A Novel Series of Flavone‐Cinnamic Acid Hybrid Derivatives as Hypoglycemic Agents: Design, Synthesis, and Activity Studies

ABSTRACT

In this work, α‐glucosidase was chosen as the main therapeutic target. Novel flavone‐cinnamic acid hybrid derivatives were designed, synthesized, and structurally identified based on flavone prototypes with intrinsic α‐glucosidase inhibitory effects. Considering the multi‐target pathological features of diabetes, α‐glucosidase inhibition, lipase inhibition, DPPH radical scavenging capacities, and insulin‐resistant HepG2 cell assays were systematically evaluated to clarify their hypoglycemic mechanisms. Most acacetin‐cinnamic acid hybrid derivatives exerted strong dual inhibitory effects on the two enzymes, with 6c and 6l showing the best α‐glucosidase and lipase inhibitory activity, respectively, at the concentration of 50 µM. In the baicalein‐cinnamic acid hybrid derivatives, only 7a possessed prominent lipase inhibition, while other analogues were weak in enzyme inhibition but superior to parent baicalein. Notably, baicalein‐cinnamic acid hybrid derivatives presented outstanding antioxidant activity, and significantly enhanced glucose uptake in insulin‐resistant HepG2 cells, among which 7f and 7i were the most promising candidates. Enzyme inhibition and antioxidant properties jointly determined their cellular hypoglycemic effects. Overall, these hybrids exert anti‐diabetic effects via synergistic multi‐pathway actions, and 7f and 7i deserve further in‐depth mechanistic research.

Novel flavone‐cinnamic acid hybrid derivatives were rationally designed, synthesized, and comprehensively evaluated through α‐glucosidase, lipase, DPPH antioxidant, and insulin‐resistant HepG2 cell assays. Their hypoglycemic effects are presumed to stem from such synergistic bioactivities. Compounds 7f and 7i displayed superior glucose consumption capacity. They can be chosen as promising hypoglycemic candidates for future mechanism investigations.

1. Introduction

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance (IR) and inadequate insulin secretion, resulting in persistent hyperglycemia [1]. As the most common form of diabetes, T2DM accounts for approximately 90% to 95% of all diagnosed cases. While T2DM primarily affects adults, its incidence has increased significantly among adolescents and children in recent years [2]. The primary goal of therapeutic intervention is to maintain blood glucose within physiological ranges, prevent complications such as nephropathy, cardiovascular disease, and neuropathy, and improve patients’ quality of life [3]. Glycemic control should initially be achieved through lifestyle interventions, including dietary changes, regular physical activity, and weight management [4]. If lifestyle interventions do not provide sufficient glycemic control, pharmacological agents should be introduced under medical supervision. These agents include biguanides (e.g., metformin), sulfonylureas (e.g., glibenclamide), glinides (e.g., repaglinide), thiazolidinediones (e.g., rosiglitazone), α‐glucosidase inhibitors (e.g., acarbose), incretin‐based therapies such as glucagon‐like peptide‐1 (GLP‐1) agonists (e.g., exenatide), dipeptidyl peptidase‐4 inhibitors (e.g., sitagliptin), and GLP‐1/glucose‐dependent insulinotropic polypeptide dual receptor agonists (e.g., tirzepatide) [5], as well as sodium‐glucose cotransporter 2 inhibitors (e.g., dapagliflozin) [6, 7]. If these pharmacotherapies are ineffective or in cases of severe hyperglycemia, insulin therapy should be initiated according to individual blood glucose profiles [4].

Metformin is currently recognized as the first‐line therapeutic agent for T2DM worldwide [8]. Its developmental origin can be traced back to the early 20th century, when German researchers isolated galegine from the natural plant Galega officinalis, a bioactive compound that serves as the precursor of metformin [9]. To date, the exploration of hypoglycemic agents derived from natural plants remains a crucial and enduring strategy in the development of therapeutic drugs for T2DM [10].

Flavones, a subclass of polyphenolic compounds widely distributed in the plant kingdom, belong to the broader flavonoid family [11]. These bioactive molecules exhibit diverse pharmacological properties, including anti‐diabetic, anti‐cancer, neuroprotective, anti‐inflammatory, antioxidant, anti‐asthmatic, anti‐ulcer, cardiovascular protective, antimicrobial, and anti‐malarial activities [12]. Recent studies have increasingly focused on the anti‐diabetic properties of flavones. These effects are mainly mediated by targeting key molecular components, including α‐glucosidase, aldose reductase, protein tyrosine phosphatases, protein kinase C, peroxisome proliferator activated receptor‐γ, and advanced glycation end products [13].

α‐Glucosidase is an intracellular glycoside hydrolase that plays a pivotal role in the catabolism of complex carbohydrates, facilitating the absorption of ingested carbohydrates in the gastrointestinal tract. This enzymatic activity directly contributes to the elevation of postprandial glycemia and subsequent insulin spikes, which are critical pathological features of T2DM [14]. α‐Glucosidase inhibitors exert their therapeutic effects by inhibiting dietary carbohydrate degradation via α‐glucosidase, thereby slowing the production and absorption of monosaccharides (e.g., glucose). This mechanism effectively reduces postprandial blood glucose peaks and attenuates blood glucose fluctuations, rendering α‐glucosidase inhibitors significant clinical value in the management of T2DM [15].

