Synthesis, characterization and vasculoprotective effects of nitric oxide-donating derivatives of chrysin

Article abstract of DOI:10.1016/j.bmc.2010.03.056

Vascular complications are major causes of disability and death in patients with diabetes mellitus. It is often characterized by endothelial dysfunction. Studies have shown that either the loss of nitric oxide bioactivity or the decreased biosynthesis of

Full text of DOI:10.1016/j.bmc.2010.03.056

Bioorganic & Medicinal Chemistry 18 (2010) 3020–3025
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, characterization and vasculoprotective effects of nitric
oxide-donating derivatives of chrysin
Xiao-Qing Zou a,b, Sheng-Ming Peng b, Chang-Ping Hu a, Li-Feng Tan b, Qiong Yuan a, Han-Wu Deng a,
Yuan-Jian Li a,
*
a Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha 410078, China
b Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Vascular complications are major causes of disability and death in patients with diabetes mellitus. It is
often characterized by endothelial dysfunction. Studies have shown that either the loss of nitric oxide
bioactivity or the decreased biosynthesis of NO is a central mechanism in endothelial dysfunction. As
such, the delivery of exogenous NO is an attractive therapeutic option that has been used to slow the pro-
gress of diabetic vascular complications. In this paper, a novel group of hybrid nitric oxide-releasing chry-
sin derivatives was synthesized. The results indicated that all these chrysin derivatives exhibited in vitro
inhibitory activities against aldose reductase and advanced glycation end-products formation. And some
of them were even found to increase the glucose consumption of HepG2 cells. Furthermore, all com-
pounds released NO upon incubation with phosphate buffer at pH 7.4. These hybrid ester NO donor pro-
drugs offer a potential drug design concept for the development of therapeutic or preventive agents for
vascular complications due to diabetes.
Received 6 February 2010
Revised 22 March 2010
Accepted 23 March 2010
Available online 27 March 2010
Keywords:
Hybrid
NO donor
Chrysin derivatives
Vasculoprotection
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
on grafting an NO-releasing moiety to chrysin derivatives via a vari-
ablespacer. Wesupposethatthenew pharmacodynamichybridswill
Diabetes is a chronic metabolic disorder characterized by
hyperglycemia and followed by micro and macrovascular compli-
cations. Vascular endothelial dysfunction has been implicated in
the development and progress of diabetic vascular complications.1
An early marker of endothelial dysfunction is the reduction of
endothelium-dependent vasodilation due to the reduced bioavail-
ability of nitric oxide (NO).2 Many common time-honored drugs
(e.g., glycerol trinitrate) act via the release of exogenous NO. Hy-
brid NO donor drugs represent a novel type of drug that retains
the pharmacological activity of the parent compound but also
has the biological actions of NO.3
Chrysin (5,7-dihydroxyflavone), a natural, widely distributed
flavonoid, has been reported to possess diverse biologically active
properties, including antioxidant,4,5 anticancer,6 anxiolytic,7 anti-
inflammatory,8,9 anti-diabetic10 and anti-glucosidase characteris-
tics.11 Recently, several researchers have attempted to modify the
profile of chrysin and found that some chrysin derivatives have di-
verse activities including anti-diabetic effects.12–17 Shin et al. synthe-
sizeda seriesofchrysin derivatives inwhichthe butylcompoundwas
found to have the most potent hypoglycemic effect on diabetic
mice.17 This study aims to investigate a synthesis approach based
present the combined advantages of hypoglycemic effect with a slow
release of NO, thus mimicking the effects of endogenous NO, which
can improve endothelial dysfunction and prevent the progression
of diabetic complications.
Research has indicated that no matter what is the cause of dia-
betes, the result is always hyperglycemia.18 Hyperglycemia in-
creases the risk of both micro and macrovascular diseases in
patients with diabetes.19 In both type 1 and type 2 diabetes, large,
prospective clinical studies have shown a strong relation between
time-averaged mean values of glycemia, measured as glycated
hemoglobin A1c (HbA1c), and diabetic complications.20,21 These
studies are the basis for the American Diabetes Association’s cur-
rent recommended treatment goal that HbA1c should be <7%.22
Selvin performed a meta-analysis of 13 cohort studies and indi-
cated that for type 2 diabetes, a 1% point increase in HbA1c was
associated with a significant 18% increase in the risk of coronary
heart disease or stroke and a 28% increase in the risk of peripheral
vascular disease.23 The metabolism of excess glucose drives several
damage pathways, such as oxidative stress, generation of advanced
glycation end-products (AGE), increased aldose reductase (AR)-re-
lated polyol pathway flux, activation of protein kinase C, increased
hexosamine pathway flux, which leads to endothelial dysfunction.
