The first total synthesis of 7-O-β-d-glucopyranosyl-4′-O-α-l-rhamnopyranosyl apigenin via a hexanoyl ester-based protection strategy

Article abstract of DOI:10.1016/j.carres.2008.12.016

The first total synthesis of 7-O-β-d-glucopyranosyl-4′-O-α-l-rhamnopyranosyl apigenin 1, which exhibits good anti-hepatitis B virus and anti-stroke activities, was accomplished in six steps and 20% overall yield from apigenin. Another synthetic route, in which the target was obtained in seven steps, was also developed to prove the utility of a hexanoyl ester-based orthogonal protection strategy. The hexanoyl protection strategy provided all the flavonoid intermediates with good solubility and reactivity, enabled efficient selective protection and glycosylation, and provided a practical and effective synthetic strategy for flavonoids, starting from commercially available flavone.

Full text of DOI:10.1016/j.carres.2008.12.016

Carbohydrate Research 344 (2009) 511–515
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
The first total synthesis of 7-O-b-D-glucopyranosyl-40-O-
a-L-rhamnopyranosyl
apigenin via a hexanoyl ester-based protection strategy
Qi Gao a, Gaoyan Lian b, Feng Lin a,
*
a Shanghai Institute of Pharmaceutical Industry, 1111 Zhongshanbeiyi Road, Shanghai 200437, China
b State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, CAS, 354 Fenglin Road, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
The first total synthesis of 7-O-b-D a-L-rhamnopyranosyl apigenin 1, which exhibits
-glucopyranosyl-40-O-
Article history:
Received 24 October 2008
Received in revised form 11 December 2008
Accepted 15 December 2008
Available online 25 December 2008
good anti-hepatitis B virus and anti-stroke activities, was accomplished in six steps and 20% overall yield
from apigenin. Another synthetic route, in which the target was obtained in seven steps, was also devel-
oped to prove the utility of a hexanoyl ester-based orthogonal protection strategy. The hexanoyl protec-
tion strategy provided all the flavonoid intermediates with good solubility and reactivity, enabled
efficient selective protection and glycosylation, and provided a practical and effective synthetic strategy
for flavonoids, starting from commercially available flavone.
Keywords:
Total synthesis
Flavonoid glycoside
Hexanoyl
Ó 2008 Elsevier Ltd. All rights reserved.
Glycosylation
Glycosylated flavonoids occur widely in human food and drinks,
especially in fruits and vegetables.1 They are also major compo-
nents of many plant drugs2 and exhibit a broad spectrum of
biological activities, such as antimicrobial,3 antiviral, and anti-
inflammatory activities.4 Their biological activities against cancer,
cardiovascular, and neurodegenerative diseases also have attracted
increasing interest.4,5
eral solution for these old problems has been developed. During
our continuous medicinal research on flavonoids to support the
investigation of many important physiology processes and drug
discoveries, we tried to develop a practical general strategy for
orthogonal protection of polyphenols and flavonoid glycosylation,
which will serve as a basis for the preparation of flavonoid
libraries.
Recently, the hexanoyl group was used occasionally by Yu and
co-workers20 and Botting and Al-Maharik21 for the selective depro-
tection or solubility improvement in flavonoid synthesis. We envi-
sioned that the lipophilic hexanoyl group would effectively
improve the solubility of flavonoids, which would result in im-
proved reactivity, and the increased reactivity of the hexanoyl
group would enable selective protection. In this work we show that
starting from trihexanoyl apigenin 3, all the three hydroxyl groups
of apigenin could be discriminated effectively with very economi-
cal methods. We thus developed a general strategy to introduce
7-O-b-
D
-Glucopyranosyl-40-O-
a-
L-rhamnopyranosyl apigenin 1
(Fig. 1) is one of the main effective components of Ranunculus sie-
boldii, a traditional Chinese herb medication for hepatitis patients.
The compound exhibits inhibitory activity against hepatitis B virus
(HBV) replication, and its inhibition effect is comparable with that
of lamivudine.6 Recently, our colleagues reported its remarkable
activity against cerebral thrombosis and stroke.7 However, the
mechanism of these interesting biological activities remains un-
clear, and further investigation was hindered by its limited avail-
ability from natural sources.8 Thus, an efficient total synthesis
route of 1 is needed.
The major impediments to flavonoid synthesis are the poor sol-
ubility of most flavonoids in common organic solvents, which in
turn results in poor reactivity, and the lack of effective orthogonal
protecting strategies for polyphenols.9–13 Although some of mono-
glycosyl flavonoids have been synthesized in the last decade, and
some protection strategies were introduced,14–16 bis-glycosyl
flavonoids synthesis is still recognized as a challenging task (only
four syntheses have been reported to date),13,17–19 because no gen-
O
OH
O
HO
HO
O
O
O
O
OH
OH
OH
HO
OH
1
* Corresponding author. Tel.: +86 21 55514600x226; fax: +86 21 65169893.
