Synthesis and biological evaluation of dihydrofuran-fused perhydrophenanthrenes as a new anti-influenza agent having novel structural characteristic

Article abstract of DOI:10.1016/j.bmc.2006.09.046

Dihydrofuran-fused perhydrophenanthrenes were synthesized by means of o-quinodimethane chemistry with high generality and stereoselectivity, and found to exhibit potent anti-influenza activity. These compounds exerted an inhibitory effect on various strains of influenza virus growth, including influenza A and B, with a concentration dependent manner, and direct cytotoxicity was low. Several biological experiments suggested that these new drugs affected a virus replication process before mRNA synthesis stage. Novel rigid cage-type of structural characteristic of the compounds has not been found in hitherto anti-influenza drugs, and will provide new basis and motif for exploring promising and unprecedented anti-influenza agents.

Full text of DOI:10.1016/j.bmc.2006.09.046

Bioorganic & Medicinal Chemistry 15 (2007) 424–432
Synthesis and biological evaluation of dihydrofuran-fused
perhydrophenanthrenes as a new anti-influenza agent having novel
structural characteristic
Yuji Matsuya,a Kazushige Sasaki,a Hiroshi Ochiaib and Hideo Nemotoa,*
aFaculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
bDepartment of Human Sciences and Fundamental Nursing, Faculty of Medicine,
University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
Received 31 August 2006; revised 19 September 2006; accepted 21 September 2006
Available online 10 October 2006
Abstract—Dihydrofuran-fused perhydrophenanthrenes were synthesized by means of o-quinodimethane chemistry with high gener-
ality and stereoselectivity, and found to exhibit potent anti-influenza activity. These compounds exerted an inhibitory effect on var-
ious strains of influenza virus growth, including influenza A and B, with a concentration dependent manner, and direct cytotoxicity
was low. Several biological experiments suggested that these new drugs affected a virus replication process before mRNA synthesis
stage. Novel rigid cage-type of structural characteristic of the compounds has not been found in hitherto anti-influenza drugs, and
will provide new basis and motif for exploring promising and unprecedented anti-influenza agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
for new anti-influenza drug discovery, there remains a
considerable and urgent demand for improved therapeu-
tic agents having different mechanisms of action, due to
rapid emergence of mutant viral antigens and drug resis-
tance as well as undesirable adverse effects of the present
treatment options.
Influenza has been a contagious respiratory disease typ-
ically caused by influenza A and B viruses. These respi-
ratory tract infections have been a major cause of
pandemic mortality and morbidity all over the world,
being a significant medical problem.1 In addition to vac-
cination as a prophylactic method, several therapeutic
treatments have been employed for decades, representa-
tively including amantadine (1) and rimantadine (2).2
These agents block an ion channel of the viral M2 pro-
tein, which has a vital role for the replication process;
however, lack of the M2 protein in the influenza B virus
limits their utility to treating influenza A virus infec-
tions.3 Recent advances in the understanding of the rep-
lication mechanism of the influenza viruses have resulted
in the identification of potential molecular targets for
pharmaceutical intervention. For example, endonucle-
ase4 and neuraminidase5 inhibitors have been newly
developed and have become available such as zanamivir
(3)6 and oseltamivir (4).7 Despite such continuous efforts
In conjunction with our recent research program direct-
ed toward the development of new anti-viral agents, we
have reported that the dihydrofuran-fused tetracyclic
compounds (5) exhibit a notable growth inhibitory
activity against hemagglutinating virus of Japan (HVJ)
in rhesus monkey kidney (LLC-MK2) cells.8 These com-
pounds have an interesting structural feature character-
ized by a highly rigid cage-type conformation (Fig. 1),
which is suggested to play a crucial role for showing
the anti-viral activity by a preliminary survey using the
hemagglutinin aggregation (HA) assay method. These
results prompted us to investigate a possibility of the
dihydrofuran-fused compounds as a new class of anti-
influenza agents possessing a novel structural character-
istic. Here, we describe the synthesis and biological eval-
uation of variously substituted derivatives, containing
the common dihydrofuran-fused perhydrophenanthrene
core structure, as new potential influenza therapeutic
agents.
Keywords: Perhydrophenanthrenes; Anti-influenza agents; Cage struc-
ture; mRNA.
*
Corresponding author. Tel: +81 76 434 7530; fax: +81 76 434 5047;
e-mail: nemotoh@pha.u-toyama.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.046

Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
425
synthetic method of the tetracyclic compounds (5) with
a high stereoselectivity.8,10 Among target compounds
in this study, listed in Figure 2, the compounds 6, 7,
and 8 have been synthesized and reported in our previ-
ous paper,8 taking advantage of a high generality of
the above method based on successive thermal electrocy-
clic reactions of benzocyclobutenes via o-quinodime-
thanes. Biological examination of this series of
compounds has revealed that the TIPS (triisopropylsilyl)
derivative (8) exhibits the most potent anti-viral activity,
at least against HVJ growth.8 Therefore, a simple regio-
isomer (9) of the compound 8 was synthesized according
to Scheme 1, which presents good exemplification of a
general synthetic scheme for the series of dihydrofu-
ran-fused tetracyclic compounds.
Me
NH2
NH2
rimantadine (2)
amantadine (1)
HO
H
O
CO2H
NH2
HO
O
CO2Et
Me
HO
HN
Me HN
Me
HN
Me
O
NH2
O
NH
zanamivir (3)
oseltamivir (4)
O
NC
H
Readily available benzocyclobutene derivative 1711 was
subjected to alkylation, introduction of a TIPS group,
and then deprotection to give an alcohol 18, which
was transformed into a furan-containing derivative 19
by an oxidation-addition sequence. Re-oxidation of
the alcohol 19 followed by ketalization afforded a com-
pound 20, a substrate for the key thermal electrocyclic
reactions. This compound was heated in refluxing o-di-
chlorobenzene to form an o-quinodimethane intermedi-
ate, which subsequently participated in [4 + 2]
cycloaddition with the furan moiety in an intramolecu-
lar fashion with exclusive endo selectivity to furnish
the tetracyclic compound 9 (Scheme 1).
O
NC
H
H
O
H
O
O
O
RO
RO
5
Figure 1. Structures of representative known anti-influenza agents (1–
4) and dihydrofuran-fused perhydrophenanthrenes (5) having rigid
cage-type conformation.
