Discovery and anti-diabetic effects of novel isoxazole based flavonoid derivatives

Fitoterapia 142 (2020) 104499

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Discovery and anti-diabetic effects of novel isoxazole based flavonoid derivatives

T

Jiang-Ping Niea, Zhen-Ni Qua, Ying Chena, Jia-Hao Chena, Yue Jianga,b, Mei-Na Jina, Yang Yua, ⁎ ⁎ Wen-Yan Niuc, Hong-Quan Duana,d, , Nan Qina, a

School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics (Theragnostic), Tianjin, People's Republic of China b Department of Pharmacy, Tianjin Children's Hospital, People's Republic of China c Department of Immunology, Key Laboratory of Immune Microenvironment and Disease of the Educational Ministry of China, People's Republic of China d Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, People's Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Kaempferol Isoxazole derivative AMP-activated protein kinase Phosphoenol pyruvate carboxykinase Glucose 6-phosphatase Anti-diabetes

3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl] kaempferol is a novel lead compound to discover anti-diabetic and anti-obesity drugs. The present study reported the scaffold hopping of the lead compound to obtain a new isoxazole derivative, C45, which has improved glucose consumption at the nanomolar level (EC50 = 0.8 nM) in insulin resistant (IR) HepG2 cells. Western blotting showed that C45 markedly enhanced the phosphorylation of AMPK (AMP-activated protein kinase) and reduced the levels of the gluconeogenesis key enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose 6-phosphatase) in HepG2 cells. The potential molecular mechanism of C45 may be activation of the AMPK/PEPCK/G6Pase pathways. We concluded that C45 might be a valuable candidate to discover anti-diabetic drugs.

1. Introduction Diabetes mellitus (DM) is currently a major health problem for the people all over the world and the global prevalence is increasing [1,2]. Noninsulin-dependent diabetes mellitus (type II diabetes mellitus, T2DM) is a metabolic endocrine disorder characterized by hyperglycemia, which is caused by abnormalities in insulin secretion, insulin resistance (IR) in target tissues, increased hepatic glucose production, and decreased tissue glucose uptake [3,4]. The increased rate of hepatic glucose production is significantly associated with enzyme levels associated with carbohydrate metabolism, such as glucose-6-phosphatase (G6Pase), phosphoenol pyruvate carboxy kinase (PEPCK) and AMPactivated protein kinase (AMPK) [5–7]. Therefore, inhibition or activation of enzyme expression is one of the most important approaches to prevent chronic diabetes. In recent years, there is growing interest in the utilization of natural products as potential therapeutic agents for glucose regulation [8–10]. Previous investigation in our lab revealed that, a semi-synthesized flavonoid derivative of tiliroside, 3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3en-1-yl] kaempferol (Fla-CN, Fig. 1), was able to significantly enhance glucose consumption in insulin-resistant (IR) HepG2 cells, compared

with that of metformin [11]. Fla-CN reduced whole-body adiposity, ameliorated metabolic lipid disorder, improved insulin sensitivity and relieved other disorders characterized by insulin resistance in high fat diet induced obesity mice [12]. Fla-CN could effectively inhibit the differentiation of 3 T3-L1 pre-adipocyte by up-regulating miR-27 and activating AMPK [13]. In the present study, we focused on the structural optimization of Fla-CN to obtain the new derivatives with enhanced effects on glucose metabolism. Furthermore, we explored the possible molecular mechanism for the activity of Fla-CN derivatives. 2. Results and discussion 2.1. Chemistry Carbonyl product with sodium borohydride (NaBH4) resulted in a reduction reaction to produce the C3 in 56.8% yield or C4 in 78.5% (Scheme 1). The double bond elimination of Fla-CN was done with Pd − C under H2 atmosphere to obtain C5 in 43.7% yield [14]. The hydroxy derivatives were subjected by dimethyl sulfate to form methylated products (Scheme 2)[15–17]. Anhydrous aluminum bromide in acetonitrile was selected as the most suitable demethylating

⁎ Corresponding authors at: School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics (Theragnostic), Tianjin, People's Republic of China. E-mail addresses: [email protected] (H.-Q. Duan), [email protected] (N. Qin).

https://doi.org/10.1016/j.fitote.2020.104499 Received 3 December 2019; Received in revised form 6 February 2020; Accepted 7 February 2020 Available online 10 February 2020 0367-326X/ © 2020 Elsevier B.V. All rights reserved.

Fitoterapia 142 (2020) 104499

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2.2.2. Effects on C45 on gluconeogenesis in HepG2 cells To investigate the effect of C45 on gluconeogenesis in HepG2 cells, we examined the glucose production in HepG2 cells treated with different concentrations of C45. As shown in Fig. 2, metformin, a prescription drug, decreased glucose production at 1.0 mM (P < .01). C45 also decreased glucose production in a dose dependent manner, especially at 5 (P < .05) and 12.5 μM (P < .01), respectively. Fig. 1. The chemical structure of 3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1yl] kaempferol.

2.2.3. Potential molecular mechanism on glucose metabolism in response to flavonoid derivative C45 AMPK is a key player in regulating energy balance at both the cellular and whole-body levels [24,25]. A number of pharmacological compounds that increase AMPK activity indirectly or through direct binding have been identified [26]. Two existing classes of antidiabetic drugs, biguanides (for example, metformin) and thiazolidinediones (for example, rosiglitazone), both have been shown to activate AMPK. A number of plant-derived natural products, including berberine [27] and quercetin [28], also activated AMPK indirectly [6]. Our previous study reported a flavonoid derivative that could enhance glucose consumption via the AMPK pathway in HepG2 cells [11]. Gluconeogenesis refers to the transformation of non-glucose substances such as amino acid, lactic acid, pyruvate and glycerin into glucose. In T2DM, the rate of glucose production exceeds that of glucose consumption in the circulation. Moreover, gluconeogenesis, rather than glycogenolysis, was the primary source of hepatic glucose production, resulted in hyperglycemia. Gluconeogenesis in the liver is regulated by multiple enzymes including PEPCK and G6Pase, which are rate- limiting enzymes in gluconeogenesis. According to the previous reports, activated AMPK could suppress gluconeogenesis through the inhibition of the transcription of PEPCK and G6Pase [29]. Furthermore, liver-specific AMPK knockout mice were glucose intolerant and displayed fasting hyperglycemia, presumably because of elevated gluconeogenesis associated with increased PEPCK and G6Pase activity [30]. In this study, compound C45 decreased glucose production in a dose dependent manner (Fig. 2). In addition, HepG2 cells were treated with C45 (10 μM) alone or combined with AMPK inhibitor Compound C (4 μM). At the same time, AICAR (0.1 mM), an AMPK agonist, was used as a positive control. As illustrated in Fig. 3A and B, C45 increased phosphorylation of AMPK remarkably, and this effect was blocked by Compound C. Derivative C45 and AICAR both significantly downregulated protein expressions of G6Pase and PEPCK in HepG2 cells. Moreover, the down regulation of PEPCK and G6Pase by C45 was blocked by Compound C. (Fig. 3A and C). These results suggested that the inhibitory action of C45 on key enzyme during hepatic gluconeogenesis was at least partly mediated by AMPK activation.

condition to afforded the corresponding 3-hydroxyflavones (Scheme 3) [18]. 3-substituted derivatives (C1, C2, C7) and C14 were prepared through bromine reagents. To obtain triazole products C10, C39, C42 and C43, a click reaction was used. The isoxazole derivatives C45 and C49 were synthesized using chloro-benzaldoxime. Various hydroxy flavones were prepared by boron tribromide [19–21].

