Synthesis of novel 3,7-substituted-2-(3',4'-dihydroxyphenyl)flavones with improved antioxidant activity

Article abstract of DOI:10.1021/jm000951n

A series of 3,7-disubstituted-2-(3',4'-dihydroxyphenyl)flavones was synthesized as potential cardioprotective agents in doxorubicin antitumor therapy. The influence of substituents on the 3 and 7 positions of the flavone nucleus on radical scavenging and

Full text of DOI:10.1021/jm000951n

3752
J . Med. Chem. 2000, 43, 3752-3760
Syn th esis of Novel 3,7-Su bstitu ted -2-(3′,4′-d ih yd r oxyp h en yl)fla von es w ith
Im p r oved An tioxid a n t Activity
Fre´de´rique A. A. van Acker,*,†,‡ J os A. Hageman,‡ Guido R. M. M. Haenen,# Wim J . F. van der Vijgh,†
Aalt Bast,# and Wiro M. P. B. Menge‡
Department of Medical Oncology, University Hospital Vrije Universiteit, De Boelelaan 1117,
1081 HV Amsterdam, The Netherlands, LACDR, Department of Pharmacochemistry, Faculty of
Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, and
Department of Pharmacology and Toxicology, University Maastricht, P.O. Box 616,
6200 MD Maastricht, The Netherlands
Received March 24, 2000
A series of 3,7-disubstituted-2-(3′,4′-dihydroxyphenyl)flavones was synthesized as potential
cardioprotective agents in doxorubicin antitumor therapy. The influence of substituents on
the 3 and 7 positions of the flavone nucleus on radical scavenging and antioxidant properties
was explored to improve the antioxidant activity of our lead compound monoHER. In the TEAC
assay most compounds had a similar potency (3.5-5 times as potent as trolox), but in the LPO
assay IC50 values ranged from 0.2 to 37 µM. In general, the 3-substituted flavones (9a -j) were
the most potent compounds in the LPO assay. The number of hydroxyl groups is not the only
prerequisite for antioxidant activity. Substitution in ring A of the flavonoid is not necessary
for high activity, but the presence of a 7-OH group significantly modifies the antioxidant activity.
The compounds are good antioxidants, which makes it interesting to evaluate them as
cardioprotective agents.
In tr od u ction
Recently, our group has demonstrated that 7-monohy-
droxyethylrutoside (monoHER; Chart 1), a semisyn-
thetic flavonoid belonging to a class of hydroxyethylru-
tosides, provides dose-dependent protection against the
doxorubicin-induced cardiotoxicity.13
Flavonoids form a class of benzo-γ-pyrone derivatives
which are ubiquitous in plants. The immediate family
members of flavonoids include flavones, flavanes, fla-
vonols, anthocyanidins, and catechins. They possess a
wide spectrum of biological activities. Some flavonoids
have been found to possess anticancer,1,2 antiischemic,3
antiallergic, antiinflammatory,4-6 and several other
activities. Recent interest in these substances has been
stimulated by the potential health benefits arising from
the antioxidant activity of these polyphenolic com-
pounds. These are the result of their high propensity
to transfer electrons, to chelate ferrous ions, and to
scavenge reactive oxygen species.7 Because of these
properties, flavonoids have been considered as potential
protectors against chronic cardiotoxicity caused by the
cytostatic drug doxorubicin.
Doxorubicin is a very effective antitumor agent, but
its clinical use is limited by the occurrence of a cumula-
tive dose-related cardiotoxicity, resulting in, for ex-
ample, congestive heart failure. Although the mecha-
nism causing this chronic cardiotoxicity has not been
fully elucidated, it is generally believed that the forma-
tion of oxygen free radicals plays a crucial role (for
reviews, see refs 8-12). In the presence of traces of iron,
doxorubicin facilitates the formation of radicals. More-
over, the iron-catalyzed Haber-Weiss reaction (eq 1) or
the Fenton reaction (eq 2) is induced, leading to the
formation of the extremely reactive hydroxyl radical.
H2O2 + O2•-+ Fe2+ f HO• + HO- + O2 + Fe2+ (1)
H2O2 + Fe2+ f HO• + HO- + Fe3+
(2)
In the present study monoHER has been used as a
lead structure for the development of new flavones with
improved antioxidant activity. The general structure of
the new compounds consists of a flavone backbone with
a C2-C3 double bond and a catechol moiety on ring B.
According to previously published structure-activity
relationships (SARs) of commercially available fla-
vonoids, incorporation of these structural elements leads
to potent antioxidants.14-18 All new compounds were
tested for their antioxidant activity in two different
assays. First by the trolox equivalent antioxidant capa-
city assay (TEAC) which is a chemical assay that is a
one-phase system containing 2,2′-azinobis(3-ethylben-
zthiazoline-6-sulfonic acid) radicals (ABTS). This assay
allows us to study the radical scavenging activity
directly without interference by other factors. In addi-
tion to this chemical assay, the compounds were tested
in a lipid peroxidation assay using rat liver microsomes.
In this two-phase system with a lipid and an aqueous
phase, not only radical scavenging but, for example, also
iron chelation and lipophilicity of the compounds play
a role. By comparing the results of these two assays,
the influence of the iron chelation and lipophilicity on
the antioxidant activity can be estimated.
* Corresponding author: F. A. A. van Acker, Dept. of Medical
Oncology, BR-232, University Hospital Vrije Universiteit, De Boelelaan
1117, 1081 HV Amsterdam, The Netherlands. Tel: +31204442773.
Fax: +31204443844. E-mail: f.vanacker@azvu.nl.
† University Hospital Vrije Universiteit.
Previously SAR studies have been performed using
either commercially available flavonoids or flavonoids
‡ Vrije Universiteit.
#
University Maastricht.
10.1021/jm000951n CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/15/2000

3,7-Substituted-2-(3′,4′-dihydroxyphenyl)flavones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3753
Ch a r t 1. Chemical Structure of
Sch em e 1a
Monohydroxyethylrutoside (monoHER)
or mixtures thereof isolated from plants.14,16,17,19-21
These sets of compounds form a structurally highly
diverse group of compounds, and consequently it has
not been possible to obtain a detailed SAR. In the
present study we have focused on the 3,7-disubstituted-
2-(3′,4′-dihydroxyphenyl)flavones and studied the influ-
ence of the 3 and 7 substituents on the antioxidant
activity in a more systematic way. To obtain compounds
with an improved cardioprotective profile in vivo com-
pared to monoHER, we have selected substituents that
not only modify antioxidant activity but also improve
water solubility and cardioselectivity. In 1991, Petty and
Grisar prepared an R-tocopherol analogue with im-
proved cardioselectivity by the introduction of a qua-
ternary ammonium group at the 2 position of R-toco-
pherol.22,23 We decided to apply this approach and
prepared some compounds with a quaternary am-
monium group at the 3 or 7 position. The effect of the
different substituents at the 3 and 7 positions on the
antioxidant profile was evaluated with the TEAC and
LPO assays.
a
(a) LiHMDS, THF; (b) DOWEX, 2-propanol; (c) EtOH, 1,4-
dioxane, 40% w/v KOH; (d) 1,4-dioxane, EtOH, 5.4% w/v NaOH,
35% H2O2.
Biologica l Eva lu a tion
All compounds were tested in a lipid peroxidation
assay to determine their inhibitory effect on the oxida-
tive degradation of membrane lipids. Attack by reactive
oxygen species (ROS) produces a lipid peroxy radical,
which can either abstract a hydrogen atom from an
adjacent lipid to form a lipid hydroperoxide or form a
lipid endoperoxide. The formation of lipid endoperoxides
in unsaturated fatty acids with at least three double
bonds separated by methylene groups leads to the
formation of malondialdehyde (MDA) as a breakdown
product. In vitro the formed MDA is detected as the
Ch em istr y
pink-colored thiobarbituric acid (TBA) adduct (λmax
535 nm).
)
The substituted flavonoids were obtained via the four-
or five-step synthesis route shown in Scheme 1. Ap-
propriately protected hydroxyacetophenones were coupled
to 3,4-dibenzyloxybenzaldehyde according to Pfister et
al.24 to give the corresponding chalcones. The benzyl
group was our group of choice for the protection of the
hydroxyl groups because of its stability under various
reaction conditions and ease of deprotection. The chal-
cones were subjected to the Algar-Flynn-Oyamada
reaction,25,26 to give the corresponding flavon-3-ols 4b-d
in moderate yields.
The compounds were also tested in a chemical radical
scavenging assay (TEAC assay). This assay is based on
the generation and detection of a colored long-lived
specific radical cation; ABTS is oxidized by azobis-
(amidinepropane) (ABAP) to give the relatively stable
ABTS radical. The concentration of ABTS•+ is measured
at 734 nm. Thus the antioxidant-induced reduction of
the ABTS•+ concentration is directly related to the
antioxidant capacity of the compound being tested.
In the next step the free hydroxyl group was reacted
with a variety of electrophiles (Scheme 2) to give
compounds 6b-7d . The glycosylated flavonoids 9i,j
were prepared via a silver oxide-assisted coupling of an
R-bromoperacetylated mono- or disaccharide.27 The
acetyl protective groups were removed with potassium
tert-butoxide and methanol prior to debenzylation. In
the final step the benzyl protective groups were removed
either using hydrochloric acid in glacial acetic acid
(method A)28 or by hydrogenolysis in methanol (method
B).29 Method B is strongly preferred over method A,
because of the ease of isolation and the higher yield.
