Optimization of novel acyl pyrrolidine inhibitors of hepatitis C virus RNA-dependent RNA polymerase leading to a development candidate

Article abstract of DOI:10.1021/jm061207r

Optimization of a pyrrolidine-based template using structure-based design and physicochemical considerations has provided a development candidate 20b (3082) with submicromolar potency in the HCV replicon and good pharmacokinetic properties.

Full text of DOI:10.1021/jm061207r

© Copyright 2007 by the American Chemical Society
Volume 50, Number 5
March 8, 2007
Letters
Optimization of Novel Acyl Pyrrolidine Inhibitors
of Hepatitis C Virus RNA-Dependent RNA
Polymerase Leading to a Development Candidate
Martin J. Slater,* Elizabeth M. Amphlett,
David M. Andrews, Gianpaolo Bravi, George Burton,
Anne G. Cheasty, John A. Corfield, Malcolm R. Ellis,
Rebecca H. Fenwick, Stephanie Fernandes,
Rossella Guidetti, David Haigh, C. David Hartley,
Peter D. Howes, Deborah L. Jackson, Richard L. Jarvest,
Victoria L. H. Lovegrove, Katrina J. Medhurst,
Nigel R. Parry, Helen Price, Pritom Shah,
Figure 1.
Scheme 1. Synthesis of C4 Acids and C4 Primary Amidesa
Onkar M. P. Singh, Richard Stocker, Pia Thommes,
Claire Wilkinson, and Alan Wonacott
Infectious Diseases CEDD and Medicinal DiscoVery Research,
GlaxoSmithKline, GSK Medicines Research Centre, Gunnels Wood
Road, SteVenage SG1 2NY, United Kingdom
ReceiVed October 16, 2006
Abstract: Optimization of a pyrrolidine-based template using structure-
based design and physicochemical considerations has provided a
development candidate 20b (3082) with submicromolar potency in the
HCV replicon and good pharmacokinetic properties.
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.
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.2 The current gold standard
therapies are based upon the use of pegylated interferon-R in
combination with ribavirin. These therapies provide a sustained
virological response in around 50% of patients infected with
genotype 1 and have the disadvantage of frequent and severe
side effects.3,4 The development of new therapies to treat HCV
infection effectively is, therefore, of paramount importance and
is currently an intensive area of research.5
a Reagents: (a) (i) 2-thiophenecarboxaldehyde for 3a or 2-thiazolecar-
boxaldehyde for 4a, DCM; (ii) tert-butyl acrylate, LiBr, Et3N, THF; (b) (i)
2-thiazolecarboxaldehyde, DCM; (ii) acrylamide, LiBr, Et3N, THF; (c) (i)
4-CF3 benzoyl chloride or 4-tert-butyl benzoyl chloride, Et3N, DCM; (ii)
TFA, DCM; (d) (i) (-)-di-O,O′-p-tolyl-L-tartaric acid, DCM; (ii) NaHCO3;
(e) (i) benzoyl chloride derivative, Et3N, DCM; (ii) TFA, DCM.
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 structural
viral proteins, whereas viral proteases (NS2, NS3) are respon-
* To whom correspondence should be addressed. Phone: +44 1438
763416. Fax: +44 1462 763620. E-mail: martin.j.slater@gsk.com.
10.1021/jm061207r CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/02/2007

898 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Letters
Table 1. Enzyme, Replicon, and Selected Rat PK Data for 4-Carboxy and 4-Amido Acyl Pyrrolidine Derivatives
HCV polymeraseb
1b FL (RB01)
IC50/µM
HCV replicon
1b ELISA
EC50/µM
Vero
toxicity
CCID50/µM
ratc
F
%
ratc
iv clearance
mL/min/kg
cmpd
chiralitya
ClogD14
1a
6a
7a
8b
9b
10b
11b
rac
rac
rac
h
h
h
20
1.1
3.8
0.97
0.014
0.020
0.005
>20
>20
>20
>20
0.57
1.2
>500
>500
>500
430
>500
>500
>500
-0.8
-0.1
-0.5
0.6
0.6
0.5
2
2
5
0.6
21
24
29
37
h
0.61
0.2
a rac ) racemic and h ) homochiral. b Filtermat assay using full length 1b enzyme and polyUrA homopolymer substrate.15 c Dosed at 2 mg/kg oral and
1 mg/kg iv as a solution in 10% aqueous DMSO, n ) 3 rats.
