Discovery of pyrano[3,4-b]indoles as potent and selective HCV NS5B polymerase inhibitors

Article abstract of DOI:10.1021/jm0401255

A novel series of HCV NS5B RNA-dependent RNA polymerase inhibitors containing a pyrano-[3,4-b]indole scaffold is described leading to the discovery of compound 16, a highly potent and selective inhibitor that is active in the replicon system.

Full text of DOI:10.1021/jm0401255

J. Med. Chem. 2004, 47, 6603-6608
6603
Discovery of Pyrano[3,4-b]indoles as Potent and Selective HCV NS5B
Polymerase Inhibitors
Ariamala Gopalsamy,* Kitae Lim, Gregory Ciszewski, Kaapjoo Park, John W. Ellingboe, Jonathan Bloom,
Shabana Insaf, Janis Upeslacis, Tarek S. Mansour, Girija Krishnamurthy, Murthy Damarla, Yelena Pyatski,
Douglas Ho,† Anita Y. M. Howe,‡ Mark Orlowski,‡ Boris Feld,‡ and John O’Connell‡
Chemical and Screening Sciences and Infectious Diseases, Wyeth Research, Pearl River, New York 10965, and
Princeton University, Princeton, New Jersey 08544
Received July 2, 2004
A novel series of HCV NS5B RNA-dependent RNA polymerase inhibitors containing a pyrano-
[3,4-b]indole scaffold is described leading to the discovery of compound 16, a highly potent and
selective inhibitor that is active in the replicon system.
Introduction
Virally encoded polymerases are essential enzymes
as targets for therapeutic intervention. Inhibitors of
polymerases of human cytomegalovirus, human immu-
nodeficiency virus (HIV), human herpes simplex virus
type I, and hepatitis B virus are currently in clinical
use.1-4 Hepatitis C virus (HCV) is emerging as one of
the most significant infections in humans. Current
estimates of approximately 2.7-4 million people of the
U.S. population and 150 million worldwide as HCV
carriers represent a significant medical problem with
negative economic implications.5 The current approved
treatments, interferon monotherapy or interferon and
ribavirin combination therapy, are effective in 10-40%
of the patients. Considerable side effects are associated
Figure 1. HCV NS5B polymerase inhibitors.
with these regimens, causing up to 20% of the patients
to discontinue the therapy.6 As a result, there is an
unmet need for developing a safe and effective antiviral
agent.
The HCV genome encodes the RNA-dependent RNA
polymerase NS5B as a 65 KDa protein essential for viral
replication. The activities of this enzyme have been
extensively characterized in vitro.7 Recently, a cell-
Figure 2. Pyranoindole lead 5 for HCV NS5B polymerase.
culture model system in Huh7 cells containing a sub-
genomic replicon capable of supporting HCV replication
Our effort toward identifying a HCV polymerase
has been developed.8 The availability of these in vitro
inhibitor started with the high throughput screening
systems makes it possible to screen for inhibitors that
(HTS) of the various compound libraries. The HTS
might have clinical utility for treatment of diseases
consisted of an in vitro nucleotide incorporation assay
caused by HCV.
in which a functionally active recombinant NS5B en-
zyme and a hairpin-like primed RNA substrate were
used. A positive control consisted of the enzyme in the
absence of compound, and a negative control consisted
Both base-modified nucleoside analogues such as â-D-
N4-hydroxycytidine,9 sugar-modified 2′-alkyl nucleosides
analogues,10 and 3′-deoxyribonucleotides11 have been
reported to block HCV replication in replicon systems.
of the enzyme in the presence of basilen blue, a dye
In analogy to HIV, various nonnucleosides from differ-
shown to inhibit NS5B polymerase activity in this assay.
ent chemical classes were recently reported12 (Figure
The effort culminated in the identification of pyrano-
1). The diketo acid inhibitor 1 is reported to interact
indole 5 (Figure 2), which had an IC50 of 3.0 µM. It was
with the metal ions present in the enzyme active site,
whereas benzimidazole inhibitors 3 and N,N-disubsti-
found to be selective against human polymerase â (IC50
> 100 µM), calf thymus polymerase R (IC50 > 100 µM),
tuted phenylalanine inhibitors 4 are reported to display
helicase (IC50 > 75 µM), as well as HIV reverse tran-
allosteric interactions.
scriptase (IC50 > 100 µM). Compound 5 was not cyto-
toxic in rapidly dividing and stationary Vero and Huh7
cells as measured by a standard MTS metabolic assay.
On the basis of these results preliminary SAR studies
and biological characterization of this novel lead were
* To whom correspondence should be addressed. Tel: (845) 602-
2841. Fax: (845) 602-3045. E-mail: gopalsa@wyeth.com.
† Princeton University.
‡ Infectious Diseases, Wyeth Research.
10.1021/jm0401255 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/19/2004

6604 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Scheme 1. Synthesis of Pyranoindole Derivativesa
Gopalsamy et al.
