Biological active analogues of the opioid peptide biphalin: Mixed α/β3-peptides

Article abstract of DOI:10.1021/jm301456c

Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-β³ amino acids. The derivative 1 containing hβ³ Phe in place of Phe showed good μ- and δ-receptor affinities (K_i^δ =

Full text of DOI:10.1021/jm301456c

Brief Article
pubs.acs.org/jmc
Biological Active Analogues of the Opioid Peptide Biphalin: Mixed
α/β3‑Peptides
Adriano Mollica,*,† Francesco Pinnen,† Roberto Costante,† Marcello Locatelli,† Azzurra Stefanucci,†
Stefano Pieretti,‡ Peg Davis,§ Josephine Lai,§ David Rankin,§ Frank Porreca,§ and Victor J. Hruby∥
†Dipartimento di Farmacia, Universita
‡Istituto Superiore di Sanita,
Dipartimento del Farmaco, Viale Regina Elena 299, 00161, Rome, Italy
̀
di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy
̀
§Department of Pharmacology, ∥Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United
States
S
* Supporting Information
ABSTRACT: Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-β3 amino acids.
The derivative 1 containing hβ3 Phe in place of Phe showed good μ- and δ-receptor affinities (Kiδ = 0.72 nM; Kiμ = 1.1 nM) and
antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.
INTRODUCTION
■
Biphalin is a unique dimeric opioid peptide composed of two
enkephalin-based tetrapeptides (Tyr-D-Ala-Gly-Phe) linked by
a hydrazide bridge between the two C-terminal phenylalanine
moieties. Biphalin displays a strong affinity for both μ and δ
receptors, with an EC50 in the nanomolar range. When
administered intracerebroventricularly (icv) it shows higher
potency than morphine and etorphine in animal models of
pain.1 The remarkable activity is due primarily to its dimeric
structure which improves the ability to meet the topographical
requirements of involved receptors.2 Furthermore, this opioid
octapeptide produced less physical dependence than other
Figure 1. Structure of biphalin and analogues 1−4.
opioid agonists.1a,3
A major problem concerning the use of peptides, including
vivo thermal models of nociception by icv and iv admin-
istrations in mice, with human plasma stability also evaluated.
the opioid peptides, as drugs, is their susceptibility to enzymatic
hydrolysis when administered in vivo.4 Approaches that have
been explored in an effort to overcome this problem in opioid
peptides include the use of D-amino acids, β-amino acids,
various types of synthetic residues, and backbone cyclization.5
A well-known and useful approach is the design and synthesis
of α/β hybrid peptides, where one or more native residues of
the backbone sequence is replaced by the corresponding β2- or
β3-amino acid analogues.6 Hybrid α/β peptides fold the
backbone in a manner more similar to the natural α-peptides
than full β-peptides and together with high their intrinsic
metabolic stability make them good candidates for drug design.
β3-amino acids have been used in the design of several classes of
ligands, including platelet aggregation factors, angiotensin II,
chemotactic agents, and neuropeptides, including opioid
peptides.7−10
This work reports the synthesis and in vitro biological
evaluation of four biphalin analogues (Figure 1) containing β3
homoamino acids (hβ3Xaa). hβ3Tyr and hβ3Phe were
incorporated in place of the tyrosine1−1ˈand phenylalanine4−4ˈ
residues, respectively, and D-alanine2−2ˈand glycine3−3ˈ were
replaced with a β-alanine residue. The most active product of
this series, which contains hβ3 Phe (1), was further tested in in
BIOLOGICAL EVALUATION
■
Binding Affinities at μ/δ Opioid Receptors.11 To
determine the affinity to the μ-opioid (MOR) and the δ-opioid
receptors (DOR) of compounds 1−4, tritiated opioid peptides
DAMGO ([3H]-[D-Ala(2), N-Me-Phe-(4), Gly-ol(5)] enke-
phalin) and Deltorphin (selective agonists for MOR and DOR,
respectively) were used. Binding IC50 and Ki values are shown
in Table 1.
