Discovery of α-Amidobenzylboronates as Highly Potent Covalent Inhibitors of Plasma Kallikrein

Abstract

Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin swelling in subcutaneous and submucosal membranes in various locations of the body. Herein, we describe a series of potent α-amidobenzylboronates as potential covalent inhibitors of PKa. These compounds exhibited time-dependent inhibition of PKa (compound 20 IC₅₀ 66 nM at 1 min, 70 pM at 24 h). Further compound dissociation studies demonstrated that 20 showed no apparent reversibility comparable to d-Phe-Pro-Arg-chloromethylketone (PPACK) (23), a known nonselective covalent PKa inhibitor.

Figure 1

Schematic of (left) contact activation system (CAS) and (right) kallikrein–kinin system (KKS). Both initiated by factor XIIa, the cascade leading to bradykinin production and the positive feedback mechanism within the KKS are shown.

Figure 2

Chemical structures of berotralstat and sebetralstat.

Figure 3

Examples of literature boron-containing compounds under FDA approval or in clinical development.

Figure 4

Chemical structure of the only known nonselective boronic acid containing PKa inhibitor compound 10.

Figure 5

(A) Truncation of 11 to the structure of interest 12. (B) Docking of 12 into PKa active site (PDB 6O1S) with key residues shown in bold. (C) Overlay of docked structure of 11 (teal) with 12 (green) in PKa. Docking experiments were performed using OEDOCKING Hybrid docking.

Scheme 1. Synthesis of Compounds 15a–b

Reagents and conditions: a) n-BuLi, CH₂Cl₂, THF, −78 °C, 16 h; b) LiHMDS (1 M in THF) THF, −78 °C, 16 h; c) 4 N HCl in dioxane, Et₂O, −60 °C, 3 h. Yield: 40–45% (3 steps).

Scheme 2. Synthesis of Compound 18

Reagents and conditions: a) 16a, HATU, DIPEA, MeCN, 0 °C to rt, 3 h, 55–58%; b) 3 N HCl, C₅H₁₁B(OH)₂, MeOH/cyclohexane (1:1), rt, 16 h, 23%.

Scheme 3. Synthesis of Compounds 19–22

Reagents and conditions: a) carboxylic acids 16a–c HATU, DIPEA, MeCN, 0 °C to rt, 3 h; for 16b, see ref (36); for 16c, see ref (9).

Figure 6

Extended time course for 20 against plasma kallikrein. ΔF = change in fluorescence, and RFU = relative fluorescence units.

Scheme 4. Synthesis of Compounds 24–27

Reaction conditions: a) carboxylic acids 16b–c, HATU, DIPEA, MeCN, 0 °C to rt, 3 h.

Scheme 5. Synthesis of Compounds 28–32

Reagents and conditions: a) K₂CO₃, DMF, rt, 80%; b) 3-Cl benzaldehyde or 2-F, 4-Me benzaldehyde, NaOH, methanol, rt, 3 h, 70–81%; b) B₂Pin₂, DPEPhos (0.1 mol %), CuCl (0.03 mol %), KO^tBu, MeOH, THF, rt, 16 h, 34–56%.

Figure 7

LHS Dissociation curves of 19–20, covalent control 23, and compounds 24 and 26. RHS Dissociation curves of covalent control 23, compounds 31–32, and noncovalent control 33.

Scheme 6. Hydrolysis of 17b to 34 and 35 in pH7.4 Phosphate Buffer and Proposed Hydrolysis of 20 to 36 and 37

Figure 8

Docking of boronic acid 37 into the PKa active site (Ala-195 mutant). Docking results were subsequently visualized in PKa. Docking experiments were performed using OEDOCKING Hybrid with results visualized in PKa active site (PDB 6O1S) using the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.