Influence of the side chain next to C-terminal benzimidazole in opioid pseudopeptides containing the Dmt-Tic pharmacophore

Article abstract of DOI:10.1021/jm900686q

To improve the structure-activity studies of the lead δ opioid agonist H-Dmt-Tic-Asp*-Bid, we synthesized and pharmacologically characterized a series of analogues in which the side chain next to 1H-benzimidazole-2-yl (Bid) was substituted by those endowe

Full text of DOI:10.1021/jm900686q

5556 J. Med. Chem. 2009, 52, 5556–5559
DOI: 10.1021/jm900686q
Influence of the Side Chain Next to C-Terminal Benzimidazole in Opioid Pseudopeptides Containing the
Dmt-Tic Pharmacophore
Gianfranco Balboni,*,† Claudio Trapella,‡ Yusuke Sasaki,§ Akihiro Ambo,§ Ewa D. Marczak, Lawrence H. Lazarus, and
Severo Salvadori*,‡
†Department of Toxicology, University of Cagliari, I-09124, Cagliari, Italy, ‡Department of Pharmaceutical Sciences and Biotechnology Center,
University of Ferrara, I-44100 Ferrara, Italy, §Department of Pharmacology, Tohoku Pharmaceutical University, 4-1 Komatsushima 4-chome,
Aoba-Ku, Sendai 981-8558, Japan, and Medicinal Chemistry Group, Laboratory of Pharmacology, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina 27709
Received May 22, 2009
To improve the structure-activity studies of the lead δ opioid agonist H-Dmt-Tic-Asp*-Bid, we
synthesized and pharmacologically characterized a series of analogues in which the side chain next to
1H-benzimidazole-2-yl (Bid) was substituted by those endowed with different chemical properties.
Interesting results were obtained: (1) only Gly, Ala, and Asp resulted in δ agonism, (2) Phe yielded δ
antagonism, (3) and all other residues except Glu (devoid of any activity) gave μ agonism.
Introduction
benzimidazole heterocycles (Bid) by cyclization/dehydration
in acetic acid at 60 °C for 1 h. Each intermediate, after Boc
deprotection with TFA, was condensed with Boc-Tic-OH via
WSC/HOBt. Subsequently, Boc deprotection (TFA) and the
final condensation (WSC/HOBt) with Boc-Dmt-OH gave the
fully protected compounds. Final deprotections of side chains
(if present) and N-terminal amine functions (by TFA
treatment) gave the crude final compounds (9-18), which
were purified by preparative reverse-phase HPLC.
The dipeptide Dmt-Tic1 represents a versatile opioid phar-
macophore able to induce a wide range of activities by
interaction with μ and/or δ receptors.2-5 δ Opioid receptor
agonists are known to produce many pharmacological effects
in rodents, including analgesia,6 antidepressant,7 neuropro-
tection/neurogenesis,8 and anti-Parkinson9 activities. On the
basis of the potential utility of δ agonists in different pharma-
cological fields, here we report a structure-activity study
related to the side chain adjacent to Bid in our lead δ agonist
H-Dmt-Tic-Asp*-Bid. In preceding studies, we demonstrated
that side chain of Asp can be substituted by the methyl side
chain of Ala5 or removed (Gly)3 with the maintenance of δ
agonism. Interestingly, the introduction of a protected or
unprotected Lys side chain resulted in μ agonism without δ
agonism.4 To gain a better understanding of the importance
of the side chain in the definition of the opioid activity in
Dmt-Tic analogues, we synthesized a series of compounds in
which the side chain next to the C-terminal Bid is derived from
the principal natural amino acids. Further, on the basis of our
previous results, the asymmetric carbon carrying the side
chain seems to be unimportant for activity.5,10
Results and Discussion
Receptor Affinity Analysis. Receptor binding data for
δ- and μ-receptors and δ-selectivity (Kiμ/ Kiδ) are reported
in Table 1. All new compounds (9-18) had subnanomolar
affinities for δ-opioid receptors (Kiδ=0.035-0.457 nM),
which are in quite good accordance with the reference
compounds containing the Dmt-Tic pharmacophore (1-6).
As expected, the lack of a negative charge decreased δ-selectiv-
ity essentially by increasing μ-affinity (Kiμ = 0.077-20.2 nM).
