Synthesis and biological activity of bimorpholine and its carbanucleoside

Article abstract of DOI:10.1080/15257770.2011.621919

A new enantiomerically pure carbacyclic nucleoside analogue with bimorpholine as a nonaromatic nucleobase was synthesized. The nucleoside analogue and bimorpholine were tested for cytotoxicity using an MTT assay and the xCELLigence System. Both assays revealed that compound 3 was highly cytotoxic at a 50 μM concentration while the cytotoxic effect of compound 1 was much less prominent. No antiretroviral activity was detected for this compound. In contrast, it acted as a potent inhibitor of hepatitis C virus (HCV) replication. Most likely this effect originates largely from the cytotoxicity of the compound; however, it is possible that a specific mechanism of HCV inhibition also exists. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.621919

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Synthesis and Biological Activity of
Bimorpholine and its Carbanucleoside
Kerti Ausmees a , Anastasia Selyutina c , Kristel Kütt a , Kristin Lippur
a , Tõnis Pehk b , Margus Lopp a , Eva Žusinaite c d , Andres Merits c &
Tõnis Kanger a
a Department of Chemistry, Tallinn University of Technology, Tallinn,
Estonia
b National Institute of Chemical Physics and Biophysics, Tallinn,
Estonia
c Institute of Technology, University of Tartu, Tartu, Estonia
d Baltic Technology Development, Tallinn, Estonia
Published online: 07 Nov 2011.
To cite this article: Kerti Ausmees , Anastasia Selyutina , Kristel Kütt , Kristin Lippur , Tõnis Pehk ,
Margus Lopp , Eva Žusinaite , Andres Merits & Tõnis Kanger (2011): Synthesis and Biological Activity of
Bimorpholine and its Carbanucleoside, Nucleosides, Nucleotides and Nucleic Acids, 30:11, 897-907
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Nucleosides, Nucleotides and Nucleic Acids, 30:897–907, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.621919
SYNTHESIS AND BIOLOGICAL ACTIVITY OF BIMORPHOLINE
AND ITS CARBANUCLEOSIDE
Kerti Ausmees,1 Anastasia Selyutina,3 Kristel Ku¨ tt,1 Kristin Lippur,1
2
1
To˜ nis Pehk, Margus Lopp, Eva Zusinaite,3,4 Andres Merits,3
ˇ
and To˜ nis Kanger1
1Department of Chemistry, Tallinn University of Technology, Tallinn, Estonia
2National Institute of Chemical Physics and Biophysics, Tallinn, Estonia
3Institute of Technology, University of Tartu, Tartu, Estonia
4Baltic Technology Development, Tallinn, Estonia
A new enantiomerically pure carbacyclic nucleoside analogue with bimorpholine as a nonaro-
2
matic nucleobase was synthesized. The nucleoside analogue and bimorpholine were tested for cyto-
toxicity using an MTT assay and the xCELLigence System. Both assays revealed that compound 3
was highly cytotoxic at a 50 µM concentration while the cytotoxic effect of compound 1 was much
less prominent. No antiretroviral activity was detected for this compound. In contrast, it acted as a
potent inhibitor of hepatitis C virus (HCV) replication. Most likely this effect originates largely from
the cytotoxicity of the compound; however, it is possible that a specific mechanism of HCV inhibition
also exists.
Keywords Bimorpholines; carbacyclic nucleoside analogue; hepatitis C virus
INTRODUCTION
Substituted morpholines are widely occurring structural motifs in various
bioactive natural products, as well as in several pharmaceutically important
compounds.[1] The spectrum of the biological properties of morpholine-
containing compounds is very wide and includes antiviral activity.[2–4] On
the other hand, nucleosides are a very important group of antiviral com-
pounds. In recent decades, carbocyclic nucleosides have received a great
deal of attention because they are recognized by enzymes and receptors that
Received 8 July 2011; accepted 1 September 2011.
The authors are grateful to the Estonian Ministry of Education and Research (Grant No:
0142725s06), the Centre of Excellence in Chemical Biology, the EU European Regional Development
Fund 3.2.0101.08-0017, and the Estonian Science Foundation (Grants No. 8289, 8880).
