From the potent and selective μ opioid receptor agonist H-Dmt-D-Arg-Phe-Lys-NH2 to the potent δ antagonist H-Dmt-Tic-Phe-LyS(Z)-OH

Article abstract of DOI:10.1021/jm0504959

H-Dmt-D-Arg-Phe-Lys-NH₂ ([Dmt¹]DALDA) binds with high affinity and selectivity to the μ opioid receptor and is a potent and long-acting analgesic. Substitution of D-Arg in position 2 with Tic and masking of the lysine amine side chai

Full text of DOI:10.1021/jm0504959

5608
J. Med. Chem. 2005, 48, 5608-5611
From the Potent and Selective µ Opioid Receptor Agonist
H-Dmt-D-Arg-Phe-Lys-NH2 to the Potent δ Antagonist H-Dmt-Tic-Phe-Lys(Z)-OH
Gianfranco Balboni,*,#,† Maria Teresa Cocco,# Severo Salvadori,† Romeo Romagnoli,† Yusuke Sasaki,‡
Yoshio Okada,| Sharon D. Bryant,§ Yunden Jinsmaa,§ and Lawrence H. Lazarus§
Department of Toxicology, University of Cagliari, I-09124, Cagliari, Italy, Department of Pharmaceutical Sciences and
Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy, Tohoku Pharmaceutical University, 4-1, Komatsushima
4-chome, Aoba-Ku, Sendai 981-8558, Japan, Department of Medicinal Chemistry, Faculty of Pharmaceutical Science and High
Tecnology Research Center, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan, and Medicinal Chemistry Group,
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina 27709
Received May 26, 2005
H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) binds with high affinity and selectivity to the µ
opioid receptor and is a potent and long-acting analgesic. Substitution of D-Arg in position 2
with Tic and masking of the lysine amine side chain by Z protection and of the C-terminal
carboxylic function instead of the amide function transform a potent and selective µ agonist
into a potent and selective δ antagonist H-Dmt-Tic-Phe-Lys(Z)-OH. Such a δ antagonist could
be used as a pharmacological tool.
The development of tolerance and physical depen-
dence induced by chronic morphine administration
limits its prolonged use in the treatment of pain.1
Analgesia and tolerance to morphine are abolished in
µ opioid receptor knock-out mice, implicating the µ
opioid receptor as the primary receptor type mediating
both of these effects.2-4 However, several lines of
evidence suggest the additional involvement of the δ
opioid receptor in morphine tolerance. Initial studies
using δ opioid receptor antagonists5 and more recent
studies using δ opioid receptor knock-out mice6 were
shown to disrupt the development of tolerance. In
pharmacological studies, the selective δ receptor an-
tagonist naltridole has been shown to interact with
alternative receptors because naltridole binding was
still detected in the µ/δ/κ triple knock-out mice.7 Fur-
thermore, at high concentrations, NTI has been shown
to lose its δ selectivity and act as an agonist in some
cell types.8
The N-terminal dimethylation of opioid peptides
containing the Dmt-Tic pharmacophore drastically de-
creases δ opioid receptor agonism while enhancing δ
antagonism.9,10 Fortunately, during an attempt to pre-
pare N-terminal dimethylated analogues of selective µ
agonists dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-
NH2), endomorphin-1 (H-Tyr-Pro-Trp-Phe-NH2), endo-
morphin-2 (H-Tyr-Pro-Phe-Phe-NH2), and H-Dmt-D-
Arg-Phe-Lys-NH2 ([Dmt1]DALDA) (reference compound,
Table 1),11 we found a side chain protected [Dmt1]-
DALDA (H-Dmt-D-Arg(NO2)-Phe-Lys(Z)-NH2) (com-
pound 1, Table 1) analogue with potent activity on GPI
and MVD assays (IC50 ) 0.509 and 1.69 nM, respec-
tively). Considering the correlation between side chain
positive charge masking of D-Arg2 and Lys4 and the
potency increase for δ receptors (binding and agonist
functional bioactivity), we decided to examine thor-
oughly other modifications with the aim of converting
the potent µ agonist [Dmt1]DALDA into a potent δ
antagonist. In this work, starting from side chain
positive charge masking, we also considered the pres-
ence of a C-terminal carboxylic group (2 and 4, Table
1) and the substitution of D-Arg2 with Tic (3 and 4, Table
1) to enhance the δ opioid agonist activity and reduce µ
opioid agonist activity. The substitution of D-Arg2 with
Tic gives peptides containing the Dmt-Tic pharmaco-
phore identified by us as a potent δ opioid antagonist.12
The same modification (substitution of the second amino
acid with Tic) was reported by some of us for Leu-
enkephalin and dermorphin13 to yield quite potent δ
antagonists (pA2 ) 6.5-7.5).
