Preparation method and antibacterial application of honokiol derivative

Abstract

The invention discloses a preparation method and antibacterial application of honokiol derivatives with antibacterial activity. The structure of the honokiol derivatives is shown as a general formula I. The natural product honokiol is taken as a starting raw material, the amine functional group is introduced through Mannich reaction to prepare the novel derivative, and the synthesis method is simple and convenient to operate, few in steps and easy for large-scale production. In-vitro activity evaluation shows that the derivative not only has good antibacterial activity on gram-positive and gram-negative bacteria, but also can effectively inhibit the formation of a biofilm, and the water solubility of the derivative is also improved. The invention is of great significance to development and research of new antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a new method for preparing natural and magnolol derivatives and their antibacterial applications. Background Technology

[0002] Bacteria pose a pervasive threat to human and animal health, not only causing serious systemic infections but also often lurking through daily contact and diet. For example, Staphylococcus aureus can rapidly multiply and produce heat-resistant enterotoxins in protein-rich foods (such as cakes and cooked meat products). These toxins are difficult to destroy even with high-temperature cooking, and ingestion can cause symptoms such as projectile vomiting and abdominal cramps, and in severe cases, even dehydration or collapse. According to the World Health Organization, on average, one in ten hospitalized patients worldwide develops a hospital-acquired infection. In some low- and middle-income countries, the mortality rate is as high as 40%, especially in intensive care units, where mortality rates associated with drug-resistant bacterial infections are significantly increased. With the increasing overuse and misuse of antibiotics, the problem of bacterial resistance is becoming more severe, resulting in extremely limited clinical treatment options, thus prolonging the course of illness, increasing mortality, and imposing a heavy economic burden on healthcare. In addition, bacterial infections in animal husbandry also cause huge economic losses. For example, pathogenic Escherichia coli in birds can cause large-scale disease outbreaks in livestock and poultry, resulting in global losses of hundreds of billions of dollars annually in the livestock industry, seriously impacting food safety and economic stability. Therefore, developing novel antibacterial drugs with broad-spectrum antibacterial activity and the ability to overcome drug resistance has become an urgent task in the global public health field.

[0003] Natural products have long been a crucial source for the discovery of novel drug molecules. Magnolol, a natural bisphenol active ingredient extracted from the traditional Chinese medicine Magnolia officinalis, possesses broad-spectrum antibacterial activity. However, the poor water solubility and low antibacterial rate of natural product active molecules limit their application in pharmaceuticals and food preservation. Structural modification of magnolol through chemical synthesis holds promise for enhancing its antibacterial activity while improving its physicochemical properties, opening up broader prospects for the theoretical expansion and practical application of natural products in the field of antibacterial therapy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a series of new magnolol derivatives;

[0005] The second objective of this invention is to provide a method for synthesizing magnolol derivatives.

[0006] A third objective of this invention is to provide applications of magnolol derivatives.

[0007] The objective of this invention is achieved through the following technical solution: and magnolol derivatives having a chemical structure as represented by general formula I:

[0008]

[0009] R1 or R2 represents amine compounds that are substituted on the benzene ring.

[0010] Furthermore, the amine-containing group is any one of the following:

[0011]

[0012] Furthermore, the aforementioned magnolol derivatives have the following chemical structures:

[0013]

[0014] The synthetic method and synthetic route for magnolol derivatives are as follows:

[0015]

[0016] Preferably, the molar ratio of magnolol to formaldehyde aqueous solution is 1:1 to 1:6.

[0017] Preferably, the molar ratio of magnolol to amine reagent is 1:1 to 1:6.

[0018] Preferably, the volume ratio of ethanol to acetic acid is 1:10 to 10:1.

[0019] Preferably, the substitution reaction is carried out at a temperature of 25–120°C for a time of 1–10 h.

