HCV NS5B polymerase inhibitors 2: Synthesis and in vitro activity of (1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.05.022

(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

Full text of DOI:10.1016/j.bmcl.2009.05.022

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4480–4483
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
HCV NS5B polymerase inhibitors 2: Synthesis and in vitro activity
of (1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine
and azolo[1,5-a]pyrimidine derivatives
b
b
b
b
c
Guangyi Wang a, , Huoxing Lei , Xiaofang Wang , Debasis Das , Jian Hong , Colin H. Mackinnon ,
Thomas S. Coulter c, Christian A. G. N. Montalbetti c, Richard Mears c, Xinjie Gai c, Sarah E. Bailey c,
Donald Ruhrmund a, Lisa Hooi a, Shawn Misialek a, P. T. Ravi Rajagopalan a, Robert K. Y. Cheng c,
John J. Barker c, Brunella Felicetti c, Dorian L. Schönfeld c, Antitsa Stoycheva 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
c Department of Medicinal Chemistry, Evotec (UK) Ltd, 114 Milton Park, Abingdon, Oxfordshire, OX14 4SA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives
have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase 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 cirrhosis.2
Thus far, there is no universally effective therapy for all HCV geno-
types. The current treatment for patients infected with genotype 1
machinery responsible for synthesis and replication of the viral
RNA.4 NS5B is essential for viral replication and has been clinically
validated.6 Along with HCV protease NS3/4A, NS5B is recognized 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 mimic
natural polymerase substrates and allosteric inhibitors that bind
to less conserved sites outside the active site and impair the
enzyme’s catalytic efficiency.
HCV is 48 weeks of pegylated interferon-
a
(Peg-IFN-
a) and ribavirin
(RBV). One of the main antiviral effects from Peg-IFN-
a
and RBV
comesfromaboostofthenaturalimmuneresponse. Thesuccessrate
for achieving a sustained viral response for genotype 1 patients in
the US, Europe and Japan is ꢀ40%.3 The long duration of treatment
(48 weeks for genotype 1) is difficult for patients to tolerate owing
H
N
H
N
O
OH N
O
O
OH N
O
S
S
S
S
O O
O O
N
H
N
H
to side effects associated with Peg-IFN-a and RBV that include flu-
N
N
O
N
O
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
2
1
IC50
<10 nM (1b)
IC50 = 0.3 nM (1b)
EC50 = 3 nM (1b)
EC50 = 8.5 nM (1b)
H
N
O
N
O
O
N
O
H
N
S
S
OH
S
OH
N
S
O O
O O
X
Y
N
H
N
H
X
N
Y
O
O
* Corresponding authors at present address: Alios Biopharma, Inc., South San
Francisco, CA 94080, USA.
E-mail addresses: gyiwang@yahoo.com, gwang@intermune.com (G. Wang),
lbeigelman@intermune.com (L. Beigelman).
3
4
X,Y = N, CH
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.022

G. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4480–4483
4481
O
Oi-Bu
O
H2N
O
H
N
EtOOC
N
COOEt
O
COOEt
f
S
a,b,c
d
e
O
O
S
+
N
H2N
N
N
COOMe
COOH
O O
O
O
H2N
10
9
6
7
8
5
O
O
O
H
N
H
N
H
N
O
OH N
O
S
S
OH N
S
O
N
O
S
S
O O
O O
O O
h
g
N
H
N
H
N
H
+
S O
O
N
N
O H2N
O
3a
3b
11
Scheme 1. Reagents and conditions: (a) 2,5-(MeO)2THF, KOAc, AcOH, H2O, reflux, quant. (crude); (b) i-Pent-Br, LDA, THF, ꢁ78 °C, rt, overnight, 22%; (c) LiOH, MeOH, H2O, rt,
6 h, 59%; (d) i-BuOC(O)Cl, TEA, DCM, 0 °C, 4 h, 82%; (e) diethyl malonate, NaH, THF, rt, 2 h, 57%; (f) CH3SO3H, rt, overnight, 29%; (g) toluene, reflux, 2 h; (h) PPSE, 160 °C, 3 h,
15% (two steps).
14 was isolated as the minor product. C-Methylation of 14 and sub-
sequent hydrolysis gave the acid 16, which was converted to the
anhydride 17 by treatment with ethoxytrimethylsilylacetylene.
