Potent δ-opioid receptor agonists containing the Dmt-Tic pharmacophore

Article abstract of DOI:10.1021/jm020336e

Conversion of δ-opioid receptor antagonists containing the 2′,6′-dimethyl-L-tyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) pharmacophore into potent δ-agonists required a third heteroaromatic nucleus, such as 1H-benzimidazole-2-yl (

Full text of DOI:10.1021/jm020336e

5556
J . Med. Chem. 2002, 45, 5556-5563
P oten t δ-Op ioid Recep tor Agon ists Con ta in in g th e Dm t-Tic P h a r m a cop h or e
Gianfranco Balboni,† Severo Salvadori,‡ Remo Guerrini,‡ Lucia Negri,§ Elisa Giannini,§ Yunden J insmaa,|
Sharon D. Bryant,| and Lawrence H. Lazarus*,|
Department of Toxicology, University of Cagliari, I-09126 Cagliari, Italy, Department of Pharmaceutical Science and
Biotechnology Center, University of Ferrara, I-441000 Ferrara, Italy, Department of Human Physiology and Pharmacology
“Vitorio Erspamer”, University La Sapienza, I-00185 Rome, Italy, and Peptide Neurochemistry, LCBRA,
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received August 6, 2002
Conversion of δ-opioid receptor antagonists containing the 2′,6′-dimethyl-L-tyrosine (Dmt)-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) pharmacophore into potent δ-agonists
required a third heteroaromatic nucleus, such as 1H-benzimidazole-2-yl (Bid) and a linker of
specified length both located C-terminally to Tic in the general formula H-Dmt-Tic-NH-
CH(R)-R′. The distance between Tic and Bid is a determining factor responsible for the
acquisition of δ agonism (2, 2′, 3, 4, 6) or δ antagonism (8). Compounds containing a C-terminal
Ala (1, 1′), Asp (5), or Asn (7) with an amide (1, 1′, 5) or free acid group (7) served as δ-antagonist
controls lacking the third heteroaromatic ring. A change in chirality of the spacer (2, 2′) or
inclusion of a negative charge via derivatives of Asp (4, 6) resulted in potent δ agonism and
moderate µ agonism, although δ-receptor affinity decreased about 10-fold for 4 while µ affinity
fell by over 2 orders of magnitude. Repositioning of the negative charge in the linker altered
activity: H-Dmt-Tic-NH-CH(CH2-Bid)COOH (6) maintained high δ affinity (Ki ) 0.042
nM) and δ agonism (IC50 ) 0.015 nM), but attachment of the free acid group to Bid [H-Dmt-
Tic-NH-CH2-Bid(CH2-COOH) (9)] reconstituted δ antagonism (Ke ) 0.27 nM). The data
demonstrate that a linker separating the Dmt-Tic pharmacophore and Bid, regardless of the
presence of a negative charge, is important in the acquisition of opioids exhibiting potent δ
agonism and weak µ agonism from a parent δ antagonist.
In tr od u ction
lished after modifications to the aromatic ring of Tic
extensively weakened opioid activity.14,17 Thus, the
Dmt-Tic pharmacophore plays a significant role in
activation (agonism) or inhibition (antagonism) of the
biological response mechanism of opioid receptors that
is mediated through a G-protein-coupled second mes-
senger system.19-24
The simple pharmacophore Dmt-Tic1 can acquire
differential bioactivity through N- or C-terminal modi-
fications; namely, N,N-methylation enhanced δ affinity,
while C-terminal modifications by various hydrophobic
groups or a linker and a Bid group increased binding
to µ-opioid receptors by orders of magnitude, resulting
in compounds exhibiting δ agonism in addition to
substances with both δ antagonism and µ agonism.2-5
Although the Dmt-Tic pharmacophore is the prototype
for a family of highly selective δ-opioid receptor antago-
nists,6 this remarkable pseudodipeptide can be trans-
formed into a potent δ agonist by the C-terminal
addition of a linker and a 1H-benzimidazole-2-yl (Bid)
group.4 Numerous data in the literature demonstrate
the capacity of Dmt as the N-terminal residue to
enhance and modify the biological activity of a vast
array of opioid compounds, including transforming
deltorphin B into a high dual affinity δ/µ agonist and
enhancing the activity of DPDPE, DALDA, endomor-
phin-2 analogues, enkephalin, which also exhibited
extraordinary stability, and [Dmt1,Tic2]dynorphin A(1-
11)-NH2, which became a δ antagonist.4-18 Moreover,
the requirement of an unaltered Tic residue was estab-
Ra tion a le
The remarkable transformation of the Dmt-Tic phar-
macophore from a δ antagonist into a potent δ agonist4
initiated further investigations to examine our original
hypothesis; namely, the length of the linker between Tic
and Bid or another aromatic nucleus mediates the
alteration of functional bioactivity.4 It is well-known
that the coupling of various hydrophobic substituents
at the C terminus of Tic, a reaction that simultaneously
removes the negative charge, promotes interaction with
µ-opioid receptors3,5,25 to produce bifunctional mol-
ecules.3,4 Whereas studies on the TIPP family of opioid
peptides indicated that hydrophobic C-terminal exten-
sions resulted in analogues producing δ agonism or δ
antagonism with µ agonism, it was only a Dmt deriva-
tive (DIPP-NH2[ψ]) that exhibited the highest activity
toward both δ- and µ-opioid receptors.13 On the other
hand, extensive studies by Salvadori et al.3 and Page´
et al.5 verified the appearance of dual bioactivity profiles
(δ agonism/δ antagonism with µ agonism) using a series
of Dmt-Tic analogues, which contained hydrophobic or
aromatic substituents at the C terminus.
* To whom correspondence should be addressed. Address: Peptide
Neurochemistry, MD C3-04, NIEHS, P.O. Box 12233, Research
Triangle Park, NC 27709. Phone: 919-541-3238. Fax: +1-919-541-
0626. E-mail: lazarus@niehs.nih.gov.
† University of Cagliari.
‡ University of Ferrara.
This study unveils a new series of H-Dmt-Tic-NH-
CH(R)-R′ analogues (Figure 1) in which a third het-
§ University La Sapienza.
| National Institute of Environmental Health Sciences.
