A betulinic acid derivative, its preparation and use

Abstract

This invention discloses a betulinic acid derivative, its preparation method, and its uses. Using betulinic acid, a natural lupin-type pentacyclic triterpenoid, as the parent compound, a series of betulinic acid derivatives based on C-2 and C-3 position structural modifications were synthesized sequentially via oxidation and Claisen condensation reactions. These betulinic acid derivatives exhibit excellent α-glucosidase inhibitory activity. Their chemical structures differ significantly from existing clinical drugs, and their parent compounds are derived from natural products. This invention holds promise for overcoming the gastrointestinal adverse reactions and other drawbacks of traditional α-glucosidase inhibitors, providing new drug candidates for diabetes treatment. Furthermore, the raw materials for this invention are relatively widely available, facilitating subsequent scale-up production and process optimization, and possessing the potential for further development into clinical drug candidates.

Description

Technical Field

[0001] This invention relates to organic synthesis, specifically to a betulinic acid derivative, its preparation method, and its uses, belonging to the field of organic synthesis technology. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia caused by multiple etiologies. The most common pathophysiological causes of diabetes are defects in insulin secretion, defects in insulin action (insulin resistance), or a combination of both. Insulin is the only blood glucose-lowering hormone secreted by pancreatic β-cells, and its core function is to promote anabolism and maintain blood glucose homeostasis.

[0003] Based on etiology and pathogenesis, diabetes is mainly divided into four categories: type 1 diabetes, type 2 diabetes, gestational diabetes, and special types of diabetes. Type 1 diabetes accounts for about 5-10% of cases, and its core pathological feature is an absolute deficiency of insulin caused by autoimmune destruction of pancreatic β cells. It is more common in children and adolescents. [1] Type 2 diabetes accounts for 90-95% of all diabetes cases and is the most common type of diabetes. Its pathogenesis is primarily insulin resistance, accompanied by a progressive decline in pancreatic β-cell function, leading to a relative insufficiency of insulin secretion. In recent years, the global prevalence of type 2 diabetes has continued to rise, becoming a major public health problem. ] Gestational diabetes mellitus (GDM) typically occurs in the second and third trimesters, with a global prevalence of approximately 16.7%. The cumulative incidence of developing type 2 diabetes within 10 years postpartum is approximately 12.08%, with a long-term risk about 7 times higher than in women without gestational diabetes. Special types of diabetes include drug-induced diabetes, diabetes caused by pancreatic exocrine disorders, and monogenic diabetes. Globally, there are 9.5 million people with type 1 diabetes, with 513,000 new cases and approximately 174,000 premature deaths projected in 2025, of which about 17.2% are due to delayed diagnosis after clinical onset. These epidemiological data reveal the severe disease burden of diabetes, the root cause of which lies in the multi-system complications resulting from prolonged hyperglycemia. The harm of diabetes primarily stems from systemic metabolic disorders caused by insulin deficiency or impaired insulin action. Its complications can affect multiple areas, including the eyes, kidneys, nerves, cardiovascular system, feet, mouth, and cognitive function, making it a leading cause of disability and death.

[0004] The treatment of diabetes follows the principle of "comprehensive management," encompassing lifestyle interventions, drug therapy, and prevention and treatment of complications. Treatment plans must be individualized based on the type of diabetes and individual patient differences. Drug therapy, according to its mechanism of action, is mainly divided into the following categories: drugs that promote insulin secretion (sulfonylureas, meglitinides, DPP-4 inhibitors), drugs that improve insulin sensitivity (metformin, thiazolidinediones), drugs that delay carbohydrate absorption (alpha-glucosidase inhibitors), drugs that increase urinary glucose excretion (SGLT-2 inhibitors), and drugs based on incretins (GLP-1 receptor agonists). Alpha-glucosidase is one of the important targets for diabetes treatment. This enzyme is located at the brush border of the small intestine and is responsible for breaking down complex carbohydrates into monosaccharides. Alpha-glucosidase inhibitors competitively inhibit the activity of this enzyme, delaying carbohydrate absorption and lowering postprandial blood glucose. Currently, widely used alpha-glucosidase inhibitors in clinical practice include acarbose, voglibose, and miglitol. These drugs reduce postprandial blood glucose spikes by competitively binding to the active site of α-glucosidase, thus slowing down the digestion and absorption of carbohydrates. However, because unabsorbed carbohydrates are fermented by bacteria in the intestines to produce gas, these drugs often cause gastrointestinal adverse reactions such as bloating, diarrhea, and flatulence. Approximately 20-30% of patients discontinue use due to intolerance. In addition, issues such as significant individual differences in efficacy and the impact of three-times-daily dosing on long-term adherence urgently need to be addressed.

