Design and synthesis of 2,3,4,9-tetrahydro-1H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[b]indole derivatives as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase

Article abstract of DOI:10.1016/j.bmcl.2006.01.105

A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 2,3,4,9-tetrahydro-1H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[b] indole scaffolds were designed and synthesized. Optimization of the aromatic region showed preference for 5,8

Full text of DOI:10.1016/j.bmcl.2006.01.105

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2532–2534
Design and synthesis of 2,3,4,9-tetrahydro-1H-carbazole
and 1,2,3,4-tetrahydro-cyclopenta[b]indole derivatives as
non-nucleoside inhibitors of hepatitis C virus NS5B
RNA-dependent RNA polymerase
Ariamala Gopalsamy,a,* Mengxiao Shi,a Gregory Ciszewski,a Kaapjoo Park,a
John W. Ellingboe,a Mark Orlowski,b Boris Feldb and Anita Y. M. Howeb
aChemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965, USA
bInfectious Diseases, Wyeth Research, Pearl River, NY 10965, USA
Received 23 November 2005; revised 18 January 2006; accepted 19 January 2006
Available online 15 February 2006
Abstract—A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 2,3,4,9-tetrahydro-1H-carbazole
and 1,2,3,4-tetrahydro-cyclopenta[b]indole scaffolds were designed and synthesized. Optimization of the aromatic region showed
preference for 5,8-disubstitution pattern in both the scaffolds examined while favoring the n-propyl moiety for the C-1 position.
1,2,3,4-tetrahydro-cyclopenta[b]indole scaffold was slightly more potent than the corresponding 2,3,4,9-tetrahydro-1H-carbazole
and analogue 36 displayed an IC50 of 550 nM against HCV NS5B enzyme.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is the major etiological agent of
post-transfusion and sporadic non-A, non-B hepatitis.1
HCV infection is one of the causes for liver cirrhosis
and hepatocellular carcinoma leading to liver failure.
Current estimates of approximately 2–3% of the word
population as HCV carriers represent a significant med-
ical problem with economic burden implications.2 The
present approved therapy involves pegylated interfer-
on-a as a single agent or in combination with the
broad-spectrum anti-viral ribavirin.3 The severe side ef-
fects associated with these therapies significantly reduce
patient’s compliance. This approach is not aimed at any
particular viral target. Prevalence of various HCV sub-
types underscores the need for direct inhibition of viral
targets.
tion cycle. Small molecule inhibitors of this target have
received much attention in the recent past as potential
therapeutic agents for treatment of HCV infection.5
Both allosteric and active site inhibitors of NS5B poly-
merase have been reported. We recently reported6a pyr-
ano[3,4-b]indole 1 as a potent (IC50 = 0.33 lM) and
selective inhibitor of NS5B polymerase. While the preli-
minary structure–activity relationship was explored
retaining the pyrano[3,4-b]indole intact, we were inter-
ested in expanding our optimization efforts to scaffold
modification6b as well. As a first step we were interested
in identifying the role of the pyran oxygen in 1. Toward
that end 2,3,4,9-tetrahydro-1H-carbazole 2 was consid-
ered. 1,2,3,4-tetrahydro-cyclopenta[b]indole 3 was cho-
sen to shed more light on the ring size requirement for
this region of the molecule (Fig. 1).
HCV is a positive strand RNA virus and the genome
consists of 9600 base pairs that encode several structural
and non-structural proteins.4 NS5B RNA-dependent
RNA polymerase is a central enzyme in the viral replica-
In this communication, we report the synthesis of these
scaffolds and explore the structure–activity relationship
requirements for these new scaffolds.
As shown in Scheme 1, the synthesis7 of the representa-
tive compound 12 for the 2,3,4,9-tetrahydro-1H-carba-
zole scaffold started from 2-allyl cyclohexanone 4,
which upon deprotonation followed by alkylation with
Keywords: Tetrahydro-1H-carbazole; Tetrahydro-cyclopenta[b]indole;
NS5B RNA-dependent RNA polymerase inhibitor; HCV inhibitor.
