Synthesis of rottlerone analogues and evaluation of their -glucosidase and DPP-4 dual inhibitory and glucose consumption-promoting activity

Article abstract of DOI:10.3390/molecules26041024

Our previous study found that desmethylxanthohumol (1) inhibited α-glucosidase in vitro. Recently, further investigations revealed that dehydrocyclodesmethylxanthohumol (2) and its dimer analogue rottlerone (3) exhibited more potent α-glucosidase inhibito

Full text of DOI:10.3390/molecules26041024

molecules
Article
Synthesis of Rottlerone Analogues and Evaluation of Their
α-Glucosidase and DPP-4 Dual Inhibitory and Glucose
Consumption-Promoting Activity
1
1
1
1
1
1,2
1
1,
Yinan Zhang , Haibo Wang , Yan Wu , Xue Zhao , Zhihong Yan , Robert H. Dodd , Peng Yu , Kui Lu *
1
,
and Hua Sun *
1
China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal
Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China;
15302138535@mail.tust.edu.cn (Y.Z.); 1411502122@mail.tust.edu.cn (H.W.); wuyan6@163.com (Y.W.);
zhaoxue1632014@163.com (X.Z.); yanzhihongzs@163.com (Z.Y.); robert.dodd@cnrs.fr (R.H.D.);
yupeng@tust.edu.cn (P.Y.)
Centre de recherche de Gif-sur-Yvette, Institut de Chimie des Substances Naturelles, UPR 2301, CNRS,
Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
2
*
Correspondence: lukui@tust.edu.cn (K.L.); sunhua@tust.edu.cn (H.S.); Tel.: +86-22-60912562 (K.L. & H.S.)
Abstract: Our previous study found that desmethylxanthohumol (
Recently, further investigations revealed that dehydrocyclodesmethylxanthohumol (
analogue rottlerone ( ) exhibited more potent -glucosidase inhibitory activity than
of this study was to synthesize a series of rottlerone analogues and evaluate their -glucosidase
and DPP-4 dual inhibitory activity. The results showed that compounds 4d and 5d irreversibly
and potently inhibited -glucosidase (IC50 = 0.22 and 0.12 M) and moderately inhibited DPP-4
IC50 = 23.59 and 26.19 M), respectively. In addition, compounds 4d and 5d significantly promoted
1
) inhibited
α
-glucosidase in vitro
) and its dimer
. The aim
.
2
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
3
α
1
ꢀ
ꢁꢂꢃꢄꢅꢆ
α
Citation: Zhang, Y.; Wang, H.;
Wu, Y.; Zhao, X.; Yan, Z.; Dodd, R.H.;
Yu, P.; Lu, K.; Sun, H. Synthesis of
Rottlerone Analogues and Evaluation
of Their α-Glucosidase and DPP-4
Dual Inhibitory and Glucose
Consumption-Promoting Activity.
Molecules 2021, 26, 1024.
α
µ
(
µ
glucose consumption, with the activity of 5d at 0.2 µM being comparable to that of metformin at a
concentration of 1 mM.
Keywords: rottlerone;
α-glucosidase inhibitor; DPP-4 inhibitor; inhibitory mechanism; glucose
https://doi.org/10.3390/
consumption
molecules26041024
Academic Editors: Marisa Freitas,
Daniela Ribeiro, Eduarda Fernandes
and Andrzej Kutner
1
. Introduction
Diabetes mellitus is a multi-gene metabolic disease that is characterized by hyper-
Received: 27 January 2021
Accepted: 9 February 2021
Published: 15 February 2021
glycemia that is caused by a relative (Type 2 Diabetes) or absolute (Type 1 Diabetes) insulin
deficiency. The number of diabetes patients has been increasing rapidly in recent years
due to changes in people’s living standards and dietary habits. The number of adult
diabetes patients worldwide is about 463 million, and this number may reach 578 million
by 2030, according to the statistical results of the International Diabetes Federation (IDF) in
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
2019 [
1]. Diabetes is becoming one of the most widespread health burning problems in the
elderly [2].
α
-Glucosidase plays an important role in the physiological process of postprandial
digestion and the absorption of carbohydrates in food in patients with Type 2 Diabetes,
and it is the key enzyme involved in the hydrolysis of dietary carbohydrates [
Glucosidases specifically hydrolyze the -1,4-glucopyranosidic bond to release glucose.
Therefore, the inhibition of digestive -glucosidase is one therapeutic approach to slowing
down carbohydrate digestion and glucose absorption, thereby stabilizing blood glucose
level and preventing hyperglycemia in diabetic patients. -Glucosidase inhibitors have
become a better choice in the case of Asian diabetic patients that are associated with
high consumption of carbohydrates in their staple diet [ ]. Dipeptidyl peptidase-4 (DPP-
3]. α-
α
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
α
α
4
4) inhibitors enhance the plasma level of active glucagon-like peptide-1 (GLP-1) and
glucose-dependent insulinotropic polypeptide (GIP), which results in increased insulin
4
.0/).
Molecules 2021, 26, 1024. https://doi.org/10.3390/molecules26041024
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1024
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secretion and decreased glucagon secretion. It has been reported that the addition of a
DPP-4 inhibitor to patients with type 2 diabetes that is inadequately controlled by an
α
-glucosidase inhibitor achieved better glycemic control without further increasing the
risk of weight gain and hypoglycemia [ ]. Thus, the development of dual inhibitors of
glucosidase and DPP-4 may provide a novel approach to the discovery of new antidiabetic
agents. To the best of our knowledge, such -glucosidase and DPP-4 dual inhibitors
have never been reported. It should be noted that the development of new -glucosidase
5
α-
α
α
inhibitors has been largely neglected, since the introduction of acarbose, voglibose, and
miglitol leave much room for the improvement of anti-diabetic therapies [3].
Desmethylxanthohumol (
anticancer and antioxidant activity [
anthohumol inhibits -glucosidase in vitro
revealed that dehydrocyclodesmethylxanthohumol (
) [ ] exhibited more potent -glucosidase inhibitory activity than
we prepared a series of rottlerone derivatives 4a 4d and 5a 5d for the purpose of studying
structure-activity relationships and improving the -glucosidase inhibitory potencies of
compounds . In addition, the synthesized compounds were also evaluated for DPP-4
1
, Figure 1), isolated from hops, has been found to display
]. In a previous study, we found that desmethylx-
]. Furthermore, our recent investigations
) and its dimer analogue rottlerone
(Figure 1). Therefore,
6,7
α
[8
2
(
3
9
α
1
–
–
α
1–3
inhibition with the goal of identifying potential dual-targeted anti-diabetic drugs.
