Pyrrolidine-5,5-trans-lactams. 4. Incorporation of a P3/P4 Urea Leads to Potent Intracellular Inhibitors of Hepatitis C Virus NS3/4A Protease

Article abstract of DOI:10.1021/ol035826v

(Matrix presented) In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a

Full text of DOI:10.1021/ol035826v

ORGANIC
LETTERS
2003
Vol. 5, No. 24
4627-4630
Pyrrolidine-5,5-trans-lactams. 4.
Incorporation of a P3/P4 Urea Leads to
Potent Intracellular Inhibitors of
Hepatitis C Virus NS3/4A Protease
Martin J. Slater,* Elizabeth M. Amphlett, David M. Andrews, Paul Bamborough,
Seb J. Carey, Martin R. Johnson, Paul S. Jones, Gail Mills, Nigel R. Parry,
Donald O’N. Somers, Alan J. Stewart, and Tadeusz Skarzynski
GlaxoSmithKline Medicines Research Center, Gunnels Wood Road,
SteVenage, SG1 2NY, U.K.
martin.j.slater@gsk.com
Received September 22, 2003
W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/orglett.
ABSTRACT
In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency
of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a 100 nM IC in the replicon cell-based surrogate
50
HCV assay.
An estimated 3% of the global human population is infected
by hepatitis C virus (HCV),1 an infection that 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-R, alone or in combina-
tion with ribavirin. 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 anticipated introduction
of pegylated interferon offers the potential to improve
sustained response rates against certain genotypes.5 The
development of new therapies to treat HCV infection
effectively is therefore of paramount importance and is
currently an intensive area of research.6
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 polyprotein is
processed by cellular signal peptidases to produce the
(4) (a) Reichard, O.; Scvarcz, R.; Weiland, O. Hepatology 1997, 26, 108S.
(b) Davis, G. L.; Esteban-Mur, R.; Rustgi, V.; Hoefs, J.; Gordon, S. C.;
Trepo, C.; Schiffman, M. L.; Zeuzem, S.; Craxi, A.; Ling, M. H.; Albrecht,
J. N. Eng. J. Med. 1998, 339, 1493. (c) McHutchinson, J. G.; Gordon, S.
C.; Schiff, E. R.; Schiffman, M. L.; Lee, W. M.; Rustgi, V.; Goodman, Z.
D.; Ling, M. H.; Cort, S.; Albrecht, J. N. Eng. J. Med. 1998, 339, 1485.
(5) (a) Luxon, B. A.; Grace, M.; Brassard, D.; Bordens, R. Clin. Ther.
2002, 24, 1363. (b) Youngster, S.; Wang, Y.-S.; Grace, M.; Bausch, J.;
Bordens, R.; Wyss, D. F. Curr. Pharm. Des. 2002, 8, 2139.
(1) World Health Organization Weekly Epidemiological Record 1997,
72, 65.
(2) (a) Gerber, M. A. J. Hepatol. 1993, 17 (suppl 3), 108S. (b) Alter,
M. J. Hepatology 1997, 26, 62S. (c) Hoofnagle, J. H. Hepatology 1997,
26, 15S. (d) Kwong, A. D. AntiViral Res. 1998, 40, 1. (e) Hoofnagle, J. H.
Digestion 1998, 59, 563.
(6) (a) Moradpour, D.; Brass, V.; Gosert, R.; Rainer, Wolk, B.; Blum,
H. E. Trends Mol. Med. 2002, 8, 476. (b) Tan, S.-L, Pause, A.; Shi, Y.;
Sonenberg, N. Nat. ReV. Drug DiscoVery 2002, 11, 867.
(3) (a) Di Bisceglie, A. M. Lancet 1998, 351, 351. (b) Brown, J. L. Lancet
1998, 351, 83.
10.1021/ol035826v CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/06/2003

