Modulation of P-glycoprotein-mediated multidrug resistance by flavonoid derivatives and analogues

Article abstract of DOI:10.1021/jm021099i

Flavonoid derivatives were synthesized and tested for their ability to modulate P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) in vitro. These compounds belong to various flavonoid subclasses, namely: chromones, azaisoflavones, and aurones. Among the investigated compounds, three showed potent reversing activity. 2-(4-Methylpiperazin-1-ylcarbonyl)-5-hydroxychromone (4a), 5,7-dimethoxy-3-phenyl-4-quinolone (5), and 4,6-dimethoxyaurone (6) potentiated daunorubicin cytotoxicity on resistant K562 cells. They were also able to increase the intracellular accumulation of rhodamine-123, a fluorescent molecule which acts as a probe of P-glycoprotein-mediated MDR. This suggests that these compounds act, at least in part, by inhibiting P-glycoprotein activity. The most active compound, 5-hydroxy-2-(4-methylpiperazin- 1-ylcarbonyl)chromone (4a) was found to be a powerful reversal agent, more potent than cyclosporin A, used as the reference molecule. No effect was observed on MRP transport nor on cell proliferation. Little apoptosis was induced on K562S cells with 4a compared to K562R, probably due to the extrusion of the compound by Pgp.

Full text of DOI:10.1021/jm021099i

J . Med. Chem. 2003, 46, 2125-2131
2125
Mod u la tion of P -Glycop r otein -Med ia ted Mu ltid r u g Resista n ce by F la von oid
Der iva tives a n d An a logu es
Mohamed Hadjeri,† Magali Barbier,‡ Xavier Ronot,‡ Anne-Marie Mariotte,† Ahce`ne Boumendjel,*,† and
J ean Boutonnat‡
De´partement de Pharmacochimie Mole´culaire, UMR CNRS 5063, Faculte´ de Pharmacie de Grenoble,
38706 La Tronche, France, and Laboratoire de Dynamique Cellulaire de l’EPHE, UMR CNRS 5525,
Institut Albert Boniot, 38706 La Tronche, France
Received November 20, 2002
Flavonoid derivatives were synthesized and tested for their ability to modulate P-glycoprotein
(Pgp)-mediated multidrug resistance (MDR) in vitro. These compounds belong to various
flavonoid subclasses, namely: chromones, azaisoflavones, and aurones. Among the investigated
compounds, three showed potent reversing activity. 2-(4-Methylpiperazin-1-ylcarbonyl)-5-
hydroxychromone (4a ), 5,7-dimethoxy-3-phenyl-4-quinolone (5), and 4,6-dimethoxyaurone (6)
potentiated daunorubicin cytotoxicity on resistant K562 cells. They were also able to increase
the intracellular accumulation of rhodamine-123, a fluorescent molecule which acts as a probe
of P-glycoprotein-mediated MDR. This suggests that these compounds act, at least in part, by
inhibiting P-glycoprotein activity. The most active compound, 5-hydroxy-2-(4-methylpiperazin-
1-ylcarbonyl)chromone (4a ) was found to be a powerful reversal agent, more potent than
cyclosporin A, used as the reference molecule. No effect was observed on MRP transport nor
on cell proliferation. Little apoptosis was induced on K562S cells with 4a compared to K562R,
probably due to the extrusion of the compound by Pgp.
In tr od u ction
The treatment of cancer with anticancer drugs is
frequently impaired by the intrinsic or acquired resis-
tance of tumor cells. Broad-spectrum resistance to
structurally and mechanistically diverse anticancer
agents constitutes the multidrug resistance (MDR)
phenotype.1 The overexpression of a particular class of
transmembrane glycoprotein, encoded by a small family
of mdr genes, is a common mechanism by which tumor
cells acquire MDR.2 The best known are P-glycoprotein
(Pgp),3 and, to a lesser extent, the multidrug resistance-
associated protein (MRP).4 Pgp which features ATPase
activity decreases the intracellular drug concentration
F igu r e 1. Chemical structure of flavonoid subclasses.
below its active threshold by rapidly pumping the
anticancer drug out of the MDR-tumor cells.5 Various
attempts are being made to develop noncytotoxic com-
pounds which will interfere with the P-gp function, thus
allowing intracellular accumulation of the cytotoxic
drug. In this context, several molecules including the
calcium channel blocker verapamil, the calmodulin
inhibitor trifluoroperazine, and cyclosporin have been
used to reverse multidrug resistance.6 However, the
clinical application of these agents have met with
limited success mainly because of adverse effects when
used at concentrations required to overcome multidrug
resistance.7 Therefore, new potent and noncytotoxic
reversal agents with minimal adverse effects are strongly
hoped for.
