Inhibitors of HCV NS5A: From iminothiazolidinones to symmetrical stilbenes

Article abstract of DOI:10.1021/ml1002647

The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV replication in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue, BMS-824 (18), was used in resistance mapping studies, which rev

Full text of DOI:10.1021/ml1002647

LETTER
pubs.acs.org/acsmedchemlett
Inhibitors of HCV NS5A: From Iminothiazolidinones to Symmetrical
Stilbenes
Jeffrey L. Romine,*,† Denis R. St. Laurent,† John E. Leet,‡ Scott W. Martin,† Michael H. Serrano-Wu,†,^
Fukang Yang,† Min Gao,§ Donald R O’Boyle II,§ Julie A. Lemm,§ Jin-Hua Sun,§ Peter T. Nower,§
Xiaohua (Stella) Huang,|| Milind S. Deshpande,†,# Nicholas A. Meanwell,*,† and Lawrence B. Snyder†
†Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut
06492, United States
‡Synthesis & Analysis Technology Team, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford,
Connecticut 06492, United States
§Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492,
United States
Discovery Analytical Sciences, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492,
United States
S Supporting Information
b
ABSTRACT: The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV repli
cation in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue,
BMS-824 (18), was used in resistance mapping studies, which revealed that inhibitory activity was related
to disrupting the function of the HCV nonstructural protein 5A. Despite the development of coherent and
interpretable SAR, it was subsequently discovered that in DMSO 18 underwent an oxidation and
structural rearrangement to afford the thiohydantoin 47, a compound with reduced HCV inhibitory
activity. However, HPLC bioassay fractionation studies performed after incubation of 18 in assay media
led to the identification of fractions containing a dimeric species 48 that exhibited potent antiviral activity.
Excision of the key elements hypothesized to be responsible for antiviral activity based on SAR
observations reduced 48 to a simplified, symmetrical, pharmacophore realized most effectively with the
stilbene 55, a compound that demonstrated potent inhibition of HCV in a genotype 1b replicon with an
EC50 = 86 pM.
KEYWORDS: HCV NS5A inhibitor, iminothiazolidinone, stilbene
lmost 200 million individuals worldwide are infected with
hepatitis C virus (HCV), a disease for which the current,
A
optimal therapy consists of weekly subcutaneous injections of
pegylated interferon-R (PEG-IFN) in combination with twice-
daily oral doses of ribavirin (RBV).1-3 The inadequacies of this
treatment regimen are evident in the low response rates for
genotype 1-infected patients and the significant incidence of side
effects. The development of direct acting antiviral agents (DAAs)
to treat HCV has focused primarily on inhibitors of NS3 protease
and the NS5B RNA-dependent RNA polymerase, and several
compounds are presently in clinical trials as add-on to PEG-IFN/
RBV therapy.4 We have recently reported the discovery of the
HCV NS5A inhibitor BMS-790052 (1), a compound that
demonstrates a potent antiviral effect in HCV genotype 1-in-
fected subjects following single oral doses.5 In this article we
describe the preliminary structure-activity relationships (SAR)
developed for the HTS screening hit 2 (BMS-858) and delineate
a chemical reactivity inherent to this class of compound that led
to the elucidation of a structurally complex dimeric NS5A
inhibitor from which a simplified, symmetrical pharmacophore
was excised as a prelude to the design of BMS-790052 (1).
Using a dual HCV genotype 1b/bovine viral diarrhea virus
replicon screen in high throughput mode, the thiazolidinone 2
Received: November 3, 2010
Accepted: December 20, 2010
Published: January 11, 2011
r
2011 American Chemical Society
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Scheme 1. Synthetic Approach to Iminothiazolidinones
Using Parallel Solution Phase Chemistry
Table 1. Structure-Activity Relationships Associated with
Thiazolidinones as Inhibitors of HCV 1b Replicon Activity
was identified as a potent HCV inhibitor, EC50 = 0.58 μM, that
demonstrated a good therapeutic window with respect to
cytotoxicity (CC50 = >100 μM) and showed no significant
inhibitory activity in counterscreens.6,7 This compound had been
prepared as part of a prospective library according to the syn-
thetic route depicted in Scheme 1, where regiocontrol during
ring closure occurred as anticipated based on literature prece-
dent.8-10 Assignment of the 2-arylimino Z-configuration is also
based on prior studies, a function of minimizing unfavorable
steric interactions,11,12 while the C-5 position is configurationally
unstable and cannot be defined.13 Screening results for additional
library members were available (see compounds 3-7) and gave
indication that antiviral activity was closely associated with the
embedded amino acid moiety (Table 1).7 To expand upon this
observation, a survey of amino acid analogues was conducted that
confirmed that the L-configuration was essential for antiviral
activity since the unnatural, D-configured 8 was over 150-fold
weaker, an observation reproduced in the proline derivatives 9
and 10. Lastly, the data obtained for compounds 12-15 com-
municate a clear preference for smaller CR substituents.14
Based on the results obtained for 2 and 9 in Table 1, L-alanine
and L-proline were selected as substrates to probe the SAR at the
carboxybenyl (Cbz) terminus (R3), the 3-fluorophenyl ring (R2),
and furan heterocycle (R1), as collated in Tables 2 and 3.