*
Corresponding author. Tel.: +1 845 602 2841; fax: +1 845 602
3045; e-mail: gopalsa@wyeth.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.105

A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2532–2534
2533
A simple unsubstituted aromatic system 14, even with
the optimal substitution of n-propyl in the C-1 position,
was completely devoid of any activity indicating the
need for aromatic substitutions for the polymerase
activity. Mono chloro analog 15 or various difluoro ana-
logs 16–18 did not satisfy the requirements. However, as
observed previously for the pyranoindole series, a 5,8-di-
substitution pattern was found to be regaining substan-
tial amount of potency, with preference for a halogen or
cyano group (analogs 13, 20, and 21 in Table 1). We also
investigated the requirement for the acetic acid moiety in
this series. On a direct comparison of analog 13 with
analog 23, where the acetic acid was replaced with for-
mic acid, a 7-fold loss in potency was observed. Howev-
er, by placing the appropriate 5,8-aromatic substituents
equivalent potency could be regained even with formic
acid (analog 26) as the C-1 substituent.
Figure 1. Scaffold substitution for pyrano[3,4-b]indole.
The second scaffold of interest, 1,2,3,4-tetrahydro-cyclo-
penta[b]indoles, was evaluated by using the most favor-
able aromatic substitutions for the C-5 and C-8
positions in the formic acid derivative as the basis for
modification. The analogs explored are shown in Table
2. Comparing direct analogs 23 and 27 from both the
scaffolds of interest, it was clear that there is an im-
proved potency with 1,2,3,4-tetrahydro-cyclopenta[b]in-
dole. This was further validated with analog 36, which
exhibited submicromolar potency compared to the car-
bazole analog 26. However, the overall structure–activ-
ity relationship revealed by both scaffolds tracked well
with that of the parent pyranoindole scaffold. Of the
biased small set of C-1 alkyl groups investigated (n-pro-
pyl, n-butyl, and allyl), good tolerance was shown for
these groups in this region. The most potent inhibitor
36 from this SAR study was selected for further
characterization of its ability to inhibit hepatitis C virus
replicon9b in human liver cell. Although the compound
Scheme 1. Reagents and conditions: (a) NaH, toluene, 100 ꢁC, 6 h,
68%; (b) RuO2–NaIO4, CCl4–acetone, 0 ꢁC to rt, 3 h, 77%; (c) EtI,
Na2CO3, 8 h, 66%; (d) NaOAC, MeOH, 60 ꢁC, overnight, 62%; (e)
BF3–Et2O, HOAc, 100 ꢁC, 2 h, 58%; (f) CuCN, NMP, microwave,
220 ꢁC, 15 min, 70%; (g) 1 N NaOH, EtOH, THF, 8 h, 72%.
bromopropane afforded 5. Ruthenium oxide–sodium
metaperiodate oxidation of the allyl group gave the acid
6. The acid was converted to the ester 7, which was con-
densed with the required phenyl hydrazine 8 to give the
hydrazone 9. Lewis acid-catalyzed cyclization of 9 affor-
ded the required tricyclic carbazole scaffold 10. The aro-
matic bromo substituent was converted to cyano group
under microwave condition and the ester was hydro-
lyzed to give the acid 12 of interest. The synthesis of
Table 1. HCV NS5B inhibitory activity of 2,3,4,9-tetrahydro-1H-
carbazole derivatives
1,2,3,4-tetrahydro-cyclopenta[b]indole 3 was carried
out in an analogous manner following Scheme 1.
Compound R1
R2 R3 R4
X
n
IC50
(lM)8,9
Our initial efforts were focused on extrapolating the
SAR that we had observed in the case of pyrano[3,4-
b]indole to 2,3,4,9-tetrahydro-1H-carbazole scaffold.
