A polyethylene glycol-Rakicidin B1 derivative, its preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a polyethylene glycol-Rakicidin B1 derivative, and further discloses its preparation method and application. The polyethylene glycol-Rakicidin B1 derivative of the present invention connects polyethylene glycols with different degrees of polymerization to the structure of the Rakicidin B1 esterified derivative through click chemistry reactions, realizing the structural modification of the Rakicidin B1 compound, and effectively solving the problem that the poor solubility of existing Rakicidins compounds and esterified derivatives affects their applications. The activity of the polyethylene glycol-Rakicidin B1 derivative of the present invention shows that the derivative has obvious anti-neuroinflammatory effects, and also has certain in vitro inhibitory effects on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 under normoxic and hypoxic cultures.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a polyethylene glycol-Rakicidin B1 derivative, and further discloses a preparation method and application thereof. Background Art

[0002] At present, hundreds of bioactive substances have been isolated from the metabolites of marine microorganisms. Some of them are antibiotics with antitumor or antibacterial activities that may have clinical application value. Rakicidins compounds are an important class of antitumor antibiotics with application value. At present, a series of Rakicidins compounds with antitumor or antibacterial activities have been reported to be found from Micromonospora and Streptomyces.

[0003] In 2016, our research group first reported internationally that the new compound Rakicidin B1 has strong activity against Clostridium difficile and is sensitive to metronidazole and vancomycin-resistant strains. At the same time, it has characteristics such as a narrow antibacterial spectrum and poor oral absorption, making it have the advantages of not harming the intestinal microbiota when playing a role (the normal intestinal microbiota can antagonize the growth of Clostridium difficile to reduce the recurrence rate) and low systemic toxicity. It can overcome problems such as high recurrence rate and high systemic toxicity brought by some marketed or in-development varieties during clinical use, and is an ideal candidate compound for the development of anti-drug-resistant Clostridium difficile drugs, opening a new "window" for the successful development of new, highly efficient, low-toxic and anti-drug-resistant CDI drugs.

[0004]

[0005] Rakicidin B1 brings opportunities for the development of new anti-CDI drugs. However, due to factors such as its relatively high cytotoxicity, poor solubility and unstable structure, since 2017, our research group has carried out semi-synthetic modification of Rakicidin B1 derivatives in order to obtain new derivatives with high efficiency, low toxicity and significantly improved physical and chemical properties through chemical modification means. For example, the water-soluble nitrogen heterocyclic group introduced by the carbonate and carbamate linkage methods at the C-3 hydroxyl site disclosed in Chinese Patent CN110437313A synthesized a new Rakicidin B1 derivative, and through in vitro anti-Clostridium difficile activity screening, compounds with antibacterial activity slightly stronger or equivalent to Rakicidin B1 and reduced toxicity were obtained. The above findings provide more possibilities for finding new anti-Clostridium difficile drugs of Rakicidin B1 with high efficiency and low toxicity, which is worthy of in-depth study. However, through further pharmaceuticability evaluation research, it was found that the esterified derivatives of Rakicidin B1 have similar solubility and stability problems to Rakicidin B1, resulting in obstacles to its preclinical research.

[0006] Therefore, in the process of developing new derivatives of Rakicidin as novel anti-CDI drugs, how to apply mature, reliable, efficient and novel chemical modification strategies to obtain novel derivatives of Rakicidin B1 with low toxicity, strong activity and excellent physicochemical properties (solubility and stability) is the key to the successful development of Rakicidin B1 as a novel anti-drug-resistant CDI drug. The field expects more in-depth structural modification and performance research based on new derivatives of Rakicidin B1, which is of great significance for its clinical development and application. Summary of the Invention

[0007] To this end, the technical problem to be solved by the present invention is to provide a polyethylene glycol-Rakicidin B1 derivative, which shows good solubility and significant effects on neuritis;

[0008] The second technical problem to be solved by the present invention is to provide a preparation method and application of the above-mentioned polyethylene glycol-Rakicidin B1 derivative.

[0009] To solve the above technical problems, a polyethylene glycol-Rakicidin B1 derivative according to the present invention is characterized in that the derivative has a structure shown in the following formula (Ⅰ):

[0010]

[0011] Wherein, the A group has a structure shown in *, indicating the connection site, and n is selected from polyethylene glycols with different degrees of polymerization.

[0012] Specifically, for the polyethylene glycol-Rakicidin B1 derivative, the Q group has a structure shown as -Q1-CH2-Q2-; wherein,

[0013] the Q1 group is selected from a substituted or unsubstituted amino group, a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group;

[0014] the Q2 group is selected from a substituted or unsubstituted heterocyclic group;

[0015] The substitution means that it can be optionally substituted by a halogen or an alkyl group.

[0016] Specifically, for the polyethylene glycol-Rakicidin B1 derivative, the Q1 group is selected from a substituted or unsubstituted amino group or a substituted or unsubstituted phenyl group;

[0017] the Q2 group is selected from a substituted or unsubstituted N-containing heterocyclic group;

[0018] Preferably, the Q1 group is selected from an amino group or a phenyl group, and the Q2 group is selected from a triazole group.

[0019] Specifically, for the polyethylene glycol-Rakicidin B1 derivative, the derivative has a structure shown in the following formula (P1) or (P2):

[0020]

[0021] Preferably, in the derivative, the molecular weight of the polyethylene glycol is 500-3000;

[0022] Preferably, in the derivative, the molecular weight of the polyethylene glycol is 750-2000.

