Concise synthesis and antidiabetic activity of natural flavonoid glycosides, oroxins C and D, isolated from the seeds of Oroxylum indium

Article abstract of DOI:10.1177/1747519820927966

The first concise synthesis of natural flavonoid glycosides, oroxins C (1) and D (2), which were isolated from the seeds of Oroxylum indicum, was efficiently achieved by a convergent strategy. The synthesized natural products 1 and 2 were evaluated for th

Full text of DOI:10.1177/1747519820927966

927966CHL0010.1177/1747519820927966Journal of Chemical ResearchLi et al.
research-article2020
Research Paper
Journal of Chemical Research
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Concise synthesis and antidiabetic activity of
natural flavonoid glycosides, oroxins C and D,
isolated from the seeds of Oroxylum indium
https://doi.org/10.1177/1747519820927966
DOI: 10.1177/1747519820927966
journals.sagepub.com/home/chl
Gang Li1*, Guanghui Wang1*, Yangliu Tong2, Junheng Zhu2,
Tongtong Yun2, Xiaoping Ye2, Fahui Li3, Shengli Yuan3
and Qingchao Liu2
Abstract
The first concise synthesis of natural flavonoid glycosides, oroxins C (1) and D (2), which were isolated from the
seeds of Oroxylum indicum, was efficiently achieved by a convergent strategy. The synthesized natural products 1 and 2
were evaluated for their inhibitory activities against α-glucosidase, α-amylase, and lipase. Compound 1 showed strong
α-amylase and lipase inhibition, with IC50 values of 210 and 190μM, respectively, but exhibited no inhibitory activity
against α-glucosidase. Compound 2 showed strong inhibition against α-glucosidase and lipase, with the respective
IC50 values of 180 and 80μM.
Keywords
antidiabetic activity, flavonoid glycosides, glycosylation, oroxin C, oroxin D
Date received: 21 February 2020; accepted: 29 April 2020
The first concise synthesis of natural flavonoid glycosides, oroxins C (1) and D (2), which were isolated from the seeds
of Oroxylum indicum, was efficiently achieved by a convergent strategy. Preliminary pharmacological research exhibited
positive response against α-glucosidase, α-amylase, and lipase. Notably, the flavonoid glycoside 2 showed stronger lipase
inhibitory activity (IC50 =80μM), which was two times than positive control orlistat.
HO
O
HO
O
HO
O
HO
HO
O
HO
HO
O
O
HO
O
HO
HO
O
O
O
HO
HO
HO
OH
OH
O
baicalein
orox n
i
1
C
HO
HO
HO
(
)
OH
O
OCH3
O
HO
HO
O
O
HO
HO
O
HO
HO
HO
OH
scutellarein
O
OH
oroxin D (2)
O
Introduction
1Weifang University of Science and Technology, Weifang, P.R. China
2Department of Pharmaceutical Engineering, Northwest University,
Xi’an, P.R. China
Flavonoid glycosides, glycosylated secondary metabolites
widely distributed in the plant kingdom (such as vegetables,
fruits, and medicinal plants),1–3 having tremendous struc-
tural diversity, exhibit a multitude of biological and physi-
ological activities, such as antimicrobial,4 antitumor,5
anti-inflammatory,6 antidiabetic,7 and hepatoprotectant
activities.8 Compared with flavonoids, flavonoid glycosides
possess similar stability, bioactivity, and better solubility
and are often more efficacious than their aglycones in phar-
maceutical studies.9 Although flavonoid glycosides are
widely distributed and have been demonstrated to possess
important biological activities, access to this class of natural
3Department of Oncology, Qingdao Municipal Hospital, Qingdao, P.R. China
*Gang Li and Guanghui Wang contributed equally to this work.
Corresponding author:
Qingchao Liu, Department of Pharmaceutical Engineering, Northwest
University, Xi’an 710069, Shaanxi, P.R. China.
Email: liuqc21@nwu.edu.cn
Fahui Li, Weifang University of Science and Technology, Weifang, P.R.
China.
Email: fahuili@163.com

2
Journal of Chemical Research 00(0)
H
O
O
H
O
H
O
O
H
O
O
O
H
OC
H
H
O
O
3
H
O
O
O
H
H
O
O
O
O
H
HO
O
H
O
H
O
O
O
O
O
O
O
O
O
H
O
H
O
H
O
H
O
H
H
H
O
H
O
1
oroxin C ( )
2
oroxin D ( )
Figure 1. Chemical structure of flavonoid glycosides, oroxins C (1) and D (2).
products by separation and purification, especially in desir- NH2NH2·HOAc in dimethylformamide (DMF) at room tem-
able amounts, presents a formidable task. This results in a perature, to obtain 2,3,4,6-tetra-O-benzoyl-β-d-gl-
great obstacle for further pharmacological studies of homo- ucopyranosyl-(1→6)-2, 3, 4-tri-O-benzoyl-d -
geneous flavonoid glycosides. Chemical synthesis could glucopyranoside 8, followed by CBr4 and PPh3 in CH2Cl2 to
provide a feasible way to solve the problem, and many provide the desired donor 5 in 90% yield. The preparation of
groups have made great efforts to provide sufficient amounts donor 3 began with 1,2,3,4-tetra-O-benzoyl-6-O-trityl-
of flavonoid glycosides through chemical synthesis.10–12
d-glucopyranoisde10.ThelatterwastreatedwithFeCl3·6H2O
Recently, Wu et al.13 reported two novel flavonoid glyco- in CH2Cl2 at room temperature to afford 1,2,3,4-tetra-O-ben-
sides, oroxin C (baicalein 7-O-β-d-glucopyranosyl-(1→6)-β- zoyl-d-glucopyranoisde 11, which was coupled with
d-glucopyranosyl-(1→6)-β-d-glucopyranoside, 1) and oroxin 2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl-(1→6)-2,3,4-
D (scutellarein 4'-methyl ether 7-O-β-d-glucopyranosyl- tri-O-benzoyl-α-d-glucopyranosyl trichloroacetimidate 9 in
(1→6)-β-d-glucopyranoside, 2), isolated from the seeds of anhydrous CH2Cl2 in the presence of trimethylsilyl trifluo-
Oroxylum indicum as a part of a program to search for potent romethanesulfonate (TMSOTf) to obtain the trisaccharide 12
and effective antidiabetic agents. However, the formidable in 89% yield. Treatment of 12 with NH2NH2·HOAc in DMF,
task of isolating these two natural products turned out to be an followed by CBr4 and PPh3 in CH2Cl2, provided the trisac-
obstacle for further pharmacological research, and so it is use- charide bromide donor 3 in 83% yield.
