Synthesis and opioid activity of N,N-Dimethyl-Dmt-Tic-NH-CH(R)-R′ analogues: Acquisition of potent δ antagonism

Article abstract of DOI:10.1016/j.bmc.2003.09.039

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035-0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′-8′) elicited a greater reduction in μ-affinity (40-70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′-7′) (pA₂=8.14-9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.

Full text of DOI:10.1016/j.bmc.2003.09.039

Bioorganic & Medicinal Chemistry 11 (2003) 5435–5441
Synthesis and Opioid Activity of N,N-Dimethyl-Dmt-Tic-NH-
CH(R)-R0 Analogues: Acquisition of Potent ꢀ Antagonism
Gianfranco Balboni,a Severo Salvadori,b Remo Guerrini,b Lucia Negri,c Elisa Giannini,c
Sharon D. Bryant,d Yunden Jinsmaad and Lawrence H. Lazarusd,*
aDepartment of Toxicology, University of Cagliary, I-09126, Cagliary, Italy
bDepartment of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy
cDepartment of Human Physiology and Pharmacology ‘Vittorio Erspamer,’ University La Sapienza, I-00185 Rome, Italy
dPeptide Neurochemistry, LCBRA, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
Received 13 May 2003; accepted 17 September 2003
Abstract—N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R0 series of compounds produced no significant affect on the high
d-opioid receptor affinity (Ki=0.035–0.454 nM), but dramatically decreased that for the m-opioid receptor. The effect of N-methyl-
ation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the
third aromatic group (R0): phenyl (Ph) (50–80) elicited a greater reduction in m-affinity (40–70-fold) compared to analogues con-
taining 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss
of d-agonism coupled with the appearance of potent d antagonism (40–70) (pA2=8.14–9.47), while 1 exhibited only a 160-fold
decreased d agonism (10) and the d antagonism of 8 enhanced >10-fold (pA2=10.62, 80); and (ii) a consistent loss of m-affinity
resulted in enhanced d-opioid receptor selectivity. With the exception of compound 10, the change in the hydrophobic environment
at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced
potent d-selective antagonists.
Published by Elsevier Ltd.
Introduction
piperidine, pyrrolidine or pyrrole, were detrimental to d
and m bioactivity as well as affinity;7 similar effects were
N-Alkylation of opioid peptides containing the Dmt-Tic
pharmacophore were initially designed to increase sta-
bility against cyclization between the C-terminal car-
boxylate and the N-terminal amine leading to
diketopiperazine formation,1,2 which drastically reduced
both opioid receptor affinity and bioactivity.3 While
earlier studies demonstrated that N,N-methylation of
enkephalin analogues yielded d antagonists,4,5 N-mono-
or N,N-dimethylation of the Dmt-Tic pharmacophore
also altered the pharmacologically defined profiles for d-
and m-bioactivity in some analogues without substantially
effecting d-opioid receptor affinity.6,7 The combined effect
of N,N-dimethylation and C-terminal modification in the
Dmt-Tic pharmacophore played a variable role on the
acquisition of m affinity and bioactivity profile. N-Alkyl-
ation with bulky, cyclic hydrophobic groups, such as
observed
when
cyclopropylmethyl,
dicyclopro-
pylmethyl, benzyl or diallyl groups were incorporated
into dynorphin A(1–11) analogues.8
It has been well documented that N-alkylation of a wide
variety biologically relevant peptides9ꢀ15 and model
peptides produced physicochemical perturbations that
affected their solution structure, conformation16ꢀ20 and
biological activities.13,14,21 However, X-ray crystal-
lography analyses of three Dmt-Tic analogues verified
that N-methylation did not substantially effect the
N-terminal conformations of these compounds.22 In
other compounds, the presence of N-alkylated amino
acids influenced hydrogen bonding, cis/trans orienta-
tions, electrostatic interactions and stereochemistry,18 as
well as aggregation rates, conformational freedom and
flexibility.11,16 These alterations invariably affected their
pharmacological properties,13,14,21 as noted for other
opioid peptides,4,5,7,8,23ꢀ25 and the enhanced bioactivity in
some N-alkylated peptide hormones resulted in resistance
*Corresponding author. Tel.: +1-919-541-3238; fax: +1-919-541-
0696; e-mail: lazarus@niehs.nih.gov
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2003.09.039

5436
G. Balboni et al. / Bioorg. Med. Chem. 11 (2003) 5435–5441
to proteolytic degradation.9 However, the loss of
bioactivity in other peptides was attributed to a sub-
stantial conformational change,13 or steric hindrance
within the receptor binding site by the bulky N-terminal
alkylated amine.7,8 Nonetheless, the requirement for an
N-terminal amine in opioid peptides was demonstrated
by replacing it with a methyl group that obliterated
d-receptor affinity in analogues of the Dmt-Tic phar-
macophore.26 In contrast, the same modification in
[Dmt1]dynorphin A(1–11)-NH2, a k-opioid agonist,
produced a k antagonist.27
exceptional d agonist.28,29 This communication further
explores the effect of increased hydrophobicity through
N,N-dimethylation in a series of H-Dmt-Tic-NH-
CH(R)-R0 compounds on opioid receptor binding and
functional bioactivity (Fig. 1).
