Design, syntheses and lipid accumulation inhibitory activities of novel resveratrol mimics

Article abstract of DOI:10.1016/j.ejmech.2017.11.017

Hispidine was initially discovered from Ficus Hispida for cardiovascular protection. In this paper, hispidine derivatives, which contain a novel resveratrol-like scaffold, have been designed, synthesized, and assayed as agents against lipid accumulations in 3T3-L1 pre-adipocytes. Six hispidine derivatives have the activity of reducing TG in 3T3-L1 adipocytes in dosage-dependent manner. The most active compound can reduce the lipid accumulation up to 78.4% at 10 μM qPCR and Western blotting results demonstrate that the two most active compounds inhibit both lipodenesis and adipogenesis in 3T3-L1 cells through (1) increasing the phosphorylations of AMPK and ACC, promoting SIRT1 expression. These three proteins are key regulators for lipogenesis and energy metabolism. (2) Decreasing the expressions of PPARγ, sREBP-1c, and FABP4, which are pivotal regulators for adipogenesis. Overall, this work proves that hispidine derivatives diminish the lipid accumulation in 3T3-L1 cell line by downregulating lipogenic and adipogenic pathways.

Full text of DOI:10.1016/j.ejmech.2017.11.017

Accepted Manuscript
Design, syntheses and lipid accumulation inhibitory activities of novel resveratrol
mimics
Chanjuan Li, Bao Cheng, Sai Fang, Huihao Zhou, Qiong Gu, Jun Xu
PII:
S0223-5234(17)30910-8
10.1016/j.ejmech.2017.11.017
EJMECH 9892
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2017
Revised Date: 20 October 2017
Accepted Date: 5 November 2017
Please cite this article as: C. Li, B. Cheng, S. Fang, H. Zhou, Q. Gu, J. Xu, Design, syntheses and lipid
accumulation inhibitory activities of novel resveratrol mimics, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.11.017.
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Design, Syntheses and Lipid Accumulation Inhibitory Activities of Novel Resveratrol
Mimics
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Chanjuan Li†, Bao Cheng†, Sai Fang, Huihao Zhou, Qiong Gu*, and Jun Xu*
School of Pharmaceutical Sciences, Sun Yat-Sen University, 132 East Circle Road at University
City, Guangzhou, 510006, China.

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Abstract
Hispidine was initially discovered from Ficus Hispida for cardiovascular protection. In this paper,
hispidine derivatives, which contain a novel resveratrol-like scaffold, have been designed,
synthesized, and assayed as agents against lipid accumulations in 3T3-L1 pre-adipocytes. Six
hispidine derivatives have the activity of reducing TG in 3T3-L1 adipocytes in dosage-dependent
manner. The most active compound can reduce the lipid accumulation up to 78.4% at 10 µM.
qPCR and Western blotting results demonstrate that the two most active compounds inhibit both
lipodenesis and adipogenesis in 3T3-L1 cells through (1) increasing the phosphorylations of
AMPK and ACC, promoting SIRT1 expression. These three proteins are key regulators for
lipogenesis and energy metabolism. (2) Decreasing the expressions of PPARγ, sREBP-1c, and
FABP4, which are pivotal regulators for adipogenesis. Overall, this work proves that hispidine
derivatives diminish the lipid accumulation in 3T3-L1 cell line by downregulating lipogenic and
adipogenic pathways.
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Key words: lipid accumulation, AMPK pathway, PPARγ pathway, SIRT1, adipocyte

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1. Introduction
Hispidine, which is extracted from Ficus Hispida, and resveratrol are structurally mimics
(Figure 1). Local people make Ficus Hispida juice as a substitute for milk. The stem and leaf of
the plant are also used as natural therapeutic agents against hypoglycemia, diarrhoeal, and
cardiovascular risk [1-4]. The mechanisms of actions of hispidine derivatives are not well
understood.
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Figure 1. Structures of hispidine and resveratrol.
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Resveratrol, the naturally occurring polyphenol found in red wine and grape skin, was
initially reported as a potent anti-tumor agent [5]. From then on, resveratrol was further reported
for many pharmacological activities such as, cardio-protection, anti-aging, anti-oxidation,
anti-cancer, anti-inflammation [6-8], anti-obesity and, anti-diabetes [9]. The mechanism of
actions of resveratrol are reported to be associated with Silent information regulator 1 (SIRT1)
activation [10], Adenosine 5-monophosphate-activated protein kinase (AMPK) phosphorylation
and, Acetyl CoA carboxylase (ACC) phosphorylation [11].
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Considering the structures and physiological benefits of both resveratrol and hispidine,
derivatives, which contain a novel resveratrol and hispidine-like scaffold, were designed and
synthesized in pursuing to find lipid accumulation inhibitory agents. 3T3-L1 cell line, the in vitro
model recapitulating both pre-adipocyte differentiation (adipogenesis) and lipid accumulation
(lipogenesis) of adipocyte differentiation, was adopted in this study for screening [12]. The
differentiation of 3T3-L1 pre-adipocytes is mainly regulated by peroxisome
proliferator-activated receptor γ (PPARγ) pathway [13]. Increased PPARγ expression can
stimulate sterol regulatory element-binding protein-1c (sREBP-1c) and fatty acid-binding protein
4 (FABP4) expressions, which induce adipocyte differentiation in the early stage [14-16]. The

