Potent inhibitors of HCV-NS3 protease derived from boronic acids

Article abstract of DOI:10.1016/j.bmcl.2008.10.124

Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

Full text of DOI:10.1016/j.bmcl.2008.10.124

Bioorganic & Medicinal Chemistry Letters 19 (2009) 180–183
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent inhibitors of HCV-NS3 protease derived from boronic acids
*
Srikanth Venkatraman , Wanli Wu, Andrew Prongay, Viyyoor Girijavallabhan, F. George Njoroge
Schering Plough Research Institute, K15-MS 3545, 2015, Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma,
leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single posi-
tive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhi-
bition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous
efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine
traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine
trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound
to NS3 protease is also discussed.
Received 1 August 2008
Revised 27 October 2008
Accepted 28 October 2008
Available online 5 November 2008
Keywords:
HCV
NS3 protease
Boronic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Hepatitis C virus is the primary etiological agent responsible for
chronic HCV infections of liver leading to liver cirrhosis and hepa-
tocellular carcinoma.1 Nearly 200 million people are infected
worldwide and up to 80% of them turn chronic infections. Current
the compounds that is currently undergoing Phase III clinical trials
and shown to be efficacious in humans (Fig. 1).
As a possible back-up to Sch 503034 (1), we explored inhibitors
that contained alternative electrophiles to ketoamides. Boronic
acids have been extensively investigated as possible electrophiles
that could react with nucleophilic serine in the context of develop-
ing inhibitors of thrombin receptor and other serine proteases
including HCV NS3 protease.5 ValcadeÒ, a boronic acid derived
dipeptide is an inhibitor of 26S proteosome that has been success-
fully developed into a drug for treatment of multiple myeloma.6
We therefore decided to investigate the replacement of ketoamide
group in 1 with a boronic acid derivative. From our previous SAR
exploration of inhibitors spanning from P6–P1, it was determined
that an ethyl group at P1 was well tolerated. We therefore decided
to explore compound 2 as our first boronic acid target (Fig. 2).
Intermediates for the synthesis of the inhibitors of type 2 were
accomplished using methods outlined in Scheme 1. Deprotonation
of dichloromethane with BuLi at À100 °C, followed by treatment
with trimethylborate resulted in compound 4. Methyl ester 4
was converted to the pinene diol ester 5 by transesterification with
(+)-pinenediol. Treatment of 5 with ethyl or cyclobutyl Grignard
therapeutic regimens include
a-interferon and its combinations
with RibavirinÒ, which is effective only in 25–40% of patients for
sustained response. The newly introduced pegylated interferon
has reduced the frequency of injection and has a sustained re-
sponse in lowering viral titers and improved histological growth.2
The lack of efficient methods for the treatment of chronic HCV
infections requires the identification of new chemical entities.
Hepatitis C virus is a Flavivirdae family virus that encodes a
polyprotein of ꢀ3000 amino acids from a positive strand RNA gen-
ome. This polyprotein is post translationally spliced to produce
various proteins essential for viral replication. Autocatalytic cleav-
age at the NS2–NS3 junction followed by cleavage of NS3–NS4A,
NS4A–NS4B, NS4B–NS5A and NS5A–NS5B by the NS3 protease
produces functionally mature virions. The inhibition of NS3 prote-
ase would possibly impair the ability of the enzyme to process the
polyprotein, thus arresting maturation of virus and inhibiting viral
production.3
HCV NS3 protease is a serine protease which enables the lesion
of a Cys-Ser bond with the assistance of the cofactor NS4A. The
hydrolysis of the amide bond proceeds with the attack of Ser-139
followed by the stabilization of the tetrahedral transition state by
His-57 and Asp-81. We and others have developed various novel
O
H
N
NH2
Ki* =14nM
N
inhibitors containing a a-ketoamide moiety as an electrophilic trap
H
O
O
that trap Ser-139 reversibly. Sch 503034, Boceprevir (1),4 is one of
HN
N
HNE/HCV=2200
RepliconEC 90 =0.35
O
µ
M
O
Me
Me
Me
1, Boceprevir
* Corresponding author. Tel.: +1 908 740 3758; fax: +1 908 740 7152.