Results and Discussion
The receptor affinity constants (Ki values) for the eight
pairs of peptides revealed that d affinity essentially
remained unaltered upon N,N-methylation of the Dmt-
Tic pharmacophore, whereas m affinity decreased sub-
stantially (Table 1) as seen with Dmt-Tic analogues
containing C-terminal NH-tBu, NHMe, 1-adamantyl6
or a free acid functional group.7 In our present series of
compounds (Table 1), N,N-dimethylation decreased m
affinity by 40- (50), 50- (60), 60- (80) and 70-fold (70)
relative to their non-methylated analogues (5–8), which
was similar to data acquired in derivatives of the Dmt-
Tic pharmacophore containing a variety of C-terminal
hydrophobic groups.7,30,31 This effect appeared to
depend on the aromatic moiety (Bid or Ph) at the
C-terminal of Dmt-Tic. Peptides with Ph (5–8, 50–80)
As a general principle, the increase in overall hydro-
phobicity of the Dmt-Tic pharmacophore greatly
enhanced receptivity to m receptors with minimal effect
on d-receptor affinity although the spectrum of bioac-
tivity in vitro changed dramatically. Recent data indi-
cated the importance of a linker between the C-terminus
of Tic and a third aromatic center [1H-benzimidazole-2-
yl (Bid) or phenyl (Ph)] enhanced not only m-receptor
affinity, but also converted a potent d antagonist into an
displayed
N-alkylation compared to the derivatives containing Bid
a dramatic decrease in m-affinity after
with similar alkyl linkers (2–4, 20–40) (Table 1).
Studies reported that the length of the linker and com-
position of the C-terminal aromatic substituents caused
a major shift in the spectrum of biological activity of the
Figure 1. General structure of the reported compounds. R=H or
CH3; linker=none, CO–NH, CO–NH–CH2, CO–NH–CH2–CH2, CO-
Gly-NH, CO-Gly-NH–CH2.
Table 1. Opioid receptor affinity of H-Dmt-Tic-NH-CH(R)-R0 analogues with and without N,N-dimethylation
No.
Compound
d (nM)
ꢁ
ꢁ
SE
n
m (nM)
ꢁ
ꢁ
SE
N
m/d
Ref
28
1
H-Dmt-Tic-NH–CH2-Bid
0.035
0.006
(3)
0.50
0.054
(3)
14
10
2
N,N(Me)2-Dmt-Tic-NH–CH2-Bid
H-Dmt-Tic-NH–CH2–CH2-Bid
N,N(Me)2-Dmt-Tic-NH–CH2–CH2-Bid
H-Dmt-Tic-Gly-NH–CH2-Bid
N,N(Me)2-Dmt-Tic-Gly-NH–CH2-Bid
H-Dmt-TIB
0.037
0.067
0.074
0.058
0.057
0.13
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
0.004
0.015
0.007
0.005
0.004
0.04
(3)
(4)
(3)
(3)
(4)
(4)
(3)
(4)
(4)
(3)
(3)
(3)
(3)
(3)
(3)
3.35
5.49
18.6
20.5
27.7
7.22
66.2
0.95
38.4
1.45
72.1
0.155
10.7
0.163
9.44
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
0.39
0.093
1.1
(3)
(3)
(3)
(3)
(3)
(4)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
91
82
28
28
20
3
251
353
486
56
2.4
30
4
1.1
0.96
4.7
28, 35
40
5
N,N(Me)2-Dmt-TIB
0.454
0.069
0.049
0.093
0.099
0.042
0.051
0.031
0.034
0.018
0.01
146
14
H-Dmt-Tic-NH–CH2–Ph
0.23
3.4
50
6
N,N(Me)2-Dmt-Tic-NH-CH2-Ph
H-Dmt-Tic-NH–Ph
0.007
0.015
0.21
711
16
0.23
6.8
60
7
N,N(Me)2-Dmt-Tic-NH–Ph
H-Dmt-Tic-Gly-NH–Ph
728
0.007
0.008
0.002
0.003
0.003
0.33
0.018
0.11
3.6
210
5.3
278
28
28
70
8
N,N(Me)2-Dmt-Tic-Gly-NH–Ph
H-Dmt-Tic-Gly-NH–CH2–Ph
N,N(Me)2-Dmt-Tic-Gly-NH–CH2–Ph
80
Me, methyl; Bid, 1H-benzimidazol-2-yl; TIB, 3-(1H-benzimidazol-2-yl)-1,2,3,4-tetrahydroisoquinolin-2-yl; Ph, phenyl. The number of independent
repetitions n (in parentheses) used different synaptosomal preparations as summarized in the Experimental.