Kerti Ausmees and Anastasia Selyutina have contributed equally.
Address correspondence to To˜nis Kanger, Department of Chemistry, Tallinn University of Tech-
nology, Akadeemia tee 15, 12618 Tallinn, Estonia. E-mail: kanger@chemnet.ee; Andres Merits, Institute
of Technology, University of Tartu, Nooruse, Estonia. E-mail: andres.merits@ut.ee
897

898
K. Ausmees et al.
O
O
O
O
H
O
N
NH
HN
NH
1
3
FIGURE 1 Tested compounds.
also respond to standard nucleosides.[5,6] Carbocyclic nucleosides are inert
against the action of enzymes that cleave glycosidic bonds, making them
metabolically more stable. In connection with our ongoing project on the
synthesis[7,8] and investigation of antiviral compounds,[9] we present here
the results of our studies of the nonnucleoside antiviral compound 1. Our
preliminary screening of 5,5ꢁ-disubstituted-2,2ꢁ-bimorpholine derivatives re-
vealed no cytotoxicity (at a 50 µM concentration) using a standard MTT
assay; no clear activity against retroviruses (human immunodeficiency virus
type 1) was detected in a biological assay (data not shown). Surprisingly, one
of the compounds had potent anti-HCV (hepatitis C virus) activity at 50 µM
and that prompted us to derivatize it with sugar analogues. Thus, we used this
potential antiviral compound as a nucleobase and designed and synthesized
the new carbocyclic nucleoside analogue 3 (Figure 1). The compounds were
tested for the ability to suppress replication of HCV, a positive single-strand
RNA virus that has infected an estimated 3% of the world’s population and
is one of the main causes of liver cirrhosis and cancer.[10,11]
RESULTS AND DISCUSSION
Synthesis of the Compounds
The synthesis of bimorpholine 1 was described by us previously.[12,13]
Although glycosyl derivatives with secondary amines are known,[14] the re-
action of bimorpholine with ribose (acylated or nonacylated) resulted in an
unstable hemiaminal. Carbocyclic nucleosides, on the other hand, give a sta-
ble C–N bond. The synthetic scheme to 2,3-dideoxy carbocyclic nucleosides
with bimorpholine as a base is very straightforward (Scheme 1).
O
O
R
R
H
O
N
NH
H
O
O
O
O
R
R
HN
NH
3
1
2
SCHEME 1 Retrosynthetic route to nonaromatic nucleoside carba-analogue 3.

Synthesis and Biological Activity of Bimorpholine and Its Carbanucleoside
899
The target 3 can be obtained by the reductive amination of cyclopen-
tanone 2 with bimorpholine 1. Our approach started with 3-oxocyclopent-
anecarboxylic acid 5 that was prepared using the literature procedure re-
ported by Stetter and Kuhlmann[15] (Scheme 2). The skeleton of the carbo-
cycle was formed via a cascade of reactions that involved a conjugate addition
of sodium cyanide to ethyl acrylate, an attack of the formed ester enolate
on the second molecule of ethyl acrylate and cyclization. This one-pot pro-
cedure gave a cyclopentanone derivative 4 in a 42% yield. Simultaneous
acidic hydrolysis and decarboxylation afforded racemic acid 5 in a 64%
yield.
N
O
O
10
%
H
C
l
O
a
N
N
C
O
O
O
H
O
DM
O
r
SO
e u
x
fl
5
4
ruc ne
B
i
r s a za on
t lli ti
y
C
O
O
+
O
O
H
O
H
O
5b
a
5
SCHEME 2 Synthesis and resolution of enantiomers of keto acid 5.
The treatment of racemic acid 5 with (−)-brucine and the crystallization
of diastereoisomeric salts from water[16] enabled us to resolve enantiomers.
R-enantiomer 5b was obtained after four sequential crystallizations in 98%
of ee. The enantiomeric purity of acid 5b was determined after derivatization
with 2-bromo-2ꢁ-acetonaphthone by chiral HPLC on column Chiralcel ODH.