Many flavones and their derivatives have been reported to exert hypoglycemic effects by inhibiting α‐glucosidase [16, 17]. Chrysin (1), acacetin (2), and baicalein (3) (Figure 1) are representative flavones characterized by multiple hydroxyl substitutions on the A ring. These compounds not only possess inherent hypoglycemic activities but also contain derivatizable hydroxyl groups, making them suitable candidates for structural modification [18, 19, 20]. Such modifications can enhance hypoglycemic potency by introducing functional groups and improve metabolic stability, a key parameter in drug development. As a result, chrysin, acacetin, and baicalein were chosen as the lead compounds for developing novel α‐glucosidase inhibitors in our project.

FIGURE 1.

The structures of chrysin, acacetin, baicalein, and cinnamic acid.

Cinnamic acid (4a, Figure 1) and its derivatives, which are principal bioactive constituents of Cinnamomum cassia Presl, demonstrate a wide range of pharmacological activities, such as anti‐tumor, antibacterial, anti‐inflammatory, anti‐aging, and anti‐diabetic effects [21]. Structurally, these compounds contain acrylic acid moieties and benzene rings with conjugated π‐electron systems. This structural configuration facilitates effective interactions with hydrophobic pockets in target proteins, making them suitable molecular scaffolds for enhancing the biological activities of natural compounds through structural modification [22]. Shin et al. introduced cinnamic acid into the structure of chrysin and evaluated the in vivo hypoglycemic activity of the target compound, which was slightly higher than that of the control group [23]. For acacetin, although no studies regarding its cinnamoylation modifications have been reported, Nguyen et al. explored the hypouricemic effects of acacetin and 4,5‐O‐dicaffeoylquinic acid methyl ester (a compound bearing a cinnamic acid moiety) isolated from Chrysanthemum morifolium. Their findings verified the therapeutic potential of these two compounds against metabolic disorders, which provides credible evidence for the design and hypoglycemic research of novel acacetin‐cinnamic acid hybrid derivatives [24]. In our previous work, baicalein was structurally modified via aminomethylation at the C‐8 position and cinnamoylation at the 7‐hydroxyl group. Biological evaluation demonstrated that the resulting baicalein derivative possessed moderate CDK1/cyclin B inhibitory activity and exerted a prominent antiproliferative effect on MCF‐7 tumor cells [25].

Against this background, three novel series of flavone‐cinnamic acid hybrid derivatives were designed and synthesized by esterifying the 7‐hydroxyl groups of flavones with the carboxyl groups of cinnamic acid or its derivatives. The biological activities of these compounds were systematically evaluated by α‐glucosidase, lipase, and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH). While α‐glucosidase is involved in carbohydrate metabolism, lipase is crucial for lipid metabolism. These enzymes are functionally interconnected and operate in a coordinated manner within the metabolic network. Based on this regulatory relationship, lipase inhibitory activity was also conducted for all target compounds after α‐glucosidase assay. Since compounds showing potent α‐glucosidase and lipase inhibitory activities, especially those with antioxidant properties, may ameliorate the health status of patients with metabolic syndrome, DPPH radical scavenging assays were subsequently performed on all target compounds. Subsequently, cellular activity assays were further performed on compounds with favorable biological activities.

2. Results and Discussion

2.1. Chemistry

The general synthetic strategy for all target compounds is outlined in Scheme 1. Initially, cinnamic acid derivatives containing free phenolic hydroxyl groups (4b–e) were treated with acetic anhydride [26] or ethyl chloroformate [27] to afford the hydroxyl‐protected intermediates bearing acetoxy or ethoxycarbonyloxy substituents (4j–q). Together with commercially available fluoro‐ and trifluoromethyl‐substituted cinnamic acids, all the cinnamic acid derivatives containing electron‐withdrawing groups can modulate the electron density of the cinnamic acid benzene ring, regulate acylation reactivity and regioselectivity, and affect product yield and by‐product formation. Furthermore, they can enrich the structural diversity of flavone‐cinnamic acid hybrid derivatives for subsequent structure‐activity relationship investigations and simultaneously improve the lipophilicity, chemical stability, and purification performance of the target compounds. Subsequently, cinnamic acid and its derivatives (4) were subjected to esterification reactions with flavone scaffolds, including chrysin (1), acacetin (2), and baicalein (3), in dichloromethane (DCM). The esterification was catalyzed by a combination of 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4‐dimethylaminopyridine (DMAP), resulting in the target esterified products (5–7) [28]. Among the three series of target compounds, baicalein‐cinnamic acid hybrid derivatives exhibit the lowest yields in the final reaction (28%–46%). This can be attributed to the fact that the 6‐phenolic hydroxyl group on baicalein can also undergo acylation with cinnamic acid derivatives, leading to increased by‐product formation and a consequent reduction in yield. Within each series, derivatives bearing an electron‐withdrawing substituent on the para‐position of the benzene ring in the cinnamic acid moiety show lower yields, with the para‐ethoxycarbonyloxy substituent analogues possessing the lowest yields (28%–34%). The reason may be that although para‐electron‐withdrawing substitution on the benzene ring of the cinnamic acid moiety enhances acylation reactivity, it leads to non‐selective acylation of the muti‐hydroxylated flavone skeleton, increases by‐product formation, and consequently reduces the target product yield. The chemical structures of all target compounds (detailed in Table 1) were comprehensively characterized and unambiguously confirmed by ¹H and ¹³C nuclear magnetic resonance (NMR) spectra and high‐resolution mass spectrometry (HRMS) (see Supplementary File 1, Figure S1–S47).