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lipogenesis and energy metabolism are mainly orchestrated by AMPK pathway [17]. Activated
AMPK down-regulates ACC activity through phosphorylating ACC, resulting in inhibited
lipogenesis and increased energy metabolism in SIRT1 related manner [18].
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To understand the mechanisms of actions of these hispidine derivatives on 3T3-L1
pre-adipocytes differentiation, experiments were further designed to study how hispidine
derivatives diminish lipid accumulation and, through which pathway.
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2. Chemistry
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The synthetic route for hispidine derivatives are shown in Scheme 1 and Scheme 2.
Commercially available 4-hydroxy-6-methyl-2H-pyran-2-one was first reacted with K2CO3 and
(CH3)2SO4 at room temperature in acetone overnight to give compound 2. Compound 2 was then
reacted with different aldehydes in the presence of Mg(OCH3)2 and was refluxed under N2 to
give compounds 3-14. Compounds 3-14 were reacted with excessive liquid NH3 (28%-30%) in
ethanol at 120 °C in sealed tube to provide target compounds 15-23.
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3 R=ph-
4 R=2-MeO-ph-
8 R=4-Cl-ph-
5 R=3-MeO-ph-
9 R=4-N, N-dimethyl-ph- 10 R=cyclohexyl-
14 R=thiophen-2-yl-
6 R=4-MeO-ph-
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7 R=2-Cl-ph-
11 R=Styryl- 12 R=2-methyl-furan-2-yl- 13 R=furan-2-yl-
Scheme 1. Syntheses of 1-14. Reagents and conditions: (a) K2CO3, acetone, (CH3)2SO4, r.t.,
overnight, yield: 85%. (b) aldehydes Mg(OCH3)2, reflux, N2, overnight, yield: 32%-59%.
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15 R=ph-
16 R=2-MeO-ph- 17 R=3-MeO-ph-
18 R=4-MeO-ph-

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19 R=2-Cl-ph- 20 R=cyclohexyl- 21 R=2-methyl-furan-2-yl- 22 R=furan-2-yl-
23 R=thiophen-2-yl-
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Scheme 2. Syntheses of 15-23. Reagents and conditions: (c) excessive liquid NH3, EtOH,
120°C, in the sealed tube, 36h, yield: 19%-51%.
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3. Experiments
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3.1 Chemistry
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All chemicals and solvents were purchased from commercial sources and were used without
further purification. Column chromatography (CC) was carried out on silica gel (200–300 mesh,
Qingdao Ocean Chemical Company, China), and thin-layer chromatography (TLC) analyses
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were carried out on silica gel GF254 glass plates. H NMR and 13C NMR were recorded in
CDCl3, Acetone-d6, or Methanol-d4 at room temperature on the Bruker spectrometer.
Electrospray ionization–mass spectrometry (ESI-MS) spectra were recorded on an Agilent
G1946D mass spectrometer.
3.1.1. 4-methoxy-6-methyl-2H-pyran-2-one (2)
A mixture of 4-hydroxy-6-methyl-2H-pyran-2-one (2.60 mmol) and K2CO3 (2.60 mmol)
and (CH3)2SO4 (2.60 mmol) in acetone (15 mL) was stirred at room temperature overnight. The
mixture was filtered. The filtrate was concentrated in vacuum. The residue was purified with
silica gel chromatography using chloroform as eluent to obtain target compound 2 as white solid.
Yield: 86%; 1H NMR (400 MHz, CDCl3) δ 5.71 (s, 1H), 5.34 (s, 1H), 3.73 (s, 3H), 2.14 (s, 3H).
3.1.2 General procedure for the preparation of compounds 3-14
Compound 2 (2.40 mmol) and appropriate aldehydes (2.40 mmol) in the presence of
Mg(OCH3)2 (10 mL) was refluxed under N2 overnight. After the reaction was completed as
indicated by TLC, the mixture was concentrated under reduced pressure. The residue was
purified with silica gel chromatography to obtain target compounds 3-14.
3.1.2.1 (E)-4-methoxy-6-styryl-2H-pyran-2-one (3), Yield 56%; m.p. 147.2 – 149.3 °C; white
solid; 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.1 Hz, 2H), 7.44 (s, 1H), 7.37 (d, J = 7.6 Hz,
1H), 7.31 (s, 2H), 6.59 (dd, J = 16.0, 4.1 Hz, 1H), 5.95 (s, 1H), 5.50 (s, 1H), 3.82 (s, 3H).

