Improved Synthesis of Effective 3-(Indolin-6-yl)-4-(N-pyrazole-sulfonamide)-1 H-pyrrolo[2,3- b]pyridine-Based Inhibitors of NADPH Oxidase 2

Abstract

NADPH oxidase enzymes (NOXs) are a family of enzymes generating superoxide, which form reactive oxygen species. NOX2 activity is a causative agent for the progression of many diseases: neurodegenerative, cardiovascular, immune dysregulations, and even hereditary diseases and cancer. Administering antioxidants helps in inhibiting NOX2 activity; however, the development of selective inhibitors may provide greater improvement in the therapy of diseases. Here, an optimized synthesis of two most promising NOX2 inhibitors based on the 3-(indolin-6-yl)-4-(N-pyrazole-sulfonamide)-1H-pyrrolo [2,3-b]pyridine structure, namely, GSK2795039 and NCATS-SM7270, and an isomeric derivative of the same class, IMBIOC-1, is reported. The new modified procedures simplify the isolation, reduce byproduct formation, and improve the yields in 0.1-1 g scale preparations. Molecular modeling of the structures of NOX2 complexes with inhibitors validated their binding at the same site as NADPH, with IMBIOC-1 forming the largest number of intermolecular interactions with the NOX2 active site. Testing the effects of the compounds on amyloid beta-induced oxidative stress and toxicity in HMC3 microglial cells showed that all three inhibitors completely prevented the pathological amyloid-beta effect. At the same time, NCATS-SM7270 and IMBIOC-1 provided a stronger protective effect on microglial cell survival than GSK2795039, which allowed us to assert the potential of those compounds as neuroprotective agents.

Keywords: Alzheimer’s disease; GSK2795039; NOX2 inhibitor; microglial cells; reactive oxygen species.

Figure 1

Examples of the inhibitors of NADPH oxidase 2 (NOX2) based on 3-(indolin-6-yl)-4-(N-pyrazole-sulfonamide)-1H-pyrrolo[2,3-b]pyridine skeleton: GSK2795039 [12,18], NCATS-SM7270 [3], and a newly found inhibitor isomeric to GSK2795039.

Figure 2

General retrosynthetic analysis for synthesis of the GSK2795039-type NOX2 inhibitors (top) and the proposed pathway of synthesis (below).

Scheme 1

Synthesis of (Bpin)-indoline and (Bpin)-indole derivatives (blocks A).

Scheme 2

Synthesis of dihalogenoindole-based building blocks C.

Scheme 3

Synthesis of pyrazole-based building blocks E.

Scheme 4

The Suzuki cross-coupling between blocks A and B.

Scheme 5

Final Buchwald–Hartwig cross-coupling for synthesis of all three NOX2 inhibitors.

Figure 3

Binding sites of the inhibitors of GSK2795039, NCATS-SM7270, IMBIOC-1, and NADPH with NOX2: (A) The docking site of NADPH (gray area) and inhibitors GSK2795039 (green), NCATS-SM7270 (blue), and IMBIOC-1 (magenta). (B) Amino acid residues of the NOX2 interacting with GSK2795039 inhibitor. (C) Amino acid residues of NOX2 interacting with the NCATS-SM7270 inhibitor. (D) Amino acid residues of NOX2 interacting with the IMBIOC-1 inhibitor.

Figure 4

Effect of GSK2795039 (commercial), GSK2795039* (synthesized), NCATS-SM7270, and IMBIOC-1 on cell death and ROS level in HMC3 microglial cells in the absence and presence of Aβ: (A) MTT analysis of viability of HMC3 cells treated with NOX2 inhibitors for 24 h. (B) Flow cytometry analysis of dead cells in HMC3 population after 24 h incubation with 10 µM of Aβ and 25 µM inhibitors. (C) Flow cytometry analysis of ROS level in HMC3 cells treated with 10 µM of Aβ and 25 µM inhibitors for 24 h. (D) Determination of IC50 values for NOX2 inhibitors in HMC3 cells treated with 10 µM of Aβ for 24 h. Data are presented as mean ± SD of at least three independent experiments with triplicate measurements. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.