The design, synthesis and biological evaluations of C-6 or C-7 substituted 2-hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus

Article abstract of DOI:10.1016/j.bmc.2011.10.058

C7-Substituted 2-hydroxyisoquinoline-1,3-diones inhibit the strand transfer of HIV integrase (IN) and the reverse-transcriptase-associated ribonuclease H (RNH). Hepatitis C virus (HCV) NS5B polymerase shares a similar active site fold to RNH and IN, sugge

Full text of DOI:10.1016/j.bmc.2011.10.058

Bioorganic & Medicinal Chemistry 20 (2012) 467–479
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The design, synthesis and biological evaluations of C-6 or C-7 substituted
2-hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus
Yue-Lei Chen , Jing Tang, Matthew J. Kesler, Yuk Y. Sham, Robert Vince, Robert J. Geraghty,
⇑
Zhengqiang Wang
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St. SE, MMC 204, Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
C7-Substituted 2-hydroxyisoquinoline-1,3-diones inhibit the strand transfer of HIV integrase (IN) and the
reverse-transcriptase-associated ribonuclease H (RNH). Hepatitis C virus (HCV) NS5B polymerase shares
a similar active site fold to RNH and IN, suggesting that N-hydroxyimides could be useful inhibitor scaf-
folds of HCV via targeting the NS5B. Herein we describe the design, chemical synthesis, replicon and bio-
chemical assays, and molecular docking of C-6 or C-7 aryl substituted 2-hydroxyisoquinoline-1,3-diones
as novel HCV inhibitors. The synthesis involved an improved and clean cyclization method, which
allowed the convenient preparation of various analogs. Biological studies revealed that the C-6 analogs,
a previously unknown chemotype, consistently inhibit both HCV replicon and recombinant NS5B at low
micromolar range. Molecular modeling studies suggest that these inhibitors may bind to the NS5B active
site.
Received 26 August 2011
Revised 13 October 2011
Accepted 18 October 2011
Available online 24 October 2011
Keywords:
2-Hydroxyisoquinoline-1,3-dione
Hepatitis C virus (HCV)
NS5B polymerase
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
HCV is an enveloped RNA virus belonging to the Hepacivirus
genus of the Flaviviridae family. Its genome is a 9.6 kb single-
Discovered as the causative agent of non-A/non-B hepatitis,1
HCV infects an estimated 170 million people globally2 and approx-
imately 4 million in the USA.3 Individuals with chronic HCV infec-
tion are at high risk of developing progressive liver injury, fibrosis,
cirrhosis and hepatocellular carcinoma.4,5 Current standard of care
for HCV infection, the combination of subcutaneous pegylated
interferon (peg-IFN) alpha and oral ribavirin (RBV),6 has proved
effective in only about 50% the patients infected with HCV geno-
type 1,6,7 the predominant genotype in North America and Europe.
In addition, the peg-IFN/RBV regimen is associated with severe ad-
verse effects that are often found intolerable.8 Therefore, there is
an unmet clinical need to develop novel chemotherapy against
HCV with better efficacy and tolerability. Experimental drugs spe-
cifically targeting HCV replication have consistently yielded im-
proved sustained virological response (SVR) rates, suggesting that
these direct-acting antivirals (DAAs) would provide a better treat-
ment option for HCV infection.9
stranded positive sense RNA encoding a polyprotein precursor of
about 3000 amino acids. This polyprotein is processed by host pep-
tidases and viral proteases to yieldfour structural proteins (core pro-
tein, envelope proteins E1 and E2, and p7) and six nonstructural (NS)
proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Among these pro-
teins the NS3-4A serine-protease10 and the NS5B viral RNA-depen-
dent RNA polymerase11 have been the primary viral targets for
current efforts in HCV chemotherapy development. Major inhibitors
in clinical development are highlighted inFigure 1. The two protease
inhibitors, telaprevir (1)12,13 and boceprevir (2)14,15 (Fig. 1), were ap-
proved by FDA recently. These antivirals effect significantly im-
proved SVR rates, though the response could be compromised by
the quick development of drug resistance. Concerted efforts have
been made to develop second-generation HCV protease inhibi-
tors.16–21 Similarly, the nonnucleoside inhibitors (NNIs)22–24 of
HCV NS5B polymerase are also confronted with the resistance issue
as they bind to highly mutable allosteric sites. GS-919025 (3, Fig. 1),
an NNI demonstrating exceptional antiviral activity in vitro, is cur-
rently in phase II clinical development. By contrast, nucleoside
NS5B inhibitors, such as the uridine analog PSI-7977 (4)26 and the
guanosine analog PSI-938 (5)27, target the highly conserved active
site, thus have a high genetic barrier to resistance development
and a pan-genotypic antiviral activity. Recently, a third HCV-en-
coded protein, NS5A, has emerged as a novel target for HCV chemo-
therapeutic intervention.28–30 Although the exact role of NS5A
remains unclear, the first-in-class inhibitor BMS-790052 (6)28,30
Abbreviations: IN, integrase; RNH, ribonuclease H; HCV, hepatitis C virus; peg-
INF, pegylated interferon; RBV, ribavirin; SVR, sustained virological response; DAA,
direct-acting antiviral; NNI, nonnucleoside inhibitor; DKA, diketoacid; Luc, lucif-
erase; 2 mA, 20-C-methyl adenosine; SAR, Structure–activity-relationship.
⇑
Corresponding author. Tel.: +1 612 626 7025; fax: +1 612 625 8154.
E-mail address: wangx472@umn.edu (Z. Wang).
Present address: Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Room 5407, 555 Zuchongzhi Road, Shanghai 201203, PR China.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.058

468
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
O
O
NH
NH2
H
N
O
O
O
N
H
NH
H
H
N
H
N
O
O
O
H
N
O
N
N
N
H
N
O
O
H
1, Telaprevir
2, Boceprevir
O
N
F
NH
O
O
HN P
O
N
CF3
N
O
O
O
N
N
O
N
OH F
F3C
3, GS-9190
4, PSI-7977
N
O
H
N
O
N
O
MeO
O
N
N
O
P
N
N
N
H
N
O
N
N
H
OMe
O
O
HN
NH2
F
O
O
5, PSI-938
6, BMS-790052
Figure 1. Representative HCV inhibitors: 1 and 2, protease inhibitors; 3, nonnucleoside polymerase inhibitor; 4 and 5, nucleoside polymerase inhibitors; 6, NS5A inhibitor.
exhibits exceptional in vitro potency and is currently under clinical
development. Nevertheless, even if some of these DAAs progress to
market, continued search for novel HCV inhibitor scaffolds is war-
ranted to combat all genotypes and resistant viral strains.
5-hydroxy-pyrimidinone compounds 1035 and 1136 containing a
chelating triad similar to DKA were found to inhibit both HIV IN
and HCV NS5B. A similar chemotype, 2-hydroxyisoquinoline-1,3-
diones, is a well-known inhibitor scaffold for HIV RNH.37 Of partic-
ular interest is the recent report of C-7-substituted 2-hydroxyiso-
quinoline-1,3-diones (12 and 13) as dual inhibitors of HIV RNH
and IN.38–40 Based on these observations, we hypothesized that
N-hydroxyimides 14 could inhibit HCV replication by targeting
the NS5B (Fig. 2).
Intriguingly, HCV NS5B polymerase shares a similar active site
fold to RNH and IN,31 which is manifested by the resemblance
among their inhibitor pharmacophores (Fig. 2). Particularly,
compounds 7,32 833 and 934 featuring
a
diketoacid (DKA)
chelating functionality inhibit all three enzymes. Similarly,
HIV IN Inhibitors
HIV RNH Inhibitors
HCV NS5B Inhibitors
CN
O
OH
Cl
O
OH
O
OH
OH
OH
OH
S
O
OH
HN
O
O
O
Cl
O
7, IC50 = 0.10 µM
8, IC50 = 3.2 µM
9, IC50 = 0.045 µM
O
OH
OH
N
N
N
H
N
N
N
O
OH
N
O
F
O
HN
S
O
10, IC50 = 0.015 µM
11, IC50 = 2.6 µM
7
NH
O
NH
O
Ar
6
O
O
O
N OH
O
N OH
O
N OH
O
µ
12, IC50 = 0.09 µM
13, IC50 = 5.7
M
14, novel inhibitors?
Figure 2. Design of novel NS5B inhibitor scaffold 14 based on the pharmacophore resemblance among HIV IN inhibitors, HIV RNH inhibitors and HCV NS5B inhibitors.

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
469
Notably, NS5B inhibitors with a chelating triad generally also
have an aromatic ring directly connected to the chelating core,
for example, the thiophene ring of compound 11. This prompted
us to synthesize analogs with a C-7 aromatic substitution (14, sub-
stitution at C-7). Additionally, aromatic substitution at C-6 position
would generate a similar chemotype (14, substitution at C6) with a
different overall molecular shape which is of medicinal chemistry
interest. This latter chemotype has never been reported in litera-
ture, possibly due to the synthetic challenge posed by the unfa-
vored directing effects from both the carbonyl and the methylene
groups.
largely to their easy deprotection. However, the cyclization of dia-
cid 16 with hydroxylamines containing acid labile groups turned
out to be particularly problematic. Many conventional dehydrating
acylation conditions with activating reagents and the reported aze-
otropic condition all failed to achieve a workable cyclization.
Through extensive reaction condition screening, we finally discov-
ered that adding a dehydrating reagent, preferably carbonyldiimid-
azole (CDI), into a refluxing mixture of diacid 17 and THPONH2 in
chlorobenzene provided an extremely rapid access to 18. The reac-
tion typically completed in 30 s by TLC and the purification proved
particularly easy. Filtering the mixture through a short silica gel
plug and evaporation of solvents typically yielded essentially pure
intermediates 18a–g, which were used directly for the final depro-
tection. Removal of O-THP was effected under a catalytic amount of
p-TSA monohydrate yielding desired compounds 19a–g in pure
form. Although this synthetic sequence is still compromised by
the low yield of the cyclization step, it does provide a rapid and
broadly applicable access to chemotype 19 due to the high func-
tional group compatibility and ease of product separation.
This cyclization method also allowed us to prepare the unprec-
edented C-6 substituted 2-hydroxyisoquinoline-1,3-diones
(Scheme 2). In this case, the requisite amino diacid intermediate
23 was synthesized starting from commercially available chloride
20. A nucleophilic aromatic substitution reaction and a subsequent
saponification produced the nitro substituted diacid 22 which was
reduced via a catalytic hydrogenation furnishing the desired amino
diacid 23.
2. Results and discussion
2.1. Chemistry
2-Hydroxyisoquinoline-1,3-diones with a substitution at C-7
position were prepared based on a reported procedure38 as de-
picted in Scheme 1. Diacid 15 was subjected to standard Sandmey-
er conditions for iodo substitution. After the addition of excessive
urea, the diazonium salt intermediate was found particularly sen-
sitive to temperature, hence a careful monitoring of the mixture
temperature is recommended. Suzuki coupling of the common
intermediate 16 with various aromatic boronic acids was found
to proceed smoothly with a combination of Pd(PPh3)4, K2CO3, and
H2O: EtOH (v/v = 1:1) under microwave. For the synthesis of target
structure 19, the reported method38 used BnONH2 to cyclize with
diacids 17 under azeotropic condition followed by the removal of
the Bn protection with BBr3. However, attempts to effect this reac-
tion sequence with our substrates were confronted with inconsis-
tent yields. Moreover, the forcing deprotection conditions would
also compromise our ability to install substituents of various nat-
ures potentially useful for medicinal chemistry.
2.2. HCV replicon assay
Until recently, detection of a complete HCV replication cycle in
cell culture was not possible. To study more limited aspects of HCV
replication, subgenomic viral RNAs (replicons) capable of self-
replication in human liver cells were engineered.41,42 An HCV
genotype 1b replicon stably maintained in Huh7 liver cells
To optimize this reaction sequence, commercially available
O-silyl and O-THP protected hydroxylamines were employed due
H2N
CO2H
CO2H
R
CO2H
CO2H
I
CO2H
CO2H
a
b
d
15
16
17a-f
O
O
R
OTHP
c
R
OH
O
N
N
O
18a-g
19a-g
Scheme 1. Synthesis of C-7 substituted 2-hydroxyisoquinoline-1,3-diones. Reagents and conditions: (a) 4 N aq sulfuric acid, NaNO2, ꢀ10 °C, then urea, ꢀ10 to 0 °C, KI, rt
overnight, 44%; (b) boronic acid, Pd(PPh3)4, K2CO3, H2O: EtOH (v/v = 1:1), microwave, 150 °C, 30 min, 87%-quant; (c) THPONH2, ClPh, CDI, reflux 30 s; (d) p-TSA hydrate,
MeOH, 6–22% over two steps.
CO2H
CO2H
CO2H
CO2R
CO2H
Cl
a
c
d
H2N
O
O2N
O2N
21, R = Me
22, R = H
20
b
23
O
CO2H
CO2H
CO2H
CO2H
e
f
OTHP
O
OH
O
g
N
N
R
I
R
R
24
25a-n
26a-n
27a-n
Scheme 2. Synthesis of C-6 substituted 2-hydroxyisoquinoline-1,3-diones. Reagents and conditions: (a) dimethyl malonate, CuBr, NaOMe, 92%; (b) NaOH, MeOH, rt
overnight, 49%; (c) 10% Pd/C, H2, EtOH, 85%; (d) 4 N aq sulfuric acid, NaNO2, ꢀ10 °C, then urea, ꢀ10 to 0 °C, KI, rt overnight, 58%; (e) boronic acid, Pd(PPh3)4, K2CO3, H2O: EtOH
(v/v = 1:1), microwave, 150 °C, 30 min, 59%-quant.; (f) THPONH2, ClPh, CDI, reflux 30 s; (g) p-TSA hydrate, MeOH, 10–24% over two steps.

