Design, synthesis and structure-activity relationships of 4-phenyl-1H-1,2,3-triazole phenylalanine derivatives as novel HIV-1 capsid inhibitors with promising antiviral activities

Abstract

HIV-1 CA is involved in different stages of the viral replication cycle, performing essential roles in both early (uncoating, reverse transcription, nuclear import, integration) and late events (assembly). Recent efforts have demonstrated HIV-1 CA protein as a prospective therapeutic target for the development of new antivirals. The most extensively studied CA inhibitor, PF-3450074 (PF-74, discovered by Pfizer), that targets an inter-protomer pocket within the CA hexamer. Herein we reported the design, synthesis, and biological evaluation of a series of 4-phenyl-1H-1,2,3-triazole phenylalanine derivatives as HIV-1 CA inhibitors based on PF-74 scaffold. Most of the analogues demonstrated potent antiviral activities, among them, the anti-HIV-1 activity of 6a-9 (EC₅₀ = 3.13 μM) is particularly prominent. The SPR binding assay of selected compounds (6a-9, 6a-10, 5b) suggested direct and effective interaction with recombinant CA proteins. The mechanism of action studies also demonstrated that 6a-9 displays the effects in both the early and late stages of HIV-1 replication. To explore the potential binding mode of the here presented analogues, 6a-9 was analyzed by MD simulation to predict its binding to the active site of HIV-1 CA monomer. In conclusion, this novel series of antivirals can serve as a starting point for the development of a new generation of HIV-1 treatment regimen and highlights the potentiality of CA as a therapeutic target.

Keywords: 1,2,3-Triazole; CA protein; HIV-1; MD simulation; Phenylalanine derivatives; SPR assay.

Fig.1

The co-crystallized structure of PF-74 complexed with CA hexamer (A, PF-74 in green, PDB ID: 5HGL) and monomer (B, PF-74 in yellow, PDB ID: 2XDE), respectively. H-bond interactions are indicated by red dashed lines. Hydrogens (nonpolar) are not shown. Figures were prepared with the PyMOL visualization program (http://www.pymol.org). (C) Chemical structure of PF-74 and overlapped PF-74 conformations from Fig.1 A (PF-74 in green) and B (PF-74 in yellow). (D) The design of novel 4-phenyl-1H-1,2,3-triazole phenylalanine derivatives as HIV-1 CA inhibitors.

Fig. 2

SPR isotherms of target compounds 6a-9 (B), 6a-10 (C), 5b (D) and PF-74 (A) binding to CA proteins (monomer left, hexamer right), respectively.

Fig. 2

SPR isotherms of target compounds 6a-9 (B), 6a-10 (C), 5b (D) and PF-74 (A) binding to CA proteins (monomer left, hexamer right), respectively.

Fig.3

The effect of compound 6a-9 on viral production. Standard deviation represents 2 replicates performed in duplicate. Performed with NL_4-3 Env pseudo-typed HIV-I virus at 10 μM compound concentration.

Fig.4

The effect of 6a-9 on the NL_4-3 capsid assembly in vitro at 3M NaCl. (A) Capsid assembly was monitored by an increase in turbidity using a spectrophotometer at 350 nm over 19 minutes. Capsid was used at a final concentration of 30 μM, and compounds 6a-9 and PF-74 at a final concentration of 50 μM. (B) Slope/velocity quantification of capsid assembly during the first 2 minutes. Experiments were performed in triplicate. (AU) Absorption unit.

Fig.5

(A) RMSD (heavy atoms) of amino acids of CA HIV-1 monomer in reference to the first frame of the MD simulation. (B) RMSD (heavy atoms) of the bound 6a-9 in reference to the docked conformer.

Fig.6

Binding interactions of 6a-9 in the first (A) and second (C) clusters. Expanded views of the representative structures of the first (B) and second (D) clusters.

Scheme 1.

The synthetic route of target compounds. Reagents and Conditions: (i) 4-methoxy-N-methylaniline, PyBop, DIEA, CH₂Cl₂, 0 °C to r.t.; (ii) CF₃COOH, CH₂Cl₂, r.t.; (iii) N₃CH₂COOH, HATU, DIEA, CH₃CN, 0 ° C to r.t.; (iv) corresponding substituted aminophenylacetylene, L-ascorbic acid sodium salt, CuSO₄‧5H₂O. THF/H₂O (v:v=1:1), r.t.; (v) corresponding acyl chloride, TEA, CH₂Cl₂, 0 ° C to r.t..