The identification of α-ketoamides as potent inhibitors of hepatitis C virus NS3-4A proteinase

Article abstract of DOI:10.1016/S0960-894X(00)00654-5

Peptides based upon the non-prime side residues of the NS4A-4B cleavage site of hepatitis C virus (HCV) NS3-4A proteinase containing an α-ketoamide moiety in place of the scissile amide bond are potent inhibitors of this enzyme.

Full text of DOI:10.1016/S0960-894X(00)00654-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 355±357
The Identi®cation of ꢀ-Ketoamides as Potent Inhibitors of
Hepatitis C Virus NS3-4A Proteinase
J. M. Bennett,b A. D. Campbell,a A. J. Campbell,a M. G. Carr,a R. M. Dunsdon,a
J. R. Greening,a D. N. Hurst,a N. S. Jennings,a P. S. Jones,a S. Jordan,a P. B. Kay,a
M. A. O'Brien,a J. King-Underwood,a T. M. Raynham,a C. S. Wilkinson,b
T. C. I. Wilkinsonb and F. X. Wilsona,*
aDepartment of Chemistry, Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AY, UK
bDepartment of Viral Diseases, Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AY, UK
Received 13 September 2000; revised 27 October 2000; accepted 16 November 2000
AbstractÐPeptides based upon the non-prime side residues of the NS4A-4B cleavage site of hepatitis C virus (HCV) NS3-4A pro-
teinase containing an a-ketoamide moiety in place of the scissile amide bond are potent inhibitors of this enzyme. # 2001 Elsevier
Science Ltd. All rights reserved.
Hepatitis C virus (HCV) is the cause of the majority of
cases of transfusion-associated hepatitis and a sig-
ni®cant proportion of community-acquired hepatitis
worldwide. Infection by HCV can lead to a range of
clinical conditions including an asymptomatic carrier
state, severe chronic active hepatitis, cirrhosis and, in
some cases, hepatocellular carcinoma.1 Current thera-
pies for HCV infection include treatment with inter-
feron-a in combination with ribavirin, but this therapy
is of only limited ecacy. Hence, a new treatment for
the disease would be of great interest.2
The HCV NS3 protein encodes a serine proteinase
which is responsible for the cleavage at the NS3-4A,
NS4A-4B, NS4B-5A, and NS5A-5B junctions in the
viral polyprotein. The 54 amino acid NS4A protein is a
co-factor which binds to the NS3 protein and enhances
its proteolytic activity.3 This NS3-4A proteinase is one
of the most intensively studied targets for HCV antiviral
therapy.2
These compounds incorporate an electrophile at the C-
terminus of the P1-residue and are potent inhibitors of
this enzyme.4 6 The use of aldehydes and boronic acids
to terminate the inhibitors at that point prevented
exploration of the structure±activity relationships of the
prime-side. Potent a-keto acids have been reported7 but
again these terminate the inhibitor. The introduction of
an a-keto amide functionality, to give inhibitors such as
3, should a€ord an electrophilic carbonyl, whilst retaining
the opportunity of prime-side exploration.8,9 We there-
fore undertook the synthesis of this class of inhibitors to
determine their ecacy against HCV NS3-4A protei-
nase.10 The synthesis of one such inhibitor11 is outlined
in Scheme 1.12
We recently described the design and synthesis of mole-
cules such as 1 and 2, derived from the non-prime side
residues of the NS4A-4B cleavage site of the proteinase.
*Corresponding author. Tel.: +44-1707-366464; fax: +44-1707-373-
504; e-mail: francis.wilson@roche.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00654-5

