cis-âC), 31.46 (s, Tmp
trans-âC), 30.97 (s, Dmt trans-âC), 30.41 (s, Dmt cis-âC), 29.81
(s, Pro cis-âC), 28.66 (s, Pro trans-âC), 28.17, 28.00, 27.47 (3p,
Boc), 24.36 (s, Pro trans-γC), 20.87 (s, Pro cis-γC), 20.36, 20.31
(2p, Tmp CH3), 20.11 (p, Dmt trans-CH3), 19.75 (p, Tmp CH3),
19.52 (p, Dmt cis-CH3).
Nr-tert-Butyloxycarbonyltyrosylprolyl-2′-isopropyl-6′-methyl-
L-phenylalanylphenylalanylamide. Yield 269 mg (83.8%); mp
222-224 °C; Rf3 ) 0.62; [R] 2D5 -45.51° (c 0.31, MeOH). 1H
NMR (400.1 MHz, DMSO-d6) δ: 9.21 (s, 0.21H, Tyr cis-OH),
9.15 (s, 0.76H, Tyr trans-OH), 8.46 (d, 0.21H, J ) 8.89 Hz, Phe
cis-NH), 7.84 (d, 0.76H, J ) 7.84 Hz, Phe trans-NH), 7.73 (d,
0.77H, J ) 8.12 Hz, Imp trans-NH), 7.66 (d, 0.21H, J ) 8.07 Hz,
Imp cis-NH), 7.30-7.10 (m, 5.0H, Phe Ar-H), 7.10-6.83 (m,
7.9H, CONH2, Imp Ar-H, Tyr Ar-H, Dmt NH), 6.68-6.56 (m,
2.1H, Tyr Ar-H, Dmt NH), 4.58-4.47 (m, 0.21H, Phe cis-RH),
4.45-4.10 (m, 3.60H, Pro trans-RH, Imp RH, Tyr RH, Phe trans-
RH), 3.90 (d, 0.19H, J ) 7.67 Hz, Pro cis-RH), 3.67-3.50 (m,
0.65H, Pro trans-δH), 3.55-3.40 (m, 0.73H, Pro trans-δH), 3.28-
3.11 (m, 1.62H, Pro cis-δH, CH(CH3)2), 3.11-2.92 (m, 2.0H, Phe
âH, Imp âH), 2.92-2.67 (m, 3.0H, Phe âH, Tyr âH, Imp âH),
Nr-tert-Butyloxycarbonyltyrosylprolyl-2′-ethyl-6′-methyl-L-
phenylalanylphenylalanylamide. Yield 242 mg (76.7%); mp 222-
224 °C; Rf3 ) 0.66; [R] 2D5 -48.52° (c 0.34, MeOH). H NMR
1
(400.1 MHz, DMSO-d6) δ: 9.20 (s, 0.26H, Tyr cis-OH), 9.15 (s,
0.73H, Tyr trans-OH), 8.50 (d, 0.23H, J ) 8.97 Hz, Phe cis-NH),
7.99 (d, 0.75H, J ) 7.87 Hz, Phe trans-NH), 7.81 (d, 0.75H, J )
8.13 Hz, Emp trans-NH), 7.73 (d, 0.24H, J ) 8.10 Hz, Emp cis-
NH), 7.27-7.13 (m, 5.0H, Phe Ar-H), 7.10-6.88 (m, 7.12H, Emp
Ar-H, Tyr Ar-H, CONH2), 6.82-6.72 (m, 0.7H, CONH2), 6.70-
6.57 (m, 2.10H, Tyr Ar-H, Tyr NH), 4.60-4.52 (m, 0.26H, Phe
cis-RH), 4.46-4.10 (m, 3.5H, Pro trans-RH, Phe trans-RH, Tyr RH,

Bifunctional Endomorphin-2 Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2761
2.67-2.55 (m, 0.82H, Tyr trans-RH), 2.25 (s, 3.0H, Imp Ar-CH3),
2.05-1.90 (m, 0.81H, Pro trans-âH), 1.90-1.77 (m, 1.63H, Pro
trans-γH), 1.77-1.63 (m, 0.96H, Pro trans-âH), 1.47-1.36 (m,
0.18H, Pro cis-γH), 1.36-1.02 (m, 15.23H, Pro cis-âH, Boc, Imp
CH(CH3)2), 0.90-0.88 (m, 0.18H, Pro cis-γH). 13C NMR (100.6
MHz, DMSO-d6) δ: 172.27, 172.20, 171.39, 170.75, 170.51,
170.27, 170.02, 155.96, 155.67, 155.47, 155.25, 147.38, 137.76,
137.57, 136.68, 132.99 (16q), 130.38, 130.06, 129.07, 129.00,
127.90, 127.51, 126.39, 126.09, 122.79, 114.77 (10t, Ar-C), 78.38,
77.86 (2q, Boc), 59.50 (t, Pro RC), 54.32 (t, Imp RC), 54.17 (t,
Tyr RC), 53.71 (t, Phe trans-RC), 53.46 (t, Phe cis-RC), 46.62 (s,
Pro trans-δC), 46.14 (s, Pro cis-δC), 37.61 (s, Pro cis-âC), 37.24
(s, Pro trans-âC), 36.74 (s, Tyr cis-âC), 35.53 (s, Tyr trans-âC),
30.48 (s, Imp trans-âC), 29.88 (s, Imp cis-âC), 29.74 (s, Pro cis-
âC), 28.66 (s, Pro trans-âC), 28.07 (p, trans-Boc), 27.95 (t, CH-
(CH3)2), 27.73 (p, cis-Boc), 24.43 (s, Pro trans-γC), 24.31, 23.76,
23.65 (3p, CH(CH3)2), 21.00 (s, Pro cis-γC), 20.46 (p, Imp cis-
Ar-CH3), 20.23 (p, Imp trans-Ar-CH3).
CONH2), 7.34-7.02 (m, 11.90H, Ar-H, CONH2), 6.95, 6.93 (2s,
0.75H, Ar-H), 6.74-6.67 (m, 2.0H, Ar-H), 4.50-4.33 (m, 2.66H,
Pro trans-RH, Mmp RH, Phe RH), 4.15 (t, 0.64H, J ) 6.40 Hz,
Tyr trans-RH), 3.60-3.48 (m, 1.36H, Pro cis-RH, Tyr cis-RH, Pro
trans-δH), 3.35-3.20 (m, 0.72H, Pro cis-δH), 3.10-2.77 (m, 6.64H,
Pro trans-δH, Phe âH, Tyr âH, Mmp âH), 2.29, 2.26 (2s, 3.0H,
Mmp CH3), 2.0-1.88 (m, 0.64H, Pro trans-âH), 1.82-1.56 (m,
2.28H, Pro cis-âH, Pro trans-âH, Pro γH), 1.56-1.38 (m, 0.72H,
Pro cis-âH, Pro cis-γH), 1.34-1.18 (m, 0.36H, Pro cis-γH). 13C
NMR (100.6 MHz, DMSO-d6) δ: 172.95, 172.38, 170.66, 170.60,
169.99, 167.00, 166.85, 156.67, 156.45, 137.74, 136.06, 135.99,
135.68, 135.60 (14q), 130.75, 130.26, 129.86, 129.75, 129.25,
129.16, 129.12, 127.91, 127.83, 126.37, 126.20, 126.10, 125.58,
125.45 (14t), 124.51, 123.94 (2q), 115.34, 115.18 (2t), 59.44 (t,
Pro trans-RC), 59.12 (t, Pro cis-RC), 53.67 (t, Mmp RC), 53.20 (t,
Phe RC), 52.36 (t, Tyr trans-RC), 52.31 (t, Tyr cis-RC), 46.70 (s,
Pro trans-δC), 46.44 (s, Pro cis-δC), 37.45 (s, Phe cis-âC), 37.24
(s, Phe trans-âC), 36.21 (s, Tyr cis-âC), 35.21 (s, Tyr trans-âC),
34.68 (s, Mmp trans-âC), 34.25 (s, Mmp cis-âC), 31.04 (s, Pro
cis-âC), 28.25 (s, Pro trans-âC), 24.32 (s, Pro trans-γC), 21.23 (s,
Pro cis-γC), 19.03 (p, Mmp trans-CH3), 18.95 (p, Mmp cis-CH3).
