X-ray crystallography.

<p>Data collection and refinement statistics.</p><p>¹Values in parentheses refer to highest-resolution shell.</p><p>X-ray crystallography.</p

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

<div><p>Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC₅₀ of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.</p></div

Synthesis of P1’-P2’ fragments.

<p>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.</p

Synthesis of 3 substituted dihydroquinolinone 21.

<p>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.</p

Nomenclature for FXIa substrates and corresponding binding sites.

<p>(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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113705#pone.0113705.ref082" target="_blank">82</a>]). 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.</p

Synthesis of 3-substituted quinolinone 23.

<p>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.</p

Structures with associated FXIa potencies and selected physicochemical and ADME parameters.

<p>^<i>a</i>logD measured by liquid chromatography. ^<i>b</i>Caco2 A to B permeability measured at pH 6.5. ^<i>c</i>Efflux ratio determined in Caco2 cells by dividing P_app B to A with P_app A to B permeabilities measured at pH 7.4 at side A. ^<i>d</i>Rat intrinsic clearance determined from measurements in rat hepatocytes. ^<i>e</i>Rat <i>in vivo</i> clearance. ^fCaco2 A to B permeability measured at pH 7.4 at side A.</p

Crystal structures of compounds 9, 12 and 13 in complex with FXIa.

<p>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.</p

Structures with associated selectivity data.

<p>^<i>a</i><i>K</i>_i values are shown in parenthesis. ^<i>b</i>% inhibition of the compounds at 99 μM are given if the IC₅₀ was above 99 μM.</p

Crystal structure of compound 9 [31] in complex with FXIa.

<p>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.</p