The synthesis of puerarin derivatives and their protective effect on the myocardial ischemia and reperfusion injury

Article abstract of DOI:10.1080/10286020.2010.505563

Puerarin is a naturally occurring isoflavone and is frequently used for the treatment of cardiovascular symptoms in China. By the structural modification of the puerarin molecule at different positions, seven new puerarin derivatives were obtained, and their cardioprotective activities (in vitro and in vivo) were respectively evaluated. The finding that the activities of 3 and 8 markedly exceeded puerarin suggested that the acylated modification of phenolic hydroxyl at C-7 in the puerarin molecule may improve the cardioprotective activity, which will be an important reference for further structural optimization.

Full text of DOI:10.1080/10286020.2010.505563

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The synthesis of puerarin derivatives
and their protective effect on the
myocardial ischemia and reperfusion
injury
Zhi-Qiang Feng a , Ying-Yu Wang a , Zong-Ru Guo a , Feng-Ming Chu
a & Piao-Yang Sun b
a Key Laboratory of Bioactive Substances and Resources Utilization
of Chinese Herbal Medicine, Ministry of Education & Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College , Beijing, 100050, China
b Hengrui Pharmaceutical Company , Lianyungang, 222002, China
Published online: 28 Sep 2010.
To cite this article: Zhi-Qiang Feng , Ying-Yu Wang , Zong-Ru Guo , Feng-Ming Chu & Piao-Yang
Sun (2010) The synthesis of puerarin derivatives and their protective effect on the myocardial
ischemia and reperfusion injury, Journal of Asian Natural Products Research, 12:10, 843-850, DOI:
10.1080/10286020.2010.505563
To link to this article: http://dx.doi.org/10.1080/10286020.2010.505563
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Journal of Asian Natural Products Research
Vol. 12, No. 10, October 2010, 843–850
ORIGINAL ARTICLE
The synthesis of puerarin derivatives and their protective effect on the
myocardial ischemia and reperfusion injury
Zhi-Qiang Fenga, Ying-Yu Wanga, Zong-Ru Guoa*, Feng-Ming Chua and Piao-Yang Sunb
aKey Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine,
Ministry of Education & Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China; Hengrui Pharmaceutical Company,
b
Lianyungang 222002, China
(Received 24 May 2010; final version received 28 June 2010)
Puerarin is a naturally occurring isoflavone and is frequently used for the treatment of
cardiovascular symptoms in China. By the structural modification of the puerarin
molecule at different positions, seven new puerarin derivatives were obtained, and their
cardioprotective activities (in vitro and in vivo) were respectively evaluated. The
finding that the activities of 3 and 8 markedly exceeded puerarin suggested that the
acylated modification of phenolic hydroxyl at C-7 in the puerarin molecule may
improve the cardioprotective activity, which will be an important reference for further
structural optimization.
Keywords: puerarin derivatives; myocardial ischemia; reperfusion injury
1. Introduction
However, the reperfusion through thrombo-
lysis, percutaneous coronary angioplasty, or
bypass surgery is the standard treatment in
impending acute myocardial infarction,
meaning that reperfusion injury will be a
major clinical problem in the near future [4].
This reperfusion injury involves the
activation of an inflammatory cascade and
is manifested as functional impairment,
arrhythmia, and accelerated progression
of cell death in certain critically injured
myocytes. The major mediators of reper-
fusion injury are oxygen radicals, calcium
loading, and neutrophils [5]. The oxygen
radicals are generated by injured myocytes
and endothelial cells in the ischemic zone,
as well as by neutrophils that enter the
ischemic zone, and become activated on
reperfusion. These oxygen radicals exacer-
bate membrane damage, which leads to
Acute myocardial ischemia and myocardial
infarction are the leading cause of morbidity
and mortality in the Western World, and
according to the World Health Organiz-
ation, it will be the major cause of death in
the world by the year 2020 [1]. Early
restoration of blood flow (reperfusion) is an
absolute prerequisite for the survival of
ischemic myocardium. However, reperfu-
sion has been referred by Braunwald and
Kloner [2] as the ‘double edged sword’
because reperfusion itself may lead to
accelerated and additional myocardial
injury beyond that generated by ischemia
alone, and this results in a spectrum of
reperfusion-associated pathologies, ranging
from reversible dysfunction (‘myocardial
stunning’) to irreversible necrosis [2],
collectively called reperfusion injury [3].
