Synthesis of (2-amino)ethyl derivatives of quercetin 3-O-methyl ether and their antioxidant and neuroprotective effects

Article abstract of DOI:10.1016/j.bmc.2015.05.023

Reactive oxygen species have been implicated in several diseases, particularly in ischemia-reperfusion injury. Quercetin 3-O-methyl ether has been reported to show potent antioxidant and neuroprotective activity against neuronal damage induced by reactive

Full text of DOI:10.1016/j.bmc.2015.05.023

Accepted Manuscript
Synthesis of (2-amino)ethyl derivatives of quercetin 3-O-methyl ether and their
antioxidant and neuroprotective effects
Young Hun Lee, Hyoung Ja Kim, Ho Yoo, Seo Yun Jung, Bong Jin Kwon, Nam-
Jung Kim, Changbae Jin, Yong Sup Lee
PII:
S0968-0896(15)00423-X
http://dx.doi.org/10.1016/j.bmc.2015.05.023
BMC 12319
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
5 March 2015
9 May 2015
11 May 2015
Please cite this article as: Lee, Y.H., Kim, H.J., Yoo, H., Jung, S.Y., Kwon, B.J., Kim, N-J., Jin, C., Lee, Y.S.,
Synthesis of (2-amino)ethyl derivatives of quercetin 3-O-methyl ether and their antioxidant and neuroprotective
effects, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.023
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Synthesis of (2-amino)ethyl derivatives of quercetin 3-
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neuroprotective effects
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Young Hun Lee, Hyoung Ja Kim, Ho Yoo, Seo Yun Jung, Bong Jin Kwon, Nam-Jung Kim, Changbae Jin, and Yong Sup
Lee*
1

Bioorganic & Medicinal Chemistry
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Synthesis of (2-amino)ethyl derivatives of quercetin 3-O-methyl ether and their
antioxidant and neuroprotective effects
c
c
c
a,b
a
Young Hun Leea,b†, Hyoung Ja Kim , Ho Yoo , Seo Yun Jung , Bong Jin Kwon , Nam-Jung Kim ,
†
Changbae Jinc, and Yong Sup Leea,b*
aMedicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
bDepartment of Life and Nanophamaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
cMolecular Recognition Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Reactive oxygen species have been implicated in several diseases, particularly in ischemia-
reperfusion injury. Quercetin 3-O-methyl ether has been reported to show potent antioxidant and
neuroprotective activity against neuronal damage induced by reactive oxygen species. Several
aminoethyl-substituted derivatives of quercetin 3-O-methyl ether have been synthesized to
increase water solubility while retaining antioxidant and neuroprotective activity. Among such
derivatives, compound 3a shows potent and well-balanced antioxidant activity in three types of
cell-free assay systems and has in vivo neuroprotective effects on transient focal ischemic injury
induced by the occlusion of the middle cerebral artery in rats.
Received in revised form
Accepted
Available online
Keywords:
Quercetin 3-O-methyl ether
Aminoethyl
2009 Elsevier Ltd. All rights reserved.
Antioxidant
Neuroprotection
MCAO model
1. Introduction
Since no medical treatment is currently approved for the
treatment of stroke to reduce brain infarction or neurological
In aerobic organisms, most cells have mitochondria, which
produce energy for cells in the form of ATP through an electron
transport chain. During respiration, oxygen is normally reduced
to water, but when this process is incomplete, some reactive
oxygen species (ROS) are produced, including superoxide anion
radical, hydrogen peroxide, peroxynitrite, and hydroxyl radical.1
Because ROS are highly reactive, they can damage cells and
cause diseases such as cardiovascular disease,2 ischemic stroke,3
and Alzheimer’s disease.4 In particular, close attention has been
paid to the involvement of oxidative stress in brain dysfunction
and neurodegenerative disorders.5 The brain may be particularly
vulnerable to oxidative stress caused by ROS because of its high
polyunsaturated lipid content, regions of high iron
concentrations, and moderate levels of antioxidant molecules.6 In
ischemic stroke, which is caused by some blockage in the artery
to the brain, ROS can exacerbate cell injury and death because
the re-supply of oxygen to the brain through reperfusion is
relevant during the ischemic phase.7,8 Therefore, antioxidants that
can scavenge oxygen free radicals have the therapeutic potential
disability beyond tissue plasminogen activator, a thrombolytic
agent restricted to administration within 3 hours after stroke, it is
most important to develop neuroprotective drugs, which can be
given to minimize the neuronal damage which follows a stroke.
Quercetin 3-O-methyl ether (1, Q-3ME) has been identified as
a potent antioxidant from Opuntia ficus-indica var. saboten (Fig
1).11 Compound 1 has been found to inhibit neuronal damage
induced by H2O2 or superoxide anion radicals in primary cultured
rat cortical cells12 and to have in vivo neuroprotective effects on
transient focal ischemic brain injury in a middle cerebral artery
occlusion (MCAO) rat model.13 In addition, the synthesis of 7-O-
glucosyl flavones (for example, 2) has been reported to enhance
the water solubility of 1. However, an excessive increase in
hydrophilicity by introducing the polar glucose group reduces
inhibitory activity against lipid peroxidation.14
The introduction of the alkylamino group to the structure of
compounds is a general strategy for improving water solubility.15-
17
The alkylamino group can increase the water solubility of
compounds through the formation of hydrochloride salts. On the
other hand, the lipophilic characteristic can be retained by
to treat neuronal injury following ischemia and reperfusion.9,10
———
*Corresponding author.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
†
These authors contributed equally.
2

position of the substituents on antioxidant activity and how
antioxidant activity profiles influence in vivo neuroprotective
effects. The B-ring of Q-3ME was also varied through the
introduction of an additional hydroxyl group to provide myricetin
3-O-methyl ether derivatives (R1 = OH). Finally, in vivo
neuroprotective effects of selected compounds on transient focal
ischemic brain injury were investigated using an MCAO rat
model to examine whether the neuroprotective effects correlate to
their antioxidant activities and are influenced by the introduction
of aminoethyl substituents.
2. Results and discussion
2.1. Chemistry
The synthesis of 3a–3h and 4a–4h started from O-benzyl-
protected 3-methoxyflavones 6a and 6b (Scheme 1). The
regioselective 7-O-alkylation of 6a and 6b with several
alkylaminoethyl chlorides in the presence of K2CO3 produced
7a–7h.19 Benzyl-protecting groups in 7a–7h were removed by
hydrogenolysis with Pd(OH)2 and cyclohexene in EtOH and then
transformed into C-7 aminoethyl-substituted Q-3ME derivatives
3a–3h as hydrochloride salts by treating with HCl gas in ethanol.
C-5 aminoethyl-substituted compounds 4a–4h were prepared
using a procedure similar to that for the synthesis of 3a–3h. The
selective 7-O-benzylation of 6a and 6b with benzyl bromide in
the presence of K2CO3, followed by 5-O-alkylation with
alkylaminoethyl chlorides, produced 9a–9h. Finally, the removal
of the benzyl-protecting group in 9a–9h with Pd(OH)2 and
cyclohexene and then treatment with HCl provided C-5
aminoethyl derivatives 4a–4h as hydrochloride salts.
Figure 1. Structures of quercetin 3-O-methyl ether (1), its 7-O-
glucoside (2), luteolin derivative 5, and target compounds 3a–3h and
4a–4h.
introducing diverse alkyl groups. To prove whether this strategy
works on flavone compounds, we introduced aminoethyl groups
at the C-7 position of luteolin derivatives as a preliminary work
and found that the synthesized 7-O-aminoethyl luteolin
derivatives (e.g. 5) exhibited a similar level of lipid peroxidation
inhibitory activities to luteolin.18 Based on these findings, we
expanded this strategy to Q-3ME (1), which has a methoxy
substituent at the C-3 position in the structure of luteolin, since
this compound had been already proven to show in vivo
neuroprotective effects on transient focal ischemic brain injury in
a MCAO rat model.13 Accordingly, aminoethyl-substituted Q-
3ME derivatives 3a–3h and 4a–4h were derived in hydrochloride
salt forms to improve water solubility without affecting
antioxidant activities and neuroprotective effects of 1 (Fig 1).
Additionally, an aminoethyl group was attached at the C-5 or C-7
position of the Q-3ME structure to examine effects of the
2.2. Antioxidant activity of aminoethyl-substituted Q-3ME
derivatives
Synthesized compounds were assessed for antioxidant activity
Scheme 1. Reagents and conditions: (a) R3R2NCH2CH2Cl, K2CO3, acetone, 60 °C; (b) i) Pd(OH)2/C, EtOH/cyclohexene (1:1), 60–70 °C, ii) HCl, MeOH;
(c) benzyl bromide, K2CO3, acetone, 55 °C; (d) R3R2NCH2CH2Cl, K2CO3, DMF, 70 °C.
3

Table 1. The antioxidant activity of aminoethyl derivatives 3a–3h, 4a–4h, and their parent compound 1.