E-mail address: linfeng@mail.sioc.ac.cn (F. Lin).
Figure 1. Structure of 7-O-b-
nin, 1.
D a-L-rhamnopyranosyl apige-
-glucopyranosyl-40-O-
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.12.016

512
Q. Gao et al. / Carbohydrate Research 344 (2009) 511–515
OR1
OH
OR
O
HO
O
O
R1O
HO
O
O
b
a
OR1
O
OH
OR
3 R1= Hexanoyl, 88%
7 R1= Ac, 80%
4
Apigenin, 2
OBz
O
OBz
O
OR
BzO
BzO
BzO
BzO
R= Hexanoyl
O
O
BzO
Br
5
BzO
c
6
OR
O
Scheme 1. Reagents and conditions: (a) For 3: hexanoyl chloride, NEt3, DMAP, DMF, 88%; for 7: Ac2O, pyridine, 80%; (b) 0.5 equiv K2CO3, CH2Cl2–MeOH (1:1), 1.5 h, 93%;
(c) 5, K2CO3, Aliquat 336, CHCl3–H2O, 45 °C, 24 h, 78%.
glycosyl groups selectively to flavones, and applied this strategy to
the first total synthesis of 7-O-b-
rhamnopyranosyl apigenin 1.
a significant amount of 7,40-dihydroxyl apigenin was obtained after
2 h. In addition, when these optimized deprotection conditions
were applied to tri-O-acetyl apigenin, 7, poor selectivity was ob-
tained.22 The excellent selectivity in our cases (and below) demon-
strated that hexanoyl protection helped to discriminate the
reactivity of each position in an effective way. Furthermore, our
new method provided compounds in good purity after simple work
up without column chromatography. With 4 in hand, glycosylation
with bromide 5 under phase-transfer-catalyzed (PTC)11,23 condi-
tions provided 6 with b selectivity in 78% yield; Aliquat 336
(CH3(C8H17)3N+Clꢀ) was the best PTC reagent.
D a-L-
-glucopyranosyl-40-O-
Commercially available apigenin was treated with hexanoyl
chloride in DMF (Scheme 1). After recrystallization from ethanol,
tri-O-hexanoyl apigenin, 3, was obtained in 88% yield in a purity
of >95%. Compared with tri-O-acetyl apigenin, 7, hexanoyl deriva-
tive 3 shows remarkably improved solubility in common organic
solvents (Table 1). Selective hydrolysis of the 7-acyl group was first
achieved in 78% yield upon treatment with PhSH at ꢀ20 °C in
anhydrous N-methyl pyrolidinone following Yu’s procedure.20
However, this procedure, which requires strict reaction conditions
(ꢀ20 °C for 24 h) with toxic and odorous PhSH, is not suitable to
scale up.
Thus, we developed a new efficient selective deprotection
method as described below. Treatment of 3 with 0.5 equiv K2CO3
in CH2Cl2–MeOH provided the 7-OH selectively deprotected prod-
uct 4 in 93% yield (Scheme 1), no 40-O deprotection was observed.
A mixed solvent (CH2Cl2–MeOH, 1:1) was optimized for this selec-
tive deprotection. Use of one or the other of these solvents alone
resulted in either poor yield or non-selective deprotection. The
amount of base was also important; if 1 equiv of K2CO3 was used,
Treatment of compound 6 with the previously described K2CO3-
promoted deprotection conditions satisfyingly gave the 40-OH
product 8 in 84% yield, with the 5-O-hexanoate and the four ben-
zoates on the glucosyl moiety intact (Scheme 2).20,23 These results
were in accordance with the reported reactivity order: 7-O-
acyl > 40-O-acyl > 5-O-acyl,24 and demonstrate that the appropriate
reactivity of the hexanoyl group helped us to realize the selective
deprotection in a very effective and economical way.
