Synthesis, cytotoxicity, and antioxidative activity of minor prenylated chalcones from Humulus lupulus

Article abstract of DOI:10.1021/np800188b

The minor hop (Humulus lupulus) chalcones 3′-geranylchalconaringenin (3), 5′-prenylxanthohuraol (4), flavokawin (5), xanthohumol H (8), xanthohumol C (9), and 1″,2″-dihydroxanthohumol C (10) were synthesized. The non-natural chalcones 3′-geranyl-6′-O- methylchalconaringenin (2), 3′-methylflavokawin (6), and 2′-0-methyl-3′-prenylchalconaringenin (7) were also synthesized. Cytotoxicity was investigated in HeLa cells, and these compounds all had IC ₅₀ values comparable to xanthohumol (8.2-19.2 μM). The ORAC-fluorescein assay revealed potent antioxidative activity for 7 and 8 with 5.2 and 4.8 Trolox equivalents, respectively.

Full text of DOI:10.1021/np800188b

J. Nat. Prod. 2008, 71, 1237–1241
1237
Synthesis, Cytotoxicity, and Antioxidative Activity of Minor Prenylated Chalcones from
Humulus lupulus
Susanne Vogel and Jo¨rg Heilmann*
Chair of Pharmaceutical Biology, Institute of Pharmacy, UniVersity of Regensburg, UniVersita¨tsstrasse 31, 93053 Regensburg, Germany
ReceiVed March 25, 2008
The minor hop (Humulus lupulus) chalcones 3′-geranylchalconaringenin (3), 5′-prenylxanthohumol (4), flavokawin (5),
xanthohumol H (8), xanthohumol C (9), and 1′′,2′′-dihydroxanthohumol C (10) were synthesized. The non-natural
chalcones 3′-geranyl-6′-O-methylchalconaringenin (2), 3′-methylflavokawin (6), and 2′-O-methyl-3′-prenylchalconar-
ingenin (7) were also synthesized. Cytotoxicity was investigated in HeLa cells, and these compounds all had IC50 values
comparable to xanthohumol (8.2-19.2 µM). The ORAC-fluorescein assay revealed potent antioxidative activity for 7
and 8 with 5.2 and 4.8 Trolox equivalents, respectively.
Hop (Humulus lupulus L., Cannabaceae) cones contain many
structurally related prenylated chalcones, with xanthohumol (1)
being the most abundant one. The minor compounds include
xanthogalenol, 4′-O-methylxanthohumol, 5′-prenylxanthohumol (4),
and xanthohumols B, C (9), D, E, and H (8).1,2 Due to the good
availability of 1, which can be isolated from hop cones or
synthesized in good yields,3,4 its pharmacological characterization
advanced significantly, and it has been shown to exhibit an
interesting spectrum of pharmacological effects. In addition to
antiproliferative activity against different cancer cell lines,5–7 1 also
exhibited apoptotic6,8 and chemopreventive activity due to protec-
tive effects against carcinogens or pro-carcinogens.9,10 Several in
vitro studies substantiated effects on enzymes and transcription
factors involved in the genesis of cancer,8,11–15 and in vivo growth
inhibition of a vascular tumor has been reported.13 Pharmacological
data concerning the minor related compounds are scarce due to
limited availability via isolation. Recently we described a synthetic
route to the minor hop compounds desmethylxanthohumol, xan-
thogalenol, and 4′-methylxanthohumol,3 and Lee and Xia reported
the synthesis of 9.16 Manageable synthetic approaches are needed
to provide quantities of minor hop chalcones and potential phase I
metabolites available for pharmacological testing. Here we report
on the synthesis of 3′-geranylchalconaringenin (3), 5′-prenylxan-
thohumol (4), xanthohumol H (8), xanthohumol C (9), 1′′,2′′-
dihydroxanthohumol C (10), and the non-prenylated chalcone
flavokawin (5). The structurally related non-natural compounds 3′-
geranyl-6′-O-methylchalconaringenin (2), 3′-methylflavokawin (6),
and 2′-O-methyl-3′-prenylchalconaringenin (7) were synthesized to
analyze the relevance of varying substitution for the pharmacologi-
cal activity of prenylated chalcones. Compounds 8 and 10 have
been isolated from hops,2 but were also reported as metabolites
from rat feces after oral application of 1.17 The cytotoxic and
antioxidative activity of all compounds was determined against
HeLa cells and in the ORAC assay, respectively.
