Synthesis of New Derivatives of Berberine Canagliflozin and Study of Their Antibacterial Activity and Mechanism

The isoquinoline alkaloid berberine, derived from Coptidis rhizoma, exhibits antibacterial, hypoglycemic, and anti-inflammatory properties. Canagliflozin is a sodium–glucose cotransporter 2 (SGLT2) inhibitor. We synthesized compounds B9OC and B9OBU by conjugating canagliflozin and n-butane at the C9 position of berberine, aiming to develop antimicrobial agents for combating bacterial infections worldwide. We utilized clinically prevalent pathogenic bacteria, namely Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, to investigate the antibacterial efficacy of B9OC. This was accomplished through the determination of the MIC80 values, analysis of bacterial growth curves, evaluation of biofilm formation using crystal violet staining, assessment of impact on bacterial proteins via SDS-PAGE analysis, and observation of alterations in bacterial morphology utilizing field emission scanning electron microscopy. Meanwhile, the ADMET of compound B9OC was predicted using a computer-aided method. The findings revealed that B9OC exhibited lower minimal inhibitory concentrations against all three bacteria compared to berberine alone or in combination with canagliflozin. The minimal inhibitory concentrations (MICs) of B9OC against the three experimental strains were determined to be 0.035, 0.258, and 0.331 mM. However, B9OBu exhibited a lower level of antimicrobial activity compared to berberine. The compound B9OC exhibits a broad spectrum of antibacterial activity by disrupting the integrity of bacterial cell walls, leading to cellular rupture and the subsequent degradation of intracellular proteins.


Introduction
Berberine (BBR, C 20 H 18 NO 4 , MW 336.37, Figure 1), a natural isoquinoline alkaloid, is a pivotal component in numerous traditional Chinese medicines.It can be derived from plants such as the three-leaf Coptis of the Berberidaceae family, Huanglian of the Ranunculaceae family, and Huangbo of the Rutaceae family [1].Commonly encountered as yellow needle-shaped crystals, berberine hydrochloride represents a salt form widely utilized as an oral broad-spectrum antibiotic for gastrointestinal infections.Moreover, its heat-clearing and detoxifying properties have been extensively harnessed over prolonged durations.With its cost-effectiveness and well-established safety and efficacy profile, berberine hydrochloride emerges as an invaluable naturally derived extract in traditional medicine.The extensive pharmacological activities of berberine are attributed to its potent antibacterial and antiviral properties, as well as its ability to regulate blood glucose levels, protect cardiovascular health, modulate metabolism, and exhibit anti-cancer effects.Consequently, it finds application in the treatment of various diseases [2][3][4][5].Canagliflozin (CAN, C 24 H 25 FO 5 S, MW 444.52, Figure 1) is a sodium-glucose co-transporter 2 (SGLT2) inhibitor Molecules 2024, 29, 273 2 of 14 commonly used in the treatment of diabetes.Studies in the literature have demonstrated that the combination of berberine (BBR) and canagliflozin (CAN) exhibits synergistic effects in reducing hyperglycemia associated with diabetes while minimizing adverse effects [6].Therefore, in our previous study, we synthesized a novel compound BC (Figure 2) by chemically linking canagliflozin at the C13 position of berberine to enhance its hypoglycemic efficacy.Surprisingly, the results revealed that compound BC did not exhibit significant hypoglycemic activity; however, it displayed potent antibacterial properties against Pseudomonas aeruginosa [7].Currently, approximately 700,000 individuals succumb to bacterial infections annually worldwide.Based on the prevailing trajectory, it is projected that by 2050, around 10 million people will perish due to superbug infections [8].Concurrently, there is a continuous rise in bacterial resistance towards antibiotics [9].Consequently, the urgent imperative within the global healthcare domain lies in the discovery of novel antibiotics capable of combating superbugs.Recently conducted studies on the antibacterial activity of berberine derivatives have revealed that modifications at positions C8, 9, and 12, in addition to position C13, can significantly enhance the efficacy of berberine against bacteria.Based on our comprehensive understanding of the properties of both berberine and canagliflozin, as well as extensive literature research, we propose a hypothesis that conjugating canagliflozin at position C9 of berberine may result in a compound exhibiting remarkably potentiated antibacterial effects (see Figure 3) [10][11][12][13].Building upon this premise, we have devised and synthesized a derivative of berberine known as B9OC, with an aim to explore its antibacterial activity and underlying mechanism.
strated that the combination of berberine (BBR) and canagliflozin (CAN) e gistic effects in reducing hyperglycemia associated with diabetes while m verse effects [6].Therefore, in our previous study, we synthesized a novel c (Figure 2) by chemically linking canagliflozin at the C13 position of berber its hypoglycemic efficacy.Surprisingly, the results revealed that compoun exhibit significant hypoglycemic activity; however, it displayed potent antib erties against Pseudomonas aeruginosa [7].Currently, approximately 700,00 succumb to bacterial infections annually worldwide.Based on the prevailin is projected that by 2050, around 10 million people will perish due to super [8].Concurrently, there is a continuous rise in bacterial resistance towards Consequently, the urgent imperative within the global healthcare domain covery of novel antibiotics capable of combating superbugs.Recently cond on the antibacterial activity of berberine derivatives have revealed that m positions C8, 9, and 12, in addition to position C13, can significantly enhan of berberine against bacteria.Based on our comprehensive understanding ties of both berberine and canagliflozin, as well as extensive literature rese pose a hypothesis that conjugating canagliflozin at position C9 of berberine a compound exhibiting remarkably potentiated antibacterial effects (see Fig Building upon this premise, we have devised and synthesized a derivativ known as B9OC, with an aim to explore its antibacterial activity and unde nism.strated that the combination of berberine (BBR) and canagliflozin (CAN) e gistic effects in reducing hyperglycemia associated with diabetes while m verse effects [6].Therefore, in our previous study, we synthesized a novel (Figure 2) by chemically linking canagliflozin at the C13 position of berber its hypoglycemic efficacy.Surprisingly, the results revealed that compoun exhibit significant hypoglycemic activity; however, it displayed potent antib erties against Pseudomonas aeruginosa [7].Currently, approximately 700,00 succumb to bacterial infections annually worldwide.Based on the prevailin is projected that by 2050, around 10 million people will perish due to super [8].Concurrently, there is a continuous rise in bacterial resistance towards Consequently, the urgent imperative within the global healthcare domain covery of novel antibiotics capable of combating superbugs.Recently cond on the antibacterial activity of berberine derivatives have revealed that m positions C8, 9, and 12, in addition to position C13, can significantly enhan of berberine against bacteria.Based on our comprehensive understanding ties of both berberine and canagliflozin, as well as extensive literature res pose a hypothesis that conjugating canagliflozin at position C9 of berberine a compound exhibiting remarkably potentiated antibacterial effects (see Fig Building upon this premise, we have devised and synthesized a derivativ known as B9OC, with an aim to explore its antibacterial activity and unde nism.

