Discovery of Thienoimidazole-Based HCV NS5A Genotype 1a and 1b Inhibitors

Article abstract of DOI:10.1021/ml300461f

The discovery of potent thienoimidazole-based HCV NS5A inhibitors is herein reported. A novel method to access the thienoimidazole [5, 5]-bicyclic system is disclosed. This method gave access to a common key intermediate (6) that was engaged in Suzuki or

Full text of DOI:10.1021/ml300461f

Letter
pubs.acs.org/acsmedchemlett
Discovery of Thienoimidazole-Based HCV NS5A Genotype 1a and 1b
Inhibitors
Simon Giroux,*,† Jinwang Xu,† T. Jagadeeswar Reddy,‡ Mark Morris,† Kevin M. Cottrell,†
Caroline Cadilhac,‡ James A. Henderson,† Oliver Nicolas,‡ Darius Bilimoria,‡ Francois Denis,‡
Nagraj Mani,† Nigel Ewing,† Rebecca Shawgo,† Lucille L’Heureux,‡ Subajini Selliah,‡ Laval Chan,‡
Nathalie Chauret,‡ Francoise Berlioz-Seux,† Mark N. Namchuk,† Anne-Laure Grillot,†
Youssef L. Bennani,‡ Sanjoy K. Das,‡ and John P. Maxwell†
†Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, Massachusetts 02139, United States
‡Vertex Pharmaceuticals Canada Incorporated, 275 Boul. Armand-Frappier, Laval, QC, H7V 4A7, Canada
S
* Supporting Information
ABSTRACT: The discovery of potent thienoimidazole-based HCV
NS5A inhibitors is herein reported. A novel method to access the
thienoimidazole [5,5]-bicyclic system is disclosed. This method
gave access to a common key intermediate (6) that was engaged in
Suzuki or Sonogashira reactions with coupling partners bearing
different linkers. A detailed study of the structure−activity
relationship (SAR) of the linkers revealed that aromatic linkers
with linear topologies are required to achieve high potency for both
1a and 1b HCV genotypes. Compound 20, with a para-phenyl
linker, was identified as a potential lead displaying potencies of 17
and 8 pM against genotype 1a and 1b replicons, respectively.
KEYWORDS: HCV, NS5A, thienoimidazoles
he hepatitis C virus (HCV) is a pathogen currently
1
Taffecting 170 million people worldwide.
Since the
discovery of HCV in 1989, intense research efforts to find a
cure have been pursued on both the academic and the
pharmaceutical fronts.2,3 The standard of care (SOC) has
recently evolved from a two-drug combination of intravenous
pegylated interferon-α (PEG-IFN) and oral ribavirin (RBV)4 to
a three-drug combination of PEG-IFN, RBV, plus one of two
recently approved direct-acting antivirals (DAAs), Incivek or
Victrelis. These new DAAs act as reversible covalent inhibitors
of the NS3/4A protease.5−8 When combined with PEG-IFN/
RBV, either of these two agents can significantly increase
sustained virological response (SVR) and dramatically reduce
treatment duration.9 However, new treatments with all-oral
regimens that would eliminate the need for intravenous PEG-
IFN are desirable.
Figure 1. Chemical structures of BMS-790052 (1) and 2.
reduction of HCV RNA in 24 h with a 10 mg dose (genotypes
1a and 1b).13
To this end, the nonstructural protein NS5A has recently
emerged as a potential therapeutic target for HCV.10−12
Although NS5A has no known enzymatic activity and its
exact role remains elusive, evidence suggests that it plays an
essential function in the regulation of the HCV replication.10
To date, several NS5A inhibitors are in various stages of
development, most notably, daclatasvir (BMS-790052, 1, Figure
1), which has emerged as the benchmark molecule in this
area.11,12 In clinical trials, 1 displayed an impressive 3 log10
C2 symmetric bis-benzimidazole analogues with aryl linkers,
such as 2, have been shown to be potent inhibitors of NS5A in
both genotype 1a and 1b replicons.14 In this manuscript, we
Special Issue: HCV Therapies
Received: December 18, 2012
Accepted: January 27, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
describe our results at replacing one of the benzimidazoles with
an isosteric thienoimidazole moiety, thereby breaking the C2
symmetry of the molecules. We also provide the details of the
linker structure−activity relationship (SAR) studies of two
unsymmetrical series, namely, the benzimidazole and the
imidazole series.
