Letters
To replace the sulfonylacetic acid moiety, several
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 21
(2) Brown, D. R.; Bryan, J . T.; Cramer, H.; Fife, K. H. Analysis of
Human Papillomavirus Types in Exophytic Condylomata Acumi-
nata by Hybrid Capture and Southern Blot Techniques. J . Clin.
Microbiol. 1993, 2667-2673.
(3) Yang, L.; Mohr, I.; Fouts, E.; Lim, D. A.; Nohaile, M.; Botchan,
M. The E1 Protein of Bovine Papillomavirus 1 Is an ATP-
Dependent DNA Helicase. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 5086-5090.
(4) IC50 values were determined at room temperature using 3.5 nM
HPV6 E1 and serial 2-fold dilutions of inhibitors in a buffer
containing 1 µM ATP with a trace amount of [γ-33P]ATP and
500 µM Mg(OAc)2 at pH 7.5. (a) J effery, J . A.; Sharom, J . R.;
Fazekas, M.; Rudd, P.; Welchner, E.; Thauvette, L.; White, P.
W. An ATPase Assay Using Scintillation Proximity Beads for
High-Throughput Screening or Kinetic Analysis. Anal. Biochem.
2002, 304 (1), 55-62. (b) White, P. W.; Pelletier, A.; Brault, K.;
Titolo, S.; Welchner, E.; Thauvette, L.; Fazeka, M.; Cordingley,
M.; Archambault, J . Characterization of Recombinant HPV6 and
11 E1 Helicases. J . Biol. Chem. 2001, 276 (25), 22426-22438.
(5) The ATPase assay used in the screen and for the determination
of IC50 presented in this report is carried out using an ATP
concentration (1 µM) well below the Km (12 µM)4b to maximize
its sensitivity to inhibitors.
(6) (a) Rishton, G. M. Nonleadlikeness and Leadlikeness in Bio-
chemical Screening. Drug Discovery Today 2003, 8 (2), 86-96.
(b) Teague, S. J .; Davis, A. M.; Leeson, P. D.; Oprea, T. The
Design of Leadlike Combinatorial Libraries. Angew. Chem., Int.
Ed. 1999, 38 (24), 3743-3747.
(7) (a) A detailed account of the experiments performed to clarify
the mechanism of inhibition of the compounds in this series will
be published separately. White, P. W.; et al. Manuscript in
preparation. (b) Segel, I. H. Enzyme Kinetics; J ohn Wiley & Sons:
New York, 1975; pp 161-226. (c) As a consequence of the
hyperbolic competitive mechanism, compounds in this series are
less active at higher ATP concentration. For example, IC50 values
are approximately 10-fold higher at 50 µM ATP.
known carboxylate and phosphate isosteres were
screened. Two surrogates, ketodifluoromethylphospho-
nic acid derivative 8b and nitromethyl sulfone 8c,
exhibited the desired level of potency (Table 4, entries
3 and 4). One of the two, the nitromethyl sulfone 8c,
was moderately permeable14 but not potent enough to
show cell-based activity. Nevertheless, 8b and 8c con-
stitute excellent starting points for lead optimization.
Con clu sion . The hit-to-lead exercise presented herein
led to the discovery of low nanomolar inhibitors of the
ATPase activity of HPV6 E1 helicase. Preliminary SAR
studies have revealed a permissive area of inhibitor 1,
which suggested the existence of an exploitable binding
site or pocket in HPV6 E1. Parallel synthesis of diverse
libraries produced analogues with improved potency
compared to the initial hit 1 supporting the hypothesis
of an additional binding site. Subsequent focused librar-
ies were generated, producing analogues such as 6g,
which is nearly 500-fold more potent than 1. In addition,
the sulfonacetic acid pharmacophore could be replaced
with more stable surrogates such as a ketodifluoro-
methylphosphonic acid or a nitromethyl sulfone. Future
work should focus on further optimization of the new
surrogates for binding to E1 and improvement of the
overall biopharmaceutical profile of this lead series.
(8) Crute, J .; Grygon, C. A.; Hargrave, K. D.; Simoneau, B.; Faucher,
A.-M.; Bolger, G.; Kibler, P.; Liuzzi, M.; Cordingley, M. G. Herpes
Simplex Virus Helicase-Primase Inhibitors are Active in Animal
Models of Human Disease. Nat. Med. 2002, 8 (4), 386-391.
