The protonation states of residues in the allosteric site were adjusted to the dominant ionic forms at pH 7.4. potent NS5B inhibitors. NS5B RNA dependent RNA polymerase (RdRp) inhibition assay was carried out as described in Methods section.27,36 The RdRp reaction was performed on poly rA/U12 template-primer (TP), employing recombinant HCV NS5B (genotype 1b) with an N-terminal His-tag and C-terminal 21-amino acid deletion (NS5BC21).27,36 LQB34, a coumestan derivative, previously characterized by us as a NS5B inhibitor was included as an internal reference standard.27 To identify candidates belonging to a wider range of structural scaffolds, preliminary screening was conducted at a concentration of 250 M for each compound. This analysis yielded four compounds exhibiting 50% inhibition of NS5B RdRp activity (Table 1), thus demonstrating a 17% hit rate. Of these, compounds 3 (rhodanine analog) and 4 (imidazocoumarin analog) exhibited IC50 values of 55.2 M and 60.2 M, respectively. Further exploration of SAR around imidazocoumarin analog 4 resulted in either inactive or poorly active analogs (Supplementary data, compounds 39-43, Table S1). Thus we pursued compound Benzophenonetetracarboxylic acid 3 for further SAR investigations. Table 1 Anti-NS5B RdRp activity of compounds 1-4 to the position (compound 5, IC50 = 50.9 M) led to a marginal enhancement of inhibitory activity. Bioisosteric replacement of the CCF3 group in compound 5 with CBr group (compound 6, IC50 = 20.2 M) resulted in a Rabbit Polyclonal to ME1 2.5-fold increase in inhibitory activity. Further improvement in inhibitory activity was obtained when 3-CF3 benzylidene was replaced with 2,4-dichlorobenzylidene moiety (compound Benzophenonetetracarboxylic acid 7, IC50 = 17.9 M). We purchased compounds 8-11, to explore the effect of 3-position substituents on rhodanine ring in the presence of 2,4-dichlorobenzylidene substituent at 5-position of the rhodanine ring. Separating Ccarboxyl group from the rhodanine core by ethylene bridge resulted in a marginal decrease in activity (compound 8, IC50 = 19.9 M). Further decrease in activity was observed when carboxyl group in compound 8 was replaced with bioisosteric sulfonic acid group (compound 9, IC50 = 23.5 M). Separation of carboxyl group from the rhodanine core by a propylene linker resulted in substantial loss of activity (compound 10, IC50 = 58.0 M), in contrast the activity improved by 3.6-fold when the pentylene linker was used (compound 11, IC50 = 16.1 M). Several additional analogs such as: 5-(2,4-dichlorobenzylidene)-3-ethyl-2-thioxothiazolidin-4-one, 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid, and 2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic acid lacking (a) a carboxyl group at N3 substituent, (b) benzylidene moiety at C5-position, and (c) substituents on benzylidene moiety, respectively, were found to be either inactive or poorly active (Supplementary data, compounds 29-34, Table S1). In addition, analogs with electron donating substituents around the benzylidene moiety (Supplementary data, compounds 35 and 36, Table S1) exhibited poor activity, whereas electron withdrawing 3-cyano and 3-carboxy substituents for the benzylidene moiety (Supplementary data, substances 37 and 38, Desk S1) exhibited higher NS5B inhibitory activity. The 1-naphthylidene substituent at 5-placement from the rhodanine band was harmful to the NS5B inhibitory activity once the N3-substituent was either an acetic acidity (substance 12, IC50 = 61.4 M) or -methyl acetic acidity (substance 13, IC50 = 68.1 M), as opposed to a bulkier -isopropyl acetic acidity substituent (chemical substance 14, IC50 = 16.9 M) that was beneficial. We following acquired substances 15-17, which much like substance 14 transported the -isopropyl acetic acidity substituent at 3-placement from the rhodanine band, but harbored some Benzophenonetetracarboxylic acid substituted benzylidenes instead of 1-naphthylidene moiety. While 4-fluorobenzylidene (substance 15, IC50 = 42.9 M) and 2-chlorobenzylidene (chemical substance 16, IC50 = 36.1 M) analogs became approximately 2-3 fold much less active in comparison to chemical substance 14, similar inhibitory activity was seen in the current presence of 2,4-dichlorobenzylidene substituent (chemical substance 17, IC50 = 14.1 M). Predicated on these results, we procured substances 18-22 bearing 2,4-dichlorobenzylidene substituent at 5-placement and a different substituents at 3-placement from the rhodanine band. While -propylacetic acidity (substance 18, IC50 = 28.2 M) and -methylthioethyl acetic acidity (chemical substance 19, IC50 = 29.2 M) substituents led to two-fold reduction in inhibitory activity, the bulkier -isobutyl acetic acidity analog (chemical substance 20, IC50 = 18.9.