C-isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance

Article abstract of DOI:10.1021/jm991128y

Previous studies have shown that flavones bind to P-glycoprotein (Pgp) with higher affinity than isoflavones, flavanones, and glycosylated derivatives. In the present work, a series of C- or O-substituted hydrophobic derivatives of chrysin were synthesized to further investigate structural requirements of the A ring toward Pgp modulation. Increasing hydrophobicity at either position 6, 8, or 7 increased the affinity of in vitro binding to a purified cytosolic domain of Pgp, but only benzyl and 3,3-dimethylallyl C-substitution produced a high maximal quenching of the protein intrinsic fluorescence. Inhibition of membrane Pgp within leukemic cells, characterized by intracellular drug accumulation, was specifically produced by isoprenylated derivatives, with 8-(3,3-dimethylallyl)chrysin being even more efficient than the commonly used cyclosporin A.

Full text of DOI:10.1021/jm991128y

J . Med. Chem. 2001, 44, 763-768
763
C-Isop r en yla tion of F la von oid s En h a n ces Bin d in g Affin ity tow a r d
P -Glycop r otein a n d Mod u la tion of Ca n cer Cell Ch em or esista n ce
Gilles Comte,# J ean-Baptiste Daskiewicz,# Christine Bayet,# Gwenae¨lle Conseil,‡ Armelle Viornery-Vanier,§
Charles Dumontet,§ Attilio Di Pietro,‡ and Denis Barron*,#
Laboratoire des Produits Naturels, CNRS-UMR 5013, UFR de Chimie-Biochimie, Universite´ Claude Bernard Lyon 1,
43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France, Institut de Biologie et Chimie des Prote´ines,
CNRS-UPR 412, 7 Passage du Vercors, 69367 Lyon Cedex 07, France, and Laboratoire de Cytologie Analytique,
Faculte´ de Me´decine Rockefeller, INSERM-U 453, 8 Avenue Rockefeller, 69880 Lyon, France
Received August 28, 2000
Previous studies have shown that flavones bind to P-glycoprotein (Pgp) with higher affinity
than isoflavones, flavanones, and glycosylated derivatives. In the present work, a series of C-
or O-substituted hydrophobic derivatives of chrysin were synthesized to further investigate
structural requirements of the A ring toward Pgp modulation. Increasing hydrophobicity at
either position 6, 8, or 7 increased the affinity of in vitro binding to a purified cytosolic domain
of Pgp, but only benzyl and 3,3-dimethylallyl C-substitution produced a high maximal quenching
of the protein intrinsic fluorescence. Inhibition of membrane Pgp within leukemic cells,
characterized by intracellular drug accumulation, was specifically produced by isoprenylated
derivatives, with 8-(3,3-dimethylallyl)chrysin being even more efficient than the commonly
used cyclosporin A.
In tr od u ction
activity of lipophilic flavonoids substituted by either
methyl, isopropyl, benzyl, or isoprenyl groups. Isopre-
nylated flavonoids are natural compounds which con-
stitute a minor class of plant secondary metabolites
recently reviewed.12 As starting material, we have
chosen the simplest 5,7-dihydroxyflavone named chrysin.
In this paper, we describe the effects of ring A alkylation
on the interaction of flavonoids with Pgp. Mono- and
disubstituted chrysin derivatives were prepared to study
the effects of hydrophobicity and substituent position
on the affinity of binding to the transporter and on
related inhibition leading to intracellular drug ac-
cumulation.
Multidrug resistance (MDR) of cancer cells is often
correlated with the overexpression of P-glycoprotein
(Pgp), an ABC-type plasma membrane transporter that
rejects chemotherapeutic drugs out of the cell by using
ATP hydrolysis as an energetic source.1,2 Tumor cells
expressing Pgp are resistant to a number of major
cytotoxic agents, including anthracyclines, vinca alca-
loids, taxanes, and epipodophyllotoxins. Therefore, it is
of high priority to develop molecules that can inhibit
Pgp activity.
Pgp is able to bind a number of compounds, but most
of them are transported.3 Hydroxylated steroids such
as cortisol, dexamethasone, aldosterone, or corticoster-
one can be effluxed by Pgp,4,5 whereas more hydrophobic
ones such as progesterone and antiprogestin RU 486 are
not transported and behave as efficient modulators of
cellular MDR by inhibiting anticancer drug efflux.4,6,7
Unfortunately, it appears very difficult to use such
compounds at a therapeutic level because of their
hormonal consequences.
Flavonoids have also been reported as modulators of
Pgp.8-10 We recently showed that flavones and flavonols
bind more strongly to Pgp cytosolic site than isoflavones,
flavanones, and glycosylated derivatives.11 Moreover,
the hydroxylation at position 5, in addition to ketone
at position 4, is essential for the ability of these
modulators to mimic the adenine moiety of ATP. We also
showed that such flavonoids are able to interact with
both ATP and steroid binding sites within the cytosolic
domain of the transporter. Since most Pgp effectors are
rather hydrophobic, it appeared of interest to test the
Resu lts a n d Discu ssion
Although O-alkylated flavonoids are of easier access
than C-alkylated ones, the latter compounds are ex-
pected to be more advantageous because phenolic hy-
droxyls remain free and can play a role in the interac-
tion. There are two main methods to obtain C-prenylated
flavonoids. The first one13 consists of a sigmatropic
rearrangement of O-prenylated compounds which gives
a high specificity in the prenylation position. The second
one is a direct C-alkylation process in alkali medium.14
This method may afford a mixture of isomers but is a
one-step reaction because no protecting group is re-
quired. As a general alkylation process we retained the
latter method since it affords the highest diversity of
isomers in a single experiment (Scheme 1). All alkyla-
tions were performed in a solution of aqueous tetram-
ethylammonium hydroxide/MeOH in the presence of
tetraethylammonium iodide. C-Methylation and C-iso-
propylation of chrysin were performed under rather
forced conditions, by refluxing chrysin with 5 equiv of
methyl iodide or isopropyl bromide, respectively. In each
case, mixtures of C-monoalkylated (1, 4) and O- plus
C-polyalkylated isomers (3, 6, 7) were obtained. Both
* To whom correspondence should be addressed. Tel: 33 4 72 43 14
21. Fax: 33 4 72 43 15 57. E-mail: denis.barron@univ-lyon1.fr.
#
CNRS-UMR 5013.
