Synthesis and evaluation of non-dimeric HCV NS5A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.02.023

Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new monodentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a showed picomolar range activity along with an excellent selectivity index (SI > 90,000).

Full text of DOI:10.1016/j.bmcl.2013.02.023

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2031–2034
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of non-dimeric HCV NS5A inhibitors
Franck Amblard a, Hongwang Zhang a, Longhu Zhou a, Junxing Shi b, Drew R. Bobeck b, James H. Nettles a,
Satish Chavre a, Tamara R. McBrayer b, Philip Tharnish b, Tony Whitaker b, Steven J. Coats b,
Raymond F. Schinazi a,
⇑
a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Veterans Affairs Medical Center, Decatur,
GA 30033, USA
b RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new mono-
dentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along
with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a
showed picomolar range activity along with an excellent selectivity index (SI > 90,000).
Ó 2013 Elsevier Ltd. All rights reserved.
Received 20 December 2012
Revised 22 January 2013
Accepted 1 February 2013
Available online 13 February 2013
Keywords:
NS5A
Antiviral agent
HCV
According to the World Health Organization, HCV has infected
an estimated 170 million individuals worldwide and more than
three million people contract HCV each year.1 Of the six major
HCV genotypes known, type 1a/b accounts for approximately 70%
of all infections in the US, Europe, China and Japan.2 Although often
asymptomatic, it can progress to chronic hepatitis leading to liver
cirrhosis and in many cases hepatocellular carcinoma. Until re-
cently, HCV therapy, utilizing interferon/ribavirin (IFN/RBV), was
effective in only 40–60% of patients and was often associated with
significant side effects. In May 2011, two HCV protease inhibitors,
Incivek and Victrelis, were approved by the US FDA for use in com-
bination with the standard of care (IFN/RBV) and provided a sus-
tained virologic response (SVR) of about 70–80% in genotype 1
HCV-infected subjects.3 However, these drugs have limitations that
include the need of response-guided therapy and the management
of severe skin rashes and anemia.
In the search for new therapeutic agents interfering with HCV
replication, non-structural (NS) viral proteins have been primarily
targeted including NS2, NS3 (protease), NS4A, NS4B, NS5B (poly-
merase) and NS5A. NS5A is a large phosphoprotein (49 kDa) re-
quired for HCV replication and particle assembly. Although its
ultimate role in the HCV replication cycle is not fully understood,4
it has been shown to be a required component of the HCV RNA
replication and several molecules have been recently identified
as potent inhibitors. For example, BMS-790052 has subnanomolar
EC50 values against genotype 1 replicons and a phase I clinical trial,
in patients chronically infected with HCV, produced a 3.3 log10
reduction in mean viral load 24 h after administration of a single
100 mg dose.5 Unfortunately, for both subtypes 1a and 1b, treat-
ment with BMS-790052 quickly lead to the emergence of several
drug-induced mutations.
We hypothesized that lower molecular weight unsymmetrical
compounds could be developed to have greater activity toward
known NS5A mutations and therefore, a higher barrier to resis-
tance (Fig. 1). As part of our SAR study, modifications were com-
bined on various positions of the monodentate NS5A scaffold (A,
B, C and D) and served as probes for 3D-exploration of the NS5A
binding area (Fig. 1). Based on this strategy, we have developed a
second generation of unsymmetrical, still highly potent, NS5A
inhibitors.
Modifications on the various targeted positions have been
achieved through general Scheme 1 by adapting chemistry previ-
ously described.6 Commercially available phenyl methyl ketones
1 were brominated in moderate to excellent yields then condensed
with N-Boc protected proline in presence of DIPEA to afford com-
pound 2. Further, cyclization of the keto-ester of 2 to the corre-
sponding imidazole was accomplished in presence of ammonium
acetate followed by Boc deprotection under acidic conditions gave
compound 3. Modification of Part C and D was achieved by reac-
tion of 3 with various protected amino acids under traditional pep-
tidic coupling conditions and generated the analogs 4a–o.
Secondary modifications of Part A were explored using Pd-cata-
lyzed cross coupling between the bromo compound 4b and various
⇑
Corresponding author.