R3R2N-
a
b
c
d
DPPH radicalsa
O2- radicalsb
Lipid peroxidationc
R1
R3R2N-
Aqueous solubility
(mM)f
cLogPe
Entry
IC50 (µM)d
3a
3b
3c
3d
3e
3f
H
H
a
b
c
d
a
b
c
d
a
b
c
d
a
b
c
d
20.74 4.55
18.87 3.05
47.16 11.66
27.35 3.10
26.32 5.42
16.79 1.52
25.98 6.74
22.93 2.63
> 100
8.15 0.15
11.36 1.84
7.41 0.28
8.14 1.64
> 100
19.18 0.19
21.80 1.15
16.23 0.79
15.78 1.90
10.54 0.92
10.11 0.66
10.15 1.24
11.54 0.54
25.09 1.34
23.29 0.55
37.37 2.85
23.32 1.06
11.88 0.47
10.92 0.96
11.96 0.93
11.24 0.90
19.00 0.51
3.03
2.68
1.86
2.71
2.68
2.34
1.52
2.36
3.03
2.68
1.86
2.71
2.68
2.34
1.52
2.36
1.92
> 50
-
H
-
H
-
OH
OH
OH
OH
H
-
> 100
-
3g
3h
4a
4b
4c
4d
4e
4f
> 100
-
> 100
> 50
5.80 0.54
4.58 0.32
11.25 3.07
6.53 2.52
> 100
-
H
84.00 15.93
> 100
-
H
-
H
> 100
-
OH
OH
OH
OH
32.42 8.42
27.04 9.82
23.18 7.28
22.06 1.07
14.17 0.69
-
> 100
-
4g
4h
1
> 100
-
-
> 100
0.011
17.39 0.50
40.99 6.19
-
Ascorbic acid
Trolox
>50
-
-
71.44 5.54
a DPPH radical scavenging activity.
b Superoxide anion radical scavenging activity generated in the xanthine/xanthine oxidase system.
c Iron-dependent lipid peroxidation inhibition activity using a rat liver homogenate.
d IC50 values with standard deviations are from three independent experiments and expressed as the mean S.D.
e Values of the free base form, calculated by OSIRIS property explorer.
f Solubility in water at room temperature, determined by HPLC.
in three assay systems for DPPH scavenging, superoxide anion
radical scavenging, and lipid peroxidation inhibition.14 The
putative antioxidants, ascorbic acid and trolox were used as
positive controls. The parent compound Q-3ME (1) was also
used to assess the variation of their antioxidant activity by the
aminoalkyl substituent and the number of hydroxyl groups in the
B-ring. First, the effects of the C-7 aminoethyl substituent on
antioxidant activity were investigated (Table 1). The DPPH
radical scavenging activity (IC50 = 18.87–47.16 µM) of C-7
aminoethyl derivatives 3a–3d, which have two hydroxyl groups
at the B-ring, was similar to or slightly lower than that of Q-3ME
(1, IC50 = 14.17 µM). The lipid peroxidation inhibitory activity
(IC50 = 15.78–21.80 µM) of these compounds was comparable to
that of the parent compound (IC50 = 19.00 µM), indicating that
the substitution of the aminoethyl group at C-7 had little effect on
antioxidant activity. In addition, the superoxide anion radical
scavenging activity (IC50 = 8.14–11.36 µM) of these compounds
was two times more potent than that of Q-3ME (IC50 = 17.39
µM) in the xanthine/xanthine oxidase assay system. By contrast,
the lipid peroxidation inhibitory activity (IC50 = 10.11–11.54
µM) of C-7 aminoethyl derivatives 3e–3h, which have three
hydroxyl groups at the B-ring (myricetin 3-O-methyl ether
derivatives), was two times more potent than that of Q-3ME.
However, to our surprise, these four derivatives showed no
noticeable superoxide anion radical scavenging activity under a
concentration of 100 µM. From our previous findings, the C-7
aminoethyl luteolin derivatives possessing three hydroxyl groups
at the B-ring showed superoxide anion radical scavenging
activities to some extent although their potency was 2- to 4-fold
less (IC50 = 17.56–30.12 µM) than that of luteolin (IC50 = 8.66
µM).18 It is noticeable in that slight difference of a substituent
such as the presence or absence of 3-methoxy substituent at the
flavones structure can influence the antioxidant activity profile.
The relationship between the structure and antioxidant activity
of C-5 aminoethyl derivatives 4a–4h was similar to that for C-7
aminoethyl derivatives, except for DPPH radical scavenging
activity (Table 1). The lipid peroxidation inhibitory activity of
compounds 4a–4d, which have two hydroxyl groups at the B-
4

potent and well-balanced antioxidant activity in the three assay
systems.
Aqueous solubilities of 1 and selected compound 3a and 3h
are determined. The parent compound Q-3ME showed very poor
water solublilty (11.4 µM), however, compound 3a and 3h
showed much greater aqueous solubilities (> 50 mM) indicating
that aminoethyl analogues prepared as HCl salts has more
favorable pharmacokinetic properties than the parent compound
1. (Table 1).
Currently, there are many softwares predicting a lipophilic
property of a compound. Calculated LogP values (cLogP) of the
free base form of 3a–3h and 4a–4h were obtained from the
Osiris program20 and included in the Table 1. Except compounds
containing morpholinoethyl substituent (cLogP = 1.52–1.86),
every compound showed increased predicted lipophilicity (cLogP
= 2.34–3.03) than Q-3ME (cLogP = 1.92). Based on these data, it
is expected that the lipophilic characteristic can be increased by
introducing aminoethyl groups on the structure of Q-3ME.
In summary, the type of aminoethyl substituents exhibited
little effect on antioxidant activity of Q-3ME while the position
of aminoethyl substituents exhibit significant effect on DPPH
radical scavenging activity. On the other hand, additional
hydroxyl group at the B ring increased the lipid peroxidation
inhibitory activity and decreased superoxide anion radical
scavenging activity regardless of type and position of aminoalkyl
substituent. Furthermore, aminoethyl-substituted Q-3ME
derivatives showed improved pharmacokinetic properties
including water solubility and lipophilicity.
Figure 2. Representative TTC staining showing effects of 3a, 3h, and 1
administered at a dose of 10 mg/kg (i.v.) to rats subjected to middle
cerebral artery occlusion (MCAO) for 2 h. Each brain was cut into seven
serial slices 2 mm thick starting 1 mm from the frontal pole 24 h after
MCAO. The posterior surface of each slice was imaged. TTC stained
normal brain areas deep red but did not stain infarcted tissue. Values on
the left-hand side indicate the distance from the frontal pole.
2.3. Neuroprotective effects of compounds 3a and 3h
ring, was similar to or slightly lower than that of Q-3ME, but
there was a 2- to 3-fold increase in the superoxide anion radical
scavenging activity as were found on C-7 aminoethyl derivatives.
However, there was a sharp decrease in their DPPH radical
scavenging activity. Noteworthy is that this DPPH radical
scavenging activity recovered through the introduction of an
additional hydroxyl group at the B-ring (4e–4h). Compounds 4e–
4h showed ca. 2-fold increase in their lipid peroxidation
inhibitory activity and no superoxide anion radical scavenging
activity under a concentration of 100 µM, as in the case of
compounds 3e–3h. This result indicates that the position of
aminoethyl substituents and the presence of an additional
hydroxyl group at the B-ring had considerable influence on the
antioxidant activity profile. The mechanism underlying the
effects of these modifications of Q-3ME on antioxidant activity,
particularly with respect to DPPH and superoxide anion radical
scavenging activity, remains to be investigated. However, the
results clearly indicate that C-7 aminoethyl derivatives 3a–3d,
which have two hydroxyl groups at the B-ring, showed more
Compound 3a was selected to further examine its in vivo
neuroprotective activity in a transient focal cerebral ischemic rat
model because it showed potent and well-balanced antioxidant
activity in the three assay systems. The data of in vivo
neuroprotective activity of Q-3ME (1)13 was also included in the
Table 2 and 3 for comparisons. Compound 3h, which showed no
superoxide anion radical scavenging activity under a 100 µM
concentration, was also included in the experiment to compare
and examine whether this activity would be correlated with the
neuroprotective effect. The transient MCAO rat model was used
to assess neuroprotective effects because most cases of ischemic
stroke occur in the MCA region and ROS generated by
reperfusion can damage neuronal cells and provoke infarction.
Transient focal cerebral ischemia was induced by the occlusion
of the right MCA for 2 h with a silicone-coated 4-0 nylon
monofilament in male Sprague-Dawley rats.13 Compound 3a and
3h dissolved in 0.9% saline were administered intravenously 30
Table 2. Neuroprotective effects of 3a, 3h, and 1 administered intravenously 30 min after the onset of ischemia at a dose of 10 mg/kg in a rat model
of transient focal cerebral ischemia for 2 h.
