Design and Synthesis of Small Molecule RhoA Inhibitors: A New Promising Therapy for Cardiovascular Diseases?

Abstract

RhoA is a member of Rho GTPases, a subgroup of the Ras superfamily of small GTP-binding proteins. RhoA, as an important regulator of diverse cellular signaling pathways, plays significant roles in cytoskeletal organization, transcription, and cell-cycle progression. The RhoA/ROCK inhibitors have emerged as a new promising treatment for cardiovascular diseases. However, to date, RhoA inhibitors are macromolecules, and to our knowledge, small molecular-based inhibitors have not been reported. In this study, a series of first-in-class small molecular RhoA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. Virtual screening of ∼200,000 compounds, followed by SPR-based binding affinity assays resulted in three compounds with binding affinities to RhoA at the micromolar level (compounds 1-3). Compound 1 was selected for further structure modifications in considering binding activity and synthesis ease. Fourty-one new compounds (1, 12a-v, 13a-h, and 14a-j) were designed and synthesized accordingly. It was found that eight (12a, 12j, 14a, 14b, 14d, 14e, 14 g, and 14h) showed high RhoA inhibition activities with IC(50) values of 1.24 to 3.00 μM. A pharmacological assay indicated that two compounds (14g and 14 h) demonstrated noticeable vasorelaxation effects against PE-induced contraction in thoracic aorta artery rings and served as good leads for developing more potent cardiovascular agents.

Multi-gram Preparation of 7-Nitroquinoxalin-2-amine

“…Chemical shifts from 1 H and 13 C nuclear magnetic resonance (NMR) spectra of the obtained isomer are in accordance with values previously described for the 7-nitroquinoxalin-2-ol. 5 NOE (nuclear Overhauser effect) difference experiment also confirmed obtainment of regioisomer 3. By using phosphoryl chloride under refluxing conditions, we prepared the corresponding aryl chloride 2-chloro-7-nitroquinoxaline (4) in good yields by filtration after slowly addition of the reaction mixture into a mixture of ice and water.…”

“…The precipitate was filtrated and washed with water to obtain 13.43 g of a red/cherry solid. Procedure for the synthesis of N-(4-methoxybenzyl)-7-nitroquinoxalin-2-amine (5) 20 mL of triethylamine (3 eq.) and 6.8 mL of p-methoxybenzylamine (1.05 eq.)…”

Direct Modulation of Small GTPase Activity and Function

“…[101][102][103][104] Structure-based virtual screening identified several small molecules as potential RhoA inhibitors. [101] After binding was verified by SPR measurements,4 1a nalogues were synthesized and their biological activity evaluated. Tw ocompounds showed low micromolar inhibition of RhoA activity (IC 50 = 1.2-1.7 mm)and impaired phenylephrine (PE) induced thoracic aorta artery ring contraction.…”

Discovery of novel chemical scaffolds as RhoA inhibitors using virtual screening, synthesis, and bioactivity evaluation

“…Within the RhoA signaling module, two strategies of intervention have been proposed for effective suppression of RhoA activities. One strategy is targeting GTP binding sites to compete with GTP leading to RhoA inactivation, 14 while the other strategy is targeting the GEF-RhoA interactive surface essential for the guanine nucleotide exchange reaction also leading to RhoA inactivation. 15,16 To date, both strategies have led to effective small molecular compounds targeting RhoA (Fig.…”