Berberine derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides a berberine derivative as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing 3-cyclopentylhexanoic acid, a first organic solvent, oxalyl chloride and anhydrous DMF (Dimethyl Formamide), and reacting to obtain an intermediate I; performing high-temperature negative-pressure pyrolytic reaction on berberine hydrochloride, mixing and dissolving berberine hydrochloride with alcohol, and performing free radical substitution reaction to obtain an intermediate II; and mixing the intermediate II, a second organic solvent, the intermediate I and pyridine, and reacting to obtain the berberine derivative. The berberine derivative has broad-spectrum antibacterial activity and high biological safety, has excellent antibacterial and bacteriostatic activity on drug-resistant staphylococcus aureus, pseudomonas aeruginosa and escherichia coli, can overcome the limitation of traditional berberine drugs, becomes a new-generation antibacterial drug, meets the requirements of current clinical antibiotic substitutes, and has broad application prospects. And an innovative treatment scheme is provided for solving the problem of bacterial drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a berberine derivative and a preparation method and application thereof. Background Art

[0002] With the increasing bacterial resistance worldwide, the research and development of antibacterial drugs faces severe challenges. The widespread use of traditional antibiotics has led to drug resistance in many pathogens, especially the rapid spread of drug-resistant strains in hospital environments, which greatly reduces the effectiveness of clinical treatment. The existing antibacterial drugs have reduced therapeutic effects when facing new drug-resistant strains, and many common infectious diseases have become more difficult to control. Therefore, the development of new antibacterial drugs has become an urgent task for the pharmaceutical industry and global public health today. Berberine is a natural alkaloid widely found in Chinese herbal medicines. It has multiple pharmacological activities, especially in antibacterial, antiviral and anti-inflammatory aspects. It is expected to be a potential antibacterial drug. However, due to the poor water solubility, poor absorption and rapid metabolism of berberine in the intestine, its effective concentration in the body is low, resulting in low bioavailability, which greatly limits the antibacterial activity and application of berberine. Secondly, berberine has a narrow antibacterial spectrum, mainly targeting Gram-negative bacteria in the intestine, and has a weak effect on Gram-positive bacteria and other bacteria. Therefore, the application of berberine in antibacterial therapy still has great limitations.

[0003] Therefore, there is an urgent need to provide a new berberine derivative with broad-spectrum antibacterial activity, high bioavailability, effectiveness against drug-resistant strains, and low toxicity, so as to solve the problem of bacterial resistance existing in clinical treatment of existing antibacterial drugs. Summary of the invention

[0004] In view of this, the present invention provides a berberine derivative and a preparation method and application thereof to solve the problem of bacterial resistance in clinical treatment.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides a berberine derivative, and the structural formula of the berberine derivative is as follows:

[0007]

[0008] The present invention also provides a method for preparing a berberine derivative, comprising the following steps:

[0009] 1) mixing 3-cyclopentanehexylpropionic acid, a first organic solvent, oxalyl chloride and anhydrous DMF, and reacting to obtain intermediate I;

[0010] 2) subjecting berberine hydrochloride to a high temperature negative pressure pyrolysis reaction, and then dissolving it in alcohol to undergo a free radical substitution reaction to obtain intermediate II;

[0011] 3) mixing the intermediate II, the second organic solvent, the intermediate I and pyridine, and reacting them to obtain the berberine derivative of formula I;

[0012] There is no order restriction for step 1) and step 2);

[0013] The structural formula of the intermediate I is

[0014] The structural formula of the intermediate II is

[0015] Preferably, the mass volume ratio of 3-cyclopentanehexylpropionic acid, the first organic solvent, oxalyl chloride and anhydrous DMF in step 1) is 100 mg: 5-10 mL: 178 mg: 5-10 mL; the first organic solvent includes one or more of anhydrous dichloromethane, anhydrous dichloroethane, anhydrous ether and anhydrous chloroform.

