5590 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Brief Articles
(8) Achilefu, S.; Srinivasan, A.; Schmidt, M. A.; J imenez, H. N.;
Bugaj, J . E.; Erion, J . L. Novel Bioactive and Stable Neurotensin
Peptide Analogues Capable of Delivering Radiopharmaceuticals
and Molecular Beacons to Tumors. J . Med. Chem. 2003, 46,
3403-3411.
(9) Luca, S.; White, J . F.; Sohal, A. K.; Filippov, D. V.; van Boom,
J . H.; Grisshammer, R.; Baldus, M. The conformation of neuro-
tensin bound to its G protein-coupled receptor. Proc. Nat. Acad.
Sc. 2003, 100, 10706-10711.
(10) Pang, Y.-P.; Cusack, B.; Groshan, K.; Richelson, E. Proposed
Ligand Binding Site of the Transmembrane Receptor for Neu-
rotensin(8-13). J . Biol. Chem. 1996, 271, 15060-15068.
(11) Barroso, S.; Richard, F.; Nicolas-Ethe`ve, D.; Reversat, J .-L.;
Bernassau, J .-M.; Kitabgi, P.; Labbe´-J ullie´, C. Identification of
Residues Involved in Neurotensin Binding and Modeling of the
Agonist Binding Site in Neurotensin Receptor 1. J . Biol. Chem.
2000, 275, 328-336.
(12) Takeuchi, Y.; Marshall, G. R. Conformational Analysis of
Reverse-Turn Constraints by N-Methylation and N-Hydroxyla-
tion of Amide Bonds in Peptides and Non-Peptide Mimetics. J .
Am. Chem. Soc. 1998, 120, 5363-5372.
(13) Ward, P.; Ewan, G. B.; J ordan, C. C.; Ireland, S. J .; Hagan, R.
M.; Brown, J . R. Potent and Highly Selective Neurokinin
Antagonists. J . Med. Chem. 1990, 33, 1848-1851.
(14) Hinds, M. G.; Welsh, J . H.; Brennand, D. M.; Fisher, J .; Glennie,
M. J .; Richards, N. G. J .; Turner, D. L.; Robinson, J . A. Synthesis,
Conformational Properties, and Antibody Recognition of Peptides
Containing â-Turn Mimetics Based on R-Alkylproline Deriva-
tives. J . Med. Chem. 1991, 34, 1777-1789.
(15) Genin, M. J .; Ojala, W. H.; Gleason, W. B.; J ohnson, R. L.
Synthesis and Crystal Structure of a Peptidomimetic Containing
the (R)-4.4-Spiro Lactam Type-II â-Turn Mimic. J . Org. Chem.
1993, 58, 2334-2337.
removed by the treatment with piperidine/DBU (2%/2% in
DMF). Subsequently, Boc-Tos(Arg) was attached two times as
described above. Upon completion, the N-termini were de-
blocked (TFA) and the HF-cleavage from the resin using
anisole as the scavenger (HF/anisole 9/1, 2 h, 0 °C) was
performed. HF was evaporated, and the resin was washed with
tert-butyl methyl ether. The pure peptides were obtained by
extraction of the resin with acetic acid, followed by lyophiliza-
tion and purification via preparative HPLC (gradient elution:
5-35% CH3CN + 0.1% TFA/H2O + 0.1% TFA) on a ZORBAX
300SB-C18 PrepHT (21.2 × 250 mm, 7 µm) column.
Recep tor Bin d in g Exp er im en ts. Receptor binding data
were determined utilizing homogenates of membranes from
porcine striatum which were prepared from fresh brains
obtained from the local slaughterhouse. Dissection of the
striatum and preparation of membrane homogenates were
performed as described previously.26
The neurotensin receptor binding assay was run at a final
volume of 1.5 mL on 24-well plates. The experiment was
started by adding membranes which were diluted with binding
buffer (50 mM TrisHCl, 1 mM EDTA, 0.2 mM bacitracin, 0.1%
BSA; pH 7.4) to a final concentration of 660 µg/tube to a
mixture of 0.2 nM [3H]neurotensin (specific activity 91 Ci/
mmol; Perkin-Elmer, Boston, MA) and the test compound at
eight different concentrations (in the range from 0.001 nM to
100 µM). Incubation was continued for 30 min. at 37 °C and
stopped by rapid filtration through GF/B filters precoated with
0.3% polyethyleneimine. Filters were washed five times with
ice-cold washing buffer (50 mM TrisHCl, 1 mM EDTA; pH 7.4)
and dried, and the radioactivity was counted in a Microbeta
Trilux (Perkin-Elmer, Freiburg, Germany). Unspecific binding
was determined in the presence of 2 µM neurotensin (Sigma-
Aldrich). Protein concentration was established by the method
of Lowry using bovine serum albumin as standard.27
(16) Genin, M. J .; J ohnson, R. L. Design, Synthesis, and Conforma-
tional Analysis of a Novel Spiro-Bicyclic System as a Type II
â-Turn Peptidomimetic. J . Am. Chem. Soc. 1992, 114, 8778-
8783.
(17) Seebach, D.; Boes, M.; Naef, R.; Schweizer, W. B. Alkylation of
Amino Acids without Loss of the Optical Activity: Preparation
of R-Substituted Proline Derivatives. A Case of Self-Reproduction
of Chirality. J . Am. Chem. Soc. 1983, 105, 5390-5398.
