Discovery of potent macrocyclic HCV NS5A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.05.042

HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Merck's effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure.

Full text of DOI:10.1016/j.bmcl.2016.05.042

Accepted Manuscript
Discovery of potent macrocyclic HCV NS5A inhibitors
Wensheng Yu, Bancha Vibulbhan, Stuart B. Rosenblum, Gregory S. Martin, A.
Samuel Vellekoop, Christian L. Holst, Craig A. Coburn, Michael Wong, Oleg
Selyutin, Tao Ji, Bin Zhong, Bin Hu, Lei Chen, Michael P. Dwyer, Yueheng
Jiang, Anilkumar G. Nair, Ling Tong, Qingbei Zeng, Sony Agrawal, Donna
Carr, Laura Rokosz, Rong Liu, Stephanie Curry, Patricia McMonagle, Paul
Ingravallo, Fred Lahser, Ernest Asante-Appiah, James Fells, Joseph A.
Kozlowski
PII:
S0960-894X(16)30528-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.042
BMCL 23901
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 April 2016
13 May 2016
14 May 2016
Please cite this article as: Yu, W., Vibulbhan, B., Rosenblum, S.B., Martin, G.S., Samuel Vellekoop, A., Holst, C.L.,
Coburn, C.A., Wong, M., Selyutin, O., Ji, T., Zhong, B., Hu, B., Chen, L., Dwyer, M.P., Jiang, Y., Nair, A.G., Tong,
L., Zeng, Q., Agrawal, S., Carr, D., Rokosz, L., Liu, R., Curry, S., McMonagle, P., Ingravallo, P., Lahser, F., Asante-
Appiah, E., Fells, J., Kozlowski, J.A., Discovery of potent macrocyclic HCV NS5A inhibitors, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.042
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Discovery of potent macrocyclic HCV NS5A inhibitors
Wensheng Yua,*, Bancha Vibulbhana, Stuart B. Rosenbluma, Gregory S. Martinb, A. Samuel Vellekoopb, Christian L. Holstb,
Craig A. Coburnc, Michael Wonga, Oleg Selyutina, Tao Jid, Bin Zhongd, Bin Hud, Lei Chena, Michael P. Dwyera, Yueheng
Jianga, Anilkumar G. Naira, Ling Tonga, Qingbei Zenga, Sony Agrawale, Donna Carre, Laura Rokosze, Rong Liuf, Stephanie
Curryf, Patricia McMonaglef, Paul Ingravallof, Fred Lahserf, Ernest Asante-Appiahf, James Fellsg, Joseph A. Kozlowskia
aDepartment of Medicinal Chemistry, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway NJ 07065, USA
bAlbany Molecular Research Inc., 26 Corporate Circle, Albany, NY 12203, USA
cDepartment of Medicinal Chemistry, Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA
dWuXi AppTec, 288 Fute Zhong Road, Shanghai, 200131, China
eDepartment of In Vitro Pharmacology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
fDepartment of Infectious Diseases, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
gDepartment of Structural Chemistry, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway NJ 07065, USA
This is where the receipt/accepted dates will go; Received Month XX, 2015; Accepted Month XX, 2015 [BMCL RECEIPT]
Abstract—HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA
titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the
treatment of HCV infection. Merck’s effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe
the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure. [BMCL ABSTRACT] ©2000
Elsevier Science Ltd. All rights reserved.
The World Health Organization (WHO) estimates that approximately 3% of the world's
population are infected with Hepatitis C virus (HCV).1 Consequently, HCV has become the leading
cause of chronic liver disease and liver transplants in the developed world. The diversity in the virus is
impressive with seven major genotypes (GT1-7) present in the population, with multiple subtypes (a, b,
c, etc.) identified for majority of them. However, currently ~90% of all HCV infections can be
associated with genotypes 1, 2, and 3. Interferon-based standard-of-care (SOC) therapy for chronic
hepatitis C infection was limited by both efficacy and tolerability.2 Adding HCV protease inhibitors,
such as Telaprevir or Boceprevir, to the therapy improved response rates for genotype 1 patients,3 but
unfortunately left many patients without a cure, leaving open a need for improved treatment regimens.
Current HCV therapy consists of interferon-free, direct-acting antiviral agents (DAAs), with reduced
overall treatment durations, broader coverage of HCV patient sub-populations and increased rates of
sustained virologic response (SVR). Typically, treatment in the clinic involves use of two or more
DAAs, each targeting a different mechanism of action in the life cycle of HCV. SVR rates have been
improved with this approach and it continues to be an important strategy for improving clinical
outcomes. 4,5
The HCV NS5A protein is a multifunctional RNA binding protein essential for HCV replication.6
NS5A encodes no known enzymatic function but has been shown to be important at various stages of the
HCV life cycle, including involvement in both viral replication and virion assembly.6 Over the past
decade, NS5A has become an attractive pharmacological target and inhibitors of this protein have
become an integral part of fixed-dose combination (FDC) regimens.7 To date, four NS5A inhibitors are
available for use in the treatment of chronic HCV infection. 8~12
Our early work in this area identified MK-4882 (Figure 1) as a potent NS5A inhibitor of GT1.11
Continued efforts in the development of NS5A inhibitors led to MK-8325, a unique silicon containing
analog with an improved PK profile13 (Figure 1). While MK-8325 demonstrated robust potency,
combined with an improved rat PK profile,13 we sought to determine if it was possible to identify

