Structure-based design, synthesis, and biological evaluation of 1,1-dioxoisothiazole and benzo[b]thiophene-1,1-dioxide derivatives as novel inhibitors of hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2008.05.083

A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS

Full text of DOI:10.1016/j.bmcl.2008.05.083

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4181–4185
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design, synthesis, and biological evaluation
of 1,1-dioxoisothiazole and benzo[b]thiophene-1,1-dioxide derivatives
as novel inhibitors of hepatitis C virus NS5B polymerase
*
Sun Hee Kim , Martin T. Tran, Frank Ruebsam, Alan X. Xiang, Benjamin Ayida, Helen McGuire, David Ellis,
Julie Blazel, Chinh V. Tran, Douglas E. Murphy, Stephen E. Webber, Yuefen Zhou, Amit M. Shah, Mei Tsan,
Richard E. Showalter, Rupal Patel, Alberto Gobbi, Laurie A. LeBrun, Darian M. Bartkowski, Thomas G. Nolan,
Daniel A. Norris, Maria V. Sergeeva, Leo Kirkovsky, Qiang Zhao, Qing Han, Charles R. Kissinger
Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thio-
phene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based
design led to the identification of a number of potent NS5B inhibitors with nanomolar IC50 values. The
most potent compound exhibited IC50 less than 10 nM against the genotype 1b HCV polymerase and
EC50 of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds
were also evaluated.
Received 26 March 2008
Revised 19 May 2008
Accepted 19 May 2008
Available online 24 May 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
1,1-Dioxoisothiazole
Benzo[b]thiophene-1,1-dioxide
Hepatitis C virus (HCV)
NS5B inhibitors
X-ray co-crystal structures
DMPK properties
Hepatitis C virus (HCV) is a major cause of acute hepatitis and
chronic liver disease.1 About 170 million people, 3% of the world’s
population, are infected with HCV and an estimated 3 million indi-
viduals become newly infected each year.2 Currently there are no
vaccines available to prevent hepatitis C. The existing combination
palm-binding NS5B inhibitors, which might exhibit improved
pharmacokinetic (PK) properties.
O O
S
O O
S
treatment of pegylated interferon-a and ribavirin is costly, incon-
R"
N
OH
N
venient, not well-tolerated, and has inadequate response rates,
HO
especially for genotype 1 patients.3
R
N
H
N
R
One promising avenue for treating HCV is to inhibit its replica-
tion by disabling the NS5B polymerase, a virally encoded protein
essential for the HCV life cycle.
H
N
N
R'
O
N
R'
O
In 2006 GSK reported a series of benzothiadiazine analogs (1,
Fig. 1) as potent HCV polymerase inhibitors.4 These molecules bind
to the ‘‘palm” region of NS5B in one of the three binding sites
where non-nucleoside inhibitors typically interact with the pro-
tein.5 Recently, we disclosed a related series of pyridazinone-con-
taining NS5B inhibitors (2, Fig. 1), which also bind to the palm
region of the enzyme.6 The pyridazinone-containing compounds
were found to display oral bioavailabilities in animals. In
continuation, we sought to identify other structurally diverse,
1
2
O
S
O
HO
N
N
O
O
HO
R1
S
X
R4
7
R2
O
6
N
O
R3
3 (IC50 1b = 1.4 μM)
4
* Corresponding author. Tel.: +1 858 453 7200; fax: +1 858 453 7210.
E-mail address: sdsunhee@yahoo.com (S.H. Kim).
Figure 1. HCV NS5B polymerase inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.083

4182
S. H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4181–4185
Herein, we report a novel series of inhibitors of HCV polymerase
in which the benzothiadiazine fragment present in 1 is replaced
with a 1,1-dioxoisothiazole moiety. An early lead compound 3
Boc
N
Me
S
O
S
O
S
O
O
O O
(Fig. 1) exhibited reasonable potency (IC50 = 1.4 lM) against the
a-b
genotype 1b NS5B enzyme and provided a good starting point for
SAR development. In the present study, we focused on optimizing
the lead structure via variation of R1, R2, R3, R4, and X (4, Fig. 1). As
described below, the design of these target molecules was aided by
several co-crystal structures we obtained of various pyridazinone-
containing inhibitors complexed with the NS5B protein.6
The syntheses of these analogs began with the assembly of key
synthetic precursors 8 and 13 (Scheme 3). Precursors 8 containing
various R1, R2, and R3 groups were prepared according to the gen-
eral synthetic route outlined in Scheme 1. Reductive alkylation of
amino acid esters 5 gave the corresponding secondary amines 6,
which were then subjected to an EDC-mediated coupling with
mono-ethyl malonate to afford amides 7.
