Further studies on the effect of lysine at the C-terminus of the Dmt-Tic opioid pharmacophore

Article abstract of DOI:10.1016/j.bmc.2007.02.039

A wide range of activities are induced by Lys when introduced at C-terminus of the δ-opioid Dmt-Tic pharmacophore through the α-amine group, including: improved δ-antagonism, μ-agonism and μ-antagonism. Here we report the synthesis of a new series of comp

Full text of DOI:10.1016/j.bmc.2007.02.039

Bioorganic & Medicinal Chemistry 15 (2007) 3143–3151
Further studies on the effect of lysine at the C-terminus of the
Dmt-Tic opioid pharmacophore
a
a
b
Gianfranco Balboni,a,b, Valentina Onnis, Cenzo Congiu, Margherita Zotti,
*
Yusuke Sasaki,c Akihiro Ambo,c Sharon D. Bryant,d Yunden Jinsmaa,d
Lawrence H. Lazarus,d Ilaria Lazzari,b Claudio Trapellab and Severo Salvadorib
aDepartment of Toxicology, University of Cagliari, I-09124 Cagliari, Italy
bDepartment of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy
cTohoku Pharmaceutical University, 4-1, Komatsushima 4-chome, Aoba-Ku, Sendai 981-8558, Japan
dMedicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 22709, USA
Received 11 January 2007; revised 13 February 2007; accepted 20 February 2007
Available online 22 February 2007
Abstract—A wide range of activities are induced by Lys when introduced at C-terminus of the d-opioid Dmt-Tic pharmacophore
through the a-amine group, including: improved d-antagonism, l-agonism and l-antagonism. Here we report the synthesis of a new
series of compounds with the general formula H-Dmt-Tic-NH-(CH2)4-CH(R)-R0 (R = -NH2, -NH-Ac, -NH-Z; R0 = CO-NH-Ph,
-CO-NH-CH2-Ph, -Bid) in which Lys is linked to Dmt-Tic through its side-chain amine group. All new compounds (1–9) displayed
potent and selective d-antagonism (MVD, pA2 = 7.81–8.27), which was independent of the functionalized a-amine and carboxylic
groups of C-terminal Lys. This behaviour suggests a direct application as a prototype intermediate, such as Boc-Dmt-Tic-e-Lys(Z)-
OMe, which could be successfully applied in the synthesis (after Z or methyl ester removal) of unique designed multiple ligands con-
taining the pharmacophore of the quintessential d-antagonist Dmt-Tic and another opioid or biologically active non-opioid ligand.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
neuroprotection and neurogenesis,9 regulation of food in-
take10 and in the treatment of alcoholism.11
Extensive structure–activity studies on the prototype
d-opioid receptor antagonist, H-Dmt-Tic-OH,ꢀ,1 revealed
that even minor chemical modifications changed its phar-
macological profile,2 including enhanced d-antagonism,3
the reversal to d-agonism,4 the appearance of mixed
l-agonism/d-agonism,5 as well as formation of mixed
l-agonism/d-antagonism,5 l-agonism6 and -antagonism.6
Each pharmacological profile indicated interesting poten-
tial for therapeutic applications, such as analgesia with
low tolerance and dependence,5 antidepressantactivity,7,8
Recently, we demonstrated that the substitution of C-ter-
minal amino acids in tri- and tetrapeptides containing the
Dmt-Tic pharmacophore with side-chain protected Lys
improved d-antagonist potency.12,13 On the basis of these
results, we extended the substitution of the side-chain pro-
tected or unprotected Lys to other biologically active
compounds previously developed by us [H-Dmt-Tic-
NH-CH(CH2-COOH)-Bid
(Bid = 1H-benzimidazole-
2-yl) a d-agonist; H-Dmt-Tic-Gly-NH-Ph a l-agonist/
Keywords: d-Opioid receptors; Dmt-Tic pharmacophore; Designed multiple ligands; Opioid peptides.
*
ꢀ
Corresponding author. Tel.: +39 532 291 275; fax: +39 532 291 296; e-mail addresses: gbalboni@unica.it; bbg@unife.it
Abbreviations. In addition to the IUPAC-IUB Commission on Biochemical Nomenclature (J. Biol. Chem. 1985, 260, 14–42), this paper uses the
following additional symbols and abbreviations: Ac, acetyl; Bid, 1H-benzimidazole-2-yl; Boc, tert-butyloxycarbonyl; DAMGO, [D-Ala2,N-Me-
Phe4,Gly-ol5]enkephalin; Deltorphin II, H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2; DMF, N,N-dimethylformamide; DMSO-d6, hexadeuteriodim-
ethyl sulfoxide; Dmt, 20,60-dimethyl-L-tyrosine; EtOAc, ethyl acetate; GPI, guinea-pig ileum; HOBt, 1-hydroxybenzotriazole; HPLC, high
performance liquid chromatography; IBCF, isobutyl chloroformate; MALDI-TOF, matrix assisted laser desorption ionization time-of-flight;
MeOH, methanol; MVD, mouse vas deferens; NMM, 4-methylmorpholine; pA2, negative log of the molar concentration required to double the
agonist concentration to achieve the original response; Pe, petroleum ether; TFA, trifluoroacetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid; TLC, thin-layer chromatography; WSC, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide; Z, benzyloxycarbonyl.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.039

3144
G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
d-agonist and H-Dmt-Tic-Gly-NH-CH2-Ph a l-agonist/
d-antagonist] with a quite surprising array of interesting
results. Lysine, when introduced in place of the C-termi-
nal amino acid in the above reference compounds, did
not produce a simple improvement in the original phar-
macological activities but provided opioid ligands which
exhibited mixed properties ranging from d-antagonism,
l-agonism and interestingly, l-antagonism.6 Considering
the variety of biological effects induced by Lys in tripep-
tides and pseudotripeptides of the general formula
H-Dmt-Tic-Lys(R)-R0, the studies described herein ex-
tend our initial investigations on the synthesis and biolog-
ical evaluation of a new series of constitutional isomers
developed on the framework of H-Dmt-Tic-e-Lys(R)-
R0, where Lys is linked to the Dmt-Tic dipeptide through
the e-amine group, in order to further evaluate the impor-
tant influence of Lys on opioid receptor interactions and
functional bioactivities to produce opioid ligands for
potential translation into human health initiatives.
