A ursolic acid derivative, and a synthetic method and application thereof

Abstract

This invention discloses an ursolic acid derivative, its synthesis method, and its applications. This invention modifies ursolic acid to obtain three ursolic acid derivatives. Derivative B1 exhibits the best antibacterial activity, inhibiting the growth of both Staphylococcus aureus and Escherichia coli. Derivative B10 shows antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis; derivative B14 exhibits antibacterial activity against Staphylococcus aureus and Escherichia coli. Compared with the parent ursolic acid backbone, these three derivatives (B1, B10, and B14) show significantly enhanced antibacterial efficacy. Experimental results show that derivative B1 inhibits the formation of biofilms in Staphylococcus aureus and Escherichia coli.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to an ursolic acid derivative, its synthesis method and application, and more specifically, to a C-28 substituted ursolic acid derivative, its synthesis method and its application in the antibacterial field. Background Technology

[0002] Ursolic acid (UA), also known as ursolic acid, is a natural pentacyclic triterpenoid compound widely distributed in nature. Ursolic acid derivatives possess various biological activities, including antitumor, anti-inflammatory, antibacterial, antiviral, antioxidant, and liver-damaging effects. Current research indicates that chemical modification studies of ursolic acid derivatives primarily focus on three key structural sites: the hydroxyl group at C-3, the unsaturated double bond at C12-C13, and the carboxylic acid group at C-28. Vishwaka et al. modified the structure of ursolic acid at two sites, C-3 and C-28, and successfully synthesized eight ursolic acid aryl ester derivatives. The minimum inhibitory concentration (MIC) of the derivatives was determined by the agar dilution method against attenuated Mycobacterium tuberculosis strains. The results showed that compound UA-1H had the strongest activity, with a MIC of 12.5 μg / mL, which was far superior to the original ursolic acid (Vishwakarma S, Srivastava SK, Khare NK, et al. Synthesis and Structural Activity Relationship Study of Ursolic Acid Derivatives as Antitubercular Agent [J]. Med. Chem, 2024, 20(6): 630-645.). Dwivedi et al. synthesized a series of derivatives by modifying the C-3 site of ursolic acid. When derivatives UA-4 and UA-5 were used in combination with antibiotics as resistance reversal agents, they exhibited a significant synergistic effect (Dwivedi GR, Maurya A, Yadav DK, et al. Drug Resistance Reversal Potential of Ursolic Acid Derivatives against Nalidixic Acid‐and Multidrug‐Resistant Escherichia Coli [J]. Chem. Biol. Drug Des,2015, 86(3): 272-283.). However, ursolic acids suffer from low water solubility, which not only reduces their bioavailability but also increases the difficulty of formulation development. Therefore, further exploration of new strategies for structural modification of ursolic acids is needed to provide new ideas for the structural modification of novel ursolic acid antibacterial drugs. Summary of the Invention

[0003] To address the shortcomings of the existing technology, this invention provides an ursolic acid derivative, its synthesis method, and its applications. The ursolic acid derivative of this invention possesses potent antibacterial activity.

[0004] The specific technical solution is as follows:

[0005] One object of the present invention is to provide an ursolic acid derivative or a pharmaceutically acceptable salt thereof, said ursolic acid derivative having the structure shown in formula (I) or formula (II):

[0006] Equation (Ⅰ) is: ;

[0007] Where R is (Derivative B1) or (Derivative B10);

[0008] Equation (II) is: ;

[0009] Where R1 is (Derivative B14).

[0010] Experiments have confirmed that these three derivatives (B1, B10, and B14) exhibit significantly enhanced antibacterial efficacy compared to the parent UA skeleton. Derivative B1 showed the best antibacterial activity, inhibiting the growth of both Staphylococcus aureus and Escherichia coli, with a MIC of 37.5 μg / mL against Staphylococcus aureus. Derivative B10 showed antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis; derivative B14 showed antibacterial activity against Staphylococcus aureus and Escherichia coli.