additional HCV NS5A inhibitors with improved potency against resistance-associated variants (RAV’s),
such as GT1a mutant Y93H.
Figure 1. MK-4882 and MK-8325 and their in vitro potency profiles13
Genotypes, EC50 (nM)
1a
1b
2a
3a
1a Y93H
MK-4882
MK-8325
0.01
0.01
0.001
0.003
0.4
0.002
4
0.4
24
47
Early in the development of our NS5A inhibitors, we observed that the amino acid moiety
attached to each of the proline rings was a necessary component for potent activity. For discussion
purposes we designate these as the “caps”. Over recent years, much effort has been devoted to exploring
different amino acid residues, aiming to determine what is tolerated as well as to improve the virologic
profile of the inhibitors. These efforts included constraining the conformation of amino acid moiety by
macrocycle formation. For example, Qiu and coworkers prepared macrocyclic NS5A inhibitors where
the amino acid cap was connected to either the imidazole moiety, or the core of the molecule.14~17 In
addition, NS5A inhibitors which used a macrocycle to replace both the methoxycarbonyl-L-valine cap
(Moc-Val) and the L-proline were reported.18 In a different effort, Or and coworkers attached
cyclosporine-type of macrocycle to the amino acid cap.19 While only GT1a or GT1b data was reported
for these molecules, the analogs did show reasonable potency, suggesting that more changes might be
tolerated. So far we have not noticed reports of macrocyclic NS5A inhibitors bearing a linkage between
the amino acid residue and the L-proline. Thus, we decided to selectively prepare some macrocyclic
NS5A inhibitors in the MK-8325 and MK-4882 scaffolds. For simplicity we chose to prepare MK-8325
analogs with unsubstituted L-proline, as it would speed the synthesis and will provide similar in vitro
data to the silicon containing analogs, as indicated by compound 1 (Table 1) which showed a similar
potency profile to MK-8325.13
In the absence of a crystal structure of the bioactive conformation of compound 1, we conducted
conformational overlays which indicated that cyclizing the Moc-Val moiety back to either the core
(northern cyclization) or the proline (southern cyclization) would mimic 1, without imparting an energy
penalty (Figure 2).20 For the northern cyclization, the C3’ position of the 7-phenyl-1H-naphtho[1,2-
d]imidazole core seemed to be the optimal cyclization point. Molecular modeling of this option
indicated a 19-member ring (2) could overlay reasonably well with compound 1 (Figure 2). For the
southern cyclization, we identified two modes of cyclization between the L-valine and proline. In the
first mode, the L-valine could cyclize back to the C5 position of the proline, via a carbonyl, to form a six
member ring (3). In the second mode, modeling predicted a 15-membered ring (4) to be ideal to link
between the L-valine and the proline C4 position (Figure 2). Based upon the modeling, it seemed that at
the proline connection, an “S” configuration of C5 in compound 3 and a “R” configuration of C4 in
compound 4 would be preferred since these configurations would deliver the rings away from the core.
While these constrained NS5A inhibitors were intriguing, it was important at the outset of this effort to
establish whether one could substitute the core, proline, and cap regions of compound 1 and retain a
reasonable virologic potency.

