C-6 aryl substituted 4-quinolone-3-carboxylic acids as inhibitors of hepatitis C virus

Article abstract of DOI:10.1016/j.bmc.2012.05.066

Quinolone-3-carboxylic acid represents a highly privileged chemotype in medicinal chemistry and has been extensively explored as antibiotics and antivirals targeting human immunodeficiency virus (HIV) integrase (IN). Herein we describe the synthesis and anti-hepatitis C virus (HCV) profile of a series of C-6 aryl substituted 4-quinlone-3-carboxylic acid analogues. Significant inhibition was observed with a few analogues at low micromolar range against HCV replicon in cell culture and a reduction in replicon RNA was confirmed through an RT-qPCR assay. Interestingly, evaluation of analogues as inhibitors of NS5B in a biochemical assay yielded only modest inhibitory activities, suggesting that a different mechanism of action could operate in cell culture.

Full text of DOI:10.1016/j.bmc.2012.05.066

Bioorganic & Medicinal Chemistry 20 (2012) 4790–4800
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C-6 aryl substituted 4-quinolone-3-carboxylic acids as inhibitors
of hepatitis C virus
⇑
Yue-Lei Chen , Jeana Zacharias, Robert Vince, Robert J. Geraghty, Zhengqiang Wang
Center for Drug Design, Academic Health Center, University of Minnesota, 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:
Quinolone-3-carboxylic acid represents a highly privileged chemotype in medicinal chemistry and has
been extensively explored as antibiotics and antivirals targeting human immunodeficiency virus (HIV)
integrase (IN). Herein we describe the synthesis and anti-hepatitis C virus (HCV) profile of a series of
C-6 aryl substituted 4-quinlone-3-carboxylic acid analogues. Significant inhibition was observed with a
few analogues at low micromolar range against HCV replicon in cell culture and a reduction in replicon
RNA was confirmed through an RT-qPCR assay. Interestingly, evaluation of analogues as inhibitors of
NS5B in a biochemical assay yielded only modest inhibitory activities, suggesting that a different mech-
anism of action could operate in cell culture.
Received 27 March 2012
Revised 21 May 2012
Accepted 29 May 2012
Available online 7 June 2012
Keywords:
Hepatitis C virus inhibitors
6-Aryl-4-quinolone-3-carboxylic acids
NS5B polymerase
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
HCV is an enveloped, positive-sense and single-stranded RNA
virus belonging to the Hepacivirus genus of the Flaviviridae family.
HCV1 infects an estimated 180 million people globally2 and
approximately 4 million in the USA.3 Chronic HCV infection pro-
gresses through liver injury, fibrosis, cirrhosis and can lead to
hepatocellular carcinoma.4,5 Until the recent approval of two pro-
tease inhibitors, the standard of care (SOC) for treating HCV infec-
tion has been the combination of subcutaneous pegylated
interferon (peg-IFN) alpha and oral ribavirin (RBV).6 By boosting
the host immune system this SOC has proved only moderately suc-
cessful in patients infected with HCV genotype 1,6,7 the predomi-
nant genotype in North America and Europe, with a 40ꢀ50%
sustained virological response (SVR) rates. In addition, the peg-
IFN/RBV regimen is associated with severe adverse effects includ-
ing fatigue, hemolytic anemia, depression, and flu like symptoms.8
Developing novel HCV chemotherapy with better efficacy and tol-
erability continues to be an endeavor of tremendous interest from
both academia and industry. Direct-acting antivirals (DAAs) specif-
ically targeting HCV replication have consistently yielded im-
proved antiviral profiles and would provide better treatments for
HCV infection.9
Its genomic RNA encodes a polyprotein precursor of about 3000
amino acids, which is processed first by host peptidases at the N-
terminus to release four structural proteins (core, E1, E2 and P7),
and then by the virally encoded NS2 zinc dependent protease
and NS3-4A serine proteases to yield six nonstructural (NS) pro-
teins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).10 Apparently the
NS3-4A serine-protease is essential for viral replication and repre-
sents an important antiviral target11 as exemplified by the success-
ful development of telaprevir (1)12,13 and boceprevir (2)14,15
(Fig. 1), the two recently approved protease inhibitors. Combined
with PEG-IFN/RBV, these protease inhibitors treat HCV infection
with significantly improved SVR rates and reduced duration,
though the response is largely limited to genotype 1 HCV infection
and the regimens are susceptible to resistance development.10
Second-generation HCV protease inhibitors with improved effica-
cies against resistant viral strains are being actively pursued.16–21
NS5B is the RNA dependent RNA polymerase, thus another major
target for HCV chemotherapy development. Numerous nucleoside
inhibitors (NIs)10,22 and non-nucleoside inhibitors (NNIs)23–25 of
NS5B are in current HCV antiviral pipeline. The NIs such as the uri-
dine analogue PSI-7977 (3, Fig. 2)26 and the guanosine analogue
PSI-938 (4, Fig 2),27 target the highly conserved active site, thus
pose a high genetic barrier to resistance development and confer
a pan-genotypic antiviral activity. By contrast, the NNIs bind to
mutable allosteric sites and are more susceptible to resistance
mutations. GS-919028 (5, Fig. 2) isan established NNI with excep-
tional in vitro antiviral activityA third HCV-encoded protein,
NS5A, is also becoming an increasingly important target for HCV
drug discovery.29–31 NS5A is a phosphoprotein associated with
Abbreviations: HIV, human immunodeficiency virus; IN, integrase; HCV, hepa-
titis C virus; SOC, standard of care; peg-INF, peglylated interferon; RBV, ribavirin;
SVR, sustained virological response; DAA, direct-acting antiviral; NNI, non-nucle-
oside inhibitor; EVG, elvitegravir; Luc, luciferase; 20-C-Me-A, 20-C-methyl adeno-
sine; SAR, structure-activity-relationship; RT-qPCR, reverse transcription and
quantitative polymerase chain reaction.
⇑
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 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.066

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
4791
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
2, Boceprevir
1, Telaprevir
Figure 1. The first FDA-approved DAAs: protease inhibitors telaprevir (1) and boceprevir (2)
O
N
O
O
NH
O
O
HN P
O
O
N
N
O
O
O
P
N
N
O
O
N
O
NH2
F
O
OH F
3, PSI-7977
4, PSI-938
F
H
N
N
N
O
MeO
O
N
CF3
N
N
N
H
N
N
OMe
N
N
O
HN
H
F3C
O
6, BMS-790052
5, GS-9190
Figure 2. Representative HCV inhibitors in clinical development: 3, non-nucleoside polymerase inhibitor; 4 and 5, nucleoside polymerase inhibitors; 6, NS5A inhibitor.
Quinolones are among the most privileged molecular scaffolds
O
N
O
in medicinal chemistry.34 Of particular importance is the 4-quino-
lone-3-carboxylic acid chemotype that constitutes a major class of
antibacterial agents. Four generations of quinolone-3-carboxylic
acid antibiotics (Fig. 3) have been developed for Gram-positive,
Gram-negative and anaerobic bacterial infections.35 Obviously this
chemotype possesses highly favorable pharmacokinetic properties
and provides an attractive scaffold for designing inhibitors of other
medicinally interesting targets, a notable example being HIV IN
inhibitor elvitegravir (EVG, 13, Fig. 4).36 EVG builds on the 4-quin-
olone-3-carboxylic acid core and mimics the canonical diketoacid
pharmacophore for chelating Mg2+ ions.37 Significantly, HCV
NS5B polymerase shares a similar active site fold to HIV IN,38
which is manifested by the resemblance between their inhibitor
pharmacophores (Fig. 4). Numerous chemotypes including the
5-hydroxy-pyrimidinones (1139 and 1440) and 2-hydroxyisoquino-
line-1,3-diones (12,41–43 15–1644) have shown potent inhibition
against both HIV IN and HCV NS5B. The major pharmacophore dif-
ference, however, is that IN inhibition requires a flexible terminal
benzyl group whereas a rigid aromatic group, typically a thiophene
or furan moiety,23 is immediately connected to the core of NS5B
inhibitors (Fig. 4). Naturally we have decided to synthesize and test
scaffold 17 as HCV inhibitors. During the course of our work, a
quinolone-3-carboxylic acid ester series was reported to inhibit
HCV through targeting NS5B.45
O
N
O
F
F
OH
OH
N
HN
8, Ciprofloxacin
7, Flumequine
O
O
O
O
F
F
OH
OH
H
N
N
N
N
O
N
O
NHH
9, Levofloxacin
10, Moxifloxacin
Figure 3. Four generations of clinical quinolone antibiotics, all featuring
quinolone-3-carboxylic acid core: 7, first generation; 8, second generation; 9, third
generation; and 10, fourth generation.
a
host cell membranes without apparent enzymatic activity. By
binding directly to the NS5B polymerase, NS5A constitutes an
essential component of the HCV replication complex and is be-
lieved to modulate the activity of NS5B.32,33 BMS-790052 (6),29,31
the first-in-class NS5A inhibitor is currently under clinical develop-
ment. These experimental drugs and many others in the pipeline
have the great potential to enhance current regimens. However,
the challenges of HCV chemotherapy in achieving pan-genotypic
efficacy, large barrier to resistance selection, interferon-free regi-
men and efficacy in hard-to-treat populations necessitate contin-
ued efforts in search for novel inhibitors. We report herein the
design, chemical synthesis and biological evaluations of quino-
lone-3-carboxylic acid analogues as HCV inhibitors.