10.1021/jm020336e CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/09/2002

δ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5557
Sch em e 1. Synthesis of Compound 2
[H-Dmt-Tic-NH-CH(CH3)-Bid]
F igu r e 1. General structures of H-Dmt-Tic-NH-CH(R)-
Rd analogues. The asterisk (/) denotes chirality.
cyclization and dehydration in acetic acid (AcOH), as
outlined in Schemes 1 and 2, respectively. N-Alkylation
of Bid in position 1 (9) was obtained by treatment of
Boc-NH-CH2-Bid4 with K2CO3 and Br-CH2-COOEt
(bromoacetic acid ethyl ester). After N-terminal depro-
tection with trifluoroacetic acid (TFA), each derivative
was condensed with Boc-Tic-OH (Boc was deprotected
with TFA) and then with Boc-Dmt-OH via WSC/HOBt
(1-ethyl-3-[(3′-dimethyl)aminopropyl]carbodiimide/1-hy-
droxy-1,2,3-benzotriazole). In the final compounds, ben-
zyl esters, ethyl ester, and Boc protecting groups were
removed by catalytic hydrogenation (Pd/C, 5%), 1 N
NaOH, and TFA treatment, respectively. H-Dmt-Tic-
D-Ala-NH2 was prepared as previously reported for
H-Dmt-Tic-Ala-NH2.27 H-Dmt-Tic-Asn-OH was
prepared in a stepwise procedure starting from H-Asn-
OBzl28 that was condensed with Boc-Tic-OH and then
with Boc-Dmt-OH via WSC/HOBt.
eroaromatic center (Bid) was used to evaluate the
potential of converting the δ-opioid antagonist pharma-
cophore, H-Dmt-Tic-R (R ) amino acid, -OH, -NH2,
-CHOH), into a δ-agonist pharmacophore. Analogues
containing Ala and R-aminocarboxylic acids, such as Asp
and Asn, were further tested as control compounds to
explore the biological effect with δ- and µ-opioid recep-
tors in the absence of Bid. In addition, the effect of a
negative charge on bioactivity was investigated because
previous studies with opioid peptides revealed that the
presence of a negative charge repels ligands from the µ
receptor with minimal effect on δ affinity.26 Finally, four
aspects regarding the linker between Tic2 and Bid were
examined: (i) the addition of linear and branched alkyl
groups between the amide and Bid (2, 2′, 3, 8); (ii) the
stereochemistry of the carbon atom between the amide
and Bid (2, 2′); (iii) the position of Bid in the molecule
(R or R′, Figure 1; 4, 6); and (iv) the addition of a
carboxylic function to the linker or the nitrogen of Bid
(4, 6, 9, Figure 1).
Resu lts
Recep tor In ter a ction s. While all the peptides listed
in Table 1 exhibited high δ affinity, several analogues
(2, 2′, 3, 6, 8, 9) demonstrated exceptional Ki values
(<0.1 nM). On the other hand, only three Bid-containing
peptides had high µ affinities (Ki < 1 nM; 2, 2′, 3); the
µ affinities of the other compounds ranged from 5 to 80
nM. Alkylation of the carbon atom between the amide
and Bid (2, Figure 1) did not substantially modify δ-
and µ-receptor affinities and pharmacological activities
(infra vide) compared to the reference δ agonist, Dmt-
Tic-NH-CH2-Bid (3). The corresponding diastereomer
Ch em istr y
All peptides and pseudopeptides were prepared in a
stepwise procedure by standard solution peptide syn-
thesis methods. Mixed carbonic anhydride coupling of
tert-butyloxycarbonyl (Boc)-Ala-OH, Boc-D-Ala-OH,
Boc-Asp-OBzl, or Boc-Asp(OBzl)-OH with o-phenyl-
endiamine gave the corresponding crude intermediate
monoamides, which were converted without purification
to the desired 1H-benzimidazol-2-yl (Bid) derivatives by

5558 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Balboni et al.
Ta ble 1. Receptor Affinity and Functional Bioactivity of Analogues Containing the Dmt-Tic Pharmacophore
functional bioactivityb
receptor affinitya (nM)
MVD
GPI
IC50 (nM)
peptide
Ki(δ)
Ki(µ)
µ/δ
IC50 (nM)
Ke (nM)
1, H-Dmt-Tic-Ala-NH2
0.241 ( 0.02 (5)
0.301 ( 0.021 (4)
0.063 ( 0.004 (3)
0.047 ( 001 (3)
0.035 ( 0.006 (3)
0.443 ( 0.043 (4)
0.289 ( 0.018 (3)
0.042 ( 0.017 (4)
1.10 ( 0.26 (5)
47.1 ( 3.4 (4)
47.5 ( 4.6 (3)
0.411 ( 0.016 (3)
0.233 ( 0.01 (3)
0.50 ( 0.054 (3)
53.9 ( 4.4 (3)
2,565 ( 290 (3)
21.68 ( 5.5 (3)
80.3 ( 8.2 (4)
195
158
6.5
10.0 ( 1.3c
25.1 ( 1.1
4740 ( 345c
>10000
71.7 ( 8.2
6.36 ( 0.7
40.7 ( 5
1′, H-Dmt-Tic-D-Ala-NH2
2, H-Dmt-Tic-NH-CH(CH3)-Bid
2′, H-Dmt-Tic-NH(R)CH(CH3)-Bid
3, H-Dmt-Tic-NH-CH2-Bid
4, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid
5, H-Dmt-Tic-Asp-NH2
6, H-Dmt-Tic-NH-CH(CH2-Bid)-COOH
7, H-Dmt-Tic-Asn-OH
8, H-Dmt-Tic-NH-CH2-CH2-Bid
9, H-Dmt-Tic-NH-CH2-Bid(CH2-COOH) 0.021 ( 0.0025 (4)
deltorphin C
dermorphin
0.26 ( 0.03
0.026 ( 0.002
0.035 ( 0.003
0.12 ( 0.02
5
14d
122
1724 ( 150
>10000
8875
516
73
7.58 ( 0.31
0.015 ( 0.003
1558 ( 153
>10000
5.88 ( 0.21
4.78 ( 0.37e
0.27 ( 0.015 3193 ( 402
>1200
0.067 ( 0.015 (4)
5.49 ( 0.03 (3)
6.92 ( 0.25 (4)
272 ( 50 (11)
82
107.5 ( 11.4e
330
1135f
146g
0.24 ( 0.06 (6)
178.6 ( 18 (15)
0.30 ( 0.012
16.5 ( 1.8
1.22 ( 0.13 (22)
1.21 ( 0.18
a
The Ki values (nM) were determined according to the Chang and Prusoff43 as detailed in Experimental Section. The mean ( SE with
n repetitions in parentheses is based on independent assays with five to eight peptide doses using multiple synaptosome preparations.
b
Agonist activity was expressed as IC50 (Ke) obtained from concentration-response curves. These values represent the mean ( SE of at
least five fresh tissue samples. [D-Ala2]Deltorphin I (deltorphin C)11 and dermorphin were the internal standards for MVD (δ bioactivity)
and GPI (µ bioactivity) tissue preparations, respectively. c Data from Salvadori et al.6 Receptor binding data from Balboni et al.4 These
d
bioassay data were not reported previously. e Data taken from Balboni et al.4 f Binding assay data from Salvadori et al.44 Receptor
g
data from Lazarus et al.41
Sch em e 2. Synthesis of Compound 6
[H-Dmt-Tic-NH-CH(CH2-Bid)-COOH)]
function (4, Figure 1) improved δ selectivity relative to
2, 2′, and 3 even though δ affinity fell by about 10-fold
(Table 1). Compound 5 with a very weak Ki(µ) (2565 nM)
had the highest δ selectivity, comparable to many Dmt-
Tic analogues with a free C-terminal carboxyl group.2,3,5,6
In the Bid analogues containing carboxylate groups
(4, 6, 9), µ affinities diminished and δ selectivities
increased, which verified the hypothesis that a negative
charge in an opioid ligand retards binding to µ receptors
without detrimental effects on δ affinity.11,26 However,
the position of the carboxyl group relative to the Bid
moiety (compare 6 and 9) influenced µ binding to a
greater extent than that for the δ receptor. Bioactivity
data for analogues containing a different chirality at the
C-terminal of Dmt-Tic indicated that the change in
chirality had essentially no influence on the bioactivity
profiles (compare pairs of compounds 1 + 2 and 1′ +
2′).
