Total synthesis of 8-(6″-umbelliferyl)-apigenin and its analogs as anti-diabetic reagents

Article abstract of DOI:10.1016/j.ejmech.2016.07.015

The naturally occurring flavone 8-(6″-umbelliferyl)apigenin, a hybrid structure of apigenin and coumarin, as well as seven of its analogues were synthesized for the first time by using iodination and Suzuki coupling reactions as key steps. The synthesis of 8-(6″-umbelliferyl)-apigenin was achieved in seven linear steps from the commercially available 1-(2,4,6-trihydroxyphenyl)ethan-1-one and 7-hydroxyl coumarine with 31% overall yield. Effects of these compounds on glucose disposal were investigated in adipocytes. All of the flavonoid and coumarin hydrids were found to have better bioactivities than their corresponding flavonoid cores. The most potent compound 15 (10?μΜ) could promote glucose consumption by 57% which exhibited similar effect as the positive control metformin at 1?mM. Moreover, fluorescence microscopy showed that four 8-(6″-umbelliferyl)apigenin analogues 2, 15, 30 and 31 could promote the 2-NBDG uptake into 3T3-L1 cells, which consist with those observed in the regulation of glucose.

Full text of DOI:10.1016/j.ejmech.2016.07.015

European Journal of Medicinal Chemistry 122 (2016) 674e683
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
00
Total synthesis of 8-(6 -umbelliferyl)-apigenin and its analogs as
anti-diabetic reagents
a
a
b
a
a
c
Guojun Pan , Lianbo Zhao , Na Xiao , Ke Yang , Yantao Ma , Xia Zhao ,
Zhenchuan Fan , Yongmin Zhang , Qingwei Yao , Kui Lu , Peng Yu a
China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Lab of Industrial Fermentation
Microbiology, Tianjin Key Lab of Industrial Microbiology, Sino-French Joint Lab of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology,
Tianjin University of Science and Technology, Tianjin, 300457, PR China
d
a
e
a,
*
, **
a
b
State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, 211198, PR
China
c
College of Chemistry, Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Tianjin Normal University, Tianjin, 300387, PR China
College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China
d
e
3
82 Longvalley Lane, DeKalb, IL 80115, USA
a r t i c l e i n f o
a b s t r a c t
00
The naturally occurring flavone 8-(6 -umbelliferyl)apigenin, a hybrid structure of apigenin and
Article history:
Received 1 June 2016
Received in revised form
coumarin, as well as seven of its analogues were synthesized for the first time by using iodination and
00
Suzuki coupling reactions as key steps. The synthesis of 8-(6 -umbelliferyl)-apigenin was achieved in
8
July 2016
seven linear steps from the commercially available 1-(2,4,6-trihydroxyphenyl)ethan-1-one and 7-
hydroxyl coumarine with 31% overall yield. Effects of these compounds on glucose disposal were
investigated in adipocytes. All of the flavonoid and coumarin hydrids were found to have better bio-
Accepted 9 July 2016
Available online 11 July 2016
activities than their corresponding flavonoid cores. The most potent compound 15 (10
mМ) could pro-
Keywords:
8
Flavonoid
00
-(6 -umbelliferyl)-apigenin
mote glucose consumption by 57% which exhibited similar effect as the positive control metformin at
00
mM. Moreover, fluorescence microscopy showed that four 8-(6 -umbelliferyl)apigenin analogues 2, 15,
1
Coumarins
Suzuki coupling reaction
Anti-diabetic
30 and 31 could promote the 2-NBDG uptake into 3T3-L1 cells, which consist with those observed in the
regulation of glucose.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
and coumarin, were isolated from Gnidia socotrana by Frank et al.
(Fig. 1) [10].
Apigenin, a naturally occurring flavonoid and abundantly pre-
sent in common fruits, vegetables and herbs such as parsley, on-
ions, oranges, chamomile, tea and wheat sprouts, has been shown
to process many bioactivities including anti-cancer [1], anti-
inflammatory [2], antioxidant [3] and anti-diabetic properties [4].
Coumarins are a prominent class of benzopyrones of either natural
or synthetic origins with diverse biological activities including
antimicrobial [5], monoamine oxidase (MAO) inhibition [6], anti-
tumor [7], antioxidant [8] and anti-diabetic properties [9]. In 2002,
Due to their structural uniqueness and good bioactivities of
apigenin and coumarin, along with our on-going interest in the
synthesis of natural flavonoids and chalcones, we embarked on a
synthetic effort towards these types of molecules [11]. In this paper,
we would like to report a facile synthesis of compound 2 and its
analogs by using iodination and Suzuki coupling reactions as key
steps, as well as anti-diabetic activity evaluation of these new
flavonoids.
00
00
two novel flavonoids, 6-(8 -umbelliferyl)apigenin (1) and 8-(6 -
umbelliferyl)apigenin (2), both belonging to the hybrids of apigenin
2
. Chemistry
00
Our retro-synthetic analysis for the synthesis of 8-(6 -umbelli-
feryl)apigenin was depicted in Scheme 1. The target molecule could
be synthesized from iodo apigenin derivative 3 and coumarin
boronic ester 4 by Suzuki coupling followed by deprotection. The
required iodo compound 3 can be readily prepared from a
*
Corresponding author.
** Corresponding author.
E-mail addresses: lukui@tust.edu.cn (K. Lu), yupeng@tust.edu.cn (P. Yu).
http://dx.doi.org/10.1016/j.ejmech.2016.07.015
223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
0

G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
675
commercially
available
starting
material
1-(2,4,6-
of the natural product and the analogues were evaluated and
compared to apigenin (16), chrysin (17), quercetin (18), luteolin
(19), 7-hydroxyl coumarin (6) (Fig. 2) as well as Metformin which
was used as positive control.
trihydroxyphenyl)ethan-1-one (5) and the boronic ester fragment
4
can be derived from 7-hydroxyl coumarin (6).
Compound 3 was synthesized in four steps from 5 via a regio-
selective iodination of the apigenin derivative 9 according to our
previously reported procedures (Scheme 2) [11a]. The coumarin
boronic ester intermediates 4 was prepared from iodide 13 by
coupling with bis(pinacolato)diboron in the presence of
PdCl2(dppf). The key intermediate 13 was synthesized from 7-
hydroxyl coumarin 6 through methylation, transesterification,
regio-selective iodination and relactonization in 62% overall yield
according to literature procedures (Scheme 3) [12].
The bioactivities of this compounds were summarized in Fig. 3.