This indicates that hyperglycemia is a major cause of the vascular
complications in diabetes cases.
* Corresponding author. Tel./fax: +86 731 82355078.
E-mail address: yuan_jianli@yahoo.com (Y.-J. Li).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.056

X.-Q. Zou et al. / Bioorg. Med. Chem. 18 (2010) 3020–3025
3021
A great deal of evidence indicates that the non-enzymic glyca-
tion of proteins is implicated in a number of biochemical abnor-
malities associated with diabetes. The glycation reaction occurs
between the carbonyl group of sugars and the amino group of pro-
teins, which finally results in the formation of AGE. The increased
serum concentration of AGE in patients with diabetes has been
associated with the dysfunction of the vascular endothelium.24
Since studies have shown the role of AGE in promoting diabetic
complications, it is believed that the inhibition of AGE formation
may prevent the progression of diabetic complications.
The synthesis of compounds 7a–c and 8a–c was accomplished
according to the general pathway illustrated in Scheme 2. An alkyl
chain with a bromide group was introduced to the 7-position of
chrysin 1 by a selective reaction with an appropriate dibromo al-
kane, yielding the 7-bromo alkane derivatives 6a–c, which were
subsequently converted to the nitro esters 7a–c via treatment with
silver nitrate (AgNO3) in anhydrous acetonitrile. These compounds
were then reacted with acetic anhydride to produce the final
substituted nitro esters 8a–c.
AR is the first, rate-limiting enzyme in the polyol pathway of
glucose metabolism, which converts glucose into sorbitol. The
hyperactivity of AR has been linked to the development of cardio-
vascular and neurological complications in diabetic patients.25 In
line with this, patients treated with AR inhibitors showed an
improvement of vascular and other complications of diabetes, such
as peripheral neuropathy, retinopathy, and cataracts.25 Studies of
this nature make the development of a potent AR inhibitor an obvi-
ous and attractive strategy to prevent or delay the onset and pro-
gression of diabetic complications. In this light, some research
has shown that NO donors can prevent AR activation and sorbitol
accumulation during the onset of diabetes.26,27
We report herein the synthesis and characterization of several
NO-donating derivatives of chrysin. The NO-releasing capacities,
glucose consumption promotion, AR and AGE inhibitory effects of
theses derivatives were explored in vitro. We hope the results to
be of value in further understanding the potential uses of chrysin
NO-donating derivatives for the development of therapeutic and
preventive agents for vascular complications of diabetes.
3. Results and discussion
In the present study, we investigated AR and AGE assays, NO-
releasing capacities were also determined for all the target com-
pounds in vitro. Structure–activity relationships acquired for the
assays indicated that the hybrid nitrooxyl carboalkoxy methyl
chrysin (5a–c) resulted in high inhibitory activities on AGE forma-
tion (IC50 = 43.89 6.76 to 137.6 8.86
high inhibitory effects on AR (IC50 = 0.13 0.03 to 0.30 0.01
M). The potency of AGE formation inhibition was 6–18.8 times
lM, Table 1), as well as
l
that of the positive reference compound aminoguanidine. AR inhi-
bition showed a potency that was over 10 times higher than the
positive reference compound quercetin. Derivatives 5b, 7b, and
8b exerted higher AR inhibition activities than the other serial
compounds, indicating that modified compounds with butyl
groups were more effective in inhibiting AR. NO appeared to be
released from all the chrysin derivatives upon incubation with
phosphate-buffered saline solution (PBS at pH 7.4) in the presence
of
L-cysteine. The percentage of released NO varied from
1.21 0.20% to 1.77 0.10%, indicating a slow release. The amount
of NO released from sodium nitroprusside (SNP), however, was
substantially higher (10.42 1.80%). These results should be as-
sessed based on the actual additional amount of NO needed by
the body. The concentrations of NO required to mediate primarily
protective effects are extremely low (i.e., picomolar to nanomolar
range). At higher concentrations, particularly under conditions of
oxidative stress, NO is highly cytotoxic, a feature that is exploited
by inflammatory cells in response to invading pathogens.28 In our
2. Chemistry
The preparation of the nitrate derivatives follows the synthetic
routes illustrated in Scheme 1. Chrysin 1 was reacted with bromo-
acetate to produce compound 2. Subsequent hydrolysis of this
compound and reaction with a dibromo alkane produced com-
pounds 4a–c. These compounds were then reacted with AgNO3
(silver nitrate), producing products 5a–c.