Biphalin was used as reference.1b Analogue 1 has a very good
opioid receptor affinity, showing subnanomolar affinity at the
DOR and a potent Ki value at the MOR (0.72 and 1.1 nM,
respectively). Analogues 2−4 display poor affinity for opioid
receptors, with a mild μ-selectivity for peptides 3 and 4 (Kiμ/Kiδ
= 0.13). Peptide 2 showed little MOR/DOR discrimination,
with Ki values higher than 100 nM at both receptors.
GPI and MVD in Vitro Bioassay.12 The biological activity
was also investigated through isolated tissue-based functional
Received: October 16, 2012
Published: April 2, 2013
© 2013 American Chemical Society
3419
dx.doi.org/10.1021/jm301456c | J. Med. Chem. 2013, 56, 3419−3423

Journal of Medicinal Chemistry
Brief Article
Table 1. Binding Affinities and in Vitro Activities for Peptide Derivatives at δ/μ Opioid Receptors
a
b
a
c
hDOR , [3H]Deltorphin
rMOR , [3H]DAMGO
MVD (δ)
GPI (μ)
d
d
e
,
f
e,f
compd
log IC50
Ki (nM)
log IC50
Ki (nM)
Kiμ/Kiδ
IC50 (nM)
33 9.4
IC50 (nM)
IC50 (GPI)/IC50 (MVD)
1
2
3
4
−8.84 0.11
−4.97 0.16
−8.75 0.11
−8.27 0.14
0.72
240
450
480
2.6
−8.64 0.07
−6.42 0.08
−6.90 0.08
−6.87 0.11
1.1
172
57
1.53
0.72
0.13
0.13
0.54
50 6.5
1.5
>0.75
0.9
1300 221
1800 266
710 97
9.2% at 1 μM
1700 306
670 139
8.8 0.3
62
0.9
g
Bph
1.4
2.7 1.5
3.2
a
b
c
Displacement of [3H]Deltorphin (μ-selective) and [3H]DAMGO (δ-selective) from rat brain membrane binding site. Kd = 0.50 0.3 nM. Kd =
d
0.50 0.1 nM. The log IC50 standard error are expressed as logarithmic values determined from the nonlinear regression analysis of data
collected from at least two independent experiments performed in duplicate. The Ki values are calculated using the Cheng and Prusoff equation to
e
correct for the concentration of the radioligand used in the assay. Concentration at 50% inhibition of muscle contraction in electrically stimulated
f
g
isolated tissues (n = 4). SEM. Biphalin (Bhp), refs 1b and 13.
Figure 2. Antinociceptive results of hot plate and tail-flick in vivo bioassays for compound 1, Biphalin, and morphine sulfate. Compounds were
injected by icv administration (A,B) at a dose of 0.6 nmol, and systemic iv administration (C,D) at a dose of 3000 nmol. The data represent the
mean SEM. The statistical significance among groups was determined, in comparison with vehicle-treated animals with the analysis of variance
(two-way ANOVA test) followed by Bonferroni’s posthoc comparisons using the statistical software SPSS. Statistical significance was P < 0.05 (*P <
0.05; ***P < 0.001).
assay using guinea pig ileum/longitudinal muscle myenteric
plexus (GPI) and mouse vas deferens (MVD) tissues (Table
1). Biphalin was used as a reference.13 Compound 1 showed
the greatest potency, with no relevant selectivity for μ- and δ-
receptor (with Ki values of 33 and 50 nM, respectively, for
MVD and GPI). Analogue 4 showed near identical IC50 around
700 nM in both MVD and GPI assays, while 2 and 3 had weak
activity. These data are in agreement with results of binding
assay.
In Vitro Metabolic Stability.15 The enzymatic stability of
both Biphalin and the Biphalin analogue 1 was tested by
incubation in human plasma at 37 °C. Degradation curves
(Figure 3) were obtained by plotting the total amount of
remaining parent compound (expressed as μg/mL) versus time
(expressed as minutes), demonstrating increased stability for
the modified compound due to lack of recognition by
degrading enzymes present in human plasma.