*Corresponding author. Email: zrguo@imm.ac.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020.2010.505563
http://www.informaworld.com

844
Z.-Q. Feng et al.
injury, the specific bioactivity in the
OH OH
4″
laboratory and the efficacy in clinical trials
remain low, and the solubility and bioavail-
ability need to be improved. Therefore,
optimizing the pharmacological activities
against myocardial ischemia and reperfu-
sion injury by modifying the structure of 1
is essential to improve its efficacy in
clinical trials. Herein, seven derivatives of
1 were synthesized and their cardioprotec-
tive effects on the myocardial ischemia and
reperfusion injury were evaluated.
3″
OH
6″
5″
2″
O
1″
7
OH
H
8
1
O
HO
2
2′
1′
6
3′
OH
3
4
5
O
6′
4′
5′
1
Figure 1. The structure of puerarin (1).
calcium loading. The neutrophils accumu-
late in the microcirculation, release
inflammatory mediators, and contribute
to microvascular obstruction and the no-
reflow phenomenon in the reperfused
myocardium [5].
2. Results and discussion
There are two phenolic hydroxyl groups
and four alcoholic hydroxyl groups in the
structure of 1, which could respectively be
modified according to the reaction activity
distinction resulting from different pos-
itions in the structure. In order to probe the
key part of the molecular structure that
results in the protective effect on myocar-
dial ischemia and reperfusion injury,
search for the relevance between the
activity in reducing myocardial I/R injury
and the structure modified for the phenolic
hydroxyl and alcoholic hydroxyl located at
different positions in the molecule, four
acylates of different alcoholic hydroxyls
(4–7) and three acylates of different
phenolic hydroxyls (2, 3 and 8) were
designed and synthesized (Scheme 1).
The phenolic hydroxyl group at C-7
could selectively be acylated at an alkaline
medium such as the solution of anhydrous
K2CO3 in acetone, on the basis of the
unequal acidity (the acidity of phenolic
hydroxyl at C-7 is larger than at C-40).
When the molar ratio of 1, K2CO3, and
acylating agent was 1:1:1, the acylate (2
and 3) of phenolic hydroxyl at C-7 was
afforded as the main acylate. However,
when the molar ratio was 1:2:2, the two
phenolic hydroxyls at C-7 and C-40 were
together acylated, such as 8. It is interest-
ing that two sets of signals are observed in
the 1H NMR measurements of the acylates
of phenolic hydroxyl at C-7 at room
Puerarin (1, daidzein-8-C-glucoside)
(Figure 1) is a naturally occurring
isoflavone, isolated from the root of
Pueraria lobata (Willdenow) Ohwi, one
of the most popular Chinese herbal
medicines. Studies showed that 1 is an
effective scavenger of active oxygen
radicals such as hydroxyl and superoxide
radicals, and this may result from the
isoflavone’s polyphenolic structure [6].
