Regioselective synthesis of di-C-glycosylflavones possessing anti-inflammation activities

Article abstract of DOI:10.1039/c0ob00011f

Three methods are utilized to synthesize a variety of 6,8-di-C- glycosylflavones bearing identical or distinct glycosyl moieties. Some C-glycosylation compounds are found to have better anti-inflammation activities than the parent flavones. Among them, 6,8-di-C-glucosylapigenin (known as vicenin-2) shows inhibition of TNF-α expression and NO production with IC₅₀ values of 6.8 and 5.2 μM, respectively.

Full text of DOI:10.1039/c0ob00011f

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Regioselective synthesis of di-C-glycosylflavones possessing anti-inflammation
activities†
Jiun-Jie Shie,a Chih-An Chen,b Chih-Chien Lin,c Angela Fay Ku,b Ting-Jen R. Cheng,a Jim-Min Fang*a,b and
Chi-Huey Wong*a
Received 16th April 2010, Accepted 28th June 2010
DOI: 10.1039/c0ob00011f
Three methods are utilized to synthesize a variety of 6,8-di-C-glycosylflavones bearing identical or
distinct glycosyl moieties. Some C-glycosylation compounds are found to have better anti-inflammation
activities than the parent flavones. Among them, 6,8-di-C-glucosylapigenin (known as vicenin-2) shows
inhibition of TNF-a expression and NO production with IC50 values of 6.8 and 5.2 mM, respectively.
the Sc(OTf)3-promoted glycosylation of ( )-naringenin with D-
glucose gave a low yield (<20%) of the desired 6,8-di-C-b-D-
Introduction
Dendrobium huoshanense1 (Orchidaceae) is a valuable herbal
plant used in traditional Chinese medicine.1–3 The polysaccharide
constituent of stem mucilage is found to exhibit specific functions
in activating murine splenocytes to produce several cytokines,
including IFN-g, IL-10, IL-6, and IL-1a, as well as hematopoi-
etic growth factors GM-CSF and G-CSF.4 The bioactive small
molecules in D. huoshanense are reported to include the bibenzyl,5
phenanthrene,6 fluorene,7 coumarin,8 sesquiterpene,9 flavanone,10
and alkaloid11 structural types. We recently also isolated four 6,8-
di-C-glycosylflavones with a core structure of apigenin bearing
pentoside (arabinoside or xyloside) and rhamnosyl–hexoside
(glucoside or galactoside) substituents.12
The constituents of C-glycosylflavones are rich in Rutaceous,
Compositous and Fabaceous plants.13 The naturally-occurring
C-glycosides are generally linked at C-6 and/or C-8 on the A-
ring of flavonoid nucleus. Monosaccharides of D-Glc, D-Gal, D-
Ara, L-Ara, D-Xyl and L-Rha are commonly found in natural
glycosylapigenins.14 It has been difficult to isolate di-C-glycosyl
flavonoids from nature sources;12,14d,14f thus, organic synthesis is an
alternative and effective method to obtain sufficient quantities
of these bioactive compounds. In this study, a series of 6,8-
di-C-glycosyl flavonoids bearing identical or distinct glycosyl
substituents were synthesized and their biological activities were
examined.
glucosylnaringenin (2aa)16 along with a significant amount (~15%)
of mono-C-glycosylation product and other unidentified side
products (Scheme 1). Purification of 6,8-di-C-glucosylnaringenin
or the peracetylation derivative 2aaAc was only realized by
repeated chromatography to remove undesired side products.
Oxidation of the peracetylated flavanone 2aaAc with DDQ,
followed by saponification, yielded 6,8-di-C-b-D-glucosylapigenin
(3aa, vicenin-2).15,17 Attempts to direct C-glycosylation on the
A-ring of apigenin failed, presumably because the electron-
withdrawing effect in the conjugated system of flavone disfavored
the electrophilic aromatic substitution reaction.
In another approach, Sato and coworkers have carried out
the Sc(OTf)3-promoted glycosylation of phloroacetophenone (4)
with D-glucose.18 Accordingly, the reaction gave 43% and 38%
yields of mono- and di-C-glycosyl phloroacetophenones under
optimized conditions. In our hands, the Sc(OTf)3-promoted
glycosylation of phloroacetophenone with D-xylose, followed by
benzylation of the phenolic groups, afforded pure 6,8-di-C-xylosyl
phloroacetophenone (5bbBn) in 16% isolated yield (Scheme 2).
Condensation of 5bbBn with substituted benzaldehydes in the
presence of KOH gave diglycosylchalcones 7bb1–3 in 60–70%
yields. Without prior benzylation, the phenoxide ions would be
generated in the presence of KOH base, and thus prevent the
desired aldol reaction.19 By acid catalysis, diglycosylchalcones
7bb1–3 underwent intramolecular Michael reaction smoothly to
give 6,8-di-C-glycosylflavanones 8bb1–3 after debenzylation. To
avoid undesired oxidation of the phenolic moieties, flavanones
8bb1–3 were protected as the peracetylates prior to oxidation with
Me2SO/I2.19a,20 Reacetylation was performed to facilitate isolation
of the products because some acetyl groups were cleaved by HI
(generated in situ) under the reaction conditions. By subsequent
saponification, the target compounds of 6,8-di-C-glycosylflavones
9bb1–3 having different substituents on the B-ring were obtained
in reasonable yields.
Results and discussion
1. Synthesis
At the first sight, direct di-C-glycosylation of naringenin (1)
looks attractive to attain di-C-glycosylflavones having identical
glycosyl substituent, in particular, ( )-naringenin is commercially
available. However, Sato’s15 and our current studies showed that
aThe Genomics Research Center, Academia Sinica, Taipei, 115,
Taiwan. E-mail: chwong@gate.sinica.edu.tw; Fax: (8862)27898771;
Tel: (8862)27899400
This synthetic approach can be applied to build libraries
of 6,8-di-C-glycosylflavanones (e.g. 8bb1–3) and 6,8-di-C-
glycosylflavones (e.g. 9bb1–3) containing different substituents on
the B-ring because the precursors of di-C-glycosylchalcone are
easily prepared by using a variety of substituted benzaldehydes
(e.g. 6a–c). In the case of the di-C-glycosylchalcone bearing two
different glycosyl moieties at 3¢- and 5¢-positions, both 2¢- and
bDepartment of Chemistry, National Taiwan University, Taipei, 106, Taiwan.
E-mail: jmfang@ntu.edu.tw; Fax: (8862)23637812; Tel: (8862)33661663
cInstitute of Biochemistry Sciences, National Taiwan University, Taipei, 106,
Taiwan
† Electronic supplementary information (ESI) available: Experimental
details and NMR spectra. See DOI: 10.1039/c0ob00011f
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Scheme 1 Synthesis of 6,8-di-C-glucosylapigenin by the Sc(OTf)3-promoted di-C-glycosylation of ( )-naringenin with D-glucose. Reagents and conditions:
(a) Sc(OTf)3, EtOH, H2O, reflux, 16 h; (b) Ac2O, DMAP, pyridine, 0 to 25 ◦C, 24 h; (c) DDQ, PhCl, 130 ◦C, 24 h; (d) NaOMe, MeOH, 25 ◦C, 1 h.
Scheme 2 Stepwise synthesis of di-C-glycosylflavones via diglycosylation of phloroacetophenone with D-xylose. Reagents and conditions: (a) Sc(OTf)3,
EtOH, H2O, reflux 14 h; (b) BnBr, K2CO3, DMF, 25 ◦C, 12 h; 16% from 4; (c) KOH, MeOH, 45 ◦C; 60–70%; (d) HCl, MeOH, reflux, 25 min; (e) H2,
Pd/C, MeOH, EtOAc, 25 ◦C, 3 h; (f) Ac2O, pyridine, 25 ◦C, 12 h; (g) cat. I2, DMSO, 130 ◦C, 3 h; (h) Ac2O, pyridine, 25 ◦C, 4 h; (i) MeONa, MeOH,
25 ◦C, 3 h.
6¢-alkoxy groups can act as the Michael donors in the
acid-catalyzed cyclization, giving two regioisomers of di-C-
glycosylflavanone. To circumvent this problem, a regioselective
synthesis of 6,8-di-C-glycosyl flavonoids was thus explored by tan-
dem C-glycosylations of flavans to introduce individual glycosyl
residues at the designated 6- or 8-positions (Scheme 3).
Comparing the three phenol groups in naringenin, the 5-
OH is the least reactive due to its intramolecular bonding
with the C-4 carbonyl group, whereas the 7-OH is activated
by conjugation with the carbonyl group at the para position.
Thus, alkylation of ( )-naringenin (1) with benzyl bromide (1.3
equiv.) in the presence of K2CO3 (1 equiv.) occurred selectively
at the most acidic 7-OH group. Flavanone 10 was obtained in
97% overall yield by acetylation of the remaining 4¢- and 5-
OH groups. Furthermore, flavanone 10 was transformed into
flavan 11 in order to avoid difficulty in C-glycosylations caused
by the electron-withdrawing effect of the C-4 carbonyl group.
Flavan 11 was obtained in 90% yield by reduction of 10 with
NaBH4 (2.0 equiv.).21 Deacetylation at C-5 along with removal
of the C-4 carbonyl is rationalized.21 Accordingly, the acetyl
group of 5-OAc is readily transferred to the 4-OH group that
resulted from an initial reduction of the C-4 carbonyl in flavanone
10. Such transesterification thus facilitates the elimination of
an HOAc molecule to form an o-quinonemethide intermediate,
which is reduced further by NaBH4 to give the observed flavan
product 11.
