Design, synthesis and α-glucosidase inhibition study of novel embelin derivatives

Article abstract of DOI:10.1080/14756366.2020.1715386

Embelin is a naturally occurring para-benzoquinone isolated from Embelia ribes (Burm. f.) of the Myrsinaceae family. It was first discovered to have potent inhibitory activity (IC₅₀ = 4.2 μM) against α-glucosidase in this study. Then, four seri

Full text of DOI:10.1080/14756366.2020.1715386

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and α-glucosidase inhibition
study of novel embelin derivatives
Xiaole Chen, Min Gao, Rongchao Jian, Weiqian David Hong, Xiaowen Tang,
Yuling Li, Denggao Zhao, Kun Zhang, Wenhua Chen, Xi Zheng, Zhaojun Sheng
& Panpan Wu
To cite this article: Xiaole Chen, Min Gao, Rongchao Jian, Weiqian David Hong, Xiaowen Tang,
Yuling Li, Denggao Zhao, Kun Zhang, Wenhua Chen, Xi Zheng, Zhaojun Sheng & Panpan Wu
(2020) Design, synthesis and α-glucosidase inhibition study of novel embelin derivatives, Journal of
Enzyme Inhibition and Medicinal Chemistry, 35:1, 565-573, DOI: 10.1080/14756366.2020.1715386
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 565–573
https://doi.org/10.1080/14756366.2020.1715386
SHORT COMMUNICATION
Design, synthesis and a-glucosidase inhibition study of novel embelin derivatives
a
a
a
Xiaole Chen , Min Gao , Rongchao Jian , Weiqian David Honga,b,c, Xiaowen Tanga, Yuling Lia, Denggao Zhaoa,b
,
ꢀ
ꢀ
Kun Zhanga,b,d, Wenhua Chena,b, Xi Zhenga,b, Zhaojun Shenga,b and Panpan Wua,b,d
b
aSchool of Biotechnology and Health Sciences, Wuyi University, Jiangmen, P.R. China; International Healthcare Innovation Institute (Jiangmen),
c
d
Jiangmen, P.R. China; Department of Chemistry, University of Liverpool, Liverpool, UK; School of Chemical Engineering and Light Industry,
Guangdong University of Technology, Guangzhou, P.R. China
ABSTRACT
ARTICLE HISTORY
Received 9 September 2019
Revised 12 December 2019
Accepted 7 January 2020
Embelin is a naturally occurring para-benzoquinone isolated from Embelia ribes (Burm. f.) of the Myrsinaceae
family. It was first discovered to have potent inhibitory activity (IC50 ¼ 4.2 lM) against a-glucosidase in this
study. Then, four series of novel embelin derivatives were designed, prepared and evaluated in a-glucosidase
inhibition assays. The results show that most of the embelin derivatives synthesised are effective a-glucosidase
inhibitors, with IC50 values at the micromolar level, especially 10d, 12d, and 15d, the IC50 values of which are
1.8, 3.3, and 3.6 lM, respectively. Structure–activity relationship (SAR) studies suggest that hydroxyl groups in
the 2/5-position of para-benzoquinone are very important, and long-chain substituents in the 3-position are
highly preferred. Moreover, the inhibition mechanism and kinetics studies reveal that all of 10d, 12d, 15d,
and embelin are reversible and mixed-type inhibitors. Furthermore, docking experiments were carried out to
study the interactions between 10d and 15d with a-glucosidase.
KEYWORDS
Embelin; a-glucosidase
inhibitor; anti-diabetes;
hypoglycaemic agent;
benzoquinone
Introduction
hydroxyl groups15,16
. Embelin and its derivatives have been
reported to possess anti-cancer17,18, antimicrobial19,20, antioxi-
dant21, analgesic22, anti-inflammatory22, anxiolytic23, antifertility24
activities, etc. Thanks to these diverse biological activities, embelin
is considered as the “second solid gold of India” next to curcu-
min25. In the last decade, several studies have reported antidia-
betic activity of embelin26–28. In 2016, Sharanbasappa et al.29
reviewed the antidiabetic activity of embelin and its derivatives. It
was concluded from this review and meta-analysis that the E. ribes
extract, embelin and its derivatives have positive effects on blood
glucose, HbAlc, insulin, and lipid profiles. In addition, heart rate,
systolic blood pressure, lactate dehydrogenase, creatinine kinase
and oxidative stress markers in diabetic rats return to normal after
treatment with E. ribes extract and embelin. Moreover, Dang
et al.30,31 reported that the methanolic extract of E. ribes and sev-
eral compounds isolated from the leaves of E. ribes have signifi-
cant a-glucosidase inhibitory activity. These results inspire us to
investigate a-glucosidase inhibition of embelin, which is the main
constituent of E. ribes. In this study, four series of novel embelin
derivatives were designed and prepared in 5- to 6-step chemical
reactions. Their a-glucosidase inhibitory activity was evaluated,
and the mode of action and SAR analysis were described by
means of kinetic and molecular modelling evaluations.