have been demonstrated to possess MDR reversing
activity through high affinity binding with P-gp.8-10
Isoflavones have been described as inactive on Pgp-
mediated MDR.11 Finally, aurones have never been
investigated as reversal agents. The only data available
regarding aurones, in the field of MDR, is their binding
affinity with the C-terminal nucleotide-binding domain
(NBD2) of Pgp.12 Studies of structure-activity relation-
ships made on chalcones, flavones, and flavonols have
concluded that a hydroxyl group on position 5 (position
6′ in chalcones) is important. 5-OH methylation leads
to slightly less active compounds. Because of the chelat-
ing effect induced by the adjacent carbonyl group, the
hydroxyl loses its acidic properties and therefore does
not affect the activity which can be decreased by the
presence of acidic groups.6 Hydroxylation on position 7
(position 4′ in chalcones) was deleterious for activity,
probably due to the acidic group influence, whereas
methoxylation was slightly beneficial. The 2,3 double
bond (the R,â-double bound in chalcones) and the
In this context, flavonoids were investigated as a new
class of MDR modulators (chemical structures are
shown in Figure 1). Chalcones, flavones, and flavonols
* Corresponding author. Tel.: (33) 4 76 51 86 88. Fax: (33) 4 76 63
71 65. E-mail: Ahcene.Boumendjel@ujf-grenoble.fr.
† Faculte´ de Pharmacie de Grenoble.
‡ Institut Albert Boniot.
10.1021/jm021099i CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/19/2003

2126 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Hadjeri et al.
F igu r e 2. Structure of studied molecules: piperazinylchromones 4, 3-phenyl-4-quinolone 5, and aurone 6.
Sch em e 1a
a
Reagents: (a) MeI, K2CO3, acetone, reflux, 4 h; (b) diethyl oxalate, NaOEt, EtOH, 100 °C, 10 h (c) NaHCO3 (20% in H2O), 80 °C, 3 h;
(d) HBr/AcOH, 100 °C, 2 h; (e) N-alkylpiperazine, N-acylpiperazine, piperidine, morpholine, or benzylamine, EDC, Et3N, THF, 24 h.
carbonyl group are also essential for MDR modula-
tion.13-15 On another hand, a parameter shared by many
MDR modulators is the presence of at least one basic
nitrogen. A survey on MDR modulators concluded that
this last criterion was frequently a piperazine unit.6,16-19
On the basis of these data from literature, we wanted
to report the synthesis and MDR modulation of a variety
of flavonoid derivatives, namely piperazinylcarbonyl-
chromones, 3-phenyl-4-quinolones (azaisoflavone), and
aurones (Figure 2). Piperazinylcarbonylchromones (com-
pounds 4) were designed by combining a chromone unit
(5-hydroxy-4-chromanone containing all structural ele-
ments identified among flavonoids) with a piperazine
unit, so as to obtain a synergistic effect on the MDR
modulation. 3-Phenyl-4-quinolone (azaisoflavone, 5) was
investigated as an analogue of inactive isoflavones, to
check the importance of nitrogen. Finally we report the
effect of 4,6-dimethoxyaurone (6) on MDR modulation
for the first time.
of the latter with methyl iodide gave 2-hydroxy-6-
methoxyacetophenone (1).23 Condensation of 1 with
diethyl oxalate in the presence of sodium ethoxide in
EtOH and then acidic cyclization afforded ester 2.22,24
Saponification of the ester with NaHCO3 followed by a
demethylation with HBr in AcOH provided 2-carboxy-
5-hydroxychromone (3).25 The target amides 4a -i were
synthesized by reacting acid 3 with either a piperazine
derivative, piperidine, morpholine, or a benzylamine
using EDC as a coupling agent. N-Benzoylpiperazine,
is not commercially available; therefore, it was prepared
according to a recently reported method.26
Resu lts a n d Discu ssion
In this study, we investigated compounds 4a -i, 5, 5a ,
and 6 (Figure 2 and Scheme 1). Although isoflavones
are inactive on Pgp-mediated MDR, we included isofla-
vone 5a with the aim to compare it with azaisoflavone
5. These compounds were meant to be tested in vitro to
assess their ability to restore intracellular accumulation
of daunorubicin (DNR). Since DNR cytotoxicity experi-
ments cannot assess the effect of the tested compounds
on Pgp transport activity, we started our screening by
an assay of Pgp transport function, using rhodamine-
123. The latter is a fluorescent dye and probe of Pgp
transport activity. After this, the most active compounds
on the transport activity were selected and tested for
their ability to restore intracellular accumulation of
DNR.
Ch em istr y
The synthesis of azaisoflavone 5, isoflavone 5a , and
aurone 6 (Figure 2) investigated in this study has been
already reported; therefore, it will not discussed here.20,21
The synthetic strategy chosen for the preparation of
piperazinylcarbonylchromones and analogues (4) re-
quired the preparation of 2-carboxy-5-hydroxychromone
3.22 Chromone 3 was obtained in four steps starting
from 2,6-dihydroxyacetophenone (Scheme 1). Treatment

Modulation of Pgp-Mediated Multidrug Resistance
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2127
Ta ble 1. Mean of Rhodamine-123 Fluorescence, in Arbitrary
Unit (a.u.), in K562 Cells with or without Expression of PGP
Proteina
to the number of cell divisions, and labeling with PKH67
does not alter cell cycle distribution nor cell growth.32,33
Mean PKH67 fluorescence intensity on K562S and
K562R treated with 4a , 5, or 6 was evaluated for 2 days.
