Discovery and Preclinical Characterization of Fulacimstat (BAY 1142524), a Potent and Selective Chymase Inhibitor As a New Profibrinolytic Approach for Safe Thrombus Resolution

Abstract

Chymase is a serine-protease produced by mast cells. In the past few decades, its role in fibrotic diseases triggered the search for orally available chymase inhibitors. Aiming at reducing adverse cardiac remodeling after myocardial infarction, our research efforts resulted in the discovery of fulacimstat (BAY 1142524). While clinical trials did not demonstrate efficacy in this indication, the recent discovery of a new unexpected biological role of chymase spurred a revival of interest in chymase inhibition: chymase was shown to inactivate plasmin within fibrin-rich clots. Chymase inhibitors are now considered as potential profibrinolytic drugs with low bleeding risk and therefore exceptional safety for the treatment of acute thrombosis settings such as stroke, pulmonary embolism, or venous thrombosis. This article describes the chemical optimization journey from a screening hit to the discovery of fulacimstat (BAY 1142524), a selective chymase inhibitor with a good safety profile, as well as its preclinical in vitro and in vivo characterization.

Figure 1

Selected previously known nonpeptidic chymase inhibitors.

Figure 2

X-ray structure analysis of compound 1 bound to human chymase (resolution 2.4 Å; PDB ID 9GBH). The protease is shown in a stick representation (periwinkle-blue) with transparent Connolly-like surface; the ligand (gray) is shown as balls and sticks. Heteroatoms are colored as follows: oxygen, red; nitrogen, blue; and fluorine, cyan. Hydrogen bonds are depicted as dashed yellow lines. Hydrogen bond lengths are indicated in Å next to the bond. The 1,2,4-triazine-3,5-dione core interacts with the protein mainly via the carbonyl and carboxyl groups, while the N-1 nitrogen has no crucial binding interaction with the protein.

Scheme 1. Synthesis of Compound 12

Scheme 2. Synthesis of Compound 16

Figure 3

X-ray structure analysis of 8 bound to human chymase (resolution 2.0 Å; PDB ID 9GC1). (A) Overall binding mode from a similar viewpoint as in Figure 2. Compound 8 interacts with Gly216 on the rim of the S1 subsite and with Lys192, which points toward the solvent. Furthermore, compound 8 interacts through water bridges with Ser214 on the rim of the S1 site and with Asp175 in S4. (B) Detail side view with emphasis on S1 subpocket. The 2-methyl-3-trifluorobenzyl group of 8 follows the shape of the S1 pocket.

Figure 4

X-ray structure analysis of 27 bound to human chymase (resolution 2.2 Å; PDB ID 9GC9).

Figure 5

X-ray structure analysis of compound 47 bound to human chymase (resolution 1.8 Å; PDB ID 9GCC).

Scheme 3. Synthesis of Fulacimstat (BAY 1142524, 86)

Figure 6

X-ray structure analysis of fulacimstat (BAY 1142524, 86) bound to human chymase (resolution 1.8 Å; PDB ID 9GCD). The (R)-configurated chiral center of the indane-moiety can be recognized on the right-hand side, orienting the trifluoromethyl-phenyl part thereof into the S1-pocket (in the back).

Figure 7

Representative dose–response curves of fulacimstat (BAY 1142524, 86) in biochemical chymase activity assays. Chymase activity is measured as the fluorescence intensity of the cleaved peptidic substrate and is depicted as percentage of the activity observed in the absence of inhibitor.

Figure 8

Antifibrotic effects of fulacimstat (BAY 1142524, 86) in an animal model of cardiac fibrosis in hamsters. n = 9–15 per group. Data are presented as mean ± SEM; ** = P < 0.01; *** = P < 0.001 vs isoprenaline 20 mg/kg.

Figure 9

Activity of human plasmin in vitro measured by fluorescence of 7-aminomethylcoumarin (AMC) resulting from specific cleavage of d-Ala-Leu-Lys-(7-amino-4-methylcoumarin). (A) Kinetics of recombinant human chymase-induced inactivation of purified human plasmin in the absence or presence of increasing concentrations of fulacimstat. Each point represents the mean ± SEM (n = 6–15). One-way ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance with * = p < 0.05, ** = p < 0.01, *** = p < 0.001. (B) This graph depicts the maximal fluorescence obtained in each group at the end of the experiment (60 min).

Figure 10

Chymase inhibition reduces thrombus weight in the FeCl₃-induced injury model in the vena femoralis in a hamster. Data are presented as mean ± SEM; * = P < 0.05; *** = P < 0.001 vs vehicle.