2-Morpholinoisoflav-3-enes as flexible intermediates in the synthesis of phenoxodiol, isophenoxodiol, equol and analogues: Vasorelaxant properties, estrogen receptor binding and Rho/RhoA kinase pathway inhibition

Article abstract of DOI:10.1016/j.bmc.2012.02.008

Isoflavone consumption correlates with reduced rates of cardiovascular disease. Epidemiological studies and clinical data provide evidence that isoflavone metabolites, such as the isoflavan equol, contribute to these beneficial effects. In this study we developed a new route to isoflavans and isoflavenes via 2-morpholinoisoflavenes derived from a condensation reaction of phenylacetaldehydes, salicylaldehydes and morpholine. We report the synthesis of the isoflavans equol and deoxygenated analogues, and the isoflavenes 7,4′-dihydroxyisoflav-3-ene (phenoxodiol, haganin E) and 7,4′-dihydroxyisoflav-2-ene (isophenoxodiol). Vascular pharmacology studies reveal that all oxygenated isoflavans and isoflavenes can attenuate phenylephrine-induced vasoconstriction, which was unaffected by the estrogen receptor antagonist ICI 182,780. Furthermore, the compounds inhibited U46619 (a thromboxane A₂ analogue) induced vasoconstriction in endothelium-denuded rat aortae, and reduced the formation of GTP RhoA, with the effects being greatest for equol and phenoxodiol. Ligand displacement studies of rat uterine cytosol estrogen receptor revealed the compounds to be generally weak binders. These data are consistent with the vasorelaxation activity of equol and phenoxodiol deriving at least in part by inhibition of the RhoA/Rho-kinase pathway, and along with the limited estrogen receptor affinity supports a role for equol and phenoxodiol as useful agents for maintaining cardiovascular function with limited estrogenic effects.

Full text of DOI:10.1016/j.bmc.2012.02.008

Bioorganic & Medicinal Chemistry 20 (2012) 2353–2361
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Morpholinoisoflav-3-enes as flexible intermediates in the synthesis
of phenoxodiol, isophenoxodiol, equol and analogues: Vasorelaxant
properties, estrogen receptor binding and Rho/RhoA kinase pathway inhibition
Andrew J. Tilley a, Shannon D. Zanatta a, Cheng Xue Qin b, In-Kyeom Kim c, Young-Mi Seok c,
Alastair Stewart b, Owen L. Woodman d, Spencer J. Williams a,
⇑
a School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
b Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia
c Cardiovascular Research Institute and Department of Pharmacology, Kyungpook National University School of Medicine, Daegu, Republic of Korea
d School of Medical Sciences and Health Innovations Research Institute, RMIT University, Bundoora, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoflavone consumption correlates with reduced rates of cardiovascular disease. Epidemiological studies
and clinical data provide evidence that isoflavone metabolites, such as the isoflavan equol, contribute to
these beneficial effects. In this study we developed a new route to isoflavans and isoflavenes via 2-mor-
pholinoisoflavenes derived from a condensation reaction of phenylacetaldehydes, salicylaldehydes and
morpholine. We report the synthesis of the isoflavans equol and deoxygenated analogues, and the isofla-
venes 7,40-dihydroxyisoflav-3-ene (phenoxodiol, haganin E) and 7,40-dihydroxyisoflav-2-ene (isophe-
noxodiol). Vascular pharmacology studies reveal that all oxygenated isoflavans and isoflavenes can
attenuate phenylephrine-induced vasoconstriction, which was unaffected by the estrogen receptor
antagonist ICI 182,780. Furthermore, the compounds inhibited U46619 (a thromboxane A2 analogue)
induced vasoconstriction in endothelium-denuded rat aortae, and reduced the formation of GTP RhoA,
with the effects being greatest for equol and phenoxodiol. Ligand displacement studies of rat uterine
cytosol estrogen receptor revealed the compounds to be generally weak binders. These data are consis-
tent with the vasorelaxation activity of equol and phenoxodiol deriving at least in part by inhibition of
the RhoA/Rho-kinase pathway, and along with the limited estrogen receptor affinity supports a role for
equol and phenoxodiol as useful agents for maintaining cardiovascular function with limited estrogenic
effects.
Received 20 December 2011
Revised 26 January 2012
Accepted 2 February 2012
Available online 11 February 2012
Keywords:
Phytoestrogens
Kinase inhibitor
Vascular pharmacology
Polyphenols
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ing ability to produce 3d upon isoflavone consumption. 30–50% of
the adult human population cannot produce 3d upon isoflavone
Epidemiological studies have correlated high consumption of
leguminous foodstuffs, such as soy, with a reduced rate of cardio-
vascular disease.1 While early attention focussed on isoflavones
such as genistein and daidzein (1) as the major contributors to
the positive biological activities, in recent years focus has moved
to the metabolites formed from ingestion of these isoflavones
(Fig. 1).2 Metabolism of 1 or the related isoflavone formonetin 2
leads to the production of the isoflavan (S)-equol (3d),2 and possi-
bly the isoflavene phenoxodiol (4) (also known as dehydroequol
and haganin E).3 There is growing realization that 3d has a range
of beneficial effects on vascular function including as an antioxi-
dant and as a vasorelaxant.4 However, inter-individual variation
in intestinal microflora between humans results in a greatly vary-
consumption5 and there is evidence that equol-producer status
correlates with improved responses to isoflavone consumption in
the treatment of cardiovascular disease, menopause and osteopo-
rosis.6 Phenoxodiol itself possesses significant anticancer activities
and can act as a chemosensitizer for a variety of anticancer agents.7
Isoflavones and isoflavans possess structural similarities with
steroidal hormones such as estrogen and estradiol and are known
to bind to estrogen receptors.8 Alternative sources of estrogenic
compounds that do not possess the hormonal properties of estro-
gen are of interest for their therapeutic potential in the prevention
of cardiovascular disease, particularly in men and in postmeno-
pausal women as long term hormone-replacement therapy for pre-
vention of coronary heart disease is no longer recommended.9,10
Given the metabolism of dietary daidzein (1) to equol (3d), it is
likely that many of the cardiovascular benefits ascribed to soy
and red clover isoflavones in fact result from equol.4 A recent clin-
ical trial demonstrated that (S)-equol can alleviate hot flushes and
⇑
Corresponding author.
E-mail address: sjwill@unimelb.edu.au (S.J. Williams).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.008

2354
A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
reagents.15–18 Semi-synthetic routes to equol (3d) and phenoxodiol
(4) rely upon partial hydrogenation of daidzein (1) and are limited
in their ability to access analogues by the availability of the neces-
sary isoflavone precursors.19 Other de novo syntheses also suffer
from problems with long synthetic routes.20,21 Our attention was
drawn to the work of Dean and Varma who reported a two-step
synthesis of 2-morpholinoisoflav-3-ene (9a) in 55% overall
yield.22,23 In this approach reaction of morpholine and phenylacet-
aldehyde (5a) affords N-styrylmorpholine (6), which in a second
step reacts with salicylaldehyde (7a) (Fig. 2; upper pathway). Gi-
ven the structural similarity of such 2-morpholinoisoflav-3-enes
and isoflavans, this route seemed a promising avenue for synthesis
of equol and analogues. One-pot reaction of 5a, 7a and morpholine
afforded 9a directly in 62% yield (Fig. 2; middle pathway). This
yield is a modest improvement on that obtained through the step-
wise approach. Under these conditions it is possible that the reac-
tion proceeds by an alternative pathway benefitting from
organocatalysis by morpholine (Fig. 2; lower pathway). Thus, acti-
vation of phenylacetaldehyde by morpholine leads to 6; reaction of
morpholine with salicylaldehyde generates an iminium ion (8);
and in situ Mannich-type condensation of these reactive species,
with elimination of morpholine, affords 9a.