structural viral proteins (C, E1, E2, p7), whereas viral
proteases (NS2, NS3) are responsible for the production of
mature nonstructural replicative proteins. The multifunctional
70-kD NS3 protein has been extensively investigated.7 The
amino-terminal third of the protein is a trypsin-like serine
protease that cleaves the NS3-4A, NS4A-NS4B, NS4B-
NS5A, and NS5A-NS5B junctions.
Although isolated NS3 is enzymatically active, it forms a
heterodimer with the NS4A cofactor constituting an integral
structural component of the enzyme.8 It has been reported
that when appropriate mutations were made in the NS3
protease region of the HCV genome, the infectivity of these
RNAs in chimpanzees was abolished.9 NS3 protease is thus
an essential viral function and an excellent target for the
development of novel anti-HCV agents.
Our initial report described the design of R or â ethyl-
substituted pyrrolidine trans-lactams such as 1.10 Subse-
quently, we published the further elaboration of the template
to provide compound 2, in which the symmetrical spiro-
cyclobutyl group has replaced the ethyl P1 substituent and
cyclopropylcarbonyl has replaced the methanesulfonyl sub-
stituent on the lactam. (Figure 1).11 Herein we describe the
substituted. Even the most promising leads from this ap-
proach were reduced in potency by approximately 3-fold
compared with the standard Boc-valine derivative (2) and
could not be improved upon, either with further Boc
replacements or cyclic substituents (data not shown). This
is consistent with earlier amino acid scanning using Boc and
CBZ-protected amino acids and confirms that valine is the
optimal choice for P3.11 Our attention then focused on the
linker from the valine nitrogen. Carbamate derivatives of this
type have been previously disclosed, and indeed there is a
strong preference for small hydrophobic groups such as tert-
butyl.11
Commercially available isocyanates were employed to
synthesize an initial small set of ureas. To gain access to a
larger and more diverse set of ureas such as 4, we prepared
a p-nitrophenyl carbamate derivative of the amine 3 in situ
and reacted this with a set of amines (R1R2NH) (Scheme 1).
Scheme 1. Initial Array Synthesis of P3-P4 Ureasa
Figure 1. Pyrrolidine-5,5-trans-lactams as inhibitors of HCV NS3/
4A protease.
optimization of the interactions in the S3 and S4 protein
subsites leading to sub-micromolar inhibitors in the HCV
replicon assay.
In the course of the optimization of 2, a diverse range of
analogues was prepared wherein the pyrrolidine nitrogen of
the homochiral template is amide- and sulfonamide-
a Reagents and conditions: (i) HCl, dioxan; (ii) p-nitrophenyl
chloroformate (1.5 equiv), EtiPr2N (2 equiv), CH2Cl2, THF; then
R1R2NH (3 equiv), MeCN in situ then aqueous sodium carbonate
workup.
(7) (a) Clarke, B. E. J. Gen. Virol. 1997, 78, 2397. (b) Major, M. E.;
Feinstone, S. H. Hepatology 1997, 25, 1527.
(8) Kim, J. L.; Morgenstern, K. A.; Lin, C.; Fox, T.; Dwyer, M. D.;
Landro, J. A.; Chambers, S. P.; Markland, W.; Lepre, C. A.; O’Malley, E.
T.; Harbeson, S. L.; Rice, C. M.; Murcko, M. A.; Caron, P. R.; Thomson,
J. A. Cell 1996, 8, 344.
(9) Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M. J.
Virol. 2000, 74, 2046.
(10) Slater, M. J.; Andrews, D. M.; Baker, G.; Bethell, S.; Carey, S.;
Chaignot, H.; Clarke, BE.; Coomber, B.; Ellis, M.; Good, A.; Gray, N.;
Hardy, G.; Jones, P.; Mills G.; Robinson, E. Bioorg. Med. Chem. Lett. 2002,
12, 3359.
(11) (a) Andrews, D. M.; Carey, S. J.; Chaignot, H.; Coomber, B. A.;
Gray, N. M.; Hind, S. L.; Jones, P. S.; Mills, G.; Robinson, J. E.; Slater,
M. J. Org. Lett. 2002, 4, 4475. (b) Andrews, D. M.; Chaignot, H.; Coomber,
B. A.; Good, A. C.; Hind, S. L.; Johnson, M. R.; Jones, P. S.; Mills, G.;
Robinson, J. E.; Skarzynski, T.; Slater, M. J.; Somers, D. O’N. Org. Lett.
2002, 4, 4479. (c) Andrews, D. M.; Chaignot, H. M.; Coomber, B. A.;
Dowle, M. D.; Hind, S. L.; Johnson, M. R.; Jones, P. S.; Mills, G.; Patikis,
A.; Pateman, T. J.; Robinson, E.; Slater, M. J.; Trivedi, N. Eur. J. Med.
Chem. 2003, 38, 339.
We noted that compounds such as the dimethylaminopropyl
urea 4a were more active than the standard (2) when tested
initially, whereas the highly purified sample of 4a was
consistently less active than the standard (see Table 1). On
further investigation by LC-MS, we noted that the “active
sample” contained a higher molecular weight impurity
consistent with the trans-lactam dimer 5. We reasoned that
formation of the carbamate of 3 had been incomplete and
that remaining 3 was available to react with the p-nitrophenyl
carbamate and form the dimer 5. Indeed, on repeating the
synthesis with 2 equiv of trans-lactam 3 and in the absence
of an additional amine, 5 became the major product. The
activity of 5 was striking (Kobs/I ) 1530 M-1 s-1) and the
most potent inhibitor of NS3/4A we had encountered, which
4628
Org. Lett., Vol. 5, No. 24, 2003

