Novel Triazolopyridine-Based BRD4 Inhibitors as Potent HIV-1 Latency Reversing Agents

Abstract

Bromodomain-containing protein 4 (BRD4) inhibitors have been proven to be a promising option for anti-HIV-1 latency therapeutics. We herein describe the design, synthesis, and anti-HIV-1 latency bioevaluation of triazolopyridine derivatives as BRD4 inhibitors. Among them, compound 13d displayed favorable HIV-1 reactivation and prominent safety profile without triggering abnormal immune activation. It exerted strong synergism when combined with the PKC activator prostratin and has the same BRD4-targeting latency mechanism as observed with JQ1, by stimulating Tat-dependent HIV-1 elongation. Besides, it neither affected the antiviral efficacies of antiviral drugs nor caused secondary infections to uninfected cells and the latency reversing potency of 13d, in turn, was not affected by different classes of antiviral drugs.

Figure 1

Design pipeline of triazolopyridine-based derivatives.

Scheme 1. Synthetic Protocol for Triazolopyridine Derivatives 12a–g and 13a–u

Reagents and conditions: (a) triethyl orthoacetate, 130°C, 40 min; (b) 2-fluoro-5-nitrophenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane/H₂O, 95 °C, 12 h; (c) substituted phenol, Cs₂CO₃, DMSO, 100 °C, 2 h; (d) Fe, NH₄Cl, THF/EtOH/H₂O, 100 °C; or 10% Pd/C, H₂, 12 h; (e) R⁴COCl, pyridine, CH₂Cl₂, rt, 24 h; (f) R⁴SO₂Cl, pyridine, CH₂Cl₂, rt, 24 h.

Scheme 2. Synthetic Route of Compounds 22–24

Reagents and conditions: (a) 2,4-difluorophenol, Cs₂CO₃, DMSO, 100 °C, 24 h; (b) Fe, NH4Cl, THF/EtOH/H2O, 100 °C, 2 h; (c) bis(pinacolato)diboron, Na₂CO₃, Pd(dppf)Cl2, 1,4-dioxane, 100°C, 2 h; (d) N₂H₄·H₂O, EtOH, 80 °C, 1 h; (e) triethyl orthoacetate, 130 °C, 1 h; (f) Na₂CO₃, Pd(dppf)Cl2, 1,4-dioxane, 100 °C, 6 h; (g) R₄SO₂Cl, pyridine, CH₂Cl₂, r.t, 24 h; (h) isopropyl chloroformate, pyridine, CH₂C_l2, rt, 24 h.

Figure 2

Neither 13d nor JQ1 affected T-cell immune activation state and HIV-1 coreceptors. (A) PBMCs were treated with 13d (10 μM) or JQ1 (1 μM) for 48 h, cells were collected, and the expression of three cell surface receptors CD25, CD69, and HLA-DR in CD4⁺ T cells (up) or CD8⁺ T cells (down) was detected by the flow cytometry method. (B, C) PBMCs were treated with 13d (10 μM) or JQ1 (1 μM) for 48 h, cells were collected, and the expression HIV-1 coreceptors CXCR4 and CCR5 on the cell surface in PBMC cells was detected by flow cytometry (*p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001). NC: negative control.

Figure 3

Effect of 13d on activation and recruitment of BRD4 and P-TEFb. (A) J-Lat cells were treated with different concentrations of 13d (2, 10, and 50 μM) for 24 h, cells were collected, the total protein was extracted, and the expression of BRD4 and phosphorylated BRD4 was checked. (B) Statistical analysis of the expression of protein in (A). (C) J-Lat cells were treated with different concentrations of 13d (2, 10, and 50 μM) for 24 h, cells were collected and the total protein was extracted, and the expression of Cyclin T1, CDK9, and phosphorylated CDK9 were measured by Western blot assay. (D) Statistical analysis of the expression of protein in (B). (E–G) J-Lat cells were treated with 13d (10 μM) or JQ1 (1 μM) for 6 h, 1% formaldehyde was added for cross-linking of DNA–protein, the relative enrichment ratios of BRD4 (E), CDK9 (F) and RNA pol II (G) in HIV-1 LTR were measured by ChIP assay (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). NC: negative control.

Figure 4

Overall performance of 13d combined with other LRAs or anti-HIV drugs. J-Lat cells were treated with 13d or combined with SAHA, prostratin, and JQ1 for 48 h, cells were collected, and (A) the percentage of GFP positive J-Lat cells was measured by flow cytometry. (B) Then the CI values were calculated using Chou’s median-effect equation and summarized. (C) J-Lat cells were treated with 13d (10 μM) alone or combined with EFV, RA,L and DRV for 48 h; cells were then collected and the percentage of GFP positive J-Lat cells was measured by flow cytometry. (D) HIV-1_IIIB acute infected C8166 cells were treated with EFV (1 nM), RAL (50 nM), and DRV (25 nM) alone or combined with 13d (10 μM), respectively, for 72 h; cell culture supernatant was collected and the expression of HIV-1 p24 antigen in the supernatant was detected by ELISA assay.