Development of carboxylic acid replacements in indole-N-acetamide inhibitors of hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2007.06.093

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here 2 series of indole-N-acetamides, bearing

Full text of DOI:10.1016/j.bmcl.2007.06.093

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5143–5149
Development of carboxylic acid replacements in indole-N-
acetamide inhibitors of hepatitis C virus NS5B polymerase
Ian Stansfield,* Marco Pompei, Immacolata Conte, Caterina Ercolani,
Giovanni Migliaccio, Mark Jairaj, Claudio Giuliano, Michael Rowley and Frank Narjes
IRBM (Merck Research Laboratories Rome), Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy
Received 16 May 2007; revised 28 June 2007; accepted 29 June 2007
Available online 7 July 2007
Abstract—Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently
emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here 2 series of indole-
N-acetamides, bearing physicochemically diverse carboxylic acid replacements, which show potent affinity for the NS5B enzyme
with reduced potential for formation of glucuronide conjugates. Preliminary optimization of these series furnished compounds that
are potent in the blockade of subgenomic HCV RNA replication in HUH-7 cells.
Ó 2007 Elsevier Ltd. All rights reserved.
HCV is a major human pathogen associated with
chronic hepatitis and liver disease, cirrhosis, hepato-cel-
lular carcinoma, and liver failure.1 Worldwide, there are
an estimated 170 million chronic carriers,2 whilst in the
US alone, 4 million have antibodies to HCV, indicating
an on-going or previous infection with the virus. For
over 10 years, frontline therapies have been based
around interferon-a, commonly dosing in conjunction
with ribavirin. Despite progress with such therapies
(e.g., introduction of pegylated interferon),3 sustained
viral response (SVR) rates are still typically poor, partic-
ularly for genotype-1 infections that predominate in
Europe, Japan, and the U.S.4 In addition, therapy is of-
ten accompanied by significant adverse side effects5—
consequently, there is a pressing need for new and
broadly effective therapeutics to combat HCV.3,6
viral genomic RNA. NS5B is the viral RNA-dependent
RNA polymerase (RdRp). The RdRp activity of NS5B
is essential for viral replication7 and has no functional
equivalent in uninfected mammalian cells—thus making
the NS5B protein an attractive target for drug
discovery.8
The NS5B RdRp comprises the palm, fingers, and
thumb subdomains common to nucleotide polymerizing
enzymes.9 Inhibition of NS5B can be achieved through
interaction at the active site, for which both nucleoside
ligands10,11 and non-nucleoside inhibitors have been de-
scribed.11–15 Alternatively, several allosteric inhibitor
binding sites on NS5B have been identified distal to
the catalytic center.11,16 Recent reports from our labora-
tories documented the development of N-acetamido-in-
dole-6-carboxylates, such as 1, as potent inhibitors
interacting at one such allosteric site that lies close to
a conserved amino acid, proline 495, on the surface of
the thumb domain.17–19 An issue with compounds from
this class, however, was that glucuronide conjugates of
the carboxylic acid were frequently observed as major
circulating metabolites (Fig. 1).
HCV is a small, enveloped, single stranded positive
RNA virus in the Flaviviridae family. The genome is
approximately 10,000 nucleotides and encodes a single
polyprotein of about 3000 amino acids. This polyprotein
comprises the structural (C, E1, and E2) and non-struc-
tural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) pro-
teins that are required for replication and packaging of
To moderate the formation of glucuronide conjugates in
the systemic circulation, viable alternatives to the
C6-carboxylate were sought. There are a number of
moieties documented in the literature as mimetics for
carboxylic acids, with diverse physical attributes and
spanning a multi-log unit range of pKa values. In this
Keywords: Hepatitis C virus; NS5B polymerase; Allosteric inhibition;
Indoles; Acid replacements.