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Scheme 3 Synthesis of di-C-glycosylapigenins via regioselective tandem glycosylations of flavans. Reagents and conditions: (a) BnBr, K2CO3 (1 equiv.),
DMF, 25 ◦C, 12 h; 75%; (b) Ac2O, pyridine, DMAP, 25 ◦C, 4 h; 95% from 1; (c) NaBH4, THF–H2O, 0 ◦C, 45 min; 90%; (d) cat. TMSOTf, CH2Cl2, -15 ◦C
(30 min) to 25 ◦C (3 h); 55–79%; (e) H2, Pd/C, EtOAc, MeOH, 25 ◦C, 1 h; 83–92%; (f) BnBr, K2CO3, DMF, 50 ◦C, 5 h; (g) CAN, CH3CN, H2O, 25 ◦C,
2 h; (h) PDC, CH2Cl2, reflux, 4 h; (i) NaOMe, MeOH, 25 ◦C, 12 h; 79–91%; (j) DDQ, PhCl, 140 ◦C, 24 h; (k) Ac2O, pyridine, DMAP, 25 ◦C, 4 h; (l) cat.
I2, Me2SO, 140 ◦C, 4 h.
In this synthetic route, D-glucose (series a), D-xylose (series b), D-
arabinose (series c) and L-arabinose (series d) were prior elaborated
to the corresponding peracetylglycosyl trichloroacetimidates to
act as the glycosyl donors. The first C-glycosylation of 11
occurred selectively at the C-6 position, giving 12aBn–12dBn,
by using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as
the reaction promoter. The regioselective C-glycosylation could
be attributable to a Fries-type O→C glycoside rearrangement
of the initially formed O-glycosylation compound.22 However,
the possibility of direct Friedel–Crafts C-glycosylation at the
most activated position (C-6) of the aromatic A-ring is not
excluded.22 After removal of the benzyl group in 12aBn–12dBn by
hydrogenolysis, the second C-glycosylation with another glycosyl
donor, either the same with or different from that in the first
glycosylation, proceeded smoothly at C-8 to give a series of 6,8-
di-C-glycosylflavans (13aa, 13ab and other analogs).
(e.g. 14aaBn) in good yields. Di-C-glycosylflavanols 14bdBn and
14bdBn were further oxidized by pyridinium dichromate (PDC)
to give the corresponding di-C-glycosylflavanones, which were
subjected to hydrogenation and saponification to give 6,8-di-
C-glycosylnaringenins 2bd and 2db. On the other hand, di-C-
glycosylflavanols (e.g. 14aaBn) were directly oxidized by excess
amounts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
to afford the corresponding di-C-glycosylflavones. After debenzy-
lation, the product was converted to the peracetate derivative for
isolation and structural characterization. The final saponification
culminated in the desired 6,8-di-C-glucosylapigenins (3aa). Di-
C-glycosylflavanones (e.g. 15aa) were also oxidized in I2/Me2SO,
followed by removal of the protecting groups, to give 6,8-di-C-
glycosylapigenins (e.g. 3aa). A series of 6,8-di-C-glycosylapigenin
compounds bearing the same or distinct glycosyl moieties at
the designated positions (C-6 and C-8) were thus synthesized by
similar procedures (Scheme 3).
The acetates of 6-C-glycosylflavans 12aBn, 12bBn and 12cBn
could be oxidized by excess cerium(IV) ammonium nitrate
(CAN, see Scheme 5);23 however, attempts to oxidize 6,8-di-C-
glycosylflavans (e.g. 13aa) and their peracetylation derivatives
failed. Finally, the bis-benzylation derivatives were found to be
smoothly oxidized by CAN to give 6,8-di-C-glycosylflavanols
Chemical elaboration of the B-ring in 6,8-di-C-
glycosylflavanone was also feasible by mimicking the enzymatic
oxidation of phenol in the biosynthesis of flavonoids.23c For
example, flavanone 15dbBn was selectively hydrolyzed by K2CO3
(1 equiv.) in CH2Cl2–MeOH under mild conditions to expose the
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Scheme 4 Elaboration of the B-ring in 6,8-di-C-glycosylflavone. Reagents and conditions: (a) K2CO3 (1 equiv.), CH2Cl2–MeOH, 25 ◦C, 1 h; (b) IBX (2
equiv.), DMSO, 25 ◦C, 5 h; then Na2SO3, 25 ◦C, 4 h; (c) Ac2O, pyridine, DMAP, 25 ◦C, 6 h; (d) H2, Pd/C, THF–MeOH, 25 ◦C, 3 h; (e) cat. I2, DMSO,
140 ◦C, 4 h; (f) NaOMe, MeOH, 25 ◦C, 2 h.
Scheme 5 Synthesis of 6-C-glycosylflavones that support the structural determination of 6,8-di-C-glycosylflavones. Reagents and conditions: (a) AcCl,
Et3N, DMAP, CH2Cl2, 25 ◦C, 10 h; (b) CAN, MeCN–AcOH–H2O, 50 ◦C, 10 h; (c) cat. I2, DMSO, 140 ◦C, 1 h; (d) H2, Pd/C, 25 ◦C, 1 h; (e) Ac2O,
pyridine, 25 ◦C, 10 h; (f) MeONa, MeOH, 25 ◦C, 5 h.
phenolic group on the B-ring (Scheme 4). The phenolic moiety
was then oxidized with 2-iodoxybenzoic acid (IBX),24 followed
by reductive workup of the o-quinone intermediate with sodium
dithionite (Na2S2O4), to give a catechol product. After acetylation
and debenzylation, flavanone 17db was further oxidized with
I2/Me2SO and isolated as a peracetylated product, which was
subjected to saponification to afford 6,8-di-C-glycosylluteolin
18db.
glycosylapigenins 3a, 3b and 3c (Scheme 5) were thus synthesized
and fully characterized to provide supporting evidence for the
structural assignments of the 6,8-di-C-glycosylapigenins.
All the glycosyl substituents in 3a–c were found to exist in the
pyranoside forms with the aglycone in an equatorial position, i.e.
glucoside and xyloside in the b-configuration, and arabinoside in
the a-configuration. The “anomeric” proton (H-1¢¢) in the axial
position in compound 3a occurred at dH 4.89 as a doublet with a
large coupling constant (J = 10 Hz). The structure of 3a was unam-
biguously assigned as 6-C-(b-D-glucopyranosyl)apigenin because
the synthetic sample exhibited the physical and spectral properties
([a], IR, HRMS, 1H and 13C NMR) consistent with those reported
for a natural product, isovitexin.25 Compound 3b contained a b-
xylopyranoside, rather than furanoside, because the H-1¢¢ at dH
2. Structure elucidation
As shown in the above-delineated schemes, various 6,8-di-C-
glycosylflavones bearing substituents of D-glucoside, D-xyloside,
D-arabinoside and L-arabinoside were synthesized. The substi-
tutions at C-6 and C-8 were confirmed by the disappearance
of the signals for H-6 and H-8 in the 1H NMR spectra.
However, structural determination of the sterically demanding
molecule of 6,8-di-C-glycosylflavone is not trivial at all by NMR
analysis because it often exists as a mixture of rotamers. More-
over, C-glycosylation of ( )-naringenin would produce 6,8-di-C-
glycosylnaringenins (e.g. 2bd) and 6,8-di-C-glycosylflavans (e.g.
13aa) as mixtures of diastereomers, of which structure elucidation
was further complicated by the existence of rotamers. Three 6-C-
3
4.79 (d, J = 9.9 Hz) showed a JH,C correlation with the C-5¢¢ at
dC 70.6 in the HMBC spectrum. The a-arabinopyranoside in 3c
was also inferred from the HMBC correlation of C-5¢¢ (at dC 69.8)
with the axial H-1¢¢ (at dH 4.79, d, J = 9.8 Hz).
The 1H and 13C resonances of the sugar moieties in di-C-
glycosylflavones were assigned according to their 1H, 13C, DEPT,
COSY, HSQC and HMBC spectra. The a- or b-configurations
of sugar residues could be deduced by the coupling constants of
the “anomeric” protons H-1¢¢ and H-1¢¢¢, if their resonances were
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Table 1 Anti-inflammation activities of water-, ethanol-, and methanol-
soluble extracts of Dendrobium huoshanense
Table 2 Anti-inflammation activities of apigenin and (di)glyco-
sylapigenins
Pretreated
extract
Induced TNF-a
Nitric oxide
Cell growth
(%)b
IC50/mMa
(%)a
production (%)a
Compound
C-6
C-8
TNF-a
NO
DH-H2O
DH-MeOH
DH-EtOH
129.3c/139.2d
92.7c/66.8d
88.2c/44.8d
NDe/NDe
42.9h
115f/120g
102f/100g
94f/94g
Apigenin
3a
3b
3c
H
H
H
H
H
D-Glc
D-Xyl
D-Ara
D-Xyl
D-Ara
L-Ara
D-Xyl
D-Ara
D-Xyl
18.5 3.5
9.7 3.0
35 7.0
32 5.6
6.8 2.5
32 4.2
27.5 2.1
19.5 6.3
24.5 0.7
>100
19 6.8
5.2 1.3
11 4.3
9.5 0.4
3.9 0.9
8.1 0.3
9.9 1.6
6.9 1.9
6.7 0.5
>100
32.2h
D-Glc
D-Xyl
D-Ara
D-Glc
D-Glc
D-Glc
D-Xyl
D-Xyl
D-Xyl
D-Ara
D-Ara
L-Ara
a The value detected in cells by treatment only with LPS (100 ng mL-1)
was defined as 100%. b The value detected in cells with no treatment was
defined as 100%. c The extract (100 mg mL-1) was added prior to treatment
with LPS. d The extract (250 mg mL-1) was added prior to treatment with
LPS. e Not determined. f The extract (100 mg mL-1) was added to cells for
6 h. g The extract (250 mg mL-1) was added to cells for 6 h. h The extract
(50 mg mL-1) was added prior to treatment with LPS.
