Javamycin derivative as well as preparation method and application thereof

Abstract

The invention discloses a josamycin derivative as well as a preparation method and application thereof. The josamycin derivative represented by formula I is separated from josamycin fermentation broth through acidification, solid-liquid separation, extraction, reverse extraction, crystallization, high performance liquid preparation and other methods, and has excellent antibacterial activity # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, and particularly to a josamycin derivative, a preparation method thereof, and uses thereof. Background Art

[0002] Josamycin is a macrolide antibiotic produced by Streptomyces narbonensis var. Josamyceticus. It is a broad-spectrum antibiotic with an antibacterial spectrum similar to that of erythromycin. By inhibiting the protein synthesis process of pathogens, it has high activity against Gram-positive bacteria and some Gram-negative bacteria, including Staphylococcus aureus, hemolytic streptococcus, gonococcus, diplococcus meningitidis, Bordetella pertussis, mycoplasma, chlamydia, etc. It is also relatively effective against spirochetes, rickettsiae, and large viruses. Clinically, it is used for respiratory tract infections, bronchitis, pneumonia, paranasal sinusitis, otitis media, skin infections, postoperative infections, urinary tract, otolaryngology, and dental infections. Josamycin was discovered by Professor Umezawa Hamao of the Institute of Microbial Chemistry, Japan, and was developed by Yamanouchi Pharmaceutical Co., Ltd. (renamed Astellas after acquiring Fujisawa Pharmaceutical in 2005) in 1967.

[0003] The structural formula of josamycin is shown as follows:

[0004]

[0005] Due to the widespread use and even abuse of antibiotics, many bacteria have developed increasingly serious multidrug resistance. The rise of antibiotic resistance has become a major threat to human health. Therefore, developing antibiotics with good bioavailability and low toxicity is the key to current drug development. Exploring natural derivatives of josamycin is of great significance for developing new pharmacological effects of such antibiotics. Summary of the Invention

[0006] Based on the above defects in the prior art, the object of the present invention is to provide a josamycin derivative represented by Formula I, which has excellent antibacterial activity.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The first object of the present invention is to provide a josamycin derivative represented by Formula I,

[0009]

[0010] The second object of the present invention is to provide a preparation method of a josamycin derivative, comprising the following steps:

[0011] A. Acidify the josamycin fermentation broth with an acid, and obtain an acidified solution after solid-liquid separation;

[0012] B. Adjust the pH of the acidified solution obtained in step A to alkaline, add an organic solvent for extraction, and separate out the organic phase; Back-extract the organic phase with an aqueous acid solution to obtain a back-extract solution;

[0013] C. Adjust the pH of the back-extract solution obtained in step B to 7.0 - 12.0, and obtain a crude product after solid-liquid separation;

[0014] D. Purify and separate the crude product by high performance liquid preparative chromatography to obtain the compound of formula I.

[0015] Furthermore, in step A, the acid used in the acidification process of the fermentation broth is selected from any one or more of sulfuric acid, phosphoric acid, citric acid, and oxalic acid;

[0016] The pH of the obtained acidified solution is 1.0 - 4.0;

[0017] Preferably, the pH of the acidified solution is 2.0 - 3.0;

[0018] More preferably, the pH of the acidified solution is 2.3.

[0019] Furthermore, in step B, adjust the pH of the acidified solution to 7.0 - 12.0;

[0020] Preferably, adjust the pH of the acidified solution to 8.0 - 10.0;

[0021] More preferably, adjust the pH of the acidified solution to 9.8.

[0022] Furthermore, in step B, the organic solvent used for extraction is selected from any one or more of ethyl acetate, butyl acetate, toluene, dichloromethane, and methyl isobutyl ketone;

[0023] Furthermore, in step B, the aqueous acid solution is selected from any one or more of sulfuric acid aqueous solution, phosphoric acid aqueous solution, citric acid aqueous solution, and oxalic acid aqueous solution;

[0024] Furthermore, in step B, the aqueous acid solution is selected from any one or more of sulfuric acid aqueous solution with a volume ratio of 0.1 - 5%, phosphoric acid aqueous solution with a volume ratio of 0.1 - 5%, citric acid aqueous solution with a mass ratio of 0.1 - 4%, and oxalic acid aqueous solution with a mass ratio of 0.1 - 4%;

[0025] Preferably, the mass ratio of the citric acid aqueous solution or the oxalic acid aqueous solution is 1%.