Diabetes mellitus (DM) is a group of chronic metabolic disorders
characterised by hyperglycaemia resulting from defects in insulin
secretion, insulin action, or both1,2. According to WHO data, DM
was the 7th cause of death worldwide in 2016, when it killed 1.6
million people3. There are two major forms of the disease: Type 1
and Type 2 diabetes. The latter accounts for 90 ꢁ 95% of all cases,
formerly called non-insulin-dependent diabetes mellitus (NIDDM)
or adult-onset diabetes, and usually occurs after age 40, becoming
more common with increasing age1,2,4,5
.
The management of Type 2 diabetes includes hyperglycaemia
treatment, diabetic comorbidity prevention, and metabolism
adjustment6,7. One therapeutic approach is to retard the absorp-
tion of glucose via inhibition of enzymes, such as a-glucosidase
and a-amylase, in the digestive organs8–11. a-Glucosidase is an
exo-type carbohydrase widely distributed in microorganisms,
plants, and animal tissues. Inhibiting a-glucosidase slows the ele-
vation of blood sugar after meals8,12. a-Glucosidase inhibitors
(AGIs) are a unique class of oral hypoglycaemic agents approved
for the prevention and management of Type
2 diabetes.
Nowadays, there are four AGIs, including acarbose, miglitol, vogli-
bose, and emiglitate, used in the clinical treatment of Type 2 dia-
betes9,13. However, current approaches including AGIs have some
shortcomings such as safety concerns, limited efficacy, failure in
metabolism adjustment, and the prevention of diabetic complica-
tions6,9,14. Thus, developing new therapeutic drugs to treat Type 2
diabetes is necessary, and has received wide attention.
Materials and methods
Procedure for the synthesis of 1,2,4,5-tetramethoxybenzene
(compound 3)
Embelin is a naturally occurring para-benzoquinone isolated
from Embelia ribes (Burm. f.) of the Myrsinaceae family, and con- 2,5-Dihydroxycyclohexa-2,5-diene-1,4-dione (5 g, 3.6 mmol) was
tains two carbonyl groups, an active methylene group and two dissolved in methanol (200 ml), and HCl solution (12 M, 6 ml) was
CONTACT Zhaojun Sheng
wyuchemszj@126.com
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, 529020, P.R. China; Panpan Wu
wyuchemwpp@126.com
These authors contributed equally to this work.
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, 529020, P.R. China
ꢀ
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

566
X. CHEN ET AL.
Scheme 1. Synthesis of novel embelin derivatives.
added slowly. The mixture was stirred overnight. The reaction was General procedure for the synthesis of 4a–17a, 4b–17b,
and 4c–17c
monitored by TLC. After reaction completion, the mixture was fil-
tered to obtain 2,5-dimethoxycyclohexa-2,5-diene-1,4-dione 1 as a
yellow solid. Compound 1 was dissolved in H2O (50 ml), and
Na2S2O4 (10 g, 57.4 mmol) was added. The mixture was heated to
reflux for 8 ꢁ 15 min. Then the mixture was cooled to crystallise,
and filtered to furnish 2,5-dimethoxybenzene-1,4-diol 2 as a white
crystalline solid. Compound 2 (1.7 g, 10 mmol) was dissolved in
DMSO (10 ml), and KOH (1.4 g, 25 mmol) was added. The mixture
was stirred for 15 min, and then MeI (1.88 ml, 25 mmol) was
added. The mixture was stirred overnight. After reaction comple-
tion, the pH value was adjusted to 5 ꢁ 6 by saturated NH4Cl solu-
tion. The mixture was washed with saturated NaCl solution and
H2O and dried over MgSO4. The solution was condensed under
reduced pressure to give crude product, which was purified by sil-
ica gel column chromatography (PE/EtOAc ¼ 10/1), yielding
1,2,4,5-tetramethoxybenzene 3 (1.05 g, 53%).
