Fig 1.
Nomenclature for FXIa substrates and corresponding binding sites.
(A) FIX sequences that are substrates for FXIa. The scissile bonds cleaved by FXIa are marked with a red dashed line. Residues N- and C-terminal of the scissile bond are referred to as P1, P2 etc. and P1’, P2’ etc., respectively. (B) Depiction of FXIa active site in complex with FIXa substrate residues (from PDB entry 1XXD [82]). According to standard nomenclature, the substrate P1 residue binds the enzyme S1 site, the P1’ residue binds the S1’ site, and so on. The scissile bond is marked with a red dashed line.
Fig 2.
Structures with associated FXIa potencies.
aKi values are shown in parenthesis. bPrecipitates at higher concentration than 111 μM. cKD value from Biacore binding experiment.
Fig 3.
Ligand detected one dimensional NMR spectroscopy.
Part of 1H T1rho NMR spectra of p-methyl-benzamidine (pMeBza): (A) 100 μM pMeBza in buffer; (B) after addition of 5 μM FXIa CD; and (C) after addition of 10 μM compound 2. Shown is the doublet from the protons in the ortho position. The extra signals in C are from the inhibitor.
Fig 4.
Crystal structure of compound 1 in complex with FXIa.
The surface exposed point for linker attachment used to develop compound 4 is highlighted. The KD of the compound 4 was determined to 390 nM. Compound 4 was linked to the sensor chip via the primary amine.
Fig 5.
Crystal structures of compounds 5 (A) and 6 (B) in complex with FXIa.
Hydrogen bonds are depicted as dashed lines and the refined 2FoFc electron density maps are contoured at 1σ. Compound 6 is depicted in the charged form to satisfy the hydrogen bond to Gly218.
Fig 6.
Structures with associated selectivity data.
aKi values are shown in parenthesis. b% inhibition of the compounds at 99 μM are given if the IC50 was above 99 μM.
Fig 7.
Crystal structure of compound 9 [31] in complex with FXIa.
Compound 9 is displayed in thick sticks with green carbons, selected binding site residues in thin sticks with grey carbons, water oxygens in red spheres, and hydrogen bond interactions with dashed lines. The arrows indicate: (1) the central water molecule, (2) the hydrogen acceptor of the P1-P1’ amide and (3) the hydrogen donor of the P1’-P2’ amide.
Fig 8.
Crystal structures of compounds 9, 12 and 13 in complex with FXIa.
Compounds 9 (green), 12 (magenta) and compound 13 (yellow) are overlaid. The protein surface from the FXIa CD:compound 9 complex is shown as grey surface and the central water molecule that interacts with both amides is shown as sphere.
Fig 9.
Illustration of potency SAR for initial expansion/linking.
Fig 10.
Synthesis of intermediate 13C and routes to compounds 13, 14, 25, 26 and 28.
i) Methyl acetoacetat, DMAP, Pyridine, xylenes, reflux, 8h, ii) AcOH, Br2, r.t, 3h, iii) Neat H2SO4, 45°C, 46h, iv) Hexamine, DCM, reflux, 5h, then conc. HCl, reflux, 2h, v) Diethylmalonate, THF, NaH, reflux, 1h, vi) 20% HCl, reflux, 12h, vii) TBTU, TEA, DMF, 20B, r.t, 16h, viii) 15B, T3P, 13E, TEA, 120°C, 20 min., ix) HOBt, EDAC, 13E, BOC-L-phenylalanine, TEA, DMF, r.t, 16h, x) TFA, DCM, r.t, 16h, xi) AcOH, TBTU, 14B, pyridine, DMF, r.t, 16h, xii) 2M MeNH2, TEA, THF, 50°C, 1h, xiii) (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid, HOBt, EDAC, DIPEA, DMF, r.t, 16h, xiv) 4M HCl in dioxane, r.t, 2h, xv) 15A, HOBt, EDAC, DIPEA, DMF, xvi) (S)-2-(tert-butoxycarbonyl(methyl)amino)-3-phenylpropanoic acid, HOBt, EDAC, DIPEA, DMF, r.t, 16h, xvii) 4M HCl in dioxane, r.t, 3h, xviii) 15A, TBTU, EDAC, DIPEA, DMSO, r.t, 48h, xix) 4-methyl-3-phenyl-1H-pyrazole-5-carboxylic acid, HOBt, EDAC, DIPEA, DMSO, r.t, 16h.
