Analogues of xanthones——Chalcones and bis-chalcones as α-glucosidase inhibitors and anti-diabetes candidates

Article abstract of DOI:10.1016/j.ejmech.2017.02.007

Two series of compounds (chalcones and bis-chalcones) were designed, synthesized, and evaluated as α-glucosidase inhibitors (AGIs) with 1-deoxynojirimycin as positive control in vitro. Most of the compounds with two or four hydroxyl groups showed better inhibitory activities than 1-deoxynojirimycin towards α-glucosidase with noncompetitive mechanism. Moreover, most of the hydroxy bis-chalcones exhibit good α-glucosidase inhibitory activities in enzyme test. Inspiringly, bis-chalcones 2g (at 1 μM concentration) has stronger effect than 1-deoxynojirimycin on reducing the glucose level in HepG-2 cells (human liver cancer cell line).

Full text of DOI:10.1016/j.ejmech.2017.02.007

Accepted Manuscript
Analogues of xanthones——chalcones and bis-chalcones as α-glucosidase inhibitors
and anti-diabetes candidates
Chao-Yun Cai, Li Rao, Yong Rao, Jin-Xuan Guo, Zhi-Zun Xiao, Jing-Yu Cao, Zhi-Shu
Huang, Bo Wang
PII:
S0223-5234(17)30073-9
10.1016/j.ejmech.2017.02.007
EJMECH 9203
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 October 2016
Revised Date: 11 January 2017
Accepted Date: 4 February 2017
Please cite this article as: C.-Y. Cai, L. Rao, Y. Rao, J.-X. Guo, Z.-Z. Xiao, J.-Y. Cao, Z.-S. Huang,
B. Wang, Analogues of xanthones——chalcones and bis-chalcones as α-glucosidase inhibitors
and anti-diabetes candidates, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.02.007.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Analogues of Xanthones——Chalcones and Bis-chalcones as α-Glucosidase
Inhibitors and anti-Diabetes Candidates
Chao-Yun Cai a, Li Rao a, Yong Rao b, Jin-Xuan Guo a, Zhi-Zun Xiao a, Jing-Yu Cao a, Zhi-Shu
Huang b, Bo Wang a,*
a School of Chemistry, Sun Yat-sen University, 135 Xingang West Road, Guangzhou, 510275, P R
China
b School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510080, P R China
Two series of chalcones and bis-chalcones were synthesized and exhibited α-glucosidase
inhibition via a non-competitive mechanism. Moreover, the compound 2g could significantly
reduce the glucose level in HepG-2 cells.

ACCEPTED MANUSCRIPT
Analogues of Xanthones——Chalcones and Bis-chalcones as
α
-Glucosidase Inhibitors and Anti-Diabetes Candidates
Chao-Yun Cai a, Li Rao a, Yong Rao b, Jin-Xuan Guo a, Zhi-Zun Xiao a, Jing-Yu Cao a,
Zhi-Shu Huang b, Bo Wang a,*
a School of Chemistry, Sun Yat-sen University, 135 Xingang West Road, Guangzhou, 510275, P R
China
b School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510080, P R China
Abstract
Two series of compounds (chalcones and bis-chalcones) were designed,
synthesized, and evaluated as
positive control in vitro. Most of the compounds with two or four hydroxyl groups
showed better inhibitory activities than 1-deoxynojirimycin towards -glucosidase with
noncompetitive mechanism. Moreover, most of the hydroxy bis-chalcones exhibit good
-glucosidase inhibitory activities in enzyme test. Inspiringly, bis-chalcones 2g (at 1 µM
α-glucosidase inhibitors (AGIs) with 1-deoxynojirimycin as
α
α
concentration) has stronger effect than 1-deoxynojirimycin on reducing the glucose level
in HepG-2 cells (human liver cancer cell line).
Keywords：α-glucosidase inhibitors; chalcones; bis-chalcones; noncompetitive.
1. Introduction
Diabetes mellitus (DM), a worldwide public health problem, is a group of
metabolic diseases that feature with high blood sugar levels in patients. Thus, discovery of
hypoglycemic agents with strong potency and weak side effect is highly desirable. Of all
anti-diabetic drugs,
post-prandial hyperglycemia [1]. The well-developed
α
-glucosidase inhibitors seem to be the most effective in reducing
-glucosidase inhibitors (AGIs),
α
such as acarbose, miglitol and voglibose, have been clinically used. However, their
adverse effects (e.g. gastrointestinal symptoms) [2-3] in some cases and newly discovered
additive effects (like stabling carotid plaques, and reducing inflammation) [1] make the
discovery of novel AGIs still attractive and challenging.
Our previous efforts on AGIs led to the discovery of that xanthones are potent
α-glucosidase inhibitors and H-bond, extendπsystem, and flexibility are key factors
influencing the inhibitory activities [4-6]. We reasonably speculated that chalcones, as the
analogues of xanthones (Figure 1), would have better α-glucosidase inhibitory activity
due to the flexibility of the framework of chalcones.
Figure 1 The structures of benzoxanthone, chalcone and bis-chalcone
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Chalcones, also known as
α
-β-unsaturated ketones, display various biological
activities, such as anti-cancer [7], anti-diabetic [8-9], anti-oxidant, anti-malarial,
antimicrobial, anti-HIV, antifilarial [10] and anti-inflammatory effects [11]. Bis-chalcones
also have many biological activities. As is reported, bis-chalcone analogues are the potent
NO production inhibitors and cytotoxic agents against four human cancer cell lines (A549,
DU145, KB, and KB-VIN) [12]. Biphenyl based bis-chalcones have anticancer activity
against human breast cancer MCF-7 and MDA-MB-231 cell lines, HeLa cell line, and
human embryonic kidney (HEK-293) cells [13]. Some bis-chalcones have appreciable
antibacterial and antifungal activities [14]. However, the
activities of bis-chalcones are still unknown. In this article, a series of chalcones and
bis-chalcones (with extend π system) were synthesized and evaluated as -glucosidase
α-glucosidase inhibitory
α
inhibitors, aimed at clarifying the structure-activity relationship and finding suitable
anti-diabetes candidates.
