Structure-activity relationship studies of flavonoids as potent inhibitors of human platelet 12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2

Article abstract of DOI:10.1016/j.bmc.2007.07.036

Human lipoxygenase (hLO) isozymes have been implicated in a number of disease states and have attracted much attention with respect to their inhibition. One class of inhibitors, the flavonoids, have been shown to be potent lipoxygenase inhibitors but their study has been restricted to those compounds found in nature, which have limited structural variability. We have therefore carried out a comprehensive study to determine the structural requirements for flavonoid potency and selectivity against platelet 12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2. We conclude from this study that catechols are essential for high potency, that isoflavones and isoflavanones tend to select against 12-hLO, that isoflavans tend to select against 15-hLO-1, but few flavonoids target 15-hLO-2.

Full text of DOI:10.1016/j.bmc.2007.07.036

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7408–7425
Structure–activity relationship studies of flavonoids as
potent inhibitors of human platelet 12-hLO, reticulocyte
15-hLO-1, and prostate epithelial 15-hLO-2
Yesseny Vasquez-Martinez,a, Rachana V. Ohri,b, Victor Kenyon,b
Theodore R. Holmanb,* and Silvia Sepu´lveda-Bozaa,*
aLaboratorio de Investigacio´n Cientı´fica Emory Black, Escuela de Medicina, Facultad de Ciencias Me´dicas,
Universidad de Santiago de Chile, Casilla 442, Correo 2 Santiago, Chile
bDepartment of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA
Received 28 May 2007; revised 11 July 2007; accepted 13 July 2007
Available online 22 August 2007
Abstract—Human lipoxygenase (hLO) isozymes have been implicated in a number of disease states and have attracted much atten-
tion with respect to their inhibition. One class of inhibitors, the flavonoids, have been shown to be potent lipoxygenase inhibitors but
their study has been restricted to those compounds found in nature, which have limited structural variability. We have therefore
carried out a comprehensive study to determine the structural requirements for flavonoid potency and selectivity against platelet
12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2. We conclude from this study that catechols are essential for high
potency, that isoflavones and isoflavanones tend to select against 12-hLO, that isoflavans tend to select against 15-hLO-1, but few
flavonoids target 15-hLO-2.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
pro-neoplastic effects, which contribute to prostatic ade-
nocarcinoma by promoting cell proliferation and angio-
genesis.14–16 Prostate epithelial 15-hLO-2,17 however, is
thought to inhibit the growth of prostatic adenocarci-
noma16 because its loss contributes to increased prolifer-
ation and reduced differentiation of cancerous cells.18,19
For these reasons, there is considerable interest in the
discovery and development of selective inhibitors of
12-hLO and 15-hLO-1, which do not inhibit 15-hLO-2.
Lipoxygenases (LO) catalyze the first step in the conver-
sion of polyunsaturated fatty acids to hydroxylated fatty
acids.1–3 They contain a non-heme iron (Fe+3) that pro-
motes hydrogen atom abstraction, dioxygenation of the
1,4-diene moiety, and reduction of the peroxyl radical to
form mono-peroxyl fatty acids.4 A wide variety of hu-
man lipoxygenase (hLO) isozymes are found in nature,
which are named after the oxygenated carbon atom of
the fatty acid product.5 The three human lipoxygenase
isozymes of this investigation, platelet 12-human lipoxy-
genase (12-hLO), reticulocyte 15-human lipoxygenase-1
(15-hLO-1) and prostate epithelial 15-human lipoxyge-
nase-2 (15-hLO-2), are involved in a number of diseases.
Platelet 12-hLO6,7 is implicated as a critical signaling
molecule in tumor metastasis,8 skin disease,9 and neuro-
nal degeneration.10 Reticulocyte 15-hLO-111 has been
implicated in atherogenic processes,12,13 and several
The search for selective lipoxygenase inhibitors has
uncovered several classes of natural products with inhib-
itory activity. Known inhibitors against 12-hLO include,
the alkaloids, such as chelerythrine and sanguinarine,20
the tropolones, such as hinokitiol,21 the arachidonic acid
analogues, such as falcarindiol and panaxynol,22 and the
phenolics, such as rosmarinic acid, caffeic acid,23 and
anthracenones.24 Known 15-hLO-1 inhibitors include
the marine-derived brominated phenol esters,25 the
marine sponge-derived terpenes,26,27 the nord-
ihydroguaiaretic acid (NDGA) derivatives,28 and the
anthraquinones.29 Nevertheless, the most effective inhib-
itors against both 12-hLO and 15-hLO-1 are found in the
broad class of phenolic compounds,30 especially among
flavonoids such as baicalein,31 quercetin,32 luteolin,33
Keywords: Lipoxygenase; Flavonoids; Reductive inhibition; IC50
.
Corresponding authors. Tel.: +1 831 459 5884; fax: +1 831 459
2935; e-mail: tholman@chemistry.ucsc.edu
*
These authors contributed equally to this research.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.036

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7409
anadanthoflavone,34 esculetin,35 and epicatechin.36
Interestingly, members of this class of potent inhibitors
have rarely been simultaneously investigated to deter-
mine their relative potency and selectivity against
12-hLO, 15-hLO-1 and 15-hLO-2. This fact, together
with the limited structural variability of natural flavo-
noids, highlighted the need to investigate the structural
requirements of this broad class of flavonoid inhibitors
against each of these three hLO isozymes.
synthesized analogously (Scheme 1), starting from 2,4-
dihydroxyacetophenone (2). The 2-alkyl-substituted
6,7-methylenedioxybenzopyran-4-ones (12a–c) were
obtained from the corresponding 2-hydroxy-4,5-
methylene-dioxyacetophenone (10) using the method
previously described,37 and the 6,7-dihydroxy-2-t-butyl-
benzopyran-4-one (14a) was prepared from 12a as
shown in Scheme 1. The deprotection of the methylen-
edioxy group could not be achieved with 12b and 12c,
because a complex mixture was obtained that we were
unable to separate and identify. The 2-substituted
3-hydroxychromen-4-ones (21a–f) were prepared
according to Fourgerousse et al.38 by acylation of the
2-hydroxyacetophenone (15) in pyridine solution at
room temperature for 2 h. Selective bromination at the
alpha position with regard to the carbonyl group was
achieved using phenyltrimethylammonium tribromide
(PTT). In the next step, the functional chromanol oxy-
gen atom was introduced with potassium benzoate, un-
der vigorous stirring (48 h) at room temperature. The
Baker–Venkataraman rearrangement was carried out
using a non-nucleophilic base, sodium hydride, in
DMF at 0–5 ꢁC. The product was washed with water
and used immediately in the cyclization step, which took
place in a 0.5% solution of sulfuric acid in glacial acetic
acid. Finally, the hydroxyl groups were deprotected by
saponification in a 5% alcoholic solution of sodium
hydroxide, for 2 h at 60 ꢁC (Scheme 2). The isoflavo-
noids (25a–j) were obtained by electrophilic substitution
of appropriate phenols with benzyl cyanides (Houben
Hoesch reaction). The resulting hydroxylketones were
cyclized to the isoflavones using DMF/MeSO2Cl as a
carbon atom donor in the presence of BF3Et2O (Scheme
3).39 The isoflavanone (26a–b) and isoflavane (7a–g)
were obtained through catalytic hydrogenation with
Pd/C (10%) from the corresponding isoflavones. The
reduction of isoflavones to the corresponding isoflava-
In the current investigation, we have used the basic
structure of flavonoids, the benzopyran skeleton, as a
structural template for the design of novel, potent, and
selective inhibitors of hLO. We present the synthesis
of new benzopyran-4-one analogues with different alkyl
and/or aryl substitution at positions C-2 and C-3, and
with the scaffold decorated with hydroxyl groups at dif-
ferent positions. This diverse library of flavonoid deriv-
atives was subsequently screened in vitro against 12-
hLO, 15-hLO-1, and 15-hLO-2 and compared with
known inhibitors in order to determine the relative
structural determinants for each isozyme’s inhibition.
2. Results and discussion
2.1. Synthesis
The 2-substituted 6-hydroxychromen-4-ones (7a–f) were
prepared according to Nussbaumer et al.37 by acylation
of 2,5-dihydroxyacetophenone (1) to introduce the de-
sired substituent. The doubly acylated intermediate
(3a–g) underwent migration of acyl residues when trea-
ted with sodium hydride. The resulting crude products
(5a–g) were then cyclized to the corresponding 6-hydroxy-
chromen-4-ones (7a–f) with sulfuric and acetic acids.
The 2-substituted 7-hydroxychromen-4-ones (8a–e) were
Scheme 1. Synthesis of 6-and 7-hydroxybenzopyran-4-ones. (i) 2.5 eq. R2COCl/pyridine/rt; (ii) NaH/DMF/0 ꢁC; (iii) AcOH/H2SO4/60 ꢁC; (iv) Ac2O/
BF3OEt/90 ꢁC; (v) 2.5 eq. R2COCl/pyridine/rt; (vi) NaH/DMF/0 ꢁC; (vii) AcOH/H2SO4/60 ꢁC; (viii) PCl5/CH2Cl2; (ix) H2O.

7410
Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
Scheme 2. Synthesis of 3-hydroxy-benzopyran-4-ones. (i) 2.5 eq R2COCl/pyridine/rt; (ii) PhMe3N+Br3ꢀ/THF/rt; (iii) PhCOOK/CH3CN/rt; (iv) NaH/
DMF/0 ꢁC; (v) AcOH/H2SO4/60 ꢁC; (vi) NaOH 5%/EtOH-H2O/60 ꢁC.
Scheme 3. Synthesis of isoflavonoids. (i) ZnCl2/H2O/H+; (ii) DMF/MeSO2Cl in BF3OEt; (iii) H2/Pd/C/EtOH-Dioxane/6 h; (iv) H2/Pd/C/HAc—0.1%
conc. H2SO4/14 h.
nones (26a–b) was done by catalytic hidrogenation over
Pd/C (10%) in dioxane/ethanol for 6 h. To obtain the
isoflavanes (27a–g), the corresponding isoflavones were
hydrogenated over Pd/C (10%) in acetic acid containing
0.1% concentrated sulfuric acid, for 14 h.
tency of known flavonoid inhibitors, quercetin, baica-
lein, and fisetin, in conjunction with a library of
synthetic flavonoid compounds against the activity of
three human lipoxygenases, 12-hLO, 15-hLO-1, and
15-hLO-2.
2.2. Biological evaluation
Initial HTP screening of all forty-seven flavonoid com-
pounds was performed against 12-hLO, 15-hLO-1, and
15-hLO-2, using the Fe3+/xylenol orange screening
method40 in a 384-well format and compared these re-
sults with our well-established manual screen.31,41 The
HTP screen uncovered twelve flavonoids with IC50 val-
Many flavonoids have been investigated over the years
as lipoxygenase inhibitors, however, they are rarely
screened simultaneously with a variety of hLO isozymes
to determine specificity. We present the inhibitory po-

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7411
ues lower than 50 lM for 12-hLO, thirteen for 15-hLO-
1, and three for 15-hLO-2. Our manual screen of the en-
tire library confirmed these results, with one additional
inhibitor found for 12-hLO and 3 for 15-hLO-1. These
data indicate that our one-point HTP screen was able
to detect hLO inhibitors exceptionally well, with over
95% predictive power for all three of the hLO isozymes,
12-hLO, 15-hLO-1, and 15-hLO-2.
duction of a second hydroxyl group at C-5 (34) led to a
20-fold improvement of the IC50, with an excellent selec-
tivity for 15-hLO-1 (IC50 15-hLO-1/12-hLO < 0.03).
The next class of compounds investigated were the 2-
alkylbenzopyran-4-ones, 7a–f, 8a–e, 12a–c, and 14 (Ta-
ble 2). The first conclusion derived from these data is
that the catechol moiety is important for activity. This
is clearly seen when comparing 7e, 8d, 12a, and 14, be-
cause all have the same substituent attached to the pyr-
anone ring, but only 14 has a catechol moiety and only
14 is a lipoxygenase inhibitor. This is consistent with our
previous results regarding NDGA derivatives, in which
the catechol moiety was critical for inhibition due to
its ability to reduce the active site ferric species of soy-
bean lipoxygenase to its inactive ferrous form, thus
interrupting the catalytic cycle.45,46 The second conclu-
sion is that this class of compounds, along with the
3-hydroxybenzopyran-4-ones (Table 3), are in general
poor inhibitors. The only exception, aside from the cat-
echol containing 14, is 7f, which selectively inhibits
15-hLO-1 (IC50 = 38 10 lM). Interestingly, the two
features that are critical for the inhibitory potency of
7f are the position of the alcohol (8e is inactive) and
the length and bulk of the alkane side group (7b and
7d are inactive), indicating a specific binding mode for
7f. Compounds 21a–f (Table 3) are all poor inhibitors,
consistent with the conclusion that a catechol moiety
is important for inhibitor potency.
Full IC50 values were determined for all the potent flavo-
noid inhibitors found. As seen in Table 1, the known LO
inhibitors, nordihydroguaiaretic acid (NDGA), querce-
tin (28), fisetin (29), and baicalein (30) are potent inhib-
itors of 12-hLO and 15-hLO-1, which is consistent with
previous data.42,31,32 Nevertheless, despite the potency
of these inhibitors against 12-hLO and 15-hLO-1, only
NDGA had some effect on 15-hLO-2, albeit stronger
than the published value of 76 lM.43 Interestingly, quer-
cetin, fisetin, and baicalein are reductive inhibitors
against 15-hLO-1, even though the positions of the alco-
hols are dramatically different among the three. This im-
plies that multiple binding orientations are possible for
all three compounds, affording each inhibitor’s catechol
moiety access to the iron to perform an inner sphere
reduction.44 The conclusion that there are multiple
inhibitor binding modes is supported by the fact that
both 29 and 30 are potent, reductive inhibitors but 36
is not. The catechol is therefore required for activity
but it can be on either ring B or ring C, suggesting at
least two spatially distinct orientations of flavonoids to
achieve ferric ion binding and reduction.
Another critical finding, beyond the presence of a cate-
chols being present, is that the aromaticity and oxida-
tion state of ring C are important for the potency and
selectivity of these compounds against LO (Tables 4a,
4b and 4c). Among the 6,7-catechol compounds, the
parent structure (25a) is not potent against either 12-
hLO or 15-hLO-1 (Table 4a). However, if the double
bond is removed from ring C, 26a, the potency and
selectivity against 12-hLO increase dramatically (IC50
We also investigated commercial flavonoids, 31–36, to
examine the influence of hydroxyl groups on the inhibi-
tory activity of the flavone skeleton (Table 1). We found
that none of the monohydroxylated and dihydroxylated
flavonoids inhibited 12-hLO or 15-hLO-2. Compound
31, however, with a single hydroxyl group at the C-3 po-
sition, was a weak inhibitor of 15-hLO-1, and the intro-
Table 1. Inhibitory activity (IC50 SD) of commercial flavonoidsa
0
0
R3
R5
R6
R7
R3
—
R4
—
12-hLO
IC50 (lM)
15-hLO-1
IC50(lM)
15-hLO-1/12-hLO
15-hLO-2
IC50 (lM)
Reduction
15-hLO-1
NDGA
28
29
—
—
OH
H
—
H
—
2.6 0.3
0.44 0.08
0.95 0.2
0.86 0.3
>100
0.25 0.02
2.2 0.3
1.4 0.2
9.1 0.8
73 18
>100
0.1
5
11 0.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
Yes
Yes
Yes
Yes
No
No
—
OH
OH
H
OH
OH
OH
H
OH
OH
H
OH
OH
H
H
1.5
12
30
31
OH
H
OH
H
OH
H
H
H
< 0.73
—
—
32
33
H
OH
H
H
H
H
>100
>100
H
H
OH
H
H
H
>100
34
35
OH
OH
OH
OH
H
H
H
H
>100
>100
>100
3.3 1.6
>100
>100
< 0.03
—
—
No
—
OH
H
H
OH
H
H
36
H
H
H
—
a All IC50 values are expressed in micromolar units.