All peptides were prepared in a stepwise procedure
by standard solution peptide synthesis as outlined in
Scheme 1. As an example, in this scheme is reported
the synthesis of H-Dmt-Tic-Phe-Lys(Z)-OH (4). H-Lys-
(Z) OMe or H-Lys(Z)-NH2 was condensed with Boc-Phe-
OH via WSC/HOBt. After Boc N-terminal deprotection
with TFA, each derivative was condensed with Boc-D-
Arg(NO2)-OH or Boc-Tic-OH and finally, after Boc
deprotection, with Boc-Dmt-OH (WSC/HOBt). Final
C-terminal amide compounds were obtained by Boc
deprotection with TFA, while C-terminal free carboxylic
acid was obtained by hydrolysis of the methyl ester with
1 N NaOH and then TFA treatment. Crude peptides
were purified by preparative reversed-phase HPLC.
Receptor Affinity Analysis
Receptor binding and functional bioactivity are re-
ported in Table 1. [Dmt1]DALDA, the reference com-
pound, shows high affinity and selectivity for µ recep-
tors. D-Arg2 and Lys4 side chain protection with NO2
and Z, respectively, (1) induces an 87-fold increase in δ
receptor affinity, while µ receptor affinity remains
* To whom correspondence should be addressed. Phone: +39-532-
291-275. Fax: +39-532-291-296. E-mail: gbalboni@unica.it; bbg@unife.it.
# University of Cagliari.
† University of Ferrara.
‡ Tohoku Pharmaceutical University.
| Kobe Gakuin University.
§ National Institute of Environmental Health Sciences.
10.1021/jm0504959 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/30/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5609
Table 1. Receptor Binding and Functional Bioactivity
receptor affinitya
selectivity
functional bioactivity
GPI
MVD
MVD
b
compd
structure
Kiδ (nM)
2100 ( 310
Kiµ (nM)
δ/µ
µ/δ
IC50 (nM)c
IC50 (nM)c
pA2
d
H-Dmt-D-Arg-Phe-Lys-NH2
H-Dmt-D-Arg(NO2)-Phe-Lys(Z)-NH2 24.2 ( 1.2 (3)
H-Dmt-D-Arg(NO2)-Phe-Lys(Z)-OH
H-Dmt-Tic-Phe-Lys(Z)-NH2
H-Dmt-Tic-Phe-Lys(Z)-OH
0.143 ( 0.015
0.15 ( 0.055 (6)
1.45 ( 0.12 (4)
1.14 ( 0.18 (4)
14700
161
1.41 ( 0.29
23.1 ( 2.0
1
2
3
4
0.509 ( 0.151 1.69 ( 0.26
47.6 ( 18 (3)
0.23 ( 0.04 (4)
0.019 ( 0.009 (4) 2.75 ( 0.34 (4)
33
63.0 ( 15.5
1887 ( 887
682 ( 147
>10000
>10000
5
8.42
11.43
145 >10000
a The Ki values were determined according to Chang and Prusoff,27 as detailed in the Supporting Information. The mean ( SE with n
repetitions in parentheses is based on independent duplicate binding assays with five to eight peptide doses using several different
synaptosomal preparations. b pA2 is the negative logarithm to the base 10 of the molar concentration of an antagonist that is necessary
to double the concentration of agonist needed to elicit the original submaximal response. The antagonist properties of these compounds
were tested using deltorphin C (δ opioid receptor agonist) or dermorphin (µ opioid receptor agonist). c Agonist activity was expressed as
IC50 obtained from dose-response curves. These values represent the mean ( SE for at least five fresh tissue samples. Deltorphin C and