[0005] In summary, although existing α-glucosidase inhibitors play an important role in the treatment of diabetes, their clinical application is limited by unresolved issues such as gastrointestinal adverse reactions and individual differences in efficacy. Developing novel α-glucosidase inhibitors, especially selective inhibitors derived from natural products or compounds that act on new binding sites, has significant clinical value and application prospects for enriching diabetes treatment options and improving patients' quality of life. Summary of the Invention

[0006] This invention provides a betulinic acid derivative, its preparation method, and its uses. Using betulinic acid, a natural lupin-type pentacyclic triterpenoid, as the parent compound, a series of betulinic acid derivatives based on C-2 and C-3 position structural modifications were synthesized sequentially via oxidation and Claisen condensation reactions. These betulinic acid derivatives exhibit excellent α-glucosidase inhibitory activity. Their chemical structures differ significantly from existing clinical drugs, and their parent compounds are derived from natural products. They are expected to overcome the gastrointestinal adverse reactions and other drawbacks of traditional α-glucosidase inhibitors, providing new candidate drugs for diabetes treatment.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] According to a first embodiment of the present invention, a betulinic acid derivative is provided:

[0009] A betulinic acid derivative, the general structural formula of which is shown below:

[0010] (I).

[0011] In the general structural formula (I), R is one of styryl, fluorostyryl, chlorostyryl, bromostyryl, nitrostyryl, alkoxystyryl, thiophene, furanyl, and pyridinyl.

[0012] Preferably, betulinic acid derivatives having the general structural formula (I) are selected from one or more of the following compounds:

[0013]

[0014] According to a second embodiment of the present invention, a method for preparing betulinic acid derivatives is provided:

[0015] A method for preparing betulinic acid derivatives or preparing betulinic acid derivatives as described in the first embodiment, the method comprising the following steps:

[0016] S1. Betulinic acid is oxidized in the presence of an oxidizing agent to obtain betulinic acid:

[0017] .

[0018] S2. Betulinic acid is reacted with an aldehyde having general structural formula (II) in the presence of a base catalyst to obtain a betulinic acid derivative having general structural formula (I):

[0019] .

[0020] Wherein: R is one of styryl, fluorostyryl, chlorostyryl, bromostyryl, nitrostyryl, alkoxystyryl, thiophene, furanyl, and pyridinyl.

[0021] Preferably, R is one of styrene, p-fluorostyrene, p-chlorostyrene, p-bromostyrene, p-nitrostyrene, p-methoxystyrene, thiophene, furanyl, and pyridinyl, with R preferably being one of styrene, p-fluorostyrene, and p-methoxystyrene.

[0022] Preferably, in step S1, the oxidant is 2-iodobenzoic acid (IBX). Preferably, the amount of oxidant used is 2 to 5 times the molar amount of betulinic acid.

[0023] Preferably, in step S2, the alkaline catalyst is NaH. Preferably, the amount of alkaline catalyst used is 0.5 to 2 times the molar amount of betulinic acid.

[0024] Preferably, in step S2, the molar ratio of betulinic acid to an aldehyde having the general structural formula (II) is 1:1 to 3, more preferably 1:1.2 to 2.

[0025] Preferably, step S1 specifically involves: dissolving betulinic acid in a solvent (e.g., THF) to obtain a betulinic acid solution, and dissolving the oxidant in a solvent (e.g., DMSO) to obtain an oxidant solution. Then, the betulinic acid solution is slowly added dropwise (e.g., 1-3 drops / s) to the oxidant solution while stirring. The reaction temperature is 15-30°C, and the reaction time is 3-24 hours. After the reaction is complete, the solvent is removed by rotary evaporation (mainly to remove THF), and the concentrated solution is slowly introduced into a saturated Na₂S₂O₃ aqueous solution and ice water, and stirred for 20-30 minutes (IBX and its reduction product (2-iodobenzoic acid) have extremely low solubility in water and are thus removed). Solid-liquid separation is performed (e.g., vacuum filtration), and the filtrate is extracted 2-3 times with ethyl acetate. The organic phases are combined and washed 1-3 times each with Na₂S₂O₃ solution and saturated brine. The organic phase was dried with anhydrous sodium sulfate, and then concentrated (e.g., vacuum concentration) and purified by column chromatography (e.g., by silica gel column chromatography with a mesh size of 60-120) to obtain betulinic acid (i.e., intermediate BA=O).

[0026] Preferably, step S2 is as follows: under a protective atmosphere (e.g., nitrogen and / or inert gas), betulinic acid is first dissolved in a solvent (e.g., THF), and then a base catalyst is added in multiple portions (e.g., 2-10 times) at 0°C. After adding the base catalyst, the reaction proceeds for 15-30 minutes, followed by the addition of an aldehyde with general structural formula (II) and stirring. The reaction temperature is 20-30°C, and the reaction time is 1-2 hours (preferably monitored by TLC to determine the reaction endpoint). After the reaction is complete, the reaction mixture is diluted with twice the volume of cold water, and then saturated sodium bisulfite is added to treat any unreacted aldehyde. The mixture is then extracted multiple times (e.g., 2-3 times) with ethyl acetate. The combined organic phases are dried with anhydrous sodium sulfate, and finally, the betulinic acid derivative with general structural formula (I) is obtained by concentration (e.g., cyclone vapor concentration) and purification (e.g., purification by 60-120 mesh silica gel column chromatography).