DISCUSSION AND CONCLUSION
■
In Vivo “Hot Plate” and “Tail-Flick” Bioassays.14
Antinociceptive properties of compound 1 were further
investigated using the mouse hot plate and tail-flick assays,
following both local (icv, 0.6 nmol) and systemic (iv, 3000
nmol) injections. Morphine and Biphalin were used as
reference compounds (Figure 2).
In this work, four new biphalin analogues were synthesized and
investigated. The novel compounds were evaluated for their μ/
δ receptor activity, and the results are shown in Table 1.
Compound 1, containing hβ3Phe residues in position 4 and 4′,
showed remarkable binding affinity, with Ki values of 0.72 and
1.1 nM, respectively, for DOR and MOR, resulting in a
3420
dx.doi.org/10.1021/jm301456c | J. Med. Chem. 2013, 56, 3419−3423

Journal of Medicinal Chemistry
Brief Article
icv administration, the antinociceptive profile of the analogue 1
was very similar to Biphalin, producing 100% of the MPE 30
and 45 min after administration. In both in vivo models, the
maximum effect is reached 15−30 min after drug injection, and
no significant decrease is observed for the next 30 min.
Following iv administration (hot plate and tail-flick tests),
compound 1 displayed a higher (ranging from 40 to 140% 15−
60 min after administration) and more long lasting
antinociceptive effect than Biphalin, thus confirming the
improved plasma stability, in full accord with in vitro stability
data, reported in Figure 3 (for detailed experimental data see
Supporting Information).
The improved metabolic stability paired with good
antinociceptive activity confirms that Phe moiety modifica-
tion16c,d is a promising strategy in the field of Biphalin
analogues development.
Figure 3. Stability curves for Biphalin (green line) and the Biphalin
derivative 1 (purple line). The samples were tested in three
independent experiments (n = 3) and represent the mean
SEM.
The significance among groups was evaluated with the analysis of
variance (two-way ANOVA test) followed by Bonferroni’s posthoc
comparisons between compound 1 and Biphalin using the statistical
software GraphPad Prism v.4. Statistical significance was P < 0.05 (**P
< 0.01; ***P < 0.001); times from t0 to t45 present no statistical
significance (P > 0.05).
EXPERIMENTAL SECTION
■
Chemistry. Synthesis of all new analogues was performed in
solution phase using the Nα-Boc strategy. All synthesis began with
hydrazine, with repeated steps of coupling/purification/deprotection
of the intermediate products, until the final products were obtained as
TFA salts (Scheme 1).
a
Scheme 1. Synthesis of Biphalin Analogues 1−4
receptor affinity comparable with that of Biphalin. In the GPI
and MVD in vitro bioassays (Table 1), compound 1 was still
the most potent of the series, but all the peptides showed lower
potency than Biphalin, suggesting that the activities of the
compounds do not totally reflect the ability to induce a
biological response.
The high affinity and the strong in vitro activity of 1,
confirmed by in vivo nociception tests, may be explained by the
fact that the distance between the two aromatic rings of Tyr
and Phe is the same as the parent peptide, therefore the
additional methylene group increases the flexibility of the
compound without compromising side chain arrangements.