P h a r m a cologica l Activity in Vitr o. Several pep-
tides (2, 2′, 3, 4, 6) acted as potent δ-opioid agonists.
The IC50 values ranged nearly 20-fold, from 0.015 to 0.26
nM in isolated organ preparations (Table 1), and they
were many times more effective in inhibiting electrically
evoked contractions in the mouse vas deferens (MVD)
than in the guinea-pig ileum (GPI). Interestingly, the
analogues containing carboxylic acids (4, 6, 9) exhibited
very weak µ agonism relative to 2, 2′, and 3. In addition,
compound 9, which displayed the highest δ affinity, was
inactive as an agonist in the MVD preparation at a
concentration as high as 137 µM. In fact, compound 9
behaved as a potent antagonist. displacing the [D-Ala2]-
deltorphin I dose response curve to the right (Ke ) 0.27
nM) with a weak bioactive response on GPI (IC50 ) 3193
nM).
Analogues 1, 1′, 5, and 7 were pure δ antagonists that
lacked the third aromatic center, Bid. These control
compounds exhibited micromolar activity on GPI, while
in contrast, compound 8, a potent δ antagonist contain-
ing Bid, displayed weak µ agonism (Table 1). None of
the peptide analogues (Table 1) exhibited µ antagonism.
Molecu la r Mod elin g. H-Dmt-Tic-NH-CH2-Bid,
a δ-opioid receptor agonist (3), was modeled using
coordinates for Dmt and Tic from the X-ray crystal
of 2 (2′, Figure 1) maintained comparable δ selectivity
and potent agonist activity even though µ affinity and
agonist potency increased. The control compounds,
containing Ala-NH2 instead of Bid (1, 1′, Figure 1),
exhibited equivalent δ affinities and selectivities. In
addition, the tripeptide analogue controls with C-
terminal Asp and Asn (5 and 7, respectively) had
relatively good δ affinities (0.289 and 1.10 nM, respec-
tively) and δ-antagonist bioactivities [Ke(mouse vas
deferens) ) 7.58 and 5.88 nM, respectively]. The alkyl-
ation of the side chain of compound 2 with a carboxylic

δ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5559
chemical modification applied to 4 to obtain analogue 2
elevated µ affinity about 130-fold, since it is known that
hydrophobic peptides enhance µ-receptor interaction.3,5
However, a change in pharmacological activity was not
observed; in other words, both 4 and 2 retained strong
δ agonism. In a similar chemical approach, deletion of
the COOH function of TIP gave weak δ agonists,36 while
in analogues containing Trp, removal of the COOH
function failed to modify δ antagonism.37
Finally, alkylation with CH2-COOH on the nitrogen
of Bid (9) was carried out in order to determine if a
negative charge on the heteroaromatic function could
modify δ selectivity as well as the biological properties.
Comparison of the biological data of 9 with data of 6
and 4 (Figure 1 and Table 1) indicated a δ affinity
relatively similar to that of 6 but 20-fold greater than
that of 4; compound 9 quite unexpectedly revealed
potent δ-antagonist activity with Ke ) 0.27 nM. These
data suggest that the alkylation of the nitrogen of Bid
can affect the acquisition of agonism or antagonism. A
previous study exploring the chemical composition and
length of the linker between Tic and Bid indicated that
the distance between the aromatic groups may be
responsible for the manifestation of δ agonism (compare
3 and 8), but the present study demonstrated that
compound 6 with the same distance as that of 8 was a
potent δ agonist.4
Molecular modeling of 3 revealed that a number of
low-energy forms were accessible with unique orienta-
tions of the Bid pharmacophore (Figure 2) and the
addition of an acetate group to Bid may limit the
conformational freedom, thereby resulting in δ antago-
nism. The adaptability of the ligand to the δ-receptor
binding pocket may be an important factor for receptor
activation as seen in the X-ray diffraction analysis of
three Dmt-Tic analogues,29b which were, along with
other opioid molecules, docked into a model of the
δ-opioid receptor based on the crystalline structure of
bovine rhodopsin.29c Those docking studies29c further
demonstrated that binding sites for δ agonists and δ
antagonists were contained in distinct but overlapping
regions of the δ-opioid receptor. The molecular modeling
demonstrated the accessible conformational space of the
C-terminal portion of these peptides based on the Dmt -
Tic pharmacophore generated from the X-ray crystal-
lography data.29b These conformation studies provide
valuable information for docking to opioid receptors29c
in comparison with other bioactive opioids in order to
develop a working hypothesis for the mechanism of
activating their respective receptors. Most importantly,
however, the models provide a molecular template upon
which to design new opioid compounds that interact
with specific opioid receptors.
F igu r e 2. Backbone superimpositions of four low-energy
conformers of the δ-opioid receptor agonist H-Dmt-Tic-NH-
CH2-Bid (3).
structure of N,N-(Me)2-Dmt-Tic-OH (δ antagonist)29b
(Figure 2). Features of the Dmt-Tic pharmacophore
include cis orientation around the peptide bond, ap-
proximately 5 Å between the aromatic rings, and near-
parallel orientation of the aromatic ring structures.29
Conformations describing the C-terminal modifications
were generated by extensive conformational searching
and energy minimization using InsightII. One-hundred
forty-seven low-energy conformers were generated by
the search, and four of the lowest energy conformations
with unique C-terminal orientations were superimposed
for comparison and evaluation as potential bioactive
forms (Figure 2). The relative energies of the four
unique low-energy structures ranged from 67 to 97 kcal/
mol, and centroid distances between the aromatic rings
of Tic and Bid ranged from 6.7 to 9.9 Å. The most
notable structural variations were observed around
bonds between the amide and methylene linker, as well
as between the methylene linker and Bid (Figure 2).
Discu ssion
Opioid peptide agonists for the δ-opioid receptor
include a vast array of enkephalin derivatives,30 as well
as J OM-13 cyclic analogues,31-33 the potent deltorphin
family of amphibian skin peptides,11 and the unique
peptide H-Dmt-Tic-NH-CH2-Bid4 (Figure 1 and
Table 1). These new pseudopeptides containing Dmt and
Tic, well-known for their δ antagonism25,29 and potent
δ-inverse agonism,34 provided templates for the forma-
tion of δ opioids endowed with potent δ-agonist activity
(Table 1).