In fact, compounds 3 and 18 containing Asp and Glu side
chains exhibited δ-selectivity 122 and 36, respectively. All other
analogues, except 10, containing Bid at the C-terminal were
essentially nonselective (Kiμ/ Kiδ or Kiδ/ Kiμ = 1.1-14). Com-
pound 10, in which Tyr substitutes for Dmt, exhibited a
behavior in line with results obtained by Schiller et al. concern-
ing the pharmacological characterization of H-Dmt-Tic-Phe-
Phe-NH2 and the corresponding Tyr analogue H-Tyr-Tic-Phe-
Phe-NH2; Tyr increases δ selectivity especially through the
lowering of μ affinity.12
Functional Bioactivity. Compounds 9-18 were tested in
the electrically stimulated MVD and GPI pharmacological
assays for intrinsic functional bioactivity (Table 1). As is
quite usually observed with opioids containing the Dmt-Tic
pharmacophore, a close correlation between binding and
functional bioactivity data is often lacking.10 A similar lack
of correlation was also observed by Hruby et al. in a series
of 4-anilidopiperidine analogues.13 On the other hand,
Chemistry
All pseudopeptides (9-18) were prepared stepwise in solu-
tion using conventional synthetic methods as outlined in
Scheme 1 (Supporting Information). Carboxylic functions
of Boc protected amino acids were transformed into Bid
according to the procedure of Nestor et al.11 Briefly, mixed
carbonic anhydride coupling of Boc protected amino acids
with o-phenylendiamine gave the crude intermediate amides,
which were converted without purification to the desired
*To whom correspondence should be addressed. For G. B.: phone,
(þ39)-70-675-8625; fax, (þ39)-70-675-8612; E-mail, gbalboni@unica.it;
bbg@unife.it. For S. S.: phone, (þ39)-532-455-918; fax, (þ39)-532-455-953;
E-mail: sal@unife.it.
r
pubs.acs.org/jmc
Published on Web 07/30/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5557
Table 1. Receptor Binding Affinities and Functional Bioactivities of Compounds 1-18
H-Dmt-Tic-Xaa*-Bid
receptor affinityb (nM)
Kiδ Kiμ
selectivity
functional bioactivity
MVD IC50c (nM)
d
Xaa*
Kiμ/ Kiδ
MVD pA2
GPI IC50c (nM)
Reference Compounds
1
2
3
4
5
6
7
8
Gly*e
Ala*f
Asp*f
Lys*g
0.035
0.063
0.443
0.49
0.50
14
6.5
0.13
0.26
0.12
26.92
71.7
1724
39.7
375
0.411
53.9
0.16
0.37
0.13
0.143
1.33
122
3.1L
na
na
Lys(Z)*g
0.64
0.18
1.7L
9049
Lys(Ac)*g
[Dmt1]DALDA h
E-2i
1.4L
53.9
1.41
6.9
2100
6085
14685L
4575L
23.1
344
Compound
9
Phe*
[Tyr1]Phe*
Leu*
0.049 ( 0.008 (3)
0.457 ( 0.074 (4)
0.082 ( 0.007 (4)
0.065 ( 0.005 (4)
0.138 ( 0.047 (3)
0.051 ( 0.009 (3)
0.135 ( 0.051 (3)
0.035 ( 0.004 (5)
0.179 ( 0.003 (3)
0.04 ( 0.006 (3)
0.25 ( 0.024 (5)
20.2 ( 1.7 (5)
5.1
44
8.79
8.60
na
273 ( 48
398 ( 19.3
23.4 ( 1.5
106 ( 36
10.2 ( 2.6
8.5 ( 0.13
35.4 ( 6.5
7.84 ( 0.32
34.9 ( 9.2
494 ( 19.3
10
11
12
13
14
15
16
17
18
0.12 ( 0.04 (4)
0.095 ( 0.008 (5)
0.13 ( 0.02 (5)
0.23 ( 0.02 (3)
0.30 ( 0.03 (3)
0.077 ( 0.007 (5)
0.13 ( 0.016 (5)
1.45 ( 0.39 (3)
1.5
1.5
1.1L
4.5
2.2
2.2
1.4L
36
Arg(NO2)*
Arg*
Asn*
na
na
na
na
Trp*
Ser(Ac)*
Ser*
Glu*
na
na
na
b The Ki values (nM) were determined according to Cheng and Prusoff.31 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. c Agonist activity was expressed as IC50
obtained from dose-response curves. These values represent the mean ( SE for at least four tissue samples. DPDPE and PL017 were the internal
standards for MVD (δ-opioid receptor bioactivity) and GPI (μ-opioid receptor bioactivity) tissue preparation, respectively. d The pA2 values of opioid
antagonists against the δ and μ agonists (deltorphin II and endomorphin-2, respectively) were determined by the method of Kosterlitz and Watt.32 e Data
taken from Lazarus et al.3 f Data taken from Lazarus et al.5 g Data taken from Balboni et al.4 h Data taken from Schiller et al.14 i Data taken from Zadina
et al.18 Lμ-receptor selectivity Kiδ/ Kiμ. na = no antagonism.