The results of laboratory and clinical
studies [7] demonstrated that 1 can
attenuate ischemia–reperfusion (I/R)
injury and promote recovery of myocardial
function by mechanisms such as inhibiting
myocardial apoptosis [8], increasing SOD
activity [9], downregulating Bax
expression, upregulating Bcl-2 [10], inhi-
biting the production of proinflammatory
cytokines [11], improving the myocardial
ultramicrostucture [12], refraining the
calcium overload [13], etc. It has also
been reported that 1 protects the myocar-
dium against ischemia and reperfusion
injury via inhibiting mitochondrial per-
meability transition pore opening and
activating the mitochondrial ATP-sensitive
potassium channel [14]. Although 1 has
beneficial effects in cases of myocardial

Journal of Asian Natural Products Research
845
OR6 OR5
OR4
O
O
O
OR3
H
OH
R2O
O
O
OH
O
H
O
HO
OR1
5
2: R1 = R3 = R4 = R5 = R6 = H, R2 = C(O)CH3
O
OH
a
b
c
3: R1 = R3 = R4 = R5 = R6 = H, R2 = C(O)C(CH3)3
4: R1 = R2 = R4 = R5 = R6 = H, R3 = C(O)OCH3
O
O
d
Puerarin
g
OH
e
O
6: R2 = R6 = H, R1 = R3 = R4 = R5 = C(O)CH3
f
OH
O
H
HO
8: R3 = R4 = R5 = R6 = H, R1 = R2 = CH2OC(O)C(CH3)3
7
O
OH
Scheme 1. The synthesis of derivatives 2–8. Reagent and conditions: (a) benzoic aldehyde, tosic
acid, acetone, rt; (b) acetyl chloride, K2CO3, acetone, rt; (c) pivalyl chloride, K2CO3, acetone, rt; (d)
methylchloroformate, K2CO3, acetone, rt; (e) acetic anhydride, boron trifluoride, acetone, rt; (f)
chloromethyl pivalate, NaI, K2CO3, acetone, rt; (g) 2-methoxyprop-1-ene, tosic acid, acetone, rt.
temperature, which suggested that the
acylation of phenolic hydroxyl at C-7
leads to the formation of two main
conformational isomers, resulting from
the crowding between the acylate group
and the glucose group [15]. The alcoholic
hydroxyl could also selectively be acylated
at an acidic medium such as the solution of
BF3 in acetone. When a suitable reaction
condition was selected, the acylate of
alcoholic hydroxyl groups located at
different positions could be obtained, such
as 4–7. In order to discover the structure
that will show the more protective effect on
myocardial ischemia and reperfusion
injury, seven compounds synthesized
were respectively evaluated at the Langen-
dorff perfusion model of isolated rat heart
and the models of the SD rat myocardial
ischemia induced by posterior pituitary
hormones. Some of them showed pro-
nounced cardioprotective activity.
rat. The experimental groups consist of
model group, sham group, and medication
group. The level of lactate dehydrogenase
(LDH) in the effluent from the perfused
heart was used to monitor the damage of
myocardial tissues. The statistical signifi-
cance was compared between the medi-
cation groups and the model group by
Student’s t-test. The results are presented
in Table 1.
LDH, one of important metabolic
enzymes in cardiomyocytes, has been
regarded as reliable indices with which to
evaluate myocardial I/R injury. As shown
in Table 1, I/R caused a significant
increase in the level of LDH compared
with the sham group. The release of LDH
was reduced by treatment with 1 and its six
derivatives except for 8, and, especially, 3
and 2 among them exhibit a significant
decrease in the level of LDH compared
with the positive control, which showed
that the cardioprotective activities of 3 and
2 markedly exceed 1. This indicated that
At the Langendorff perfusion model,
the isolated hearts were prepared from SD

846
Z.-Q. Feng et al.
Table 1. The effect of puerarin and its derivatives on I/R injury in the model of isolated rat heart.
LDH activity (U/l g wet wt)
LDH active ratio
Group
C, 10–4 mol/l
N
Pre-I
Post-RP
Post-RP/Pre-I
Blank control
Ischemic model
2
3
3
4
5
7
5
12
5
5
5
5
5
5
5
273 ^ 77
222 ^ 69
318 ^ 64
256 ^ 120
210 ^ 32
193 ^ 39
204 ^ 50
199 ^ 102
180 ^ 42
273 ^ 34
216 ^ 68
638 ^ 164
677 ^ 102
418 ^ 174
634 ^ 124
507 ^ 96
542 ^ 9
494 ^ 136
536 ^ 109
752 ^ 222
0.80 ^ 0.13
3.04 ^ 0.83
2.21 ^ 0.62
1.74 ^ 0.89
3.01 ^ 0.26
2.67 ^ 0.49
2.32 ^ 0.46
2.75 ^ 0.98
3.21 ^ 1.26
2.85 ^ 1.03
2.18
2.00
0.20
2.11
1.98
2.19
1.55
2.40
*
8
Puerarin
5
Notes: C, concentration; LDH, lactate dehydrogenase; Pre-I, pre-ischemia; Post-RP, post-reperfusion. P , 0.05
*
vs. Ischemic model.
