Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic cores

Article abstract of DOI:10.1016/j.bmcl.2014.12.042

Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.

Full text of DOI:10.1016/j.bmcl.2014.12.042

Accepted Manuscript
Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic
cores
James A. Henderson, Darius Bilimoria, Monica Bubenik, Caroline Cadilhac,
Kevin M. Cottrell, Francois Denis, Evelyne Dietrich, Nigel Ewing, Guy
Falardeau, Simon Giroux, Lucille L’Heureux, Bingcan Liu, Nagraj Mani, Mark
Morris, Olivier Nicolas, Oswy Z. Pereira, Carl Poisson, T. Jagadeeswar Reddy,
Subajini Selliah, Rebecca S. Shawgo, Louis Vaillancourt, Jian Wang, Jinwang
Xu, Nathalie Chauret, Francoise Berlioz-Seux, Laval C. Chan, Sanjoy K. Das,
Anne-Laure Grillot, Youssef L. Bennani, John P. Maxwell
PII:
S0960-894X(14)01350-X
http://dx.doi.org/10.1016/j.bmcl.2014.12.042
BMCL 22287
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 October 2014
10 December 2014
12 December 2014
Please cite this article as: Henderson, J.A., Bilimoria, D., Bubenik, M., Cadilhac, C., Cottrell, K.M., Denis, F.,
Dietrich, E., Ewing, N., Falardeau, G., Giroux, S., L’Heureux, L., Liu, B., Mani, N., Morris, M., Nicolas, O., Pereira,
O.Z., Poisson, C., Jagadeeswar Reddy, T., Selliah, S., Shawgo, R.S., Vaillancourt, L., Wang, J., Xu, J., Chauret, N.,
Berlioz-Seux, F., Chan, L.C., Das, S.K., Grillot, A-L., Bennani, Y.L., Maxwell, J.P., Synthesis and evaluation of
NS5A inhibitors containing diverse heteroaromatic cores, Bioorganic & Medicinal Chemistry Letters (2014), doi:
http://dx.doi.org/10.1016/j.bmcl.2014.12.042
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and evaluation of NS5A inhibitors containing diverse
heteroaromatic cores
James A. Henderson*,a, Darius Bilimoriab, Monica Bubenikb, Caroline Cadilhacb, Kevin M.
Cottrella, Francois Denisb, Evelyne Dietrichb, Nigel Ewinga, Guy Falardeaub, Simon Girouxa,
Lucille L’Heureuxb, Bingcan Liub, Nagraj Mania, Mark Morrisa, Olivier Nicolasb, Oswy Z.
Pereirab, Carl Poissonb, T. Jagadeeswar Reddyb, Subajini Selliahb, Rebecca S. Shawgoa, Louis
Vaillancourtb, Jian Wanga, Jinwang Xua, Nathalie Chauretb, Francoise Berlioz-Seuxa, Laval C.
Chanb, Sanjoy K. Dasb,+, Anne-Laure Grillota, Youssef L. Bennania, and John P. Maxwella
a Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210, USA
b Vertex Pharmaceuticals Canada Incorporated, 275 Boul. Armand-Frappier, Laval, QC, H7V 4A7, Canada
+ Present address: AngioChem, 201 President-Kennedy Ave., Suite PK-2880, Montreal, QC H2X 3Y7, Canada
Abstract
Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical
potential in the treatment of hepatitis C when used in combination with other direct-
acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir,
and ombitasvir share a common pharmacophore that features a pair of (S)-
methoxycarbonylvaline capped pyrrolidines linked to various cores by amides,
imidazoles and/or benzimidazoles. In this letter, we describe the evaluation of NS5A
inhibitors which contain alternative heteroaromatic replacements for these amide
mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing
benzoxazoles was parlayed toward the identification of potent NS5A inhibitors
containing other heteroaromatic replacements such as indoles and imidazopyridines.
Letter
Hepatitis C virus (HCV) is currently one of the most significant world health
concerns. It is estimated that 130-170 million individuals (~3%) worldwide and more
than 5 million Americans (1.8%) are infected with HCV.1 In the majority of cases (80%),
the viral infection leads to a chronic form of hepatitis and without therapeutic
intervention can lead to morbidity or mortality through either cirrhosis and liver failure or
hepatocellular carcinoma.2 An extended, moderately efficacious and poorly tolerated
*
Corresponding author. Tel.: +1 617 961 7605
E-mail address: james_henderson@vrtx.com (J. A. Henderson)

