Synthesis and biological evaluation of a series of flavone derivatives as potential radioligands for imaging the multidrug resistance-associated protein 1 (ABCC1/MRP1)

Article abstract of DOI:10.1016/j.bmc.2005.10.009

Multidrug resistance (MDR) is one of the major problems affecting the treatment of cancer. In vivo visualization and quantification of MDR proteins would be of great value to better select the therapeutic strategy. Six flavone-based compounds were synthesized and evaluated for their cytotoxic activity and MDR-reversing capacity using hMRP1 or hMDR1 overexpressing cell lines for in vitro assays. All the flavone derivatives were highly selective for hMRP1-expressing cell lines. These derivatives each used at 4 μM (a non-cytotoxic concentration) enhance significantly the sensitivity of hMRP1-mediated MDR cell line toward doxorubicin toxicity. Their MDR-reversing capacity suggests that, in particular, the 4′-fluoroalkyloxy and 4′-iodo apigenin derivatives are potential new radiopharmaceuticals to visualize in vivo MRP1-mediated MDR phenomenon by PET or SPECT.

Full text of DOI:10.1016/j.bmc.2005.10.009

Bioorganic & Medicinal Chemistry 14 (2006) 1599–1607
Synthesis and biological evaluation of a series of flavone
derivatives as potential radioligands for imaging the
multidrug resistance-associated protein 1 (ABCC1/MRP1)
Sylvie Mavel,a,* Branko Dikic,b Somchit Palakas,c, Patrick Emond,a Ivan Greguric,b
Adrienne Gomez de Gracia,c Filomena Mattner,b Manuel Garrigos,c
Denis Guilloteaua and Andrew Katsifisb
aUniversite´ Franc¸ois Rabelais, Faculte´ de Pharmacie, Lab. de Biophysique Me´dicale et Pharmaceutique,
INSERM U619, 31 av. Monge, 37200 Tours, France
bANSTO, Radiopharmaceuticals Research Institute, New Illawarra Road Lucas Heights, NSW 2234, Australia
cUnite´ de Recherche Associe´e 2096 (CNRS), Service de Biophysique des Fonctions Membranaires (DBJC, CEA),
CEA-Saclay, 91191 Gif-sur-Yvette cedex, France
Received 13 July 2005; revised 5 October 2005; accepted 6 October 2005
Available online 2 November 2005
Abstract—Multidrug resistance (MDR) is one of the major problems affecting the treatment of cancer. In vivo visualization and
quantification of MDR proteins would be of great value to better select the therapeutic strategy. Six flavone-based compounds were
synthesized and evaluated for their cytotoxic activity and MDR-reversing capacity using hMRP1 or hMDR1 overexpressing cell
lines for in vitro assays. All the flavone derivatives were highly selective for hMRP1-expressing cell lines. These derivatives each used
at 4 lM (a non-cytotoxic concentration) enhance significantly the sensitivity of hMRP1-mediated MDR cell line toward doxorubi-
cin toxicity. Their MDR-reversing capacity suggests that, in particular, the 40-fluoroalkyloxy and 40-iodo apigenin derivatives are
potential new radiopharmaceuticals to visualize in vivo MRP1-mediated MDR phenomenon by PET or SPECT.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ers. In particular, flavone-based compounds have been
shown in vitro to modulate the binding affinity of nucle-
otide on cytosolic domains of MDR1.3,4 However, dif-
ferences in binding affinities were observed between the
various classes of flavonoid compounds. It seems that
halogen or long alkyl chain on the B-ring of chalcones
or flavones increases the binding affinities toward the
NBD2 recombinant domain of hMDR1.5,6 In addition,
flavone-based compounds have been shown in vitro to
modulate the hMRP1 transport and ATPase activi-
ties.7–10 In this respect, apigenin is among the most
potent flavone-based modulator. Therefore, we antici-
pated that apigenin-based compounds might be good
candidates to target hMDR1 or hMRP1 and have syn-
thesized a series of apigenin derivatives, introducing
iodine or fluorine atoms in order to be potentially used
as imaging agents to monitor MDR phenotype in tumors
by single-photon emission tomography (SPECT) or pos-
itron emission tomography (PET). At this moment, most
studies are performed with 99mTc-labeled substrates11 as
SPECT radiotracers. However, the interest in studying
Multidrug resistance (MDR) is a major problem in the
treatment of cancer. This phenomenon is in part medi-
ated by the overexpression of plasma membrane trans-
porters like the P-glycoprotein1 (MDR1 or ABCB1) or
the multidrug resistance-associated protein2 (MRP1 or
ABCC1) which both possess a broad capacity to trans-
port compounds of chemical diversity. It is of great
interest to identify MDR cells and the development of
radiopharmaceuticals targeted toward MDR1 and/or
MRP1 should be useful for improving chemotherapeutic
strategies. A great number of compounds belonging to
various chemical structural classes have been demon-
strated to interact with these two membrane transport-
Keywords: Halogenated apigenin derivatives; Flavone; Multidrug
resistance; MRP1.
*
Corresponding author. Tel.: +33 2 47 36 72 20; fax: +33 2 47 36 72
24; e-mail: sylvie.mavel@univ-tours.fr
S.P. is the recipient of a Thai Research Program Doctoral Award.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.009

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S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
MDR with PET is increasing, and thus the development
of a new radiopharmaceutical labeled with 11C or 18F is
being pursued.
lective methoxymethylation of the 2- and 4-hydroxyls
was achieved with the use of the weak base K2CO3 to give
the bis-MOM ether,12 whereas the triMOM-ether 1b re-
quired the addition of the stronger base, NaOH, and the
phase transfer catalyst tetrabutyl ammonium chloride.13
The chalcones 3a–e were prepared in high yield by base-
catalyzed condensation of the protected acetophenone
1b with the substituted benzaldehydes 2a–e. Hydrolysis
of the MOM-protecting groups with dilute HCl in meth-
anol followed by cyclization with solid sodium acetate un-
der reflux gave the desired dihydro-flavonoid derivatives
4a–e. Oxidation of the crude intermediate with iodine in
pyridine14 led to the target flavones 5a–e (Scheme 1).
Thereafter, we evaluate their putative interaction with
either hMDR1 or hMRP1 through their respective ability
to reverse the transporter-mediated MDR phenotype
using cell cytotoxic assays performed on well-character-
ized cell lines expressing either human MDR1
(hMDR1-NIH3T3) or human MRP1 (GLC4/Adr). We
found that among the apigenin derivatives studied most
of them appear to modulate selectively the hMRP1-asso-
ciated MDR.
