Inhibition of subgenomic hepatitis C virus RNA replication by acridone derivatives: Identification of an NS3 helicase inhibitor

Article abstract of DOI:10.1021/jm801608u

We report the synthesis and structure-activity relationship (SAR) of a large series of acridones and acridone-fragment derivatives designed on the basis of the selective antihepatitis C virus (HCV) activity shown by acridone 2, previously studied as a pot

Full text of DOI:10.1021/jm801608u

3354
J. Med. Chem. 2009, 52, 3354–3365
Inhibition of Subgenomic Hepatitis C Virus RNA Replication by Acridone Derivatives:
Identification of an NS3 Helicase Inhibitor
Giuseppe Manfroni,§ Jan Paeshuyse,† Serena Massari,§ Samantha Zanoli,# Barbara Gatto, Giovanni Maga,# Oriana Tabarrini,*,§
Violetta Cecchetti,§ Arnaldo Fravolini,§ and Johan Neyts*,†
Dipartimento di Chimica e Tecnologia del Farmaco, UniVersita` degli Studi di Perugia, Via del Liceo, 1, 06123 Perugia, Italy, Rega Institute for
Medical Research, Katholieke UniVersiteit LeuVen, B-3000 LeuVen, Belgium, Istituto di Genetica Molecolare, CNR-PaVia, Via Abbiategrasso,
207, 27100 PaVia, Italy, and Dipartimento di Scienze Farmaceutiche, UniVersita` di PadoVa, Via Marzolo 5, 35131, PadoVa, Italy
ReceiVed December 19, 2008
We report the synthesis and structure-activity relationship (SAR) of a large series of acridones and acridone-
fragment derivatives designed on the basis of the selective antihepatitis C virus (HCV) activity shown by
acridone 2, previously studied as a potential antibovine viral diarrhea virus (BVDV) compound. The evaluation
of their ability to inhibit the HCV replication in Huh-5-2 cells led to the identification of new, selective
inhibitors. This indicates that the acridone skeleton, when properly functionalized, is a suitable scaffold to
obtain potential anti-HCV agents. Interestingly, during identification of possible cellular and viral targets,
it was discovered that compound 23 exerts inhibitory activity on the HCV NS3 helicase, a very promising
target for the development of anti-HCV drugs.
Introduction
the derivatives, even if no strict correlation was observed
between the anti-BVDV and anti-HCV activity. In particular,
compound 1 exhibited an anti-HCV activity that was comparable
to that for BVDV but proved to be somewhat more toxic against
the human hepatoma Huh-5-2 cells than on the bovine kidney
cells. On the other hand, the 1,3-dimethoxy analogue 2, which
was completely inactive against BVDV, proved to be equally
potent in the replicon assay, but above all, it was the most
selective, being devoid of any toxicity. In several independent
experiments, this compound was shown to inhibit viral RNA
synthesis in a dose-dependent manner (Figure 1). This interesting
biological profile made 2 a valid hit compound worthy of an
in-depth investigation aimed at identifying the structural phar-
macophoric requirements and increasing the HCV replicon
inhibitory activity while maintaining the selectivity. Here we
describe the design, synthesis, and structure-activity relation-
ship (SAR) of an enlarged series of acridone derivatives (7-42
Table 2) obtained by modifying the substitution pattern on the
acridone nucleus. To explore the structural features responsible
for the selective anti-HCV activity of 2, more incisive modifica-
tions were made on the acridone scaffold itself, giving rise to
derivatives 43-48 (Figure 2). Various viral and cellular proteins
were considered as the target for this class of molecules; HCV
NS3 helicase was the only enzyme that was found to be inhibited
by our compounds, albeit with moderate potency.
Worldwide, an estimated 170 million people are chronically
infected with hepatitis C virus (HCVa) and are at high risk for
developing chronic hepatitis, liver cirrhosis, and hepatocellular
carcinoma.1 Current treatment consists of a combination of
subcutaneously administrated pegylated interferon-R with the
orally dosed nucleoside analogue ribavirin. This combination
is generally poorly tolerated, is contraindicated for many
patients, and is only effective in controlling the disease in a
fraction of the individuals who are eligible for therapy.2
Therefore, there is an obvious, pressing need to develop more
effective and tolerated treatments. Today, a number of molecules
are being studied in clinical trials,3 but none of them have yet
been approved.
In an earlier paper,4 we reported the design, synthesis, and
antiviral evaluation of a small series of acridone derivatives
(compounds 1-6, Table 1), identifying some selective inhibitors
of bovine viral diarrhea virus (BVDV, a virus related to HCV5,6
)
replication. 1,3-Dihydroxy derivative 1, characterized by an
amino group at the C-7 position and the 2-pyridinylpiperazine
at the C-6 position, emerged as the most interesting compound
displaying anti-BVDV activity (EC50) of 0.5 ( 0.2 µg/mL with
no cytotoxicity at a concentration 200 times higher (CC50 > 100
µg/mL).
As part of our efforts to identify new anti-HCV agents,
acridones 1-6 have now been evaluated in the HCV genotype
1 subgenomic replicon system. The biological results reported
in Table 1 revealed an interesting antiviral activity for some of
Chemistry
Acridone derivatives 7-11 variously functionalized at the
C-ring were synthesized as reported in Scheme 1, analogous to
our previously reported analogues.4 Briefly, an Ullman reaction
of 2,4-dichloro-5-nitrobenzoic acid7 with various anilines gave
diphenylamine carboxylic acids 49, 50, 51,8 and 52, which were
then cyclized using PPA at 120 °C to give the nitroacridones
53-57. The ring closure of monomethoxy anthranylate 49 led
to the formation of two regioisomers, 53 and 54; the mixture
was difficult to separate, so it was used in the next step and
then separated. The successive sequential N-methylation to
58-60, 61,9 and 62, nucleophilic reaction with 1-(2-pyridi-
nyl)piperazine to 63-67, and nitro reduction afforded the target
compounds 7-11. With few exceptions, SnCl2 ·2H2O under acid
* To whom correspondence should be addressed. For O.T.: phone, +
39 075 585 5139; fax, +39 075 585 5115; e-mail, oriana.tabarrini@unipg.it.
For J.N.: phone, + 32 16 337353; fax, + 32 16 337340; e-mail,
johan.neyts@rega.kuleuven.be.
§
Universita` degli Studi di Perugia.
Katholieke Universiteit Leuven.
Istituto di Genetica Molecolare.
†
#
Universita` di Padova.
a Abbreviations: HCV, hepatitis C virus; BVDV, bovine viral diarrhea
virus; SAR, structure-activity relationship; PPA, polyphosphoric acid;
IMPDH, inosine monophosphate dehydrogenase; MTS/PMS, (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium)/phenazinemethosulfate.
10.1021/jm801608u CCC: $40.75
2009 American Chemical Society
Published on Web 04/23/2009

Acridones as HCV Replication Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3355
Table 1. Structure, Anti-BVDV Activity, Anti-HCV Activity, and Cytoxic/Cytostatic Effect of Reference Compounds
a Reference 4. b Data from ref 4. c EC50 values were obtained as described in the Experimental Section. All the data represent mean values for at least
three independent experiments ( standard deviation. d CC50 values were obtained as described in the Experimental Section. All the data represent mean
values for at least three independent experiments ( standard deviation.
1-iodo-3-methylbutane, with 1-(2-pyridinyl)piperazine followed
by reduction of the nitro group with SnCl2. Following the same
procedure, N-methyl acridones 19-26 were also prepared by
reacting precursor 754 with selected 4-arylpiperazines or with
NaOMe for 26.
The 1,3-dimethoxyacridones 22, 24, and 26 were converted
to the corresponding 1-hydroxy-3-methoxy (27, 28, and 29) and
1,3-dihydroxy derivatives (30, 31, and 32) using 48% HBr in
the presence of traces of AcOH. The progress of the reaction
was strictly monitored to avoid the formation of the sole
dihydroxy derivatives.
N-Isopentyl derivative 39 was prepared by reducing acridone
77 (Scheme 3). The N-10 unsubstituted C-6 chlorine derivatives
40-42 were directly prepared starting from precursor 74 through
Figure 1. Effect of 2 on HCV replicon replication in Huh-5-2 cells
reduction (to 40) and successive de-O-methylation (to 41 and
(measured as luciferase signal, bars) and on the proliferation of
42) (Scheme 3).
exponentially growing cells (diamonds). Data are expressed as percent-
Target compounds 34 and 35 (Table 2) were both prepared
(not shown) starting from the hit compound 2.4 In particular,
N,N-dimethylamino derivative 34 was synthesized by methy-
lation of the C-7 amino group with a large excess of MeI in the
presence of K2CO3. The C-7 unsubstituted derivative 35 was
prepared by converting the amino group into the diazonium salt
and then decomposed in the presence of 50% H3PO2 aqueous
solution.
Diphenylamine 43 was synthesized as shown in Scheme 4
by closely following the procedure illustrated for the synthesis
of acridones 7-11, bypassing the cyclization step. Thus,
antranilic acid 7812 was N-methylated, the obtained methyl ester
79 was coupled with 1-(2-pyridinyl)piperazine to form inter-
mediate 80, which was then reduced to the corresponding amino
derivative 81 and finally hydrolyzed in mild basic condition to
the target compound 43.
age of untreated controls (UTC) and are mean values ( SD for four
independent experiments: (*) P < 0.05.
condition was used as a general procedure to reduce the nitro
group, even if the aminoderivative was obtained in very low
yield mainly due to the formation of the side o-chloroamino
derivative. It is known that SnCl2 can promote the insertion of
a chlorine atom at the o-position of anilines;10,11 however, the
very low solubility of the nitroacridones in the common solvents
did not permit the use of other reduction methods.
Following the synthetic pathway described above, the reaction
of 2-chloro-4,5-difluorobenzoic acid with 3,5-dimethoxyaniline
(not shown) gave 7-fluoroacridone 33 (Table 2).
Acridone derivatives 12-15 (Scheme 2) were prepared by
O-alkylation with the selected alkyl halide of the 1-hydroxy-
7-nitro derivative 69,4 in turn conveniently prepared by selective
de-O-methylation of 684 with LiCl, followed by catalytic
reduction of nitro intermediates 70-73.
Scheme 3 depicts the synthesis of the 7-amino-1,3-dimethoxy
derivatives 16-26 variously functionalized at the N-10 or C-6
position of the acridone nucleus. The target compounds 16-18
were prepared by reacting the corresponding precursors 74,12
76,12 and 77, the latter prepared by alkylation of 7412 with
For the synthesis of diphenylamine 44, the route depicted in
Scheme 5 was employed. Starting with 2,4-difluoronitrobenzene,
there were two nucleophilic displacements in succession: the
first with 1-(2-pyridinyl)piperazine, using toluene as solvent,
to give ortho-substituted 82 (assigned by NOESY experiments)
as the major regioisomer, the second with 3,5-dimethoxy-N-