2.2. Pharmacology 2.2.1. The effects of flavonoid derivatives on glucose consumption The glucose consumption of IR HepG2 cells in response to all the derivatives was evaluated and EC50 values were used to assess their potencies. Compounds C1-C7, C10-C13, C15-C20 and C28-C51 significantly enhanced glucose consumption in IR HepG2 cells (Table 1). In this optimization study, two derivatives (C42 and C45) showed higher potency than the lead compound Fla-CN. In general, hydroxyl group was a donor of H bond which was a major interaction between the ligand and receptor. As the lead compound had three hydroxyl groups, we firstly evaluated the activities of methylation derivatives (C6, C28, C29). These results revealed that the three derivatives had the similar effect as Fla-CN at the same order of magnitude (Table 1). Based on these results, we speculated that the methylation of hydroxyl groups made no significant difference on the activity, compared to the lead compound. Our previous study showed that the benzonitrile fragment was a crucial group for the activity. In this study, we changed the unsaturated ketone linker while retaining the flavonoid and benzonitrile fragment. The activities were significantly decreased when the unsaturated ketone linker was changed. These changes included the reduction of unsaturated ketone and the shortening of carbon chain. In these derivatives, the α, β-unsaturated ketone might give a suitable conformation for their activities. Although the α, β-unsaturated ketone was an important group for the activities, it was also a Michael addition receptor which was a kind of alert groups in medicinal chemistry. Considering this problem, we decided to investigate the scold-hopping of the α, β-unsaturated ketone. In recent years, isoxazole or triazole rings were used to replace the active groups in the field of structure modification [22]. The isoxazole or triazole derivatives (C10-C13 and C39-C51) were prepared. The results showed that the activities were retained upon the replacement of the α, β-unsaturated ketone fragment with triazole or isoxazole group. It was interesting that the different carbon chains of two series derivatives (triazole derivative: CH2 × 2; isoxazole derivative: CH2 × 1) both exhibited better activities. It means that the number of rotation bond could affect the activity of triazole or isoxazole derivatives. It could be further speculated that the covalent attachment of an unsaturated ketone with target proteins was not the major mode of their interaction. The isoxazole derivative C45 exhibited better activity than the lead compound. This was not surprising, because isoxazole ring, as an important nitrogen heterocycle, has been shown to be an essential structural component for many pharmaceuticals. Isoxazole derivatives are reported to elicit a broad spectrum of pharmacological activities viz. antimicrobial, antiviral, anticancer, anti-inflammatory, immunomodulatory, anticonvulsant or anti-diabetic properties [23].

3. Materials and methods 3.1. Materials and apparatus All reagents and solvents were obtained from commercial sources and used without purification unless mentioned. Reactions were monitored by thin-layer chromatography (TLC) using precoated silica gel glass plates containing a fluorescence indicator. The column chromatography was carried out on silica gel (300–400 mesh) (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong province, China). 1H, 13 C NMR spectra were taken using a Bruker AVANCE III 400 instrument (Bruker Biospin AG, Fallanden, Switzerland). 1H NMR spectra were recorded in CDCl3 and DMSO-d6 using tetramethylsilane and chemical shifts were quoted in ppm, referenced to tetramethylsilane (TMS). Peak multiplicities are reported as follow: s (singlet), d (doublet), t (triplet), q (quartet), m (multiple). The high-resolution mass spectra were recorded on an Agilent 1200 LC-MS in ESI mode (Agilent, Palo Alto, CA, USA).

2

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Scheme 1. Synthesis of compounds C3-C6 and C28-C38.

Scheme 2. Synthesis of compounds C1-C2 and C15-C20.

concentrated and purified by silica gel chromatography to obtain yellow solid C3 or C4. (E)-4-(4-((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen3-yl)oxy)-3-hydroxybut-1-en-1-yl)benzonitrile (C3), yellow solid; Yield, 56.8%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.66 (s, 1H, OH), 10.84 (s, 1H, OH), 10.22 (s, 1H, OH), 8.02 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 16.0 Hz, 1H), 6.55 (dd, J = 16.0, 5.2 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 5.45 (d, J = 4.8 Hz, 1H), 4.53–4.48 (m, 1H), 4.03 (d, J = 5.6 Hz, 2H); 13C NMR (100 MHz,

3.2. Chemistry 3.2.1. General procedure for compoundsC3 and C4 Sodium borohydride (NaBH4, 12 mg, 0.31 mmol) was added slowly to the solution of Compound Fla-CN (120 mg, 0.26 mmol) or C5 (119 mg, 0.26 mmol) in CH2Cl2/MeOH (1:1, 10 mL). After being stirred at room temperature for 3 h, the reaction mixture was quenched with saturated aqueous NH4Cl (15 mL). After the removal of MeOH under reduced pressure, the resulting aqueous solution was extracted with CH2Cl2 (15 mL × 3), washed with brine, dried over Na2SO4, 3

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Scheme 3. Synthesis of compounds C10-C14 and C39-C51.

Table 1 The effects of Fla-CN derivatives on the glucose consumption in IR HepG2 cells (n = 3). No.

EC50 (nM)

No.

EC50 (nM)

No.

EC50 (nM)

C1 C2 C3 C4 C5 C6 C10 C11 C12 C13 C15 C16 C17 C18

184 ± 27.9 197 ± 31.5 126 ± 11.6 112 ± 15.1 79 ± 24.6 4.6 ± 1.9 34 ± 7.1 41 ± 6.9 78 ± 8.1 155 ± 19.7 146 ± 23.3 99 ± 8.9 59 ± 6.5 92 ± 5.9

C19 C20 C28 C29 C30 C31 C32 C33 C34 C35 C36 C37 C38 C39

146 ± 14.9 187 ± 28.7 2.3 ± 1.1 7.8 ± 2.1 61 ± 10.7 118 ± 19.2 147 ± 21.3 137 ± 18.6 78 ± 12.4 56 ± 0.6 134 ± 23.6 71 ± 14.8 99 ± 11.4 3.7 ± 1.8

C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 C51 Fla-CN Met

2.5 ± 1.4 1.78 ± 0.7 1.2 ± 0.6 2.1 ± 0.9 1.9 ± 1.0 0.8 ± 0.3 1.3 ± 0.9 2.9 ± 1.4 46 ± 8.6 89 ± 24.5 94 ± 22.3 82 ± 18.9 3.3 ± 1.7 258 ± 34.3

Fig. 2. The effects on gluconeogenesis of C45 in HepG2 cells. The measurements of glucose production were described in Methods. Data are expressed as means ± SEM; n = 4. (⁎P < .05, ⁎⁎P < .01 compared with control group).

4-(4-((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl) oxy)-3-hydroxybutyl)benzonitrile (C4), yellow solid; Yield, 78.5%; Yield 78.5%, yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ: 12.66 (s, 1H, OH), 10.87 (s, 1H, OH), 10.27 (s, 1H, OH), 8.01 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 2.0 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H),

DMSO‑d6) δ: 177.8, 164.1, 161.2, 160.1, 156.3, 155.6, 141.4, 136.6, 134.5, 132.5, 130.5, 128.2, 126.9, 120.5, 118.9, 115.4, 109.5, 104.1, 98.6, 93.7, 75.5, 69.6; HR-ESIMS (m/z): [M + Na]+ calcd. For C26H19NNaO7: 480.1059, found: 480.1058. 4

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Fig. 3. Flavonoid derivative C45 exerted an inhibitory effect on hepatic gluconeogenesis via the AMPK signaling pathway. HepG2 hepatocytes were incubated with 1 mM AICAR, 10 μM C45 and/or 10 μM Compound C. Total protein was extracted after 24 h incubation. (A) Protein expression of G6Pase, PEPCK, p-AMPK, total AMPK and β-actin in HepG2 hepatocytes. (B) Relative protien level of p-AMPK/t-AMPK was expressed relative to the control. (C) Protein levels of G6Pase and PEPCK were expressed relative to the control. Data was expressed as means ± SEM (n = 3). ⁎P < .05, ⁎⁎P < .01 compared with control group. #P < .05 compared to Compound C group.

