Discovery of N-Arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide Derivatives as HCV NS5B Polymerase Inhibitors

Article abstract of DOI:10.1002/cmdc.201200058

The metal ion chelating β-N-hydroxy-γ-ketocarboxamide pharmacophore was integrated into a quinazolinone scaffold, leading to N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as hepatitisC virus (HCV) NS5B polymerase inhibitors. Lead optimization led to the identification of N-phenylpropyl carboxamide 9k (IC₅₀=8.8μM). Compound 9k possesses selectivity toward HCV1b replicon Ava.5 cells (EC₅₀=17.5μM) over parent Huh-7 cells (CC₅₀=187.5μM). Compound 9k effects a mixed mode of NS5B inhibition, with NTP-competitive displacement properties. The interaction between 9k and NS5B is stabilized by the presence of magnesium ions. Docking studies showed that the binding orientation of 9k occupies the central portions of both magnesium-mediated and NTP-ribose-response binding sites within the active site region of NS5B. As a result, 3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives are disclosed herein as novel, mainly active site inhibitors of HCV NS5B polymerase.

Full text of DOI:10.1002/cmdc.201200058

MED
DOI: 10.1002/cmdc.201200058
Discovery of N-Arylalkyl-3-hydroxy-4-oxo-3,4-
dihydroquinazolin-2-carboxamide Derivatives as HCV
NS5B Polymerase Inhibitors
Ravindra Ramesh Deore,[a] Grace Shiahuy Chen,[a, b] Pei-Teh Chang,[a] Ting-Rong Chern,[a, c]
Shin-Yu Lai,[a] Ming-Hsieh Chuang,[b] Jung-Hsin Lin,[a, c, d] Fan-Lu Kung,[a] Chien-Shu Chen,[a]
Chun-Tang Chiou,[e] and Ji-Wang Chern*[a, f]
The metal ion chelating b-N-hydroxy-g-ketocarboxamide phar-
macophore was integrated into a quinazolinone scaffold, lead-
ing to N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide derivatives as hepatitis C virus (HCV) NS5B poly-
merase inhibitors. Lead optimization led to the identification
of N-phenylpropyl carboxamide 9k (IC50 =8.8 mm). Compound
9k possesses selectivity toward HCV1b replicon Ava.5 cells
(EC50 =17.5 mm) over parent Huh-7 cells (CC50 =187.5 mm).
Compound 9k effects a mixed mode of NS5B inhibition, with
NTP-competitive displacement properties. The interaction be-
tween 9k and NS5B is stabilized by the presence of magnesi-
um ions. Docking studies showed that the binding orientation
of 9k occupies the central portions of both magnesium-medi-
ated and NTP-ribose-response binding sites within the active
site region of NS5B. As a result, 3-hydroxy-4-oxo-3,4-dihydro-
quinazolin-2-carboxamide derivatives are disclosed herein as
novel, mainly active site inhibitors of HCV NS5B polymerase.
Introduction
Hepatitis C virus (HCV) is the major causative agent of chronic
non-A, non-B hepatitis.[1] It is a hepatotropic single-stranded
RNA virus that belongs to the genus Hepacivirus, family Flavi-
viridae. Chronic HCV infection leads progressively to major liver
disorders such as cirrhosis, end-stage liver disease, and hepato-
cellular carcinoma.[2,3] Among six major genotypes that encom-
pass nine different subtypes, HCV genotype 1 causes the ma-
jority of HCV infections.[4–6] Neither a vaccine nor an effective
therapy against all HCV genotypes is available.[7] Current thera-
py involves a combination of ribavirin and PEGylated interferon
(IFN)-a, which can enhance sustained virological response
(SVR) rates to 54–56%.[8,9] However, this combination therapy
does not show lasting improvement in 40–50% of HCV pa-
tients, and other disadvantages such as high cost and unde-
sired side effects persist.[10]
Most non-nucleoside NS5B polymerase inhibitors have been
characterized as allosteric inhibitors and demonstrate potent
inhibitory activity in cell-based assays. Although allosteric in-
hibitors provide favorable pharmacokinetic profiles, the emer-
gence of resistant mutant viral strains limits their progress in
the treatment of patients with chronic HCV infections.[15] How-
ever, the active site of NS5B polymerase still represents a po-
tentially viable target. Active site residues Asp318, Asp319,
and Asp220 are important for NS5B catalytic activity.[16] The
active site forms complexes with two Mg2+ ions and coordi-
[a] Dr. R. R. Deore,+ Prof. G. S. Chen,+ Dr. P.-T. Chang,+ T.-R. Chern, Dr. S.-Y. Lai,
Prof. J.-H. Lin, Prof. F.-L. Kung, Prof. C.-S. Chen, Prof. J.-W. Chern
School of Pharmacy, National Taiwan University, Taipei 10051 (Taiwan ROC)
E-mail: jwchern@ntu.edu.tw
[b] Prof. G. S. Chen,+ M.-H. Chuang
The HCV NS5B polymerase is not expressed in host cells,
and it is therefore considered as a potential target for HCV
therapeutics. HCV NS5B polymerase is an RNA-dependent RNA
polymerase that catalyzes the synthesis of both negative-
strand copies of the incoming viral RNA and positive-strand
progeny RNA genomes.[11] NS5B polymerase has a tertiary
structure with three constitutive peptide domains known as
the palm, fingers, and thumb.[12] Two magnesium ions, coordi-
nated in the palm domain, facilitate the catalytic activity of
HCV NS5B. A number of HCV NS5B polymerase inhibitors with
structurally diverse scaffolds have been reported,[13] and inhibi-
tors such as ANA598, BMS791325, Filibuvir, GS9190, PSI7977,
RG7128, VX222, and VX759 have been advanced to phase II
clinical trials.[14]
Department of Applied Chemistry
Providence University, Taichung 43301 (Taiwan ROC)
[c] T.-R. Chern, Prof. J.-H. Lin
Division of Mechanics, Research Center for Applied Sciences
Academia Sinica, Taipei 11529 (Taiwan ROC)
[d] Prof. J.-H. Lin
Institute of Biomedical Sciences
Academia Sinica, Taipei 11529 (Taiwan ROC)
[e] Dr. C.-T. Chiou
Division of Herbal Drugs and Natural Products
National Research Institute of Chinese Medicine, Taipei 11221 (Taiwan ROC)
[f] Prof. J.-W. Chern
Department of Life Science
National Taiwan University, Taipei 10617 (Taiwan ROC)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200058.
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HCV NS5B Polymerase Inhibitors
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nates with the b- and g-phosphate groups of incoming ribonu-
cleotide triphosphate (NTP) substrates to activate the 3’-hy-
droxy group of the primer strand toward nucleophilic attack
by the a-phosphate.[17] Until now, only a few non-nucleoside
NS5B active site inhibitors have been reported. Most of these
are involved in divalent metal ion chelation and act as pyro-
phosphate mimics. HCV NS5B active site inhibitors such as a,g-
diketo acid (DKA) 1,[18] monoethyl ester of meconic acid 2,[19]
dihydroxypyrimidine carboxylic acid 3,[20] and isothiazole 4[21]
are divalent metal ion chelators (Figure 1). The HIV integrase
inhibitors hydroxypyrimidinone 5[22] (Raltegravir) and bicyclic
pyrimidinone 6[23] are also divalent metal ion chelators.
As part of our studies on anti-HCV agents,[24] 2-hydroxy-1-
oxo-4-phenyl-1,2-dihydroisoquinolin-3-carboxylic acid (7a) was
identified as an HCV NS5B inhibitor with moderate antiviral ac-
tivity (EC50 =15.7 mm) and good selectivity in HCV genotype 1b
replicon Ava.5 cells.[25] However, 4-unsubstituted 2-hydroxy-1-
oxo-1,2-dihydroisoquinolin-3-carboxylic acid (7b) is inactive
toward HCV NS5B polymerase. In view of bicyclic pyrimidinone
6[23] (an HIV integrase inhibitor) and N-hydroxypyridinone 8[26]
(an anthrax lethal factor inhibitor) as metal ion chelators, we
anticipated that integration of the b-N-hydroxy-g-ketocarboxa-
mide pharmacophore into a bicyclic system would result in co-
ordination with the magnesium ions. Thus, derivatization of
carboxylic acid 7b into N-benzyl carboxamide and scaffold
hopping of isoquinoline to quinazoline led to quinazolinone
derivatives 9 (Figure 2), which would serve as active site inhibi-
tors of NS5B polymerase. Herein we report the synthesis and
structure–activity relationship (SAR) of 3-hydroxy-4-oxo-3,4-di-
hydroquinazolin-2-carboxamide derivatives 9 as HCV NS5B
polymerase inhibitors.