Application of our one-pot approach to oxygenated substrates
required the synthesis of the corresponding phenylacetaldehydes.
The oxidation of 2-arylethanols using chromium reagents has been
reported to be capricious, being complicated by carbon–carbon
bond cleavage.24 Instead, Dess–Martin oxidation of 4-benzyloxy-
phenylethanol (10) or IBX oxidation of 4-tert-butyldimethylsilyl-
oxyphenyl)ethanol (11) afforded the respective phenylacetaldehy-
des 5b and 5c quantitatively (Fig. 3). One-pot synthesis of 2-
morpholinoisoflav-3-enes 9b–d bearing electron-donating benzyl-
oxy substituents on either the phenylacetaldehyde or salicylalde-
hyde components, in the presence of 4 Å molecular sieves
proceeded in good yield (Table 1). In particular, use of 4-benzyl-
oxysalicyladehyde (7b) and 4-benzyloxyphenylacetaldehyde (5b)
Figure 1. Equol, dehydroequol and their metabolism from dietary isoflavones.
other menopausal symptoms in postmenopausal equol-non-pro-
ducing Japanese women.11 Equol can inhibit noradrenaline-in-
duced contraction of rat aorta and can relax pre-contracted
aorta12 and cerebral arteries both in vitro and in vivo.13 In addition,
equol has superior superoxide-scavenging activity to the isoflav-
ones genistein and daidzein, and can preserve NO levels, thereby
preventing oxidation of low density lipoprotein.14 Phenoxodiol
shares with daidzein the capacity to antagonize the contractile ef-
fects of noradrenaline, enhance vasodilatation and protect against
endothelium damage by oxidized low density lipoprotein, and is
comparable to 17b-estradiol in this application.12 In rat carotid
artery the vasorelaxant activity of equol is endothelium indepen-
dent13 whereas phenoxodiol causes endothelium-dependent relax-
ation of rat aorta.12 In the present study we sought to develop a
new route to equol and phenoxodiol allowing the synthesis of
structural analogues, and to investigate structure–activity relation-
ships for effective vasodilatation and the mechanism of vasodilata-
tion compared to the hormone estrogen. As previous vascular
studies with isoflavones have highlighted the importance of the
presence of hydroxyl groups on the isoflavone core, this study fo-
cussed on the importance of hydroxylation on activity.
2. Results and discussion
2.1. Chemical synthesis
Published routes to homochiral isoflavan derivatives suffer from
lengthy routes and the requirement for air and moisture-sensitive
Figure 3. Preparation of phenylacetaldehydes.
Figure 2. Upper pathway: Two-step synthesis of 2-morpholinoisoflav-3-ene 9a. Middle pathway: one-pot synthesis of 9a. Lower pathway: proposed mechanism of one-pot
approach.

A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
2355
Table 1
Synthesis of 2-morpholinoisoflav-3-enes
7a X = H
7a X = H
7b X = OBn
7b X = OBn
7c X = OTBS
5a Y = H
5b Y = OBn
5a Y = H
5b Y = OBn
5c Y = OTBS
9a X = Y = H
62%
58%
61%
80%
30%
9b X = H, Y = OBn
9c X = OBn, Y = H
9d X = Y = OBn
9e X = Y = OTBS
afforded the 40,7-dibenzyloxyisoflavan (9d) in excellent yield
(80%), isolated by recrystallization from the crude reaction mix-
ture. On the other hand, condensation of the tert-butyldimethylsi-
lyl protected salicylaldehyde (7c) and phenylacetaldehyde (5c)
afforded the 40,7-di-tert-butyldimethylsilyl isoflavan (9e) in low
yield (30%), and required the omission of molecular sieves,
most likely owing to the acid sensitivity of the silyl protecting
groups.
for the reduction of the oxygenated congeners, affording mixtures
of the isoflav-2-enes and -3-enes 12b–d in good yields with isola-
tion effected by recrystallization. Reduction of the isoflavene mix-
tures with H2 and Pd–C or Pd(OH)2–C effected double bond
saturation and, in the cases of 12b–d, protecting group removal,
affording the isoflavans 3a–d in good yields (Table 2). Notably, in
the case of equol (3d) the product was purified simply by
recrystallization.
Reduction of the hemiaminal of the 2-morpholinoisoflav-3-ene
(9a) by Pd-catalyzed hydrogenolysis met with only limited suc-
cess.22 By contrast, ionic hydrogenation of 9a, via an intermediate
isoflavylium ion,25 proved more effective. While this transforma-
tion could be effected by warming 9a in formic acid in pyridine26
to give 12a, a mixture of isoflav-2-ene and 3-ene (83%, 1:2), this
procedure was less successful on the oxygenated analogues. Alter-
natively, the reduction could be achieved by treatment with trieth-
ylsilane and BF3ꢀEt2O affording 86% of a 1:4 mixture of the 2-ene
and 3-ene 12a (Table 2). These latter conditions proved effective
Initially, it was anticipated that phenoxodiol (4) and isophe-
noxodiol (15) could be prepared from the isomeric benzyl-pro-
tected isoflavenes 9d. However, these compounds possessed poor
solubility in organic solvents suitable for chromatographic resolu-
tion. Instead, tert-butyldimethylsilyl protecting groups were inves-
tigated with the aim of improving their organic solubility.
Reduction of 9e using triethylsilane and BF3ꢀEt2O afforded a readily
separable mixture of the 2-ene 13 and 3-ene 14 (1:5) (Fig. 4). Final-
ly, individual deprotection of each isomer with HFꢀpyridine affor-
ded 15 and 4.
Table 2
Synthesis of isoflavans
9a X = Y = H
12a X = Y = H
1:4
1:4
1:3
1:3
86%
80%
74%
80%
3a X = Y = H
86%
63%
71%
75%
9b X = H, Y = OBn
9c X = OBn, Y = H
9d X = Y = OBn
12b X = H, Y = OBn
12c X = OBn, Y = H
12d X = Y = OBn
3b X = H, Y = OH
3c X = OH, Y = H
3d X = Y = OH
Dashed bonds indicate the presence of two regioisomeric alkenes, in the ratio shown in the table.
Figure 4. Synthesis of 7,40-dihydroxyisoflav-2-ene (isophenoxodiol) 15 and -3-ene (phenoxodiol) 4.

2356
A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
Figure 5. Effect of equol and analogues on tension in rat isolated aortae precon-
tracted with phenylephrine (n = 3–5). Error bars represent standard error mean.
2.2. Vascular activity
Vasorelaxant activity was assessed by measuring inhibition of
contraction of rat thoracic aorta induced by phenylephrine (PE),
as it has been shown that isoflavones can act as functional antag-
onists of PE.27 Equol (3d), phenoxodiol (4), isophenoxodiol (15)
and both 7-hydroxy (3c) and 40-hydroxy (3b) isoflavans caused
concentration-dependent relaxation of rat isolated aortae, whereas
isoflavan (3a) caused only contraction at high concentrations
(Fig. 5). The responses to each of the compounds were unaffected
by the estrogen receptor antagonist ICI 182,780 (0.1 lM, data not
shown), demonstrating that the vasorelaxant activity is estrogen-
receptor independent.