[0023] The present invention also discloses a method for preparing the polyethylene glycol-Rakicidin B1 derivative. The method includes the step of connecting the polyethylene glycol modified with an azide group through a click chemical reaction on the alkynyl-modified Rakicidin B1 esterification derivative; or the step of connecting the polyethylene glycol modified with an alkyne group through a click chemical reaction on the azide group-modified Rakicidin B1 esterification derivative.

[0024] Specifically, for the preparation method of the polyethylene glycol-Rakicidin B1 derivative, the preparation method of the compound P1 includes the following steps:

[0025]

[0026] (1) Take the compound Rakicidin B1 and add p-nitrophenyl chloroformate. React at -10 to 0 °C in an organic solvent system. Collect the reactant, add water and mix, and collect the precipitated solid for standby;

[0027] (2) Take the product obtained in step (1), add triethylamine and propargylamine and mix. React successively at -10 to 0 °C and at room temperature in an organic solvent system. Collect the reactant, add saturated brine and mix, and collect the precipitated solid for standby;

[0028] (3) Take the product obtained in step (2), add the polyethylene glycol modified with an azide group and mix. React in an organic solvent system in the presence of a catalyst. Collect the reaction solution and prepare the required compound P1 by semi-preparative liquid phase;

[0029] Preferably, the catalyst includes a mixture of copper sulfate pentahydrate and sodium ascorbate.

[0030] Preferably, in steps (1), (2), and (3), the organic solvents are independently selected from pyridine or DMF.

[0031] Specifically, for the preparation method of the polyethylene glycol-Rakicidin B1 derivative, the preparation method of the compound P2 comprises the following steps:

[0032]

[0033] (1) Take the compound Rakicidin B1 and mix it with 4-chloromethylbenzoyl chloride, react in an organic solvent system, collect the reactant, add water, and collect the precipitated solid for standby;

[0034] (2) Take the product obtained in step (1), mix it with sodium azide, react in an organic solvent system in the presence of a catalyst at 50-60 °C, collect the reactant, add saturated brine and mix, and collect the precipitated solid for standby;

[0035] Preferably, the catalyst includes potassium iodide;

[0036] (3) Take the product obtained in step (2), add alkynyl-modified polyethylene glycol, react in an organic solvent system in the presence of a catalyst, and collect the reaction solution, and prepare the required compound P2 by semi-preparative liquid phase;

[0037] Preferably, the catalyst includes a mixture of copper sulfate pentahydrate and sodium ascorbate.

[0038] Preferably, in steps (1), (2), and (3), the organic solvents are independently selected from pyridine or DMF.

[0039] Specifically, for the preparation method of the polyethylene glycol-Rakicidin B1 derivative, the semi-preparative liquid phase preparation step specifically includes: separating the collected reaction solution by semi-preparative liquid chromatography on a YMC ODS C18 column, eluting with methanol-water with a volume ratio of 830:170 as the eluent, monitoring the peak elution time range of the target product at a wavelength of 262 nm by the DAD ultraviolet detector of the preparative liquid chromatography, collecting the eluent within this time range, and concentrating and freeze-drying the eluent to obtain the product. The present invention also discloses the use of the polyethylene glycol-Rakicidin B1 derivative and its stereoisomers, tautomers, amorphous substances, isotopomers, polymorphs, solvates or pharmaceutically acceptable salts thereof for the preparation of a drug with anti-neuritis activity and / or for the treatment of neuritis.

[0040] The present invention also discloses a drug with anti-neuritis activity and / or for the treatment of neuritis, and the active ingredient of the drug includes the polyethylene glycol-Rakicidin B1 derivative and its stereoisomers, tautomers, amorphous substances, isotopomers, polymorphs, solvates or pharmaceutically acceptable salts thereof.

[0041] Specifically, the derivatives for use in the present invention can be made into pharmaceutical compositions and their unit dosage forms together with conventional additives or diluents. Such forms include solids (especially in the form of tablets, filled capsules, powders, and pills), and liquids (especially aqueous solutions or non-aqueous solutions, suspensions, emulsions, elixirs), and capsules filled with the above forms, all forms for oral administration, suppositories for rectal administration, and sterile injectable solutions for parenteral administration. Such pharmaceutical compositions and their unit dosage forms may include conventional components in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient corresponding to the required daily dosage range.

[0042] The compounds for use in the present invention can be administered in various oral and parenteral (e.g., injection) dosage forms. To those skilled in the art, the following dosage forms may contain the compounds of the present invention or their pharmaceutically acceptable salts as active ingredients.

[0043] To prepare the compounds for use in the present invention into corresponding drugs, the pharmaceutically acceptable carrier can be solid or liquid. Solid forms of preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier can be one or more substances that also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrants, or encapsulating materials. Liquid preparations include solutions, suspensions, and emulsions, for example, aqueous solutions or water-propylene glycol solutions. The compounds for use in the present invention can be formulated into preparations for parenteral administration (e.g., injection, such as rapid bolus injection or continuous infusion), and can be present in ampoules, pre-filled syringes, small-volume infusion bags, or multi-dose containers in unit dosage forms together with added preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous carrier, and may contain formulation components such as suspending agents, stabilizers, and / or dispersing agents. Additionally, the active ingredient can be in the form of a powder, which can be obtained by sterile isolation of the solid or lyophilization from a solution, for reconstitution with a suitable carrier such as sterile, pyrogen-free water immediately before use. Aqueous solutions suitable for oral administration can be prepared by dissolving the active ingredient in water and adding the required coloring agents, flavoring agents, stabilizers, and thickening agents. Aqueous suspensions suitable for oral administration can be prepared by dispersing the finely divided active ingredient in water containing a viscous substance, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

[0044] The polyethylene glycol-Rakicidin B1 derivative of the present invention uses the prodrug design theory to connect polyethylene glycols with different degrees of polymerization to the structure of the Rakicidin B1 ester derivative through click chemistry reactions on the Rakicidin B1 ester derivative, realizing the structural modification of the Rakicidin B1 compound. Compared with the original Rakicidin B1 compound, the polarity of the derivative is reduced and the solubility is enhanced, effectively solving the problem that the poor solubility of existing Rakicidins compounds and their ester derivatives affects their applications.