Exchange of the Cbz for either benzoyl (16 and 17) or the iso-
steric phenylpropionoyl (20 and 21) moieties resulted in re-
duced potency, whereas a phenacetyl proved to be optimal in
both the alanine 18 and proline 19 analogues, enhancing potency
by ∼100-fold compared to the cases of 2 and 9, respectively. The
high level of potency observed for 18 (BMS-824), EC50
=
0.006 μM, facilitated its use in resistance mapping studies
designed to illuminate aspects of the mode of antiviral activity.
These studies implicated the NS5A protein as the target of this
class of inhibitor based on the identification of a Y93H change
that, when introduced into the replicon, significantly compro-
mised the potency of 18, EC50 > 5 μM.5,15
The parallel SAR observed between the two amino acid series
extended to variation of substituents attached to the iminothia-
zolidinone ring, as shown in Table 3. For example, 2-pyridyl and
3-pyridyl heterocycles in place of the furan enhanced potency by
>15-fold compared to the case of 2 in both the alanine (23 and
25) and proline (24 and 26) series, whereas the more lipophilic
2,3,4-trifluorophenyl ring merely preserved potency (28 and 29).
Somewhat analogously, lipophilic substituents appended to the
3-phenyl ring (compounds 30-37) generally resulted in re-
duced antiviral activity compared to the case of 2, while more
a EC50 values are averages of at least two independent determinations.
polar substituents were beneficial in improving potency, as
illustrated by compounds 38-45.
Although the data reported in Tables 1-3 provide a coherent
and interpretable SAR, we subsequently discovered C-5 phenyl-
iminothiazolidinones 2 and 18 were consumed during the course
of the 3 day replicon assay. The first indication of an inherent
chemical reactivity emerged when it was observed that, on
standing in DMSO, compound 2 underwent an oxidative re-
arrangement to generate the thiohydantoin 46 (Scheme 2).16,17
The structure of 46 was elucidated by correlation spectroscopy,
which established the proximity of the furanyl-methylene pro-
tons (δ5.2, dd, 2H) to the amide carbonyl carbon (δ 171.4 ppm)
and the thiourea carbon (δ 182.5 ppm) atoms. In addition,
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nuclear Overhauser effects (nOe) were observed between the
ortho protons on the two phenyl rings at δ 9.09 (d, 1H) and δ
7.30 (d, 2H), as depicted in structure 46.16
Thiohydantoin 46, however, exhibited poor activity in the
replicon assay, with an EC50 = 13 μM, suggesting that other
species in the cell culture media may be responsible for the
observed inhibitory activity of 2, and this prompted a more
detailed analysis of compound 18.17 While stable indefinitely in
solid form and quite stable in solvents such as MeOH and
CH3CN, the iminothiazolidinone 18 underwent the same
oxidative transformation in DMSO as 2, to form 47 (EC50
>
20 μM).16 Moreover, preincubation of 18 in assay media until
completely consumed, as determined by HPLC, followed by
analysis of the replicon inhibitory activity of the solution,
produced an equivalent antiviral effect to that recorded for
parent compound 18 under standard assay conditions. HPLC
analysis of the preincubated media detected the thiohydantoin
47 as the predominant product; however, it was observed to
undergo degradation over time to the thiourea starting material
depicted in Scheme 1, presumably via hydrolysis of intermediate
C (Scheme 2). As a result of these findings, HPLC biogram
fractionation experiments were performed, an exercise that
traced potent HCV inhibitory activity to two minor components
in the media identified as isomers of the dimeric species 48
(Figure 1).17 The connectivity of the dimer was established by 1H
and 13C NMR to be at the C-5 methine carbon, with the less
mobile isomer on reversed phase LC analysis being the more
potent compound, EC50 = 0.6 nM, and which converted to the
more mobile but less potent (EC50 = 43 nM) isomer upon
heating at 55 ꢀC.17
Table 2. Comparison of the Effect of Structural Variation of
the Amino Acid N Substituent on Replicon Potency in the
L-Alanine and L-Proline Series
In considering how compounds 46-48 might be generated, it
was recognized that hydrogen abstraction at the C-5 methine
carbon of 18 would form a classic captodative radical stabilized by
the electron donating sulfur atom and the electron withdrawing
carbonyl group.18,19 A radical mechanism is consistent with the
formation of 47 by a process involving combination of a C-5
radical with oxygen that would give hydroperoxide A (Scheme 2).