Figure 2. Macrocycle strategy based on compound 1. Overlays of 1 (purple) and 2 (blue), or 3 (yellow), or 4 (green).
Initial SAR focused on exploring substitution on the core of compound 1 to aid in defining if our
targeted position for macrocycle construction would be tolerated. The synthetic route for substituting the
core of compound 1 is shown in Scheme 1. Acid 5 was converted to the corresponding amine
intermediate, followed by protection of the amine with an acetyl group to give acetamide 6. Nitration of
6 using HNO3/AcOH afforded 7 which was hydrolyzed followed by reduction of the nitro group to
afford diamine 8. A N-Boc-L-proline was introduced via HATU-mediated amide coupling to give
amide 9, which was subjected to imidazole formation using acid to afford 10. Bromide 10 was
converted to the pinacol boronate ester 11 under Pd-coupling conditions. 3-Bromophenol (12) was
treated with acetic anhydride to give acylated compound 13 which underwent an AlCl3-promoted
rearrangement to give methyl ketone 14. The hydroxyl group of 14 was methylated under MeI/K2CO3
condition to give compound 15. Bromination of 15 followed by introduction of N-Ac-L-proline afforded
16 which was treated with NH4Ac to give imidazole 17. Demethylation of 17 under AlBr3/EtSH
conditions followed by deacetylation in acidic condition gave phenol 18. The Moc-Val group was
introduced under HATU coupling condition to give bromide 19 which was ready to couple with boronate
11. The coupling product of 19 and 11 was treated with acid to remove the Boc group and followed by
HATU coupling with Moc-Val to give the final compound 20. Other 3’-substituted derivatives (21 and
22) were prepared by the route shown in Scheme 1 except substituting the appropriately substituted
acetophenone for compound 14. In addition, the 3’-hydroxyl group of compound 20 could also be
functionalized (23,Table 1).

.
Scheme 1. Preparation of compound 20
Conditions: (a) (PhO)2P(O)N3, Et3N, t-BuOH, reflux, 15h; (b) HCl, MeOH, rt, 15h, 91% for two steps; (c) Acetic anhydride, Et3N, DCM, rt, 1.5h, 79%; (d)
HNO3, AcOH, 0 oC to rt, 6.5h, 83%; (e) HCl, MeOH, 75 oC, 6h, 95%; (f) Fe, NH4Cl, THF, water, 60 oC, 3h, 95%; (g) N-Boc-L-proline, HATU, DIPEA,
DMF, rt, 9h, 90%; (h) AcOH, 60 oC, 1h, 100%; (i) bis(pinacolato)diboron, Pd(dba)2, (c-hexyl)3P, KOAc, dioxane, 100oC, 4h, 82%; (j) Ac2O, pyridine, DCM;
(k) AlCl3, 160 oC; (l) MeI, K2CO3, DMF, rt, 91%; (m) CuBr2, EtOAc, reflux, 73%; (n) N-Ac-L-proline, Et3N, EtOAc, rt, 80%; (o) NH4OAc, xylene, 100 oC,
64%; (p) AlBr3, EtSH, 0 oC, 49%; (q) HCl (6N), 90 oC, 93%; (r) (methoxycarbonyl)-L-valine, HATU, DIPEA, DMF, rt, 100%; (s) 11, Pd(dppf)Cl2, K2CO3,
DME, H2O, 90oC, 74%; (t) TFA, DCM; (u) (methoxycarbonyl)-L-valine, HATU, DIPEA, DMF, rt, 49% for two steps.
The in vitro virologic profiles of compounds bearing C3’ substitutions on the core are
summarized in Table 1. Introduction of a hydroxyl group at the C3’ position was tolerated as compound
20 was similar in activity to compound 1 except a 10x loss in GT2a potency. Similar results were
observed for the corresponding methyl (21) and cyclopropyl (22) substituted analogs. However, larger
groups, such as the 2-morpholinoethoxy group found in 23, hurt the potency across the genotypes tested.
It was encouraging that substitution was tolerated at the C3’ position for this would be a critical
attachment point for the northern cyclization macrocycles.
Table 1. In vitro potency profile of compounds 1, 20~2321
Genotypes, EC50 (nM)a
ID
1
20
21
22
R
H
OH
Me
cPr
1a
1b
2a
0.02
0.2
0.09
0.2
1a Y93H
119
201
nt
0.01
0.08
0.02
0.1
0.004
0.004
0.002
0.002
nt
23
>1
2
>10
1339
a nt = not tested.

