Acridone derivatives: Design, synthesis, and inhibition of breast cancer resistance protein ABCG2

Article abstract of DOI:10.1016/j.bmc.2007.02.017

The breast cancer resistance protein (BCRP, ABCG2) is among the latest discovered ABC proteins to be involved in MDR phenotype and for which only few inhibitors are known. In continuing our program aimed at discovering efficient multidrug resistance modul

Full text of DOI:10.1016/j.bmc.2007.02.017

Bioorganic & Medicinal Chemistry 15 (2007) 2892–2897
Acridone derivatives: Design, synthesis, and inhibition
of breast cancer resistance protein ABCG2
Ahcene Boumendjel,a,* Sira Macalou,b Abdelhakim Ahmed-Belkacem,b
Madeleine Blanca and Attilio Di Pietrob
aDe´partement de Pharmacochimie Mole´culaire, UMR 5063 CNRS/Universite´ Joseph Fourier-Grenoble I,
5 avenue de Verdun BP 138, 38243 Meylan, France
bInstitut de Biologie et Chimie des Prote´ines, UMR 5086 CNRS/Universite´ Lyon 1, IFR128 BioSciences Gerland-Lyon Sud,
7 passage du Vercors, 69367 Lyon Cedex 07, France
Received 20 September 2006; revised 1 February 2007; accepted 9 February 2007
Available online 13 February 2007
Abstract—The breast cancer resistance protein (BCRP, ABCG2) is among the latest discovered ABC proteins to be involved in
MDR phenotype and for which only few inhibitors are known. In continuing our program aimed at discovering efficient multidrug
resistance modulators, we conceived and synthesized new acridones as ABCG2 inhibitors. The design of target molecules was based
on earlier results dealing with ABCG2 inhibition with flavone and chromone derivatives. The human wild-type (R482) ABCG2-
transfected cells were used for rational screening of inhibitory acridones. The synthesis of target compounds, the inhibitory activity
against ABCG2, and structure–activity relationships are described. One of the acridones was even more potent than the reference
inhibitor, GF120918, as shown by its ability to inhibit mitoxantrone efflux.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
is able to transport P-gp substrates but is not inhibited
by most P-gp inhibitors, cardiovascular drugs, and
immunosuppressors.12,13 Since the discovery of BCRP
in 1998, considerable efforts have been made to conceive
inhibitors of this protein for acting in vivo as MDR
modulators.14,15 The ultimate therapeutical goal of such
agents is a co-administration with the anticancer drugs
to make the treatment more effective, with minimal drug
side-effects.
The decreased accumulation of anticancer drugs in can-
cer cells may be mediated by several membrane proteins
including the ABC (ATP-Binding Cassette) family of
transporters.1 The best studied are P-glycoprotein
(P-gp/ABCB1) encoded by the mdr1 gene2 and multi-
drug resistance protein (MRP1/ABCC1) encoded by
the mrp1 gene.3 More recently, the breast cancer resis-
tance protein ABCG2 has been described as a new drug
transporter.4–6
GF120918 (Fig. 1) is an acridone-derived compound
which inhibits both P-gp and ABCG2.16–19 The anti-
fungal agent, fumetrimorgin C,20,21 and derivatives have
been reported as specific inhibitors of ABCG2.22 In
recent reports, we have shown that 6-prenylchrysin
and piperazine chromones act as powerful and specific
inhibitors of ABCG2.23,24
The structure of ABCG2 consists of 655 aminoacids
with a molecular mass of 72.1 kDa. In contrast to
P-gp/ABCB1 and MRP1/ABCC1, ABCG2 is a half-
transporter containing only one ATP-binding site and
six transmembrane domains.7 However, ABCG2 is also
able to induce cross resistance against various mole-
cules, such as doxorubicin, topotecan, SN38, mitoxan-
trone, methotrexate, flavopiridol, zidovudine, and
lamivudine.8–11 It is interesting to highlight that ABCG2
Acridones are naturally occurring alkaloids which can
be considered as aza-analogs of xanthones.25 Acridones
have been studied as P-gp, and to a lesser extent as
MRP, inhibitors. In our continuing efforts to discover
new MDR modulators, we targeted new acridone deriv-
atives as inhibitors of the BCRP efflux pump, with the
aim to determine structure–activity relationships. The
Keywords: ABCG2; Acridones; MDR; BCRP.