C2H5OOCCH2O
O
HO
O
K2CO3 CH3COCH3
BrCH2COOC2H5
OH O
OH O
2
1
HOOCCH2O
O
C2H5OOCCH2O
O
KOH
CH3OH
OH O
OH O
3
2
Br(CH2)nOOCCH2O
O
Br(CH2)nBr
HOOCCH2O
O
4a, n = 2
4b, n = 4
4c , n = 6
OH O
Et3N, CH3COCH3
OH O
3
4a-4c
AgNO3
CH3CN
O2NO(CH2)nOOCCH2O
O
Br(CH2)nOOCCH2O
O
5a, n = 2
5b, n = 4
5c , n = 6
OH O
OH O
5a-5c
4a-4c
Scheme 1. Synthesis of chrysin derivatives 5a–c.

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X.-Q. Zou et al. / Bioorg. Med. Chem. 18 (2010) 3020–3025
Br(CH2)nO
Br(CH2)nBr
HO
O
O
O
O
6a, n = 2
6b, n = 4
6c, n = 6
K2CO3, acetone
OH
OH
6a-6c
1
AgNO3
CH3CN, lucifugous
O2NO(CH2)nO
O
O2NO(CH2)nO
O
acetic anhydride
K2CO3, acetone
H3C
C
O
O
O
OH
O
8a-8c
7a-7c
8a, n = 2
8b, n = 4
8c, n = 6
7a, n = 2
7b, n = 4
7c , n = 6
Scheme 2. Synthesis of chrysin derivatives 7a–c and 8a–c.
Table 1
Structure and in vitro AR, AGE inhibition and NO-releasing properties of the target compounds
R2
O
O
R1
Compound
Structure
R2
AR inhibitiona
AGE inhibitionb
IC50 (lmol/L)
%NO releasedc
R1
0.1%DMSO
Chrysin
5a
5b
5c
7a
7b
7c
8a
8b
OH
OH
OH
OH
OH
OH
OH
CH3COO
CH3COO
CH3COO
OH
7.79 0.57
0.30 0.01
0.13 0.03
0.21 0.07
0.49 0.006
0.29 0.009
0.71 0.06
0.86 0.02
0.39 0.04
0.42 0.03
63.54 6.42
43.89 6.76
52.47 3.73
137.6 8.86
P300
75.5 8.07
P300
P300
NO3(CH2)2COOCH2O
NO3(CH2)4COOCH2O
NO3(CH2)6COOCH2O
NO3(CH2)2O
NO3(CH2)4O
NO3(CH2)6O
NO3(CH2)2O
NO3(CH2)4O
NO3(CH2)6O
1.77 0.10
1.26 0.09
1.38 0.06
1.67 0.15
1.43 0.04
1.21 0.20
1.68 0.11
1.48 0.16
1.43 0.22
P300
P300
8c
OH
OH
HO
O
O
Quercetind
2.85 0.04
OH
OH
NH
Aminoguanidinee
826.22 9.26
H2N HN
NH2
SNPf
10.42 1.80
Each value represents the mean S.D. (n = 3).
a
The concentration required for a 50% inhibition of the decrease in the optical density of NADPH at 340 nm relative to 0.1% DMSO. IC50 values were calculated from the
dose inhibition curve.
b
The concentration required for a 50% inhibition of the fluorescence intensity of AGE relative to 0.1% DMSO, IC50 values were calculated from the dose inhibition curve.
Percent of nitric oxide released based on a theoretical maximum release of 1 mol of NO/mol of the target compounds.
Quercetin was used as positive control for AR inhibition test.
Aminoguanidine (AG) was used as positive control for AGE inhibition test.
c
d
e
f
SNP (sodium nitroprusside) was used as positive control for NO releasing test.

X.-Q. Zou et al. / Bioorg. Med. Chem. 18 (2010) 3020–3025
3023
8
6
4
2
0
study, the release of adequate amounts of NO required to provide
protective effects were balanced with the concentration range re-
quired for the sufficient activity of chrysin derivatives.