E-mail address: Srikanth.Venkatraman@spcorp.com (S. Venkatraman).
Figure 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.124

S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 180–183
181
OH
H
N
a
B
COOCH3
N
OH
N
H
N
H
N
COOCH3
.HCl
N
H
O
BocHN
O
O
8
O
2
9
Figure 2.
c
COOCH3
b
N
H
N
H
N
Cl
Cl
a
b
O
OCH3
Cl
Cl
O
B
3
OCH3
10
4
O
B
O
O
Cl
H
B
N
O
Cl
Cl
O
2; R1 = CH3
c
d
N
B
H
N
H
N
; R1 =cyclobutyl
O
O
R1
15
R1
; R1 = CH3
12; R1 =cyclobutyl
O
6a R1 = CH3
11
O
5
1
6b
R =cyclobutyl
Scheme 2. Reagents and conditions: (a) Boc-tert-Leu, HATU, NMM, 0 °C ? rt.
(b) i—4 M HCl in dioxane, rt. 1 h. ii—tert-C4H9NCO, NMM, CH2Cl2, 0 °C ? rt. 12 h. (c)
i—aq LiOH, THF, rt, 2 h. ii—IBCF, (C2H5)3N, À15 °C, 7a, À15 °C ? rt., 3 h. (d) NaIO4,
acetone/0.1 M soln. NH4OAc, rt, 48 h.
O
H2N
d
.HCl
B
O
R1
boronate ester compound 12 (Ki = 10 nM) as well as the boronic
acid derivative 15 (Ki = 10 nM), a 50-fold improvement in binding
over 2. Our studies in the ketoamide series of inhibitors had previ-
ously demonstrated that the replacement of the P3 tert-butyl urea
cap with various modified urea moieties enhanced binding of
ketoamide inhibitors. To evaluate the effect of this novel capping
group we decided to synthesize the sulfonamide derived com-
pound 16. The synthesis of inhibitor 16 is outlined in Scheme 3.
Table 1 summarizes the activity of these compounds.
7a; R1 = CH3
1
7b
; R =cyclobutyl
Scheme 1. Reagents and conditions: (a) BuLi, À100 °C ? rt. (CH3O)3B. (b) (+)-
Pinenediol, rt., THF (c) R1MgCl, THF, À78 °C? rt., 12 h (d) i—LiHMDS, THF, rt. ii—HCl
in ether.
reagent resulted in compounds of type 6 in excellent yields. Fur-
ther displacement of the chloride of 6 with LiHMDS followed by
treatment with HCl yielded amine salts of type 7.
Completion of synthesis of inhibitor 2 was accomplished using
reactions outlined in Scheme 2. Thus, coupling of Boc protected
tert-leucine with 3,4-dimethylcyclopropyl fused proline 87 using
HATU and NMM resulted in dipeptide 9 in 60% yield. The Boc group
of the dipeptide 9 was deprotected using 4 M HCl in dioxane and
further treatment of the resultant amine salt with tert-butyl isocy-
anate yielded tert-butyl urea 10. Hydrolysis of methyl ester of com-
pound 10 with aq LiOH, followed by coupling of the resulting acid
with the amine salt 7a using isobutyl chloroformate and Et3N
yielded inhibitor precursor 11, which was converted to 2 by reac-
tion with NaIO4.