[0045] The activity of the polyethylene glycol-Rakicidin B1 derivative of the present invention shows that, on the basis of retaining the potential anti-Clostridium difficile activity, the derivative has obvious anti-neuroinflammatory effects and can be used as an anti-Clostridium difficile active compound of a new structural type; and the derivative has certain in vitro inhibitory effects on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 under normoxic and hypoxic cultures, and can be developed and utilized as a new type of anti-cancer or antibacterial alternative drug, with further potential for development value.

[0046] The polyethylene glycol-Rakicidin B1 derivative of the present invention is beneficial to giving full play to the cost controllable advantages of its upstream raw materials through the structural modification of the compound Rakicidin B1, making the added value of Rakicidins compounds greater, contributing to the industrialization prospect of the product brand and creating greater market economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, wherein,

[0048] Figure 1 It is a typical diagram of neutrophils at the inflammatory site of zebrafish after treatment with samples FIMP1-2000 (P1) and FIMP2-2000 (P2); among them, the white arrow indicates the analysis site, and the green fluorescent particles are neutrophils;

[0049] Figure 2 It is the number of neutrophils at the inflammatory site of zebrafish after treatment with samples FIMP1-2000 (P1) and FIMP2-2000 (P2); compared with the model control group, ***p < 0.001;

[0050] Figure 3 It is the anti-neuroinflammatory efficacy of samples FIMP1-2000 (P1) and FIMP2-2000 (P2) on zebrafish;

[0051] Figure 4The relative expression level of IL-1β gene after treatment with sample FIMP2-2000 (P2); compared with the model control group, *p<0.05, ***p<0.001;

[0052] Figure 5 The relative expression level of IL-6 gene after treatment with sample FIMP2-2000 (P2); compared with the model control group, ***p<0.001. Specific implementation manners

[0053] In the following examples of the present invention, the Rakicidin B1 compound used was fermentatively synthesized by a method known in the prior art.

[0054] In Examples 1-3 of the present invention, the synthesis of polyethylene glycol-Rakicidin B1 derivatives was carried out according to the following equations respectively. The polyethylene glycols selected in Examples 1-3 were polyethylene glycols with molecular weights of 750, 1000, and 2000:

[0055]

[0056] In Example 4 of the present invention, the synthesis of polyethylene glycol-Rakicidin B1 derivative was carried out according to the following equation, and the polyethylene glycol with a molecular weight of 2000 was selected:

[0057]

[0058] Example 1

[0059] This example is used to synthesize the above-mentioned polyethylene glycol-Rakicidin B1 derivative FIMP1-750, and specifically includes the following steps:

[0060] (1) Add compound Rakicidin B1 (0.5 g, 0.8 mmol) and pyridine (10.0 mL) into a 50 mL eggplant-shaped flask, and slowly drop p-nitrophenyl chloroformate (1.0 g, 5 mmol) under stirring at -5°C. After the dropping is completed, react for about 0.5 hours, and after detecting the completion of the reaction by TLC; then add a large amount of water until a grayish-white insoluble substance precipitates. After suction filtration, place the filter cake in a vacuum drying oven and dry it at 50°C for 1 hour for standby;

[0061] (2) Add 12.0 mL of pyridine into a 50 mL eggplant-shaped flask. While stirring, add the product (0.5 g) from step (1). Slowly dropwise add triethylamine (0.69 mL, 5 mmol) and propargylamine (0.20 mL, 3 mmol) under stirring at -5°C. After stirring and reacting for 10 minutes at -5°C, transfer it to room temperature and react for another 10 minutes. During the reaction, stop the reaction after tracking the completion of the reaction by HPLC (Agilent C18 column, 5 μm, 250 mm×4.6 mm, methanol-water = 850:150, column temperature 40°C, flow rate 1.0 mL / min, wavelength 262 nm); Pour the reaction solution into 200 mL of saturated brine, and a light brown insoluble substance precipitates. After suction filtration and washing with water, place the filter cake in a vacuum drying oven and dry it at 50°C for 1 hour, and directly use it for the next step of the reaction;

[0062] (3) Add 10 mL of DMF solution into a 50 mL eggplant-shaped flask. While stirring, add the product (0.51 g) from step (2), the commercially available azide-modified polyethylene glycol MPEG750-N3 (1.0 g), and a small amount of CuSO4·5H2O (50 mg, 0.2 mmol) and sodium ascorbate (100 mg, 0.5 mmol) as reaction catalysts. Stir and react at room temperature for 2 hours. During the reaction, stop the reaction after tracking the completion of the reaction by HPLC (Agilent C18 column, 5 μm, 250 mm×4.6 mm, methanol-water = 850:150, column temperature 40°C, flow rate 1.0 mL / min, wavelength 262 nm). After the reaction is completed, separate the collected reaction solution by semi-preparative liquid chromatography using a YMCODS C18 column (50 μm, 190 mm×60.8 mm), use methanol-water with a volume ratio of 830:170 as the eluent, control the eluent flow rate at 15 ml / min for elution, monitor the elution peak time range of the target product at a wavelength of 262 nm through the DAD ultraviolet detector of the preparative liquid chromatography, and collect the eluent within this time range (135 - 165 min). The eluent is concentrated and then freeze-dried to obtain the final product polyethylene glycol-RakicidinB1 derivative, denoted as FIMP1-750.