*
Corresponding author. Tel.: +39 06 91093286; fax: +39 06
91093654; e-mail: ian_stansfield@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.093

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I. Stansfield et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5143–5149
O
BBr3 or hydrolytic cleavage of the methyl ester, forma-
tion of the primary amide and dehydration to the nitrile,
followed by alkylation with NaH/tert-butyl bromoace-
tate afforded intermediate 3. Preparation of the amide
oxime and a one-pot acylation/cyclization protocol
using carbonyldiimidazole generated the oxadiazolone.
Pd-mediated cross-coupling with the appropriate aryl-
boronic acid then installed the aromatic functionality
at C2 in 4. Unmasking of the tert-butyl ester and
TBTU-promoted amide bond formation yielded the fi-
nal compounds 5. Clearly, this strategy is well suited
to modifications to the acetamide dimension in the final
step, however, the synthesis could be readily tailored to
fit final diversification steps at alternative positions
around the indole. Hence, for example, manipulation
at C6 could be performed equally well after diversifica-
tion at C2 and in the N1-acetamide moieties, simply
by moving steps in the synthetic sequence. Initial Suzuki
coupling on 2 followed by the 3-step alkylation, depro-
tection, amide bond formation sequence, and final ester
cleavage afforded C6 carboxylic acid 6. The carboxylate
moiety could then either be manipulated as before to set
up the oxadiazolone ring, or subjected to EDAC medi-
ated coupling with sulfonamides or sulfamides to yield
the corresponding acylsulfonamides or sulfonylureas 7.
NEt2
N
N
IC50 < 0.006 μM
EC50 0.15 μM
O
HO2C
OMe
1
Chx = cyclohexyl
Chx
Figure 1. Potent N-acetamido-indole-6-carboxylic acid inhibitor of
HCV NS5B.
paper, we describe the discovery and initial optimization
of 2 such structural classes (oxadiazolones and acyl sul-
fonamides/sulfonyl ureas) chosen for their disparate
pKa’s and physical characteristics—leading to indole
N-acetamides with reduced potential regarding forma-
tion of glucuronide conjugates, that retain intrinsic affin-
ity for the NS5B enzyme and potency as inhibitors of
HCV replication in a surrogate cell-based assay.
The compounds described herein were assessed for
activity (IC50) against the purified DC55 NS5B enzyme
in the presence of heterogeneous template RNA. Inhibi-
tion of replication of subgenomic HCV RNA was mea-
sured in HUH-7 cells using a modification of the
procedure of Bartenschlager.20 Unless otherwise stated,
cell-based data (EC50) were measured in the presence
of 10% fetal calf serum. Pharmacokinetic studies in rats
were performed with n = 3, and the following dosing
parameters were used: iv 3 mg/kg (60% DMSO + 20%
PEG400 + 20% H2O), po 3 mg/kg (PEG400).
As illustrated in Scheme 2, taking bromo derivative 8
through the analogous synthetic sequence allows func-
tionalization at C2 as the final step.
In the field of angiotensin II receptor antagonists, het-
erocycles such as oxadiazolones have been reported as
replacements for carboxylic acid moieties.21 Although
2 tautomeric forms can be envisaged, studies suggest
that generally the non-aromatic oxadiazolone form is
The indoles reported in Tables 1–4 were prepared from
the methyl 2-bromo-3-cyclohexylindole-6-carboxylate
precursor17 2, as outlined in Scheme 1. Thus, either
Table 1. HCV NS5B polymerase enzyme inhibition (IC50), cell-based efficacy (EC50), and in vivo rat pharmacokinetic properties for diverse C6 acid
moieties
NMe2
O
R
N
Chx
Frat (%)b,c/ t1/2 (h)d
Cl (ml/min/kg)e/ AUC (lM h)f
a
Compound
R
IC50/EC50 (lM)
O
O
9
0.086/1.5
45/5
32/2
HO
N
O
10
11
0.068/4.4
0.046/2.1
20/13
24/2
12/2
51/1
N
H
O
O
O
S
N
H
a IC50/EC50 values are means from at least 2 experiments.
b Compounds were dosed as trifluoroacetate salts. po/iv 3 mg/kg body weight; n = 3. Vehicle iv 60% DMSO/20% PEG400/20% H2O, po PEG400.
c Oral bioavailability.
d Terminal phase plasma halflife following iv administration.
e Plasma clearance.
f After oral dosing.