[0026] Furthermore, in step C, adjust the pH of the back-extract solution to 7.0 - 12.0;

[0027] Preferably, adjust the pH of the back-extract solution to 8.0 - 10.0;

[0028] More preferably, the pH of the stripping solution is adjusted to 9.5.

[0029] Furthermore, in step D, the purification and separation packing material for high performance liquid chromatography preparation is C18, and the mobile phase is an aqueous solution of sodium acetate at 5 g / L - acetonitrile - tetrahydrofuran, and the volume ratio of the aqueous solution of sodium acetate at 5 g / L, acetonitrile, and tetrahydrofuran is 1000:1000:25.

[0030] Furthermore, the josamycin fermentation broth in step A) is prepared by the following method:

[0031] 1) Inoculate the strain into the seed medium and culture to obtain a seed solution;

[0032] 2) Inoculate the seed solution into the fermentation medium and culture to obtain the fermentation broth.

[0033] Furthermore, the formula of the seed medium is: soybean cake powder 3 - 8 g, yeast extract powder 3 - 8 g, corn steep liquor dry powder 3 - 8 g, corn protein powder 3 - 8 g, soybean oil 3 - 8 ml, calcium carbonate 2 - 5 g, drinking water 1 L, and the pH is 6.9 - 7.5;

[0034] Preferably, the culture conditions are a temperature of 28 - 32 °C and a culture time of 20 - 30 h.

[0035] Furthermore, the ratio of the fermentation medium is: soybean oil 60 - 100 ml, corn protein powder 20 - 28 g, sesame cake powder 8 - 14 g, leucine 4 - 8 g, magnesium sulfate heptahydrate 0.1 - 0.5 g, ferrous sulfate heptahydrate 0.1 - 0.5 g, manganese sulfate monohydrate 0.1 - 0.5 g, calcium carbonate 1 - 5 g, drinking water 1 L, and the pH is 6.5 - 7.0;

[0036] Preferably, the culture conditions are a temperature of 28 - 32 °C and a culture time of 104 - 152 h.

[0037] Furthermore, the strain used for fermentation is Streptomyces narbonensis.

[0038] The third object of the present invention is to provide a pharmaceutical composition comprising the josamycin derivative shown in formula I,

[0039]

[0040] The fourth object of the present invention is to provide the use of the josamycin derivative as described above, or the pharmaceutical composition as described above in the preparation of a drug for treating or preventing fungal infectious diseases;

[0041] Preferably, the fungus is Bacillus subtilis or Staphylococcus aureus.

[0042] Effect of the Invention

[0043] The josamycin derivative represented by Formula I provided by the present invention has excellent antibacterial activity, and is simple to prepare and low in cost. Description of the Drawings

[0044] Figure 1 1H NMR spectrum of Compound 1 obtained in Example 1;

[0045] Figure 2 13C NMR spectrum of Compound 1 obtained in Example 1;

[0046] Figure 3 2D NMR (DEPT) spectrum of Compound 1 obtained in Example 1;

[0047] Figure 4 High-resolution mass spectrum of Compound 1 obtained in Example 1;

[0048] Figure 5 Infrared scanning spectrum of Compound 1 obtained in Example 1;

[0049] Figure 6 Inhibitory effect of Compound 1 on Bacillus subtilis;

[0050] Figure 7 Inhibitory effect of Compound 1 on Staphylococcus aureus. Detailed Description of the Invention

[0051] In order to make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following provides a detailed description by listing specific examples. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0052] The present invention provides a method for preparing a josamycin derivative, which comprises the following steps:

[0053] A. Acidify the josamycin fermentation broth with acid, and obtain an acidified solution after solid-liquid separation;

[0054] B. Adjust the acidified solution obtained in step A to alkaline, add an organic solvent for extraction, and separate out the organic phase; back-extract the organic phase with an acid aqueous solution to obtain a back-extract solution;

[0055] C. Adjust the pH of the back-extract solution obtained in step B to 7.0 - 12.0, and obtain a crude product after solid-liquid separation;

[0056] D. Purify and separate the crude product by high-performance liquid preparative chromatography to obtain the compound of Formula I.