J = 9.2 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 9.2 Hz, 2H), 6.46 (d, J = 2.0 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H), 5.13 (s, 2H), 3.91 (s, 3H), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 177.5, 164.5, 160.9, 160.8, 155.8, 140.9, 135.9, 131.1, 129.4, 127.7, 121.6, 117.7, 112.9, 110.9, 104.9, 96.9, 91.3, 71.9, 54.8, 54.4; HRESIMS (m/z): [M + Na]+ calcd. For C25H19NNaO6: 452.1110, found: 452.1112. 4-(((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)methyl)benzonitrile (C17), yellow solid; Yield, 25.4%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.53 (s, 1H, OH), 10.44 (s, 1H, OH), 7.83 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.53 (d, J = 7.2 Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 6.63 (s, 1H), 6.30 (s, 1H), 5.04 (s, 2H), 3.81 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ: 177.7, 165.1, 160.8, 160.1, 156.7, 156.2, 142.2, 136.1, 132.1, 130.3, 128.5, 120.3118.7, 115.4, 110.5, 105.2, 97.8, 92.2, 72.3, 55.9; HR-ESIMS (m/ z): [M + Na]+ calcd. For C24H17NNaO6: 438.0954, found: 438.0957. 4-(2-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3yl)oxy)acetyl)benzonitrile (C18), yellow solid; Yield, 37.4%; 1H NMR (400 MHz, CDCl3) δ: 8.25 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 6.54 (s, 1H), 6.37 (s, 1H), 5.39 (s, 2H), 3.97 (s, 3H), 3.91 (s, 3H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 194.0, 173.8, 164.2, 161.4, 160.9, 158.8, 153.2, 138.9, 137.8, 132.4, 130.2, 129.3, 122.6, 118.0, 116.6, 113.9, 109.1, 96.0, 92.5, 74.2, 56.5, 55.9, 55.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C27H21NNaO7: 494.1216, found: 494.1213. 4-(2-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)acetyl)benzonitrile (C19), yellow solid; Yield, 32.3%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.43 (s, 1H, OH), 8.13 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 7.99 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.80 (s, 1H), 6.39 (s, 1H), 5.61 (s, 2H), 3.87 (s, 6H), 3.85 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ: 193.8, 177.6, 165.2, 161.5, 160.8, 156.2, 154.9, 137.5, 136.6, 132.7, 130.4, 128.5, 121.9, 118.0, 115.4, 114.0, 105.0, 97.9, 92.4, 74.0, 56.1, 55.04; HR-

5.01 (d, J = 5.2 Hz, 1H), 3.92–3.85 (m, 2H), 3.73–3.66 (m, 1H), 2.84–2.77 (m, 1H), 2.71–2.64 (m, 1H), 1.83–1.74 (m, 1H), 1.65–1.56 (m, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 177.9, 164.1, 161.2, 160.1, 156.3, 155.7, 148.4, 136.6, 132.1, 130.4, 129.4, 120.5, 119.0, 115.4, 108.5, 104.1, 98.5, 93.7, 76.0, 68.1, 48.5, 34.6, 31.2; HR-ESIMS (m/z): [M + Na]+ calcd. For C26H21NNaO7: 482.1216, found: 482.1213. 3.2.2. General procedure for compounds C6, C15-C17, C18-C20 and C28-C38 Compound C1 (240 mg, 0.60 mmol) and anhydrous potassium carbonate (K2CO3, 124 mg, 0.90 mmol) were weighed in a reaction flask, and 10 mL of acetone was added. Then dimethyl sulfate (85 μL, 0.90 mmol) was added dropwise and the mixture was stirred at room temperature for 12 h. The reaction was quenched by adding appropriate amount of diluted sodium hydroxide solution (removing the toxic dimethyl sulfate), and the pH was adjusted to neutral with dilute hydrochloric acid. The reaction mixture was concentrated, and washed with CH2Cl2. The crude product was purified by gel column chromatography to yield the desired products C15-C17. The procedure described above (using the same quantities of reagents) was followed to yield C6, C18-C20 and C28-C38. 4-(((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3-yl) oxy)methyl)benzonitrile (C15), yellow solid; Yield, 35.3%; 1H NMR (400 MHz, CDCl3) δ: 7.95 (d, J = 8.8 Hz, 2H), 7.56 (m, 4H), 6.95 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 5.10 (s, 2H), 3.96 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 173.8, 164.0, 161.3, 160.9, 158.9, 153.6, 142.6, 139.2, 131.9, 130.4, 128.9, 122.9, 118.9, 113.8, 111.5, 109.3, 95.9, 92.5, 72.5, 56.4, 55.8, 55.4. HR-ESIMS (m/z): [M + Na]+ calcd. for C26H21NNaO6: 466.1267, found: 466.1266. 4-(((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)methyl)benzonitrile (C16), yellow solid; Yield, 31.1%; 1H NMR (400 MHz, CDCl3) δ: 12.59 (s, 1H, OH), 7.96 (d, 5

