HCV NS5A replication complex inhibitors. Part 3

Article abstract of DOI:10.1016/j.bmcl.2012.11.086

In a recent disclosure,¹ we described the discovery of dimeric, prolinamide-based NS5A replication complex inhibitors exhibiting excellent potency towards an HCV genotype 1b replicon. That disclosure dealt with the SAR exploration of the peripheral region of our lead chemotype, and herein is described the SAR uncovered from a complementary effort that focused on the central core region. From this effort, the contribution of the core region to the overall topology of the pharmacophore, primarily vector orientation and planarity, was determined, with a set of analogs exhibiting <10 nM EC ₅₀ in a genotype 1b replicon assay.

Full text of DOI:10.1016/j.bmcl.2012.11.086

Bioorganic & Medicinal Chemistry Letters 23 (2013) 779–784
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
HCV NS5A replication complex inhibitors. Part 31: discovery of potent
analogs with distinct core topologies
Omar D. Lopez a, , Van N. Nguyen a, Denis R. St. Laurent a, Makonen Belema a, Michael H. Serrano-Wu a,
⇑
Jason T. Goodrich a, Fukang Yang a, Yuping Qiu a, Amy S. Ripka a, Peter T. Nower b, Lourdes Valera b,
Mengping Liu b, Donald R. O’Boyle II b, Jin-Hua Sun b, Robert A. Fridell b, Julie A. Lemm b, Min Gao b,
Andrew C. Good c, Nicholas A. Meanwell a, Lawrence B. Snyder a
a Department of Medicinal Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA
b Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA
c Department of Computer-Aided Drug Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA
a r t i c l e i n f o
a b s t r a c t
In a recent disclosure,1 we described the discovery of dimeric, prolinamide-based NS5A replication com-
plex inhibitors exhibiting excellent potency towards an HCV genotype 1b replicon. That disclosure dealt
with the SAR exploration of the peripheral region of our lead chemotype, and herein is described the SAR
uncovered from a complementary effort that focused on the central core region. From this effort, the
contribution of the core region to the overall topology of the pharmacophore, primarily vector orientation
and planarity, was determined, with a set of analogs exhibiting <10 nM EC50 in a genotype 1b replicon
assay.
Article history:
Received 3 October 2012
Revised 14 November 2012
Accepted 20 November 2012
Available online 3 December 2012
Keywords:
HCV
NS5A inhibitors
Stilbene replacement
Antivirals
Ó 2012 Elsevier Ltd. All rights reserved.
Daclatasvir
Hepatitis C virus (HCV) infection is a worldwide health problem
that affects an estimated 170 million individuals and more than 4
million Americans.2 In about 80% of the patients, the disease pro-
gresses to a chronic state that in many cases is asymptomatic,
but without treatment places the patients at risk of developing
more serious liver disease. Liver failure resulting from chronic
HCV infection is now considered to be the leading cause of liver
transplantations in the US.2 HCV is an exceptionally heterogeneous
virus and has been classified into six major genotypes and with a
total of over 100 subtypes.3 This, coupled with the high replication
rate of the virus and its error-prone replicase enzyme, provide pos-
sible explanations for the lack of protective immunity from HCV
infection.4 For many years, the standard of care has been a combi-
agents (DAAs) have shown the potential for eliminating interferon
from current therapies and, therefore, distinct classes of inhibitors
with orthogonal resistance profiles are currently being explored in
an effort to identify the optimal compounds for use in combination
therapy.7
NS5A appears to play an important function in regulating HCV
replication and, although its precise role is largely unknown, it
has been implicated in HCV resistance to IFN-
a
therapy.8 The
NS5A protein exists in multiple phosphorylation states, designated
p56 (basally phosphorylated) and p58 (hyperphosphorylated), and
though some NS5A adaptive mutations affect the phosphorylation
state of NS5A in HCV replicons, a definitive understanding of the
role of NS5A phosphorylation in replication has not been estab-
lished.9 NS5A is comprised of three domains and the crystal struc-
ture of the well-conserved amino-terminal domain I has been
determined by two independent groups.10 Both structures are that
of a dimer, but their mode of dimerization is different, a phenom-
enon of unknown consequence. However, biochemical studies
have provided further evidence for the formation of dimers, there-
by corroborating the crystallographic data.11
nation of pegylated interferon-a (IFN-a) and ribavarin. This treat-
ment is not only poorly tolerated but also less effective toward
genotype 1, the most prevalent strain in Europe, the USA and Asia,
with cure rates of only 40–50%.5
The recent addition of protease inhibitors to this regimen
greatly improved the response rates (67–75% among genotype 1
treatment-naïve patients) although the tolerability of these thera-
pies is less than desirable.6 Combinations of direct acting antiviral
Clinical proof-of-concept of NS5A as a viable target was ob-
tained with the potent HCV inhibitor daclatasvir (1).12 Herein we
describe the initial investigation of the core region of the stilbene
lead (2),13 showing that subtle changes to planarity, vectorial
⇑
Corresponding author. Tel.: +1 203 677 6003; fax: +1 203 677 77200.
E-mail address: omar.lopez@bms.com (O.D. Lopez).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.086