*
Corresponding author. Tel.: +33 4 76 04 10 06; fax: +33 4 76 04 10
07; e-mail: Ahcene.Boumendjel@ujf-grenoble.fr
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.017

A. Boumendjel et al. / Bioorg. Med. Chem. 15 (2007) 2892–2897
2893
OMe
OMe
O
OH
O
N
N
H
MeO
O
OMe
O
N
H
tectochrysin
GF120918
O
O
R1
R2
R3
R = H, OMe
OMe
-HN-(CH )
Y =
2 2
R
N
H
N
H
O
Y
series I
series II
Figure 1. Structures of tectochrysin, GF120918, and the targeted acridones.
target compounds were designed from the following
observations: (a) acridones, as represented by
GF120918, have proved to be interesting inhibitors of
the ABCG2 pump; (b) in our previous studies related
to the investigation of flavonoids as potent MDR mod-
ulators, we have reported that tectochrysin (5-hydroxy-
7-methoxyflavone) was an efficient inhibitor of
ABCG2.23 Having these elements in hand we conceived
two series of acridones (Fig. 1): series I to check the sub-
stitution pattern found in tectochrysin, and series II
related to the structure of GF120918 with targeted acri-
dones having simple and easily accessible carboxamides
at the 4-position.
agents27,28 led us to investigate a number of structurally
related acridones as potential inhibitors, with the aim to
identify MDR modulators and to establish structure–ac-
tivity relationships for the inhibition of mitoxantrone
efflux by ABCG2-transfected cells. The GF120918-sensi-
tive drug efflux activity of human wild-type (R482)
ABCG2-transfected cells was used for rational screening
of inhibitory acridones and establishment of structure–
activity relationships.
The inhibitory activity against ABCG2 is reported in
Table 1, expressed as IC50 values. One of the first target-
ed acridone compounds was the 1-hydroxy-3-methoxy-
acridone (2c), which possesses
a
comparable
substitution pattern as tectochrysin (Fig. 1), but this
compound turned out to be inactive. However, methyl-
ation of the 5-OH group (1c) was highly beneficial since
the IC50 dropped down to 7.9 2.8 lM. The 1,3-posi-
tion of the two methoxy groups (present in acridone
1c) seemed to be important, since the 1,2-methoxylation
(acridone 1d) dramatically decreased the activity. It is
worthwhile mentioning that the presence of a single
methoxy group at C-2 (1b) was even more beneficial
than the two methoxy groups at C-1 and C-3 (1c). As
shown in Table 1, the perturbation of the substitution
pattern of 1c (compounds 2a–2d) was harmful for the
inhibition activity.
2. Chemistry
Acridones of series I were prepared in two steps (Scheme
1): condensation of 2-chlorobenzoic acid with substitut-
ed aniline in the presence of copper as a catalyst, and
then cyclization under acidic conditions according to
Tercio et al.26 The selective demethylation of methoxyl
group adjacent (ortho-position) to the carbonyl group
was conducted in the presence of AlCl3 in acetonitrile.
When the methoxyl group was not at the ortho-position,
then the demethylation required the use of BBr3 in
CH2Cl2.
The preparation of acridones of series II required the
synthesis of carboxylic acid 3 (Scheme 1). The latter
was obtained by reacting 2-chlorobenzoic or 2-chloro-
4-methoxybenzoic acid with 2-aminobenzoic acid
according to the same procedure mentioned above.
Finally, target acridones (4) were obtained by coupling
acids 3 with conveniently substituted amines in the pres-
ence of DCC as a coupling agent.
The highest activity of this series was reached with
1-methoxy-3-trifluoromethyl acridone (1e) (IC50
=
0.77 0.07 lM). However, all these acridones were less
efficient than GF120918 both in terms of affinity
(IC50 = 0.41 0.21 lM) and maximal inhibition (one-
fifth to one-third lower). Here again, attempts to deme-
thylate the methoxy group (2d) led to a complete loss of
activity and hence confirmed the importance of a meth-
oxy group at position C-1. In order to check the N–H
role, we N-methylated the most active compounds in
this series and found that N-methylation led to inactive
compounds. These results indicated that the N–H func-
tion was probably acting as a hydrogen-bond donor.