Effects of B9OC on E. Coli, S. aureus, and P. aeruginosa Growth
The effects of B9OC, BBR, CAN, B9OBU, and BBR + CAN on the growth of S. aureus, E. coli, and P. aeruginosa were evaluated (refer to Figure 4).Among these drugs, B9OC, BBR, and B9OBU demonstrated significant inhibitory effects on the growth of all three bacterial strains.Notably, B9OC exhibited the strongest bacteriostatic effect.Compared to BBR, drug B9OC displayed a more potent inhibition against the growth of S. aureus and E. coli.No significant differences were observed in the effects of either BBR or its combination with CAN (BBR + CAN) on the growth of these bacterial strains.Furthermore, CAN did not exert any notable impact on the growth of any experimental bacteria strains.
Molecules 2024, 29, x FOR PEER REVIEW 5 of 15 E. coli.No significant differences were observed in the effects of either BBR or its combination with CAN (BBR + CAN) on the growth of these bacterial strains.Furthermore, CAN did not exert any notable impact on the growth of any experimental bacteria strains.

Antibiofilm Activity of B9OC
The anti-biofilm efficacy of B9OC was investigated on three test strains using the crystal violet staining method (Figures 5 and 6).B9OC, BBR, and BBR + CAN exhibited inhibitory effects on biofilm formation of all three test strains at MIC and 1/2MIC concentrations.Among them, B9OC demonstrated the most potent inhibitory effect.The combination of BBR + CAN showed a stronger impact on bacterial biofilms compared to that of BBR alone.CAN had no significant effect on S. aureus biofilms, while its influence on E. coli and P. aeruginosa biofilms was weaker.At 1/2MIC concentration, B9OBU did not exhibit a significant effect on S. aureus and E. coli biofilms.Figure 5 visually presents the crystal violet staining results of biofilms adhered to the pore walls, highlighting that B9OC has the greatest impact on S. aureus biofilms.Therefore, it can be inferred that the antibacterial mechanism of action for B9OC is associated with disrupting bacterial biofilms.