[0063] Upon observation and identification, the target compound polyethylene glycol-RakicindinB1 derivative FIMP1-750 synthesized in this example is a white amorphous powder. Since polyethylene glycol is a polymer and polyethylene glycol with different degrees of polymerization is connected to RakicindinB1 through chemical synthesis, the final product obtained is a polymer.

[0064] As shown by high-resolution mass spectrometry (HR-ESI-MS), its average molecular weight is 1329.8005, and its nuclear magnetic data are as follows: 1HNMR (600 MHz, DMSO) δ 8.25 (1H, s), 7.86 (4H), 7.78 (1H, br.s), 7.22 (1H, s), 6.90 (1H, m), 6.18 (1H, br.s), 5.45 (1H, s), 5.35 (4H), 5.17 (1H), 4.49 (12H), 4.36 (6H), 4.25 (16H), 3.79 (28H), 2.08 (3H), 1.98 (5H), 1.48 (9H), 1.24 (22H, m), 1.07 (6H, d, J = 6.6 Hz), 0.92 (2H, m), 0.83 (3H, m). It can be seen that the structure of the reaction product is correct.

[0065] Example 2

[0066] This example is used to synthesize the above polyethylene glycol-Rakicidin B1 derivative FIMP1-1000, which specifically includes the following steps:

[0067] (1) Add compound Rakicidin B1 (0.5 g, 0.8 mmol) and pyridine (10.0 mL) to a 50 mL eggplant-shaped flask. Slowly add p-nitrophenyl chloroformate (1.0 g, 5 mmol) dropwise under stirring at -5 °C. After the addition is complete, react for about 0.5 hours. After the reaction is completed by TLC detection; then add a large amount of water until a grayish-white insoluble substance precipitates. After suction filtration, place the filter cake in a vacuum drying oven and dry it at 50 °C for 1 hour for later use;

[0068] (2) Add 12.0 mL of pyridine to a 50 mL eggplant-shaped flask. Add the product of step (1) (0.5 g) under stirring. Slowly add triethylamine (0.69 mL, 5 mmol) and propargylamine (0.20 mL, 3 mmol) dropwise under stirring at -5 °C. Stir and react at -5 °C for 10 minutes, then transfer it to room temperature and react for 10 minutes. During the reaction, use HPLC (Agilent C18 column, 5 μm, 250 mm × 4.6 mm, methanol-water = 850:150, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm) to track the reaction. After the reaction is complete, stop the reaction; pour the reaction solution into 200 mL of saturated brine, and a light brown insoluble substance precipitates. After suction filtration and washing with water, place the filter cake in a vacuum drying oven and dry it at 50 °C for 1 hour and directly use it for the next step of the reaction;

[0069] (3) Add 10 mL of DMF solution to a 50 mL eggplant-shaped flask. While stirring, add the product from step (2) (0.51 g), then add the commercially available azide-modified polyethylene glycol MPEG1000-N3 (1.0 g), as well as a small amount of CuSO4·5H2O (50 mg, 0.2 mmol) and sodium ascorbate (100 mg, 0.5 mmol) as reaction catalysts. Stir and react at room temperature for 2 hours. During the reaction, monitor the reaction by HPLC (Agilent C18 column, 5 μm, 250 mm × 4.6 mm, methanol-water = 850:150, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm). Stop the reaction after the reaction is complete. After the reaction is over, separate the collected reaction solution by semi-preparative liquid chromatography using a YMCODS C18 column (50 μm, 190 mm × 60.8 mm), with methanol-water with a volume ratio of 830:170 as the eluent, control the eluent flow rate at 15 ml / min for elution, monitor the elution time range of the target product through the DAD ultraviolet detector of the preparative liquid chromatography at a wavelength of 262 nm, and collect the eluent within this time range (135 - 165 min). The eluent is concentrated and then freeze-dried to obtain the final product, polyethylene glycol-RakicidinB1 derivative, denoted as FIMP1-1000.

[0070] Upon observation and identification, the target compound polyethylene glycol-RakicindinB1 derivative FIMP1-750 synthesized in this example is a white amorphous powder. Since polyethylene glycol is a polymer and polyethylene glycol with different degrees of polymerization is connected to RakicindinB1 through chemical synthesis, the final product obtained is a polymer.

[0071] High-resolution mass spectrometry (HR-ESI-MS) shows that its average molecular weight is 1618.0304, and its nuclear magnetic data are as follows: 1HNMR(600 MHz, DMSO) δ 8.96 (1H, s), 8.57 (1H, d, J = 8.4 Hz), 8.17 (4H, m), 7.79 (1H, s), 7.44 (1H, d, J = 7.2 Hz), 7.25 (1H, s), 6.92 (1H, d, J = 15.0 Hz), 6.19 (1H, d, J = 15.0 Hz), 5.72 (3H, m), 5.49 (1H, s), 5.42 (2H, s), 5.32 (1H, s), 5.12 (1H, d, J = 9.6 Hz), 5.11 (6H, d, J = 6.6 Hz), 4.53 (4H, m), 4.36 (1H, s), 3.79 (52H), 1.58 (1H, br.s), 1.24 (22H, m), 1.11 (4H, m), 1.07 (3H, d, J = 6.6 Hz), 1.06 (3H, d, J = 6.0 Hz), 0.93 (4H, m), 0.83 (6H, m). It can be seen that the structure of the product is correct.

