The discovery and structure-activity relationships of pyrano[3,4-b]indole based inhibitors of hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2010.03.002

We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.

Full text of DOI:10.1016/j.bmcl.2010.03.002

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2968–2973
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery and structure–activity relationships of pyrano[3,4-b]indole
based inhibitors of hepatitis C virus NS5B polymerase
c
c
c
c
Matthew G. LaPorte a, , Tandy L. Draper , Lori E. Miller , Charles W. Blackledge , Lara K. Leister ,
*
Eugene Amparo c, Alison R. Hussey c, Dorothy C. Young d, Srinivas K. Chunduru d, Christopher A. Benetatos d,
Gerry Rhodes e, Ariamala Gopalsamy f, Torsten Herbertz g, Christopher J. Burns c, Stephen M. Condon b,
*
a University of Pittsburgh, Center for Chemical Methodologies and Library Development, Pittsburgh, PA 15260, USA
b TetraLogic Pharmaceuticals, 343 Phoenixville Pike Malvern, PA 19355, USA
c Department of Medicinal Chemistry, ViroPharma Incorporated 397 Eagleview Boulevard Exton, PA 19341, USA
d Department of Biology, ViroPharma Incorporated 397 Eagleview Boulevard Exton, PA 19341, USA
e Department of Pharmacology, ViroPharma Incorporated 397 Eagleview Boulevard Exton, PA 19341, USA
f Department of Infectious Diseases, Wyeth, Pearl River, NY 10965, USA
g ViroPharma Incorporated, Department of Medicinal Chemistry, 397 Eagleview Boulevard Exton, PA 19341, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the structure–activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors
of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the sig-
nificantly more potent compound 12.
Received 2 February 2010
Accepted 1 March 2010
Available online 6 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HCV NS5B polymerase
Pyranoindoles
Hepatitis C virus (HCV) is a serious disease which often leads to
chronic hepatitis, cirrhosis, and hepatocellular carcinoma.1 These
complications are responsible for about 10,000–12,000 deaths/year
in the U.S.1 The necessity for liver transplantation in the U.S. is
most often the result of chronic HCV infection.1b Recent worldwide
estimates indicate that approximately 1–3% of the total population
may be infected with HCV.2
replication is the NS5B RNA-dependent RNA polymerase.7,8 The HCV
NS5B is an attractive target owing to the success of other antiviral
agents that inhibit viral polymerases.9 One such class is the non-
nucleoside reverse transcriptase inhibitors (NNRTI’s) that target
HIV RT. This communication will describe our optimization efforts
and structure–activity relationships on the novel pyranoindole ser-
ies of allosteric HCV NS5B enzyme inhibitors.
Current therapies for HCV involve a combination of ribavirin
Several thumb domain sites of HCV NS5B have been identified.8
The compounds depicted in Scheme 1 occupy the same allosteric
binding site (Thumb Pocket II) of HCV NS5B with their acidic func-
tionalities participating in hydrogen bonding to the Ser476 and
Tyr477 residues.
and interferon-a
.3 This treatment regimen causes unfavorable side
effects which often results in poor patient compliance. Further-
more, only about 40% of patients infected with HCV achieve a sus-
tained medical benefit.3a,4,5 Recently, pegylated forms of
interferon-
a
(Pegasys™ and PEG-INTRON™) which provide im-
Another critical feature of these ligands is the ability to occupy a
proved patient tolerabilities and response rates of over 50% have
been approved.3 Clearly there is a medical need for additional
HCV antiviral agents.
Hepatitis C belongs to the Flaviviridae family of positive-single
stranded RNA viruses.6 The HCV genome encodes a 3000 amino acid
polyprotein which is processed into structural and non-structural
proteins.6 Oneofthenon-structuralproteinsthatis essentialforviral
hydrophobic ‘dimple’ region defined by residues Leu419, Trp528
,
Tyr477, and Arg422. Gopalsamy has reported a series of tetrahydro-
pyrano[3,4-b]indole-containing compounds exemplified by struc-
ture 1.10 Compound 1 displayed potent inhibitory activity against
the HCV NS5B
D lM). Co-crystallographic
21 polymerase (IC50 = 0.3
analysis has determined that the hydrophobic ‘dimple’ on the pro-
tein is occupied by the n-propyl group of these pyranoindole-based
inhibitors (see Fig. 1).