2. Results and discussion
2.1. Chemistry
Synthesis of quinolone scaffold is well documented.46 In our
case, the key quinolone-3-carboxylic intermediates 23–24 were
synthesized via the classic Gould–Jacobs approach.47 This reaction

4792
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
HIV IN inhibitors HCV NS5B inhibitors
O
OH
OH
OH
OH
N
N
N
N
H
N
O
O
N
N
F
HN
O
S
O
11, Raltegravir
14, IC50 = 2.6 µM
NH
O
O
O
X
N OH
N OH
O
O
15: X = O, IC50 = 1.3 µM
16: X = S, IC50 = 7.3 µM
12
F
O
O
O
O
Cl
OH
OH
Ar
O
N
N
R
OH
17, novelinhibitors?
13, Elvitegravir
Figure 4. Design of novel NS5B inhibitor scaffold 17 based on the pharmacophore resemblance between HIV IN inhibitors and HCV NS5B inhibitors.
maintained in Huh7 human liver cells (Huh-7/HCV1b-Rluc) was
EtO2C
EtO
CO2Et
H
H
used. HCV subgenomic viral RNAs (replicons) capable of self-
replication in human liver cells are commonly used to identify
and characterize inhibitors of HCV replicative enzymes.49–51 The
replicon RNA expresses renilla luciferase (Luc) thereby permitting
the measurement of Luc activity as an indication of the level of
replicon RNA.
X
a
CO2Et
CO2Et
X
+
N
H
NH2
18: X = 4-I
19: X = 3-Br
21
: X = 4-I
22: X = 3-Br
20
O
O
O
O
The initial testing of all quinolone-3-carboxylic acid analogues in
the Huh7 replicon assay was at a single concentration of 10
and 20-C-methyl adenosine (20-C-Me-A)52 were included in each
experiment as control compounds and known inhibitors at 10
and 0.5 M, respectively. Replicon cells were also treated with vehi-
cle alone (DMSO) and cell culture medium in each assay. Com-
pounds showing apparent Luc reduction (P20%) were tested for
cell viability using an MTS-based colorimetric assay. Results from
these testings are summarized in Table 1.
These preliminary screening results revealed a few discernible
structure–activity-relationship (SAR) trends. First of all, the substi-
tution position of the aromatic ring appears to greatly impact
potency as all C7 analogues were inactive (25o–r, Table 1) whereas
a number of C6 isomers showed significant activity (25a, 25c, 25e,
25g, 25i, 25k and 25l, Table 1). This trend could reflect the influ-
ence of different molecular shapes between C6 and C7 isomers
on target binding. Secondly, the N-1 substituent also appears cru-
cial for inhibition, with unsubstituted (25d, 25h) and simple alkyl
substituted (25m) analogues showing no activity while hydroxy-
ethyl and hydroxypropyl groups are substantially better for inhibi-
tion. Thirdly, for the aromatic substituent both 2-thiophene (25e)
and 2-furan (25i) seem to be tolerated. However, the best inhibi-
tion was achieved with 2-benzothiophene (25g) and 2-benzofuran
(25k, 25l) substituted analogues. Other aromatic substituents,
including 5-acylthiophene (25f), 3-furan (25j) and para-methoxy-
phenyl (25n), failed to yield appreciable inhibitory activity against
HCV replicon.
lM. RBV
b
c
OEt
OH
X
R1
N
H
N
l
M
R2
l
23
: X = 6-I
25a-r
24: X = 7-Br
Scheme 1. Synthesis of C-6/C-7 substituted quinolone-3-carboxylic acids. Reagents
and conditions: (a) toluene, reflux, 58–91%; (b) Ph2O, reflux, 42–96%; (c) (i) K2CO3,
DMF, 100 °C oil bath, 30 min, then alkylating reagent (R2-Br),
l
W 110 °C, 2.8 h; (ii)
R1B(OH)2, K2CO3, PdCl2,
lW 130 °C, 35 min; (iii) pTSA/MeOH (O-THP protection) or
K2CO3/H2O (O-Ac protection), 11–99% over three steps.
sequence began with an addition-elimination reaction between
halogen substituted anilines 18–19 and ethoxy ethylenemalonate
20 to produce malonate intermediates 21–22, which upon intra-
molecular cyclization yielded the desired quinolone-3-carboxylic
intermediates 23–24. The halogen in these intermediates serves
as a synthetic handle for the introduction of a panel of aromatic
rings onto the C-6 or C-7 site. This was achieved by first alkylating
the N-1 position followed by a Suzuki coupling reaction48 with
commercial aromatic boronic acids. The alkylating reagents con-
taining a hydroxyl group are preferably protected by THP for opti-
mal Suzuki coupling step. O-Ac protection also allowed the Suzuki
coupling reaction, albeit with a lower yield. The resulting esters
were converted to acid analogues 25a–r via removal of O-protec-
tion and saponification.
With the preliminary results, four compounds with significant
inhibition (P40%) were then selected for further testing in dose re-
sponse fashion. Both RBV and 20-C-Me-A were used as controls for
this assay. The results are shown in Figure 5 and summarized in
2.2. HCV replicon assay
To evaluate the ability of quinolone-3-carboxylic acid analogues
to inhibit HCV replication, an HCV genotype 1b replicon stably

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
4793
Table 1
Preliminary screening results of compounds 25a-r against HCV replicon
O
O
OH
R1
N
R2
25a-r
Compd
C6/C7
R1
R2
Luc reduction% (10
l
M)a
Cell viability% (10 lM)
25a
25b
6
6
I
I
H
Bn
22
0
100
99
OH
25c
6
I
21
100
25d
6
H
0
100
S
OH
OH
OH
25e
25f
6
6
40
0
100
100
S
O
S
25g
6
58
100
S
25h
25i
25j
6
6
6
H
0
100
100
100
O
O
OH
OH
OH
20
0
O
25k
25l
6
6
6
63
44
4
100
100
91
O
O
O
OH
25m
OH
OH
25n
25o
6
7
0
95
MeO
14
100
S
S
O
OH
25p
25q
7
7
12
15
97
OH
OH
100
25r
7
14
94
O
RBV
20-C-Me-A
—
—
—
—
—
—
44
98
92
87
a
Reduction of Luc activity, indicating inhibitory activity of a compound.
Table 2. Notably compounds with a benzo fused aromatic substitu-
ent (25g and 25k) were found to inhibit HCV replicon at low micro-
molar range, while the 2-thiophene analogue (25e) and the N-1
hydroxypropyl substituted analogue (25l) were considerably less
potent. This finding is consistent with the SAR trends observed
from the preliminary screening. Nevertheless, the two best com-
pounds (25g and 25k) inhibit HCV clearly better than RBV, suggest-
ing that quinolone-3-carboxylic acid scaffold has the potential for
antiviral discovery against HCV. Future work needs to address
the toxicity and the low therapeutic indices associated with these

4794
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
CN
Cl
Cl
O
OH
O
OH
O
26
Figure 7. Evaluation of compounds as inhibitors of NS5B primer-dependent RNA
polymerase activity. Primer-dependent incorporation of [3H]UTP on
poly(A)
template by recombinant NS5B 55 was measured in duplicate samples in the
presence of compound or DMSO and mean value compared to that of DMSO. The
mean value and standard deviation of two independent experiments are shown. A
known NS5B inhibitor 2654 was used as a control.
a
Figure 5. Representative dose–response curves for compound inhibition of HCV
replicon-containing cells. The percent (%) inhibition of each compound compared to
DMSO alone is shown. Doses were performed in triplicate and repeated 2–3
independent times. Average values for each dose plus standard deviation for one
representative experiment are shown.
D
measured using the TaqManÒ Gene Expression Cells-to-CT system
(Applied Biosystems) and TaqManÒ primers/probes. The primers/
probes to measure HCV replicon cDNA were custom synthesized
to NS5B (Applied Biosystems). The control primers/probe recog-
nize GAPDH cDNA (Applied Biosystems). Parallel wells of lysate
were analyzed for either HCV replicon RNA or GAPDH RNA and
relative levels of HCV RNA calculated using the comparative
method.53 The percent reduction in replicon RNA for cells treated
with 25k compared to cells treated with DMSO alone is shown in
Table 2
Dose response antiviral results of selected compounds against HCV replicon
Compd
EC50
(
l
M)a
CC50
>50
15 3.5
34 22
25 2.1
32
(
l
M)b
TIc
25e
25g
25k
25l
36 3.5
5.9 2.2
5.8 1.7
23 4.2
14 4.5
0.20 0.11
>1.4
2.5
5.9
1.1
2.3
RBVd
20-C-Me-Ad
55 15
270
Figure 6. Compound 25k was tested at 10
l
M and reduced replicon
a
RNA approximately 70%. 20-C-Me-A treatment (0.5
lM) reduced
Concentration inhibiting virus replication by 50%, mean value standard
deviation from at least two independent determinations.