Figure 1. Divalent metal ion chelators as enzyme inhibitors.
Figure 2. The design of b-N-hydroxy-g-ketocarboxamide inbuilt quinazoli-
nones 9 as pyrophosphate mimics for NS5B polymerase inhibitors.
Results and Discussion
Chemistry
Treatment of isatoic anhydride (10) with O-benzylhydroxya-
mine in the presence of triethylamine afforded 2-amino-N-ben-
zyloxybenzamide 11 (Scheme 1). Compound 11 reacted with
diethyl oxalate in the presence of catalytic amounts of para-
toluenesulfonic acid, leading to ethyl N-benzyloxy-4-oxo-3,4-di-
hydroquinazolin-2-carboxylate (12), which underwent catalytic
debenzylation over hydrogen in the presence of Pd/C to give
b-N-hydroxy-g-ketocarboxylate 13. The ethyl carboxylate 13
was then treated with various amines to afford target com-
pounds 9a–e and 9g–k. Alternatively, O-benzyl compound 12
could be derivatized to carboxamides 14a–d, which were then
debenzylated over hydrogen to give N-hydroxy derivatives 9.
In addition, nitro compound 9d was reduced to its amino ana-
logue 9 f by catalytic hydrogenation.
Biological evaluation
HCV NS5B polymerase inhibitory activity
Scheme 1. Reagents and conditions: a) PhCH2ONH2·HCl, Et3N, THF, reflux, 6 h;
b) diethyl oxalate, p-TSA (cat.), reflux, 18 h; c) H2, Pd/C (10%), EtOAc, RT,
30 min; d) arylalkylamines, EtOH, RT or reflux, 14–96 h; e) H2, Pd/C (10%),
EtOAc/MeOH (1:1), RT, 1 h; f) H2, Pd/C (10%), EtOH, RT, 2 h.
HCV NS5B polymerase activity was measured by the release of
inorganic pyrophosphate (PPi), a nonradioactive assay for RNA-
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dependent RNA polymerase ac-
tivity.[27] Carboxamides 9 were
evaluated for their inhibitory ac-
tivity toward HCV NS5B poly-
merase by PPi inhibition assay,
and the results are listed in
Table 1. The reference com-
pound DKA 1 showed moderate
inhibitory activity, with an IC50
value of 20 mm. Compound 9a
possesses NS5B inhibitory activi-
ty (IC50 =28 mm) similar to that
of DKA 1. The substituent effect
on the N-benzyl group was ex-
amined. Derivatives with N-(4-
fluorobenzyl) (9b), N-(4-chloro-
benzyl) (9c), and N-(4-nitroben-
zyl) (9d) groups lost inhibitory
activity, with IC50 values of 45 mm
or higher. N-(4-Methoxybenzyl)
derivative 9e showed a twofold
enhancement in NS5B inhibitory
activity (IC50 =14 mm) relative to
the parent compound 9a, and
N-(4-aminobenzyl) derivative 9 f
Table 1. Inhibitory activities against HCV NS5B polymerase and cytotoxicity toward parental Huh-7 cells and
HCV replicon Ava.5/Huh-7 cells.
Compd
R
n
IC50 [mm][a,b]
CC50 [mm][b]
EC50 [mm][b]
SI[c]
9a
9b
9c
9d
9e
9 f
9g
9h
9i
H
4-F
1
1
1
1
1
1
1
1
1
2
3
–
28.2Æ4.4
>50.0
44.5Æ6.5
>50.0
13.9Æ2.9
29.9Æ3.2
32.8Æ3.3
25.6Æ2.7
28.1Æ1.4
46.1Æ6.3
8.8Æ1.6
19.9Æ0.3
>250
59.4Æ8.1
54.4Æ1.9
76.3Æ8.8
50.8Æ2.9
32.9Æ5.2
21.4Æ3.7
36.3Æ8.7
29.6Æ1.7
33.8Æ5.3
28.7Æ5.4
17.5Æ2.5
127.6Æ5.1
>4.2
3.6
2.8
>4.9
5.4
>11.9
6.2
>8.5
6.0
>8.7
10.7
1.5
194.1Æ16.5
215.7Æ24.9
>250
175.0Æ8.3
>250
224.8Æ10.7
>250
202.1Æ23.3
>250
4-Cl
4-NO2
4-OCH3
4-NH2
2-Cl
3-Cl
3,4-diCl
H
9j
9k
1
H
–
187.5Æ14.6
186.8Æ19.1
[a] NS5B inhibitory activity based on PPi generation. [b] IC50 (concentration required for 50% inhibition of NS5B
activity), CC50 (cytotoxicity against Huh-7 cells), and EC50 (cytotoxicity against Ava.5, HCV replicon cells) values
represent the mean ÆSD of three independent experiments, each carried out in triplicate. [c] Selectivity index:
CC50/EC50.
was found to be roughly equipotent (IC50 =30 mm) with 9a.
Whereas N-(4-chlorobenzyl)carboxamide 9c is inactive toward
the polymerase, N-(2-chlorobenzyl) (9g) and N-(3-chlorobenzyl)
(9h) carboxamides possess inhibitory activity, with IC50 values
of 33 and 26 mm, respectively. The N-(3,4-dichlorobenzyl) deriv-
ative 9i is also active (IC50 =28 mm). Thus, a chloro substituent
at the ortho or meta positions of the N-benzyl group is tolerat-
ed, but not at the para position.
In addition, the linker chain of the N-arylalkyl carboxamide
was extended. Compound 9j, with an ethylene linker, showed
decreased inhibitory activity (IC50 =46 mm). Upon increasing
the chain length to propylene, compound 9k turned out to be
the most potent inhibitor (IC50 =9 mm) in this series of 3-hy-
droxy-4-oxoquinazolin-2-carboxamides.
Anti-HCV activities
Compounds 9a–k and DKA 1 were found to be non-cytotoxic
to the parental Huh-7 cell line (CC50 >175 mm, Table 1). DKA
1 is not cytotoxic to HCV1b replicon Ava.5 cells (EC50 =128 mm)
despite its inhibitory activity toward NS5B polymerase (IC50 =
20 mm, Table 1). Compounds 9a–d showed very weak cytotox-
icity (EC50 >50 mm) toward Ava.5 cells. Compounds 9e–k exhib-
ited better antiviral activity, with EC50 values ranging from 17
to 36 mm toward the Ava.5 cell line. The most potent inhibitor
9k also demonstrated the highest antiviral activity (EC50 =
17 mm). As a result, compound 9k possesses significant selec-
tivity (SI>10.7, Table 1) for HCV replicon Ava.5 cells. The trend
in anti-HCV activity of compounds 9a–k toward Ava.5/Huh-7
cells was observed microscopically through decreased cell
number and cell swelling (Figure 3). The antiviral effect was
also investigated at the HCV RNA level in HCV replicon Ava.5
Figure 3. The effects on Huh-7 and Ava.5 cells. Microscopic morphologies
(20ꢁ) of Huh-7 (right side) and Ava.5 (left side) cells after treatment with the
indicated compounds followed by Giemsa staining. Images are from a repre-
sentative experiment; at least three independent experiments with similar
results were performed.
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cells. Compound 9k effectively decreased the RNA levels in
HCV replicons at concentrations of 10 mm and above (Figure 4).
In agreement with the results of cytotoxicity assessments with
Ava.5 cells, DKA 1 showed no effect on RNA levels.
tion constants (kic =0.64 and kiu =3.94, respectively) of 9k
were estimated according to the Lineweaver–Burk plots (Fig-
ure 5c,d), indicating that 9k most likely acts as a competitive
inhibitor (kic <kiu). The kinetics of competitive inhibition was
further examined by varying the concentration of 9k in the
presence of a fixed concentration of fluorescein-labeled GTP.
Not only does compound 9k effectively compete with GTP
binding at NS5B, it displaces NTP binding as well (Figure 6).
