W.-H. Song et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4528–4531
4529
O
OH
O
O
H
N
OH
OH
COOH
O
N
O
HO
N
N
O
N
COOH
O
F
3
4
5 (S-1360)
Cl
O
O
O
O
H
N
H
N
N
N
N
N
HN
N
O
F
6 (5-ClTEP)
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.
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.
>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
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
50 lM, which indicated that the substituents and sort of the aryl
cell counting kit-8 (CCK8) as previously reported.12
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
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
(500
zole derivative 30 was the most potent molecule with an EC50 va-
lue of 3.9 M. Additionally, compound 30 did not show clear
cytotoxicity at 500 M. Therefore, we further clarified the inhibi-
lM). The results as shown in Table 2 suggested that the tria-
l
O
O
O
"one-pot" synthesis
H
N
l
H
N
EtO
Ar
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-
X
X
N
N
N
N
X = N 10-22
7
X = C 23-33
a
c
OMe
OMe
X
b
O
O
O
N
N
EtO
Ar
X
N
N
N
N
9
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.
ity with an EC50 of 3.9
128. Moreover, to confirm the antiviral activity of compound 30,
lM and a selectivity index greater than
Song, Wu-Hui