Scheme 2. Resolution of Pyrano[3,4-b]indole
Enantiomersa
a Reagents and conditions: method A, (1) cinchonine, 2-bu-
tanone/water; (2) 1 N HCl/EtOAc; method B, HPLC, CHIRAL-
PACK-AD (250 × 20 mm) column, 10% isopropyl alcohol in
heptane (0.1% TFA) eluant; (h) EDCI, HOBT, DIEA, DMF, rt, 88%.
Table 1. HCV NS5B Polymerase Inhibitory Activity of
Pyranoindole Analogues
IC50
(µM)
compd
R1
R2
R3
R4
R5
a Reagents and conditions: (a) Fe, NH4Cl, 100 °C, 3 h, 92%; (b)
NaNO2/HCl, SnCl2, 0-10 °C, 75%; (c) 9, THF-H2O; (d) ZnCl2,
ethylene glycol, 25% from 8; (e) BF3·Et2O, CH2Cl2, 93%; (f) CuCN,
NMP, microwave, 88%; (g) 1 N NaOH, 98%.
5
18
19
20
14
15
21
22
Et
H
H
H
H
H
H
H
F
Cl
Cl
Cl
H
H
H
H
H
Et
H
H
H
propyl
ethyl
3.0
Et
>30
Cl
Cl
CN
CN
CN
CN
propyl
Propyl
propyl
propyl
propyl
propyl
0.27
6.0
>30
Me
Me
F
undertaken. Herein, we report on the synthesis, chemi-
cal resolution, and biological activity of pyrano[3,4-b]-
indoles as promising inhibitors of HCV NS5B enzyme.
0.57
1.0
0.12
Me
Chemistry
pattern to improve the potency. It was found that
replacing the ethyl group on C5 with chlorine to give
the 5,8-dichloro compound 19 increased the enzymatic
potency significantly. Of the other substituents placed
on the C-5 position, a cyano group was well-tolerated.
A substituent on the C-8 position was found to be
essential for activity, since compound 20 showed a
significant loss of potency in comparison to 19. A small
substituent like methyl in 15 or fluoro in 21 was
sufficient to regain the activity. The trisubstituted
aromatic pattern led to a slight improvement in the
potency, as shown by compound 22 (Table 1).
Since the hit identified from HTS was racemic, it was
of importance to establish if there is a preference for
binding one enantiomer over the other and, if found to
be the case, to identify the absolute stereochemistry of
the active enantiomer. Toward this end, acid 15 was
separated by chiral HPLC method, and the fast-eluting
active enantiomer was converted to the amide derivative
17. X-ray crystallography of the amide identified the
eutomer to be the R isomer.
The chiral separation protocol using a CHIRALPACK-
AD column was utilized to resolve compounds 5, 19, 15,
21, and 22 for further biological evaluation. The enan-
tiomeric excess (ee) was in the range of 86-100%. For
each pair of enantiomers, greater activity of 7-20-fold
was found with the R isomer (Table 2). In the case of
compounds 25 and 26, good inhibitory activity was
found for the enantiomers; however, these compounds
were less desirable than others, due to their activity in
the MTS assay.
The pyranoindole analogues were synthesized as
shown in Scheme 1 starting from the appropriately
substituted nitrobenzenes, such as 6. Reduction to the
aniline 7 followed by diazotization using sodium nitrite
and tin chloride reduction resulted in hydrazine 8.
Reaction of the hydrazine with 2,3-dihydrofuran 9 gave
the alcohol 10, which underwent cyclization upon treat-
ment with zinc chloride to give the tryptophol 11.
Condensation of the tryptophol with â-keto esters 12
gave the pyranoindole 13. The bromo compound was
converted to the nitrile 14 by treating with CuCN.
Hydrolysis of the ester gave the acid 15. The enanti-
omers were separated by a chiral HPLC method or by
resolution using cinchonine to form the salt, which was
separated and hydrolyzed to give pure enantiomers
(>99% enantiomeric excess) (Scheme 2). The free acid
was converted to the 4-bromobenzyl amide derivative
17 and was used for structural confirmation by X-ray
crystallography.