This assumption was supported by the fact that the
3. Results and discussion
Due to its recent discovery, we know only few inhibitors
of ABCG2.13 Our extensive studies on MDR-reversal

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A. Boumendjel et al. / Bioorg. Med. Chem. 15 (2007) 2892–2897
Series I:
R1
R1
O
R2
R3
R2
R3
COOH
a, b
+
Cl
O
N
H
H2N
1
O
c
OMe
OH
N
H
N
H
2
Series II :
O
COOH
a, b
+
Cl
R
H2N
R
N
H
CO2H
O
CO2H
3
R = H, OMe
d
R
N
H
O
4
X
Scheme 1. Reagents and conditions: (a) Cu, K2CO3, isoamylic alcohol, 160 °C, 10 h; (b) H3PO4/P2O5, 110 °C, 3 h; (c) i—AlCl3, CH3CN, 110 °C, 14 h
(for demethylation of OMe at 1-position); ii—BBr3, CH2Cl2, 24 h (demethylation of OMe at other positions); (d) H2NR2, DCC, Et3N.
replacement of the N–H by an oxygen to obtain
xanthones led to completely inactive compounds (results
not shown).
4. Experimental
4.1. Materials and methods
In series II, the coupling of a methoxylated phenethyl
group led to acridone analogs with IC50 values in the
submicromolar range, comparable to GF120918
(0.41 0.21 lM). Table 1 and Figure 2 show that (4c)
1H and 13C NMR spectra were recorded on a Bruker
1
AC-200 (200 MHz for H, 50 MHz for 13C) and Bruker
AC-400 instrument (400 MHz for 1H, 100 MHz for
13C). Chemical shifts are reported as d values (ppm)
relative to Me4Si as an internal standard. EI and DCI
mass spectra were recorded on a Fisons Trio 1000
instrument. Elemental analyses were performed by the
Analytical Department of CNRS, Vernaison, France.
Thin-layer chromatography (TLC) was carried out
using Merck silica gel F-254 plates (thickness
0.25 mm). Flash chromatography was carried out using
Merck silica gel 60, 200–400 mesh. All solvents were dis-
tilled prior to use. Chemicals and reagents were obtained
either from ALDRICH or ACROS companies and used
as obtained.
even
induced
a
higher
maximal
inhibition
(112.3 8.9%), with respect to (4a) (71.3 12.0%), indi-
cating a positive effect of the methoxy group at position
6. This might be compared to the increased inhibition
observed for Ko143 versus Ko134.29 In contrast, a
second methoxy group at position 3 of the lateral phenyl
group (4d) was detrimental for affinity (IC50
=
1.38 0.22 lM) and also for maximal inhibition
(94.9 6.1%). The role played by a monomethoxyphen-
ethyl and a dimethoxyphenethyl group was already
reported in an earlier study with benzopyranone deriva-
tives, showing the requirement for at least one methoxy
group.24 Methoxylation at the left-aromatic ring seemed
not to be important. When the monomethoxy or
dimethoxyphenethyl group was linked to the carbonyl
group via a piperazine unit, fluorescent compounds were
obtained.
4.1.1. 1,3-Dimethoxy-10H-acridin-9-one (1c). A mixture
of 2-chlorobenzoic acid (1.56 g, 10 mmol), 3,5-dime-
thoxyaniline (1.53 g, 10 mmol), powdered copper
(25 mg), and K2CO3 (1.5 g) in isoamylic alcohol
(10 mL) was heated at 160 °C for 10 h. After cooling,
the alcohol was evaporated under vacuum, and the res-
idue was dissolved in hot water (120 mL) and acidified
with concentrated HCl. The precipitate was washed with
hot water, dissolved in ethyl acetate, and the solution
was dried over Na2SO4 and then evaporated. The crude
product was purified over silica gel chromatography
On the basis of the structure–activity elements drawn
from the present study, it can be concluded that it is pos-
sible to conceive simple and potent ABCG2 inhibitors.
The easy and short synthesis of such compounds makes
them attractive for further development.

A. Boumendjel et al. / Bioorg. Med. Chem. 15 (2007) 2892–2897
2895
Table 1. Inhibition of mitoxantrone efflux by wild type ABCG2-transfected cells
R1
O
R2
R3
N
H
R1
R2
R3
IC50 (lM)
% maximal inhibition versus GF120918a
a
Compound
1a
1b
1c
1d
1e
2a
2b
2c
2d
OMe
H
H
H
6.3 2.1
4.8 2.3
7.9 2.8
>20
74 7.2
66 6.6
65 7.0
OMe
H
H
OMe
OMe
OMe
OH
H
OMe
H
OMe
H
CF3
H
0.77 0.07
>20
>20
81 8.2
H
OH
H
H
OH
OH
OMe
CF3
>20
>20
H
O
R
N
H
O
X
a
Compound
X
R
H
IC50 (lM)
% maximal inhibition versus GF120918a
71.3 12.