Scheme 2. Synthesis of Valyl Urea Pyrrolidine-5,5-trans-lactamsa
a Reagents and conditions: (i) (a) sodium hydrogen carbonate/CH2Cl2 extraction to generate free base; (b) p-nitrophenyl chloroformate,
pyridine, CH2Cl2, (86%); (ii) valine derivative, EtiPr2N, MeCN or Et3N, CH2Cl2; (iii) H2, 5% Pd/C, EtOH (92%) when X ) PhCH2O2C;
(iv) 4-(2-pyrimidinyl)piperazine, HATU, EtiPr2N, MeCN; (v) RNH2, Et3N, CH2Cl2.
also translated into potent inhibition in the surrogate cellular
assay, the HCV replicon.12
A small program of work was undertaken based upon the
bisvalyl urea amides, particularly with a view to improving
aqueous solubility. Thus, the benzyl ester in 7b was removed
by standard hydrogenolysis, and the resulting acid 8 was
coupled to a range of amines. The 4-(2-pyrimidinyl)-
piperazinyl amide 9 illustrates the results of this approach
and is one of the most potent enzyme inactivators of this
series, with sub-micromolar potency in the HCV replicon
(Table 2).
Table 1. NS3/4A Protease and HCV Replicon Activities
NS3/4A
protease
IC50 (µM)
NS3/4A
protease
Kobs/I (M-1 s-1
)
HCV
replicon12
compound
IC50 (µM)
2
0.5
0.66
nt
400
ntb
1530
4.0
14
0.39
We then investigated replacing the carbonyl moiety in the
urea by a sulfonyl group. Treatment of the amine 3 with
sulfuryl chloride followed by the tert-butyl ester of valine
4a a
5
a Purified sample of 4a. b Not tested.
Table 2. NS3/4A Protease and HCV Replicon Activities of
Urea Pyrrolidine-5,5-trans-lactams
Further SAR to exploit this observation was carried out,
and our initial objective was to assess the requirement for
two trans-lactam units. To prevent the formation of 5, the
carbamate 6 was purified by silica column chromatography
and then subjected to a separate reaction step with amine
nucleophiles (Scheme 2). Three simple esters of valine and
valinol were selected and provided the bisvalyl ureas 7a-
d. Biochemical potency data (Table 2) clearly illustrated that
the second trans-lactam unit could be dispensed with, and
the critical role of the second valine unit was evident, since
these molecules possessed similar or better potency than 5.
Moreover, activity in the HCV replicon assay was
maintained, with 7b, the benzyl ester, providing excellent
cellular activity (IC50 ) 100 nM). Additionally, the valinol
derivative 7d demonstrated good activity in both assays,
indicating that the ester could also be dispensed with.
NS3/4A protease
HCV replicon
compound
Kobs/I (M-1 s-1
)
IC50 (µM)
5
7a
7b
7c
7d
9
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
11
1530
1750
1335
2290
1072
7761
395
376
324
167
390
853
912
922
543
458
89
0.39
0.48
0.10
0.38
0.50
0.30
2.2
4.5
1.6
8.5
6.3
2.8
2.6
1.2
1.6
3.5
(12) (a) Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilmann,
L.; Bartenschlager, R. Science 1999, 285 (5424), 110. (b) Blight, K. J.;
Kolykhalov, A. A.; Rice, C. M. Science 2000, 290 (5498), 1972. (c)
Bartenschlager, R.; Lohmann, V. AntiViral Res. 2001, 52, 1 (d) Parry, N.
R.; Chung, V.; Viner, K. C.; Carroll, A. R. AntiViral Res. 2002, 53 (3)
A73.
nta
12
340
nta
a Not tested.
Org. Lett., Vol. 5, No. 24, 2003
4629