The styrylchromone was prepared by a Claisen condensa-
tion of the 2-hydroxyacetophenone 1a and the cinnamic
ester 2f, giving the b-diketone 3f as a non-purified inter-
mediate (Scheme 2). The 1H NMR spectra of this interme-
diate 3f revealed that it was a mixture of tautomers.15,16
Cyclodehydration and simultaneous deprotection of dim-
ethoxymethylated diketone with amberlyst acidic cation
exchange resin in refluxing propan-2-ol16 gave 5f in 82%
yield.
2. Results
2.1. Synthesis
To prepare the desired compounds in high yields, the
hydroxyl groups in the starting acetophenone were pro-
tected as the MOM-ether derivatives (1) in a step-wise
manner with methoxymethyl (MOM) chloride. Regiose-
HO
OH
O
HO
a
O
O
O
O
O
1a
HO
b
R
R
MOMO
OMOM
O
O
OMOM
O
MOMO
O
O
HO
HO
R
R
H
+
e
f
g
MOMO
1b
O
O
OH
MOMO
OH
3a: R = 4-O(CH2)9F
5a: R = 4-O(CH2)9F
4a: R = 4-O(CH2)9F
2a: R = 4-O(CH2)9F
3b: R = 4-O(CH2)10
3c: R = 4-Br
F
5b: R = 4-O(CH2)10
5c: R = 4-Br
F
4b: R = 4-O(CH2)10
4c: R = 4-Br
F
2b: R = 4-O(CH2)10
2c: R = 4-Br
F
d
5d: R = 3-Br
3d: R = 3-Br
4d: R = 3-Br
2d: R = 3-Br
3e : R = 4-I
5e: R = 4-I
4e: R = 4-I
2e : R = 4-I
O
(CH2)n
HO
H
n =9
n= 10
O
c
HO
H
O
Scheme 1. Synthesis of flavones 5a–e. Reagents and conditions: (a) MOMCl, K2CO3, acetone, reflux; (b) tetrabutyl ammonium chloride, NaOH,
MOMCl; (c) K2CO3, HO(CH2)nBr (n = 9, 10), DMF, reflux; (d) PBSF/NEt3(HF)3/iPr2NEt (1.2/1.3/3.8 equiv, respectively), MeCN, rt; (e) KOH,
DMF, 0 ꢁC; (f) HCl, MeOH, reflux, NaOAc, reflux; (g) I2, pyridine, reflux.

S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
1601
MOMO
OMOM
O
O
O
HO
OMOM
MOMO
b
a
MeO
+
OH
OH
O
OH
O
O
3f
5f
1a
2f
Scheme 2. Synthesis of the styrene derivative 5f. Reagents and conditions: (a) NaH, THF, reflux; (b) amberlyst, iC3H5OH, reflux.
2.2. In vitro evaluation
derivatives and from chemosensitization experiments
in which these compounds were tested at a non-toxic
concentration in combination with several concentra-
tions of doxorubicin or colchicine depending on the cell
Tables 1 and 2 summarize the cytotoxicity data from
dose–response curves obtained for each of the flavone
Table 1. Determination of compounds 5a–f toxicity for parental GLC4 and hMRP1-expressing GLC4/Adr cells
a
Compound added
IC50 (lM)
GLC4
RRc
1
GLC4/Adr
RRc
340
Doxorubicin (DOX) (clogPd2.29)
MK571 (clogPd 6.13)
5a (clogPd 6.99)
0.009 0.003b
3.1 0.3
37 14
25
12
2
4
17
>100
3
5b (clogPd 7.52)
>100
22
5c (clogPd 4.42)
2
36 13
26
30 10
>50
5d(clogPd 4.42)
29 2.5
23
7
5e (clogPd 4.69)
2
5f (clogPd 3.76)
>50
DOX + MK571 (10 lM)
DOX + 5a (4 lM)
DOX + 5b (4 lM)
DOX + 5c (4 lM)
DOX + 5d (4 lM)
DOX + 5e (4 lM)
DOX + 5f (4 lM)
0.009 0.003
0.0095 0.001
0.009 0.001
0.0085 0.002
0.0095 0.001
0.011 0.002
0.009 0.001
1
1
1
1
1
1
1
0.70 0.09
1.5 0.1
1.1 0.1
0.70 0.06
1.1 0.1
1.0 0.1
1.4 0.1
78
167
122
78
122
111
156
a Cell viability was determined by the MTT assay, IC50 is the compound concentration value (lM) required to kill 50% of cells tested.
b Values are means SD of at least three experiments.
c RR: relative resistance (doxorubicin IC50 values for GLC4 or CLC4/Adr cells in the absence or presence of a nontoxic concentration of another
compound were divided by the doxorubicin IC50 value for GLC4).
d Calculated log P (clogP, ChemDraw Ultra 6.0.1, 2000).
Table 2. Determination of compound 5a–f toxicity for parental NIH3T3 and hMDR1-expressing NIH3T3 cells
a
Compound added
IC50 (lM)
NIH3T3
RRc
1
hMDR1-NIH3T3
RRc
21
Colchicine (COL) (clogPd1.03)
Cyclosporine A (clogPd 14.0)
5a (clogPd 6.99)
0.0017 0.0002b
0.036 0.002
9.1 0.35
6
9
0.4
1
9
10
20
30
20
40
1
1
2
3
2
4
5b (clogPd 7.52)
10
20
30
20
40
1
5c (clogPd 4.42)
2
5d(clogPd 4.42)
3
5e (clogPd 4.69)
2
5f (clogPd 3.76)
4
COL + cyclosporine A (1 lM)
COL + 5a (0.3 lM)
COL + 5b (0.3 lM)
COL + 5c (1 lM)
COL + 5d (1 lM)
COL + 5e (1 lM)
0.0006 0.00003
0.0019 0.0001
0.0017 0.0001
0.0017 0.0001
0.0019 0.0001
0.0017 0.0002
0.0015 0.0001
0.3
1
0.0035 0.0001
0.036 0.003
0.036 0.002
0.046 0.004
0.042 0.003
0.042 0.003
0.036 0.002
6
21
21
27
25
25
21
1
1
1
1
COL + 5f (1 lM)
1
a Cell viability was determined by the MTT assay, IC50 is the compound concentration value (lM) required to kill 50% of cells tested.
b Values are means SD of at least three experiments.
c RR: relative resistance (colchicine IC50 values for NIH3T3 or hMDR1-NIH3T3 cells in the absence or presence of a nontoxic concentration of
another compound were divided by the colchicine IC50 value for NIH3T3).
d Calculated logP (clogP, ChemDraw Ultra 6.0.1, 2000).

