Design, synthesis chalcone derivatives as AdipoR agonist for type 2 diabetes

Article abstract of DOI:10.1111/cbdd.13319

Two structurally novel series of chalcone derivatives were designed and synthesized as potential agents against type 2 diabetes. As a result of the antidiabetic biological evaluation in streptozotocin (STZ)-induced type 2 diabetes animal model, 13e, 13g, and 19f showed more significant reduction in serum Glu, TG, TC levels by contrast to the positive control AdipoRon. In addition to upregulating the expression of AdipoR1 and AdipoR2, 13e and 19f treatment also increased expression of AMPK and PPAR-α. Taken together, these results suggested that 13e and 19f might be a promising compound for type 2 diabetes treatment.

Full text of DOI:10.1111/cbdd.13319

MR. PANHU ZHU (Orcid ID : 0000-0001-5440-4391)
MR. WEIJUN HUANG (Orcid ID : 0000-0003-2202-0574)
PROFESSOR JIAMING LI (Orcid ID : 0000-0003-2141-7561)
Article type
: Research Article
Corresponding author mail id:lijiaming2017@sina.com
Design, synthesis chalcone derivatives as AdipoR agonist for
type 2 diabetes
Panhu Zhu1, Weijun Huang1, Jiaming Li1*, Xiaodong Ma1, Mengqi Hu1, Yujun Wang1,
Qinlong Xu2, Xianna Wang1.
(1 Department of Medicinal Chemistry, Anhui University of Chinese Medicine, 103 Meishan Road,
Hefei 230031, PR China; 2. Hefei Industrial Pharmaceutical Institute Co, Ltd, Hefei 230059, PR
China.)
Abstract
Two structurally novel series of chalcone derivatives were designed and synthesized
as potential agents against type 2 diabetes. As a result of the antidiabetic biological
evaluation in streptozotocin (STZ) induced type 2 diabetes animal model, 13e, 13g
and 19f showed more significant reduction in serum Glu, TG, TC levels by contrast to
the positive control AdipoRon. In addition to upregulating the expression of AdipoR1
and AdipoR2, 13e and 19f treatment also increased expression of AMPK and PPAR-α.
Taken together, these results suggested that 13e and 19f might be a promising
compounds for type 2 diabetes treatment.
Keywords: chalcone derivatives; type 2 diabetes; AdipoR ; expression
1 Introduction
Type II diabetes has become a major global health challenge of the 21st century,
it is associated with population aging and increasing incidence of obesity. Besides,
type II diabetes also lead to a marked increase in cardiovascular risk.[1] Predominantly
resulted from insulin resistance and impaired beta cell function,[2,3] type II diabetes is
commonly treated by enhancing the sensitivity of tissue to insulin and improving the
production of insulin from β-cell of pancreas.[4] Other factors that facilitating the
development of type 2 diabetes embraces overweight, less exercise and a high-calorie
diet lifestyle.[5]
*Corresponding author: Jiaming Li, Emails: lijiaming2017@sina.com
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not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/cbdd.13319
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As an adipokine secreted by the adipose tissue, adiponectin[6-8] plays an essential
role in ameliorates insulin resistance, and harbors anti-diabetic, anti-atherogenic, as
well as anti-inflammatory properties. In respect of its mechanism of action,
adiponectin functions as the endogenous agonist of adiponectin receptors, which
comprise two subtypes, known as AdipoR1 and AdipoR2. While the former
predominantly uses the adenosine monophosphate-activated kinase (AMPK)
pathways in skeletal muscle, the latter action is mainly mediated by peroxisome
proliferator-activated receptor alpha (PPAR-α) pathways in the liver[9-10]
AdipoRon[11] (Figure 1) is a chemically synthesized small-molecule agonist
against adiponectin receptors, which was discovered by Miki Okada-Iwabu and
co-workers. It exhibited a similar effect to adiponectin in liver and muscle tissues.
Upon interaction with adiponectin receptors, adipoRon activated PPAR-α and AMPK
pathways, thereby lowering insulin resistance and glucose intolerance.
Chalcones, as the precursors of flavonoids, have been extensively exploited for
the development of anti-diabetic agents.[12-13] Some chalcone derivatives exhibited
anti-diabetic activity via affecting the level of adiponectin. Among them, naringenin
chalcone (1), as an isolated derivative of chalcone, was proven to increase the
expression of AdipoR2 and adiponectin gene in adipocytes, hence minimizing insulin
resistance.[14] Alternatively, 4-hydroxyderricin (2) compromised insulin resistance and
boosted glucose uptake via promoting adipocyte differentiation, stimulating
adiponectin secretion, and increasing the expression of PPAR-α and the
phosphorylation of AMPK.[15-17] Besides, elafibranor (3), a dual PPAR-α/δ agonist,
was reported to have the potential for treating cardiometabolic diseases such as type 2
diabetes, dyslipidemia, insulin resistance and etc.[18-21] Compound 4, discovered by us,
was capable of reducing serum TG and TC levels in high-fat-diet (HFD) induced
hyperlipidemia C57/BL6 mice.[22] Comparing with pioglitazone, compounds 5 and 6
developed by Hsieh, C.T et al, displayed superior efficacy in reducing glucose
medium concentration.[23] (Figure 2)
During our continuous efforts to explore chalcone derivatives anti-diabetic
agents, the key structural features of both chalcone and adipoRon were merged into a
single chemical entity via insertion of carbon-carbon double bond between the
carbonyl functionality and the terminal benzene ring. Upon this strategy, compounds
of series I with structural novelty were obtained. On the basis of this, compounds of
series II were designed via reversing the acryloyl moiety of compounds of series I for
SAR study. Additionally, for both series, different substituents on the terminal
benzene ring A were introduced to investigate their impact on activity (Figure 3).
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2. Results and discussion
2.1 Chemistry
The synthetic approach of chalcone derivatives is outlined in Scheme 1.