[0020] The present invention has the following advantages:

[0021] (1) The magnolol derivatives disclosed in this invention are a series of novel compounds, which are proposed for the first time. In addition, this invention also provides a method for synthesizing the magnolol derivatives, which uses magnolol as a raw material and obtains the magnolol derivatives through a series of reactions. The yield is high, the derivatives are easy to separate, and the water solubility of the derivatives is improved.

[0022] (2) Compared with magnolol derivatives synthesized in this invention, magnolol showed better antibacterial activity against Gram-positive and Gram-negative bacteria. Attached Figure Description

[0023] Figure 1 The target compound 5 has a hydrogen spectrum.

[0024] Figure 2 Carbon 5 spectrum data of the target compound;

[0025] Figure 3 Mass spectrum data for target compound 5;

[0026] Figure 4 The target compound 5 was used to inhibit the MBC of Staphylococcus aureus.

[0027] Figure 5 The target compound 5 inhibited the inhibition zone of Staphylococcus aureus;

[0028] Figure 6 Time-growth curve of target compound 5 inhibiting Staphylococcus aureus;

[0029] Figure 7 The target compound 5 was used to inhibit biofilm formation in Staphylococcus aureus.

[0030] Figure 8 The results of laser confocal microscopy observation of the effect of target compound 5 on inhibiting the biofilm of Staphylococcus aureus;

[0031] Figure 9 To inhibit the MBC value of MRSA by target compound 5;

[0032] Figure 10 The inhibition zone of target compound 5 inhibits MRSA;

[0033] Figure 11 The time-growth curve for the inhibition of MRSA by target compound 5;

[0034] Figure 12 Compound 5 was used to inhibit biofilm formation in MRSA.

[0035] Figure 13 To observe the results of target compound 5 on inhibiting MRSA biofilm using laser confocal microscopy; Detailed Implementation

[0036] Example 1: Synthesis of Compound 1

[0037] Take a three-necked flask, add magnolol (1.00 g, 3.76 mmol), and dissolve it in ethanol and acetic acid (20 mL: 4 mL). Add formaldehyde aqueous solution (0.50 g, 15.02 mmol) and ethanolamine (0.65 g, 11.28 mmol). Under nitrogen protection, heat the resulting mixture under reflux and stir for 10 h. After the solution cools to room temperature, remove the solvent by rotary evaporation under reduced pressure. Neutralize acetic acid with saturated sodium bicarbonate, extract with ethyl acetate, dry and concentrate, and then perform rapid silica gel column chromatography to give compound 1 (yellow solid, 95%).

[0038] 1H NMR (600 MHz, Chloroform-d) δ 7.17 (d, J = 2.2 Hz, 1H), 7.08 (d, J= 2.2 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.70 (d, J = 2.2 Hz, 1H), 6.02 –5.83 (m, 2H), 5.06 – 4.96 (m, 4H), 4.84 (d, J = 18.7 Hz, 2H), 3.95 (d, J =14.6 Hz, 4H), 3.67 (dt, J = 15.8, 5.0 Hz, 4H), 3.36 (d, J = 6.6 Hz, 2H), 3.23(d, J = 6.8 Hz, 2H), 2.75 (dt, J = 18.1, 5.1 Hz, 4H). 13 C NMR (151 MHz, Chloroform-d) δ 152.11, 136.99, 136.27, 129.37, 129.11, 128.77, 127.99,126.56, 126.24, 125.84, 121.57, 120.51, 114.36, 114.16, 60.16, 51.48, 49.20,49.10, 38.45, 33.13, 28.68. HRMS (ESI) C 24 H 32 N₂O₄ [M+H] + calcd = 413.2435; found =413.2435.

