Substituted tetrahydro-β-carbolines as potential agents for the treatment of human papillomavirus infection

Article abstract of DOI:10.1016/j.bmcl.2009.10.123

The identification and optimization of a series of substituted tetrahydro-β-carbolines with potent activity against human papillomavirus is described. Structure-activity studies focused on the substitution pattern and chirality of the β-carboline ring system are discussed. Optimization of these parameters led to compounds with antiviral activities in the low nanomolar range.

Full text of DOI:10.1016/j.bmcl.2009.10.123

Bioorganic & Medicinal Chemistry Letters 20 (2010) 256–259
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted tetrahydro-b-carbolines as potential agents for the treatment
of human papillomavirus infection
a
a
a
a
John F. Miller a, , Elizabeth M. Turner , Ronald G. Sherrill , Kristjan Gudmundsson , Andrew Spaltenstein ,
*
Phiroze Sethna b, Kevin W. Brown b, Robert Harvey b, Karen R. Romines b, Pamela Golden c
a Department of Medicinal Chemistry, Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive,
Research Triangle Park, NC 27709-3398, USA
b Department of Virology, Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline Research and Development, Five Moore Drive, Research Triangle Park,
NC 27709-3398, USA
c Department of DMPK, Infectious Diseases Center for Excellence in 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 identification and optimization of a series of substituted tetrahydro-b-carbolines with potent activity
against human papillomavirus is described. Structure–activity studies focused on the substitution pattern
and chirality of the b-carboline ring system are discussed. Optimization of these parameters led to com-
pounds with antiviral activities in the low nanomolar range.
Received 28 September 2009
Accepted 27 October 2009
Available online 30 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Tetrahydro-b-carboline
Human papillomavirus
Antiviral
Human papillomaviruses (HPVs) comprise a family of small
double-stranded DNA viruses that are implicated as the causal
agents in a variety of benign and pre-malignant epithelial tumors.
Several HPV variants are known to infect the genital mucosa and
HPV infection is now known to be the most common sexually
transmitted disease worldwide.1 HPVs associated with genital
infections are classified as either low or high risk depending on
their particular disease associations.2 Low risk HPVs (i.e., HPV 6
and 11) cause genital warts whereas the high variants (HPV 16
and 18) are clearly linked with genital cancers including cervical
carcinoma. The majority of HPV infections are transient, however,
a high proportion of persistent infections with high risk types are
known to progress to cervical dysplasia, a precursor to cervical
cancer.
Currently employed treatments for HPV-associated genital
lesions include various ablative and cytodestructive therapies
as well as topical treatment with the immunostimulator imiqui-
mod (AldaraÒ). Two anti-HPV vaccines (CervarixÒ and GardasilÒ)
were recently approved, however, the long term effect of these
agents on cancer incidence remains to be seen.3 There are cur-
rently no small molecule oral agents available for the treatment
of HPV, therefore this constitutes a significant unmet medical
need.
Researchers from our laboratories recently reported the lead
optimization of a series of substituted tetrahydrocarbazoles that
originated from a high throughput screen.4 The screen employed
a human cervical keratinocyte cell line (W12-20850) isolated from
a low grade cervical lesion containing HPV 16 episomal DNA.5 In
addition to the tetrahydrocarbazole hits, we also identified several
substituted tetrahydro-b-carbolines with single digit micromolar
potencies. These compounds, which possessed aryl and acyl sub-
stituents at the 2- and 3-positions respectively (Fig. 1), show a
close structural resemblance to the previously reported tetrahyd-
rocarbazoles. Herein we report our efforts toward the optimization
of the b-carboline series.
In our initial structure–activity studies we chose to hold the
2-substituent constant as p-tolyl and examine the effect of varying
the substituent on the nitrogen atom at the 3-position. These com-
pounds were prepared as shown in Scheme 1. Tryptamine 1 was
5
6
4
7
O
N
3
8
1
R
X
2
N
9
H
A
* Corresponding author. Tel.: +1 919 483 2750; fax: +1 919 315 6923.
E-mail address: john.6.miller@gsk.com (J.F. Miller).
Figure 1. Tetrahydro-b-carboline lead series (A).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.123

