Substituted tetrahydrocarbazoles with potent activity against human papillomaviruses

Article abstract of DOI:10.1016/j.bmcl.2009.05.003

The synthesis and SAR of a series of substituted 1-aminotetrahydrocarbazoles with potent activity against human papillomaviruses are described. Synthetic approaches allowing for variation of the substitution pattern of the tetrahydrocarbazole are outlined and resulting changes in antiviral activity are highlighted. Several compounds with in vitro antiviral activity (W12 antiviral assay) in the low nanomolar range were identified and (1R)-6-bromo-N-[(1R)-1-phenylethyl]-2,3,4,9-tetrahydro-1H-carbazole-1-amine was selected for further evaluation.

Full text of DOI:10.1016/j.bmcl.2009.05.003

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3489–3492
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted tetrahydrocarbazoles with potent activity against human
papillomaviruses
a
a
a
Kristjan S. Gudmundsson a, , Paul R. Sebahar , Leah D’Aurora Richardson , John G. Catalano ,
Sharon D. Boggs a, Andrew Spaltenstein a, Phiroze B. Sethna b, Kevin W. Brown b,
Robert Harvey b, Karen R. Romines b
*
a Department of Medicinal Chemistry, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive,
Research Triangle Park, NC 27709-3398, USA
b Department of Virology, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive, Research Triangle Park,
NC 27709-3398, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of a series of substituted 1-aminotetrahydrocarbazoles with potent activity against
human papillomaviruses are described. Synthetic approaches allowing for variation of the substitution
pattern of the tetrahydrocarbazole are outlined and resulting changes in antiviral activity are highlighted.
Several compounds with in vitro antiviral activity (W12 antiviral assay) in the low nanomolar range were
identified and (1R)-6-bromo-N-[(1R)-1-phenylethyl]-2,3,4,9-tetrahydro-1H-carbazole-1-amine was
selected for further evaluation.
Received 3 March 2009
Revised 1 May 2009
Accepted 4 May 2009
Available online 7 May 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Tetrahydrocarbazole
Human papillomavirus
Antiviral
Human papillomaviruses (HPVs) are small nonenveloped DNA
viruses that cause a wide variety of benign and pre-malignant epi-
thelial tumors. Several HPVs infect the genital mucosa. HPV infec-
tion is the most common sexually transmitted disease throughout
the world, with an incidence roughly twice that of herpes simplex
infection.1 There are over 5.5 million new cases of sexually trans-
mitted HPV infection that occur in the US each year, with at least
20 million people currently infected.2
target the viral infection. Recently, vaccines (CervarixÒ and Garda-
silÒ) have been developed to prevent some types of HPV infection,
but the impact of these vaccines on cancer incidence remains to be
seen.5
A human cervical keratinocyte cell line, W12-20850, isolated
from a low grade cervical lesion and containing HPV-16 episomal
DNA, served as a primary assay for anti-HPV activity.6 This cell-
based HPV screen (W12 assay) was used to screen in excess of
Papillomaviruses that cause genital infections are classified as
either low risk or high risk HPVs.3 The low risk HPVs (such as
HPV 6 and 11) cause genital warts, but the high risk HPVs (such
as HPV 16 and 18) can cause genital cancers including cervical car-
cinoma. The role of HPV as the principal agent in the etiology of
cervical cancer has been clearly established4 with a lifetime risk
of invasive cancer in the range of 5–10% for untreated infections.
While most HPV infections are transient, lasting less than 1 year,
a high proportion of persistent infections with high risk types pro-
gress to cervical dysplasia, the precursor to cervical cancer.
Currently available treatments for genital warts and cervical
dysplasia involve surgical removal or chemical destruction of the
infected tissue. The only drug available is the immunomodulator
imiquimod (AldaraÒ), which is only approved for the treatment
of external genital warts. None of the present treatments directly
500 K compounds, resulting in identification of several 5–10 lM
inhibitors that contained a 1-aminotetrahydrocarbazole core,7
substituted at the C6 and 1-amine positions (Fig. 1).