RNA levels approximately 95%. We conclude from this data that
25k inhibits replicon RNA production as expected.
b
Concentration resulting in 50% cell death.
c
Therapeutic index, defined by CC50/EC50
.
d
Ref.44
2.4. Biochemical NS5B assay
Quinolone-3-carboxylic acid scaffold is known to inhibit HIV IN
based on its ability to chelate Mg2+ ions at the enzyme active
site.36,37 HCV NS5B catalysis also involves Mg2+ chelation as well
as a similar fold of active conformation. Therefore, we had hypoth-
esized that our quinolone analogues will inhibit NS5B, likely via
binding to the active site. To test this hypothesis, two compounds
(25e and 25g) active in the replicon assay were selected for study
in the biochemical NS5B assay measuring primer-dependent RNA
polymerase activity from a heterologous template. A known potent
DKA NS5B inhibitor 2654 was used as a control compound. The as-
say results are shown in Figure 7. Notably while the control com-
pound inhibits NS5B effectively at 1
inhibitors showed only modest inhibition at concentrations up to
50 M. In addition, the inhibition observed at 10 M and 50 lM
lM in our assay, our HCV
l
l
did not appear to occur in a strict dose-dependent fashion for
either 25e or 25g, especially 25e where the increase in dose did
not significantly improve the percent inhibition. Furthermore, vir-
Figure 6. Compound 25k inhibits HCV replicon RNA production. Relative HCV
replicon RNA level for cells treated with control compound 20-C-Me-A (0.5
M), 25k
(10 M) or DMSO alone for 3 days prior to RNA isolation and RT-qPCR.
l
l
tually no inhibition was observed with compound 25k at 10
lM
and 50 M doses (data not shown). The lack of robust and dose-
l
dependent inhibition of NS5B in this assay may indicate that these
analogues are not NS5B inhibitors. There are, however, established
NS5B inhibitors that do not demonstrate inhibition in biochemical
assays measuring recombinant NS5B polymerase activity.55 Such
inhibitors may require NS5B expression in the cell with other viral
and cellular proteins to manifest inhibition. Therefore further
experimentation is required to provide a better indication of mech-
anism of action.
compounds, which might reflect their ability to bind to DNA topo-
isomerase, the underlying mechanism of action for quinolone
antibiotics.35
2.3. RT-qPCR assay
To further confirm that these quinolone compounds actually in-
hibit HCV sub-genomic replication, we have also performed an as-
say to directly detect the viral replicon RNA level using RT-qPCR.
This was done by lysing cells in a 96-well dish, converting RNA
in that lysate to cDNA, and conducting real-time PCR to quantify
cDNA levels. Given the high cost of this assay, a representative
compound 25k was chosen and the relative level of replicon RNA
for compound-treated cells compared to DMSO-treated cells was
3. Conclusions
Quinolone-3-carboxylic acid is a highly privileged molecular
scaffold, particularly as antibacterial agents and HIV IN inhibitors.
Based on the similar fold of active site shared by HIV IN and HCV

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
4795
NS5B and the pharmacophore resemblance between their inhibi-
tors, we designed C-6 aromatic substituted quinolone-3-carboxylic
acids as a novel inhibitor scaffold for HCV. These analogues were
synthesized following a classic Gould-Jacobs reaction sequence
and featuring a Suzuki coupling reaction. All synthetic analogues
were tested in a primary screening assay using HCV replicon 1b
Huh-7/HCV1b-Rluc cells. Four active compounds were further
tested in a dose response fashion, which identified 25k as the best
(Cq,aryl), 133.42(Ct,aryl), 128.71(Ct,aryl), 125.35(Ct,aryl), 115.90(Ct,aryl),
113.66(Cq,aryl), 95.50(N-C=C), 60.57(Et), 60.25(Et), 14.38(Et),
14.32(Et); HRMS (ESI+) calcd for C14H17BrNO4+(M+H+) 342.0335,
found 342.0310; HPLC tR 15.9 min; Mp: 61–62 °C.
4.1.4. Ethyl 6-iodo-4-oxo-1H-quinoline-3-carboxylate (23)56
3 mL of diphenyl ether was vigorously stirred in a test tube and
heated to reflux with a sand bath, and to which, 1.0 g (2.6 mmol) of
starting material (21) was added. The resulting solution was re-
fluxed for another 30 min, cooled to rt, and triturated with diethyl
ether to give the product as a precipitation, which was collected by
filtration. The solid was washed with diethyl ether for several
times to give 0.86 g (2.5 mmol) of grey powder 23 (96%). 1H NMR
(400 MHz, DMSO-d6, measured at 100 °C due to solubility issue)
d 12.39 (s, 1H, NH), 8.55 (s, 1H, H-2), 8.41 (d, J = 2.0 Hz, 1H, Haryl),
7.97 (dd, J = 8.6, 2.1 Hz, 1H, Haryl), 7.42 (d, J = 8.6 Hz, 1H, Haryl),
4.20 (q, J = 7.1 Hz, 2H, Et), 1.26 (t, J = 7.1 Hz, 3H, Et). 13C NMR
(100 MHz, DMSO-d6, measured at 100 °C due to solubility issue)
HCV inhibitor of this series (EC50 = 5.8 lM, CC50 = 34 lM). The abil-
ity of 25k to inhibit HCV viral RNA production was verified through
an RT-qPCR assay. However, the biochemical NS5B polymerase as-
say yielded poor inhibition suggesting that a mechanism of action
other than NS5B inhibition is possible.
4. Experimental
4.1. Chemistry
@
@
4.1.1. General procedures
d 172.40(C O), 164.97(C O), 145.64(C-2), 140.93(CHaryl), 139.02
@
@
All commercial chemicals were used as supplied unless other-
wise indicated. Dry solvents (THF, Et2O, CH2Cl2 and DMF) were
dispensed under argon from an anhydrous solvent system with
two packed columns of neutral alumina or molecular sieves. Flash
chromatography was performed on a Teledyne Combiflash RF-200
with RediSep columns (silica) and indicated mobile phase. All reac-
tions 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
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 ꢁ 4.6 mm). HPLC condi-
tions: solvent A = H2O, solvent B = MeCN; flow rate = 1.0 mL/min;
Gradient (B%): 0–13 min (10–95); 13–20 min (95); 20–23 min
(95–10); 23–25 min (10). All tested compounds have a purity P
96%.
(Cq,aryl), 134.51(CHaryl), 129.36(Cq,aryl), 121.65(CHaryl), 110.72
(Cq,aryl), 90.09(Cq,aryl), 60.13(Et), 14.76(Et); HRMS (ESI-) calcd for
C12H9INO3-(M-H+) 341.9633, found 341.9641; HPLC: Solubility
is too poor to make an HPLC analysis; Mp: 322–324 °C (decomp.).
4.1.5. Ethyl 7-bromo-4-oxo-1H-quinoline-3-carboxylate (24)56
3 mL of diphenyl ether was vigorously stirred in a test tube and
heated to reflux with
a sand bath, and to which, 800 mg
(2.35 mmol) of starting material (22) was added. The resulting
solution was refluxed for another 30 min, cooled to rt, and tritu-
rated with diethyl ether to give the product as a precipitation,
which was collected by filtration. The solid was washed with
diethyl ether for several times to give 497 mg of white powder
as the crude product, which is sufficiently pure for the further cou-
pling reactions. For analytical purpose, the crude product was
washed with 20% MeOH in DCM for three times by means of cen-
trifuge. The resulting solid was dissolved in a minimum amount of
boiling DMSO, and then cooled to rt to give the pure product as a
white powder 24 (291 mg, 0.99 mmol, 42%). 1H NMR (400 MHz,
DMSO-d6, measured at 100 °C due to solubility issue) d 11.88
(s, 1H, NH), 8.45 (s, 1H, H-2), 8.07 (d, J = 8.6 Hz, 1H, Haryl), 7.80
(d, J = 1.7 Hz, 1H, Haryl), 7.51 (dd, J = 8.6, 1.7 Hz, 1H, Haryl), 4.24
(q, J = 7.1 Hz, 2H, Et), 1.29 (t, J = 7.1 Hz, 3H, Et). 13C NMR
(100 MHz, DMSO-d6, measured at 100 °C due to solubility issue)
4.1.2. Ethyl a
-carbethoxy-b-p-iodoanilinoacrylate (21)56
21.9 g (0.1 mol) of 4-iodoaniline and 21.6 g (0.1 mol) of diethyl
ethoxymethylene malonate were dissolved in 150 mL of toluene
and refluxed for 1.0 h. The mixture was then evaporated to dry-
ness. The residue was adsorbed on silica gel and purified by stan-
dard flash chromatography method with EtOAc and hexanes to
give 22.7 g (0.058 mol) of product 21 (58%) as a light grey crystal.