the acylated modifications of phenolic
hydroxyl at C-7 in 1 may improve the
cardioprotective activity, and the larger the
bulk of the acylating agent, the greater the
effect on activity. The reason could be that
the bulky volume of the group linking with
phenolic hydroxyl at C-7 leads to the
larger change of conformation of 1 due to
the crowding between the acylating group
and the glucose group.
5 min, and the ST-segment changes were
observed. In two medication groups, the
SD rats were, respectively, administered
by hypodermic injection of 20 and
40 mg/kg derivatives of 1, everyday once,
lasting for 7 days, and after 30 min of the
eventual time, pituitrin (0.5 U/kg) was
administered i.v. to induce myocardial
ischemia. The ST-segment elevation
(ST-E) in the ECG was used to detect
and quantify myocardial ischemia. The
results are presented in Tables 2 and 3.
Assessment of the ST-segment of the
ECG plays a pivotal role in the diagnosis
and management of ischemic heart dis-
ease. The presence of ST-E is a diagnostic
In the model of myocardial ischemia
induced by pituitrin, the SD rats were
anesthetized and their normal electrocar-
diogram (ECG) II leads were recorded.
Then, hypophysin was administered i.v.,
and ECGs were continuously recorded for
Table 2. The effect of puerarin derivatives (20 mg/kg, hypo. 7 days) on the ST elevation in ECG in
SD rats of myocardial ischemia induced by pituitrin.
ST-E (mV)
Compound
N
Pre-ischemia
Post-ischemia
ST-E change
Model
2
3
4
5
15
5
5
5
5
5
5
5
5
0.091 ^ 0.015
0.093 ^ 0.015
0.1 ^ 0
0.096 ^ 0.01
0.086 ^ 0.018
0.0864 ^ 0.019
0.093 ^ 0.015
0.1 ^ 0
0.26 ^ 0.05
0.21 ^ 0.03
0.167 ^ 0.053
0.118 ^ 0.018
0.206 ^ 0.017
0.263 ^ 0.03
0.23 ^ 0.019
0.22 ^ 0.038
0.21 ^ 0.021
0.17 ^ 0.04
0.106 ^ 0.017
0.172 ^ 0.028
0.147 ^ 0.034
0.131 ^ 0.023
0.115 ^ 0.03
0.072 ^ 0.036
0.098 ^ 0.023
*
6
7
8
Puerarin
**
*
0.104 ^ 0.009
0.198 ^ 0.023
Note: P , 0.05, P , 0.01 vs. blank control.
**
*

Journal of Asian Natural Products Research
847
Table 3. The effect of puerarin derivatives (40 mg/kg, hypo. 7 days) on the ST elevation in ECG in
SD rats of myocardial ischemia induced by pituitrin.
ST-E (mV)
Compound
N
Pre-ischemia
Post-ischemia
ST-E change
Model
2
3
4
5
15
5
5
5
5
5
5
5
5
0.091 ^ 0.015
0.093 ^ 0.015
0.1 ^ 0.004
0.086 ^ 0.018
0.093 ^ 0.015
0.080 ^ 0.018
0.096 ^ 0.009
0.089 ^ 0.017
0.1 ^ 0
0.26 ^ 0.05
0.264 ^ 0.052
0.19 ^ 0.028
0.231 ^ 0.04
0.231 ^ 0.023
0.21 ^ 0.019
0.27 ^ 0.05
0.167 ^ 0.053
0.171 ^ 0.048
0.092 ^ 0.028
0.145 ^ 0.037
0.138 ^ 0.027
0.13 ^ 0.024
0.175 ^ 0.05
0.092 ^ 0.015
0.099 ^ 0.028
*
6
7
8
Puerarin
0.178 ^ 0.018
0.198 ^ 0.028
**
*
Note: P , 0.05, P , 0.01 vs. blank control.