Recently, several classes of non-nucleoside allosteric NS5B inhib-
itors have been reported,5–7 some of which achieved low nanomolar
inhibition in both enzyme and replicon assays. Among the most po-
The crude 17 was used directly for reaction with diethyl malonate.
tent NS5B inhibitors are 1,1-dioxo-2H-benzo[1,2,4]thiadiazines, of
The resulting 18 was condensed with 10 to give the racemic mix-
which compounds 1 and 2 shown above exhibited promising activ-
ture of 3c and 3d.
ities against HCV NS5B polymerase.8,9 Compound 1 also exhibited a
Synthesis of 3e and 3f is shown in Scheme 3. Benzyl 2-amino-
propionate (19) was reacted with benzaldehyde to give the Schiff
base 20. Alkylation of 20 and subsequent acidic hydrolysis yielded
21, which was condensed with ethyl glyoxylate to form 22. Treat-
good DMPK profile achieving high plasma and liver concentration
and showed anti-HCV activity in chimpanzee model.10
In search of potent allosteric inhibitors of HCV NS5B polymerase
at InterMune, we explored several series of benzothiadiazine com-
pounds. In this report we describe synthesis and in vitro anti-HCV
ment of 22 with tosylmethylisocyanide (TosMIC) in the presence of
base yielded the imidazole derivative 23. Hydrogenation/hydrog-
activities of azolo[1,5-a]pyridine-benzothiadiazines 3 and azol-
enolysis of 23 over palladium reduced 2-methylbutenyl group to
o[1,5-a]pyrimidine-benzothiadiazines 4. An X-ray crystal structure
of 4a bound to HCV NS5B polymerase is also presented.
isopentyl and deprotected benzyl group concomitantly to give
the acid 24. The b-ketoacetate 25 was prepared by conversion of
24 to the acyl chloride and subsequent reaction with ethyl acetate
in the presence of LiHMDS. Compound 25 was cyclized in the pres-
Synthesis of pyrrolo[1,5-a]pyridine-benzothiadiazine 3a and
3b11 is shown in Scheme 1. 2-Aminopropanoic acid (5) was reacted
with 2,5-dimethoxytetrahydrofuran in the presence of KOAc, fol-
ence of DBU and the resulting 26 was condensed with 10 to afford
lowed by alkylation and hydrolysis, to yield the acid 6. Treatment
the racemic mixture of 3e and 3f.
of 6 with isobutyl chloroformate yielded the anhydride 7. Reaction
Synthesis of the azolo[1,5-a]pyrimidines 4a–h is shown in
of 7 with diethyl malonate yielded 8, which was cyclized to afford
Scheme 4. Compounds 27a–b were converted to the corresponding
the key intermediate 9. Condensation of 9 with the 2-aminoben-
secondary amines 28a–b, respectively, in moderate yields by reac-
tion with isopentanoyl chloride and subsequent treatment with
alane–dimethylethylamine complex while 27c was converted to
28c by reaction with isovaleraldehyde and subsequent reduction.
zenesulfonamide 108 gave the coupling product 11, which was cy-
clized in polyphosphoric acid trimethylsilyl ester (PPSE) according
to a published procedure.12 The racemic mixture of 3a and 3b was
separated using a chiral supercritical prep LC13 and their stereo-
Condensation of 28a–c with triethyl methanetricarboxylate and
chemical assignment achieved by X-ray crystallography.14
the following spontaneous cyclization gave 29a–c, respectively,
Synthesis of 3c and 3d is shown in Scheme 2. Alkylation of 12
in moderate yields. Condensation of 29b–c with 10 in PPSE affor-
with the bromide 13 yielded two N-alkylated products, of which
ded 4b–c, respectively, in low yields while condensation of 29a
O
O
O
O
O
O
d
b
a
OEt
COOMe
OEt
COOMe
c
OH
COOH
O
OEt
OMe
N
N
N
N
N
N
N
N
+
O
N
N
H
Br
13
12
14
17
15
16
O
OH N
O
H
S
N
S
OH O
O O
e
f
N
H
OEt
10
+
N
N
N
N
O
O
3c+3d
18
Scheme 2. Reagents and conditions: (a) K2CO3, NMP, 80 °C, 2 h, 11%; (b) MeI, LiHMDS, THF, ꢁ78 °C, 4 h, then rt, 74%; (c) NaOH, EtOH, H2O, reflux, 4 h, 90%; (d) TMS–OEt–
acetylene, ClCH2CH2Cl, 70 °C, 3 days; (e) diethyl malonate, NaH, DMA, 120 °C, 4 h, 11% (two steps); (f) PPSE, 160 °C, 1.5 h, 13%.