[0016] Preferably, the reaction temperature in step 1) is -20 to 0°C and the reaction time is 1 to 2 hours.

[0017] Preferably, in step 2), the mass volume ratio of berberine hydrochloride to alcohol is 1 g: 50-100 mL; the alcohol includes one or both of anhydrous ethanol and anhydrous methanol.

[0018] Preferably, the temperature of the high-temperature negative pressure pyrolysis reaction in step 2) is 200 to 300° C., the pressure is -0.01 to -0.05 MPa, and the time is 30 to 60 min; the time of the free radical substitution reaction is 3 to 5 min.

[0019] Preferably, in step 3), the mass volume ratio of intermediate II, the second organic solvent, intermediate I and pyridine is 100 mg: 5-10 mL: 0.2-0.4 mL: 0.2-0.3 mL; the second organic solvent comprises one or more of anhydrous acetonitrile, anhydrous DMF and anhydrous dioxane.

[0020] Preferably, the reaction temperature in step 3) is 20-30°C and the reaction time is 1-2h.

[0021] The present invention also provides a use of the berberine derivatives prepared by the preparation method of the berberine derivatives in the preparation of antibacterial drugs.

[0022] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention transforms the structure of berberine so that the obtained berberine derivatives have broad-spectrum antibacterial activity and have excellent antibacterial and antibacterial activity against drug-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P.aeruginosa) and Escherichia coli (E.coli). In addition, compared with berberine (BBR) and the current first-line clinical drugs ampicillin (Ampicillin) and vancomycin (Vancomycin), the berberine derivatives of the present invention have more excellent antibacterial activity against MRSA, P.aeruginosa and E.coli. At the same time, the berberine derivatives of the present invention have higher biosafety than berberine (BBR), better promotion of the repair ability of MRSA-infected wounds in vitro and the therapeutic effect of pneumonia infected by P.aeruginosa bacteria in vivo.

[0024] The berberine derivatives of the present invention can overcome the limitations of traditional berberine drugs, become a new generation of antibacterial drugs, meet the current clinical demand for antibiotic substitutes, and provide an innovative treatment plan for solving the problem of bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The wound healing diagram of 9-(3'-cyclopentylpropionyloxy)berberine prepared in Example 1 on MRSA infected wound repair, wherein: Figure 1 a in the figure is a diagram of the wound healing process. Figure 1 b in the figure is the wound closure trajectory diagram;

[0027] Figure 2 This is a quantitative analysis chart of the change in wound area of ​​MRSA-infected wound repair by 9-(3'-cyclopentylpropionyloxy)berberine prepared in Example 1;

[0028] Figure 3 This is a graph showing the bacterial count on the wound surface of mice infected with MRSA in Experimental Example 2;

[0029] Figure 4 This is a graph showing the bacterial viability of 9-(3'-cyclopentylpropionyloxy)berberine prepared in Example 1 on the 11th day of MRSA-infected wound repair;

[0030] Figure 5This is a graph showing the bacterial load in the lungs of mice infected with P. aeruginosa bacterial pneumonia after treatment with 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1, wherein the ordinate represents the bacterial load;

[0031] Figure 6 This is a graph showing the total protein concentration in the bronchoalveolar lavage fluid of mice infected with P. aeruginosa bacterial pneumonia after treatment with 9-(3'-cyclopentylpropionyloxy)berberine prepared in Example 1;

[0032] Figure 7 This is a graph showing the total number of cells in the bronchoalveolar lavage fluid of mice infected with P. aeruginosa bacterial pneumonia after treatment with 9-(3'-cyclopentylpropionyloxy)berberine prepared in Example 1;

[0033] Figure 8 This is a graph showing the change in wet / dry weight ratio of the lungs of mice infected with P. aeruginosa bacterial pneumonia after treatment with 9-(3'-cyclopentylpropionyloxy)berberine prepared in Example 1;