Generally, the flavonoids including apigenin (16), chrysin (32),
quercetin (18) and luteolin (19) could promote glucose consump-
tion in adipocytes to some extent, but 7-hydroxyl coumarin (6) did
not showed this kind of activity. Most of the hybrid compounds
including 7-methoxyl coumarin substituted flavonoids and 7-
hydroxyl coumarin substituted flavonoids show stronger glucose
disposal activity than their corresponding flavonoids cores (15 and
2 versus apigenin, 29 and 32 versus chrysin, 30 versus quercetin, 31
and 34 versus luteolin), which suggests that introducing a
coumarin substitution at 8-position of flavonoids could increase the
glucose disposal activity.
With the key intermediates 3 and 4 in hand, the Suzuki coupling
reaction was initially attempted using Pd(PPh3)4 as the catalyst and
ꢀ
Cs
2
CO
3
as the base in DMF at 100 C. The desired coupling product
14 was obtained in 14% yield. To improve the yield, the reaction was
optimized by screening a variety of solvents and palladium cata-
Moreover, 7-methoxyl coumarin substituted flavonoids showed
stronger glucose disposal activity than the corresponding 7-
hydroxyl coumarin substituted flavonoids (15 versus 2, 29 versus
32, 30 versus 33, 31 versus 34), which indicated that 7-phenol
group on the coumarin diminished the bioactivity (Fig. 3).
Although the electronic effect and steric effect of the methoxyl
group did not have much difference with the phenol group, an
intramolecular hydrogen bond between 7-phenol group on the
coumarin and 7-phenol group on the flavonoid may undermine the
intermolecular hydrogen bond between target proteins with the
latter.
lysts. (Entries 1e4, Table 1). This led us to the identification of
ꢀ
Pd(PPh
3
)
4
along with Cs
2
CO
3
in dioxane at 100 C as the optimal
reaction conditions for the formation of 14, which was isolated in
7
0% yield.
The final deprotection was tested by using the common reagent
BCl . Unfortunately, no formation of the desired product was
3
detected and the reaction gave the partially deprotected interme-
diate 15 resulting from selective cleavage of the isopropyl groups
was obtained. However, when a stronger dealkylation reagent BBr3
00
was employed, the final target, 8-(6 -umbelliferyl)-apigenin (2)
was formed in nearly quantitative yield (Scheme 4).
However, the analogues bearing alkyl protection groups
including 14, 26, 27 and 28 have no glucose disposal activity, which
suggested that the phenol groups on the flavonoids of the hybrids
were required for the activity (Fig. 4).
3
. Results and discussion
In order to further confirm the beneficial effects of four com-
pounds 2, 15, 30 and 31 on glucose homeostasis, we then detected
their influence on 2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)-amino-
With the natural product 2 and its methyl ether 15 in hand, the
anti-diabetic activities of these two compounds were evaluated by
using glucose disposal bioassay in adipocytes [13]. To our delight,
0
0
2-deoxy-D-glucose (2-NBDG) uptake under basal condition by us-
the 8-(6 -umbelliferyl)-apigenin (2) (10
10 М) could promote glucose consumption by 48% and 57%
respectively.
mМ) and compound 15
ing Metformin as control. As shown in Fig. 5, these four compounds
promoted basal glucose uptake in adipocytes evidenced by stronger
fluorescence in cytoplasma. These findings were in agreement with
those observed in the regulation of glucose consumption.
(
m
Encouraged by the results of compounds 2 and 15, we further
designed and synthesized three series of analogous compounds by
replacing apigenin with other flavonoids and changing the pro-
tection pattern of the 7-hydroxyl on the coumarin moiety. These
compounds were synthesized in a similar way as shown in Scheme
4. Conclusions
5
, that N-iodosuccinimide (NIS) was used as a highly efficient re-
In summary, the apigenin and coumarin hybrid natural product
0
0
agent for the regio-selective iodination when chrysin, luteolin and
kaempferol isopropyl ethers were used as substrates according to
our previous research [11a].
8-(6 -umbelliferyl)-apigenin (2) were synthesized for the first time
in seven liner steps from the commercially available 1-(2,4,6-
trihydroxyphenyl)ethan-1-one (5) and 7-hydroxyl coumarin in
31% overall yield by employing iodination and the Suzuki coupling
To gain SAR information of this type of compounds, the activities
0
0
00
Fig. 1. 6-(8 -umbelliferyl)-apigenin and 8-(6 -umbelliferyl)-apigenin.

676
G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
00
Scheme 1. Retro-synthetic analysis of 8-(6 -umbelliferyl)apigenin (2).
Scheme 2. Synthesis of key intermediate 3.
reaction as key steps. Moreover seven analogues were synthesized
by similar procedures. The biological evaluations of glucose
disposal activities in adipocytes for these compounds were per-
formed. The results reveal that attaching 7-methoxyl coumarin to
the C-8 position of apigenin, chrysin, quercetin and luteolin would
increase the bioactivities compared to those with a flavonoid core.
The bioactivity of four compounds 2, 15, 30 and 31 was confirmed
by detection of 2-NBDG uptake in 3T3-L1 cells with fluorescence
Scheme 3. Synthesis of key intermediate 4.

G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
677
00
Scheme 4. Synthesis of 8-(6 -umbelliferyl)-apigenin 2 and its methyl ether 15.
00
microscopy. The syntheses of other 8-(6 -umbelliferyl)-apigenin
derivatives and analogues and other biological evaluations for
these compounds are ongoing in our laboratory.
acidified with 2.5 N H
give the mixture of 12 and 13. Then diphenyl ether (8 mL) was
added and the mixture was heated under N
2
SO
4
. The mixture was filtered and dried to
ꢀ
2
at 190 C for 4 h. After
cooling, the mixture purified by column chromatography on silica
gel (Hexane: EA ¼ 9: 1) to afford the desired product 13 (600 mg,
5
. Experimental section
1
6
9% for two steps) as a white solid. H NMR (400 MHz, CDCl
3
)
d
7.88
(
3
s, 1H), 7.58 (d, J ¼ 12.0 Hz, 1H), 6.77 (s, 1H), 6.27 (d, J ¼ 12.0 Hz, 1H),
5.1. Chemistry
.95 (s, 3H).
