4554 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Torino et al.
7.90 (1H, d, J = 8.4, Phe3 NH), 9.18 (1H, s, Tyr OH), 9.7 (1H, s,
ΔZPhe4 NH). HRMS for [M þ H]þ: m/z calcd 670.3241; found
670.3248.
gates in position 2. Bioorg. Med. Chem. 2009, 17, 3789–3794.
(b) Torino, D.; Mollica, A.; Pinnen, F.; Lucente, G.; Feliciani, F.; Peg,
D.; Lai, J.; Ma, S.-w.; Porreca, F.; Hruby, V. J. Synthesis of new
endomorphin analogues modified at the Pro2 residue. Bioorg. Med.
Chem. Lett. 2009, 19, 4115–4118.
Boc-Tyr-Pro-ΔZPhe-ΔZPhe-NH2 (11). Eluent mixture:
1
chloroform/methanol, 95:5; 56% yield. H NMR (400 MHz,
(6) 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.
(7) (a) Honda, T.; Shirasu, N.; Isozaki, K.; Kawano, M.; Shigehiro,
D.; Chuman, Y.; Fujita, T.; Nose, T.; Shimohigashi, Y. Differential
receptor binding characteristics of consecutive phenylalanines in
μ-opioid specific peptide ligand endomorphin-2. Bioorg. Med.
Chem. 2007, 15, 3883–3888. (b) Shao, X.; Gao, Y.; Zhu, C.; Liu, X.;
Yao, J.; Cui, Y.; Wang, R. Conformational analysis of endomorphin-2
analogs with phenylalanine mimics by NMR and molecular modeling.
Bioorg. Med. Chem. 2007, 16, 3539–3547.
(8) Fichna, J.; do-Rego, J.-C.; Chung, N. N.; Lemieux, C.; Schiller,
P. W.; Poels, J.; Vanden Broeck, J.; Costentin, J.; Janecka, A.
Synthesis and characterization of potent and selective μ-opioid re-
ceptor antagonists, [Dmt1, D-2-Nal4] EM-1 (Antanal-1) and [Dmt1,
D-2-Nal4] EM-2 (Antanal-2). J. Med. Chem. 2007, 50, 512–520.
(9) Gao, Y.; Liu, X.; Liu, W.; Qi, Y.; Liu, X.; Zhou, Y.; Wang, R.
Opioid receptor binding and antinociceptive activity of the analo-
gues of endomorphin-2 and morphiceptin with phenylalanine
mimics in the position 3 or 4. Bioorg. Med. Chem. Lett. 2006, 16,
3688–3692.
DMSO-d6): δ 1.3 [(9H, s, C(CH3)3], 1.71-2.26 (4H, m, Pro C3H2
and Pro C4H2), 2.62-2.75 (2H, m, Tyr CβH2), 3.48-3.72 (2H, m,
Pro C5H2), 4.25 (1H, m, Tyr CRH), 4.46 (1H, m, Pro CRH),
6.6-7.8 (19H, m, aromatics, Tyr NH, ΔZPhe3 CβH, ΔZPhe4 CβH,
and CONH2), 9.22 (1H, s, Tyr OH), 9.52 (1H, s, ΔZPhe4 NH),
10.12 (1H, s, ΔZPhe3 NH). HRMS for [M þ H]þ: m/z calcd
668.3084; found 668.3091.
Boc-Tyr-Pro-Phe-ΔZPhe-NH2 (16). Eluent mixture: chloro-
form/methanol, 95:5; 55% yield. 1H NMR (400 MHz, DMSO-d6):
δ 1.3 [(9H, s, C(CH3)3], 1.7-2 (4H, m, Pro C3H2 and Pro C4H2),
2.5-3.1 (4H, m, Phe4 CβH2 and Tyr CβH2), 3.48 and 3.58 (2H,
m, Pro C5H2), 4.21 (1H, m, Tyr CRH), 4.32 (1H, m, Pro CRH),
4.53 (1H, m, Phe4 CRH), 6.6-7.51 (18H, m, aromatics, Tyr NH,
ΔZPhe3 CβH and CONH2), 8.31 (1H, d, J = 7.2, Phe4 NH), 9.66
(1H, s, ΔZPhe3 NH), 9.45 (1H, s, Tyr OH). HRMS for [M þ H]þ:
m/z calcd 670.3241; found 670.3238.
Binding and Functional Assays. All binding assays used crude
membrane preparations from transfected HEK293 cells expres-
sing the human δ-opioid receptor or HN9.10 cells expressing
the rat μ-opioid receptor. Binding affinities of the compounds
were determined by competitive binding analysis against the
δ-selective agonist [3H]DPDPE and the μ-selective agonist
[3H]DAMGO in the respective membrane preparations. Data
from three independent experiments were fitted by nonlinear
regression analysis using GraphPad Prism. Ki values were
calculated from IC50 by the Cheng and Prusoff equation.21
The in vitro tissue bioassays (MVD and GPI/LMMP) were
performed as described previously.22 IC50 values represent mean
values from no less than four experiments. IC50 values, relative
potency estimates, and their associated standard errors were
determined by fitting the data to the Hill equation by a compu-
terized nonlinear least-squares method.
(10) Fichna, J.; do-Rego, J.-C.; Kosson, P.; Costentin, J.; Janecka, A.
Characterization of antinociceptive activity of novel endomorphin-
2 and morphiceptin analogs modified in the third position. Bio-
chem. Pharmacol. 2005, 69, 179–185.
€
€
€ ꢁ
ꢁ
ꢁ
(11) Tomboly, Cs.; Kover, K. E.; Peter, A.; Tourwe, D.; Biyashev, D.;
ꢁ
ꢁ
Benyhe, S.; Borsodi, A.; Al-Khrasani, M.; Ronai, A. Z.; Toth, G.
