P1 Phenethyl peptide boronic acid inhibitors of HCV NS3 protease

Article abstract of DOI:10.1016/S0960-894X(02)00682-0

A series of peptide boronic acids containing extended, hydrophobic P1 residues was prepared to probe the shallow, hydrophobic S1 region of HCV NS3 protease. The p-trifluoromethylphenethyl P1 substituent was identified as optimal with respect to inhibitor potency for NS3 and selectivity against elastase and chymotrypsin.

Full text of DOI:10.1016/S0960-894X(02)00682-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3199–3202
P1 Phenethyl Peptide Boronic Acid Inhibitors of
HCV NS3 Protease
E. Scott Priestley,* Indawati De Lucca, Bahman Ghavimi, Susan Erickson-Viitanen
and Carl P. Decicco
Bristol-Myers Squibb Pharmaceutical Research Institute, Experimental Station, Wilmington, DE 19880-0500, USA
Received 21 June 2002; accepted 16 July 2002
Abstract—A series of peptide boronic acids containing extended, hydrophobic P1 residues was prepared to probe the shallow,
hydrophobic S1 region of HCV NS3 protease. The p-trifluoromethylphenethyl P1 substituent was identified as optimal with respect
to inhibitor potency for NS3 and selectivity against elastase and chymotrypsin.
# 2002 Published by Elsevier Science Ltd.
Hepatitis Cvirus (HCV) chronically infects approxi-
mately 170 million persons worldwide and causes ser-
ious progressive liver disease. In a substantial fraction
of cases, HCV infection leads to cirrhosis and hepato-
cellular carcinoma.1 The best treatment for HCV infec-
tion is a combination of pegylated interferon and
ribavirin, which produces sustained virological response
in only 54% of patients and causes significant side
effects, such as anemia, neutropenia, and depression.2
The unsatisfactory efficacy and safety profile of current
therapies, together with the highly variable course of
disease progression, makes clinical treatment of HCV
infection difficult.1 There is a clear need for substantially
improved antiviral therapies for HCV.
the chimpanzee disease model9 have demonstrated that
functional NS3 is required for HCV infectivity,10 vali-
dating the enzyme as a target for drug discovery.
Early in the evolution of our HCV NS3 protease med-
icinal chemistry program, we focused on optimizing the
P1 residue11 in peptide boronic acid inhibitors.12 Lit-
erature reports indicated that the protease prefers small,
hydrophobic P1 side chains (ethyl, allyl, CH2SH) in
peptide substrates13,14 and inhibitors.15À17 Inspection of
the three-dimensional structure of NS3 protease shows
that the S1 site is very hydrophobic, as it is encom-
passed by the side chains of Phe 154, Ala 157, Lys 136,
Val 132, and Leu 135. Moreover, S1 is not well defined,
but rather exists as a broad surface extending to S3. In
order to further probe structure/activity relationships in
this region of the protease active site, we have prepared
peptide boronic acids with extended, hydrophobic P1
substituents. Initially, a series of linear alkyl, terminally
branched alkyl, and aralkyl P1 residues was investi-
gated. Subsequently, an extensive series of substituted
phenethyl substituents were examined. We report that a
p-trifluoromethylphenethyl P1 residue affords optimal
inhibitor potency for NS3 and specificity with respect to
other serine proteases.
Although HCV replication is poorly characterized due
to an inability to grow the virus in cell culture, the steps
involved in processing the viral polyprotein are under-
stood in reasonable detail.3 HCV has a 9.5 kb RNA
genome that encodes a polyprotein of 3010 to 3040 aa.
Processing of the polyprotein by cellular and viral pro-
teases generates at least ten mature viral structural and
nonstructural (NS) proteins. One of these, NS3, is a
serine protease that is responsible for cleaving the four
carboxy terminal cleavage sites in the polyprotein. NS3
is well characterized biochemically,4 and has been crys-
tallized alone,5 in complex with its cofactor peptide
NS4A,6,7 and with peptide inhibitors.8 Experiments in
Peptide boronic acid inhibitors were synthesized using
the methodology for asymmetric homologation of
boronic acid pinanediol esters developed by Matteson
and co-workers.18,19 As shown in Scheme 1, reaction of
a Grignard reagent with triisopropyl borate, followed
by esterification with (+)-pinanediol affords a boronic
*Corresponding author. Tel.:+1-302-467-6881; fax:+1-302-467-6881;
e-mail: scott.priestley@bms.com
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00682-0

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E. S. Priestley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3199–3202
ester. Homologation with dichloromethyllithium20 dia-
stereoselectively provides an (S)-a-chloroboronic ester.