The mean PKH67 fluorescence ratio (day0/day2) of
controls and cells treated with one of the compounds
revealed that there was no difference between controls
and treated cells. However, we found that the three
compounds were able to induce little apoptosis, ranging
from 30% to 35% for necrosis, and from 4% to 10% for
apoptosis, especially at 10-6 M after 2 days in K562S
and at a lesser degree on K562R.31 No cell division effect
was observed, which correlates to cell cycle results.
rhodamine-123 accumulation
at selected concentrations of compoundb-d
compound
(in K562R)
4a
4b
4c
4d
4e
4f
4g
4h
4i
5
5a
1 µM
0.1 µM
0.01 µM
1 nM
207
85
82
136
38
42
45
42
46
82
138
47
57
95
37
47
44
44
45
55
46
203
87
134
44
40
72
43
45
46
41
42
43
54
178
80
76
40
39
45
41
38
43
39
50
36
40
40
72
3. Cell Cyt ot oxicit y Assa y Usin g TOTO-3 Dye
In cor p or a tion . Compounds 4a , 5, and 6, the most
active identified by the rhodamine-123 accumulation
assay, were tested for their ability to potentiate DNR
cytotoxicity on K562S and K562R cells, which were
shown to overexpress P-glycoprotein. Flow cytometry,
a highly sensitive method, was used to detect the effect
of modulators on MDR cells in heterogeneous cell
population; potency of the compounds was compared
with that of cyclosporin A, an established Pgp inhibitor.
The dose-response curves which show the percentage
of viable cells (which correspond to cells with low level
of TOTO-3 fluorescence) are reported in Figures 3 and
4.34,35 At different concentrations, compounds 4a and 6
are much more active than cyclosporin A. Figures 3 and
4 indicate that K562S viability was reduced to 20%
when treated with DNR and cyclosporin A, 4a , or 6
compared to K562R cells which was less reduced (vi-
ability at 40%). Quinolone 5 induced a lower decrease
of cellular viability (65%) for both K562S and K562R
cells. Finally, the ability of these compounds to induce
necrosis in sensitive and resistant cells was evaluated,
and no significant difference between cyclosporin A and
4a , 5, or 6 was found. As an example and as shown in
Figure 5, a mixture of DNR and cyclosporin A at 10-6
M induced 78% of necrosis in K562S cells, where 4a
induced 82% as detected by TOTO-3. At the same
concentration, DNR and cyclosporin A induced 58% of
necrosis in K562R cells whereas a mixture of DNR and
4a induced 61% of necrosis.
49
249
102
6
cyclosporin A
a
b
Controls (without modulator): K562S, 200; K562R, 40. An
arbitrary unit (au) which represents the fluorescence intensity at
590 nm. c Results are the mean of at least 3 independent experi-
d
ments. Interexperimental variations were inferior to 25%.
1. R h od a m in e-123 Accu m u la t ion Assa y. Rhod-
amine-123 is a mitochondrial membrane potential-
sensitive fluorescent probe used for the functional assay
of Pgp-mediated cell resistance.27,28 The reversing prop-
erties were tested at four concentrations (10-6 M, 10-7
M, 10-8 M, and 10-9 M) on K562S (sensitive) and K562R
(adriamycin resistant) cells. Cyclosporin A, one of the
MDR modulators used clinically which acts on Pgp
transport activity, was used as a reference. As shown
in Table 1, the results are expressed by the mean of
rhodamine-123 fluorescence on cells expressing Pgp
(arbitrary unit, a.u). At 1 µM, except for the isoflavone
5a and benzylchromone 4i, all compounds showed a
good activity, although the maximum activity was
observed for 4a , 4d , and 6. At 0.01 µM, compounds 4a
and 6 were still strongly active and even more active
than cyclosporin A. Finally at 10 nM, N-methylpiper-
azinylcarbonylchromone (4a ) was as active as cyclospor-
in A. These compounds are Pgp specific since MRP
transport activity evaluation by using the calcein-AM
method did not show any inhibitory activity (data not
shown).29,30
2. Effect on Cell P r olifer a tion a n d Necr osis. 2.1.
Cell Cycle Assa y. To complete our study, the effect of
each compound on the cell cycle distribution of the
different phases was investigated. K562S and K562R
cell distribution in G0/G1, S, and G2+M phases was
analyzed at a 10-6 M concentration of each compound.
We concluded that there was no significant difference
between controls and cells treated with various modula-
tors.31 On the basis of the rhodamine-123 accumulation
assay and the lack of cell cycle modification, we selected
4a , 5, and 6 for further experiments.
2.2. Cell Division , Ap op tosis, a n d Necr osis Assa y.
The balance between cell proliferation and drug-induced
cell death by apoptosis or necrosis plays a major role in
determining the response to chemotherapy. To check the
balance factor, a triple labeling using PKH67, annexin
biotin (streptavidin AMCA), and propidium iodide were
used to simultaneously follow cell division, apoptosis,
and necrosis, respectively. PKH is a green fluorescent
membrane-intercalating dye which has the advantage
that its fluorescence intensity decreases proportionally
Str u ctu r e-Activity Rela tion sh ip s
The structures investigated in this study were struc-
turally modified flavonoids, and they were chosen to
answer structural criteria identified among MDR re-
versal agents. In the chromones series (compounds 4),
the accumulation assay showed that at 10-6 M, com-
pounds 4a -d enhanced rhodamine-123 accumulation
in which 4a was the most active. This higher activity
was confirmed at lower concentrations (10-8 M). At
nanomolar concentrations, 4a was still active, as well
as cyclosporin A. It seems that the nature of the
substituent on the piperazine ring was important. From
these data, we can conclude that the basicity of the
alkylated nitrogen was involved in rhodamine-123 ac-
cumulation. The stronger activity of 4d compared to 4c
might be due to the basicity of nitrogen. The nitrogen
in 4c is less basic because its lone pair is delocalized to
the phenyl ring., We synthesized N-acetyl and N-
benzoyl analogues (compounds 4e and 4f) to confirm the
importance of the basicity factor, when both nitrogen

2128 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Hadjeri et al.