3356 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Manfroni et al.
Table 2. Anti-HCV Activity and Cytostatic Effect of Acridone Derivatives

Acridones as HCV Replication Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3357
Table 2. Continued
a EC50 values were obtained as described in the Experimental Section and are shown as mean values of two to three determinations. b CC50 values were
obtained as described in the Experimental Section and are shown as mean values of two to three determinations. c Reference 4. d EC50 and CC50 are shown
as single determination. e Reference 12.
obtained. However, when the reaction was carried out using
microwaves and AlCl3 as catalyst, according to a recent
publication,14 compound 84 was obtained in 65% yield. The
reaction conditions also caused a simultaneous de-O-methylation
which occurred at the C-2 position as expected and confirmed
by NOESY experiments. The successive nucleophilic reaction
with 1-(2-pyridinyl)piperazine followed by catalytic reduction
gave monomethoxy derivative 46 through the intermediate 85.
The O-methylation of intermediate 85 gave 86 which was
catalytically reduced to the desired dimethoxy derivative 45.
Phenylquinolone 47 (Scheme 7) was synthesized by starting
with nitro intermediate 87, prepared as previously reported by
us,15 which was coupled with 1-(2-pyridinyl)piperazine to give
88 and then reduced under catalytic conditions.
Results and Discussion
All the target compounds synthesized in this study were
evaluated for their ability to inhibit HCV replication in Huh-
Figure 2. Acridone scaffold disconnection.
5-2 cells, a Huh-7 derived cell line containing a genotype 1b
HCV replicon with the luciferase report gene. INF-R was
included as a positive control. The cytostatic potential of the
compounds was evaluated in parallel in the same cell line. Data
are summarized in Tables 2 and 3.
With selection of acridone 2 as the hit compound, about 40
analogues were synthesized. Of these, derivatives 12, 14, 19,
23, and 35 were found to selectively inhibit HCV RNA synthesis
in the context of the intact cell, in a dose-dependent manner.
Other compounds proved to be cytostatic or did not inhibit the
methylaniline13 in the presence of t-BuOK to give dipheny-
lamine intermediate 83 which was finally reduced to 44.
In an initial attempt to obtain benzophenone 45 (Scheme 6),
a Friedel-Crafts reaction of 4-chloro-3-nitrobenzoyl chloride
was carried out with 1,3-dimethoxybenzene, but under the usual
conditions (AlCl3/CH2Cl2 at room temperature or reflux; AlCl3/
nitrobenzene at reflux; FeCl3/Bi2O3 in CH2Cl2 at room temper-
ature or reflux) the expected dimethoxybenzophenone was not

3358 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Manfroni et al.
Scheme 1a
a Reagents and conditions: (i) Cu(OAc)2 ·2H2O, KOAc, Et3N, 2-propanol, reflux; (ii) PPA, 120 °C; (iii) MeI, K2CO3, dry DMF, 90 °C; (iv)
1-(2-pyridinyl)piperazine, dry DMF, 90 °C; (v) SnCl2 ·2H2O, 8 N HCl, reflux.
Scheme 2a
thiazol-2-yl)piperazine, can also be a suitable RV side chain
resulting in compounds 19 and 23 which are endowed with an
antiviral activity comparable to that of the reference compound.
All the other 4-arylpiperazines (derivatives 20, 21, 22, 24, and
25) were unsuitable as RV substituent. When a methoxy group
was employed as RV substituent (compounds 26, 29, and 32),
a slight decrease in antiviral activity was observed without a
dose-dependent effect.
Good anti-HCV activity was shown by RV chloro derivatives
36, 37, and 38 (Table 2), previously prepared by us as potential
antitumoral agents.12 Besides having a chlorine atom as an RV
substituent instead of the pyridinylpiperazine, compounds 36-38
differ from 2 bearing an RIV ethyl group. When the chlorine
atom was coupled with an RIV methyl group (see derivatives
4-64 in Table 1), contrasting anti-HCV results were obtained.
Therefore, to further explore the role of the RIV substituent in
the chloroacridone series, new analogues were prepared (com-
pounds 39-42). Unfortunately this novel set of data did not
contribute to the delineation of an SAR and discouraged the
synthesis of further analogues.
The deletion of the RVI amino group from compound 2 was
fruitful; in fact, compound 35 emerged as one of the best
molecules synthesized in this study. On the contrary, when the
amino group was replaced by a fluorine atom, there was a loss
of activity (compound 33) while its N,N-alkylation (compound
34) greatly increased the cytostatic effect.
Regarding the incisive structural modifications made on the
acridone scaffold itself (Figure 2), the opening of the B ring to
produce diphenylamines was detrimental; 43 was inactive while
44 proved highly cytostatic (Table 3). An increase in toxicity
was also noted in benzophenone 45, derived from a different
acridone skeleton opening; however, the C-2 de-O-methylated
derivative 46 resulted in appreciable anti-HCV activity (EC50
) 3.7 µg/mL) and was not cytostatic. The 2-phenylquinolone
47, derived from the shifting of the A ring to the C-2 position
of the dimethoxyquinolone nucleus, was neither active nor toxic,
while a weak antiviral activity (EC50 ) 11 µg/mL) and no
cytostatic activity were displayed by quinolone derivative 48,16
in which the acridone structure was further simplified by the
deletion of the C ring.
a Reagents and conditions: (i) LiCl, DMF, reflux; (ii) RX, Cs2CO3, dry
DMF, 90 °C; (iii) H2, Raney Ni, DMF, 1 atm, room temp. For RI of the
target compounds, see Table 2.
replication of the replicon. Some insights in the SAR were
derived from this data set. Regarding the modifications made
on the C-ring, both monomethoxy derivatives 7 and 8, deriva-
tive 9 bearing vicinal RII and RIII methoxy groups, and derivative
10 which lacks both methoxy groups proved to be more toxic
than the parent compound 2 without appreciable selective
inhibition of HCV subgenomic replication. On the other hand,
the presence of RI and RIII methyl groups gave compound 11
whose activity was comparable to that of 2 but without a dose-
dependent effect. The RI substituent was further studied by
replacing the methoxy group with longer side chains such as
ethoxy (12), propoxy (13), and isobutyloxy (14) as well as with
an acetic side chain (15). The antiviral activity was maintained
by derivatives 12 and 14, although the latter was somewhat more
cytostatic.
Overall, the above data show that the coupled presence of RI
and RIII methoxy groups is the best substitution pattern for the
C-ring. The biological profile of a few analogues bearing the
RI hydroxyl group (27 and 28) or RI and RIII hydroxyl groups
(30 and 31) also supports this observation.
Regarding the RIV substituent, the methyl group present in
the hit compound 2 was confirmed to be the best one and cannot
be omitted as in compound 16 or replaced with ethyl (17) or
isopentyl group (18), as this results in an increase in cytostatic
effect.
Besides 1-(2-pyridinyl)piperazine, which characterizes 2, its
regioisomer 1-(4-pyridinyl)piperazine, as well as the 1-(1,3-
The acridone moiety is thus the most suitable for granting
selective antiviral activity, but the benzophenone scaffold, when

Acridones as HCV Replication Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3359
Scheme 3a
a Reagents and conditions: (i) 1-iodo-3-methylbutane, K2CO3, dry DMF, 90 °C; (ii) RVH, dry DMF, 90 °C (for 26, MeONa, dry DMF); (iii) SnCl2 ·2H2O,
8 N HCl, reflux; (iv) H2, Raney Ni, DMF, 1 atm, room temp; (v) 48% HBr, AcOH, reflux. For RIII, RIV, and RV, see Table 2.
Scheme 4a
Scheme 6a
a Reagents and conditions: (i) AlCl3, microwaves, 57% potency, 7 min;
(ii) 1-(2-pyridinyl)piperazine, dry DMF, 90 °C; (iii) H2, Raney Ni, DMF,
1 atm, room temp; (iv) MeI, Cs2CO3, dry DMF, 90 °C.
Scheme 7a
a Reagents and conditions: (i) MeI, K2CO3, dry DMF, 90 °C; (ii) 1-(2-
pyridinyl)piperazine, dry DMF, 90 °C; (iii) H2, Raney Ni, EtOAc, 1 atm,
room temp; (iv) LiOH ·2H2O, dioxane/H2O (3:1).
Scheme 5a
a Reagents and conditions: (i) 1-(2-pyridinyl)piperazine, dry DMF, 90
°C; (ii) H2, Raney Ni, DMF, 1 atm, room temp.
Table 3. Anti-HCV and Cytostatic Effect of the Molecules Synthesized
by Disconnecting the Acridone Scaffold
a
b
compd
EC50 (µg/mL)
CC50 (µg/mL)
43
44
45
46
47
48c
>50
4.1
6.6
3.7
>50
11
>50
7.2
15
>50
>50
>33
a Reagents and conditions: (i) 1-(2-pyridinyl)piperazine, Et3N, toluene;
(ii) 3,5-dimethoxy-N-methylaniline, t-BuOK, DMSO, 0 °C; (iii) H2, Raney
Ni, EtOAc, 1 atm, room temp.
a EC50 values were obtained as described in the Experimental Section
and are shown as single determination. b CC50 values were obtained as
described in the Experimental Section and are shown as single determination.
c Reference 16.
properly functionalized as in compound 46, is also a valid hit
in an effort to obtain new anti-HCV agents.
In summary, the structural modifications made on the hit
compound 2 yielded new analogues including 12, 14, 19, 23,
and 35 which inhibited HCV RNA replication in a selective
and dose-dependent manner. However, in order for these new
chemotypes to be developed as anti-HCV agents, their potency
and selectivity must be increased. The identification of their
molecular target could help the optimization of this class of
HCV replication inhibitors. Therefore, the effect of the com-
pounds on a number of potential cellular and viral targets was
investigated.
Acridone-based compounds have recently been reported
to be potent reversible and uncompetitive inosine monophos-
phate dehydrogenase (IMPDH) inhibitors17 that are effective
on a rat adjuvant arthritis model. IMPDH is recognized as
being one of the potential cellular targets in anti-HCV
therapy.18,19 Ribavirin exerts its antiviral activity against
many viruses by inhibiting (as its 5′-monophosphate) this
enzyme,20,21 similar to the uncompetitive IMPDH inhibitor
mycophenolic acid21-23 and the Vertex Pharmaceutical
compound VX-497.21,24 The latter was not further developed

