Synthesis and characterization of 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide-based compounds targeting the PA-PB1 interface of influenza A virus polymerase

Abstract

Influenza viruses (Flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. Antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-Flu armamentarium still remains inadequate. In search for new anti-Flu drugs, our group has focused on viral RNA-dependent RNA polymerase (RdRP) developing disruptors of PA-PB1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. By merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. In particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important SAR insight and identify new active compounds showing both the ability to inhibit PA-PB1 interaction and viral replication in the micromolar range and at non-toxic concentrations. For few compounds, the ability to efficiently inhibit PA-PB1 subunits interaction did not translate into anti-Flu activity. Chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. Finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, H-bonds enhanced its binding within the PA_C cavity.

Keywords: Influenza virus; PA-PB1 heterodimerization; Protein-protein interaction; RNA-Dependent RNA polymerase.

Graphical abstract

Fig. 1

Structures and biological activities of compounds 1–4 previously reported [27,32].

Fig. 2

Structure of the compounds synthesized in this study. Compounds 5–18 are analogues of compound 3 and compounds 19–26 are analogues of compound 4.

Scheme 2

Synthetic route of the target compounds 5–8, 19 and 20. Reagents and conditions: (a) sulphur, N,N-diethylamine, EtOH, rt or sulphur, morpholine, EtOH, reflux; (b) compound 30 or 31, CH₂Cl₂, DIPEA, rt; (c) p-chloranil, 1,4-dioxane, 90 °C.

Scheme 3

Synthetic route of the target compound 10. Reagents and conditions: (a) DMF, POCl₃, from 0 °C to r. t.; (b) NMP, NH₂OH hydrochloride, 115 °C; (c) K₂CO₃, MeOH/THF (5:1), ethyl thioglycolate, reflux; (d) NH₂NH₂ hydrate, 80 °C; (e) Ni-Raney, DMF, 90 °C; (f) compound 30, CH₂Cl₂, DIPEA, rt.

Scheme 4

Synthetic route of the target compound 11. Reagents and conditions: (a) ethyl thioglycolate, DMF, KOH, from 0 °C to 80 °C; (b) NH₂NH₂ hydrate, 80 °C; (c) Ni-Raney, DMF, 90 °C; (d) compound 30, CH₂Cl₂, DIPEA, rt.

Scheme 5

Synthetic route of the target compounds 13 and 22. Reagents and conditions: (a) NH₂NH₂ hydrate, 80 °C; (b) Ni-Raney, DMF, 90 °C; (c) compound 30 or 31, CH₂Cl₂, DIPEA, rt.

Scheme 6

Synthetic route of the target compound 12 and 21. Reagents and conditions: (a) thiourea, EtOH, reflux; (b) 2-bromoacetamide, DIPEA, DMF, rt; (c) compound 30 or 31, CH₂Cl₂, DIPEA, rt.

Scheme 7

Synthetic route of the target compounds 15, 24, 18 and 26. Reagents and conditions: (a) sulphur, N,N-diethylamine, EtOH, rt; (b) compound 30 or 31, CH₂Cl₂, DIPEA, rt; (c) LiOH, H₂O/THF (1:1), 50 °C; (d) Ac₂O, 100 °C; (e) DIPEA, EDC, HOBt, CH₂Cl₂, from 0 °C to rt.

Scheme 8

Synthetic route of the target compounds 17 and 25. Reagents and conditions: (a) compound 30 or 31, CH₂Cl₂, DIPEA, rt.

Scheme 1

Synthetic route of the intermediates 30 and 31. Reagents and conditions: (a) glacial acetic acid, reflux; (b) NaOH, MeOH, reflux; (c) oxalyl chloride, CH₂Cl₂, DMF, room temperature (r.t.).

Fig. 3

Asymmetric unit of 14 and 23, showing a partial atom-numbering scheme. Displacement ellipsoids are drawn at 50% probability level.

Fig. 4

FLAP binding poses for compounds 23 (A,B) and 14 (C,D). Two orientations of the same pose in the PA cavity are illustrated to better visualize the predicted interactions. Compounds 14 and 23 are shown in sticks mode and in green color, while reference compounds 3 and 4 are shown in lines style and purple color. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)