The isoflavones daidzein (1) and genistein attenuate vascular
contraction through inhibition of the RhoA/Rho-kinase signalling
pathway.28 In order to investigate attenuation of vascular contrac-
tion by this pathway, we studied the effects of the isoflavans and
isoflavenes in endothelium-denuded rat aortic rings. After pre-
treatment with the isoflavenes or isoflavans, or vehicle for
Figure 6. (A) Inhibitory effect of isoflavans and an isoflavene on U46619-induced
vasoconstriction in rat aorta (n = 2). All experiments including vehicle contained
30 nM U46619. (B) Effect of compounds on U46619-induced increase in GTP-RhoA
(n = 3). Error bars represent standard error mean. ꢃ significance of p <0.01 (ANOVA,
Dunnett’s post test) relative to vehicle plus U46619.
30 min, the addition of 11,9-epoxymethano-prostaglandin F2
(U46619), a thromboxane A2 analog, increased vascular tension,
which was inhibited by pretreatment with 100 M of each of the
compounds (Fig. 6A). We utilized 300 M flavone as a positive con-
a
l
l
uterine cytosol preparations was isophenoxodiol (15) > phenoxo-
diol (4) > equol (3d) > 7-OH isoflavan (3c) ꢁ 4-OH-isoflavan
(3b) ꢂ isoflavan (3a) (Table 3). Affinities for the ER were several
orders of magnitude lower than for the ER antagonist ICI
182,780. It is well established that the presence and distance be-
tween the hydroxyls of estradiol and analogues are key determi-
nants of ER binding affinity.8 This data is in accord with these
established trends, with the dihydroxy compounds isophenoxodiol
(15), phenoxodiol (4) and equol (3d) possessing the highest poten-
cies, the mono-hydroxy isoflavans 3b and 3c showing less affinity,
and isoflavan (3a), without hydroxy substituents, having no
trol as this compound has previously been shown to be an inhibitor
of U46619-induced vasoconstriction.29 Limited inhibition of con-
traction was seen for isoflavan (3a) and the monohydroxylated
isoflavans 3b and 3c, moderate inhibition for equol (3d), and the
greatest effect was seen for phenoxodiol (4).
In order to examine the effect of the compounds on the RhoA/
Rho-kinase pathway, we examined the levels of guanosine triphos-
phate (GTP)-RhoA. In its active GTP-bound form, RhoA activates
Rho-kinase, which then phosphorylates and inactivates the down-
stream target myosin light chain (MLC) phosphatase.30 The in-
crease in GTP-RhoA over control therefore serves as a marker of
Rho-kinase activation. The rank order of inhibition of U46619-in-
duced GTP RhoA largely follows the trend in inhibition of
U46619-induced vasoconstriction (Fig. 6A). Limited inhibition is
seen for isoflavans 3b and 3c, moderate inhibition for isoflavan
(3a) and equol (3d), with the greatest effect being seen for phe-
noxodiol (4).
detectable affinity at 10 lM. The affinity of equol (3d) determined
here is similar to that previously reported for rat uterine cytosol
preparations.31
3. Conclusion
We have described an exceptionally simple method for the syn-
thesis of equol 3d and related isoflavans 3a–c. This method was
used to synthesize the isoflavenes phenoxodiol (4) and isophe-
noxodiol 15. The key step is a hybrid organocatalytic three-compo-
nent coupling wherein morpholine acts both as an organocatalyst
in a Mannich-type reaction, and stoichiometrically, affording 2-
morpholinoisoflav-3-enes, which can be reduced to isoflavene
mixtures, and thence to the isoflavans.
2.3. Displacement of [3H]-estradiol binding
In order to assess the potential hormonal properties of equol
and analogues we examined their ER affinity using estrogen recep-
tor isolated from rat uterine cytosol (Fig. 7). The rank order of po-
tency of the analogues in displacing [3H]-estradiol binding from rat

A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
2357
benefits with reduced side-effects. However, variability in metabo-
lism between individuals suggests that administration of metabo-
lites or their structural variants may prove a more reliable
therapeutic approach. This study shows that structural variation
of the metabolic products of isoflavones can tune their vascular
activities, and therefore that modified isoflavans and/or isoflavenes
may prove to be alternative agents for maintaining cardiovascular
function while limiting direct hormonal activities.
4. Experimental
4.1. General chemical methods
All solvents were distilled prior to use. Petroleum spirits refers
to the fraction boiling range 40–60 °C. Thin layer chromatography
(t.l.c.) was performed on aluminium sheets pre-coated with Merck
Silica Gel 60, using mixtures of EtOAc/petroleum spirits and EtOAc/
petroleum spirits/Et3N. Detection was achieved via irradiation by
UV light. Flash chromatography was performed using the method
of Still et al. using Scharlau Silica Gel 60.38 NMR data was recorded
on Varian Inova 400 and 500 instruments. Chemical shifts are ex-
pressed in parts per million (d) using residual solvent (1H NMR d
7.26 ppm for CDCl3,
d d
2.50 ppm for DMSO-d6; 13C NMR
77.36 ppm for CDCl3, 39.52 ppm for DMSO-d6) or TMS as an inter-
nal standard (d 0.00 ppm), and J values are reported in Hz. Melting
points were obtained using a Riechert-Jung hot-stage melting
point apparatus and are corrected. High resolution electrospray
ionization mass spectra were obtained on a Finnigan LTQ FT mass
spectrometer (The University of Melbourne). Microanalysis was
performed by CMAS (Belmont, Victoria).
Figure 7. [3H]-Estradiol displacement by the ER antagonist, ICI 182,780 and by
isoflavans and isoflavenes in rat uterine cytosol estrogen receptor (n = 3). Error bars
represent standard error mean.
Table 3
LogIC50 values for displacement of [3H]-estradiol in rat uterine
cytosol estrogen receptor by isoflavans and isoflavenes (n = 3)
4.2. Chemical synthesis
Compound
LogIC50 mean SEM
4.2.1. 2-Morpholinoisoflav-3-ene (9a)
Equol (3d)
5.78 0.11
6.18 0.13
6.63 0.13
>5
5.12 0.10
5.44 0.10
8.23 0.12
Phenoxodiol (4)
Isophenoxodiol (15)
Isoflavan (3a)
4-OH isoflavan (3b)
7-OH isoflavan (3c)
ICI 182,780
A mixture of phenylacetaldehyde 5a (0.29 mL, 2.5 mmol), sali-
cylaldehyde 7a (0.27 mL, 2.5 mmol), morpholine (0.22 mL,
2.5 mmol) and 4 Å molecular sieves in toluene (2 mL) was stirred
at reflux for 24 h. The mixture was filtered and the filtrate diluted
with toluene (15 mL). The organic phase washed with 1 M NaOH
(5 ꢄ 10 mL), satd aq NaHCO3 (4 ꢄ 10 mL) and brine (3 ꢄ 10 mL).
The organic phase was dried (MgSO4), filtered and concentrated
under reduced pressure to give a light brown crystalline solid.
