Quinolones as HCV NS5B polymerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.11.068

Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin. To address the limitations of this therapy, numerous small molecule agents are in development, which act by directly affecting key steps in the viral

Full text of DOI:10.1016/j.bmcl.2010.11.068

Bioorganic & Medicinal Chemistry Letters 21 (2011) 82–87
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinolones as HCV NS5B polymerase inhibitors
Dange V. Kumar a, Roopa Rai b, , Ken A. Brameld c, John R. Somoza d, Ravi Rajagopalan e, James W. Janc f,
⇑
Yu M. Xia g, Tony L. Ton h, Michael B. Shaghafi i, Huiyong Hu j, Isabelle Lehoux k, Nhat To l, Wendy B. Young j,
Michael J. Green m
a Myriad Pharmaceuticals, 305 Chipeta Way, Salt Lake City, UT 84108, USA
b Alios Biopharma, 260 E. Grand Ave., South San Francisco, CA 94080, USA
c Drug By Design, Menlo Park, CA 94027, USA
d Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA 94404, USA
e InterMune Inc., 3280 Bayshore Blvd, Brisbane, CA 94005, USA
f Theravance, Inc., South San Francisco, CA 94080, USA
g Crown Bioscience, No. 6 Beijing West Road, Taicang City, Jiangsu Province 215400, PR China
h 3399 Lake Arrowhead Ave., Fremont, CA 94555, USA
i Department of Chemistry, University of California, Irvine, CA 92697, USA
j Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
k Allosteros Therapeutics, 1230 Bordeaux Dr., Sunnyvale, CA 94089, USA
l Department of Anesthesiology, University of California-Los Angeles, CA 90095, USA
m Virobay, Inc., 1490 O’Brien Drive, Menlo Park, CA 94025, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin.
To address the limitations of this therapy, numerous small molecule agents are in development, which
act by directly affecting key steps in the viral life-cycle. Herein we describe our discovery of quinolone
derivatives, novel small-molecules that inhibit NS5b polymerase, a key enzyme of the viral life-cycle.
A crystal structure of a quinoline analog bound to NS5B reveals that this class of compounds binds to allo-
steric site-II (non-nucleoside inhibitor-site 2, NNI-2) of this protein.
Received 12 September 2010
Revised 10 November 2010
Accepted 16 November 2010
Available online 21 November 2010
Keywords:
HCV
Ó 2010 Elsevier Ltd. All rights reserved.
NS5B polymerase inhibitors
Non-nucleoside inhibitors
Quinolone derivatives
Cocrystal structure
HCV NS5B NNI-2 binder
Hepatitis C virus (HCV), a member of the Flaviviridae family, is
estimated to infect 170 million individuals worldwide.1,2 The viral
genome, approximately 9.6 kb in size, is a single-stranded, posi-
tive-sense RNA that encodes structural and non-structural pro-
teins.3 Chronic infection by HCV often leads to severe liver disease
including hepatocellular carcinoma, cirrhosis, and liver failure.4,5
The current standard of care is a combination of peginterferon
alfa-2a/b and Ribavirin, which successfully eradicates the virus in
only about 50% of the patients infected with genotype 1.6–8 Conse-
quently, considerable pharmaceutical resources have been focused
upon the discovery of novel therapies, particularly direct-acting
agents specifically targeting essential viral proteins.9
promising results in clinical trials11–13 and the polymerase is con-
sidered a well validated drug target. Reported inhibitors are either
nucleoside analogs which target the active site or non-nucleoside
inhibitors (NNIs) that bind to one of four allosteric sites (Fig. 1).
Two of these allosteric sites are located in the thumb region
(NNI-1 and NNI-2) and two are in the palm region (NNI-3 and
NNI-4) as determined by X-ray crystallography and in vitro resis-
tance studies.14–16
Recognizing the value of additional therapies, we initiated a
program directed towards the discovery of novel inhibitors of
NS5B polymerase. This Letter describes our discovery of small-
molecules that inhibit HCV replication by impairing the function
of NS5B polymerase.
The viral RNA dependent RNA polymerase (RdRp) encoded by
the non-structural protein 5b (NS5B) is essential for viral replica-
tion.10 Several specific NS5B polymerase inhibitors have shown
We employed a SPA (Scintillation Proximity Assay)18a format
for our HTS campaign to screen for inhibitors of NS5B polymerase.