*
*
Hyperglycemia is the primary cause of vascular complications
in diabetes. To date, the benefit of glycemic control with respect
to the macrovascular complications in diabetes remains controver-
sial. Nevertheless, the importance of glycemic control in prevent-
ing the microvascular complications of retinopathy, nephropathy,
and neuropathy in patients with type 2 diabetes has been consis-
tently demonstrated over the past 20 years.29 Some researchers
demonstrated that NO could stimulate glucose metabolism or
transport in adipocytes or skeletal muscle.30–32 Shin et al. have
found that a series of chrysin alkyl derivatives have potent hypo-
glycemic effect in vivo.17 To evaluate the hypoglycemic effects of
the target compounds, we determined the glucose consumption
of HepG2 cells treated with chrysin, chrysin derivatives, and posi-
tive reference rosiglitazone (Fig. 1). The results showed that 5b, 5c,
7a, 7b, 7c, and rosiglitazone significantly promoted the glucose
consumption of HepG2 cells compared to the blank vehicle 0.1%
DMSO (P <0.05). In contrast, chrysin significantly reduced glucose
consumption (P <0.05 vs 0.1% DMSO, Fig. 2). It is of note that 5a,
8a, 8b, 8c had no effect on the glucose consumption of HepG2 cells.
An O7-nitrooxyalkyl nitric oxide donor moiety is attached to the
chrysin parent compound and its O7-[(nitrooxyl)alkyloxycarbonyl]
methyl analogs were synthesized (5a–c). Furthermore, a novel
class of hybrid ester prodrugs (7a–c) was synthesized. AR, AGE for-
mation, and glucose consumption assays, as well as NO-releasing
capacities, showed that: (i) the [(nitrooxyl)ethoxycarbonyl] methyl
derivatives are more potent inhibitors of AR and AGE, (ii) the par-
ent compound chrysin is a potent AGE inhibitor, (iii) all the hybrid
ester prodrugs release NO slowly upon incubation with PBS in the
*
*
*
*
*
Figure 2. Effects of 10 lmol/L chrysin, chrysin derivatives (5a–c, 7a–c and 8a–c)
and rosiglitazone on glucose consumption of HepG2 cells (values are mean S.D.;
n = 3; *P <0.05 vs 0.1% DMSO).
blank. After the cells reached 80–90% confluence, the medium was
replaced by DMEM supplemented with 1% FBS. Twelve hours later,
the medium was removed and the same culture medium contain-
ing target compounds, chrysin or rosiglitazone (standard product)
was added to wells. After 24 h of treatment, glucose was assayed in
10 lL of medium by enzymatic methods with diagnostic kits (Nan-
jing Jiancheng Bioengineering Inst.). Data were expressed as glu-
cose concentrations of blank wells subtracting the remaining
glucose in the cell plated wells. Following the glucose measure-
ment in the medium, a MTT assay was used to monitor the cell
number and to adjust the glucose consumption values.
presence of L-cysteine, and (iv) the O7-nitrooxyethyl chrysin deriv-
atives (7a–c), O7-[(nitrooxyl)butoxycarbonyl] methyl analog (5b),
and O7-[(nitrooxyl)hexoxycarbonyl] methyl analog (5c) signifi-
cantly promotes the glucose consumption of HepG2 compared to
the blank vehicle 0.1% DMSO. In order to test our hypothesis that
the 5-OH group contributes to the observed activities, compounds
8a–c or O5-acetyl, O5-butyryl, and O5-caproyl, respectively, were
designed in a flavone skeleton. The changes made decreased the
inhibition of AR and AGE formation, as well as glucose consump-
tion promotion, which suggests that the 5-OH group is the key
group that determines the biological activities.
4.1.2. AR inhibitory activity34
After homogenization, centrifugation, (NH4)2SO4 fractionation
(30–75%) and dialysis, the crude AR from calf lenses was obtained.
The inhibitory activity of the compounds on aldose reductase was
carried out using a 200
(750 g/mL protein), and different concentrations of the com-
pounds (0.1–10 mol/L) in 50 mM PBS (pH 6.0) containing 5 mM
b-mercaptoethanol, 0.24 mM NADPH, 0.4 M Li2SO4 and 2.5 mM of
glyceraldehyde (substrate) were added. The reaction was initiated
by addition of glyceraldehyde and the decrease in the optical den-
sity of NADPH at 340 nm was recorded for 3 min. IC50 of the com-
pounds was calculated using Sigmaplot software and expressed as
mean S.D. of triplicate experiments. The flavonoid quercetin was
used as a reference in the AR assay.
lL volume optimized amount of enzyme
l
l
In conclusion, these hybrid ester NO donor prodrugs offer a po-
tential drug design concept for the development of therapeutic or
preventive agents for vascular complications of diabetes.