Results and Discussion
The general strategy utilized 6-hydroxy-2,4-dimethoxymethyl-
acetophenone (11), prepared from 2,4,6-trihydroxyacetophenone,
as the starting compound for all nine chalcones. By changing the
sequence of the standard reactions, reflux with prenyl or geranyl
bromide in acetone-K2CO3, Claisen rearrangement in N,N-dim-
ethylaniline, methylation with dimethyl sulfate by using the phase
transfer catalyst tetrabutylammonium iodide, MOM protection and
deprotection, most of the B ring fragments were synthesized. A
further concluding reaction was necessary only for xanthohumol
H (8), xanthohumol C (9), and 1′′,2′′-dihydroxanthohumol C (10)
to synthesize the ring fragments 25, 27, and 28 (Schemes 1 and 2,
* Corresponding author. Tel: +49 941 943 4759. Fax: +49 941 943
4990. E-mail: joerg.heilmann@chemie.uni-regensburg.de.
10.1021/np800188b CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/09/2008

1238 Journal of Natural Products, 2008, Vol. 71, No. 7
Vogel and Heilmann
Scheme 1. Key Steps of the Synthetic Route for Compounds
2-4a
methylpropionamide) dihydrochloride (AAPH) as free radical
initiator.20 All compounds showed moderate to high activities of
1.7 to 5.2 Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid) equivalents in a concentration range between 0.1 and
1.0 µM (Table 1). For the most active compounds, 5, 7, and 8 (4.0
( 0.5, 5.2 ( 0.8, and 4.8 ( 0.1 Trolox equivalents, respectively),
this is comparable to other strong antioxidants such as ferulic acid
(4.4 ( 0.2 Trolox equivalents, concentration range 0.4-1.3 µM),
but lower in comparison to the very potent quercetin (10.5 ( 0.4
Trolox equivalents, concentration range 0.2-0.6 µM) and caffeic
acid (6.6 ( 0.2 Trolox equivalents, concentration range 0.2-1.0
µM).20
Interestingly, some minor secondary metabolites and phase I
metabolites of 1 are more active in comparison to 1 itself. This
makes their broad pharmacological characterization as well as the
synthesis of further metabolites and structurally related non-natural
derivatives worthwhile. Compound 6 was synthesized as a first
representative of a set of chalcones with other substituents on 3′,
and ongoing investigations will focus on the synthesis of non-natural
chalcones with increased antioxidant activity. Of special interest
is the pharmacological activity of 8, which is not only a minor
secondary metabolite in hop cones but also a phase I metabolite of
1 detected in rat feces after oral application of 1000 mg/kg 1.17
Introduction of an OH group increased the activity in comparison
to 1, and since this reaction often occurs in the metabolism of 1 (at
different positions of the molecule),17,21 synthesis and pharmaco-
logical testing of these compounds could be interesting. It is also
noteworthy that the presence of only 6′-O- and 4′-O-methylated
prenylated chalcones has been reported in hop cones, whereas 2′-
O-methyl derivatives generally do not occur. The biological activity
of 7, which we named xanthoflorianol, showed antioxidant activity
in the ORAC test significantly higher in comparison to 1, xan-
thogalenol, and 4′-O-methylxanthohumol.3 Thus, the present in-
vestigation revealed a strategy for the synthesis of various prenylated
hop chalcones, xanthohumol phase I metabolites, and structurally
related non-natural chalcones, and all investigated compounds
showed interesting pharmacological activity.
Experimental Section
General Experimental Procedures. 2,4,6-Trihydroxyacetophenone
hydrate (98%), MOM-Br (tech. 90%), prenyl (96%) and geranyl
bromide (95%), and 2,3-dichloro-5,6-dicyan-1,4-benzochinone (DDQ,
98%) were obtained from Aldrich. Dimethyl sulfate (99%) was
purchased from Merck, and N,N-dimethylaniline (99%) from Janssen.
Melting points were measured on a Bu¨chi melting point B-545 apparatus
(uncorrected). UV spectra were recorded on a Cary 50 Scan (Varian;
in MeOH, UVA-sol). All 1H and 13C NMR experiments were recorded
in acetone-d6 (Deutero GmbH, purity 99.8%) or CDCl3 (Deutero GmbH,
purity 99.8%) on a Bruker Avance 300 (operating at 300.13 MHz for
1H and 75.47 MHz for 13C) at 300.0 K and referenced against residual
non-deuterated solvent. The 2D spectra were measured on a Bruker
a (a) Acetone, K2CO3, geranyl or prenyl bromide, 24 h (reflux); (b) N,N-
dimethylaniline, 3 h (reflux), argon atmosphere, 200 °C; (c) dimethyl sulfate,
NaOH, CH2Cl2-H2O, 3:2, tetrabutylammonium iodide (phase transfer catalyst),
24 h (room temperature); (d) acetone, 3 N HCl, 4 h (0 °C).
1
Avance 400 (operating at 400.13 MHz for H and 100.61 MHz for
13C) at 300.0 K. HR- and LREIMS (70 eV) were measured on a MAT
710A. Column chromatography (CC) was always performed with
normal phase silica gel (Firma Merck, 0.063-0.200 mm); TLC analysis
was done with silica gel 60 F254 plates (Merck) using a UV lamp for
detection (254 and 365 nm). The optical density in the MTT cytotoxicity
assay was measured at 560 nm using a microplate reader (Tecan).