Figure 1. Structures of 1–3 and rottlerone analogues.
2. Results
2.1. Synthesis and Characterisation of Rottlerone Analogues
The synthesis of rottlerone analogues 4a
shown in Scheme 1. The mono-MOM protected 2’,4’,6’-trihydroxyacetophenone (
synthesized from 2’,4’,6’-trihydroxyacetophenone according to a known procedure [10].
Compound was converted to by cyclization with 3-methyl-2-butenal, followed by
removal of the MOM protection to provide . Conversion of the latter to dimer was
achieved by reaction with formaldehyde [11]. The rottlerone derivatives 4a 4h were then
obtained by base-catalyzed aldol condensation of with the corresponding substituted
benzaldehydes. Deprotection of 4e 4h using 2M hydrochloride acid afforded 5a 5d. Fi-
–4d
,
5a–
5d, and 12
–13 was accomplished, as
6
) was
6
7
8
9
–
9
–
–
nally, methylated rottlerone derivatives 12 and 13 were obtained by aldol condensation of
compound 9 with benzaldehyde followed by O-methylation using CH N in DCM.
2
2

Molecules 2021, 26, 1024
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Scheme 1. Synthesis of rottlerone derivatives. Reagents and conditions: (a) (1) MOMCl, DIPEA, DCM, r.t. 6 h, 82%. (2) 2 M
◦
◦
◦
HCl, MeOH, 30 C, 3 h, 61%. (b) 3-methyl-2-butenal, pyridine, 120 C, 75%. (c) 3 M HCl, MeOH, 30 C, 6 h, 75%. (d) HCHO,
1
◦
◦
DCM, reflux, 8 h, 71%. (e) R CHO, 30% KOH, EtOH, 50 C, 6 h, 56–88 %. (f) 2M HCl, MeOH, THF, 45 C, 24 h, 70–81%. (g)
CH N , DCM, r.t. 5 h, 30–44%.
2
2
2
.2. Rottlerone Analogues Inhibited α-Glucosidase and DPP-4
The -glucosidase and DPP-4 inhibitory activities of the synthesized compounds were
evaluated. Acarbose and sitagliptin, clinically useful -glucosidase and DPP-4 inhibitors,
respectively, were chosen as the reference compounds for activity comparison. Dimer was
first evaluated and compared with the inhibitory activity of compound and rottlerone
), as shown in Table 1. Interestingly, replacement of the styrene moiety of rottlerone by
a methyl group ( ) led to a reduction in -glucosidase inhibition. The variation of the
electronic properties of para substituents on the phenyl group did not significantly alter the
-glucosidase inhibitory activity (4a and 4b vs. ). However, the introduction of a hydroxy
group at this para position (5a) resulted in a 10-fold improvement in activity. Furthermore,
α
α
9
2
(3
9
α
α
3

Molecules 2021, 26, 1024
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the
α
-glucosidase inhibitory activities were found to be two-fold and four-fold higher
than 5a in the case of the o,p-dihydroxyphenyl and o,m,p-trihydroxyphenyl derivatives
and 5d, respectively. The lower activity of the o,m-dihydroxy derivative 5c is perhaps
5b
due to the formation of an intramolecular hydrogen bond between the adjacent hydroxy
groups. Notably, compound 5d also showed a modest DPP-4 inhibitory activity with an
IC50 = 26.19 µM. Altogether, these results revealed that free phenolic hydroxy groups are
crucial for obtaining the desired activities.
Table 1. α-Glucosidase and DPP-4 inhibitory activities of rottlerone derivatives.
IC50 (µM) a
Entry
α-Glucosidase
DPP-4
1
2
3
22.42 ± 3.10
9.74 ± 0.73
5.91 ± 0.19
6.41 ± 0.33
8.71 ± 0.54
1.84 ± 0.41
0.22 ± 0.07
0.51 ± 0.19
0.24 ± 0.02
1.07 ± 0.06
0.12 ± 0.02
7.11 ± 0.05
3.39 ± 0.13
0.37 ± 0.04
-
>50
>50
>50
>50
>50
4
a
4
4
b
c
21.48 ± 0.09
23.59 ± 0.14
>50
4
5
d
a
5
5
b
c
>50
>50
5
d
26.19 ± 0.22
9
>50
1
1
2
3
>50
>50
Sitagliptin
Acarbose
0.08 ± 0.01
188.57 ± 5.52
-
a
Results are the average of three independent experiments, each performed in duplicate.
Compounds 4c and 4d, in which the phenyl ring is replaced by a furan or a thiophene
ring, possessed three-fold and 26-fold higher
α-glucosidase inhibitory activity than 3. Note-
worthy, compound demonstrated some degree of DPP-4 inhibitory activity (Table 1). This
3
suggests that the phenyl ring of the rottlerone analogues can be advantageously replaced
by a heterocyclic ring and it opens the way to a wide variety of further possible structural
modifications to improve activity.
The methylation of the phenol hydroxyls on the 2H-chromene nucleus was also found
to have considerable influence on
α-glucosidase inhibitory activity. In particular, the
trimethylated derivative 13 demonstrated a 10-fold enhancement of this activity when
compared to the demethylated analogue 12 and comparable activity to that of 4d and 5d
.
We hypothesize that the 7-methoxy group prevents the formation of an H-bond between
0
the 7- and 7 -hydroxyls, resulting in a vastly different steric environment favorable to
α-glucosidase inhibitory activity.
2.3. The Enzyme Kinetics of Compounds 4d and 5d
The most active -glucosidase inhibitors of our series, 4d and 5d, were then used for
α
kinetic analyses in order to shed light on their mechanism of action. The plots of the velocity
versus enzyme concentrations in the presence of different concentrations of 4d and 5d
gave a family of parallel straight lines (Figure 2a,b). Increasing the inhibitor concentrations
did not affect the slopes of the lines, indicating that 4d and 5d are irreversible inhibitors.
Moreover, the value of 1/V increased with increasing concentrations of 5d, but the intercept
on the X-axis and Y-axis remained constant. This result indicates that compound 5d is a
mixed competitive α-glucosidase inhibitor.

Molecules 2021, 26, 1024
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0
.008
0.006
0.004
0
0
2
μM
0 μM
0
0
.2 μM
.5 μM
.05 μM
.5 μM
0.006
0.004
0.002
0.002
0
.000
0.000
0
2
4
6
8
0
2
4
6
8
Enzyme Concentration (mU)
Enzyme Concentration (mU)
(a)
(b)
4
000
0
0
0
μM
.2 μM
.5 μM
3000
2000
1000
-3
-2
-1
0
1
2
3
1
/(S, mM)
c)
(
Figure 2. Inhibitory kinetics of 4d and 5d on
glucosidase ( , 0 M; , 0.05 M; , 2.5 M). (
, 0.2 M; , 0.5 M). ( ) Lineweaver–Burk plots for inhibition of 5d
α
-glucosidase. (
) The interactions of 5d and
, 0 M;
a
) The interactions of 4d and
-glucosidase ( , 0
, 0.2 M; , 0.5
α-
M;
M).