The solid was recrystallized to afford compound 9a as a yellow
crystalline solid (0.457 g, 62%), mp 101–103 °C (EtOAc/petroleum
spirits; lit.22 105–106 °C). 1H-NMR (400 MHz, CDCl3) d 2.65 (dt,
2H, 3J = 4.6, 2J = 11.8, CH2NCH2), 3.10 (dt, 2H, 3J = 4.6, 2J = 11.9,
CH2NCH2), 3.63 (t, 4H, J = 4.8, CH2OCH2), 5.78 (s, 1H, H2), 6.91–
6.96 (m, 2H, H30,50), 7.04–7.46 (m, 6H, Ar), 7.70–7.73 (m, 2H,
H20,60). 13C-NMR (500 MHz, CDCl3) d 47.1, 67.2, 90.7, 114.9,
120.8, 121.2, 123.5, 126.1, 127.1, 127.8, 128.3, 128.5, 129.7,
137.7, 153.7.
All mono- and dihydroxylated isoflavans and isoflavenes caused
concentration-dependent relaxation of rat isolated aortae, whereas
the unsubstituted isoflavan caused only contraction at high con-
centrations. Vasorelaxation was ER-independent, as demonstrated
by the lack of effect of the ER alpha and beta antagonist ICI 182,780
on vasorelaxant responses to the isoflavenes and isoflavans. RhoA/
Rho-kinase activation plays an important role in the tonic compo-
nent of U46619-induced contraction and MLC20 phosphoryla-
tion.32,33 U46619 activates RhoA, and was inhibited by both
equol and phenoxodiol. ER receptor binding assays revealed mod-
erate affinities for the dihydroxylated compounds equol, phenoxo-
diol and isophenoxodiol. This study suggests that the vascular
activity of the most potent analogues, equol, phenoxodiol and
isophenoxodiol, is ER independent and derives, at least in part,
from inhibition of the RhoA/Rho-kinase pathway. In this context
it is noteworthy that the vasorelaxant activity of other polycyclic
aromatic compounds such as the isoflavones daidzein and gene-
stein,28 the flavones flavone29 and 30,40-dihydroxyflavonol,34 and
the polyphenols honokiol,35 glyceollin I36 and gomisin A37 derive
in part or wholly from inhibition of the RhoA/Rho-kinase pathway.
Dietary isoflavones and their metabolites may have potential
benefits in the maintenance of human health. Owing to the risks
of long-term administration of estrogen therapy, phytoestrogens
have emerged as a possible alternative that may provide similar
4.2.2. Mixture of isoflav-2-ene and isoflav-3-ene (12a)
BF3ꢀEt2O (1.12 mL, 8.80 mmol) was added dropwise to a stirred
solution of 2-morpholinoisoflav-3-ene 9a (1.17 g, 4.00 mmol) and
Et3SiH (1.28 mL, 8.00 mmol) in CH2Cl2 (20 mL) at 0 °C. After
30 min, the reaction mixture was allowed to warm to room tem-
perature and stirring was continued for an additional 22 h. The
reaction was quenched with satd aq NaHCO3 (20 mL), diluted with
CH2Cl2 (20 mL) and the organic phase was separated and washed
with satd aq NaHCO3 (3 ꢄ 30 mL), brine (3 ꢄ 30 mL) and the com-
bined aqueous layers re-extracted with additional CH2Cl2 (50 mL).
The combined organic layers were dried (MgSO4), filtered and con-
centrated under reduced pressure to give a yellow solid. The yel-
low solid was purified by passing through a plug of silica (20%

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A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
EtOAc/petroleum spirits + 1% NEt3) to afford a crystalline yellow
powder (0.718 g, 86%). A small portion was recrystallized from
EtOAc/petroleum spirits to afford a mixture of isoflav-2-ene and -
3-ene 12a (1:4) as
(500 MHz, CDCl3) d 3.79 (s, 2H, H4,4, 2-ene), 5.18 (d, 2H, J2,4 1.4,
organic layers were dried (MgSO4), filtered and concentrated under
reduced pressure to give a light yellow solid, which was recrystal-
lized from EtOAc/petroleum spirits to afford a mixture of isoflav-2-
ene and -3-ene 12b (1:4) as a colourless solid (0.277 g, 80%). Partial
1H NMR (500 MHz, CDCl3) d 3.75 (s, 2H, H4,4, 2-ene), 5.14 (d, 2H,
J2,4 = 1.4, H2,2, 3-ene).
a
colourless powder. Partial 1H NMR
H2,2, 3-ene).
4.2.3. 3-Phenylchroman (3a)
4.2.7. 3-(40-Hydroxyphenyl)chroman (3b)
An isomeric mixture of isoflavenes 12a (157 mg, 0.754 mmol)
and Pd(OH)2 (20 mg) in EtOAc (5 mL) was treated with H2 at atmo-
spheric pressure for 28 h. The mixture was filtered (Celite), and the
filtrate concentrated under reduced pressure to give a yellow oil.
The yellow oil was recrystallized to afford the chroman 3a as a col-
ourless solid (135 mg, 86%), mp 52–54 °C (petroleum spirits; lit.22
53–54 °C). 1H NMR (400 MHz, CDCl3) d 2.97–3.10 (m, 2H, H4,4),
3.19–3.28 (m, 1H, H3), 4.03 (t, 1H, J = 10.6, H2), 4.27–4.33 (m,
1H, H2), 6.83–6.89 (m, 2H, H30,50), 7.06–7.14 (m, 2H, Ar), 7.21–
7.28 (m, 3H, H20,40,60), 7.29–7.37 (m, 2H, Ar). 13C NMR (500 MHz,
CDCl3) d 32.6, 38.8, 71.0, 116.7, 120.5, 122.1, 127.2, 127.5, 127.6,
128.9, 129.9, 141.5, 154.5.
The isomeric mixture of isoflavenes 12b (126 mg, 0.400 mmol),
Pd/C (25 mg) and AcOH (0.1 mL) in 1:1 THF/EtOAc (10 mL) was
treated with H2 (10 atm) for 24 h. The mixture was filtered (Celite),
and the filtrate concentrated under reduced pressure to give a light
brown solid which was purified by flash chromatography (20%
EtOAc/petroleum spirits + 1% AcOH), affording 3b as a colourless
solid (57 mg, 63%), mp 102–103 °C (EtOAc/petroleum spirit; lit.39
120–122 °C); (C15H14O2 requires C, 79.6; H, 6.2. Found: C, 79.5;
H, 6.2). 1H NMR (500 MHz, CDCl3) d 2.96–3.06 (m, 2H, H4,4),
3.17–3.23 (m, 1H, H3), 3.99 (t, 1H, J = 10.6, H2), 4.31–4.34 (m,
1H, H2), 4.78–4.80 (br s, 1H, OH), 6.81–6.89 (m, 4H, H30,50,Ar),
7.08–7.15 (m, 4H, H20,60,Ar). 13C NMR (500 MHz, CDCl3) d 32.7,
37.9, 71.2, 115.8, 116.7, 120.5, 122.2, 127.6, 128.7, 129.9, 133.7,
154.4, 154.7. HRMS (ESI+) m/z 249.0886 (C15H14NaO2 [M+Na]+ re-
quires 249.0891).
4.2.4. 4-Benzyloxyphenylacetaldehyde (5b)
Dess–Martin periodinane (1.103 g, 2.60 mmol) was added to a
solution of 2-(4-benzyloxyphenyl)ethanol (0.457 g, 2.00 mmol) in
CH2Cl2 (15 mL) at room temperature, and the resultant mixture al-
lowed to stir for 0.5 h. A 1:1 mixture of Na2S2O3 (1.5 M) and satd aq
NaHCO3 (30 mL) was added and the mixture stirred until two clear
layers formed. The organic phase was taken up in ether (20 mL),
washed with water (3 ꢄ 20 mL) and brine (3 ꢄ 20 mL), dried
(MgSO4), filtered and concentrated under reduced pressure to af-
ford the crude aldehyde 5b as a clear yellow oil which was used
in the subsequent step without purification.