ful for syntheses of the two novel flavonoid glycosides 1 and
The partially protected baicalein and scutellarein accep-
2 to be completed to enable further SAR investigation. In this tors 4 and 6 were readily prepared in a concise manner as
article, we report the first, concise synthesis of oroxin C (1) depicted in Scheme 3. Baicalein was treated with Ac2O and
and oroxin D (2) (Figure 1); the two flavonoid glycosides are AcONa to obtain fully acetylated compound 14, which was
also evaluated for in vitro inhibition activity against α- sequentially benzylated with BnBr and hydrogenolysed
glucosidase, α-amylase, and lipase. These investigations are with Pd(OH)2/C to provide acceptor 4 in 90% yield for the
valuable for the design and preparation of stronger inhibitors two steps.11 The synthesis of the acceptor 6 was started with
and the elucidation of structure–activity relationships (SARs) scutellarein, which was treated with dichlorodiphenylmeth-
for the treatment of type 2 diabetes.
ane in diphenyl ether to produce the desired compound 15
in 83% yield.19 Subsequently, the C4'-OH of compound 15
was selectively reacted with MeI to afford 16,20 and then
the benzophenone ketal group was removed under the
hydrolysis conditions with 80% AcOH in H2O to obtain 17
in 95% yield.20 Similar to the aforementioned route to the
baicalein acceptor 4, compound 17 was first treated with
Ac2O and AcONa to obtain 18, followed by selective sub-
stitution of the most reactive C7-OAc by a benzyl group
using BnBr and KI to produce C7-OBn scutellarein deriva-
tive, which was reduced with Pd(OH)2/C and H2 to remove
the benzyl group affording the desired scutellarein acceptor
6 in 78% yield for the two steps.
With an effective synthetic access to the bromide donors
(3 and 5) and baicalein and scutellarein acceptors (4 and 6),
we then set about to assemble the target flavonoid glycosides
1 and 2. According to Koenigs–Knorr protocol, coupling of
baicalein acceptor 4 or scutellarein acceptor 6 with the bro-
mide donor 3 or 5 was carried out in quinoline in the presence
of a catalytic amount of Ag2O at room temperature to afford
the desired flavonoid derivatives 19 and 20 with β-isomeric
glycosydic linkage formed highly stereoselectively. The ben-
zoyl groups was then removed with Mg(OMe)2 in MeOH-
CH2Cl2 to provide the target flavonoid glycosides, oroxins C
(1) and D (2) (Scheme 4). All the analytical data
Results and discussion
In terms of their chemical structures, the glycoside resi-
dues of the target compounds 1 and 2 are attached to a
phenolic hydroxyl group rather than to an alkyl hydroxyl
group. Because of their reduced nucleophilicity, phenolic
hydroxyl groups are known as low-yielding glycosylation
acceptors compared to alkyl hydroxyl groups.14 Glycosyl
bromides were chosen as efficient donors for the construc-
tion of this kind of glycosydic bond, and glycosyl bro-
mides were the first type of donor applied to flavonoid
glycoside synthesis.15 A bisglycosyl kaempferol was effi-
ciently synthesized through a phase-transfer-catalyzed
(PTC) protocol with glycosyl bromides as the donor by our
group,16 and the Zemplen-Farkas protocol and Koenigs–
Knorr protocols have also been developed for this kind of
donor.17 Herein, the target molecules 1 and 2 were obtained
through the condensation of the glycosyl bromides 3 and 5
with partially protected baicalein and scutellarein accep-
tors 15 and 20 as a key step (Scheme 1).
As shown in Scheme 2, disaccharide bromide 5 was read-
ily prepared in a straightforward manner from compound 7
previously reported by our group18 and was treated with

Li et al.
3
HO
HO
HO
O
O
HO
HO
HO
O
O
OCH3
HO
HO
O
O
HO
O
O
HO
HO
HO
HO
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
OH
OH
oroxin C (1)
oroxin D (2)
BzO
BzO
BzO
O
BzO
BzO
BzO
BzO
OCH3
O
O
BzO
HO
O
O
BzO
O
BzO
HO
O
O
O
BzO
BzO
O
BzO
BzO
AcO
BzO
AcO
BzO
Br
OAc O
BzO
Br
OAcO
3
5
4
6
OH
HO
HO
O
HO
HO
O
OH O
baicalein
OH O
scutellarein
Scheme 1. Retrosynthesis of target flavonoid glycosides, oroxins C (1) and D (2).