F igu r e 3. Viability of K562S controls and treatment with cyclosporin A, 4a , 5, or 6 at different concentrations, after 72 h.
F igu r e 4. Viability of K562R control and treated with cyclosporin A, 4a , 5, or 6 at different concentrations, after 72 h.
F igu r e 5. Viability of K562S and K562R, controls, and treatment with cyclosporin A or 4a , at 10-6 M after 72 h, as measured
by CMF using TOTO-3 dye. Two populations were observed: viable cells with a low level of TOTO-3 fluorescence, and necrotic
cells with a high level of TOTO-3 fluorescence. (A) K562S cells treated with DNR and cyclosporin A. (B) K562S cell treated with
DNR and 4a . (C) K562R cells treated with DNR and cyclosporin A. (D) K562R cells treated with DNR and 4a .

Modulation of Pgp-Mediated Multidrug Resistance
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2129
1H, 50 MHz for 13C). Chemical shifts were reported as δ 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 Depart-
ment of CNRS, Vernaison, France. Thin-layer chromatography
(TLC) was carried out using E. 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
distilled prior to use. Chemicals and reagents were obtained
either form Aldrich or ACROS companies and used without
modification.
2-Hyd r oxy-6-m eth oxya cetop h en on e (1). The title com-
pound was prepared according to Lau et al.:23 mp 58-62 °C
(lit. 57-58 °C); 1H NMR (CDCl3): δ 13.21 (s, OH); 7.34 (t, 1H,
J ) 8.3 Hz, H4′); 6.58 (d, 1H, J ) 8.3 Hz, H5′); 6.39 (d, 1H, J
) 8.3 Hz, H3′); 3.90 (s, 3H, OCH3); 2.68 (s, 3H, H2). 13C NMR
(CDCl3): δ 205.0 (C1); 164.6 (C6′); 161.5 (C2′); 136.0 (C4′); 111.3
(C1′); 110.7 (C5′); 101.1 (C3′); 55.6 (OCH3); 33.6 (C2). MS (EI,
m/z) 166 (M+). Anal. (C9H10O3) C, H.
2-E t h yloxy-5-m et h oxych r om on e (2). A solution of 1
(10.68 g, 64.33 mmol) in ethyl oxalate (35 mL, 250 mmol) was
added to a freshly prepared solution of EtONa in EtOH
(prepared by addition of 8.87 g of sodium to 100 mL of absolute
EtOH). The mixture was stirred at 100 °C for 10 h, after which,
the mixture was cooled to room temperature, ethanol was
evaporated, and water was added. The solution was acidified
with HCl (2 M) and extracted with EtOAc. The organic layer
was separated, washed with brine, dried (Na2SO4), and
concentrated. The resulting residue was dissolved in ethanol
(50 mL) and heated at 100 °C for 15 min; concentrated HCl (5
mL) was added, and the solution was stirred at 100 °C for 1
h. Ethanol was evaporated, and the residue was kept in the
refrigerator (4 °C) overnight and yielded 11.32 g (71%) of 2,
as brown crystals: mp 125-126 °C (crystallization from
MeOH:CH2Cl2. lit. 130-131° C);24 1H NMR (CDCl3): δ 7.88 (t,
1H, J ) 8.4 Hz, H7); 7.41 (d, 1H, J ) 8.4 Hz, H8); 7.26 (s, 1H,
H3); 7.12 (d, 1H, J ) 8.1 Hz, H6); 4.71 (q, 2H, J ) 7.1 Hz, OCH2-
CH3); 4.25 (s, 3H, OCH3); 1.70 (t, 3H, J ) 7,1 Hz, OCH2CH3).
13C NMR (CDCl3): δ 177.7 (C4); 160.2 (CO2Et); 159.5 (C2); 157.7
(C5); 150.1 (C9); 134.5 (C7); 116 (C3); 114 (C10); 110.2 (C6); 106.7
(C8); 62.6 (CO2CH2CH3); 56.2 (OCH3); 13.8 (CO2CH2CH3). MS
(DCI, m/z) 249 (M + H)+. Anal. (C13H12O5) C, H.
2-Ca r boxy-5-h yd r oxych r om on e (3). Ester 2 (5 g, 20.16
mmol) was dissolved in a solution of NaHCO3 (100 mL, 20%
in H2O) and heated at 80 °C for 3 h. After cooling, the solution
was acidified with concentrated HCl and extracted with
EtOAc. The organic layer was washed with water, dried over
Na2SO4, and evaporated to yield acid as a beige powder (90%).
