Tetrahydrocarbazole amides with potent activity against human papillomaviruses

Article abstract of DOI:10.1016/j.bmcl.2009.06.001

Synthesis of a series of tetrahydrocarbazole amides with potent activity against human papillomaviruses is described. Synthetic approaches allowing for variation of the substitution pattern of the tetrahydrocarbazole and the amide are outlined and resulti

Full text of DOI:10.1016/j.bmcl.2009.06.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4110–4114
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrahydrocarbazole amides with potent activity against human
papillomaviruses
a
a
a
Kristjan S. Gudmundsson a, , Sharon D. Boggs , Paul R. Sebahar , Leah D’Aurora Richardson ,
Andrew Spaltenstein a, Pamela Golden c, Phiroze B. Sethna b, Kevin W. Brown b,
Kelly Moniri b, Robert Harvey b, Karen R. Romines b
*
a Department of Medicinal Chemistry, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Research & 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 & Development, Five Moore Drive,
Research Triangle Park, NC 27709-3398, USA
c Department of DMPK, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Research & 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:
Synthesis of a series of tetrahydrocarbazole amides with potent activity against human papillomaviruses
is described. Synthetic approaches allowing for variation of the substitution pattern of the tetrahydroc-
arbazole and the amide are outlined and resulting changes in antiviral activity and certain developability
parameters are highlighted. Several compounds with in vitro antiviral activity (W12 antiviral assay) in
the single digit nanomolar range were identified and N-[(1R)-6-chloro-2,3,4,9-tetrahydro-1H-carbazol-
1-yl]-2-pyridinecarboxamide was selected for further evaluation.
Received 9 May 2009
Revised 27 May 2009
Accepted 1 June 2009
Available online 6 June 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 that occur in the US each year, with at least 20 million
people currently infected.2
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 while 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 treatment for genital warts and cervical dys-
plasia involve surgical removal or chemical destruction of the in-
fected 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
target the viral infection, and indeed due to the simplicity of the
virus it provides few unique viral targets for drug development. Re-
cently, vaccines (CervarixÒ and GardasilÒ) have been developed to
prevent some types of HPV infection, but the impact of these vac-
cines 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 optimization of anti-HPV activ-
ity.6 Previously we described optimization of the 1-aminotetrahyd-
rocarbazole scaffold, where we showed that C6-substitution of the
tetrahydrocarbazole core with a lipophilic halogen (Cl or Br), and
a
-methylbenzylamine substitution with (R)-stereochemistry at
the 1-amine, were optimal for anti-HPV activity (compound 1).7
Compound 1 (Fig. 1) showed good antiviral activity (IC50 30 nM)
and selectivity (SI >100 in multiple cell lines). However, upon fur-
ther studies we found it to have potential cytochrome P450 inhibi-
tion (against CYP2D6, IC50 0.16
lM) as well as modest hERG
potassium channel inhibition (IC50 1.7
lM). Inhibition of both
CYP2D68 as well as the hERG channel9 have been linked to com-
pounds with charged amines and a heterocyclic (aromatic) moiety.
We thus became interested in changing the basicity of the 1-amine
and investigating how this would affect the anti-HPV activity.
* Corresponding author. Tel.: +44 (0) 1784482810.
E-mail address: ksgudmundsson@gmail.com (K.S. Gudmundsson).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.001

K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4110–4114
4111
Table 1
3
4
HPV activity and cytotoxicity of tetrahydrocarbazole amides and sulfonamides
5
6
Br
Br
H
NH
1
N
R
H
N
H
N
H
H
1
a
b
Compd
8
R
IC50
(
lM)
CC50
(lM)
Figure 1. (1R)-6-Bromo-N-[(1R)-1-phenylethyl]-2,3,4,9-tetrahydro-1H-carbazole-
1-amine (1).
0.01
0.04
0.09
50.8
6-Bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one (6) was pre-
paredfromp-bromoaniline(2)and2-(hydroxymethylene)cyclohex-
anone (4),10 via cyclohexane-1,2-dione(p-bromophenyl)hydrazone
(5) as outlined in Scheme 1. The reaction, known as the Borsche syn-
thesis, is a special case of the Fischer indole synthesis.11 Tetra-
hydrocarbazolone 6 was used to make aniline derivative 8 and
aminopyridine derivative 10 via two step reductive amination as
shown in Scheme 1.