The subsequent 40-O-glycosylation proved to be a challenging
task, because of the poor nucleophilicity of the flavone 40-hydroxyl
group.13 According to our investigation for the preparation of 15
(see below), direct 40-O-rhamnosylation of 8 was achieved via
Mitsunobu reaction, which provided 10 exclusively as the
a-ano-
Table 1
The solubility data of apigenin per-hexanoyl 3 and per-acetyl 7 (25 °C)
mer, in 40% yield in DMF as reaction solvent. It is noteworthy that
there are very few examples of direct flavone 40-O-glycosyla-
tion,11,13,25 Those that are reported either suffer from low yields
or relied upon expensive reagents, our work provides a practical
method for this problem. Global deprotection of 10 with sodium
Solvent
EtOAc
CH2Cl2
Acetone
Toluene
3
7
330 mg/mL
<1 mg/mL
>1 g/mL
39 mg/mL
>1 g/mL
5 mg/mL
170 mg/mL
<1 mg/mL
OBz
O
OBz
O
OR
OH
BzO
BzO
BzO
BzO
O
O
O
O
O
a
BzO
BzO
6
OR
O
OR
8
R = Hexanoyl
BzO
BzO
OH
O
OBz
OR
BzO
BzO
c
O
O
BzO
OBz
1
9
O
O
O
OBz
OBz
b
BzO
O
10
Scheme 2. Reagents and conditions: (a) 0.5 equiv K2CO3, CH2Cl2–MeOH (1:1), 2 h, 84%; (b) DEAD, PPh3, DMF, ꢀ20?0 °C, 2 h, then 0?20 °C, 8 h, 40%; (c) NaOMe, MeOH, 88%.

Q. Gao et al. / Carbohydrate Research 344 (2009) 511–515
513
OR
OR
b
OH
RO
O
O
a
BnO
O
BnO
O
O
OR
OR
O
OR
12
3
R= Hexanoyl
11
X
O
9 X= OH
13 X= Br
BzO
BzO
BzO
O
OBz
c, d
e
O
g
f
HO
O
O
BnO
O
O
O
10
OBz
OBz
OBz
OBz
NPh
CF3
BzO
BzO
O
OR
OR
O
O
15
16
BzO
OBz
14
Scheme 3. Reagents and conditions: (a) BnCl, K2CO3, KI, acetone 24 h, 85%; (b) 0.5 equiv K2CO3, CH2Cl2–MeOH (1:1), 10 h, 92%; (c) for 9, DEAD, PPh3, DMF, ꢀ20?0 °C, 2 h,
then 0?20 °C, 8 h, 63%; (d) for 13, K2CO3, Aliquat 336, CHCl3–H2O, 45 °C, 24 h, 35%; (e) for 14, TMSOTf or BF3ꢁEt2O, CH2Cl2, 4 Å MS; (f) Pd/H2, CHCl3–EtOH, 40?45 °C, 9 h, 79%;
(g) 5, TBAB, K2CO3, CHCl3–H2O, 45 °C, 24 h, 71%.
methoxide gave 1 in 88% yield. The synthetic compound had spec-
tral data in full agreement with the reported natural one,6 and
exhibited the same anti-stroke activity.7
and provided good yields of the transformations, especially the
direct glycosylation of inert 40-hydroxyl. This method provided a
practical flavonoid synthesis strategy starting from commercially
available flavone. Further application studies will probe the gener-
ality of this method for flavone glycoside synthesis. These investi-
gations are ongoing in our laboratory and will be reported in due
course.
Another synthetic route was also developed to prove the appli-
cation of hexanoyl orthogonal protection strategy (Scheme 3).
Compound 3 was transformed to 7-O-benzyl 11, and the 40-O-
hexanoyl group was readily hydrolyzed with the K2CO3-promoted
conditions to obtain 12 in 10 h; these two steps proceeded in high
yield and in good selectivity. The electron-donating 7-O-benzyl
group, in contrast to the electron-withdrawing 7-O-tetrabenzoyl-
glucosyl group of compound 6, made the 40-O-hexanoyl group of
11 resistant to hydrolysis. It is noteworthy that all the three hydro-
xyl groups of apigenin 12 were readily discriminated from each
other in three steps in high yield. The orthogonal protecting pat-
tern in this compound make it a valuable intermediate for future
flavonoid glycoside synthesis.
1. Experimental
1.1. General methods
Reagents and solvents were reagent grade and used without
further purification. Methylene dichloride was distilled from cal-
cium hydride, and DMF was dried over 4 Å molecular sieves. All
reactions involving air or moisture sensitive reagents or intermedi-
ates were performed under a nitrogen or argon atmosphere. Flash
chromatography was performed on Qingdao Haiyang silica gel
(300ꢀ400 mesh). Analytical TLC was performed using 0.25 mm
EM Silica Gel 60 F250 plates that were visualized by irradiation
(254 nm) or by staining with H2SO4–methanol solution. 1H and
13C NMR spectra were obtained using 300 MHz Bruker AM300
and 400 MHz Varian instruments. Chemical shifts are reported in
parts per million (ppm d) referenced to the residual 1H resonance
of the solvent (CDCl3, 7.26 ppm; DMSO-d6, 2.49 ppm). 13C spectra
were referenced to the residual 13C resonance of the solvent
(CDCl3, 77.0 ppm; DMSO-d6, 39.5 ppm). Splitting patterns are des-
ignated as follows: s, singlet; br, broad; d, doublet; dd, doublet of
doublets; t, triplet; q, quartet; m, mulitplet. Elemental analysis
was performed on a Foss-Heraeus Vario EL instrument. Mass spec-
tra (ESI) were performed on Shimadzu LCMS-2010EV. High resolu-
tion mass spectra (MALDI/DHB) were performed on IonSpec 4.7
Tesla FTMS from Varian.
