4-Hydroxy-6-methoxyaurones with high-affinity binding to cytosolic domain of P-glycoprotein

Article abstract of DOI:10.1248/cpb.50.854

A series of 4-hydroxy-6-methoxyaurones and 4,6-dimethoxyaurones has been synthesised and tested for their binding affinity toward the nucleotide-binding domain of P-glycoprotein, an ABC (ATP-Binding Cassette) transporter which mediates the resistance of cancer cells to chemotherapy. These compounds differ from each other by the nature of the substituent on the aurone B-ring. The binding affinity seems to be linked to the nature of the substituent, as well as to the presence or the absence of a hydroxy group at position 4. The most active compounds were 4′-bromo-4-hydroxy-6-methoxyaurone and 4-hydroxy-4′-iodo-6-methoxyaurone.

Full text of DOI:10.1248/cpb.50.854

854
Notes
Chem. Pharm. Bull. 50(6) 854—856 (2002)
Vol. 50, No. 6
4-Hydroxy-6-methoxyaurones with High-Affinity Binding to Cytosolic
Domain of P-Glycoprotein
Ahcène BOUMENDJEL, ,a Chantal BENEY,a Nabajyoti DEKA,a Anne-Marie MARIOTTE,a
*
b
Martin Ata LAWSON,a Doriane TROMPIER,b Hélène BAUBICHON-CORTAY,b and Attilio DI PIETRO
a Laboratory of Pharmacognosy, UMR-CNRS 5063, Grenoble Pharmacy School; 38706 La Tronche, France: and b Institute
for Protein Biology and Chemistry (IBCP); UMR-CNRS 5086, 7 Passage du Vercors 69367 Lyon cedex 07 France.
Received December 17, 2001; accepted February 13, 2002
A series of 4-hydroxy-6-methoxyaurones and 4,6-dimethoxyaurones has been synthesised and tested for
their binding affinity toward the nucleotide-binding domain of P-glycoprotein, an ABC (ATP-Binding Cassette)
transporter which mediates the resistance of cancer cells to chemotherapy. These compounds differ from each
other by the nature of the substituent on the aurone B-ring. The binding affinity seems to be linked to the nature
of the substituent, as well as to the presence or the absence of a hydroxy group at position 4. The most active
compounds were 4؅-bromo-4-hydroxy-6-methoxyaurone and 4-hydroxy-4؅-iodo-6-methoxyaurone.
Key words aurone; binding affinity; P-glycoprotein; multidrug resistance
Multidrug resistance (MDR) of tumor cells to cytotoxic phloroglucinol 1 with chloroacetonitrile in the presence of
drugs is a major problem in cancer chemotherapy. Cells se- ZnCl2, and the obtained imine was then hydrolysed to give
lected for MDR are characterized by cross-resistance to a the ketone 2, which was cyclised in basic medium to provide
wide variety of compounds, either natural or synthetic.1) The the benzofurane 3. Protection of the hydroxyls provided the
pharmacological basis for MDR appears to be a decreased 4,6-dimethoxybenzofurane 4, and condensation of the latter
accumulation and retention of drugs by these cells. It has with a benzaldehyde derivative gave the aurone 5. The last
been reported that the mechanism by which tumor cells ac- step was a selective demethylation utilising BBr3 to obtain
quire this MDR phenotype is the overexpression of an ATP- the target aurone 6. Under the same conditions, deprotection
dependent membrane glycoprotein called P-glycoprotein of aurones 5g and 5h gave a complex of several compounds
(Pgp), that may be capable of binding a drug and expelling it which were difficult to purify. Other methods used (Me3SI;
from the cell.2—5) The driving force of drug efflux is Pgp-me- Py·HCl; HBr/AcOH) failed to yield the desired compounds.