3360 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Manfroni et al.
unknotting and relaxation assays were performed for hit
compound 2 along with derivatives 14 and 44 which are
characterized by a different degree of toxicity. Doxorubicin and
m-amsacrine (m-AMSA), respectively, were included as positive
controls in the two assays. From the analyses of the gel (data
not shown) it appeared that the tested acridones were inactive
against the human enzyme, indicating that their cytostatic effect
in Huh-5-2 cells was not dependent on the topoisomerase II
interaction.
Conclusions
Starting from hit compound 2, a large series of acridones
and acridone-fragment derivatives were synthesized and
evaluated for their ability to inhibit the HCV replication in
Huh-5-2 cells. Some new selective anti-HCV compounds
were identified revealing that the acridone skeleton, when
properly functionalized, represents a suitable scaffold for
obtaining potential anti-HCV agents. Structure features
responsible for the HCV replication inhibition are a methoxy,
ethoxy, or isobutyloxy group as RI substituent, a 4-arylpi-
perazine as RV side chain with 1-(2-pyridinyl)-, 1-(4-pyri-
dinyl)-, and 1-(1,3-thiazol-2-yl)piperazine which confer the
best activity, a RIV methyl group, and an amino group or
hydrogen atom as an RVI substituent. Searching for their
molecular target, we have identified the thiazolpiperazinyl
derivative 23 which inhibited, albeit with low potency, the
HCV NS3 helicase. Studies are in progress to gain insight
into the precise mechanism of action of 23, which would
ultimately provide a structural basis for the discovery of
improved anti-NS3 helicase acridones.
Figure 3. Dose-response curve for the inhibition of NS3 helicase
activity by compound 23. Values are the mean of three independent
replicates. Error bars are (SD. Data were fitted to the equation (% obs)
) (% max)/(1 + [I]/IC50), where (% obs) is the observed percentage
of unwound substrate, (% max) is the maximal unwinding percentage
in the absence of the inhibitor, [I] is the inhibitor concentration, and
IC50 is the 50% inhibitory dose.
as an HCV inhibitor.3 To determine if IMPDH was the target
of the acridones reported in this study, the cytostatic effect
in Vero cells alone and in combination with guanosine was
tested for the best molecules (2, 14, 19, 23, and 35). There
was no evidence that the cytostatic effect was reversed in
the presence of guanosine (data not shown).
When the activity of the same molecules was tested on the
HCV RNA polymerase NS5B, one of the most relevant key
enzyme of the HCV replication, none of the compounds showed
inhibitory activity at concentrations up to 100 µM. These data
are in agreement with what we previously reported for acridone
1, which had little or no inhibitory effect on the BVDV NS5B
polymerase activity.4
None of the above compounds were able to inhibit the NS3
NTPase activity (IC50 > 1 mM). However, as shown in Figure
3, the thiazolpiperazinyl derivative 23 inhibited the helicase
activity with an IC50 of 50 ( 5 µg/mL (110 ( 12 µM). Its
antienzymatic activity was approximately 10 times less than the
anti-HCV activity in the cellular assay. However, such discrep-
ancies are not uncommon because of the differences between
the in vitro conditions (for example, high enzyme-to-substrate
ratio) and the replicon assay. Thus, so far, our data support the
reasonable hypothesis that the target of this compound is likely
the HCV NS3 helicase. NS3 has long been regarded as a very
promising target for the development of anti-HCV drugs. It is
an essential enzyme for viral replication and bears little
homology to cellular helicases. However, to date, only a few
examples of inhibitors of this activity have been reported,
particularly in the non-nucleoside class.25 Interestingly, a series
of compounds strictly related to our acridones have recently
been reported to be good inhibitors of HCV helicase activity.26
Although for these compounds the inhibitory effect in the
enzymatic assay did not translate into antiviral activity in the
replicon assay, this study lends support to our observation that
acridones may have the potential to inhibit HCV replication by
targeting the helicase.
For all the other selective acridones identified in this study,
which did not recognize the NS3 helicase, the anti-HCV replicon
activity could be due to either a different unknown mechanism
of action or a polypharmacological effect. The latter interpreta-
tion is in line with what has been previously reported by
Bastow29 for other antiviral acridones, for which more than one
biochemical target has been identified.
Experimental Section
All reactions were routinely checked by thin-layer chromatog-
raphy (TLC) on silica gel 60F254 (Merck) and visualized by using
UV or iodine. Column chromatography separations were carried
out on Merck silica gel 60 (mesh 70-230), flash chromatography
on Merck silica gel 60 (mesh 230-400), and reverse phase
chromatography on Merck RP C18 (40-63 µm, Lichroprep).
Melting points were determined in capillary tubes (Bu¨chi Electro-
termal model 9100) and are uncorrected. Elemental analyses were
performed on a Carlo Erba elemental analyzer, model 1106, and
data for C, H, and N are within (0.4% of the theoretical values.
1H NMR spectra were recorded at 200 MHz (Bruker Avance DPX-
1
200), while H-1H NMR NOESY spectra were recorded at 400
MHz (Bruker Avance DRX-400) using residual solvent such as
chloroform (δ ) 7.26) or dimethyl sulfoxide (δ ) 2.48) as an
internal standard. The 2D NOESY experiments were run in phase-
sensitive mode at 298 K. Data processing was performed with
standard Bruker software XwinNMR. Chemical shifts are given in
ppm (δ), and the spectral data are consistent with the assigned
structures. GC/MS analyses were carried out with an HP 6890 gas
chromatograph (25 m dimethylsilicone capillary column) equipped
with an HP 5973 mass selective detector. Microwave reactions were
carried out using a domestic unmodified microwave oven (Daewoo
KOR-63F7, 2.45 MHz, 700 W). Reagents and solvents were
purchased from common commercial suppliers and were used as
such. After extraction, organic layers were dried over anhydrous
Na2SO4, filtered, and concentrated with a Bu¨chi rotary evaporator
at reduced pressure. Yields were of purified product and were not
Further studies are in progress to determine the precise
mechanism by which 23 inhibits the NS3 helicase activity. In
particular, we are addressing whether the inhibition is due to
direct enzyme-drug interaction or is mediated by the compound
binding to the nucleic acid substrate.
Finally, since some of the known antiviral acridone deriva-
tives are able to inhibit the topoisomerase II enzyme,27,28 we
wanted to ascertain whether this enzyme was recognized by
the compounds reported here. Therefore, topoisomerase II

