Flavonoid derivatives synthesis and anti-diabetic activities

Article abstract of DOI:10.1016/j.bioorg.2019.103501

In high fat diet-induced obese mice, the flavonoid derivative of tiliroside, Fla-CN, has antihyperglycemic effects, can improve insulin sensitivity, ameliorate metabolic lipid disorders, and benefits certain disorders characterized by insulin resistance.

Full text of DOI:10.1016/j.bioorg.2019.103501

Journal Pre-proofs
Flavonoid derivatives synthesis and anti-diabetic activities
Ying Chen, Feng-Bo Cheng, Xiao-Ran Wu, Wen Zhu, Jian-Wen Liao, Yue
Jiang, Chang Zhang, Wen-Yan Niu, Yang Yu, Hong-Quan Duan, Nan Qin
PII:
S0045-2068(19)31367-7
DOI:
https://doi.org/10.1016/j.bioorg.2019.103501
YBIOO 103501
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
28 August 2019
30 October 2019
7 December 2019
Please cite this article as: Y. Chen, F-B. Cheng, X-R. Wu, W. Zhu, J-W. Liao, Y. Jiang, C. Zhang, W-Y. Niu, Y.
Yu, H-Q. Duan, N. Qin, Flavonoid derivatives synthesis and anti-diabetic activities, Bioorganic Chemistry
(2019), doi: https://doi.org/10.1016/j.bioorg.2019.103501
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Inc.

Flavonoid derivatives synthesis and anti-diabetic activities
Ying Chen1, Feng-Bo Cheng1, Xiao-Ran Wu1, Wen Zhu1,2, Jian-Wen Liao1,3,
Yue Jiang1,2, Chang Zhang1, Wen-Yan Niu4, Yang Yu1, Hong-Quan Duan1,5*,
and Nan Qin1*
1School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on
Technologies Enabling Development Clinical Therapeutics and Diagnostics
(Theragnostic), Tianjin, People’s Republic of China
2Department of Pharmacy, Tianjin Children's Hospital, People’s Republic of
China
3Tianjin Neurosurgery Institute, Tianjin Cerebral Vascular and Neural
Degenerative Disease Key Laboratory, Tianjin Huanhu Hospital, Tianjin,
People’s Republic of China
4Department of Immunology, Key Laboratory of Immune Microenvironment
and Disease of the Educational Ministry of China, People’s Republic of China
5Research Center of Basic Medical Sciences, Tianjin Medical University,
Tianjin, People’s Republic of China
Corresponding authors:
Hong-Quan Duan, duanhq@tmu.edu.cn
Nan Qin, qinnan@tmu.edu.cn

Abstract
In high fat diet-induced obese mice, the flavonoid derivative of tiliroside,
Fla-CN, has antihyperglycemic effects, can improve insulin sensitivity,
ameliorate metabolic lipid disorders, and benefits certain disorders
characterized by insulin resistance. Fla-CN is a novel lead compound to
discovery anti-diabetic and anti-obesity drugs. The present study reported the
optimization of Fla-CN to obtain a new derivative, 10b, which has improved
glucose consumption at the nanomolar level (EC50 = 0.3 nM) in insulin
resistant (IR) HepG2 cells. 10b also increased the glycogen content and
glucose uptake, and concurrently inhibited gluconeogenesis in HepG2 cells.
Western blotting showed that 10b markedly enhanced the phosphorylation of
AMPK (AMP-activated protein kinase) and AS160 (protein kinase B
substrate of 160 kDa) and reduced the levels of the gluconeogenesis key
enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6P (glucose
6-phosphatase) in HepG2 cells. The potential molecular mechanism of 10b
may be activation of the AMPK/AS160 and AMPK/PEPCK/G6P pathways.
We concluded that 10b might be a valuable candidate to discover
anti-diabetic drugs.
Keywords: Flavonoids, Structure-Activity Relationship, Glucose Metabolism
Disorders, ratio of AMP/ATP, AMPK pathway

1. Introduction
Hyperglycemia is driven by impaired rates of peripheral glucose uptake and
elevated rates of hepatic glucose output. The production of glucose is one of
the most important functions of the liver. This process is under complex
control by substrates, hormones, and nerves [1]. This control is achieved by
diverse regulatory mechanisms that tune the activities of enzymes and
transporters in a concerted fashion. In the rat liver, significant changes in the
carbohydrate metabolism-related enzyme levels occur between 5 and 100
hours during a starvation-refeeding cycle [2, 3]. In a given metabolic system,
not all enzymes are equally important for the regulation of systemic behavior.
Rather, only specific key enzymes carry the burden of regulatory control. For
example,
glucose-6-phosphatase
(G6P)
and
phosphoenolpyruvate
carboxykinase (PEPCK), which have the key roles in hepatic glucose
production regulation and could be applied therapeutically to treat
hyperglycemia. AMP-activated protein kinase (AMPK) is an energy-sensing
enzyme that controls glucose and lipid metabolism. AMPK activation
increases glucose uptake, fatty acid oxidation, and mitochondrial biogenesis,
which helps to ameliorate different aspects of metabolic dysregulation,
including hyperglycemia and insulin resistance [4]. Therefore, to overcome
chronic diabetes, researchers are seeking novel classes of anti-hyperglycemia
factors with multiple effects or targets.

