Discovery of proline sulfonamides as potent and selective hepatitis C virus NS5b polymerase inhibitors. Evidence for a new NS5b polymerase binding site

Article abstract of DOI:10.1021/jm060168g

Through high throughput screening, substituted proline sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA polymerase inhibitor. Optimization of various regions of the lead molecule resulted in compounds that displayed good potency and selectivity.

Full text of DOI:10.1021/jm060168g

3052
J. Med. Chem. 2006, 49, 3052-3055
Chart 1. Examples of NS5b Polymerase Inhibitors. Diketo
Acid 1,6a Phenylalanine Derivative 2,6b Thiophene Carboxylic
Acid 3,6c Pyranone 4,6d and Pyranoindole (HCV-371) 55
Discovery of Proline Sulfonamides as Potent and
Selective Hepatitis C Virus NS5b Polymerase
Inhibitors. Evidence for a New NS5b Polymerase
Binding Site
Ariamala Gopalsamy,*,‡ Rajiv Chopra,‡ Kitae Lim,‡
Gregory Ciszewski,‡ Mengxiao Shi,‡ Kevin J. Curran,‡
Steven F. Sukits,‡ Kristine Svenson,‡ Joel Bard,‡
John W. Ellingboe,‡ Atul Agarwal,‡ Girija Krishnamurthy,‡
Anita Y. M. Howe,† Mark Orlowski,† Boris Feld,†
John O’Connell,† and Tarek S. Mansour‡
Chemical and Screening Sciences and Infectious Diseases, Wyeth
Research, 401 N. Middletown Road, Pearl RiVer, New York 10965
ReceiVed February 14, 2006
Abstract: Through high throughput screening, substituted proline
sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA
polymerase inhibitor. Optimization of various regions of the lead
molecule resulted in compounds that displayed good potency and
selectivity. The crystal structure of 6 and NS5b polymerase complex
confirmed the binding near the active site region. The optimization
approach and SAR are discussed in detail.
of the enzyme upon inhibitor binding, at the emission and
excitation wavelengths of 340 and 295 nm, respectively. From
these experiments and NMR binding studies, the stoichiometry
of binding was found to be a 1:1 ratio, indicative of specific
binding. This compound was then considered as a lead for
further optimization.
Hepatitis C virus (HCV) infection is one of the causes for
liver cirrhosis and hepatocellular carcinoma leading to liver
failure and as such is a growing medical problem worldwide.1
Currently approved therapies involve pegylated interferon-R as
a single agent or in combination with ribavirin.2 The current
therapeutic approach is not aimed at any particular viral target.
Lack of specificity of current therapies against the known HCV
subtypes underscores the need for direct inhibition of viral
targets in an effort to significantly improve patient outcomes.
HCV is a positive strand RNA virus, and the genome consists
of 9600 base pairs that encode several structural and non-
structural proteins.3 Recently, BILN-2061, the NS3/4A serine
protease inhibitor, has provided proof-of-concept clinical data.4
Non-nucleoside inhibitors of NS5b RNA dependent RNA
polymerase such as HCV-3715 have advanced to the clinic, and
several other distinct chemical classes6 (Chart 1) have received
much attention as potential therapeutic agents for treatment of
HCV infection. An important feature of allosteric inhibitors of
HCV NS5b polymerase is defined by the nature of the binding
pocket in the target enzyme. HCV-371 5 and phenylalanine
analogues 2 have been shown to bind at a region distant from
the catalytic site. In this manuscript we disclose the discovery
of certain substituted proline sulfonamides as potent inhibitors
of HCV NS5b polymerase with the evidence for a new binding
site close to the catalytic site.
All compounds described in this study were prepared in
parallel either by solid-phase or solution-phase routes as
described in Schemes 1 and 3. Solid-phase methodology was
employed for varying the amino acid region of the molecule
while the solution phase route was used for varying the