Results and Discussions
Attempts to understand the SAR of the lead molecule
started with the acid moiety. Conversion to ester or
amide derivatives rendered the molecules inactive as
NS5B inhibitors, indicating the importance of the free
acid. Replacement of the n-propyl group in the C1-
position of the pyran unit with an ethyl group 18
decreased the NS5B potency significantly. On the basis
of this preliminary structure-activity information, it
was decided to focus on the aromatic substitution

Pyrano[3,4-b]indoles as Polymerase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6605
Table 2. HCV NS5B Polymerase Inhibitory Activity of
gel GHLF 250 M prescored plates. The terms “concentrated”
and “evaporated” refer to removal of solvents using a rotary
evaporator at water aspirator pressure with a bath tempera-
ture equal to or less than 60 °C. Unless otherwise noted,
reagents were obtained from commercial sources and were
used without further purification
Separated Enantiomers
IC50
(µM)
MTS
compd
R1
R2
R3
R5
stereo
(µM)
23
24
25
26
16
27
28
29
30
31
Et
H
H
H
H
H
H
H
H
F
Cl
Cl
Cl
Cl
Me
Me
F
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
R
S
R
S
R
S
R
S
R
S
2.0
>50
>50
33
Et
>15
5-Bromo-2-methylaniline (7). A mixture of Fe powder
(9.31 g, 167 mmol) and NH4Cl (2.48 g, 46.3 mmol) in water
(50 mL) was refluxed for 30 min. To this hot mixture was
added 4-bromo-2-nitrotoluene (6) (10 g, 46.3 mmol) slowly and
then the reaction mixture was refluxed for 48 h. The mixture
was cooled to room temperature and extracted with ethyl
acetate (3 × 100 mL). The organic solution was washed with
water (3 × 200 mL) and brine (200 mL), dried (Na2SO4), and
concentrated. The residue was purified by flash chromatog-
raphy (silica, 15% EtOAc in hexanes) to give 7.9 g (92%) of
Cl
Cl
0.06
1.1
0.33
2.4
0.44
5.2
49
CN
CN
CN
CN
CN
CN
>50
>50
>50
>50
>50
>50
F
Me
Me
0.08
0.63
F
1
On the basis of these findings, compound 16 was
selected for further characterization and advancement.
By employing fluorescence spectroscopy techniques, the
apparent KD of 0.8 µM was determined from the changes
in the tryptophan fluorescence intensity at the emission
wavelength of 340 nm in comparison to the NS5B-
inhibitor complex excitation wavelength of 295 nm.
From these experiments the stoichiometry of binding
was found to be in a 1:1 ratio. The details of binding of
16 to an allosteric site of NS5B12a will be reported
elsewhere.
Compound 16 displayed broad inhibitory activities
against the NS5B enzyme derived from HCV genotypes
1a, 1b, and 3a with IC50 values ranging from 0.3 to 1.4
µM for 90% of the genotypes. It showed no inhibitory
activity against a panel of human polymerases, includ-
ing mitochondrial DNA polymerase γ, and other unre-
lated viral polymerases up to 80 µM, demonstrating its
specificity for the HCV polymerase. Moreover, a single
administration to Huh7 cells containing the HCV sub-
genomic replicon for 3 days resulted in a dose-dependent
reduction of the steady-state levels of viral RNA and
protein (EC50 ) 4.8 µM for HCV RNA).
the title compound as a pale yellow oil. H NMR (300 MHz,
CDCl3): δ 6.88 (m, 1H), 6.81 (m, 2H), 3.63 (bs, 2H), 2.09 (s,
3H). Anal. Calcd for C7H8BrN·HCl: C, 37.79; H, 4.08; N, 6.29.
Found: C, 37.91; H, 3.92; N, 6.22.
5-Bromo-2-methylphenylhydrazine Hydrochloride (8).
To a suspension of 5-bromo-2-methylaniline (7) (4.80 g, 25.8
mmol) in concentrated HCl (16 mL) was added dropwise a
solution of sodium nitrite (1.96 g, 28.4 mmol) in water (10 mL)
over 30 min at 0 °C. To the mixture was added dropwise a
solution of SnCl2‚2H2O (17.46 g, 77.4 mmol) in concentrated
HCl (15 mL) over 50 min. After stirring for 1 h at 0 °C, the
reaction mixture was basified with 50% NaOH (30 mL). The
mixture was further diluted with water (20 mL) and treated
with another portion of 50% NaOH (10 mL) and then crushed
ice (100 g). The reaction mixture was extracted with ether (3
× 100 mL), and the combined organic phases were washed
with brine, dried over Na2SO4, and filtered. The filtrate was
acidified by adding an anhydrous solution of HCl in ether (1
N in ether, 31 mL, 31 mmol). The precipitate was collected
and dried under reduced pressure to give 4.57 g (75%) of the
title compound as a white amorphous solid. 1H NMR (300
MHz, DMSO): δ 10.31 (bs, 3H), 8.11 (bs, 1H), 7.12 (s, 1H),
7.06 (m, 2H), 2.14 (s, 3H). Anal. Calcd for C7H9BrN2·HCl: C,
35.40; H, 4.24; N, 11.79. Found: C, 35.40; H, 4.16; N, 11.78.
4-Bromo-7-methyltryptophol (11). To a solution of 5-bro-
mo-2-methylphenylhydrazine hydrochloride (8) (4.57 g, 19.2
mmol) in 30% aqueous THF (100 mL) at 0 °C was added
dropwise a solution of 2,3-dihydrofuran (9) (1.60 mL, 21.2
mmol) in THF (10 mL). After stirring for 2 h at 0 °C and 12 h
at room temperature, the reaction mixture was diluted with
ether (100 mL). The organic solution was washed with
saturated NaHCO3 (2 × 100 mL) and brine (100 mL), dried
(Na2SO4), and concentrated. The residue (10) was dissolved
in ethylene glycol (30 mL), treated with ZnCl2 (5.76 g, 42.2
mmol), and heated at 170 °C for 4 h. The reaction mixture
was cooled to room temperature and 6 N HCl (100 mL) was
added. The mixture was extracted with ether (3 × 100 mL)
and washed with water (200 mL) and brine (200 mL). The
organic solution was dried over Na2SO4 and concentrated. The
residue was purified by flash chromatography (silica, 40%
EtOAc in hexanes) to give 1.22 g (25%) of the title compound
It is interesting to note that Mobilio et al. reported
analgesic activity for certain cis-1,4-disubstituted tet-
rahydropyrano[3,4-b]indoles residing in S-stereoiso-
mers.13 Compound 16 is devoid of analgesic activity.
