Design and synthesis of lactam-thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.06.031

Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.

Full text of DOI:10.1016/j.bmcl.2014.06.031

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of lactam–thiophene carboxylic acids as potent
hepatitis C virus polymerase inhibitors
David Barnes-Seeman a, Carri Boiselle b, Christina Capacci-Daniel b, Rajiv Chopra a, Keith Hoffmaster b,
Christopher T. Jones c, , Mitsunori Kato a, Kai Lin c, Sue Ma c, , Guoyu Pan b, Lei Shu a, Jianling Wang b,
Leah Whiteman c, Mei Xu a, Rui Zheng a, Jiping Fu a, ,
⇑
a Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, United States
b Metabolism & Pharmacokinetics, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, United States
c Department of Infectious Diseases, Novartis Institutes for BioMedical Research, 500 Technology Square, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the successful incorporation of a lactam as an amide replacement in the design of
hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay
and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead
compound has a favorable pharmacokinetic profile in rats and dogs.
Received 23 April 2014
Revised 8 June 2014
Accepted 11 June 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
HCV
NS5B polymerase
PXR
CYP induction
Lactam
Thiophene
Hepatitis C virus (HCV), which chronically infects an estimated
3% of the world’s population, is an important causative agent of
liver fibrosis, cirrhosis and hepatocellular carcinoma.1,2 In the last
decade, the focus of anti-HCV drug discovery has been on develop-
ing interferon-free treatment regimens.3,4 In order to effectively
suppress viral resistance, it is necessary to use combination ther-
apy with agents that target multiple viral proteins or host path-
ways. The HCV RNA-dependent RNA polymerase (NS5B) is an
attractive drug target that is essential for the replication of the viral
genome.5,6 A number of nucleoside-based and non-nucleoside
inhibitors of NS5B have demonstrated efficacy in clinical trials.7–9
Sofosbuvir, a nucleotide-based HCV polymerase inhibitor and the
first interferon-free treatment for HCV genotypes 2 and 3, was
approved by the FDA in 2013.10
active site.16 The interaction between this class of inhibitors and
the NS5B enzyme is largely hydrophobic, with the only hydrogen
bonding occurring between the carboxylic acid and the amide
nitrogens of S476 and Y477 (Fig. 2).17 The amide linkage and the
isopropyl group are important structural elements that maintain
an optimal dihedral angle between the amide and thiophene
groups. These interactions ensure that the trans-methyl-cyclohexyl
group is well positioned in the hydrophobic pocket.
Recently, a series of thumb Site II thiophene carboxylic acid
inhibitors (exemplified by compound 3) was reported that features
aryl and heteroaryl groups as replacements for the amide link-
age.18 In these compounds, it is expected that the hetero aromatic
group will maintain similar geometric, electronic and hydrogen
bonding properties as the amide group. Although inhibitors such
Thiophene carboxylic acids, with structures represented by
compounds 1 and 2 (Fig. 1), are characteristic of a class of polymer-
ase inhibitors that bind to the allosteric Thumb Site II of the NS5B
protein.11–15 This binding site is located near the base of the thumb
domain and is approximately 35 Å away from the polymerase
as 3 were potent inhibitors of the purified NS5B enzyme (IC50
0.1 M), the compounds had weak potency in the replicon assay
(EC50, 15 M). In this Letter, we describe the successful implemen-
tation of a lactam group as a replacement for the amide linkage,
yielding thiophene–lactam carboxylic acids as potent NS5B Site II
inhibitors.19 These compounds were further optimized to reduce
activation of the pregnane X receptor (PXR), the nuclear hormone
receptor responsible for CYP3A4 induction.
,
l
l
⇑
Corresponding author. Tel.: +1 510 923 2043.
E-mail address: jiping.fu@novartis.com (J. Fu).
Our initial design of Site II inhibitors, similar to designs
Current address: Novartis Institutes for BioMedical Research, 5300 Chiron way,
Emeryville, CA 94608, United States.
described previously,18 incorporated heterocycles as amide isoster-
http://dx.doi.org/10.1016/j.bmcl.2014.06.031
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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2
D. Barnes-Seeman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
N
N
N
N
N
OH
O
O
O
S
S
S
OH
OH
OH
1
2
3
Figure 1. Thiophene carboxylic acid Site II inhibitors.