It has been previously shown that n-propyl substitution is
greatly preferred over an ethyl group at this position.10 However,
no other variation of substituents at this position has been
described. We were therefore interested in further examining the
* Corresponding authors at present address: University of Pittsburgh, Center for
Chemical Methodologies and Library Development, Pittsburgh, PA 15260, USA.
E-mail addresses: mgl12@pitt.edu (M.G. LaPorte), scondon@tetralogicpharma.-
com (S.M. Condon).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.002

M. G. LaPorte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2968–2973
2969
S
CN
Me
CO2H
O
N
O
CO2H
OH
N
H
1
2
Et
Me
OH
O
N
N
CO2H
O
N
N
N
N
F3C
O
Me
Et
Cl
3
4
Cl
Scheme 1. Reported allosteric thumb pocket II HCV NS5B inhibitors.
studies have determined that the R-isomer retains all of the desired
biological activity.10a,15,16
The SAR of the alkyl-containing C1 substituents indicates toler-
ance for several functionalities including small, branched alkyl
groups. Compound 8h (R = sec-butyl), prepared as a mixture of four
stereoisomers, was found to be the most potent analog in this ser-
ies. Subsequent investigation determined that all the biological
activity resided in the (S)-sec-butyl isomer (vide infra).17
Several analogs were prepared which incorporated heteroatoms
into this C1 substituent (Table 2) however all of these analogs
exhibited poor enzyme inhibition.
Despite several of these 5,8-dichloro-indole analogs displaying
modest activity in the HCV replicon assay (data not shown), many
of these analogs also demonstrated general cellular toxicity.10 We
have observed that the 5-cyano-8-methyl-indole-containing ana-
logs exhibited greater separation of replicon activity and cytotoxic-
ity and therefore we selected this template for further optimization
(Scheme 3).10,18
The most active compounds from this series are listed in
Table 3. It was found that the (S)-sec-butyl containing group was
greatly preferred over the (R)-isomer (cf. 11 vs 10). Compound 11
also showed a twofold improvement in replicon cellular activity
compared to the n-propyl containing compound 1. The cellular
activity was further enhanced when the (S)-sec-butyl substitution
was incorporated onto the ethoxy-pyrazole containing scaffold (12
vs 13). Compound 12 has recently been reported to have in vivo
activity in the chimeric mouse model of hepatitis C infection.19,20
While exploring the SAR around the C1 group which occupies
the hydrophobic ‘dimple’ which occupies the C1-groups, we were
also interested in the hydrogen bond interactions of the acetic acid
group with the Ser476 and Tyr477 protein residues.
Incorporation of
a-ketoesters into the synthetic process pro-
Figure 1. (a) Crystal structure of NS5B with tetrahydro[3,4-b]indole inhibitor.
Electrostatic potential representation: Red-negative charge; Blue-positive charge;
White-non-polar; Ser476 & Tyr477 highlighted in Green. (b) Crystal structure of NS5B
with tetrahydro[3,4-b]indole inhibitor-side view indicating R1 group in hydropho-
bic ‘dimple’.
vided formate-based analogs (18, Scheme 4) with HCV polymerase
inhibition comparable to the acetate analogs (1) but with poor
activity against other NS5B isolates (data not shown).14,18,21
These formic acid analogs, however, showed poorer cellular rep-
licon activities and high potential for efflux as exemplified by high
basolateral to apical permeabilities in Caco-2 assays (Table 4).22,23
Co-crystallographic analysis of 1, and related pyranoindole-
based ligands, bound to HCV NS5B indicated an open region lo-
cated proximal to the C1 position of 1 (Fig. 2). We reasoned that
structure–activity relationship at the C1-position of these
pyranoindole inhibitors.10 The desired pyranoindole compounds
8 were readily assembled by the Lewis acid-catalyzed cyclization
of the substituted tryptophol 5 with a variety of b-ketoesters 6
(Scheme 2).10–14 It is important to note that the compounds
depicted in Table 1 were prepared as racemic mixtures. Previous
a suitable substituent tethered from the
a-carbon of the acetate
moiety of 1 would allow access to this region of the binding site.