7412
Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
Table 2. Inhibitory activity (IC50 SD) of 2-alkylbenzopyran-4-onesa
R2
R6
R7
12-hLO
IC50 (lM)
15-hLO-1
IC50 (lM)
15-hLO-1/12hLO
15-hLO-2
IC50 (lM)
Reduction
15-hLO-1
7a
7b
7c
CH2CH3
OH
OH
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
38 10
>100
>100
>100
>100
>100
>100
>100
> 100
18 3.5
—
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
—
—
—
—
No
—
—
—
—
—
—
—
—
No
CH2CH2CH3
CH(CH3)2
CH2CH(CH3)2
C(CH3)3
H
OH
OH
H
—
—
7d
7e
H
OH
OH
H
—
7f
CH2(CH2)3CH3
CH2CH2CH3
CH(CH3)2
CH2CH(CH3)2
C(CH3)3
H
< 0.38
—
—
8a
8b
8c
H
OH
OH
OH
OH
OH
H
H
—
—
8d
8e
H
CH2(CH2)3CH3
C(CH3)3
H
—
—
12a
12b
12c
14
O–CH2–O
O–CH2–O
O–CH2–O
OH
CH2CH(CH3)2
Ph
C(CH3)3
—
—
OH
<0.18
a All IC50 values are expressed in micromolar units.
Table 3. Inhibitory activity (IC50 SD) of 3-hydroxybenzopyran-4-
onesa
Compound 25b is a potent inhibitor for 12-hLO, but not
as potent against 15-hLO-1. Removal of the double
bond from ring C, that is, 26b, increases the potency to-
ward 12-hLO, while inhibition of 15-hLO-1 is lost (IC50
15-hLO-1/12-hLO > 26) (Table 4b). If the ketone is also
removed, that is, 27b, then the potency against 15-hLO-
1 increases dramatically, while the potency against 12-
hLO decreases, shifting the IC50 15-hLO-1/12-hLO ratio
from 26 for 26b to 0.03 for 27b. Compound 25d is not
potent against either 12-hLO or 15-hLO-1, however, re-
moval of the double bond and ketone from ring C, that
is, 27c, improves inhibition of both 12-hLO and 15-
hLO-1 (IC50 15-hLO/12-hLO-1 = 0.032) (Table 4c).
R2
12-hLO
IC50 (lM)
15-hLO-1
IC50 (lM)
15-hLO-2
IC50 (lM)
21a
21b
21c
21d
21e
21f
CH2CH3
CH2CH2CH3
CH(CH3)2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH2CH(CH3)2
C(CH3)3
CH2(CH2)3CH3
These observations indicate that the selectivity is af-
fected by the planarity of the flavonoid, with 27a–c
and 27d being selective against 15-hLO-1, while 25b,
25c, 26a, and 26b are selective against 12-hLO. These
trends suggest that 6,7-dihydroxyisoflavones and 6,7-
dihydroxyisoflavanones tend to be selective against 12-
hLO, while 6,7-dihydroxyisoflavans tend to be selective
a All IC50 values are expressed in micromolar units.
15-hLO-1/12-hLO = 12) (Table 4b). If the ketone is also
removed, that is, 27a, the compound remains as a potent
inhibitor, but its selectivity is reversed (IC50 15-hLO/12-
hLO-1 = 0.019) (Table 4c).
Table 4a. Inhibitory activity (IC50 SD) of 6,7-dihydroxyisoflavonesa
0
0
R3
R4
12-hLO IC50 (lM)
15-hLO-1 IC50 (lM)
15-hLO-1/12-hLO
15-hLO-2 IC50 (lM)
Reduction 15-hLO-1
25a
25b
25c
25d
H
H
OCH3
OH
NO2
>100
>100
—
6
>100
>100
>100
>100
NA
No
8.7 0.95
5.8 0.54
>100
49 7.8
>100
>100
OH
>17
—
NA
NA
O–CH2–O
a All IC50 values are expressed in micromolar units.

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
Table 4b. Inhibitory activity (IC50 SD) of 6,7-dihydroxyisoflavanonesa
7413
0
0
R3
R4
12-hLO IC50 (lM)
15-hLO-1 IC50 (lM)
15-hLO-1/12-hLO
15-hLO-2 IC50 (lM)
Reduction 15-hLO-1
26a
26b
26c
H
H
OCH3
OH
H
1.6 0.1
3.8 0.29
14 1.4
19 1.9
>100
0.21 0.02
12
>26
>100
>100
>100
No
NA
Yes
CH3
0.014
a All IC50 values are expressed in micromolar units.
Table 4c. Inhibitory activity (IC50 SD) of 6,7-dihydroxyisoflavansa
0
0
R3
R4
12-hLO IC50 (lM)
15-hLO-1 IC50 (lM)
15-hLO-1/12-hLO
15-hLO-2 IC50 (lM)
Reduction 15-hLO-1
27a
27b
27c
27d
H
H
OCH3
OH
7.6 0.6
17 1.7
11 1.2
15 1.4
0.15 0.01
0.51 0.12
0.35 0.06
0.21 0.02
0.019
0.031
0.032
0.014
>100
Yes
Yes
Yes
Yes
71 28
16 2.2
8.3 0.92
O–CH2–O
CH3
H
a All IC50 values are expressed in micromolar units.
against 15-hLO-1. These inhibitor results for 12-hLO
and 15-hLO-1 are consistent with previous inhibition
data against porcine 5-lipoxygenase, which indicates
that some isoflavans are better inhibitors of the three
LO isozymes, 5, 12, and 15-hLO-1, than the correspond-
ing isoflavones.47 An exception to this trend is 26c,
which is selective against 15-hLO, even though it is an
isoflavanone, indicating that certain substituents on ring
B can counter the effect of the ring C structure. Finally,
the role of ring C also affects the mechanism of inhibi-
tion, with 27a being a reductive inhibitor, while 26a is
not. These data suggest that the non-planarity of ring
C in 27a allows better access to the iron for an inner
sphere reduction.
tural flexibility of the isoflavan but not by the more rigid
isoflavones and isoflavanones.
The data in Tables 5a and 5b indicate that the 7,8-cate-
chol compounds are potent inhibitors of both 12-hLO
and 15-hLO-1 and that in general, shifting the catechol
does not greatly affect the inhibitor’s ability to reduce
the iron. One exception to this trend is the 10-fold de-
crease in potency of 27e compared to 27a. Another
observation is that the 7,8-isoflavones are more selective
against 12-hLO, while the 7,8- dihydroxyisoflavans are
more selective against 15-hLO-1. This is seen by com-
paring 25e with 27g, whose potencies are comparable
but with reversed selectivity. These trends are similar
to those observed for 6,7-dihydroxyflavonoids (Tables
4a, 4b and 4c) and support our hypothesis that the struc-
ture of ring C is important for the selectivity for both
6,7-and 7,8-dihydroxyflavonoids.
Modifications on ring B also have effects on the inhibi-
tion potency of both isoflavones and isoflavanones (Ta-
bles 4a and 4b). This is seen by inspecting the structures
of compounds 25a and 25d, which do not inhibit either
12-hLO or 15-hLO-1, while 25b and 25c are both potent
and selective inhibitors of 12-hLO (IC50 15-hLO-1/12-
hLO = 6 and 17, respectively). Among the isoflava-
nones, 26a and 26b are selective against 12-hLO, while
26c is selective against 15-hLO-1 (vide supra). However,
inspection of the four isoflavan compounds (27a–d),
whose ring C lacks both the double bond and ketone
(Table 4c), demonstrates that their potency and selectiv-
ity are relatively unaffected by R03 and R40 modifications.
This structure–activity relationship difference between
the three classes of inhibitors, isoflavones, isoflavanones,
and isoflavans, suggests different binding modes, where
the ring B substituent’s effects are nullified by the struc-
In the preceding discussion, we have not mentioned
reduction potential as a variable because it is exceed-
ingly hard to establish a trend for two reasons. First,
it is difficult to estimate the effect on the reduction po-
tential for substituents that are on ring B because any ef-
fect would be weak due to their relative distance from
the catechols. This assertion is supported by the fact that
25g and 25h have comparable inhibitory potencies, even
though the electron-withdrawing chlorine is on the elec-
tronically distinct para- and meta-positions, respectively.
Second, it was previously shown that the potency of very
simple catechols does not track with their reduction po-
tential,44 indicating that it would be even harder to dif-

7414
Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
Table 5a. Inhibitory activity (IC50 SD) of 7,8-dihydroxyisoflavonesa
0
0
R3
R4
12-hLO IC50 (lM)
15-hLO-1 IC50 (lM)
15-hLO-1/12-hLO
15-hLO-2 IC50 (lM)
Reduction 15-hLO-1
25e
25f
25g
25h
25i
25j
H
H
H
Cl
CH3
H
1.6 0.3
6.4 0.6
7.8 0.8
13 1.6
9.0 1.0
6.2 0.7
11 0.7
8.3 0.8
5
2
>100
>100
>100
>100
>100
>100
Yes
Yes
Yes
Yes
Yes
Yes
Cl
H
0.48 0.09
0.78 0.08
3.6 0.3
10
8
CH3
CF3
H
3
13
H
0.62 0.06
a All IC50 values are expressed in micromolar units.
Table 5b. Inhibitory activity (IC50 SD) of 7,8-dihydroxyisoflavansa
0
0
R3
H
R4
12-hLO IC50 (lM)
15-hLO-1 IC50 (lM)
15-hLO-1/12-hLO
15-hLO-2 IC50 (lM)
Reduction 15-hLO-1
27e
27f
27g
OCH3
OCH3
CH3
70 8.7
>100
26 4.5
5.7 1.0
>100
3.7 0.66
0.08
—
>100
>100
>100
Yes
Yes
Yes
OCH3
H
0.14
a All IC50 values are expressed in micromolar units.
ferentiate between steric and reduction potential effects
with the complex compounds presented in this
investigation.
2.3. Molecular modeling analysis
As discussed above, our data suggest that there are
multiple modes in which flavonoids can bind to the
active site of the hLO isozymes, depending on the
position of the catechol moiety with respect to the
flavonoid scaffold. To probe this possibility further,
we flexibly docked four distinct catechol inhibitors
into the active site of the 15-hLO-1 homology model
to visualize their mode of binding.41 Given that
these particular catechols are known to directly bind
and reduce the ferric center of LO,44 we analyzed
the docking behavior of compounds 25b, 25e, 29,
and 30, each representing one of the four classes
of catechols studied. The docking poses of these
four inhibitors (Fig. 1) demonstrate that even if we
constrain the close proximity of the catechol to the
iron (i.e., to act as a reductive inhibitor), the active
site of 15-hLO-1 is large enough to accommodate
four distinct binding modes of the flavonoid scaffold.
This modeling result supports the experimental con-
clusion that for small catechols, no single binding
mode predominates. It should be noted that we were
unable to relate the GLIDE score of these inhibitors
to their inhibitory potency, most likely due to their
small size relative to the 15-hLO-1 active site and
the poor estimation of the metal–catechol bond by
the docking program.
One of the most intriguing aspects of this study is the
weak potency of most of these compounds against 15-
hLO-2, even though many are potent against either
12-hLO or 15-hLO-1. The only inhibitors that are po-
tent against 15-hLO-2 are those that have a 6,7-catechol
but lack the double bond and ketone group in ring C
(27b–d). Interestingly, modifications of ring B affect
the potency of the isoflavans against 15-hLO-2, with
27d being more than 10-fold more potent than 27a. This
trend is opposite to that of the 7,8-isoflavans, whose
changes in ring B do not affect potency, illustrating a dif-
ference in their structure–activity relationship. In addi-
tion, the 15-hLO-2 inhibitors are more potent against
15-hLO-1 than 12-hLO, which may suggest a greater
similarity in the active sites between 15-hLO-2 and 15-
hLO-1, however, differences in the access or exit chan-
nels cannot be completely ruled out. This is reasonable
given the similar regio-specificity of product generation
between 15-hLO-2 and 15-hLO-1. This explanation,
however, does not account for the fact that the majority
of the 15-hLO-1 and 12-hLO inhibitors do not affect 15-
hLO-2, possibly due to the fact that the protein sequence
of 15-hLO-2 has limited sequence identity to both 12-
hLO (37%) or 15-hLO-1 (37%).

