5348
G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5345–5349
assay plate. After 5 days, medium was removed, and
90 lL of MEM-FCS supplemented with 10 lL of 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium/phenazinemethosulfate
(MTS/PMS) solution (Promega, Leiden, The Nether-
VIRGIL, the European Network of Excellence on Anti-
viral Drug Resistance (Grant LSHM-CT-2004-503359
from the Priority 1 ‘Life Sciences, Genomics and
Biotechnology’), and by a grant (S-6146-Sectie1) from
the Belgian Government (‘Federale Overheidsdienst
Volksgezondheid, Veiligheid van de Voedselketen en
Leefmilieu’).
lands) was added to each well. Following
a 2 h
incubation period at 37 ꢁC, the optical density of each
well was read at 490 nm in a microplate reader (signal
to noise ratio = 5). The percentage CPE was calculated
as follows: % CPE = ((ODtreated)BVDV
((ODcontrol mock ); in which (ODtreated BVDV
ꢀ(ODcontrol BVDV
)
)
)/
=
References and notes
)
ꢀ (ODVC BVDV
)
the OD490nm of cells infected with BVDV and treated
with a certain dilution of compound, (ODcontrol BVDV
the OD490nm of cells infected with BVDV and left
untreated, and (ODcontrol mock = the OD490nm of cells
)
=
1. Lindenbach, B. D.; Rice, C. M. In Fields Virology; Knipe,
D. M., Howley, P. M., Griffin, D. E., Martin, M. A.,
Lamb, R. A., Roizman, B., Straus, S. E., Eds.; Lippincott
Williams & Wilkins: Philadelphia, 2001; pp 991–1041.
2. Houe, H. Biologicals 2003, 31, 137.
3. Edwards, S.; Fukusho, A.; Lefevre, P. C.; Lipowski, A.;
Pejsak, Z.; Roehe, P.; Westergaard, J. Vet. Microbiol.
2000, 73, 103.
)
mock infected and left untreated. The 50% effective
concentration (EC50) was defined as the concentration of
compound that offered 50% protection of the cells
against virus-induced cytopathic effect (CPE) and was
calculated using logarithmic interpolation. Cytostatic
assay. MDBK cells were seeded at a density of 5 · 103
cells per well of a 96-well plate (confluency 10–15%) in
MEM-FCS; 24 h later, serial dilutions of the test
compounds were added. Cells were allowed to prolifer-
ate for 3 days at 37 ꢁC, after which the cell number was
determined by means of the MTS/PMS (Promega)
method (signal to noise ratio = 5). The % cell growth
was calculated as follows: (ODtreated/ODcontrol); in which
(ODtreated) = the OD490nm of cells treated with a certain
dilution of compound, (ODcontrol) = the OD490nm of cells
left untreated. The 50% cytostatic concentration (CC50)
was defined as the concentration that inhibited the
proliferation of exponentially growing cells by 50% and
was calculated using linear interpolation.
4. Greiser-Wilke, I.; Grummer, B.; Moennig, V. Biologicals
2003, 31, 113.
5. Stegeman, A.; Elbers, A.; de Smit, H.; Moser, H.; Smak,
J.; Pluimers, F. Vet. Microbiol. 2000, 73, 183.
6. Stringfellow, D. A.; Riddell, K. P.; Givens, M. D.; Galik,
P. K.; Sullivan, E.; Dykstra, C. C.; Robl, J.; Kasinathan,
P. Theriogenology 2005, 63, 1004.
7. Durantel, D.; Carrouee-Durantel, S.; Branza-Nichita, N.;
Dwek, R. A.; Zitzmann, N. Antimicrob. Agents Chemo-
ther. 2004, 48, 497.
8. Givens, M. D.; Stringfellow, D. A.; Dykstra, C. C.;
Riddell, K. P.; Galik, P. K.; Sullivan, E.; Robl, J.;
Kasinathan, P.; Kumar, A.; Boykin, D. W. Antiviral Res.
2004, 64, 113.
16. Paeshuyse, J.; Leyssen, P.; Mabery, E.; Boddeker, N.;
Vrancken, R.; Froeyen, M.; Ansari, I. H.; Dutartre,
H.; Rozenski, J.; Gil, L. H. V. G.; Letellier, C.;
Lanford, R.; Canard, B.; Koenen, F.; Kerkhofs, P.;
Donis, R. O.; Herdewijn, P.; Watson, J.; De
Clercq, E.; Puerstinger, G.; Neyts, J. J. Virol. 2006,
80, 149.