4.2.8. 7-Benzyloxy-2-morpholinoisoflav-3-ene (9c)
A mixture of phenylacetaldehyde 5a (0.18 mL, 1.5 mmol), 4-
benzyloxysalicylaldehyde 7b (0.343 g, 1.50 mmol), morpholine
(0.13 mL, 1.5 mmol) and 4 Å molecular sieves in toluene (2.0 mL)
was stirred at reflux for 24 h. The mixture was filtered and the fil-
trate was diluted with toluene (25 mL). The organic phase was
washed with 1 M NaOH (4 ꢄ 20 mL), satd aq NaHCO3 (4 ꢄ 20 mL)
and brine (2 ꢄ 20 mL). The organic phase was dried (MgSO4), fil-
tered and concentrated under reduced pressure to give a yellow/
brown crystalline solid. The solid was recrystallized to afford 9c
as a light-brown crystalline solid (0.37 g, 61%), mp 143–144 °C
(EtOAc/petroleum spirits); (C26H25NO3 requires C, 78.2; H, 6.3; N,
3.5. Found: C, 78.2; H, 6.4; N, 3.5). 1H NMR (400 MHz, CDCl3) d
2.65 (dt, 2H, 3J = 4.8, 2J = 11.9, CH2NCH2), 3.05 (dt, 2H, 3J = 4.5,
2J = 12.1, CH2NCH2), 3.62 (t, 4H, J = 4.8, CH2OCH2), 5.06 (d, 1H,
J = 11.4, CH2Ph), 5.09 (d, 1H, J = 11.4, CH2Ph), 5.75 (s, 1H, H2),
6.55–6.60 (m, 2H, H30,50), 7.06–7.07 (m, 2H, Ar), 7.27–7.47 (m,
8H, Ar), 7.68 (d, 2H, J = 8.4, H20,60). 13C NMR (500 MHz, CDCl3) d
47.2, 67.4, 70.3, 91.1, 101.4, 108.1, 115.1, 123.3, 125.8, 126.0,
127.6, 127.7, 128.0, 128.2, 128.6, 128.7, 136.9, 138.0, 155.2,
160.5. HRMS (ESI+) m/z 422.1727 (C26H25NNaO3 [M+Na]+ requires
422.1732).
4.2.5. 40-Benzyloxy-2-morpholinoisoflav-3-ene (9b)
A mixture of salicylaldehyde 7a (0.21 mL, 2.0 mmol), morpho-
line (0.18 mL, 2.0 mmol), 2-(4-benzyloxyphenyl)acetaldehyde 5b
(0.453 g, 2.00 mmol) and 4 Å molecular sieves in toluene (10 mL)
was stirred at reflux for 24 h. The mixture was cooled, filtered
and the filtrate was diluted with toluene (25 mL). The organic
phase was washed with 1 M NaOH (4 ꢄ 25 mL), satd aq NaHCO3
(4 ꢄ 25 mL) and brine (2 ꢄ 25 mL). The organic extract was dried
(MgSO4), filtered and concentrated under reduced pressure to af-
ford the isoflavene 9b as an off-white crystalline solid (0.460 g,
58%), mp 134–136 °C (EtOAc/petroleum spirit); (C26H25NO3 re-
quires C, 78.2; H, 6.3; N, 3.5. Found: C, 78.2; H, 6.3; N, 3.6). 1H
NMR (500 MHz, CDCl3) d 2.63 (dt, 2H, 3J = 4.4, 2J = 11.8, CH2NCH2),
3.05 (dt, 2H, 3J = 4.4, 2J = 12.0, CH2NCH2), 3.61 (t, 4H, J = 5.1,
CH2OCH2), 5.11 (s, 2H, CH2Ph), 5.73 (s, 1H, H2), 6.90–6.94 (m,
2H, Ar), 6.98–7.01 (m, 3H, H4,30,50), 7.11 (dd, 1H, J = 1.6, J = 7.4,
Ar), 7.15–7.19 (m, 1H, Ar), 7.33–7.46 (m, 5H, Ph), 7.64 (d, 2H,
J = 9.0, H20,60). 13C NMR (500 MHz, CDCl3) d 47.2, 67.6, 70.2,
90.80, 90.81, 115.0, 120.9, 121.5, 122.1, 127.0, 127.5, 128.0,
128.1, 128.2, 128.8, 129.5, 130.7, 137.0, 153.7, 158.8. HRMS
(ESI+) m/z 400.1906 (C26H26NO3 [M+H]+ requires 400.1913).
4.2.9. Mixture of 7-benzyloxyisoflav-2-ene and 7-benzyloxyiso-
flav-3-ene (12c)
BF3ꢀEt2O (0.28 mL, 2.2 mmol) was added dropwise to a stirred
solution of 7-benzyloxy-2-morpholinoisoflav-3-ene 9c (0.398 g,
0.996 mmol) and Et3SiH (0.32 mL, 2.0 mmol) in CH2Cl2 (7 mL) at
0 °C. After 30 min, the reaction mixture was allowed to warm to
room temperature and stirring was continued for an additional
21 h. The reaction was quenched with satd aq NaHCO3 (10 mL), di-
luted with CH2Cl2 (20 mL) and the organic phase was separated
and washed with satd aq NaHCO3 (3 ꢄ 25 mL), brine (2 ꢄ 25 mL)
and the combined aqueous layers re-extracted with additional
CH2Cl2 (50 mL). The combined organic extract was dried (MgSO4),
filtered and concentrated under reduced pressure to give a yellow
solid. The yellow solid was purified by passing through a plug of
silica (20% EtOAc/petroleum spirits + 1% NEt3) to afford a mixture
of isoflav-2-ene and -3-ene 12c (1:3) as a yellow powder (0.23 g,
74%). Partial 1H NMR (500 MHz, CDCl3) d 3.72 (s, 2H, H4,4, 2-
ene), 5.15 (d, 2H, J2,4 1.4, H2,2, 3-ene).