dermorphin were the internal standards for MVD (δ opioid receptor bioactivity) and GPI (µ opioid receptor bioactivity) tissue preparations,
respectively. d Data taken from Schiller et al.11
Scheme 1. Synthesis of H-Dmt-Tic-Phe-Lys(Z)-OH
unchanged, with a loss of 90-fold selectivity. The
substitution of the C-terminal primary amide (1) with
a carboxylic acid (2) causes a small drop in δ affinity
(2-fold), µ affinity (10-fold), and selectivity (5-fold). The
replacement of D-Arg(NO2)2 with Tic (3) produces a 106-
fold increase of δ binding and an 8-fold decrease of µ
binding compared to 1, giving a compound that is
essentially nonselective (µ/δ ) 5). The change of C-

5610 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Brief Articles
terminal amide in 3 with a carboxylic acid gives 4,
endowed with high δ affinity (0.019 nM) with an
increase of 12 and more than 100000-fold compared to
3 and [Dmt1]DALDA, respectively. The affinity for µ
receptors remains in the same order of magnitude for 2
and 3. Compound 4 is quite selective for δ receptor (µ/δ
) 145). Despite the κ receptor affinity reported for
) 18.2 nM; MVD, Ke ) 0.209 nM, pA2 ) 9.68) and
H-Dmt-Tic-Phe-Phe-OH (DIPP-OH) (Ke ) 0.196 nM, pA2
) 9.71).21 Finally, considering the structures of H-Dmt-
Tic-Phe-Phe-OH and H-Dmt-Tic-Phe-Lys(Z)-OH and
their δ antagonist potency (Ke ) 0.196 nM and Ke )
0.0037 nM), the increase of about 50-fold activity must
to be assigned to the protected Lys at the C-terminal
position. Further studies are in progress about this
position. H-Dmt-Tic-Phe-Lys(Z)-OH is likely to find wide
use as a pharmacological tool in opioid research and
may also have potential as a therapeutic agent.
κ
[Dmt1]DALDA (Ki ) 22.3 nM),11 c1-4 did not show
affinity at concentrations up to 10 µM for this receptor.
Functional Bioactivity
Compounds 1-4 were tested in the electrically stimu-
lated MVD and GPI assays for intrinsic activity (Table
1). We and other investigators have previously discussed
the discrepancy of the correlation between receptor
binding affinities and functional bioactivity. Unfortu-
nately, we have neither a definitive nor comprehensive
answer for this observation.12 Our data reveal that all
analogues were inactive as antagonists in the GPI
assay. Compound 1 shows agonist activities for GPI and
MVD comparable to that of the reference compound. The
change of the C-terminal amide with the carboxylic
function (2) reduces the agonist activity for GPI and
MVD (120- and 400-fold, respectively) compared to 1.
The substitution of D-Arg(NO2)2 with Tic drastically
transforms the compound’s behavior. In fact, 3 and 4
show interesting δ antagonist activity with pA2 of 8.42
and 11.43, respectively. At the same time, they dem-
onstrate negligible or no agonist activity for the µ
receptor. As expected, the C-terminal amide (3) main-
tains a little µ agonist activity (IC50 ) 1887 nM)
compared to the C-terminal carboxylic acid (4).