[0039] Example 2 Synthesis of Compound 5

[0040] Take a three-necked flask, add magnolol (1.00 g, 3.76 mmol), and dissolve it in ethanol and acetic acid (20 mL: 4 mL). Add formaldehyde aqueous solution (0.37 g, 11.28 mmol) and N,N-diethylethylenediamine (0.65 g, 5.63 mmol). Under nitrogen protection, heat the resulting mixture under reflux and stir for 4 h. After the solution cools to room temperature, remove the solvent by rotary evaporation under reduced pressure. Neutralize acetic acid with saturated sodium bicarbonate, extract with ethyl acetate, dry and concentrate, and then perform rapid silica gel column chromatography to give compound 5 (yellow oil, 67%). NMR characterization of compound 5 (… Figure 1-2 ):

[0041] 1H NMR (600 MHz, Chloroform-d) δ 7.19 (d, J = 1.5 Hz, 1H), 7.05 (d, J= 2.1 Hz, 1H), 6.98 – 6.91 (m, 2H), 6.91 – 6.79 (m, 2H), 6.01 – 5.85 (m, 2H),5.06 – 4.93 (m, 4H), 4.05 (q, J = 7.1 Hz, 1H), 3.96 (s, 2H), 3.36 (d, J = 6.7Hz, 2H), 3.27 (d, J = 6.8 Hz, 2H), 2.76 – 2.51 (m, 8H), 1.03 (t, J = 7.3 Hz, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 154.97, 149.97, 136.88, 135.82, 130.93, 129.18, 128.45, 127.46, 127.44, 127.13, 127.03, 126.04, 121.83, 114.54, 114.50, 114.42, 57.38, 57.36, 51.44, 50.71, 45.90, 44.70, 38.41, 32.99, 17.38, 17.37, 10.40. Mass spectrometric characterization of compound 5 ( Figure 3 HRMS (ESI) C 25 H 34 N₂O₂ [M+H] + calcd= 395.2693; found =395.2705.

[0042] Example 3 Synthesis of Compound 10

[0043] In a three-necked flask, add magnolol (0.50 g, 1.88 mmol) dissolved in ethanol and acetic acid (10 mL: 2 mL), then add formaldehyde aqueous solution (0.17 g, 3.76 mmol) and 4-(4-piperidinyl)morpholine (0.33 g, 2.82 mmol). Under nitrogen protection, heat the resulting mixture under reflux with stirring for 6 h. After cooling to room temperature, remove the solvent by rotary evaporation under reduced pressure. Neutralize acetic acid with saturated sodium bicarbonate, extract with ethyl acetate, dry and concentrate, and then perform rapid silica gel column chromatography to give compound 10 (yellow oil, 72%).

[0044] 1H NMR (600 MHz, Chloroform-d) δ 7.28 (d, J = 8.1 Hz, 1H), 7.25 (s,1H), 6.96 (s, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.67 (s, 1H), 6.03 – 5.94 (m,1H), 5.93 – 5.83 (m, 1H), 5.17 – 4.89 (m, 4H), 3.66 (d, J = 5.4 Hz, 4H), 3.62(s, 2H), 3.38 (t, J = 4.8 Hz, 2H), 3.25 – 3.21 (m, 2H), 3.01 (d, J = 11.4 Hz,2H), 2.49 (d, J = 5.7 Hz, 4H), 2.26 (d, J = 11.9 Hz, 1H), 2.04 (t, J = 12.2Hz, 2H), 1.79 (s, 1H), 1.52 (q, J = 12.3 Hz, 2H). 13 C NMR (151 MHz, Chloroform-d) δ 155.56, 150.82, 137.87, 136.65, 132.10, 130.16, 129.64,128.63, 127.98, 127.96, 127.38, 127.25, 121.73, 115.71, 115.52, 115.49,65.89, 49.67, 39.44, 34.07, 15.30. HRMS (ESI) C 28 H 36 N₂O₃ [M+H] + calcd =449.2799; found =449.2797.