Synthetically, we envisaged that a disconnection involving
the coupling of a linker bearing an aryl boronic acid (Suzuki) or
an acetylene derivative (Sonogashira) with a common
intermediate (6) could provide us a versatile method giving
access to the full carbon framework of these analogues. Finally,
a two-step sequence involving an acid-induced N-Boc
deprotection and the capping of both pyrrolidine nitrogens
with the requisite valine carbamate moieties would give access
to the desired analogues. Our synthetic approach is presented
in Figure 2.
tion in 72% yield over two steps. Iodoimidazole 4 was coupled
to 4-bromophenylacetylene under the Sonogashira conditions,
and the resulting intermediate was smoothly iodinated with N-
iodosuccinimide to afford alkyne 5 in 76% yield over two steps.
The key cyclization leading to the required thienoimidazole
core was then attempted. Much to our delight, thienoimidazole
6 could be isolated in ca. 40% yield after exposure of 5 to
Na2S·9H2O and CuI in DMSO. To the best of our knowledge,
this reaction represents a novel entry to the strained [5,5]-
bicyclic thienoimidazole core.16
The key intermediate 6 was then used to synthesize all
compounds described herein. Generally, a three-step sequence
gave access to all final compounds:17 (1) Suzuki or Sonogashira
coupling of the corresponding partner, (2) N-Boc deprotection,
and (3) coupling of the required valine N-methylcarbamate
moiety11,12,14 (Schemes 2 and 3).
Scheme 2. General Preparations of NS5A Inhibitors 7−10
a
via the Suzuki and Sonogashira Reactions
a
Figure 2. Retrosynthetic analysis.
Reagents and conditions: (a) Pd(OAc)2, sodium 2′-dicyclohexyl-
phosphino-2,6-dimethoxy-1,1′-biphenyl-3-sulfonate hydrate, NaHCO3,
IPA/H2O, 120 °C, μW. (b) TFA, CH2Cl2 or HCl, dioxane, RT. (c)
t3p, DIEA, (S)-N-methoxycarbonyl valine, CH2Cl2.
First, we undertook the synthesis of the thienoimidazole key
intermediate 6 following the sequence in Scheme 1. To this
end, imidazole 315 was converted to iodoimidazole 4 via a bis-
iodination using N-iodosuccinimide and a proto-dehalogena-
Compounds 7−10 are unsymmetrical benzimidazole-thie-
noimidazoles (Table 1). Excellent genotype 1b (G1b) replicon
potency was achieved with or without a linker, and good to
modest genotype 1a (G1a) replicon potency was observed for
compounds 7−9 (Table 1). However, the addition of the
ethylene linker resulted in a 16-fold enhancement of G1a
potency (0.038 nM, 10) as compared to 7 that bears no linker
(0.619 nM, 7). We postulate that the ethylene linker of 10
could facilitate the optimal orientation of the essential valine-
proline moieties for optimal contact with the G1a protein. The
flexibility of 10 as compared to the more rigid and linear
arrangement of phenyl (8) and acetylene (9) derivatives may
be responsible for the increased G1a activity.
Scheme 1. Preparation of Key Intermediate 6 via a Novel
Entry to the Thienoimidazole Core
a
To assess if additional flexibility around the benzimidazole
moiety is a requirement to achieve G1a potency, we replaced
the [5,6]-benzimidazole system with an imidazole, directly
attached to different linkers (Scheme 3). The change of the
benzimidazole moiety for an imidazole would give rise to
compounds with topologies reminiscent of BMS-790052 (1,
G1a = 50 pM, G1b = 9 pM) and ultimately could lead to better
activities against genotype 1a.
a
Reagents and conditions: (a) NIS, CH2Cl2, 0 °C to RT. (b) LiCl,
MeMgCl, i-PrMgCl, THF, −20 °C to RT (c) 4-Bromophenylacety-
lene, CuI, PdCl2·(dppf), NEt3, DMF, 80 °C. (d) NIS, CH2Cl2, RT. (e)
Na2S·9H2O, CuI, DMSO, 150 °C.