Next, the selective reduction of a carboxylic group in the presence of a
carbonyl group was performed (Scheme 3). The esterification and acetal-
ization of keto acid 5b with trimethyl orthoformate, followed by a reduction
of the ester in compound 6, and the deprotection of the carbonyl group in
compound 7 afforded the hydroxy ketone 2 in 85% overall yield.[17]
LiAlH4
H+
O
O
O
e
M
)
3
H
C O
O
(
O
H
O
O
O
H
O
O
O
H
O
H
l
C
3COC
7
2
5b
6
SCHEME 3 Synthesis of hydroxy ketone 2.
The final step of the synthesis was reductive amination (Scheme 4).
There was no reaction between bimorpholine 1 and ketone 2 with NaBH4
in the presence of acetic acid. However, the reaction with zinc-modified
cyanoborohydride[18] led to the mixture of isomers of the target carbocyclic
nucleoside analogue 3 with 40% of the unreacted starting bimorpholine.
Column chromatography on silica gel afforded pure single more stable cis

900
K. Ausmees et al.
a
N
NBH
3
C
O
O
n
Z
l
C 2
O
O
O
+
H
O
M
S
HN
NH
H
O
N
NH
D
M
C
s
T
p
H
O
2
1
3
SCHEME 4 Reductive amination of hydroxy ketone 2.
isomer of 3 in a 15% yield. Configuration of this isomer was determined
by a detailed 1H and 13C NMR analysis with the help of 2D FT experiments
and with a comparison of present results with NMR data from cis and trans
isomers of dideoxynucleoside analogues.[7]
BIOLOGICAL ACTIVITIES OF SYNTHESIZED COMPOUNDS
Analysis of the Cytotoxicity of Compounds
The observed inhibition of the replication of HCV in a cell culture can
result from the direct antiviral effect of the compound. Alternatively, it may
be related to its ability to specifically inhibit the activity of the essential cel-
lular co-factor of HCV (an example of such an inhibitor is the cyclophilin
inhibitor Alisporivir),[19] or may originate from a mild general cytotoxic
effect, as the replication of HCV in a cell culture is very sensitive to toxic
compounds, even in concentrations that are not toxic to the cells. As com-
pounds 1 and 3 lack obvious similarities with known HCV inhibitors, either
indirect mechanism of action was suggested.
An analysis performed by use of the MTT assay demonstrated that com-
pound 3 is toxic for Huh7-Lunet and Huh-Luc/neo-ET (human hepatoma
cells, which carry an autonomously replicating subgenomic HCV replicon,
expressing Firefly luciferase as a reporter protein)[20] at a 50 µM concentra-
tion. The analysis of compound 1 was compromised by a large experiment-
to-experiment variation in results; overall, no cytotoxicity (which would be
statistically significant) was observed at 50 µM (data not shown). Such be-
havior, however, suggested that the compound had some effect on the cells,
and hence, more sensitive methods were required. Therefore, the effects
of both compounds on the cells were analyzed using the xCELLigence Sys-
tem (Roche), which measures electrical impedance across interdigitated
microelectrodes integrated on the bottom of tissue culture E-Plates. The
impedance measurement provides quantitative information about the bio-
logical status of cells, including cell number and viability. Most importantly,
this system makes it possible to measure the same sample over the entire
experiment, greatly reducing the variation in the results.

Synthesis and Biological Activity of Bimorpholine and Its Carbanucleoside
901
TABLE 1 Results of the analysis of cytotoxic properties of the compounds
Compound
concentration
6 hours
24 hours
48 hours
72 hours
1, 50 µM
1, 10 µM
1, 1 µM
3, 50 µM
3, 10 µM
3, 1 µM
1.01
1.04
1.00
0.50
0.95
0.95
0.79
0.91
0.97
0.14
0.77
0.90
0.75
0.83
0.97
0.04
0.71
0.83
0.78
0.76
0.95
< 0.01
0.59
0.74
Note: Normalized cell indexes (values are normalized to the time point of the compound administra-
tion) are shown. Cell viability is presented as a ratio of the Normalized cell index of cells treated with
the tested compounds to the Normalized cell index of the cells treated with relevant concentrations of
DMSO (taken as 1).