The improvement in δ selectivity of 4, 6, and 9 relative
to other Bid-containing peptides and the elevated
selectivity of 5 confirm earlier observations that a
negative charge in an opioid peptide is detrimental for
µ-receptor interaction.26 Furthermore, the absence of the
Bid pharmacophore (1, 1′, 5, 7) (Figure 1 and Table 1)
resulted in δ-antagonist activity as observed previ-
ously.6,35 On the other hand, Bid is not exclusively
associated with δ agonism because compounds 8 and 9
were potent δ antagonists (Table 1). Quite interestingly,
deletion of the carboxylic acid function of 6 to yield 8
(Figure 1, Table 1) not only improved µ affinity as
expected but also shifted the pharmacological activity
from a δ agonist to a δ antagonist. Similarly, the same
Con clu sion s
Several analogues in the H-Dmt-Tic-NH-CH(R)-
R′ series of peptides containing Bid have exceptionally
high δ agonism in vitro, while slight modifications
reverted the peptide back to its original state, namely,
to a potent δ antagonist. Peptides 2, 2′, 3, and 4
contained a relatively short linker, -NH-CH2- (Figure
1), between the Dmt-Tic pharmacophore and the het-
eroaromatic ring Bid that yielded δ agonism regardless
of the side chain (2, 2′, 4) or chirality (2, 2′). As seen

5560 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Balboni et al.
Ta ble 2. Analysis Results of Dmt-Tic Pharmacophore Compoundsa
compound
MH+, m/z
analytical
New Compounds
TFA‚H-Dmt-Tic-D-Ala-NH2 (1′)
439
512
512
556
483
556
483
(C24H30N4O4‚TFA) C, H, N
(C30H33N5O3‚2TFA) C, H, N
(C30H33N5O3‚2TFA) C, H, N
(C31H33N5O5‚2TFA) C, H, N
(C25H30N4O6‚TFA) C, H, N
(C31H33N5O5‚2TFA) C, H, N
(C25H30N4O6‚TFA) C, H, N
(C31H33N5O5‚2TFA) C, H, N
2TFA‚H-Dmt-Tic-NH-(S)CH(CH3)-Bid (2)
2TFA‚H-Dmt-Tic-NH-(R)CH(CH3)-Bid (2′)
2TFA‚H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4)
TFA‚H-Dmt-Tic-Asp-NH2 (5)
2TFA‚H-Dmt-Tic-NH-(S)CH(CH2-Bid)-COOH (6)
TFA‚H-Dmt-Tic-Asn-OH (7)
2TFA‚H-Dmt-Tic-NH-CH2-Bid(N1-CH2-COOH) (9)556
Published Compounds
TFA‚H-Dmt-Tic-Ala-NH2 (1)6
2TFA‚H-Dmt-Tic-NH-CH2-Bid (3)4
TFA‚H-Dmt-Tic-NH-CH2-CH2-Bid (8)4
a
The analysis of the new compounds detailed in Experimental Section are included. The data on those compounds previously published
are referenced.
previously4 and with compound 8 (Table 1), increasing
the length of the linker reconstituted δ antagonism;
however, the addition of a carboxyl group restored δ
agonism (6). Furthermore, the alkylation of the Bid
nitrogen (9) caused an unanticipated reversion from δ
agonism to δ antagonism (Table 1). These data signify
that the addition of Bid and the carboxylic group to
improve δ-opioid receptor selectivity of the Dmt-Tic
pharmacophore does not guarantee δ agonism. Instead,
the precise location and special orientation of these
chemical groups is critical for exhibiting agonist or
antagonist behaviors. These stimulating data serve as
a springboard to launch the design of new opioid
analogues containing other types of linkers, functional
groups, and heteroaromatic nuclei that affect bioactivity
in a similar manner that may lead to answers regarding
opioid receptor activation and the development of
compounds suitable for clinical and therapeutic applica-
tions.
tography (2 cm × 70 cm, 0.7-1 g of material) was run on silica
gel 60 (70-230 mesh, Merck) using the same eluent systems.
Melting points were determined on a Kofler apparatus and
are uncorrected. Optical rotations were determined at 10 mg/
mL in methanol with a Perkin-Elmer 241 polarimeter with a
1
10 cm water-jacketed cell. All H nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker 200 MHz spectrom-
eter. MALDI-TOF (matrix-assisted laser desorption ionization
time-of-flight) mass spectrometry of peptides was conducted
using a Hewlett-Packard G 2025 A LD-TOF system. The
samples were analyzed in the linear mode with 28 kV
accelerating voltage, mixing them with a saturated solution
of R-cyano-4-hydroxycinnamic acid matrix. The analysis re-
sults for the new compounds are in Table 2.
Boc-Tic-NH-(S)CH(CH3)-Bid . To a solution of Boc-
Tic-OH (0.55 g, 2 mmol) and 2 TFA‚H2N-(S)CH(CH3)-Bid
[1-(1H-benzimidazol-2-yl)ethylamine]39 (0.8 g, 2 mmol) in DMF
(10 mL) at 0 °C were added NMM (0.44 mL, 4 mmol), HOBt
(0.34 g, 2.2 mmol), and WSC (0.42 g, 2.2 mmol). The reaction
mixture was stirred for 3 h at 0 °C and for 24 h at room
temperature. After DMF was evaporated, the residue was
solubilized in EtOAc and washed with NaHCO3 (5%) and brine.
The organic phase was dried and evaporated to dryness. The
residue was crystallized from Et2O/Pe (1:9, v/v): yield 0.7 g
Exp er im en ta l Section
(83%); Rf(B) ) 0.81; HPLC K′ ) 7.42; mp 215-217 °C; [R]20
D
Ma ter ia ls. H-Dmt-OH was prepared according to Dygos
et al.,38 and the purity and chirality were compared to a sample
generously donated by J . H. Dygos. Boc-Tic-OH was pur-
chased from Bachem (Heidelberg, Germany). The radioactive
opioid ligands were from commercial sources: [3H]DPDPE
(32.0 Ci/mmol; NEN-DuPont, Billerica, MA) and [3H]DAGO
(58.0 Ci/mmol, Amersham, Arlington Heights, IL).
-18.5°; MH+ 421; 1H NMR (DMSO) δ 1.38-1.42 (d, 9H), 1.56
(d, 3H), 3.08-3.15 (m, 2H), 4.22-4.53 (m, 4H), 7.05-7.71 (m,
9H), 7.97 (s, 1H).