3aa
3ab
3ac
3bb
3bc
3bd
3cb
3cc
27 1.4
27 5.6
>100
8.8 0.8
13 2.1
>100
3db
distinguishable from other protons. For example, compound 3db,
identical to a natural product isolated from Viola yedoensis,14f
showed the H-1¢¢ and H-1¢¢¢ at dH 5.03 (d, J = 10 Hz) and 4.78 (d,
J = 9.6 Hz), respectively, consistent with the a-configuration of
L-arabinoside and the b-configuration of D-xyloside. From time to
time, the assignments were also assisted by NMR analysis of the
peracetylation derivatives of di-C-glycosylflavones. For example,
peracetylation of 3aa (vicenin-2) gave a derivative 3aaAc,15 which
displayed two axial protons H-1¢¢ and H-1¢¢¢ at dH 4.78 and 4.55
with large coupling constant (J = 10 Hz) corresponding to the
6b- and 8b-oriented glucosides. Though H-1¢¢ and H-1¢¢¢ in 9bb1
were covered by the signals of methanol (small amount often
found in CD3OD solvent), the “anomeric” protons were diagnostic
in the peracetylation derivative 9bb1Ac to deduce the 6b- and
8b-configurations of xylopyranosides. Compound 9bb1Ac also
displayed the correlations of C-1¢¢ (at dC 74.2) with H-5¢¢ (at dH
4.40) as well as C-1¢¢¢ (at dC 72.8) with H-5¢¢¢ (at dH 4.15) to support
the assignment of 9bb1 as 6,8-di-C-b-D-xylopyranosylluteolin.
a Concentration of the indicated compound required for 50% inhibition of
TNF-a expression or NO production.
the 3¢-OH group to methoxy (9bb2) or addition of extra methoxy
groups (9bb3) on the B-ring did not improve the anti-inflammation
activities. In contrast, the corresponding peracetates (9bb1Ac,
9bb2Ac and 9bb3Ac) greatly suppressed TNF-a expression to 27–
50%, indicating that acetylation may help the availability of the
compounds to the cells.
As shown in Table 2, compound 3a, having a glucose moiety on
the apigenin scaffold, but not 3b or 3c with xylose or arabinose
moieties, increased anti-inflammation activities, as the IC50 for
TNF-a expression decreased from 18.5 mM (for apigenin) to
9.7 mM. Diglycosylapigenin 3aa with an extra glucose on the C-
8 position further enhanced the potency up to an IC50 value of
6.8 mM. Correlating with inhibition of TNF-a expression, both 3a
and 3aa also inhibited iNOS expression (data not shown) and then
NO production, with IC50 values of 5.2 and 3.9 mM, respectively.
3. Biological activities
Flavonoids possess several biological activities such as anti-
cancer, antibacterial, anti-inflammatory, immunomodulatory and
antioxidants.14g,26 D. huoshanense was claimed to have anti-
inflammation activities in traditional Chinese medicinal practice.
When various fractions from D. huoshanense were tested for
anti-inflammation activities in our preliminary studies, we also
found that ethanol- or methanol-soluble fractions (DH-EtOH and
DH-MeOH, respectively), but not water-soluble fractions (DH-
H2O), can inhibit expression of TNF-a and other inflammatory
cytokines in lipopolysaccharide (LPS)-activated RAW264.7 cells
(Table 1). In addition to cytokine expression, we also monitored
the expression of nitric oxide (NO), which is involved in in-
flammation and immunoregulation.27 NO production appeared
to decrease upon DH-EtOH and DH-MeOH treatment in LPS-
activated cells. The fraction was later identified to contain 6,8-di-
C-glycosyl flavonoids.12
To further dissect the structure-and-activity relationship, mono-
glycosyl and di-glycosyl flavonoids were synthesized as mentioned
above. The synthesized analogues with various sugars were further
tested for their anti-inflammation activities by monitoring TNF-a
expression as well as NO production rate. It was found that TNF-a
expression in Raw264.7 cells decreased to ~75% on treatment with
50 mM of 6,8-di-C-b-D-xylopyranosylluteolin (9bb1). Changing
Conclusion
Three methods were applied to the synthesis of di-C-
glycosylflavones. The first method (Scheme 1) used a Lewis
acid, Sc(OTf)3, to promote the glycosylations of ( )-naringenin
with unmodified monosaccharides to give the di-C-glycosylation
products accompanied by mono-C-glycosylation and unidentified
compounds. This synthetic procedure is straightforward; however,
it is tedious to obtain pure di-C-glycosylapigenins by repeated
chromatography.
The second method (Scheme 2) for the synthesis of di-C-
glycosylflavones starts with the Sc(OTf)3-promoted diglycosyla-
tion of phloroacetophenone. The subsequent aldol condensations
with substituted benzaldehydes gave diglycosylchalcones, which
underwent intramolecular Michael reactions to afford a series
of 6,8-di-C-glycosylflavanones and 6,8-di-C-glycosylflavones with
various substituents on the B-ring. This method is limited to
the synthesis of those compounds bearing the same glycosyl
moieties because the intramolecular Michael reactions would lack
regiochemical control in cases where the di-C-glycosylchalcone
bears two different glycosyl moieties.
Finally, the problems encountered in the first and sec-
ond methods were circumvented by regioselective tandem
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glycosylations of flavans (Scheme 3). Flavan is more electron-rich
than flavanone and flavones, rendering facile C-glycosylation. To
an appropriate flavan, the first glycosylation was introduced to
the C-6 position, and the second glycosylation with the same or
distinct glycosyl donor occurred at the C-8 position. The prepared
6,8-di-C-glycosylflavans were then elaborated to a series of 6,8-di-
C-glycosylflavones by modification at the B- and C-rings.
The prepared 6,8-di-C-glycosylflavones were found to exhibit
anti-inflammation activities by inhibiting TNF-a expression and
NO production. Introduction of glucosyl moieties as in 3aa
improved the anti-inflammation activities compared with the
parent compound of apigenin. Our study also indicates that
acetylation may help the availability of the compounds (e.g.
9bb1Ac) to suppress TNF-a expression.
H2O–HOAc, 15 : 30 : 2 : 1 to 30 : 30 : 5 : 1) to give a crude sample of
3,5-di-C-b-D-xylopyranosylphloroacetophenone (5bb).
For analytical purposes, the crude sample of 5bb was treated
with Ac2O (5 mL) in pyridine (5 mL) for 12 h at room temperature.
The reaction mixture was partitioned between 1 M HCl and
EtOAc. The organic phase was washed with 1 M HCl and brine,
dried over anhydrous MgSO4, filtered, and then concentrated
by rotary evaporation. The residue was purified by column
chromatography on silica gel (EtOAc–hexane, 1 : 1 to 1.5 : 1) to
give 5bbAc (320 mg, 20%).
To a stirred solution of compound 5bbAc (188 mg, 0.23 mmol)
in dry methanol (6 mL) was added dropwise sodium methoxide
(60 mg, 1.1 mmol) in dry methanol (3 mL). The resulting solution
was stirred at room temperature for 1 h. Dowex 50W¥8 (H+) was
added to the stirred reaction mixture until the solution became
neutral. The resulting mixture was filtered and washed with
methanol. The filtrate was evaporated in vacuo, and washed by
ether and hexane to give compound 5bb (91 mg, 92%). C18H24O12;
colorless solid, mp 154–155.5 ◦C; TLC (Me2CO–EtOAc–H2O–
HOAc, 30 : 30 : 5 : 1) Rf 0.25; 1H NMR (CD3OD, 400 MHz) d 4.81
(2 H, d, J = 10 Hz, a-anomeric H), 4.08 (2 H, dd, J = 11.6, 5.6 Hz),
3.70–3.60 (4 H, m), 3.44 (2 H, t, J = 9 Hz), 3.37–3.30 (2 H, m),
2.64 (3 H, s); 13C NMR (CD3OD, 100 MHz) d 205.5, 163.4 (2 ¥),
162.6, 106.5, 104.3 (2 ¥), 79.4 (2 ¥), 77.7 (2 ¥), 74.2 (2 ¥), 71.8
(2 ¥), 71.4 (2 ¥), 33.4; HRMS (ESI) calcd for C18H23O12: 431.1190,
found: m/z 431.1195 [M - H]-.
Experimental
General
All the reagents and solvents were reagent grade and were
used without further purification unless otherwise specified. All
solvents were anhydrous grade unless indicated otherwise. All
non-aqueous reactions were carried out in oven-dried glassware
under a slight positive pressure of argon unless otherwise noted.