2′,6′-Dimethyl-L-tyrosylprolyl-2′-methyl-L-phenylalanylphe-
nylalanylamide Hodrochloride (2′). Yield 153.8 mg (93.9%); mp
180-182 °C. 1H NMR (400.1 MHz, DMSO-d6) δ: 9.30 (s, 0.7H,
Dmt cis-OH), 9.15 (s, 0.3H, Dmt trans-OH), 8.53 (br, 3.0H, NH3+),
8.30-8.20 (m, Mmp NH, Phe cis-NH), 7.94 (d, 0.3H, J ) 8.22
Hz, Phe trans-NH), 7.62 (s, 0.7H, cis-CONH2), 7.34-7.00 (m,
10.3H, CONH2, Ar-H), 6.43 (s, 2.0H, Dmt Ar-H), 4.51 (dt, 0.7H,
J ) 4.77, 8.98 Hz, Phe cis-RH), 4.47-4.32 (m, 1.6H, Pro trans-
RH, Mmp RH, Phe trans-RH), 4.10 (dd, 0.3H, J ) 5.23, 10.08 Hz,
Dmt trans-RH), 3.61 (dd, 0.7H, J ) 4.49, 10.82 Hz, Dmt cis-RH),
3.38-3.24 (m, 1.0H, Pro cis-δH, Pro trans-δH), 3.24-3.13 (m,
0.7H, Pro cis-δH), 3.08-2.75 (m, 6.7H, Pro cis-RH, Phe âH, Mmp
âH, Dmt âH), 2.35-2.20 (m, 3.3H, Pro trans-δH, Mmp Ar-CH3),
2.17, 2.05 (2s, 6.0H, Dmt Ar-CH3), 1.87-1.74 (m, 0.3H, Pro trans-
âH), 1.67-1.50 (m, 1.7H, Pro cis-âH, Pro trans-âH, Pro trans-
γH), 1.50-1.37 (m, 0.7H, Pro cis-γH), 1.25-1.0 (m, 1.3H, Pro
cis-âH, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6) δ: 173.13,
172.36, 170.76, 170.55, 170.33, 169.91, 167.83, 167.28, 155.93,
155.62, 138.43, 138.15, 137.68, 137.64, 135.97, 135.84, 135.77,
135.70 (18q), 129.87, 129.71, 129.32, 129.11, 129.01, 128.72,
127.89, 127.81, 126.29, 126.12, 125.59, 125.48 (12t, Ar-C),
121.38, 120.98 (2q), 115.05, 114.94 (2t, Ar-C), 59.63 (t, Pro trans-
RC), 59.04 (t, Pro, cis-RC), 53.71 (t, Mmp cis-RC), 53.54 (t, Phe
RC), 53.06 (t, Mmp trans-RC), 49.74 (t, Dmt RC), 46.61 (s, Pro
cis-δC), 46.12 (s, Pro trans-δC), 37.59 (s, Phe cis-âC), 37.29 (s,
Phe trans-âC), 34.78 (s, Mmp trans-âC), 33.55 (s, Mmp cis-âC),
31.04 (s, Pro cis-âC), 30.70 (s, Dmt cis-âC), 30.01 (s, Dmt trans-
âC), 28.85 (s, Pro trans-âC), 24.14 (s, Pro trans-γC), 21.18 (s, Pro
cis-γC), 20.02 (p, Dmt trans-CH3), 19.29 (p, Dmt cis-CH3), 19.00
(p, Mmp trans-CH3), 18.91 (p, Dmt cis-CH3).
Nr-tert-Butyloxycarbonyl-2′,6′-dimethyl-L-tyrosylprolyl-2′-iso-
propyl-6′-methyl-L-phenylalanylphenylalanylamide. Yield 252
mg (75.6%); mp 143-145 °C; Rf3 ) 0.67; [R] 25 -35.88° (c 0.44,
D
1
MeOH). H NMR (400.1 MHz, DMSO-d6) δ: 9.06 (br, 0.35H,
Dmt cis-OH), 8.86 (br, 0.37H, Dmt trans-OH), 8.25 (d, 0.37H, J
) 8.71 Hz, Phe cis-NH), 8.17-7.98 (m, 0.47H, Phe trans-NH),
7.77-7.53 (m, 0.47H, Imp trans-NH), 7.50 (d, 0.38H, J ) 8.09
Hz, Imp cis-NH), 7.30-7.12 (m, 5.5H, Phe Ar-H, Dmt NH), 7.12-
6.86 (m, 5.0H, Imp Ar-H, CONH2), 6.75 (d, 0.40H, J ) 8.46 Hz,
Dmt cis-NH), 6.44-6.30 (m, 2.1H, Dmt Ar-H, Dmt NH), 4.50-
4.25 (m, 2.46H, Pro trans-RH, Phe trans-RH, Imp RH, Dmt trans-
RH), 4.25-4.17 (m, 0.51H, Phe cis-RH), 4.15-4.07 (m, 0.45H,
Dmt cis-RH), 3.56-3.42 (m, 0.56H, Pro cis-δH), 3.26-2.62 (m,
9.0H, Pro cis-RH, Pro trans-δH, Pro cis-δH, Phe âH, Dmt âH,
Imp âH, Imp CH(CH3)2), 2.36-2.00 (m, 9.0H, Imp Ar-CH3, Dmt
Ar-CH3), 2.00-1.85 (m, 0.59H, Pro trans-âH), 1.85-1.57 (m,
2.0H, Pro cis-âH, Pro trans-âH, Pro trans-γH), 1.45-1.00 (m,
15.52H, Boc, Imp CH(CH3)2, Pro cis-γH), 1.00-0.85 (m, 0.51H,
Pro cis-âH), 0.70-0.55 (m, 0.38H, Pro cis-γH). 13C NMR (100.6
MHz, DMSO-d6) δ: 172.32, 172.01, 171.22, 170.50, 170.10,
169.94, 155.63, 155.45, 154.90, 154.68, 147.37, 147.12, 138.11,
137.85, 137.75, 137.44, 136.64, 136.53, 132.95, 132.85 (20q),
129.08, 128.99, 127.96, 127.89, 127.50, 127.40, 126.37, 126.30,
126.15, 126.11 (10t, Ar-C), 124.20 (q), 122.92, 122.79, 114.83,
114.61 (4t, Ar-C), 78.63, 77.91 (2q, Boc), 59.77 (t, Pro trans-
RC), 59.24 (t, Pro cis-RC), 54.36 (t, Phe cis-RC), 53.84 (t, Phe
trans-RC), 53.76 (t, Imp trans-RC), 53.45 (t, Imp cis-RC), 51.53
(t, Dmt cis-RC), 51.49 (t, Dmt, trans-RC), 46.55 (s, Pro cis-δC),
46.35 (s, Pro trans-δC), 38.04 (s, Phe cis-âC), 37.24 (s, Phe trans-
âC), 31.30 (s, Dmt cis-âC), 30.85 (s, Dmt trans-âC), 30.49 (s, Imp
trans-âC), 29.82 (s, Pro cis-âC), 29.40 (s, Imp cis-âC), 28.73 (s,
Pro trans-âC), 28.17 (p, Boc), 27.98 (p, Boc; t, CH(CH3)2), 27.45
(p, Boc), 24.37 (s, Pro trans-γC), 24.33, 23.78, 23.70 (3p, CH-
(CH3)2), 20.89 (s, Pro cis-γC), 20.58 (p, Imp cis-Ar-CH3), 20.21
(p, Imp trans-Ar-CH3), 20.07 (p, Dmt cis-Ar-CH3), 19.49 (p, Dmt
trans-Ar-CH3).