Rf 0.58 (EtOAc:hexanes = 1:2); 1H NMR (400 MHz, CDCl3) d 10.96
(d, J = 13.5 Hz, 1H, NH), 8.44 (d, J = 13.5 Hz, 1H, Halkene), 7.68–7.61
(m, 2H, Haryl), 6.92–6.84 (m, 2H, Haryl), 4.26 (pseudo-dq, J = 21.6,
7.1 Hz, 4H, Et), 1.36 (t, J = 7.1 Hz, 3H, Et), 1.31 (t, J = 7.1 Hz, 3H,
@
@
d 173.17 (C O), 164.99(C O), 145.27(C-2), 140.86(Cq,aryl), 128.36
@
@
(CHaryl), 127.94(CHaryl), 126.76(Cq,aryl), 126.00(Cq,aryl), 121.55
(CHaryl), 112.01(Cq,aryl), 60.03(Et), 14.62(Et); HRMS (ESI-) calcd for
C12H9BrNO3-(M-H+) 293.9771, found 293.9772; HPLC: Solubility
is too poor to make an HPLC analysis; Mp: 334–335 °C (decomp.,
recrystallized from DMSO).
Et). 13C NMR (100 MHz, CDCl3) d 169.23(C O), 165.82(C O),
@
@
@
@
@
151.47(N–C C), 139.35(2C, Ct,aryl), 139.01(2C, Ct,aryl), 119.21(Cq,aryl),
@
@
94.73(N–C C), 88.23(C Cq,aryl), 60.83(Et), 60.53(Et), 14.72(Et),
4.2. General procedure for preparation of compounds 25a–r36
@
14.58(Et); HRMS (ESI+) calcd for C14H17INO4+(M+H+) 390.0197,
found 390.0202; HPLC tR 16.2 min; Mp: 110–111 °C.
(a) Alkylation and Suzuki coupling in one pot: A mixture of
0.58 mmol of halogen derivative 23 or 24, 100 mg (0.72 mmol,
1.2 equiv) of K2CO3 and 3 mL of DMF was stirred in a 100 °C oil bath
for 30 min, cooled to rt, and treated with alkylating reagent (1.2 equiv
to halogen derivative). The resulting mixture was subjected to micro-
wave reactor at 110 °C for 10,000 s, cooled to rt, charged with a mix-
ture of boronic acid (1.2 equiv to halogen derivative), 115 mg of
K2CO3 (1.2 equiv to boronic acid), and 15 mg of PdCl2, and again sub-
jected to microwave reactor at 130 °C for 35 min. The resulting sus-
pension was evaporated to dryness. The residue was adsorbed on
silica gel and purified by standard flash chromatography method
with MeOH and DCM to give the crude product (bearing protected
and unprotected hydroxyls) as a mixture.
4.1.3. Ethyl a
-carbethoxy-b-m-bromoanilinoacrylate (22)56
Starting from 8.6 g (50 mmol) of 3-bromoaniline and 11.0 g
(51 mmol) of diethyl ethoxymethylene malonate, the same proce-
dure as described for the preparation of compound (21) gave
15.58 g (45.7 mmol) of white solid 22 (91%). Rf 0.40 (EtOAc:hex-
anes = 1:5); 1H NMR (400 MHz, CDCl3) d 11.23 (d, J = 13.2 Hz, 1H,
NH), 8.46 (d, J = 13.3 Hz, 1H, Halkene), 7.55 (dd, J = 8.0, 1.2 Hz, 1H,
Haryl), 7.37–7.20 (m, 2H, Haryl), 6.97 (td, J = 8.0, 1.5 Hz, 1H, Haryl),
4.32 (q, J = 7.1 Hz, 2H, Et), 4.23 (q, J = 7.1 Hz, 2H, Et), 1.36 (t,
J = 7.1 Hz, 3H, Et), 1.31 (t, J = 7.1 Hz, 3H, Et). 13C NMR (100 MHz,
@ @
168.31(C O), 165.57(C O), 150.46(N-C=C), 137.67
@ @
CDCl3)
d

4796
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
(b) Removal of O-THP and saponification in one pot: In the case of
(400 MHz, DMSO-d6) d 13.44 (s, 1H, NH), 8.84 (s, 1H, H-2), 8.36 (d,
J = 2.1 Hz, 1H, Haryl), 8.17 (dd, J = 8.7, 2.1 Hz, 1H, Haryl), 7.82
using O-THP alkylating reagents, the crude product was mixed with
150 mg of pTSA hydrate in 3 mL of MeOH. The mixture was stirred at
rt until TLC indicated disappearance of the protected compound. All
volatile components were removed under vacuum. The residue was
mixed with 1.2 g of K2CO3 and 3 mL of water and subjected to micro-
wave reactor at 130 °C for 35 min. The resulting mixture (at ca. 70–
90 °C) was filtered through glass wool, which was washed with boil-
ing aq. K2CO3 (10% w/v) for several times. Combined filtrates were
cooled to rt and acidified (to pH = 1) with 2 N HCl to give the product
25a–r as a precipitation, which was collected by filtration, washed
with water for several times, and dried at rt overnight.
(d, J = 8.7 Hz, 1H,
Haryl), 7.64 (m, 2H, 2x Hthiophene), 7.17
(dd, J = 5.1, 3.6 Hz, 1H, Hthiophene). 13C NMR (101 MHz, DMSO-d6) d
@
@
178.41(C O), 166.76(C O), 145.43(C-2), 142.11(Cq,aryl), 139.16
@
@
(Cq,aryl), 132.04(Cq,aryl), 131.53(CHaryl), 129.32(CHthiophene), 127.36
(CHthiophene), 125.52(CHthiophene), 125.27(Cq,aryl), 121.25(CHaryl),
120.65(CHaryl), 108.23(Cq,aryl); HRMS (ESI-) calcd for C14H8NO3S
ꢀ(MꢀH+) 270.0230, found 270.0234; HPLC tR 10.0 min; Mp: >400 °C.
4.2.5. 1-(2-Hydroxyethyl)-6-(thiophen-2-yl)-4-oxo-quinoline-3-
carboxylic acid (25e)
(c) Removal of O-Ac, if applicable, and saponification in one pot:
In the case of using O-Ac alkylating reagents or alkylating reagent
without protected hydroxyls, The residue was directly mixed with
1.0 g of K2CO3 and 3 mL of water and subjected to microwave reac-
tor at 130 °C for 35 min. The resulting mixture (at ca. 70–90 °C)
was filtered through glass wool, which was washed with boiling
aq K2CO3 (10% w/v) for several times. Combined filtrates were
cooled to rt and acidified (to pH = 1) with 2 N HCl to give the prod-
uct 25a–r as a precipitation, which was collected by filtration,
washed with water for several times, and dried at rt overnight.
Prepared from (1) THPOCH2CH2Br and 24, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yellow
powder, yield: 51% for three steps. 1H NMR (400 MHz, DMSO-d6) d
8.87 (s, 1H, H-2), 8.35 (d, J = 8.5 Hz, 1H, Haryl), 8.13 (s, 1H, Haryl),
7.90 (d, J = 3.0 Hz, 1H, Hthiophene), 7.86 (dd, J = 8.5, 1.2 Hz, 1H, Haryl),
7.75 (d, J = 4.3 Hz, 1H, Hthiophene), 7.24 (dd, J = 4.3, 3.0 Hz, 1H,
Hthiophene), 5.07 (br s, 1H, -OH), 4.70 (t, J = 4.5 Hz, 2H, NCH2CH2OH),
3.80 (br s, 2H, NCH2CH2OH). 13C NMR (100 MHz, DMSO-d6) d
@
@
177.70(C O), 166.56(C O), 151.20(C-2), 141.72(Cq,aryl), 140.63
@
@
(Cq,aryl), 139.47(Cq,aryl), 129.41(CHthiophene), 129.06(CHthiophene),
127.54(CHaryl), 127.44(CHthiophene), 124.70(Cq,aryl), 124.17(CHaryl),
4.2.1. 6-Iodo-4-oxo-quinoline-3-carboxylic acid (25a)
113.87(CHaryl),
107.68(Cq,aryl),
59.10(NCH2CH2OH),
56.31
Prepared from saponification of 23, white solid, yield: 99%. 1H
NMR (400 MHz, DMSO-d6) d 13.44 (s, 1H, -NH), 8.88 (s, 1H, H-2),
8.51 (d, J = 2.0 Hz, 1H, Haryl), 8.12 (dd, J = 8.7, 2.0 Hz, 1H, Haryl),
7.58 (d, J = 8.7 Hz, 1H, Haryl). 13C NMR (100 MHz, DMSO-d6) d
(NCH2CH2OH); HRMS (ESI-) calcd for C16H12NO4Sꢀ(MꢀH+)
314.0493, found 314.0486; HPLC tR 10.0 min; Mp: 237–240 °C.