4. Experimental
4.1. Biological activity
4.1.3. Inhibitory activity of AGE formation35
To prepare the AGE reaction solution, 10 mg/mL of bovine ser-
um albumin in 50 mM PBS (pH 7.4) was added to 0.2 M glucose,
and 0.02% sodium azide was added to prevent bacterial growth.
4.1.1. Glucose consumption assay33
HepG2 cells (obtained from the Cell Culture Center of the Cen-
tral South University, Changsha, China) grown in high glucose
DMEM (purchased from Hyclone) containing 10% FBS (obtained
from Sijiqing Biological Engineering Materials, Hangzhou, China)
were plated into 96-well tissue culture plates with some wells left
The reaction mixture (950
lL) was then mixed with various con-
centrations of the target compounds (1–300
l
mol/L). After incu-
bating at 37 °C for 14 d, the fluorescence intensity of AGE was
determined by a LS-55 fluorospectrophotometer (PE, USA) with
excitation and emission wavelengths at 350 nm and 420 nm,
respectively. IC50 of the compounds was calculated using sigma-
plot software and expressed as mean S.D. of triplicate experi-
ments. Aminoguanidine hydrochloride was used as a reference
compound.
4.1.4. Detection of nitrite36
A solution of the appropriate compound (20
lL) in dimethyl-
Figure 1. The structure of rosiglitazone.
sulfoxide (DMSO) was added to 2 mL of 1:1 v/v mixture of

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X.-Q. Zou et al. / Bioorg. Med. Chem. 18 (2010) 3020–3025
50 mM PBS (pH 7.4) with MeOH, containing of 5 Â 10À4
M
L
-cys-
carded. Recrystallization was performed with ethyl acetate/petro-
leum ether (4a: 2.01 g, 48% yield). Mp 122–124 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 12.75 (s, 1H), 7.86 (d, 2H, J = 8 Hz),
7.53–7.55 (m, 3H), 6.64 (s, 1H), 6.53 (s, 1H), 6.36 (s, 1H), 4.71 (s,
2H), 4.46–4.48 (m, 2H), 3.37–3.39 (m, 2H).
teine. The final concentration of target compounds was 10À4 M.
After 1 h at 37 °C, 1 mL of the reaction mixture was treated with
250 lL of Griess reagent [sulfanilamide (4 g), N-naphthylethylen-
ediamine dihydrochloride (0.2 g), 85% phosphoric acid (10 mL) in
distilled water (final volume: 100 mL)]. After 10 min at room tem-
perature, the absorbance was measured at 540 nm. Sodium nitrite
standard solutions (10–80 nmol/mL) were used to construct the
calibration curve. The results were expressed as the percentage
of NO released (n = 3) relative to a theoretical maximum release
of 1 mol NO/mol of test compound.
Compounds 4b and 4c were synthesized according to the meth-
od for 4a.
Compound 4b: 46.0% yield, mp 127–128 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.76 (s, 1H), 7.85 (d, 2H, J = 8 Hz), 7.53–7.57 (m,
3H), 6.63 (s, 1H), 6.54 (s, 1H), 6.38 (s, 1H), 4.68 (s, 2H), 4.39 (s,
2H), 4.14 (m, 2H), 1.76 (s, 4H).
Compound 4c: 34.7% yield, mp 100–102 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.77 (s, 1H), 7.86 (d, 2H, J = 8 Hz), 7.50–7.52 (m,
3H), 6.64 (d, 1H, J = 2.8 Hz), 6.50 (s, 1H), 6.33 (s, 1H), 4.68 (s, 2H),
4.37–4.39 (s, 2H), 4.11–4.12 (m, 2H), 1.81 (s, 4H), 1.61–1.68 (m,
4H), 1.23–1.37 (s, 4H).
4.1.5. Statistical analysis
Data were shown as mean S.D. Differences between individual
groups were analyzed by using ANOVA followed by Dunett’s test. A
difference with a P value of <0.05 was considered to be significant.
4.2. Chemical synthesis
4.2.2.4. O7-[(Nitrooxyl)ethoxycarbonyl] methyl chrysin (5a–c). A
solutionof 4a(2.09 g, 5 mmol)wasmixedwithanhydrousacetonitrile
(30 mL) and heated to50 °C. Tothissolution, acetonitrile(20 mL) con-
taining silver nitrate (0.43 g, 2.5 mmol) was dribbled, followed by
heating to 70 °C. The reaction was allowed to proceed for 1 h in the
dark. Filtration was performed to remove any silver bromide precipi-
tates. The filtrate was concentrated and a yellow solid was produced.