**
*
criterion of acute myocardial ischemia. In
this study, the impact of the precondition-
ing by derivatives of 1 on the amounts of
elevation of the ST-segment was assessed
in a model of SD rat myocardial ischemia
induced by pituitrin. As shown in Tables 2
and 3, the myocardial ischemia induced by
pituitrin caused a significant increase in
the degree of ST-E compared with pre-
ischemia, and the degree of elevation of
the ST-segments was markedly reduced in
rats pretreated by some derivatives of 1;
especially 3 and 8, at both 20 and 40 mg/kg
doses, exhibited a significant decrease in
the degree of ST-E, which showed the
potential cardioprotective activity of 3 and
8. For 3, the results evaluated in the
cardioprotective activity in a model of SD
rat myocardial ischemia induced by
pituitrin were conformed to the results at
the Langendorff perfusion model of
isolated rat heart. However, as for 8, the
results evaluated at two models were
inconsistent, which may be explained by
the metabolite of 8. In the molecular
structure of 8, two phenolic hydroxyls at
C-7 and C-40 had been acylated together by
chloromethyl pivalate. In the presence of
the esterolytic enzyme, the pivalate group
at C-40 in the molecule of 8 was easily
hydrolyzed, but the hydrolysis of the
pivalate group at C-7 was difficult due to
the barrier shield from the bulky glucosyl
unit. In other words, the cardioprotective
activity of 8 at the ischemia model of SD
rat may result from the metabolite of 8, not
from 8 itself. The hypothesis needs to be
further confirmed by the study on the
metabolite of 8.
3. Conclusion
In summary, by the structural modification
of 1 at different positions, seven new
derivatives of 1 were obtained, and their
cardioprotective activities were, respect-
ively, evaluated in the model of myocar-
dial I/R injury of Langendorff isolated rat
heart and the model of SD rat myocardial
ischemia induced by pituitrin. According
to the results, the cardioprotective activi-
ties of 3 and 8 markedly exceeded 1, which
suggested that the acylated modification of
phenolic hydroxyl at C-7 in 1 may improve
the cardioprotective activity, and the larger
the bulk of the acylating agent, the greater
the effect on activity, which will be an
important reference for further structural
optimization.
4. Experimental
4.1 General experimental procedures
Melting points were recorded on a Yanaco
melting point apparatus and are uncor-
rected. 1H NMR spectra were recorded on
a Varian Oxford 300, using TMS as an

848
Z.-Q. Feng et al.
internal standard. Chemical shifts (d) are
expressed in ppm. Mass spectra were
obtained on an Autospec-Ultima ETOF
mass spectrometer for FAB-MS. HR-MS
was recorded on Agilent LC/MSD TOF.
acetate, and washed, respectively, with
water and sat. aqueous solution of NaCl.
The isolated organic layer was dried with
anhydrous Na2SO4. The evaporation of
the organic solvent gave the crude
product, which was purified by chromato-
graphy on a column of silica gel (ethyl
acetate/methanol ¼ 10/1) to afford 7-O-
pivalyl puerarin (0.345 g, 69%), a white
solid, mp 216–2188C. 1H NMR
(300 MHz, CD3OD, ppm) d: 8.26 and
8.25 (1H, s, H-2), 8.17 (1H, d, J ¼ 9.0 Hz,
H-5), 7.35 (2H, d, J ¼ 8.4 Hz, H-20, 60),
7.17 and 7.10 (1H, d, J ¼ 9.0 Hz, H-6),
6.80 (2H, d, J ¼ 8.4 Hz, H-30, 50), 1.37
(9H, s, ZCH3), 5.05 and 4.66 (1H, d,
J ¼ 9.9 Hz, H-100), 3.22–4.16 (6H, m, H-
200-H-600). FAB-MS: m/z 50 [M þ H]þ.