G. Balboni et al. / Bioorg. Med. Chem. 11 (2003) 5435–5441
5437
peptides, from being very strong d antagonists to highly
potent d agonists.28,29 As seen in Table 2, the functional
bioactivity data indicated that N,N-dimethylation was
detrimental to the observed d agonist bioactivity of
compounds 1, 4–7. Interestingly, 4, 5 and 6 initially
displayed weak multifunctional bioactivities, that is,
weak d agonism/d antagonism and weak m agonism;
they became much more potent d antagonists upon
N,N-dimethylation while losing all bioactivity toward
the m-opioid receptor (GPI) and d agonism as well
(Table 2), except 10 which had only a 160-fold
decrease in d agonism. In general, d antagonism
increased more than 10-fold for 20, 30 and 80, and at
the same time m bioactivity decreased, whereas with 50
and 60 d-receptor antagonism increased by more than
two orders of magnitude and 80 displayed a remark-
ably high d-antagonism (pA2=10.62), which accom-
panied a 1,300-fold decrease in m-agonist activity
(Table 2). Thus, the data indicated that m agonism
decreased substantially in all the analogues studied
(10–80) (Table 2), which contrasted to the effects gen-
erated by non-N,N-dimethylated derivatives of the
H-Dmt-Tic series of pseudopeptides.7,28,29
inhibited the mechanism(s) of activating the d-opioid
receptor that gave rise to d- or m-receptor agonism.
Conclusions
N,N-Dimethylation of C-terminally extended Dmt-Tic
pharmacophore analogues containing an alkyl linker to
a third aromatic center, either Ph or a Bid, had the fol-
lowing effects: (i) Minimal alteration of d-opioid recep-
tor affinity; (ii) reduction in the m-receptor affinity
relative to non-methylated analogues; (iii) drastically
decreased d-opioid receptor agonism while enhancing d
antagonism; and (iv) eliminated or greatly reduced the m
bioactivity for all compounds. The major consequence
was the acquisition of potent d-opioid receptor antag-
onism in several analogues, the degree to which this
occurred involved the chemical composition of the third
aromatic residue. These compounds may play a role
determining the function of distinct opioid receptors in
non-knockout animal models and might find potential
clinical and therapeutic application as stable and highly
selective d-opioid receptor antagonists with enhanced
hydrophobicity required for passage through membrane
barriers.26
The effect of N,N-dimethylation on the biological
activity of Dmt-Tic-containing peptides apparently
involves several interrelated factors: (i) Stabilization
against cyclization (diketopiperazine formation),1,2
which is a spontaneous reaction that greatly reduced the
interaction with d receptors and eliminated biological
activity.3 (ii) Limitation in the rotation of the N-term-
inal amine due to steric hindrance by the bulky N,N-
dimethyl groups16,18 allowing the potential formation of
more stable hydrogen bonds.6 (iii) Increasing the
hydrophobic interactions between the ligand and the
receptor binding site could involve the spatial position-
ing of the pharmacophore32 deduced from X-ray dif-
fraction analyses of three Dmt-Tic pharmacophore
analogues, two of which contained N,N-dimethyla-
tion.22 (iv) Enhancement of the d antagonist activity
suggesting that N,N-dimethylation interfered with or
Experimental
Chemistry
Scheme 1 summarizes the basic synthesis of N,N-dime-
thyl-Dmt-Tic-NH-CH(R)-R0 analogues. H-l-Dmt-OH
was prepared according to Dygos et al.,33 and purity
and chirality were compared to a sample generously
donated by Dygos et al. Boc-Tic-OH was purchased
from Bachem (Heidelberg, Germany). All pseudopep-