4-hydroxy acetophenone, as the starting material, was subjected to O-alkylation in the
presence of K2CO3 and KI to afforded the intermediate 8. Subsequent hydrolysis
furnished the carboxylic derivative 9. Afterwards, aldolic condensation of 9 and
substituted aldehydes 10a-h provided 11a-h as the intermediates. Finally, compounds
13a-h were attained as the desired compounds upon condensation of 11a-h and
compound 12 in the presence of EDCl and HOBt (Scheme 1).
Compounds 19a-h were prepared according to a similar synthetic procedure
(Scheme 2), but p-Hydroxybenzaldehyde was employed as the starting material. It
underwent O-alkylation, hydrolysis, aldolic condensation and condensation to afford
corresponding chalcone derivatives. The structures of all the compounds were
characterized by melting points, mass spectrometry, 13C NMR and 1H NMR.
2.2 Biological evaluation
2.2.1 The TG TC and Glu lowering activity of chalcone derivatives
To investigate the antidiabetic effecacy of these chalcone derivatives in type 2
diabetes mice, we firstly evaluated the capability of these chalcone derivatives to
lowering TG, TC and Glu levels in STZ induced type 2 diabetes model with
AdipoRon employed as the positive control at the dose of 50 mg/kg. As summarized
in table 1, in general, all the compounds effectively reversed the increase in TG, TC
and Glu levels induced by STZ. Among them, compounds 13f and 19e exhibited
comparable potency to AdipoRon. Particularly, compounds 13c and 13h exerted
comparable efficacy in lowering TG and TC levels, but superior efficacy in lowering
Glu level when compared to AdipoRon. Moreover, compounds 13e, 13g and 19f
surpass AdipoRon in lowering the levels of TG, TC and Glu (Table 1).
From the data for TG TC and Glu lowering activity of these chalcone
derivatives, some valuable SARs can be deduced, the key structural features of both
chalcones and adipoRon were merged into a single chemical entity via insertion of
carbon-carbon double bond between the carbonyl functionality and the terminal
benzene ring, which promote activity. As for the A ring, introduction of
electron-withdrawal group was detrimental for activity. Such as compounds 13a, 13b
and 19a-d, 19h. Interestingly, on the A ring, the electron-donating group may have
improved the TG, TC and Glu lowering activity. When the substitutions on the A ring
are electron-donating groups such as 4-OCH3 and 3, 4-(OCH3)2, the activity is higher,
we also found that via reversing the acryloyl moiety of compounds have no effect on
activity.
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2.2.2 Effects of compounds on AdipoR1 and AdipoR2.
Encouraged by the remarkable efficacy of 13e and 19f in lowering TG, TC and
Glu levels in STZ induced type 2 diabetes model, we further examined their efficacy
in modulating the biological function of AdipoR1 and AdipoR2. According to Figure
4, it was evident that both 13e and 19f effectively reversed the
high-glucose(HG)-induced decrease in the expression of AdipoR1 and AdipoR2 in a
dose-dependent manner. Besides, Compound 13e (fluorescence intensity: 89.57)
treatment led to more obvious increase in AdipoR2 expression in LO2 cells at the
concentration of 2μM when compared to AdipoRon (Figure 4).
2.2.3 Effects of compounds on AMPK and phospho-Thr172 AMPK
Based on the aforementioned effects of 13e and 19f on AdipoR, we therefore
examined their capability of upregulating AMPK signaling cascade. In the performed
assay, higher fluorescence intensity monitored indicated increase in the level of
AMPK and phospho-Thr172 AMPK. As shown in Figure 5, HG treatment led to the
decrease in the expression of AMPK and Phospho-Thr172 AMPK in both C2C12 and
LO2 cells. In comparison, both 13e and 19f remarkably reversed HG-induced
decrease in AMPK and Phospho-Thr172 AMPK at the concentration as low as 0.25
μM, which was consistent with the impact of them on AdipoR (Figure 5).
2.2.4 Effects of compounds on PPAR-α
Similarly, to investigate whether the effects on AdipoR2 was transformed to the
signaling pathway modulation, we performed assays to examine PPAR-α expression
in the LO2 cells at high glucose concentration. As a result, HG-induced decrease in
PPAR-α level was reversed by either 13e or 19f treatment at both low and high
dosage, thereby verifying their impact on signaling pathway (Figure 6).
3 CONCLUSIONS
In summary, two novel series of anti-diabetic agents were designed and
synthesized via merging structural features of chalcone and AdipoRon. In STZ
induced type 2 diabetes model, compounds 13e, 13g and 19f exhibited more favorable
activities than the reference AdipoRon in lowering the levels of TG, TC and Glu.
Further biological evaluation utilizing 13e and 19f as representatives demonstrated
their capability of facilitating both AdipoR1 and AdipoR2 expression. Consistent with
their effect on the receptor, both 13e and 19f treatment culminated in upregulation of
downstream AMPK and PPARα pathways. These results suggested that 13e and 19f
held the promise to be extensively developed as potential anti-type II diabetic agents.
4 EXPERIMENTAL SECTION
4.1 General information
All reagents, chemicals, and solvents were obtained from commercial sources
and used without further purification unless noted otherwise. Chromatography was
carried on silica gel (200–300 mesh). Melting points were taken on a WRS-1B
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melting point apparatus. NMR spectra were recorded on Bruker Avance (300 MHz) or
Agilent VNMRS600 (600 MHz), using TMS as internal standard. EI-MS were
recorded on LCQ ADVANTAGE MAX (FINNIGA) apparatus.
4.2.1 Synthesis of 2-(4-acetylphenoxy)acetic acid (9)
A solution of p-Hydroxyacetophenone (5g, 37.0mmol), K2CO3 (6.76g, 49mmol),
ethyl chloroacetate (5.98g, 49mmol), KI (0.83g, 5mmol), in DMF (80mL) and stirred
for 2 h at 88 °C, the reaction mixture was cooled to room temperature, water (100ml)
was added and filtered, the filtrate was extracted with EtOAc (50mL×3). The organic
layer was washed with water (100mL×3), brine (100mL×3), dried with anhydrous
sodium sulfate, filtered, and evaporated in vacuum to give the crude compound 8, as a
yellow oil.