2. Results and discussion
2.1. Chemistry
In the course of our research on the o-quinodimethane
chemistry,9 we have established a concise and efficient
To facilitate the consideration of the effects of substitu-
ents on the aromatic ring to the bioactivities, com-
pounds 10–13 were synthesized through simple
derivatizations of the phenolic compound 6. Among
them, for instance, trifluoromethyl and tert-butyl deriv-
atives (10 and 11) are imitative compounds of the TIPS
derivative 8 with regard to the lipophilic and sterically
congested nature, respectively. Synthetic methods of
these compounds are presented in Section 4, and briefly,
reagents and conditions are as follows; for 10: S-(trifluo-
romethyl)dibenzothiophenium triflate, K2CO3 (54%);
for 11: t-BuOC(=NH)CCl3, BF3ÅOEt2 (47%); for 12:
Ac2O, pyridine (79%); for 13: PhCH2COCl, Et3N,
DMAP (61%).
O
NC
H
O
NC
H
R1
R2
O
R
O
H
H
R = OTIPS (14)
R = OCF3 (15)
R
R
1 = H, R2 = OH (6)
1 = H, R2 = OMe (7)
O
R1 = H, R2 = OTIPS (8)
R
1 = OTIPS, R2 = H (9)
1 = H, R2 = OCF3 (10)
NC
H
O
R
R1 = H, R2 = OtBu (11)
O
R
R
R
1 = H, R2 = OAc (12)
1 = H, R2 = OCOBn (13)
H
R = OTIPS (16)
Two congeners (14 and 15) having a double bond in-
stead of the ethylene ketal structure were also synthe-
sized, taking our previous finding into account that
Figure 2. Chemical structures of dihydrofuran-fused perhydrophe-
nanthrenes investigated in this study.
OH
CN
CN
HO
CN
TIPSO
TIPSO
d, e
O
a – c
OH
O
18
17
19
O
CN
O
O
O
NC
O
TIPSO
f, g
NC
H
H
h
TIPSO
O
O
O
20
OTIPS
9
Scheme 1. Synthesis of compound 9. Reagents and conditions: (a) LDA, then Br(CH2)3OTHP (82%); (b) TIPSOTf (91%); (c) PPTs (78%); (d) TPAP,
NMO (62%); (e) 3-lithiofuran (73%); (f) TPAP, NMO (68%); (g) TMSO(CH2)2OTMS, TMSOTf (88%); (h) o-dichlorobenzene, reflux (59%).

426
Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
such type of compound showed a relatively high inhibi-
tory activity against HVJ growth.8 The compound 21,
which was prepared by the same synthetic procedure
for 19, was subjected to the thermal reaction to afford
the compound 14 as a result of b-elimination of the
hydroxyl group after the cyclization.8 This compound
was further derivatized to the compound 15 in two steps
(Scheme 2). Additionally, a ring-expanded derivative
(16) was prepared for the comparison of the bioactivi-
ties, the synthesis of which had already been reported.10
these three compounds were subjected to the examina-
tions of dose-dependence and cytotoxicity.
Figure 4 clearly shows that these compounds influenced
the influenza A/Aichi/2/68 virus yields at 72-h postinfec-
tion in a concentration-dependent manner. The cytotox-
ic activity was evaluated by the standard MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay method,13 and the OD (optical density) values
for 24 h cultured MDCK cells after treatment with
100 lM or 200 lM of the test compounds are summa-
rized in Table 1. These results indicated that these com-
pounds did not exhibit any direct cytotoxicity, at least at
100 lM drug concentration, and were expected to have
good safety indexes.
2.2. Biology
Initially, we examined the inhibitory activity of the test
compounds (6–16, Fig. 2) against the virus growth in
Madin-Darby canine kidney (MDCK) cells using influ-
enza A/Aichi/2/68 (H3N2 subtype) virus strain at
10 lM drug concentration. The virus yields as a percent
of control were estimated by a plaque titration meth-
od,12 and the results are shown in Figure 3, including
amantadine as a positive control (PC). This survey dis-
closed that several compounds inhibited the virus
growth and could have potential as a new anti-influenza
agent. In particular, the compounds 10, 14, and 16
exhibited a potent activity, suppressing the virus prolif-
eration up to ca. 30% of control, and consequently,
The scope of applicability of the compounds was
investigated using a variety of influenza virus strains,
including A/PR/8/34 (H1N1), A/USSR/92/77 (H1N1),
A/NWS/33 (H1N1), B/Lee, and B/Singapore/222/79.
As shown in Figure 5, it was found that these com-
pounds were effective for both influenza A and B type
viruses, implying that the mechanism of action differs
from that of amantadine. These data suggest that a ser-
ies of dihydrofuran-fused perhydrophenanthrenes inves-
tigated in this study are likely to have a broad spectrum
OH
CN
NC
H
H
8
: compound 10
: compound 14
a
O
: compound 16
TIPSO
O
7
R
21
R = OTIPS (14)
R = OH (22)
R = OCF3 (15)
b
c
6
5
4
3
Scheme 2. Synthesis of compounds 14, 15, and 22. Reagents and
conditions: (a) o-dichlorobenzene, reflux (30%); (b) TBAF, THF
(94%); (c) S-(trifluoromethyl)dibenzothiophenium triflate, K2CO3
(43%).
0
25
50
100
(Control)
100
80
60
40
20
Concentration (μM)
Figure 4. Examination of concentration-dependence of the com-
pounds 10, 14, and 16 on the growth of influenza A/Aichi/2/68 virus
in MDCK cells. Data are expressed as log of PFU/mL (means of three
experiments). For clarity of the figure, error bars are omitted, which
are presented in Supplementary material in tabular form.
Table 1. MTT assay for evaluation of the direct cytotoxicity of the
compounds 10, 14, and 16a
Compound
OD value
100 lM
200 lM
6
7
8
9
10 11 12 13 14 15 16 PC
(Compound No.)
10
14
1.018 0.044
1.035 0.054
1.010 0.066
1.071 0.050
1.062 0.029
1.085 0.065
0.722 0.018
16
Control
Figure 3. Inhibitory effect of the test compounds 6–16 on the growth
of influenza A/Aichi/2/68 virus in MDCK cells at 10 lM drug
concentration. Amantadine was used as a positive control (PC). Data
are expressed as means SD of three experiments (percent of control
yielding 2.77 · 104 plaque forming unit (PFU)/mL).
a Optical density (OD) for cultured MDCK cells after treatment with
the test compound and then MTT solution was measured at 570 nm,
and expressed as mean SD of three measurements.

Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
427
100
80
60
40
20
viral mRNA
: compound 10
: compound 14
: compound 16
Lane 1
Lane 2
Lane 3
Figure 7. Electrophoresis of RNA derived from MDCK cells infected
with influenza A/Aichi/2/68 virus (lane 1: no drug treatment, lane 2:
100 lM drug 10 treatment) or without infection (lane 3). After
isolation of total RNA, reverse transcription, and PCR, the mRNA
preparations were electrophoresed in 1.6% agarose gel.
endocytosis, whose processes are mediated by one of the
viral glycoproteins, hemagglutinin. After sequential fu-
sion process between the viral envelope and the endo-
some constituting the cell membrane, and uncoating
process, synthesis of virus mRNA occurs in the nucleus,
which is catalyzed by a virally encoded polymerase.
After replication, progeny viruses can be released from
the infected cell with the aid of another glycoprotein,
neuraminidase, and spread the infection. In the light
of the time-related effect above described, we investigat-
ed whether mRNA synthesis occurred after the drug
treatment. The experiments were performed by isolation
of RNA from MDCK cells infected with influenza A/Ai-
chi/2/68 virus, reverse transcription, followed by PCR
and electrophoresis, whose results are shown in Figure
7. Lanes 1 and 2 represent RNA derived from the infect-
ed cells without drug treatment and with drug treatment
(10, 100 lM), respectively, and lane 3 represents RNA
derived from non-infected cells. The virus mRNA band
found in lane 1 obviously disappeared in lane 2, indicat-
ing that the drug affected virus proliferation at the
mRNA synthesis stage or before it.
Figure 5. Examination of anti-viral activity of the compounds 10, 14,
and 16 against various strains of influenza A and B viruses. Data are
expressed as means SD of three experiments (percent of control) at
100 lM drug concentration. Viruses used are A/PR/8/34 (PR-8), A/
USSR/92/77 (USSR), A/NWS/33 (NWS), B/Lee (Lee), and B/Singa-
pore/222/79 (Sing).
of anti-influenza activity and may be useful for the man-
agement of outbreaks with pandemic potential.
In conjunction with a mode of action, several experi-
ments were performed using the compound 10 as a rep-
resentative. At first, time-related effect of the test
compound was investigated, which includes a compari-
son among the drug treatments initiated at various times
postinfection. The results, shown in Figure 6, indicated
that the anti-influenza effect was observed only when
the drug treatment was initiated within 1-h postinfec-
tion, and the virus yields were comparable to that of
control culture in the cases of 2 h or later after the virus
infection. These facts suggest that the drug affects the
influenza virus growth at a relatively early stage in the
replication process.
Low pH environments in endosomes and lysosomes
have been known to play an important role for uncoat-
ing process of the viral RNA during influenza infec-
tion.14 For example, bafilomycin A1 has been reported
to exert an inhibitory effect on the influenza virus
growth through a specific inhibition of vacuolar-type
proton pump to raise the pH in endosomes and lyso-
somes.15 To examine whether the drug 10 could exert
a similar effect, acidification of intracellular compart-
ments under influence of the drug was monitored using
a vital fluorescence microscope with acridine orange.12b
This examination revealed that the amount and intensity
of fluorescence in the drug-treated cells were almost
identical with the control cells, implying that the drug
did not affect the acidic environment of intracellular
compartments, which causes the uncoating process.
Although further studies may be necessary to elucidate
a conclusive mechanism of action, we overall consider
that the dihydrofuran-fused perhydrophenanthrenes
investigated in this study exhibit the anti-influenza activ-
ity by affecting a process before mRNA synthesis.
The cycle of influenza infection is initiated by binding of
virus particles to host cell surface receptors followed by
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9 10
time (h) post infection
Figure 6. Time-related effect of the compound 10 on the growth of
influenza A/Aichi/2/68 virus in MDCK cells at 100 lM drug concen-
tration. After infection under drug-free condition, the cells were
treated after various delays of 0–10 h at 37 ꢁC. After a total of 24 h
incubation, data are expressed as means (% of control) SD of three
experiments.
3. Conclusion
In this paper, we disclosed that dihydrofuran-fused per-
hydrophenanthrenes could have potential for new type

428
Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
of an anti-influenza agent. Novel structural features of
these compounds may serve for a new therapeutic op-
tion against influenza infections. Broad generality of
the synthetic method to the core structure by means of
the o-quinodimethane chemistry will facilitate prepara-
tion of a wide variety of analogous derivatives, which
can contribute further in-depth SAR considerations.
oil. 1H NMR (CDCl3, 300 MHz): d 1.53–2.19 (m,
10H), 3.17 (d, 1H, J = 14 Hz), 3.44–3.54 (m, 2H), 3.60
(d, 1H, J = 14 Hz), 3.79–3.87 (m, 2H), 4.59 (br, 1H),
6.69 (s, 1H), 6.78 (d, 1H, J = 8.0 Hz), 6.96 (d, 1H,
J = 8.0 Hz); 13C NMR (CDCl3, 75 MHz): d 19.8, 25.5,
30.9, 34.3, 42.1, 62.8, 67.0, 99.3, 109.0, 117.4, 121.8,
125.2, 132.0, 143.9, 156.5; IR (neat) cmꢀ1: 3375, 2235;
MS m/z 287 (M+); HRMS Calcd for C17H21NO3:
287.1521 (M+). Found: 287.1523.