R'
Y477
NH2
N
S476
O
Me
OH
a, b
Me
OMe
+
R'
S
S
O
O
O
7
8
4
Scheme 1. Synthesis of pyrrole analogs. Reagents and conditions: (a) R0 = c-hexyl,
150 °C; or R0 = 4-trans-Me-c-hexyl, I2, THF; (b) NaOH or LiOH, THF, MeOH, water.
es (Fig. 3). In addition to heteroaryl groups, such as pyrrole, we also
examined saturated lactams as amide replacements. These two
structures are complementary in that they have different hybrid-
ization and therefore offer the possibility of tuning the trajectory
of the hydrophobic group.
The pyrrole ring was constructed by a Paal–Knorr condensation
reaction between 3-amino-thiophene 7 and diketone 8 either with
heat or using I2 as a promoter (Scheme 1).20 Hydrolysis under basic
conditions gave thiophene carboxylic acid 4. The synthesis of lac-
Figure 2. Co-crystal structure of 1 and HCV polymerase enzyme complex.17
sp2
sp3
O
amide
X
R'
replacement
N
R'
( )n=0, 1
N
N
R''
O
O
O
O
R1
R1
S
S
S
OH
OH
4: R1 = Ph
OH
X = CH2, or O
5: R1 = Ph
6: R1
=
t-Bu
Figure 3. Replacement of amide with pyrrole and lactam.
NH2
OMe
S
O
7
O
O
O
O
a
b
BrZn
+
R'
Cl
R'
( )n
( )n
OEt
OEt
9
10
( )n
N
R'
( )n
NH
OEt
R'
c, d ,e
O
O
OH
OMe
S
S
O
O
11
5
Scheme 2. Synthesis of lactam analogs. Reagents and conditions: (a) Pd(PPh3)4, THF, rt; (b) Bu2SnCl2, Ph3SiH, THF; (c) LiOH, THF, water, MeOH; (d) Py, (Boc)2O, CH2Cl2; (e)
LiOH, THF, MeOH, water.
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D. Barnes-Seeman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
X
tam–thiophenes is illustrated in Scheme 2. Various acyl chlorides
were coupled with zinc bromide 9 in the presence of catalytic
Pd(PPh3)4.21 A reductive amination between ketoester 10 and
3-aminothiophene 7 was accomplished using PhSiH3 as the reduc-
ing agent and catalytic Bu2SnCl2.22 The ethyl ester on the alkyl
chain in 11 was then selectively hydrolyzed, and the resulting car-
boxylic acid cyclized to form the desired lactam with Boc2O and
pyridine. Finally, the thiophene carboxylic ester was hydrolyzed
with LiOH to give 5. All lactam analogs were prepared in racemic
form using this synthetic route.
X
R'
R'
Br
13
HN
N
O
O
OH
OMe
R1
S
R1
S
a, b
O
O
1
12a:
12b:
R
= Ph
X = CH2 or O
5:
1
t-Bu
R =
R
1 = Ph
t-Bu
6: R1
=
Scheme 3. Synthesis of lactam analogs by CuI catalyzed cross coupling. Reagents
and conditions: (a) CuI, trans-cyclohexanediamine, K2CO3, dioxane, 110 °C; (b) LiOH
or NaOH, THF, MeOH, water.
Thiophene lactams could also be synthesized by a Cu-catalyzed
coupling reaction between thiophene bromide 12 and lactam 13
(Scheme 3).23 In this route, enantiomerically pure products could
be obtained by using enantiomerically pure lactams.
O
H
O
H
O
N
N
N
a
O
O
O
O
+
O
O
S
S
S
OMe
OMe
OMe
16a
14
16b
16a/16b = 1/4
b
O
CH3
O
CH3
N
N
O
O
O
O
+
S
S
OMe
17b
OMe
17a
17a/17b = 3/1
O
R
O
N
R
N
OH
c, d, e, f
O
O
O
O
S
S
OH
OMe
18: R = H
16: R = H
17
19
: R = Me
: R = Me
g, f
O
R
N
O
OH
O
S
OH
20: R = H
21
: R = Me
O
H
O
H
O H
N
N
OH
N
OH
c, d, h, f
O
O
O
O
O
O
S
S
S
OMe
16a
a-substituted morpholinone analogs. Reagents and conditions: (a) LDA, allyl iodide, THF; (b) LDA, MeI, THF; (c) OsO4, NMO, acetone, water; (d) NaIO4,
OH
OH
22a
22b
Scheme 4. Synthesis of
THF, water; (e) NaBH4, MeOH; (f) LiOH, THF, MeOH, water; (g) 9-BBN, THF; H2O2, NaHCO3, water; (h) MeMgBr, THF.