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ESIMS (m/z): [M + Na]+ calcd. For C26H19NNaO7: 480.1059, found: 480.1055. 4-(2-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)acetyl)benzonitrile (C20), yellow solid; Yield, 17.4%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.46 (s, 1H, OH), 10.31 (s, 1H, OH), 8.07 (m, 4H), 8.01 (d, J = 7.6 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.77 (s, 1H), 6.37 (s, 1H), 5.59 (s, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ: 193.8, 177.5, 165.1, 160.7, 160.3, 156.2, 155.4, 137.6, 136.3, 132.7, 130.6, 128.5, 120.3, 118.1, 115.4, 104.9, 97.8, 92.3, 74.05, 56.1, 55.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C25H17NNaO7: 466.0903, found: 466.0907. (E)-4-(4-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen3-yl)oxy)-3-hydroxybut-1-en-1-yl)benzonitrile (C6), yellow solid; Yield, 43.4%; 1H NMR (400 MHz, CDCl3) δ: 8.08 (d, J = 9.2 Hz, 2H), 7.75 (d, J = 16.4 Hz, 1H), 7.67–7.62 (m, 4H), 7.30 (d, J = 16.4 Hz, 1H), 6.99 (d, J = 9.2 Hz, 2H), 6.53 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 2.4 Hz, 1H), 4.89 (s, 2H), 3.96 (s, 3H), 3.91 (s, 3H), 3.86 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 195.6, 173.6, 164.1, 161.4, 160.9, 158.8, 153.0, 141.0, 139.4, 139.0, 132.5, 130.1, 128.9, 125.2, 122.7, 118.4, 114.0, 113.4, 109.2, 95.9, 92.5, 75.8, 56.5, 55.8, 55.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H23NNaO7: 520.1372, found: 520.1374. (E)-4-(4-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)-3-oxobut-1-en-1-yl)benzonitrile (C28), yellow solid; Yield, 27.1%; 1H NMR (400 MHz, CDCl3) δ: 12.44 (s, 1H, OH), 8.09 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 16.4 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 16.4 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 2.0 Hz, 1H), 6.37 (d, J = 2.0 Hz, 1H), 4.90 (s, 2H), 3.88 (s, 3H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 194.9, 178.1, 165.6, 161.9, 156.7, 155.9, 141.3, 138.8, 137.2, 132.6, 130.5, 128.8, 124.6, 122.4, 118.4, 114.1, 113.6, 105.8, 98.1, 92.3, 75.9, 55.9, 55.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C28H21NNaO7: 506.1216, found: 506.1219. (E)-4-(4-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)-3-oxobut-1-en-1-yl)benzonitrile (C29), yellow solid; Yield, 22.5%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.51 (s, 1H, OH), 10.30 (s, 1H, OH), 8.08 (d, J = 8.4 Hz, 2H), 7.91–7.86 (m, 4H), 7.70 (d, J = 16.4 Hz, 1H), 7.19 (d, J = 16.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 2.0 Hz, 1H), 6.37 (d, J = 2.0 Hz, 1H), 5.13 (s, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ: 194.5, 177.6, 165.2, 160.8, 160.4, 156.2, 155.5, 140.3, 138.8, 136.5, 132.7, 130.6, 129.1, 125.4, 120.4, 118.5, 115.5, 112.4, 104.9, 97.9, 92.4, 75.3, 56.1; HR-ESIMS (m/z): [M + Na]+ calcd. for C27H19NNaO7: 492.1059, found: 492.1056. (E)-4-(4-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen3-yl)oxy)-3-hydroxybut-1-en-1-yl)benzonitrile (C30), yellow solid; Yield, 28.7%; 1H NMR (400 MHz, CDCl3) δ: 8.05 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H), 7.41 (d, J = 7.6 Hz, 2H), 7.02 (d, J = 7.6 Hz, 2H), 6.84 (d, J = 15.6 Hz, 1H), 6.53 (s, 1H), 6.38 (s, 1H), 6.30 (dd, J = 15.6 Hz, 4.4 Hz, 1H), 5.91 (br s, 1H), 4.69–4.66 (m, 1H), 4.07–4.04 (m, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.75–3.71 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 174.8, 164.4, 161.5, 161.0, 158.9, 154.0, 141.5, 139.7, 132.3, 131.8, 129.9, 129.6, 126.9, 122.6, 119.0, 114.2, 110.6, 108.8, 96.1, 92.5, 70.8, 56.6, 55.8, 55.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H25NNaO7: 522.1529, found: 522.1526. (E)-4-(3-hydroxy-4-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4oxo-4H-chromen-3-yl)oxy)but-1-en-1-yl)benzonitrile (C31), yellow solid; Yield, 31.6%; 1H NMR (400 MHz, CDCl3) δ: 12.24 (s, 1H, OH), 8.07 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 16.0 Hz, 1H), 6.48 (d, J = 2.0 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 6.29 (dd, J = 16.0, 5.2 Hz, 1H), 4.69–4.67 (m, 1H), 4.12–4.09 (m, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.78–3.74 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 173.6, 160.6, 156.8, 156.6, 151.7, 151.6, 135.9, 132.5, 127.1, 125.9, 125.0, 124.6, 121.7, 117.0, 113.6, 109.1, 105.6, 100.4, 92.9, 87.2, 65.5, 50.6, 50.2; HRESIMS (m/z): [M + Na]+ calcd. For C28H23NNaO7: 508.1372, found: 508.1371.

(E)-4-(3-hydroxy-4-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4oxo-4H-chromen-3-yl)oxy)but-1-en-1-yl)benzonitrile (C32), yellow solid; Yield, 24.3%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.64 (s, 1H, OH), 10.26 (s, 1H, OH), 8.06 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.73–6.70 (m, 2H), 6.55 (dd, J = 16.0 Hz, 5.2 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 4.51 (t, J = 5.2 Hz, 1H), 4.06 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ: 183.2, 170.3, 166.1, 165.5, 161.2, 146.6, 142.1, 139.7, 131.7, 135.8, 133.4, 132.2, 125.7, 124.2, 120.7, 119.2, 114.7, 110.3, 102.9, 97.5, 80.7, 74.9, 61.3; HR-ESIMS (m/z): [M + Na]+ calcd. for C27H21NNaO7: 494.1216, found: 494.1215. 4-(4-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3yl)oxy)-3-hydroxybutyl)benzonitrile (C33), yellow solid; Yield, 33.1%; 1 H NMR (400 MHz, CDCl3) δ: 8.02 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 7.29 (d, J = 7.6 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.52 (s, 1H), 6.36 (s, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 3.89 (s, 5H), 3.71–3.67 (m, 1H), 2.95–2.88 (m, 1H), 2.82–2.74 (m, 1H), 1.81–1.74 (m, 1H), 1.68–1.62 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 174.7, 164.3, 161.5, 160.9, 158.9, 153.8, 148.1, 139.8, 132.1, 129.9, 129.4, 122.7, 119.2, 114.3, 109.5, 108.9, 96.1, 92.5, 69.0, 56.5, 55.8, 55.4, 34.2, 32.0; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H27NNaO7: 524.1685, found: 524.1689. 4-(3-hydroxy-4-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4oxo-4H-chromen-3-yl)oxy)butyl)benzonitrile (C34), yellow solid; Yield, 27.5%; 1H NMR (400 MHz, CDCl3) δ: 12.26 (s, 1H, OH), 8.02 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 7.6 Hz, 2H), 7.30 (d, J = 7.6 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.45 (s, 1H), 6.36 (s, 1H), 4.15–4.09 (m, 2H), 3.89 (s, 3H), 3.87 (s, 4H), 3.68–3.66 (m, 1H), 2.92–2.90 (m, 1H), 2.81–2.75 (m, 1H), 1.79–1.77 (m, 1H), 1.66–1.64 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 178.9, 171.1, 165.8, 162.0, 161.8, 156.9, 147.8, 137.9, 132.2, 130.2, 129.3, 122.4, 119.1, 114.3, 109.7, 105.6, 98.2, 92.4, 78.2, 69.0, 60.4, 55.9, 55.5, 53.5, 33.9, 31.9; HR-ESIMS (m/z): [M + Na]+ calcd. For C28H25NNaO7: 510.1529, found: 510.1532. 4-(3-hydroxy-4-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo4H-chromen-3-yl)oxy)butyl)benzonitrile (C35), yellow solid; Yield, 20.7%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.64 (s, 1H, OH), 10.28 (s, 1H, OH), 8.04 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 2.0 Hz, 1H), 6.37 (d, J = 2.0 Hz, 1H), 3.92–3.90 (m, 2H), 3.86 (s, 3H), 3.72–3.68 (m, 1H), 2.84–2.77 (m, 1H), 2.72–2.64 (m, 1H), 1.83–1.75 (m, 1H), 1.66–1.57 (m, 1H); 13C NMR (100 MHz, DMSO‑d6) δ: 178.0, 165.1, 160.9, 160.2, 156.3, 156.0, 148.4, 136.9, 132.1, 130.5, 129.4, 120.5, 119.0, 115.4, 108.5, 105.1, 97.7, 92.3, 76.0, 68.1, 56.1, 48.6, 34.6, 31.2; HR-ESIMS (m/z): [M + Na]+ calcd. for C27H23NNaO7: 496.1372, found: 496.1376. 4-(4-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3yl)oxy)-3-oxobutyl)benzonitrile (C36), yellow solid; Yield, 38.5%; 1H NMR (400 MHz, CDCl3) δ: 8.01 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 6.4 Hz, 2H), 7.32 (d, J = 6.4 Hz, 2H), 6.99 (d, J = 7.6 Hz, 2H), 6.53 (s, 1H), 6.36 (s, 1H), 4.60 (s, 2H), 3.97 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.16 (br s, 2H), 3.02 (br s, 2H); 13C NMR (100 MHz, CDCl3) δ: 206.8, 137.6, 146.1, 161.4, 160.9, 158.8, 152.9, 146.9, 139.3, 132.2, 129.9, 129.3, 122.7, 119.1, 113.3, 109.7, 109.1, 95.9, 92.5, 76.1, 56.5, 55.8, 55.4, 39.8, 32.0, 29.0; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H25NNaO7: 522.1529, found: 522.1526. 4-(4-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)-3-oxobutyl)benzonitrile (C37), yellow solid; Yield, 25.7%; 1H NMR (400 MHz, CDCl3) δ: 12.37 (s, 1H, OH), 8.01 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 6.44 (s, 1H), 6.35 (s, 1H), 4.61 (s, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.03 (s, 4H); 13C NMR (100 MHz, CDCl3) δ: 205.6, 177.9, 165.6, 156.7, 155.7, 146.7, 137.1, 132.3, 130.4, 129.3, 122.4, 118.9, 114.1, 110.1, 105.8, 98.0, 92.3, 76.4, 55.8, 55.5, 39.7, 29.1; HRESIMS (m/z): [M + Na]+ calcd. For C28H23NNaO7: 508.1372, found: 508.1376. 4-(4-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4H6