2970
M. G. LaPorte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2968–2973
Cl
Cl
Cl
OH
O
CO2Et
O
O
a
+
N
H
N
H
R
EtO
R
Cl
5
6
7
Cl
O
b
CO2H
N
H
R
Cl
8
Scheme 2. Reagents: (a) BF3ÁOEt2, DCM; (b) NaOH, EtOH.
substituent into this area of the protein.8d Our strategy opted to
incorporate a nitrogen atom at the -position that would allow dif-
Table 1
HCV NS5B inhibitory activities of ( ) pyranoindole analogs 8
a
ferent trajectories via amide, carbamate, and sulfonamide
Compd
R
HCV pol (BK) IC50, lM
substitutions.
8a
8b
8c
8d
8e
8f
8g
8h*
8i
8j
8k
8l
8m
8n
8o
Methyl
Ethyl
n-Pr
3,3,3-Trifluoropropyl
n-Bu
1-Butenyl
2-Propyl
rac-sec-Butyl
1-Cyanoethyl
c-Pr
c-Bu
c-Pent
c-Hex
c-Propylmethyl
c-Pentylmethyl
27
For the preparation of the amino-substituted pyranoindoles, we
employed chemistry described by Maurer et al. to synthesize the
ketone component (Scheme 5).24 Opting to prepare the two diaste-
reomers in racemic form, we treated dl-Cbz-serine (19) with an ex-
cess of n-propylmagnesium chloride affording the desired n-propyl
ketone (20) in good yield. The key transformation was performed
using 4-cyano-7-methyltryptophol 14 and 1 equiv of BF3ÁEt2O in
DCM at ambient temperature to give 21 and 22 in an ꢀ2:1 ratio
by TLC, 1H NMR analysis, and isolated yield.
Interestingly, the diastereoselection of this cyclization is under
thermodynamic control since independent treatment of either
pure 21 or 22 under the BF3ÁEt2O conditions reproduced 21 and
22 in an ꢀ2:1 ratio suggesting the intermediacy of carbocationic
species 23 (Fig. 3).
6.3
0.37
0.4
0.76
1.7
7
0.15
0.3
7.3
0.35
3.3
>30
1.9
9.4
*
Mixture of four stereoisomers.
The assignment of relative stereochemistry was established by
the synthetic intersection of 22 beginning from L-N-phenylsulfo-
nylserine (not shown) and later confirmed by single crystal X-ray
analysis (Supplementary data).
Table 2
HCV NS5B inhibitory activities of ( ) pyranoindole analogs 8
Compd
R
HCV pol (BK) IC50, lM
Having secured a reliable synthetic route to the racemates (21
and 22), we next sought to functionalize the amino groups within
each isomer series. For illustrative purposes, the methanol group in
21 (or 22) was oxidized in two steps ((i) IBX; (ii) NaClO2) and ester-
ified. The Cbz group was removed under standard conditions (H2,
Pd/C, Scheme 6).
8p
8q
8r
8s
8t
Methoxymethyl
Methylthiomethyl
Ethoxymethyl
Ethylthiomethyl
Methoxyethyl
>30
7
26
3.7
>24
An initial set of simple amides and sulfonamides were prepared
We were encouraged when it was later disclosed that the structur-
ally-unrelated ligand (4, PF00868554) projected a heteroaromatic
to identify the stereochemical preference at the a-position. In vitro
inhibition of HCV NS5B was evaluated using the truncated BB7D21
CN
Me
CN
Me
CN
O
O
O
CO2H
CO2H
CO2H
N
H
N
H
N
H
Me
9
10
11
CN
Me
CN
Me
O
O
CO2H
CO2H
N
N
H
O
N
N
H
N
O
N
12
13
Scheme 3. 5-Cyano-8-methylpyranoindoles.

