Betulinic acid derivative as well as synthesis method and application thereof

Abstract

The invention discloses a betulinic acid derivative as well as a synthesis method and application thereof. According to the betulinic acid derivative, the carboxyl position of betulinic acid C-28 is subjected to structural modification, and four halogenated aniline derivatives are obtained. Wherein the derivatives b, d and e show strong activity on staphylococcus aureus, the derivatives d and e also show strong activity on vancomycin-resistant staphylococcus aureus (VRSA), and the derivative o strongly inhibits streptococcus pneumoniae. Compared with a parent skeleton, the four derivatives show significantly enhanced antibacterial efficacy. Experimental data prove that the derivatives d and e and further derivatives thereof may provide a promising strategy for resisting drug-resistant gram-positive pathogens, and the derivative d can be further developed into a novel therapeutic agent for biofilm-mediated drug-resistant infection.

Description

Technical Field

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

[0002] Pathogenic bacterial infections have long posed a significant threat to global public health, causing millions of deaths annually and significantly increasing the global disease burden. Fleming's accidental discovery of penicillin in 1928 ushered in the golden age of antibiotics, revolutionizing medical practice and enabling complex interventions such as organ transplantation and chemotherapy. However, since the 1940s, the widespread use of antibiotics in clinical and agricultural settings has accelerated the emergence of antimicrobial resistance (AMR). Today, AMR has become a critical global health challenge in the 21st century, driven by factors including horizontal gene transfer and genomic mutations in pathogens.

[0003] Multidrug-resistant (MDR) and extensively drug-resistant (XDR) "superbugs" are spreading globally, rendering traditional antibiotics ineffective against common infections such as urinary tract infections and sepsis. According to the World Health Organization, AMR-related infections cause more than 2.3 million deaths annually in North America and more than 33,000 direct deaths in Europe. A 2019 systematic analysis attributed 4.95 million deaths to drug-resistant infections, with Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa causing 929,000 deaths. Without intervention, AMR-related deaths could reach 10 million annually by 2050, with projected economic losses of $100 trillion.

[0004] The antibiotic development pipeline remains severely inadequate. As of 2019, only 42 candidate drugs were in clinical trials, of which only 4 showed novel mechanisms of action. This crisis stems from three key factors: (1) the high cost of antibiotic development and the rapid emergence of drug resistance; (2) the technical bottleneck in discovering new structural frameworks; and (3) the ongoing global overuse of antibiotics.

[0005] Natural products and their derivatives have historically been the cornerstone of novel drug discovery. Plant-derived metabolites exhibit diverse pharmacological properties, including antibacterial, antiviral, and antitumor activities. Notably, artemisinin (antimalarial) and vinblastine (anticancer) are exemplary models of successful therapeutic drugs derived from natural products. Plant antimicrobial agents, particularly pentacyclic triterpenoids, provide uniquely structured, low-toxicity skeletons that can be used to combat drug-resistant pathogens. Betulinic acid (BA), a lupinane-type triterpenoid widely distributed in Betulaceae plants, possesses broad biological activities, including antimicrobial activity. Although natural BA exhibits moderate antimicrobial potency, its low eukaryotic cytotoxicity and modifiable C-3 / C-28 sites make it an ideal skeleton for structural optimization. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides a betulinic acid derivative, its synthesis method, and its applications. The derivative obtained by this invention exhibits significantly enhanced antibacterial efficacy, particularly potent activity against Staphylococcus aureus.

[0007] The specific technical solution is as follows:

[0008] One object of the present invention is to provide a betulinic acid derivative or a pharmaceutically acceptable salt thereof, said betulinic acid derivative having the structure shown in formula (I):

[0009] ;

[0010] Equation (I)

[0011] Wherein, R is selected from one of the following groups:

[0012] (Derivative b);

[0013] (Derivative d);

[0014] (Derivative e);

[0015] (Derivative o).

[0016] Experiments have confirmed that the above-mentioned betulinic acid derivatives possess potent antibacterial activity. Derivatives b, d, and e exhibited potent activity against Staphylococcus aureus; derivatives d and e also showed potent activity against vancomycin-resistant Staphylococcus aureus (VRSA); while derivative o strongly inhibited Streptococcus pneumoniae. Compared to the parent BA skeleton, these four derivatives (b, d, e, and o) showed significantly enhanced antibacterial efficacy.