The activity found for compounds 1−4 is compatible with
previous biological studies on Biphalin analogues with non-
hydrazine linkers. Those studies state that an increased distance
between the two pharmacophores of Biphalin, obtained by
using short diamine bridges (containing one or two methylene
groups) or cyclic linkers (e.g., piperazine) is well tolerated or
can even improve the in vitro affinity, and our data here
confirm those conlcusions.16 On the other hand, the distance
between Phe4,4ˈ and Tyr1,1ˈ side chains, as well as the reciprocal
pharmacophores arrangement of the dimeric structure, are
crucial for the activity. Thus, the use of β-residues in position
1,1′, 2,2′, and 3,3′ of the backbone led to an evident loss of
affinity and activity (see products 2−4), whereas the
introduction of the additional methylene group in position
4,4′ is well tolerated (see product 1).16b These data support the
importance of D-Ala and Gly as keystructural residues, in
addition to the well-known role of tyrosine. The lack of activity
and affinity of compounds 2−4 is probably due to the β-
residues that affect the spacing between the pharmacophoric
Tyr and Phe residues. Interestingly, compounds 3 and 4
showed a significant selectivity for MOR (about 8-fold),
suggesting a higher sensitivity of the DOR for modifications
induced by β-residues. The antinociceptive in vivo profile of
compound 1 clearly indicates that 1 is endowed with good
activity, several times higher than morphine tested under the
same conditions (for icv), but slightly lower than Biphalin, as
expected from the MVD/GPI tests. In the hot plate test, after
a
(1) Xaa4 = hβ3Phe; Xaa3 = Gly; Xaa2 = D-Ala; Xaa1 = Tyr; (2) Xaa4
= Phe; Xaa3 = βAla; Xaa2 = D-Ala; Xaa1 = Tyr; (3) Xaa4 = Phe; Xaa3
= Gly; Xaa2 = βAla; Xaa1 = Tyr; (4) Xaa4 = Phe; Xaa3 = Gly; Xaa2 =
D-Ala; Xaa1 = hβ3Tyr.
All coupling reactions were performed with the standard method of
HOBt/EDC/NMM in DMF.16b Deprotection of Nα-tert-butyloxycar-
bonyl group was performed using TFA/CH2Cl2 1:1 for 1 h, under
nitrogen atmosphere. The intermediate TFA salts were used for
subsequent reactions without further purification. Boc protected
intermediate products were purified by silica gel column chromatog-
raphy, or in the case of scarcely soluble products, the purification was
performed by trituration in EtOAc.16c Final products 1−4 were
purified by RP-HPLC using a Waters XBridge Prep BEH130 C18, 5.0
μm, 250 mm × 10 mm column at a flow rate of 4 mL/min on a Waters
Binary pump 1525, using as eluent a linear gradient of H2O/
acetonitrile 0.1% TFA ranging from 5% acetonitrile to 90% acetonitrile
in 45 min. The purity of the Nα-Boc-protected products was confirmed
by NMR analysis on a Varian VXR 300 MHz and mass spectrometry
ESI-HRMS. The purity of all final TFA salts was confirmed by NMR
analysis, ESI-HRMS, and by analytical RP-HPLC (C18-bonded 4.6 mm
× 150 mm) at a flow rate of 1 mL/min, using as eluent a gradient of
H2O/acetonitrile 0.1% TFA ranging from 5% acetonitrile to 95%
acetonitrile in 50 min, and was found to be ≥95%. All synthetic
procedure and intermediates’ characterizations are reported in the
Supporting Information.
2 TFA·(Tyr-D-Ala-Gly-hβ3Phe-NH)2 (1). Rf = 0.61 (n-Bu-OH/
1
AcOH/H2O 4:1:1). Rt (HPLC) = 20.24 min. HNMR (DMSO-d6)
3421
dx.doi.org/10.1021/jm301456c | J. Med. Chem. 2013, 56, 3419−3423

Journal of Medicinal Chemistry
Brief Article
δ: 1.05 (6H, d, D-Ala CH3), 2.53 (4H, t, hβ3Phe hβCH2), 2.71−2.85
(4H, m, Tyr βCH2), 2.73−2.80 (4H, m, hβ3Phe βCH2), 3.53−3.61
(4H, m, Gly αCH2), 3.85 (2H, t, Tyr αCH), 4.15 (2H, m, hβ3Phe
αCH), 4.34 (2H, t, D-Ala αCH), 6.65−7.01 (8H, m, Tyr Ar), 7.12−
7.27 (10H, m, hβ3Phe Ar), 7.88 (2H, d, hβ3Phe NH), 8.05 (6H, d, Tyr
REFERENCES
■
(1) (a) Horan, P. J.; Mattia, A.; Bilsky, E. J.; Weber, S.; Davis, T. P.;
Yamamura, H. I.; Malatynska, E.; Appleyard, S. M.; Slaninova, J.;
Misicka, A. Antinociceptive profile of Biphalin, a dimeric enkephalin
analog. J. Pharmacol. Exp Ther. 1993, 265, 1446−1454. (b) Lipkowski,
A. W.; Misicka, A.; Davis, P.; Stropova, D.; Janders, J.; Lachwa, M.;
Porreca, F.; Yamamura, H. I.; Hruby, V. Biological activity of fragments
and analogues of the potent dimeric opioid peptide, Biphalin. Bioorg.