Preparation of Compounds: MOM Protection. A mixture of 2,4,6-
trihydroxyacetophenone (4.7 g, 1 equiv), anhydrous K2CO3 (24.2 g, 7
equiv), and MOM bromide (dropwise addition of 5.0 g, 2.5 equiv) was
stirred and refluxed in acetone (160 mL) for 3 h. The reaction mixture
was cooled to room temperature and filtered. The filtrate was evaporated
and the residue subjected to CC with petroleum ether-EtOAc, 1:1, as
eluent to yield intermediate 11 (68%, colorless oil, 1H and EIMS
identical to literature data.4 The same procedure was applied for
protection of 4-hydroxybenzaldehyde (2.5 g, 1 equiv) using 1.3 equiv
of MOM bromide and 4 equiv of anhydrous K2CO3 to the protected
aldehyde in 90% yield.3
Experimental Section). For 4 a detour via deprotection of 15 to 16
was necessary to enable the double Claisen rearrangement of the
diether 17. The complete 1H and 13C NMR data of 4 have not been
reported, probably due to its isolation in very limited amounts,22
and are given in the Experimental Section. For 8 we report here
the hitherto unpublished 13C NMR data. All assignments were
justified by 2D NMR (1H,1H COSY, 1H,13C HSQC, 1H,13C HMBC,
1
and H,1H NOESY) experiments.
Cytotoxicity of all synthesized chalcones was tested against a
HeLa cell line using the MTT cell proliferation assay and compared
to the positive control 1 (Table 1).18,19 Activity of all chalcones
was similar, with 3′-geranyl-6′-O-methylchalconaringenin (2) being
the most (IC50 8.2 ( 1.2 µM) and flavokawin (5) the least toxic
compound (IC50 19.2 ( 1.2 µM).
To assay the antioxidative activity of the chalcones, the ORAC
(oxygen radical absorbance capacity)-fluorescein assay was used,
generating peroxyl radicals by the application of 2,2′-azobis(2-
Aldol Coupling. Respective ring fragments (1 equiv) were stirred
in cold (0 °C) EtOH-H2O, 3:2, or EtOH-THF, 1:1 (for 7 and 9),

Prenylated Chalcones from Humulus lupulus
Journal of Natural Products, 2008, Vol. 71, No. 7 1239
Scheme 2. Key Steps of the Synthetic Route for Compounds 7-10a
a (a) 3 N HCl, MeOH, 15 min (reflux); (b) acetone, K2CO3, Br-CH2-O-CH3, 3 h (reflux); (c) dimethyl sulphate, NaOH, CH2Cl2-H2O, 3:2, tetrabutylammonium
iodide (phase transfer catalyst), 24 h (room temperature); (d) Hg(OAc)2-H2O, tetrahydrofuran, NaBH4, 3 N NaOH, 60 min (<30 °C); (e) benzene, dioxane (both
dried), DDQ, 3 h (reflux); (f) tetrahydrofuran, formic acid, 3 h (reflux).
160.6 (C-4), 143.2 (C-ꢀ), 134.8 (C-3′′), 131.6 (C-8′′), 131.3 (C-2,6),
128.2 (C-1), 125.5 (C-R), 125.2 (C-7′′), 123.9 (C-2′′), 116.8 (C-3,5),
108.9 (C-3′), 106.3 (C-1′), 91.6 (C-5′), 56.2 (OCH3-6′), 40.6 (C-4′′),
27.5 (C-6′′), 25.8 (C-10′′), 21.8 (C-1′′), 17.7 (C-9′′), 16.3 (C-4′′); EIMS
(pos mode) m/z 422 [M]+ (78), 407 [M - CH3]+ (6), 379 [M - C3H7]+
(8), 353 [M - C5H9]+ (49), 299 (75), 233 (87), 179 (100); HREIMS
m/z 422.2093 (calcd for C26H30O5 422.2093).
Table 1. Cytotoxic (HeLa cells, 150.000 cells/mL, 72
h
incubation, IC50 values in µM ( SD, n ) 8) and Antioxidative
(Trolox equivalents, concentration range 0.1-1 µM) Activity of
Chalcones 1-10
compound
IC50 (µM)
Trolox equiv
1
2
3
4
5
6
7
8
9
10
9.4 ( 1.4
8.2 ( 1.2
18.2 ( 1.7
9.7 ( 2.1
19.2 ( 1.2
12.2 ( 1.5
10.4 ( 1.5
9.2 ( 1.5
12.5 ( 1.7
15.4 ( 1.4
2.3 ( 0.2
3.4 ( 0.2
2.3 ( 0.1
1.9 ( 0.1
4.0 ( 0.5
2.1 ( 0.4
5.2 ( 0.8
4.8 ( 0.1
1.8 ( 0.1
1.7 ( 0.2
Geranylbromide (2.54 g, 1.5 equiv), K2CO3 (4.31 g, 4 equiv), and
11 (2 g, 1 equiv) were refluxed in 150 mL of acetone for 24 h with
stirring to give 12 after cooling to room temperature, filtration, and
CC (petroleum ether-EtOAc, 6:1) of the evaporated filtrate (yield 78%).