[0057] In some embodiments, the josamycin fermentation broth in step A) is prepared by the following method:

[0058] 1) Inoculate the strain into a seed medium and culture to obtain a seed solution;

[0059] 2) Inoculate the seed solution into a fermentation medium and culture to obtain a fermentation broth.

[0060] In some embodiments, the formula of the seed medium is: soybean cake powder 3 - 8 g, yeast extract powder 3 - 8 g, corn steep liquor dry powder 3 - 8 g, corn protein powder 3 - 8 g, soybean oil 3 - 8 ml, calcium carbonate 2 - 5 g, drinking water 1 L, pH 6.9 - 7.5; the culture conditions are temperature 28 - 32 °C and culture for 20 - 30 h.

[0061] In some embodiments, the proportion of the fermentation medium is: soybean oil 60 - 100 ml, corn protein powder 20 - 28 g, sesame cake powder 8 - 14 g, leucine 4 - 8 g, magnesium sulfate heptahydrate 0.1 - 0.5 g, ferrous sulfate heptahydrate 0.1 - 0.5 g, manganese sulfate monohydrate 0.1 - 0.5 g, calcium carbonate 1 - 5 g, drinking water 1 L, pH 6.5 - 7.0; the culture conditions are temperature 28 - 32 °C and culture for 104 - 152 h.

[0062] In some embodiments, the acid used in the acidification process of the fermentation broth in step A) is selected from any one or more of sulfuric acid, phosphoric acid, citric acid, and oxalic acid.

[0063] In some embodiments, the acid used in the acidification process of the fermentation broth is sulfuric acid.

[0064] In some embodiments, the pH of the acidified liquid obtained in step A) is 1.0 - 4.0, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0.

[0065] In some embodiments, the pH of the acidified liquid obtained in step A) is 2.0 - 3.0.

[0066] In some embodiments, the pH of the acidified liquid obtained in step A) is 2.3.

[0067] In some embodiments, in step B, the pH of the acidified solution is adjusted to 7.0 to 12.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0.

[0068] In some embodiments, in step B, the pH of the acidified solution is adjusted to 8.0 to 10.0.

[0069] In some embodiments, in step B, the pH of the acidified solution is adjusted to 9.8.

[0070] In some embodiments, in step B, the organic solvent used for extraction is selected from any one or more of ethyl acetate, butyl acetate, toluene, dichloromethane, and methyl isobutyl ketone.

[0071] In some embodiments, in step B, the organic solvent used for extraction is selected from butyl acetate.

[0072] In some embodiments, in step B, the aqueous acid solution is selected from any one or more of sulfuric acid aqueous solution, phosphoric acid aqueous solution, citric acid aqueous solution, and oxalic acid aqueous solution.

[0073] In some embodiments, in step B, the aqueous acid solution is selected from sulfuric acid aqueous solutions with a volume ratio of 0.1 to 5%, such as sulfuric acid aqueous solutions with a volume ratio of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%.

[0074] In some embodiments, in step B, the aqueous acid solution is selected from aqueous phosphoric acid solutions with a volume ratio of 0.1 to 5%, such as aqueous phosphoric acid solutions with a volume ratio of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%.

[0075] In some embodiments, in step B, the aqueous acid solution is selected from aqueous citric acid solutions with a mass ratio of 0.1 to 4%, such as aqueous citric acid solutions with a mass ratio of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%.

[0076] In some embodiments, in step B, the aqueous acid solution is selected from aqueous oxalic acid solutions with a mass ratio of 0.1 to 4%, such as aqueous oxalic acid solutions with a mass ratio of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%.

[0077] In some embodiments, in step C, the pH of the stripping solution is adjusted to 7.0 - 12.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0.