Compound 8 (6.3g, 31mmol) was dissolved in methanol (30 mL), 10% NaOH
(1.9g, 48mmol, 20mL) was added dropwise at room temperature, and the solution was
stirred for 2 h at room temperature. The solvent was removed under reduced pressure
and the solid formed diluted with water, hydrochloric acid was slowly added dropwise
to pH = 4-5 to give a white solid, filtration and drying, then recrystallized with
anhydrous ethanol to afford pure compound 9. (5.1g, yield 85.0%);
mp.166.5-167.5 °C; 1H NMR (300 MHz, DMSO-d6): δ 13.16 (s, 1H, COOH), 7.93 (d,
J = 8.9 Hz, 2H, ArH), 7.02 (d, J = 8.9 Hz, 2H, ArH), 4.81 (s, 2H, OCH2), 2.52 (s, 3H,
COCH3); 13C NMR (75 MHz, DMSO-d6): δ 196.8, 170.2, 162.0, 130.8, 130.7, 114.8,
65.0, 26.9; ESI-MS (m/z) for C10H10O4: 195.06 (M+H)+.
4.2.2 Synthesis of (E)-2-(4-(3-(4-fluorophenyl)acryloyl)phenoxy)acetic acid (11a)
A
mixture
of
2-(4-acetylphenoxy)acetic
acid
(1g,
5.2mmol),
4-fluorobenzaldehyde (0.77g, 6.2mmol), in ethanol (10mL), 10% aqueous NaOH (1g,
25 mmol, 9mL) was added dropwise under an ice-cooled, and the solution was stirred
for 30 h at 0 °C. The solvent was removed under reduced pressure and the solid
formed diluted with water, hydrochloric acid was slowly added dropwise to pH = 4-5
to give a white solid, filtration and drying, then recrystallized with anhydrous ethanol
to afford pure compound 11a (1.27g, yield 81.2 %); mp: 186.2-188.1 ℃; 1H NMR (600
MHz, DMSO-d6): δ 13.25 (s, 1H, COOH), 8.14 (d, J = 8.7 Hz, 2H, ArH), 7.94 (t, J =
8.7 Hz, 2H, ArH), 7.89 (d, J = 15.6 Hz, 1H, =CH), 7.69 (d, J = 15.6 Hz, 1H, =CH),
7.27 (t, J = 8.3 Hz, 2H, ArH), 7.05 (d, J = 8.7 Hz, 2H, ArH), 4.81 (s, 2H, OCH2); 13C
NMR (151 MHz, DMSO-d6): δ 190.4, 172.9, 166.4, 164.6, 145.1, 134.6, 134.3, 133.9,
133.8, 124.9, 119.1, 117.6, 67.8; ESI-MS (m/z) for C17H13FO4: 299.01 (M-H)+.
4.2.3
Synthesis
of
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-fluorophenyl)acryloyl)phenoxy)acetami
de (13a)
A 100 mL round-bottomed flask containing CH2Cl2 (20ml), DMF (10ml), was
cooled in an ice-water bath, compound 11a (1.0g, 3.3mmol), EDCI (0.86g, 4.5mmol),
HOBt (0.61g, 4.5mmol) were added. The mixture was stirred at 0 °C for 2 h and then
triethylamine (0.9mL, 7.1mmol), compound 12 (0.67g, 3.5mmol) were added to the
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mixture, the mixture was stirred at 0 °C for 20 h. The reaction was poured into water
(100mL) and extracted with CH2Cl2 (50mL×3). The organic layers was washed with
water (100mL×3), saturated sodium carbonate (50mL×3), brine (100mL×3), dried
with anhydrous Na2SO4, filtered, and concentrated. The residue was purified by
column chromatography (CH2Cl2/MeOH = 80:1−70:1) to afford yellow solid (0.98g,
yield 63.1 %); mp: 141.2-141.9 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.16 (d, J = 8.8
Hz, 2H, ArH), 8.03 (d, J = 7.9 Hz, 1H, NH), 7.94 (dd, J = 8.4, 5.7 Hz, 2H, ArH), 7.90
(d, J = 15.6 Hz, 1H, =CH), 7.70 (d, J = 15.6 Hz, 1H, =CH), 7.30 – 7.19 (m, 7H, ArH),
7.08 (d, J = 8.8 Hz, 2H, ArH), 4.59 (s, 2H, OCH2), 3.66-3.60 (m, 1H, pip-CH), 3.40 (s,
2H, ArCH2), 2.72 (d, J = 11.2 Hz, 2H, pip-CH2), 1.97 (t, J = 11.1 Hz, 2H, pip-CH2),
1.68 (d, J = 10.4 Hz, 2H, pip-CH2), 1.49 (m, 2H, pip-CH2); 13C NMR (151 MHz,
DMSO-d6): δ 190.35 , 169.35 , 165.59 , 164.86 , 145.10 , 141.72 , 134.58 , 134.26 ,
133.93 , 131.77 , 131.23 , 129.92 , 124.96 , 119.05 , 118.91 , 117.81 , 70.05 , 65.21 ,
55.07 , 49.17 , 34.51; Purity: 99.4% by HPLC; ESI-MS (m/z) for C29H29FN2O3:
473.36 (M+H)+.
4.2.4
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-chlorophenyl)acryloyl)phenoxy)acetami
de (13b)
1
White solid, yield 51.9 %; mp: 173.4-175.8 ℃; H NMR (300 MHz, DMSO-d6): δ
8.19 (d, J = 8.8 Hz, 2H, ArH), 8.07 (d, J = 7.9 Hz, 1H, NH), 7.96 (d, J = 4.4 Hz, 2H),
7.92 (d, J = 15.3 Hz, 1H, =CH), 7.70 (d, J = 15.6 Hz, 1H, =CH), 7.53 (d, J = 8.4 Hz,
2H, ArH), 7.35–7.26 (m, 5H, ArH), 7.10 (d, J = 8.8 Hz, 2H, ArH), 4.61 (s, 2H,
OCH2), 3.70–3.60 (m, 1H, pip-H), 3.45 (s, 2H, ArCH2), 2.76 (d, J = 12.7 Hz, 2H,
pip-CH2), 2.01 (t, J = 10.6 Hz, 2H, pip-CH2), 1.71 (d, J = 10.0 Hz, 2H, pip-CH2),
13
1.57–1.45 (m, 2H, pip-CH2); C NMR (75 MHz, DMSO-d6): δ 187.2, 166.2, 161.8,
141.7, 134.9, 133.8, 130.9, 130.7, 130.5, 128.9, 128.7, 128.1, 126.8, 122.7, 114.7,
66.90 , 62.02, 51.90, 45.99, 31.32; Purity: 100% by HPLC; ESI-MS (m/z) for
C29H29ClN2O3: 489.16 (M+H)+.