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
O
O
O
a
O
O
O
b
O
O
O
BzO
BzO
OBz
BzO
H
O
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
Br
7
8
5
c
z
B
O
O
z
B
O
O
z
B
O
O
z
BzO
z
B
O
B
O
NH
CC 3
z
O
O
BzO
B
BzO
O
z
B
O
BzO
TrO
BzO
BzO
l
O
O
BzO
9
HO
BzO
d
O
O
O
BzO
zO
B
O
e
BzO
BzO
BzO
BzO
10
BzO
11
OBz
OBz
BzO
OBz
12
a
BzO
BzO
BzO
BzO
BzO
BzO
O
O
b
O
O
O
O
BzO
BzO
BzO
BzO
O
O
BzO
BzO
O
O
BzO
BzO
BzO
H
O
BzO
13
BzO
BzO
BzO
r
BzO
B
3
Scheme 2. Reagents and conditions: (a) NH2NH2·HOAc, DMF, 81% for 8, 78% for 13; (b) CBr4, PPh3, CH2Cl2, 90% for 5, 83% for
3; (c) NC.CCl3, DBU, CH2Cl2, 88%; (d) FeCl3·6H2O, CH2Cl2, 93%; (e) TMSOTf, CH2Cl2, 0 °C, 89%.
(see supplemental material) for the synthesized flavonoid were active. Compound 1 showed strong α-amylase and
glycosides 1 {[α]D25 = −73.1 (c 0.12, MeOH); ref: lipase inhibition, with IC50 values of 210 and 190μM,
[α]D20 = −72.0 (c 0.1, MeOH)} and 2 {[α]D25 =−168.2 (c respectively, but it exhibited no inhibitory activity against
0.11, MeOH); ref: [α]D20 =−170.0 (c 0.1, MeOH)} were α-glucosidase. Compound 2 showed strong inhibition
identical in all respects to those reported in the literaure.13
against α-glucosidase and lipase, with the respective IC50
To examine the potential antidiabetic ability of the natu- values of 180 and 80μM. Notably, the lipase inhibitory
ral flavonoid glycosides, oroxins C (1) and D (2), their in activity of Compound 2 was twofold stronger than orlistat, a
vitro inhibitory activities against α-glucosidase, α-amylase, widely used clinically useful drug, used here as positive
and lipase were evaluated, and the results are summarized control. These results demonstrated that glycosyl fragment
in Table 1. The data indicate that two flavonoid glycosides favorably enhances the lipase inhibitory activity.

4
Journal of Chemical Research 00(0)
H
H
O
O
O
AcO
AcO
O
O
H
O
O
a
b
O
AcO
c
A
O
H
O
O
4
OAc
baicalein
14
H
OC
H
O
H
O
3
O
O
O
O
O
O
O
O
H
O
O
O
Ph
Ph
Ph
Ph
e
d
H
O
H
O
H
O
H
O
16
scutellarein
15
f
H
b
H
3
OC
OC
H
OC
3
3
AcO
AcO
H
O
O
O
O
H
H
O
O
a
AcO
O
O
OAc
H
O
O
OAc
6
18
17
Scheme 3. Reagents and conditions: (a) Ac2O, AcONa, 80 °C, 90% for 14, 91% for 18; (b) BnBr, K2CO3, acetone, reflux;
(c) Pd(OH)2/C, H2, THF, 80% for 4, 78% for 6; (d) dichlorodiphenylmethane, Ph2O, 175 °C, 83%; (e) MeI, K2CO3, DMF, 91%;
(f) 80% AcOH, reflux, 95%.
Table 1. Inhibitory activities of 1 and 2 for α-glucosidase, α-amylase, and lipase.
Compounds
IC50 (μM)a
α-Glucosidase
α-Amylase
Lipase
1
2
NA
210.3±19.1
NA
579.2±30.7
-
190.1±18.2
80.0±9.5
-
180.4±25.7
449.3±38.9
-
b
Acarbose
Orlistat
158.4±26.6
NA: not active.
aThe IC50 values in μM were calculated from the dose response curve of six concentrations of each test compound in triplicate.
b“-”: not determined.
on silica gel (200–300 mesh). Optical rotations were deter-
mined with a Perkin–Elmer Model 241 MC polarimeter.
1H NMR and 13C NMR spectra were taken on a JEOL
JNM-ECP 600 spectrometer with tetramethylsilane as the
internal standard, and chemical shifts are recorded in δ val-
ues. Mass spectra were recorded on a Q-TOF Global mass
spectrometer.
Conclusion
In conclusion, we have succeeded in the first concise syn-
thesis of the natural flavonoid glycosides, oroxins C (1) and
D (2) using a convergent strategy. Preliminary pharmaco-
logical research exhibited positive responses against α-
glucosidase, α-amylase, and lipase. Notably, the flavonoid
glycoside 2 showed stronger lipase inhibitory activity
(IC50 =80μM), which was twice that of the positive control
orlistat. Further study on preparation and bioactivity evalu-
ation of analogs and derivatives of these compounds is in
progress and will be reported in due course.
2,3,4,6-Tetra-O-benzoyl-β-d-glucopyranosyl-(1→6)-2,3,4-
tri-O-benzoyl-α-d-glucopyranosyl bromide (5). A solution
of 8 (1.20g, 1.12mmol), triphenylphosphine (1.46g,
5.60mmol), and carbon tetrabromide (1.85g, 5.60mmol)
in dichloromethane (20mL) was stirred at room tempera-
ture for 3h. The organic solution was diluted with ethyl
acetate, and then washed with saturated sodium bicarbon-
ate twice and brine in sequence, dried with anhydrous
Na2SO4, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(petroleum ether-EtOAc, 5:1) to obtain 5 (1.14g, 90%) as
a colorless syrup. [α]D25 =+40.3 (c=1.0, CHCl3); 1H NMR
(400MHz, CDCl3) δ 7.20–8.03 (m, 35 H), 6.75 (d, J=3.6Hz,
Experimental
Chemistry
Commercial reagents were used without further purifica-
tion unless specialized. Solvents were dried and redistilled
prior to use in the usual way. Thin-layer chromatography
(TLC) was performed on precoated E. Merck Silica Gel 60
F254 plates. Flash column chromatography was performed

Li et al.