tides were prepared by standard solution step-by-step
peptide synthesis. Mixed carbonic anhydride coupling
of Boc-Gly-OH, Boc-bAla-OH or Boc-Gly-Gly-OH
with o-phenylendiamine gave the corresponding crude
intermediate amides, which were converted without
purification to the desired heteroaromatic derivatives by
cyclization and dehydration in acetic acid. After depro-
tection with TFA, each derivative was condensed with
Boc-Tic-OH and then with Boc-Dmt-OH via WSC/
HOBt. Di- and tripeptides containing C-terminal benzyl
amide or phenyl amide were obtained in a similar man-
ner starting from the condensation of the appropriate
Boc-amino acid with benzyl amine or aniline via WSC/
HOBt, respectively. N,N-dimethylation was accom-
plished with 37% aqueous formaldehyde and
NaBH3CN according to the procedure of Borch and
Hassid as outlined in Scheme 1.34 Crude pseudopeptides
were purified by preparative reversed-phase high per-
formance liquid chromatography (HPLC) using a
Waters Delta Prep 4000 system with Waters Prep LC 40
mm Assembly C18 column (30ꢂ4 cm; 15 mm particle
size). 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%
solvent B (60% acetonitrile in 0.1% TFA, v/v) in 25 min
was adopted for the elution of the products. Analytical
Table 2. Functional Bioactivity of H-Dmt-Tic-NH-CH(R)-R0 analo-
gues with and without N,N-dimethylation
No. MVD IC50
(nM)
ꢁ
SE
MVD GPI IC50
pA2
ꢁ
SE
Ref
(nM)
1
0.035
5.6
—
—
—
—
52.5
—
111.0
—
ꢁ
ꢁ
0.003
0.4
—
—
8.32
40.7
132.6
107.5
ꢁ
ꢁ
ꢁ
ꢁ
5
15
11.4
28
28
28
10
2
20
3
9.67 7152
9.00 400
9.90 >10 mM
7.90 30
8.14 >10 mM
6.01 850
9.51 >10 mM
6.81
9.07
—
350
30
4
ꢁ
ꢁ
1.5 28, 35
110
40
5
ꢁ
ꢁ
13.4
8.07
50
6
171.6
—
3.0
110
683
2.57
ꢁ
ꢁ
13
55
60
7
28
28
70
8
9.47 1720
9.25 2.69
10.62 3652
ꢁ
ꢁ
112
202
—
—
80
Antagonist activity is defined by pA2. Inactivity is denoted by a dash
(—).

5438
G. Balboni et al. / Bioorg. Med. Chem. 11 (2003) 5435–5441
Scheme 1. Synthesis of N,N-(Me)2-Dmt-Tic-Gly-NH–CH2-Bid (30).
HPLC analyses were performed with a Beckman System
Gold with a Beckman ultrasphere ODS column (250ꢂ4.6
mm, 5 mm). Analytical determinations and capacity fac-
tor (K0) of the products were determined using the HPLC
in the above solvent systems (solvents A and B) pro-
grammed at flowrates 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 nm.
hydrin (1%, Merck), fluorescamine (Hoffman-La
Roche) and chlorine reagents were used as sprays. Open
column chromatography (2ꢂ70 cm, 0.7–1 g 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 10 cm water-jacketed cell. All 1H
NMR spectra were recorded on a Bruker 200 MHz spec-
trometer. MALDI-TOF analyses (matrix assisted laser
desorption ionization time-of-flight mass spectrometry)
of peptides were conducted using a Hewlett Packard G
TLC was performed on precoated plates of silica gel
F254 (Merck, Darmstadt, Germany) using the following
solvent systems: (A) 1-butanol/AcOH/H2O (3:1:1, v/v/v);
and (B) CH2Cl2/toluene/methanol (17:1:2, v/v/v). Nin-

G. Balboni et al. / Bioorg. Med. Chem. 11 (2003) 5435–5441
5439
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 a-cyano-4-hydroxy-
cinnamic acid matrix.
yield 0.31 g. (98%); Rf (A) 0.52; HPLC K0=6.51; mp
155–157 ꢃC; [a]2D0 ꢀ31.8; MH+ 267.
Boc - Dmt - Tic - NH–CH2–Ph. This compound was
obtained by condensation of Boc-Dmt-OH with
.
TFA H-Tic-NH–CH2–Ph as reported for Boc-Dmt-Tic-
Peptide synthesis
Gly-NH–CH2–Ph:28 yield 0.15 g. (83%); Rf (B) 0.73;
HPLC K0=10.9; mp 145–147 ꢃC; [a]2D0 ꢀ22.6; MH+
558; 1H NMR (DMSO) d=1.40, 1.42 (2ꢂs, 9H), 2.17 (s,
6H), 3.08–3.15 (m, 4H), 4.22–4.92 (m, 5H), 6.37 (s, 2H),
6.91–7.31 (m, 10H), 8.25 (m, 1H).