A
semi-synthesized
flavonoid
derivative
of
tiliroside,
3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl]kaempferol (Fla-CN, Fig. 1),
caused significantly enhanced glucose consumption compared with that of
metformin in HepG2 cells, which are an insulin-resistant (IR) human
hepatocellular carcinoma cell line [5]. An in vivo study in high fat diet
induced obese mice showed that Fla-CN had antihyperglycemic effects, could
improve insulin sensitivity, ameliorate metabolic lipid disorders, and benefit
certain disorders characterized by insulin resistance [6]. The present study
investigated the structural optimization of Fla-CN to obtain the new
derivatives with enhanced effects on glucose metabolism. Furthermore, we
explored that the possible molecular mechanism of the activity of Fla-CN
derivatives.
OH
HO
O
O
CN
O
OH
O
Fig. 1 The chemical structure of
3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl]kaempferol (Fla-CN)
2. Results
2.1 Chemistry
Derivatives of Fla-CN were synthesized according to a previous report with
some major adaptations [5]. All derivatives were prepared through aldol

condensation. In comparison with our earlier study, morpholinium
trifluoroacetate was used to catalyze this aldol condensation, with reference to
Zumbansen’s report[7]. To obtain alkynyl substituted derivatives 4a-4d, a
Sonogashira reaction was used in the first step (Scheme 1). Nitrogen
heterocycle derivatives 8h-8u and 10a-10b were synthesized using building
blocks 9h-9u, which were constructed from fluorine-substituted
formylbenzonitrile (Scheme 2).
R
R
Br
a
b
c
CHO
CHO
O
1
2
4a: R=H
4b: R=
4c: R=
OH
R
R
HO
O
O
d
Br
O
OH
O
OH
O
4d: R=
3
OH
4a-4d
Reagents and conditions: a.alkyne, Pd(PPh3)2Cl2, CuI, TEA,THF, r.t.; b. Morpholine TFA salts, acetone, reflux;
c.Pyrrolidone hydrotribromide, THF, r.t.; d. Kaempferol, K2CO3, 1,4-dioxane, 80°C;
Scheme 1. Synthesis of compounds 4a-4d

NC
R
NC
R
NC
R
a
b
c
CHO
Br
O
O
5
7
6
OH
NC
N
NC
CN
R
HO
O
d
CHO
O
CHO
F
OH
O
O
8a-8u
5a-5d
9h-9u
Reagents and conditions: a. Morpholine TFA salts, acetone, reflux; b. Pyrrolidone hydrotribromide, THF, r.t.;
c. Kaempferol, K2CO3, 1,4-dioxane, 80°C; d. amine, K2CO3, THF, 65°C.
No. Substituted Group No. Substituted Group No. Substituted Group
8a
8b
8c
8d
8e
8f
8h
8i
8o
8p
8q
8r
2-pyrrolidinyl,4-CN
2-pyrrolidinyl,5-CN
3-pyrrolidinyl,4-CN
3-CN,5-pyrrolidinyl
2-piperidinyl,4-CN
2-piperidinyl,5-CN
3-piperidinyl,4-CN
3-CN,4-piperidinyl
2-morpholinyl,4-CN
2-morpholinyl,5-CN
3-morpholinyl,4-CN
3-CN,4-morpholinyl
2-F,4-CN
2-F,5-CN
8j
3-F,4-CN
3-CN,4-F
8k
8l
8s
8t
3-F,5-CN
2-Cl,3-CN
2-(N-Boc-
piperazineyl),4-CN
8m
8n
2-(N-Boc-
piperazineyl),5-CN
8g
8u
2-Cl,5-CN
OH
OH
CN
CN
HO
O
O
HO
O
O
3 mol/L HCl, MeOH,
reflux.
O
O
OH
O
N
N
OH
O
N
.
N
H
HCl
Boc
8t-8u
10a: 4-CN; 10b:5-CN
Scheme 2. Synthesis of compounds 8a-8u and 10a-10b
2.2 Pharmacology
2.2.1 Effects of the flavonoid derivatives on glucose consumption
The glucose consumption of IR HepG2 cells in response to all the derivatives

were evaluated and EC50 values were used to assess their potencies.
Compounds 4b-4d, 8a-8s, and 10a-10b significantly enhanced glucose
consumption in IR HepG2 cells (Table 1). In this optimization study, four
derivatives (4b, 8m, 10a, and 10b) showed higher potency than the lead
compound Fla-CN. The chemical structure of 4b was different from the other
three compounds; therefore, 4b and 10b were chosen for the more detailed
research associated with glucose metabolism.
Table 1. Effects of Fla-CN derivatives on IR HepG2 cells’ glucose
consumption (n = 3).
No.
4a
4b
4c
4d
8a
8b
8c
8d
8e
8f
EC50 (nM) No.
63.9 ± 8.59 8g
0.4 ± 0.11 8h
9.3 ± 1.63 8i
15.1 ± 1.57 8j
1.2 ± 0.36 8k
2.3 ± 0.49 8l
6.5 ± 0.74 8m
5.9 ± 0.77 8n
5.0 ± 0.85 8o
5.4 ± 0.65 8p
EC50 (nM) No.
10.8 ± 1.46 8q
8.3 ± 0.93 8r
6.1 ± 0.87 8s
11.8 ± 1.38 8t
18.3 ± 2.36 8u
1.8 ± 0.27 10a
0.9 ± 0.15 10b
10.1 ± 1.28 Fla-CN
9.5 ± 1.31 Met
1.2 ± 0.67
EC50 (nM)
1.5 ± 0.34
8.7 ± 1.33
7.6 ± 1.38
52.3 ± 7.62
45.2 ± 6.61
0.8 ± 0.36
0.3 ± 0.07
3.3 ± 0.54
258.1 ± 33.36

Our previous study found that the cyan-substituted flavonoid derivative
(Fla-CN) showed potential anti-diabetic activity [5]. Cyan groups contain C-N
triple bonds; therefore, our first thought was to substitute C-N triple bonds by
C-C triple bonds. Based on this notion, 4a-4d were designed, synthesized, and
evaluated for their activities. Compared with other alkyne-containing
derivatives, 4b exhibited higher activity than Fla-CN (Table 1). Combining
the results of 4a-4d and those of a previous study[5], we speculated that the
cyan group was the most active group. Halogen bonding had been
successfully harnessed for lead identification and optimization [8]; therefore,
multi substituted groups, such as a fluorine atom or chlorine atom, were
introduced into the Fla-CN derivatives that also contained a cyan group
(8a-8g). Most of these derivatives exhibited the same effects as Fla-CN at the
same order of magnitude. Moreover, the results showed that the effects had
good tolerance at the substituted positions of the cyan groups. Aromatic
amines usually appear in marketed drugs; therefore, a C-N bond was also
introduced into Fla-CN (8h-8s) to discover more potent derivatives.
Fortunately, six-membered heterocyclic nitrogen derivatives at the
2-subsititued position (8l, 8m 8p, and 8q) exhibited higher activities than
Fla-CN (Table 1). Based on this discovery, we designed other two
six-membered heterocyclic nitrogen derivatives at the 2-subsititued position
(10a and 10b). To our surprise, both 10a and 10b showed higher potency,
with EC50 values below 1.0 nM (Table 1).