[0045] Application Example 1: Determination of MIC values ​​of compounds in Examples 1-3 against Staphylococcus aureus, MRSA, and Escherichia coli

[0046] Accurately pipette 8 μL from the cultured bacterial suspension and add 8 mL of the corresponding liquid culture medium, then thoroughly vortex to prepare a bacterial suspension. Perform serial dilutions of the compounds using the two-fold dilution method. In A1–A... 12 Add 100 μL of liquid culture medium to each well. Add 100 μL of the initial drug solution (1024 μg / mL) to well A1, mix well, and then transfer 100 μL to well A2. Perform serial dilutions to the final concentration in well A2 as needed.10 Kong, discard A 10 100 μL of liquid in the well. A 11 As a blank control, A 12 Wells served as negative controls. 100 μL of bacterial suspension was added to each well, resulting in final working concentrations of the drug in each well of 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL, respectively. The concentrations for negative and blank controls were both 0 μg / mL. Three replicates were set for each concentration. The 96-well plate was incubated at 37°C for 12 h. After incubation, 45 μL of 400 μg / mL resazurin solution was added to each well, and the plate was incubated at 37°C in the dark for an appropriate time. Bacterial survival was assessed by observing the color changes in each well, and the minimum inhibitory concentration (MIC) of the compound was determined accordingly (blue indicates no bacterial growth, red indicates bacterial growth).

[0047] The MIC values ​​of the raw material and magnolol against Staphylococcus aureus, MRSA, and Escherichia coli were 64, 32, and 128 µg / mL, respectively; the MIC values ​​of compound 1 against Staphylococcus aureus, MRSA, and Escherichia coli were 64, 32, and 128 µg / mL, respectively; the MIC values ​​of compound 5 against Staphylococcus aureus, MRSA, and Escherichiacoli were 16, 16, and 128 µg / mL, respectively; and the MIC values ​​of compound 10 against Staphylococcus aureus, MRSA, and Escherichiacoli were 32, 32, and 128 µg / mL, respectively. From the above MIC values, compound 5 showed the best antibacterial effect, exhibiting good antibacterial activity against Staphylococcus aureus and MRSA. The MIC values ​​of compound 5 were 4 times and 2 times higher than those of magnolol, respectively.

[0048] Application Example 2: Determination of MBC value of compound 5 in Staphylococcus aureus

[0049] The minimum bactericidal concentration (MBC) of the compound against Staphylococcus aureus was determined using the broth dilution method. The MIC experiment was repeated, with 100 μL of the solution from each well of a 96-well plate (with no bacterial growth) spread onto TSB agar plates. After complete absorption, the plates were transferred to an incubator and incubated upside down at 37°C for 24 h. The minimum drug concentration on the plate without bacterial growth was the MBC of the compound. The MBC experiment (…) Figure 4It can be seen that compound 5 inhibits Staphylococcus aureus by an MBC value of 2 MIC (32 µg / mL), and can reduce the number of viable MRSA bacteria by 99.9%.

[0050] Application Example 3: Determination of the inhibition zone of compound 5 against Staphylococcus aureus

[0051] The diameter of the inhibition zone of the compound against Staphylococcus aureus was determined using the Oxford cup method. Two sterile Oxford cups with an inner diameter of 6 mm were placed on a plate. Staphylococcus aureus culture grown to the logarithmic growth phase was mixed into TSB agar medium pre-cooled to 50°C, and the bacterial concentration was controlled at 1.0 × 10⁻⁶. 6 CFU / mL, pour plates and allow them to solidify, then remove the Oxford cups. Add 100 μL of a 4 MIC compound solution to the left well and 100 μL of culture medium to the right well. Place the plates in the refrigerator's chiller compartment for 1 h to diffuse, then incubate at 37°C for 24 h. Measure the diameter of the inhibition zone using calipers. The criteria for judging the antibacterial effect are as follows: an inhibition zone diameter (D-DBlank, mm) greater than 2 mm indicates antibacterial activity; an inhibition zone equal to or less than 2 mm indicates no antibacterial activity; an inhibition zone greater than 2 mm and less than 5 mm indicates moderate antibacterial activity; and an inhibition zone greater than 5 mm indicates high antibacterial activity. (Based on the inhibition zone experiment...) Figure 5 It can be seen that the inhibition zone diameter of compound 5 against Staphylococcus aureus is 19.80–24.60 mm.