A solution of ceric ammonium nitrate (CAN, 1.4 mmol) in MeCN/
H2O (7/3, 5 ml) was added to a solution of compounds 4–17
(0.55 mmol) in MeCN (4 ml) in a salt-ice bath.
Method A. The suspension was stirred for 10 min at ꢂ5 ꢃC. The
organic solvent was removed and the residue was redissolved in
EtOAc (5 ml). The organic layer was washed with saturated brine,
dried over MgSO4 and condensed. The crude product was purified
by flash chromatography (PE/EtOAc ¼ 10/1), furnishing first pure
4a–17a (32%ꢁ40%). Then, the eluent was changed to PE/EtOAc ¼
5/1, giving pure 4c–17c (23%ꢁ30%).
Method B. The suspension was stirred for 2 h at room tem-
perature. The organic solvent was removed and the residue was
redissolved in EtOAc (5 ml). The organic layer was washed with
saturated brine, dried over MgSO4 and condensed. The crude
product was purified by flash chromatography (PE/EtOAc ¼ 10/1),
furnishing first pure 4a–17a (35%ꢁ43%). Then, the eluent was
changed to PE/EtOAc ¼ 3/1, giving pure 4b–17b (18%ꢁ27%).
Reagents and conditions: (i) conc. HCl, MeOH, rt., overnight; (ii)
Na2S2O4, H2O, reflux, 8 ꢁ 15 min; (iii) KOH, MeI, DMSO, rt., over-
night; (iv) n-BuLi, HMPA, THF, ꢂ40 ꢃC to rt., overnight; (v) CAN,
MeCN/H2O, ꢂ5 ꢃC, 10 min; (vi) CAN, MeCN/H2O, ꢂ5 ꢃC to rt., 2 h;
(vii) NaOH, EtOH, 80 ꢃC, 2 ꢁ 3 h.
General procedure for the synthesis of 4d–17d
2 M NaOH solution (8 ml) was added to a solution of compounds
4a–17a or 4b–17b (0.35 mmol) in EtOH (2 ml). The mixture was
heated to reflux for 2 ꢁ 3 h. Next, the organic solvent was
removed. The pH value of the aqueous solution was adjusted to
5 ꢁ 6. Then, the solution was diluted with EtOAc (3 ml). The
organic layer was washed with saturated brine, dried over MgSO4,
and condensed. The crude product was then washed with a low-
polar organic solvent to remove impurities, and filtered to obtain
pure 4d–17d (20%ꢁ45%).
General procedure for the synthesis of 4–17
Hexamethylphosphoric triamide (HMPA, 353 lL, 2 mmol) was
added to a solution of compound 3 (1 g, 5.1 mmol) in dry THF
(60 ml) under N2 atmosphere. Next, n-BuLi (2.44 ml, 6.12 mmol)
was added slowly to the mixture at ꢂ40 ꢃC and stirred at this tem-
perature for 10 min. Then the mixture was allowed to heat to
ꢂ10 ꢃC, and various halogenated hydrocarbons (5.5 mmol) were
added dropwise. The mixture was stirred at room temperature
overnight. THF in the reaction mixture was removed under
reduced pressure, and the residue was redissolved in EtOAc. The
organic solution was washed with 1 M HCl solution and saturated
brine, then dried over MgSO4 and evaporated. The crude product
was chromatographed (PE/EtOAc ¼ 15/1 to 5/1) to supply pure
compound 4–17 (20%ꢁ60%).
a-Glucosidase inhibition
The a-glucosidase inhibitory activity was determined on a Thermo
Scientific Multiskan GO Microplate Reader. All compounds were
dissolved in DMSO. p-Nitrophenol glucoside (PNPG) as a substrate
and a-glucosidase (from Saccharomyces cerevisiae) were used for
the bioassay. First, 0.7 U/ml a-glucosidase solution and 1 mmol/L

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
567
PNPG solution in 0.1 M PBS (pH 6.8) were prepared. Next, 35 ml of solutions was prepared, at constant PNPG concentration (1 mM).