2.2.2 Effects of the flavonoid derivatives on glucose uptake
To investigate whether flavonoid derivatives could promote glucose uptake in
HepG2
cells,
we
evaluated
the
uptake
of
2-NBDG
by
(2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose)
HepG2 cells treated with different concentrations of Fla-CN, 4b, 10b, and
insulin (Fig. 2A–C). Compared with that of the control group, the cells treated
with Fla-CN, 4b, and 10b showed significantly enhanced 2-NBDG uptake, in
a dose-dependent manner (P < 0.05).
2.3 Effects of the flavonoid derivatives on the glycogen content
Compared with that of the control group, the glycogen content in the
insulin-treated group was significantly increased (P < 0.001). Compared with
the control group, 4b at 12.5 μM effectively increased the glycogen content
(P < 0.01) in HepG2 cells (Fig. 2E). Compared with the control group, 10b
and Fla-CN also effectively increased the glycogen content (2.5 μM, P <
0.01; 12.5 μM, P < 0.001, respectively) in HepG2 cells (Fig. 2D, F). These
results suggested that 4b, 10b, and Fla-CN promoted glycogen synthesis,
supporting the hypothesis that treatment with these flavonoid derivatives
improved glucose metabolism in HepG2 cells.

Fig. 2 The effects of Fla-CN derivatives on glucose uptake and the
glycogen content. The effects on glucose uptake of Fla-CN (A), 4b (B), and
10b (C) in HepG2 cells. Glucose uptake activity was measured using
fluorescent
2-NBDG
The
(2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose).
effects of Fla-CN (D), 4b (E) and 10b (F) on the glycogen contents of HepG2
cells. Data represent the mean ± the standard error of the mean (SEM); nꢀ=ꢀ
4. (* P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control group).

2.3.1 Effects of the flavonoid derivatives on gluconeogenesis
Fig. 3A–C shows that metformin, a prescription drug, decreased glucose
production at 1.0 mM (P < 0.01). Fla-CN also decreased glucose production
at 2.5 (P < 0.05) and 12.5 μM (P < 0.001), respectively (Fig. 3A). Derivative
4b decreased glucose production only at 12.5 μM (P< 0.01, Fig. 3B). Fig. 3C
shows that 10b treatment resulted in decreased glucose production in a dose
dependent manner.
2.3.2 Effects on the flavonoid derivatives on glycolysis
To investigate the effects of 4b, 10b, or Fla-CN on anaerobic respiration,
lactate release from HepG2 hepatocytes was measured. Insulin at 100 nM was
used as positive control. HepG2 cells were treated for 24 h with 0.5, 2.5, 12.5
mmol/L 4b, 10b or Fla-CN (together with no compound controls). Lactate
release from HepG2 hepatocytes was increased by insulin (P < 0.001; Fig.
3D–F). The concentration of lactate showed no significant difference between
flavonoid derivatives and the control group (Fig. 3D–3F). Lactate is the end
product of glycolysis; therefore, these data suggested that 4b, 10b, or Fla-CN
did not affect glycolysis.

Fig. 3 The effects of Fla-CN derivatives on gluconeogenesis and
glycolysis. The effects on gluconeogenesis of Fla-CN (A), 4b (B), and 10b
(C) in HepG2 cells. The effects on glycolysis of Fla-CN (D), 4b (E) and 10b
(F) in HepG2 cells. Data represent the mean ± the standard error of the mean
(SEM); nꢀ=ꢀ4. (*P < 0.05, **P < 0.01, ***P < 0.001 compared with the control
group).

2.4 Potential molecular mechanism on glucose metabolism in response to
of flavonoid derivative 10b
The above evaluations of Fla-CN, 4b and 10b suggested that 10b exhibited
more potency than Fla-CN and 4b on glucose metabolism. Fla-CN activated
AMPK in vitro and in vivo [7,8]; therefore, AMPK may be a key factor in the
molecular mechanism of glucose metabolism. As shown in Fig. 4, in HepG2
cells, 10b treatment at 0.5 to 12.5 μM led to markedly enhanced levels of
phosphorylated AMPK and AS160 (protein kinase B substrate of 160 kDa)
compared with those of the control group, in a dose dependent manner.
Fig. 4 The effects of 10b on the levels of AMPK, phospho (p)-AMPK and
p-AS160 in HepG2 cells. The positive control comprised

5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside, (AICAR; 0.1 mM),
which is a known activator of AMPK. (A) Western blotting assays detected
the levels of AMPK, p-AMPK, AS160, and p-AS160 in HepG2 cells; (B) The
relative value of p-AMPK was quantified using densitometry and normalized
with that of AMPK; (C) The relative level of p-AS160 was quantified using
densitometry and normalized to that of β-actin. Data represent the mean ± the
standard error of the mean (SEM); nꢀ=ꢀ3. (*P < 0.05, ** P < 0.01, ***P < 0.001
compared with the control group). AMPK, AMP-activated protein kinase;
AS160, protein kinase B substrate of 160 kDa.
Phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase
(G6P) are key enzymes in gluconeogenesis. The results shown in Fig. 5
indicate that 10b, Fla-CN, and the AMPK activator AICAR could
significantly decrease the protein levels of PEPCK and G6P in HepG2 cells
(Fig. 5).
Fig. 5 The effects of 10b on PEPCK and G6P protein levels in HepG2