All commercial materials and reagents were used without
further purification, unless otherwise stated. All solvents were
distilled prior to use. The solvents for reaction were distilled to
5.1.3. 7-methoxy-6-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-
yl)-coumarin (4)
remove water over Na or CaH
2
. All reactions were carried out in
Compound 13 (4.0 g, 13.24 mmol, 1.0 equiv), bis(pinacolato)
oven-dried glassware under an inert atmosphere (nitrogen or
diboron (6.73 g, 26.48 mmol, 2.0 equiv), PdCl
2
(dppf) (969 mg,
argon). For chromatography, 200e300 mesh silica gel (Qingdao,
1.32 mmol, 0.1 equiv) and Cs CO (8.63 g, 26.48 mmol, 2.0 equiv)
2
3
1
13
China) was employed. H and C NMR spectra were recorded at
00 MHz and 100 MHz with a Brucker ARX 400 spectrometer. The
were dissolved in DMSO (60 mL) under argon and the resulting
ꢀ
4
suspension was heated to 80 C with stirring for 24 h. After cooled
1
chemical shifts (
million (ppm) referenced to the residual proton signal of the
deuterated solvent (CDCl at at
¼ 7.26 ppm and DMSO-d
¼ 2.50 ppm); coupling constants were expressed in hertz (Hz).
NMR spectra were referenced to the carbon signal of CDCl
d
) for H NMR spectra were given in parts per
to room temperature, the reaction mixture was poured into ice-
water (200 mL) and extracted with EtOAc (3 ꢁ 50 mL). The com-
bined organic layers were washed with brine (3 ꢁ 200 mL), dried
3
d
6
13
d
C
2 4
over Na SO and concentrated under reduced pressure. The crude
3
product was purified by column chromatography on silica gel
(
d
¼ 77.0 ppm) and DMSO-d
6
(
d
¼ 40.0 ppm). The following ab-
(petroleum ether/ethyl acetate 10:1) to afford compound 4 (2.92 g,
1
breviations are used to describe NMR signals: s ¼ singlet,
d ¼ doublet, t ¼ triplet, m ¼ multiple, and dd ¼ doublet of doublets.
HRMS were recorded on Bruker Daltonics, Inc. APEXIII 7.0 TESLA
FTMS (ESI). Known products were characterized by comparing with
their corresponding H NMR and
literature.
9.67 mmol, 73%) as a white solid. H NMR (400 MHz, CDCl
3
)
d
7.81
(s, 1H), 7.64 (d, J ¼ 8.0 Hz, 1H), 6.76 (s, 1H), 6.23 (d, J ¼ 8.0 Hz, 1H),
13
3
3.90 (s, 3H), 1.37 (s, 12H); C NMR (100 MHz, CDCl ) d 167.3, 161.1,
158.1, 143.5, 137.0, 112.9, 112.1, 98.6, 83.9, 56.2, 24.8; LRMS (ESI) m/z
1
13
þ
[MþH]þ
C NMR reported in the
302 [MþH] ; HRMS (ESI)m/z: Calcd for
16 5
C H20BO
302.1434; found, 302.1435.
5
.1.1. 7-Methoxycoumarin (10)
5.1.4. 1-(2-hydroxy-4,6-diisopropoxyphenyl)ethanone (7)
CO (9.86 g, 71.37 mmol) and (CH CHI (12.13 g, 71.37 mmol)
K
2
CO (2.0 g, 14.5 mmol) and MeI (3.42 g, 24.1 mmol) were
3
K
2
3
3 2
)
added to a solution of 7-hydroxyl coumarin (6) (2.0 g, 12.3 mmol) in
acetone (100 mL) and the mixture was reacted for 5 h. After
filtration and dilution with EtOAc, the resulting solution was
were added to a stirred solution of 5 (5.00 g, 29.74 mmol) in dry
DMF (10 mL). After the addition, the mixture was heated to 60 C
ꢀ
and stirred for 10 h. The reaction was cooled to room temperature,
filtered and diluted with ethyl acetate (100 mL) and the resulting
solution was poured into aqueous HCl (1 M, 100 mL). The organic
layer was separated, washed with brine (3 ꢁ 100 mL) and dried
2 4
washed with brine and dried over anhydrous Na SO . The solvent
was removed to afford compound 10 (2.1 g, 97%) as a crystalline
1
solid. H NMR (400 MHz, CDCl
3
)
d
7.95 (d, J ¼ 16.1 Hz, 1H), 7.40 (d,
J ¼ 8.7 Hz, 1H), 6.53e6.48 (m, 2H), 6.38 (s, 1H), 3.81 (s, 3H).
2 4
over anhydrous Na SO . After concentrated to dryness under
reduced pressure, the crude product was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 100:1)
to afford the known compound 7 (6.90 g, 92%) as a yellow oil.
5.1.2. 6-Iodo-7-methoxycoumarin (13)
A solution of freshly prepared MeONa [made from Na (1.0 g) and
dried methanol (25 mL)] were added to a suspension of 10 (1.0 g,
.7 mmol) in dry methanol (15 mL) under N , and the mixture was
refluxed for 4.5 h. After cooling and neutralization with 2 N HCl, the
mixture was filtered and dried to give 11 (1.1 g, 93%) as a white
5
2
5.1.5. 5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4H-chromen-4-
one (9)
4-isopropoxybenzaldehyde (1.65 g, 9.99 mmol) and compound
7 (2.52 g, 9.99 mmol) were added to ethanol (3 mL) with stirring for
5 min. Then a solution of 50% KOH (aq.) (2.80 g, 49.94 mmol) was
solid. NH
4
OH (12.5 mL 25% aq) was added to a solution of 11 (0.6 g,
2
3
.9 mmol) in dioxane (5 mL). Then a solution of iodine (0.79 g,
ꢀ
.1 mmol) in 25 mL of aqueous KI (5% w/v) was added dropwised
added to the reaction mixture and heated to 60 C with stirring for
ꢀ
with stirring at 0 C. After stirring for 1 h, the mixture was slightly
6 h. After cooled to room temperature, the mixture was poured into

678
G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
00
Scheme 5. Synthesis of 8-(6 -umbelliferyl)-apigenin analogues 29e34.
ice water and acidized with concentrated hydrochloric acid to
pH ¼ 7.0. Then the suspension was extracted with DCM (3 ꢁ 50 mL),
the combined organic layers were washed with brine (100 mL),
dried over Na SO and concentrated under reduced pressure. The
2 4
crude products was purified by column chromatography on silica
gel (petroleum ether/ethyl acetate 100:1) to afford intermediate 8
afford compound 9 (3.45 g, 87%) as a yellow solid. 1H NMR
(400 MHz, CDCl
3
)
d
7.79 (d, J ¼ 8.8 Hz, 2H), 6.97 (d, J ¼ 8.8 Hz, 2H),
6.52 (d, J ¼ 2.4 Hz, 1H), 6.50 (s, 1H), 6.35 (d, J ¼ 2.4 Hz, 1H),
4.69e4.55 (m, 3H), 1.45 (d, J ¼ 6.1 Hz, 6H), 1.40 (d, J ¼ 6.1 Hz, 6H),
1
3
1.38 (d, J ¼ 6.1 Hz, 6H); C NMR (100 MHz, CDCl
3
) d 177.4, 162.0,
160.5, 160.4, 159.9, 159.4, 127.6, 123.7, 115.8, 110.1, 107.6, 100.8, 94.5,
72.5, 70.5, 70.1, 22.0, 21.9; LRMS (ESI) m/z 397 [MþH] .
