New acridone-4-carboxylic acid derivatives as potential inhibitors of Hepatitis C virus infection

Article abstract of DOI:10.1016/j.bmc.2008.08.074

A new class of compounds-acridone derivatives-was tested using the direct fluorometric helicase activity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C virus (HCV). The compounds were also tested as

Full text of DOI:10.1016/j.bmc.2008.08.074

Bioorganic & Medicinal Chemistry 16 (2008) 8846–8852
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New acridone-4-carboxylic acid derivatives as potential inhibitors
of Hepatitis C virus infection
b
b
Anna Stankiewicz-Drogon a, Larisa G. Palchykovska b, , Valentina G. Kostina , Inna V. Alexeeva ,
*
Anatoly D. Shved b, Anna M. Boguszewska-Chachulska a,
*
a Institute of Biochemistry and Biophysics PAS, Pawinskiego 5a, 02-106 Warsaw, Poland
b Institute of Molecular Biology and Genetics NAS, Zabolotnoho 150, 03143 Kyev, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of compounds—acridone derivatives—was tested using the direct fluorometric helicase activ-
ity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C
virus (HCV). The compounds were also tested as putative transcription inhibitors of in vitro transcription
based on the DNA-dependent T7 RNA polymerase. Most of the acridone derivatives tested were transcrip-
tion inhibitors; however, only four of them inhibited the NS3 helicase at low concentrations (IC50 from
Received 4 June 2008
Revised 22 August 2008
Accepted 28 August 2008
Available online 31 August 2008
3 lM to 20 lM) and were therefore selected for further studies on the mechanism of inhibition. The acri-
Keywords:
done derivatives probably act via intercalation into double-stranded nucleic acids but they may also
interact directly with viral enzymes. Selected carboxamides were tested in the subgenomic HCV replicon
system. Two of the compounds: N-(pyridin-4-yl)-amide and N-(pyridin-2-yl)-amide of acridone-4-car-
boxylic acid are efficient RNA replication inhibitors with selectivity indexes of 19.4 and 40.5, respectively,
proving that the acridone derivatives may be regarded as potential antiviral agents.
Ó 2008 Elsevier Ltd. All rights reserved.
Hepatitis C virus
Acridone derivative
NS3 helicase
Fluorometric assay
Intercalation
Subgenomic HCV replicon
Anti-HCV drugs
1. Introduction
As the three-dimensional structure of the HCV NS5B protein is
similar to that of the T7 phage RNA polymerase, and since both
About 180 million people worldwide are affected with chronic
hepatitis caused by Hepatitis C virus (HCV; WHO, 2007). As the
current therapy is long and ineffective in about 50% of individuals
affected with genotype 1b,1–3 there is an urgent need for more
effective, better tolerated and less expensive treatments.
polymerases contain the same structural domains, as well as the
conserved GDD sequence motif,10,11 an in vitro transcription assay
based on the T7 RNA polymerase was used to test possible tran-
scription inhibition capacities of selected compounds. This assay
is routinely applied to examine potential inhibitors of RNA synthe-
sis that may interact with DNA or RNA, such as acridine or acridone
derivatives,12,13 and therefore seemed a good tool to perform a par-
allel pre-screening of acridone-4-carboxylic acid derivatives to
identify compounds with anti-polymerase activities.
Acridone derivatives have been known for many years as anti-
microbial agents.14 Recently their anticancer14,15 and antiviral
properties have been revealed. An acridone derivative, cycloferon,
is known to act against Influenza virus and adenoviruses,16 whereas
other acridone derivatives were shown to inhibit Human immuno-
deficiency virus,17 West Nile virus 18 and Herpes simplex virus replica-
tion.19–21
Since acridones are not nucleoside analogs, their specificity
towards viral enzymes may be higher6 as was demonstrated by
recent studies in cell cultures on the replication of viruses of the
Flaviviridae family. The studies of Tabarrini et al. led to the identi-
fication of acridone compounds that inhibit both Bovine viral
diarrhea virus and HCV replication, and show selective antiviral
activities.22
The development of antiviral agents directly targeting the viral
life cycle seems to be the most promising therapeutic strategy, as it
should block HCV replication and spread of infection. This goal
could be achieved by direct inhibition of viral enzymes involved
in the replication process, such as the NS3 protein, exerting both
protease and RNA helicase/NTP-ase activities, or the NS5B protein,
an RNA-dependent RNA polymerase.4–6 One advantage of this
approach is that these proteins have no close homologs among cel-
lular enzymes.6,7 Our studies focused on the potential inhibitors of
the activity of the HCV RNA helicase—the C-terminal portion of the
NS3 protein indispensable for viral replication—an enzyme that
unwinds double-stranded (ds) forms of RNA and thus allows viral
replication and translation to occur.5,8,9
* Corresponding authors. Tel.: +48 22 592 24 17; fax: +48 22 658 46 36 (A.M.B.-C.);
fax: +38 044 526 07 59 (L.P.).
E-mail addresses: l.palchykovska@imbg.org.ua (L.G. Palchykovska), annach@ibb.
waw.pl (A.M. Boguszewska-Chachulska).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.074

A. Stankiewicz-Drogon et al. / Bioorg. Med. Chem. 16 (2008) 8846–8852
8847
Here, we demonstrate that a different approach—to study the
at concentrations lower than 100
lM were subsequently tested in
anti-helicase activity of a large group of acridone derivatives so
as to select potential specific inhibitors of HCV replication—may
also ultimately yield effective anti-HCV drugs. Their antiviral activ-
ity may be further enforced by inhibition of the RNA-dependent
RNA polymerase, NS5B.4,6
smaller increments near the presumed 50% inhibitory concentra-
tions (IC50).
