HCV NS5B polymerase inhibitors 3: Synthesis and in vitro activity of 3-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.05.021

3-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

Full text of DOI:10.1016/j.bmcl.2009.05.021

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4484–4487
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
HCV NS5B polymerase inhibitors 3: Synthesis and in vitro activity
of 3-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-
2H-quinolizin-2-one derivatives
b
b
b
a
a
Guangyi Wang a, , Laiguo Zhang , Xiaomin Wu , Debasis Das , Donald Ruhrmund , Lisa Hooi ,
*
Shawn Misialek a, P. T. Ravi Rajagopalan a, Brad O. Buckman a, Karl Kossen a, Scott D. Seiwert a,
Leonid Beigelman a,
*
a Discovery Research, InterMune, Inc., Brisbane, CA 94005, USA
b Chemistry Department, WuXi AppTec Co., Ltd, Shanghai 200131, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
3-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives as potential
anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymer-
ase inhibition, and replicon activity are discussed.
Received 6 March 2009
Revised 5 May 2009
Accepted 6 May 2009
Available online 9 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Benzothiadiazine
HCV NS5B polymerase inhibitors
Hepatitis C is a major cause of end-stage liver disease as well as
the leading cause of liver transplantations.1 About 3% of the world’s
population has been infected with HCV and, of these, 60–80% may
progress to chronic liver disease, and 20% of these develop cirrho-
sis.2 Thus far, there is no universally effective therapy for all HCV
genotypes. The current treatment for patients infected with geno-
nized as the most viable protein target for HCV drug discovery.5–7
Two classes of NS5B inhibitors have been well developed: active
site inhibitors such as nucleoside or nucleotide inhibitors that mi-
mic natural polymerase substrates and allosteric inhibitors that
bind to less conserved sites outside the active site and impair the
enzyme’s catalytic efficiency.
type 1 HCV is 48 weeks of pegylated interferon-
ribavirin (RBV). One of the main antiviral effects from Peg-IFN-
and RBV comes from a boost of the natural immune response.
The success rate for achieving a sustained viral response for geno-
type 1 patients in the US, Europe and Japan is ꢀ40%.3 The long
duration of treatment is difficult for patients to tolerate owing to
a
(Peg-IFN-
a
) and
Recently, several classes of non-nucleoside allosteric NS5B inhib-
itors5–7 have been reported, some of which achieved low nanomolar
inhibition in both enzyme and replicon systems. Among the most
potent inhibitors are 1,1-dioxo-2H-benzothiadiazine compounds,
of which compound 1 shown below was one of the first benzothiadi-
azinecompoundsfoundactiveagainstHCVNS5Bpolymerase.8 Com-
pound 1 binds in a site that is located in the palm domain of the
polymerase in the proximity of the active site (see Ref. 8 for a crystal
structure of a benzothiadiazine compound bound to NS5B). Recent
work from a different institution identified the low nanomolar
inhibitors,9,10 which exhibited a good DMPK profile and showed
anti-HCV activity in chimpanzee model.11
a
side effects associated with Peg-IFN-a and RBV that include flu-like
symptoms, fatigue, depression, gastrointestinal symptoms, pul-
monary effects, and others.3 These limitations have led to intense
interest in the discovery and development of novel compounds
that target the viral and host proteins.
HCV NS5B polymerase is an RNA dependent RNA polymerase
that resides at the C-terminal domain of a polypeptide of several
structural and nonstructural proteins and contains the catalytic
machinery responsible for synthesis and replication of the viral
RNA.4 NS5B is essential for the viral replication and has been clin-
ically validated.6 Along with HCV protease NS3/4A, NS5B is recog-
O
OH N
O
H
N
O
O
S
R4
S
R3 OH N
S
R2
O O
N
H
N
N
H
N
O
O
R1
1
2
* Corresponding authors.
E-mail addresses: gyiwang@yahoo.com, gwang@intermune.com (G. Wang),
lbeigelman@intermune.com (L. Beigelman).