The effect of 9k on NS5B might result in a decreased associa-
tion between NS5B and NTP, from the NTP-free state of NS5B
and the rate of NTP hydrolysis in the NTP-bound state of NS5B.
Inhibition mode on NS5B polymerase
The inhibitory profile of HCV NS5B polymerase activity was de-
termined by using a PPi-based NS5B activity assay in the pres-
ence of NTPs and 9k. As shown in Figure 5a,b, dose–response
curves at the non-saturated NTP phase indicate that a substan-
tial increase in NTP concentration affects the inhibitory activity
of 9k toward NS5B. The competitive and uncompetitive inhibi-
Binding affinity of 9k for NS5B polymerase
A quartz crystal microbalance (QCM) system was used to moni-
tor the specific interaction between 9k and NS5B. Compound
9k has no adsorptions with a control and reference-protein-
modified sensor (data not shown). Importantly, compound 9k
showed specific adsorption behavior with concentration-de-
pendent frequency shifts (from 5 to 15 Hz) on the NS5B-coated
sensor (Supporting Information). The extensive concentration
range used allowed determination of the dissociation constant
(Kd) of 9k toward the NS5B binding site (Figure 7a). The Kd
value (22 mm) was calculated from the double-reciprocal plot
(Figure 7b). Although this result does not allow a determination
as to whether 9k binds NS5B solely at the active site, or at an
allosteric site functionally coupled to the active site, this find-
ing does provide compelling evidence that 9k interacts with
Figure 4. Effect of 9k on HCV RNA levels in HCV replicon Ava.5 cells. A fully
confluent monolayer of Ava.5 cells in a 10-cm Petri dish were treated with
compounds 1 or 9k at various concentrations for 24 h. After incubation,
total RNA was extracted, and HCV or GAPDH mRNA levels were determined
by semi-quantitative RT-PCR. The HCV/GAPDH ratio represents intensity rela-
tive to control (vehicle). Images were taken from a representative experi-
ment; data represent the mean of three independent experiments, each per-
formed in triplicate.
Figure 5. Model of inhibition and inhibition constants for compound 9k toward NS5B polymerase kinetics. a) Michaelis–Menten and b) Lineweaver–Burk plots
depict the effect of 9k at concentrations of 0, 0.5, 1.0, 2.5, 5.0, and 10 mm on NS5B activity (LA: luminescence activity). Secondary plots were constructed and
used to calculate the c) uncompetitive and d) competitive inhibition constants for 9k on NS5B according to nonlinear regression analysis; data represent the
mean ÆSD from at least three independent experiments.
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dominated by 9k, suggesting that the binding of 9k at NS5B
likely results in the occlusion or interference with the associa-
tion of NTP with the NS5B active site region. Furthermore, the
interaction between 9k and NS5B is significantly affected by
the presence of magnesium ions (Figure 7d). The magnesium-
dependent binding of 9k and this compound’s ability to
impede the association of NTP with NS5B further confirm that
9k binds NS5B principally at the active site region.
Molecular modeling
A docking study was performed to explore the interactions be-
tween 9k and NS5B polymerase. As shown in Figure 8, com-
pound 9k interacts with one magnesium ion present in the
active site (Asp318–Asp319–Asp220), which is indispensable
to the polymerase-catalyzed nucleotidyl transfer reaction. Su-
perimposition of 9k and crystallographic UTP (Figure 8a)
shows that 9k chelates the same magnesium ion that is che-
lated by the b- and g-phosphate groups of UTP. The binding
pose is consistent with one previously reported.[28] Unlike the
hydrogen bonding interactions established between the pyri-
midine nucleobase of UTP and polar residues of NS5B, the hy-
drophobic phenylpropylene group of 9k is inserted into a hy-
drophobic region in the enzyme consisting of Leu159 and
Val52. There are several residues involved in the interaction
(Figure 8b,c). The amide group of 9k forms a hydrogen bond
with the backbone group of Asp225, the same residue that is
Figure 6. Competition and displacement of fluorescein-labeled GTP from
NS5B polymerase. a) The binding of fluorescein-labeled GTP on NS5B was
*
*
monitored simultaneously in the presence of 9k ( ) or 1 ( ) at various con-
centrations. b) The preformed fluorescein-labeled NTP–NS5B complex was
*
*
incubated with 9k ( ) or 1 ( ) at various concentrations. Unbound fluores-
cein-labeled NTP was removed, and the fluorescence intensity of NS5B-
bound NTP was measured; fluorescence from the group without NS5B inhib-
itors was taken as 100%, and fluorescence from the group without fluores-
cein–GTP was taken as 0%. Data represent the mean ÆSD from three inde-
pendent experiments, each performed in triplicate.
the NS5B active site. Both the level and rate of total binding at
NS5B in the presence of biotin-11-GTP (Mr =914 Da) and 9k
(Mr =323 Da) were lower than those in the presence of biotin-
11-GTP alone (Figure 7c). The changes in both early association
rate (twofold increase in slope during the first 50 s) and kinetic
level (24% frequency increase at 200 s) for NS5B binding were
Figure 7. The binding of compound 9k to NS5B as measured by QCM coated with NS5B (5 mgmLÀ1). The a) dose–response binding curve and subsequent
b) linear reciprocal plot for 9k binding with NS5B are plotted according to the frequency changes at 200 s. c) Biotin–NTP (blue, 200 mm) and 9k (black,
200 mm) were added individually or simultaneously (red, 200 mm each) to identify the total NS5B binding changes during the combination of NTP and 9k. Ex-
periments shown in panels a)–c) were carried out in the presence of magnesium. d) Compounds 9k (red) and 1 (black) were added to NS5B at conditions
with (solid lines) or without (dashed lines) magnesium; vehicle controls (ctrl) are shown in blue. Experiments were carried out in triplicate and gave similar re-
sulting frequency shifts.
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hydrogen bonded to the 2’-hydroxy group of UTP. The 3-hy-
droxy group of 9k is also hydrogen bonded to Phe224, which
interacts with the triphosphate group of NTP substrates. The
phenylpropylene group of 9k forms hydrophobic interactions
with Leu159, which interacts with the pyrimidine base of UTP.
We therefore reasoned that these hydrogen bonding and hy-
drophobic interactions, together with magnesium chelation,
contribute to the overall binding of 9k to NS5B.
Physicochemical studies
N-Arylalkyl-3-hydroxy-4-oxoquinazolin-2-carboxamides 9 were
designed as divalent metal ion chelating pyrophosphate
mimics, like the DKA prototype. Accordingly, the interaction of
1
compound 9k with magnesium ions was examined by H NMR
spectroscopy (Table 2 and Supporting Information). Incubation
of compound 9k (0.01m in [D6]DMSO) with MgCl2·6H2O
1
(2 equiv) had little or no effect on the H NMR spectrum of 9k.
Table 2. Change in chemical shift values of 9k upon incubation with
MgCl2·6H2O, H2O, Mg(OAc)2·4H2O, and NaOAc.
Proton
Dd [ppm]
Mg(OAc)2·4H2O
MgCl2·6H2O
H2O
NaOAc
H-5
H-6
H-7
H-8
NH
H-a
H-b
H-c
0.00
0.00
0.00
À0.01
+0.03
0.00
À0.01
À0.01
À0.02
À0.01
0.00
À0.02
+0.02
À0.02
À0.02
À0.03
À0.05
À0.01
À0.04
+0.12
+0.74
+0.06
À0.01
+0.06
À0.26
À0.15
À0.20
À0.08
+0.46
+0.02
+0.01
+0.02
To confirm the effect of hydrate in MgCl2·6H2O, the 1H NMR
spectrum of 9k in the presence of 10% water was recorded
with negligible modifications.[25,29] Thus, compound 9k did not
chelate the magnesium ion under neutral conditions. However,
the OH peak (d=12.05 ppm) disappeared, and the NH peak
(d=8.95 ppm) shifted downfield by 0.74 ppm with loss of reso-
lution upon addition of Mg(OAc)2·4H2O (2 equiv) to 9k (0.01m
in [D6]DMSO). Propylene protons were consequently shifted
Figure 8. Docking model of 9k into the NS5B active site. a) The superim-
posed predicted binding pose of compound 9k (green) and the crystallo-
graphically determined orientation of NTP (yellow) in the catalytic site of
NS5B. The two Mg2+ ions are indicated as magenta spheres, oxygen atoms
are shown in red, nitrogen in blue, and phosphorus in orange. Interactions
of b) compound 9k and c) UTP with NS5B active site residues. Residues
within 4.5 ꢂ of 9k are presented in the 2D interaction diagram. Purple cir-
cles on the ligand represent the intensity of solvent exposure: the larger the
circle, the greater the exposure of ligand to the solvent. Green, blue, and
magenta dashed lines represent side chain hydrogen bonds, backbone hy-
drogen bonds, and metal chelation interactions, respectively. The two
oxygen atoms of compound 9k are chelated with magnesium ions. The
bond lengths of chelating interactions between molecular atoms and metal
ions range between 2.1 and 3.7 ꢂ. Residues in green circles are hydropho-
bic; residues circled in red and blue represent acidic and basic properties, re-
spectively.