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7415
Figure 1. Representative poses of compound docking simulations for 15-hLO-1 with the closest phenolic distance to the iron, for inner sphere
˚
reduction, indicated with a dashed line. 25b, 3.2 A (a), 25e, 3.2 A (b), 29, 4.2 A (c), and 30, 3.1 A (d). The first coordination sphere ligands that
˚
˚
˚
surround the active site iron are also shown, with Thr593 shown in the lower right quadrant.
In summary, the data presented here outline a number
of important discoveries. First, the aromaticity and oxi-
dation state of ring C in our flavonoid compounds ap-
pear to be important for both the inhibitory potency
and selectivity against hLO, with isoflavones and iso-
flavanones preferentially inhibiting 12-hLO and isofla-
vanes preferentially inhibiting 15-hLO-1. Second,
modification of the basic flavonoid structure has pro-
duced a number of selective inhibitors of both 12-hLO
(25c, 26b, and 25j) and 15-hLO-1 (26c, 27a, and 27d),
indicating that the flavonoid skeleton is a viable scaffold
for selective inhibitor development. Third, a variety of
binding modes are possible for flavonoids in lipoxygen-
ases as seen by the fact that compounds 25b, 25e, 29,
and 30 are all potent, reductive inhibitors but their rela-
tive positioning of ring B to their catechol moiety is dif-
ferent. Finally, it appears that the structural
requirements for 15-hLO-2 inhibition are dramatically
different from those of 15-hLO-1 and 12-hLO, which
indicates that if 15-hLO-2 has a beneficial role in cancer
prevention, inhibitors can be developed that do not tar-
get its activity.
ported in parts per million, using tetramethylsilane as
an internal reference. High-resolution mass spectra were
recorded with an EI MS-50 AEI instrument at Bonn
University, Germany, and with a MAT 95XP, Ther-
mo-Finnigan spectrometer at the University of Chile,
Santiago. All starting materials were commercially
available, >98% purity, and used without further purifi-
cation. Quercetin, baicalein, fisetin, 3-hydroxyflavone,
6-hydroxyflavone, 7-hydroxyflavone, 3,5-dihydroxyflav-
one, 3,6-dihydroxyflavone, and 3,7-dihydroxyflavone
were purchased from Aldrich.
The HPLC analyses of the compounds were performed
using a Merck-Hitachi Intelligent L-6200A Pump, an
L-4250 UV-Vis Detector, and a D-7000 HSM System
Manager Report, a C18 reverse phase column (Hypersil
ODS-5, 250 · 4 mm), and a flow rate of 1 mL/min.
Compounds 7a–14 were detected at 260 nm, 21a–f at
319 nm, 25a–j, 26a, 26b, 27b, 27f, and 27g at 255 nm,
and 27a and 27c–e at 295 nm. Two different solvent sys-
tems were used: system 1: (A) acetonitrile and (B) 1%
acetic acid and system 2: (A) acetonitrile and (B) a 1:1
mixture of 1% acetic acid/methanol. A gradient of
30 min of duration was used in both cases, beginning
with 30% of (A), reaching 99% at 30 min, and (B) start-
ing with 70% and ending with 1% in the same period. In
both systems all the compounds used were found to be
more than 95% pure.
3. Experimental
3.1. Chemical synthesis
Melting points were determined on an Electrothermal
apparatus and are not corrected. UV spectra were re-
3.2. General procedure for the synthesis of 2-alkyl-6-
hydroxy-4-H-benzopyran-4-one (7a–f)
1
corded on a Spectronic Genesys 5 instrument. H and
13C NMR spectra were recorded at 300 or 400 MHz
(1H) and 75 or 100 MHz (13C), respectively (Bruker
AMX 300 and Bruker AMX 400 spectrometers), with
(CH3)4Si as internal standard. Chemical shifts are re-
3.2.1. Synthesis of intermediate compounds 3a–g, 4a–f. A
solution of 1 (5 g, 32.8 mmol) in dry pyridine (35 mL)
was treated with propionyl chloride (10 g, 83 mmol) un-

7416
Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
der cooling in an ice-bath to keep the reaction tempera-
ture at about 20 ꢁC. The mixture was stirred for 18 h at
room temperature; and then the solvent was partially re-
moved under reduced pressure. The residue was poured
onto ice and hydrochloric acid (32%, 30 mL) and ex-
tracted with diethyl ether three times. The combined or-
ganic layers were washed with aqueous sodium
carbonate solution and water, dried (MgSO4), and con-
centrated to obtain the crude title compound 3a, which
was purified by chromatography on silica gel eluting
with dichloromethane to give 3a.
3.2.1.9. 20,40-Dibutanoyloxyacetophenone (4b) (72%).
1H NMR (CDCl3) dH 7.81 (1H, d, J = 8.6 Hz), 7.06 (1H,
dd, J = 8.6 Hz, J = 2.3 Hz), 6.92 (1H, d, J = 2.3 Hz),
2.57 (2H, t, J = 7.4 Hz), 2.52 (2H, t, J = 7.3 Hz), 2.51
(3H, s), 1.77 (4H, m), 1.03 (3H, t, J = 7.4 Hz), 1.02
(3H, t, J = 7.4 Hz).
3.2.1.10. 20,40-Di-(2-methylpropanoyloxy)-acetophe-
none (4c) (97%). 1H NMR (CDCl3) dH 7.80 (1H, d,
J = 8.6 Hz), 7.06 (1H, d, J = 8.6 Hz, J = 2.3 Hz), 6.89
(1H, d, J = 2.3 Hz), 2.80 (3H, s), 2.51 (2H, m), 1.32 (6H,
d, J = 7.1 Hz), 1.21 (6H, d, J = 7.1 Hz).
Compounds 3b–g and 4a–f were prepared in a similar
fashion using 1 or 2 and 2.5 eq of the requisite acyl
chloride.
3.2.1.11. 2040-Di-(3-methylbutanoyloxy)-acetophenone
(4d) (93%). d1 H NMR (CDCl3) 7.78 (1H, d, J = 8.6 Hz),
H
7.04 (1H, dd, J = 8.6 Hz, J = 2.3 Hz), 6.89 (1H, d,
J = 2.3 Hz), 2.49 (3H, s), 2.46 (2H, d), 2.41 (2H, d),
2.198 (2H, m), 1.03 (6H, s), 1.02 (6H, s).
3.2.1.1. 20,50-Dicyloxypropanoyloxyacetophenone (3a)
(77%). H NMR (CDCl3) dH 7.50 (1H, d, J = 2.8 Hz),
1
7.24 (1H, dd, J = 8.6 Hz, J = 2.8 Hz), 7.09 (1H, d,
J = 8.6 Hz), 2.60 (4H, m), 2.51 (3H, s), 1.25 (3H, t,
J = 7.4 Hz), 1.25 (3H, t, J = 7.5 Hz).
3.2.1.12. 20,40-Di-(2,2-dimethylpropanoyloxy)-acetophe-
none (4e) (89%). 1H NMR (CDCl3) dH 7.77 (1H, d,
J = 8.6 Hz), 7.02 (1H, dd, J = 8.6 Hz, J = 2.3 Hz), 6.81
(1H, d, J = 2.3 Hz), 2.50 (3H, s), 1.35 (9H, s), 1.32 (9H, s).
3.2.1.2. 20,50-Dibutanoyloxyacetophenone (3b) (91%).
1H NMR (CDCl3) dH 7.49 (1H, d, J = 2.8 Hz), 7.23 (1H,
dd, J = 8.6 Hz; J = 2.8 Hz), 7.09 (1H, d, J = 8.6 Hz),
2.55 (4H, m), 2.50 (3H, s), 1.77 (4H, m), 1.02 (6H, t,
J = 7.4 Hz).
3.2.1.13. 20,40-Dihexanoyloxyacetophenone (4f) (76%).
1H NMR (CDCl3) dH 7.81 (1H, d, J = 8.6 Hz), 7.06 (1H,
dd, J = 8.6 Hz, J = 2.3 Hz), 6.92 (1H, d, J = 2.3 Hz),
2.58 (4H, m), 2.51 (3H, s), 1.72 (4H, m), 1.36 (8H, m),
0.91 (6H, t, J = 7.1 Hz).
3.2.1.3. 20,50-Di-(2-methylpropanoyloxy)-acetophenone
(3c) (85%). 1H NMR (CDCl3) dH 7.46 (1H, d, J = 2.6 Hz),
7.22 (1H, dd, J = 8.9 Hz, J = 2.8 Hz), 7.06 (1H, d,
J = 8.7 Hz), 2.80 (2H, m), 2.50 (3H, s), 1.31 (6H, d,
J = 6.9 Hz), 1.29 (6H, d, J = 6.9 Hz).
3.2.2. Synthesis of the intermediate 3-(1,3-dioxopentyl)-
4-hydroxyphenyl propanoate (5a–g, 6a–f). Crude 3a
(5.2 g, 20 mmol) was dissolved in dry DMF (30 mL)
and added slowly at 0 ꢁC under argon to a suspension
of sodium hydride (95% pure, 870 mg, 34.4 mmol) in
dry DMF (30 mL). Acetic acid was added to the mix-
ture with caution and then, it was poured into water
3.2.1.4. 2050-Di-(3-methylbutanoyloxy)-acetophenone (3d)
(91%). 1H NMR (CDCl3) dH 7.47 (1H, d, J = 2.8 Hz), 7.23
(1H, dd, J = 8.6 Hz, J = 2.8 Hz), 7.09 (1H, d, J = 8.8 Hz),
2.51 (3H, s), 2.47 (2H, d, J = 7.1 Hz), 2.42 (2H,
d,J = 7.1 Hz), 2.22 (2H, m), 1.04 (12H, d, J = 6.6 Hz).
(300 mL)
and
extracted
with
ethyl
acetate
(3 · 70 mL). The combined organic layers were washed
with saturated aqueous sodium chloride solution, dried
(MgSO4), and concentrated to yield the crude title
compound, which was used in the next step without
further purification.
3.2.1.5. 20,50-Di-(2,2-dimethylpropanoyloxy)-acetophe-
none (3e) (95%). 1H NMR (CDCl3) dH7.42 (1H, d,
J = 2.6 Hz), 7.19 (1H, dd, J = 8.7 Hz, J = 2.6 Hz), 7.01
(1H, d, J = 8.7 Hz), 2.50 (3H, s), 1.35 (9H, s), 1.33
(9H, s).
3.2.3. Synthesis of the 2-alkyl-6-hydroxy-4H-benzopyran-
4-ones (7a–f). A solution of 4.3 mmol of crude 5a in
methanol (30 mL) was treated with 15 mL of concen-
trated aqueous hydrochloric acid in 20 mL of dioxane.
The mixture was heated to 60 ꢁC and then poured into
water, and extracted with ethyl acetate. Subsequent dry-
ing over magnesium sulfate and evaporation yielded
(7a), which was crystallized in methanol.
3.2.1.6. 20,50-Dihexanoyloxyacetophenone (3f) (82%).
1H NMR (CDCl3) dH 7.49 (1H, d, J = 2.8 Hz), 7.24 (1H,
dd, J = 8.6 Hz, J = 2.8 Hz), 7.09 (1H, d, J = 8.6 Hz) 2.58
(4H, m), 2.54 (3H, s), 1.74 (4H, m), 1.37 (8H, m), 0.90 (6H,
m).
3.2.1.7. 20,50-Dicinnamoyloxyacetophenone (3g) (87%).
1H NMR (CDCl3) dH 7.83 (1H, d, J = 15.9 Hz), 7.82 (1H, d,
J = 15.9 Hz), 7.57 (1H, d, J = 2.8 Hz), 7.51 (4H, m), 7.40
(6H, m), 7.30 (1H, dd, J = 8.8 Hz, J = 3.0 Hz), 7.15 (1H,
d, J = 8.8 Hz), 6.59 (1H, d, J = 16.1 Hz), 6.56 (1H, d,
J = 16.1 Hz), 2.49 (1H, s).
3.2.3.1. 2-Ethyl-6-hydroxy-4H-benzopyran-4-one (7a)
(60%). 1H NMR (DMSO-d6) dH 9.90 (1H, s, OH-6), 7.46
(1H, d, J = 9.0 Hz, H-5), 7.27 (1H, d, J = 2.8 Hz, H-8),
7.18 (1H, dd, J = 9.0 Hz, J = 3.0 Hz, H-7), 6.11 (1H, s,
H-3), 2.63 (2H, c, J = 7.5 Hz, Ar–CH2–), 1.21 (3H, t,
J = 7.5 Hz, Ar–CH2CH3). 13C NMR (DMSO-d6)
dC177.3, 171.2, 154.9, 150.1, 124.3, 123.1, 119.7, 107.8,
27.0, 11.2. UV: 328 and 226 nm. HRMS (M+H)+ calcd
for C11H10O3, 190.0629; found 190.0622, mp 164–
165 ꢁC, lit.,48 mp 165 ꢁC.
3.2.1.8. 20,40-Dipropanoyloxyacetophenone (4a) (88%).
1H NMR (CDCl3) dH 7.82 (1H, d, J = 8.6 Hz), 7.08 (1H,
dd, J = 8.6 Hz, J = 2.3 Hz), 6.94 (1H, d, J = 2.3 Hz), 2.52
(3H, s), 2.32 (3H, s), 2.28 (3H, s).