[0017] A second objective of this invention is to provide a method for synthesizing the above-mentioned betulinic acid derivative, which includes the following steps:

[0018] S1. An R-substituted ammonia undergoes a condensation reaction with chloroacetyl chloride to yield an intermediate amide;

[0019] S2. The intermediate amide undergoes a nucleophilic substitution reaction with the carboxylic acid moiety of betulinic acid to obtain a betulinic acid derivative.

[0020] Further, step S1 includes: dissolving the R-substituted ammonia in solvent A to obtain reaction mixture A; adding triethylamine to reaction mixture A and adding chloroacetyl chloride, and reacting at -10 to -2°C for 8 to 15 h.

[0021] In step S1, the molar ratio of the R-group-substituted ammonia to chloroacetyl chloride is preferably 1:(1~1.5).

[0022] In step S1, the preferred ratio of the amount of R-substituted ammonia to triethylamine is 20 mmol:(2~5) mL.

[0023] In step S1, solvent A is preferably dichloromethane.

[0024] In step S1, chloroacetyl chloride is first dissolved in solvent A, and then added to reaction mixture A.

[0025] Specifically, in step S1: the reaction mixture A is cooled to -10~-2℃, then triethylamine is added, and the mixture is stirred for 20~50 minutes, and then chloroacetyl chloride is added.

[0026] Further, step S2 includes: dissolving betulinic acid in solvent B to obtain reaction mixture B; adding potassium carbonate to reaction mixture B, and adding the intermediate amide obtained in step S1, and reacting at 70-80°C for 8-15 h.

[0027] In step S2, the preferred molar ratio of betulinic acid to intermediate amide is 1:(1~2).

[0028] In step S2, the molar ratio of betulinic acid to potassium carbonate is 1:(2~5).

[0029] In step S2, solvent B is preferably acetonitrile.

[0030] In step S2, the intermediate amide is first dissolved in solvent B, and then added to reaction mixture B.

[0031] Specifically, in step S2: potassium carbonate is added to reaction mixture B, and the mixture is stirred for 0.5 to 1.5 hours, followed by the addition of the intermediate amide. After the intermediate amide is added, the reaction is carried out under reflux conditions.

[0032] A third objective of this invention is to provide the use of the above-mentioned betulinic acid derivatives or their pharmaceutically acceptable salts in non-therapeutic, non-diagnostic antibacterial and / or sterilization applications.

[0033] Furthermore, the aforementioned antibacterial or sterilization refers to inhibiting or killing Gram-positive bacteria.

[0034] Furthermore, the application of the aforementioned betulinic acid derivatives or their pharmaceutically acceptable salts in non-therapeutic and / or sterilization targeting biofilm-mediated non-therapeutic and non-diagnostic antibacterial and / or sterilization methods is provided. Experiments have demonstrated that the aforementioned betulinic acid derivatives, especially derivative d, can persistently inhibit biofilm accumulation in bacterial strains and effectively disrupt the biofilm formation process.

[0035] Specifically, it provides the use of at least one of derivatives b, d, e, or a pharmaceutically acceptable salt thereof in the non-therapeutic, non-diagnostic inhibition or killing of Staphylococcus aureus.

[0036] Specifically, it provides the use of at least one of derivatives d and e, or a pharmaceutically acceptable salt thereof, in the non-therapeutic, non-diagnostic inhibition or eradication of vancomycin-resistant Staphylococcus aureus (VRSA).

[0037] Specifically, it provides the use of derivative o, or a pharmaceutically acceptable salt thereof, in the non-therapeutic, non-diagnostic inhibition or killing of Streptococcus pneumoniae (S. pneumoniae).

[0038] A fourth objective of this invention is to provide the use of the above-mentioned betulinic acid derivatives or pharmaceutically acceptable salts thereof in the preparation of antibacterial and / or sterilizing drugs.

[0039] Furthermore, the use of the above-mentioned betulinic acid derivatives or pharmaceutically acceptable salts thereof in the preparation of antibacterial and / or sterilizing agents targeting biofilm-mediated antibacterial and / or sterilizing agents is provided.

[0040] Specifically, the use of at least one of derivatives d and e, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for inhibiting or killing Staphylococcus aureus is provided.

[0041] Specifically, the use of at least one of derivatives d and e, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for inhibiting or killing vancomycin-resistant Staphylococcus aureus (VRSA).