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S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
line. For each flavone derivative tested sensitive and
resistant cells of each cell type share similar chemosensi-
tive profiles (see Tables 1 and 2). The simplest explana-
tion for these results is that the flavone derivatives tested
are probably not transported by hMDR1 or hMRP1 be-
cause if they were one would expect to observe a relative
resistance in MDR cells compared to the sensitive cells
which is not the case (see Tables 1 and 2). Since the bind-
ing of a molecule to a MDR transporter can take place
without necessarily the bound molecule being transport-
ed, as exemplified by many MDR modulators, we first
examined the effect of each flavone derivative on colchi-
cine sensitivity of hMDR1-NIH3T3 cells that express
human MDR1 but not MRP1. For this purpose,
hMDR1-NIH3T3 cells were cultured in the continuous
presence of a non-toxic concentration of each flavone
derivative and treated for three days with increasing
concentrations of colchicine. The results presented in
Table 2 indicate that none of the flavone derivatives test-
ed was able to significantly sensitize hMDR1-NIH3T3
cells to colchicine. Moreover, control experiments dem-
onstrate that none of these flavone derivatives had any
significant effect on the colchicine chemosensitivity of
the parental NIH3T3 cells (Table 2). In contrast, we ob-
served that each flavone derivative added to the culture
medium at a non-toxic concentration was able to sensi-
tize GLC4/Adr cells to doxorubicin, with the 40-bromo
flavone being the most potent modulator (5c, Table 1).
Incubation of GLC4/Adr cells with the 40-bromo fla-
vone 5c (at 4 lM) caused a fourfold decrease in the
doxorubicin IC50 value (see Table 1). This enhancement
of doxorubicin toxicity is similar to the one observed
when a non-toxic concentration of MK571 (at 10 lM)
is added to the cell culture medium (see Table 1). None
of the flavone derivatives tested neither MK571 (at
10 lM) had any significant effect on the doxorubicin
toxicity toward the parental GLC4 cells (see Table 1),
suggesting a role for hMRP1 in determining the capacity
of these apigenin derivatives to enhance the toxicity of
doxorubicin.
the field of MDR, are its binding to the cytosolic C-termi-
nal nucleotide-binding domain (NBD2) of mouse MDR1
or Leishmania ltrmdr1.3,4 Thus even if we cannot rule out
the possibility that some of the apigenin derivatives
investigated in the present study might interact with
hMDR1, it remains that this putative interaction is likely
not interfering with either colchicine binding or efflux
since colchicine resistance of hMDR1-NIH3T3 cells is
not perturbed by the presence of either of these com-
pounds. Concerning MRP1-mediated resistance, our re-
sults are reminiscent of a recent study performed on
hMRP1 transfected HeLa cells in which it was found that
hMRP1 does not confer resistance to a series of bioflavo-
noids and in particular to apigenin.20 The fact that these
two studies were performed on different hMRP1-express-
ing cell types gives strength to the common conclusion
that apigenin and the apigenin derivatives tested in the
present study are likely not transported by hMRP1. In
this respect, direct transport studies using radiolabeled
flavone derivatives would certainly be helpful in drawing
a definitive conclusion. Interestingly, we found a reduc-
tion in long-term viability of hMRP1-expressing cells
treated with apigenin derivatives (4 lM) when compared
to their sensitive counterpart. Evidently, this potentia-
tion of doxorubicin toxicity was not a result of non-spe-
cific membrane perturbation induced by either of these
flavone derivatives, since it was not observed for sensitive
cell lines or hMDR1-expressing cell line. In particular,
compound 5c (at 4 lM) appeared to sensitize hMRP1-ex-
pressing cells to doxorubicin as efficiently as MK571 (at
10 lM). It seems clear that growth inhibition was
brought about by modulation of hMRP1 function by
each of the various flavone derivatives studied and raises
questions concerning the mechanism(s) involved. It can
be noticed that, independently of the chemical modifica-
tions introduced in the B ring of compounds 5a–e, it
seems that the common apigenin backbone of these five
molecules represents a structural pattern which precludes
the transport of these compounds by either hMDR1 or
hMRP1. This is an interesting structural property com-
mon to these halogenated apigenin derivatives because
if they turn out to bind selectively to hMRP1 they will
stay associated longer than if they were expelled from
the cell by hMRP1 therefore making easier the observa-
tion of hMRP1-related MDR by SPECT or PET.
3. Discussion
In the present study, we have investigated the effects of a
series of new synthesized flavone derivatives on mem-
brane-mediated drug resistance. Growth inhibition as-
says with continuous exposure to various compounds
were performed using two well-characterized MDR cell
lines to test whether the hMDR1 or hMRP1 membrane
proteins can efflux these compounds and therefore confer
resistance to these cells when compared with their sensi-
tive counterpart. We found that hMDR1 as well as
hMRP1 was unable to confer cross-resistance to the six
synthesized compounds which suggests that these flavone
derivatives are likely not effluxed by either of these two
transporters. Flavonoids have long been investigated as
a class of MDR1 modulators and molecules belonging
to various flavonoid chemical subclasses were demon-
strated to bind with high affinity to MDR1.17–19 Howev-
er, to our knowledge, the flavone apigenin has not been
reported to modulate drug accumulation in hMDR1 can-
cer cells. The only data available regarding apigenin, in
4. Conclusion
In conclusion, according to cytotoxicity experiments, we
found that halogenated apigenin derivatives constituted
a class of hMRP1-related MDR modulators. These data
open the way for investigation in new potential radio-
pharmaceuticals (5a,b as potent radiofluorinated, 5e can
be radioiodinated) in the field of MDR imaging. The rel-
atively short and easy synthesis of these molecules makes
them potential candidates for further development.
5. Experimental
General details: NMR spectra were recorded on a
Bruker 400 MHz spectrometer. Mass spectra were

S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
1603
performed on a Waters Micromass 2MD Quadropole.
Elemental analysis was performed by Microanalytical
and the analytical results obtained were within 0.4%
of the theoretical values.
CH2), 25.8, 28.9, 29.0, 29.1, 29.3 (5CH2), 30.3 (d,
J = 19 Hz, CH2), 68.3 (CH2), 84.0 (d, J = 164 Hz,
CH2), 114.7 (2CHar), 129.7 (Car), 131.9 (2CHar), 164.2
(Car), 190.7 (CHO). MS: (ES+): 267.37.
All chemicals were obtained from Sigma Aldrich unless
stated otherwise. Doxorubicin was dissolved at 5 mM in
ethanol:PBS (v/v). All the drug stock solutions were
stored at 4 ꢁC and diluted in culture medium before use.
5.3.2. 4-(10-Fluorodecyloxy)benzaldehyde 2b. Compound
2b was purified by column chromatography (silica gel,
ethyl acetate–hexane: 30/70) to afford a white solid.