470
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
(Huh-7/HCV1b-Rluc) was used for this study. The replicon RNA ex-
presses renilla luciferase (Luc) and the Luc activity can be mea-
sured as a surrogate for measuring the level of replicon RNA.
All newly synthesized 2-hydroxylisoquinoline-1,3-dione ana-
(Fig. 3). Our inhibitors contain a chelating triad capable of binding
to both Mg2+ ions at the active site, thus could represent a rare type
of active site binding NNIs. Similar to nucleoside inhibitors, active
site binding NNIs would be less prone to resistance development
and may have the advantage of pan-genotypic activity when com-
pared to typical NNIs.
logs were first tested at a single concentration of 10 lM in this rep-
licon assay with RBV and 20-C-methyl adenosine (2 mA)43 as
control compounds. Replicon cells receiving vehicle alone (DMSO)
and cell culture medium were also included in the assay. The cell
viability was measured using an MTS-based colorimetric assay.
Compounds consistently producing excellent Luc reduction
The most active compound observed in our biochemical assay,
27a, was docked into the binding pocket of NS5B active site. The
standard protocol for docking using the divalent metal cofactors
as a required constraint identified the expected chelating geometry
of the triad to the two metal cofactors (Fig. 4). This observed mode
of binding suggested R48 and K155 may play a crucial role in the
recognition of the 6- or 7- substitutions. Interestingly, both R48
ad K155 residues were also observed to form crucial salt bridge
interactions with the terminal phosphate of UTP in the original
X-ray crystallographic structure.45 For compound 27a, this mode
of binding allows the furan oxygen to form a critical H-bond to
either K155 or R48 depending on its orientation. For 6-thiophene
analog (27f), this H-bond interaction was expected to be weaker
due to the lower electronegativity of the sulfur atom, resulting in
slight decrease in the inhibitory activity. Modeling of compounds
19a and 27c further showed the loss of this crucial H-bond interac-
tion and could also explain the relative decrease of their inhibitory
activity. Substitution at the C-7 position for compounds 19a and
19d led to the loss of this crucial H-bond. For compound 19d, the
presence of the dihalo-substituted phenyl group at the C-7 position
resulted in optimal overlap of its aromatic ring with the guanidi-
nium side chain of R48 (not shown), resulting in likely favorable
(P72%) and high viability (P86%) at 10 lM compared to DMSO
alone were chosen for dose response studies. Both the EC50 and
CC50 values were determined from these experiments (Table 1).
As clearly shown in Table 1, except for 19d, all 2-hydroxyiso-
quinoline-1,3-diones with a C-7 aryl substituent showed signifi-
cant cytotoxicity (viability 637%) in Huh-7/HCV1b-Rluc cells,
rendering them unsuitable as an inhibitor scaffold for HCV. Inter-
estingly, when the C-7 aromatic group is replaced with iodine,
the compound (19g) becomes a viable HCV inhibitor with an
EC50 of 3.9 lM and a decent selectivity ratio. This iodo compound
serves as a valuable handle for further structure–activity-relation-
ship (SAR) explorations. On the other hand, relocating the aromatic
substituent from C-7 to C-6 position generates an isochemotype
which shows drastically improved antiviral activity as well as cyto-
toxicity profile. This is exemplified with compounds 27a and 27f,
where a furan or thiophene substitution at C-6 position confers
low micromolar (1.9 and 1.7 lM) inhibitory activity with good
therapeutic index (6.8 and 11), while their corresponding C-7 re-
gio-isomers (19a and 19b) were found toxic (21% viability at
p–p overlapping interactions for inhibitory binding.
10 lM for both). These differences in activity and toxicity between
regio-isomers may reflect a fundamental change in their binding
mode to the protein target as their molecular shape can be signif-
icantly different. Nevertheless, these significant improvements led
to the synthesis and testing of numerous analogs differing primar-
ily in the aromatic substituent. In the end, one C-7 analog (19g)
and four C-6 isomers (27a, 27b, 27f and 27l) were found consider-
ably more potent than RBV, validating this scaffold as a potentially
useful HCV inhibitor type.
3. Conclusions
Based on the similar fold of active site shared among HIV IN,
RNH and HCV NS5B and the pharmacophore resemblance among
their inhibitors, we designed C-6 and C-7 aromatic substituted 2-
hydroxyisoquinoline-1,3-diones as a novel inhibitor scaffold for
HCV NS5B. Difficulties presented in the synthesis of these analogs
prompted us to discover an improved synthetic method for the key
cyclization step with greater functional group compatibility and
easier product separation. All synthetic analogs were tested in a
primary screening assay using HCV replicon 1b Huh-7/HCV1b-Rluc
cells and the C-6 isomers consistently showed significantly better
inhibitory activity than RBV at low micromolar range. Biochemical
assay against recombinant NS5B confirmed that these analogs tar-
get viral polymerase with low micromolar IC50. Pharmacophore
model and molecular docking suggested that these new NS5B
inhibitors may bind to the active site. Collectively these studies
have led to the identification of a molecular scaffold potentially
useful as HCV inhibitors, and laid a foundation for further SAR to
diversify the C-6 or C-7 substituents.
2.3. Mechanism of action
Our analogs were designed based on the binding mode and
pharmacophore model of NS5B inhibitors. As mentioned earlier,
2-hydroxyisoquinoline-1,3-diones are known to inhibit HIV IN
and RNH,38–40 two enzymes sharing a similar fold of active confor-
mation to HCV NS5B. Therefore, we hypothesized that the inhibi-
tory activity of our compounds against HCV replicon can be
attributed to NS5B inhibition. To test this hypothesis, active com-
pounds selected from the replicon assay were further evaluated
against recombinant NS5B using a scintillation proximity assay.
The known DKA compound 934 was used as a control compound.
As shown in Table 2, all selected compounds were found active
4. Experimental
4.1. Chemistry
against NS5B at low
furan, demonstrated a much higher potency (IC50 = 1.3
the C7 regio-isomer 19a (IC50 = 18 M). These observations corre-
l
M concentrations and 27a, which has a C-6
lM) than
l
late positively with the observed inhibitory activity in HCV repli-
con, strongly supporting NS5B as the enzyme target of our
inhibitors.
4.1.1. General procedures
All commercial chemicals were used as supplied unless other-
wise indicated. Dry solvents (THF, Et2O, CH2Cl2 and DMF) were dis-
pensed under argon from an anhydrous solvent system with two
packed columns of neutral alumina or molecular sieves. Flash chro-
matography was performed on a Teledyne Combiflash RF-200 with
RediSep columns (silica) and indicated mobile phase. All reactions
were performed under inert atmosphere of ultra-pure argon with
oven-dried glassware. 1H and 13C NMR spectra were recorded on
a Varian 600 MHz or a Varian 400 MHz spectrometer. Mass data
2.4. Mode of binding
Superpositioning of all available NS5B X-ray crystallographic
structures bound with known inhibitors revealed that besides the
active site where endogenous substrates and nucleoside inhibitors
bind, this enzyme also has at least four allosteric sites22,44 (thumb
I, thumb II, palm I and palm II) for the binding of various NNIs

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
471
Table 1
Assay results for C6- or C7-substituted analogs in HCV replicon genotype 1b
O
OH
O
7
N
R
6
Compd
Substitution position
R
Single concentration (10
Luc reduction (%)a
l
M)
Dose response
Cell viability (%)
EC50
(l
M)b
CC50
—
(l
M)c
TId
—
19a
19b
7
7
51
99
21
21
—
O
s
—
—
—
F
F
19c
19d
19e
7
7
7
100
30
33
83
37
—
—
—
—
—
—
—
F
Cl
54
—
—
—
—
MeO
19f
19g
27a
7
7
6
83
79
89
33
86
96
—
O
I
3.9 1.5
27
6.9
6.8
1.9 0.81
13 0.58
O
27b
27c
6
6
76
87
52
4.3 0.35
11 2.6
—
2.6
—
O
e
100
—
O
27d
27e
6
6
98
73
28
46
—
—
—
—
—
—
O
O
27f
6
6
93
72
100
100
1.7 0.46
44 32
18 8.0
98
11
s
27g
2.2
S
27h
27i
6
6
95
95
34
32
—
—
—
—
—
—
s
S
O
O
27j
6
28
79
—
—
—
F3C
F
27k
27l
6
6
93
73
29
90
—
—
—
9.6 2.5
11 2.7
1.2
F
F
27m
6
78
34
27n
27o
6
6
95
3
23
—
—
—
—
—
—
O
S
H3C
100
O
(continued on next page)

472
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
Table 1 (continued)
Compd
Substitution position
R
Single concentration (10
Luc reduction (%)a
l
M)
Dose response
EC50
M)b
Cell viability (%)
(
l
CC50
32
(
l
M)c
TId
RBV
2 mA
—
—
—
—
—
—
—
—
14 4.5
0.20 0.11
2.3
270
55 15
a
Reduction of Luc activity, indicating inhibitory activity of a compound.
Concentration inhibiting virus replication by 50%, mean value standard deviation from at least two independent determinations.
Concentration resulting in 50% cell death.
b
c
d
e
Therapeutic index, defined by CC50/EC50
Not determined.
.
Table 2
Biochemical assay results against recombinant HCV NS5B
O
OH
7
N
R
6
O
Compd
Substitution position
7
R
IC50 (l
M)a
19a
18 7.2
7.1 0.7
O
F
19d
7
Cl
O
19g
27a
7
6
I
15 1.4
1.3 1.0
Figure 4. Docking of 27a (green) in HCV NS5B polymerase active site. The chelation
of both Mg2+ ions (light blue sphere) allows the placement of the C-6 furan ring in
close proximity to R48 and K155, allowing the formation key H-bond for potent
27b
27c
27f
27g
6
6
6
6
18 13
11 4.0
7.3 3.3
9.0 3.7
O
inhibition. Both UTP (cyan) (taken from PDB: 1NB645
) and benzothiadiazine
O
S
(brown) (taken from PDB:3HHK46) are shown to highlight the unique mode of
binding of compound 27a.
s
were acquired on an Agilent TOF II TOS/MS spectrometer capable
of ESI and APCI ion sources. Analysis of sample purity was per-
formed on a Varian Prepstar SD-1 HPLC system with a Varian
Microsorb-MW 100-5 C18 column (250 mm ꢁ 4.6 mm). HPLC con-
ditions: solvent A = H2O, solvent B = MeCN; flow rate = 1.0 mL/min;
Gradient (B%): 0–13 min (1–5); 13–20 min (95); 20–23 min (95–
10); 23–25 min (10). All tested compounds have a purity P96%.
27l
9b
6
F
20 12
—
—
0.015 0.0070
a
Concentration inhibiting enzyme by 50%, mean value standard deviation from
at least two independent determinations.
Reported IC50 = 0.045 l
M.34
b
4.1.1.1. 2-(Carboxymethyl)-5-iodobenzoic acid (16)38
.
Ten
grams (52 mmol) of amine 15 was dissolved in 100 mL of 4 N aq
sulfuric acid and cooled to ꢀ15 °C. To this, a solution of NaNO2
(10 g, 145 mmol) in 100 mL of water was added dropwise. The
mixture was then stirred between ꢀ10 and 0 °C for 1 h, before a
solution of 10 g of urea in 30 mL of water was introduced dropwise
(internal temperature must be maintained below 0 °C). Upon ceas-
ing of nitrogen generation, a solution of KI (15 g, 90 mmol) in
30 mL of water was added, and the mixture was warmed to rt
gradually and stirred at rt overnight. The precipitate was separated
by filtration, washed with cold water several times and dried at rt
overnight to give 6.9 g (22.5 mmol, 44%) of product as a yellow so-
lid: 1H NMR (400 MHz, DMSO-d6) d 12.69 (br s, 2H, 2 ꢁ COOH),
8.14 (d, J = 2.0 Hz, 1H, HAr), 7.83 (dd, J = 8.0, 2.0 Hz, 1H, HAr), 7.12
(d, J = 8.1 Hz, 1H, HAr), 3.87 (s, 2H, CH2).
4.1.1.2.
(21).
2-(2-Methoxy-2-oxoethyl)-4-nitrobenzoic
A solution of 9.0 g (44.8 mmol) 2-chloro-4-nitrobenzoic
acid
acid 20 in 100 mL of dimethyl malonate was bubbled with Ar for
10 min, followed by addition of 500 mg of CuBr (3.5 mmol) and
6.0 g (111 mmol) of NaOMe. The mixture was stirred in a 100 °C
Figure 3. Binding sites of HCV NS5B inhibitors.