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3.95 (s, 3H), 3.88 (s, 3H), 2.33 (t, J = 2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ: 173.8, 163.9, 161.2, 160.9, 158.8, 153.8, 138.2, 130.3, 123.2, 113.7, 109.2, 95.8, 92.4, 59.0, 56.4, 55.8, 55.4, 29.7; HR-ESIMS (m/z): [M + Na]+ calcd. For C21H18NaO6: 389.1001, found: 389.1003. 3-(but-3-yn-1-yloxy)-5,7-dimethoxy-2-(4-methoxyphenyl)-4Hchromen-4-one (C14), yellow solid; Yield, 46.7%; 1H NMR (400 MHz, CDCl3) δ: 8.10 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz 2H), 6.51 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 4.17 (t, J = 6.8 Hz, 2H), 3.96 (s, 3H), 3.89 (s, 3H), 2.67–2.63 (m, 2H), 1.93 (t, J = 2.6 Hz, 1H); 13 C NMR (100 MHz, CDCl3) δ: 173.8, 163.9, 161.2, 160.9, 158.8, 152.9, 139.7, 130.1, 123.1, 113.8, 109.3, 95.7, 92.4, 81.0, 69.9, 69.5, 56.4, 55.8, 55.4, 20.8; HR-ESIMS (m/z): [M + Na]+ calcd. For C22H20NaO6: 403.1158, found: 403.1155.

chromen-3-yl)oxy)-3-oxobutyl)benzonitrile (C38), yellow solid; Yield, 19.5%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.47 (s, 1H, OH), 10.31 (s, 1H, OH), 8.02 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.75 (s, 1H), 6.37 (s, 1H), 4.80 (s, 2H), 3.86 (s, 3H), 2.91 (s, 4H); 13C NMR (100 MHz, DMSO‑d6) δ: 205.0, 177.5, 165.1, 160.8, 160.3, 156.2, 155.4, 147.2, 136.4, 132.1, 130.5, 129.4, 120.3, 118.9, 115.5, 108.7, 104.9, 97.8, 92.3, 75.6, 56.0, 28.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C27H21NNaO7: 494.1216, found: 494.1217. 3.2.3. 4-(4-((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3yl)oxy)-3-oxobutyl)benzonitrile C5 The compound Fla-CN (1.0 eq.) was taken in anhydrous THF (5 mL), and Pd − C (0.1 eq.) was added followed by shaking at hydrogen atmosphere. On completion of the reaction, the catalyst was filtered and the solvent was removed to get crude flavones C5, which was purified through column chromatography. A yellow solid; Yield, 43.7%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.49 (s, 1H, OH), 10.90 (s, 1H, OH), 10.29 (s, 1H, OH), 7.98 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 1.6 Hz, 1H), 6.21 (d, J = 1.6 Hz, 1H), 4.77 (s, 2H), 2.90 (s, 4H); 13C NMR (100 MHz, DMSO‑d6) δ: 205.1, 177.4, 164.2, 161.1, 160.2, 156.3, 155.1, 147.2, 136.2, 132.2, 130.4, 129.4, 120.4, 118.9, 115.5, 108.7, 103.9, 98.6, 93.7, 75.7, 38.7, 28.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C26H19NNaO7: 480.1059, found: 480.1055.

3.2.6. General procedure for compounds C42 and C43 A small flask (25 mL) was charged with C7 or C14 (1.0 eq.), 4iodobenzonitrile (1.0 eq.), Na2CO3 (0.1 eq.), L-proline (0.1 eq.), NaN3 (1.5 eq.), and CuSO4•5H2O (0.05 eq.). DMSO/H2O 9:1 (8 mL) was added to the mixture, followed by freshly prepared 1 M aqueous solution of L‑sodium ascorbate (0.1 eq.). The mixture was heated at 65 °C for 48 h with vigorous stirring. After cooling to room temperature, the suspension was extracted with CH2Cl2 (10 mL × 3), and the combined organic layer was washed by brine for three times, dried over Na2SO4 and concentrated under reduced pressure to give a yellow residue, which was purified by silica gel chromatography to afford C42 or C43. 4-(4-(((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3yl)oxy)methyl)-1H-1,2,3-triazol-1-yl) benzonitrile (C42), yellow solid; Yield, 62.5%; 1H NMR (400 MHz, CDCl3) δ: 8.47 (s, 1H), 8.05 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 2.0 Hz, 1H), 6.37 (d, J = 2.0 Hz, 1H), 5.27 (s, 2H), 3.98 (s, 3H), 3.90 (s, 3H), 3.85 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 172.1, 164.1, 163.9, 161.6, 161.3, 160.9, 158.9, 153.6, 146.1, 139.8, 139.4, 133.8, 130.5, 124.7, 122.8, 122.4, 120.6, 117.8, 113.6, 113.0, 122.2, 95.9, 92.6, 65.0, 56.4, 55.8, 55.4; HRESIMS (m/z): [M + Na]+ calcd. for C28H22N4NaO6: 533.1437, found: 533.1437. 4-(4-(2-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen3-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitrile (C43), yellow solid; Yield, 53.6%; 1H NMR (400 MHz, CDCl3) δ: 8.65 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.46 (d, J = 2.0 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 4.26 (t, J = 6.2 Hz, 2H), 3.91 (s, 3H), 3.83 (s, 3H), 3.76 (s, 3H), 3.18 (t, J = 6.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 174.4, 164.1, 161.3, 160.9, 158.9, 153.7, 146.9, 140.1, 139.7, 133.8, 130.1, 129.7, 122.8, 120.9, 120.5, 118.0, 113.9, 111.7, 109.3, 95.9, 92.5, 69.8, 56.5, 55.8, 55.3, 52.3; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H24N4NaO6: 547.1594, found: 547.1591.

3.2.4. General procedure for compounds C1 and C2 A mixture of kaempferol (175 mg, 0.61 mmol), 4-cyanobenzyl bromide (100 mg, 0.51 mmol) or 4-(2-bromoacetyl)benzonitrile (114 mg, 0.51 mmol), and K2CO3 (84 mg, 0.61 mmol) in 1,4-dioxane (10 mL) was heated at 80 °C for 24 h. The mixture was cooled, filtered, concentrated, and purified on gel column chromatography to give a yellow solid C1 or C2. 4-(((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl) oxy)methyl)benzonitrile (C1), yellow solid; Yield, 34.6%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.63 (s, 1H, OH), 10.88 (s, 1H, OH), 10.24 (s, 1H, OH), 7.85 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 2.0 Hz, 1H), 6.22 (d, J = 2.0 Hz, 1H), 5.10 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 177.7, 164.2, 161.2, 160.2, 156.5, 156.4, 142.3, 136.1, 132.1, 130.3, 128.6, 120.4, 118.7, 115.5, 110.6, 104.2, 98.7, 93.8, 72.4; HR-ESIMS (m/z): [M + Na]+ calcd. for C23H15NNaO6: 424.0797, found: 424.0795. 4-(2-((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl) oxy)acetyl)benzonitrile (C2), yellow solid; Yield, 37.5%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.48 (s, 1H, OH), 10.88 (s, 1H, OH), 10.24 (s, 1H, OH), 8.08 (d, J = 8.4 Hz, 2H), 8.02–7.99 (m, 4H), 6.89 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 2.0 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 5.55 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 193.9, 177.4, 164.2, 161.1, 160.2, 156.3, 155.1, 137.6, 136.1, 132.7, 130.5, 128.5, 120.4, 118.0, 115.4, 103.9, 98.7, 93.7, 74.0; HR-ESIMS (m/z): [M + Na]+ calcd. For C24H15NNaO7: 452.0746, found: 452.0747.