[0042] Specifically, the use of derivative O or a pharmaceutically acceptable salt thereof in the preparation of medicaments for inhibiting or killing Streptococcus pneumoniae is provided.

[0043] Specifically, the drug may also include pharmaceutically acceptable excipients.

[0044] 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.

[0045] Specifically, the drug can be administered orally, sublingually, intravenously, by injection, or by inhalation.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention modifies the C-28 carboxyl group of betulinic acid (BA) to obtain four halogenated aniline BA derivatives. Derivatives b, d, and e exhibit potent activity against Staphylococcus aureus; derivatives d and e also show potent activity against vancomycin-resistant Staphylococcus aureus (VRSA); while derivative o strongly inhibits Streptococcus pneumoniae. Compared with the parent BA backbone, these four derivatives (b, d, e, and o) show significantly enhanced antibacterial efficacy. Notably, derivatives d and e both show significant inhibitory activity against VRSA, indicating that these compounds and their further derivatives may provide a promising strategy for combating drug-resistant Gram-positive pathogens. Through a series of comprehensive experiments including MIC determination, Oxford cup method, time-bacterial kinetics, and biofilm inhibition, derivative d demonstrates enhanced broad-spectrum efficacy against both drug-sensitive and drug-resistant Staphylococcus aureus. Molecular docking proposes inhibition of SarA protein as a possible mechanism for its anti-biofilm activity, which could be further developed into a novel therapeutic agent against biofilm-mediated drug-resistant infections. Attached Figure Description

[0048] Figure 1 Derivative b obtained in Example 1 1 H NMR spectrum;

[0049] Figure 2 The derivative d obtained in Example 2 1 H NMR spectrum;

[0050] Figure 3 For derivative e obtained in Example 3 1 H NMR spectrum;

[0051] Figure 4 The derivative o obtained in Example 4 1 H NMR spectrum;

[0052] Figure 5 Representative images showing the inhibitory effect of derivative d in test 2 on the growth of Staphylococcus aureus;

[0053] Figure 6 Representative images showing the inhibitory effect of derivative d in test 2 on VRSA growth;

[0054] Figure 7 To test the time-bactericidal curve of derivative d in part 3 against Staphylococcus aureus;

[0055] Figure 8 To test the time-sterilization curve of derivative d in group 3 against VRSA;

[0056] Figure 9 To test the effect of derivative d in group 4 on Staphylococcus aureus biofilm formation;

[0057] Figure 10 To test the effect of derivative d in group 4 on VRSA biofilm formation;

[0058] Figure 11 To test the docking conformation of derivative d in SarA (2FRH) within the binding pocket. Detailed Implementation

[0059] 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.

[0060] Example 1

[0061] A betulinic acid derivative was synthesized, and the betulinic acid derivative has the structure shown in the following chemical formula:

[0062] ;

[0063] Where R is .

[0064] The synthesis path is as follows:

[0065]

[0066] The specific synthesis method is as follows:

[0067] S1. Synthesis of the intermediate amide: 20.0 mmol of R-substituted ammonia R-NH2 (4-chloro-3-fluoroaniline) and 60 mL of dichloromethane (DCM) were added to a dry glass reactor and stirred at room temperature until completely dissolved to obtain reaction mixture A. Reaction mixture A was then cooled to -5°C, followed by the dropwise addition of 3.5 mL of triethylamine. After stirring for 30 min, 24.0 mmol of chloroacetyl chloride dissolved in 30 mL of dichloromethane was slowly added over 30 min via a dropping funnel. The reaction was vigorously stirred at -5°C for 12 h. The reaction was monitored for completion by thin-layer chromatography. The resulting mixture was washed successively with saturated NaCl solution (3 × 50 mL) and distilled water (3 × 50 mL). The organic phase was dried over anhydrous Na2SO4, concentrated under vacuum, and purified by silica gel (200–300 mesh) column chromatography using a gradient eluent to obtain the pure intermediate amide.

