Synthesis and evaluation of novel HCV replication inhibitors

Article abstract of DOI:10.1007/s11030-017-9733-z

Abstract: Direct acting antiviral agents to cure hepatitis C virus (HCV) infection has emerged as the gold standard therapy. Along with protease inhibitors, nucleoside polymerase inhibitors and non-nucleoside polymerase inhibitors, the inhibition of NS5a

Full text of DOI:10.1007/s11030-017-9733-z

Mol Divers
DOI 10.1007/s11030-017-9733-z
SHORT COMMUNICATION
Synthesis and evaluation of novel HCV replication inhibitors
David C. McGowan1 · Mourad D. Khamlichi2 · Alex De Groot1 ·
Frederik Pauwels1 · Frédéric Delouvroy1 · Kristof Van Emelen1 ·
Kenneth Simmen1 · Pierre Raboisson1
Received: 13 October 2016 / Accepted: 20 February 2017
© Springer International Publishing Switzerland 2017
Abstract Direct acting antiviral agents to cure hepatitis C
virus (HCV) infection has emerged as the gold standard
therapy. Along with protease inhibitors, nucleoside poly-
merase inhibitors and non-nucleoside polymerase inhibitors,
the inhibition of NS5a has proved to be an effective way
to treat HCV patients. Here we report on novel HCV NS5a
inhibitors which were synthesized and evaluated in the HCV
replicon assay. A series of inhibitors were formed by a
cycloaddition reaction in parallel to establish new leads and
explore the effects of unsymmetrical cap substitution. This
led to the identification of several triazoles with picomolar
potency in vitro against hepatitis C virus.
Keywords Hepatitis C · HCV NS5a · Replication inhibitor ·
Click chemistry
Introduction
Over 4 million people in the USA and 180 million world-
wide are infected with hepatitis C virus (HCV) [1,2]. Chronic
infection is the leading cause of cirrhosis and hepatocel-
lular carcinoma globally [3]. Increased survival rates and
improved clinical outcome are associated with sustained
virologic response (SVR) [4,5] which has been shown to
be achieved in clinic trials upon administration of approved
direct acting antivirals (DAA) [6]. Clinical scenarios encom-
pass the combination of medicaments to enhance the antiviral
effect resulting in faster cure rates [7]. Among the therapeu-
tic targets are the replication inhibitors effecting an RNA
binding protein which also interacts with other HCV non-
structural proteins known as HCV NS5a [8,9]. Daclatasvir is
a first-in-class HCV NS5a inhibitor that has shown a robust
decline in viral HCV RNA in patients infected with HCV
G1 [1,10,11]. This highly selective inhibitor has in vitro
potencyinthepicomolarrange. Itischaracterizedbyacentral
biphenyl scaffold further substituted at the 4,4ꢀ-positions by
2-(pyrrolidin-2-yl)-1H-imidazoles that are in turn acylated
with amino acid derived moieties, termed caps. Modeling
suggests that the biphenyl scaffold of Daclatasvir behaves
as a spacer between the substituted heterocycles [12] and
that there is evidence for an asymmetric binding to NS5a
[13]. In this communication, we describe our synthetic effort
toward novel inhibitors and their structure–activity relation-
ship (Fig. 1).
Graphical Abstract
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-017-9733-z) contains supplementary
material, which is available to authorized users.
David C. McGowan
dmcgowan@its.jnj.com
B
1
Janssen Infectious Diseases BVBA, Turnhoutseweg 30,
2340 Beerse, Belgium
2
Villapharma Research, Parque Tecnológico de Fuente Álamo,
Ctra. El Estrecho-Lobosillo, Km. 2,5- Av. Azul, 30320
Fuente Álamo de Murcia, Murcia, Spain
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Fig. 1 Approved HCV NS5a inhibitors Daclatasvir [10,11], Ombitasvir [14] and Elbasvir [15]
Design strategy
that would allow a combinatorial array of monomers with
different caps on either side leading to a rapid assembly of
unsymmetrical inhibitors (Fig. 2). Another advantage of the
triazole central scaffold is that it does not contain additional
chiral centers.