N
N
O
O
9
10
11
c
H
H
N
N
S
S
O
O
S
O
O
O O
O O
S
d
N
HN
Cl
O
12
Scheme 2. Reagents and conditions: (a) NBS, (BzO)2, CCl4, reflux, 1 d, 49%; (b)
NHBocMs, Cs2CO3, DMF, 35–70 °C, 6 h, 81%; (c) AcOH, reflux, 1–2 d, 94%; (d) POCl3,
DMF, reflux, 10 min, 70%.
Cyclization of 7 under basic condition followed by the decar-
boxylation of the resulting ester intermediate provided the desired
2,4-pyrrolidiones 8.
Different approaches were adopted for the construction of frag-
ment 13 depending on the identity of R4 and X.7 Herein we discuss
the synthesis of reactive fragment 12 as one example. Starting
material 9 was prepared by using slightly modified literature pro-
cedures.8 Benzylic bromination of 9 using one equivalent of NBS
with benzoyl peroxide as an initiator gave predominantly the mon-
obrominated product. Nucleophilic displacement of the bromine
with Boc-protected methane sulfonamide resulted in the forma-
tion of 10. Hot glacial acetic acid was then employed to simulta-
neously remove the tert-butyl and the Boc protecting groups to
give rise to 11. Subsequent chlorination with POCl3 and catalytic
amount of DMF afforded compound 12 (Scheme 2).
The key fragments 8 and 13 were successfully coupled by using
NaH in THF as shown in Scheme 3. Nucleophilic displacement of
the halogen atom of fragment 13 by the enolate of pyrrolidiones
8 provided the desired products 4 as yellow solids.9
No attempts were made to rigorously determine the enantio-
meric purity of final compounds 4. It is therefore possible that
racemization can occur during the synthesis of 8 (Scheme 1) or
during the coupling reaction with 13 (Scheme 3).
R4
O
O
S
O
R1
R2
+
4
X
O
N
R3
Hal
8
13
Scheme 3. Reagent and condition: (a) NaH, THF, 25 °C, 1 h. Hal = Cl or Br; X = N or
CH.
substituents properly appended to the benzothiadiazine ring could
dramatically improve inhibitor potency.6 Accordingly, we sought
to introduce similar polar fragments into our lead inhibitor 3 to
determine whether analogous potency improvements could be
realized. Due to the different NS5B-binding geometries adopted
by the benzothiadiazine and 1,1-dioxoisothiazole-containing
inhibitors, the 7-position of the latter ring system (R4) appeared
to be the ideal location to introduce such modifications. Adding
an OEt fragment in this manner to 3 resulted in a slight decrease
of NS5B enzyme inhibition (14, Table 1).
The above-synthesized 1,1-dioxoisothiazole and benzo[b]thio-
phene-1,1-dioxide derivatives 4 were evaluated for in vitro activi-
ties against NS5B enzymes derived from HCV genotypes 1b6a and
against an HCV genotype 1b replicon.6a The results of these assess-
ments are shown in Table 1.
However, the corresponding hydroxy analog (15) displayed im-
proved anti-NS5B characteristics and incorporating other polar R4
moieties (16–21) afforded several compounds with improved
NS5B inhibition properties relative to 3 (e.g., 16 and 21). Although
the 7-OEt substituent was not the optimal R4 fragment identified
by this initial SAR effort, it was synthetically convenient and there-
fore was employed in our exploration of R1 and R3 modifications.