Potent inhibitory activity towards the NS3 helicase was exhib-
ited only by derivatives with pyridine heterocyclic tailpieces (20,
21, 22 and 27; Table 1). Compound 27 appeared to be the best
inhibitor of helicase activity with an IC50 of 3.82
pound 20 had slightly weaker inhibitory properties with an IC50
of 8.9 M. Addition of CH3 substituents to the pyridine ring of 27
also slightly decreased the anti-helicase activity of 21 and 22,
resulting in IC50 values of 6.2 M and 20.2 M, respectively. The
lM, while com-
2. Results
l
2.1. Chemistry
l
l
length of the linker at the pyridine-bearing amide fragment
appears crucial for anti-helicase activity since addition of -CH2–
For an extensive structure-activity relationship study of acri-
done-4-carboxylic acid amides, we first synthesized N-(carboxy-
phenyl)anthranilic acid (1) on a preparative scale, using the
method of Jourdan–Ulmann copper-catalysed condensation of
anthranilic acid with ortho-bromobenzoic acid. Cyclization to the
acridone-4-carboxylic acid (ACA) (2) was achieved by heating com-
pound 1 with excess polyphosphoric acid at 120 °C.23,24 The syn-
thesis of ACA (2) and its amides is shown in Scheme 1.
Table 1
Inhibitory activities of acridone-4-carboxylic acid derivatives
Compound
MW
NS3 helicase inhibition
Transcription inhibition
IC99 (lM)
IC50 SDc
(l
M)
a
b
N-Substituted acridone-4-carboxamides were synthesized by
amide bond formation between an activated form of ACA and an
appropriate amine. A convenient technique for carboxylic group
activation was developed that allowed us to obtain high yields of
the products. Following this methodology, we prepared an essen-
tially complete set of acridone-4-carboxamides whose amide frag-
ments were formed by the alkyl groups or aryl-, heteryl-ring
systems bearing exocyclic groups at different ring positions. The
structures of the compounds studied are presented in Scheme 1.
12
13
14
15
16
17
18
19
20
21
22
27
27a
328.37
328.37
328.37
309.37
306.36
308.34
314.34
365.43
315.3
329.3
329.3
315.3
329.3
>100
>100
>100
>100
>100
>100
>100
>100
8.9 0.4
6.2 1.3
20.2 2.3
3.8 0.3
>100
10
40
80
80
>100
>100
40
>100
20
40
80
40
>100
2.2. Helicase inhibition assay
a
Concentration of inhibitor necessary to reduce the NS3 helicase activity by 50%.
Inhibitor concentration necessary to reduce the T7 RNA polymerase activity by
b
To test the library of new acridone derivatives, the fluorometric
helicase activity assay was applied.25,26 The compounds were ini-
99%.
c
Standard deviation.
tially tested at 10–100
lM and those showing anti-helicase activity
COOH
+
COOH
i
H2N
Br
N
H
COOH
COOH
ii
1
O
O
iii
N
H
N
H
COOH
R
12-22, 27, 27a
2
Me
R:
Me
N
N
O
N
14=
N
15=
Me
12=
13=
N
O
O
O
O
O
16=
20=
17=
Me
19=
N
N
O
18=
N
N
N
N
O
O
O
O
O
O
N
N
21=
22=
27=
27a=
N
N
N
Me
N
N
N
N
O
O
Scheme 1. Synthesis of acridone-4-carboxylic acid derivatives. Reagents and conditions: (i) Cu, K2CO3/DMF, 60–120 °C, 2 h; (ii) PPA, 120 °C, 1 h; (iii) SOCl2/Py, toluene, amine,
Et3N, room temperature, 6–7 h.

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A. Stankiewicz-Drogon et al. / Bioorg. Med. Chem. 16 (2008) 8846–8852
in compound 27a led to complete loss of its inhibitory properties
(Table 1).
2.3. Mechanism of anti-helicase action
A series of experiments were designed to establish the mech-
anism of action of ACA derivatives on the helicase activity. Inter-
action/competition of helicase inhibitors (20, 21, 22 and 27) with
the enzyme or a ds DNA substrate was studied in helicase activity
assays performed at increasing enzyme or substrate concentra-
tions and constant inhibitor concentrations, close to their IC50 val-
ues (10, 5, 33 and 5 lM, respectively). Inhibition was independent
of enzyme concentration, suggesting lack of competition and
interaction (Fig. 1A), while in the presence of increasing substrate
concentrations a decrease of inhibition was observed (Fig. 1B),
clearly indicating interaction of the compounds with the
substrate.
Figure 2. Inhibition of the NS3 helicase activity by compound 21 at three ATP
concentrations. The results are presented as the percent activity of the helicase
tested in the same conditions but with DMSO instead of inhibitor.
To check if inhibition of helicase activity by acridone-4-carbox-
amides depends on the ATP concentration, the IC50 of 21 was mea-
sured at three ATP concentrations (Fig. 2). The three inhibition
curves and the IC50 values did not differ, proving that the ATPase
activity is not inhibited. This was further confirmed for all four
inhibitors in the ATPase radioactive assay (data not shown).
tion products,12 we used it as an in vitro model to study
polymerase inhibition by the set of ACA derivatives.