[0027] According to a third embodiment of the present invention, a use of a betulinic acid derivative is provided:

[0028] Use of a betulinic acid derivative, or a betulinic acid derivative prepared by the method described in the first embodiment, or a betulinic acid derivative prepared by the method described in the second embodiment, for the preparation of an α-glucosidase inhibitor.

[0029] In this invention, betulinic acid, a natural lupinane-type pentacyclic triterpenoid compound, possesses multi-target anti-diabetic potential. Studies have shown that betulinic acid exerts its hypoglycemic effect through multiple pathways: on the one hand, it can directly inhibit the activity of α-glucosidase and α-amylase, delaying carbohydrate digestion and absorption; on the other hand, it can improve insulin resistance through mechanisms such as activating the AMPK signaling pathway, upregulating PGC-1α expression, and promoting GLUT4 translocation, while also stimulating ATP-sensitive potassium channels (K-ATP) to promote insulin secretion. This multi-target characteristic makes the betulinic acid skeleton an ideal template for anti-diabetic drug development. However, there is still considerable room for improvement in the inhibitory activity of the betulinic acid matrix, and its poor water solubility and low bioavailability limit its direct application. Research has revealed that the modification of the C-3 hydroxyl group in betulinic acid is a key site affecting the inhibitory activity of α-glucosidase. Introducing different structural fragments can significantly enhance the binding affinity between the compound and the enzyme's active site. This invention first oxidizes the C-3 hydroxyl group to a ketone carbonyl group (O=C-), and then introduces a benzene ring or heterocyclic methylene structure (R2-CH=C-) at the C-2 position via Claisen condensation to construct an α,β-unsaturated ketone system, thereby enhancing the interaction with key amino acid residues within the α-glucosidase active site. Specifically, the benzene ring or heterocycle in the introduced aromatic methylene structure can form a stable bond with aromatic amino acid residues (such as tryptophan and tyrosine) in the enzyme's active site through π-π stacking and hydrophobic interactions. Simultaneously, the carbonyl oxygen atom in the α,β-unsaturated ketone structure can act as a hydrogen bond acceptor, forming hydrogen bonds with the hydroxyl groups of serine or threonine residues at the enzyme's active site, further enhancing the binding affinity. Furthermore, the introduction of different substituents (such as halogen atoms like fluorine, chlorine, and bromine, and methoxy and nitro groups) can regulate the electron cloud density and molecular conformation of compounds through electronic effects (electron withdrawal or donation) and steric effects, thereby optimizing their binding energy and selectivity with enzymes and enhancing inhibitory activity. This structural synergistic modification strategy based on the C-2 and C-3 positions not only significantly improves the inhibitory activity against α-glucosidase, but also, because the parent structure is derived from natural products, its biocompatibility and safety are expected to be improved, providing a possible structural basis for reducing the gastrointestinal adverse reactions of traditional α-glucosidase inhibitors.

[0030] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0031] 1. Highly efficient α-glucosidase inhibitory activity: The betulinic acid derivative provided by this invention exhibits excellent inhibitory activity against α-glucosidase, with a half-maximal inhibitory concentration (IC50) of [missing value]. 50The levels of glucose and ketones are significantly lower than or close to those of the existing clinical drug acarbose. By introducing different aromatic methylene or heterocyclic methylene structures at the C-2 position and forming a ketone carbonyl group at the C-3 position, an α,β-unsaturated ketone system was constructed. This system can tightly bind to key amino acid residues (such as tryptophan, tyrosine, and serine) in the active site of α-glucosidase through various interactions such as π-π stacking, hydrophobic interactions, and hydrogen bonds, thereby competitively inhibiting enzyme activity, effectively delaying carbohydrate breakdown and absorption, and reducing postprandial blood glucose levels.

[0032] 2. Reduced Potential Gastrointestinal Adverse Reactions: Since the derivative of this invention is based on the natural product betulinic acid, it possesses inherent biocompatibility. Compared to traditional α-glucosidase inhibitors (such as acarbose), the derivative of this invention has an optimized structure, potentially exhibiting better intestinal mucosal permeability or less impact on the gut microbiota. This could reduce gastrointestinal adverse reactions such as bloating, diarrhea, and flatulence caused by the fermentation of unabsorbed carbohydrates in the intestine. This advantage stems from its unique chemical structure and enzyme binding mode, as well as the potentially better tolerability afforded by its natural product parent.