Absolute infarct volume (mm3)a
Corrected total infarct
Treatment
n
Edema (%)[a]
volume (mm3)[a]
Striatum
Cortex
Total
Vehicle
13
8
41.9 2.9
30.1 5.4
39.3 5.1
27.2 4.9
195.6 11.5
93.6 18.8*
123.4 20.6*
79.9 9.7*
237.4 13.1
128.9 25.1*
164.0 23.1*
108.8 13.1*
163.2 9.4
92.1 18.3*
108.9 15.3*
78.8 9.7*
13.0 0.7
6.7 1.1*
9.4 1.3*
5.5 0.8*
3a
3h
1
14
12
a Data is presented as the mean S.E.M.
* Significantly different from the vehicle-treated group based on Duncan's multiple-range test (p < 0.05)
5

min after the onset of ischemia at a dose of 10 mg/kg. Infarct size
and % edema were measured 24 h after the onset of ischemia by
Table 3. Effects of 3a, 3h, and 1 (10 mg/kg, i.v., 30 min after the onset
of ischemia) on neurological scores measured 30 min and 24 h after the
onset of MCAO.
using
a 2,3,5-triphenyltetrazolium chloride (TTC) staining
method. The area of the absolute infarct in the cortex and
striatum and the total area of both hemispheres were measured
for each slice by using a computerized image analysis system
(Optimas). Absolute infarct volume was calculated by
multiplying the area by the slice thickness and summing the
volume. The corrected infarct area in a slice was calculated to
compensate for the effect of brain edema by subtracting the area
of normal tissue in the ipsilateral hemisphere from the total area
of the contralateral hemisphere. Corrected total infarct volume
was then calculated by multiplying the area by the slice thickness
and summing the volume. Hemispheric swelling representing
tissue edema was expressed as a percentage increase in the size
of the ipsilateral hemisphere in comparison to the contralateral
hemisphere (% edema). In addition, neurological scoring was
performed 30 min and 24 h after MCAO. The neurobehavioral
test consisted of scoring the degree of left-forelimb flexion (0 to
3), the duration of left-forelimb flexion (0 to 4), and the
symmetry of movement/forepaw outstretching (0 to 3). Rats were
scored on a ranking scale of 0 to 10, which reflected the
cumulative score on individual tests with a score of 10 reflecting
normal behavior. Fig. 2 shows representative TTC staining to
illustrate neuroprotective effects of compound 3a and 3h. The
area stained red with TTC was considered normal, whereas the
rest, infarcted. As shown in Table 2, treatment with 10 mg/kg
(i.v.) of compound 3a significantly reduced the absolute infarct
volume of the cortex by 52.2% (93.6 vs. 195.6 mm3) and absolute
total infarct volume by 45.7% (128.9 vs. 237.4 mm3) in
comparison to the vehicle-treated group. In addition, there were
significant decreases in corrected total infarct volume and %
edema by 43.6% (92.1 vs. 163.2 mm3) and 48.5% (6.7% vs.
13.0% edema), respectively. This also produced a significant
behavioral recovery effect when measured at 24 h after MCAO,
increasing the neurological score to 4.87 0.58, whereas it was
Neurological scoresa
Treatment
30 min
24 h
Vehicle
2.32 0.11
2.25 0.16
2.21 0.11
2.29 0.12
2.53 0.21
4.87 0.58*
3.64 0.32
5.29 0.46*
3a
3h
1
a Data is presented as the mean S.E.M.
* Significantly different from the vehicle-treated group based on
Duncan's multiple-range test (p < 0.05).
permeability.23 Accordingly, it remains to measure permeability
across the BBB or brain concentration of the compounds.
3. Conclusion
C-7 or C-5 aminoethyl-substituted Q-3ME derivatives 3a–3h
and 4a–4h were synthesized in hydrochloride salt forms to
improve water solubility while retaining antioxidant activities. C-
7 aminoethyl derivatives 3a–3d, which have two hydroxyl
groups at the B-ring, showed well-balanced antioxidant activity.
These compounds showed nearly equipotent DPPH radical
scavenging and lipid peroxidation inhibitory activity and a 2-fold
increase in superoxide anion radical scavenging activity in
comparison to Q-3ME, the parent compound. On the other hand,
the substitution of the aminoethyl group at C-5 position or the
introduction of an additional hydroxyl group at the B-ring
increased or reduced the antioxidant activity of Q-3ME
depending on the assay system. Compound 3a, which showed
potent and well-balanced antioxidant activity in the three assay
systems, had a neuroprotective effect on transient focal ischemic
brain injury in an MCAO rat model. More specifically, it
significantly reduced corrected total infarct volume and % edema
by 43.6% and 48.5%, respectively, with a significant behavioral
recovery effect at a dose of 10 mg/kg. The neuroprotective
effects of 3a were generally comparable to those of Q-3ME with
minor variations, while the introduction of an aminoethyl group
to Q-3ME increased its water solubility. On the other hand,
compound 3h, which showed no superoxide anion radical
scavenging activity under a concentration of 100 µM, had a
weaker neuroprotective effect than 3a in the MCAO rat model,
suggesting that superoxide anion radical scavenging activity
plays an important role in neuroprotective effects.
2.53
0.21 for the control group (Table 3). This in vivo
neuroprotective potency of 3a is consistent with the in vitro
antioxidant activity examined in the cell-free assay system. These
neuroprotective effects of 3a were comparable to those of Q-
3ME, which significantly reduced the corrected total infarct
volume and % edema by approximately 50% at a dose of 10
mg/kg.13 However, treatment with 3h produced weaker
neuroprotective effects on ischemic brain injury than that with 3a.
Compound 3h significantly reduced absolute total infarct volume
by 30.9% (164.0 vs. 237.4 mm3) in comparison to the vehicle-
treated group (Table 2). In addition, corrected total infarct
volume and % edema were significantly reduced by 33.3%
(108.9 vs. 163.2 mm3) and 27.7% (9.4% vs. 13.0% edema),
respectively. However, treatment with 3h did not produce a
significant behavioral recovery effect (Table 3). According to a
comparison of neuroprotective effects of treatment between 3a
and 3h with respect to infarct volume, edema size, and behavioral
recovery, the superoxide anion radical scavenging effect was an
important parameter. Finally, ability of the compounds to cross
the blood-brain barrier (BBB) should be considered in eliciting in
vivo neuroprotective activity against ischemic brain injury. It has
been known that the penetration rate of flavonoids is dependent
on the lipophilicity and interactions with efflux transporters.21,22
However, the predicted lipophilicity of 3a (cLogP = 3.03) is not
so higher than that of 3h (cLogP = 2.36) to explain clearly the
differences in their neuroprotective effects by the passage rate of
compound through BBB. It is also known that cerebral ischemia
induces disruption and hyperpermeability of the BBB, thus
enhancing brain penetration of compounds with poor
4. Experimental
4.1. General instrumentation and chemicals
NMR spectra were recorded on a Gemini Varian-300
operating at 300 MHz for H NMR and 75 MHz for 13C NMR
spectra. High resolution mass spectra (HR-MS) were recorded on
Jeol accuTOF (JMS-T100TD) equipped with a DART (direct
analysis in real time) ion source from Ionsense (Tokyo, Japan) in
the positive modes. Analytical thin-layer chromatography (TLC)
was performed on precoated silica gel (E. Merck Kiesegel 60F254
layer thickness 0.25 mm). Column chromatography was
performed with Merck Kiesegel 60 Art 9385 (230-400 mesh). All
1
6

2.60 (2H, t, J = 5.9 Hz), 2.33–2.31 (4H, m), 1.45–1.39 (4H, m),
1.35–1.32 (2H, m); 13C NMR (75 MHz, CDCl3) δ 179.1, 165.1,
162.3, 157.0, 155.5, 153.0, 141.1, 139.8, 137.9, 137.2, 129.0,
128.7, 128.4, 127.8, 125.9, 108.8, 106.4, 98.8, 93.1, 75.7, 71.7,
67.2, 60.5, 57.9, 55.5, 26.4, 24.5.
solvents, chemicals and reagents were purchased from Sigma-
Aldrich, Acros or TCI.