I. Stansfield et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5143–5149
Table 2. C6 hydroxyoxadiazole SAR: intrinsic potency and cell-based activity
5145
NR1R2
O
O
N
O
N
N
H
Ar
Chx
NR1R2
IC50 (lM)
EC50 (lM)
hPPB (% free)
a
a
Compound
Ar
10
NMe2
0.068
4.4
0.1
—
—
—
—
—
12
13
14
15
16
NMe2
0.78
20
N
0.059
0.053
0.3
1.3
2.4
17
N
N
N
N
N
N
N
N
F
OMe
0.067
3.7
17
18
0.017
0.17
1.9
—
Cl
N
N
N
N
N
N
>50
9.4
N
19
20
0.075
0.047
1.60
1.1
—
N
N
2.1
N
N
O
21
0.026
1.1
—
N
O
a IC50/EC50 values are means from at least 2 experiments.
predominant over the hydroxyoxadiazole.22 As a conse-
quence, the pKa is significantly elevated (and so the % of
the neutral form at physiological pH) with respect to
carboxylic acids. In contrast, simple acyl sulfonamides/
sulfonyl ureas have pKa values that approach more clo-
sely that of the parent carboxylic acid, but offer a handle
from which to explore structural space on the enzyme
surface. (Measured values: 9 pKa = 4.8; 10 pKa = 6.1;
23 pKa = 5.2.)23
each class, they both show acceptable oral bioavailabil-
ity and systemic exposure in rat.
Most significantly, in vitro studies with 10 and 11 in rat
liver microsomes demonstrated that both these series
potentially offer a means by which to radically reduce
the extent of glucuronidative metabolism in the indole
N-acetamide class (Fig. 2). In vivo, elevated clearance
for 11 was not associated with the formation of conju-
gates, but rather a greater first pass effect due to oxida-
tive metabolism and biliary excretion.
Pleasingly, despite diverse physical properties, both oxa-
diazolone 10 and acylsulfonamide 11 display excellent
intrinsic potency on the isolated enzyme, being if any-
thing marginally more potent than the parent carboxylic
acid 9, with comparable activity in the cell based assay
(Table 1). Furthermore, as prototypical molecules for
Having shown that it was possible to address glucuron-
idation without compromising significantly intrinsic
potency, attention then focused on SAR to optimize
cell-based activity (Table 2). In the oxadiazolone class,

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I. Stansfield et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5143–5149
Table 3. C6 acylsulfonamide SAR: intrinsic potency and cell-based activity
NMe2
N
O
O
R
Ar
Chx
a
a
Compound
R
Ar
Ph
IC50 (lM)
EC50 Standard/(no serum) (lM)
hPPB (% free)
0.8
O
O
S
11
0.046
2.1/(0.52)
3.9/(0.13)
N
H
S
O
O
22
Ph
0.011
0.3
N
H
O
O
23
24
Ph
Ph
0.031
0.64
5.0/(0.15)
NAb
<0.1
—
S
N
H
O
O
O
S
S
N
O
25
26
27
28
29
30
31
Ph
Ph
Ph
0.031
0.009
0.029
0.011
0.022
0.019
0.014
5.4/(2.0)
0.71
1.6
1.9
0.8
—
N
N
H
O
O
S
N
N
H
O
O
HO2C
O
S
N
H
O
OMe
S
1.5
—
N
H
O
O
O
O
O
S
1.5
0.8
1.0
1.1
N
H
O
O
HO2C
O
S
0.56
0.38
N
H
O
S
N
N
H
a IC50/EC50 values are means from at least 2 experiments.
b NA, not active.
a consequence of the tautomeric preference and charge
delocalization upon ionization is a greater lipophilicity
for this moiety over a carboxylic acid. In light of this,
the possibility to introduce polarity in other regions of
the structure would be highly desirable as a means of
reducing the overall lipophilicity and improving param-
eters such as plasma protein binding (PPB).