[0011] A second objective of this invention is to provide a method for synthesizing the above-mentioned ursolic acid derivative, comprising the following steps:

[0012] S1. Obtain intermediates or ;

[0013] S2. React ursolic acid with the intermediate obtained in step S1 to obtain ursolic acid derivative;

[0014] Furthermore, in step S1: Alternatively, R1-NH2 reacts with chloroacetyl chloride to yield an intermediate.

[0015] Furthermore, in step S1: triethylamine is added to the reaction.

[0016] Furthermore, in step S1, the reaction temperature is preferably 0℃ to -20℃, more preferably -2℃ to -10℃.

[0017] Specifically, in step S1: ... Alternatively, R1-NH2 can be dissolved in a solvent, triethylamine can be added, and after mixing thoroughly, chloroacetyl chloride can be added dropwise; after the addition is complete, the reaction can proceed for 3-5 hours.

[0018] In step S1: The molar ratio of R1-NH2 to chloroacetyl chloride is preferably 1:(1~3), more preferably 1:(1~1.5).

[0019] In step S1: The molar ratio of R1-NH2 to triethylamine is preferably 1:(1~4), more preferably 1:(1.2~2).

[0020] In step S1, the solvent used is preferably dichloromethane.

[0021] Furthermore, in step S2: potassium carbonate is added to the reaction.

[0022] Furthermore, in step S2, the preferred reaction temperature is 60~90℃.

[0023] Specifically, in step S2: ursolic acid is dissolved in a solvent, potassium carbonate is added, and the reaction is carried out at 60~90℃ for 20~50 min. Then the intermediate obtained in step S1 is added, and the reaction is carried out at the same temperature for 8~12 h.

[0024] In step S2, the molar ratio of ursolic acid to the intermediate is preferably 1:(1~3), more preferably 1:(1~1.5).

[0025] In step S2, the molar ratio of ursolic acid to potassium carbonate is preferably 1:(1~4), more preferably 1:(1.2~2).

[0026] In step S2, the preferred solvent is acetonitrile.

[0027] A third objective of this invention is to provide the use of the above-mentioned ursolic acid derivatives or their pharmaceutically acceptable salts in non-therapeutic, non-diagnostic antibacterial and / or sterilization applications.

[0028] Specifically, the use of at least one of derivatives B1, B10, and B14 in the non-therapeutic, non-diagnostic inhibition or killing of Staphylococcus aureus is provided.

[0029] Specifically, the use of derivative B1 and / or derivative B14 in the non-therapeutic, non-diagnostic inhibition or killing of Escherichia coli (E. coli) is provided.

[0030] Specifically, the application of derivative B10 in the non-therapeutic, non-diagnostic inhibition or killing of Staphylococcus epidermidis is provided.

[0031] Furthermore, the above-mentioned ursolic acid derivatives or their pharmaceutically acceptable salts are used in the preparation of antibacterial and / or sterilizing drugs.

[0032] Specifically, at least one of derivatives B1, B10, and B14 is used in the preparation of drugs that inhibit or kill Staphylococcus aureus.

[0033] Specifically, derivative B1 and / or derivative B14 are used in the preparation of drugs that inhibit or kill Escherichia coli (E. coli).

[0034] Specifically, derivative B10 is used in the preparation of drugs that inhibit or kill Staphylococcus epidermidis.

[0035] Specifically, the drug may also include pharmaceutically acceptable excipients.

[0036] Specifically, the drug is not limited by dosage form, which may be a capsule, tablet, granule, gel, sustained-release agent, oral liquid, drop pill, emulsion, injection or nano-formulation.

[0037] Specifically, the drug can be administered orally, sublingually, intravenously, by injection, or by inhalation.