2. Chemistry
Peptides (1–6) and pseudopeptides (7–9) were prepared
stepwise by solution peptide synthetic methods, as out-
lined in Schemes 1 and 2, respectively. Boc-Tic-OH was
(R = Ac; Z)
R
HN
O
O
N
O
R
O
O
N
O
H2N
HN
O
O
O
O
N
H
OH
WSC/HOBt
Boc-Tic-OH
NaOH 1N
(R' = -CH2-Ph; -Ph)
R'-NH2
O
N
O
R
O
O
N
O
R
HN
HN
H
N
WSC/HOBt
O
OH
O
N
H
R'
N
H
O
HO
TFA
Boc-Dmt-OH
WSC/HOBt
O
O
HN
O
R
N
O
R
O
HN
N
H
HN
H
N
R'
H
O
N
N
H
N
H
R'
O
TFA
(R = Z)
(R' = -CH2-Ph; -Ph)
(R = Ac; Z)
(R' = -CH2-Ph; -Ph)
HO
H2; C/Pd
HO
N
R
H2N
HN
H
N
R'
O
O
O
O
N
H
N
N
H
NH2
H
O
O
N
O
N
R'
H-Dmt-Tic-ε-Lys(R)-R'
H
(Comp. 1, 2, 4, 5)
O
TFA
HO
N
H2N
NH2
O
H
N
O
O
N
R'
H
H-Dmt-Tic-ε-Lys-R'
(Comp. 3. 6)
Scheme 1. Synthesis of compounds 1–6.

G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
3145
(R = Ac; Z)
H2N
O
N
O
R
O
N
O
R
O
H2N
IBCF
HN
HN
NH2
H
N
O
OH
O
N
H
N
H
O
CH3COOH
65°C; 1h
O
N
R
HN
H
HN
R
N
TFA
HN
H
O
N
N
O
N
O
N
H
H
N
Boc-Dmt-OH
WSC/HOBt
HO
HO
TFA
O
O
N
O
R
HN
H
N
O
R
H2N
N
HN
H
N
H
O
O
N
N
H
N
H
N
N
H-Dmt-Tic-NH-(CH2)4-CH(NH-R)-Bid
Comp. 7, 8
H2; C/Pd
(R = Z)
HO
HO
TFA
O
O
N
O
N
N
H
NH2
H2N
NH2
H
N
H
N
O
O
N
H
O
N
H
N
N
H-Dmt-Tic-NH-(CH2)4-CH(NH2)-Bid
Comp. 9
Scheme 2. Synthesis of compounds 7–9.
condensed with commercially available Z-Lys-OMe or
Ac-Lys-OMe via WSC/HOBt obtaining the correspond-
ing Boc-Tic-e-Lys(Z)-OMe or Boc-Tic-e-Lys(Ac)-OMe.
C-Terminal methyl ester protecting groups were removed
by hydrolysis with 1 N NaOH and then each pseudodi-
peptide was condensed with benzylamine or aniline via
WSC/HOBt. N-terminal Boc-protected pseudodipeptide
amides were treated with TFA and condensed with Boc-
Dmt-OH via WSC/HOBt. Final N-terminal Boc depro-
tection with TFA gave compounds 1, 2, 4 and 5 (Scheme
1). Catalytic hydrogenation (10% Pd/C) and TFA treat-
ment of Boc-Dmt-Tic-e-Lys(Z)-amides gave the final
products 3 and 6 (Scheme 1). Pseudopeptides (7–9), con-
taining C-terminal 1H-benzimidazol-2-yl (Bid), were syn-
thesized in a similar manner (Scheme 2). Mixed carbonic
anhydride coupling of Boc-Tic-e-Lys(Z)-OH or Boc-Tic-
e-Lys(Ac)-OH with o-phenylendiamine gave the corre-
sponding crude intermediate monoamides, which were
converted without purification to the desired heteroaro-
matic derivatives by cyclization and dehydration in acetic
acid. As detailed in Scheme 1, after Na deprotection with
TFA, each derivative was condensed with Boc-Dmt-OH
via WSC/HOBt. Final N-terminal Boc deprotection with
TFA gave compounds 7, 8 (Scheme 2). Catalytic hydroge-
nation (10% Pd/C) and TFA treatment of Boc-Dmt-Tic-
NH-(CH2)4-CH(NH-Z)-Bid provided the product 9
(Scheme 2). Final compounds (1–9) were purified by pre-
parative HPLC as described in Experimental Section.
3. Results and discussion
3.1. Receptor affinity analysis
Receptor binding and functional bioactivities are re-
ported in Table 1. All the compounds (1–9) exhibited

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G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
Table 1. Receptor binding affinities and functional bioactivities of compounds 1–9
Compound
Structure
Receptor affinitya (nM)
Selectivity
Functional bioactivity
Kil
MVD pA2
c
b
GPI IC50 (nM)
Kid
d
H-Dmt-Tic-NH2
1.22
277
227
5.9
7.2
>10,000
1
2
3
4
5
6
7
8
9
H-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph
H-Dmt-Tic-e-Lys(Ac)-NH-CH2-Ph
H-Dmt-Tic-e-Lys-NH-CH2-Ph
H-Dmt-Tic-e-Lys(Z)-NH-Ph
H-Dmt-Tic-e-Lys(Ac)-NH-Ph
H-Dmt-Tic-e-Lys-NH-Ph
H-Dmt-Tic-NH-(CH2)4-CH(NH-Z)-Bid
H-Dmt-Tic-NH-(CH2)4-CH(NH-Ac)-Bid
H-Dmt-Tic-NH-(CH2)4-CH(NH2)-Bid
0.53 0.08 (4)
0.21 0.01 (3)
1.00 0.02 (4)
0.47 0.04 (4)
0.22 0.005 (3)
2.02 0.20 (4)
0.86 0.06 (3)
0.21 0.01 (3)
2.64 0.18 (3)
3.15 0.39 (4)
3.43 0.54 (4)
0.60 0.11 (4)
2.67 0.41 (4)
2.57 0.34 (4)
0.89 0.12 (4)
2.78 0.27 (4)
0.99 0.06 (4)
1.02 0.08 (3)
8.27
8.07
7.81
8.23
8.18
7.92
7.82
8.12
8.09
1451 200
1990 674
553 173
711 194
486 63
16.3
1.7*
5.7
11.7
2.3*
3.2
515 73
618 140
610 185
434 26
4.7
2.6*
a The Ki values (nM) were determined according to Cheng and Prusoff.24 The means SE with n repetitions in parentheses is based on independent
duplicate binding assays with 5–8 peptide doses using several different synaptosomal preparations.
b Agonist activity was expressed as IC50 obtained from dose–response curves. These values represent means SE for at least 5–6 fresh tissue samples.