IC50 = 32 nM (1b)
EC50 = 417 nM (1b)
R1 = alkyl or ArCH2
R2,R3,R4 = H, F or alkyl
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.021

G. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4484–4487
4485
OEt
O
OEt
O
OEt
O
c
O
a
b
O
O
O
d
N
OH
N
e
OEt
N
OEt
N
N
O
R
R
R
R
3
4a R = isopentyl
5a-c
6a-c
7a-c
4b R = neohexyl
4c
R = 4-fluorobenzyl
H
O
OH N
O
O
O
H
N
S
N
S
R4
OH O
S
OH N
S
H
N
O
O
O O
R
O O
S
f
H2N
S
N
OEt
N
N
H
N
N
H
O O
+
H2N
O
R
O
O
R1
2a R1 = isopentyl
2b R1 = neohexyl
2c R1 = 4-fluorobenzyl
9
4
R = Me
2d
2e
8a-c
R = i-Pr
R = c-Pr
10 R = i-Pr
11 R = c-Pr
4
Scheme 1. Reagents and conditions: (a) i-Pent-Br, LDA, THF, ꢁ78 °C, rt, overnight, 64–70%; (b) NaOH, H2O, rt, overnight; (c) (i) CDI, THF, rt, 3 h; (ii) KOCOCH2COOEt, MgCl2,
TEA, THF, rt, 2 days, 45–55% (2 steps); (d) EtOCOCl, NaH, THF, rt, 2 h; (e) DMSO, 120 °C, 2 h, 25–38% (2 steps); (f) PPSE, 160 °C, 2.5 h, 18–43%.
In search of potent inhibitors of HCV polymerase NS5B at Inter-
OH O
OH
O
O
Mune, we explored several series of benzothiadiazine compounds.
In this Letter, we describe synthesis and in vitro anti-HCV activity
of compounds 2.
a
N
O
OEt
N
OEt
O
Target compounds 2a–e were synthesized, in six steps, as
shown in Scheme 1. Alkylation of the ester 3 with isopentyl bro-
mide, neohexyl triflate and 4-fluorobenzyl chloride yielded the
alkylated products 4a–c, respectively, in good yields, which were
hydrolyzed to the acids 5a–c. An attempt to prepare 7a from 5a
and diethyl malonate via the corresponding acyl chloride or anhy-
dride failed, and a complicated mixture was obtained. A possible
reason was that the alpha proton of 5-methyl-2-(pyridin-2-
yl)hexanoyl chloride or anhydride may have comparable acidity
as the methylene of diethyl malonate, which may have slowed
down the desired alkylation and caused side reactions. Finally,
7a–c were prepared, respectively, by a two-step sequence: conver-
sion of the acids 5a–c to the ketoacetates 6a–c by reaction with
monoethyl malonate potassium salt and subsequent reaction with
ethyl chloroformate. The 1-substituted quinolizinones 8a–c were
prepared, respectively, through cyclization of 7a–c by heating in
DMSO. Condensation of 8a–c with the methylsulfonamide 99 in
polyphosphoric acid trimethylsilyl ester (PPSE)12 gave the desired
benzothiadiazines 2a–c. Similarly, 2d and 2e were prepared by
condensation of 8a with the isopropylsulfonamide 10 and the
cyclopropylsulfonamide 11, respectively.
8a
12
O
OH N
H
N
S
S
O O
b
N
N
H
12 + 9
O
2f
O
OH N
O
H
S
N
S
O O
c
N
N
H
2c
O
2g
F
Scheme 2. Reagents and conditions: (a) H2, 10% Pd/C, AcOH, 60 °C, 71%; (b) same as
(f) in Scheme 1; (c) H2, 10% Pd/C, AcOH, rt, 40%.