4. Experimental
4.1. Chemistry
To a solution of this compound (700 mg, 2.44 mmol)
and triethylamine (407 lL, 2.92 mmol) in dry CH2Cl2
(8 mL) was added TIPSOTf (938 lL, 2.68 mmol) at
0 ꢁC, and the mixture was stirred at the same tempera-
ture for 45 min. The reaction mixture was diluted with
H2O at 0 ꢁC, and extracted with CH2Cl2. The combined
organic layer was dried over MgSO4 and concentrated
in vacuo. The residue was purified by column chroma-
tography to afford TIPS derivative (985 mg, 91%) as a
Reagents were purchased from commercial sources and
used as received. Anhydrous solvents were obtained
from commercial sources or prepared by distillation
over CaH2 or P2O5. 1H and 13C NMR spectra were
obtained on a Varian Gemini 300 (300 MHz for 1H
and 75.46 MHz for 13C), using chloroform as an internal
reference. Mass spectra were measured on a JEOL
D-200 or a JEOL AX 505 mass spectrometer, and the
ionization method was electron impact (EI, 70 eV). IR
spectra were recorded on a Perkin-Elmer 1600 spectrom-
eter. Column chromatography was carried out by
employing Cica Silica Gel 60 N (spherical, neutral, 40–
50 lm). Synthetic procedures for the compounds 6–8,8
16,10c, and 2110c have already been reported previously.
NMR charts of the final compounds subjected to bio-
logical examinations are provided as Supplementary
material.
1
colorless oil. H NMR (CDCl3, 300 MHz): d 1.02–1.10
(m, 18H), 1.22–1.29 (m, 3H), 1.52–2.06 (m, 10H), 3.19
(d, 1H, J = 14 Hz), 3.42–3.51 (m, 2H), 3.62 (d, 1H,
J = 14 Hz), 3.76–3.86 (m, 2H), 4.57 (br, 1H), 6.75 (s,
1H), 6.82 (d, 1H, J = 8.0 Hz), 6.97 (d, 1H, J = 8.0 Hz);
13C NMR (CDCl3, 75 MHz): d 12.9, 18.2, 19.9, 25.7,
26.9, 30.9, 34.5, 41.9, 42.1, 62.6, 66.8, 99.0, 113.4,
121.9, 125.0, 133.0, 144.1, 156.2; IR (neat) cmꢀ1: 2236;
MS m/z 443 (M+); HRMS Calcd for C26H41NO3Si:
443.2856 (M+). Found: 443.2853.
To
a solution of the TIPS derivative (900 mg,
4.1.1. 5-Hydroxybenzocyclobutene-1-carbonitrile (17). To
a solution of 5-methoxybenzocyclobutene-1-carbonitri-
le11 (2.0 g, 12.6 mmol) in MeCN (38 mL) was added
TMSI (7.2 mL, 50.3 mmol) at room temperature. After
stirring for 5 min, the mixture was refluxed for 18 h.
The reaction mixture was cooled, and then diluted with
H2O, and the mixture was extracted with AcOEt. The
combined organic layer was dried over MgSO4 and con-
centrated in vacuo. The residue was purified by column
chromatography to afford 17 (1.6 g, 86%) as a colorless
solid. Mp 84–85 ꢁC; 1H NMR (CDCl3, 300 MHz): d
3.43 (dd, 1 H, J = 14, 2.8 Hz), 3.57 (dd, 1H, J = 14,
5.5 Hz), 4.17 (dd, 1H, J = 5.5, 2.8 Hz), 6.73 (s, 1H),
6.75 (s, 1H), 6.82 (d, 1H, J = 7.9 Hz), 6.96 (d, 1H,
J = 7.9 Hz); 13C NMR (CDCl3, 75 MHz): d 28.2, 35.5,
109.9, 117.5, 119.9, 124.7, 134.0, 138.9, 156.4; IR
(KBr) cmꢀ1: 3382, 2245; MS m/z 145 (M+); HRMS
Calcd for C9H7NO: 145.0528 (M+) Found: 145.0531.
2.03 mmol) in dry EtOH (5 mL) was added PPTs
(102 mg, 0.41 mmol) at room temperature, and the mix-
ture was refluxed for 3 h. The reaction mixture was
diluted with H2O and then extracted with Et2sO. The
combined organic layer was dried over MgSO4 and con-
centrated in vacuo. The residue was purified by column
chromatography to afford 18 (569 mg, 78%) as a color-
1
less oil. H NMR (CDCl3, 300 MHz): d 1.06–1.08 (m,
18H), 1.18–1.26 (m, 3H), 1.74–1.81 (m, 2H), 1.97–2.02
(m, 2H), 2.70 (s, 1H), 3.15 (d, 1H, J = 14 Hz), 3.58 (d,
1H, J = 14 Hz), 3.63 (t, 1H, J = 6.0 Hz), 6.73 (d, 1H,
J = 1.7 Hz), 6.82 (dd, 1H, J = 8.0, 1.7 Hz), 6.95 (d, 1H,
J = 8.0 Hz); 13C NMR (CDCl3, 75 MHz): d 12.9, 18.2,
29.6, 34.1, 42.0, 42.2, 62.3, 113.4, 121.9, 122.0, 125.1,
133.0, 144.0, 156.3; IR (neat) cmꢀ1: 3433, 2235; MS
m/z 359 (M+); HRMS Calcd for C21H33NO2Si:
359.2281 (M+). Found: 359.2290.
4.1.3. 1-[3-(Furan-3-yl)-3-hydroxyprop-1-yl]-5-(triisopro-
pylsilyloxy)benzocyclobutene-1-carbonitrile (19). To a
solution of 18 (300 mg, 0.83 mmol) and NMO
(117 mg, 1.00 mmol) in dry CH2Cl2 (5 mL) was added
TPAP (catalytic amount) at room temperature. After
stirring for 10 min, the solution was filtered through
Celite, and the solvent was evaporated off to leave a res-
idue, which was subjected to column chromatography to
give a corresponding aldehyde (185 mg, 62%) as a color-
4.1.2.
1-(3-Hydroxyprop-1-yl)-5-(triisopropylsilyloxy)-
benzocyclobutene-1-carbonitrile (18). To a solution of
17 (500 mg, 3.14 mmol) in dry THF (6.3 mL) was added
LDA (1.0 M THF solution, 6.91 mL, 6.91 mmol) at
ꢀ78 ꢁC. After stirring for 10 min, a solution of
Br(CH2)3OTHP (770 mg, 3.45 mmol) in dry THF
(4 mL) was added dropwise to the mixture, which was
then stirred for 30 min at the same temperature. The
reaction was quenched with H2O at ꢀ78 ꢁC and extract-
ed with Et2O. The combined organic layer was dried
over MgSO4 and concentrated in vacuo. The residue
was subjected to column chromatography to afford
C-alkylated compound (740 mg, 82%) as a colorless
1
less oil. H NMR (CDCl3, 300 MHz): d 1.04–1.10 (m,
18H), 1.15–1.25 (m, 3H), 2.18–2.29 (m, 2H), 2.68–2.76
(m, 2H), 3.14 (d, 1H, J = 14 Hz), 3.58 (d, 1H,
J = 14 Hz), 6.70 (d, 1H, J = 1.6 Hz), 6.80 (dd, 1H,
J = 7.9, 1.6 Hz), 6.91 (d, 1H, J = 7.9 Hz).

Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
429
To a solution of the aldehyde (202 mg, 0.56 mmol) in
dry THF (5 mL) were added 3-lithiofuran (1.0 M THF
solution, 0.62 mL, 0.62 mmol, prepared from 3-bro-
mofuran and n-butyllithium) at ꢀ78 ꢁC, and the mixture
was stirred for 1 h. The reaction was quenched with
H2O, and the aqueous mixture was extracted with
Et2O. The combined organic layer was dried over
MgSO4 and concentrated in vacuo. The residue was
purified by column chromatography to afford 19
(174 mg, 73%) as a colorless oil. 1H NMR (CDCl3,
300 MHz): d 1.08–1.10 (m, 18H), 1.20–1.30 (m, 3H),
1.85–2.13 (m, 5H), 3.16 (d, 1H, J = 14 Hz), 3.62 (d,
1H, J = 14 Hz), 4.73 (t, 1H, J = 5.8 Hz), 6.39–6.40 (m,
1H), 6.74 (d, 1H, J = 1.9 Hz), 6.83 (dd, 1H, J = 8.0,
1.9 Hz), 6.97 (d, 1H, J = 8.0 Hz), 7.38–7.40 (m, 2H);
13C NMR (CDCl3, 75 MHz): d 12.9, 18.2, 33.6, 34.5,
41.9, 42.2, 66.5, 108.3, 113.4, 121.8, 122.0, 125.1,
128.5, 133.0, 139.2, 143.7, 143.9, 156.3; IR (neat)
cmꢀ1: 3434, 2234; MS m/z 425 (M+); HRMS Calcd for
C25H35NO3Si: 425.2386 (M+). Found: 425.2389.
vent was evaporated to leave a residue, which was puri-
fied by column chromatography to afford 9 (24 mg,
59%) as a colorless solid. Mp 62–64 ꢁC; 1H NMR
(CDCl3, 300 MHz): d 1.02–1.10 (m, 18H), 1.17–1.29
(m, 3H), 1.83–1.92 (m, 2H), 2.47–2.58 (m, 1H), 2.78–
2.82 (m, 1H), 3.10 (dd, 1H, J = 16, 2.7 Hz), 3.20 (dd,
1H, J = 16, 2.5 Hz), 3.86–3.96 (m, 4H), 4.02 (d, 1H,
J = 10 Hz), 5.28 (ddd, 1H, J = 10, 2.7, 2.5 Hz), 6.00 (s,
1H), 6.81 (d, 1H, J = 8.2 Hz), 6.84 (s, 1H), 7.12 (d,
1H, J = 8.2 Hz); 13C NMR (CDCl3, 75 MHz): d 12.9,
18.2, 30.0, 31.6, 31.9, 33.2, 35.9, 51.2, 63.9, 65.5, 80.0,
104.8, 110.5, 117.6, 120.0, 127.5, 131.3, 131.8, 143.1,
155.5; IR (KBr) cmꢀ1: 2364; MS m/z 467 (M+); HRMS
Calcd for C27H37NO4Si: 467.2492 (M+). Found:
467.2493.
4.1.6. 2,3,5a,10c-Tetrahydro-1H,6H-5-oxa-3-oxo-8-(tri-
fluoromethoxy)acephenanthrylene-10b-carbonitrile ethyl-
ene ketal (10). To a solution of 6 (50 mg, 0.16 mmol) in
dry DMF (1 mL) were added K2CO3 (27 mg,
0.19 mmol) and S-(trifluoromethyl)dibenzothiophenium
trifluoromethanesulfonate (71 mg, 0.18 mmol) at room
temperature, and the mixture was stirred for 14 h. The
reaction mixture was diluted with H2O and then extract-
ed with AcOEt. The combined organic layer was dried
over MgSO4 and evaporated. The residue was purified
by column chromatography to afford 10 (33 mg, 54%)
4.1.4. 1-[3-(Furan-3-yl)-3-oxo-prop-1-yl]-5-(triisopropyl-
silyloxy)benzocyclobutene-1-carbonitrile ethylene ketal
(20). To a solution of 19 (100 mg, 0.23 mmol) and
NMO (33 mg, 0.28 mmol) in dry CH2Cl2 (5 mL) was
added TPAP (catalytic amount) at room temperature.
According to the work-up procedure mentioned above,
a corresponding ketone (66 mg, 68%) was obtained as
1
as a colorless solid. Mp 176–178 ꢁC; H NMR (CDCl3,
1
a colorless oil. H NMR (CDCl3, 300 MHz): d 1.06–
300 MHz): d 1.79–1.94 (m, 2H), 2.45–2.63 (m, 1H),
2.81–2.90 (m, 1H), 3.07 (dd, 1H, J = 16, 3.2 Hz), 3.25
(dd, 1H, J = 16, 2.9 Hz), 3.82–3.97 (m, 4H), 3.99–4.04
(m, 1H), 5.28–5.29 (m, 1H), 6.02 (s, 1H), 6.75–6.93 (m,
2H), 7.19 (d, 1H, J = 8.8 Hz); 13C NMR (acetone-d6,
75 MHz): d 30.5, 32.2, 34.5, 36.0, 51.9, 65.7, 80.2,
101.2, 105.1, 111.4, 114.7, 117.8, 122.8, 123.0, 127.7,
137.8, 143.4, 146.8, 158.1; IR (KBr) cmꢀ1: 2269; MS
m/z 379 (M+); HRMS Calcd for C19H16F3NO4:
379.1031 (M+). Found: 379.1043; Anal. Calcd for
C19H16F3NO4: C, 60.16; H, 4.25; N, 3.69. Found: C,
60.29; H, 4.32; N, 3.72.
