Design and synthesis of ethyl pyrrolidine-5,5-trans-lactams as inhibitors of hepatitis C virus NS3/4A protease.

Article abstract of DOI:10.1016/S0960-894X(02)00789-8

Using a pyrrolidine-5,5-trans-lactam template, we have designed small, neutral, mechanism-based inhibitors of hepatitis C NS3/4A protease. Compound 11a, with an alpha-ethyl P1 substituent and a Boc-valine substituent at the pyrrolidine nitrogen, has an IC(50)=30 microM.

Full text of DOI:10.1016/S0960-894X(02)00789-8

Bioorganic& Medicinal Chemistry Letters 12 (2002) 3359–3362
Design and Synthesis of Ethyl Pyrrolidine-5,5-trans-lactams as
Inhibitors of Hepatitis C Virus NS3/4A Protease
Martin J. Slater,* David M. Andrews, Graham Baker, Susanne S. Bethell, Seb Carey,
Helene Chaignot, Berwyn Clarke, Barry Coomber, Malcolm Ellis, Andrew Good,
Norman Gray, George Hardy, Paul Jones, Gail Mills and Ed Robinson
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
Received 1 July 2002; revised 24 August 2002; accepted 11 September 2002
Abstract—Using a pyrrolidine-5,5-trans-lactam template, we have designed small, neutral, mechanism-based inhibitors of hepatitis
C NS3/4A protease. Compound 11a, with an alpha-ethyl P1 substituent and a Boc-valine substituent at the pyrrolidine nitrogen,
has an IC50=30 mM.
# 2002 Elsevier Science Ltd. All rights reserved.
An estimated 3% of the global human population is
infected by hepatitis C virus (HCV),1 an infection which
often leads to cirrhosis, hepatocellular carcinoma and
liver failure in later life.2 It has been estimated that of
those currently infected, 20% and 4% are likely to
develop liver cirrhosis and liver cancer, respectively, in
the next decade.3 Current therapies are based upon the
use of interferon-a, alone or in combination with riba-
virin. Although patients sustained response rates are
markedly improved using combination therapies, at
least 50% of patients fail to show a sustained response.
Additionally, these therapies have the disadvantage of
frequent and severe side-effects.4 The development of new
therapies to treat HCV infection effectively is therefore
of paramount importance, and is currently an intensive
area of research.5
terminal third of the protein is a trypsin-like serine pro-
tease that cleaves the NS3-4A, NS4A-NS4B, NS4B-
NS5A, and NS5A-NS5B junctions.
Although NS3 is enzymatically active on its own, NS4A
interacts with it to form an integral structural compo-
nent of the enzyme.7 The NS3/4A heterodimer mod-
ulates protease activity, and is essential for cleavage at
the NS3–4A and NS4B-NS5A junctions. It has been
reported that when appropriate mutations were made in
the NS3 protease region of the HCV genome, the infec-
tivity of these RNAs in chimpanzees was abolished.8 NS3
protease is thus an essential viral function and an excel-
lent target for the development of novel anti-HCV agents.
A number of peptidicand non-peptidicinhibitors of the
serine protease have been reported.5,9 The former have
been mainly competitive inhibitors designed from pep-
tide substrates or cleavage products, whilst the non-
peptidicmoleucles have emerged through random
screening and have displayed non-competitive mechan-
isms of action. As a starting point for the design of
inhibitors we sought to take advantage of the pyrro-
lidine-5,5-0trans-lactam template which allows access to
the S1, S1 and S3–S4 specificity pockets, and traps the
active site serine hydroxyl group by acylation (Fig. 1).
These proven templates, developed as thrombin,10
human neutrophil elastase (HNE)11 and human cyto-
megalovirus (HCMV) inhibitors,12 are active intracellu-
larly, stable in plasma, and orally active in vivo. We
HCV is a small enveloped virus, the genome of which is
a 9.5 kb single stranded RNA that encodes for a single
large polyprotein of 3010–3030 amino acids. This poly-
protein is processed by cellular signal peptidases to
produce the structural viral proteins (C, E1, E2, p7),
whereas viral proteases (NS2, NS3) are responsible for
the production of mature non-structural replicative
proteins. The multifunctional 70kD NS3 protein is the
most extensively studied viral protein.6 The amino-
*Corresponding author. Tel.:+44-1438-763416; fax:+44-1438-
763620; e-mail: mjs40312@gsk.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00789-8