From our earlier SAR work around the pyranoindole
molecule, C-1 n-propyl group was found to be ideal with
some flexibility for the substituents in the aromatic re-
gion. Based on this knowledge we synthesized carbazole
analog 13 incorporating the exact aromatic substituents
as that of the pyranoindole 1. The compound was found
to be slightly less potent than the pyranoindole 1,
although it was tested as racemic mixture. Extending
the study to other carbazoles with varying aromatic sub-
stituents did not improve the potency. However, the
structure–activity correlation observed in the pyranoin-
dole series was found to be extendable to the carbazole
series.
1
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Me
Me
H
H
H
H
H
F
H
H
H
Cl
H
H
H
Br
H
H
H
H
H
H
H
CN
CN
H
O
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0.57
2.1
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
>33
>33
30
H
H
H
F
F
F
H
>33
>33
>33
2.0
F
Cl
H
H
H
H
H
H
H
H
H
F
H
F
CN
CN
CN
CN
CN
CN
CONH2
Me
F
2.3
>33
15
10
5.8
Cl
Cl
CF3 CH2
Cl CH2
3.2

2534
A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2532–2534
Table 2. HCV NS5B inhibitory activity of 1,2,3,4-tetrahydro-cyclo-
penta[b]indole derivatives
Margolis, H. S. N. Engl. J. Med. 1999, 341, 556; (b) Cohen,
J. Science 1999, 285, 26; (c) Memom, M. I.; Memom, M. A.
J. Viral Hepat. 2002, 9, 84; (d) Hanazaki, K. Curr. Med.
Chem. Anti-infect. Agents 2003, 2, 103.
3. Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi,
V. K.; Shiffman, M.; Reindollar, R.; Goodman, Z. D.;
Koury, K.; Ling, M.; Albrecht, J. K. Lancet 2001, 358, 958.
4. (a) Beaulieu, P. L.; Montse, L.-B. Curr. Med. Chem. Anti-
Infect. Agents 2002, 1, 163; (b) Tan, S.-L.; Pause, A.; Shi,
Y.; Sonenber, N. Nature 2002, 1, 867; (c) Adachi, T.; Ago,
H.; Habuka, N.; Okuda, K.; Komatsu, M.; Ikeda, S.;
Yatsunami, K. Biochim. Biophys. Acta 2002, 1601, 39.
5. (a) Condon, S. M.; LaPorte, M. G.; Herbertz, T. Curr.
Med. Chem. Anti-Infect. Agents 2005, 4, 99; (b) Harper, S.;
Avolio, S.; Pacini, B.; Di Filippo, M.; Altamura, S.; Tomei,
L.; Paonessa, G.; Di Marco, S.; Carfi, A.; Giuliano, C.;
Padron, J.; Bonelli, F.; Migliaccio, G.; De Francesco, R.;
Laufer, R.; Rowley, M.; Narjes, F. J. Med. Chem. 2005, 48,
4547; (c) Shipps, G. W.; Deng, Y.; Wang, T.; Popovici-
Muller, J.; Curran, P. J.; Rosner, K. E.; Cooper, A. B.;
Girijavallabham, V.; Butkiewicz, N.; Cable, M. Bioorg.
Med. Chem. Lett. 2005, 15, 115; (d) Gordon, C. P.; Keller,
P. A. J. Med. Chem. 2005, 48, 1; (e) Pfefferkorn, J. A.;
Greene, M. L.; Nugent, R. A.; Gross, R. J.; Mitchell, M.
A.; Finzel, B. C.; Harris, M. S.; Wells, P. A.; Shelly, J. A.;
Anstadt, R. A.; Kilkuskie, R. E.; Kopta, L. A.; Schwende,
F. J. Bioorg. Med. Chem. Lett. 2005, 15, 2481; (f) 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. Bioorg. Med. Chem. Lett.
2005, 15, 1553.