E-mail address: rschina@emory.edu (R.F. Schinazi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.023

2032
H3CO
F. Amblard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2031–2034
H
H
N
N
N
N
H
N
NH
O
a
O
N
N3
H2N
N
N
N
O
O
O
O
HN
HN
N
N
N
H
O
O
4c
8
HN
OCH3
OCH3
BMS-790052
OCH3
H
N
N
O
RHN
b
N
O
HN
9a
R = CO2Me
R = SO2Me
H
OCH3
C
N
N
9b
A
B
N
O
O
Scheme 3. Reagents and conditions: (a) H2, Pd/C, MeOH, rt, 15 h, 91%; (b)
MeOC(O)Cl, pyridine, rt or MeSO2Cl, pyridine, rt, 15 h, 60–75%.
HN
D
OCH3
Vero (African green monkey kidney epithelial cells), and PBM (pri-
mary human peripheral blood mononuclear cells). While the first
simple truncated compound 4a and modified derivatives 4b and
4d–o did not show any activity against HCV in the replicon system
Figure 1. Structures of BMS-790052 and targeted monodentate compounds.
R1
R1
O
c , d
a, b
when tested up to 10 lM (Table 1), functionalization of the phenyl
N
Boc
O
O
O
ring with an azido group lead to compound 4c, exhibiting a median
effective concentration (EC50) of 8 nM against HCV with no appar-
ent toxicity in four different cell lines. Interestingly, halogenation
of 4c on the imidazole moiety afforded even more potent com-
pounds, including bromo derivative 5a that displayed an EC50 of
0.7 nM. Elongation of compound 4a by addition of a second phenyl
ring lead to compound 4j that displayed an EC50 of 300 nM. Inter-
estingly, substitution of this second ring with a 4-CF3- or 4-OH-
group lead to compounds 6c and 6e displaying EC50 values of 4.6
and 5 nM, respectively. However, unlike for compound 4c bearing
only one phenyl ring, halogenation of the imidazole ring of com-
pound 6c led to loss of anti-HCV activity (compound 7).
Drug–drug interactions have become an important issue in
modern health care. It is now apparent that many drug–drug inter-
actions can be explained by alterations in the metabolic enzymes
that are present in the liver and other extra-hepatic tissues. Many
of the major pharmacokinetic interactions between drugs are due
to hepatic cytochrome P450 (CYP450) enzymes being affected by
one or more of the drugs present in a given individual. Further-
more, drug interactions can be a result of inhibition or induction
of CYP450 enzymes. The potential drug–drug interaction liabilities
of the most active compounds were investigated by a CYP450
reversible inhibition assay. Compounds 4c, 5a, 6c and 6j did not
appear to inhibit the major CYP450 enzymes at the IC90 level and
had a favorable CYP profile, which is suggestive of no drug–drug
interactions (Table 2). Due to the large therapeutic window, the
CYP inhibitions observed in the micromolar ranges are expected
not to be clinically relevant at therapeutic doses.
1
2
H
N
H
N
R1
N
O
R1
N
H
R3
e
N
N
O
HN
3
R4
4a-o
H
R1
N
N
R3
f
N
O
O
HN
R2
R4
5a-c
Scheme 1. Reagents and conditions: (a) Br2, CH2Cl2, rt, overnight, 70–95%; (b) Boc-
Pro-OH, DIPEA, CH3CN, rt, 5 h; (c) NH4OAc, toluene, 95 °C, 15–20 h, 62–2%, 2 steps;
(d) 6 N HCl, CH3OH, 50 °C; (e) R4-(CO)NH-CHR3-COOH, DIPEA, HOBt, EDC, CH3CN,
0 °C to rt, 15 h, 45–65%, 2 steps; (f) NXS, CH2Cl2, rt, 0.5–15 h, 42–65% (X = Br, Cl, I).
substituted phenyl boronic acids (Scheme 2, compounds 6a–l) but
also by alkylation of amino derivative 8 obtained by hydrogenation
of azido compound 4c (Scheme 3, compounds 9a–b). Because we
recently determined that halogenation of BMS-790052’ imidazole
ring using NBS, NCS or NIS lead to highly potent compounds exhib-
iting excellent overall profiles,7 we restricted our efforts on Part B
to the preparation of compounds 5a–c and 7 (Schemes 1 and 2).