In the subsequent 40-O-rhamnosylation, we encountered some
difficulties because the nucleophilicity of the flavone 40-hydroxyl
group is poor. We first tried with donor 14,20 but under various
conditions (promoted by TMSOTf or BF3ꢁEt2O, with or without
4 Å MS), no desired product was obtained. The best 40-O-rham-
nosylation was via a Mitsunobu reaction, which provided 15 exclu-
sively as the
yield was reduced to 42% in CH2Cl2. PTC condensation of the bro-
mide 13 and 12 with Aliquat 336 gave 15 as the -anomer in
a-anomer, in 63% yield in DMF as reaction solvent; the
a
35% yield; when Aliquat 336 was substituted by n-Bu4NBr (TBAB),
no product was obtained. The PTC conditions not only confirmed
the
a-glycoside linkage of the product obtained with Mitsunobu
reaction, but also showed that the choice of PTC is vital for the syn-
thesis. Due to the fact that there are very few examples of direct
flavone 40-O-glycosylation, our results are significant and worthy
of further investigation.
To obtain the final target, the 7-O-benzyl ether of 15 was hydro-
genated with Pd/C to provide 16. Compound 16 was reacted with
tetrabenzoyl glucosyl bromide 5 under PTC conditions to obtain
bis-glycosyl flavonoid 10, whose structure was confirmed by 1H
and 13C NMR data. Thus, compound 1 could be prepared by this
route in seven steps and in 16% overall yield from apigenin.
1.2. 5,7,40-Tri-O-hexanoyl apigenin (3)
Apigenin (1.08 g, 4 mmol), Et3N (2.8 mL, 20 mmol), and DMAP
(155 mg, 1.2 mmol) were dissolved in DMF (10 mL). The mixture
was cooled under ice bath, and hexanoyl chloride (3 mL, 23 mmol)
was slowly added. The mixture was further stirred. After about 8 h,
TLC showed no remaining apigenin, and the solution was diluted
with CH2Cl2 and washed with 1 M HCl, water, satd aq NaHCO3,
and brine. The organic phase was dried (Na2SO4) and the solvent
removed under vacuum. The residue was purified by recrystalliza-
In conclusion, the first total synthesis of 7-O-b-
D-glucopyrano-
syl-40-O-
a-L-rhamnopyranosyl apigenin, 1, which exhibits good
anti-HBV and anti-stroke activities, was accomplished in six steps
and 20% overall yield from apigenin. A hexanoyl-based protection
strategy was developed, which greatly improved the solubility of
flavonoid intermediates, allowed the efficient selective protection,

514
Q. Gao et al. / Carbohydrate Research 344 (2009) 511–515
tion (EtOH) to obtain 1.99 g of 3, in 88% yield as a white solid.
Rf = 0.45 (petroleum ether–EtOAc 6:1); 1H NMR (400 MHz, CDCl3):
d 7.87 (d, 2H, J = 8.4 Hz), 7.34 (s, 1H), 7.25 (d, 2H, J = 8 Hz), 6.83 (s,
1H), 6.62 (s, 1H), 2.67 (m, 6H), 1.79 (m, 6H), 1.41 (m, 12H), 0.93 (m,
9H); 13C NMR (100 MHz, CDCl3): d 176.1, 171.9, 171.6, 170.8, 161.6,
157.6, 154.1, 153.5, 150.4, 128.5, 127.4, 127.4, 122.3, 122.3, 114.9,
113.7, 108.7, 108.5, 34.3, 34.2, 34.1, 31.3, 31.2, 31.1, 24.4, 24.4,
24.1, 22.3, 22.3, 22.2, 13.8, 13.8, 13.7. Anal. Calcd for C33H40O8: C,
70.19; H, 7.14. Found: C, 69.95; H, 7.17. ESI-MS (m/z): 565.2
[M+H]+.