Acridones as HCV Replication Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3361
optimized. All starting materials were commercially available,
unless otherwise indicated.
7-Fluoro-1,3-dimethoxy-10-methyl-6-[4-(2-pyridinyl)-1-piper-
azinyl]-9(10H)-acridinone (33). The title compound was prepared
following the general Ullman (replacing 2,4-dichloro-5-nitrobenzoic
acid with 2-chloro-4,5-difluorobenzoic acid), cyclization, alkylation,
and coupling (120 °C, 120 h) procedures. After crystallization by
EtOH, 33 was obtained in 8% overall yield as a gray solid: mp
General Procedure for Ullman Reaction. 4-Chloro-2-[(3,4-
dimethoxyphenyl)amino]-5-nitrobenzoic Acid (50). A mixture of
2,4-dichloro-5-nitrobenzoic acid7 (5.00 g, 21.20 mmol), 3,4-
dimethoxyaniline (4.87 g, 31.80 mmol), KOAc (6.24 g, 63.60
mmol), Cu(OAc)2 ·2H2O (0.13 g, 0.64 mmol), and Et3N (8.92 mL,
63.60 mmol) in 2-propanol (150 mL) was refluxed for 12 h. The
solvent was then concentrated under reduced pressure, the mixture
was poured into ice-water, acidified with 2 N HCl to pH ∼3, and
the solid formed was filtered, collected, and dissolved in CHCl3.
The organic solution was extracted with water and evaporated to
dryness. The solid obtained was triturated with EtOH and filtered
1
118-119 °C; H NMR (DMSO-d6) δ 3.30-3.40 and 3.70-3.80
(m, each 4H, piperazine CH2), 3.65 (s, 3H, NCH3), 3.90 and 3.95
(s, each 3H, OCH3), 6.25 and 6.30 (d, J ) 2.0 Hz, each 1H, H-2
and H-4), 6.65-6.85 (m, 3H, H-5 and pyridine CH), 7.55 (ddd, J
) 1.8, 8.5, and 10.3 Hz, 1H, pyridine CH), 8.10 (d, J ) 13.3 Hz,
1H, H-8), 8.22 (dd, J ) 1.8 and 5.0 Hz, 1H, pyridine CH). Anal.
(C25H25FN4O3) C, H, N.
1-Hydroxy-3-methoxy-10-methyl-7-nitro-6-[4-(2-pyridinyl)-1-
piperazinyl]-9(10H)-acridinone (69).4 A mixture of 684 (0.46 g,
0.99 mmol) and LiCl (0.20 g, 4.80 mmol) in DMF (15 mL) was
refluxed with stirring for 10 h. The solvent was then concentrated
under reduced pressure, and the residue was poured into ice-water,
made acidic (pH 6) with 2 N HCl, and extracted with CHCl3. The
solvent was evaporated to dryness and the crude product was
purified by flash chromatography, eluting with CHCl3/MeOH (98:
2) to give 69 (0.30 g, 68%) as an orange solid. Melting point and
spectral data are in agreement with those previously reported.4
General Procedure for O-Alkylation Reaction. 1-Ethoxy-3-
methoxy-10-methyl-7-nitro-6-[4-(2-pyridinyl)-1-piperazinyl]-
9(10H)-acridinone (70). A mixture of 694 (0.17 g, 0.37 mmol),
Cs2CO3 (1.20 g, 3.70 mmol), and 1-iodoethane (0.09 mL, 1.10
mmol) in dry DMF (20 mL) was heated at 90 °C for 5 h. The
solvent was concentrated under reduced pressure, and the residue
was poured into ice-water. The precipitate obtained was filtered,
dried, and purified by flash chromatography, eluting with benzene/
acetone (80:20) to give 70 (0.10 g, 55%) as a yellow solid: mp
>300 °C (dec); 1H NMR (CDCl3) δ 1.62 (t, J ) 7.0 Hz, 3H,
OCH2CH3), 3.30-3.37 (m, 4H, piperazine CH2), 3.75 (s, 3H,
NCH3), 3.76-3.84 (m, 4H, piperazine CH2), 3.96 (s, 3H, OCH3),
4.18 (q, J ) 7.0 Hz, 2H, OCH2CH3), 6.33 and 6.40 (d, J ) 2.1 Hz,
each 1H, H-2 and H-4), 6.65-6.75 (m, 3H, H-5 and pyridine CH),
7.55 (ddd, J ) 2.0, 8.1, and 9.1 Hz, 1H, pyridine CH), 8.25 (dd, J
) 1.9 and 5.7 Hz, 1H, pyridine CH), 9.00 (s, 1H, H-8).
1
to give 50 (4.0 g, 54%) as a brown solid: mp 248-249 °C; H
NMR (DMSO-d6) δ 3.70 and 3.80 (s, each 3H, OCH3), 6.80-7.10
(m, 4H, aromatic CH), 8.70 (s, 1H, H-6), 10.10 (s, 1H, NH), 14.00
(bs, 1H, CO2H).
General Procedure for Cyclization Reaction. 3-Chloro-6,7-
dimethoxy-2-nitro-9(10H)-acridinone (55). A mixture of 50 (2.50
g, 7.09 mmol) and PPA (12.5 g) was heated in an oil bath at 120
°C for 7 h. After cooling, the mixture was triturated with ice-water
and neutralized with 10% NaOH, and the dark precipitate was
collected by filtration and then recrystallized by DMF to give
1
compound 55 (0.5 g, 21%) as a yellowish solid: mp >300 °C; H
NMR (DMSO-d6) δ 3.87 and 3.94 (s, each 3H, OCH3), 6.90 (s,
1H, H-4), 7.48 and 7.52 (s, each 1H, H-5 and H-8), 8.75 (s, 1H,
H-1), 12.10 (s, 1H, NH).
General Procedure for N-Alkylation Reaction. 3-Chloro-6,7-
dimethoxy-10-methyl-2-nitro-9(10H)-acridinone (60). MeI (0.33
mL, 5.37 mmol) was added to a mixture of compound 55 (0.45 g,
1.34 mmol) and K2CO3 (0.74 g, 5.37 mmol) in dry DMF (15 mL).
The reaction mixture was heated at 90 °C for 4 h. After cooling,
the mixture was poured into ice-water and the solid obtained was
filtered, washed with water, and recrystallized by EtOH/DMF to
give compound 60 (0.35 g, 74%) as a brown solid: mp >300 °C;
1H NMR (DMSO-d6) δ 3.78 and 3.83 (s, each 3H, OCH3) 3.90 (s,
3H, NCH3), 7.09 (s, 1H, H-4), 7.41 (s, 1H, H-5), 8.00 (s, 1H, H-8),
8.70 (s, 1H, H-1).
General Procedure for Reduction of NO2 Group. Method
B. 7-Amino-1-ethoxy-3-methoxy-10-methyl-6-[4-(2-pyridinyl)-
1-piperazinyl]-9(10H)-acridinone (12). A stirred solution of 70
(0.10 g, 0.204 mmol) in DMF (15 mL) was hydrogenated over a
catalytic amount of Raney nickel at room temperature and
atmospheric pressure for 1 h. The mixture was then filtered over
Celite, and the filtrate was evaporated to dryness to give a residue
which was crystallized by EtOH/EtOAc to afford 12 (0.046 g, 49%)
General Procedure for Coupling Reaction. 2,3-Dimethoxy-
10-methyl-7-nitro-6-[4-(2-pyridinyl)-1-piperazinyl]-9(10H)-acri-
dinone (65). A mixture of compound 60 (0.31 g, 0.88 mmol) and
1-(2-pyridinyl)piperazine (0.4 mL, 2.64 mmol) in dry DMF (10
mL) was heated at 90 °C for 7 h. The solution was concentrated
under reduced pressure, and after the mixture was cooled, the
precipitate was collected by filtration, washed with EtOH, and dried
to give 65 (0.31 g, 73%) as a yellow solid: mp >300 °C; 1H NMR
(DMSO-d6) δ 3.25-3.40 and 3.60-3.75 (m, each 4H, piperazine
CH2), 3.85 (s, 3H, NCH3), 4.00 and 4.05 (s, each 3H, OCH3),
6.60-6.70 and 6.75-690 (m, each 1H, pyridine CH), 7.15 and 7.25
(s, each 1H, H-1 and H-4), 7.50-7.60 (m, 1H, pyridine CH), 7.70
(s, 1H, H-5), 8.20-8.25 (m, 1H, pyridine CH), 8.75 (s, 1H, H-8).
General Procedure for Reduction of NO2 Group. Method
A. 2-Amino-6,7-dimethoxy-10-methyl-3-[4-(2-pyridinyl)-1-pip-
erazinyl]-9(10H)-acridinone (9). A solution of SnCl2 ·2H2O (0.35
g, 1.56 mmol) in 8 N HCl (15 mL) was added, at room temperature
with stirring, to a solution of nitro derivative 65 (0.25 g, 0.52 mmol)
in 8 N HCl (10 mL). The mixture was heated under reflux for 1 h
and after cooling it was made basic to pH ∼9 with 10% NaOH
solution to obtain a solid which was collected, washed several times
with water, dried, purified by flash column chromatography, eluting
with CHCl3/MeOH (98:2), and then recrystallized by DMF to give
derivative 9 (0.07 g, 33%) as a yellow solid: mp 291-293 °C; 1H
NMR (DMSO-d6) δ 2.95-3.12 and 3.57-3.68 (m, each 4H,
piperazine CH2), 3.74 (s, 3H, NCH3), 3.80 and 3.89 (s, each 3H,
OCH3), 4.82 (s, 2H, NH2), 6.58 (dd, J ) 4.91 and 7.90 Hz, 1H,
pyridine CH), 6.80 (d, J ) 8.6 Hz, 1H, pyridine CH), 7.00 and
7.08 (s, each 1H, H-1 and H-4), 7.40-7.58 (m, 3H, H-5, H-8, and
pyridine CH), 8.08 (dd, J ) 1.4 and 4.6 Hz, 1H, pyridine CH).
Anal. (C25H27N5O3) C, H, N.
1
as a yellow solid: mp 215-216 °C; H NMR (DMSO-d6) δ 1.40
(t, J ) 6.7 Hz, 3H, OCH2CH3), 3.09-3.20 (m, 4H, piperazine CH2),
3.70-3.82 (m, 7H, piperazine CH2 and NCH3), 3.92 (s, 3H, OCH3),
4.09 (q, J ) 6.8 Hz, 2H, OCH2CH3), 6.30 and 6.57 (d, J ) 2.1 Hz,
each 1H, H-2 and H-4), 6.70 (m, 1H, pyridine CH), 6.95 (d, J )
8.1 Hz, 1H, pyridine CH), 7.09 (s, 1H, H-5), 7.54 (s, 1H, H-8),
7.58-7.67 (m, 1H, pyridine CH), 8.12-8.18 (m, 1H, pyridine CH).
Anal. (C26H29N5O3) C, H, N.
7-Amino-1,3-dimethoxy-6-[4-(2-pyridinyl)-1-piperazinyl]-
9(10H)-acridinone (16). The title compound was prepared from
7412 according to the general procedure for coupling reaction
followed by reduction (method A). After flash chromatography
purification, eluting with CHCl3/MeOH (97:3), 16 was obtained in
1
35% overall yield as a yellow solid: mp 196-197 °C; H NMR
(DMSO-d6) δ 3.05-3.15 and 3.65-3.75 (m, each 4H, piperazine
CH2), 3.85 and 3.90 (s, each 3H, OCH3), 5.80 (bs, 2H, NH2), 6.15
and 6.35 (bs, each 1H, H-2 and H-4), 6.60-6.80 (m, 1H, pyridine
CH), 6.85-7.00 (m, 2H, H-5 and pyridine CH), 7.45 (s, 1H, H-8),
7.50-7.70 (m, 1H, pyridine CH), 8.15-8.25 (m, 1H, pyridine CH),
11.00 (s, 1H, NH). Anal. (C24H25N3O3) C, H, N.
7-Amino-10-ethyl-1,3-dimethoxy-6-[4-(2-pyridinyl)-1-piper-
azinyl]-9(10H)-acridinone (17). The title compound was prepared
from 7612 according to the general procedure for coupling reaction
followed by reduction (method B, using DMF/2-methoxyethanol