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7417
3.2.3.2.
6-Hydroxy-2-propyl-4H-benzopyran-4-one
CH2–), 1.67 (2H, m, Ar–CH2CH2-), 0.93 (3H, t,
J = 7.4 Hz, Ar–CH2CH2CH3). 13C NMR (DMSO-d6)
dC 176.6, 169.0, 162.9, 158.2, 126.9, 116.3, 115.1,
109.3, 102.6, 35.5, 20.1, 13.7. UV: 292, 247 and
214 nm. HRMS (M+H)+ calcd for C12H12O3,
204.0786; found 204.0778. Mp 150–152 ꢁC, lit.,37 mp
143 ꢁC.
1
(7b) (58%). H NMR (DMSO-d6) dH 9.89 (1H, s, OH-
6), 7.44 (1H, d, J = 8.8 Hz, H-5), 7.27 (1H, d,
J = 3.0 Hz, H-8), 7.18 (1H, dd, J = 8.8 Hz; J = 3.0 Hz,
H-7), 6.12 (1H, s, H-3), 2.57 (2H, t, J = 7.4 Hz, Ar–
CH2–), 1.66 (2H, m, Ar–CH2CH2–), 0.93 (3H, t,
J = 7.3 Hz, Ar–CH2CH2CH3). 13C NMR (DMSO-d6)
dC 177.2, 169.5, 155.0, 150.1, 124.4, 123.1, 119.8, 108.8,
108.0, 35.6, 20.2, 13.7. UV: 328 nm, 226 nm. HRMS
(M+H)+ calcd for C12H12O3, 204.0786; found 204.0792,
mp 155–156 ꢁC, lit.,37 mp 150 ꢁC.
3.2.3.8. 7-Hydroxy-2-isopropyl-4H-benzopyran-4-one
1
(8b) (45%). H NMR (DMSO-d6) dH10.68 (1H, s, OH-
7), 7.82 (1H, d, J = 8.6 Hz, H-5), 6.87 (1H, dd,
J = 8.8 Hz, J = 2.3 Hz, H-6), 6.80 (1H, d, J = 2.0 Hz,
H-8), 6.04 (1H, s, H-3), 2.83 (1H, m, Ar–CH–) 1.23
(6H, d, J = 7.1 Hz, Ar–CH(CH3)2). 13C NMR
(DMSO-d6) dC 176.9, 173.6, 163.1, 157.8, 126.9, 116.5,
114.9, 107.0, 102.7, 32.8, 20.5. UV: 295, 247 and
214 nm. HRMS (M+H)+ calcd for C12H12O3,
204.0786; found 204.0791. Mp 156–158 ꢁC.
3.2.3.3. 6-Hydroxy-2-isopropyl-4H-benzopyran-4-one
(7c) (56%). H NMR (DMSO-d6) dH 9.89 (1H, s, 6-
1
OH), 7.46 (1H, d, J = 9.0 Hz, H-5), 7.27 (1H, d,
J = 2.8 Hz, H-8), 7.18 (1H, dd, J = 9.0 Hz, J = 2.8 Hz,
H-7), 6.10 (1H, s, H-3), 2.87 (1H, m, Ar–CH–), 1.24
(6H, d, J = 7.0 Hz, Ar–CH(CH3)2). 13C NMR (DMSO-
d6) dC 177.4, 174.0, 155.0, 150.0, 124.4, 123.2, 119.8,
107.9, 106.4, 32.9, 20.3. UV: 328 and 226 nm. HRMS
(M+H)+ calcd for C12H12O3, 204.0786; found 204.0790.
Mp 182–183 ꢁC.
3.2.3.9. 7-Hydroxy-2-isobutyl-4H-benzopyran-4-one
(8c) (52%). 1H NMR (DMSO-d6) dH 10.68 (1H, s,
OH-7), 7.83 (1H, d, J = 8.6 Hz, H-5), 6.87 (1H, dd,
J = 8.8 Hz, J = 2.3 Hz, H-6), 6.80 (1H, d, J = 2.0 Hz,
H-8), 6.06 (1H, s, H-3), 2.46 (2H, d, J = 7.3 Hz, Ar–
CH2), 2.05 (1H, m, Ar–CH2CH–), 0.93 (6H, d,
J = 6.8 Hz, Ar–CH2CH(CH3)2). 13C NMR (DMSO-d6)
dC 176.6, 168.2, 162.9, 158.3, 126.9, 116.5, 115.2,
110.3, 102.7, 42.5, 26.9, 22.1. UV: 292, 247 and
214 nm. HRMS (M+H)+ calcd for C13H14O3,
218.0942; found 218.0936. Mp 152–153 ꢁC.
3.2.3.4. 6-Hydroxy-2-isobutyl-4H-benzopyran-4-one
(7d) (50%). H NMR (DMSO-d6) dH 9.90 (1H, s, OH-
1
6), 7.44 (1H, d, J = 8.8 Hz, H-5), 7.27 (1H, d,
J = 3.0 Hz, H-8), 7.17 (1H, dd, J = 8.8 Hz; J = 3.0 Hz,
H-7), 6.12 (1H, s, H-3), 2.48 (2H, d, J = 7.3 Hz, Ar–
CH2), 2.06 (1H, m, Ar–CH2CH–), 0.93 (6H, d,
J = 6.6 Hz, Ar–CH2CH(CH3)2). 13C NMR (DMSO-d6)
dC 177.1, 168.7, 155.0, 150.1, 124.4, 123.1, 119.8,
109.7, 108.0, 42.7, 27.0, 22.4. UV: 328 and 229 nm.
HRMS (M+H)+ calcd for C13H14O3, 218.0942; found
218.0942. Mp 145–146 ꢁC.
3.2.3.10. 7-Hydroxy-2-t-butyl-4H-benzopyran-4-one
(8d) (40%). 1H NMR (DMSO-d6) dH 10.69 (1H, s,
OH-7), 7.82 (1H, d, J = 8.9 Hz, H-5), 6.87 (1H, dd,
J = 8.7 Hz, J = 2.3 Hz, H-6), 6.82 (1H, d, J = 2.0 Hz,
H-8), 6.07 (1H, s, H-3), 1.28 (9H, s, –C(CH3)3). 13C
NMR (DMSO-d6) dC 177.0, 175.2, 163.0, 158.1, 126.8,
116.0, 115.2, 106.0, 102.6, 36.4, 27.9. UV: 295, 247 and
214 nm. HRMS (M+H)+ calcd for C13H14O3,
218.0942; found 218.0943. Mp 222–223 ꢁC.
3.2.3.5.
6-Hydroxy-2-t-butyl-4H-benzopyran-4-one
1
(7e) (50%). H NMR (DMSO-d6) dH 9.90 (1H, s, OH-
6), 7.48 (1H, d, J = 8.8 Hz, H-5), 7.26 (1H,
d,J = 2.8 Hz, H-8), 7.19 (1H, dd, J = 8.8 Hz;J = 2.8
Hz, H-7), 6.13 (1H, s, H-3), 1.30 (9H, s, C(CH3)3). 13C
NMR (DMSO-d6) dC 177.6, 175.8, 162.3, 154.9, 150.1,
123.3, 119.7, 107.8, 105.4, 36.5, 28.1. UV: 328, 226 nm.
HRMS (M+H)+ calcd for C13H14O3, 218.0942; found
218.0937, mp 179–180 ꢁC, lit.,37 mp 170 ꢁC.
3.2.3.11.
7-Hydroxy-2-pentyl-4H-benzopyran-4-one
(8e) (45%). 1H NMR (DMSO-d6) dH 10.65 (1H, s,
OH-7), 7.82 (1H, d, J = 8.7 Hz, H-5), 6.87 (1H, dd,
J = 8.9 Hz, J = 2.3 Hz, H-6), 6.80 (1H, d, J = 2.3 Hz,
H-8), 6.07 (1H, s, H-3), 2.58 (2H, t, J = 7.5 Hz, Ar–
CH2), 1.64 (2H, m, Ar-CH2CH2CH2–), 1.31 (4H, m,
Ar–CH2CH2CH2CH2–), 0.86 (3H, m, Ar–(CH2)4CH3).
13C NMR (DMSO-d6) dC 176.6, 169.3, 162.9, 158.2,
126.9, 116.3, 115.1, 109.2, 102.6, 33.6, 31.0, 26.3, 22.2,
14.2. UV: 247 and 214 nm. HRMS (M+H)+ calcd for
C14H16O3, 232.1099; found 232.1094, mp 135–136 ꢁC.
3.2.3.6. 6-Hydroxy-2-pentyl-4H-benzopyran-4-one (7f)
1
(40%). H NMR (DMSO-d6) dH 9.89 (1H, s, OH-6),
7.44 (1H, d, J = 9.1 Hz, H-5), 7.27 (1H, d, J = 3.0 Hz,
H-8), 7.16 (1H, dd, J = 9.1 Hz, J = 3.0 Hz, H-7), 6.13
(1H, s, H-3), 3.31 (2H, t, J = 7.6 Hz, Ar–CH2–), 1.64
(4H, m, Ar–CH2CH2CH2CH2–), 1.31 (2H, m, Ar–
CH2CH2CH2–), 0.85 (3H, t, J = 6.8 Hz, Ar–
(CH2)4CH3). 13C NMR (DMSO-d6) dC 177.1, 169.7,
155.0, 150.1, 124.4, 123.1, 119.8, 108.7, 108.0, 33.7,
31.0, 26.4, 22.2, 14.2. UV: 328 and 226 nm. HRMS
(M+H)+ calcd for C14H16O3, 232.1099; found
232.1093, mp 118–120 ꢁC.
3.3. General procedure for the synthesis of 2-alkyl-6,7-
methylenedioxybenzopyran-4-ones (12a–c)
3.3.1. Synthesis of 2-hydroxy-4,5-methylenedioxyace-
tophenone (10) (77%). To a solution of sesamol (2.76 g,
0.02 mol) in Ac2O (10 mL) was added 5 mL of the boron
trifluoride etherate complex at 0 ꢁC, under argon. The
resulting solution was heated to 80–90 ꢁC for 1 h and
poured into a saturated NaOAc solution (20 mL). After
extracting with Et2O, the Et2O solution was washed
3.2.3.7.
7-Hydroxy-2-propyl-4H-benzopyran-4-one
(8a) (42%). 1H NMR (DMSO-d6) dH 10.68 (1H, s,
OH-7), 7.82 (1H, d, J = 8.5 Hz, H-5), 6.87 (1H, dd,
J = 8.5 Hz; J = 2.5 Hz, H-6), 6.79 (1H, d, J = 2.5 Hz,
H-8), 6.06 (1H, s, H-3), 2.56 (2H, t, J = 7.4 Hz, Ar–