þ
(
3.58 g, 90%) as a yellow oil.
8
(3.98 g, 9.99 mmol) and I2 (254 mg, 1.0 mmol) were added to a
ꢀ
flask with DMSO (5 mL). The reaction mixture was stirred at 130 C
for 7 h. After completion of the reaction, the mixture was extracted
with DCM (3 ꢁ 50 mL), the combined organic layers were washed
5.1.6. 5,7-diisopropoxy-2-phenyl-4H-chromen-4-one (20)
2 3 3 2
K CO (3.45 g, 24.98 mmol) and (CH ) CHI (4.25 g, 24.98 mmol)
were added to a stirred solution of 17 (2.54 g, 9.99 mmol) in dry
ꢀ
with brine (100 mL), dried over Na
2
SO
4
and concentrated under
DMF (10 mL). After the addition, the mixture was heated to 45 C
reduced pressure. The crude products was purified by column
and stirred for 30 h. The reaction was cooled to room temperature,
chromatography on silica (petroleum ether/ethyl acetate 40:1) to
filtered and diluted with ethyl acetate (100 mL) and the resulting

G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
679
Fig. 2. The structures of apigenin, chrysin, quercetin, luteolin, metformin and 7-hydroxyl coumarin.
Fig. 3. Apigenin (16), chrysin (17), quercetin (18), luteolin (19) metformin, 7-hydroxyl coumarin (6), natural product (2) and its analogues promoted glucose consumption in
adipocytes under basal condition. Data are mean ± SEM (n ¼ 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Blank.
solution was poured into aqueous HCl (1 M, 100 mL). The organic
compound 18 (3.02 g, 9.99 mmol) as substrate to give known
compound 21 (4.0 g, 78%) as a pale yellow solid.
layer was separated, washed with brine (3 ꢁ 100 mL) and dried
2 4
over anhydrous Na SO . After concentrated to dryness under
reduced pressure, the crude product was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 100:1)
to afford the known compound 20 (3.08 g, 91%) as a pale yellow
solid.
5
4
.1.8. 2-(3,4-diisopropoxyphenyl)-5,7-diisopropoxy-4H-chromen-
-one (22)
The procedure is the same as the preparation of 20 by using
compound 19 (2.86 g, 9.99 mmol) as substrate to give compound 22
1
(2.91 g, 64%). H NMR (400 MHz, CDCl
3
)
d
7.47 (dd, J ¼ 8.4, 2.4 Hz,
5.1.7. 2-(3,4-diisopropoxyphenyl)-3,5,7-triisopropoxy-4H-
1H), 7.41 (d, J ¼ 2.0 Hz, 1H), 6.98 (d, J ¼ 8.4 Hz, 1H), 6.53 (d,
J ¼ 2.4 Hz, 1H), 6.50 (s, 1H), 6.36 (d, J ¼ 2.4 Hz, 1H), 4.68e4.51 (m,
4H),1.46 (d, J ¼ 6.4 Hz, 6H),1.41 (d, J ¼ 6.0 Hz, 6H),1.38 (d, J ¼ 6.0 Hz,
chromen-4-one (21)
The procedure is the same as the preparation of 20 by using

680
G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
compound 20 (3.38 g, 1.0 mmol) as substrate to give compound 23
1
(
3
4.22 g, 91%). H NMR (400 MHz, CDCl ) d 8.02e7.99 (m, 2H),
7
4
.53e7.51 (m, 3H), 6.66 (s, 1H), 6.50 (s, 1H), 4.75e4.66 (m, 1H),
13
.64e4.55 (m,1H),1.46 (dd, J ¼ 6.0,1.4 Hz,12H); C NMR (101 MHz,
3
CDCl ) d
177.1,160.7,158.8,158.4,155.6,131.4,131.3,129.0,126.2,111.
4
, 108.3, 99.9, 93.4, 73.6, 72.6, 22.0, 21. 9; LRMS (ESI) m/z 465
þ
I [MþH]þ 465.0557;
[MþH] ; HRMS (ESI)m/z: Calcd for C21
21 4
H O
found, 465.0568.
5.1.11. 8-iodo-2-(3,4-diisopropoxyphenyl)-3,5,7-triisopropoxy-4H-
chromen-4-one (24)
The procedure is the same as the preparation of 3 by using
compound 21 (5.13 g, 10.0 mmol) as substrate to give compound 24
(6.25 g, 98%).
5.1.12. 8-iodo-2-(3,4-diisopropoxyphenyl)-5,7-diisopropoxy-4H-
chromen-4-one (25)
The procedure is same as the preparation of 3 by using com-
pound 22 (4.54 g, 10.0 mmol) as substrate to give compound 25
1
Fig. 4. Apigenin (16), chrysin (17), quercetin (18), luteolin (19), metformin and ana-
logues bearing alkyl protection groups promoted glucose consumption in adipocytes
under basal conditions.
(4.81 g, 83%) as a yellow solid. H NMR (400 MHz, CDCl
3
)
d
7.69 (d,
J ¼ 2.0 Hz, 1H), 7.62 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H),
6
.58 (s, 1H), 6.45 (s, 1H), 4.71 (m, 1H), 4.66e4.54 (m, 3H), 1.47 (d,
1
3
J ¼ 6.0 Hz, 12H), 1.41 (d, J ¼ 6.0 Hz, 6H), 1.39 (d, J ¼ 6. Hz, 6H);
C
3
NMR (100 MHz, CDCl ) d 177.1, 161.0, 160.0, 157.9, 152.1, 149.0, 124.1,
6
H), 1.38 (d, J ¼ 6.0 Hz, 6H).
1
2
20.5, 116.7, 115.7, 111.4, 107.0, 99.2, 73.6, 72.6, 72.5, 72.0, 22.3, 22.1,
þ
2.1, 21.9; LRMS (ESI) m/z 581 [MþH] ; HRMS (ESI)m/z: Calcd for
5.1.9. 8-iodo-5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4H-
I [MþNa]þ 603.1214; found, 603.1247.
