Discovery of the first series of inhibitors of human papillomavirus type 11: Inhibition of the assembly of the E1-E2-Origin DNA complex

Article abstract of DOI:10.1016/S0960-894X(03)00510-9

We have discovered a series of inhibitors of the assembly of the HPV11 E1-E2-origin DNA complex, which incorporate an indandione fused to a substituted tetrahydrofuran.

Full text of DOI:10.1016/S0960-894X(03)00510-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2539–2541
Discovery of the First Series of Inhibitors of Human
Papillomavirus Type 11: Inhibition of the Assembly of the
E1–E2–Origin DNA Complex
Christiane Yoakim,a,* William W. Ogilvie,a,y Nathalie Goudreau,a Julie Naud,a
Bruno Hache,a Jeff A. O’Meara,a Michael G. Cordingley,b Jacques Archambaultb
and Peter W. Whiteb
a
´
Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Quebec, Canada H7S 2G5
´
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Quebec, Canada H7S 2G5
b
Received 6 March 2003; accepted 2 May 2003
Abstract—We have discovered a series of inhibitors of the assembly of the HPV11 E1–E2–origin DNAcomplex, which incorporate
an indandione fused to a substituted tetrahydrofuran.
# 2003 Elsevier Ltd. All rights reserved.
Infection with human papillomaviruses (HPV) is wide-
spread in the population and is associated with a broad
spectrum of disease ranging from benign warts to
malignant lesions.1 More than 25 types of HPV infect
the anogenital region. The two types most often detec-
ted in benign genital warts (condyloma acuminata) are
HPV6 and 11, known as ‘low-risk’ types whereas pre-
cancerous cervical lesions contain mostly the ‘high-risk’
HPV types 16, 18, 31 and 45. Current therapies of HPV-
induced lesions rely on surgical removal of the infected
tissues or their destruction with cytotoxic agents or by
cryotherapy.2 The immunomodulator imiquimod
(Aldara, 3M) is also widely used for the treatment of
external genital warts.3 However, there is a need for
virus-specific drug therapy.4
the replicative helicase of papillomaviruses.8 E2 binds
with high affinity to specific sites in the viral origin of
DNAreplication and, by forming a complex with E1,
enhances binding of E1 specifically to the origin. For-
mation of this complex between E1, E2 and origin DNA
is an essential step in HPV DNAreplication as is needed
for the assembly of larger oligomeric E1 complexes that
have helicase activity. This activity is required during
the initiation and elongation phases of DNAreplication
to unwind the origin as well as the DNAahead of the
replication fork.6 Once bound to the origin, E1 and E2
also interact with components of the host DNArepli-
cation machinery, including the polymerase a primase
complex and human single-stranded binding protein
(hSSB/RPA), to promote replication.6 The essential
roles of the E1 and E2 in HPV DNAreplication make
them attractive targets for antiviral therapies.4
HPV are small, non-enveloped viruses containing a
double-stranded covalently closed circular DNAgen-
ome (approximately 8 kb in length) that encodes five
well-characterized early gene products and two late
capsid proteins.5 Only two virally encoded proteins, E1
and E2, are required for replication of the genome.6 E2
is a sequence-specific DNA-binding protein that is
required for transcription and replication of the viral
genome as well as its segregation during mitosis.7 E1 is
The lack of structural information on the E1 and E2
proteins from HPV type 11 led us to rely on high
throughput screening for the identification of inhibitors.
During the HTS campaign, we screened 140,000 com-
pounds from our corporate collection, which includes
combinatorial libraries, compounds from medicinal
chemistry programs, as well as external acquired com-
pounds. Approximately 1200 initial hits were obtained,
however, after characterization and further profiling for
selectivity we have identified only one lead structure.
This paper will outline the initial discovery and early
SAR optimization of the first inhibitors of the assembly
*Corresponding author. Tel.: +1-450-682-4640; fax: +1-450-682-
4189; e-mail: cyoakim@lav.boehringer-ingelheim.com
yPresent address: Department of Chemistry, University of Ottawa, 10
Marie Curie St., Ottawa, Canada.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00510-9

