Induction of a novel conformation in the progesterone receptor by ZK299 involves a defined region of the carboxyl-terminal tail.

Progesterone receptor antagonists are a promising class of therapeutic drugs indicated for the treatment of a variety of reproductive conditions. Understanding their mechanism of action at the molecular level is an important prerequisite for the development of future generations of these drugs. Using limited proteolytic analysis to monitor conformational changes in the progesterone receptor, we can detect three distinct classes of progestin antagonist. The effect of the first, RU486, on the conformation of the carboxyl terminus of the receptor has been previously described. The second, exemplified by RWJ 47626, a nonsteroidal compound with in vitro antiprogestin activity, induces a proteolytic fragment pattern indistinguishable from that induced by the agonist R5020. Finally, ZK299 induces a fragment pattern intermediate between that induced by R5020 and RU486. Site-directed mutagenesis of the carboxyl-terminal tail of the progesterone receptor indicates that the region containing the putative activation function AF-2 is differentially exposed to proteolytic attack depending on the nature of the antagonist bound. The differentially exposed region is most accessible when the antagonist RU486 is bound, less accessible when the antagonist ZK299 is bound, and least accessible when the antagonist RWJ47626 or agonist R5020 is bound. The results suggest that multiple types of antiprogestin can be defined in terms of their effects on the conformation of the carboxyl-terminal activation function of the progesterone receptor.

Heteroatom analogues of bemoradan: chemistry and cardiotonic activity of 1,4-benzothiazinylpyridazinones.

A series of close analogues of the potent, long-acting cardiotonic bemoradan (2a) was synthesized and examined in both in vitro and in vivo test systems. Changing the oxygen heteroatom at the 1-position of the benzoxazine ring of bemoradan to sulfur gave 4a, a more potent enzyme inhibitor and in vivo cardiotonic compound by the iv route. Intraduodenal administration of bemoradan, however, showed a superior response compared to its sulfur analogue, possibly due to oxidation of sulfur followed by a facile Pummerer rearrangement. Model studies were performed to examine the effect of the oxidation state of sulfur. Lack of a heteroatom at the 1-position, 3a (Y-590), afforded a compound with activity and potency very similar to those of bemoradan while the 1-selena compound gave a much less potent analogue 5. Analogues having a methyl group on the 4-nitrogen (2b, 3b, and 4b) were less potent than the desmethyl compounds, but all of these compounds have potent PDE III inhibiting activity and the ability to increase cardiac force in an anesthetized dog preparation when given iv.

gamma-Aminobutyrate-A receptor modulation by 3-aryl-1-(arylsulfonyl)- 1,4,5,6-tetrahydropyridazines.

A series of 3-aryl-1-(arylsulfonyl)-1,4,5,6-tetrahydropyridazine allosteric modulators of the GABAA receptor was synthesized, and biological activity was examined in vitro and in vivo. Beginning with 1a, stepwise modification of the substituents and conservation of the scaffold yielded a chemical series in which the modulatory activity was enhanced by the presence of GABA. The SAR suggests, but does not establish, that the compounds bind to the steroid binding site on the GABAA receptor. The GABA shift for each compound indicates that all compounds in this series are either agonists or partial agonists.

6-Benzoxazinylpyridazin-3-ones: potent, long-acting positive inotrope and peripheral vasodilator agents.

A series of 6-benzoxazinylpyridazin-3-ones was prepared and evaluated for inhibition of cardiac phosphodiesterase (PDE) fraction III in vitro and for positive inotropic activity in vivo. 6-[3,4-Dihydro-3-oxo-1,4(2H)-benzoxazin-7-yl]-2,3,4,5-tetrahydro-5 - methylpyridazin-3-one (bemoradan) was found to be an extremely potent and selective inhibitor of canine PDE fraction III and a long-acting, potent, orally active inotropic vasodilator agent in various canine models. Additional benzoxazin-6-yl and -8-yl compounds were also prepared. Altering the pyridazinone substitution from the 6-position to the 7-position produced a 14-fold increase in the iv cardiotonic potency (ED50) from 77 to 5.4 micrograms/kg while substitution at the 8-position reduced potency. Methyl substitution at various sites in the molecule was also examined. Positive inotropic activity was maintained for between 8 and 24 h after a single oral dose (100 micrograms/kg) of bemoradan in dogs, thus making it one of the most potent and long-acting orally effective inotropes yet described. Bemoradan is currently under development as a cardiotonic agent for use in the management of congestive heart failure.

Nonsteroidal progesterone receptor ligands. 2. High-affinity ligands with selectivity for bone cell progesterone receptors.

