1,2-Dibenzamidobenzene Inhibitors of Human Factor Xa

Abstract

High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound 4, fXa apparent K(ass) = 0.64 x 10(6) L/mol). SAR studies in this novel structural series of fXa inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tert-butyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced slightly higher levels of activity than other substituents when present in combination with favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds, having fXa apparent K(ass) = 25.5 x 10(6) L/mol, about 40x more active than the original lead. This lead series does not show potent inhibition of human thrombin. A model for the binding of these ligands to the fXa active site is proposed. The model is consistent with the observed SAR and can serve to guide future SAR studies.

A new paradigm for pattern recognition of drugs

“…The analysis of fXa inhibitors (Table 4) [43] with the ConGO algorithm shows significant superimposition of the most active molecules due to closely similar electron structures (Fig. 12a).…”

Developments in factor Xa inhibitors for the treatment of thromboembolic disorders

“…33). 198 K ass represents the apparent association constant and is approximately equal to 1/K i . 199 During follow-on SAR studies, it was discovered that the 4-hydroxy substitution was not essential to the antifactor Xa activity and that a wide range of substituents were also tolerated at the 4-position, as shown in 180-182.…”

The Minimum Significant Ratio: A Statistical Parameter to Characterize the Reproducibility of Potency Estimates from Concentration-Response Assays and Estimation by Replicate-Experiment Studies

“…The apparent K ass values were obtained in a high-volume testing protocol [13][14][15] using automated dilutions of inhibitors (performed in triplicate at 4-8 inhibitor concentrations) into 96-well plates and chromogenic substrate hydrolysis rates determined at 405 nm using a Thermomax plate reader from Molecular Devices (San Francisco, CA). The assay protocol was 50 µl buffer (0.06 M tris, 0.3 M NaCl, pH 7.4), 25 µl inhibitor test solution (in MeOH), 25 µl human fXa (32 nM in 0.03 M tris, 0.15 M NaCl, 1 mg/ml HSA), and, finally, 150 µl substrate (0.3 mM in water) added within 2 min to start hydrolysis.…”