Displacement of chloride by lithium bis(tri-
methylsilyl)amide,21 followed by acidolysis gives the
(R)-a-aminoboronic ester as a stable hydrochloride salt.
Coupling to the protected pentapeptide Boc-Asp(OBut)-
Glu(OBut)-Val-Val-Pro-OH and deprotection with tri-
fluoroacetic acid afforded the desired peptide boronic
acids 1–12 (See Table 1 for definition of the R groups).22
required boronates were prepared by hydroboration of
a substituted styrene with catecholborane, followed by
transesterification with (+)-pinanediol.23 Subsequent
homologation, nitrogen substitution, and peptide cou-
pling afforded hexapeptides 13–32 (See Table 2 for
definition of the R groups).
A series of substituted phenethyl containing peptides
was prepared as shown in Scheme 2. In this case, the
Scheme 2. Synthesis of 13–32. Reagents and conditions: (a) catechol-
borane, 70 ꢀC; (+)-pinanediol, THF; (32–82%); (b) Cl2CHLi, THF,
À100 ꢀC; ZnCl2, À100 to 25 ꢀC; (14–92%); (c) LHMDS, THF, À78 to
25 ꢀC; 4 N HCl/dioxane, À78 ꢀC; (10–95%); (d) Boc–Asp(OBut)-
Glu(OBut)-Val-Val-Pro-OH, PyAOP, DIEA, DMF, or EDC, HOAt,
NaHCO3, C HCl2/DMF (5:1), 0 ꢀC; (8–56%); (e) 90% TFA, 5% tri-
2
isopropylsilane, 5% CH2Cl2; (87–95%). Compound 30 was prepared
from 4-tert-butoxystyrene.
Table 2. Inhibition of NS3 protease, human leukocyte elastase, and
human pancreatic chymotrypsin by P1 phenethyl peptide boronic
acidsa
Scheme 1. Synthesis of 1–12. Reagents and conditions: (a) triisopropyl
borate, Et2O, À78 to 25 ꢀC ; HSO4, 0 ꢀC; (+)-pinanediol; (21–76%);
2
(b) Cl2CHLi, THF, À100 to 25 ꢀC; (63–83%) (c) LHMDS, THF, À78
to 25 ꢀC; 4 N HCl/dioxane, À78 ꢀC; (22–90%); (d) Boc-Asp(OBut)-
Glu(OBut)-Val-Val-Pro-OH, PyAOP, DIEA, DMF; (23–62%); (e)
90% TFA, 5% triisopropylsilane, 5% CH2Cl2; (87–95%). Compounds
2 and 9 were prepared as pinacol esters, and thus are a 1:1 mixture of
diastereomers at P1. The synthesis of 8 began by esterification of
commercially available phenylboronic acid with (+)-pinanediol.
Compd
R
NS3
Elastase
Chymotrypsin
IC50 (mM)
Table 1. Inhibition of NS3 protease, human leukocyte elastase, and
human pancreatic chymotrypsin by peptide boronic acidsa
Ki (mM) IC50 (mM)
10
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
H
2-Methyl
3-Methyl
4-Methyl
2,4-Dimethyl
2,5-Dimethyl
2-Fluoro
3-Fluoro
4-Fluoro
2,6-difluoro
3-Trifluoromethyl
4-Trifluoromethyl
4-Chloro
0.008
0.82b
0.034
0.017
0.53b
1.0b
3.5
NT
5.7
0.075
NT
NT
3.7
NT
NT
NT
NT
5.0
NT
NT
NT
NT
0.8
NT
NT
1.8
1.4
1.6
0.9
0.45
0.40
0.34
0.9
0.56
0.22
0.018
0.009
0.006
0.93b
0.025
0.002
0.002
0.004
0.007
0.005
0.003
0.003
0.008
0.003
0.003
Compd
R
NS3
Ki (mM)
Elastase
IC50 (mM)
Chymotrypsin
IC50 (mM)
0.050
NT
NT
1
Ethyl
n-Butyl
n-Pentyl
n-Hexyl
i-Butyl
0.008
0.011
0.012
0.013
0.008
0.039
0.007
0.9
0.020
NT
NT
NT
0.060
NT
7.3
NT
NT
3.5
>60
2.1
0.38
0.42
NT
0.30
0.28
NT
0.070
0.075
NT
2b
3
16
0.065
NT
48
>60
>60
>60
NT
4
5
6
7
4-Bromo
4-Phenyl
i-Amyl
4-Isopropyl
4-Cyclohexyl
4-tert-Butyl
4-Hydroxy
4-Methoxy
4-Phenoxy
4-Methylpentyl
Phenyl
Benzyl
Phenethyl
Phenpropyl
Phenbutyl
8
9b
10
11
12
0.5
0.008
0.20
0.010
20
>60
NT
0.4
1.9
aEach Ki is the average of at least three independent determinations,
while each IC50 is the average of two determinations.