Deltorphin II and endomorphin-2 were the internal standards for MVD (d-opioid receptor bioactivity) and GPI (l-opioid receptor bioactivity)
tissue preparation, respectively.
c The pA2 values of opioid antagonists against the agonist deltorphin II were determined by the method of Kosterlitz and Watt.25
d Data taken from Salvadori et al.1
* l-Opioid receptor selectivity Kid=Kli .
nanomolar affinity for d-opioid receptors ðKdi ¼
0:21–2:64 nMÞ. As expected, the lack of a free carboxylic
function in molecules containing the Dmt-Tic pharma-
cophore induces a substantial increase in l-opioid recep-
tor affinity (Kli ¼ 0:60–3:43 nM).12,4 Compounds (1, 2,
4, 5, 7, 8) containing a Lys residue protected at the
a-amine function (Z, Ac) had weak d-opioid receptor
selectivity (Kli =Kid ¼ 16:3–3:2); the acetyl protecting
group confers marginally better selectivity than the Z
group. Removal of the a-amine protecting group of
Lys (3, 6, 9) shifted the d-opioid selectivity to a very
weak l-selective ligands essentially attributable to the
presence of the additional positive charge.14 The same
general behaviour was observed previously in the series
of Dmt-Tic containing peptides with Lys in the third
position.6
Bid, no interesting l-agonism activity was observed.
Interestingly, all these compounds (1–9) had about the
same order of magnitude of d-antagonism (MVD, pA2
7.82–8.27), which was independent of the substitutions
and modifications made on the a amine and carboxylic
functions of Lys, but in quite good agreement with the
reference dipeptide H-Dmt-Tic-NH2.1
4. Conclusions
Considering the new derivatives (1–9) as analogues of the
published reference compounds [H-Dmt-Tic-NH-CH2-
Bid d-agonist, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid
d-agonist, H-Dmt-Tic-Gly-NH-Ph l-agonist/d-agonist
and H-Dmt-Tic-Gly-NH-CH2-Ph l-agonist/d-antago-
nist] the introduction of Lys (linked through its e amine
group to the Dmt-Tic pharmacophore) in place of the
C-terminal amino acid failed to maintain the original
pharmacological activity, as previously reported for
the corresponding isomers containing Lys linked
through its a-amine group.6 While isomers containing
the C-terminal a-Lys revealed a variety of opioid effects
(d-antagonism, l-agonism and l-antagonism),6 the iso-
mers containing C-terminal e-Lys demonstrated a un-
ique d-opioid antagonism of about the same order of
magnitude, and independent of the substituents linked
on the a position. Without taking into considerations
the different behaviour of Lys when coupled to the
Dmt-Tic pharmacophore through its a- or e-amine
group, it is of notable significance to utilize these new
isomers as potential precursors in the synthesis of ‘de-
signed multiple ligands’, where one of the two pharma-
cophores is represented by the d-selective antagonist
dipeptide Dmt-Tic.15,16 The four methylene groups of
Lys side chain can be considered the spacer linked to
the first pharmacophore (Dmt-Tic) and, as reported by
Neumeyer et al., its length generally does not influence
the biological activity of either pharmacophores.17,18
3.2. Functional bioactivity
Compounds 1–9 were tested in the electrically stimu-
lated MVD and GPI assays for intrinsic functional bio-
activity (Table 1). We and other investigators have
previously discussed the discrepancy in the correlation
between receptor binding affinities and functional bioac-
tivity. Unfortunately, we have neither definitive nor
comprehensive explanations for these observations.12,6
In comparison to the Dmt-Tic peptides containing a
protected or unprotected Lys residue at the C-terminus6
all the analogues are inactive as d-opioid agonists in the
MVD assay (Table 1). Furthermore, they exhibited a
weak or very weak l-agonism in the GPI assay (GPI,
IC50 434–1990 nM), which is in quite good agreement
with the previous studies, except analogues containing
Bid at C-terminus.6 When Lys was linked to Dmt-Tic
through the a-amine group and its carboxylic function
transformed into Bid, the pseudopeptides predominately
had selective l-agonism. On the other hand, when Lys
was linked to Dmt-Tic through the e-amine group and
its carboxylic function once again transformed into

G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
3147
The second pharmacophore, required to complete the
potential designed multiple ligands, preferentially
should be selected among the compounds in the opioid
field, but other pharmacophores endowed with activity
for other receptors would be readily inserted as well.16,19
More importantly, the second pharmacophore can be
conveniently inserted using the deprotected a-amino or
carboxylic function depending on the required final bio-
active product. Furthermore, very similar compounds
were obtained when using fluorescent chromophores in
place of the second pharmacophore.20,21 Recently, Oka-
da et al. reported the synthesis of similar compounds
with the general formula H-Dmt-Tic-NH-(CH2)6-NH-
R (where R = Dmt, Phe, Tic and Tic-Dmt) in which
all the opioids displayed increased d antagonism attrib-
utable to the additional aromatic amino acids.22 How-
ever, the same enhancement in d-antagonist activity
could be derived, at least in part, by the presence of
the spacer as reported here and in preceding studies.20,21
ing 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 polar-
imeter 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
1
acid as a matrix. H NMR (d) spectra were measured,
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-Tic-e-Lys(Z)-OMe. To a solution of Boc-Tic-
OH (0.9 g, 3.24 mmol) and HClÆZ-Lys-OMe (0.95 g,
3.24 mmol) in DMF (10 mL) at 0 ꢁC, NMM (0.35 mL,
3.24 mmol), HOBt (0.54 g, 3.56 mmol) and WSC
(0.68 g, 3.56 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 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 1.4 g
(78%); Rf (B) 0.89; HPLC K0 5.43; mp 101–103 ꢁC;
In summary, we suggest the possibility to use a unique
intermediate [for example Boc-Dmt-Tic-e-Lys(Z)-OMe]
for the synthesis of designed multiple ligands containing:
(a) the d-antagonist pharmacophore Dmt-Tic, (b) a
spacer of defined length and (c) two different protected
functionalities (amine and carboxylic functions) for the
linkage to a variety of second pharmacophores. As fur-
ther explorations of this proposal, the synthesis of multi-
ple ligands derived from the coupling of the selectively
deprotected d-antagonist intermediate Boc-Dmt-Tic-e-
Lys(Z)-OMe with salvinorin A23a (j-agonist); and the
synthesis of H-Dmt-Tic-e-Lys(4-Fluorobenzoyl)-OH as
a potential pharmacological tool for PET imaging of d
receptors,23b,c are currently in progress in our
laboratory.