[0038] Furthermore, the application of the aforementioned ursolic acid derivatives or their pharmaceutically acceptable salts in non-therapeutic, non-diagnostic antibacterial and / or sterilization targeting biofilm-mediated processes is provided. Experiments have demonstrated that the aforementioned ursolic acid derivatives, particularly derivative B1, can persistently inhibit biofilm formation in bacterial strains.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention modifies the C-28 position of ursolic acid (UA) to obtain three ursolic acid derivatives. Compared with the parent UA skeleton, these three derivatives (B1, B10, and B14) exhibit significantly enhanced antibacterial efficacy. Derivative B1 showed the best antibacterial activity, inhibiting the growth of both Staphylococcus aureus and Escherichia coli, with a MIC value of 37.5 μg / mL against Staphylococcus aureus. Derivative B10 showed antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis; derivative B14 showed antibacterial activity against Staphylococcus aureus and Escherichia coli. Experimental results showed that derivative B1 inhibited the formation of biofilms from Staphylococcus aureus and Escherichia coli. Molecular docking results showed that derivative B1 spontaneously binds to SarA protein with a binding energy of -7.39 kcal / mol. During the interaction, three hydrogen bonds are formed (the interaction sites are LysB163 and LysA127, respectively), accompanied by π-π stacking, which together maintain the stability of the compound structure. This provides strong structural biological evidence for the antibacterial and anti-biofilm activities of ursolic acid derivatives. Attached Figure Description

[0041] Figure 1 For derivative B1 in Example 1 1 H NMR spectrum;

[0042] Figure 2 For derivative B1 in Example 1 13 CNMR spectrum;

[0043] Figure 3 For derivative B10 in Example 2 1 H NMR spectrum;

[0044] Figure 4 For derivative B10 in Example 2 13 CNMR spectrum;

[0045] Figure 5 For derivative B14 in Example 3 1 H NMR spectrum;

[0046] Figure 6 For derivative B14 in Example 3 13 CNMR spectrum;

[0047] Figure 7 Kinetic diagram of the antibacterial activity of derivative B1 in test 2 co-cultured with Staphylococcus aureus for different time periods;

[0048] Figure 8 The kinetic diagram of the antibacterial activity of derivative B1 in test 2 co-cultured with Escherichia coli for different time periods;

[0049] Figure 9 To test the time-bactericidal curves of derivative B1 and tetracycline against Staphylococcus aureus;

[0050] Figure 10 To test the time-bactericidal curves of derivative B1 and tetracycline against Escherichia coli;

[0051] Figure 11 To test the inhibitory effect of derivative B1 in group 4 on Staphylococcus aureus biofilm formation;

[0052] Figure 12 To test the inhibitory effect of derivative B1 in group 4 on Escherichia coli biofilm formation;

[0053] Figure 13 To test the interaction mechanism between derivative B1 in group 5 and SarA protein. Detailed Implementation

[0054] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] Example 1

[0056] A ursolic acid derivative was synthesized, and the ursolic acid derivative has the structure shown in the following chemical formula:

[0057] ;

[0058] Where R is .

[0059] The synthesis path is as follows:

[0060] ;

[0061] .

[0062] The specific synthesis method is as follows:

[0063] S1. Synthetic intermediate: Weigh 1.00 g The substance was added to a 250 mL three-necked flask, and 30 mL of dichloromethane was added as a reaction solvent. The reaction was carried out in a cold trap, and the temperature was lowered to -5 °C. After the substance was completely dissolved in dichloromethane, triethylamine was slowly added to the three-necked flask. After adding triethylamine, the mixture was stirred for 10 min to ensure that the triethylamine was fully mixed with the system. Chloroacetyl chloride was dissolved in dichloromethane, transferred to a constant pressure dropping funnel, and added dropwise to the flask. After the addition was complete, the reaction was continued for 4 h. During the reaction, the temperature of the reaction system was maintained at -5 °C. The molar ratio of chloroacetyl chloride to chloroacetyl chloride is 1:1.2; The molar ratio with triethylamine is 1:1.5.

[0064] The reaction process was monitored by TLC, using V 石油醚 V 乙酸乙酯 The reaction was determined by using a 1:1 ratio as the developing solvent. After the reaction was completed, saturated saline solution was added in small amounts several times using a separatory funnel to separate the organic layer and the aqueous layer. Anhydrous sodium sulfate was added to the separated organic layer and the mixture was sealed and dried to remove the water. The solid was then removed by filtration, and the solvent in the organic layer was removed by vacuum evaporation to obtain the crude product. The crude product was further purified by column chromatography to obtain the intermediate.