Schiller et al. observed the opposite behavior for the lead μ
agonist [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2) (7),
which had very high selectivity in the receptor affinity (Kiδ/
Kiμ = 14685) but only minor difference in functional bio-
activity (GPI IC50 =1.41 nM; MVD IC50 =23.1 nM).14,15
Starting from the first identified δ selective antagonist con-
taining the Tyr-Tic pharmacophore H-Tyr-Tic-Phe-Phe-OH
(TIPP),16 and our initial findings on the correlation between
the introduction of 1H-benzimidazol-2-yl (Bid) at the C-
terminal of peptides containing the Dmt-Tic pharmacophore
and the induction of δ agonism,3,5 we synthesized com-
pounds 9 and 10 containing Phe* next to Bid. As seen in
Table 1, these derivatives exhibited δ antagonist activity
(pA2=8.79 and 8.60, respectively) despite the introduction
of Bid at their C-terminal. Furthermore, the difference in
receptor affinity and selectivity derived from the substitution
of Tyr with Dmt, as noted above, is not confirmed by
functional bioactivity; in fact, whereas 9 and 10 had compar-
able pA2 values, H-Tyr-Tic-Phe-Phe-OH (pA2 =8.32) and
H-Tyr-Tic-Phe-Phe-NH2 (pA2=7.74) appear less active than
the corresponding analogue containing Dmt: H-Dmt-Tic-
Phe-Phe-OH (pA2 =9.70)17 and H-Dmt-Tic-Phe-Phe-NH2
(pA2=9.68).12 Compounds (1,3 2,5 4,4 6,4 11-17) character-
ized by different chemical functions in their side chains, are
all endowed with μ agonist activity with IC50 values ranging
from 7.84 to 106 nM, whereas reference compounds (1-3)
showed potent δ agonism and compounds (9, 10) were δ anta-
gonists, confirming the activity reported by Schiller et al.16 The
other reference substances (4-6) and new compounds (11-18)
completely lacked any activity toward δ receptors. Quite un-
expectedly, compound 18 containing the negatively charged
side chain of Glu in place of Asp (3, our lead δagonist),5 did not
exhibit δ agonism, but only a low degree of μ agonism. It seems
that bulky side chains are not allowed for δ agonism and they
are also detrimental for μ agonism (4 and 6 vs 5, 12 vs 13).
Finally, compound (14) H-Dmt-Tic-Asn*-Bid appears to be an
interesting μ agonist, which is endowed with activity compar-
able to the endogenous opioid endomorphin-2 (8)18 and only
slightly less active than the lead μ agonist [Dmt1]DALDA.14 Its
potential importance is related to the spontaneous deamidation
reaction of Asn residues that can occur under physiological
conditions;19 in this case, the μ agonist (14) has the potential to
be transformed into a potent and selective δ agonist H-Dmt-
Tic-Asp*-Bid (3). As suggested by Kieffer et al., this behavior
could be important in the regulation of δ-opioid receptor
trafficking via μ-opioid receptor stimulation, which may be
able to increase the potency of δ agonists with some potentially
important clinical implications.20,21
Conclusions
On the basis of the importance which δ opioid agonists
assume in different pharmacological fields,22 we extended the
correlation between the presence of the C-terminal 1H-benzi-
midazol-2yl chemical function and the induction of agonist
activity in pseudopeptides containing the Dmt-Tic pharma-
cophore of general formula H-Dmt-Tic-(L)or(D)Xaa-OH/-
NH2. Previously, we demonstrated that these tripeptides
are all endowed of δ antagonist activity (pA2=7.60-10.07),
except H-Dmt-Tic-Glu-NH2, which resulted a δ agonist
(IC50=2.5 nM).23 The majority of these peptides were
transformed at their C-terminal to the corresponding Bid
analogues to yield a wide range of activities: only Gly (1),
Ala (2), and Asp (3) gave δ agonism; Phe (9, 10) yielded δ

5558 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Balboni et al.