5
BzO
BzO
BzO
BzO
O
O
O
BzO
O
O
BzO
BzO
BzO
O
O
BzO
BzO
BzO
O
O
BzO
BzO
BzO
O
BzO
BzO
BzO
O
O
O
HO
O
O
BzO
BzO
3
Br
BzO
AcO
AcO
a
OAc
OAc
4
19
1
b
BzO
BzO
O
BzO
O
BzO
O
OCH3
H
OC
BzO
BzO
3
O
O
BzO
BzO
O
BzO
BzO
H
O
O
O
BzO
O
O
BzO
BzO
Br
5
a
BzO
AcO
AcO
OAc
O
OAc
6
20
2
b
Scheme 4. Reagents and conditions: (a) Ag2O, quinoline, 79% for 21, 85% for 22; (b) Mg(OMe)2, MeOH-CH2Cl2, 83% for 1, 86%
for 2.
1 H, H-1), 6.24 (t, J=9.9Hz, 1 H, H-3), 5.95 (t, J=9.6Hz, 5.7Hz, 1 H, H-6''-2), 4.23 (m, 2 H, H-5', H-5''), 4.17 (dd,
1 H, H-3'), 5.66 (t, J=9.6Hz, 1 H, H-4'), 5.58 (dd, J=9.6, J=11.9, 2.1Hz, 1 H, H-6-1), 3.95 (dd, J=11.9, 5.8Hz, 1 H,
7.8 Hz, 1 H, H-2'), 5.53 (t, J = 9.9 Hz, 1 H, H-4), 5.43 H-6-2). 13C NMR (100MHz, CDCl3) δ 166.1, 165.9, 165.8,
(t, J=9.9, 3.6Hz, 1 H, H-2), 5.07 (d, J=7.8Hz, 1 H, H-1'), 165.7, 165.6, 165.5, 165.3, 165.1, 164.7, 133.3, 133.1,
4.63 (t, J=12.4, 3.2Hz, 1 H, H-6'-1), 4.53 (m, 1 H, H-5), 133.0, 130.1, 130.0, 129.9, 129.8, 129.7, 129.5, 129.3,
4.51 (t, J=12.4, 5.3Hz, 1 H, H-6'-2), 4.21 (m, 1 H, H-5'), 129.1, 128.9, 128.7, 128.3, 101.1 (C-1''), 100.5 (C-1'), 90.5
4.19 (dd, J=11.6, 1.8Hz, 1 H, H-6-1), 3.91 (dd, J=11.6, (C-1), 73.5 (C-3''), 72.9 (C-3'), 72.3 (C-5'), 72.1 (C-5''),
6.0Hz, 1 H, H-6-2). 13C NMR (100MHz, CDCl3) δ 166.0, 71.9 (C-5), 71.7 (C-2''), 71.5 (C-2'), 70.5 (C-2), 70.1 (C-3),
165.9, 165.8, 165.7, 165.5, 165.3, 165.2, 133.1, 133.0, 69.9 (C-4''), 69.6 (C-4'), 68.6 (C-4), 68.3 (C-6'), 67.1 (C-6),
130.1, 130.0, 129.9, 129.8, 129.6, 129.5, 129.1, 128.9, 63.0 (C-6''). HRMS (MALDI) calcd for C95H76O27Na
128.3, 100.6 (C-1'), 90.6 (C-1), 72.9 (C-3'), 72.1 (C-5'), [M+Na]+ 1671.4466, found 1671.4461.
71.9 (C-5), 71.7 (C-2'), 70.5 (C-2), 70.1 (C-3), 69.6 (C-4'),
68.6 (C-4), 67.1 (C-6), 63.0 (C-6'). HRMS (ESI) calcd for 2,3,4,6-Tetra-O-benzoyl-β-d-glucopyranosyl-(1→6)-2,3,4-
C61H49O17BrNa [M+Na]+ 1155.2045, found 1155.2049.
