Hepatitis C NS5B polymerase inhibitors: Functional equivalents for the benzothiadiazine moiety

Article abstract of DOI:10.1016/j.bmcl.2010.12.067

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.

Full text of DOI:10.1016/j.bmcl.2010.12.067

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1876–1879
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hepatitis C NS5B polymerase inhibitors: Functional equivalents
for the benzothiadiazine moiety
⇑
Douglas K. Hutchinson , Charles A. Flentge, Pamela L. Donner, Rolf Wagner, Clarence J. Maring,
Warren M. Kati, Yaya Liu, Sherie V. Masse, Tim Middleton, Hongmei Mo, Debra Montgomery, Wen W. Jiang,
Gennadiy Koev, David W. A. Beno, Kent D. Stewart, Vincent S. Stoll, Akhteruzzaman Molla, Dale J. Kempf
Department of Antiviral Research, Department of Drug Metabolism and Pharmacokinetics, Department of Structural Biology, Global Pharmaceutical Research and
Development, Abbott Laboratories, Abbott Park, IL 60064, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothi-
adiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited
potent activity in enzymatic and replicon assays.
Received 1 November 2010
Revised 9 December 2010
Accepted 15 December 2010
Available online 13 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C
NS5B polymerase inhibitors
Benzothiadiazine
Bioisosteres
Benzothiadiazine derivatives, such as 11 and 2,2 shown in Figure
1, are of great interest since they have been found to be potent
inhibitors of the RNA-dependent RNA polymerase (RdRp) of the
hepatitis C virus (HCV).
A number of SAR studies have shown that modification of the
N-alkyl-4-hydroxy-3,4-dihydroquinoline-2-one moiety of 1,1 such
as that of the gem-dialkyl derivative 2,2 can have profound effects
on both the antiviral activity of these compounds, and on their
pharmacological properties.
sulfonyl group participates in a water-mediated hydrogen bond
with the hydroxyl group of serine-288 in the palm site of the
HCV NS5B polymerase.1b,5b,c To probe the importance of this inter-
action to the in vitro activity, we attempted to prepare quinazoline
derivative 10. As shown in Scheme 1, borane reduction6 of nitro
O
O
O
O
H
S
N
S
OH
N
S
OH
N
While these and other studies3 have focused primarily on
changes to the AB ring system of 1, much less has appeared con-
cerning changes to the benzothiadiazine CD ring system. Notably,
disclosures by Roche4 and more recently by Anadys5 have de-
scribed compounds such as 3 and 4 (see Fig. 1), which exhibit po-
tent antiviral activity in both enzymatic and cell culture assays,
despite the replacement of one of the nitrogen atoms of the benzo-
thiadiazine moiety with a carbon atom, as in 3, or with the saccha-
rin moiety, as in 4. In view of the great portent of these findings, we
wish to describe our own efforts in this area.
As part of our efforts to understand the importance of each of
the structural features of benzothiadiazine 2, we sought to under-
stand qualitatively how much binding energy was gained by hav-
ing the sulfonyl moiety in the CD ring system. A number of
groups have published crystal structures suggesting that the C-ring
D
C
O O
N
H
N
H
A
B
O
N
O
1
2
O
O
O
H
O
S
S
N
N
O
OH
N
S
HO
O
O
F
O
N
O
H
N
S
N
O
4
3
F
F
⇑
Corresponding author. Tel.: +1 847 838 5899.
E-mail address: hutsmi@sbcglobal.net (D.K. Hutchinson).
Figure 1. Benzothiadiazine analog series and CD ring replacements.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.067