The inhibition rates were measured by the above method with
different concentrations of a-glucosidase (0.00, 0.35, 0.7, and
1.05 U/ml).
The inhibition type of the enzyme was assayed by
Lineweaver–Burk plots. A series of diluted inhibitor solutions was
prepared, at constant a-glucosidase concentration (0.7 U/ml). The
inhibition rates were measured by the above method with differ-
ent concentrations of PNPG (1, 0.5, 0.4, 0.3, 0.25, and 0.2 mM).
0.1 M PBS (pH 6.8), 10 ml of 0.7 U/ml a-glucosidase solution (final
concentration: 0.07 U/ml), and 5 ml of sample solution were added
to the 96-well plate. The mixture was pre-incubated for 10 min at
37 ꢃC. Then, 50 ml of 1 mM PNPG (final concentration: 0.5 mM) was
rapidly added to initiate the reaction. Then the 96-well plate was
quickly transferred into the microplate reader, incubated at 37 ꢃC
for 30 min (1 min shake þ 9 min incubation, repeated 3 times).
After that, 50 ml of 1 M of Na2CO3 solution was added to each well
to stop the reaction. The 96-well plate was quickly transferred into
the microplate reader and shaken for 30 s. In the blank reference
group, 5 ml DMSO replaced the sample solution, other operations
were the same. The OD value was measured at 405 nm. The inhib-
ition rate was calculated by Equation (1).
Molecular docking
The molecular modelling and docking simulation were imple-
mented by using Sybyl 2.0. The 3D structure of a-glucosidase
(PDB code: 1UOK) was obtained from RCSB Protein Data Bank and
the two most potent embelin derivatives (10d and 15d) were
built by molecular modelling package in Sybyl. All the water mole-
cules were removed and hydrogen atoms were added to make
Inhibition rate ð%Þ ¼ ½ðA0 ꢂ A1Þ=A0ꢄ ꢅ 100
(1)
where A0 is the OD of the control, and A1 is the OD of the sample.
The inhibition mechanism of 10d, 12d, and 15d was deter-
mined by the following method: A series of diluted inhibitor protein and ligand protonation. Tripos force field was adopted to
Figure 1. Design of novel embelin derivatives.
Figure 2. 13C-NMR spectrum of compound 5d.

568
X. CHEN ET AL.
make the energy of protein and ligand to be minimised. the core structure but failed due to its large steric hindrance.
Ligand–protein docking simulations were processed by the Although b-naphthyl and biphenyl were introduced successfully,
Surflex-Dock within Sybyl. The catalytic pocket of acarbose dock- the synthetic yields of 16 and 17 were low. The oxidation of
4 ~ 17 by CAN produced different oxidised products depending
on the reaction time. At the beginning of the oxidation reaction, a
mixture of 4a~17a and 4b~17b was obtained. As the reaction
proceeded, mixtures of 4a~17a and 4c~17c were obtained as the
main products. Finally, the demethylation of 4a~17a and 4b~17b
under basic conditions gave 4d~17d. The final products 4a~17a,
4b~17b and 4c~17c were purified by flash chromatography, and
4d~17d were purified by washing with petroleum ether or
dichloromethane.
ing with a-glucosidase was defined as active site. Other settings
were kept default.
Results and discussion
Compound design and synthesis
In our previous study, embelin displayed potent a-glucosidase
inhibitory activity with an IC50 value of 4.20 lM. In order to investi-
gate structure–activity relationships (SAR) and obtain more active
AGIs, four series (a, b, c, and d) of novel embelin derivatives were
designed (Figure 1). First, in order to study the function of the
hydroxyl groups in the 2/5-position, they were monomethylated
or dimethylated, giving series a and b. Second, the core structure
was varied from para-benzoquinone to ortho-benzoquinone, giv-
ing series c. Third, because the long C11H23 tail in the 3-position
results in the poor bioavailability of embelin, new groups were
introduced at this position, giving series d. The structures of R
groups are shown in Figure 1. In order to study the length of the
substituents, C3- to C15-n-alkyl groups were introduced
(Compounds 4 ~ 10). In addition to the straight-chain groups,
branched alkyl groups or straight-chain groups containing a heter-
oatom were introduced. Moreover, aromatic rings or cycloalkanes
were introduced in the terminal group.
All final compounds were characterised by 1H-NMR, 13C-NMR,
and HRMS. It is noteworthy that there is an interesting phenom-
enon in the 13C-NMR spectra of compounds 4d~17d. Taking 5d
as an example (Figure 2), six different carbons exist in the para-
benzoquinone ring. However, there are only two carbon signals at
Table 2. IC50 values of selected compounds against a-glucosidase.