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4
D. Barnes-Seeman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1
Amide replacement SAR
The a-position of morpholinone could be further substituted to
generate an additional series of analogs 18–21 (Scheme 4). Mor-
pholinone 14 was alkylated with an allyl group to give 16a and
16b in a 1:4 ratio. The two diastereomers were separated by silica
gel chromatography and converted to the thiophene carboxylic
acids separately. The stereochemical assignment of the alkylation
product 16a was assigned based on a cocrystal structure of its
derivative 22a and the NS5B enzyme.24 The major product 16a
derived from the allyl group reacting on the face of enolate oppo-
site to the cyclohexane group. This observation is consistent with
the diastereoselectivity previously reported on morpholinone alky-
lations.25 Morpholinone 17 with a quaternary carbon center at the
X
O
R1
S
OH
4, 5: R1 = Ph
t-Bu
6: R1
=
Compound Heterocycle X
NS5B IC50
Replicon 1b EC50
(l
M)
(
lM)
a-position was prepared by methylation of 16 with LDA as the
1
0.060
0.27
base. The methylation gave a 3:1 mixture of diastereomers.
The major diastereomer was putatively assigned to be 17a, with
the methyl group adding from the face opposite to cyclohexane
group.
The diastereomers in both 16 and 17 were further converted to
the desired thiophene carboxylic acids with tethered hydroxyl
groups. The allyl group was converted to ethyl alcohols 18 and
19 or propyl alcohols 20 and 21 through standard olefin cleav-
age/reduction or hydroboration sequences followed by basic
hydrolysis. Secondary alcohols 22a and 22b were synthesized by
addition of methyl Grignard to the olefin cleavage product of 16a
followed by hydrolysis.
Me
4a
1.5
>33
>33
N
N
Me
4b
5a
10.5
N
0.74
11.6
O
The potencies of pyrroles 4 and lactams 5 and 6 against purified
NS5B enzyme and in the cell-based GT-1b replicon system are
summarized in Table 1. Replacing the amide in 1 with pyrroles,
as in compound 4a and 4b, resulted in significant reductions in
potency against the enzyme. Neither 4a nor 4b had measurable
inhibition in the replicon assay. In this scaffold, similar to the thi-
ophene amide series, a trans methyl group on the cyclohexyl ring
was found to improve potency ten-fold in the enzyme assay.
In the lactam series, our initial attempt was to replace the
amide in 1 with a piperidinone. Unfortunately, compound 5a,
which contains a 4-trans-methyl-cyclohexyl group, showed low
potency in both enzymatic and replicon assays. We hypothesized
that the positioning and trajectory of the cyclohexyl group might
be different in 1 and 5a, and therefore, the fit of the trans-
methyl-cyclohexyl in the pocket might not be optimal. Thus, we
also prepared compound 5b with a cyclohexyl group. Remarkably,
this analog showed promising enzyme inhibition and potency in
the replicon assay. Having observed how subtle changes in this
lipophilic R group can result in dramatic effects on inhibitory activ-
ity, we went on to survey other substituents, including cyclopentyl,
THP and phenyl groups (Table 1, compounds 5c, 5d, 5e). Unfortu-
nately, these modifications uniformly resulted in a significant loss
in potency. Finally, the pyrrolidinone 5f was slightly less potent
compared to the piperidinone analog 5b.
Compounds 5a–f were all synthesized in racemic form. Com-
pound 5f was further separated into two enantiomers 5g and 5h.
Only one enantiomer (5g) was responsible for the observed inhibi-
tion. The absolute configuration of 5g was established based on a
co-crystal structure with NS5B enzyme (Fig. 4).26 This structure
showed that the cyclohexyl group overlaps very well with the
methyl-cyclohexyl group of 1 and fully occupies the hydrophobic
pocket. This observation explains why the addition of a methyl
group to the cyclohexyl ring of 5g would be detrimental to the
potency of the compound against the enzyme. Incidentally, in
another class of thumb Site II inhibitor, namely dihydropyran-2-
ones, discovered by Pfizer, the cyclopentane group also occupies
this hydrophobic pocket and is the optimal substituent for enzy-
matic potency.27–29
5b
5c
5d
0.073
0.72
0.33
N
N
O
O
3.7
>10
>33
N
O
O
5e
0.87
5.8
N
O
O
O
O
5f
0.18
0.12
0.64
0.39
N
5g
5h
N
N
>10
>33
6a
6b
0.010
0.003
0.012
0.017
N
O
O
N
O
N
O
O
6c
0.002
0.005
the lactam analogs were prepared in enantiomerically pure form
(6a–6c). Compound 6a has an EC50 of 12 nM in the replicon assay,
which is significantly more potent than the corresponding phenyl
analog 5b. The piperidinone could be replaced by morpholinone
Further potency improvements were achieved by replacing the
phenyl group in the thiophene ring with a t-butyl alkynyl group.13
With the knowledge that an S-configuration is required for potency,
(6b) without losing potency. Dimethyl substitution in the
a position
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D. Barnes-Seeman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Figure 4. (a) Co-crystal structure of 5g and HCV polymerase enzyme; (b) Overlay co-crystal structure of 1 and 5g complexed with the HCV polymerase enzyme.