SCHEME 1.

Synthetic routes of flavone‐cinnamic acid hybrid derivatives (5a–m, 6a–m, and 7a–m). Reagents and conditions: (a) Ac₂O, H₂SO₄, rt, 0.5 h; (b) EtOCOCl, pyridine, rt, 8 h; (c) EDCI, DMAP, DCM, rt, 0.5 h.

TABLE 1.

The substituents of compounds 5a–m, 6a–m, and 7a–m.

2.2. Enzyme Activity Assays

2.2.1. α‐Glucosidase Inhibition Results

The anti‐α‐glucosidase activities of all target compounds were evaluated using acarbose as the positive control. Acarbose is a well‐established clinical α‐glucosidase inhibitor, and the corresponding results were presented in Table 2. Referring to the concentrations applied in our previous studies, three gradient concentrations, including 100, 50, and 25 µM, were selected for pre‐experiments. Finally, 50 µM was determined as the optimal concentration for subsequent activity verification. The IC₅₀ value of acarbose was determined to be 71.29 ± 0.47 nM, aligning with the literature‐reported value of 21.82 ± 3.26 nM [29]. Assays indicated that none of the target compounds exhibited greater inhibitory potency than acarbose. Among the three flavone lead compounds, baicalein displayed the highest intrinsic activity. Following structural modification, derivatives of chrysin and acacetin showed marked improvements in activity compared to their respective parent compounds. Specifically, 9 of 13 chrysin derivatives and 8 of 13 acacetin derivatives exhibited higher activities than their leads. In contrast, only 5 of 13 baicalein derivatives showed increased activities, with limited improvement. Overall, the acacetin‐cinnamic hybrid derivatives (6a–m) demonstrated stronger inhibitory activities than the chrysin‐cinnamic hybrid derivatives (5a–m), which in turn surpassed the baicalein‐cinnamic hybrid derivatives (7a–m). These results suggest that a methoxy group on the B ring of the flavone scaffold enhances α‐glucosidase inhibition. Additionally, within acacetin‐cinnamic hybrid derivatives, compounds 6b–d bearing a fluorine substituent on the benzene ring of the cinnamic acid moiety exhibited the most potent inhibitory activities. This finding indicates that introducing a fluorine substituent at this position enhances α‐glucosidase inhibitory efficacy.

TABLE 2.

The inhibitory rates of all target compounds against α‐glucosidase at the concentration of 50 µM.

Cpds.	Inhibition rate/%	Cpds.	Inhibition rate/%	Cpds.	Inhibition rate/%
5a	32.41 ± 1.18	6a	19.85 ± 1.20	7a	32.29 ± 2.67
5b	27.68 ± 1.83	6b	40.37 ± 3.56	7b	31.68 ± 3.37
5c	18.50 ± 3.36	6c	44.96 ± 3.44	7c	22.88 ± 2.25
5d	36.09 ± 3.29	6d	40.55 ± 2.63	7d	28.28 ± 1.65
5e	25.38 ± 2.25	6e	30.95 ± 4.50	7e	29.71 ± 2.61
5f	8.97 ± 3.55	6f	24.85 ± 4.67	7f	24.04 ± 2.16
5g	27.75 ± 0.89	6g	12.61 ± 1.09	7g	23.88 ± 2.25
5h	39.30 ± 1.24	6h	16.98 ± 0.43	7h	27.76 ± 1.32
5i	26.06 ± 2.22	6i	9.72 ± 4.15	7i	21.55 ± 0.46
5j	23.84 ± 3.60	6j	6.34 ± 2.12	7j	35.15 ± 1.63
5k	15.67 ± 5.22	6k	14.32 ± 3.09	7k	31.46 ± 2.76
5l	8.35 ± 2.74	6l	21.84 ± 3.34	7l	27.78 ± 1.12
5m	21.07 ± 2.16	6m	32.57 ± 4.49	7m	24.42 ± 2.22
Chrysin	19.82 ± 5.04	Acacetin	19.83 ± 1.72	Baicalin	28.68 ± 2.34
Acarbose	99.84 ± 0.54 IC50 = 71.29 ± 0.47nM

2.2.2. Lipase Inhibition Results

Following the α‐glucosidase inhibitory assay, all target compounds were further evaluated for their lipase inhibitory activity, with orlistat serving as the positive control. Orlistat is a clinically applied gastrointestinal lipase inhibitor widely used for the treatment of obesity and hyperlipidemia. The results are presented in Table 3. The tested concentration was referenced from a previous α‐glucosidase assay. The IC₅₀ value of orlistat was 3.27 ± 0.06 µM, consistent with the literature‐reported value of 0.99 ± 0.11 µM [30]. Anti‐lipase activity assays revealed that none of the target compounds exhibited greater inhibitory potency than orlistat. However, most target compounds exhibited higher lipase inhibitory activity than their respective parent flavone lead compounds. Among the three flavone derivative series, the descending order was acacetin derivatives (6a–m), chrysin derivatives (5a–m), and baicalein derivatives (7a–m), mirroring the results from the α‐glucosidase activity assays. In particular, compound 6l showed the optimal lipase inhibitory activity with an inhibition rate of 71.38% ± 1.24% at the concentration of 50 µM. Compounds 6c, 6f, 6g, 6h, and 6m exhibited favorable inhibition rates ranging from 52.44% ± 2.56% to 59.03% ± 0.82%. Further structure‐activity relationship analysis of the acacetin‐cinnamic acid hybrid derivatives revealed that substitution of the benzene ring in the cinnamic acid moiety with small to medium‐sized groups (e.g., compounds 6f–h) had minimal impact on inhibitory activity depending on substituent position. In contrast, derivatives with larger substituents and para‐substitution (e.g., compounds 6j–l) exhibited superior activity compared to other positional isomers. Furthermore, disubstituted derivatives on the benzene ring of the cinnamic acid moiety exhibited reduced activity compared to their monosubstituted counterparts, as evidenced by the pairs 6h versus 6i and 6l versus 6m.