‡ CNRS-UPR 412.
§ INSERM-U 453.
10.1021/jm991128y CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/26/2001

764 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Sch em e 1. Alkylated Chrysin Derivatives
Comte et al.
the KD value measured for the most hydrophobic
compound 16 was more than 500-fold lower than that
of chrysin. It can be anticipated that alkylation strength-
ened the flavonoid interaction at the modulatory site
that binds hydrophobic steroids.
However, only mono-C-substituted benzyl (8, 9) or 3,3-
dimethylallyl (12, 13) derivatives displayed a high
maximal quenching (∆Fmax) comparable to that observed
with chrysin. Indeed relatively low values were pro-
duced by 6- or 8-C-monosubstituted methyl (1), isopropyl
(4), or geranyl (15, 16) derivatives, as well as by 6,8-C-
disubstituted compounds (10, 14), indicating a different
modification of the tryptophan environment. A more
complex situation occurred in the case of 7-O-monosub-
stituted derivatives since both very low (11) or moderate
values (2, 5) were observed. A high maximal quenching
might be indicative of ATP site overlapping that would
be lowered by most hydrophobic substitutions, possibly
due to a shift toward the steroid-interacting region.
Only isoprenylated derivatives were able to inhibit
Pgp-mediated daunomycin efflux from leukemic K562/
R7 cells, leading to enhanced intracellular accumulation
of the drug (Table 1, right column). Whereas chrysin
produced a limited inhibition of the daunomycin efflux
(20% as compared to cyclosporin A), a much higher effect
was observed with dimethylallyl substitution at position
6 (12) or even higher at position 8 (13). As a matter of
fact, the effect of 8-dimethylallylchrysin (13) was stron-
ger than that of cyclosporin A, which is considered to
be one of the most potent modulators available. The
difference observed between the two positions of dim-
ethylallyl substitution might indicate some difference
in binding orientation. The inhibition was not further
increased by increasing the hydrophobicity of the sub-
stituent(s). This might indicate that such compounds
(14-16) could be hydrophobically trapped by the mem-
brane and exhibit lower availability for Pgp. On the
other hand, it was surprising that the mono-C-benzyl
derivatives (8, 9) did not produce any modulating effect,
suggesting that they do not reach the Pgp target. Either
penetration through, or retention by, the membrane or
interaction with other more reactive targets might be
responsible for this apparent lack of efficiency in Pgp-
overexpressing leukemic cells.
C-benzylation and C-isoprenylation of chrysin required,
as expected, smoother conditions (1 equiv of benzyl
chloride, prenyl bromide, or geranyl bromide). While
mono-C-benzylflavones (8, 9) as well as di-C-benzyl-
chrysin (10) were produced after 90 min reaction at
room temperature, the isoprenylated flavones (12-16)
were obtained under microwave irradiation, which
allowed us first to improve yields and second to decrease
the reaction times to a few minutes.15,16 Microwave-
induced synthesis was not used for C-benzylation be-
cause of degradation processes. O-Alkylations were
realized in DMF at room temperature in the presence
of K2CO3 and 2-bromopropane or benzyl chloride as
alkylating reagent, yielding O-isopropyl- (5) and O-
benzylchrysin (11), respectively. The reaction mixtures
were submitted to several chromatographic stages
yielding pure alkylated chrysin derivatives. The com-
pounds were identified on the basis of their spectroscopic
data. The alkylation position was determined by com-
It is important to mention that the modulating effects
of C-isoprenylated chrysin derivatives were observed at
concentrations which were not toxic for the cells, sug-
gesting that these compounds should be investigated in
vivo as potential Pgp modulators in tumor cells. Ad-
ditional works are in progress to further characterize
the inhibitory flavonoid-binding site on Pgp at the
molecular and cellular levels, with the aim to design
even more potent and specific inhibitors.
1
paring the H and 13C NMR chemical shifts with those
of the literature13-14,17 and by the 1H-13C heteronuclear
long-range correlations obtained in the 2D-NMR maps.
The binding of chrysin derivatives to the purified
C-terminal cytosolic domain, NBD2, of Pgp was directly
measured by quenching of tryptophan-related intrinsic
fluorescence, as described previously for unsubstituted
flavonoids11 or halogenated chalcones.18 Table 1 shows
that the increase in hydrophobicity of chrysin by alky-
lation with either methyl, isopropyl, benzyl, 3,3-dim-
ethylallyl, or geranyl substituents was correlated with
an increase in affinity for in vitro binding to the Pgp
cytosolic domain, as monitored by a lowering in KD
values. This effect was observed among compounds
monoalkylated at either position 6 (1 < 4, 8, 12 < 15),
position 8 (9 < 13 < 16), or position 7 (2 < 5 < 11). A
similar situation was encountered in the case of di- or
trisubstituted derivatives (3 < 6 < 7, 10, 14). In fact,
Exp er im en ta l Section
Gen er a l Ch em istr y Meth od s. NMR spectra were recorded
on DPX 300 (300 MHz for 1H, 75 MHz for 13C) instruments.