[0065] S2. Synthesis of ursolic acid derivative: Weigh 0.5 g of ursolic acid and add it to a 100 mL round-bottom flask. Add 40 mL of acetonitrile as the reaction solvent. Set the temperature to 80℃ and start heating and stirring. After the substance dissolves, slowly add anhydrous potassium carbonate to the round-bottom flask and react for 30 min to allow the potassium carbonate to react completely. Then add the intermediate obtained in step S1 to the reaction system, maintain the temperature at 80℃, and continue the reaction for 10 h. The molar ratio of ursolic acid to intermediate is 1:1.2; the molar ratio of ursolic acid to potassium carbonate is 1:1.5.

[0066] The reaction process was monitored by TLC, using V 石油醚 V 乙酸乙酯 The reaction was determined by using a solvent ratio of 70:1 as the developing solvent; after the reaction was completed, the solid was removed by filtration, and the acetonitrile solvent was removed by vacuum evaporation to obtain the crude product; the crude product was further purified by column chromatography to obtain the final product derivative B1.

[0067] The final product was a white solid with a yield of 62%.

[0068] The target compound was analyzed by nuclear magnetic resonance hydrogen spectroscopy (NMR 1H spectroscopy). 1 H NMR, carbon spectrum ( 13 Characterized by C NMR and mass spectrometry (MS), as follows:

[0069] 1 H NMR (400 MHz, DMSO-d6) δ 6.91 (d, J = 9.1 Hz, 2H), 6.83 (d, J = 9.1Hz, 2H), 5.15 (s, 1H), 4.77 (d, J = 14.5 Hz, 1H), 4.68 (d, J = 14.5 Hz, 1H), 4.28 (d, J = 5.1 Hz, 1H), 3.68 (s, 3H), 3.51 (dt, J = 10.3, 5.2 Hz, 4H), 2.99 (dd, J = 13.9, 8.8 Hz, 5H), 2.17 (d, J = 11.2 Hz, 1H), 2.07-1.75 (m, 4H), 1.74–1.43 (m, 10H), 1.35–1.19 (m, 4H), 1.04–0.64 (m, 25H). Derivative B1 1 HNMR spectrum as follows Figure 1 As shown.

[0070] 13CNMR (101 MHz, DMSO-d6) δ 175.59, 164.78, 153.33, 145.07, 137.81,124.87(2C), 118.04(2C), 114.25, 76.78, 60.71, 55.13(2C), 54.74, 52.37, 50.13,49.69, 47.40, 46.98, 43.87, 41.56, 41.21, 38.44, 38.34, 38.22, 36.46(2C),36.15, 32.63, 30.09, 28.22, 27.43, 26.97, 23.81, 23.22, 22.86, 21.00, 17.95, 16.96, 16.80, 16.06, 15.20. Derivative B1... 13 CNMR spectrum as shown Figure 2 As shown.

[0071] ESI-MS (m / z): 689.48 [M+H] + .

[0072] Example 2

[0073] The ursolic acid derivative was synthesized according to Example 1. The difference from Example 1 is that R is... Other technical features are the same as in Example 1. Derivative B10 was obtained. The product was a yellow solid with a yield of 13%.

[0074] The target compound was analyzed by nuclear magnetic resonance hydrogen spectroscopy (NMR 1H spectroscopy). 1 H NMR, carbon spectrum ( 13 Characterized by C NMR and mass spectrometry (MS), as follows:

[0075] 1H NMR (400 MHz, DMSO-d6) δ 8.12 – 8.03 (m, 2H), 7.07 – 6.98 (m, 2H), 5.14 (d, J = 3.9 Hz, 1H), 4.79 (d, J = 14.6 Hz, 1H), 4.71 (d, J = 14.6 Hz,1H), 4.29 (d, J = 5.1 Hz, 1H), 3.64 – 3.43 (m, 8H), 3.00 (dt, J = 10.4, 5.3Hz, 1H), 2.16 (d, J = 11.3 Hz, 1H), 2.07 – 1.99 (m, 1H), 1.95 – 1.76 (m, 3H),1.71 – 1.42 (m, 10H), 1.37 – 1.22 (m, 4H), 1.05 – 0.65 (m, 25H). Derivative B10 1 HNMR spectrum as follows Figure 3 As shown.