4.2. Synthesis
4.2.1. 2-(3,4-Bis(benzyloxy)phenyl)-5-hydroxy-3-methoxy-7-
(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one (7a). To a stirred
solution of compound 6a18 (1.0 mmol) in acetone (60 mL) was
added K2CO3 (4.0 mmol) and 1-(2-chloroethyl)piperidine
hydrochloride (1.3 mmol). The reaction mixture was heated at
reflux for 6 h. The mixture was concentrated and the residue was
treated with EtOAc and water. The organic layer was separated,
dried over anhydrous MgSO4, filtered, and concentrated. The
residue was purified by flash column chromatography
4.2.6. 5-Hydroxy-3-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-2-
(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one (7f). The
compound 7f was prepared from 6b (1.0 mmol) and 1-(2-
chloroethyl)pyrrolidine hydrochloride (1.3 mmol) using the
1
procedure described for 7a. Yield 31%. H NMR (300 MHz,
CDCl3) δ 7.52–7.31 (17H, m), 6.45 (1H, d, J = 2.1 Hz), 6.40 (1H,
d, J = 2.1 Hz), 5.24 (6H, s), 4.23 (2H, t, J = 5.7 Hz), 3.70 (3H, s),
2.99 (2H, t, J = 5.7 Hz), 2.73–2.69 (4H, m), 1.90–1.86 (4H, m);
13C NMR (75 MHz, CDCl3) δ 178.6, 164.6, 161.8, 156.5, 155.2,
152.6, 140.7, 139.4, 137.4, 136.7, 128.6, 128.5, 128.2, 127.9,
127.3, 125.4, 108.4, 105.9, 98.3, 92.6, 75.2, 71.3, 67.7, 60.0, 54.7,
23.5.
1
(CH2Cl2/MeOH = 10:1) to afford 7a (89%). H NMR (300 MHz,
CDCl3) δ 7.79 (1H, d, J = 1.4 Hz), 7.70 (1H, d, J = 8.4 Hz), 7.53–
7.29 (10H, m), 7.06 (1H, d, J = 8.4 Hz), 6.42 (1H, d, J = 2.1 Hz),
6.36 (1H, d, J = 2.1 Hz), 5.29 (2H, s), 5.28 (2H, s), 4.21 (2H, t, J
= 6.0 Hz), 3.73 (3H, s), 2.86 (2H, t, J = 6.0 Hz), 2.59–2.56 (4H,
m), 1.68–1.63 (4H, m), 1.51–1.47 (2H, m); 13C NMR (75 MHz,
CDCl3) δ 178.6, 164.8, 162.2, 156.8, 155.8, 151.5, 148.6, 139.3,
137.2, 136.9, 128.9, 128.8, 128.3, 128.2, 127.5, 127.4, 123.5,
122.8, 115.3, 113.9, 106.3, 98.6, 92.9, 71.6, 71.1, 66.8, 60.3, 57.7,
55.3, 26.0, 25.7, 24.3.
4.2.7. 5-Hydroxy-3-methoxy-7-(2-morpholinoethoxy)-2-(3,4,5-
tris(benzyloxy)phenyl)-4H-chromen-4-one
compound 7g was prepared from 6b (1.0 mmol) and 4-(2-
(7g).
The
chloroethyl)morpholine hydrochloride (1.3 mmol) using the
1
procedure described for 7a. Yield 51%. H NMR (300 MHz,
CDCl3) δ 7.51–7.30 (17H, m), 6.43 (1H, d, J = 2.1 Hz), 6.38 (1H,
d, J = 2.1 Hz), 5.23 (6H, s), 4.25 (2H, t, J = 5.4 Hz), 3.83–3.79
(4H, m), 3.68 (3H, s), 2.91 (2H, t, J = 5.4 Hz), 2.69–2.65 (4H,
m); 13C NMR (75 MHz, CDCl3) δ 178.7, 164.4, 161.9, 156.6,
155.3, 152.6, 139.4, 137.4, 136.7, 128.6, 128.5, 128.3, 128.0,
127.4, 125.4, 108.4, 106.1, 98.7, 93.0, 75.3, 71.3, 66.7, 60.1, 57.2,
54.0, 25.2, 24.8.
4.2.2. 2-(3,4-Bis(benzyloxy)phenyl)-5-hydroxy-3-methoxy-7-
(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one
compound 7b was prepared from 6a (1.0 mmol) and 1-(2-
(7b).
The
chloroethyl)pyrrolidine hydrochloride (1.3 mmol) using the
1
procedure described for 7a. Yield 71%. H NMR (300 MHz,
CDCl3) δ 7.75 (1H, d, J = 2.0 Hz), 7.69 (1H, d, J = 8.7 Hz), 7.49–
7.33 (10H, m), 7.31 (1H, d, J = 8.7 Hz), 6.39 (1H, d, J = 2.1 Hz),
6.34 (1H, d, J = 2.1 Hz), 5.26 (2H, s), 5.25 (2H, s), 4.16 (2H, t, J
= 6.0 Hz), 3.69 (3H, s), 2.93 (2H, t, J = 6.0 Hz), 2.66–2.62 (4H,
m), 1.83–1.80 (4H, m); 13C NMR (75 MHz, CDCl3) δ 179.1,
165.0, 162.3, 157.0, 155.9, 151.7, 148.7, 139.4, 137.3, 137.0,
129.0, 128.5, 128.3, 127.7, 127.6, 123.7, 122.9, 115.4, 114.1,
106.4, 98.7, 93.1, 71.7, 71.3, 68.1, 60.5, 55.1, 23.9, 23.1.
4.2.8.
7-(2-(Diethylamino)ethoxy)-5-hydroxy-3-methoxy-2-
(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one (7h). The
compound 7h was prepared from 6b (1.0 mmol) and 2-chloro-
N,N-diethylethanamine hydrochloride (1.3 mmol) using the
1
procedure described for 7a. Yield 46%. H NMR (300 MHz,
CDCl3) δ 7.52–7.31 (17H, m), 6.44 (1H, d, J = 2.1 Hz), 6.40 (1H,
d, J = 2.1 Hz), 5.25 (6H, s), 4.17 (2H, t, J = 6.0 Hz), 3.71 (3H, s),
2.97 (2H, t, J = 6.0 Hz), 2.71 (4H, q, J = 7.2 Hz), 1.15 (6H, t, J =
7.2 Hz); 13C NMR (75 MHz, CDCl3) δ 178.7, 164.7, 161.9, 156.5,
155.1, 152.6, 140.7, 139.4, 137.4, 136.7, 128.6, 128.2, 127.9,
127.3, 125.4, 108.4, 105.9, 98.7, 93.4, 75.2, 71.3, 60.1, 51.4, 47.9,
11.8.
4.2.3. 2-(3,4-Bis(benzyloxy)phenyl)-5-hydroxy-3-methoxy-7-
(2-morpholinoethoxy)-4H-chromen-4-one (7c). The compound
7c was prepared from 6a (1.0 mmol) and 4-(2-
chloroethyl)morpholine hydrochloride (1.3 mmol) using the
1
procedure described for 7a. Yield 93%. H NMR (300 MHz,
4.2.9.
(piperidin-1-yl)ethoxy)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-5-hydroxy-3-methoxy-7-(2-
hydrochloride
CDCl3) δ 7.79 (1H, d, J = 2.1 Hz), 7.69 (1H, d, J = 8.7 Hz), 7.53–
7.35 (10H, m), 7.06 (1H, d, J = 8.7 Hz), 6.42 (1H, d, J = 1.2 Hz),
6.37 (1H, d, J = 1.2 Hz), 5.29 (2H, s), 5.28 (2H, s), 4.20 (2H, t, J
= 6.0 Hz), 3.80–3.76 (4H, m), 3.74 (3H, s), 2.87 (2H, t, J = 6.0
Hz), 2.64–2.61 (4H, m).
(3a). To a solution of 7a in EtOH and cyclohexene (1:1) was
added a catalytic amount of Pd(OH)2/C. The reaction mixture
was refluxed (60–70 °C) for the period of time (TLC monitoring),
filtered through Celite and concentrated. The residue was
dissolved in absolute EtOH and then saturated with HCl gas at
0 °C until the mixture become acidic. The resulting precipitate
4.2.4.
2-(3,4-Bis(benzyloxy)phenyl)-7-(2-
(diethylamino)ethoxy)-5-hydroxy-3-methoxy-4H-chromen-4-
one (7d). The compound 7d was prepared from 6a (1.0 mmol)
and 2-chloro-N,N-diethylethanamine hydrochloride (1.3 mmol)
1
was filtered and dried to afford 3a (66%). H NMR (300 MHz,
1
DMSO-d6) δ 7.61 (1H, d, J = 1.8 Hz), 7.49 (1H, d, J = 8.4 Hz),
6.95 (1H, d, J = 8.4 Hz), 6.80 (1H, d, J = 2.1 Hz), 6.45 (1H, d, J
= 2.1 Hz), 4.49 (2H, br s), 3.80 (3H, s), 3.67–3.63 (4H, m), 3.51
(2H, br s), 1.80–1.78 (4H, m), 1.51–1.49 (2H, m); 13C NMR (75
MHz, DMSO-d6) δ 178.32, 163.40, 160.94, 156.46, 148.96,
145.37, 138.29, 120.98, 116.00, 115.98, 105.83, 98.56, 93.41,
60.03, 54.74, 53.01, 47.87, 22.68, 21.35; HRMS (ESI) m/z: calcd
for C23H25NO7+ ([M-HCl]+) 427.1626, found 427.1653.
using the procedure described for 7a. Yield 78%. H NMR (300
MHz, CDCl3) δ 7.77 (1H, d, J = 2.0 Hz), 7.69 (1H, d, J = 8.6 Hz),
7.51–7.34 (10H, m), 7.03 (1H, d, J = 8.6 Hz), 6.40 (1H, d, J = 2.1
Hz), 6.34 (1H, d, J = 2.1 Hz), 5.27 (2H, s), 5.26 (2H, s), 4.09 (2H,
t, J = 6.2 Hz), 3.71 (3H, s), 2.90 (2H, t, J = 6.2 Hz), 2.66 (4H, q,
J = 7.1 H), 1.07 (6H, t, J = 7.1 Hz); 13C NMR (75 MHz, CDCl3) δ
179.1, 165.1, 162.3, 157.0, 155.9, 151.6, 148.7, 139.4, 137.3,
137.0, 129.0, 128.5, 128.3, 127.6, 123.7, 122.9, 115.4, 114.1,
106.4, 98.7, 93.0, 71.7, 71.3, 67.7, 60.5, 51.8, 48.3, 12.3.