sible for the production of mature nonstructural proteins. These
include the RNA-dependent RNA polymerase (RdRp, NS5B),
which synthesizes the new viral RNA strands. It is a well
characterized enzyme, essential for viral replication, with no
known mammalian equivalent and thus represents an attractive
target for the development of novel anti-HCV agents.6
A diverse range of non-nucleoside inhibitors of the HCV
NS5B polymerase have been reported, including dihydroxypy-
rimidine carboxylic acids, benzimidazole derivatives, benzothia-
diazines, thiophene carboxylic acids, phenylalanine derivatives,
dihydropyrones, aminothiazoles, and pyranoindoles.7 Recently,
we disclosed the acyl pyrrolidine series identified through a high
throughput screening program against the enzyme8 (e.g., 1a,9
Figure 1). Coordinates for the structures of 1a and 7a bound to
HCV polymerase BKd21 have been deposited in the Protein
Data Bank under accession codes 2jc0 and 2jc1, respectively,
Figure 2. X-ray crystal structure of compound 7a (orange) in the palm
binding site of HCV polymerase; the catalytic aspartic acid residues
are indicated in black.
and herein, we describe further optimization of the series leading
to the identification of the development candidate 20b (3082).
The previous report described a solid-phase approach to
exploration of the structure-activity relationships (SAR) of acyl
pyrrolidines.8 These contained two carboxylic acid motifs,
providing the lead molecule bearing a 4-trifluoromethyl benzoyl
group at N1 and a 2-thiophene at C5 (1a, Figure 1). Separation
of the racemic compound by chiral HPLC demonstrated that
the enzyme activity resided with the (2S,4S,5R) enantiomer.8,9
Although the dicarboxylic acids displayed good inhibitory
activity in the polymerase biochemical assay, they were all
inactive in the cellular HCV replicon assay.10 The initial
objective we set was to achieve significant inhibition in the HCV
replicon, and our first approach was to replace the carboxylic
acid at C4 by a primary amide. We exploited the solution-phase
cycloaddition procedures described by Grigg and co-workers.11
Imines derived from amino acids and aldehydes were reacted
with tert-butyl acrylate or acrylamide as the dipolarophiles and
Figure 3. Comparison of the X-ray structures of compounds 1a (green)
and 7a (orange), showing the different binding modes and movement
of Leu384 to accommodate the tert-butyl group in 7a. For clarity, the
protein surface is shown only for 7a (except for the side chain of
Leu384).
with lithium bromide and triethylamine in THF. This procedure
formed the racemic pyrrolidines in good yields, with excellent
regiochemical and stereochemical control (Scheme 1). Standard
acylation with a benzoyl chloride derivative and triethylamine
followed by TFA deprotection of the tert-butyl esters afforded
the racemic targets (1a, 6a, and 7a). Replacement of the 4-CF3
group by a bulkier 4-tert-butyl in the benzamide gave a
significant improvement in potency against the enzyme (com-
pare 1a and 6a, Table 1). Another key SAR finding was that
combining a 2-thiazole at C5 (in place of 2-thiophene) with a
primary amide at C4 also provided good polymerase activity
(7a vs 8b). This indicated that we could dispense with the C4
carboxylic acid, although activity in the HCV replicon assay
was still elusive.
close to the catalytic site (Figure 2). Moreover, 1a and 7a have
different binding modes in which the bulkier tert-butyl sub-
stituent on the benzamide causes movement in the side chain
of Leu384 and allows 7a to bind deeper into the pocket (Figure
3). This is consistent with the improved potency of the tert-
butyl containing compounds. A consequence of this binding
mode is that a small lipophilic pocket is created, which is
potentially accessible from the 3-position of the benzamide,
which we sought to exploit.