[0072] Example 3

[0073] This example is used to synthesize the above-mentioned polyethylene glycol-Rakicidin B1 derivative FIMP1-2000, which specifically includes the following steps:

[0074] (1) Add compound Rakicidin B1 (0.5 g, 0.8 mmol) and pyridine (10.0 mL) into a 50 mL eggplant-shaped flask. Slowly add p-nitrophenyl chloroformate (1.0 g, 5 mmol) dropwise under stirring at -5 °C. After the addition is complete, react for about 0.5 hour, and stop the reaction after detection by TLC. Then add a large amount of water until a grayish-white insoluble substance precipitates. After filtration, place the filter cake in a vacuum drying oven and dry it at 50 °C for 1 hour for later use.

[0075] (2) Add 12.0 mL of pyridine into a 50 mL eggplant-shaped flask. Add the product from step (1) (0.5 g) under stirring. Slowly add triethylamine (0.69 mL, 5 mmol) and propargylamine (0.20 mL, 3 mmol) dropwise under stirring at -5 °C. Stir and react at -5 °C for 10 minutes, then transfer to room temperature and react for another 10 minutes. Stop the reaction after tracking the reaction to completion by HPLC (Agilent C18 column, 5 μm, 250 mm × 4.6 mm, methanol-water = 850:150, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm). Pour the reaction solution into 200 mL of saturated brine, and a light brown insoluble substance will precipitate. Filter it, wash it with water, and place the filter cake in a vacuum drying oven and dry it at 50 °C for 1 hour for direct use in the next step of the reaction.

[0076] (3) Add 10 mL of DMF solution to a 50 mL eggplant-shaped flask. While stirring, add the product from step (2) (0.51 g), then add commercially available azide-modified polyethylene glycol MPEG2000-N3 (1.0 g), as well as a small amount of CuSO4·5H2O (50 mg, 0.2 mmol) and sodium ascorbate (100 mg, 0.5 mmol) as reaction catalysts. Stir the reaction at room temperature for 2 hours. During the reaction, monitor the reaction by HPLC (Agilent C18 column, 5 μm, 250 mm × 4.6 mm, methanol-water = 850:150, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm). Stop the reaction after the reaction is complete. After the reaction, separate the collected reaction solution by semi-preparative liquid chromatography using a YMCODS C18 column (50 μm, 190 mm × 60.8 mm), with methanol-water at a volume ratio of 830:170 as the eluent. Control the eluent flow rate at 15 ml / min for elution. Monitor the elution peak time range of the target product at a wavelength of 262 nm through the DAD ultraviolet detector of the preparative liquid chromatography, and collect the eluent within this time range (135 - 165 min). The eluent is concentrated and then freeze-dried to obtain the final product, polyethylene glycol-RakicidinB1 derivative, denoted as FIMP1-2000.

[0077] Upon observation and identification, the target compound polyethylene glycol-RakicindinB1 derivative FIMP2-2000 synthesized in this example is a white amorphous powder. Since polyethylene glycol is a polymer and polyethylene glycol with different degrees of polymerization is connected to RakicindinB1 through chemical synthesis, the final product obtained is a polymer.

[0078] As shown by high-resolution mass spectrometry (HR-ESI-MS), its average molecular weight is 2695.6167, and its NMR data is shown in Table 1 below.

[0079] Table 1 NMR data of FIMP1-2000 (600M, DMSO)

[0080]

[0081]

[0082] a Recorded for 1H NMR and 150MHz for 13C NMR at 600MHz in DMSO-d6, δH in ppm, J in Hz.

[0083] It can be seen that the structure of the compound synthesized in this example is correct.

[0084] Example 4

[0085] This example is used to synthesize the above-mentioned polyethylene glycol-Rakicidin B1 derivative FIMP2, and specifically includes the following steps:

[0086] (1) Add compound Rakicidin B1 (0.5 g, 0.8 mmol) and pyridine (10.0 mL) into a 50 mL eggplant-shaped flask. Slowly add 4-chloromethylbenzoyl chloride (0.76 mL, 5 mmol) dropwise under stirring at -5 °C. After the addition is complete, react for about 0.5 hours and then detect the completion of the reaction by TLC. Then add a large amount of water until a light pink insoluble substance precipitates. After suction filtration, place the filter cake in a vacuum drying oven and dry it at 50 °C for 1 hour for later use;

[0087] (2) Add 20 mL of DMF solution into a 100 mL eggplant-shaped flask. Add the product (1 g) from step (1), NaN3 (0.487 g, 7.5 mmol), and KI (0.096 g, 0.58 mmol) under stirring, and react under stirring at 55 °C for 0.5 hours. During the reaction, track the completion of the reaction with HPLC (Agilent C18 column, 5 μm, 250 mm × 4.6 mm, methanol-water = 850:150, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm), and then stop the reaction. Pour the reaction solution into 200 mL of saturated brine, and a light yellowish-brown insoluble substance precipitates. After suction filtration, wash it with water and ether respectively, and then dry the filter cake and directly use it for the next step of the reaction;