2. Result and Discussion
2.1 Chemistry
2.1.1 Synthesis of chalcones and bis-chalcones
Two series of compounds (chalcones and bis-chalcones), including four new
compounds (2g, 2i, 2j and 2k), were synthesized respectively, the structures of all
compounds were confirmed with IR, 1H NMR, 13C NMR, and HRMS techniques.
Initially, we tried to synthesize compounds 2a-2d using SOCl2/EtOH as catalytic
system according to literature [15]. Unfortunately, the yields were lower than 10%
because substrates contain multi-hydroxyl groups. Therefore, we switched to two steps
methods, that is, synthesizing compounds 1 and then demethylating to generate target
compounds 2. As expected, 1a-1d (with only one hydroxyl group) were provided through
the reaction catalyzed by SOCl2 with moderate yields (30%~57%). 1e and 1f, without
hydroxyl groups, were easily synthesized via Aldol condensation reactions in the presence
of aqueous KOH with good yields (71%~78%). Then compounds 2a-2m were obtained
by the demethylation of 1a-1m in the presence of BBr3/CH2Cl2, respectively.
It should be noted that the demethylation conditions would be substrate-dependent.
For example, when we synthesized 2d, 2f, 2g, 2h, 2j and 2k from 1d, 1f, 1g, 1h, 1j and
1k, some by-products with one or two methoxy groups retaining were obviously observed,
which led to the low yields (15%~31%) of desired 2d, 2f, 2g, 2h, 2j and 2k.
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Scheme 1 Synthesis of the target compounds 1a-1m and 2a-2m
Conceptually, the demethylation proceeds via an adduct 1 formed between BBr3
and methoxyl chalcone followed by the loss of bromide. Then the methoxyl group of the
cationic intermediate 2 is attacked by the free bromide cleaving the C-O bond (Scheme 2)
[16]. Then ArOBBr2 would be hydrolyzed to generate ArOH. Interestingly, for chalcones
with 3, 4-methoxyl groups, a cyclic borate can be formed when the demethylation is
proceeded (Scheme 3) [17].
Scheme 2 Conceptual reaction mechanism for demethylation
Scheme 3 Conceptual reaction mechanism for demethylation of dimethoxyl groups
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2.1.2 Stereochemistry of chalcones and bis-chalcones
Generally, an AB two-spin system overlapping with a typical trans coupling of
3JAB =12~18 Hz reveals a CC double bond with protons in trans configuration (Figure 2)
[18]. Take 1a as example, the AB two-spin system ( δA=7.20 and δB=6.75) with a typical
trans coupling of 3JAB =16.0 Hz reveals the CC double bond of chalcone 1a in trans
configuration. From the NMR spectroscopy of chalcones and bis-chalcones, the methoxyl
chalcones and bis-chalcones 1a-1m are all in trans configuration. The configuration of
hydroxyl chalcones and bis-chalcones 2b, 2f, 2i, 2l and 2m could not be confirmed by 1H
NMR because the spectral peaks are overlapped. Since these five compounds were
synthesized from methoxyl chalcones and bis-chalcones 1b, 1f, 1i, 1l and 1m which are in
trans configuration and the demethylation would not change the configuration of these
compounds. It can be inferred that 2b, 2f, 2i, 2l and 2m are in trans configuration.
Figure 2 The stereochemistry of chalcones
2.2 α-Glucosidase inhibitory activity
As shown in Table 1, nearly all the hydroxyl chalcones and bis-chalcones (21-2m)
showed higher activities than 1-deoxynojirimycin, one of the well-known AGIs.
Compound 2k showed the highest inhibitory activity. The IC50 value was only 1.0 µM. On
the contrary, the chalcones and bis-chalcones containing methoxyl groups (1a-1m)
showed much lower inhibitory activities, and due to the poor solubility, only the
inhibition ratio at 40 µM level were obtained for some compounds. Therefore, it is
reasonable to consider that the H-bond donor effect of the hydroxyl group is the key effect
during the interaction of these compounds with the enzyme.
Table 1 The IC50 value or inhibition of the compounds
Inhibition or
Compounds
Structure
Compounds
Structure
IC50/µM
IC50/µM
1a
>200
2a
71.1±2.6
1b
1c
>200
2b
2c
35.2±0.2
13.2±2.7
58.7±3.0
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1d
1e
1f
>200
83.2±1.0
123.1±9.9
27.2% a
27% a
2d
2e
2f
42.0±6.0
12.5±2.1
15.6±2.8
23.7±0.3
22.5±3.2
10.1±1.7
5.5±1.2
1g
1h
1i
2g
2h
2i
OH
O
OH
HO
O
OH
32.0% a
55.2% a
1j
2j
1k
66.9±2.5
2k
1.0±0.1
1l
16.0% a
29% a
2l
6.5±0.2
1m
2m
18.3±0.7
a : the inhibition of the compounds at the concentration of 40 µM (The solubility of these methoxyl
bis-chalcones in PBS are not good ). The IC50 value of positive control (1-deoxynojirimycin, PG) is
21.3±8.7µM.
The position of the hydroxyl groups in chalcones is another key factor for their
AG inhibitory activities. As shown in Figure 3, in ring A, the 4-hydroxyl group was
stronger than the 3-hydroxyl group in enhancing the inhibitory effect, with the IC50 values
of 2c (13.4±2.7 µM) vs. 2d (42.0±6.0 µM). However, introducing one more hydroxyl
groups to the ring A at the C3 position of 2b (35.2±0.2 µM) led to stronger AGIs 2f
(15.6±2.8 µM). In addition, the 2’-hydroxyl group is more effective than the 3’-hydroxyl
group in ring B to improve the inhibitory activity towards chalcones (2b and 2c).