We became interested in exploring the SAR of these 1-amino-
tetrahydrocarbazoles and herein we describe optimization of the
substitution pattern of the tetrahydrocarbazole core. Substituted
2,3,4,9-tetrahydro-1H-carbazol-1-ones (E) were prepared from
anilines (A) and 2-(hydroxymethylene)cyclohexanone (C),8 via
cyclohexane-1,2-dione(phenyl)hydrazone (D) as outlined in
4
3
5
8
R1
6
2
R2
1
N
N
7
H
H
* Corresponding author. Tel.: +44 (0)1784 482810.
E-mail address: ksgmjl@aol.com (K.S. Gudmundsson).
Figure 1. Aminotetrahydrocarbazole, where R1 and R2 are hydrophobic groups.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.003

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K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3489–3492
O
OH
Br
H
R1
C
R1
12
a
b
a
b
+
NH2
N
H
NH2
11
N2
A
B
Br
Br
13
R1
R1
c
O
N
H
N
N
H
d
c
O
D
I
E
N
H
NH2
14
R1
Br
Br
d or e
15
R2
N
H
N
H
e
F
NHCH2Ph
NHCH2Ph
N
H
N
H
Scheme 1. Reagents and conditions: (a) NaNO2, aq HCl, 20 °C, 15 h; (b) NaOAc, aq
MeOH, reflux, 2 h (49% from A for R1 = Br); (c) concd HCl in AcOH; 120 °C for 20 min
Br
Br
(88% for
R
1 = Br); (d) R2NH2, pTsOH, toluene then BH3 in THF; (e) R2NH2,
16
17
NaBH(OAc)3, AcOH, DCE (31% for R1 = Br, R2 = Bn). (Compound F is racemic at
1-amine position.)
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 75 °C for 16 h (73%); (b) ZnCl2,
xylenes, reflux, 6 h (23%); (c) I2 (4 equiv), NaHCO3 (8 equiv), CH2Cl2, 20 °C for 3 h
(49%); (d) NaHCO3, benzylamine (neat), 150 °C in SmithCreator microwave for
10 min (54%); (e) DDQ (1.5 equiv), CH2Cl2, 16 h at rt (26%).
Scheme 1. The reaction, known as the Borsche synthesis, is a spe-
cial case of the Fischer indole synthesis.9 Desired amines F were
prepared from tetrahydrocarbazolones (E) by either a two step
(imine formation followed by reduction) or one pot reductive
amination.
The activity of tetrahydrocarbazoles where the C6 substituent is
varied is shown in Table 1 (compounds 1–9). A lipophilic electron
withdrawing substituent appeared to be preferred at C6, with the
bromo derivative 9 showing the best activity. To investigate if
the substitution pattern was optimal we next explored the effect
of moving the bromine to the C7 and C8 positions. The 7-bromo
derivative 10 was synthesized from 3-bromoaniline in a similar
fashion as outlined in Scheme 1. To access the 8-bromo derivative
a different methodology was employed (Scheme 2).
Condensation of aniline 11 and 3-bromocyclohexene 12 gave
compound 13. Lewis acid catalyzed aza-Claisen rearrangement of
13 gave 14. Treatment of 14 with iodine under basic conditions10
affected ring closure via an iodonium intermediate and resulted
in formation of hexahydrocarbazole 15. Nucleophilic substitution
of iodine with benzylamine in a microwave gave 16. Oxidation of
16 by DDQ gave the desired tetrahydrocarbazole 17. The effect of
moving the bromine from the C6-position (9) to C7 or C8-position
(10 and 17, respectively) resulted in reduced anti-HPV activity of
nearly two orders of magnitude.11 Next we investigated optimizing
the amine substituent of the 1-aminotetrahydrocarbazole. We
chose the 6-bromo and the 6-methyl as the C6 substituents for
these studies. The N-benzyl-6-bromotetrahydrocarbazoleamine 9
showed the best anti-HPV activity of the C6 substituted com-
pounds, while the N-benzyl-6-methyltetrahydrocarbazole 4 was
about 10-fold less active than 9. We wanted to see if this difference
in activity between the 6-bromo and 6-methyl substituents was
maintained when the 1-amine was changed from benzylamine
(Table 2 shows activity of several of those amines). In general,
compounds with small alkylamine substituents (18) or polar
amine substituents (19 and 20) showed limited activity. More lipo-
philic alkylaryls, such as benzylamine (4) or phenethylamine (21),
Table 1
HPV activity and cytotoxicity of tetrahydrocarbazoles
R1
6
7
N
H
N
H
8
were more potent. The a-methylbenzylamine (22) and the indane-
amine (23) showed still better activity, indicating that a-substitu-
tion of the alkylaryl was optimal.