Having demonstrated the ability to substitute the C3’ position of the core, attention was turned
towards exploring substitution off the methoxycarbonyl-L-valine cap (Moc). Compound 24, which had
a chloro substitution on the naphthoimidazole moiety, was used as the comparator. 24 was 5x more
potent against GT2a than compound 1. A number of other groups were inserted in place of the methyl
group on both Moc groups and in vitro virologic profiles of these compounds are summarized in Table
2.22, 23 Replacing the methyl group of the Moc group with an isopropyl group (25) resulted in > 100x
loss in potency on GT1a and GT1b as compared to the corresponding methyl analog 24. In the same
vein, installation of either a methoxyethyl (26) or methylcyclopropyl (27) resulted in a significant loss in
activity across all genotypes assayed. Based upon this scan of the terminal end of the cap, larger
substituents beyond methyl did not appear to be well-tolerated at this position.
Table 2. In vitro potency profile of compounds 24~2721
Genotypes, EC50 (nM)a
ID
24
25
26
R
Me
i-Pr
1a
0.01
>1
1b
0.003
35
2a
0.004
nt
1a Y93H
121
nt
>1000
>1
15
>1
27
>1
4
nt
nt
a nt = not tested.
Having explored both core and cap substitution, it remained to examine the ability to substitute
the proline portion of the molecule. Analogs bearing substituents at the C4 position of the proline were
prepared and in vitro virologic profiles of these analogs are summarized in Table 3.24 The (4R)-NH2-
proline analog 28 showed >10x loss in potency against all genotypes tested. However, the
corresponding methyl amide 29 and methyl carbamate 30 showed potency similar to 1, suggesting either
an amide or a carbamate functionality could be tolerated at this position. Hydroxyl substitution on the
proline was explored as well. Compound 31 which bearing a (1R,4S)-2-azabicyclo[2.2.1]heptane on the
left-hand side of the molecule was used for comparison. (1R,4S)-2-azabicyclo[2.2.1]heptane was known
for HCV NS5a inhibitors9 and it showed comparable potency when replacing L-proline (31 vs.1). Both
(4R)-OH-proline analog 32 and (4R)-MeO-proline analog 33 demonstrated comparable virologic profiles
to 31 is indicative that an ether linkage may be worth exploring in the macrocyclic targets. Interestingly,
larger substituents, such as (1R)-methoxyethyl group (34), showed >20x loss in potency against GT1a
and GT1a Y93H in Table 3.

Table 3. In vitro potency profile of compounds 28~3421
Genotypes, EC50 (nM)
1a
Y93H
A
B
1a
1b
2a
2
28
29
0.4
0.03
987
225
0.07
0.2
0.01
0.1
30
0.009
0.05
80
31
32
33
34
0.02
0.2
0.005
0.007
0.007
0.01
0.02
0.4
93
21
0.05
>1
0.02
0.1
43
404
Based upon the SAR summarized in Tables 1~3, it had been established that C3’ position of the
core and the C4 position of the proline could be substituted and retain reasonable virologic profiles.
However, extending off the methoxycarbonyl-L-valine caps with larger groups was not well-tolerated
leading to analogs with reduced genotype activity. With this data in mind we proceeded toward
preparation of the macrocyclic targets shown in Figure 2. The northern cyclization route, toward
macrocyclic inhibitor 2, is outlined in Scheme 2. Intermediate 35 was prepared based upon the
chemistry outlined in Scheme 1. Acid 36 was prepared according to a similar method used to prepare
methoxycarbonyl-L-valine.23 Deprotection of 35 followed by HATU-mediated amide coupling with acid
36 and deprotection of TBDMS group afforded diol 37. The compound was subjected to an
intramolecular Mitsunobu reaction to yield the macrocyclic intermediate 38 which was taken through the
sequence outlined in Scheme 1 to afford compound 2. The in vitro potency profile of 2 was listed in
Table 4. Unfortunately, it showed reduced potency against genotypes 1a, 1b, and 1a Y93H, compared to
compound 1. It seemed that cyclization to the core might not be tolerated, so efforts were directed onto
the southern cyclization analogs.