Overall, 36 monodentate compounds were evaluated in an
in vitro HCV replicon assay and their potential toxic effects as-
sessed in multiple cell lines (Table 1), including: Huh-7 (a human
hepatocellular carcinoma cell line), primary human hepatocytes,
In conclusion, extremely potent non-dimeric NS5A inhibitors
(picomolar activity) with a wide therapeutic window (>104) have
H
H
R5
N
N
N
N
a
Br
N
N
O
O
O
O
HN
HN
4b
OCH3
6a-l
OCH3
H
N
R5
N
b
N
O
O
HN
R2
7
OCH3
Scheme 2. Reagents and conditions: (a) R5-Ph-B(OH)2, PdCl2(PPh3)2, Na2CO3, THF, rt, 50–77%; (b) NCS, CH2Cl2, rt, 15 h, 58%.

F. Amblard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2031–2034
2033
Table 1
Structures, anti-HCV activity and cytotoxicity of BMS-790052 and compounds 4a–o, 5a–c, 6a–l, 7, 8, 9a–b
H
R1
N
N
R3
N
O
O
HN
R2
R4
a
Compd
R1
R2
R3
R4
HCV replicon in Huh-7 cells
Cytotoxicity, CC50
CEM
(lM)
EC50 (nM)
EC90 (nM)
PBM
Vero
BMS
4a
4b
4c
4d
4e
4f
4g
4h
4i
N/Ab
H
4-Br
4-N3
4-CN
4-NO2
4-CF3O
4-CF3
4-OMe
4-Me
4-Ph
4-Ph
4-Ph
N/A
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Cl
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
N/A
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iBu
Me
allyl
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
N/A
0.01
0.03
N/A
N/A
52
19
9.6
>100
48
>100
33
>100
30
26
>100
>100
18
25
13
21
15
>100
16
12
14.2
33
14
14
13
12
10
13
50
13
>100
17
59
6
11
10
>100
>100
13
21
>100
50
>100
98
>100
>100
>100
>100
>100
70
61
11
73
>100
>100
52
>100
>100
98
16
12
75
32
10
55
>100
30
>100
61
67
54
11
10
>100
>100
>100
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OtBu
Me
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
>1000
>100
8
>100
>100
>100
>100
>1000
>1000
300
>100
>1000
>1000
1000
>100
0.7
>100
>100
>100
>100
>100
49
>100
>100
>100
14
16
16
100
56
>100
35
N/A
N/A
N/A
N/A
N/A
N/A
900
>1000
N/A
N/A
N/A
N/A
2.9
4j
4k
4l
4m
4n
4o
5a
5b
5c
6a
6b
6c
6d
6e
6d
6e
6f
6g
6h
6i
6j
6k
6l
4-Ph
2,4-diCl
4-PhSO2–
4-N3
4-N3
4-N3
4
2
14
3
94
64.2
>100
90
>100
24
44
11
70
>100
85
>100
14
>100
>100
37
4-CN-Ph
4-Me-Ph
4-CF3-Ph
2-CF3-Ph
3-CF3-Ph
4-CF3O-Ph
4-HO-Ph
3-OH-Ph
4-MeO-Ph
4-NH2C(O)-Ph
4-MeOOC-Ph
4-HO-CH2-Ph
4-MeSO2-Ph
4-N3-Ph
4-CF3-Ph
4-NH2
>100
>100
4.6
>100
>100
>100
5
>1000
>100
21
>100
2
>100
>100
>100
200
>100
>100
N/A
N/A
9.6
N/A
N/A
N/A
17
N/A
N/A
76
N/A
9
N/A
N/A
N/A
300
N/A
N/A
7
8
9a
9b
11
>100
>100
>100
4-NHCO2Me
4-NHSO2Me
a
All compounds had a CC50 of >1
N/A, not applied.
lM in Huh-7 cells.