2TF A‚H-Tic-NH-(S)CH(CH3)-Bid . Boc-Tic-NH-(S)-
CH(CH3)-Bid (0.4 g, 0.95 mmol) was treated with TFA (1 mL)
for 0.5 h at room temperature. Et2O/Pe (1:1, v/v) was added to
the solution until the product precipitated: yield 0.49 g (95%);
Gen er a l Meth od s. Crude peptides were purified by pre-
parative reversed-phase high-performance liquid chromatog-
raphy (HPLC) using a Waters Delta Prep 4000 system with a
Waters PrepLC 40 mm assembly column C18 (30 cm × 4 cm,
300 Å, 15 µm particle size column). The column was perfused
at a flow rate of 40 mL/min with mobile-phase solvent A (10%
acetonitrile in 0.1% TFA, v/v), and a linear gradient from 0%
to 50% of solvent B (60%, acetonitrile in 0.1% TFA, v/v) in 25
min was adopted for the elution of the products. Analytical
HPLC analyses were performed with a Beckman System Gold
with a Beckman ultrasphere ODS column (5 µm; 4.6 mm ×
250 mm). Analytical determinations and capacity factor (K′)
of the products were determined using HPLC conditions in
the above solvent systems (solvents A and B) programmed at
a flow rate of 1 mL/min using the following linear gradients:
(a) from 0% to 100% B in 25 min and (b) from 10% to 70% B
in 25 min. All analogues showed less than 1% impurities when
monitored at 220 and 254 nm.
Rf(A) ) 0.36; HPLC K′ ) 5.32; mp 180-182 °C; [R]20 -20.9°;
D
MH+ 321.
Boc-Dm t-Tic-NH-(S)CH(CH3)-Bid . To a solution of
Boc-Dmt-OH (0.11 g, 0.37 mmol) and 2TFA‚H-Tic-NH-
(S)CH(CH3)-Bid (0.2 g, 0.37 mmol) in DMF (10 mL) at 0 °C
were added NMM (0.08 mL, 0.74 mmol), HOBt (0.063 g, 0.41
mmol), and WSC (0.079 g, 0.41 mmol). The reaction mixture
was stirred for 3 h at 0 °C and for 24 h at room temperature.
After DMF was evaporated, the residue was solubilized in
EtOAc and washed with NaHCO3 (5%) and brine. The organic
phase was dried and evaporated to dryness. The residue was
crystallized from Et2O/Pe (1:9, v/v): yield 0.18 g (81%); Rf(B)
) 0.77; HPLC K′ ) 7.8; mp 143-144 °C; [R]20 -24.6°; MH+
D
1
611; H NMR (DMSO) δ 1.32-1.39 (d, 9H), 1.55 (d, 3H), 2.17
(s, 6H), 2.85-3.10 (m, 4H), 4.22-4.65 (m, 5H), 6.37 (s, 2H),
7.05-7.71 (m, 10H), 7.97 (s, 1H).
2TF A‚H -Dm t -Tic-NH -(S)CH (CH 3)-Bid (2). Boc-
Dmt-Tic-NH-(S)CH(CH3)-Bid (0.061 g, 0.1 mmol) was
treated with TFA (1 mL) for 0.5 h at room temperature. Et2O/
Pe (1:1, v/v) was added to the solution until the product
precipitated: yield 0.09 g (93%); Rf(A) ) 0.46; HPLC K′ ) 4.34;
Thin-layer chromatography (TLC) was performed on pre-
coated plates of silica gel F254 (Merck, Darmstadt, Germany)
using the following solvent systems: (A) 1-butanol/AcOH/H2O
(3:1:1, v/v/v); (B) CH2Cl2/toluene/methanol (17:1:2, v/v/v). Nin-
hydrin (1%, Merck), fluorescamine (Hoffman-La Roche), and
chlorine reagents were used as sprays. Open column chroma-
mp 158-160 °C; [R]20 -26.1°; MH+ 512. Anal. (C30H33N5O3‚
D
2TFA) C, H, N.

δ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5561
Boc-Tic-NH-(R)CH(CH3)-Bid . This intermediate was
obtained by condensation of Boc-Tic-OH with 2TFA‚H2N-
(R)CH(CH3)-Bid via WSC/HOBt as reported for Boc-Tic-
filtration, the solution was evaporated to dryness. The residue
was crystallized from Et2O/Pe (1:9, v/v): yield 0.24 g (94%);
Rf(B) ) 0.76; HPLC K′ ) 7.1; mp 175-177 °C; [R]20 +4.8°;
D
NH-(S)CH(CH3)-Bid: yield 0.68 g (82%); Rf(B) ) 0.79; HPLC
MH+ 656.
1
K′ ) 7.38; mp 215-217 °C; [R]20 +3.5°; MH+ 421; H NMR
2TF A‚H-Dm t-Tic-NH-(S)CH(CH2-Bid )-COOH (6).
Boc-Dmt-Tic-NH-(S)CH(CH2-Bid)-COOH was treated
with TFA as reported for 2TFA‚H-Dmt-Tic-NH-(S)CH-
(CH3)-Bid: yield 0.17 g (96%); Rf(A) ) 0.87; HPLC K′ ) 2.93;
D
(DMSO) δ 1.38-1.40 (d, 9H), 1.56 (d, 3H), 3.08-3.17 (m, 2H),
4.20-4.54 (m, 4H), 7.05-7.71 (m, 9H), 7.97 (s, 1H).
2TF A‚H-Tic-NH-(R)CH(CH3)-Bid . Boc-Tic-NH-(R)-
CH(CH3)-Bid was treated with TFA as reported for 2TFA‚
H-Tic-NH-(S)CH(CH3)-Bid: yield 0.36 g (94%); Rf(A) )
0.41; HPLC K′ ) 5.75; mp 180-182 °C; [R]20D +7.9°; MH+ 321.
Boc-Dm t-Tic-NH-(R)CH(CH3)-Bid . This compound
was obtained by condensation of Boc-Dmt-OH with 2TFA‚
H-Tic-NH-(R)CH(CH3)-Bid via WSC/HOBt as reported for
Boc-Dmt-Tic-NH-(S)CH(CH3)-Bid: yield 0.17 g (79%); Rf-
(B) ) 0.85; HPLC K′ ) 7.2; mp 142-144 °C; [R]20D +12.6°; MH+
mp 165-167 °C; [R]20 +6.2°; MH+ 556. Anal. (C31H33N5O5‚
D
2TFA) C, H, N.
3-(1H-Ben zim id a zol-2-yl)-3-ter t-bu toxyca r bon yla m in o-
p r op ion ic Acid Ben zyl Ester [Boc-NH-(S)CH(CH2-
COOBzl)-Bid ]. This compound was obtained by condensation
of Boc-Asp(OBzl)-OH with o-phenylendiamine via IBCF as
reported for Boc-NH-(S)CH(CH2-Bid)-COOBzl: yield 1.62
g (82%); Rf(B) ) 0.52; HPLC K′ ) 6.5; mp 136-138 °C; [R]20
D
+15.8°; MH+ 396; 1H NMR (DMSO) δ 1.40 (s, 9H), 2.80 (d,
4H), 5.34-5.51 (m, 3H), 7.19-7.70 (m, 10H), 7.99 (s, 1H).