HR-ESI-MS: m/z 501.1743 [M þ H]þ
(calcd for C26H29O10, 501.1755).
4.2 Compound 2
To a solution of puerarin (0.416 g, 1 mmol)
in acetone (50 ml), anhydrous K2CO3
(0.138 g, 1 mmol) was added. After stirring
for 10 min, acetyl chloride (1 mmol) was
added dropwise at room temperature, and
the mixture was stirred until the reaction
was complete (monitored by TLC). After
the removal of the solvent, the residue was
diluted with ethyl acetate, and washed,
respectively, with water and sat. aqueous
solution of NaCl. The isolated organic
layer was dried with anhydrous Na2SO4.
The evaporation of the organic solvent
gave the crude product, which was purified
by chromatography on a column of silica
gel (ethyl acetate/methanol ¼ 10/1) to
afford 7-O-acetyl puerarin (0.289 g,
4.4 Compound 4
To a solution of puerarin (0.416 g, 1 mmol)
in acetone (50 ml), anhydrous K2CO3
(0.138 g, 1 mmol) was added. After stirring
for 10 min, methyl chloroformate
(1 mmol) was added dropwise at room
temperature, and the mixture was stirred
until the reaction was complete (monitored
by TLC). After the removal of the solvent,
the residue was diluted with ethyl acetate,
and washed, respectively, with water and
sat. aqueous solution of NaCl. The isolated
organic layer was dried with anhydrous
Na2SO4. The evaporation of the organic
solvent gave the crude product, which
was purified by chromatography on a
column of silica gel (ethyl acetate/
methanol ¼ 10/1) to afford 10-O-methoxy
formyl puerarin (0.25 g, 53%), a white
solid, mp 180–1828C. 1H NMR
(300 MHz, CD3OD, ppm) d: 8.16 (1H, s,
H-2), 7.98 (1H, d, J ¼ 8.7 Hz, H-5), 7.33
(2H, d, J ¼ 8.4 Hz, H-20, 60), 6.89 (1H, d,
J ¼ 8.7 Hz, H-6), 6.78 (2H, d, J ¼ 8.4 Hz,
H-30, 50), 3.39 (3H, s, ZOCH3), 5.38 (1H,
dd, J ¼ 9.9, 9.0 Hz, H-200), 5.15 (1H, d,
J ¼ 9.9 Hz, H-100), 3.42–3.85 (5H, m,
1
63%), a white solid, mp 194–1968C. H
NMR (300 MHz, CD3OD, ppm) d: 8.28
and 8.26 (1H, s, H-2), 8.18 (1H, d,
J ¼ 8.7 Hz, H-5), 7.36 (2H, d,
J ¼ 7.8 Hz, H-20, 60), 7.18 and 7.15 (1H,
d, J ¼ 8.7 Hz, H-6), 6.80 (2H, d,
J ¼ 7.8 Hz, H-30, 50), 1.37 (3H, s,
ZCH3), 5.05 and 4.67 (1H, d,
J ¼ 9.9 Hz, H-100), 3.30–4.15 (6H, m, H-
200-H-600). FAB-MS: m/z 459 [M þ H]þ.
HR-ESI-MS: m/z 459.1277 [M þ H]þ
(calcd for C23H23O10, 459.1285).
4.3 Compound 3
To a solution of puerarin (0.416 g, 1 mmol)
in acetone (50 ml), anhydrous K2CO3
(0.138 g, 1 mmol) was added. After stirring
for 10 min, the pivalyl chloride (1 mmol)
was added dropwise at room tempera-
ture, and the mixture was stirred until
the reaction was complete (monitored
by TLC). After the removal of the solvent,
the residue was diluted with ethyl

Journal of Asian Natural Products Research
849
H-300-H-600). FAB-MS: m/z 475 [M þ H]þ.
