Cyclophilin J PPIase Inhibitors Derived from 2,3-Quinoxaline-6 Amine Exhibit Antitumor Activity

Abstract

Cyclophilin J (CyPJ), also called peptidylprolyl isomerase like 3, has been identified as a novel member of the cyclophilin family. Our previous research has resolved the three-dimensional structure of CyPJ and demonstrated the peptidylprolyl cis-trans isomerase (PPIase) activity of CyPJ, which can be inhibited by the common immunosuppressive drug cyclosporine A (CsA). Importantly, CyPJ is upregulated in hepatocellular carcinoma (HCC) and promotes tumor growth; CyPJ inhibition by CsA- or siRNA-based knockdown results in a remarkable suppression of HCC. These findings suggest that CyPJ may be a potential therapeutic target for HCC, and discovery of relevant inhibitors may facilitate development of a novel CyPJ-based targeting therapy. However, apart from the common inhibitor CsA, CyPJ has yet to be investigated as a target for cancer therapy. Here, we report structure-based identification of novel small molecule non-peptidic CyPJ inhibitors and their potential as antitumor lead compounds. Based on computer-aided virtual screening, in silico, and subsequently surface plasmon resonance analysis, 19 potential inhibitors of CyPJ were identified and selected for further evaluation of PPIase CyPJ inhibition in vitro. Thirteen out of 19 compounds exhibited notable inhibition against PPIase activity. Among them, the compound ZX-J-19, with a quinoxaline nucleus, showed potential for tumor inhibition; thus, we selected it for further structure-activity optimization. A total of 22 chemical derivatives with 2,3-substituted quinoxaline-6-amine modifications were designed and successfully synthesized. At least 2 out of the 22 derivatives, such as ZX-J-19j and ZX-J-19l, demonstrated remarkable inhibition of tumor cell growth, comparable to CsA but much stronger than 5-fluorouracil. These results indicate that these two small molecules represent novel potential lead compounds for CyPJ-based antitumor drug development.

Keywords: CyPJ; PPIL3; PPIase; cyclophilin J; cyclophilin J inhibitor; hepatocellular carcinoma; peptidylprolyl isomerase; quinoxaline derivative.

FIGURE 1

Flow chart of virtual screening and identification of CyPJ inhibitors. SPECS and CNPD are chemical libraries containing 200,000 and 50,000 compounds, respectively. SPR, surface plasmon resonance. PPIase, peptidylprolyl cis–trans isomerase.

FIGURE 2

Chemical structures of 19 potential CyPJ inhibitors identified with in silico screening and SPR analysis. Following in silico screening and SPR analysis, 19 ZX-J compounds numbered 1–19 were identified and selected for further investigation. The numbers in the compound ZX-J-19 represent the positions of the carbons in the quinoxaline ring. CsA, cyclosporine A, is a well-established cyclophilin protein inhibitor.

FIGURE 3

The synthesis route of 2,3-substituted-quinoxaline-6-amine derivatives. (A) The synthetic production of ethanediones (including intermediate chemicals 1a, 1b, 1c). Reagents and conditions: (a) Vitamin B1 (VB1), EtOH/H₂O 2:1, NaOH, room temperature, 12 h; (b) Cu(NO₃)₂, HOAc/H₂O 1:1, 70°C, 6 h; (c) carbon disulfide, –70°C, pyridine, 4 h. (B) The synthetic process of 2,3-substituted-quinoxaline-6-amine derivatives (i.e., ZX-J-19 derivatives). Compound 1 was referred to as 1a, 1b, or 1c obtained from (A). Reagents and conditions: (a) HOAc, reflux, 8 h; (b) Na₂Sx, x = 2–6, EtOH, reflux, 12 h; (c) RCOCl, pyridine, acetone, reflux, 4 h. The numbers in the ZX-J-19 derivatives represent the position of the carbons in the quinoxaline ring. R₁ refers to the side residuals at the positions 2 and 3 of the quinoxalines. R₂ refers to the side residual linked to the amide group at the position 6 of the quinoxalines.