The corresponding flavones were obtained via the
Baker-Venkataraman30,31 procedure starting from the
appropriately protected hydroxyacetophenones and eth-
yl 3,4-dibenzyloxybenzoate to give the corresponding
flavones in approximately 50% yield. The subsequent
functionalization and deprotection procedures were
similar to those for the preparation of the flavonols.
Resu lts a n d Discu ssion
Table 1 displays the lipid peroxidation (LPO) and
radical scavenging (TEAC) data for compounds 8a -11a .
As can be seen from this table, the compounds show a
completely different SAR profile for radical scavenging
and lipid peroxidation. In the chemical TEAC assay
most compounds have a similar potency (approximately
3.5-5 times more potent than trolox). However, in the
LPO assay the IC50 values range from 0.2 to 37.3 µM.
The differences between these two assays result from
the different experimental circumstances.
In the TEAC assay the radical scavenging activity is
measured in an aqueous environment. Therefore, the
antioxidant capacity is only dependent on the amount
of radicals that can be scavenged by the flavonoid.
However, the LPO assay consists of a two-phase system.
The combination of Fe2+, vitamin C, and oxygen present
in the aqueous phase causes the formation of reactive

3754 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
van Acker et al.
Sch em e 2
oxygen species (ROS). The resulting degradation of
membrane lipids by these ROS is quantified via the
amount of MDA-TBA complex formed. This renders the
LPO assay a very complex system.
In general, substitution on the 3 position leads to very
potent compounds, which are more potent than the
7-substituted flavones. However, the introduction of an
acidic moiety (9d ) at the 3 position causes a 10-fold
decrease in the activity in the LPO assay. Under
physiological conditions this acidic group is deproto-
nated and this negative charge is expected to decrease
the ability to penetrate the membrane.
The introduction of a quaternary ammonium group
at the 3 position (9f-h ) does not influence the antioxi-
dant activity. Apparently, introduction of a positively
charged substituent at C3 is allowed. As a result of the
positive charge these compounds dissolve well in water.
The fact that these compounds still have potent anti-
oxidant activity in the LPO assay suggests that they
also can be found in the membrane. The charged amino
group combined with the flavonoid backbone probably
allows these compounds to act as amphiphilic com-
pounds, such as the phosphatidylcholines, and this
might explain their good antioxidant activity.
The introduction of a sugar moiety at the 3 position
is less well tolerated. If the 3-hydroxyethyl group (9c)
is replaced by a glucose (9i) or a rutinose (9j) moiety,
the activity decreases 5- and 26-fold, respectively. The
introduction of a bulky substituent at the 3 position
induces an increase in the torsion angle between ring
B and the rest of the molecule. This results in a loss of
conjugation and a decrease in activity.33 Furthermore,
the introduction of a sugar moiety will also change the
lipophilicity and this might affect membrane penetra-
tion.
From the results of the TEAC assay we conclude that
all listed compounds have a similar radical scavenging
capacity. The catechol moiety and the flavone backbone,
the two structural elements present in all our com-
pounds, are clearly the functional groups involved in
radical scavenging. Compound 9d has a significantly
higher radical scavenging activity than the others. The
favorable interaction between the negatively charged
substituent and the positively charged ABTS radical
probably leads to an overestimation of the radical
scavenging capacity of this compound.
The differences in potency of the compounds in the
LPO assay are most probably the result of additional
properties such as iron chelating ability and lipophili-
city. When comparing the LPO IC50 values of the new
compounds with monoHER, it is clear that almost all
new compounds are more potent in protecting against
lipid peroxidation. This is in good agreement with
previous findings that a catechol moiety in ring B, the
2,3-double bond, and the 4-oxo function are the domi-
nant factors for antioxidant activity.14-18
To study the influence of a substituent at the 7
position, we have synthesized a series of compounds
with different groups at this position (8a -e). Substitu-
tion of the 7-hydroxyl group (8a ) by other groups (8b-
e) showed that the methoxy-substituted flavone (8b)
was the most active compound of this series. The high
activity of this compound is surprising as it has been
reported that a (substituted) 3-hydroxyl group is re-
quired for high activity.15-17 Moreover, this finding is
also in sharp contrast with the findings of Cao19 and
Arora32 who stated that O-methylation of the hydroxyl
substituents suppresses the antioxidant activity of the
flavonoids. The introduction of a charged quaternary
ammonium group at the 7 position (8d ) is highly
unfavorable.
Surprisingly, and in contrast to other reports,17,32,33
a hydroxyl substituent at the 7 position in combination
with a substituent at the 3 position, although of no
influence on the radical scavenging capacity, decreases
the antioxidant properties (compounds 10a -d ). An
exception is the 3-hydroxy compound (10a ) where the
addition of a hydroxyl group improves the antioxidant
activity.
A small electron-donating group on position 7 in

3,7-Substituted-2-(3′,4′-dihydroxyphenyl)flavones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3755
Ta ble 1. Yields, Melting Points, and Antioxidant Activities in the Lipid Peroxidation Assay and TEAC Assay
substituent
LPO IC50 (µM)
TEAC (mM)
compd
8a
8b
8c
8d
8e
9a
9b
R3
R5
R7
yielda (%)
mp (°C)
300 dec
>240
206.7-208.7
>210
173.6
(mean ( SEM, n ) 3-5) (mean ( SD, n ) 3-5)
H
H
H
H
H
OH
OCH3
H
OH
82
88
37
74
59
69
41
42
25
20
27
33
40
75
44
11.0 ( 1.3
2.5 ( 0.8
16.4 ( 1.7
37.3 ( 2.4
8.1 ( 1.1
1.8 ( 1.0
1.3 ( 0.7
0.6 ( 0.1
6.5 ( 1.0
1.0 ( 0.1
1.2 ( 0.3
1.4 ( 0.3
1.0 ( 0.3
2.8 ( 0.9
15.7 ( 4.8
0.2 ( 0
4.3 ( 0.2
3.3 ( 0.2
4.3 ( 0.7
1.9 ( 0.3
2.9 ( 0.4
4.5 ( 0.5
4.4 ( 0.3
4.4 ( 0.4
7.6 ( 0.6
3.4 ( 0.4
4.2 ( 0.2
4.3 ( 0.6
3.3 ( 0.6
4.9 ( 0.3
4.3 ( 0.3
5.6 ( 0.4
5.9 ( 0.7
3.4 ( 0.2
5.1 ( 0.3
4.9 ( 0.4
3.0 ( 0.2c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH2CH2OH
O(CH2)3N+(CH3)3
O(CH2)3N(CH3)2
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
280 dec
198.5-199.7
186.2-186.8
253.3-254.9
98.5-99.8
234.4-235.4
230.8-232.8
196.7-198.5
166.6-168.6
174 dec
9c
9d
9e
9f
9g
9h
9i
9j
OCH2CH2OH
OCH2COOH
O(CH2)3N(CH3)2
O(CH2)3N+(CH3)3
O(CH2)6N+(CH3)3
O(CH2)8N+(CH3)3
O-glucosyl
O-rutinosyl
OH
10a b
10b
10c
10d
11a
OCH3
86
77
10
92
281.4
>300
200 dec
300 dec
1.6 ( 0.7
21.7 ( 6.1
3.8 ( 0.1
13.8 ( 4.4
12.7 ( 3.7
OCH2CH2OH
O(CH2)3N+(CH3)3
OH
OCH2CH2OH
monoHER O-rutinosyl
OH OCH2CH2OH
a
b
Yield determined from intermediate to product. Fisetin. c van den Berg, R.; Haenen, G. R. M. M.; van den Berg, H.; van der Vijgh,
W. J . F.; Bast, A. The predictive value of the antioxidant capacity of structurally related flavonoids using the trolox equivalent antioxidant
capacity (TEAC) assay. Food Chem., in press.
were recorded on a Bruker AC-200 (200 MHz) spectrometer.
Chemical shifts for 1H and 13C NMR are given in ppm (δ)
relative to tetramethylsilane (TMS) as internal standard.
Melting points (not corrected) were measured on an Electro-
thermal AI-9100 apparatus. THF was dried over LiAlH4 and
distilled before use. 4-Benzyloxy-2-hydroxyacetophenone,35
â-bromoethylbenzyl ether36 and ethyl 3,4-dibenzyloxyben-
zoate37 were prepared according to literature procedures.
combination with substituents on the 3 position is
allowed for activity; see also compounds 8a ,b.
The influence of the 5-OH group present in monoHER
has not been evaluated. These 3,5,7-trisubstituted
compounds are synthetically not easily accessible, and
the present study shows that substituents at C5 are not
required for excellent antioxidant activity.
3′,4′-Dib en zyloxy-7-h yd r oxyfla von e (4a ). 32.28 g (200
mmol) of 1,1,1,3,3,3-hexamethyldisilazan (HMDS) was dis-
solved in dry THF (250 mL) and cooled to -60 °C under an
atmosphere of dry nitrogen. 125 mL (200 mmol) of butyllithium
(1.6 M in hexane) was added dropwise followed by 50 mmol of
2,4-dihydroxyacetophenone (1a ) in dry THF (100 mL) and
stirred for 45 min at -30 °C. The reaction mixture was cooled
to -60 °C and 19.4 g (50 mmol) of 2 in dry THF (100 mL) was
added dropwise. This mixture was stirred for another 45 min
at -60 °C and at room temperature overnight. The reaction
mixture was poured on ice and acidified with 3.0 M HCl. The
THF was removed under reduced pressure and the residue
was extracted with chloroform (2 × 150 mL). The chloroform
layers were dried over sodium sulfate and concentrated in
vacuo. The crude 1-(2,4-dihydroxyphenyl)-3-(3,4-dibenzyloxy-
phenyl)-1,3-propanedione was dissolved in 2-propanol (100
mL), 25 g of Dowex W50 × 8 (H+ form) was added and heated
under reflux for 16 h under an atmosphere of dry nitrogen.