The phenylamide 18 and tolylamides bearing exocyclic methyl
groups at positions meta-, ortho- and para- (13, 14 and 12) exhib-
ited significant, position-dependent activity on cell-free RNA tran-
scription (Table 1). Compound 12 appeared to be the best
transcription inhibitor, almost completely suppressing RNA syn-
2.4. Inhibition of T7 polymerase transcription
Taking into account the topological and functional similarity of
polymerases, and the fact that the T7 RNA polymerase does not re-
quire a radioactive technique to evaluate and visualize transcrip-
thesis at 10 lM (Fig. 3). The derivatives with pyridine heterocyclic
tailpieces (20, 21, 22 and 27) exhibited comparable inhibition of
RNA synthesis that was also substituent-dependent, compound
20 being the best inhibitor of this group of compounds (concentra-
tion of the inhibitor necessary to reduce the enzyme activity by
99%, IC99, equals 20 lM). The carboxamide 27a with the short
CH2 linker at the pyridine-bearing amide fragment completely lost
the capacity to inhibit RNA synthesis (Table 1).
Figure 1. Inhibition of NS3 helicase activity by compounds 20, 21, 22 and 27, (A) at
increasing enzyme concentrations. (B) At increasing dsDNA substrate concentra-
tions. The results are presented as the percent activity of the helicase tested in the
same conditions but with DMSO instead of inhibitor.
Figure 3. T7 polymerase transcription assay for compound 12. Inhibition of
transcription is reflected by the disappearance of the transcription product in an
agarose gel (A), presented as percent of the positive control C (B).

A. Stankiewicz-Drogon et al. / Bioorg. Med. Chem. 16 (2008) 8846–8852
8849
Table 2
2.5. Intercalation studies
Inhibition of HCV replication in Huh-7 cells carrying the subgenomic replicona
b
c
ACA derivatives exerting anti-helicase activity (20, 21, 22 and
27) and structurally most similar compound (27a) as well as the
most potent transcription inhibitors (12, 13, 14 and 18) were
tested for their intercalatory properties. Initially, a dsDNA gel
migration retardation approach was applied with a large excess
of inhibitors.27 In this assay the compounds that intercalate into
dsDNA inhibit ethidium bromide (EtBr) intercalation and the
dsDNA band disappears partially or totally. This approach revealed
Compound
EC50
(lM)
CC50
(
l
M)
SId = CC50/EC50
12
13
14
18
20
21
22
27
>100
5.6
>100
5.5
99.3
90.9
174.8
7.0
39.2
411.9
10.2
>200
n/ae
1.0
5.2
7.1
19.4
1.1
3.5
40.5
2.4
19.0
12.9
9.0
6.5
11.1
10.2
4.3
27a
weak dsDNA intercalation properties of the compounds at 100
lM
4’-Azidocytidine
1.4
n/a
or 500 M (Fig. 4A), much weaker than that of epidoxorubicin used
l
a
as control. Compounds 20, 22 and 27 seem to be the strongest
dsDNA intercalators of all the acridone derivatives analysed; a
slightly lower effect was observed for 18.
The intercalatory properties were further studied using dsRNA,
the cellular target of the HCV helicase. The results (Fig. 4B) demon-
strate that of the nine compounds compared, 20, 22 and 27 have
the strongest intercalatory properties, visible at 20 lM; 14, 18
and 21 are slightly weaker intercalators, while the other com-
pounds do not intercalate into dsRNA. Partial reappearance of the
Each experiment was performed at least three times independently.
Inhibitor concentration needed to reduce viral replication to 50%.
Inhibitor concentration that inhibits cell growth by 50%.
Selectivity index.
b
c
d
e
Not applicable.
compound that inhibited cell growth by 50%, CC50 above
150 M), reducing the HCV RNA level in a dose-dependent manner,
with selectivity index (SI = CC50/EC50 of 19.4 and 40.5,
l
a
)
respectively.
ds nucleic acid (NA) band at 500
itation of the compound.
lM 20 is probably due to precip-
3. Discussion
2.6. HCV replicon studies
Natural and synthetic acridone drugs are known to share a com-
mon property of NA intercalation with low selectivity towards NA
sequence. The specific activity of the compound is most often due
to side-chain substitutions protruding into the major or minor
grooves of the NA.13,19,28 As the structure of the DNA duplex plays
an important role in the unwinding process conducted by the NS3
helicase,29 the inhibition of enzyme functions may be due to inter-
action of acridone-4-carboxamides with the DNA and subsequent
disruption of the helical structure of the DNA that leads to inhibi-
tion of protein translocation along the DNA. It was also proposed
that the biological activities of acridone drugs are not only due to
Selected carboxamides were tested at concentrations ranging
from 1 M to 200 M both in the replicon RNA amplification and
l
l
cytotoxicity studies. Of nine compounds tested, eight inhibited
RNA replication with EC50 (50% effective concentration defined as
the inhibitor concentration that reduced luminescence by 50%)
ranging from 5 lM to 20 lM, but most of them were cytotoxic at
similar concentrations (Table 2). Nevertheless, two compounds,
20 and 27, exhibited antiviral activity together with low cytotoxic-
ity (50% cytotoxic concentration defined as the concentration of
Figure 4. Intercalation assay for selected acridone-4-carboxylic acid derivatives. Their intercalatory properties are reflected by the disappearance of dsDNA (A) or dsRNA (B)
bands. DMSO: dsNA incubated with DMSO, Epi: dsNA incubated with epidoxorubicin; 12–27a: dsNA incubated with selected compounds at given concentrations.

8850
A. Stankiewicz-Drogon et al. / Bioorg. Med. Chem. 16 (2008) 8846–8852
their binding to DNA but also to their direct interaction with topoi-
somerases and telomerases,14,19,30 and thus interaction with other
enzymes (e.g. helicase or polymerase) cannot be excluded. The po-
sition and nature of the substituent plays an important role in the
antiviral activity of acridones15,21 and our data point to the pyri-
dine heterocyclic tailpieces as key pharmacophores for the anti-
helicase activity of the compounds, while the tolyl and pyridine
amide fragments determine the anti-polymerase activity.