sulfonamide region of the molecule.
As shown in Scheme 1, Fmoc-protected proline was attached
to the Wang resin using standard peptide coupling conditions.
Fmoc was then removed, and the free amine was reacted with
suitably protected sulfonyl chlorides to form the resin-bound
Figure 1. High throughput hit for NS5b polymerase.
Scheme 1a
High throughput screening of various libraries to identify
NS5b polymerase inhibitors led to the proline sulfonamide 6
(Figure 1) with an IC50 of 3.1 µM. This compound was found
to be selective for HCV NS5b polymerase over human poly-
merase â (IC50 > 100 µM), calf thymus polymerase R (IC50
>
100 µM), HCV helicase ((IC50 > 75 µM), and HIV reverse
transcriptase (IC50 > 100 µM). Employing fluorescence spec-
troscopy techniques, we determined the apparent KD of 1.6 µM
from the changes in the endogenous tryptophan fluorescence
* To whom correspondence should be addressed. Tel: 845-602-2841.
Fax: 845-602-3045. E-mail: gopalsa@wyeth.com.
‡ Chemical and Screening Sciences.
a Method A. Reagents: (a) EDCI, HOBT, DIEA, DMF, RT, 6 h; (b)
20% piperidine in DMF, RT, 20 min; (c) pyridine, RT, 12 h; (d) 1:1 TFA-
DCM, RT, 2 h.
† Infectious Diseases.
10.1021/jm060168g CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/06/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3053
Scheme 2a
Table 2. NS5b Polymerase Activity of Proline Sulfonamides:
Optimization of Proline Region
a Method B. Reagents: (e) NaHCO3, H2O, Fmoc-Cl, THF, 12 h; (f)
SOCl2, DMF, RT, 4 h.
Scheme 3a
compd
W
stereochem
X
n
IC50(µM)
21
26
27
28
29
30
31
32
33
34
COOH
COOH
COOMe
CN
tetrazole
H
COOH
COOH
COOH
COOH
S
R
S
S
S
-
S
R
S
R
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
S
0
0
0
0
0
0
1
1
0
0
0.77
7.9
>20
1.4
0.56
4.8
2.0
7.1
a Reagents: (g) Pyridine, THF, RT, 8 h; (h) aq NaOH, EtOH, RT, 8 h.
Table 1. NS5b Polymerase Activity of Proline Sulfonamides:
Optimization of Sulfonamide Region
0.24
1.7
S
Since the original hit was derived from L-proline, it was of
interest to see if the D-proline analogue had any impact on the
potency of the molecule. Toward this end, compound 21 was
compared with its antipode 26. This resulted in a 10-fold loss
of potency. Capping the carboxylic acid as an ester 27 was not
favorable. However, a carboxylic acid isostere such as tetrazole
29, was equipotent, while the cyano intermediate 28 was less
potent. Complete removal of the acid reduced the activity
significantly (compound 30). Expanding the proline ring to a
six-membered piperidine ring 31 was not preferred, but sub-
stituting the proline ring with a thioproline was well tolerated.
We have consistently found 3-10-fold greater activity for the
L-amino acids stereoisomers (Table 2). Despite the excellent
potency against NS5b polymerase of compounds 21, 24, and
25, they displayed moderate activity in cellular replicon.
Compounds exhibited very low permeability in the PAMPA
assay (PAMPA @ pH 7.4 for compound 25 Pe ) 0.02 × 10-6
cm/s), and the compounds showed lower permeability than
atenolol in the Caco-2 measurement (AfB for compound 25
Pe ) 0.4 × 10-6 cm/s). However, the compounds were not
cytotoxic in the MTS assay of growing Vero cells or MTS assay
of growing clone A cells (compound 25 IC50 value >100 µM
in both assays).5b
compd
R1
NH2
NH2
NH2
NHCOMe
NHCOPh
OH
H
OH
R2
R3
R4
R5
IC50(µM)
6
H
H
H
H
Cl
H
Me
Me
Me
H
H
H
H
Cl
Me
H
H
H
H
H
H
H
H
H
H
H
3.1
>20
5.95
>20
>20
0.77
>20
2.1
17
18
19
20
21
22
23
24
25
Cl
Cl
Cl
Cl
Cl
F
H
Cl
Cl
Cl
Br
Cl
OH
OH
Cl
Cl
0.26
0.08
sulfonamides. The desired compound was cleaved from the resin
using TFA cleavage conditions. The Fmoc-protected sulfonyl
chlorides employed were synthesized as shown in Scheme 2.
Solution-phase parallel synthesis employed proline ester 14
as the starting material. A number of sulfonyl chlorides were