Antibiofilm Activity of B9OC
The anti-biofilm efficacy of B9OC was investigated on three test strains using the crystal violet staining method (Figures 5 and 6).B9OC, BBR, and BBR + CAN exhibited inhibitory effects on biofilm formation of all three test strains at MIC and 1/2MIC concentrations.Among them, B9OC demonstrated the most potent inhibitory effect.The combination of BBR + CAN showed a stronger impact on bacterial biofilms compared to that of BBR alone.CAN had no significant effect on S. aureus biofilms, while its influence on E. coli and P. aeruginosa biofilms was weaker.At 1/2MIC concentration, B9OBU did not exhibit a significant effect on S. aureus and E. coli biofilms.Figure 5 visually presents the crystal violet staining results of biofilms adhered to the pore walls, highlighting that B9OC has the greatest impact on S. aureus biofilms.Therefore, it can be inferred that the antibacterial mechanism of action for B9OC is associated with disrupting bacterial biofilms.

Sds-Page Analysis
The effects of B9OC, BBR, CAN, and BBR + CAN on the protein levels of Staphylococcus aureus were analyzed using SDS-PAGE (Figures 7 and 8).It can be observed that the protein bands of Staphylococcus aureus treated with B9OC exhibited a noticeable decrease in intensity.Therefore, it can be inferred that B9OC exerts a detrimental effect on the proteins within Staphylococcus aureus.The changes in protein bands after treatment with BBR and CAN were not statistically significant.Although the protein bands slightly decreased in intensity after treatment with BBR + CAN, it was not as pronounced as observed with B9OC.Based on the analysis of the experimental results, it can be concluded that the antibacterial mechanism of action for B9OC is associated with its disruption of bacterial proteins.
Molecules 2024, 29, x FOR PEER REVIEW 5 of 15 E. coli.No significant differences were observed in the effects of either BBR or its combination with CAN (BBR + CAN) on the growth of these bacterial strains.Furthermore, CAN did not exert any notable impact on the growth of any experimental bacteria strains.

Antibiofilm Activity of B9OC
The anti-biofilm efficacy of B9OC was investigated on three test strains using the crystal violet staining method (Figures 5 and 6).B9OC, BBR, and BBR + CAN exhibited inhibitory effects on biofilm formation of all three test strains at MIC and 1/2MIC concentrations.Among them, B9OC demonstrated the most potent inhibitory effect.The combination of BBR + CAN showed a stronger impact on bacterial biofilms compared to that of BBR alone.CAN had no significant effect on S. aureus biofilms, while its influence on E. coli and P. aeruginosa biofilms was weaker.At 1/2MIC concentration, B9OBU did not exhibit a significant effect on S. aureus and E. coli biofilms.Figure 5 visually presents the crystal violet staining results of biofilms adhered to the pore walls, highlighting that B9OC has the greatest impact on S. aureus biofilms.Therefore, it can be inferred that the antibacterial mechanism of action for B9OC is associated with disrupting bacterial biofilms.

Sds-Page Analysis
The effects of B9OC, BBR, CAN, and BBR + CAN on the protein levels of Staphylococcus aureus were analyzed using SDS-PAGE (Figures 7 and 8).It can be observed that the protein bands of Staphylococcus aureus treated with B9OC exhibited a noticeable decrease in intensity.Therefore, it can be inferred that B9OC exerts a detrimental effect on the proteins within Staphylococcus aureus.The changes in protein bands after treatment with BBR and CAN were not statistically significant.Although the protein bands slightly decreased in intensity after treatment with BBR + CAN, it was not as pronounced as observed with B9OC.Based on the analysis of the experimental results, it can be concluded that the antibacterial mechanism of action for B9OC is associated with its disruption of bacterial proteins.

