We prepared nine analogues (1-9) of MCPA-D-Phe-Phe-Ile-Asn-Cys-Pro-Arg-Gly-NH2, [MCPA1, D-Phe2, Phe3, Ile4, Arg8]oxytocin (MCPA = beta-mercapto-beta,beta-pentamethylenepropionic acid), a potent antagonist of the rat uterotonic action of oxytocin (OT). We replaced D-Phe with D-Trp and made [MCPA1,D-Trp2,Phe3,Ile4,Arg8]OT (1), which had OT pA2 of 7.51, somewhat higher than that of the D-Phe2 antagonist which has OT pA2 = 7.35 in our rat uterotonic assay. Both compounds are equipotent as antagonists of [Arg8]vasopressin in the rat antidiuretic assay, with pA2 = 8.1. Other substitutions gave [MCPA1,D-Trp2,4-Cl-Phe3,Ile4,Arg8]OT, (2), OT pA2 7.44; [MCPA1,D-Trp2,Phe3,Ile4,3,4-dehydro-Pro7,Arg8]OT (3), OT pA2 = 7.42; [MCPA1,D-Trp2,Phe3,Arg8]OT (4), OT pA2 = 7.58; [MCPA1,D-Trp2,Phe3,Arg8,Gly9-NHEt]OT (5), OT pA2 = 7.49; [MCPA1,D-Trp2,Ile4,Arg8]OT (6), OT pA2 = 7.46; [MCPA1,D-Trp2,Val4,Arg8]OT (7), OT pA2 = 7.58; [MCPA1,D-Trp2,Thr4,Arg8]OT (8), OT pA2 = 7.48; and finally, [MCPA1,D-Trp2,Arg8]OT (9), which was a more potent and more selective OT antagonist, with OT pA2 = 7.77 in the uterotonic assay and ADH pA2 less than 5.9 in the antidiuretic assay and hence is an important lead for the design of OT antagonists.
Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O. Since the more hydrophilic NH and C=O substitutions showed a sharply decreased antagonistic potency (rat uterotonic test in vitro), additional modifications were made to reduce their hydrophilicity. Acylation of the NH group with various acyl groups, and ketalization or thioketalization of C=O with more or less bulky substituents led to a partial restoration of potency, the N-carbamyl- and the 2-mercapto-2-adamantaneacetyl analogues being equipotent with PA. Internal cyclization by amidation of the NH-group with Gly-9, resulted in a bicyclic analogue, (cyclo 1-9)[(HN)Pmp1, Gly9]PA which was equipotent with PA. When Pen-6 was introduced into the bicyclic derivative instead of Cys-6, to reduce the flexibility of the rings, the resulting (cyclo 1-9)[(HN)Pmp1, Pen6, Gly9]PA had somewhat better potency (pA2 = 8.17) in the uterotonic test and no detectable activity in the antidiuretic assay. In the case of substitution of PA with beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, (S)Pmp, there was also an increase in inhibitory potency in the uterotonic test (pA2 = 8.08): the analogue had extremely weak antidiuretic activity. To establish the importance of the steric effects of the Pen-6 substitution, analogues [Pen6]PA and [(S)Pmp1, Pen6]PA were made and found to be very potent, with a pA2 of 8.72 and 8.86, respectively. The high potency of the latter analogue and its extremely weak action in the diuretic assay makes it an attractive candidate for studies on the inhibition of the biological effects of oxytocin and for the prevention of preterm labour.