The first NS5a inhibitors were discovered by screening in
an in vitro assay for anti-HCV activity. The inhibitors later
evolved into dimeric species [16] that offered exceptional
potencyandbettergenotypecoverageandithasbeenrecently
described that unsymmetrical inhibitors could offer advan-
tages in optimizing potency and pharmacokinetics [17]. Our
goal was to discover an unsymmetrical series with high
potency that could be assembled rapidly using combinato-
rial techniques.
In our design strategy, we proposed that it would be
advantageous to design several monomeric unit permuta-
tions that could be easily fused together. We were interested
to first modify the structural motifs to afford functionalized
monomers with two different reactive handles. In principle,
the monomers containing complimentary reactive handles
could be combined together under mild conditions that would
not affect the functional groups present. These modified frag-
ments could then be linked to resemble a dimeric scaffold
The Cu(I) catalyzed Huisgen azide–alkyne 1,3-dipolar
cycloaddition was chosen because of facile access to the
functional groups required for the reactive pairs, and the mild
reactionconditionsusedtocombinethealkyneandazide. Tri-
azoles with symmetrical substitution have been previously
described as inhibitors of HCV [18] but were not assembled
via cycloaddition. Our effort was to explore the unsymmet-
rical 1,3-disubstituted triazole central core and to combine
that with the combinatorial strategy for the end groups. To
this point, three functionalized benzimidazole azides were
prepared and cross-reacted with three terminal alkyne deriva-
tives, asoutlinedinScheme1. Thesamethreecappinggroups
were used across each axis as shown in Table 1. The product
combination via click chemistry under Sharpless conditions
[19] therefore produced six products that were unsymmet-
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Fig. 2 Design strategy for novel HCV NS5a inhibitors
Scheme 1 Synthesis of the triazole series. Reagents and condi-
tions: (i) Boc proline, CDI, pyridine/DMF 45◦C then 4-nitro-1,2-
phenylenediamine, rt, 18h, HOAc, 100◦C, 1h; (ii) Pd/C, MeOH;
(iii) NaNO2, HCl, NaN3, water; (iv) DIC, DIPEA, DMA, HOBt; (v)
HCl; (vi) HATU, DIPEA, DMF; (vii) TMSCCH, PdCl2(PPh3)2, CuI,
Et3N; (viii) K2CO3, CH3OH; (ix) Na L-ascorbate, CuSO4 5H2O,
t−BuOH/water(1/1), rt, 18h
rically capped and three that bore the same cap on either
side.
pyridin-1-ylmethylene]-N-methylmethanaminium hexaflu-
orophosphate N-oxide (HATU) coupling agents in dimethy-
lacetamide (DMA) to yield the corresponding amides 4a, b,
c after flash chromatography in 70–90% yields.
The route toward the three alkyne derivatives started with
arylbromide 5 [20], subsequent deprotection under acidic
conditions (6) followed by installation of the capping groups
(a, b and c) via HATU in DMF provided 7a, b, c. Typical
Sonogashira reaction conditions produced the TMS acety-
lene derivatives, followed by deprotection of the TMS group
under basic conditions to give 9a, b, c. Combination of the
azides (4a, 4b, 4c) and the acetylenes (9a, 9b, 9c) via click
chemistry under Sharpless conditions afforded triazoles 10–
18 and their activities are shown in Table 1. The combination
provided nine compounds with sub-nanomolar potency with
the exception of 10 having a half-maximal effective concen-
tration (EC50) of 2.5 nM, bearing cap a on both sides.
Results and discussion
Formation of the aryl azides began with the reaction of
Boc proline and carbonyldiimidazole (CDI), followed by the
addition of 4-nitro-1,2-phenylenediamine to afford the pro-
tected benzimidazole 1 in 80% yield. Reduction of the nitro
group via catalytic hydrogenation led to the aniline deriva-
tive 2. Treatment of 2 with sodium nitrite in an aqueous HCl
solution generated the arylazonium salt, which was reacted
with NaN3 to give the corresponding azide with concomitant
Boc deprotection (3). The liberated amine 3 was condensed
with three caps (a, b and c) using diisopropylcarbodi-
imide (DIC) or [(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]
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Table 1 Activity of the combinatorial array of triazole products 10–18 in the HCV replicon assay
Compound
EC50 /EC90 (nM)
9b
9c
9a
10
2.5 / 5.8
11
0.04 / 0.20
12
0.17 / 0.65
4a
13
0.2 / 0.7
14
0.5 / 1.5
15
0.6 / 2.5
4b
16
0.1 / 0.6
17
0.02 / 0.07
18
0.02 / 0.07
4c
The product structures are not shown but represented in the table (10–18). The 50% cytotoxic concentration (CCx50) in Huh-7 and MT-4 cells was
greater than the highest concentration tested (>0.1 µM). Daclatasvir displayed an EC50 of 0.01 nM (reported data 0.009 0.004 nM [21]) in our
assay
This series of novel HCV inhibitors explored the combinatorial approach where fragments were combined in a cycloaddition reaction in parallel.