First, we compared the action of compounds 1, 3 and DMSO as a diluent
control to Huh-Luc/neo-ET cells. Three concentrations of the substances,
50, 10, and 1 µM, and relevant DMSO concentrations (0.5%, 0.1%, and
0.01%) were used. The normalized results from one of the three repro-
ducible experiments are shown in Table 1.
As is evident from the presented data, compound 1 was moderately toxic
at high concentrations (50 and 10 µM) and had no or almost no toxic effects
at 1 µM. Consistent with the results from the MTT assays, compound 3 was
found to be extremely toxic at high concentrations (50 µM), and moderately
toxic at lower concentrations (10 and 1 µM).
To confirm or rule out the possibility that toxic effects of compound
1 were related to the presence of the HCV replicon in cells, we compared
the effects of compound 1 to Huh-Luc/neo-ET cells and Huh7-Lunet cells
(the parental line for Huh-Luc/neo-ET, which did not contain the HCV
replicon) using a similar assay. The results of one reproducible experiment
are shown in Table 2.
Though some slight experiment-to-experiment differences (probably re-
lated to slightly different cell densities and growth conditions) were ob-
served, the tendency always remained the same (the differences between
TABLE 2 Results of the analysis of cytotoxic action of compound 1 to the Huh-7 Lunet cells, with or
without HCV replicon.
Compound
concentration
Huh7-Lunet
24 hours
Huh-Luc/neo-
ET 24 hours
Huh7-Lunet
48 hours
Huh-Luc/neo-
ET 48 hours
1, 50 µM
1, 10 µM
1, 1 µM
0.78
0.83
1.04
0.79
0.94
1.01
0.62
0.62
0.86
0.61
0.87
1.01
Note: Cell viability is presented as a ratio of the normalized cell index of cells treated with the tested
compound to the normalized cell index of the cells treated with relevant concentrations of DMSO (taken
as 1 for each concentration).

902
K. Ausmees et al.
TABLE 3 Effect of compound 1 on the HCV replication.
Concentration
6 hours
24 hours
48 hours
72 hours
50 µM
10 µM
1 µM
1.03
1.23
0.88
0.51
0.75
0.83
0.25
0.88
1.19
0.12
0.85
1.68
Note: Luciferase activity, which is proportional to the HCV RNA copy number, was measured at the in-
dicated time points and normalized to the total protein content. At each time point, the luciferase activity
in control cells treated with relevant amounts of DMSO is taken as 1. The data from one reproducible
experiment is shown.
the HCV replicon containing cells and Huh7-Lunet cells were statistically
nonsignificant). Thus, it is evident from the data that the presence of the
HCV replicon does not make Huh-7 Lunet cells more susceptible to it nor
does the cytotoxic effects protect cells against the cytotoxic effects of the
compound.
Anti-HCV Activity of Compound 1
Based on the cytotoxicity analysis, we excluded compound 3 from subse-
quent analysis and tested next the action of compound 1 on HCV replication.
In this experiment, Huh-Luc/neo-ET cells were incubated with the indicated
concentrations of compound 1 or relevant concentrations of the DMSO used
as a control. At indicated time points, Luc activity was measured and normal-
ized to the total protein in the cell lysate; the results are presented in Table
3.
As is evident from the presented data, compound 1 drastically inhibited
HCV replication at a 50 µM concentration. At 10 µM, the inhibition was
almost completely lost and even some enhancement of the HCV replication
was observed at a 1 µM concentration.
