Diamino benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors. Part 6: further focus on the contracted C4'-side chain analogues.

Novel benzo[b]thiophene diamine thrombin inhibitors were investigated, focusing on a contracted C4'-side chain series. SAR studies identified compounds with either a pyrrolidino or morpholino group as potent, active site directed thrombin inhibitors when the amino group was connected to the C3-phenyl ring with a methylene linker at the C4' position of the phenyl ring.

Diamino benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors. 5. Potency, efficacy, and pharmacokinetic properties of modified C-3 side chain derivatives.

A systematic investigation of the structure-activity relationships of the C-3 side chain of the screening hit 1a led to the identification of the potent thrombin inhibitors 23c, 28c, and 31c. Their activities (1240, 903, and 1271 x 10(6) L/mol, respectively) represent 2200- and 2900-fold increases in potency over the starting lead 1a. This activity enhancement was accomplished with an increase of thrombin selectivity. The in vitro anticoagulant profiles of derivatives 28c and 31c were determined, and they compare favorably with the clinical agent H-R-1-[4aS, 8aS]perhydroisoquinolyl-prolyl-arginyl aldehyde (D-Piq-Pro-Arg-H; 32). The more potent members of this series have been studied in an arterial/venous shunt (AV shunt) model of thrombosis and were found to be efficacious in reducing clot formation. However, their efficacy is currently limited by their rapid and extensive distribution following administration.

Synthesis and in vitro biological activity of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol: a 24,25-dehydro analog of the hypocholesterolemic agent 4alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol.

4Alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol (LY295427) was previously identified from a Chinese hamster ovary (CHO) cell-based low density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. To investigate the effect of the 24,25-unsaturation in the D-ring side chain (desmosterol D-ring side chain) on antagonizing the repressing effect of 25-hydroxycholesterol, 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol (17), a 24,25-dehydro analog of LY295427, was thus synthesized from lithocholic acid via the formation of 3alpha-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4alpha- (2-propenyl)-5alpha-cholan-24-al (15). Test results showed that 17 had an EC30 value of 2.6 microM, comparable to 2.9 microM of LY295427, in the CHO cell-based LDLR/Luc assay in the presence of 25-hydroxycholesterol. Apparently, the built-in 24,25-unsaturation in the D-ring side chain of 17 had added little effect to antagonizing the repressing effect of 25-hydroxycholesterol. In the [1-14C-acetate]cholesterol biosynthesis inhibition assay, 17 at 10 microg/ml (23 microM) has been shown to inhibit the cholesterol biosynthesis in CHO cells by 38% relative to the vehicle control; whereas LY295427 showed no inhibition in the same assay in our previous studies. In contrast to LY295427, the built-in 24,25-unsaturation in the D-ring side chain of 17 has conferred an inhibitory effect on cholesterol biosynthesis in CHO cells. In summary, the observed LDL receptor promoter activity of 17 is related to its ability to prevent 25-hydroxycholesterol from exerting the repressing effect via an undetermined mechanism and, in part, to inhibit the cholesterol biosynthesis.

Dibasic benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors: 2. Sidechain optimization and demonstration of in vivo efficacy.

Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.

Synthesis of 4 alpha-(2-propenyl)-5,6-secocholestan-3 alpha-ol, a novel B-ring seco analog of the hypocholesterolemic agent 4 alpha-(2-propenyl)-5 alpha-cholestan-3 alpha-ol.

4 alpha-(2-Propenyl)-5 alpha-cholestan-3 alpha-ol (LY295427) was previously identified from a CHO cell-based assay to be a potent LDL receptor up-regulator and had demonstrated to be an effective agent in lowering plasma cholesterol levels in hypercholesterolemic hamsters. In order to investigate the effect of flexibility of the 3 alpha-hydroxy-bearing A-ring on the activity, 4 alpha-(2-propenyl)-5,6-secocholestan-3 alpha-ol (11), a B-ring seco analog of LY295427, is thus synthesized from cholest-4-en-3-one. Test results indicate that 11 is not active in the CHO cell-based LDL receptor/luciferase assay at concentrations up to 20 micrograms/mL. The result underlines the importance of maintaining the A-B-C-D ring rigidity of the 3 alpha-sterols in terms of binding to the putative oxysterol receptor.