[0088] (3) Add 10 mL of DMF solution to a 50 mL eggplant-shaped flask. While stirring, add the product from step (2) (0.56 g), then add commercially available alkynyl-modified polyethylene glycol MPEG2000-ALK (1.0 g), as well as a small amount of CuSO4·5H2O (50 mg, 0.2 mmol) and sodium ascorbate (100 mg, 0.5 mmol) as reaction catalysts. Stir and react at room temperature for 2 hours. During the reaction, monitor the reaction by HPLC (Agilent C18 column, 5 μm, 250 mm×4.6 mm, methanol-water = 850:150, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm). Stop the reaction after the reaction is complete. After the reaction, separate the collected reaction solution by semi-preparative liquid chromatography using a YMCODS C18 column (50 μm, 190 mm×60.8 mm), with methanol-water at a volume ratio of 830:170 as the eluent. Control the eluent flow rate at 15 ml / min for elution. Monitor the elution peak time range of the target product through the DAD ultraviolet detector of the preparative liquid chromatography at a wavelength of 262 nm, and collect the eluent within this time range (135 - 165 min). The eluent is concentrated and then freeze-dried to obtain the final product polyethylene glycol-RakicidinB1 derivative, denoted as FIMP2-2000.

[0089] Upon observation and identification, the target compound polyethylene glycol-RakicindinB1 derivative FIMP2-2000 synthesized in this example is a white amorphous powder. Since polyethylene glycol is a polymer and polyethylene glycol with different degrees of polymerization is connected to RakicindinB1 through chemical synthesis, the final product obtained is a polymer.

[0090] As shown by high-resolution mass spectrometry (HR-ESI-MS), its average molecular weight is 2610.5410, and its nuclear magnetic data is shown in Table 2 below.

[0091] Table 2 Nuclear magnetic data of FIMP2-2000 (600M DMSO)

[0092]

[0093]

[0094]

[0095] a Recorded for 1 H NMR and 150MHz for 13 C NMR at 600MHz in DMSO-d6, δ H in ppm, J in Hz.

[0096] It can be seen that the structure of the synthesized compound in this embodiment is correct.

[0097] As can be seen from the results of the above embodiments, the solution of the present invention is based on the Rakicidin B1 esterified derivative, and polyethylene glycol with different degrees of polymerization is connected to the structure of the Rakicidin B1 esterified derivative through click chemical reaction to obtain the polyethylene glycol-Rakicidin B1 derivative with the corresponding structure, thereby realizing the structural modification of the Rakicidin B1 compound.

[0098] Experimental Example

[0099] 1. Evaluation of anti-tumor (Panc-1 cells) efficacy

[0100] Human pancreatic cancer (Panc-1) cells were labeled with a red fluorescent dye and transplanted into the yolk sac of 2dpf wild-type AB strain zebrafish eggs by microinjection, with about 200 cells transplanted into each fish, to establish a zebrafish-human pancreatic cancer transplantation model; the zebrafish injected with human pancreatic cancer were placed at 35 °C and cultured until 3dpf.

[0101] At 3dpf, zebrafish with better consistency of tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 zebrafish in each well (experimental group). The compound "B1" was administered at concentrations of 13.9, 41.7, and 125 ng / mL, "FIMP1-2000" and "FIMP2-2000" were both administered at concentrations of 27.8, 83.3, and 250 μg / mL, the positive control gemcitabine hydrochloride was administered at a concentration of 20.0 μg / mL, and a model control group was set up at the same time, with a volume of 3 mL in each well.

[0102] After treatment at 35 °C for 48 h, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing. The NIS-Elements D 3.20 advanced image processing software was used to collect data and analyze the fluorescence intensity (S) of tumor cells. The anti-tumor efficacy of the samples was evaluated based on the statistical analysis results of this index. The statistical processing results were expressed as mean ± SE, and the anti-tumor efficacy calculation formula was as follows:

[0103]

[0104] Statistical analysis was performed using SPSS software, and p < 0.05 indicated a significant difference.

[0105] The test results are shown in Table 3 below.

[0106] Table 3 Experimental results of the anti-tumor (Panc-1 cells) efficacy evaluation of the samples (n = 10)

[0107]

[0108] Compared with the model control group, *p < 0.05, ***p < 0.001

[0109] The above results indicate that the polyethylene glycol-Rakicidin B1 derivative FIMP1-2000 of the present invention has antitumor efficacy against tumors (Panc-1 cells), and the polyethylene glycol-Rakicidin B1 derivative FIMP2-2000 has obvious antitumor efficacy against tumors (Panc-1 cells).

[0110] 2. Evaluation of the antitumor (HCT-8) efficacy of sample FIMP1-20000

[0111] Human colon cancer (HCT-8) cells were labeled with a red fluorescent dye and transplanted into the yolk sac of 2 dpf wild-type AB strain zebrafish by microinjection, with about 200 cells transplanted into each fish, to establish a zebrafish human colon cancer transplantation model; the zebrafish injected with human colon cancer were placed in an incubator at 35 °C until 3 dpf.

[0112] At 3 dpf, zebrafish with relatively consistent tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 zebrafish in each well (experimental group). The compound "B1" was administered at concentrations of 13.9, 41.7, and 125 ng / mL, "FIMP1-2000" at concentrations of 27.8, 83.3, and 250 μg / mL, and the positive control 5-FU at a concentration of 130 μg / mL by water solution. At the same time, a model control group was set up, and the volume of each well was 3 mL.