diated ATP hydrolysis, the liberated energy of which is used
for the drug transport.6)
Results and Discussion
During the past decade, a tremendous effort has been
Aurones appear attractive from the perspective of drug de-
made to find compounds which are able to overcome MDR sign. Besides their contribution to the flowers of some popu-
by restoring the intracellular accumulation of antitumor lar ornamental plants, such as snapdragon and cosmos,14)
agents in resistant cells.7,8) Our group, has investigated the they possess a wide range of biological activities. They have
field of flavonoids which interact with the C-terminal nu- been described as antifungal agents and as phytoalexins.16)
cleotide-binding domain of P-glycoprotein and may consti- The naturally-occurring aureusidin (4,6,3Ј,4Ј-tetrahydroxy-
tute potential MDR modulators. We have studied flavones, aurone) was found to be a potent inhibitor of iodothyronine-
flavonols, chalcones and other phenolic compounds, and deiodinase.17) Recently, Kayser et al. reported the drug-po-
have identified the structural criteria required for a high bind- tential of aurones in Leishmania infections.18) The advantage
ing-affinity.9—13)
Continuing our efforts in identifying Pgp-mediated MDR
modulators, we wish to report here for the first time the bind-
ing of aurones, which constitute a naturally-occurring sub-
class of flavonoids frequently found in plants, where they
contribute to the coloration of fruits and flowers (Fig. 1).14)
In previous studies we reported that the presence of a 2–3
double bond, a 4-ketone group, a 7-methoxy group, and a 5-
hydroxy or methoxy group are favourable for high binding-
Fig. 1
affinity of flavones toward the C-terminal nucleotide-binding
domain (NBD2) of Pgp. Halogenation on the B-ring of
flavones (A-ring in chalcones) significantly increases the
binding affinity toward NBD2 of P-glycoprotein.9—12) There-
fore, we decided to identify, synthesise and test aurones
where these structural criteria are conserved (Fig. 2).
Chemistry
We earlier reported a general and convenient synthesis
of aurones.15) It employed (Chart 1) the condensation of Fig. 2. Structure of Aurones Targeted in This Study
To whom correspondence should be addressed. e-mail: Ahcene.Boumendjel@ujf-grenoble.fr
© 2002 Pharmaceutical Society of Japan

June 2002
855
(i) a. ZnCl2, HCl, Et2O, 0 °C ; b. 1 N HCl, 100 °C. (ii) MeONa, MeOH, reflux, 2 h. (iii) MeI, K2CO3, DMF, 80 °C, 1 h. (iv) KOH, MeOH/H2O, rt, 1 h. (v) BBr3/CH2Cl2, 24 h.
Chart 1
Table 1. Role of Aurone Substituents on the Affinity for NBD2 of Pgp
Substituent
where the B-ring substituted by the hydrophobic halogen
would bind to the steroid-interacting region and the A/C
rings would interact with the ATP-binding site. Efforts are
under way to synthesise aurones possessing more hydropho-
bic (alkyl chains) on the B-ring in order to obtain derivatives
with high binding affinity.
Aurone
KD (mM)
DFmax (%)
at position 4Ј
5a
5b
5c
5d
5e
5f
H
F
Cl
Br
7.0Ϯ1.1
2.9Ϯ0.7
0.99Ϯ0.2
0.82Ϯ0.08
0.54Ϯ0.04
20Ϯ4.6
99.9Ϯ5.8
88.6Ϯ0.9
88.5Ϯ6.3
84.1Ϯ2.5
85Ϯ2.1
Experimental
I
General Melting points were measured on a Fisher micromelting point
apparatus and are uncorrected. MS spectra were determined on a JEOL HX-
CN
100Ϯ8
5g
5h
6a
6b
6c
6d
6e
6f
–N(CH3)2
3Ј,4Ј,6Ј-OCH3
2.6Ϯ0.4
92Ϯ43
72Ϯ3.5
1
110 spectrometer. H- and 13C-NMR spectra were measured on Bruker AC-
87.6Ϯ28
63.4Ϯ6.3
85.1Ϯ10.5
74.5Ϯ3.4
80Ϯ7.9
200 spectrometer and were referenced to internal standards, tetramethylsi-
lane (dHϭ0.00, dCϭ0.0); CDCl3 (dHϭ7.27, dCϭ77.0). Electron-impact
mass spectra were obtained at 70 eV using a Trio 1000 instrument. Elemen-
tal analyses were performed by the analytical department of CNRS, Vernai-
son, France. Thin-layer chromatography (TLC) was carried out using Merck
silica gel F-254 plates (0.25 mm thick). Flash chromatography was carried
out using Merck silica gel 60, 200—400 mesh. All solvents were distilled
prior to use. The spectral and analytical data of 2Ј,4Ј,6Ј-trihydroxy-2-
chloroacetophenone (2); 4,6-dihydroxybenzofuran-3(2H)-one (3); 4,6-di-
methoxybenzofuran-3(2H)-one (4) and aurones 5a, 5b, 5c, 5d, 5g, 5h have
been reported (ref. 15).