M. G. LaPorte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2968–2973
2971
Table 3
HCV NS5B inhibitory and cellular activities of pyranoindoles
Compd HCV pol (BK) HCV pol (BB7) HCV NS5A HCV
GAPDH
IC50
,
lM
IC50
,
l
M
EC50
,
lM
RNA,
l
M
lM
1
9
0.3
0.4
3.2
0.13
0.009
0.012
0.4
1.1
4.9
0.26
0.002
0.005
13.1
29.7
nd
2.2
0.12
0.88
4.8
nd
nd
2.1
0.02
0.14
>160
nd
nd
>10
>20
>90
( ) 10
11
12
13
(genotype 1b) NS5B enzyme and cellular activity was evaluated
using a replicon-containing cell line (Huh7-BB7, genotype 1b,
Apath, LLC, St. Louis, MO); inhibition of viral replication was mon-
itored using an ELISA assay (HCV NS5A).10c Replicon results were
qualified with a counter assay for general cytotoxicity (BrdU
incorporation).
Figure 2. Crystal structure of NS5B with tetrahydro[3,4-b]-indole.
of the amino substituents were tolerated but only the acetate ana-
log (26a) exhibited NS5B inhibition comparable to the lead 1.
Table 5 lists the results from a small set of analogs prepared
beginning with the major diastereomer rac-21. In general, each
CN
CN
OH
O
O
CO2Et
a
+
R
N
H
N
H
R
EtO
Me
14
Me
O
15
16
CN
Me
CN
O
O
b
chiral HPLC
separation
CO2H
CO2H
N
H
R
N
H
Me
17
18
Scheme 4. Reagents: (a) BF3ÁOEt2, DCM; (b) NaOH, EtOH.
Table 4
Formic acid versus acetate analogs
Compd
HCV pol (BK)
HCV pol (BB7)
HCV RNA
GAPDH RNA
Papp (A–B) cm/s
Papp (B–A) cm/s
Papp(B–A)//Papp(A–B)
IC50
,
l
M
IC50
,
lM
EC50
,
lM
CC50
,
lM
(10À6
)
(10À6
)
cm/s (10À6
)
1
12
18
0.3
0.009
0.7
0.4
0.002
3.4
4.8
0.02
12
>30
>20
>30
24
nt
3.2
2.2
nt
24.2
0.09
nt
7.5
OH
CN
OH
NHCbz
OH
N
H
14
n-PrMgCl
Me
Me
HO
NHCbz
BF3-OEt2
O
O
19
20
CN
CN
Me
O
O
NHCbz
OH
NHCbz
OH
+
N
H
N
H
Me
Me
Me
rac-21 (major)
rac-22 (minor)
Scheme 5. Preparation of amino-alcohol intermediates.

2972
M. G. LaPorte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2968–2973
Table 5
BzCHN
O
OH
Me
Bioactivities of amides and sulfonamides derived from rac-21
CN
Me
Compd
R
HCV pol (BB7)
HCV NS5A
EC50
BrdU CC50
lM
,
IC50,
l
M
,
l
M
N
H
1
—
0.4
1.7
5
4.5
6
13
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
26a
26b
26c
26d
26e
26f
–C(O)CH3
–C(O)(CH2)3CH3
–C(O)Ph
–SO2Me
–SO2(CH2)3CH3
–SO2Ph
23
Figure 3. Putative intermediate 23 in acid-mediated cyclization.
3.6
4
An analogous set of analogs was prepared beginning with the
minor isomer rac-22 (Table 6). It is apparent that these analogs
were much less active than those listed in Table 5 suggesting that
Table 6
Bioactivities of amides and sulfonamides derived from rac-22
the S-configuration is preferred at the
a-position. None of these
amino-substituted analogs displayed activity in the whole cell as-
say of HCV replication suggesting that, in addition to their modest
anti-polymerase activity, these analogs suffered from either high
protein binding and/or poor membrane transport properties.