Compound 2f was prepared from 8a by a two-step procedure
(Scheme 2). Hydrogenation over palladium selectively reduced
the left ring of 8a to give 12 that contains a saturated 6-membered
ring. Condensation of 12 with 9 afforded the desired benzothiadi-
azine 2f. The hydrogenation was also successfully applied to the
quinolizinone–benzothiadiazine compound 2c, providing 2g in
40% yield.
Synthesis of 2h–j is shown in Scheme 3. Reaction of the com-
mercially available 13 with t-butyl cyanoacetate in the presence
of t-BuOK and Pd(PPh3)4 gave the ester 14, which was then alkyl-
ated with isopentyl bromide. Decarboxylation of 15 followed by
the hydrolysis of the cyano moiety under acidic condition yielded
the acid 16, which was subjected to a 4-step conversion described
in Scheme 1 (c–f) to afford the desired 2h. In the similar fashion the
reaction of the commercially available 17a and 17b with diethyl
malonate in the presence of cuprous iodide and cesium carbonate
gave 18a and 18b, respectively. Alkylation and subsequent decar-
boxylation of 18a and 18b yielded the acids 20a and 20b, respec-
tively. Compounds 20a and 20b were converted to 2i and 2j,
respectively, by the 4-step procedure shown in Scheme 1.
Benzothiadiazine compounds 2a–j were tested in HCV NS5B
genotype 1b polymerase and replicon assays,13,14 and the results
are presented in Table 1. All the compounds exhibited very potent
inhibition of HCV NS5B polymerase, with all IC50 values less than
5 nM, which was lower limit imposed by the enzyme concentra-
tion. The most active compound in the replicon system was com-
pound 2c with EC50 value of 2.3 nM and CC50 value of greater
than 100 lM, which translated to a CC50/EC50 ratio of more than
40,000. Other four compounds 2a, 2b, 2i and 2j also exhibited
excellent replicon activity with EC50 values of 5–17 nM. The rest
of the compounds in Table 1 also showed significant replicon activ-
ity. R1 of the structure 2 was selected from a group of alkyls based
on the excellent activity of the quinolinone–benzothiadiazine com-
pounds bearing these alkyl groups,8 however, the activity order in
the quinolizinone–benzothiadiazine series in this Letter seems dif-
ferent from that in the quinolinone–benzothiadiazine series. In the
quinolizinone–benzothiadiazine series the best R1 so far was 4-flu-
orobenzyl group, as shown by the EC50 value (2.3 nM) of com-

4486
G. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4484–4487
H
N
O
OH N
O
O
S
S
O
O
O O
c
4 steps*
b
a
N
N
N
H
COOH
N
OH
CN
N
Ot-Bu
N
N
Cl
Br
O
CN
14
13
16
2h
15
O
OH N
H
S
N
S
R
R
R
O O
O
R
R
4 steps*
b'
a'
c'
COOEt
COOEt
N
H
COOH
N
N
N
OEt
OEt
O
N
O
18a-b
17a R=F
2i R=F
20a-b
19a-b
17b R=Me
2j R=Me
Scheme 3. Reagents and conditions: (a) N„CCH2COOt-Bu, t-BuOK, Pd(PPh3)4, dioxane, 70 °C, overnight, 46%; (b) isopentyl bromide, Cs2CO3, DMF, rt, overnight, 70%; (c) HCl,
H2O, 100 °C, overnight, quant.; (a0) H2C(COOEt)2, picolinic acid, CuI, Cs2CO3, dioxane, 100 °C, overnight, 42–48% from 17b; (b0) isopentyl bromide, K2CO3, DMF, 50–60 °C,
overnight, 58–74%; (c0) NaOH, EtOH, H2O, 100 °C, 1 h, 84%. *Four steps include steps c, d, e and f in Scheme 1.