[0034] Fig. 9 This is a diagram of the alveolar structure of mice infected with P. aeruginosa bacterial pneumonia after treatment with 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1, wherein: Fig. 9 a in the figure is the alveolar structure with a scale bar of 50 μm. Fig. 9 b is a local enlarged view of a, and the scale bar is 20 μm;

[0035] Fig.10 This is a diagram showing the degree of lung damage in mice infected with P. aeruginosa bacterial pneumonia after treatment with 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1;

[0036] Fig.11 This is a graph showing changes in body weight of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4;

[0037] Fig.12 This is a graph showing the heart weight of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4;

[0038] Fig.13 This is a graph showing the liver weight of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4;

[0039] Fig.14 This is a graph showing the spleen weight of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4;

[0040] Fig.15 This is a graph showing the lung weight of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4;

[0041] Fig.16 This is a graph showing the kidney weight of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4;

[0042] Fig.17 This is a graph showing changes in the level of alanine aminotransferase (ALT) in the serum of mice that received different doses of 9-(3'-cyclopentylpropionyloxy) berberine in Experimental Example 4;

[0043] Fig.18 This is a graph showing changes in serum aspartate aminotransferase (AST) levels in mice that received different doses of 9-(3'-cyclopentylpropionyloxy) berberine in Experimental Example 4;

[0044] Fig.19 The figure is a graph showing changes in serum creatinine (Cr) content in mice receiving different doses of 9-(3'-cyclopentylpropionyloxy) berberine in Experimental Example 4;

[0045] Fig. 20 The morphological changes of liver and kidney tissues of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy)berberine in Experimental Example 4 are shown in FIG. DETAILED DESCRIPTION

[0046] The present invention provides a berberine derivative, wherein the berberine derivative is 9-(3'-cyclopentylpropionyloxy) berberine, and its structural formula is as follows:

[0047]

[0048] The present invention also provides a method for preparing a berberine derivative, comprising the following steps:

[0049] 1) mixing 3-cyclopentanehexylpropionic acid, a first organic solvent, oxalyl chloride and anhydrous DMF, and reacting to obtain intermediate I;

[0050] 2) subjecting berberine hydrochloride to a high temperature negative pressure pyrolysis reaction, and then dissolving it in alcohol to undergo a free radical substitution reaction to obtain intermediate II;

[0051] 3) mixing the intermediate II, the second organic solvent, the intermediate I and pyridine, and reacting them to obtain the berberine derivative of formula I;

[0052] There is no order restriction for step 1) and step 2);

[0053] The structural formula of the intermediate I is

[0054] The structural formula of the intermediate II is

[0055] In the present invention, the synthesis route of the berberine derivative is as follows:

[0056]

[0057] In the present invention, the mass volume ratio of 3-cyclopentanehexylpropionic acid, the first organic solvent, oxalyl chloride and anhydrous DMF in step 1) is 100 mg: 5-10 mL: 178 mg: 5-10 mL, more preferably 100 mg: 5 mL: 178 mg: 5 mL.

[0058] In the present invention, in step 1), the first organic solvent comprises one or more of anhydrous dichloromethane, anhydrous dichloroethane, anhydrous ether and anhydrous chloroform.

[0059] In the present invention, the reaction temperature in step 1) is -20 to 0°C, preferably -18 to -1°C, more preferably -15 to -2°C; the reaction time is 1 to 2h, more preferably 1.5h.

[0060] In the present invention, the intermediate I in step 1) is in oily form.

[0061] In the present invention, the mass volume ratio of berberine hydrochloride and alcohol in step 2) is 1g:50-100mL, preferably 1g:55-85mL, more preferably 1g:60-70mL; the alcohol includes one or both of anhydrous ethanol and anhydrous methanol, wherein the alcohol can provide hydroxyl groups for the reaction.