Using zwitterionic indole oxadiazolone 13 as a starting
point, SAR around the C2 phenyl was explored. Substi-
tution in this series showed that although small moieties
in the ortho position such as F (14) were tolerated, bulk-
ier substituents (15) proved deleterious—presumably a
steric clash between the C2 and N1 elements perturbing
their orientations and compromising interactions with
the enzyme. Electron donating functionality at the meta
position was acceptable (16), whilst the para-position
could accommodate substitution with a modest gain in
enzyme affinity (17). Once more, polar electron deficient
heteroaromatics at C2 proved incompatible with enzyme
affinity (18). C2/N1-acetamide moiety optimization,
using amines from commercial sources and in-house
synthesis, afforded potent enzyme inhibitors spanning
Regrettably, polar heterocycles at C2 (e.g., 12) proved
largely incompatible with the primarily lipophilic
interactions of this element and the enzyme surface. In
contrast, introducing a basic centre in the acetamide
sidechain (e.g., 13) to afford formally zwitterionic
species gave a moderate enhancement in cell-based
potency.

I. Stansfield et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5143–5149
5147
Table 4. Optimized C6 acylsulfonamide and acylsulfonylurea: intrinsic potency and cell-based activity
a
a
Compound
Structure
IC50 (lM)
EC50 (lM)
O
NEt2
N
N
O
O
O
O
32
0.009
0.34
S
N
OMe
H
NEt2
N
N
O
O
O
O
33
0.004
0.12
S
N
N
H
O
a IC50/EC50 values are means from at least 2 experiments.
OtBu
NR1R2
NR1R2
O
O
O
O
O
O
O
O
O
H
MeO2C
NC
S
S
N
N
N
N
a, b, c
R
N
H
R
N
H
a
Br
Br
Br
Ar
2
3
Chx
Chx
Chx
Chx
8
7
d, e, f
Scheme 2. Reagents and condition: (a) ArB(OH)2, Pd(PPh3)2Cl2,
Na2CO3 (aq), 1,4-dioxane, reflux, 75–85%.
f, c, g, h, a
NR1R2
OtBu
O
O
O
N
140%
120%
100%
80%
60%
40%
20%
0%
O
HO2C
N
N
N
Ar
H
Ar
6
4
Chx
Chx
b, d, e, j
i, j
g, h, j
NR1R2
NR1R2
N
O
O
O
O
N
O
O
O
S
N
N
R
N
Ar
H
Ar
H
7
5
Chx
Chx
Scheme 1. Reagents and conditions: (a) BBr3, CH2Cl2, or NaOH,
dioxane, H2O, 60 °C, 75–100%; (b) (i) (BOC)2O, H4NHCO3, pyridine,
DMF, 24 h; (ii) (CF3CO)2O, Et3N, CH2Cl2, CHCl3, 0 °C, 3 h, 75%; (c)
NaH, tert-butyl bromoacetate, DMF, 60 °C, 95%; (d) H2NOHÆHCl,
iPr2NEt, MeOH, 48 h, 80%; (e) carbonyldiimidazole, dioxane, 70 °C,
40%; (f) ArB(OH)2, Pd(PPh3)2Cl2, Na2CO3 (aq), 1,4-dioxane, reflux,
75–85%; (g) CF3CO2H, CH2Cl2, 2 h, 100%; (h) TBTU, iPr2NEt,
HNR1R2, CH2Cl2, 16 h, 50–75%; (i) EDCI, DMAP, RSO2NH2, 35–
70%; (j) RP-HPLC, 50–85%.
(9)
(10)
(11)
Figure 2. Turnover in liver microsomes from diverse species in the
presence of UDPGA (% dose remaining at 1 h).
difficult to break into sub-lM levels of cell based activ-
ity. This would suggest that plasma protein binding may
not be the key determinant restricting cell-based activity
for this series. Potentially log D (which for the oxadiazo-
lone series generally remains around a log unit higher
than for the analogous examples in the C6 carboxylic
acid class), coupled with the higher H-bond acceptor
a 20–80 nM activity range. Although it was possible to
improve physical properties (as judged by hPPB) with-
out compromising intrinsic potency (e.g., 20), it proved

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I. Stansfield et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5143–5149
count, may contribute to limiting the free intracellular
levels of compound attainable. In accord with this
hypothesis, there was minimal impact on cell-based
activity when the assay was performed in the absence
of serum proteins. (e.g., compound 19 EC50 (no serum)
1.0 vs 1.6 lM in standard conditions).