780
O. D. Lopez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 779–784
Table 1
orientation and polarity in this part of the molecule contribute in
an important way to the ability of these molecules to inhibit HCV
replication.
Activity of phenylacetamide analogs
O
O
O
O
R
N
N
H
N
O
O
N
N
O
N
H
N
H
NH
N
H
HN
O
N
N
O
O
a,b
1 (daclatasvir)
R
Compound
1b EC50 (nM)
O
2
0.09
12.4
O
NH
N
HN
O
N
O
2
3
During sample handling, it was observed that 2 was susceptible
to cis/trans isomerization when stored in DMSO under ambient
light. Although the exact cis/trans composition under the assay
conditions was not monitored, an independent synthesis and pro-
filing of the cis isomer 3 revealed a 140-fold reduction in potency,
suggesting a strong preference for the trans isomer (Table 1).14 In
an effort to avoid isomerization issues, we prepared a series of ole-
fin isosteres of fixed geometries that sought to approximate both
the vectorial orientation and molecular length of 2 and 3 (see
examples 4–12 in Table 1). From this series, the trans-cyclopropyl
derivative 4, although weaker than the stilbene analog 2, was 140-
fold more potent than its cis-cyclopropyl counterpart (5), consis-
tent with the relative potency observed between the cis and
trans-stilbenes 2 and 3. Replacement of the trans olefin with an
amide linkage yielded compound 6, with an EC50 of 11 nM, N-
methylation (7) of which resulted in a significant potency loss
(568-fold). These results agree with structural studies by Shudo
et al. that show that unsubstituted benzanilides prefer to exist in
a transoid conformation, whereas N-methyl benzanilides exist in
H
H
4c
5.66
H
H
5
6
796
O
N
11.0
H
O
CH3
N
7
6250
8
1.74
13.9
N
N
a predominantly cisoid conformation, stabilized by favorable
p–p
interactions between the phenyl rings.15,16
To further probe the association of the trans geometry with en-
hanced potency, conformationally restricted analogs 8 and 9 that
could mimic the trans disposition of stilbene 2 were prepared,
and indeed, both demonstrated good inhibitory potency in the
genotype 1b replicon. Moreover, the introduction of polarity into
the linker region offered a means to improve the pharmaceutics
properties of the lead series if, for example, solubility were to be-
come an issue.1,17
The more flexible bibenzyl analog 11 exhibited a 67-fold po-
tency loss in comparison to 2 while the ether-linked analog 12 suf-
fered further potency erosion when compared to its all-carbon
counterpart 11. The potency disparity between 11 and 12 was sur-
prising in light of the tolerance for polarity observed with 6. Anal-
ysis of X-ray data previously reported in the literature of relevant
core structural fragments suggests that the biaryl rings in 2, 11
and 12 may favor different conformational topologies.18 The ero-
sion in potency may be explained by a preference of the aromatic
rings to be coplanar as in 2 or in parallel planes, as in 11 (Fig. 1).
However, ether 12 has a preferred conformation where the aro-
matic rings are in orthogonal planes, likely resulting in a different
topology of the peripheral pharmacophoric elements, particularly
the amide NH, as compared to 2 and 11. It is noteworthy that al-
kyne 13, where overall planarity is restored, exhibits an EC50 of
6.24 nM.
9c
N
N
CH3
10
11
1050
6.01
N
H
12
13
97.8
6.24
O
Genotype-1b CC50 and BVDV EC50 >104 nM in all cases.
Values are means of at least two experiments.
Mixture of trans-diastereomers.
a
b
c
sought to explore this more fully by examining 1,2-disubstituted,
five-membered heterocyclic linkers (see Table 2, examples 14–
16). These analogs were found to possess better potency than 5
and 7 and analyses of X-ray data of relevant core structural frag-
ments of these analogs reported in the literature further corrobo-
rates the preference for planarity in this region. Indeed, these
data reveal a difference in preferred topology in analogs containing
a cis-cyclopropyl linker (5) or an N-methyl amide linker (7) in
The reduced inhibitory activity associated with the cis-disposed
aryl core moieties, as in 5 and 7, suggested that this configuration
was less preferred. In addition, the reduced planarity across this re-
gion of the linker may also be a contributing factor. Therefore, we