CDCl3): d 176.6, 173.3, 166.2, 165.7, 165.2, 165.0, 162.6, 160.0,
159.9, 158.1, 150.4, 133.7, 133.7, 133.5, 133.2, 129.9, 129.9, 129.9,
129.8, 129.8, 129.8, 129.6, 129.6, 129.6, 129.2, 129.2, 128.7, 128.7,
128.7, 128.7, 128.5, 128.5, 128.5, 128.4, 128.4, 128.4, 127.8, 122.1,
116.0, 116.0, 112.6, 109.3, 106.2, 102.7, 98.3, 72.9, 72.4, 71.4, 69.2,
63.0, 34.2, 31.4, 24.0, 22.3, 13.9. Anal. Calcd for C55H46O15: C,
69.76; H, 4.90. Found: C, 69.55; H, 4.80. ESI-MS (m/z): 969.3 [M+Na]+.
1.6. 5-O-Hexanoyl-7-O-(2,3,4,6-tetra-O-benzoyl-b-
D-glucopy-
ranosyl)-40-O-(2,3,4-tri-O-benzoyl-
a-L-rhamnopyranosyl)
apigenin (10)
1.3. 5,40-Di-O-hexanoyl-apigenin (4)
Route A: DEAD (47 lL, 0.3 mmol) was added to a stirred solution
To a solution of 3 (2 g, 3.54 mmol) in 100 mL CH2Cl2 and 100 mL
methanol was added K2CO3 (240 mg, 1.77 mmol) at 0 °C. After
1.5 h, TLC showed no remaining 3, and the reaction was quenched
by the addition of HCl–MeOH (2 M) to pH 6–7. The mixture was fil-
tered through Celite and the solvent was removed under vacuum,
the resulting oil was purified by column chromatography (CH2Cl2–
acetone 30:1) to provide 1.52 g of 4, in 92% yield as a white solid.
Rf = 0.4 (silica, CH2Cl2–acetone 30:1) 1H NMR (400 MHz, CDCl3): d
8.52 (s, 1H), 7.74 (d, 2H, J = 8.4 Hz), 7.17 (d, 2H, J = 8.8 Hz), 6.71
(d, 1H, J = 2.4 Hz), 6.51 (d, 1H, J = 2.8 Hz), 6.50 (s, 1H), 2.71 (t, 2H,
J = 7.2 Hz), 2.56 (t, 2H, J = 7.6 Hz), 1.78 (m, 4H), 1.39 (m, 8H), 0.93
(m, 6H); 13C NMR (100 MHz, CDCl3): d 177.7, 173.5, 172.3, 162.4,
161.9, 158.9, 153.6, 150.7, 128.6, 127.7, 127.6, 122.6, 122.5,
110.4, 109.9, 107.6, 101.7, 34.6, 34.5, 31.5, 31.4, 24.7, 24.4, 22.6,
22.5, 14.2, 14.1. Anal. Calcd for C27H30O7: C, 69.51; H, 6.48. Found:
C, 69.60; H, 6.48. ESI-MS (m/z): 465.2 [MꢀH]ꢀ.
of 8 (189 mg, 0.2 mmol), 9 (79 mg, 0.167 mmol), PPh3 (78 mg,
0.3 mmol), and 4 Å MS (30 mg) in anhydrous DMF (5 mL) at –20 °C
under N2. The reaction mixture was stirred for 2 h at –20 °C, and
then was stirred for 6 h at rt. After TLC showed no remaining 9,
the solution was diluted with CH2Cl2 and washed by 1 M HCl, water,
aq satd NaHCO3, and brine. The organic phase was dried (Na2SO4)
and the solvent removed under vacuum. The residue was purified
by chromatography on silica gel (petroleum ether–EtOAc–CH2Cl2
7:1:2) to obtain 118 mg of 10 in 42% yield, as a yellow syrup.