For the 6-bromo substituted compounds the activity trends
were the same. Notable is the very good anti-HPV potency of the
a
b
Compd
R1
H
IC50
(lM)
CC50 (lM)
1
2
3
4
5
6
7
8
9
6.7
19.7
7.6
1.9
1.0
0.23
1.6
0.33
0.15
11.1
11.4
40.4
44.5
57.9
21.3
6-CO2CH3
6-OCH3
6-CH3
6-CF3
6-NO2
6-F
6-Cl
6-Br
7-Br
8-Br
a-methylbenzylamine 26 in that it is 5-fold more active than the
>100
20.4
35.9
27.9
18.8
>40
benzylamine 9 and over 10-fold more potent than the dimethylb-
enzylamine compound 27. For all analogs, the C6 bromo substi-
tuted compounds showed about 10-fold better activity than the
C6 methyl substituted compounds. The promising activity of the
10
17
a-methylbenzylamine derivatives 22 and 26 prompted us to syn-
>40
thesize their stereoisomers from the chiral (R)- and (S)-a-methylb-
enzylamines, respectively.
a
HPV activity measured in W12-20850 cells containing episomal HPV-16 DNA.
IC50 is the concentration at which 50% efficacy in the W12 assay is observed using a
hybrid capture method.
Use of chiral
facial selectivity in the reductive amination of the tetra-
hydrocarbazolones. Thus, reductive amination of 6-
a-methylbenzylamines resulted in high diastereo-
b
CC50 is the concentration at which 50% cytotoxicity is observed in human
foreskin fibroblasts (HFF cells).

K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3489–3492
3491
Table 2
NH2
(R)
Br
HPV activity and cytotoxicity of tetrahydrocarbazoles with different amine
substitutions
H
(R)
NH
H
N
H
R1
(R)
Br
30
a and b
H
31 (major)
R2
N
H
N
H
O
N
H
R1
R2
IC50
>20
(l
M)
CC50
>20
(lM)
a
b
Br
Compd
29
NH
(S)
N
H
H
18
Me
(R)
H
32 (minor)
OH
N
19
20
Me
Me
>30
>30
>30
>30
Scheme 3. Reagents and conditions: (a) NaBH(OAc)3, AcOH, DCE, rt 12 h (52%,
90:10 ratio of R,R-diastereomer 31 vs S,R-diastereomer 32); (b) separation of
diastereomers via Supercritical Fluid Chromatography using
a Berger Amine
column from Chiral Technologies, 10% MeOH (with 2% diethylamine, 10% CHCl3)
as mobile phase at 1500 psi, 50 °C, 2 mL/min. Retention time for R,R-diastereomer
31 17.5 min, for S,R-diastereomer 32 19.8 min. HCl salt of 31 obtained by dissolving
31 in Et2O and dropwise addition of 1 M HCl in Et2O.