introduced, and the resulting esters 16 were hydrolyzed to
provide analogues with variation on the sulfonamide region of
the molecule.
Following the determination of the 1:1 binding stoichiometry
from 1D NMR experiments for the protein and compound 6
complex, the crystal structure of 6 and NS5b polymerase
complex was determined by soaking of preformed NS5b-delta
21 crystals (PDB ID: 2GC8). With resolution of 2.2 Å, the
compound was seen to bind approximately 10 Å from the Motif
D catalytic aspartic acids in the active site (Figure 2), making
interactions with residues from both the palm and thumb domain
of NS5b. The most significant interactions are made from the
loop containing Try448. This displays hydrogen bonds to the
sulfonamide and carboxylic acid regions of compound 6. The
sulfonamide group is oriented such that one sulfonamide oxygen
forms a hydrogen bond to the backbone amide of Tyr448 (Figure
3) and the other to a bound water molecule (not shown).
Carboxylic group oxygen forms two hydrogen bonds to the
backbone amide of Gly449 and to the side chain of Gln446.
The aryl group of the sulfonamide region in compound 6 packs
between the hydrophobic side-chains of Tyr448 and Tyr415 and
above the side chain of Met414. The R3 chloro substituent is
within 3.6 Å of the guanidinium group of Arg200. The R4
methyl group is within 3.4 Å of the side-chain of Leu384. The
importance of both these interactions is negligible given the
Initial SAR studies were focused on determining the impor-
tance of the substituents on the aryl group (Table 1). Removal
of the lipophilic substituents as in compound 17 rendered the
molecule inactive. However, switching the position of the chloro
and methyl group did not alter the potency substantially as
shown by compound 18. Acylating the amino group with a small
acetyl group or a bulky benzoyl group as in compounds 19 and
20 displayed greatly reduced activity. However, a significant
boost in activity was obtained by replacing the amino group
with a hydroxyl group in conjunction with the dichloro
substituents as in compound 21.
Compound 21 with ortho substitution demonstrated the
importance of the hydroxyl group since the activity was rather
weak in the absence of the hydroxyl group, even in the presence
of the dichloro substituents. Replacing one of the chloro groups
with a more electron-withdrawing smaller fluoro group (com-
pound 23) reduced the activity while a bromo group was well
tolerated (compound 24). Another large increase in potency was
observed when an additional chloro substituent was added to
the aromatic group as in compound 25 which displayed an IC50
value of 80 nM.

3054 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Letters
In summary, we have identified a novel series of proline
sulfonamide derivatives as potent and selective inhibitors of
HCV NS5b-RNA dependent RNA polymerase. Importantly,
these compounds bind in a new binding site close to the catalytic
site of NS5b polymerase. Specific amino acid polymerase
interactions are reported for compound 6.
Acknowledgment. We thank Wyeth Discovery Analytical
Chemistry Department, in particular Dr. Melissa Lin for the
spectral data and Dr. Patric Stenberg for Caco-2 data. We thank
Dr. Jonathan Bloom, Janis Upeslacis, and our collaborators at
Viropharma Inc. for their support of this work.
Supporting Information Available: Experimental details for
the biological assay and the synthetic procedures. This material is
available free of charge via the Internet at http://pubs.acs.org
Figure 2. Ribbon diagram of NS5b complex crystal structure indicating
compound 6 (red space-filling atoms) between the thumb (colored
green) and palm (colored purple) domains. The fingers domain is
colored blue, and active site Motif D is colored yellow.
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JM060168G