The solids were filtered, suspended in DMF and filtered to
remove the Dowex. The filtrate was concentrated under
reduced pressure to give 11.0 g (49%) 4a as a white solid; mp
245.4-245.8 °C. 1H NMR (DMSO): δ 5.25 (s, 2H, OCH2Ph),
5.29 (s, 2H, OCH2Ph), 6.87 (s, 1H, C3H), 6.91 (dd, 2H, J ) 9
Hz, 2 Hz, C5′H), 7.0 (d, 1H, J ) 2 Hz, C8H), 7.21 (d, J ) 8 Hz,
C6H), 7.3-7.6 (m, 10H, OCH2Ph × 2), 7.64 (dd, 1H, J ) 7 Hz,
2 Hz, C6′H), 7.70 (t, J ) 2 Hz, C2′H), 7.86 (d, 1H, J ) 9 Hz,
C5H), 10.9 (br s, 1H, OH).
3′,4′-Diben zyloxy-3-h yd r oxyfla von e (4b). A suspension
of 31.4 mmol of 3,4-dibenzyloxybenzaldehyde (3) and 31.4
mmol of 2-hydroxyacetophenone (1b) in ethanol (80 mL) and
dioxane (50 mL) was cooled to 10 °C and 25 mL of 40% w/v
KOH solution was added dropwise. The reaction mixture was
stirred for 66 h at room temperature. CH2Cl2 (400 mL) was
added and the organic layer was washed with H2O (3 × 50
mL), dried over sodium sulfate and concentrated in vacuo. The
oily residue was dissolved in dioxane (110 mL) and ethanol
(300 mL), and 5.4% (w/v) NaOH solution (100 mL). 11.4 mL
of 35% H2O2 was added dropwise. The reaction mixture was
Con clu sion s
We have successfully developed a synthesis route for
the preparation of 3-subsituted, 7-substituted, and 3,7-
disubstituted-2-(3′, 4′-dihydroxyphenyl)flavones. All new
compounds are potent radical scavengers. In the LPO
assay the 3-substituted compounds are superior to the
7-substituted compounds. We found that the catechol
moiety in combination with a C2-C3 double bond and
a 4-keto function are the essential structural elements
for potent antioxidant activity. From previous SAR
studies19,34 it was concluded that the antioxidant activ-
ity correlates with the number of phenolic OH groups
present. With our series of compounds we have shown
that this is not necessarily true, as our 3-substituted
flavones without substituents in ring A are the most
potent compounds. Introduction of an additional OH on
the 7 position in combination with a 3 substituent does
not improve activity unless an OH group is present at
the 3 position. This finding is in contrast to results
obtained from the highly diverse sets of commercially
available flavonoids that are usually investigated. Ad-
ditionally we have shown that substitution of C7
significantly modifies the antioxidant activity of the
molecule, in contrast to earlier findings.17,32
We successfully prepared new flavonoids which are
of interest as potential cardioprotectors; they might be
more active than monoHER in vivo.
Exp er im en ta l Section
In str u m en ts a n d An a lyses. Elemental analyses were
performed for C, H, N (Department of Microanalysis, Gronin-
gen University, The Netherlands). 1H and 13C NMR spectra

3756 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
van Acker et al.
stirred in an ice bath for 2 h and subsequently at room
temperature overnight, resulting in a yellow suspension. After
acidification with 2 M HCl (100 mL) the precipitate was
filtered and washed with H2O (500 mL). The crude product
was recrystallized from ethanol to give 7.0 g (49%) of a light
+ C7H + C6′H), 7.82 (d, 1H, J ) 2 Hz, C2′H), 8.23 (dd, 1H, J
) 7 Hz, 2 Hz, C5H).
3-Meth oxy-7,3′,4′-tr iben zyloxyfla von e (7b). 4c was re-
1
acted as described for 5b; yield 1.0 g (96%). H NMR (CDCl3):
δ 3.70 (s, 3H, OCH3), 5.14 (s, 2H, C7OCH2Ph), 5.25 (s, 4H,
C3′OCH2Ph + C4′OCH2Ph), 6.90 (d, 1H, J ) 2 Hz, C8H), 7.0-
7.1 (m, 2H, C5′H + C6H), 7.28-7.49 (m, 15H, 3× OCH2Ph),
7.66 (dd, 1H, J ) 7 Hz, 2 Hz, C6′H), 7.77 (d, 1H, J ) 2 Hz,
C2′H), 8.12 (d, 1H, J ) 9 Hz, C5H).
1
yellow solid; mp 145.8-146.8 °C. H NMR (CDCl3): δ 5.22 (s,
2H, OCH2Ph), 5.25 (s, 2H, OCH2Ph), 7.01 (d, J ) 8 Hz, C5′H),
7.08 (br s, 1H, C3OH), 7.3-7.5 (m, 12 H, OCH2Ph × 2 + C6H
+ C8H), 7.64 (dt, 1H, J ) 7 Hz, 1.5 Hz, C7H), 7.82 (dd, 1H, J
) 9 Hz, 2 Hz, C6′H), 7.92 (d, 1H, J ) 2 Hz, C2′H), 8.20 (dd,
1H, J ) 8 Hz, 2 Hz, C5H).
3′,4′-Diben zyloxy-7-(h ydr oxyeth oxy)flavon e (5c). A mix-
ture of 2.2 mmol of 4a and 44 mmol of ethylenecarbonate was
heated to 95 °C, 23 mmol of powdered K2CO3 was added and
the reaction mixture was stirred at 95 °C for 1 h. Chloroform
(50 mL) was added and the K2CO3 was filtered. The filtrate
was washed with 5 N HCl (3 × 10 mL), dried over sodium
sulfate and concentrated under reduced pressure. The product
was recrystallized from ethanol to give 0.5 g (45%) of 5c as a
yellow powder. 1H NMR (DMSO): δ 3.75 (t, 2H, J ) 6 Hz,
OCH2CH2OH), 4.17 (t, 2H, J ) 6 Hz, OCH2CH2OH), 5.23 (s,
2H, C3′OCH2Ph), 5.29 (s, 2H, C4′OCH2Ph), 6.92 (s, 1H, C3H),
7.08 (dd, 1H, J ) 9 Hz, 2 Hz, C5′H), 7.25 (d, 1H, J ) 7 Hz,
C8H), 7.3-7.6 (m, 10H, OCH2Ph × 2), 7.73 (dd, 1H, J ) 9 Hz,
2 Hz, C6′H), 7.78 (d, 1H, J ) 2 Hz, C2′H), 7.94 (d, 1H, J ) 7
Hz, C5H).
3-Hyd r oxy-7,3′,4′-tr iben zyloxyfla von e (4c). 3,4-Diben-
zyloxybenzaldehyde (3) was reacted with 4-benzyloxy-2-hy-
droxyacetophenone (1c) in a similar way as described for 4b.
The brown oil resulting from step 1 was crystallized from
ethanol/chloroform (5:1) to give 1.8 g (42%); mp 120.3-121.3
°C. In the second step the product was extracted with CH2Cl2
(2 × 100 mL). The organic layers were dried over sodium
sulfate and evaporated under reduced pressure. The product
was crystallized form ethanol/CH2Cl2 (100 mL, 2:1) to give 1.82
g (41%) of 4c; mp 160.6-161.2 °C. 1H NMR (CDCl3): δ 5.16
(s, 2H, C7OCH2Ph), 5.24 (s, 4H, C3′OCH2Ph + C4′OCH2Ph),
6.9 (br s, 1H, C3OH), 6.96-7.04 (m, 3H, C5′H + C6H + C8H),
7.23-7.47 (m, 15H, 3× OCH2Ph), 7.77 (dd, 1H, J ) 9 Hz, 2
Hz, C6′H), 7.87 (d, 1H, J ) 2 Hz, C2′H), 8.10 (d, 1H, J ) 9 Hz,
C5H).
3′,4′-Diben zyloxy-3-(h yd r oxyeth oxy)fla von e (6c). The
reaction was carried out with â-bromoethylbenzyl ether and
4b, using the same method as described for 4d (step 2); yield
7-(2-Ben zyloxyeth oxy)-3-h yd r oxy-3′,4′-d iben zyloxyfla -
von e (4d ). A stirred suspension of 20 mmol of 2,4-dihydroxy-
acetophenone, 20 mmol of â-bromoethylbenzyl ether and 24
mmol of K2CO3 in acetone (60 mL) was heated to reflux for 16
h. The solvent was evaporated and the residue resuspended
in H2O (100 mL) and extracted with CHCl3 (2 × 100 mL). The
combined layers were dried over sodium sulfate and concen-
trated under reduced pressure. The crude product was purified
by column chromatography (CH2Cl2) to give 3.7 g (65%) of a
colorless liquid. 1H NMR (CDCl3): δ 2.53 (s, 3H, CH3), 3.81 (t,
2H, J ) 6 Hz, BnOCH2CH2O), 4.14 (t, 2H, J ) 6 Hz,
BnOCH2CH2O), 4.60 (s, 2H, OCH2Ph), 6.40 (m, 2H, C3H +
C5H), 7.33 (s, 5H, OCH2Ph), 7.60 (d, 1H, J ) 9 Hz, C6H), 12.71
(s, 1H, OH).