4.2.6. Mixture of 40-benzyloxyisoflav-2-ene and 40-benzyloxyiso-
flav-3-ene (12b)
BF3ꢀEt2O (0.31 mL, 2.4 mmol) was added dropwise to a stirred
solution of 40-benzyloxy-2-morpholinoisoflav-3-ene 9b (0.439 g,
1.10 mmol) and Et3SiH (0.35 mL, 2.2 mmol) in CH2Cl2 (10 mL) at
0 °C. After 30 min, the reaction mixture was allowed to warm to
room temperature and stirring was continued for an additional
16 h. The reaction was quenched with satd aq NaHCO3 (10 mL), di-
luted with CH2Cl2 and the organic phase was washed with satd aq
NaHCO3 (3 ꢄ 25 mL), brine (2 ꢄ 25 mL) and the combined aqueous
layers re-extracted with additional CH2Cl2 (30 mL). The combined

A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
2359
4.2.10. 7-Hydroxy-3-phenylchroman (3c)
water; lit.19 158–160 °C). 1H NMR (500 MHz, DMSO-d6) d2.74–2.86
(m, 2H, H4,4), 2.98–3.04 (m, 1H, H3), 3.89 (t, 1H, J = 10.5, H2), 4.13–
4.15 (m, 1H, H2), 6.19 (d, 1H, J6,8 = 2.2, H8), 6.28 (dd, 1H, J5,6 = 8.2,
An isomeric mixture of isoflavenes 12c (157 mg, 0.499 mmol)
and Pd(OH)2 (25 mg) and AcOH (0.1 mL) in 1:1 THF/EtOAc (5 mL)
was treated with H2 (10 atm) for 24 h. The mixture was filtered
(Celite), and the filtrate was concentrated under reduced pressure
to give a colourless solid. The solid was recrystallized to afford the
chroman 3c as a colourless powder (80 mg, 71%), mp 132–134 °C
(EtOAc/petroleum spirits; lit.40 130 °C); (C15H14O2 requires C,
79.6; H, 6.2. Found: C, 79.8; H, 6.4). 1H NMR (500 MHz, CDCl3) d
2.92–3.02 (m, 2H, H4,4), 3.19–3.25 (m, 1H, H3), 4.02 (t, 1H,
J = 10.6, H2), 4.32–4.35 (m, 1H, H2), 4.63–4.88 (br s, 1H, OH),
6.37–6.41 (m, 2H, Ar), 6.95 (d, 1H, J = 8.2, H5), 7.24–7.37 (m, 3H,
Ar), 7.34–7.37 (m, 2H, Ar). 13C NMR (500 MHz, CDCl3) d 31.9,
38.9, 71.0, 103.4, 108.2, 114.5, 127.2, 127.5, 128.9, 130.6, 141.5,
155.1, 155.2. HRMS (ESI+) m/z 227.1067 (C15H15O2 [M+H]+ requires
227.1072).
J6,8 = 2.3, H6), 6.71 (d, 2H, J2 ,3 = 8.4, H30,50), 6.86 (d, 1H, J5,6 = 8.2,
0
0
H5), 7.10 (d, 2H, J2 ,3 = 8.4, H20,60), 9.00–9.56 (br s, 2H, OH). 13C
NMR (500 MHz, DMSO-d6) d 31.3, 37.1, 70.2, 102.5, 108.0, 112.5,
115.2, 128.3, 130.0, 131.6, 154.5, 156.1, 156.6.
0
0
4.2.14. 2-(4-tert-Butyldimethylsiloxyphenyl)acetaldehyde (5c)
A mixture of IBX (1.31 g, 4.69 mmol) and 2-(4-tert-butyldim-
ethylsilyloxyphenyl)ethanol (11)41 (847 mg, 3.35 mmol) in DMSO
(8 mL) was stirred at rt for 4.5 h. EtOAc (20 mL) was then added
followed by 1.5 M Na2S2O3 (10 mL) and satd NaHCO3 (10 mL).
The aqueous layer was extracted with EtOAc (10 mL ꢄ 2) and the
combined organic extracts washed with water (2 ꢄ 10 mL), brine
(10 mL), dried (MgSO4) and concentrated. The crude 2-(4-tert-
butyldimethylsiloxyphenyl)acetaldehyde 5c was used without
purification (734 mg, 87%). 1H NMR (400 MHz, CDCl3) d 0.20 (s,
12H, Si(CH3)2(C(CH3)3)), 0.98 (s, 9H, Si(CH3)2(C(CH3)3)), 3.61 (d,
2H, J = 2.8, CH2), 6.83 (app. d, 2H, J = 8.4, Ar-H), 7.07 (app. d, 2H,
J = 8.4, Ar-H), 9.71 (t, 1H, J = 2.8, CHO).
4.2.11. 40,7-Dibenzyloxy-2-morpholinoisoflav-3-ene (9d)
A mixture of morpholine (0.14 mL, 1.5 mmol), 2-(4-benzyloxy-
phenyl)acetaldehyde 5b (0.339 g, 1.50 mmol), 4-benzyloxysalicyl-
aldehyde 7b (0.342 g, 1.50 mmol) and 4 Å molecular sieves in
toluene (10 mL) was stirred at reflux for 20 h. The mixture was fil-
tered and the filtrate was diluted with toluene. The organic phase
was washed with 1 M NaOH (4 ꢄ 30 mL), satd aq NaHCO3
(3 ꢄ 30 mL) and brine (30 mL). The organic phase was dried
(MgSO4), filtered and concentrated under reduced pressure to give
a light brown solid. The solid was recrystallized to afford 9d as a
light brown crystalline powder (0.606 g, 80%), mp 162–164 °C
(EtOAc/petroleum spirits); (C33H31NO4 requires C, 78.4; H, 6.2; N,
2.8. Found: C, 78.4; H, 6.1; N, 2.7). 1H NMR (500 MHz, CDCl3) d
2.64 (dt, 2H, 3J = 4.5, 2J = 11.8, CH2NCH2), 3.05 (dt, 2H, 3J = 4.6,
2J = 11.9, CH2NCH2), 3.61 (t, 4H, J = 4.8, CH2OCH2), 5.05 (d, 1H,
J = 11.5, CH2Ph), 5.08 (d, 1H, J = 11.6, CH2Ph), 5.10 (s, 2H, CH2Ph),
5.70 (s, 1H, H2), 6.56 (dd, 1H, J6,8 = 2.5, J5,6 = 8.3, H6), 6.59 (d, 1H,
J6,8 = 2.5, H8), 6.95 (s, 1H, H4), 6.98 (d, 2H, J = 9.0, H30,50), 7.02 (d,
1H, J = 8.3, H5), 7.32–7.45 (m, 10H, Ph). 13C NMR (500 MHz, CDCl3)
d 31.1, 47.2, 67.4, 70.2, 70.3, 91.2, 101.4, 108.0, 115.0, 115.2, 121.8,
125.4, 127.3, 127.6, 127.7, 127.8, 128.2, 128.7, 128.8, 130.9, 136.9,
137.1, 154.9, 158.5, 160.3. HRMS (ESI+) m/z 506.2324 (C33H32NO4
[M+H]+ requires 506.2331).
4.2.15. 40,7-Di-tert-butyldimethylsilyloxy-2-morpholinoisoflav-
3-ene (9e)
A solution of morpholine (252 mg, 2.93 mmol), 4-[(tert-butyldi-
methylsilyl)oxy]-2-hydroxybenzaldehyde
(5c)42
(729 mg,
2.93 mmol) and 2-(4-tert-butyldimethylsiloxyphenyl) acetalde-
hyde (734 mg, 2.93 mmol) in toluene (10 mL) was heated at reflux
for 18 h. The reaction was then cooled to rt, filtered (Celite) and
concentrated. 40,7-Di-tert-butyldimethylsilyloxy-2-morpholinoi-
soflav-3-ene 9e was obtained as a brown oil after flash chromatog-
raphy (10% EtOAc/petroleum spirits + 1% triethylamine) (483 mg,
30%). 1H NMR (400 MHz, CDCl3) d 0.23 (s, 12H, Si(CH3)2(C(CH3)3) ꢄ
2), 1.00 (s, 9H, Si(CH3)2(C(CH3)3)), 1.01 (s, 9H, Si(CH3)2(C(CH3)3),
2.64 (m, 2H, CH2NCH2), 3.05 (m, 2H, CH2OCH2), 3.62 (t, 4H,
J = 4.0, CH2OCH2), 5.68 (s, 1H, H2), 6.41 (dd, 1H, J5,6 = 10, J6,8 = 2.4,
H6), 6.45 (d, 1H, J6,8 = 2.4, H8), 6.84 (app. d, 2H, J = 8.6, H30,50),
6.94 (s, 1H, H4), 6.97 (d, 1H, J5,6 = 10, H5), 7.55 (app. d, 2H, J =
8.6, H20,60). 13C NMR (100 MHz, CDCl3) d ꢅ4.4, ꢅ0.02, 18.2, 25.5,
25.7, 47.0, 67.2, 90.9, 106.6, 112.9, 115.5, 120.0, 126.7 121.7,
127.1, 127.4, 131.1, 154.5, 155.3, 156.9.
