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  • In this context we initiated a program to develop


    In this context, we initiated a program to develop new antiviral chemotypes towards a host factor implicated in Moxidectin replication: Moxidectin the host (human) dihydrofolate reductase (DHFR) [21]. We recently identified this enzyme as the molecular target of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil (1a, Fig. 1) [21]. This class of host DHFR inhibitors possessed intriguing dual activity against influenza A and B viruses and RSV. For the most promising derivatives (11a, 13a, 14a and 16a: range EC50 = 0.015–0.10 μM), the potency against influenza B was comparable to that of zanamivir (antiviral EC50 = 0.060 μM) [21], a neuraminidase inhibitor that is globally advised for influenza therapy [22]. These DHFR inhibitors also exhibited nanomolar activity (mean EC50 ∼0.008 μM) against RSV with a SI (ratio of cytotoxic to antiviral concentration) above 10,000 for compounds 11a, 14a and 16a, far surpassing the potency and safety profile of the anti-RSV drug ribavirin (EC50 = 5.8 μM, SI > 43). This report details our investigations to probe the antiviral activity of two novel azaspiro-2,4-diamino-1,6-dihydrotriazine scaffolds (compounds 1–21 and 22, 23; see Fig. 1) against both Orthomyxovirus (influenza A and B) and Paramyxovirus (RSV) representatives. These new dihydrotriazines were designed in order to better explore the chemical space around these scaffolds that are well tolerated for targeting the host (human) DHFR enzyme, with a view to evaluate its effect on virus inhibition. The new compounds confirmed the inhibitory profile against influenza viruses, especially type B. In order to verify the host-factor DHFR as the molecular target also for the azaspiro dihydrotriazines, the most promising derivatives were assayed against the recombinant hDHFR. These investigations were accompanied by molecular modeling studies of the title compounds in complex with the hDHFR, which predicted the most important features underlying protein/ligand binding. These information suggested the key molecular interactions required to achieve pharmacological activity and helped to rationalize the observed SAR of dihydrotriazine-containing derivatives acting as host (human) DHFR inhibitors.
    Results and discussion
    Biological evaluation 3.1. Inhibition of influenza virus. The antiviral activity of compounds 1–23 was evaluated in cellular assays with influenza A/PR/8/34 (A/H1N1) and B/Ned/537/05 virus. The antiviral activity data obtained by microscopic inspection of the viral CPE (data not shown) were in agreement with those obtained by the colorimetric MTS cell viability test (Table 1). The EC50 values were similar whether based on the MTS or microscopic method, i.e. for compounds 4 and 6 the EC50 values were 0.29 and 0.19 μM, based on the MTS method, versus 0.42 and 0.35 μM, for the microscopic method. In parallel, the cytotoxicity of the compounds was determined by microscopy and MTS assay, yielding the MCC and CC50 values, respectively (Table 1). In addition, the target compounds were tested against a diverse panel of other viruses, including RSV (Table 1) which was highly susceptible to the previously reported series of cycloguanil-like dihydrotriazines [21] depicted in Fig. 1. Although the test compounds were directed towards a host cell enzyme, they produced no or marginal cytotoxicity at 100 μM in human cervix carcinoma HeLa or african green monkey Vero (results not shown) cell cultures, which were not highly proliferating due to addition of only 2% fetal calf serum (FCS) in the infection medium. Somewhat higher cytotoxicity was observed in the MDCK cells, which were actively dividing in the influenza assays due to the use of Ultra-MDCK® medium. This specialized medium enables to work under FCS-free conditions when adding trypsin to achieve multicycle influenza replication [23]. For instance, for the two most potent antiviral compounds 4 and 6, the MCC was 0.8 μM (4) and 0.16 μM (6). Yet, their 50% cytotoxic concentration values (i.e. CC50 assessed by MTS cell viability assay), were 18 and 51 μM for 4 and 6, respectively, which is 22- and 319-fold higher than the corresponding MCC values.