Conclusions
In summary, we have identified a novel series of
1,3,4,9-tetrahydropyrano[3,4-b]indole derivatives as po-
tent selective inhibitors of HCV NS5B RNA dependent
RNA polymerase.
Experimental Section
General Procedures. Melting points were determined in
1
as a light brown oil. H NMR (300 MHz, CDCl3): δ 8.23 (bs,
an open capillary tubes on a Meltemp melting point apparatus
1
1H), 7.18 (d, J ) 7.65 Hz, 1H), 7.08 (d, J ) 2.16 Hz, 1H), 6.81
(d, J ) 7.65 Hz, 1H), 3.95 (t, J ) 6.42 Hz, 2H), 3.27 (t, J )
6.42 Hz, 2H), 2.40 (s, 3H), 1.69 (bs, 1H). Anal. Calcd for C11H12-
BrNO: C, 51.99; H, 4.76; N, 5.51. Found: C, 51.74; H, 4.92;
N, 5.28.
and are uncorrected. H NMR spectra were determined with
a Bruker DPX-300 spectrometer at 300 MHz. Chemical shifts
are reported in parts per million (δ) relative to residual
chloroform (7.26 ppm), TMS (0 ppm), or dimethyl sulfoxide
(2.49 ppm) as an internal reference with coupling constants
(J) reported in hertz (Hz). The peak shapes are denoted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad. Electrospray (ES) mass spectra were recorded in
positive or negative mode on a Micromass Platform spectrom-
eter. Electron impact and high-resolution mass spectra were
obtained on a Finnegan MAT-90 spectrometer. Combustion
analyses were obtained using a Perkin-Elmer Series II 2400
CHNS/O analyzer. The combustion analysis was conducted on
the free base. Chromatographic purifications were performed
by flash chromatography using Baker 40-µm silica gel. Thin-
layer chromatography (TLC) was performed on Analtech silica
5-Bromo-8-methyl-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indole-1-acetic Acid Ethyl Ester (13). To a solution
of 4-bromo-7-methyltryptophol (11) (1.12 g, 4.41 mmol) and
ethyl butyryl acetate (12) (0.71 mL, 4.41 mmol) in CH2Cl2 (20
mL) was added BF3·OEt2 (0.56 mL, 4.41 mmol) dropwise at
room temperature. The solution was stirred for 2 h and then
washed with saturated aqueous NaHCO3 (15 mL) and brine
(15 mL). The organic phase was dried (Na2SO4) and filtered
through a pad of silica gel. The filter cake was washed with
additional CH2Cl2 and the combined organic layer was evapo-
rated to provide 1.62 g (93%) of the title compound as a white

6606 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Gopalsamy et al.
1
solid. H NMR (300 MHz, CDCl3): δ 9.33 (bs, 1H), 7.11 (d, J
second crop of 0.4 g was obtained (>99% ee). The mother liqour
was concentrated and gave a third crop of 0.5 g (>99% ee).
) 7.65 Hz, 1H), 6.76 (d, J ) 7.65 Hz, 1H), 4.19 (m, 2H), 4.03
(m, 1H), 3.90 (m, 1H), 3.15 (m, 2H), 3.03 (d, J ) 16.6 Hz, 1H),
2.89 (d, J ) 16.6 Hz, 1H), 2.43 (s, 3H), 2.08 (m, 1H), 1.96 (m,
1H), 1.38 (m, 1H), 1.27 (t, J ) 7.14 Hz, 3H), 1.18 (m, 1H), 0.87
(t, J ) 7.29 Hz, 3H). Anal. Calcd for C19H24BrNO3: C, 57.88;
H, 6.14; N, 3.55. Found: C, 57.63; H, 5.91; N, 3.74
Method B. Alternatively, preparative HPLC using CHIRAL-
PACK-AD (250 × 20 mm) and 10% isopropyl alcohol in
heptane (0.1% TFA) as eluant gave R- and S-enantiomers of
5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyrano[3,4-b]-
indole-1-acetic acid as white solids. HRMS (ESI): [M + H]+
calcd for C18H21N2O3 313.1547, found 313.1545 (R-enantiomer)
and 313.1547 (S-enantiomer). Chiral HPLC: HP 1100 with
spiderlink CHIRALPACK-AD, 250 × 4.6 mm, isopropyl alcohol/
heptane containing 0.1% TFA (10:90), 1.0 mL/min, DAD 215
5-Cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indole-1-acetic Acid Ethyl Ester (14). 5-Bromo-8-
methyl-1-propyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-ace-
tic acid ethyl ester (13) (1.27 g, 3.22 mmol) and CuCN (0.433
g, 4.83 mmol) were dissolved in N-methyl-2-pyrrolidinone (15
mL) and the solution was divided into four microwave reaction
vessels (3.75 mL of each). The reaction vessels were heated in
a microwave at 220 °C for 15 min. The reaction mixtures were
combined and then diluted with water (30 mL). The crude
mixture was extracted with EtOAc (3 × 50 mL). The combined
organic phase was washed with brine (100 mL), dried over Na2-
SO4, and concentrated. The residue was purified by flash
chromatography (silica, 20% EtOAc in hexanes) to give 0.959
nm; tR ) 6.98 min (R-enantiomer), 9.37 min (S-enantiomer).