A novel series of nonsteroidal heterocycles was discovered which display cell-type selective, high-affinity (nanomolar) binding to the progesterone receptors from TE85 osteosarcoma cells but > 1 microM binding affinity to the progesterone receptors from T47D and ZR75 human breast carcinoma cells. Structure-activity relationships were developed for a set of these compounds, and a representative analog 1-(3,4-dichlorobenzoyl)-3-phenyl-1,4,5,6-tetrahydropyridazine++ + (1i, RWJ 25333) was chosen for further evaluation. RWJ 25333 stimulated the in vitro proliferation of human osteoblast-like cells but not human breast cells.

Synthesis and cardiotonic activity of a series of substituted 4-alkyl-2(1H)-quinazolinones.

The synthesis, cardiac fraction III cyclic nucleotide phosphodiesterase (PDE-III) inhibition, and positive inotropic activity of a series of 2(1H)-quinazolinones are reported. A general synthesis of the series involved the cyclization of 2-aminoacetophenones with potassium cyanate in acetic acid. Modifications at the 4-position of the quinazoline nucleus were best achieved by formation of the intermediate N1-acyl-N3-phenylurea from the substituted phenyl isocyanate and appropriate carboxamide. PPA was used to ring close to the quinazoline product. Generally the SAR for the series paralleled the five-point model previously published for PDE-III inhibition. The most active analogue of the series was 5,6-dimethoxy-4-methyl-2(1H)-quinazolinone (1) (ORF 16600), which had about twice the intravenous potency of amrinone. Compound 1 is currently under development as an orally active cardiotonic.

Bemoradan--a novel inhibitor of the rolipram-insensitive cyclic AMP phosphodiesterase from canine heart tissue.

Canine cardiac muscle contains a type IV cyclic AMP (cAMP) phosphodiesterase (PDE) that is composed of two subtypes. One subtype is sensitive to rolipram inhibition (RSPDE), whereas the other is not inhibited significantly by rolipram (RIPDE). The RIPDE is inhibited by several cardiotonic agents operating by a PDE-inhibitory mechanism. Bemoradan [RWJ-22867; 7-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-2H-1,4-benzoxazin -3(4H)-one], a novel, potent positive inotropic agent, demonstrated biphasic inhibition of the fraction III enzyme from canine cardiac muscle. Inhibition by rolipram of the RSPDE converted the IC50 curves of bemoradan, indolidan, pimobendan, and imazodan to sigmoidal, monophasic curves. Lineweaver-Burk analysis yielded competitive inhibition KI values of 0.023, 0.09, 0.065 and 0.60 microM, respectively, for these compounds. The cardiotonic compounds, however, were not potent inhibitors of the Type I and Type II cAMP PDEs found in canine ventricular muscle. The order of potency for inhibiting the RIPDE cAMP PDE subtype was bemoradan greater than pimobendan greater than indolidan greater than imazodan. Bemoradan is, therefore, a potent inhibitor of the cardiac muscle cAMP PDE which could, in part, be responsible for its cardiotonic activity.

Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity.

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.

Design, synthesis and bronchodilatory activity of a series of quinazoline-3-oxides.

A synthetic program of rational drug design was undertaken to develop a series of quinazoline-3-oxides as pulmonary-selective inhibitors of ovalbumin-induced, leukotriene-mediated bronchoconstriction. The most active and selective compounds contained a methyl group at the 4-position, a medium sized branched alkyl group at the 2-position, and a small electron donating group on the phenyl ring. Significant enhancement in selectivity was observed in comparing the pulmonary versus cardiovascular effects of these new bronchodilators with the effects of theophylline.

Discovery of the first non-peptide antagonist of the motilin receptor.

A first-in-class non-peptide antagonist of the motilin receptor was identified through electronic screening of our corporate database against a 3D pharmacophore. The pharmacophore was developed from the motilin 22 residue endogenous peptide using NMR structural data, principles of peptide folding, and peptide structure activity relationships. The NMR data supported helical content within the peptide, and both the hydrophobic staple and N-capping box motifs were identified in the motilin sequence. The conformational features of these motifs were imposed on the peptide structure, providing a constrained conformer as a starting point for database searching. A trisubstituted cyclopentene lead was identified directly from the electronic search. Compounds in this series inhibit the binding of 125I-motilin to human antral smooth muscle membrane and antagonize motilin-induced intracellular calcium mobilization in cells expressing the human motilin receptor. A potent compound developed through optimization, RWJ 68023, is active in binding and cell-based functional assays and is also effective in inhibiting motilin-induced contractility in segments of rabbit duodenum. This orally active compound is currently undergoing clinical evaluation for the treatment of gastrointestinal disorders associated with altered motility.