[0068] S2. In a round-bottom flask, 1.0 mmol of betulinic acid was dissolved in 30 mL of acetonitrile at room temperature and stirred magnetically to obtain reaction mixture B. 3.0 mmol of potassium carbonate was added to reaction mixture B, and the mixture was stirred at room temperature for 1 h. Then, an intermediate amide solution dissolved in 10 mL of acetonitrile was added dropwise, and the mixture was heated to reflux at 80 °C for 12 h. The progress was monitored by thin-layer chromatography. After the reaction was complete, the resulting reaction mixture was cooled to room temperature, filtered under vacuum, and concentrated under vacuum. The residue was purified by silica gel (200–300 mesh) column chromatography using a gradient eluent to give derivative b of the target compound. The product was a white solid in 60% yield.

[0069] The target compound was passed through 1 H NMR, 13 Characterized by C NMR and high-resolution mass spectrometry (HRMS), as follows:

[0070] 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 7.87 (dd, J = 6.6, 2.6 Hz,1H), 7.50-7.33 (m, 2H), 4.73-4.60 (m, 3H), 4.56 (s, 1H), 4.25 (d, J = 5.1 Hz,1H), 2.93 (ddt, J = 28.2, 11.0, 5.3 Hz, 2H), 2.31-2.13 (m, 2H), 1.99 (s, 1H),1.93-1.79 (m, 1H), 1.65 (s, 3H), 1.62-1.21 (m, 14H), 1.20-1.07 (m, 2H), 1.00 (m, 1H), 0.94 (s, 3H), 0.86 (d, J = 9.3 Hz, 7H), 0.75 (s, 3H), 0.65 (s, 4H). Derivative b 1 H NMR spectrum as shown Figure 1 As shown.

[0071] 13 C NMR (101 MHz, DMSO-d6) δ 174.77, 165.85, 151.91, 150.06, 135.75,120.48, 119.30, 117.09, 116.88, 109.64, 76.75, 62.00, 55.77, 54.87, 49.89,48.71, 46.44, 41.97, 40.22, 38.44, 38.24, 37.43, 36.66, 36.19, 33.85, 31.32,29.89, 29.05, 28.03, 27.12, 25.00, 20.38, 18.88, 17.90, 15.85, 15.72, 15.54, 14.32.

[0072] HRMS (ESI) m / z: calculated C 38 H 52 ClFNO4 [MH] - 640.3574, found:640.3603.

[0073] Example 2

[0074] The betulinic acid derivative was synthesized according to Example 1, the difference being that R is... That is, the R-substituted ammonia R-NH2 is 4-fluoro-3-(trifluoromethyl)aniline. Other technical features are the same as in Example 1. Derivative d is obtained. The product is a white solid with a yield of 58%.

[0075] The target compound was passed through 1 H NMR, 13 Characterized by C NMR and high-resolution mass spectrometry (HRMS), as follows:

[0076] 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.07 (dd, J = 6.6, 2.6 Hz, 1H), 7.81 (dt, J = 7.3, 3.4 Hz, 1H), 7.51 (t, J = 9.8 Hz, 1H), 5.77 (s, 1H),4.68 (s, 2H), 4.58 (s, 1H), 4.33-4.24 (m, 1H), 3.03-2.86 (m, 2H), 2.30-2.14(m, 2H), 2.05-1.78 (m, 2H), 1.67 (s, 3H), 1.64-1.23 (m, 14H), 1.13 (td, J = 12.4, 3.9 Hz, 2H), 1.02 (m, 1H), 0.96 (s, 3H), 0.87 (d, J = 12.8 Hz, 7H), 0.76 (s, 3H), 0.67 (s, 4H). Derivative d... 1 H NMR spectrum as shown Figure 2 As shown.

[0077] 13 C NMR (101 MHz, DMSO-d6) δ174.80, 166.06, 155.61, 150.05, 135.30,124.99, ,117.86, ,116.97, 116.33, 109.62, 76.73, 62.06, 55.76, 54.85, 51.63,49.88, 48.72, 46.44, 41.96, 40.21, 38.43, 38.22, 37.43, 36.65, 36.15, 33.84,31.32, 29.88, 29.02, 28.02, 27.10, 24.99, 20.36, 18.86, 17.85, 15.79, 15.70, 15.47, 14.30.

[0078] HRMS (ESI) m / z: calculated C 39 H 52 F4NO4 - [MH] - 674.3838, found: 674.3756.

[0079] Example 3

[0080] The betulinic acid derivative was synthesized according to Example 1, the difference being that R is... That is, the R-substituted ammonia R-NH2 is 2-bromo-4-fluoroaniline. Other technical features are the same as in Example 1. Derivative e is obtained. The product is a white solid with a yield of 60%.