1
Mp: 225-227 °C. [R]25 ) -11 ( 1° (c ) 1.1%, acetone). H
D
NMR (300 MHz, acetone-d6): δ 10.37 (bs, 1H), 7.35 (d, J )
7.50 Hz, 1H), 7.03 (d, J ) 7.50 Hz, 1H), 4.05 (m, 2H), 3.08-
2.91 (m, 4H), 2.54 (s, 3H), 2.09 (m, 2H), 1.45 (m, 1H), 1.03 (m,
1H), 0.84 (t, J ) 7.26 Hz, 3H). MS (ESI): m/z 311.2. Anal.
Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97. Found: C,
69.19; H, 6.32; N, 8.90.
[(S)-5-Cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropy-
rano[3,4-b]indol-1-yl]acetic Acid (27). Mp: 224-226 °C.
1
g (88%) of the title compound as a white solid. H NMR (300
[R]25 ) +11 ( 1° (c ) 1%, acetone). 1H NMR (300 MHz,
MHz, CDCl3): δ 9.75 (bs, 1H), 7.33 (d, J ) 7.52 Hz, 1H), 6.93
(d, J ) 7.52 Hz, 1H), 4.21 (m, 2H), 4.11 (m, 1H), 4.03 (m, 1H),
3.08 (t, J ) 5.52, 2H), 2.99 (d, J ) 4.17 Hz, 2H), 2.57 (s, 3H),
2.06 (m, 2H), 1.42 (m, 1H), 1.26 (t, J ) 7.16 Hz, 3H), 1.18 (m,
1H), 0.88 (t, J ) 7.32 Hz, 3H). Anal. Calcd for C20H24N2O3: C,
70.57; H, 7.11; N, 8.23. Found: C, 70.27; H, 6.92; N, 8.18.
D
acetone-d6): δ 10.37 (bs, 1H), 7.35 (d, J ) 7.50 Hz, 1H), 7.03
(d, J ) 7.50 Hz, 1H), 4.05 (m, 2H), 3.08-2.91 (m, 4H), 2.54 (s,
3H), 2.09 (m, 2H), 1.45 (m, 1H), 1.03 (m, 1H), 0.84 (t, J ) 7.26
Hz, 3H). MS (ESI): m/z 311.2. Anal. Calcd for C18H20N2O3:
C, 69.21; H, 6.45; N, 8.97. Found: C, 68.94; H, 6.42; N, 9.28.
1-(R)-N-(4-Bromobenzyl)-2-(5-cyano-8-methyl-1-propyl-
1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetamide (17).
To a solution of 1-(R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tet-
rahydropyrano[3,4-b]indole-1-acetic acid (16) (20.0 mg, 0.064
mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
drochloride (EDCI, 15.0 mg, 0.077 mmol), and 1-hydroxyben-
zotriazole (10.4 mg, 0.077 mmol) in DMF (4 mL) was added
N,N-diisopropylethylamine (67 µL, 0.384 mmol) followed by
4-bromobenzylamine hydrochloride (17.1 mg, 0.077 mmol) at
room temperature. The reaction mixture was stirred for 20 h
at ambient temperature. Water (5 mL) was added to the
mixture and the resulting mixture was extracted with ethyl
acetate (3 × 10 mL). The combined organic phase was washed
with brine (20 mL), dried over Na2SO4, and concentrated. The
residue was purified by flash chromatography (silica, 40%
EtOAc in hexanes) to give 27 mg (88%) of the title compound
as a white solid. The solid was crystallized from ethyl acetate
for X-ray crystallography. Mp: 173-175 °C. 1H NMR (300
MHz, CDCl3): δ 10.15 (bs, 1H), 7.33 (m, 3H), 6.97 (m, 2H),
6.88 (m, 1H), 4.42 (dd, J ) 11.2, 4.6 Hz, 1H), 4.29 (dd, J )
11.2, 4.6 Hz, 1H), 4.03 (m, 2H), 3.11-2.95 (m, 4H), 2.24 (s,
3H), 2.07 (m, 1H), 1.91 (m, 1H), 1.35 (m, 2H), 0.89 (t, J ) 5.4
Hz, 3H). HRMS (ESI): [M + H]+ calcd for C25H27BrN3O2
480.1281, found 480.1285.