4.2.10. 2-(3,4-Dihydroxyphenyl)-5-hydroxy-3-methoxy-7-(2-
(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one hydrochloride
(3b). The compound 3b was prepared from 7b using the
4.2.5. 5-Hydroxy-3-methoxy-7-(2-(piperidin-1-yl)ethoxy)-2-
(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one (7e). The
compound 7e was prepared from 6b (1.0 mmol) and 1-(2-
chloroethyl)piperidine hydrochloride (1.3 mmol) using the
1
procedure described for 3a. Yield 95%. H NMR (300 MHz,
DMSO-d6) δ 7.57 (1H, s), 7.48 (1H, d, J = 6.5 Hz), 6.85 (1H, d, J
= 6.5 Hz), 6.72 (1H, s), 6.34 (1H, s), 4.16 (2H, t, J = 5.6 Hz),
3.79 (3H, s), 3.78–3.76 (4H, m), 2.79 (2H, t, J = 5.6 Hz), 1.69–
1.67 (4H, m); 13C NMR (75 MHz, DMSO-d6) δ 179.3, 164.4,
160.7, 157.2, 156.9, 149.2, 145.4, 139.1, 122.9, 122.3, 117.1,
1
procedure described for 7a. Yield 77%. H NMR (300 MHz,
CDCl3) δ 7.27–7.09 (17H, m), 6.19 (1H, d, J = 1.8 Hz), 6.14 (1H,
d, J = 1.8 Hz), 4.99 (6H, s), 3.97 (2H, t, J = 5.9 Hz), 3.44 (3H, s),
7

116.6, 106.4, 99.6, 94.2, 64.8, 61.2, 55.9, 54.6, 24.1; HRMS
(ESI) m/z: calcd for C22H23NO7+ ([M-HCl]+) 413.1469, found
413.1522.
filtered, and concentrated. The residue was solidified by treating
EtOH to afford 8a. (85%). H NMR (300 MHz, CDCl3) δ 12.68
1
(1H, s, OH), 7.80 (1H, s), 7.70 (1H, d, J = 8.7 Hz), 7.54–7.35
(15H, m), 7.06 (1H, d, J = 8.7 Hz), 6.50 (1H, d, J = 1.5 Hz), 6.46
(1H, d, J = 1.5 Hz), 5.30 (4H, s), 5.15 (2H, s), 3.75 (3H, s); 13C
NMR (75 MHz, CDCl3) δ 178.6, 164.4, 161.9, 156.6, 155.5,
151.3, 148.3, 138.9, 136.9, 136.6, 135.7, 128.7, 128.6, 128.5,
128.3, 127.9, 127.8, 127.4, 127.2, 127.1, 123.2, 122.5, 115.1,
113.7, 106.1, 98.5, 92.9, 71.3, 70.8, 70.4, 60.0.
4.2.11. 2-(3,4-Dihydroxyphenyl)-5-hydroxy-3-methoxy-7-(2-
morpholinoethoxy)-4H-chromen-4-one hydrochloride (3c).
The compound 3c was prepared from 7c using the procedure
described for 3a. Yield 35%. 1H NMR (300 MHz, CDCl3
+
CD3OD) δ 7.67 (1H, s), 7.55 (1H, d, J = 8.5 Hz), 6.95 (1H, d, J =
8.5 Hz), 6.47 (1H, s), 6.33 (1H, s), 4.20 (2H, br s), 3.83 (3H, s),
3.77–3.75 (4H, m), 2.87 (2H, br s), 2.63–2.60 (4H, m); 13C NMR
(75 MHz, D2O) δ 177.8, 162.8, 159.3, 155.4, 155.2, 147.7, 143.7,
137.4, 121.5, 120.5, 115.5, 114.9, 104.8, 98.1, 92.8, 63.9, 61.8,
59.6, 55.6, 52.2; HRMS (ESI) m/z: calcd for C22H23NO8+ ([M-
HCl]+) 429.1418, found 429.1457.
4.2.18.
tris(benzyloxy)phenyl)-4H-chromen-4-one
7-(Benzyloxy)-5-hydroxy-3-methoxy-2-(3,4,5-
(8b). The
compound 8b was prepared from 6b (2.0 mmol) using the
1
procedure described for 8a. Yield 94%. H NMR (300 MHz,
DMSO-d6) δ 12.56 (1H, b, OH), 7.58–7.30 (22H, m), 6.90 (1H, d,
J = 2.4 Hz), 6.52 (1H, d, J = 2.4 Hz), 5.26 (6H, s), 5.11 (2H, s),
3.70 (3H, s).
4.2.12. 7-(2-(Diethylamino)ethoxy)-2-(3,4-dihydroxyphenyl)-
5-hydroxy-3-methoxy-4H-chromen-4-one hydrochloride (3d).
The compound 3d was prepared from 7d using the procedure
4.2.19.
7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-
1
described for 3a. Yield 31%. H NMR (300 MHz, D2O) δ 7.11
methoxy-5-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one (9a).
To a stirred solution of compound 8a (1.0 mmol) in acetone (60
(1H, s), 7.07 (1H, d, J = 8.3 Hz), 6.60 (1H, d, J = 8.3 Hz), 5.96
(1H, s), 5.86 (1H, s), 3.94 (2H, br s), 3.46 (3H, s), 3.23 (2H, br s),
3.13–3.04 (4H, m), 1.12 (6H, t, J = 7.1 Hz); 13C NMR (75 MHz,
D2O) δ 178.3, 163.1, 159.8, 156.3, 155.9, 148.2, 144.3, 137.9,
121.2, 115.9, 115.4, 105.3, 98.2, 93.5, 66.3, 59.9, 57.8, 47.5, 8.4;
HRMS (ESI) m/z: calcd for C22H25NO7+ ([M-HCl]+) 415.1626,
found 415.1649.
mL)
was added K2CO3 (4.0 mmol) and 1-(2-
chloroethyl)piperidine hydrochloride (1.3 mmol). The reaction
mixture was heated at reflux for 6 h. The mixture was
concentrated and the residue was treated with EtOAc and water.
The organic layer was separated, dried over anhydrous MgSO4,
filtered, and concentrated. The residue was purified by flash
column chromatography (CH2Cl2/MeOH = 10:1) to afford 9a
4.2.13. 5-Hydroxy-3-methoxy-7-(2-(piperidin-1-yl)ethoxy)-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(3e). The compound 3e was prepared from 7e using the
1
(50%). H NMR (300 MHz, CDCl3) δ 7.81 (1H, d, J = 1.8 Hz),
7.67 (1H, d, J = 8.7 Hz), 7.53–7.34 (15H, m), 7.04 (1H, d, J = 8.7
Hz), 6.55 (1H, d, J = 1.8 Hz), 6.46 (1H, d, J = 1.8 Hz), 5.29 (4H,
s), 5.15 (2H, s), 4.27 (2H, t, J = 6.0 Hz), 3.73 (3H, s), 3.05 (2H, t,
J = 6.0 Hz), 2.70–2.68 (4H, m), 1.71–1.67 (4H, m), 1.52–1.50
(2H, m); 13C NMR (75 MHz, CDCl3) δ 173.7, 162.7, 159.9,
158.5, 152.3, 150.6, 148.2, 141.1, 136.9, 136.6, 135.6, 128.7,
128.5, 128.4, 128.3, 127.9, 127.8, 127.4, 127.2, 127.1, 114.9,
113.7, 109.6, 97.2, 93.6, 71.2, 70.8, 70.3, 67.2, 59.8, 57.1, 54.9,
25.5, 23.7.
1
procedure described for 3a. Yield 65%. H NMR (300 MHz,
D2O) δ 6.44 (2H, s), 5.60 (2H, s), 4.69 (2H, t, J = 9.0 Hz), 3.47
(3H, s), 3.33 (2H, s), 3.10 (2H, s), 2.80 (2H, t, J = 9.0 Hz), 1.77–
1.34 (6H, m); 13C NMR (75 MHz, D2O) δ 177.7, 162.7, 159.2,
154.9, 144.5, 137.4, 136.1, 119.5, 107.7, 104.3, 97.8, 92.6, 79.6,
59.5, 54.9, 53.6, 22.6, 21.0; HRMS (ESI) m/z: calcd for
C23H25NO8+ ([M-HCl]+) 443.1575, found 443.1581.
