A new opioid designed multiple ligand derived from the μ opioid agonist endomorphin-2 and the δ opioid antagonist pharmacophore Dmt-Tic

Article abstract of DOI:10.1016/j.bmc.2007.08.047

Opioid compounds with mixed μ agonist/δ antagonist properties could be used as analgesics with low propensity to induce tolerance and dependence. Here we report the synthesis of a new designed multiple ligand deriving from the μ selective agonist endomorp

Full text of DOI:10.1016/j.bmc.2007.08.047

Bioorganic & Medicinal Chemistry 15 (2007) 6876–6881
A new opioid designed multiple ligand derived from the l opioid
agonist endomorphin-2 and the d opioid antagonist
pharmacophore Dmt-Tic
Severo Salvadori,a Claudio Trapella,a Stella Fiorini,a Lucia Negri,b Roberta Lattanzi,b
Sharon D. Bryant,c Yunden Jinsmaa,c Lawrence H. Lazarusc and Gianfranco Balbonid,a,
*
aDepartment of Pharmaceutical Science and Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy
bDepartment of Human Physiology and Pharmacology ‘‘Vittorio Erspamer’’, University La Sapienza, I-00185 Rome, Italy
cMedicinal Chemistry Group, Laboratory of Pharmacology and Chemistry,
National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
dDepartment of Toxicology, University of Cagliari, I-09126 Cagliari, Italy
Received 7 May 2007; revised 9 August 2007; accepted 17 August 2007
Available online 29 August 2007
Abstract—Opioid compounds with mixed l agonist/d antagonist properties could be used as analgesics with low propensity to
induce tolerance and dependence. Here we report the synthesis of a new designed multiple ligand deriving from the l selective ago-
nist endomorphin-2 and the d selective antagonist pharmacophore Dmt-Tic. As predicted, the resulting bivalent ligand showed a l
agonist/d antagonist profile deriving from the corresponding activities of each pharmacophore.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
agonist in some cell types.7 These observations sug-
gest that the development of opioid ligands possess-
The development of tolerance and physical dependence
induced by chronic morphine administration limits its
prolonged use in the treatment of pain. Analgesia and
tolerance to morphine are abolished in l-opioid receptor
knock-out mice, implicating the l-opioid receptor as the
primary receptor type mediating both these effects.1–3
However, several lines of evidence suggest the additional
involvement of the d-opioid receptor in morphine toler-
ance. Initial studies using d-opioid receptor antagonists4
and more recent studies using d-opioid receptor knock-
out mice5 were shown to disrupt the development of
tolerance. In pharmacological studies, the selective d
receptor antagonist naltrindole has been shown to inter-
act with alternative receptors because naltrindole bind-
ing was still detected in the l/d/j triple knock-out mice.6
ing mixed
l
agonist/d antagonist activity may
provide a novel approach for the development of
analgesic agents with low propensity to produce tol-
erance, physical dependence and other side effects. In
this context, interesting compounds were reported.
As an example, the pseudotetrapeptide DIPP[W] dis-
played mixed
l
agonist/d antagonist properties
in vitro, and analgesia with reduced physical depen-
dence and tolerance when administered icv in rats.8
In our previous SAR studies, we demonstrated that
the C-terminal elongation of the Dmt-Tic (Dmt,
20,60-dimethyl-L-tyrosine; Tic, 1,2,3,4-tetrahydroiso-
quinoline3-carboxylic acid)
d
selective dipeptide
antagonist pharmacophore yielded the mixed l ago-
nist/d antagonist compound, H-Dmt-Tic-Gly-NH-Bzl
[pEC50 (Guinea Pig Ileum, GPI) = 8.57 and pA2
(Mouse Vas Deferens, MVD) = 9.25].9
Furthermore, at high concentrations, naltrindole has
been shown to lose its d selectivity and act as an
A new interesting strategy in the synthesis of such com-
pounds is actually developed through the designed mul-
tiple ligands (DML) obtained by the linkage of two
different selective pharmacophores.10 For example, a
mixed l agonist/d antagonist pseudopeptide was ob-
tained by linking tail to tail through an ethylene spacer,
Keywords: Designed multiple ligand; Endomorphin-2; Dmt-Tic phar-
macophore; Analgesia; Physical dependence; Opioid peptides; d Opi-
oid receptors; l Opioid receptors.