Solvents were used as internal references (CDCl3, DMSO-d6,
acetone-d6 and methanol-d4). EI-MS were obtained at 70 eV
using a Micromass ZAB2-SEQ spectrometer. The ionization
current and the chamber temperature were 200 µA and 200
°C, respectively. FAB- and CI-positive MS were recorded on a
Finnigan MAT 95 XL spectrometer. Elemental analyses were
performed by the Analytical Department of Marseille Univer-
sity, St. J e´roˆme, France. UV spectra were recorded in ethanol
using a Shimadzu UV-vis spectrophotometer model 1240.

C-Isoprenylation of Flavonoids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 765
Ta ble 1. Effects of Chrysin Alkylation on the Binding to Pgp Cytosolic Domain and on the Inhibition of Daunomycin Cellular Efflux
substituents
R2
binding to Pgpb
R3 hydrophobicity indexa log Pa ∆Fmax (%)
KD (µM)
flavone
R1
relative daunomycin accumulationc
chrysin
H
Me
H
Me
i-Pr
H
i-Pr
i-Pr
Bn
H
Bn
H
H
H
H
H
H
H
H
i-Pr
H
H
H
0.358
0.512
0.719
0.842
0.608
0.898
1.248
1.479
0.754
0.754
1.060
1.076
0.812
0.853
1.296
1.288
1.350
5.70
5.85
6.06
6.18
5.95
6.24
6.60
6.84
6.10
6.10
6.41
6.42
6.15
6.20
6.56
6.64
6.71
94.6 ( 3.1
46.6 ( 7.2
68.1 ( 9.9
57.3 ( 3.3
39.0 ( 1.6
53.2 ( 4.7
44.5 ( 2.9
13.1 ( 0.9
3.10 ( 0.97
6.3 ( 4.3
1.30 ( 0.35
0.21 ( 0.06
1.32 ( 0.34
0.28 ( 0.18
0.20
0.20
0.23
0.30
0.24
0.30
0.29
0.29
0.09
0.25
0.39
0.27
0.83
1.52
0.84
0.81
0.90
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Me
Me
H
i-Pr
i-Pr
i-Pr
H
H
H
Bn
H
H
38.4 ( 1.1 0.033 ( 0.008
72.4 ( 1.4
99.1 ( 3.8
0.34 ( 0.04
0.99 ( 0.14
Bn
Bn
H
40.9 ( 1.9 0.036 ( 0.017
29.3 ( 2.3 0.068 ( 0.044
3,3-DMA
H
3,3-DMA 3,3-DMA
Ger
H
H
83.4 ( 1.5
81.0 ( 1.9
0.30 ( 0.03
0.28 ( 0.04
3,3-DMA
H
H
H
21.5 ( 0.5 0.015 ( 0.003
48.2 ( 1.1 0.045 ( 0.005
67.0 ( 2.1 0.025 ( 0.005
H
Ger
a
Hydrophobicity indexes were established using the function RM ) log(1/Rf - 1) after TLC on C18 reverse-phase silica gel 20- × 20-cm
plates in methanol:water (8:2). Log P values were estimated by extrapolation from the curve log P ) f(RM) established with data obtained
b
for quercetine and platanetine according to previous results.22 Direct binding to Pgp recombinant cytosolic domain was measured by
quenching of intrinsic fluorescence. Data analysis with the Grafit program allowed the determination of dissociation constant and percent
maximal quenching of the intrinsic fluorescence. c Pgp-positive leukemic K562/R7 cells were incubated with daunomycin and assayed by
flow cytometry for intracellular remaining fluorescent drug in the absence or presence of cyclosporin A. Results of daunomycin-efflux
inhibition, leading to its intracellular accumulation, are expressed as a ratio of the effect obtained with 10 µM chrysin derivatives to the
effect of cyclosporin A (taken as a reference).
TLC was carried out using Merck silica gel Si60 F254 20- ×
20-cm plastic sheets and RP-18 F254s 20- × 20-cm aluminum
sheets and HPTLC on DIOL-F254s 10- × 10-cm sheets.
Analytical HPLC was carried out on a Thermo Separation
Products system equipped with a P-4000 quaternary gradient
pump system and a UV-6000LP photodiode array detector
using analytical 250- × 4.6-mm columns packed with Merck
Lichrospher 100Si 5 µm, Merck Lichrospher 100RP18 5 µm
or Merck Lichrospher 100DIOL 5 µm. Flash chromatography
was carried out using Merck silica gel 60, 70-230 mesh, or
Pharmacia Sephadex LH-20. MPLC were carried out using
Merck silica gel 15-25 µm, Lichroprep 100DIOL 40-63 µm,
or Lichroprep 100RP18 25-40 µm with UV detection at 254
and 366 nm. Microwave irradiation were realized in a Bluewind
1797 microwave oven at 120 and 240 W by successive periods
of 1 and 2.5 min.
was chromatographed on silica gel using a gradient of ethyl
acetate in hexane (0 to 100% in 300 min) affording pure
compounds 4 (29.8 mg; yield 0.85%), 6 (124 mg; yield 11.1%)
and 7 (12.8 mg; yield 2.5%).
O-Isop r op yla ted Ch r ysin Der iva tive 5. To 100 mg of
chrysin (0.392 mmol), 81 mg K2CO3 (0.590 mmol; 1.5 equiv)
and 12.6 mg of tetrabutylammonium bromide (0.0392 mmol;
0.1 equiv) in 10 mL of dimethylformamide was added 46 µL
of isopropyl bromide (0.49 mmol; 1.25 equiv). After 8 days at
room temperature under stirring, the mixture was diluted with
water, acidified with HCl (6 N), and extracted with ethyl
acetate. The organic layer was then concentrated to dryness
affording a brown residue; the latter was chromatographed
on silica gel using hexane:ethyl acetate (95:5 to 0:100 in 150
min) as mobile phase affording pure compound 5 (68.8 mg;
yield 59.1%).