Pyridylamides 20, 22 and 27 appeared to be the most potent
dsDNA intercalators and this could explain their anti-helicase
and anti-polymerase activities. On the other hand phenylamide
18 exhibited no anti-helicase activity in spite of its intercalatory
properties, while the tolylamide 12, the most potent polymerase
cient to explain the inhibition of either transcription, NA unwind-
ing by the helicase, or the cytotoxicity of acridone derivatives in
Huh-7 cells. However, intercalation of the acridone derivatives into
dsRNA may play an important role in inhibition of HCV replication
in the replicon system. Pyridylamides 20 and 27 are efficient HCV
replication inhibitors with SI values of 19.4 and 40.5, respectively.
Both compounds are the best helicase inhibitors (27 has the
lowest IC50 value) and are moderate transcription inhibitors.
Therefore in the replicon system they can act via inhibition of both
NS3 and NS5B activities.
Compound 27 was designed on the basis of first results of heli-
case inhibition by pyridyl derivatives. Demonstration of its effi-
ciency as the helicase and RNA replication inhibitor proves that
the approach adopted in search for antiviral agents was appropri-
ate. The molecules selected could be further modified to improve
their biological activity and obtain specific anti-HCV drugs.
inhibitor did not show any intercalatory properties up to 500 lM
(Table 1 and Fig. 4). Thus we conclude that intercalation is not a
sufficient mechanism for potent inhibition of either helicase or
polymerase activities.
All the intercalating compounds showed a preference for dsRNA,
an advantageous feature for inhibitors of amplification of RNA
viruses since such molecules could modify secondary structures of
viral untranslated regions (UTRs) and of dsRNA intermediates
formed during genome replication without affecting cellular DNA.
All four helicase inhibitors (20, 21, 22 and 27, Table 1) are replica-
tion inhibitors in the HCV replicon system based on their EC50 values
but the cell viability assay revealed significant cytotoxicity of 21 and
22, which may be the main reasons of the antiviral effect observed
(Table 2). Cytotoxicity may be due to the presence of the –CH3 sub-
stituent in the pyridyl ring that could favour a specific conformation
of the compound and thus cause strong interaction of acridone
derivatives with dsDNA thereby interfering with cellular processes.
Intercalation of acridone derivatives into dsRNA seems to be the
most important mechanism of the inhibition of HCV RNA replication
in the cell as all dsRNA intercalators studied had EC50 values below
5. Experimental
5.1. Chemistry
1H NMR spectra were measured with a Mercury 400 (400 MHz)
spectrometer; chemical shifts are expressed in d units using tetra-
methylsilane as standard (NMR peak description: s, singlet; d, dou-
blet; t, triplet; m, multiplet; and br, broad peak). Mass spectra
were recorded with an Agilent 1100 (HPLC APCI MS) spectrometer
in a positive mode. Reactions were monitored by TLC on Merck silica
gel plates (60F254) and visualised by UV light. Melting points of com-
pounds were determined with a Boetius apparatus. All commercial
reagents and organic solvents were used with further purification.
5.1.1. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid p-tolylamide
(12)
20 lM (Table 2). A high specificity for dsRNA structures as well as
Thionyl chloride (81.3
lL, 1.13 mmol) and freshly dried pyri-
lack of methyl group in the pyridyl ring may be the main reasons
dine (91.3 L, 1.13 mmol) were added to the suspension of com-
l
for the very low cytoxicities of 20 and 27 (Table 2).
pound 2 (193.5 mg, 0.81 mmol) in 10 ml of dry toluene. The
reaction mixture was stirred at room temperature for 3–4 h, com-
bined with excess (267 mg, 2.5 mmol) p-toluidine, triethylamine
We presume, however, that the mechanism of inhibition of viral
replication cannot be based solely on the dsRNA intercalatory
properties of the acridone derivatives. As both carboxamides 20
and 27 are good inhibitors of T7 polymerase transcription and heli-
case-mediated NA unwinding (Table 1), their inhibition of HCV
RNA replication in the replicon system may be due to a double ef-
fect of inhibition of two HCV enzymes, the NS3 helicase and the
NS5B polymerase (Table 2). This possibility is further supported
by the fact that the full-length NS3 activity is dependent on its
interaction with NS5B.31,32 These possibilities will be verified by
NS5B polymerase inhibition studies that are currently in progress.
Preliminary computer modeling analyses revealed that the po-
sition of the nitrogen atom in the pyridyl ring considerably affects
the spatial location of the ligand in the hydrophobic pocket of the
T7 polymerase catalytic site. The ligand-receptor complex is stabi-
lized by the formation of relatively short (2–2.5 Å) and effective
hydrogen bonds between the ligand and amino acid residues, NA
or NTPs. These studies, together with computer modeling of li-
gand-NS3 helicase interactions, will be the subject of a future
publication.
(337 lL, 2.4 mmol) and stirred for another 3 h. The reaction was
monitored by TLC. The following day the solvent was removed un-
der vacuum and water was added to the solid residue. The precip-
itate formed was filtered, washed with water and dried. The crude
product was crystallized several times from n-butanol–DMF (19:1)
yielding 193 mg of analytically pure compound 12. Yield: 73%; mp
338–342 °C. 1H NMR (DMSO-d6) d 2.36 (3 H, CH3, s), 7.16 (2H, d,
J = 8.0 Hz), 7.27 (1 H, t, J = 6.8), 7.34 (1H, t, J = 7.8), 7.65–7.69 (4H,
m), 8.24 (1H, d, J = 7.6 Hz), 8.37 (1H, d, J = 7.2 Hz), 8.48 (1H, d,
J = 7.6 Hz), 10.47 (1H, s, CONH), 12.04 (1H, s, NH); LC–MS m/z
[M+H]+ 329.