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Figure 3 The structure of chalcone
The effect of the number of hydroxyl groups on the inhibitory activity of
chalcones was much intriguing. Compound 2a with 2’, 4’, 6’-trihydroxyl groups in ring B,
showed much weaker activity than 2c does, which has 2’, 4’-dihydroxyl groups in ring B;
while 2c with 4-hydroxyl group in ring A, displayed almost the same activity to 2e with
2,4-hydroxyl groups in ring A. On the other hand, bis-chalcones 2j (5.5±1.2 µM) and 2l
(6.5±0.4 µM) showed a much strong inhibitory activity than 2m (18.3±0.7 µM), which
indicated that introducing more hydroxyl groups in bis-chalcones were very important in
discovering stronger AGIs.
Surprisingly, compound 2g and 2h had lower inhibitory activities than compound
2k, probably due to the formation of intramolecular hydrogen bonds in 2g and 2h (Figure
4), which devastates their binding to the enzyme as hydrogen bond donors. Similarly,
exemplified by the fact that, methoxy groups are hydrogen bond acceptors, chalcones and
bis-chalcones with methoxy groups all provided lower inhibitory activities than hydroxyl
groups containing counterparts.
Figure 4 The structure of 2g
2.3 The inhibition kinetics of a-glucosidase
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Figure 5 The inhibition types of compounds 2c, 2g, 2j and 2l
To further explore interaction mechanism of chalcones and bis-chalcones with
a-glucosidase, the inhibition types of typical compounds 2c, 2g, 2j and 2l were
7

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determined from Lineweavere Burk plots according to the methods reported in literatures
[19-20]. As shown in Figure 5, the straight lines with the same Michaelis-Menten constant
Km suggested that these compounds were non-competitive a-glucosidase inhibitors by
binding to the non-competitive sites of the enzyme.
2.4 The analysis of glucose level and glucose up-take in cell
To further investigate the α-glucosidase inhibitory activities of chalcones and
bis-chalcones, the glucose level in HepG-2 cells were evaluated.
Figure 6 The effect of the compounds (1µM) on the glucose level in HepG-2 cells
As shown in Figure 6, 1-deoxynojirimycin (PG) served as a positive control. PG
exerted subtle effect on reducing glucose level in HepG-2 cells at 5 µM, with the evidence
of an approximately 50% decrease in glucose level in HepG-2 after the PG administration.
Compared with PG at 5µM, compound 2g exerted a similar effect with the PG treatment
at 1 µM for 24 h, suggesting that compound 2g had a better effect than
1-deoxynojirimycin on reducing the glucose level in cell.
Figure 7 The effect of the compounds (1 µM) on glucose uptake activity in HepG-2 cells
Furthermore, the effect of the compounds on glucose uptake activity was
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evaluated. As shown in Figure 7, PG showed no major effect on the glucose uptake
activity in HepG-2 cells. Similar with positive control PG, most compounds except 2j
showed no major effect on glucose uptake activity, demonstrating that compound 2g
reducing glucose level in HepG-2 cells via inhibiting a-glucosidase activity.
Interestingly, compound 2k exhibited the best inhibitory activities (1.0±0.1µM) in
the enzyme assays in vitro, compound 2g (at 1 µM concentration) has stronger effect on
reducing the glucose level in HepG-2 cells. To elucidate this bias, the Log P value of
compound 2g and 2k was tested. The results were shown in Table 2. Compound 2g
showed a higher log P value than 2k, which means compound 2g could be easier to cross
cell membranes. This may reveal the reason of ambivalent resulted in the enzyme assay
and glucose levels in cell.
Table 2 The log P values for selected compounds
compounds
Log P
1.60
2g
2k
1.10
3. Conclusion
As the analogues of xanthones, twenty-six chalcones and bis-chalcones were
designed, synthesized, and evaluated as AGIs. Most of the hydroxyl chalcones and
bis-chalcones showed better inhibitory activities than 1-deoxynojirimycin, and
bis-chalcone 2k showed the highest inhibitory activity, the IC50 value was only 1.0µM.
The structure and activity relationship revealed that, the number and the position of the
hydroxyl group(s) are the key factors to determining the a-glucosidase inhibitory activity
of chalcones probably because that the hydroxyl groups of the compounds bound to
a-glucosidase as hydrogen-bond donors. Moreover, kinetic analysis revealed that
compounds 2c, 2g, 2j and 2l are noncompetitive inhibitors. Inspiringly, compound 2g
displayed stronger activity than 1-deoxynojirimycin in reducing the glucose level in
HepG-2 cells, which means that compound 2g could be a potential drug candidates for
treating diabetes.
4. Experimental and methods
4.1 General
Melting points were determined using a SGW X-4 digital melting point apparatus,
and the temperatures are not corrected. IR spectra were determined on an EQUINOX 55
1
13
Fourier transformation infrared spectrometer. H and C NMR spectra were recorded on
Varian INOVA 500, Bruker AVANCE AV400 or Mercury-Plus 300 NMR spectrometer
in CDCl3, DMSO-d6 or Acetone-d6 and TMS as an internal standard. High resolution (HR)
mass spectra were measured on Thermo MAT95XP mass spectrometer. UV spectra were
recorded
on
a
Shimadzu
UV-2450
scanning.
1-Deoxynojirimycin,
p-nitrophenyl-a-D-glucopyranoside (PNP) and a-glucosidase (from Saccharomyces
cerevisiae) were purchased from Sigma (St. Louis, MO, USA). All the other reagents
9

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were used as purchased without further purification.