tri-O-benzoyl-β-d-glucopyranosyl -(1→6)-2,3,4-tri-O-
benzoyl-α-d-glucopyranosyl bromide (3). A solution of 12
2,3,4,6-Tetra-O-benzoyl-β-d-glucopyranosyl-(1→6)-2,3,4- (0.80g, 0.49mmol) and NH2NH2·HOAc (45mg, 0.49mmol,
tri-O-benzoyl-β-d-glucopyranosyl -(1→6)-2,3,4-tri-O- 1.0 eq) in DMF (10mL) were stirred for 6h at room tem-
benzoyl-α-d-glucopyranoside (12). A solution of 11 perature. The reaction mixture was evaporated. The residue
(0.50g, 0.84mmol), disaccharide trichloroacetimidate 9 was purified by silica gel column chromatography (petro-
(1.53g, 1.26mmol, 1.5 eq), and 4 Å powdered molecular leum ether-EtOAc, 3:1) to obtain 13. To a solution of 13 in
sieves were stirred for 30min at room temperature in dry dichloromethane (15mL), triphenylphosphine (0.64g,
CH2Cl2 (20mL). The mixture was cooled to 0 °C for 2.45mmol) and carbon tetrabromide (0.81g, 2.45mmol)
30min, followed by the dropwise addition of TMSOTf was added, and the reaction mixture was stirred at room
(50μL, 0.04mmol, 0.05 eq). After 1h, the reaction was temperature for 4h. The organic solution was diluted with
quenched with triethylamine, filtered through Celite and ethyl acetate, and then washed with saturated sodium bicar-
evaporated. The residue was purified by silica gel column bonate twice and brine in sequence, dried with anhydrous
chromatography (petroleum ether-EtOAc, 4:1) to obtain 12 Na2SO4, and concentrated under reduced pressure. The
(1.23g, 89%) as a white solid. [α]D25 =+61.8 (c=1.0, residue was purified by silica gel column chromatography
1
CHCl3); H NMR (400MHz, CDCl3) δ 7.18–8.05 (m, 55 (petroleum ether-EtOAc, 4:1) to obtain 3 (512mg, 65% for
H), 6.79 (d, J=3.6Hz, 1 H, H-1), 6.32 (t, J=9.9Hz, 1 H, two steps) as a colorless syrup. [α]D25 =+56.7 (c=1.0,
H-3), 5.99 (t, J=9.6Hz, 1 H, H-3'), 5.87 (t, J=9.7Hz, 1 H, CHCl3); 1H NMR (400MHz, CDCl3) δ 7.19–8.06 (m, 50 H),
H-3''), 5.66 (t, J=9.6Hz, 1 H, H-4'), 5.61 (t, J=9.7Hz, 1 H, 6.73 (d, J=3.7Hz, 1 H, H-1), 6.31 (t, J=9.9Hz, 1 H, H-3),
H-4''), 5.58 (dd, J=9.6, 7.8Hz, 1 H, H-2'), 5.55 (dd, J=9.7, 5.95 (t, J=9.7Hz, 1 H, H-3'), 5.89 (t, J=9.7Hz, 1 H, H-3''),
7.7 Hz, 1 H, H-2''), 5.51 (t, J = 9.9 Hz, 1 H, H-4), 5.41 5.67 (t, J=9.7Hz, 1 H, H-4'), 5.63 (t, J=9.7Hz, 1 H, H-4''),
(t, J=9.9, 3.6Hz, 1 H, H-2), 5.13 (d, J=7.8Hz, 1 H, H-1'), 5.58 (dd, J=9.7, 7.7Hz, 1 H, H-2'), 5.54 (dd, J=9.7, 7.8Hz,
5.09 (d, J=7.7Hz, 1 H, H-1''), 4.75 (t, J=12.3, 3.3Hz, 1 H, 1 H, H-2''), 5.51 (t, J=9.9Hz, 1 H, H-4), 5.45 (t, J=9.9,
H-6'-1), 4.63 (t, J=12.0, 3.2Hz, 1 H, H-6''-1), 4.55 (m, 1 H, 3.7 Hz, 1 H, H-2), 5.19 (d, J = 7.7 Hz, 1 H, H-1'), 5.07
H-5), 4.51 (t, J=12.3, 5.5Hz, 1 H, H-6'-2), 4.49 (t, J=12.0, (d, J=7.8Hz, 1 H, H-1''), 4.79 (t, J=12.5, 3.2Hz, 1 H,

6
Journal of Chemical Research 00(0)
H-6'-1), 4.63 (t, J=12.1, 3.3Hz, 1 H, H-6''-1), 4.55 (m, 1 H, solid. [α]D25 =+80.9 (c=1.0, CHCl3); 1H NMR (400MHz,
H-5), 4.50 (t, J=12.5, 5.6Hz, 1 H, H-6'-2), 4.49 (t, J=12.0, CDCl3) δ 7.13–8.03 (m, 55 H), 6.91 (s, 1 H, H-8), 6.79
5.7Hz, 1 H, H-6''-2), 4.29 (m, 2 H, H-5', H-5''), 4.17 (dd, (d, J=7.8Hz, 1 H, H-1"), 6.61 (s, 1 H, H-3), 6.36 (t, J=9.9Hz,
J=12.3, 2.5Hz, 1 H, H-6-1), 3.99 (dd, J=12.3, 5.1Hz, 1 H, 1 H, H-3"), 5.95 (t, J=9.6Hz, 1 H, H-3"'), 5.88 (t, J=9.7Hz,
H-6-2). 13C NMR (100MHz, CDCl3) δ 166.0, 165.9, 165.8, 1 H, H-3"''), 5.71 (t, J=9.6Hz, 1 H, H-4"'), 5.65 (t, J=9.6Hz,
165.7, 165.6, 165.5, 165.3, 165.2, 164.9, 133.2, 133.1, 1 H, H-4''"), 5.59 (dd, J=9.6, 7.7Hz, 1 H, H-2'"), 5.55 (dd,
133.0, 130.1, 130.0, 129.9, 129.8, 129.7, 129.5, 129.3, J=9.6, 7.8Hz, 1 H, H-2"''), 5.52 (t, J=9.6Hz, 1 H, H-4"),
129.1, 128.9, 128.7, 128.5, 128.3, 101.3 (C-1''), 100.5 5.45 (t, J=9.6, 7.8Hz, 1 H, H-2"), 5.23 (d, J=7.7Hz, 1 H,
(C-1'), 90.3 (C-1), 73.5 (C-3''), 72.9 (C-3'), 72.6 (C-5'), 72.1 H-1"'), 5.11 (d, J=7.8Hz, 1 H, H-1"''), 4.85 (t, J=12.1,
(C-5''), 71.7 (C-5), 71.6 (C-2''), 71.5 (C-2'), 70.7 (C-2), 70.1 3.3Hz, 1 H, H-6'"-1), 4.73 (t, J=12.5, 3.6Hz, 1 H, H-6"''-
(C-3), 69.8 (C-4''), 69.3 (C-4'), 68.5 (C-4), 68.3 (C-6'), 66.9 1), 4.61 (m, 1 H, H-5"), 4.56 (t, J=12.1, 5.7Hz, 1 H, H-6'"-
(C-6), 63.1 (C-6''). HRMS (MALDI) calcd for C88H71O- 2), 4.49 (t, J=12.5, 5.7Hz, 1 H, H-6"''-2), 4.33 (m, 2 H,
25BrNa [M+Na]+ 1629.3360, found 1629.3366.