Reactions were magnetically stirred and monitored by thin-layer
chromatography on silica gel using aqueous p-anisaldehyde as
visualizing agent. Silica gel (0.040–0.063 mm particle sizes) and
LiChroprep RP-18 (0.040–0.063 mm particle sizes) were used for
column chromatography. Flash chromatography was performed
on silica gel of 60–200 mm particle size. Molecular sieves were
activated under high vacuum at 220 ◦C over 6 h.
2,4,6,3¢,4¢-Pentabenzyloxy-3,5-di-C -(b-D-xylopyranosyl)chal-
cone (7bb1). To a solution of crude 5bb (112 mg, 0.26 mmol) in
dry DMF (3 mL) were added PhCH2Br (200 mg, 1.17 mmol) and
K2CO3 (161 mg, 1.17 mmol). The mixture was stirred at room
temperature for 12 h, and complete consumption of 5bb was
shown by TLC analysis. The mixture was filtered, concentrated
by evaporation under reduced pressure, and purified by column
chromatography on silica gel (CHCl3–MeOH, 8 : 1) to give 5bbBn
(160 mg, 88%).
Melting points were recorded on a Yanaco or Electrothermal
R
MEL-TEMPꢀ 1101D apparatus in open capillaries and are
not corrected. Infrared (IR) spectra were recorded on Nicolet
Magna 550-II or Thermo Nicolet 380 FT-IR spectrometers.
Nuclear magnetic resonance (NMR) spectra were obtained on
Varian Unity Plus-400 (400 MHz) or Bruker AVANCE (400
and 600 MHz) spectrometers. Chemical shifts (d) are given in
parts per million (ppm) relative to dH 7.26/dC 77.0 (central
line of t) for CHCl3/CDCl3, dH 4.80 for H2O/D2O, dH 3.31/dC
48.2 for CD3OD-d4, or dH 2.49/dC 39.5 for DMSO-d6. The
splitting patterns are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), dd (double of doublets) and
br (broad). Coupling constants (J) are given in Hz. Distortionless
enhancement polarization transfer (DEPT) spectra were taken to
determine the types of carbon signals. The ESI-MS experiments
were conducted on a Bruker Daltonics BioTOF III high-resolution
mass spectrometer. Optical rotations were measured on digital
polarimeter of Japan JASCO Co. DIP-1000. [a]D values are given
in units of 10-1 deg cm2 g-1.
Potassium hydroxide (200 mg, 3.6 mmol) was added to
a
solution of 5bbBn (280 mg, 0.4 mmol) and 3,4-di-
benzyloxybenzaldehyde (380 mg, 1.2 mmol) in MeOH (5 mL).
◦
The solution was stirred at 45 C for 24 h. After confirming the
disappearance of 5bbBn by TLC analysis, 1 M HCl was added
to neutralize the reaction mixture. The volatiles were removed by
rotary evaporation under reduced pressure, and the residue was
partitioned between water and EtOAc. The organic phase was
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated by rotary evaporation. The residue was purified by
column chromatography on silica gel (CHCl3–MeOH, 13 : 1) to
give 7bb1 (245 mg, 61%). C60H58O14; yellow prisms, mp 121.8–
123.8 ◦C; TLC (CHCl3–MeOH, 10 : 1) Rf 0.13; [a]2D5 -38.08 (c 2.1,
EtOAc); IR vmax (neat) 3400, 2921, 1576, 1508, 1454, 1268, 1134,
1085 cm-1; 1H NMR (a mixture of rotamers, CDCl3, 400 MHz) d
7.44–7.02 (27 H, m), 6.96 (1 H, d, J = 8.4 Hz), 6.79 (2 H, dd, J =
12.2, 3.8 Hz), 5.09 (2 H, s), 5.04 (2 H, s), 4.95 (2 H, t, J = 12.4 Hz),
4.78–4.66 (4 H, m), 4.55–4.49 (2 H, m), 4.22 (1 H, t, J = 9.2 Hz),
4.08 (1 H, t, J = 8.8 Hz), 3.87 (2 H, d, J = 6 Hz), 3.36–3.32 (1 H,
m), 3.25–3.16 (3 H, m), 3.14–3.06 (2 H, m); 13C NMR (CDCl3,
100 MHz) d 193.9, 160.9, 157.6, 157.0, 151.3, 148.5, 146.2, 136.5,
136.3, 136.1, 128.4–127.0 (5 ¥), 125.5, 124.0, 123.3, 122.2, 113.8,
79.8 (2 ¥), 78.9 (2 ¥), 78.4 (2 ¥), 75.9 (2 ¥), 75.7 (2 ¥), 71.2, 71.1, 70.7
Representative synthetic procedures
3,5-Di-(C-b-D-xylopyranosyl)acetophenone (5bb). A mixture
of phloroacetophenone (4, 372 mg, 2 mmol), D-xylose (900 mg,
6 mmol), and Sc(OTf)3 (197 mg, 0.4 mmol) in EtOH (6 mL)/H2O
(3 mL) was heated at reflux for 14 h. The reaction mixture was
cooled, and concentrated under reduced pressure. The residue was
subject to column chromatography on silica gel (acetone–EtOAc–
4456 | Org. Biomol. Chem., 2010, 8, 4451–4462
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(2 ¥), 69.9; HRMS (ESI) calcd for C60H59O14: 1003.3905, found:
m/z 1003.3917 [M + H]+.
70.6 (2 ¥), 70.2 (2 ¥); HRMS (ESI) calcd for C25H25O14: 549.1244,
found: m/z 549.1247 [M - H]-.
4¢-Acetoxy-7-benzyloxy-5-hydroxyflavan (11). A suspension
mixture of ( )-naringenin (1,7.5 g, 27.6 mmol) and K2CO3 (3.82 g,
27.6 mmol) in anhydrous DMF (100 mL) was stirred at room
temperature for 10 min, and then benzyl bromide (4.3 mL,
35.8 mmol) was added dropwise. The mixture was warmed to
room temperature, and stirred for 12 h at room temperature. The
reaction was quenched by addition of saturated aqueous NH4Cl,
and the mixture was concentrated in vacuo. The residual solid
was washed with water and evaporated to dryness under reduced
pressure. The crude product (~10.9 g) was used in the next step
without further purification.
5,7,3¢,4¢-Tetrahydroxy-6,8-di-C -(b-D-xylopyranosyl)flavanone
(8bb1). A solution of 7bb1 (153 mg, 0.152 mmol) in concentrated
HCl (1.5 mL) and MeOH (3 mL) was heated at reflux for 25 min.
After the consumption of 7bb1 as shown by TLC analysis, the
reaction mixture was concentrated by rotary evaporation under
reduced pressure. The residue dissolved in MeOH–EtOAc (1 : 1,
6 mL) was vigorously stirred with 10% Pd–C (40 mg) under an
H2 atmosphere at room temperature for 2 h. The mixture was
filtered through a Celite pad, and rinsed with MeOH. The filtrate
was concentrated, and purified by column chromatography on
silica gel (acetone–EtOAc–H2O–HOAc, 15 : 30 : 2 : 1) to give 8bb1
(62 mg, 74%). C25H28O14; TLC (Me2CO–EtOAc–H2O–HOAc,
A
solution of the above-prepared crude product and
Ac2O (20 mL) in pyridine (30 mL) was treated with 4-
dimethylaminopyridine (DMAP, 60 mg, 0.5 mmol). The solution
was stirred for 4 h at room temperature; the mixture was diluted
with EtOAc and washed with 1 M HCl. After neutralization with
saturated aqueous NaHCO3, the organic layer was dried over
anhydrous MgSO4, filtrated and concentrated. The residue was
purified by flash column chromatography (EtOAc–hexane, 1 : 4 to
1 : 2.5) to afford 10 (11.7 g, 95% for two steps).
1
30 : 30 : 5 : 1) Rf 0.25; H NMR (CD3OD, 400 MHz) d 6.95 (1
H, d, J = 8.8 Hz), 6.84–6.77 (2 H, m), 5.35–5.30 (1 H, m), 4.80–
4.70 (2 H, m), 4.03–3.93 (4 H, m), 3.69–3.56 (2 H, m), 3.50–3.22 (4
H, m), 3.11–3.02 (1 H, m), 2.83–2.71 (1 H, m); HRMS (ESI) calcd
for C25H28O14Na: 575.1371, found: m/z 575.1379 [M + Na]+.
6,8-Di-C -(b-D -xylopyranosyl)-5,7,3¢,4¢-tetrahydroxyflavone
(9bb1). A solution of 8bb1 (200 mg, 0.36 mmol) in Ac2O (6 mL)
and pyridine (6 mL) was stirred 12 h at room temperature.
The mixture was partitioned between 1 M HCl and EtOAc.
The organic phase was washed with 1 M HCl and brine, dried
over anhydrous MgSO4, filtered, and then concentrated by rotary
evaporation. The residue was purified by column chromatography
on silica gel (EtOAc–hexane, 1 : 2 to 1 : 1) to give 8bb1Ac (260 mg)
as a diastereomeric mixture.