9. Buckwold, V. E.; Beer, B. E.; Donis, R. O. Antiviral Res.
2004, 60, 1.
10. Lindenbach, B. D.; Evans, M. J.; Syder, A. J.; Wolk, B.;
Tellinghuisen, T. L.; Liu, C. C.; Maruyama, T.; Hynes, R.
O.; Burton, D. R.; McKeating, J. A.; Rice, C. M. Science
2005, 309, 623.
11. Wakita, T.; Pietschmann, T.; Kato, T.; Date, T.; Miyam-
oto, M.; Zhao, Z.; Murthy, K.; Habermann, A.; Krauss-
lich, H. G.; Mizokami, M.; Bartenschlager, R.; Liang, T.
J. Nat. Med. 2005, 11, 791.
17. All new compounds were fully characterized by NMR,
MS, and HRMS spectra. Synthesis of compound 23
(BPIP):
A mixture of 3,4-diaminopyridine (2.000 g),
benzoic acid (1 equiv), and polyphosphoric acid (50 g)
was heated at 190 ꢁC for 3 h with stirring. Then the
mixture was cooled to ambient temperature and poured
into ice/water. The resulting mixture was neutralized by
addition of solid Na2CO3. The crude product was
collected by filtration, washed with water, and dried.
Recrystallized from water; off-white crystals; mp: 229–
230 ꢁC; yield: 96%; 1H NMR (200 MHz, DMSO-d6) d
8.95 (d, 1H, H4, J = 1.0 Hz), 8.31 (d, 1H, H6,
J = 5.4 Hz), 8.28–8.17 (m, 2H, arom. H), 7.64–7.50 (m,
4H, arom. H). 2-Phenyl-1(3)H-imidazo[4,5-c]pyridine
(0.500 g) was dissolved in dry DMF (5 mL) and the
resulting solution was cooled to 0 ꢁC. Aqueous 50%
sodium hydroxide (1.5 equiv) was added and the mixture
was stirred for 15 min. Then 4-bromobenzyl bromide
(1.2 equiv) was added and the resulting mixture was
stirred for 24 h at room temperature. Finally, water
(50 mL) was added, the precipitate was collected by
filtration and dried to give the crude product mixture.
12. Zhong, J.; Gastaminza, P.; Cheng, G.; Kapadia, S.; Kato,
T.; Burton, D. R.; Wieland, S. F.; Uprichard, S. L.;
Wakita, T.; Chisari, F. V. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 9294.
13. Thomson, B. J.; Finch, R. G. Clin. Microbiol. Infect. 2005,
11, 86.
14. Lauer, G. M.; Walker, B. D. N. Eng. J. Med. 2001, 345,
41.
15. Cells and viruses. Madin-Darby bovine kidney (MDBK)
cells were grown in MEM (Gibco) supplemented with
5% heat-inactivated FCS (Integro). FCS was shown to
be free of BVDV-1 and BVDV-2 by RT-PCR.20 First-
passage BVDV NADL stock was generated from
pNADLp15a as previously described.21 Anti-BVDV
assay for cp strains (MTS). MDBK cells were seeded
at a density of 5 · 103 per well in 96-well cell culture
plates (confluency 10–15%) in MEM-FCS. Following
24 h incubation at 37 ꢁC and 5% CO2 medium was
removed and 5-fold serial dilutions of the test com-
pounds were added in a total volume of 100 lL, after
which the cp BVDV virus inoculum (MOI = 2) was
added to each well. This inoculum resulted in a greater
than 90% destruction of the cell monolayer after 3 days
of incubation at 37 ꢁC. Uninfected cells and cells
receiving virus without compound were included in each
Recrystallized from
a mixture of diisopropyl ether
(10 mL) and ethyl acetate (26 mL); colorless crystals;
mp: 212–214 ꢁC; yield: 45%; 1H NMR (200 MHz,
DMSO-d6) d 9.09 (br s, 1H, H4), 8.40–8.33 (m, 2H,
arom. H), 8.17 (dd, 1H, H6, J = 6.8, 1.5 Hz), 7.73 (d,
1H, H7, J = 6.8 Hz), 7.64–7.58 (AA0BB0, 2H, arom. H),