C H O
27 33 6
chromen-4-one(3)
NIS (2.47 g, 10.99 mmol) was added to a solution of 9 (3.97 g,
0
0
5
.1.13. 5 ,7’-diisopropoxy-7-methoxy-2’-phenyl-2H,4 H-[6,8’-
1
0 mmol) in dry DMF (25 mL) and the resulting solution was stirred
bichromene]-2,4’-dione (26)
Compound 23 (464 mg, 1.0 mmol), 4 (393 mg, 1.3 mmol), tet-
rakis(triphenylphosphine)palladium (116 mg, 0.1 mmol) and
ꢀ
at 70 C for 10 h. The reaction mixture was then poured into water
ꢀ
(
200 mL) at 0 C and extracted with DCM (3 ꢁ 50 mL), the com-
bined organic layers were washed with brine (3 ꢁ 100 mL), dried
Cs
2
CO
3
(652 mg, 2.0 mmol) were dissolved in dioxane (5 mL) under
over Na SO and concentrated under reduced pressure. The crude
2
4
ꢀ
argon and the resulting suspension was heated to 100 C with
stirring for 12 h. After cooled to room temperature, the reaction
mixture was poured into ice-water (50 mL) and extracted with
EtOAc (3 ꢁ 20 mL). The combined organic layers were washed with
products was purified by column chromatography on silica (pe-
troleum ether/ethyl acetate 30:1) to afford compound 3 (4.49 g,
1
8
6%) as a yellow solid. H NMR (400 MHz, CDCl
3
)
d
7.97 (d,
J ¼ 8.4 Hz, 2H), 6.97 (d, J ¼ 8.4 Hz, 2H), 6.54 (s, 1H), 6.43 (S, 1H),
brine (3 ꢁ 50 mL), dried over anhydrous Na
2 4
SO and concentrated
4
.71e4.55 (m, 3H), 1.44 (d, J ¼ 6.0 Hz, 12H), 1.36 (d, J ¼ 6.0 Hz, 6H);
under reduced pressure. The crude product was purified by column
1
3
C NMR (100 MHz, CDCl
3
) d 177.1, 161.1, 161.0, 160.7, 159.9, 157.9,
chromatography on silica (petroleum ether/ethyl acetate 20:1 to
1
28.2, 123.2, 115.9, 111.5, 106.7, 99.3, 73.6, 72.6, 70.1, 22.1, 22.0, 21.9;
1
0:1) to get crude product, then washed with PE:EA ¼ 1:1 to afford
þ
LRMS (ESI) m/z 523 [MþH] ; HRMS (ESI)m/z: Calcd for C24
H
27
O
5
I
1
a new compound 26 (328 mg, 0.64 mmol, 64%) as a yellow solid. H
NMR (400 MHz, CDCl
7.68 (d, J ¼ 9.5 Hz, 1H), 7.44e7.37 (m, 4H),
.32 (m, 2H), 6.97 (s, 1H), 6.62 (s, 1H), 6.53 (s, 1H), 6.32 (d, J ¼ 9.4 Hz,
1H), 4.63 (m, 2H), 3.77 (s, 3H), 1.51 (d, J ¼ 6.1 Hz, 6H), 1.28 (d,
þ
[
MþNa] 545.0795; found, 545.0820.
3
) d
7
5
.1.10. 8-iodo-5,7-diisopropoxy-2-phenyl-4H-chromen-4-one (23)
The procedure is the same as the preparation of 3 by using
13
J ¼ 6.0 Hz, 3H), 1.24 (d, J ¼ 6.0 Hz, 3H); C NMR (100 MHz, CDCl
3
)
Fig. 5. Metformin as well as the best four compounds 2, 15, 30 and 31 regulated 2-NBDG uptake in 3T3-L1 adipocytes under basal condition.

G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
681
Table 1
Suzuki coupling reaction of compound 3 under different conditions.
a
Entry
Solvent
Catalyst
Base
Yieldb (%)
1
2
3
4
DMF
Pd(PPh
Pd(PPh
Pd(PPh
3
3
3
)
)
)
4
4
4
2
Cs CO
2
Cs CO
2
Cs CO
2
Cs CO
3
3
3
3
14
16
70
11
DMSO
Dioxane
Dioxane
2
PdCl (dppf)
a
ꢀ
Reaction conditions: 3 (1.0 mmol), 4 (1.2 mmol), catalyst (0.1 mmol) and base (2.0 mmol) in solvent (3 mL) heated to 100 C for 12 h.
Isolated yield after purification by silica gel chromatography.
b
d
177.6, 161.3, 161.0, 160.0, 159.6, 159.2, 156.6, 155.7, 143.5, 131.5,
31.5, 131.1, 128.9, 125.5, 119.5, 113.2, 112.1, 110.4, 108.2, 107.9, 99.0,
(400 MHz, CDCl
3
)
d
7.68 (d, J ¼ 9.5 Hz, 1H), 7.40 (s, 1H), 7.08 (dd,
1
9
J ¼ 8.5, 1.8 Hz, 1H), 6.95 (s, 1H), 6.86 (d, J ¼ 1.7 Hz, 1H), 6.81 (d,
J ¼ 8.4 Hz, 1H), 6.50 (d, J ¼ 4.0 Hz, 2H), 6.30 (d, J ¼ 9.6 Hz, 1H),
4.66e4.46 (m, 3H), 4.11e4.02 (m, 1H), 3.77 (s, 3H), 1.49 (d,
J ¼ 6.0 Hz, 6H), 1.30 (d, J ¼ 6.0 Hz, 6H), 1.24 (dd, J ¼ 13.6, 6.0 Hz, 6H),
8.9, 73.3, 71.4, 56.0, 22.1, 22.1, 22.0, 21.9; LRMS (ESI) m/z 513
þ
[MþNa]þ 535.1727;
[
MþH] ; HRMS (ESI)m/z: Calcd for C31
28 7
H O
found, 535.1742.
13
1
.16 (d, J ¼ 6.0 Hz, 3H), 1.13 (d, J ¼ 6.0 Hz, 3H); C NMR (100 MHz,
0
5
2
.1.14. 5 ,7’-diisopropoxy-2’-(4-isopropoxyphenyl)-7-methoxy-
3
CDCl ) d 177.6, 161.2, 161.1, 160.1, 159.4, 159.1, 156.5, 155.6, 152.0,
0
H,4 H-[6,8’-bichromene]-2,4’-dione (14)
148.6, 143.6, 131.4, 124.1, 119.8, 119.7, 116.3, 115.1, 113.1, 112.0, 110.3,
107.7, 106.8, 98.9, 98.8, 73.2, 72.3, 71.8, 71.3, 56.1, 22.1, 22.1, 22.0,
The procedure is same as the preparation of 26 by using com-
þ
pound 3 (522 mg, 1.0 mmol) as substrate to give compound 14
21.9, 21.9; LRMS (ESI) m/z 629 [MþH] ; HRMS (ESI)m/z: Calcd for
1
[MþNa]þ 651.2565; found, 651.2592.