1
downfield. The H NMR spectrum of 9k (0.01m in [D6]DMSO) in
the presence of sodium acetate resulted in a downfield shift of
the NH signal by d=0.46 ppm and upfield shifts of all protons
on quinazolinone ring. On the basis of these results, it was pro-
posed that the weak base acetate ion causes partial deproto-
nation of 9k, which then chelates magnesium ions upon addi-
tion of magnesium acetate.
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(200 MHz, [D6]DMSO): d=8.25 (d, J=7.8 Hz, 1H), 7.93 (dd, J=7.2,
7.8 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.68 (dd, J=7.2, 7.8 Hz, 1H),
7.45 (m, 5H), 5.32 (s, 2H, CH2), 4.41 (q, J=7.0 Hz, 2H), 1.29 ppm (t,
J=7.0 Hz, 3H); 13C NMR (50 MHz, [D6]DMSO): d=160.0, 156.9,
145.9, 145.6, 135.6, 133.7, 130.1, 129.8, 129.1, 128.9, 128.3, 126.8,
123.8, 79.7, 63.7, 14.2 ppm; Anal. calcd for C18H16N2O4·0.1hexane: C
67.10, H 5.27, N 8.41, found: C 67.41, H 4.95, N 8.75.
Conclusions
N-Arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxa-
mide derivatives 9a, 9e–i, and 9k were identified as HCV
NS5B polymerase inhibitors. Compound 9k was discovered as
a mixed-type competitive inhibitor against NS5B polymerase,
with an IC50 value of 8.77 mm; it possesses selective cytotoxici-
ty, with an EC50 value of 17.5 mm toward HCV1b replicon Ava.5
cells. Competitive displacement and docking studies illustrated
that the 3-hydroxy-4-oxo-2-carboxamide moiety of 9k might
interact with two magnesium ions like pyrophosphate mimics
in the NS5B polymerase active site. As a result, integration of
the b-N-hydroxy-g-ketocarboxamide pharmacophore into a bi-
cyclic system such as that represented by 3-hydroxy-4-oxo-3,4-
dihydroquinazolin-2-carboxamide derivatives could give rise to
a series of HCV NS5B polymerase inhibitors.
Ethyl 3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxylate (13):
A solution of 12 (0.5 g, 1.54 mmol) and 10% Pd/C (50 mg) in
EtOAc (10 mL) was subjected to hydrogenation for 30 min. The re-
action mixture was passed through Celite, followed by evaporation
and recrystallization (hexane/EtOAc) to yield pale-brown crystals of
1
13 (0.212 g, 59%); mp: 149–1518C; H NMR (200 MHz, [D6]DMSO):
d=12.43 (bs, 1H), 8.19 (d, J=7.8 Hz, 1H), 7.89 (dd, J=7.2, 7.4 Hz,
1H), 7.75 (d, J=7.8 Hz, 1H), 7.63 (dd, J=7.6, 7.2 Hz, 1H), 4.42 (q,
J=7.0 Hz, 2H), 1.32 ppm (t, J=7.0 Hz, 3H); 13C NMR (50 MHz,
[D6]DMSO): d=160.2, 157.6, 146.8, 145.9, 135.1, 128.5, 128.1, 126.6,
122.9, 63.3, 14.3 ppm; Anal. calcd for C11H10N2O4: C 56.41, H 4.30, N
11.96, found: C 56.37, H 4.24, N 11.97.
Experimental Section
N-Benzyl-3-benzyloxy-4-oxo-3,4-dihydroquinazolin-2-carboxa-
mide (14a): To a solution of 12 (0.2 g, 0.62 mmol) in EtOH (10 mL)
was added benzylamine (0.33 g, 3.08 mmol). The mixture was
heated at reflux for 40 h. After evaporation, the mixture was
passed through a flash column (15ꢁ3 cm, hexane/EtOAc 4:1) to
yield white crystals of 14a (0.202 g, 85%); mp: 163–1658C;
1H NMR (200 MHz, [D6]DMSO): d=9.53 (t, J=5.4 Hz, 1H), 8.25 (d,
J=8.2 Hz, 1H), 7.93 (dd, J=8.2, 7.2 Hz, 1H), 7.80 (d, J=8.2 Hz, 1H),
7.70–7.62 (m, 1H), 7.42–7.27 (m, 10H), 5.31 (s, 2H), 4.51 ppm (d,
J=5.6 Hz, 2H); 13C NMR (50 MHz, [D6]DMSO): d=160.7, 157.2,
149.7, 146.1, 138.5, 135.5, 133.9, 130.1, 129.6, 129.0, 128.8, 128.4,
128.2, 128.0, 127.6, 126.7, 123.4, 79.8, 42.7 ppm; Anal. calcd for
C23H19N3O3: C 71.67, H 4.97, N 10.90, found: C 71.90, H 4.94, N
10.84.
Chemistry
All chemical reagents and solvents were obtained from commercial
sources (Merck, Mallinckrodt, Acros, Lancaster, Aldrich, and TCI)
and were used without further purification. Reaction progress was
monitored by thin-layer chromatography (TLC) analysis on silica
gel 60 F254 plates (Merck), visualized by UV light. Chromatographic
purification was carried out on silica gel columns (Silica gel Si 60,
40–63 mm, Merck). Melting points were determined with a Mel-
Temp melting point apparatus, and are uncorrected. NMR spectra
were recorded on AMX-400 (Bruker) and DPX-200 (Bruker) spec-
trometers. MS (ESI) spectra were recorded on a Finnigan TSQ 7000
with ESI mode negative ion trap detector. HRMS data were ob-
tained with ESI mode positive ion trap. Elemental analyses were re-
corded on Thermo Flash 2000 (for N, C, S, H). Purity (>95%) deter-
minations were performed by HPLC (column: Merck LiChrospher
100 RP-18e; pump A type: L-7100; injection volume: 20 mL
(100 mgmLÀ1 for each compound); detector: Hitachi UV/Vis L-7420,
l 254 nm).
3-(Benzyloxy)-N-(4-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-
carboxamide (14b): To a solution of 12 (0.3 g, 0.9 mmol) in EtOH
(10 mL) was added 4-fluorobenzylamine (0.463 g, 3.7 mmol), and
the reaction mixture was heated at reflux for 96 h. After evapora-
tion, the mixture was passed through a flash column (16ꢁ3.5 cm,
hexane/EtOAc 4:1). Recrystallization (EtOAc/hexane) gave white
2-Amino-N-benzyloxybenzamide (11): To a mixture of isatoic an-
hydride (10) (10 g, 61.3 mmol) and O-benzylhydroxyamine hydro-
chloride (11.74 g, 73.6 mmol) in anhydrous THF (100 mL), Et3N
(12.39 g, 122.6 mmol) was added. The mixture was heated at reflux
for 6 h. After evaporation, EtOAc (150 mL) was added to the resi-
due followed by addition of H2O (100 mL) with vigorous shaking.