1
240 mg (49%) of 6c as a light-yellow solid. H NMR (CDCl3):
δ 3.69 (t, 2H, J ) 6 Hz, CH2OCH2Ph), 4.27 (t, 2H, J ) 6 Hz,
C3OCH2), 4.40 (s, 2H, OCH2Ph), 5.14 (s, 2H, OCH2Ph), 5.27
(s, 2H, OCH2Ph), 6.86 (d, 1H, J ) 9 Hz, C5′H), 7.2-7.5 (m,
12H, OCH2Ph × 3 + C6H + C8H), 7.64 (dt, 1H, J ) 7 Hz, 2
Hz, C7H), 7.8-7.9 (m, 2H, C2′H + C6′H), 8.20 (dd, J ) 7 Hz,
2 Hz, C5H).
3-Hyd r oxyeth oxy-7,3′,4′-tr iben zyloxyfla von e (7c). The
reaction was carried out with 4c, using the same method as
described for 5c. The product was recrystallized from ethanol
and chloroform to give 1.15 g (96%) of 7c as a light-yellow solid;
mp 136.7 °C. 1H NMR (CDCl3): δ 3.6-3.8 (m, 4H, OCH2CH2O),
5.15 (s, 2H, OCH2Ph), 5.25 (s, 2H, OCH2Ph), 5.26 (s, 2H, OCH2-
Ph), 6.92 (d, 1H, J ) 2 Hz, C8H), 7.0-7.1 (m, 2H, C6H +
C5′H), 7.2-7.5 (m, 10H, OCH2Ph × 2), 7.63 (dd, 1H, J ) 8
Hz, 2 Hz, C6′H), 7.74 (d, 1H, J ) 2 Hz, C2′H), 8.12 (d, 1H, J
) 9 Hz, C5H).
3,4-Dibenzyloxybenzaldehyde (3) was reacted with 2-hy-
droxy-4-(2-benzyloxyethoxy)acetophenone (1d ) in a similar way
as described for 4b. The brown oil resulting from step 1 was
crystallized from ethanol/diethyl ether to give 2.0 g (49%). In
the second step 70 mL of 0.8% w/v NaOH instead of 100 mL
of 5.4% NaOH was used and the product was extracted with
CHCl3 (2 × 100 mL). The organic layers were dried over
sodiumsulfate and evaporated under reduced pressure. The
product was crystallized from methanol/CHCl3 (4:1) to give 1.2
g (59%) of 4d . 1H NMR (CDCl3): δ 3.87 (t, 2H, J ) 6 Hz,
BnOCH2CH2O), 4.25 (t, 2H, J ) 6 Hz, BnOCH2CH2O), 4.64
N-(3-(3′,4′-Diben zyloxyfla von -7-yl)oxyp r op yl)-N,N,N,-
tr im eth yla m m on iu m Ch lor id e (5d ). To a solution of 6.0
mmol of 4a in DMSO (30 mL) were added 17.4 mmol of K2-
CO3 and 1.2 mL (12.0 mmol) of 1-bromo-3-chloropropane and
the mixture was stirred at room temperature for 5 h. The
DMSO was evaporated under reduced pressure. The residue
was suspended in CH2Cl2 (100 mL), filtered and concentrated
in vacuo to give 3.0 g (95%) of 7-(3-chloropropoxy)-3′,4′-
(s, 2H, PhCH2-OCH2CH2O), 5.24 (s, 4H, C3′OCH2Ph
+
1
dibenzyloxyflavone; mp 152.6-153.4 °C. H NMR (CDCl3): δ
C4′OCH2Ph), 6.88-7.04 (m, 3H, C8H + C5′H + C6H), 7.24-
7.51 (m, 15H, PhOCH2CH2, C3′OCH2Ph + C4′OCH2Ph), 7.78
(dd, 1H, J ) 9 Hz, 2 Hz, C6′H), 7.85 (d, 1H, J ) 2 Hz, C2′H),
8.05 (d, 1H, J ) 9 Hz, C5H).
2.29 (m, 2H, CCH2C), 3.76 (t, 2H, J ) 6 Hz, CH2Cl), 4.22 (t,
2H, J ) 6 Hz, OCH2), 5.23 (s, 4H, OCH2Ph × 2), 6.59 (s, 1H,
C3H), 6.89-7.02 (m, 3H, C6H + C8H + C5′H), 7.30-7.50 (m,
12H, OCH2Ph × 2 + C2′H + C6′H), 8.08 (d, 1H, J ) 9 Hz,
C5H).
3′,4′-Diben zyloxy-7-m et h oxyfla von e (5b ). 2.2 mmol of
potassium tert-butoxide was added to a suspension of 2.0 mmol
of 4a in dry THF (50 mL), followed by 8.8 mmol of CH3I and
the mixture was stirred at room temperature for 16 h. H2O
(100 mL) was added and the mixture was extracted with CH2-
Cl2 (2 × 100 mL). The combined organic layers were dried over
sodium sulfate and concentrated under reduced pressure to
A mixture of 2.5 mmol of 7-(3-chloropropoxy)-3′,4′-diben-
zyloxyflavone, 15 mL of trimethylamine (33% in ethanol) and
2 mmol of K2CO3 in dioxane (15 mL) was heated at 100 °C for
56 h in a stainless steel bomb. The mixture was filtered and
the solvents were evaporated under reduced pressure. The
product was recrystallized twice from 2-propanol/ethanol to
1
1
give 0.9 g (100%) of 5b as a white solid. H NMR (CDCl3): δ
give 1.26 g (86%) of 5d as a white solid. H NMR (DMSO): δ
3.87 (s, 3H, OCH3), 5.19 (s, 4H, OCH2Ph × 2), 6.55 (s, 1H,
C3H), 6.8-7.0 (m, 3H, C5′H + C6H + C8H), 7.26-7.44 (m,
12H, OCH2Ph × 2 + C2′H +C6′H), 8.04 (d, 1H, J ) 9 Hz, C5H).
2.27 (m, 2H, CCH2C), 3.12 (s, 9H, N(CH3)3), 3.82 (t, 2H, J ) 6
Hz, CH2N), 4.24 (t, 2H, J ) 6 Hz, OCH2), 5.24 (s, 2H, C3′OCH2-
Ph), 5.27 (s, 2H, C4′OCH2Ph), 6.93 (s, 1H, C3H), 7.0-8.0 (m,
16H, C6′H + C2′H + C6H + C8H + C5′H + C3′OCH2Ph +
C4′OCH2Ph + C5H).
3′4′-Diben zyloxy-3-m et h oxyfla von e (6b ). The reaction
was carried out with 4b, using the same method as described
for 5b. The product was crystallized twice from ethyl acetate
and hexane to give 240 mg (47%) as a white solid. 1H NMR
(CDCl3): δ 3.74 (s, 3H, OCH3), 5.28 (s, 4H, OCH2Ph), 6.98 (d,
J ) 7 Hz, C5′H), 7.3-7.7 (m, 14H, OCH2Ph × 2 + C6H + C8H
N-(3-(3′,4′-Dib en zyloxyfla von -3-yl)oxyp r op yl)-N,N,N-
tr im eth yla m m on iu m Br om id e (6f). 4.5 mL (44.3 mmol) of
1,3-dibromopropane was added to a solution of 11.1 mmol of
4b and 11.8 mmol of potassium tert-butoxide in dry THF (250

3,7-Substituted-2-(3′,4′-dihydroxyphenyl)flavones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3757
mL). The reaction mixture was stirred at room temperature
for 19 h and subsequently heated to reflux for 5 h under an
atmosphere of dry nitrogen. CH2Cl2 (250 mL) was added and
the mixture was washed with H2O. The organic layer was dried
over magnesium sulfate and evaporated under reduced pres-
sure. The crude product was purified by column chromatog-
raphy (ethyl acetate/hexane 1:1 (v/v)) and recrystallized from
ethyl acetate and hexane to give 2.71 g (42%) of 3-(3-
7,3′,4′-tribenzyloxyflavone; mp 136.1-137.3 °C. 1H NMR
(CDCl3): δ 2.06 (m, 2H, OCH2CH2CH2Cl), 3.59 (t, 2H, J ) 7
Hz, OCH2CH2CH2Cl), 4.08 (t, 2H, J ) 7 Hz, OCH2CH2CH2-
Cl), 5.15 (s, 2H, C7OCH2Ph), 5.22 (s, 2H, C3′OCH2Ph), 5.24
(s, 2H, C4′OCH2Ph), 6.89 (d, 1H, J ) 1 Hz, C8H), 7.0-7.1 (m,
2H, C5′H + C6H), 7.3-7.8 (m, 17H, 3× OCH2Ph +C2′H +
C6′H), 8.12 (d, 1H, J ) 7 Hz, C5H).