0
red solution of 2TFA H-Dmt-Tic-NH–CH2-Bid (0.29
.
2TFA N,N-(Me)2-Dmt-Tic-NH-CH2-Bid (1 ). To a stir-
28
.
g, 0.40 mmol) in acetonitrile/H2O (1:1, v/v) (10 mL)
were added NMM (0.09 mL, 0.80 mmol), 37% aqueous
formaldehyde (0.32 mL, 4 mmol) and sodium cyanobor-
ohydride (0.08 g, 1.2 mmol). Glacial acetic acid (0.04
mL) was added over 10 min and the reaction was stirred
at room temperature for 15 min. The reaction mixture
was acidified with TFA (0.1 mL) and directly purified by
preparative HPLC: yield 0.26 g. (88%); Rf (A) 0.35;
HPLC K0=5.70; mp 158–160 ꢃC; [a]2D0 ꢀ31.8; MH+ 527.
.
TFA H - Dmt - Tic - NH–CH2–Ph. Boc-Dmt-Tic-NH–
.
CH2–Ph was treated with TFA as reported for TFA H-
Dmt-Tic-Gly-NH–CH2–Ph:28 yield 0.07 g. (96%); Rf
(A) 0.49; HPLC K0=9.03; mp 158–160 ꢃC; [a]2D0 ꢀ23.4;
MH+ 458.
0
.
TFA N,N-(Me)2-Dmt-Tic-NH–CH2–Ph (5 ). This was
0
was obtained by exhaustive methylation of 2TFA H-
.
2TFA N,N-(Me)2-Dmt-Tic-NH–CH2–CH2-Bid (2 ). This
.
obtained by exhaustive methylation of TFA H-Dmt-
Tic-NH-CH2-Ph as reported for TFA.N,N-(Me)2-Dmt-
Tic-Gly-NH-Ph: yield 0.66 g. (91%); Rf (A) 0.52; HPLC
K0=9.36; mp 164–166 ꢃC; [a]2D0 ꢀ21.9; MH+ 487.
.
Dmt-Tic-NH–CH2–CH2-Bid28
.
as
reported
for
2TFA N,N-(Me)2-Dmt-Tic-NH–CH2-Bid: yield 0.048 g.
(81%); Rf (A) 0.38; HPLC K0=5.29; mp 165–167 ꢃC; [a]2D0
ꢀ27.2; MH+ 541.
Boc-Tic-NH–Ph. This compound was obtained by con-
densation of Boc-Tic-OH with aniline as reported for
Boc-Gly-NH–CH2–Ph:28 yield 0.78 g. (85%); Rf (B)
0.78; HPLC K0=11.5; mp 139–141 ꢃC; [a]2D0 ꢀ31.5;
0
.
2TFA N,N-(Me)2-Dmt-Tic-Gly-NH–CH2-Bid (3 ). This
.
was obtained by exhaustive methylation of 2TFA H-
Dmt-Tic-Gly-NH–CH2-Bid28 as reported for 2TFA.N,N-
(Me)2-Dmt-Tic-NH–CH2-Bid: yield 0.048 g. (89%); Rf
(A) 0.39; HPLC K0=4.76; mp 159–161 ꢃC; [a]2D0 ꢀ27.2;
MH+ 584.
MH+ 353; H NMR (DMSO) d=1.40, 1.42 (2ꢂs, 9H),
1
3.06–3.15 (m, 2H), 4.22–4.92 (m, 3H), 6.95–7.15 (m,
9H), 8.96 (bs, 1H).
.
TFA H-Tic-NH–Ph. Boc-Tic-NH–Ph was treated with
TFA as reported for TFA.H-Gly-NH–CH2–Ph:28 yield
0.27 g. (96%); Rf (A) 0.48; HPLC K0=6.27; mp
150–152 ꢃC; [a]2D0 ꢀ33.1; MH+ 253.
.