4
Me
Me
Me
Me
Br
1.9
2.7
21.3
13.5
40
bromotetrahydrocarbazolone 29 with (R)-a-methylbenzylamine
(30) gave the R,R-diastereomer 31 in 9:1 excess over the S,R-diaste-
reomer 32 (Scheme 3). These diastereomers could be separated
using supercritical fluid chromatography. Chirality of the 1-amine
chiral center was initially assigned by Ab Initio Vibrational Circular
Dichroism (VCD) Spectroscopy12 and later confirmed by X-ray
crystallography for 31. The R,S-diastereomer (33) as well as the
21
22
23
24
9
0.54
0.47
6.83
0.15
0.41
0.03
0.6
S,S-diastereomer (34) were also prepared from (S)-a-methylben-
zylamine, in this case the S,S-isomer 34 was the major diastereo-
mer (34:33 in 9:1 ratio). Finally, all diastereomers of the 6-
methyltetrahydrocarbazoles were prepared and their anti-HPV
activity tested (Table 3). Antiviral testing of the diastereomers re-
40
vealed that while the chirality of the
a-methyl group had little
influence on the antiviral activity, the stereochemistry of the 1-
amine was very important. Thus diastereomers with R-stereo-
chemistry at the 1-amine showed good potency (31, 33, 35 and
37), while the corresponding 1-amino-S-isomers (32, 34, 36 and
38) were virtually inactive. Again the 6-bromo compounds showed
better potency than the corresponding 6-methyl derivatives.
OH
20.1
18.8
11.4
22.9
>40
30.8
Br
Table 3
HPV activity and cytotoxicity of chiral tetrahydrocarbazoles
1-amine
25
26
27
Br
chirality
R1
α-methyl
CH3
chirality
N
H
N
H
Br
a
b
Compd
R1
Chirality
(1-amine)
Chirality
IC50
CC50
M)
(a
-methyl)
(l
M)
(
l
31
32
33
34
35
36
37
38
Br
Br
Br
Br
Me
Me
Me
Me
R
S
R
S
R
S
R
S
R
R
S
S
R
R
S
S
0.03
>0.5
0.02
2.85
0.3
7.1
0.2
6.7
9.0
>30
15
28.5
29
41
29
36
Br
28
Br
0.12
a
a
HPV activity measured in W12-20850 cells containing episomal HPV-16 DNA.
IC50 is the concentration at which 50% efficacy in the W12 assay is observed using a
hybrid capture method.
HPV activity measured in W12-20850 cells containing episomal HPV-16 DNA.
IC50 is the concentration at which 50% efficacy in the W12 assay is observed using a
hybrid capture method.
b
b
CC50 is the concentration at which 50% cytotoxicity is observed in human
CC50 is the concentration at which 50% cytotoxicity is observed in human
foreskin fibroblasts (HFF cells).
foreskin fibroblasts (HFF cells).

3492
K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3489–3492
4. (a) Bosch, F. X.; Lorincz, A.; Munoz, N.; Meijer, C. J. L. M.; Shah, K. V. J. Clin.
Compound 31 was selected for further studies based on potency
Pathol. 2002, 55, 244; (b) zur Hausen, H. Nat. Rev. 2002, 2, 342.
5. (a) Schmiedeskamp, M. R.; Kockler, D. R. Ann. Pharmacother. 2006, 40, 1344; (b)
Haug, C. J. N. Engl. J. Med. 2008, 359, 861.
6. For a description of the W12 cell line see: (a) Stanley, M. A.; Browne, H. M.;
Appleby, M.; Minson, A. C. Int. J. Cancer 1989, 43, 672; (b) For assay see: Boggs,
S.; Catalano, J.; Gudmundsson, K. S.; Richardson, L. D.; Sebahar, P. R. WO 2005/
023245.
7. For previous syntheses of the tetrahydrocarbazole core see: (a) Chakraborty, D.
P.; Islam, A.; Bhattacharya, P. J. Org. Chem. 1973, 38, 2728; (b) Chakraborty, D.
P.; Chowdhury, B. K. J. Org. Chem. 1968, 32, 1265; (c) Shoeb, A.; Anwer, F.; Kapil,
R. S.; Popli, S. P. J. Med. Chem. 1973, 16, 425; (d) Mahboobi, S.; Kuhr, S.; Meindl,
W. Arch. Pharm. 1994, 327, 611; (e) Balamurali, R.; Rajendra Prasad, K. J. Il
Farmaco 2001, 56, 229.