Yield (81%). 1H NMR (CD3CN): d 1.36–1.50 (m,
6CH2), 1.68–1.75 (m, CH2), 1.80 (m, CH2), 4.09 (t,
J = 6.2 Hz, CH2), 4.46 (dt, J = 47.6, 6.2 Hz, CH2F),
7.08 (d, J = 8.75 Hz, 2Har), 7.86 (d, J = 8.75 Hz, 2Har),
9.89 (s, CHO). 13C NMR (CD3CN): 26.1 (d, J = 5 Hz,
CH2), 26.8, 29.9, 30.1, 30.2, 30.4, 30.4 (6CH2), 31.3 (d,
J = 19 Hz, CH2), 69.6 (CH2), 84.3 (d, J = 162 Hz,
CH2), 116.0 (2CHar), 131.1 (Car), 132.9 (2CHar), 165.4
(Car), 192.1 (CHO). MS: (ES+): 281.43.
5.1. 2-Hydroxy-4,6-di-O-methoxymethylacetophenone 1a
and 2,4,6-tri-O-methoxymethylacetophenone 1b
Compounds 1a,b were synthesized as previously
described.12,21
5.2. 4-(Hydroxyalkyloxy)benzaldehydes. General method
To 9-bromononan-1-ol (20.9 g, 0.1 mol) or 10-bromod-
ecan-1-ol (20.9 g, 0.1 mol) in dry DMF (40 mL) were
added 4-hydroxybenzaldehyde (12.2 g, 0.1 mol) and
dry K2CO3 (20.7 g, 0.15 mol) and refluxed overnight un-
der N2. The DMF was evaporated under reduced pres-
sure, diluted with H2O (50 mL), extracted with DCM
(5 · 20 mL), dried over anhydrous MgSO4, and filtered.
The solvent was evaporated to dryness and the crude
product was purified by column chromatography (silica
gel, ethyl acetate–hexane: 30/70) to afford a white solid.
5.4. 1-(2,4,6-Tris(methoxymethyl)phenyl)-3-(substituted-
phenyl)prop-2-enones 3a–e. General method
To the 4-substituted benzaldehydes 2a–e (0.1 mol) was
added
2,4,6-tri-O-methoxymethylacetophenone
1b
(28.63, 0.1 mol) in DMF (100 mL) at 0 ꢁC followed by
powdered KOH (8.42 g, 0.15 mol). After stirring for
4 h at 0 ꢁC, the reaction mixture was diluted with H2O
(250 mL) and extracted with DCM (100 mL · 3). The
organic layer was washed with H2O (50 mL · 2), dried
over anhydrous MgSO4, and filtered. The product was
purified by column chromatography (silica gel, ethyl
acetate–hexane: 30/70) to afford a pale yellow solid.
5.2.1. 4-(9-Hydroxynonyloxy)benzaldehyde. Yield 64% 1H
NMR (CDCl3): d 1.38 (m, 4CH2), 1.49 (m, CH2), 1.61 (m,
CH2), 1.84 (m, CH2), 3.67 (t, J = 6.6 Hz, 2H, CH2), 4.07
(t, J = 6.5 Hz, 2H, CH2), 7.02 (d, J = 8.75 Hz, 2Har),
7.85 (d, J = 8.75 Hz, 2Har), 9.91 (s, CHO). 13C NMR
(CDCl3): 26.9, 27.1, 30.2, 30.4, 30.5, 30.7, 33.9, 64.2,
69.6 (9CH2), 116.0 (2CHar), 131.0 (Car), 133.2 (2CHar),
165.5 (Car), 192.0 (CHO). MS: (ES+): 265.0.
5.4.1. 1-(2,4,6-Tris(methoxymethyl)phenyl)-3-(4-(9-fluor-
ononyloxy)phenyl)prop-2-en-1-one 3a. Yield (88%). 1H
NMR (CD3CN): d 1.33–1.43 (m, 5CH2), 1.70–1.75 (m,
2CH2), 3.32 (s, 6H, OCH3), 3.46 (s, 3H, OCH3), 3.99
(t, J = 6.6 Hz, 2H, CH2), 4.41 (dt, J = 47.5, 6.2 Hz,
2H, CH2F), 5.10 (s, 4H, CH2O), 5.18 (s, 2H, CH2O),
6.52 (s, 2Har), 6.83 (d, J = 16.2 Hz, 1H, HC@C), 6.91
(d, J = 8.8 Hz, 2Har), 7.27 (d, J = 16.2 Hz, 1H, Hb),
7.52 (d, J = 8.8 Hz, 2Har). 13C NMR (CD3CN): d 26.0
(d, J = 5 Hz, CH2), 26.8, 30.0, 30.1, 30.1, 30.3 (5CH2),
31.3 (d, J = 19 Hz, CH2), 56.8 (2OCH3), 56.8 (OCH3),
69.3 (CH2), 85.3 (d, J = 164 Hz, CH2), 95.7 (2OCH2),
95.7 (OCH2), 98.2 (2CHar), 116.1 (2CHar), 116.2 (Car),
127.8 (HC@C), 128.2 (Car), 131.4 (2CHar), 146.3
(CHb), 156.7 (Car), 160.5 (2Car), 162.5 (Car), 194.8
(C@O). MS: (ES+): 549.37.
5.2.2. 4-(10-Hydroxydecyloxy)benzaldehyde. Yield 55%
1H NMR (CDCl3): d 1.28 (m, 5CH2), 1.48 (m, CH2),
1.59 (m, CH2), 1.80 (m, CH2), 3.67 (t, J = 6.6 Hz, 2H,
CH2), 4.07 (t, J = 6.6 Hz, 2H, CH2), 7.02 (d, J = 8.5 Hz,
2Har), 7.86 (d, J = 8.5 Hz, 2Har), 9.91 (s, CHO). 13C
NMR (CDCl3): d 26.9, 27.1, 30.2, 30.5, 30.6, 30.6, 30.7,
34.0, 64.3, 69.6 (10CH2), 115.96 (Car), 130.9 (2CHar),
133.2 (Car), 165.5 (2CHar), 192.0 (CHO). MS: (ES+):
279.0.