4.2.6. 1-(2-Hydroxyethyl)-6-(4-acetylthiophen-2-yl)-4-oxo-
quinoline-3-carboxylic acid (25f)
@
@
177.35(C O),
166.45(C O),
146.05(C-2),
142.34(CHaryl),
@
@
139.23(Cq,aryl), 133.77(CHaryl), 126.51(Cq,aryl), 122.28(CHaryl),
108.54(Cq,aryl), 91.97(Cq,aryl); HRMS (ESI-) calcd for C10H5I-
NO3-(M-H+) 313.9320, found 313.9311; HPLC tR 9.3 min; Mp:
335–340 °C (decomp.).
Prepared from (1) THPOCH2CH2Br and 23, (2) corresponding boro-
nic acid, and (3) removal of O-THP and saponification, yellow solid,
yield: 81% for three steps. 1H NMR (400 MHz, DMSO-d6) d 8.86 (s,
1H, H-2), 8.52 (d, J = 2.2 Hz, 1H, Haryl), 8.25 (m, 1H, Haryl), 8.10 (d, J =
9.0 Hz, 1H,
Haryl), 7.95 (d, J = 3.9 Hz, 1H, Hthiophene), 7.81 (d,
J = 3.9 Hz, 1H, Hthiophene), 5.04 (t, J = 5.2 Hz, 1H, -CH2OH), 4.62 (d,
J = 4.6 Hz, 2H, NCH2CH2OH), 3.77 (m, 2H, NCH2CH2OH), 2.54 (s, 3H,
4.2.2. 1-Benzyl-6-iodo-4-oxo-quinoline-3-carboxylic acid (25b)
Prepared from (1) BnBr and 23, and (2) saponification, white so-
lid, yield: 61% for two steps. 1H NMR (400 MHz, DMSO-d6) d 9.26 (s,
1H, H-2), 8.60 (d, J = 2.1 Hz, 1H, Haryl), 8.11 (dd, J = 9.1, 2.1 Hz, 1H,
MeC O). 13C NMR (101 MHz, DMSO-d6) d
@
@
191.07(MeC O),
@
@
@
@
177.90(C O), 166.34(C O), 150.82(C-2), 149.26 (Cq,aryl), 144.18(Cq,ar-
@
@
yl), 139.89(Cq,aryl), 135.63(CHthiophene), 131.69 (CHaryl), 130.79(Cq,aryl),
126.94(CHthiophene), 126.37(Cq,aryl), 122.54 (CHaryl), 119.99(CHaryl),
H
aryl), 7.62 (d, J = 9.1 Hz, 1H, Haryl), 7.36–7.20 (m, 5H, Bn), 5.83 (s,
2H, Bn). 13C NMR (101 MHz, DMSO-d6)
d
177.06(C O),
@
@
107.98(Cq,aryl), 59.05(NCH2CH2OH), 56.42 (NCH2CH2OH), 26.85(Me
@
166.06(C O), 150.92(C-2), 142.53(CHaryl), 139.34(Cq,aryl), 135.61
@
@
C
O); HRMS (ESI-) calcd for C18H14NO5Sꢀ(MꢀH+) 356.0598, found
@
(Cq,aryl), 134.63(CHaryl), 129.40(2C, CHBn), 128.51(CHBn), 127.82
(Cq,aryl), 127.00(2C, CHBn), 121.24(CHaryl), 109.04(Cq,aryl), 92.57
(Cq,aryl), 56.82(CH2Bn); HRMS (ESI-) calcd for C17H11INO3-(M-H+)
403.9789, found 403.9808; HPLC tR 13.9 min; Mp: 277–280 °C.
356.0584; HPLC tR 10.0 min; Mp: 286–288 °C.
4.2.7. 1-(2-Hydroxyethyl)-6-(benzo[b]thiophen-2-yl)-4-oxo-
quinoline-3-carboxylic acid (25g)
Prepared from (1) THPOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yellow
powder, yield: 49% for three steps. 1H NMR (600 MHz, DMSO-d6) d
8.89 (s, 1H, H-2), 8.58 (d, J = 2.3 Hz, 1H, Haryl), 8.36 (dd, J = 9.0,
2.3 Hz, 1H, Haryl), 8.17 (d, J = 9.0 Hz, 1H, Haryl), 8.11 (s, 1H, Haryl),
8.04–7.97 (m, 1H, Haryl), 7.91–7.85 (m, 1H, Haryl), 7.45–7.35 (m,
4.2.3. 1-(2-Hydroxyethyl)-6-iodo-4-oxo-quinoline-3-carboxylic
acid (25c)
Prepared from (1) AcOCH2CH2Br and 23, and (2) removal of O-
Ac and saponification, white solid yield: 57% for two steps. 1H
NMR (400 MHz, DMSO-d6) d 8.88 (s, 1H, H-2), 8.60 (d, J = 2.1 Hz,
1H, Haryl), 8.18 (dd, J = 9.0, 2.1 Hz, 1H, Haryl), 7.85 (d, J = 9.0 Hz,
1H, Haryl), 5.00 (br s, 1H, –OH), 4.58 (t, J = 4.8 Hz, 2H, NCH2CH2OH),
3.72 (m, 2H, NCH2CH2OH). 13C NMR (101 MHz, DMSO-d6) d
2H, Haryl), 4.66 (t, J = 5.0 Hz, 2H, NCH2CH2OH), 3.79 (t, J = 5.0 Hz,
13
@
2H, NCH2CH2OH). C NMR (150 MHz, DMSO-d6) d 177.21(C O),
@
@
167.11(C O), 165.69(Cq,aryl), 150.00(Ct,aryl), 140.68(Cq,aryl), 140.12
@
@
@
176.89(C O), 166.21(C O), 151.06(C-2), 142.40(CHaryl), 139.35
@
@
(Cq,aryl), 138.89(Cq,aryl), 138.57(Cq,aryl), 131.02(Cq,aryl), 125.71(Ct,aryl),
124.96(Ct,aryl), 124.78(Cq,aryl), 123.86(Ct,aryl), 122.31(Ct,aryl), 121.75
(Ct,aryl), 121.68(Ct,aryl), 119.26(Ct,aryl), 107.17(Ct,aryl), 58.29
(NCH2CH2OH), 55.72(NCH2CH2OH); HRMS (ESI-) calcd for
(Cq,aryl), 134.52(CHaryl), 127.60(Cq,aryl), 120.96(CHaryl), 108.08
(Cq,aryl), 92.35(Cq,aryl), 58.93(NCH2CH2OH), 56.37(NCH2CH2OH);
HRMS (ESI-) calcd for
357.9573; HPLC tR 9.0 min; Mp:251–254 °C (decomp.).
C
12H9INO4-(MꢀH+) 357.9582, found
C
20H14NO4Sꢀ(MꢀH+) 364.0649, found 364.0648; HPLC tR
13.2 min; Mp: 238–245 °C (decomp.).
4.2.4. 6-(Thiophen-2-yl)-4-oxo-quinoline-3-carboxylic acid
(25d)
4.2.8. 6-(Furan-2-yl)-4-oxo-quinoline-3-carboxylic acid (25h)
Prepared from (1) THPOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, light
Prepared from (1) corresponding boronic acid and 23, and (2)
saponification, light yellow solid, yield: 39% for two steps. 1H NMR

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
4797
grey powder, yield: 47% for three steps. 1H NMR (400 MHz, DMSO-
d6) d 13.45 (s, 1H, –NH), 8.84 (s, 1H, H-2), 8.44 (d, J = 1.9 Hz, 1H,
aryl), 8.16 (dd, J = 8.6, 1.9 Hz, 1H, Haryl), 7.81 (m, 2H, Haryl, Hfuran),
7.13 (d, J = 3.3 Hz, 1H, Hfuran), 6.63 (dd, J = 3.3, 1.8 Hz, 1H, Hfuran).
4.2.12. 1-(3-Hydroxypropyl)-6-(benzo[B]furan-2-yl)-4-oxo-
quinoline-3-carboxylic acid (25l)
H
Prepared from (1) THPOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yellow
powder, yield: 48% for three steps. 1H NMR (400 MHz, DMSO-d6) d
8.99 (s, 1H, H-2), 8.77 (d, J = 1.4 Hz, 1H, Hbenzofuran), 8.46 (d,
J = 7.9 Hz, 1H, Haryl), 8.15 (d, J = 9.0 Hz, 1H, Haryl), 7.69 (br s, 3H,
2 ꢁ Hbenzofuran, 1xHaryl), 7.37 (t, J = 7.6 Hz, 1H, Hbenzofuran), 7.29 (t,
J = 7.4 Hz, 1H, Hbenzofuran), 4.75 (br s, 1H, -OH), 4.64 (t, J = 6.9 Hz,
2H, NCH2CH2CH2OH), 3.48 (br s, 2H, NCH2CH2CH2OH), 2.07–1.91
13C NMR (101 MHz, DMSO-d6) d 178.52(C O), 166.75(C O),
@
@
@
@
152.05(Cq,aryl), 145.28(C-2), 144.34(CHfuran), 138.96 (Cq,aryl),
129.74(CHaryl), 128.49(Cq,aryl), 125.23(Cq,aryl), 121.04(CHaryl),
118.79(CHaryl), 112.88(CHfuran), 108.21(Cq,aryl), 108.05(CHfuran);
HRMS (ESI-) calcd for
254.0468; HPLC tR 9.7 min; Mp: 300–304 °C (decomp.).