Starting from the well-known µ opioid agonist tet-
rapeptide [Dmt1]DALDA, it is possible to change its
activity from µ agonist to a very potent δ antagonist by
applying modifications inducing δ antagonism, such as
the following: (1) Masking side chain positive charges
decreases µ and κ affinity. (2) Introduction of a carboxy-
lic function, especially at the C-terminal, increases δ
selectivity, and most important, (3) introduction of a Tic
residue in position 2 gives Tyr/Dmt-Tic pharmacophore
derivatives (Dmt induces opioid affinity and potency
increase without modification of receptor selectivity).14
Schiller P. W. et al. reported the synthesis of [Tic2]-
deltorphin I endowed with δ selective partial agonist
activity.15 The same introduction of Tic in position 2
(combined with the other modifications reported above)
transforms [Dmt1]DALDA into more potent δ antago-
nists such as naltridole (Ke ) 0.21 nM, pA2 ) 9.68),16
TIPP[Ψ] (Ke ) 2.89 nM, pA2 ) 8.54),17 N(Me)2Dmt-Tic-
OH (Ke ) 0.28 nM, pA2 ) 9.55),10 and H-Dmt-Tic-NH-
CH2-Bid(CH2COOH) (Ke ) 0.27 nM, pA2 ) 9.57).18
Experimental Section
Chemistry. Boc-D-Arg(NO2)-Phe-Lys(Z)-NH2. To a solu-
tion of Boc-D-Arg(NO2)-OH (0.13 g, 0.40 mmol) and TFA‚H-
Phe-Lys(Z)-NH222 (0.22 g, 0.40 mmol) in DMF (10 mL) at 0 °C
were added NMM (0.04 mL, 0.40 mmol), HOBt (0.07 g, 0.44
mmol), and WSC (0.08 g, 0.44 mmol). The mixture was stirred
for 3 h at 0 °C and for 24 h at room temperature. After DMF
was evaporated, the residue was dissolved in EtOAc and
washed with citric acid (10% in H2O), NaHCO3 (5% in H2O),
and brine. The organic phase was dried by Na2SO4 and then
by evaporation. The residue was precipitated from Et2O/Pe (1:
9, v/v): yield 0.27 g (93%); Rf(B) ) 0.84; HPLC K′ ) 6.55; mp
141-143 °C; [R]20 +28.2; MH+ 729.
D
Boc-D-Arg(NO2)-Phe-Lys(Z)-OMe. Yield 0.32 g (90%); Rf-
(B) ) 0.92; HPLC K′ 7.49; mp 150-152 °C; [R]20D +25.4°; MH+
744.
Boc-Tic-Phe-Lys(Z)-NH2. Yield 0.48 g (91%); Rf(B) ) 0.88;
HPLC K′ ) 7.98; mp 157-159 °C; [R]20 +29.3°; MH+ 687.
D
Boc-Tic-Phe-Lys(Z)-OMe. Yield 0.55 g (94%); Rf(B) ) 0.94;
HPLC K′ ) 8.64; mp 141-143 °C; [R]20 +28.5; MH+ 702.
D
TFA‚H-D-Arg(NO2)-Phe-Lys(Z)-NH2. Boc-D-Arg(NO2)-Phe-
Lys(Z)-NH2 (0.27 g, 0.37 mmol) was treated with TFA (2 mL)
for 30 min at room temperature. Et2O/Pe (1:1, v/v) was added
to the solution until the product precipitated: yield 0.27 g
(96%); Rf(A) ) 0.80; HPLC K′ ) 5.68; mp 155-157 °C; [R]20
D
+30.4; MH+ 629.
TFA‚H-D-Arg(NO2)-Phe-Lys(Z)-OMe. Yield 0.29 g (95%);
Rf(A) ) 0.89; HPLC K′ ) 6.29; mp 162-164 °C; [R]20D +27.4°;
MH+ 644.
TFA‚H-Tic-Phe-Lys(Z)-NH2. Yield 0.45 g (95%); Rf(A) )
0.84; HPLC K′ ) 6.11; mp 165-167 °C; [R]20 +32.7°; MH+
D
587.
TFA‚H-Tic-Phe-Lys(Z)-OMe. Yield 0.54 g (96%); Rf(A) )
0.77; HPLC K′ ) 7.00; mp 153-155 °C; [R]20D +30.4; MH+ 602.