Meth yla ted Ch r ysin Der iva tives 1-3. To 1 g of chrysin
(3.92 mmol) dissolved in 10 mL of methanol and 15 mL of 10%
aqueous tetramethylammonium hydroxide was added 1 g of
tetraethylammonium iodide (3.89 mmol; 1 equiv). After ho-
mogenization, 1.22 mL of methyl iodide (19.65 mmol; 5 equiv)
was added dropwise to the solution. The latter was refluxed
for 2 h. After return to room temperature, the mixture was
diluted with water, acidified with HCl (6 N), and extracted
with ethyl acetate. The organic layer was then concentrated
to dryness affording a brown residue; the latter was chromato-
graphed on silica gel using chloroform:acetic acid (95:5) as
mobile phase and was submitted to a DIOL MPLC using first
a gradient of chloroform in hexane (0 to 100% in 180 min) and
then a gradient of methanol in chloroform (0 to 50% in 60 min)
affording pure compounds 1 (47.4 mg; yield 4.5%) and 3 (138.7
mg; yield 12.4%). Compound 2 was obtained by methylation
of chrysin using anhydrous K2CO3 and dimethyl sulfate in
acetone. Its spectroscopic data were in accordance with those
of literature.17
Isop r op yla ted Ch r ysin Der iva tives 4, 6, 7. To 1 g of
chrysin (3.92 mmol) dissolved in 8 mL of methanol and 12 mL
of 10% aqueous tetramethylammonium hydroxide was added
1 g of tetraethylammonium iodide (3.89 mmol; 1 equiv). After
homogenization, 1.85 mL of isopropyl bromide (19.65 mmol; 5
equiv) was added dropwise to the solution. The latter was
refluxed for 48 h. After return to room temperature, the
mixture was diluted with water, acidified with HCl (6 N), and
extracted with ethyl acetate. The organic layer was then
concentrated to dryness affording a brown residue; the latter
C-Ben zyla ted Ch r ysin Der iva tives 8-10. To 1 g of
chrysin (3.92 mmol) dissolved in 10 mL of methanol and 20
mL of 10% aqueous tetramethylammonium hydroxide was
added 1 g of tetraethylammonium iodide (3.89 mmol; 1 equiv).
After homogenization, 0.45 mL of benzyl chloride (3.93 mmol;
1 equiv) was added dropwise to the solution. After 90 min at
room temperature, the mixture was diluted with water,
acidified with HCl (6 N), and extracted with ethyl acetate. The
organic layer was then concentrated to dryness affording a
brown residue; the latter was chromatographed on silica gel
using a gradient of ethyl acetate in hexane (10 to 100% in 350
min) affording two fractions, A and B. Fraction A was pure
compound 10 (36.1 mg; yield 2.1%). Fraction B was then
submitted to a DIOL MPLC using first a gradient of chloroform
in hexane (0 to 100% in 230 min) and then a gradient of
methanol in chloroform (0 to 50% in 60 min) affording two
fractions, C and D. Fraction C was pure compound 8 (75.4 mg;
yield 5.6%). Fraction D was submitted to another DIOL MPLC
using first a gradient of chloroform in hexane (0 to 100% in
300 min) affording pure compound 9 (56.3 mg; yield 4.2%).
O-Ben zyla ted Ch r ysin Der iva tive 11. To 100 mg of
chrysin (0.39 mmol), 81 mg K2CO3 (0.590 mmol, 1.5 equiv) and
12.6 mg of tetrabutylammonium bromide (0.039 mmol; 0.1
equiv) dissolved in 4 mL of dimethylformamide was added 56
µL of benzyl chloride (0.49 mmol; 1.25 equiv) in a 25-mL PTFE
flask. The latter was submitted to 8 successive 2.5-min
microwave irradiations at 240 W (pause time between 2
irradiation periods to return to 25 °C). The mixture was diluted
with water, acidified with HCl (6 N), and extracted with ethyl

766 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Comte et al.
acetate. The organic layer was then concentrated to dryness
affording a brown residue; the latter was chromatographed
on silica gel using hexane:ethyl acetate (95:5 to 6:4 in 180 min)
as mobile phase affording pure compound 11 (60.5 mg; yield
44.7%).
(C1a), 95.51 (C8), 100.95 (C6), 106.96 (C10), 107.25 (C3),
128.28 (C2′ + C6′), 131.01 (C3′ + C5′), 133.23 (C1′), 133.78
(C4′), 159.88 (C9), 164.11 (C5), 165.81 (C2), 166.20 (C7), 184.18
(C4); UV (λ nm (log ꢀ)) 269 (4.46), 311 (4.10); MS (CI+) m/z
297 [M + H]+; HRMS (C18H17O4) calcd 297.1128, found
297.1126. Anal. (C18H16O4) C, H.
P r en yla ted Ch r ysin Der iva tives 12-14. To 1 g of chrysin
(3.92 mmol) dissolved in 10 mL of methanol and 20 mL of 10%
aqueous tetramethylammonium hydroxide was added 1 g of
tetraethylammonium iodide (3.89 mmol; 1equiv). After ho-
mogenization, 450 µL of 3,3-dimethylallyl bromide (3.91 mmol;
1 equiv) was added dropwise to the solution. The latter was
submitted to 10 successive 1-min microwave irradiations at
120 W (pause time between 2 irradiation periods to return to
25 °C). The mixture was diluted with water, acidified with HCl
(6 N), and extracted with ethyl acetate. The organic layer was
then concentrated to dryness affording a brown residue; the
latter was chromatographed on silica gel using hexane:ethyl
acetate (8:2) as mobile phase affording two fractions, A and
B. Fraction A was pure compound 14 (35.5 mg; yield 2.3%).
Fraction B (269 mg) was submitted to a DIOL MPLC using a
gradient of i-PrOH in hexane (5 to 40% in 150 min) as mobile
phase, which afforded pure compounds 12 (90.2 mg; yield 7.2%)
and 13 (139.6 mg; yield 11%).