3.2.7. General procedure for compounds C10 and C39 To 4-(azidomethyl)benzonitrile (6.9 eq.) and C7 or C14 (5.7 eq.) in CH2Cl2/MeOH 1:1 (10 mL) were added CuSO4•5H2O (1.1 eq.) and L‑sodium ascorbate (2.3 eq.) and the mixture was stirred at 35 °C for 12 h. After cooling to room temperature, then the suspension was extracted with dichloromethane (10 mL × 3), and the combined organic layer was washed by brine for three times, dried over Na2SO4 and concentrated under reduced pressure to give a yellow residue, which was purified by silica gel chromatography to give C10 or C39. 4-((4-(((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C10), yellow solid; Yield, 87.4%; 1H NMR (400 MHz, CDCl3) δ: 8.06 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.27 (s, 1H), 6.97 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 1.6 Hz, 1H), 6.36 (d, J = 1.6 Hz, 1H), 5.56 (s, 2H), 5.22 (br s, 2H), 3.96 (s, 3H), 3.91 (s, 3H), 3.88 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 174.1, 164.0, 161.3, 160.9, 158.8, 153.4, 139.9, 132.8, 129.9, 128.4, 124.7, 122.8, 118.2, 113.9, 112.6, 109.2, 95.9, 92.5, 65.1, 56.5, 55.8, 55.4, 53.3; HR-ESIMS (m/z):

3.2.5. General procedure for compounds C7 and C14 To a mixture of compound 9 (1.5 g, 4.6 mmol), K2CO3 (3.16 g, 23 mmol) and acetone (50 mL) in 100 mL round-bottomed flask, 3bromopropyne (394 μL, 4.6 mmol) or 4-bromobut-1-yne (429 μL, 4.6 mmol) was added dropwise and then it was refluxed for 2 h (monitored by TLC). The reaction mixture was cooled, filtered, concentrated, and purified by column chromatography over silica gel to yield yellow solid C7 or C14. 5,7-dimethoxy-2-(4-methoxyphenyl)-3-(prop-2-yn-1-yloxy)-4Hchromen-4-one (C7), yellow solid; Yield, 96.3%; 1H NMR (400 MHz, CDCl3) δ: 8.12 (d, J = 9.2 Hz, 2H), 6.99 (d, J = 9.2 Hz, 2H), 6.50 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 4.97 (d, J = 2.0 Hz, 2H), 7

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[M + Na]+ calcd. For C29H24N4NaO6: 547.1594, found: 547.1596. 4-((4-(2-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen3-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C39), yellow solid; Yield, 75.2%; 1H NMR (400 MHz, DMSO‑d6) δ: 8.07 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 6.80 (s, 1H), 6.49 (s, 1H), 5.68 (s, 2H), 4.18 (br s, 2H), 3.89 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.03(br s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 172.1, 163.6, 160.7, 160.3, 158.1, 151.9, 141.6, 139.0, 132.6, 129.5, 128.5, 122.4, 118.5, 113.8, 110.8, 108.4, 70.2, 56.0, 55.9, 55.3, 52.0, 26.3; HR-ESIMS (m/z): [M + Na]+ calcd. For C30H26N4NaO6: 561.1750, found: 561.1748.

505.1126. 4-((4-(((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C11), yellow solid; Yield, 26.7%; 1H NMR (400 MHz, CDCl3) δ: 12.55 (s, 1H, OH), 8.03 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 9.2 Hz, 2H), 6.44 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 5.55 (s, 2H), 5.25 (s, 2H), 3.89 (s, 3H), 3.88 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 178.6, 165.5, 161.9, 161.8, 156.7, 156.6, 144.6, 139.6, 136.9, 132.9, 130.4, 128.3, 124.2, 122.5, 118.1, 114.0, 112.8, 105.8, 97.9, 92.2, 65.1, 55.8, 55.5, 53.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C28H22N4NaO6: 533.1437, found: 533.1437. 4-((4-(((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C12), yellow solid; Yield, 21.2%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.73 (s, 1H, OH), 10.34 (s, 1H, OH), 8.20 (s, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 2.0 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 5.65 (s, 2H), 5.23 (s, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ: 178.0, 165.1, 160.9, 160.1, 156.4, 156.2, 142.5, 141.4, 135.5, 132.6, 130.3, 128.3, 125.6, 120.3, 118.5, 115.3, 110.8, 105.5, 97.8, 92.3, 63.9, 56.1, 51.9; HR-ESIMS (m/z): [M + Na]+ calcd. for C27H20N4NaO6: 519.1281, found: 519.1284. 4-((4-(2-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C40), yellow solid; Yield, 28.1%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.62 (s, 1H), 8.04 (s, 1H), 7.98 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.76 (s, 1H), 6.39 (s, 1H), 5.67 (s, 2H), 4.26 (t, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.05 (t, J = 6.4 Hz, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 178.1, 165.2, 161.3, 160.9, 158.0, 156.3, 143.8, 141.6, 136.9, 132.6, 130.1, 128.6, 128.4, 123.3, 122.0, 114.0, 110.8, 105.2, 97.8, 92.3, 70.8, 55.4, 55.3, 52.0, 26.2; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H24N4NaO6: 547.1594, found: 547.1595. 4-((4-(2-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C41), yellow solid; Yield, 26.3%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.66 (s, 1H, OH), 10.26 (s, 1H, OH), 8.03 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 5.67 (s, 2H), 4.24 (t, J = 6.4 Hz, 2H), 3.86 (s, 3H), 3.04 (t, J = 6.4 Hz, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 178.0, 165.1, 160.9, 160.2, 156.3, 156.1, 143.8, 141.6, 136.6, 132.7, 130.2, 128.6, 123.4, 120.3, 118.5, 115.4, 110.8, 105.1, 97.7, 92.3, 70.7, 56.1, 52.0, 48.5, 26.2; HRESIMS (m/z): [M + Na]+ calcd. For C28H22N4NaO6: 533.1437, found: 533.1437. 4-(5-(((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3yl)oxy)methyl)isoxazol-3-yl)benzonitrile (C44), yellow solid; Yield, 23.5%; 1H NMR (400 MHz, DMSO‑d6) δ: 12.59 (s, 1H, OH), 10.93 (s, 1H, OH), 10.23 (s, 1H, OH), 7.99 (s, 4H), 7.85 (d, J = 8.8 Hz, 2H), 7.19 (s, 1H), 6.85 (d, J = 8.8 Hz, 2H), 6.46 (d, J = 2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H), 5.28 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 177.5, 168.7, 164.3, 161.2, 160.6, 160.2, 156.6, 156.4, 135.4, 133.1, 132.6, 130.3, 127.4, 120.2, 118.4, 115.3, 112.7, 104.1, 103.3, 98.7, 93.8, 64.9, 63.2; HR-ESIMS (m/z): [M + Na]+ calcd. for C26H16N2NaO7: 491.0855, found: 491.0856. 4-(5-(((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)methyl)isoxazol-3-yl)benzonitrile (C46), yellow solid; Yield, 46.6%; 1H NMR (400 MHz, CDCl3) δ: 12.50 (s, 1H, OH), 7.97 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.57 (s, 1H), 6.46 (d, J = 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 5.28 (s, 2H), 3.88 (s, 3H), 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 178.2, 169.1, 165.7, 161.9, 161.8, 160.8, 157.0, 156.8, 136.4, 133.1, 132.7, 133.0, 127.3, 122.3, 118.3, 113,9, 113.6, 105.9, 102.3, 98.1, 92.4, 63.8, 55.9, 55.4, 53.4; HR-ESIMS (m/ z): [M + Na]+ calcd. For C28H20N2NaO7: 519.1168, found: 519.1166.