TABLE 3.

The inhibitory rates of all target compounds against lipase at the concentration of 50 µM.

Cpds.	Inhibition rate/%	Cpds.	Inhibition rate/%	Cpds.	Inhibition rate/%
5a	55.22 ± 1.00	6a	48.78 ± 3.35	7a	64.44 ± 4.98
5b	44.07 ± 1.68	6b	46.86 ± 3.40	7b	17.82 ± 2.62
5c	45.45 ± 2.57	6c	55.10 ± 2.33	7c	11.82 ± 4.45
5d	56.60 ± 3.79	6d	24.83 ± 0.85	7d	39.71 ± 5.44
5e	32.54 ± 3.80	6e	45.50 ± 3.04	7e	8.34 ± 1.06
5f	21.59 ± 1.92	6f	57.68 ± 2.57	7f	26.04 ± 5.29
5g	34.47 ± 13.21	6g	56.94 ± 3.96	7g	14.72 ± 2.63
5h	16.94 ± 1.49	6h	52.44 ± 2.56	7h	—
5i	13.92 ± 2.45	6i	35.21 ± 1.94	7i	22.60 ± 3.43
5j	35.09 ± 5.29	6j	23.69 ± 5.15	7j	6.33 ± 3.75
5k	17.23 ± 3.03	6k	28.71 ± 4.22	7k	15.93 ± 2.41
5l	27.29 ± 1.85	6l	71.38 ± 1.24	7l	27.00 ± 4.43
5m	10.57 ± 1.03	6m	59.03 ± 0.82	7m	39.33 ± 1.59
Chrysin	18.45 ± 2.09	Acacetin	17.76 ± 0.53	Baicalin	12.24 ± 1.90
Orlistat	93.86 ± 0.39 IC50 = 3.27 ± 0.06µM

2.3. DPPH Radical Scavenging Results

Oxidative stress is closely linked to the pathogenesis of metabolic diseases, including diabetes mellitus and hyperlipidemia. It plays a pivotal role in driving IR progression and impairing pancreatic β‐cell function in diabetes. Meanwhile, excessive oxidative stress disrupts lipid metabolism, promotes abnormal lipid deposition, and exacerbates inflammatory responses, thereby accelerating the occurrence and development of hyperlipidemia. Many flavone compounds possess inherent antioxidant properties, making them potentially valuable for regulating glucose and lipid homeostasis. Based on previous enzyme inhibitory activity experiments, all target compounds were subjected to DPPH radical scavenging assays for antioxidant capacity evaluation, with vitamin C (VC) employed as the positive control due to its classic antioxidant property, potent and stable DPPH radical scavenging ability, and excellent experimental reproducibility. The results are summarized in Table 4. The concentration used for DPPH radical scavenging assay was also determined based on our previous experimental conditions and was fixed at 500 µM. The IC₅₀ value of VC was 35.58 ± 0.38 µM, which is within the same order of magnitude as the literature‐reported value (10.75 ± 0.66 µM) [31]. The DPPH assay results demonstrated that all target compounds had weaker antioxidant activities than VC. However, among the three series of target compounds, baicalein‐cinnamic acid hybrid derivatives (7a–m) exhibited the highest DPPH radical scavenging activities, with baicalein itself showing the strongest antioxidant potency among all tested samples. After determining the scavenging rates at 500 µM, the IC₅₀ values of baicalein derivatives were further evaluated. The trend of IC₅₀ values was consistent with that of the scavenging rates at 500 µM. Compounds 7j and 7k, bearing ethoxycarbonyloxy substituent on the benzene ring of the cinnamic acid moiety, showed the best activities with IC₅₀ values of 88.67 ± 5.28 and 71.53 ± 0.88 µM, respectively. However, both were less potent than the lead compound baicalein (IC₅₀ = 43.75 ± 2.88 µM). These findings indicate that phenolic hydroxyls on the flavone scaffold are critical structural determinants governing DPPH radical scavenging activity. Baicalein exhibited markedly stronger antioxidant activity than chrysin and acacetin, owing to its extra phenolic hydroxyl group. This additional hydroxyl facilitates hydrogen proton donation to neutralize free radicals, thereby effectively enhancing antioxidant potency.

TABLE 4.

The DPPH radical scavenging rates of compounds 5 and 6 at the concentration of 500 µM and IC₅₀ values of compound 7.

