Author's personal copy W.-H. Song et aL/ Bioorg Med. Chem. Lett. 23(2013)4528-4531 O COOH N COOH 6 (5-CITEP) Figure 2. Structures of aryl diketo acid mimics such as monoethyl ester of meconic acid (3) dihydroxypyrimidine carboxylic acid (4), S-1360(5). 5-CITEP(6)and our most active compound 30. inhibitors as anti-HIV agents leading to the discovery of S-1360 (5. >50%)at 50 HM. The brief structure activities relationships (SArs Fig. 2)and 5-CITEP (6. Fig. 2), 6 related homologues of DKAs have were summarized as follows. The substitute of benzyloxy in the not been tested for anti-HCV activity to our knowledge. aryl ring is essential for the biological activity. The introduction As shown in scheme 1, the designed compounds tetrazole deriv- of chloride atom at the opposite position of benzyloxy can increase atives 10-22 and triazole derivatives 23-33 were synthesized via a the anti-HCV activities, when the benzyloxy was at 4-position of facile'one-pot' reaction as previously reported. 17,18 Reaction of the phenyl ring. in both tetrazole and triazole derivatives. but the ef- tarting material, 1H-tetrazol-5-ethyl formate(7), with 2-meth- fect of the fluoride atom on the Hcv activities was more compl xyproene in the presence p-TSA and followed by Claisen conden- cated. For the tetrazole derivatives, the introduce fluoride atom ition with various substituted aryl methyl ketone catalyzed by can increase the activities, when the benzyloxy was at 3-positie sodium ethoxide afford diketo tetrazole and triazole intermediate of phenyl ring. In comparison, the fluoride atom was more appro- 9 which was then deprotected by 4N hydrochloric acid to obtain priate when the benzyloxy was at 3-position of phenyl ring in the target compounds 10-33 triazole series. Moreover, HIV integrase inhibitors 5 and 6 were All synthesized diketo triazole and tetrazole derivatives were also synthesized according to the reference and their anti-HCV initially evaluated for their anti-HCV activities and cytotoxicity at activities evaluated. Unfortunately, both compounds possess the single concentration of 50 HM in an authentic HCV infection/ much weaker anti-HCV activities with less than 50% inhibition eplication system in the human hepatoma cell lines Huh-7, using 50 HM, which indicated that the substituents and sort of the aryl cell counting kit-8( CCK8)as previously reported. group played a critical role for the antiviral activities, even though The preliminary results of antiviral effect and cytotoxicity effect all the compounds contained aryl diketo tetrazole or aryl diketo are shown in Table 1, respectively. RG7128 was used as positive triazole groups control, which is a nucleoside clinical candidate in phase 2b. Ate Further, three of the nine active compounds( 14, 30, and 33) I of nine compounds exhibited more than 50% inhibition and all were selected to determine the ecso values of their anti-HCV activ- unds showed low cytotoxic activity(cell viability ratio ities and to test their cytotoxicity in higher concentration (500 HM). The results as shown in Table 2 suggested that the tria zole derivative 30 was the most potent molecule with an ECso va- lue of 3.9 HM. Additionally, compound 30 did not show clear "oneo -pot" synthesis cytotoxicity at 500 HM. Therefore, we further clarified the inhibi tory effect of compound 30 on the synthesis of viral protein and the replication of viral genome. Cell lysates were subjected to wes- tern blot analysis with the antibody of viral non-structural protein 7 X=N1022 X=C23-3 NS5A, in which the level of tubulin served as a loading control. As shown in Fig 3, the synthesis of HCV NS5A proteins was inhibited by compound 30 in a dose-dependent manner. Moreover, quantita tive rT-PCR was also employed to examine the RNa level of HCv genome, which was normalized by cellular GAPDH mRNA. A OMe dose-dependent reduction of HCv RNa levels by compound 30 was also observed(Fig. 4), which confirmed compound 30 as a promising lead with anti-HCV activity. In conclusion, we designed and synthesized dike nd triazole derivatives to take advantage of known bioisosteres of carboxylic acids, starting from diketo acid, known active site inhibitors of NS5B. Among the synthesized compounds, 4-(4-fluo- robenzyloxy )phenyl diketo triazole(30)exhibited anti-HCV activ Scheme 1. Reagents and conditions: (a)2-methoxypropene, p-TSA, THF, rt, 1 h:(b) ity with an ECso of 3.9 HM and a selectivity index greater than aryl methyl ketone, NaoEt, 60C, 3 h: (C)4NHCI(aq)l rt too"C, h: 62-89]overalL. 