D. K. Hutchinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1876–1879
1877
NO2
O
S
OH
N
O
O
NO2
NO2
a
b
H2N
H2N
S
N
H
H2N
H2N
O
8
5
7
6
c
H
H
N
S
O O
N
OH
N
S
OH
N
O
O O
N
N
H
O
9
10
Scheme 1. Reagents and conditions: (a) (i) BH3–Me2S, THF; (ii) HCl, MeOH, 30% overall; (b) dioxane, Et3N, 100 °C, 38%; (c) (i) H2, Pt/C, EtOAc; (ii) MsCl, pyridine, 55% overall.
amide 57 afforded diamine 6 in low yield as its bis-hydrochloride.
Treatment of this material with ketenedithioacetal 72b,d afforded
the corresponding dihydroquinazoline 8 in moderate yield.
After reduction and methanesulfonamide formation, dihydro-
quinazoline 9 was obtained in 55% overall yield. Unfortunately,
using a variety of oxidation conditions (DDQ, etc.), we were unable
to oxidize 9 to the desired quinazoline 10. Testing of 9, however,
against HCV polymerases from genotypes 1a and 1b showed IC50
Consequently, we utilized relatively unknown chemistry pub-
lished by Schnekenburger,9 whereby an N-methoxy quinoline salt
is allowed to react with a 1,3-dicarbonyl system in the presence of
an amine base. Thus, amine oxides 13 were treated with methyl
triflate, and after a salt exchange with hexafluorophosphoric
acid,10 the N-methoxy quinoline salts 14 could be isolated in good
overall yield. Treatment of a cold DMF solution of diketone 15 and
Hünig’s base with a solution of salts 14 cleanly afforded quinoline
derivatives 16 in 48–78% yields (see Supplementary data for a de-
tailed preparation of 16a). Unsubstituted compound 18 was pre-
pared in an analogous manner from N-methoxyquinoline
hexafluorophosphate (17). All of the compounds 16 and 18 were
bright yellow in color, due to the favored hydrogen bond-stabilized
enaminedione tautomer shown, which predominates in both solu-
tion and the solid state.11
values of 0.83 and 4.2 lM, respectively.
Surmising that the modest activity of 9 was due in part to
lowered propensity of the four rings to assume coplanarity, we
decided to prepare the corresponding quinoline derivatives. The
general synthetic route is summarized below in Scheme 2. As
shown, commercially available nitro quinoline derivatives 11
were reduced to corresponding amine and treated with meth-
anesulfonyl chloride to afford methanesulfonamides 12 in good
overall yield. Oxidation with MCPBA readily afforded amine oxi-
des 13. While amine oxides 13 could be treated with diketone
152b in the presence of warm acetic anhydride, to afford quino-
line derivatives 16 directly,8 such a process in our hands seldom
afforded the desired products in yields higher than 20%, and
purification was complicated by the presence of many unidenti-
fied side products.
Compounds 16 were evaluated in biochemical assays against
HCV NS5B polymerases from genotypes 1a and 1b. In addition,
the activities of these compounds in cell culture against the HCV
subgenomic replicons from genotypes 1a and 1b were also deter-
mined. Results are summarized above in Table 1 (see Supplemen-
tary data for biological methods).
Prototypical compound 16a (R = H) showed surprisingly
good activity in both enzymatic and cell culture assays, being
-
PF6
R
N
R
R
R
N
H
H
H
N
N
N
NO2
b
d
a
S
S
S
O
O
O
O
O O
N+
O-
N+
O
14a-i
12a-i
11a-i
13a-i
c
15
-
PF6
R
N+
O
H
N
O
S
O
OH
O
O
15
17
N
H
N
H
f
e
O
O
O
15
18
16a-i
Scheme 2. Reagents and conditions. (a) (i) H2, Pd/C, EtOAc; (ii) MsCl, pyridine, 70% overall; (b) MCPBA, CH2Cl2, 92%; (c) 15, Ac2O,
EtOH–H2O, 56%; (e) 15, DIEA, DMF, 0 °C, 48–78%; (f) DIEA, DMF, 0 °C, 69%.
D, <25%; (d) (i) MeOTf, CH2Cl2; (ii) HPF6,