Tyrosylprolyl-3′,5′-dimethyl-L-phenylalanylphenylalanyla-
mide Hydrochoride (3). Yield 142.5 mg (87.2%); mp 170-172
1
°C. H NMR (400.1 MHz, DMSO-d6) δ: 9.53 (s, 0.34H, Tyr cis-
+
General Procedure for Synthesis of H-Tyr/Dmt-Pro-Xaa-Phe-
NH2‚HCl (Xaa ) Mmp, 3,5Dmp, Dmp, Dmt, Tmp, Emp, Imp).
Boc-Tyr/Dmt-Pro-Xaa-Phe-NH2 (0.25 mmol) was treated with TFA
(0.6 mL, 7.79 mmol) and anisole (40 µL) for 1 h at room
temperature. The reaction solution was diluted with hexane, and
the solid was collected by filtration, dried over KOH pellets, and
purified by semipreparative RP-HPLC. The purified peptide was
lyophilized from water (3 × 10 mL) containing 1 M HCl (0.25
mL, 0.25 mmol) to give amorphous powder. Elemental analysis
results of H-Tyr/Dmt-Pro-Xaa-Phe-NH2 are summarized in Sup-
porting Information (Table 4).
OH), 9.43 (s, 0.66H, Tyr trans-OH), 8.40-8.16 (m, 3.68H, NH3
,
3,5Dmp cis-NH, Phe cis-NH), 8.12 (d, 0.66H, J ) 8.22 Hz, 3,5Dmp
trans-NH), 7.88 (d, 0.66H, J ) 7.91 Hz, Phe trans-NH), 7.47 (s,
0.34H, cis-CONH2), 7.33-7.05 (m, 8.0H, Ar-H, CONH2), 6.94
(d, 0.66H, J ) 8.31 Hz, Ar-H), 6.83-6.67 (m, 5.0H, Ar-H),
4.50-4.30 (m, 2.66H, Pro trans-RH, 3,5Dmp RH, Phe RH), 4.15 (t,
0.66H, J ) 6.53 Hz, Tyr trans-RH), 3.70 (dd, 0.36H, J ) 5.68,
8.92 Hz, Tyr cis-RH), 3.60-3.48 (m, 0.66H, Pro trans-δH), 3.48-
3.43 (m, 0.34H, Pro cis-RH), 3.35-3.20 (m, 0.68H, Pro cis-δH),
3.07-2.70 (m, 6.66H, Pro trans-âH, Phe âH, 3,5Dmp âH, Tyr âH),
2.20 (s, 6.0H, 3,5Dmp CH3), 2.0-1.87 (m, 0.66H, Pro trans-âH),
1.78-1.59 (m, 1.98H, Pro trans-âH, Pro trans-γH), 1.59-1.44 (m,
0.68H, Pro cis-âH, Pro cis-γH), 1.44-1.32 (m, 0.34H, Pro cis-
âH), 1.32-1.16 (m, 0.34H, Pro cis-γH). 13C NMR (100.6 MHz,
DMSO-d6) δ: 173.01, 172.55, 170.67, 170.44, 170.36, 169.97,
167.12, 167.08, 156.70, 156.49, 137.76, 137.50, 137.15, 136.80,
136.76 (15q), 130.69, 130.20, 129.25, 129.12, 127.93, 127.86,
Tyrosylprolyl-2′-methyl-L-phenylalanylphenylalanylamide Hy-
1
drochloride (2). Yield 136.5 mg (83.7%); mp 170-172 °C. H
NMR (400.1 MHz, DMSO-d6) δ: 9.51 (s, 0.36H, Tyr cis-OH),
9.41 (s, 0.64H, Tyr trans-OH), 8.49-8.43 (m, 0.36H, Phe cis-NH),
8.43-8.07 (m, 4.0H, NH3+, Phe trans-NH, Mmp cis-NH), 8.07-
7.98 (m, 0.64H, Mmp trans-NH), 7.47-7.40 (m, 0.36H, cis-

2762 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Li et al.
127.61, 126.89, 126.66, 126.12 (10t, Ar-C), 124.63, 123.98 (2q),
115.38, 115.24 (2t, Ar-C), 59.60 (s, Pro trans-RC), 59.35 (t, Pro
cis-RC), 55.12 (t, 3,5Dmp cis-RC), 54.05 (t, 3,5Dmp trans-RC), 53.76
(t, Phe RC), 52.43 (t, Tyr trans-RC), 52.36 (t, Tyr cis-RC), 46.74
(s, Pro trans-δC), 46.40 (s, Pro cis-δC), 37.50 (s, Phe cis-âC), 37.38
(s, Phe trans-âC), 37.29 (s, 3,5Dmp trans-âC), 36.63 (s, 3,5Dmp cis-
âC), 36.16 (s, Tyr cis-âC), 35.30 (s, Tyr trans-âC), 30.97 (s, Pro
cis-âC), 28.85 (s, Pro trans-âC), 24.21 (s, Pro trans-γC), 21.13 (s,
Pro cis-γC), 20.81 (p, 3,5Dmp CH3).