Cpds.	Scavenging rate/%	Cpds.	Scavenging rate/%	Cpds.	IC50/µM
5a	16.75 ± 0.39	6a	30.90 ± 4.80	7a	122.3 ± 2.51
5b	8.52 ± 0.91	6b	13.22 ± 2.99	7b	707.2 ± 56.01
5c	14.83 ± 1.08	6c	7.63 ± 1.16	7c	621.3 ± 88.81
5d	16.37 ± 2.61	6d	15.84 ± 2.40	7d	197.0 ± 16.65
5e	7.27 ± 0.98	6e	9.14 ± 1.67	7e	492.5 ± 75.47
5f	10.32 ± 0.95	6f	20.94 ± 2.86	7f	171.6 ± 36.4
5g	18.76 ± 1.04	6g	19.87 ± 2.97	7g	231.6 ± 23.89
5h	22.22 ± 0.78	6h	22.3 ± 2.01	7h	269.6 ± 8.73
5i	18.76 ± 3.40	6i	8.55 ± 2.98	7i	207.7 ± 30.88
5j	11.45 ± 0.93	6j	4.86 ± 1.20	7j	88.67 ± 5.28
5k	6.77 ± 1.95	6k	22.45 ± 3.13	7k	71.53 ± 0.88
5l	7.06 ± 1.94	6l	14.58 ± 1.57	7l	932.4 ± 137
5m	13.32 ± 1.44	6m	18.80 ± 1.98	7m	378.6 ± 17.24
Chrysin	9.11 ± 0.41	Acacetin	3.5 ± 1.40	Baicalin	43.75 ± 2.88
VC	93.35 ± 0.11 IC50 = 35.58 ± 0.38 µM

The results from α‐glucosidase inhibitory, lipase inhibitory, and DPPH radical scavenging assays revealed distinct activity profiles among the three flavone derivative series. Acacetin‐cinnamic hybrid derivatives (6a–m) showed the highest inhibitory activities in both enzyme assays, while baicalein‐cinnamic hybrid derivatives (7a–m) exhibited the lowest potency in these evaluations. In contrast, 7a–m displayed the strongest antioxidant activities in the DPPH radical scavenging assay. Although both enzyme inhibitory activity and antioxidant capacity are mechanistically linked to potential hypoglycemic effects, the comparative hypoglycemic efficacy of the three flavone‐cinnamic hybrid derivatives requires further validation through additional in vitro cellular studies. These investigations are essential to determine the translational significance of the observed in vitro enzyme and antioxidant activities for hypoglycemic effects in biological systems.

2.4. Cell Assay Results

2.4.1. Cytotoxicity Assay Results

To preclude potential interference with the insulin‐resistant cell‐based activity assay, it is indispensable to evaluate the cytotoxicities of target compounds. Non‐specific cytotoxic effects on HepG2 cells can impair cell viability and perturb basal glucose consumption, thereby skewing the quantification of hypoglycemic activity. Thus, the cytotoxicities of all target compounds were evaluated as a prerequisite before conducting anti‐IR assays. As presented in Tables 5, 6, and 7, none of the tested compounds displayed significant cytotoxicities under the employed experimental conditions, confirming that the subsequent measurements of hypoglycemic effects would not be confounded by compound‐induced cytotoxicity.

TABLE 5.

The hypoglycemic activities of compounds 5 against insulin‐resistant cells in vitro.

Cpds.	Cytotoxicity/µM	The increased rate of glucose consumption
		10 µM	1 µM	0.1 µM
5a	> 50	36.11 ± 1.27	24.73 ± 1.46	14.63 ± 2.43
5b	> 27.18	9.317 ± 1.22	8.07 ± 0.52	4.98 ± 0.34
5c	> 22.5	23.04 ± 2.02	11.74 ± 0.48	3.87 ± 0.51
5d	> 25	43.57 ± 0.80	36.64 ± 0.86	24.37 ± 0.90
5e	> 25	41.44 ± 1.19	34.02 ± 1.10	29.26 ± 0.83
5f	> 50	37.45 ± 0.70	37.81 ± 1.32	35.47 ± 0.39
5g	> 25	12.59 ± 0.47	9.70 ± 2.10	7.44 ± 2.61
5h	> 25	18.36 ± 2.66	16.71 ± 1.54	15.26 ± 1.01
5i	> 50	39.04 ± 0.32	33.32 ± 0.78	29.96 ± 0.58
5j	> 25	34.16 ± 0.45	30.63 ± 1.42	28.13 ± 1.07
5k	> 50	31.22 ± 1.92	32.05 ± 0.87	19.93 ± 1.90
5l	> 12.5	6.97 ± 2.00	10.66 ± 1.22	7.23 ± 4.43
5m	> 31.25	33.92 ± 0.60	29.00 ± 0.62	22.45 ± 1.53
Chrysin	> 25	24.96 ± 1.61	16.93 ± 0.75	9.49 ± 1.07
Metformin		26.54 ± 0.92	19.09 ± 1.47	13.52 ± 1.42

TABLE 6.

The hypoglycemic activities of compounds 6 against insulin‐resistant cells in vitro.

Cpds.	Cytotoxicity/µM	The increased rate of glucose consumption
		10−5 M	10−6 M	10−7 M
6a	> 25	31.71 ± 1.53	22.70 ± 1.42	7.73 ± 1.43
6b	> 10	22.94 ± 3.22	7.97 ± 1.88	4.66 ± 0.95
6c	> 16.65	40.44 ± 1.54	32.7 ± 0.32	25.67 ± 2.91
6d	> 16.5	41.98 ± 0.31	32.06 ± 2.93	26.36 ± 0.58
6e	> 16.5	12.63 ± 1.82	6.553 ± 1.63	5.90 ± 0.69
6f	> 16.5	20.40 ± 1.64	24.75 ± 0.90	19.79 ± 0.35
6g	> 50	39.42 ± 0.29	30.02 ± 0.48	25.8 ± 1.14
6h	> 100	26.95 ± 2.31	12.14 ± 0.673	5.347 ± 0.97
6i	> 25	24.19 ± 1.30	21.16 ± 0.11	15.54 ± 1.29
6j	> 50	9.04 ± 0.41	8.08 ± 0.31	4.41 ± 0.10
6k	> 50	14.22 ± 2.51	9.39 ± 1.91	6.64 ± 0.77
6l	> 16.5	13.37 ± 2.06	4.42 ± 1.72	4.59 ± 3.76
6m	> 50	25.22 ± 3.64	26.06 ± 3.02	17.22 ± 2.42
acacetin	> 20	24.20 ± 1.25	20.90 ± 1.25	9.80 ± 1.49
Metformin		29.30 ± 2.87	17.72 ± 1.08	12.51 ± 3.18

