Discovery and Evaluation of Anti-Fibrinolytic Plasmin Inhibitors Derived from 5‑(4-Piperidyl)isoxazol-3-ol (4-PIOL)

Inhibition of plasmin has been found to effectively reduce fibrinolysis and to avoid hemorrhage. This can be achieved by addressing its kringle 1 domain with the known drug and lysine analogue tranexamic acid. Guided by shape similarities toward a previously discovered lead compound, 5-(4-piperidyl)­isoxazol-3-ol, a set of 16 structurally similar compounds was assembled and investigated. Successfully, <i>in vitro</i> measurements revealed one compound, 5-(4-piperidyl)­isothiazol-3-ol, superior in potency compared to the initial lead. Furthermore, a strikingly high correlation (R² = 0.93) between anti-fibrinolytic activity and kringle 1 binding affinity provided strong support for the hypothesized inhibition mechanism, as well as revealing opportunities to fine-tune biological effects through minor structural modifications. Several different ligand-based (Freeform, shape, and electrostatic-based similarities) and structure-based methods (e.g., Posit, MM/GBSA, FEP+) were used to retrospectively predict the binding affinities. A combined method, molecular alignment using Posit and scoring with <i>T</i>_combo, lead to the highest coefficient of determination (R² = 0.6)

Discovery and Evaluation of Anti-Fibrinolytic Plasmin Inhibitors Derived from 5‑(4-Piperidyl)isoxazol-3-ol (4-PIOL)

Inhibition of plasmin has been found to effectively reduce fibrinolysis and to avoid hemorrhage. This can be achieved by addressing its kringle 1 domain with the known drug and lysine analogue tranexamic acid. Guided by shape similarities toward a previously discovered lead compound, 5-(4-piperidyl)­isoxazol-3-ol, a set of 16 structurally similar compounds was assembled and investigated. Successfully, <i>in vitro</i> measurements revealed one compound, 5-(4-piperidyl)­isothiazol-3-ol, superior in potency compared to the initial lead. Furthermore, a strikingly high correlation (R² = 0.93) between anti-fibrinolytic activity and kringle 1 binding affinity provided strong support for the hypothesized inhibition mechanism, as well as revealing opportunities to fine-tune biological effects through minor structural modifications. Several different ligand-based (Freeform, shape, and electrostatic-based similarities) and structure-based methods (e.g., Posit, MM/GBSA, FEP+) were used to retrospectively predict the binding affinities. A combined method, molecular alignment using Posit and scoring with <i>T</i>_combo, lead to the highest coefficient of determination (R² = 0.6)

Potent Fibrinolysis Inhibitor Discovered by Shape and Electrostatic Complementarity to the Drug Tranexamic Acid

Protein–protein interfaces provide an important class of drug targets currently receiving increased attention. The typical design strategy to inhibit protein–protein interactions usually involves large molecules such as peptides and macrocycles. One exception is tranexamic acid (TXA), which, as a lysine mimetic, inhibits binding of plasminogen to fibrin. However, the daily dose of TXA is high due to its modest potency and pharmacokinetic properties. In this study, we report a computational approach, where the focus was on finding electrostatic potential similarities to TXA. Coupling this computational technique with a high-quality low-throughput screen identified 5-(4-piperidyl)-3-isoxazolol (4-PIOL) as a potent plasminogen binding inhibitor with the potential for the treatment of various bleeding disorders. Remarkably, 4-PIOL was found to be more than four times as potent as the drug TXA

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)­alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)­alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not

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

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

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

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