4.1.1 General methods for the synthesis of 1a-1d
Acetyl benzenes (1.0 mmol) were reacted with substituted benzaldehydes (1.2
mmol) at 80℃ for 36 hours in the presence of SOCl2 (1.42 mmol) using ethanol as a
solvent. Then the solution was poured into water, the precipitate after filtered and dried
was purified by flash chromatography.
4.1.1.1. (E)-4-hydroxy-2’,4’,6’-trimethoxychalcone (1a)
Yellow solid, 30% yield, m.p. 208-211℃. 1H NMR (300 MHz, Acetone-d6) δ =8.85 (s, 1H,
ArOH), 7.50 (d, J = 8.7Hz, 2H), 7.20 (d, J = 16.0Hz, 1H), 6.87 (d, J = 8.7Hz, 2H), 6.75 (d,
J = 16.0Hz, 1H), 6.30 (s, 2H), 3.87 (s, 3H), 3.75 (s, 6H); LRMS (EI) m/z 314 M+
4.1.1.2. (E)-4-hydroxy-3’,4’-dimethoxychalcone (1b)
Yellow solid, 57% yield, m.p. 165-168℃. 1H NMR (300 MHz, DMSO-d6) δ 10.01 (s, 1H),
7.84 (dd, J = 8.4, 1.8 Hz, 1H), 7.71 (m, 3H), 7.62 (d, J = 15.6, 1H), 7.59 (d, J = 2.1 Hz,
1H), 7.07 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 3.85 (d, J = 3.3 Hz, 6H); LRMS
(EI) m/z 284 M+
4.1.1.3. (E)-4-hydroxy-2’,4’-dimethoxychalcone (1c)
Yellow solid, 50% yield, mp 153~155℃. 1H NMR (300 MHz, Acetone-d6) δ 8.88 (s, 1H),
7.65 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.57 (m, 3H), 7.45 (d, J = 15.0 Hz, 1H),
6.90 (d, J = 8.7 Hz, 2H), 6.67 (d, J = 2.1 Hz, 1H), 6.62 (dd, J = 8.1, 2.7 Hz, 1H), 3.97 (s,
3H), 3.90 (s, 3H) ; LRMS (EI) m/z 284 M+
4.1.1.4. (E)-3-hydroxy-2’,4’-dimethoxychalcone (1d)
Orange solid, 40% yield, m.p. 159-160℃. 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.8
Hz, 1H), 7.67 (d, J = 15.6 Hz, 1H), 7.52 (d, J = 15.6 Hz, 1H), 7.31, 7.27, 7.20 (d, J = 7.6
Hz, 1H), 7.14 (s, 1H), 6.90 (dd, J = 8.0, 2.4 Hz, 1H), 6.59 (dd, J = 8.8, 2.0 Hz, 1H), 6.52
(d, J = 2.0 Hz, 1H), 3.93 (s, 3H), 3.90 (s, 3H); LRMS (EI) m/z 284 M+
4.1.2 General methods for the synthesis of 1e-1f
To a stirred solution of benzaldehydes (0.2 mmol) and acetyl benzenes (0.25 mmol)
in methanol was added aqueous KOH (50%). The mixture was stirred at 70℃ for 3 hours,
then the solution was poured into water and methanol was removed under reduced
pressure. The solution was extracted with two portions of CH2Cl2 and the organic layer
was separated and evaporated under reduced pressure. The residue after evaporation was
purified by flash chromatography.
4.1.2.1. (E)-2,4,2’,4’-tetramethoxychalcone (1e)
Pale yellow solid, 78% yield, mp 134~136℃. 1H NMR (300 MHz, DMSO-d6) δ 7.71 (d, J
= 15.9 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 15.9 Hz,
1H), 6.65 (d, J = 2.1 Hz, 1H), 6.63 – 6.54 (m, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H),
3.81 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 188.6, 153.1, 151.3, 149.3, 144.1, 131.6,
128.1, 122.9, 119.7, 111.2, 110.9, 110.3, 110.0, 56.1, 56.0; LRMS (EI) m/z 328 M+
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4.1.2.2. (E)-3,4,3’,4’-tetramethoxychalcone (1f)
Yellow solid, 71% yield, mp 114~116℃. 1H NMR (400 MHz, DMSO-d6) δ 7.93 (dd, J =
8.4, 2.0 Hz, 1H), 7.84 (d, J = 15.2 Hz, 1H), 7.68 (d, J = 15.6 Hz, 1H), 7.61 (d, J = 2.0 Hz,
1H), , 7.54 (d, J = 2.0 Hz, 1H) 7.41 (dd, J = 8.4, 2.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H),
13
7.03 (d, J = 8.0 Hz, 1H), 3.89-3.87 (m, 9H), 3.83(s, 3H). C NMR (101 MHz, CDCl3) δ
188.6, 153.1, 151.3, 149.3, 144.1, 131.6, 128.1, 122.9, 119.7, 111.2, 110.9, 110.3, 110.0,
56.1, 56.0; LRMS (EI) m/z 328 M+
4.1.3 General methods for the synthesis of 1g-1i
Phthalaldehyde (0.1 mmol) reacted with acetyl benzenes (0.21 mmol) in methanol
at 70℃ for 3 hours after adding aqueous KOH (50%). The post treatment is the same
with the synthesis of 1e-1f.