20
1
½aꢀ ꢁ20.1; m/z 554 (M+H)+; H NMR (DMSO-d6) d
D
1.29–1.90 (m, 15H), 3.05–3.67 (m, 7H), 4.22–4.42 (m,
3H), 4.92–5.34 (m, 3H), 6.96–7.19 (m, 9H).
5.2.2. Boc-Tic-e-Lys(Z)-OH. To a solution of Boc-Tic-e-
Lys(Z)-OMe (1.4 g, 2.53 mmol) in MeOH (10 mL) was
added 1 N NaOH (2.8 mL). The reaction mixture was
stirred for 24 h at room temperature. After solvent evap-
oration, the residue was dissolved 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 precipitated from Et2O/Pe (1:1, v/v):
5. Experimental
5.1. Chemistry
yield 1.1 g (81%); Rf (B) 0.42; HPLC K0 3.71; mp 120–
20
122 ꢁC; ½aꢀ ꢁ21.2; m/z 540 (M+H)+.
D
5.1.1. General methods. Crude peptides and pseudopep-
tides were purified by preparative reversed-phase HPLC
[Waters Delta Prep 4000 system with Waters Prep LC
40 mm Assembly column C18 (30 · 4 cm, 15 lm parti-
cle)] 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%, ace-
tonitrile + 0.1% TFA in H2O, v/v) in 25 min. Analytical
HPLC analyses were performed with a Beckman System
5.2.3. Boc-Tic-e-Lys(Z)-NH-CH2-Ph. To a solution of
Boc-Tic-e-Lys(Z)-OH (0.52 g, 0.96 mmol) and benzyl-
amine (0.1 mL, 0.96 mmol) in DMF (10 mL) at
0 ꢁC, HOBt (0.16 g, 1.06 mmol) and WSC (0.2 g,
1.06 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 dis-
solved in EtOAc and washed with citric acid (10%
in H2O), NaHCO3 (5% in H2O) and brine. The or-
ganic phase was dried (Na2SO4) and evaporated to
dryness. The residue was precipitated from Et2O/Pe
Gold
(Beckman
ultrasphere
ODS
column,
250 · 4.6 mm, 5 lm particle). 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 gradients from 0% to 100% B in
25 min. Analogues had less than 1% impurities at 220
and 254 nm.
(1:9, v/v): yield 0.53 g (88%); Rf (B) 0.85; HPLC K0
20
5.37; mp 102–104 ꢁC; ½aꢀ ꢁ17.7; m/z 629 (M+H)+;
1H NMR (DMSO-d6) dD1.29–1.79 (m, 15H), 3.05–
3.20 (m, 4H), 4.22–4.53 (m, 5H), 4.92–5.34 (m, 3H),
6.96–7.14 (m, 14H).
TLC was performed 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
(Hoffman-La Roche) and chlorine spray reagents. Melt-
5.2.4. TFAÆH-Tic-e-Lys(Z)-NH-CH2-Ph. Boc-Tic-e-
Lys(Z)-NH-CH2-Ph (0.47 g, 0.75 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

3148
G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
precipitated: yield 0.34 g (87%); Rf (A) 0.51; HPLC
5.2.11. Boc-Dmt-Tic-e-Lys(Ac)-NH-CH2-Ph. This inter-
mediate was obtained by condensation of Boc-Dmt-
OH with TFAÆH-Tic-e-Lys(Ac)-NH-CH2-Ph via WSC/
HOBt as reported for Boc-Dmt-Tic-e-Lys(Z)-NH-CH2-
20
K0 4.3; mp 115–117 ꢁC; ½aꢀ ꢁ19.8; m/z 529 (M+H)+.
D
5.2.5. Boc-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph. To a solution
of Boc-Dmt-OH (0.075 g, 0.24 mmol) and TFAÆH-Tic-e-
Lys(Z)-NH-CH2-Ph (0.15 g, 0.24 mmol) in DMF
(10 mL) at 0 ꢁC, NMM (0.03 mL, 0.24 mmol), HOBt
(0.04 g, 0.26 mmol) and WSC (0.05 g, 0.26 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
Ph: yield 0.14 g (83%); Rf (B) 0.75; HPLC K0 4.92; mp
20
D
135–137 ꢁC; ½aꢀ ꢁ17.5; m/z 729 (M+H)+; 1H NMR
(DMSO-d6) d 1.29–1.79 (m, 15H), 2.02 (s, 3H), 2.35 (s,
6H), 2.92–3.20 (m, 6H), 4.41–4.92 (m, 7H), 6.29 (s,
2H), 6.96–7.14 (m, 9H).