OMe
4a
0.35 0.16
0.45 0.07
HN
HN
OMe
4b
H
90.2 4.9
OMe
OMe
4c
OMe
OMe
0.56 0.16
1.38 0.22
0.41 0.21
112.3 8.9
94.9 6.1
(100)
HN
OMe
OMe
4d
HN
GF120918
—
a The values are averages from three-independent experiments.
column eluted with ethylacetate/hexane (1:1) to obtain
acridone 1c as a yellow powder.
The cyclization was performed as following:26 a solu-
tion of P2O5 (3.75 g) in H3PO4 (1.5 mL) was heated
under N2 steam at 110 °C during 1 h. To the latter
solution was added the compound to be cyclized,
and the solution was heated at 110 °C for 3 h. After
cooling, ice was added and the mixture was alkalin-
ized by adding NaOH (3 N). A beige precipitate was
formed; it was washed with H2O and crystallized in
H2O–CH3OH. (53% yield); mp (H2O/CH3OH) 259–
1
261 °C; H NMR (DMSO-d6) : d 11.36 (s, 1H); 8.08
(d, 1H, J = 8 Hz); 7.60 (t, 1H, J = 8.4 Hz); 7.37 (d,
1H, J = 8 Hz); 7.16 (t, 1H, J = 7.2 Hz); 6.45 (d, 1H,
J = 2 Hz); 6.24 (d, 1H, J = 2 Hz); 3.86 (s, 3H); 3,82
(s, 3H). MS (EI): m/z 255 [M]+Å. Anal. Calcd for
C15H13NO3: C, 70.58; H, 5.13; N, 5.49. Found: C,
70.49; H, 5.01; 5.39.
Figure 2. Concentration-dependent mitoxantrone intracellular accu-
mulation as a result of ABCG2-mediated efflux inhibition. Effects of
(4a), (4c), and (4d) as compared to GF120918.

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A. Boumendjel et al. / Bioorg. Med. Chem. 15 (2007) 2892–2897
1
4.1.2. 1-Hydroxy-3-methoxy-10H-acridin-9-one (2c).30
To a solution of 1c (0.64 g, 2.5 mmol) in acetonitrile
(90 mL) was added AlCl3 (9 g), and the mixture was
heated at 110 °C for 14 h. After cooling to room temper-
ature, the solution was poured into HCl (2%, 180 mL)
and heated at 80 °C for 30 min. The solution was evap-
orated to around 200 mL. The precipitate formed was
washed with H2O and crystallized in EtOH (95%).
(50% yield); mp 233–235 °C (EtOH); 1H NMR
(DMSO-d6): d 11.97 (s, 1H); 8.17 (dd, 1H, J1 = 8 Hz,
J2 = 1.2 Hz); 7.74 (dt, 1H, J1 = 1.6 Hz, J2 = 7.2 Hz);
7.49 (d, 1H, J = 8.4 Hz); 7.29 (dt, 1H, J1 = 0.8 Hz,
J2 = 7.2 Hz); 6.38 (d, 1H, J = 2.4 Hz); 6.16 (d, 1H,
J = 2.4 Hz); 3.85 (s, 3H). MS (EI): m/z 241 [M]+Å. Anal.
Calcd for C14H11NO3: C, 69.70; H, 4.60; N, 5.81.
Found: C, 69.66; H, 4.52; N, 5.77.
221 °C; H NMR (DMSO-d6): d 11.48 (s, 1H); 10.47
(1H, s); 8.16 (d, 1H, J = 8.4 Hz); 8.05 (d, 1H,
J = 8.8 Hz); 7.65 (t, 1H, J = 8 Hz); 7.44 (d, 1H,
J = 8 Hz); 7.19 (t, 1H, J = 8 Hz); 6.78 (s, 1H); 6.71 (d,
1H, J = 8.8 Hz); 3.37 (s, 3H). MS (EI): m/z 211 [M]+Å.
Anal. Calcd for C13H9NO2: C, 73.92; H, 4.29; N, 6.63.
Found: C, 73.84; H, 4.19; N, 6.54.