cells. (A) Western blotting analysis was used to determine the PEPCK protein
levels in HepG2 cells; (B) The relative level of PEPCK was quantified using
densitometry and normalized to that of β-actin; (C) Western blotting analysis
was used to determine the G6P protein level in HepG2 cells; (D) The relative
level of G6P was quantified using densitometry and normalized to that of
β-actin. Data represent the mean ± the standard error of the mean (SEM); nꢀ=
**
ꢀ3. (*P < 0.05, P < 0.01, ***P < 0.001 compared with the control group).
PEPCK, phosphoenolpyruvate carboxykinase; G6P, glucose 6-phosphatase
(G6P).
2.5 Effects of 10b on the AMP/ATP ratio in HepG2 cells
An increase in the ratio of AMP to ATP indicated that several natural
compounds and metformin could active AMPK [9-11]. Therefore, we
examined the ATP and AMP levels in cultured HepG2 cells after 10b (10
μM) or vehicle treatment. The results showed that 10b decreased the ATP
content and increased AMP content (Table. 2). Thus, the ratio of AMP to
ATP was elevated by treatment with 10b (Table. 2).
Table 2.
Effects of 10b on the AMP/ATP ratio in HepG2 cells
AMP（μg·mL-1）
ATP（μg·mL-1）
Group
AMP/ATP
Control
1.425 ± 0.0423
1.411 ± 0.0382
1.482 ± 0.0195
1.476 ± 0.0360
1.670 ± 0.0160
44.942 ± 1.0331
44.120 ± 1.1706
43.508 ± 0.1891
41.475 ± 0.5798
42.196 ± 1.0337
0.031/1
0.032/1
0.034/1
0.036/1*
0.039/1*
10b (0.01 μM)
10b (0.05 μM)
10b (8 μM)
10b (12 μM)

N = 3, *P < 0.05 compared with control group
3. Discussion
Among all the Fla-CN derivatives, 4b and 10b exhibited excellent
enhancement of glucose consumption. Furthermore, in the glucose uptake,
gluconeogenesis, glycogen production, and glycolysis experiments, both 4b
and 10b could improve glucose uptake and glycogen production in HepG2
cells (Fig. 2). Meanwhile, these two compounds could inhibit
gluconeogenesis in HepG2 cells (Fig. 3A, 3B, 3C). However, neither 4b nor
10b affected glycolysis (Fig. 3D–F). Compared with 4b and Fla-CN, 10b had
a lower effective concentration, such as 0.5 or 2.5 μM (Fig. 2C, 2F, 3C).
This phenomenon was attribute to the structural difference of these two
derivatives. Compared with 4b and Fla-CN, 10b has a nitrogen heterocycles
fragment. A cursory glance at any of pharmaceutical paper reveals that
nitrogen heterocycles are common drug fragments [12]. In this study, five- or
six-membered rings were introduced into the lead compound. The results
showed that six-membered nitrogen heterocycle derivatives exhibited more
potency than five-membered nitrogen heterocycle derivatives (Table 1). The
ring size might be a crucial factor for the activity. The binding site of the
potential targets might require a larger group at the benzonitrile fragment.
Lipophilicity is another underlying structural property that affects higher-level

physicochemical and biochemical properties. It often is an effective guide for
modifying the structure of a lead series to improve a property. In this study,
the most potency piperazine derivatives 10a and 10b exhibit the lowest cLogP
values of 3.14 in all of these derivatives. The cLogP value of Fla-CN is 3.30.
It was concluded that a lower cLogP might be a better manner of the
derivative’s activity. Among all the six-membered nitrogen heterocycle
derivatives, piperazine derivatives showed excellent potency. This indicated
that piperazine is an important group in these derivatives. This was not
surprising, because piperazine, as an important nitrogen heterocycle, has been
shown to be an essential structural component for many pharmaceuticals.
Piperazine derivatives are reported to elicit a broad spectrum of
pharmacological activities viz. antidepressant, anticancer, anthelmintic,
antibacterial, antifungal, antimycobacterial, antimalarial, and anticonvulsant
activities [13]. In this study, derivatives 10a and 10b exhibited activity at the
nanomole levels when they contained a piperazine group. It was possible that
the binding pocket of target proteins might have some acidic amino acid
residues, some hydrogen bond receptors, or some aromatic amino acid
residues. These amino acid residues could interact with the piperazine group.
These results were helpful for identifying the binding site of 10b with its
potential target proteins.
AMPK is a highly conserved sensor of cellular energy [4]. A number of
pharmacological compounds that increase AMPK activity indirectly, or

through direct binding, have been identified [14]. The first such direct
activator to be identified was 5-aminoimidazole-4-carboxamide
ribonucleotide (also known as ZMP), the monophosphate derivative of the
cell permeable precursor AICAR [15, 16]. This finding led to the wide use of
AICAR as a pharmacological tool to investigate the effect of AMPK
activation in cells[14]. AMPK is activated in response to an increase in AMP
and ADP levels, and this underpins the mechanism of action of a wide
number of compounds that indirectly activate AMPK. The biguanide
metformin, a widely used oral anti-diabetic drug, inhibits complex I in the
mitochondria, leading to a reduction in mitochondrial respiration and ATP
production [17]. Metformin has been shown to activate AMPK[9]. A number
of plant-derived natural products, which are used in traditional herbal
medicine, also activate AMPK indirectly by inhibiting mitochondrial
respiration [4], for example, berberine [18] and quercetin [19]. Our previous
study reported that a flavonoid derivative could enhance glucose consumption
via the AMPK pathway in HepG2 cells [5]. AMPK is a sensor of cellular
energy status that becomes activated under conditions of a high cellular
AMP/ATP ratio through allosteric binding with AMP and subsequent
phosphorylation by upstream AMPK-kinases [20]. Therefore, we analyzed the
contents of AMP and ATP levels in 10b treated HepG2 cells and found that
10b increased the AMP:ATP ratio in HepG2 cells (Table 2). Thus, derivative
10b could activate AMPK by increasing the AMP:ATP ratio, as confirmed

using western blotting (Fig. 4). AS160 is also known as a Rab-GTPase
activating protein (GAP). The Rab-GAP activity of AS160 maintains Rab
substrates in their inactive, GDP-bound, form, which causes intracellular
retention of GLUT4 [21]. AS160 phosphorylation by AMPK inactivates its
Rab-GAP activity, thereby relieving the inhibition on its Rab substrates.
Subsequently, GLUT4 storage vesicles are mobilized to the cell surface under
the regulation of activated Rabs [22, 23]. These effects on the AMPK/AS160
pathway might lead directly to enhanced glucose uptake.
Some studies using nonspecific AMPK activators such as AICAR [24, 25]
indicated that AMPK repressed key gluconeogenic genes, such as PEPCK
and G6P, both of which have been shown to contribute to aberrant hepatic
glucose production in metabolic disorders [26]. In the present study, the
AMPK activator AICAR and 10b could decrease the protein levels of PEPCK
and G6P in HepG2 cells (Fig. 5). These results agreed with those of previous
study, which demonstrated reduced PEPCK and G6P levels in
streptozotocin-induced and ob/ob diabetic mice after adenovirus-induced
transfer of constitutively active AMPK [27]. Thus, derivative 10b might
downregulate gluconeogenesis via the AMPK/PEPCK/G6P pathway.
4. Conclusion
In the present study, we produced semi-synthesized flavonoid
derivatives, most of which could improve glucose consumption in IR HepG2