This solid was washed in water several times until the runoff was
clear. The solid was then dried and dissolved in dichloromethane.
Any undissolved solids were discarded. The solution was spun and
dried to produce a yellow solid product (5a: 0.53 g, 44.9% yield). Mp
127–129 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 12.76 (s, 1H), 67.89
(d, 2H, J = 8 Hz), 7.53–7.58 (m, 3H), 6.69 (s, 1H), 6.55 (s, 1H), 6.39 (s,
1H), 4.77 (s, 2H), 4.54–4.57 (m, 2H), 3.55–3.57 (m, 2H). EI-MS (m/z):
401 [M]+. Anal. Calcd for C19H15NO9: C, 56.86; H, 3.77; N, 3.49. Found:
C, 56.53; H, 3.95; N, 3.12.
4.2.1. Materials and instrumentation
All chemicals were of reagent grade and commercially available.
Chrysin (>98%) was purchased from ShanXi Huike Co., Ltd, Jiangsu,
China, and used without further purification. All the 1H NMR spec-
tra were recorded on a Bruker AV 400 model Spectrometer in
DMSO-d6. Chemical shifts (d) for 1H NMR spectra were reported
in parts per million of residual solvent protons. ESI-MS spectra
were recorded on a ThermoFinnigan LCD-Advantage mass spec-
trometer. Melting points were measured using a Beijing Biotech
X-4 micromelting point apparatus.
4.2.2. General method for synthesis of compounds 5a–c, 7a–c
and 8a–c
4.2.2.1. O7-(Ethoxycarbonyl) methyl chrysin (2). Anhydrous
potassium carbonate (0.69 g, 5.0 mmol) was added to a solution
of 1 (2.54 g, 10 mmol) in anhydrous acetone (150 mL), followed
by refluxing until the solution became clear. Methyl bromoacetate
(2.3 mL, 20 mmol) was then dribbled into the mixture, and acetone
(10 mL) was added. The solution was refluxed for 24 h and vacuum
filtered. The filter liquor was concentrated to obtain a yellow solid,
which was subsequently washed with petroleum, 1% NaOH, and
water. The solid was dried (2.3 g, 71% yield). Mp 160–162 °C. 1H
NMR (400 MHz, CDCl3) d (ppm): 12.75 (s, 1H), 7.87–7.89 (m, 2H),
7.51–7.57 (m, 3H), 6.68 (s, 1H), 6.53 (d, 1H, J = 2.3 Hz), 6.37 (d,
1H, J = 2.3 Hz), 4.7 (s, 2H), 4.27–4.32 (m, 2H), 1.30–1.34 (m, 3H).
Compounds 5b and 5c were synthesized according to the meth-
od for 5a.
Compound 5b: 75.2% yield, mp 122–123 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.78 (s, 1H), 7.89 (d, 2H, J = 8 Hz), 7.53–7.57 (m,
3H), 6.69 (s, 1H), 6.53 (s, 1H), 6.36 (s, 1H), 4.72 (s, 2H), 4.47 (s,
2H), 4.28 (m, 2H), 1.81 (s, 4H). EI-MS (m/z): 429 [M]+. Anal. Calcd
for C21H19NO9: C, 58.74; H, 4.46; N, 3.26. Found: C, 58.57; H,
4.82; N, 3.01.
Compound 5c: 55.0% yield, mp 97–98 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.77 (s, 1H), 7.89 (d, 2H, J = 8 Hz), 7.53–7.57 (m,
3H), 6.67–6.69 (d, 1H, J = 2.3 Hz), 6.53 (s, 1H), 6.37 (s, 1H), 4.72
(s, 2H), 4.41–4.44 (s, 2H), 4.22–4.26 (m, 2H), 1.81 (s, 4H), 1.68–
1.73 (m, 4H), 1.40 (s, 4H). EI-MS (m/z): 457 [M]+. Anal. Calcd for
C23H23NO9: C, 60.39; H, 5.07; N, 3.06. Found: C, 60.02; H, 5.34; N,
3.01.
4.2.2.2. O7-Carboxymethyl chrysin (3). Potassium hydroxide
(1.68 g, 30 mmol) was added to a solution of 2 (1.70 g, 5.0 mmol)
in anhydrous methanol (120 mL). The solution was then mechani-
cally stirred and heat refluxed for 8 h until a yellow solid that does
not dissolve in methanol was obtained. The solid was dried with
methanol and dissolved in water. Then, the pH was adjusted to
the desired acidity with hydrochloric acid. The solution underwent
vacuum filtration, as well as water washing and drying. (3: 1.4 g,
90% yield). Mp 284–285 °C. 1H NMR (400 MHz, CDCl3) d (ppm):
12.78 (s, 1H), 8.09 (d, 2H, J = 7 Hz), 7.55–7.63 (m, 3H), 7.03 (s,
1H), 6.81 (d, 1H, J = 2.3 Hz), 6.39 (d, 1H, J = 2.3 Hz), 4.83 (s, 2H).