4.2.5
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-bromophenyl)acryloyl)phenoxy)acetam
ide (13c)
White solid, yield 67.1 %; mp: 176.6-177.8 ℃; 1H NMR (300 MHz, DMSO-d6): δ 8.19
(d, J = 8.7 Hz, 2H, ArH), 8.05 (d, J = 8.4 Hz, 1H, NH), 7.97 (d, J = 15.3 Hz, 1H,
=CH), 7.87 (d, J = 8.4 Hz, 2H, ArH), 7.72 (d, J = 15.3 Hz, 1H, =CH), 7.66 (d, 2H,
ArH), 7.31 (t, J = 6.8 Hz, 5H, ArH), 7.11 (d, J = 8.7 Hz, 2H, ArH), 4.62 (s, 2H,
OCH2), 3.68–3.61 (m, 1H, pip-H), 3.46 (s, 2H, ArCH2), 2.77 (d, J = 13.1 Hz, 2H,
pip-CH2), 2.05 (d, J = 15.4 Hz, 2H, pip-CH2), 1.65-1.78 (m, 2H, pip-CH2), 1.57–1.47
(m, 2H, pip-CH2); 13C NMR (75 MHz, DMSO-d6): δ 187.7, 166.7, 162.3, 142.3,
134.6, 132.3, 131.7, 131.5, 131.4, 131.2, 129.2, 128.6, 127.4, 124.3, 123.3, 115.2,
67.4, 62.5, 52.4, 46.5, 31.8; Purity: 98.4% by HPLC; ESI-MS (m/z) for
C29H29BrN2O3: 533.24 (M+H) +.
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4.2.6 (E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(p-tolyl)acryloyl)phenoxy)acetamide
(13d)
1
Yellow solid, yield 78.4 %, mp: 162.3-163.5 ℃; H NMR (600 MHz, DMSO-d6): δ
8.16 (d, J = 8.4 Hz, 2H, ArH), 8.02 (d, J = 7.2 Hz, 1H, NH), 7.90 (d, J = 15.5 Hz, 1H,
=CH), 7.69 -7.62(m, 3H, ArH, =CH), 7.32-7.20 (m, 7H, ArH), 7.08 (d, J = 8.4 Hz,
2H, ArH), 4.59 (s, 2H, OCH2), 3.68 – 3.58 (m, 1H, pip-CH), 3.40 (s, 2H, ArCH2),
2.72 (d, J = 9.1 Hz, 2H, pip-CH2), 2.33 (s, 3H, CH3), 1.97 (t, J = 9.2 Hz, 2H,
pip-CH2), 1.68 (d, J = 11.4 Hz, 2H, pip-CH2), 1.54 – 1.44 (m, 2H, pip-CH2); 13C
NMR (75 MHz, CDCl3): δ 188.6, 166.6, 160.6, 144.7, 138.6, 138.3, 134.9, 132.5,
131.4, 131.0, 129.1, 129.0, 128.9, 128.3, 127.1, 125.7, 121.4, 114.5, 67.3, 63.0, 52.1,
46.3, 32.1, 21.4; Purity: 99.5% by HPLC; ESI-MS (m/z) for C30H32N2O3: 469.28
(M+H) +.
4.2.7
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-methoxyphenyl)acryloyl)phenoxy)aceta
mide (13e)
1
Yellow solid, yield 78.4 %; mp: 162.3-163.5 ℃; H NMR (600 MHz, DMSO-d6): δ
8.14 (d, J = 7.0 Hz, 2H, ArH), 8.02 (brs, 1H, NH), 7.82 (d, J = 7.2 Hz, 2H, ArH), 7.79
(d, J = 15.0 Hz, 1H, =CH), 7.67 (d, J = 15.0 Hz, 1H, =CH), 7.27–7.21 (m, 5H, ArH),
7.07 (d, J = 7.2 Hz, 2H, ArH), 6.99 (d, J = 7.2 Hz, 2H, ArH), 4.58 (s, 2H, OCH2), 3.80
(s, 3H, OCH3), 3.71–3.56 (m, 1H, pip-CH), 3.41 (s, 2H, ArCH2), 2.72 (brs, 2H,
pip-CH2), 1.97 (brs, 2H, pip-CH2), 1.68 (d, J = 10.8 Hz, 2H, pip-CH2), 1.55–1.43 (m,
2H, pip-CH2); 13C NMR (75 MHz, CDCl3): δ 188.6, 166.7, 161.7, 160.5, 144.3,
138.2, 132.7, 130.9, 130.2, 129.1, 128.3, 127.6, 127.1, 119.3, 114.5, 114.4, 67.3, 63.0,
55.4, 52.1, 46.3, 32.1; Purity: 99.4% by HPLC;ESI-MS (m/z) for C30H32N2O4: 485.29
(M+H) +
4.2.8 (E)-N-(1-benzylpiperidin-4-yl)-2-(4-cinnamoylphenoxy)acetamide (13f)
1
Yellow solid, yield 66 %, mp: 158.8-160.1 ; H NMR (600 MHz, DMSO-d6): δ
8.16 (d, J = 8.6 Hz, 2H, ArH), 8.04 (d, J = 7.8 Hz, 1H, NH), 7.93 (d, J = 15.6 Hz, 1H,
=CH), 7.86 (d, J = 8.6 Hz, 2H, ArH), 7.70 (d, J = 15.6 Hz, 1H, =CH), 7.48–7.39 (m,
3H, ArH), 7.31–7.22 (m, 5H, ArH), 7.08 (d, J = 8.6 Hz, 2H, ArH), 4.59 (s, 2H, OCH2),
3.68–3.59 (m, 1H, pip-CH), 3.42 (s, 2H, ArCH2), 2.74 (brs, 2H, pip-CH2), 1.99 (brs,
2H, pip-CH2), 1.69 (d, J = 11.3 Hz, 2H, pip-CH2), 1.53–1.47 (m, 2H, piperidine-CH2);
13C NMR (75 MHz, CDCl3): δ 188.6, 166.6, 160.6, 144.5, 138.0, 134.9, 132.4, 131.0,
130.5, 129.1, 129.0, 128.4, 128.3, 127.2, 121.6, 114.6, 67.3, 62.9, 52.1, 46.3, 32.0;
Purity: 100% by HPLC; ESI-MS (m/z) for C29H30N2O3: 455.27 (M+H) +.