antagonism, Glu (18) and Lys(Z) (5) were inactive, and all
other aminoacids (11-17) exhibited μ agonist activity ,which
in some cases (13, 14, 16) was comparable to the endogenous
μ agonist endomorphin-2 (8). Surprisingly, all the new ana-
logues(11-18) weredevoidofany δactivity. Interestingly, the
μ agonist H-Dmt-Tic-Asn*-Bid (IC50=8.5 nM) (14) in which
Bid was formed from the R carboxylic function of the
C-terminal Asn could represent a new tool in the opioid field.
Asn and Gln side chains are known to undergo spontaneous
nonenzymatic deamidation to form Asp and Glu residues
under physiological conditions, with mechanisms including
direct hydrolysis and/or succinimide-mediated deamidation,
depending on pH of the environment.19 Whether specific
deamidases exist to further accelerate this process or not is
presently unknown.24 However, on the basis of this assump-
tion, the μ agonist H-Dmt-Tic-Asn*-Bid could potentially be
deamidated to form the potent and selective lead δ agonist
H-Dmt-Tic-Asp*-Bid with some important pharmacological
implications. For example, mixtures of selective μ and δ
agonists have been found to produce additive or synergistic
antinociceptive effects in both rodents and nonhuman pri-
mates without enhancing or attenuating most of each other’s
nonantinociceptive effects.25 As described by Schiller, bi- or
multifunctional drugs may have improved potency due to
synergistic effects or may produce fewer side effects than
compounds acting at a single target.15 Bifunctional μ ago-
nists/δ agonists and their usefulness were described by Anan-
than26 and more recently by Zhang et al.27 Considering that
opioid bivalent ligands reported herein were designed for the
simultaneous interaction with both receptors, H-Dmt-Tic-
Asn*-Bid and some other published opioids such as H-Dmt-
Tic-Asp-Bid(N1-Me),10 RWJ-394674,28 and N-desmethylclo-
zapine29 could represent a new class of “timed” bifunctional
ligands able to interact at different times with different
receptors. These may be suitable for example in trafficking
of opioid receptors.30
3TFA H-Dmt-Tic-Arg*-Bid (13). Boc-Dmt-Tic-Arg*-Bid
3
was treated with TFA as reported for 2TFA H-Dmt-Tic-
Phe*-Bid: yield 0.07 g (97%); Rf(A) 0.32; HPLC K0 4.43; mp
3
169-171 °C; [R]20 þ15.7; m/z 598 (M þ H)þ. 1H NMR
D
(DMSO-d6) δ 1.55-2.65 (m, 12H), 2.92-3.17 (m, 4H), 3.95-
4.51 (m, 3H), 4.87-4.92 (m, 2H), 6.29 (s, 2H), 6.96-7.70 (m,
8H). Anal. C39H43F9N8O9: C; H; N.
2TFA H-Dmt-Tic-Asn*-Bid (14). Boc-Dmt-Tic-Asn*-Bid
3
was treated with TFA as reported for 2TFA H-Dmt-Tic-
Phe*-Bid: yield 0.1 g (97%); Rf(A) 0.35; HPLC K0 4.3; mp
3
155-157 °C; [R]20 þ19.2; m/z 556 (M þ H)þ. 1H NMR
D
(DMSO-d6) δ 2.35 (s, 6H), 2.61-3.17 (m, 6H), 3.95-4.51 (m,
3H), 4.92-5.10 (m, 2H), 6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal.
C35H36F6N6O8: C; H; N.
2TFA H-Dmt-Tic-Trp*-Bid (15). Boc-Dmt-Tic-Trp*-Bid
3
was treated with TFA as reported for 2TFA H-Dmt-Tic-
3
Phe*-Bid: yield 0.06 g (94%); Rf(A) 0.42; HPLC K0 5.06; mp
178-180 °C; [R]20 þ30.5; m/z 627 (M þ H)þ. 1H NMR
D
(DMSO-d6) δ 2.35 (s, 6H), 2.86-3.17 (m, 6H), 3.95-4.51 (m,
3H), 4.92-5.26 (m, 2H), 6.29 (s, 2H), 6.80-7.70 (m, 13H). Anal.