CONCLUSIONS
Nonaromatic nucleoside carba-analogue 3 is extremely cytotoxic at high
concentrations. Compound 1 is a potent inhibitor of HCV replication at
a 50 µM concentration; in contrast, at 10 µM, its ability to inhibit HCV is
greatly reduced. As at the same concentration, some cytotoxic effects were
observed (Table 1), it is likely that the action of compound 1 against HCV
is not direct but is mediated by its general cytotoxicity. However, it should
be noted that while the cytotoxic effects of compound 1 on the cells were
nearly identical at both the 50 and 10 µM concentrations (Table 1), the
HCV replication was inhibited only at the higher (50 µM) concentrations;
similarly, the cytotoxic effects were relatively mild (nearly undetectable using
an MTT assay) and the effect against HCV was more prominent. Therefore,
it is possible that the cytotoxicity of compound 1 is not its only mechanism of

Synthesis and Biological Activity of Bimorpholine and Its Carbanucleoside
903
action against HCV and that some specific mechanism of inhibition exists.
Additional research is needed to verify this possibility, and if the specific
inhibition exists, to unravel its mechanism.
EXPERIMENTAL
1
Full assignment of H and 13C chemical shifts is based on the 1D and
2D FT NMR spectra on a Bruker AMX500, AvanceIII 400. Deuterosolvent
peaks (CHCl3 δ = 7.27, CDCl3 δ = 77.00) or the TMS peak was used as
chemical shift references. High-resolution mass spectra were recorded on
an LTQ Orbitrap (Thermo Electron). Optical rotations were obtained using
a Anton Paar GWB Polarimeter MCP 500. Chiral HPLC was performed using
the Chiralcel OD-H (250 × 4.6 mm).
Reactions sensitive to oxygen or moisture were conducted under an
argon atmosphere in flame-dried glassware. Anhydrous dichloromethane
was freshly distilled over CaH2 and anhydrous diethyl ether, over LiAlH4.
Commercial reagents were generally used as received. The petroleum ether
used had bp 40◦C–60◦C.
Ethyl 2-Cyano-3-oxocyclopentanecarboxylate 4
NaCN (18.4 g, 0.380 mol) is suspended in DMSO (74.5 mL) at 80◦C
under Ar. Ethyl acrylate (81.5 mL, 0.750 mol) is added to the reaction
mixture slowly over 2.5 hours and the mixture is heated for an additional 2
hours at 80◦C. After stirring at room temperature for 17 hours, the reaction
is finished with the addition of glacial acetic acid (23.6 mL) and water and
stirred for 1 hour at room temperature. The reaction mixture is extracted
with CH2Cl2, back-extracted with water and sat. NaCl solution, and dried over
Na2SO4. The solvent is removed and the mixture is purified by Kugelrohr
distillation to afford the desired product as a colorless oil (28.5 g, 42% yield).
1H NMR (400 MHz, CDCl3) δ 4.27 (m, 2H), 3.64 (d, J = 10.9 Hz, 1H),
3.35 (td, J = 10.6, 6.8 Hz, 1H), 2.60–2.32 (m, 3H), 2.11–1.98 (m, 1H), 1.33
(t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 203.84, 171.03, 115.21,
62.17, 45.73, 42.25, 36.52, 25.02, 14.12.
3-Oxocyclopentanecarboxylic Acid 5
Ethyl 2-cyano-3-oxocyclopentanecarboxylate 4 (28.5 g, 0.157 mol) is dis-
solved in an 18% HCl solution (660 mL) and heated to reflux for 1 hour.
The mixture is concentrated and extracted with CH2Cl2. After removal of
the solvent, the desired carboxylic acid 5 is obtained (20.1 g, 64% yield).
Racemic carboxylic acid (6 g, 0.05 mol) and (−)-brucine (22.2 g, 0.056
mol) are dissolved in water (107 mL) and heated to reflux until a clear
solution is obtained. The mixture is allowed to cool to room temperature.

904
K. Ausmees et al.
The formed crystals are filtered and washed with water and dried. After four
sequential crystallizations, 98% ee is obtained.
Enantiomerically pure (R)-3-oxocyclopentanecarboxylic acid (−)-bruc-
ine salt is dissolved in water and heated to reflux until a clear solution is
obtained. NH4OH is added and filtered, the crystals are washed with water,
and the filtrate is concentrated, acidified with 1M HCl, and extracted with
Et2O. (R)-3-oxocyclopentanecarboxylic acid 5b is obtained as soft yellow
crystals (1.1 g, 18% yield, mp = 48◦C).