5.2.12. TFAÆH-Dmt-Tic-e-Lys(Ac)-NH-CH2-Ph (2).
Boc-Dmt-Tic-e-Lys(Ac)-NH-CH2-Ph was treated with
TFA as reported for TFAÆH-Dmt-Tic-e-Lys(Z)-NH-
CH2-Ph: yield 0.08 g (96%); Rf (A) 0.45; HPLC K0
20
1
from Et2O/Pe (1:9, v/v): yield 0.17 g (87%); Rf (B)
3.21; mp 131-133 ꢁC; ½aꢀ ꢁ15.5; m/z 629 (M+H)+; H
D
20
0.81; HPLC K0 5.23; mp 129–131 ꢁC; ½aꢀ ꢁ16.6; m/z
NMR (DMSO-d6) d 1.29–1.79 (m, 6H), 2.02 (s, 3H),
2.35 (s, 6H), 2.92–3.20 (m, 6H), 3.95–4.92 (m, 7H),
6.29 (s, 2H), 6.96–7.14 (m, 9H). Anal Calcd for
C38H46F3N5O7: C, 61.53; H, 6.25; N, 9.44. Found: C,
61.77; H, 6.39; N, 9.15.
D
820 (M+H)+; 1H NMR (DMSO-d6) d 1.29–1.79 (m,
15H), 2.35 (s, 6H), 3.05–3.20 (m, 6H), 4.46–4.53 (m,
5H), 4.92–5.34 (m, 4H), 6.29 (s, 2H), 6.96–7.19 (m,
14H).
5.2.6. TFAÆH-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph (1). Boc-
Dmt-Tic-e-Lys(Z)-NH-CH2-Ph (0.11 g, 0.13 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 solu-
tion until the product precipitated: yield 0.09 g
5.2.13. Boc-Dmt-Tic-e-Lys-NH-CH2-Ph. To a solution
of Boc-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph (0.1 g, 0.12
mmol) in methanol (30 mL) was added Pd/C (10%,
0.07 g), and H2 was bubbled for 1 h at room tempera-
ture. After filtration, the solution was evaporated to
dryness. The residue was precipitated from Et2O/Pe
(96%); Rf (A) 0.47; HPLC K0 3.63; mp 125–127 ꢁC;
20
½aꢀ ꢁ14.6; m/z 721 (M+H)+; 1H NMR (DMSO-d6)
(1:9, v/v): yield 0.07 g (85%); Rf (B) 0.58; HPLC K0
D
20
4.98; mp 144–145 ꢁC; ½aꢀ ꢁ18.7; m/z 687 (M+H)+.
d 1.29–1.79 (m, 6H), 2.35 (s, 6H), 3.05–3.20 (m,
6H), 3.95–4.53 (m, 6H), 4.92–5.34 (m, 3H), 6.29 (s,
2H), 6.96–7.19 (m, 14H). Anal. Calcd for
C44H50F3N5O8: C, 63.37; H, 6.04; N, 8.40. Found:
C, 63.22; H, 5.98; N, 8.21.
D
5.2.14. 2TFAÆH-Dmt-Tic-e-Lys-NH-CH2-Ph (3). Boc-
Dmt-Tic-e-Lys-NH-CH2-Ph was treated with TFA as
reported for TFAÆH-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph:
yield 0.07 g (95%); Rf (A) 0.39; HPLC K0 3.32; mp
20
D
5.2.7. Boc-Tic-e-Lys(Ac)-OMe. This intermediate was
obtained by condensation of Boc-Tic-OH with
HClÆAc-Lys-OMe via WSC/HOBt, as reported for
148–150 ꢁC; ½aꢀ ꢁ16.2; m/z 587 (M+H)+; 1H NMR
(DMSO-d6) d 1.29–1.79 (m, 6H), 2.35 (s, 6H), 2.92–
3.20 (m, 6H), 3.56–4.92 (m, 7H), 6.29 (s, 2H), 6.96–
7.14 (m, 9H). Anal Calcd for C38H45F6N5O8: C, 56.08;
H, 5.57; N, 8.61. Found: C, 56.30; H, 5.68; N, 8.70.
Boc-Tic-e-Lys(Z)-OMe: yield 1.6 g (82%); Rf (B) 0.77;
20
HPLC K0 5.32; mp 127–129 ꢁC; ½aꢀ ꢁ20.5; m/z 463
D
1
(M+H)+; H NMR (DMSO-d6) d 1.29–1.90 (m, 15H),
2.02 (s, 3H), 2.92–3.67 (m, 7H), 4.17–4.92 (m, 4H),
6.96–7.02 (m, 4H).
5.2.15. Boc-Tic-e-Lys(Z)-NH-Ph. This intermediate was
obtained by condensation of Boc-Tic-e-Lys(Z)-OH with
aniline via WSC/HOBt as reported for Boc-Tic-e-
5.2.8. Boc-Tic-e-Lys(Ac)-OH. This intermediate was ob-
tained by hydrolysis of Boc-Tic-e-Lys(Ac)-OMe as re-
Lys(Z)-NH-CH2-Ph: yield 0.52 g (88%); Rf (B) 0.81;
20
D
HPLC K0 5.61; mp 94–96 ꢁC; ½aꢀ ꢁ19.4; m/z 615
1
ported for Boc-Tic-e-Lys(Z)-OH: yield 1.26 g (82%); Rf
(M+H)+; H NMR (DMSO-d6) d 1.29–1.89 (m, 15H),
2.92–3.20 (m, 4H), 4.17–5.34 (m, 6H), 6.96–7.64 (m, 14H).
20
(B) 0.45; HPLC K0 5.18; mp 135–137 ꢁC; ½aꢀ ꢁ22.3;
D
m/z 449 (M+H)+.
5.2.16. TFAÆH-Tic-e-Lys(Z)-NH-Ph. Boc-Tic-e-Lys(Z)-
5.2.9. Boc-Tic-e-Lys(Ac)-NH-CH2-Ph. This intermedi-
ate was obtained by condensation of Boc-Tic-e-Ly-
s(Ac)-OH with benzylamine via WSC/HOBt as
reported for Boc-Tic-e-Lys(Z)-NH-CH2-Ph: yield
NH-Ph was treated with TFA as reported for TFAÆH-
Tic-e-Lys(Z)-NH-CH2-Ph: yield 0.37 g (95%); Rf (A)
20
0.46; HPLC K0 4.32; mp 111–113 ꢁC; ½aꢀ ꢁ19.9; m/z
D
515 (M+H)+.