[0033] 3. Structural Novelty and Diversity: Using betulinic acid as the parent compound, this invention successfully introduced various substituents with different structures, such as styryl (and its para-substituted derivatives such as fluorine, chlorine, bromine, nitro, and methoxy), thiophene, furanyl, and pyridyl, through C-3 oxidation and C-2 Claisen condensation reactions, synthesizing a series of novel betulinic acid derivatives. This structural diversity not only provides a rich compound library for screening highly active and selective α-glucosidase inhibitors, but also lays the foundation for in-depth research on structure-activity relationships and elucidating their inhibitory mechanisms.

[0034] 4. Good drug development potential: The preparation method of this invention is relatively simple. Using betulinic acid as the starting material, the target derivative can be obtained through two key reactions. The source of the raw material is relatively wide (betulinic acid exists in a variety of plants), which is conducive to subsequent scale-up production and process optimization. At the same time, preliminary physicochemical studies on the derivatives show that some compounds have certain improvements in water solubility and bioavailability compared to the parent betulinic acid, and have the potential to be further developed into clinical drug candidates.

[0035] 5. Possibility of multi-target synergistic effect: While retaining some structural features of the parent compound, the derivative of this invention enhances the inhibitory effect on α-glucosidase through structural modification. Therefore, it is expected to inherit and synergize with other pharmacological activities of the parent compound (such as improving insulin resistance and promoting insulin secretion) to achieve multi-target comprehensive treatment of diabetes, thereby improving the therapeutic effect and reducing the drug resistance problem that may be caused by single-target drugs. Attached Figure Description

[0036] Figure 1 This is a synthetic circuit diagram of betulinic acid derivatives having the general structural formula (I) of the present invention.

[0037] Figure 2 This is a standard curve diagram of p-nitrophenol according to the present invention.

[0038] Figure 3 The photon spectrum of betulinic acid derivative I-1 obtained in Example 1 of this invention is shown.

[0039] Figure 4 This is the carbon spectrum of betulinic acid derivative I-1 obtained in Example 1 of the present invention.

[0040] Figure 5 The hydrogen spectrum of betulinic acid derivative I-2 obtained in Example 2 of this invention.

[0041] Figure 6 This is the carbon spectrum of betulinic acid derivative I-2 obtained in Example 2 of the present invention.

[0042] Figure 7 This is the fluorine spectrum of betulinic acid derivative I-2 obtained in Example 2 of the present invention.

[0043] Figure 8 The hydrogen spectrum of betulinic acid derivative I-3 obtained in Example 3 of this invention.

[0044] Figure 9 This is the carbon spectrum of betulinic acid derivative I-3 obtained in Example 3 of the present invention.

[0045] Figure 10 The photoluminescence spectrum of betulinic acid derivative I-4 obtained in Example 4 of this invention is shown.

[0046] Figure 11 This is the carbon spectrum of betulinic acid derivative I-4 obtained in Example 4 of the present invention.

[0047] Figure 12 The photoluminescence spectrum of betulinic acid derivative I-5 obtained in Example 5 of this invention is shown.

[0048] Figure 13 This is the carbon spectrum of betulinic acid derivative I-5 obtained in Example 5 of the present invention.

[0049] Figure 14 The photoluminescence spectrum of betulinic acid derivative I-6 obtained in Example 6 of this invention is shown.

[0050] Figure 15 This is the carbon spectrum of betulinic acid derivative I-6 obtained in Example 6 of the present invention.

[0051] Figure 16The photoluminescence spectrum of betulinic acid derivative I-7 obtained in Example 7 of this invention is shown.

[0052] Figure 17 This is the carbon spectrum of betulinic acid derivative I-7 obtained in Example 7 of the present invention.

[0053] Figure 18 The hydrogen spectrum of betulinic acid derivative I-8 obtained in Example 8 of this invention.

[0054] Figure 19 This is the carbon spectrum of betulinic acid derivative I-8 obtained in Example 8 of the present invention.

[0055] Figure 20 The hydrogen spectrum of betulinic acid derivative I-9 obtained in Example 9 of this invention.

[0056] Figure 21 This is the carbon spectrum of betulinic acid derivative I-9 obtained in Example 9 of the present invention. Detailed Implementation

[0057] The technical solution of the present invention will be illustrated below with examples. The scope of protection sought by the present invention includes, but is not limited to, the following embodiments.

[0058] Example 1

[0059] Preparation of intermediate BA=O: (BA=O).

[0060] Betulinic acid (5 mmol) was dissolved in THF (40 mL) to obtain a betulinic acid solution. IBX (15 mmol) was dissolved in DMSO (30 mL) to form an IBX solution. The betulinic acid solution was slowly added dropwise (1 drop / s) to the IBX solution at 23 °C with stirring for 12 h. After the reaction was completed, the temperature was raised to 35 °C and rotary evaporation was used to remove most of the THF, retaining the DMSO phase. The concentrate was then slowly introduced into a saturated Na2S2O3 aqueous solution and ice water and stirred for 25 minutes to precipitate IBX and its reduction product (2-iodobenzoic acid), which was removed by filtration. The resulting filtrate was extracted three times with ethyl acetate, and the organic phases were combined and washed once each with Na2S2O3 solution (50 mL) and saturated brine (50 mL). The organic phase was then dried with anhydrous sodium sulfate, and finally purified by vacuum concentration and 100-mesh silica gel column chromatography to obtain a white solid intermediate BA=O (2.05 g, yield approximately 90%).