Among our confirmed ‘hits’ was compound 1 (Fig. 2), which inhib-
ited NS5B polymerase enzymatic function with an IC50 of 1.15
against the wild-type (genotype 1b) enzyme. Upon testing this
lM
⇑
Corresponding author.
E-mail address: roopa.rai@hotmail.com (R. Rai).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.068

D. V. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 82–87
83
Cl
N
N
O
F
N
N
O
HO
HO2C
O
N
N
S
O
N
O
JTK-109
GSK-625433
NNI-1 site binder
NNI-3 site binder
OH
HN
O
N
N
N
N
O
O
F
HO
O
N
N
S
O
Filibuvir
O
HCV-796
NNI-2 site binder
NNI-4 site binder
Figure 1. Representative non-nucleoside HCV polymerase inhibitors that have progressed to clinical trials and their respective allosteric binding sites.
O
N
O
4
O
O
6
7
3
2
1
Compound 1
Fold shift in IC50 Measured for Mutant NS5b
Relative to the Wild Type Value
WT
IC50
(µM)
P495L/WT
(NNI-1)
M423F/WT
(NNI-2)
M414T/WT
(NNI-3)
1.15
1
> 200
2
Figure 2. Analysis of hit compound 1 through site-directed mutagenesis and IC50
shifts led to the conclusion that this class of compound binds at allosteric site II of
NS5B polymerase.
Figure 3. Model of 1 bound to NNI-2 site observed for X-ray structure 1NHU.
Potential hydrogen bonding interactions are shown as dashed lines. Solvent
accessible region is marked by an arrow.
compound against a panel of recombinant NS5B enzymes designed
with targeted mutations in allosteric sites NNI-1, NNI-2, and NNI-3
and evaluating the fold-change in enzyme inhibition over wild-
type, we were able to conclude that compound 1 was inhibiting
enzymatic activity by binding at site NNI-2 of the polymerase.
Twopreviously reportedcrystal structures of NNI-2 allostericsite
binders (PDB codes 1NHU19 and 1OS520) were used to develop a
bindingmodehypothesis forcompound1. TheseX-raystructuresre-
vealed the presence of two critical recognition elements in the bind-
ing pocket: (1) a conserved hydrogen bonding motif to the backbone
amide NH of Ser 476 and/or Tyr 477 and (2) a hydrophobic pocket
formed predominantly by Leu 419, Met 423 and Trp 528. Manual
docking of compound 1, while maintaining these interactions, sug-
gested a reasonable fit (Fig. 3). The C-4 carbonyl may form a hydro-
gen bond to the backbone amide NH of Tyr 477, the C-3 ketone
carbonyl forms a hydrogen bond to Ser 476, and the primary hydro-
phobic pocket is occupied by the phenyl of the C-3 phenyl ketone
group. This pose would place the 6- and 7-position substituents of
the quinolone oriented towards solvent. To improve potency by
optimizing hydrophobic interactions, we began our SAR investiga-
tions at the 1- and 3-positions of the quinolone ring.
analogs (2) was important, with the bulky tert-butyl and methyl sul-
fone derivatives bringing the binding potency down to sub-micro-
molar levels. A polar amine substituent at the para-position results
in loss of potency, while adding a 2- or 3-substituent to a para-
substituted benzyl analog is well tolerated (compounds 3 and 4).
This SAR is consistent with the hypothesis that 1-position substitu-
ents fit in a small hydrophobic pocket. Among the non-aromatic sub-
stituents, the cyclopropyl-methyl compound 5 is inactive with
potency being regained to 9 lM with the larger cyclohexyl methyl
group in compound 6. The data obtained with the phenethyl substi-
tution (compound 7) suggests that the vector into the pocket is quite
narrow and requires a more linear substituent as potency is regained
with a phenylpropyl substitution (compound 8).
The poor replicon potency observed for compounds in Table 1
underscored the need to improve the binding potency and physical
properties of the series. Therefore, the 3-position phenyl ketone
was subjected to an exploratory SAR analysis. The proposed bind-
ing mode of compound 1 suggested that the ketone carbonyl
formed an important hydrogen bond to Ser 476. Analogs were syn-
thesized that retained this critical hydrogen bond acceptor, but
varied the distance and geometry between the quinolone ring H-
bond acceptor, and the terminal phenyl ring (Table 2).