H-5"', H-5"''), 4.26 (dd, J=12.0, 2.6Hz, 1 H, H-6"-1), 3.97
(dd, J=12.0, 5.1Hz, 1 H, H-6-2"), 2.33, 2.43 (s each, 3 H
5,6,7-Tri-O-acetyl-4'-O-methyl scutellarein (18). A solution each, 2×COCH3). 13C NMR (100MHz, CDCl3) δ 178.5
of compound 17 (3.00g, 10mmol) and NaOAc (1.13g, (C-4), 166.0, 165.9, 165.8, 165.7, 165.6, 165.5, 165.3,
14mmol) in acetic anhydride (15mL) was stirred at 75 °C 165.2, 164.6, 163.5 (C-2), 159.1 (C-7), 150.3 (C-5), 133.3,
until start material disappear. The reaction mixture was 133.2, 133.0, 130.1, 130.0, 129.9, 129.8, 129.7, 129.5,
poured into ice water, and the precipitate was collected by 129.4, 129.2, 128.9, 128.5 (C-6), 128.3, 104. 3 (C-3), 101.6
filtration and washed by EtOH to give compound 18 (3.88g, (C-1"''), 100.1 (C-1"'), 90.9 (C-1"), 73.7 (C-3"''), 72.8
91%) as a yellow solid. 1H NMR (400MHz, CDCl3) δ 7.69 (C-3"'), 72.5 (C-5"'), 72.3 (C-5"''), 71.9 (C-5"), 71.7
(d, J=8.5Hz, 2 H, H-2', H-6'), 6.94 (d, J=8.5Hz, 2 H, H-3', (C-2"''), 71.4 (C-2"'), 70.6 (C-2"), 70.3 (C-3"), 69.7 (C-4"''),
H-5'), 6.79 (s, 1 H, H-8), 6.65 (s, 1 H, H-3), 3.86 (s, 3 H, 69.5 (C-4"'), 68.4 (C-4"), 68.1 (C-6"'), 66.7 (C-6"), 63.1
OCH3), 2.34, 2.36, 2.43 (s each, 3 H each, 3×COCH3); (C-6'"'). HRMS (MALDI) calcd for C107H84O32Na
HRMS (ESI) calcd for C22H18O9Na [M+Na]+ 449.0843, [M+Na]+ 1903.4838, found 1903.4833.
found 449.0849.
5,6-Di-O-acetyl-7-O-[2,3,4,6-tetra-O-benzoyl-β-d-
5,6-Di-O-acetyl-4'-O-methyl scutellarein (6). A solution of glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-d-
compound 18 (2.13g, 5mmol), benzyl bromide (1.78mL, glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-d-
15mmol), and anhydrous K2CO3 (2.76g, 20mmol) in acetone glucopyranosyl]-4'-O-methyl scutellarein (20). Similar
(120mL) was refluxed for 24h with stirring. The reaction procedure as that used for the synthesis of 19 was used to
mixture was cooled down to room temperature, filtered, and get 20. Thus, 6 (200mg, 0.52mmol) coupled with 5
the solvent was evaporated under reduced pressure to give the (884mg, 0.78mmol) under the effect of silver oxide
crude compound as a yellow solid. A solution of above crude (181mg, 0.78mmol), after silica gel column chromatogra-
compound and Pd(OH)2/C (1.12g) in anhydrous THF phy (petroleum ether-EtOAc, 2:1) to afford 20 (635mg,
1
(100mL) was stirred at room temperature for 3h until start 85%) as a white solid. [α]D25 =+56.5 (c=1.0, CHCl3); H
material was disappeared. The precipitate was collected by NMR (400 MHz, CDCl3) δ 7.19–8.03 (m, 37 H), 6.94
filtration and purified by silica gel column chromatography (d, J = 8.6 Hz, 2 H, H-3', H-5'), 6.73 (s, 1 H, H-8), 6.69
(petroleum ether-EtOAc, 3:1) to obtain 6 (1.50g, 78%) as a (d, J=7.7Hz, 1 H, H-1"), 6.61 (s, 1 H, H-3), 6.25 (t, J=9.6Hz,
yellow solid. 1H NMR (400MHz, CDCl3) δ7.65 (d, J=8.6Hz, 1 H, H-3"), 5.93 (t, J=9.6Hz, 1 H, H-3"'), 5.67 (t, J=9.6Hz,
2 H, H-2', H-6'), 6.95 (d, J=8.6Hz, 2 H, H-3', H-5'), 6.75 (s, 1 H, H-4"'), 5.55 (dd, J = 9.6, 7.8 Hz, 1 H, H-2"'), 5.51
1 H, H-8), 6.63 (s, 1 H, H-3), 3.87 (s, 3 H, OCH3), 2.35, 2.43 (t, J=9.6Hz, 1 H, H-4"), 5.40 (t, J=9.6, 7.7Hz, 1 H, H-2"),
(s each, 3 H each, 2×COCH3); HRMS (ESI) calcd for 5.05 (d, J=7.8Hz, 1 H, H-1'"), 4.61 (t, J=12.0, 3.3Hz, 1 H,
C20H16O8Na [M+Na]+ 407.0737, found 407.0731.