A final set of carbamate analogs were prepared beginning with
the more active diastereomer rac-21 (Table 7). The racemic Cbz-
capped amine (24, Scheme 6) was the most active analog in the
Compd
R
HCV pol (BB7)
HCV NS5A
EC50
BrdU CC50,
lM
IC50,
l
M
,
l
M
27a
27b
27c
27d
27e
27f
–C(O)CH3
–C(O)(CH2)3CH3
–C(O)Ph
–SO2Me
–SO2(CH2)3CH3
–SO2Ph
17
>30
>30
13
22
16
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
enzymatic (IC50 = 0.72 lM) and cellular assays (EC50 = 35 lM).
Unfortunately, inhibition of replication in the cellular assay does
not appear to be specific for HCV since incorporation of bromode-
oxyuridine (BrdU) into cellular DNA was also inhibited at compara-
ble levels. From a ligand design perspective, however, the enzyme
inhibition may suggest that the carbamate moiety projects the
phenyl ring of the Cbz group into the targeted sub-site in a fashion
analogous to 4.8d
Table 7
Bioactivities of carbamates derived from rac-21
Compd
R
HCV pol (BB7)
HCV NS5A
EC50
BrdU CC50,
lM
IC50
,
lM
,
l
M
We have described the SAR of the C1-position of tetrahydropyr-
ano[3,4-b]indole based inhibitors of HCV NS5B polymerase.
Through structure-based design, we have identified and detailed
the preparation and biological activities of novel series containing
either formic acid functionalities or a-amino-substituted-acetates.
From these investigations on the pyranoindole template, a potent
26g
26h
26i
26i
26k
26l
24
–C(O)OMe
–C(O)OEt
5.6
7.2
5.9
11
9.5
18
>100
>100
>100
>100
>100
>100
35
>100
>100
>100
>100
>100
>100
45
–C(O)OAllyl
–C(O)OiPr
–C(O)OiBu
–C(O)OtBu
–C(O)OBn
0.72
analog 12 was realized which was significantly more potent than
CN
CN
O
O
NHCbz
NHCbz
CO2H
Me
1. IBX, DCM
2. NaHClO2
OH
N
H
N
H
Me
Me
Me
rac-21(major)
rac-24 (major)
CN
CN
H
N
O
O
NH2
5. R-Cl
6.NaOH
3. CH2N2
4. H2, Pd
R
CO2Me
N
H
CO2
Me
N
H
Me
Me
Me
rac-25
rac-26
CN
Me
CN
Me
H
O
O
6-steps
N
NHCbz
OH
R
CO2H
Me
N
H
N
H
Me
rac-27
rac-22 (minor)
Scheme 6. Preparation of rac-amino-ester intermediate.

M. G. LaPorte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2968–2973
2973
Conference, March 2005, San Diego, California.; (c) Howe, A. Y. M.; Bloom, J.;
Baldick, C. J.; Benetatos, C. A.; Cheng, H.; Christensen, J. S.; Chunduru, S. K.;
Coburn, G. A.; Feld, B.; Gopalsamy, A.; Gorczyca, W. P.; Herrmann, S.; Johann,
S.; Jiang, X.; Kimberland, M. L.; Krisnamurthy, G.; Olson, M.; Orlowski, M.;
Swanberg, S.; Thompson, I.; Thorn, M.; Del Vecchio, A.; Young, D. C.; van Zeijl,
M.; Ellingboe, J. W.; Upeslacis, J.; Collett, M.; Mansour, T. S.; O’Connell, J. F.
Antimicrob. Agents Chemother. 2004, 48, 4813.
HCV-371 (1) and showed in vivo activity in chimeric mouse model
of HCV infection.19
Acknowledgments
We wish to thank our collaborators at Wyeth for their assis-
tance. We also wish to thank Victor Young (University of Minne-
sota) for solving the crystal structure of rac-22.
11. The tryptophol intermediate was constructed by Fischer-Indole cyclization of
the corresponding hydrazone, see Ref. 10.