2TF A‚H 2N-(S)CH (CH 2-COOBzl)-Bid . Boc-NH-(S)-
CH(CH2-COOBzl)-Bid was treated with TFA as reported for
2TFA‚H2N-(S)CH(CH2-Bid)-COOBzl: yield 1.26 g (95%); Rf-
(A) ) 0.78; HPLC K′ ) 4.2; mp 142-144 °C; [R]20D +17.3°; MH+
296.
1
611; H NMR (DMSO) δ 1.32-1.39 (d, 9H), 1.55 (d, 3H), 2.17
(s, 6H), 2.85-3.15 (m, 4H), 4.20-4.65 (m, 5H), 6.34 (s, 2H),
7.05-7.72 (m, 10H), 7.97 (s, 1H).
2TFA‚H-Dm t-Tic-NH(R)CH(CH3)-Bid (2′). Boc-Dmt-
Tic-NH-(R)CH(CH3)-Bid was treated with TFA as reported
for 2TFA‚H-Dmt-Tic-NH-(S)CH(CH3)-Bid: yield 0.088 g
(96%); Rf(A) ) 0.40; HPLC K′ ) 3.78; mp 152-154 °C; [δ]20
D
+16.1°; MH+ 512. Anal. (C30H33N5O3‚2TFA) C, H, N.
Boc-Tic-NH -(S)CH (CH 2-COOBzl)-Bid . This com-
pound was obtained by condensation of Boc-Tic-OH with
2TFA‚H2N-(S)CH(CH2-COOBzl)-Bid via WSC/HOBt as re-
ported for Boc-Tic-NH-(S)CH(CH3)-Bid: yield 0.76 g (84%);
3-(1H-Ben zim id a zol-2-yl)-2-ter t-bu toxyca r bon yla m in o-
p r op ion ic Acid Ben zyl Ester [Boc-NH-(S)CH(CH2-
Bid )-COOBzl]. A solution of Boc-Asp-OBzl (1 g, 3.1 mmol)
and 4-methylmorpholine (NMM, 0.34 mL, 3.1 mmol) in N,N-
dimethylformamide (DMF, 10 mL) was treated at -20 °C with
isobutyl chloroformate (IBCF, 0.4 mL, 3.1 mmol). After 10 min
at -20 °C, o-phenylendiamine (0.33 g, 3.1 mmol) was added.
The reaction mixture was allowed to stir while slowly warming
to room temperature (1 h) and was then stirred for 3 h. The
solvent was evaporated, and the residue was partitioned
between ethyl acetate (EtOAc) and H2O. The EtOAc layer was
washed with 5% NaHCO3 and brine and dried over Na2SO4.
The solution was filtered, the solvent was evaporated, and the
residual solid was dissolved in glacial AcOH (10 mL). The
solution was heated at 65 °C for 1 h. After the solvent was
evaporated, the residue was crystallized from diethyl ether
(Et2O)/petroleum ether (Pe) (1:9, v/v): yield 0.98 g (80%); Rf-
(B) ) 0.79; HPLC K′ ) 6.6; mp 141-143 °C; [R]20D +12.1°; MH+
Rf(B) ) 0.76; HPLC K′ ) 4.05; mp 159-161 °C; [R]20 +10.3°;
D
MH+ 555; 1H NMR (DMSO) δ 1.40 (s, 9H), 2.80-3.05 (m, 4H),
4.22-5.51 (m, 6H), 6.96-7.70 (m, 14H), 7.98 (s, 1H).
2TFA‚H-Tic-NH-(S)CH(CH2-COOBzl)-Bid. Boc-Tic-
NH-(S)CH(CH2-COOBzl)-Bid was treated with TFA as
reported for 2TFA‚H-Tic-NH-(S)CH(CH3)-Bid: yield 0.62
g (91%); Rf(A) ) 0.67; HPLC K′ ) 3.49; mp 152-154 °C; [R]20
D
+11.8°; MH+ 455.
Boc-Dm t -Tic-NH-(S)CH(CH 2-COOBzl)-Bid . This
compound was obtained by condensation of Boc-Dmt-OH
with 2TFA‚H2N-(S)CH(CH2-COOBzl)-Bid via WSC/HOBt
as reported for Boc-Dmt-Tic-NH-(S)CH(CH3)-Bid: yield
0.47 g (83%); Rf(B) ) 0.63; HPLC K′ ) 9.11; mp 171-173 °C;
[R]20D +4.3°; MH+ 746; 1H NMR (DMSO) δ 1.40 (s, 9H), 2.35-
3.05 (m, 12H), 4.46-5.51 (m, 7H), 6.29 (s, 2H), 6.96-7.70 (m,
13H), 7.99 (s, 1H).
1
396; H NMR (DMSO) δ 1.40 (s, 9H), 3.09 (d, 2H), 4.86-5.30
(m, 3H), 7.19-7.70 (m, 10H), 7.98 (s, 1H).
Boc-Dm t -Tic-NH -(S)CH (CH 2-COOH )-Bid . Boc-
Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid was treated with
C/Pd and H2 as reported for Boc-Dmt-Tic-NH-(S)CH(CH2-
Bid)-COOH: yield 0.28 g (92%); Rf(B) ) 0.79; HPLC K′ ) 6.9;
2TF A‚H 2N-(S)CH (CH 2-Bid )-COOBzl. Boc-NH-(S)-
CH(CH2-Bid)-COOBzl (0.9 g, 2.28 mmol) was treated with
TFA (1 mL) for 0.5 h at room temperature. Et2O/Pe (1:1, v/v)
was added to the solution until the product precipitated; yield
1.11 g (93%); Rf(A) ) 0.83; HPLC K′ ) 4.85; mp 152-154 °C;
mp 173-175 °C; [R]20 +5.2°; MH+ 656.
D
2TF A‚H-Dm t-Tic-NH-(S)CH(CH2-COOH)-Bid (4).
Boc-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid was treated
with TFA as reported for 2TFA‚H-Dmt-Tic-NH-(S)CH-
(CH3)-Bid: yield 0.17 g (90%); Rf(A) ) 0.67; HPLC K′ ) 2.90;
[R]20 +15.3°; MH+ 296.
D
Boc-Tic-NH -(S)CH (CH 2-Bid )-COOBzl. This sub-
stance was obtained by condensation of Boc-Tic-OH with
2TFA‚H2N-(S)CH(CH2-Bid)-COOBzl via WSC/HOBt as re-
ported for Boc-Tic-NH-(S)CH(CH3)-Bid: yield 0.74 g (79%);
mp 164-166 °C; [R]20 +7.9°; MH+ 556. Anal. (C31H33N5O5‚
D
2TFA) C, H, N.