Sds-Page Analysis
The effects of B9OC, BBR, CAN, and BBR + CAN on the protein levels of Staphylococcus aureus were analyzed using SDS-PAGE (Figures 7 and 8).It can be observed that the protein bands of Staphylococcus aureus treated with B9OC exhibited a noticeable decrease in intensity.Therefore, it can be inferred that B9OC exerts a detrimental effect on the proteins within Staphylococcus aureus.The changes in protein bands after treatment with BBR and CAN were not statistically significant.Although the protein bands slightly decreased in intensity after treatment with BBR + CAN, it was not as pronounced as observed with B9OC.Based on the analysis of the experimental results, it can be concluded that the antibacterial mechanism of action for B9OC is associated with its disruption of bacterial proteins.

The Morphology of Bacteria Observed by FESEM
The morphology of the bacteria was examined using field emission scanning electron microscopy (FESEM), as depicted in Figure 9.It is evident that the untreated bacterial cells exhibited a smooth and intact surface devoid of any wrinkles or grooves.In contrast, the B9OC-treated bacteria displayed pronounced wrinkling and indentation on their cellular

The Morphology of Bacteria Observed by FESEM
The morphology of the bacteria was examined using field emission scanning electron microscopy (FESEM), as depicted in Figure 9.It is evident that the untreated bacterial cells exhibited a smooth and intact surface devoid of any wrinkles or grooves.In contrast, the B9OC-treated bacteria displayed pronounced wrinkling and indentation on their cellular surfaces, ultimately leading to cellular rupture.This observation strongly suggests that B9OC inflicted damage upon the bacterial cell wall, which aligns with the findings obtained from Sections 2.7 and 2.8.

The Morphology of Bacteria Observed by FESEM
The morphology of the bacteria was examined using field emission scanning electron microscopy (FESEM), as depicted in Figure 9.It is evident that the untreated bacterial cells exhibited a smooth and intact surface devoid of any wrinkles or grooves.In contrast, the B9OC-treated bacteria displayed pronounced wrinkling and indentation on their cellular surfaces, ultimately leading to cellular rupture.This observation strongly suggests that B9OC inflicted damage upon the bacterial cell wall, which aligns with the findings obtained from Sections 2.7 and 2.8.

Synthesis of Berberrubine
The berberine was accurately weighed and transferred into a flask.Subsequently, the flask was introduced into a vacuum drying oven under a vacuum pressure of 20 to 30 mmHg and subjected to heating at 195 • C for a duration of 0.5-1 h.During this process, the yellow solid transformed into a dark red color and subsequently cooled down to room temperature [14][15][16].Following that, the samples were dissolved in methanol and dichloromethane before being subjected to silica gel column chromatography (eluate: DCM:MeOH = 10:1).The resulting product was collected, yielding red solid berberrubine (Figure 10).

Synthesis of Berberrubine
The berberine was accurately weighed and transferred into a flask.Subseque flask was introduced into a vacuum drying oven under a vacuum pressure of mmHg and subjected to heating at 195 °C for a duration of 0.5-1 h.During this the yellow solid transformed into a dark red color and subsequently cooled down temperature [14][15][16].Following that, the samples were dissolved in methanol chloromethane before being subjected to silica gel column chromatography DCM:MeOH = 10:1).The resulting product was collected, yielding red solid berbe (Figure 10).

Synthesis of Canagliflozin Bromide (Br-C)
Canagliflozin (1 equivalent) was dissolved in anhydrous acetonitrile, followe addition of N-Bromosuccinimide NBS (2.5 equivalents) and Triphenylphosphine P equivalents) under cooled conditions.The temperature was then raised to 50 stirred for 5 h.After cooling to room temperature, the acetonitrile solvent was r through spin evaporation, and the product was further purified using silica gel chromatography with a mixture of DCM:MeOH = 40:1 as the eluent.Finally, foamy solid, canagliflozin bromide (Br-C), was obtained (Figure 11).

Synthesis of Canagliflozin Bromide (Br-C)
Canagliflozin (1 equivalent) was dissolved in anhydrous acetonitrile, followed by the addition of N-Bromosuccinimide NBS (2.5 equivalents) and Triphenylphosphine PPh 3 (3.5 equivalents) under cooled conditions.The temperature was then raised to 50 • C and stirred for 5 h.After cooling to room temperature, the acetonitrile solvent was removed through spin evaporation, and the product was further purified using silica gel column chromatography with a mixture of DCM:MeOH = 40:1 as the eluent.Finally, a white foamy solid, canagliflozin bromide (Br-C), was obtained (Figure 11).