4.2.14. 5-Hydroxy-3-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(3f). The compound 3f was prepared from 7f using the procedure
4.2.20.
7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-
methoxy-5-(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one
(9b). The compound 9b was prepared from 8a (1.0 mmol) and 1-
(2-chloroethyl)pyrrolidine hydrochloride (1.3 mmol) using the
1
described for 3a. Yield 32%. H NMR (300 MHz, D2O) δ 6.59
(2H, s), 5.84 (2H, s), 4.64 (2H, t, J = 5.4 Hz), 3.85 (2H, br s),
3.49 (3H, s), 3.30–3.28 (4H, m), 1.95–1.93 (4H, m); HRMS
(ESI) m/z: calcd for C22H23NO8+ ([M-HCl]+) 429.1418, found
429.1412.
1
procedure described for 9a. Yield 68%. H NMR (300 MHz,
CDCl3) δ 7.78 (1H, s), 7.65 (1H, d, J = 8.4 Hz), 7.47–7.32 (15H,
m), 7.03 (1H, d, J = 8.4 Hz), 6.53 (1H, s), 6.44 (1H, s), 5.27 (4H,
s), 5.13 (2H, s), 4.27 (2H, t, J = 5.7 Hz), 3.70 (3H, s), 3.18 (2H, t,
J = 5.7 Hz), 2.85–2.83 (4H, m), 1.87–1.85 (4H, m); 13C NMR (75
MHz, CDCl3) δ 174.1, 163.2, 160.3, 159.0, 151.1, 148.6, 141.6,
137.4, 137.1, 136.1, 129.2, 129.0, 128.8, 128.4, 128.3, 128.0,
127.6, 124.1, 122.4, 115.3, 114.2, 97.7, 94.2, 71.7, 71.3, 70.9,
68.6, 60.3, 55.2, 54.7, 23.9.
4.2.15.
5-Hydroxy-3-methoxy-7-(2-morpholinoethoxy)-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(3g). The compound 3g was prepared from 7g using the
1
procedure described for 3a. Yield 16%. H NMR (300 MHz,
DMSO-d6) δ 7.11 (2H, s), 6.59 (1H, s), 6.50 (1H, s), 4.49 (2H, s),
4.04–3.62 (10H, m), 3.73 (3H, s); HRMS (ESI) m/z: calcd for
C22H23NO9+ ([M-HCl]+) 445.1367, found 445.1492.
4.2.21.
methoxy-5-(2-morpholinoethoxy)-4H-chromen-4-one
7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-
(9c).
4.2.16. 7-(2-(Diethylamino)ethoxy)-5-hydroxy-3-methoxy-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(3h). The compound 3h was prepared from 7h using the
The compound 9c was prepared from 8a (1.0 mmol) and 4-(2-
chloroethyl)morpholine hydrochloride (1.3 mmol) using the
1
1
procedure described for 9a. Yield 49%. H NMR (300 MHz,
procedure described for 3a. Yield 85%. H NMR (300 MHz,
D2O) δ 6.51 (2H, s), 5.63 (1H, s), 5.60 (1H, s), 4.70–4.66 (2H, m),
3.50 (3H, s), 3.06–3.02 (6H, m), 1.12 (6H, t, J = 7.2 Hz); 13C
NMR (75 MHz, D2O) δ 177.6, 167.87, 162.66, 159.16, 154.86,
144.59, 137.42, 136.26, 119.59, 107.68, 104.36, 97.90, 92.40,
79.7, 59.49, 49.80, 48.04, 8.17; HRMS (ESI) m/z: calcd for
C22H25NO8+ ([M-HCl]+) 431.1575, found 431.1604.
CDCl3) δ 7.81 (1H, d, J = 2.1 Hz), 7.67 (1H, d, J = 8.7 Hz), 7.53–
7.34 (15H, m), 7.05 (1H, d, J = 8.7 Hz), 6.56 (1H, d, J = 1.8 Hz),
6.45 (1H, d, J = 1.8 Hz), 5.29 (4H, s), 5.17 (2H, s), 4.23 (2H, t, J
= 6.0 Hz), 3.79–3.76 (4H, m), 3.72 (3H, s), 3.01 (2H, t, J = 6.0
Hz), 2.74–2.71 (4H, m); 13C NMR (75 MHz, CDCl3) δ 173.6,
162.7, 159.9, 158.5, 152.3, 150.6, 148.2, 141.1, 136.9, 136.6,
135.6, 128.7, 128.5, 128.4, 128.3, 127.9, 127.8, 127.5, 127.1,
127.0, 123.6, 121.9, 114.9, 113.7, 109.7, 97.3, 93.6, 71.2, 70.8,
70.4, 67.6, 66.9, 59.8, 56.9, 54.1.
4.2.17.
hydroxy-3-methoxy-4H-chromen-4-one (8a). To
7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-5-
stirred
a
solution of compound 6a (2.0 mmol) in acetone (40 mL) was
added K2CO3 (3.0 mmol) and benzyl bromide (2.6 mmol). The
reaction mixture was heated at reflux for 4 h. The mixture was
concentrated and the residue was treated with EtOAc and water.
The organic layer was separated, dried over anhydrous MgSO4,
4.2.22.
(diethylamino)ethoxy)-3-methoxy-4H-chromen-4-one
7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-5-(2-
(9d).
The compound 9d was prepared from 8a (1.0 mmol) and 2-
chloro-N,N-diethylethanamine hydrochloride (1.3 mmol) using
8

1
the procedure described for 9a. Yield 67%. H NMR (300 MHz,
was filtered to afford 4a (71%). 1H NMR (300 MHz, D2O) δ 6.88
(1H, s), 6.76 (1H, d, J = 8.4 Hz), 6.34 (1H, d, J = 8.4 Hz), 5.88
(1H, s), 5.86 (1H, s), 4.02 (2H, br s), 3.50–3.40 (4H, m), 3.43
(3H, s), 2.96 (2H, t, J = 7.2 Hz), 1.92–1.43 (6H, m); HRMS (ESI)
m/z: calcd for C23H25NO7+ ([M-HCl]+) 427.1626, found 427.1795.
4.2.28. 2-(3,4-Dihydroxyphenyl)-7-hydroxy-3-methoxy-5-(2-
CDCl3) δ 7.60 (1H, s), 7.46 (1H, d, J = 8.7 Hz), 7.30–7.13 (15H,
m), 6.83 (1H, d, J = 8.7 Hz), 6.34 (1H, s), 6.27 (1H, s), 5.08 (4H,
s), 4.95 (2H, s), 3.97 (2H, t, J = 6.3 Hz), 3.53 (3H, s), 2.89 (2H, t,
J = 6.3 Hz), 2.53 (4H, q, J = 6.9 Hz), 0.92 (6H, t, J = 6.9 Hz); 13
C
NMR (75 MHz, CDCl3) δ 174.8, 163.2, 160.6, 159.0, 152.7,
151.1, 148.6, 141.6, 137.4, 137.1, 136.1, 129.2, 129.0, 128.8,
128.4, 128.3, 128.0, 127.6, 124.2, 122.4, 115.3, 114.2, 110.1,
97.6, 94.0, 71.7, 71.3, 70.8, 68.5, 60.3, 51.8, 48.3, 12.2.
(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one
(4b). The compound 4b was prepared from 9b using the
hydrochloride
1
procedure described for 4a. Yield 60%. H NMR (300 MHz,
D2O) δ 6.57 (1H, s), 6.45 (1H, d, J = 8.4 Hz), 6.06 (1H, d, J = 8.4
Hz), 5.66 (1H, s), 5.56 (1H, s), 3.81 (2H, t, J = 7.2 Hz), 3.50–
3.36 (4H, m), 3.24 (3H, s), 2.98 (2H, br s), 2.01–1.83 (4H, m);
13C NMR (75 MHz, D2O) δ 174.8, 161.4, 157.7, 156.9, 152.7,
146.7, 143.3, 139.0, 120.3, 114.9, 114.3, 106.7, 97.6, 96.1, 64.1,
4.2.23. 7-(Benzyloxy)-3-methoxy-5-(2-(piperidin-1-yl)ethoxy)-
2-(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one (9e). The
compound 9e was prepared from 8b (1.0 mmol) and 1-(2-
chloroethyl)piperidine hydrochloride (1.3 mmol) using the
1
procedure described for 9a. Yield 53%. H NMR (300 MHz,
+
CDCl3) δ 7.48–7.30 (22H, m), 6.54 (1H, d, J = 1.5 Hz), 6.48 (1H,
d, J = 1.5 Hz), 5.23 (6H, s), 5.17 (2H, s), 4.28 (2H, t, J = 6.3 Hz),
3.70 (3H, s), 3.05 (2H, t, J = 6.3 Hz), 2.70–2.68 (4H, m), 1.70–
1.68 (4H, m), 1.52–1.50 (2H, m); 13C NMR (75 MHz, CDCl3) δ
173.6, 162.8, 159.9, 158.5, 152.5, 152.0, 141.5, 137.5, 136.8,
135.6, 128.7, 128.5, 128.1, 127.8, 127.5, 127.3, 125.8, 109.6,
108.2, 97.2, 93.6, 75.1, 71.3, 70.4, 67.3, 59.8, 57.2, 54.9, 25.6,
23.8.