Table 2
Anti-HCV activity and PXR activation profiles of piperidinone and morpholinones
O
R1
R2
N
O
N
O
O
O
S
S
OH
OH
6a
Cpd
R1
R2
NS5B IC50
(
lM)
Replicon 1b EC50
(l
M)
PXR activation at 10 lM
6a
6b
6c
0.01
0.012
0.017
0.005
0.007
0.066
0.003
0.020
0.014
0.083
0.011
0.025
141%
87%
145%
3%
H
Me
H
H
Me
CH2CH2OH
H
CH2CH2OH
Me
CH2CH2CH2OH
H
0.003
0.002
0.002
0.012
0.002
0.006
0.002
0.035
0.002
0.004
18a
18b
19a
19b
20a
20b
21a
21b
CH2CH2OH
Me
CH2CH2OH
H
CH2CH2CH2OH
Me
CH2CH2CH2OH
NA
77%
29%
5%
NA
34%
8%
CH2CH2CH2OH
Me
OH
22a
22b
H
H
0.003
0.002
0.008
0.030
9%
4%
OH
pola rgroup
X
tether
N
O
O
S
OH
(a)
(b)
Figure 5. (a) Strategy to reduce PXR induction. (b) Model of morpholinone 6c binding to the NS5B enzyme.
of morpholinone further improved replicon EC50 to 5 nM (6c). Since
the polymerase enzymatic assay has a lower limit of detection of
2 nM, the most potent compounds could not be differentiated based
on this method.
Further profiling of 6a–6c in off-target safety assays indicated that
these compounds were strong activators of PXR, a key transcription
factor responsible for upregulation of drug metabolizing enzymes,
notably CYP3A4.30–34 In a PXR reporter gene assay, at a 10
lM
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6
D. Barnes-Seeman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3
concentration, compounds 6a–6c activated PXR by 87–145% relative
to rifampicin (Table 2). Morpholinone 6b had slightly lower PXR acti-
vation than piperidinone 6a. Adding two methyl groups at the
Comparison of pharmacokinetic parameters of 18a, 20a and 22a in rats and dogs
Compound
18a
20a
22a
a
-position regained PXR activation. The potential for 6b to induce
Rat IV (1 mg/kg)
AUC [nmol h/L]
Cl [mL/min/kg]
Vss [L/kg]
T1/2 (h)
AUC [nmol h/L]
F%
AUC [nmol h/L]
Cl [ml/min/kg]
Vss [L/kg]
T1/2 (h)
AUC [nmol h/L]
F%
1420
27
2.1
2
3374
48
2077
5.6
0.3
3.8
5684
27
984
38
1.2
1.1
2108
43
2343
5.4
0.1
2.4
2924
12
2758
14
3.0
5.2
10580
77
1452
7.7
0.7
5
6302
43
CYP3A4 was further confirmed by measuring testosterone metabo-
lism (a CYP3A4 substrate) and CYP3A4 mRNA expression in human
hepatocytes. Pretreatment of hepatocytes with 10 lM of 6b
increased CYP3A4-mediated testosterone metabolism by 61% and
increased CYP3A4 mRNA expression level by 66% as compared to
treatment with 10 lM rifampicin.
From a regulatory perspective, a negative in vitro result for
CYP3A4 induction in the PXR assay eliminates the need for addi-
tional in vitro or in vivo induction studies for CYP3A4.32 In early
drug discovery, PXR data, as expressed in the percentage of activa-
tion compared with a positive control such as rifampicin at a par-
Rat PO (5 mg/kg)
Dog IV (0.3 mg/kg)
Dog PO (3.0 mg/kg)
ticular concentration (e.g., 10 lM), have been widely used to rank
and 2% increases, respectively, as compared to treatment with
10 M rifampicin. These compounds are therefore anticipated to
have a low risk of CYP3A4 induction.
order of the CYP3A4 induction potential of test compounds. Miti-
gating the risk of CYP3A4 induction early in drug discovery can
help streamline the development process, and maximize the ability
to combine our NS5B inhibitors with other important HCV thera-
pies. Thus, we sought to reduce PXR activation in the reporter gene
l
Based on their potency in the NS5B enzymatic and replicon
assays, low PXR and CYP3A4 induction liability, and limited off-
target liabilities, the pharmacokinetic profiles of 18a, 20a and
22a were characterized. The in vivo pharmacokinetic parameters
in Sprague–Dawley rats and beagle dogs are summarized in Table 3.
