Potent gastrin-releasing peptide (GRP) antagonists derived from GRP (19-27) with a C-terminal DPro psi [CH2NH]Phe-NH2 and N-terminal aromatic residues.

We have previously reported that octapeptides with a -DPro psi[CH2NH]Phe- NH2 C-terminus are potent GRP antagonists and have greatly enhanced in vivo stability. Now we report the detailed syntheses of such peptides and additional attempts to further increase metabolic stability. Replacement of the -DPro psi[CH2NH]Phe-NH2 with a "-DPro-statine"-Phe-NH2 led to less potent antagonistic activity. The introduction of ThiAla and BzthAla, to replace His and Trp, respectively, did not increase activity. A series of analogs having different aromatic residues at the N-terminal, other than 3-phenylpropionic acid, are equally potent. These residues show increased activity when hydrophilic substitutions are added to the aromatic ring. Replacement of the C-terminal Phe by DPhe and D2Nal is tolerated. Even though none of these peptides have higher activity than the original lead peptide, they are potentially more metabolically stable.

Development of potent gastrin-releasing peptide antagonists having a D-Pro-psi(CH2NH)-Phe-NH2 C terminus.

Gastrin-releasing peptide (GRP) is a 27-amino acid neuroendocrine hormone that may play a role in the pathophysiology of small cell lung carcinoma. GRP and bombesin, a structurally related peptide, stimulate the growth of some cultured cell types. C-terminal GRP peptide analogs were developed that inhibited 6 nM bombesin-induced [3H]thymidine incorporation into quiescent murine Swiss 3T3 cells, which routinely produced a 6-fold stimulation over the basal extent of incorporation. The peptides were also analyzed for their capacity to inhibit the binding of 50 pM 125I-labeled GRP to Swiss 3T3 cells. The combination of two chemical modifications, each antagonistic in itself, led to the creation of antagonists with orders of magnitude greater potency than either modification alone. (i) Antagonist analogs of the form -Leu26-psi(CH2NH)-Xaa27-NH2 [where Xaa is Leu, norleucine (Nle), or Phe; residues numbered after GRP], similar to those introduced by Coy and coworkers [for review, see Jensen, R. T. & Coy, D. H. (1991) Trends Pharmacol. Sci. 12, 13-19], were found to have nanomolar potencies. (ii) We found that an octapeptide C-terminal GRP analog having D-Pro adjacent to the C-terminal amino acid amide was antagonistic, with a potency of 40 nM. By combining both modifications, specific analogs were found with potencies > 1000-fold greater than our lead structure--[(4'-hydroxy)-3-phenylpropanoyl]-Pro-Arg-Gly-Asn-His-Tr p-Ala-Val - Gly-His-Leu-psi(CH2NH)-Nle-NH2--and greater than any antagonist previously reported. The analogs [(4'-hydroxy)-3-phenylpropanoyl]-His-Trp-Ala-Val-D-Ala-His-D-Pro- psi(CH2NH)-Phe-NH2 and 1-naphthoyl-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi(CH2NH)-Phe-NH2 antagonized [3H]thymidine incorporation with IC50 values of approximately 0.3 nM and inhibited the binding of 125I-labeled GRP with IC50 values of approximately 1 pM. These peptides may be of use in the study of the physiology of GRP.

Conveyance of partial agonism/antagonism to bombesin/gastrin-releasing peptide analogues on Swiss 3T3 cells by a carboxyl-terminal leucine insertion.

Gastrin-releasing peptide (GRP) is a neuroendocrine hormone that may be involved in the pathophysiology of small cell lung carcinoma. We describe carboxylterminal peptide analogues of GRP and bombesin, a 14-residue amphibian homologue, that were modeled after the antagonist [Leu13-psi(CH2NH)-Leu14]bombesin and retained the psi bond. Three novel peptides contained a Leu insertion amino to the psi bond, i.e. ... Leu13Leu14 psi X (residues numbered after bombesin) where X = LeuNH2 or norleucine-NH2). The Leu-insertion analogues behaved as pure partial agonists/antagonists when examined for the ability to stimulate [3H]thymidine incorporation into quiescent Swiss 3T3 cells (agonist activity) and to diminish the agonist response of GRP (antagonist activity). A time course of [3H]thymidine incorporation into quiescent cells indicated maximal incorporation at 20-h post-peptide addition for bombesin and GRP and a Leu-insertion peptide, but the extent of the incorporation for the Leu-insertion peptide was half that of GRP and bombesin. The agonist dose responses of the Leu-insertion peptides (EC50 values of 1-10 nM) paralleled GRP and bombesin, but the maximal response of the Leu-insertion peptides, even at concentrations as high as 10(-4) M, was half the maximal value of GRP or bombesin. High concentrations of the Leu-insertion peptides antagonized 10 nM GRP (a concentration that produced a near-maximal GRP response) yielding a response that was half the maximal value of GRP and equivalent to the maximal response of the Leu-insertion peptides alone. Analogues of the form ... Leu13 psi X behaved as complete antagonists. The KD values of the Leu-insertion peptides for competitive binding versus 125I-GRP (2-50 nM) were as potent as parent ... Leu14 agonists. Stability studies indicated that peptide potencies for both agonist and antagonist activities diminished upon peptide incubation in medium or on cells. The results suggested that, for the Leu-insertion peptides, degradation into distinct products with different activities was not responsible for their partial agonist/antagonist behavior. Computer-generated molecular modeling studies indicated that the novel structures could adopt energy minimized conformations for either an agonist or an antagonist as proposed earlier (Coy, D.H., Heinz-Erian, P., Jiang, N.-Y., Sasaki, Y., Taylor, J., Moreau, J.-P., Wolfrey, W.T., Gardner, J.D., and Jensen, R. T. (1988) J. Biol. Chem. 263, 5056-5060).