C
14H8NO4ꢀ(MꢀH+) 254.0459, found
(m, 2H, NCH2CH2CH2OH). 13C NMR (100 MHz, DMSO-d6)
d
@
@
177.91(C O), 166.29(C O), 154.91(Cq,aryl), 153.72(Cq,aryl), 150.00
@
@
4.2.9. 1-(2-Hydroxyethyl)-6-(furan-2-yl)-4-oxo-quinoline-3-
carboxylic acid (25i)
(C-2), 139.49(Cq,aryl), 130.74(CHaryl), 129.04(Cq,aryl), 127.78(Cq,aryl),
126.34(Cq,aryl), 125.67(CHbenzofuran), 123.88(CHbenzofuran), 121.88
(CHaryl), 121.15(CHbenzofuran), 119.59(CHaryl), 111.72(CHaryl),
108.34 (Cq,aryl), 104.39(CHaryl), 57.84(NCH2CH2CH2OH), 51.74
(NCH2CH2CH2OH), 31.88(NCH2CH2CH2OH); HRMS (ESI-) calcd
Prepared from (1) THPOCH2CH2Br and 24, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yellow
powder, yield: 60% for three steps. 1H NMR (400 MHz, DMSO-d6) d
8.86 (s, 1H, H-2), 8.34 (d, J = 8.5 Hz, 1H, Haryl), 8.10 (s, 1H, Haryl),
7.92 (d, J = 8.5 Hz, 1H, Haryl), 7.89 (br s, 1H, Hfuran), 7.38 (d,
J = 3.3 Hz, 1H, Hfuran), 6.70 (m, 1H, Hfuran), 5.07 (br s, 1H, -OH),
4.65 (d, J = 4.3 Hz, 2H, NCH2CH2OH), 3.79 (br s, 2H, NCH2CH2OH).
for
C
21H16NO5ꢀ(MꢀH+) 362.1034, found 362.1025; HPLC tR
11.3 min; Mp: 211–214 °C.
13C NMR (100 MHz, DMSO-d6) d 177.66(C O), 166.56(C O),
4.2.13. 1-Propyl-6-(benzo[B]furan-2-yl)-4-oxo-quinoline-3-
carboxylic acid (25bm)
@
@
@
@
151.78(Cq,aryl),
151.09(C-2),
145.40(CHfuran),
140.59(Cq,aryl),
135.45(Cq,aryl), 127.24(CHaryl), 124.53(Cq,aryl), 121.88(CHaryl),
113.21(CHfuran), 111.76(CHaryl), 110.72(CHfuran), 107.69(Cq,aryl),
59.03(NCH2CH2OH), 56.27(NCH2CH2OH); HRMS (ESI-) calcd for
Prepared from (1) CH3CH2CH2Br and 23, (2) corresponding
boronic acid, and (3) saponification, light grey powder, yield: 52%
for three steps. 1H NMR (400 MHz, DMSO-d6) d 8.94 (s, 1H, H-2),
8.77 (d, J = 1.9 Hz, 1H, Hbenzofuran), 8.37 (dd, J = 9.0, 2.0 Hz, 1H, Haryl),
C
16H12NO5ꢀ(MꢀH+) 298.0721, found 298.0729; HPLC tR 9.9 min;
Mp: 260–262 °C.
8.08 (d, J = 9.0 Hz, 1H, Haryl), 7.66 (dd, J = 10.8, 8.2 Hz, 2H, Haryl,
Hbenzofuran), 7.56 (s, 1H, Hbenzofuran), 7.35 (t, J = 7.3 Hz, 1H, Hbenzofuran),
7.27 (t, J = 7.4 Hz, 1H,
Hbenzofuran), 4.52 (t, J = 7.3 Hz, 2H,
4.2.10. 1-(2-Hydroxyethyl)-6-(furan-3-yl)-4-oxo-quinoline-3-
carboxylic acid (25j)
NCH2CH2CH3), 3.03 (s, 1H, –OH), 1.97–1.81 (m, 2H, NCH2CH2CH3),
0.96 (t, J = 7.3 Hz, 3H, NCH2CH2CH3). 13C NMR (100 MHz, DMSO-d6)
Prepared from (1) THPOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yel-
low powder, yield: 40% for three steps. 1H NMR (400 MHz,
DMSO-d6) d 8.85 (s, 1H, H-2), 8.49 (d, J = 2.1 Hz, 1H, Haryl), 8.43
(dd, J = 1.4, 0.9 Hz, 1H, Hfuran), 8.17 (dd, J = 9.0, 2.1 Hz, 1H, Haryl),
8.07 (d, J = 9.0 Hz, 1H, Haryl), 7.83–7.78 (m, 1H, Hfuran), 7.13 (dd,
J = 1.9, 0.9 Hz, 1H, Hfuran), 5.04 (br s, 1H, –CH2OH), 4.63 (t,
J = 4.7 Hz, 2H, NCH2CH2OH), 3.77 (dd, J = 9.2, 4.7 Hz, 2H,
@
@
d 178.01(C O), 166.05(C O), 155.19(Cq,aryl), 153.95(Cq,aryl), 149.67
@
@
(C-2), 139.71(Cq,aryl), 130.73(CHaryl), 129.20(Cq,aryl), 128.03(Cq,aryl),
126.64(Cq,aryl), 125.60(CHbenzofuran), 123.81(CHbenzofuran), 121.86
(CHaryl),
121.65(CHbenzofuran),
119.50(CHaryl),
111.64(CHaryl),
108.73 (Cq,aryl), 104.30(CHbenzofuran), 55.58(NCH2CH2CH3), 22.53
(NCH2CH2CH3), 10.86(NCH2CH2CH3); HRMS (ESI-) calcd for
C
21H16NO4ꢀ(MꢀH+) 346.1085, found 346.1083; HPLC tR 15.6 min;
Mp: 250–254 °C.
NCH2CH2OH). 13C NMR (101 MHz, DMSO-d6) d 178.08(C O),
@
@
@
166.65(C O), 150.29(C-2), 145.28(CHfuran), 141.19(CHfuran),
@
4.2.14. 1-(2-Hydroxyethyl)-6-(p-methoxyphenyl)-4-oxo-
quinoline-3-carboxylic acid (25n)
138.73(Cq,aryl), 132.00(CHaryl), 130.54(Cq,aryl), 126.44(Cq,aryl),
124.79(Cq,aryl), 121.75(CHaryl), 119.44(CHaryl), 109.08(CHfuran),
107.54(Cq,aryl), 59.01(NCH2CH2OH), 56.42(NCH2CH2OH); HRMS
(ESI-) calcd for C16H12NO5ꢀ(MꢀH+) 298.0721, found 298.0710;
HPLC tR 10.0 min; Mp: 193–194 °C.
Prepared from (1) THPOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, white
solid, yield: 67% for three steps. 1H NMR (400 MHz, DMSO-d6) d
8.88 (s, 1H, H-2), 8.54 (d, J = 2.1 Hz, 1H, Haryl), 8.23 (dd, J = 8.9,
2.1 Hz, 1H, Haryl), 8.11 (d, J = 8.9 Hz, 1H, Haryl), 7.42 (m, 1H, Hmethoxy-
phenyl), 7.33 (m, 1H, Hmethoxyphenyl), 7.30–7.26 (m, 1H, Hmethoxyphenyl),
6.99 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H, Hmethoxyphenyl), 5.05 (t, J = 5.5 Hz,
1H, -OH), 4.70–4.60 (m, 2H, NCH2CH2OH), 3.83 (d, J = 2.8 Hz, 3H,
OMe), 3.81–3.74 (m, 2H, NCH2CH2OH). 13C NMR (101 MHz,
4.2.11. 1-(2-Hydroxyethyl)-6-(benzo[B]furan-2-yl)-4-oxo-
quinoline-3-carboxylic acid (25k)
Prepared from (1) THPOCH2CH2Br and 24, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yellow
powder, yield: 13% for three steps. 1H NMR (400 MHz, DMSO-d6) d
8.93 (s, 1H, H-2), 8.47 (d, J = 8.5 Hz, 1H, Haryl), 8.40 (pseudo-s, 1H,
@
@
DMSO-d6) d 178.22(C O), 166.59(C O), 160.35(Cq,aryl), 150.62(C-
@
@
2), 140.06(Cq,aryl), 139.25(Cq,aryl), 138.11(Cq,aryl), 133.07(CHaryl),
130.77(CHmethoxyphenyl), 126.29(Cq,aryl), 123.43(CHaryl), 119.71
(CHmethoxyphenyl), 119.46(CHaryl), 114.32(CHmethoxyphenyl), 112.85
Haryl), 8.17 (dd, J = 8.5, 1.3 Hz, 1H, Haryl), 7.91 (s, 1H, Hbenzofuran),
7.75 (d, J = 7.6 Hz, 1H, Hbenzofuran), 7.70 (d, J = 8.2 Hz, 1H, Hbenzofuran),
7.44–7.38 (m, 1H, Hbenzofuran), 7.31 (t, J = 7.6 Hz, 1H, Hbenzofuran), 5.08
(br s, 1H, -OH), 4.74 (t, J = 4.9 Hz, 2H, NCH2CH2OH), 3.83 (t,
J = 4.9 Hz, 2H, NCH2CH2OH). 13C NMR (101 MHz, DMSO-d6) d
(CHmethoxyphenyl),
107.64(Cq,aryl),
59.00(NCH2CH2OH),
56.43
(NCH2CH2OH), 55.68(OMe); HRMS (ESI-) calcd for C19H16NO5
ꢀ(MꢀH+) 338.1034, found 338.1039; HPLC tR 13.4 min; Mp: 202–
203 °C.