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3.1.2.2 (E)-4-methoxy-6-(2-methoxystyryl)-2H-pyran-2-one (4) Yield: 57%; m.p. 134.0 –
136.1 °C; white solid; 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 16.1 Hz, 1H), 7.49 (d, J = 7.0
Hz, 1H), 7.30 (dd, J = 15.5, 8.3 Hz, 2H), 7.11 – 6.85 (m, 3H), 6.69 (t, J = 14.5 Hz, 1H), 5.91 (s,
1H), 5.46 (s, 1H), 3.90 (s, 3H), 3.83 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 171.2, 164.2, 159.4,
158.0, 131.4, 130.5, 128.2, 124.3, 120.7, 119.4, 111.1, 100.9, 88.6, 55.8, 55.4. LC−MS (ESI) m/z:
258.1 [M + H]+.
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3.1.2.3 (E)-4-methoxy-6-(3-methoxystyryl)-2H-pyran-2-one (5) Yield: 54%; white solid; H
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NMR (400 MHz, CDCl3) δ 7.46 – 7.33 (m, 1H), 7.19 (dd, J = 11.1, 8.5 Hz, 1H), 7.03 (d, J = 7.6
Hz, 1H), 6.93 (d, J = 10.9 Hz, 1H), 6.82 (dd, J = 8.2, 2.2 Hz, 1H), 6.49 (d, J = 15.9 Hz, 1H), 5.87
(d, J = 1.8 Hz, 1H), 5.42 (d, J = 1.8 Hz, 1H), 3.76 (s, 3H), 3.75 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 171.0, 163.9, 159.9, 158.5, 136.6, 135.7, 129.9, 120.1, 118.9, 115.2, 112.5, 101.4, 88.9,
55.9, 55.3. LC−MS (ESI) m/z: 258.1 [M + H]+.
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3.1.2.4 (E)-4-methoxy-6-(4-methoxystyryl)-2H-pyran-2-one (6) Yield 59%; m.p. 152.5 –
155.3 °C; white solid; 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 5.9 Hz, 1H), 7.32 (s, 2H),, 6.82
(d, J = 8.2 Hz, 2H), 6.37 (d, J = 15.9 Hz, 1H), 5.81 (s, 1H), 5.39 (s, 1H), 3.75 (s, 3H), 3.74 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 171.2, 164.1, 160.7, 159.1, 135.4, 135.4, 128.9, 128.0,
116.4, 114.4, 114.4, 100.4, 88.3, 55.8, 55.3. LC−MS (ESI) m/z: 258.1 [M + H]+.
3.1.2.5 (E)-6-(2-chlorostyryl)-4-methoxy-2H-pyran-2-one (7) Yield 43%; m.p. 115.6 - 116.8 °C;
light yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 16.0 Hz, 1H), 7.51 (dd, J = 6.1, 3.4
Hz, 1H), 7.33 (dd, J = 5.8, 3.5 Hz, 1H), 7.20 (d, J = 2.7 Hz, 1H), 7.18(d, J = 2.9 Hz, 1H), 6.51 (d,
J = 16.0 Hz, 1H), 5.92 (d, J = 2.0 Hz,1H), 5.44 (d, J = 2.0 Hz, 1H) 3.78 – 3.54 (m, 3H). 13C
NMR (100 MHz, CDCl3) δ 169.8, 162.7, 157.2, 133.5, 132.5, 130.6, 129.1, 126.0, 120.4, 101.0,
88.3, 54.9. LC−MS (ESI) m/z: 263.1 [M + H]+.
3.1.2.6 (E)-6-(4-chlorostyryl)-4-methoxy-2H-pyran-2-one (8) Yield 47%; m.p. 139.0 – 141.2 °C;
light yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.9 Hz, 1H), 7.34 (s, 2H), 7.27 (d, J
= 8.4 Hz, 2H), 6.47 (d, J = 16.0 Hz, 1H), 5.87 (d, J = 1.8 Hz, 1H), 5.42 (d, J = 1.8 Hz, 1H), 3.82
– 3.63 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 170.9, 163.7, 158.2, 135.2, 134.3, 133.7, 129.1,

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129.1, 128.5, 128.5, 119.1, 101.6, 89.0, 55.9. LC−MS (ESI) m/z: 263.1 [M + H]+.
3.1.2.7 (E)-6-(4-(dimethylamino)styryl)-4-methoxy-2H-pyran-2-one (9) Yield 32%; m.p. 145.0
– 146.7 °C; yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 2.9 Hz, 1H), 7.31 (s, 2H),,
6.62 (d, J = 4.4 Hz, 2H), 6.31 (dd, J = 15.8, 4.2 Hz, 1H), 5.77 (s, 1H), 5.36 (s, 1H), 3.75 (d, J =
4.4 Hz, 3H), 3.15 – 2.79 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 171.4, 164.4, 159.9, 151.2,
136.3, 128.9, 128.9, 123.2, 113.6, 112.0, 112.0, 99.2, 87.6, 55.7, 40.1, 40.1. LC−MS (ESI) m/z:
272.1 [M + H]+.
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3.1.2.8 (E)-6-(2-cyclohexylvinyl)-4-methoxy-2H-pyran-2-one (10) Yield 57%; white solid; H
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NMR (400 MHz, CDCl3) δ 6.57 (dd, J = 15.7, 7.0 Hz, 1H), 5.82 (d, J = 15.7 Hz, 1H), 5.70 (d, J
= 2.0 Hz, 1H), 5.35 (d, J = 2.0 Hz, 1H), 3.74 (s, 3H), 2.04 (dd, J = 10.9, 7.0 Hz, 1H), 1.67 (d, J =
10.8 Hz, 4H), 1.59 (d, J = 12.7 Hz, 1H), 1.22 (m, 3H), 1.07 (m, 3H). 13C NMR (100 MHz, CDCl3)
δ 171.2, 164.0, 158.9, 145.0, 118.9, 99.5, 88.0, 55.7, 40.7, 32.0, 25.7. LC−MS (ESI) m/z: 235.1
[M + H]+.
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3.1.2.9 4-methoxy-6-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)-2H-pyran-2-one (11) Yield: 43%;
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light yellow solid; H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 7.2 Hz, 2H), 7.27 (d, J = 7.7 Hz,
3H), 7.25 – 7.15 (m, 3H), 6.86 – 6.71 (m, 2H), 6.08 (d, J = 15.1 Hz, 1H), 5.80 (s, 1H), 5.40 (s,
1H), 3.74 (d, J = 2.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.0, 163.0, 157.6, 137.2, 135.4,
135.2, 127.7, 127.7, 127.6, 126.0, 125.9, 125.9, 121.0, 99.9, 87.7, 54.8. LC−MS (ESI) m/z: 255.1
[M + H]+.
3.1.2.10 (E)-4-methoxy-6-(2-(5-methylfuran-2-yl)vinyl)-2H-pyran-2-one (12) Yield 43%; m.p.
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148.5 – 150.4 °C; light yellow solid; H NMR (400 MHz, CDCl3) δ 7.11 (d, J = 15.6 Hz, 1H),
6.33 (dd, J = 9.2, 6.2 Hz, 2H), 5.99 (d, J = 2.7 Hz, 1H), 5.80 (s, 1H), 5.38 (d, J = 1.9 Hz, 1H),
3.73 (d, J = 10.1 Hz, 3H), 2.27 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 171.1, 164.0, 158.9, 154.6,
150.2, 122.7, 114.9, 108.8, 100.5, 88.2, 55.8, 13.8. LC−MS (ESI) m/z: 233.2 [M + H]+.
3.1.2.11 (E)-6-(2-(furan-2-yl)vinyl)-4-methoxy-2H-pyran-2-one (13) Yield 41%; m.p. 143.0 –
144.7 °C; light yellow solid; 1 HNMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 7.21 (t, J = 4.7 Hz, 1H),
7.17 (d, J = 4.8 Hz, 1H), 6.50 (d, J = 15.7 Hz, 2H), 6.40 (d, J = 3.1 Hz, 1H), 5.86 (s, 1H), 5.41 (d,