Compound 12 (1.4 g) was refluxed and stirred for 3 h (200 °C) in
N,N-dimethylaniline (4 mL) under argon atmosphere. After cooling to
room temperature, the reaction mixture was acidified with 3 N HCl
and extracted three times with EtOAc. The organic phases were
combined, washed with a solution of Na2CO3, dried over Na2SO4, and
evaporated. CC of the residue with petroleum ether-EtOAc, 4:1,
yielded 13 (23%). A mixture of 13 (220 mg, 1 equiv), dimethyl sulfate
(1.1 equiv, dropwise addition), tetrabutylammonium iodide (0.1 equiv),
and NaOH (1.4 equiv) was stirred in 10 mL of CH2Cl2-H2O, 3:2, for
24 h at room temperature. Separation of organic and aqueous phases,
extraction of the aqueous phase with CH2Cl2, and CC of the residue of
(all dried) the combined and evaporated CH2Cl2 phases with petroleum
ether-EtOAc, 2:1, yielded 14 (89%).
under an argon atmosphere together with the MOM-protected 4-hy-
droxybenzaldehyde (1.1 equiv) and KOH (dropwise addition of 50
equiv) initially for 1 h in an ice bath and afterward for 72 h at room
temperature. The reaction mixture was poured into ice-water acidified
with 3 N HCl and extracted three times with CH2Cl2 or EtOAc. The
organic phases were combined, washed with water, dried over Na2SO4,
and evaporated. The residue was subjected to CC (different petroleum
ether-EtOAc mixtures: 1:1, 3:2, 2:1, and 3:1, respectively) and yielded
the respective protected chalcones.3
Deprotection. For deprotection, all compounds were dissolved in
MeOH, and 3 N HCl was added to give a ratio of 5:1 MeOH-3 N
HCl. After 15 min under reflux the reaction mixture was poured into
ice-water and extracted three times with EtOAc or CH2Cl2. The organic
phases were combined, washed with water, and dried over Na2SO4.
After evaporation, CC of the residue on silica gel using CH2Cl2-EtOAc
(6:1 and 4:1) or petroleum ether-EtOAc (1:1, 1:2, and 1:3) gave the
corresponding chalcones.3
3-Geranyl-2,4-dimethoxymethyl-6-methoxyacetophenone (14):
1
yellowish oil; H NMR (CDCl3, 300 MHz) δ 1.57 (3H, s, H3-10′′),
1.64 (3H, s, H3-9′′), 1.74 (3H, s, H3-4′′), 2.06 (4H, m, H2-5′′,6′′), 2.50
(3H, s, COCH3), 3.31 (2H, d, J ) 6.8, H2-1′′), 3.47 (3H, s, MOM-
CH3), 3.48 (3H, s, MOM-CH3), 3.79 (3H, s, OCH3), 4.89 (2H, s,
OCH2O), 5.05 (1H, t, J ) 6.7, H-7′′), 5.14 (1H, t, J ) 6.7, H-2′′), 5.20
(2H, s, OCH2O), 6.55 (1H, s, H-5). Aldol coupling of 14 with MOM-
protected 4-hydroxybenzaldehyde and deprotection resulted in 2 after
CC with petroleum ether-EtOAc, 1:1.
2′,4,4′,6′-Tetrahydroxy-3′-geranylchalcone (3′-geranylchalcon-
aringenin) (3): yield 25%; yellow-orange, amorphous powder; mp
74-79 °C; UV (MeOH) λmax 365 nm; 1H and 13C NMR identical with
literature data;22 EIMS (pos mode) m/z 408 [M]+ (40), 365 [M -
C3H7]+ (2), 323 (100), 285 (81), 203 (53), 165 (60); HREIMS m/z
408.1931 (calcd for C25H28O5 408.1937). Aldol coupling of 13 with
MOM-protected 4-hydroxybenzaldehyde and deprotection yielded 3
after CC with petroleum ether-EtOAc, 1:2.
3′-Geranyl-2′,4,4′-trihydroxy-6′-methoxychalcone (3′-geranyl-6′-
O-methylchalconaringenin) (2): yield 53%; dark yellow, amorphous
powder; mp 107-112 °C; UV (MeOH) (log ε) λmax 370 nm (4.51); 1H
NMR (acetone-d6, 300 MHz) δ 1.61 (3H, s, H3-10′′), 1.64 (3H, s, H3-
9′′), 1.78 (3H, s, H3-4′′), 1.98 (4H, m, H2-5′′,6′′), 3.31 (2H, d, J ) 6.9,
H2-1′′), 3.93 (3H, s, -OCH3-6′), 5.08 (1H, t, J ) 6.9, H-7′′), 5.27 (1H,
t, J ) 7.1, H-2′′), 6.14 (1H, s, H-5′), 6.91 (2H, d, J ) 8.5, H-3,5), 7.61
(2H, d, J ) 8.5, H-2,6), 7.75 (1H, d, J ) 15.6, H-ꢀ), 7.89 (1H, d, J )
15.6, H-R), 9.04 (1H, s, OH), 14.70 (1H, s, OH-2′); 13C NMR (acetone-
d6, 75 MHz) δ 193.3 (CO), 166.5 (C-2′), 162.8 (C-4′), 161.9 (C-6′),
3-Geranyl-6-hydroxy-2,4-dimethoxymethylacetophenone (13): yel-
1
lowish oil; H NMR (CDCl3, 300 MHz) δ 1.57 (3H, s, H3-10′′), 1.65

1240 Journal of Natural Products, 2008, Vol. 71, No. 7
Vogel and Heilmann
(3H, s, H3-9′′), 1.75 (3H, s, H3-4′′), 2.08 (4H, m, H2-5′′,6′′), 2.70 (3H,
s, COCH3), 3.31 (2H, d, J ) 6.6, H2-1′′), 3.45 (3H, s, MOM-CH3),
3.50 (3H, s, MOM-CH3), 4.95 (2H, s, OCH2O), 5.06 (1H, t, J ) 6.7,
H-7′′), 5.15 (1H, t, J ) 6.3, H-2′′), 5.20 (2H, s, OCH2O), 6.47 (1H, s,
H-5), 12.95 (1H, s, OH).