Route B: To a solution of acceptor 16 (165 mg, 0.2 mmol) and do-
nor 5 (197 mg, 0.3 mmol) in 5 mL chloroform was added 5 mL H2O,
and then K2CO3 (69 mg, 0.5 mmol) and TBAB (16 mg, 0.05 mmol)
were added. The mixture was stirred at 40–45 °C for 24 h when
TLC showed most of 16 was consumed, and then the reaction was
then quenched by the addition of 1 M HCl solution. The solution
was diluted with CH2Cl2 and washed with brine. The organic phase
was dried (Na2SO4) and the solvent removed under vacuum. The res-
iduewaspurified bychromatographyon silicagel (petroleumether–
EtOAc–CH2Cl2 8:1:2) to obtain 200 mg of 10, in 71% yield as yellow
syrup. 1H NMR (400 MHz, CDCl3): d 8.15 (m, 2H), 7.97 (m, 8H),
7.87 (m, 4H), 7.46 (m, 25H), 7.03 (d, 1H, J = 1.7 Hz), 6.71 (d, 1H,
J = 1.7 Hz), 6.55 (s, 1H), 6.04 (m, 2H), 5.83 (m, 5H), 5.64 (d, 1H,
J = 7.2 Hz), 4.78 (d, 1H, J = 9.3 Hz), 4.53 (m, 2H), 4.29 (m, 1H), 2.67
(m, 2H), 1.77 (m, 2H), 1.38 (m, 7H), 0.93 (m, 3H); 13C NMR
(100 MHz, CDCl3): d 172.2, 166.1, 165.7, 165.6, 165.6, 165.6, 165.2,
165.0, 161.7, 160.0, 158.5, 133.7, 133.6, 133.6, 133.4, 133.3, 133.0,
130.0, 130.0, 129.9, 129.9, 129.9, 129.8, 129.8, 129.7, 129.7, 129.6,
129.6, 129.6, 129.3, 129.3, 129.3, 129.1, 129.1, 129.1, 129.0, 129.0,
129.0, 128.7, 128.7, 128.7, 128.6, 128.6, 128.6, 128.6, 128.5, 128.5,
128.5, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.0, 128.0,
128.0, 128.0, 125.5, 116.8, 113.0, 109.4, 107.7, 102.5, 98.3, 95.7,
73.0, 72.5, 71.4, 71.4, 70.4, 69.6, 69.3, 67.9, 63.1, 34.1, 31.3, 24.0,
22.4, 17.7, 13.9. ESI-MS (m/z): 1427.4 [M+Na]+.
1.4. 5,40-Di-O-hexanoylapigenin-7-yl 2,3,4,6-tetra-O–benzoyl-b-
D-glucopyranoside (6)
To a solution of acceptor 4 (94 mg, 0.2 mmol) and donor 5
(197 mg, 0.3 mmol) in 4 mL chloroform was added 4 mL H2O, and
then K2CO3 (69 mg, 0.5 mmol) and Aliquat 336 (8 mg, 0.02 mmol)
were added. The mixture was stirred at 40–45 °C. After 24 h, TLC
showed most of 4 was consumed, and the reaction was quenched
byadditionof 1 MHClsolution. ThesolutionwasdilutedwithCH2Cl2
and washed by brine. The organic phase was dried (Na2SO4) and the
solvent removed under vacuum. The resulting oil was purified by
chromatography on silica gel (petroleum ether–EtOAc–CH2Cl2
6:1:2) to obtain 163 mg of 6, in 78% yield as yellow syrup. 1H NMR
(400 MHz, CDCl3): d 7.95 (m, 9H), 7.38 (m, 15H), 7.01 (s, 1H),
6.71(s, 1H), 6.68 (s, 1H), 6.02 (t, 1H, J = 9.2 Hz), 5.84 (t, 1H,
J = 8.8 Hz), 5.64 (t, 1H, J = 9.1 Hz), 5.63 (d, 1H, J = 7.1 Hz), 4.78 (d,
1H, J = 9.7 Hz), 4.50 (m, 2H), 2.67 (m, 4H), 1.77 (m, 4H), 1.40 (m,
8H), 0.94 (m, 6H); 13C NMR (100 MHz, CDCl3): d 176.2, 172.2,
171.8, 166.1, 165.6, 165.2, 165.0, 161.4, 160.0, 158.3, 153.3, 150.9,
133.6, 133.6, 133.4, 133.2, 129.9, 129.9, 129.8, 129.8, 129.5, 129.5,
129.3, 129.3, 128.7, 128.7, 128.6, 128.6, 128.6, 128.6, 128.5, 128.5,
128.5, 128.4, 128.4, 128.4, 127.4, 127.4, 127.4, 122.3, 122.3, 113.0,
109.6, 108.4, 102.4, 98.3, 73.0, 72.5, 71.4, 69.3, 63.1, 34.4, 34.1,
31.3, 31.2, 24.5, 24.0, 22.4, 22.3, 13.9,13.9. Anal. Calcd for
C61H56O16: C, 70.10; H, 5.40. Found: C, 70.31; H, 5.25. ESI-MS
(m/z): 1083.3 [M+K]+.
1.7. 7-O-Benzyl-5,40-di-O-hexanoyl apigenin (11)
To a solutionof 3 (4.4 g, 7.8 mmol) in acetone (50 mL)were added
K2CO3 (10 g, 72 mmol) and KI (390 mg, 2.3 mmol). To this well-stir-
red mixture, benzyl chloride (5.8 mL, 51 mmol) was added, and the
mixture was stirred at 55 °C. After 24 h, TLC showed no remaining
3, and the mixture was filtered and the solvent removed under vac-
uum. Theresultingoilwaspurifiedbycrystallization(EtOAc–hexane
13:120 mL) to obtain 3.69 g of 11 in 85% yield, as a pale yellow solid.