b
Table 2
Cytochrome P450 inhibition data for compounds 4c, 5a, 6c, 6j and BMS-790052a
Compd
CYP1A2 (lM)
CYP3A4 (lM)
CYP2D6 (
lM)
CYP2C9 (lM)
IC50
IC90
IC50
IC90
IC50
>100
50.5 6.8
62.1 32.9
>100
IC90
IC50
IC90
4c
5a
6c
6j
>100
>100
>100
>100
>100
>100
5.1 0.6
1.5 1.6
6.5 0.6
6.9 2.1
7.2 1.7
96.1
46.1 23.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
50.5 19.7
9.1 1.9
31.2 26.0
59.5 13.6
>100
>100
>100
>100
>100
80.9 21.1
82.9 13.4
56.7 12.4
>100
BMS-790052
>100
a
Mean SD of at least two independent assays; IC50 = 50% inhibitory concentration. The following positive controls were used (IC50
,
l
M):
a
-naphthoflavone for 1A2 (>93%
at 3
l
M); ketoconazole for 3A4 (>93% at 10 M); PH-053 (proprietary) for 2D6 (>70% at 100 M); sulfaphenazole for 2C9 (>90% at 10
l
l
lM).
been discovered. Out of all compounds prepared, compound 5a ap-
peared to be the most promising with an EC50 of 0.7 nM and no
toxicity or inhibition of major human CYP enzymes at therapeuti-
cally relevant concentrations. In vitro resistance profile of com-
pound 5a was established by mutation selection in HCV
subgenomic replicon containing Huh-7 cells. After 2 months expo-
sure, Y93H and Q30E were among the selected resistant virus, sim-
ilar to those observed with BMS-790052 treatment, which
confirmed that this monodentate compound acts as an NS5A inhib-
itor. Through this work, we demonstrated for the first time that, a
bidentate structure (i.e., BMS-790052) was not a sine qua non con-
dition for a molecule to inhibit HCV NS5A.

2034
F. Amblard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2031–2034
Romine, J. L.; Snyder, L. B.; St. Laurent, D. R.; Wang, G.; Xu, N.-N.; Belema, M. PCT
Acknowledgments
Int. Appl. 2010, WO 2010117635 A1 20101014.; (f) Belema, M.; Nguyen, V. N.;
Serrano-Wu, M.; St. Laurent, D. R.; Qiu, Y.-P., Ding, M.; Meanwell, N. A.; Snyder,
L. B. U.S. Pat. Appl. Publ. 2010, US 20100080772 A1 20100401.; (g) Bachand, C.;
Belema, M.; Deon, D. H.; Good, A. C.; Goodrich, J.; Hamann, L. G.; James, C. A.;
Langley, D. R.; Lavoie, R.; Lopez, O. D.; Martel, A.; Meanwell, N. A.; Nguyen, V. N.;
Romine, J. L.; Ruediger, E. H.; Snyder, L. B.; St. Laurent, D. R.; Yang, F.-K., Wang, G.
PCT Int. Appl. 2008, WO 2008144380 A1 20081127.; (h) Milbank, J. B. J.; Tran, T.
D.; Wakenhut, F. PCT Int. Appl. 2011, WO 2011154871 A1 20111215.; (i)
Vandyck, K.; Verschueren, W. G.; Raboisson, P. J.-M. B. PCT Int. Appl. 2012, WO
2012013643 A1 20120202.; (j) Li, L.-P.; Zhong, M. PCT Int. Appl. 2011, WO
2011156543 A2 20111215.; (k) Qiu, Y.-L.; Wang, C.; Ying, L.; Peng, X.-W.; Or, Y.
S. PCT Int. Appl. 2010, WO 2010091413 A1 20100812.; (l) Li, L.-P.; Zhong, M. PCT
Int. Appl. 2011, WO 2011150243 A1 20111201.; (m) Kim, S.; Gao, Q.; Yang, F.-K.
U.S. Pat. Appl. Publ. 2010, US 20100158862 A1 20100624.; (n) Bachand, C.;
Belema, M.; Deon, D. H.; Good, A. C.; Goodrich, J.; James, C. A.; Lavoie, R.; Lopez,
O. D.; Martel, A.; Meanwell, N. A.; Nguyen, V. N.; Romine, J. L.; Ruediger, E. H.;
Snyder, L. B.; St. Laurent, D. R.; Yang, F.-K.; Langley, D. R.; Wang, G.; Hamann, L.