[0078] In some embodiments, in step C, the pH of the stripping solution is adjusted to 8.0 - 10.0.

[0079] In some embodiments, in step C, the pH of the stripping solution is adjusted to 9.5.

[0080] In some embodiments, in step D, the purification and separation packing material for high performance liquid chromatography preparation is C18, and the mobile phase is 5 g / L sodium acetate aqueous solution - acetonitrile - tetrahydrofuran, and the volume ratio of the 5 g / L sodium acetate aqueous solution, acetonitrile, and tetrahydrofuran is 1000:1000:25.

[0081] The present invention also provides a pharmaceutical composition, which contains the foregoing josamycin derivative.

[0082] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg - 1000 mg.

[0083] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01% - 99.99% of the foregoing josamycin derivative. In some embodiments, the pharmaceutical composition contains 0.1% - 99.9% of the foregoing josamycin derivative. In some embodiments, the pharmaceutical composition contains 0.5% - 99.5% of the foregoing josamycin derivative. In some embodiments, the pharmaceutical composition contains 1% - 99% of the foregoing josamycin derivative. In some embodiments, the pharmaceutical composition contains 2% - 98% of the foregoing josamycin derivative.

[0084] In certain embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent, or excipient.

[0085] The compounds, mixtures, compositions, etc. involved in the present invention can be formulated into single doses, which contain the active compounds of the present invention as well as carriers, excipients, etc. The dosage forms for administration can be tablets, capsules, injections, granules, powders, suppositories, pills, creams, pastes, gels, powders, oral solutions, inhalants, suspensions, dry suspensions, patches, or lotions, etc. These dosage forms can contain commonly used components in pharmaceutical preparations, such as diluents, absorbents, wetting agents, binders, disintegrants, colorants, pH regulators, antioxidants, isotonicity regulators, or anti-adhesives, etc.

[0086] The present invention also provides the use of the josamycin derivatives or the pharmaceutical composition in the preparation of drugs for treating or preventing fungal infectious diseases.

[0087] Term Explanation:

[0088] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0089] "Pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs, as well as other chemical components, and other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0090] As used herein, the term "pharmaceutically acceptable" means that these compounds, materials, compositions, and / or dosage forms are suitable for contact with patient tissues within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reaction, or other problems or complications, have a reasonable benefit / risk ratio, and are effective for the intended use.

[0091] As used herein, the singular forms "a", "an", and "the" include plural references, and vice versa, unless the context clearly indicates otherwise.

[0092] The above terms related to the present invention have been defined, and those skilled in the art can also understand the above terms in combination with the prior art. The following further describes based on the content of the present invention and the definition of the terms.

[0093] The following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, they are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0094] Furthermore, the NMR spectra were obtained using a Varian 400 MHz nuclear magnetic resonance spectrometer or equivalent equipment. CDCl3 and DMSO-d6 were often used as solvents, and chemical shifts were reported in ppm.

[0095] Preparation of the Compound of Formula I in Example 1

[0096]

[0097] Preparation of the seed solution: Inoculate the strain into the seed medium and culture to obtain the seed solution;

[0098] Seed medium: 3 - 8 g of soybean cake powder, 3 - 8 g of yeast extract powder, 3 - 8 g of corn steep liquor dry powder, 3 - 8 g of corn protein powder, 3 - 8 ml of soybean oil, 2 - 5 g of calcium carbonate, 1 L of drinking water, pH 6.9 - 7.5; Culture conditions: Culture temperature 28 - 32 °C, culture time 20 - 30 h;

[0099] Preparation of the josamycin fermentation broth: Inoculate the seed solution into the fermentation medium and culture to obtain 30 L of the fermentation broth;

[0100] Fermentation medium: 80 ml of soybean oil, 24 g of corn protein powder, 12 g of sesame cake powder, 6 g of leucine, 0.3 g of magnesium sulfate heptahydrate, 0.3 g of ferrous sulfate heptahydrate, 0.3 g of manganese sulfate monohydrate, 3 g of calcium carbonate, 1 L of drinking water, pH 6.5 - 7.0; Culture conditions: Culture temperature 28 - 32 °C, culture time 104 - 152 h;