2540
C. Yoakim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2539–2541
Scheme 1. (a) EtOH, piperidine (cat) (91%); (b) H2O2, NaOH, MeOH (95%); (c) toluene, reflux.
of the HPV11 E1–E2–origin ternary complex. Com-
pound 1 represents our initial hit showing activity in an
E2-dependent E1/DNAbinding assay (IC 50=11 mM).9
In early studies to address the stability of our hit in the
assay buffer, it was found that compound 1 is rapidly
converted to a single product. This product was identi-
fied as the hydrolyzed compound 5, which was also
obtained after treatment of 1 with sodium hydroxide
under slow addition conditions. Similarly, the trans/cis
isomer (4) was converted to the corresponding sodium
salt under dilute conditions as shown in Scheme 2.
The syntheses of all compounds were carried out
according to Schemes 1 and 2. Satisfactory MS, 1H
NMR, IR, and homogeneity data (>95% by HPLC)
were obtained for all inhibitors. The biochemical assay
used for screening was reported previously.9 All IC50
values reported are the average of at least three separate
determinations.
Confirmation of our initial hit structure as well as the
relative stereochemistry of the substituent on the furan
ring was accomplished via an independent synthesis.
Synthesis of 1 was achieved based on a literature pro-
cedure10 and is outlined in Scheme 1. Commercially
available indan-1,3-dione was condensed with p-chloro-
benzaldehyde in the presence of a catalytic amount of
piperidine to give the corresponding benzylidene. Base-
catalyzed oxidation with peroxide afforded epoxide 2.11
Epoxide 2 undergoes thermal 1,3-dipolar cycloaddition
in the presence of the maleimide 3 to give, after puri-
fication, a racemic 1:1 mixture of the cis/cis isomer (1),
and the trans/cis isomer (2). Comparison with these two
synthetic isomers allowed us to confirm by NMR that
the initial hit has the all cis relative stereochemistry. In
addition, the trans/cis isomer 4 was found not to be an
inhibitor (IC50>80 mM).
Having confirmed that the active form of our hit was
indeed represented by compound 5, with a 7.8 mM
activity, and that the relative stereochemistry was
important as shown by the inactivity of 6 (IC50 >80
mM), we undertook our initial SAR study. We first
turned our attention to the phenyl bearing the chlorine
atom. As shown in Table 1, removal of the chlorine
atom resulted in a loss of approximately 10-fold in
potency (compound 7 vs 5). Afurther decrease in
activity was observed when the chlorine is at the ortho
Table 1.
Compd
R
IC50 (mM)
5
7
4-Cl
H
7.8
71
8
9
10
2-Cl
3-Cl
3,4-Cl2
>80
2.6
0.35
Scheme 2.

C. Yoakim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2539–2541
2541
position (compound 8, IC50 >80 mM). However a 3-fold
References and Notes
increase in potency was observed with a meta-chloro
substituent (compound 9). Furthermore, it was gratify-
ing to observe that the beneficial effects were additive.
Indeed, the 3,4-dichlorocompound (10) shows a 10-fold
improvement in activity over our original hit
(IC50=0.35 mM).
1. Zur Hausen, H.; De Villiers, E. M. Annu. Rev. Microbiol.
1994, 48, 427.
2. Beutner, K. R.; Ferenczy, A. Am. J. Med. 1997, 102,
28.
3. Beutner, K. R.; Tyring, S. K.; Trofatter, K. F., Jr.;
Douglas, J. M., Jr; Spruance, S.; Owens, M. L.; Fox,
T. L.; Hougham, A. J.; Schmitt, K. A. Antimicrob. Agents
Chemother. 1998, 42, 789.
4. Phelps, W. C.; Barnes, J. A.; Lobe, D. C. Antiviral Chem.
Chemother. 1998, 9, 359.
5. Howley, P. M. Fields Virology, 3rd ed.; Fields, B. N.;
Knipe, D. M. Eds.; Raven: New York, 1996; p 2045. Shah, K.
V.; Howley, P. M. Virology; 3rd ed.; Fields, B. N.; Knipe, D.
M. Eds.; Raven: New York, 1996; p 2077.
6. Chow, L. T.; Broker, T. R. Intervirology 1994, 37, 150.
7. McBride, A.; Myers, G. Human Papillomaviruses. In The-
oretical Biology and Biophysics; Los Alamos, New Mexico,
USA, 1997; p 54.
In studies to be published elsewhere,12 this series of
compounds was found to be devoid of any DNAinter-
calating activity and to be inactive in unrelated bio-
chemical assays of protein–DNAinteractions, suggesting
that they are specific inhibitors of HPV11 E1 and/or E2.
It was also found that our best compound is able to inhi-
bit HPV DNAreplication in a surrogate cellular assay. 12
However, because HPV or other papillomaviruses can
not be propagated easily in vitro, there is no assay to
evaluate the antiviral activity of this series of compounds.
In summary, the first series of inhibitors capable of
antagonizing the assembly of the HPV11 E1–E2–origin
DNAcomplex have been described. They incorporate
an indandione system spiro-fused to a substituted tetra-
hydrofuran ring. Future publications will describe the
full SAR of this series of inhibitors and will address
their exact mechanism of action.
8. Sverdrup, F.; Myers, G. Human papillomaviruses 1997. In
Theoretical Biology and Biophysics; Los Alamos, New Mexico,
USA, 1997; p 37.
9. White, P. W.; Pelletier, A.; Brault, K.; Titolo, S.; Thau-
vette, L.; Fazekas, M.; Cordingley, M. G.; Archambault, J. J.
Biol. Chem. 2001, 276, 22426.
10. Krysin, M. Y.; Anohina, I. K.; Zalukaev, L. P. Khimiya
Geterotsiklicheskikh Soedinenii 1987, 11, 1463.
11. Weigele, M.; Tengi, J. P.; De Bernardo, S.; Czajkowski,
R.; Leimgruber, W. J. Org. Chem. 1976, 41, 388.
12. White, P.W.; Titolo, S.; Brault, K.; Thauvette, L.; Pelle-
tier, A.; Welchner, E.; Bourgon, L.; Doyon, L.; Ogilvie,
W.W.; Yoakim, C.; Cordingley, M.G.; Archambault J. JBC
In press.
Acknowledgements
We are grateful to C. Boucher and S. Valois for their
excellent analytical support. We extend our gratitude to
Michael Bos for his support to the program.