[0076] 13 C NMR (101 MHz, CDCl3) δ 177.07, 165.71, 154.27, 139.43, 138.10, 126.03(2C), 125.73, 113.28(2C), 79.04, 60.72, 55.24, 52.84, 48.39, 47.59,47.04(2C), 44.00, 42.14, 41.18, 39.62, 39.16, 38.81(2C), 38.67, 37.04, 36.68,33.06, 30.73, 28.20, 28.09, 27.27, 24.33, 23.58, 23.37, 21.26, 18.38, 17.18, 17.09, 15.72, 15.54. Derivative B10... 13 CNMR spectrum as shown Figure 4 As shown.

[0077] ESI-MS (m / z): 704.46 [M+H] + .

[0078] Example 3

[0079] A ursolic acid derivative was synthesized, and the ursolic acid derivative has the structure shown in the following chemical formula:

[0080]

[0081] Where R1 is .

[0082] The synthesis path is as follows:

[0083] ;

[0084] .

[0085] The synthesis method is the same as in Example 1, except that: [the following is a partial translation of the original text, which is incomplete and requires further context.] The equimolar amount was replaced with R1-NH2; other technical characteristics were the same as in Example 1. Derivative B14 was obtained. The product was a white solid with a yield of 58%.

[0086] The target compound was analyzed by nuclear magnetic resonance hydrogen spectroscopy (NMR 1H spectroscopy). 1 H NMR, carbon spectrum ( 13 Characterized by C NMR and mass spectrometry (MS), as follows:

[0087] 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 7.87 (ddd, J = 10.0, 6.2,3.2 Hz, 1H), 7.32 (td, J = 9.8, 5.1 Hz, 1H), 6.97 (tt, J = 7.6, 3.3 Hz, 1H), 5.16 (d, J = 3.7 Hz, 1H), 4.71 (d, J = 15.0 Hz, 1H), 4.64 (d, J = 14.9 Hz, 1H), 4.29 (d, J = 5.1 Hz, 1H), 3.00 (dt, J = 10.5, 5.5 Hz, 1H), 2.18 (d, J =11.3 Hz, 1H), 2.08 – 1.72 (m, 4H), 1.70 – 1.42 (m, 10H), 1.29 (td, J = 17.7, 14.8, 6.8 Hz, 4H), 1.07 – 0.67 (m, 25H). Derivative B14 1 H NMR spectrum as shown Figure 5 As shown.

[0088] 13C NMR (101 MHz, CDCl3) δ 175.78, 165.67, 138.22, 126.32, 126.06,115.49, 115.37, 110.88, 109.22, 108.92, 79.05, 63.13, 55.26, 53.28, 48.59,47.52, 42.17, 39.57, 39.17, 38.97, 38.81, 38.64, 36.99, 36.86, 32.93, 30.58,28.19, 27.98, 27.26, 24.56, 23.71, 23.32, 21.18, 18.33, 17.08, 17.01, 15.67, 15.40. Derivative B14... 13 CNMR spectrum as shown Figure 6 As shown.

[0089] ESI-MS (m / z): 624.38 [MH] - .

[0090] Comparative Examples 1-10

[0091] Referring to Example 1, the difference from Example 1 is that R is a group shown in Table 1. Other technical features are the same as in Example 1.

[0092] Table 1. R groups of Comparative Examples 1-10

[0093]

[0094] Comparative Examples 11-16

[0095] Referring to Example 3, the difference from Example 3 is that R1 is a group shown in Table 2. Other technical features are the same as in Example 3.