Scheme 2. Preparation of compound 2
Conditions: (a) HCl, DCM, rt, 16h, 100%; (b) 36, HATU, DIPEA, DMF, rt, 16h, 59%; (c) TBAF, THF, 34%; (d) DEAD, PPh3, THF, 0 oC, 21%.
The preparation of compound 3 is outlined in Scheme 3. Hydrogenation of dimethyl (2S,5S)-1-
((S)-1-phenylethyl)pyrrolidine-2,5-dicarboxylate 39 afforded the corresponding amine 40 to which a
Boc-L-valine was introduced to give compound 41. Compound 41 was deprotected to yield amine 42
which underwent an intramolecular cyclization to give bicycle 43 under basic conditions. LiOH-
mediated hydrolysis of 43 gave acid 44 which was taken through Scheme 1 to afford compound 3.
Scheme 3. Preparation of compound 3
Conditions: (a) H2 (40psi), Pd(OH)2, MeOH; (b) Boc-L-valine, HATU, DIPEA, DMF, rt, 16h; (c) HCl, MeOH, 68% for three steps; (d) Et3N, DCM, 16h,
54% for two steps; (e) LiOH, THF, water, 100%.
The preparation compound 4 is shown in Scheme 4. Compound 45 was prepared according to
the chemistry outlined in Scheme 1. An allyl group was introduced to the (5R)-OH of proline 45
followed by Boc deprotection to afford 46. In a separate reaction, pent-4-en-1-ol (48) was converted to
pent-4-en-1-yl carbonochloridate which was reacted with L-valine under basic conditions to yield acid
49. HATU-mediated amide coupling of 46 and 49 afforded diene 50 which was subjected to Grubb’s 1st
generation catalyst to promote ring closure to give macrocyclic intermediate 51. Compound 4 was
prepared from 51 via the chemistry outlined in Scheme 1. Hydrogenation of 4 afforded the
corresponding hydrogenated analog 61 (Table 4).

Scheme 4. Preparation of compound 4
Conditions: (a) allyl bromide, NaH, THF, rt; (b) TFA, DCM, rt, 4h; (c) 48, phosgene, pyridine, Et2O, rt, 4h; (d) 47, NaOH, Na2CO3, THF, rt, 4h, 86%; (e) 46,
HATU, DIPEA, DMF, rt, 4h, 73%; (f) 1st generation Grubbs catalyst, DCM, rt, 16h, 73%.
For comparison sake, the corresponding macrocyclic analogs bearing the MK-4882 core were
also prepared (62 and 63) according to Scheme 5. It is important to note that the double bond in
compound 62 is in a different location than compound 4. Compound 53 was treated with 2-bromo-1-(4-
iodophenyl)ethan-1-one (52) in the presence of base to afford keto-ester 54, which was converted to
imidazole 55 under standard ammonium acetate conditions. Suzuki coupling reaction between 55 and
boronic acid 56 led to bromide 57, which was converted to a pinacol boronate followed by Suzuki
coupling reaction with imidazole bromide 58 to yield compound 59. The Boc group of 59 was removed
under acidic conditions and an ((allyloxy)carbonyl)-L-valine group was introduced via HATU coupling
condition to give diene 60. Compound 60 was subjected to a ring-closing metathesis reaction to yield
macrocycle 62. The corresponding hydrogenated macrocycle 63 was obtained via the reduction of the
double bond in 62 under hydrogenation conditions.