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
473
oil bath overnight. It was observed that the mixture turned first
into solid and then gradually became a dark brown suspension.
The resulting mixture was cooled to rt, mixed with 100 mL of
water and 100 mL of hexanes, and filtered. The water phase was
then separated, washed first with hexanes, and then with toluene
several times. The resulting water phase was acidified (to pH 1) by
2 N aq HCl and extracted with DCM several times. Combined or-
ganic phases were evaporated to dryness, and the residue was
co-evaporated with toluene several times to give a yellow solid
as crude product (9.9 g, 41 mmol, 92%) which is sufficiently pure
for the following reaction. The crude product was recrystallized
from diethyl ether and hexanes for an analytically pure sample.
Rf 0.82 (MeOH:DCM = 1:10); 1H NMR (400 MHz, CDCl3) d 10.86
(br s, 1H, COOH), 8.29 (d, J = 8.6 Hz, 1H, HAr), 8.26–8.21 (m, 1H,
General procedure for Suzuki coupling: 450 mg (1.47 mmol) of
iodo derivative 16 or 24, boronic acid (2.4 mmol, 1.6 equiv),
40 mg (0.035 mmol) of Pd(PPh3)4 and 750 mg (5.4 mmol) of
K2CO3 were mixed with 3 mL of aq EtOH (v/v = 1:1) and subjected
to microwave reactor at 150 °C for 30 min. The resulting mixture
was then evaporated to remove EtOH. The residue as an aqueous
solution was filtered through glass wool while hot. The glass wool
was washed several times with boiling aq K2CO3 (10% w/v). Com-
bined filtrates were acidified (to pH 1) with 2 N aq HCl to give pre-
cipitate, which was collected by filtration, washed with water and
dried overnight at rt.
4.1.1.6.
(17a).
2-(Carboxymethyl)-5-(furan-2-yl)benzoic
acid
Yield 98%. 1H NMR (400 MHz, DMSO-d6) d 12.64 (br s,
H
Ar), 8.16 (d, J = 2.1 Hz, 1H, HAr), 4.17 (s, 2H, CH2), 3.73 (s, 3H,
2H, 2 ꢁ COOH), 8.17 (d, J = 2.0 Hz, 1H, HAr), 7.79 (dd, J = 8.0,
2.0 Hz, 1H, HAr), 7.75 (dd, J = 1.8, 0.7 Hz, 1H, Hfuran), 7.36 (d,
J = 8.0 Hz, 1H, HAr), 6.99 (dd, J = 3.4, 0.7 Hz, 1H, Hfuran), 6.59 (dd,
J = 3.4, 1.8 Hz, 1H, Hfuran), 3.93 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d 172.8 (C@O), 168.5 (C@O), 152.5 (Cq,Ar), 143.7 (CHfuran),
135.9 (Cq,Ar), 133.5 (CHAr), 131.8 (Cq,Ar), 129.6 (Cq,Ar), 126.8 (CHAr),
125.5 (CHAr), 112.6 (CHAr), 106.8 (CHAr), 40.2 (CH2, overlapped with
CH3-DMSO); HRMS (ESIꢀ) calcd for C13H9Oꢀ5 [MꢀH]ꢀ 245.0455,
found 245.0457; Mp: 216–218 °C; HPLC tR 2.7 min.
Me); 13C NMR (100 MHz, CDCl3) d 170.9 (ArCH2COOH), 170.0 (Ar-
COOH), 150.1(Cq,Ar), 138.5 (Cq,Ar), 134.2 (Cq,Ar), 133.0 (CHAr), 127.1
(CHAr), 122.4 (CHAr), 52.4 (CH3), 40.3 (CH2); HRMS (ESIꢀ) calcd
for C10H8NOꢀ6 [MꢀH]ꢀ 238.0357, found 238.0383; Mp: 100–101 °C
(diethylether-hexanes); HPLC tR 2.9 min.
4.1.1.3. 2-(Carboxymethyl)-4-nitrobenzoic acid (22).
The
crude product 21 (9.9 g, 41 mmol) from last step was stirred with
a solution of 12 g (0.3 mol) of NaOH in 100 mL of MeOH at rt over-
night. The mixture was then acidified (to pH 1) with conc. aq HCl,
evaporated to dryness and co-evaporated with toluene several
times. The residue was extracted with 20% MeOH in DCM several
times. Combined organic phases were evaporated to dryness and
the residue was recrystallized with minimum amount of EtOAc
to give the product 4.5 g (20 mmol, 49%) of off-white solid. 1H
NMR (400 MHz, DMSO-d6) d 8.26 (d, J = 2.4 Hz, 1H, HAr), 8.19 (dd,
J = 8.6, 2.4 Hz, 1H, HAr), 8.08 (d, J = 8.6 Hz, 1H, HAr), 4.08 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 172.2 (C@O), 167.4 (C@O),
149.2 (Cq,Ar), 138.8 (Cq,Ar), 137.2 (Cq,Ar), 132.0 (CHAr), 127.1 (CHAr),
122.4 (CHAr), 39.8 (CH2, overlapped with CH3-DMSO); HRMS (ESIꢀ)
calcd for C9H6NOꢀ6 [MꢀH]ꢀ 224.0201, found 224.0201; Mp: 171–
172 °C (EtOAc); HPLC tR 2.2 min.
4.1.1.7.
(17b).
2-(Carboxymethyl)-5-(thiophen-2-yl)benzoic
acid
Quantitative yield. 1H NMR (400 MHz, DMSO-d6) d
12.66 (br s, 2H, 2 ꢁ COOH), 8.08 (d, J = 2.2 Hz, 1H, HAr), 7.78 (dd,
J = 8.0, 2.2 Hz, 1H, HAr), 7.55 (ddd, J = 4.8, 4.3, 1.2 Hz, 2H, 2 ꢁ
H
H
thiophene), 7.36 (d, J = 8.0 Hz, 1H, HAr), 7.14 (dd, J = 5.1, 3.6 Hz, 1H,
thiophene), 3.92 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
172.8 (C@O), 168.4 (C@O), 142.6 (Cq,Ar), 136.1 (Cq,Ar), 133.7 (CHAr),
133.0 (Cq,Ar), 132.0 (Cq,Ar), 129.1 (CHthiophene), 128.8 (CHAr), 127.5
(CHAr), 126.6 (CHthiophene), 124.6 (CHthiophene), 40.1 (CH2, overlapped
with CH3-DMSO); HRMS (ESIꢀ) calcd for C13H9O4Sꢀ[MꢀH]ꢀ
261.0227, found 261.0235; Mp: 227–228 °C; HPLC tR 3.2 min.
4.1.1.8. 4-(Carboxymethyl)-3’,4’-difluorobiphenyl-3-carboxylic
acid (17c).
Yield 97%. 1H NMR (400 MHz, DMSO-d6) d 12.67
4.1.1.4. 4-Amino-2-(carboxymethyl)benzoic acid (23).
Fif-
(br s, 2H, 2 ꢁ COOH), 8.10 (d, J = 2.2 Hz, 1H, HAr), 7.82–7.74 (m,
2H, HAr and Hdifluorophenyl), 7.54–7.49 (m, 2H, 2 ꢁ Hdifluorophenyl),
7.41 (d, J = 8.0 Hz, 1H, HAr), 3.95 (s, 2H, CH2); 13C NMR (100 MHz,
teen grams (66.7 mmol) of starting material 22 and 1.5 g of 10%
Pd/C (purchased from Aldrich) were suspended 100 mL of EtOH
and subjected to hydrogenation at 6 bar pressure for 3 h at rt.
The resulting suspension was mixed with 100 mL of water, re-
fluxed for 10 min, and filtered through Celite 545 (Aldrich) while
hot. The filtrate was then evaporated to a minimum amount of sol-
vent and the resulting solid was collected by filtration. The solid
was washed with water, dried at rt overnight and weights at
11.1 g (56.9 mmol, 85%). 1H NMR (400 MHz, DMSO-d6) d 7.65 (d,
J = 8.5 Hz, 1H, HAr), 6.49–6.30 (m, 2H, HAr), 5.79 (br s, 2H), 3.76 (s,
2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 173.1 (C@O), 168.4
(C@O), 152.7 (Cq,Ar), 139.3 (Cq,Ar), 133.4 (CHAr), 117.2 (CHAr), 116.5
(Cq,Ar), 111.7 (CHAr), 41.0 (CH2); HRMS (ESIꢀ) calcd for
C9H8NOꢀ4 [MꢀH]ꢀ 194.0459, found 194.0459; Mp: 208–209 °C
(water-ethanol); HPLC tR 2.2 min.
DMSO-d6)
d 172.8 (C@O), 168.6 (C@O), 150.2 (dd, J = 245.4,
12.8 Hz, CF), 149.7 (dd, J = 246.5, 12.7 Hz, CF), 137.2 (Cq,Ar), 136.6
(Cq,Ar), 133.5 (CHAr), 131.9 (Cq,Ar), 130.3 (CHAr), 129.2 (d,
J = 11.8 Hz,
Cq,Ar), 128.9 (CHAr), 123.9 (dd, J = 6.5, 3.2 Hz,
CHdifluorophenyl), 118.4 (d, J = 17.1 Hz, CHdifluorophenyl), 116.2 (d,
J = 17.8 Hz, CHdifluorophenyl), 40.0 (CH2); 19F NMR (376 MHz, DMSO-
d6)
C
d
ꢀ131.5 (m), ꢀ133.8 (m); HRMS (ESIꢀ) calcd for
15H9F2Oꢀ4 [MꢀH]ꢀ 291.0474, found 291.0506; Mp: 192–195 °C;
HPLC tR 3.4 min.
4.1.1.9. 4-(Carboxymethyl)-3’-chloro-4’-fluorobiphenyl-3-car-
boxylic acid (17d).
Quantitative yield. 1H NMR (400 MHz,
DMSO-d6) d 12.72 (br s, 2H, 2 ꢁ COOH), 8.09 (d, J = 2.1 Hz, 1H, HAr),
7.89 (dd, J = 7.1, 2.3 Hz, 1H, Hchlorofluorophenyl), 7.79 (dd, J = 8.0,
4.1.1.5. 2-(Carboxymethyl)-4-iodobenzoic acid (24).
Start-
2.1 Hz, 1H,
HAr), 7.68 (ddd, J = 8.6, 4.6, 2.3 Hz, 1H,
ing from 8.0 g (41 mmol) of amine 23, the same procedure as de-
scribed for the preparation of compound 16 gave 7.29 g
(23.8 mmol, 58%) of yellow solid. 1H NMR (400 MHz, DMSO-d6) d
12.64 (br s, 2H, 2 ꢁ COOH), 7.76–7.73 (m, 2H, 2 ꢁ HAr), 7.63–7.61
(m, 1H, HAr), 3.89 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
172.6 (C@O), 168.3 (C@O), 141.2 (CHAr), 139.2 (Cq,Ar), 136.4 (CHAr),
132.5 (CHAr), 130.6 (Cq,Ar), 100.1 (Cq,Ar), 40.0 (CH2, overlapped with
H
chlorofluorophenyl), 7.53–7.45 (m, 1H, Hchlorofluorophenyl), 7.41 (d,
J = 8.0 Hz, 1H, HAr), 3.95 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-
d6) d 172.8 (C@O), 168.6 (C@O), 157.4 (d, J = 247.3 Hz, CF), 137.4
(d, J = 3.7 Hz, Cq,Ar), 137.0 (Cq,Ar), 136.6 (Cq,Ar), 133.5 (CHAr), 132.1
(Cq,Ar), 130.3 (CHAr), 129.1 (CHchlorofluorophenyl), 129.0 (CHAr), 127.8
(d, J = 7.5 Hz, CHchlorofluorophenyl), 120.6 (d, J = 17.8 Hz, Cq,Ar), 117.8 (d,
J = 21.0 Hz, CHchlorofluorophenyl), 40.1 (CH2); 19F NMR (376 MHz,
CH3-DMSO); HRMS (ESI+) calcd for C9H6IOꢀ[MꢀH]ꢀ 304.9316,
4
DMSO-d6) d ꢀ112.7 (m); HRMS (ESIꢀ) calcd for C15H9ClFOꢀ[MꢀH]
found 304.9321; Mp: 198–199 °C (EtOAc-hexnaes); HPLC tR
4
ꢀ 307.0179, found 307.0206; Mp: 186–188 °C; HPLC tR 5.5 min.
2.4 min.