4.1.3.1. (2E,2'E)-3,3'-(1,3-phenylene)bis(1-(2,4-dimethoxyphenyl)prop-1-en-2-one) (1g)
1
Yellow solid, 56% yield, mp 169~170℃. H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.8
Hz, 3H), 7.72 (d, J =15.6 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 15.6 Hz, 2H),
7.45 (t, J = 7.6 Hz, 1H), 6.60 (dd, J = 8.4, 2.0 Hz, 2H), 6.53 (d, J = 2.0 Hz, 2H), 3.95 (s,
6H), 3.90 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 190.2, 164.4, 160.5, 141.1, 136.2, 133.0,
129.3, 129.3, 128.3, 127.9, 122.1, 105.3, 98.6, 55.8, 55.6; LRMS (EI) m/z 458 M+
4.1.3.2. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(2,4-dimethoxyphenyl)prop-1-en-2-one) (1h)
1
Yellow solid, 60% yield, mp 215~217℃. H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.8
Hz, 2H), 7.70 (d, J = 15.6 Hz, 2H), 7.64 (s, 4H), 7.59 (d, J = 15.6 Hz, 2H), 6.60 (dd, J =
8.8, 2.4 Hz, 2H), 6.53 (d, J = 2.4 Hz, 2H), 3.95 (s, 6H), 3.91 (s, 6H) ; LRMS (EI) m/z 458
M+
4.1.3.3. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(3,4-dimethoxyphenyl)prop-2-en-1-one) (1i)
Yellow solid, 33% yield, mp 231~232℃. 1H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 15.6
Hz, 2H), 7.73 – 7.71 (m, 6H), 7.66-7.61 (m, 4H), 6.96 (d, J = 8.4 Hz, 2H), 4.00 (d, J = 1.6
Hz, 12H). 13C NMR (101 MHz, CDCl3) δ 188.3, 153.5, 149.4, 142.7, 136.9, 131.2, 128.9,
123.1, 122.6, 110.8, 110.0, 56.1, 56.1; LRMS (EI) m/z 458 M+
4.1.4 General methods for the synthesis of 1j-1m
Diacetylbenzene (0.1 mmol) reacted with substituted benzaldehydes (0.21 mmol)
in methanol at 70℃ for 3 hours after adding aqueous KOH (50%). The post treatment is
the same with the synthesis of 1e-1f.
4.1.4.1. (2E,2'E)-1,1'-(1,3-phenylene)bis(3-(2,4-dimethoxyphenyl)prop-2-en-1-one) (1j)
Yellow solid, 43% yield, mp 160~162℃. 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.21
(dd, J = 7.6, 1.6 Hz, 2H), 8.12 (d, J = 15.6 Hz, 2H), 7.63 (m, 5H), 6.56 (dd, J = 8.8, 2.4
Hz, 2H), 6.50 (d, J = 2.4 Hz, 2H), 3.92 (s, 6H), 3.88 (s, 6H). 13C NMR (101 MHz, CDCl3)
δ 190.5, 163.3, 160.5, 141.2, 139.1, 132.0, 131.1, 128.8, 128.3, 120.1, 117.0, 105.5, 98.5,
55.6, 55.5; LRMS (EI) m/z 458 M+
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4.1.4.2. (2E,2'E)-1,1'-(1,4-phenylene)bis(3-(2,4-dimethoxyphenyl)prop-2-en-1-one) (1k)
1
Yellow solid, 37% yield, mp 220~221℃. H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 4H),
8.05 (d, J = 15.6 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 15.6 Hz, 2H), 6.68-6.65
13
(m, 4H), 3.93 (s, 6H), 3.87 (s, 6H). C NMR (101 MHz, CDCl3) δ 190.9, 163.3, 160.6,
141.7, 141.4, 131.1, 128.5, 120.4, 117.0, 108.4, 105.5, 98.5, 55.6, 55.5; LRMS (EI) m/z
458 M+
4.1.4.3. (2E,2'E)-1,1'-(1,3-phenylene)bis(3-(3,4-dimethoxyphenyl)prop-2-en-1-one) (1l)
Yellow solid, 33% yield, mp 120~121℃. 1H NMR (300 MHz, DMSO-d6) δ 8.67 (s, 1H),
8.39 (dd, J = 7.5, 1.5 Hz, 2H), 7.89 (d, J = 15.6 Hz, 2H), 7.78 – 7.72 (m, 3H), 7.56 (d, J =
1.8 Hz, 2H), 7.43 (dd, J = 8.1, 1.8 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 3.86 (s, 6H), 3.82 (s,
6H). 13C NMR (101 MHz, CDCl3) δ 189.9, 151.7, 149.3, 145.9, 138.9, 132.2, 129.0,
128.2, 127.7, 123.5, 119.6, 111.2, 110.2, 56.1; LRMS (EI) m/z 458 M+
4.1.4.4. (2E,2'E)-1,1'-(1,3-phenylene)bis(3-(4-methoxyphenyl)prop-2-en-1-one) (1m)
1
Yellow solid, 59% yield, mp 192~194℃. H NMR (400 MHz, CDCl3) δ 8.65 (t, J = 1.6
Hz, 1H), 8.24 (dd, J = 7.6, 1.6 Hz, 2H), 7.87 (d, J = 15.6 Hz, 2H), 7.69-7.66 (m, 5H), 7.50
(d, J = 15.6 Hz, 2H), 7.00 – 6.97 (m, 4H), 3.90 (s, 6H); LRMS (EI) m/z 398 M+
4.1.5 General methods for the synthesis of 2a-2m
The solution of methoxychalcone (0.4 mmol) in dried dichloromethane was added
to the stirred boron tribromide BBr3 (1 mol/L, 5 mL) at 0℃. The mixture was stirred for
24 or 48 hours at room temperature, after that the solution was poured into ice water. The
precipitate after filtered and dried was purified by flash chromatography.