Rf = 0.48 (silica, petroleum ether–EtOAc 6:1); 1H NMR (400 MHz,
CDCl3): d 7.84 (dd, 2H, J = 6.8 Hz, 2 Hz), 7.42 (m, 5H), 7.23 (dd, 2H,
J = 6.8 Hz, 1.6 Hz), 6.93 (d, 1H, J = 2.4 Hz), 6.68 (d, 1H, J = 2.4 Hz),
6.55 (s, 1H), 5.16 (s, 2H), 2.73 (t, 2H, J = 7.6 Hz), 2.58 (t, 2H,
J = 7.2 Hz), 1.78 (m, 4H), 1.44 (m, 8H), 0.93 (m, 6H); 13C NMR
(100 MHz, CDCl3): d 176.2, 172.2, 171.6, 162.5, 161.1, 158.7, 153.2,
150.8, 135.4, 128.7, 128.6, 128.5, 128.4, 127.4, 127.4, 127.4, 127.3,
122.2, 122.2, 111.6, 108.9, 108.3, 99.9, 70.7, 34.3, 34.2, 31.3, 31.2,
24.6, 24.1, 22.3, 22.1, 13.8, 13.7. Anal. Calcd for C34H36O7: C, 73.36;
H, 6.52. Found: C, 73.56; H, 6.38. ESI-MS (m/z): 579.3 [M+Na]+.
1.5. 5-O-Hexanoyl-apigenin-7-yl 2,3,4,6-tetra-O-benzoyl-b-D-
glucopyranoside (8)
The procedure is the same as that for compound 4, column chro-
matography (CH2Cl2–acetone 20:1) provided 8, in 84% yield as a pale
yellow solid: 1H NMR (400 MHz, CDCl3): d 7.82 (m, 8H), 7.40 (m,
15H), 6.90 (m, 1H), 6.72 (m, 3H), 6.39 (s, 1H), 6.03 (m, 1H), 5.85 (m,
2H), 5.63 (d, 1H, J = 10 Hz), 4.76 (m, 1H), 4.50 (m, 2H), 2.67 (m,
2H), 1.76 (m, 2H), 1.36 (m, 4H), 0.91 (m, 3H); 13C NMR (100 MHz,

Q. Gao et al. / Carbohydrate Research 344 (2009) 511–515
515
1.8. 7-O-Benzyl-5-O-hexanoyl apigenin (12)
133.3, 133.2, 130.0, 130.0, 129.7, 129.7, 129.1, 129.1, 129.0, 129.0,
128.6, 128.6, 128.6, 128.4, 128.4, 128.3, 128.3, 128.0, 128.0, 128.0,
125.5, 116.8, 116.8, 110.4, 109.6, 106.7, 101.5, 95.7, 71.6, 70.5,
69.8, 67.9, 34.3, 31.3, 24.1, 22.3, 17.7, 13.8. ESI-MS (m/z): 849.3
[M+Na]+.
To a solution of 11 (2 g, 3.6 mmol) in 100 mL CH2Cl2 and 100 mL
methanol, K2CO3 (246 mg, 1.8 mmol) was added at 0 °C. After 10 h,
TLC showed no remaining 11, and the reaction was quenched by
the addition of HCl–MeOH (2 M) to pH 6–7. The mixture was fil-
tered through Celite and the solvent removed under vacuum, and
the resulting oil was purified by chromatography on silica gel
(CH2Cl2–acetone 20:1) to obtain 1.51 g of 12, in 92% yield as a pale
yellow solid. Rf = 0.5 (silica, CH2Cl2–acetone 20:1); 1H NMR
(400 MHz, CDCl3): d 9.23 (s, 1H), 7.89 (dd, 2H, J = 6.8 Hz, 2 Hz),
7.42 (m, 5H), 7.20 (d, 1H, J = 2 Hz), 7.02 (dd, 2H, J = 6.8 Hz, 2 Hz),
6.73 (d, 1H, J = 2 Hz), 6.49 (s, 1H), 5.30 (s, 2H), 2.76 (m, 2H), 1.75
(m, 2H), 1.43 (m, 4H), 0.94 (m, 3H); 13C NMR (100 MHz, CDCl3):
d 177.1, 173.7, 162.7, 162.6, 160.1, 158.6, 150.3, 135.3, 128.8,
128.7, 128.5, 128.3, 127.8, 127.5, 127.4, 122.2, 116.2, 116.1,
111.0, 108.9, 105.9, 99.9, 70.8, 34.3, 31.3, 24.1, 22.3, 13.9. Anal.
Calcd for C28H26O6: C, 73.35; H, 5.72. Found: C, 73.19; H, 5.60.
ESI-MS (m/z): 457.2 [MꢀH]ꢀ.