Precedent for peroxide formation and reductive cleavage in/by
AA = L-Alanine
EC50 (μM)a
AA = L-Proline
EC50 (μM)a
R3
cmpd
cmpd
Ph
CH2Ph
CH2CH2Ph 20
16
1.9
17
19
21
7.9
18 (BMS-824)
0.006
0.023
3.1
5.1
a EC50 values are averages of at least two independent determinations.
Table 3. Effect of Variation of the 2-Furyl and 3-Fluorophenyl Moieties in Both the L-Alanine and L-Proline Series on Antiviral
Activity in a HCV Genotype 1b Replicon
R1
R2
3-F-C6H4
cmpd
AA = L-alanine EC50 (μM)a
cmpd
AA = L-proline EC50 (μM)a
2-tetrahydrofuranyl
2-pyrazinyl
2-pyridinyl
3-pyridinyl
2,3,4-F3-C6H2
2-furyl
22
23
25
27
28
30
32
34
36
38
40
0.65
0.025
0.035
0.026
0.55
2.2
3-F-C6H4
24
26
0.038
0.002
3-F-C6H4
3-F-C6H4
3-F-C6H4
29
31
33
35
37
39
41
0.6
4-CF3-C6H4
3-F-4-CF3-C6H3
4-Cl-C6H4
4-Et-C6H4
4-CN-C6H4
0.98
2.2
2-furyl
2.5
2-furyl
0.51
1.5
1.17
1.8
2-furyl
2-furyl
0.037
0.01
0.086
0.009
2-furyl
2-furyl
2-furyl
5-quinolinyl
42
44
0.02
43
45
0.01
0.02
4-morpholinylphenyl
a EC50 values are averages of at least two independent determinations.
0.015
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Figure 1. Dimeric species identified in replicon assay media and pharmacophore hypothesis.
Scheme 2. Oxidative Rearrangement of the Iminothiazolidinones 2 and 18
DMSO, which in this case affords B, is available in the literature
for other systems.20,21 Unique to B, however, is the potential for
ring-opening to the R-ketoamide C, which can reclose in a
complementary fashion to form the 2-thiohydantoin 47, with
experimental support for this sequence of events obtained as
follows. Snider has reported that radicals generated in the
presence of Mn(OAc)3/Cu(OAc)2 can undergo further oxida-
tion in which the resulting cation is trapped by acetic acid.22,23
The phenyl acetamide 49 was exposed to these conditions
(Scheme 3) and gave 50, an isolable derivative of B, in 59%
isolated yield together with some of the rearranged thiohydan-
toin 51 in 16% yield. The structure of compound 50 was
established by N-H long-range NMR correlation in order to
rule out the acetoxy derivative of 51, which may result simply
from exchange of the hydroxyl group with acetic acid.24
Exposure of 2 and 18 to the same reaction conditions gave the
acetoxy derivatives 52 (48%) and 53 (78%), respectively, with no
evidence of thiohydantoin formation. In the latter case, a small
amount of dimeric product 48 was obtained (12 mg, 2.3%),
which was shown by 1H NMR and LC to be identical with the
more mobile dimeric isomer isolated from media.25,26 In a further
experiment, acetates 52 and 53 were subjected to basic methanol
(K2CO3/MeOH), which effected rapid conversion to thiohy-
dantoins 46 and 47.27
radical that can either react with oxygen to produce hydroperoxide
A or combine to produce dimer 48. The absolute requirement for
an L-configured amino acid coupled with the high sensitivity of
potency to changes in the amine capping element suggested that
this region of the molecule made an important contribution to
the antiviral activity observed with dimer 48. This appreciation
led to the hypothesis that excision of the iminothiazolidinone
heterocycles would simplify the NS5A-inhibiting pharmaco-
phore in 48 to that of the palindromic bibenzyl derivative 54,
as depicted in Figure 1. Synthetic accessibility facilitated the
testing of this hypothesis, and 54, a compound quite stable to
replicon assay conditions, demonstrated potent inhibition,
EC50 = 30 nM.28 Furthermore, the more rigidly disposed stilbene
55 proved to be an even more potent inhibitor of the 1b HCV
replicon with an EC50 = 0.086 ( 10 nM (n = 4).28,29 As is evident
from Table 4, SAR for this new class of stilbene inhibitors closely
resembled that of the iminothiazolidinones. Replacing the phe-
nylacetic acid amine cap with Cbz (compound 57) effected a
100-fold loss in potency, similar in magnitude to that observed
between 18 and 2, while the L-configuration was again essential
for potency based on the comparison of 56 and 58 with the
natural proline derivatives 55 and 57. Additional profiling
of stilbene 55 in the Y93H replicon revealed reduced potency,
EC50 = 4 μM, confirming its association of inhibition with the
NS5A protein. In a genotype 1a replicon, stilbene 55 was found
to be inactive, EC50 > 10 μM, providing a clear objective for the
next phase of optimization.