cells. Among them, 10b exhibited powerful potency at a nanomolar
concentration. Derivative 10b upregulated glucose uptake and glycogen
production, and downregulated gluconeogenesis in HepG2 cells. Its molecular
mechanism might be activation the AMPK pathway by upregulating the
AMP:ATP ratio. Derivative 10b could represent a valuable lead compound
for the discovery of anti-diabetic drugs. The target identification study of 10b
is undergoing in our lab.
5. Experimental section
5.1. Chemistry
5.1.1. General instrumentation and reagents
All the solvents and reagents used in the present study came from commercial
sources and were used without purification, unless otherwise stated. Reactions
were monitored by Thin-layer chromatography (TLC) with precoated silica
gel glass plates containing a fluorescence indicator were used to monitor the
reactions. Column chromatography was carried out on silica gel (SiO2; 300–
400 mesh) and a Toyopeal HW-40 (Tosoh Co., Ltd, Tokyo, Japan). The
nuclear magnetic resonance (NMR) spectra were obtained using a Bruker
1
AVANCE III 400 instrument (Bruker, Billerica, MA, USA; H NMR, 400
13
MHz; C NMR, 100 MHz). The high-resolution mass spectra were recorded
in ESI mode using an Agilent 1200 LC-MS instrument (Agilent, Palo Alto,
CA, USA).

5.1.2. General procedure for intermediates synthesis
Compound 1 was prepared in accordance with the Sonogashira coupling
reaction [28]. The α, β-unsaturated ketos 2 and 6 were synthesized using
morpholine trifluoroacetic acid salts catalyzed by the Classin-Schimitt
reaction [7]. The α-bromine substituted ketos 3 and 7 were obtained using
pyrrolidone hydrotribromide [5].
5.1.3. General procedure for the synthesis of compounds 4 and 8
Target compounds 4 and 8 were semi-synthesized from kaempferol with α-
bromine substituted ketos using an etherification reaction. A solution of the
bromine substituted ketos (1.0 eq) in 1,4-dioxane (5 ml) was added to a
mixture of kaempferol (1.2 eq) and K2CO3 (1.2 eq) in 1,4-dioxane (10 ml;
stirred for 90 min while maintaining gentle reflux) and mixed for 30 min.
Stirring of the reaction mixture at 80 C was continued until TLC indicated
that the starting material had disappeared (CH2Cl2: MeOH, 95:5). Under
reduced pressure, the solvent was removed from the reaction mixture, water
was added, 1 M HCl was added to the aqueous phase for neutralization (to pH
7), and the reaction was extracted using ethyl acetate. The organic phase was
dried over anhydrous magnesium sulfate, filtered, and concentrated.
Gel-permeation chromatography (HW-40; CH2Cl2: MeOH, 1:1) and

preparative-TLC were used to purify the crude product, which appeared as a
yellow solid product.
5.1.3.1.
3-O-[(E)-4-(4-ethynyphenyl)-2-oxobut-3-en-1-yl]kaempferol (4a)
Yield: 5.8%. 1H NMR (400 MHz, DMSO-d6): δ 12.53 (1H, s, OH), 10.87 (1H,
s, OH), 10.24 (1H, s, OH), 8.03 (2H, d, J = 8.8 Hz), 7.68 (2H, d, J = 8.0 Hz),
7.65 (1H, d, J = 16.4 Hz), 7.52 (2H, d, J = 8.0 Hz), 7.07 (1H, d, J = 16.4 Hz),
6.92(2H, d, J = 8.8 Hz), 6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz),
13
5.07 (2H, s), 4.36 (1H, s). C NMR (100 MHz, DMSO-d6): δ 194.5, 177.4,
164.2, 161.1, 160.2, 156.3, 155.2, 141.4, 136.2, 134.6, 132.2, 130.5, 128.7,
123.7, 123.3, 120.5, 115.5, 104.0, 98.7, 93.7, 83.2, 83.0, 75.3. HR-ESI-MS
m/z: found 477.0950, calculated 477.0941 [M+Na]+.
5.1.3.2.
3-O-[(E)-4-(4-(3,3-dimethylbut-1-yn-1-yl)phenyl)-2-oxobut-3-en-
1-yl]kaempferol (4b)
1
Yield: 8.1%. H NMR (400 MHz, DMSO-d6): δ 12.54 (1H, s, OH), 10.88
(1H, s, OH), 10.26 (1H, s, OH), 8.04 (2H, d, J = 8.4 Hz), 7.64 (1H, d, J = 16.0
Hz), 7.63 (2H, d, J = 8.0 Hz), 7.39 (2H, d, J = 8.0 Hz), 7.05 (1H, d, J = 16.0
Hz), 6.92(2H, d, J = 8.4 Hz), 6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0
13
Hz), 5.06 (2H, s), 1.30 (9H, s) . C NMR (100 MHz, DMSO-d6): δ 194.4,
177.5, 164.2, 161.1, 160.2, 156.3, 155.2, 141.6, 136.3, 133.6, 131.7, 130.5,
128.6, 125.3, 122.7, 120.5, 115.5, 104.0, 101.0, 98.7, 93.7, 78.8, 75.3, 30.6,
27.7. HR-ESI-MS m/z: found 533.1507, calculated 533.1576 [M+Na]+.
5.1.3.3.
3-O-[(E)-4-(4-hydroxybut-1-yn-1-yl)phenyl)-2-oxobut-3-en-1-yl]k