Table 1
Inhibition of HCV NS5B polymerase genotype 1b by compounds 2a–2j and replicon activity
a
a
Compound
IC50
(
lM)
EC50
(
lM)
CC50
(lM)
Compound
IC50
(
lM)
EC50
0.21
0.036
0.20
0.005
0.015
(l
M)
CC50 (lM)
2a
2b
2c
2d
2e
<0.005
<0.005
<0.005
<0.005
<0.005
0.017
0.010
0.0023
0.13
30
2f
2g
2h
2i
<0.005
<0.005
<0.005
<0.005
<0.005
>1
>10
>1
>1
>1
>100
>100
>10
0.057
>10
2j
a
The initial CC50 values of 2a–j were either >10
lM or >1
lM, limited by the compound concentrations used, but the CC50 values of 2a–c were re-determined at higher
compound concentrations.
4. Lindenbach, B. D.; Rice, C. M. Nature 2005, 436, 933.
5. Beaulieu, P. L. Curr. Opin. Invest. Drugs 2007, 8, 614.
6. Rönn, R.; Sandström, A. Curr. Top. Med. Chem. 2008, 8, 533.
7. Tramontano, E. Mini-Rev. Med. Chem. 2008, 8, 1298.
pound 2c which is several fold lower than 2a and 2b (EC50: 17 and
10 nM) in which R1 is isopentyl and neohexyl group while the best
R1 was cyclopropylethyl, neohexyl and isopentyl in quinolinone–
benzothiadiazine series.8 Further R1 optimization may be useful
for enhancement of potency as well as CC50/EC50 ratio and possibly
for improvement of other drug-like properties such as solubility
and cell-permeability. The data in Table 1 indicate that methylsulf-
onamide at the R4 position of the structure 2 is superior to the
cyclopropylsulfonamide and isopropylsulfonamide, probably
implicating the effect of substituent size. It seems that a small R2
is well tolerated, as indicated by excellent IC50 and EC50 values of
2i and 2j (R2 = F or Me). However, compared with 2a, the methyl
group at the R3 position decreased the replicon activity by more
than 10-fold (17 nM for 2a vs 200 nM for 2h), indicating the intol-
erance of a substituent at this position. It is interesting to note that
compounds 2f and 2g in which the left ring is saturated still exhib-
ited good activity in both NS5B and replicon assays. Overall, the
data in Table 1 clearly demonstrate that the quinolizin-2-one-ben-
zothiadiazine ring system is a very promising scaffold for further
derivatization and optimization.
8. Tedesco, R.; Shaw, A. N.; Bambal, R.; Chai, D.; Concha, N. O.; Darcy, M. G.;
Dhanak, D.; Fitch, D. M.; Gates, A.; Gerhardt, W. G.; Halegoua, D. L.; Han, C.;
Hofmann, G. A.; Johnston, V. K.; Kaura, A. C.; Liu, N.; Keenan, R. M.; Lin-Goerke,
J.; Sarisky, R. T.; Wiggall, K. J.; Zimmerman, M. N.; Duffy, K. J. J. Med. Chem.
2006, 49, 971.
9. Krueger, A. C.; Madigan, D. L.; Green, B. E.; Hutchinson, D. K.; Jiang, W. W.; Kati,
W. M.; Liu, Y.; Maring, C. J.; Masse, S. V.; McDaniel, K. F.; Middleton, T. R.; Mo,
H.; Molla, A.; Montgomery, D. A.; Ng, T. I.; Kempf, D. J. Bioorg. Med. Chem. Lett.
2007, 17, 2289.
10. Krueger, A. C.; Madigan, D. L.; Jiang, W. W.; Kati, W. M.; Liu, D.; Liu, Y.; Maring,
C. J.; Masse, S.; McDaniel, K. F.; Middleton, T.; Mo, H.; Molla, A.; Montgomery,
D.; Pratt, J. K.; Rockway, T. W.; Zhang, R.; Kempf, D. J. Bioorg. Med. Chem. Lett.
2006, 16, 3367.
11. Chen, C. M.; He, Y.; Lu, L.; Lim, H. B.; Tripathi, R. L.; Middleton, T.; Hernandez, L.
E.; Beno, D. W. A.; Long, M. A.; Kati, W. M.; Bosse, T. D.; Larson, D. P.; Wagner,
R.; Lanford, R. E.; Kohlbrenner, W. E.; Kempf, D. J.; Pilot-Matias, T. J.; Molla, A.