NMR (400.1 MHz, DMSO-d6) δ: 9.52 (s, 0.28H, Tyr cis-OH),
9.41 (s, 0.69H, Tyr trans-OH), 9.00 (s, 0.90H, Dmt OH), 8.40-
8.10 (m, 4.0H, -NH3+, Dmt NH, Phe cis-NH), 7.95 (d, 0.73H, J
) 8.18 Hz, Phe trans-NH), 7.30-7.04 (m, 8.0H, CONH2, Tyr and
Phe Ar-H), 6.99 (d, 0.64H, J ) 8.36 Hz, Tyr Ar-H), 6.93 (s,
0.68H, trans-CONH2), 6.73 (d, 0.62H, J ) 8.22 Hz, Tyr cis-Ar-
H), 6.69 (d, 1.40H, J ) 8.31 Hz, Tyr trans-Ar-H), 6.38 (s, 2.0H,
Dmt Ar-H), 4.50-4.33 (m, 2.0H, Pro trans-RH, Phe RH, Dmt
cis-RH), 4.30 (q, 0.73H, J ) 7.30, 14.84 Hz, Dmt trans-RH), 4.15
(t, 0.70H, J ) 6.52 Hz, Tyr trans-RH), 3.72-3.65 (m, 0.35H, Pro
cis-RH), 3.65-3.50 (m, 1.0H, Tyr cis-RH, Pro trans-âH), 3.10-
2.55 (m, 6.7H, Pro trans-δH, Phe âH, Tyr âH, Dmt âH), 2.19,
2.16 (2s, 6.0H, Dmt CH3), 2.04-1.90 (m, 0.70H, Pro trans-âH),
1.83-1.69 (m, 1.40H, Pro trans-γH), 1.69-1.36 (m, 1.9H, Pro cis-
âH, Pro trans-âH, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6)
δ: 172.70, 172.78, 170.96, 170.69, 170.32, 169.90, 167.04, 166.84,
156.67, 156.43, 155.02, 154.98, 137.87, 137.82, 137.77, 137.75
(16q), 130.78, 130.36, 129.18, 129.09, 127.87, 127.80, 126.04 (9t,
Ar-C), 124.74, 124.49, 124.04 (3q), 115.32, 115.17, 114.76 (3t,
Ar-C), 59.48 (t, Pro trans-RC), 59.07 (t, Pro cis-RC), 53.80, 53.70
(2s, Phe RC and Dmt RC), 52.40 (s, Tyr RC), 46.73 (s, trans Pro
δC), 46.63 (s, Pro cis-δC), 37.31 (s, Phe cis-âC), 37.25 (s, Phe
trans-âC), 36.18 (s, Tyr cis-âC), 35.25 (s, Tyr trans-âC), 31.60 (s,
Dmt trans-âC), 31.14 (s, Dmt cis-âC, Pro cis-âC), 28.83 (s, Pro
trans-âC), 24.41 (s, Pro trans-γC), 21.41 (s, Pro cis-γC), 20.13 (p,
Dmt cis-CH3), 20.04 (p, Dmt trans-CH3).
2′,6′-Dimethyl-L-tyrosylprolyl-2′,6′-dimethyl-L-tyrosylpheny-
lalanylamide Hydrochloride (5′). Yield 130 mg (67.8%); mp 209-
211 °C. 1H NMR (400.1 MHz, DMSO-d6) δ: 9.31 (s, 0.57H, Dmt1
cis-OH), 9.12 (s, 0.39H, Dmt1 trans-OH), 8.99, 8.96 (2s, 0.97H,
Dmt3 OH), 8.47 (br, 2.8H, NH3+), 8.20-8.07 (m, 1.58H, Dmt3 NH,
Phe cis-NH), 7.86 (d, 0.38H, J ) 8.13 Hz, Phe trans-NH), 7.48 (s,
0.54H, cis-CONH2), 6.50-6.30 (m, 4.0H, Dmt1 and Dmt3 Ar-H),
4.49 (td, 0.59H, J ) 4.89, 8.81 Hz, Phe cis-RH), 4.43-4.32 (m,
0.82H, Pro trans-RH, Phe trans-RH), 4.32-4.20 (m, 1.0H, Dmt3
âH), 4.10 (dd, 0.39H, J ) 5.09, 10.0 Hz, Dmt1 trans-RH), 3.68-
3.57 (m, 0.54H, Dmt1 cis-RH), 3.40-3.16 (m, 1.18H, Pro cis-δH),
3.10-2.60 (m, 6.6H, Pro cis-RH, Phe âH, Dmt1 âH, Dmt3 âH),
2.30-2.20 (m, 12.82H, Pro trans-δH, Dmt1 and Dmt3 CH3), 1.87-
1.73 (m, Pro trans-âH), 1.67-1.38 (m, 2.64H, Pro cis-âH, Pro trans-
âH, Pro trans-γH, Pro cis-γH), 1.32-1.07 (m, 1.24H, Pro cis-âH,
Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6) δ: 172.94, 172.22,
170.92, 170.80, 169.91, 169.82, 167.94, 167.21, 155.99, 155.59,
155.01, 154.98, 138.47, 138.16, 137.80, 137.67, 137.55 (18q),
129.29, 129.04, 127.84, 127.80, 126.08, 126.02 (6t, Ar-C), 124.94,
124.69, 121.37, 121.07 (4q), 115.08, 114.90, 114.75 (3t, Ar-C),
59.67 (t, Pro trans-RC), 59.08 (t, Pro cis-RC), 54.83 (t, Dmt1 RC),
53.56 (t, Phe RC), 49.77 (t, Dmt3 RC), 46.81 (s, Pro cis-δC), 46.08
(s, Pro trans-δC), 37.51 (s, Phe cis-âC), 37.28 (s, Phe trans-âC),
31.08 (s, Dmt3 trans-âC), 31.04 (s, Pro cis-âC), 30.73 (s, Dmt1
cis-âC), 30.48 (s, Dmt3 cis-âC), 30.09 (s, Dmt1 trans-âC), 28.89
(s, Pro trans-âC), 24.20 (s, Pro trans-γC), 21.37 (s, Pro cis-γC),
20.09, 20.02, 19.99, 19.36 (4p, Dmt CH3).
2′,6′-Dimethyl-L-tyrosylprolyl-3′,5′-dimethyl-L-phenylalanylphe-
nylalanylamide Hydrochloride (3′). Yield 141.6 mg (86.3%); mp
180-182 °C. 1H NMR (400.1 MHz, DMSO-d6) δ: 9.30 (s, 0.71H,
Dmt cis-OH), 9.18 (s, 0.29H, Dmt trans-OH), 8.50 (br, 3.0H, NH3+),
8.36 (d, 0.71H, J ) 8.47 Hz, Phe cis-NH), 8.12 (d, 0.71H, J )
8.35 Hz, 3,5Dmp cis-NH), 8.01 (d, 0.29H, J ) 8.22 Hz, Phe trans-
NH), 7.84 (d, 0.29H, J ) 7.99 Hz, 3,5Dmp trans-NH), 7.63 (s, 0.71H,
cis-CONH2), 7.37-7.05 (m, 6.29H, CONH2, Ar-H), 6.84-6.75
(m, 3.0H, Ar-H), 6.45, 6.43 (2s, 2.0H, Dmt Ar-H), 4.51 (dt,
0.71H, J ) 4.90, 8.75 Hz, Phe cis-RH), 4.46-4.33 (m, 0.87H, Pro
trans-RH, 3,5Dmp trans-RH, Phe trans-RH), 4.33-4.22 (m, 0.71H,
3,5Dmp cis-RH), 4.11 (dd, 0.29H, J ) 5.61, 9.50 Hz, Dmt trans-
RH), 3.66 (dd, 0.71H, J ) 4.01, 11.08 Hz, Dmt cis-RH), 3.44-
3.28 (m, 0.8H, Pro cis-δH), 3.25-3.14 (m, 0.8H, Pro cis-δH),
3.10-2.68 (m, 6.7H, Pro cis-RH, Dmt âH, 3,5Dmp âH, Phe âH),
2.88-2.80 (m, 0.4H, Pro trans-δH), 2.20, 2.06 (2s, 12.0H, Dmt
and 3,5Dmp CH3), 1.87-1.75 (m, 0.29H, Pro trans-âH), 1.67-1.40
(m, 2.29H, Pro cis-âH, Pro trans-âH, Pro trans-γH, Pro cis-γH),
1.28-1.14 (m, 0.71H, Pro cis-âH), 1.14-1.0 (m, 0.71H, Pro cis-
γH). 13C NMR (100.6 MHz, DMSO-d6) δ: 173.17, 172.48, 170.83,
170.31, 170.25, 169.98, 167.94, 167.60, 155.95, 138.43, 138.14,
137.74, 137.65, 137.25, 136.78 (15q), 129.28, 129.11, 127.92,
127.82, 127.59, 126.73, 126.61 (8t, Ar-C), 121.43, 121.02 (2q),
115.05 (t, Ar-C), 59.81 (t, Pro trans-RC), 59.08 (t, Pro cis-RC),
55.67 (t, 3,5Dmp cis-RC), 54.02 (t, 3,5Dmp trans-RC), 53.61 (t, Phe
RC), 49.81 (t, Dmt RC), 46.65 (s, Pro cis-δC), 46.21 (s, Pro trans-
3,5
δC), 37.60 (s, Phe cis-âC), 37.41 (s, Phe trans-âC), 37.24 (s,
-
Dmp trans-âC), 36.01 (s, 3,5Dmp cis-âC), 31.11 (s, Pro cis-âC),
30.58 (s, Dmt cis-âC), 30.03 (s, Dmt trans-âC), 28.83 (s, Pro trans-
3,5
âC), 23.98 (s, Pro trans-γC), 21.09 (s, Pro cis-γC), 20.81 (p,
Dmp CH3), 20.07 (p, Dmt trans-CH3), 19.23 (p, Dmt cis-CH3).