HR-ESI-MS: m/z 475.1226 [M þ H]þ
(calcd for C23H23O11, 475.1234).
complete (monitored by TLC). Ethanol
(5 ml) was added and the mixture was
stirred for 1 h. Then, a solution of NaHCO3
was added. After the removal of the
solvent, the residue was diluted with ethyl
acetate, and washed, respectively, with
water and a sat. aqueous solution of
NaHCO3. The isolated organic layer was
dried with anhydrous Na2SO4. The evap-
oration of the organic solvent gave the
crude product, which was purified by
chromatography on a column of silica gel
(ethyl acetate/methanol ¼ 10/1) to afford
6 (0.210 g, 36%), a solid, mp 126–1288C.
1H NMR (300 MHz, CD3OD, ppm) d: 8.34
(1H, s, H-2), 7.99 (1H, d, J ¼ 9.0 Hz, H-5),
7.55 (2H, d, J ¼ 8.4 Hz, H-20, 60), 6.92
(1H, d, J ¼ 9.0 Hz, H-6), 7.12 (2H, d,
J ¼ 8.4 Hz, H-30, 50), 5.80 (1H, d,
J ¼ 10.2 Hz, H-100), 5.31 (1H, m, H-200),
5.25 (1H, m, H-300), 3.98 (1H, m, H-400),
3.54–3.84 (3H, m, H-500-H-600), 2.23 (3H,
s, COCH3), 1.96 (3H, s, COCH3), 1,49
(3H, s, COCH3), 1.32 (3H, s, COCH3).
FAB-MS: m/z 585 [M þ H]þ. HR-ESI-
MS: m/z 585.1586 [M þ H]þ (calcd for
C29H29O13, 585.1602).
4.5 Compound 5
To a solution of puerarin (0.416 g, 1 mmol)
and p-toluenesulfonic acid (two grains) in
acetone (50 ml), benzaldehyde (1.2 mmol)
was added dropwise at 08C under stirring.
The mixture was stirred at room tempera-
ture until the reaction was complete
(monitored by TLC). Then, 5 ml solution
of NaHCO3 was added for the termination
of the reaction. After the removal of the
solvent, the residue was diluted with ethyl
acetate, and washed, respectively, with
water and aqueous solution of NaHCO3.
The isolated organic layer was dried with
anhydrous Na2SO4. The evaporation of the
organic solvent gave the crude product,
which was purified by chromatography on
a
column of silica gel (ethyl
acetate/methanol ¼ 10/1) to afford 5
(0.24 g, 48%), a solid, mp 199–2018C.
1H NMR (300 MHz, CD3OD, ppm) d: 8.16
(1H, s, H-2), 8.00 (1H, d, J ¼ 9.0 Hz, H-5),
7.31 (2H, d, J ¼ 8.4 Hz, H-20, 60), 6.95
(1H, d, J ¼ 9.0 Hz, H-6), 6.79 (2H, d,
J ¼ 8.4 Hz, H-30, 50), 7.48 (2H, m, ArH),
7.29 (3H, m, ArH), 5.59 (1H, s,
PhCH(O)2), 4.32 (1H, dd, J ¼ 10.2,
8.1 Hz, H-200), 5.15 (1H, d, J ¼ 10.2 Hz,
H-100), 4.22 (1H, dd, J ¼ 10.5, 4.8 Hz, H-
600), 3.76 (1H, dd, J ¼ 10.5, 6.6 Hz, H-600),
3.69 (1H, dd, J ¼ 8.1, 7.8 Hz, H-300), 3.63
(1H, dd, J ¼ 9.3, 7.8 Hz, H-400), 3.57 (1H,
m, H-500). FAB-MS: m/z 505 [M þ H]þ.
HR-ESI-MS: m/z 505.1479 [M þ H]þ
(calcd for C28H25O9, 505.1493).