3.2.8. General procedure for compounds C45 and C49 To a solution of (Z)-4-cyano-N-hydroxybenzimidoyl chloride (1.5 eq.) and compound C7 or C14 (1.0 eq.) in CH2Cl2 (10 mL), triethylamine (1.5 eq.) was added slowly at 0 °C. Then the mixture was stirred at room temperature for 48 h. After completion of the reaction, the mixture was dissolved in 20 mL water and extracted with CH2Cl2. Separated the organic layer, dried over anhydrous Na2SO4, concentrated under reduced pressure and purified by silica gel chromatography to yield product C45 or C49. 4-(5-(((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-3yl)oxy)methyl)isoxazol-3-yl)benzonitrile (C45), yellow solid; Yield, 51.2%; 1H NMR (400 MHz, CDCl3) δ: 7.98 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.64 (s, 1H), 6.51 (d, J = 1.6 Hz, 1H), 6.37 (d, J = 1.6 Hz, 1H), 5.30 (s, 2H), 3.98 (s, 3H), 3.90 (s, 3H) 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 173.6, 169.8, 164.1, 161.4, 160.9, 160.8, 158.8, 153.8, 138.7, 133.3, 132.6, 130.1, 127.6, 122.6, 118.3, 113.8, 113.5, 109.2, 102.2, 96.0, 92.5, 63.6, 56.4, 55.8, 55.4; HR-ESIMS (m/z): [M + Na]+ calcd. For C29H22N2NaO7: 533.1325, found: 533.1328. 4-(5-(2-((5,7-dimethoxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen3-yl)oxy)ethyl)isoxazol-3-yl)benzonitrile (C49), yellow solid; Yield, 47.8%; 1H NMR (400 MHz, CDCl3) δ: 7.92 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.64 (s, 1H), 6.50 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 4.43 (t, J = 6.0 Hz, 2H), 3.97 (m, 3H), 3.90 (s, 3H), 3.76 (m, 3H), 3.24 (t, J = 6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 173.9, 172.0, 164.0, 161.2, 160.9, 160.9, 158.8, 153.4, 139.4, 133.7, 132.6, 129.9, 127.4, 122.9, 118.4, 113.8, 113.3, 109.3, 100.4, 95.9, 92.5, 68.3, 56.4, 55.8, 55.2, 48.6, 27.6; HR-ESIMS (m/z): [M + Na]+ calcd. for C30H24N2NaO7: 547.1481, found: 547.1480. 3.2.9. General procedure for compounds C11-C13, C40-C41, C44, C46C48, and C50-C51 Compound C10 (1.0 eq.) was dissolved in CH2Cl2 (10 mL) and added 1.0 M dichloromethane solution of boron tribromide (BBr3) under nitrogen and ice cooling. Stirring was continued for 4 h at room temperature, and then the reaction was quenched by the slow addition of cooled water and extracted with CH2Cl2. The combined organic layers were washed with brine, and then dried over Na2SO4, concentrated under reduced pressure and purified by silica gel chromatography to obtain C11-C13. The procedure described above (using the same quantities of reagents) was followed to afford C40-C41, C44, C46-C47, C48 and C50C51. 4-((4-(((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (C13), yellow solid; Yield, 18.3%; 1H NMR (400 MHz, DMSO-d6) δ: 12.69 (s, 1H, OH), 10.86 (s, 1H, OH), 10.24 (s, 1H, OH), 8.19 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 2.0 Hz, 1H), 6.22 (d, J = 2.0 Hz, 1H), 5.65 (s, 2H), 5.20 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 177.9, 164.1, 161.2, 160.0, 156.3, 156.1, 142.6, 141.4, 135.4, 132.6, 128.3, 125.5, 120.4, 118.5, 115.3, 110.8, 104.1, 98.6, 93.7, 64.0, 51.9; HRESIMS (m/z): [M + Na]+ calcd. for C26H18N4NaO6: 505.1124, found: 8

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cells were then solubilized in 100% dimethyl sulfoxide (DMSO) for 5 min. The absorbance was measured using microplate reader (BioTek, Winooski, VT, USA) at 570 nm.

4-(5-(((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)methyl)isoxazol-3-yl)benzonitrile (C47), yellow solid; Yield, 20.1%; 1H NMR (400 MHz, DMSO-d6) δ: 12.58 (s, 1H, OH), 10.25 (s, 1H, OH), 7.99 (br s, 4H), 7.89 (d, J = 7.2 Hz, 2H), 7.20 (s, 1H), 6.86 (d, J = 7.2 Hz, 2H), 6.76 (d, J = 2.0 Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 5.29 (s, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSOd6) δ: 177.6, 168.7, 165.2, 160.9, 160.6, 160.3, 156.9, 156.3, 135.6, 133.1, 132.5, 130.4, 127.3, 120.1, 118.4, 115.4, 112.7, 105.1, 103.3, 97.9, 92.5, 63.2, 56.1; HR-ESIMS (m/z): [M + Na]+ calcd. For C27H18N2NaO7: 505.1012, found: 505.1015. 4-(5-(2-((5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3yl)oxy)ethyl)isoxazol-3-yl)benzonitrile (C48), yellow solid; Yield, 27.1%; 1H NMR (400 MHz, DMSO-d6) δ: 12.66 (s, 1H, OH), 10.86 (s, 1H, OH), 10.16 (s, 1H, OH), 7.99 (s, 4H), 7.82 (d, J = 8.8 Hz, 2H), 6.99 (s, 1H), 6.78 (d, J = 8.8 Hz, 2H), 6.43 (d, J = 2.0 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H), 4.36 (t, J = 6.0 Hz, 2H), 3.24 (t, J = 6.0 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 177.8, 171.8, 164.1, 161.2, 160.6, 160.0, 156.3, 155.9, 136.0, 133.0, 130.2, 127.2, 120.3, 118.4, 115.4, 112.4, 104.1, 100.8, 98.6, 93.7, 68.7, 48.5, 27.3; HR-ESIMS (m/z): [M + Na]+ calcd. For C27H18N2NaO7: 505.1012, found: 505.1015. 4-(5-(2-((5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4-oxo-4Hchromen-3-yl)oxy)ethyl)isoxazol-3-yl)benzonitrile (C50), yellow solid; Yield, 38.4%; 1H NMR (400 MHz, CDCl3) δ: 12.59 (s, 1H, OH), 7.91 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.58 (s, 1H), 6.43 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 4.41 (t, J = 6.0 Hz, 2H), 3.87 (s, 3H), 3.75 (s, 3H), 3.24 (t, J = 6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 178.6, 171.6, 165.5, 161.9, 160.9, 156.7, 156.5, 137.1, 133.5, 132.6, 130.3, 127.3, 122.4, 119.4, 113.9, 113.4, 105.9, 100.4, 97.9, 92.2, 68.8, 55.8, 55.2, 53.4, 28.0; HR-ESIMS (m/z): [M + Na]+ calcd. for C29H22N2NaO7: 533.1325, found: 533.1328. 4-(5-(2-((5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-4Hchromen-3-yl)oxy)ethyl)isoxazol-3-yl)benzonitrile (C51), yellow solid; Yield, 32.9%; 1H NMR (400 MHz, DMSO-d6) δ: 12.64 (s, 1H, OH), 10.19 (s, 1H, OH), 7.99 (s, 4H), 7.85 (d, J = 8.8 Hz, 2H), 6.99 (s, 1H), 6.79 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 2.0 Hz, 1H), 6.37 (d, J = 2.0 Hz, 1H), 4.38 (t, J = 6.0 Hz, 2H), 3.85 (s, 3H), 3.24 (t, J = 6.0 Hz, 2H); 13C NMR (100 MHz, DMSO‑d6) δ: 177.9, 171.7, 165.1, 160.9, 160.6, 160.2, 156.2, 136.3, 133.0, 130.2, 127.2, 120.2, 118.4, 115.4, 112.4, 105.1, 100.8, 97.8, 92.3, 68.7, 56.0, 24.3; HR-ESIMS (m/z): [M + Na]+ calcd. for C28H20N2NaO7: 519.1168, found: 519.1166.