8. Tautomer of 2-formylcyclohexanone prepared as described by: Tishler, M.; Gal,
G.; Stein, G. A. Org. Synth. 1959, 39, 27.
9. (a) Borsche, W.; Witte, A.; Bothe, W. Justus Liebigs Ann. Chem. 1908, 359, 49; (b)
Borsche, W.; Kienitz, G. A. Chem. Ber. 1910, 43, 2335.
10. Gataullin, R. R.; Minnigulov, F. F.; Fatykhov, A. A.; Spirikhin, L. V.;
Abdrakhmanov, I. B. Russ. J. Org. Chem. 2001, 37, 1289.
11. Due to the poor activity of C-7 (and C-8) substituted compounds, C-5 and other
alternative substitution patterns were not pursued.
and ease of synthesis.13 Cytotoxicity testing in additional cell lines
(human keratinocytes, Vero cells) showed good separation be-
tween the demonstrated anti-HPV activity, as measured by the
W12 assay and cytotoxicity (SI >300 in human keratinocyte cells,
SI >500 in Vero cells). When the compound was dosed (10 mg/
kg) as a suspension (0.5% HPMC:0.1% Tween 80) to female rats it
showed good pharmacokinetics (t1/2 4.4 h; Vd 1.9 L/kg; F 47%; Cl
13.4 mL/min/kg), indicating it might be suitable for oral dosing.
Furthermore, screening against a panel of enzymes and receptors
(PanLab) showed little risk of unwanted enzyme or receptor inhi-
bition. Given the very potent anti-HPV activity in the W12 assay,
low cytotoxicity and suitable pharmacokinetic profile, compound
31 was progressed for testing in animal models.
References and notes
12. Experimental VCD spectra acquired in CDCl3 using Bomem Chiral RTM VCD
spectrometer operating between 2000 and 800 cmÀ1. The GAUSSIAN 98 Suite of
computational programs was used to calculate model VCD spectra.
Stereochemical assignments made by comparing calculated and measured
spectra. For reference: (a) Chesseman, J. R.; Frisch, M. J.; Devlin, F. J.; Stephens,
P. J. Chem. Phys. Lett. 1996, 252, 211; (b) Stephens, P. J.; Devlin, F. J. Chirality
2000, 12, 172.
1. (a) Howley, P. M. In Fundamental Virology; Howley, P. M., Ed.; Lippincott-Raven:
Philadelphia, PA, 1996; p 947; (b) Kroutsky, L. Am. J. Med. 1997, 102, 3.
2. (a) Wilson, F. X. Expert Opin. Emerging Drugs 2001, 6, 199; (b) Department of
Health and Human Services Center for Disease Control and Prevention. Genital
HPV infection—CDC factsheet. www.cdc.gov/std/HPV/STDFact-HPV.htm#
common.; (c) Dunne, E. F.; Unger, E. R.; Sternberg, M.; McQuillan, G.; Swan, D.
C.; Patel, S. S.; Markowitz, L. E. J. Am. Med. Assoc. 2007, 297, 813.
3. (a) Syrjanen, K.; Mantyjarvi, R.; Saarikoski, S.; Vayrynen, M.; Syrjanen, S.;
Parkkinen, S.; Yliskoski, M.; Saastamoinen, J.; Castren, O. Br. J. Obstet. Gynaeccol.
1988, 95, 1096; (b) Wilson, V. G.; Rosas-Acosta, G. Curr. Drug Targets—Infect.
Disord. 2003, 3, 221; (c) Severson, J.; Evans, T. Y.; Lee, P.; Chan, T.-S.; Arany, I.;
Tyring, S. K. J. Cutan. Med. Surg. 2001, 5, 43.
13. While compound 33 shows similar potency and selectivity as 31 it is only
obtained as
bromotetrahydrocarbazolone and (S)-
a
minor isomer from the reductive amination of 6-
-methylbenzylamine.
a