3′), 87.8 (C-5′), 56.4 (OCH3-6′), 56.1 (OCH3-4′), 7.5 (CH3); EIMS (pos
mode) m/z 314 [M]+ (100), 221 (21), 194 (41); HREIMS m/z 314.1152
(calcd for C18H18O5 314.1154).
Coupling of 2-hydroxy-3-methyl-4,6-dimethoxyacetophenone with
MOM-protected 4-hydroxybenzaldehyde yielded the MOM-protected
chalcone after CC with petroleum ether-EtOAc, 2:1 (59% yield).
Deprotection resulted in pure 6 after CC with petroleum ether-EtOAc,
1:1.
2′,4,4′-Trihydroxy-6′-methoxy-3′,5′-diprenylchalcone (5′-prenyl-
xanthohumol) (4): yield 61%; yellow-orange, amorphous powder; mp
1
62-68 °C; UV (MeOH) λmax 370 nm; H (acetone-d6, 300 MHz) δ
4,4′,6′-Trihydroxy-2′-methoxy-3′-prenylchalcone (2′-O-methyl-3′-
prenylchalconaringenin, xanthoflorianol) (7): yield 41%; yellow-
orange, amorphous powder; mp 57-60 °C; UV (MeOH) λmax (log ε)
370 nm (4.50); 1H NMR (acetone-d6, 300 MHz) δ 1.67 (3H, s, H3-5′′),
1.79 (3H, s, H3-4′′), 3.33 (2H, d, J ) 6.9, H2-1′′), 3.72 (3H, s, OCH3-
2′), 5.24 (1H, t, J ) 6.9, H-2′′), 6.25 (1H, s, H-5′), 6.95 (2H, d, J )
8.5, H-3,5), 7.64 (2H, J ) 8.5, H-2,6), 7.82 (1H, d, J ) 15.6, H-ꢀ),
7.89 (1H, d, J ) 15.6, H-R), 8.99 (1H, s, OH), 9.47 (1H, s, OH), 13.50
(1H, s, OH-6); 13C NMR (acetone-d6 75 MHz) δ 193.3 (CO), 165.6
(C-6′), 163.8 (C-4′), 162.3 (C-2′), 160.8 (C-4), 144.4 (C-ꢀ), 131.3 (C-
2,6), 131.3 (C-3′′), 127.9 (C-1), 124.3 (C-R), 123.9 (C-2′′), 116.9 (C-
3,5), 115.5 (C-3′), 109.4 (C-1′), 100.2 (C-5′), 63.4 (OCH3-2′), 25.8
(C-5′′), 23.0 (C-1′′), 18.0 (C-4′′); EIMS (pos mode) m/z 354 [M]+ (100),
339 [M - CH3]+ (20), 235 (31), 234 (38), 219 (60), 179 (100);
HREIMS m/z 354.1463 (calcd for C21H22O5 354.1467). 6-Hydroxy-
2,4-dimethoxymethyl-3-prenylacetophenone (20) was synthesized ac-
cording Vogel et al.3 20 (450 mg; colorless oil, 1H and EIMS identical
to literature data4) was dissolved in 45 mL of MeOH and deprotected
1.65 (3H, s, prenyl-CH3), 1.68 (3H, s, prenyl-CH3), 1.77 (3H, s, prenyl-
CH3), 1.79 (3H, s, prenyl-CH3), 3.38 (4H, m, H2-1′′,1′′′), 3.69 (3H, s,
OCH3-6′), 5.19 (2H, m, H-2′′,2′′′), 6.95 (2H, d, J ) 8.5, H-3,5), 7.63
(2H, d, J ) 8.5, H-2,6), 7.83 (1H, d, J ) 15.4, H-ꢀ), 7.90 (1H, d, J )
15.4, H-R), 8.03 (1H, s, OH), 8.97 (1H, s, OH), 13.89 (1H, s, OH-2′);
13C NMR (acetone-d6, 75 MHz) δ 193.8 (CO), 163.2 (C-2′,4′), 160.9
(C-4), 159.9 (C-6′), 144.4 (C-ꢀ), 132.3 and 131.4 (C-3′′,3′′′), 131.4
(C-2,6), 127.9 (C-1), 124.4 (C-R), 124.1 and 123.3 (C-2′′,2′′′), 116.9
(C-3,5), 115.0 (C-5′), 112.4 (C-3′), 109.4 (C-1′), 63.5 (OCH3-6′), 25.9
(C-5′′,5′′′), 23.3 and 23.2 (C-1′′,C-1′′′), 18.1 (C-4′′,4′′′); EIMS (pos
mode) m/z 422 [M]+ (100), 407 [M - CH3]+ (39), 379 [M - C3H7]+
(22), 351 (35), 287 (45), 247 (49), 231 (77); HREIMS m/z 422.2091
(calcd for C26H30O5 422.2093).