O. D. Lopez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 779–784
781
Table 3
Activity of shortened analogs
H
H
HN
HN
N
H
NH
H H
2
11
O
O
O
R
H
O
H
HN
N
N
O
N
H
N
N
H
H
12
Figure 1. Relative disposition of the phenyl rings in analogs 2, 11 and 12.
a,b
R
Compound
1b EC50 (nM)
17
18
1700
1990
O
O
Table 2
Activity of 1,2-heterocyclic analogs
19
20
3950
638
O
O
O
R
O
N
N
N
N
H
H
Genotype-1b CC50 and BVDV EC50 >104 nM in all cases.
Values are means of at least two experiments.
a
b
a,b
R
Compound
1b EC50 (nM)
HN
N
N
14
20.9
ken in aggregate, these results suggest that replacing the alkene
moiety with a simple methylene, as in 17, is significantly detri-
mental to potency. Similar results were observed with polar vari-
ants 18 and 19, as well as with the lipophilic dimethyl
homologue 20, and were consistent with a preferred linear topol-
ogy of the pharmacophore.21
N
HN
N
15
16
8.18
4.57
H
N
Results from an examination of longer central linkers are illus-
trated in Table 4. Extending the two carbon atom linker of 11
(EC50 = 6 nM) by one carbon was detrimental to inhibitory potency
(21, EC50 = 36 nM), although the level of potency of 21 suggests
that some flexibility in length is tolerated. While introducing con-
formational constraints and overt basicity, as in 22 and 23, was
detrimental to potency, a subset of compounds with more rigid
and planar linkers exhibited enhanced potency. Two analogs, 25
and 26, had EC50s <10 nM while exhibiting some tolerance for
polarity. Certain facets of the SAR that emerged, for example, the
potency disparity between analogs 24 and 25, and the poor po-
tency observed with the benzimidazole 10 are not well under-
stood. However, the 180-fold potency spread between pyrazoles
29 and 30 may be due to steric effects associated with the N-
methyl group distorting the adjacent phenyl group out of planarity,
thereby affecting the relative disposition of the peripheral pharma-
a
Genotype-1b CC50 and BVDV EC50 >104 nM in all cases.
Values are means of at least two experiments.
b
which both phenyl rings are facing each other presenting ring
planes that are orthogonal to that of the linker.15,19 On the other
hand, when the linker is a Z alkene (e.g., 3) or a 1,2-disubstituted
five membered ring (e.g., 14), one phenyl ring is coplanar with
the linker while the other ring adopts an orthogonal plane
(Fig. 2).20 These topographical differences will affect the presenta-
tion of the pharmacophoric elements to NS5A and may provide an
explanation for the observed variations in potency.
While the topological disposition observed in 2 is preferred, the
optimal distance between the peripheral moieties was not known.
Our attention was therefore directed towards examining this as-
pect, and the outcome of this investigation is shown in Table 3. Ta-
20,22
cophoric elements.
In general, it appears that the impact on potency stemming
from changes to the central core of the pharmacophore is related
to a combination of molecular length, planarity, and polarity. In
addition, it is noteworthy that there was not a considerable differ-
ence in potency when the phenyl rings were attached in a 1-2 or in
a 1-3 fashion to the heterocycle. For example, 3,4-substituted pyr-
azole 16 and 3,5-substituted pyrazole 29 had similar potency in
spite of the significantly different vectorial orientation of the two
molecules. While the more potent inhibitors indicate a preference
for planarity in the central linker region, molecules with a remark-
ably diverse set of topologies exhibit nearly comparable levels of
inhibitory activity, as indicated by analogs 4, 16 and 25. The struc-
tural basis for these observations remains enigmatic and may only
be illuminated by more detailed mechanistic studies or the co-
crystallization of an inhibitor-NS5A complex in order to be fully
appreciated.
O
H
H
N
HN
HN
HN
HN
7
5
NH
N
H
H
Notably, almost all of these analogs of 2 exhibited weak potency
toward a genotype 1a replicon (EC50 = 1–10 lM), with the excep-
HN
HN
HN
HN
tion of compound 25 which showed modest levels of inhibition,
with an EC50 of 345 nM. All compounds were also tested for target
specificity by evaluation in a bovine viral diarrhea virus (BVDV)
14
Figure 2. Proposed relative disposition of the phenyl rings in analogs 3, 5, 7 and 14.
3