-
2′,6′-Dimethyl-L-tyrosylprolyl-2′,6′-dimethyl-L-phenylalanylphe-
nylalanylamide Hydrochloride (4′). Yield 168 mg (69.5%); mp
194-196 °C. 1H NMR (400.1 MHz, DMSO-d6) δ: 9.33 (s, 0.6H,
Dmt cis-OH), 9.15 (s, 0.4H, Dmt trans-OH), 8.58, 8.40 (2br, 3.0H,
-NH3+), 8.25 (d, 1.6H, J ) 8.62 Hz, Dmp NH, Phe cis-NH), 7.95
(d, 0.4H, J ) 8.38 Hz, Phe trans-NH), 7.45 (s, 0.6H, cis-CONH2),
7.37-7.09 (m, 5.6H, Ar-H, CONH2), 7.03 (s, 0.4H, trans-CONH2),
6.98-6.84 (m, 3.0H, Ar-H), 6.68 (s, 0.4H, trans-CONH2), 6.46
(s, 1.2H, Dmt cis-CH3), 6.40 (s, 0.8H, Dmt trans-CH3), 4.50 (dt,
0.6H, J ) 4.71, 8.95 Hz, Phe cis-âH), 4.44-4.28 (m, 1.8H, Pro
trans-RH, Dmt RH, Phe trans-RH), 4.10 (dd, 0.4H, J ) 5.02, 9.59
Hz, Dmt trans-RH), 3.63 (dd, 0.6H, J ) 4.30, 10.43 Hz, Dmt cis-
RH), 3.38-3.28 (m, 0.6H, Pro cis-δH), 3.28-3.17 (m, 0.6H, Pro
cis-δH), 3.08-2.70 (m, 6.6H, Pro cis-RH, Phe âH, Dmt âH, Dmp
âH), 2.37-2.0 (m, 12.6H, Pro cis-RH, Dmt and Dmp CH3), 1.87-
1.74 (m, 0.4H, Pro trans-âH), 1.67-1.43 (m, 2.4H, Pro cis-âH,
Pro trans-âH, Pro trans-γH, Pro cis-γH), 1.30-1.10 (m, 1.2H, Pro
cis-âH, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6) δ: 172.97,
172.20, 170.69, 170.61, 169.93, 169.81, 167.92, 167.20, 155.99,
155.58, 138.44, 138.15, 137.77, 137.70, 136.76, 136.52, 134.66,
134.30 (18q), 129.30, 129.02, 127.84, 127.79, 126.07, 126.04,
126.02, 125.98 (8t, Ar-C), 121.34, 121.03 (2q), 115.08, 114.90
(2t, Ar-C), 59.62 (t, Pro trans-RC), 59.09 (t, Pro cis-RC), 54.26
(t, Dmp cis-RC), 53.63 (t, Phe RC), 53.01 (t, Dmp trans-RC), 49.72
(t, Dmt RC), 37.47 (s, Pro cis-âC), 37.24 (s, Pro trans-âC), 32.48
(s, Dmp trans-âC), 31.09 (s, Dmp cis-âC), 31.03 (s, Pro cis-âC),
30.69 (s, Dmt cis-âC), 30.10 (s, Dmt trans-âC), 28.90 (s, Pro trans-
âC), 24.17 (s, Pro trans-âC), 21.30 (s, Pro cis-âC), 20.04 (p, Dmp
CH3), 19.81 (p, Dmt trans-CH3), 19.40 (p, Dmt cis-CH3).
Tyrosylprolyl-2′,4′,6′-trimethyl-L-phenylalanylphenylalany-
lamide Hydrochloride (6). Yield 156.1 mg (95.3%); mp 179-
1
181 °C. H NMR (400.1 MHz, DMSO-d6) δ: 9.53 (s, 0.3H, Tyr
cis-OH), 9.41 (s, 0.7H, Tyr trans-OH), 8.49 (d, 0.3H, J ) 8.75 Hz,
Tmp cis-NH), 8.40-8.10 (br, 4.0H, NH3+, Tmp trans-NH, Phe cis-
NH), 8.00 (d, 0.7H, J ) 8.30 Hz, Phe trans-NH), 7.30-6.91 (m,
8.3H, Ar-H, CONH2), 6.80-6.60 (m, 4.7H, Ar-H, CONH2),
4.50-4.32 (m, 2.7H, Pro trans-RH, Tmp RH, Phe RH), 4.15 (t,
0.7H, J ) 6.36 Hz, Tyr trans-RH), 3.82-3.75 (m, 0.3H, Pro cis-
RH), 3.69 (t, 0.3H, J ) 7.08 Hz, Tyr cis-RH), 3.60-3.50 (m, 0.7H,
Pro trans-δH), 3.40-3.25 (m, 0.6H, Pro cis-δH), 3.10-2.72 (m,
6.7H, Pro trans-δH, Phe âH, Tyr âH, Tmp âH), 2.30-2.10 (m,
9.0H, Tmp Ar-CH3), 2.02-1.90 (m, 0.7H, Pro trans-âH), 1.85-
1.69 (m, 1.4H, Pro trans-γH), 1.69-1.38 (m, 1.9H, Pro cis-âH,
Pro trans-âH, Pro cis-γH). 13C NMR (100.6 MHz, DMSO-d6) δ:
172.66, 172.25, 170.76, 170.54, 170.32, 169.97, 166.94, 166.78,
156.69, 156.44, 137.87, 137.81, 136.64, 136.49, 134.71, 131.16,
131.06 (18q), 129.15, 129.04, 128.62, 127.86, 127.79, 126.02 (6t,
Tyrosylprolyl-2′,6′-dimethyl-L-tyrosylphenylalanylamide Hy-
1
drochloride (5). Yield 143.0 mg (87.3%); mp 188-190 °C. H

Bifunctional Endomorphin-2 Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2763
Ar-C), 124.40, 124.01 (2q), 115.31, 115.15 (2t, Ar-C), 59.41 (t,
Pro trans-RC), 59.04 (t, Pro cis-RC), 53.67 (t, Phe RC), 53.43 (t,
Pro cis-RC), 53.29 (t, Tmp trans-RC), 52.32 (t, Tyr RC), 46.72 (s,
Pro trans-δC), 46.64 (s, Pro cis-δC), 37.15 (s, Phe âC), 36.05 (s,
Tyr cis-âC), 35.21 (s, Tyr, trans-âC), 32.01 (s, Tmp trans-âC), 31.62
(s, Tmp cis-âC), 31.13 (s, Pro cis-âC), 28.85 (s, Pro trans-âC),
24.39 (s, Pro trans-γC), 21.39 (s, Pro cis-γC), 20.36, 19.86, 19.78
(3p, Tmp Ar-CH3).