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M. J. Slater et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3359–3362
Figure 1. Generic5,5 pyrrolidine trans-lactam template and part of the 5A/5B substrate.
now report the design of such mechanism-based, low
molecular weight, non-peptidic 5,5-trans-lactam mole-
cules as inhibitors of NS3/4A protease.
coupling conditions to provide a diverse set of compounds
(data not shown). To our surprise, none of these were
active up to concentrations of 300 mM, even with long pre-
incubation times (up to 18 h). Compounds 5, 6 and 7 were
similarly inactive. At this point we chose to append short
peptides to 7 which mimic the P3–P6 amino acid residues in
the natural substrate. Compound 1a, containing the nat-
ural P3–P6 sequence found at the 5A–5B cleavage site (Ac-
E-D-V-V), was thus prepared and did indeed inhibit the
protease in a time-dependent manner (Table 1).14 The IC50
for 1a was 19 mM when the compound was pre-incubated
with enzyme for 18 h and 69 mM with 2 h pre-inc ubation.
Whilst cysteine is established as the preferred P1 amino
acid for intermolecular cleavage of substrates by NS3,
the ethyl group has been shown to be an acceptable
inert replacement in substrates and inhibitors,9 and was
chosen as the substituent 0adjacent to the carbonyl group
in the lactam. For the P1 substituent, we initially selec-
ted methanesulphonyl to mimicthe serine or alanine
0
commonly found at P1 , and this functionality also
serves to activate the lactam moiety. The remaining
binding pockets on the non-primed side of the cleavage
site (Pn) may then be accessed via substituents at the
pyrrolidine nitrogen (Fig. 1). The 5,5-trans lactam com-
pound 7 (Scheme 1) thus became our initial target.
By making single amino acid replacements and trun-
cating the peptidic portion, we were able to delineate
SAR and in Table 1 present key findings. The key role
of the l-valine at P3 is illustrated by the weak activity of
the corresponding glycine analogue (1b), d-valine ana-
logue (1c) and proline analogue (1d). A key and surpris-
ing observation was that on truncating the side-chain, we
observed that a simple Boc-valine substituent (compound
1g) had similar activity to the multiple-acid-containing-
peptide 1a, and was more active than 1h (valine).
The key intermediate 2 was prepared by reported meth-
ods11 and the flexible acyliminium ion route developed
by Macdonald and co-workers11 was adapted to intro-
duce the ethyl side-chain. With 3 in hand, selective
hydrolysis of the trifluoroacetamide group (K2CO3/
EtOH), ring closure with tBuMgCl to form the trans-
lactam 5, lactam activation (MeSO2Cl/LiHMDS/THF),
and deprotection of the pyrrolidine 6 (H2/10%Pd,C/
HCl/iPrOH) proceeded smoothly to provide the pyrro-
lidine trans-lactam 7 as the hydrochloride salt.13
The data in Table 1 were generated using our initial
chromogenic assay to evaluate protease inhibition,
employing NS3 protease domain, synthetic18-mer 4A
cofactor, and Ac-E-D-V-V-P-C-pNO2 anilide as sub-
strate.15 We then moved to a fluorogenicassay system
using full-length NS3/4A protein and Abz-E-D-V-V-P-
C-S-M-S-(3-NO2)-Y-H as substrate,15 as this assay has
the advantage of delivering linear kinetics and greater
In order to demonstrate the utility of this template against
NS3 protease and explore SAR, a large range of car-
boxylicacids ( ꢀ200) were coupled to 7 under standard
Scheme 1. (i) BF3.OEt2, DCM; (ii) K2CO3, EtOH, 37% from 2; (iii) tBuMgCl, THF; (iv) LiHMDS, MeSO2Cl, THF; (v) H2, Pd/C, iPrOH, HCl;
(vi) HATU, DIPEA, RCO2H, MeCN or DMF; (vii) HCl, dioxan.