The latter was dissolved in 40 mL of HBr:AcOH (1:2) and
heated at 100 °C for 2 h. After being cooled to room temper-
ature, the solution was extracted with CH2Cl2. The organic
layer was washed with H2O, brine, dried over Na2SO4, and
evaporated to yield 3.61 g (87%) of 3 as a brown solid: mp
253-255 °C (lit. 264 °C);37 1H NMR (CDCl3): δ 12.2 (s, 1H,
OH); 7.75 (t, 1H, J ) 8.5 Hz, H7); 7.15 (d, 1H, J ) 8.7 Hz, H8);
6.95 (s, 1H, H3); 6.85 (d, 1H, J ) 8.3 Hz, H6). 13C NMR (CDCl3):
δ 184.1 (C4), 174.0 (CO2H); 161.6 (C2); 160.4 (C5); 154.8 (C9);
136.6 (C7); 113.6 (C3); 111.9 (C6); 108 (C8); 106.9 (C10). MS (DCI,
m/z) 207 (M + H)+. Anal. (C10H6O5) C, H.
atoms are not basic, or compounds 4g and 4h , in which
the nitrogen was replaced with a carbon (4g) or with
an oxygen (4h ). In all cases, the rhodamine-123 ac-
cumulation assay did not lead to any significant activity
(Table 1).
Substitution of the piperazine group with p-chloro-
benzylamine group (4i) led to a significantly less active
compound. Due to insolubility problems, p-chlorobenz-
ylamine derivative was the only compound tested among
a series of benzylamines. We also synthesized analogues
of 4a -d when the piperazine unit was linked to the
chromone via a methylene groupe (data not shown).
Unfortunately, these compounds were insoluble in all
systems used, therefore they were not tested.
On the basis of these data, we synthesized and tested
azaisoflavone 5 (3-phenyl-4-quinolone) and compared it
to the parent isoflavone 5a . Isoflavones were shown to
have no effect on rhodamine-123 accumulation.11 We
hypothesized that the substitution of the intracyclic
oxygen by a nitrogen would confer one of the structural
features. As shown in both assays (accumulation and
cytotoxicity), quinolone 5 was strongly active compared
to isoflavone. This stronger activity could not only be
attributed to the quinolone nitrogen atom (weakly
basic), but also to its ability of forming hydrogen bonds
on the active site of Pgp. Finally, we tested 4,6-
dimethoxyaurone (6) which isastructural isomer of
flavone. Aurones are natural compounds, frequently
found in vegetables, especially in fruit and flowers in
which they contribute to coloration.36 Methoxylation at
positions 4 and 6 was chosen, because, according to the
numbering system and the biosynthesis of aurones,
positions 4 and 6 are equivalent to positions 5 and 7 in
flavones. Aurone 6 was found to be as active as N-
methylpiperazinylcarbonylchromone (4a ) when using
the rhodamine-123 accumulation assay. This strong
activity, even in the absence of any nitrogen atom, is
quite interesting. It may indicate the importance of the
aurone 3D structure, because its isomer, 5,7-dimethoxy-
flavone, is almost inactive.37 Therefore, it is conceivable
that the introduction of any nitrogen atom would
enhance the activity. For example, azaaurones, in which
the intracyclic oxygen is replaced by nitrogen, are
interesting.
More interesting still, the three most active com-
pounds (4a , 5, and 6) potentiate DNR cytotoxicity as
much as cyclosporin A (Figures 3, 4, and 5). Apoptosis
induced by compounds 4a , 5, and 6 on K562S and, at a
lesser degree on K562R, which may lead to an extrusion
of the compounds by Pgp, so this activity might be in
competition with anticancer drugs.
In conclusion, according to both accumulation and
cytoxicity data, we found that 5-hydroxychromone linked
to a piperazine unit constituted a class of highly active
compounds as MDR modulators. We also reported for
the first time MDR modulation by one aurone and one
3-phenyl-4-quinolone. These data opens the way for
investigation in a new potential pharmacophores in the
field of MDR modulators. The relatively short and easy
synthesis of these molecules make them potential
candidates for further development.
Am id es 4. Acid 3 was dissolved in THF (2 mL/mmol),
treated with EDC (1.2 equiv), and stirred at room temperature
for 1 h. Benzylamine or piperazine derivative (1.5 equiv) was
slowly added, followed by addition of Et3N (1.2 equiv). After
the mixture was stirred at room temperature for 24 h, water
was added, and the mixture was extracted with EtOAc. The
organic layer was washed with water, brine, dried over Na2-
SO4, and concentrated. The product was purified by chroma-
tography on silica gel eluted with a gradient of cyclohexane/
EtOAc (9/1) to (6/4) to yield amides 4 as yellow solids.
5-H yd r oxy-2-(4-m et h ylp ip er a zin -1-ylca r b on yl)ch r o-
m on e (4a ): 22%; mp 96-98 °C; 1H NMR (CD3OD): δ 7.61 (t,
1H, J ) 8.4 Hz, H7); 7.01 (d, 1H, J ) 8.4 Hz, H8); 6.81 (d, 1H,
J ) 8.3 Hz, H6); 6.49 (s, 1H, H3); 3.72-3,60 (m, 4H, CONCH2);
2.50-2,43 (m, 4H, CONCH2CH2); 2.32 (s, 3H, NCH3). 13C NMR
Exp er im en ta l Section
Gen er a l Ch em istr y Meth od s. 1H and 13C NMR spectra
were recorded on a Bruker AC-200 instrument (200 MHz for

2130 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Hadjeri et al.