59.0, 54.3, 53.4, 22.8; HRMS (ESI) m/z: calcd for C22H23NO7
([M-HCl]+) 413.1469, found 413.1605.
4.2.29. 2-(3,4-Dihydroxyphenyl)-7-hydroxy-3-methoxy-5-(2-
morpholinoethoxy)-4H-chromen-4-one hydrochloride (4c).
The compound 4c was prepared from 9c using the procedure
1
described for 4a. Yield 74%. H NMR (300 MHz, D2O) δ 6.75
(1H, s), 6.65 (1H, d, J = 8.7 Hz), 6.21 (1H, d, J = 8.7 Hz), 5.87
(1H, s), 5.79 (1H, s), 4.13–4.08 (4H, m), 3.84 (2H, t, J = 7.2 Hz),
3.53–3.50 (4H, m), 3.42 (3H, s), 3.26 (2H, t, J = 7.2 Hz); 13C
NMR (75 MHz, D2O) δ 174.7, 161.4, 157.5, 157.0, 152.7, 146.7,
143.3, 139.0, 120.3, 114.9, 106.7, 97.5, 96.2, 63.9, 62.1, 59.3,
55.8, 52.1; HRMS (ESI) m/z: calcd for C22H23NO8+ ([M-HCl]+)
429.1418, found 429.1479.
4.2.24.
7-(Benzyloxy)-3-methoxy-5-(2-(pyrrolidin-1-
yl)ethoxy)-2-(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one
(9f). The compound 9f was prepared from 8b (1.0 mmol) and 1-
(2-chloroethyl)pyrrolidine hydrochloride (1.3 mmol) using the
1
procedure described for 9a. Yield 68%. H NMR (300 MHz,
CDCl3) δ 7.50–7.32 (22H, m), 6.54 (1H, d, J = 1.5 Hz), 6.47 (1H,
d, J = 1.5 Hz), 5.23 (6H, s), 5.18 (2H, s), 4.29 (2H, t, J = 5.4 Hz),
3.69 (3H, s), 3.19 (2H, t, J = 5.4 Hz), 2.86–2.84 (4H, m), 1.90–
1.88 (4H, m); 13C NMR (75 MHz, CDCl3) δ 173.7, 162.8, 159.8,
158.5, 152.5, 152.1, 141.5, 140.2, 137.4, 136.8, 135.5, 128.7,
128.4, 128.1, 127.8, 127.4, 127.2, 125.8, 109.5, 108.2, 97.2, 93.7,
75.1, 71.2, 70.4, 68.1, 59.8, 54.7, 54.2, 50.5, 23.4.
4.2.30. 5-(2-(Diethylamino)ethoxy)-2-(3,4-dihydroxyphenyl)-
7-hydroxy-3-methoxy-4H-chromen-4-one hydrochloride (4d).
The compound 4d was prepared from 9d using the procedure
1
described for 4a. Yield 74%. H NMR (300 MHz, D2O) δ 6.61
(1H, s), 6.44 (1H, d, J = 8.7 Hz), 6.07 (1H, d, J = 8.7 Hz), 5.68
(1H, s), 5.54 (1H, s), 4.67–4.66 (2H, m), 3.27–3.26 (2H, m), 3.15
(3H, s), 3.13–3.03 (4H, m), 1.13 (6H, t, J = 7.2 Hz); 13C NMR
(75 MHz, D2O) δ 174.6, 161.4, 157.5, 156.9, 152.7, 146.7, 143.4,
139.1, 127.1, 120.4, 114.4, 106.7, 100.2, 97.7, 96.0, 62.7, 59.2,
50.9, 47.5, 8.4; HRMS (ESI) m/z: calcd for C22H25NO7+ ([M-
HCl]+) 415.1626, found 415.1640.
4.2.25. 7-(Benzyloxy)-3-methoxy-5-(2-morpholinoethoxy)-2-
(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one (9g). The
compound 9g was prepared from 8b (1.0 mmol) and 4-(2-
chloroethyl)morpholine hydrochloride (1.3 mmol) using the
1
procedure described for 9a. Yield 56%. H NMR (300 MHz,
4.2.31. 7-Hydroxy-3-methoxy-5-(2-(piperidin-1-yl)ethoxy)-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(4e). The compound 4e was prepared from 9e using the
CDCl3) δ 7.48–7.32 (22H, m), 6.54 (1H, d, J = 2.1 Hz), 6.45 (1H,
d, J = 2.1 Hz), 5.22 (6H, s), 5.17 (2H, s), 4.24 (2H, t, J = 5.7 Hz),
3.79–3.76 (4H, m), 3.68 (3H, s), 2.99 (2H, t, J = 5.7 Hz), 2.74–
2.72 (4H, m); 13C NMR (75 MHz, CDCl3) δ 173.5, 162.8, 159.9,
158.5, 152.5, 152.1, 141.5, 140.2, 137.5, 136.8, 135.6, 128.7,
128.5, 128.1, 127.8, 127.5, 127.2, 125.8, 109.7, 108.2, 97.4, 93.6,
75.1, 71.3, 70.4, 67.7, 66.9, 59.8, 57.0, 54.2, 36.4, 31.3.
1
procedure described for 4a. Yield 77%. H NMR (300 MHz,
D2O) δ 6.15 (2H, s), 5.52 (2H, s), 4.68–4.65 (2H, m), 3.50–3.24
(4H, m), 3.49 (3H, s), 2.82 (2H, br s), 1.75–1.33 (6H, m); HRMS
(ESI) m/z: calcd for C23H25NO8+ ([M-HCl]+) 443.1575, found
443.1665.
4.2.26. 7-(Benzyloxy)-5-(2-(diethylamino)ethoxy)-3-methoxy-
2-(3,4,5-tris(benzyloxy)phenyl)-4H-chromen-4-one (9h). The
compound 9h was prepared from 8b (1.0 mmol) and 2-chloro-
N,N-diethylethanamine hydrochloride (1.3 mmol) using the
4.2.32. 7-Hydroxy-3-methoxy-5-(2-(pyrrolidin-1-yl)ethoxy)-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(4f). The compound 4f was prepared from 9f using the procedure
1
described for 4a. Yield 43%. H NMR (300 MHz, D2O) δ 6.14
1
procedure described for 9a. Yield 63%. H NMR (300 MHz,
(2H, s), 5.56 (2H, s), 4.70–4.65 (2H, m), 3.52–3.35 (4H, m), 3.26
(3H, s), 2.99–2.98 (2H, m), 2.01–1.84 (4H, m); 13C NMR (75
MHz, D2O) δ 174.7, 161.4, 157.5, 156.8, 152.2, 143.9, 139.2,
135.1, 119.3, 107.1, 106.4, 97.1, 95.8, 79.7, 63.7, 59.1, 54.5, 53.4,
22.7; HRMS (ESI) m/z: calcd for C22H24NO8+ ([M-Cl]+) 430.1496,
found 430.1485.
CDCl3) δ 7.52–7.33 (22H, m), 6.54 (1H, d, J = 2.1 Hz), 6.49 (1H,
d, J = 2.1 Hz), 5.24 (6H, s), 5.19 (2H, s), 4.19 (2H, t, J = 6.6 Hz),
3.72 (3H, s), 3.11 (2H, t, J = 6.6 Hz), 2.74 (4H, q, J = 7.2 Hz),
1.14 (6H, t, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3) δ 173.6,
162.8, 160.2, 158.5, 152.5, 151.9, 141.5, 140.2, 137.5, 136.8,
135.6, 128.7, 128.5, 128.3, 128.1, 127.8, 127.4, 127.3, 125.9,
109.6, 108.2, 97.1, 93.6, 75.3, 71.3, 70.4, 68.2, 59.9, 51.4, 47.9,
11.8.
4.2.27. 2-(3,4-Dihydroxyphenyl)-7-hydroxy-3-methoxy-5-(2-
(piperidin-1-yl)ethoxy)-4H-chromen-4-one hydrochloride (4a).
To a solution of 9a in EtOH and cyclohexene (1:1) was added a
catalytic amount of Pd(OH)2/C. The reaction mixture was
refluxed (60–70 °C) for the period of time (TLC monitoring),
filtered through Celite and concentrated. The residue was
dissolved in absolute EtOH and then saturated with HCl gas at
0 °C until the mixture become acidic. The resulting precipitate
4.2.33.