5.1.2. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid m-tolylamide
(13)
Compound 13 was prepared from ACA (2) and m-toluidine as a
yellow solid, using an approach similar to that described for com-
pound 12. Yield: 54%; mp 276–277 °C (toluene–DMF); H NMR
(DMSO-d6) d 2.39 (3H, s, CH3), 6.95 (1H, d, J = 7.6 Hz), 7.22–7.29
(2H, m), 7.35 (1H, t, J = 7.6 Hz), 7.54 (1H, d, J = 8.0 Hz), 7.65–7.71
(3H, m), 8.24 (1H, d, J = 8.0 Hz), 8.36 (1H, d, J = 7.2), 8.59 (1H, d,
J = 7.6 Hz), 10.46 (1H, s, CONH), 12.02 (1H, s, NH); LC–MS m/z
[M+H] 329.
4. Conclusions
A series of acridone derivatives were synthesized and the anti-
HCV activity of the compounds was evaluated. The anti-helicase
activity of the compounds studied is determined by the pyridine
heterocyclic tailpieces while the tolyl and pyridine amide frag-
ments are key pharmacophores for the anti-polymerase activity.
The ability of the compounds to intercalate into dsNA is not suffi-
5.1.3. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid o-tolylamide
(14)
Compound 14 was prepared from ACA (2) and o-toluidine as a
yellow solid, using an approach similar to that described for com-

A. Stankiewicz-Drogon et al. / Bioorg. Med. Chem. 16 (2008) 8846–8852
8851
pound 12. Yield: 45%; mp 256–258 °C (n-butanol); 1H NMR
5.1.10. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid (4-
methyl-pyridin-2-yl)-amide (21)
(DMSO-d6) d 2.33 (3H, s, CH3), 7.21–7.30 (4H, m), 7.36 (2H, m),
7.62 (1H, d, J = 8.0 Hz), 7.69 (1H, t, J = 6.8), 8.34 (1H, d, J = 8.0 Hz),
8.48 (2H, dd, J = 7.6), 10.29 (1H, s, CONH), 12.32 (1H, s, NH); LC–
MS m/z [M+H]+ 329.
Compound 21 was prepared from ACA (2) and 4-methyl-2-ami-
nopyridine as a pale yellow solid, using an approach similar to that
described for compound 12. Yield: 76%; mp 207–210 °C (n-buta-
nol); 1H NMR (DMSO-d6) d 2.45 (3H, s, CH3), 7.00 (1H, d,
J = 4.8 Hz), 7.25–7.33 (2H, m), 7.64 (1H, d, J = 8.0 Hz), 7.72 (1H, t,
J = 7.2 Hz), 8.12 (1H, s), 8.25 (2H, d, J = 8.0 Hz), 8.50 (2H, d,
J = 7.6 Hz), 11.00 (1H, s, CONH), 12.00 (1H, s, NH); LC–MS m/z
[M+H]+ 330.
5.1.4. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid (15)
Compound 15 was prepared from ACA (2) and N,N-(dimethyl-
ethylene)diamine as a yellow solid, using an approach similar to
that described for compound 12. Yield: 42%; mp 171–173 °C (tolu-
ene); 1H NMR (DMSO-d6) d 2.27 (6H, s, N(CH3)2); 2.58 (2H, t,
NHCH2); 3.44–3.49 (2H, m, CH2CH2); 7.23–7.29 (2H, m); 7.58
(1H, d, J = 8.4 Hz); 7.69 (1H, t, J = 8.4 Hz); 8.23 (2H, d, J = 8.4 Hz);
8.42 (1H, d, J = 8.0 Hz), 8.77 (1H, br s, CONH); 12.62 (1H, br s,
NH); LC-MS m/z [M+H]+ 310.
5.1.11. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid (3-
methyl-pyridin-2-yl)-amide (22)
Compound 22 was prepared from ACA (2) and 6-methyl-2-ami-
nopyridine as a yellow solid, using an approach similar to that de-
scribed for compound 12. Yield 19%; mp 233–235 °C (n-butanol);
1H NMR (DMSO-d6) d 2.50 (3H, s, CH3), 7.01(1H, d, J = 7.6 Hz),
7.29–7.32 (2H, m), 7.63–7.73 (3H, m), 8.05 (1H, d, J = 8.4 Hz),
8.24 (1H, d, J = 7.6 Hz), 8.48 (2H, d, J = 7.6 Hz), 10.97 (s, 1H, CONH),
12.00 (1H, s, NH); LC–MS m/z [M+H]+ 330.
5.1.5. 4-(Piperidine-1-carbonyl)-10H-acridin-9-one (16)
Compound 16 was prepared from ACA (2) and piperidine as a
pale yellow solid, using an approach similar to that described for
compound 12. Yield: 46%; mp 234–236 °C (ethanol); 1H NMR
(DMSO-d6) d 1.67–1.68 (6H, CH2, m), 3.24 (2H, CH2, m), 3.74 (2H,
CH2, m), 7.21–7.26 (2H, m), 7.56 (1H, d, J = 8.4 Hz), 7.66 (1H, t,
J = 7.2 Hz), 7.83 (1H, d, J = 8.4), 8.21 (1H, d, J = 8.0), 8.32 (1H, d,
J = 7.2), 10.67 (1H, s, NH); LC–MS m/z [M+H]+ 307.