(Z)-4,6-Dimethoxy-(4Ј-iodoobenzylidene)benzofuran-3(2H)-one (5e): mp
165—167 °C (EtOAc). 1H-NMR (CDCl3, 200 MHz) d: 3.91 (3H, s), 3.95
(3H, s), 6.14 (1H, d, Jϭ1.8 Hz), 6.38 (1H, d, Jϭ1.8 Hz), 6.65 (1H, s), 7.55
(2H, d, Jϭ8.4 Hz), 7.75 (2H, d, Jϭ8.6 Hz). MS m/z: 409 (Mϩ1)ϩ. Anal.
Calcd for C17H13IO4: C, 50.02; H, 3.21; I, 15.68. Found: C, 49.98; H, 3.17; I,
15.64.
H
F
Cl
Br
I
1.32Ϯ0.33
2.7Ϯ0.8
0.46Ϯ0.08
0.15Ϯ0.07
0.26Ϯ0.03
2.9Ϯ1
78.1Ϯ2.6
46.2Ϯ7.6
CN
of such molecules is their high stability, as compared to chal-
cones which can easily undergo a cyclisation to yield inactive
flavanones.
The binding of aurones to the purified C-terminal cytoso-
lic domain of Pgp was measured by the quenching of protein
intrinsic fluorescence, as previously described.9) The dissoci-
ation constant (KD) and the maximal fluorescence quenching
(DFmax) were determined using the Grafit program.19) As
(Z)-(4Ј-Cyanobenzylidene)-4,6-dimethoxybenzofuran-3(2H)-one (5f): mp
195—198 °C (EtOAc). 1H-NMR (CDCl3, 200 MHz) d: 3.96 (3H, s), 4.18
shown in Table 1, 4,6-dimethoxyaurones are less active than (3H, s), 6.53 (1H, d, Jϭ1.4 Hz), 6.69 (1H, d, Jϭ1.4 Hz), 7.20 (1H, s), 7.69
(2H, d, Jϭ8.5 Hz), 7.94 (2H, d, Jϭ8.5 Hz). MS m/z: 307 (Mϩ). Anal. Calcd
their corresponding 4-hydroxy-6-methoxyaurones, and halo-
genated aurones are the most active. The binding affinity of
the latter increases with the size (hydrophobicity) of the halo-
for C18H13NO4: C, 70.35; H, 4.26; N, 4.56. Found: C, 70.31; H, 4.23; N,
4.53.
4-Hydroxy-6-methoxyaurones (6a—f), General Procedure A solu-
gen. Substitution of the 4-hydroxyaurone hydrogen at posi-
tion 4Ј with a bromo atom increases the binding affinity by
nearly 8.8-fold. Overall, the data clearly show that the bind-
ing affinity is most likely correlated to the lipophilicity of the
halogen rather than to hydrogen-bond capacity. The size of
the halogen may also contribute to the binding affinity. The
presence of heteroatoms other than halogens is unfavourable
for high-binding affinity as can be deduced from KD of 5g
and 5h.
tion of aurone (5) in CH2Cl2 was treated with BBr3 (1 M in CH2Cl2, 2 eq) at
room temperature for 24 h, after which, the solution was poured into ice
water and extracted with CH2Cl2. The CH2Cl2 was dried over Na2SO4 and
concentrated and the crude was purified by column chromatography eluted
with CH2Cl2 to yield pure aurone (6). Under the same conditions, aurones
(5g) and (5h) gave a complex mixture of several compounds.
