Evaluation of the Dmt-Tic Pharmacophore
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 719
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albumin. (Presoaking filters in 0.1% polyethyeneimine is only
required in assays with peptides carrying an overall positive
charge, such as dynorphin or nociceptin.)52 Nonspecific binding
was determined using 2 µM unlabeled DPDPE or DAGO for
δ- or µ-assays, respectively. The Ki data, determined according
to Cheng and Prusoff,53 represent the means ( standard error
(SE) from three or more independent assays using at least
three different synaptosomal preparations and testing the
analogues with 5-8 graded dosages over 2-3 orders of
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F u n ction a l P h a r m a cologica l Bioa ssa ys. The in vitro
pharmacological assays used a single MVD for δ receptors and
a 2-3 cm segment of GPI for µ receptors with each suspended
in 20 mL organ baths containing balanced salt solutions in a
physiological buffer.2 Peptide analogues were assayed for
µ-agonist activity by the inhibition of electrically stimulated
contractions in comparison to dermorphin; δ-antagonist activ-
ity used deltorphin C (δ1-agonist).12 Data were obtained from
four independent assays and tissue samples. Agonism is
expressed as the IC50 (nM) value and antagonism as the pA2
value.1
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Refer en ces
(1) In addition to the IUPAC-IUB Commission on Biochemical
Nomenclature (J . Biol. Chem. 1985, 260, 14-42), this paper uses
the following symbols and abbreviations: AcOH, acetic acid; Bid,
1H-benzimidazol-2-yl; Boc, tert-butyloxycarbonyl; Bzl, CH2-Ph;
DAGO, [D-Ala2, N-Me-Phe4, Gly ol5] enkephalin; DMF, N,N-
dimethylforamide; Dmt, 2′,6′-dimethyl-L-tyrosine; DPDPE, cy-
clic[D-Pen2,5]enkephalin; Et2O, diethyl ether; EtOAc, ethyl ac-
etate; GPI, guinea pig ileum; HOBt, 1-hydroxy-1,2,3-benzotriazole;
HPLC, high-performance liquid chromatography; MeOH, metha-
nol; MVD, mouse vas deferens; NMM, 4-methyl morpholine; pA2,
negative log of the molar concentration required to double the
agonist concentration to achieve the original response; Pe,
petroleum ether; pEC50, negative log of the molar concentration
of an agonist to produce 50% of the maximum effect; Ph, phenyl;
TEA, triethylamine; TFA, trifluoroacetic acid; Tic, 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid; TIPP, H-Tyr-Tic-
Phe-(Phe)-OH; TLC, thin-layer chromatography; WSC, 1-ethyl-
3-[3′-dimethyl)aminopropyl] carbodiimide.
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