4.7 Compound 7
To a solution of puerarin (0.416 g, 1 mmol)
and p-toluenesulfonic acid (two grains) in
acetone (50 ml), 2-methoxyl propylene
(1.2 mmol) was added dropwise at 08C
under stirring. The mixture was stirred at
room temperature until the reaction was
complete (monitored by TLC). Then, 5 ml
solution of NaHCO3 was added. After the
removal of the solvent, the residue was
diluted with ethyl acetate, and washed,
respectively, with water and sat. aqueous
solution of NaHCO3. The isolated organic
layer was dried with anhydrous Na2SO4.
The evaporation of the organic solvent
gave the crude product, which was purified
by chromatography on a column of silica
gel (ethyl acetate/methanol ¼ 10/1) to
afford 7 (0.27 g, 59%), a solid, mp 230–
2328C. 1H NMR (300 MHz, CD3OD, ppm)
4.6 Compound 6
To a solution of puerarin (0.416 g, 1 mmol)
in acetone (50 ml), acetic anhydride
(5 mmol) and a solution of boron trifluor-
ide in ether (0.2 ml) were added at 08C
under stirring. The mixture was stirred at
room temperature until the reaction was

850
Z.-Q. Feng et al.
d: 8.17 (1H, s, H-2), 8.01 (1H, d,
J ¼ 9.0 Hz, H-5), 7.33 (2H, d,
J ¼ 8.7 Hz, H-20, 60), 6.94 (1H, d, J ¼
9.0 Hz, H-6), 6.79 (2H, d, J ¼ 8.7 Hz,
H-30, 50), 4.26 (1H, t, J ¼ 9.6 Hz, H-200),
5.06 (1H, d, J ¼ 9.6 Hz, H-100), 3.80
(2H, m, H-600), 3.69 (1H, dd, J ¼ 9.6,
9.3 Hz, H-300), 3.54 (1H, dd, J ¼ 9.3,
9.0 Hz, H-400), 3.39 (1H, m, H-500), 1.51
(3H, s, CH3), 1.36 (3H, s, CH3). FAB-MS:
m/z 457 [M þ H]þ. HR-ESI-MS: m/z
457.1479 [M þ H]þ (calcd for C24H25O9,
457.1493).
ZCH3). FAB-MS: m/z 645 [M þ H]þ.
HR-ESI-MS: m/z 645.2531 [M þ H]þ
(calcd for C33H41O13, 645.2541).
References
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4.8 Compound 8
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To a solution of chloromethyl pivalate
(2.05 g, 13.6 mmol) in anhydrous acetone
(30 ml),
anhydrous
NaI
(2.04 g,
13.6 mmol) was added, and the mixture
was sealed overnight, then was added to a
solution of puerarin (2.8 g, 6.8 mmol) and
anhydrous K2CO3 (1.9 g, 13.6 mmol) in
acetone (150 ml) under stirring, and the
mixture was stirred until the reaction was
complete (monitored by TLC). After the
removal of the solvent, the residue was
diluted with ethyl acetate (150 ml), and
washed, respectively, with water and sat.
aqueous solution of NaCl. The isolated
organic layer was dried with anhydrous
Na2SO4. The evaporation of the organic
solvent gave the crude product, which
was purified by chromatography on a
column of silica gel (ethyl acetate/
methanol ¼ 10/1) to afford 8 (1.37 g,
31.3%), a white solid, mp 176–1788C.
1H NMR (300 MHz, CD3OD, ppm) d: 8.19
(1H, s, H-2), 8.21 (1H, d, J ¼ 7.5 Hz, H-5),
7.36 (2H, d, J ¼ 8.4 Hz, H-20, 60), 7.38
(1H, d, J ¼ 7.5 Hz, H-6), 6.80 (2H, d,
J ¼ 8.4 Hz, H-30, 50), 6.01 (2H, s, ZOCH2-
OZ), 5.90 (2H, m, ZOCH2OZ), 5.02 and
4.96 (1H, d, J ¼ 9.9 Hz, H-100), 3.25–4.17
(6H, m), 1.10 (9H, s, ZCH3), 1.16 (9H, s,
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