12. The b-ketoesters were either commercially available or made by literature
procedures. See: Lokot, I. P.; Pashkovsky, F. S.; Lakhvich, F. A. Tetrahedron 1999,
55, 4783.
13. All new compounds gave satisfactory 1H NMR and mass spectral analyses.
14. The pyranoindoles depicted in Tables 1, 2 and 4 were prepared using the following
general method: To a solution of the substituted tryptophol in DCM (0.2 M) was
Supplementary data
Supplementary data (structure derived from single crystal X-ray
analysis of 22) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2010.03.002.
added the corresponding b- (or
a-) ketoester (1–1.1 equiv) followed by BF3ÁOEt2
(1–1.2 equiv) at room temperature. The reactions were generally complete
within 2–4 h as determined by TLC (SiO2) analysis. The reaction mixture was
diluted with DCM, washed with saturated NaHCO3 and brine. The organic phase
was dried over Na2SO4, filtered, and concentrated. The crude products were
purified by chromatography (SiO2). The resulting esters were hydrolyzed using
aqueous NaOH in THF/EtOH to give the desired pyranoindoles.
References and notes
15. The IC50’s are an average of multiple experiments with the standard deviations
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amplified by PCR from BB7 plasmid DNA containing HCV genotype 1b (BB7),
cloned expressed, and purified by Escherichia coli. The RNA dependent RNA
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1
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9. For HCV NS5B inhibitors demonstrating antiviral effects in early clinical trials,
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Relationship of Substituted Pyranoindoles as HCV RNA Dependent RNA
Polymerase Inhibitors’. 229th American Chemical Society National
was initiated by adding 3 nM pOF transcribed RNA substrate, 0.4 U/
ll RNasin,
and 0.125 Ci ll
l
[a
-
33P]GTP (indicated are final concentrations in the 50
reaction mix). After 120 min at room temperature, the amount of RNA
synthesized was quantified by collecting the radiolabeled product RNA on
Millipore multiscreen membrane filter plates. The filters containing the
reaction products were allowed to dry at room temperature and counted in a
Wallac Microbeta after an addition of 50 ll of Optiphase™ scintillant.
Inhibition data were analyzed using the sigmoidal dose–response (variable
slope) equation in GraphPad Prism (GraphPad Software Inc., San Diego, CA).
The 50% inhibitory concentration (IC50) was the drug concentration that
decreased the enzyme activity by 50% relative to control samples incubated
without compound.
17. The chiral b-ketoester derived from (S)-(+)-2-methylbutyric acid was prepared
to afford diastereomeric pyranoindoles which were separated using chiral
chromatography.
18. Chiral HPLC separation using CHIRALPACK AD, mobile phase Heptane/IPA.
19. LaPorte, M. G.; Jackson, R. W.; Draper, T. L.; Gaboury, J. A.; Galie, K.; Herbertz,
T.; Hussey, A. R.; Rippin, S. R.; Benetatos, C. A.; Chunduru, S. K.; Christensen, J.
S.; Coburn, G. A.; Rizzo, C. J.; Rhodes, G.; O’Connell, J.; Howe, A. Y. M.; Mansour,
T.; Collett, M. S.; Pevear, D. C.; Young, D. C.; Gao, T.; Tyrrell, D. L. J.; Kneteman,
N. M.; Burns, C. J.; Condon, S. M. ChemMedChem 2008, 3, 1508.
20. At this point, the reason for the increased activities of the pyrazole containing
compounds is not well understood.
21. For the preparation of a-ketoesters 15, See: Wasserman, H. H.; Ho, W. B. J. Org.
Chem. 1994, 59, 4364.
22. Hillgren, K. M.; Kato, A.; Borchardt, R. T. Med. Res. Rev. 1995, 15, 83.
23. Efflux is considered significant if: Papp(B–A) P1.0 Â 10À6 cm/s and, Papp(B–A)/
Papp(A–B) P3.0 Â 10À6 cm/s.
24. Maurer, P. J.; Takahata, H.; Rapoport, H. J. Am. Chem. Soc. 1984, 106, 1095.