(
410 mg, 0.72 mmol, 72%) as a yellow solid. H NMR (400 MHz,
CDCl
7.68 (d, J ¼ 9.6 Hz, 1H), 7.41 (s, 1H), 7.33 (d, J ¼ 8.8 Hz, 2H),
.97 (s, 1H), 6.79 (d, J ¼ 8.8 Hz, 2H), 6.52 (d, J ¼ 7.2 Hz, 2H), 6.32 (d,
J ¼ 9.6 Hz, 1H), 4.68e4.54 (m, 3H), 3.77 (s, 3H), 1.50 (d, J ¼ 6.0 Hz,
H), 1.32 (dd, J ¼ 5.9, 2.5 Hz, 6H), 1.28 (d, J ¼ 6.0 Hz, 3H), 1.24 (d,
C
37
40
H O
9
3
) d
0
0
6
5.1.17. 5 ,7’-dihydroxy-7-methoxy-2’-phenyl-2H,4 H-[6,8’-
bichromene]-2,4’-dione (29)
6
Boron tri-chloride (0.7 mL, 0.7 mmol, 1 M solution in hexane)
was added to a stirred solution of 26 (51 mg, 0.1 mmol) in anhy-
drous DCM (5 mL) under 0 C for 30 min. After addition, the reac-
13
J ¼ 6.0 Hz, 3H); C NMR (100 MHz, CDCl
3
) d 177.6, 161.4, 161.0,
ꢀ
1
60.4, 160.2, 159.4, 159.1, 156.5, 155.6, 143.5, 131.5, 127.2, 123.2,
1
5
19.6,115.8,113.1,112.1,110.3,107.8,106.6, 99.0, 98.9, 73.3, 71.3, 70.1,
tion mixture was heated to room temperature for 4 h. Then excess
ice-water was added to the reaction mixture with stirring for
10 min. Then the suspension was filtrated and the residue was
þ
6.0, 22.1, 22.1, 22.0, 21.9; LRMS (ESI) m/z 571 [MþH] ; HRMS (ESI)
m/z: Calcd for C34
H
34
O
8
[Mþ Na]þ 593.2146; found, 593.2177.
recrystallized in methanol to afford compound 29 (41 mg, 96%) as a
0
0
1
5.1.15. 2’-(3,4-diisopropoxyphenyl)-3 ,5 ,7’-triisopropoxy-7-
yellow solid. H NMR (400 MHz, DMSO-d
6
)
d
12.97 (s, 1H), 10.93 (s,
0
methoxy-2H,4 H-[6,8’-bichromene]-2,4’-dione (27)
The procedure is same as the preparation of 26 by using com-
pound 24 (638 mg, 1.0 mmol) as substrate to afford a new com-
1H), 8.06 (d, J ¼ 9.6 Hz, 1H), 7.70 (s, 1H), 7.66 (d, J ¼ 7.3 Hz, 2H), 7.53
(d, J ¼ 6.6 Hz, 1H), 7.47 (d, J ¼ 7.0 Hz, 2H), 7.27 (s, 1H), 7.04 (s, 1H),
13
6.45 (s, 1H), 6.37 (d, J ¼ 9.1 Hz, 1H), 3.80 (s, 3H); C NMR (100 MHz,
1
pound 27 (473 mg, 0.69 mmol, 69%) as a light brown solid. H NMR
6
DMSO-d ) d 182.64, 163.4, 162.5, 161.1, 161.1, 160.8, 155.8, 155.0,
(
(
6
400 MHz, CDCl
3
)
d
7.67 (d, J ¼ 12.0 Hz,1H), 7.48 (d, J ¼ 8.0, 1H), 7.39
144.7, 132.5, 131.1, 129.6, 126.4, 118.3, 113.2, 112.4, 105.4, 104.3, 103.7,
þ
s, 1H), 7.30 (s, 1H), 6.93 (s, 1H), 6.75 (d, J ¼ 8.0 Hz, 1H), 6.49 (s, 1H),
99.8, 99.2, 56.8; LRMS (ESI) m/z 429 [MþH] ; HRMS (ESI)m/z: Calcd
for C25H
16
O
7
[MꢂH]ꢂ 427.0823; found, 427.0836.
.28 (d, J ¼ 8.0 Hz, 1H), 4.80e4.63 (m, 1H), 4.58e4.49 (m, 3H),
.13e4.09 (m, 1H), 3.75 (s, 3H), 1.49 (d, J ¼ 8.0 Hz, 6H), 1.28 (dd,
4
13
0
0
J ¼ 8.0 Hz, J ¼ 28.0 Hz, 6H), 1.21e1.15 (m, 18H); C NMR (100 MHz,
5.1.18. 5 ,7’-dihydroxy-2’-(4-hydroxyphenyl)-7-methoxy-2H,4 H-
[6,8’-bichromene]-2,4’-dione (15)
CDCl
1
1
2
3
) d 174.5, 161.3, 161.1, 159.3, 159.2, 155.6, 155.5, 152.7, 151.0,
47.8, 143.6, 138.4, 131.5, 124.2, 123.0, 119.7, 118.4, 115.2, 113.0, 112.1,
10.3, 107.3, 99.0, 98.2, 73.9, 73.1, 72.5, 71.4, 71.3, 56.0, 22.4, 22.4,
2.2, 22.1, 22.1, 22.0, 21.9; LRMS (ESI) m/z 687 [MþH] ; HRMS (ESI)
The procedure is same as the preparation of 29 by using 14
(57 mg, 0.1 mmol) as substrate, Boron tri-chloride (0.8 mL,
þ
0.8 mmol, 1 M solution in hexane) to afford compound 15 (43 mg,
þ
1
m/z: Calcd for C40
H
47
O
10 [MþH] 687.3161; found, 687.3164.