2′,6′-Dimethyl-L-tyrosylprolyl-2′-ethyl-6′-methyl-L-phenyla-
lanylphenylalanylamide Hydrochloride (7′). Yield 143.4 mg
(87.0%); mp 176-178 °C. H NMR (400.1 MHz, DMSO-d6) δ:
1
9.32 (s, 0.65H, Dmt cis-OH), 9.14 (s, 0.35H, Dmt trans-OH), 8.52
(br, 3.0H, NH3+), 8.27-8.17 (m, 1.65H, Emp NH, Phe cis-NH),
7.89 (d, 0.35H, Phe trans-NH), 7.51 (s, 0.65H, cis-CONH2), 7.36-
7.28 (m, 1.30H, Ar-H), 7.25-7.12 (m, 4.6H, Ar-H), 7.06-6.89
(m, 3.1H, CONH2, Ar-H), 6.65 (s, 0.35H, trans-CONH2), 6.45,
6.40 (2s, 2.0H, Dmt Ar-H), 4.51 (dt, 0.65H, J ) 4.75, 9.02 Hz,
Phe cis-RH), 4.43-4.26 (m, 1.70H, Pro trans-RH, Emp RH, Phe
trans-RH), 4.10 (dd, 0.35H, J ) 5.15, 10.02 Hz, Dmt trans-RH),
3.64 (dd, 0.65H, J ) 4.01, 10.94 Hz, Dmt cis-RH), 3.36-3.18 (m,
1.65H, Pro cis-δH, Pro trans-δH), 3.10-2.50 (m, 8.65H, Pro cis-
RH, Phe âH, Emp âH, Dmt âH, Emp CH2CH3), 2.34-2.18 (m,
3.35H, Emp Ar-CH3, Pro trans-δH), 2.14, 2.09 (2s, 6.0H, Dmt
Ar-CH3), 1.87-1.76 (m, 0.35H, Pro trans-âH), 1.68-1.43 (m,
2.35H, Pro cis-âH, Pro trans-âH, Pro trans-γH, Pro cis-γH), 1.27-
1.02 (m, 4.30H, Pro cis-âH, Pro cis-γH, Emp CH2CH3). 13C NMR
(100.6 MHz, DMSO-d6) δ: 173.03, 172.15, 170.58, 169.95, 169.83,
167.96, 167.20, 156.01, 142.77, 142.64, 138.46, 138.15, 137.74,
137.68, 136.87, 136.61, 133.90, 133.44 (14q), 129.34, 129.02,
127.84, 127.77, 126.31, 126.26, 126.11, 126.02 (8t, Ar-C), 121.36,
121.04 (2q), 115.08, 114.90 (2t, Ar-C), 59.63 (t, Pro trans-RC),
59.09 (t, Pro cis-RC), 54.93 (t, Emp RC), 53.57 (t, Phe RC), 49.76
(t, Dmt RC), 46.76 (s, Pro cis-δC), 46.10 (s, Pro trans-δC), 37.52
(s, Phe cis-âC), 37.24 (s, Phe trans-âC), 31.70 (s, Emp trans-âC),
31.02 (s, Pro cis-âC), 30.68 (s, Dmt cis-âC), 30.27 (s, Emp cis-
âC), 30.10 (s, Dmt trans-âC), 28.95 (s, Pro trans-âC), 25.41 (s,
Emp cis-CH2CH3), 25.04 (s, Emp trans-CH2CH3), 24.18 (s, Pro
trans-γC), 21.30 (s, Pro cis-γC), 20.13 (p, Emp trans-CH2CH3),
20.01 (p, Emp cis-CH2CH3), 19.97 (p, Dmt cis-Ar-CH3), 19.36 (p,
Dmt trans-Ar-CH3).
2′,6′-Dimethyl-L-tyrosylprolyl-2′,4′,6′-trimethyl-L-phenylala-
nylphenylalanylamide Hydrochloride (6′). Yield 137.6 mg (80.6%);
1
mp 184-186 °C. H NMR (400.1 MHz, DMSO-d6) δ: 9.32 (s,
0.6H, Dmt cis-OH), 9.14 (s, 0.4H, Dmt trans-OH), 8.48 (br, 3.0H
NH3+), 8.24-8.14 (m, 1.6H, Tmp NH, Phe cis-NH), 7.93 (d, 0.4H,
J ) 8.38 Hz, Phe trans-NH), 7.40 (s, 0.60H cis-CONH2), 7.33-
7.10 (m, 5.6H, Phe Ar-H, cis-CONH2), 7.05 (s, 0.4H, trans-
CONH2), 6.75, 6.74 (2s, Tmp Ar-H), 6.60 (s, 0.4H, trans-CONH2),
6.46 (s, 1.2H, Dmt cis-Ar-CH3), 6.40 (s, 0.8H, Dmt trans-Ar-
CH3), 4.49 (td, 0.6H, J ) 4.76, 8.9 Hz, Phe cis-RH), 4.42-4.24
(m, 1.8H, Pro trans-RH, Tmp RH, Phe trans-RH), 4.00 (dd, 0.4H,
J ) 5.08, 10.06 Hz, Dmt trans-RH), 3.64 (dd, 0.6H, J ) 4.36, 10.88
Hz, Dmt cis-RH), 3.46-3.27 (m, 1.4H, Pro cis-δH, Pro trans-δH),
3.27-3.18 (m, 0.6H, Pro cis-δH), 3.08-2.68 (m, 6.6H, Pro cis-
âH, Phe âH, Tmp âH, Dmt âH), 2.30-2.00 (m, 15.4H, Pro trans-
δH, Dmt and Tmp Ar-CH3), 1.87-1.75 (m, 0.4H, Pro trans-âH),
1.70-1.43 (m, 2.4H, Pro cis-âH, Pro trans-âH, Pro trans-γH, Pro
cis-γH), 1.30-1.15 (m, 1.2H, Pro cis-âH, Pro cis-γH). 13C NMR
(100.6 MHz, DMSO-d6) δ: 172.96, 172.23, 170.74, 170.68, 169.91,
169.83, 167.98, 167.22, 156.00, 155.59, 138.45, 138.16, 137.75,
136.57, 136.33, 134.73, 134.66, 131.55, 131.16 (19q), 129.29,
129.02, 128.59, 127.84, 127.79, 126.07, 126.01 (7t, Ar-C), 121.37,
121.07 (2q), 115.09, 114.90 (2t, Ar-C), 59.62 (t, Pro trans-RC),
59.09 (t, Pro cis-RC), 54.50 (t, Tmp cis-RC), 53.59 (t, Phe RC),
53.10 (t, Tmp trans-RC), 49.78 (t, Dmt cis-RC), 49.73 (t, Dmt trans-
RC), 46.80 (s, Pro cis-δC), 46.10 (s, Pro trans-δC), 37.47 (s, Phe
cis-âC), 37.18 (s, Phe trans-âC), 32.21 (s, Tmp trans-âC), 31.03
(s, Pro cis-âC), 30.86 (s, Tmp cis-âC), 30.74 (s, Dmt trans-âC),
30.12 (s, Dmt cis-âC), 28.92 (s, Pro trans-âC), 24.18 (s, Pro trans-
γC), 21.36 (s, Pro cis-γC), 20.38 (p, Tmp trans-Ar-CH3), 20.35
(p, Tmp, cis-Ar-CH3), 20.01 (p, Tmp trans-Ar-CH3), 19.94 (p, Dmt
trans-Ar-CH3), 19.73 (p, Tmp cis-Ar-CH3), 19.36 (p, Dmt cis-Ar-
CH3).