2TFA N,N-(Me)2-Dmt-3-(1H-benzimidazol-2-yl)-1,2,3,4-
tetrahydroisoquinoline-2-yl (TIB) (40). This was obtained
by exhaustive methylation of 2TFA.H-Dmt-TIB35,28 as
.
reported for 2TFA N,N-(Me)2-Dmt-Tic-NH–CH2-Bid:
yield 0.058 g. (90%); Rf (A) 0.48; HPLC K0=3.92; mp
Boc-Dmt-Tic-NH–Ph. This compound was obtained by
168–170 ꢃC; [a]2D0 ꢀ35.1; MH+ 470.
condensation of Boc-Dmt-OH with TFA H-Tic-NH–Ph
.
as reported for Boc-Dmt-Tic-Gly-NH–CH2–Ph;28 yield
0.16 g. (82%); Rf (B) 0.69; HPLC K0=10.1; mp 140–142 ꢃC;
[a]2D0 ꢀ24.1; MH+ 544; 1H NMR (DMSO) d=1.40, 1.42
(2ꢂs, 9H), 2.17 (s, 6H), 3.08–3.15 (m, 4H), 4.22–4.92
(m, 4H), 6.37 (s, 2H), 6.91–7.31 (m, 10H), 8.95 (bs, 1H).
0
solution of TFA H-Dmt-Tic-Gly-NH–Ph (0.1 g, 0.17
.
TFA N,N-(Me)2-Dmt-Tic-Gly-NH–Ph (7 ). To a stirred
28
.
mmol) in acetonitrile/H2O (1:1, v/v) (10 mL) were
added NMM (0.02 mL, 0.17 mmol), 37% aqueous
formaldehyde (0.14 mL, 1.7 mmol) and sodium cyano-
borohydride (0.032 g, 0.51 mmol). Glacial acetic acid
(0.04 mL) was added over 10 min and the reaction was
stirred at room temperature for 15 min. The reaction
mixture was acidified with TFA (0.1 mL) and directly
purified by preparative HPLC: yield 0.08 g. (84%); Rf
(A) 0.48; HPLC K0=9.36; mp 155–157 ꢃC; [a]2D0 ꢀ18.3;
MH+ 487.
.
TFA H - Dmt - Tic - NH–Ph. Boc-Dmt-Tic-NH–Ph was
.
treated with TFA as reported for TFA H-Dmt-Tic-Gly-
NH–CH2–Ph:28 yield 0.065 g. (97%); Rf (A) 0.46;
HPLC K0=8.25; mp 146–148 ꢃC; [a]2D0 ꢀ25.8; MH+ 445.
0
obtained by exhaustive methylation of TFA H-Dmt-
.
TFA N,N-(Me)2-Dmt-Tic-NH–Ph (6 ). This product was
.
.
Tic-NH–Ph as reported for TFA N,N-(Me)2-Dmt-Tic-
Boc-Tic-NH–CH2–Ph. This compound was obtained by
condensation of Boc-Tic-OH with benzylamine as
reported for Boc-Gly-NH–CH2–Ph:28 yield 0.81 g.
(87%); Rf (B) 0.81; HPLC K0=12.1; mp 132–134 ꢃC;
[a]2D0 ꢀ40.8; MH+ 367; 1H NMR (DMSO) d=1.39, 1.46
(2ꢂs, 9H), 3.06 (m, 2H), 4.22–4.96 (m, 5H), 6.95–7.15
(m, 9H), 8.26 (m, 1H).
Gly-NH–Ph: yield 0.58 g (89%); Rf (A) 0.51; HPLC
K0=8.39; mp 149–151 ꢃC; [a]2D0 ꢀ27.5; MH+ 472.
Boc-Tic-Gly-NH–Ph. This compound was obtained by
condensation of Boc-Tic-OH with TFA.H-Gly-NH–Ph
as reported for Boc-Tic-Gly-NH–Bzl: yield 0.24 g.
(85%); Rf (B) 0.67; HPLC K0=10.73; mp 131–133 ꢃC;
[a]2D0 ꢀ36.8; MH+ 410; 1H NMR (DMSO) d=1.39, 1.41
(2ꢂs, 9H), 3.08–3.15 (m, 2H), 3.61 (d, 2H), 4.22–4.92
(m, 3H), 6.91–7.31 (m, 10H), 8.96 (bs, 1H).
.
TFA H-Tic-NH–CH2–Ph. Boc-Tic-NH–CH2–Ph was
treated with TFA as reported for TFA.H-Gly-NH–Bzl:28

5440
G. Balboni et al. / Bioorg. Med. Chem. 11 (2003) 5435–5441
.
TFA H-Tic-Gly-NH–Ph. Boc-Tic-Gly-NH–Ph was trea-
2. Capasso, S.; Sica, F.; Mazzarella, L.; Balboni, G.;
Guerrini, R.; Salvadori, S. Int. J. Pept. Prot. Res. 1995, 45,
567.