•
µ
N
c
µ
ꢀ
µ
b
α
•
µ
N
µ
ꢀ
µ
(
•
µ
N
µ
ꢀ
µ
2.4. Glucose Consumption of Compounds 4d and 5d
In order to further study the anti-diabetic activity of compounds 4d and 5d, their
effects on cell-based glucose consumption were evaluated using metformin as a positive
control. Compound 5d was observed to significantly promote glucose consumption at 0.2
and 0.04
µM, while compound 4d showed similar potency at 0.2
µM, as shown in Figure 3.
5
4
3
2
1
0
***
*
**
**
*
Figure 3. Compounds 4d and 5d promoted glucose consumption in HepG2 cells. The data are the
average of three independent experiments, each performed in duplicated. * p < 0.05, ** p < 0.01, and
*
** p < 0.001 vs. control.

Molecules 2021, 26, 1024
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3. Materials and Methods
3.1. Chemistry
All of the reagents and solvents were commercially available and used without further
1
13
purification. The H-NMR and C-NMR spectra were recorded on a Bruker AM-400 NMR
spectrometer (Billerica, Middlesex, MA, USA) in CDCl or d -DMSO (dimethyl sulfoxide).
3
6
Mass spectra were obtained on a Q-TOF mass spectrometer (Agilent, Santa Clara, CA,
USA) and high resolution mass spectra were obtained on a hybrid IT-TOF mass spectrome-
ter (Shimadzu LCMS-IT-TOF, Kyoto, Japan). Compound purity was determined by UV
absorbance at 254 nm during tandem liquid chromatography/mass spectrometry (Agilent,
Santa Clara, CA, USA). All of the final products had a purity of
this method.
≥
95%, as determined using
3.1.1. 1-(2,6-Dihydroxy-4-(methoxymethoxy)phenyl)ethan-1-one (6)
1
-(2,4,6-Trihydroxyphenyl)ethan-1-one (1.68 g, 10 mmol) in dry CH Cl (100 mL)
2
2
were added N,N-diethylpropan-2-amine (3.93 g, 25 mmol) and chloro(methoxy)methane
◦
(
1.77 g, 22 mmol) at 0 C. The reaction mixture was warmed to room temperature and
then stirred for 6 h. The reaction was quenched with NH Cl (aq) and extracted with
4
CH Cl (500 mL) three times. The combined organic layers were washed with brine,
2
2
dried over Na SO , and concentrated under reduced pressure. The crude product was
2
4
purified by column chromatography on silica (petroleum ether/ethyl acetate 20:1) to afford
-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-1-one (2.1 g, 82%) as a colorless oil. 1-
1
(
2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-1-one (1.28 g, 5 mmol) in MeOH (50 mL)
◦
was added 2 N HCl (5 mL) at 0 C. After addition, the reaction mixture was warmed to
4
◦
0 C and then stirred for 3 h. The reaction was quenched with NaHCO (aq) and extracted
3
with EtOAc (100 mL) three times. The combined organic layers were washed with brine,
dried over Na SO , and concentrated under reduced pressure. The crude product was
2
4
purified by column chromatography on silica (petroleum ether/ethyl acetate 10:1) to afford
1
a compound 6 (0.65 g, 61%) as a white solid. H-NMR (400 MHz, d -DMSO) 12.26 (s, 2H),
6
6
.02 (s, 2H), 5.17 (s, 2H), 3.36 (s, 3H), 2.58 (s, 3H). 13C-NMR (100 MHz, d -DMSO) 203.8,
6
164.4, 163.6, 105.8, 95.1, 93.9, 56.3, 33.0.
3
.1.2. 1-(7-Hydroxy-5-(methoxymethoxy)-2,2-dimethyl-2H-chromen-8-yl) ethan-1-one (
Compound (2.12 g, 10.0 mmol) in dry pyridine (20 mL) was added 3-methyl-2-
butenal (1.26 g, 15.0 mmol). The reaction was heated to 120 C for 16 h. The mixture was
cooled and diluted with CH Cl (20 mL). The mixture was poured into 5% HCl (aq, 20 mL).
7)
6
◦
2
2
The organic phase was washed with NaHCO (aq) (20 mL) and brine (3
×
20 mL). The
3
organic phase was dried over Na SO and then concentrated under reduced pressure. The
2
4
crude product was purified by column chromatography on silica (petroleum ether/ethyl
1
acetate 50:1) to afford a compound
7
(2.09 g, 75%) as a yellow oil. H-NMR (400 MHz,
CDCl ) 13.64 (s, 1H), 6.58 (d, J = 10.0 Hz, 1H), 6.20 (s, 1H), 5.43 (d, J = 10.0 Hz, 1H), 5.20
3
13
(
s, 2H), 3.47 (s, 3H), 2.67 (s, 3H), 1.50 (s, 6H). C-NMR (100 MHz, CDCl ) 202.4, 164.9,
3
157.4, 155.4, 123.8, 115.5, 105.5, 102.2, 93.9, 93.2, 77.0, 55.5, 32.2, 26.9.
3.1.3. 1-(5,7-Dihydroxy-2,2-dimethyl-2H-chromen-8-yl)ethan-1-one (8)
Compound
7 (2.0 mmol) in MeOH (10 mL) was added 3 N HCl (2.5 mL) and then
◦
heated to 60 C for 6 h. The mixture was poured in cold NaHCO (aq) and extracted with
3
EtOAc (3
×
20 mL). The combined organic layers were washed with brine, dried over
Na SO , and concentrated under reduced pressure. The crude product was purified by
2
4
column chromatography on silica (petroleum ether/ethyl acetate 20:1) in order to afford a
1
compound
8
(250 mg, 53%) as a yellow solid. H-NMR (400 MHz, d -DMSO) 13.55 (s, 1H),
6
1
1
1
0.83 (s, 1H), 6.47 (d, J = 10.0 Hz, 1H), 5.90 (s, 1H), 5.52 (d, J = 10.0 Hz, 1H), 2.58 (s, 3 H),
.45 (s, 6H). 13C-NMR (100 MHz, d -DMSO) 203.0, 165.3, 160.4, 157.0, 125.2, 116.7, 105.1,
6
02.3, 95.8, 78.3, 33.3, 27.8.