J. F. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 256–259
257
Table 1
a
Effect of the 3-position substituent on HPV activity and cytotoxicity
NH2
NH
N
N
p-tol
H
H
1
R1
p-tol
IC50
N
2
N
b for R1 = acyl
c for R1 = sulfonyl
d for R1 = alkyl
H
a
b
R1
p-tol
N
Compound
R1
(lM)
CC50 (lM)
N
O
O
H
3
1.3
2.3
20
>100
>100
>100
3 - 14
Ph
Ph
Scheme 1. Reagents and conditions (a) p-tolualdehyde, glacial AcOH, 80 °C (86%);
(b) RCOCl, DMAP (1.5 equiv), CH2Cl2 (73% for 4) or RCOOH, EDCI, HOBt, DMF (85%
for 5); (c) RSO2Cl, DMAP (1.5 equiv), CH2Cl2 (63% for 11); (d) RCHO, NaBH(OAc)3,
1,2-dichloroethane (83% for 13).
4
5
O
Ph
O
O
subjected to Pictet-Spengler cyclization with p-tolualdehyde to
give intermediate 2.6 Access to amides 3–7 and carbamates 8–10
was provided by acylation with acid chlorides/chloroformates or
by coupling with carboxylic acids under standard carbodiimide
coupling conditions. Similarly, sulfonamide derivatives 11 and 12
were prepared by reaction with the appropriate sulfonyl chloride.
Finally, amine derivatives 13 and 14 were prepared by reductive
alkylation of 2 with isobutyraldehyde and benzaldehyde, respec-
tively.
Table 1 illustrates HPV activity and cytotoxicity data for the 3-
position analogs 3–14. The phenylpropionyl derivative 3 provided
a 1.3 lM IC50 in the W12 assay and an appreciable 100-fold cyto-
toxicity window. Unsaturation of the 3-substituent sidechain to af-
ford cinnamoyl derivative 4 led to little change in activity whereas
the analogous linear acetylene derivative 5 displayed a 10-fold loss
of activity. The basic sidechain analogs 6 and 7 showed modest
6
7
18
15
94
40
N
N
O
O
8
9
1.7
0.9
31
OMe
O
>100
Ph
O
10
23
>100
O
activities in the 15 lM range accompanied by marked erosion of
the cytotoxicity windows, indicating that polar and/or ionizable
groups are not well tolerated. Carbamate derivatives 8 and 9 pro-
vided low micromolar activities while maintaining good cytotoxic-
ity windows whereas the sterically more demanding naphthyl
carbamate 10 showed a 20-fold reduction in potency. The sulfon-
amide derivatives 11 and 12 showed relatively weak activities with
some preference for the smaller methyl analog 11. The isobutyl
O
O
O
11
12
23
66
>100
>100
S
S
Me
O
OMe
and benzyl amine analogs 13 and 14 showed 25 and 5.8 lM activ-
13
14
25
50
94
ities respectively. It is interesting that compound 14 maintains the
low micromolar potency characteristic of the generally more active
acyl derivatives even though it possesses a different hybridization
geometry (sp3 vs sp2) at the ring attachment point.
5.8
Having explored the 3-position SAR in the b-carboline series, we
turned our attention to the 2-position. In this exercise we held the
3-substituent constant as phenylpropionyl and varied the 2-substi-
tuent. Our synthetic approach is illustrated in Scheme 2.
Compounds 15–19 were prepared as described earlier by Pictet-
Spengler cyclization of 1 with the appropriate aldehydes followed
by acylation with phenylpropionyl chloride. Catalytic hydrogenol-
ysis of 19 afforded alcohol 20 which was subjected to Mitsunobu
reaction with phenol to give phenyl ether 21. Alternatively, 20
was converted to the PNP active carbonate by treatment with
p-nitrophenyl chloroformate followed by reaction with pyrrolidine
to give carbamate 22. Compounds 24 and 25 were synthesized
from ester intermediate 23 which itself was obtained via Pictet-
Spengler reaction of 1 with glyoxylic acid followed by Fisher ester-
ification and then acylation with phenylpropionyl chloride.7
Saponification of 23 with LiOH followed by EDCI coupling of the
resulting carboxylic acid with pyrrolidine afforded amide deriva-
tive 24. DIBAL reduction of 23 gave an aldehyde intermediate
which was subjected to reductive amination with pyrrolidine to
give 25.
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.
b
CC50 is the concentration at which 50% cytotoxicity is observed in human
foreskin fibroblasts (HFF cells).
Table 2 shows SAR data for the 2-position analogs 15–25. Phe-
nyl derivative 15 was equipotent with the p-tolyl analog 3 whereas
insertion of a nitrogen atom into the aromatic ring to give pyridine
16 led to a complete loss of activity. The phenethyl derivative 17
illustrates that chain extension of the aromatic unit maintains
HPV activity. Insertion of an oxygen atom into the ethylene tether
of 17 affords benzyl ether 19 which shows a modest loss of activity.
However, replacement of one of the methylene units in 17 with an
oxygen atom leads to a nearly complete loss of potency as illus-
trated by phenyl ether 21. Interestingly, the isobutyl derivative
18 was the most potent analog in the entire set showing a
330 nM IC50 while maintaining a reasonable cytotoxicity window.
Methyl carbamate 23 showed a relatively weak 20 lM IC50 while