3. Balboni, G.; Guerrini, R.; Salvadori, S.; Tomatis, R.; Bry-
ant, S. D.; Bianchi, C.; Attila, M.; Lazarus, L. H. Biol. Chem.
1997, 378, 19.
4. Cotton, R.; Giles, M. G.; Miller, L.; Shaw, J. S.; Timms, D.
Eur. J. Pharmacol. 1984, 97, 331.
5. Lovett, J. A.; Portoghese, P. S. J. Med. Chem. 1987, 30, 1144.
6. 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.
.
ted with TFA as reported for TFA H-Tic-Gly-NH–Bzl:
yield 0.18 g. (96%); Rf (A) 0.35; HPLC K0=6.07; mp
165–167 ꢃC; [a]2D0 ꢀ32.5; MH+ 310.
Boc-Dmt-Tic-Gly-NH–Ph.
obtained by condensation of Boc-Dmt-OH with
This
compound
was
.
TFA H-Tic-Gly-NH–Ph as reported for Boc-Dmt-Tic-
Gly-NH–Bzl: yield 0.14 g. (84%); Rf (B) 0.64; HPLC
0
ꢃ
1
K =9.9; mp 144—146 C; [a]2D0 ꢀ19.7; MH+ 601; H
NMR (DMSO) d=1.40, 1.42 (2ꢂs, 9H), 2.17 (s, 6H),
3.08–3.15 (m, 4H), 3.61 (d, 2H), 4.22–4.92 (m, 3H), 6.37
(s, 2H), 6.91–7.31 (m, 11H), 8.94 (bs, 1H).
7. Salvadori, S.; Guerrini, R.; Balboni, G.; Bianchi, C.; Bry-
ant, S. D.; Cooper, P. S.; Lazarus, L. H. J. Med. Chem. 1999,
42, 5010.
8. Soderstrom, K.; Choi, H.; Berman, F. W.; Aldrich, J. V.;
Murray, T. F. Eur. J. Pharmacol. 1997, 338, 191.
9. Turk, J.; Needleman, P.; Marshall, G. R. J. Med. Chem.
1975, 18, 1139.
10. Turk, J.; Needleman, P.; Marshall, G. R. Mol. Pharmacol.
1976, 12, 217.
11. Patel, D.; Tonelli, A. Biopolymers 1976, 15, 1623.
12. Jeffs, P.; Heald, S.; Chodosh, D.; Eggleston, D. Int. J.
Pept. Prot. Res. 1984, 24, 442.
.
TFA H-Dmt-Tic-Gly-NH–Ph. Boc-Dmt-Tic-Gly-NH-
.
Ph was treated with TFA as reported for TFA H-Dmt-
Tic-Gly-NH-Bzl: yield 0.07 g. (97%); Rf (A) 0.41; HPLC
K0=7.18; mp 155–157 ꢃC; [a]2D0 ꢀ21.8; MH+ 444.
0
was obtained by exhaustive methylation of TFA H-
.
TFA N,N-(Me)2-Dmt-Tic-Gly-NH–CH2–Ph (8 ). This
.
Dmt-Tic-Gly-NH–CH2–Ph28 as reported for TFA N,N-
.
13. Ogawa, H.; Burke, G. T.; Chanley, J. D.; Kasoyannis,
P. G. Int. J. Pept. Prot. Res. 1987, 30, 460.
14. Dive, V.; Yiotakis, A.; Rounestand, C.; Gilquin, B.;
Labadie, J.; Toma, F. Int. J. Pept. Prot. Res. 1992, 39, 506.
15. He, Y.-B.; Huang, Z.; Raynor, K.; Reisine, T.; Goodman,
M. J. Am. Chem. Soc. 1993, 115, 8066.
(Me)2-Dmt-Tic-Gly-NH–Ph: yield 0.07 g. (86%); Rf (A)
0.45; HPLC K0=9.19; mp 158–160 ꢃC; [a]2D0 ꢀ20.4;
MH+ 473.
Biological assays
16. Tonelli, A. E. Biopolymers 1976, 15, 1615.
17. Vitoux, B.; Aubry, A.; Cung, M.; Boussard, G.; Marraud,
M. Int. J. Pept. Prot. Res. 1981, 17, 469.
18. Vitoux, B.; Aubry, A.; Cung, M.; Marraud, M. Int. J.
Pept. Prot. Res. 1986, 27, 617.
19. Vitoux, B.; Cung, M.; Marraud, M. J. Chim. Phys. Phys-
Chim. Biol. 1988, 85, 339.