@
@
177.71(C O), 166.48(C O), 155.18(Cq,aryl), 153.74(Cq,aryl), 151.34
@
@
(C-2), 140.58(Cq,aryl), 135.10(Cq,aryl), 128.89(Cq,aryl), 127.39(CHaryl),
126.34(CHbenzofuran), 125.53(Cq,aryl), 124.12(CHbenzofuran), 122.77
(CHaryl), 122.29(CHbenzofuran), 113.69(CHaryl), 111.91(CHbenzofuran),
4.2.15. 1-(2-Hydroxyethyl)-7-(thiphen-2-yl)-4-oxo-quinoline-3-
carboxylic acid (25o)
Prepared from (1) AcOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-Ac and saponification, yellow
powder, yield: 11% for three steps. 1H NMR (400 MHz, DMSO-d6)
107.88
Cq,aryl), 106.70(CHbenzofuran), 59.13(NCH2CH2OH), 56.32
(NCH2CH2OH); HRMS (ESI-) calcd for C20H14NO5-(M-H+)
348.0877, found 348.0869; HPLC tR 12.5 min; Mp: 346–355 °C
(decomp.).

4798
Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
d 8.86 (s, 1H, H-2), 8.48 (d, J = 2.1 Hz, 1H, Haryl), 8.23 (m, 1H, Haryl),
8.09 (m, 1H, Haryl), 7.73 (m, 1H, Hthiophene), 7.65 (dd, J = 5.0, 0.8 Hz,
1H, Hthiophene), 7.18 (dt, J = 13.8, 6.9 Hz, 1H, Hthiophene), 5.04 (t,
J = 5.3 Hz, 1H, -OH), 4.63 (br s, 2H, NCH2CH2OH), 3.77 (d,
J = 4.2 Hz, 2H, NCH2CH2OH). 13C NMR (101 MHz, DMSO-d6) d
4.3. Biology
4.3.1. Cells and recombinant protein
The HCV genotype 1b replicon-containing cells, Huh-7/HCV1b-
Rluc cells (Y-L Chen, et al, Bioorg Med Chem, 2012) (obtained from
G. Luo, University of Kentucky), were maintained in DME supple-
@
@
177.96(C O),
166.50(C O),
150.49(C-2),
141.66(Cq,aryl),
@
@
139.01(Cq,aryl), 132.08(Cq,aryl), 131.47(CHaryl), 129.40(CHthiophene),
127.63(CHthiophene), 126.47(Cq,aryl), 125.88(CHthiophene), 121.42
(CHaryl), 119.84(CHaryl), 107.71(Cq,aryl), 59.02(NCH2CH2OH), 56.45
(NCH2CH2OH); HRMS (ESI-) calcd for C16H12NO4Sꢀ(MꢀH+)
314.0493, found 314.0499; HPLC tR 9.7 min; Mp: 209–211 °C.
mented with 10% fetal bovine serum, 500 lgm/ml G418 (Invitro-
gen), 100 IU streptomycin and 100 IU penicillin, 1ꢁ non-essential
amino acids (Invitrogen), and 1 mM sodium pyruvate (Invitrogen).
The HCV genotype 1b NS5B polymerase missing the C-terminal 55
amino acids (NS5B
D55) was expressed from the plasmid pET-
21d(+)-NS5B
D
55 and purified, excluding the heparin-agarose
chromatography, from bacteria as described.57 The enzyme was
stored at ꢀ80 °C in storage buffer (50 mM Tris pH 7.5, 50% glycerol,
1 mM b-ME).
4.2.16. 1-(3-Hydroxypropyl)-7-(thiophen-2-yl)-4-oxo-
quinoline-3-carboxylic acid (25p)
Prepared from (1) THPOCH2CH2Br and 24, (2) corresponding
boronic acid, and (3) removal of O-THP and saponification, yellow
powder, yield: 47% for three steps. 1H NMR (400 MHz, DMSO-d6) d
8.96 (s, 1H, H-2), 8.32 (d, J = 8.5 Hz, 1H, Haryl), 8.12 (d, J = 1.2 Hz, 1H,
4.3.2. HCV replicon assay
Approximately 6 ꢁ 103 replicon-containg cells per well were
plated in an opaque 96-well tissue culture plate (BD Falcon) in
the absence of G418. Cells were exposed to culture medium con-
taining compound (dissolved in DMSO), DMSO alone, or nothing
added the next day and incubated at 37 °C/5% CO2 for three days.
The culture medium was removed and renilla luciferase activity
measured using ViVi-Ren Live Cell Substrate (Promega) as de-
scribed.44 Compounds and controls were performed in triplicate
and each experiment repeated independently at least twice. For
H
H
aryl), 7.89–7.82 (m, 2H, Haryl, Hthiophene), 7.74 (dd, J = 5.0, 1.0 Hz, 1H,
thiophene), 7.23 (dd, J = 5.0, 3.7 Hz, 1H, Hthiophene), 4.82 (t, J = 4.6 Hz,
1H, –OH), 4.66 (t, J = 7.0 Hz, 2H, NCH2CH2CH2OH), 3.47 (dd, J = 10.1,
5.4 Hz, 2H, NCH2CH2CH2OH), 2.05–1.88 (m, 2H, NCH2CH2CH2OH).
13C NMR (100 MHz, DMSO-d6) d 177.60(C O), 166.43(C O),
@
@
@
@
150.53 (C-2), 141.71(Cq,aryl), 140.37(Cq,aryl), 139.53(Cq,aryl), 129.44
(CHthiophene), 129.07(CHthiophene), 127.48(CHaryl), 127.38(CHaryl),
124.67(Cq,aryl), 124.10(CHthiophene), 113.74(CHaryl), 108.10(Cq,aryl),
the intial 10
C-Me-A (0.5
l
M compound evaluation, ribavirin (10
l
M) and 20-
57.82(NCH2CH2CH2OH),
51.41(NCH2CH2CH2OH),
31.87
(NCH2CH2CH2OH); HRMS (ESI-) calcd for C17H14NO4Sꢀ(MꢀH+)
328.0649, found 328.0638; HPLC tR 11.4 min; Mp: 192–195 °C
(decomp.).
lM) were included as controls. The concentration of
compound that reduced luciferase activity by 50% was defined as
the 50% effective concentration (EC50). The EC50 was determined
by comparing luciferase activity for eight serial dilutions of com-
pound and vehicle treated cells using GraphPad Prism software.
4.2.17. 1-(2-Hydroxyethyl)-7-(furan-2-yl)-4-oxo-quinoline-3-
carboxylic acid (25q)
4.3.3. Cell proliferation assay
Prepared from (1) AcOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-Ac and saponification, yellow
powder, yield: 63% for three steps. 1H NMR (400 MHz, DMSO-d6)
d 8.85 (s, 1H, H-2), 8.55 (d, J = 2.1 Hz, 1H, Haryl), 8.22 (dd, J = 9.0,
2.1 Hz, 1H, Haryl), 8.09 (d, J = 9.1 Hz, 1H, Haryl), 7.84 (dd, J = 1.7,
0.6 Hz, 1H, Hfuran), 7.21 (d, J = 3.2 Hz, 1H, Hfuran), 6.65 (dd, J = 3.2,
1.7 Hz, 1H, Hfuran), 5.05 (br s, 1H, –CH2OH), 4.62 (t, J = 4.8 Hz, 2H,
NCH2CH2OH), 3.77 (br s, 2H, NCH2CH2OH). 13C NMR (101 MHz,
Approximately 6 ꢁ 103 replicon-containg cells per well were
plated in a clear 96-well tissue culture plate (Corning) in the ab-
sence of G418. Cells were exposed to culture medium containing
compound (dissolved in DMSO), DMSO alone, or nothing added
the next day and incubated at 37 °C/5% CO2 for three days. CellTiter
96 AQueous One Solution Cell Proliferation reagent (Promega) was
added and measured according to manufacturer’s instructions by
spectrometry at 450 nm with a SpectraMax E5 (Molecular De-
vices). Compounds and controls were performed in triplicate and
each experiment repeated independently at least twice. For the in-
@
@
DMSO-d6) d 178.03(C O), 166.54(C O), 151.75(Cq,aryl), 150.41
@
@
(C-2), 144.52(CHfuran), 138.87(Cq,aryl), 129.71(CHaryl), 128.46(Cq,aryl),
126.44(Cq,aryl), 119.67(CHaryl), 119.52(CHaryl), 112.94(CHfuran),
tial 10
(0.5 M) were included as controls. The concentration of com-
lM compound evaluation, ribavirin (10 l
M) and 20-C-Me-A
108.44(CHfuran),
107.68(Cq,aryl),
59.01(NCH2CH2OH),
56.45
(NCH2CH2OH); HRMS (ESI-) calcd for C16H12NO5ꢀ(MꢀH+)
298.0721, found 298.0712; HPLC tR 10.3 min; Mp: 233–235 °C.
l
pound that reduced cell proliferation by 50% was defined as the
50% cytotoxic concentration (CC50). The CC50 was determined by
comparing absorbance readings from eight serial dilutions of com-
pound and vehicle treated cells using GraphPad Prism software.