4.2.2.5. O7-Bromethyl chrysin (6a). Anhydrous potassium car-
bonate (0.69 g, 5.0 mmol) was added to a solution of 1 (2.54 g,
10 mmol) in 150 mL of anhydrous acetone, followed by refluxing
until the solution became clear. Then, 1,2-dibromo ethylene
(9.4 g, 50 mmol) was added dropwise, followed by refluxing for
24 h and vacuum filtration. This procedure yielded concentrated
filter liquor, from which a yellow solid was obtained. The solids
were collected, washed with petroleum, 1% NaOH and water,
respectively, then dried (6a: 1.5 g, 42% yield). Mp 157–158 °C.
1H NMR (400 MHz, DMSO-d6) d (ppm): 12.81 (s, 1H), 8.10 (d,
2H, J = 8 Hz), 7.53–7.62 (m, 3H), 7.05 (s, 1H), 6.87 (d, 1H,
J = 2.3 Hz), 36.43 (d, 1H, J = 2.3 Hz), 4.45–4.51 (m, 2H), 3.82–3.84
(m, 2H).
4.2.2.3. O7-[(Bromoethoxyl)carbonyl] methyl chrysin (4a–c). Tri-
ethylamine (3.03 g, 30 mmol) was added to a solution of 3 (3.12 g,
10 mmol) in acetone (100 mL). The mixture was then refluxed for
30 min. 1,2-Dibromoethane (9.40 g, 50 mmol) was dribbled into
the mixture, followed by refluxing for 5 h and filtration to remove
any precipitates that may have formed. The filter liquor was concen-
trated to obtain a solid, which was then washed twice with petro-
leum and ethanol. A light yellow solid was produced. This solid
was dissolved with dichloromethane. Any insoluble solids were dis-
Compounds 6b and 6c were synthesized according to the meth-
od for 6a.
Compound 6b: 74.5% yield, mp 146–147 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.71 (s, 1H), 7.87–7.89 (m, 2H), 7.52–7.56 (m,

X.-Q. Zou et al. / Bioorg. Med. Chem. 18 (2010) 3020–3025
3025
3H), 6.74 (s, 1H), 6.50 (d, 1H, J = 2.3 Hz), 6.36 (d, 1H, J = 2.3 Hz),
4.07–4.10 (m, 2H), 3.48–3.51 (m, 2H), 1.98–2.12 (m, 4H).
Compound 6c: 60% yield, mp 126–127 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.64 (s, 1H), 7.81–7.83 (m, 2H), 7.44–7.51 (m,
3H), 6.60 (s, 1H), 6.43 (d, 1H, J = 2.3 Hz), 6.30 (d, 1H, J = 2.3 Hz),
3.96–3.99 (m, 2H), 3.35–3.38 (m, 2H), 1.76–1.86 (m, 4H), 1.45–
1.46 (m, 4H).
EI-MS (m/z): 414 [M+H]+. Anal. Calcd for C21H19NO8: C, 61.02; H,
4.63; N, 3.39. Found: C, 61.32; H, 4.84; N, 3.12.
Compound 8c: 36.3% yield, mp 87–88 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 7.84–7.86 (m, 2H), 7.50–7.51 (m, 3H), 6.87 (d,
1H, J = 2.3 Hz), 6.60 (s, 2H), 4.46–4.49 (m, 2H), 4.05–4.08 (m, 2H),
2.44 (s, 3H), 1.76–1.87 (m, 4H), 1.52 (s, 4H). EI-MS (m/z): 442
[M+H]+. Anal. Calcd for C23H23NO8: C, 62.58; H, 5.25; N, 3.17.
Found: C, 62.86; H, 5.56; N, 3.02.