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Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
with 10% aqueous NaHCO3 solution and H2O, dried
(Na2SO4), and concentrated under reduced pressure.
The residue was recrystallized from absolute EtOH to
obtain the acetophenone 10. 1H NMR (CDCl3) dH
12.99 (1H, s), 7.03 (1H, s), 6.43 (1H,s), 5.95 (2H,s),
2.50 (3H,s). 13C NMR (CDCl3) dC 201.9. 162.1,
1154.4, 140.5, 112.3, 107.2, 101.9, 98.7, 26.4.
131.8, 129.8, 118.9, 106.9, 103.5, 101.5, 99.2. HRMS
(M+H)+ calcd for C16H10O4, 266.5792; found
265.99224 (The molecular ion does not appear with suf-
ficient intensity) mp 210–212 ꢁC.
3.3.3. Synthesis of 6,7-dihydroxy-2-t-butylbenzopyran-4-
one (14). A solution of 12c (0.7 g, 0.65 mmol) in chloro-
form (8 mL) was treated with PCl5 (0.3 g, 1.3 mmol).
The mixture was refluxed for 7 h and after cooling the
solvent was removed under reduced pressure. The resi-
due was hydrolyzed in water refluxing for 2 h, and after
cooling was extracted twice with ethyl acetate. The sol-
vent was removed and the product was purified through
a silica gel column, eluting with a 2:1 CH2Cl2/EtOAc in
3.3.2. Synthesis of 2-alkyl benzopyran-4-ones (12a–c). A
solution of 10 (5 g, 32.8 mmol) in dry pyridine (35 mL)
was treated with trimethylacetyl chloride (10 g,
83 mmol) under cooling with an ice-bath to keep the
reaction temperature at about 20 ꢁC. The mixture was
stirred for 18 h at room temperature; and the solvent
was partially distilled off under reduced pressure. The
residue was poured onto ice and hydrochloric acid
(32%, 30 mL) and extracted with diethyl ether. The com-
bined organic layers were washed with aqueous sodium
carbonate solution and water, dried (MgSO4), and con-
centrated to give the crude title compound 11a.
1
a 2:1 proportion as eluent to give (14) (53%). H NMR
(CDCl3) dH 8.06 (1H, s, OH-7), 7.98 (1H, s, OH-6), 7.55
(1H, s, H-5), 7.26 (1H, s, H-8), 6.29 (1H, s, H-3), 1.35
(9H, s, –C(CH3)3). 13C NMR (CDCl3) dC 179.1, 177.0,
150.5, 150.3, 128.2, 123.1, 119.1, 110.6, 106.1, 37.8,
28.1. HRMS (M+H)+ calcd for C13H14O4, 234.0892;
found 234.0894, mp 148–149 ꢁC.
Crude 11a (5.2 g, 19 mmol) was dissolved in dry DMF
(30 mL) and added slowly to a suspension of sodium hy-
dride (95% pure, 870 mg, 34.4 mmol) in dry DMF
(30 mL), at 0 ꢁC, under argon. Acetic acid was added
to the mixture with caution and then, it was poured into
water (300 mL) and extracted with ethyl acetate
(3 · 70 mL). The combined organic layers were washed
with saturated aqueous sodium chloride solution, dried
(MgSO4), and evaporated to yield the crude title com-
pound. A solution of this crude compound in methanol
(30 mL) was treated with concentrated aqueous hydro-
chloric acid in dioxane. The mixture was heated to
60 ꢁC and then poured into water, and washed with
ethyl acetate. Subsequent drying over magnesium sulfate
and evaporation yielded (12a).
3.3.4. General procedure for the synthesis of the interme-
diate compounds (16a–f). Synthesis of 20-propanoyloxy
acetophenone (16a). A mixture of (15) (1.5 g, 11 mmol)
and propionyl chloride (2.62 mL, 22 mmol) was stirred
in dry pyridine (5 mL) at room temperature for 2 h.
The mixture was then poured into a mixture of crushed
ice (25 mL) and concentrated HCl (1.5 mL), extracted
twice with dichloromethane, and the combined organics
were washed three times with water. The solvent was re-
moved under reduced pressure. The residue was purified
through a silica gel column using CH2Cl2 as eluent to
1
give (16a) (2.3 g, 97%). H NMR (CDCl3) dH7.78 (1H,
dd, J = 8.1 Hz, J = 2.1 Hz), 7.50 (1H, ddd, J = 7.7 Hz,
J = 8.1 Hz, J = 1.7 Hz), 7.29 (1H, ddd, J = 7.7 Hz,
J = 8.1 Hz, J = 1.7 Hz), 7.08 (1H, dd, J = 8.5 Hz,
J = 1.1 Hz), 2.63 (2H, q), 2.52 (3H, s), 1.26 (3H, t,
J = 7.5 Hz).
3.3.2.1. 6,7-Methylenedioxy-2-t-butylbenzopyran-4-
1
one (12a). H NMR (CDCl3) dH 7.46 (1H,s, H-5), 6.84
(1H, s, H-8), 6.27 (1H,s, H-3), 6.06 (2H, s, O–CH2–O),
1.31 (9H, s, C(CH3)3). 13C NMR (CDCl3) dC 177.9,
175.5, 153.9, 152.8, 146.1, 118.1, 106.2, 102.4, 102.2,
97.9, 36.4, 28.0. UV: 319, 277, 235 nm. HRMS
(M+H)+ calcd for C14H14O4, 246.0892; found
246.0897, mp 214–215 ꢁC.
1
3.3.4.1. 20-Butanoyloxyacetophenone (16b) (90%). H
NMR (CDCl3) dH7.76 (1H, dd, J = 7.8 Hz, J = 1.5 Hz),
7.50 (1H, ddd, J = 7.8 Hz,J = 7.6 Hz, J = 8.0 Hz,
J = 1.8 Hz), 7.29 (1H, ddd, J = 7.8 Hz, J = 7.6 Hz,J =
7.6 Hz, J = 1.0 Hz), 7.06 (1H, dd, J = 8,1 Hz, J =
1.3 Hz), 2.84 (1H, m), 2.53 (3H, s), 1.33 (6H, d,
J = 6.8 Hz).
3.3.2.2. 2-Isobutyl-6,7-methylenedioxybenzopyran-4-
1
one (12b). H NMR (CDCl3) dH 7.48 (1H,s, H-5), 6.82
(1H, s, H-8), 6.19 (1H, s, H-3), 6.07 (2H, s, O–CH2–
O), 2.44 (2H, d, J = 7.1 Hz, Ar–CH2–), 2.10 (1H, m,
Ar–CH2CH–), 0.98 (6H, d, J = 6.6 Hz, Ar–
CH2CH(CH3)2). 13C NMR (CDCl3) dC 177.3, 168.5,
154.0, 152.8, 146.1, 118.4, 110.0, 102.3, 97.9, 43.3,
27.2, 22.3. UV: 319, 277 and 235 nm. HRMS (M+H)+
calcd for C14H14O4, 246.0892; found 246.0890, mp 90–
92 ꢁC.
3.3.4.2. 20-(2-Methylpropanoyloxy)-acetophenone (16c)
(98%). H NMR (CDCl3) dH7.77 (1H, dd, J = 7.8 Hz,
J = 1.8 Hz), 7.50 (1H, ddd, J = 7.5 Hz, J = 8.1 Hz,
J = 1.8 Hz, J = 1.5 Hz), 7.29 (1H, ddd, J = 7.6 Hz,
J = 7.5 Hz, J = 7.7 Hz, J = 1.3 Hz), 7.08 (1H, dd,
J = 8.1 Hz,J = 1.0 Hz), 2.58 (2H, t, J = 7.4 Hz), 2.53 (3H,
s), 1.79 (2H, m), 1.04 (3H, t, J = 7.3 Hz).
1
3.3.4.3. 20-(3-Methylbutanoyloxy)-acetophenone (16d)
(80%). 1H NMR (CDCl3) dH 7.76 (1H, dd,
J = 7.8 Hz,J = 1.8 Hz), 7.49 (1H, ddd, J = 7.5 Hz,
J = 8.0 Hz, J = 8.2 Hz, J = 1.8 Hz), 7.28 (1H, ddd,
J = 7.6 Hz,J = 7.5 Hz, J = 7.8 Hz, J = 1.3 Hz), 7.08 (1H,
dd, J = 8.1 Hz, J = 1.5 Hz), 2.52 (3H, s), 2.48 (2H, d,
J = 7.1 Hz), 2.24 (1H, m), 1.05 (6H, d, J = 6.6 Hz).
3.3.2.3.
6,7-Methylenedioxy-2-phenylbenzopyran-4-
one (12c). 1H NMR (CDCl3) dH8.03, (2H, dd,
J = 8.0 Hz, J = 2.1 Hz, H-20, H-60), 7.56 (3H, m, H-30,
H-40, H-50), 7.37 (1H, s, H-5), 7.31 (1H, s, H-8), 6.94
(1H, s, H-3), 6.21 (2H, s, O–CH2–O). 13C NMR
(CDCl3) dC 176.6, 162.5, 153.6, 153.3, 146.8, 132.2,

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7419
3.3.4.4. 20-(2,2-Dimethylpropanoyloxy)-acetophenone
(16e) (98%). 1H NMR (CDCl3) dH 7.74 (1H, dd,
J = 7.8 Hz, J = 1.8 Hz), 7.49 (1H, ddd, J = 7.3 Hz,
J = 8.1 Hz,J = 8.1 Hz, J = 1.8 Hz), 7.28 (1H, ddd,
J = 7.6 Hz,J = 7.6 Hz, J = 7.6 Hz, J = 1.3 Hz), 7.02
(1H, dd, J = 8.1 Hz, J = 1.0 Hz), 2.5 (3H, s), 1.4 (9H, s).
J = 7.4 Hz, J = 1.1 Hz), 7.16 (1H, dd, J = 8.1 Hz,
J = 1.1 Hz), 4.39 (2H, s), 2.61 (2H, m), 1.75 (2H, m), 1.37
(4H, m), 0.91 (3H,t, J = 7.0 Hz).
3.3.6. General procedure for the synthesis of 18a–f. Synthesis
of 2-(2-phenoxyacetyl) phenyl propionate (18a). A mixture of
(18a) (3.0 g, 11 mmol) and potassium benzoate (2.7 g,
17 mmol) was stirred in acetonitrile (48 mL) at room tem-
perature for 48 h. The salts were filtered off, and the solvent
was removed under reduced pressure. The residue was taken
up in dichloromethane, washed with aqueous sodium car-
bonate, and then washed with water. The solvent was re-
moved under reduced pressure, and the residue was
recrystallized from ethanol to give 18a (53%).
3.3.4.5. 20-Hexanoyloxyacetophenone (16f) (49%). 1H
NMR (CDCl3) dH 7.77 (1H, dd, J = 7.8 Hz, J = 1.8 Hz),
7.50 (1H, ddd, J = 7.4 Hz, J = 8.0 Hz, J = 8.0 Hz,
J = 1.8 Hz), 7.28 (1H, ddd, J = 7.6 Hz, J = 7.6 Hz,
J = 7.6 Hz, J = 1.3 Hz), 7.08 (1H, dd, J = 8.1 Hz,
J = 1.3 Hz), 2.59 (2H, t, J = 7.6 Hz), 2.52 (3H, s), 1.75
(2H, m), 1.38 (4H, m), 0.91 (3H, t, J = 7.1 Hz).
3.3.5. General procedure for the synthesis of 17a–f. Synthesis
of 20-propanoyloxy-2-bromoacetophenone (17a). To a solu-
tion of (16a) (2.2 g, 11.5 mmol) in anhydrous tetrahydrofu-
ran (10 mL), PTT (4.3 g, 11.5 mmol) was added in portions
over a period of 10 min. The reaction mixture was stirredun-
til a colorless precipitate was formed. Theresiduewasrecrys-
3.3.7. General procedure for the synthesis of (20a–f).
Synthesis of 2-ethyl-4-oxo-4 H-chromen-3-yl benzoate
(20a). To a suspension of sodium hydride (148 mg,
6.2 mmol) in dry DMF (2.5 mL) was added 18a (1.8 g,
5.6 mmol) in DMF (2.5 mL). The reaction mixture was
refluxed for 90 min with stirring, and then the cooled
mixture was poured into a mixture of ice (120 g) and
concentrated HCl (2 mL). The crude b-diketone (19a)
precipitated subsequently was washed with water and
was used in the following ring closure step without fur-
ther purification.
1
tallized from ethanol to give (17a) (3.0 g, 97%), H NMR
(CDCl3) dH 7.80 (1H, dd, J = 7.7 Hz, J = 1.5 Hz), 7.56
(1H, ddd, J = 7.8 Hz, J = 7.9 Hz, J = 7.7 Hz J = 1.5 Hz),
7.32 (1H, ddd, J = 7.6 Hz, J = 7.7 Hz, J = 7.5 Hz,
J = 1.1 Hz), 7.18 (1H, dd, J = 8.1 Hz, J = 0.8 Hz), 4.39
(2H, s), 2.65 (2H, c, J = 7.5 Hz), 1.27 (3H, t, J = 7.5 Hz).
To a solution of crude b-diketone (19a) in acetic acid
(10 mL), concentrated sulfuric acid (0.25 mL) was added
dropwise. The reaction mixture was heated at 60 ꢁC for
90 min with stirring, and the solution was poured over ice
(60 g). The precipitate was filtered, washed with water,
and recrystallized from ethanol to yield the pure (20a)
3.3.5.1. 20-Butanoyloxy-2-bromoacetophenone (17b)
(98%). H NMR (CDCl3) dH 7.77 (1H, dd, J = 7.9 Hz,
J = 1.7 Hz), 7.55 (1H, ddd, J = 7.5 Hz, J = 8.2 Hz,
J = 8.1, HzJ = 1.7 Hz), 7.31 (1H, ddd, J = 7.6 Hz,
J = 7.5 Hz, J = 7.7 Hz, J = 1.1 Hz), 7.13 (1H, dd,
J = 8.1 Hz, J = 1.1 Hz), 4.38 (2H, s), 2.84 (1H, m),
1.33 (6H, d, J = 7.2 Hz).
1
1
(556 mg, 60%) as colorless needles. H NMR (CDCl3) dH
8.17 (1H, dd, J = 7.6 Hz, J = 1.8 Hz), 8.16 (1H, dd,
J = 8.3 Hz, J = 1.3 Hz), 7.61 (1H, ddd, J = 7.8 Hz,
J = 7.8 Hz, J = 7.8 Hz, J = 1.5 Hz), 7.56 (1H, dd), 7.43
(3H, m), 7.33 (1H, ddd,J = 7.6 Hz, J = 7.6 Hz, J = 7.6 Hz,
J = 1.0 Hz), 2.70 (2H, c, J = 7,6 Hz), 1.27 (3H, t,
J = 7,6 Hz).
3.3.5.2. 20-(2-Methylpropanoyloxy)-2-bromoacetophe-
none (17c) (98%). 1H NMR (CDCl3) 7.79 (1H, dd,
J = 7.7 Hz, J = 1.7 Hz), 7.55 (1H, ddd, J = 7.5 Hz,
J = 8.2 Hz, J = 7.9 Hz,
J =1.5 Hz), 7.31(1H, ddd,
J = 7.7 Hz, J = 7.6 Hz, J = 8.1 Hz, J = 1.1Hz), 7.16 (1H,
dd, J = 8.3 Hz, J = 1.3 Hz), 4.39 (2H, s), 2.59 (2H, t,
J = 7.4 Hz), 1.78 (2H, m), 1.03 (3H, t, J = 7.3 Hz).
3.3.7.1. 2-Isopropyl-4-oxo-4H-chromen-3-yl benzoate
(20b) (80%). 1H NMR (CDCl3) dH8.15 (3H, m), 7.59 (2H,
m), 7.45 (3H, m), 7.32 (1H, ddd, J = 7.5 Hz, J = 7.5 Hz,
J = 7.6 Hz, J = 1.0 Hz), 3.21 (1H, q, J = 6.8 Hz), 1.3
(6H, d, J = 6.8 Hz).
3.3.5.3. 20-(3-Methylbutanoyloxy)-2-bromoacetophe-
1
none (17d) (98%). H NMR (CDCl3) dH 7.78 (1H, dd,
J = 7.9 Hz, J = 1.7 Hz), 7.55 (1H, ddd, J = 7.5 Hz,
J = 8.1 Hz, J = 7.7 Hz, J = 1.7 Hz), 7.31(1H, ddd,
J = 7.7 Hz, J = 7.8 Hz, J = 7.5 Hz, J = 1.1 Hz), 7.15
(1H, dd, J = 8.1 Hz, J = 1.1 Hz), 4.39 (2H, s), 2.50
(2H, d, J = 7.0 Hz), 2.25 (1H, m), 1.05 (6H, s).
3.3.7.2. 4-Oxo-2-propyl-4H-chromen-3-yl benzoate
(20c) (55%). H NMR (CDCl3) dH 8.22 (3H, m), 7.65
(2 H, m), 7.50 (3H, m), 7.40 (1H, ddd, J = 7.6 Hz,
J = 7.5 Hz, J = 7.7 Hz, J = 1.0 Hz), 2.70 (2H, t,
J = 7.3 Hz), 1.81 (2H, m), 1.01 (3H, t, J = 7.3 Hz).
1
3.3.5.4. 20-(2,2-Dimethylpropanoyloxy)-2-bromoacetoph-
1
enone (17e) (96%). H NMR (CDCl3) dH 7.75 (1H, dd,
3.3.7.3. 2-Isobutyl-4-oxo-4H-chromen-3-yl benzoate (20d)
1
J = 7.7 Hz, J = 1.7 Hz), 7.54 (1H, ddd, J = 7.5 Hz,
J = 8.1 Hz, J = 8.1 Hz, J = 1.7 Hz),7.33 (1H, ddd, J =
7.0 Hz, J = 7.4 Hz, J = 7.6 Hz, J = 1.1 Hz), 7.07 (1H, dd,
J = 8.1 Hz, J = 1.1 Hz), 4.37 (2H, s), 1.37 (9H, s).
(92%). H NMR (CDCl3) dH 8.17 (3H, m), 7.59 (2H, m),
7.43 (3H, m), 7.34 (1H, ddd, J = 7.6 Hz, J = 7.5 Hz,
J = 7.7 Hz, J = 1.0 Hz), 2.55 (2H, d, J = 7.3 Hz), 2.16 (1H,
m), 0.96 (6H, d, J = 6.8 Hz).
3.3.5.5. 20-Hexanoyloxy-2-bromoacetophenone (17f)
(97%). H NMR (CDCl3) dH 7.79 (1H, dd, J = 7.9 Hz,
J = 1.7 Hz), 7.56 (1H, ddd, J = 7.4Hz, J = 8.3 Hz, J =
8.1 Hz, J = 1.7 Hz), 7.32 (1H, ddd, J = 7.6 Hz, J = 7.9 Hz,
3.3.7.4. 2-t-Butyl-4-oxo-4H-chromen-3-yl benzoate
1
1
(20e) (61%). H NMR (CDCl3) dH 8.12 (3H, m), 7.60
(2H, m), 7.44 (3H, m), 7.31 (1H, ddd, J = 7.6 Hz,
J = 7.6 Hz, J = 7.6 Hz, J = 1.01 Hz), 1.36 (1H, s).