Compound
R
IC50 (lM)
The target compounds were prepared according to a published
method32 with slight modification. 2,5-Dihydroxycyclohexa-2,5-
diene-1,4-dione was used as starting material: first, the two
hydroxyl groups were protected by methylation to yield com-
pound 1. Then the benzoquinone core structure was reduced by
Na2S2O4, giving compound 2. Further methylation produced
1,2,4,5-tetramethoxybenzene 3. Next, n-BuLi was used to abstract
the hydrogen atom from the benzene ring, and then various halo-
genated hydrocarbons were added to furnish 4 ~ 17. The yield of
this step depended on the steric hindrance of the halogenated
hydrocarbon. For example, we tried to introduce a-naphthyl into
6d
7d
embelin
9d
10d
12d
n-C7H15
n-C9H19
n-C11H23
n-C13H27
n-C15H31
126.8
5.7
4.2
2.3
1.8
3.3
14d
15d
45.3
3.6
16d
17d
74.8
11.2
Acarbose
Ursolic acid
584.0
4.3
Figure 3. Tautomerism between two forms of para-benzoquinone.
Table 1. Inhibition rate of all compounds at 250 lM against a-glucosidase.
Comp.
Inhibition rate
Comp.
Inhibition rate
Comp.
Inhibition rate
Comp.
Inhibition rate
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
9.0%
11.5%
1.5%
13.5%
12.5%
nt
nt
1.5%
nt
0%
70.1%
nt
6.0%
5.1%
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
15b
16b
17b
10.4%
14.9%
5.4%
20.9%
12.0%
nt
nt
2.6%
nt
0%
71.1%
nt
4.3%
14.3%
4c
5c
6c
7c
8c
9c
10c
11c
12c
13c
14c
15c
16c
17c
14.9%
16.4%
16.1%
33.2%
0.6%
nt
nt
3.3%
nt
0%
74.7%
nt
37.2%
59.7%
4d
5d
6d
7d
Embelin
9d
10d
11d
12d
13d
14d
15d
16d
17d
18.1%
19.0%
80.8%
99.4%
100%
100%
100%
37.6%
100%
0%
85.2%
100%
81.4%
99.5%
nt: not tested.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
569
>90 ppm, which belong to the carbons in the 3/6-position. This concentration of 250 lM. Most compounds showed an obvious
inhibitory effect on a-glucosidase. Among the four series, com-
pounds from series d showed much better inhibitory activity than
those from the other three series, which indicates that the
hydroxyl groups in the 2/5-positions are very important for the
a-glucosidase inhibitory activity. Moreover, the modification of the
core structure from para-benzoquinone to ortho-benzoquinone
resulted in a significant reduction of activity.
may be due to keto-enol tautomerism of para-benzoquinone
(Figure 3). This rapid transfer between two tautomers makes it dif-
ficult to record the corresponding carbon signals. Once one of the
hydroxyl groups is methylated, the 13C-NMR signals of the core
structure return to be normal.
Then, the inhibition rates of the compounds which inhibited
more than 80% of a-glucosidase in the initial screening were further
evaluated at more than eight concentrations. The IC50 values were
calculated by GraphPad Prism 7.0 software. The results are summar-
ised in Table 2 and the inhibition curves are shown in Figure 4. The
length of the chain in the 3-position is a key factor affecting the
activity. The compounds with longer chains display better activity.
For example, compounds 4d and 5d have a very low inhibitory
effect, while 6d~10d have potent activity, which suggests the
length of the chain should be longer than n-C7H15. Moreover, when
the length was similar, branched groups slightly enhance the activ-
ity. For example, the lengths of the chain in the 3-position of com-
pounds 5d and 11d, 7d, and 12d were similar, but 11d exhibited
better inhibitory activity than 5d, and 12d showed slightly better
activity than 7d. However, the introduction of an oxygen atom in
the chain results in the loss of activity (compounds 13a~13d). In
addition, the introduction of large hydrophobic groups such as
cyclohexyl, phenyl, b-naphthyl, and 4-biphenyl (compounds 14d,
15d, 16d, and 17d) in the terminal, seems to have positive influ-
ence on the activity. Meanwhile, the length of these large hydro-
phobic groups is closely related to the activity.
a-Glucosidase inhibitory activity and SAR analyses
The inhibitory activities of embelin and its derivatives on a-gluco-
sidase from baker’s yeast were determined by the reported
method6,11 with slight modification using acarbose and ursolic
acid as the positive reference compounds. As depicted in Table 1,
the inhibitory activities of all compounds were screened at the
Embelin
7d
100
80
60
40
20
0
9d
10d
12d
15d
-1
0
1
2
3
logC (mM)
Figure 4. Inhibition curves of embelin and its derivatives.