3362 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Manfroni et al.
(1:3) as solvent). After flash chromatography purification, eluting
solution. The precipitate so obtained was filtered and dried, and
after purification by flash chromatography (CHCl3/MeOH, 98:2),
compound 27 was obtained in 31% yield, followed by compound
30 in 11% yield.
with CHCl3/MeOH (97:3), 17 was obtained in 14% overall yield
1
as a yellowish solid: mp 250-252 °C; H NMR (DMSO-d6) δ
1.25-1.45 (m, 3H, CH2CH3), 3.00-3.20 and 3.60-3.70 (m, each
4H, piperazine CH2), 3.70 and 3.80 (s, each 3H, OCH3), 4.20-4.40
(m, 2H, CH2CH3), 4.85 (bs, 2H, NH2), 6.20 and 6.40 (bs, each 1H,
H-2 and H-4), 6.55-6.60 (m, 1H, pyridine CH), 6.80-6.90 (m,
1H, pyridine CH), 7.00 (s, 1H, H-5), 7.40-7.60 (m, 2H, pyridine
CH and H-8), 8.10-8.20 (m, 1H, pyridine CH). Anal. (C26H29N5O3)
C, H, N.
Compound 27: mp 287-289 °C; 1H NMR (DMSO-d6) δ
3.10-3.20 and 3.30-3.40 (m, each 4H, piperazine CH2), 3.60 (s,
3H, NCH3), 3.70 (s, 3H, OCH3), 5.00 (bs, 2H, NH2), 6.10 and 6.40
(d, J ) 2.1 Hz, 1H, H-2 and H-4), 6.80-6.85 (m, 1H, aromatic
CH), 6.90-7.05 (m, 2H, aromatic CH), 7.15-7.30 (m, 3H, H-5
and aromatic CH), 7.50 (s, 1H, H-8), 15.25 (s, 1H, OH). Anal.
(C25H26N4O3) C, H, N.
7-Amino-10-isopentyl-1,3-dimethoxy-6-[4-(2-pyridinyl)-1-pip-
erazinyl]-9(10H)-acridinone (18). The title compound was pre-
pared from 77 according to the general procedure for coupling
reaction followed by reduction (method A). After flash chroma-
tography purification, eluting with CHCl3/MeOH (98:2), followed
by crystallization by EtOH, 18 was obtained in 20% overall yield
as a yellow solid: mp 246-248 °C; 1H NMR (CDCl3) δ 1.15 (d, J
) 3.4 Hz, 6H, CH(CH3)2), 1.75-2.00 (m, 3H, NCH2CH2-
CH(CH3)2), 3.10-3.25 and 3.60-3.80 (m, each 4H, piperazine
CH2), 3.90 and 4.00 (s, each 3H, OCH3), 4.15-4.25 (m, 2H,
NCH2CH2), 6.26 and 6.40 (d, J ) 2.1 Hz, each 1H, H-2 and H-4),
6.60-6.75 (m, 2H, pyridine CH), 6.90 (s, 1H, H-5), 7.50-7.60
(m, 1H, pyridine CH), 7.90 (s, 1H, H-8), 8.23-8.27 (m, 1H,
pyridine CH). Anal. (C29H35N5O3) C, H, N.
Compound 30: mp >300 °C; 1H NMR (DMSO-d6) δ 3.20-3.25
and 3.30-3.35 (m, each 4H, piperazine CH2), 4.70 (s, 3H, NCH3),
6.00 and 6.30 (d, J ) 1.8 Hz, each 1H, H-2 and H-4), 6.75-6.80
(m, 1H, aromatic CH), 6.90-7.10 (m, 2H, aromatic CH), 7.20 (s,
1H, H-5), 7.20-7.25 (m, 2H, aromatic CH), 7.50 (s, 1H, H-8),
10.40 and 14.50 (s, each 1H, OH). Anal. (C24H24N4O3) C, H, N.
6-Chloro-10-isopentyl-1,3-dimethoxy-7-nitro-9(10H)-acri-
done (77). The title compound was prepared from 7412 employing
1-iodo-3-methylbutane according to the general procedure for
N-alkylation. After purification by column chromatography (CHCl3/
MeOH, 97:3), the title compound was obtained as a yellow solid
in 24% yield: mp 209-211 °C; 1H NMR (DMSO-d6) δ 1.05 (d, J
) 4.3 Hz, 6H, CH(CH3)2), 1.50-1.70 (m, 2H, NCH2CH2),
1.80-1.95 (m, 1H, NCH2CH2CH), 3.85 and 3.95 (s, each 3H,
OCH3), 4.25-4.45 (m, 2H, NCH2CH2), 6.50 and 6.55 (bs, each
1H, H-2 and H-4), 7.85 (s, 1H, H-5), 8.75 (s, 1H, H-8).
7-Amino-6-chloro-10-isopentyl-1,3-dimethoxy-9(10H)-acridi-
none (39). The title compound was obtained from nitro derivative
77 according to general reduction procedure (method A). After
crystallization by DMF, the title compound was obtained as a yellow
solid in 74% yield: mp 299-300 °C; 1H NMR (CDCl3) δ 1.15 (d,
J ) 4.3 Hz, 6H, CH(CH3)2), 1.60-1.90 (m, 3H, NCH2CH2CH),
3.90 and 4.00 (s, each 3H, OCH3), 4.10-4.25 (m, 2H, NCH2CH2),
6.25 and 6.35 (d, J ) 2.0 Hz, each 1H, H-2 and H-4), 7.30 (s, 1H,
H-5), 7.90 (s, 1H, H-8). Anal. (C20H23ClN2O3) C, H, N.
7-(Dimethylamino)-1,3-dimethoxy-10-methyl-6-[4-(2-pyridi-
nyl)-1-piperazinyl]-9(10H)-acridinone (34). A mixture of 24 (0.23
g, 0.52 mmol), MeI (0.3 mL, 5.2 mmol), and K2CO3 (0.21 g, 1.55
mmol) in dry DMF (20 mL) was heated at 90 °C for 15 h. After
cooling, the reaction mixture was poured into ice-water and the
yellowish precipitate was collected by filtration, dried, and purified
by column chromatography (CHCl3/MeOH, 98:2) followed by
crystallization with Et2O/EtOH to give 34 (0.08 g, 35%): mp
205-206 °C; 1H NMR (DMSO-d6) δ 2.80 (s, 6H, N(CH3)2),
3.35-3.50 (m, 4H, piperazine CH2), 3.60-3.75 (m, 7H, NCH3 and
piperazine CH2), 3.85 and 3.90 (s, each 3H, OCH3), 6.25 and 6.35
(d, J ) 2.1 Hz, each 1H, H-2 and H-4), 6.65-6.75 (m, 3H, pyridine
CH and H-5), 7.55 (ddd, J ) 2.0, 7.2, and 8.9 Hz, 1H, pyri-
dine CH), 8.05 (s, 1H, H-8), 8.20 (dd, J ) 1.9 and 5.0 Hz, 1H,
pyridine CH). Anal. (C27H31N5O3) C, H, N.
1,3-Dimethoxy-10-methyl-6-[4-(2-pyridinyl)-1-piperazinyl]-
9(10H)-acridinone (35). A solution of NaNO2 (0.04 g, 0.6 mmol)
in water (2 mL) was added portionwise to a stirred solution of 24
(0.23 g, 0.5 mmol) in 6 N HCl, cooled at 0 °C. After 30 min, a
solution of NaBF4 (0.10 g, 0.9 mmol) in water (3 mL) was added
and the reaction mixture was kept at 0 °C for 30 min. The borine
diazonium salt formation was detected by using the ꢀ-naphthol
assay. The 6,8-dimethoxy-10-methyl-9-oxo-3-[4-(2-pyridinyl)-1-
piperazinyl]-9,10-dihydro-2-acridinediazonium tetrafluoroborate red
precipitate was filtered and washed with a few drops of water and
successively with Et2O. The crude product, without further purifica-
tion, was suspended in 50% H3PO2 (4 mL), and the mixture was
stirred for 4 h, then poured into ice-water, made basic (pH 8) with
10% NaOH, and extracted several times with CHCl3. The organic
layers were then evaporated to dryness, giving a residue which was
purified by flash chromatography (CHCl3/MeOH, 97:3) followed
by trituration with EtOAc to give 35 (0.11 g, 50%) as a white solid:
mp 205-207 °C; 1H NMR (CDCl3) δ 3.40-3.60 (m, 4H, piperazine
CH2), 3.70-3.85 (m, 7H, piperazine CH2, and NCH3), 3.90 and
4.10 (s, each 3H, OCH3), 6.25 and 6.30 (bs, each 1H, H-2 and
7-Amino-1,3-dimethoxy-10-methyl-6-[4-(4-pyridinyl)-1-piper-
azinyl]-9(10H)-acridinone (19). The title compound was prepared
from 7512 according to the general procedure for coupling reaction
employing the 1-(4-pyridinyl)piperazine followed by reduction
(method A). After flash chromatography purification, eluting with
CHCl3/MeOH (97:3), 19 was obtained in 18% overall yield as a
yellow solid: mp 297-300 °C (dec); 1H NMR (DMSO-d6) δ
3.10-3.20 and 3.50-3.60 (m, each 4H, piperazine CH2), 3.75 (s,
3H, NCH3), 3.80 and 3.95 (s, each 3H, OCH3), 4.90 (bs, 2H, NH2),
6.25 and 6.50 (d, J ) 1.6 Hz, each 1H, H-2 and H-4), 6.80-6.90
(m, 2H, pyridine CH), 7.00 (s, 1H, H-5), 7.50 (s, 1H, H-8),
8.10-8.20 (m, 2H, pyridine CH). Anal. (C25H27N5O3) C, H, N.
7-Amino-1,3,6-trimethoxy-10-methyl-9(10H)-acridinone (26).
A mixture of 7512 (0.70 g, 2.0 mmol) and freshly prepared MeONa
(0.34 g, 6.0 mmol) in dry DMF (40 mL) was stirred at room
temperature for 40 min. After water addition (1.0 mL), the solvent
was concentrated under reduced pressure to give a solid which was
filtered, dried, and crystallized by DMF to give 1,3,6-trimethoxy-
10-methyl-7-nitro-9(10H)-acridinone (0.45 g, 63%) as a yellow
solid: mp 275-277 °C; 1H NMR (DMSO-d6) δ 3.75 (s, 1H, NCH3),
3.85, 3.90, and 4.00 (s, each 3H, OCH3), 6.45 and 6.65 (d, J ) 2.0
Hz, each 1H, H-2 and H-4), 7.10 (s, 1H, H-5), 8.65 (s, 1H, H-8).
The nitro intermediate so obtained was then reduced employing
the general procedure (method A) to give after flash chromatography
purification (CHCl3/MeOH, 97:3) the title compound 26 in 25%
1
yield as a brown solid: mp 295-297 °C; H NMR (DMSO-d6) δ
3.75 (s, 3H, NCH3), 3.79, 3.87, and 3.90 (s, each 3H, OCH3), 4.85
(bs, 2H, NH2), 6.30 and 6.57 (bs, each 1H, H-2 and H-4), 6.95 (s,
1H, H-5), 7.43 (s, 1H, H-8). Anal. (C17H18N2O4) C, H, N.
7-Amino-6-chloro-1,3-dimethoxy-9(10H)-acridinone (40). The
title compound was prepared from 7412 according to the general
reduction procedure (method A, 2 h). After purification by flash
chromatography, eluting with gradient of CHCl3/MeOH (100:0 to
95:5), the title compound was obtained in 35% yield as a brown
1
solid: mp 290-293 °C; H NMR (DMSO-d6) δ 3.60 and 3.70 (s,
each 3H, OCH3), 6.10 and 6.25 (bs, each 1H, H-2 and H-4), 7.20
(s, 1H, H-5), 7.40 (s, 1H, H-8), 10.90 (s, 1H, NH). Anal.
(C15H13ClN2O3) C, H, N.
General Procedure for De-O-methylation. 7-Amino-1-hy-
droxy-3-methoxy-10-methyl-6-(4-phenyl-1-piperazinyl)-9(10H)-
acridinone (27) and 7-Amino-1,3-dihydroxy-10-methyl-6-(4-
phenyl-1-piperazinyl)-9(10H)-acridinone (30). A mixture of 22
(0.10 g, 0.22 mmol) in 48% HBr (1.5 mL) and a few drops AcOH
was heated at reflux until starting material disappeared and two
products formed (8 h). After the mixture was cooled, the bromo-
hydrate precipitate was collected by filtration and solubilized in
water and the solution made basic (pH 8) with saturated Na2CO3