The combination of the valine carbamate cap c, on the benzimidazole side, with the (R)-mandelic amide cap b, on the acetylene side, identified 17
as a promising new lead. This finding was comparable with the combination of two valine carbamate caps (18). Conversely, swapping the caps on
the same triazole, thus the valine carbamate cap c, on the alkyne side, with the (R)-mandelic amide cap b, on the benzimidazole fragment, led to a
30-fold less potent analog (15). This finding indicated that compounds 17 and 18 offered favorable disposition of the capping groups for optimal
activity. This series warrants further exploration as potential anti-HCV agents
Experimental
1H: 2.5 ppm, 13C: 39.51 ppm) was used as internal standard
for calibration of spectra. The following standard abbrevia-
tions were used for signal multiplicities: singlet (s), doublet
(d), triplet (t), multiplet (m) and broad signal (br. s.). Predom-
inant forms are described. Minor signals of both tautomeric
and rotameric forms were observed as well. High-resolution
mass spectrometry was performed on a Waters Acquity®
IClass UPLC®-DAD and Xevo G2-S QTOF. The samples
were run on a Waters CSH C18 (1.7 µm, 2.1 × 50mm, at
50◦C) column, with a flow rate of 1 mL/min, using reverse
phase chromatography with a gradient from 95% A to 5%
A in 4.6 min, and held for 0.4min (A: 95% CH3COONH4
6.5 mM + 5% CH3CN, B: CH3CN). Melting points were
measured with a differential scanning calorimeter (DSC)
Mettler-Toledo DSC823e, applying a temperature gradient of
10 ◦C/minute, reaching a maximum temperature of 300 ◦C.
Compounds 10 to 18 did not display a melting point (or tem-
perature of glass transition).
Reagents and solvents were purchased from commercial
sources and used without purification. Anhydrous solvents
were obtained from Aldrich and used directly. Silica gel
chromatography was generally performed with prepacked
silica gel cartridges (from Biotage, Interchim, or Teledyne
Isco). The purity of all compounds was determined by UPLC,
conducted on Waters: Acquity® UPLC®-DAD and SQD sys-
tems, Waters HSS T3 column (1.8 µm, 2.1 × 100 mm, at
40◦ C). Themobilephaseconsistedof 10mMCH3COONH4
in 95% H2O + 5% CH3CN (A) and CH3CN (B). A gradient
program was used as follows: From 100% A to 5% A in 2.10
min, to 0% A in 0.90 min, to 5% A in 0.5 min, at a flow rate
of 0.8 mL/min. All compounds exhibited greater than 95%
purity. 1H and 13C (APT) NMR experiments were performed
at 27 ◦C on a Bruker Avance I—600 MHz. TMS or residual
solvent shift of deuterated dimethyl sulfoxide (DMSO-d6,
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Procedure for the synthesis of 2-((S)-pyrrolidin-2-yl)-6-
3.69–3.82 (m, 4 H), 3.84 - 3.88 (m, 1 H), 4.07 (br t, J = 8.3
Hz, 1 H), 5.05–5.09 (m, 1 H), 5.21 (dd, J = 8.4, 2.3 Hz, 1
H), 7.20 (t, J = 7.4 Hz, 1 H), 7.24 (d, J = 7.6 Hz, 2 H),
7.26–7.31 (m, 2 H), 7.53 (s, 1 H), 7.65 (d, J = 8.5 Hz, 1 H),
7.73–7.76 (m, 1 H), 7.82 (br d, J = 7.9 Hz, 2 H), 7.88–7.92
(m, 2 H), 8.05–8.08 (m, 1 H), 9.18–9.23 (m, 1 H), 11.79 (br
s, 1 H), 12.43–12.62 (m, 1 H). 13C NMR (DMSO-d6) δ 18.6,
19.0, 24.3, 29.9, 31.0, 31.3, 31.4, 40.9, 46.9, 47.2, 51.5, 54.4,
55.0, 55.1, 58.1, 110.3, 112.2, 112.7, 114.9, 124.6, 125.5,
126.4, 127.8, 128.2, 129.6, 131.4, 134.0, 135.0, 135.5, 138.9,
143.4, 147.3, 149.4, 156.9, 158.6, 169.5, 170.5. ESI-HRMS
(TOF) m/z: 741.3617 (Calcd. for C41H44N10O4[M+H]+:
741.3625).