Ger a n yla ted Ch r ysin Der iva tives 15, 16. To a solution
of 1 g of chrysin (3.92 mmol) in 10 mL of methanol and 20 mL
of 10% aqueous tetramethylammonium hydroxide was added
1 g of tetraethylammonium iodide (3.89 mmol; 1 equiv). After
homogenization, 800 µL of geranyl bromide (3.92 mmol; 1
equiv) was added dropwise to the solution. The latter was
submitted to 6 successive 1-min microwave irradiations at 120
W (pause time between 2 irradiation periods to return to 25
°C). The mixture was diluted with water, acidified with HCl
(6 N), and extracted with ethyl acetate. The organic layer was
then concentrated to dryness affording a brown residue, which
was chromatographed on Sephadex LH-20 using acetone as
mobile phase and DIOL MPLC with hexane:CHCl3 (70:30 to
0:100 in 70 min) affording pure compounds 15 (320 mg; 0.8197
mmol; yield 20.91%) and 16 (150.5 mg; 0.3855 mmol; yield
9.83%).
1
6-C-7-O-Diisop r op ylch r ysin (6): H NMR (acetone-d6) δ
1.32 (d, 6H, H2b + H3b, J ) 7.1 Hz), 1.42 (d, 6H, H2a + H3a,
J ) 6.0 Hz), 3.62 (h, H1b, J ) 7.1 Hz), 4.85 (h, H1a, J ) 6.0
Hz), 6.81 (s, H3), 6.82 (s, H8), ca. 7.60 (m, 3H, H3′ + H4′ +
H5′), 8.08 (m, 2H, H2′ + H6′), 13.26 (s, 5OH); 13C NMR
(acetone-d6) δ 21.62 (C2b + C3b), 23.16 (C2a + C3a), 25.25
(C1b), 72.52 (C1a), 93.53 (C8), 106.76 (C10), 107.18 (C3),
120.40 (C6), 128.19 (C2′ + C6′), 130.96 (C3′ + C5′), 133.29
(C1′), 133.67 (C4′), 158.00 (C9), 160.76 (C5), 164.21 (C7), 165.42
(C2), 184.36 (C4); UV (λ nm (log ꢀ)) 274 (4.37), 318 (4.06); MS
(CI+) m/z 339 [M + H]+; HRMS (C21H23O4) calcd 339.1597,
found 339.1596. Anal. (C21H22O4) C, H.
6,8-Di-C-7-O-tr iisop r op ylch r ysin (7): 1H NMR (CDCl3)
δ 1.35 (d, 6H, H2a + H3a, J ) 6.2 Hz), 1.41 (d, 6H, H2b +
H3b, J ) 7.0 Hz), 1.48 (d, 6H, H2c + H3c, J ) 7.2 Hz), 3.41
(h, H1b, J ) 7.0 Hz), 3.65 (h, H1c, J ) 7.2 Hz), 4.21 (h, H1a,
J ) 6.2 Hz), 6.70 (s, H3), ca. 7.55 (m, 3H, H3′ + H4′ + H5′),
7.95 (m, 2H, H2′ + H6′), 13.19 (br s, 5OH); 13C NMR (CDCl3)
δ 20.03 (C2b + C3b), 21.30 (C2c + C3c), 22.34 (C2a + C3a),
25.53 (C1c), 26.28 (C1b), 77.25 (C1a), 105.89 (C3), 108.29 (C10),
119.50 (C8), 124.51 (C6), 126.45 (C2′ + C6′), 129.20 (C3′ +
C5′), 131.67 (C4′), 131.81 (C1′), 154.11 (C9), 158.60 (C5), 158.98
(C7), 164.07 (C2), 183.71 (C4); UV (λ nm (log ꢀ)) 279 (4.53);
MS (CI+) m/z 381 [M + H]+; HRMS (C24H29O4) calcd 381.2067,
found 381.2066. Anal. (C24H28O4‚0.40hexane‚0.60H2O) C, H.
6-C-Ben zylch r ysin (8): 1H NMR (DMSO-d6) δ 3.89 (s, 2H,
H7a), 6.61 (s, H8), 6.97 (s, H3), ca. 7.13 (m, H4a), ca. 7.24 (m,
4H, H2a + H3a + H5a + H6a), 7.57 (m, 3H, H3′ + H4′ + H5′),
8.06 (m, 2H, H2′ + H6′), 11.05 (s, 7OH), 13.20 (s, 5OH); 13C
NMR (DMSO-d6) δ 27.36 (C7a), 93.37 (C8), 103.70 (C10),
105.03 (C3), 110.79 (C6), 125.51 (C4a), 126.31 (C2′ + C6′),
127.99 (C2a + C6a), 128.16 (C3a + C5a), 129.05 (C3′ + C5′),
130.68 (C1′), 131.87 (C4′), 140.57 (C1a), 155.45 (C9), 158.63
(C5), 162.19 (C7), 162.94 (C2), 181.85 (C4); UV (λ nm (log ꢀ))
273 (4.39), 320 (4.05); MS (FAB+) m/z 345 [M + H]+; HRMS
(C22H17O4) calcd 345.1128, found 345.1126. Anal. (C22H16O4‚
0.80MeOH) C, H.