Structure activity study on the Phe side chain arrangement of
endomorphins using conformationally constrained analogues.
J. Med. Chem. 2004, 47, 735–743.
(12) Hruby, V. J.; Al-Obeidi, F.; Kazmierski, W. Emerging approaches
in the molecular design of receptor-selective peptide ligands: con-
formational, topographical and dynamic considerations. Biochem.
J. 1990, 268, 249–262.
(13) Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich, M.
Design of peptides, proteins and peptidomimetics in chi space.
Biopolymers. 1997, 43, 219–266.
(14) Schwyzer, R. ACTH: a short introductory review. Ann. N.Y. Acad.
Sci. 1977, 297, 3–26.
Acknowledgment. This research was supported in part by
grants of the U.S. Public Health Service and National Institutes
of Health, Grants DA006284 and DA13449 (V.J.H.).
(15) Goel, V. K.; Guha, M.; Baxla, A. P.; Dey, S.; Singh, T. P. Design of
peptides with R,β-dehydro-residues: synthesis, crystal structure
and molecular conformation of a peptide N-tertiary-butyloxycar-
bonyl-L-Leu-ΔPhe-L-Ile-OCH3. J. Mol. Struct. 2003, 658, 135–141.
(16) Biondi, B.; Giannini, E.; Negri, L.; Melchiorri, P.; Lattanzi, R.;
Rosso, F.; Ciocca, L.; Rocchi, R. Opioid peptides: synthesis and
biological activity of new endomorphin analogs. Int. J. Pept. Res.
Ther. 2006, 12, 145–151.
(17) Flippen-Anderson, J. L.; Deschamps, J. R.; George, C.; Reddy,
P. A.; Lewin, A. H.; Brine, G. A.; Sheldrick, G.; Nikiforovich, G.
X-ray structure of Tyr-D-Tic-Phe-Phe-NH2(D-TIPP-NH2), a highly
potent μ-receptor selective opioid agonist: comparison with pro-
posed model structures. J. Pept. Res. 1997, 49, 384–393.
(18) Doi, M.; Asano, A.; Komura, E.; Ueda, Y. The structure of
endomorphin analogue incorporating 1-aminicyclohexane-1-car-
boxylic acid for proline is similar to the β-turn of Leu-enkephalin.
Biochem. Biophys. Res. Commun. 2002, 297, 138–142.
(19) In, Y.; Minoura, K.; Tomoo, K.; Sasaki, Y.; Lazarus, L. H.;
Okada, Y.; Ishida, T. Structural function of C-terminal amidation
of endomorphin. FEBS J. 2005, 272, 5079–5097.
(20) Piazzesi, A. M.; Bardi, R.; Crisma, M.; Bonora, G. M.; Toniolo, C.;
Chauhan, V. S.; Kaur, P.; Uma, K.; Balaram, P. Conformational
restrictions of peptides via backbone modification: solution and
crystal-state analysis of Boc-L-Pro-ΔZPhe-Gly-NH2. Gazz. Chim.
Ital. 1991, 121, 1–7.
Supporting Information Available:
Elemental analysis of
final products, details on experimental procedures for the
synthesis of intermediates, X-ray crystallographic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Zadina, J. E.; Hackler, L.; Ge, L. J.; Kastin, A. J. A potent and
selective endogenous agonist for the μ-opiate receptor. Nature
1997, 386, 499–502.
(2) Hackler, L.; Zadina, J. E.; Ge, L. J. Isolation of relatively large
amounts of endomorphin-1 and endomorphin-2 from human brain
cortex. Peptides 1997, 18, 1635–1639.
(3) Recent reviews: (a) Janecka, A.; Staniszewska, R.; Fichna, J.
Endomorphin analogs. Curr. Med. Chem. 2007, 14, 3201–3208.
(b) Fichna, J.; Janecka, A.; Costentin, J.; do Rego, J.-C. The endomor-
phin system and its evolving neurophysiological role. Pharmacol. Rev.
2007, 59, 88–123.
(4) (a) Leitgeb, B. Structural investigation of endomorphins by experi-
mental and theoretical methods: hunting for the bioactive con-
formation. Chem. Biodiversity 2007, 4, 2703–2724. (b) Yu, Y.; Shao,
X.; Cui, Y.; Liu, H.-m.; Wang, C.; Fan, Y.; Liu, J.; Dong, S.; Cui, Y.-x.;
Wang, R. Structure activity study on the spatial arrangement of the third
aromatic ring of endomorphins 1 and 2 using an atypical constrained C
terminus. ChemMedChem 2007, 2, 309–317.
(21) Wang, Z.; Gardell, L. R.; Ossipov, M. H.; Vanderah, T. W.;
Brennan, B. B.; Hochgeschwender, U.; Hruby, V. J.; Malan,
T. P., Jr.; Lai, J.; Porreca, F. Pronociceptive actions of dynor-
phin maintain chronic neurophatic pain. J. Neurosci. 2001, 21,
1779–1786.
(22) Kramer, T. H.; Davis, P.; Hruby, V. J.; Burks, T. F; Porreca, F.
In vitro potency, affinity and agonist efficacy of highly selective
delta opioid receptor ligands. J. Pharmacol. Exp. Ther. 1993, 266,
577–584.
(5) (a) Perlikowska, R.; Gach, K.; Fichna, J.; Toth, G.; Walkowiak, B.;
do Rego, J.-C.; Janecka, A. Biological activity of endomorphin and
[Dmt1]endomorphin analogs with six-membered proline surro-