(R)-2-Hydroxy-2-phenyl-1-((S)-2-(6-(4-(4-(2-((S)-1-(2-phen
ylacetyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)-1H-1,2,
3-triazol-1-yl)-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)et
hanone (13) White solid, 6% yield. 1H NMR (DMSO-d6)δ
ppm 1.84–2.19 (m, 8 H), 3.13–3.19 (m, 1 H), 3.57–3.62 (m,
1 H), 3.72 (s, 2 H), 3.78–3.83 (m, 1 H), 3.88–3.92 (m, 1 H),
5.09 (dd, J = 7.9, 1.9 Hz, 1 H), 5.21 (dd, J = 7.9, 2.6
Hz, 1 H), 5.32 (d, J = 6.0 Hz, 1 H), 5.47 (br d, J = 6.0
Hz, 1 H), 7.20–7.24 (m, 2 H), 7.27–7.35 (m, 4 H), 7.37–7.41
(m, 2 H), 7.41–7.44 (m, 2 H), 7.55 (d, J = 1.9 Hz, 1 H),
7.68–7.71 (m, 1 H), 7.75–7.80 (m, 1 H), 7.87 (d, J = 8.3
Hz, 2 H), 7.89–7.96 (m, 2 H), 8.07 - 8.11 (m, 1 H), 9.25 (br d,
J = 5.7 Hz, 1 H), 11.82 (br s, 1 H), 12.51–12.72 (m, 1 H).13C
NMR (DMSO-d6) δ 24.1, 24.1, 30.9, 31.2, 40.9, 46.4, 46.9,
(4-(4-(2-((S)-pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)-1
H-1,2,3-triazol-1-yl)-1H-benzo[d]imidazoles 10 to 18. An
alkyne (0.5 mmol), aryl azide (0.66 mmol), sodium L-
ascorbate (1 mmol) and copper sulfate pentahydrate (0.1
mmol) were dissolved in t-butanol:water (1:1, 2 mL). The
mixture was stirred overnight at room temperature. The sol-
vent was removed under reduced pressure and the resulting
crude material was purified via silica column chromatogra-
phy using a CH2Cl2 to CH2Cl2 : CH3OH (9:1) gradient.
2-Phenyl-1-((S)-2-(5-(4-(1-(2-((S)-1-(2-phenylacetyl)pyrro
lidin-2-yl)-1H-benzo[d]imidazol-6-yl)-1H-1,2,3-triazol-4-
yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)ethanone (10)
Yellow solid, 6% yield.1H NMR (DMSO-d6) δ ppm 1.92–
2.18 (m, 7 H), 2.26–2.32 (m, 1 H), 3.55–3.62 (m, 1 H),
3.63–3.69 (m, 1 H), 3.71–3.82 (m, 5 H), 3.88 (t, J = 7.7
Hz, 1 H), 5.09 (dd, J = 7.7, 2.2 Hz, 1 H), 5.22 (dd, J = 8.5,
2.7 Hz, 1 H), 7.19–7.33 (m, 10 H), 7.54 (d, J = 1.9 Hz, 1 H),
7.66–7.68 (m, 1 H), 7.75–7.79 (m, 1 H), 7.86 (br d, J = 8.3
Hz, 2 H), 7.89–7.97 (m, 2 H), 8.08 (s, 1 H), 9.20–9.26 (m,
1 H), 11.83 (br s, 1 H). 13C NMR (DMSO-d6)δ 24.1, 24.2,
31.2, 31.3, 40.7, 40.9, 46.9, 47.1, 54.5, 55.0, 110.2, 112.1,
112.5, 114.8, 119.6, 124.6, 125.4, 126.3, 127.7, 128.0, 128.1,
129.4, 129.5, 131.3, 133.9, 134.8, 135.3, 135.5, 139.0, 143.2,
147.2, 149.6, 158.5, 169.2, 169.4. ESI-HRMS (TOF) m/z:
702.3301 (Calcd. for C42H39N9O2[M+H]+: 702.3305).