Molecules 2021, 26, 1024
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3
.1.4. 1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
ethan-1-one) (9)
Compound
8 (1.0 mmol) in CH Cl (10 mL) was added polyformaldehyde (0.5 mmol)
2 2
and refluxed for 8 h. Solvent was removed under reduced pressure and the residue was
purified by column chromatography on silica (petroleum ether/ethyl acetate 200:1) to
1
afford a compound
9
(170 mg, 71%) as a yellow solid. H-NMR (400 MHz, CDCl ) 15.81
3
(
(
1
s, 2H), 9.43(s, 2H), 6.63 (d, J = 10.0 Hz, 2H), 5.43 (d, J = 10.0 Hz, 2H), 3.75 (s, 2H), 2.67
s, 6H), 1.47 (s, 12H). 13C-NMR (100 MHz, CDCl ) 202.8, 160.2, 157.3, 154.9, 124.0, 116.0,
3
+
04.9, 104.0, 102.4, 77.1, 31.7, 26.9, 14.4. HRMS (ESI) m/z: calcd for C27H28O8 [M + H] ,
481.2656; found, 481.2604.
3.1.5. General Procedure for the Synthesis of Compounds 3 and 4a–4h
Compound
9 (0.5 mmol) in EtOH (3 mL) was added 30% KOH (aq, 1 mL) and
◦
◦
substituted benzaldehyde (1.2 mmol) at 0 C. The reaction was heated to 50 C for 6 h.
After cooling down to 0 C, 2N HCl was added to the reaction mixture until the pH = 5–6.
◦
The precipitate was filtered and then purified by column chromatography on silica to give
compounds 3 and 4a–4d.
3
.1.6. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-phenylprop-2-en-1-one) (3)
1
A red solid (0.266 g, yield 81%). H-NMR (400 MHz, CDCl ) 9.67 (s, 2H), 8.19 (d,
3
J = 15.6 Hz, 2H), 7.83 (d, J = 15.6 Hz, 2H), 7.62–7.60 (m, 4H), 7.42–7.40 (m, 6H), 6.68 (d,
J = 12.0 Hz, 2H), 5.47 (d, J = 12.0 Hz, 2H), 3.82 (s, 2H), 1.54 (s, 12H). 1 C-NMR (100 MHz,
3
CDCl ) 192.9, 162.8, 158.9, 155.4, 143.3, 135.5, 130.4, 129.0, 128.4, 126.8, 125.1, 117.3, 106.5,
3
105.3, 103.8, 78.2, 28.0, 15.7.
3
.1.7. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(4-methoxyphenyl)prop-2-en-1-one) (4a)
1
A red solid (0.270 g, yield 75%). H-NMR (400 MHz, CDCl ) 9.70 (s, 2H), 8.10 (d,
3
J = 15.6 Hz, 2H), 7.83 (d, J = 15.6 Hz, 2H), 7.57 (d, J = 8.8 Hz, 4H), 6.95 (d, J = 8.8 Hz, 4H),
6
.69 (d, J = 9.6 Hz, 2H), 5.48 (d, J = 10.0 Hz, 2H), 3.86 (s, 6H), 3.82 (s, 2H), 1.54 (s, 12H).
1
3
C-NMR (100 MHz, CDCl ) 192.7, 162.9, 161.6, 158.7, 155.3, 143.4, 130.2, 128.3, 124.9, 124.4,
3
1
17.4, 114.5, 106.6, 105.3, 78.0, 55.4, 28.0, 15.8. HRMS (ESI) m/z: calcd for C43H40O10
+
[
M + H] , 717.3394; found, 717.3366.
3
.1.8. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(4-fluoro phenyl)prop-2-en-1-one) (4b)
A red solid (0.270g, yield 78%). 1H-NMR (400 MHz, CDCl ) 9.66 (s, 2H), 8.10 (d,
3
J = 15.6 Hz, 2H), 7.79 (d, J = 15.6 Hz, 2H), 7.57–7.61 (m, 4H), 7.09-7.13 (d, 4H), 6.69 (d,
1
3
J = 9.6 Hz, 2H), 5.48 (d, J = 10.0 Hz, 2H), 3.86 (s, 6H), 3.82 (s, 2H), 1.54 (s, 12H). C-NMR
100 MHz, CDCl ) 192.7, 162.8, 158.9, 155.4, 142.0, 131.8, 130.2, 130.1, 126.6, 125.1, 117.3,
(
3
1
16.3, 116.1, 106.5, 105.2, 103.8, 78.2, 28.0, 15.7. HRMS (ESI) m/z: calcd for C41H34F2O8
+
[
M + H] , 693.2394; found, 693.2301.
3
.1.9. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(furan-3-yl)prop-2-en-1-one) (4c)
1
A white solid (0.253 g, yield 75%). H-NMR (400 MHz, CDCl ) 9.62 (s, 2H), 8.38 (d,
3
J = 15.6 Hz, 2H), 7.69 (d, J = 15.2 Hz, 2H), 7.61 (d, J = 15.2 Hz, 2H), 7.47 (d, J = 8.2 Hz, 2H),
.37 (t, J =7.6 Hz, 2H), 7.25(d, J = 14.4 Hz, 2H), 7.01 (s, 2H), 6.68 (d, J =7.2 Hz, 2H), 5.51 (d,
7
13
J = 10.0 Hz, 2H), 3.83 (s, 2H), 1.60 (s, 12H). C-NMR (100 MHz, CDCl3)
δ
192.0, 162.8, 159.0,
1
1
55.6, 155.6, 153.7, 129.4, 128.6, 127.5, 126.5, 125.3, 123.3, 121.8, 117.1, 111.8, 111.2, 106.4,
05.4, 103.9, 78.4, 27.8, 15.7. HRMS (ESI) m/z: calcd for C41H36O8 [M + H] , 657.0782;
+
found, 657.0760.

Molecules 2021, 26, 1024
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3
.1.10. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(thiophen-3-yl)prop-2-en-1-one) (4d)
1
A red solid (0.271 g, yield 85%). H-NMR (400 MHz, CDCl ) 9.68 (s, 2H), 8.02 (q,
3
J = 18.0 Hz, 4H), 7.47–7.29 (m, 2H), 7.09 (d, J = 3.6 Hz, 2H), 6.67 (d, J = 9.6 Hz, 2H), 5.48 (d,
J = 9.6 Hz, 2H), 3.81 (s, 2H), 1.56 (s, 12H). 13C-NMR (100 MHz, CDCl ) 192.0, 162.9, 158.8,
3
1
55.3, 141.4, 136.0, 131.9, 128.6, 128.4, 125.9, 125.1, 117.1, 106.5, 105.1, 103.7, 78.2, 27.9, 15.7.