474
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
4.1.1.10. 4-(Carboxymethyl)-3’-methoxybiphenyl-3-carboxylic
acid (17e).
Quantitative yield. 1H NMR (400 MHz, DMSO-d6)
8.05 (d, J = 8.1 Hz, 1H, HAr), 7.93 (dd, J = 8.1, 1.9 Hz, 1H, HAr), 7.84
(d, J = 1.9 Hz, 1H, HAr), 7.75–7.70 (m, 2H, 2 ꢁ Hdibenzofuran), 7.52
(ddd, J = 15.4, 8.5, 4.5 Hz, 2H, 2 ꢁ Hdibenzofuran), 7.44–7.38 (m, 1H,
d 12.63 (br s, 2H, 2 ꢁ COOH), 8.10 (d, J = 2.1 Hz, 1H, HAr), 7.78 (dd,
J = 7.9, 2.1 Hz, 1H, HAr), 7.43–7.34 (m, 2H, HAr and Hmethoxyphenyl),
7.25–7.19 (m, 1H, Hmethoxyphenyl), 7.20–7.15 (m, 1H, Hmethoxyphenyl),
6.95 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H, Hmethoxyphenyl), 3.95 (s, 2H, CH2),
3.81 (s, 3H, OMe); 13C NMR (100 MHz, DMSO-d6) d 172.9 (C@O),
168.7 (C@O), 160.3 (Cq,Ar), 141.1 (Cq,Ar), 139.2 (Cq,Ar), 136.2 (Cq,Ar),
133.4 (CHAr), 130.6 (CHmethoxyphenyl), 130.4 (CHAr), 128.9 (CHAr),
119.4 (CHmethoxyphenyl), 113.8 (CHmethoxyphenyl), 112.6 (CHmethoxyphenyl),
55.6 (OMe), 40.1 (CH2, overlapped with CH3-DMSO); HRMS (ESIꢀ)
calcd for C16H13Oꢀ5 [MꢀH]ꢀ 285.0786, found 285.0795; Mp: 167–
170 °C; HPLC tR 3.5 min.
H
dibenzofuran), 4.05 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
172.9 (C@O), 168.6 (C@O), 155.9 (Cq,Ar), 153.0 (Cq,Ar), 139.2 (Cq,Ar),
137.5 (Cq,Ar), 132.5 (CHAr), 131.4 (CHAr), 128.3 (CHdibenzofuran),
127.5 (2C, CHdibenzofuran and CHAr), 125.0 (Cq,Ar), 124.2 (CHdibenzofuran),
124.2 (Cq,Ar), 123.8 (CHdibenzofuran), 123.8 (Cq,Ar), 121.7 (CHdibenzofuran),
121.6 (Cq,Ar), 121.6 (CHdibenzofuran), 112.3 (CHdibenzofuran), 40.8 (CH2);
HRMS (ESIꢀ) calcd for
C
21H13Oꢀ5 [MꢀH]ꢀ 345.0768, found
345.0778; Mp: 207–208 °C; HPLC tR 6.9 min.
4.1.1.16. 4-(Benzofuran-2-yl)-2-(carboxymethyl)benzoic acid
(25e).
Yield 85%. 1H NMR (400 MHz, DMSO-d6) d 13.88 (br s,
4.1.1.11. 5-(Benzofuran-2-yl)-2-(carboxymethyl)benzoic acid
2H, 2 ꢁ COOH), 7.90 (d, J = 8.1 Hz, 1H, HAr), 7.86–7.81 (m, 2H,
2 ꢁ HAr), 7.69–7.61 (m, 2H, 2 ꢁ Hbenzofuran), 7.52 (d, J = 0.9 Hz, 1H,
Hbenzofuran), 7.37–7.30 (m, 1H, Hbenzofuran), 7.30–7.23 (m, 1H,
(17f).
2H, 2 ꢁ COOH), 8.39 (d, J = 2.0 Hz, 1H, HAr), 8.03 (dd, J = 8.0,
2.0 Hz, 1H,
Ar), 7.68–7.62 (m, 2H, 2 ꢁ Hbenzofuran), 7.50 (d,
Yield 87%. 1H NMR (400 MHz, DMSO-d6) d 12.68 (br s,
H
benzofuran), 3.89 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
H
J = 0.9 Hz, 1H, Hbenzofuran), 7.46 (d, J = 8.0 Hz, 1H, HAr), 7.35–7.29
(m, 1H, Hbenzofuran), 7.29–7.23 (m, 1H, Hbenzofuran), 3.98 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 172.7 (C@O), 168.3 (C@O),
154.7 (Cq,Ar), 154.6 (Cq,Ar), 137.5 (Cq,Ar), 133.7 (CHAr), 131.9 (Cq,Ar),
129.2 (Cq,Ar), 129.0 (Cq,Ar), 128.2 (CHAr), 126.7 (CHAr), 125.3
(CHbenzofuran), 123.8 (CHbenzofuran), 121.7 (CHbenzofuran), 111.7 (CHben-
zofuran), 103.0 (CHbenzofuran), 40.2 (CH2, overlapped with CH3-DMSO);
172.9 (C@O), 169.2 (C@O), 154.9 (Cq,Ar), 154.6 (Cq,Ar), 136.9 (Cq,Ar),
132.1 (Cq,Ar), 131.7 (CHAr), 129.2 (Cq,Ar), 129.1 (Cq,Ar), 127.8 (CHAr),
125.5 (CHbenzofuran), 123.8 (CHbenzofuran), 123.3 (CHAr), 121.9 (CHben-
zofuran), 111.7 (CHbenzofuran), 104.0 (CHbenzofuran), 41.9 (CH2); HRMS
(ESIꢀ) calcd for C ꢀ 17H11O5ꢀ[MꢀH]ꢀ 295.0612, found 295.0622;
Mp: 242–243 °C; HPLC tR 5.4 min.
HRMS (ESIꢀ) calcd for
C
17H11Oꢀ5 [MꢀH]ꢀ 295.0612, found
4.1.1.17.
(25f).
2-(Carboxymethyl)-4-(thiophen-2-yl)benzoic
acid
Yield 72%. 1H NMR (400 MHz, DMSO-d6) d 12.54 (br s,
295.0623; Mp: 237–240 °C; HPLC tR 5.4 min.
2H, 2 ꢁ COOH), 7.92 (d, J = 8.7 Hz, 1H, HAr), 7.66–7.62 (m, 3H, HAr
2 ꢁ Hthiophene), 7.61 (s, 1H, HAr), 7.16 (dd, J = 4.8, 3.9 Hz, 1H,
thiophene), 3.98 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
,
4.1.1.12.
(25a).
2-(Carboxymethyl)-4-(furan-2-yl)benzoic
acid
Yield 88%. 1H NMR (400 MHz, DMSO-d6) d 12.53 (br s,
H
2H, 2 ꢁ COOH), 7.93 (d, J = 8.8 Hz, 1H, HAr), 7.80 (dd, J = 1.8,
0.7 Hz, 1H, Hfuran), 7.67 (m, 2H, 2 ꢁ HAr), 7.07 (dd, J = 3.4, 0.7 Hz,
1H, Hfuran), 6.63 (dd, J = 3.4, 1.8 Hz, 1H, Hfuran), 3.97 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) d 172.8 (C@O), 168.2 (C@O), 152.4
(Cq,Ar), 144.4 (CHfuran), 138.0 (Cq,Ar), 133.5 (Cq,Ar), 131.8 (CHAr),
129.4 (Cq,Ar), 127.5 (CHAr), 122.1 (CHAr), 112.9 (CHfuran), 108.4
(CHfuran), 40.5 (CH2, overlapped with CH3-DMSO); HRMS (ESIꢀ)
calcd for C13H9Oꢀ5 [MꢀH]ꢀ 245.0455, found 245.0458; Mp: 202–
204 °C; HPLC tR 2.8 min.
172.8 (C@O), 168.2 (C@O), 142.4 (Cq,Ar), 138.2 (Cq,Ar), 137.2 (Cq,Ar),
132.0 (CHAr), 129.6 (Cq,Ar), 129.5 (CHthiophene), 129.2 (CHthiophene),
127.6 (CHAr), 125.6 (CHAr), 124.0 (CHthiophene), 40.4 (CH2, overlapped
with CH3-DMSO); HRMS (ESIꢀ) calcd for C13H9O4Sꢀ[MꢀH]ꢀ
261.0227, found 261.0238; Mp: 209–210 °C; HPLC tR 3.3 min.
4.1.1.18.
(25g).
2-(Carboxymethyl)-4-(thiophen-3-yl)benzoic
acid
Yield 83%. 1H NMR (400 MHz, DMSO-d6) d (2 ꢁ COOH
peak is not presented) 7.93–7.91 (m, 1H, HAr), 7.71–7.73 (m, 1H,
H
Ar), 7.64–7.62 (m, 1H, HAr), 7.60–7.58 (m, 1H, HAr), 7.57–7.55
(m, 2H, HAr), 3.70 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
173.2 (C@O), 170.5 (C@O), 141.0 (Cq,Ar), 136.8 (Cq,Ar), 135.7 (2C,
Cq,Ar), 131.5 (CHAr), 128.5 (CHAr), 127.7 (CHAr), 126.6 (CHAr), 124.5
(CHAr), 122.3 (CHAr), 43.3 (CH2).
4.1.1.13.
acid (25b).
2-(Carboxymethyl)-4-(5-methylfuran-2-yl)benzoic
Yield 80%. 1H NMR (600 MHz, CD3OD-d4) d 8.02
(d, J = 8.4 Hz, 1H, HAr), 7.60 (d, J = 8.4, 1.2 Hz, 1H, HAr), 7.55 (s,
1HAr), 6.77 (d, J = 3.6 Hz, 1Hfuran), 6.10 (d, J = 2.4 Hz, Hfuran), 4.03
(s, 2H, CH2), 2.36 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6) d
174.3 (C@O), 169.2 (C@O), 153.1 (CHfuran), 150.9 (Cq,Ar), 136.7
(Cq,Ar), 134.4 (Cq,Ar), 131.6 (Cq,Ar), 128.2 (CHAr), 126.3, (CHAr), 121.2
(CHAr), 108.2 (CHfuran), 107.9 (CHfuran), 40.4 (CH2), 12.4 (CH3); MS
(ESIꢀ) [MꢀH]ꢀ 259.25.
4.1.1.19. 4-(Benzo[b]thiophen-3-yl)-2-(carboxymethyl)benzoic
acid (25h).
(br s, 2H, 2 ꢁ COOH), 8.08 (m, 1H,
Yield 92%. 1H NMR (400 MHz, DMSO-d6) d 12.57
H
benzothiophene), 8.03 (d,
J = 7.9 Hz, 1H, HAr), 7.95–7.89 (m, 2H, HAr and Hbenzothiophene),
7.64–7.58 (m, 2H, HAr and Hbenzothiophene), 7.50–7.39 (m, 2H,
2 ꢁ Hbenzothiophene), 4.04 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-
d6) d 172.9 (C@O), 168.5 (C@O), 140.6 (Cq,Ar), 138.9 (Cq,Ar), 137.8
(Cq,Ar), 137.3 (Cq,Ar), 136.3 (Cq,Ar), 132.6 (CHbenzothiophene), 131.6
(CHAr), 130.1 (Cq,Ar), 127.2 (CHAr), 126.4 (CHAr), 125.3
(CHbenzothiophene), 125.2 (CHbenzothiophene), 123.8 (CHbenzothiophene),
122.8 (CHbenzothiophene), 40.0 (CH2, overlapped with CH3-DMSO);
4.1.1.14.
(25c).
2-(Carboxymethyl)-4-(furan-3-yl)benzoic
acid
Yield 95%. 1H NMR (400 MHz, DMSO-d6) d 12.50 (br s,
2H, 2 ꢁ COOH), 8.27 (dd, J = 1.5, 0.9 Hz, 1H, Hfuran), 7.93–7.88 (m,
1H, HAr), 7.77–7.74 (m, 1H, Hfuran), 7.60 (m, 2H, 2 ꢁ HAr), 7.00 (dd,
J = 1.9, 0.9 Hz, 1H, Hfuran), 3.95 (s, 2H, CH2). 13C NMR (100 MHz,
DMSO-d6) d 172.9 (C@O), 168.4 (C@O), 145.1 (CHfuran), 141.0
(CHfuran), 137.9 (Cq,Ar), 135.8 (Cq,Ar), 131.7 (CHAr), 129.8 (CHAr),
129.1 (Cq,Ar), 125.3 (Cq,Ar), 124.3 (CHAr), 109.0 (CHfuran), 40.6 (CH2,
overlapꢀped with CH3-DMSO); HRMS (ESIꢀ) calcd for
C13H9O5 [MꢀH]ꢀ 245.0455, found 245.0462; Mp: 208–209 °C;
HPLC tR 2.8 min.
HRMS (ESIꢀ) calcd for
C
17H11O4Sꢀ[MꢀH]ꢀ 311.0384, found
311.0403; Mp: 197–198 °C; HPLC tR 5.8 min.
4.1.1.20. 2-(Carboxymethyl)-4-(dibenzo[b,d]thiophen-4-yl)ben-
zoic acid (25i).
Yield 59%. 1H NMR (400 MHz, DMSO-d6) d
12.67 (br s, 2H, 2 ꢁ COOH), 8.45–8.35 (m, 2H, 2 ꢁ Hdibenzothiophene),
8.07 (d, J = 8.0 Hz, 1H, HAr), 8.04–7.97 (m, 1H, Hdibenzothiophene),
7.76 (dd, J = 8.0, 1.8 Hz, 1H, HAr), 7.71 (d, J = 1.8 Hz, 1H, HAr),
7.68–7.56 (m, 2H, 2 ꢁ Hdibenzothiophene), 7.56–7.48 (m, 2H, 2 ꢁ
4.1.1.15. 2-(Carboxymethyl)-4-(dibenzo[b,d]furan-4-yl)benzoic
acid (25d).
d 12.83 (br s, 2H, 2 ꢁ COOH), 8.21–8.15 (m, 2H, 2 ꢁ Hdibenzofuran),
Quantitative yield. 1H NMR (400 MHz, DMSO-d6)
H