5.3. 4-(Fluoroalkyloxy)benzaldehydes 2a,b. General
method
5.4.2. 3-(4-(10-Fluorodecyloxy)phenyl)-1-(2,4,6-tris(meth-
oxymethy)phenyl)prop-2-enone 3b. Yield (81%). 1H NMR
(CD3CN): d 1.38–1.44 (m, 6CH2), 1.60–1.75 (m, 2CH2),
3.32 (s, 6H, OCH3), 3.46 (s, 3H, OCH3), 3.99 (t, J = 6.8,
2H, CH2), 4.41 (dt, J = 47.6, 6.0 Hz, 2H, CH2F), 5.09 (s,
4H, CH2O), 5.18 (s, 2H, CH2O), 6.53 (s, 2Har), 6.83 (d,
J = 16.2 Hz, 1H, HC@C), 6.90 (d, J = 8.8 Hz, 2Har),
7.27 (d, J = 16.2 Hz, 1H, C@CHb), 7.52 (d, J = 8.8 Hz,
2Har). 13C NMR (CD3CN): d 26.51 (d, J = 6Hz, CH2),
27.2, 30.4, 30.5, 30.6, 30.8, 30.8 (6CH2), 31.7 (d,
J = 19 Hz, CH2), 57.3 (2OCH3), 57.3 (OCH3), 69.7
(CH2), 85.8 (d, J = 162 Hz, CH2F), 96.1 (2CH2O), 96.2
(CH2O), 98.6 (2CHar), 116.5 (2CHar), 116.6 (Car), 128.2
(HC@C), 128.6 (Car), 131.8 (2CHar), 146.7 (CHb), 157.1
4-(9-Fluorononyloxy)benzaldehyde 2a and 4-(10-fluo-
rodecyloxy)benzaldehyde 2b were synthesized as per
the method of Yin et al.22
5.3.1. 4-(9-Fluorononyloxy)benzaldehyde 2a. Compound
2a was purified by column chromatography (silica gel,
ethyl acetate–hexane: 30/70) to afford a white solid.
Yield (88%). 1H NMR (MeOH-d4): d 1.38–1.51 (m,
5CH2), 1.68 (m, CH2), 1.81 (m, CH2), 4.08 (t,
J = 6.1 Hz, CH2), 4.41 (dt, J = 47.6, 6.1 Hz, CH2F),
7.07 (d, J = 8.7 Hz, 2Har), 7.85 (d, J = 8.7 Hz, 2Har),
9.83 (s, CHO). 13C NMR (CDCl3): 25.1 (d, J = 5.3 Hz,

1604
S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
(Car), 161.0 (2Car), 162.9 (Car), 195.27 (C@O). MS: (ES+):
563.38.
13.50 (s, OH). 13C NMR (CDCl3): 56.2, 56.5 (2CH3),
93.9 (CH2), 94.9 (CHenol), 94.9 (CH2), 97.6, 102.9 (C-
3, C-5), 106.8 (Car), 122.8 (Ca), 127.7 (C-20, C-60),
128.7 (C-30, C-50), 130.7 (C-40), 135.1 (Car), 138.6 (Cb),
159.1 (Car), 162.8 (Car), 166.2 (Car), 173.4 (Car), 193.9
(Car).
5.4.3. 1-(2,4,6-Tris(methoxymethoxy)phenyl)-3-(4-brom-
ophenyl)prop-2-enone 3c. Yield (63%). 1H NMR
(CD3CN): d 3.36 (s, 6H, OCH3), 3.49 (s, 3H, OCH3),
5.13 (s, 4H, OCH2), 5.23 (s, 2H, OCH2), 6.56 (s, 2Har),
7.01 (d, J = 16.2 Hz, 1H, HC@C), 7.34 (d, J = 16.2 Hz,
1H, C=CHb), 7.55 (d, J = 8.6 Hz, 2Har), 7.6 (d,
J = 8.6 Hz, 2Har). 13C NMR (CD3CN): d 56.4 (2OCH3),
56.3 (OCH3), 95.4 (2OCH2), 95.6 (OCH2), 98.0 (2CHar),
115.6 (Car), 125.1 (Car), 130.5 (HC@C), 131.1 (2CHar),
133.1 (2CHar), 135.0 (Car), 144.4 (CHb), 156.6 (2Car),
160.6 (Car), 194.6 (C@O). MS: (ES+): 467.20/470.22.
5.6. 2-(Substituted-phenyl)-2,3-dihydro-5,7-dihydroxy-
chromen-4-ones 4a–e. General method
To the substituted 1-(2,4,6-Tris(methoxymethyl)phenyl)-
3-(substituted-phenyl)prop-2-enone 3a–e (0.0032 mol) in
MeOH (60 mL) was added 10% HCl (6 mL) and the
mixture was refluxed for 1 h. Then NaOAc (5.3 g,
0.064 mol) was added and then the resulting mixture
was refluxed for 3 h. The mixture was cooled and H2O
(50 mL) added and extracted with EtOAc (50 mL · 2),
dried over anhydrous MgSO4, filtered, and evaporated
to dryness. The solid was purified by column chromatog-
raphy (silica gel, ethyl acetate–hexane: 30/70) to afford a
white solid.
5.4.4. 1-(2,4,6-Tris(methoxymethoxy)phenyl)-3-(3-brom-
ophenyl)prop-2-enone 3d. Yield (63%). 1H NMR (CDCl3):
d 3.36 (s, 6H, OCH3), 3.49 (s, 3H, OCH3), 5.15 (s, 4H,
OCH2), 5.23 (s, 2H, OCH2), 6.56 (s, 2Har), 7.02 (d,
J = 8.5 Hz, 1H, CHa), 7.33 (d, J = 8.5 Hz, 1H, CHb),
7.36 (t, J = 7.6 Hz, 1Har), 7.60 (t, J = 6.8 Hz, 1Har), 7.65
(t, J = 6.8 Hz, 1Har), 7.84 (s, 1Har). 13C NMR (CD3CN):
d 56.3 (2OCH3), 56.4 (OCH3), 95.4 (OCH2), 95.6
(2OCH2), 97.9 (2CHar), 115.03 (Car), 123.85 (Car), 128.0
(CHar), 131.0 (CHa), 131.6 (CHar), 131.9 (CHar), 134.2
(CHar), 138.1 (Car), 145.0 (CHb), 157.1 (2Car), 161.2
(Car), 196.3 (C@O). MS: (ES+): 467.19/469.19.
5.6.1. 2-(4-(9-Fluorononyloxy)phenyl)-2,3-dihydro-5,7-dihydr-
1
oxychromen-4-one 4a. Yield 55%. H NMR (DMSO-d6): d
1.3–1.41 (m, 5CH2), 1.57–1.71 (m, 2CH2), 2.71 (dd,
J = 17.1, 3.1 Hz, 1H, H-3), 3.25 (dd, J = 17.1, 12.6 Hz,
1H, H-3), 3.97 (t, J = 6.6 Hz, CH2), 4.42 (dt, J = 44.6,
6.1 Hz, CH2F), 5.50 (dd, J = 12.7, 3.0 Hz, 1H, H-2), 5.89
(s, 1Har), 5.89 (s, 1Har) 6.95 (d, J = 8.7 Hz, 2Har), 7.41 (d,
J = 8.7 Hz, 2Har), 10.78 (s, OH). 13C NMR (DMSO-d6):
24.5 (d, J = 5 Hz, CH2), 25.3, 28.4, 28.5, 28.5, 28.7
(5CH2), 29.7 (d, J = 19 Hz, CH2), 41.9 (C-3), 67.4 (CH2),
78.1 (C-2), 83.7 (d, J = 162 Hz, CH2F), 94.9 (CHar), 95.7
(CHar), 101.7 (Car), 114.2 (2CHar), 128.1 (2CHar), 130.3
(Car), 158.7 (Car), 162.7 (Car), 163.4 (Car), 166.5 (Car),
196.1 (CO). MS: (ES+): 431.3.