1.10 (m, 18H), 1.19–1.27 (m, 3H), 2.35–2.40 (m, 2H),
3.02–3.08 (m, 2H), 3.22 (d, 1H, J = 14 Hz), 3.65 (d,
1H, J = 14 Hz), 6.73–6.77 (m, 2H), 6.85 (d, 1H,
J = 8.2 Hz), 6.99 (d, 1H, J = 8.2 Hz), 7.44–7.46 (m,
1H), 8.04–8.09 (m, 1H); 13C NMR (CDCl3, 75 MHz):
d 12.9, 18.1, 31.5, 40.0, 41.4, 42.0, 108.6, 113.3, 121.5,
122.2, 125.2, 127.4, 132.8, 143.3, 144.4, 147.3, 156.4,
192.5; IR (neat) cmꢀ1: 2233, 1682; MS m/z 423 (M+);
HRMS Calcd for C25H33NO3Si: 423.2230 (M+). Found:
423.2229.
To a solution of the ketone (60 mg, 0.14 mmol) and
ethylenedioxybis(trimethylsilane) (42 lL, 0.17 mmol) in
dry CH2Cl2 (2 mL) was added trimethylsilyl trifluorom-
ethanesulfonate (catalytic amount) at 0 ꢁC, and the mix-
ture was stirred at the same temperature for 2 h. The
solvent was evaporated off, and the residue was purified
by column chromatography to afford 20 (58 mg, 88%) as
a colorless oil. 1H NMR (CDCl3, 300 MHz): d 1.07–1.10
(m, 18H), 1.20–1.29 (m, 3H), 2.00–2.06 (m, 2H), 2.11–
2.21 (m, 2H), 3.15 (d, 1H, J = 14 Hz), 3.60 (d, 1H,
J = 14 Hz), 3.87–3.92 (m, 2H), 3.97–4.01 (m, 2H),
6.31–6.33 (m, 1H), 6.72 (d, 1H, J = 2.2 Hz), 6.82 (dd,
1H, J = 8.0, 2.2 Hz), 6.96 (d, 1H, J = 8.0 Hz), 7.37–
7.40 (m, 2H); 13C NMR (CDCl3, 75 MHz): d 12.9,
18.2, 31.7, 36.3, 41.7, 42.1, 65.0, 106.9, 108.6, 113.4,
122.0, 125.1, 127.3, 133.0, 140.1, 143.6, 143.9, 156.3;
IR (neat) cmꢀ1: 2233; MS m/z 467 (M+); HRMS Calcd
for C27H37NO4Si: 467.2492 (M+). Found: 467.2493.
4.1.7. 8-tert-Butoxy-2,3,5a,10c-tetrahydro-1H,6H-5-oxa-
3-oxo-acephenanthrylene-10b-carbonitrile ethylene ketal
(11). To a solution of 6 (100 mg, 0.32 mmol) in dry hex-
ane (1.3 mL) and dry CH2Cl2 (0.3 mL) were added BF3
etherate (catalytic amount) and tert-butyl trichloroace-
timidate (187 lL, 0.96 mmol) at room temperature,
and the mixture was stirred for 20 h. After addition of
H2O, the mixture was extracted with AcOEt. The com-
bined organic layer was dried over MgSO4 and concen-
trated in vacuo. The residue was purified by column
chromatography to afford 11 (55 mg, 47%) as a colorless
1
solid. Mp 191–193 ꢁC; H NMR (CDCl3, 300 MHz): d
1.38 (s, 9H), 1.80–1.97 (m, 2H), 2.44–2.60 (m, 1H),
2.81–2.92 (m, 1H), 3.10 (dd, 1H, J = 16, 3.2 Hz), 3.24
(dd, 1H, J = 16, 2.9 Hz), 3.83–3.95 (m, 4H), 3.98–4.03
(m, 1H), 5.30 (ddd, 1H, J = 10, 3.2, 2.9 Hz), 6.00 (s,
1H), 6.88–6.95 (m, 2H), 7.19 (d, 1H, J = 9.0 Hz); 13C
NMR (CDCl3, 75 MHz): d 29.2, 30.0, 31.6, 34.2, 35.6,
51.4, 60.6, 63.9, 65.5, 79.0, 79.9, 104.8, 110.3, 122.3,
122.4, 125.2, 125.7, 126.3, 128.4, 136.2, 143.1, 155.7;
IR (KBr) cmꢀ1: 2235; MS m/z 367 (M+); HRMS Calcd
for C22H25NO4: 367.1784 (M+), found: 367.1786; Anal.
4.1.5. 2,3,5a,10c-Tetrahydro-1H,6H-5-oxa-3-oxo-9-(tri-
isopropylsilyloxy)acephenanthrylene-10b-carbonitrile eth-
ylene ketal (9). A solution of 20 (40 mg, 0.09 mmol) in
o-dichlorobenzene (3 mL) was refluxed for 2 h. The sol-

430
Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
Calcd for C22H25NO4: C, 71.91; H, 6.86; N, 3.81.
Found: C, 71.81; H, 6.99; N, 3.70.
(dd, 1H, J = 8.8, 2.1 Hz), 6.03 (s, 1H), 6.69 (dd, 1H,
J = 8.5, 2.0 Hz), 6.78 (s, 1H), 6.85 (d, 1H, J = 8.5 Hz);
13C NMR (CDCl3, 75 MHz): d 12.9, 18.1, 32.1, 34.5,
35.4, 49.1, 55.7, 109.7, 112.1, 115.0, 118.4, 121.0,
122.9, 123.9, 126.4, 136.7, 142.4, 159.1; IR (neat)
cmꢀ1: 2241; MS m/z 407 (M+); HRMS Calcd for
C25H33NO2Si: 407.2281 (M+). Found: 407.2284.
4.1.8. 8-Acetoxy-2,3,5a,10c-tetrahydro-1H,6H-5-oxa-3-
oxo-acephenanthrylene-10b-carbonitrile ethylene ketal
(12). To a solution of 6 (100 mg, 0.32 mmol) in dry pyr-
idine (2 mL) was added acetic anhydride (33 lL,
0.35 mmol) at room temperature, and the mixture was
stirred for 12 h. The reaction mixture was diluted with
H2O and extracted with AcOEt. The combined organic
layer was washed with 10% HCl and saturated
NaHCO3, successively, then dried over MgSO4. Evapo-
ration of the solvent gave a residue, which was purified
by column chromatography to afford 12 (96 mg, 79%)
4.1.11.