Structure-based design played a key role in further potency
enhancements. Soaking the acids 1a and 7a into polymerase
crystals (1b BK∆21) showed that these molecules bind in the
palm region of the polymerase enzyme in an allosteric pocket
Racemic pyrrolidine 5a was efficiently resolved using (-)-
di-O,O′-p-tolyl-L-tartaric acid in dichloromethane. The chiral
pyrrolidine 5b was acylated under standard conditions with the

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 899
Scheme 2. Synthesis of C4 Hydroxymethyl Derivativesa
a Reagents: (a) (i) 2-thiazolecarboxaldehyde, DCM; (ii) methyl acrylate, LiBr, Et3N, THF; (b) 3-methoxy-4-tert-butyl benzoyl chloride, Et3N, DCM; (c)
LiBH4, THF; (d) LiAlH4, -40 °C, THF; (e) TFA, DCM; for 16b and 20b, (f) MeI, NaH, DMF then TFA, DCM; for 17a and 21b, (f) EtI, NaH, DMF then
TFA, DCM; (g) chiral resolution 12a into 12b, (R)-(-)-1,1′-binaphthyl-2,2′-diyl-hydrogen phosphate, i-PrOH, 90 °C, then Et3N, Et2O to form the free base.
Table 2. Enzyme, Replicon, and Selected Rat PK Data for 4-Alkoxymethyl and 4-Hydroxymethyl Acyl Pyrrolidine Derivatives
HCV polymeraseb
1b FL (RB01)
IC50/µM
HCV replicon
1b ELISA
EC50/µM
Vero
toxicity
CCID50/µM
ratc
F
%
ratc
iv clearance
mL/min/kg
cmpd
chiralitya
ClogD14
15a
16b
17a
19a
20b
21b
rac
h
rac
rac
h
1.0
2.3
3.2
0.73
0.44
0.43
1.3
5.7
12.8
0.21
0.39
0.47
482
265
214
358
240
142
11
44
39
12
0.6
1.2
1.7
0.6
12
59
120
36
5
5
1.2 (2.2)d
1.7 (2.7)d
h
a rac ) racemic and h ) homochiral. b SPA assay using full length 1b enzyme and rCrG homopolymer substrate.15 c Dosed at 2 mg/kg oral and 1 mg/kg
iv as a solution in aqueous 10% DMSO, n ) 3 rats. d Measured logD.
3-bromo, 3-chloro, and 3-methoxy derivatives of 4-tert-butyl
benzoyl chloride, and the esters deprotected to form 9b, 10b,
and 11b (Scheme 1). These compounds were very potent in
the enzyme assay, with IC50 values 50-100-fold better than
the unsubstituted compound 8b and additionally delivered very
good potency in the HCV replicon assay, with the 3-bromo and
3-methoxy containing analogues (9b and 11b) now achieving
submicromolar EC50 values (Table 1).
All of the amides 8b-11b had poor oral bioavailability in
the rat (F e 5%, Table 1). Because clearance from the plasma
compartment was moderate (24-42% of liver blood flow), we
considered that poor membrane permeability may be an issue.
Although formally these molecules comply with Lipinski’s rule
of 5,12 calculated logD (ClogD) values are in the range
0.2-0.6. We reasoned that replacing the C4-amide with a more
lipophilic moiety while maintaining molecular weight below
500 may improve permeability and deliver improved PK
properties. C4-Methyl ether derivatives were selected as ap-
propriate targets that met these criteria.