0.33 g (81%); Rf (B) 0.79; HPLC K0 5.32; mp 108–
20
110 ꢁC; ½aꢀ ꢁ18.6; m/z 537 (M+H)+; 1H NMR
5.2.17. Boc-Dmt-Tic-e-Lys(Z)-NH-Ph. This intermedi-
ate was obtained by condensation of Boc-Dmt-OH with
TFAÆH-Tic-e-Lys(Z)-NH-Ph via WSC/HOBt as re-
ported for Boc-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph: yield
D
(DMSO-d6) d 1.29–1.79 (m, 15H), 2.02 (s, 3H), 2.92–
3.20 (m, 4H), 4.17–4.92 (m, 6H), 6.96–7.14 (m, 9H).
5.2.10. TFAÆH-Tic-e-Lys(Ac)-NH-CH2-Ph. Boc-Tic-e-
Lys(Ac)-NH-CH2-Ph was treated with TFA as reported
for TFAÆH-Tic-e-Lys(Z)-NH-CH2-Ph: yield 0.21 g
0.17 g (87%); Rf (B) 0.76; HPLC K0 5.73; mp 124–
20
D
126 ꢁC; ½aꢀ
ꢁ15.9; m/z 807 (M+H)+; 1H NMR
(DMSO-d6) d 1.29–1.89 (m, 15H), 2.35 (s, 6H), 2.92–
3.20 (m, 6H), 4.41–5.34 (m, 7H), 6.29 (s, 2H), 6.96–
7.64 (m, 14H).
(97%); Rf (A) 0.48; HPLC K0 3.92; mp 121–123 ꢁC;
20
½aꢀ ꢁ20.7; m/z 437 (M+H)+.
D

G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
3149
5.2.18. TFAÆH-Dmt-Tic-e-Lys(Z)-NH-Ph (4). Boc-Dmt-
Tic-e-Lys(Z)-NH-Ph was treated with TFA as re-
ported for TFAÆH-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph:
5.2.25. Boc-Tic-NH-(CH2)4-CH(NH-Z)-Bid. A solution
of Boc-Tic-e-Lys(Z)-OH (0.5 g, 0.93 mmol) and NMM
(0.1 mL, 0.93 mmol) in DMF(10 mL) was treated at
ꢁ20 ꢁC with IBCF (0.12 mL, 0.93 mmol). After 10 min
at ꢁ20 ꢁC, o-phenylendiamine (0.1 g, 0.93 mmol) was
added. The reaction mixture was allowed to stir while
slowly warming to room temperature (1 h) and was then
stirred for an additional 3 h. The solvent was evaporated
and the residue was partitioned between EtOAc and
H2O. The EtOAc layer was washed with NaHCO3 (5%
in H2O) and brine and dried over Na2SO4. The solution
was filtered, the solvent evaporated, and the residual so-
lid was dissolved in glacial acetic acid (10 mL). The solu-
tion was heated at 65 ꢁC for 1 h. After the solvent was
evaporated, the residue was precipitated from Et2O/Pe
(1:9, v/v): yield 0.47 g (82%); Rf (B) 0.66; HPLC K0
yield 0.09 g (90%); Rf (A) 0.40; HPLC K0 3.70; mp
20
133–135 ꢁC; ½aꢀ ꢁ13.9; m/z 707 (M+H)+; 1H NMR
(DMSO-d6) d D 1.29–1.89 (m, 6H), 2.35 (s, 6H),
2.92–3.20 (m, 6H), 3.95–5.34 (m, 7H), 6.29 (s, 2H),
6.96–7.64 (m, 14H). Anal Calcd for C43H48F3N5O8:
C, 62.99; H, 5.90; N, 8.54. Found: C, 62.95; H,
5.76; N, 8.41.
5.2.19. Boc-Tic-e-Lys(Ac)-NH-Ph. This intermediate
was obtained by condensation of Boc-Tic-e-Lys(Ac)-
OH with aniline via WSC/HOBt as reported for Boc-
Tic-e-Lys(Z)-NH-CH2-Ph: yield 0.32 g (81%); Rf (B)
20
0.74; HPLC K0 4.21; mp 100–102 ꢁC; ½aꢀ ꢁ19.9; m/z
D
20
524 (M+H)+; 1H NMR (DMSO-d6) d 1.29–1.89 (m,
15H), 2.02 (s, 3H), 2.92–3.20 (m, 4H), 4.17–4.92 (m,
4H), 6.96–7.64 (m, 9H).
4.92; mp 134–136 ꢁC; ½aꢀ ꢁ12.8; m/z 613 (M+H)+; H
1
D
NMR (DMSO-d6) d 1.29–1.84 (m, 15H), 2.92–3.20 (m,
4H), 4.17–5.34 (m, 6H), 6.96–7.70 (m, 13H).
5.2.20. TFAÆH-Tic-e-Lys(Ac)-NH-Ph. Boc-Tic-e-Ly-
s(Ac)-NH-Ph was treated with TFA as reported for
5.2.26. 2TFAÆH-Tic-NH-(CH2)4-CH(NH-Z)-Bid. Boc-
Tic-NH-(CH2)4-CH(NH-Z)-Bid was treated with TFA
as reported for TFAÆH-Tic-e-Lys(Z)-NH-CH2-Ph: yield
TFAÆH-Tic-e-Lys(Z)-NH-CH2-Ph: yield 0.27 g (96%);
20
D
Rf (A) 0.43; HPLC K0 3.47; mp 117–119 ꢁC; ½aꢀ
0.33 g (96%); Rf (A) 0.41; HPLC K0 3.57; mp 137–
20
139 ꢁC; ½aꢀ ꢁ14.1; m/z 513 (M+H)+.
ꢁ20.8; m/z 424 (M+H)+.