782
O. D. Lopez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 779–784
Table 4
Activity of elongated analogs
H
N
H
N
i
NH2
H2N
N
N
X
X
Boc
Boc
O
O
O
O
O
O
31
32
R
N
N
N
H
N
H
ii
H
N
H
N
a,b
R
Compound
1b EC50 (nM)
N
N
X
O
O
R
R
O
O
21
36.1
159
2-30
N
Scheme 1. Reagents and conditions: (i) Boc-
L-proline, EEDQ, CH2Cl2; (ii) (a) 4 N HCl,
22
23
dioxane; (b) RCOOH, EDCI, DIEA, THF.
3890
N
i
H2N
NH2
N
N
N
H2N
NH2
24
25
149
O
33
31a
Scheme 2. Reagents and conditions: (i) Lindlar catalyst, H2 (1 atm), EtOAc.
2.38
O
Br
H2N
O
26
27
28
1.32
14.7
67.2
N
i
ii, iii
S
H2N
Br
NH
trans-34
34
trans-31b
trans-35
35
31b
cis-
cis-
cis-
N
Scheme 3. Reagents and conditions: (i) NBS, DME, H2O; (ii) LiHMDS, Pd2dba3,
2-dicyclohexylphosphophinobiphenyl; (iii) 1 N HCl.
R
N
N
29 R = H
30 R = Me
0.71
181
i
O2N
NO2
a
Genotype-1b CC50 and BVDV EC50 >104 nM in all cases.
Values are means of at least two experiments.
H2N
NH2
36
31c
b
Scheme 4. Reagents and conditions: (i) SnCl2ꢀ2H2O, EtOH, 70 °C; (ii) H2 (1 atm), Pd/
replicon as well as for cellular toxicity in genotype 1b replicons
and, in all cases, EC50 or CC50 values remained over 10 M.
Compounds 2–30 were synthesized in a straightforward man-
ner by coupling the respective bis-anilines 31 with Boc- -proline.
After deprotection of the Boc group in acidic conditions, the prod-
ucts were coupled with phenylacetic acid under standard condi-
tions and purified by reverse phase preparatory HPLC
(Scheme 1). The bis-aniline precursors of 2, 3, 11, 12, 13, 18, 19
and 20 were obtained from commercial sources while 31a was de-
rived from 4,40-(ethyne-1,2-diyl)dianiline (Scheme 2).
Cyclopropyl derivatives trans-31b and cis-31b (Scheme 3) were
obtained from the trans and cis stilbene, respectively, by bromina-
tion and palladium-mediated amination (for trans-34, the resulting
racemic mixture was used for the synthesis of final compound 4).
Bis-aniline 31c was obtained via direct reduction of bis(4-nitro-
phenyl)methane by standard methods (Scheme 4), while 4-ami-
no-N-(4-aminophenyl)-N-methylbenzamide 31d was readily
obtained by standard methods as shown in Scheme 5.
Precursor 31e was prepared as follows: Heck coupling of aryl
bromide 38 and styrene derivative 39 afforded carbamate 40
(Scheme 6). After deprotection of the primary amine, intermediate
41 was cyclized by heating under microwave irradiation to afford
the desired tetrahydro-isoquinoline 42. Methylation followed by
C, MeOH.
l
hydrogenation afforded racemic 31e. Intermediates 33f, 33g and
33h were obtained by direct coupling of tetrahydro-isoquinolines
44a–b with their corresponding bromides, followed by reduction
of the nitro groups.
Condensation of 48 and 49 followed by reduction, readily affor-
ded benzimidazole 31i (Scheme 7).
The heterocyclic-linked templates 31j–q were synthesized fol-
lowing well-known procedures in the literature, as exemplified
in Scheme 8.23
In summary, a series of new cores have been identified as suit-
able replacements for the alkene moiety present in the original stil-
bene-based lead series of HCV NS5A inhibitors represented by 2.
Several linkers that explored aspects of varied geometry, length
and polarity were prepared, with many exhibiting excellent po-
tency against a genotype 1b HCV replicon. Moreover, these com-
pounds exhibit minimal activity in a BVDV replicon inhibition
assay, indicating good virus specificity, and possess an excellent
therapeutic index. These results demonstrate the flexibility of the
binding site on NS5A as well as a preference for an overall planar
topography while accommodating a diverse set of linker elements.
In the particular case of example 25, modest potency towards a
L