B
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Scheme 3. General Preparations of NS5A Inhibitors 11−21
Table 2. SAR Studies of the Linker Moiety (Imidazole
Series)
a
via the Suzuki Reaction
a
Reagents and conditions: (a) Pd(OAc)2, sodium 2′-dicyclohexyl-
phosphino-2,6-dimethoxy-1,1′-biphenyl-3-sulfonate hydrate, NaHCO3,
IPA/H2O, 120 °C, μW. (b) TFA, CH2Cl2 or HCl, dioxane, RT. (c)
t3p, DIEA, (S)-N-methoxycarbonyl valine, CH2Cl2.
Table 1. SAR Studies of the Linker Moiety (Benzimidazole
Series)
790052 (1) and other NS5A inhibitors.10,19 Additionally, all
analogues from Tables 1 and 2 were noncytotoxic displaying
CC50 values greater than 20 μM.20
Indeed, very strong G1a potencies (EC50 < 0.1 nM) could be
achieved with a bis-fused thiophene, thiophene, and phenyl-
based linker (Table 2). Remarkably low ratios of 1a/1b (≤6)
were obtained for 11, 13, 17, and 20 with compound 20
displaying replicon EC50 values of 0.008 and 0.017 nM against
G1b and G1a, respectively. The para arrangement of the linker
seems to be critical as the meta-substituted phenyl linker
displayed very low potency against G1a (compare compounds
20 and 21), further confirming the critical spatial arrangement
of the proline-valine moiety required to achieve G1a potency.
We postulate that these compounds bind at the dimeric NS5A
interface, thereby preventing the formation of the active NS5A
homodimer. This is supported by the identification of
mutations in the replicon assay (G1a: M28, Q30, L31, and
Y93; G1b: Y93), which are proximal to or at the dimer
interface.18 These mutations have also been reported for BMS-
With good G1a and G1b potencies in hand, the ADME
properties of 20 were investigated further. Metabolic stability
was evaluated in rat (RLM) and human (HLM) liver
microsomes in the presence of NADPH and UDPGA as
cofactors. Compound 20 was stable in rat and human
microsomes with 91 and 100% parent intact, respectively,
after incubation for 1 h at a concentration of 5 μM. The
observed Caco-2 ratio (BA/AB ratio) of 2.0 suggested that
compound 20 is not a substrate for the Pgp efflux transporter.
Importantly, the pharmacokinetic profile of 20 in rats indicated
low clearance (Cl 5.3 mL/min/kg) and moderate bioavailability
(F = 25%) after dosing (3 mg/kg) as a solution in 30% aqueous
PEG-400. Key pharmacokinetic data for selected compounds
are included in Table 3. Compounds 10, 11, and 21 also display
C
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protease and NS5B polymerase inhibitors. Curr. Opin. Pharmacol.
2008, 8, 522−531.
low Cl below 15 mL/min/kg and half-lives (T1/2) ranging from
5 to 11 h.
(9) Poordad, F.; Dieterich, D. Treating hepatitis C: Current standard
of care and emerging direct-acting antiviral agents. J. Viral Hepatitis
2012, 19, 449−464.
Table 3. Pharmacokinetic Profiles for Selected Compounds
(10) Schmitz, U.; Tan, S.-L. NS5AFrom Obscurity to New Target
for HCV Therapy. Recent Pat. Anti-infect. Drug Discovery 2008, 3, 77−
92.