aempferol (4c)
1
Yield: 5.3%. H NMR (400 MHz, DMSO-d6): δ 12.54 (1H, s, OH), 10.89
(1H, s, OH), 10.27 (1H, s, OH), 8.04 (2H, d, J = 8.8 Hz), 7.64 (1H, d, J = 16.0
Hz), 7.64 (2H, d, J = 8.0 Hz), 7.43 (2H, d, J = 8.0 Hz), 7.05 (1H, d, J = 16.0
Hz), 6.92(2H, d, J = 8.8 Hz), 6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0
13
Hz), 5.06 (2H, s), 4.91(1H, m, OH), 3.59(2H, m), 2.58(2H, m). C NMR
(100 MHz, DMSO-d6): δ 194.4, 177.5, 164.2, 161.1, 160.2, 156.3, 155.2,
141.6, 136.3, 133.7, 131.8, 130.5, 128.6, 125.3, 122.8, 120.5, 115.5, 104.0,
98.7, 93.7, 91.3, 80.9, 75.3, 59.6, 23.4.HR-ESI-MS m/z: found 421.1214,
calculated 421.1212 [M+Na]+.
5.1.3.4.
3-O-[(E)-4-(4-(3-hydroxybut-1-yn-1-yl)phenyl)-2-oxobut-3-en-1-y
l]kaempferol (4d)
1
Yield: 5.6%. H NMR (400 MHz, DMSO-d6): δ 12.54 (1H, s, OH), 10.89
(1H, s, OH), 10.29 (1H, s, OH), 8.04 (2H, d, J = 8.8 Hz), 7.67 (2H, d, J = 8.4
Hz), 7.65 (1H, d, J = 16.4 Hz), 7.44 (2H, d, J = 8.4 Hz), 7.06 (1H, d, J = 16.4
Hz), 6.92(2H, d, J = 8.8 Hz), 6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0
Hz), 5.07 (2H, s), 5.52(1H, d, J = 5.2 Hz, OH ), 4.60(1H, dd, J1 = 5.2 Hz, J2 =
13
6.8 Hz), 1.38(3H, d, J = 6.8 Hz). C NMR (100 MHz, DMSO-d6): δ 194.5,
177.4, 164.2, 161.1, 160.2, 156.3, 155.2, 141.5, 136.2, 134.0, 131.7, 130.5,
128.7, 124.5, 123.0, 120.5, 115.5, 104.0, 98.7, 95.7, 93.7, 81.9, 75.3, 56.7,
24.5.HR-ESI-MS m/z: found 421.1225, calculated 421.1212 [M+Na]+.
5.1.3.5.
3-O-[(E)-4-(4-cyano-2-fluorophenyl)-2-oxobut-3-en-1-yl]kaempfe

rol (8a)
1
Yield: 4.6%. H NMR (400 MHz, DMSO-d6): δ 12.51 (1H, s, OH), 10.90
(1H, s, OH), 10.25 (1H, s, OH), 8.01 (2H, d, J = 8.8 Hz), 7.99-7.76 (3H, m),
7.66 (1H, d, J = 16.4 Hz), 7.27 (1H, d, J = 16.4 Hz), 6.91 (2H, d, J = 8.8 Hz),
6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.10 (2H, s). 13C NMR (100
MHz, DMSO-d6): δ 195.0, 177.4, 164.3, 161.2(d, J = 252.0 Hz), 161.1, 160.2,
156.3, 155.2, 136.2, 132.2, 130.4(d, J = 17.3 Hz), 129.0, 127.6, 127.5,
127.1(d, J = 12.0 Hz), 120.4, 120.2(d, J = 25.0 Hz), 117.4, 115.5, 113.8(d, J =
12.0 Hz), 103.9, 98.7, 93.8, 75.6. HR-ESI-MS m/z: found 496.0811,
calculated 496.0808 [M+Na]+.
5.1.3.6.
3-O-[(E)-4-(5-cyano-2-fluorophenyl)-2-oxobut-3-en-1-yl]kaempfe
rol (8b)
1
Yield: 2.2%. H NMR (400 MHz, DMSO-d6): δ 12.52 (1H, s, OH), 10.93
(1H, s, OH), 10.26 (1H, s, OH), 8.03 (2H, d, J = 8.8 Hz), 8.50-7.50 (3H, m),
7.63 (1H, d, J = 16.4 Hz), 7.29 (1H, d, J = 16.4 Hz), 6.90 (2H, d, J = 8.8Hz),
6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.08 (2H, s). 13C NMR (100
MHz, DMSO-d6): δ 194.5, 177.4, 164.2, 161.1, 160.2, 156.3, 155.2, 136.1,
134.1, 131.9, 130.5, 126.8, 120.4, 117.7, 115.4, 104.0, 98.7, 93.7,
75.5.HR-ESI-MS m/z: found 496.0818, calculated 496.0808 [M+Na]+.
5.1.3.7.
3-O-[(E)-4-(4-cyano-3-fluorophenyl)-2-oxobut-3-en-1-yl]kaempfe
rol (8c)
1
Yield: 12.6%. H NMR (400 MHz, DMSO-d6): δ 12.52 (1H, s, OH), 10.90