TABLE 7.

The hypoglycemic activities of compounds 7 against insulin‐resistant cells in vitro.

Cpds.	Cytotoxicity/µM	The increased rate of glucose consumption
		10−5 M	10−6 M	10−7 M
7a	> 50	45.28 ± 0.45	42.99 ± 1.70	23.07 ± 2.63
7b	> 100	25.33 ± 1.52	21.99 ± 0.80	16.02 ± 0.67
7c	> 100	31.42 ± 2.72	26.74 ± 1.22	24.15 ± 0.67
7d	> 250	34.10 ± 1.96	32.29 ± 1.61	20.20 ± 0.92
7e	> 250	50.57 ± 2.31	36.85 ± 2.453	23.83 ± 2.53
7f	> 200	58.67 ± 1.25	53.21 ± 1.71	38.38 ± 1.35
7g	> 100	12.44 ± 1.56	17.07 ± 0.83	11.91 ± 1.19
7h	> 100	5.13 ± 0.39	12.51 ± 1.61	9.37 ± 0.76
7i	> 50	50.93 ± 1.33	52.56 ± 1.94	15.57 ± 1.40
7j	> 50	32.48 ± 2.25	26.38 ± 1.32	15.02 ± 0.55
7k	> 125	8.391 ± 1.21	11.97 ± 1.32	2.66 ± 1.97
7l	> 100	41.02 ± 1.32	28.54 ± 3.11	23.83 ± 2.06
7m	> 100	12.65 ± 0.61	10.43 ± 0.86	4.55 ± 0.96
Baicalin	> 50	23.27 ± 2.46	12.16 ± 1.30	12.17 ± 2.55
Metformin		27.21 ± 2.02	19.34 ± 1.49	8.90 ± 1.86

2.4.2. Anti‐IR Results

In vitro anti‐IR assays were performed on all target compounds, with metformin used as the positive control, which possesses definite anti‐IR activity, reliable pharmacological effect, and stable performance in in vitro evaluation models. The results are summarized in Tables 5, 6, and 7. Based on the results of enzyme activity assays and the commonly used concentration of metformin, anti‐IR assays were carried out at three concentrations: 10, 1, and 0.1 µM. Collectively, all three series of flavone derivatives showed promising anti‐IR activities, with most compounds enhancing the glucose consumption capacity of insulin‐resistant cells more effectively than metformin at three concentration levels. The experimental results revealed a descending order of anti‐IR activity: baicalein‐cinnamic hybrid derivatives (7a–m) > acacetin‐cinnamic hybrid derivatives (6a–m) > chrysin‐cinnamic hybrid derivatives (5a–m). This trend differs from the activity hierarchy observed in the α‐glucosidase and lipase inhibitory assays, suggesting that the antioxidant mechanism may play a more prominent role than α‐glucosidase and lipase inhibition in mediating the hypoglycemic effects. Among baicalein derivatives, compounds 7f and 7i exhibited the strongest activities. Notably, although baicalein derivatives showed weaker DPPH radical scavenging activities compared to the parent compound baicalein, their anti‐IR activities were significantly superior. This finding indicates that the hypoglycemic effects of the target compounds are mediated by a multi‐targeted mechanism, including α‐glucosidase inhibition, lipase inhibition, and antioxidant activity. The precise molecular mechanisms underlying this multi‐targeted activity await further elucidation through subsequent mechanistic investigations.

3. Conclusions

Three series of flavone‐cinnamic acid hybrid derivatives, comprising 39 compounds, were systematically designed and synthesized to evaluate their potential hypoglycemic activities. In α‐glucosidase and lipase inhibition assays, none of the synthesized compounds surpassed the positive controls. However, most showed greater inhibitory potency than their respective parent flavone leads. Among the three series, acacetin‐cinnamic acid hybrids (6a–m) displayed favorable inhibitory activities against the two enzymes, especially compounds 6c against α‐glucosidase and 6l against lipase, whereas baicalein‐cinnamic acid hybrids (7a–m) showed the weakest effects. In the DPPH radical scavenging assay, 7a–m showed the highest antioxidant activities among the three series. Compounds 7j and 7k exhibited the strongest potency. Nevertheless, their activities remained lower than that of the lead compound baicalein. In vitro cellular experiments revealed that most target compounds enhanced glucose consumption in insulin‐resistant cells more effectively than metformin. 7a–m exhibited the strongest anti‐IR activities, with the majority outperforming baicalein. Compounds 7f and 7i were identified as the most active. Collectively, these findings suggest that the hypoglycemic effects of the target compounds are mediated by multiple mechanisms, including α‐glucosidase inhibition, lipase inhibition, and antioxidant activity. The divergent activity rankings between enzyme assays and cellular evaluations imply that the antioxidant mechanism may contribute more substantially to the hypoglycemic effects. Further investigations are warranted to elucidate the precise molecular mechanisms involved.