Tyrosylprolyl-2′-isopropyl-6′-methyl-L-phenylalanylphenyla-
lanylamide Hydrochloride (8). Yield 155.7 mg (94.9%); mp 173-
1
175 °C. H NMR (400.1 MHz, DMSO-d6) δ: 9.53 (s, 0.3H, Dmt
cis-OH), 9.40 (s, 0.7H, Dmt trans-OH), 8.44 (d, 0.3H, J ) 8.81
Hz, Imp cis-NH), 8.37-8.05 (m, 4.0H, NH3+, Imp trans-NH, Phe
cis-NH), 7.91 (d, 0.7H, J ) 8.05 Hz, Phe trans-NH), 7.32-6.87
(m, 12.0H, Ar-H, CONH2), 6.64-6.76 (m, 2.0H, Ar-H), 4.51-
4.36 (m, 2.0H, Pro trans-RH, Imp cis-RH, Phe RH), 4.32 (dd, 0.7H,
J ) 7.23, 15.13 Hz, Imp trans-RH), 4.13 (t, 0.7H, J ) 6.33 Hz,
Tyr trans-RH), 3.67-3.50 (m, 1.3H, Pro cis-RH, Tyr cis-RH, Pro
trans-δH), 3.37-3.16 (m, 1.6H, Pro cis-δH, Imp CH(CH3)2), 3.10-
2.78 (m, 6.7H, Pro trans-δH, Phe ââH, Tyr âH, Imp âH), 2.31,
2.29 (2s, 3.0H, Imp Ar-CH3), 2.07-1.96 (m, 0.7H, Pro trans-âH),
1.82-1.64 (m, 2.1H, Pro trans-âH, Pro trans-γH), 1.64-1.51 (m,
0.6H, Pro cis-âH, Pro cis-γH), 1.51-1.41 (m, 0.3H, Pro cis-âH),
1.41-1.27 (m, 0.3H, Pro cis-γH), 1.20-1.04 (m, 6.0H, CH(CH3)2).
13C NMR (100.6 MHz, DMSO-d6) δ: 172.75, 172.23, 170.57,
17052, 170.37, 169.94, 167.11, 166.82, 156.70, 156.44, 147.45,
147.24, 137.81, 137.65, 136.76, 136.68, 132.97, 132.71 (18q),
130.73, 130.33, 129.25, 129.17, 127.84, 127.79, 127.56, 127.47,
126.48, 126.41, 126.06 (11t, Ar-C), 124.55, 124.00 (2q), 122.83,
115.33, 115.18 (3t, Ar-C), 59.49 (t, Pro trans-RC), 59.17 (t, Pro
cis-RC), 54.46 (t, Imp RC), 53.66 (t, Phe cis-RC), 53.56 (t, Phe
trans-âC), 52.44 (t, Tyr trans-âC), 52.32 (t, Tyr cis-âC), 46.72 (s,
Pro trans-δC), 46.53 (s, Pro cis-δC), 37.39 (s, Phe âC), 36.20 (s,
Tyr cis-âC), 35.34 (s, Tyr trans-âC), 31.05 (s, Pro cis-âC), 30.88
(s, Imp trans-âC), 30.45 (s, Imp cis-âC), 28.93 (s, Pro trans-âC),
27.87 (t, Imp CH(CH3)2), 24.42 (s, Pro trans-γC), 24.36 (p, Imp
trans-Ar-CH3), 23.86 (p, Imp cis-Ar-CH3), 21.33 (s, Pro cis-γC),
20.61 (p, Imp cis-CH(CH3)2), 20.27 (p, Imp trans-CH(CH3)2).
2′,6′-Dimethyl-L-tyrosylprolyl-2′-isopropyl-6′-methyl-L-phe-
nylalanylphenylalanylamide Hydrochloride (8′). Yield 131.3 mg
Tyrosylprolyl-2′-ethyl-6′-methyl-L-phenylalanylphenylalany-
lamide Hydrochloride (7). Yield 158.3 mg (96.6%); mp 167-
169 °C. 1H NMR (400.1 MHz, DMSO-d6) δ: 9.53 (s, 0.33H, Tyr
cis-OH), 9.41 (s, 0.67H, Tyr trans-OH), 8.43 (d, 0.33H, J ) 8.96
Hz, Emp cis-NH), 8.37-8.10 (m, 4.0H, NH3+, Emp trans-NH, Phe
cis-NH), 7.97 (d, 0.67H, J ) 8.30 Hz, Phe trans-NH), 7.30-6.90
(m, 11.33H, Ar-H, CONH2), 6.80-6.64 (m, 2.67H, Ar-H, trans-
CONH2), 4.52-4.33 (m, 2.67H, Pro trans-RH, Emp RH, Phe RH),
4.14 (t, 0.67H, J ) 6.26 Hz, Tyr trans-RH), 3.68-3.63 (m, 0.33H,
Pro cis-RH), 3.60-3.50 (m, 1.0H, Tyr cis-RH, Pro trans-δH), 3.36-
3.24 (m, 0.66H, Pro cis-δH), 3.08-2.76 (m, 6.67H, Pro trans-δH,
Phe âH, Tyr âH, Emp âH), 2.67-2.55 (m, 2.0H, Emp CH2CH3),
2.31, 2.28 (2s, 3.0H, Emp Ar-CH3), 2.03-1.90 (m, 0.67H, Pro
trans-γH), 1.83-1.70 (m, 1.34H, Pro trans-γH), 1.70-1.30 (m,
2.0H, Pro cis-âH, Pro trans-âH, Pro cis-γH), 1.14-1.03 (m, 3.0H,
Emp CH2CH3). 13C NMR (100.6 MHz, DMSO-d6) δ: 172.71,
172.18, 170.62, 170.42, 170.38, 169.86, 166.99, 166.85, 156.70,
156.44, 142.84, 142.71, 137.83, 137.75, 136.95, 136.80, 133.53,
133.44 (18q), 130.76, 130.34, 129.21, 130.34, 127.85, 127.78,
126.38, 126.30, 126.04 (9t, Ar-C), 124.47, 123.96 (2q), 115.33,
115.16 (2t, Ar-C), 59.45 (t, Pro trans-RC), 59.08 (t, Pro cis-RC),
53.79 (t, Emp RC), 53.67 (t, Phe RC), 52.38 (t, Tyr, trans-RC),
52.29 (t, Tyr cis-RC), 46.71 (s, Pro trans-δC), 46.58 (s, Pro cis-
δC), 37.34 (s, Phe cis-âC), 37.22 (s, Phe, trans-âC), 36.15 (s, Tyr
cis-âC), 35.32 (s, Tyr trans-âC), 31.47 (s, Emp trans-âC), 31.13
(s, Emp cis-âC), 30.95 (s, Pro cis-âC), 28.84 (s, Pro trans-âC),
25.30 (s, Emp cis-CH2CH3), 25.12 (s, Emp trans-CH2CH3), 24.38
(s, Pro trans-γC), 21.32 (s, Pro, cis-γC), 20.09 (p, Emp cis-Ar-
CH3), 19.99 (p, Emp, trans-Ar-CH3), 15.06 (p, Emp CH2CH3).