5-Cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indole-1-acetic Acid (15). To a solution of 5-cyano-8-
methyl-1-propyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-ace-
tic acid ethyl ester (14) (0.959 g, 2.82 mmol) in THF/MeOH (7
mL/15 mL) was added 1 N NaOH (5.64 mL, 5.64 mmol). The
reaction mixture was stirred at ambient temperature over-
night. Most of the THF/MeOH was removed under reduced
pressure and the resulting mixture was acidified with 1 N HCl.
The mixture was extracted with EtOAc (3 × 30 mL). The
combined organic phase was washed with brine (60 mL), dried
over Na2SO4, and concentrated to provide 0.868 g (99%) of the
title compound as a white solid. 1H NMR (300 MHz, acetone-
d6): δ 10.37 (bs, 1H), 7.35 (d, J ) 7.50 Hz, 1H), 7.03 (d, J )
7.50 Hz, 1H), 4.05 (m, 2H), 3.08-2.91 (m, 4H), 2.54 (s, 3H),
2.09 (m, 2H), 1.45 (m, 1H), 1.03 (m, 1H), 0.84 (t, J ) 7.26 Hz,
3H). Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97.
Found: C, 68.97; H, 6.84; N, 8.80.
[(R)-5-Cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropy-
rano[3,4-b]indol-1-yl]acetic Acid (16). Method A. [(R)-5-
Cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-
1-yl]acetic acid was obtained by resolution with cinchonine
according to the following procedure. (()-5-Cyano-8-methyl-
1-propyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic acid (6.4
g, 20.5 mmol) and cinchonine (5.9 g, 20.0 mmol) were dissolved
in a mixture of 2-butanone (125 mL) and water (5 mL) with
heating. The clear solution was stirred and allowed to cool to
room temperature overnight. The resulting solid was isolated,
washed with 10 mL of 2-butanone, and dried to give 2.4 g (20%
yield, >98% ee). The mother liquor was concentrated and
dissolved again in a mixture of 2-butanone (100 mL) and water
(1.5 mL) with heating. The solution was stirred and allowed
to cool to room temperature overnight. The resulting solid was
isolated, washed with 10 mL of 2-butanone, and dried to give
a second crop of salt (2.3 g, 18% yield, >98% ee). The two crops
(total 4.7 g) were combined and treated with 50 mL of 1 N
HCl and 100 mL of ethyl acetate. The ethyl acetate layer was
washed with 1 N HCl (30 mL) and water (50 mL). The aqueous
layers were combined and extracted with ethyl acetate (50
mL). This ethyl acetate layer was washed with water (50 mL).
The combined ethyl acetate layers were dried over sodium
sulfate, filtered, and concentrated in vacuo to give 2.25 g. This
material was triturated with 10 mL of ethyl acetate and the
precipitate was collected, rinsed with 5 mL of ethyl acetate,
and dried to give 1.27 g (>98% ee). The mother liquor was
concentrated to a volume of 5 mL and the precipitate was
collected, rinsed with 2 mL of ethyl acetate, and dried. A
The following compounds were prepared by following the
above procedure.
(8-Chloro-5-ethyl-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indol-1-yl)acetic Acid (5). Mp: 152-154 °C. 1H NMR
(400 MHz, DMSO-d6) δ 11.99 (s, 1H), 10.91 (s, 1H), 7.01 (d, J
) 8.0 Hz, 1H), 6.75 (d, J ) 8.2 Hz, 1H), 3.98-3.84 (m, 2H),
2.96 (d, J ) 13.6 Hz, 2H), 2.94-2.82 (m, 2H), 2.78 (d, J ) 13.6
Hz, 2H), 2.10-1.93 (m, 2H), 1.33-1.19 (m, 1H), 1.21 (t, J )
7.6 Hz, 3H), 0.81-0.74 (m, 4H). HRMS: calcd for C18H22ClNO3
- H, 334.12154, found (ESI-, [M - H]-), 334.12124.
8-Chloro-1,5-diethyl-1,3,4,9-tetrahydro-pyrano[3,4-b]-
1
indole-1-acetic Acid (18). Mp: 148-150 °C. H NMR (400
MHz, DMSO): δ 12.0 (s, 1Η), 10.9 (s, 1H), 7.05 (d, J ) 8 Hz,
1H), 6.8 (d, J ) 8 Hz, 1H), 4.0 (m, 2H), 3.02-3.0 (m, 2H), 2.7-
2.95 (m, 4H), 2.05 (m, 2H), 1.4 (t, J ) 7.4 Hz 3H), 0.5 (t, J )
7.4 Hz 3H). HRMS: calcd for C17H20ClNO3 + H, 322.12045,
found (ESI-FTMS, [M + H]), 322.12023.