4.2.2.6. O7-Nitrooxyethyl chrysin (7a). Compound 6a (1.81 g,
5 mmol) was dissolved in anhydrous acetonitrile, followed by
heating to 50 °C. AgNO3 (2.5 mmol) and acetonitrile (20 mL) were
added. The mixture was stirred by heating to 70 °C in the dark
for 1 h. The precipitate was filtered out, and the solvent was evap-
orated under a vacuum. The resulting residue was dissolved in
ethyl ether and water (7a: 1.44 g, 84% yield). Mp 135–136 °C. 1H
NMR (400 MHz, DMSO-d6) d (ppm): 12.78 (s, 1H), 8.07–8.09 (d,
2H, J = 8 Hz), 7.55–7.61 (m, 3H), 7.03 (s, 1H), 6.81 (d, 1H,
J = 2.3 Hz), 6.39 (d, 1H, J = 2.3 Hz), 4.59(s, 2H), 4.14 (s, 2H). EI-MS
(m/z): 343 [M]+. Anal. Calcd for C17H13NO7: C, 59.48; H, 3.82; N,
4.08. Found: C, 59.12; H, 4.01; N, 4.07.
References and notes
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Compounds 7b and 7c were synthesized according to the meth-
od for 7a.
ˇ
ˇ ˇ
10. Lukacínová, J. M.; Benacka, R.; Keller, J.; Maguth, T.; Kurila, P.; Vaško, L. Acta Vet.
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Compound 7b: 93% yield, mp 122–123 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.73 (s, 1H), 7.87–7.89 (m, 2H), 7.53 (d, 3H,
J = 8 Hz), 6.67 (s, 1H), 6.493 (d, 1H, J = 8 Hz), 6.354–6.36 (d, 1H,
J = 2.3 Hz), 4.56 (s, 2H), 4.09 (s, 2H), 1.96 (s, 4H). EI-MS (m/z):
371 [M]+. Anal. Calcd for C19H17NO7: C, 61.45; H, 4.61; N, 3.77.
Found: C, 61.17; H, 4.86; N, 3.59.
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Compound 7c: 91% yield, mp 105–106 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 12.71 (s, 1H), 7.87–7.89 (m, 2H), 7.50–7.57 (m,
3H), 6.67 (s, 1H), 6.50 (d, 1H, J = 2.3 Hz), 6.36 (d, 1H, J = 2.3 Hz),
4.46–4.4 9 (m, 2H), 4.03–4.06 (m, 2H), 1.74–1.87 (m, 4H), 1.50–
1.53 (m, 4H). EI-MS (m/z): 399 [M]+. Anal. Calcd for C21H21NO7:
C, 63.15; H, 5.30; N, 3.51. Found: C, 62.83; H, 5.48; N, 3.26.
4.2.2.7. O5-Acetyl-O7-nitrooxyethyl chrysin (8a). The com-
pound 7a (0.34 g, 1 mmol) was placed into a 250 mL flask and
the following were added sequentially: anhydrous potassium car-
bonate (0.14 g, 1 mmol) and anhydrous acetone (50 mL). The mix-
ture was then heated via reflux. About 4.7 mL acetic anhydride was
then dribbled into the mixture. Reflux was continued for 24 h, after
which vacuum filtration, de-sludging, filter evaporation, ethanol
rinsing, and ethyl acetate recrystallization were carried out. A
beige, solid O5-acetyl-O7-nitrooxyethyl chrysin product was ob-
tained (8a: 0.11 g, 28.6% yield). Mp 112–113 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 7.85 (d, 2H, J = 8 Hz), 7.52 (d, 3H,
J = 2.3 Hz), 6.90 (s, 1H), 6.64 (d, 2H, J = 2.3 Hz), 4.39 (s, 2H), 3.68
(s, 2H), 2.44 (s, 3H). EI-MS (m/z): 386 [M+H]+. Anal. Calcd for
C19H15NO8: C, 59.22; H, 3.92; N, 3.64. Found: C, 59.58; H, 4.12;
N, 3.46.
Compounds 8b and 8c were synthesized according to the meth-
od for 8a.
Compound 8b: 51% yield, mp 106–107 °C. 1H NMR (400 MHz,
CDCl3) d (ppm): 7.85 (d, 2H, J = 2.3 Hz), 7.52 (s, 3H), 6.87 (s, 1H),
6.61 (s, 2H), 4.56 (s, 2H), 4.11 (s, 2H), 2.44 (s, 3H), 1.97 (s, 4H).
35. Ardestani, A.; Yazdanparast, R. Int. J. Biol. Macromol. 2007, 41, 572.
36. Bhandari, S. V.; Bothara, K. G.; Patil, A. A.; Chitre, T. S.; Sarkate, A. P.; Gore, S. T.;
Dangre, S. C.; Khachane, C. V. Bioorg. Med. Chem. 2009, 17, 390.