Corresponding author. Tel.: +39 532 291 275; fax: +39 532 291
296; e-mail addresses: gbalboni@unica.it; bbg@unife.it
*
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.047

S. Salvadori et al. / Bioorg. Med. Chem. 15 (2007) 6876–6881
6877
a selective d antagonist (H-Tyr-TicW[CH2-NH]Cha-
Phe-OH) with a selective l agonist (H-Dmt-D-Arg-
Phe-Lys-NH2).11 Recently, Neumeyer et al. reported
the synthesis and pharmacological evaluation of homo-
and heterodimeric designed multiple ligands deriving
from morphinans,12 and from the l/j agonist morphi-
nan derivative butorphan, and the d antagonist dipep-
tide Dmt-Tic.13 With the aim to extend our studies in
this field, we now report the synthesis of the first
DML obtained through the tail to tail condensation of
the d selective Dmt-Tic pharmacophore with the endog-
enous opioid l selective agonist endomorphin-2, con-
nected by an ethylenediamine spacer. Although opioid
bivalent ligands were developed by tail to tail condensa-
tion, such as the l-opioid selective dermorphin
analogues14 and the opioid mimetics containing the
bis-[H-Dmt-NH(CH2)n]-2(1H)pyrazinone,15 or the d-
opioid selective enkephalin16 and the 1,6-bis-(N,N-di-
methyl-Dmt-Tic-NH)hexyl,17 this is the first report of
a C-terminally extended endomorphin-2 with the Dmt-
Tic pharmacophore in which both moieties of the new
DML retained their inherent opioid receptor preference
and biological activities. Bifunctional ligands were also
formed between disparate receptor systems, for example
combining the weakly l-opioid selective casomorphine
and a substance P antagonist.18
using a rat brain synaptosome (P2) fraction.9,21 In this
assay, (1) displayed high l-receptor binding affinity
(Kli ¼ 1:03 nM), which is similar to the l selective refer-
ence compound endomorphin-2 (3) (Kli ¼ 0:69 nM),22
and high d receptor binding affinity (Kdi ¼ 1:45 nM)
comparable to the reference d selective antagonist H-
Dmt-Tic-NH2 (Kil ¼ 1:22 nM).21 Compound (1) did
not show significant
j receptor binding affinity
(Kji ¼ 1 lM).
3.2. Functional bioactivity
The GPI and MVD bioassays were carried out as re-
ported in Section 5.9,21 The functional bioactivity of
compound (1) showed a l agonist activity (IC50,
GPI = 25.1 nM) in the same order of magnitude of the
reference endomorphin-2 (IC50, GPI = 15 nM).22 At
the same time, its
d antagonist activity (pA2,
MVD = 8.9) was 50 times higher than the reference d
antagonist H-Dmt-Tic-NH2 (pA2, MVD = 7.2).21 This
new DML compound endowed of l agonist/d antago-
nist activity showed a functional bioactivity that is in
quite good accord with our best l agonist/d antagonist
compound derived from the Dmt-Tic pharmacophore
(H-Dmt-Tic-Gly-NH-Bzl; IC50, GPI = 8.57 nM; pA2,
MVD = 9.25).9 With the aim to demonstrate that l
activity is independent from the presence of the ethy-
lenediamine spacer we synthesized compound (4) which
maintained the d antagonism but is endowed with only
poor l agonism.
2. Chemistry
The DML pseudopeptide was prepared by standard
solution peptide synthesis reactions as outlined in
Scheme 1. Boc-N-protected endomorphin-2 tetrapeptide
(Boc-Tyr-Pro-Phe-Phe-OH)15e was obtained by a 2 + 2
condensation via WSC/HOBt (WSC, N-(3-dimethyami-
nopropyl)-N0-ethylcarbodiimide; HOBt, 1-hydrox-
ybenzotriazole) starting from the corresponding
dipeptides Boc-Tyr-Pro-OH19 and H-Phe-Phe-OBzl.20
Z-N-monoprotected ethylenediamine was condensed
with Boc-Tic-OH via WSC/HOBt to give Z-NH-CH2-
CH2-NH Tic Boc. After Boc deprotection (TFA,
trifluoroacetic acid), it was condensed with Boc-Dmt-
OH (WSC/HOBt) to obtain Z-NH-CH2-CH2-NH
Tic Dmt Boc. This compound was Z (benzyloxy-
carbonyl) deprotected by catalytic hydrogenation and
then condensed (WSC/HOBt) with the Boc-N-protected
endomorphin-2 tetrapeptide previously deprotected at
the C-terminus by catalytic hydrogenation. Removal
of Boc (tert-butyloxycarbonyl) protecting groups
(TFA) gave the final pseudopeptides DML that was
purified by preparative reverse phase HPLC. The final
compounds (1, 4) were identified by HPLC, elemental
analysis, mass spectrometry and NMR spectroscopy.