1
(79.6%); mp 185-187 °C. H NMR (400.1 MHz, DMSO-d6) δ:
9.31 (s, 0.54H, Dmt cis-OH), 9.12 (s, 0.46H, Dmt trans-OH), 8.47
(br, 3.0H, NH3+), 8.30 (0.46H, d, J ) 8.57 Hz, Imp trans-NH),
8.21 (d, 1.08H, J ) 8.72 Hz Imp cis-NH, Phe cis-NH), 7.82 (d,
0.46H, J ) 8.31 Hz, Phe trans-NH), 7.55 (s, 0.46H, trans-CONH2),
7.34, 7.32 (2s, 1.08H, Ar-H), 7.26-6.85 (m, 8.46H, Ar-H,
CONH2), 6.45, 6.39 (2s, Dmt Ar-H), 4.51 (dt, 0.54H, J ) 4.71,

2764 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Li et al.
8.98 Hz, Phe cis-RH), 4.45-4.24 (m, 1.92H, Pro trans-RH, Imp
RH, Phe trans-RH), 4.09 (dd, 0.46H, J ) 5.30, 9.98 Hz, Dmt trans-
RH), 3.69 (dd, 0.54H, J ) 4.08, 11.20 Hz, Dmt cis-RH), 3.37-
3.10 (m, 2.54H, Pro cis-δH, Pro trans-δH, Imp CH(CH3)2), 3.10-
2.75 (m, 6.54H, Pro cis-RH, Phe âH, Imp âH, Dmt âH), 2.34-
2.17 (m, 3.46H, Pro trans-δH, Imp Ar-CH3), 2.11, 2.09 (2s, 6.0H,
Dmt Ar-CH3), 1.90-1.75 (m, 0,46H, Pro trans-âH), 1.68-1.40
(m, 2.46H, Pro cis-âH, Pro trans-âH, Pro trans-γH, Pro cis-γH),
1.27-1.00 (m, 7.08H, Pro cis-âH, Pro cis-γH, Imp CH(CH3)2).
13C NMR (100.6 MHz, DMSO-d6) δ: 173.06, 172.18, 170.54,
170.50, 170.03, 169.85, 168.05, 167.17, 156.03, 155.55, 147.35,
147.14, 138.44, 138.13, 137.74, 137.57, 136.69, 136.50, 133.12,
132.72 (20q), 129.35, 129.11, 127.83, 127.78, 127.53, 127.45,
126.42, 126.36, 126.09, 126.05, 122.87, 122.82 (12t, Ar-C),
121.34, 121.05 (2q), 115.08, 114.90 (2t, Ar-C), 59.59 (t, Pro trans-
RC), 59.12 (t, Pro cis-RC), 55.36 (t, Imp cis-RC), 54.12 (t, Imp
trans-RC), 53.62 (t, Phe cis-RC), 53.42 (t, Phe trans-RC), 49.80 (t,
Dmt, cis-RC), 49.72 (t, Dmt, trans-RC), 46.72 (s, Pro cis-δC), 46.06
(s, Pro, cis-δC), 37.49 (s, Phe, cis-âC), 37.40 (s, Phe trans-âC),
31.11 (s, Imp, trans-âC), 30.95 (s, Pro trans-âC), 30.67 (s, Imp,
cis-âC), 30.09 (s, Dmt, trans-âC), 29.87 (s, Dmt cis-âC), 28.94 (s,
Pro, cis-âC), 27.93 (t, Imp cis-CH(CH3)2), 27.83 (t, Imp trans-CH-
(CH3)2), 24.41 (p, Imp trans-CH(CH3)2), 24.17 (p, Imp cis-CH-
(CH3)2), 24.12 (s, Pro trans-γC), 23.89 (p, Imp trans-CH(CH3)2),
23.82 (p, Imp cis-CH(CH3)2), 21.34 (s, Pro cis-γC), 20.73 (p, Imp
cis-Ar-CH3), 20.24 (p, Imp trans-Ar-CH3), 19.96 (p, Dmt trans-
Ar-CH3), 19.34 (p, Dmt cis-Ar-CH3).
Opioid Receptor Binding Assays. Opioid receptor affinities
were determined under equilibrium conditions (2.5 h at room
temperature (22 °C)) in a competition assay using brain P2
synaptosomal membranes prepared from 150-160 g Sprague-
Dawley rats.29,40 Synaptosomes were preincubated to remove
endogenous opioids for 60 min at 22 °C, washed in excess ice-
cold buffer (50 mM Tris-HCl, pH 7.5) containing protease inhibitor
(soybean trypsin inhibitor), and stored in a 20% glycerol-containing
buffer (50 mM HEPES, pH 7.5) with protease inhibitor at -80 °C
as described previously.29,40 The δ- and µ-opioid receptors were
radiolabeled with 1.9 nM [3H]deltorphin-II (Tyr-D-Ala-Phe-Glu-
Val-Val-Gly-NH2) and 3.5 nM [3H]DAMGO ([D-Ala2,N-Me-Phe4,-
Gly-ol5]enkephalin), respectively,29,36,40 and excess unlabeled pep-
tide (2 µM) established the level of nonspecific binding. After
incubation, the radiolabeled membranes containing both radioli-
gands were rapidly filtered on Whatman GF/C glass fiber filters
presoaked in 0.1% polyetheneimine in order to optimize the signal-
to-noise ratio, washed with ice-cold Tris-HCl-BSA (bovine serum
albumin) buffer, and dried at 75-80 °C for 1 h. Radioactivity was
determined using EcoLume (ICN, Costa Mesa, CA). All compounds
were analyzed in duplicate using five to eight peptide dosages and
several synaptosomal preparations in independent repetitions (n
values noted in Table 2) to ensure statistical significance. The
affinity constants (Ki) were calculated according to Cheng and
Prusoff54 using published Kd values for [3H]deltorphin-II (1.4 nM)
and [3H]DAMGO (3.5 nM).
evoked contraction and expressed as IC50 (nM) obtained from the
dose-response curves. The IC50 values represent the mean ( SE of
five to six separate assays. δ-Opioid antagonist potencies in the
MVD assay were determined against the δ agonist deltorphin-II
and are expressed as pA2 determined using the Schild plot.57 The
Schild slopes of all analogues were 0.88-1.07.
Acknowledgment. These studies were supported in part by
a Grant-in-Aid for Japan Society for the Promotion of the
Science (JSPS) Fellows (1503306) to T.L. and in part by the
Intramural Research Program of the NIH and NIEHS. The
assistance and expertise of the library staff at NIEHS are greatly
appreciated.
Supporting Information Available: Tables 1-4 listing el-
emental analysis results of all the new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
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