H-6'"-1), 4.55 (m, 1 H, H-5"), 4.51 (t, J=12.0, 5.5Hz, 1 H,
H-6'"-2), 4.23 (m, 1 H, H-5"'), 4.18 (dd, J=12.1, 3.0Hz, 1
5,6-Di-O-acetyl-7-O-[2,3,4,6-tetra-O-benzoyl-β-d- H, H-6"-1), 3.91 (dd, J=12.1, 5.8Hz, 1 H, H-6"-2), 3.87
glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-d- (s, 3 H, OCH3), 2.35, 2.43 (s each, 3 H each, 2×COCH3).
glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-d- 13C NMR (100MHz, CDCl3) δ 176.5 (C-4), 166.0, 165.9,
glucopyranosyl] baicalein (19). To a solution of 4 (300mg, 165.8, 165.7, 165.5, 165.3, 165.2, 163.6 (C-2), 159.7 (C-4'),
0.85mmol), 3 (2.04g, 1.27mmol) and silver oxide (296mg, 158.8 (C-7), 145.3 (C-5), 133.1, 133.0, 130.1, 130.0, 129.9,
1.27mmol) in dry quinoline (30mL) at room temperature 129.8, 129.6, 129.5, 129.1, 128.9, 128.0 (C-6), 128.3, 114.3
was added activated 4 Å MS (ca. 500mg) under argon (C-3'), 114.2(C-5'), 104. 3 (C-3), 100.9 (C-1"'), 91.7 (C-1"),
atmosphere. The mixture was stirred for 24h, and then 72.7 (C-3"'), 72.3 (C-5"'), 71.9 (C-5"), 71.6 (C-2'"), 70.7
diluted with dichloromethane followed by filtration to (C-2"), 70.3 (C-3"), 69.5 (C-4"'), 68.3 (C-4"), 67.2 (C-6"),
remove insoluble substances. The solution was washed 63.0 (C-6"'), 55.9 (C4-OCH3). HRMS (MALDI) calcd for
with 1M HCl three times, saturated sodium bicarbonate C81H64O25Na [M+Na]+ 1459.3629, found 1459.3621.
and brine in sequence, dried with anhydrous Na2SO4, and
then concentrated under reduced pressure. The residue was Baicalein7-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl
purified by silica gel column chromatography (petroleum -(1→6)-β-d-glucopyranoside (1). To a solution of 19
ether-EtOAc, 2:1) to obtain 19 (1.26g, 79%) as a white (100mg, 0.053mmol) in methanol (2mL) and CH2Cl2

Li et al.
7
(0.5mL) was added 7% magnesium methoxide (1.2mL). p-nitrophenyl-α-d-glucopyranoside (PNPG) as a substrate.
The solution was stirred at room temperature overnight. In brief, 20μL of enzyme solution (0.8U/mL α-glucosidase
The solution was neutralized with the weakly acidic ion- in 0.01M potassium phosphate buffer (pH 6.8) containing
exchange resin (amberlite IRC 76, H+ form) and filtered. 0.2% of bovine serum albumin (BSA)) and 120μL of the
The filtrate was concentrated in vacuo and purified by synthetic compound in 0.5% DMSO of 0.01M potassium
RP-18 column chromatography (methanol–water, 1:2) to phosphate buffer were mixed and was preincubated at
afford oroxin C (1) (33mg, 83%) as a yellow solid. 37°C prior to the initiation of the reaction by adding the
[α]D25 =−73.1 (c 0.12, MeOH); 1H NMR (400MHz, substrate. After 15min of preincubation, PNPG solution
dimethyl sulfoxide (DMSO)-d6) δ 12.09 (s, 1 H, C5-OH), (20μL; 5.0mM PNPG in 0.1M potassium phosphate buf-
8.58 (s, 1 H, C6-OH), 8.09 (d, J=8.5Hz, 2 H, H-2', H-6'), fer (pH 6.8)) was added and then incubated together at
7.63 (d, J=8.5Hz, 2 H, H-3', H-5'), 7.62 (m, 1 H, H-4'), 37°C. After 15min of incubation, 0.2M Na2CO3 (80μL)
7.08 (s, 1 H, H-8), 6.99 (s, 1 H, H-3), 5.01 (d, J=7.3Hz, 1 in 0.1M potassium phosphate buffer was added to the test
H, H-1"), 4.23 (d, J=7.5Hz, 1 H, H-1"'), 4.03 (dd, J=11.2, tube to stop the reaction. Acarbose was used as positive
1.9Hz, 1 H, H-6"-1), 3.70 (t, J=9.1Hz, 1 H, H-4"'), 3.68 control. The increment in absorption at 410nm due to the
(dd, J=11.2, 4.1Hz, 1 H, H-6"-2), 3.66 (m, 3 H, H-6"'-1, hydrolysis of PNPG by α-glucosidase was monitored con-
H-6"'-2, H-4""), 3.51 (dd, J=11.2, 4.3Hz, 1 H, H-6""-1), tinuously with an auto multi-functional microplate reader
3.45 (m, 2 H, H-2"", H-6""-2), 3.42 (t, J=9.0Hz, 1 H, (BIORAD680).