4. Conclusion
The C-terminal joining of endomorphin-2 and the
H-Dmt-Tic pharmacophore via an ethylenediamine lin-
ker yielded a new DML pseudopeptide, that maintained
the high receptor affinity and in vitro biological potency
of the parent peptide ligands. In summary, here we
confirmed once more the usefulness of the d selective
Dmt-Tic pharmacophore in the synthesis of DML com-
pounds;13,15e,24 but, more important, here we reported
for the first time the possibility to use endomorphin-2
(an endogenous ligand for l-opioid receptors) in the
synthesis of a new DML. As an example, endomor-
phin-2 could be used in the synthesis of compounds
useful for magnetic resonance imaging or PET imaging.
H-Tyr-Pro-Phe-Phe-NH-CH2-CH2-NH-CO-C6H4-pF
could represent a potential pharmacological tool for
PET imaging of l-opioid receptors.25
5. Experimental
5.1. Chemistry
3. Results and discussion
5.1.1. General methods. Crude peptides were purified by
preparative reverse phase HPLC [Waters Delta Prep
4000 system with Waters Prep LC 40 mm Assembly col-
umn C18 (30 · 4 cm, 15 lm particle size)] and eluted at a
flow rate of 25 mL/min with mobile phase solvent A
(10% acetonitrile + 0.1% TFA in H2O, v/v), and a linear
gradient from 25% to 75% B (60%, acetonitrile + 0.1%
3.1. Receptor affinity analysis
Receptor binding and functional bioactivities of H-Tyr-
Pro-Phe-Phe-NH-CH2-CH2-NH Tic Dmt-H (1)
and the reference compounds (2–4) are reported in
Table 1. Opioid receptor binding studies were performed

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S. Salvadori et al. / Bioorg. Med. Chem. 15 (2007) 6876–6881
Boc-Tic-OH
NH2
Z
N
WSC/HOBt
H
H
H
Boc-Dmt-OH
WSC/HOBt
H
TFA
Z
N
Z
N
Z
N
N
Tic-Dmt-Boc
H2; C/Pd
N
Tic-H
N
Tic-Boc
H
H
H
H
H
N
Boc-Tyr-Pro-Phe-Phe-OH
WSC/HOBt
Boc-Tyr-Pro-Phe-Phe
N
N
Tic-Dmt-Boc
H2N
Tic-Dmt-Boc
H
TFA
TFA
OH
O
H
N
H2N
Tic-Dmt-H
(4)
O
O
H
N
H
H2
N
N
O
N
N
N
N
H
O
H
O
NH2
(1)
OH
Scheme 1. Synthesis of H-Tyr-Pro-Phe-Phe-NH-CH2-CH2-NH Tic Dmt-H.
Table 1. Receptor binding and functional bioactivity
Compound
Receptor affinityc (nM)
Selectivity
Functional bioactivity
Kil
d/l
l/d
MVD (IC50) (nM) MVD pA2
GPI (IC50)e (nM)
d
Kid
1
H-Tyr-Pro-Phe-Phe-NH-CH2-
CH2-NH Tic Dmt-H
1.45 0.20 (3) 1.03 0.17 (4) 1.41
—
8.9
25.1
3
a
2
3
4
H-Dmt-Tic-NH2
1.22 0.09
9230 200
H-Dmt-Tic-NH-CH2-CH2-NH2 1.81 0.12 (4) 2.72 0.18 (3)
277 26
—
13,400
227
—
—
344 93
—
7.2
—
>10,000
15
285.6 21.4
b
H-Tyr-Pro-Phe-Phe-NH2
0.69 0.16
2
3.36
7.6
a Ref. 21.
b Ref. 22.
c The Ki values (nM) were determined according to Chang and Prusoff.23 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.
d pA2 is the negative logarithm to 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 II as a d selective opioid
agonist.
e Agonist activity was expressed as IC50 obtained from dose–response curves using guinea-pig ileum (GPI). These values represent means SE for at
least five fresh tissue samples. Deltorphin II and dermorphin were the internal standards for mouse vas deferens (MVD, d-opioid receptor
bioactivity) and GPI (l-opioid receptor bioactivity) tissue preparations, respectively.