4482
G. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4480–4483
O
EtO
c
OBn
e
b
d
a
OBn
N
EtO
OBn
H2N
EtO
OBn
OBn
N
N
H2N
21
O
20
O
19
O
O
O
S
O
N
22
23
H
N
O
OH N
O
S
OH O
O O
O
OEt
O
f
g
EtO
h
N
H
N
N
N
10
+
O
N
OH
OEt
O
N
N
O
O
O
3e+3f
N
N
26
25
24
Scheme 3. Reagents and conditions: (a) PhCHO, 4 Å MS, CHCl3, rt, 24 h, quant; (b) i. 3,3-dimethylallyl bromide, LiHMDS, THF, ꢁ78 °C to rt, 16 h, ii. 1 M HCl/H2O, rt, 2 h, 80%;
(c) EtOC(O)C(O)H, 4 Å MS, THF; (d) TosMIC, LiHMDS, THF, ꢁ78 °C to rt, 45% (two steps); (e) H2, Pd/C, THF, rt, quant; (f) i. (COCl)2, cat. DMF, THF, rt, 5 h; ii. EtOAc, LiHMDS, THF,
ꢁ78 to ꢁ20 °C, 1 h, 44%; (g) DBU, toluene, 50 °C, 4 h, quant; (h) PPSE, 140 °C, 2 h, 45%.
O
N
O
H
N
O
N
O
H
N
S
S
OH
S
OH
N
O
O
OH
N
S
O O
f or g
O O
N
X
Y
X
Y
e
X
Y
a,b
X
Y
N
N
H
NH
NH
R1
NH
NH2
N
OEt
+ 10
N
N
H
N
O
or c,d
O
R2
1
2
4d
R =Me, R =i-Pent
27a X=CH, Y=N
27b X=Y=N
4e R1=cyclo-Pr, R
4f R1=H, R2=Bn
2=i-Pent
27c
X=N, Y=CH
29a-c
4a X=CH, Y=N
4b X=Y=N
28a-c
4g R1=H, R2=4-F-Bn
1
2
4c X=N, Y=CH
4h
R =H, R =4-F-PhCH2CH2
Scheme 4. Reagents and conditions: (a) i-BuO(CO)Cl, pyridine, rt, 16 h; (b) alane–NMe2Et complex, THF, 50 °C, 2 d for 27a–b; (c) isovaleraldehyde, THF, AcOH, 4 Å MS, rt,
16 h; (d) NaBH4, rt, 3 h, for 27c; (e) CH(COOEt)3, toluene, Microwave, 120–130 °C, 30 min; (f) PPSE, 140 °C, 2 h, for 29b–c; (g) i—DMF, 110 °C, 10 h; ii—DBU, pyridine, 120 °C,
16 h, for 29a.
with 10 in DMF with DBU gave 4a in low yield. Compounds 4d–h
were prepared using the same procedures as described for 4c.
We obtained a co-crystal structure of compound 4a in complex
the protein. An ordered water molecule participates in a network
of polar interactions with one of the sulfonamide oxygens of the
benzothiadiazine and the backbone amide nitrogen of Ser556, as
well as the side chain OH of Ser288. An additional ordered water
molecule bridges interactions between the enolic OH of the pyrim-
idone ring with the backbone amide nitrogen of Gly449. This water
molecule is located within 3.3 Å of the second benzothiadiazine
sulfonamide oxygen. The methyl sulfonamide substituent on the
benzothiadiazine ring is involved in additional polar interactions
with the protein. The amide NH of this group is within hydrogen
bonding distance of the Asp318 side chain, while one of the sulfon-
amide oxygens is within 3 Å or less of two hydrogen-bond donors
on the protein: the backbone NH of Asp318 and the side-chain N-
atom of Asn291. The isopentyl substituent on the pyrimidone ring
with HCV NS5B polymerase genotype 1b
D21N-His-tagged at 1.9 Å
resolution (Fig. 1).15 The ligand binds to the enzyme at an allosteric
site located in the palm domain of the polymerase, similarly to
other benzothiadiazines reported in the literature9,16 The dihedral
angle between the azolopyrimidine ring and the benzothiadiazine
moiety is close to 22°. The ligand core is anchored in the binding
site via several hydrogen bonds and water-mediated contacts with
Table 1
Inhibition of HCV NS5B polymerase genotype 1b and replicon by compounds 3a–f and
4a–h
Compound
IC50
(lM)
EC50
(lM)
CC50 (lM)
3a + 3b
3a
3b
<0.005
<0.005
0.15
0.23
0.15
4.9
>100
19.5
170
3c + 3d
3e + 3f
4a
4b
4c
4d
4e
4f
4g
<0.005
<0.005
<0.005
<0.005
<0.005
<0.005
0.009
<0.005
<0.005
0.018
1.1
6.6
0.55
6.7
0.55
1.7
>20
4.0
12
>100
>100
>1
>100
>100
>100
>100
>100
>100
>100
Figure 1. Co-crystal X-ray structure of compound 4a bound to HCV NS5B
polymerase at 1.9 Å resolution. Hydrogen bonds and polar interactions discussed
in the text are shown as dashed lines. Figure rendered with PyMOL.
4h
>100

G. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4480–4483
4483
Showalter, R. E.; Lardy, M.; Shah, A. M.; Tsan, M.; Patel, R.; LeBrun, L. A.;
Kamran, R.; Sergeeva, M. V.; Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.;
Kirkovsky, L. Bioorg. Med. Chem. Lett. 2008, 18, 3616.
occupies a deep hydrophobic pocket formed by the side chains of
Pro197, Leu384, Met414, Tyr415, and Arg200.