258
J. F. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 256–259
c for R2 =
CH2OBn
Table 2
O
Effect of the 2-position substituent on HPV activity and cytotoxicity
a,b
N
1
O
N
N
H
R2
Ph
15 - 19
N
R2
H
Ph
d for 21
e,f for 22
O
O
a
b
Compound
R2
IC50
(lM)
CC50 (lM)
N
N
N
N
OH
OR'
H
Ph
H
Ph
15
1.2
>100
>100
20
O
N
N
21: R' = Ph 22: R' =
16
17
>100
2.7
j,k for 24
l,m for 25
O
>100
g,h,i
N
1
N
H
CO2Me
Ph
18
19
0.33
7.3
22
23
O
N
O
O
24: R2 =
25: R2 =
>100
N
N
R2
N
H
Ph
21
22
72
>100
>100
O
O
Scheme 2. Reagents and conditions: (a) RCHO, glacial AcOH, 80 °C or TFA, CH2Cl2, rt
(80% for R = Ph); (b) phenylpropionyl chloride, (i-Pr)2EtN, CH2Cl2 (86% for 16); (c)
H2, Pd(OH)2, MeOH (62%); (d) PhOH, di-t-butyl azodicarboxylate, Ph3P, CH2Cl2
(30%); (e) 4-nitrophenyl chloroformate, pyridine, CH2Cl2 (91%); (f) pyrrolidine, (i-
Pr)2EtN, CH2Cl2 (83%); (g) glyoxylic acid, aqueous HCl (88%); (h) HCl, MeOH, reflux,
97%; (i) phenylpropionyl chloride, (i-Pr)2EtN, CH2Cl2 (66%); (j) LiOH, THF, H2O
(95%); (k) pyrrolidine, EDCI, HOBt, DMF (78%); (l) DIBAL, ether, À78 °C; (m)
pyrrolidine, NaBH(OAc)3, 1,2-dichloroethane, (25% over two steps).
O
>100
N
O
O
23
24
20
>100
>100
OMe
N
the carbamate and amide derivatives 22 and 24 lost all activity.
The basic pyrrolidine analog 25 showed 8.5 lM activity in the
>100
HPV assay and little separation between antiviral activity and
cytotoxicity.
N
The next structural feature we were interested in exploring was
substitution on the left-hand benzo ring of the b-carboline ring
system. The syntheses in this case were very straightforward
involving Pictet-Spengler cyclization of commercially available
substituted tryptamines with p-tolualdehyde followed by acyla-
tion with phenylpropionyl chloride (Scheme 3).
Table 3 shows the SAR data for the benzo substituted analogs
27–33. Collectively, the benzo ring substitutions are well tolerated
with several conferring substantial increases in HPV activity rela-
tive to the unsubstituted parent compound 3. In addition, all of
the analogs in this set maintain >100-fold cytotoxicity windows.
Of particular interest is the 7-chloro analog 30 which shows a
62 nM anti-HPV IC50. This activity trend is consistent with observa-
tions in the previously discussed, structurally related tetrahydroc-
arbazole series.4
25
8.5
16
a,b
See footnotes a and b in Table 1.
R3
O
R3
a,b
NH2
N
N
H
N
p-tol
Ph
H
26
27 - 33
Scheme 3. Reagents and conditions: (a) p-tolualdehyde; glacial AcOH, 80 °C for
compounds 27–32; TFA, CHCl3, reflux for compound 33 (73% for 30); (b)
phenylpropionyl chloride, DMAP (1.5 equiv), CH2Cl2 (91% for 30).
The final structural feature that we were interested in exploring
was the absolute configuration at the 2-position of the b-carboline
ring system. Therefore, the racemic 7-chloro analog 30 was sub-
jected to enantiomeric separation via chiral supercritical fluid
chromatography.8 The absolute stereochemistries were then
assigned using Ab Initio Vibrational Circular Dichroism (VCD)
Spectroscopy.9 In addition, we combined the optimal 2-position
isobutyl substituent with the 7-chloro feature by synthesizing
racemic 34 which was also subjected to chiral separation and
VCD-based absolute stereochemical assignment.8,9 The SAR data
for these four compounds is shown in Table 4. A clear trend
emerged with the S absolute configuration being preferred afford-
ing 23 nM and 18 nM activities for compounds 30b and 34b,
respectively.
In order to assess their viability as potential drug candidates,
compounds 30b and 34b were subjected to additional biological
profiling including rat pharmacokinetic analysis. Following IV
dosing of 1 mg/kg in rats, compound 30b showed a 6.2 h t1/2
,
and a 3.6 mL/min/kg clearance value while 34b showed a 0.8 h
t1/2, and a 17.3 mL/min/kg clearance value. Unfortunately, both
compounds showed negligible oral bioavailability in rat when
dosed at 10 mg/kg. However, 30b showed a 73% oral bioavailabil-
ity in mouse following a 5 mg/kg dose. Screening of 30b against a
panel of enzymes and receptors (PanLabs) showed little risk of
undesirable off-target activity. In addition, 30b was found to be
devoid of deleterious cytochrome-P450 activity following screen-
ing against a CYP panel. Thus, compound 30b has been identified