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J = 2.9 Hz, 1H), 3.76 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.0, 162.9, 157.4, 150.5, 142.9,
121.5, 115.5, 112.3, 111.3, 100.2, 87.6, 54.9. LC−MS (ESI) m/z: 219.1 [M + H]+.
3.1.2.12 (E)-4-methoxy-6-(2-(thiophen-2-yl)vinyl)-2H-pyran-2-one (14) Yield 37%; m.p. 166.1
- 168.2 °C; light yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 15.6 Hz, 1H), 7.24 (d, J
= 4.9 Hz, 1H), 7.11 (d, J = 3.0 Hz, 1H), 6.96 (t, J = 3.8 Hz, 1H), 6.30 (d, J = 15.6 Hz, 1H), 5.83
(s, 1H), 5.40 (s, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 171.0, 163.8, 158.3, 140.6,
129.5, 128.6, 128.1, 127.1, 117.7, 100.9, 88.6, 55.9. LC−MS (ESI) m/z: 235.2 [M + H]+.
3.1.3 General procedure for the preparation of compounds 15-23
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Compounds 3-14 (1.2 mmol) were reacted with excessive liquid NH3 (28%), stirred in the
ethanol at 120 °C, in sealed tubes, for 36h. Each mixture was concentrated in vacuum. The
residue was purified with silica gel chromatography using chloroform/methanol (V: V = 20:1) as
eluent to obtain target compounds 15-23.
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3.1.3.1 (E)-4-hydroxy-6-styrylpyridin-2(1H)-one (15) Yield: 28%; light yellow solid; H NMR
(400 MHz, Methanol-d4) δ 7.57 (d, J = 7.1 Hz, 2H), 7.42 (s, 1H), 7.38 (dd, J = 9.6, 4.8 Hz, 3H),
7.33 (dd, J = 5.2, 1.8 Hz, 1H), 6.79 (d, J = 16.0 Hz, 1H), 6.09 (d, J = 1.9 Hz, 1H), 5.14 (d, J =
1.9 Hz, 1H). 13C NMR (100 MHz, Methanol-d4) δ 167.8, 162.6, 160.0, 136.9, 136.0, 130.3,
129.9, 129.9, 128.5, 128.5, 120.5, 101.9, 83.5. LC−MS (ESI) m/z: 214.1 [M + H]+.
3.1.3.2 (E)-4-hydroxy-6-(2-methoxystyryl)pyridin-2(1H)-one (16) Yield: 27%; m.p. 248.0 –
249.7 °C; light yellow solid; 1H NMR (400 MHz, Methanol-d4) δ 7.57 (d, J = 7.3 Hz, 2H), 7.31
(t, J = 7.4 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.96 (m, 1H), 6.86 (d, J = 16.6 Hz, 1H), 6.10 (s, 1H),
5.48 (s, 1H), 3.90 (s, 3H). 13C NMR (100 MHz, Methanol-d4) δ 165.4, 164.2, 159.2, 157.2, 143.9,
129.6, 127.9, 126.6, 124.0, 120.3, 120.0, 110.5, 97.7, 54.2. LC−MS (ESI) m/z: 244.2 [M + H]+.
3.1.3.3 (E)-4-hydroxy-6-(3-methoxystyryl)pyridin-2(1H)-one (17) Yield: 23%; m.p. 168.4 –
171.1 °C; light yellow solid; 1H NMR (400 MHz, Acetone-d6) δ 7.33 (s, 1H), 7.30 (d, J = 7.3 Hz,
1H), 7.21 (d, J = 6.7 Hz, 2H), 6.91 (d, J = 7.4 Hz, 1H), 6.86 (d, J = 16.0 Hz, 1H), 6.28 (s, 1H),
6.08 (s, 1H), 5.08 (s, 1H), 3.84 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ 163.4, 161.1, 160.0,
159.3, 138.1, 134.4, 130.6, 121.4, 120.7, 115.7, 113.1, 101.6, 84.4, 55.5. LC−MS (ESI) m/z:

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244.1 [M + H]+.
3.1.3.4 (E)-4-hydroxy-6-(4-methoxystyryl)pyridin-2(1H)-one (18) Yield: 31%; m.p. 144.6 -
146.5 °C; light yellow solid; 1H NMR (400 MHz, Acetone-d6) δ 7.58 (d, J = 7.9 Hz, 2H), 7.29 (d,
J = 15.9 Hz, 1H), 6.96 (d, J = 7.7 Hz, 2H), 6.69 (d, J = 15.9 Hz, 1H), 6.24 (s, 2H), 6.02 (s, 1H),
5.05 (s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ 163.5, 161.5, 160.2, 159.7, 134.1,
129.7, 129.7, 129.3, 118.8, 115.1, 115.1, 100.6, 84.0, 55.6. LC−MS (ESI) m/z: 244.1 [M + H]+.
3.1.3.5 (E)-6-(2-chlorostyryl)-4-hydroxypyridin-2(1H)-one (19) Yield: 19%; m.p. 213.1 –
215.4 °C; light yellow solid; 1H NMR (400 MHz, Acetone-d6) δ 7.84 (s, 1H), 7.70 (d, J = 5.9 Hz,
1H), 7.48 (d, J = 2.9 Hz, 1H), 7.37 (d, J = 2.6 Hz, 2H), 6.91 (d, J = 7.7 Hz, 1H), 6.33 (s, 2H),
6.16 (s, 1H), 5.11 (d, J = 5.4 Hz, 1H). 13C NMR (100 MHz, Acetone-d6) δ 163.2, 159.9, 158.7,
134.5, 131.0, 130.8, 129.4, 128.3, 128.0, 124.0, 102.6, 84.7. LC−MS (ESI) m/z: 248.1 [M + H]+.
3.1.3.6 (E)-6-(2-cyclohexylvinyl)-4-hydroxypyridin-2(1H)-one (20) Yield: 51%; m.p. 173.2 –
175.3 °C; light yellow solid; 1H NMR (400 MHz, CDCl3) δ 6.60 (dd, J = 15.7, 7.0 Hz, 1H), 5.83
(d, J = 15.7 Hz, 1H), 5.61 (s, 1H), 5.11 (s, 1H), 4.94 (s, 2H), 2.08 (d, J = 7.3 Hz, 1H), 1.71 (d, J
= 10.2 Hz, 4H), 1.23 (t, J = 16.8 Hz, 3H), 1.11 (t, 3H). 13C NMR (100 MHz, CDCl3) δ 164.7,
159.4, 158.7, 144.8, 119.3, 98.4, 84.9, 40.8, 32.1, 32.1, 25.9, 25.7, 25.7. LC−MS (ESI) m/z:
220.1 [M + H]+.
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3.1.3.7 (E)-4-hydroxy-6-(2-(5-methylfuran-2-yl)vinyl)pyridin-2(1H)-one (21) Yield: 34%; m.p.
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181.7 – 183.5 °C; yellow solid; H NMR (400 MHz, Acetone-d6) δ 6.78 (d, J = 15.7 Hz, 1H),
6.30 (d, J = 2.7 Hz, 1H), 6.18 (d, J = 15.7 Hz, 1H), 5.99 (s, 2H), 5.88 (s, 1H), 5.76 (s, 1H), 4.77
(s, 1H), 2.61 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ 163.4, 160.2, 159.3, 155.0, 151.4,
121.8, 117.0, 115.0, 109.5, 100.8, 84.0, 13.7. LC−MS (ESI) m/z: 218.1 [M + H]+.
3.1.3.8 (E)-6-(2-(furan-2-yl)vinyl)-4-hydroxypyridin-2(1H)-one (22) Yield: 36%; m.p.
174.5-176.9 °C; yellow solid; 1H NMR (400 MHz, Acetone-d6) δ 7.62 (s, 1H), 7.12 (d, J = 15.8
Hz, 1H), 6.72 (t, J = 11.6 Hz, 1H), 6.56 (d, J = 15.7 Hz, 2H), 6.28 (s, 2H), 6.07 (d, J = 1.2 Hz,
1H), 5.06 (d, J = 1.7 Hz, 1H). 13C NMR (100 MHz, Acetone-d6) δ 163.4, 160.1, 158.9, 152.8,
144.9, 121.7, 118.7, 113.4, 113.1, 101.4, 84.3. LC−MS (ESI) m/z: 204.1 [M + H]+.