4.1.5.1. (E)-4-hydroxy-2’,4’,6’-trihydroxychalcone (2a)
1
Yellow solid, 11% yield, m.p. 235-236℃. H NMR (300 MHz, DMSO-d6) δ = 12.50 (s,
2H, ArOH), 10.38 (s, 1H, ArOH), 10.04 (s, 1H, ArOH), 7.94 (d, J = 15.3 Hz, 1H), 7.62 (d,
J = 15.6 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.81 (s, 2H); LRMS
(ESI) m/z 271 [M-H]-
4.1.5.2. (E)-4-hydroxy-3’,4’-dihydroxychalcone (2b)
Brown solid, 86% yield, m.p. 199-201℃. 1H NMR (300 MHz, DMSO-d6) δ = 9.98 (s, 1H,
ArOH), 9.84 (s, 1H, ArOH), 9.29 (s, 1H, ArOH), 7.66 (d, J = 8.7 Hz, 2H), 7.53 ~ 7.68 (m,
3H), 7.46 (d, J = 2.1 Hz, 1H), 6.76 ~ 6.85 (m, 3H); LRMS (ESI) m/z 255 [M-H]-
4.1.5.3. (E)-4-hydroxy-2’,4’-dihydroxychalcone (2c)
1
Yellow solid, 48% yield, m.p. 198~200℃. H NMR (500 MHz, Acetone-d6) δ 13.65 (s,
1H), 9.49 (s, 1H), 9.03 (s, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 15.0 Hz, 1H), 7.81 –
7.75 (m, 3H), 6.95 (d, J = 8.5 Hz, 2H), 6.49 (dd, J = 9.0, 2.5 Hz, 1H), 6.39 (d, J = 2.5 Hz,
1H); LRMS (ESI) m/z 255 [M-H]-
12

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4.1.5.4. (E)-3-hydroxy-2’,4’-dihydroxychalcone (2d)
1
Yellow solid, 21% yield, m.p. 222-223℃. H NMR (300 MHz, DMSO-d6) δ = 13.34 (s,
1H, ArOH), 10.71 (s, 1H, ArOH), 9.60 (s, 1H, ArOH), 8.16 (d, J = 9.0 Hz, 1H), 7.85(d, J
= 15.3Hz, 1H), 7.67 (d, J = 15.6 Hz, 1H), 7.18 ~ 7.33 (m, 3H), 6.85 (d, J = 7.5 Hz, 1H),
6.39 (dd, J = 9.0, 2.4 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H); LRMS (ESI) m/z 255 [M-H]-
4.1.5.5. (E)-2,4,2’,4’-tetrahydroxychalcone (2e)
Orange solid, 30% yield, Td 151~152℃. 1H NMR (400 MHz, Acetone-d6) δ 13.80(s,1H),
9.40(s,1H), 9.22(s,1H), 8.91(s,1H), 8.24(dd, J=15.6 Hz,1.6 Hz, 1H), 8.05(d, J=9.2Hz, 1H),
7.82(d, J=15.2Hz 1H), 7.73(d, J=8.4Hz 1H), 6.53 (d, J=1.6Hz, 1H), 6.49(d, J=2.0Hz, 1H),
6.47(d, J=2.4Hz, 1H), 6.38(s, 1H); LRMS (ESI) m/z 271 [M-H]-
4.1.5.6. (E)-3,4,3’,4’-tetrahydroxychalcone (2f)
Yellow solid, 26 % yield, Td 199~200℃. 1H NMR (400 MHz, Acetone-d6) δ 8.66 (s, 1H),
8.49 (s, 1H), 8.31 (s, 1H), 8.12 (s, 1H), 7.66 – 7.55 (m, 4H), 7.32 (d, J = 2.0 Hz, 1H), 7.19
(dd, J = 8.0, 2.0 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H); LRMS (ESI)
m/z 271 [M-H]-
4.1.5.7. (2E,2'E)-3,3'-(1,3-phenylene)bis(3-(2,4-dihydroxyphenyl)prop-1-en-2-one) (2g)
Yellow solid, 25% yield, mp 213~215℃. IR (KBr) 3163, 2922, 2851, 1637, 1594, 1577,
1520, 1435, 1361, 1310, 1288, 1227, 1173, 1129, 1029, 996, 975, 838, 781, 750, 663, 631,
583, 560, 526, 501, 484, 452 cm-1. 1H NMR (400 MHz, Acetone-d6) δ 13.48 (s, 2H), 9.59
(s, 2H), 8.43 (s, 1H), 8.21 (d, J = 8.9 Hz, 2H), 8.12 (d, J = 15.5 Hz, 2H), 8.02 – 7.90 (m,
4H), 7.61 (t, J = 7.8 Hz, 1H), 6.53 (dd, J = 8.9, 2.4 Hz, 2H), 6.42 (d, J = 2.4 Hz, 2H). 13C
NMR (126 MHz, Acetone-d6) δ 191.8, 166.9, 165.4, 143.1, 135.9, 132.8, 130.7, 129.6,
128.8, 121.8, 113.5, 108.2, 102.9; HRMS (ESI) m/z 401.10296 (calcd for C24H17O6,
401.10306)
4.1.5.8. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(2,4-dihydroxyphenyl)prop-2-en-1-one) (2h)
Yellow solid, 15% yield, mp 252~254℃. 1H NMR (400 MHz, Acetone-d6) δ 13.48 (s, 2H),
9.66 (s, 2H), 8.21 (d, J = 9.2 Hz, 2H), 8.08 (d, J = 15.6 Hz, 2H), 8.00 (s, 4H), 7.93 (d, J =
15.2 Hz, 2H), 6.52 (dd, J = 8.8, 2.4 Hz, 2H), 6.42 (d, J = 2.0 Hz, 2H); LRMS (ESI) m/z
401 [M-H]-
4.1.5.9. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(3,4-dihydroxyphenyl)prop-2-en-1-one) (2i)
Brown solid, 33% yield, IR (KBr) 3477, 3244, 3086, 2749, 1653, 1608, 1571, 1528, 1512,
1437, 1377, 1327, 1283, 1246, 1184, 1113, 1050, 977, 936, 898, 865, 830, 799, 785, 740,