J. F. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 256–259
259
Table 3
Acknowledgments
Effect of the benzo ring substituent on HPV activity and cytotoxicity
We would like to thank Doug Minick for the VCD stereochemi-
cal assignments and Amanda Culp and Manon Villeneuve for chiral
purification.
6
7
O
R3
N
8
N
9
p-tol
References and notes
Ph
H
R3
IC50
(lM)
CC50 (lM)
a
b
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) 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.
3. (a) Schmiedeskamp, M. R.; Kockler, D. R. Ann. Pharmacother. 2006, 40, 1344; (b)
Snoeck, R. Antiviral Res. 2006, 71, 181; (c) Haug, C. J. N. Eng. J. Med. 2008, 359,
861.
Compound
27
28
29
30
31
32
33
7-Me
7-MeO
7-F
7-Cl
8-MeO
8-F
0.35
0.57
0.44
0.062
5.8
>100
>100
>100
>100
>100
>100
>100
1.7
2.2
9-Me
a,b
See footnotes a and b in Table 1.
4. (a) 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.
Med. Chem. Lett. 2009, 19, 3489; (b) Gudmundsson, K. S.; Boggs, S. D.; Sebahar, P.
R.; Richardson, L.; Spaltenstein, A.; Golden, P.; Sethna, P. B.; Brown, K. W.;
Moniri, K.; Harvey, R.; Romines, K. R. Bioorg. Med. Chem. Lett. 2009, 19, 4110.
5. 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 a description of the
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.; Singer, S.; Strum, J.; Gudmundsson, K.; Biron, K.; Romines, K. R.; Sethna, P.
Antiviral Res. 2009, 82, 1.
Table 4
Effect of chirality in the 7-chloro series on HPV activity and cytotoxicity
Cl
O
N
*
N
R2
Ph
H
6. For previous syntheses of tetrahydro-b-carbolines see: Cox, E. D.; Cook, J. M.
Chem. Rev. 1995, 95, 1797.
R2
Chirality
IC50
7.7
(
lM)
CC50 (lM)
a
b
7. For Pictet-Spengler reaction with glyoxylic acid see: Ho, B. T.; Walker, K. E. Org.
Synth. 1971, 51, 136.
8. Separated on a Berger analytical SFC with HP1100 diode array detector using a
Compound
30a
30b
34a
34b
p-Tol
p-Tol
i-Butyl
i-Butyl
R
S
R
S
>100
>100
>100
>100
0.023
0.88
0.018
Diacel OD-H column (Chiral Technologies, 4.6 Â 250 mm, 5
lM). Compound 30b
eluted with 30% MeOH in CO2, flow rate 2 mL/min at 1500 psi, 40 °C. Compound
34b eluted with 20% MeOH in CO2, flow rate 2 mL/min at 1500 psi, 27 °C.
9. 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 references: (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.
a,b
See footnotes a and b in Table 1.
as a potential new agent for the treatment of human papilloma-
virus infection.