+
HRMS (ESI) m/z: calcd for C37H32O8S2 [M + H] , 669.3011; found, 669.3024.
3
.1.11. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(4-(methoxymethoxy)phenyl)prop-2-en-1-one) (4e)
1
A red solid (0.343g, 88%). H-NMR (400 MHz, CDCl ) 9.69 (s, 2H), 8.11 (d, J = 15.6 Hz,
3
2
H), 7.82 (d, J = 15.6 Hz, 2H), 7.56 (d, J = 8.4 Hz, 4H), 7.08 (d, J = 8.8 Hz, 4H), 6.68 (d,
J = 10.0 Hz, 2H), 5.48 (d, J = 10.0 Hz, 2H), 5.22 (s, 4H), 3.82 (s, 2H), 3.49 (s, 6H), 1.53 (s, 12H).
13
C-NMR (100 MHz, CDCl ) 192.7, 162.9, 159.1, 158.7, 155.3, 143.2, 130.1, 129.3, 125.0, 124.9,
3
117.4, 116.6, 106.6, 105.3, 103.8, 94.2, 78.1, 56.2, 28.0, 15.8.
3
.1.12. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(2,4-bis(methoxymethoxy)phenyl)prop-2-en-1-one) (4f)
1
A red solid (0.250g, 56%). H-NMR (400 MHz, CDCl ) 9.71 (s, 2H), 8.26 (d, J = 15.6 Hz,
3
2
H), 8.09 (d, J = 16.0 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 2.4 Hz, 2H), 6.76 (dd,
J = 8.8, 2.4 Hz, 2H), 6.68 (d, J = 10.0Hz, 2H), 5.47 (d, J = 10.0 Hz, 2H), 5.27 (s, 4H), 5.20 (s,
H), 3.82 (s, 2H), 3.52 (s, 6H), 3.50 (s, 6H), 1.52 (s, 12H). 13C-NMR (100 MHz, CDCl ) 192.9,
4
3
1
1
63.0, 160.5, 159.0, 158.6, 157.8, 155.3, 138.2, 128.4, 125.0, 124.7, 119.3, 117.4, 109.6, 106.6,
05.3, 103.7, 103.6, 94.8, 94.3, 78.0, 56.4, 56.3, 28.0, 15.8.
3
.1.13. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(2,3-bis(methoxymethoxy)phenyl)prop-2-en-1-one) (4g)
1
A red solid (0.367g, 82%). H-NMR (400 MHz, CDCl ) 9.70 (s, 2H), 8.30 (d, J = 15.6 Hz,
3
2
7
4
H), 8.17 (d, J = 16.0 Hz, 2H), 7.34 (dd, J = 8.0, 1.2 Hz, 2H), 7.20 (dd, J = 8.0, 1.2 Hz, 2H),
.09 (t, J = 8.0 Hz, 2H), 6.67 (d, J = 10.0 Hz, 2H), 5.48 (d, J = 10.0 Hz, 2H), 5.22 (s, 4H), 5.20 (s,
H), 3.82 (s, 2H), 3.66 (s, 6H), 3.52 (s, 6H), 1.52 (s, 12H). 13C-NMR (100 MHz, CDCl ) 192.9,
3
1
1
63.0, 158.8, 155.4, 150.5, 146.5, 138.4, 130.7, 127.7, 125.1, 124.6, 120.1, 118.2, 117.2, 106.6,
05.3, 103.8, 99.5, 95.2, 78.2, 58.0, 56.3, 28.0, 15.8.
3
.1.14. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(2,3,4-tris(methoxymethoxy)phenyl)prop-2-en-1-one) (4h)
1
A red solid (0.397g, 76%). H-NMR (400 MHz, CDCl ) 9.72 (s, 2H), 8.24 (d, J = 15.6 Hz,
3
2
H), 8.13 (d, J = 15.6 Hz, 2H), 7.40 (d, J = 9.2 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.66 (d,
J = 10.0 Hz, 2H), 5.47 (d, J = 10.0 Hz, 2H), 5.25 (s, 4H), 5.21 (s, 4H), 5.16 (s, 4H), 3.81 (s, 2H),
.65 (s, 6H), 3.62 (s, 6H), 3.52 (s, 6H), 1.52 (s, 12H). 13C-NMR (100 MHz, CDCl ) 192.7, 163.1,
3
3
1
1
58.7, 155.3, 153.2, 151.1, 139.7, 138.5, 125.9, 125.0, 124.6, 122.6, 117.3, 112.0, 106.6, 105.2,
03.7, 100.0,98.8, 95.0, 78.1, 58.2, 57.4, 56.4, 28.0, 15.8.
3.1.15. General Procedure for the Synthesis of Compounds 5a–5d
Compounds 4e
–
4h (0.10 mmol) in MeOH (3 mL) and THF (3 mL) was added 2 N HCl
◦
◦
(
1 mL) at 0 C. After addition, the mixture was heated to 45 C for 24 h. The mixture was
poured in cold NaHCO (aq, 10 mL) and then extracted with EtOAc (3
×
20 mL). The com-
3
bined organic layers were washed with brine, dried over Na SO , and then concentrated
2
4
under reduced pressure. The crude product was purified by column chromatography on
silica (petroleum ether/ethyl acetate 4:1) to afford a compounds 5a–5d.
3
.1.16. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(4-hydroxyphenyl)prop-2-en-1-one) (5a)
1
A red solid (55 mg, 81%). H-NMR (400 MHz, d -DMSO) 15.36 (s, 2H), 10.17 (s,
6
2
H), 7.92 (d, J = 15.6 Hz, 2H), 7.72 (d, J = 15.6 Hz, 2H), 7.56 (d, J = 8.8 Hz, 4H), 6.87 (d,

Molecules 2021, 26, 1024
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J = 8.8 Hz, 4H), 6.63 (d, J = 10.0 Hz, 2H), 5.60 (d, J = 10.0 Hz, 2H), 3.78 (s, 2H), 1.50 (s, 12H).
13
C-NMR (100 MHz, d -DMSO) 192.5, 163.6, 160.7, 154.5, 143.9, 130.9, 126.4, 125.7, 123.6,
6
1
17.2, 116.6, 107.3, 105.2, 103.0, 78.0, 27.9, 16.3. HRMS (ESI) m/z: calcd for C41H36O10
+
[
M + H] , 689.2181; found, 689.2145.
