Each peptide bond in leuprolide (1), deslorelin (13), and nafarelin (24) was separately substituted with N-methyl. The synthesized compounds were tested for in vitro receptor binding, LH release, and stability against chymotrypsin and intestinal degradation. The NMe-Ser4 (30), NMe-Leu7 (33), and Sar10 (35) analogues of nafarelin had pD2 values 2-, 20-, 9-fold higher than their respective parent. All the other N-methyl agonists were less active. For the first time, conversion of LHRH agonists to antagonists was observed as a result of N-methyl substitution in the peptide backbone. [NMe-Phe2,DLeu6,Pro9NHEt]LHRH (4), [NMe-1Nal3,DLeu6,Pro9NHEt]LHRH (6), [NMe-His2,DTrp6,Pro9NHEt]LHRH (14), [NMe-Phe2,DNal6]LHRH (27), and [D2Nal6,NMe-Arg8]LHRH (34) exhibited antagonist responses. Substitutions of NMe-1Nal3, NMe-Ser4, or NMe-Tyr5 in leuprolide rendered the 3-4 peptide bond in these compounds completely stable to chymotrypsin. Examination of the three-dimensional structure of leuprolide when bound to the active site of chymotrypsin, reveals the NH's of residues 3 and 5 are involved in hydrogen bond interactions with the enzyme. N-Methylation at these positions is not only disrupting the hydrogen bond interactions, but is also sterically preventing the substrate from fitting in the enzyme's active site. All the compounds in the leuprolide series were also tested against intestinal degradation using an in vitro rat jejunum sac assay. In this model the pattern of stabilization was similar, but not identical, to that against chymotrypsin. The pharmacokinetics of all the analogues in the leuprolide series and of several others in the deslorelin and nafarelin series were determined. The clearance values of all the three NMe-Tyr5 analogues, 8, 20, and 31 were lower than their respective parents. These slower clearances suggest lower rates of metabolism.

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To stabilize leuprolide (1) against chymotrypsin and intestinal degradation several agonists of LHRH (2-12), modified at position 1, 2, or 3 and/or containing N-alpha-methyl at positions 1, 2, or 4, were synthesized by SPPS. These agonists were tested in vitro for (a) rat pituitary LHRH receptor binding, (b) LH release from rat pituitary cells, (c) stability against chymotrypsin, and (d) stability against rat intestinal degradation. The clearances of the compounds in the rat were determined using a RIA. Complete stabilization against chymotrypsin (t1/2) and lumenal degradation (T1/2) was achieved with substitution of NMe-Ser4 in leuprolide; however, with an increase in clearance. Substitution with 1-Nal3 increased both t1/2 and T1/2, while substitution with NAc-Sar1 increased only T1/2. [NAcSar1,NMeSer4,D-Trp6,Pro9NHEt]LHRH (12), the doubly stabilized analogue, was tested in the rat by both iv and id administrations, and its bioavailabilities were measured. No significant improvement in id absorption over leuprolide was observed.

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.

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