A mixture of 2.0 mmol of 3-(3-chloropropoxy)-7,3′,4′-triben-
zyloxyflavone in 15 mL of trimethylamine (33% in ethanol)
was heated to 100 °C for 64 h in a stainless steel bomb. The
solvents were evaporated under reduced pressure and the
product was crystallized from toluene to give 167 mg (12%) of
1
bromopropoxy)-3′,4′-dibenzyloxyflavone. H NMR (CDCl3): δ
218 (m, 2H, CCH2C), 3.47 (t, 2H, J ) 6 Hz, CH2Cl), 4.13 (t,
2H, J ) 6 Hz, OCH2), 5.23 (s, 2H, OCH2Ph), 5.27 (s, 2H, OCH2-
Ph), 7.04 (s, 1H, C3H), 7.25-7.5 (m, 3H, C6H + C8H + C5′H),
7.6-7.8 (m, 12H, OCH2Ph × 2 + C2′H + C6′H), 8.23 (dd, 1H,
J ) 7 Hz, 2 Hz, C5H).
1
a white solid; mp 174.7-174.8 °C. H NMR (DMSO): δ 2.08
(m, 2H, OCH2CH2CH2N(CH3)3), 3.06 (s, 9H, N+(CH3)3), 3.52
(m, 2H, OCH2CH2CH2N(CH3)3), 4.08 (t, 2H, J ) 7 Hz, OCH2-
CH2CH2N(CH3)3), 5.23 (s, 2H, C7OCH2Ph), 5.27 (s, 4H, 2x
OCH2Ph), 7.14 (dd, 1H, J ) 9 Hz, 2 Hz, C5′H), 7.20-7.70 (m,
19H, 3× OCH2Ph + C6H + C8H + C6′H + C2′H), 7.98 (d,
1H, J ) 9 Hz, C5H).
A mixture of 4.7 mmol of 3-(3-bromopropoxy)-3′,4′-dibenzyl-
oxyflavone and 25 mL of trimethylamine (33% in ethanol) in
dioxane (15 mL) was heated to 100 °C for 17 h in a stainless
steel bomb. The solvents were evaporated under reduced
pressure and the product was recrystallized from ethanol and
hexane to give 2.1 g (71%) of 6f as an off-white powder. 1H
NMR (DMSO): δ 2.03-2.18 (m, 2H, CH2), 3.38 (s, 9H,
N(CH3)3), 3.45-3.55 (m, 2H, CH2N), 4.0 (t, 2H, J ) 7 Hz,
OCH2), 5.24 (s, 2H, OCH2Ph), 5.28 (s, 2H, OCH2Ph), 7.26-
7.55 (m, 11H, 2× OCH2Ph + C5′H), 7.70-7.92 (m, 5H, C6H +
C8H + C7H + C6′H + C2′H), 8.10 (d, 1H, J ) 9 Hz, C5H).
N-(6-(3′,4′-Diben zyloxyfla von -3-yl)oxyh exyl)-N,N,N-tr i-
m eth yla m m on iu m Ch lor id e (6g). 6g was prepared by
alkylation of 4b with 1-chloro-6-iodohexane, using the same
method as described for 5d . The crude product was purified
by column chromatography (CH2Cl2) to give 1.6 g (94%) of 3-(6-
chlorohexyloxy)-3′,4′-dibenzyloxyflavone. 1H NMR (CDCl3): δ
1.36 (m, 4H, CH2), 1.68 (m, 4H, CH2), 3.47 (t, 2H, J ) 6 Hz,
CH2Cl), 3.99 (t, 2H, J ) 6 Hz, OCH2), 5.23 (s, 2H, OCH2Ph),
5.27 (s, 2H, OCH2Ph), 7.03 (d, 1H, J ) 8 Hz, C5′H), 7.25-7.5
(m, 12H, 2× OCH2Ph + C6H, C8H), 7.66 (dt, 1H, J ) 7 Hz, 2
Hz, C7H), 7.71 (dd, 1H, J ) 8 Hz, 1 Hz, C6′H), 7.80 (d, 1H, J
) 1 Hz, C2′H), 8.23 (dd, 1H, J ) 7 Hz, 2 Hz, C5H).
3′,4′-Dib e n zyloxy-7-(3-d im e t h yla m in op r op oxy)fla -
von e (5e). 4a was reacted in a similar way as described for
5d using dimethylamine (33% in ethanol). The residue was
suspended in acidic water (pH ) 1-2), washed with chloroform
(2 × 100 mL), basified with 1 M NaOH and extracted with
chloroform (2 × 150 mL). The organic layers were dried over
sodium sulfate and concentrated in vacuo to give 0.8 g (60%)
of 5e. 1H NMR (CDCl3 + 20% DMSO): δ 2.37 (m, 2H, CCH2C),
2.86 (d, 6H, J ) 7 Hz, N(CH3)2), 3.30 (m, 2H, CH2N), 4.30 (t,
2H, J ) 6 Hz, OCH2), 5.21 (s, 2H, C3′OCH2Ph), 5.23 (s, 2H,
C4′OCH2Ph), 6.70 (s, 1H, C3H), 6.98-7.13 (m, 3H, C6H + C8H
+ C5′H), 7.33-7.56 (m, 12H, OCH2Ph × 2 + C2′H + C6′H),
7.98 (d, 1H, J ) 9 Hz, C5H).
3′,4′-Dib e n zyloxy-3-(3-d im e t h yla m in op r op oxy)fla -
von e (6e). 4b was prepared using the method described for
5d . After evaporation of the DMSO the residue was dissolved
in H2O (100 mL) and extracted with chloroform (2 × 100 mL).
The organic layers were dried over sodium sulfate and
evaporated under reduced pressure to give 1.5 g (95%) of 3-(3-
chloropropoxy)-3′,4′-dibenzyloxyflavone. 1H NMR (CDCl3): δ
2.08 (m, 2H, OCH2CH2CH2Cl), 3.60 (t, 2H, J ) 7 Hz, OCH2-
CH2CH2Cl), 4.11 (t, 2H, J ) 7 Hz, OCH2CH2CH2Cl), 5.26 (s,
4H, C3′OCH2Ph + C4′OCH2Ph), 7.12 (d, 1H, J ) 9 Hz, C5′H),
7.26-7.52 (m, 17H, 3× OCH2Ph + C6H + C8H), 7.60-7.70
(m, 3H, C2′H + C6′H + C7H), 8.20 (d, 1H, J ) 9 Hz, C5H).
A mixture of 2.0 mmol of 3-(3-chloropropoxy)-3′,4′-dibenzy-
loxyflavone in ethanol (10 mL) and dioxane (10 mL), 2.0 mmol
of NaI, 4.0 mmol of Na2CO3 and 25 mL of dimethylamine (33%
in ethanol) was heated to 60 °C for 56 h in a stainless steel
bomb. The solvents were evaporated under reduced pressure.
The resulting brown oil was used directly in the next step.
(3′4′-Diben zyloxyfla von -3-yl)oxya cetic Acid (6d ). To a
stirred solution of 2.0 mmol of 4b and 4.0 mmol of potassium
tert-butoxide in dry THF (25 mL) was added 6.1 mmol of ethyl
chloroacetate. The reaction mixture was stirred at room
temperature for 16 h. After acidification with concentrated
HCl, ice-cold H2O (100 mL) was added and the mixture was
extracted with CH2Cl2 (4 × 25 mL). The organic layers were
dried over magnesium sulfate and evaporated in vacuo. The
product was crystallized from ethyl acetate to give 0.62 g (1.15
mmol 57%) of ethyl (3′,4′-dibenzyloxyflavon-3-yl)oxyacetate as
an off-white powder.
3-(6-Chlorohexyloxy)-3′,4′-dibenzyloxyflavone was reacted in
a similar way as described for 5d , without the addition of K2-
CO3. The product was crystallized from ethanol and ether to
give 1.14 g (69%) 6g. 1H NMR (CDCl3): δ 1.2-1.7 (m, 8H, CH2),
3.28 (s, 9H, N+(CH3)3), 3.42 (t, 2H, J ) 7 Hz, CH2N), 3.86 (t,
2H, J ) 7 Hz, OCH2), 5.14 (s, 2H, OCH2Ph), 5.19 (s, 2H, OCH2-
Ph), 7.03 (d, 1H, J ) 9 Hz, C5′H), 7.10-7.70 (m, 15H, 2×
OCH2Ph + C6H + C8H + C7H + C6′H + C2′H), 8.13 (dd, 1H,
J ) 9 Hz, C5H).
N-(8-(3′,4′-Diben zyloxyfla von -3-yl)oxyoctyl)-N,N,N-tr i-
m eth yla m m on iu m Br om id e (6h ). 6h was prepared by
alkylation of 4b with 1,8-dibromooctane, using the same
method as described for 5d ; yield 1.05 g (82%) of 3-(8-
1
bromooctyloxy)-3′,4′-dibenzyloxyflavone. H NMR (CDCl3): δ
1.2-1.5 (m, 8H, O(CH2)3(CH2)4CH2Br), 1.5-1.9 (m, 4H, OCH2-
(CH2)2(CH2)5Br), 3.34 (t, 2H, J ) 7 Hz O(CH2)7CH2Br), 3.97
(t, 2H, J ) 7 Hz, OCH2(CH2)7Br), 5.22 (s, 2H, C3′OCH2Ph),
5.25 (s, 2H, C4′OCH2Ph), 7.02 (d, 1H, J ) 9 Hz, C5′H), 7.25-
7.55 (m, 12H, C3′OCH2Ph + C4′OCH2Ph + C6H + C8H),
7.59-7.73 (m, 2H, C6′H + C7H), 7.81 (d, 1H, J ) 2 Hz, C2′H),
8.22 (dd, 1H, J ) 8.0 Hz, 2 Hz, C5′H).