(CD3OD): δ 184.6 (C4); 162.1 (CON); 160.5 (C2); 157.5 (C5, C9);
137.6 (C7); 112.9 (C3); 111.3 (C8); 108.6 (C6); 55.9 and 55.2
(CONCH2); 50.4 and 45.9 (CONCH2CH2); 43.2 (NCH3). MS
(DCI, m/z) 289 (M + H)+. Anal. Calcd for C15H16N2O4: C, 62.49;
H, 5.59; N, 9.72. Found. C, 62.02; H, 5.77; N, 9.43.
(m, 4H, CONCH2CH2). 13C NMR (CDCl3): δ 182.8 (C4); 160.9
(CON); 160.0 (C2); 158.5 (C5); 153.0 (C9); 136.2 (C7); 112.4 (C3);
110.8 (C8); 107.2 (C6); 66.7 (CONCH2CH2); 48.0 and 42.9
(CONCH2). MS (DCI, m/z) 276 (M + H)+. Anal. (C14H13NO5)
C, H, N.
2-(4-E t h ylp ip e r a zin -1-ylca r b on yl)-5-h yd r oxych r o-
m on e (4b): 36%; mp 112-114 °C; H NMR (CDCl3): δ 12.16
2-[N-(4-Ch lor oben zyla m in oca r bon yl)]-5-h yd r oxych r o-
m on e (4i): 29%; mp 174-177 °C; H NMR (CDCl3): δ 7.65
1
1
(s, 1H, OH); 7.52 (t, 1H, J ) 8,4 Hz, H7); 6.90 (d, 1H, J ) 8.3
Hz, H8); 6.78 (d, 1H, J ) 8.3 Hz, H6); 6.41 (s, 1H, H3), 3.73-
3.52 (m, CONCH2); 2.55-2,49 (m, 4H, (CONCH2CH2); 2.38 (q,
2H, J ) 7.1 Hz, NCH2CH3); 1.06 (t, 3H, J ) 7.2 Hz,
N-CH2CH3). 13C NMR (CDCl3): δ 182.8 (C4); 160.8 (CON);
160.2 (C2); 159.1 (C5); 155.8 (C9); 136,0 (C7); 112.1 (C3); 111.3
(C10); 110.4 (C8); 107.2 (C6); 52.9 and 51.2 (CONCH2); 52.1
(NCH2CH3); 47.2 and 42.5 (CONCH2CH2); 11.7 (NCH2CH3).
MS (DCI, m/z) 303 (M + H)+. Anal. (C16H18N2O4) C, H, N.
5-H yd r oxy-2-(4-p h en ylp ip er a zin -1-ylca r b on yl)ch r o-
(t, 1H, J ) 8,4 Hz, H7); 7.35 (m, 4H, -C6H5); 7.10 (d, 1H, J )
8.4 Hz, H8); 6.92 (s, 1H, H3); 6.85 (d, 1H, J ) 8.4 Hz, H6); 4.56
(s, 2H, CH2Ph). 13C NMR (CDCl3): δ 183.4 (C4); 161.0 (CON);
158.6 (C2); 155.3 (C5); 155.1 (C9); 136.2 (C7′); 135.3 (C4′); 134
(C1′); 128.1-129.4 (C2′, C3′, C5′, C6′); 112.3, 112,4 (C3, C10); 111.3
(C8); 106.9 (C6); 43.3 (CONCH2). MS (DCI, m/z) 330 (M + H)+.
Anal. (C17H12ClNO4) C, H, Cl, N.
Cell Lin es a n d Cu ltu r e Con d ition s. The human myeloid
leukemic K562 cell line, derived from a chronic myeloid
leukemia, and its adriamycin resistant sub-line, K562/R which
expresses high level of Pgp, were generously donated by Pr.
J . P. Marie (Hotel Dieu, Paris). Parent resistant K562 cells
were named in the text as K562S and K562R, respectively.
The human cell line HL60, derived from an acute myeloid
leukemia and adriamycin resistant form, HL60/R, which
expressed moderate level of MRP, were donated by the M. S.
Center (Division of Biology, Kansas State University). All cells
were cultured at 37 °C, in a humidified atmosphere, with 5%
CO2, in RPMI-1640 medium supplemented with 10% FBS, 2
mM glutamine, 100 IU/mL penicillin, and 100 ng/mL strep-
tomycin. Chemoresistance was maintained by continuous
exposure to adriamycin at 10-6 M for K562R and HL60/MRP.
1
m on e (4c): 28%; mp 159-161 °C; H NMR (CDCl3): δ 12.2
(s, 1H, OH); 7.57 (t, 1H, J ) 8.4 Hz, H7); 7.3 (d, 1H, J ) 7.8
Hz, H8); 7.26 (d, 1H, J ) 7.6 Hz, H6); 6.91 (m, 5H, -C6H5);
6.51 (s, 1H, H3); 3.91-3.71 (m, 4H, CONCH2); 3.27 (m, 4H,
CONCH2CH2). 13C NMR (CDCl3): δ 182.8 (C4); 160.8 (CON);
160.3 (C2); 158.7 (C5); 155.7 (C9); 150.9 (C1′); 136.1 (C7); 129.5
(C3′, C5′); 121 (C4′); 116.7 (C2′, C6′); 112.2 (C3); 111.2 (C10); 110.6
(C8); 107.1 (C6); 50.0 and 49.7 (CONCH2); 46.9 and 42.5
(CONCH2CH2). MS (DCI, m/z) 351 (M + H)+. Anal. (C20H18N2O4)
C, H, N.