Synthesis of Berberrubine
The berberine was accurately weighed and transferred into a flask.Subsequently flask was introduced into a vacuum drying oven under a vacuum pressure of 20 t mmHg and subjected to heating at 195 °C for a duration of 0.5-1 h.During this pro the yellow solid transformed into a dark red color and subsequently cooled down to r temperature [14][15][16].Following that, the samples were dissolved in methanol and chloromethane before being subjected to silica gel column chromatography (elu DCM:MeOH = 10:1).The resulting product was collected, yielding red solid berberru (Figure 10).

Synthesis of Canagliflozin Bromide (Br-C)
Canagliflozin (1 equivalent) was dissolved in anhydrous acetonitrile, followed by addition of N-Bromosuccinimide NBS (2.5 equivalents) and Triphenylphosphine PPh3 equivalents) under cooled conditions.The temperature was then raised to 50 °C stirred for 5 h.After cooling to room temperature, the acetonitrile solvent was remo through spin evaporation, and the product was further purified using silica gel col chromatography with a mixture of DCM:MeOH = 40:1 as the eluent.Finally, a w foamy solid, canagliflozin bromide (Br-C), was obtained (Figure 11).

Synthesis of 9-Berberrubine-(9→6 ′ )-O-canagliflozin Derivative (B9OC)
After consulting the synthetic methodologies employed by other researchers [17][18][19] without yielding any positive outcomes, we endeavored to modify the experimental conditions.Following numerous attempts, a suitable synthesis approach was eventually established.Berberrubine (1.2 equivalents), canagliflozin bromide (1 equivalent), and sodium tert-butyl alcohol (2 equivalents) were dissolved in 20 mL of anhydrous acetonitrile under argon protection, with the temperature set at 60 • C for stirring over an 8 h period.The solvent was subsequently removed via rotary evaporation.Purification was accomplished using dichloromethane as the eluent through silica gel column chromatography.This process led to the isolation of the yellow solid product known as 9-berberrubine-(9→6 ′ )-O-canagliflozin derivative (B9OC) (Figure 12).dium tert-butyl alcohol (2 equivalents) were dissolved in 20 mL of anhydrous acetonitrile under argon protection, with the temperature set at 60 °C for stirring over an 8 h period.The solvent was subsequently removed via rotary evaporation.Purification was accomplished using dichloromethane as the eluent through silica gel column chromatography.This process led to the isolation of the yellow solid product known as 9-berberrubine-(9→6′)-O-canagliflozin derivative (B9OC) (Figure 12).

Synthesis of Berberine 9 Oxybutyl Derivative (B9OBU)
The berberrubine (1 equivalents), n-butane bromide (3 equivalents), and potassium carbonate (3 equivalents) were dissolved in anhydrous DMF and stirred at a temperature of 80 °C for a duration of 4 h [20,21].Following the removal of the solvent, purification was carried out using silica gel column chromatography to obtain a yellow solid derivative known as berberine 9 oxybutyl derivative (B9OBU) (Figure 13).

Determination of Minimum Inhibitory Concentrations 80 (MIC80)
The bacterial strains Staphylococcus aureus (S. aureus, 0485U), Escherichia coli (E.coli, 0335U), and Pseudomonas aeruginosa (P.aeruginosa, BNCC125486) were cultured in LB medium and incubated overnight at 37 °C in a shaking incubator.The culture was

Synthesis of Berberine 9 Oxybutyl Derivative (B9OBU)
The berberrubine (1 equivalents), n-butane bromide (3 equivalents), and potassium carbonate (3 equivalents) were dissolved in anhydrous DMF and stirred at a temperature of 80 • C for a duration of 4 h [20,21].Following the removal of the solvent, purification was carried out using silica gel column chromatography to obtain a yellow solid derivative known as berberine 9 oxybutyl derivative (B9OBU) (Figure 13).

Synthesis of Berberine 9 Oxybutyl Derivative (B9OBU)
The berberrubine (1 equivalents), n-butane bromide (3 equivalents), and potassium carbonate (3 equivalents) were dissolved in anhydrous DMF and stirred at a temperature of 80 °C for a duration of 4 h [20,21].Following the removal of the solvent, purification was carried out using silica gel column chromatography to obtain a yellow solid derivative known as berberine 9 oxybutyl derivative (B9OBU) (Figure 13).