Organic layer was separated, dried using anhydrous Na2SO4, and
concentrated to give the crude product, which was purified by
flash column (20ꢁ4 cm, hexane/EtOAc 4:1) to afford white crystals
of 11 (12.8 g, 86%); mp: 104–1068C (Lit.[30]: 103–1048C); 1H NMR
(200 MHz, [D6]DMSO): d=11.44 (bs, 1H), 7.43–7.28 (m, 6H), 7.14
(dd, J=8.2, 7.2 Hz, 1H), 6.70 (d, J=8.2 Hz, 1H), 6.47 (dd, J=7.6,
7.2 Hz, 1H), 6.29 (bs, 2H), 4.89 ppm (s, 2H).
1
crystals of 14b (0.323 g, 86%); mp: 192–1948C; H NMR (200 MHz,
[D6]DMSO): d=9.53 (bs, 1H), 8.24 (d, J=7.8 Hz, 1H), 7.96–7.62 (m,
3H), 7.41–7.32 (m, 7H), 7.08–7.06 (m, 2H), 5.30 (s, 2H, CH2),
4.49 ppm (d, J=5.4 Hz, 2H); 13C NMR (50 MHz, [D6]DMSO): d=
164.1, 160.7, 159.3, 157.2, 149.6, 146.0, 135.5, 134.8, 134.7, 133.9,
130.1, 129.9, 129.6, 128.9, 128.4, 128.1, 128.7, 123.4, 115.7, 115.3,
109.5, 79.7, 42.0 ppm; Anal. calcd for C23H18N3O3: C 68.48, H 4.50, N
10.42, found: C 68.65, H 4.47, N 10.48.
3-(Benzyloxy)-N-(4-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-
carboxamide (14c): To a solution of 12 (0.3 g, 0.9 mmol) in EtOH
(10 mL) was added 4-chlorobenzylamine (0.5 g, 3.7 mmol), and the
reaction mixture was heated at reflux for 72 h. Upon cooling, the
crystals were filtered and washed with EtOH to give white crystals
of 14c (0.265 g, 68%); mp: 203–2048C; 1H NMR (200 MHz,
[D6]DMSO): d=9.56 (t, J=6.0 Hz, 1H), 8.25 (dd, J=8.0, 1.0 Hz, 1H),
7.93–7.89 (m, 1H), 7.80–7.78 (m, 1H), 7.70–7.66 (m, 1H), 7.40–7.25
(m, 9H), 5.29 (s, 2H), 4.49 ppm (d, J=6.0 Hz, 2H); 13C NMR
(50 MHz, [D6]DMSO): d=160.7, 157.1, 149.6, 146.0, 137.6, 135.5,
133.8, 132.1, 130.1, 129.8, 129.6, 128.9, 128.7, 128.4, 128.1, 126.7,
123.4, 79.7, 42.0 ppm; Anal. calcd for C23H18N3O3: C 65.79, H 4.32, N
10.01, found: C 65.90, H 4.27, N 10.02.
Ethyl
3-benzyloxy-4-oxo-3,4-dihydroquinazolin-2-carboxylate
(12): To a mixture of 2-amino-N-benzyloxybenzamide 11 (2 g,
8.3 mmol) in diethyl oxalate (10 mL), para-toluenesulfonic acid (p-
TSA, 100 mg) was added. The mixture was held at reflux for 10 h.
After cooling, the reaction mixture was passed through a silica gel
column (6ꢁ3 cm) using 200 mL hexane (to remove excess diethyl
oxalate) and then 200 mL EtOAc. The crude product was passed
through a flash column (16ꢁ4 cm, hexane/EtOAc 4:1) to yield
white crystals of 12 (1.64 g, 61%); mp: 123–1248C; 1H NMR
856
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HCV NS5B Polymerase Inhibitors
MED
3-(Benzyloxy)-N-(4-methoxybenzyl)-4-oxo-3,4-dihydroquinazolin-
2-carboxamide (14d): To a solution of 12 (0.3 g, 0.9 mmol) in EtOH
(10 mL) was added 4-methoxybenzylamine (0.5 g, 3.7 mmol). The
reaction mixture was heated at reflux for 48 h. After evaporation,
the mixture was passed through a flash column (16ꢁ3.5 cm,
hexane/EtOAc 4:1). Recrystallization (EtOAc/hexane) gave white
311.9 (100) [MÀOH]À; HRMS (ESI+) calcd for C16H12ClN3O3: [MH+
ÀOH]+ 314.0696, found: 314.0693; Analytical HPLC 97.03% purity,
tR =6.86 min.
3-Hydroxy-N-(4-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide (9d): To a solution of 13 (0.3 g, 1.28 mmol) in EtOH
(15 mL) were added 4-nitrobenzylamine hydrochloride (0.3 g,
1.54 mmol) and Et3N (0.168 g, 1.67 mmol), and the reaction mixture
was stirred at room temperature for 48 h. After evaporation, 0.1n
HCl (15 mL) was added to the residue, followed by extraction with
EtOAc. Organic extract was dried by anhydrous Na2SO4, followed
by evaporation and recrystallization (EtOAc) to yield pale-yellow
crystals of 9d (0.326 g, yield: 75%); mp: 226–2288C; 1H NMR
(400 MHz, [D6]DMSO): d=12.14 (bs, 1H), 9.59 (t, J=6.0 Hz, 1H),
8.24 (d, J=8.4 Hz, 2H), 8.20 (d, J=8.0 Hz, 1H), 7.89–7.84 (m, 1H),
7.75 (d, J=8.0 Hz, 1H), 7.66–7.59 (m, 3H), 4.62 ppm (d, J=6.0 Hz,
2H); 13C NMR (100 MHz, [D6]DMSO): d=160.1, 157.8, 149.8, 147.1,
146.8, 146.2, 134.9, 128.7, 128.0, 127.9, 126.4, 124.0, 122.7,
42.0 ppm; Anal. calcd for C16H12N4O5: C 56.47, H 3.55, N 16.46,
found: C 56.44, H 3.58, N 16.04.
1
crystals of 14d (0.288 g, 75%); mp: 162–1648C; H NMR (200 MHz,
[D6]DMSO): d=9.45 (t, J=6.0 Hz, 1H), 8.24 (dd, J=8.0, 1.0 Hz),
7.97–7.88 (m, 1H), 7.80–7.76 (m, 1H), 7.69–7.61 (m, 1H), 7.42 (s,
5H), 7.26–7.22 (m, 2H), 6.82–6.77 (m, 2H), 5.30 (s, 2H), 4.43 (d, J=
6.0 Hz, 2H), 3.71 ppm (s, 3H); 13C NMR (50 MHz, [D6]DMSO): d=
160.5, 158.8, 157.1, 149.7, 146.1, 135.4, 133.9, 130.4, 130.1, 129.6,
129.4, 128.9, 128.3, 128.1, 126.7, 123.3, 114.1, 79.7, 55.5, 42.1 ppm;
Anal. calcd for C11H10N2O4: C 69.39, H 5.10, N 10.11, found: C 69.48,
H 5.08, N 10.14.
N-Benzyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide
(9a): To a solution of 13 (0.1 g, 0.427 mmol) in EtOH (10 mL) was
added benzylamine (0.183 g, 1.708 mmol). The mixture was heated
at reflux for 14 h. After evaporation, hexane (20 mL) was added to
the residue, and the crystals formed were filtered. The crude prod-
uct was passed through a flash column (15ꢁ3 cm, CHCl3/MeOH
49:1). Recrystallization (EtOAc/hexane) gave white crystals of 9a
(81 mg, 64%). Alternatively, 9a can be prepared as follows: to a so-
lution of 14a (60 mg, 0.156 mmol) in EtOAc (10 mL) was added
Pd/C (10%, 15 mg) and subjected to hydrogenation for 5 h. The re-
action mixture was passed through Celite. The crude product was
passed through a flash column (15ꢁ2 cm, CHCl3/MeOH 49:1). Re-
crystallization (EtOAc/hexane) gave white crystals of 9a (11 mg,
23.9%); mp: 203–2058C; 1H NMR (200 MHz, [D6]DMSO): d=12.29
(bs, 1H), 9.58 (t, J=5.7 Hz, 1H), 8.16 (d, J=7.8 Hz, 1H), 7.91–7.75
(m, 2H), 7.63–7.56 ppm (m, 1H), 7.34–7.23 (m, 5H), 4.48 (d, J=
6.2 Hz, 2H); 13C NMR (50 MHz, [D6]DMSO): d=161.4, 160.0, 147.5,
146.2, 139.2, 135.2, 128.8, 128.5, 128.2, 128.0, 127.4, 126.7, 123.2,
43.1 ppm; LRMS (ESIÀ) m/z (%) 278.0 (100) [MÀOH]À; HRMS (ESI+)
calcd for C16H13N3O3: [MH+ÀOH]+ 280.1086, found: 280.1085; Ana-
lytical HPLC 99.88% purity, tR =5.14 min.