Accordingly, we replaced the R3 benzyl substituent present in
14 with a 3,3-dimethylbutyl fragment that had imparted good
NS5B inhibition to the previously studied pyridazinone–benzothi-
adiazines.6 The resulting compound (22) displayed slightly weaker
NS5B inhibition levels relative to 14. However, replacement of the
R1 iso-propyl group of compound 22 with a tert-butyl moiety (23)
resulted in an increase in activity in the enzymatic assay. Addi-
tional analogs of compound 23 were therefore synthesized. Incor-
poration of larger aliphatic or aromatic groups at the R1 position
impaired inhibitor potency (24–26). However, combination of an
R1 tert-butyl entity with substituted R3 benzyl moieties further im-
proved potency (27 and 28) with the 3-Cl, 4-F-benzyl group prov-
ing to be the optimal R3 fragment. Inclusion of a less bulky
aliphatic moiety at the R3 position was also explored but was found
to be detrimental to inhibitor potency (compare29 to 23 and 28).
As expected from our initial SAR studies, removal of the OEt group
present in 23 resulted in a slight improvement in NS5B inhibition
properties (compound 30). But compound 30 is clearly much more
Our previous SAR studies, which were conducted with pyridaz-
inone–benzothiadiazine NS5B inhibitors, demonstrated that polar
O
O
R1
R2
R1
a or b
R2
OMe
OMe
NH
NH2 • HCl
R3
5
6
c
O
O
R1
R2
R1
R2
OMe
d-e
O
N
OEt
N
R3
R3
O
O
8
7
Scheme 1. Reagents and conditions: (a) for aromatic aldehydes: i—RCHO, TEA,
MgSO4, THF, 25 °C, 12 h; ii—NaBH4, MeOH, 25 °C, 1 h; (b) for aliphatic aldehydes:
RCHO, NaBH3CN, MeOH, 25 °C, 12 h; (c) HO2CCH2CO2Et, EDCꢀHCl, TEA, DCM, 25 °C,
12 h; (d) NaOEt, EtOH, 25 °C, 12 h; (e) 1 M H2SO4 (aq), reflux, 1 h.

S. H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4181–4185
4183
Table 1
1,1-Dioxoisothiazole analogs (4)a
R1
R2
R3
R4
X
IC50 1b (
lM)
EC50 1b (
l
M)
HLM T1/2 (min)
3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
H
H
H
H
H
H
H
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
H
OEt
OH
N
N
N
N
N
N
N
1.4
3.6
0.47
0.52
1.2
24
18
47
>60
>60
>60
>60
—
—
14
15
16
17
18
19
OCH2CONH2
OCH2CH2CONH2
OCH2COC(CH3)3
OCH2CN
88
c
100
>33
12
21
1.6
>60
O
N
20
CH(CH3)2
H
CH2Ph
N
3.2
>33
>60
>60
O
O
O
N
21
CH(CH3)2
H
CH2Ph
N
0.35
15
N
O
22
23
24
25
26b
27
28
29
30
31
32
CH(CH3)2
C(CH3)3
CH2CH(CH3)2
C6H11
2-Thiophene
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
H
H
H
H
H
H
H
H
H
H
H
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2(3-Cl-Ph)
CH2(3-Cl-4-F-Ph)
CH2CH2CH(CH3)2
CH2CH2C(CH3)3
CH2(3-Cl-4-F-Ph)
CH2(3-Cl-4-F-Ph)
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
N
N
N
N
N
N
N
N
N
N
N
9.0
1.5
5.1
4.0
2.9
1.6
0.76
5.1
0.42
0.033
0.07
—
—
—
—
—
—
—
—
—
>60
—
—
—
>60
>60
—
10
>60
>60
H
4.3
7.9
0.7
OCH2CONH2
OCH2CN
O
N
33
C(CH3)3
H
CH2(3-Cl-4-F-Ph)
N
0.18
1.8
>60
N
O
34
35
36
37
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
H
H
H
H
CH2(3-Cl-4-F-Ph)
CH2(3-Cl-4-F-Ph)
CH2(3-Cl-4-F-Ph)
CH2CH2C(CH3)3
CH2NHSO2CH3
N
N
N
N
<0.010
0.023
0.094
0.2
0.07
0.09
13
>60
>60
>60
48
CH2N(CH3)SO2CH3
d
NHSO2CH3
OCH2CONH2
11
O
N
38
C(CH3)3
H
CH2CH2C(CH3)3
N
0.65
2
44
N
O
39
40
41
42
43
44
45
46
47
C(CH3)3
C(CH3)3
–CH2CH2–
–CH2CH2–
CH2CH3
CH3
CH(CH3)2
C(CH3)3
C(CH3)3
H
H
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2(3-Cl-4-F-Ph)
CH2(3-Cl-4-F-Ph)
CH2(3-Cl-4-F-Ph)
CH2(3-Cl-4-F-Ph)
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2NHSO2CH3
CH2CH2SO2CH3
CH2NHSO2CH3
CH2N(CH3)SO2CH3
CH2N(CH3)SO2CH3
CH2N(CH3)SO2CH3
CH2N(CH3)SO2CH3
CH2N(CH3)SO2CH3
CH2NHSO2CH3
N
N
N
N
N
N
CH
CH
CH
0.018
0.045
0.13
0.62
0.41
0.46
13
0.35
2.6
>33
—
—
—
—
5.8
6.4
>60
>60
>60
>60
>60
>60
>60
>60
>60
CH3
CH3
CH3
H
0.92
0.24
H
a
b
c
CC50 (GAPDH) > 33
Racemic amino acid ester 5 was employed in synthesis.