Rf(B) ) 0.74; HPLC K′ ) 4.5; mp 166-168 °C; [R]20 +8.2°;
D
[2-(ter t-Bu toxyca r bon yla m in om eth yl)ben zim id a zol-1-
yl]a cetic Acid Eth yl Ester [Boc-NH-CH2-Bid (N1-CH2-
COOEt)]. To a solution of Boc-NH-CH2-Bid4 (0.5 g, 1.63
mmol) in DMF (10 mL) at room temperature, K2CO3 (0.79 g,
5.72 mmol) and, after 1 h, bromoacetic acid ethyl ester (0.19
mL, 1.73 mmol) were added. The reaction mixture was stirred
for 86 h at room temperature. After DMF was evaporated, the
residue was solubilized in EtOAc and washed with NaHCO3
(5%) and brine. The organic phase was dried and evaporated
to dryness. The residue was crystallized from Et2O/Pe (1:9,
v/v): yield 0.46 g (84%); Rf(B) ) 0.93; HPLC K′ ) 4.95; mp
139-141 °C; MH+ 334; 1H NMR (DMSO) δ 1.30-1.40 (m, 12H),
4.20-4.69 (m, 6H), 7.26-7.70 (m, 4H), 8.00 (bs, 1H).
2TFA‚H2N-CH2-Bid(N1-CH2-COOEt). Boc-NH-CH2-
Bid(N1-CH2-COOEt) was treated with TFA as reported for
2TFA‚H2N-(S)CH(CH2-Bid)-COOBzl: yield 0.51 g (98%); Rf-
(A) ) 0.88; HPLC K′ ) 2.50; mp 222-224 °C; MH+ 234.
Boc-Tic-NH-CH2-Bid (N1-CH2-COOEt). This com-
pound was obtained by condensation of Boc-Tic-OH with
2TFA‚H2N-CH2-Bid(N1-CH2-COOEt) via WSC/HOBt as
MH+ 555; 1H NMR (DMSO) δ 1.40 (s, 9H), 3.04-3.17 (m, 4H),
4.22-5.32 (m, 6H), 7.01-7.70 (m, 14H), 7.98 (s, 1H).
2TFA‚H-Tic-NH-(S)CH(CH2-Bid)-COOBzl. Boc-Tic-
NH-(S)CH(CH2-Bid)-COOBzl was treated with TFA as
reported for 2TFA‚H-Tic-NH-(S)CH(CH3)-Bid: yield 0.73
g (92%); Rf(A) ) 0.71; HPLC K′ ) 5.31; mp 158-160 °C; [R]20
D
+10.4°; MH+ 455.
Boc-Dm t -Tic-NH-(S)CH(CH 2-Bid )-COOBzl. This
compound was obtained by condensation of Boc-Dmt-OH
with 2TFA‚H-Tic-NH-(S)CH(CH2-Bid)-COOBzl via WSC/
HOBt as reported for Boc-Dmt-Tic-NH-(S)CH(CH3)-Bid:
yield 0.44 g (81%); Rf(B) ) 0.65; HPLC K′ ) 9.7; mp 165-167
1
°C; [R]20 +3.5°; MH+ 746; H NMR (DMSO) δ 1.40 (s, 9H),
D
2.35-3.14 (m, 8H), 4.48-5.33 (m, 7H), 6.32-7.70 (m, 16H),
7.98 (s, 1H).
Boc-Dm t-Tic-NH-(S)CH(CH2-Bid )-COOH. To a so-
lution of Boc-Dmt-Tic-NH-(S)CH(CH2-Bid)-COOBzl (0.3
g, 0.4 mmol) in MeOH (30 mL) was added C/Pd (10%, 0.05 g),
and H2 was bubbled for 1 h at room temperature. After

5562 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
reported for Boc-Tic-NH-(S)CH(CH3)-Bid: yield 0.42 g
Balboni et al.
Conformations describing the C-terminal modifications were
generated by extensive conformational searching and energy
minimization. The Dmt-Tic portion of the molecule was
constrained by tethering with a force constant of 1000 kcal
mol-1 Å-1 to preserve the X-ray coordinates and minimized
for 1000 steps or until the root-mean-square (rms) gradient
was 0.0002 kcal mol-1 Å-2 using the cff91 force field and the
conjugate gradient algorithm. Minimization parameters in-
cluded a distant-dependent dielectric [ꢀ ) 1.0(r)], and all scale
terms except nonbonded and Coulombic terms were set to 1.0;
the latter terms were 2.0. Conformational searches were
performed via a torsion force with a force constant of 100 kcal
mol-1 Å-1 that was applied to three dihedrals labeled C1
(peptide bond between Tic and NH2), C2 (bond between -NH-
and -CH2-), and C3 (bond between -CH2- and -Bid). The
region evaluated ranged from 0° and 360°: C1 for two intervals
(180°); C2 and C3 for six intervals (60°). All final conformers
were minimized for an additional 200 iterations. The C-
terminal region was evaluated using conformational searching
techniques, which forces angles of rotation and does not require
solvation.
(86%); Rf(B) ) 0.92; HPLC K′ ) 9.12; mp 148-150 °C; [R]20
D
-11.6°; MH+ 493; 1H NMR (DMSO) δ 1.30-1.40 (m, 12H), 3.05
(m, 2H), 4.12-4.92 (m, 9H), 6.96-7.70 (m, 8H), 8.00 (bs, 1H).
2TF A‚H -Tic-NH -CH 2-Bid (N1-CH 2-COOE t ). Boc-
Tic-NH-CH2-Bid(N1-CH2-COOEt) was treated with TFA
as reported for 2TFA‚H-Tic-NH-(S)CH(CH3)-Bid: yield
0.36 g (96%); Rf(A) ) 0.93; HPLC K′ ) 6.11; mp 187-189 °C;
[R]20 -14.3°; MH+ 393.
D
Boc-Dm t-Tic-NH-CH2-Bid (N1-CH2-COOEt). This
compound was obtained by condensation of Boc-Dmt-OH
with 2TFA‚H-Tic-NH-CH2-Bid(N1-CH2-COOEt) via WSC/
HOBt as reported for Boc-Dmt-Tic-NH-(S)CH(CH3)-Bid:
yield 0.33 g (92%); Rf(B) ) 0.96; HPLC K′ ) 9.8; mp 154-156
°C; [R]20D -22.0°; MH+ 684; 1H NMR (DMSO) δ 1.30-1.40 (m,
12H), 2.35 (s, 6H), 3.05 (m, 4H), 4.12-4.92 (m, 10H), 6.29 (s,
2H), 6.96-7.70 (m, 8H), 8.00 (m, 2H).
2TF A‚H -Dm t -Tic-NH -CH 2-Bid (N1-CH 2-COOE t ).