[0081] The target compound was passed through 1 H NMR, 13 Characterized by C NMR and high-resolution mass spectrometry (HRMS), as follows:

[0082] 1 H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 7.64 (td, J = 9.4, 8.9, 4.3Hz, 2H), 7.28 (td, J = 8.6, 2.9 Hz, 1H), 4.79-4.64 (m, 3H), 4.56 (s, 1H), 4.25 (d, J = 5.1 Hz, 1H), 3.01-2.86 (m, 2H), 2.21 (dd, J = 18.1, 9.2 Hz, 2H), 1.99 (s, 2H), 1.65 (s, 3H), 1.62-1.22 (m, 14H), 1.20-1.10 (m, 2H), 1.00 (m, 1H), 0.94 (s, 3H), 0.86 (d, J = 8.0 Hz, 7H), 0.75 (s, 3H), 0.65 (s, 4H). Derivative e 1 H NMR spectrum as shown Figure 3 As shown.

[0083] 13C NMR (101 MHz, DMSO-d6) δ 174.53, 166.08, 150.07, 132.29, 127.94,119.67, 119.42, 115.16, 114.94, 109.63, 76.72, 61.78, 55.81, 54.85, 49.87,48.71, 46.44, 41.96, 40.21, 38.43, 38.21, 37.41, 36.66, 36.17, 33.82, 31.29,29.85, 29.06, 28.03, 27.10, 25.00, 20.36, 18.86, 17.88, 15.86, 15.72, 15.60.

[0084] HRMS (ESI) m / z: calculated C 38 H 52 BrFNO4 - [MH] - 684.3069, found: 684.2917.

[0085] Example 4

[0086] The betulinic acid derivative was synthesized according to Example 1, the difference being that R is... That is, the R-substituted ammonia R-NH2 is 4-fluoroaniline. Other technical features are the same as in Example 1. Derivative o is obtained. The product is a white solid with a yield of 62%.

[0087] The target compound was passed through 1 H NMR, 13 Characterized by C NMR and high-resolution mass spectrometry (HRMS), as follows:

[0088] 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 7.57 (dd, J = 8.9, 5.0 Hz, 2H), 7.16 (t, J = 8.9 Hz, 2H), 4.68 (d, J = 2.5 Hz, 1H), 4.64 (d, J = 2.0 Hz,2H), 4.56 (d, J = 2.5 Hz, 1H), 4.26 (d, J = 5.1 Hz, 1H), 2.93 (ddt, J = 29.3,11.1, 5.4 Hz, 2H), 2.21 (qd, J = 12.2, 10.8, 3.3 Hz, 2H), 2.05-1.79 (m, 2H), 1.65 (s, 3H), 1.62-1.22 (m, 14H), 1.21-1.06 (m, 2H), 1.00 (dd, J = 13.0, 4.7Hz, 1H), 0.94 (s, 3H), 0.86 (d, J = 8.3 Hz, 7H), 0.75 (s, 3H), 0.65 (s, 4H). Derivative o 1 H NMR spectrum as shown Figure 4 As shown.

[0089] 13 C NMR (101 MHz, DMSO-d6) δ 174.78, 165.44, 156.79, 150.10, 134.93,120.80, 120.72, 115.45, 115.23, 109.64, 76.74, 62.00, 55.78, 54.86, 49.88,48.70, 46.46, 41.98, 40.22, 38.45, 38.22, 37.42, 36.67, 36.20, 33.83, 31.31,29.87, 29.05, 28.05, 27.12, 25.01, 20.37, 18.89, 17.91, 15.88, 15.74, 15.55, 14.33.

[0090] HRMS (ESI) m / z: calculated C 38 H 53 FNO4 - [MH] - 606.3964, found: 606.4013.

[0091] Comparative Examples 1-16

[0092] 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.

[0093] Table 1 R-base of each comparative example

[0094]

[0095]

[0096] Test 1

[0097] The minimum inhibitory concentration (MIC) of betulinic acid derivatives obtained in each example and comparative example was determined.

[0098] The in vitro antibacterial activity of each example and each comparative example against six pathogenic strains was evaluated by standardized minimum inhibitory concentration (MIC) determination, with betulinic acid (BA), levofloxacin, and tetracycline as positive controls.