Acridones as HCV Replication Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3363
H-4), 6.55-6.65 (m, 3H, pyridine CH, H-5, and H-7), 6.85 (d, J )
9.0 Hz, 1H, pyridine CH), 7.50-7.60 and 8.15-8.20 (m, each 1H,
pyridine CH), 8.30 (d, J ) 8.9 Hz, 1H, H-8). Anal. (C25H26N4O3)
C, H, N.
Methyl 4-Chloro-2-[N-(3,5-dimethoxyphenyl)-N-methylamino]-
5-nitrobenzoate (79). The title compound was prepared starting
from 7812 and using MeI, according to the general procedure for
N-alkylation (24 h). After purification by flash chromatography
(cycloexane/EtOAc, 70:30) followed by treatment with Et2O/EtOH,
the title compound was obtained in 20% yield as an orange solid:
mp 138-140 °C; 1H NMR (CDCl3) δ 3.40 (s, 3H, NCH3), 3.50 (s,
3H, OCH3), 3.75 (bs, 6H, OCH3 and CH3), 6.15-6.20 (d, J ) 2.1
Hz, 2H, H-2′ and H-6′), 6.20 (t, J ) 2.1 Hz, 1H, H-4′), 7.25 (s,
1H, H-3), 8.30 (s,1H, H-6).
Methyl 2-[N-(3,5-Dimethoxyphenyl)-N-methylamino]-5-nitro-
4-[4-(2-pyridinyl)-1-piperazinyl]benzoate (80). The title com-
pound was obtained starting from 79 according to the general
procedure for coupling reaction. The reaction mixture was cooled
obtaining a precipitate which was filtered to give 80 as an orange
solid in 79% yield: mp 145-147 °C; 1H NMR (CDCl3) δ 3.23-3.32
(m, 4H, piperazine CH2), 3.35 and 3.58 (s, each 3H, NCH3 and
OCH3), 3.72 (bs, 6H, OCH3 and CH3), 3.75-3.82 (m, 4H,
piperazine CH2), 6.00-6.10 (m, 3H, H-2′, H-4′, and H-6′),
6.63-6.75 (m, 3H, pyridine CH and H-3), 7.58 (dt, J ) 1.9 and
7.0 Hz, 1H, pyridine CH), 8.30 (dd, J ) 1.9 and 5.4 Hz, 1H,
pyridine CH), 8.41 (s, 1H, H-6).
purified by flash chromatography (cyclohexane/EtOAc, 70:30) to
give 83 (0.50 g, 56%) as a yellowish solid: mp 152-154 °C; H
1
NMR (CDCl3) δ 3.10-3.20 (m, 4H, piperazine CH2), 3.35 (s, 3H,
NCH3), 3.70-3.75 (m, 4H, piperazine CH2), 3.80 (bs, 6H, OCH3),
6.25-6.40 (m, 5H, pyridine CH, H-2′, H-6′, H-2, and H-6),
6.55-6.70 (m, 2H, pyridine CH and H-4′), 7.50 (ddd, J ) 2.0,
7.1, and 8.7 Hz, 1H, pyridine CH), 8.00 (d, J ) 9.0 Hz, 1H, H-5),
8.15 (ddd, J ) 0.7, 2.0, and 5.0 Hz, 1H, pyridine CH).
N4-(3,5-Dimethoxyphenyl)-N4-methyl-2-[4-(2-pyridinyl)-1-pip-
erazinyl]-1,4-benzenediamine (44). The title compound was
prepared starting from 83 according to the general reduction
procedure (method B). After purification by column chromatography
(cyclohexane/EtOAc, 60:40), 44 was obtained in 82% yield as a
pale-yellow solid: mp 126-128 °C; 1H NMR (CDCl3) δ 2.90-310
(m, 4H, piperazine CH2), 3.20 (s, 3H, NCH3), 3.65-3.75 (m, 10H,
OCH3 and piperazine CH2), 4.00 (bs, 2H, NH2), 5.80-5.90 (m,
3H, H-2′, H-6′, and H-3), 6.55-6.80 (m, 5H, H-4′, H-5, H-6, and
pyridine CH), 7.50-7.60 and 8.25-8.30 (m, each 1H, pyridine CH).
Anal. (C24H29N5O2) C, H, N.
(4-Chloro-3-nitrophenyl)(2-hydroxy-4-methoxyphenyl)metha-
none (84). A mixture of 1,3-dimethoxybenzene (0.25 g, 1.80 mmol),
4-chloro-3-nitrobenzoyl chloride (0.30 g, 1.48 mmol), and AlCl3
(0.29 g, 1.80 mmol) in an open tube was irradiated employing
microwaves (domestic microwave oven Daewoo KOR-6377) for
7 min at 57% potency. The mixture was then poured into ice-water,
and the solid obtained was filtered, washed with water, then with
Et2O, and dried to give 84 (0.45 g, 65%) as a yellow solid: mp
Methyl 5-Amino-2-[N-(3,5-dimethoxyphenyl)-N-methylamino]-
4-[4-(2-pyridinyl)-1-piperazinyl]benzoate (81). The title com-
pound was obtained starting from 80 following the general reduction
procedure (method B). After crystallization by EtOH, the title
compound was obtained as a white solid in 45% yield: mp 136-138
°C; 1H NMR (methanol-d4) δ 3.00-3.10 (m, 4H, piperazine CH2),
3.56 (s, 3H, NCH3), 3.63-3.70 (m, 13H, OCH3, CH3 and piperazine
CH2), 4.75 (s, 2H, NH2), 5.63 (d, J ) 2.1 Hz, 2H, H-2′ and H-6′),
5.78 (t, J ) 2.1 Hz, 1H, H-4′), 6.63-6.75 (m, 1H, pyridine CH),
6.84-6.90 (m, 2H, H-3 and pyridine CH), 7.26 (s, 1H, H-6),
7.53-7.63 (m, 1H, pyridine CH), 8.10-8.20 (m, 1H, pyridine CH).
5-Amino-2-[N-(3,5-dimethoxyphenyl)-N-methylamino]-4-[4-
(2-pyridinyl)-1-piperazinyl]benzoic Acid (43). LiOH·2H2O (0.02
g, 0.54 mmol) was added to a solution of compound 81 (0.13 g,
0.27 mmol) in a dioxane/H2O (3:1) mixture. The reaction mixture
was stirred at room temperature for 2 days and then poured into
water and acidified with AcOH until a precipitate formed. The solid
was filtered, dried, and purified by flash chromatography (CHCl3/
MeOH, 99:1), giving 43 (0.012 g, 11%) as a white solid: mp
1
144-145 °C; H NMR (CDCl3) δ 3.90 (s, 3H, OCH3), 6.45 (dd,
1H, J ) 2.3 and 9.0 Hz, H-5′), 6.55 (d, J ) 2.3 Hz, 1H, H-3′),
7.40 (d, J ) 9.0 Hz, 1H, H-6′), 7.65 (d, J ) 8.2 Hz, 1H, H-5), 7.80
(dd, J ) 1.9 and 8.2 Hz, 1H, H-6), 8.15 (d, J ) 1.9 Hz, H-2),
12.20 (bs, 1H, OH). GC-MS: m/z ) 307 (42) [M+], 277 (20), 261
(14), 242 (6), 207 (7), 151 (100). The qualitative analysis of NOESY
spectra showed two relevant NOE cross-peaks: 4-OCH3/H-3;
4-OCH3/H-5.
(2-Hydroxy-4-methoxyphenyl){3-nitro-4-[4-(2-pyridinyl)-1-
piperazinyl]phenyl}methanone (85). The title compound was
prepared starting from 84 by using the general procedure for the
coupling reaction (70 °C for 2 h). The solvent was concentrated
under reduced pressure and the orange oil was triturated with EtOH,
giving a solid which was filtered and dried to give 85 (0.55 g, 97%):
1
mp 145-147 °C; H NMR (CDCl3) δ 3.25-3.40 and 3.65-3.80
(m, each 4H, piperazine CH2), 3.90 (s, 3H, OCH3), 6.45 (dd, J )
2.4 and 8.8 Hz, 1H, H-5′), 6.53 (d, J ) 2.4 Hz, 1H, H-3′),
6.60-6.70 (m, 2H, pyridine CH), 7.20 (d, J ) 8.8 Hz, 1H, H-6′),
7.45-7.55 (m, 2H, H-5 and pyridine CH), 7.80 (dd, J ) 2.0 and
8.7 Hz, 1H, H-6), 8.20-8.30 (m, 2H, H-2 and pyridine CH), 12.50
(bs, 1H, OH).
1
136-138 °C; H NMR (CDCl3) 2.95-3.05 (m, 4H, piperazine
CH2), 3.20 (s, 3H, NCH3), 3.63-3.75 (m, 10H, piperazine CH2
and OCH3), 4.76 (bs, 2H, NH2), 6.00-6.10 (m, 2H, H-2′ and H-6′),
6.20-6.30 (m, 1H, H-4′), 6.63-6.75 (m, 3H, H-3 and pyridine
CH), 7.48-7.60 (m, 1H, pyridine CH), 7.65 (s, 1H, H-6), 8.23-8.25
(m, 1H, pyridine CH). Anal. (C25H29N5O4) C, H, N.
{3-Amino-4-[4-(2-pyridinyl)-1-piperazinyl]phenyl}(2,4-
dimethoxyphenyl)methanone (45). The title compound was
prepared starting from 85 and using MeI according to the general
procedure for O-alkylation to give intermediate 86 which was
reduced by the general reduction procedure (method B). After
crystallization by EtOH/Et2O, compound 45 was obtained in 57%
1-(5-Fluoro-2-nitrophenyl)-4-(2-pyridinyl)piperazine (82). A
solution of 2,4-difluoronitrobenzene (2.1 mL, 18.0 mmol), dry Et3N
(4.5 mL, 32.0 mmol), and 1-(2-pyridinyl)piperazine (4.8 mL, 31.5
mmol) in dry toluene (10 mL) was stirred at room temperature for
2.5 h. The obtained precipitate was filtered, dried, and purified by
flash chromatography (cyclohexane/EtOAc, 60:40) to give 82 (3.73
g, 58%) as an orange solid: mp 111-112 °C; 1H NMR (CDCl3) δ
3.10-3.20 and 3.65-3.80 (m, each 4H, piperazine CH2), 6.60-6.85
(m, 4H, H-6, H-4, and pyridine CH), 7.45-7.60 (m, 1H, pyridine
CH), 7.85 (dd, J ) 6.0 and 9.0 Hz, 1H, H-3), 8.15 (ddd, J ) 0.8,
2.0, and 5.0 Hz, 1H, pyridine CH). The qualitative analysis of
NOESY spectra showed one relevant NOE cross-peak: piperazine
CH2/H-6.
N-(3,5-Dimethoxyphenyl)-N-methyl-4-nitro-3-[4-(2-pyridinyl)-
1-piperazinyl]aniline (83). t-BuOK (1.12 g, 10.0 mmol) was added
portionwise to a stirred solution of N-methyl-3,5-dimethoxyaniline13
(0.83 g, 5.0 mmol) in dry DMSO (15 mL) cooled at 0 °C. A solution
of compound 82 (1.50 g, 5.00 mmol) in dry DMSO (8 mL) was
added dropwise to this mixture. After 5 min the mixture was poured
into ice-water and the solid obtained was filtered, dried, and
1
overall yield as a yellow crystalline solid: mp 141-143 °C; H
NMR (CDCl3) δ 3.00-3.20 (m, 4H, piperazine CH2), 3.60-3.75
(m, 7H, OCH3 and piperazine CH2), 3.80 (s, 3H, OCH3), 4.10 (bs,
2H, NH2), 6.50-6.60 (m, 2H, H-3′ and H-5′), 6.65-6.75 (m, 2H,
pyridine CH), 6.95 (d, J ) 8.2 Hz, 1H, H-6′), 7.20 (dd, J ) 2.0
and 8.2 Hz, 1H, H-6), 7.25-7.35 (m, 2H, H-5 and H-2), 7.45-7.55
(m, 1H, pyridine CH), 8.20 (m, 1H, pyridine CH). Anal.
(C24H26N4O3) C, H, N.
{3-Amino-4-[4-(2-pyridinyl)-1-piperazinyl]phenyl}(2-hydroxy-
4-methoxyphenyl)methanone (46). The title compound was
prepared from 85 according to the general reduction procedure
(method B). After crystallization by Et2O/EtOH compound 46 was
obtained in 60% yield as a yellow crystalline solid: mp 168-170
1
°C; H NMR (CDCl3) δ 3.00-3.20 and 3.55-3.65 (m, each 4H,
piperazine CH2), 3.80 (s, 3H, OCH3), 4.20 (bs, 2H, NH2), 6.40 (dd,
J ) 2.51 and 8.76 Hz, 1H, H-5′), 6.50 (d, J ) 2.51 Hz, 1H, H-3′),
6.65-6.80 (m, 2H, pyridine CH), 7.00-7.10 (m, 3H, H-5, H-6′,