Scheme 5. Preparation of compound 62 and 63
Conditions: (a) TEA, EtOAc, rt, 18h, 84%; (b) NH4OAc, xylene, 140 oC, 5h, 90%; (c) Pd(PPh3)4, KF, dioxane, reflux, 16h, 51%; (d) 57,
bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMF, 110 oC, 1h; (e) 58, Pd(dppf)Cl2, Na2CO3, THF/H2O (10:1), reflux, 16h, 39% for two steps; (f) TFA/DCM
(1:5), rt, 3h, 100%; (g) ((allyloxy)carbonyl)-L-valine, HATU, CH3CN, DIPEA, rt, 75%; (h) Grubbs catalyst, DCM, 43 oC, 16h, 55%; (i) Pd/C, MeOH, H2,
0.5h, 15%.
The in vitro potency profiles of compound 2~4 and 61~63 are summarized in Table 4.
Compound 3, containing the six membered ring between the cap and proline, although showed similar
potency to compound 1 against GT1b, suffered >100x loss in potency against GT3a and 30x loss in
potency against GT1a (Table 4). Interestingly, the 15-membered macrocyclic derivative 4 showed pretty
similar potency to compound 1, indicating the macrocyclic moiety was well tolerated. To our surprise,
the corresponding hydrogenated analog 61 resulted in a 10x loss in GT3a potency and a 4x loss in GT1a
Y93H potency than 4, suggesting that the olefin conformation in compound 4 might be preferred over
the corresponding alkyl conformation in compound 61. The macrocyclic compound bearing the MK-
4882 core (62) showed similar potency against GT 3a and GT1a Y93H to MK4882. However, it was
40x weaker against both GT1a and GT1b. The potency loss against GT1a and GT1b of compound 62 vs.
MK-4882 was not observed in the case of compound 4 vs. compound 1. Interestingly, in both case
scenario, the potency on both GT3a and GT1a Y93H was minimally affected by the installation of the
macrocyle. Again, converting 61 to its hydrogenated analog 62 resulted in an weaker NS5a inhibitor
with 3x loss in GT1a potency and 5x loss in GT1b potency. With the potency data of compounds 4 and
61 in hand, it was interesting to notice that the macrocyclic moiety were well tolerated while small
modification to the methyl group of the Moc cap resulted in a significant loss in activity across all
genotypes assayed (Table 2). Based on the conformational overlay shown in Figure 2, compound 1 and
4 overlay with each other reasonablely well in the Moc region. Modification to the methyl group of the
Moc cap of compound 1 with a larger group will result in a substitutent extended outside that region and
that might not be tolerated.

Table 4. In vitro potency profile of compounds 1~4, MK-4882, and 61~6320
Genotypes, EC50 (nM)
ID
core
R
1a
1b
3a
1A_Y93H
119
1
A
0.01
0.004
0.07
2
A
1
2
-
857
3
4
A
A
0.3
0.007
0.006
12
460
43
0.01
0.04
61
A
B
0.006
0.01
0.01
0.5
4
188
24
MK-
4882
0.001
62
B
B
0.4
1.4
0.04
0.2
2
45
nt
63b
nt
a nt = not tested, b with 10% FBS added in the assay.
In conclusion, several macrocyclic NS5A inhibitors bearing either the core of compound 1 (L-
proline analog of MK-8325) or MK-4882 were prepared and evaluated. While the northern cyclized
macrocycle showed reduced potency versus the acyclic analogs, the southern cyclized macrocyclic
NS5A inhibitors bearing the core of compound 1 showed comparable potency profile to compound 1 and
MK-8325. While compounds 4 and 61 displayed similar virologic potency profiles to MK-8325, these
analogs did not demonstrate improved potency against resistance-associated variants (RAV’s), such as
GT1a Y93H. Additional efforts directed towards the identification of HCV NS5A inhibitors with
improved potency profiles will be reported in future publications.

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20.Alignment was performed using Rapid Overlay of Chemical Structures (ROCS v. 3.2.0.4) software. A single low-energy
conformation of compound 1 was used as the query molecule to align the compounds. Omega software v. 2.5.1.4
(OpenEye Scientific Software, Inc., Santa Fe, NM, USA) was used to generate up to 500 conformations of each compound
using default parameters. ROCS was then employed to superimpose compound 1 and other compounds. Results were
visualized using PYMOL (v. 1.7).
21.For the assay methods used for this publication, see example 41 in reference 20. Assay variation is up to 3-fold. For
additional references, see (a) Tong X.; Bogen, S.; Chase, R.; Girijavallabhan V.; Guo, Z.; Njoroge, F. G.; Prongay, A.;
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Handt, L.; Koeplinger, K.; Zhang, N. R.; Graham, D.; Davies, M. E.; MacCoss, M.; Hazuda, D. J.; Olsen, D. B.
Antimicrob. Agents Chemother. 2009, 926.
22.Compounds in Table 2 were prepared by using the chemistry outlined in Scheme 1 starting from different L-valine
derivatives.
23.For detailed prepration procedures of the L-valine derivatives, see: Dwyer, M.; Keertikar, K.; Zeng, Q.; R. Mazzola; J.
Caldwell; H. Tang; Nair, A. G.; Shankar, B.; Rosenblum, S. B.; Kozlowski, J. A. Patent WO2013039878 A1, 20130321.
24.Compounds in Table 3 were prepared by using the chemistry outlined in Scheme 1.
*Corresponding author E-mail: wensheng.yu@merck.com (W.Yu)