Reacting methyl acrylate with the imine derived from leucine
tert-butyl ester (2b) and 2-thiazole carboxaldehyde gave racemic
pyrrolidine 12a in excellent yield in our standard cycloaddition
conditions (Scheme 2). Initially this was processed in racemic
form to give the benzamide derivative 13a. We selected the
3-methoxy substituent over the equipotent 3-bromo substituent
as the 3-methoxy series had superior solubility properties and
lower molecular weights. To resolve the racemic pyrrolidine
12a, in this case, (R)-(-)-1,1′-binaphthyl-2,2′-diyl-hydrogen
phosphate in hot isopropanol was effective, providing the
required salt in good yield with high ee (98.8%).13 As the
phosphate salt had poor solubility in dichloromethane and many
other solvents, prior to the acylation step it was necessary to
liberate the pyrrolidine base 12b using triethylamine in ether.
Standard reaction conditions with 3-methoxy-4-tert-butyl ben-
zoyl chloride then afforded 13b. A number of reducing reagents
were evaluated to convert the methyl ester to the desired alcohol.
Lithium borohydride in THF gave a mixture of alpha and beta
methyl alcohols, in which the beta methyl alcohol 14a was the
major product (70% yield) and could be separated from the
minor alpha epimer using silica chromatography. Lithium
aluminum hydride reduction of 13a at -40 °C gave a good
yield of the alpha methyl alcohol 18a. It was crucial to maintain
the temperature around -40 °C for this reaction, as at higher
temperatures, a significant quantity of the beta epimer 14a was
formed due to epimerisation of the C4-ester prior to reduction.
Additionally, partial reduction of the benzamide (giving 3-meth-
oxy-4-tert-butyl benzoic acid after work up) was observed at
0 °C. The racemic alcohols 14a and 18a were deprotected to
give the C4-methyl alcohols 15a (beta) and 19a (alpha),
respectively. The methyl ether derivatives were prepared by
alkylation of the alcohols with methyl or ethyl iodide using
sodium hydride in DMF and subsequently deprotected in the
usual manner (Scheme 2).16
Potency and rat pharmacokinetic properties for these com-
pounds are summarized in Table 2. In all cases, the alpha
epimers are more potent than the corresponding beta epimers.15
The racemic alcohols (15a and 19a) have good activity in the
replicon assay, especially the alpha methyl alcohol (19a) with
an EC50 of 0.21 uM. However, both alcohols possess PK profiles
in the rat similar to the C4-amides (poor absorption and
moderate clearance). The beta methyl and ethyl ethers (16b and
17a) have moderate activities in the replicon assay. Gratifyingly,
the alpha methyl (20b) and ethyl ethers (21b) have very good
potencies in this cellular assay, with EC50 values of 0.39 and

900 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Letters
Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R. L.;
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0.47 uM, respectively. Moreover, both 20b and 21b displayed
excellent pharmacokinetic profiles in the rat, with good oral
bioavailabilities and low iv clearance values. The stereochem-
istry of 21b was confirmed as (2S,4S,5R) by single molecule
X-ray crystallography. It is noteworthy that the beta methyl ether
16b also demonstrated a good PK profile in the rat.
(7) (a) Beaulieu, L. P.; Tsantrizos, Y. S. Inhibitors of the HCV NS5B
polymerase: New hope for the treatment of hepatitis C infections.