O
NHBoc
a
NHBoc
b
HO
H2N
19
18
OCH
N
O
O
N
O
H
N
H
N
O
N
S
NHBoc
O
S
d
O
O
Compound 2 was tested for its inhibitory activity in a HCV pro-
tease continuous assay. Its ability to prevent the NS3 protease cat-
alyzed hydrolysis of chromogenic 4-phenylazophenyl [PAP] ester
from peptide fragment Ac-DTEDVVP(Nva)-O-4-PAP was measured
using a spectrophotometric assay.8 The binding constant for the
inhibitor 2 was determined as Ki = 380 nM. We also evaluated
the pinene ester 11 for its ability to inhibit HCV NS3 protease
and determined its binding constant Ki = 500 nM. Since these com-
pounds were less potent than the corresponding first generation
compound 1, we decided to synthesize the corresponding cyclobu-
tylmethyl containing amino boronic acid 7b and incorporate it into
the inhibitor. The synthesis of cyclobutyl derived amino acid 7b
was accomplished in a similar manner to that of 7a except that
the cyclobutylmethyl was introduced using the appropriate Grig-
nard reagent. The synthesis of subsequent inhibitors was also
similar to that of 2 and is as shown in Scheme 2. The introduction
of a lipophilic cyclobutyl group at P1 enhanced binding of both the
20
21
OH
B
H
N
OH
N
d
H
H
N
O
O
N
CH3
O
N
R1
S
O
CH3
O
16;R 1 =CH
3
;R 1 =cyclobutyl
17
Scheme 3. Reagents and conditions: (a) i—NH4Cl, EDCI, DMF, NMM, rt,
ii—BH3ÁDMS, THF, reflux, 2 h. (b) i—CH3SO2Cl, pyridine, À78 °C ? rt, 12 h.
ii—Cs2CO3, DMF, CH3I (c) i—4 M HCl, dioxane, ii—amine salt9, CH3CN, (C2H5)3N, rt.
(d) i—aq LiOH, THF, rt, 2 h. ii—IBCF, (C2H5)3N, À15 °C, 7, À15 °C?rt, 3 h. iii—NaIO4,
acetone/0.1 M soln. NH4OAc, rt, 48 h.

182
S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 180–183
Table 1
The coupling of Boc protected tert-leucine with ammonium
chloride using EDCI resulted in Boc protected tert-leucine amide,
which was further reduced to amine 19 using BH3ÁSMe2. The amine
19 was reacted with methanesulfonyl chloride and the resulting
sulfonamide was methylated with methyl iodide and Cs2CO3 to
yield N-methylated sulfonamide 20. Deprotection of Boc group of
20 followed by treatment of formed amine salt with nitrophenyl
carbamate of intermediate 9 yielded compounds 21. The methyl
ester of intermediate 21 was hydrolyzed with aq lithium hydroxide
and the intermediate acid was coupled with amine salt of type 7
using mixed anhydride method with IBCF and Et3N. The formed
pinene diol ester was once again hydrolyzed to the corresponding
boronic acid using NaIO4 to yield compounds of type 16.
The replacement of the P3 tert-butyl urea cap with sulfonamide
derived cap 20 had a profound effect on binding. Compound 13 ob-
tained by the replacement of tert-butyl urea P3 cap of compound
11 with sulfonamide demonstrated a Ki = 34 nM, a fifteen folds
improvement in potency. Similarly replacement of P3 tert-butyl
cap in the P1 cyclobutylmethyl derived compound 12 resulted in
inhibitor 14 (Ki = 500 pM); a twenty fold improvement in activity
over 12. This was the first time we had identified a compound that
bound to NS3 protease in the picomolar range. We next evaluated
the boronic acid analogs of compounds 13 and 14. The correspond-
ing boronic acid analogs of compound 13, compound 16 demon-
strated a Ki = 52 nM; whereas the boronic acid derivative of
compound 14, compound 17 had a Ki = 200 pM.
OR2
B
H
N
OR2
N
H
N
H
N
O
R1
R3
O
O
Entry
Structure
Ki (nM)
O
B
H
N
O
N
11
500
H
N
H
O
N
O
O
O
B
H
N
O
N
12
10
H
N
H
N
O
O
O
O
H
N
B
Comparison of binding activities of the pinene diol esters 12–14
to their corresponding boronic acid inhibitors 15–17 suggested
that the boronate esters were readily hydrolyzed in the assay con-
ditions and the activity of the boronate esters probably arose from
the corresponding boronic acid derivatives.