We report twelve analogues of [Pmp1,D-Trp2,Arg8]oxytocin, ANTAG (Pmp = beta, beta-pentamethylene-beta-mercaptopropionic acid), which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats, as measured in a uterotonic assay. Nine of the following analogues were designed by replacement of each of the nine residues in ANTAG with an L-tryptophan residue: [Ac-Trp1,D-Trp2,Val6,Arg8]OT, [Pmp1,Trp2,Arg8]OT, [Pmp1,D-Trp2,Trp3,Arg8]OT, [Pmp1,D-Trp2,Trp4,Arg8]OT, [Pmp1,D-Trp2,Trp5,Arg8]OT, [Aaa1,D-Trp2,Trp6,Arg8]OT, [Aaa1,D-Trp2,Val6,Arg8]OT, [Pmp1,D-Trp2,Ica7,Arg8]OT, [Pmp1,D-Trp2,Trp7,Arg8]OT, [Pmp1,D-Trp2,Trp8]OT, [Pmp1,D-Trp2,Arg8,Trp9]OT (11), [Pmp1,D-Trp2,Arg8,Trp(For)9]OT (12). In these analogues Aaa = 1-adamantaneacetic acid, and Ica = indoline-2-carboxylic acid. All linear analogues and analogues featuring Trp substitutions in the ring sequence of ANTAG were OT antagonists of lower potency than the parent peptide. All the analogues featuring Trp substitutions in the tail sequence of ANTAG were OT antagonists of equal or better potency than the parent peptide. Replacement with Ica7 gave analogue 8, equipotent with ANTAG, but replacement with Trp7 gave analogue 9, which shows almost a two-fold increase in potency (pA2 = 8.06). Replacement with Trp9 gave analogue 11 (pA2 = 8.03) which is about 1.8 times more potent than the parent antagonist, although Trp(For)9 had lower potency. Of great interest is that substitution with Trp8 leads to a more potent analogue, 10 (pA2 = 8.22), which, unlike most antidiuretic hormone antagonists, lacks any cationic charge in the molecule. The antidiuretic assay shows antagonists 9-11 to be weak antagonists of [Arg8]vasopressin, the antidiuretic hormone, with pA2 less than or equal to 6.0; hence, they may be interesting leads for future design of more potent and specific OT antagonists.
We report analogues of N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Ilys-Pro-D-Ala- NH2, the parent antagonist (PA), which is a potent antagonist of LHRH. To simplify future radioactive labeling we prepared N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Arg-Pro-D-Ala-NH2 (4), [Arg8]PA, which had good activity in the antiovulatory assay (AOA). Other analogues were designed at first by substituting with Arg at positions 5, 6, 7, 9, and 10, and Trp or Leu at position 8. Subsequent analogues were prepared in attempts to improve the AOA of the initial ones. Substitutions with Arg9 or Arg10 led to analogues 1-3 with no AOA activity at 5 micrograms/rat. However, substitution with Arg7 gave 9, [Arg7,Leu8]PA, with significant activity in the AOA at 5 micrograms/rat and borderline activity at 2.5 micrograms/rat, and substitution with Ilys7 gave 13, [Ilys7,Leu8]PA, with borderline activity at 2 micrograms/rat, both analogues showing much weaker activity than PA in the histamine release assay (HRA) and therefore being potentially safer. Substitutions with D-Arg6 or Arg5 led to analogues with either good AO activity at 5 micrograms/rat (analogue 7) or with borderline activity at 5 micrograms/rat (analogue 8), although both were more potent than 6 in the HRA. Combinations of Ilys or Arg at positions 7 and 8 led to 10 and 11, both of which were tested at 2 micrograms/rat and found to have either good AO activity (analogue 10) or borderline activity (analogue 11) but unsuitably potent in HR. Substitutions using Ilys7 and neutral amino acids at position 8 led to 14-17 which were inactive in the AOA. Of great significance is the substitution with Arg7 yielding analogue 9, which was much safer in the HRA than analogue 4, [Arg8]PA. Analogues 9 and 13, featuring substitutions with the Arg7-Leu8 or Ilys7-Leu8 sequences were even safer than PA or 6 in the HRA. Analogue 12, [D-Trp3,Tyr5,D-Arg6,Arg7,Leu8]PA, featuring the Arg7-Leu8 sequence, had much lower potency in the HRA than [D-Trp3,Tyr5,D-Arg6,Leu7,Arg8]PA, which has the normal Leu7-Arg8 sequence. Ilys7 together with neutral amino acids at position 8 led to analogues 14-17 which were also very weak (safer) in the HRA, with the smaller amino acids Ala8 and Abu8 being the weakest of all analogues prepared.(ABSTRACT TRUNCATED AT 400 WORDS)
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