Tetrahydrocarbazole-1-amine 11 was prepared by treatment of
6 with NH4OAc and sodium cyanoborohydride in methanol. Con-
densation of 2-chloropyrimidine (12) and 11 in a microwave at
150 °C was used to prepare pyrimidine derivative 13.
10
13
14
15
20.7
89.5
>50
50
N
N
N
O
O
12.9
CH3
Anti-HPV activity for compounds 8, 10 and 13 is shown in Table
1. These compounds showed potent anti-HPV activity as well as lit-
tle CYP2D6 (32
lM for 8) and hERG (>30 lM for 8) inhibition. This
0.4
demonstrated that by changing the basicity of the 1-amine we
O
O
OH
16
17
0.03
0.58
>20
>50
H
Br
4
Br
a
b
+
O
O
NH2
N2
2
3
Br
Br
c
O
N
N
N
18
19
20
21
1.2
>50
>40
>20
>50
>20
H
O
H
6
5
O
O
Br
e
NH2
0.32
7
N
N
H
H
d
8
6
d
e
0.045
2.26
N
Br
O
NH2
9
N
N
N
H
H
O
S
10
CH3
N
O
Cl
12
N
g
Br
Br
f
N
O
S
6
22
0.024
NH2
N
N
N
N
H
H
H
O
11
13
a
HPV activity measured in W12-20850 cells containing episomal HPV-16 DNA.
IC50 is the concentration at which 50% efficacy in the W-12 assay is observed using a
hybrid capture method.
Scheme 1. Reagents and conditions: (a) NaNO2, aq HCl, 20 °C, 15 h; (b) NaOAc, aq
MeOH, reflux, 2 h, (49% from 2); (c) concd HCl in AcOH; 120 °C for 20 min, (88%); (d)
p-TsOH (cat), toluene (Dean Stark), reflux, 16 h; (e) NaBH4, MeOH, 30 min (yellow
solid, 51% for 8); (f) NH4OAc, NaCNBH3, MeOH, 60 °C, 15 h (52%); (g) DMF,
Microwave (Smith Synthesizer), 150 °C, 15 min (26%).
b
CC50 is the concentration at which 50% cytotoxicity is observed in human
foreskin fibroblasts (HFF cells).12

4112
K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4110–4114
Table 2
could modulate CYP2D6 and hERG inhibition. However modulation
of the basicity of the 1-amine reduced solubility and resulted in
poor bioavailability in rats (bioavailability <5% from a solution for-
mulation of 8).
HPV activity and cytotoxicity of tetrahydrocarbazoles benzamides
1-amine
chirality
X
We thus turned our attention to synthesis of amides and sulfon-
amides. Amides could be conveniently prepared from amine 11
and acid chlorides as outlined in Scheme 2. While simple alkyla-
mides, like acetamide 14, showed little activity, more lipophilic
amides like cyclohexanecarboxamide 15 and especially benzamide
16, showed much better anti-HPV activity. The benzamide 16 was
about 2 orders of magnitude more potent than the acetamide 14.
This prompted us to change the distance between the amide and
the phenyl moiety by synthesis of the phenylacetamide 17 and
phenylpropaneamide 18, as well as phenylpropenamide 19 and fi-
nally the benzyl carbamate 20. Moving the amide and the phenyl
moiety 1 or 2 ‘carbons’ apart did not improve activity. In fact these
derivatives were, with the exception of the benzyl carbamate 20,
about an order of magnitude less potent than 16. We then synthe-
sized a couple of sulfonamide derivatives by condensing amine 11
with sulphonyl chlorides under basic conditions as outlined in
Scheme 2. Sulfonamides 21 and 22 showed similar activity trends
to the amides, thus the simple methylsulfonamide 21 showed poor
activity while toluyl sulfonamide 22 was equally potent to the
benzamide 16. For further optimization we chose to look at explor-
ing the substitution pattern of the benzamide moiety of 16, as the
molecular weight of the amides is lower than the weight of the sul-
fonamides, and more benzoyl chlorides are commercially available
than sulphonyl chlorides.