[0062] In the present invention, the temperature of the high-temperature negative pressure pyrolysis reaction in step 2) is 200-300°C, preferably 220-280°C, and more preferably 230°C; the pressure of the high-temperature negative pressure pyrolysis reaction is -0.01-0.05Mpa, preferably -0.02-0.04Mpa, and more preferably -0.03Mpa; the time of the high-temperature negative pressure pyrolysis reaction is 30-60min, preferably 35-55min, and more preferably 40min; wherein the high-temperature negative pressure pyrolysis reaction can break the methyl bond of the benzyl ring methoxy group.

[0063] In the present invention, the time of the free radical substitution reaction in step 2) is 3 to 5 minutes, more preferably 4 minutes.

[0064] In the present invention, the intermediate II in step 2) is in the form of a red solid.

[0065] In the present invention, the mass volume ratio of intermediate II, the second organic solvent, intermediate I and pyridine in step 3) is 100 mg: 5-10 mL: 0.2-0.4 mL: 0.2-0.3 mL, more preferably 100 mg: 10 mL: 0.4 mL: 0.3 mL.

[0066] In the present invention, the second organic solvent in step 3) comprises one or more of anhydrous acetonitrile, anhydrous DMF and anhydrous dioxane.

[0067] In the present invention, the reaction temperature in step 3) is 20-30°C, preferably 22-28°C, more preferably 25°C; the reaction time is 1-2h, more preferably 1.5h.

[0068] In the present invention, after the reaction in step 3) is completed, the process further includes filtering and washing the product 9-(3'-cyclopentylpropionyloxy)berberine.

[0069] In the present invention, the washing solvent preferably includes one or more of anhydrous dichloromethane, anhydrous ethyl acetate and anhydrous ether; the number of washings is preferably 2 to 5 times, more preferably 3 times.

[0070] In the present invention, the 9-(3'-cyclopentylpropionyloxy)berberine is in the form of a yellow solid.

[0071] The present invention also provides a use of the berberine derivatives prepared by the preparation method of the berberine derivatives in the preparation of antibacterial drugs.

[0072] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0073] Example 1

[0074] 1) 3-Cyclopentanepropionic acid (100 mg) was added to 5 mL of anhydrous CH2Cl2, stirred at 0°C for 5 min, and then oxalyl chloride (178 mg) and anhydrous DMF (5 mL) were added. After reacting at 0°C for 1 h, the mixture was dried using a vacuum rotary evaporator to obtain an oily intermediate I;

[0075] 2) Berberine hydrochloride (100 mg) was placed in a round-bottom flask and stirred at 200°C under negative pressure (-0.01 MPa) for 30 min. After the reaction was completed, it was cooled to room temperature, and 5 mL of anhydrous ethanol was added to mix and dissolve to carry out free radical substitution reaction for 4 min. It was then concentrated by vacuum rotary evaporator and purified by column chromatography to obtain a red solid intermediate II.

[0076] 3) Dissolve 50 mg of intermediate II in 5 mL of anhydrous acetonitrile and transfer to a round-bottom flask containing 0.2 mL of intermediate I. Add 3 drops of pyridine and react at 25°C for 1 h. Filter and wash the precipitate with anhydrous dichloromethane solvent 3 times to obtain a yellow solid product 9-(3'-cyclopentylpropionyloxy)berberine.

[0077] The NMR data of 9-(3'-cyclopentylpropionyloxy)berberine are:

[0078] 1 H NMR (400 MHz, DMSO-d 6) δ10.00(s,1H),9.06(s,1H),8.27(d,J=9.3Hz,1H),8.21(d,J=9.2Hz,1H),7. 81(s,1H),7.09(s,1H),6.17(s,2H),4.98(t,J=5.9Hz,2H),4.02(s,3H),3.22 (t,J=6.1Hz,2H),2.88(t,J=7.6Hz,2H),1.98–1.89(m,1H),1.87–1.79(m,2H) ,1.79–1.72(m,2H),1.67–1.59(m,2H),1.58–1.49(m,2H),1.23–1.13(m,2H).

[0079] 13 C NMR(101MHz,DMSO-d6)δ171.20,150.86,150.45,148.19,144.93,138.55,134.10,133.42,131.33,127.15, 126.36,121.66,121.08,120.85,108.91,106.03,102.62,57.72,55.73,33.05,32.44,30.76,26.67,25.23.