10d
12d
20
15
10
5
20
15
10
5
0uM
1.5uM
2uM
0uM
2uM
3uM
6uM
3uM
0
0
0.00
0.35
0.70
1.05
1.40
0.00
0.35
0.70
1.05
1.40
[E] (U/mL)
[E] (U/mL)
15d
embelin
20
15
10
5
20
15
10
5
0uM
2uM
3uM
4uM
0uM
1.5uM
2uM
2.5uM
0
0
0.00
0.35
0.70
1.05
1.40
0.00
0.35
0.70
1.05
1.40
[E] (U/mL)
[E] (U/mL)
Figure 5. Determination of the mechanism of the inhibition of a-glucosidase by 10d, 12d, 15d, and embelin.

570
X. CHEN ET AL.
Table 3. Ki, Kis, and inhibition type of selected compounds
against a-glucosidase.
Compound Ki value (lM) Kis value (lM) Inhibition type
Embelin
10d
12d
3.72
1.24
3.71
2.40
1.37
0.60
1.93
0.33
Mixed-type
Mixed-type
Mixed-type
Mixed-type
15d
investigated using PNPG as the substrate. The relationship
between enzyme activity and concentration in the presence of dif-
ferent concentrations of selected compounds was studied. As
shown in Figure 5, the plots of enzyme activity versus enzyme
concentration give a set of straight lines, which all pass through
the origin. The increase in the inhibitor concentration resulted in
the reduction of the slope of the line, which suggests that the
inhibitors reduce the activity of a-glucosidase. Therefore, the
inhibition of compounds 10d, 12d, 15d, and embelin against
a-glucosidase is reversible.
In order to elucidate the action mode, the most potent com-
pounds 10d, 12d, 15d, and embelin were selected for enzyme
kinetic studies. The kinetic data were expressed by
Lineweaver–Burk double-reciprocal plots. As shown in Figure 6,
the plots of 1/v versus 1/[S] give a group of straight lines with dif-
ferent slopes that intersect at the third quadrant (Figure
6(A1–D1)), suggesting that all of them are mixed-type inhibitors.
Thus, these compounds bind not only with the free a-glucosidase
but also with the a-glucosidase-PNPG complex. In other words,
they inhibit the function of a-glucosidase by not only directly
binding to free enzyme (EI), but also by interfering with the for-
mation of the a-glucosidase-PNPG (ES) intermediate through pro-
ducing an a-glucosidase-PNPG-inhibitor (ESI) complex in a non-
competitive manner33. The inhibition constant for the inhibitor
binding with free enzyme (Ki) was determined from a plot of the
slope (Km/Vm) versus the inhibitor concentration (Figure 6(A2–D2)),
and the inhibition constant for the inhibitor binding with enzy-
me–substrate complex (Kis) was obtained from the vertical inter-
cept (1/Vm) versus the inhibitor concentration (Figure 6(A3–D3))34.
The results are collected in Table 3: the Kis values of all of them
are smaller than their Ki values, which mean that they have higher
affinity with the enzyme-substrate complex than with the
free enzyme.
Molecular docking
Compound 10d and 15d have the strongest inhibition against
free a-glucosidase (Table 3), and molecular docking was intro-
duced to confirm the binding mode of the two compounds with
the active site of a-glucosidase. The protomol of the active site
integrate with 10d is as shown in Figure 7(A), which reveals that
compound 10d can be effectively inserted into the protomol. The
results show that the long-chain n-C15H31 group can be inserted
into the hydrophobic region of the active site selectively (Figure
7(B,C)). The hydrophobic pocket is long and relatively narrow,
which explain why long-chain substituents in the 3-position are
highly preferred. As depicted in Figure 7(D–F), the two carbonyl
groups and two hydroxyl groups from para-benzoquinone core
structure interact with several amino acid residues including
Figure 6. A1–D1: Lineweaver–Burk double-reciprocal plots; A2–D2: Plots of slope
versus concentration of inhibitors for the determination of the inhibition constant
Ki; A3–D3: Plots of Y-intercept versus concentration of inhibitors for the determin-
ation of the inhibition constant Kis.