Benzothiadiazine compounds 3a–f and 4a–h were tested in
HCV NS5B genotype 1b polymerase and replicon assays.17,18 As
can be seen from Table 1, optically pure 3a and the racemic mix-
tures 3a + 3b, 3c + 3d, and 3e + 3f all showed very potent NS5B
inhibition with IC50 < 5 nM, which was the lower limit imposed
by the enzyme concentration. However, the compounds exhibited
significant difference in replicon activity. EC50 value of the racemic
10. 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.
11. Compounds 3a and 3b were synthesized and evaluated independently before a
patent application (Ruebsam, F.; Dragovich, P. US Patent Application 2008/
0227774 A1) was published.
12. Imai, Y.; Mochizuki, A.; Kakimoto, M. Synthesis 1983, 10, 851.
13. Chiral separation was achieved using ChiralPak AD,
5
lM column, ID
TM
3a + 3b was 0.23
the racemic mixtures 3c + 3d (EC50: 1.1
6.6 M), respectively. Why additional nitrogen in the azole ring
lM, which is 5-fold and 29-fold more active than
150 ꢂ 30 mm (Daicel Industries) on Berger MultiGram SFC instrument.
Mobile phase: A: supercritical CO2, B: IPA (0.25% DEA), A:B = 60:40 at 40 ml/
min. Column temp.: 35 °C.
l
M) and 3e + 3f (EC50
:
l
14. Tan, H.; Wang, G.; Moy, C.; Kossen, K.; Rajagopalan, R.; Misialek, S.; Ruhrmund,
D.; Hooi, L.; Snarskaya, N.; Stoycheva, A.; Buckman, B.; Seiwert, S.; Beigelman,
L. A poster presentation (# 936) at 44th Annual Meeting of the European
Association for the Study of the Liver, Copenhagen, Denmark, 2009. A co-
crystal structure of 3a and NS5B was presented.
has detrimental effect on replicon activity remains unclear and
needs further investigation. Compounds 4a–d and 4f–g all exhib-
ited potent NS5B inhibition with IC50 values less than 5 nM. Com-
pounds 4a and 4c exhibited submicromolar replicon activity while
other compounds in 4 series mostly had EC50 values in low micro-
molar range. Compounds 4e and 4h exhibited lower potency in
both enzyme and replicon assays. Lower potency of 4h compared
to 4g is probably due to the introduction of the bulky 4-fluoro-
phenethyl group in place of the smaller 4-fluorobenzyl moiety
while lower potency of 4e compared to 4d is likely due to replace-
ment of the methyl group on the azole ring by a larger cyclopropyl
group.
In summary, four novel azolo[1,5-a]pyridine and azolo[1,5-
a]pyrimidine scaffolds have been described. Most of the com-
pounds derived from these scaffolds demonstrated very potent
NS5B inhibition (<5 nM) and exhibited promising replicon potency
reaching submicromolar EC50 values as exemplified by compounds
3a, 4a and 4c. These scaffolds have been selected for further deriv-
atization and optimization, which are underway.
15. A PDB file for the NS5B polymerase/inhibitor complex (PDB identifier 3H98)
has been deposited with the Protein Data Bank.
16. 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.
17. 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 inhibitors in DMSO were prepared and
added to 5 nM NS5b
5 min of incubation, reactions were initiated by the addition of a buffered
substrate mix containing 250 nM 50-biotinylated-rU12 RNA primer, 1
g/mL
poly-rA RNA template, 1 M UTP and 0.625
Ci 5,6-3H-UTP. Total reaction
volumes were 100 L with 5% DMSO (v/v). The reaction was stopped after 2 h
by adding 20 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
D21 (genotype 1b, J4 strain) enzyme in above buffer. After
l
l
l
l
l
l
l
incubated for 1 h. Plates were then read using a Wallac MicroBeta reader.
Inhibition data were plotted and fit to a 4-parameter logistic equation to
extract IC50 values. Z prime values under these conditions were >0.6.
References and notes
18. HCV replicon assay (EC50, lM): A modified assay based on a published 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 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
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9. Ruebsam, L.; Webber, S. E.; Tran, M. T.; Tran, C. V.; Murphy, D. E.; Zhao, J.;
Dragovich, P. S.; Kim, S. H.; Li, L.-S.; Zhou, Y.; Han, Q.; Kissinger, C. R.;
monolayers were lysed by addition of 100 lL of either BrightGLO (Promega) or
ATPlite reagent (Perkin–Elmer) 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 luminescence versus log compound
concentration were fit to a 4-parameter logistic equation to determine EC50
and CC50 values.