6. (a) Gopalsamy, A.; Lim, K.; Ciszewski, G.; Park, K.;
Ellingboe, J. W.; Bloom, J.; Insaf, S.; Upeslacis, J.;
Mansour, T. S.; Krishnamurthy, G.; Damarla, M.; Pyatski,
Y.; Ho, D.; Howe, A. Y. M.; Orlowski, M.; Feld, B.;
O’Connell, J. J. Med. Chem. 2004, 47, 6603; (b) Gopals-
amy, A.; Aplasca, A.; Ciszewski, G.; Park, K.; Ellingboe, J.
W.; Orlowski, M.; Feld, B.; Howe, A. Y. M. Bioorg. Med.
Chem. Lett. 2006, 16, 457.
7. (a) Mobilio, D.; Demerson, C. A.; Humber, L. G. US
4687860, 1987.; (b) Failli, A. F.; Steffan, R. J.; Kreft, A. F.;
Caggiano, T. J.; Caufield, C. E. US 5830911, 1998.;
8. Compounds were purified by HPLC and the purity was
>95%. Compounds were tested as racemic mixtures.
9. (a) The recombinant C-terminally truncated NS5B enzyme
used in the assay was derived from genotype 1b, BK strain.
Inhibitors were pre-incubated with the enzyme for 15 min
followed by an addition of an RNA template, NTPs, and
[a-32P]GTP. The reaction was carried out at room temper-
ature for 2 h. Product RNA containing incorporated
radioactive nucleotides was collected by filtration and the
amount of radioactivity was quantified using a scintillation
counter. The IC50 values reported are mean values for more
than two independent measurements; (b) Howe, A. Y. M.;
Feld, B.; Bloom, J.; Gopalsamy, A.; Krishnamurthy, G.;
Chunduru, S.; Young, D.; O’Connell, J. F. Antimicrob.
Agent Chemother. 2004, 48, 4813.
Compound
R
R1
R3
R4
IC50 (lM)8,9
27
28
29
30
31
32
33
34
35
36
37
38
n-Pr
Me
Me
Me
F
H
H
H
H
H
H
F
CN
CN
CN
CN
CN
CN
CN
CN
CN
Cl
4.7
4.6
n-Butyl
Allyl
n-Pr
11.0
8.1
n-Butyl
Allyl
n-Pr
F
5.4
F
14.5
1.8
Me
Me
Me
Cl
n-Butyl
Allyl
n-Pr
F
1.5
6.3
F
H
H
H
0.55
1.9
n-Butyl
Allyl
Cl
Cl
Cl
Cl
1.2
was found to be permeable (PAMPA: 6.48 · 10À6 cm/s
at pH 7.4), it showed modest activity (IC50 = 27 lM)
in this assay, it showed a narrow therapeutic window
with measurable cytotoxicity in the standard MTS met-
abolic assay (IC50 = 43 lM).
In conclusion, we have explored 2,3,4,9-tetrahydro-1H-
carbazole and 1,2,3,4-tetrahydro-cyclopenta[b]-indole
derivatives as a novel class of HCV NS5B RNA-depen-
dent RNA polymerase inhibitors. The structure–activity
requirement for this class of inhibitors seems to track
very well with the pyrano[3,4-b]indole series. 1,2,3,4-tet-
rahydro-cyclopenta[b]-indole scaffold is slightly more
potent than the corresponding 2,3,4,9-tetrahydro-1H-
carbazole and is a novel chemo type for HCV polymer-
ase for further exploration.
Acknowledgments
The authors thank Discovery Analytical Chemistry
group at Wyeth Research, Pearl River, NY, for spectral
data. We thank Drs. Tarek Mansour, Jonathan Bloom,
and John O’Connell for their support for the work.
References and notes
1. Choo, Q. L.; Kuo, G.; Weiner, A. J.; O’Verby, L. R.;
Bradley, D. W.; Houghton, M. Science 1989, 244, 359.
2. (a) Alter, M. J.; Kruszon-Moran, D.; Nainan, O. V.;
McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.;