Boc-Dmt-D-Arg(NO2)-Phe-Lys(Z)-NH2. To a solution of
Boc-Dmt-OH (0.10 g, 0.32 mmol) and TFA.H-D-Arg(NO2)-Phe-
Lys(Z)-NH2 (0.24 g, 0.32 mmol) in DMF (10 mL) at 0 °C were
added NMM (0.03 mL, 0.32 mmol), HOBt (0.05 g, 0.35 mmol),
and WSC (0.07 g, 0.35 mmol). The mixture was stirred for 3 h
at 0 °C and for 24 h at room temperature. After DMF was
evaporated, the residue was dissolved in EtOAc and washed
with citric acid (10% in H2O), NaHCO3 (5% in H2O), and brine.
The organic phase was dried (Na2SO4) and evaporated to
dryness. The residue was precipitated from Et2O/Pe (1:9,
v/v): yield 0.27 g (91%); Rf(B) ) 0.75; HPLC K′ ) 6.57; mp
144-146 °C; [R]20 +23.1; MH+ 920.
D
Boc-Dmt-D-Arg(NO2)-Phe-Lys(Z)-OMe. Yield 0.32 g (90%);
In 1992, Schiller et al. reported a very similar study
without knowing the starting point endomorphin-2 (H-
Tyr-Pro-Phe-Phe-NH2), a potent and selective µ agonist
identified in 1997.19 In fact, the introduction of Tic in
position 2 gave H-Tyr-Tic-Phe-Phe-NH2 (TIPP-NH2)
(GPI, IC50 ) 1700 nM; MVD, Ke ) 18.0 nM, pA2 ) 7.74).
The substitution of the C-terminal amide with the
carboxylic acid gave the first δ antagonist containing
Tic, H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) (Ke ) 4.80 nM,
pA2 ) 8.32).20 The introduction of Dmt1 in place of Tyr
transforms TIPP-NH2 and TIPP-OH into the more
potent H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) (GPI, IC50
Rf(B) ) 0.83; HPLC K′ ) 7.55; mp 141-143 °C; [R]20 +20.4;
D
MH+ 935.
Boc-Dmt-Tic-Phe-Lys(Z)-NH2. Yield 0.18 g (85%); Rf(B)
) 0.84; HPLC K′ ) 7.74; mp 161-163 °C; [R]20D +35.6°; MH+
878.
Boc-Dmt-Tic-Phe-Lys(Z)-OMe. Yield 0.38 g (83%); Rf(B)
) 0.89; HPLC K′ ) 8.53; mp 154-156 °C; [R]20 +33.6; MH+
D
893.
Boc-Dmt-D-Arg(NO2)-Phe-Lys(Z)-OH. To a solution of
Boc-Dmt-Tic-Phe-Lys(Z)-OMe (0.17 g, 0.22 mmol) in EtOH (10
mL) at room temperature, 1 N NaOH (0.27 mL, 0.27 mmol)
was added. The mixture was stirred for 3 h at room temper-
ature. After EtOH was evaporated, the residue was dissolved

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5611
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in EtOAc and washed with citric acid (10% in H2O) and brine.
The organic phase was dried (Na2SO4) and evaporated to
dryness. The residue was crystallized from Et2O/Pe (1:9, v/v):
yield 0.17 g (87%); Rf(B) ) 0.64; HPLC K′ ) 7.25; mp 154-
156 °C; [R]20 +22.8; MH+ 921.
D
Boc-Dmt-Tic-Phe-Lys(Z)-OH. Yield 0.18 g (89%); Rf(B) )
0.68; HPLC K′ ) 8.03; mp 165-167 °C; [R]20D +24.1; MH+ 879.
