B-ring substituted 5,7-dihydroxyflavonols with high-affinity binding to P-glycoprotein responsible for cell multidrug resistance

Article abstract of DOI:10.1016/S0960-894X(00)00595-3

Starting from the interaction of galangin (3,5,7-trihydroxyflavone) with a cytosolic nucleotide-binding domain of P-glycoprotein, a series of flavonol derivatives was synthesized and tested for their binding affinity towards the same target. The 5,7-dihyd

Full text of DOI:10.1016/S0960-894X(00)00595-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 75±77
B-ring Substituted 5,7-Dihydroxy¯avonols with High-Anity
Binding to P-Glycoprotein Responsible for Cell Multidrug
Resistance
Ahcene Boumendjel,a,* Frederic Bois,a Chantal Beney,a
Anne-Marie Mariotte,a Gwenaelle Conseilb and Attilio Di Pietrob
a
Â
Â
Departement de Pharmacochimie Moleculaire UMR-CNRS 5063, UFR de Pharmacie de Grenoble 38706, La Tronche, France
b
 Â
Institut de Biologie et Chimie des Proteines, UMR 5086 CNRS/Universite Claude Bernard-Lyon I, 7 Passage du Vercors,
69367 Lyon, France
Received 28 July 2000; revised 11 October 2000; accepted 23 October 2000
AbstractÐStarting from the interaction of galangin (3,5,7-trihydroxy¯avone) with a cytosolic nucleotide-binding domain of P-gly-
coprotein, a series of ¯avonol derivatives was synthesized and tested for their binding anity towards the same target. The 5,7-
dihydroxy-40-iodo¯avonol and 5,7-dihydroxy-40-n-octyl¯avonol derivatives displayed much higher binding anities, with respective
increases of 6- and 93-fold as compared to galangin. # 2000 Elsevier Science Ltd. All rights reserved.
There is now general consensus that one of the major
mechanisms by which tumor cells become resistant to
several of the most ecient anticancer drugs presently
available is a decreased intracellular drug bioavail-
ability. Such a multidrug resistance (MDR) phenotype
has often been correlated to the increased expression of
a 170-kDa plasma membrane glycoprotein (P-gp) which
acts as a cellular `pump' by extruding the anticancer
compounds and preventing their antitumor e€ect.1 Two
intracellular ATP-binding sites have been described in
P-gp and the active export of cytotoxic compounds
requires the energy derived from their ATPase activity.2
Di€erent ways have been undertaken to restore chemo-
sensitivity of the MDR tumor cells to cytotoxic drugs.
Besides attempts to discover anticancer drugs which
would not be transported substrates for P-gp through
maintaining a sucient activity towards their molecular
target, another pharmacological approach consists of
trying to suciently inhibit P-gp pump functioning in
order to restore cell sensitivity to anticancer drugs.3
By using a puri®ed recombinant protein corresponding
to a cytosolic nucleotide-binding domain of P-gp, we
have previously demonstrated that ¯avonoids are e-
cient P-gp inhibitors, by binding to cytosolic sites which
partly overlap the ATP-binding site and the modulators
interacting region.4 Studies of structure±activity rela-
tionships have concluded that ¯avones are more ecient
than iso¯avones, while ¯avonols and chalcones are even
more active.
Figure 1.
*Corresponding author. Tel.: +33-4-7651-8688; fax: +33-4-7663-
7165; e-mail: ahcene.boumendjel@ujf-grenoble.fr
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00595-3