(18) Wang, H.; Germanas, J . P. 4-Alkyl-2-trichloromethyloxazolidin-
5-ones: Valuable Precursors to Enantionmerically Pure C- and
N-Protected R-Alkyl Prolines. Synlett 1999, 7, 33-36.
(19) Hoffmann, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Rational
Molecular Design and EPC Synthesis of a Type VI â-Turn
Inducing Peptide Mimetic. Angew. Chem., Int. Ed. 2001, 40,
3361-3364.
(20) Ward, P.; Ewan, G. B. Spirolactam containing peptides. United
States Patent Application US 5166136, 1992.
(21) Casimir, J . R.; Tourwe´, D.; Iterbeke, K.; Guichard, G.; Briand,
J .-P. Efficient Synthesis of (S)-4-Phthalimido-1,3,4,5-tetrahydro-
8-(2,6-dichlorobenzyloxy)-3-oxo-2H-2-benzazepin-2-acetic Acid
(Pht-Hba-(2,6-Cl2-Bn)-Gly-OH). J . Org. Chem. 2000, 65, 6487-
6492.
Data analysis of the resulting competition curves was
accomplished by nonlinear regression analysis using the
algorithms in PRISM (GraphPad Software, San Diego, CA).
Ki values were derived from the corresponding EC50 data
utilizing the equation of Cheng and Prusoff.28
Su p p or tin g In for m a tion Ava ila ble: Methods and ma-
terials, synthetic procedures, and analytical data of 5, ent-6,
ent-7, 8, ent-8, ent-9, 10, and ent-10, analytical data of 1-4,
6, 7, 9, and [NMeTyr11]NT(8-13), and elemental analysis data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(22) O-(2,6-Dichlorobenzyl)tyrosine methyl ester hydrochloride was
obtained from O-(2,6-dichlorobenzyl)tyrosine prepared as de-
scribed in ref 13 by esterification with MeOH/SOCl2.
(23) Carpino, L. A. 1-Hydroxy-7-azabenzotriazole. An Efficient Pep-
tide Coupling Additive. J . Am. Chem. Soc. 1993, 115, 4397-4398.
(24) Falb, E.; Yechezkel, T.; Salitra, Y.; Gilon, C. In situ generation
of Fmoc-amino acid chlorides using bis-(trichloromethyl)carbon-
ate and its utilization for difficult couplings in solid-phase
peptide synthesis. J . Pept. Res. 1999, 53, 507-517.
(25) Cusack, B.; McCormick, D. J .; Pang, Y.-P.; Souder, T.; Garcia,
R.; Fauq, A.; Richelson, E. Pharmacological and Biochemical
Profiles of Unique Neurotensin 8-13 Analogs Exhibiting Species
Selectivity, Stereoselectivity, and Superagonism. J . Biol. Chem.
1995, 31, 18359-18366.
(26) Hu¨bner, H.; Haubmann, C.; Utz, W.; Gmeiner, P. Conjugated
Enynes as Nonaromatic Catechol Bioisosteres: Synthesis, Bind-
ing Experiments, and Computational Studies of Novel Dopamine
Receptor Agonists Recognizing Preferentially the D3 Subtype.
J . Med. Chem. 2000, 43, 756-762.
(27) Lowry, O. H.; Rosebrough, N. J .; Farr, A. L.; Randall, R. J .
Protein measurement with the folin phenol reagent. J . Biol.
Chem. 1951, 193, 265-275.
Refer en ces
(1) Binder, E. B.; Kinkead, B.; Owens, M. J .; Nemeroff, C. B.
Neurotensin and Dopamine Interactions. Pharmacol. Rev. 2001,
53, 453-486 (and references therein).
(2) Tyler-McMahon, B.; Boules, M.; Richelson, E. Neurotensin:
Peptide for the next millennium. Regul. Pept. 2000, 93, 125-
136 (and references therein).
(3) Shilling, P. D.; Richelson, E.; Feifel, D. The effects of systemic
NT69L, a neurotensin agonist, on baseline and drug-disrupted
prepulse inhibition. Behav. Brain Res. 2003, 143, 7-14.
(4) Sefler, A. M.; He, J . X.; Sawyer, T. K.; Holub, K. E.; Omecinsky,
D. O.; Reily, M. D.; Thanabal, V.; Akunne, H. C.; Cody, W. L.
Design and Structure-Activity Relationships of C-Terminal
Cyclic Neurotensin Fragment Analogues. J . Med. Chem. 1995,
38, 249-257.
(5) Hong, F.; Cusack, B.; Fauq, A.; Richelson, E. Peptidic and
Nonpeptidic Neurotensin Analogs. Curr. Med. Chem. 1997, 4,
412-434 (and references therein).
(6) Lundquist, J . T., IV; Dix, T. A. Preparation and Receptor Binding
Affinities of Cyclic C-Terminal Neurotensin (8-13) and (9-13)
Analogues. Bioorg. Med. Chem. Lett. 1999, 9, 2579-2582.
(7) Lundquist, J . T., IV; Dix, T. A. Synthesis and Human Neuro-
tensin Receptor Binding Activities of Neurotensin(8-13) Ana-
logues Containing Position 8 R-Azido-N-alkylated Derivatives
of Ornithine, Lysine, and Homolysine. J . Med. Chem. 1999, 42,
4914-4918.
(28) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (IC50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
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