[0061]

[0062] Preparation of target product I-1: (I-1).

[0063] Intermediate BA=O (1 mmol) was added to a round-bottom flask. Under an argon atmosphere, THF (5 mL) was added to dissolve the starting material. Then, NaH (1.2 mmol) was added in three portions at 0 °C. After reacting for 20 minutes, trans-cinnamaldehyde (1.5 mmol) was added, and the reaction was stirred at room temperature until complete (TLC monitoring). After the reaction was complete, the reaction mixture was diluted with twice the volume of cold water. Unreacted aldehydes were treated with saturated sodium bisulfite, followed by extraction with ethyl acetate three times. The combined organic phases were dried over anhydrous sodium sulfate, concentrated by cyclone vaporization, and purified by 100-mesh silica gel column chromatography to obtain the white solid target product I-1 (yield approximately 83%, mp 236.2~236.6 °C).

[0064]

[0065] Example 2

[0066] Repeat Example 1, except that trans-cinnamaldehyde was replaced with p-fluorinated trans-cinnamaldehyde, to obtain the target product I-2 as a white solid (yield approximately 86%, mp 249.0~249.1℃): (I-2).

[0067]

[0068] Example 3

[0069] Repeat Example 1, except that trans-cinnamaldehyde was replaced with p-chlorotrans-cinnamaldehyde, to obtain the target product I-3 as a white solid (yield approximately 76%, mp 253.0~253.2℃): (I-3).

[0070]

[0071] Example 4

[0072] Repeat Example 1, except that trans-cinnamaldehyde was replaced with p-bromotrans-cinnamaldehyde, to obtain the target product I-4 as a white solid (yield approximately 75%, mp 248.2~248.7℃): (I-4).

[0073]

[0074] Example 5

[0075] Repeat Example 1, except that trans-cinnamaldehyde was replaced with p-nitrotrans-cinnamaldehyde, to obtain the target product I-5 as a yellow solid (yield approximately 79%, mp 294.1~294.3℃): (I-5).

[0076]

[0077] Example 6

[0078] Repeat Example 1, except that trans-cinnamaldehyde was replaced with p-methoxytrans-cinnamaldehyde, to obtain the target product I-6 as a yellow solid (yield approximately 82%, mp 230.6~230.9℃): (I-6).

[0079]

[0080] Example 7

[0081] Repeat Example 1, except that trans-cinnamaldehyde was replaced with 2-thiophenecaraldehyde, to obtain the target product I-7, a white solid (yield approximately 82%, mp 3 13.2~313.9℃): (I-7).

[0082]

[0083] Example 8

[0084] Repeat Example 1, except that trans-cinnamaldehyde was replaced with 2-furanaldehyde (i.e., furfural), to obtain the target product I-8 as a white solid (yield approximately 84%, mp 232.2~232.8℃): (I-8).

[0085]

[0086] Example 9

[0087] Repeat Example 1, except that trans-cinnamaldehyde was replaced with 3-pyridinecarboxaldehyde, to obtain the target product I-9 as a white solid (yield approximately 81%, mp 214.2~214.6℃): (I-9).

[0088]

[0089] Activity test

[0090] (1) Experimental Method: p-nitrophenyl-α-D-glucopyranoside (pNPG) was used as the substrate for the enzyme reaction to determine the inhibitory activity of the target compounds (I-1 to I-9) against α-glucosidase. The detection principle of this method is as follows: under constant temperature conditions of 37 ℃, α-glucosidase can catalyze the decomposition of pNPG to generate glucose and p-nitrophenol (pNP). The latter is pale yellow in the reaction system and has a characteristic absorption peak at a wavelength of 405 nm. By monitoring the change in absorbance at this wavelength, the inhibitory ability of the sample against α-glucosidase can be quantitatively reflected. The reaction principle of the pNPG method is as follows:

[0091] .

[0092] (2) Reagent preparation:

[0093] Preparation of 0.01 mmol / L phosphate buffer solution (PBS, pH=6.8): Weigh 0.2 g KCl, 8 g NaCl, 3.58 g Na2HPO4·12H2O and 0.27 g KH2PO4, dissolve in distilled water to obtain a phosphate buffer solution with pH=7.4, adjust the pH to 6.8, and make up to 1000 mL in a volumetric flask.

[0094] Preparation of 5 mmol / L pNPG solution: Weigh 0.0753 g pNPG, dissolve in 0.01 mmol / L PBS solution, and dilute to a final volume of 50 mL in a volumetric flask.