To a solution of 10 (8.58 g, 19.32 mmol) in THF (70 mL) and
H2O (35 mmol) was slowly added NaBH4 (1.47 g, 38.64 mmol)
at 0 ◦C. The mixture solution was stirred for 45 min at 0 ◦C,
and then quenched by addition of saturated aqueous NH4Cl. The
organic layer was separated and the aqueous layer was extracted
with EtOAc (5 ¥). The combined organic extracts were dried over
MgSO4, filtered and concentrated. The crude product was purified
by flash column chromatography (20% to 30% EtOAc in hexane)
to afford 11 (6.75 g, 90%). C24H22O5; colorless solid; mp 165.5–
A solution of flavanone 8bb1Ac (56 mg, 0.057 mmol) and I2
(4.4 mg, 0.017 mmol) in DMSO (4 mL) was stirred at 130 ◦C for
◦
167 C; TLC (EtOAc–hexane, 3 : 7) Rf 0.3; IR (film) 3360, 2928,
3 h. The mixture was cooled, quenched by addition of Na2S2O3(aq)
,
1732, 1655, 1129 cm-1; 1H NMR (600 MHz, CDCl3) d 7.41–7.28
(7 H, m), 7.08 (2 H, d, J = 8.5 Hz), 6.17 (1 H, d, J = 2.4 Hz),
6.06 (1 H, d, J = 2.4 Hz), 4.98–4.96 (3 H, m), 4.81 (1 H, s, OH),
2.72–2.63 (2 H, m), 2.29 (3 H, s), 2.21–2.18 (1 H, m), 2.03–1.97 (1
H, m); 13C NMR (150 MHz, CDCl3) d 169.5, 158.4, 156.6, 154.5,
150.2, 139.0, 136.9, 128.5 (2 ¥), 128.4, 127.9, 127.4 (2 ¥), 127.2
(2 ¥), 121.6 (2 ¥), 102.0, 95.5, 95.3, 70.0, 29.4, 21.1, 18.9; HRMS
calcd for C24H23O5: 391.1545, found: m/z 391.1549 [M + H]+.
and partitioned with water and EtOAc. The organic phase was
washed with water and brine, dried over anhydrous MgSO4, and
filtered. After the volatiles were removed by rotary evaporation
under reduced pressure, the residue dissolved in pyridine (3 mL)
and Ac2O (3 mL) was stirred 3 h at room temperature. The mixture
was partitioned between 1 M HCl(aq) and EtOAc. The organic
phase was washed with 1 M HCl and brine, dried over anhydrous
MgSO4, filtered, and then concentrated by rotary evaporation.
The residue was purified by column chromatography on silica gel
(EtOAc–hexane, 1 : 1 to 1.5 : 1) to give 9bb1Ac (48 mg, 87%).
To a stirred solution of 9bb1Ac (9 mg, 0.009 mmol) in dry
methanol (3 mL) was added dropwise a solution of sodium
methoxide (10 mg, 0.18 mmol) in dry methanol (1 mL). The
mixture was stirred at room temperature for 3 h, and neutralized
by addition of Dowex 50W¥8 (H+). The mixture was filtered
and washed with methanol. The filtrate was evaporated in vacuo,
washed by ether and hexane to give 9bb1 (4.5 mg, 90%). C25H26O14;
colorless solid, mp 203–205 ◦C; TLC (acetone–EtOAc–H2O–
HOAc, 30 : 30 : 5 : 1) Rf 0.24; IR vmax (KBr) 3399, 2922, 2856, 1645,
4¢-Acetoxy-6-C-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-7-
benzyloxy-5-hydroxyflavan (12aBn). To a solution of flavan 11
(1.95 g, 5 mmol) and 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl
trichloroacetimidate (2.71 g, 5.5 mmol) in anhydrous CH2Cl2
(40 mL) at -15 ◦C was added dropwise trimethylsilyl triflate
(110 mL, 0.5 mmol). The mixture solution was stirred for
30 min, and then warmed to room temperature within 3 h. The
reaction was quenched by addition of saturated aqueous NaHCO3
(15 mL), and the mixture was extracted with EtOAc (5¥). The
combined organic extracts were dried over MgSO4, filtered, and
concentrated. The crude product was purified by flash column
chromatography (30% EtOAc in hexane) to afford 12aBn as a
white foam (2.81 g, 79%), which contained an inseparable mixture
1
1625, 1578, 1440, 1351, 1299, 1221, 1087, 1056 cm-1; H NMR
(CD3OD, 400 MHz) d 7.42 (1 H, d, J = 7.8 Hz), 7.41 (1 H, s),
6.92 (1 H, d, J = 7.8 Hz), 6.57 (1 H, s), 4.85 (2 H, covered by the
signal of methanol), 4.14–3.98 (4 H, m), 3.81 (1 H, br s), 3.71–3.65
(2 H, m), 3.54–3.42 (3 H, m);13C NMR (CD3OD, 100 MHz) d
182.7, 165.1, 161.6, 159.9, 155.4, 149.7, 145.6, 122.5, 119.5, 115.5,
113.3, 107.6, 104.2, 102.8 (2 ¥), 78.8, 78.6, 75.4, 72.1, 70.7 (2 ¥),
1
of diastereomers (existing as rotamers) as shown by the H and
13C NMR spectra. TLC (EtOAc–hexane, 1 : 2) Rf 0.35; IR (film)
1
3512, 2942, 1752, 1623, 1321, 1229 cm-1; H NMR (600 MHz,
CDCl3) d 7.51 (1 H, br s), 7.37–7.29 (7 H, m), 7.06–7.04 (2
H, m), 6.06 (0.5 H, s), 6.04 (0.5 H, s), 5.39–5.37 (1 H, m),
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5.32–5.24 (2 H, m), 5.20–5.18 (1 H, m), 4.94–4.85 (3 H, m), 4.28–
4.25 (1 H, m), 4.10–4.08 (1 H, m), 3.81 (1 H, d, J = 9.8 Hz),
2.78–2.75 (1 H, m), 2.63–2.61 (1 H, m), 2.24 (3 H, s), 2.13–2.97 (11
H, m), 1.77 (3 H, s); 13C NMR (150 MHz, CDCl3) d 170.6, 170.3,
169.4, 169.0, 168.9, 156.88/156.87, 155.26/155.18, 155.13/155.08,
150.26/150.23, 139.0, 136.8, 128.7 (2 ¥), 128.0, 127.29/127.24
(2 ¥), 127.1/127.0 (2 ¥), 121.6 (2 ¥), 104.2, 101.6/101.5,
92.9/92.8, 77.5/77.3, 76.1/76.0, 74.1, 73.94/73.90, 70.49/70.46,
70.34/70.31, 67.9, 61.49/61.45, 29.5/29.3, 21.1, 20.79, 20.6, 20.5,
20.4/20.3, 19.2/19.0; HRMS calcd for C38H40NaO14: 743.2316,
found: m/z 743.2325 [M + Na]+.
to give 14aaBn (296 mg, 71% for two steps), which contained a
diastereomeric mixture (existing as rotamers) as shown by the 1H
and 13C NMR spectra. C59H64O24; pale yellow foam; TLC (EtOAc–
hexane, 1 : 1) Rf 0.30; 1H NMR (600 MHz, CDCl3) d 7.63–7.35 (12
H, m), 7.18–7.15 (2 H, m), 6.09–5.86 (1.5 H, m), 5.46–5.18 (3.5 H,
m), 5.15–5.00 (4.8 H, m), 4.98–4.71 (5.2 H, m), 4.27–3.81 (4.5 H,
m), 3.75–3.56 (1 H, m), 3.36–3.28 (0.5 H, m), 2.32–1.67 (29 H, 9 ¥
OAc; C3–Ha and Hb); HRMS calcd for C59H64NaO24: 1179.3685,
found: m/z 1179.3692 [M + Na]+.
4¢-Acetoxy-6-C-(tri-O-acetyl-b-D-xylopyranosyl)-8-C-(tri-O-
acetyl-a-L-arabinopyranosyl)-5,7-di-hydroxyflavanone (15bd).
A
4¢-Acetoxy-6,8-di-C-(2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
syl)-5,7-dihydroxyflavan (13aa). Compound 12aBn (720 mg,
1 mmol) was subjected to hydrogenolysis on Pd/C (10%, 50 mg)
in CH3OH (10 mL)/EtOAc (10 mL) for 1 h at room temperature
under an atmosphere of hydrogen. The mixture was filtered
through Celite; the filtrate was concentrated to yield a crude
product, which was chromatographed on a short silica gel column
(EtOAc–hexane (1 : 1)) to afford 12a (580 mg, 92%) containing
an inseparable mixture of diastereomers (existing as rotamers) as
shown by the 1H and 13C NMR spectra.
solution of 14bdBn (773 mg, 0.763 mmol) and PDC (1.15 g,
3.052 mmol) in CH2Cl2 (30 mL) was heated at reflux for 4 h.
The mixture was concentrated by rotary evaporation, filtered
through a short silica pad, and rinsed with EtOAc. The filtrate was
concentrated; the residue was rinsed with Et2O–hexane to afford
15bdBn (760 mg, 98%) as an inseparable mixture of diastereomers
(existing as rotamers).