(R)-2-Hydroxy-2-phenyl-1-((S)-2-(5-(4-(1-(2-((S)-1-(2-phen
ylacetyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-6-yl)-1H-1,2, 54.5, 55.5, 72.1, 110.3, 112.1, 112.5, 114.9, 119.6, 124.6,
3-triazol-4-yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)eth
anone (11) Yellow solid, 28% yield. 1H NMR (DMSO-d6)δ
ppm 1.80–1.86 (m, 1 H), 1.96–2.19 (m, 6 H), 2.26–2.32 (m, 1
H), 3.08–3.14 (m, 1 H), 3.63–3.69(m, 1 H), 3.73 (d, J = 15.8
Hz, 1 H), 3.79 (d, J = 15.8 Hz, 1 H), 3.81–3.85 (m, 1 H),
3.85–3.91 (m, 1 H), 5.08 (br dd, J = 7.1, 3.0 Hz, 1 H),
5.22 (br dd, J = 8.4, 2.7 Hz, 1 H), 5.27 (d, J = 6.4 Hz,
1 H), 7.15–7.20 (m, 1 H), 7.20–7.24 (m, 1 H), 7.25–7.28
(m, 2 H), 7.28–7.32 (m, 2 H), 7.36–7.40 (m, 2 H), 7.40–
7.43 (m, 2 H), 7.65–7.71 (m, 1 H), 7.72–7.79 (m, 2 H), 7.86
(br d, J = 7.8 Hz, 2 H), 7.95 (br d, J = 7.8 Hz, 2 H),
8.03–8.11 (m, 1 H), 9.22–9.28 (m, 1 H), 11.97 (br s, 1 H),
12.43–12.66 (m, 1 H). 13C NMR (DMSO-d6)δ 24.0, 24.3,
31.0, 31.3, 40.8, 46.2, 47.2, 55.0, 55.1, 72.2, 110.3, 112.2,
114.9, 119.7, 124.7, 125.5, 126.4, 127.6, 128.0, 128.2, 128.5,
129.6, 131.4, 134.1, 135.4, 139.7, 143.4, 147.3, 149.6, 158.5,
169.5, 170.8. ESI-HRMS (TOF) m/z: 718.3255 (Calcd. for
C42H39N9O3[M+H]+: 718.3254).
125.4, 126.2, 127.6, 127.8, 128.0, 128.1, 128.4, 129.4, 131.3,
133.9, 134.8, 135.5, 139.0, 139.6, 143.3, 147.3, 149.6, 158.3,
169.2, 170.9. ESI-HRMS (TOF) m/z: 718.3247 (Calcd. for
C42H39N9O3[M+H]+: 718.3254).