Prenylbromide (2.83 g, 1.5 equiv), K2CO3 (6.99 g, 4 equiv), and 11
(3.24 g, 1 equiv) were refluxed in 150 mL of acetone for 24 h with
stirring to give 15 (colorless oil; 1H NMR and EIMS identical to
literature data4) after cooling to room temperature, filtration, and CC
(petroleum ether-EtOAc, 2:1) of the evaporated filtrate (yield 91%).
Then, 10 drops of 3 N HCl were successively added to a cooled (0 °C)
solution of 15 (1.16 g, 1 equiv) in 20 mL of acetone (pH ) 2) and
stirred for 4 h in an ice bath. After evaporation the residue was
distributed between EtOAc and H2O, and the aqueous layer was
extracted three times with EtOAc. The organic phases were combined,
washed with water, dried over Na2SO4, and evaporated to give known
16 after CC (petroleum ether-EtOAc, 4:1) in 68% yield. Prenylbromide
(1.07 g, 1.5 equiv) was added to a refluxing mixture of K2CO3 (2.64 g,
4 equiv) and 16 (1.34 g, 1 equiv) in 50 mL of acetone. Stirring for
24 h, cooling to room temperature, filtration, and CC (petroleum
ether-EtOAc, 4:1) of the evaporated filtrate yielded 17 (79%).
Compound 17 (1.29 g) was refluxed in N,N-dimethylaniline (4 mL)
under argon to yield 18 (40%) after CC with petroleum ether-EtOAc,
6:1. Compound 18 was methylated according to intermediate 13,
resulting in 19 (CC with petroleum ether-EtOAc, 5:1) in 71% yield.
2-Hydroxy-4-methoxymethyl-6-methoxy-3,5-diprenylacetophe-
1
to yield 21 (40%; yellow solid, H and EIMS identical to literature
data24). MOM protection of 21 gave, probably due to the strong
hydrogen bond for the OH at position 2 (δH 14.0), the 2-hydroxy-4,6-
dimethoxymethyl-3-prenylacetophenone 22 (45% yield), which was
methylated according to 13 to give 2-methoxy-4,6-dimethoxymethyl-
3-prenylacetophenone (23, 50% yield).
2-Methoxy-4,6-dimethoxymethyl-3-prenylacetophenone (23): yel-
1
lowish oil; H NMR (CDCl3, 300 MHz) δ 1.66 (3H, s, prenyl-CH3),
1.76 (3H, s, prenyl-CH3), 2.51 (3H, s, COCH3), 3.29 (2H, d, J ) 5.8,
H2-1′), 3.46 (3H, s, MOM-CH3), 3.47 (3H, s, MOM-CH3), 3.71 (3H,
s, OCH3), 5.13 (2H, s, OCH2O), 5.16 (3H, m, OCH2O and H-2′), 6.70
(1H, s, H-5). Aldol coupling and deprotection yielded 7 after CC with
petroleum ether-EtOAc, 1:1.
Xanthohumol H (8): yield 65%; yellow-orange, amorphous powder;
mp 212-214 °C; UV (MeOH) λmax 370 nm; 1H identical with literature
data;2 13C NMR (acetone-d6, 75 MHz) δ 193.3 (CO), 166.4 (C-2′), 163.2
(C-4′), 161.9 (C-6′), 160.6 (C-4), 143.1 (C-ꢀ), 131.3 (C-2,6), 128.2
(C-1), 125.5 (C-R), 116.8 (C-3,5), 110.1 (C-3′), 106.2 (C-1′), 92.0 (C-
5′), 70.8 (C-3′′), 56.1 (OCH3-6′), 43.2 (C-2′′), 29.7 (C-1′′,5′′), 17.9 (C-
4′′); EIMS (pos mode) m/z 372 [M]+ (49), 354 [M - H2O]+ (13), 339
[M - H2O - CH3]+ (12), 179 (100); HREIMS m/z 372.1569 (calcd
for C21H24O6 372.1573).
1
none (19): yellowish oil; H NMR (CDCl3, 300 MHz) δ 1.69 (6H, s,
2 × prenyl-CH3), 1.78 (6H, s, 2 x prenyl-CH3), 2.71 (3H, s, COCH3),
3.34 (4H, d, J ) 6.3, 2 × prenyl-CH2), 3.58 (3H, s, MOM-CH3), 3.70
(3H, s, OCH3), 4.98 (2H, s, OCH2O), 5.20 (2H, t, J ) 6.4, 2 × CHd).