5.1.12. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid pyridin-
2-ylamide (27)
Compound 27 was prepared from ACA (2) and 2-aminopyridine
as a yellow solid, using an approach similar to that described for
compound 12. Yield 42%; mp 285–287 °C (n-butanol–DMF); 1H
NMR (DMSO-d6) d 7.17 (1 h, t, J = 6.8 Hz), 7.27–7.33 (2H, m),
7.66–7.70 (2H, m), 7.85 (1H, t, J = 7.6 Hz), 8.25 (2H, t, J = 6.8 Hz),
8.40 (2H, d, J = 8.40, 8.47–8.50 (3H, m), 11.11 (1H, s, CONH),
11.91 (1H, s, NH); LC–MS m/z [M+H]+ 316.
5.1.6. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid morpholylamide
(17)
Compound 16 was prepared from ACA (2) and morpholine as a
yellow solid, using an approach similar to that described for com-
pound 12. Yield: 46%; mp 196–198 °C (ethanol); 1H NMR (DMSO-
d6) d 3.21–3.40 (2H, CH2, m), 3.55–3.80 (6H, CH2, m), 7.22–7.28
(2H, m), 7.61 (1H, d, J = 7.2 Hz), 7.66 (1H, t, J = 7.2 Hz), 7.83 (1H,
d, J = 8.4 Hz), 8.22 (1H, d, J = 8.0 Hz), 8.34 (1H, d, J = 7.6 Hz), 10.76
(1H, s, NH); LC–MS m/z [M+H]+ 309.
5.1.13. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid (pyridin-
2-yl-methyl)-amide (27a)
Compound 27a was prepared from ACA (2) and 2-(amino-
methyl)pyridine as a pale yellow solid, using an approach similar
to that described for compound 12. Yield 31%; mp 211–214 °C
(n-butanol–DMF); 1H NMR (DMSO-d6) d 4.68 (2H, d, J = 4.8 Hz,
CH2), 7.23–7.32 (3H, m), 7.41 (1H, d, J = 8.0 Hz), 7.59 (1H, d,
J = 8.0 Hz), 7.67–7.75 (3H, m), 8.23 (1H, d, J = 7.6 Hz), 8.38 (1H, d,
J = 7.2), 8.46 (1H, d, J = 8.0 Hz), 8.52 (1H, d, J = 8.0 Hz), 9.52 (1H, t,
J = 4.8 Hz, NH); LC–MS m/z [M+H]+ 330.
5.1.7. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid phenylamide
(18)
Compound 18 was prepared from ACA (2) and phenylamine
as a yellow solid, using an approach similar to that described
for compound 12. Yield: 78%; mp 255–258 °C (n-butanol); 1H
NMR (DMSO-d6)
d
7.14 (1H, t, J = 7.2 Hz), 7.28 (1H, t,
J = 7.6 Hz), 7.38 (3H, m), 7.69 (2H, m), 7.79 (1H, d, J = 8.0 Hz),
8.24 (1H, d, J = 8.0 Hz), 8.37 (1H, d, J = 7.6 Hz), 8.49 (1H, d,
J = 7.6 Hz), 10.55 (1H, s, CONH), 11.98 (1H, s, NH); LC-MS m/z
[M+H]+ 315.
5.2. Biology
5.2.1. Helicase assay
The fluorometric helicase activity assay was performed as de-
scribed in Boguszewska-Chachulska et al. with minor modifica-
tions concerning the reaction temperature and volume (37 °C and
5.1.8. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid N-[(morphol-
4-yl)propyl]amide (19)
Compound 19 was prepared from ACA (2) and 4-(aminopro-
pyl)morpholine as a yellow solid, using an approach similar to that
described for compound 12. Yield: 69%; mp 161–162 °C (n-propa-
nol); 1H NMR (DMSO-d6) d 1.78–2.05 (2H, m, NCH2), 2.42–2.49 (6H,
CH2, m), 3.48–3.52 (2H, CH2, m), 3.61–3.68 (4H, CH2, m), 7.23–7.30
(2H, m), 7.59 (1H, d, J = 8.4 Hz), 7.68–7.73 (1H, m), 8.23 (2H, d,
J = 8.4 Hz), 8.43 (1H, d, J = 8.4), 8.92 (1H, t, CONH), 12.66 (1H, s,
NH); LC-MS m/z [M+H]+ 366.
60 l
l).25,26
5.2.2. In vitro transcription assay
The in vitro transcription reactions of plasmid DNAs were per-
formed as described in Melton et al.33 Nucleoside triphosphates,
T7 RNA polymerase and ribonuclease inhibitor (RiboLockTM) were
from Fermentas. Each reaction mixture contained 0.5
RI-restricted plasmid (pTZ19R with a 341 bp insert, cloned into
the Ecl136II site), 2 mM of each NTP, 1 U/ l of RNase inhibitor
lg of Eco-
l
5.1.9. 9-Oxo-9,10-dihydro-acridine-4-carboxylic acid pyridin-4-
ylamide (20)
and 12 U of T7 RNA polymerase in reaction buffer (40 mM Tris–
HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl and
10 mM DTT).
Compound 20 was prepared from ACA (2) and 4-aminopyridine
as a pale yellow solid, using an approach similar to that described
for compound 12. Yield: 75%; mp 373–376 °C (DMF); 1H NMR
(DMSO-d6) d 7.30 (1H, t, J = 7.0 Hz), 7.36 (1H, t, J = 7.6 Hz), 7.70–
7.72 (2H, m), 7.81–7.83 (2H, m), 8.25 (1H, d, J = 8.0 Hz), 8.36 (1H,
d, J = 6.8 Hz), 8.47–8.54 (3H, m), 10.86 (1H, s, CONH), 11.75 (1H,
s, NH); LC-MS m/z [M+H]+ 316.