C42H40F6N6O7: C; H; N.
2TFA H-Dmt-Tic-Ser(Ac)*-Bid (16). Boc-Dmt-Tic-Ser-
3
(Ac)*-Bid was treated with TFA as reported for 2TFA H-
3
Dmt-Tic-Phe*-Bid: yield 0.07 g (96%); Rf(A) 0.37; HPLC
K0 4.25; mp 143-145 °C; [R]20D þ20.7; m/z 570 (M þ H)þ. 1H
NMR (DMSO-d6) δ 2.01 (s, 3H), 2.35 (s, 6H), 2.92-3.17 (m,
4H), 3.93-3.97 (m, 1H), 4.41-4.76 (m, 4H), 4.92-5.47 (m, 2H),
6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal. C36H37F6N5O9: C; H; N.
2TFA H-Dmt-Tic-Ser*-Bid (17). Boc-Dmt-Tic-Ser*-Bid was
3
treated with TFA as reported for 2TFA H-Dmt-Tic-Phe*-Bid:
3
yield 0.06 g (95%); Rf(A) 0.31; HPLC K0 3.92; mp 150-152 °C;
[R]20D þ22.6; m/z 529 (M þ H)þ. 1H NMR (DMSO-d6) δ 2.35
(s, 6H), 2.92-3.17 (m, 4H), 3.95-4.20 (m, 3H), 4.41-4.51
(m, 2H), 4.92-4.96 (m, 2H), 6.29 (s, 2H), 6.96-7.70 (m, 8H).
Anal. C34H35F6N5O8: C; H; N.
2TFA H-Dmt-Tic-Glu*-Bid (18). Boc-Dmt-Tic-Glu*-Bid
was treated with TFA as reported for 2TFA H-Dmt-Tic-
3
3
Phe*-Bid: yield 0.16 g (97%); Rf(A) 0.69; HPLC K0 5.11;
mp 173-175 °C; [R]20 þ21.5; m/z 571 (M þ H)þ. H NMR
1
D
(DMSO-d6) δ 2.23-2.25 (m, 4H), 2.35 (s, 6H), 2.92-3.17 (m,
4H), 3.93-3.97 (m, 1H), 4.41-4.51 (m, 2H), 4.87-4.92 (m, 2H),
6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal. C36H37F6N5O9: C; H; N.
Experimental Section
Chemistry. 2TFA H-Dmt-Tic-Phe*-Bid (9). Boc-Dmt-Tic-
3
Phe*-Bid (0.14 g, 0.2 mmol) was treated with TFA (1 mL) for
0.5 h at room temperature. Et2O/Pe (1:1, v/v) were added to the
solution until the product precipitated: yield 0.16 g (96%); Rf (A)
0.43; HPLC K0 5.1; mp 149-151 °C; [R]20D þ29.7; m/z 589 (Mþ
H)þ. 1H NMR (DMSO-d6) δ 2.35 (s, 6H), 2.92-3.17 (m, 6H),
3.95-4.51 (m, 3H), 4.92-5.26 (m, 2H), 6.29 (s, 2H), 6.96-7.70
(m, 13H). Anal. C40H39F6N5O7: C; H; N.
Acknowledgment. This study was supported in part by the
University of Cagliari (GB), the University of Ferrara (SS),
and in part by the Intramural Research Program of the NIH
and NIEHS (L.H.L. and E.D.M.).
Supporting Information Available: Abbreviations, chemistry
general methods, synthesis of intermediates, and pharmacology.
This material is available free of charge via the Internet at http://
pubs.acs.org.
2TFA H-Tyr-Tic-Phe*-Bid (10). Boc-Tyr-Tic-Phe*-Bid was
3
treated with TFA as reported for 2TFA H-Dmt-Tic-Phe*-Bid:
03
yield 0.1 g (95%); Rf (A) 0.40; HPLC K 4.82; mp 145-147 °C;
[R]20D þ13.8; m/z 561 (M þ H)þ. 1H NMR (DMSO-d6) δ 2.92-
3.22 (m, 6H), 3.95-4.51 (m, 3H), 4.90-5.23 (m, 2H), 6.68-7.70
(m, 17H). Anal. C38H35F6N5O7: C; H; N.
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