dibenzothiophene), 4.05 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
475
172.8 (C@O), 168.4 (C@O), 143.4 (Cq,Ar), 138.8 (Cq,Ar), 137.9 (Cq,Ar),
137.7 (Cq,Ar), 136.4 (Cq,Ar), 135.6 (Cq,Ar), 135.5 (Cq,Ar), 132.3 (CHAr),
131.6 (CHAr), 130.9 (Cq,Ar), 127.8 (CHdibenzothiophene), 127.6
(CHdibenzothiophene), 126.8 (CHAr), 126.1 (CHdibenzothiophene), 125.4
(CHdibenzothiophene), 123.3 (CHdibenzothiophene), 122.7 (CHdibenzothiophene),
122.2 (CHdibenzothiophene), 40.5 (CH2, overlapped with CH3-DMSO);
127.3 (CHAr), 126.9 (CHAr), 126.6 (CHAr), 125.9 (CHAr), 125.7 (CHAr),
125.6 (CHAr), 40.3 (CH2); MS (ESIꢀ) [MꢀH]ꢀ 331.24.
4.1.1.26. 3-(Carboxymethyl)-40-(methylsulfonyl)biphenyl-4-car-
boxylic acid (25o).
Yield 80%. 1H NMR (600 MHz, DMSO-d6) d
8.09 (d, J = 6.6 Hz, 2HAr), 8.05 (d, J = 7.2 Hz, 2HAr), 7.99 (d, J = 7.2 Hz,
1HAr), 7.78 (d, J = 7.2 Hz, 1H, HAr), 7.77(s, 1H), 3.97 (s, 2H, CH2); MS
(ESIꢀ) [MꢀH]ꢀ 333.34.
HRMS (ESIꢀ) calcd for
C
21H13O4Sꢀ[MꢀH]ꢀ 361.0540, found
361.0531; Mp: 187–188 °C; HPLC tR 7.5 min.
General procedure for cyclization and deprotection: 0.8 mmol of
diacid, 1.7 mmol (2.1 equiv) of THPONH2 and 3 mL of PhCl were re-
fluxed in a 130 °C oil bath for several minutes with vigorous stir-
ring. To the mixture, a solution of CDI in DCM (3 mL, 0.4 M,
1.2 mmol) was added dropwise. The suspension turned immedi-
ately to clear and the reflux was kept for another 30 s. The resulting
solution was evaporated to dryness, re-dissolved in DCM and
loaded to a silica gel plug, which was then rinsed with diethyl ether
for several times. The filtrates were combined and evaporated to
dryness. The residue could be purified by silica gel chromatogra-
phy with EtOAc and hexanes. Otherwise it was directly stirred with
50 mg of p-TSA hydrate and 3 mL MeOH. Upon disappearance of
starting material by TLC, the mixture was again evaporated to dry-
ness under vacuum below 40 °C. The residue was triturated with
DCM and water to give the precipitate, which was collected by fil-
tration and washed with DCM and water several times, and dried
at rt overnight.
4.1.1.21.
zoic acid (25j).
4-(Benzo[d][1,3]dioxol-5-yl)-2-(carboxymethyl)ben-
Yield 88%. 1H NMR (400 MHz, DMSO-d6) d
12.49 (br s, 2H, 2 ꢁ COOH), 7.92 (d, J = 8.8 Hz, 1H, HAr), 7.60 (m,
2H, 2 ꢁ HAr), 7.30 (d, J = 1.8 Hz, 1H, Hmethylenedioxyphenyl), 7.21 (dd,
J = 8.1, 1.8 Hz, 1H, Hmethylenedioxyphenyl), 7.01 (d, J = 8.1 Hz, 1H,
Hmethylenedioxyphenyl), 6.06 (s, 2H, CH2-methylenedioxyphenyl), 3.98
(s, 2H, CH2-hydroximide); 13C NMR (100 MHz, DMSO-d6) d 172.9
(C@O), 168.5 (C@O), 148.5 (Cq,Ar), 147.9 (Cq,Ar), 143.3 (Cq,Ar), 137.8
(Cq,Ar), 133.4 (Cq,Ar), 131.6 (CHAr), 130.7 (CHAr), 129.3 (Cq,Ar), 125.1
(CHAr), 121.1 (CHmethylenedioxyphenyl), 109.2 (CHmethylenedioxyphenyl),
107.6 (CHmethylenedioxyphenyl), 101.8 (CH2-methylenedioxyphenyl),
40.6 (CH2, overlapped with CH3-DMSO); HRMS (ESIꢀ) calcd for
C
16H11Oꢀ6 [MꢀH]ꢀ 299.0561, found 299.0577; Mp: 229–230 °C;
HPLC tR 3.4 min.
4.1.1.22. 3-(Carboxymethyl)-40-(trifluoromethyl)biphenyl-4-car-
boxylic acid (25k).
Yield 81%. 1H NMR (600 MHz, CD3OD-d6)
d 8.03 (d, J = 7.8 Hz, 1H, HAr), 7.72 (d, J = 7.2 Hz, 2HAr), 7.63(d,
J = 7.8 Hz, 1H, HAr), 7.54 (d, J = 7.2 Hz, 1H, HAr), 7.49 (s, 1H, HAr),
4.00 (s, 2H, CH2); 13C NMR (101 MHz, DMSO-d6) d 174.1 (C@O),
168.7 (C@O), 143.3 (Cq,Ar), 142.9 (CHAr), 137.4 (Cq,Ar), 131.6 (Cq,Ar),
130.9 (CHAr), 129.9 (CHAr), 127.4 (CHAr), 125.5 (CHAr), 125.4 (CHAr),
125.3 (Cq,Ar), 40.0 (CH2); MS (ESIꢀ) [MꢀH]ꢀ 323.25.
4.1.1.27.
dione (19a).
7-(Furan-2-yl)-2-hydroxyisoquinoline-1,3(2H,4H)-
Yield 20% (for two steps from 17a). 1H NMR
(400 MHz, DMSO-d6) d 10.45 (s, 1H, –NOH), 8.25 (d, J = 1.9 Hz,
1H, HAr), 7.95 (dd, J = 8.2, 1.9 Hz, 1H, HAr), 7.78 (dd, J = 1.7, 0.5 Hz,
1H, Hfuran), 7.42 (d, J = 8.2 Hz, 1H, HAr), 7.08 (dd, J = 3.3, 0.5 Hz,
1H, Hfuran), 6.61 (dd, J = 3.3, 1.7 Hz, 1H, Hfuran), 4.24 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) d 166.7 (C@O), 162.0 (C@O), 152.1
(Cq,Ar), 143.9 (CHfuran), 134.0 (Cq,Ar), 129.9 (Cq,Ar), 128.9 (CHAr),
128.6 (CHAr), 126.1 (Cq,Ar), 122.2 (CHAr), 112.7 (CHfuran), 107.3
(CHfuran), 37.2 (CH2); HRMS (ESIꢀ) calcd for C13H8NOꢀ4 [MꢀH]ꢀ
242.0459, found 242.0466; Mp: 203–208 °C (decomp.); HPLC tR
9.9 min.
4.1.1.23.
acid1 (25l).
3-(Carboxymethyl)-40-fluorobiphenyl-4-carboxylic
Yield 78%. 1H NMR (600 MHz, CD3OD-d4) d 8.03
(d, J = 7.8 Hz, 1H, HAr), 7.62 (td, J = 8.4, 3.0 Hz, 2H, 2 ꢁ HAr), 7.53
(d, J = 7.8 Hz, 1H, Hyl), 7.47 (s, 1H, HAr), 7.13 (t, J = 8.4 Hz, 2H,
2 ꢁ HAr), 4.03 (s, 2H, CH2); 13C NMR (150 MHz,CD3OD) d 174.1
(C@O), 168.7 (C@O), 163.7 (CF), 162.1 (CHAr), 143.7 (CHAr), 137.3
(Cq,Ar), 135.9 (Cq,Ar), 131.5 (Cq,Ar), 130.5 (CHAr), 128.7 (d, J = 8.0 Hz,
4.1.1.28. 2-Hydroxy-7-(thiophen-2-yl)isoquinoline-1,3(2H,4H)-
dione (19b).
Yield 21% (for two steps from 17b). 1H NMR
C
q,Ar), 125.1 (CHAr), 115.4(d, J = 6.8 Hz, CHAr), 40.2 (CH2); MS (ESIꢀ)
[MꢀH]ꢀ 273.24.
(400 MHz, DMSO-d6) d 10.46 (s, 1H, –NOH), 8.16 (d, J = 1.9 Hz,
1H, HAr), 7.94 (dd, J = 8.0, 1.9 Hz, 1H, HAr), 7.60 (dd, J = 7.3, 4.3 Hz,
2H, 2 ꢁ Hthiopene), 7.42 (d, J = 8.0 Hz, 1H, HAr), 7.15 (dd, J = 5.0,
3.7 Hz, 1H, Hthiopene), 4.25 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d 166.7 (C@O), 162.0 (C@O), 142.2 (Cq,Ar), 134.2 (Cq,Ar),
133.3 (Cq,Ar), 130.6 (CHAr), 129.2 (CHthiophene), 129.1 (CHAr), 126.9
(CHthiophene), 126.2 (Cq,Ar), 125.0 (CHthiophene), 124.1 (CHAr), 37.2
(CH2); HRMS (ESIꢀ) calcd for C13H8NO3Sꢀ[MꢀH]ꢀ 258.0230, found
258.0230; Mp: 211–219 °C (decomp.); HPLC tR 10.0 min.
4.1.1.24. 3-(Carboxymethyl)-30,40-difluorobiphenyl-4-carboxylic
acid (25m).
Yield 84%. 1H NMR (400 MHz, DMSO-d6) d 13.58
(br s, 2H, 2 ꢁ COOH), 7.88 (d, J = 8.0 Hz, 1H, HAr), 7.81 (ddd,
J = 12.3, 7.8, 2.1 Hz, 1H, Hdifluorophenyl), 7.67–7.60 (m, 2H, 2 ꢁ HAr),
7.60–7.48 (m, 2H, 2 ꢁ Hdifluorophenyl), 3.89 (s, 2H, CH2); 13C NMR
(100 MHz, DMSO-d6) d 172.9 (C@O), 169.1 (C@O), 150.2 (dd,
J = 245.3, 12.7 Hz, CF), 149.8 (dd, J = 245.4, 14.3 Hz, CF), 140.6
(Cq,Ar), 137.4–136.6 (m, 3C, 3 ꢁ Cq,Ar), 131.6 (CHAr), 130.4 (CHAr),
125.4 (CHAr), 124.1 (dd, J = 6.6, 3.2 Hz, CHdifluorophenyl), 118.5 (d,
J = 17.1 Hz, CHdifluorophenyl), 116.4 (d, J = 17.9 Hz, CHdifluorophenyl),
4.1.1.29.
1,3(2H,4H)-dione (19c).
7-(3,4-Difluorophenyl)-2-hydroxyisoquinoline-
Yield 22% (for two steps from 17c).
41.5 (CH2); 19F NMR (376 MHz, DMSO-d6)
d
ꢀ131.55(m),
1H NMR (400 MHz, DMSO-d6) d 10.45 (s, 1H, –NOH), 8.22 (d,
J = 2.0 Hz, 1H, HAr), 7.96 (dd, J = 8.0, 2.1 Hz, 1H, HAr), 7.85 (m, 1H,
ꢀ133.34(m); HRMS (ESIꢀ) calcd for C15H9F2Oꢀ4 [MꢀH]ꢀ 291.0474,
found 291.0499; Mp: 188–190 °C; HPLC tR 3.5 min.
H
difluorophenyl), 7.54 (m, 3H, HAr and 2 ꢁ Hdifluorophenyl), 4.28 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 166.7 (C@O), 162.0 (C@O),
150.3 (dd, J = 245.7, 12.6 Hz, CF), 149.8 (dd, J = 246.8, 12.5 Hz,
CF), 137.5 (Cq,Ar), 136.8 (dd, J = 6.2, 3.7 Hz, 1C, Cq,Ar), 134.8 (Cq,Ar),
132.1 (CHAr), 129.0 (CHAr), 126.1 (Cq,Ar), 125.9 (CHAr), 124.1 (dd,
J = 6.6, 3.3 Hz, 1C, CHdifluorophenyl), 118.5 (d, J = 17.2 Hz, 1C,
CHdifluorophenyl), 116.4 (d, J = 18.1 Hz, 1C, CHdifluorophenyl), 37.2 (CH2);
19F NMR (376 MHz, DMSO-d6) d ꢀ131.42(m), ꢀ133.48(m); HRMS
(ESIꢀ) calcd for C15H8F2NO3ꢀ[MꢀH]ꢀ 288.0478, found 288.0497;
Mp: 196–198 °C; HPLC tR 11.7 min.
4.1.1.25. 3-(Carboxymethyl)-30-(phenyl)biphenyl-4-carboxylic
acid (25n).
Yield 82%. 1H NMR (600 MHz, CD3OD-d4) d 8.15
(d, J = 8.4 Hz, 1H, HAr), 7.84 (s, 1H), 7.66 (m, 3H, HAr), 7.62 (d,
J = 7.8 Hz, 2H), 7.58 (s, 1H), 7.53 (t, J = 7.8 Hz, 1H, HAr), 7.46 (t,
J = 7.8 Hz, 2HAr), 7.37 (t, J = 7.2 Hz, 1HAr), 4.12 (s, 2H, CH2); 13C
NMR (150 MHz, DMSO-d6) d 174.2 (C@O), 169.0 (C@O), 144.9
(Cq,Ar), 141.9 (Cq,Ar), 140.8 (Cq,Ar), 140.2 (Cq,Ar), 137.1 (Cq,Ar), 131.7
(Cq,Ar), 130.9 (CHAr), 129.2 (CHAr), 128.9 (CHAr), 128.6 (CHAr),

