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  • As for antiviral activity eight out of


    As for antiviral activity, eight out of 23 synthesized compounds were active against influenza B virus with EC50 values in the range of 0.19–39 μM. Four of the eight influenza B hits were active against influenza A and two were also active against RSV (4 and 6, EC50 = 0.40 and 1.8 μM, respectively). Compound 4 exhibited similar activity for influenza B and RSV (EC50 values of 0.29 and 0.40 μM, respectively; P-value >0.1; two-sided Student\'s t-test [24]); its anti-influenza potency was comparable to that of zanamivir (EC50 = 0.14 μM) and was 11-fold higher than that of ribavirin. For influenza B, 4 exhibited a favorable SI (ratio of CC50 to EC50) of 62. Even higher activity and, in particular, selectivity were noted for compound 6 (EC50 = 0.19 μM; SI: 268), which similarly to 4 was also active against RSV. This result indicates that the DHFR enzyme is equally important for supporting influenza replication in MDCK cells as it is in allowing RSV replication in HeLa cells. MDCK and HeLa are the prototypic cell lines for antiviral research on influenza and RSV, respectively, since they give robust virus replication and CPE. The previous set of cycloguanil-like derivatives bore at N (1) of dihydrotriazine core a variously substituted aromatic ring, where lipophilic electron-withdrawing groups in position 3 properly conditioned the antiviral activity, as demonstrated by the sub-micromolar range potency of 11a-16a (EC50 = 0.015–0.10 μM) against influenza B virus [21]. Analyzing the role of the substituent at N (1) position of the new dihydrotriazines, a phenyl ring (2–6, 8, 9) provided again activity against influenza B, and in some cases, influenza A virus, whereas a 3-chlorobenzyl moiety or n-propyl or methoxyethyl chain abolished the activity. The only exception was derivative 20, which displayed an EC50 of 3.0 μM against influenza B virus; this molecule combined a methoxyethyl substitution at N (1) with an ethylsulfonic group, such as decoration of spiropiperidine nitrogen. Basing on our positive results for the previous series of cycloguanil analogs, the 3-Cl or 3-OCH3 substituents were included on the phenyl ring at N (1) position of dihydrotriazine (Fig. 2). The data confirmed that a 3-Cl NMS-E973 (2–6) enhances the antiviral potency for influenza B, whereas the 3-OCH3 group is less effective (8, 9). In place of the two methyl groups of the most potent cycloguanil-like derivatives (11a-16a), the new dihydrotriazines bore the spiropiperidine ring, whose nitrogen atom was differently functionalized. Regarding the influence of the R1 variation at the piperidine nitrogen, carbamic (2, 8), amide (3–5 and 9) and ureido (6) functions were permitted, while the lack of a substituent abolished the activity (1 and 7). Among the active compounds bearing an amide function (3–5), the 4-tolyl ring (4) provided the highest potency for influenza B (EC50 = 0.29 μM), 26- and 14-fold better than the analogs 5 and 3, which carry a naphthyl and cyclobutyl moiety, respectively. It is worth noting that the antiviral activity was mainly determined by the presence of the phenyl ring at N (1) rather than the substituent on the piperidine nitrogen. For four 1-phenyl substituted dihydrotriazines (i.e. 2, 3, 5 and 8), the favorable activity against influenza B was confirmed for influenza A virus. It was remarkable that 4 and 6, the two best compounds for influenza B, were found inactive against influenza A virus. These 1-(3-chlorophenyl) derivatives (4 and 6) compare favorably for potency and safety profile (EC50 = 0.29 and 0.19 μM; SI = 62 and 268, respectively) with the 3-Cl- and 3,4-diCl-phenyl substituted cycloguanil analogs 11a and 16a, which showed EC50 values against influenza B virus equal to 0.10 and 0.080 μM, and SI of 14 and 84, respectively [21]; however, the 3-Br (13a) and 3-CF3 (14a) cycloguanil-like derivatives remain unsurpassed, providing better antiviral activity and selectivity indexes (EC50∼ 0.016 μM; SI = 1812 and 407, respectively). On the other hand, 4 and 6 were the only two compounds in the entire series with activity against RSV, for which a superior SI (ratio of MCC to EC50) was noted (250 and 56 for 4 and 6, respectively). In terms of anti-RSV potency, these two molecules were 15-fold (4) and 3-fold (6) superior to the licensed drug ribavirin (EC50 = 5.8 μM), a non-specific anti-RSV drug [25], whose administration can be burdened by toxicity. Nevertheless, 4 and 6 result significantly less effective anti-RSV agents than cycloguanil-like dihydrotriazines (11a-16a), which exhibited EC50 values in the range 0.0075–0.080 μM, and markedly superior SI values between 1250 and 13333 [21]. None of the compounds investigated here displayed activity against DNA viruses (i.e., herpes simplex virus, adenovirus or vaccinia virus), enveloped RNA viruses (i.e., HIV, coronavirus, parainfluenza-3 virus, vesicular stomatitis virus, sindbis virus, yellow fever virus or Punta Toro virus) or non-enveloped RNA viruses (i.e., Coxsackievirus B4 and reovirus-1) (data not shown).