Not determined.
This substitution is at the 6-position of the isothiazole ring.
l
M for all analogs.
d
labile in the HLM assay compared to the other compounds in the
series.
the sulfonamide NH present in 34 could be methylated with min-
imal loss of in vitro enzymatic potencies (compare 35 with 34).
This result was consistent with the absence of an H-bond observed
between the polymerase and the sulfonamide NH in the NS5B-34
co-crystal structure. As predicted by X-ray analysis, directly attach-
ing the sulfonamide to the 6-position of the 1,1-dioxoisothiazole
ring system, in analogy to the optimal functionalization of the
pyridazinone–benzothiadiazines we previously described,6 was
highly detrimental to anti-NS5B potencies (compare 36 to 34).
The activity loss was further magnified in the replicon assay, prob-
ably due to the poor cell permeability of 36 (comparing with 34).
An additional set of R4 variations was also investigated using a
combination of tert-butyl and 3,3-dimethylbutyl moieties as R1
and R3 substituents, respectively (37–40). As observed previously,
the most potent of these analogs incorporated a CH2NHSO2CH3
moiety at the R4 position (39). The minimal activity loss, when
Having identified the tert-butyl and 3-Cl, 4-F-benzyl groups as
optimal R1 and R3 moieties, respectively, we then examined
appending highly polar substituents to the 1,1-dioxoisothiazole
ring system. As shown in Table 1, inclusion of such fragments into
the inhibitor design afforded several compounds with excellent
NS5B inhibitory properties (31–36). Many of these molecules also
exhibited good activity in the replicon assay that was clearly dis-
tinct from cytotoxic effects. In particular, a compound containing
a CH2NHSO2CH3 R4 substituent (34) displayed NS5B inhibition
activity that was more potent than the quantitation limit of the
enzymatic assay (<10 nM). As described in the X-ray analysis sec-
tion below, the methylene (CH2) moiety represents an optimal
spacer between the polar NHSO2CH3 fragment and the 1,1-dioxo-
isothiazole ring system. Additional SAR studies demonstrated that

4184
S. H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4181–4185
Figure 4. X-ray co-crystal structure showing hydrogen-bonding interaction of
compound 34 with Asn 291 (2.2 Å).
Figure 2. X-ray co-crystal structure of compound 3 (orange) bound to NS5B poly-
merase (2.3 Å). A pyridazinone–benzothiadiazine compound (cf, structure 2, R = Ph,
R0 = CH2CH2CH(CH3)2, R00 = OMe) as observed in an independently determined
co-crystal structure6 is superimposed in purple.
Ser 556
Gly 449
HN
Ser 288
N
H
O
O
the R4 sulfonamide NH was methylated (compare 35 with 34) in
the previous set of analogs, led us to explore whether the NH group
of the R4 sulfonamide present in 39 could be replaced with a meth-
ylene linker. Interestingly, this modification reduced the potency
minimally despite the significant structural change.
N
O
H
O
O
H
Tyr 448
O
N
O
H
O
H
H
H
H
O
N
O
S
H
O
O
O
N
Asn 291
Some limited efforts were then expended to determine whether
the sp3 carbon present in the 4-hydroxy-1,5-dihydropyrrol-2-one
moiety of the above inhibitors could tolerate geminal substitution.