In addition to determining the NS5b inhibition potential, com-
pounds were also evaluated in a cell-based viral replication surro-
gate assay known as the replicon system.17,18b In parallel,
compounds were also evaluated for their cytotoxic effects in the
same cell line. Table 1 shows selected examples of analogs at the
1-position of the quinolone ring. Para-substitution on the benzyl
The highlight of this SAR exploration was the discovery of the 3-
position benzyl ester which imparted improved binding potency.

84
D. V. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 82–87
Table 1
1-Position SAR of quinolone series
O
O
O
18b,c
18b,c
#
IC50
(
lM)
EC50
(l
M)
GI50
(lM)
O
N
R1
–H
–Cl
–t-Bu
–NH2
–SO2Me
–SO2NH2
170
1.1
0.68
140
0.5
na
11.7
9.5
na
25.3
>50
na
15.5
>25
na
>50
>25
R
2
0.85
F
3
4
0.41
1.1
>12
na
>25
na
CF3
Cl
Cl
5
6
120
9
na
38
na
22
Ph
7
8
110
2.2
na
na
na
na
Ph
Table 2
3-Position SAR of quinolone series; discovery of benzyl esters
O
R2
R1
N
#
IC50
(l
M)
EC50
(l
M)
GI50(lM)
Cl
R1
R2
O
1
9
6,7-Dimethoxy
6,7-Dimethoxy
1.7
16
11.7
>15
15.5
>15
O
O
O
O
10
6,7-Dimethoxy
0.02
1.2
28.3
Cl
O
N
R
11
12
13
6-Fluoro, 7-morpholinyl
6,7-Dimethoxy
16 (R = H) 330 (R = Me)
na
na
na
na
na
na
Cl
Cl
O
100
O
O
S
6,7-Dimethoxy
> 150
Cl
R
H
N
H
N
14
15
6-Fluoro, 7-N-methyl piperazinyl
6-Fluoro, 7-N-methyl piperazinyl
26 (R = H) >150 (R = Cl)
4.6 (R = H) 2 (R = Cl)
na
na
na
na
O
O
H
N
R

D. V. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 82–87
85
The 4-chlorobenzyl ester (compound 10) with a binding potency of
20 nM provided us with a lead compound, notwithstanding the
presumed in vivo metabolic liability of the ester functionality.
The 4-chlorobenzyl group apparently occupies a critical pocket of
access this putative pocket using linkers other than an ester group
resulted in potency losses of >100-fold and was not pursued
further.
The benzyl ester 10 proved to have poor solubility in physiolog-
ical media. Introducing a methyl-piperazine group at the 7-posi-
the protein as the smaller ethyl ester (compound 9, IC50 16 lM))
had poor activity. We explored alternative ways to access this
putative pocket using linkers other than an ester group. Selected
examples shown in Table 2 include amide, reverse amide, sulfone,
ether, and urea linkers (11–15). Exploration of alternative ways to
tion afforded compound 16 (IC50 0.016 lM) with improved
binding affinity and solubility. The structure of this compound
was solved in complex with a construct of NS5B from which the
C-terminal 21 residues had been removed.
O
N
O
O
N
O
Analoging
O
O
F
O
O
Cl
N
Cl
N
10
Cl
16
SO2Me
The structure was refined using data to 2.2 Å resolution
(R = 0.219; FreeR = 0.264; RMSbonds = 0.005 Å; RMSangles = 0.74°,
PDB accession code: 3PHE). There were four independent mole-
cules in the asymmetric unit and each showed clear electron den-
sity for the inhibitor. The crystal structure revealed a binding mode
consistent with our earlier hypothesis. The ligand occupies the
NNI-2 binding site located in the thumb region (Fig. 4a). Overall,
the protein structure is very similar to that observed for 1OS5
and 1NHU. Two hydrogen bonds exist between the ligand carbonyl
oxygen atoms and the backbone amide NH atoms of Ser 476 and
Tyr 477. The benzyl ester occupies a hydrophobic pocket lined by
Leu 419, Met 423, and Trp 528. Residues Leu 497 and Met 423
adopt conformations similar to those observed in 1OS5, not
1NHU ( Fig. 4b). This allows for a pocket to accommodate the
O
O
O
F
F
NH2
EtOOC
F
F
diphenyl ether
(reflux)
OEt
EtO
EtO
OEt
N
H
O
O
O
N
O
1. 4-Methylpiperazine,
F
F
F
F
Cl
acetonitrile, reflux
OEt
OEt
MeO2
S
2. NaOH, EtOH, reflux
N
H
K2CO3
O
O
F
SO2Me
OH
Br
Cl
N
N
16
N
K2CO3
Figure 4. (a) X-ray structure of 16 bound to NNI-2 site (PDB accession code: 3PHE).