4. Materials and Methods

4.1. General

Analytical grade solvents of commercial origin were used without further purification. The progress of all reactions was monitored by thin‐layer chromatography (TLC) on silica gel precoated glass plates incorporated with a fluorescent indicator. Melting points were measured using an X‐5 microscopic melting point apparatus (Yuhua Instrument Co., Ltd., Gongyi, China). ¹H and ¹³C‐NMR spectra were recorded on Bruker AV‐III‐400 or Bruker AV‐III‐600 spectrometers. HRMS data were collected on an Agilent 6540 UHD accurate mass quadrupole time‐of‐life mass spectrometer (Q‐TOF/MS) operating in the low‐resonance electrospray ionization (ESI) mode. α‐Glucosidase, lipase, and DPPH were purchased from Beijing Solarbio Science & Technology Co., Ltd. The Cell Counting Kit‐8 (CCK‐8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). HepG2 cells (RRID:CVCL_0027) were provided by the National Collection of Authenticated Cell Cultures. Enzymatic and cellular activity assays were performed using an enzyme‐linked immunosorbent assay (ELISA) microplate reader of Tecan Trading AG (Switzerland).

4.2. Chemical Synthesis

4.2.1. Synthesis of Compounds 4j–m

One of the cinnamic acid derivatives (4b–e, 1 mmol) was accurately weighed out and transferred into a 50‐mL flask. Acetic anhydride (9.4 mL) was then added to dissolve the substrates. Under continuous magnetic stirring, concentrated sulfuric acid (0.2 mL) was added dropwise. The reaction mixture was then heated to 50°C and stirred for 0.5 h, with reaction progress closely monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature and cautiously poured into ice water under vigorous stirring to quench the reaction and induce precipitation. After standing for 20 min, the resulting precipitate was collected by vacuum suction filtration [26]. The obtained solid was dried thoroughly and further purified either by recrystallization from ethyl acetate or by column chromatography using DCM/methanol (100:1, v/v) as the eluent to obtain the target products 4j–m (supplementary file 2, 4j–m).

4.2.2. Synthesis of Compounds 4n–q

One of the cinnamic acid derivatives (4b–e, 1 mmol) was accurately weighed out and transferred into a 50‐mL flask, to which pyridine (1.5 mmol) was subsequently added. Under continuous magnetic stirring, ethyl chloroformate (2 mmol) was then added dropwise. The reaction mixture was stirred at room temperature for 3 h, with progress closely monitored by TLC. After reaction completion, concentrated hydrochloric acid was added to adjust the mixture to pH 1‐2. The resulting solution was then transferred into ice water and stirred vigorously to induce precipitation. After standing for 0.5 h, the formed precipitation was filtered [27]. The obtained filter cake was dried thoroughly and further purified either by recrystallization from ethyl acetate or by column chromatography using DCM/methanol (100:1, v/v) as the eluent to obtain the target products 4n–q (supplementary file 2, 4n–q).

4.2.3. Synthesis of Compounds 5a–m

Cinnamic acid or its derivatives (4a, f–q, 1.2 mmol) were accurately weighed out and transferred into a 50‐mL flask. DCM (12 mL) was used as the reaction medium. Under continuous magnetic stirring, EDCI (0.2300 g, 1.2 mmol), DMAP (0.0610 g, 0.5 mmol), and chrysin (0.2542 g, 1 mmol) were successively added. The reaction mixture was stirred at room temperature for 3 h under a nitrogen atmosphere, with reaction progress closely monitored by TLC [28]. Upon completion, the target products were isolated via column chromatography using a DCM/methanol (100:1, v/v) eluent and further purified by recrystallization from ethyl acetate to yield compounds 5a–m (supplementary file 2, 5a–m).

4.2.4. Synthesis of Compounds 6a–m

The synthetic procedures of compounds 6a–m (supplementary file 2, 6a–m) were the same as those of compounds 5a–m.

4.2.5. Synthesis of Compounds 7a–m

The synthetic procedures of compounds 7a–m (supplementary file 2, 7a–m) were the same as those of compounds 5a–m.

4.3. Biology

4.3.1. α‐Glucosidase Inhibition Assay

p‐Nitrophenyl‐β‐D‐glucopyranoside (PNPG) serves as a glycosidic substrate for α‐glucosidase. Upon hydrolysis by α‐glucosidase, PNPG is converted into glucose and p‐nitrophenol (PNP), where PNP exhibits a maximum absorbance at 405 nm. Measurement of the optical density (OD) at this specific wavelength enables quantification of the α‐glucosidase inhibition rate for each sample. The half‐maximal inhibitory concentration (IC₅₀) is then determined from the inhibition rate data using the formula provided below [32]:

$$\mathrm{Inhibition}\mathrm{rate}(\%)=\left(1-\frac{{\mathrm{OD}}_{\mathrm{sample}}-\mathrm{O}{\mathrm{D}}_{\mathrm{sample}\mathrm{blank}}}{\mathrm{O}{\mathrm{D}}_{\mathrm{enzyme}}-\mathrm{O}{\mathrm{D}}_{\mathrm{enzyme}\mathrm{blank}}}\right)\times 100\%$$

4.3.2. Lipase Inhibition Assay

Sodium p‐nitrophenyl phosphate (PNPP) functions as a lipase substrate that contains a phosphodiester bond. Under the catalysis of lipase, PNPP undergoes hydrolysis to generate phosphate ions and PNP. The OD value of the PNP produced in the lipase‐catalyzed reaction was measured at 405 nm. The lipase inhibition rate and corresponding IC₅₀ values for each sample were calculated using the same formula as that applied in the α‐glucosidase inhibition assay [33].