4.1.7.
1-Methoxy-3-trifluoromethyl-10H-acridin-9-one
(1e). Yield 31%; mp 259–261 °C; 1H NMR (DMSO-
d6): d 11.97 (s, 1H); 8.17 (d, 1H, J = 8 Hz); 7.71 (t, 1H,
J = 8 Hz); 7.49 (d, 1H, J = 8.4 Hz); 7.28 (t, 1H,
J = 8 Hz); 7.22 (d, 1H, J = 2.4 Hz); 7.18 (d, 1H,
J = 2.4 Hz); 3.95 (s, 3H). MS (EI): m/z 293 [M]+Å. Anal.
Calcd for C15H10F3NO2: C, 61.44; H, 3.44; N, 4.78.
Found: C, 61.37; H, 3.30; N, 4.70.
4.1.3. 2-Methoxy-10H-acridin-9-one (1b). It was pre-
pared by following the same procedure as for 1c. (27%
yield); mp > 270 °C (H2O–CH3OH); 1H NMR
(DMSO-d6): d 11.78 (s, 1H); 8.23 (dd, 1H, J1 = 8.4 Hz,
J2 = 1.6 Hz); 7.69 (dt, 1H, J1 = 1.6 Hz, J2 = 6.8 Hz);
7.63 (d, 1H, J = 2.8 Hz); 7.54–7.50 (m, 2H); 7.41 (dd,
1H, J1 = 2.8 Hz, J2 = 8.8 Hz); 7.22 (dt, 1H,
J1 = 1.3 Hz, J2 = 6.8 Hz); 3.88 (s, 3H). MS (EI): m/z
225 [M]+Å. Anal. Calcd for C14H11NO2: C, 74.65; H,
4.92; N, 6.22. Found: C, 74.53; H, 4.85; N, 6.13.
4.1.8.
1-Hydroxy-3-trifluoromethyl-10H-acridin-9-one
(2d). A solution of 1e (0.23 g, 0.7 mmol) in HBr (48%,
2.5 mL) was refluxed for 24 h under a N2 steam, then
added to ice water and alkalinized with NaHCO3. The
precipitate formed was chromatographed over silica
gel column eluted with ethyl acetate/hexane 1:1. (20%
1
yield); mp 259–261 °C; H NMR (DMSO-d6): d 12.47
(s, 1H); 8.22 (d, 1H, J = 8 Hz); 7.84 (t, 1H,
J = 7.2 Hz); 7.58 (d, 1H, J = 8.4 Hz); 7.37 (t, 1H,
J = 7.2 Hz); 7.22 (s, 1H); 6.74 (s, 1H). MS (EI): m/z
279 [M]+Å. Anal. Calcd for C14H8F3NO2: C, 60.22; H,
2.89; N, 5.02. Found: C, 60.11; H, 2.80; N, 4.97.
4.1.4. 2-Hydroxy-10H-acridin-9-one (2b). It was prepared
starting from 1b as follows. To an ice cooled solution of 1b
(0.45 g, 2 mmol) in CH2Cl2(10 mL) was slowly added
BBr3 (10 mL from a 1 M solution in CH2Cl2). The mix-
ture was stirred at room temperature for 24 h and then
added to ice water which led to the formation of green pre-
cipitate. The latter was filtered off, washed with water, and
the dried under vacuum to yield 2b as a yellow powder.
(54% yield); mp > 270 °C; 1H NMR (DMSO-d6): d
11.64 (s, 1H); 8.19 (d, 1H, J = 7.6 Hz); 7.67 (t, 1H,
J = 7.2 Hz); 7.55 (d, 1H, J = 2.8 Hz); 7.50 (d, 1H,
J = 8.4 Hz); 7.46 (d, 1H, J = 8.8 Hz); 7.28 (dd, 1H,
J1 = 2.4 Hz, J2 = 8.8 Hz); 7.19 (t, 1H, J = 8 Hz). MS
(EI): m/z 211 [M]+Å. Anal. Calcd for C13H9NO2: C,
73.92; H, 4.29; N, 6.63. Found: C, 73.89; H, 4.22; N, 6.57.