(9) Recently, an X-ray structure of the helicase domain of SV40 large
T antigen (a helicase belonging to the same superfamily as HPV
E1 but with very low homology) has been published: Li, D.;
Zhao, R.; Lilyestrom, W.; Gai, D.; Zhang, R.; DeCaprio, J . A.;
Fanning, E.; J ochimiak, A.; Szakonyi, G.; Chen, X. Structure of
the Replicative Helicase of the Oncoprotein SV40 Large Tumor
Antigen. Nature 2003, 423, 512-518.
(10) Presumably due to a fragmentation reaction analogous to that
of the J ulia coupling of alkylsulfonylbenzothiazolyl to aldehydes.
Baudin, J . B.; Hareau, G.; J ulia, S. A. A Direct Synthesis of
Olefins by Reaction of Carbonyl Compounds with Lithio Deriva-
tives of 2-[Alkyl- or (2′-Alkenyl)- or Benzyl-sulfonyl]-benzothia-
zoles. Tetrahedron Lett. 1991, 32 (9), 1175-1178.
(11) Diverse amines were selected using the “Molecular Operating
Environment” MOE software available from Chemical Comput-
See Supporting Information for a sample of the amine building
blocks used in this library.
(12) Assay described in the following. White, P. W.; Titolo, S.; Brault,
K.; Thauvette, L.; Pelletier, A., Welchner, E.; Bourgon, L.; Doyon,
L.; Ogilvie, W. W.; Yoakim, C.; Cordingley, M. G.; Archambault,
J . Inhibition of Human Papillomavirus DNA Replication by
Small Molecules Antagonists of the E1-E2 Protein Interaction.
J . Biol. Chem. 2003, 278 (29), 26765-26772.
Ack n ow led gm en t. The authors are grateful to the
following colleagues for their support of the project, their
help in the preparation of the manuscript, and their
valuable assistance in the preparation and character-
ization of analogues: Jacques Archambault, Paul Ander-
son, Michael Bo¨s, Michael Cordingley, Pierre Lavalle´e,
J ianmin Duan, Nathalie Goudreau, Araz J akalian,
Marc-Andre´ Poupart, Kimberley Thompson, Norman
Aubry, Marie-Eve Beaudoin, Sylvain Bordeleau, Colette
Boucher, Karine Brault, Michel Garneau, Michael Little,
Miguel Nantel, Serge Valois, Ewald Welchner, and
Dominik Wernick.
Su p p or tin g In for m a tion Ava ila ble: Figures showing a
sample list of amine building blocks used in libraries and
additional sulfonylacetic acid replacements tested; chemistry
section containing synthetic description, schemes, and ad-
ditional references; experimental procedures and characteriza-
tion of all compounds described in this paper. This material
acs.org.
(13) The sulfonylacetic acid methyl ester prodrugs were inactive in
the cell-based assay, but their low aqueous solubility limited the
concentration at which they could be tested to 1-10 µM.
(14) A to B permeability of 5 × 10-6 cm/s in Caco2 monolayer assay
modified (cell density of 125 000/cm2 in 12-well plates from
Costar no. 3401) from the following. Liang, E.; Proudfoot, J .;
Yazdanian, M. Mechanisms of Transport and Structure-Perme-
ability Relationship of Sulfasalazine and Its Analogs in Caco-2
Cell Monolayers. Pharm. Res. 2000, 17 (10), 1168-1174.
Refer en ces
(1) (a) Howley, P. M. Papillomavirinae: The Viruses and Their
Replication. In Fields Virology, 3rd ed.; Field, B. N., Knipe, D.
M., Howley, P. M., Eds. Lippincott-Raven: Philadelphia, PA,
1996; pp 2045-2076 and references therein. (b) Shah, K. V.;
Howley, P. M. Papillomaviruses. In Fields Virology, 3rd ed.;
Field, B. N., Knipe, D. M., Howley, P. M., Eds.; Lippincott-
Raven: Philadelphia, PA, 1996; pp 2077-2109 and references
therein.
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