3-Hydroxy-N-(4-methoxybenzyl)-4-oxo-3,4-dihydroquinazolin-2-
carboxamide (9e): To a solution of 14d (45 mg) in MeOH (5 mL)
Pd/C (10%, 10 mg) was added. The mixture was subjected to hy-
drogenation for 20 min. The reaction mixture was passed through
Celite. The crude product was passed through a flash column (15ꢁ
3 cm, CHCl3/MeOH 19:1) to give white crystals of 9e (26 mg, 74%);
1
mp: >3008C; H NMR (200 MHz, [D6]DMSO): d=12.18 (t, J=5.7 Hz,
1H), 8.03 (d, J=8.2 Hz, 1H), 7.71–7.57 (m, 2H), 7.43–7.35 (m, 1H),
7.30 (d, J=8.6 Hz, 2H), 6.89 (d, J=8.6 Hz, 2H), 4.45 (d, J=5.4 Hz,
2H), 3.72 ppm (s, 3H); 13C NMR (50 MHz, [D6]DMSO): d=162.1,
161.9, 158.7, 147.8, 144.4, 131.4, 131.3, 129.2, 128.0, 125.7, 125.1,
120.7, 114.2, 55.5, 42.3 ppm; Anal. calcd for C17H15N3O4·0.5CHCl3: C
54.59, H 4.06, N 10.91, found: C 54.65, H 3.74, N 10.78.
N-(4-Aminobenzyl)-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide (9 f): To a solution of 9d (0.1 g, 0.29 mmol) in EtOH
(10 mL) Pd/C (10%, 20 mg) was added. The mixture was subjected
to hydrogenation for 2 h. The reaction mixture was passed through
Celite. The crude product which was passed through a flash
column (10ꢁ3 cm, CHCl3/MeOH 49:1) to afford yellow crystals of
9 f (21 mg, 23%); mp: 243–2458C; 1H NMR (400 MHz, [D6]DMSO):
d=12.22 (bs, 1H), 9.28 (t, J=6.2 Hz, 1H), 8.16 (d, J=8.0 Hz, 1H),
7.89–7.85 (m, 1H), 7.76 (d, J=8.0 Hz, 1H), 7.61–7.57 (m, 1H), 7.01
(d, J=8.0 Hz, 2H), 6.50 (d, J=8.4 Hz, 2H), 4.98 (s, 2H), 4.28 ppm (d,
J=6.4 Hz, 2H); LRMS (ESIÀ) m/z (%) 293.0 (100) [MÀOH]À; HRMS
(ESI+) calcd for C16H14N4O3: [MH+ÀOH]+ 295.1195, found:
295.1193; Analytical HPLC 96.23% purity, tR =5.51 min.
N-(4-Fluorobenzyl)-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide (9b): To a solution of 13 (0.2 g, 0.854 mmol) in EtOH
(10 mL) was added 4-fluorobenzylamine (0.32 g, 2.562 mmol), and
the mixture was heated at reflux for 24 h. After evaporation, the
mixture was passed through a flash column (15ꢁ3 cm, CHCl3/
MeOH 49:1) to yield white crystals of 9b (0.143 g, 54%); mp: 217–
1
2198C; H NMR (200 MHz, [D6]DMSO): d=12.27 (bs, 1H), 9.60 (t, J=
6.2 Hz, 1H), 8.16 (d, J=7.8 Hz, 1H), 7.92–7.75 (m, 2H), 7.64–7.56
(m, 1H), 7.43–7.35 (m, 2H), 7.20–7.08 (m, 2H), 4.45 ppm (d, J=
6.2 Hz, 2H); 13C NMR (50 MHz, [D6]DMSO): d=164.1, 161.3, 160.0,
159.3, 147.5, 146.1, 135.4, 135.3, 135.1, 130.1, 129.9, 128.5, 128.1,
126.6, 123.1, 115.6, 115.2, 42.4 ppm; LRMS (ESIÀ) m/z (%) 296.0
(100) [MÀOH]À; HRMS (ESI+) calcd for C16H12FN3O3: [MH+ÀOH]+
298.0992, found: 298.0994; Analytical HPLC 99.84% purity, tR =
5.04 min.
N-(2-Chlorobenzyl)-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide (9g): To a solution of 13 (0.2 g, 0.854 mmol) in EtOH
(10 mL) was added 2-chlorobenzylamine (0.363 g, 2.562 mmol),
and the reaction mixture was heated at reflux for 24 h. After evap-
oration, the mixture was passed through a flash column (15ꢁ3 cm,
CHCl3/MeOH 49:1) to afford white crystals of 9g (0.155 g, 55%);
N-(4-Chlorobenzyl)-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide (9c): To a solution of 13 (0.2 g, 0.854 mmol) in EtOH
(10 mL) was added 4-chlorobenzylamine (0.363 g, 2.562 mmol),
and this was heated at reflux for 24 h. After evaporation, the mix-
ture was passed through a flash column (15ꢁ2 cm, CHCl3/MeOH
49:1) to afford white crystals of 9c (0.165 g, 59%); mp: 212–2148C;
1H NMR (200 MHz, [D6]DMSO): d=12.29 (bs, 1H), 9.63 (t, J=6.3 Hz,
1H), 8.16 (d, J=7.8 Hz, 1H), 7.92–7.75 (m, 2H), 7.64–7.56 (m, 1H),
7.37 (bs, 4H), 4.46 ppm (d, J=6.4 Hz, 2H); 13C NMR (50 MHz,
[D6]DMSO): d=161.3, 160.1, 147.5, 146.1, 138.2, 135.2, 131.9, 129.8,
128.7, 128.5, 128.1, 126.6, 123.1, 42.5 ppm; LRMS (ESIÀ) m/z (%)
1
mp: 191–1938C; H NMR (200 MHz, [D6]DMSO): d=12.24 (bs, 1H),
9.58 (t, J=6.0 Hz, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.93–7.77 (m, 2H),
7.65–7.57 (m, 1H), 7.47–7.27 (m, 4H), 4.56 ppm (d, J=6.2 Hz, 2H);
13C NMR (50 MHz, [D6]DMSO): d=161.3, 160.3, 147.5, 146.0, 135.9,
135.2, 132.2, 129.5, 129.1, 129.0, 128.5, 128.1, 127.6, 126.6, 123.2,
41.2 ppm; LRMS (ESIÀ) m/z (%) 311.9 (100) [MÀOH]À; HRMS (ESI+)
calcd for C16H12ClN3O3: [MH+ÀOH]+ 314.0696, found: 314.0699;
Analytical HPLC 97.22% purity, tR =6.93 min.
N-(3-Chlorobenzyl)-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-car-
boxamide (9h): To a solution of 13 (0.2 g, 0.854 mmol) in EtOH
ChemMedChem 2012, 7, 850 – 860
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857