3-(8-Bromooctyloxy)-3′,4′-dibenzyloxyflavone was reacted in
a similar way as described for 6f, with the modification that
3-(8-bromooctyloxy)-3′,4′-dibenzyloxyflavone was dissolved in
ethanol and 1,4-dioxane; yield 0.93 g (91%) of a yellow solid.
1H NMR (CDCl3): δ 1.2-1.7 (m, 12H, CH2), 3.23 (s, 9H, N+-
(CH3)3), 3.38 (t, 2H, J ) 9 Hz, O(CH2)7CH2N+(CH3)3), 3.97 (t,
2H, J ) 6 Hz, OCH2), 5.18 (s, 2H, C3′OCH2Ph), 5.21 (s, 2H,
C4′OCH2Ph), 7.07 (d, 1H, J ) 9 Hz, C5′H), 7.33-7.50 (m, 12H,
C3′OCH2Ph + C4′OCH2Ph + C6H + C8H), 7.61-7.72 (m, 2H,
C6′H + C7H), 7.79 (d, 1H, J ) 2 Hz, C2′H), 8.18 (dd, 1H, J )
7 Hz, 2 Hz, C5H).
N-(3-(7,3′,4′-Tr iben zyloxyflavon -3-yl)oxypr opyl)-N,N,N-
tr im eth yla m m on iu m Ch lor id e (7d ). 4c was alkylated using
1-bromo-3-chloropropane, employing the same method as
described for 5d . The product was purified by column chro-
matography (CH2Cl2) to give 3.2 g (84%) of 3-(3-chloropropoxy)-
The powder was dissolved in 1,4-dioxane and a solution of
2.07 mmol of NaOH dissolved in 7.5 mL of methanol was
added. The reaction mixture was stirred at room temperature
for 16 h. After acidification with glacial acetic acid H2O (20
mL) was added and the mixture was extracted with CH2Cl2
(2 × 20 mL). The organic layers were dried over sodium sulfate
and evaporated under reduced pressure. The resulting yellow
powder was used without further purification. 1H NMR
(CDCl3): δ 4.0 (s, 2H, OCH2COOH), 5.21 (s, 2H, C3′OCH2Ph),
5.23 (s, 2H, C4′OCH2Ph), 7.00 (d, 1H, J ) 9 Hz, C5′H), 7.18-
7.74 (m, 15H, 2× OCH2Ph + C2′H + C6′H + C6H + C7H +
C8H), 8.2 (dd, 1H, J ) 8.0 Hz, 1 Hz, C5H).
3′,4′-Diben zyloxy-3-tetr a a cetylglu cosylfla von e (6i). To

3758 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
van Acker et al.
a solution of 1 mmol of 4b and 1.5 mmol of acetobromoglucose
in dry pyridine (10 mL) (+ molecular sieves 5 Å) was added
2.2 mmol of silver oxide. After stirring at room temperature
for 1 h the silver salts were removed by filtration over Celite
and silica. The solvent was removed in vacuo and the crude
product was purified by column chromatography (ether) to give
700 mg (90%) of a green glassy solid. 1H NMR (CDCl3): δ 1.78
(s, 3H, Ac), 1.98 (s, 3H, Ac), 1.99 (s, 3H, Ac), 2.07 (s, 3H, Ac),
3.58 (dt, 1H, J ) 10 Hz, 1 Hz, CH-O), 3.9-4.1 (m, 2H, CH2-
OAC), 5.0-5.3 (m, 3H, 3× CH-O), 5.24 (s, 2H, OCH2Ph), 5.28
(s, 2H, OCH2Ph), 5.68 (d, 1H, J ) 8 Hz, acetal-H), 6.99 (d, 1H,
J ) 9 Hz, C5′H), 7.3-7.8 (m, 15H, 2× OCH2Ph + C2′H + C6′H
+ C6H + C7H + C8H), 8.18 (dd, 1H, J ) 8 Hz, 2 Hz, C5H).
3′,4′-Diben zyloxy-3-h exa a cetylr u tin osylfla von e (6j). A
suspension of 5 g of rutine in 50% acetic acid (150 mL) was
heated to reflux under an atmosphere of dry nitrogen for 6 h.
H2O (100 mL) was added and the reaction mixture was kept
at 4 °C overnight and filtered. The filtrate was concentrated
in vacuo. The residue was dissolved in pyridine (15 mL) and
acetic acid anhydride (15 mL) and stirred for 3 h at room
temperature. CH2Cl2 was added and the organic layer was
washed with H2O (2×), dried over sodium sulfate and evapo-
rated to dryness. The product was purified by column chro-
matography (ether) to give 1.28 g (27%) of â-heptaacetylruti-
nose as a white ‘glass’.
This material was dissolved in dry CH2Cl2 (12 mL), 4.9
mmol of TiBr4 in dry CH2Cl2 (12 mL) was added and the
reaction mixture was stirred for 17 h at room temperature
under an atmosphere of dry nitrogen. The reaction mixture
was neutralized with NaOAc and filtered over Celite and
concentrated under reduced pressure. The resulting orange/
brown oil (1.6 g) was used without further purification. 4b was
reacted with R-acetobromorutinose as described for 6i to give
634 mg (46%) 6j as a white-greenish ‘glass’. 1H NMR (CDCl3):
δ 0.97 (d, 3H, J ) 6 Hz, CH3), 1.86 (s, 3H, Ac), 1.88 (s, 3H,
Ac), 1.96 (s, 3H, Ac), 1.97 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.03 (s,
3H, Ac), 3.3-3.7 (m, 4H, 2× CH-O + CH2O), 4.49 (s, 1H,
acetal-H), 4.8-5.3 (m, 6H, CH-O), 5.21 (s, 2H, OCH2Ph), 5.23
(s, 2H, OCH2Ph), 5.65 (d, 1H, J ) 8 Hz, acetal-H), 7.03 (d, 1H,
J ) 9 Hz, C5′H), 7.2-7.7 (m, 15H, 2× OCH2Ph + C2′H + C6′H
+ C6H + C7H + C8H), 8.15 (d, J ) 8 Hz, C5H).
suspension was filtered and the residue gave 0.5 g (93%) of
8e; mp 173.6 °C. H NMR (D2O): δ 2.02 (m, 2H, CH2) 2.88 (s,
6H, N(CH3)2), 3.18 (t, 2H, J ) 7 Hz, CH2N), 3.60 (m, 2H,
C7OCH2), 5.70 (s, 1H, C3H), 6.10 (s, 1H, C8H), 6.30-6.60 (m,
4H, C6H + C2′H + C5′H + C6′H), 7.13 (d, 1H, J ) 9 Hz, C5H).
Anal. (C20H21NO5‚HCl) C, H, N.
1
3,3′,4′-Tr ih yd r oxyfla von e (9a ). Washed with hexane and
1
ether (twice); yield 69%; mp dec at 280 °C. H NMR (DMSO):
δ 6.89 (d, 1H, J ) 9 Hz, C5′H), 7.44 (t, 1H, J ) 8 Hz, C6H),
7.61 (dd, 1H, J ) 9 Hz, 2 Hz, C6′H), 7.65-7.75 (m, C7H +
C8H + C2′H), 8.09 (d, 1H, J ) 8 Hz, C5H), 9.4 (br s, 3H, OH).
Anal. (C15H10O5) C, H.
(3′,4′-Dih yd r oxyfla von -3-yl)oxya cetic Acid (9d ). Yield
1
0.21 g (43%) of 9d as a green powder; mp 253.3-254.9 °C. H
NMR (DMSO): δ 4.74 (s, 2H, OCH2COOH), 6.89 (d, 1H, J )
9 Hz, C5′H), 7.48 (t, 1H, J ) 7.4 Hz, C6H), 7.58-7.86 (m, 4H,
C6′H + C7H + C8H + C2′H), 8.09 (dd, 1H, J ) 8 Hz, 2 Hz,
C5H), 9.32 (s, 1H, C3′OH), 9.79 (s, 1H, C4′OH), 12.92 (br s,
1H, CH2COOH). Anal. (C17H12O7) C, H.
N-(3-(3′,4′-Dih yd r oxyfla von -3-yl)oxyp r op yl)-N,N,N-tr i-
m eth yla m m on iu m Mixed Br om id e/Ch lor id e Sa lt (9f).
1
Crystallized from ethanol; yield 91%; mp 234.4-235.4 °C. H
NMR (D2O): δ 1.86 (m, 2H, CH2), 2.98 (s, 9H, N(CH3)3), 3.12
(m, 2H, CH2N), 3.45 (t, 2H, J ) 6 Hz, OCH2), 6.49 (d, 1H, J )
9 Hz, C5′H), 6.74 (d, 1H, J ) 8 Hz, C8H), 6.8-6.9 (m, 2H,
C2′H + C6′H), 7.10 (t, 1H, J ) 8 Hz, C6H), 7.37 (t, 1H, J ) 8
Hz, C7H), 7.46 (d, 1H, J ) 8 Hz, C5H). Anal. (C21H24NO5Cl0.62
Br0.38) C, H, N.