5a,10c-Dihydro-1H,6H-8-hydroxy-5-oxa-ace-
phenanthrylene-10b-carbonitrile (22). To a solution of
14 (30 mg, 0.07 mmol) in CH2Cl2 (1 mL) was added
TBAF (1.0 M THF solution, 80 lL, 0.08 mmol) at
0 ꢁC, and the mixture was stirred for 0.5 h at room tem-
perature. The reaction mixture was diluted with H2O,
extracted with Et2O, and then dried over MgSO4.
Evaporation of the solvent followed by column chroma-
tography afforded 22 (17 mg, 94%) as a colorless oil. 1H
NMR (CDCl3, 300 MHz): d 2.91–3.02 (m, 2H), 3.20–
3.31 (m, 2H), 3.88 (d, 1H, J = 11 Hz), 4.90–5.01 (m,
1H), 5.25–5.38 (m, 1H), 5.42–5.49 (m, 1H), 6.01 (dd,
1H, J = 8.8, 1.9 Hz), 6.12 (s, 1H), 6.64 (dd, 1H,
J = 8.7, 2.5 Hz), 6.77 (s, 1H), 6.98 (d, 1H, J = 8.9 Hz);
13C NMR (CDCl3, 75 MHz): d 32.0, 34.5, 35.6, 49.2,
55.2, 109.7, 112.9, 115.1, 118.4, 120.8, 122.9, 124.0,
126.5, 136.3, 142.4, 159.0; IR (neat) cmꢀ1: 2232; MS
m/z 251 (M+); HRMS Calcd for C16H13NO2: 251.0946
(M+). Found: 251.0958.
1
as a colorless solid. Mp 121–123 ꢁC; H NMR (CDCl3,
300 MHz): d 1.86 (ddd, 1H, J = 12, 6.5, 3.2 Hz), 1.90
(ddd, 1H, J = 12, 3.5, 3.0 Hz), 2.29 (s, 3H), 2.56 (ddd,
1H, J = 15, 6.5, 3.0 Hz), 2.88 (ddd, 1H, J = 15, 3.5,
3.2 Hz), 3.14 (dd, 1H, J = 16, 3.5 Hz), 3.29 (dd, 1H,
J = 16, 2.4 Hz), 3.86–3.96 (m, 4H), 4.02 (d, 1H,
J = 11 Hz), 5.30 (ddd, 1H, J = 11, 3.5, 2.4 Hz), 6.01 (s,
1H), 7.05 (s, 1H), 7.08 (d, 1H, J = 9.3 Hz), 7.32 (d, 1H,
J = 9.3 Hz); 13C NMR (CDCl3, 75 MHz): d 21.4, 29.9,
31.5, 34.1, 35.6, 51.3, 63.9, 65.5, 79.6, 104.7, 110.3,
120.5, 121.8, 123.5, 126.9, 128.4, 137.2, 143.2, 150.7,
169.1; IR (KBr) cmꢀ1: 2230, 1760; MS m/z 353 (M+);
HRMS Calcd for C20H19NO5: 353.1263 (M+). Found:
353.1253; Anal. Calcd for C20H19NO5: C, 67.98; H,
5.42; N, 3.96. Found: C, 67.99; H, 5.42; N, 3.98.
4.1.12. 5a,10c-Dihydro-1H,6H-5-oxa-8-(trifluorometh-
oxy)acephenanthrylene-10b-carbonitrile (15). According
to the synthetic procedure for 10, the compound 22
(17 mg, 0.07 mmol) afforded 15 (9 mg, 43%) as a color-
less solid. Mp 167–169 ꢁC; 1H NMR (CDCl3,
300 MHz): d 2.84–2.98 (m, 2H), 3.17–3.27 (m, 2H),
3.83 (d, 1H, J = 11 Hz), 5.23–5.32 (m, 1H), 5.39–5.46
(m, 1H), 5.97 (dd, 1H, J = 8.8, 2.2 Hz), 6.08 (s, 1H),
6.63 (dd, 1H, J = 8.7, 2.4 Hz), 6.77 (s, 1H), 6.94 (d,
1H, J = 9.1 Hz); 13C NMR (CDCl3, 75 MHz): d 32.1,
34.6, 35.5, 49.2, 55.1, 109.7, 112.8, 115.3, 118.3, 120.8,
122.7, 124.5, 126.1, 136.3, 142.2, 147.0, 158.9; IR
(KBr) cmꢀ1: 2243; MS m/z 319 (M+); HRMS Calcd
for C17H12F3NO2: 319.0820 (M+). Found: 319.0828;
Anal. Calcd for C17H12F3NO2: C, 63.95; H, 3.79; N,
4.39. Found: C, 63.82; H, 3.71; N, 4.50.
4.1.9. 2,3,5a,10c-Tetrahydro-1H,6H-5-oxa-3-oxo-8-(phe-
nylacetoxy)acephenanthrylene-10b-carbonitrile ethylene
ketal (13). To a solution of 6 (50 mg, 0.16 mmol) in
dry CH2Cl2 (1 mL) were added phenylacetyl chloride
(23 lL, 0.18 mmol) and DMAP (catalytic amount) at
room temperature, and the mixture was stirred for 2 h.
According to the work-up procedure just mentioned
above, the compound 13 (42 mg, 61%) was obtained as
a colorless solid. Mp 72–74 ꢁC; 1H NMR (CDCl3,
300 MHz): d 1.83 (ddd, 1H, J = 11, 4.3, 3.5 Hz), 1.88
(ddd, 1H, J = 11, 7.9, 3.2 Hz), 2.17 (s, 2H), 2.55 (ddd,
1H, J = 15, 7.9, 4.3 Hz), 2.88 (ddd, 1H, J = 15, 3.5,
3.2 Hz), 3.12 (dd, 1H, J = 16, 2.1 Hz), 3.26 (dd, 1H,
J = 16, 3.5 Hz), 3.82–3.95 (m, 4H), 3.99–4.03 (m, 1H),
5.29 (ddd, 1H, J = 10, 3.5, 2.1 Hz), 5.99 (s, 1H), 7.03–
7.06 (m, 2H), 7.28–7.38 (m, 6H); 13C NMR (CDCl3,
75 MHz): d 29.9, 31.5, 34.1, 35.6, 41.6, 51.3, 63.9, 65.5,
79.6, 104.6, 110.3, 120.4, 121.8, 123.4, 126.9, 127.5,
128.8, 129.3, 133.3, 137.2, 143.2, 150.7, 169.6; IR
(KBr) cmꢀ1: 2229, 1755; MS m/z 429 (M+); HRMS
Calcd for C26H23NO5: 429.1576 (M+). Found:
429.1565; Anal. Calcd for C26H23NO5: C, 72.71; H,
5.40; N, 3.26. Found: C, 72.77; H, 5.60; N, 3.29.