[0099] The test method is as follows: Culture medium and test drug solution are added sequentially to a sterile 96-well plate, with a total volume of 100 μL per well. Then, 100 μL of bacterial suspension (1.5 × 10⁻⁶) is added to each well. 6 The CFU / mL concentrations were mixed thoroughly. The final concentrations of the test compounds in the wells were 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL. The plates were incubated at 37°C for 24 hours, and then the optical density (OD600) at 600 nm was measured using a microplate reader. The bacterial strains used in the tests included Staphylococcus aureus (S. aureus) ATCC25923, vancomycin-resistant Staphylococcus aureus (VRSA), Streptococcus pneumoniae (S. pneumoniae) ATCC49619, Staphylococcus epidermidis (S. epidermidis) ATCC12228, Escherichia coli (E. coli) ATCC25922, and Pseudomonas aeruginosa (P. aeruginosa) ATCC27853. All experiments were performed in triplicate. The test results are shown in Table 2.

[0100] Table 2. Betulinic acid derivatives and their MIC values ​​(μg / mL) against six bacterial pathogens.

[0101]

[0102] As shown in Table 2, derivatives b, d, and e exhibited potent activity against Staphylococcus aureus, with MIC values ​​of 125, 31.25, and 62.5 μg / mL, respectively. Derivatives d and e also showed potent activity against vancomycin-resistant Staphylococcus aureus (VRSA) (MIC = 62.5 μg / mL). Derivative o inhibited Streptococcus pneumoniae (MIC = 125 μg / mL). Compared to the parent BA backbone, these four derivatives (b, d, e, and o) showed significantly enhanced antibacterial efficacy.

[0103] The examples and comparative examples include twenty novel BA analogs, categorized into halogenated anilines (a-o), amino polycyclic heterocyclic compounds (p, q), and piperazines (r-t). Structure-activity relationship (SAR) analysis showed that all active derivatives exhibited broad-spectrum activity against Gram-positive bacteria, but no activity was detected against selected Gram-negative strains within the tested concentration range. Aniline substituents with electron-withdrawing groups, particularly 4-chloro-3-fluoroaniline (b), 4-fluoro-3-(trifluoromethyl)aniline (d), 2-bromo-4-fluoroaniline (e), and 4-fluoroaniline (o), significantly enhanced antibacterial efficacy, indicating a positive correlation between electron loss and bioactivity. Notably, derivatives d and e both showed significant inhibitory activity against VRSA, suggesting that these compounds and their further derivatives may provide a promising strategy for combating drug-resistant Gram-positive pathogens.

[0104] Test 2

[0105] The antibacterial effect of the betulinic acid derivative (derivative d) obtained in Example 2 was evaluated using the Oxford cup method.

[0106] Derivative d exhibited the strongest antibacterial activity in all examples and comparative examples, and was further evaluated. Its efficacy against Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus (VRSA) was evaluated using the Oxford cup method, with tetracycline as a positive control.

[0107] The test method is as follows: Under a laminar flow hood, 100 μL of Staphylococcus aureus and VRSA (1.5 × 10⁻⁶) were added. 6 Bacterial suspensions (CFU / mL) were spread onto sterile agar plates. Using a sterile swab, the plate was rotated 60° between each spread, moving in a circular motion along the edge to ensure even distribution of each inoculum. Sterile Oxford cups were then aseptically placed onto the agar surface using sterile forceps, ensuring complete contact. 200 μL of the test compound at 8×MIC was added to each cup, along with an equal volume of solvent as a negative control. The plates were incubated at 37°C for 24 hours, and the diameter of the inhibition zone was measured. The entire experiment was performed in triplicate.

[0108] The test results for Staphylococcus aureus are shown in [link to test results]. Figure 5 The test results for VRSA can be found in [link to VRSA test results]. Figure 6 . Figure 5 , Figure 6 In the diagram: A, B, and C represent the negative control, derivative d, and tetracycline, respectively. The vertical axis represents the diameter of the inhibition zone, where d is the diameter of derivative d and TC is the diameter of tetracycline. Figure 5 As shown, treatment with derivative d at a concentration of 8×MIC produced an inhibition zone of 15.5 mm in diameter against Staphylococcus aureus (P<0.001), indicating a good inhibitory effect. Figure 6 As shown, a distinct inhibition zone with a diameter of 11.8 mm was observed in VRSA (P<0.001), confirming its antimicrobial potential against this drug-resistant strain.