[0080] Experimental Example 1

[0081] The minimum inhibitory concentration (MIC) of 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1 was determined, and the specific determination steps were as follows:

[0082] In fresh MH broth medium, overnight cultured methicillin-resistant Staphylococcus aureus MRSA, Pseudomonas aeruginosa P.aeruginosa and E.coli culture fluids were inoculated at a ratio of 1% of bacteria to MH broth to obtain three culture fluids containing different bacteria, and then cultured at 37°C and 200rpm to mid-exponential phase; then, all three culture fluids were diluted to 7.5×10 5CFU / mL for standby; then, in a 96-well plate, a fresh MH broth medium was used to dilute the 25.6 mg / mL 9-(3'-cyclopentylpropionyloxy) berberine solution by two-fold gradient, with a volume of 100 μL per well, and then 100 μL of bacterial suspension (the above three culture solutions containing different bacteria) was added to each well separately, and after gently shaking and mixing, it was cultured at 37°C and horizontally for 12 hours; finally, the bottom of the well was observed with the naked eye to see if there was indicator bacteria growing, and the lowest concentration without indicator bacteria growth was used as the minimum inhibitory concentration (MIC) of 9-(3'-cyclopentylpropionyloxy) berberine. In addition, the same experimental process was used to determine the minimum inhibitory concentration of berberine, ampicillin and vancomycin. The experimental results are shown in Table 1.

[0083] Table 1 Minimum inhibitory concentration

[0084]

[0085]

[0086] As shown in Table 1 above, the antibacterial activity of 9-(3'-cyclopentylpropionyloxy)berberine against drug-resistant Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli is significantly better than that of berberine and the current first-line clinical drugs ampicillin and vancomycin.

[0087] Experimental Example 2

[0088] The therapeutic effect of 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1 on MRSA-infected wound repair was determined, and the specific determination steps were as follows:

[0089] ICR male mice aged 6 to 8 weeks were raised for one week, and after they adapted to the environment, they were randomly divided into 5 groups (n=7 mice in each group), namely, control group, negative control group (referred to as blank gel group), 0.5 μg / mL 9-(3'-cyclopentylpropionyloxy) berberine treatment group (referred to as low-dose group containing gel group), 1 μg / mL 9-(3'-cyclopentylpropionyloxy) berberine treatment group (referred to as medium-dose group containing gel group), and 2 μg / mL 9-(3'-cyclopentylpropionyloxy) berberine treatment group (referred to as high-dose group containing gel group). According to the weight of the mice, a 1% sodium pentobarbital solution was intraperitoneally injected at a dose of 0.1 mL / 10 g to give anesthesia, and then a 2×2 cm hair shaver was used to shave the middle and rear part of the back of the mice. 2 Remove hair in a certain area, then apply depilatory cream with a cotton swab, leave it on for 10 minutes, and then clean it with a cotton ball dipped in water. Place the mouse on its side, use forceps to pull the depilated skin symmetrically, and use a 6mm diameter skin biopsy device to puncture; use a cotton swab to dip 2×10 8CFU / mL of MRSA bacterial solution was evenly smeared on the wound for inoculation; 1 hour later, the remaining groups except the control group were given corresponding treatments; the healing status of the mouse skin wounds was photographed and recorded every two days.

[0090] During the treatment, the bacterial count on the wound was also measured. The specific measurement steps were as follows: 1. Cut and weigh 50 mg of the skin around the wound and put it into a 1.5 mL centrifuge tube, and add 1 mL of PBS buffer to homogenize the skin tissue; 2. After homogenization, perform gradient dilution with PBS buffer, then take 40 μL to the TSB agar plate and use an L-shaped smear stick to spread the bacteria; 3. Place the coated plate in a 37°C oven and culture it for 12 hours, then take it out and count it.