Med. Chem. Lett. 1999, 9, 2763−2766. (c) Silbert, B. S.; Lipkowski, A.
W.; Cepeda, M. S.; Szyfelbein, S. K.; Osgood, P. F.; Carr, D. B.
Analgesic activity of a novel bivalent opioid peptide compared to
morphine via different routes of administration. Agents Actions 1991, 3,
382−387.
(2) (a) Flippen-Anderson, J. L.; Deschamps, J. R.; George, C.; Hruby,
V. J.; Misicka, A.; Lipkowski, A. W. Crystal structure of Biphalin
sulfate: a multireceptor opioid peptide. J. Pept. Res. 2002, 59, 123−133.
(b) Abbruscato, T. J.; Thomas, S. A.; Hruby, V. J.; Davis, T. P. Brain
and spinal cord distribution of Biphalin: correlation with opioid
receptor density and mechanism of CNS entry. J. Neurochem. 1997, 69,
1236−1245.
(3) Yamazaki, M.; Suzuki, T.; Narita, M.; Lipkowski, A. W. The
opioid peptide analogue Biphalin induces less physical dependence
than morphine. Life Sci. 2001, 69, 1023−1028.
(4) (a) Koda, Y.; Del Borgo, M.; Wessling, S. T.; Lazarus, L. H.;
Okada, Y.; Toth, I.; Blanchfieldm, J. T. Synthesis and in vitro
evaluation of a library of modified endomorphin 1 peptides. Bioorg.
́ ́
Med. Chem. 2008, 16, 6286−6296. (b) Tomboly, C.; Peter, A.; Toth,
G. In vitro quantitative study of the degradation of endomorphins.
Peptides 2002, 23, 1573−1580.
(5) (a) Mollica, A.; Davis, P.; Ma, S. W.; Porreca, F.; Lai, J.; Hruby, V.
J. Synthesis and biological activity of the first cyclic Biphalin analogues.
Bioorg. Med. Chem. Lett. 2006, 16, 367−372. (b) Mollica, A.;
Guardiani, G.; Davis, P.; Ma, S. W.; Porreca, F.; Lai, J.; Mannina, L.;
Sobolev, A. P.; Hruby, V. J. Synthesis of stable and potent δ/μ opioid
peptides: analogues of H-Tyr-c[D-Cys-Gly-Phe-D-Cys]-OH by ring-
closing metathesis. J. Med. Chem. 2007, 50, 3138−3142. (c) Zieleniak,
A.; Rodziewicz-Motowidło, S.; Rusak, L.; Chung, N. N.; Czaplewski,
C.; Witkowska, E.; Schiller, P. W.; Ciarkowski, J.; Izdebski, J.
Deltorphin analogs restricted via a urea bridge: structure and opioid
activity. J. Pept. Sci. 2008, 14, 830−837. (d) Weltrowska, G.;
Berezowska, I.; Lemieux, C.; Chung, N. N.; Wilkes, B. C.; Schiller,
P. W. N-Methylated cyclic enkephalin analogues retain high opioid
receptor binding affinity. Chem. Biol. Drug. Des. 2010, 75, 82−88.
(e) Hruby, V. J. Designing peptide receptor agonists and antagonists.
Nature Rev. Drug Discovery 2002, 1, 847−858.
+
NH3 ), 8.17 (2H, t, Gly NH), 8.52 (2H, d, D-Ala NH), 9.44 (2H, s,
OH), 10.03 (2H, s, NH−NH). ESI-HRMS calcd for C52H62F6N10O14
m/z: 1165.4429 [M + H]+; found 1165.4431.
