Solution and solid-phase synthesis of potent inhibitors of hepatitis C virus NS3 proteinase.

Article abstract of DOI:10.1016/S0960-894X(01)00816-2

A versatile route for the synthesis of homochiral alpha-ketoamide analogues of amino acids is described. Incorporation of this functionality into peptide sequences using either solution or solid-phase chemistry resulted in potent inhibitors of the Hepatitis C Virus NS3 proteinase.

Full text of DOI:10.1016/S0960-894X(01)00816-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 641–643
Solution and Solid-Phase Synthesis of Potent Inhibitors of
Hepatitis C Virus NS3 Proteinase
Rebekah Beevers, Maria G. Carr, Philip S. Jones, Steven Jordan, Paul B. Kay,
Robert C. Lazell and Tony M. Raynham*
Department of Chemistry, Roche Discovery Welwyn, 40 Broadwater Road,
Welwyn Garden City, Hertfordshire AL7 3AY, UK
Received 24 September 2001; revised 20 November 2001; accepted 3 December 2001
Abstract—A versatile route for the synthesis of homochiral a-ketoamide analogues of amino acids is described. Incorporation of
this functionality into peptide sequences using either solution or solid-phase chemistry resulted in potent inhibitors of the Hepatitis
C Virus NS3 proteinase. # 2002 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-
nificant proportion of cases of community-acquired
hepatitis worldwide. Infection by HCV frequently leads
to persistent infections that result in a range of clinical
conditions including an asymptomatic carrier state,
severe chronic active hepatitis, cirrhosis and, in some
cases, hepatocellular carcinoma.1 The viral proteinase
derived fromthe NS3 gene has proved a popular target
for drug discovery programmes in this area2,3 and
indeed work in these laboratories, amongst others, has
identified potent inhibitors based on the NS4A-4B clea-
vage site of the proteinase, for example, 1–3.4,5
significantly higher potency than had been previously
observed. Our interest in a-ketoamides extended beyond
those derived fromreadily available a-amino acids and
therefore we sought to develop a simple synthesis of this
functionality which did not require pre-synthesis of the
amino acid. We also required a route which could be
relied upon to introduce the stereochemistry in a con-
trolled manner, would be sufficiently flexible to allow
introduction of a variety of groups in either the N- or C-
terminal directions and which could be adapted for use
on solid support.
We were attracted by the work of Davies et al.,6 who
had devised a stereospecific route to b-amino-a-hyd-
roxyesters and we modified this chemistry to suit our
needs. Thus, Michael addition of lithium( S)-N-benzyl-
a-methylbenzylamide to tert-butyl hept-2-enoate
4
followed by quenching of the resultant enolate with
the Davis (1S)-(+)-(10-camphorsulphonyl)oxaziridine
introduced all the required functionality and set up the
required stereocentre7 in one pot. Hydrogenolysis then
afforded 5 which could be functionalised on either the
N- or C-terminus. For example, cleavage of the tert-
butyl ester followed by N-protection produced b-amino-
a-hydroxyacid 6. This was readily coupled to a variety
of amines (exemplified here (Scheme 1) with (S)-(À)-1-
phenylpropylamine) and the hydroxyl group then oxi-
dised using Dess–Martin periodinane.8 Thus, the
desired a-ketoamide functionality was introduced with-
out epimerisation of the sensitive adjacent chiral centre.
Removal of the N-tert-butyloxycarbonyl protecting
The development of a-ketoamide based inhibitors
enabled exploration of space occupied by the prime-side
residues of the substrate and resulted in inhibitors with
*Corresponding author. Tel.: +44-1698-736153; fax: +44-1698-
736187; e-mail: p.jones@organon.nhe.akzonobel.nl
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00816-2

642
R. Beevers et al. / Bioorg. Med. Chem. Lett. 12 (2002) 641–643
Scheme 1. (a) (i) (S)-N-Benzyl-a-methylbenzylamine, n-BuLi, THF, 0 ꢀC, 45 min; (ii) add 4, À78 ꢀC, 2 h; (iii) (1S)-(+)-(10-camphorsulphonyl)oxa-
ziridine, À78 ꢀC, 1 h, 85%; (b) (i) H2, Pd/C, 0.5 MPa, AcOH, 16 h; (ii) NaHCO3 wash, 98%; (c) TFA, 30 min, 70%; (d) (Boc)2O, NaHCO3, H2O,
.
dioxan, 16 h, 100%; (e) (S)-(À)-1-phenylpropylamine, HOBt, Me2N(CH2)3NCNEt HCl, CH2Cl2, 3 h, 76%; (f) Dess–Martin periodinane, CH2Cl2,
t
t
t
.
30 min, 70%; (g) TsOH; MeCN, 20 min; (h) Succ(O Bu)-Asp(O Bu)-Glu(O Bu)-Phe(2-Me)-Val(3-Me)-Leu-OH, HOBt, Me2N(CH2)3NCNEt HCl,
NEM, CH2Cl2, 16 h, 32% over steps g and h; (i) TFA, 30 min, 47%.
Scheme 2. (a) Fmoc-NSu, NaHCO3, pH 9–10, water, dioxan; (b) TFA, CH2Cl2 (1:1), 30 min, 61% over steps a and b; (c) RINK amide MBHA
resin, TBTU, NEM, DMF, 1 h; (d) AcOH, TBTU, NEM, DMF, 1 h; (e) piperidine, DMF (1:4), 5 min; (f) Fmoc-amino acid, TBTU, NEM, DMF, 1
h; (g) repeat steps e and f as required; (h) Dess–Martin periodinane, CH2Cl2, 1 h; (i) TFA, water (19:1), 10 min.
group, coupling to the C-terminus of a peptide (exem-
plified here with a protected pentapeptide) followed by
global deprotection with TFA afforded the desired
potent inhibitor 8 (IC50 11 nM).9
we required a more flexible route to allow straight-
forward modification of the peptide residues. Solid-phase
technology seemed particularly suited to our needs.10
Hydroxyester 5 was converted to hydroxyacid 9 which
was then coupled to RINK amide MBHA resin11 using
TBTU in DMF. Any unfunctionalised linker amines
were then capped as their acetamides. Removal of the
This route allowed rapid access to peptides containing
a-ketoamides and was particularly useful if the peptides
required for coupling were readily available. However,