4.1.9. 1,2-Dimethoxy-10H-acridin-9-one (1d). Yield 40%;
mp > 270 °C; 1H NMR (DMSO-d6): d 8.20 (d, 1H,
J = 7.6 Hz); 7.66 (t, 1H, J = 8.4 Hz); 7.57 (s, 1H); 7.49
(d, 1H, J = 8.4 Hz); 7.21 (t, 1H, J = 7.6 Hz); 6.97 (s,
1H); 3.92 (s, 3H); 3.85 (s, 3H). MS (EI): m/z 255
[M]+Å. Anal. Calcd for C15H13NO3: C, 70.58; H, 5.13;
N, 5.49. Found: C, 70.56; H, 5.07; N, 5.42.
4.1.10. 9-Oxo-9,10-dihydroacridine-4-carboxylic acid 4-
methoxyphenethyl amide (4a). Yield 5%; mp 225–227 °C;
1H NMR (CDCl3): d 8.63 (d, 1H, J = 8 Hz); 8.45 (d, 1H,
J = 8 Hz); 7.75-7.67 (m, 2H); 7.43 (d, 1H, J = 8.4 Hz);
7.32–7.15 (m, 4H); 6.91–6.86 (m, 2H); 6.57 (br s, 1H);
3.85–3.76 (m, 2H); 3.81 (s, 3H); 2.96 (t, 2H,
J = 6.8 Hz). MS (EI): m/z 372 [M]+Å. Anal. Calcd for
C23H20N2O3: C, 74.18; H, 5.41; N, 7.41. Found: C,
74.00; H, 5.33; N, 7.41.
4.1.5. 1-Methoxy-10H-acridin-9-one (1a). It was pre-
pared following the same procedure as for 1c. The final
compound was purified by silica gel column chromato-
graphy eluted with ethyl acetate:hexane 1:1 to yield 1a
as a yellow powder. Yield 41%; mp 267–269 °C; 1H
NMR (DMSO-d6): d 11.62 (s, 1H); 8.19 (d, 1H, J = 8
Hz); 8.13 (d, 1 H, J = 8.8 Hz); 7.69 (t, 1H, J = 8.4 Hz);
7.48 (d, 1H, J = 8 Hz); 7.23 (t, 1H, J = 8 Hz); 6.89 (d,
1H, J = 2.4 Hz); 6.86 (dd, 1H, J1 = 2.4 Hz,
J2 = 8.8 Hz); 3.95 (s, 3H). MS (EI): m/z 225 [M]+Å. Anal.
Calcd for C14H11NO2: C, 74.65; H, 4.92; N, 6.22. Found
: C, 74.56; H, 4.88; N, 6.09.
4.1.11. 9-Oxo-9,10-dihydroacridine-4-carboxylic acid 3,4-
dimethoxyphenethyl amide (4b). Yield 7%; mp 205–
207 °C; H NMR (CDCl3): d 8.60 (d, 1H, J = 8 Hz);
8.44 (d, 1H, J = 8 Hz); 7.78 (d, 1H, J = 7.2 Hz); 7.70
(t, 1H, J = 8 Hz); 7.43 (d, 1H, J = 8.4 Hz); 7.31 (d, 1H,
J = 8 Hz); 7.14 (t, 1H, J = 7.6 Hz); 6.86–6.80 (m, 2H);
6.74 (br s, 1H); 3.88 (s, 3H); 3.87 (s, 3H); 3.82–3.77
(m, 2H); 2.97 (t, 2H, J = 6.8 Hz). MS (EI): m/z 402
[M]+Å. Anal. Calcd for C24H22N2O4: C, 71.63; H, 5.51;
N, 6.96. Found: C, 71.55; H, 5.39; N, 6.92.
1
4.1.6. 1-Hydroxy-10H-acridin-9-one (2a). This com-
pound was prepared starting from 1a and following
the same procedure as for 2b. After treatment with ice,
the product was extracted with ethyl acetate and the res-
idue was chromatographed over silica gel column eluted
with ethyl acetate/hexane 1:1. Yield 71%; mp 219–
4.1.12. 6-Methoxy-9-oxo-9,10-dihydroacridine-carboxyl-
ic acid-(4-methoxyphenethyl) amide (4c). Yield 30%; mp
219–221 °C; H NMR (CDCl3): d 12.20 (s, 1H); 8.60
(d, 1H, J = 8 Hz); 3.39 (d, 1H, J = 8 Hz); 7.75 (br s,
1

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1H); 7.30–6.70 (m, 7H); 3.95 (br s, 2H); 3.80 (s, 6H); 3.0
(br s, 2H). MS (EI): m/z 402 [M]+Å. Anal. Calcd for
C24H22N2O4: C, 71.63; H, 5.51; N, 6.96. Found: C,
71.49; H, 5.38; N, 6.89.