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Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
3.3.7.5. 4-Oxo-2-pentyl-4H-chromen-3-yl benzoate
(20f) (85%). H NMR (CDCl3) dH 8.13 (3H, m), 7.59
(2H, m), 7.43 (3H, m), 7.33 (1H, ddd, J = 7.6 Hz,
J = 7.6 Hz, J = 7.6 Hz, J = 0.8 Hz), 2.55 (2H, t,
J = 7.6 Hz), 1.68 (2H, t, J = 7,0 Hz), 1.29 (4H, m),
(0.82, 3H, t, J = 7.1 Hz).
37.9, 27.2, 22.5. HRMS (M+H)+ calcd for C13H14O3,
1
218.0942; found 218.0947. Mp 130–131 ꢁC, UV (MeOH)
kmax 319, 283, 232, 205 nm.
3.4.1.4. 3-Hydroxy-2-t-butyl-4H-benzopyran-4-one (21e)
1
(40%). H NMR (CDCl3) dH 8.13 (1H, dd, J = 8.1 Hz,
J = 1.5 Hz, H-5), 7.57 (1H, ddd, J = 8.6 Hz, J = 7.0 Hz,
J = 1.6 Hz, H-7), 7.41 (1H, dd, J = 8.5 Hz, J = 0.9 Hz, H-
8), 7.30 (1H, ddd, J = 8.0 Hz, J = 7.0 Hz, J = 1.1 Hz, H-
6), 6.45 (1H, s, OH-3), 1.41 (9H, s, C(CH3)3). 13C NMR
(CDCl3) dC 173.3, 162.6, 155.5, 137.1, 133.1, 125.4, 124.4,
121.1, 118.3, 36.5, 27.6. HRMS (M+H)+ calcd for
C13H14O3, 218.0942; found 218.0948. Mp 169–171 ꢁC, UV
(MeOH) kmax 319, 283, 232 and 205 nm.
3.4. General procedure for the synthesis of (21a–f)
3.4.1. 2-Ethyl-3-hydroxy-4 H-benzopyran-4-one (21a).
Compound(20a)(500 mg, 1.7 mmol)washydrolyzedwith
5% aqueous sodium hydroxide solution (5 mL) in ethanol
(40 mL) at 60 ꢁC for 2 h. The reaction mixture was poured
into a mixture of crushed ice (60 g) and concentrated HCl
(1 mL). It was then extracted with ethyl acetate and
washed with aqueous sodium hydrogen carbonate and
water. The solvent was removed under reduced pressure,
and the residue was recrystallized from aqueous ethanol
3.4.1.5. 3-Hydroxy-2-pentyl-4H-benzopyran-4-one (21f)
1
(65%). H NMR (CDCl3) dH 8.15 (1H,dd, J = 8.3 Hz,
J = 1.5 Hz, H-5), 7.57 (1H, ddd, J = 8.6 Hz, J = 7.0 Hz,
J = 1.7 Hz, H-7), 7.40 (1H, dd, J = 8.5 Hz, J = 0.6 Hz, H-
8), 7.30 (1H, ddd, J = 8.1 Hz, J = 7.0 Hz, J = 1.1 Hz, H-
6), 6.18 (1H, s, OH-3), 2.77 (2H, t, J = 7.6 Hz,Ar–CH2–),
1.71 (2H, m, Ar–CH2CH2CH2–), 1.32 (4H, m, Ar–
CH2CH2CH2CH2–), 0.84 (3H, m, Ar–(CH2)4CH3). 13C
NMR dC 172.5, 155.6, 152.5, 138.2, 133.1, 125.5, 124.3,
121.5, 118.1, 31.4, 28.9, 26.4, 22.4, 14.0. HRMS (M+H)+
calcd for C14H16O3, 232.1099; found 232.1105. Mp 96–
99 ꢁC. UV (MeOH) kmax 319, 283, 232 and 205 nm.
1
to give 21a (170 mg, 53%) H NMR (CDCl3) dH 8.15
(1H, dd, J = 7.3 Hz, J = 1.8 Hz, H-5), 7.57 (1H, ddd,
J = 8.7 Hz, J = 7.2 Hz, J = 1.5 Hz, H-7), 7.40 (1H, dd,
J = 8.6 Hz, J = 1.0 Hz, H-8), 7.30 (1H, ddd, J = 8.1 Hz,
J = 7.1 Hz, J = 1.0 Hz, H-6), 6.08 (1H, s, OH-3), 2.81
(2H, c, Ar–CH2), 1.28 (3H, t, J = 7.7 Hz, CH2CH3). 13
C
NMR (CDCl3) dC 172.5, 155.7, 153.3, 137.7, 133.0,
125.5, 124.3, 121.5, 118.1, 22.4, 10.9. HRMS (M+H)+
calcd for C11H10O3, 190.0629; found 190.0630. Mp 99–
101 ꢁC, UV (MeOH) kmax 319, 283, 232 and 205 nm.
3.5. General procedure for the synthesis of compounds
25a–j
3.4.1.1. 2-Isopropyl-3-hydroxy-4H-benzopyran-4-one
(21b) (61%). 1H NMR (CDCl3) dH 8.15 (1H, dd,
J = 8.1 Hz, J = 1.8 Hz, H-5), 7.57 (1H, ddd, J = 8.7 Hz,
J = 7.1 Hz, J = 1.8 Hz, H-7), 7.42 (1H, dd, J = 8.8 Hz,
J = 1.0 Hz, H-8), 7.30 (1H, ddd, J = 8.0 Hz J = 7.0 Hz,
J = 1.2 Hz, H-6), 6.27 (1H, s, OH-3), 3.41 (1H, m, Ar–
CH–), 1.28 (6H, d, J = 6.8 Hz, Ar–CH(CH3)2). 13C
NMR (CDCl3) dC 172.7, 156.0, 155.7, 136.9, 132.9,
125.5, 124.3, 121.4, 118.1, 28.2, 19.4. HRMS (M+H)+
calcd for C12H12O3, 204.0786; found 204.0788. Mp 152–
153 ꢁC, UV (MeOH) kmax 319, 283, 232 and 205 nm.
Dry HCl (g) was passed into a cooled (0 ꢁC), stirred mix-
ture of the substituted phenylacetonitrile (22) (0.34 mol)
and anhydrous ZnCl2 (30 g, 0.22 mol) in dry diethyl
ether (200 mL). The corresponding polyhydroxybenzene
(23) (0.28 mol) was added portionwise with constant
bubbling of gaseous HCl for a further 4–6 h. The reac-
tion mixture was then stirred at room temperature for
4–20 h, until the complete disappearance of the reagents,
as shown by thin-layer chromatography.
3.4.1.2.
3-Hydroxy-2-propyl-4H-benzopyran-4-one
The ketiminium chloride intermediate that separated as
an oil after 3–4 days was washed with diethyl ether and
hydrolyzed by refluxing in 5% HCl (1 L) for 4–5 h. The
ketone (24), which separated upon cooling, was filtered
and recrystallized in the appropriate solvent.
(21c) (63%). 1H NMR (CDCl3) dH 8.16 (1H, dd,
J = 7.8 Hz, J = 1.5 Hz, H-5), 7.56 (1H, ddd, J = 8.7 Hz,
J = 7.0 Hz, J = 1.8 Hz, H-7), 7.39 (1H, dd, J = 8.6 Hz,
J = 1.3 Hz, H-8), 7.30 (1H, ddd,J = 8.1 Hz, J = 7.1 Hz,
J = 1.1 Hz, H-6), 6.23 (1H, s, OH-3), 2.75 (2H, t,
J = 7.4 Hz, Ar–CH2), 1.74 (2H, m, Ar–CH2CH2–), 0.86
(3H, t, J = 7.4 Hz, Ar–CH2CH2CH3). 13C NMR (CDCl3)
dC 172.5, 155.7, 152.3, 138.3, 132.9, 125.5, 124.3, 121.4,
118.1, 30.8, 20.1, 13.7. HRMS (M+H)+ calcd for
C12H12O3 204.0786; found 204.0790. Mp 102–104 ꢁC.
UV (MeOH) kmax 319, 283, 232, 205 nm.
To a solution of benzylketone (24) (0.18 mol) in dry DMF
(200 mL) BF3Et2O (0.88 mol) was added dropwise. This
solution was warmed to 50 ꢁC and a solution of metha-
nesulfonyl chloride (0.56 mol) in DMF (100 mL) was
slowly added. The resulting mixture was then heated to
100 ꢁC for 2 h. After cooling, it was poured into water
(400 mL) and left overnight to give a precipitate, which
was then stirred for 2 h in cold methanol (50 mL), filtered,
and crystallized in a mixture of water/ethanol.
3.4.1.3. 2-Isobutyl-3-hydroxy-4H-benzopyran-4-one (21d)
1
(45%). H NMR (CDCl3) dH 8.21 (1H, dd, J = 8.1 Hz,
J = 1.7 Hz, H-5), 7.62 (1H, ddd, J = 8.6 Hz, J = 7.0 Hz,
J = 1.7 Hz, H-7), 7.44 (1H, dd, J = 8.5 Hz, J = 0.9 Hz, H-
8) 7.35 (1H, ddd, J = 8.1 Hz, J = 7.1 Hz, J = 1.0 Hz, H-6),
6.23 (1H, s, OH-3), 2.71 (2H, d, J = 7.2 Hz, Ar–CH2–),
2.21 (1H, m, Ar–(CH2CH(CH3)2)), J = 1.01 (6H, d,
J = 6.6 Hz, Ar–(CH2CH(CH3)2)). 13C NMR (CDCl3) dC
172.4, 155.7, 151.7, 138.8, 133.0, 125.4, 124.2, 121.4, 118.1,
3.5.1. 6,7-Dihydroxy-40-methoxyisoflavone (25a) (72%).
1H NMR (DMSO-d6) dH 10.38 (1H, s, 7-OH), 9.75 (1H,
s, 6-OH), 8.27 (1H, s, 2-H), 7.49 (2H, d, J = 8.6 Hz,
20-H, 60-H), 7.37 (1H, s, 5-H), 6,97 (2H, d, J = 8.6 Hz,
30-H, 50-H), 6.89 (1H, s, H-8), 3.76 (3H, s, OCH3). 13C
NMR (DMSO-d6) dC 174.9, 159.5, 153.3, 152.9, 151.5,