A solution of 15bdBn (223 mg, 0.22 mmol) in EtOAc–CH3OH
(1 : 1, 10 mL) was subjected to hydrogenolysis by vigorously
stirring with 10% Pd/C (50 mg) under an atmosphere of H2 at
room temperature for 2 h. The mixture was filtered through a pad
of Celite, rinsed with CH3OH, and concentrated under reduced
pressure. The residue was rinsed with Et2O–n-pentane to give
15bd (172 mg, 99%) as an inseparable mixture of diastereomers
To a solution of monoglycosylflavan 12a (300 mg, 0.47 mmol)
and 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl trichloroacetimidate
(296 mg, 0.6 mmol) in anhydrous CH2Cl2 (25 mL) at -15 ◦C was
added dropwise trimethylsilyl triflate (6 mL, 0.024 mmol). The
mixture solution was stirred for 30 min, and then warmed to room
temperature over a period of 3 h. The reaction was quenched by
addition of saturated aqueous NaHCO3 (15 mL), and the mixture
was extracted with EtOAc (5¥). The combined organic extracts
were dried over MgSO4 and evaporated in vacuo. The crude
product was purified by flash column chromatography (EtOAc–
hexane (2 : 5)) to give the di-C-glycosylflavan 13aa (347 mg, 77%),
which contained a diastereomeric mixture (existing as rotamers)
◦
(existing as rotamers). C39H42O20; white prisms, mp 174–176 C;
TLC (EtOAc–hexane, 3 : 2) Rf 0.35; IR vmax (neat) 3308, 2924, 1749,
1
1632, 1370, 1220 cm-1; H NMR (CDCl3, 400 MHz) d 12.76 (1
H, br, OH), 8.76 (1 H, br, OH), 7.68 (0.7 H, d, J = 8 Hz), 7.40
(1.3 H, d, J = 8 Hz), 7.18 (2 H, d, J = 8 Hz), 6.00–5.89 (1 H,
m), 5.64 (1 H, d, J = 11.6 Hz), 5.43–5.24 (3 H, m), 5.14–4.86 (4
H, m), 4.30–4.23 (1 H, m), 4.14–3.97 (1 H, m), 3.81–3.71 (1 H,
m), 3.51–3.41 (1 H, m), 3.17–2.72 (2 H, m), 2.33 (3 H, s), 2.27
(3 H, s), 2.05 (3 H, s), 2.03 (3 H, s), 1.99 (3 H, s), 1.88 (3 H,
s), 1.85 (3 H, s); 13C NMR (CDCl3, 100 MHz) d 196.2/194.9,
170.1, 169.8, 169.6, 169.3, 168.8, 168.5, 168.1, 163.2/162.5, 160.9,
158.8, 150.4, 135.4/135.2, 127.9/126.3 (2 ¥), 121.9/121.5 (2 ¥),
104.1, 103.0, 101.9/100.9, 78.7, 74.1, 73.4, 72.6/72.2, 71.4, 71.0,
69.9/69.0, 68.6/68.3, 67.9, 67.4/67.1, 66.9/66.3, 43.5/42.4, 20.8
(2 ¥), 20.4 (3 ¥), 20.3, 20.0; HRMS (ESI) calcd for C39H41O20:
829.2191, found: m/z 829.2195 [M - H]-.
1
as shown by the H NMR spectrum. C45H52O23; colorless foam;
TLC (EtOAc–hexane, 1 : 1) Rf 0.40; 1H NMR (600 MHz, CDCl3)
d 7.43 (1 H, d, J = 8.2 Hz), 7.39–7.36 (3 H, m), 7.13–7.10 (2 H, m),
5.44–5.18 (6 H, m), 5.15–4.76 (2.5 H, m), 4.32–4.28 (2 H, m), 4.14–
3.98 (2.5 H, m), 3.91–3.81 (2 H, m), 2.82–2.74 (1 H, m), 2.65–2.56
(1 H, m), 2.31–1.81 (29 H, 9 ¥ OAc; C3–Ha and Hb); HRMS calcd
for C45H52NaO23 (M+ + Na): 983.2797, found: m/z 983.2800.
4¢-Acetoxy-6,8-di-C-(2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
syl)-5,7-dibenzyloxy-4-hydroxyflavan (14aaBn). A mixture of
13aa (347 mg, 0.36 mmol), benzyl bromide (170 mg, 1.08 mmol)
and K2CO3 (174 mg, 1.26 mmol) in anhydrous DMF (6 mL)
was heated at 50 ◦C for 5 h. The mixture was cooled to room
temperature, diluted with EtOAc (10 mL), and filtered. The filtrate
was concentrated by rotary evaporation under reduced pressure,
and the residue was partitioned with EtOAc (20 mL) and water
(8 mL). The organic layer was separated, washed with water
(8 mL), dried over MgSO4, filtered and concentrated by rotary
evaporation under reduced pressure.
6-C-b-D-xylopyranosyl-8-C-a-L-arabinopyranosyl-4,5,7-trihy-
droxyflavanone (2bd). To
a solution of 15bd (60.6 mg,
0.073 mmol) in CH3OH (5 mL) was added sodium methoxide
(50 mg, 0.93 mmol) at room temperature. The mixture was stirred
for 2 h, neutralized with Dowex 50 W ¥ 8 (H+), filtered and
evaporated in vacuo. The residue was washed with CH2Cl2 and
Et2O to give 2bd (35 mg, 89%) as an inseparable diastereomeric
mixture (existing as rotamers). C25H28O13; yellow prisms, mp 192–
193.5 ◦C; IR vmax (neat) 3364, 2913, 1625, 1518, 1455, 1342, 1206,
1
1088 cm-1; H NMR (CD3OD, 400 MHz) d 7.35 (2 H, d, J =
The crude product of 13aaBn was dissolved in CH3CN–water
(5 : 1, 24 mL) and stirred with cerium(IV) ammonium nitrate
(CAN, 6 mmol) for 2 h at room temperature. The mixture was
partitioned between EtOAc (50 mL) and water (20 mL). The
aqueous layer was extracted with EtOAc (3¥). The combined
organic extracts were dried over MgSO4, concentrated, and pu-
rified by flash column chromatography (EtOAc–hexane (45 : 55))
8.4 Hz), 6.80 (2 H, d, J = 8.4 Hz), 5.35 (1 H, d, J = 12.8 Hz),
4.72 (1 H, d, J = 10.4 Hz), 4.68 (1 H, d, J = 9.2 Hz), 4.20 (1 H,
br), 4.04 (1 H, t, J = 9.2 Hz), 3.95–3.90 (3 H, m), 3.63–3.60 (2
H, m), 3.54 (1 H, dd, J = 9.2, 2.4 Hz), 3.37 (1 H, t, J = 9.2 Hz),
3.30–3.24 (1 H, m), 3.00 (1 H, dd, J = 17.2, 12.8 Hz), 2.72 (1 H,
dd, J = 17.2, 1.6 Hz); 13C NMR (CD3OD, 100 MHz) d 198.6,
165.6, 163.9, 161.7, 158.8, 130.9, 129.1/128.9 (2 ¥), 116.3 (2 ¥),
4458 | Org. Biomol. Chem., 2010, 8, 4451–4462
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106.4, 105.2, 103.1, 80.7, 80.4, 76.9, 75.8, 75.3, 72.2, 71.7, 71.6,
71.5, 71.1, 70.4, 44.4; HRMS (ESI) calcd for C25H27O13: 535.1452,
found: m/z 535.1453 [M - H]-.
was added, and the mixture was stirred for an additional 4 h.
The mixture was quenched by addition of H2O (20 mL), and
then partitioned between 1 M HCl(aq) and EtOAc (3 ¥ 10 mL).
The combined organic layers were washed with brine, dried
over anhydrous MgSO4, filtered and concentrated under reduced
pressure.
6,8-Di-C-(b-D-glucopyranosyl)-5,7,4¢-trihydroxyflavone
(vic-
enin-2) (3aa)15,17
.
A solution of 14aaBn (231 mg, 0.2 mmol) and
DDQ (227 mg, 1 mmol) in chlorobenzene (10 mL) was stirred
for 24 h at 140 ◦C. The reaction mixture was evaporated in vacuo
and the residue was partitioned between EtOAc (20 mL) and
saturated aqueous NaHCO3 (20 mL). The organic layer was
dried over MgSO4, filtered and concentrated to give a crude
diglycosylapigenin derivative (210 mg), which was subjected
to hydrogenolysis by stirring with Pd/C (30 mg) in CH3OH
(10 mL)/EtOAc (10 mL) for 1 h at room temperature under an
atmosphere of hydrogen. The mixture was filtered through Celite,
and the filtrate was concentrated to yield the crude debenzylated
product (181 mg) as a pale yellow solid.
To a solution of this debenzylated crude product in pyridine
(5 mL) was added Ac2O (5 mL). The mixture was stirred for
10 h, and then concentrated under reduced pressure. The residue
was partitioned between EtOAc (30 mL) and water (20 mL).
The organic layer was washed with 1 M aqueous HCl (10 mL)
and water (10 mL). After neutralization with saturated aqueous
NaHCO3, the organic layer was dried over MgSO4, filtered
and concentrated. The residue was purified by flash column
chromatography (60% EtOAc in hexane) to afford 3aaAc (142 mg,
66% overall yield).
The crude product having a benzenediol moiety was treated
with Ac2O (3 mL) in pyridine (5 mL) and DMAP (10 mg,
0.08 mmol) at room temperature for 6 h. The mixture was
quenched with CH3OH, concentrated under reduced pressure, and
partitioned between 1 M HCl(aq) and EtOAc. After neutralization
with saturated aqueous NaHCO3, the organic layer was separated,
washed with brine, dried over anhydrous MgSO4, filtered and
concentrated. The residual solid was subjected to hydrogenolysis
under an atmosphere of H2 by vigorous stirring with 10% Pd/C
(20 mg) in THF–CH3OH (2 : 1, 30 mL) at room temperature for
3 h. The mixture was filtered through a pad of Celite, rinsed with
CH3OH and concentrated. The residue was purified by column
chromatography on silica gel (EtOAc–hexane, 2 : 3 to 1 : 1) to
afford 17db as a mixture of diastereomers (41 mg, 74% overall
yield).