[0091] The experimental results of this experiment are as follows Figures 1 to 4 As shown, Figure 1 Schematic diagram of wound healing within 11 days of 9-(3'-cyclopentylpropionyloxy)berberine on MRSA-infected wound repair, where: Figure 1 a in the figure is a diagram of the wound healing process. Figure 1 b in the figure is the wound closure trajectory diagram. Figure 1 It can be seen that with the increase of time after trauma, the wound area of ​​the five groups gradually decreased, but the wound contraction rate of the control group and the blank gel group was significantly slower than that of the 9-(3'-cyclopentylpropionyloxy) berberine group from the first day, and the speed of wound repair in the three treatment groups was positively correlated with the treatment dose of 9-(3'-cyclopentylpropionyloxy) berberine.

[0092] Figure 2 This is a quantitative analysis chart of the wound area change. Figure 2 It can be seen that on the 11th day, the wound healing rate of the control group was 30.18±1.62%, the wound healing rate of the blank gel group was 60.21±2.01%, the wound healing rate of the low-dose group containing Ι gel was 75.49±0.87%, the wound healing rate of the medium-dose group containing Ι gel was 83.09±2.67%, and the wound healing rate of the high-dose group containing Ι gel was 91.62±3.01%, which shows that the administration of 9-(3'-cyclopentylpropionyloxy) berberine has the function of promoting the repair of MRSA / ATCC33591 infected wounds.

[0093] Figure 3 The results of the determination of bacterial count on the wound surface during treatment are as follows: Figure 3It can be seen that on the 11th day after the trauma, the bacterial colonies of the control group's skin basically covered the entire culture dish, with a colony formation rate of 100%; the colony formation rate of the blank gel group's skin was 62%; the colony formation rate of the low-dose group containing Ι gel was 37%, the colony formation rate of the medium-dose group containing Ι gel was 15%, and the colony formation rate of the high-dose group containing Ι gel was 8%. Figure 4 Figure 11 shows the viability of each group of bacteria on the 11th day after the wound. Figure 4 It can be seen that the bacterial viability of the 9-(3'-cyclopentylpropionyloxy) berberine administration group is weak, and the bacterial viability is weakened as the dosage of 9-(3'-cyclopentylpropionyloxy) berberine increases. In summary, the 9-(3'-cyclopentylpropionyloxy) berberine of the present invention has excellent antibacterial properties.

[0094] Experimental Example 3

[0095] The therapeutic effect of 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1 on bacterial pneumonia infected by P. aeruginosa was determined, and the specific determination steps were as follows:

[0096] ICR male mice aged 6 to 8 weeks were raised for one week, and after they adapted to the environment, they were randomly divided into 5 groups (n=7 mice in each group), namely, the control group, the Pseudomonas aeruginosa infection group (referred to as the Pseudomonas aeruginosa modeling group), the 50 μg / mL 9-(3'-cyclopentylpropionyloxy) berberine treatment group (referred to as the low-dose Ι administration group), the 100 μg / mL 9-(3'-cyclopentylpropionyloxy) berberine treatment group (referred to as the medium-dose Ι administration group), and the 200 μg / mL 9-(3'-cyclopentylpropionyloxy) berberine treatment group (referred to as the high-dose Ι administration group). According to the weight of mice, 0.1 mL / 10 g of sodium pentobarbital solution with a mass concentration of 1% was intraperitoneally injected for anesthesia, and then the trachea was exposed. The control group mice were injected with 50 μL of 1×PBS solution through the trachea, and the Pseudomonas aeruginosa modeling group and treatment groups (low-dose Ι administration group, medium-dose Ι administration group, and high-dose Ι administration group) were injected with 50 μL of 1×10 6 CFU / mL of Pseudomonas aeruginosa bacterial solution (resuspended in 50 μL 1× PBS), the mice were stood upright for 2 minutes to allow the bacteria to enter the lungs smoothly, and the wounds were sutured; 2 hours later, the treatment group was intraperitoneally injected with corresponding concentrations of 9-(3'-cyclopentylpropionyloxy) berberine solution, and the mice were anesthetized and killed 24 hours after bacterial infection, and the serum, bronchoalveolar lavage fluid and lung tissue were collected and stored in a -80℃ refrigerator.