97%) as a yellow solid. H NMR (400 MHz, DMSO-d )
d
13.10 (s, 1H),
6
1
0.84 (s, 1H), 10.34 (s, 1H), 8.05 (d, J ¼ 9.6 Hz, 1H), 7.67 (s, 1H), 7.49
0
5
2
.1.16. 2’-(3,4-diisopropoxyphenyl)-5 ,7’-diisopropoxy-7-methoxy-
(d, J ¼ 8.8 Hz, 2H), 7.27 (s, 1H), 6.82e6.78 (m, 3H), 6.41 (s, 1H), 6.37
0
13
H,4 H-[6,8’-bichromene]-2,4’-dione (28)
(d, J ¼ 9.2 Hz, 1H), 3.79 (s, 3H); C NMR (100 MHz, DMSO-d
6
)
The procedure is same as the preparation of 26 by using com-
d 182.5, 164.0, 162.2, 161.6, 161.1, 161.1, 160.9, 155.7, 154.9, 144.7,
pound 25 (580 mg, 1.0 mmol) as substrate to afford a new com-
pound 28 (446 mg, 0.71 mmol, 71%) as a yellow solid. H NMR
132.5, 128.5, 121.6, 118.4, 116.4, 113.1, 112.4, 104.0, 103.5, 103.0, 99.7,
99.0, 56.8; LRMS (ESI) m/z 445 [MþH] ; HRMS (ESI)m/z: Calcd for
1
þ

682
G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
C
25
H
17
O
8
[MꢂH]ꢂ 443.0772; found, 448.0810.
(69 mg, 0.1 mmol) as substrate, Tribromoborane (250 mg,
1
1.0 mmol) to afford compound 33 (43 mg, 93%). H NMR (400 MHz,
0
0
5.1.19. 2’-(3,4-dihydroxyphenyl)-3 ,5 ,7’-trihydroxy-7-methoxy-
DMSO-d 12.63 (s, 1H), 11.46 (s, 1H), 9.52 (s, 1H), 9.30 (s, 1H), 9.04
6
) d
0
2H,4 H-[6,8’-bichromene]-2,4’-dione (30)
(s, 1H), 7.94 (d, J ¼ 8.0 Hz, 1H), 7.65 (s, 1H), 7.52 (s, 1H), 7.16 (d,
The procedure is same as the preparation of 29 by using 27
J ¼ 8.0 Hz, 1H), 6.82 (s, 1H), 6.69 (d, J ¼ 8.0 Hz, 1H), 6.26 (s, 1H), 6.19
1
3
(
1
69 mg, 0.1 mmol) as substrate, Boron tri-chloride (1.0 mL,
.0 mmol, 1 M solution in hexane) to afford compound 30 (47 mg,
(d, J ¼ 8.0 Hz, 1H); C NMR (100 MHz, DMSO-d
6
) d 176.3, 162.3,
161.2, 160.1,155.3,154.0,147.9,146.8,145.3,145.1, 135.9,132.7, 122.8,
1
9
1
1
8%) as a yellow solid. H NMR (400 MHz, DMSO-d
0.76 (s, 1H), 9.62 (s,1H), 9.42 (s, 1H), 9.06 (s,1H), 8.04 (d, J ¼ 8.0 Hz,
H), 7.64 (s,1H), 7.39 (s,1H), 7.23 (s,1H), 7.02 (d, J ¼ 8.0 Hz,1H), 6.70
d, J ¼ 8.0 Hz, 1H), 6.39 (s, 1H), 6.35 (d, J ¼ 12.0 Hz, 1H), 3.77 (s, 3H);
6
)
d
12.61 (s, 1H),
119.9, 118.9, 116.0, 115.8, 111.1, 110.8, 104.5, 103.3, 102.8, 99.6; LRMS
þ
10 [MþH]þ
(ESI) m/z 463 [MþH] ; HRMS (ESI)m/z: Calcd for C24
15
H O
463.0660; found, 463.0668.
(
1
3
0
0
C NMR (100 MHz, DMSO-d
6
)
d184.5, 176.5, 161.8, 161.2, 160.9,
5.1.24. 2’-(3,4-dihydroxyphenyl)-5 ,7,7’-trihydroxy-2H,4 H-[6,8’-
bichromene]-2,4’-dione (34)
160.3, 155.7, 153.8,148.1, 147.3, 145.4,144.8, 136.1, 132.5, 122.5,119.7,
1
18.5,115.8,113.0,112.4,103.4,103.1, 99.7, 98.4, 56.7; LRMS (ESI) m/z
The procedure is same as the preparation of 32 by using 28
(63 mg, 0.1 mmol) as substrate, Tribromoborane (225 mg,
0.9 mmol) to afford compound 34 (44 mg, 98%). H NMR (400 MHz,
þ
10 [MþH]þ
477.0 [MþH] ; HRMS (ESI)m/z: Calcd for C25
16
H O
1
477.0816; found, 477.0808.
DMSO-d
6
)
d
7.98 (d, J ¼ 9.6 Hz, 1H), 7.58 (s, 1H), 7.09 (d, J ¼ 8.0 Hz,
0
5
2
.1.20. 2’-(3,4-dihydroxyphenyl)-5 ,7’-dihydroxy-7-methoxy-
1H), 7.04 (s, 1H), 6.98 (s, 1H), 6.78 (d, J ¼ 8.0 Hz, 1H), 6.69 (s, 1H),
0
13
H,4 H-[6,8’-bichromene]-2,4’-dione (31)
The procedure is same as the preparation of 29 by using 28
6.42 (s, 1H), 6.26 (d, J ¼ 9.2 Hz, 1H). C NMR (100 MHz, DMSO-d
6
)
d
182.5, 164.4, 162.2, 161.0, 160.0, 155.3, 155.1, 150.1, 146.1, 145.0,
(
0
9
63 mg, 0.1 mmol) as substrate, Boron tri-chloride (0.9 mL,
132.9, 122.2, 119.2, 117.4, 116.2, 114.1, 112.0, 111.7, 104.1, 103.8, 103.0,
þ
.9 mmol, 1 M solution in hexane) to afford compound 31 (44 mg,
102.6, 99.0; LRMS (ESI) m/z 447 [MþH] ; HRMS (ESI)m/z: Calcd for
1
[MꢂH]ꢂ 445.0565; found, 445.0596.
5%). H NMR (400 MHz, DMSO-d
6
)
d
13.13 (s,1H),10.83 (s,1H), 9.99
24 14 9
C H O
(
7
s, 1H), 9.20 (s, 1H), 8.05 (d, J ¼ 8.8 Hz, 1H), 7.66 (s, 1H), 7.25 (s, 1H),
.02 (s, 1H), 6.96 (s, 1H), 6.77 (d, J ¼ 8.0 Hz, 1H), 6.70 (s, 1H), 6.41 (s,
5.2. Biology evaluation
13
1
d
1
1
H), 6.36 (d, J ¼ 8.8 Hz,1H), 3.80 (s, 3H); C NMR (100 MHz, DMSO-
182.4, 164.3, 162.1, 161.1, 161.1, 160.9, 155.8, 154.9, 150.2, 146.1,
44.8, 132.4, 122.0, 119.0, 118.4, 116.3, 113.9, 113.1, 112.4, 104.1, 103.5,
6
)
d
5.2.1. Cell culture and differentiation
3T3-L1 cells (a cell line of preadipocyte from ATCC) were
cultured in Dulbecco’s Minimum Essential Medium (DMEM, Gibco)
þ
03.1, 99.8, 99.0, 56.7; LRMS (ESI) m/z 461 [MþH] ; HRMS (ESI)m/z:
Calcd for C25
H
16
O
9
[MꢂH]ꢂ 459.0722; found, 459.0751.