20. Bello, J. Biopolymers 1992, 32, 491.
21. Toth, G.; Peter, A.; Tourwe, D.; Jaspers, H.; Verheyden,
P. M. F.; Toth, Z. Peptides 1994; Maia, H. L. S., Ed.;
ESCOM: Leiden, 1995; p 335.
22. Bryant, S. D.; George, C.; Flippen-Anderson, J.; Salva-
dori, S.; Balboni, G.; Guerrini, R.; Lazarus, L. H. J. Med.
Chem. 2002, 45, 5506.
23. Kawai, M.; Fukuta, N.; Ito, N.; Kagami, T.; Butsugan,
Y.; Maruyama, M.; Kudo, Y. Int. J. Pept. Prot. Res. 1990, 35,
452.
Receptor binding. Receptor affinities of the peptides
were obtained using rat brain synaptosome preparations
under equilibrium binding conditions as described pre-
viously6,7 (Table 1). The d-opioid receptors were labeled
with [3H]DPDPE (32.0 Ci/mmol; NEN-DuPont, Bill-
erica, MA, USA) and m-opioid receptors with [3H]DAGO
(58.0 Ci/mmol: Amersham, Arlington Heights, IL, USA)
in which 2 mM unlabeled peptide established the level of
nonspecific binding. The Ki values were determined
according to Cheng and Prusoff36 and given as the mean-
ꢁstandard error (SE) with n (in parentheses) to indicate
between three and five independent repetitions with dif-
ferent synaptosomal preparations for each analogue.
Functional bioactivity
24. Choi, H.; Murray, T. F.; DeLander, G. E.; Caldwell, V.;
Aldrich, J. J. Med. Chem. 1992, 35, 4638.
Functional bioassays in vitro utilized guinea-pig ileum
(myenteric plexus longitudinal muscle from the small
intestine; GPI) for m receptors and mouse vas deferens
(MVD) for d receptors. Agonism was determined by
inhibition of electrically stimulated relaxation of muscle
twitch as published previously.29 The IC50 values repre-
sent the meanꢁSE of five tissue samples based on del-
torphin B and dermorphin as the internal standards for
MVD and GPI assays, respectively. The pA2 is the
negative log of the molar concentration necessary to
double the agonist concentration (deltorphin B) to
achieve the original response.
25. Maeda, D. Y.; Ishmael, J. E.; Murray, T. F.; Aldrich, J. V.
J. Med. Chem. 2000, 43, 3941.
26. Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.;
Balboni, G.; Salvadori, S. Drug Discor. Today 1998, 3, 284.
27. Lu, Y.-F.; Nguyen, T. M.-D.; Weltroska, G.; Berezowska,
I.; Lemieux, C.; Chung, N. N.; Schiller, P. W. J. Med. Chem.
2001, 44, 3048.
28. Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi, C.; Rizzi,
D.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2002, 45, 713.
29. Balboni, G.; Salvadori, S.; Guerinni, R.; Negri, L.; Gion-
nini, Y.; Yunden, J.; Bryant, S. D.; Lazarus, L. H. J. Med.
Chem. 2002, 45, 5556.
30. Page, D.; Naismith, A.; Schmidt, R.; Coupal, M.;
Labarre, M.; Gosselin, M.; Bellemare, D.; Payza, K.; Brown,
W. J. Med. Chem. 2001, 44, 2387.
31. Page, D.; McClory, A.; Mischki, T.; Schmidt, R.; Butter-
worth, J.; St-Onge, S.; Labarre, M.; Payza, K.; Brown, W.
Bioorg. Med. Chem. Lett. 2000, 10, 167.
References and Notes
1. Carpenter, K. A.; Weltrowska, C.; Wilkes, B. C.; Schmidt,
R.; Schiller, P. W. J. Am. Chem. Soc. 1994, 116, 8450.
32. Bryant, S. D.; Balboni, G.; Guerrini, R.; Salvadori, S.;
Tomatis, R.; Lazarus, L. H. Biol. Chem. 1997, 378, 107.

G. Balboni et al. / Bioorg. Med. Chem. 11 (2003) 5435–5441
5441
33. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson,
O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R. Synthesis
1992, 8, 741.
35. Balboni, G.; Salvadori, S.; Guerrini, R.; Bianchi, C.;
Santagada, V.; Calliendo, G.; Bryant, S. D.; Lazarus, L. H.
Peptides 2000, 21, 1663.
34. Borch, R. F.; Hassid, A. I. J. Org. Chem. 1972, 37,
1673.
36. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.