(5,8-Dichloro-1-propyl-1,3,4,9-tetrahydropyrano[3,4-b]-
indol-1-yl)acetic Acid (19). Mp: 166-167 °C. 1H NMR (300
MHz, CDCl3): δ 9.12 (bs, 1H), 7.03 (d, J ) 8.26 Hz, 1H), 6.96
(d, J ) 8.26 Hz, 1H), 4.04 (m, 2H), 3.14 (m, 2H), 3.06 (m, 2H),
2.03 (m, 2H), 1.42 (m, 1H), 1.21 (m, 1H), 0.89 (t, J ) 7.34 Hz,

Pyrano[3,4-b]indoles as Polymerase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6607
3H). HRMS: calcd for C16H17Cl2NO3 + H, 342.06582, found
(ESI-FTMS, [M + H]), 342.06567.
DMSO): δ 12.02 (bs, 1H), 11.33 (bs, 1H), 7.00 (d, J ) 9.00 Hz,
1H), 3.96 (m, 2H), 2.95 (d, J ) 10.3 Hz, 1H), 2.83 (t, J ) 3.9
Hz, 1H), 2.72 (d, J ) 10.3 Hz, 1H), 2.54 (s, 3H), 1.99 (m, 2H),
1.28 (m, 1H), 0.85 (m, 1H), 0.79 (t, J ) 5.41 Hz, 3H). MS (ESI)
m/z 329.1. Anal. Calcd for C18H19FN2O3‚0.25H2O: C, 64.56;
H, 5.87; N, 8.37. Found: C, 64.61; H, 5.93; N, 8.38.
5-Chloro-1,3,4,9-tetrahydro-1-propylpyrano[3,4-b]in-
dole-1-acetic Acid (20). 1H NMR (400 MHz, DMSO): δ 12.0
(s, 1Η), 11.0 (s, 1H), 7.2 (d, J ) 8 Hz, 1H), 6.9-7.0 (m, 2H),
4.0 (m, 2H), 3.2-2.7 (m, 3H), 2.05 (m, 2H), 1.4 (m, 1H), 0.7
(m, 4H). MS: m/z 307. Anal. Calcd for C16H18ClNO3: C, 62.44;
H, 5.89; N, 4.55. Found: C, 62.43; H, 5.62; N, 4.39.
[(1S)-5-Cyano-6-fluoro-8-methyl-1-propyl-1,3,4,9-tet-
rahydropyrano[3,4-b]indol-1-yl]acetic Acid (31). Mp: 241-
242 °C. [R]25 ) +19 ( 1° (c ) 1%, acetone). 1H NMR (300
(5-Cyano-8-fluoro-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indol-1-yl)acetic Acid (21). Mp: 228-230 °C.1H NMR
(300 MHz, DMSO): δ 12.02 (bs, 1H), 11.33 (bs, 1H), 7.00 (d, J
) 9.00 Hz, 1H), 3.96 (m, 2H), 2.95 (d, J ) 10.3 Hz, 1H), 2.83
(t, J ) 3.9 Hz, 1H), 2.72 (d, J ) 10.3 Hz, 1H), 2.54 (s, 3H),
1.99 (m, 2H), 1.28 (m, 1H), 0.85 (m, 1H), 0.79 (t, J ) 5.41 Hz,
3H). MS (ESI): m/z 315.1. Anal. Calcd for C17H17FN2O3: C,
64.55; H, 5.42; N, 8.86. Found: C, 64.61; H, 5.27; N, 8.86.
(5-Cyano-6-fluoro-8-methyl-1-propyl-1,3,4,9-tetrahy-
dropyrano[3,4-b]indol-1-yl)acetic Acid (22). Mp: 249-251
D
MHz, DMSO): δ 12.02 (bs, 1H), 11.33 (bs, 1H), 7.00 (d, J )
9.00 Hz, 1H), 3.96 (m, 2H), 2.95 (d, J ) 10.3 Hz, 1H), 2.83 (t,
J ) 3.9 Hz, 1H), 2.72 (d, J ) 10.3 Hz, 1H), 2.54 (s, 3H), 1.99
(m, 2H), 1.28 (m, 1H), 0.85 (m, 1H), 0.79 (t, J ) 5.41 Hz, 3H).
HRMS: calcd for C18H19FN2O3 + H, 331.14580, found (ESI+,
[M + H]+), 331.14515. Anal. Calcd for C18H19FN2O3: C, 65.44;
H, 5.80; N, 8.48. Found: C, 65.45; H, 5.75; N, 8.47.
Supporting Information Available: Analysis data for
the compounds synthesized. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
°C. H NMR (300 MHz, DMSO): δ 12.02 (bs, 1H), 11.33 (bs,
1H), 7.00 (d, J ) 9.00 Hz, 1H), 3.96 (m, 2H), 2.95 (d, J ) 10.3
Hz, 1H), 2.83 (t, J ) 3.9 Hz, 1H), 2.72 (d, J ) 10.3 Hz, 1H),
2.54 (s, 3H), 1.99 (m, 2H), 1.28 (m, 1H), 0.85 (m, 1H), 0.79 (t,
J ) 5.41 Hz, 3H). MS (ESI): m/z 329.2. Anal. Calcd for C18H19-
FN2O3: C, 65.44; H, 5.80; N, 8.48. Found: C, 65.43; H, 5.86;
N, 8.44.