4.2.12. Mixture of 40,7-dibenzyloxyisoflav-2-ene and 40,7-diben-
zyloxyisoflav-3-ene (12d)
4.2.16. 40,7-Di-tert-butyldimethylsilyloxyisoflav-2-ene (13) and
40,7-di-tert-butyldimethylsilyloxyisoflav-3-ene (14)
BF3ꢀEt2O (0.17 mL, 1.3 mmol) was added dropwise to a stirred
solution of 40,7-dibenzyloxy-2-morpholinoisoflav-3-ene 9d
(0.290 g, 0.574 mmol) and Et3SiH (0.19 mL, 1.2 mmol) in CH2Cl2
(10 mL) at 0 °C. After 30 min, the reaction mixture was allowed
to warm to room temperature and stirring was continued for an
additional 15 h. The reaction was quenched with satd aq NaHCO3
(15 mL), diluted with CH2Cl2 (20 mL) and the organic phase
washed with satd aq NaHCO3 (3 ꢄ 25 mL), brine (3 ꢄ 25 mL) and
the combined aqueous layers re-extracted with additional CH2Cl2
(50 mL). The combined organic layers were dried (MgSO4), filtered
and concentrated under reduced pressure to give a light brown so-
lid, which was recrystallized from THF/petroleum spirits to afford a
mixture of isoflav-2-ene and -3-ene 12d (1:4) as a light brown
powder (0.199 g, 80%). Partial 1H NMR (500 MHz, CDCl3) d 3.67
(s, 2H, H4,4, 2-ene), 5.11 (d, 2H, J2,4 = 1.4, H2,2, 3-ene).
BF3ꢀOEt2 (849
of 40,7-di-tert-butyldimethylsilyloxy-2-morpholinoisoflav-3-ene
9e (1.68 g 3.03 mmol) and triethylsilane (970 L, 6.07 mmol) in
lL, 6.67 mmol) was added dropwise to a solution
l
CH2Cl2 (50 mL) at 0 °C. The resulting mixture was stirred at rt for
18 h. The mixture was diluted with EtOAc (10 mL) and water
(10 mL) and the aqueous layer was extracted with EtOAc (2 ꢄ 10
mL) and the combined organic extracts washed with water
(2 ꢄ 10 mL), brine (10 mL), dried (MgSO4) and concentrated. 40,7-
Di-tert-butyldimethylsilyloxyisoflav-2-ene 13 was obtained as a
colourless solid after flash chromatography (10% CH2Cl2/petroleum
spirits) (288 mg) and recrystallisation from EtOAc/petroleum spir-
its (78 mg, 5%); mp 89 °C; (C27H40O3Si2 requires C, 69.18; H, 8.60.
Found C, 69.30; H, 8.67). 1H NMR (500 MHz, CDCl3) d 0.216 (s,
6H, Si(CH3)2(C(CH3)3), 0.22 (s, 6H, Si(CH3)2(C(CH3)3), 1.00 (s, 9H,
Si(CH3)2(C(CH3)3), 1.004 (s, 9H, Si(CH3)2(C(CH3)3), 3.67 (s, 2H,
H4), 6.45 (d, 1H, J6,8 = 2.5, H8), 6.55 (dd, 1H, J5,6 = 10, J6,8 = 2.5,
H6), 6.48 (app. d, 2H, J5,6 = 9.0, H30,50), 6.91 (t, 1H, J = 1.5, H2),
6.99 (d, 1H, J5,6 = 8.5, H5), 7.25 (app. d, 2H, J5,6 = 9.0, H20,60). 13C
NMR (125 MHz, CDCl3) d ꢅ4.5, ꢅ4.4, ꢅ0.01, 18.2, 25.7, 25.8,
107.6, 112.4, 115.5, 120.1, 125.4, 129.6, 130.8, 136.7, 151.0,
4.2.13. Equol (3d)
An isomeric mixture of isoflavenes 12d (180 mg, 0.428 mmol),
Pd(OH)2 (50 mg) and AcOH (0.1 mL) in 1:1 THF/EtOAc (10 mL)
was treated with H2 (10 atm) for 36 h. The mixture was filtered
(Celite), and the filtrate concentrated under reduced pressure to
give a light brown solid, which was recrystallized to afford racemic
equol 3d as a off-white plates (78 mg, 75%), mp 157–158 °C (EtOH/
154.8, 155.0. IR
m .
2955, 1610, 1509, 1253, 1162, 836, 779 cmꢅ1
Further elution (15–25% CH2Cl2/petroleum spirits) gave 40,7-di-

2360
A. J. Tilley et al. / Bioorg. Med. Chem. 20 (2012) 2353–2361
tert-butyldimethylsilyloxyisoflav-3-ene 14 as a colourless solid
after flash chromatography (10% CH2Cl2/petroleum spirits)
(733 mg) and recrystallisation from EtOAc/petroleum spirits
(400 mg, 28%) mp 81 °C; (C27H40O3Si2 requires C, 69.18; H, 8.60.
Found C, 69.16; H, 8.65). 1H NMR (500 MHz, CDCl3) d 0.221 (s,
6H, Si(CH3)2(C(CH3)3), 0.22 (s, 6H, Si(CH3)2(C(CH3)3), 0.99 (s, 9H,
Si(CH3)2(C(CH3)3 1.00 (s, 9H, Si(CH3)2(C(CH3)3), 5.10 (s, 2H, H2),
6.40 (d, 1H, J6,8 = 2.5, H8), 6.41 (dd, 1H, J6,8 = 2.5, J5,6 = 10, H6),
6.67 (s, 1H, H4), 6.85 (app. d, 2H, J = 8.5, H30,50), 6.93 (d, 1H,
J5,6 = 8.5, H5), 7.29 (app. d, 2H, J = 8.5, H20,60). 13C NMR (125 MHz,
CDCl3) d ꢅ4.4, ꢅ4.39, ꢅ0.01, 18.2, 25.7, 67.2, 107.5, 113.4, 117.0,
length. The aortic rings were mounted between two stainless steel
wires, one of which was linked to an isometric force transducer
(model FT03, Grass Medical Instruments, Quincy, MA, USA) con-
nected to a MacLab/8 (model MKIII, AD Instrument Co., Sydney,
Australia), and the other end anchored to a glass rod submerged
in a standard 10 mL organ bath. The organ bath was filled with
Krebs-bicarbonate solution [composition (mM): NaCl, 118.0; KCl,
4.7; KH2PO4, 1.2; MgSO4ꢀ7H2O, 1.2;
D-glucose, 11.0; NaHCO3,
25.0; CaCl2ꢀ2H2O, 2.5]. The bath medium was maintained at
37 °C, pH 7.4 and continuously aerated with 95% O2, 5% CO2. Aortic
rings were equilibrated for 1 h at a resting tension of 1 g, and then
were precontracted with an isotonic, high potassium physiological
salt solution (KPSS), in which all of the NaCl of the normal Krebs
solution was replaced with KCl (122.7 mM), to achieve maximal
contraction. After re-equilibration, the rings were sub-maximally
118.3 120.3, 125.7, 127.3, 128.7 130.2, 154.0, 155.4, 156.4. IR
2956, 1509, 1269, 1170, 838, 781 cmꢅ1
m
.