Aldol coupling of 19 with MOM-protected 4-hydroxybenzaldehyde and
deprotection yielded pure 4 after CC with petroleum ether-EtOAc,
1:1.
Intermediate 20 was methylated according to the procedure for 13
to give 24 in 89% yield (450 mg; yellowish oil, 1H and EIMS identical
to literature data4). THF (10 mL) and intermediate 24 (590 mg, 1 equiv)
were added to a solution of Hg(OAc)2 (2.22 g, 4 equiv) in 5 mL of
H2O and stirred (reaction temperature <30 °C, stirring until the
dropwise addition of 3 N NaOH did not result in precipitation of Hg).
Subsequently, 5 mL of 3 N NaOH and a solution of 99 mg NaBH4 in
5 mL of 3 N NaOH were carefully added and stirred for a further 60
min. After separation of the precipitated Hg and saturation with NaCl,
the reaction mixture was extracted three times with diethyl ether. The
organic phases were combined, dried over Na2SO4, and evaporated.
CC of the residue with EtOAc as eluent yielded 25 (70% yield).
3-(3-Hydroxy-3-methylbutyl)-6-methoxy-2,4-dimethoxymethyl-
acetophenone (25): colorless oil; 1H NMR (CDCl3, 300 MHz) δ 1.25
(6H, s, 2 × CH3), 1.66 (2H, dt, J ) 6.9, 8.4, H2-2′), 1.77 (1H, s, OH),
2.47 (3H, s, COCH3), 2.68 (2H, dt, J ) 6.9, 8.4, H2-1′), 3.48 (3H, s,
MOM-CH3), 3.50 (3H, s, MOM-CH3), 3.78 (3H, s, OCH3), 4.91 (2H,
s, OCH2O), 5.20 (2H, s, OCH2O), 6.53 (1H, s, H-5). 25 was coupled
with protected 4-hydroxybenzaldehyde and deprotected to give pure 8
(CC with petroleum ether-EtOAc, 1:2).
2′,4-Dihydroxy-4′,6′-dimethoxychalcone (flavokawin) (5): yield
70%; yellow-orange, amorphous powder; mp 179-182 °C; UV
(MeOH) λmax 365 nm; 1H and 13C NMR identical with literature data;1,23
EIMS (pos mode) m/z 300 [M]+ (100), 207 (42), 181 (64); HREIMS
m/z 300.0998 (calcd for C17H16O5 300.0998). A mixture of 2,4,6-
trihydroxyacetophenone (2.12 g, 1 equiv), tetrabutylammonium iodide
(841 mg, 0.2 equiv), NaOH (1.14 g, 2.5 equiv), and dimethyl sulfate
(3.16 g, 2.2 equiv) was stirred in 15 mL of CH2Cl2-H2O, 3:2, for 24 h
at room temperature. Separation of phases, extraction of the aqueous
phase with CH2Cl2, and CC of the residue with petroleum ether-EtOAc,
2:1, yielded 2-hydroxy-4,6-dimethoxyacetophenone and 2-hydroxy-4,6-
dimethoxy-3-methylacetophenone (30% each). Coupling of 2-hydroxy-
4,6-dimethoxyacetophenone with MOM-protected 4-hydroxybenzal-
dehyde yielded the corresponding protected chalcone (83%) after CC
with petroleum ether-EtOAc, 2:1. Deprotection resulted in pure 5 after
CC with the same eluent mixture.
2′,4-Dihydroxy-4′,6′-dimethoxy-3′-methylchalcone (6): yield 73%;
yellow-orange, amorphous powder; mp 173-177 °C; UV (MeOH) λmax
(log ε) 365 nm (4.48); 1H NMR (acetone-d6, 300 MHz) δ 1.97 (3H, s,
H3-3′), 3.96 (3H, s, OCH3-4′), 4.05 (3H, s, OCH3-6′), 6.31 (1H, s, H-5′),
6.92 (2H, d, J ) 8.8, H-3,5), 7.62 (2H, J ) 8.8, H-2,6), 7.76 (1H, d,
J ) 15.6, H-ꢀ), 7.90 (1H, d, J ) 15.6, H-R), 8.99 (1H, s, OH-4), δ
14.33 (1H, s, OH); 13C NMR (acetone-d6, 75 MHz) δ 193.7 (CO), 165.0
(C-2′), 164.6 (C-4′), 162.3 (C-6′), 160.7 (C-4), 143.4 (C-ꢀ), 131.3 (C-
2,6), 128.0 (C-1), 125.4 (C-R), 116.8 (C-3,5), 106.7 (C-1′), 105.8 (C-
Xanthohumol C (9): yield 87%; yellow-orange, amorphous powder;
1
mp 90-98 °C; UV (MeOH) λmax 370 nm; H and 13C NMR identical
with literature data;22 EIMS (pos mode) m/z 352 [M]+ (34), 337 [M -
CH3]+ (37), 217 (100); HREIMS m/z 352.1312 (calcd for C21H20O5
352.1311). Intermediate 24 was deprotected to yield 2,4-dihydroxy-6-
methoxy-3-prenylacetophenone (26, 69% white powder, identical to

Prenylated Chalcones from Humulus lupulus
Journal of Natural Products, 2008, Vol. 71, No. 7 1241
literature data25). DDQ (354 mg, 1 equiv) and 26 (390 mg, 1 equiv)
were dissolved in dried benzene and 10 drops of dried dioxane. The
reaction mixture was heated under reflux for 3 h, cooled to room
temperature, and filtered. Evaporation and CC of the residue with
petroleum ether-EtOAc, 6:1, resulted in 27 (90% yield; yellow solid,
identical to literature data25). Intermediate 27 was coupled with
protected 4-hydroxybenzaldehyde and deprotected to yield pure 9 after
CC with petroleum ether-EtOAc, 1:1.