2 TFA·(Tyr-D-Ala-βAla-Phe-NH)2 (2). Rf = 0.41 (n-Bu-OH/AcOH/
1
H2O 4:1:1). Rt (HPLC) = 19.26 min. HNMR (DMSO-d6) δ: 1.21
(6H, d, D-Ala CH3), 2.16−2.27 (4H, m, βAla CH2−CO), 2.70−2.80
(4H, m, Tyr βCH2), 2.77−2.86 (4H, m, Phe βCH2), 3.54−3.71 (4H,
m, βAla CH2−N), 3.85 (2H, t, Tyr αCH), 4.38−4.49 (2H, m, D-Ala
αCH), 4.52 (2H, t, Phe αCH), 6.69−6.96 (8H, m, Tyr Ar), 7.09−7.25
+
(10H, m, Phe Ar), 7.76 (2H, d, βAla NH), 8.04 (6H, d, Tyr NH3 ),
8.20 (2H, d, Phe NH), 8.36 (2H, d, D-Ala NH), 9.36 (2H, s, OH),
10.21 (2H, s, NH−NH). ESI-HRMS calcd for C52H62F6N10O14 m/z:
1165.4429 [M + H]+; found 1165.4431.
2 TFA·(Tyr-βAla-Gly-Phe-NH)2 (3). Rf = 0.39 (n-Bu-OH/AcOH/
1
H2O 4:1:1). Rt (HPLC) = 19.55 min. HNMR (DMSO-d6) δ: 2.27−
2.32 (4H, m, βAla CH2−CO), 2.67−2.81 (4H, m, Tyr βCH2), 2.87−
2.92 (4H, m, βAla CH2−N), 2.91−2.99 (4H, m, Phe βCH2), 3.28−
3.39 (4H, m, Gly αCH2), 3.77 (2H, t, Tyr αCH), 4.24 (2H, t, Phe
αCH), 6.65−6.95 (8H, m, Tyr Ar), 7.11−7.28 (10H, m, Phe Ar), 7.71
+
(2H, d, βAla NH), 8.07 (6H, d, Tyr NH3 ), 8.16 (2H, t, Gly NH),
8.22 (2H, d, Phe NH), 9.25 (2H, s, OH), 10.19 (2H, s, NH−NH).
ESI-HRMS calcd for C50H58F6N10O14 m/z: 1137.4116 [M + H]+;
found 1137.4120.
̈
̈
2 TFA·(hβ3Tyr-D-Ala-Gly-Phe-NH)2 (4). Rf = 0.53 (n-Bu-OH/
1
AcOH/H2O 4:1:1). Rt (HPLC) = 29.48 min. HNMR (DMSO-d6)
δ: 1.12 (6H, d, D-Ala CH3), 2.61 (4H, t, hβ3Tyr βCH2), 2.78 (4H, t,
hβ3Tyr hβCH2), 2.96−3.01 (4H, m, Phe βCH2), 3.37−3.49 (4H, m,
Gly αCH2), 3.66−3.74 (2H, m, hβ3Tyr αCH), 4.19−4.23 (2H, m, D-
Ala αCH), 4.60 (2H, m, Phe αCH), 6.60−6.93 (8H, m, hβ3Tyr Ar),
+
7.07−7.20 (10H, m, Phe Ar), 7.81 (6H, d, hβ3Tyr NH3 ), 8.07 (2H, t,
Gly NH), 8.21 (2H, d, Phe NH), 8.33 (2H, d, D-Ala NH), 9.38 (2H, s,
OH), 10.15 (2H, s, NH−NH). ESI-HRMS calcd for C52H62F6N10O14
m/z: 1165.4429 [M + H]+; found 1165.4426.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures, characterization of intermediates, bio-
logical assays, and stability assay. This material is available free
of charge via the Internet at http://pubs.acs.org
(6) Seebach, D.; Beck, A. K.; Bierbaum, D. J. The world of β- and γ-
peptides comprised of homologated proteinogenic amino acids and
other components. Chem. Biodiversity 2004, 1, 1111−1239.