-
N-(6-(3′,4′-Dih yd r oxyfla von -3-yl)oxyh exyl)-N,N,N-t r i-
m eth yla m m on iu m Ch lor id e (9g). Crystallized from ethanol;
yield 51%; mp 230.8-232.8 °C. 1H NMR (DMSO): δ 1.15-1.8
(m, 8H, CH2), 3.04 (s, 9H, N+(CH3)3), 3.36 (m, 2H, CH2N), 3.98
(t, 2H, J ) 6 Hz, OCH2), 6.96 (d, 1H, J ) 8 Hz, C5′H), 7.4-7.9
(m, 5H, C8H + C2′H + C6′H + C6H + C7H), 8.08 (dd, 1H, J
) 8 Hz, 1 Hz, C5H), 9.44 (s, 1H, OH), 9.91 (s, 1H, OH). Anal.
(C24H30ClNO5) C, H, N.
N-(8-(3′,4′-Dih yd r oxyfla von -3yl)oxyoctyl)-N,N,N-tr im -
eth yla m m on iu m Br om id e (9h ). Debenzylation was carried
out with HBr instead of HCl; washed with ethyl acetate; yield
1
54%; mp 196.7-198.5 °C. H NMR (DMSO): δ 1.15-1.75 (m,
12H, CH2), 3.04 (s, 9H, N+(CH3)3), 3.28 (m, 2H, CH2N), 3.97
(t, 2H, J ) 6 Hz, OCH2), 6.91 (d, 1H, J ) 8 Hz, C5′H), 7.4-7.9
(m, 5H, C8H + C2′H + C6′H + C6H + C7H), 8.07 (dd, 1H, J
) 8 Hz, 1 Hz, C5H), 9.44 (br s, 1H, OH), 9.67 (br s, 1H, OH).
Anal. (C26H34BrNO5) C, H, N.
Gen er a l P r oced u r e for Deben zyla tion (Meth od A). A
suspension of 2 mmol of the protected product in HCl (36%,
50 mL) and glacial acid (50 mL) was heated to 100 °C under
an atmosphere of dry nitrogen for 2-3 h. The solvents were
evaporated under reduced pressure and the product was
recrystallized.
3-Meth oxy-7,3′,4′-tr ih yd r oxyfla von e (10b). Crystallized
from ethanol/chloroform (4:1) to give 0.5 g (90%) of a yellow
powder; mp 281.4 °C. 1H NMR (DMSO): δ 3.76 (s, 3H, OCH3),
6.8-7.0 (m, 3H, C5′H + C6H + C8H), 7.42 (dd, 1H, J ) 8 Hz,
1 Hz, C6′H), 7.55 (d, 1H, J ) 1 Hz, C2′H), 7.86 (d, 1H, J ) 8
Hz, C5H), 9.90 (br s, 1H, C7OH). Anal. (C16H12O6) C, H.
3-Hydr oxyeth oxy-7,3′,4′-tr ih ydr oxyflavon e (10c). Washed
with CHCl3 to give 0.5 g (80%) of an orange powder; mp >300
3′,4′,7-Tr ih yd r oxyfla von e (8a ). Crystallized from ethanol;
1
yield 82%; mp dec at 300 °C. H NMR (CDCl3 + 20% DMSO):
δ 6.35 (br s, OH + H2O), 6.59 (s, 1H, C3H), 6.8-6.9 (m, 3H,
C8H, C6H, C5′H), 7.26 (d, 1H, J ) 9 Hz, C6′H), 7.34 (s, 1H,
C2′H), 7.86 (d, 1H, J ) 8 Hz, C5H). Anal. (C15H10O5) C, H.
3′,4′-Dih ydr oxy-7-m eth oxyflavon e (8b). Crystallized from
ethanol/chloroform 4:1 to give 0.5 g (88%) of yellow powder;
1
°C. H NMR (DMSO): δ 4.66 (m, 2H, OCH2CH2OH), 5.00 (m,
1
mp > 240 °C. H NMR (CDCl3 + 20% DMSO): δ 3.87 (s, 3H,
2H, OCH2CH2OH), 7.05 (d, 1H, J ) 8 Hz, C5′H), 7.35 (d, 1H,
J ) 8 Hz, C6H), 7.46 (s, 1H, C8H), 7.88-8.00 (m, 2H, C6′H +
C2′H), 8.06 (d, 1H, J ) 9 Hz, C5H), 9.88 (br s, 1H, C7OH),
10.85 (br s, 1H, C3′OH), 12.35 (br s, 1H, C4′OH). Anal.
(C17H14O7) C, H.
OCH3), 6.50 (s, 1H, C3H), 6.8-7.0 (m, 3H, C5′H + C6H +
C8H), 7.2-7.4 (m, 2H, C6′H + C2′H), 7.93 (d, 1H, J ) 9 Hz,
C5H). Anal. (C16H12O5) C, H.
3′,4′-Dih ydr oxy-7-h ydr oxyeth oxyflavon e (8c). Yield 83%;
mp 206.7-208.7 °C. 1H NMR (DMSO): δ 3.76 (t, 2H, J ) 5
Hz, CH2OH), 4.13 (t, 2H, J ) 5 Hz, C7OCH2), 6.66 (s, 1H,
C3H), 6.90 (d, 1H, J ) 8 Hz, C5′H), 7.07 (d, 1H, J ) 7 Hz,
C6H), 7.28 (s, 1H, C8H), 7.35-7.50 (m, 2H, C2′H + C6′H), 7.89
(d, 1H, J ) 8 Hz, C5H). Anal. (C17H14O6) C, H.
N-(3-(7,3′,4′-Tr ih yd r oxyfla von -3-yl)oxyp r op yl)-N,N,N-
tr im eth yla m m on iu m Ch lor id e (10d ). Crystallized from
methanol/ether (1:1) to give 95 mg (95%) of a brown-white
powder; mp dec >200 °C. 1H NMR (DMSO): δ 2.11 (m, 2H,
CH2), 3.07 (s, 9H, N+(CH3)3), 3.48 (m, 2H, CH2N), 3.97 (t, 2H,
J ) 6 Hz, OCH2), 6.9-7.0 (m, 3H, C5′H + C6H + C8H), 7.43
(dd, 1H, J ) 8 Hz, 2 Hz, C6′H), 7.54 (s, 1H, J ) 2 Hz, C2′H),
7.88 (d, 1H, J ) 9 Hz, C5H), 9.56 (s, 1H, OH), 9.76 (s, 1H,
OH), 10.93 (s, 1H, OH). Anal. (C21H24ClNO6) C, H, N.
Gen er a l P r oced u r e for Deben zyla tion (Meth od B). To
a suspension/solution of 1 mmol of the required compound in
methanol (100 mL) or a mixture of ethyl acetate/methanol (2:
1) was added 150 mg of 10% Pd/C. The reaction mixture was
stirred at room temperature for 30 min to 1 h under an
atmosphere of 1.5 atm H2(g) after replacement of the air by
N-(3-(3′,4′-Dih yd r oxyfla von -7-yl)oxyp r op yl)-N,N,N-tr i-
m eth yla m m on iu m Ch lor id e (8d ). Crystallized from etha-
1
nol/diethyl ether yielding 0.8 g (91%) of 8d ; mp > 210 °C. H
NMR (DMSO + D2O): δ 2.22 (m, 2H, (CH3)3NCH2CH2CH2O),
3.12 (s, 9H, (CH3)3NCH2CH2CH2O), 3.50 (m, 2H, (CH3)3NCH2-
CH2CH2O), 3.89 (m, 2H, (CH3)3NCH2CH2CH2O), 6.26 (s, 1H,
C3H), 6.55-7.2 (m, 5H, C5′H + C6′H + C8H + C5H + C6H),
7.58 (d, 1H, J ) 9 Hz, C5H). Anal. (C21H24ClNO5) C, H, N.
3′,4′-Dih yd r oxy-7-(3-d im et h yla m in op r op oxy)fla von e
(8e). The residue was dissolved in CH2Cl2. The resulting

3,7-Substituted-2-(3′,4′-dihydroxyphenyl)flavones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3759
nitrogen. The Pd/C was filtered and the solvent was evaporated
under reduced pressure.
LP O Assa y. Microsomes were thawed and resuspended in
ice-cold Tris-HCl buffer (50 mM, pH ) 7.4 at 37 °C) and
washed by centrifugation (45 min, 115000g). The pellet was
resuspended in 2 mL of 50 mM Tris-HCl buffer and heated to
100 °C for at least 2 min to remove all enzymatic factors just
before resuspending and diluting it in ice-cold Tris buffer to
1.5 mg/mL.
3′,4′-Dih yd r oxy-3-m eth oxyfla von e (9b). Yield 88%; mp
198.5-199.7 °C. 1H NMR (DMSO): δ 3.83 (s, 3H, OCH3), 6.95
(d, 1H, J ) 9 Hz, C5′H), 7.45-7.88 (m, 5H, C7H + C6H +
C8H + C6′H + C2′H), 8.1 (d, 1H, J ) 7 Hz, C5H). Anal.
(C16H12O5) C, H.
Stock solutions of the flavonoids were freshly prepared in
nitrogen-purged DMSO and Nanopure water (1:1) just before
use, as some of the flavonoids tended to oxidize very quickly.