646, 622, 573, 548, 508 cm-1.1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 2H), 9.40 (s, 2H),
7.97 – 7.93 (m, 6H), 7.70 – 7.67 (m, 4H), 7.54 (d, J = 2.0 Hz, 2H), 6.88 (d, J = 8.4 Hz,
13
2H). C NMR (126 MHz, DMSO-d6) δ187.6, 151.5, 146.0, 142.0, 137.1, 130.0, 129.6,
123.5, 122.8, 115.9, 115.6; HRMS (ESI) m/z 401.10303 (calcd for C24H17O6, 401.10306)
4.1.5.10. (2E,2'E)-1,1'-(1,3-phenylene)bis(3-(2,4-dihydroxyphenyl)prop-2-en-1-one) (2j)
Reddish brown solid, 31% yield, mp 138~140℃. IR (KBr) 3185, 2973, 2926, 2601, 1643,
13

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1597, 1550, 1514, 1449, 1383, 1316, 1253, 1176, 1099, 1064, 1038, 979, 847, 796, 771,
1
737, 682, 693, 602, 570, 534, 499, 450 cm-1. H NMR (500 MHz, DMSO-d6) δ 10.26 (s,
2H), 10.01 (s, 2H), 8.56 (s, 1H), 8.32 – 8.27 (m, 2H), 8.03 (d, J = 15.5 Hz, 2H), 7.79 –
13
7.65 (m, 5H), 6.40 (d, J = 2.0 Hz, 2H), 6.34 (dd, J = 8.5, 2.0 Hz, 2H). C NMR (126
MHz, DMSO-d6) δ 188.9, 161.7, 159.3, 140.9, 138.8, 131.8, 130.7, 129.2, 127.3, 117.0,
113.3, 108.1, 102.5; HRMS (ESI) m/z 401.10300 (calcd for C24H17O6, 401.10306)
4.1.5.11. (2E,2'E)-1,1'-(1,4-phenylene)bis(3-(2,4-dihydroxyphenyl)prop-2-en-1-one) (2k)
Black solid, 17% yield, IR (KBr) 3150, 2608, 1643, 1576, 1539, 1501, 1444, 1390, 1341,
1
1318, 1258, 1214, 1134, 1035, 1010, 981, 846, 801, 771, 701, 500, 444 cm-1; H NMR
(400 MHz, DMSO-d6) δ 10.28 (s, 2H), 10.05 (s, 2H), 8.18 (s, 4H), 8.03 (d, J = 15.6 Hz,
2H), 7.73 (d, J = 8.8 Hz, 2H), 7.69(d, J = 15.2, 2H), 6.41 (d, J = 2.0 Hz, 2H), 6.34 (dd, J =
8.4, 2.4 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ 189.76, 162.20, 159.84, 141.65,
141.58, 131.11, 128.88, 117.62, 113.76, 108.67, 102.94. HRMS (ESI) m/z 401.10301
(calcd for C24H17O6, 401.10306)
4.1.5.12. (2E,2'E)-1,1'-(1,3-phenylene)bis(3-(3,4-dihydroxyphenyl)prop-2-en-1-one) (2l)
Dark yellow solid, 61% yield, mp 236~237℃. 1H NMR (300 MHz, DMSO-d6) δ 9.74 (s,
2H), 9.13 (s, 2H), 8.61 (s, 1H), 8.32 (dd, J = 7.8, 1.5 Hz, 2H), 7.71 (t, J = 7.8 Hz, 1H),
7.65 (s, 4H), 7.28 (d, J = 1.8 Hz, 2H), 7.19 (dd, J =8.4, 2.1 Hz, 2H), 6.80 (d, J = 8.1 Hz,
13
2H). C NMR (126 MHz, DMSO-d6) δ 189.2, 149.5, 146.1, 139.0, 132.8, 129.8, 128.1,
126.7, 123.1, 118.8, 116.3, 116.1; LRMS (ESI) m/z 401 [M-H]-
4.1.5.13. (2E,2'E)-1,1'-(1,3-phenylene)bis(3-(4-hydroxyphenyl)prop-2-en-1-one) (2m)
Yellow solid, 57% yield, mp 271~273℃. 1H NMR (400 MHz, Acetone-d6) δ 9.00 (s, 2H),
8.76 (t, J = 1.6 Hz, 1H), 8.37 (dd, J = 7.6, 1.6 Hz, 2H), 7.86 – 7.74 (m, 9H), 6.96 (d, 4H);
LRMS (ESI) m/z 369 [M-H]-
4.2. Biological study
4.2.1 Enzyme assays
The inhibitory activities of all the chalcones and bis-chalcones were measured by
using the methods similar to those articles previously. Typically, a-glucosidase activity
was assayed in 50 mM phosphate buffer (pH 6.8) containing 5% v/v dimethylsulfoxide.
The inhibitors were pre-incubated with the enzyme in phosphate buffer at 37℃ for 30
minutes. PNP glycoside, the substrate, was then added and the enzymatic reaction was
carried out at 37℃ for 60 seconds. The reaction was monitored spectrophotometrically
by measuring the absorbance at 400 nm. The assay was performed in triplicate with five
different concentrations around the IC50 values, and the mean values were calculated
[19-21].
4.2.2 Kinetics of enzyme inhibition
The inhibition types of compounds 2c, 2h, 2j and 2l were determined from
Lineweavere Burk plots, using the methods similar to those reported in literatures [18-19].