6-C-Meth ylch r ysin (1): 1H NMR (DMSO-d6) δ 1.99 (s, 3H,
H1a), 6.59 (s, H8), 6.95 (s, H3), ca. 7.58 (m, 3H, H3′ + H4′ +
H5′), 8.05 (m, 2H, H2′ + H6′), 13.06 (s, 5OH); 13C NMR
(DMSO-d6) δ 7.35 (C1a), 93.12 (C8), 103.59 (C10), 105.06 (C3),
107.04 (C6), 126.33 (C2′ + C6′), 129.12 (C3′ + C5′), 130.78
(C1′), 131.91 (C4′), 155.04 (C9), 158.45 (C5), 162.36 (C7), 162.88
(C2), 181.80 (C4); UV (λ nm (log ꢀ)) 272 (4.29), 319 (4.01); MS
(CI+) m/z 269 [M + H]+; HRMS (C16H13O4) calcd 269.0815,
found 269.0813. Anal. (C16H12O4‚0.60H2O) C, H.
8-C-Ben zylch r ysin (9): 1H NMR (DMSO-d6) δ 4.12 (s, 2H,
H7a), 6.38 (s, H6), 6.96 (s, H3), ca. 7.13 (m, H4a), ca. 7.24 (m,
4H, H2a + H3a + H5a + H6a), 7.57 (m, 3H, H3′ + H4′ + H5′),
7.94 (m, 2H, H2′ + H6′), 11.01 (s, 7OH), 12.83 (s, 5OH); 13C
NMR (DMSO-d6) δ 27.74 (C7a), 98.50 (C6), 103.89 (C10),
104.98 (C3), 105.74 (C8), 125.74 (C4a), 126.20 (C2′ + C6′),
127.82 (C2a + C6a), 128.22 (C3a + C5a), 129.09 (C3′ + C5′),
130.80 (C1′), 131.90 (C4′), 140.43 (C1a), 154.76 (C9), 159.46
(C5), 162.18 (C7), 162.99 (C2), 182.09 (C4); UV (λ nm (log ꢀ))
274 (4.29); MS (FAB+) m/z 345 [M + H]+; HRMS (C22H17O4)
calcd 345.1128, found 345.1126. Anal. (C22H16O4‚MeOH) C, H.
6-C-7-O-Dim eth ylch r ysin (3): 1H NMR (acetone-d6) δ 2.06
(s, 3H, H1a), 4.00 (s, 3H, H1b), 6.82 (s, H8), 6.83 (s, H3), ca.
7.61 (m, 3H, H3′ + H4′ + H5′), 8.09 (m, 2H, H2′ + H6′), 13.00
(s, 5OH); 13C NMR (acetone-d6) δ 8.44 (C1a), 57.64 (C1b), 91.74
(C8), 106.91 (C10), 107.30 (C3), 110.17 (C6), 128.19 (C2′ + C6′),
130.98 (C3′ + C5′), 133.25 (C1′), 133.70 (C4′), 158.01 (C9),
160.22 (C5), 165.52 (C2), 165.59 (C7), 184.20 (C4); UV (λ nm
(log ꢀ)) 272 (4.23), 313 (3.94); MS (CI+) m/z 283 [M + H]+;
HRMS (C17H15O4) calcd 283.0971, found 283.0970. Anal.
(C17H14O4) C, H.
1
6,8-C-Diben zylch r ysin (10): H NMR (DMSO-d6) δ 4.04
(s, 2H, H7b), 4.27 (s, 2H, H7a), 6.99 (s, H3), ca. 7.14 (m, 2H,
H4a + H4b), ca. 7.25 (m, 8H, H2a + H3a + H5a + H6a +H2b
+ H3b + H5b + H6b), 7.55 (m, 3H, H3′ + H4′ + H5′), 7.91 (m,
2H, H2′ + H6′), 10.16 (s, 7OH), 13.22 (s, 5OH); 13C NMR
(DMSO-d6) δ 27.60 (C7b), 28.10 (C7a), 104.10 (C10), 104.97
(C3), 105.31 (C8), 111.22 (C6), 125.60 (C4b), 125.81 (C4a),
126.21 (C2′ + C6′), 127.73 (C2a + C6a), 128.05 (C2b + C6b or
C3a + C5a), 128.08 (C3a + C5a or C2b + C6b), 128.26 (C3b +
C5b), 129.08 (C3′ + C5′), 130.80 (C1′), 131.91 (C4′), 140.26
(C1a), 140.39 (C1b), 153.23 (C9), 157.08 (C5), 159.97 (C7),
162.99 (C2), 182.24 (C4); UV (λ nm (log ꢀ)) 275 (4.16); MS (CI+)
m/z 435 [M + H]+; HRMS (C29H23O4) calcd 435.1597, found
435.1596. Anal. (C29H22O4‚0.60H2O‚2hexane) C, H.
1
6-C-Isop r op ylch r ysin (4): H NMR (DMSO-d6) δ 1.28 (d,
6H, H2a + H3a, J ) 7.1 Hz), 3.47 (h, H1a, J ) 7.1 Hz), 6.55
(s, H8), 6.94 (s, H3), ca. 7.57 (m, 3H, H3′ + H4′ + H5′), 8.05
(m, 2H, H2′ + H6′), 10.89 (s, 7OH), 13.29 (s, 5OH); 13C NMR
(DMSO-d6) δ 20.99 (C2a + C3a), 23.91 (C1a), 94.49 (C8),
104.64 (C10), 105.90 (C3), 117. 40 (C6), 127.20 (C2′ + C6′),
130.01 (C3′ + C5′), 131.61 (C1′), 132.80 (C4′), 155.91 (C9),
159.77 (C5), 163.36 (C7), 163.68 (C2), 182.94 (C4); UV (λ nm
(log ꢀ)) 273 (4.06), 322 (3.82); MS (CI+) m/z 297 [M + H]+;
HRMS (C18H17O4) calcd 2971128, found 297.1126. Anal.
(C18H16O4‚0.40hexane) C, H.