The compounds dissolved in DMSO were added at four concen-
trations (3.125, 6.25, 12.5 and 25
lg/ml) prior to the addition of T7
RNA polymerase. The final reaction mixture (20
l
l) was incubated
at 37 °C for 1 h to obtain a concentration-dependent response. The
reactions were stopped by cooling to ꢀ20 °C. The reaction products
were visualized by 1 h electrophoresis in a 1.2% agarose gel, in TBE

8852
A. Stankiewicz-Drogon et al. / Bioorg. Med. Chem. 16 (2008) 8846–8852
buffer supplemented with EtBr. The concentration of inhibitor nec-
essary to reduce the enzyme activity by 99% (IC99) was determined
by densitometric evaluation of the intensity of the electrophoresis
bands using the Scion Image for Windows, Release Beta 4.0.3.
References and notes
1. Hayashi, N.; Takehara, T. J. Gastroenterol. 2006, 41, 17.
2. Fried, M. W.; Shiffman, M. L.; Reddy, K. R.; Smith, C.; Marinos, G.; Goncales, F. L.,
Jr.; Haussinger, D.; Diago, M.; Carosi, G.; Dhumeaux, D.; Craxi, A.; Lin, A.;
Hoffman, J.; Yu, J. N. Engl. J. Med. 2002, 347, 975.
3. Baker, D. E. Rev. Gastroenterol. Disord. 2003, 3, 93.
4. Huang, M.; Deshpande, M. Expert Rev. Anti Infect Ther. 2004, 2, 375.
5. Frick, D. N. Curr. Issues Mol. Biol. 2007, 9, 1.
6. Walker, M. P.; Hong, Z. Curr. Opin. Pharmacol. 2002, 2, 534.
7. Lam, A. M.; Rypma, R. S.; Frick, D. N. Nucleic Acids Res. 2004, 32, 4060.
8. Lam, A. M.; Frick, D. N. J. Virol. 2006, 80, 404.
5.2.3. Nucleic acid intercalation
The intercalatory properties of selected compounds were stud-
ied by the dsNA migration retardation assay.27 dsDNA was pre-
pared by PCR amplification of a cDNA fragment of the HCV con1
isolate coding for the 30 UTR,34 and dsRNA by annealing products
of in vitro transcription reactions performed on the PCR product
of the con1 30 UTR (Krawczyk et al., in preparation). dsDNA and
dsRNA (both 15 nM, composed of 490 bp) were incubated for
9. Gordon, C. P.; Keller, P. A. J. Med. Chem. 2005, 48, 1.
10. Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R. L.; Mathieu, M.; De
Francesco, R.; Rey, F. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13034.
11. Tunitskaya, V. L.; Kochetkov, S. N. Biochemistry (Mosc) 2002, 67, 1124.
12. Piestrzeniewicz, M.; Studzian, K.; Wilmanska, D.; Plucienniczak, G.;
Gniazdowski, M. Acta Biochim. Pol. 1998, 45, 127.
45 min at room temperature with 20, 100 and 500 lM compounds
in 20 mM Tris pH 7.5, and then submitted to electrophoresis in 1%
agarose gel in 1ꢁ TAE. The gel was subsequently stained with EtBr.
13. Piestrzeniewicz, M. K.; Wilmanska, D.; Studzian, K.; Szemraj, J.; Czyz, M.;
Denny, W. A.; Gniazdowski, M. Z. Naturforsch. [C] 1998, 53, 359.
14. Demeunynck, M.; Charmantray, F.; Martelli, A. Curr. Pharm. Des. 2001, 7,
1703.
15. Belmont, P.; Bosson, J.; Godet, T.; Tiano, M. Anticancer Agents Med. Chem. 2007,
7, 139.
16. Zarubaev, V. V.; Slita, A. V.; Krivitskaya, V. Z.; Sirotkin, A. K.; Kovalenko, A. L.;
Chatterjee, N. K. Antiviral Res. 2003, 58, 131.
17. Fujiwara, M.; Okamoto, M.; Okamoto, M.; Watanabe, M.; Machida, H.; Shigeta,
S.; Konno, K.; Yokota, T.; Baba, M. Antiviral Res. 1999, 43, 189.
18. Goodell, J. R.; Puig-Basagoiti, F.; Forshey, B. M.; Shi, P. Y.; Ferguson, D. M. J. Med.
Chem. 2006, 49, 2127.
5.2.4. HCV replicon and cells
The human hepatoma cell line Huh-7, carrying the subgenomic
HCV genotype 1 replicon with the luc-ubi-neo (reporter/selective)
fusion gene,35 was kindly provided by Dr. Ralf Bartenschlager (Uni-
versity of Heidelberg, Heidelberg). The cells were grown as de-
scribed in Gozdek et al.36
19. Goodell, J. R.; Madhok, A. A.; Hiasa, H.; Ferguson, D. M. Bioorg. Med. Chem. 2006,
14, 5467.
5.2.5. Anti-HCV replicon studies
20. Bastow, K. F. Curr. Drug Targets Infect. Disord. 2004, 4, 323.
21. Bernardino, A. M.; Castro, H. C.; Frugulhetti, I. C.; Loureiro, N. I.; Azevedo, A. R.;
Pinheiro, L. C.; Souza, T. M.; Giongo, V.; Passamani, F.; Magalhaes, U. O.;
Albuquerque, M. G.; Cabral, L. M.; Rodrigues, C. R. Bioorg. Med. Chem. 2008, 16,
313.
22. Tabarrini, O.; Manfroni, G.; Fravolini, A.; Cecchetti, V.; Sabatini, S.; De Clercq, E.;
Rozenski, J.; Canard, B.; Dutartre, H.; Paeshuyse, J.; Neyts, J. J. Med. Chem. 2006,
49, 2621.