Inhibition mechanism
Compounds 9d, 10d, 12d, and 15d displayed the most potent
activity in the a-glucosidase inhibitory assays. Because the chem-
ical structures of compounds 9d and 10d are very similar, com- MET284, LYS413, SER288, and LYS293, which locate in the
pound 10d was selected as representative in the next entrance of the active pocket. These hydrophilic interactions
a
mechanism studies. The inhibition mechanism of the selected reduce the binding free energy of inhibitor and target protein,
compounds 10d, 12d, 15d, and embelin on a-glucosidase was resulting in the increase of inhibitory function. Thus, the hydroxyl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
571
Figure 7. Docking binding model of 10d with yeast a-glucosidase. (A): Binding mode of 10d docked with the prototype molecular of the active site. (B) and (C):
Active site MOLCAD surface representation of lipophilic potential. (D): The interaction of 10d with the surrounding amino acids. (E) and (F): Active site MOLCAD surface
representation of hydrogen bonding.
Figure 8. Docking binding model of 15d with yeast a-glucosidase. (A): Binding mode of 15d docked with the prototype molecular of the active site. (B) and (C):
Active site MOLCAD surface representation of lipophilic potential. (D): The interaction of 15d with the surrounding amino acids. (E) and (F): Active site MOLCAD surface
representation of hydrogen bonding.
groups of embelin and its derivatives are more helpful to improve 10d is more effective of interaction with the active site. This differ-
ence may be contributed to 10d possessing more potent inhibi-
tory activity.
the inhibitory activity against a-glucosidase, which also is in
accordance with the in vitro assay.
The docking binding mode of 15d with a-glucosidase is shown
in Figure 8. The results suggest that 10d and 15d have similar
binding mode with the active site. However, the flexibility of the
substituent (n-heptylphenyl) in the 3-position of 15d is less than
that of 10d (n-pentadecyl), thus the hydrophobic portion of 15d
Conclusions
Embelin was first discovered to have potent inhibitory activity
against a-glucosidase in this study. Based on the structure of
only interact with a part of the active hydrophobic pocket, while embelin, four series of embelin derivatives were designed and

572
X. CHEN ET AL.
prepared. All final compounds were characterised by H-NMR, 13C-
NMR, and HRMS. Enzyme inhibition bioassay results show that
most of these embelin derivatives have potent inhibitory activity.
The SAR study suggests that the two hydroxyl groups and long-
chain substituents are the key factors affected the inhibitory activ-
ity. The most active derivatives 10d, 12d, 15d, and embelin were
selected for the further mechanism and kinetic study. The results
reveal all of them are reversible and mixed-type inhibitors.
Molecular docking study was introduced to insight into the bind-
ing mode of 10d and 15d with a-glucosidase. The docking results
suggest that the formation of hydrogen bonds between the
hydrophilic groups and several amino acids at the active site, as
well as the hydrophobic interaction between the hydrophobic
substituents in the 3-position of para-benzoquinone and hydro-
phobic pocket, are important mechanism of inhibition. This study
provides several leading structures for designing and developing
novel AGIs. These findings encourage us to continue our efforts
towards the optimisation of the pharmacological profile of these
embelin derivatives.
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Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Natural Science Foundation of
Guangdong Province [No. 2016A030310441], National Natural 16. Sheng Z, Ge S, Gao M, et al. Synthesis and biological activity
of embelin and its derivatives: an overview. Mini Rev Med
Chem 2019. E-pub ahead of print. doi:10.2174/
1389557519666191015202723
Science Foundation of China [No. 81803390], the Special Fund
Project of Science and Technology Innovation Strategy in
Guangdong Province [No. Jiangke(2018)352], Jiangmen Science and
Technology Project of Basic and Theoretical Science Research [No.
2019030102100008866], Science Foundation for Young Teachers of
Wuyi University [No. 2016td01], the Foundation of Department of
Education of Guangdong Province [Nos. 2017KQNCX200 and
2017KSYS010] and Innovation and Entrepreneurship Training
Program for Undergraduates of Guangdong Province.
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