(1H, s, OH), 10.26 (1H, s, OH), 8.03 (2H, d, J = 8.8 Hz), 8.00-7.70 (3H, m),
7.67 (1H, d, J = 16.4 Hz), 7.25 (1H, d, J = 16.4 Hz), 6.91 (2H, d, J = 8.8Hz),
6.46 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.11 (2H, s). 13C NMR (100
MHz, DMSO-d6): δ 194.5, 177.4, 164.2, 162.0(d, J = 254.0 Hz), 161.1, 160.2,
156.3, 155.2, 142.1(d, J = 9.0 Hz), 139.1, 136.2, 134.3, 130.5, 126.7, 125.4(d,
J = 3.0 Hz), 120.4, 115.6, 115.4(d, J = 9.0 Hz), 113.9, 104.0, 101.1(d, J =
15.0 Hz), 98.7, 93.7, 75.4. HR-ESI-MS m/z: found 496.0823, calculated
496.0808 [M+Na]+.
5.1.3.8.
3-O-[(E)-4-(3-cyano-4-fluorophenyl)-2-oxobut-3-en-1-yl]kaempfe
rol (8d)
1
Yield: 6.1%. H NMR (400 MHz, DMSO-d6): δ 12.53 (1H, s, OH), 10.92
(1H, s, OH), 10.28 (1H, s, OH), 8.04 (2H, d, J = 8.8 Hz), 8.35-7.50 (3H, m),
7.65 (1H, d, J = 16.4 Hz), 7.16 (1H, d, J = 16.4 Hz), 6.90 (2H, d, J = 8.8Hz),
6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.08 (2H, s). 13C NMR (100
MHz, DMSO-d6): δ 194.4, 177.4, 164.2, 161.1, 160.2, 156.3, 155.2, 142.1,
139.1, 136.2, 135.8(d, J = 9.0 Hz), 133.9, 130.5, 124.4, 120.5, 117.3(d, J =
20.0 Hz), 115.4, 113.6, 104.0, 98.7, 93.7, 75.3.HR-ESI-MS m/z: found
496.0810, calculated 496.0808 [M+Na]+.
5.1.3.9.
3-O-[(E)-4-(5-cyano-3-fluorophenyl)-2-oxobut-3-en-1-yl]kaempfe
rol (8e)
1
Yield: 9.8%. H NMR (400 MHz, DMSO-d6): δ 12.53 (1H, s, OH), 10.90
(1H, s, OH), 10.26 (1H, s, OH), 8.04 (2H, d, J = 8.8 Hz), 8.10-7.90 (3H, m),

7.64 (1H, d, J = 16.4 Hz), 7.26 (1H, d, J = 16.4 Hz), 6.91 (2H, d, J = 8.8Hz),
6.46 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.10 (2H, s). 13C NMR (100
MHz, DMSO-d6): δ 194.4, 177.4, 164.2, 162.0(d, J = 246.0 Hz), 161.1, 160.2,
156.3, 155.1, 138.8, 138.3(d, J = 9.0 Hz), 136.2, 130.5, 128.8, 125.8, 120.4,
120.0(d, J = 12.0 Hz), 117.2(d, J = 3.0 Hz), 115.4, 113.4(d, J = 9.0 Hz),
104.0, 98.6, 93.7, 75.3. HR-ESI-MS m/z: found 496.0821, calculated
496.0808 [M+Na]+.
5.1.3.10. 3-O-[(E)-4-(3-cyano-2-chlorophenyl)-2-oxobut-3-en-1-yl]kaempfe
rol (8f)
1
Yield: 2.5%. H NMR (400 MHz, DMSO-d6): δ 12.51 (1H, s, OH), 10.91
(1H, s, OH), 10.26 (1H, s, OH), 8.02(2H, d, J = 8.8 Hz), 8.20-7.50 (3H, m),
7.82 (1H, d, J = 16.0 Hz), 7.22 (1H, d, J = 16.0 Hz), 6.91 (2H, d, J = 8.8Hz),
13
6.47 (1H, s), 6.21 (1H, s), 5.10 (2H, s). C NMR (100 MHz, DMSO-d6): δ
194.4, 177.4, 164.2, 161.1, 160.2, 156.3, 155.2, 136.2, 136.0, 135.7, 133.7,
132.6, 130.5, 128.4, 127.0, 120.4, 115.8, 115.5, 104.0, 98.7, 93.7, 75.6.
HR-ESI-MS m/z: found 512.0512, calculated 512.0513 [M+Na]+.
5.1.3.11. 3-O-[(E)-4-(5-cyano-2-chlorophenyl)-2-oxobut-3-en-1-yl]kaempfe
rol (8g)
1
Yield: 2.3%. H NMR (400 MHz, DMSO-d6): δ 12.52 (1H, s, OH), 10.92
(1H, s, OH), 10.27 (1H, s, OH), 8.04(2H, d, J = 8.8 Hz), 8.50-7.70 (3H, m),
7.79 (1H, d, J = 16.4 Hz), 7.33 (1H, d, J = 16.4 Hz), 6.90 (2H, d, J = 8.8Hz),
6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.09 (2H, s). 13C NMR (100

MHz, DMSO-d6): δ 194.4, 164.2, 161.1, 160.2, 156.3, 155.2, 138.8, 136.2,
134.7, 134.6, 133.4, 132.1, 131.3, 130.5, 127.1, 120.5, 117.6, 115.4, 111.0,
104.0, 93.7, 75.6. HR-ESI-MS m/z: found 512.0520, calculated 512.0513
[M+Na]+.
5.1.3.12. 3-O-[(E)-4-(4-cyano-2-(pyrrolidin-1-yl)phenyl)-2-oxobut-3-en-1-y
l]kaempferol (8h)
1
Yield: 4.6%. H NMR (400 MHz, DMSO-d6): δ 12.54 (1H, s, OH), 10.37
(2H, s, OH), 8.02 (2H, d, J = 8.8 Hz), 7.85 (1H, d, J = 16.0 Hz), 7.55 (1H, d, J
= 8.0 Hz), 7.21 (1H, d, J = 1.2 Hz), 7.15 (1H, d, J = 8.0 Hz), 6.91 (2H, d, J =
8.8 Hz), 6.84 (1H, d, J = 16.0 Hz), 6.46 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J =
2.0 Hz), 5.04 (2H, s), 3.28(4H, t), 1.87(4H, t). 13C NMR (100 MHz,
DMSO-d6): δ 194.5, 177.4, 164.3, 161.1, 160.2, 156.3, 155.2, 149.6, 140.8,
136.2, 130.5, 130.0, 126.7, 122.9, 120.9, 120.5, 119.0, 118.2, 115.5, 112.6,
104.0, 98.7, 93.7, 75.3, 52.0, 25.2. HR-ESI-MS m/z: found 547.1489,
calculated 547.1481 [M+Na]+.
5.1.3.13. 3-O-[(E)-4-(5-cyano-2-(pyrrolidin-1-yl)phenyl)-2-oxobut-3-en-1-y
l]kaempferol (8i)
1
Yield: 2.5%. H NMR (400 MHz, DMSO-d6): δ 12.56 (1H, s, OH), 10.90
(1H, s, OH), 10.26 (1H, s, OH), 8.03 (2H, d, J = 8.8 Hz), 7.90 (1H, d, J = 16.0
Hz), 7.83 (1H, d, J = 2.0 Hz), 7.56 (1H, dd, J1 = 2.0 Hz, J2 = 8.8 Hz), 6.91
(2H, d, J = 8.8Hz), 6.85 (1H, d, J = 8.8 Hz), 6.77 (1H, d, J = 16.0 Hz), 6.47
(1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.04 (2H, s), 3.40(4H, t),