23. Gaidukevich, A. N.; Levitin, E. Y.; Kravchenko, A. A.; Kazakov, G. P.; Mikitenko,
E. E.; Arsen’eva, T. I.; Pinchuk, V. V.; Beletskaya, O. V.; Zakharova, T. I. Chem.-
Pharm. J. 1985, 19, 180.
24. Rewcastle, G. W.; Denny, W. A. Synthesis 1985, 217.
25. Boguszewska-Chachulska, A. M.; Krawczyk, M.; Najda, A.; Kopanska, K.;
Stankiewicz-Drogon, A.; Zagorski-Ostoja, W.; Bretner, M. Biochem. Biophys.
Res. Commun. 2006, 341, 641.
The conditions of the assay used to test the antiviral activity of
the compounds were described in Paeshuyse et al. and Gozdek
et al.36,37 The experiments were carried out at least three times
with three replicates for each inhibitor concentration; for compar-
ison 40-azidocytidine38 (kindly provided by J. Neyts, Katolik Univer-
sität, Leuven) was tested at concentrations from 0.1 lM to 200 lM.
The data were calculated as the percentage of the luminescence of
control samples containing 1–2% DMSO. The 50% effective concen-
tration (EC50) is defined as the inhibitor concentration that reduced
luminescence by 50%.
5.2.6. Cytotoxicity assay
26. Boguszewska-Chachulska, A. M.; Krawczyk, M.; Stankiewicz, A.; Gozdek, A.;
Haenni, A. L.; Strokovskaya, L. FEBS Lett. 2004, 567, 253.
27. Dunstan, H. M.; Ludlow, C.; Goehle, S.; Cronk, M.; Szankasi, P.; Evans, D. R.;
Simon, J. A.; Lamb, J. R. J. Natl. Cancer Inst. 2002, 94, 88.
28. Adams, A. Curr. Med. Chem. 2002, 9, 1667.
Huh-7 cells carrying the HCV replicon were seeded and grown
with serial dilutions of the compounds as described for the repli-
con assay.36 After 3 days of incubation at 37 oC, the medium was
29. Tackett, A. J.; Wei, L.; Cameron, C. E.; Raney, K. D. Nucleic Acids Res. 2001, 29,
565.
30. Dziegielewski, J.; Slusarski, B.; Konitz, A.; Skladanowski, A.; Konopa, J. Biochem.
Pharmacol. 2002, 63, 1653.
removed and 100 ll of Dulbecco modified Eagle’s medium (DMEM;
Invitrogen) without phenol red supplemented with 0.2 mg/ml XTT
(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)-car-
31. Jennings, T. A.; Chen, Y.; Sikora, D.; Harrison, M. K.; Sikora, B.; Huang, L.;
Jankowsky, E.; Fairman, M. E.; Cameron, C. E.; Raney, K. D. Biochemistry (Mosc)
2008, 47, 1126.
32. Zhang, C.; Cai, Z.; Kim, Y. C.; Kumar, R.; Yuan, F.; Shi, P. Y.; Kao, C.; Luo, G. J.
Virol. 2005, 79, 8687.
33. Melton, D. A.; Krieg, P. A.; Rebagliati, M. R.; Maniatis, T.; Zinn, K.; Green, M. R.
Nucleic Acids Res. 1984, 12, 7035.
34. Lohmann, V.; Korner, F.; Koch, J.; Herian, U.; Theilmann, L.; Bartenschlager, R.
Science 1999, 285, 110.
35. Vrolijk, J. M.; Kaul, A.; Hansen, B. E.; Lohmann, V.; Haagmans, B. L.; Schalm, S.
W.; Bartenschlager, R. J. Virol. Methods 2003, 110, 201.
36. Gozdek, A.; Zhukov, I.; Polkowska, A.; Poznanski, J.; Stankiewicz-Drogon, A.;
Pawlowicz, J. M.; Zagorski-Ostoja, W.; Borowski, P.; Boguszewska-Chachulska,
A. M. Antimicrob. Agents Chemother. 2008, 52, 393.
bonyl-2 H-tetrazolium hydroxide; Serva) and 25
methosulfate (PMS; Serva) was added. Wells without cells contain-
ing 100 l of culture medium with XTT-PMS staining solution
(‘blank’) were also prepared. The plates were incubated for 3–4 h
at 37 oC. The absorbance was measured with Synergy HT (Biotek)
at 450 nm and at 690 nm as a reference; the ‘blank’ absorbance val-
ues were subtracted from the sample values. Data were calculated
as the percentage of cell growth in the control samples containing
1–2% DMSO. The 50% cytotoxic concentration (CC50) is the concen-
tration of compound that inhibited cell growth by 50%.
lM phenazine
l
37. Paeshuyse, J.; Coelmont, L.; Vliegen, I.; Van hemel, J.; Vandenkerckhove, J.;
Peys, E.; Sas, B.; De Clercq, E.; Neyts, J. Biochem. Biophys. Res. Commun. 2006,
348, 139.
Acknowledgments
38. Klumpp, K.; Leveque, V.; Le Pogam, S.; Ma, H.; Jiang, W. R.; Kang, H.;
Granycome, C.; Singer, M.; Laxton, C.; Hang, J. Q.; Sarma, K.; Smith, D. B.;
Heindl, D.; Hobbs, C. J.; Merrett, J. H.; Symons, J.; Cammack, N.; Martin, J. A.;
Devos, R.; Najera, I. J. Biol. Chem. 2006, 281, 3793.
The authors thank Ludmila Strokovska for her help in starting
the work with the HCV helicase and Anne-Lise Haenni for helpful
suggestions. This work was partially supported by the MNiSW
Grants 2P05A 03829 and N N401 2329 33.