5-H yd r oxy-2-(4-b en zylp ip er a zin -1-ylca r b on yl)ch r o-
1
m on e (4d ): 28%; mp 109-113 °C; H NMR (CDCl3): δ 12.1
(s, 1H, OH); 7.51 (t, 1H, J ) 8.4 Hz, H7); 7.30-7,26 (m, 5H,
C6H5); 6.86 (d, 1H, J ) 8.5 Hz, H8); 6.78 (d, 1H, J ) 8.3 Hz,
H6); 6.4 (s, 1H, H3); 3.71 (ls, 2H, CH2Ph); 3,41 (m, 4H,
CONCH2); 2.45 (m, 4H, CONCH2CH2). 13C NMR (CDCl3): δ
184.1 (C4); 161.9 (CO-N); 161.4 (C2); 160.1 (C5); 156.9 (C9);
138.4 (C1′); 137.2 (C7); 130.1 (C2′, C6′ or C3′, C5′); 129.4 (C2′, C6′
or C3′, C5′); 128.6 (C4′); 113.3 (C10); 112.4 (C3); 111.5 (C8); 108.3
(C6); 63.8 (NCH2Ph); 54.4 and 53.4 (CONCH2); 48.2 and 43.6
(CONCH2CH2). MS (DCI, m/z) 365 (M + H)+. Anal. Calcd for
F u n ction a l Asp ect of P gp a n d MRP on Cells Tr ea ted
w ith Rever sa l Agen ts. Detection of Pgp activity using
rhodamine-123 efflux. K562S/R were washed twice and sus-
pended at a density of 106 cell/mL in RPMI medium. The
modulator was added to K562R at various concentrations,
ranging from 10-6 M to 10-9 M, for 15 min at 37 °C. For each
resistant line, cyclosporin A was added at 10 µM for 15 min
at 37 °C. Rhodamine-123 was diluted in order to obtain a final
concentration of 0.05 µg/mL. Cells were incubated 1 h at 37
°C, then analyzed by flow cytometry (FCM). For MRP detec-
tion, calcein-AM efflux was used. HL60S/MRP were washed
twice and suspended at a density of 106 cell/mL and then
incubated with cyclosporin A for 15 min at 10 µM in order to
block MRP protein. Modulator was added to HL60/MRP at
various concentration ranging from 10-6 M to 10-9 M for 15
min at 37 °C. HL60S/MRP cells were incubated with calcein-
AM at final concentration of 0.05 µM during 10 min at 37 °C
then analyzed by FCM.
Cell Cycle. K562 and HL60 cell lines were incubated with
the reversal agent at 10-6 M for 24 and 48 h. The cell cycle
was evaluated using Becton Dickinson’s Cycle Test Plus. Cells
were incubated with trypsin in a spermine tetrahydrochloride
detergent buffer for 10 min at room temperature. Trypsin
inhibitor and ribonuclease A were added for 10 min to cells
without any washing step. Finally, PI was added and incu-
bated for 10 min, and then cells were immediately analyzed
using a FACSCalibur.
Tr ip le La belin g w ith P KH67, An n exin Bitotin Str ep ta -
vid in AMCA, a n d P r op id iu m Iod id e. PKH67 labeling was
performed as described previously.29 An amount of 2 × 107 cells
was suspended in 1 mL of Diluent C and stained by rapidly
admixing with a working PKH67 solution prepared by diluting
20 µL of 10-3 M ethanolic dye stock in 1.0 mL of Diluent C
(final staining concentrations: 10 µM PKH67, 5 × 106 cells/
mL). PKH67 fluorescence decrease was followed during 3 days.
Every day, annexinV biotin and streptavidin/AMCA were used
to evaluate apoptotic cells, and PI was used to detect necrotic
cells. Cells were incubated with modulator at 10-6 M for 24
and 48 h. Cells were pelleted and labeled with annexinV/biotin,
revealed by streptavidin/AMCA and PI after 15 min at 4 °C,
and finally analyzed with FACSVantage.
C
21H20N2O4. C, 69.22; H, 5.53; N, 7.69. Found. C, 68.98; H,
5.69; N, 7.54.
2-(N-Acet ylp ip er a zin -1-ylca r b on yl)-5-h yd r oxych r o-
m on e (4e): 35%; amorphous; 1H NMR (CDCl3): δ 7.59 (t, 1H,
J ) 8.4 Hz, H7); 6.91 (dd, 1H, J 1 ) 0.8 Hz, J 2 ) 8.6 Hz, H8);
6.86 (dd, 1H, J 1 ) 0.8 Hz, J 2 ) 8.5 Hz, H6); 6.52 (s, 1H, H3);
3.77-3.59 (m, 8H, H-piperazinyl); 2.15 (s, 3H, CH3CO). 13C
NMR (CDCl3): δ 182.8 (C4); 169.2 (NCOCH3); 160.9 (C2CO-
piperazinyl); 160.8 (C2); 158.3 (C5); 155.7 (C9); 136.2 (C7); 112.4
(C3); 111.3 (C10); 111.0 (C8); 107.1 (C6); 46.7 and 45.6 (CONCH2);
42.6 and 41.4 (CONCH2); 21.3 (CH3CO). MS (DCI, m/z) 317
(M + H)+. Anal. (C16H16N2O5) C, H, N.