[0109] Test 3

[0110] The time-bactericidal kinetics of the betulinic acid derivative obtained in Example 2 against Staphylococcus aureus and VRSA were investigated.

[0111] Time-bacterial action curves provide a reliable method for evaluating the pharmacodynamic properties of antimicrobial drugs. To systematically evaluate the bactericidal potential of the synthesized betulinic acid derivative, time-bacterial action kinetics experiments were conducted to study the inhibitory and bactericidal effects of derivative d obtained in Example 2 against Staphylococcus aureus and its vancomycin-resistant Staphylococcus aureus (VRSA), with tetracycline as a positive control.

[0112] The testing method is as follows: Single colonies of Staphylococcus aureus and VRSA were inoculated separately into brain heart and brain extract (BHI) broth and incubated at 37°C with shaking (200 rpm) for 16–18 hours. The resulting bacterial suspension was adjusted to approximately 1.5 × 10⁻⁶ using sterile broth. 6 CFU / mL. The test derivative d and tetracycline (positive control) were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water to a stock solution of 8×MIC. For the time-sterilization assay, twenty-one sterile 5 mL tubes were divided into three groups: negative control, sample, and positive control, corresponding to time points of 0, 0.5, 1, 2, 4, 8, and 24 hours. The reaction mixture was prepared as follows: the negative control consisted of 1 mL of bacterial suspension and 1 mL of broth; the sample group consisted of 1 mL of bacterial suspension and 1 mL of 8×MIC test compound; the positive control group consisted of 1 mL of bacterial suspension and 1 mL of 8×MIC tetracycline. All tubes were incubated at 37°C. At each specified time point, 100 μL aliquots were taken from the tubes and serially diluted in sterile broth (10 μL / mL). -1 Up to 10 -7The samples were then spread onto agar plates. After incubating at 37°C for 24 hours, viable colonies were counted on plates producing 30–300 colonies. All experiments were performed in duplicate.

[0113] The time-bactericidal curve of derivative d against Staphylococcus aureus is shown in the figure. Figure 7 The time-bactericidal curve of derivative d against VRSA is shown in [reference needed]. Figure 8 . Figure 7 , Figure 8 In the text: d represents derivative d, and TC represents tetracycline. For example... Figure 7 As shown, exposure to the 8×MIC derivative d resulted in a 1.31 log reduction in Staphylococcus aureus within the first hour. 10 CFU / mL, and decreased by a cumulative 4.27 log over 24 hours. 10 CFU / mL, with no bacterial regrowth observed during this period. Notably, derivative d also exhibited significant inhibitory activity against VRSA, reducing bacterial count by 0.80 log [value missing] within 1 hour. 10 CFU / mL, and decreased by 4.04 log after 24 hours. 10 CFU / mL, with no regrowth detected within 24 hours. These kinetic results are consistent with the previously described Oxford cup assay, jointly confirming that derivative d possesses stable and effective antibacterial activity against both drug-sensitive and drug-resistant strains.

[0114] Test 4

[0115] Given that derivative d exhibits potent antibacterial activity against both drug-sensitive and drug-resistant Staphylococcus aureus strains, its impact on biofilm formation was further evaluated.

[0116] The assay method was as follows: To assess biofilm formation, Staphylococcus aureus and VRSA suspension (100 μL) were co-cultured with 100 μL of derivative d (8×MIC) in 96-well plates; sterile BHI was used as a blank control, and both conditions were repeated three times. After incubation at 37°C for 4, 8, 12, 16, 20, and 24 hours, the airborne cells and supernatant were aspirated. The attached biofilm was then washed three times with sterile distilled water. The biofilm was quantified by staining with 200 μL of crystal violet for 15 minutes (room temperature). After removing unbound dye and repeating the washing steps, the plates were air-dried. Subsequently, the crystal violet bound to the biofilm was dissolved in 30% acetic acid for 20 minutes, and the absorbance was measured at 570 nm using a microplate reader.

