Chemical formulae of the FX-2212a inhibitor (2S)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid and the DX9065a (2S)-{4-[1-acetimidoyl-(3S)-pyrrolidinyl]oxyphenyl}-3-(7-amidino-2-naphthyl)propionic acid. Schematic drawing of the interactions between two inhibitors, DX9065a and FX-2212a, and factor Xa. Hydrogen bonds are shown as thin dashed lines, and hydrophobic interactions are shown as thick dashed lines. In the case of Q192, the aliphatic chain portion of Q192 makes the hydrophobic interaction. The symbol “⊥” indicates that the two interacting aromatic groups are not stacked but are perpendicular to each other.

The scheme of the synthesis of FX-2212. N-tert-Butoxycarbonyl-N-3-(tert-butyldimethylsilyloxy)propyl-4-pyridylamine 3: 2 was prepared from 3-bromo-1-propanol and tert-butyldimethylsilyl chloride. To a solution of 4-tert-butoxycarbonylaminopyridine (400 mg, 2.1 mmol) in N,N′-dimethylformamide (DMF) (4 ml) was added 60% NaH (83 mg, 2.1 mmol) and 2 (1.0 g, 4.2 mmol) in DMF (6 ml), and the mixture was stirred for 12 h. After work up and purification, 375 mg of 3 was obtained. N-tert-Butoxycarbonyl-N-3-hydroxypropyl-4-pyridylamine 4: To a solution of 3 (375 mg, 1.0 mmol) in tetrahydrofuran (THF) (10 ml) was added acetic acid (175 μl, 3.1 mmol) and 1 M tetrabutylammonium fluoride in THF (3.1 ml, 3.1 mmol) at room temperature, and the mixture was stirred for 3 h. After work up and purification, 213 mg of 4 was obtained. N-tert-Butoxycarbonyl-N-3-iodopropyl-4-pyridylamine 5: 4 (212 mg, 0.84 mmol) in CH₂Cl₂ (6 ml) was treated with triphenylphosphine (550 mg, 2.1 mmol), iodine (426 mg, 1.7 mmol), and imidazole (143 mg, 2.1 mmol) at 0°C for 0.5 h. After work up and purification, 272 mg of 5 was obtained. Ethyl 5-(N-tert-butoxycarbonyl-N-4-pyridylamino)-2-(3′-cyano-3-biphenylyl)pentanoate 6: After ethyl 3′-cyanophenyl-3-phenylacetate (295 mg, 1.1 mmol) in THF (3 ml) was added dropwise to a mixture of 1 M lithium bis(trimethylsilyl) amide in THF (1.16 ml, 1.2 mmol) and hexamethylphosphoramide (750 μl, 4.3 mmol) in THF (3 ml) and stirred for 0.5 h at −78°C, 5 in THF (4 ml) was added to the mixture, and the reaction mixture was allowed to reach room temperature. After work up and purification, 153 mg of 6 was obtained. Ethyl 2-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoate dihydrochloride 7: After treating 6 (40 mg, 0.08 mmol) in ethanol (1 ml) with 4 M HCl–dioxane (10 ml) at room temperature for 2 days, the mixture was concentrated. The residue was dissolved in ethanol (10 ml) and bubbled with NH₃ gas until saturation at 0°C. After stirring at room temperature for 3 days, the reaction mixture was concentrated in vacuo. The residue was purified to give 25 mg of 7. FX-2212, 2-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid dihydrochloride 8: 7 (20 mg, 0.04 mmol) was dissolved in 2 M HCl (4 ml), and the mixture was refluxed for 2 h. After concentration and purification, 15 mg of FX-2212 was obtained as white solids. The identity of the compound was checked by NMR and MS.

Ribbon drawing of the Des[1–44] factor Xa-FX-2212a complex structure (only one molecule in the form 1 crystal is shown). The light chain consists of the first (yellow) and the second EGF domains (orange). The trypsin-like catalytic domain is shown in blue. FX-2212a (red ball and stick) is bound to the active site. One calcium ion (pink) each is bound to the first EGF domain and the catalytic domain. The binding region for the effector protease receptor-1 is shown in magenta.

The extended flexible structures of the effector protease receptor-1 binding site in three crystallographically independent molecules: two molecules (green and red) in the form 1 crystal and one molecule (blue) in the form 2 crystal. The second EGF domains were superimposed.

(a) Stereo view of the electron density for FX-2212a in difference electron density maps (contoured at 1.6σ) calculated after modeling the first EGF domain and the simulated annealing refinement. The final structure is superimposed. (b) Binding interactions of FX-2212a (magenta ball and stick) with Des[1–44] factor Xa in the form 1 crystal. The Cα backbone is shown in blue, and residues involved in interaction are shown as a yellow ball-and-stick model. Conserved hydrogen bonds in the three crystallographically independent molecules are shown in green and a unique hydrogen bond in this interaction is shown in orange.