(R)-2-Hydroxy-1-((S)-2-(5-(4-(1-(2-((S)-1-((R)-2-hydroxy-2
-phenylacetyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-6-yl)-
1H-1,2,3-triazol-4-yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-
1-yl)-2-phenylethanone (14) Yellow solid, 9% yield. 1H
NMR (DMSO-d6) δ ppm 1.80–1.93 (m, 2 H), 1.95–2.19 (m,
6 H), 3.08–3.14 (m, 1 H), 3.14–3.19 (m, 1 H), 3.80–3.85 (m,
1 H), 3.87–3.92 (m, 1 H), 5.08 (dd, J = 7.3, 2.6 Hz, 1 H),
5.21 (dd, J = 7.9, 2.6 Hz, 1 H), 5.27 (d, J = 6.3 Hz, 1 H),
5.32 (d, J = 6.3 Hz, 1 H), 5.45 (br d, J = 6.5 Hz, 1 H), 5.47
(d, J = 6.6 Hz, 1 H), 7.31–7.35 (m, 2 H), 7.37–7.44 (m, 8
H), 7.51–7.56 (m, 1 H), 7.67–7.72 (m, 1 H), 7.74–7.80 (m,
1 H), 7.86 (br d, J = 7.3 Hz, 2 H), 7.95 (br d, J = 7.9 Hz,
2 H), 8.06–8.12 (m, 1 H), 9.25 (br s, 1 H), 11.96 (br s, 1 H),
12.57–12.76 (m, 1 H). 13C NMR (DMSO-d6) δ 24.0, 24.2,
Methyl((S)-3-methyl-1-oxo-1-((S)-2-(5-(4-(1-(2-((S)-1-(2-ph 31.0, 46.3, 46.5, 55.1, 55.6, 72.1, 72.2, 110.4, 112.2, 115.0,
enylacetyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-6-yl)-1H-1, 119.4, 119.8, 124.7, 125.6, 127.6, 127.7, 127.9, 128.0, 128.5,
2,3-triazol-4-yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)bu 128.5, 131.4, 134.0, 139.7, 139.7, 143.4, 147.3, 149.6, 158.4,
tan-2-yl)carbamate (12) Yellow solid, 14% yield. 1H NMR
(DMSO-d6)δ ppm 0.85 (br d, J = 6.6 Hz, 3 H), 0.90 (br
d, J = 6.9 Hz, 3 H), 1.91–2.04 (m, 4 H), 2.07–2.17 (m, 4
H), 2.24–2.30 (m, 1 H), 3.53 (s, 3 H), 3.62–3.67 (m, 1 H),
170.8, 171.0. ESI-HRMS (TOF) m/z: 734.3205 (Calcd. for
C42H39N9O4[M+H]+: 734.3203).
Methyl ((S)-1-((S)-2-(5-(4-(1-(2-((S)-1-((R)-2-hydroxy-2-ph
enylacetyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-6-yl)-1H-
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1,2,3-triazol-4-yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)
149.5, 156.8, 158.2, 170.6, 170.7. ESI-HRMS (TOF) m/z:
-3-methyl-1-oxobutan-2-yl)carbamate(15)Yellowsolid, 15% 757.3578 (Calcd. for C41H44N10O5[M+H]+: 757.3574).
yield. 1H NMR (DMSO-d6)δ ppm 0.86 (br d, J = 6.6 Hz,
3 H), 0.92 (br d, J = 6.3 Hz, 3 H), 1.85–1.90 (m, 1 H),
1.93–2.05 (m, 4 H), 2.09–2.19 (m, 4 H), 3.13–3.19 (m, 1 H),
3.55 (s, 3 H), 3.79–3.84 (m, 2 H), 3.87–3.92 (m, 1 H), 4.08
(br t, J = 8.3 Hz, 1 H), 5.09 (dd, J = 7.2, 3.5 Hz, 1 H), 5.21
(dd, J = 7.9, 2.6 Hz, 1 H), 5.32 (d, J = 6.2 Hz, 1 H), 5.47
(br d, J = 6.5 Hz, 1 H), 7.31–7.35 (m, 1 H), 7.39 (t, J = 7.8
Hz, 2 H), 7.41–7.44 (m, 2 H), 7.53–7.56 (m, 1 H), 7.69 (br d,
J = 8.5 Hz, 1 H), 7.74–7.79 (m, 1 H), 7.81–7.87 (m, 2 H),
7.89–7.96 (m, 2 H), 8.09 (s, 1 H), 9.20–9.28 (m, 1 H), 11.81
(br s, 1 H), 12.57–12.73 (m, 1 H). 13C NMR (DMSO-d6)δ
18.6, 19.0, 24.1, 24.3, 29.9, 31.0, 46.5, 46.9, 51.5, 54.3, 55.6,
58.1, 72.1, 110.4, 112.2, 112.7, 115.0, 119.7, 124.6, 125.5,
127.7, 128.0, 128.5, 131.4, 134.0, 135.0, 139.7, 143.3, 147.4,
149.4, 156.9, 158.4, 170.5, 171.0. ESI-HRMS (TOF) m/z:
757.3571 (Calcd. for C41H44N10O5[M + H]+: 757.3574).