4.3.3. DPPH Radical Scavenging Activity Experiment

DPPH is a stable, nitrogen‐centered free radical with an unpaired electron, enabling it to accept electrons or hydrogen atoms from antioxidants. In solution, DPPH exhibits maximum absorbance at 517 nm and appears deep purple. When a free radical scavenger is present, DPPH's unpaired electron is captured, causing the solution's color to fade and the absorbance at 517 nm to decrease. This decrease is linearly correlated with the concentration of the free radical scavenger. Lower absorbance at 517 nm indicates stronger free radical scavenging activity, which reflects the antioxidant capacity of the sample. The antioxidant capacity is quantified as an inhibition rate, calculated using the same formula as the α‐glucosidase inhibition assay [34].

4.3.4. Cell Experiments

4.3.4.1. Cytotoxicity Assay

Cytotoxicity assays are crucial for evaluating the potential adverse effects of target compounds on cell viability. In this project, HepG2 cells were used as the model and maintained under optimal conditions. Upon reaching the desired confluence, the cells were treated with various concentrations of the test compounds. After incubation, cell viability was evaluated using the CCK‐8 assay, which measures cellular metabolic activity. Cell viability rates and IC₅₀ values were calculated by comparing the absorbance of treated cells to untreated controls, following the provided formula [35]:

$$\mathrm{Cell}\mathrm{viability}\mathrm{rate}(\%)=\left(\frac{\mathrm{O}{\mathrm{D}}_{\mathrm{sample}}-\mathrm{O}{\mathrm{D}}_{\mathrm{blank}}}{\mathrm{O}{\mathrm{D}}_{\mathrm{negtive}\mathrm{control}}-\mathrm{O}{\mathrm{D}}_{\mathrm{blank}}}\right)\times 100\%$$

4.3.4.2. Anti‐IR Activity Assay

To investigate the effects of target compounds on IR, an IR model was established in HepG2 cells using glucosamine, a known inducer of IR. Elevated intracellular glucosamine activates the hexosamine biosynthesis pathway (HBP), a key branch of glucose metabolism, which disrupts insulin signaling and impairs glucose homeostasis, thereby worsening IR phenotypes. Four experimental groups were established: a blank control (untreated HepG2 cells), a model control (IR‐induced HepG2 cells without treatment), a positive drug control (IR‐induced HepG2 cells treated with metformin), and target compound groups (IR‐induced HepG2 cells treated with different concentrations of the test compounds). After co‐incubation, a glucose uptake assay was conducted. Cells were cultured with a fluorescently labeled glucose analog, and the fluorescence intensity was measured using a microplate reader. The relative glucose uptake for each group was quantified based on these measurements, with higher fluorescence indicating greater glucose uptake [36]:

$$\begin{array}{ccc}\hfill \mathrm{Concentration}\mathrm{of}\mathrm{glucose}& =& \mathrm{standard}\mathrm{concentration}\mathrm{of}\mathrm{glucose}\hfill \\ & & \times \frac{\mathrm{O}{\mathrm{D}}_{\mathrm{sample}}-\mathrm{O}{\mathrm{D}}_{\mathrm{blank}}}{\mathrm{O}{\mathrm{D}}_{\mathrm{standard}}-\mathrm{O}{\mathrm{D}}_{\mathrm{blank}}}\hfill \end{array}$$

$$\begin{array}{ccc}& & \mathrm{Glucose}\mathrm{consumption}=\mathrm{concentration}\mathrm{of}\mathrm{glucose}\mathrm{in}\mathrm{medium}\hfill \\ & & -\mathrm{concentration}\mathrm{of}\mathrm{glucose}\mathrm{in}\mathrm{suspension}\hfill \end{array}$$

$$\begin{array}{ccc}& & \mathrm{Glucose}\mathrm{consumption}\mathrm{increase}\mathrm{rate}\left(\%\right)\hfill \\ & & =[(\mathrm{glucose}\mathrm{consumption}\mathrm{in}\mathrm{model}\mathrm{group}\hfill \\ & & -\mathrm{glucose}\mathrm{consumption}\mathrm{in}\mathrm{sample}\mathrm{group})/\hfill \\ & & \mathrm{concentration}\mathrm{of}\mathrm{glucose}\mathrm{in}\mathrm{medium}]\times 100\%\hfill \end{array}$$

Author Contributions

Jiajia Mou, Lili Wang, and Yanru Deng designed the experiments. Xiangxiang Zhao and Jiali Zuo performed the experiments. Guizhi Xiao and Yanru Deng analyzed the data. Jiajia Mou and Lili Wang wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71501‐sup‐0001‐SuppMat.docx

Supporting File 1: cbdv71501‐sup‐0002‐SuppMat.docx

Supporting File 3: cbdv71501‐sup‐0003‐SuppMat.docx

Supporting File 4: cbdv71501‐sup‐0004‐SuppMat.docx

Data Availability Statement

The data that support the findings of this project are available from the corresponding author upon reasonable request.