2-(N-Ben zoylp ip er a zin -1-ylca r bon yl)-5-h yd r oxych r o-
m on e (4f): 23%; amorphous; 1H NMR (CDCl3): δ 7.57 (t, 1H,
J ) 8.4 Hz, H7); 7.45-7.40 (m, 5H, H-benzoyl); 6.90 (dd, 1H,
J 1 ) 0.7 Hz, J 2 ) 8.6 Hz, H8); 6.85 (dd, 1H, J 1 ) 0.7 Hz, J 2
)
8.6 Hz, H6); 6.51 (s, 1H, H3); 3.75-3.71 (m, 8H, COCH2). 13C
NMR (CDCl3): δ 182.7 (C4); 170.7 (NCOC6H5); 160.8 (C2CO-
piperazinyl); 160.6 (C2); 158.3 (C5); 155.7 (C9); 136.2 (C7); 134.7
(C1′); 130.3 (C4′); 128.7 (C3′, C5′); 127.1 (C2′, C6′); 112.4 (C3); 111.1
(C10); 111.0 (C8); 107.1 (C6); 46.9 and 42.7 (CONCH2). MS (DCI,
m/z) 379 (M + H)+. Anal. (C21H18N2O5) C, H, N.
5-H yd r oxy-2-(p ip er id in -1-ylca r b on yl)ch r om on e (4g):
31%; mp 104-106 °C; 1H NMR (CDCl3): δ 7.51 (t, 1H, J ) 8.4
Hz, H7); 6.92 (dd, 1H, J 1 ) 0.8 Hz, J 2 ) 8.4 Hz, H8); 6.84 (dd,
1H, J 1 ) 0.8 Hz, J 2 ) 8.3 Hz, H6); 6.43 (s, 1H, H3); 3.70-3.43
(m, 4H, CONCH2); 1.90-1.65 (m, 6H, CONCH2CH2); 13C NMR
(CDCl3): δ 183.0 (C4); 160.9 (-CO-); 160.3 (C2); 156.1 (C5); 136.5
(C9); 135.9 (C7); 113.6 (C10); 112.1 (C3); 109.9 (C8); 107.2 (C6);
48.1 and 43.5 (CONCH2); 26.5 and 25.3 (CONCH2CH2); 24.3
(CONCH2CH2CH2). MS (DCI, m/z) 274 (M + H)+. Anal. (C15H15
NO4) C, H, N.
-
5-Hyd r oxy-2-(m or p h olin -1-ylca r bon yl)ch r om on e (4h ):
25%; mp 151-153 °C; 1H NMR (CDCl3): δ 7.57 (t, 1H, J ) 8.4
Hz, H7); 6.92 (d, 1H, J ) 8.4 Hz, H8); 6.85 (d, 1H, J ) 8.3 Hz,
H6); 6.49 (s, 1H, H3); 3.79-3.75 (m, 4H, CONCH2); 3.61-3.57
Cytotoxicity Assa y. To avoid spectral overlapping, TO-
TO-3 was chosen to follow necrosis because DNR emits a red-
orange fluorescence after excitation at 488 nm. Even if both
are able to label DNA and DNR, the fluorescence emission

Modulation of Pgp-Mediated Multidrug Resistance
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2131
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spectrum partially overlaps the TOTO-3 excitation spectrum,
and no quenching phenomenon was observed. K562S and
K562R at 106 cells/mL were incubated with different concen-
trations of modulators (4a , 5, 6, and cyclosporin A) at (10-6
,
10-7, 10-8, and 10-9 M) for 15 min at 37 °C before DNR
addition. A control without DNR was made for each modulator.
Cell lines were diluted at 105 and plated in 24-well microplates,
and then DNR was added at final concentration of 10-6 M.
After 72 h of incubation at 37 °C, TOTO-3 at 5 nM final
concentration was added to each cell suspension, 5 min prior
to FCM analysis.
Ack n ow led gm en t. This work was supported by a
grant from the “Ligue Nationale contre le Cancer
(Commite´ de la Haute-Savoie)” and the “GEFLUC
(De´le´gation de l′Ise`re)” whom we thank warmly. M.
Barbier is a fellowship recipient for the “Ligue Nationale
contre le Cancer (Commite´ de la Haute-Savoie)”. M.
Hadje´ri is a fellowship recipient for the “Fondation pour
la Recherche Me´dicale”.
Su p p or tin g In for m a tion Ava ila ble: The biological data
(tables and figures) concerning the effect of compounds 4a -e,
5, 5a , and 6 on cell cycle and the effect of compounds 4a , 5,
and 6 on cell division, apoptosis, and necrosis. This material
is available free of charge via the Internet at http://pubs.
acs.org.
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