regimen of pegylated interferon-α (PEG-IFN-α) and ribavirin (RBV) had served as the
HCV standard of care until recently. Improvements in cure rates and treatment durations
of the most dominant form of the virus, genotype 1, were realized through the addition of
the direct-acting antiviral (DAA) agents telaprevir, boceprevir, simeprevir (NS3ꢀ4a
protease) and/or sofosbuvir (NS5B polymerase) to the standard of care.3,4 Although
further optimization in the areas of resistance, tolerability, and pan-genotypic efficacy is a
necessity, ongoing clinical trials employing all oral interferon-free combinations of
DAAs are yielding promising results.5
The HCV NS5A protein has evolved into an attractive drug target. NS5A is a
large phosphoprotein (49 kDa) with no apparent enzymatic activity or homologues in
prokaryotes and eukaryotes. Although the mechanistic role of NS5A in the HCV
replication cycle currently is unknown, it is thought to play important roles in viral
replication, virion assembly and secretion from infected cells.6 The elucidation of
NS5A’s mechanistic role remains an active area of investigation.7 Proof of concept for
this target was initially demonstrated by daclatasvir (BMS-790052, Figure 1), which
displayed an impressive 3log10 reduction of HCV RNA in 24 h with one 10 mg oral dose
(genotypes 1a and 1b); a viral load drop that was considerably faster than had been
observed with HCV inhibitors acting by other mechanisms.8 These results stimulated
considerable interest in the design and development of NS5A replication complex
inhibitors (RCIs) and several related clinical candidates are currently being evaluated.9,10
The pharmacophore of the NS5A RCIs in Figure 1 is comprised of a pair of (S)-
methoxycarbonylvaline capped pyrrolidines attached to various cores by anilides,
imidazoles and/or benzimidazoles, suggesting the importance of a group with H-bond
donating and accepting properties (Figure 1). In this manuscript, we expand upon
previously reported NS5A RCIs containing amides, imidazoles and benzimidazoles to
describe inhibitors with alternate heteroaromatic replacements.
C2-symmetric bis-benzimidazole analogs with aryl linkers, such as 1 (Figure 1),
have been shown to be potent inhibitors of NS5A genotype 1a and 1b replicons.11
Subsequent efforts to desymmetrize these scaffolds led to the identification of the
thienoimidazole isostere as an attractive surrogate for benzimidazole.12 We therefore set
out to design nonsymmetrical inhibitors with diverse heteroaromatic cores, with an initial

focus on analogs containing 2,5- and 2,6-substituted benzoxazoles.
Figure 1. Structures of NS5A inhibitors

Scheme 1: General preparation of benzoxazole-containing analogs, exemplified by
6,7,10, and 11. Reagents and conditions: (a) (4-bromophenyl)boronic acid (1.0 eq),
Pd(PPh3)4, K3PO4, 5:1 PhMe:MeOH, 70 °C; (b) bis(pinacolato)diboron, PdCl2dppf,
KOAc, DMF, 85 °C; (c) 2 (1.0 eq), Pd(OAc)2, SSPhos, NaHCO3(aq), i-PrOH, 100 °C; (d)
TFA, DCM, rt; (e) (S)-2-((methoxycarbonyl)-amino)-3-methylbutanoic acid, HATU,
DIPEA, DMF, rt; (f) 4,4,5,5-tetramethyl-2-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
S
yl)thieno[3,2-b]thiophen-2-yl]-1,3,2-dioxaborolane (1.5 eq), Pd(OAc)2, SPhos, K2CO3,
3:1 2-MeTHF:H2O, 90 °C
Symmetrical and nonsymmetrical benzoxazole-containing compounds were
prepared by the general approach shown in Scheme 1. Coupling of iodo-benzoxazole 2
and iodo-benzimidazole 3 with (4-bromophenyl)boronic acid and reaction of the resulting
aryl bromide with bis(pinacolato)diboron under Suzuki conditions provided phenyl-
boronate intermediates 4 and 5, respectively. The boronates 4 and 5 were coupled with
iodo-oxazole 2, Boc-deprotected under acidic conditions, and coupled with N-
(methoxycarbonyl)-L-valine in the presence of HATU to give analogs 6 and 7.
Compounds containing the thienothiophene (e.g., 10 and 11) or biphenyl-imidazole linker
(12) were prepared by analogous sequences.13
In comparison to 1, bis-benzoxazole analogs 6 and 10 exhibited a marked
reduction in activity against both genotype 1a (G1a) and genotype 1b (G1b) replicons

(Table 1).12 The inclusion of benzimidazole in the nonsymmetrical analogs 7, 11, and 12
resulted in the return of potent G1b activity (EC50 = 17 pM). Notably, replacement of the
phenyl linker in compound 7 with thienothiophene (11) resulted in a 1800-fold increase
in G1a activity (EC50 = 32 pM). Further lengthening of the scaffold with a biphenyl-
imidazole linker, as in 12, resulted in diminished G1a potency (11 nM).