[0113] After treatment at 35 °C for 48 h, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing. The NIS-Elements D 3.20 advanced image processing software was used to collect data and analyze the fluorescence intensity (S) of tumor cells. The statistical analysis results of this index were used to evaluate the antitumor efficacy of the sample. The results of statistical processing were expressed as mean ± SE, and the formula for calculating the antitumor efficacy was as follows:

[0114]

[0115] Statistical analysis was performed using SPSS software, and p < 0.05 indicated a significant difference.

[0116] The test results are shown in Table 4 below.

[0117] Table 4 Experimental results of the evaluation of the antitumor (HCT-8 cells) efficacy of sample FIMP1-2000 (n = 10)

[0118]

[0119] Compared with the model control group, **p < 0.01, ***p < 0.001

[0120] The above results indicate that the polyethylene glycol-Rakicidin B1 derivative FIMP1-2000 of the present invention has obvious anti-tumor effects against tumor (HCT-8).

[0121] 3. Evaluation of the anti-tumor (HCT-8) efficacy of sample FIMP2-2000

[0122] Human colon cancer (HCT-8) cells were labeled with a red fluorescent dye and transplanted into the yolk sac of 2 dpf wild-type AB strain zebrafish by microinjection, with about 200 cells transplanted into each fish, to establish a zebrafish-human colon cancer transplantation model; the zebrafish injected with human colon cancer were placed at 35°C and cultured until 3 dpf.

[0123] At 3 dpf, zebrafish with better consistency of tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 zebrafish in each well (experimental group). The compound "FIMP2-2000" was administered in water at concentrations of 27.8, 83.3, and 250 μg / mL, and the positive control 5-FU at a concentration of 130 μg / mL. At the same time, a model control group was set up, with a volume of 3 mL in each well.

[0124] After treatment at 35°C for 48 h, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing. The NIS-Elements D 3.20 advanced image processing software was used to collect data and analyze the fluorescence intensity (S) of tumor cells. The anti-tumor efficacy of the sample was evaluated based on the statistical analysis results of this index. The statistical processing results were expressed as mean ± SE, and the anti-tumor efficacy calculation formula was as follows:

[0125]

[0126] Statistical analysis was performed using SPSS software. p < 0.05 indicates a significant difference.

[0127] The test results are shown in Table 5 below.

[0128] Table 5 Experimental results of the anti-tumor (HCT-8 cells) efficacy evaluation of sample FIMP2-2000 (n = 10)

[0129]

[0130] Compared with the model control group, *p < 0.05, **p < 0.01

[0131] From the above results, it can be seen that compared with the model control group, the polyethylene glycol-Rakicidin B1 derivative FIMP2-2000 of the present invention had p>0.05, p<0.05, and p>0.05 respectively, and its anti-tumor efficacies were 19%, 26%, and 18% respectively, indicating that the compound FIMP2-2000 has anti-tumor (HCT-8) efficacy.

[0132] 4. Evaluation of the anti-neuroinflammatory efficacy of the samples

[0133] Randomly select 360 3-dpf transgenic neutrophil green fluorescent MPX strain zebrafish and place them in a 6-well plate, with 30 zebrafish in each well (experimental group).

[0134] Respectively, dissolve and administer the compound "B1" at concentration doses of 34.7, 104, and 313 ng / mL, "FIMP1-2000" at concentration doses of 222, 667, and 2000 μg / mL, "FIMP2-2000" at concentration doses of 222, 667, and 2000 μg / mL, and the positive control indomethacin at a concentration of 28.6 μg / mL. At the same time, set up a normal control group and a model control group, and the volume of each well is 3 mL.

[0135] After pretreatment for 1 h, except for the normal control group, the other experimental groups were all dissolved and administered copper sulfate pentahydrate to establish a zebrafish neuroinflammatory model. After treatment at 28°C for 2 h, randomly select 10 zebrafish from each experimental group and take pictures under a fluorescence microscope. The typical pictures of neutrophils in the inflammatory sites of zebrafish treated with the samples FIMP1-2000 (P1) and FIMP2-2000 (P2) in this experimental example are shown in the appendix Figure 1 As shown; among them, the white arrow indicates the analysis site, and the green fluorescent particles are neutrophils.

[0136] Use Nikon NIS-Elements D 3.20 advanced image processing software to collect data and analyze the number of neutrophils (N) in the inflammatory sites of zebrafish. Evaluate the anti-neuroinflammatory efficacy of the samples based on the statistical analysis results of this index. The statistical processing results are expressed as mean±SE, and the anti-neuroinflammatory efficacy calculation formula is as follows:

[0137]

[0138] Perform statistical analysis using SPSS software. p<0.05 indicates a significant difference.

[0139] The test results are shown in Table 6 below. The number of neutrophils in the inflammatory sites of zebrafish treated with the samples FIMP1-2000 (P1) and FIMP2-2000 (P2) is shown in the appendix Figure 2As shown, the results of the anti-neuroinflammatory effects of the samples FIMP1-2000 (P1) and FIMP2-2000 (P2) on zebrafish are shown in the appendix Figure 3 as shown.