Accordingly, several spirocyclic or quaternary analogs of com-
pounds 34 and 35 were prepared (41–44), but all of them exhibited
considerably decreased anti-NS5B activities relative to the parent
compounds. We also explored replacing the 1,1-dioxoisothiazole
present in the current study with a benzo[b]thiophene-1,1-dioxide
moiety. Unfortunately, the three examples that were synthesized
(45–47) displayed relatively weak NS5B inhibition properties.
Therefore additional benzo[b]thiophene-1,1-dioxide-containing
molecules were not prepared (for the most direct assessment of
this change, compare 47 with 39).
X-ray analysis. Prior to conducting the analog synthesis
described above, we utilized the analysis of several NS5B inhibitor
co-crystal structures to identify the optimal location to append R4
substituents to the 1,1-dioxoisothiazole ring system. As shown in
Figure 2, we superimposed10 the co-crystal structure of our early
1,1-dioxoisothiazole lead (311) with that of a pyridazinone-con-
taining compound (2,6a R = Ph, R0 = CH2CH2CH(CH3)2, R00 = OMe)
S
H
N
H
H
Met 414
N
Tyr 415
Gln 446
Cl
F
Met 414
Leu 384
Cys 366
Tyr 448
Pro 197
Tyr 415
Arg 200
Figure 5. Schematic representation of the interactions between compound 34 and
the NS5B protein. Hydrogen bonds that are shorter than 3.2 Å are represented as
dashed lines, and the residues which make up the enzyme-binding subsites are
shown.
whose anti-NS5B potency had been significantly improved by
increasing the polarity of the R0 OCH3 group.6a This exercise sug-
gested that derivatizing the 1,1-dioxoisothiazole 7-position would
better orient any appended fragments for interaction with the
NS5B protein relative to performing similar modifications to the
corresponding 6-location. Based on this observation, we synthe-
sized most of the inhibitors described in this work with the R4 sub-
stitution appended to the 1,1-dioxoisothiazole 7-position.
Figure 3 shows the X-ray co-crystal structure of compound 34
complexed with the NS5B protein. It clearly illustrates how the
inclusion of a CH2 spacer between the 1,1-dioxoisothiazole ring
system and the sulfonamide moiety allows the functionality to
appropriately access the far right side of the palm-binding pock-
et.12 As depicted in Figures 4 and 5, the sulfonamide oxygen
atoms form direct and water-mediated hydrogen bonds with
NS5B residues Asn291 and Ser288, respectively. These interac-
tions are likely responsible for the significant potency improve-
ments exhibited by compounds 34 and 39. Similar H-bonds
were also noted in X-ray co-crystal structures of optimized
pyridazinone–benzothiadiazine inhibitors complexed with the
Figure 3. X-ray co-crystal structure of compound 34 bound to the NS5B polymerase
(2.2 Å).

S. H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4181–4185
4185
F(%)b
Table 2
PK data for selected analogs (30, 34, 35)
a
Compound
Papp [(cm/s) ꢁ 10ꢂ6
]
(B-to-A)/(A-to-B) ratio
AUC24 (h ng/mL)b
C12 (ng/mL)b
A-to-B
B-to-A
30
34
35
9.9
0.1
0.2
19
15
29
2
150
132
4685
38
193
5
0.5
3.1
NDc
1
4
a
See Ref.6c for assay conditions. Controls: Papp Atenolol (low) = 0.4–0.8 (cm/s) ꢁ 10ꢂ6, Papp Propanolol (high) = 10–16 (cm/s) ꢁ 10ꢂ6
Cynomolgus monkeys; 1 mg/kg single oral dose; 1% DMSO, 9.9% Cremophor EL in 50 mM PBS, pH 7.4.
Not determined.13
.
b
c
NS5B protein.6c In contrast with the pyridazinone–benzothiadi-
azine co-crystal structures previously examined, the sulfonamide
NH moiety present in 34 did not form a hydrogen bond with the
NS5B protein, suggesting that it could be modified without sig-
nificantly sacrificing inhibitor potency.