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3.1.3.9 (E)-4-hydroxy-6-(2-(thiophen-2-yl)vinyl)pyridin-2(1H)-one (23) Yield: 32%; m.p.
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201.7- 203.8 °C; yellow solid; H NMR (400 MHz, Methanol-d4) δ 7.52 (d, J = 15.7 Hz, 1H),
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7.44 (d, J = 5.0 Hz, 1H), 7.27 (d, J = 3.2 Hz, 1H), 7.06 (dd, J = 5.0, 3.7 Hz, 1H), 6.53 (d, J =
15.7 Hz, 1H), 6.04 (d, J = 1.7 Hz, 1H), 5.11 (d, J = 1.9 Hz, 1H). 13C NMR (100 MHz,
Methanol-d4) δ 167.8, 162.5, 159.7, 142.0, 130.5, 129.1, 128.9, 128.3, 119.4, 101.6, 83.4.
LC−MS (ESI) m/z: 220.1 [M + H]+.
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3.2 Cell culture and adipocyte differentiation
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Cells (HEK293T, 3T3-L1) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL
streptomycin at 37 °C in 5% CO2. Adipocyte differentiation was carried out on 3T3-L1 in
accordance with Ji-Ming Ye [19]. Briefly, 2 days after full confluence (day 0), the medium was
changed to differentiation medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin,
100 µg/mL streptomycin, 2 µg/mL insulin, 100 ng/mL dexamethasone, 0.5 mM
3-isobutyl-1-methylxanthine (IBMX) and 10 ng/mL biotin) for 3 days. Then (day 3),
differentiation medium was switched to post-differentiation medium (DMEM supplemented with
10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 µg/mL insulin) for additional 3 days.
At the end (day 6), cells were used for further analysis. Tested compounds were dissolved in
dimethyl sulfoxide (DMSO) and supplemented at indicated concentrations throughout the course
of differentiation. Berberine (BBR) was used as positive control. 0.1% DMSO was used as
vehicle control.
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3.3 Triglycerides (TGs) assay
3T3-L1 cells were collected at the end of differentiation and washed twice with ice-cold
PBS buffer (0.2 M NaCl, 10 mM Na2HPO4, 3 mM KCl, 2 mM KH2PO4, pH 7.4). Then the cells
were lysed, the levels of intracellular triglycerides were determined using commercial TG assay
kit (Jiancheng Bioengineering Institution, Nanjing, China) according to the manufacture’s
protocol. Results were presented as the relative TG content compared to the positive control
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3.4 Oil red O (ORO) staining
Cells were fixed with 10% (v/v) formaldehyde in PBS for 1 hour at room temperate and
stained with freshly prepared ORO working solution for 30 min followed by three washes with
water. Plates were pictured using a microscope (Nikon, Tokyo, Japan) equipped with a CCD
digital camera (Nikon, Tokyo, Japan). Constant condenser and light intensity settings were used
throughout the imaging process.
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3.5 Cytotoxicity assay
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HEK293T cells were seeded at the density of 5000 per well into 96-well plates to grow
overnight. Then the cells were treated with various concentrations of compounds for 24h. 0.5%
DMSO as vehicle control. MTT (0.05 mg) was added to each well and incubated for 4h, after
which 100 µL DMSO was added to each well. The absorbance of wells was recorded at 492 nm
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after 20 min of shaking. The inhibition percentage was calculated as (Aexperiment –Ablank)/(Acontrol
–
Ablank).
3.6 Real-time quantitative PCR
Total RNA was isolated using RNAiso plus (TaKaRa, Dalian, China) according to the
manufacturer’s protocol. Total RNA (1 µg) was converted to cDNA using ReverTra Ace qPCR
RT Master Mix (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. cDNA and
SYBR Green was used to amplify specific target genes by Real-time PCR Master Mix (Toyobo,
Osaka, Japan). The following PCR primer sequences were used: β-actin, sense
5’-TGGAATCCTGTGGCATCCATGAAA-3’
5’-TAAAACGCAGCTCAGTAACAGTCC-3’;
and
antisense
sense
AMPK,
5’-AAGCCGACCCAATGACATCA-3’ and antisense 5’-CTTCCTTCGTACACGCAAAT-3’;
ACC, sense 5’-AGGATTTGCTGTTTCTCAGAGCTT-3’ and antisense
5’-CAGGATCTACCCAGGCCACAT-3’; SIRT1, sense 5’-TTGTGAAGCTGTTCGTGGAG-3’
and antisense 5’-GCGTGGAGGTTTTTCAGTA-3’; PPARγ, sense
5’-TGCTGTTATGGGTGAAACTCTG-3’ antisense 5’-GAAATCAACTGTGGTAAAGGGC-3’;
sREBP-1c, sense 5’-CAGCTCAGAGCCGTGGTGA-3’and antisense

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5’-TGTGTGCACTTCGTAGGGTC-3’;
FABP4,
sense
5’-GTCACCATCCGGTCAGAGAGTAC-3’and antisense5’-TCGTCTGCGGTGATTTCATC-3’.
3.7 Western blotting
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Cell lysates were prepared in lysis buffer containing 1% (v/v) NP-40, 0.25% (w/v)
deoxycholate, 0.15% (w/v) SDS, protease inhibitor (Beyotime, Shanghai, China), and
phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Protein concentrations were
quantified using Pierce Rapid Gold BCA Protein Assay Kit (ThermoFisher scientific, Waltham,
USA). Protein samples were denature in SDS sample buffer and then subjected to SDS-PAGE
and blotted onto polyvinylidene difluoride (Millipore, Darmstadt, Germany) membranes. The
membranes were then blocked with 5% (w/v) skim milk in TBST (Tris-buffered saline
containing 0.1% (v/v) Tween 20) for 2 h and then incubated with primary antibodies overnight at
4 °C. After three times washing in TBST, the membranes were then incubated with anti-mouse or
anti-rabbit IgG secondary antibodies (conjugated to Horseradish Peroxidase) at room
temperature for 2 h. Immunoreactive signals were detected using enhanced chemiluminescence
reagents with Chemiluminescence imager (Tanon 5200, Shanghai, China). Densitometry analysis
was performed using Quantity One Software (Bio-Rad Laboratories, USA) and quantified to the
loading control α-Tublin. Antibodies used including phospho (Thr172)- and total-AMPKα (Cell
Signaling, Danvers, USA), phospho (Ser79)- and total- ACC (Cell Signaling, Danvers, USA),
SIRT1 (Cell Signaling, Danvers, USA), PPARγ (Sigma, St. Louis, USA), sREBP-1c (Santa Cruz,
Dallas, USA), FABP4 (Abcam, Cambridge, USA), α-Tublin (Sigma, St. Louis, USA).
4. Results
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4.1 Identifying hispidine derivatives as lipid-lowering agents through 3T3-L1 cell-based
assays.
The 3T3-L1 cells were treated with differentiation cocktail together with 21 compounds at
the concentration of 10 µM. 11 compounds exhibited the ability of the down-regulating TG
accumulations in 3T3-L1 cells (Figure 2A). Among the 11 active compounds, 6 compounds
significantly exhibited greater activities than BBR (a well-known positive control agent for