1H NMR (400 MHz, CDCl3) δ 10.36 (s, 1H), 3.25–3.13 (m, 1H), 2.55–2.14
(m, 6H). 13C NMR (101 MHz, CDCl3) δ 216.76, 180.25, 40.92, 40.70, 37.35,
25
25
26.46. 5b [α]D = +20 (c 1.9, MeOH), (lit. [α]D = +22 (c 1.9, MeOH).[16]
Chiral HPLC (Chiralcel OD-H, n-Hexane:iPrOH 8:2, 1 mL/minutes, UV
254 nm, tR = 25.07 minutes (R-enantiomer) and tR = 26.8 minutes (S-
enantiomer).
(R)-Methyl 3,3-dimethoxycyclopentanecarboxylate (6)
(R)-3-oxocyclopentanecarboxylic acid 5b (978 mg, 6.0 mmol) is dissolved
in methanol (12 mL); orthoformate (6.0 mL) and acetylchloride (0.4 mL,
6.0 mmol) are added and the mixture is stired for 5 hours. Na2CO3 (3 g)
and petroleum ether (15 mL) are added and the mixture is stired for an
additional hour. After filtration and solvent removal, the crude product is
obtained as a light yellow oil (1.1 g, quantitative yield).
1H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.22 (s, 3H), 3.20 (s, 3H),
2.94–2.83 (m, 1H), 2.12 (dd, J = 23.4, 8.6 Hz, 1H), 2.06 (dd, J = 13.4, 8.8
Hz, 1H), 2.02–1.77 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 175.84, 110.86,
51.80, 49.68, 49.07, 41.07, 37.21, 33.52, 26.60.
(R)-(3,3-Dimethoxycyclopentyl)methanol 7
Anhydrous Et2O (10 mL) is added to a flame-dried, round bottom flask
under Ar, LiAlH4 (294 mg, 7.8 mmol) is added, and the mixture is cooled to
0◦C. (R)-methyl 3,3-dimethoxycyclopentanecarboxylate 6 (1.1 g, 6 mmol) is
dissolved in anhydrous Et2O (10 mL), slowly added to the reaction mixture,
and heated to reflux for 3 hours. The reaction mixture is cooled to 0◦C and
quenched with H2O and 4N NaOH and dried over Na2SO4. After filtration
and solvent removal, the crude product is obtained as a light yellow oil (911
mg, 95% yield).
1H NMR (400 MHz, CDCl3) δ 3.59–3.48 (m, 2H), 3.21 (s, 6H), 2.41–2.17
(m, 2H), 2.00 (dd, J = 13.3, 8.8 Hz, 1H), 1.95–1.70 (m, 3H), 1.56 (ddd, J =
13.3, 7.5, 1.7 Hz, 1H), 1.50–1.37 (m, 1H). 13C NMR (101 MHz, CDCl3) δ
111.42, 66.73, 49.36, 49.25, 38.95, 37.39, 33.47, 25.76.

Synthesis and Biological Activity of Bimorpholine and Its Carbanucleoside
905
(R)-3-(Hydroxymethyl)cyclopentanone 2
(R)-(3,3-dimethoxycyclopentyl)methanol 7 (911 mg, 5.7 mmol) is dis-
solved in an Et2O/H2O (1:1, 20 mL) mixture, a few drops of 1M HCl is
added and the mixture is stired overnight. The solvent is removed, and
the mixture is purified by silica gel column chromatography (petroleum
ether:acetone 16%) to give product 2 as a colorless oil (610 mg, 94% yield).
1H NMR (400 MHz, CDCl3) δ 3.73–3.64 (m, 2H), 2.52–2.42 (m, 1H),
2.42–2.35 (m, 1H), 2.35–2.27 (m, 1H), 2.27–2.18 (m, 1H), 2.18–2.10 (m,
2H), 2.03 (ddd, J = 10.0, 8.4, 0.9 Hz, 1H), 1.75 (m, 1H). 13C NMR (101
25
MHz, CDCl3) δ 219.63, 65.75, 41.67, 38.99, 38.02, 25.63. [α]D = +84 (c
0.85, acetone).