[0095] Preparation of 0.5 mmol / L p-nitrophenol (pNP) standard solution: Weigh 0.00695 g of p-nitrophenol, dissolve it in 0.01 mmol / L PBS solution, and dilute to a volumetric flask of 100 mL.

[0096] Preparation of 1 mg / mL glutathione (GSH) solution: Weigh 0.05 g of glutathione, dissolve it in 0.01 mmol / L PBS solution, and dilute to a final volume of 50 mL in a volumetric flask.

[0097] Preparation of 0.1 mol / L sodium carbonate solution: Weigh 1.0599 g Na2CO3, dissolve it in 0.01 mmol / L PBS solution, and dilute to a volumetric flask of 100 mL.

[0098] Preparation of α-glucosidase solution: Dissolve 100 U of α-glucosidase in 1 mL of pH 7 PBS buffer to prepare a 100 U / mL α-glucosidase stock solution. Then pipette 30 μL and dilute with PBS buffer to a 10 mL volumetric flask. Both the stock solution and the diluent should be stored at 2–8 °C.

[0099] Sample solution preparation: Weigh 0.02 mmol of sample, dissolve it in DMSO, and prepare sample solutions with concentration gradients of 10 mmol / L, 5 mmol / L, 2.5 mmol / L, 1.25 mmol / L, and 0.625 mmol / L (I-1 to I-9 are prepared in the same manner to obtain each sample solution).

[0100] Preparation of standard acarbose solutions: Weigh 0.02 mmol (0.0129 g) of acarbose and dissolve it in DMSO. Prepare standard acarbose solutions with concentration gradients of 10 mmol / L, 5 mmol / L, 2.5 mmol / L, 1.25 mmol / L, and 0.625 mmol / L.

[0101] (3) Enzyme activity determination:

[0102] Preparation of the p-nitrophenol standard curve: Prepare solutions of p-nitrophenol at concentrations of 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, and 50 μmol / L. Measure the absorbance at a wavelength of 405 nm. Plot a linear regression equation with the concentration of p-nitrophenol on the x-axis and the absorbance on the y-axis (e.g., p-nitrophenol concentration on the x-axis and absorbance on the y-axis). Figure 2 (As shown).

[0103] As shown in Table 1, take two 10 mL centrifuge tubes and add the corresponding solutions in the order listed in the table, then mix well. Place the centrifuge tubes in a 37℃ constant temperature and humidity incubator for 20 minutes. After that, transfer 1 mL of the mixture from one centrifuge tube to a 5 mL centrifuge tube, then add 4 mL of Na2CO3 solution to each tube and mix well. Use a 100 μL pipette to transfer 100 μL of the mixture to each well of a 96-well plate, and measure the absorbance at 405 nm using a microplate reader.

[0104] Table 1. Determination of α-glucosidase activity

[0105]

[0106] (4) Determination of α-glucosidase inhibitory activity

[0107] The experiment was divided into four groups: test group (A), enzyme-free group (B), sample-free group (C), and blank group (D). Twelve 5 mL centrifuge tubes were used for each sample, numbered, and the corresponding solutions were added in the order shown in Table 2 to obtain final sample concentrations of 12.5 mmol / L, 25 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L, respectively. 0.3 U / mL of α-glucosidase was added to the corresponding centrifuge tubes, mixed well, and incubated at 37°C for 10 minutes. Then, 5 mmol / L of pNPG solution was added, mixed well, and incubated for another 20 minutes. After the incubation period, 250 μL of the mixture was transferred to 5 mL centrifuge tubes, 1 mL of Na₂CO₃ solution was added, and the mixture was mixed. 100 μL of the mixture was then transferred to a 96-well plate using a 100 μL pipette, and the absorbance was measured at 405 nm using a microplate reader.

[0108] Table 2. Determination of α-glucosidase inhibitory activity

[0109]

[0110] When performing α-glucosidase inhibition activity experiments, if the test sample has its own color, it will interfere with the absorbance measurement. Therefore, an enzyme-free group should be set up as a background control for each sample. Meanwhile, the reaction time has a significant impact on the results; therefore, the incubation time must be strictly controlled, and parallel experiments should be set up to reduce errors. The α-glucosidase activity inhibition rate is calculated as follows: Inhibition rate (%) = [(A1-A2) / A1] × 100%, A1 = CD, A2 = (AD) - (BD), where A, B, C, and D represent the absorbance values ​​of the test group, enzyme-free group, sample-free group, and blank group, respectively. IC50 50 The values ​​were obtained by fitting the data using the software GraphPad Prism 8.0: a curve was plotted with the concentration of the sample to be tested as the x-axis and the inhibition rate as the y-axis. The concentration corresponding to the inhibition rate of 50% is the half-maximal inhibition concentration.