H-3"), 3.41 (t, J=9.1Hz, 1 H, H-3"), 3.34 (m, 1 H, H-5"),
3.33 (m, 2 H, H-5"', H-3""), 3.23 (t, J=9.0Hz, 1 H, H-4"), Assay for α-amylase inhibitory activities. The α-amylase inhib-
3.17 (m, 2 H, H-2"', H-5""), 3.15 (dd, J=9.0, 7.2Hz, 1 H, itory activities were measured with the method reported by
H-2"); 13C NMR (100MHz, DMSO-d6) δ 182.6 (C-4), Xiao et al.21 and Yoshikawa et al.22 with slight modifica-
163.8 (C-2), 151.6 (C-7), 149.2 (C-9), 146.4 (C-5), 132.1 tions. Substrate was prepared by heating starch (250mg) in
(C-4'), 130.9 (C-1'), 130.5 (C-6), 129.3 (C-3', C-5'), 126.7 12mL of 0.4M NaOH solution for 5min at 100°C, and then
(C-2', C-6'), 106.3 (C-10), 104.5 (C-3), 103.9 (C-1"'), 101.0 cooled to 0°C and adjusted to pH 7 with 2M HCl. Sample
(C-1"), 98.5 (C-1'"'), 94.3 (C-8), 76.8 (C-5'"'), 75.6 (C-5"), solutions were prepared by dissolving each solution in ace-
75.5 (C-5"'), 75.0 (C-3'"'), 73.6 (C-3"'), 73.3 (C-3"), 73.2 tate buffer (pH 6.5). The sample (20μL) and the substrate
(C-2'"'), 72.4 (C-2"'), 72.1 (C-2"), 70.2 (C-4"), 70.1 (C-4"'), (40μL) were mixed in a microplate well. After preincuba-
69.8 (C-4'"'), 69.2 (C-6"), 66.9 (C-6"'), 60.6 (C-6'"'). HRMS tion at 37°C for 15min, 5mg/mL α-amylase solution
(ESI) calcd for C33H41O20 [M+H]+ 757.2186, found (20mL) was added and the solution was incubated at 37°C
757.2193.
for 15min. The reaction was stopped by adding 50mL 1M
HCl, and then 50mL iodine solution was added. The absor-
Scutellarein 4'-methyl ether 7-O-β-d-glucopyranosyl-(1→6) bances were measured at 650nm by a microplate reader.
-β-d-glucopyranoside (2). Similar procedure as that used Acarbose was used as positive control.
for the synthesis of 1 from 19 was used to provide 2, after
RP-18 column chromatography (methanol–water, 1:2), Assay for lipase inhibitory activities. Lipase inhibitory activi-
(37mg, 86%) as a yellow solid. [α]D25 =−168.2 (c 0.11, ties were measured according to the method of Han et al.23
1
MeOH); H NMR (400MHz, DMSO-d6) δ 12.63 (s, 1 H, with slight modifications. Substrate was prepared by soni-
C5-OH), 8.55 (s, 1 H, C6-OH), 8.07 (d, J=8.8Hz, 2 H, H-2', cation of a mixture of glyceryl trioleate (80mg), lecithin
H-6'), 7.19 (d, J=8.8Hz, 2 H, H-3', H-5'), 7.13 (s, 1 H, (10mg), and sodium cholate (5mg) suspended in 9mL of
H-8), 6.89 (s, 1 H, H-3), 5.05 (d, J=7.3Hz, 1 H, H-1"), 4.21 0.1M TES buffer (pH 7.0). Samples were prepared by dis-
(d, J=7.8Hz, 1 H, H-1"'), 4.04 (dd, J=11.2, 2.2Hz, 1 H, solving each sample in 0.1M TES buffer. The sample
H-6"-1), 3.86 (s, 3 H, C4'-OCH3), 3.78 (m, 1 H, H-5"), 3.66 (20μL) and the substrate (20μL) were mixed in microplate
(m, 2 H, H-6"-2, H-6'"-1), 3.38 (dd, J=9.0, 7.3Hz, 1 H, wells. After preincubated for 5min, 10μL of lipase solution
H-2"), 3.35 (dd, J=11.3, 4.1Hz, 1 H, H-6'"-2), 3.31 (dd, (20μg/mL) was added to each reaction mixture and incu-
J=9.3, 7.8Hz, 1 H, H-2"'), 3.21 (t, J=9.5Hz, 1 H, H-4'"), bated for 30min at 37°C. The amount of released fatty acid
3.18 (m, 2 H, H-3", H-5'"), 3.14 (t, J=9.4Hz, 1 H, H-4''), 3.09 was measured at 405nm. Inhibition of lipase activity was
(t, J=9.5Hz, 1 H, H-3'"); 13C NMR (100MHz, DMSO-d6) δ expressed as the percentage decrease in the absorbance
182.4 (C-4), 163.8 (C-2), 162.4 (C-4'), 151.5 (C-7), 149.1 when porcine pancreatic lipase was incubated with the test
(C-9), 146.4 (C-5), 130.5 (C-6), 128.5 (C-2', C-6'), 122.9 compounds. Orlistat was used as positive control.
(C-1'), 114.8 (C-3', C-5'), 106.0 (C-10), 103.0 (C-3), 103.9
(C-1"'), 101.0 (C-1"), 94.4 (C-8), 77.4 (C-5'"), 76.7 (C-3"), Declaration of conflicting interests
75.8 (C-2"'), 75.6 (C-5"), 73.6 (C-3"'), 73.1 (C-2"), 70.2
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
(C-4"), 69.7 (C-4"'), 69.5 (C-6"), 61.2 (C-6"'). HRMS (ESI)
calcd for C28H33O16 [M+H]+ 625.1763, found 625.1770.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
project was financial supported by the Foundation of Facility
Horticulture Laboratory Project of Shandong Universities
Biological activities assay
Assay for α-glucosidase inhibitory activities. Inhibitory α-
glucosidase activities were determined spectrophoto-
metrically in a 96-well microtiter plate based on (2018YY011, 2018YY042).

8
Journal of Chemical Research 00(0)
10. Gaspar A, Matos MJ, Garrido J, et al. Chem Rev 2014; 114:
4960.
11. Li YF, Yu B, Sun JS, et al. Tetrahedron Lett 2015; 56: 3816.
12. Han ZY, Zheng ZW, Cai L, et al. Tetrahedron Lett 2018; 59:
3773.
ORCID iD
Qingchao Liu
https://orcid.org/0000-0003-3319-7498
Supplemental material
13. Wu BL, Wu ZW, Yang F, et al. Phytochemistry Lett 2019;
32: 66.
Supplemental material for this article is available online.
14. Jacobsson M, Malmberg J and Ellervidk U. Carbohydr Res
2006; 341: 1266.
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