J. W. Chern et al.
MED
(10 mL) was added 3-chlorobenzylamine (0.24 g, 1.71 mmol), and
the reaction mixture was heated at reflux for 20 h. After evapora-
tion, the mixture was passed through a flash column (15ꢁ3 cm,
CHCl3/MeOH 49:1) to yield white crystals of 9h (0.17 g, 60%); mp:
BL21(DE3) competent cells (Yeastern Biotech, Taiwan). The bacterial
cells were grown in LB plus the appropriate antibiotic at 378C until
an OD600 value of 0.6 was reached. The bacterial cells were then
grown at 258C with shaking at 300 rpm, and the expression of
NS5B was initiated by the addition of glucose (0.1%) and isopro-
pyl-b-d-thiogalactopyranoside (IPTG; 0.5 mm, Fermentas). Follow-
ing incubation for 4–6 h, cells were harvested by centrifugation,
and bacterial pellets were washed once with 0.5 mL ice-cold buf-
fer A (10 mm Tris pH 8.0, 150 mm NaCl, 1 mm EDTA). The cells were
resuspended in buffer A with imidazole (5 mm), NaN3 (0.02%),
DNase I (1 mgmLÀ1), and aliquots of EDTA-free protease inhibitor
cocktail tablet (Roche); they were then incubated on ice for
30 min, and the cell suspension was sonicated on ice to lyse cells
(four cycles: six times for 10 s, each time with 5 s pauses in be-
tween on ice). The crude lysates were centrifuged at 13000 rpm
(15000 g) at 48C for 30 min to pellet cellular debris. The lysate su-
pernatants were then harvested and passed through a 0.22 mm
filter (Millipore), and proteins >100 kDa were removed by a molec-
ular weight cutoff membrane (Pall Life Science). The clear lysate
(protein solution) was batch loaded into a nickel affinity resin
(Sigma) and washed successively with a buffer containing imida-
zole (first 5 mm and then 25 mm) with centrifugation at 1000 rpm
(140 g) at 48C for 5 min. The protein was eluted with the applica-
tion of imidazole buffer (200–300 mm) with centrifugation (140 g)
at 48C for 5 min. The proteins were concentrated up to 5 mgmLÀ1
by dialysis with dialysis tubing (Pierce), followed by lyophilization,
and finally exchanged into buffer containing glycerol (50%), HEPES
(25 mm, pH 7.5), NaCl (600 mm), and NaN3 (0.1%) and stored at
À808C. The protein concentration was determined with the bicin-
choninic acid approach by using BCA protein assay reagent
(Pierce).
1
213–2158C; H NMR (200 MHz, [D6]DMSO): d=12.3 (bs, 1H), 9.64 (t,
J=5.4 Hz, 1H), 8.16 (d, J=7.6 Hz, 1H), 7.92–7.76 (m, 2H), 7.64–7.57
(m, 1H), 7.41–7.32 (m, 4H), 4.47 ppm (d, J=6.0 Hz, 2H); 13C NMR
(50 MHz, [D6]DMSO): d=161.5, 160.2, 147.3, 146.2, 141.8, 135.1,
133.4, 130.6, 128.4, 127.9, 127.8, 127.3, 126.6, 123.1, 42.7 ppm;
LRMS (ESIÀ) m/z (%) 311.9 (100) [MÀOH]À; HRMS (ESI+) calcd for
C16H12ClN3O3: [MH+ÀOH]+ 314.0696, found: 314.0705; Analytical
HPLC 96.42% purity, tR =5.14 min.
N-(3,4-Dichlorobenzyl)-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-
carboxamide (9i): To a solution of 13 (0.2 g, 0.853 mmol) in EtOH
(10 mL) was added 3,4-dichlorobenzylamine (0.15 g, 0.853 mmol),
and the mixture was stirred at room temperature for 48 h. After
evaporation, the mixture was passed through a flash column (10ꢁ
3 cm, CHCl3/MeOH 19:1) to afford white crystals of 9i (0.183 g,
59%); mp: 226–2288C; 1H NMR (400 MHz, [D6]DMSO): d=12.15 (bs,
1H), 9.50 (t, J=6.0 Hz, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.89–7.85 (m,
1H), 7.74 (d, J=8.0 Hz, 1H), 7.64–7.58 (m, 3H), 7.38–7.35 (m, 1H),
4.49 ppm (d, J=6.0 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
161.0, 157.8, 149.8, 146.2, 140.0, 134.8, 131.4, 131.0, 130.0, 129.6,
128.0, 127.9, 127.8, 126.4, 122.7, 41.4 ppm; Anal. calcd for
C16H11Cl2N3O3: C 52.77, H 3.04, N 11.54, found: C 52.81, H 3.04, N
11.29.
3-Hydroxy-4-oxo-N-phenethyl-3,4-dihydroquinazolin-2-carboxa-
mide (9j): To a solution of 13 (0.1 g, 0.426 mmol) in EtOH (10 mL)
was added phenethylamine (0.155 g, 1.28 mmol), and the reaction
mixture was heated at reflux for 24 h. Upon cooling, the mixture
was passed through a flash column (15ꢁ3 cm, CHCl3/MeOH 19:1)
to afford white crystals of 9j (0.105 g, 80%); mp: 298–3008C;
1H NMR (200 MHz, [D6]DMSO): d=11.84 (bs, 1H), 8.04 (d, J=8.0 Hz,
1H), 7.73–7.58 (m, 2H), 7.43–7.19 (m, 6H), 3.56–3.46 (m, 2H),
2.83 ppm (t, J=7.6 Hz, 2H); 13C NMR (50 MHz, [D6]DMSO): d=
162.1, 161.8, 147.6, 144.4, 139.8, 131.2, 129.0, 128.8, 127.9, 126.5,
125.6, 125.1, 120.7, 41.0, 35.5 ppm; Anal. calcd for C17H15N3O3·0.4
CHCl3: C 58.53, H 4.35, N 11.91, found: C 58.90, H 4.53, N 11.89.
HCV NS5B polymerase inhibition assay
HCV NS5B RNA-dependent RNA polymerase activity based on the
generation of inorganic pyrophosphate (PPi) was performed in
Luimtrac 96-well microplates (Greiner Bio-one) as follows: Reaction
conditions for NS5B polymerase: HEPES (50 mm, pH 8.0), MgCl2
(2.5 mm), RNAsin (20 UmLÀ1, Promega), random heteropolymer
primer (0.5 mgmLÀ1, MDbio), RNA template isolated from HepG2
cells (5 mgmLÀ1), NTP mixture (1 mm, Invitrogen), DTT (4 mm,
3-Hydroxy-4-oxo-N-(3-phenylpropyl)-3,4-dihydroquinazolin-2-
carboxamide (9k): To a solution of 13 (0.1 g, 0.43 mmol) in EtOH
(10 mL) was added 3-phenylpropan-1-amine (0.173 g, 1.28 mmol),
and the reaction mixture was stirred at room temperature for 36 h.
After evaporation, the mixture was passed through a flash column
(15ꢁ3 cm, CHCl3/MeOH 19:1) to afford white crystals of 9k
Sigma), and recombinant His-tagged NS5B polymerase (1 mgmLÀ1
)
in the presence or absence of compounds. The reaction mixture
was supplemented with ATP sulfurylase and luciferase-coupled
enzyme (each 30 mL, Cambrex Bio Science Roackland) after incuba-
tion at 378C for 60 min. The initiated sulfurylase-/luciferase-based
reactions were incubated at 378C for 60 min and transferred to the
Luminometer Orion II (Berthold DS, Germany) for detection of the
signal generated over time at a 0.2 s reading every 30–60 s. Read-
ings were monitored and compared with NS5B reaction without
treatment as positive control (100% NS5B activity), and no NS5B
reaction as negative control (0% NS5B activity means no PPi gen-
eration).
1
(0.116 g, 85%); mp: 108–1118C; H NMR (400 MHz, [D6]DMSO): d=
12.06 (bs, 1H), 8.94 (t, J=5.6 Hz, 1H), 8.17 (d, J=7.2 Hz, 1H), 7.88–
7.84 (m, 1H), 7.74 (d, J=8.0 Hz, 1H), 7.58 (dd, J=6.8, 7.2 Hz, 1H),
7.31–7.16 (m, 5H), 3.18–3.24 (m, 2H), 2.66 (t, J=7.6 Hz, 2H),
1.81 ppm (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=160.7, 157.8,
150.1, 146.2, 142.0, 134.7, 128.7, 128.6, 127.8, 127.7, 126.3, 126.2,
122.6, 38.6, 32.7, 31.0 ppm; Anal. calcd for C18H17N3O3: C 66.86, H
5.30, N 13.00, found: C 66.76, H 5.28, N 12.80.
Determination of the NS5B polymerase inhibition constant
and mode of inhibition
Biology
According to the PPi-based HCV NS5B RNA-dependent RNA poly-
merase activity assay, the reaction levels in the presence or ab-
sence of 9k were determined by using varying concentrations of
NTP (10–200 mm) with a fixed concentration of RNA template–
primer as mentioned in the previous section. Inhibition data of
these reactions were analyzed by Michaelis–Menten plots and
double-reciprocal plots of the polymerase activity versus the con-
Expression and purification of recombinant His-tagged
NS5B polymerase
The pET expression plasmid (pET15b) encoding the His-tagged N-
terminal full-length non-structural protein 5B (NS5B from HCV gen-
otype 1b, provided by Prof. Shin Chang, Institute of Microbiology,
National Taiwan University, Taiwan), and was transformed into
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centration of the variable substrate for each concentration of 9k.