[0111] Based on the experimental data, the standard linear regression equation for p-nitrophenol was obtained (e.g., Figure 2 As shown (in the diagram): y = 2.61x + 0.0517, R 2 =0.9962. The calculated enzyme activity value of α-glucosidase is as follows: A 测 =0.123, A 空 =0.058, ∆A=A 测 -A 空=0.065. According to the standard equation for p-nitrophenol, the concentration of p-nitrophenol is approximately C = 0.00510 mmol / L, which means approximately 0.177 μg of p-nitrophenol is produced per minute. If enzyme activity is defined as the amount of p-nitrophenol produced per minute under specific conditions, i.e., 1 U = 1 μg / min, then the enzyme activity unit of 1 mL of enzyme solution is 3.55 U.

[0112] Based on the α-glucosidase activity inhibition rate, the IC50 values ​​of each compound were calculated using the software GraphPad Prism 8.0. 50 The values ​​are shown in Table 3 below:

[0113] Table 3. Inhibitory activities of target compounds I-1 to I-9 on α-glucosidase

[0114]

[0115] As shown in Table 3, the synthesized betulinic acid C-2 and C-3 enone derivatives both exhibited extremely strong α-glucosidase inhibitory activity, IC50... 50 The activity values ​​ranged from 0.03 to 21.5 μM, which was far superior to the positive control acarbose (49.26 μM). This leap in activity was mainly attributed to the α,β-unsaturated ketone skeleton introduced by aldol condensation. On the one hand, this structure enhances the rigidity and hydrophobicity of the molecule by elongating the conjugated system, which is conducive to forming a stable π-π stacking and hydrophobic interaction with the enzyme active pocket. On the other hand, the ketone carbonyl group may act as a Michael acceptor and reversibly covalently bind to the nucleophilic residues in the enzyme active center, thereby greatly improving the inhibitory efficacy.

[0116] In the trans-cinnamaldehyde series (I-1 to I-6), the structure-activity relationship can be analyzed from the perspective of the electronic effects of substituents, as follows: First, the compounds with the strongest inhibitory activity are I-6 (-OCH3) and I-1 (unsubstituted), whose IC50 values ​​are... 50 The concentrations of I-6 and Cl were 0.03 μM and 0.031 μM, respectively. The high activity of I-6 is attributed to the methoxy group's electron-donating effect, which increases the electron cloud density of the enone system, potentially enhancing hydrogen bond interactions with polar enzyme residues. Its hydrophobicity also contributes to stable positioning of the molecule within the active pocket. Secondly, halogen-substituted derivatives exhibited moderate inhibitory activity, in the order F (0.07 μM) > Br (1.17 μM) > Cl (13.84 μM). In the conjugated enone skeleton, although the F atom is small, its strong electron-withdrawing ability effectively reduces the electron cloud density of the carbonyl group, enhancing interactions with nucleophilic enzyme residues. Br has high polarizability, hence its moderate activity. Cl, with its larger atom, easily creates steric hindrance in this rigid skeleton, thus exhibiting the lowest activity. Finally, the IC50 of I-5 (-NO2) was... 50The value is 13.8 μM because although the strong electron-withdrawing effect of the nitro group can further reduce the electron cloud density of the carbonyl group, its strong polarity leads to excessive overall hydrophilicity of the molecule, which is not conducive to penetrating the hydrophobic active pocket of the enzyme.

[0117] In the aromatic heterocyclic series (I-7 to I-9), the inhibitory activities of different aromatic heterocyclic substituents vary significantly, and are discussed in stratification according to activity strength as follows: First, the strongest inhibitory activity is that of 3-pyridyl-substituted I-9, with an IC50 of [missing value]. 50 At 1.63 μM, the nitrogen atom on the pyridine ring can act as a hydrogen bond acceptor, forming additional interactions with the enzyme's polar residues; secondly, the 2-thienyl-substituted I-7 (IC) 50 =5.76 μM) has moderate activity, and the thiophene ring also has certain aromaticity and polarizability; finally, the 2-furanyl-substituted I-8 (IC 50 The weakest activity (at 21.5 μM) is due to the weak aromaticity of the furan ring and the hydrophilicity of the oxygen atom, which hinders its fit with the hydrophobic pocket, leading to a decrease in binding energy. Overall, the α,β-unsaturated ketone skeleton is key to achieving a significant leap in activity. Electron-donating groups, moderately electron-withdrawing fluorine atoms, and naphthalene rings with large hydrophobic surface areas are advantageous directions for structural optimization of this type of inhibitor.

[0118] In summary, all synthesized betulinic acid derivatives inhibited α-glucosidase activity, and their inhibitory effects were stronger than those of acarbose. Among them, I-6 showed the best inhibitory effect on α-glucosidase activity at an IC50 value of [missing value]. 50 With a concentration of 0.03 µM, which is 1642 times that of acarbose, this compound is expected to become a lead compound for α-glucosidase inhibitors.