5.4.5. 1-(2,4,6-Tris(methoxymethoxy)phenyl)-3-(4-iodophe-
nyl)prop-2-enone 3e. Yield (57%). 1H NMR (DMSO-d6): d
3.28 (s, 6H, OCH3), 3.43 (s, 3H, OCH3), 5.15 (s, 4H,
OCH2), 5.22 (s, 2H, OCH2), 6.54 (s, 2Har), 7.07 (d,
J = 16.2 Hz, 1H, CHa), 7.24 (d, J = 16.2 Hz, 1H, CHb),
7.50 (d, J = 8.4 Hz, 2Har), 7.78 (d, J = 8.4 Hz, 2Har). 13
C
NMR (DMSO-d6): d 55.8 (2OCH3), 55.9 (OCH3), 94.1
(2OCH2), 94.0 (OCH2), 96.6 (2CHar), 97.66 (Car), 114.2
(Car), 129.3 (CHa), 130.3 (2CHar), 133.8 (2CHar), 137.7
(Car) 143.2 (CHb), 155.1 (2Car), 158.9 (Car), 193.1
(C@O). MS: (ES+): 515.15.
5.6.2. 2-(4-(10-Fluorodecyloxy)phenyl)-2,3-dihydro-5,7-
dihydroxychromen-4-one 4b. Yield 50%. 1H NMR
(DMSO-d6): d 1.22–1.45 (m, 6CH2), 1.56–1.71 (m,
2CH2), 2.71 (dd, J = 17.2, 3.2 Hz, 1H, H-3), 3.26 (dd,
J = 17.2, 12.6 Hz, 1H, H-3), 3.97 (t, J = 6.3 Hz, CH2),
4.41 (dt, J = 47.6, 6.1 Hz, CH2F), 5.50 (dd, J = 12.6,
2.9 Hz, H-2), 5.89 (s, 1Har), 5.89 (s, 1Har), 6.95 (d,
J = 8.7 Hz, 2Har), 7.41 (d, J = 8.7 Hz, 2Har), 10.78 (s,
OH). 13C NMR (DMSO-d6): d 24.6 (d, J = 5 Hz,
CH2), 25.4, 28.6, 28.6, 28.7, 28.8, 28.8 (6CH2), 29.8 (d,
J = 19 Hz, CH2), 42.0 (C-3), 67.5 (CH2), 78.2 (C-2),
83.7 (d, J = 162 Hz, CH2F), 95.0 (CHar), 95.8 (CHar),
101.8 (Car), 114.3 (2CHar), 128.2 (2CHar), 130.4 (Car),
158.9 (Car), 162.8 (Car), 163.4 (Car), 166.6 (Car), 196.2
(CO). MS: (ES+): 417.3.
5.5. 1-(2-Hydroxy-4,6-di-O-methoxymethylphenyl)-5-
phenyl-1,3-pent-4-enedione 3f
A solution of the ester 2f (4.05 g, 25 mmol) in anhydrous
THF (10 mL) was added to a solution of NaH (60% in
mineral oil 1.6 g, 40 mmol) in anhydrous THF (10 mL)
under nitrogen. After stirring under reflux for 15 min,
the mixture was treated dropwise with a solution of 2-
hydroxy-4,6-di-O-methoxymethylacetophenone
1a
(229 mg, 10 mmol) in anhydrous THF (15 mL) over
30 min. The mixture was heated to reflux and monitored
by TLC until the reaction was complete (4 h). The
cooled mixture was hydrolyzed with H2O (200 mL),
then acidified with HCl diluted and extracted with
CH2Cl2. The organic phase was dried and evaporated
to dryness. The diketone 3f was recrystallized from
methanol in 72% yield.
5.6.3. 2-(4-Bromophenyl)-2,3-dihydro-5,7-dihydroxychro-
1
men-4-one 4c. Yield 55%. H NMR (DMSO-d6): d 2.78
(dd, J = 17.1, 3.2 Hz, 1H, H-3), 3.21 (dd, J = 17.1,
12.6 Hz, 1H, H-3), 5.57 (dd, J = 12.6, 3.1 Hz, 1H, H-
2), 5.90 (s, 1Har), 5.92 (s, 1Har), 7.48 (d, J = 8.5 Hz,
2Har), 7.63 (d, J = 8.5 Hz, 2Har), 10.83 (br s, OH). 13C
NMR (DMSO-d6): d 41.8 (CH2-3), 77.5 (CH-2), 94.9
(CHar), 95.9 (CHar), 101.7 (Car), 121.6 (C–Br), 128.7
(2CHar), 131.4 (2CHar), 138.0 (Car), 162.4 (Car), 163.4
(Car), 166.6 (Car), 195.6 (CO). MS: (ESꢀ): 335.1/336.1.
The enol derivative: 1H NMR (CDCl3): d 3.52 (s, CH3),
3.60 (s, CH3), 5.22 (s, CH2), 5.33 (s, CH2), 6.32 (d,
J = 2.4 Hz, H-3), 6.34 (d, J = 2.4 Hz, H-5), 6.58 (d,
J = 15.6 Hz, Hb), 6.81 (s, CHenol), 7.41–7.45 (m,
3Har), 7.58–7.69 (m, 3H, Ha, 2Har), 13.24 (s, OH),

S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
1605
5.6.4. 2-(3-Bromophenyl)-2,3-dihydro-5,7-dihydroxychro-
men-4-one 4d. Yield 50%. H NMR (DMSO-d6): d 2.79
30.6, 34.2 (8CH2), 64.2, 69.6 (2CH2), 95.7 (C-8), 100.6
(C-6), 105.1, 105.4 (C-3, C-4a), 116.7 (C-30, C-50),
124.3 (C-10), 130.0 (C-20–C-60), 159.0, 163.1, 163.3,
165.0, 166.0 (C-8a, C-5, C-2, C-7, C-40), 183.4 (C-4).
MS: (ES+): 429.42.