4.2.9
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenoxy)a
cetamide (13g)
1
Yellow solid, yield 61 %; mp: 66.8-67.9 ℃; H NMR (300 MHz, DMSO-d6): δ 8.19
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(d, J = 8.8 Hz, 2H, ArH), 8.07 (d, J = 8.1 Hz, 1H, NH), 7.85 (d, J = 15.5 Hz, 1H,
=CH), 7.70 (d, J = 15.5 Hz, 1H, =CH), 7.55 (s, 1H, ArH), 7.39 (d, J = 8.3 Hz, 1H,
ArH), 7.33–7.24 (m, 5H, ArH), 7.11 (d, J = 8.8 Hz, 2H, ArH), 7.02 (d, J = 8.4 Hz, 1H,
ArH), 4.62 (s, 2H, OCH2), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.68–3.58 (m,
1H, pip-CH), 3.44 (s, 2H, ArCH2), 2.76 (d, J = 11.6 Hz, 2H, pip-CH2), 2.01 (t, J =
10.4 Hz, 2H, pip-CH2), 1.72 (d, J = 10.1 Hz, 2H, pip-CH2), 1.57–1.45 (m, 2H,
pip-CH2); 13C NMR (75 MHz, DMSO-d6) δ: 187.2, 166.2, 161.6, 151.1, 148.9, 143.7,
131.1, 130.7, 128.7, 128.1, 127.6, 126.8, 123.8, 119.5, 114.6, 111.5, 110.71, 66.9,
62.0, 55.6, 55.5, 51.9, 46.0, 31.3; Purity: 98.3% by HPLC; ESI-MS (m/z) for
C31H34N2O5: 515.24 (M+H) +.
4.2.10
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(3,5-dimethoxyphenyl)acryloyl)phenoxy)a
cetamide (13h)
Yellow solid, yield 66.7 %; m.p.88.5-89.8 ; 1H NMR (600 MHz, DMSO-d6): δ 8.17
(d, J = 8.4 Hz, 2H, ArH), 8.04 (brs, 1H, NH), 7.93 (d, J = 15.5 Hz, 1H, =CH), 7.64 (d,
J = 15.5 Hz, 1H, =CH), 7.31 – 7.22 (m, 5H, ArH), 7.08 (d, J = 8.4 Hz, 2H, ArH), 7.05
(s, 2H, ArH), 6.56 (s, 1H, ArH), 4.59 (s, 2H, OCH2), 3.79 (s, 6H, 2×OCH3), 3.64–3.57
(m, 1H, pip-CH), 3.41 (s, 2H, ArCH2), 2.73 (d, J = 10.2 Hz, 2H, pip-CH2), 1.97 (t, J =
10.2 Hz, 2H, pip-CH2), 1.69 (d, J = 10.2 Hz, 2H, pip-CH2), 1.52–1.46 (m, 2H,
pip-CH2); 13C NMR (75 MHz, CDCl3): δ 188.5, 166.6, 161.1, 160.7, 144.5, 138.1,
136.8, 132.3, 131.0, 129.1, 128.3, 127.2, 122.1, 114.6, 106.4, 102.7, 67.3, 62.9, 55.5,
52.1, 46.3, 32.0; Purity: 99.6% by HPLC; ESI-MS (m/z) for C31H34N2O5: 515.27
(M+H) +.
4.2.11
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl)phe
noxy)acetamide (19a)
Yellow solid, yield 50.9 %; mp: 148.2-149.7 ; H NMR (300 MHz, DMSO-d6): δ
1
8.26 (dd, J = 8.7, 5.7 Hz, 2H, ArH), 8.04 (d, J = 7.8 Hz, 1H, NH), 7.88 (d, J = 8.7 Hz,
2H, ArH), 7.86 (d, J = 15.6 Hz, 1H, =CH), 7.74 (d, J = 15.6 Hz, 1H, =CH), 7.40–7.22
(m, 7H, ArH), 7.05 (d, J = 8.7 Hz, 2H, ArH), 4.57 (s, 2H, OCH2), 3.73–3.58 (m, 1H,
pip-CH), 3.45 (s, 2H, ArCH2), 2.77 (d, J = 10.9 Hz, 2H, pip-CH2), 2.02 (t, J = 10.2
Hz, 2H, pip-CH2), 1.71 (d, J = 10.2 Hz, 2H, pip-CH2), 1.58–1.47 (m, 2H, pip-CH2);
13C NMR (75 MHz, DMSO-d6): δ 187.5, 166.4, 164.9, 159.9, 144.0, 134.4, 131.4,
130.7, 128.7, 128.1, 127.8, 126.8, 119.5, 115.6, 115.1, 66.9, 62.0, 51.9, 46.0, 31.3;
Purity: 99.7% by HPLC; ESI-MS (m/z) for C29H29FN2O3: 473.26 (M+H) +.
4.2.12
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)phe
noxy)acetamide (19b)
White solid, yield 51.9 %; mp: 164.2-165.3 ; H-NMR (300 MHz, DMSO-d6): δ
1
8.18 (d, J = 8.4 Hz, 2H, ArH), 8.03 (d, J = 7.8 Hz, 1H, NH), 7.88 (d, J = 8.7 Hz, 2H,
ArH), 7.84 (d, J = 15.3 Hz, 1H, =CH), 7.74 (d, J = 15.3 Hz, 1H, =CH), 7.64 (d, J =
This article is protected by copyright. All rights reserved.