[0096] Table 2. R1 groups of Comparative Examples 11-16

[0097]

[0098] Test 1

[0099] The minimum inhibitory concentration (MIC) of ursolic acid derivatives and ursolic acid (UA) obtained in each example and comparative example was determined.

[0100] The in vitro antibacterial activity of each example and each comparative example against four pathogenic strains was evaluated by standardized minimum inhibitory concentration (MIC) determination, with tetracycline (TC) as a positive control.

[0101] The MIC values ​​of all compounds were determined using the standard microbroth dilution method. Single colonies were inoculated into LB broth and cultured at 37°C and 180 rpm with shaking for 16–18 h. The cultured bacterial suspension was then removed and its turbidity was adjusted to 0.5 McFarland units using a McFarland turbidimeter. The adjusted suspension was then diluted with culture medium to obtain a concentration of 1.5 × 10⁻⁶. 6 Prepare a bacterial suspension at CFU / mL. Add the culture medium and test drug solution sequentially to a sterile 96-well plate, with a total volume of 100 μL per well. Then, add 100 μL of bacterial suspension (1.5 × 10⁻⁶) to each well. 6 The culture medium was mixed thoroughly with CFU / mL; only 200 μL of culture medium was added to the negative control group; and only 200 μL of bacterial suspension was added to the bacterial control group. The final concentrations of the test compound in the wells were 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, and 1.17 μg / mL. The plates were incubated at 37°C for 20 hours. After incubation, the 96-well plates were observed under light with a black background. The lowest drug concentration of the compound was indicated by diffuse turbidity or a net-like sediment at the bottom of the wells when bacteria grew, and by clear and transparent wells with no bacterial growth. The bacterial strains used in the test included Staphylococcus aureus ATCC29213, Staphylococcus epidermidis ATCC12228, Escherichia coli ATCC25922, and Pseudomonas aeruginosa CGMCC10104. Each experimental procedure was repeated three times in parallel. The test results are shown in Table 3.

[0102] Table 3. Ursolic acid derivatives and MIC values ​​(μg / mL) of ursolic acid against four bacterial pathogens.

[0103]

[0104] As shown in Table 3, the MICs of derivatives B1 and B10 against Staphylococcus aureus were 37.5 μg / mL and 150 μg / mL, respectively; the MIC of derivative B10 against Staphylococcus epidermidis was 150 μg / mL; and the MIC of derivative B1 against Escherichia coli was 75 μg / mL. The introduction of the R-aniline 2,5-difluoroaniline enabled derivative B14 to exhibit antibacterial activity, with an MIC of 75 μg / mL against Staphylococcus aureus and 150 μg / mL against Escherichia coli. Derivative B1 showed the best antibacterial activity against all target derivatives.

[0105] Test 2

[0106] The antibacterial kinetics of ursolic acid derivative B1 obtained in Example 1 against Staphylococcus aureus and Escherichia coli were investigated.

[0107] The test method is as follows: Take a sterile 96-well plate and add 100 μL of the prepared bacterial solution (1.5 × 10⁻⁶) to each well. 6 The control group received 100 μL of the prepared bacterial culture (1.5 × 10⁻⁶ CFU / mL) and 100 μL of the compound solution (4 × MIC; DMSO concentration < 2%). 6 The 96-well plate was incubated with 100 μL of LB medium (CFU / mL) and placed in a 37°C incubator. The plates were then removed at 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. The absorbance of each well at 600 nm was measured using a microplate reader, and the inhibition rate was calculated. The above experimental procedure was repeated three times in parallel.

[0108] The results of the antibacterial kinetic test against Staphylococcus aureus are shown in [the table below]. Figure 7 (***: P<0.001 compared with the control group). The results showed that derivative B1 (4×MIC) had an inhibitory effect on the growth of Staphylococcus aureus during the culture period (P<0.001). Compared with the control group, the bacterial growth rate in the experimental group was significantly reduced, and the inhibitory effect gradually increased within the first 12 h, reaching a maximum inhibition rate of 58.52% at 12 h, and maintaining a significant inhibition rate within 24 h.