356
J. M. Bennett et al. / Bioorg. Med. Chem. Lett. 11 (2001) 355±357
Scheme 1. (i) N,O-Dimethylhydroxylaminehydrochloride, diisopropylethylamine, hydroxybenzotriazole (HOBT), 1-(3-diethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI), tetrahydrofuran, room temperature (rt), 16 h, 91%; (ii) LiAlH4, tetrahydrofuran, 0 ꢀC, 30 min, 87%; (iii)
acetone cyanohydrin, Et3N, CH2Cl2, rt, 1.5 h, 93%; (iv) HCl, re¯ux, 17 h; (v) Boc2O, NaHCO3, H2O, 3 days, 84% over two steps; (vi) BnNH2,
HOBT, EDCI, CH2Cl2, rt, 2 h, 76%; (vii) Dess±Martin periodinane, CH2Cl2, rt, 1 h, 65%; (viii) TsOH, MeCN, rt, 1 h, 91%; (ix) protected penta-
peptide fragment EDCI, HOBT, N-ethyl morpholine, CH2Cl2, rt, 6 h, 32%; (x) TFA, 30 min, 76%.
Table 1.
Gratifyingly, as can be seen in Table 1, the activities13 of
the optimal a-ketoamides are all in the nanomolar range
and are approximately 10-fold more potent than their
corresponding aldehyde or boronic acid analogues. The
primary amide in 4 can be substituted with benzyl or (2-
naphthyl)-methyl moieties, which in turn can be sub-
stituted around the aromatic ring. The stereospeci®c
nature of the interaction is clearly indicated by the e€ect
of substitution at the 1-position where a 250-fold dis-
crimination is observed for introduction of a chiral
methyl group (compare compounds 6 and 7). Replace-
ment of the CH2CF3 sidechain at P1 by butyl had little
e€ect on potency, but improved the plasma stability of
the compound.
Compound
P1
NHR
NH2
IC50 (nM)
4
5
CH2CF3
CH2CF3
7
4
6
7
CH2CF3
CH2CF3
4
These inhibitors also display selectivity for HCV NS3-
4A proteinase over other important human serine pro-
teinases. The data for inhibitor 14 is shown in Table 2.
1100
8
9
CH2CF3
CH2CF3
7
7
In summary, the introduction of the a-ketoamide elec-
trophile has led to the identi®cation of highly potent
inhibitors of the NS3-4A proteinase. Further work on
this exciting class of inhibitors will be reported else-
where.17
10
11
12
13
14
CH2CF3
CH2CF3
CH2CF3
CH2CF3
Bu
4
6
References and Notes
4
1. Cuthbert, J. A. Clin. Microbiol. Rev. 1994, 7, 505.
2. Dymock, B. W.; Jones, P. S.; Wilson, F. X. Antiviral Chem.
Chemother. 2000, 11, 79.
3. Bartenschlager, R. Antiviral Chem. Chemother. 1997, 8, 281
and references cited therein.
8
NH2
11
4. Attwood, M. R.; Bennett, J. M.; Campbell, A. D.; Can-
ning, G. G. M.; Carr, M. G.; Dunsdon, R. M.; Greening,
J. R.; Jones, P. S.; Kay, P. B.; Handa, B. K.; Hurst, D. N.;
Jennings, N. S.; Jordan, S.; Keech, E.; O'Brien, M. A.;
Overton, H. A.; King-Underwood, J.; Raynham, T. M.;
Stenson, K. P.; Wilkinson, C. S.; Wilkinson, T. C. I.; Wilson,
F. X. Antiviral Chem. Chemother. 1999, 10, 259.
Table 2. Activity of 14 against important human serine proteinases
Enzyme
IC50 (nM)
1.
2.
3.
4.
HCV NS3-4A Proteinase
Elastase14
11
12,000
300
Chymotrypsin15
Trypsin16
5. Attwood, M. R.; Hurst, D. N.; Jones, P. S.; Kay, P. B.;
Raynham, T. M.; Wilson, F. X. Patent WO9822496A2.
>200,000

J. M. Bennett et al. / Bioorg. Med. Chem. Lett. 11 (2001) 355±357
357
6. Attwood, M. R.; Hurst, D. N.; Jones, P. S.; Kay, P. B.;
Raynham, T. M.; Wilson, F. X. Patent GB2337262A1.
7. Di Marco, S.; Rizzi, M.; Volpari, C.; Walsh, M. A.; Narjes,
F.; Colarusso, S.; De Francesco, R.; Matassa, V. G.; Sollazzo,
M. J. Biol. Chem. 2000, 275, 7152.
11. All novel compounds were characterised by 1H NMR and
HPLC.
12. The protected peptide fragment is Succ(OtBu)-
Asp(OtBu)-Glu(OtBu)-(oMe)Phe-tBuGly-Leu-OH prepared
as described in ref 10.
8. Edwards, P. D.; Bernstein, P. R. Med. Res. Rev. 1994, 14,
127 and references cited therein.
13. The inhibition of HCV NS3-4A proteinase activity was
determined as reported in ref 4.
9. In the course of this work others have reported a-keto-
amides as HCV NS3-4A proteinase inhibitors: Han, W.; Hu,
Z.; Jiang, X.; Decicco, C. P. Biorg. Med. Chem. Lett. 2000, 10,
711. Also ref 2 and references cited therein.
10. Hurst, D. N.; Jones, P. S.; Kay, P. B.; Raynham, T. M.;
Wilson, F. X. Patent GB2338482A1.
14. Castillo, M. J.; Nakajima, K.; Zimmerman, M.; Powers,
J. C. Anal. Biochem. 1979, 99, 53.
15. Oleksyszyn, J.; Powers, J. C. Biochemistry 1991, 30, 485.
16. Kanaoka, Y.; Takahashi, T.; Nakayama, H.; Takada, K.;
Kimura, T.; Sakakibara, S. Chem. Pharm. Bull. 1977, 25, 3126.
17. Manuscript in preparation.