3
.1.17. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(2,4-dihydroxyphenyl)prop-2-en-1-one) (5b)
1
A red solid (50 mg, 70%). H-NMR (400 MHz, d -DMSO) 16.20 (s, 2H), 10.41 (s, 2H),
6
1
(
2
0.06 (s, 2H), 8.16 (d, J = 15.6 Hz, 2H), 7.94 (d, J = 15.6 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 6.60
d, J = 10.0 Hz, 2H), 6.43 (d, J = 2.4 Hz, 2H), 6.34 (dd, J = 8.4, 2.0 Hz, 2H), 5.57 (d, J = 9.6 Hz,
H), 3.75 (s, 2H), 1.49 (s, 12H). 13C-NMR (100 MHz, d -DMSO) 192.8, 163.8, 162.0, 160.2,
6
1
54.7, 141.7, 132.52 125.7, 122.7, 117.2, 114.3, 108.7, 107.2, 105.1, 103.2, 103.1, 78.1, 27.7, 16.0.
+
HRMS (ESI) m/z: calcd for C41H36O12 [M + H] . 721.2279; found, 721.2280.
3
.1.18. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(2,3-dihydroxyphenyl)prop-2-en-1-one) (5c)
1
A red solid (52 mg, 73%). H-NMR (400 MHz, d -DMSO) 15.30 (s, 2H), 9.71 (s, 2H),
6
9
(
3
.22 (s, 2H), 8.15 (d, J = 15.6 Hz, 2H), 7.99 (d, J = 15.6 Hz, 2H), 7.02 (d, J = 7.2 Hz, 2H), 6.86
d, J = 7.6 Hz, 2H), 6.72 (t, J = 7.8 Hz, 2H), 6.61 (d, J =10.0 Hz, 2H), 5.55 (d, J = 9.6 Hz, 2H),
.76 (s, 2H), 1.48 (s, 12H). 13C-NMR (100 MHz, d -DMSO) 193.0, 163.7, 154.6, 146.6, 146.3,
6
1
39.8, 126.5, 125.9, 122.7, 119.8, 119.4, 117.6, 117.2, 107.2, 105.3, 103.0, 79.6, 78.2, 27.8, 16.3.
−
HRMS (ESI) m/z: calcd for C41H36O12 [M − H] , 719.2134; found, 719.2112.
3
.1.19. (2Z,2’Z)-1,1’-(Methylenebis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
3-(2,3,4-trihydroxyphenyl)prop-2-en-1-one) (5d)
1
A red solid (59 mg, 78%). H-NMR (400 MHz, d -DMSO)
δ
16.07 (s, 2H), 9.89 (s, 2H),
6
9
.32 (s, 2H), 8.66 (s, 2H), 8.09 (d, J = 15.6 Hz, 2H), 7.98 (d, J = 15.6 Hz, 2H), 6.93 (d, J = 8.8 Hz,
H), 6.60 (d, J = 10.0 Hz, 2H), 6.42 (d, J = 8.8 Hz, 2H), 5.56 (d, J = 10.0 Hz, 2H), 3.74 (s, 2H),
192.8, 163.7, 154.7, 149.9, 148.5, 133.5, 126.0,
2
1
1
.48 (s, 12H). 13C-NMR (100 MHz, d -DMSO)
δ
6
22.7, 121.2, 117.1, 115.2, 108.5, 107.1, 105.2, 103.0, 79.6, 78.2, 27.7, 16.2. HRMS (ESI) m/z:
−
calcd for C41H35O14 [M − H] , 751.2032; found, 751.2030.
3.1.20. General Procedure for the Synthesis of Compounds 10–11
A solution of KOH (20% aq, 20 mL) and ether (20 mL) was added methyl nitrosourea
◦
(
1 g) at 0 C and then stirred for 10 min. Compound
9
(1.0 mmol) in CH Cl (10 mL)
2
2
◦
was dripped above ether solution at 0 C. The reaction mixture was stirred for 6 h at
r.t. The reaction was quenched with glacial acetic acid (0.1 mL) and then extracted with
EtOAc (3
×
100 mL). The combined organic layers were washed with brine, dried over
Na SO , and then concentrated under reduced pressure. The crude product was purified
2
4
by column chromatography on silica (petroleum ether/ethyl acetate 100:1 and 50:1) to
afford compounds 10 and 11.
3
.1.21. 1,1’-(Methylenebis(7-hydroxy-5-methoxy-2,2-dimethyl-2H-chromene-6,8-diyl))bis
(
ethan-1-one) (10)
1
A yellow solid (0.150 g, yield 30%). H-NMR (400 MHz, CDCl ) 13.79 (s, 2H), 6.46
3
(
1
d, J = 10.0 Hz, 2H), 5.46 (d, J = 9.6 Hz, 2H), 3.87 (s, 2H), 3.64 (s, 6H), 2.67 (s, 6H), 1.48 (s,
13
2H). C-NMR (100 MHz, CDCl ) 204.1, 163.7, 161.2, 155.0, 125.4, 117.7, 114.4, 108.0, 106.2,
3
65.8, 61.8, 33.5, 27.7, 15.2.
3
5
.1.22. 1-(6-((8-Acetyl-5,7-dimethoxy-2,2-dimethyl-2H-chromen-6-yl)methyl)-7-hydroxy-
-methoxy-2,2-dimethyl-2H-chromen-8-yl)ethan-1-one (11)
1
A yellow solid (0.230 g, yield 44%). H-NMR (400 MHz, CDCl ) 13.83 (s, 1H), 6.49 (d,
3
J = 10.0 Hz, 1H), 6.44 (d, J = 10.0 Hz, 1H), 5.57-5.53 (d, J= 10.0 Hz, 1H), 5.47 (d, J = 10.0 Hz,
1
1
H), 3.91 (s, 2H), 3.61 (s, 6H), 3.52 (s, 3H), 2.67 (d, J = 5.6 Hz, 3H), 2.50 (s, 3H), 1.48 (s, 6H),
.40 (s, 6H). 13C-NMR (100 MHz, CDCl ) 204.1, 201.9, 163.6, 161.1, 156.4, 156.3, 155.1, 149.1,
3

Molecules 2021, 26, 1024
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1
29.0, 125.5, 121.4, 120.6, 117.6, 117.1, 114.5, 111.2, 107.9, 106.3, 77.5, 76.4, 62.9, 62.1, 61.6,
33.5, 32.6, 27.6, 27.6, 17.6.