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3T3-L1 assay). Compound 20 was the best TG accumulation reducer, which reduced 78.4% TG
accumulation. The chemical structures of the 6 most active compounds are depicted in Figure
2B.
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Figure 2. (A) TG assay results for 11 active hispidine derivatives. 3T3-L1 pre-adipocytes
were treated with 10 µM of 21 compounds on days 0 and 3, for 3 days during the differentiation.
The levels of intracellular TG were determined using commercial TG assay kit. Data are
represented as mean ± SD of three independent experiments (*p < 0.05 vs vehicle, **p < 0.01 vs
vehicle). (B) The structures of compounds with best TG-lowing effect.
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4.2 Measuring the inhibitory efficacies of lipid accumulation in 3T3-L1 cells.
To further evaluate the inhibitory efficacies of these compounds on lipid accumulation, the
3T3-L1 pre-adipocytes were treated with differentiation cocktail together with 6 active
compounds using different concentrations (10, 3.3, 1.1, and 0.37 µM) for 6 days. All 6 active

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compounds significantly reduced the intracellular lipid accumulation in a dosage-dependent
manner (Figure 3A). Compounds 20 and 21 are superior to others in terms of reducing TG
accumulation. ORO staining was applied for visual inspecting the quantity of lipid, which was
stained in red. The agents tested in 3T3-L1 assays were BBR, resveratrol, and compounds 20 and
21. In Figure 3B, there is almost no lipid (red spots) can be observed in the cells treated with
BBR, resveratrol, compound 20 or 21 at 10 µM. There is significantly reduced lipid in the cells
treated with those agents at 1 µM. Previous cytotoxicity tests demonstrated that 1 or 10 µM are
far away below the CC50 of these agents. Specifically, compound 21 is not toxic to 3T3-L1 cells
at up to 50 µM (Figure 3B).
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Figure 3. (A) Hispidine derivatives dose-dependently reduce lipid accumulation in 3T3-L1
cells. (B) Compounds 20 and 21 are superior to BBR and resveratrol in terms of reducing TG
accumulation. Pictures were taken on day-6 with 10× magnification. UN: undifferentiated cells.
Vehicle: differentiated cells without compounds. 293T cells received treatment of compounds for
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24 h to test their cytotoxicity. Data are represented as mean ± SD of three independent
experiments (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle).
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4.3 Hispidine derivatives suppressed adipogenic transcript factors expressions.
PPARγ and sREBP-1c are the main transcription factors involved in adipocyte
differentiation. FABP4 is a marker of adipocyte differentiation. To elucidate the mechanism of
actions of compounds 20 and 21 in the differentiation of 3T3-L1 pre-adipocyte, we investigated
compounds 20 and 21 effects on the mRNA and protein expressions of adipogenic genes PPARγ,
sREBP-1c, and downstream transcriptional factor FABP4 through qPCR and Western blotting
analyses. The experiments demonstrate that when treated with differentiation cocktail, PPARγ,
sREBP-1c and FABP4 mRNA were significantly up-regulated. After treated with compound 20
or 21 for 3 days, the mRNA expressions of PPARγ, sREBP-1c and FABP4 were significantly
suppressed comparing with that of vehicle (Figures 4C - 4E). Consistently, PPARγ, sREBP-1c
and FABP4 protein expression levels were increased when treated with differentiation cocktail.
After treated with compound 21, PPARγ, sREBP-1c and FABP4 protein expression levels were
significantly attenuated (Figures 4A, 4B). These results indicate that compounds 20 and 21 can
suppress the differentiation of 3T3-L1 pre-adipocytes by inhibiting the mRNA and protein
expressions of adipogenic genes.
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Figure 4. Hispidine derivatives blocked PPARγ-FABP4 pathway. 3T3-L1 pre-adipocytes
were treated with differentiation cocktail with or without compound 20 or 21 at day 0 for 3 days.
Then the cells were harvested for qPCR and Western blotting analyses. Un: undifferentiated cells.
Dif. cocktail: differentiation cocktail. (A) PPARγ, sREBP-1c, and FABP4 protein levels. (B)
Quantitative PPARγ, sREBP-1c, and FABP4 protein levels. (C) - (E) mRNA levels for PPARγ
(C), sREBP-1c (D), and FABP4 (E). Data are represented as mean ± SD of 3 independent
experiments (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle).
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4.4 Hispidine derivatives suppress lipogenesis by up-regulating AMPK pathway.
AMPK activation can inhibit lipogenesis and induce energy metabolism. To prove
compounds 20 and 21 up-regulating AMPK pathway, qPCR and Western blotting analyses were
performed on 3T3-L1 cells treated with compound 20 or 21. In figure 5, the mRNA and protein
levels of t-AMPK were up-regulated when treated with differentiation cocktail. Treatment with
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compound 20 or 21 caused increased AMPKα phosphorylation. Consequently, ACC (the
substrate of AMPK) phosphorylation was increased. Specifically, compounds 20 and 21
increased the AMPKα phosphorylation by 3.45 and 3.10 fold, and that of ACC by 2.98 and 4.12
fold. In short, the mRNA expression was not increased, the phosphorylation was increased
instead.
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AMPKα activation has many therapeutic benefits because of its enhancing oxidative
metabolism and mitochondrial biogenesis. This process relies on SIRT1 [10], which is required
for AMPKα activation during mitochondrial biogenesis as well as energy metabolism [17]. Here,
the mRNA and SIRT1 protein expressions were also studied. Results indicate that SIRT1 mRNA
and protein expression levels in differentiated 3T3-L1 cells are significantly higher than
pre-adipocytes. After the 3T3-L1 cells were treated with compounds 20 or 21, SIRT1 mRNA and
protein expression levels are even higher. This means that SIRT1 involves in AMPK activation
during pre-adipocytes differentiation (Figures 5A, 5B).
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Figure 5. Hispidine derivatives suppress lipogenesis by up-regulating AMPK pathway.
3T3-L1 pre-adipocytes were treated with differentiation cocktail with or without compounds 20
or 21 at day 0 for 3 days. Then the cells were harvested for qPCR and Western blotting analysis.
Un: undifferentiated cells. Dif. cocktail: differentiation cocktail. (A) Protein expression levels of
the phosphorylated AMPKα, total AMPKα, phosphorylated ACC, total ACC, and SIRT1. (B)
Quantitative AMPKα, total AMPKα, phosphorylated ACC, total ACC, and SIRT1 protein levels.
(C) The mRNA levels for AMPK, ACC, and SIRT1. Data are represented as mean ± SD of 3
independent experiments (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle).
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5. Discussion
In this study, hispidine derivatives, which contain a novel resveratrol-like scaffold, have
been designed, synthesized, and assayed as agents against lipid accumulations in 3T3-L1
pre-adipocytes. The structure-activity relationship can be drawn through 3D and 2D molecular