476
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
4.1.1.30. 7-(3-Chloro-4-fluorophenyl)-2-hydroxyisoquinoline-
366.0770, found 366.0781; Rf 0.60 (EtOAc:hexanes = 1:1); Mp:
148–149 °C; HPLC tR 14.6 min.
1,3(2H,4H)-dione (19d).
Yield 21% (for two steps from 17d).
1H NMR (400 MHz, DMSO-d6) d 10.46 (s, 1H, –NOH), 8.21 (d,
J = 2.0 Hz, 1H, HAr), 7.99–7.91 (m, 2H, HAr and Hchlorofluorophenyl), 7.73
(m, 1H, Hchlorofluorophenyl), 7.49 (m, 2H, HAr and Hchlorofluorophenyl), 4.28
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.6 (C@O), 162.0
(C@O), 157.6 (d, J = 247.7 Hz, CF), 137.3 (Cq,Ar), 137.01 (d, J = 3.7 Hz,
4.1.1.35.
dione (27a).
6-(Furan-2-yl)-2-hydroxyisoquinoline-1,3(2H,4H)-
Yield 22% (for two steps from 25a). 1H NMR
(400 MHz, DMSO-d6) d 10.41 (s, 1H, –NOH), 8.04 (d, J = 8.3 Hz,
1H, HAr), 7.85 (d, J = 1.4 Hz, 1H, Hfuran), 7.78 (d, J = 8.3 Hz, 1H,
Cq,Ar), 134.8 (Cq,Ar), 132.2 (CHAr), 129.2 (CHAr), 129.0 (CHchlorofluorophe-
HAr), 7.69 (s, 1H, HAr), 7.17 (d, J = 3.3 Hz, 1H, Hfuran), 6.66 (dd,
nyl), 127.9 (d, J = 7.5 Hz, CHchlorofluorophenyl), 126.1 (Cq,Ar), 125.9 (CHAr),
120.7 (d, J = 17.8 Hz, Cq,Ar), 117.9 (d, J = 21.1 Hz, CHchlorofluorophenyl),
37.2 (CH2); 19F NMR (376 MHz, DMSO-d6) d ꢀ112.34(m); HRMS
(ESIꢀ) calcd for C15H8ClFNO3ꢀ[MꢀH]ꢀ 304.0182, found 304.0191;
Mp: 220–224 °C (decomp.); HPLC tR 12.0 min.
J = 3.3, 1.4 Hz, 1H, Hfuran), 4.29 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d 166.7 (C@O), 161.9 (C@O), 152.0 (Cq,Ar), 144.9 (CHfuran),
136.0 (Cq,Ar), 134.8 (Cq,Ar), 128.9 (CHAr), 124.0 (Cq,Ar), 122.6 (CHAr),
122.3 (CHAr), 113.0 (CHfuran), 109.4 (CHfuran), 37.4 (CH2); HRMS
(ESIꢀ) calcd for C13H8NO4ꢀ[MꢀH]ꢀ 242.0459, found 242.0466;
Mp: 190–193 °C (decomp.); HPLC tR 10.0 min.
4.1.1.31. 2-Hydroxy-7-(3-methoxyphenyl)isoquinoline-1,3(2H,
4H)-dione (19e).
Yield 12% (for two steps from 17e). 1H
NMR (400 MHz, DMSO-d6) d 10.42 (br s, 1H, –NOH), 8.21 (d,
J = 2.0 Hz, 1H, HAr), 7.96 (dd, J = 8.0, 2.0 Hz, 1H, HAr), 7.47 (d,
J = 8.0 Hz, 1H, HAr), 7.44–7.37 (m, 1H, Hmethoxyphenyl), 7.31–7.24 (m,
1H, Hmethoxyphenyl), 7.23–7.19 (m, 1H, Hmethoxyphenyl), 6.97 (ddd,
J = 8.2, 2.5, 0.9 Hz, 1H, Hmethoxyphenyl), 4.28 (s, 2H, CH2), 3.82 (s, 3H,
4.1.1.36. 2-Hydroxy-6-(5-methylfuran-2-yl)isoquinoline-1,3(2H,
4H)-dione (27b).
(600 MHz, DMSO) d 10.41 (s, 1H, –NOH), 8.00 (d, J = 7.8 Hz, 1H,
Ar), 7.72 (d, J = 7.8 Hz, 1H, Hfuran), 7.61 (s, 1H, HAr), 7.04 (d,
Yield 22% (for two steps from 25b). 1H NMR
H
J = 3.0 Hz, Hfuran), 6.27 (d, J = 2.4 Hz, 1H, Hfuran), 4.26 (s, 2H, CH2),
2.35 (s, 3H, CH3); HRMS (ESIꢀ) calcd. for C14H11NO4 [MꢀH]ꢀ
256.0610, found 256.0569; HPLC tR 13.1 min.
OMe); 13C NMR (100 MHz, DMSO-d6)
d 166.7 (C@O), 162.1
(C@O), 160.3 (Cq,Ar), 140.7 (Cq,Ar), 139.6 (Cq,Ar), 134.4 (Cq,Ar), 132.2
(CHAr), 130.7 (CHmethoxyphenyl), 128.9 (CHAr), 126.0 (Cq,Ar), 125.8
(CHAr), 119.4 (CHmethoxyphenyl), 114.1 (CHmethoxyphenyl), 112.6
(CHmethoxyphenyl), 55.7 (OMe), 37.2 (CH2); HRMS (ESIꢀ) calcd for
4.1.1.37.
dione (27c).
6-(Furan-3-yl)-2-hydroxyisoquinoline-1,3(2H,4H)-
Yield 14% (for two steps from 25c). 1H NMR
C
16H12NOꢀ4 [MꢀH]ꢀ 282.0772, found 282.0780; Mp: 132–137 °C
(400 MHz, DMSO-d6) d 10.40 (s, 1H, –NOH), 8.34 (s, 1H, HAr),
7.99 (d, J = 8.2 Hz, 1H, Hfuran), 7.79 (s, 1H, HAr), 7.72 (d,
J = 8.2 Hz, 1H, Hfuran), 7.63 (s, 1H, Hfuran), 7.03 (s, 1H, HAr), 4.24
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.7 (C@O),
162.0 (C@O), 145.3 (CHAr), 141.6 (CHAr), 137.3 (Cq,Ar), 135.8
(Cq,Ar), 128.8 (CHfuran), 125.2 (Cq,Ar), 124.8 (CHfuran), 124.6 (CHAr),
123.8 (Cq,Ar), 109.0 (CHfuran), 37.4 (CH2); HRMS (ESIꢀ) calcd for
(decomp.); HPLC tR 11.0 min.
4.1.1.32. 7-(Benzofuran-2-yl)-2-hydroxyisoquinoline-1,3(2H,4
H)-dione (19f).
Yield 6% (for two steps from 17f). 1H NMR
(400 MHz, DMSO-d6) d 10.52 (br s, 1H, –NOH), 8.47 (d, J = 1.8 Hz,
1H, HAr), 8.19 (dd, J = 8.1, 1.8 Hz, 1H, HAr), 7.67 (d, J = 8.1 Hz, 2H,
2 ꢁ Hbenzofuran), 7.58 (s, 1H, Hbenzofuran), 7.52 (d, J = 8.1 Hz, 1H, HAr),
7.34 (dd, J = 11.2, 4.4 Hz, 1H, Hbenzofuran), 7.27 (t, J = 7.8 Hz, 1H,
C
13H8NOꢀ4 [MꢀH]ꢀ 242.0459, found 242.0456; Mp: 194–198 °C
(decomp.); HPLC tR 9.7 min.
H
benzofuran), 4.30 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.6
(C@O), 162.1 (C@O), 154.7 (Cq,Ar), 154.2 (Cq,Ar), 135.5 (CHAr), 129.8
(Cq,Ar), 129.4 (CHAr), 129.1 (Cq,Ar), 129.1 (Cq,Ar), 126.4 (Cq,Ar), 125.5
(CHbenzofuran), 123.9 (CHbenzofuran), 123.5 (CHAr), 121.8 (CHbenzofuran),
111.7 (CHbenzofuran), 103.5 (CHbenzofuran), 37.4 (CH2); HRMS (ESIꢀ)
calcd for C17H10NOꢀ4 [MꢀH]ꢀ 292.0615, found 292.0631; Mp: 241–
249 °C (decomp); HPLC tR 12.0 min.
4.1.1.38.
1,3(2H,4H)-dione (27d)
6-(Dibenzo[b,d]furan-4-yl)-2-hydroxyisoquinoline-
.
Yield 24% (for two steps from 25d).
1H NMR (400 MHz, DMSO-d6) d 10.46 (s, 1H, –NOH), 8.26–8.14 (m,
3H, HAr, 2 ꢁ Hdibenzofuran), 8.03 (dd, J = 8.2, 1.5 Hz, 1H, HAr), 7.92 (d,
J = 1.5, 1H, HAr), 7.78–7.70 (m, 2H, 2 ꢁ Hdibenzofuran), 7.59–7.49 (m,
2H, 2 ꢁ Hdibenzofuran), 7.43 (td, J = 7.5, 0.9 Hz, 1H, Hdibenzofuran), 4.37
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.8 (C@O), 162.0
(C@O), 155.9 (Cq,Ar), 153.0 (Cq,Ar), 140.9 (Cq,Ar), 135.6 (Cq,Ar), 128.6
(CHAr), 128.4 (CHdibenzofuran), 127.9 (CHAr), 127.9 (CHAr), 127.6
(CHdibenzofuran), 125.1 (Cq,Ar), 124.9 (Cq,Ar), 124.3 (CHdibenzofuran),
123.9 (CHdibenzofuran), 123.9 (CHdibenzofuran), 123.7 (Cq,Ar), 122.0 (Cq,Ar),
121.8 (CHdibenzofuran), 112.3 (CHdibenzofuran), 37.6 (CH2); HRMS (ESIꢀ)
4.1.1.33.
(19g).
2-Hydroxy-7-iodoisoquinoline-1,3(2H,4H)-dione
Yield 11% (for two steps from 16). 1H NMR (400 MHz,
DMSO-d6) d 10.47 (br s, 1H, –NOH), 8.24 (d, J = 1.8 Hz, 1H, HAr),
7.96 (dd, J = 8.1, 1.8 Hz, 1H, HAr), 7.18 (d, J = 8.1 Hz, 1H, HAr), 4.18
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.4 (C@O), 161.0
(C@O), 142.0 (CHAr), 136.0 (CHAr), 134.8 (Cq,Ar), 130.3 (CHAr),
127.5 (Cq,Ar), 92.9 (Cq,Ar), 37.2 (CH2); HRMS (ESIꢀ) calcd for
C9H5INOꢀ3 [MꢀH]ꢀ 301.9320, found 301.9317; Mp: 166–174 °C
(decomp.); HPLC tR 9.3 min.
calcd for
C
21H12NOꢀ4 [MꢀH]ꢀ 342.0772, found 342.0754; Mp:
185–195 °C (decomp.); HPLC tR 13.6 min.
4.1.1.39.
4H)-dione (27e).
6-(Benzofuran-2-yl)-2-hydroxyisoquinoline-1,3(2H,
4.1.1.34. 2-((Tetrahydro-2H-pyran-2-yl)oxy)-6-(thiophen-2-yl)is
Yield 22% (for two steps from 25e). 1H
oquinoline-1,3(2H,4H)-dione (26f).
Yield 20% from com-
pound 25f. 1H NMR (400 MHz, DMSO-d6) d 8.01 (d, J = 8.3 Hz, 1H,
NMR (400 MHz, DMSO-d6) d 10.45 (br s, 1H, –NOH), 8.10 (d,
J = 8.3 Hz, 1H, HAr), 7.99 (d, J = 8.3 Hz, 1H, HAr), 7.90 (pseudo-s,
1H, HAr), 7.72–7.60 (m, 3H, 3 ꢁ Hbenzofuran), 7.37 (dd, J = 11.1,
4.2 Hz, 1H, Hbenzofuran), 7.28 (t, J = 7.3 Hz, 1H, Hbenzofuran), 4.32 (s,
2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.6 (C@O), 161.8
(C@O), 155.0 (Cq,Ar), 154.0 (Cq,Ar), 136.1 (Cq,Ar), 134.3 (Cq,Ar),
129.0 (Cq,Ar), 128.9 (CHAr), 126.0 (CHbenzofuran), 125.2 (Cq,Ar), 124.0
(CHbenzofuran), 123.9 (CHAr), 123.7 (CHAr), 122.2 (CHbenzofuran),
111.7 (CHbenzofuran), 105.3 (CHbenzofuran), 37.5 (CH2); HRMS (ESIꢀ)
calcd for C17H10NOꢀ4 [MꢀH]ꢀ 292.0615, found 292.0614; Mp:
262–265 °C (decomp.); HPLC tR 12.0 min.
H
H
Ar), 7.77 (d, J = 8.3 Hz, 1H, HAr), 7.68 (m, 3H, HAr and 2 ꢁ
thiophene), 7.19 (dd, J = 5.0, 3.7 Hz, 1H, Hthiophene), 5.19 (s, 1H,
H2THP), 4.31 (pseudo-s, 3H, H6a and CH2-hydroximide), 3.50 (d,
THP
6b
THP
3a
3b
THP
J = 10.9 Hz, 1H, H ), 1.92 (m, 1H, H ), 1.85–1.65 (m, 2H, H
and H ), 1.58 (m, 3H, H
THP
4a
THP
4b
5a;b
THP
and H ); 13C NMR (100 MHz,
THP
DMSO-d6) d 166.6 (C@O), 161.8 (C@O), 141.9 (Cq,Ar), 138.9 (Cq,Ar),
136.3 (Cq,Ar), 129.4 (CHthiophene), 129.3 (CHAr), 128.4 (CHthiophene),
126.4 (CHAr), 124.6 (CHAr), 124.3 (CHthiophene), 124.2 (Cq,Ar), 102.0
(C2THP), 62.0 (CT6HP), 37.9 (CH2-hydroximide), 28.0 (C3THP), 25.0
(C5THP), 18.0 (CT4HP); HRMS (ESI+) calcd for C18H17NNaO4S+[M+Na]+