Taken together, these results are consistent with a mechanism
involving hydrogen atom abstraction from C-5 of the thiazolidi-
none ring, presumably mediated by oxygen, to afford a stabilized
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Scheme 3. Radical Generation, Oxidation, and Trapping with Acetic Acid
site for viral RNA.30 The residues conferring resistance to
inhibitor 55 are proximal to the amino terminus of NS5A and
are located between the protein and the membrane, suggesting
that the symmetrical compounds bind across the dimer inter-
face.5 Although symmetry is not essential for inhibition of HCV
replication in genotype 1b replicon, as reported elsewhere,
genotype 1a inhibition is most optimally achieved in symme-
trically disposed compounds.32
Table 4. SAR Associated with Stilbene-Based HCV NS5A
Inhibitors
In summary, we describe here fundamental aspects asso-
ciated with the HCV inhibitory activity of a series of
C-5-phenylated iminothiazolidinones discovered in a high through-
put replicon screen, compounds that target the NS5A protein.
The SAR observed for this chemotype was highly sensitive to
the absolute configuration of the amino acid and to structural
changes in the capping element at the amino terminus, and it
was consistent between the alanine and proline series. Chemical
reactivity at the C-5 position of iminothiazolidinones in DMSO
was based on a propensity to form radicals at this site, resulting
in oxidation via reaction with oxygen or the formation of
dimeric species upon radical combination. The precise SAR
associated with the amino acid moiety suggested that this
element was a critical determinant of biological activity and
lead to a hypothesis that the pharmacophore inherent in 48
could be simplified to that of a symmetrical bibenzyl derivative,
an idea initially vindicated upon synthesis and evaluation of 54.
The potent inhibition of HCV replication in the replicon assay
by symmetrically disposed stilbene 55 ultimately lead to the
design of BMS-790052 (1), the compound that established
proof-of-concept for HCV NS5A inhibition as a treatment of
HCV infection.
’ ASSOCIATED CONTENT
a EC50 values are averages of at least two independent determinations.
S
Supporting Information. Experimental details for syn-
b
thetic procedures and associated chemical data for compounds
2-58 and the experimental protocol used to evaluate inhibitors
in the replicon assay. This material is available free of charge via
the Internet at http://pubs.acs.org.
BMS-790052 (1) is a first-in-class inhibitor for HCV NS5A
with high selectivity, picomolar potency, and broad genotype
coverage in vitro that likely acts by disrupting a replication-
protein assembly process critical to viral replication.5 The
synthesis of stilbene 55 was a critical step in the design of
BMS-790052 (1), a compound that has established the clinical
relevance of interfering with the function of NS5A as an
approach to the treatment of HCV infection.5 Interestingly,
the discovery of symmetry within HCV NS5A inhibitors
coincides well with X-ray crystallographic data subsequently
published for the amino terminus of the NS5A protein.30,31 In
one report, NS5A residues 36-198 crystallized as a dimer
forming a U-shaped structure with the inner surface lined with a
preponderance of basic amino acids proposed to form a binding
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: 203-677-6694. Fax: 203-677-7702. E-mail: Jeffrey.
Romine@bms.com.
Present Addresses
^Novartis Institute for BioMedical Research, Cambridge, MA
02139.
#Achillion Pharmaceuticals, New Haven, CT 06511.
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’ ACKNOWLEDGMENT
Martin, S. W.; Serrano-Wu, M. H.; Meanwell, N. A.; Snyder, L. B.; Gao,
M. Discovery, Isolation and Synthesis of Potent Hepatitis C Virus NS5A
Inhibitors with Dimeric Structures. Manuscript submitted.
(18) Giese, B. Radicals in Organic Synthesis: Formation of Carbon-
Carbon Bonds; Pergamon Press: New York, 1986.
We wish to express our gratitude to the Bristol-Myers Squibb
Research and Development, Department of Analytical Services,
in general, and the mass spectroscopy group, in particular.
(19) Viehe, H. G.; Janousek, Z.; Merenyi, R.; Stella, L. The Capto-
dative Effect. Acc. Chem. Res. 1985, 18, 148–154.
(20) Schmidt, V. A.; Alexanian, E. J. Metal-Free, Aerobic Dioxygena-
tion of Alkenes Using Hydroxamic Acids. Angew. Chem., Int. Ed. 2010,
49, 4491–4494.
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in DMSO to evaluate in the replicon assay.
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