[0052] Application Example 4: Effect of Compound 5 on the time-growth curve of Staphylococcus aureus

[0053] First, Staphylococcus aureus was activated and passaged, and then diluted to 1.0 × 10⁻⁶. 6 Prepare a bacterial suspension using CFU / mL. Add CFU / mL to each test tube. 6 Staphylococcus aureus bacterial suspension (CFU / mL) and different concentrations of honokiol solution were used to prepare test tubes with final honokiol concentrations of 1 / 2, 1, and 2 MIC, respectively. A control group without honokiol was also included. The test tubes were incubated at 37℃ with shaking (150 r / min), and absorbance at 600 nm was measured at 0, 2, 4, 6, 8, 10, 12, and 24 h. The absorbance was then analyzed using time-growth curves. Figure 6It was found that compound 5 inhibited the growth of *Staphylococcus aureus*, and the inhibitory effect was positively correlated with the concentration of compound 5. Compound 5 at 1 / 2 MIC showed a certain inhibitory effect on the growth of *Staphylococcus aureus*. MIC concentrations of both compound 5 and magnolol significantly prolonged the time for *Staphylococcus aureus* to enter the lag and logarithmic growth phases, and the maximum specific growth rate also decreased significantly. However, no *Staphylococcus aureus* grew in the 2 MIC concentration treatment group because the concentration of compound 5 at this point reached the minimum bactericidal concentration, effectively killing *Staphylococcus aureus*.

[0054] Application Example 5: Effect of Compound 5 on the biofilm formation ability of Staphylococcus aureus

[0055] The concentration added to each well of the 96-well plate was 1.0 × 10⁻⁶. 6 180 μL of a Staphylococcus aureus bacterial suspension (CFU / mL) was mixed with 20 μL of Compound 5 solution by pipetting to achieve a final concentration of magnolol within the range of 1 / 16 MIC to MIC. A Staphylococcus aureus bacterial suspension without Compound 5 was used as a control group. The 96-well plate was then incubated at 37°C for 48 h, after which the bacterial suspension was discarded. Each well was washed three times with sterile PBS buffer and air-dried. The Staphylococcus aureus biofilm was fixed with methanol for 30 min, the methanol was discarded, and the plate was stained with crystal violet for 30 min. After staining, excess stain was rinsed with water, and the plate was air-dried. 33% glacial acetic acid was added to the wells and shaken for 30 min to fully dissolve the dye bound to the biofilm. Finally, the OD was measured using a microplate reader. 595 .Depend on Figure 7 It was found that compounds 1 / 16, 1 / 8, 1 / 4, 1 / 2, and MIC compound 5 all inhibited the formation of biofilm in *Staphylococcus aureus*, and the inhibitory effects of 1 / 2 and MIC compound 5 on biofilm formation were not significantly different. CLSM results ( Figure 8 It was observed that in the untreated control group, Staphylococcus aureus formed a thick and uniform biofilm. However, in the experimental group with compound 5 added, the thickness and density of the biofilm were significantly reduced, indicating that compound 5 inhibited bacterial proliferation in the Staphylococcus aureus biofilm and reduced bacterial activity.

[0056] Application Example 6: Determination of MBC value of Compound 5 against MRSA bacteria

[0057] The steps are as shown in Application Example 2, from the MBC experiment ( Figure 9 It can be seen that compound 5 inhibits MRSA with an MBC value of 2 MIC (32 µg / mL).

[0058] Application Example 7: Determination of the inhibition zone of compound 5 against MRSA bacteria

[0059] The steps are as shown in Application Example 3, based on the inhibition zone experiment ( Figure 10 It can be seen that the diameter of the inhibition zone of compound 5 inhibiting MRSA is 18.80–23.60 mm.