bIndicates inhibition data is from an IC50 measurement, not Ki.
aEach Ki is the average of at least three independent determinations,
while each IC50 is the average of two determinations.
bCompound is a 1:1 mixture of diastereomers at P1.

E. S. Priestley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3199–3202
3201
Our initial experiments focused on peptides containing
linear alkyl (1–4), terminally branched alkyl (5–7), and
aralkyl (8–12) P1 residues. Table 1 shows enzyme
inhibition data for these compounds against the cataly-
tic domain of NS3 protease and two other serine pro-
teases, human leukocyte elastase and human pancreatic
chymotrypsin.24À27
theless, our results will facilitate the design of next-gen-
eration HCV protease inhibitors based on both cyclic
peptide and peptidomimetic structures.
Acknowledgements
The authors thank Dr. Charles Kettner for generously
providing the pinacol esters of boronorleucine and
borophenylalanine and Larry Mersinger for measuring
the NS3 Ki values. We thank Lorraine Gorey-Feret and
Marina Bukhityarova for determining IC50 values for
NS3, elastase, and chymotrypsin.
The extended linear alkyl residues (2–4) each give com-
parable potency to ethyl (1) for NS3 protease, and have
only modest selectivity over chymotrypsin (30- to 190-
fold). The branched alkyls isobutyl (5) and 4-methyl-
pentyl (7) also afford inhibitors with potency compar-
able to 1, while isoamyl (6) induces a 5-fold loss in
potency. In addition, 7 has 1000-fold selectivity over
elastase and 40-fold selectivity over chymotrypsin. The
aralkyls display a more defined structure–activity rela-
tionship than either the linear or branched alkyls. Pep-
tides containing P1 phenyl (8), benzyl (9), and
phenpropyl (11) are 25- to 100-fold less potent than (1),
while the phenethyl (10) and phenbutyl (12) peptides
maintain low nanomolar potency. Compound 10
has more than 400-fold selectivity over elastase, but
only 9-fold selectivity over chymotrypsin. Inhibition of
other serine proteases has been previously observed by
peptide boronic acids with P1 phenethyl residues.28,29
References and Notes
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11. For a description of the protease nomenclature, see:-
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We subsequently investigated the properties of a series
of peptides with substituted phenethyl residues at P1 in
order to improve the potency and selectivity of the lead
structure 10 (Table 2). Substitution of the phenethyl
group at the para position is clearly preferred over ortho
substitution and equivalent or superior to meta sub-
stitution (compare 13, 14, and 15; 18, 19, and 20; 22 and
23). Disubstitution is unacceptable, even with a para
substituent (16, 17, and 21). At the para position, all
substituents tested afforded potent inhibitors, including
alkyl (15, 27, 28, and 29), aryl (26), electron-with-
drawing (20, 23, 24, and 25), and electron-donating (30,
31, and 32). Compounds with para substituted P1 resi-
dues show modest potency for elastase (IC50=0.2–5
mM) and highly variable potency for chymotrypsin
(IC50=0.050 to >60 mM). Two conclusions regarding
chymotrypsin inhibition may be drawn: A para halogen
substituent improves chymotrypsin affinity (20 and 24)
and a sterically bulky para substituent abrogates chy-
motrypsin binding (26, 27, 28, 29, and 32). Finally, the
p-trifluoromethylphenethyl residue (23) appears optimal
in this series in terms of NS3 potency (4-fold more
potent than 1) and selectivity (900-fold versus elastase
and 8000-fold vs chymotrypsin).