G. PCT Int. Appl. 2009, WO 2009102318 A1 20090820.; (o) Bachand, C.; Belema,
M.; Deon, D. H.; Good, A. C.; Goodrich, J.; James, C. A.; Lavoie, R.; Lopez, O. D.;
Martel, A.; Meanwell, N. A.; Nguyen, V. N.; Romine, J. L.; Ruediger, E. H.; Snyder,
L. B.; St. Laurent, D. R.; Yang, F.-K., Langley, D. R.; Hamann, L. G. PCT Int. Appl.
2008, WO 2008021936 A2 20080221.; (p) Belema, M.; Romine, J. L.; Nguyen, V.
N.; Wang, G.; Lopez, O. D.; St. Laurent, D. R.; Chen, Q.; Bender, J. A.; Yang, Z.;
Hewawasam, P.; Xu, N.-N.; Meanwell, N. A.; Easter, J. A.; Su, B.-N.; Smith, M. J.
PCT Int. Appl. 2011, WO 2011075439 A1 20110623.; (q) Corte, J. R.; Fang, T.;
Decicco, C. P.; Pinto, D. J. P.; Rossi, K. A.; Hu, Z.-L.; Jeon, Y.; Quan, M. L.;
Smallheer, J. M.; Wang, Y.-F.; Yang, W. PCT Int. Appl. 2011, WO 2011100401 A1
20110818.
This work was supported in part by NIH Grant 5P30-AI-50409
(CFAR), 5R01-AI-071846-03 and by the Department of Veterans Af-
fairs. Dr. Schinazi is the founder and a major shareholder of RFS
Pharma, LLC. Emory received no funding from RFS Pharma, LLC to
perform this work and vice versa.
References and notes
1. Hepatitis C-global prevalence (update). WHO wkly. Epidemio. rec 1999, 74, 425.
2. Kim, A. I.; Saab, S. Am. J. Med. 2005, 118, 808.
3. Sheridan, C. Nat. Biotechnol. 2011, 29, 553.
4. Lemon, S. M.; McKeating, J. A.; Pietschmann, T.; Frick, D. N.; Glenn, J. S.;
Tellinghuisen, T. L.; Symons, J.; Furman, P. A. Antivir. Res. 2010, 86, 79.
5. (a) 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. Nature 2010, 465, 96; (b) Asselah, T. J. Hepatol. 2011, 54, 1069.
6. (a) Sun, J.-H.; Gao, M.; O’Boyle, D. R. II.; Lemm, J. A.; Roberts, S. B.; Belema, M.;
Meanwell, N. A. PCT Int. Appl. 2012, WO 2012009394 A2 20120119.; (b) Lopez, O.
D.; St. Laurent, D. R.; Goodrich, J.; Romine, J. Lee; Serrano-Wu, M.; Yang, F.-K.;
Kakarla, R.; Yang, X.-J.; Qiu, Y.-P.; Snyder, L. B. U.S. Pat. Appl. Publ. 2011, US
20110294819 A1 20111201.; (c) Belema, M.; Romine, J. L.; Nguyen, V. N.; Wang,
G.; Lopez, O. D.; St. Laurent, D. R.; Chen, Q.; Bender, J. A.; Yang, Z.; Hewawasam,
P.; Xu, N.-N.; Meanwell, N. A.; Easter, J. A.; Su, B.-N.; Smith, M. J. U.S. Pat. Appl.
Publ. 2011, US 20110286961 A1 20111124.; (d) Belema, M.; Hewawasam, P. U.S.
Pat. Appl. Publ. 2011, US 20110237636 A1 20110929.; (e) Bender, J. A.;
Hewawasam, P.; Kadow, J. F.; Lopez, O. D.; Meanwell, N. A.; Nguyen, V. N.;
7. Zhang, H.; Zhou, L.; Amblard, F.; Shi, J.; Bobeck, D. R.; Tao, S.; McBrayer, T. R.;
Tharnish, P. M.; Whitaker, T.; Coats, S. J.; Schinazi, R. F. Bioorg. Med. Chem. Lett.
2012, 22, 4864.