[0097] The collected serum, alveolar lavage fluid and lung tissue were tested, and the experimental results were as follows: Figures 5 to 10 As shown. Among them, Figure 5Figure 2 shows the lung bacterial load of mice infected with Pseudomonas aeruginosa pneumonia in the control group, Pseudomonas aeruginosa model group and three treatment groups after treatment. Figure 5 It can be seen that compared with the Pseudomonas aeruginosa modeling group, the three treatment groups can significantly reduce the lung bacterial load; Figure 6 and Figure 7 The total protein concentration and total cell count of bronchoalveolar lavage fluid in mice with acute lung injury induced by Pseudomonas aeruginosa are statistically analyzed. Figures 6-7 It can be seen that the 9-(3'-cyclopentylpropionyloxy) berberine treatment group can significantly inhibit the inflammatory pathological progression such as inflammatory cell infiltration in bronchoalveolar lavage fluid; Figure 8 is a graph of the wet / dry weight ratio of mouse lungs, from Figure 8 It can be seen that the 9-(3'-cyclopentylpropionyloxy) berberine treatment group can also reduce pulmonary edema. Fig. 9 This is the result of pathological experiment. Fig. 9 It can be seen that the alveolar structure of the lung tissue of the normal control group mice is intact without obvious lesions, while the lung tissue morphology of the Pseudomonas aeruginosa modeling group mice is significantly changed, the alveolar wall is significantly thickened, and a large number of inflammatory cell infiltrations can be observed; after treatment with 9-(3'-cyclopentylpropionyloxy) berberine, the morphology of the mouse lung tissue is significantly improved, the thickness of the alveolar wall shows a concentration-dependent thinning, and in the high-concentration treatment group (high-dose Ι administration group), the lung tissue morphology has basically recovered to the same level as that of the normal control group mice. Fig.10 The experimental results of lung injury degree are as follows: Fig.10 It can be clearly seen that 24 hours after bacterial infection, there was a significant difference between the Pseudomonas aeruginosa modeling group and the normal control group (***p<0.001, where * represents significant significance, the more *s, the stronger the significant difference, and ns represents no significant difference), indicating that the modeling was successful; after treatment with 9-(3'-cyclopentylpropionyloxy), the degree of lung injury in the treatment group of mice was much lower than that in the Pseudomonas aeruginosa modeling group, and the degree of lung injury was inversely proportional to the dosage.

[0098] Experimental Example 4

[0099] The biosafety assessment of 9-(3'-cyclopentylpropionyloxy) berberine prepared in Example 1 was performed, and the specific steps were as follows:

[0100] 1. Select mice with similar weight, gender and health conditions and randomly divide them into four groups (n=10 mice in each group). They are the group receiving 100 μL 9-(3'-cyclopentylpropionyloxy) berberine at a dose of 30 mg / kg (referred to as the administration group (30 mg / kg)), the group receiving 100 μL 9-(3'-cyclopentylpropionyloxy) berberine at a dose of 60 mg / kg (referred to as the administration group (60 mg / kg)), the group receiving 100 μL 9-(3'-cyclopentylpropionyloxy) berberine at a dose of 120 mg / kg (referred to as the administration group (120 mg / kg)) and the group receiving 100 μL normal saline (referred to as the control group). 2. Monitor the clinical symptoms, toxicity and mortality of the four groups of mice for 24 hours. 3. The weight and physical condition of the mice were observed and recorded every day until day 7, when the mice were euthanized and their organs (heart, liver, spleen, lungs and kidneys) were removed.