M. J. Slater et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3359–3362
Table 1. NS3/4A protease activities of compounds 1a–1h in the chromogenic assay
3361
Compd
R
% Inhibition at 100 mM 2 h
pre-incubation
% Inhibition at 100 mM 18 h
pre-incubation
1a
1b
1c
1d
1e
1f
Ac-E-D-V-V-
Ac-E-D-V-G-
Ac-E-D-V-(D)V-G-
Ac-E-D-V-P-
Ac-D-V-V-
Ac-V-V-
IC50=69 mM
IC50=19 mM
24
10
24
5
68
21
52
11
85
11
94
45
IC50=27 mM
41
1g
1h
Boc-V-
H-V-
Table 2. NS3/4A protease inhibition
Compd
R
Chromogenicassay
Fluorogenicassay
IC50/mM 4 h
pre-incubation
IC50/mM 18 h
pre-incubation
IC50/mM 4 h
pre-incubation
IC50/mM 18 h
pre-incubation
1a
1g
11a
13a
11b
13b
SO2Me
SO2Me
SO2Me
SO2Me
COMe
COMe
69
62
30
99
88
>400
19
27
22
28
45
147
>400
182
284
140
341
193
216
172
194
447
sensitivity. Cross-checking with this fluorogenic assay
showed that the SAR was consistent between assays,
although compounds generally displayed higher IC50
values in this system (see Table 2).
mer (containing 25% of the beta isomer).13 A much
more efficient separation involved derivatisation of the
lactam 5 with the tert-butyldimethylsilyl group, as the
isomers 8 and 9 could be completely separated by Bio-
tage chromatography (cyclohexane:ethyl acetate, 6:1).
Fluoride deprotection using tetrabutyl ammonium fluo-
ride buffered with acetic acid, reaction with methane-
sulphonyl chloride or acetyl chloride, and exchange of
CBZ for Boc-valine was then performed on the indivi-
dual isomers in the standard manner, to deliver the tar-
gets 11a, 11b, 13a, 13b.
As 1g was a 1:1 mixture of alpha and beta ethyl diastereo-
isomers, we sought to separate these and determine the
preferred stereochemistry of the ethyl substituent for
inhibition. In addition to the methanesulphonyl group,
we chose also to explore acetyl substitution at the lac-
0
tam to exemplify a different small activating P1 sub-
stituent (Scheme 2). The isomers of the CBZ-protected
pyrrolidine 5 were partially separated by crystallisation
from ethyl acetate, which afforded the beta isomer
(containing 20% of the alpha isomer). Silica column
chromatography of the mother liqueur (ethyl acetate:
cyclohexane, 2:1 as eluant) then provided the alpha iso-
Both alpha and beta ethyl isomers were active, although
the alpha–ethyl compounds (11a, 11b) were found to be
more rapid enzyme inhibitors than the beta-counter-
parts (13a, 13b). Methanesulphonyl was a more active
lactam substituent than acetyl (Table 2).
Scheme 2. (i) tBDMSCl, LiHMDS, THF; (ii) nBu4NF, AcOH, THF; (iii) MeSO2Cl for a, MeCOCl for b, LiHMDS, THF; (iv) H2, Pd/C, iPrOH,
HCl; (v) HATU, DIPEA, Boc-valine, MeCN.

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M. J. Slater et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3359–3362
In conclusion, we have designed low molecular weight,
neutral inhibitors of the NS3/4A protease using a
pyrrolidine trans-lactam template. These provide excel-
lent starting points for the design of potential HCV
drugs. The further optimisation of these inhibitors will
be reported in due course.
M. D.; Landro, J. A.; Chambers, S. P.; Markland, W.; Lepre,
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13. The relative stereochemistry is assigned on the basis of
coupling constants and NOE experiments in the 1H NMR
spectrum, and by comparison with compounds described in
references 11 and 12.
14. The detailed kinetics of enzyme inhibition and mechanism
of action will be the subject of a separate publication.
15. Chromogenic assay: Enzyme concentration was 0.56 mM,
4A concentration was 0.8 mM, substrate concentration was
1.5mM, substrate Km=1.85mM. Flourogenicassay: Enzyme
concentration was 20 nM, NS3/4A ratio 1:1, substrate con-
centration was 25 mM, substrate: Km=5 mM.
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