All compounds were added on ice, after which the incubates
were transferred to a water bath (37 °C), where 200 µM
ascorbate was added. The reaction was started by adding 10
µM freshly prepared FeSO4 in nitrogen-purged Nanopure
water. The maximum DMSO concentration in the final incu-
bation mixture was 2.5%, which was found to have no influence
on the assay. LPO was assayed by measuring thiobarbituric
acid (TBA) reactive substances according to Haenen et al.38
At t ) 0, 5, 10, 15, 30, 45 and 60 min a 0.3-mL aliquot of the
incubation was mixed with 2 mL of an ice-cold TBA-trichlo-
roacetic acid-HCl-butylhydroxytoluene (BHT) solution to stop
the reaction. After heating (15 min, 80 °C) and centrifugation
(15 min) the absorbance at 535-600 nm was determined. The
TBA-trichloroacetic acid-HCl-BHT solution was prepared
by dissolving 41.6 mg of TBA/10 mL of trichloroacetic acid
(16.8% w/v in 0.125 N HCl). To 10 mL of TBA-trichloroacetic
acid-HCl was added 1 mL of BHT (1.5 mg/mL ethanol).
The IC50 was determined by measuring the % LPO inhibi-
tion at several concentrations and interpolating the 50%
inhibition point. The results are expressed as mean ( SEM
(n ) 3-5).
TEAC Assa y. A solution of 2,2′-azinobis(3-ethylbenzthia-
zoline-6-sulfonic acid) (ABTS; Sigma, St. Louis, MO) (1.23 mg/
mL) and azobis(amidinepropane) (ABAP; Polysciences Inc.,
Warrington, MA) (3.96 mg/mL) in 50 mM phosphate buffer
(pH ) 7.4) was heated at 70 °C for 20 min. The amount of
ABTS radicals was measured by determination of the absor-
bance at 734 nm.
Stock solutions of the flavonoids were freshly prepared in
nitrogen-purged DMSO and Nanopure water (1:1) just before
use. A callibration curve of trolox ((()-6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid; Aldrich, Milwaukee,
WI) was made in a concentration range from 0 to 20 µM. The
maximum DMSO concentration in the final incubation mixture
was 0.25%, which was found to have no influence on the assay.
A mixture of 50 µL of test compound and 950 µL of ABTS•+
solution was incubated at 37 °C for 5 min. Subsequently the
absorbance at 734 nm was determined on a UV/VIS spectro-
photometer (Ultrospec 2000, Amersham Pharmacia Biotech,
Sweden).
3′,4′-Dih yd r oxy-3-(h yd r oxyeth oxy)fla von e (9c). Yield
85%; mp 186.2-186.8 °C. 1H NMR (DMSO): δ 3.65 (t, 2H,
OCH2CH2OH), 4.05 (t, 2H, OCH2CH2OH), 6.9 (d, 1H, J ) 9
Hz, C5′H), 7.45 (dt, 1H, J ) 7 Hz, 1 Hz, C6H), 7.6-7.85 (m,
4H, C7H + C8H + C6′H + C2′H) 8.08 (dd, 1H, J ) 7 Hz, 1
Hz, C5H). Anal. (C17H14O6) C, H.
3′,4′-Dih ydr oxy-3-(3-dim eth ylam in opr opoxy)flavon e Hy-
d r och lor id e (9e). Crystallized from methanol and washed
with ether; yield 150 mg (21% over 2 steps); mp 98.5-99.8
1
°C. H NMR (CDCl3 + 20% DMSO): δ 2.11 (t, 2H, OCH2CH2-
CH2N(CH3)2), 2.74 (s, 6H, N(CH3)2), 3.24 (m, 2H, CH2N), 3.85
(t, 2H, J ) 6 Hz, OCH2CH2CH2N(CH3)2), 6.79 (d, 1H, J ) 8
Hz, C5′H), 7.2-7.6 (m, 4H, C6H + C8H + C6′H + C7H), 7.70
(d, 1H, J ) 2 Hz, C2′H), 7.95 (dd, 1H, J ) 8 Hz, 1 Hz, C5H),
8.56 (s, 1H, OH), 8.89 (s, 1H, OH), 11.35 (br s, 1H, N+H). Anal.
(C20H21NO5‚HCl) C, H, N.
3′,4′-Dih yd r oxy-3-glu cosylfla von e (9i). Before the de-
benzylation procedure, the acetyl groups were removed. 0.6
mmol of potassium tert-butoxide was added to a solution of
380 mg (0.49 mmol) of 6i in MeOH (50 mL) and the reaction
mixture was stirred for 1.5-2 h. H2O was added and the water
layer was extracted twice with ethyl acetate. The crude product
was purified by column chromatography (ethyl acetate/
methanol 9:1) to give 280 mg (94%). Subsequently the com-
1
pound was debenzylated.; yield 89%; mp 166.6-168.6 °C. H
NMR (DMSO): δ 3.0-3.7 (m, 6H, 4× CH + 1× CH2 sugar),
5.55 (m, 1H, acetal-H), 6.86 (d, 1H, J ) 9 Hz, C5′H), 7.48 (t,
1H, J ) 7 Hz, C6H), 7.6-7.9 (m, 4H, C2′H + C6′H + C7H +
C8H), 8.08 (d, 1H, J ) 8 Hz, C5H). 13C NMR (DMSO): 60.7
(CH2), 69.6 (CH), 73.7 (CH), 76.3 (CH), 77.3 (CH), 100.5 (acetal-
CH), 114.9 (CH), 116.2 (CH), 117.9 (CH), 121.2 (C), 121.4 (CH),
123.0 (C), 124.7 (2× CH), 133.6 (CH), 135.4 (C), 144.6 (C), 148.1
(C), 154.2 (C), 155.7 (C), 173.1 (CO). Anal. (C21H20O10) C, H.
3′,4′-Dih yd r oxy-3-r u tin osylfla von e (9j). Before the de-
benzylation procedure the acetyl groups were removed as
described for 9i; yield 95%; mp dec at 174 °C. 1H NMR
(DMSO): δ 0.97 (d, 3H, J ) 6 Hz, CH3) 3.0-3.7 (m, 10H, 8×
CH + 1× CH2 sugar), 5.46 (m, 2H, 2× acetal-H), 6.86 (d, 1H,
J ) 9 Hz, C5′H), 7.46 (t, 1H, J ) 7 Hz, C6H), 7.6-7.9 (m, 4H,
C2′H + C6′H + C7H + C8H), 8.08 (d, 1H, J ) 8 Hz, C5H). 13
C
NMR (DMSO): δ 17.5 (CH3), 66.6 (CH2), 68.0 (CH), 69.8 (CH),
70.1 (CH), 70.2 (CH), 71.6 (CH), 73.7 (CH), 75.7 (CH), 76.4
(CH), 100.4 (acetal-CH), 100.8 (acetal-CH), 115.0 (CH), 116.2
(CH), 117.9 (CH), 121.2 (C), 121.5 (CH), 122.9 (C), 124.7 (2×
CH), 133.6 (CH), 135.4 (C), 144.5 (C), 148.0 (C), 154.3 (C), 156.0
(C), 173.2 (CO). Anal. (C27H30O14) C, H.
The antioxidant-induced decrease in absorbance is directly
related to the antioxidant capacity of the compound (solution)
being tested. The trolox equivalent antioxidant capacity
(TEAC) is defined as the concentration (mM) of trolox having
an antioxidant capacity equivalent to 1 mM test compound.
The data are expressed as mean ( SD (n ) 3-5).
7-(2-Hydr oxyoxyeth oxy)-3,3′,4′-tr ih ydr oxyflavon e (11a).
Yield 240 mg (92%); mp dec at 300 °C. 1H NMR (DMSO): δ
3.77 (q, 2H, J ) 6 Hz, HOCH2CH2O), 4.15 (t, 2H, J ) 6 Hz,
HOCH2CH2O), 4.99 (t, 1H, J ) 6 Hz, HOCH2CH2O), 6.89 (d,
1H, J ) 9, C5′H), 7.05 (dd, 1H, J ) 9 Hz, 2 Hz, C6H), 7.22 (d,
1H, J ) 2 Hz, C8H), 7.55 (dd, 1H, J ) 8 Hz, 2 Hz, C6′H), 7.74
(d, 1H, J ) 2 Hz, C2′H), 7.97 (d, 1H, J ) 9 Hz, C5H), 9.17
(1H, s, C3OH), 9.27 (s, 1H, C3′OH), 9.57 (s, 1H, C4′OH). Anal.
(C17H14O7) C, H.
Ack n ow led gm en t. The authors thank Dr. Rob van
der Steen for his help with the syntheses and valuable
discussions and Mrs. Ing. Andrea van de Stolpe for the
determination of the melting points. We thank Ms.
J anneke W. Hulshof and Ms. Olga Schouten for their
assistance with the lipid peroxidation assay.
P r ep a r a tion of Micr osom es. Male Wistar rats (200-220
g) obtained from Harlan Olac CPB (Horst, The Netherlands)
were sacrificed by decapitation. Livers were removed and
homogenized in ice-cold phosphate buffer (50 mM phosphate
+ 0.1 mM EDTA, pH ) 7.4; 1:2 w/v). The homogenate was
centrifuged at 10000g (20 min at 4 °C). Subsequently, the
supernatant was centrifuged at 100000g for 60 min (4 °C). The
pellet was resuspended in phosphate buffer and centrifuged
again at 100000g (60 min, 4 °C). Finally the microsomal pellet
was resuspended in phosphate buffer (microsomes originating
from 2 g liver/mL buffer) and 1-mL aliquots were stored at
-80 °C.
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