Antimicrob. Agents Chemother. 2007, 51, 4290.
12. Imai, Y.; Mochizuki, A.; Kakimoto, M. Synthesis 1983, 10, 851.
13. NS5B assay: A modified assay based on
a published method (McKercher,
G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.; Lefebvre, S.; Pellerin, C.;
Thauvette, L.; Kukolj, G. Nucleic Acids Res. 2004, 32, 422) was used. Assays
were performed at room temperature in 96-well (white, round bottom)
plates in 20 mM Tris, pH 7.5 buffer containing 5 mM MgCl2, 5 mM KCl,
1 mM EDTA, 2 mM DTT, 0.01% BSA. Appropriate serial dilutions of
In summary, we have successfully built a novel quinolizinone–
benzothiadiazine scaffold. Ten compounds based on this scaffold
have been synthesized and tested for their anti-HCV activity. All
the compounds inhibited HCV NS5B polymerase at less than
5 nM concentration and exhibited good to excellent replicon activ-
ity. Particularly, compound 2c has EC50 value of 2.3 nM and its
CC50/EC50 ratio exceeds 40,000. Further optimization and biological
studies are underway and will be reported in the due time.
inhibitors in DMSO were prepared and added to 5 nM NS5b
(genotype 1b, J4 strain) enzyme in above buffer. After 5 min of
incubation, reactions were initiated by the addition of buffered
substrate mix containing 250 nM 50-biotinylated-rU12 RNA primer,
g/
Ci 5,6-3H-UTP. Total
with 5% DMSO (v/v). The reaction was
L of 164 g/mL yeast RNA and 10 mg/mL
streptavidin PVT SPA beads in 0.5 M EDTA, pH 8.0. After 30 min, 80 L of
5 M CsCl was added and incubated for 1 h. Plates were then read using a
Wallac MicroBeta reader. Inhibition data was plotted and fit to 4-
prime values under
D21
a
1
l
mL poly-rA RNA template,
reaction volumes were 100
1 lM UTP and 0.625 l
lL
stopped after 2 h by adding 20
l
l
l
a
parameter logistic equation to extract IC50 values.
these conditions were >0.6.
Z
References and notes
14. HCV replicon assay (EC50, lM): A modified assay based on a published
1. Shepard, C. W.; Finelli, L.; Alter, M. J. Lancet Infect. Dis. 2005, 5, 558.
2. WHO, Hepatitis C. Incidence/Epidemiology under Section Surveillance and
Control. 2002. http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/
en/index.html.
method (Vrolijk, J. M.; Kaul, A.; Hansen, B. E.; Lohmann, V.; Haagmans, B.
L.; Schalm, S. W.; Bartenschlager, R. J. Virol. Methods 2003, 110, 201) was
used. Exponentially growing Huh-7 cells stably transfected with luc/neo ET
replicon were maintained in DMEM media supplemented with 10% fetal
3. Manns, M. P.; Wedemeyer, H.; Cornberg, M. Gut 2006, 55, 1350.

G. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4484–4487
4487
calf serum and seeded at a density of 5 ꢂ 103 cells/well in white 96-well
plates. Compounds were dissolved in DMSO, diluted in DMSO in a serial
fashion to create an appropriate range of concentrations, and added to
cells approximately 24 h after plating. The final DMSO concentration in
the cell plate was 1%. After 46–50 h exposure, the media was discarded
from the assay plate and the cell monolayers were lysed by addition of
100 lL of either BrightGLO (Promega) or ATPlite reagent (PerkinElmer)
with incubation at 20 °C for 2 min on an orbital shaker. Following
incubation, luminescence was assessed on a SpectraMax M5 plate reader
(Molecular Devices). Plots of luminescesce versus log compound
concentration were fit to
EC50 and CC50 values.
a 4-parameter logistic equation to determine