TFA in H2O, v/v) in 25 min. Analytical HPLC analyses
were performed with a Beckman System Gold (Beckman
ultrasphere ODS column, 250 · 4.6 mm, 5 lm particle
size). Analytical determinations and capacity factor
(K0) of the products used HPLC in solvents A and B
programmed at flow rate of 1 mL/min with linear gradi-
ent from 0% to 100% B in 25 min. Analogues had less
than 1% impurities at 220 and 254 nm. TLC was per-
formed on precoated plates of silica gel F254 (Merck,
Darmstadt, Germany): (A) 1-butanol/AcOH/H2O
(3:1:1, v/v/v); (B) CH2Cl2/toluene/methanol (17:1:2).
Ninhydrin (1% ethanol, Merck), fluorescamine (Hoff-
man-La Roche) and chlorine spray reagents. Melting
points were determined on a Kofler apparatus and are
uncorrected. Optical rotations were assessed at 10 mg/
mL in methanol with a Perkin-Elmer 241 polarimeter
in a 10 cm water-jacketed cell. Molecular weights of
the compounds were determined by a MALDI-TOF
analysis (Hewlett Packard G2025A LD-TOF system
mass spectrometer) and a-cyano-4-hydroxycinnamic

S. Salvadori et al. / Bioorg. Med. Chem. 15 (2007) 6876–6881
6879
1
acid as a matrix. H NMR (d) spectra were measured,
(10 mL) at 0 ꢁC, NMM (0.06 mL, 0.59 mmol), HOBt
(0.1 g, 0.65 mmol) and WSC (0.12 g, 0.65 mmol) were
added. The reaction mixture was stirred for 3 h at 0 ꢁC
and 24 h at room temperature. After DMF was evapo-
rated, 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
when not specified, in DMSO-d6 solution using a Bruker
AC-200 spectrometer, and peak positions are given in
parts per million downfield from tetramethylsilane as
internal standard.
5.2. Peptide synthesis
5.2.1. Boc-Tyr-Pro-Phe-Phe-OBzl. To a solution of Boc-
Tyr-Pro-OH19 (0.37 g, 0.97 mmol) and TFAÅH-Phe-Phe-
OBzl20 (0.5 g, 0.97 mmol) in DMF (10 mL) at 0 ꢁC,
NMM (4-methylmorpholine) (0.11 mL, 0.97 mmol),
HOBt (0.16 g, 1.07 mmol) and WSC (0.2 g, 1.07 mmol)
were added. The reaction mixture was stirred for 3 h
at 0 ꢁC and 24 h at room temperature. After DMF
(N,N-dimethylformamide) was evaporated, the residue
was dissolved in EtOAc (ethyl acetate) and washed with
citric acid (10% in H2O), NaHCO3 (5% in H2O) and
brine. The organic phase was dried (Na2SO4) and evap-
orated to dryness. The residue was precipitated from
Et2O/Pe (diethyl ether/petroleum ether) (1:9, v/v): yield
from Et2O/Pe (1:9, v/v): yield 0.33 g (87%); Rf (B)
20
0.78; HPLC K0 5.14; mp 134–136 ꢁC; ½aꢀ +13.7; m/z
D
646 (M+H)+; 1H NMR (DMSO-d6) d 1.40 (s, 9H),
2.35 (s, 6H), 2.92–3.46 (m, 8H), 4.41–4.51 (m, 2H),
4.92–5.34 (m, 4H), 6.29 (s, 2H), 6.96–7.19 (m, 9H).
5.2.6. Boc-Dmt-Tic-NH-CH2-CH2-NH2. To a solution of
Boc-Dmt-Tic-NH-CH2-CH2-NH-Z (0.30 g, 0.47 mmol)
in methanol (30 mL) was added Pd/C (10%, 0.1 g), and
H2 was bubbled for 1 h at room temperature. After
filtration, the solution was evaporated to dryness. The
residue was crystallized from Et2O/Pe (1:9, v/v): yield
0.20 g (86%); Rf (B) 0.61; HPLC K0 4.03; mp
20
132–134 ꢁC; ½aꢀ +15.9; m/z 512 (M+H)+.
