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Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Membranes from rabbit aorta and from rabbit and rat kidney cortex possess high-affinity (Kd = 10-10 M) specific binding sites for atrial natriuretic factor (ANF). Similar high-affinity sites are present in an established cell line from pig kidney, LLC-PK,. Results of fractionation studies indicate that the receptors are localized in the plasma membrane of these tissues. The binding is time-dependent and saturable. An excellent quantitative correlation was found between the affinity of synthetic ANF and analogs of intermediate activity to aorta membranes and the half-maximal concentration needed for relaxation of rabbit aorta rings contracted by addition of serotonin. Furthermore, the binding affinity of the receptor in kidney membranes is consistent with the concentration required for in vivo natriuresis in the rat. Biologically inactive synthetic ANF fragments and other peptide hormones such as angiotensin II and vasopressin do not significantly inhibit binding. These data suggest that the receptors for ANF in vascular and renal tissues are responsible for mediating the physiological actions of this peptide in these target tissues.A possible role for the cardiac atria in the regulation of extracellular fluid volume and electrolyte concentration has been shown by the induction of sodium and water excretion in response to changes in intraatrial pressure and stretch of the atrial wall (1). The cardiac atria possess granules that have the appearance of secretory granules (2-4) and whose number can be altered by manipulation of the water-electrolyte balance in experimental animals (5). Crude extracts of atria from several species have been shown to possess potent diuretic and natriuretic activity when given intravenously to rats (6). Subcellular fractionation (7) and immunocytochemistry (8) indicated that these granules are storage sites for an atrial natriuretic factor (ANF). Furthermore, in vitro, atrial extracts were shown to be potent vasorelaxants (9-14). As little as 0.0006 atrial equivalent per milliliter gave 50% relaxation of precontracted aorta tissues (12). In vivo, ANF caused lowering of mean arterial blood pressure in normal and hypertensive animals (6,15,16). Recently, a family of peptides has been isolated from acidic extracts of rat (10,11,(19)(20)(21)(22) and human (23) atria and sequenced independently by several laboratories. From rat atria, the longest peptide that has been reported contains 33 amino acids, 1, and was isolated as the free COOH-terminal tyrosine acid (20,21). Shorter rat ANF peptides are truncated at either the NH2 or COOH terminus.5 10 H-Leu -Ala-Gly-Pro -Arg-Ser -Leu-Arg-Arg-Ser-Ser-15 20Cys-Phe-Gly-Gly-Arg-Ile -Asp-Arg-Ile -Gly-Ala--30Gln -Ser-Gly-Leu-Gly-Cys -Asn-Ser-Phe-Arg-Tyr-OH 1 A peptide consisting of residues 8-33, designated sANF, has been synthesized (21, 24) and shown to possess full biological activity (12,16,21,24). The IC50 of sANF for relaxation of precontracted rabbit aorta rings is 550 pM (12). The amount of sANF required for half-maximal natriuresis wh...

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We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

A substance called atrial natriuretic factor (ANF), localized in secretory granules of atrial cardiocytes, was isolated as four homologous natriuretic peptides from homogenates of rat atria. The complete sequence of the longest form showed that it is composed of 33 amino acids. The three other shorter forms (2-33, 3-33, and 8-33) represent aminoterminally truncated versions of the 33 amino acid parent molecule as shown by analysis of sequence, amino acid composition, or both. The proposed primary structure agrees entirely with the amino acid composition and reveals no significant sequence homology with any known protein or segment of protein. The short form ANF-(8-33) was synthesized by a multifragment condensation approach and the synthetic product was shown to exhibit specific activity comparable to that of the natural ANF-(3-33).

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A series of 73 dibenzo[a,d]cycloalkenimines were synthesized and evaluated for their ability to displace (+)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]-(+)-10) from its specific binding site on rat cortical membranes. A number of the more active compounds (Ki ranging from 0.006 to 0.21 microM) were evaluated for N-methyl-D-aspartate (NMDA) antagonist activity in the rat cortical slice (Kb ranging from 0.08 to 0.9 microM) and anticonvulsant activity in the mouse against NMDA induced convulsions. The ED50 values ranged from 0.22 to 7.76 mg/kg and correlated reasonably well with the Kb determination. In the dibenzo[a,d]cyclohepten-5,10-imine series, the (+)-5S,10R enantiomer displayed consistently higher levels of biological activity. While substitution at the 3-position of (+)-10 with electronegative atoms generally increased in vitro activity, a loss of potency relative to (+)-10 (MK-801) was observed in vivo for all of the compounds tested.