yielded 11, the sulfonyl urea analogue of 7a. Employing a
molar excess of the translactam amine 3 provided a sample
of the sulfonyl urea dimer 12 after column chromatography
(Scheme 3). Biochemical potencies of 11 and 12 were
following acylation of the active site serine.11 Both of the
urea NH moieties are clearly shown to be capable of forming
a hydrogen bond with the main chain carbonyl of Ala 157,
facilitating a good fit of the valine isopropyl onto the S3
surface and the cyclopentyl moiety into the S4 subsite (Figure
2). The urea-carbonyl bidentate interaction can facilitate the
Scheme 3. Synthesis of Bisvalyl Sulfonyl Urea
Pyrrolidine-5,5-trans-lactamsa
a Reagents and conditions: (i) 2-amino-3-methyl-butyric acid
tert-butyl ester, SO2Cl2, EtiPr2N (5 equiv), CH2Cl2 (18%); (ii)
SO2Cl2, EtiPr2N (3 equiv), CH2Cl2, (9%).
Figure 2. Pyrrolidine-5,5-trans-lactam crystal structure. Numbers
indicate distances between heteroatoms in angstroms.
W A 3D rotatable image of the structure in PDB format is available.
considerably reduced, indicating a strong preference for the
planar urea linker (Table 2).
initial binding interaction and accommodate the movement
of the trans-lactam upon acylation.
Because our goal is the development of anti-HCV drug
molecules suitable for delivery by oral administration, we
were concerned that the bisvalyl urea amides have molecular
weights in the region 600 Da. We thus directed our attention
to simpler ureas more likely to achieve a combination of
good potency in the cellular assay with an acceptable
pharmacokinetic profile. Table 2 illustrates the SAR for some
of these simple ureas, again prepared from the carbamate 6
(Scheme 2). Smaller groups such as ethyl and n-propyl
demonstrated activities similar to that of the tert-butyl
carbamate standard. Many of these lipophilic P4 substituents
conferred similar levels of biochemical and replicon potency,
with cellular activities in the 1-5 µM range. Cyclohexyl
and cyclopentyl urea substituents (10g, 10h) were identified
as having the best overall profile of biochemical and cellular
potency.
In summary, we have optimized the P3 and P4 interactions
of the pyrrolidine trans-lactam template utilizing a urea
linker. Bisvalyl ureas afford sub-micromolar inhibitors in the
HCV replicon assay, and simple lipophilic ureas provide low
micromolar inhibitors with excellent developability proper-
ties. The pharmacokinetic optimization of these potent
molecules is described in the following paper.
Acknowledgment. We thank Drs. Berwyn Clarke and
George Hardy for support and encouragement; Graham
Baker, Sue Bethell, and Malcolm Ellis for provision of NS3
protease protein and initial assay systems; Michael Barnes
for the synthesis of intermediates; and Gianpaolo Bravi for
assistance with molecular modeling.
Supporting Information Available: Detailed experi-
mental procedures for synthesis of representative compounds,
characterization data for test compounds, biochemical and
replicon assay methodology, and X-ray crystallographic
methodology. This material is available free of charge via
the Internet at http://pubs.acs.org.
An X-ray structure of the cyclopentyl compound 10g was
obtained by soaking into preformed crystals of the NS3
protease with domain:NS4A cofactor.13 Features of the trans-
lactam core were consistent with those previously described
(13) Deposition of the atomic coordinates into the Protein Data Bank is
in progress.
OL035826V
4630
Org. Lett., Vol. 5, No. 24, 2003