In summary, we have investigated a series of peptide
boronic acids containing extended P1 residues as inhib-
itors of HCV NS3 protease. The 4-trifluoro-
methylphenethyl residue was identified as optimal with
respect to inhibitor potency and selectivity. Within the
P1 phenethyl series, substantial effects on inhibitor
potency and selectivity were observed with changes in
the position and identity of the aromatic ring sub-
stituents. A clear understanding of the observed struc-
ture–activity relationships awaits a crystal structure of
one of these peptides bound to NS3 protease. None-
18. Matteson, D. S.; Majumdar, D. J. Am. Chem. Soc. 1980,
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19. Matteson, D. S.; Ray, R. J. Am. Chem. Soc. 1980, 102,
7590.

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E. S. Priestley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3199–3202
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22. All intermediates gave satisfactory NMR and mass
spectral data. Peptide boronic acids 1–32 were character-
ized by high resolution mass spectrometry and analytical
HPLC.
23. Brown, H. C.; Gupta, S. K. J. Am. Chem. Soc. 1975, 97,
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[I]/Ki,app). The thermodynamic Ki was determined from Ki,app
via the relationship for competative kinetics: Ki=Ki,app/(1 +
[S]/Km) where [S]=5 mM and Km=20 mM.
26. Human neutrophil elastase was obtained from ART Bio-
chemicals, Athens, Georgia. Stock solutions of lyophilized
enzyme (1 mg/mL) were prepared in PBS buffer containing
10% glycerol and stored at À20 ꢀC. Human neutrophil elas-
tase was assayed with the Meo-Suc-Ala-Ala-Pro-Val-p-nitro-
anilide (Sigma) as a substrate (ref 12). The hydrolysis of
substrate was monitored at 405 nm on a Hewlett-Packard
spectrophotometer. Kinetic parameters were determined in
PBS buffer at room temperature with concentration of DMSO
did not exceed 2%.
27. Human pancreatic chymotrypsin was obtained from Cal-
biochem, San Diego, CA. Stock solutions of lyophilized
enzyme (20 mM) were prepared in 1 mM hydrochloric acid and
stored at À20 ꢀC. Human pancreatic chymotrypsin was
assayed with the Suc-Ala-Ala-Pro-Phe-p-nitroanilide (Calbio-
chem) as a substrate. The hydrolysis of substrate was mon-
itored at 405 nm on a Titertek Multiscan MCC/340 plate
reader. Kinetic parameters were determined in 0.1 M Tris, pH
7.8, 10 mM CaCl2 buffer at room temperature with a con-
centration of DMSO that did not exceed 2%.
24. Compounds were tested for enzyme inhibition as their
pinanediol esters, as it has been demonstrated that boronic
acid pinanediol esters are rapidly hydrolyzed to the free boro-
nic acid in dilute aqueous solution (see ref 12).
25. Inhibition of NS3 protease activity was determined using
a modification of the reported method (Taliani, M. et al. Anal.
Biochem. 1996, 240, 60). The fluorescence-based continuous
assay contained 50 mM Tris pH 7.0, 5 mM DTT, 50% gly-
cerol, 2% CHAPS, 10 mM NS4A cofactor peptide and 4 nM
NS3 protease. Inhibitors were serially diluted into the reaction
mixture followed by a 15 min preincubation with the enzyme.
Catalysis was initiated by the addition of the fluorogenic ester
substrate Ac-DED(EDANS)EEAbuÉ[COO]ASK(DABCYL)-
NH2 (final concentration=5 mM). Assays were conducted at
ambient temperature. Enzymatic activity was monitored with
a Perkin Elmer LS 50B luminescence spectrometer (excita-
tion=360 nm, emission=530 nm, both slits were set to 10
nm). Inhibition constants were determined from a nonlinear
least squares fit of the data to the equation V[I]/Vo=1/(1 +
28. Wienand, A.; Ehrhardt, C.; Metternich, R.; Tapparelli, C.
Bioorg. Med. Chem. 1999, 7, 1295.
29. Deadman, J. J.; Elgendy, S.; Goodwin, C. A.; Green, D.;
Baban, J. A.; Patel, G.; Skordalakes, E.; Chino, N.; Claeson,
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