Determination of Minimum Inhibitory Concentrations 80 (MIC80)
The bacterial strains Staphylococcus aureus (S. aureus, 0485U), Escherichia coli (E.coli, 0335U), and Pseudomonas aeruginosa (P.aeruginosa, BNCC125486) were cultured in LB medium and incubated overnight at 37 °C in a shaking incubator.The culture was

Determination of Minimum Inhibitory Concentrations 80 (MIC 80 )
The bacterial strains Staphylococcus aureus (S. aureus, 0485U), Escherichia coli (E.coli, 0335U), and Pseudomonas aeruginosa (P.aeruginosa, BNCC125486) were cultured in LB medium and incubated overnight at 37 • C in a shaking incubator.The culture was subsequently transferred to aseptic LB medium and incubated until reaching the logarithmic growth phase prior to utilization.The microdilution method was employed to determine the minimum inhibitory concentrations (MICs) of 9-berberrubine-(9→6 ′ )-O-canagliflozin derivative (B9OC), berberine (BBR), canagliflozin (CAN), berberine 9 oxybutyl derivative (B9OBU), and a combination of berberine and canagliflozin (B + C) against S. aureus, E. coli, and P. aeruginosa.In brief, 95 µL of bacterial solution containing 5 × 10 5 CFU/mL was mixed with 5 µL of B9OC, BBR, CAN, BBR + CAN, or B9OBU at various dilutions in a 96-well plate to achieve concentration gradients ranging from 0.01 mM to 5.12 mM for each drug; triplicates were performed for each concentration gradient.DMSO and dichloromethane were used as controls.The plate was then incubated at 37 • C for 24 h followed by measuring the OD 600 using an enzyme-linked immunosorbent assay reader.
MIC was defined as the drug concentration that exhibited antimicrobial activity equal to or above 80%.

Growth Curve
After the bacterial strains reached logarithmic growth phase, 95 µL of a bacterial solution (5 × 10 5 CFU/mL) and 5 µL of B9OC, BBR, CAN, BBR + CAN, and B9OBU were added to a 96-well plate in order to achieve a concentration of 1/2 MIC for each drug.This process was repeated three times in parallel.A control group containing medium with 0.1% (v/v) DMSO and dichloromethane was also included.The plate was then incubated at 37 • C for 12 h and subsequently placed on a multifunctional microplate reader to measure OD 600 at one-hour intervals over the course of 12 h.

Biofilm Growth
To investigate the antibacterial mechanism of the synthesized product B9OC, we determined its effect on bacterial biofilms.Bacterial cultures containing S. aureus, E. coli, and P. aeruginosa were adjusted to an optical density (OD 620 ) of 0.1 and added to 96-well plates.The cultures were then treated with B9OC, BBR, CAN, BBR + CAN, and B9OBU at MIC and 1/2 MIC concentrations, respectively.After incubating at 37 • C for 24 h, the bacteria in the microplate were fixed with paraformaldehyde for 30 min.Following the removal of paraformaldehyde, the plate was dried at 55 • C before adding 200 µL of a 0.1% crystal violet dye which was left to stain at room temperature for 10 min.The wells were cleaned with sterile water to remove excess dye and then dried again at 55 • C. Subsequently, we added 200 µL of a solution containing glacial acetic acid (33%) which was left at 37 • C for another half hour to fully dissolve the attached crystal violet dye, finally measuring absorbance values at a wavelength of 570 nm using a microplate reader.Each group had three biological replicates set up as samples, and statistical analysis was performed to calculate any differences.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE was employed to analyze the impact of B9OC on S. aureus proteins.The preparation of SDS-PAGE gels followed the instructions provided by the kit manufacturer.S. aureus cells, treated with PBS and 1/2MIC of CAN, BBR, B + C, B9OBU, and B9OC for 24 h, were collected through centrifugation at 10,000× g for 5 min and subsequently washed twice with PBS.Protein loading buffer was added and boiled at 100 • C for 20 min; thereafter, the supernatant was obtained via centrifugation at 10,000× g for 5 min.The protein concentration was determined using the BCA Protein Concentration Assay kit (Beyotime, Shanghai, China) and adjusted uniformly across all samples to achieve a total loading volume of 10 µg.Following sample loading, a voltage of 60 V was applied for approximately 30-35 min before increasing it to 120 V for about an additional hour.Subsequently, Coomassie Brilliant blue ultrafast staining solution was used to decolorize the electrophoresed protein bands in order to isolate them.