O. D. Lopez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 779–784
783
O
NH2
O
O
NH2
NH2
i, ii
i
Cl
H
N
CH3
+
H2N
NO2
O2N
H2N
48
49
31d
37
Scheme 5. Reagents and conditions: (i) N-methyl-4-nitroaniline, THF; (ii) H2
(1 atm), Pd/C, MeOH.
N
ii
N
NH2
NO2
N
H
H2N
N
H
H2N
H
Br
i
N
31i
50
Cbz
H
N
+
Boc
O2N
Scheme 7. Reagents and conditions: (i) N-methylmorpholine, MeOH; (ii) H2
39
38
(1 atm), 10% Pd/C, EtOAc.
Cbz
NH
Cbz
NH
H2N
NO2
ii
iii
v
H
N
N
i
H
N
ii, iii
N
NH2
O2N
Boc
O2N
O2N
40
41
Cbz
NH
O2N
Cbz
H2N
51
52
iv
31j
NH
iv
N N
N NH
N
O2N
NH2
CH3
NH
42
O2N
54a
43
53
v, iii
Bu3Sn
SnBu3
S
S
NH2
NH2
H2N
H2N
31k
55
N
H2N
CH3
Z
Z
Y
Y
31e
vi, iii
X
X
NO2
vi
vii
NH
31l
X = CH, Y = N, Z = NCH3
N
N
54a X = CH, Y = N, Z = NCH3
54b X = CH, Y = N, Z = NH
31m X = CH, Y = N, Z = NH
31n X = NH, Y = N, Z = CH
O2N
54c
O2N
X = NH, Y = N, Z = CH
31o
X = O, Y = CH, Z = N
54d X = O, Y = CH, Z = N
45
NH2
44a
NH
N
N
NH
vi, iii
N
N
H2N
31f
NH2
H2N
56
31p
R1
R1
R2
viii
NH
Br
N
viii
vii
O2N
+
R2
O2N
1
1
R = NO2, R2 = H
R = NO2, R2 = H
44a
44b
46a
46b
57
58
H
N
1
R
= H, R2 = NO2
R
1 = H, R2 = NO2
N
N
NO2
ix, iii
R1
R1
R2
R1
R2
R1
R2
vi
N
N
R2
O
59
NH2
H2N
O2N
1 = H, R2 = NH2
1 = NH2, R2 = H
31q
R
R
1 = H, R2 = NO2
1 = NO2, R2 = H
31g
31h
R
R
47a
47b
Scheme 8. Reagents and conditions: (i) 4-iodo-nitrobenzene, CuI, Pd(PPh3)Cl2,
K2CO3, Bu3N, H2O; (ii) NaN3, DMF, 200 °C, microwave; (iii) H2, Pd(OH)2, MeOH; (iv)
NaH, MeI, DMF; (v) Pd(PPh3)2Cl2, 1-bromo-4-nitrobenzene, reflux; (vi) fuming nitric
acid, 0 °C; (vii) Bredereck reagent, 100 °C; (viii) p-nitrobenzoylchloride, DIEA, THF;
(ix) NH2NH2ꢀH2O, CH2Cl2.
Scheme 6. Reagents and conditions: (i) Pd(OAc)2, Ph2P(t-Bu)2, NaOAc, DMF, 100 °C;
(ii) 10% TFA/CH2Cl2; (iii) microwave, 160 °C, 4 h, EtOH; (iv) formaldehyde, formic
acid, ClCH2CH2Cl, 70 °C; (v) H2 (1 atm), 10% Pd/C, EtOAc; (vi) 2-chloro-5-nitropyr-
idine, Et3N, EtOH; (vii) Raney nickel, NH2NH2ꢀH2O, THF/H2O; (viii) Et3N, CH2Cl2.
Acknowledgments
genotype 1a replicon was observed. The subsequent SAR investiga-
tion effort that enhanced the inhibitory potency towards genotype
1a, a prelude to the discovery of dacalatasvir (1), will be the subject
of future communications.
We thank Discovery Analytical Services (DAS) staff for obtaining
HRMS and 1H NMR data and Dawn Parker for solubility
measurements.

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O. D. Lopez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 779–784
15. (a) Itai, A.; Toriumi, Y.; Saito, S.; Kagechika, H.; Shudo, K. J. Am. Chem. Soc. 1992,
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14. Even though the cis/trans isomer composition was not monitored during
assaying, the difference in potency observed suggests that an equilibrium state
was not achieved under the assay conditions.