R. Beevers et al. / Bioorg. Med. Chem. Lett. 12 (2002) 641–643
643
Table 1.
P7
P6
P5
P4
P3
P2
Yield/20 mg resin (mg)
Purity (%)
IC50 (nM)
Succ
Succ
Succ
Succ
Succ
Succ
(2-naphthyl)-Ac
Asp
Asp
Asp
Asp
Asp
Trp
Asp
Glu
Glu
Glu
Phe(2-Me)
Phe(2-Me)
Tyr(O-Bn)
Phe(2-Me)
Phe(2-Me)
Phe(2-Me)
Phe(2-Me)
Val(3-Me)
Gly(Phe)
Ala(3-thienyl)
3.9
5.1
3.9
3.9
3.7
2.6
2.3
80
90
80
90
70
80
80
130
42
110
20
29
41
90
Leu
Leu
Leu
Leu
Leu
Leu
Val(3-Me)
Val(3-Me)
Val(3-Me)
Val(3-Me)
Val(3-Me)
d-Val
d-Ser(OBn)
Glu
Glu
References and Notes
Fmoc protecting group produced a resin loading of 0.34
mmol/g as judged by quantitation of the released
dibenzofulvene at 301 nm. The free amine could then be
functionalised using routine solid-phase peptide
chemistry12 (exemplified here (Scheme 2) using Fmoc-l-
phenylalanine, Fmoc-3-methyl-l-valine, Fmoc-2-methyl-
l-phenylalanine, Fmoc-O-tert-butyl-l-a-glutamic acid,
Fmoc-O-tert-butyl-l-a-aspartic acid and tert-butyl
hydrogen succinate). Dess–Martin oxidation introduced
the keto-functionality and subsequent TFA mediated
cleavage fromthe resin and concomitant global depro-
tection afforded the a-ketoamide inhibitor as a white
solid.
1. Bartenschlager, R. Antiviral Chem. Chemother. 1997, 8, 281
and references cited therein..
2. Dymock, B. W.; Jones, P. S.; Wilson, F. X. Antiviral Chem.
Chemother. 2000, 11, 79 and references cited therein.
3. Dymock, B. W. Emerging Drugs 2001, 6, 13 and references
cited therein.
4. Attwood, M. R.; Bennett, J. M.; Campbell, A. D.; Can-
ning, G. G. M.; Carr, M. G.; Conway, E.; 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.; 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.
Over 130 compounds were synthesised employing this
route using an Advanced ChemTech 496 synthesiser.
Typically, 20 mg of resin afforded 2–4 mg of product
with a purity of >70% as judged by LC-MS. Examples
are shown in Table 1 together with their inhibitory
activities.
5. Bennett, J. M.; Campbell, A. D.; Campbell, A. J.; Carr,
M. G.; Dunsdon, R. M.; Greening, J. R.; Hurst, D. N.; Jen-
nings, N. S.; Jones, P. S.; Jordan, S.; Kay, P. B.; O’Brien,
M. A.; King-Underwood, J.; Raynham, T. M.; Wilkinson,
C. S.; Wilkinson, T. C. I.; Wilson, F. X. Bioorg. Med. Chem.
Lett. 2001, 11, 355.
6. Davies, S.; Bunnage, M.; Chernega, A.; Goodwin, C. J.
Chem. Soc., Perkin Trans. 1 1994, 2373.
The synthetic routes described here provided rapid
access to a range of structurally diverse peptide based a-
ketoamides. The central b-amino-a-hydroxyester moiety
was introduced in one synthetic step and was readily
elaborated either in solution or on solid support. The
resultant products are inhibitors of the HCV NS3 pro-
teinase and they demonstrated a range of activities
which allowed the structure–activity relationships to be
rapidly established for this series.
7. Control of the stereochemistry of the carbon bearing the
hydroxyl functionality was not necessary since this would be
oxidised to a ketone at a later stage. However, working with a
single diastereomer facilitated monitoring the reactions and
interpreting the NMR data for the intermediates.
8. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
9. The inhibition of HCV NS3-4A proteinase activity was
determined as reported in ref 4. Figures quoted are singly
determined IC50s.
10. Dunsdon, R. M.; Greening, J. R.; Jones, P. S.; Jordan, S.;
Wilson, F. X. Bioorg. Med. Chem. Lett. 2000, 10, 1577.
11. Nova Biochem, 0.59 mmol/g loading.
Acknowledgements
12. Jones, J. Amino Acid and Peptide Synthesis; Oxford
Chemistry Primers; Davies, S. G., Series Ed.; Oxford Uni-
versity Press: New York, 1992.
We thank James Bennett and Trevor Wilkinson for
assaying these compounds.