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7421
145.4, 130.7, 125.3, 123.1, 117.2, 114.2, 108.8, 103.4,
55.8. Mp 251 ꢁC.
J = 9.0 Hz, 6-H). 13C NMR (DMSO-d6) dC 175.4,
153.3, 151.8, 137.8, 134.3, 134.0, 130.1, 128.1, 126.3,
124.4, 123.6, 118.1, 112.1. HRMS (M+H)+ calcd for
C15H9O4Cl, 288.0189; found 288.0192. Mp 258 ꢁC. UV
(MeOH) kmax 261 and 302 nm.
3.5.2. 40,6,7-Trihydroxyisoflavone (25b) (53%). 1H NMR
(DMSO-d6) dH 9.84 (1H, s, 7-OH), 8.21 (1H, s, 6-OH),
7.36 (1H, s, H-5), 7.35 (2H, d, J = 8.7 Hz, H-20, H-60),
6.87 (1H, s, H-8), 6.78 (2H, d, J = 8.7 Hz, H-30, H-50),
4.46 (1H, dd, J = 11.0 Hz, J = 13.1 Hz, H-3), 4.43 (1H,
dd, J = 5.5 Hz, J = 5.9 Hz, H-2a), 3.96 (1H, dd,
J = 5.3 Hz, J = 7.2 Hz, H-2e). 13C NMR (DMSO-d6)
dC 187.9, 174.98, 157.8, 153.1, 151.4, 145.3, 130.7,
130.3, 123.4, 117.6, 115.6, 108.6, 103.4. Mp 225 ꢁC.
1
3.5.9. 7,8-Dihydroxy-30-methylisoflavone (25i) (71%). H
NMR (DMSO-d6) dH 10.28 (1H, s, 7-OH), 9.69 (1H, s, 8-
OH), 8.42 (1H, s, 2-H), 7.51 (1H, d, J = 9.0 Hz, 5-H), 7.24-
7.53 (4H, m, 20-H, 40-H, 50-H, 60-H) 7,0 (1H, d, J = 9.0 Hz,
6-H) 2.37 (3H, s, CH3). 13C NMR (DMSO-d6) dC 175.7,
153.8, 152.9, 151.6, 138.1, 137.9, 132.4, 128.6, 126.8,
124.7, 123.4, 118.1, 113.2, 23.7. HRMS (M+H)+ calcd
for C16H12O4,268.0736; found 269.0740. Mp 268 ꢁC.
UV (MeOH) kmax 257 and 309 nm.
1
3.5.3. 6,7-Dihydroxy-40-nitroisoflavone (25c) (81%). H
NMR (DMSO-d6) dH 10.11 (1H, s, 7-OH), 8.54 (1H, s,
2-H), 8.26 (2H, d, J = 8.8 Hz, H-30, H-50), 7.89 (2H, d,
J = 8.8 Hz, H-20, H-60), 7.40 (1H, s, H-5), 6.93 (1H, s,
H-8), [signal for 8-OH not seen]. 13C NMR (DMSO-
d6) dC 174.3, 155.5, 153.3, 151.7, 147.2, 145.8, 140.4,
130.5, 123.9, 121.6, 117.1, 108.9, 103.7. Mp 168 ꢁC.
3.5.10. 7,8-Dihydroxy-30-trifluoromethylisoflavone (25j)
1
(79%). H NMR (DMSO-d6) dH 10.00 (1H, broad, s,
8-OH), 8.64 (1H, s, 2-H), 8.04 (1H, s, 20-H), 7.71-7.88
(3H, m, 40-H, 50-H, 60-H), 7.55 (1H, d, J = 9.0 Hz, 5-
H) 7.04 (1H, d, J = 9.0 Hz, 6-H) [signal for 7-OH not
seen]. 13C NMR (DMSO-d6) dC 174.7, 154.5, 150.5,
146.8, 133.5, 133.1, 132.8, 129.2, 129.0, 125.6, 124.4,
124.3, 121.6, 117.4, 115.8, 114.5. HRMS (M+H)+ calcd
for C16H9O4F3, 322.0453; found 322.0454. Mp 225 ꢁC.
3.5.4.
6,7-Dihydroxy-30,40-methylenedioxyisoflavone
1
(25d) (67%). H NMR (DMSO-d6) dH 10.1 (1H, s, 7-
OH), 9.63 (1H, s, 6-OH), 8.28 (1H, s, 2-H), 7.37 (1H,
s, H-5), 7.12 (1H, d, J = 1.6 Hz, H-20), 7.03 (1H, dd,
J = 1.6 Hz, J = 8.0 Hz, H-60), 6.94 (1H, d, J = 8.0 Hz,
H-50), 6.88 (1H, s, H-8), 6.02 (2H, s, O–CH2–O). 13C
NMR (DMSO-d6) dC 174.8, 153.6, 153.0, 151.5, 147.4,
145.4, 126.8, 123.1, 123.0, 117.2, 110.1, 108.8, 108.7,
103.4, 101.6. HRMS (M+H)+ calcd for C16H10O6,
298.0478; found 298.0480. Mp 260 ꢁC.
3.6. General procedure for the synthesis of compounds 26a
and 26b
3.6.1. Reduction of isoflavone to isoflavanone. To a stirred
solution of 1.0 g of the isoflavone in 200 ml of ethanol at
room temperature was added 0.5 g of Pd/C (10%) and
the mixture was hydrogenated at normal pressure for
6 h. Once the hydrogenation was completed, as verified
by thin-layer chromatography, the solution was filtered
and concentrated to dryness under reduced pressure.
The resulting residue was taken up with 10 mL of water,
filtered, washed with water, and dried to afford the cor-
responding isoflavanone, which was purified by chroma-
tography over a silica gel column with a 2:1 benzene/
ether mixture as eluent. The yields were 60–75%.
3.5.5. 7,8-Dihydroxy-40-methylisoflavone (25e) (78%). 1H
NMR (DMSO-d6) dH 9.81 (1H, s, 8-OH), 8.38 (1H, s, 2-
H), 7.47 (1H, d, J = 8.7 Hz, H-5), 7.45 (2H, d,
J = 8.1 Hz, H-20, H-60), 7.21 (2H, d, J = 8.1 Hz, H-30,
H-50), 6.95 (1H, d, J = 8.7 Hz, H-6), 2.32 (3H, s, CH3),
[signal for 7-OH not seen]. 13C NMR (DMSO-d6) dC
175.6, 153.9, 150.8, 147.4, 137.6, 133.6, 129.9, 129.5,
129.3, 123.5, 118.1, 116.3, 114.9, 21.5. Mp 241 ꢁC.
3.5.6. 7,8-Dihydroxyisoflavone (25f) (76%). 1H NMR
(DMSO-d6) dH9.83 (1H, s, 8-OH), 8.43 (1H, s, 2-H), 7.57
(2H, d, J = 7.0 Hz, H-30, H-50), 7.48 (1H, d, J = 8.6 Hz,
H-5), 7.42 (2H, t, J = 7.0 Hz, H-20, H-60), 7.37 (1H, d,
J = 8.6 Hz, H-6), [signal for 7-OH not seen]. 13C NMR
(DMSO-d6) dC 175.5, 154.3, 150.8, 147.4, 133.6, 132.9,
129.7, 128.7, 128.3, 123.7, 118.1, 116.4, 114.9. Mp 211 ꢁC.
3.7. General procedure for the synthesis of compounds
27a–g
3.7.1. Reduction of isoflavone to isoflavan. To a stirred
solution of 5 mmol of the isoflavone in 100 ml of acetic
acid containing 0.1% sulfuric acid was added 0.5 g of
Pd/C (10%) and the mixture was hydrogenated for 14 h
at normal pressure and room temperature. Once the
hydrogenation was completed, verified by thin-layer
chromatography, the solution was filtered and concen-
trated under reduced pressure. The resulting residue was
taken up with 50 mL of water, extracted with ether
(3 · 50 mL), and the organic lalyer dried with sodium sul-
fate to give the corresponding isoflavan, which was puri-
fied by chromatography over a silica gel column using
ether/methanol mixtures of increasing polarity as eluent.
The yields were 40–50%.
1
3.5.7. 40-Chloro-7,8-dihydroxyisoflavone (25g) (65%). H
NMR (DMSO-d6) dH10.35 (1H, s, 7-OH), 9.47 (1H, s, 8-
OH), 8.47 (1H, s, H-2), 7.61 (2H, d, J = 8.6 Hz, 20-H, 60-
H), 7.48 (1H, d, J = 8.7 Hz, H-5), 7.47 (2H, d,
J = 8.59 Hz, H-30, H-50), 6.96 (1H, d, J = 8.7 Hz, H-8).
13C NMR (DMSO-d6) dC 175.3, 154.6, 150.9, 147.4,
133.6, 133.1, 131.8, 131.4, 128.8, 122.4, 118.0, 116.4,
115.0. HRMS (M+H)+ calcd for C15H9O4Cl, 288.0189;
found 288.0191. Mp 165 ꢁC.
1
3.5.8. 30-Chloro-7,8-dihydroxyisoflavone (25h) (69%). H
NMR (DMSO-d6) dH 9.89 (2H, s, 7-OH, 8-OH), 8.42
(1H, s, 2-H), 7.46 (1H, d, J = 9.0 Hz, 5-H), 7.42-7.68
(4H, m, 20-H, 40-H, 50-H, 60-H), 7.01 (1H, d,
3.7.1.1. 6,7-Dihydroxy-40-methoxyisoflavanone (26a)
(52%). 1H NMR (DMSO-d6) dH9.80 (1H, s, 7-OH), 7.56
(1H, s, H-5), 7.14 (2H, d, J = 8.7 Hz, H-20, H-60), 7.09

7422
Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
(1H, s, H-8), 6.86 (2H, d, J = 8.6 Hz, H-30, H-50), 4.49
(1H, dd, J = 11.2 Hz, J = 13.9 H, H-3z), 4.47 (1H, dd,
J = 5.6 Hz, J = 5.7 Hz, H-2a), 3.85 (1H, dd,
J = 5.5 Hz, J = 7.6 Hz, H-2e), 3.70 (3H, s, OCH3), [sig-
nal for 6-OH not seen]. 13C NMR (DMSO-d6) dC
191.1, 159.1, 157.1, 154.9, 141.7, 130.3, 129.1, 114.5,
113.0, 111.4, 103.6, 72.0, 55.7, 50.9. Calcd for
C16H14O5, C, 67.13%; H, 4.89%, found C, 66.95%; H,
4.91%. Mp 214 ꢁC.
J = 5.5 Hz, J = 16,0 Hz, J = 1.5 Hz, H-4e).13C NMR
(DMSO-d6) dC 147.4, 146.5, 145.9, 144.3, 139.4, 135.9,
120.5, 115.7, 111.6, 108.2, 107.9, 103.4, 100.8, 69.9,
37.9, 31.4. HRMS (M+H)+ calcd for C16H14O5,
286.0841; found 286.0842, mp 162 ꢁC.
3.7.1.6. 6,7-Dihydroxy-30-methylisoflavan (27d) (90%).
1H NMR (DMSO-d6) dH8.75 (1H, s, OH-7), 8.25 (1H, s,
OH-6), 7.19 (1H, dd, J = 8.0 Hz, H-50), 7.00–7.10 (3H,
m, H-20, H-40, H-60), 6.46 (1H, s, H-5), 6.24 (1H, s, H-
8), 4.12 (1H, ddd, J = 3.0 Hz, J = 10.5, Hz J = 1.6 Hz,
H-2e), 3.86 (1H, dd, J = 16 Hz; J = 16 Hz, H-2a), 3.05
(1H, dddd, J = 10.5 Hz, J = 16.0 Hz, J = 5.5 Hz,
J = 4.6 Hz H-3a), 2.86 (1H, dd, J = 10.5 Hz,
J = 16.0 Hz, H-4a), 2.26 (1H, ddd, J = 16.0 Hz,
J = 16,0 Hz, J = 1.5 Hz, H-4e). 13C NMR (DMSO-d6)
dC 146.5, 144.3, 141.8, 139.1, 137.5, 128.6, 128.1,
127.3, 124.6, 115.7, 111.5, 103.4, 69.8, 37.3, 31.2, 21.1.
HRMS (M+H)+ calcd for C16H16O3, 256.1099; found
256.1101, mp 164 ꢁC.
3.7.1.2. 40,6,7-Trihydroxyisoflavanone (26b) (48%). 1H
NMR (DMSO-d6) dH9.84 (1H, s, 7-OH), 8.21 (1H, s, 40–
OH), 7.56 (1H, s, H-5), 6.95 (2H, d, J = 8.7 Hz, H-20, H-
60), 6.68 (2H, d, J = 8.6 Hz, H-30, H-50), 6.15 (1H, s, H-
8), 4.79 (1H, dd, J = 11.2 Hz, J = 13.9 H, H-3z), 4.55
(1H, dd, J = 5.6 Hz, J = 5.7 Hz, H-2a), 4.20 (1H, dd,
J = 5.5 Hz, J = 7.6 Hz, H-2e), 3.70 (3H, s, OCH3), [sig-
nal for 6-OH not seen]. 13C NMR (DMSO-d6) dC
193.1, 157.4, 152.3, 150.9, 138.8, 131.1, 129.0, 116.6,
116.4, 114.4, 103.4, 70.9, 56.1. Calcd for C15H12O5, C,
66.17%; H, 4.41%, found C, 66.32%; Mp 235 ꢁC.
3.7.1.7. 7,8-Dihydroxy-40-methylisoflavan (27e) (68%).
1H NMR (DMSO-d6) dH 8.54 (1H, s, OH-7), 8.09 (1H,
s, OH-8), 7.19 (2H, d, J = 8.1 Hz, H-20, H-60), 7.12 (2H,
d, J = 8.1 Hz, H-30, H-50), 6.36 (1H, d, J = 8.2 Hz, H-5),
6.28 (1H, d, J = 8.2 Hz, H-6), 4.23 (1H, ddd, J = 3.4 Hz,
J = 10.4 Hz, J = 1.7 Hz, H-2e), 3.97 (1H, dd,
J = 10.2 Hz; J = 10.2 Hz, H-2a), 3.09 (1H, ddd,
J = 4.0 Hz, J = 5.7 Hz, J = 10.0 Hz, J = 8.9 Hz, H-3),
2.89 (1H, dd, J = 10.4 Hz, J = 16.1 Hz, H-4a), 2.80
(1H, ddd, J = 5.4 Hz, J = 15.6 Hz, J = 1.2 Hz, H-4e).
13C NMR (DMSO-d6) dC144.7, 143.6, 139.5, 136.5,
133.7, 129.7, 127.9, 119.1, 113.9, 108.7, 70.5, 38.2,
32.0, 21.3. HRMS (M+H)+ calcd for C16H16O3,
256.1099; found 256.1100, mp 125–126. ꢁC.
3.7.1.3. 6,7-Dihydroxy-40-methoxyisoflavan (27a)
(49%). 1H NMR (DMSO-d6) dH8.68 (1H, s, OH-7),
8.25 (1H, s, OH-6), 7.19 (2H, d, J = 8.7 Hz, H-20, H-
60), 6.87 (2H, d, J = 8.7 Hz, H-30, H-50), 6.43 (1H, s,
H-5), 6.19 (1H, s, H-8), 4.07 (1H, ddd, J = 3.2 Hz,
J = 10.3 Hz, J = 1.6 Hz; H-2e), 3.83 (1H, dd,
J = 10.4 Hz, J = 3.2 Hz, H-2a), 3.71 (3H, s, OCH3),
3.03 (1H, dddd, J = 10.0 Hz, J = 10.1 Hz, J = 5.7 Hz,
J = 4.1 Hz, H-3a), 2.80 (1H, dd, J = 10.5 Hz,
J = 15.8 Hz, H-4a), 2.71 (1H, ddd, J = 5.5 Hz,
J = 16 Hz, J = 1.6 Hz, H-4e). 13C NMR (DMSO-d6)
dC 158.7, 147.1, 144.9, 139.7, 134.4, 129.1, 116.3,
114.6, 112.2, 104.0, 70.6, 55.7, 37.9, 31.9. HRMS
(M+H)+ calcd for C16H16O4 272.10486; found
272.10363. Mp 144 ꢁC.
3.7.1.8. 7,8-Dihydroxy-30,40-dimethoxyisoflavan (27f)
1
(49%). H NMR (DMSO-d6) dH 8.53 (1H, s, OH-7),
3.7.1.4. 406,7-Trihydroxyisoflavan (27b) (55%). 1H
NMR (DMSO-d6) dH9.22 (1H, s, OH-40), 8.66 (1H, s,
OH-7), 8,24 (1H, s, OH-6), 7.07 (2H, d, J = 8.4 Hz, H-
20, H-60), 6.69 (2H, d, J = 8.6 Hz, H-30, H-50), 6.41
(1H, s, H-5), 6.18 (1H, s, H-8), 4.05 (1H, ddd,
J = 3.2 Hz, J = 10.5 Hz, J = 1.6 Hz; H-2e), 3.78 (1H,
dd, J = 10.3 Hz, J = 10.3 Hz, H-2a), 3.06 (1H, dddd,
J = 10.1 Hz, J = 10.1 Hz, J = 5.8 Hz, J = 4.1 Hz, H-3),
2.77 (1H, dd, J = 10.4 Hz, J = 15.7 Hz, H-4a), 2.68
(1H, ddd, J = 5.6 Hz, J = 16,0 Hz, J = 1.1 Hz, H-
4e).13C NMR (DMSO-d6) dC 156.7, 147.1, 144.9,
139.7, 132.1, 128.9, 116.4, 115.9, 112.4, 104.0, 70.8,
38.0, 30.1. HRMS (M+H)+ calcd for C15H14O4
258.08920; found 258.08590. Mp 176–177 ꢁC.
8.09 (1H, s, OH-6), 6.94 (1H, d, J = 1.8 Hz, H-20), 6.88
(1H, d, J = 8.4 Hz, H-50), 6.81 (1H, dd, J = 8.4 Hz,
J = 1.8 Hz, H-60), 6.35 (1H, d, J = 8.2 Hz, H-5), 6.27
(1H, d, J = 8.2 Hz, H-6), 4.24 (1H, ddd, J = 3.2 Hz,
J = 10.2 Hz, J = 1.6 Hz, H-2e), 3.96 (1H, dd,
J = 10.2 Hz, J = 10.2 Hz, H-2a), 3.73 (3H, s, OCH3-
40), 3.71 (3H, s, OCH3-30), 3.06 (1H, dddd,
J = 10.0 Hz, J = 16.0 Hz, J = 5.4 Hz, J = 4.6 Hz, H-3),
2.90 (1H, dd, J = 10.7 Hz, J = 15.7 Hz, H-4a), 2.79
(1H, ddd, J = 5.3 Hz, J = 15.7 Hz, J = 1.1 Hz, H-4e).
13C NMR (DMSO-d6) dC 149.7, 148.4, 144,7, 143.7,
134.9, 133.8, 119.9, 118.9, 114.0, 112.8, 108.7, 70.8,
56.3, 56.2, 38.2, 32.2. HRMS (M+H)+ calcd for
C17H18O5, 302.1154; found 302.1156. Mp 159–160 ꢁC.
3.7.1.5. 6,7-Dihydroxy-30,40-methylenedioxyisoflavan
(27c) (52%). H NMR (DMSO-d6) dH8.71 (1H, s, OH-
3.7.1.9. 7,8-Dihydroxy-40-methoxyisoflavan (27g)
(56%). H NMR (DMSO-d6) dH 8.54 (1H, s, OH-7),
1
1
7), 8.26 (1H, s, OH-6), 6.91 (1H, d, J = 2.0 Hz, H-20),
6.84 (1H, d, J = 8.0 Hz, H-50), 6.76 (1H, dd,
J = 2.0 Hz, J = 8.0 Hz, H-60), 6.42 (1H, s, H-5), 6.19
(1H, s, H-8), 5.96 (2H, s, O–CH2–O), 4.06 (1H, ddd,
J = 3.5 Hz, J = 10.5 Hz, J = 1.5 Hz, H-2e), 3.84 (1H,
dd, J = 10.5 Hz, J = 10,5 Hz, H-2a), 3.02 (1H, ddd,
J = 10.5 Hz, J = 10.5 Hz, J = 5.5 Hz, H-3a), 2.79 (1H,
dd, J = 10.5 Hz, J = 16,0 Hz, H-4a), 2.71 (1H, ddd,
8.09 (1H, s, OH-8), 7.22 (2H, d, J = 8.8 Hz, H-20, H-
60), 6.89 (2H, d, J = 8.8 Hz, H-30, H-50), 6.35 (1H, d,
J = 8.2 Hz, H-5), 6.29 (1H, d, J = 8.2 Hz, H-6), 4.22
(1H, ddd, J = 3.3 Hz, J = 16 Hz, J = 1.5 Hz, H-2e),
3.92 (1H, dd, J = 10.2 Hz, J = 10.2 Hz, H-2a), 3.72
(3H, s, OCH3), 3.07 (1H, dddd, J = 10.0 Hz,
J = 9.9 Hz, J = 5.7 Hz; J = 3.9 Hz, H-3), 2.88 (1H, dd,
J = 10.1 Hz, J = 15.6 Hz, H-4a), 2.79 (1H, ddd,

Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
7423
J = 5.5 Hz, J = 15.7 Hz, J = 1.8 Hz, H-4e). 13C NMR
(DMSO-d6) dC 158.8, 144.8, 143.7, 134.3, 133.8, 129.1,
119.1, 115.3, 113.9, 108.1, 70.8, 55.7, 37.8, 32.1. HRMS
(M+H)+ calcd for C16H16O4, 272.1049; found 272.1049.
Mp 128–129 ꢁC.
from V&P Scientific. Addition of 5 lL of enzyme, which
gives a rate of approximately 0.0020 abs/s using the mul-
tidrop, started the reaction (approximately 40 nM for
12-hLO and 15-hLO-1 and 125 nM for 15-hLO-2, final
concentration). The reaction was stopped well before
completion (after approximately 5 min) by adding
appropriate amount of reagents to give a final concen-
tration of 100 lM of xylenol orange dye and 150 lM
Fe2+ in 25 mM H2SO4, with a final volume of 100 lL
in each well. The plates were stirred using a gyratory
shaker in the dark for 30 min and read at 544 nm using
Perkin-Elmer plate reader Victor2. The negative control
for the assay was 0.2 lL of DMSO (with no inhibitor)
added to reaction buffer, with no enzyme. The positive
control for the assay was 0.2 lL of DMSO added to
reaction buffer with enzyme, as mentioned above. All
the assays were done in triplicate. The percent inhibition
was calculated by subtracting the absorbance of the neg-
ative control from that of the reaction samples and
dividing by the absorbance of the positive control. The
value was then subtracted from 1 and multiplied by
100 to get the percent inhibition. In order to confirm
the accuracy of the HTP screen, all samples giving great-
er than 40% inhibition by the HTP assay were screened
manually to determine an accurate IC50 value (vida in-
fra). From this additional screening, it was determined
that compounds with less than 45% inhibition from
the HTP screen for all three hLOs had IC50 values great-
er than 100 lM.
3.8. Plasmid construction, expression, and purification of
human lipoxygenases
The histidine-tagged proteins, 12-hLO and 15-hLO-1,
were expressed and purified as described previously.42,49
The SF9 expression vector for human prostate epithelial
15-lipoxygenase-2 (15-hLO-2) was constructed as fol-
lows. The previously published plasmid, pCRII-
TOPO-15hLO-2,19 was cut with EcoRI, to liberate the
15-hLO-2 fragment. This 15-hLO-2 fragment was then
ligated into EcoRI cut, pFastBac1 (GibcoBRL), to gen-
erate the complete plasmid, pFastBac-15-hLO-2, which
was digested to determine the correct orientation. The
pFastBac-15-hLO-2 was then transposed into a recom-
binant FastBac bacmid by DH10Bac cells (GibcoBRL)
and then transfected into SF9 cells, as described in the
product literature for pFastBac1 (GibcoBRL). The virus
was subsequently amplified to ꢁ2 · 1010 plaque-forming
units (pfu), added to SF9 cells (ꢁ2 · 106 cells/ml) at a
concentration of ꢁ2 · 107 pfu/ml, and allowed to shake
for 72 h. The cells were then harvested and frozen. 15-
hLO-2 was purified by douncing the thawed cells and
loading the cell extracts onto a 20 mL column of
Macro-prep High Q, strong anion exchange column,
from Bio-Rad. The protein was eluted with a 50–
300 mM sodium chloride gradient (25 mM HEPES,
pH 8.0). The collected fractions contained 95% purified
protein and 5% glycerol and were frozen at ꢀ80 ꢁC.
Subsequent thawing produced no decrease in enzymatic
activity.
3.10. Manual absorption IC50 inhibition assay
IC50 inhibition constants for compounds with greater
than 40% inhibition from the HTP screen were deter-
mined manually as described previously,52 with the fol-
lowing changes. The reaction buffer was 25 mM Hepes
(pH 8.0) with 0.01% Triton X-100 for 12-hLO and was
25 mM Hepes (pH 7.5) with 0.01% Triton X-100 for
15-hLO-1 and 15-hLO-2. Arachidonic acid was used
as a substrate for 12-hLO (3 lM) and 15-hLO-2
(30 lM), while linoleic acid was used for 15-hLO-1
(3 lM). Enzyme rates were determined with approxi-
mately 40 nM for 12-hLO and 15-hLO-1 and 125 nM
for 15-hLO-2, final concentration, by monitoring prod-
uct formation at 234 nm (eM = 2.5 · 104) with an HP
8435 spectrophotometer at room temperature. Inhibitor
(1 mg/mL in DMSO) was added to the substrate buffer
and the reaction initiated by addition of enzyme after
a stable baseline was observed at 234 nm. Control rates
were measured using the same volume of DMSO as the
volume of inhibitor. IC50values were determined by
measuring the enzymatic rate at a variety of inhibitor
concentrations (depending on the inhibitor potency)
and plotting their percent inhibition versus inhibitor
concentration. The corresponding data were fit to a sim-
ple saturation curve using KaleidaGraph software (Syn-
ergy) on a Macintosh computer and the inhibitor
concentration at 50% activity was calculated.
13-(S)-HPOD was prepared as before,50 while LA and
AA were purchased from Aldrich Chemical, and
BWB70C and BWA4C were purchased from Sigma
Chemical. All other reagents were of reagent grade or
better and were used without further purification.
3.9. High-throughput inhibitor screen
The high-throughput (HTP) screen was developed in
384-well plates where the hydroperoxide product was
detected with iron/xylenol orange.40,51 Briefly, the lipid
peroxide formed by the human lipoxygenase enzymes re-
acts with Fe2+ present in solution and converts it to Fe3+
which in turn forms an Fe3+-xylenol orange complex
which absorbs at 560 nm. The HTP screen was per-
formed as published earlier with the following
changes.40 The buffer for 12-hLO was 25 mM Hepes
(pH 8.0) with 0.01% Triton X-100 and for 15-hLO-1
and 15-hLO-2 was 25 mM Hepes (pH 7.5) with 0.01%
Triton X-100. Arachidonic acid was used as a substrate
for 12-hLO and 15-hLO-2, while linoleic acid was used
for 15-hLO-1. The first step is the addition of 45 ll of
a 30 lM substrate buffer solution to each well using
the Multidrop 384 by Thermo Labsystems. The inhibi-
tor (1.2 ll at 2.5 mg/ml in DMSO) was then added using
a specially designed manual liquid pin transfer device
3.11. Pseudoperoxidase activity assay
The pseudoperoxidase activity was determined using a
Perkin-Elmer Lambda 40 spectrophotometer by follow-

7424
Y. Vasquez-Martinez et al. / Bioorg. Med. Chem. 15 (2007) 7408–7425
10. Zhang, Y.; Wang, H.; Li, J.; Jimenez, D. A.; Levitan, E.
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tion mixture contained 15-hLO-1 (185 lg), 25 mM
Hepes, pH 7.5, HPOD (5 lM), and inhibitor (10 lM).
The reaction was initiated by adding enzyme at room
temperature and the final volume of the reaction solu-
tion was 2 ml. BWB70C and BWA4C were used as po-
sitive controls for this experiment as they are known
redox-type inhibitors for human 5-lipoxygenase.54,55
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The 15-hLO-1 homology model was created using the
Protein Local Optimization Program (PLOP, commer-
cially distributed as Prime), which uses loop prediction,56
side chain prediction,57,58 and energy minimization to
align the target and template sequences, as previously re-
ported.41 Docking of inhibitors to this model has been
verified previously by predicting experimental inhibitor
potency based solely on docking parameters.41 The struc-
tures of compounds 25b and 25e, 29, and 30 were prepared
for docking using the LigPrep (Schro¨dinger, Inc) ligand
preparation software, which generates a minimized con-
formation of each ligand, and multiple protonation/tau-
tomerization states when appropriate. Flexible ligand
docking was performed using the Glide (Schro¨dinger,
Inc.) program,59,60 which uses a modified version of the
Chemscore energy function to score the protein-ligand
interactions.61
´
20. Vavreckova, C.; Gawlik, I.; Muller, K. Planta Med. 1996,
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Acknowledgments
25. Segraves, E. N.; Shah, R. R.; Segraves, N. L.; Johnson, T.
A.; Whitman, S.; Sui, J. K.; Kenyon, V. A.; Cichewicz, R.
H.; Crews, P.; Holman, T. R. J. Med. Chem. 2004, 47,
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26. Cichewicz, R. H.; Kenyon, V. A.; Whitman, S.; Morales,
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27. Amagata, T.; Whitman, S.; Johnson, T.; Stessmann, C. C.;
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28. Whitman, S.; Gezginci, M.; Timmermann, B. N.; Holman,
T. R. J. Med. Chem. 2002, 45, 2659–2661.
29. Malterud, K. E.; Farbrot, T. L.; Huse, A. E.; Sund, R. B.
Pharmacology 1993, 47 Suppl 1, 77–85.
T.R.H. gratefully acknowledges the gift of the original 15-
hLO-2 gene from Dr. D. Tang. The 2-alkyl-4-H-benzopy-
ran-4-ones described were synthesized by Y.V.M. during
a stay in Prof. E. Breitmaier’s laboratory at the University
of Bonn as the recipient of a DAAD fellowship. S.S.B.
thanks the MECESUP UCH 0116 program for the
HRMS. The research was supported by NASA # 13171
(S.S.B.), NIH (GM56062-6) (T.R.H.), and the American
Cancer Society (RPG-00-219-01-CDD) (TRH) Grants.
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