Boc-Dmt-Tic-NH-CH2-Bid(N1-CH2-COOEt) was treated
with TFA as reported for 2TFA‚H-Dmt-Tic-NH-(S)CH-
(CH3)-Bid: yield 0.16 g (92%); Rf(A) ) 0.81; HPLC K′ ) 6.40;
mp 162-164 °C; [R]20 -34.7°; MH+ 584.
D
2TFA‚H-Dm t-Tic-NH-CH2-Bid(N1-CH2-COOH) (9).
To a solution of 2TFA‚H-Dmt-Tic-NH-CH2-Bid(N1-CH2-
COOEt) (0.15 g, 0.22 mmol) in EtOH (10 mL) at room
temperature, 1 N NaOH (1 mL, 1 mmol) was added. The
reaction mixture was stirred for 3 h at room temperature. After
EtOH was evaporated, the residue was purified by preparative
HPLC without any treatment: yield 0.116 g (89%); Rf(A) )
0.93; HPLC K′ ) 7.8; mp 179-181 °C; [R]20D -36.2°; MH+ 556.
Anal. (C31H33N5O5‚2TFA) C, H, N.
Biologica l Activity in Isola ted Tissu e P r ep a r a tion s.
Preparations of the myenteric plexus longitudinal muscle,
obtained from the small intestine of guinea pigs (rich in
µ-opioid receptors) and preparations of mouse vas deferens
(containing δ-opioid receptors), were used for field stimulation
with bipolar rectangular pulses of supramaximal voltage.
Agonists were evaluated for their inhibition of the electrically
evoked twitch. The results were expressed as the IC50 values
obtained from concentration-response curves (Tallarida and
Murray, 1986) and converted to pEC50. The IC50 values
represent the mean ( SE of not less than five tissue samples.
[D-Ala2]Deltorphin I11 and dermorphin40 were used as internal
standards with MVD and GPI tissue preparations, respec-
tively.
Refer en ces
(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: Bid, 1H-benzimidazole-
2-yl; Boc, tert-butyloxycarbonyl; Boc-Gly-Bid, Boc-âAla-Bid,
Boc-Gly-Gly-Bid, benzimidazole derivatives obtained from the
carboxylic functions of Boc-Gly-OH, Boc-âAla-OH, and Boc-
Gly-Gly-OH, respectively; Bzl, benzyl (CH2-Ph); DAGO,
[D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; DALDA, Tyr-D-Arg-
Phe-Lys-NH2; DCC, N,N′-dicyclohexylcarbodiimide; DMF, di-
methylformamide; DMSO, dimethyl sulfoxide; Dmt, 2′,6′-dimethyl-
L-tyrosine; DPDPE, cyclic[D-Pen2,5]enkephalin; DTLET, Tyr-D-
Thr-Gly-Phe-Leu; Et2O, diethyl ether; EtPt, petroleum ether;
GPI, guinea-pig ileum; HOBt, 1-hydroxybenzotriazole; HPLC,
high-performance liquid chromatography; Ke, the antilog of pA2,
which is determined as the negative log of the molar concentra-
tion of the antagonist necessary to double the agonist (deltorphin
B) concentration; LiAlH4, lithium aluminum hydride; Me, meth-
yl; MeOH, methanol; MVD, mouse vas deferens; NaBH3CN,
sodium cyanoborohydride; NMM, 4-methylmorpholine; NTI,
naltrindole; NH-tBut, tert-butylamine; OMe, methyl ester; Pe,
petroleum ether; Ph, phenyl; TEA, triethylamine; TFA, trifluoro-
acetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
TIP(P), H-Tyr-Tic-Phe-(Phe)-OH; TLC, thin-layer chroma-
tography; WSC, 1-ethyl-3-[(3′-dimethyl)aminopropyl]carbodi-
imide hydrochloride; Z, benzyloxycarbonyl.
(2) Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.; Bianchi,
C.; Bryant, S. D.; Cooper, P. S.; Lazarus, L. H. Evolution of the
Dmt-Tic pharmacophore: N-terminal methylated derivatives
with extraordinary δ opioid antagonist activity. J . Med. Chem.
1997, 40, 3100-3108.
(3) Salvadori, S.; Guerrini, R.; Balboni, G.; Bianchi, C.; Bryant, S.
D.; Cooper, P. S.; Lazarus, L. H. Further studies on the Dmt-
Tic pharmacophore: Hydrophobic substituents at the C-termi-
nus endows δ antagonists to manifest µ agonism or µ antago-
nism. J . Med. Chem. 1999, 42, 5010-5019.
(4) Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi, C.; Rizzi, D.;
Bryant, S. D.; Lazarus, L. H. Evaluation of the Dmt-Tic
pharmacophore: conversion of a potent δ-opioid receptor an-
tagonist into a potent δ-agonist and ligands with mixed proper-
ties. J . Med. Chem. 2002, 45, 713-720.
(5) Page´, D.; Naismith, A.; Schmidt, R.; Coupal, M.; Labarre, M.;
Gosselin, M.; Bellemare, D.; Payza, K.; Brown, W. Novel C-
terminus modifications of the Dmt-Tic motif: a new class of
dipeptide analogues showing altered pharmacological profiles
toward the opioid receptors. J . Med. Chem. 2001, 44, 2387-2390.
(6) Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.; Bryant, S. D.;
Crescenzi, O.; Guerrini, R.; Picone, D.; Tancredi, T.; Temussi,
P. A.; Lazarus, L. H. δ Opioidmimetic antagonists: prototypes
for designing a new generation of ultraselective opioid peptides.
Mol. Med. 1995, 1, 678-689.
Recep tor Bin d in g Assa ys. The binding affinity of all
opioid analogues was determined under equilibrium conditions
(2 h at room temperature) in a competition assay system using
rat brain P2 synaptosomes previously preincubated in 0.1 M
NaCl, 0.4 mM GDP, 50 mM HEPES, pH 7.5, and 50 µg/mL
soybean trypsin inhibitor for 60 min at room temperature to
remove endogenous opioids.41 After extensive washing of
synaptosomes in ice-cold buffer containing protease inhibitor,
the material was resuspended in buffer, pH 7.5, containing
50 µg/mL soybean trypsin inhibitor and 20% glycerol, ali-
quoted, and stored at -80 °C. The δ- and µ-opioid receptors
were radiolabeled with [3H]DPDPE (32.0 Ci/mmol; NEN-
DuPont, Billerica, MA) and [3H]DAGO (58.0 Ci/mmol; Amer-
sham, Arlington Heights, IL), respectively, as described in
detail elsewhere.26 Excess unlabeled peptide (2 µM) established
nonspecific binding. Labeled membranes were rapidly filtered
on Whatman GF/C glass fiber filters, thoroughly washed, and
dried, and the radioactivity was determined using CytoScint
(ICN). All analogues were analyzed in duplicate using five to
eight dosages and three to five independent repetitions using
different synaptosomal preparations for each analogue (actual
n values are listed in Table 1 in parentheses) with results given
as mean ( SE. The affinity constants (Ki) were calculated
according to Cheng and Prusoff.42
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