AUTHOR INFORMATION
■
Corresponding Author
*Phone/Fax: +39-0871-3554477. E-mail: a.mollica@unich.it.
Notes
(7) (a) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar,
M. I. Design and Synthesis of Inhibitors Incorporating β-Amino Acids
of Metalloendopeptidase EC 3.4.24.15. J. Pept. Sci. 2000, 6, 470−477.
(b) Seebach, D.; Abele, S.; Gademann, K.; Guichard, G.; Hintermann,
T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V.; Oberer, L.; Hommel, U.;
Widmer, H. β2- and β3-Peptides with Proteinaceous Side Chains:
Synthesis and Solution Structures of Constitutional Isomers, a Novel
Helical Secondary Structure and the Influence of Solvation and
Hydrophobic Interactions on Folding. Helv. Chim. Acta 2003, 86,
2098−2103.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by grants from the U.S. Public
Health Service, Natural Institutes of Health, DA00624 and
DA13449.
(8) (a) Seebach, D.; Matthews, J. L. β-Peptides: a surprise at every
turn. Chem. Commun. 1997, 21, 2015−2126. (b) Seebach, D.; Abele,
S.; Gademann, K.; Guichard, G.; Hintermann, T.; Jaun, B.; Matthews,
J. L.; Schreiber, J. V.; Oberer, L.; Hommel, U.; Widmer, H. β2- And β3-
Peptides with Proteinaceous Side Chains: Synthesis and Solution
Structures of Constitutional Isomers, a Novel Helical Secondary
Structure and the Influence of Solvation and Hydrophobic Interactions
on Folding. Helv. Chim. Acta 1998, 81, 932−982.
ABBREVIATIONS USED
■
DOR, δ-opioid receptor; EDC, 1-ethyl-(3-dimethylaminoprop-
yl)-carbodiimide; ESI-HRMS, electrospray ionization−high
resolution mass spectrometry; GPI, guinea pig ileum; [3H]-
DAMGO, [3H]-[D-Ala(2), N-Me-Phe-(4), Gly-ol(5)] enkepha-
lin; HOBt, 1-hydroxybenzotriazole; icv, intracerebroventricular;
iv, intravenous; MOR, μ-opioid receptor; MVD, mouse vas
deferens; NMM, N-methylmorpholine; RP-HPLC, reversed
phase high performance liquid chromatography
(9) (a) Stachowiak, K.; Khosla, M. C.; Plucinska, K.; Khairallah, P. A.;
Bumpus, F. M. Synthesis of angiotensin II analogues by incorporating
β-homotyrosine or β-homoisoleucine residues. J. Med. Chem. 1979, 9,
3422
dx.doi.org/10.1021/jm301456c | J. Med. Chem. 2013, 56, 3419−3423

Journal of Medicinal Chemistry
Brief Article
1128−1130. (b) Zablocki, J. A.; Tjoeng, F. S.; Bovy, P. R.; Miyano, M.;
Garland, R. B.; Williams, K.; Schretzman, L.; Zupec, M. E.; Rico, J. G.;
Lindmark, R. J.; Toth, M. V.; McMakins, D. E.; Adams, S. P.; Panzer-
Knodle, S. G.; Nicholson, N. S.; Taite, B. B.; Salyers, A. K.; King, L.
W.; Campion, J. G.; Feigen, L. P. A novel series of orally active
antiplatelet agents. Bioorg. Med. Chem. 1995, 5, 539−551.
(c) Giordano, C.; Lucente, G.; Mollica, A.; Nalli, M.; Pagani
Zecchini, G.; Paglialunga Paradisi, M.; Gavuzzo, E.; Mazza, F.;
Spisani, S. Hybrid α/β3-peptides with proteinogenic side chains.
Monosubstituted analogues of the chemotactic tripeptide For-Met-
Leu-Phe-OMe. J. Pept. Sci. 2004, 10, 510−523.