1
(dd, J = 17.1, 12.7 Hz, 1H, H-3), 3.26 (dd, J = 17.1,
3.2 Hz, 1H, H-3), 5.59 (dd, J = 12.7, 3.2 Hz, 1H, H-
2), 5.91 (d, J = 2.1 Hz, 1Har), 5.95 (d, J = 2.1Hz,
1Har), 7.39 (t, J = 7.9 Hz, 1Har), 7.52 (d, J = 7.9,
1Har), 7.58 (d, J = 8.0 Hz, 1Har), 7.74 (s, 1Har), 10.85
(br s, OH). 13C NMR (DMSO-d6): d 41.8 (C-3), 77.4
(C-2), 95.0 (CHar), 96.0 (CHar), 101.6 (Car), 121.7 (C–
Br), 125.5 (CHar), 129.2 (CHar), 130.7 (CHar), 131.3
(CHar), 141.3 (Car), 162.3 (Car), 162.4 (Car), 166.6
(Car), 195.5 (CO). MS: (ESꢀ): 335.1/336.1.
1
5.7.3. 40-Bromo-5,7-dihydroxyflavone 5c. Yield 47%. H
NMR (DMSO-d6): d 6.21 (s, H-6), 6.51 (s, H-8), 6.99
(s, H-3), 7.76 (d, J = 8.6 Hz, H-30, 50), 8.00 (d,
J = 8.6 Hz, H-20, 60), 10.95 (br s, 7-OH), 12.75 (s, 5-
OH). 13C NMR (DMSO-d6): 94.1 (C-8), 99.1 (C-6),
104.0 (C-3), 105.5 (C-4a), 125.7 (C–Br), 128.3 (C-20, C-
60), 129.9 (C-10), 132.1 (C-30, C-50), 157.3, 161.4, 162.0,
164.5 (C-8a, C-5, C-2, C-7), 181.8 (C-4). MS: (ESꢀ):
333.15/334.16.
5.6.5. 2-(4-Iodophenyl)-2,3-dihydro-5,7-dihydroxychro-
1
men-4-one 4e. Yield 61%. H NMR (DMSO-d6): d 2.78
(dd, J = 17.1, 3.2 Hz, 1H, H-3), 3.21 (dd, J = 17.2,
12.5 Hz, H1, H-3), 5.57 (dd, J = 12.5, 3.2 Hz, 1H, H-2),
5.90 (s, 1Har), 5.92 (s, 1Har), 7.33 (d, J = 8.4 Hz, 2Har),
7.80 (d, J = 8.4 Hz, 2Har), 10.84 (br s, OH). 13C NMR
(DMSO-d6): d 41.9 (C-3), 77.7 (C-2), 94.7 (CHar), 95.0
(CHar), 96.0 (Car), 101.7 (Car), 128.8 (2CHar), 137.3
(2CHar), 138.5 (Car), 162.5 (Car), 163.4 (Car), 166.7
(Car), 195.7 (CO). MS: (ESꢀ): 381.1.
1
5.7.4. 30-Bromo-5,7-dihydroxyflavone 5d. Yield 50%. H
NMR (DMSO-d6): d 6.23 (s, H-6), 6.57 (s, H-8), 7.06
(s, H-3), 7.52 (m, H-50), 7.80 (d, J = 8.1 Hz, H-60), 8.08
(d, J = 7.8 Hz, H-40), 8.27 (s, H-20), 10.94 (s, 7-OH),
12.75 (s, 5-OH). 13C NMR (DMSO-d6): 94.2 (C-8),
99.5 (C-6), 103.4 (C-4a), 110.3 (C-3), 121.0 (C–Br),
128.2, 131.4, 132.7, 133.1 (C-20, C-40, C-50, C-60), 133.5
(C-10), 157.8, 161.5, 163.8, 165.9 (C-8a, C-5, C-2, C-7),
181.2 (C-4). MS: (ESꢀ): 333.17/334.18.
5.7. 40-Substituted-5,7-dihydroxyflavones 5a–e. General
method
5.7.5. 40-Iodo-5,7-dihydroxyflavone 5e. Yield 44%. 1H
NMR (DMSO-d6): d 6.21 (d, J = 1.8 Hz, H-6), 6.49 (d,
J = 1.8 Hz, H-8), 6.97 (s, H-3), 7.82 (d, J = 8.6 Hz, 2H,
H-30, 50), 7.93 (d, J = 8.6 Hz, 2H, H-20, 60), 10.92 (br
s, 7-OH). 13C NMR (DMSO-d6): 94.1 (C-8), 99.0 (C-
6), 99.8 (C–I), 104.0 (C-4a), 105.4 (C-3), 128.1 (C-20,
C-40), 130.2 (C-10), 138.0 (C-50, C-60), 157.4, 161.4,
162.3, 164.5 (C-8a, C-5, C-2, C-7), 181.7 (C-4). MS:
(ESꢀ): 379.12.
To a solution of 2-(substituted-phenyl)-2,3-dihydro-5,7-
dihydroxychromen-4-one 4a–e (0.02 mol) in anhydrous
pyridine (4 mL) was added iodine (0.53 g, 0.0021 mol)
under N2 and heated to reflux for 4 h. The solution
was then cooled and diluted with H2O (50 mL) and
was extracted with ethyl acetate (100 mL · 3). The
organic layer was washed with 10% HCl (50 mL · 2),
dried over anhydrous sodium sulfate, filtered, and evap-
orated to dryness. The solid was purified by column
chromatography (silica gel, ethyl acetate–hexane: 30/
70) to afford a pale white solid.
5.8. 5,7-Dihydroxy-2-styrylchromen-4-one 5f
A mixture of the diketone 3f (1.15 g, 3 mmol) and
amberlyst-15 resin (750 mg) in propan-2-ol (15 mL)
was stirred under reflux, whilst the reaction was moni-
tored by TLC until completion (4 h). After cooling,
the mixture was diluted with propan-2-ol (40 mL), fil-
tered, evaporated under vacuum, and the crude product
was purified by column chromatography (silica gel, ethyl
acetate–hexane: 30/70) to afford white/yellow crystals of
the flavone 5f (yield 82%).
5.7.1. 40-(9-Fluorononyloxy)-5,7-dihydroxyflavone 5a.
Yield 54%. H NMR (DMSO-d6): d 1.30 (m, 5CH2),
1
1.57–1.71 (m, 2CH2), 4.03 (t, J = 6.5 Hz, CH2), 4.41 (dt,
J = 47.6 Hz, J = 6.1 Hz, CH2F), 6.19 (d, J = 1.4 Hz, H-
6), 6.48 (d, J = 1.4 Hz, H-8), 6.82 (s, H-3), 7.07 (d,
J = 8.9 Hz, 2H, H-20, 60), 7.98 (d, J = 8.9 Hz, 2H, H-30,
50), 10.83 (br s, 7-OH), 12.90 (s, 5-OH). 13C NMR
(DMSO-d6): 24.6 (d, J = 5 Hz, CH2), 25.4, 28.5, 28.5,
28.7, 28.9 (5CH2), 29.8 (d, J = 19 Hz, CH2), 67.8 (CH2),
83.8 (d, J = 162 Hz, CH2F), 94.0 (C-8), 98.9 (C-6),
103.4, 103.7 (C-3, C-4a), 114.9 (C-30, C-50), 122.6 (C-10),
128.2 (C-20-C-60), 157.3 (C-40), 161.4, 161.7, 163.3, 164.2
(C-8a, C-5, C-2, C-7,), 181.7 (C-4). MS/EI m/z 413
[M+ꢀ1, 100%], 399 [16%], 395 [9%], 333 [10%], 314, 312
[13%].