3364 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Manfroni et al.
and pyridine CH), 7.45-7.65 (m, 2H, H-6 and H-2), 8.15-8.30
(m, 1H, pyridine CH), 12.70 (bs, 1H, OH). Anal. (C23H24N4O3) C,
H, N.
intensities of the radioactive bands corresponding to ds18:66mer
and ss18mer were quantified by densitometric scanning with
PhosphoImager.
Topoisomerase II Mediated Decatenation and Relaxation. The
topoisomerase II mediated decatenation and relaxation were
determined as previously described.32,33
5,7-Dimethoxy-1-methyl-2-{3-nitro-4-[4-(2-pyridinyl)-1-pip-
erazinyl]phenyl}-4(1H)-quinolinone (88). The title compound was
prepared starting from 8715 by using the general procedure for
coupling reaction (5 h). The addition of water gave a solid, which
was filtered, dried, and crystallized by EtOH to give 88 (0.30 g,
Acknowledgment. We thank Dr. Claudio Santi (University
of Perugia, Italy) for his help in the NOESY experiments. These
investigations were supported in part by the MIUR (PRIN 2006,
Roma, Italy). J.P. is a postdoctoral fellow of the “Fonds voor
Wetenschappelijk Onderzoek Vlaanderen”. S.Z. is the recipient
of a Fondazione Buzzati-Traverso Fellowship. We are grateful
to Roberto Bianconi, Katrien Geerts, and Marie-Henriette Stuyck
for excellent technical assistance.
1
56%) as a yellow solid: mp 215-216 °C; H NMR (CDCl3) δ
3.25-3.35 and 3.75-3.85 (m, each 4H, piperazine CH2), 3.55 (s,
3H, NCH3), 3.90 and 4.00 (s, each 3H, OCH3), 6.20 (s, 1H, H-3),
6.45 (bs, 2H, H-6 and H-8), 6.65-6.75 (m, 2H, pyridine CH), 7.20
(d, J ) 8.8 Hz, 1H, H-5′), 7.45-7.60 (m, 2H, H-6′ and pyridine
CH), 7.85 (d, J ) 2.0 Hz, 1H, H-2′), 8.20-8.25 (m, 1H, pyridine
CH).
2-{3-Amino-4-[4-(2-pyridinyl)-1-piperazinyl]phenyl}-5,7-
dimethoxy-1-methyl-4(1H)-quinolinone (47). The title compound
was prepared from 88 according to the general reduction procedure
(method B). After crystallization by EtOH, 47 was obtained in 91%
Supporting Information Available: Table containing elemental
analysis data for all the target compounds; experimental procedures
and analytical data for intermediates 49, 52-54, 56-59, 62-64,
66, 67, 71-73 and target compounds 7, 8, 10, 11, 13-15, 20-25,
28, 29, 31, 32, 41 and 42; biological protocols for HCV NS5B
assay. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
yield as a white solid: mp 133-136 °C; H NMR (DMSO-d6) δ
2.90-3.00 and 3.65-3.75 (m, each 4H, piperazine CH2), 3.50 (s,
3H, NCH3), 3.80 and 3.90 (s, each 3H, OCH3), 5.15 (s, 2H, NH2),
5.75 (s, 1H, H-3), 6.45 (d, J ) 1.6 Hz, 1H, H-6), 6.60-6.70 (m,
3H, H-8 and pyridine CH), 6.75 (d, J ) 1.6 Hz, 1H, H-2′), 6.85
(d, J ) 8.5 Hz, 1H, H-5′), 7.00 (d, J ) 8.5 Hz, 1H, H-6′), 7.50-7.60
(m, 1H, pyridine CH), 8.15 (m, 1H, pyridine CH). Anal.
(C27H29N5O3) C, H, N.
References
(1) The Global Burden of Hepatitis C Working Group. Global Burden of
Disease (GBD) for Hepatitis C. J. Clin. Pharmacol. 2004, 44, 20-
29.
(2) Pawlotsky, J.-M. Therapy of Hepatitis C: From Empiricism to
Eradication. Hepatology 2006, 43, S207–S219.
Anti-HCV Assay. Huh-5-2 cells were seeded at a density of 5
× 103 per well in a tissue culture treated white 96-well view plate
(Packard, Canberra, Canada) in complete DMEM supplemented
with 250 µg/mL G418. Following incubation for 24 h at 37 °C
(5% CO2) the medium was removed and 3-fold serial dilutions in
complete DMEM (without G418) of the test compounds were added
in a total volume of 100 µL. After 4 days of incubation at 37 °C,
cell culture medium was removed and luciferase activity was
determined using the Steady-Glo luciferase assay system (Promega,
Leiden, The Netherlands); the luciferase signal was measured using
a Luminoskan Ascent (Thermo, Vantaa, Finland). The 50% effective
concentration (EC50) was defined as the concentration of compound
that reduced the luciferase signal by 50%.
Cytostatic Effect. Huh-5-2 cells were seeded at a density of 5
× 103 cells per well of a 96-well plate in complete DMEM with
the appropriate concentrations of G418. Serial dilutions of the test
compounds in complete DMEM without of G418 were added 24 h
after seeding. Cells were allowed to proliferate for 3 days at 37
°C, after which the cell number was determined by means of the
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium)/phenazinemethosulfate (MTS/PMS) method
(Promega). The 50% cytotoxic concentration (CC50) was defined
as the concentration that inhibited the proliferation of exponentially
growing cells by 50%.
Reversal of Cytostatic Effects by Guanosine. To exclude
potential inhibition of the IMPDH, both the anti-HCV and cytostatic
assays were performed in the presence of 100 µg/mL guanosine,
similar to the method described by Neyts et al.30
HCV NS5B Assay. For details, see Supporting Information.
NS3 ATPase and Helicase Assays. Recombinant HCV NS3
cloned in the pAW3 plasmid (kindly provided by Dr. M. J.
McGarvey, Department of Medicine, Imperial College School of
Medicine, London) was expressed in E. coli and purified as
described.31
(3) http://www.hcvadvocate.org/hepatitis/hepC/HCVDrugs.html.
(4) Tabarrini, O.; Manfroni, G.; Fravolini, A.; Cecchetti, V.; Sabatini, S.;
De Clercq, E.; Rozenski, J.; Canard, B.; Dutartre, H.; Paeshuyse, J.;
Neyts, J. Synthesis and Anti-BVDV Activity of Acridones as New
Potential Antiviral Agents. J. Med. Chem. 2006, 49, 2621–2627.
(5) Buckwold, V. E.; Beer, B. E.; Donis, R. O. Bovine Viral Diarrhea
Virus as a Surrogate Model of Hepatitis C Virus for the Evaluation
of Antiviral Agents. AntiViral Res. 2003, 60, 1–15.
(6) Zitzmann, N.; Mehta, A. S.; Carroue´e, S.; Butters, T. D.; Platt, F. M.;
McCauley, J.; Blumberg, B. S.; Dwek, R. A.; Block, T. M. Imino
Sugars Inhibit the Formation and Secretion of Bovine Viral Diarrhea
Virus, a Pestivirus Model of Hepatitis C Virus: Implications for the
Development of Broad Spectrum Anti-Hepatitis Virus Agents. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 11878–11882.
(7) Goldstein, H.; Schaaf, E. 5-Nitro- and 3-Nitro-2,4-dichlorobenzoic
Acids. HelV. Chim. Acta 1957, 40, 1187–1188.
(8) Cain, B. F.; Atwell, G. J. Potential Antitumor Agents. 20. Structure-
Activity-Site Relationship for the 4′-(9-acridinylamino)alkanesulfo-
nanilides. J. Med. Chem. 1976, 19, 1409–1416.
(9) Khorti, A. G. Isoacridones and Their Derivatives. I. Salts of 2-Nitro-
10-methyl-3-acridone. Zh. Org. Khim. 1989, 25, 1970–1975.
(10) Kock, E. Entstehung Halogensubstituirter Amido-Verbindungen bei
der Reduction von Nitrokohlenwasserstoffen. Chem. Ber. 1887, 20,
1567–1569.
(11) Hurst, W. G.; Thorpe, J. F. The Formation of Chlorinated Amines by
the Reduction of Nitro-Compounds. J. Chem. Soc. 1915, 104, 934–
936.
(12) Tabarrini, O.; Cecchetti, V.; Fravolini, A.; Nocentini, G.; Barzi, A.;
Sabatini, S.; Miao, H.; Sissi, C. Design and Synthesis of Modified
Quinolones as Antitumoral Acridones. J. Med. Chem. 1999, 42, 2136–
2144.
(13) Chen, H.; Farahat, M. S.; Law, K.-Y.; Whitten, D. G. Aggregation of
Surfactant Squaraine Dyes in Aqueous Solution and Microheteroge-
neous Media: Correlation of Aggregration Behavior with Molecular
Structure. J. Am. Chem. Soc. 1996, 118, 2584–2594.
(14) Marquie`, J.; Salmoria, G.; Poux, M.; Laporterie, A.; Dubac, J.; Roques,
N. Acylation and Reaction under Microwaves. Development to Large
Laboratory Scale with a Continuous-Flow Process. Ind. Eng. Chem.
Res. 2001, 40, 4485–4490.
ATPase Activity Assays. The NTPase activity was determined
as previously described.31
Helicase Assays. The NS3h assay was performed as previously
described in a 15 µL reaction volume containing 200 nM NS3, 5
mM Tris-HCl (pH 7.5), 5 mM MgCl2, 25 nM 32P-labeled partial
duplex DNA substrate, and 5 mM ATP. The mixture was incubated
for 20 min at 25 °C and then stopped with 10 µL of termination
buffer (10% (w/v) glycerol, 0.0015% (v/v) bromophenol blue,
0.0015% (w/v) xylene cyanol FF, and 10 mM EDTA). Aliquots
were analyzed on a native 8% (w/v) polyacrylamide gel. The
(15) Manfroni, G.; Gatto, B.; Tabarrini, O.; Sabatini, S.; Cecchetti, V.;
Giaretta, G.; Parolin, C.; Del Vecchio, C.; Calistri, A.; Palumbo, M.;
Fravolini, A. Synthesis and Biological Evaluation of 2-Phenylquino-
lones Targeted at Tat-TAR Recognition. Bioorg. Med. Chem. Lett.
2009, 19, 714–717.
(16) Tabarrini, O.; Massari, S.; Daelemans, D.; Stevens, M.; Manfroni, G.;
Sabatini, S.; Balzarini, J.; Cecchetti, V.; Pannecouque, C.; Fravolini,
A. Structure-Activity Relationship Study on Anti-HIV 6-Desfluoro-
quinolones. J. Med. Chem. 2008, 51, 5454–5458.