Fig 11.
Synthesis of P1’-P2’ fragments.
i) DCM, r.t, 16h, then LiOH, water, THF, r.t, 16h, then PPA, 120°C, 2h, ii) TBTU, DIPEA, DMF, L-phenylalanine methylester, r.t, 16h, iii) TBTU, pyridine, MeNH2xHCl, DMF, r.t, 16h, iv) TBTU, (S)-2-amino-N,N-dimethyl-3-phenylpropanamide hydrochloride, TEA, DMF, r.t, 16h, v) TBTU, TEA, DCM, DMF, r.t, 16h, vi) neat TFA, r.t, 0.5h.
Fig 12.
Synthesis of the dihydroquinolinone derivatives 18 and 20.
i) DCM, TEA, r.t, 2h, ii) Methyl 2-(triphenylphosphoranylidene)acetate, toluene, reflux, 2h, iii) NaOMe, MeOH, r.t, 1h, iv) DMSO, water, NaCl, 150°C, 16h, v) 4M NaOH, r.t, 4h, vi) TBTU, TEA, 20B, THF, r.t, 16h, vii) DPPA, TEA, DMF, then 2-(trimethylsilyl)ethanol, 100°C, 10 min., viii) TBAF, CH3CN, 60°C, 16h, ix) 15B, TBTU, pyridine, DMF, r.t, 16h.
Fig 13.
Synthesis of 3 substituted dihydroquinolinone 21.
i) SnBu3H, DMSO, 100°C, 16h, ii) 4-Methoxybenzyl chloride, NaH, DMF, r.t, 2h, iii) LDA, tert-butyl 2-bromoacetate, THF, N2, -78°C, iv) Neat TFA, 80°C, 2h, v) 20B, TBTU, TEA, DMF, r.t, 16h.
Fig 14.
Synthesis of the 3-substituted chloroquinolinone 22.
i) Pd(OAc)2, PPh3, NaOAc, dry DMF, N2, 110°C, 16h, ii) 4M NaOH, r.t, 1h, iii) 20B, TBTU, TEA, THF, r.t, 12h.
Fig 15.
Synthesis of 3-substituted quinolinone 23.
i) Piperidine, EtOH, reflux, 6h, ii) DIBAL-H, Et2O, N2, r.t, iii) Neat SOCl2, reflux, 6h, iv) DEM, NaH, THF, N2, reflux, 2h, v) Conc. HCl, reflux, 16h, vi) 20B, TBTU, DIPEA, DMF, r.t, 16h.
Fig 16.
Synthesis of aminoquinoline 8.
i) (E)-3-Ethoxyacryloyl chloride, THF, 50°C, 16h, ii) Conc. HCl, 40°C, 1h, iii) Neat POCl3, 80°C, 3h, iv) Xantphos, Pd2(dba)3, tBuOK, PhMe, 100°C, 1h, then 2M HCl and THF, r.t, 2h, v) AcCl, TEA, DCM, N2, 0°C, 2h, then excess MeNH2 r.t, 16h, vi) NBS, benzoyl peroxide, (trifluoromethyl)-benzene, 80°C, 16h, then reflux 2h, vii) NaN3, DMF, 80°C, 1h, viii) Pd(OH)2 on carbon, EtOH, H2, r.t, 16h, ix) 20% NaOH, 120°C, 1h, x) DMF, DIPEA, 15B, TBTU r.t, 16h.
Fig 17.
Structures with associated FXIa potencies and selected physicochemical and ADME parameters.
alogD measured by liquid chromatography. bCaco2 A to B permeability measured at pH 6.5. cEfflux ratio determined in Caco2 cells by dividing Papp B to A with Papp A to B permeabilities measured at pH 7.4 at side A. dRat intrinsic clearance determined from measurements in rat hepatocytes. eRat in vivo clearance. fCaco2 A to B permeability measured at pH 7.4 at side A.
Table 1.
X-ray crystallography.