[0119] The main reagents and instruments used in this invention are as follows:

[0120]

[0121]

[0122] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the appended claims. For those skilled in the art, any modifications, equivalent substitutions, improvements, etc., made to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention. For example, in the preparation of betulinic acid derivatives, the aldehyde compound used can be replaced according to actual needs, as long as it can undergo an aldol condensation reaction with the intermediate to introduce an α,β-unsaturated ketone structure; reaction conditions such as temperature, time, and solvent type can also be adjusted within a reasonable range to optimize the reaction yield and product purity. At the same time, the application of betulinic acid derivatives disclosed in this invention in α-glucosidase inhibition also provides an important research foundation and potential candidate compounds for the development of novel antidiabetic drugs. Further in-depth exploration of in vivo activity evaluation, toxicity studies, and drug dosage forms can be carried out subsequently.

Claims

1. A betulinic acid derivative, characterized in that: The general structural formula of the betulinic acid derivative is shown below: （I）； In the general structural formula (I), R is one of styryl, fluorostyryl, chlorostyryl, bromostyryl, nitrostyryl, alkoxystyryl, thiophene, furanyl, and pyridinyl.

2. The betulinic acid derivative according to claim 1, characterized in that: Betulinic acid derivatives having the general structural formula (I) are selected from one or more of the following compounds:

3. A method for preparing betulinic acid derivatives or for preparing betulinic acid derivatives as described in claim 1 or 2, characterized in that: The method includes the following steps: S1. Betulinic acid is oxidized in the presence of an oxidizing agent to obtain betulinic acid: ； S2. Betulinic acid is reacted with an aldehyde having general structural formula (II) in the presence of a base catalyst to obtain a betulinic acid derivative having general structural formula (I): ； Wherein: R is one of styryl, fluorostyryl, chlorostyryl, bromostyryl, nitrostyryl, alkoxystyryl, thiophene, furanyl, and pyridinyl.

4. The method according to claim 3, characterized in that: R is one of styrene, p-fluorostyrene, p-chlorostyrene, p-bromostyrene, p-nitrostyrene, p-methoxystyrene, thiophene, furanyl, and pyridinyl, preferably one of styrene, p-fluorostyrene, and p-methoxystyrene.

5. The method according to claim 3 or 4, characterized in that: In step S1, the oxidant is 2-iodobenzoic acid; preferably, the amount of oxidant used is 2 to 5 times the molar amount of betulinic acid.

6. The method according to any one of claims 3-5, characterized in that: In step S2, the alkaline catalyst is NaH; preferably, the amount of alkaline catalyst used is 0.5 to 2 times the molar amount of betulinic acid.

7. The method according to any one of claims 3-6, characterized in that: In step S2, the molar ratio of betulinic acid to an aldehyde having the general structural formula (II) is 1:1 to 3, preferably 1:1.2 to 2.

8. The method according to any one of claims 3-7, characterized in that: Step S1 is as follows: Betulinic acid is dissolved in a solvent (e.g., THF) to obtain a betulinic acid solution, and an oxidant is dissolved in a solvent (e.g., DMSO) to obtain an oxidant solution; then the betulinic acid solution is slowly added dropwise (e.g., 1-3 drops / s) to the oxidant solution and stirred for reaction at a temperature of 15-30°C for 3-24 hours; after the reaction is completed, the solvent is removed by rotary evaporation, and the concentrated solution is slowly introduced into a saturated Na2S2O3 aqueous solution and ice water and stirred for 20-30 minutes for solid-liquid separation (e.g., vacuum filtration). The filtrate is extracted with ethyl acetate 2-3 times, and the organic phases are combined and washed 1-3 times each with Na2S2O3 solution and saturated brine; the organic phase is dried with anhydrous sodium sulfate, and finally, betulinic acid is obtained by concentration (e.g., vacuum concentration) and column chromatography purification.

9. The method according to any one of claims 3-8, characterized in that: Step S2 is as follows: under a protective atmosphere (e.g., nitrogen and / or inert gas), betulinic acid is first dissolved in a solvent (e.g., THF), and then a base catalyst is added in three portions at 0°C. After the base catalyst is added, the reaction continues for 15-30 minutes, and then an aldehyde with general structural formula (II) is added and stirred. The reaction temperature is 20-30°C and the reaction time is 1-2 hours (preferably using TLC to monitor the reaction endpoint). After the reaction is completed, the reaction mixture is first diluted with cold water, and then saturated sodium bisulfite is added to treat the unreacted aldehyde. Then, ethyl acetate is used for multiple extractions (e.g., 2-3 times). After the organic phases are combined, they are dried with anhydrous sodium sulfate. Finally, after concentration (e.g., cyclone vapor concentration) and purification (e.g., purification by 60-120 mesh silica gel column chromatography), betulinic acid derivatives with general structural formula (I) are obtained.

10. Use of a betulinic acid derivative, or the betulinic acid derivative as described in claim 1 or 2, or the betulinic acid derivative prepared by the method according to any one of claims 3-9, characterized in that: The betulinic acid derivative having the general structural formula (I) was used to prepare an α-glucosidase inhibitor.

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