4.1.13. 6-Methoxy-9-oxo-9,10-dihydroacridine-carboxy-
lic acid-(3,4-dimethoxyphenethyl) amide (4d). Yield
18%; mp 177–179 °C; 1H NMR (CDCl3): d 12.24 (s,
1H); 8.60 (d, 1H, J = 8 Hz); 8.34 (d, 1H, J = 8 Hz);
7.73 (d, 1H, J = 7.6 Hz); 7.12 (t, 1H, J = 7.6 Hz); 6.89–
6.75 (m, 5H); 3.98 (s, 3H); 3.87 (s, 6H); 3.69 (m, 2H);
2.97 (t, 1H, J = 6.4 Hz). MS (EI): m/z 432 [M]+Å. Anal.
Calcd for C25H24N2O5: C, 69.43; H, 5.59; N, 6.48.
Found: C, 69.30; H, 5.47; N, 6.43.
4.1.14. Flow cytometry. HEK-293 cells, transfected by
either ABCG2 or the empty vector (control cells), were
incubated with 5 lmol/L mitoxantrone for 30 min at
37 °C in the presence or absence of various concentra-
tions of inhibitors added as DMSO solutions (0.5% final
concentration), washed in PBS, and incubated in sub-
strate-free medium with the same inhibitor concentration
for 1 h. Intracellular drug fluorescence was monitored
with a FACscan flow cytometer (Becton–Dickinson,
Mountain View, CA). The maximal fluorescence
(100%) was the difference between mean fluorescence of
control cells and ABCG2-transfected cells, incubated
with substrate but without inhibitor. The highest inhibi-
tor concentration tested did not significantly modify the
fluorescence of control cells. Cells without drug were
included as an autofluorescence control.
15. Jekerle, V.; Klinkhammer, W.; Scollard, D. A.; Breitbach,
K.; Reilly, R. M.; Piquette-Miller, M.; Wiese, M. Int.
J. Cancer 2006, 119, 414.
16. Wallstab, A.; Koester, M.; Bohme, M.; Keppler, D. Br.
J. Cancer 1999, 79, 1053.
17. Traunecker, H. C. L.; Stevens, M. C. G.; Kerr, D. J.;
Ferry, D. R. Br. J. Cancer 1999, 81, 942.
18. Evers, R.; Kool, M.; Smith, A. J.; van Deemter, L.; de
Haas, M.; Borst, P. Br. J. Cancer 2000, 83, 366.
19. Krishnegowda, G.; Thimmaiah, P.; Hegde, R.; Dass, C.;
Houghton, P. J.; Thimmaiah, K. N. Bioorg. Med. Chem.
2002, 10, 2367.
Acknowledgments
20. Rabindran, S. K.; He, H.; Singh, M.; Brown, E.; Collins,
K. I.; Annable, T.; Greenberger, L. M. Cancer Res. 1998,
58, 5850.
This work was supported by grants from the CNRS and
´
´
´
ˆ
Universite Lyon 1 (UMR 5086), the Region Rhone-
Alpes (thematique prioritaire Cancer), the Ligue Natio-
21. Rabindran, S. K.; Ross, D. D.; Doyle, L. A.; Yang, W.;
Greenberger, L. M. Cancer Res. 2000, 60, 47.
22. van Loevezijn, A.; Allen, J. D.; Schinkel, A. H.; Koomen,
G.-J. Bioorg. Med. Chem. Lett. 2001, 11, 29.
23. Ahmed-Belkacem, A.; Pozza, A.; Munoz-Martinez, F.;
Bates, S. E.; Castanys, S.; Gamarro, F.; Di Pietro, A.;
Perez-Victoria, J. M. Cancer Res. 2005, 65, 4852.
24. Boumendjel, A.; Nicolle, E.; Moraux, T.; Gerby, B.;
Blanc, M.; Ronot, X.; Boutonnat, J. J. Med. Chem. 2005,
48, 7275.
´
ˆ
nale contre le Cancer (comites de Loire, Drome et
Savoie), and the Association pour la Recherche sur le
Cancer (ARC). S.M. and A.A.-B. were recipient of
doctoral fellowships from the Ligue Nationale contre
´
le Cancer (comite de Loire) and the ARC, respectively.
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