(1R,3S)-((2S,2ꢀS,5S,5ꢀS)-5,5ꢀ-Dibenzyl-2,2ꢀ-
bimorpholino)cyclopentyl)methanol 3
˚
Molecular sieves 4A are added to a round bottom flask and sealed, dried,
and filled with Ar. (2S,2ꢁS,5S,5ꢁS)-5,5ꢁ-dibenzyl-2,2ꢁ-bimorpholine (120 mg,
0.340 mmol) and (R)-3-(hydroxymethyl)cyclopentanone (38.8 mg, 0.340
mmol) are dissolved in dry CH2Cl2 (5 mL) and added to the reaction flask
via a syringe. In a separate flask, NaCNBH3 (32.1 mg, 0.51 mmol) and ZnCl2
(34.8 mg, 0.26 mmol) are dissolved in CH2Cl2 (5 mL), stired, and then
transferred to the reaction mixture. After 14 hours, a crystal of pTsOH is
added and the mixture is refluxed for 2 days. The reaction is quenched with
the addition of 1N NaOH, extracted with CH2Cl2, and dried over Na2SO4.
The crude product is purified by silica gel column chromatography using
CH2Cl2:NH3/MeOH 0.5% to give product 3 as a colorless, sticky oil (23 mg,
yield 15%).
1H NMR (400 MHz, CDCl3) δ 7.32–7.20 (m, 5H), 7.20–7.10 (m, 5H), 3.90
(dd, J = 11.0, 2.9 Hz, 1H), 3.67–3.58 (m, 2H), 3.58–3.53 (m, 2H), 3.53–3.45
(m, 2H), 3.33–3.19 (m, 2H), 3.10 (dt, J = 15.8, 8.0 Hz, 1H), 3.02–2.92 (m,
1H), 2.81–2.69 (m, 4H), 2.62 (dt, J = 16.2, 8.1 Hz, 1H), 2.49–2.37 (m, 3H),
2.21–2.05 (m, 2H), 1.89 (m, 1H), 1.77–1.62 (m, 2H), 1.59–1.45 (m, 1H),
1.45–1.33 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 138.59, 137.70, 129.11,
128.99, 128.60, 128.38, 126.55, 126.25, 76.38, 76.26, 72.66, 70.31, 66.79,
60.20, 59.72, 55.58, 47.57, 45.87, 39.36, 38.57, 34.81, 33.14, 26.49, 22.17. IR:
ν = 3413, 2949, 2860, 1603, 1453, 1107. HRMS (EI+) calculated 451.2955
25
[M+H], found 451.2953 [M+]. [α]D = +76 (c 0.66, CHCl3).
Analysis of Cytotoxic Activity of Compounds by xCELLigence System
Cytotoxicity of the compounds was determined using Huh-Luc/neo-ET
or Huh7-Lunet cells and the xCELLigence system (Roche). First, we put
50 µl of cell growth media (Dulbecco Modified Eagle Media, 1x Pen/Strep,
10% Foetal Calf Serum) into the well and one sweep to determine if the

906
K. Ausmees et al.
basic electrical impedance was made. Then, the cells were seeded on a plate
in the amount of 1500 cells per well. For the first 2 hours, sweeps were made
every minute (a total of 120 sweeps), and after that, every 30 minutes (a total
of 44 sweeps). Twenty-four hours later, substances in the indicated amounts
were added and the cells were incubated for 72 hours. For the first 2 hours,
sweeps were made every minute (a total of 120 sweeps), and after that, every
30 minutes (a total of 140 sweeps).
Procedure for Testing Anti-HCV Activity
About 45 000 Huh-luc/neo-ET cells were seeded on 60 mm diameter
dishes. Twenty-four hours later, compound 1 or DMSO at indicated con-
centrations was added. Cells were incubated with the compounds for the
indicated time, and after that, they were lysed, and luciferase activity in the
lysates was measured according to the protocol of Luciferase Assay systems
(Promega). The total protein concentration in the lysates was determined
using a Bradford assay (Bio-Rad) and the luminescence was normalized to
the total protein concentration.
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