0.66 g (89%); Rf (B) 0.91; HPLC K0 9.14; mp 133–
D
20
D
135 ꢁC; ½aꢀ ꢁ22.6; m/z 764 (M+H)+; 1H NMR
(DMSO-d6) d 1.40 (s, 9H), 1.92–2.34 (m, 4H), 2.92–
3.29 (m, 6H), 3.41–3.51 (m, 2H), 4.40–4.92 (m, 4H),
5.34 (s, 2H), 6.68–7.21 (m, 19H).
5.2.7. Boc-Tyr-Pro-Phe-Phe-NH-CH2-CH2-NH
Tic Dmt Boc. To a solution of Boc-Tyr-Pro-Phe-
Phe-OH (0.13 g, 0.2 mmol) and Boc-Dmt-Tic-NH-
CH2-CH2-NH2 (0.1 g, 0.2 mmol) in DMF (10 mL) at
0 ꢁC, HOBt (0.03 g, 0.22 mmol) and WSC (0.04 g,
0.22 mmol) were added. The reaction mixture was stir-
red for 3 h at 0 ꢁC and 24 h at room temperature. After
DMF was evaporated, the residue was dissolved in
EtOAc and washed with citric acid (10% in H2O), NaH-
CO3 (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 0.2 g
(84%); Rf (B) 0.92; HPLC K0 5.30; mp 147–149 ꢁC;
5.2.2. Boc-Tyr-Pro-Phe-Phe-OH15e. To a solution of
Boc-Tyr-Pro-Phe-Phe-OBzl (0.66 g, 0.86 mmol) in meth-
anol (30 mL) was added Pd/C (10%, 0.1 g), and H2 was
bubbled for 1 h at room temperature. After filtration,
the solution was evaporated to dryness. The residue
was crystallized from Et2O/Pe (1:9, v/v): yield 0.56 g
(96%); Rf (B) 0.82; HPLC K0 7.39; mp 142–144 ꢁC;
20
½aꢀ ꢁ25.1; m/z 674 (M+H)+.
D
20
D
1
5.2.3. Boc-Tic-NH-CH2-CH2-NH-Z. To a solution of
Boc-Tic-OH (0.2 g, 0.72 mmol) and HClÅH2N-CH2-
CH2-NH-Z (0.17 g, 0.72 mmol) in DMF (10 mL) at
0 ꢁC, NMM (0.08 mL, 0.72 mmol), HOBt (0.12 g,
0.79 mmol) and WSC (0.15 g, 0.79 mmol) were added.
The reaction mixture was stirred for 3 h at 0 ꢁC and
24 h at room temperature. After DMF was evaporated,
the residue was dissolved in EtOAc and washed with cit-
ric 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 0.29 g (89%); Rf (B) 0.81; HPLC K0
½aꢀ ꢁ17.2; m/z 1166 (M+H)+; H NMR (DMSO-d6) d
1.40–1.44 (m, 18H), 1.92–2.34 (m, 4H), 2.35 (s, 6H),
2.92–3.51 (m, 16H), 4.41–4.51 (m, 3H), 4.92–4.97 (m,
5H), 6.29 (s, 2H), 6.68–7.21 (m, 18H).
5.2.8. TFAÅH-Tyr-Pro-Phe-Phe-NH-CH2-CH2-NH
Tic Dmt-HÅTFA (1). Boc-Tyr-Pro-Phe-Phe-NH-CH2-
CH2-NH Tic Dmt Boc (0.17 g, 0.15 mmol) was
treated with TFA (1.5 mL) for 0.5 h at room temperature.
Et2O/Pe (1:1, v/v) were added to the solution until the
product precipitated: yield 0.17 g (95%); Rf (A) 0.46;
20
HPLC K0 5.16; mp 153–155 ꢁC; ½aꢀ ꢁ18.6; m/z 966
D
20
D
1
5.18; mp 116–118 ꢁC; ½aꢀ +7.3; m/z 455 (M+H)+; H
(M+H)+; 1H NMR (DMSO-d6) d 1.92–2.34 (m, 4H),
2.35 (s, 6H), 2.92–3.51 (m, 16H), 3.93–3.97 (m, 2H),
4.40–4.51 (m, 3H), 4.92–4.97 (m, 3H), 6.29 (s, 2H), 6.68–
7.21 (m, 18H), Anal. Calcd for C59H66F6N8O12: C,
59.39; H, 5.58; N, 9.39. Found: C, 58.82; H, 5.91; N, 9.52.