8.4 Hz, 2H, ArH), 7.35 – 7.25 (m, 5H, ArH), 7.05 (d, J = 8.7 Hz, 2H, ArH), 4.57 (s,
2H, OCH2), 3.72 – 3.57 (m, 1H, pip-CH), 3.46 (s, 2H, ArCH2), 2.77 (d, J = 10.6 Hz,
2H, pip-CH2), 2.02 (t, J = 9.2 Hz, 2H, pip-CH2), 1.71 (d, J = 10.3 Hz, 2H, pip-CH2),
1.58 – 1.47 (m, 2H, pip-CH2): 13C NMR (75 MHz, DMSO-d6): δ 187.8, 166.4, 159.9,
144.3, 137.9, 136.4, 130.8, 130.3, 128.8, 128.7, 128.1, 127.7, 126.8, 119.4, 115.1,
66.9, 62.0, 51.9, 46.0, 31.3; Purity: 99.8% by HPLC; ESI-MS (m/z) for
C29H29ClN2O3: 489.28 (M+H) +.
4.2.13
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)phe
noxy)acetamide (19c)
Yellow solid, yield 67.5 %; mp: 156.5-157℃; 1H NMR (600 MHz, DMSO-d6): δ 8.06
(d, J = 7.8 Hz, 2H, ArH), 7.98 (brs, 1H, NH), 7.84 (d, J = 7.8 Hz, 2H, ArH), 7.78 (d, J
= 15.6 Hz, 1H, =CH ), 7.74(d, J = 7.8 Hz, 2H, ArH), 7.71 (d, J = 15.6 Hz, 1H, =CH),
7.28 – 7.21 (m, 5H, ArH), 7.01 (d, J = 8.4 Hz, 2H, ArH), 4.53 (s, 2H, OCH2), 3.68 –
3.58 (m, 1H, pip-CH), 3.40 (s, 2H, ArCH2), 2.72 (d, J = 10.2 Hz, 2H, pip-CH2), 1.96
(brs, 2H, pip-CH2), 1.68 (d, J = 11.4 Hz, 2H, pip-CH2), 1.52 – 1.46 (m, 2H, pip-CH2);
13C NMR (75 MHz, CDCl3): δ 189.2, 166.8, 159.1, 144.6, 138.2, 137.0, 131.9, 130.5,
130.0, 129.1, 128.9, 128.3, 127.8, 127.1, 120.0, 115.2, 67.3, 62.98, 52.1, 46.3, 32.1;
Purity: 99.2% by HPLC; ESI-MS (m/z) for C29H29BrN2O3: 533.18 (M+H) +.
4.2.14
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-nitrophenyl)-3-oxoprop-1-en-1-yl)phen
oxy)acetamide (19d)
1
Yellow solid, yield 77.7 %; mp: 164.3-165.6℃; H NMR (600 MHz, DMSO-d6): δ
8.30-8.36 (m, 4H, ArH), 7.98 (brs, 1H, NH), 7.86 (d, J = 7.2 Hz, 2H, ArH), 7.80 (d, J
= 15.4 Hz, 1H, =CH), 7.75 (d, J = 15.4 Hz, 1H, =CH), 7.28 – 7.21 (m, 5H, ArH), 7.02
(d, J = 7.2 Hz, 2H, ArH), 4.53 (s, 2H, OCH2), 3.67 – 3.57 (m, 1H, pip-CH), 3.41 (s,
2H, ArCH2), 2.73 (brs, 2H, pip-CH2), 1.97 (brs, 2H, pip-CH2), 1.68 (d, J = 9.6 Hz,
13
2H, pip-CH2), 1.55 – 1.47 (m, 2H, pip-CH2); C NMR (75 MHz, CDCl3): δ 188.8,
166.7, 159.4, 150.0, 146.0, 143.2, 138.2, 130.7, 129.4, 129.1, 128.5, 128.3, 127.1,
123.9, 119.8, 115.3, 67.3, 63.0, 52.1, 46.3, 32.1; Purity: 98.5% by HPLC; ESI-MS
(m/z) for C29H29N3O5: 500.34 (M+H) +.
4.2.15
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)phenoxy)ace
tamide (19e)
1
Yellow solid, yield 39.0 2%, mp: 198.5-199.6 ℃; H NMR (300 MHz, DMSO-d6): δ
8.06 (d, J = 7.8 Hz, 2H, ArH), 8.02 (d, J = 8.1 Hz, 1H, NH), 7.86 (d, J = 8.1 Hz, 2H,
ArH), 7.82 (d, J = 15.3 Hz, 1H, =CH), 7.70 (d, J = 15.3 Hz, 1H, =CH), 7.37 (d, J =
7.8 Hz, 2H, ArH), 7.32–7.24 (m, 5H, ArH), 7.03 (d, J = 8.4 Hz, 2H, ArH), 4.55 (s, 2H,
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OCH2), 3.73–3.57 (m, 1H, pip-CH), 3.44 (s, 2H, ArCH2), 2.75 (d, J = 11.1 Hz, 2H,
pip-CH2), 2.41 (s, 3H, ArCH3), 2.00 (t, J = 10.9 Hz, 2H, pip-CH2), 1.70 (d, J = 10.2
Hz, 2H, pip-CH2), 1.56–1.46 (m, 2H, pip-CH2); 13C NMR (75 MHz, DMSO-d6): δ
185.8, 166.4, 159.7, 153.2, 143.4, 143.3, 138.6, 135.2, 130.6, 129.3, 128.7, 128.5,
128.0, 126.8, 119.8, 115.1, 62.0, 54.9, 51.9, 46.0, 31.4, 21.1; Purity: 99.3% by HPLC;
ESI-MS (m/z) for C30H32N2O3: m/z 469.31 (M+H) +.