[0060] Application Example 8: Effect of Compound 5 on the time-growth curve of MRSA bacteria

[0061] The steps are as shown in Application Example 4, from Figure 11 It can be seen that compound 5 inhibits the growth of MRSA, and this inhibition is positively correlated with the concentration of compound 5. Observing the growth curve trend, it can be seen that MRSA enters the lag phase at 0.88 h without drug treatment, reaches the logarithmic growth phase within 3 h, and reaches the stationary growth phase at 8 h, after which the bacterial concentration tends to stabilize, with a maximum specific growth rate of 0.36 OD / h. Compound 5 at 1 / 4 and 1 / 2 MIC concentrations has a certain inhibitory effect on MRSA growth. The MIC concentration of compound 5 significantly prolongs the time for MRSA to enter the lag and logarithmic growth phases, and the maximum specific growth rate also decreases significantly. However, no MRSA grows in the 2 MIC concentration treatment group, because at this concentration, compound 5 reaches the minimum bactericidal concentration, effectively killing MRSA.

[0062] Application Example 9: Effect of Compound 5 on the Biofilm Formation Ability of MRSA

[0063] As shown in application example 5, from Figure 12 It was found that compounds 1 / 16, 1 / 8, 1 / 4, 1 / 2, and MIC 5 significantly inhibited the formation of MRSA biofilms. The biofilm formation in the control group was significantly higher than that in each drug concentration group, indicating that the drug has an inhibitory effect on biofilm formation, and within a certain range, the higher the drug concentration, the more obvious the inhibitory effect. Meanwhile, CLSM results ( Figure 13 It was also observed that in the NC group, MRSA formed a thick and uniform biofilm. However, in the MIC group with compound 5 added, the thickness and density of the biofilm were significantly reduced. This indicates that compound 5 inhibits MRSA activity in the MRSA biofilm.

[0064] Application Example 10: Comparison of the saturated solubility of Compound 5 and magnolol

[0065] Excess amounts of magnolol and compound 5 were dissolved separately in ultrapure water to prepare saturated aqueous solutions. The resulting solutions were magnetically stirred at room temperature for 5 min. After standing overnight, the solutions were filtered through a 0.22 μm filter membrane. 0.5 mL of the filtrate was transferred to a dry sample vial, dried, and weighed. The saturated solubility in water was calculated using the following formula:

[0066] Saturated solubility (mg / mL) = (m2 - m1) / 0.5

[0067] In the formula: m1 is the mass of the sample vial (mg); m2 is the total mass of the sample vial and sample after drying (mg). Based on the solubility formula above, the solubility of magnolol is 0.01 mg / mL, and the solubility of compound 5 is 0.2 mg / mL. This indicates that the saturated solubility of compound 5 is 20 times that of magnolol, significantly improving its water solubility.

Claims

1. The structure of magnolol derivatives, characterized in that, The structural formula is as shown in Formula I: R1 or R2 represents amine compounds that are substituted on the benzene ring. Furthermore, the amine-containing group is any one of the following:

2. The structure of the magnolol derivative according to claim 1, characterized in that, The magnolol derivative was obtained by mixing magnolol, formaldehyde aqueous solution and amine reagent and reacting them.

3. The structures of the magnolol derivatives described above have the following chemical structural formulas:

4. The method for synthesizing magnolol derivatives according to claim 2, characterized in that, The synthetic route is as follows:

5. The preparation method according to claim 4, characterized in that, The molar ratio of magnolol to formaldehyde aqueous solution is 1:1 to 1:6; the molar ratio of magnolol to amine reagent is 1:1 to 1:

6.

6. The preparation method according to claim 4, characterized in that, The solvent is methanol-ethanol or acetic acid-hydrochloric acid, wherein the volume ratio is 1:10 to 10:

1.

7. The preparation method according to claim 6, characterized in that, The substitution reaction is carried out at a temperature of 25–120 °C for a time of 1–10 h.

8. The use of the magnolol derivative according to any one of claims 3, characterized in that, The aforementioned magnolol derivatives are used to combat Gram-positive and Gram-negative bacteria and to remove biofilms of Gram-positive and Gram-negative bacteria.

9. An antibacterial agent, characterized in that, The effective mass percentage of the product containing the magnolol derivative as described in claim 3 is 0.01%-99.99%.

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