1878
D. K. Hutchinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1876–1879
Table 1
Enzymatic and cell culture activities of quinoline derivatives
R
H
N
O
S
O
O
O
N
H
N
H
O
O
16a-i
18
a
a
a,b
a,b
Compound
R
Polymerase 1a IC50 (nM)
Polymerase 1b IC50 (nM)
Replicon 1a EC50 (nM)
Replicon 1b EC50 (nM)
2
16a
18
—
H
H
CH3
iso-Amyl
CONMe2
CONHMe
CO2Et
CH2OH
CHO
4
17
1680
32
4300
72
133
315
12
7
29
5550
52
1020
238
333
739
56
4
82
—
135
—
932
1040
3080
781
1200
3320
2
22
—
20
—
16b
16c
16d
16e
16f
16g
16h
16i
99
245
660
50
168
221
15
3
25
5
CO2H
a
Both IC50 and EC50 values are means of at least two independent determinations, standard deviation 10%. Detailed protocols can be found in Supplementary data.
Assay run with 5% fetal calf serum.
b
approximately 4-fold less active in enzymatic assays than highly
active comparator compound 2. In general, activity in the enzy-
matic assays decreased with increasing size of the C-ring 4-posi-
tion substituent, where activity fell off by a factor of two with
methyl substitution as with 16b, and then precipitously with the
bulky isoamyl substituent as with 16c. A similar trend with
increasing steric bulk was observed in the series of N,N-dimethyl
amide 16d and N-methylamide 16e, where all of these compounds
showed much lower activity. An interesting trend was observed in
going from ethyl ester 16f, which only showed modest activity, to
carbinol 16g and aldehyde 16h, which showed nearly identical
activity to 16a in both 1a and 1b enzymatic assays. Surprisingly,
4-position carboxylate 16i showed similar enzymatic activity to
comparator compound 2.
Unfortunately, only prototypical compound 16a showed useful
levels of activity in cell culture assays. Preliminary experiments to
determine intracellular concentration12 of highly active analog 16i
suggest that this compound (and by inference, the others) did not
readily penetrate the cell membrane of the hepatocytes used in
these assays.
group, thus forcing the molecule to bind in a less favorable orien-
tation, this being reflected in much higher IC50 values.
More subtle factors seem to come into play in the case of com-
pounds 16g–i. Not surprisingly considering the activity of carbox-
amides 16d and 16e, carboethoxy analog 16f shows only modest
activity, but both hydroxymethyl derivative 16g and aldehyde ana-
log 16h have similar activity to unsubstituted analog 16a. More
striking was the activity of carboxylic acid 16i, which was essen-
tially equipotent to comparator compound 2. Molecular mechanics
programs such as Chem3DÒ predict that the carboxyl group of 16i
and the carboxaldehyde of 16h would be co-planar with the quin-
oline ring in the absence of other interactions. In the binding site of
the enzyme, however, it is conceivable that these groups could ro-
tate so that they were oriented orthogonally to the plane of the
quinoline ring. If this were the case, they would, as shown in Figure
2, be able to project into the site on the enzyme that normally
interacts with the sulfonyl group and participate in the water-
mediated hydrogen bond with Serine 288. Similar stabilizing
interactions could also be envisioned for hydroxymethyl analog
16g, and carboxaldehyde 16h. Other interactions with the
Assuming that these compounds bind to the palm site of the
polymerase, their enzymatic activities can be rationalized by com-
parison with polymerase inhibitors whose binding modes have
been determined by X-ray crystallography studies published in
the RCSB Protein Data Bank.4c,5c The major expected binding inter-
actions are summarized below in Figure 2 for compound 16i. It can
be seen that as long as the C-4 position of the C-ring has a small
substituent (such as H with 16a) the binding will be stabilized
by, inter alia, the hydrogen bond interactions with the B-ring car-
bonyl and the NH of Tyr 448, the methanesulfonamide group with
Asn 291 and Asp 318, as well as the neohexyl side chain with the
hydrophobic pocket. Thus, without the C-ring sulfonyl group, com-
pound 16a shows less potent polymerase 1a and 1b enzymatic
activities by only a factor of 3–4-fold as compared to compound
2. Even a methyl group at C-4, as in compound 16b, can be accom-
modated by the enzyme, albeit with activity half that of 16a. When
the C-4 substituent becomes sterically larger, as with an isoamyl
group (16c), an ethoxycarbonyl group (16f), or a carboxamide
(16d and 16e), the substituent in the normal binding orientation
would force the group into making severe steric interactions with
the depression on the enzyme that interacts with the sulfonyl
Ser 556
Gly 449
Ser 288
N
N
H
H
O
H
O
H
H
H
O
H
Tyr 448
Gly 410
H
N
N
O-
O
H
O
O
S
O
H
H
N
O
N
H
Asn 291
O
O-
Met 414
Tyr 415
Phe 193
O
Asp 318
Cys 366
Pro 197
Hydrophobic Pocket
Figure 2. Graphical description of binding interactions of compound 16i with the
NS5B polymerase palm site.4c,5c

D. K. Hutchinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1876–1879
1879
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methanesulfonyl group on the D-ring would be similar to those
noted in the published crystal structure of benzothiazine 3,4c nicely
rationalizing the potent enzymatic activity of carboxylic acid 16i.
To the extent that this rationale is valid, the quinoline-4-carboxylic
acid moiety of compound 16i could be viewed as a bioisosteric
replacement for the benzothiadiazine moiety.
While compound 16i may mimic the binding site interactions of
comparator benzothiadiazine 2, it must be noted that unsubstitut-
ed compound 16a is only 5–6-fold less active than 16i. This rela-
tively modest difference considering the major loss of activity
upon removal of the methanesulfonamide moiety as with com-
pound 18, suggests that these interactions are more important to
the compound’s binding to the enzyme than those of the sulfonyl
group on the C-ring.
It can therefore be postulated that given efforts to optimize the
other structural features of this or a similar series of molecules,
good activity could be obtained without the need for the sulfonyl
group of the C-ring. Such efforts are being vigorously pursued in
our laboratories.
In conclusion, we have developed a series of compounds closely
related to our earlier gem-dialkyl benzothiadiazines that exhibited,
in some cases, excellent activity in enzymatic assays against geno-
types 1a and 1b HCV NS5b polymerases, despite radical modifica-
tion of the C-ring. In one case a 4-quinoline carboxylic acid 16i
gave essentially equivalent enzymatic activity as highly potent
benzothiadiazine derivative 2. The surprising level of activity of
unsubstituted compound 16a suggests that the contribution of
the sulfonyl group of earlier benzothiadiazine analogs is far less
important than previously appreciated.
Acknowledgments
The Authors wish to thank Pamela L. Donner for finding and
making us aware of Ref. 9, which was critical to the success of this
work.
Supplementary data
6. Brown, H. C.; Narasimhan, S.; Choi, Y. M. Synthesis 1981, 441.
7. Yale, H. L.; Kalkstein, M. J. Med. Chem. 1967, 10, 334.
8. Yousif, M. M.; Saeki, S.; Hamana, M. Chem. Pharm. Bull. 1982, 30, 2326.
9. Mehnert, J.; Schnekenburger, J. Arch. Pharm. (Weinheim) 1988, 321, 897.
10. Most literature examples of N-methoxy pyridine or quinoline salts (see for
example: Katritzky, A. R.; Lunt, E. Tetrahedron 1969, 25, 4291) describe
isolation as the corresponding perchlorates. Due to the explosive nature of
perchlorates in general, we sought to avoid such an unnecessary hazard, and as
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.067.
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