4.2.16
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl)p
henoxy)acetamide (19f)
Yellow solid, yield 46.69 %; mp: 176.5-177.8 ; H NMR (300 MHz, DMSO-d6): δ
1
8.17 (d, J = 8.4 Hz, 2H, ArH), 8.02 (d, J = 7.7 Hz, 1H, NH), 7.86 (d, J = 7.8 Hz, 2H,
ArH), 7.83 (d, J = 15.3 Hz, 1H, =CH), 7.69 (d, J = 15.3 Hz, 1H, =CH), 7.32–7.24 (m,
5H, ArH), 7.09 (d, J = 8.7 Hz, 2H, ArH), 7.04 (d, J = 8.4 Hz, 2H, ArH), 4.56 (s, 2H,
OCH2), 3.87 (s, 3H, OCH3), 3.74–3.57 (m, 1H, pip-CH), 3.44 (s, 2H, ArCH2), 2.75 (d,
J = 10.5 Hz, 2H, pip-CH2), 2.00 (t, J = 10.8 Hz, 2H, pip-CH2), 1.71 (d, J = 10.3 Hz,
2H, pip-CH2), 1.57–1.46 (m, 2H, pip-CH2); 13C NMR (75 MHz, DMSO-d6) δ: 187.2,
166.4, 163.1, 159.7, 142.9, 138.6, 130.8, 130.6, 130.5, 128.7, 128.1, 128.0, 126.8,
119.8, 115.1, 113.9, 66.9, 62.1, 55.5, 51.9, 46.0, 31.4; Purity: 98.6% by HPLC;
ESI-MS (m/z) for C30H32N2O4: 485.29 (M+H) +.
4.2.17
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-oxo-3-phenylprop-1-en-1-yl)phenoxy)aceta
mide (19g)
Yellow solid, yield 47.5 %; mp: 93.4-94.6 ; 1H NMR (300 MHz, DMSO-d6): δ 8.14
(d, J = 8.4 Hz, 2H, ArH), 8.09 (d, J = 8.7 Hz, 1H, NH), 7.86 (d, J = 8.4 Hz, 2H, ArH),
7.82 (d, J = 15.9 Hz, 1H, =CH), 7.71 (d, J = 15.9 Hz, 1H, =CH), 7.59-7.53 (m, 3H,
ArH), 7.33–7.23 (m, 5H, ArH), 7.03 (d, J = 8.4 Hz, 2H, ArH), 4.56 (s, 2H, OCH2),
3.71-3.56 (m, 1H, pip-CH), 3.43 (s, 2H, ArCH2), 2.74 (d, J = 10.7 Hz, 2H, pip-CH2),
1.98 (t, J = 10.6 Hz, 2H, pip-CH2), 1.69 (d, J = 10.8 Hz, 2H, pip-CH2), 1.56-1.46 (m,
13
2H, pip-CH2); C NMR (75 MHz, DMSO-d6): δ 189.0, 166.4, 159.9, 143.8, 138.6,
137.8, 132.9, 130.7, 128.7, 128.6, 128.4, 128.1, 127.8, 126.8, 119.8, 115.1, 66.9, 62.1,
51.9, 46.0, 31.3; Purity: 94.1% by HPLC; ESI-MS (m/z) for C29H30N2O3: 455.31
(M+H) +.
4.2.18
(E)-N-(1-benzylpiperidin-4-yl)-2-(4-(3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-e
n-1-yl)phenoxy)acetamide (19h)
Yellow solid, yield 36.16 %; mp: 143.5-144.8 ; 1H NMR (300 MHz, DMSO-d6): δ
8.31 (d, J = 7.2 Hz, 2H, ArH), 8.02 (d, J = 7.2 Hz, 1H, NH), 7.91 (d, J = 8.7 Hz, 2H,
ArH), 7.88 (d, J = 8.7 Hz, 2H, ArH), 7.81 (d, J = 15.6 Hz, 1H, =CH), 7.76 (d, J = 15.6
Hz, 1H, =CH), 7.33–7.23 (m, 5H, ArH), 7.04 (d, J = 7.2 Hz, 2H, ArH), 4.56 (s, 2H,
OCH2), 3.73–3.55 (m, 1H, pip-CH), 3.43 (s, 2H, ArCH2), 2.74 (d, J = 9.9 Hz, 2H,
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pip-CH2), 1.99 (t, J = 10.7 Hz, 2H, pip-CH2), 1.69 (d, J = 10.3 Hz, 2H, pip-CH2),
13
1.55–1.45 (m, 2H, pip-CH2); C NMR (75 MHz, DMSO-d6): δ 188.4, 166.4, 160.1,
145.0, 141.0, 138.6, 130.9, 129.1, 128.6, 128.1, 127.6, 126.8, 125.7, 119.5, 115.1,
66.9, 62.1, 51.9, 46.0, 31.4; Purity: 99.3% by HPLC; ESI-MS (m/z) for
C30H29F3N2O3: 523.30 (M+H) +.
4.3 Streptozotocin-induced diabetic rat model (STZ)
Male C57BL/6J mice of average body weight 18-22g were supplied from Model
Animal Research Center of Nanjing University, 228 male C57BL/6J mice were
randomly divided into 19 groups, 12 mice per group: a blank control group (Ordinary
feed), a model control group (high-fat diet + STZ), a AdipoRon (high-fat diet + STZ )
treatment group, 13a-h and 19a-h (high-fat diet + STZ) treatment group. Blank
control group were fed an ordinary feed, model control group and treatment group
were fed a high-fat diet. 4 weeks later, model control group and treatment group were
given a single intraperitoneal injection of STZ (50mg/kg), and the high-fat diet
feeding was continued for 2 further weeks. Then, animals of model control group and
treatment group were administered the suspension of the test sample orally at doses of
50 mg/kg-body weight every day for 10 days, after the last administration, blood
samples were collected to measure the concentrations of glucose, triglyceride and
cholesterol. Ordinary feed: corn 41.25%, wheat bran 35%, soybean meal 16%, fish
meal 6.25%, salt 0.25%, shell powder 1.14%, trace element 0.1%, compound vitamin
0.01%. High-fat diet: 88.7% ordinary feed, 1% cholesterol, 10% lard, sodium
deoxycholate 0.3%. Streptozotocin (Sigma, USA) was dissolved in 0.1 M citrate
buffer (pH 4.5) and calculated amount of freshly prepared solution was injected to
overnight fasted animals at a dose of 50 mg/kg-body weight intraperitoneally.