3.5. Glucose consumption assay HepG2 cells were cultured in α-MEM supplemented with 10% FBS. After confluence, cells were cultured in 96-well cluster plates in highglucose DMEM supplemented with 10% FBS for 24 h, and then the cells were treated with 10−7 mol/L insulin for 36 h in serum-free and phenol red-free high-glucose DMEM. After 36 h high concentration insulin stimulated, the cells were washed by high-glucose DMEM (pH = 4) for 4 times and PBS for 2 times, then add in serum-free and phenol red-free high-glucose DMEM with test compounds in different concentrations (no cytotoxic concentrations) and incubated for 24 h. After this incubation, glucose content in the culture medium was measured by glucose oxidase method using glucose assay kit to assay the remainder glucose in the culture medium. After culture, glucose content in medium was determined by glucose oxidase method. The effect of test compounds on glucose consumption of IR HepG2 cells was studied by measuring the residual glucose in the medium. The enhancement ratio of glucose consumption (GC) was calculated as follows:

GC% = (GC treatment group − GC of control group) /GC of control group × 100 The potencies of the products were expressed as median effective concentration (EC50) values. 3.6. Glucose production assay Glucose production assay was performed according to previously published methods (1). HepG2 cells were seeded in 6-well plates (5 × 105 cells per well). The cells were pre-incubated with different dosage flavonoid derivative for 24 h. The positive control was metformin of 1.0 mM. At the day of the assay, the medium was removed, and then washed twice with PBS. The cells were then incubated with glucose production buffer consisting of glucose-free DMEM (pH 7.4), without phenol red (Invitrogen, Madrid, Spain), supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate [31,32]. After a 3 h incubation, medium was collected and glucose concentration measured with a colorimetric glucose assay kit (Sigma, Madrid, Spain). The readings were then normalized to the total protein content determined from the whole-cell lysates. The bicinchoninic acid protein (BCA) assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for the whole-cell lysates.

3.3. Cell culture Human hepatic cellular carcinoma cells (HepG2) were obtained from American Type Culture Collection. The HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM-F12 medium containing 2.5% fetal bovine serum (FBS) and antibiotics (gentamicin, penicillin, and streptomycin (50 mg/L)). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2. One day after plating, the medium was changed to DMEM containing 5.5 mM D-glucose, 2 mM glutamine, and FBS, and the culture was continued. Subsequently, the experimental treatments were carried out for the indicated periods with various concentrations of glucose in serum-free media for 24 h.

3.7. Western blotting analysis Cells were lysed at 4 °C in ice-cold RIPA buffer containing protease inhibitor and phosphatase inhibitors. Cell lysates were centrifuged (12,000 rpm/min, 4 °C) for 20 min. The supernatants were collected, assayed for their protein concentration by using a Bio-Rad (Bio-Rad, Madrid, Spain) protein assay kit (according to the manufacturer's specifications). Protein samples (60 μg/lane) were separated using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Nonfat milk (5%) in TBS containing 0.1% Tween 20 (TBS-T) was used to block the membranes for 1 h. The membranes were then incubated at 4 °C overnight with primary antibodies recognizing pAMPK, AMPK, PEPCK, G6Pase, and β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The enhanced chemiluminescence western blotting detection system (GE Healthcare Life Science, Buckinghamshire, UK) was used to visualize the

3.4. Cytotoxicity assay To evaluate viability of HepG2 cells, cells were seeded at 1 × 104 cells/mL in 96 well culture plates and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. Flavonoid derivatives were added at 1–25 μM and incubated for a further 24 h. An MTT (2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay was used to test cell viability, in which 20% of MTT (purchased from Sigma Aldrich, Gillingham, UK, as a working solution at 5 mg/ml in phosphate-buffered saline (PBS) was added to the cells and incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2. The 9

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immunoreactive proteins on the membranes.

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4. Conclusions In this study, three series of derivatives were synthesized. We found that triazole or isoxazole derivatives still exhibited significant activities, and avoided the potential toxicity of the α, β-unsaturated ketone fragment. Isoxazole derivative C45 exhibited powerful potency at a nanomolar concentration. The activation of AMPK/PEPCK/G6Pase pathway was one of the molecular mechanisms of C45, which could represent a valuable compound for the discovery of anti-diabetic drugs. Author contributions Wen-Yan Niu, Hong-Quan Duan, and Nan Qin designed all of the studies. Jiang-Ping Nie, Ying Chen, Yang Yu, Hong-Quan Duan, and Nan Qin wrote this manuscript; Jia-Hao Chen and Jiang-Ping Nie prepared these compounds; Zhen-Ni Qu, Yue Jiang, Mei-Na Jin, Ying Chen finished the biological experiments. Funding This work was supported by the National Natural Science Foundation of China (NSFC) [grant number 81373297]; and the Tianjin Research Program of Applied Basic and Cutting-edge Technologies [grant number 12JCZDJC25900]; and Natural Science Foundation of Tianjin [grant number 18JCQNJC13800]. Declaration of Competing Interest There are no conflicts to declare. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2020.104499. References [1] J.P. Boyle, A.A. Honeycutt, K.M.V. Narayan, T.J. Hoerger, L.S. Geiss, H. Chen, T.J. Thompson, Projection of diabetes burden through 2050 - impact of changing demography and disease prevalence in the us, Diabetes Care 2001 (1936-1940) 24. [2] S.A. Kavatagimath, S.S. Jalalpure, Screening of ethanolic extract of diospyros malabarica desr. Bark for anti-diabetic and antioxidant potential, Indian Journal of Pharmaceutical Education and Research 50 (2016) 179–189. [3] R.A. DeFronzo, R.C. Bonadonna, E. Ferrannini, Pathogenesis of niddm. A balanced overview, Diabetes Care 15 (1992) 318–368. [4] W.T. Cefalu, Insulin resistance: cellular and clinical concepts, Exp. Biol. Med. 226 (2001) 13–26. [5] D.G. Hardie, D.A. Pan, Regulation of fatty acid synthesis and oxidation by the ampactivated protein kinase, Biochem. Soc. Trans. 30 (2002) 1064–1070. [6] D.G. Hardie, Ampk: a target for drugs and natural products with effects on both diabetes and cancer, Diabetes 62 (2013) 2164–2172. [7] K.A. Coughlan, R.J. Valentine, N.B. Ruderman, A.K. Saha, Ampk activation: a therapeutic target for type 2 diabetes? Diabetes, Metabolic Syndrome and Obesity 7 (2014) 241–253. [8] N. Kawahara, M. Satake, Y. Goda, A new acylated flavonol glycoside from the leaves of eriobotrya japonica, Chemical & Pharmaceutical Bulletin 2002 (1619-1620) 50. [9] W.L. Li, H.C. Zheng, J. Bukuru, N. De Kimpe, Natural medicines used in the traditional chinese medical system for therapy of diabetes mellitus, J.

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