TFA‚H-Dmt-D-Arg(NO2)-Phe-Lys(Z)-NH2 (1). Boc-Dmt-
D-Arg(NO2)-Phe-Lys(Z)-NH2 (0.27 g, 0.29 mmol) was treated
with TFA (2 mL) for 30 min at room temperature. Et2O/Pe
(1:1, v/v) was added to the solution until the product precipi-
tated: yield 0.26 g (96%); Rf(A) ) 0.64; HPLC K′ ) 5.17; mp
165-167 °C; [R]20D +26.7; MH+ 820; 1H NMR (DMSO) δ 1.29-
1.79 (m, 10H), 2.35 (s, 6H), 2.65-3.95 (m, 9H), 4.53-5.34 (m,
5H), 6.29 (s, 2H), 7.08-7.21 (m, 10H).
TFA‚H-Dmt-D-Arg(NO2)-Phe-Lys(Z)-OH (2). Yield 0.15
g (93%); Rf(A) ) 0.52; HPLC K′ ) 6.15; mp 170-172 °C; [R]20
D
+27.9°; MH+ 821; 1H NMR (DMSO-d6) δ 1.29-1.79 (m, 10H),
2.35 (s, 6H), 2.65-3.95 (m, 9H), 4.46-5.34 (m, 5H), 6.29 (s,
2H), 7.08-7.21 (m, 10H).
TFA‚H-Dmt-Tic-Phe-Lys(Z)-NH2 (3). Yield 0.18 g (93%);
Rf(A) ) 0.70; HPLC K′ ) 6.80; mp 157-159 °C; [R]20D +35.1°;
1
MH+ 778; H NMR (DMSO-d6) δ 1.29-1.79 (m, 6H), 2.35 (s,
6H), 2.92-3.95 (m, 9H), 4.41-5.34 (m, 7H), 6.29 (s, 2H), 6.96-
7.21 (m, 14H).
TFA‚H-Dmt-Tic-Phe-Lys(Z)-OH (4). Boc, yield 0.13 g
(95%); Rf(A) ) 0.62; HPLC K′ ) 6.83; mp 164-166 °C; [R]20
D
1
+36.7°; MH+ 779; H NMR (DMSO-d6) δ 1.29-1.78 (m, 6H),
2.35 (s, 6H), 2.92-3.95 (m, 9H), 4.41-5.34 (m, 7H), 6.29 (s,
2H), 6.96-7.21 (m, 14H).
Supporting Information Available: Experimental de-
tails, NMR data, elemental analysis results, and refs 23-29.
This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) In addition to the IUPAC-IUB Commission on Biochemical
Nomenclature (J. Biol. Chem. 1985, 260, 14-42), this paper uses
the following symbols and abbreviations: DAMGO, [D-Ala2,N-
Me-Phe4,Glyol5]enkephalin; Boc, tert-butyloxycarbonyl; DELT or
deltorphin C, [D-Ala2]deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-
Gly-NH2); dermorphin, H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2;
DMF, N,N-dimethylformamide; DMSO-d6, hexadeuteriodimethyl
sulfoxide; Dmt, 2′,6′-dimethyl-L-tyrosine; [Dmt1]DALDA, H-Dmt-
D-Arg-Phe-Lys-NH2; DPDPE, cyclo-[D-Pen2,5]enkephalin; endo-
morphin-1, H-Tyr-Pro-Trp-Phe-NH2; endomorphin-2, H-Tyr-Pro-
Phe-Phe-NH2; Et2O, ethyl ether; EtOAc, ethyl acetate; EtOH,
ethyl alcohol; GPI, guinea pig ileum; HOBt, 1-hydroxybenzo-
triazole; HPLC, high-performance liquid chromatography; MALDI-
TOF, matrix-assisted laser desorption ionization time-of-flight;
MVD, mouse vas deferens; pA2, negative log of the molar
concentration required to double the agonist concentration to
achieve the original response; Pe, petroleum ether; TFA, tri-
fluoroacetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid; TLC, thin-layer chromatography; WSC, 1-ethyl-3-(3′-
dimethylaminopropyl)carbodiimide‚HCl; Z, benzyloxycarbonyl.
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Tzavara, E.; Hanoune, J.; Roques, B. P.; Kieffer, B. L. Loss of
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