Acknowledgments
We thank Sergio Altamura and Licia Tomei for enzy-
mology; Fabrizio Fiore and Odalys Gonzalez-Paz for
PK analysis; Marina Taliani for metabolism studies; Fa-
bio Bonelli, Alan Bishop, Maria Verdirame, and Steven
Thomas for analytical work and Nadia Gennari, Mon-
ica Bisbocci, and Ottavia Cecchetti for biological test-
ing. This work was funded in part by a grant from the
MIUR.
Concomitant with the oxadiazolones, the acylsulfona-
mide series was explored. In this case, pKa and log
D can approach more closely that of the parent car-
boxylic acid class, and it proved easier to rationalize
variations in cell-based activity in terms of substitu-
ent impact on log D, PPB, and/or cell permeability
rather than an inherent limitation of the structural
class.
References and notes
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fragment (Table 3). It was found that methylation of
the acidic NH proved highly detrimental to potency,
reflecting the importance of this structural element
(e.g., 24). Although bulky functionality at C6 was tol-
erated, it was not necessary for optimal enzyme affinity
and cell-based potency, with simple acylsulfonylurea 26
affording a 10-fold boost in intrinsic enzyme affinity
over the carboxylic acid 9 along with sub-lM cell-
based activity. For overly lipophilic molecules (e.g.,
22, 23), plasma protein binding became limiting for
cell-based activity, as can be seen from the shifts be-
tween no serum and standard conditions. Polar/
charged moieties could reduce PPB, but not always
did this improve free intracellular compound concen-
tration (e.g., 25 shows reduced serum shift with respect
to 23, but raised EC50(no serum)/IC50 ratio). Intrigu-
ingly, a di-acid replacement for the C6 carboxylic acid
27 did not limit cell-based activity. SAR at C2 identi-
fied electron-rich heteroaromatics, such as furan, as
being optimal for enzyme affinity, and gratifyingly gave
further improvements in cell-based activity (e.g., 30,
31).
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At this stage, introduction of a basic charged center to
the acetamide moiety to give formally zwitterionic acyl-
sulfonamides and sulfonyl ureas gave a significant boost
in both intrinsic and cell-based potency (Table 4). N1/C2
optimization afforded compounds (e.g., 32 and 33) pos-
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rival the related C6 carboxylic acid series.18 Impor-
tantly, no cytotoxicity was observed for compounds of
this type, the CC50 values for compounds 32, 33 being
above 50 lM.
14. Crescenzi, B.; Poma, M.; Ontoria, J. M.; Marchetti, A.;
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19. A parallel evolution of allosteric indole inhibitors of NS5B
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In summary, we have described here the development
of 2 series of indole-N-acetamides, bearing physico-
chemically diverse replacements for the C6 CO2H,
that are potent allosteric inhibitors of the HCV
NS5B enzyme with reduced potential regarding forma-
tion of glucuronide conjugates. Preliminary optimiza-
tion of these series furnished compounds that are
non-cytotoxic and equipotent with their carboxylic
acid counterparts—showing 100 nM potency in the
blockade of subgenomic HCV RNA replication in
HUH-7 cells.
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23. pKa determination. Data acquired using Sirius Profiler SGA
(Sirius Analytical, Forest Row, East Sussex, UK) and built-
in software. A solution of compound in DMSO 10 mmol,
20 lL) was diluted to 2 mL using deionized water and
infused at 0.25 mL/min into a 1 mL/min pH gradient.
Changesin UV spectrum were recorded with the diode array
detector during 2 min run with pH changing from 2 to 12
and used to calculate apparent pKa of the compound.