7-O-Ben zylch r ysin (11): 1H NMR (DMSO-d6) δ 5.17 (s, 2H,
H7a), 6.48 (d, H6, J ) 2.2 Hz), 6.60 (d, H8, J ) 2.2 Hz), 6.69
(s, H3), ca. 7.44 (m, 5H, H2a + H3a + H4a + H5a + H6a),
7.54 (m, 3H, H3′ + H4′ + H5′), 7.90 (m, 2H, H2′ + H6′); 13C
NMR (DMSO-d6) δ 70.44 (C7a), 93.52 (C8), 98.94 (C6), 105.90
(C10 + C3), 126.29 (C2′ + C6′), 127.47 (C2a + C6a), 128.37
7-O-Isop r op ylch r ysin (5): 1H NMR (acetone-d6) δ 1.25 (d,
6H, H2a + H3a, J ) 6.0 Hz), 4.69 (h, H1a, J ) 6.0 Hz), 6.19
(d, H6, J ) 2.2 Hz), 6.58 (d, H8, J ) 2.2 Hz), 6.68 (s, H3), ca.
7.48 (m, 3H, H3′ + H4′ + H5′), 7.95 (m, 2H, H2′ + H6′), 12.70
(s, 5OH); 13C NMR (acetone-d6) δ 23.12 (C2a + C3a), 72.53

C-Isoprenylation of Flavonoids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 767
(C4a), 128.75 (C3a + C5a), 129.07 (C3′ + C5′), 131.29 (C1′),
131.84 (C4′), 135.72 (C1a), 157.75 (C9), 162.19 (C5), 164.02
(C2), 164.65 (C7), 182.47 (C4); UV (λ nm (log ꢀ)) 269 (4.49),
309 (4.13); MS (CI+) m/z 345 [M + H]+; HRMS (C22H17O4) calcd
345.1128, found 345.1126. Anal. (C22H16O4) C, H.
6-C-(3,3-Dim eth yla llyl)ch r ysin (12): 1H NMR (DMSO-d6)
δ 1.63 (br s, H4a), 1.73 (brs, H5a), 3.23 (d, 2H1a, J ) 7.2 Hz),
5.18 (br t, H2a, J ) 7.2 Hz), 6.58 (s, H8), 6.96 (s, H3), 7.59 (m,
3H, H3′ + H4′ + H5′), 8.06 (m, 2H, H2′ + H6′), 10.93 (s, 7OH),
13.09 (s, 5OH); 13C NMR (DMSO-d6) δ 18.55 (C5a) 21.86 (C1a),
26.34 (C4a), 94.20 (C8), 104.62 (C10), 105.92 (C3),111.99 (C6),
122.97 (C2a), 127.23 (C2′ + C6′), 130.00 (C3′ + C5′), 131.58
(C1′ or C3a), 131.65 (C1′ or C3a), 132.80 (C4′), 156.07 (C9),
159.18 (C5), 162.95 (C7), 163.75 (C2), 182.77 (C4); UV (λ nm
(log ꢀ)) 273 (4.47), 320 (4.13); MS (EI) m/e 322 [M]+; HRMS
(C20H18O4) calcd 322.1205, found 322.1208. Anal. (C20H18O4‚
0.80MeOH) C, H.
8-C-(3,3-Dim eth yla llyl)ch r ysin (13): 1H NMR (DMSO-d6)
δ 1.63 (br s, H4a), 1.75 (br s, H5a), 3.45 (d, 2H1a, J ) 6.8 Hz),
5.20 (br t, H2a, J ) 6.8 Hz), 6.31 (s, H6), 6.95 (s, H3), 7.59 (m,
3H, H3′ + H4′+ H5′), 8.04 (m, 2H, H2′ + H6′), 10.86 (s, 7OH),
12.76 (s, 5OH); 13C NMR (DMSO-d6) δ 18.68 (C5a), 22.19 (C1a),
26.30 (C4a), 99.38 (C6), 104.77 (C10), 105.84 (C3), 107.10 (C8),
123.26 (C2a), 127.15 (C2′ +C6′), 130.04 (C3′ + C5′), 131.92
(C1′ +C3a), 132.85 (C4′), 155.50 (C9), 159.94 (C5), 162.72 (C7),
163.95 (C2), 183.04 (C4); UV (λ nm (log ꢀ)) 275 (4.41); MS (EI)
m/e 322 [M]+; HRMS (C20H18O4) calcd 322.1205, found 322.1208.
Anal. (C20H18O4‚0.20MeOH) C, H.
formed at 25 °C by using either a SLM-Aminco 8000C or a
PTI QM-1 spectrofluorimeter. The tryptophan-specific intrinsic
fluorescence of 0.5 µM recombinant domain in 1.2-2.0 mL of
20 mM potassium phosphate at pH 6.8 containing 0.5 M NaCl,
20% glycerol and 0.01% methyl 6-O-(N-heptylcarbamoyl)-R-
D-glucopyranoside was scanned from 310 to 360 nm upon
excitation at 295 nm, integrated and corrected for buffer
contribution. The binding of chrysin and its derivatives was
monitored by the quenching of intrinsic fluorescence produced
by successive additions of aliquots up to 20 µM. The modifica-
tions were corrected for the inner-filter effect determined under
the same conditions with 0.5 µM N-acetyltryptophanamide.
Curve fitting of binding related to fluorescence decrease was
performed with the Grafit program (Erithacus software) as
previously described.19-21
For inhibition of Pgp-mediated drug efflux, 1 million K562/
R7 human leukemic cells expressing high levels of Pgp were
incubated for 1 h at 37 °C in 1 mL of RPMI 1640 medium
containing a final concentration of 10 µM daunomycin, in the
presence or absence of modulators. Cells were then washed
three times with ice-cold phoshate buffer saline and main-
tained on ice until analysis by flow cytometry on a FACS-II
(Becton-Dickinson Corp., Mountain View, CA). Assays were
performed in duplicate, in at least three separate experiments.