Field Emission Scanning Electron Microscopy (FESEM)
Staphylococcus aureus at a concentration of 1 × 10 8 CFU/mL was incubated with PBS and B9OC at half the minimum inhibitory concentration (MIC) for 24 h at 37 • C. The bacteria were then collected, fixed in 2.5% glutaraldehyde overnight at 4 • C, and washed three times with PBS.Subsequently, the bacterial precipitates were dehydrated using a series of ethanol concentrations (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min each before being dried at room temperature.Finally, they were observed under a field emission scanning electron microscope (SU8010, Hitachi, Tokyo, Japan).

ADMET Prediction Based on Computer Aided Prediction
We utilized the iDrug platform (Tencent, Shenzhen, China), powered by cloud computing and artificial intelligence, to input compound B9OC and predict its ADMET properties.

Statistical Analysis
GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis.Differences with statistical significance between groups were calculated by an ANOVA followed by Tukey's post hoc test.p < 0.05 was considered statistically significant.

Conclusions
This present study involved the initial pyrolysis of berberine to obtain berberrubine, followed by a classical Wilhelmson synthesis reaction where canagliflozin bromide was linked to berberine, resulting in the successful synthesis of the desired product B9OC.The minimal inhibitory concentration (MIC) of B9OC against the three bacteria demonstrated its superior antibacterial activity compared to berberine, while B9OBU exhibited lower antibacterial activity than berberine.Hence, it can be concluded that simply elongating the carbon chain length of C9 alone does not directly enhance the antibacterial activity of berberine.Compared to our previous research product BC, B9OC demonstrates enhanced bactericidal effects against Staphylococcus aureus and Escherichia coli.The findings from crystal violet staining, SDS-PAGE analysis, and FESEM observations indicate that B9OC possesses the ability to disrupt bacterial biofilms and target intracellular proteins.Moreover, it induces significant surface alterations such as wrinkling, indentation, and roughness on bacterial cell walls, ultimately leading to cell rupture (Figure 14).We employed computer-assisted prediction to evaluate the ADMET properties of compound B9OC.In the subsequent studies, we will further validate the pharmacokinetic parameters of compound B9OC through rigorous experimentation and analysis to ensure their accuracy and reliability.
for the statistical analysis.Differences with statistical significance betwee calculated by an ANOVA followed by Tukey's post hoc test.p < 0.05 was c tistically significant.

Conclusions
This present study involved the initial pyrolysis of berberine to obtain followed by a classical Wilhelmson synthesis reaction where canagliflozin linked to berberine, resulting in the successful synthesis of the desired prod minimal inhibitory concentration (MIC) of B9OC against the three bacteria its superior antibacterial activity compared to berberine, while B9OBU e antibacterial activity than berberine.Hence, it can be concluded that sim the carbon chain length of C9 alone does not directly enhance the antibact berberine.Compared to our previous research product BC, B9OC demonst bactericidal effects against Staphylococcus aureus and Escherichia coli.The crystal violet staining, SDS-PAGE analysis, and FESEM observations indi possesses the ability to disrupt bacterial biofilms and target intracellular pr ver, it induces significant surface alterations such as wrinkling, indentati ness on bacterial cell walls, ultimately leading to cell rupture (Figure 14).computer-assisted prediction to evaluate the ADMET properties of comp the subsequent studies, we will further validate the pharmacokinetic para pound B9OC through rigorous experimentation and analysis to ensure the reliability.

Discussion
Research has demonstrated that diabetes can lead to compromised immune function, resulting in urinary tract infections being one of the major complications of the disease [23].The primary pathogens responsible for these infections include Staphylococcus aureus and Escherichia coli [24].Studies have indicated that diabetic patients use SGLT2 inhibitors such as canagliflozin, dapagliflozin, and empagliflozin are at an increased risk of developing genital infections compared to those using placebo or other active treatments [25,26].In recent years, many countries and regions have been facing the challenge of an aging population, with diabetes emerging as one of the survival risks for elderly individuals.Furthermore, research has also shown that antimicrobial resistance among common pathogens plays a crucial role in reducing mortality rates among elderly patients with urinary tract infections [27,28].Therefore, our study on synthesizing a novel compound B9OC holds

Figure 3 .
Figure 3. Structures of berberine derivative with antimicrobial activity at position C9.

Table 2 .
ADMET prediction of B9OC based on computer-aided prediction.