The synthesis and bioactivities of ROCK2 inhibitors with 1,2-dithiolan-3-yl motif

Abstract

Rho-associated coiled-coil containing kinase (ROCK) plays an important role in inflammation. Herein, a series of compounds were designed and synthesized as ROCK inhibitors based on the structure-based drug design (SBDD) strategy and were evaluated for cytotoxicity, antioxidant activity and anti-inflammatory activity. Among them, compound DC24 was identified as the optimal hit in enzymatic screening with an IC₅₀ value of 0.124 μM against ROCK2 and 50-fold selectivity over ROCK1. DC24 has a novel lipid amide scaffold with a bis(4-fluorophenyl)methyl substituent, and DC24 is the first ROCK2 inhibitor interacting with the hinge region of ROCK2 via the 1,2-dithiolan-3-yl motif, which has been confirmed by the binding model of DC24 with ROCK2. In a complete Freund's adjuvant (CFA) induced acute inflammation model, DC24 at a dose of 5 mg kg^-1 exhibited an anti-inflammatory effect better than that of belumosudil. Furthermore, DC24 exhibits good safety in vivo.

Fig. 1. ROCK inhibitors launched on the market.

Fig. 2. A) 3D diagram of fasudil (pink) and co-crystal ligand Y-27632 (yellow) docking in ROCK1 (PDB: 2ETR) B) 3D diagram of fasudil (pink) and co-crystal ligand (purple) docking in ROCK2 (PDB: 6ED6).

Fig. 3. The discovery of DC24 with a lipoic amide scaffold.

Fig. 4. Docking results. A) 3D diagram of DC03 (conformation A (green), conformation B (orange), fasudil (pink) and co-crystal ligand (purple)) docking in ROCK2 (PDB: 6ED6). B) 2D diagram of the co-crystal ligand binding with ROCK2 (PDB: 6ED6). C) 2D diagram of DC03 (conformation A) binding with ROCK2 (PDB: 6ED6). D) 2D diagram of DC03 (conformation B) binding with ROCK2 (PDB: 6ED6).

Fig. 5. Docking results. A) 3D diagram of DC03 (conformation B, orange), DC24 (gray), and co-crystal ligand (purple) docking in ROCK2 (PDB: 6ED6). B) 2D diagram of DC24 binding with ROCK2 (PDB: 6ED6).

Scheme 1. Synthetic route to fasudil.

Scheme 2. Synthetic route of DC01–DC03.

Scheme 3. Synthetic route of DC04–DC15.

Scheme 4. Synthetic route of DC16–DC18.

Scheme 5. Synthetic routes of DC19–DC22.

Scheme 6. Synthetic routes of DC23–DC26.

Fig. 6. Possible metabolic pathways of compound DC01.

Fig. 7. Long-term toxicity and safety of DC01 and DC24. (A) Body weight. (B) Organ index.

Fig. 8. Evaluation of compound DC24 for the treatment of CFA-induced acute arthritis in a mouse model **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.001 compounds versus CFA.