(Z)-Benzylidene-4-hydroxy-6-methoxybenzofuran-3(2H)-one (6a): 1H-
NMR (CDCl3, 200 MHz) d: 3.92 (3H, s), 6.17 (1H, d, Jϭ1.8 Hz), 6.43 (1H,
d, Jϭ1.8 Hz), 6.78 (1H, s), 7.38—7.39 (3H, m), 7.83 (2H, dd, J1ϭ1.8 Hz,
J2ϭ8.1 Hz). MS m/z: 268 (Mϩ). Anal. Calcd for C16H12O4: C, 71.64; H,
4.51. Found: C, 71.59; H, 4.48.
It has been shown that recombinant cytosolic domains of
P-glycoprotein contain a steroid-interacting hydrophobic re-
gion in close proximity to the ATP-binding site.20) The key
roles played by the A-ring and a,b-unsaturated ketone sys-
tem of flavones in mimicking the adenine moiety of ATP
have been reported.21,22) Based on these data, we can postu-
(Z)-(4Ј-Fluorobenzylidene)-4-hydroxy-6-methoxybenzofuran-3(2H)-one
(6b): H-NMR (CDCl3, 200 MHz) d: 3.89 (3H, s), 6.16 (1H, d, Jϭ1.8 Hz),
1
6.33 (1H, d, Jϭ1.8 Hz), 6.71 (1H, s), 7.13 (2H, dd, J1ϭJ2ϭ8.7 Hz), 7.86
(2H, dd, J1ϭ5.4 Hz, J2ϭ8.7 Hz). MS m/z: 286 (Mϩ). Anal. Calcd for
C16H11FO4: C, 67.13; H, 3.87; F, 6.64. Found: C, 67.09; H, 3.83; F, 6.52.
(Z)-(4Ј-Chlorobenzylidene)-4-hydroxy-6-methoxybenzofuran-3(2H)-one
1
(6c): H-NMR (CDCl3, 200 MHz) d: 3.90 (3H, s), 6.16 (1H, d, Jϭ1.8 Hz),
late that aurones overlap the two binding sites of NBD2 6.34 (1H, d, Jϭ1.8 Hz), 6.70 (1H, s), 7.41 (2H, d, Jϭ8.5 Hz), 7.81 (2H, d,

856
Vol. 50, No. 6
Jϭ8.5 Hz). MS m/z: 302 (Mϩ). Anal. Calcd for C16H11ClO4: C, 63.48; H,
3.66; Cl, 11.71. Found: C, 63.44; H, 3.57; Cl, 11.63.
9) Conseil G., Baubichon-Cortay H., Dayan G., Jault J. M., Barron D., Di
Pietro A., Proc. Natl. Acad. Sci. U.S.A., 95, 9831—9836 (1998).
10) Bois F., Beney C., Boumendjel A., Mariotte A.-M., Conseil G., Di
Pietro A., J. Med. Chem., 41, 4161—4164 (1998).
(Z)-(4Ј-Bromobenzylidene)-4-hydroxy-6-methoxybenzofuran-3(2H)-one
1
(6d): H-NMR (CDCl3, 200 MHz) d: 3.89 (3H, s), 6.17 (1H, d, Jϭ1.8 Hz),
6.34 (1H, d, Jϭ1.8 Hz), 6.67 (1H, s), 7.57 (2H, d, Jϭ8.5 Hz), 7.73 (2H, d,
Jϭ8.5 Hz). MS m/z: 347 (Mϩ). Anal. Calcd for C16H11BrO4: C, 55.36; H,
3.19; Br, 23.02. Found: C, 55.33; H, 3.14; Br, 22.93.
11) Bois F., Boumendjel A., Mariotte A.-M., Conseil G., Di Pietro A.,
Bioorg. Med. Chem., 7, 2691—2695 (1999).
12) Boumendjel A., Bois F., Beney C., Mariotte A.-M., Conseil G., Di
Pietro A., Bioorg. Med. Chem. Lett., 11, 75—77 (2001).