[0109] The results of the antibacterial kinetic test against Escherichia coli are shown in [the table below]. Figure 8 The results showed that derivative B1 (4×MIC) inhibited the growth of Escherichia coli during the culture period (P<0.001). Compared with the control group, the bacterial growth rate in the experimental group was significantly reduced, and the inhibitory effect gradually increased within the first 16 hours, reaching a maximum inhibition rate of 52.46% at 16 hours. The inhibition rate remained significant within 24 hours.

[0110] Test 3

[0111] The time-bacterial kinetics of the ursolic acid derivative B1 obtained in Example 1 against Staphylococcus aureus and Escherichia coli were investigated.

[0112] The test method is as follows: Tetracycline was used as the positive control drug; tetracycline and compound B1 were prepared into a stock solution (8×MIC; DMSO concentration <3%) for later use. Several 10 mL sterile centrifuge tubes were prepared and divided into three groups: positive control group, negative control group, and sample group. The incubation time points were set at 0 h, 1 h, 2 h, 4 h, 8 h, and 24 h. Positive control group: 1 mL of bacterial suspension (1.5×10⁻⁶) was added to each tube.6 CFU / mL) and 1 mL tetracycline stock solution (8×MIC). Negative control group: 1 mL bacterial suspension (1.5×10⁻⁶ CFU / mL) was added to each tube. 6 Add 1 mL of LB culture medium (CFU / mL) to each tube. Sample group: Add 1 mL of bacterial suspension (1.5 × 10⁻⁶ CFU / mL) to each tube. 6 The mixture was prepared with 1 mL of compound B1 stock solution (8×MIC) and 1 mL of compound B1 stock solution. All centrifuge tubes were placed in a constant temperature shaker incubator at 37°C and 180 rpm for the specified time. At each of the set time points (0 h, 1 h, 2 h, 4 h, 8 h, and 24 h), centrifuge tubes from the positive control group, negative control group, and sample group were collected. 100 μL of the mixture was drawn from each tube and serially diluted 10-fold using culture medium (dilution gradient 10). -1 Up to 10 -8 Spread each concentration of the diluted solution onto agar plates. Incubate the plates at 37°C for 16–20 h. After incubation, select plates with colony counts between 30 and 300 and count the colonies at each dilution. Repeat the entire experiment independently three times.

[0113] The time-bactericidal curves of derivative B1 and tetracycline (TC) against Staphylococcus aureus are shown below. Figure 9 The results showed that the bacterial concentration in the experimental group continued to decrease within 24 hours of co-culture. The initial concentration of Staphylococcus aureus decreased by 0.88 log CFU / mL within 1 hour and by 1.72 log CFU / mL within 24 hours. The rate of decrease slowed down by an average of 0.071 log CFU / mL from 1 to 24 hours, and no bacterial resuscitation was observed throughout the entire process.

[0114] The time-bactericidal curves of derivative B1 and tetracycline (TC) against Escherichia coli are shown in the figure. Figure 10 The results showed that the bacterial concentration in the experimental group continued to decrease within 24 h of co-culture. The initial concentration of Escherichia coli decreased by 0.70 log CFU / mL within 1 h, and by 1.33 log CFU / mL within 24 h. The rate of decrease slowed down by an average of 0.055 log CFU / mL from 1 to 24 h, and no bacterial resuscitation was observed throughout the entire process.

[0115] Test 4

[0116] The effect of derivative B1 on bacterial biofilm formation was determined.