For a systematic look at the optimum substitution of the benz-
amide, we synthesized benzamides substituted with methyl,
methoxy and fluorine in the ortho (23–25), meta (27–29) and para
(31–33) positions, as well as 2, 3 and 4-pyridinylamides (26, 30
and 34, respectively) (Table 2). The 2-fluorosubstituted benzamide
25 and the 2-pyridinylamide 26 showed the most potent anti-HPV
activity, while the ortho-methyl derivative 23 and the ortho-meth-
oxy derivative 24 showed the worst activity. In contrast meta and
para substituents had less dramatic effect on anti-HPV activity.
Derivatives 25 and 26, being the most potent compounds, were
optimized further.
R
N
N
H
H
a
b
Compd
X
R
Chirality IC50 (lM) CC50 (lM)
O
O
O
16
Br
Racemic 0.03
Racemic 0.5
Racemic 21
>20
Me
23
24
Br
Br
40
OMe
100
O
O
F
25
26
Br
Br
Racemic 0.008
Racemic 0.005
49
72
N
O
Me
27
Br
Racemic 0.024
41
O
O
As mentioned previously for compound 1 the (R)-enantiomer at
the 1-amine showed potent activity, while the S-isomers had lim-
ited activity. As the amides described were synthesized as race-
mates we became interested in separating the enantiomers and
testing them independently. For benzamide 16, the R- and S-enan-
tiomers were separated using supercritical fluid chromatography13
and stereochemistry was assigned by Ab Initio Vibrational Circular
Dichroism (VCD) Spectroscopy.14 Again, the R-enantiomer 35 was
found to be very active, while the S-enantiomer 36 showed virtu-
ally no activity.
OMe
28
29
Br
Br
Racemic 0.021
Racemic 0.018
26
65
F
O
O
30
31
32
33
Br
Br
Br
Br
Racemic 0.19
Racemic 0.010
Racemic 0.017
Racemic 0.044
100
87
N
O
Br
Cl
O
CH3
N
N
Me
OMe
F
H
H
CH3
a
b
14
Br
O
O
NH2
Cl
N
Br
CH3
88
H
O
S
O
N
S
CH3
N
11
H
H
O
O
21
47
Scheme 2. Reagents and conditions: (a) CH3COCl added to solution of 11 and (i-
Pr)2EtN in CH2Cl2 at 0 °C, then stirred at rt for 16 h (white solid, 52%); (b) CH3SO2Cl
added to solution of 11 and (i-Pr)2EtN in CH2Cl2 at 0 °C, then stirred at rt for 5 h (tan
solid, 57%).

K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4110–4114
4113
Table 2 (continued)
Table 3
Plasma pharmacokinetics of 41
a
b
Compd
X
R
Chirality
Racemic
IC50
(lM)
CC50
100
(lM)
Parameter
Rat
Dog
Monkey
O
O
O
O
O
O
O
O
O
Cl (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
13.1
1.6
4.1
56
5
1.1
0.13
3.6
34
Br
0.2
1.7
9.5
35
N
F (%, solution)
56
Clearance (Cl) and volume of distribution (Vd ss) calculated following a 2 mg/kg iv
dose. Half life (T1/2) and oral bioavailability (F) calculated following solution doses
of 10 mg/kg (rat) and 5 mg/kg (dog and monkey).
35
36
37
38
39
40
41
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
0.008
5.7
39
52
26
55
24
36
16
61
and 41 revealed that 41 had significantly better bioavailability
in rats than 39 (56% compared with 21% from a 10 mg/kg dose
in a solution formulation), hence 41 was progressed. Cytotoxicity
testing in a number of additional cell lines (human keratinocytes,
Vero cells) showed good separation between anti-HPV activity
and cytotoxicity (SI >1000 in human keratinocytes, SI >1000 in
Vero cells). Pharmacokinetic behavior of 41 was studied in three
species and is outlined in Table 3.
Pharmacokinetic behavior of 41 indicates that this compound
might be suitable for oral administration. Further screening against
a panel of enzymes and receptors (PanLab) showed little risk of un-
wanted enzyme or receptor inhibition at concentrations close to
those demonstrating anti-HPV activities. Because of the extremely
promising anti-HPV potency and suitable pharmacokinetic profile,
compound 41 was progressed for further testing. This testing
showed that 41 had a unique profile, showing not only potent
anti-HPV activity but also activity against some other viruses,6c
while being selective (41 did not impact the growth of several pri-
mary cell lines studied). Preliminary mechanism studies indicate
that 41 most likely targets a host cell process which results in
the induction of a subset of interferon-stimulated genes.6c This
would explain its activity in some virally infected and immortal-
ized cells as well as lack of toxicity in primary cell lines. The
intriguing profile of 41 is being further evaluated and a suitable
route to prepare kilogram quantities of 41 has been developed.17
0.020
11.3
0.008
2.1
F
F
N
N
0.005
9.0
Acknowledgments
42
We thank Douglas J. Minick for VCD studies and Amanda G.