References and notes
1. McHutchison, J. G. Am. J. Manag. Care 2004, 10, S21.
2. Alter, M. J.; Kruszon-Moran, D.; Nainan, O. V.; McQuillan, G. M.; Gao, F.; Moyer,
L. A.; Kaslow, R. A.; Margolis, H. S. N. Engl. J. Med. 1999, 341, 556.
3. (a) Pawlotsky, J.-M.; McHutchinson, J. G. Hepatology 2004, 39, 554; (b) Penin, F.;
Dubuisson, J.; Rey, F. A.; Moradpour, D.; Pawlotsky, J.-M. Hepatology 2004, 39, 5.
4. Evans, K. A.; Chai, D.; Graybill, T. L.; Burton, G.; Sarisky, R. T.; Lin-Goerke, J.;
Johnston, V. K.; Rivero, R. A. Bioorg. Med. Chem. Lett. 2006, 16, 2205.
5. Koch, U.; Narjes, F. Curr. Top. Med. Chem. 2007, 7, 1302.
Pharmacokinetic studies. As part of our inhibitor optimization
effort, in vivo PK data were obtained for selected compounds in
the 1,1-dioxoisothiazole series following administration of a sin-
gle oral dose to cynomolgus monkeys (30, 34, and 35, Table 2).
The results demonstrated that as the polarity of the molecules
increased, the corresponding oral exposures drastically deterio-
rated, as measured by AUC24 and concentrations of the com-
pounds in monkey plasma 12 h after administration (C12). The
PK data are consistent with the reduction of intestinal perme-
ability with increasing compound polarity and this trend was
observed in Caco-2 assessments of the three compounds (Table
2). Similar relationships between compound polarity, Caco-2
Papp values and oral exposures were also noted during our
study of pyridazinone–benzothiadiazine NS5B inhibitors.6d Fur-
thermore, Caco-2 results indicate that compounds 34 and 35
may act as efficient efflux transporter substrates (see (B-to-A)/
(A-to-B) ratio, Table 2) which resulted in low exposures after
oral dosing. Finally, our most potent NS5B inhibitors of interest
(34 and 35) showed poor oral bioavailability values (1% and
4%, respectively) and inadequate plasma concentrations.
In summary, a novel series of NS5B inhibitors derived from
1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were
synthesized and evaluated. The compounds demonstrated potent
inhibitory activities against both genotype 1a and 1b HCV poly-
merases. Structure-based design and molecular modeling were
employed to guide the optimization of the associated SAR. The
most potent compound (34) in this series contained a R1 tert-bu-
tyl group, a R3 3-chloro-4-fluorobenzyl group, and a R4 methyl
methanesulfonamide group. However, PK results obtained from
selected compounds in this series demonstrated that although
the incorporation of polar R4 substituents was important for
enhancing the biological potencies of these molecules, such
modification also compromised their oral PK properties.14
6. (a) Zhou, Y.; Webber, S. E.; Murphy, D. E.; Li, L. S.; Dragovich, P. S.; Tran, C. V.;
Sun, Z.; Ruebsam, F.; Shah, A.; Tsan, M.; Showalter, R.; Patel, R.; Li, B.; Zhao, Q.;
Han, Q.; Hermann, T.; Kissinger, C.; LeBrun, L.; Sergeeva, M. V.; Kirkovsky, L.
Bioorg. Med. Chem. Lett. 2008, 18, 1413; (b) Zhou, Y.; Li, L. S.; Dragovich, P. S.;
Murphy, D. E.; Tran, C. V.; Ruebsam, F.; Webber, S. E.; Shah, A. M.; Tsan, M.;
Averill, D.; Showalter, R.; Patel, R.; Han, Q.; Zhao, Q.; Hermann, T.; Kissinger, C.;
LeBrun, L.; Sergeeva, M. V. Bioorg. Med. Chem. Lett. 2008, 18, 1419; (c) Li, L. S.;
Zhou, Y.; Murphy, D. E.; Stankovic, N.; Zhao, J.; Dragovich, P. S.; Bertolini, T.;
Sun, Z.; Ayida, B.; Tran, C. V.; Ruebsam, F.; Webber, S. E.; Shah, A. M.; Tsan, M.;
Showalter, R. E.; Patel, R.; LeBrun, L. A.; Bartkowski, D.; Nolan, T. G.; Norris, D.;
Kamran, R.; Brooks, J.; Sergeeva, M. V.; Kirkovsky, L.; Zhao, Q.; Kissinger, C. R.