Methyl ((S)-1-((S)-2-(6-(4-(4-(2-((S)-1-((S)-2-((methoxycar
bonyl)amino)-3-methylbutanoyl)pyrrolidin-2-yl)-1H-imida
zol-5-yl)phenyl)-1H-1,2,3-triazol-1-yl)-1H-benzo[d]imidaz
ol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbama
te (18) Yellow solid, 27% yield. 1H NMR (DMSO-d6) δ ppm
0.82–0.94 (m, 12 H), 1.92–2.32 (m, 10 H), 3.53–3.57 (m, 6
H), 3.79–3.92 (m, 4 H), 4.08 (br t, J = 8.4 Hz, 1 H), 4.10
(br t, J = 8.4 Hz, 1 H), 5.09 (dd, J = 7.2, 3.4 Hz, 1 H),
5.20–5.26 (m, 1 H), 7.29 (d, J = 8.5 Hz, 1 H), 7.32 (d,
J = 8.5 Hz, 1 H), 7.54 (br s, 1 H), 7.66 (br d, J = 8.4 Hz, 1
H), 7.76 (br dd, J = 8.3, 1.5 Hz, 1 H), 7.80–7.86 (m, 2 H),
7.92 (br s, 2 H), 8.06 (s, 1 H), 9.22–9.27 (m, 1 H), 11.81 (br
s, 1 H), 12.52–12.60 (m, 1 H). 13C NMR (DMSO-d6)δ 18.4,
18.5, 19.0, 19.0, 24.3, 24.5, 29.7, 29.8, 30.9, 30.9, 46.8, 47.0,
51.4, 51.4, 54.2, 54.8, 57.9, 58.0, 110.3, 112.1, 112.7, 114.8,
119.6, 124.5, 125.4, 127.8, 131.3, 134.0, 134.9, 138.8, 143.2,
Methyl((S)-3-methyl-1-oxo-1-((S)-2-(6-(4-(4-(2-((S)-1-(2-ph 147.2, 149.3, 156.8, 156.8, 158.3, 170.4, 170.6. ESI-HRMS
enylacetyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)-1H-1,
2,3-triazol-1-yl)-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)
butan-2-yl)carbamate (16) Yellow solid, 7% yield.1H NMR
(DMSO-d6)δ ppm 0.82–0.89 (m, 6 H), 1.91–2.00 (m, 2 H),
2.00–2.16 (m, 4 H), 2.20–2.30 (m, 3 H), 3.55 (s, 3 H), 3.57–
3.62 (m, 1 H), 3.72 (s, 2 H), 3.78–3.90 (m, 3 H), 4.10 (br
t, J = 8.4 Hz, 1 H), 5.07–5.10 (m, 1 H), 5.22 (br s, 1 H),
7.20–7.24 (m, 1 H), 7.25–7.33 (m, 4 H), 7.46–7.58 (m, 1 H),
7.66 (br d, J = 8.2 Hz, 1 H), 7.76 (br d, J = 8.5 Hz, 1 H),
7.82–7.88 (m, 2 H), 7.92–7.96 (m, 2 H), 8.06 (s, 1 H), 9.22–
9.27 (m, 1 H), 11.82 (br s, 1 H), 12.49–12.59 (m, 1 H). 13C
NMR (DMSO-d6)δ 18.3, 19.0, 24.1, 24.5, 29.6, 31.0, 31.3,
40.9, 47.0, 47.0, 51.5, 54.4, 54.8, 57.9, 110.2, 112.1, 114.9,
119.6, 124.6, 125.5, 128.0, 128.1, 129.4, 131.2, 133.9, 135.5,
143.0, 147.2, 149.5, 156.9, 158.3, 169.3, 170.6. ESI-HRMS
(TOF) m/z: 741.3629 (Calcd. for C41H44N10O4[M+H]+:
741.3625).