Cyclosporin A, a potent modulator of Pgp was used as a
positive control at a final concentration of 2 µM. Control
experiments showed that cells incubated with chrysin deriva-
tives in the absence of daunomycin did not exhibit enhanced
fluorescence (data not shown). The ability of chrysin deriva-
tives to inhibit Pgp-mediated drug efflux at a concentration
of 10 µM was quantified by comparing the shift in fluorescence
induced by these compounds to the one obtained with cy-
closporin A. In separate experiments, cell viability was tested
in cells exposed to 10 µM chrysin derivatives; it was shown to
be reduced by less than 5% in comparison to untreated cells
(data not shown).
6,8-Di-C-(3,3-dim eth ylallyl)ch r ysin (14): 1H NMR (DMSO-
d6) δ 1.63 (br s, H4a + H4b), 1.74 (s, H5a), 1.77 (s, H5b), ca.
3.33 (m, 2H1a, overlapping H2O signal), 3.56 (d, 2H1b, J )
6.1 Hz), 5.15 (m, 2H, H2a + H2b), 6.96 (s, H3), 7.59 (m, 3H,
H3′ + H4′ +H5′), 8.03 (m, 2H, H2′ + H6′), 9.77 (s, 7OH), 13.06
(s, 5OH); 13C NMR (DMSO-d6) δ 18.65 (C5a), 18.82 (C5b), 22.20
(C1a), 22.64 (C1b), 26.28 (C4b), 26.36 (C4a), 104.97 (C10),
105.77 (C3), 107.54 (C8), 112.61 (C6), 123.09 (C2a), 123.57
(C2b), 127.15 (C2′ +C6′), 130.01 (C3′ + C5′), 131.71 (C3a),
131.94 (C3b or C1′), 131.96 (C1′ or C3b), 132.81 (C4′), 153.55
(C9), 157.13 (C5), 160.08 (C7), 163.83 (C2), 183.16 (C4); UV (λ
nm (log ꢀ)) 279 (4.43), 322 (3.98); MS (EI) m/e 390 [M]+; HRMS
(C25H26O4) calcd 390.1831, found 390.1830. Anal. (C25H26O4‚
0.30MeOH) C, H.
Ack n ow led gm en t. This work was supported by
grants from the Re´gion Rhoˆne-Alpes (Emergence No.
96003891) and the European Community (Polybind
Project QLK1-1999-00505). J ean-Baptiste Daskiewicz
was the recipient of a fellowship from the French
Ministry of Research and Gwenae¨lle Conseil from the
Comite´ de Haute-Savoie de la Ligue Nationale Contre
le Cancer.
1
6-C-Ger a n ylch r ysin (15): H NMR (DMSO-d6) δ 1.51 (br
s, H10a), 1.57 (br s, H9a), 1.73 (br s, H8a), 1.93 (m, 2H4a),
1.96 (m, 2H5a), 3.23 (d, 2H1a, J ) 7.0 Hz), 5.03 (br t, H6a, J
) 6.6 Hz), 5.18 (br t, H2a, J ) 7.0 Hz), 6.58 (s, H8), 6.95 (s,
H3), 7.58 (m, 3H, H3′ + H4′+ H5′), 8.05 (dd, 2H, H2′ + H6′, J
) 8.0 and 1.8 Hz), 10.93 (s, 7OH), 13.08 (s, 5OH); 13C NMR
(DMSO-d6) δ 16.77 (C8a), 18.35 (C10a) 21.80 (C1a), 26.29
(C9a), 27.01 (C5a), ca. 40.00 (C4a, overlapping solvent signal)
94.18 (C8), 104.61 (C10), 105.91 (C3), 112.02 (C6), 122.77
(C2a), 124.93 (C6a), 127.21 (C2′ + C6′), 129.99 (C3′ + C5′),
131.48 (C7a), 131.64 (C1′), 132.78 (C4′), 135.10 (C3a), 156.06
(C9), 159.21 (C5), 162.97 (C7), 163.74(C2), 182.75 (C4); UV (λ
nm (log ꢀ)) 274 (4.42), 320 (4.12); MS (EI) m/e 390 [M]+; HRMS
(C25H26O4) calcd 390.1831, found 390.1830. Anal. (C25H26O4‚
0.90MeOH) C, H.
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8-C-Ger a n ylch r ysin (16): H NMR (DMSO-d6) δ 1.42 (br
s, H10a), 1.49 (br s, H9a), 1.75 (br s, H8a), 1.94 (m, 4H, 2H4a
+ 2H5a), 3.46 (d, 2H1a, J ) 6.6 Hz), 4.94 (m, H6a), 5.19 (br t,
H2a, J ) 6.6 Hz), 6.32 (s, H6), 6.96 (s, H3), 7.58 (m, 3H, H3′
+ H4′ + H5′), 8.04 (dd, 2H, H2′ + H6′, J ) 8.2 and 1.7 Hz),
10.86 (s, 7OH), 12.76 (s, 5OH); 13C NMR (DMSO-d6) δ 15.46
(C8a), 18.26 (C10a), 22.08 (C1a), 26.18 (C9a), 26.87 (C5a), ca.
40.00 (C4a, overlapping solvent signal), 99.36 (C6), 104.79
(C10), 105.78 (C3), 107.17 (C8), 123.20 (C2a), 124.81 (C6a),
127.15 (C2′ + C6′), 129.98 (C3′ + C5′), 131.46 (C7a), 131.89
(C1′), 132.84 (C4′), 135.41 (C3a), 155.56 (C9), 159.92 (C5),
162.68 (C7), 163.95 (C2), 183.04 (C4); UV (λ nm (log ꢀ)) 275
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