1H NMR (DMSO-d6): d 6.15 (s, 1H, H-3), 6.32 (d,
J = 1.8 Hz, 1H, H-6), 6.45 (d, J = 1.8 Hz, 1H, H-8),
6.74 (d, J = 14.6 Hz, 1H, Hb), 7.40–7.61 (m, 6H, 5Har,
Ha), 9.95 (br s, 1H, 7-OH), 12.75 (s, 1H, 5-OH). 13C
NMR (DMSO-d6): 93.7 (C-8), 99.0 (C-6), 104.4 (C-
4a), 108.2 (C-3), 119.3 (Cb), 127.2 (C-30, C-50), 128.5
(C-20-C-60), 129.4 (C-40), 134.4 (C-10), 136.6 (Ca),
157.2, 161.5, 161.7, 164.1, 181.8 (C-8a, C-5, C-2, C-7,
C-4). MS (EI) m/z 280 [M+, 100%], 262 (19%), 153
(21%), 127 (16%), 124 (20%), 96 (12%).
5.7.2. 40-(10-Fluorodecyloxy)-5,7-dihydroxyflavone 5b.
Yield 50%. 1H NMR (DMSO-d6): d 1.29–1.41 (m,
6CH2), 1.56 (m, CH2), 1.75 (m, CH2), 3.37 (t,
J = 6.4 Hz, CH2), 4.04 (dt, J = 47.6 Hz, J = 6.2 Hz,
CH2F), 6.20 (d, J = 1.4 Hz, H-6), 6.50 (d, J = 1.4 Hz,
H-8), 6.85 (s, H-3), 7.76 (d, J = 8.7 Hz, 2H, H-20, 60),
8.00 (d, J = 8.7 Hz, 2H, H-30, 50), 10.90 (br s, 7-OH),
12.90 (s, 5-OH). 13C NMR (DMSO-d6): 27.1, 30.2,
5.9. Biological evaluation: in vitro testing
5.9.1. Cell lines and culture conditions. The human GLC4
and GLC4/Adr small cell lung cancer cell lines have

1606
S. Mavel et al. / Bioorg. Med. Chem. 14 (2006) 1599–1607
been previously characterized23,24 and were kindly pro-
vided by Dr. H. J. Broxterman (Vrije Universiteit Med-
ical Centre, Amsterdam, The Netherlands) and
maintained in an exponential growth in RPMI 1640
medium supplemented with 10% heat-inactivated fetal
calf serum (FCS), penicillin G (100 U/mL), and strepto-
mycin (100 lg/mL) at 37 ꢁC in a humidified atmosphere
of 6% CO2. The GLC4/Adr cell line was cultured in the
presence of 1 lM doxorubicin. Before use in cytotoxic
experiments GLC4/Adr cells were cultured in a doxoru-
bicin-free medium for 11 days.
eral concentrations of either doxorubicin or colchicine,
the choice of the drug depending on the cell line. Fla-
vone derivatives were dissolved at 10 mM in DMSO
and dilutions prepared in culture medium. Final concen-
tration of DMSO was lower than 1% and without effect
on cell growth. To examine the effects on drug sensitivity
of either cyclosporin A (CsA) or MK571, cells were pre-
incubated with CsA (10 lM) or MK571 (10 lM) for 1 h
and then incubated with various concentrations of
drugs. Data represent means of six replicate determina-
tions and standard deviation from at least two indepen-
dent experiments.
The parental Swiss mouse embryo NIH3T3 cell line and
resistant hMDR1-NIH3T3 cell line transfected with the
human MDR1 were provided by Dr. M. M. Gottesman
(National Cancer Institute, Bethesda, MD). Both cell
lines were maintained in DulbeccoÕs modified EagleÕs
medium with 4.5 g of glucose per liter supplemented
with 10% FCS, penicillin G (50 U/mL), streptomycin
(50 lg/mL), and sodium pyruvate 1 mM at 37 ꢁC in a
humidified atmosphere of 6% CO2. The resistant cell line
was cultured in the presence of 60 ng/mL colchicine.
Acknowledgments
We thank SAVIT (Tours, France) for chemical analyses.
The authors are grateful to the Royal Thai Government
for support of S.P. This work was supported by a
French Australian Science and Technology Programme
(FAST).
5.9.2. Cell survival assay. The 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide assay (MTT) was
used to determine the cytotoxicity of each compound
in 96-well plates.25 This assay is based on the reduction
of MTT by the mitochondrial dehydrogenase of viable
cells to a blue formazan product that can be determined
spectrophotometrically. The wells situated at the periph-
ery of the microtiter plates are more subjected to evap-
oration and were not used for the cytotoxic MTT
assay but filled with 200 lL PBS. For each cell line,
equal numbers of cells were seeded into each well in a
volume of 200 lL of their respective culture media
(800 cells for GLC4; 5000 cells for GLC4/Adr; 200 cells
for NIH3T3; 300 cells for hMDR1-NIH3T3). After 12 h
incubation (37 ꢁC in a humidified atmosphere with 6%
CO2), a dose–response curve was generated for each
compound tested by adding 20 lL of compound stock
solution to each well and incubated for a further 72 h.
Nine compound concentrations were used to determine
a dose–response curve. At the end of drug exposure,
20 lL of MTT (0.5 mg/mL final concentration) was add-
ed to each well and the plates were put back in the incu-
bator for an additional 4 h. Thereafter, the microtiter
plates were centrifuged (45 min at 600g) and the culture
medium was removed by aspiration. To solubilize the
resulting formazan crystals, 200 lL of dimethylsulfoxide
(DMSO) was added in each well and the plates were
incubated at 23 ꢁC on a plate shaker for 1 h. Then the
absorbance at 540 nm was determined on a scanning
microplate reader (Labsystems Multiskan Bichromatic).
The mean absorbance of six wells for each compound
concentration was measured. IC50 was defined as being
the compound concentration that reduced this absor-
bance to 50% of control values. IC50 values were derived
by non-linear regression analysis assuming a sigmoidal
dose–response curve using SigmaPlot 2001 V.7.101 soft-
ware. Similarly, to investigate the ability of the various
compounds to chemosensitize MDR cells, each com-
pound was added at a concentration that produces less
than 5% cytoxicity and tested in combination with sev-
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