supplemented with 10% fetal bovine serum (FBS), 100
streptomycin and 100 U/mL of penicillin at 37 C in a 5% CO
m
g/mL of
ꢀ
2
at-
0
0
5
.1.21. 5 ,7,7’-trihydroxy-2’-phenyl-2H,4 H-[6,8’-bichromene]-2,4’-
mosphere. Two days after full confluence, cells were differentiated
dione (32)
via incubation in DMEM containing 0.5 mM isobutylmethylx-
Tribromoborane (175 mg, 0.7 mmol, 7.0 equiv) was added to a
anthine (IBMX), 1
mM dexamethasone (Dex), 10
mg/mL insulin for
stirred solution of 26 (51 mg, 0.1 mmol) in anhydrous DCM (5 mL)
48 h, and then for 2 days in DMEM (10% FBS) containing 10
insulin alone [13]. Adipocytes were used 8e10 days after the
initiation of differentiation.
m
g/mL
ꢀ
under ꢂ78 C for 30 min. After addition, the reaction mixture was
slowly heated to room temperature for 12 h. Then the reaction was
ꢀ
cooled to 0 C, excess ice-water was added with stirring for 10 min.
DCM was removed under reduced pressure, then the suspension
5.2.2. Glucose consumption
5
was filtrated and the residue was dried to afford the product 32 as a
Serum-starved adipocytes (2 ꢁ 10 cells per well) were cultured
1
yellow solid (40 mg, 97%). H NMR (400 MHz, DMSO-d
6
)
d
12.97 (s,
in 48-well plates for 4 h in KRH (KrebseRinger phosphateeHEPES
1
H), 10.89 (s, 1H), 10.63 (s, 1H), 8.00 (d, J ¼ 9.5 Hz, 1H), 7.73 (d,
buffer, containing 118 mmol/L NaCl, 5 mmol/L KCl, 1.3 mmol/L
J ¼ 7.4 Hz, 2H), 7.62 (s,1H), 7.54 (d, J ¼ 7.2 Hz,1H) 7.52e7.44 (m, 3H),
CaCl
HEPES, containing 0.5% BSA, pH 7.4) containing 11 mmol/L glucose
and pretreated with 10 mol/L compounds or 1 mmol/L metformin
2 4 2 4
, 1.2 mmol/L MgSO , 1.2 mmol/L KH PO , and 30 mmol/L
13
7
.00 (d, J ¼ 16.0 Hz, 2H), 6.45 (s, 1H), 6.27 (d, J ¼ 9.4 Hz, 1H);
NMR (100 MHz, DMSO-d 182.2, 163.1, 162.1, 160.5, 160.5, 159.5,
54.8, 154.7, 144.5, 132.5, 132.0, 130.8, 129.1, 126.1, 116.8, 111.6, 111.2,
C
6
)
d
m
1
respectively. The glucose concentration in KRH at 0 h and 4 h after
incubation were determined with a commercial kit based on the
glucose oxidase peroxidase (GOD-POD) method and the difference
between the two concentrations was regarded as the amount of
consumed glucose.
þ
1
04.9, 103.9, 103.5, 102.0, 98.7; LRMS (ESI) m/z 415 [MþH] ; HRMS
(
ESI)m/z: Calcd for C24
14
H O
7
[MꢂH]ꢂ 413.0667; found, 413.0677.
0
0
5.1.22. 5 ,7,7’-trihydroxy-2’-(4-hydroxyphenyl)-2H,4 H-[6,8’-
bichromene]-2,4’-dione (2)
The procedure is same as the preparation of 32 by using 14
5.2.3. Detection of 2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)-amino-
(
0
57 mg, 0.1 mmol) as substrate, Tribromoborane (200 mg,
.8 mmol, 8.0 equiv) to afford compound 2 (42 mg, 98%). H NMR
2-deoxy-
microscopy
After 1-h serum starvation, 3T3-L1 cells were incubated with
agents in KRH for 0.5 h, and cultured with 500 mol/L 2-N-7-
(nitrobenz-2-oxa-1,3-diazol-4-yl)-amino-2-deoxy- -glucose (2-
NBDG) for another 1 h. After washed with KRH for 3 times, cells
were observed by inverted fluorescence microscope (Olympus
IX81).
D-glucose uptake by 3T3-L1 cells with fluorescence
1
(
400 MHz, acetone-d
6
)
d
13.18 (s,1H), 9.47 (s, 3H), 7.94 (d, J ¼ 9.5 Hz,
1
H), 7.64 (S, 1H), 7.64 (d, J ¼ 8.8 Hz, 2H), 6.99 (s, 1H), 6.88 (d,
m
1
3
J ¼ 8.8 Hz, 2H), 6.67 (s, 1H), 6.43 (s, 1H), 6.24 (d, J ¼ 9.5 Hz, 1H);
C
D
NMR (100 MHz, DMSO-d
6
)
d
182.1, 163.7, 161.8, 161.1, 160.6, 159.6,
1
1
54.9, 154.6, 144.5, 132.5, 128.2, 121.3, 117.0, 115.9, 111.7, 111.3, 103.7,
þ
03.4, 102.7, 102.1, 98.6; LRMS (ESI) m/z 431 [MþH] ; HRMS (ESI)m/
z: Calcd for C24
14
H O
8
[MꢂH]ꢂ 429.0616; found, 429.0646.
5.2.4. Statistics
0
0
0
5
[
.1.23. 2’-(3,4-dihydroxyphenyl)-3 ,5 ,7,7’-tetrahydroxy-2H,4 H-
6,8’-bichromene]-2,4’-dione (33)
The procedure is same as the preparation of 32 by using 27
All data were expressed as mean ± SEM (standard error of the
mean), with n representing the number of independent experi-
ments. GraphPad Prism6.0 was used for statistical analysis and

G. Pan et al. / European Journal of Medicinal Chemistry 122 (2016) 674e683
683
graphs. Mean values were compared by the one-way ANOVA with
Dunnett’s post-hoc test. Values of p < 0.05 were regarded as being
statistically significant.
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3-Phenylcoumarins
versus
3-
21572158), International Science & Technology Cooperation Pro-
(
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Technology Fund Planning Project (20110504) and Youth Innova-
tion Fund of Tianjin University of Science and Technology
3
(
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