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
477
4.1.1.40. 2-Hydroxy-6-(thiophen-2-yl)isoquinoline-1,3(2H,4H)-
dione (27f).
Yield: 72% (from 26f). 1H NMR (400 MHz, DMSO-
(600 MHz, CD3OD-d4) d 8.10 (d, J = 8.4 Hz, 1H, HAr), 7.65 (m, 3H,
H
Ar), 7.55 (s, 1H), 7.14 (t, J = 8.4 Hz, 2H, HAr), 4.38 (s, 2H, CH2);
d6) d 10.39 (s, 1H, –NOH), 8.01 (d, J = 8.3 Hz, 1H, HAr), 7.75 (dd, J = 8.3,
1.6 Hz, 1H, HAr), 7.67 (m, 3H, HAr, 2 ꢁ Hthiophene), 7.18 (dd, J = 4.8,
4.0 Hz, 1H, Hthiophene), 4.27 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d 166.7 (C@O), 161.8 (C@O), 142.0 (Cq,Ar), 138.6 (Cq,Ar),
136.1 (Cq,Ar), 129.3 (CHthiophene), 129.1 (CHAr), 128.3 (CHthiophene),
126.3 (CHAr), 124.6 (CHAr), 124.3 (CHthiophene), 124.2 (Cq,Ar), 37.4
(CH2); HRMS (ESIꢀ) calcd for C13H8NO3Sꢀ[MꢀH]ꢀ 258.0230, found
258.0230; Mp: 202–210 °C (decomp.); HPLC tR 10.6 min.
HRMS (ESIꢀ) calcd. for
C
15H10FNOꢀ3 [MꢀH]ꢀ 270.0566, found
270.0544; HPLC tR 11.9 min.
4.1.1.46. 2-Hydroxy-6-(4-(trifluoromethyl)phenyl)isoquinoline-
1,3(2H,4H)-dione (27l).
10% (for two steps from 25l). 1H NMR
(600 MHz, CD3OD) d 8.27 (d, J = 8.4 Hz, 1H, HAr), 7.88 (d, J = 8.4 Hz,
2H, HAr), 7.81 (m, 3H, HAr), 7.71 (s, 1H, HAr), 4.80 (s, 2H, CH2). HRMS
(ESIꢀ) calcd. for C16H10F3NOꢀ3 [MꢀH]ꢀ 320.0535, found 320.0524;
HPLC tR 12.2 min.
4.1.1.41. 2-Hydroxy-6-(thiophen-3-yl)isoquinoline-1,3(2H,4H)-
dione (27g).
d6) d 10.43 (s, 1H, –NOH), 8.07 (m, 1H, HAr), 8.01 (m, 1H, HAr), 7.83
(m, 1H, HAr), 7.75 (m, 1H, HAr), 7.69 (m, 1H, HAr), 7.63 (m, 1H, HAr),
4.27 (s, 2H, CH2).
4.1.1.47. 6-(3,4-Difluorophenyl)-2-hydroxyisoquinoline-1,3(2H,
Yield: 6% (from 25g) 1H NMR (400 MHz, DMSO-
4H)-dione (27m).
(400 MHz, DMSO-d6) d 10.46 (br s, 1H, NOH), 8.06 (d, J = 8.2 Hz, 1H,
Ar), 7.91–7.82 (m, 1H, Hdifluorophenyl), 7.80 (d, J = 8.2 Hz, 1H, HAr), 7.74
Yield 11% (for two steps from 25m). 1H NMR
H
(s, 1H, HAr), 7.58 (m, 2H, 2 ꢁ Hdifluorophenyl), 4.28 (s, 2H, CH2); 13C NMR
(100 MHz, DMSO-d6) d 166.7 (C@O), 161.9 (C@O), 150.3 (dd,
J = 246.1, 7.7 Hz, CF), 150.1 (dd, J = 248.2, 6.9 Hz, CF), 142.8 (Cq,Ar),
136.5 (dd, J = 6.0, 3.9 Hz, Cq,Ar), 135.8 (Cq,Ar), 128.8 (CHAr), 126.3
(CHAr), 126.1 (CHAr), 124.9 (Cq,Ar), 124.4 (dd, J = 6.8, 3.2 Hz,
CHdifluorophenyl), 118.6 (d, J = 17.1 Hz, CHdifluorophenyl), 116.6 (d,
J = 18.1 Hz, CHdifluorophenyl), 37.5 (CH2); 19F NMR (376 MHz, DMSO-
4.1.1.42. 6-(Benzo[b]thiophen-3-yl)-2-hydroxyisoquinoline-1,3
(2H,4H)-dione (27h).
Yield 16% (for two steps from 25h). 1H
NMR (400 MHz, DMSO-d6) d 10.47 (br s, 1H, –NOH), 8.14 (d,
J = 8.1 Hz, 1H, HAr), 8.11–8.07 (m, 1H, Hbenzothiophene), 7.99 (s, 1H,
Hbenzothiophene), 7.98–7.91 (m, 1H, Hbenzothiophene), 7.72 (d, J = 8.1 Hz,
d6)
C
d
ꢀ131.30(m), ꢀ132.47(m); HRMS (ESIꢀ) calcd for
1H, HAr), 7.65 (s, 1H, HAr), 7.50–7.41 (m, 2H, 2 ꢁ Hbenzothiophene), 4.35
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.8 (C@O), 162.0
(C@O), 140.7 (Cq,Ar), 140.4 (Cq,Ar), 137.1 (Cq,Ar), 136.0 (Cq,Ar), 135.8
(Cq,Ar), 128.8 (CHAr), 127.8 (CHbenzothiophene), 127.7 (CHbenzothiophene),
127.1 (CHbenzothiophene), 125.4 (CHbenzothiophene), 125.3 (CHbenzothiophene),
124.6 (Cq,Ar), 123.8 (CHbenzothiophene), 122.8 (CHbenzothiophene), 37.5
(CH2); HRMS (ESIꢀ) calcd for C17H10NO3Sꢀ[MꢀH]ꢀ 308.0387, found
308.0377; Mp: 210–215 °C; HPLC tR 12.7 min.
15H8F2NOꢀ3 [MꢀH]ꢀ 288.0478, found 288.0483 Mp:149–156 °C
(decomp.); HPLC tR 11.4 min.
4.1.1.48. 6-(Biphenyl-3-yl)-2-hydroxyisoquinoline-1,3(2H,4H)-
dione (27n).
13% (for two steps from 25n). 1H NMR
(600 MHz, CD3Cl3) d 8.31 (d, J = 8.4, 1H, HAr), 7.81 (s, 1H, HAr),
7.79 (d, J = 8.4, 1H, HAr), 7.68 (m, 3H, HAr), 7.60 (m, 3H, HAr), 7.50
(t, J = 8.4, 2H, HAr), 7.41(t, J = 7.2, 1H, HAr), 4.28 (s, 2H, CH2); HRMS
(ESIꢀ) calcd. for C21H15NO3ꢀ[MꢀH] ꢀ 328.0974, found 328.0957;
HPLC tR 18.5 min.
4.1.1.43. 6-(Dibenzo[b,d]thiophen-4-yl)-2-hydroxyisoquinoline-
1,3(2H,4H)-dione (27i).
Yield 16% (for two steps from 25i). 1H
NMR (400 MHz, DMSO-d6) d 10.44 (s, 1H, –NOH), 8.43–8.32 (m, 2H,
HAr, Hdibenzothiophene), 8.14 (d, J = 8.1 Hz, 1H, HAr), 8.00–7.93 (m, 1H,
4.1.1.49. 2-Hydroxy-6-(3-(methylsulfonyl)phenyl)isoquinoline-
1,3(2H,4H)-dione (27o).
5% (for two steps from 25o). 1H NMR
Hdibenzothiophene), 7.82 (d, J = 8.2 Hz, 1H, HAr), 7.70 (s, 1H, Hdibenzothiophene),
(600 MHz, CD2Cl2) d 8.21 (d, J = 8.4 Hz, 1H, HAr), 7.97 (d, J = 8.4 Hz,
2H, HAr), 7.78 (d, J = 7.8 Hz, 2H, HAr), 7.67 (d, J = 8.4 Hz, 1H, HAr),
7.50 (s, 1H, HAr), 4.17 (s, 2H, CH2), 3.02 (s, 3H, Me); HRMS (ESIꢀ)
calcd. for C16H13NO5Sꢀ[MꢀH]ꢀ 330.0436, found 330.0425; HPLC
tR 15.2 min.
7.61 (t, J = 7.6 Hz, 1H, dibenzothiophene), 7.55 (d, J = 7.1 Hz, 1H,
H
H
dibenzothiophene), 7.48 (dd, J = 6.0, 3.1 Hz, 2H, 2 ꢁ Hdibenzothiophene), 4.30
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.7 (C@O), 161.9
(C@O), 144.9 (Cq,Ar), 138.7 (Cq,Ar), 137.6 (Cq,Ar), 136.5 (Cq,Ar), 136.0
(Cq,Ar), 135.5 (Cq,Ar), 135.2 (Cq,Ar), 128.9 (CHAr), 127.9 (CHdibenzothiophene),
127.7 (CHdibenzothiophene), 127.6 (CHdibenzothiophene), 127.3 (CHAr), 126.2
(CHdibenzothiophene), 125.4 (CHdibenzothiophene), 125.3 (Cq,Ar), 123.3
(CHdibenzothiophene), 122.8 (CHAr), 122.5 (CHdibenzothiophene), 37.5 (CH2);
4.2. Biology
4.2.1. Cells and recombinant protein
HCV genotype 1b Huh-7/HCV1b-Rluc replicon cells (obtained
from G. Luo, University of Kentucky) were maintained in DME sup-
HRMS (ESIꢀ) calcd for
C
21H12NO3Sꢀ[MꢀH]ꢀ 358.0543, found
358.0546; Mp: 190–193 °C (decomp.); HPLC tR 14.5 min.
plemented with 10% fetal bovine serum (fbs), 500 lgm/mL G418
4.1.1.44. 6-(Benzo[d][1,3]dioxol-5-yl)-2-hydroxyisoquinoline-
(Invitrogen), 100 IU streptomycin and 100 IU penicillin, 1ꢁ nones-
sential amino acids (Invitrogen), and 1 mM sodium pyruvate
(Invitrogen). The Huh-7/HCV1b-Rluc replicon RNA expresses neo-
mycin phosphotransferase to promote retention in the Huh7 cells
by selection with G418. The replicon also expresses renilla lucifer-
ase that can be measured to determine the level of replicon RNA
production. The HCV genotype 1b NS5B polymerase missing the
1,3(2H,4H)-dione (27j).
Yield 10% (for two steps from 25j).
1H NMR (400 MHz, DMSO-d6) d 10.40 (br s, 1H, –NOH), 8.02 (d,
J = 8.3 Hz, 1H, HAr), 7.71 (d, J = 8.2 Hz, 1H, HAr), 7.64 (s, 1H, HAr),
7.32 (s, 1H,
Hmethylenedioxyphenyl), 7.24 (d, J = 8.2 Hz, 1H,
Hmethylenedioxyphenyl), 7.03 (d, J = 8.1 Hz, 1H, Hmethylenedioxyphenyl), 6.07
(s, 2H, CH2-methylenedioxyphenyl), 4.27 (s, 2H, CH2-hydroximide);
13C NMR (100 MHz, DMSO-d6) d 166.8 (C@O), 162.0 (C@O), 148.6
(Cq,Ar), 148.3 (Cq,Ar), 144.8 (Cq,Ar), 135.7 (Cq,Ar), 133.0 (Cq,Ar), 128.7
(CHAr), 125.7 (2C, 2 ꢁ CHAr), 123.9 (Cq,Ar), 121.4 (CHmethylenedioxyphenyl),
109.3 (CHmethylenedioxyphenyl), 107.6 (CHmethylenedioxyphenyl), 101.9 (CH2-
methylenedioxyphenyl), 37.5 (CH2-hydroximide); HRMS (ESIꢀ)
calcd for C16H10NOꢀ5 [MꢀH]ꢀ 296.0564, found 296.0556; Mp: 232–
235 °C (decomp.); HPLC tR 10.5 min.
C-terminal 55 amino acids (NS5B
D55) was prepared by expressing
the plasmid pET-21d(+)-NS5B
D
55 in bacteria as described47 except
the heparin-agarose chromatography was omitted. The enzyme
was stored at ꢀ80 °C in storage buffer (50 mM Tris pH 7.5, 50%
glycerol, 1 mM b-ME).
4.2.2. HCV replicon assay
Replicon cells were plated out at 6 ꢁ 103 cells per well in a
white opaque 96-well tissue culture plate (BD Falcon) in the ab-
sence of G418. The next day, the cells were incubated at 37 °C/5%
4.1.1.45.
4H)-dione (27k).
6-(4-Fluorophenyl)-2-hydroxyisoquinoline-1,3(2H,
Yield 5% (for two steps from 25k). 1H NMR