4.2. Biology
4.2.1. Drugs. The test compounds were dissolved in
dimethylsulfoxide (DMSO) and then diluted with suit-
able medium to become a final concentration of DMSO
to less than 1%.
4.2.2. Virus. Influenza virus was propagated for 3 days
at 35 ꢁC in chorioallantoic cavities of 10-day-old embry-
onated hen eggs. The infected allantoic fluids were clar-
ified by centrifugation at 1000g for 20 min and stored in
small portions at ꢀ80 ꢁC as a virus stock solution.
4.1.10. 5a,10c-Dihydro-1H,6H-5-oxa-8-(triisopropylsilyl-
oxy)acephenanthrylene-10b-carbonitrile (14). A solution
of 2110c (104 mg, 0.24 mmol) in o-dichlorobenzene
(3 mL) was refluxed for 2 h. The solvent was evaporated
to leave a residue, which was chromatographed to afford
4.2.3. Cells. Madin-Darby canine kidney (MDCK) cells
were cultured as monolayers in Eagle’s minimum essen-
tial medium (MEM) supplemented with 8% fetal bovine
serum in a humidified atmosphere containing 5% CO2 at
37 ꢁC.
1
14 (30 mg, 30%) as a colorless oil. H NMR (CDCl3,
300 MHz): d 1.01–1.11 (m, 18H), 1.19–1.29 (m, 3H),
2.89–3.00 (m, 2H), 3.18–3.32 (m, 2H), 3.89 (d, 1H,
J = 11 Hz), 5.24–5.36 (m, 1H), 5.40–5.45 (m, 1H), 5.98

Y. Matsuya et al. / Bioorg. Med. Chem. 15 (2007) 424–432
431
4.2.4. Virus growth assay. A confluent monolayer of
MDCK cells in a 24-well plate was washed once with
phosphate-buffered saline (PBS) and then infected with
influenza A (Aichi/2/68) virus at a multiplicity of infec-
tion (MOI) of 5 plaque forming unit (PFU)/cell (for
experiments for Figures 3 and 6) or 5 · 10ꢀ4 PFU/cell
(for experiments for Fig. 4) for 45 min at room temper-
ature under a drug-free condition. After adsorption, the
cells were washed three times with PBS and then cul-
tured in serum free MEM (supplemented with
2.5 · 10ꢀ3% trypsin for Fig. 4) with various drugs or
1% DMSO (as a control) at 37 ꢁC. At 24 h (for Figs. 3
and 6) or 72 h (for Fig. 4) postinfection, the culture flu-
ids were collected and centrifuged at 500g for 5 min. The
virus yield in the supernatants was assayed by plaque
titration on MDCK cells.12 Reasons for employing dif-
ferent growth conditions between the experiments of
Figures 4 and 6 are as follows. In the case of influenza
virus, proliferation stages, including early and late peri-
ods, are proceeding by hour order and at least 8–12 h is
required to accomplish one-step growth cycle. There-
fore, we adopted 1-h drug treatment to clarify the target
stages of the tested drugs under one-step growth condi-
tion using a relatively higher MOI (5 PFU/cell) as shown
in Figure 6. By using such higher MOI, all cells in a dish
receive virus infection and virus growth cycle is proceed-
ing synchronously in the cells convenient to examine the
target stages in virus growth cycle. On the other hand,
the multiple growth cycle condition using the relatively
lower MOI is convenient to examine whether virus could
grow or not under a specific condition such as treatment
with drugs. Therefore, the dose-dependency in Figure 4
was examined under a multiple growth cycle condition
using 5 · 10ꢀ4 PFU/cell (compare with 5 PFU/cell in
Figure 6) and virus yields were assayed at 72-h postin-
fection at which several virus growth cycles should be
accomplished.
was isolated from collected cells by RNA-Bee Kit (Tel-
Test INC. Texas). Reverse transcription of RNA was
carried out in 20 lL reaction mixture containing
25 mM MgCl2 (2.8 lL), PCR Buffer (Mg2+ free,
2.0 lL), dNTP Mix (4.0 lL), isolated RNA (1 lg) in
DEPC-H2O (8.2 lL), RNase inhibitor (1.0 lL), reverse
transcriptase (1.0 lL), and oligo dT16 (50 pM, 1.0 lL).
Reaction was performed for 60 min at 37 ꢁC, 5 min at
99 ꢁC, and 10 min at 4 ꢁC. Subsequently, PCR was car-
ried out in 30 lL reaction mixture containing reverse
transcription product (1.0 lL), 25 mM MgCl2
(1.66 lL), PCR Buffer (Mg2+ free, 3.0 lL), DEPC-H2O
(18.84 lL), sense primer (50-AATTTTGATGCCT-
GAAACCGT-30, 1.0 lL), antisense primer (50-
GCTCTGTCCATGTTATTTGGATC-30,
1.0 lL),
dNTP Mix (3.0 lL), and Taq DNA polymerase
(0.5 lL). The mixture was denatured initially for 3 min
at 94 ꢁC, followed by 30 cycles consisted of 1 min at
94 ꢁC, 2 min at 55 ꢁC, 3 min at 72 ꢁC, and finally, for
7 min at 72 ꢁC. The products were separated in 1.6%
agarose gel electrophoresis.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.09.046.
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For experiments for Figure 5: a confluent monolayer of
MDCK cells in a 6-cm dish was washed once with PBS
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4.2.6. Evaluation of mRNA synthesis. A confluent mono-
layer of MDCK cells in a 6-cm dish was washed once
with PBS and then inoculated with 200 lL of virus solu-
tion (serum free MEM) for 45 min. After infection, the
cells were washed three times with PBS and then cul-
tured in serum free MEM with the compound 10
(100 lM) or 1% DMSO (as a control) at 37 ꢁC. At 3-h
postinfection, the cells were washed with serum free
MEM and collected to Eppendorf tubes. Total RNA

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