128. Moreover, to confirm the antiviral activity of compound 30Author's personal copy inhibitors as anti-HIV agents leading to the discovery of S-1360 (5, Fig. 2) and 5-CITEP (6, Fig. 2),16 related homologues of DKAs have not been tested for anti-HCV activity to our knowledge. As shown in scheme 1, the designed compounds tetrazole deriv￾atives 10–22 and triazole derivatives 23–33 were synthesized via a facile ‘one-pot’ reaction as previously reported.17,18 Reaction of the starting material, 1H-tetrazol-5-ethyl formate (7), with 2-meth￾oxyproene in the presence p-TSA and followed by Claisen conden￾sation with various substituted aryl methyl ketone catalyzed by sodium ethoxide afford diketo tetrazole and triazole intermediate 9 which was then deprotected by 4N hydrochloric acid to obtain target compounds 10–33. All synthesized diketo triazole and tetrazole derivatives were initially evaluated for their anti-HCV activities and cytotoxicity at the single concentration of 50 lM in an authentic HCV infection/ replication system in the human hepatoma cell lines Huh-7, using cell counting kit-8 (CCK8) as previously reported.12 The preliminary results of antiviral effect and cytotoxicity effect are shown in Table 1, respectively. RG7128 was used as positive control, which is a nucleoside clinical candidate in phase 2b.19 A to￾tal of nine compounds exhibited more than 50% inhibition and all of the compounds showed low cytotoxic activity (cell viability ratio >50%) at 50 lM. The brief structure activities relationships (SARs) were summarized as follows. The substitute of benzyloxy in the aryl ring is essential for the biological activity. The introduction of chloride atom at the opposite position of benzyloxy can increase the anti-HCV activities, when the benzyloxy was at 4-position of phenyl ring, in both tetrazole and triazole derivatives. But the ef￾fect of the fluoride atom on the HCV activities was more compli￾cated. For the tetrazole derivatives, the introduce fluoride atom can increase the activities, when the benzyloxy was at 3-position of phenyl ring. In comparison, the fluoride atom was more appro￾priate when the benzyloxy was at 3-position of phenyl ring in the triazole series. Moreover, HIV integrase inhibitors 5 and 6 were also synthesized according to the reference and their anti-HCV activities were evaluated. Unfortunately, both compounds possess much weaker anti-HCV activities with less than 50% inhibition at 50 lM, which indicated that the substituents and sort of the aryl group played a critical role for the antiviral activities, even though all the compounds contained aryl diketo tetrazole or aryl diketo triazole groups. Further, three of the nine active compounds (14, 30, and 33) were selected to determine the EC50 values of their anti-HCV activ￾ities and to test their cytotoxicity in higher concentration (500 lM). The results as shown in Table 2 suggested that the tria￾zole derivative 30 was the most potent molecule with an EC50 va￾lue of 3.9 lM. Additionally, compound 30 did not show clear cytotoxicity at 500 lM. Therefore, we further clarified the inhibi￾tory effect of compound 30 on the synthesis of viral protein and the replication of viral genome. Cell lysates were subjected to wes￾tern blot analysis with the antibody of viral non-structural protein NS5A, in which the level of tubulin served as a loading control. As shown in Fig. 3, the synthesis of HCV NS5A proteins was inhibited by compound 30 in a dose-dependent manner. Moreover, quantita￾tive RT-PCR was also employed to examine the RNA level of HCV genome, which was normalized by cellular GAPDH mRNA. A dose-dependent reduction of HCV RNA levels by compound 30 was also observed (Fig. 4), which confirmed compound 30 as a promising lead with anti-HCV activity. In conclusion, we designed and synthesized diketo tetrazole and triazole derivatives to take advantage of known bioisosteres of carboxylic acids, starting from diketo acid, known active site inhibitors of NS5B. Among the synthesized compounds, 4-(4-fluo￾robenzyloxy)phenyl diketo triazole (30) exhibited anti-HCV activ￾ity with an EC50 of 3.9 lM and a selectivity index greater than 128. Moreover, to confirm the antiviral activity of compound 30, O O N N H N O F O O COOH OH O O 3 N N OH OH COOH HO 4 O O N N H O N F 5 (S-1360) O O N N N H N HN 6 (5-ClTEP) Cl 30 Figure 2. Structures of aryl diketo acid mimics such as monoethyl ester of meconic acid (3), dihydroxypyrimidine carboxylic acid (4), S-1360 (5), 5-CITEP (6) and our most active compound 30. N N X H N O EtO N N X N O EtO OMe N N X N O OMe O Ar Ar O O N N X H N "one-pot" synthesis a b c 7 9 X = N 10-22 X = C 23-33 8 Scheme 1. Reagents and conditions: (a) 2-methoxypropene, p-TSA, THF, rt, 1 h; (b) aryl methyl ketone, NaOEt, 60 C, 3 h; (c) 4 N HCl(aq), rt to 0 C, 2 h; 62–89% overall. W.-H. Song et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4528–4531 4529