7-Hydroxy-3-methoxy-5-(2-morpholinoethoxy)-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(4g). The compound 4g was prepared from 9g using the
procedure described for 4a. Yield 42%. H NMR (300 MHz,
1
D2O) δ 6.27 (2H, s), 5.74 (2H, s), 4.02–3.93 (4H, m), 3.78 (2H,
br s), 3.46–3.40 (4H, m), 3.39 (3H, s), 3.20–3.18 (2H, m); HRMS
(ESI) m/z: calcd for C22H23NO9+ ([M-HCl]+) 445.1367, found
445.1391.
4.2.34. 5-(2-(Diethylamino)ethoxy)-7-hydroxy-3-methoxy-2-
(3,4,5-trihydroxyphenyl)-4H-chromen-4-one hydrochloride
(4h). The compound 4h was prepared from 9h using the
9

1
procedure described for 4a. Yield 71%. H NMR (300 MHz,
from the bifurcation of the internal carotid artery (ICA)
immediately after ligation of the ipsilateral common and external
carotid artery. The cylinder was made of 4-0 nylon surgical
thread 17 mm long coated with silicone mixed with a hardener to
increase the thickness of the distal 5 mm to 0.3–0.4 mm. The
proximal tip of the thread was heated, creating a globular stopper
for embolization and for easy removal of the cylinder. After
introducing the embolus, the ICA was ligated just distal to the
point of insertion. The embolus extended from the bifurcation of
the ICA to the proximal portion of the anterior cerebral artery.
The origins of the right MCA and posterior communicating
artery were occluded by the silicone rubber cylinder. Surgery was
performed within 15 min and body temperature was kept within
normal limits with a heating pad. Following surgery, anesthesia
was discontinued and only rats showing neurological deficits
characterized by left hemiparesis with upper extremity dominant
and/or circling to the left were included in cerebral ischemic
groups. After 2-h transient MCAO, reperfusion was achieved by
pulling the thread out of the ICA about 10 mm under the same
anesthetic conditions as during surgery.
D2O) δ 6.15 (2H, s), 5.56 (1H, s), 5.50 (1H, s), 4.71–4.70 (2H, m),
3.25–3.23 (2H, m), 3.23 (3H, s), 3.08–3.07 (4H, m), 1.13–1.12
(6H, m); 13C NMR (75 MHz, D2O) δ 174.6, 161.4, 157.3, 156.7,
152.1, 143.9, 139.2, 135.2, 127.1, 119.3, 107.1, 106.4, 97.2, 95.7,
62.3, 59.0, 50.9, 47.4, 8.3; HRMS (ESI) m/z: calcd for
C22H25NO8+ ([M-HCl]+) 431.1575, found 431.1672.
4.3. Antioxidant effects of compounds 3a-3h and 4a-4h
4.3.1. DPPH radical scavenging activity assay
The antioxidant activities of the synthesized compounds were
assessed by examining their abilities to scavenge the 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free radical. Reaction
mixtures containing test samples (dissolved in EtOH) and 100
µM of ethanolic DPPH solution in 96-well plates were incubated
at 37 °C for 30 min. Absorbances were measured at 515 nm.
Percent inhibitions were calculated versus ethanol-treated
controls. IC50 values denote the concentration required to
scavenge 50% of DPPH radicals.
4.4.1. Histological evaluation of brain injury
To examine cerebral infarction caused by transient MCAO,
rats were sacrificed by decapitation 24 h after onset of ischemia.
After removing brains, seven serial coronal slices of 2-mm
thickness were made starting at 1 mm from the frontal pole,
incubated in a 2% solution of TTC in normal saline at 37 °C for
60 min for vital staining, and fixed in 10% phosphate-buffered
formalin for photography. The areas of the absolute infarct area
in the cortex and striatum, and the total areas of both hemispheres
were measured for each slice using a computerized image
analysis system (Optimas). The absolute infarct volume was
calculated by multiplying the area by the slice thickness and
summing the volumes. The corrected infarct area in a slice was
calculated to compensate for the effect of brain edema by
subtracting the area of normal tissue in the ipsilateral hemisphere
from the total area of the contralateral hemisphere. Corrected
total infarct volume was then calculated by multiplying the area
by the slice thickness and summing the volumes. Hemispheric
swelling representing tissue edema was expressed as the percent
increase in the size of the ipsilateral hemisphere compared with
the contralateral hemisphere (% edema).
4.3.2. Superoxide anion radical scavenging activity assay by
xanthine oxidase system
The reaction mixture consisted of 40 mM sodium carbonate
buffer (pH 10.2) containing 0.1 mM xanthine, 0.1 mM EDTA, 50
µg protein/ml bovine serum albumin, 25 mM NBT and 1.4 ×10-8
units xanthine oxidase (EC 1.2.3.2) in final volume of 200 µl.
After incubation at 25 °C for 20 min, the reaction was terminated
by addition of 6.6 µl of 6 mM CuCl2. The absorbance of
formazan was determined at 560 nm. IC50 values denote the
concentration of samples required to scavenge 50% superoxide
anion radicals.
4.3.3. Lipid peroxidation inhibitory activity assay
The effects of the synthesized compounds on lipid
peroxidation induced by an iron-ascorbic acid mixure were
determined in rat liver homogenate. In brief, rat liver homogenate
(300 µl, 11 mg protein/ml) was incubated with 10 µM Fe2SO4,
0.4 mM ascorbic acid and various concentrations of the test
compounds in 50 mM Tris-HCl (pH 7.5) in a total volume of 1
ml at 37 °C for 30 min. After incubation, lipid peroxidation
levels were determined by measuring the formation of
thiobarbituric acid-reactive substance (TBARS). Reactions were
terminated by adding 2 ml of a solution of 0.375% thiobarbituric
acid in 15% trichloroacetic acid containing 0.25 N HCl and
0.01% butylated hydroxytoluene (TBA-TCA reagent). Mixtures
were then heated at 95 °C for 30 min, cooled, and centrifuged at
5000 × g for 10 min. The absorbances of supernatants were
measured at 535 nm. Protein contents in liver homogenates were
determined using the Bradford method using bovine serum
albumin as a standard.
4.4.2. Evaluation of neurological deficits
Neurological deficits in the rats were measured 30 min and 24
h after onset of ischemia. The neurobehavioral tests consisted of
the following. (1) In the forelimb flexion test, the rat was held in
the air by the tail, and the degree of left forelimb flexion was
scored between 0 and 3 (0, no movement on left side; 1, limited
movement on left side; 2, less extended or slower movement on
left side; 3, symmetrical movement). (2) In the duration of
forelimb flexion test, the rat was held in the air by the tail, and
the score (0 to 4) was determined by the duration of left forelimb
flexion during a 10-second period (0, 8–10 s; 1, 6–8 s; 2, 4–6 s; 3,
2–4 s; 4, 0–2 s). (3) In the symmetry of movement/forepaw
outstretching test, the rat was made to walk along the bench on
its forelimbs while being held by the tail to keep the hindlimbs in
the air. Symmetry in forepaw outstretching was observed and
scored between 0 and 3 (0, left forelimb does not move; 1, left
forelimb moves minimally and rat circles; 2, left forelimb
outstretches less than right; 3, forelimbs outstretched and rat
walks normally). Rats were scored on a ranking scale of 0 to 10,
which reflects the cumulative score of the individual tests, with a
score of 10 reflecting normal behavior.
4.4. Neuroprotective effects of compounds 3a and 3h
4.4.1. Preparation of a rat model of transient
focal cerebral ischemia
All procedures were conducted in accordance with
Institutional Animal Care and Use Committee (IACUC)
guidelines of Korea Institute of Science and Technology. All
procedures were approved by Korea Institute of Science and
Technology IACUC (Approval Number: AP-2010L1001). Under
anesthesia of male Sprague-Dawley rats (250 to 300 g) purchased
from Daehan Biolink (Chungbuk, Korea) with a gas mixture of
70% N2O and 2% isoflurane (the balance being O2), the right
MCA was occluded with a silicone rubber cylinder introduced
4.5. Determination of Aqueous Solubility
Stock solutions of drugs were made up in MeOH and used to
calibrate the HPLC (peak area in nanomoles, assuming a linear
10

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was saturated at room temperature and the saturated solution was
sonicated for 30 min at the same temperature. After standing for
an additional 30 min, the samples were centrifuged at 13000 rpm
for 3 min, and the concentration of drug in the supernatant was
determined by HPLC, using the calibration curve determined
previously.24
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This work was supported by the Basic Science Research
Program of the Korean National Research Foundation (NRF)
funded by the Ministry of Education, Science, and Technology of
Korea (#2012-006431) and by an intramural grant (2E24080)
from the Korea Institute of Science and Technology.
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