13) Comte G., Daskiewicz J. B., Bayet C., Conseil G., Vornery-Vanier A.,
Dumontet C., Di Pietro A., Barron D., J. Med. Chem., 44, 763—768
(2001).
14) Harborne J. R., “The Flavonoids: Advances in Research Since 1986,”
Chapman and Hall, London, 1994, pp. 341—399.
15) Beney C., Mariotte A.-M., Boumendjel A., Heterocycles, 55, 967—
972 (2001).
(Z)-(4Ј-Iodoobenzylidene)-4-hydroxy-6-methoxybenzofuran-3(2H)-one
1
(6e): H-NMR (CDCl3, 200 MHz) d: 3.83 (3H, s), 6.13 (1H, d, Jϭ1.8 Hz),
6.51 (1H, d, Jϭ1.8 Hz), 6.61 (1H, s), 7.67 (2H, d, Jϭ8.5 Hz), 7.84 (2H, d,
Jϭ8.5 Hz). MS m/z: 395 (Mϩ). Anal. Calcd for C16H11IO4: C, 48.75; H,
2.81; I, 32.20. Found: C, 48.71; H, 2.77; I, 32.09.
(Z)-(4Ј-Cyanobenzylidene)-4-hydroxy-6-methoxybenzofuran-3(2H)-one
1
(6f): H-NMR (CDCl3, 200 MHz) d: 3.94 (3H, s), 6.38 (1H, d, Jϭ1.6 Hz),
6.64 (1H, d, Jϭ1.6 Hz), 7.24 (1H, s), 7.70 (2H, d, Jϭ8.5 Hz), 7.92 (2H, d,
Jϭ8.5 Hz). MS m/z: 293 (Mϩ). Anal. Calcd for C17H11NO4: C, 69.62; H,
3.78; N, 4.78. Found: C, 69.57; H, 3.73; N, 4.75.
16) Brooks C. J. W., Watson D. G., Nat. Prod. Rep., 1985, 427—459.
17) Auf’mkolk M., Koerhle J., Hesch R. D., Cody V., J. Biol. Chem., 261,
11623—11627 (1986).
References
18) Kayser O., Kiderlen A. F., Folkens U., Kolodziej H., Planta Medica,
65, 316—321 (1999).
1) Gottesman M. M., Pastan I., Annu. Rev. Biochem., 62, 385—427
(1993).
2) Juliano R. L., Ling V., Biochim. Biophys. Acta, 455, 152—162 (1976).
3) Chen C., Chin J. E., Ueda K., Clark D. R., Pastan I., Gottesman M. M.,
Roninson I. B., Cell, 47, 381—389 (1986).
4) Gros P., Croop J., Housman D., Cell, 47, 371—380 (1986).
5) Gerlach J. H., Endicott J. H., Juranka P. F., Henderson G., Sarangi F.,
Deuchars K. L., Ling V., Nature (London), 324, 485—489 (1986).
6) Ashida H., Oonishi T., Uyesaka N., J. Theor. Biol., 195, 219—232
(1998).
19) Dayan G., Baubichon-Cortay H., Jault J.-M., Cortay J.-C., Deleage G.,
Di Pietro A., J. Biol. Chem., 271, 11652—11658 (1996).
20) Dayan G., Jault J.-M., Baubichon-Cortay H., Baggetto L. G., Renoir J.
M., Baulieu E. E., Gros P., Di Pietro A., Biochemistry, 36, 15208—
15212 (1997).
21) De Azevedo F. W., Mueller-Dieckmann H. J., Schulze-Gahmen U.,
Worland P. J., Sausville E., Kim S. H., Proc. Natl. Acad. Sci. U.S.A.,
93, 2735—2740 (1996).
22) Sicheri F., Moarefi I., Kuriyan J., Nature (London), 385, 602—609
(1997).
7) Ford J. M., Eur. J. Cancer, 32A, 991—1001 (1996).
8) Wiese M., Pajeva I. K., Curr. Med. Chem., 8, 685—713 (2001).