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property descriptors calculation using MOE. According to R2 and statistical significance, 8
molecular property descriptors (descriptors concerning surface area, conformation dependent
charge, pharmacophore feature, and physical properties) are found to be significantly related to
their TG inhibitory activities. Potential energy, logP, and logS are also considered related (Table
1). Increased hydrophobic surface, H-bond acceptor, potential energy, logP, and decreased logS
reduces TG inhibitory activity (Figures S44A-S44K, right), indicating that these hispidine
derivatives should keep a balance in hydrophilicity and lipophilicity (logP ~2). Compared to
hydroxyl group, hispidine derivatives with a methoxyl group at C-4 position can significantly
increase hydrophobic surface, H-bond acceptor, VDW acceptor surface, and logP, and decrease
logS (Figure S44, left). Consequently, this reduced TG inhibitory activity.
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Table 1. Correlations between molecular properties and activity.
Descriptor code
ASA
Description
R2
Significance
0.001
Water accessible surface area of
hydrophobic atoms
0.444
ASA_H
vdw_area
a_acc
Total hydrophobic surface area
Van der Waals (VDW) surface area
Number of H-bond acceptor atoms
VDW acceptor surface area
VDW volume
0.444
0.380
0.379
0.379
0.361
0.341
0.219
0.204
0.001
0.003
0.003
0.003
0.004
0.005
0.033
0.400
vsa_acc
vdw_vol
vsa_hyd
a_hyd
E
VDW hydrophobic surface area
Number of hydrophobic atoms
Potential energy
logP
Log octanol/water partition coefficient 0.128
Log solubility in water 0.120
logS
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The differentiation of pre-adipocytes into mature adipocytes consists of adipogenesis and
lipogenesis [12, 20], and compounds 20 and 21 can block both processes. Based on the results,
we can construct the pathway of compounds 20 and 21 blocking adipogenesis and lipogenesis.

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As shown in Figure 6, we conclude (1) the hispidine derivatives (compounds 20 and 21) increase
AMPK phosphorylation and SIRT1 expression, down-regulate ACC activity through
phosphorylating ACC. Consequently, the compounds inhibit lipogenesis and increase energy
metabolism in 3T3-L1 pre-adipocytes differentiation process. (2) The hispidine derivatives
inhibit PPARγ, sREBP-1c, and FABP4 expressions. The adipogenesis is mainly regulated by
PPARγ pathway [13]. sREBP-1c is a downstream factor of PPARγ, and regulates the expression
of lipogenic proteins like ACC. FABP4 is another downstream factor of PPARγ and a biomarker
of adipocyte differentiation [21]. Decreased PPARγ, sREBP-1c, and FABP4 expressions result in
inhibited adipogenesis in 3T3-L1 pre-adipocyte differentiation process.
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In conclusion, the hispidine derivatives can block both adipogenesis and lipogenesis
through increasing the phosphorylation of AMPK and ACC, up-regulating SIRT1,
down-regulating PPARγ, sREBP-1c, and FABP4. Hispidine derivatives can be promising leads
for the therapeutic solutions against obesity and type 2 diabetes.
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Figure 6. Pathway of compounds 20 and 21 blocking adipogenesis and lipogenesis.
6. Author’s Contributions
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J.X. and Q.G. designed research. CJ.L., B.C., and S.F. performed research, all authors

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analyzed data, J.X. CJ.L., and B.C. wrote the paper, all authors approved the final version.
7. Acknowledgements
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This work was funded by the Science and Technology Planning Project of Guangdong
Province (No. 2016A020217002), the Medical Scientific Research Foundation of Guangdong
Province (A2016133), the National Natural Science Foundation of China (No. 81603030), the
National Natural Science Foundation of China (No. 81473138), and the National Natural Science
Foundation of China (No. 81603026).
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8. Competing Interests
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The authors declare no competing financial interest.
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Hispidine derivatives were designed and synthesized against adipocyte lipid increase.
Hispidine derivatives inhibit both lipogenesis and adipogenesis in 3T3-L1 cells.
Hispidine derivatives increase AMPK and ACC phosphorylations and SIRT1 expression.
Hispidine derivatives downregulate adipogenic factors: PPARγ, sREBP-1c, and FABP4.