(11) Lemm, J. A.; O’Boyle, D., II; Liu, M.; Nower, P. T.; Colonno,
R.; Deshpande, M. S.; Snyder, L. B.; Martin, S. W.; St. Laurent, D. R.;
Serrano-Wu, M. H.; Romine, J. L.; Meanwell, N. A.; Gao, M.
Identification of Hepatitis C Virus NS5A Inhibitors. J. Virol. 2010, 84,
482−491.
(12) Bell, T. W. Drugs for Hepatitis C: Unlocking a New Mechanism
of Action. ChemMedChem 2010, 5, 1663−1665.
(13) Gao, M.; Nettles, R. E.; Belema, M.; Snyder, L. B.; Nguyen, V.
N.; Fridell, R. A.; Serrano-Wu, M. H.; Langley, D. R.; Sun, J.-H;
O’Boyle, D. R., II; Lemm, J. A.; Wang, C.; Knipe, J. O.; Chien, C.;
Colonno, R. J.; Grasela, D. M.; Meanwell, N. A.; Hamann, L. G.
Chemical genetics strategy identifies an HCV NS5A inhibitor with
potent clinical effect. Nature 2010, 465, 96−100.
(14) Pereira, O.; Maxwell, J.; Bennani, Y. L.; Das, S. K.; Kong, L. C.
C.; Reddy, T. J.; Giroux, S.; Cottrell, K. M.; Morris, M. A. Patent WO/
2011/119858.
(15) For the synthesis, see Kong, L. C. C.; Giroux, S.; Reddy, T. J;
Bennani, Y. L. Patent WO/2011/119870 and reference therein.
(16) For an alternative synthesis of the thienoimidazole core, see
Hartley, D. J.; Iddon, B. Use of the Vinyl Group as an Efficient
Protecting Group for Azole N-Atoms: Synthesis of Polyfunctionalized
Imidazoles and Thieno[2,3-d]-[3,2-d]imidazole. Tetrahedron Lett.
1997, 38, 4647−4650.
compds
10
iv rat PK
11
20
21
Cl (mL/min/kg)
T1/2 (h)
6.0
6.6
2.7
5.1
5.1
2.0
5.3
6.1
2.4
13.5
5.1
Vss (L/kg)
4.8
po rat PK
AUC0−8h (μg h/mL)
T1/2 (h)
0.82
5.0
0.46
1.54
8.3
0.49
10.8
5.1
Cmax (ng/mL)
% F
0.03
0.01
6.7
0.03
0.01
14.1
25.6
16
In summary, we describe the identification and optimization
of two series of nonsymmetrical thienoimidazole-containing
analogues with subnanomolar potency against both genotypes
1a and 1b replicons. This study demonstrated that
benzimidazoles can successfully be replaced by the novel
thienoimidazole isostere when a proper linker is chosen. The
optimization of compound 20, geared toward improved
potency against the mutations identified in the replicon assay,
will be reported in due course.
ASSOCIATED CONTENT
* Supporting Information
Full experimental details for representative compounds
synthesized and description of assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
(17) See the Supporting Information for the details of the syntheses
of intermediate 6 and compound 20.
(18) Large potency drops were observed when 20 was screened
against recombinant replicon cell lines harboring some of these
mutations: (1a Y93H/C, >15000-fold; 1a M28T and Q30R, >500-
fold; and 1b Y93H, 900-fold).
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-341-6934 . E-mail: simon_giroux@vrtx.com.
(19) Fridell, R. A.; Qui, D.; Wang, C.; Valera, L.; Gao, M. Resistance
Analysis of the Hepatitis C Virus NS5A Inhibitor BMS-790052 in an
In Vitro Replicon System. Antimicrob. Agents Chemother. 2010, 54,
3641−3650.
■
Notes
(20) CC50 in ET cells were determined using a [3H]-thymidine
incorporation assay from 12 concentrations of test compound in
duplicate, and data were considered accurate when the CC50 of the
positive control was within 40% of the mean value.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank James Empfield and Rene Rijnbrand for useful
comments on the manuscript as well as Ding Lu and Gregory
May for assistance on characterization.
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