Table 1
Activity of analogs containing 2,5-substituted benzoxazole
G1a
EC50
(nM)
G1b
EC50
(nM)
CC50
(µM)
Compd
Linker
6
>2000 223
7
7
58
0.017 >20
10
>2000 23
>20
11
12
0.032
11
0.003 >20
0.023 >20
The investigation of inhibitors containing 2,6-substituted benzoxazoles is shown
in Table 2. In contrast to the 2,5-substituted benzoxazole inhibitors (Table 1), both
symmetric and nonsymmetric analogs in the 2,6-substituted benzoxazole series exhibited
picomolar G1b activity. Increased G1a activity was also observed in this series and is
highlighted by a 30-fold potency difference between analogs 7 and 14 (G1a EC50 = 58
and 1.8 nm, respectively) and a 60-fold potency boost when the diphenyl-imidazole
linker was employed (compare 12 and 17). Gratifyingly, the thienothiophene-
benzimidazole linker also stood out in the 2,6-substituted benzoxazole series and
furnished inhibitor 16, which exhibited excellent potency against both G1b and 1a
replicons (EC50 = 4 and 29 pM, respectively).

Table 2.
Activity of analogs containing 2,6-substituted benzoxazole
G1a
EC50
(nM)
G1b
EC50
(nM)
CC50
(µM)
Compd
13
Linker
630
1.8
43
0.006
7
14
0.008 >20
0.019 >20
15
16
17
0.029
0.164
0.004 >20
0.005 >20
In order to evaluate if the potent activity profiles observed with 11 and 16 were
inherent to analogs containing benzoxazoles, additional heteroaromatic groups were
evaluated in the thienothiophene-benzimidazole scaffold (Table 3). This survey included
heteroaromatics with both H-bond donating and accepting capabilities, such as
benzimidazole 20 and thienoimidazole 22, as well as rings with exclusively H-bond
donating or H-bond accepting capacities.
Remarkably, the thienothiophene-
benzimidazole scaffold proved to be tolerant of heteroaromatics from each of these
donor/acceptor classes, and with the exception of indole 23 and benzthiazole 25, all
compounds exhibited potent and balanced G1a and 1b activity. Compounds in Tables 1-
3 were generally noncytotoxic, with most having CC50 values greater than 20 µM.14

Table 3.
Activity of thienothiophene-benzimidazole scaffold analogs
G1a
EC50
(nM)
G1b
EC50
(nM)
CC50
(µM)
Compd
18
HetAr
0.021
0.008
0.016
0.006
>20
>20
19
20
21
22
0.004
0.024
0.011
0.009
0.004
0.004
>20
>20
>20
23
24
25
1.3
0.006
0.006
0.025
>20
>20
>20
0.020
6.8
26
0.006
0.004
8
The thienothiophene-benzimidazole scaffold analogs discussed above generally
displayed low IV clearance and long T1/2 in rat PK experiments. Unfortunately, the low
rat oral bioavailablity observed in this series remains an area for improvement. Rat PK
details for representative compounds 16, 19, and 24 are given in Table 4.