4.2.17. 40,7-Dihydroxyisoflav-2-ene (isophenoxodiol; 15)
HFꢀpyridine (300 L) was added to a cooled (0 °C) solution of
40,7-di-tert-butyldimethylsilyloxyisoflav-2-ene
13 (70.0 mg,
l
contracted with phenylephrine (PE, 0.01–0.3
integrity was tested by a single dose of acetylcholine (ACh,
10 M). Only rings that responded to ACh (>80% relaxation) were
lM) and endothelial
0.149 mmol) in THF (1.0 mL). The reaction was then allowed to
warm to rt and stirred for 7 h. EtOAc (5 mL) was then added fol-
lowed by careful addition of satd NaHCO3 until bubbling ceased.
The organic layer was washed with satd NaHCO3 (5 mL), water
(2 ꢄ 5 mL), brine (5 mL), dried (MgSO4) and concentrated. The res-
idue was recrystallised from EtOAc/petroleum spirits to give the
40,7-dihydroxyisoflav-2-ene 15 (25 mg, 70%) as a pink solid; mp
186–190 °C. 1H NMR (400 MHz, DMSO-d6) d 3.54 (s, 2H, CH2),
6.30 (d, 1H, J = 2.4, H8), 6.47 (m, 1H, H6), 6.74 (app. d, 2H, J = 8.8,
H20,60), 6.98 (d, 1H, J = 8.0, H5), 7.07 (s, 1H, H2), 7.25 (d, 2H,
J = 8.8, H30,50), 9.40 (br s, 2H, OH ꢄ 2). 13C NMR (100 MHz,
DMSO-d6) d 24.7, 102.2, 109.5, 111.0, 112.1, 115.3, 125.3, 127.8,
l
judged to be endothelium intact and were used in the subsequent
experiments. After establishing a stable precontraction using PE
(0.01–0.3
and the analogues were determined in the absence and the pres-
ence of the estrogen receptor antagonist ICI 182,780 (0.1 M). Test
lM), cumulative concentration-response curves to equol
l
compounds were dissolved in DMSO and diluted to a final concen-
tration of a maximum of 0.1% DMSO, in Krebs-bicarbonate. All
measurements were conducted in groups of n = 3–5.
In a separate experiment for GTP-RhoA measurement, aortic
rings were denuded of endothelium by gently rubbing the internal
surface with the edge of forceps. Each ring was equilibrated in the
organ bath solution for 90 min, and contracted with 50 mM of KCl
to validate vascular function. After washout, the aortic rings were
130.0, 135.7, 150.5, 156.4, 156.7. IR
m 2956, 2929, 1509, 1269,
1170, 839 cmꢅ1. HRMS ESI+ [M+H]+ 241.0859, requires 241.0859
for C15H13O3.
pretreated with 100 lM flavonoids, 300 lM flavone (positive con-
trol), or vehicle (negative control) for 30 min, and subjected to con-
traction with U46619. Stock solutions of isoflavans and isoflavenes
4.2.18. 40,7-Dihydroxyisoflav-3-ene (phenoxodiol; 4)
HFꢀpyridine (200
l
L) was added to a cooled (0 °C) solution of
were prepared at 100
U46619 were prepared at 300 mM and 300
DMSO. The final concentrations of DMSO in assay buffer was
0.1%. Experiments were performed in duplicate.
l
M in DMSO; stock solutions of flavone and
M, respectively, in
40,7-di-tert-butyldimethylsilyloxyisoflav-3-ene 14 (150 mg, 0.320
mmol) in THF (1.5 mL). The reaction was then allowed to warm
to rt and stirred for 7 h. EtOAc (5 mL was then added followed by
careful addition of satd NaHCO3 until bubbling ceased. The organic
layer was washed with satd NaHCO3 (5 mL), water (2 ꢄ 5 mL),
brine (5 mL), dried (MgSO4) and concentrated. The residue was
recrystallised from EtOAc/petroleum spirits to give 4 (56 mg,
72%) as a pink solid mp 211–212 °C (lit.43 mp 230 °C dec). 1H
NMR (400 MHz, DMSO-d6) d 5.00 (s, 2H, CH2), 6.22 (d, 1H, J = 2.2,
H8), 6.31 (dd, 1H, J = 2.2, 8.0, H6), 6.74 (s, 1H, H4), 6.76 (app. d,
2H, J = 8.8, H20,60), 6.92 (d, 1H, J = 8.0, H5), 7.32 (d, 2H, J = 8.8,
H30,50), 9.51 (br s, 1H, OH), 9.56 (br s, 1H, OH). 13C NMR
(100 MHz, DMSO-d6) d 67.0, 103.3, 109.5, 115.8, 116.4, 117.7,
126.6, 128.2, 128.3, 128.5, 154.7, 158.0, 159.0.
l
4.5. Assay for GTP-RhoA measurement
Aortic rings were rapidly frozen by liquid nitrogen, and stored
at ꢅ80 °C. For GTP-RhoA measurement, the procedure followed
the manufacturer’s protocol for the RhoA G-LISA™ Activation As-
say (Cytoskeleton Inc, Denver, CO, USA). Briefly, previously stored
samples were homogenized in a lysis buffer, and were centrifuged
at 12,000g for 15 min at 4 °C. The supernatant, containing 37 lg of
protein, was transferred into each well of a plate which equal vol-
umes of ice-cold binding buffer mixtures were added into. The
plate was shaken on a cold orbital microplate shaker (300 rpm)
for 30 min at 4 °C and the supernatant solution removed from
wells. Diluted anti-RhoA primary antibody was added and the
plates were incubated on a microplate shaker (300 rpm) at room
temperature for 45 min, followed by addition of secondary anti-
body and incubation on a microplate shaker (300 rpm) at room
temperature for another 45 min. The plate was incubated with
an HRP detection reagent for 15 min at 37 °C. After addition of an
HRP stop buffer, the absorbance was immediately recorded at
490 nm. Experiments were performed in triplicate.
4.3. Pharmacology
All procedures were approved by the Animal Experimentation
Ethics Committee of RMIT University and conformed to the Na-
tional Health and Medical Research Council of Australia code of
practice for the care and use of animals for scientific purposes or
conducted in accordance with Guide for the Care and Use of Labo-
ratory Animals (Institutional Review Board, Kyungpook National
University School of Medicine).
4.4. Vasorelaxation
4.6. Radioligand binding
Male Sprague-Dawley rats were asphyxiated by CO2 inhalation,
followed by decapitation, and their chests opened to isolate the
thoracic aortae. After the removal of superficial connective tissues,
the aorta was cut into ring segments of approximately 2–3 mm in
Rat uterine cytosol was prepared as previously described44,45
and incubated with 0.2 nM [3H]-estradiol in a 10 mM Tris buffer
(pH 7.4, 1.5 mM EDTA, 10% w/v glycerol, 1 mM phenylmethylsulfo-
nylfluoride) in the presence and absence of ICI 182,780 to define

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