Acridones as HCV Replication Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3365
(17) Watterson, S. H.; Chen, P.; Zhao, Y.; Gu, H. H.; Murali Dhar, T. G.;
Xiao, Z.; Ballentine, S. K.; Shen, Z.; Fleener, C. A.; Rouleau, K. A.;
Obermeier, M.; Yang, Z.; McIntyre, K. W.; Shuster, D. J.; Witmer,
M.; Dambach, D.; Chao, S.; Mathur, A.; Chen, B.-C.; Barrish, J. C.;
Robl, J. A.; Townsend, R.; Iwanowicz, E. J. Acridone-Based Inhibitors
of Inosine 5′-Monophosphate Dehydrogenase: Discovery and SAR
Leading to the Identification of N-(2-(6-(4-Ethylpiperazin-1-yl)pyridin-
3-yl)propan-2-yl)-2-fluoro-9-oxo-9,10-dihydroacridine-3-carboxam-
ide (BMS-566419). J. Med. Chem. 2007, 50, 3730–3742.
(18) Dymock, B. W. Emerging Therapies for Hepatitis C Virus Infection.
Expert Opin. Emerging Drugs 2001, 6, 13–42.
(19) Nair, V.; Shu, Q. Inosine Monophosphate Dehydrogenase as a Probe
in Antiviral Drug Discovery. AntiViral Chem. Chemother. 2007, 18,
245–258.
(20) Lau, J. Y. N.; Tam, R. C.; Liang, T. J.; Hong, Z. Mechanism of Action
of Ribavirin in the Combination Treatment of Chronic HCV Infection.
Hepatology 2002, 35, 1002–1009.
(21) Zhou, S.; Liu, R.; Baroudy, B. M.; Malcolm, B. A.; Reyes, G. R. The
Effect of Ribavirin and IMPDH Inhibitors on Hepatitis C Virus
Subgenomic Replicon RNA. Virology 2003, 310, 333–342.
(22) Ramos-Casals, M.; Font, J. Mycophenolate Mofetil in Patients with
Hepatitis C Virus Infection. Lupus 2005, 14, 64–72.
(23) Henry, S. D.; Metselaar, H. J.; Lonsdale, R. C.; Kok, A.; Haagmans,
B. L.; Tilanus, H. W.; van der Laan, L. J. Mycophenolic Acid
Inhibits Hepatitis C Virus Replication and Acts in Synergy with
Cyclosporin A and Interferon-alpha. Gastroenterology 2006, 131,
1452–1462.
(24) Markland, W.; McQuaid, T. J.; Jain, J.; Kwong, A. D. Broad-Spectrum
Antiviral Activity of the IMP Dehydrogenase Inhibitor VX-497: A
Comparison with Ribavirin and Demonstration of Antiviral Additivity
with Alpha Interferon. Antimicrob. Agents Chemother. 2000, 44, 859–
866.
(25) Frick, D. N. The Hepatitis C Virus NS3 Protein: A Model RNA
Helicase and Potential Drug Target. Curr. Issues Mol. Biol. 2007, 9,
1–20.
(26) Stankiewicz-Drogon, A.; Palchykovska, L. G.; Kostina, V. G.; Alexeeva,
I. V.; Shved, A. D.; Boguszewska-Chachulska, A. M. New Acridone-4-
carboxylic Acid Derivatives as Potential Inhibitors of Hepatitis C Virus
Infection. Bioorg. Med. Chem. 2008, 16, 8846–8852.
(27) Bastow, K. F.; Itoigawa, M.; Furukawa, H.; Kashiwadaa, Y.; Bori,
I. D.; Ballas, L. M.; Lii, K.-H. Antiproliferative Actions of 7-Substi-
tuted 1,3-Dihydroxyacridones; Possible Involvement of DNA Topoi-
somerase II and Protein Kinase C as Biochemical Targets. Bioorg.
Med. Chem. 1994, 2, 1403–1411.
(28) Vance, J.; Bastow, K. F. Inhibition of DNA Topoisomerase II Catalytic
Activity by the Antiviral Agents 7-Chloro-1,3-dihydroxyacridone and
1,3,7-Trihydroxyacridone. Biochem. Pharmacol. 1999, 58, 703–708.
(29) Bastow, K. F. New Acridone Inhibitors of Human Herpes Virus
Replication. Curr. Drug Targets: Infect. Disord. 2004, 4, 323–330.
(30) Neyts, J.; Meerbach, A.; MacKenna, P.; De Clercq, E. Use of the
Yellow Fever Virus Vaccine Strain 17D for the Study of Strategies
for the Treatment of Yellow Fever Virus Infections. AntiViral Res.
1996, 30, 125–132.
(31) Maga, G.; Gemma, S.; Fattorusso, C.; Locatelli, G. A.; Butini, S.;
Persico, M.; Kukreja, G.; Romano, M. P.; Chiasserini, L.; Savini, L.;
Novellino, E.; Nacci, V.; Spadari, S.; Campiani, G. Specific Targeting
of Hepatitis C virus NS3 RNA Helicase. Discovery of the Potent and
Selective Competitive Nucleotide-Mimicking Inhibitor QU663. Bio-
chemistry 2005, 44, 9637–9644.
(32) Haldane, A.; Sullivan, D. M. DNA Topoisomerase II-Catalyzed DNA
Decatenation. In DNA Topoisomerase Protocols: Enzymology and
Drugs; Osheroff, N., Bjornsti, M.-A., Eds; Methods in Molecular
Biology, Vol. 95; Humana Press: Totowa, NJ, 2001; Vol. II, pp 13-
23.
(33) Fortune, J. M.; Osheroff, N. DNA Topoisomerase II-Catalyzed
Relaxation and Catenation of Plasmid DNA. In DNA Topoisomerase
Protocols: Enzymology and Drugs; Osheroff, N., Bjornsti, M.-A., Eds;
Methods in Molecular Biology, Vol. 95; Humana Press: Totowa, NJ,
2001; Vol. II, pp 275-281.
JM801608U