4.4 Immunocytochemistry of the C2C12 and LO2
Low expression of AdipoR1 and AdipoR2 in C2C12 cells and LO2 cells were
induced by high glucose (30 mmol/L d-glucose). The poly-lysine treated round
coverslip were placed in 24-well plates, the induced cells were inoculated onto the
round coverslip and maintained with high glucose. When the cells grew to about
50-60%, the drugs were added and incubated for 30 h. After incubation, the culture
medium was abandoned, cells were washed two times with phosphate buffered saline
(PBS), plus 4% paraformaldehyde for 15 minutes, remove the paraformaldehyde and
washed three times with PBS. Treatment with 0.5% Triton X-100 at room temperature
for 20 minutes, remove Triton X-100, washed three times with PBS. Use 10% goat
serum closed for 60 minutes. Cells were incubated with the primary antibodies (rabbit
anti-human AdipoR1, AdipoR2, Phospho-AMPK (Thr172), PPAR-alpha or mouse
anti-human AMPK) overnight at 4 °C, remove primary antibodies and washed three
times with PBS. Cells were incubated with FITC-conjugated goat anti-rabbi IgG for
30 minutes at 37
,
and then counterstained with 0.5ug/ml
4',6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature, remove DAPI
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and washed five times with PBS. Cells were imaged using a inverted microscope
(Nikon Eclipse, TS100-FDH1), the fluorescence intensity by calculating with
IMAGEJ 1.41 software (rawakorg).
AUTHOR CONTRIBUTIONS
All authors have given approval to the final version of the manuscript.
CONFLICT OF INTEREST
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was financially supported by Anhui Science and Technology key project
(15czz01077), National New Drug Innovation Program (2017ZX09309-007).
ABBREVIATIONS USED
HOBt,
1-hydroxybenzotriazole
hydrate;
hydrochloride;
EDCl,
DMF,
N-(3-dimethylaminopropyl)-N′-ethylcarbo-diimide
N,N-dimethylformamide; CDCl3, deuterochloroform;
ESI-MS, electrospray
ionization mass spectrum; NMR, nuclear magnetic resonance; δ(ppm), chemical shift;
DMSO, dimethylsulfoxide; mp, melting point.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data associated with this article can be found in the Supporting
Information, these data include HPLC spectra of target compounds in this article.
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Figure Legends
Figure 1: The structure of AdipoRon
Figure. 2 The structure of naringenin chalcone, 4-Hydroxyderricin, Elafibranor, 4, 5 and 6
Figure. 3 The design of chalcone derivatives
Scheme 1. Reagents and conditions: (I) K2CO3, DMF, KI, 88
MeOH, 20 , 1 h; (III) 10 % NaOH(aq), EtOH, 0 , 30 h; (IV) EDCI, HOBt, DMF, CH2Cl2,
Et3N, 0 , 20 h.
, 2 h; (II) 10 % NaOH(aq),
Scheme 2. Reagents and conditions: (I) K2CO3, DMF, KI, 88 , 2 h; (II) 10 % NaOH(aq),
MeOH, 20 , 1 h; (III) 10 % NaOH(aq), EtOH, 0 , 30 h; (IV) EDCI, HOBt, DMF, CH2Cl2,
Et3N, 0 , 20 h.
Table 1 Effects of chalcone derivatives on TG TC Glu levels in type 2 diabetes C57BL/6J mice.
Figure 4: LG (5 mmol/L of d-glucose), HG (30 mmol/L of d-glucose). Treatment group (0.25 μM
+ HG, or 2 μM + HG), 4',6-diamidino-2-phenylindole (DAPI), (original magnification, ×200), (A):
AdipoR1 expression in C2C12 cells, (B): AdipoR2 expression in LO2 cells, (C): fluorescence
intensity.
Figure 5: LG (5 mmol/L of d-glucose), HG (30 mmol/L of d-glucose). Treatment group (0.25 μM
+ HG, or 2 μM + HG), 4',6-diamidino-2-phenylindole (DAPI), (original magnification, ×200), (A):
AMPK, phospho-Thr172 AMPK expression in C2C12 cells, (B): AMPK, phospho-Thr172 AMPK
expression in LO2 cells, (C,): fluorescence intensity (C2C12 cells), (D): fluorescence intensity
(LO2 cells).
Figure 6: LG (5 mmol/L of d-glucose), HG (30 mmol/L of d-glucose). Treatment group (0.25 μM
+ HG, or 2 μM + HG), 4',6-diamidino-2-phenylindole (DAPI), (original magnification, ×200), (A):
PPAR-α expression in LO2 cells, (B): fluorescence intensity.
This article is protected by copyright. All rights reserved.

Figure 1: The structure of AdipoRon
Figure. 2 The structure of naringenin chalcone, 4-Hydroxyderricin, Elafibranor, 4, 5 and 6
Figure. 3 The design of chalcone derivatives
This article is protected by copyright. All rights reserved.

Scheme 1. Reagents and conditions: (I) K2CO3, DMF, KI, 88
, 2 h; (II) 10 % NaOH(aq), MeOH, 20
, 1 h;
(III) 10 % NaOH(aq), EtOH, 0
, 30 h; (IV) EDCI, HOBt, DMF, CH2Cl2, Et3N, 0 , 20 h.
Scheme 2. Reagents and conditions: (I) K2CO3, DMF, KI, 88
, 2 h; (II) 10 % NaOH(aq), MeOH, 20
, 1 h;
(III) 10 % NaOH(aq), EtOH, 0
, 30 h; (IV) EDCI, HOBt, DMF, CH2Cl2, Et3N, 0
, 20 h.
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Table 1 Effects of chalcone derivatives on TG TC Glu levels in type 2 diabetes C57BL/6J mice.
n = 10; TG (Triglyceride), TC (Total cholesterol), Glu (Glucose); AdipoRon (positive control); *P<0.05, **P<0.01 vs Blank; ▲P<0.05,
▲▲P<0.01, ▲▲▲P<0.001 vs Vehicle; # P<0.05 vs AdipoRon.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.