Table 4.
Rat pharmacokinetics of selected compounds.
Compound
16
19
24
IV Rat PK
Cl (mL/min/kg)
T1/2(h)
6.2
5.6
2.8
8.1
3.5
1.6
4.7
4.6
1.6
Vss (L/kg)
PO Rat PK
AUC0-8h(µg h/mL)
0.376
0.281
0.659
T1/2 (h)
Cmax (ng/mL)
% F
14.8
0.01
7.3
17.2
0.01
8.4
16.6
0.02
8.4
In summary, the evaluation of two series of benzoxazole-containing HCV NS5A
inhibitors led to the identification of a thienothiophene-benzimidazole scaffold, which
delivered analogs exhibiting potent and balanced G1a and 1b activity. Furthermore, it
was shown that the excellent potency profiles observed with the thienothiophene scaffold
analogs could be maintained when the benzoxazoles were replaced with a range of other
heteroaromatic rings.
Acknowledgments
The authors would like to thank Mark N. Namchuk, Ann D. Kwong and Jean Bédard
for insightful discussions.
References and notes
1. For recent reviews, see: (a) Thomas, D. L. Nat. Med. 2013, 19, 850-858, (b)
Scheel, T. K. H.; Rice, C. M. Nat. Med. 2013, 19, 837-849, (c) Lavanchy, D.
Microbiol. Infect. 2011, 17, 107-115.
2. Tong, M. J.; El-Farra, N. S.; Reikes, A. R.; Co, R. L. N. Engl. J. Med. 1995, 332,
1463-1466.
3. Lemon, S. M.; McKeating, J. A.; Pietschmann, T.; Frick, D. N.; Glenn, J. S.;
Tellinghuisen, T. L.; Symons, J.; Furman, P. A. Antiviral Res. 2010, 86, 79-92.
4. Aghemo, A.; De Francesco, R. Hepatology 2013, 58, 428-438.
5. Asselah, T.; Marcellin, P. Liver Int. 2013, 33 (Suppl. 1), 93-104.

6. Guedj, J.; Dahari, H.; Rong, L.; Sansone, N. D.; Nettles, R. E.; Cotler, S. J.;
Layden, T. J.; Uprichard, S. L.; Perelson, A. S. Proc. Natl. Acad. Sci. U.S.A.
2013, 110, 3991-3996.
7. (a) Reghellin, V.; Donnici, L.; Fenu, S.; Berno, V.; Calabrese, V.; Pagani, M.;
Abrignani, S.; Peri, F.; De Francesco, R.; Neddermann, P. Antimicrob. Agents
Chemother. 2014, doi:10.1128/AAC.03293-14; (b) Mani, N.; Yuzhakov, A.;
Yuzhakov, O.; Coll, J. T.; Black, J.; Saxena, K.; Fulghum, J. R.; Lippke, J. A.;
Govinda Rao, B.; Rijnbrand, R.; Kwong, A. D. J. Virol. 2014, 88, In Press
8. 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.; Hamman, L.G. Nature 2010, 465, 96-100.
9. Belema, M.; Lopez, O. D.; Bender, J. A.; Romine, J. L.; St. Laurent, D. R.;
Langley, D. R.; Lemm, J. A.; O’Boyle, D. R., II; Sun, J.-H.; Wang, C.; Fridell, R.
A.; Meanwell, N. A. J. Med. Chem. 2014, 57, 1643-1672.
10. On October 10th, 2014, the FDA approved Gilead Sciences’ Harvoni®
(sofosbuvir and ledipasvir) for the treatment of chronic HCV genotype 1
infection.
http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm418365.htm
11. Henderson, J. A.; Maxwell, J.; Vaillancourt, L.; Morris, M.; Grey, R., Jr.; Giroux,
S.; Kong, L. C. C.; Das, S. K.; Liu, B.; Poisson, C.; Cadilhac, C.; Bubenik, M.;
Reddy, T. J.; Falardeau, G.; Yannopoulos, C.; Wang, J.; Pereira, O. Z.; Bennani,
Y. L.; Pierce, A. C.; Bhisetti, G. R.; Cottrell, K. M.; Morone, V. Patent US
8,765,731 B2
12. For details of the NS5A replicon assays used in this letter, see: Giroux, S.; Xu, J.;
Reddy, T. J.; Morris, M.; Cottrell, K. M.; Cadilhac, C.; Henderson, J. A.; Nicolas,
O.; Bilimora, D.; Denis, F.; Mani, N.; Ewing, N.; Shawgo, R.; L’Heureux, L.;
Selliah, S.; Chan, L.; Chauret, N.; Berlioz-Seux, F.; Namchuk, M. N.; Grillot, A-
L.; Bennani, Y. L.; Das, S. K.; Maxwell, J. P. ACS Med. Chem Lett. 2014, 5, 240-
243.

13. For synthetic procedures and characterization of intermediates and biologically
evaluated compounds see: Pereira, O.; Maxwell, J. P.; Bennani, Y. B. Patent WO
2011/119858A1.
14. CC50 in Huh-7 ET cells were determined using a [3H]-thymidine incorporation
assay from 12 concentrations of the test compound in duplicate, and data were
considered accurate when the CC50 of the positive control was within 40% of the
mean value.

Figure 1

Scheme 1.

Graphical abstract