3
.1.23. (2Z,2’Z)-1,1’-(Methylenebis(7-hydroxy-5-methoxy-2,2-dimethyl-2H-chromene-6,8-
diyl))bis(3-phenylprop-2-en-1-one) (12)
Compound 10 (0.2 mmol) in EtOH (3 mL) was added 30% KOH (aq, 1 mL) and
◦
benzaldehyde (0.48 mmol). The reaction mixture was stirred at 50 C for 6 h. After cooling
◦
down to 0 C, 2 N HCl was added to the reaction mixture until the pH 5–6. The precipitate
was filtered and dried to give compound 12 (104 mg, 88%) as a red solid. H-NMR
1
(
400 MHz, CDCl ) 13.95 (s, 2H), 8.07 (d, J = 15.6Hz, 2H), 7.76 (d, J =15.6 Hz, 2H), 7.62–7.57
3
(
6
m, 4H), 7.46-7.34 (m, 6H), 6.48 (t, J = 11.4 Hz, 2H), 5.53-5.46 (m, 2H), 3.98 (s, 2H), 3.68 (s,
H), 1.54 (s, 12H). 13C-NMR (100 MHz, CDCl ) 193.7, 164.3, 161.4, 154.4, 142.1, 135.6, 130.0,
3
1
28.9, 128.2, 127.9, 125.4, 117.8, 114.8, 108.5, 106.5, 61.9, 27.8, 17.0. HRMS (ESI) m/z: calcd
+
for C H O [M + H] , 685.3995; found, 685.3970.
43
40
8
3
.1.24. (Z)-1-(6-((5,7-Dimethoxy-2,2-dimethyl-8-((Z)-3-phenylacryloyl)-2H-chromen-6-
yl)methyl)-7-hydroxy-5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-2-en-1-
one (13)
Compound 11 (0.19 mmol) in EtOH (3 mL) was added 30% KOH (aq, 1 mL) and
◦
benzaldehyde (0.46 mmol). The reaction mixture was stirred at 50 C for 6 h. After cooling
◦
down to 0 C, 2 N HCl was added to the reaction mixture until the pH 5-6. The precipitate
was filtered and dried to give compound 13 (117 mg, 87%) as a red solid. H-NMR
1
(
(
400 MHz, CDCl ) 14.00 (s, 1H), 8.07 (d, J = 15.6 Hz, 1H), 7.77 (d, J = 15.6 Hz, 1H), 7.60
dd, J = 5.6, 1.8 Hz, 2H), 7.52 (dd, J = 6.6, 3.6 Hz, 2H), 7.44–7.39 (m, 4H), 7.39–7.32 (m, 5H),
3
7
.03–6.97 (m, 1H), 6.55–6.50 (m, 1H), 6.49 (d, J = 9.8 Hz, 1H), 5.57-5.53 (m, 1H), 5.53–5.49 (m,
1
3
1
H), 3.98 (s, 2H), 3.66 (s, 3H), 3.62 (s, 3H), 3.60 (s, 3H), 1.53 (s, 6H), 1.34 (s, 6H). C-NMR
(
100 MHz, CDCl ) 194.4, 193.7, 164.3, 161.3, 157.1, 156.5, 154.5, 149.8, 144.9, 142.2, 135.5,
3
1
1
34.9, 130.2, 130.1, 129.0, 128.9, 128.8, 128.8, 128.4, 128.2, 127.7, 125.6, 120.4, 119.1, 117.8,
17.2, 115.0, 111.2, 108.5, 106.6, 77.5, 76.3, 62.6, 62.1, 61.7, 27.8, 27.6, 22.6, 17.8, 14.1. HRMS
+
(
ESI) m/z: calcd for C44H42O8 [M + Na] , 733.2785; found, 733.2770.
3.2. α-Glucosidase Inhibition Assay
The commercially available
selected as the target protein while using p-nitrophenyl-
α
-glucosidase from baker’s yeast (Sigma, G5003) was
-D-glucopyranoside (pNGP, Sigma,
α
N1377) as the substrate. The compounds and acarbose were dissolved in DMSO. The
enzyme and substrate were dissolved in potassium phosphate buffer (0.05 M, pH 6.8). The
enzymatic reaction mixture that was composed of
α
-glucosidase (0.04 U, 20
µL), substrate
(
0.5 mM, 30
µ
L), test compounds (20
µ
L), and potassium phosphate buffer (130 µ
L) was
◦
incubated at 37 C for 30 min. The enzymatic activity was detected by spectrophotometer
at the wavelength of 405 nm. The results are the average of three independent experiments,
each being performed in duplicate [8].
3.3. DPP-4 Inhibition Assay
The commercially available DPP-4 (PROSPEC, ENZ-375) was selected as the target
protein using glycyl proline p-nitroaniline as the substrate. The compounds and sitagliptin
were dissolved in DMSO. The enzyme and substrate were dissolved in Tris-HCl buffer
(0.1 M, pH 8.0). The enzymatic reaction mixture that was composed of DPP-4 (0.01 U,
5
0
µ
L), substrate (2.2 mM, 50
µ
L), test compounds (10
µ
L), and Tris-HCl buffer (90
µ
L) was
◦
incubated at 37 C for 30 min. The enzymatic activity was detected by spectrophotometer
at the wavelength of 405 nm. The results are the average of three independent experiments,
each being performed in duplicate [12].

Molecules 2021, 26, 1024
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3
.4. Kinetics of Enzyme Inhibition
The kinetics of enzyme inhibition was performed with the same assays detailed above,
in the presence of different concentrations of compounds 4d and 5d, substrate, and enzyme,
respectively. The inhibitory kinetics of 4d and 5d on -glucosidase was analyzed using the
Lineweaver-Burk plot of the substrate concentration and velocity.
α
3.5. Glucose Consumption Assay
Briefly, the HepG2 cells were cultured in Dulbecco’s-modified Eagle’s medium (DMEM)
containing 25 mmol/L D-glucose, 10% heat-inactivated fetal bovine serum (FBS), 10 U/mL
◦
penicillin, and 10 mg/mL streptomycin at 37 C, 5% CO atmosphere. The culture so-
2
lution was replaced every other day and then passaged once for 2–3 d. The cells were
seeded into 96-well plate with twenty-four wells left as blanks. After reaching 70–80%
confluence, the cells were washed with PBS twice and the medium was replaced by DMEM
containing 25 mmol/L D-glucose to hunger for 24 h. The cells were washed with PBS twice
and the medium was replaced by RPMI-1640 containing 11.1 mmol/L glucose that was
supplemented with 0.2% bovine serum albumin (BSA). To the medium was then added
1
mmol/L metformin or purified product at different concentrations, and DMSO was used
as the blank control. The glucose concentration in the medium determined a DMSO after
4 h treatment. Glucose consumption = glucose concentrations of blank wells glucose
2
−
concentrations of plated wells. The sulforhodamine B (SRB) assay was used to adjust the
glucose consumption values. The SRB assay is based on the measurement of cellular pro-
tein content. After an incubation period, the cells were fixed with 10%, 4 C trichloroacetic
◦
◦
acid, stained for 1 h at 4 C, and then washed with purified water. After drying, 60
µL SRB
(
3 mg/mL) was added into each well and then left at room temperature for 30 min. SRB
was dissolved by 1% acetic acid before air drying. Bound SRB was solubilized with 150
µL
Tris-base (10 mmol/L, pH 10.5) solution for OD determination at 546 nm while using a
microplate reader [13].