In both rats and dogs, all three compounds showed low to moder-
ate plasma clearance relative to hepatic blood. Following oral dose
studies, plasma exposures of 22a were found to be higher than 18a
and 20a in both rats and dogs. The oral bioavailability of 22a was
adequate to support further pharmacokinetic and toxicology stud-
ies in both species. Finally, in a further antiviral activity profiling,
22a had a modest shift in replicon potency (EC50, 44 nM) in the
presence of 40% human serum and was also active against HCV
G1a replicon (EC50, 11 nM).
assay to less than 25% of that induced by 10 lM rifampicin for this
series. For promising compounds that met this criteria, follow up
studies in human hepatocytes were used to confirm a lack of
CYP3A4 induction.
Several crystal structures of the human PXR ligand-binding
domain (hPXR LBD) complexed with xenobiotic ligands have been
reported.35–38 The human PXR ligand binding site is unusually
large and flexible. A pharmacophore containing one H-bond accep-
tor and three hydrophobic groups for binding to the PXR protein
has been described.39,40 Compounds 6a–6c contain a carboxylic
acid and several hydrophobic groups and therefore contain func-
tionality consistent with this pharmacophore model. One strategy
to reduce PXR activation is to introduce polar groups to the activa-
tor in order to destabilize interactions in the hydrophobic areas of
the PXR ligand-binding pocket (Fig. 5a).40 In compound 6, the t-
butyl group and cyclohexyl group form hydrophobic interactions
In summary, we have successfully incorporated lactams as
amide replacements in the design of HCV NS5B Site II thiophene
carboxylic acid inhibitors. In contrast to the amide scaffold, in
which a 4-trans-cyclohexyl group was important for replicon
potency, a cyclohexyl group on the lactam was an optimal substi-
with NS5B, and are required for binding. The position
a to the car-
tuent. Appending a tethered hydroxyl group off the
a position of
bonyl is located near the solvent opening of the binding pocket and
appears to be the ideal position to append polar groups (Fig. 5b).
Based on this hypothesis, morpholinones 18–21 with alcohols
the morpholinone lactam ring mitigated the risk of CYP3A4 induc-
tion with the lead compounds while maintaining cellular potency.
In addition, compound 22a had a favorable pharmacokinetic pro-
file in rats and dogs and is a promising candidate for the clinical
treatment of HCV infection.
attached to the
a position were prepared (Table 2).
Initial profiling revealed that the two diastereomers had mark-
edly different potencies in both the enzymatic and replicon assays
(Table 2). Compounds 18a, 19a, 20a, and 21a, which have tethered
hydroxyl groups cis to the cyclohexyl group, were more potent
than the diastereomers 18b, 19b, 20b, and 21b. Compounds 18a
Acknowledgment
We thank Dr. Zachary Sweeney and Catherine Jones for help
with the manuscript preparation.
and 20a, which are monosubstituted at the a-position, had slightly
improved replicon potency compared to 6b. Meanwhile, the iso-
mers 18b and 20b had much reduced potency in the NS5B enzyme
and replicon assays. The two potent diastereomers 18a, 20a had
much reduced PXR activation in the reporter gene assay, with 3%
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tively. Compared to compounds 18a, 18b, 20a, and 20b, the addi-
tion of a methyl group afforded compounds 19a, 19b, 21a and
21b, which had slightly improved replicon potency. However, this
change increased PXR activation especially for 19a and 19b. Sec-
ondary alcohols 22a and 22b also had much reduced PXR activa-
tion. Among these two, the more potent diastereomer 22a had an
EC50 8 nM in the replicon assay, which was comparable to the
EC50 of primary alcohol 18a.
Compounds with the most promising profile based on potency
in the replicon assay and low CYP3A4 induction risk were further
tested in a human hepatocyte CYP3A4 activity assay. Whereas
compound 6b increased CYP3A4-mediated testosterone metabo-
lism by 61%, compounds 18a, 20a, and 22a only caused 5%, 2%,
Please cite this article in press as: Barnes-Seeman, D.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.031

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