References
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[(1R)-8-Chloro-5-ethyl-1-propyl-1,3,4,9-tetrahydropy-
rano[3,4-b]indol-1-yl]acetic Acid (23). 1H NMR (400 MHz,
DMSO-d6): δ 11.99 (s, 1H), 10.91 (s, 1H), 7.01 (d, J ) 8.0 Hz,
1H), 6.75 (d, J ) 8.2 Hz, 1H), 3.98-3.84 (m, 2H), 2.96 (d, J )
13.6 Hz, 2H), 2.94-2.82 (m, 2H), 2.78 (d, J ) 13.6 Hz, 2H),
2.10-1.93 (m, 2H), 1.33-1.19 (m, 1H), 1.21 (t, J ) 7.6 Hz, 3H),
0.81-0.74 (m, 4H). HRMS: calcd for C18H22ClNO3 + H,
336.1361, found (ESI-FTMS, [M + H]), 336.13619.
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(1S)-8-Chloro-5-ethyl-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indol-1-yl]acetic Acid (24). 1H NMR (400 MHz,
DMSO-d6): δ 11.99 (s, 1H), 10.91 (s, 1H), 7.01 (d, J ) 8.0 Hz,
1H), 6.75 (d, J ) 8.2 Hz, 1H), 3.98-3.84 (m, 2H), 2.96 (d, J )
13.6 Hz, 2H), 2.94-2.82 (m, 2H), 2.78 (d, J ) 13.6 Hz, 2H),
2.10-1.93 (m, 2H), 1.33-1.19 (m, 1H), 1.21 (t, J ) 7.6 Hz, 3H),
0.81-0.74 (m, 4H). HRMS: calcd for C18H22ClNO3 - H,
334.12154, found (ESI-, [M - H]-), 334.12143.
[(1R)-5,8-Dichloro-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indol-1-yl]acetic Acid (25). Mp: 57 °C. 1H NMR (300
MHz, CDCl3): δ 9.12 (bs, 1H), 7.03 (d, J ) 8.26 Hz, 1H), 6.96
(d, J ) 8.26 Hz, 1H), 4.04 (m, 2H), 3.14 (m, 2H), 3.06 (m, 2H),
2.03 (m, 2H), 1.42 (m, 1H), 1.21 (m, 1H), 0.89 (t, J ) 7.34 Hz,
3H). HRMS: calcd for C16H17Cl2NO3 + H, 342.06582, found
(ESI-FTMS, [M + H]), 342.06637.
[(1S)-5,8-Dichloro-1-propyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indol-1-yl]acetic Acid (26). Mp: 58 °C. 1H NMR (300
MHz, CDCl3): δ 9.12 (bs, 1H), 7.03 (d, J ) 8.26 Hz, 1H), 6.96
(d, J ) 8.26 Hz, 1H), 4.04 (m, 2H), 3.14 (m, 2H), 3.06(m, 2H),
2.03 (m, 2H), 1.42 (m, 1H), 1.21 (m, 1H), 0.89 (t, J ) 7.34 Hz,
3H). HRMS: calcd for C16H17Cl2NO3 + H, 342.06582, found
(ESI-FTMS, [M + H]), 342.06576.
[(1R)-5-Cyano-8-fluoro-1-propyl-1,3,4,9-tetrahydropy-
rano[3,4-b]indol-1-yl]acetic Acid (28). Mp: 163-165 °C.1H
NMR (300 MHz, DMSO): δ 12.02 (bs, 1H), 11.33 (bs, 1H), 7.00
(d, J ) 9.00 Hz, 1H), 3.96 (m, 2H), 2.95 (d, J ) 10.3 Hz, 1H),
2.83 (t, J ) 3.9 Hz, 1H), 2.72 (d, J ) 10.3 Hz, 1H), 2.54 (s,
3H), 1.99 (m, 2H), 1.28 (m, 1H), 0.85 (m, 1H), 0.79 (t, J ) 5.41
Hz, 3H). MS (ESI): m/z 315.5. Anal. Calcd for C17H17FN2O3:
C, 64.55; H, 5.42; N, 8.86. Found: C, 64.78; H, 5.81; N, 8.70.
[(1S)-5-Cyano-8-fluoro-1-propyl-1,3,4,9-tetrahydropy-
rano[3,4-b]indol-1-yl]acetic Acid (29). Mp: 163-166 °C.1H
NMR (300 MHz, DMSO): δ 12.02 (bs, 1H), 11.33 (bs, 1H), 7.00
(d, J ) 9.00 Hz, 1H), 3.96 (m, 2H), 2.95 (d, J ) 10.3 Hz, 1H),
2.83 (t, J ) 3.9 Hz, 1H), 2.72 (d, J ) 10.3 Hz, 1H), 2.54 (s,
3H), 1.99 (m, 2H), 1.28 (m, 1H), 0.85 (m, 1H), 0.79 (t, J ) 5.41
Hz, 3H). HRMS: calcd for C17H17FN2O3 + H, 317.12960, found
(ESI-FTMS, [M + H]), 317.12936.
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