4
. Conclusions
A series of rottlerone analogues were synthesized and evaluated for their
and DPP-4 inhibitory activities. Compounds 4d and 5d were identified as new potent
-glucosidase inhibitors and moderate DPP-4 inhibitors. The kinetic analysis showed
that compounds 4d and 5d are irreversible -glucosidase inhibitors, and that 5d acts in a
α
-glucosidase
α
α
mixed competitive inhibitory mode. In addition, compounds 4d and 5d showed glucose
consumption-promoting activity in HepG2 cells. Of these two compounds, the activity of
5d
at 0.2
µM was comparable to that of metformin at a concentration of 1 mM. In view of
these significant results, compound 5d, which combines both
α
-glucosidase and DPP-4
inhibitory activities, presents great potential as a lead compound for the development of a
novel dual inhibitor treatment strategy for type 2 diabetes.
Author Contributions: Conceptualization, H.S. and K.L.; synthesis and characterization, Z.Y., H.W.
and Y.W.; activity evaluation, X.Z. and Y.Z.; Writing—original draft preparation, Y.Z.; Writing—
review and editing, H.S. and R.H.D. Funding acquisition, H.S., K.L. and P.Y. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (21502138)
and the Natural Science Foundation of Tianjin (18JCYBJC94800).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: There are no data supporting.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: All samples are available from the authors.

Molecules 2021, 26, 1024
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References
1
.
Saeedi, P.; Petersohn, I.; Salpea, P.; Malanda, B.; Karuranga, S.; Unwin, N.; Colagiuri, S.; Guariguata, L.; Motala, A.A.;
Ogurtsova, K.; et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from
the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res. Clin. Pract. 2019, 157, 107843. [CrossRef] [PubMed]
Longo, M.; Bellastella, G.; Maiorino, M.I.; Meier, J.J.; Esposito, K.; Giugliano, D. Diabetes and Aging: From Treatment Goals to
Pharmacologic Therapy. Front. Endocrinol. 2019, 10, 45. [CrossRef] [PubMed]
Ghani, U. Re-exploring promising α-glucosidase inhibitors for potential development into oral anti-diabetic drugs: Finding
needle in the haystack. Eur. J. Med. Chem. 2015, 103, 133–162. [CrossRef] [PubMed]
Hara, T.; Nakamura, J.; Koh, N.; Sakakibara, F.; Takeuchi, N.; Hotta, N. An importance of carbohydrate ingestion for the
expression of the effect of alpha-glucosidase inhibitor in NIDDM. Diabetes Care 1996, 19, 642–647. [CrossRef] [PubMed]
2.
3.
4.
5.
Min, S.H.; Yoon, J.H.; Hahn, S.; Cho, Y.M. Efficacy and safety of combination therapy with an
tidyl peptidase-4 inhibitor in patients with type 2 diabetes mellitus: A systematic review with meta-analysis. J. Diabetes Investig.
018, 9, 893–902. [CrossRef] [PubMed]
α-glucosidase inhibitor and a dipep-
2
6
.
Delmulle, L.; Bellahcène, A.; Dhooge, W.; Comhaire, F.; Roelens, F.; Huvaere, K.; Heyerick, A.; Castronovo, V.; De Keukeleire, D.
Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines.
Phytomedicine 2006, 13, 732–734. [CrossRef] [PubMed]
7
.
.
Vogel, S.; Ohmayer, S.; Brunner, G.; Heilmann, J. Natural and non-natural prenylated chalcones: Synthesis, cytotoxicity and
anti-oxidative activity. Bioorg. Med. Chem. 2008, 16, 4286–4293. [CrossRef] [PubMed]
Sun, H.; Wang, D.; Song, X.; Zhang, Y.; Ding, W.; Peng, X.; Zhang, X.; Li, Y.; Ma, Y.; Wang, R.; et al. Natural Prenylchalconaringenins
8
and Prenylnaringenins as Antidiabetic Agents: α-Glucosidase and α-Amylase Inhibition and in Vivo Antihyperglycemic and
Antihyperlipidemic Effects. J. Agric. Food Chem. 2017, 65, 1574–1581. [CrossRef] [PubMed]
9
.
Jain, S.K.; Pathania, A.S.; Meena, S.; Sharma, R.; Sharma, A.; Singh, B.; Gupta, B.D.; Bhushan, S.; Bharate, S.B.; Vishwakarma, R.A.
Semisynthesis of mallotus B from rottlerin: Evaluation of cytotoxicity and apoptosis-inducing activity. J. Nat. Prod. 2013
6, 1724–1730. [CrossRef] [PubMed]
,
7
1
0. Teng, Y.; Li, X.; Yang, K.; Li, X.; Zhang, Z.; Wang, L.; Deng, Z.; Song, B.; Yan, Z.; Zhang, Y.; et al. Synthesis and antioxidant
evaluation of desmethylxanthohumol analogs and their dimers. Eur. J. Med. Chem. 2017, 125, 335–345. [CrossRef] [PubMed]
1. Akbaba, Y.H.T.; Gksu, S.; Ertan, A.; Menzek, A. Total Synthesis of the Biologically Active, Naturally Occurring 3,4-Dibromo-5-(2-
bromo-3,4-dihydroxy-6-(methoxymethyl)benzyl)benzene-1,2-diol and Regioselective O-Demethylation of Aryl Methyl Ethers.
Helv. Chim. Acta 2010, 93, 1127–1135. [CrossRef]
1
1
2. Parmar, H.S.; Jain, P.; Chauhan, D.S.; Bhinchar, B.K.; Munjal, V.; Yusuf, M.; Choube, K.; Tawani, A.; Tiwari, V.; Manivannan, E.;
et al. DPP-IV inhibitory potential of naringin: An in silico, in vitro and in vivo study. Diabetes Res. Clin. Pract. 2012, 97, 105–111.
[CrossRef] [PubMed]
1
3. Sun, C.; Yan, Y.; Chen, Q.; Luo, F.; Zhu, X.; Li, X.; Chen, K. Purification of naringin and neohesperidin from Huyou (Citrus
changshanensis) fruit and their effects on glucose consumption in human HepG2 cells. Food Chem. 2012, 135, 1471–1478.