Bioorg. Med. Chem. Lett. 2008, 18, 3446; (d) Sergeeva, M. V.; Zhou, Y.;
Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.; Okamoto, E.; Kirkovsky, L.;
Kamran, R.; LeBrun, L. A.; Tsan, M.; Patel, R.; Shah, A. M.; Lardy, M.; Gobbi, A.; Li,
L. S.; Zhao, J.; Bertolini, T.; Stankovic, N.; Sun, Z.; Murphy, D. E.; Webber, S. E.;
Dragovich, P. S. Bioorg. Med. Chem. Lett. 2008, 18, 3421.
7. See Supplementary Data for additional syntheses of fragments 13.
8. Proudfoot, J. R.; Patel, U. R.; Dyatkin, A. B. J. Org. Chem. 1997, 62, 1851.
9. Satisfactory spectroscopic data were obtained for all new compounds. All final
analogs were purified by flash column chromatography or HPLC and
characterized by 1H NMR and LC–MS.
10. Gobbi, A.; Lardy, M.; Showalter, R.; Zhao, Q.; Zhou, Y. Abstract of Posters, 2005
Frontiers in Chemistry. Biopharmaceuticals & Biotechnology Meeting of the
American Chemical Society: San Diego, CA, 2007(Abstract 245).
11. Crystals of HCV NS5B polymerase (genotype 1b, strain BK,
D21) were grown by
the hanging drop method at room temperature using a well buffer of 20% PEG
4K, 50 mM ammonium sulfate, 100 mM sodium acetate, pH 4.7 with 5 mM
DTT. The crystals formed in space group P212121 with approximate cell
dimensions, a = 85 Å, b = 106 Å, c = 127 Å, containing two protein molecules in
the asymmetric unit. Protein-inhibitor complexes were prepared by soaking
these NS5B crystals for 4 h in solutions containing 15–20% DMSO, 20% glycerol,
20% PEG 4K, 0.1 M Hepes, 10 mM MgCl2 at pH 7.6, and inhibitors at
concentrations of 2 mM. Diffraction data were collected to a resolution of
2.3 Å for compound 3. Diffraction data for 3 were collected on beamline 14IDB
at the Advanced Photon Source at Argonne National Laboratory. Crystal
structures discussed in this paper have been deposited in the Protein Databank
(www.rcsb.org) with entry codes: 3D28 and 3BSC. Full details of structure
determination are given in the respective PDB entries.
12. Crystals of HCV NS5B polymerase (genotype 1b, strain BK,
D21) were grown by
the hanging drop method at room temperature using a well buffer of 20% PEG
4K, 50 mM ammonium sulfate, 100 mM sodium acetate, pH 4.7 with 5 mM
DTT. The crystals formed in space group P212121 with approximate cell
dimensions, a = 85 Å, b = 106 Å, c = 127 Å, containing two protein molecules in
the asymmetric unit. Protein-inhibitor complexes were prepared by soaking
these NS5B crystals for 20 h in solutions containing 15–20% DMSO, 20%
glycerol, 20% PEG 4K, 0.1 M Hepes, 10 mM MgCl2 at pH 7.6, and inhibitors at
concentrations of 5 mM. Diffraction data were collected to a resolution of 2.2 Å
for compound 34. Diffraction data for 34 were collected on beamline 14IDB at
the Advanced Photon Source at Argonne National Laboratory. Crystal
structures discussed in this paper have been deposited in the Protein
Databank (www.rcsb.org) with entry codes: 3D5M. Full details of structure
determination are given in the respective PDB entries.
Acknowledgements
The authors thank Drs. Peter Dragovich, Devron Averett, and
Steve Worland for their support and helpful discussions during
the course of this work.
Supplementary data
13. As part of a strategy to rapidly evaluate the oral PK properties of the NS5B
inhibitors under study, the corresponding PK data following IV administration
of the described compounds were not obtained.
14. During the course of this study, similar HCV NS5B inhibitors were described in
the patent literature. See: Blake, J. F.; Fell, J. B.; Fischer, J. P.; Hendricks, R. T.;
Spencer, S. R.; Stengel, P. J. U.S. Patent Application 2006/0252785, 2006.
Supplementary material describing additional syntheses of
individual 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-diox-
ides 4 can be found, in the online version, at doi:10.1016/
j.bmcl.2008.05.083.