Data from the double-reciprocal plots were fitted to equations
with linear regression for identifying Vmax and KM by using Graph-
Pad Prism 4.0 software. To determine the kic and kiu values, the two
728C for 10 min. PCR reactions were performed in 50 mL reaction
PCR buffer containing resultant cDNAs (1 mg), 10 pmol dNTPs, 1 U
Taq DNA polymerase, 20 pmol primers, and 1.25 mm MgCl2. The
set of HCV primers (sense: 5’-CTG TGA GGA ACT ACT GTC TTC-3’;
antisense: 5’-CAA CAC TAC TCG GCT AGC AGT-3’) were used to am-
plify a 221 bp region within the 5’ untranslated region (UTR) of the
HCV genome. RT-PCR for glyceraldehyde 3-phosphate dehydrogen-
ase (GAPDH) mRNA was performed in parallel to show an equal
amount of total RNA in each sample. The set of GAPDH primers
(sense: 5’-CCA TCA CCA TCT TCC AGG AGC G-3’; antisense: 5’-AAG
GCC ATG CCA GTG AGC TTC-3’) were used to amplify a 483 bp
region within GAPDH gene. All reagents for RT were purchased
from Promega. The resultant cDNAs (1 mg) were subsequently used
in PCR reactions to amplify the PCR products, which were visual-
ized with UV light after resolution in a 2% agarose gel and staining
with ethidium bromide.
secondary plots were constructed according to the KM and Vmax
,
both of which were determined from the Lineweaver–Burk plot.
The two inhibition constants are the intercepts of these lines with
the inhibitor 9k axis as determined by nonlinear regression (Graph-
Pad Prism 4.0).
MTT-based cytotoxicity studies against Huh-7 and Ava.5
cells
Antiviral activity against HCV was assessed in a three-day assay
using human hepatoma cell lines Ava.5 (Huh-7 cells containing
sub-genomic HCV replicon, genotype 1b) and parent Huh-7 cells
maintained as sub-confluent cultures on 96-well plates. The cyto-
toxic effects triggered by compounds were assessed for 48 h. Huh-
7 and Ava.5 cells were grown at a density of 5ꢁ103 cells per well
in 96-well plates. After 12 h, the cells were treated with com-
pounds (0, 1, 10 mm) for an additional 48 h. Cell survival in 96-well
plates was assessed by reduction. Cells were then incubated with
NTP competition and displacement assays
The identification of compounds that inhibit the interaction be-
tween NTP and NS5B was carried out by using fluorescein–GTP
and filtration-based competition or displacement assays. For com-
petition assays, NS5B (5 mgmLÀ1) and fluorescein–GTP (500 mm)
were incubated at 258C for 30 min in the presence of various con-
centrations of 9k in binding buffer (50 mm HEPES pH 8.0, 2.5 mm
MgCl2, 20 UmLÀ1 RNAsin, and 4 mm DTT), in a 3 kDa cutoff 96-well
filtration plate. Unbound fluorescein–GTP was then removed by fil-
tration. The fluorescence intensity of the resuspended NS5B-bound
GTP was measured. For displacement assays, NS5B was first incu-
bated with fluorescein–GTP at 258C for 30 min, and unbound GTP
was subsequently removed by filtration. Compound 9k was then
added to the resuspended GTP–NS5B complex for an additional in-
cubation at 258C for 30 min. The remaining procedures were car-
ried out as described above. Both assays had fluorescein–GTP as
positive control (100% GTP bound) and fluorescein alone as nega-
tive control (0% GTP bound).
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide
(250 mm) at 378C for 3 h, and the absorbance was measured (A570).
Values of CC50 and EC50 are the cytotoxicities against parent Huh-7
and HCV replicon Ava.5 cells, respectively. Standard deviations for
CC50/EC50 values were calculated from standard errors generated
by regression analyses. The selectivity index (SI) of compounds was
calculated [SI=CC50 (Huh-7)/EC50 (Ava.5)] for HCV assays. Experi-
ments were performed in quadruplicate.
Morphological examination for Huh-7 and Ava.5 cells
The morphologies of Huh-7 and Ava.5 cells after treatment with
compounds were investigated by Giemsa staining. Briefly, 50000
cells were grown on glass cover slips in a six-well tissue culture
plate (Corning) in FBS (10%) and antibiotic-supplemented media,
and treated with compounds (50 mm each) for 48 h. The slides
were then washed with distilled H2O and stained with Giemsa solu-
tion (0.38 g Giemsa powder in 15% MeOH and 5% glycerin) fol-
lowed by rinsing with distilled H2O and differentiated with aqueous
acetic acid (0.5%). Cells were dehydrated rapidly, cleared and
mounted. Phase-contract micrographs were taken randomly in at
least five fields of view.
Monitoring direct interactions of NS5B inhibitors with re-
combinant NS5B polymerase by QCM in the absence or
presence of NTP or Mg2+ ions
Binding analysis of the interaction between recombinant NS5B pro-
tein and 9k and 1 in the presence or absence of NTP and/or mag-
nesium was performed with a quartz crystal microbalance (QCM;
AffinixQm, Initium Inc., Tokyo, Japan). Recombinant NS5B protein
was absorbed directly onto the gold surface of a QCM crystal (QN
sensor microsystem, Initium Inc.). First, the gold surface was
cleaned by dipping in 30% H2O2/H2SO4 (1:3) for 10 min followed
by rinsing thoroughly in deionized H2O and then blowing dry
under N2 gas. This gold substrate was further used for the absorp-
tion of recombinant NS5B. NS5B proteins were spontaneously ad-
sorbed onto the gold surface of the quartz crystal from an aqueous
solution of 30 mgmLÀ1. After 20 min, the sensor was immersed,
rinsed successively with deionized H2O and dried under N2 gas.
The immobilized NS5B generated a final signal shift of ~600 Hz, in-
dicating that ~24 ng recombinant NS5B protein was absorbed. The
NS5B immobilized quartz crystal was used for the following succes-
sive experiments. To evaluate the binding affinity of compound on
NS5B, PBS (0.5 mL) with or without MgCl2 (2 mm) was added, and
then compound was added to the reaction chamber by the titrant
injections (10, 50, 100, and/or 500 mm). The interactions were moni-
tored by alterations in frequency (DF) resulting from changes in
mass at the electrode surface. Validations of nonspecific binding of
compounds on both control (unmodified) and reference protein
Semi-quantification of HCV RNA by RT-PCR
Total cellular RNA was extracted from Ava.5 cells treated with com-
pounds 9k and 1 by using TRIZOL reagent (Gibco-BRL) according
to the manufacturer’s instructions. Quantification and purity assess-
ment for RNA were performed by measuring optical density (OD)
at l 260 and 280 nm. Reverse transcription (RT) was carried out as
follows: first 1.5 mg total RNA in 10 mL DEPC-treated H2O contain-
ing 3 mg random hexamers was heated at 728C for 10 min. Then,
10 mL RT buffer containing 4 mL 5ꢁ First-Strand Buffer [250 mm
Tris-HCl pH 8.3, 375 mm KCl, 15 mm MgCl2, 10 pmol dNTPs,
0.2 pmol DTT, 20 U RNase inhibitor (RNase Out) and 200 U reverse
transcriptase (Superscript)] were added. The final mixture was incu-
bated at 378C for 1.5 h, followed by incubation at 958C for 5 min.
All the reagents for RT were purchased from Gibco-BRL. The resul-
tant complementary DNAs (cDNAs, 1 mg) were subsequently ampli-
fied with Taq DNA polymerase by PCR. The PCR conditions were as
follows: initiation at 958C for 90 s, then 30 cycles at 958C for 30 s,
588C for 1 min and 728C for 1 min, followed by a final extension at
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J. W. Chern et al.
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Acknowledgements
We thank Apath LLC (St. Louis, MO, USA) for the agreement to
use HCV replicon Ava.5 cells. This work was supported by Nation-
al Taiwan University (99R51101).
Keywords: antiviral agents
inhibitors pyrophosphate mimics
relationships
·
HCV NS5B polymerase
·
·
·
structure–activity
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Received: January 30, 2012
Published online on March 1, 2012
860
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