[0117] The effect of derivative B1 on biofilm formation in Staphylococcus aureus and Escherichia coli was quantitatively assessed using crystal violet staining. The test method was as follows: In sterile 96-well plates, 100 µL of drug stock solution (4×MIC; DMSO concentration ≤2%) and 100 µL of bacterial suspension (1.5×10⁻⁶) were added to each well of the experimental group. 6 (CFU / mL). In the control group, 100 µL of LB medium and 100 µL of bacterial suspension were added to each well. The 96-well plate was placed in a 37°C incubator and incubated for 6 h, 12 h, 18 h, and 24 h, respectively. After reaching the predetermined incubation time points, the 96-well plate was removed. The bacterial suspension in each well was discarded. The wells were gently rinsed 1-2 times along the well walls with sterile distilled water to thoroughly remove any unadhered bacteria. 200 µL of methanol was added to each well and the plate was fixed at room temperature for 20 min, then the methanol was discarded. 0.1% crystal violet staining solution was added to each well and the plate was stained at room temperature for 10 min, then the staining solution was discarded. The wells were then gently rinsed 1-2 times with sterile distilled water to remove any remaining staining solution. The plate was allowed to air dry at room temperature. 33% glacial acetic acid solution was added to each well to dissolve the crystal violet dye bound to the biofilm, and the solution was allowed to react for 25 min. The absorbance of each well was measured at 570 nm using a microplate reader.

[0118] The effect of derivative B1 on Staphylococcus aureus biofilm formation is shown in [reference needed]. Figure 11 The results showed that the biofilm in the control group was increasing, while the biofilm formation in the experimental group was significantly lower than that in the control group at the selected time points (P<0.001). This indicates that the target compound B1 can inhibit the formation of Staphylococcus aureus biofilm.

[0119] The effect of derivative B1 on Escherichia coli biofilm formation is shown in [reference needed]. Figure 12 The results showed that the biofilm amount in the control group increased over time, while the biofilm formation in the experimental group was significantly lower than that in the control group at the selected time points (P<0.001). This indicates that the target compound B1 can inhibit the formation of Escherichia coli biofilm.

[0120] Test 5

[0121] Molecular docking analysis was performed.

[0122] Staphylococcal accessory regulator A (SarA) is a core transcriptional regulator of Staphylococcus aureus virulence factors and a key factor in biofilm formation. Previous experiments have confirmed that compound B1 can effectively inhibit Staphylococcus aureus biofilm formation. To explore its structural mechanism, the SarA protein structure was obtained from the RCSB PDB database (PDB ID: 2FHR), and molecular docking studies were conducted.

[0123] Molecular docking results are shown in Figure 13 (Hydrogen bond positions: Lys B163; Lys A127). The results show that compound B1 can spontaneously bind to the active pocket of the SarA protein (binding energy -7.39 kcal / mol) through the formation of 3 hydrogen bonds and π-π stacking. This structure may stabilize the complex structure through hydrophobic / electrostatic interactions, providing a molecular-level basis for its mechanism of inhibiting biofilm formation.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An ursolic acid derivative or a pharmaceutically acceptable salt thereof, characterized in that, The ursolic acid derivatives described herein have the structure shown in formula (I) or formula (II): ； Equation (I) ； Formula (II) Where R is or ; Where R1 is .

2. A method for synthesizing the ursolic acid derivative as described in claim 1, characterized in that, Includes the following steps: S1. Obtain intermediates or ; S2. React ursolic acid with the intermediate obtained in step S1 to obtain ursolic acid derivative.

3. The synthesis method according to claim 2, characterized in that, In step S1: Alternatively, R1-NH2 reacts with chloroacetyl chloride to yield an intermediate.

4. The synthesis method according to claim 3, characterized in that, In step S1: Triethylamine is added to the reaction.

5. The synthesis method according to claim 3 or 4, characterized in that, In step S1: the reaction temperature is 0℃ to -20℃.

6. The synthesis method according to claim 2, characterized in that, In step S2: Potassium carbonate is added to the reaction.

7. The synthesis method according to claim 6, characterized in that, In step S2: the reaction temperature is 60~90℃.

8. The use of the ursolic acid derivative as described in claim 1 or a pharmaceutically acceptable salt thereof in non-therapeutic, non-diagnostic antibacterial and / or sterilization applications.

9. The application according to claim 8, characterized in that, Used in the preparation of antibacterial and / or sterilizing drugs.

10. The application according to claim 8, characterized in that, It is used for non-therapeutic and non-diagnostic antibacterial and / or sterilization mediated by biofilms.

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