Methyl ((S)-1-((S)-2-(6-(4-(4-(2-((S)-1-((R)-2-hydroxy-2-ph
enylacetyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)-1H-1,
2,3-triazol-1-yl)-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)
-3-methyl-1-oxobutan-2-yl)carbamate(17)Yellowsolid, 23%
yield. 1H NMR (DMSO-d6) δ ppm 0.84 (br d, J = 6.4 Hz,
3 H), 0.88 (br d, J = 6.4 Hz, 3 H), 1.80–1.86 (m, 1 H),
1.92–2.11 (m, 5 H), 2.18–2.31 (m, 3 H), 3.08–3.14 (m, 1 H),
3.55 (s, 3 H), 3.79–3.91 (m, 3 H), 4.10 (t, J = 8.3 Hz, 1 H),
5.06–5.11 (m, 1 H), 5.20 - 5.24 (m, 1 H), 5.27 (br s, 1 H),
5.41–5.49 (m, 1 H), 7.31–7.35 (m, 1 H), 7.39 (t, J = 7.3
Hz, 2 H), 7.41–7.43 (m, 2 H), 7.56 (br s, 1 H), 7.65–7.73
(m, 1 H), 7.71–7.80 (m, 1 H), 7.86 (br d, J = 8.3 Hz, 2 H),
7.95 (br d, J = 7.9 Hz, 2 H), 8.02–8.10 (m, 1 H), 9.26 (br
s, 1 H), 11.98 (br s, 1 H), 12.51–12.65 (m, 1 H). 13C NMR
(DMSO-d6) δ 18.4, 19.0, 23.9, 24.5, 29.6, 30.9, 46.2, 47.0,
51.5, 54.8, 55.0, 57.9, 72.1, 110.3, 112.1, 114.8, 119.6, 124.6,
125.5, 127.5, 127.8, 128.4, 131.3, 134.0, 139.7, 143.1, 147.2,
(TOF) m/z: 780.3946 (Calcd. for C40H49N11O6[M+H]+:
780.3945).
Biological assay
The in vitro antiviral activity against HCV was tested in a
cellular HCV replicon system as previously described by
Lohmann et al. [22], with the further modifications described
by Krieger et al. [23]. The assay utilized the stably trans-
fected cell line Huh-7 luc/neo (Huh-Luc). This cell line
harbors an RNA encoding a bicistronic expression construct
comprising the wild-type NS3-NS5B regions of HCV type
1b translated from an Internal Ribosome Entry Site (IRES)
from encephalomyocarditis virus (EMCV), preceded by a
reporter portion (FfL-luciferase) and a selectable marker por-
tion (neoR, neomycine phosphotransferase). The construct
is flanked by 5ꢀ and 3ꢀ NTRs (non-translated regions) from
HCV type 1b. Continued culture of the replicon cells in
the presence of G418 (neoR) is dependent on the replica-
tion of the HCV RNA. The stably transfected replicon cells
that express HCV RNA, which replicates autonomously and
to high levels, encoding inter alia luciferase, were used for
screening the antiviral compounds. The replicon cells were
plated in 384-well plates in the presence of the test and
control compounds which were added in various concentra-
tions. Followinganincubationofthreedays, HCVreplication
was measured by assaying luciferase activity (using standard
luciferase assay substrates and reagents and a PerkinElmer
ViewLuxTM ultraHTS microplate imager). Replicon cells
in the control cultures have high luciferase expression in the
absence of any inhibitor. The inhibitory activity of the com-
pound on luciferase activity was monitored on the Huh-Luc
cells, enabling a dose–response curve for each test com-
123

Mol Divers
pound. EC50 values were then calculated, which represent
the amount of compound required to decrease the level of
detected luciferase activity by 50%, or more specifically, to
reduce the ability of the genetically linked HCV replicon
RNA to replicate. CC50 values were evaluated in Huh7.5
parental cells by the MTS assay (CellTiter 96® AQueous
One Solution Cell Proliferation kit, Promega). Cells treated
with DMSO, without compound, served as control.
NS5A replication complex inhibitors: the discovery of daclatasvir.
J Med Chem 57:2013–2032. doi:10.1021/jm401836p
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CE (2011) Targeting the NS5a protein of HCV: an emerging option.
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13. Nettles JH, Stanton RA, Broyde J, Amblard F, Zhang H, Zhou L,
Shi J, McBrayer TR, Whitaker T, Coats SJ, Kohler JJ, Schinazi
RF (2014) Asymmetric binding to NS5A by Daclatasvir (BMS-
790052) and analogs suggests two novel modes of HCV inhibition.
J Med Chem 57:10031–10043. doi:10.1021/jm501291c
14. DeGoey DA, Randolph JT, Liu D, Pratt J, Hutchins C, Donner P,
Krueger AC, Matulenko M, Patel S, Motter CE, Nelson L, Keddy
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Acknowledgements The authors would like to thank the analytical
teams at Janssen Infectious Diseases and Villapharma Research, includ-
ing Alberto Fontana for HRMS data.
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