D
5.2.21. Boc-Dmt-Tic-e-Lys(Ac)-NH-Ph. This intermedi-
ate was obtained by condensation of Boc-Dmt-OH with
TFAÆH-Tic-e-Lys(Ac)-NH-Ph via WSC/HOBt as re-
ported for Boc-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph: yield
5.2.27. Boc-Dmt-Tic-NH-(CH2)4-CH(NH-Z)-Bid. To a
solution of Boc-Dmt-OH (0.075 g, 0.24 mmol) and
2TFAÆH-Tic-NH-(CH2)4-CH(NH-Z)-Bid (0.18 g,
0.24 mmol) in DMF (10 mL) at 0 ꢁC, NMM (0.05 mL,
0.48 mmol), HOBt (0.04 g, 0.26 mmol) and WSC
(0.05 g, 0.26 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 NaHCO3 (5% in H2O) and
brine. The organic phase was dried (Na2SO4) and
evaporated to dryness. The residue was precipitated
0.15 g (88%); Rf (B) 0.71; HPLC K0 5.21; mp 130–
20
D
132 ꢁC; ½aꢀ ꢁ16.8; m/z 715 (M+H)+; 1H NMR
(DMSO-d6) d 1.29–1.89 (m, 15H), 2.02 (s, 3H), 2.35 (s,
6H), 2.92–3.20 (m, 6H), 4.41–4.92 (m, 5H), 6.29 (s,
2H), 6.96–7.64 (m, 9H).
5.2.22. TFAÆH-Dmt-Tic-e-Lys(Ac)-NH-Ph (5). Boc-
Dmt-Tic-e-Lys(Ac)-NH-Ph was treated with TFA as re-
ported for TFAÆH-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph: yield
from Et2O/Pe (1:9, v/v): yield 0.16 g (85%); Rf (B)
20
0.66; HPLC K0 4.93; mp 137–139 ꢁC; ½aꢀ ꢁ14.3; m/z
D
0.08 g (98%); Rf (A) 0.37; HPLC K0 2.89; mp 127–
804 (M+H)+; 1H NMR (DMSO-d6) d 1.29–1.84 (m,
15 H), 2.35 (s, 6H), 2.92–3.20 (m, 6H), 4.41–5.34 (m,
7H), 6.29 (s, 2H), 6.96–7.70 (m, 13H).
20
D
129 ꢁC; ½aꢀ ꢁ14.8; m/z 615 (M+H)+; 1H NMR
(DMSO-d6) d 1.29–1.89 (m, 6H), 2.02 (s, 3H), 2.35 (s,
6H), 2.92–3.20 (m, 6H), 3.95–4.92 (m, 5H), 6.29 (s,
2H), 6.96–7.64 (m, 9H). Anal Calcd for C37H44F3N5O7:
C, 61.06; H, 6.09; N, 9.62. Found: C, 60.96; H, 6.03; N,
9.48.
5.2.28.
2TFAÆH-Dmt-Tic-NH-(CH2)4-CH(NH-Z)-Bid
(7). Boc-Dmt-Tic-NH-(CH2)4-CH(NH-Z)-Bid was trea-
ted with TFA as reported for TFAÆH-Dmt-Tic-e-Lys(Z)-
NH-CH2-Ph: yield 0.05 g (94%); Rf (A) 0.33; HPLC
20
5.2.23. Boc-Dmt-Tic-e-Lys-NH-Ph. Boc-Dmt-Tic-e-
Lys(Z)-NH-Ph was dissolved in methanol and treated
with Pd/C (10%) and H2 as reported for Boc-Dmt-Tic-
K0 2.95; mp 143–145 ꢁC; ½aꢀ ꢁ17.8; m/z 704 (M+H)+;
D
1H NMR (DMSO-d6) d 1.29–1.84 (m, 6H), 2.35
(s, 6H), 2.92–3.20 (m, 6H), 3.95–5.34 (m, 7H), 6.29
(s, 2H), 6.96–7.70 (m, 13H). Anal Calcd for
C45H48F6N6O9: C, 58.06; H, 5.20; N, 9.06. Found: C,
57.97; H, 5.16; N, 8.89.
e-Lys-NH-CH2-Ph: yield 0.08 g (87%); Rf (B) 0.55;
20
HPLC K0 5.12; mp 146–148 ꢁC; ½aꢀ ꢁ19.1; m/z 673
D
(M+H)+.
5.2.24. 2TFAÆH-Dmt-Tic-e-Lys-NH-Ph (6). Boc-Dmt-
Tic-e-Lys-NH-Ph was treated with TFA as reported
for TFAÆH-Dmt-Tic-e-Lys(Z)-NH-CH2-Ph: yield 0.04 g
(95%); Rf (A) 0.37; HPLC K0 2.58; mp 147–149 ꢁC;
5.2.29. Boc-Tic-NH-(CH2)4-CH(NH-Ac)-Bid. This inter-
mediate was obtained by condensation of Boc-Tic-e-Ly-
s(Ac)-OH with o-phenylendiamine via mixed anhydrides
(IBCF) as reported for Boc-Tic-NH-(CH2)4-CH(NH-
20
1
½aꢀ ꢁ14.8; m/z 573 (M+H)+; H NMR (DMSO-d6) d
Z)-Bid: yield 0.34 g (88%); Rf (B) 0.60; HPLC K0 4.31;
D
20
1.29–1.89 (m, 6H), 2.35 (s, 6H), 2.92–3.20 (m, 6H),
3.56–4.92 (m, 5H), 6.29 (s, 2H), 6.96–7.64 (m, 9H). Anal
Calcd for C37H43F6N5O8: C, 55.57; H, 5.42; N, 8.76.
Found: C, 55.82; H, 5.53; N, 8.47.
mp 140–142 ꢁC; ½aꢀ ꢁ13.7; m/z 521 (M+H)+; 1H
NMR (DMSO-d6) dD1.29–1.84 (m, 15H), 2.02 (s, 3H),
2.92–3.20 (m, 4H), 4.17–4.92 (m, 4H), 6.96–7.70 (m,
8H).