[0101] Blood and biochemical tests were performed on the removed heart, liver, spleen, lungs and kidneys. The test results were as follows: Figures 11 to 19 As shown. Among them, Fig.11 This is the weight monitoring chart of mice. Fig.11 It can be seen that compared with the control group, the specified dose of drug treatment did not affect the body weight of mice; Figures 12 to 16 The weights of the heart, liver, spleen, lung, and kidney of mice receiving different doses of 9-(3'-cyclopentylpropionyloxy) berberine are shown in Figure 2. Figures 12 to 16 As can be seen, 9-(3'-cyclopentylpropionyloxy)berberine treatment had no significant effect on the organ weights (heart, lung, liver, spleen, and kidney) of mice. Figures 17 to 19 The following are the monitoring charts of the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (Cr) in mouse serum. Figures 17 to 19 It can be seen that the levels of ALT, AST and Cr in serum were not changed under low, medium and high concentrations of 9-(3'-cyclopentylpropionyloxy) berberine treatment, indicating that 9-(3'-cyclopentylpropionyloxy) berberine treatment had no obvious adverse effects on liver and kidney function. In addition, we also used H&E staining to observe the morphological changes of mouse liver and kidney tissues, such as Fig. 20 As shown, it can be seen that after treating mice with a high dose of 9-(3'-cyclopentylpropionyloxy) berberine (120 mg / kg), the organ morphology of the mice did not change significantly. In summary, the 9-(3'-cyclopentylpropionyloxy) berberine of the present invention has high biological safety.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A berberine derivative, characterized in that The structural formula of the berberine derivative is as follows:

2. The method for preparing a berberine derivative according to claim 1, characterized in that: The steps include: 1) mixing 3-cyclopentanehexylpropionic acid, a first organic solvent, oxalyl chloride and anhydrous DMF, and reacting to obtain intermediate I; 2) subjecting berberine hydrochloride to a high temperature negative pressure pyrolysis reaction, and then dissolving it in alcohol to undergo a free radical substitution reaction to obtain intermediate II; 3) mixing the intermediate II, the second organic solvent, the intermediate I and pyridine, and reacting them to obtain the berberine derivative according to claim 1; There is no order restriction for step 1) and step 2); The structural formula of the intermediate I is The structural formula of the intermediate II is 3. The method for preparing a berberine derivative according to claim 2, characterized in that: The mass volume ratio of 3-cyclopentanehexylpropionic acid, the first organic solvent, oxalyl chloride and anhydrous DMF in step 1) is 100 mg: 5-10 mL: 178 mg: 5-10 mL; The first organic solvent includes one or more of anhydrous dichloromethane, anhydrous dichloroethane, anhydrous ether and anhydrous chloroform.

4. The method for preparing a berberine derivative according to claim 3, characterized in that: The reaction temperature in step 1) is -20 to 0°C and the reaction time is 1 to 2 hours.

5. The method for preparing a berberine derivative according to any one of claims 2 to 4, characterized in that: The mass volume ratio of berberine hydrochloride and alcohol in step 2) is 1g:50-100mL; The alcohol includes one or both of anhydrous ethanol and anhydrous methanol.

6. The method for preparing a berberine derivative according to claim 5, characterized in that: The temperature of the high temperature negative pressure pyrolysis reaction in step 2) is 200 to 300°C, the pressure is -0.01 to -0.05Mpa, and the time is 30 to 60min; The free radical substitution reaction time is 3 to 5 minutes.

7. The method for preparing a berberine derivative according to claim 6, characterized in that: The mass volume ratio of the intermediate II, the second organic solvent, the intermediate I and pyridine in step 3) is 100 mg: 5-10 mL: 0.2-0.4 mL: 0.2-0.3 mL; The second organic solvent includes one or more of anhydrous acetonitrile, anhydrous DMF and anhydrous dioxane.

8. The method for preparing a berberine derivative according to claim 7, characterized in that: The reaction temperature in step 3) is 20-30°C and the reaction time is 1-2h.

9. Use of the berberine derivatives prepared by the method for preparing berberine derivatives according to any one of claims 2 to 8 in the preparation of antibacterial drugs.

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