(b) Overlay with 1OS5 (white, also drawn in 2-D) showing overlapping regions of
the two ligands, conserved backbone conformation and rotamer differences of Ile
482 and Leu 419.
SO2Me
Scheme 2.
O
O
O
O
MeO
MeO
NH2
HC(OEt)3
Ac2O
EtO
EtO
EtO
F
Br
O
O
O
EtOOC
F3C
MeO
MeO
MeO
diphenyl ether
(reflux)
3
K2CO3
N
H
MeO
N
H
Scheme 1.

86
D. V. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 82–87
Table 3
Optimization of benzyl ester series
O
N
O
O
Ar
R3
#
IC50
(
lM)
EC50
(l
M)
GI50 (lM)
R2
Ar
R2
R3
10
17
18
19
20
21
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Tolyl
2-Tolyl
3-Tolyl
4-Cl
4-Cl
4-Cl
2-F, 4-CF3
2-F, 4-CF3
2-F, 4-CF3
6,7-Dimethoxy
0.02
1.2
1.7
28.3
>12
1.1
20
31
7.3
6-Fluoro, 7-morpholinyl
6-Fluoro, 7-piperazinyl
6,7-Dimethoxy
6,7-Dimethoxy
6,7-Dimethoxy
0.020
0.010
0.008
0.015
0.06
0.12a
0.23
0.71
1.2
a
1a replicon EC50 = 0.41 lM.
methyl sulfone, which makes mostly hydrophobic interactions
with Val 485, Leu 489, and Leu 419. Leu 419 and Ile 482 adopt dif-
ferent rotamers than seen in the 1OS5 structure, allowing for better
hydrophobic packing with the quinolone ring. Finally, the pipera-
zine is largely solvent exposed, confirming our earlier hypothesis
that the 6- and 7-positions project towards solvent.
A final round of optimization led to improvement in replicon
potency. With evidence that the 6,7-positions of the quinolone
scaffold were indeed solvent exposed, we sought to improve the
replicon potency of our lead benzyl ester compounds by attaching
a variety of polar substituents in this region of the molecule. The 7-
morpholinyl substituted analog (17) did not offer improvement in
replicon potency but the charged 7-piperazinyl substituted analog
References and notes
1. Choo, Q. L.; Richman, K. H.; Han, J. H.; Berger, K.; Lee, C.; Dong, C.; Gallegos, C.;
Coit, D.; Medina-Selby, R.; Barr, P. J., et al Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
2451.
2. Cohen, J. Science 1999, 285, 26.
3. Reed, K. E.; Rice, C. M. Curr. Top. Microbiol. Immunol. 2000, 242, 55.
4. McHutchison, J. G. Am. J. Manag. Care 2004, 10, S21.
5. Seeff, L. B. Semin Gastrointest. Dis. 1995, 6, 20.
6. Fried, M. W.; Shiffman, M. L.; Reddy, K. R.; Smith, C.; Marinos, G.; Goncales, F. L.,
Jr.; Haussinger, D.; Diago, M.; Carosi, G.; Dhumeaux, D.; Craxi, A.; Lin, A.;
Hoffman, J.; Yu, J. N. Eng. J. Med. 2002, 347, 975.
7. Hayashi, N.; Takehara, T. J. Gastroenterol. 2006, 41, 17.
8. Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi, V. K.; Shiffman, M.;
Reindollar, R.; Goodman, Z. D.; Koury, K.; Ling, M.; Albrecht, J. K. Lancet 2001,
358, 958.
9. Manns, M. P.; Foster, G. R.; Rockstroh, J. K.; Zeuzem, S.; Zoulim, F.; Houghton, M.
Nat. Rev. Drug Disc. 2007, 6, 991.
10. Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M. J. Virol. 2000, 74,
2046.
11. Erhardt, A.; Deterding, K.; Benhamou, Y.; Reiser, M.; Forns, X.; Pol, S.; Calleja, J.
L.; Ross, S.; Spangenberg, H. C.; Garcia-Samaniego, J.; Fuchs, M.; Enriquez, J.;
Wiegand, J.; Stern, J.; Wu, K.; Kukolj, G.; Marquis, M.; Beaulieu, P.; Nehmiz, G.;
Steffgen, J. Antivir. Ther. 2009, 14, 23.
12. Kneteman, N. M.; Howe, A. Y.; Gao, T.; Lewis, J.; Pevear, D.; Lund, G.; Douglas,
D.; Mercer, D. F.; Tyrrell, D. L.; Immermann, F.; Chaudhary, I.; Speth, J.; Villano,
S. A.; O’Connell, J.; Collett, M. Hepatology 2009, 49, 745.
13. Toniutto, P.; Fabris, C.; Bitetto, D.; Fumolo, E.; Fornasiere, E.; Pirisi, M. IDrugs
2008, 11, 738.
14. Hang, J. Q.; Yang, Y.; Harris, S. F.; Leveque, V.; Whittington, H. J.; Rajyaguru, S.;
Ao-Ieong, G.; McCown, M. F.; Wong, A.; Giannetti, A. M.; Le Pogam, S.; Talamas,
F.; Cammack, N.; Najera, I.; Klumpp, K. J. Biol. Chem. 2009, 284, 15517.
15. Koch, U.; Narjes, F. Infect. Disord. Drug Targets 2006, 6, 31.
16. Pauwels, F.; Mostmans, W.; Quirynen, L. M.; van der Helm, L.; Boutton, C. W.;
Rueff, A. S.; Cleiren, E.; Raboisson, P.; Surleraux, D.; Nyanguile, O.; Simmen, K.
A. J. Virol. 2007, 81, 6909.
(18) improved replicon potency to 0.12
mised safety window. This compound was also tested in the geno-
type 1a replicon and had a comparable potency of 0.41 M.
lM, albeit with a compro-
l
Improvement in binding affinity also led to improved replicon po-
tency. This is exemplified in analogs with 2-fluoro, 4-trifluoro-
methyl benzyl substitution at the 1-position (19–21). Compound
19 with the most improved enzyme potency at 8 nM also had
improved replicon potency (replicon 1b EC50 = 0.23 lM) and a
100-fold safety window. The 2-, 3- and 4-tolyl substitution off
the benzyl ester (compounds 19–21) showed that 4-substituted
benzyl ester has the best binding potency, while 2- and 3-substitu-
tion results in a relative potency loss.
Scheme 1 describes the preparationof compound3, which exempli-
fies the synthesis of keto-quinolone compounds shown in Table 1.21
Scheme 2 describes the preparation of the benzyl ester 16
which serves to exemplify syntheses of key quinolone compounds
with a benzyl ester substituent at the 3-position. The details for
synthesis of this and other key compounds in Tables 2 and 3 have
been described previously.18c
In conclusion, we have described the discovery of a novel class
of compounds that inhibit HCV viral replication. This class of com-
pounds binds to the allosteric site NNI-2 of NS5B polymerase as
confirmed through a co-crystal structure of compound 16 to
NS5B. Preliminary optimization led to attractive lead compounds
with sub-micromolar replicon potency in genotypes 1a and 1b.
Recognizing the possible in vivo liability of the key benzyl ester
substitution in these lead compounds, we initiated SAR directed
towards replacing this ester functionality. Our efforts in this area
will be the subject of a future disclosure.
17. Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager,
R. Science 1999, 285, 110.
18a. As described in: McKercher, G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.;
Lefebvre, S.; Pellerin, C.; Thauvette, L.; Kukolj, G. Nuclic Acids Res. 2004, 32, 422;
scintillation proximity assays (SPA) were performed at ambient temperature
(22 °C) in 96-well plates using 50 nM of enzyme (a C-terminal 21 residue
deletion mutant of NS5b from genotype 1b BK isolate). Reaction components
included 20 mM Tris–HCl pH 7.5, 5 mM MgCl2, 1 mM EDTA, 2 mM DTT, 5%
DMSO, 0.01% BSA and 25 mM KCl. 80
mixture of 0.5
Ci of [3H]UTP, 1 M UTP, 250 nM 50-biotinylated oligo(rU12
and 0.8 g/ml poly(rA) to assay wells containing enzyme preincubated with
inhibitor. Reactions were terminated after 120 min by the addition of 20 l of
stop solution containing 150 g/ml tRNA and 10 mg/ml streptavidin-coated
SPA beads (Amersham) in 20 mM Tris–HCl pH 7.5, 25 mM KCl, 0.5 M EDTA and
0.025% sodium azide. After incubation for 30 min, 65 l of 5 M cesium chloride
ll reactions were initiated by adding a
l
l
)
l
l
l
l
were added to the wells and further incubated for 1 h before top-counting in a
Microbeta Trilux plate reader.
18b. Replicon assay: HCV replicon-containing cells (Huh7/Clone A, genotype 1b)
were maintained in growth medium (DMEM medium, Invitrogen),
supplemented with 10% Fetal Bovine Serum, non essential amino acids and
1 mg/mL G418) (Blight, K. J.; Kolykhalov, A. A.; Rice, C. M. Science 2000, 290,
1972–1974). For the HCV replicon assay, Huh7/Clone A cells were trypsinized
from culture flasks, seeded in 1 ml of Clone A growth medium without G418 at
40,000 cells per well in 24-well plates and incubated at 37 °C in a humidified
CO2 (5%) incubator overnight. Following overnight incubation, test compound
was serially diluted in DMSO and added to the test system such that the final
Acknowledgment
The authors thank Dr. Robert Booth (Virobay Inc.) for support of
the research that went into this Letter.

D. V. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 82–87
87
concentration of DMSO was 0.5% in each well. For IC50 determinations,
compounds were tested at seven concentrations in triplicates. Plates were
incubated at 37 °C for 48 h. After incubation, cells were harvested, transferred
to 96-well plates, and subjected to total RNA extraction using the RNA
Isolation Kit (RNeasy 96, Qiagen). TaqMan quantitative PCR (RT-qPCR) was
used to quantify the amount of HCV replicon RNA in each sample. The samples
without compound treatment served as a control and the HCV replicon RNA
level from untreated cells was defined as 100%. Compound inhibitory activity
was determined as the ratio of the normalized HCV RNA amount in treated
samples relative to the untreated control. Compound IC50’s were calculated
using a standard 4 parameter curve fit model.
20. Love, R. A.; Parge, H. E.; Yu, X.; Hickey, M. J.; Diehl, W.; Gao, J.; Wriggers, H.;
Ekker, A.; Wang, L.; Thomson, J. A.; Dragovich, P. S.; Fuhrman, S. A. J. Virol. 2003,
77, 7575.
21. 3-Oxo-3-p-tolyl propionic acid ethyl ester was heated under reflux (160 °C) with
1.6 equiv of triethylorthoformate and acetic anhydride (2.5 equiv) for 4 h, then
concentrated and dried to afford the substituted acrylic ester intermediate
shown. This was dissolved in isopropanol and refluxed with an equimolar
amount of 3,4-dimethoxyaniline. Purification by silica gel chromatography
afforded
(Z)-3-(3,4-dimethoxy-phenylamino)-2-(4-methyl-benzoyl)-acrylic
acid ethyl ester in 60% yield over two steps. This compound was added as a
solid to refluxing diphenyl ether and the mixture heated at reflux for 1 h. Upon
cooling 6,7-dimethoxy-3-(4-methyl-benzoyl)-1H-quinolin-4-one crystallized
out. This compound was dissolved in DMF and alkylated by treatment with 1-
bromomethyl-2-fluoro-4-methyl-benzene (1.5 equiv) in presence of K2CO3
(3 equiv) to afford compound 3 in 70% yield.
18c. Kumar, D. V.; Rai, R. WO 2007/005779, 2007.
19. Wang, M.; Ng, K. K.; Cherney, M. M.; Chan, L.; Yannopoulos, C. G.; Bedard, J.;
Morin, N.; Nguyen-Ba, N.; Alaoui-Ismaili, M. H.; Bethell, R. C.; James, M. N. J.
Biol. Chem. 2003, 278, 9489.