3150
G. Balboni et al. / Bioorg. Med. Chem. 15 (2007) 3143–3151
5.2.30. 2TFAÆH-Tic-NH-(CH2)4-CH(NH-Ac)-Bid. Boc-
Tic-NH-(CH2)4-CH(NH-Ac)-Bid was treated with
TFA as reported for TFAÆH-Tic-e-Lys(Z)-NH-CH2-
(Whatman GFC) were soaked in 0.1% polyethylenimine
in order to enhance the signal-to-noise ratio of the
bound radiolabelled-synaptosome complex, and the fil-
ters were washed thrice in ice-cold buffered BSA.26
The affinity constants (Ki) were calculated according to
Cheng and Prusoff.24
Ph: yield 0.20 g (90%); Rf (A) 0.44; HPLC K0 3.21; mp
20
143–145 ꢁC; ½aꢀ ꢁ15.0; m/z 421 (M+H)+.
D
5.2.31. Boc-Dmt-Tic-NH-(CH2)4-CH(NH-Ac)-Bid. This
intermediate was obtained by condensation of
Boc-Dmt-OH with 2TFAÆH-Tic-NH-(CH2)4-CH(NH-
Ac)-Bid via WSC/HOBt as reported for Boc-Dmt-Tic-
5.3.2. Biological activity in isolated tissue preparations.
The myenteric plexus longitudinal muscle preparations
(2–3 cm segments) from the small intestine of male Hart-
ley strain guinea pigs (GPI) measured l-opioid receptor
agonism, and a single mouse vas deferens (MVD) was
used to determine d-opioid receptor agonism as
described previously.6,29 The isolated tissues were sus-
pended in organ baths containing balanced salt solu-
tions in a physiological buffer, pH 7.5. Agonists were
tested for the inhibition of electrically evoked contrac-
tion and expressed as IC50 (nM) obtained from the
dose–response curves. The IC50 values represent mean-
NH-(CH2)4-CH(NH-Z)-Bid: yield 0.14 g (85%); Rf (B)
20
0.60; HPLC K0 4.21; mp 132–134 ꢁC; ½aꢀ ꢁ15.2; m/z
D
712 (M+H)+; 1H NMR (DMSO-d6) d 1.29–1.84 (m,
15H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92–3.20 (m, 6H),
4.41–4.92 (m, 5H), 6.29 (s, 2H), 6.96–7.70 (m, 8H).
5.2.32. 2TFAÆH-Dmt-Tic-NH-(CH2)4-CH(NH-Ac)-Bid
(8). Boc-Dmt-Tic-NH-(CH2)4-CH(NH-Ac)-Bid was
treated with TFA as reported for TFAÆH-Dmt-Tic-e-
Lys(Z)-NH-CH2-Ph: yield 0.08 g (98%); Rf (A) 0.30;
s
SE of five or six separate assays. d-antagonist poten-
20
HPLC K0 2.63; mp 149–151 ꢁC; ½aꢀ ꢁ18.7; m/z 612
cies in the MVD assay were determined against the
d-agonist deltorphin II and are expressed as pA2 deter-
mined using the Schild Plot.30
D
1
(M+H)+; H NMR (DMSO-d6) d 1.29–1.84 (m, 6H),
2.02 (s, 3H), 2.35 (s, 6H), 2.92–3.20 (m, 6H), 3.95–4.92
(m, 5H), 6.29 (s, 2H), 6.96–7.70 (m, 8H). Anal Calcd
for C39H44F6N6O8: C, 55.84; H, 5.29; N, 10.02. Found:
C, 56.01; H, 5.38; N, 10.12.
Acknowledgments
5.2.33. Boc-Dmt-Tic-NH-(CH2)4-CH(NH2)-Bid. Boc-
Dmt-Tic-NH-(CH2)4-CH(NH-Z)-Bid was dissolved in
methanol and treated with Pd/C (10%) and H2 as
reported for Boc-Dmt-Tic-e-Lys-NH-CH2-Ph: yield
This research was supported in part by the University of
Cagliari (PRIN 2004), University of Ferrara (PRIN
2004), and the Intramural Research Program of NIH
and NIEHS. The authors appreciate the professional
expertise and assistance of the library staff and the Com-
parative Medicine Branch at NIEHS.
0.08 g (86%); Rf (B) 0.51; HPLC K0 3.56; mp 140–
20
142 ꢁC; ½aꢀ ꢁ19.3; m/z 670 (M+H)+.
D
5.2.34. 3TFAÆH-Dmt-Tic-NH-(CH2)4-CH(NH2)-Bid (9).
Boc-Dmt-Tic-NH-(CH2)4-CH(NH2)-Bid was treated
with TFA as reported for TFAÆH-Dmt-Tic-e-Lys(Z)-
NH-CH2-Ph: yield 0.06 g (95%); Rf (A) 0.29; HPLC K0
References and notes
1. Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.; Bryant,
S. D.; Crescenzi, O.; Guerrini, R.; Picone, D.; Tancredi,
T.; Temessi, P. A.; Lazarus, L. H. Mol. Med. 1995, 1, 678.
2. Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.;
Lazarus, L. H. Biopolymers 2003, 71, 86.
3. Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.;
Bianchi, C.; Bryant, S. D.; Cooper, P. S.; Lazarus, L. H.
J. Med. Chem. 1997, 40, 3100.
20
1
2.86; mp 154–156 ꢁC; ½aꢀ ꢁ19.9; m/z 570 (M+H)+; H
D
NMR (DMSO-d6) d 1.29–1.84 (m, 6H), 2.35 (s, 6H),
2.92–3.20 (m, 6H), 3.90–4.92 (m, 5H), 6.29 (s, 2H),
6.96–7.70 (m, 8H). Anal Calcd for C39H43F9N6O9: C,
51.43; H, 4.76; N, 9.23. Found: C, 51.32; H, 4.62; N, 9.12.
5.3. Pharmacology
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Giannini, E.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H.
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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 as-
say using brain P2 synaptosomal membranes prepared
from Sprague–Dawley rats.26,27 Synaptosomes were pre-
incubated to remove endogenous opioid peptides and
stored at ꢁ80 ꢁC in buffered 20% glycerol.26,28 Each
analogue was analyzed in duplicate assays using 5–8
dosages and 3–5 independent repetitions with different
synaptosomal preparations (n values are listed in Table
1 in parentheses and results are means SE). Unlabelled
peptide (2 lM) was used to determine non-specific bind-
ing 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 Bioscience, Buckinghamshire, UK;
KD = 1.5 nM) for l-opioid receptors. Glass fibre filters
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