13
1.88(4H, t). C NMR (100 MHz, DMSO-d6): δ 194.2, 177.5, 164.2, 161.1,
160.2, 156.3, 155.1, 151.5, 141.3, 136.2, 133.8, 133.5, 130.5, 122.0, 121.2,
120.5, 119.6, 115.5, 115.0, 104.0, 98.6, 98.0, 93.7, 75.1, 51.9, 25.4.
HR-ESI-MS m/z: found 547.1483, calculated 547.1481 [M+Na]+.
5.1.3.14. 3-O-[(E)-4-(4-cyano-3-(pyrrolidin-1-yl)phenyl)-2-oxobut-3-en-1-y
l]kaempferol (8j)
1
Yield: 3.5%. H NMR (400 MHz, DMSO-d6): δ 12.54 (1H, s, OH), 10.91
(1H, s, OH), 10.28 (1H, s, OH), 8.04 (2H, d, J = 8.0 Hz), 7.60 (1H, d, J = 16.0
Hz), 7.50 (1H, d, J = 8.0 Hz), 7.10 (1H, d, J = 16.0 Hz), 7.04 (1H, s), 6.99
(1H, d, J = 8.0 Hz), 6.91 (2H, d, J = 8.0 Hz), 6.47 (1H, s), 6.22 (1H, s), 5.10
13
(2H, s), 3.54(4H, s), 1.95(4H, s). C NMR (100 MHz, DMSO-d6): δ 194.5,
177.5, 164.2, 161.1, 160.2, 156.3, 155.2, 149.8, 141.6, 139.0, 136.2, 135.6,
130.5, 124.8, 120.6, 120.5, 119.6, 115.8, 115.5, 104.0, 98.6, 94.2, 93.7, 75.3,
49.6, 25.2. HR-ESI-MS m/z: found 547.1491, calculated 547.1481 [M+Na]+.
5.1.3.15. 3-O-[(E)-4-(3-cyano-5-(pyrrolidin-1-yl)phenyl)-2-oxobut-3-en-1-y
l]kaempferol (8k)
1
Yield: 6.8%. H NMR (400 MHz, DMSO-d6): δ 12.55 (1H, s, OH), 10.89
(1H, s, OH), 10.26 (1H, s, OH), 8.05 (2H, d, J = 8.8 Hz), 7.60 (1H, d, J = 16.0
Hz), 7.32 (1H, s), 7.13 (1H, d, J = 16.0 Hz), 7.09 (1H, s), 6.92 (1H, s), 6.91
(2H, d, J = 8.8Hz), 6.47 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.10
13
(2H, s), 3.27(4H, s), 1.96(4H, s). C NMR (100 MHz, DMSO-d6): δ 194.3,
177.5, 164.2, 161.1, 160.2, 156.3, 155.1, 147.8, 141.5, 136.2, 136.0, 130.5,

123.9, 120.5, 119.1, 117.4, 115.8, 115.4, 112.4, 104.0, 98.6, 93.7, 90.6, 75.2,
48.5, 24.9. HR-ESI-MS m/z: found 547.1451, calculated 547.1481[M+Na]+.
5.1.3.16. 3-O-[(E)-4-(4-cyano-2-(piperidin-1-yl)phenyl)-2-oxobut-3-en-1-yl
]kaempferol (8l)
1
Yield: 9.6%. H NMR (400 MHz, DMSO-d6): δ 12.54 (1H, s, OH), 10.85
(1H, s, OH), 10.36 (1H, s, OH), 8.03 (2H, d, J = 8.8 Hz), 7.85-7.40 (3H, m),
7.76 (1H, d, J = 16.4 Hz), 7.12 (1H, d, J = 16.4 Hz), 6.92 (2H, d, J = 8.8 Hz),
6.46 (1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.07 (2H, s), 2.88(4H, m),
13
1.66(4H, m), 1.53(2H, m). C NMR (100 MHz, DMSO-d6): δ 194.8, 177.4,
164.2, 161.1, 160.2, 156.3, 155.2, 153.7, 137.6, 136.2, 132.4, 130.5, 128.8,
125.6, 124.4, 122.6, 120.5, 118.7, 115.5, 113.0, 104.0, 98.7, 93.7, 75.4, 53.4,
25.6, 23.4. HR-ESI-MS m/z: found 561.1618, calculated 561.1638 [M+Na]+.
5.1.3.17. 3-O-[(E)-4-(5-cyano-2-(piperidin-1-yl)phenyl)-2-oxobut-3-en-1-yl
]kaempferol (8m)
1
Yield: 12.6%. H NMR (400 MHz, DMSO-d6): δ 12.55 (1H, s, OH), 10.89
(1H, s, OH), 10.26 (1H, s, OH), 8.06 (1H, d, J = 8.4 Hz), 8.04 (2H, d, J = 8.8
Hz), 7.75 (1H, dd, J1 = 2.0 Hz, J2 = 8.4 Hz), 7.65 (1H, d, J = 16.4 Hz), 7.17
(1H, d, J = 8.4 Hz), 7.12 (1H, d, J = 16.4 Hz), 6.91 (2H, d, J = 8.8 Hz), 6.47
(1H, d, J = 2.0 Hz), 6.21 (1H, d, J = 2.0 Hz), 5.07 (2H, s), 2.97 (4H, m),
13
1.67(4H, m), 1.55(2H, m). C NMR (100 MHz, DMSO-d6): δ 194.6, 177.4,
164.2, 161.1, 160.2, 156.6, 155.1, 137.8, 136.2, 134.3, 132.3, 130.5, 127.6,
123.3, 120.5, 119.0, 118.9, 115.4, 104.0, 103.6, 98.6, 93.7, 75.3, 53.0, 48.6,