[0140] Table 6 Experimental results of the anti-neuroinflammatory effects of the samples (n = 10)

[0141]

[0142] Compared with the model control group, **p < 0.01, ***p < 0.001

[0143] From the above results, it can be seen that for the polyethylene glycol-Rakicidin B1 derivative FIMP1-2000 of the present invention, compared with the model control group, p < 0.001 in both cases, and its anti-neuroinflammatory effects are 32%, 54% and 55% respectively, indicating that the compound FIMP1-2000 has obvious anti-neuroinflammatory effects under the experimental conditions; while for FIMP2-2000, compared with the model control group, p < 0.001 in both cases, and its anti-neuroinflammatory effects are 36%, 43% and 43% respectively, indicating that the compound FIMP2-2000 has obvious anti-neuroinflammatory effects under the experimental conditions.

[0144] In this experimental example, under the zebrafish neuritis model, the relative expression levels of the IL-1β gene and the IL-6 gene after the treatment with the sample FIMP2-2000 (P2) are shown in the appendix Figure 4 - 5 as shown. It can be seen that the sample FIMP2-2000 can significantly down-regulate the gene expressions of IL-1β and IL-6.

[0145] In summary, the polyethylene glycol-Rakicidin B1 derivative FIMP2-2000 of the present invention has comparable in vivo anti-Clostridium difficile infection and anti-tumor effects to Rakicidin B1, and basically solves the problems of poor solubility and stability of the original drug B1; moreover, the polyethylene glycol-Rakicidin B1 derivative FIMP2-2000 of the present invention has obvious anti-neuroinflammatory effects (zebrafish neuritis model), and the anti-neuroinflammatory effects are mainly manifested as significantly down-regulating the gene expressions of IL-1β and IL-6.

[0146] Obviously, the above embodiments are only examples given for clear illustration, and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A polyethylene glycol-Rakicidin B1 derivative, characterized in that, The derivative has a structure represented by the following formula (P1) or (P2): n is the degree of polymerization of polyethylene glycol.

2. The polyethylene glycol-Rakicidin B1 derivative according to claim 1, characterized in that, In the derivative, the molecular weight of the polyethylene glycol is 500 - 3000.

3. The polyethylene glycol-Rakicidin B1 derivative according to claim 1 or 2, characterized in that, In the derivative, the molecular weight of the polyethylene glycol is 750 - 2000.

4. A method for preparing the polyethylene glycol-Rakicidin B1 derivative according to any one of claims 1-3, characterized in that, The method includes the step of connecting azide group - modified polyethylene glycol to the alkynyl - modified Rakicidin B1 esterification derivative through click chemistry; or, connecting alkynyl - modified polyethylene glycol to the azide group - modified Rakicidin B1 esterification derivative through click chemistry; wherein, The preparation method of derivative P1 includes the following steps: (1) Take the compound Rakicidin B1 and add p - nitrophenyl chloroformate, react at - 10 to 0 °C in an organic solvent system, collect the reactant, mix it with water, and collect the precipitated solid for standby; (2) Take the product obtained in step (1), add triethylamine and propargylamine and mix, react successively at - 10 to 0 °C and at room temperature in an organic solvent system, collect the reactant, mix it with saturated brine, and collect the precipitated solid for standby; (3) Take the product obtained in step (2), add azide - modified polyethylene glycol and mix, react in an organic solvent system in the presence of a catalyst, collect the reaction solution, and prepare the required derivative P1 by semi - preparative liquid phase; The preparation method of derivative P2 includes the following steps: (1) Take the compound Rakicidin B1 and add 4 - chloromethylbenzoyl chloride and mix, react in an organic solvent system, collect the reactant, add water, and collect the precipitated solid for standby; (2) Take the product obtained in step (1), add sodium azide and mix, react at 50 - 60 °C in an organic solvent system in the presence of a catalyst, collect the reactant, mix it with saturated brine, and collect the precipitated solid for standby; (3) Take the product obtained in step (2), add alkynyl - modified polyethylene glycol, react in an organic solvent system in the presence of a catalyst, collect the reaction solution, and prepare the required derivative P2 by semi - preparative liquid phase.

5. The method for preparing the polyethylene glycol-Rakicidin B1 derivative according to claim 4, characterized in that, In the preparation method of the derivative P1, in step (3), the catalyst includes a mixture of copper sulfate pentahydrate and sodium ascorbate.

6. The method for preparing the polyethylene glycol-Rakicidin B1 derivative according to claim 4, characterized in that, In the preparation method of the derivative P2: In step (2), the catalyst includes potassium iodide; In step (3), the catalyst includes a mixture of copper sulfate pentahydrate and sodium ascorbate.

7. The method for preparing the polyethylene glycol-Rakicidin B1 derivative according to any one of claims 4-6, characterized in that, The semi - preparative liquid phase preparation step specifically includes: separating the collected reaction solution by semi - preparative liquid chromatography using a YMC ODS C18 column, eluting with methanol - water with a volume ratio of 830:170 as the eluent, monitoring the elution peak time range of the target product at a wavelength of 262 nm by the DAD ultraviolet detector of the preparative liquid chromatography, collecting the eluent within this time range, and concentrating and freeze - drying the eluent to obtain the product.

8. Use of the polyethylene glycol-Rakicidin B1 derivative according to any one of claims 1-3 and its stereoisomers, tautomers, amorphous forms, isotopomers, polymorphs, solvates or pharmaceutically acceptable salts for the preparation of a drug having anti-neuritis activity and / or for treating neuritis.

9. A drug having anti-neuritis activity and / or for treating neuritis, characterized in that, The active ingredient of the drug includes the polyethylene glycol-Rakicidin B1 derivative described in any one of claims 1-3 and its stereoisomers, tautomers, amorphous substances, isotopomers, polymorphs, solvates or pharmaceutically acceptable salts.

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