76
A. Boumendjel et al. / Bioorg. Med. Chem. Lett. 11 (2001) 75±77
Hydroxyl groups at positions 3 and 5 are essential for
high-anity binding to P-gp. In a recent study con-
ducted with chalcones, we have shown that the presence
of a hydrophobic substituent on the B-ring (at 4-posi-
tion; Fig. 1) considerably enhanced the binding a-
nity.5,6 However, the instability of chalcones (due to
their cyclization to give inactive ¯avanones) makes them
less desirables for further studies. In order to prepare
high-anity and much more stables ¯avonols to be used
for in vivo studies, we have targeted and synthesized a
series of ¯avonols (3,5,7-trihydroxy¯avones) with
di€erent hydrophobic substituents at the 40-position.
with NBD2 with a KD of 5.9 Æ 0.2 mM. Addition of a
hydroxyl group at position 40 of the B-ring produced a
slightly negative e€ect when comparing kaempferol (6.7
Æ 0.3 mM) to galangin. On the contrary, the presence of
a hydrophobic methoxy group (in kaempferide) slightly
increased the binding anity as monitored by quenching
(KD=5.0 Æ 0.2 mM).
As seen from Table 1, no signi®cant e€ect was produced
by introducing a ¯uorine in 4a (KD=6.8 Æ 0.28 mM),
whereas two chlorine atoms at positions 20 and 40 in 4b
produced a positive e€ect (KD=4.0 Æ 0.23 mM). A
much higher increase in anity was observed by intro-
ducing an iodine in 4d (KD=1.06 Æ 0.08 mM), which
was probably due to its hydrophobic character as con-
cluded previously with chalcone derivatives.5 Interest-
ingly, an intermediate 3-fold increase in anity was
observed by replacing the complete B-ring with a
diphenylmethyl substituent in 4c. Finally, the most
striking increase in binding anity was observed by an
octyl group which produced a 93-fold higher anity in
4e as compared to galangin. The choice of this sub-
stituent relies on our previous study conducted on chal-
cones which shows that an octyl group is the most
optimal substituent, and further increase in the chain
length began to alter the binding. The fact that the
maximal quenching of ¯uorescence was signi®cantly
reduced (62.1 Æ 3.3%) as compared to all other com-
pounds (ꢀ84.6 Æ 2.0%) might indicate a di€erently
orientated binding that would di€erentially modify
tryptophan environment.
Chemistry
Flavonols 4 were synthesized in two steps starting from
2,4,6-trihydroxy-o-methoxyacetophenone 1 (1 was pre-
pared from phloroglucinol and methoxyacetonitrile
according to Wahala and Hase).7 Condensation of 1
with an appropriately substituted benzoyl chloride in
the presence of K2CO3 in acetone a€orded directly the
diketone 2 which was not isolated but cyclized by treat-
ment with AcOH to give 5,7-dihydroxy-3-methoxy-
¯avones 3. In one case (R=F), the expected 5,7-
dihydroxy-3-methoxy¯avone 3a was not obtained. The
main product was the 7-¯uorobenzoyl-5-hydroxy-3-
methoxy¯avone. The latter was then treated with KOH
in MeOH to give 3a in a quantitative yield. Demethyla-
tion of methoxy¯avones 3 with BBr3 in CH2Cl2 gave
¯avonols 4 (Scheme 1).
Table 1. Role of ¯avonol B-ring substituents on the binding anity
for NBD2
Results and Discussion
Compound
R
KD
(mM)
ÁFmax
(%)
The binding of ¯avonols to the puri®ed C-terminal
nucleotide-binding domain (NBD2) of P-gp was directly
measured by the quenching of protein intrinsic ¯uores-
cence due to a single tryptophan residue, as previously
described for other classes of ¯avonoids.4 The results of
this study are listed in Table 1.
Galangin
Kaempferol
Kaempferide
4a
4b
4c
4d
4e
H
40-OH
40-OCH3
40-F
5.9 Æ 0.2
6.7 Æ 0.3
95.0 Æ 1.6
97.6 Æ 1.0
96.7 Æ 0.6
91.6 Æ 1.2
85.3 Æ 1.3
84.6 Æ 2.0
95.5 Æ 2.0
62.1 Æ 3.3
5.0 Æ 0.2
6.8 Æ 0.28
4.0 Æ 0.23
2.0 Æ 0.22
1.06 Æ 0.08
0.057 Æ 0.02
20, 40-Cl
PhR= CH(Ph)2
40-I
Our lead compound was galangin (3,5,7-trihydroxy-
¯avone) which was used as a reference and interacted
40-n-C8H17
Scheme 1.

A. Boumendjel et al. / Bioorg. Med. Chem. Lett. 11 (2001) 75±77
77
In summary, the binding anity of eight ¯avonols to
References and Notes
P-glycoprotein was evaluated by using a puri®ed
domain (NBD2). The potency was dependent on the
hydrophobic substituent on the B-ring. The two most
potent derivatives, 5,7-dihydroxy-40-iodo¯avonol and
5,7-dihydroxy-40-octyl¯avonol are being evaluated for
their ability to modulate the MDR.
1. Gottesman, M. M.; Pastan, I. Annu. Rev. Biochem. 1993,
62, 385.
2. Gros, P.; Buschman, E. Int. Rev. Cytol. 1993, 137, 169.
3. (a) Ford, J. M. Eur. J. Cancer 1996, 32A, 991. (b) Gold-
stein, L. J. Curr. Prob. Cancer 1995, 19, 65.
4. Conseil, G.; Baubichon-Cortay, H.; Dayan, G.; Jault, J.-M.;
Barron, D.; Di Pietro, A. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 9831.
Acknowledgements
5. Bois, F.; Beney, C.; Boumendjel, A.; Mariotte, A.-M.;
Conseil, G.; Di Pietro, A. J. Med. Chem. 1998, 41, 4161.
6. Bois, F.; Boumendjel, A.; Mariotte, A.-M.; Conseil, G.; Di
Pietro, A. Bioorg. Med. Chem. 1999, 7, 2691.
7. Wahala, K.; Hase, T. A. J. Chem. Soc., Perkin Trans. 1
3005, 1991.
This work was supported by the CNRS, the Universite
Joseph Fourier of Grenoble, the Universite Claude
Bernard of Lyon and the Comite du Rhone de la Ligue
Nationale contre le Cancer. G.C. was recipient of a fel-
lowship from the Comite de la Haute-Savoie.