4.2.18. 1-(2-Hydroxyethyl)-7-(benzo[B]furan-2-yl)-4-oxo-
quinoline-3-carboxylic acid (25r)
Prepared from (1) AcOCH2CH2Br and 23, (2) corresponding
boronic acid, and (3) removal of O-Ac and saponification, yellow
powder, yield: 23% for three steps. 1H NMR (400 MHz, DMSO-d6)
d 8.90 (s, 1H, H-2), 8.78 (d, J = 2.1 Hz, 1H, Haryl), 8.43 (dd, J = 9.1,
2.2 Hz, 1H, Haryl), 8.18 (d, J = 9.1 Hz, 1H, Haryl), 7.75–7.65 (m, 3H,
3xHbenzofuran), 7.42–7.34 (m, 1H, Hbenzofuran), 7.30 (m, 1H, Hbenzofuran),
5.09 (br s, 1H, -OH), 4.67 (t, J = 4.8 Hz, 2H, NCH2CH2OH), 3.81 (br s,
4.3.4. RT-qPCR Assay
Approximately 6 ꢁ 103 replicon-containg cells per well were
plated in a clear 96-well tissue culture plate (Corning) in the ab-
sence of G418. Cells were exposed to culture medium containing
compound (dissolved in DMSO) or DMSO alone and incubated at
37 °C/5% CO2 for three days. The relative level of replicon RNA for
compound-treated cells compared to DMSO-treated cells was mea-
sured using the TaqManÒ Gene Expression Cells-to-CT system (Ap-
plied Biosystems) and TaqManÒ primers/probes as per
manufacturers instructions. The cells were lysed in the wells and
stored at -80 °C. The primers/probes to measure HCV replicon
cDNA were custom synthesized to NS5B (Applied Biosystems),
LC609-NS5F 50 CCCCACATTCGGCCAGATC, LC609-NSR 50 GAT-
AGGTTCCGGACGTCCTTTG, FAM probe 50 CCCCATAGCCAAATTT.
The control primers/probe recognize GAPDH cDNA (Applied
2H, NCH2CH2OH).13C NMR (101 MHz, DMSO-d6) d 178.04(C O),
@
@
@
166.43(C O), 154.94(Cq,aryl), 153.76(Cq,aryl), 150.71(C-2), 139.75
@
(Cq,aryl), 130.64(CHaryl), 129.08(Cq,aryl), 127.81(Cq,aryl), 126.38(Cq,aryl),
125.71(CHbenzofuran), 123.92(CHbenzofuran), 121.92(CHbenzofuran), 121.12
(CHaryl), 119.83(CHaryl), 111.75(CHbenzofuran), 107.95(Cq,aryl), 104.46
(CHbenzofuran), 59.04(NCH2CH2OH), 56.48 (NCH2CH2OH); HRMS
(ESI-) calcd for C20H14NO5ꢀ(MꢀH+) 348.0877, found 348.0881;
HPLC tR 12.6 min; Mp: 247–251 °C (decomp.).

Y.-L. Chen et al. / Bioorg. Med. Chem. 20 (2012) 4790–4800
4799
Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.;
Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.;
Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay,
A.; Madison, V.; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, T. Y.; Brisson, J.-M.;
Prelusky, D.; Korfmacher, W.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky, A.;
Bradley, P.; Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.;
Njoroge, F. G. J. Med. Chem. 2006, 49, 6074.
Biosystems, catalog number 402869). The samples were analyzed
in triplicate per experiment and the experiment performed two
independent times. Samples were processed using a Mastercycler
ep realplex2 gradient S (Eppendorf) with cycling parameters of
50 °C for 2 min, 95 °C for ten min, 95 °C for 15 s, 60 °C for 1 min, re-
peat latter two steps 39 more times. Parallel wells of lysate were
analyzed for either HCV replicon RNA or GAPDH RNA and relative
levels of HCV RNA calculated using the comparative method.53 The
PCR efficiency for the two sets of primers/probe was determined
using 5-fold serial dilutions (over 3logs) of lysate from replicon
cells as described53 and was 100% (R2 0.999) and 110% (R2 0.997)
for NS5B primers/probe and GAPDH primers/probe, respectively.
16. Arasappan, A.; Bennett, F.; Bogen, S. L.; Venkatraman, S.; Blackman, M.; Chen,
K. X.; Hendrata, S.; Huang, Y.; Huelgas, R. M.; Nair, L.; Padilla, A. I.; Pan, W.;
Pike, R.; Pinto, P.; Ruan, S.; Sannigrahi, M.; Velazquez, F.; Vibulbhan, B.; Wu,
W.; Yang, M.; Saksena, A. K.; Girijavallabhan, V.; Shih, N.-Y.; Kong, J.; Meng, T.;
Jin, Y.; Wong, J.; McNamara, P.; Prongay, A.; Madison, V.; Piwinski, J. J.; Cheng,
K.-C.; Morrison, R.; Malcolm, B.; Tong, X.; Ralston, R.; Njoroge, F. G. ACS Med.
Chem. Lett. 2010, 1, 64.
17. Arasappan, A.; Padilla, A. I.; Jao, E.; Bennett, F.; Bogen, S. L.; Chen, K. X.; Pike, R.
E.; Sannigrahi, M.; Soares, J.; Venkatraman, S.; Vibulbhan, B.; Saksena, A. K.;
Girijavallabhan, V.; Tong, X.; Cheng, K.-C.; Njoroge, F. G. J. Med. Chem. 2009, 52,
2806.
18. Bennett, F.; Huang, Y.; Hendrata, S.; Lovey, R.; Bogen, S. L.; Pan, W.; Guo, Z.;
Prongay, A.; Chen, K. X.; Arasappan, A.; Venkatraman, S.; Velazquez, F.; Nair, L.;
Sannigrahi, M.; Tong, X.; Pichardo, J.; Cheng, K.-C.; Girijavallabhan, V. M.;
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Venkatraman, S.; Bennett, F.; Pan, W.; Blackman, M. L.; Padilla, A. I.; Prongay,
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1370.
4.3.5. HCV NS5B assay
Primer-dependent NS5B
using a scintillation proximity assay (SPA) as described.44 Briefly,
purified NS5B 55 (2 nM) was added to Mix 1 (20 mM Tris pH
7.5, 50 mM NaCl, 5 mM KCl, 0.5 mM MnCl2, 4U RNasin (Promega),
1 mM DTT, 1 L compound in DMSO or 1 L DMSO alone) and Mix
2 was added (450tnM Oligo-U12 primer, 30 nM UTP, 75 nM
Poly(A) template, 0.2 L reaction was incu-
i [3H]UTP). The 41
bated in 0.7 mL tubes at room temperature for 3 h and stopped
by adding 350 L of 1.2 mg streptavidin-coated SPA beads (Perkin-
D55 polymerase activity was measured
D
l
l
l
l
21. Nair, L. G.; Bogen, S.; Ruan, S.; Pan, W.; Pike, R.; Tong, X.; Cheng, K.-C.; Guo, Z.;
Doll, R. J.; Njoroge, F. G. Bioorg. Med. Chem. Lett. 2010, 20, 1689.
22. Carroll, S. S.; La Femina, R. L. Antiviral Res. 2009, 153.
23. Li, H.; Shi, S. T. Future Med. Chem. 2010, 2, 121.
24. Liu, Y.; He, Y.; Molla, A. Antiviral Res. 2009, 137.
l
Elmer) in 0.15 M EDTA. The samples were analyzed immediately
for 1 min in a scintillation counter. Each compound concentration
was performed in duplicate and repeated at least two independent
times. (see Scheme 1)
25. Beaulieu, P. L. Curr. Opin. Invest. Drugs 2007, 8, 614.
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Lam, A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. J. Med. Chem.
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Acknowledgements
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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 and Matthew Kesler for technical assistance.
29. Gao, M.; Nettles, R. E.; Belema, M.; Snyder, L. B.; Nguyen, V. N.; Fridell, R. A.;
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Supplementary data
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