Curr. Opin. InVest. Drugs 2004, 5, 838-850. (b) Gordon, C. P.;
Keller, P. A. Control of hepatitis C: A medicinal chemistry
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1H-[1]-benzothieno[2,3-c]pyran and 3,4-dihydro-1H-[1]-benzofuran
derivatives as non-nucleoside inhibitors of HCV NS5B RNA de-
pendent RNA polymerase. Bioorg. Med. Chem. Lett. 2006, 16, 457-
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Reddy, T. J.; Das, S. K.; Poisson, C.; Courchesne, M.; Proulx, M.;
Siddiqui, A.; Yannopoulos, C. G.; Nguyen-Ba, N.; Roy, C.; Nasturica,
D.; Moinet, C.; Bethell, R.; Hamel, M.; L’Heureux, L.; Maud, D.;
Nicolas, O.; Cortemanche-Asselin, P.; Brunette, S.; Bilimoria, D.;
Bedard, J. Discovery of thiophene-2-carboxylic acids as potent
inhibitors of HCV NS5B polymerase and HCV subgenomic RNA
replication. Part 2: Tertiary amides. Bioorg. Med. Chem. Letters
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Borchardt, A.; Dragovich, P.; Jewell, T.; Prins, T.; Zhou, R.; Blazel,
J.; Parge, H.; Love, R.; Hickey, M.; Doan, C.; Shi, S.; Duggal, R.;
Lewis, C.; Fuhrman, S. Identification and structure-based optimization
of novel dihydropyranones as potent HCV RNA polymerase inhibi-
tors. Bioorg. Med. Chem. Letters 2006, 16, 4834-4838. (g) Tedesco,
R.; Shaw, A. N.; Bambel, R.; Chai, D.; Concha, N. O.; Darcy, M.
G.; Dhanak, D.; Fitch, D. M.; Gates, A.; Gerhardt, W. G.; Halegoua,
D. L.; Han, C.; Hofmann, G. A.; Johnston, V. K.; Kaura, A. C.; Liu,
N.; Keenan, R. M.; Lin-Goerke, J.; Sarisky, R. T.; Wigall, K. J.;
Zimmerman, M. N.; Duffy, K. J. 3-(1,1-Dioxo-2H-(1,2,4)-benzothia-
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Compound 20b thus achieved our objective of combining
very good potency in the HCV replicon with a good PK profile
in the rat and was selected for further development. In the
marmoset, 20b had a similarly good PK profile (F ) 50%, iv
clearance ) 10 mL/min/kg, 17% of liver blood flow). In further
studies in the replicon system, negligible attenuation (∼2-fold)
of the potency was observed in the presence of human serum
albumin (30 mg/mL); additionally, 20b and interferon R-2a
demonstrated a synergistic inhibitory effect in combination
assays. A detailed biological profile of 20b will be published
in due course.17
In summary, we have described the optimization of the acyl
pyrrolidine series leading to submicromolar inhibitors in the
replicon. Replacing the polar amide at the C4-position with a
lipophilic methoxymethyl moiety resulted in 20b, a molecule
combining very good replicon potency and good pharmaco-
kinetics suitable for further development as an anti-HCV agent.
Acknowledgment. We thank Stephen Richards for NMR
spectroscopic work, Bill Leavens for accurate mass determina-
tions, Eric Hortense for chiral analysis and separations, Steve
Jackson for chiral separations, and Tadeusz Skarzynski and
Maire Convery for assistance with structural aspects.
(8) (a) Burton, G.; Ku, T. W.; Carr, T. J.; Kiesow, T.; Sarisky, R. T.;
Lin-Goerke, J.; Baker, A.; Earnshaw, D. L.; Hofmann, G. A.; Keenan,
R. M.; Dhanak, D. Identification of small molecule inhibitors of the
hepatitis C RNA-dependent RNA polymerase from a pyrrolidine
combinatorial mixture. Bioorg. Med. Chem. Lett. 2005, 15, 1553-
1556. (b) Burton, G.; Ku, T. W.; Carr, T. J.; Kiesow, T.; Sarisky, R.
T.; Lin-Goerke, J.; Hofmann, G. A.; Slater, M. J.; Haigh, D.; Dhanak,
D.; Johnson, V. K.; Parry, N. R.; Thommes, P. Studies on acyl
pyrrolidine inhibitors of HCV RNA-dependent RNA polymerase to
identify a molecule with replicon antiviral activity. Bioorg. Med.
Chem. Lett. 2007, accepted for publication.
Supporting Information Available: Experimental procedures
for the preparation of compounds, spectral and analytical character-
izing data, procedures for the biochemical assay, replicon assay,
and Vero cytotoxicity assay, and rat pharmacokinetic studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM061207R