1.11. 7-O-b-
apigenin (1)
D a-L-rhamnopyranosyl
-glucopyranosyl-40-O-
NaOMe (38 mg, 0.71 mmol) was added to a solution of 10
(250 mg, 0.178 mmol) in CH2Cl2 (3 mL) and MeOH (3 mL) at rt.
After 4 h, TLC showed no remaining 10, the reaction was quenched
by adding HCl–MeOH (2 M) to pH 6–7, and the solvent removed
under vacuum. The residue was purified by chromatography on sil-
ica gel (CH2Cl2–MeOH 3:1) to obtain 90 mg of 1 in 88% yield, as a
pale yellow solid. 1H NMR (400 MHz, C6D6N): d 8.07 (d, 2H,
J = 8.8 Hz), 7.45 (d, 2H, J = 8.8 Hz), 7.23 (d, 1H, J = 1.62 Hz), 7.07
(s, 1H), 6.96 (d, 1H, J = 2 Hz), 6.22 (s, 1H), 5.92 (d, 1H, J = 7.7 Hz),
4.74 (m, 2H), 4.36 (m, 8H), 1.67 (d, 3H, J = 6.2 Hz); 13C NMR
(100 MHz, C6D6N): d 183.2, 164.8, 164.5, 160.5, 158.3, 159.6,
129.1, 129.1, 125.1, 117.7, 117.7, 106.9, 105.3, 102.0, 101.2,
100.0, 96.0, 79.3, 78.4, 74.9, 73.8, 73.7, 72.5, 71.8, 71.5, 62.6,
18.9. ESI-MS (m/z): 601.1 [M+Na]+, 613.1 [M+Cl]ꢀ, HR-MS: m/z
calcd for C27H31O14: 579.1708, found: 579.1708.
1.9. 7-O-Benzyl-5-O-hexanoylapigenin-40-yl 2,3,
4-tri-O–benzoyl-a-L-rhamnopyranoside (15)
DEAD (47 L, 0.3 mmol) was added to a stirred solution of 12
l
(92 mg, 0.2 mmol), 9 (79 mg, 0.167 mmol), PPh3 (78 mg, 0.3 mmol),
and 4 Å MS (30 mg)in anhydrous DMF(5 mL)at –20 °C under N2. The
reaction mixture was stirred for 2 h at –20 °C, and then was stirred
for 6 h at rt. After TLC showed no remaining 9, the solution was di-
luted with CH2Cl2 and washed with 1 M HCl, water, satd aq NaHCO3,
and brine. The organic phase was dried (Na2SO4) and the solvent re-
moved under vacuum. The residue was purified by chromatography
on silica gel (petroleum ether–EtOAc–CH2Cl2 7:1:2) to obtain 96 mg
of 15 in 63% yield, as a yellow syrup. 1H NMR (400 MHz, CDCl3): d
8.15 (d, 2H, J = 8.4 Hz), 7.99 (d, 2H, J = 8 Hz), 7.87 (m, 4H), 7.67 (m,
2H), 7.63 (m, 3H), 7.31 (m, 11H), 6.97 (s, 1H), 6.70 (t, 1H), 6.57 (d,
1H), 6.06 (dd, 1H, J = 2.4 Hz, 10 Hz), 5.85 (m, 3H), 5.19 (s, 2H), 4.34
(m, 1H), 2.76 (t, 2H, J = 7.2 Hz), 1.85 (m, 2H), 1.33 (m, 4H), 1.28 (m,
3H), 0.94 (m, 3H); 13C NMR (100 MHz, CDCl3): d 176.5, 172.3,
165.8, 165.6, 162.6, 161.6, 158.9, 158.5, 150.8, 135.5, 133.7, 133.7,
133.4, 133.4, 133.2, 133.2, 130.0, 130.0, 130.0, 129.7, 129.2, 128.8,
128.8, 128.8, 128.7, 128.4, 128.4, 128.4, 128.3, 128.3, 127.9, 127.9,
127.9, 127.5, 125.9, 121.7, 116.9, 111.6, 108.9, 107.7, 100.0, 95.8,
71.5, 70.8, 70.5, 67.9, 64.2, 34.3, 31.4, 29.7, 24.2, 22.4, 17.7, 14.1,
13.9. Anal. Calcd for C55H48O13: C, 72.04; H, 5.28. Found: C, 72.31;
H, 5.15. ESI-MS (m/z): 939.3 [M+Na]+.
Acknowledgments
This work was supported by Innovation Research Grant of SIPI.
F.L. thanks the Open Grant of the State Key Lab of Bioorganic and
Natural Products Chemistry (SIOC, CAS). The authors thank Dr.
Jason C. Wong for helpful discussion.
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