C41H44O22; white prisms, mp 166–168 ◦C; TLC (EtOAc–hexane,
3 : 2) Rf 0.26; IR vmax (neat) 3246, 2916, 1731, 1637, 1374, 1218 cm-1;
1H NMR (CDCl3, 400 MHz) d 12.56 (1 H, br, OH), 8.70 (1 H, br,
OH), 7.40–7.26 (3 H, m), 5.75 (1 H, br), 5.67 (1 H, d, J = 12 Hz),
5.41 (2 H, br), 5.31–5.22 (2 H, m), 5.14 (1 H, d, J = 9.2 Hz), 4.97
(1 H, t, J = 10 Hz), 4.89 (1 H, d, J = 10.4 Hz), 4.21 (1 H, dd,
J = 10.4, 5.2 Hz), 4.10 (1 H, d, J = 12 Hz), 3.81 (1 H, d, J =
13.2 Hz), 3.36 (1 H, t, J = 9.6 Hz), 2.95 (1 H, t, J = 12.8 Hz), 2.83
(1 H, d, J = 15.6 Hz), 2.29 (6 H, s, 2 ¥ OAc), 2.23 (3 H, s, 1 ¥
OAc), 2.07–1.93 (12 H, m, 4 ¥ OAc), 1.83–1.78 (3 H, m, 1 ¥ OAc);
13C NMR (CDCl3, 100 MHz) d 195.8, 170.0, 169.8, 169.6, 169.4,
168.6, 167.9, 167.8, 167.7, 163.6, 162.1, 160.9, 142.2, 141.7, 137.2,
123.8, 123.2, 120.4, 103.0, 102.0 (2 ¥), 78.2/77.6, 74.1, 73.7, 73.3,
72.1/71.6, 71.3, 69.8/69.0, 68.7/68.6, 68.3, 67.5, 67.2, 43.1/42.6,
21.1, 21.0, 20.8, 20.8, 20.7, 20.6, 20.5, 20.4; HRMS (ESI) calcd for
C41H43O22: 887.2246, found: m/z 887.2266 [M - H]-.
A solution of compound 3aaAc (142 mg, 0.13 mmol) in dry
MeOH (5 mL) was treated with 30 wt% methanolic solution of
NaOMe (0.5 mL). The mixture was warmed to room temperature
and continuous stirred for 12 h, the mixture was then neutralized
with Amberlite IR-120 (H+), filtered, and rinsed with methanol.
The filtrate was concentrated by rotary evaporation under reduced
pressure, and the residue was washed with Et2O to afford 3aa
(66 mg, 85%). C27H31O15; pale-yellow powder, mp > 250 ◦C; [a]D20
=
+53.2 (c = 0.5, MeOH) [lit.15 [a]2D1 = +56.9 (c = 0.745, MeOH)];
IR (film) 3351, 2923, 2872, 1657, 1623, 1501 cm-1; 1H NMR
(400 MHz, DMSO-d6, at 90 ◦C) d 13.6 (1 H, br s), 13.5 (1 H,
br s), 9.20 (1 H, br s), 7.95 (2 H, d, J = 8.6 Hz), 6.94 (2 H, d,
J = 8.6 Hz), 6.69 (1 H, s), 4.89 (1 H, br d, J > 7.3 Hz, H-1¢¢¢ax,
8b-Glc), 4.80 (1 H, d, J = 9.7 Hz, H-1¢¢ax, 6b-Glc), 3.77 (1 H, d, J =
11.8 Hz), 3.74 (1 H, d, J = 11.8 Hz), 3.72–3.68 (2 H, m), 3.64–3.58
(2 H, m), 3.44 (1 H, d, J = 9.2 Hz), 3.40–3.32 (5 H, m); 13C NMR
(150 MHz, DMSO-d6, at 50 ◦C) d 181.6, 163.5, 160.6, 160.1, 157.9,
154.5, 128.3 (2 ¥), 120.8, 115.2 (2 ¥), 106.8, 104.6, 103.3, 101.9,
81.2, 80.3, 78.2, 77.2, 73.4, 72.7, 71.3, 70.3/70.0, 69.1, 68.5, 60.7,
59.2; HRMS calcd for C27H31O15: 595.1663, found: m/z 595.1668
[M + H]+.
6-C-a-L-Arabinopyranosyl-8-C-a-D-xylopyranosyl)-3¢,4¢,5,7-
tetrahydroxyflavone (18db).
A solution of 17db (47 mg,
0.053 mmol) was heated with iodine (4 mg, 0.016 mmol) in DMSO
(2 mL) at 140 ◦C for 4 h. The mixture was quenched by addition
of aqueous Na2S2O3, and extracted with EtOAc (3 ¥ 10 mL). The
combined organic extracts were washed with saturated aqueous
NaHCO3 and brine, dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The residue was treated
with added Ac2O (3 mL) in pyridine (3 mL) and DMAP (10 mg,
0.08 mmol) at room temperature for 16 h. The mixture was
quenched with CH3OH, concentrated, and partitioned between
1 M HCl(aq) and EtOAc. After neutralization with saturated
aqueous NaHCO3, the organic layer was separated, washed with
brine, dried over anhydrous MgSO4, filtered and concentrated.
The residue was purified by column chromatography on silica gel
(EtOAc–hexane, 3 : 2) to afford 18dbAc (13 mg, 25% overall yield).
Compound 18dbAc (10 mg, 0.01 mmol) was stirred with
sodium methoxide (5 mg, 0.09 mmol) in CH3OH (4 mL) at room
temperature for 2 h. The mixture was neutralized with Dowex 50
W ¥ 8 (H+), filtered and concentrated under reduced pressure. The
residue was rinsed with CH2Cl2 and Et2O to give 18db (5 mg, 90%).
3¢,4¢-Di-acetoxy-6-C-(tri-O-acetyl-a-L-arabinopyranosyl)-8-C-
(tri-O-acetyl-a-D-xylopyranosyl)-5,7-di-hydroxyflavanone (17db).
Under an atmosphere of argon, a solution of 15dbBn (63 mg,
0.062 mmol) in CH2Cl2–CH3OH (1 : 2, 6 ml) was stirred with
K2CO3 (8.6 mg, 0.062 mmol) at room temperature for 1 h. The
mixture was neutralized with Dowex 50 W ¥ 8 (H+), filtered and
concentrated under reduced pressure. The residue was treated with
2-iodoxybenzoic acid (IBX, 35 mg, 0.124 mmol) in DMSO (2 mL)
at room temperature for 5 h. As the reaction progressed, the
color changed from a translucent yellow solution to an opaque
brown solution. Sodium dithionite (Na2S2O4, 51 mg, 0.29 mmol)
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◦
C25H26O14; yellow prisms, mp > 250 C (decomposed); [a]2D5 -41
(10 mL) and water (10 mL). After neutralization with saturated
aqueous NaHCO3, the organic layer was dried over MgSO4,
filtered and concentrated. The residue was purified by flash
column chromatography (EtOAc–hexane (1 : 1)) to afford flavone
3aAc (536 mg, 71% for three steps).
A solution of 3aAc (73 mg, 0.1 mmol) in dry MeOH (5 mL)
was stirred with 30 wt% methanolic solution of NaOMe (0.2 mL)
at room temperature for 5 h. The mixture was neutralized with
Amberlite IR-120 (H+), filtered, and rinsed with methanol. The
filtrate was concentrated by rotary evaporation under reduced
pressure, and the residual yellow solids were washed with Et2O
to afford 3a (38 mg, 87%).
(c 0.33, H2O); IR vmax (neat) 3360, 2925, 1626, 1580, 1518, 1443,
1368, 1207, 1086 cm-1; H NMR (CD3OD, 400 MHz) d 7.44 (1
1
H, s), 7.41 (1 H, s), 6.92 (1 H, d, J = 8 Hz), 6.57 (1 H, s), 4.94 (1
H, d, J = 10 Hz), 4.80 (1 H, covered by the signal of methanol),
4.57 (1 H, br), 4.10–4.02 (4 H, m), 3.98 (1 H, br), 3.75 (1 H, d,
J = 12.8 Hz), 3.65–3.60 (1 H, m), 3.49 (1 H, t, J = 9.2 Hz), 3.40 (1
H, t, J = 10.8 Hz); 13C NMR (CD3OD, 100 MHz) d 184.2, 168.6,
166.8, 164.6, 157.1, 151.0, 147.0, 123.9, 120.7, 116.7, 114.6, 111.7,
105.3, 104.1 (2 ¥), 80.3, 76.5 (2 ¥), 75.4, 72.9, 72.0 (3 ¥), 71.3, 70.5;
HRMS (ESI) calcd for C25H25O14: 549.1244, found: m/z 549.1251
[M - H]-.