́
(10) (a) Wilczynska, D.; Kosson, P.; Kwasiborska, M.; Ejchart, A.;
Olma, A. Synthesis and receptor binding of opioid peptide analogues
containing β3-homo-amino acids. J. Pept. Sci. 2009, 15, 777−782.
́ ́
(b) Bozu, B.; Fulop, F.; Toth, G. K.; Toth, G.; Szucs, M. Synthesis and
̈
̈
̈
̈
opioid binding activity of dermorphin analogues containing cyclic β-
amino acids. Neuropeptides 1997, 31, 367−372. (c) Cardillo, G.;
Gentilucci, L.; Qasem, A. R.; Sgarzi, F.; Spampinato, S. Endomorphin-
1 analogues containing β-proline are μ-opioid receptor agonists and
display enhanced enzymatic hydrolysis resistance. J. Med. Chem. 2002,
45, 2571−2578.
(11) Misicka, A.; Lipkowski, A. W.; Horvath, R.; Davis, P.; Kramer, T.
H.; Yamamura, H. I.; Hruby, V. J. Topographical requirements for
delta opioid ligands: common structural features of dermenkephalin
and deltorphin. Life Sci. 1992, 51, 1025−1032.
(12) Szekeres, P. G.; Traynor, J. R. Delta opioid modulation of the
binding of guanosine-5′-O-(3-[35S]thio)triphosphate to NG108-15 cell
membranes: characterization of agonist and inverse agonist effects. J.
Pharmacol. Exp. Ther. 1997, 283, 1276−1284.
(13) Yamamoto, T.; Nair, P.; Largent-Milnes, T. M.; Jacobsen, N. E.;
Davis, P.; Ma, S. W.; Yamamura, H. I.; Vanderah, T. W; Porreca, F.;
Lai, J.; Hruby, V. J. Discovery of a potent and efficacious peptide
derivative for δ/μ opioid agonist/neurokinin 1 antagonist activity with
a 2′,6′-dimethyl-L-tyrosine: in vitro, in vivo, and NMR-based structural
studies. J. Med. Chem. 2011, 54, 2029−2038.
(14) Pieretti, S.; Di Giannuario, A.; De Felice, M.; Perretti, M.;
Cirino, G. Stimulus-dependent specificity for annexin 1 inhibition of
the inflammatory nociceptive response: the involvement of the
receptor for formylated peptides. Pain 2004, 109, 52−63.
(15) Al-Fayoumi, S. I.; Brugos, B.; Arya, V.; Mulder, E.; Eppler, B.;
Mauderli, A. P.; Hochhaus, G. Identification of stabilized dynorphin
derivatives for suppressing tolerance in morphine-dependent rats.
Pharm. Res. 2004, 21, 1450−1456.
(16) (a) Shimohigashi, Y.; Costa, T.; Chen, H. C.; Rodbard, D.
Dimeric tetrapeptide enkephalins display extraordinary selectivity for
the δ opiate receptor. Nature 1982, 297, 333−335. (b) Mollica, A.;
Davis, P.; Ma, S. W.; Lai, J.; Porreca, F.; Hruby, V. J. Synthesis and
biological evaluation of new Biphalin analogues with non-hydrazine
linkers. Bioorg. Med. Chem. Lett. 2005, 15, 2471−2475. (c) Mollica, A.;
Pinnen, F.; Feliciani, F.; Stefanucci, A.; Lucente, G.; Davis, P.; Porreca,
F.; Ma, S. W.; Lai, J.; Hruby, V. J. New potent Biphalin analogues
containing p-fluoro-L-phenylalanine at the 4,4′ positions and non-
hydrazine linkers. Amino Acids 2011, 40, 1503−1511. (d) Leone, S.;
Chiavaroli, A.; Orlando, G.; Mollica, A.; Di Nisio, C.; Brunetti, L.;
Vacca, M. The analgesic activity of Biphalin and its analog AM 94 in
rats. Eur. J. Pharmacol. 2012, 685, 70−73.
3423
dx.doi.org/10.1021/jm301456c | J. Med. Chem. 2013, 56, 3419−3423