Typically, two different concentrations of each compound lower than the IC50 values were
14

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chosen. Under each concentration, a-glucosidase activity was assayed by varying the
concentration of PNP glycoside. The enzyme reaction was performed under the
above-mentioned reaction condition. The mixtures of the enzyme and the inhibitor were
dissolved in 50mM phosphate buffer (pH 6.8) containing 5% v/v DMSO, and
pre-incubated at 37℃ for 30 min, and then the substrate was added. The enzymatic
reaction was carried out at 37℃ for 60 seconds, and monitored spectrophotometrically by
measuring the absorbance at 400 nm. Inhibition types and Ki values of the inhibitors were
determined by Double-reciprocal plots.
4.2.3 Cell cultures
HepG-2 cells (ATCC, USA) were purchased from the American Type Culture
Collection. Cells were maintained in DMEM (Gibco, USA) supplemented with 10% fetal
bovine serum (Gibco, USA) and 1% penicillin and streptomycin (BI, USA) in a
humidified atmosphere containing with 5% CO2 in air at 37℃.
4.2.4 Glucose level Analysis
Cells were seeded and cultured in serum-free medium for indicated time, and then
treated with compounds at 1 µM. After stimulation with compounds for 24 h. Cells were
collected and lysed in protein extraction buffer. Glucose content and protein level were
determined by Glucose analysis Kit (JianCheng, China) and BCA determine Kit (Thermo,
USA). The glucose level in cells was expressed as “mmol glucose / g protein”.
4.2.5 Glucose Up-take analysis
Glucose-uptake analysis was measured as follow. Cells were seeded and cultured
in serum-free medium for 2 h, and treated with compounds at 1 µM. After stimulation
with compounds for 24 h. Culture medium and cells were collected and centrifuged at
1500 rpm for 5 min, respectively. Supernatant was used to determine the glucose level.
Protein level was also determined by BCA determine Kit (Thermo, USA). The glucose
level in culture medium was expressed as “mmol glucose /g protein”.
4.2.6 The determination of the lipid-water distribution coefficient.
The distribution coefficients were determined by using the shake–flask method in
1-octanol/water system [22]. After shaking the tested compounds in 1-octanol/water (1:1)
solution for 24h, the distribution of the compounds in 1-octanol phase and water phase
was determined by HPLC analysis. The lipid–water distribution coefficient was calculated
according the following equation:
where Co is the concentration of the test compound in water and Cw is its
concentration in 1-octanol. Log P is a mean value from at least three independent tests.
Acknowledgements
15

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This research was supported by the National Natural Science Foundation of China
(21272290, J1103305) and the Fund for Innovative Chemical Experiment and Research of
School of Chemistry and Sun Yat-sen University.
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Table
Table 1 The IC50 value or inhibition of the compounds
Table 2 The logP values for selected compounds
Figure Captions
Figure 1 The structures of benzoxanthone, chalcone and bis-chalcone
Figure 2 The stereochemistry of chalcones
Figure 3 The structure of chalconeFigure 4 The structure of 2g
Figure 45 The inhibition types of compounds 2c, 2g, 2j and 2l
Figure 6 The effect of the compounds (1µM) on the glucose level in HepG-2 cells
Figure 7 The effect of the compounds (1 µM) on glucose uptake activity in HepG-2 cells
Schemes
Scheme 1 Synthesis of the target compounds 1a-1m and 2a-2m
Scheme 2 Conceptual reaction mechanism for demethylation
Scheme 3 Conceptual reaction mechanism for demethylation of dimethoxyl groups
Tables
Table 1 The IC50 value or inhibition of the compounds
Compounds
Structure
Inhibition or
Compounds
Structure
IC50/µM
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IC50/µM
1a
1b
1c
1d
1e
1f
>200
>200
2a
2b
2c
2d
2e
2f
71.1±2.6
35.2±0.2
13.2±2.7
42.0±6.0
12.5±2.1
15.6±2.8
58.7±3.0
>200
83.2±1.0
123.1±9.9
1g
1h
27.2% a
27% a
2g
2h
23.7±0.3
22.5±3.2
OH
O
OH
HO
O
OH
1i
1j
32.0% a
55.2% a
66.9±2.5
2i
2j
10.1±1.7
5.5±1.2
1.0±0.1
1k
2k
1l
16.0% a
29% a
2l
6.5±0.2
1m
2m
18.3±0.7
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a : the inhibition of the compounds at the concentration of 40 µM (The solubility of these methoxyl bis-chalcones in
PBS are low ). The IC50 value of positive control (1-deoxynojirimycin, PG) is 21.3±8.7 µM.
Table 2
compounds
2g
2k
The logP values for selected compounds
logP
1.60
1.10
Figures
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Figure 1 The structures of benzoxanthone, chalcone and bis-chalcone
Figure 2 The stereochemistry of chalcones
Figure 3 The structure of chalcone
Figure 4 The structure of 2g
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Figure 5 The inhibition types of compounds 2c, 2g, 2j and 2l
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Figure 6 The effect of the compounds (1µM) on the glucose level in HepG-2 cells
Figure 7 The effect of the compounds (1 µM) on glucose uptake activity in HepG-2 cells
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Scheme 1 Synthesis of the target compounds 1a-1m and 2a-2m
Scheme 2 Conceptual reaction mechanism for demethylation
Scheme 3 Conceptual reaction mechanism for demethylation of dimethoxyl groups
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Research Highlights
‹ Twenty-six chalcone and bis-chalcone derivatives were synthesized and four of them
are first reported.
‹ Some compounds exhibited excellent inhibitory activities against α-glucosidase, the
best IC50 value reaches to 1.0 µM.
‹ Valuable structure-activity relationship was obtained.
‹
‹
The selected compounds (2c, 2g, 2j, 2l) were showed as non-competitive inhibitors.
The compound 2g had a better effect than the positive control 1-deoxynojirimycin on
reducing the glucose level in HepG-2 cells and could be a potential drug candidates
for treating diabetes.