[0117] The effect of derivative d on biofilm formation in Staphylococcus aureus is shown in [reference needed]. Figure 9 The effect of derivative d on VRSA biofilm formation is shown in [reference needed]. Figure 10 . Figure 9 , Figure 10 In the text: d represents a derivative d. For example... Figure 9 , Figure 10 As shown, the biofilm biomass of the two Staphylococcus aureus strains in the control group increased in a time-dependent manner. In contrast, treatment with derivative d significantly inhibited biofilm formation in both strains at all time points compared to the control group (P < 0.0001). The sustained inhibition of biofilm accumulation observed at different time points suggests that derivative d may interfere with key processes in biofilm development, such as initial attachment, extracellular polymer production, or intercellular aggregation. These findings indicate that derivative d not only possesses direct antibacterial activity but also effectively disrupts biofilm formation processes, possibly through targeting regulatory mechanisms involved in biofilm maturation.

[0118] Test 5

[0119] Molecular docking analysis was performed.

[0120] Staphylococcal accessory regulatory factor A (SarA) is a key transcriptional regulator of Staphylococcus aureus virulence factors and has been identified as essential for biofilm formation. Given the aforementioned experimental evidence that derivative d significantly inhibits Staphylococcus aureus biofilm formation, SarA is hypothesized to be a potential molecular target. To elucidate its structural mechanism, molecular docking was performed, which also provides a theoretical basis for identifying targets of similar compounds.

[0121] The molecular docking method was as follows: Molecular docking was performed using Discovery Studio (DS) 2021 to predict the binding mode of the ligand with the SarA protein (PDB ID: 2FRH). The protein structure was prepared using DS Server, water molecules were removed, and hydrogen atoms with standard protonated states were distributed at pH 7.4. The ligand was constructed and energy minimization was performed. A sphere with a center coordinate of (-0.1483, -18.0931, 29.2759) and a radius of 8.3 Å was defined as the canonical binding site. Docking simulations were performed using the LibDock algorithm, and the resulting conformation was evaluated and selected based on the LibDockScore as the primary criterion.

[0122] Molecular docking results are shown in Figure 11 .like Figure 11As shown, derivative d was found to bind stably within the active pocket of the SarA protein (PDB:2FRH). A key hydrogen bond with a length of 3.02 Å and a binding energy contribution of -1.4 kcal / mol was observed between the fluorine atom of the ligand and residue LYS 127. Furthermore, several residues surrounding the binding pocket, including GLN 166, GLU 135, TYR 162, and LYS 123, may enhance the stability of the complex through hydrophobic interactions or electrostatic effects. In summary, this hydrogen bond and the microenvironment provided by the surrounding residues facilitate the efficient binding of derivative d to the target, providing a structural basis for its significant antibacterial and antibiofilm activity.

[0123] 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. A betulinic acid derivative or a pharmaceutically acceptable salt thereof, characterized in that, The betulinic acid derivative has the structure shown in formula (I): ； Equation (I) Wherein, R is selected from one of the following groups: 。 2. A method for synthesizing the betulinic acid derivative as described in claim 1, characterized in that, Includes the following steps: S1. An R-substituted ammonia undergoes a condensation reaction with chloroacetyl chloride to yield an intermediate amide; S2. The intermediate amide undergoes a nucleophilic substitution reaction with the carboxylic acid moiety of betulinic acid to obtain a betulinic acid derivative.

3. The preparation method according to claim 2, characterized in that, Step S1 includes: dissolving the R-substituted ammonia in solvent A to obtain reaction mixture A; adding triethylamine to reaction mixture A and adding chloroacetyl chloride, and reacting at -10 to -2°C for 8 to 15 hours.

4. The preparation method according to claim 3, characterized in that, In step S1, the molar ratio of the R-substituted ammonia to chloroacetyl chloride is 1:(1~1.5).

5. The preparation method according to claim 3, characterized in that, In step S1: Solvent A is dichloromethane; Chloroacetyl chloride is first dissolved in solvent A, and then added to reaction mixture A.

6. The preparation method according to claim 2, characterized in that, Step S2 includes: dissolving betulinic acid in solvent B to obtain reaction mixture B; adding potassium carbonate to reaction mixture B and adding the intermediate amide obtained in step S1; reacting at 70-80°C for 8-15 h.

7. The preparation method according to claim 6, characterized in that, In step S2, the molar ratio of betulinic acid to intermediate amide is 1:(1~2).

8. The preparation method according to claim 6, characterized in that, In step S2: Solvent B is acetonitrile; The intermediate amide is first dissolved in solvent B, and then added to reaction mixture B.

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

10. The use of the betulinic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of antibacterial and / or sterilizing agents.

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