3aAc. C35H34O17; colorless foam; TLC (EtOAc–hexane, 1 : 1)
5,4¢-Diacetoxy-6-C-(2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
syl)-7-benzyloxyflavanone (19a). Compound 12aBn (1.44 g,
2 mmol) was treated with AcCl (0.35 mL, 5 mmol), Et3N (1.4 mL,
10 mmol) and DMAP (25 mg, 0.2 mmol) in CH2Cl2 (30 mL) at
0 ◦C. The mixture was stirred for 10 h at room temperature, and
concentrated by rotary evaporation. The residue was partitioned
between EtOAc (80 mL) and water (30 mL). The organic layer was
separated, and washed with 1 M aqueous HCl (30 mL) and water
(30 mL). After neutralization with saturated aqueous NaHCO3,
the organic layer was dried over MgSO4, filtered, and concentrated
to give a crude acetylation product 12aBnAc (~ 1.60 g).
1
Rf 0.4; IR (film) 2938, 1721, 1600, 1214, 1135 cm-1; H NMR
(600 MHz, CDCl3, as mixture of rotamers) d 7.82 (2 H, d, J =
8.2 Hz), 7.29 (1 H, s), 7.22 (2 H, d, J = 8.2 Hz), 6.57 (1 H, s), 5.68
(0.7 H, t, J = 9.5 Hz), 5.61 (0.3 H, t, J = 9.5 Hz), 5.29 (1 H, t, J =
9.3 Hz), 5.14 (1 H, t, J = 9.7 Hz), 4.85–4.81 (1 H, m), 4.39 (1 H,
br d, J = 13.0 Hz), 3.96 (1 H, br d, J = 12.3 Hz), 3.79 (1 H, br d,
J = 9.4 Hz), 2.46 (3 H, s), 2.45 (3 H, s), 2.30 (3 H, s), 2.15–1.91 (9
H, m), 1.79 (3 H, s); 13C NMR (150 MHz, CDCl3, as mixture of
rotamers) d 176.0, 170.4, 170.2, 169.9, 169.6, 168.9, 168.6, 167.8,
161.7, 157.2, 153.4, 153.3, 148.7, 128.3, 127.6 (2 ¥), 122.4 (2 ¥),
119.0, 114.5, 111.8, 108.7, 76.5, 74.3, 72.3, 69.5, 68.1, 61.9, 21.3,
21.2, 21.1, 20.7, 20.67, 20.63, 20.4; HRMS calcd for C35H35O17:
727.1874, found: m/z 727.1877 [M + H]+.
The crude product 12aBnAc (~ 1.60 g) was treated with CAN
(8.16 g, 14.9 mmol) in a mixed solvents of CH3CN (20 mL),
AcOH (10 mL) ◦and water (10 mL). The mixture was stirred
for 45 min at 50 C, quenched by addition of saturated aqueous
NaHCO3, and extracted with EtOAc (5 ¥). The combined organic
layers were dried over MgSO4, filtered, and concentrated. The
residue was purified by flash column chromatography (EtOAc–
hexane (3 : 7)) to afford flavanone 19a (807 mg, 52% for two steps)
as an inseparable diastereomeric mixture (existing as rotamers).
3a. C21H20NaO10; yellow powder; mp 220–222 ◦C; [a]D20 = +28.9
(c = 1.0, MeOH) [lit.24a [a]D27 = +27.5 (c = 1.0, MeOH)]; IR (film)
3412, 2928, 1662, 1254, 1163 cm-1; 1H NMR (600 MHz, CD3OD)
d 7.77 (2 H, d, J = 8.7 Hz), 6.89 (2 H, d, J = 8.7 Hz), 6.53 (1 H, s),
6.45 (1 H, s), 4.89 (1 H, d, J = 10.0 Hz, Hanomeric, 6-b-configuration),
4.20–4.17 (1 H, m), 3.89 (1 H, dd, J = 12.2, 2.2 Hz), 3.75 (1 H,
1
C40H40O16; white foam; TLC (EtOAc–hexane, 1 : 2) Rf 0.32; H
dd, J = 12.2, 5.5 Hz), 3.50–3.48 (2 H, m), 3.44–3.41 (1 H, m); 13
C
NMR (600 MHz, CDCl3) d 7.54 (1 H, d, J = 7.5 Hz), 7.44–7.35
(6 H, m), 7.13–7.11 (2 H, m), 6.47 (0.6 H, d, J = 5.5 Hz), 6.42
(0.4 H, d, J = 3.8 Hz), 5.93–5.89 (0.6 H, m), 5.65–5.61 (0.4 H, m),
5.47–5.31 (1.3 H, m), 5.27–5.13 (2.4 H, m), 5.07–4.99 (1.7 H, m),
4.69–4.64 (0.6 H, m), 4.41–4.37 (0.3 H, m), 4.22–4.19 (0.7 H, m),
4.04 (0.7 H, d, J = 12.4 Hz), 3.95–3.92 (0.3 H, m), 3.72–3.69 (1
H, m), 3.02–2.90 (1 H, m), 2.68–2.64 (1 H, m), 2.46–1.80 (18 H,
6 ¥ OAc); HRMS calcd for C40H40NaO16: 799.2214, found: m/z
799.2217 [M + Na]+.
NMR (150 MHz, CD3OD) d 183.0, 165.2, 163.9, 161.8, 161.1,
157.7, 128.5 (2 ¥), 122.0, 116.1 (2 ¥), 108.2, 104.2, 102.8, 94.3,
81.7, 79.2, 74.3, 71.6, 70.9, 62.0; HRMS calcd for C21H20NaO10:
455.0954, found: m/z 455.0958 [M + Na]+.
Cell culture
Mice macrophage cell line Raw264.7 was obtained from ATCC
and were cultured in RPMI 1640 medium (Invitrogen) supple-
mented with 10% heat-inactivated fetal bovine serum (Invitrogen)
and penicillin/streptomycin (100 units/ml) in a 37 ◦C humidified
chamber with 5% CO2.
5,7,4¢-Trihydroxy-6-C-(b-D-glucopyranosyl)flavone
(3a,
Isovitexin)25. A mixture of flavanone 19a (807 mg, 1.04 mmol)
and iodine ◦(26 mg, 0.1 mmol) in DMSO (20 mL) was stirred for
1 h at 140 C. The mixture was poured into water (20 mL) and
extracted with EtOAc (5¥). The combined organic layers were
washed with 10% Na2S2O3 aqueous solution, water and brine.
The organic layer was dried over MgSO4 and concentrated. The
residue was dissolved in EtOAc (15 mL)/CH3OH (15 mL), and
subjected to hydrogenolysis on 10% Pd/C (80 mg) for 1 h at room
temperature under an atmosphere of hydrogen. The mixture was
filtered through a pad of Celite; the filtrate was concentrated to
afford a crude product as pale-yellow solids. The crude product
was treated with Ac2O (10 mL) in pyridine (10 mL) for 10 h
at room temperature. The mixture was concentrated in vacuo;
the residue was partitioned between EtOAc (30 mL) and water
(20 mL). The organic layer was washed with 1 M aqueous HCl
Measurement of cell viability
The effects of different compounds on cell proliferation were
R
analyzed using the CellTiter-Gloꢀ Luminescent Cell Viability
Assay (Promega). This assay uses luciferase-catalyzed reaction
to quantify ATP in the cells, which is used as an indicator of
metabolically active cells. Briefly, Raw264.7 cells were incubated
in 96-well plates at 4 ¥ 104 cells per well for 24 h, and were
subsequently treated with 100, 250, 500 and 1000 mg mL-1 of
compounds, respectively, for another 6 h. The cells were then
lysed by the addition of 20 mL of the reagent. The mixture was
diluted 1 : 1 with fresh DMEM, and luminescence in each well was
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measured 10 min after reagent addition using an EnVision 2101
multilabel reader (Perkin Elmer).
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RNA isolation and RT-PCR
Raw264.7 cells were cultured in 6-well plates at 3 ¥ 106 cells per
well for 24 h and treated with interested compounds for 30 min
followed by addition of 100 ng mL-1 LPS for another 6 h. The
cells were collected and stored at -80 ◦C until use. Total RNA was
extracted using Trizol (Invitrogen) and processed for RT-PCR as
described previously.4
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Determination of protein level of TNF-a
Raw264.7 cells were plated in 96-well plates at 4 ¥ 104 cells
per well in duplicates for 24 h. The cells were pre-treated with
compounds in various concentrations for 30 min, and then treated
with 100 ng mL-1 LPS for 6 h. The supernatants were collected by
centrifugation at 1000 rpm for 5 min at indicated time intervals,
and processed for TNF-a determination using an ELISA kit
(R&D) based on the manufacturer’s instructions. The optical
density was determined using a microplate reader at 450 nm.
The concentration of cytokine released was determined using the
standard curve. IC50 values were calculated using Graphpad Prism
(Graphpad Software Inc., San Diego, CA).
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Measurement of nitric oxide concentration
To measure the concentration of NO, a standard procedure using
Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine
dihydrochloride in 2.5% H3PO4) was used. Briefly, macrophage
RAW264.7 cells were plated in 96-well plates in 100 mL RPMI
medium without phenol-red for 24 h, and treated with compounds
for 30 min followed by a two-day treatment of 100 ng mL-1
LPS. At the indicated time, cell medium was collected and mixed
with Griess reagent in 1 : 1 ratio in wells of a 96-well plate.
Optical density at 550 nm was measured with a microplate reader
(Spectra Max, Molecular Devices) after 10 min incubation at
room temperature. The concentration of nitrite in the samples
was calculated from a sodium nitrite standard curve. IC50 values
were calculated using Graphpad Prism version software 4.0.
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Acknowledgements
We are grateful for the financial support of Yuen-Foong-Yu
Biotech Co., Taiwan, and Academia Sinica.
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