478
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
CO2 in culture medium containing compound (dissolved in DMSO),
DMSO alone, or nothing added. Three days later, the culture med-
ium was removed from the cells and renilla luciferase ViVi-Ren
Live Cell Substrate (Promega) was added in a 1:1000 dilution in
DME without phenol red and 10% fbs. Luminescence was immedi-
ately measured on a SpectraMax E5 spectrometer (Molecular
Devices). Any given compound concentration or control was
performed in triplicate and each experiment was performed inde-
pendently at least twice. For single concentration experiments,
predictor (Advanced Chemistry Development, Inc., Toronto, Can-
ada) and the presence of the two divalent metal ions within the ac-
tive site for binding. Based on our earlier studies of HIV-1 integrase
strand transfer inhibition51
, the highest scored conformation
involving the chelating triad to the divalent metal ions bichelation
was selected as the most probable mode of inhibition. All com-
pounds within Table 2 were modeled based on this observed mode
of binding to rationalize the changes in the inhibition activity. To
account for local protein flexibility, each of the final docked com-
plexes was further refined by locally restrained energy minimiza-
tion under implicit solvent condition.
ribavirin (10 lM) and 2 mA (500 nM) were included as controls.
The 50% effective concentration (EC50) was defined as the concen-
tration of compound that reduced luciferase activity by 50%. The
EC50 was determined by comparing luciferase activity for eight
serial dilutions of compound and vehicle treated cells using
GraphPad Prism software.
Acknowledgment
This research was supported by the Center for Drug Design at
the University of Minnesota. We thank Dr. Guangxiang Luo at Uni-
versity of Kentucky for providing the Huh-7/HCV1b-Rluc replicon
cells, Dr. Riccado Petrelli at University of Camerino, Italy, for pro-
viding 2 mA and the Minnesota Supercomputing Institute for com-
puting resources.
4.2.3. Cell proliferation assay
Replicon cells were plated out at 6 ꢁ 103 cells per well in a clear
96-well tissue culture plate (Corning) in the absence of G418. The
next day, the cells were incubated at 37 °C/5% CO2 in culture med-
ium containing compound (dissolved in DMSO), DMSO alone, or
nothing added for three days. CellTiter 96 AQueous One Solution Cell
Proliferation reagent (Promega) was added according to manufac-
turer’s instructions and viability measured by spectrometry at
450 nm with a SpectraMax E5 (Molecular Devices). The 50% cyto-
toxic concentration (CC50) was defined as the concentration of
compound that reduced cell proliferation by 50%. The CC50 was
determined by comparing absorbance readings from eight serial
dilutions of compound and vehicle treated cells using GraphPad
Prism software.
Supplementary data
Supplementary data (Spectral data.) associated with this article
can be found, in the online version, at doi:10.1016/j.bmc.2011.
10.058.
References and notes
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Primer-dependent NS5B
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NaCl, 5 mM KCl, 0.5 mM MnCl2, 4 U RNasin (Promega), 1 mM
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4.2.5. Modeling and docking
The study was carried out using Schrodinger modeling suite.48
The X-ray crystallographic structure of HCV NS5B in complex with
Mn2+ ions and UTP (PDB:1NB645) was taken from Protein Data
Bank. The crystallographic Mn2+ ions were replaced with Mg2+
ions. All missing hydrogen atoms were added by standard protein
preparation protocol within Maestro followed by energy minimi-
zation using OPLS 2005 forcefield49 to optimize all hydrogen bond-
ing networks. Docking of the most active compound 27a was
carried out using Glide with standard precision protocol50 with
the Mg2+ ions defined as a required constraints. The van der Waals
radii of nonpolar atoms for each of the ligands were scaled by a fac-
tor of 0.8 to account for structure variability to specific ligand bind-
ing. The ionized form of compound 27a was used based on the
estimated pKa of its hydroxyl group of 7.6 by ACDLabs pKa

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 467–479
479
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