NMR (DMSO-d6) d 1.40 (s, 9H), 2.92–3.46 (m, 6H),
4.17–4.27 (m, 2H), 4.92–5.34 (m, 3H), 6.96–7.19 (m,
9H).
5.2.4. TFAÅH-Tic-NH-CH2-CH2-NH-Z. Boc-Tic-NH-
CH2-CH2-NH-Z (0.25 g, 0.55 mmol) was treated with
TFA (2 mL) for 0.5 h at room temperature. Et2O/Pe
(1:1, v/v) were added to the solution until the product
Å
5.2.9. TFAÅH-Dmt-Tic-NH-CH2-CH2-NH2 TFA (4).
Boc-Dmt-Tic-NH-CH2-CH2-NH2 (0.07 g, 0.14 mmol)
was treated with TFA (1 mL) for 0.5 h at room tem-
perature. Et2O/Pe (1:1, v/v) were added to the solution
precipitated: yield 0.24 g (92%); Rf (A) 0.45; HPLC K0
20
3.44; mp 127–129 ꢁC; ½aꢀ þ 6:9; m/z 354 (M+H)+.
until the product precipitated: yield 0.08 g (93%); Rf
D
20
(A) 0.36; HPLC K0 3.27; mp 142–146 ꢁC; ½aꢀ +14.2;
5.2.5. Boc-Dmt-Tic-NH-CH2-CH2-NH-Z. To a solution
of Boc-Dmt-OH (0.18 g, 0.59 mmol) and TFAÅH-Tic-
NH-CH2-CH2-NH-Z (0.28 g, 0.59 mmol) in DMF
m/z 412 (M+H)+; 1H NMR (DMSO-d6) dD2.35 (s,
6H), 2.91–3.17 (m, 6H), 3.46–3.95 (m, 3H), 4.41–4.92
(m, 3H), 6.29 (s, 2H), 6.96–7.02 (m, 4H), Anal. Calcd

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S. Salvadori et al. / Bioorg. Med. Chem. 15 (2007) 6876–6881
2. Matthes, H. W.; Maldonado, R.; Simonin, F.; Valverde,
O.; Slowe, S.; Kitchen, I.; Befort, K.; Dierich, A.; Le
Meur, M.; Dolle, P.; Tzavara, E.; Hanoune, J.; Roques, B.
P.; Kieffer, B. L. Nature 1996, 383, 819.
3. Sora, I.; Funada, M.; Uhl, G. R. Eur. J. Pharmacol. 1997,
324, R1.
for C27H32F6N4O7: C, 50.78; H, 5.05; N, 8.77. Found:
C, 50.62; H, 5.18; N, 8.95.
5.3. Pharmacology
5.3.1. Radioreceptor binding assays. Opioid receptor
affinity was determined under equilibrium conditions
[2.5 h at room temperature (23 ꢁC)] in a competition
assay using brain P2 synaptosomal membranes pre-
pared from Sprague–Dawley rats.26,27 Synaptosomes
were preincubated to remove endogenous opioid pep-
tides and stored at ꢁ80 ꢁC in buffered 20% glyc-
erol.26,28 Each analogue was analyzed in duplicate
assays using 5–8 dosages and 3–5 independent repeti-
tions with different synaptosomal preparations (n val-
ues are listed in Table 1 in parentheses and results
are means SE). Unlabelled peptide (2 lM) was used
to determine non-specific binding in the presence of
1.9 nM [3H]deltorphin II (45.0 Ci/mmol, Perkin-Elmer,
Boston, MA; KD = 1.4 nM) for d-opioid receptors and
3.5 nM [3H]DAMGO (50.0 Ci/mmol, Amersham Bio-
science, Buckinghamshire, UK; KD = 1.5 nM) for l-
opioid receptors. Glass fibre filters (Whatman GFC)
were soaked in 0.1% polyethylenimine in order to en-
hance the signal-to-noise ratio of the bound radiola-
belled-synaptosome complex, and the filters were
washed thrice in ice-cold buffered BSA (bovine serum
albumin).26 The affinity constants (Ki) were calculated
according to Cheng and Prusoff.23
4. Abdelhamid, E. E.; Sultana, M.; Portoghese, P. S.;
Takemori, A. E. J. Pharmacol. Exp. Ther. 1991, 258, 299.
5. Nitsche, F.; Schuller, A. G.; King, M. A.; Zengh, M.;
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This research was supported in part by the University of
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the library staff and the Comparative Medicine Branch
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