Modification of cardiovascular actions of 2-amino-5,6-dihydroxytetralin by N,N-di-n-propyl substitution.

Effects of dopamine (DA) and the N,N-di-n-propyl derivative of 2-amino-5,6-dihydroxytetralin (dipropyl-A-5,6-DTN) were compared on renal blood flow in phenoxybenzamine pretreated dogs. Equivalent increments in renal blood flow were observed in the dose range of 12-190 nmol (ED50 of DA, 1.4 x 10(-8) mol; ED50 of dipropyl-A-5,6-DTN, 1.2 x 10(-8) mol, n = 7). Effects of 47 nmol of DA and dipropyl-A-5,6-DTN were reduced equally by simultaneous injections of 0.5 mg sulpiride (76 +/- 4 and 59 +/- 6%, respectively, n = 5). Intravenous injections of dipropyl-A-5,6-DTN in doses of 4-16 micrograms/kg elevated arterial blood pressure and decreased heart rate and cardiac contractility in open chest, vagotomized dogs, confirming its neuronal inhibitory effect mediated by DA2-presynaptic receptors. Dipropyl-A-5,6-DTN also caused dose-related femoral artery vasoconstriction in the hexamethonium pretreated dog. The hypertensive effect of intravenous dipropyl-A-5,6-DTN and its femoral vasoconstrictor effect were abolished by phenoxybenzamine. These results show that dipropyl-A-5,6-DTN possesses DA1-, DA2- and alpha-adrenergic activities. It is significant that by introducing di-n-propyl substitution on the nitrogen of 5,6-dihydroxy-2-aminotetralin, potent DA1-agonist activity can be conferred upon a molecule which is inactive in that respect. Further, beta-adrenergic activity of the primary amine is replaced by potent alpha-adrenergic activity by this substitution.

A novel inhibitor of gamma-aminobutyrate aminotransferase with anorectic activity.

1-(n-decyl)-3-Pyrazolidinone (BW357U) is a potent, selective inhibitor of gamma-aminobutyrate aminotransferase (GABA-T) in vitro and in vivo. After acute or chronic, oral or intraperitoneal administration of BW357U to rats, brain GABA levels were elevated in a dose-dependent manner. When inhibition of brain GABA-T exceeded 50%, whole brain GABA levels were elevated approximately threefold, and an anorectic effect was observed in the absence of other symptoms. This compound, because of its potency and selectivity, may be useful in studies relating to the function of GABA-containing neurons in appetite regulation.

Dopamine receptors in rat striatum and nucleus accumbens; conformational studies using rigid analogues of dopamine.

A study was made of the actions of dopamine and of some 2-amino-1,2,3,4-tetrahydronaphthalenes on dopamine-sensitive adenylate cyclase in homogenates of rat striatum and nucleus accumbens. The compounds were also tested for their ability to stimulate motor activity following bilateral injection into the nucleus accumbens of conscious rats. The most active compounds on adenylate cyclase from both striatum and nucleus accumbens were dopamine and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene(6,7-diOHATN). The 5,6-dihydroxy analogue (5,6-diOHATN) was 50 times less active than 6,7-diOHATN in striatal homogenates and 350 times less active in homogenates of nucleus accumbens. All dihydroxy compounds tested were active in causing stimulation of motor activity, the most active compounds being 6,7-and5,6diOHATN. Both dimethoxy derivatives tested were inactive on the adenylate cyclase and as locomotor stimulants.

Synthesis and dopaminergic activity of (+/-)-, (+)-, and (-)-2-dipropylamino-5-hydroxy-1,2,3,4-tetrahydronaphthalene.

In an effort to identify further the structural requirements for central dopamine receptor agonists, some monohydroxyl analogs of the known agonist 5,6-dihydroxy-2-dipropylamino-1,2,3,4-tetrahydronaphthalene were synthesized. They were examined for production of emesis in dogs and stereotyped behavior in rats. The most potent was 5-hydroxy-2-dipropylamino-1,2,3,4-tetrahydronaphthalene, which was more potent than apomorphine but less so than the dihydroxyl analog. The two enantiomers of the monohydroxyl analog were synthesized by conventional methods from an optically active intermediate, 2-benzylamino-5-methoxy-1,2,3,4-tetrahydronaphthalene. The resolution of this amine was performed with the aid of mandelic acid. Dopaminergic activity was found to be confined to the levo enantiomer. Requirements for both substitution and chirality in the tetralines were found to correspond closely to those known for the dopaminergic aporphines.

Synthesis and pharmacology of some 2-aminotetralins. Dopamine receptor agonists.

A series of 2-amino-1,2,3,4-tetrahydronaphthalene compounds bearing substituents on the nitrogen and in the aromatic ring was synthesized from beta-tetralone intermediates. Compounds were screened in vivo for dopaminergic activity using tests in which apomorphine was especially active. It was found that apparent dopaminergic activity is inherent in 2-dialkylaminotetralins, the dipropylamine substitution being the most consistently productive amine group studies. Activity was greatly enhanced by proper substitution in the aromatic ring. The 5,6-dihydroxy group was the best potentiating group found. These data support the idea that the extended conformation for the phenylethylamine moiety of ampmorphine and dopamine is favorable for dopaminergic agonist activity. They also suggest that an unetherified catechol group may not be essential for such activity.