Culp and Manon Villeneuve for chiral purification work.
a
HPV activity measured in W12-20850 cells containing episomal HPV-16 DNA.
IC50 is the concentration at which 50% efficacy in the W-12 assay is observed using a
hybrid capture method.
References and notes
b
CC50 is the concentration at which 50% cytotoxicity is observed in HFF cells.
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Appleby, M.; Minson, A. C. Int. J. Cancer 1989, 43, 672; b For description of assay
see: Boggs, S.; Catalano, J.; Gudmundsson, K.S.; Richardson, L. D.; Sebahar, P. R.
WO 2005/023245.; (c) Harvey, R.; Brown, K.; Zhang, Q.; Gartland, M.; Walton,
L.; Talarico, C.; Lawrence, W.; Selleseth, D.; Coffield, N.; Leary, J.; Moniri, K.;
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Antiviral Res. 2009, 82, 1.
As the 6-bromosubstituted tetrahydrocarbazole compounds
showed very good activity we next looked at the corresponding
6-chloro analogs in an attempt to reduce the molecular weight
of the compounds and potentially improve pharmacokinetics.
The R- and S-enantiomers were obtained via synthesis of race-
mates followed by separation by supercritical fluid chromatogra-
phy.15 The 6-chloro compounds, especially the (R)-enantiomers of
the 2-fluorosubstituted benzamide 39 and the 2-pyridinylamide
41,16 again demonstrated very impressive anti-HPV activity. Be-
cause of their lower molecular weight and otherwise similar
activity profile to the corresponding 6-bromo derivatives, these
6-chloro derivatives (39 and 41) were selected for further pro-
gression. Both 39 and 41 showed suitable cytochrome P450
(CYP2D6 >20 lM for both compounds) and hERG (IC50 >20 lM
for both compounds) profile for further development, thus solving
the potential problems associated with earlier generations of the
tetrahydrocarbazoles (such as compound 1). Head to head com-
parison of rat pharmacokinetics on solution formulations of 39
7. Gudmundsson, K. S.; Sebahar, P. R.; Richardson, L.; Catalano, J. G.; Boggs, S. D.;
Spaltenstein, A.; Sethna, P. B.; Brown, K. W.; Harvey, R.; Romines, K. R. Bioorg.
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4114
K. S. Gudmundsson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4110–4114
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Borsche, W.; Kienitz, G. A. Chem. Ber. 1910, 43, 2335.
12. Decision to compare activity (IC50) in W-12 (keratinocytes) with cytotoxicity
(CC50) in HFF cells (fibroblasts) was based on the fact that a screening of over
50 tetrahydrocarbazole showed CC50 values to be virtually identical in
keratinocytes and fibroblasts. HFF cells (fibroblasts) were amenable to high
throughput screening, whereas keratinocytes are not.
14. 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. Assignments were later confirmed by X-ray
crystallography of 41.
15. Separated on a Berger analytical SFC with HP1100 diode array detector. Eluted
with 30% MeOH in CO2; flow rate 2 mL/min at 1500 psi, 40 °C on Diacel AS-H
column (Chiral Technologies), 4.6 Â 250 mm, 5
lm; For 41 retention time
5.08 min, for 42 retention time 7.45 min.
13. Separated on a Berger analytical SFC with HP1100 diode array detector. Eluted
with 30% MeOH in CO2; flow rate 2 mL/min at 2250 psi, 50 °C on Diacel AD-H
16. Chirality confirmed by X-ray crystallography of 41.
17. Boggs, S. D.; Cobb, J. D.; Gudmundsson, K. S.; Jones, L. A.; Matsuoka, R. T.; Millar,
A.; Patterson, D. E.; Samano, V.; Trone, M. D.; Xie, S.; Zhou, X. Org. Process Res.
Dev. 2007, 11, 539.
column (Chiral Technologies), 4.6 Â 250 mm, 5
lm; R-isomer retention time
12.37 min; S-isomer retention time 16.11 min.