O
N
13
14
2
34
0.5
380
10
O
S
O
H
N
H
O
N
O
N
O
The X-ray structure of inhibitor 17 bound to HCV NS3 protease
was solved and is shown in Figure 3.10 From the structure of 17
bound to protease, it was clear that the cyclobutyl group at P1
occupy the S1 pocket. The P2 (1R,5S)-6,6-dimethyl-3-azabicy-
clo[3.1.0]hexane ring adopted a bent conformation that allowed
maximum overlap of the methylenes of proline and cyclopropyl
ring to Ala-156. The conformation adopted by cyclopropyl group
allowed the methyl group proximal to the carbonyl to interact with
His-57 and the methyl group distal to carbonyl to interact with
Ala-156 and Arg-155. The tert-butyl group of P3 sulfonamide cap-
ping occupied the S4 region of space making excellent van der
Waals contact with the protein and the oxygens of the sulfonamide
group formed a hydrogen bond with Cys-159. The oxygen of
Ser-139 is bound to the lewis acidic boron to form a tetrahedral
boronate derivative.
O
B
H
N
O
N
O
S
O
H
H
O
N
N
O
N
O
O
B
H
N
O
N
N
H
N
H
N
O
O
O
O
O
O
OH
B
H
N
OH
15
H
N
H
N
OH
H
N
B
OH
N
N
16
52
O
S
O
H
N
H
N
O
O
O
O
N
N
O
O
OH
H
N
B
OH
17
0.2
O
S
O
H
N
H
N
Figure 3. X-ray structure of inhibitor 17 bound to NS3 protease.

S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 180–183
183
2. (a) McHutchison, J. G.; Gordon, S. C.; Schiff, E. R.; Shiffman, M. L.; Lee, W. M.;
Rustgi, V. K.; Goodman, Z. D.; Ling, M.-H.; Cort, S.; Albrecht, J. K. N. Engl. J. Med.
1998, 339, 1485; (b) Davis, G. L.; Esteban-Mur, R.; Rustgi, V.; Hoefs, J.; Gordon,
S. C.; Trepo, C.; Shiffman, Mitchell L.; Zeuzem, S.; Craxi, A.; Ling, M.-H.;
Albrecht, J. N. N. Engl. J. Med. 1998, 339, 1493; (c) Zeuzem, S.; Feinman, S. V.;
Rasenack, J.; Heathcote, E. J.; Lai, M.-Y.; Gane, E.; O’Grady, J.; Reichen, J.; Diago,
M.; Lin, A.; Hoffman, J.; Brunda, M. J. N. Engl. J. Med. 2000, 343, 1666; (d)
Heathcote, E. J.; Shiffman, M. L.; Cooksley, W. G. E.; Dusheiko, G. M.; Lee, S. S.;
Balart, L.; Reindollar, R.; Reddy, R. K.; Wright, T. L.; Lin, A.; Hoffman, J.; De
Pamphilis, J. N. Engl. J. Med. 2000, 343, 1673; (e) Manns, M. P.; McHutchison, J.
G.; Gordon, S. C.; Rustgi, V. K.; Shiffman, M.; Reindollar, R.; Goodman, Z. D.;
Koury, K.; Ling, M.-H.; Albrecht, J. K. Lancet 2001, 358, 958. International
Hepatitis Interventional Therapy Group.
3. (a) Lin, C.; Kwong, A. D.; Perni, R. B. Infectious Disorders 2006, 6, 3–16; (b)
Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.;
Bös, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A.-M.;
Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagacé, L.; LaPlante, S. R.; Narjes, H.;
Poupart, M.-A.; Rancourt, J.; Sentjens, R. E.; George; T, S.; Simoneau, B.;
Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C.-L.; Llinàs-
Brunet, M. Nature 2003, 186–189.
4. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu,
Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.;
Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.;
Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.;
Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay,
A.; Madision; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, T. Y.; Brisson, J.-M.;
Prelusky, D.; Kormacher, W.; White, R.; Bogonowich-Knipp, S.; Pavlovsky, A.;
Prudence, B.; Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.;
Njoroge, F. G. J. Med. Chem. 2006, 49, 6074.
5. (a) Priestley, E. S.; De Lucca, I.; Ghavimi, B.; Erickson-Viitanen, S.; Decicco, C. P.
Bioorg. Med. Chem. Lett. 2002, 12, 3199; (b) Archer, S. J.; Camac, D. M.; Wu, Z. J.;
Farrow, N. A.; Domaille, P. J.; Wasserman, Z. R.; Bukhtiyarova, M.; Rizzo, C.;
Jagannathan, S.; Mersinger, L. J.; Kettner, C. A. Chem. Biol. 2002, 9, 79.
6. Laura, J.; Lisa, H.; William, S. D. Oncology 2004, 18, 4.
In addition to van der Waals contacts, inhibitor 17 formed a
series of specific hydrogen bonds with the protein surface. Map-
ping out the various hydrogen bonding interactions that existed
between inhibitor 17 and NS3 protease it is evident that the urea
nitrogens donated two hydrogen bonds to alanine-157 thus
improving potency and HCV specificity. Additionally, the nitrogen
of the P1 residue donated a hydrogen bond to Arg-155 and oxy-
gen of P3 carbonyl group accepted a hydrogen bond from the
Ala-157.
We next evaluated the activity of these potent inhibitors in the
replicon based cellular assay. Evaluation of these compounds dem-
onstrated an EC90 > 5.0 lM suggesting that cell permeability may
be limiting and be responsible for poor cellular activity.
In search of a possible second generation compound to Sch
503034 (1) we evaluated the possibility of replacements of the
ketoamide with a boronic acid electrophile. The replacement of
this group with boronic acid resulted in inhibitor 12 which was
equipotent to our first generation compound 1. We next evalu-
ated the replacement of P3 capping with modified P3 cap derived
such as N-(2-amino-3,3-dimethylbutyl)-N-methylmethanesulf-
onamide which resulted in compound 17 that had markedly im-
proved activity (Ki = 200 pM). This was the first time we identified
compounds in the picomolar range. Analysis of X-ray structure of
17 bound to the protease revealed binding of the boronic acid to
Ser-139, where the boronic acid forms a tetrahedral borate. Eval-
uation of these inhibitors in the replicon based cellular assay
7. (a) Mamai, A.; Zhang, R.; Natarajan, A.; Madalengoitia, J. S. J. Org. Chem. 2001,
66, 455; (b) Zhang, R.; Madalengoitia, J. S. J. Org. Chem. 1999, 64, 330.
8. Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.; Njoroge, F. G.; Windsor, W. T.;
Malcolm, B. A. Anal. Biochem. 1999, 270, 268.
showed EC90 > 5 lM suggesting that the cell penetration was
probably an issue. Further studies are on way to improve cellular
activity of these inhibitors.
9. Compound 9 was treated with 4 M HCl to deprotect the Boc group and the
resultant amine salt was treated with 4-nitrophenyl carbonate to form the 4-
nitrophenyl carbamate derivative which was used for coupling.
References and notes
10. X-ray coordinates of compound 17 bound to HCV protease has been
deposited in the PDB database with the RCSB ID code rcsb049921 and PDB
ID code 3EYD.
1. (a) World health organization Fact sheet number 164, October 2000; (b)
Wasley, A.; Alter, M. J. Semin. Liver Dis. 2000, 20, 1; (c) Brown, R. S.; Gaglio, P. J.,
et al Liver Transplant. 2003, 9, S10.