for technical assistance. Special thanks are given to Dr. T. Burgemeister
for measuring the 1D and 2D NMR spectra and to Mr. J. Kiermaier
(both University of Regensburg, Department of Analytics, NWF IV)
for mass spectra. Prof. Dr. S. Mahboobi (University of Regensburg,
Chair of Pharmaceutical Chemistry) and Dr. B. Kraus (University of
Regensburg, Chair of Pharmaceutical Biology) are gratefully acknowl-
edged for stimulating discussions.
1′′,2′′-Dihydroxanthohumol C (2′′,2′′-dimethyl-3′′,4′′-dihydro-
(2H)-pyrano[2′′,3′′:3′,4′]-2′,4-dihydroxy-6′-methoxychalcone17 (10):
yield 96%; yellow-orange, amorphous powder; mp 104-114 °C; UV
(MeOH) λmax 370 nm; 1H and 13C NMR identical with literature data;2,17
EIMS (pos mode) m/z 354 [M]+ (100), 261 (25), 234 (32), 179 (74);
HREIMS m/z 354.1465 (calcd for C21H22O5 354.1467). Intermediate
26 (100 mg, 1 equiv) was dissolved in 2 mL of THF and 3 mL of
formic acid and stirred for 3 h under reflux. The reaction mixture was
poured into ice-water and extracted three times with ethyl acetate.
The organic phases were combined, washed with water, dried over
Na2SO4, and evaporated to give 28 after CC (petroleum ether-EtOAc,
6:1) in 60% yield (white solid, identical to literature data25). 28 was
coupled with protected 4-hydroxybenzaldehyde (yield 73%) and
deprotected to yield pure 10 after CC with petroleum ether-EtOAc,
1:1.
Cell Culture and Determination of Cytotoxicity. HeLa cells
(ATCC CCL17) were cultured at 37 °C in a humidified incubator with
5% CO2. Culture medium was MEM (Biochrom AG) supplemented
with 10% FCS and 2 mM L-glutamine. Cytotoxicity was evaluated with
the colorimetric MTT assay as described by Mosman et al.18 (modified
according Heilmann et al.19 Tests were performed in duplicate and all
experiments repeated three times (n ) 8). IC50 values were calculated
from eight different concentrations, and data are reported as mean (
SD. Maximal observed (absolute) standard deviation was about 15%.
Positive control measurements were performed with xanthohumol.
ORAC-Fluorescein Assay. The ORAC-fluorescein assay was
performed according to Davalos et al.20 and Vogel et al.3 in 96-well
plates with fluorescein (final concentration 300 nM) as fluorescent probe
and 75 mM phosphate buffer (pH 7.4) for all dilution steps and as
reaction milieu. The antioxidant (chalcones or Trolox, 20 µL) was
incubated in different concentrations (chalcones, 0.1-1.0 µM; Trolox,
1-8 µM) together with a fluorescein solution (120 µL) at 37 °C for 15
min. The reaction was started by addition of 60 µL of AAPH (2,2′-
azobis(2-methylpropionamide) dihydrochloride; final concentration, 12
mM), yielding a final volume of 200 µL. After addition of AAPH, the
fluorescence was recorded every minute in a Tecan 96-plate reader (λex
485 nm, λem 536 nm, 37 °C) for 80 min. Reaction mixtures were
prepared in quadruplicate, and at least four independent assays were
performed for each sample. Samples were measured at five different
concentrations (0.1-1.0 µM). Eight calibration curves using 1-8 µM
Trolox as antioxidant were also carried out in each assay. Controls
were measured without antioxidant as well as without AAPH and
antioxidant. ORAC values were expressed as Trolox equivalents (mean
( SD) by using the standard curve calculated for each assay. The
regression coefficient between AUC and antioxidant concentration was
calculated for all samples (r2 > 0.93). Further positive control
measurements were performed with xanthohumol.
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Acknowledgment. We are grateful to Mrs. G. Brunner and Mr. S.
Pitzl (both University of Regensburg, Chair of Pharmaceutical Biology)
NP800188B