[0101] A. Add sulfuric acid to the 30 L of fermentation broth to adjust the pH to 2.3, stir for 30 minutes, filter by suction to obtain the acidified solution, and adjust the pH of the acidified solution to 9.8 with sodium hydroxide solution;

[0102] B. Add 8.5 L of butyl acetate and stir for 2 minutes, let it stand for stratification to obtain the butyl acetate extract. Add 8 L of 0.4% (V / V) phosphoric acid aqueous solution to the butyl acetate extract and stir for 2 minutes, then let it stand to separate out the aqueous phase;

[0103] C. Adjust the pH of the aqueous phase to 9.5 with sodium hydroxide solution, stir for 20 minutes, then filter by suction and dry to obtain 65 g of the crude product;

[0104] D. Take 1.5 g of the crude product, separate and purify it by preparative high performance liquid chromatography. The mobile phase is an aqueous solution of sodium acetate at 5 g / L - acetonitrile - tetrahydrofuran (1000:1000:25 V / V / V). Collect in fractions and detect by HPLC. The prepared solution of the compound obtained is extracted with dichloromethane, concentrated and dried to obtain 52 mg, which is the compound of formula I. Its HPLC purity is over 95%. The hydrogen spectrum, carbon spectrum, two-dimensional nuclear magnetic resonance (DEPT) spectrum, high-resolution mass spectrum and infrared scanning spectrum of the compound of formula I are shown in the appendix Figures 1-5 .

[0105] Study on the antibacterial activity of the compound of formula I in Example 2

[0106] I. Reagents and Instruments

[0107] 1. Reagents

[0108] Ethanol (AR grade), potassium dihydrogen phosphate (AR), dipotassium hydrogen phosphate (AR grade), antibiotic medium No. II (BR);

[0109] Culture medium: Weigh 29.5 g of antibiotic medium No. II, add 1000 ml of water, sterilize at 121 °C for 15 minutes and then use;

[0110] Phosphate buffer solution (pH 7.8): Weigh 0.2478 g of potassium dihydrogen phosphate and 3.3583 g of dipotassium hydrogen phosphate, dissolve in 600 ml of water, sterilize at 121 °C for 15 minutes and then use;

[0111] Test solution (U1, U2): Weigh 10.20 mg of the test sample precisely, place it in a 10-ml volumetric flask, dissolve it with 3 ml of ethanol, dilute it to the mark with sterilized water and shake well to obtain the test sample stock solution. Reagent preparation: Precisely measure 1 ml and 2 ml of the test sample stock solution respectively, place them in different 100-ml volumetric flasks, dilute them to the mark with phosphate buffer solution (pH 7.8) and shake well to obtain the test solution U1 with a concentration of 8.9 μg / ml and the test solution U2 with a concentration of 17.8 μg / ml.

[0112] 2. Test bacteria

[0113] Bacillus subtilis (ATCC6633), Staphylococcus aureus (CMCC(B)26003);

[0114] Preparation of Bacillus subtilis suspension: Take the nutrient agar slant culture of Bacillus subtilis, inoculate it into a culture flask containing nutrient agar medium, culture it at 35 - 37 °C for 7 days, smear and examine it under a microscope by Gram staining method, and there should be more than 85% spores. Wash the spores with sterilized water and heat them at 65 °C for 30 minutes for standby;

[0115] Preparation of Staphylococcus aureus bacterial suspension: Take the nutrient agar slant culture of Staphylococcus aureus, inoculate it into a culture flask containing nutrient agar medium, and culture it at 35 - 37°C for 20 - 22 hours. Immediately before use, wash the bacterial colonies with sterile water or 0.9% sterile sodium chloride solution and set aside.

[0116] 3. Instruments

[0117] Water bath incubator, electronic balance, inhibition zone measuring instrument, laminar flow hood, vertical autoclave.

[0118] II. Experimental methods

[0119] Take 8 flat-bottomed double dishes and divide them into 4 groups evenly; Inject 20 ml of each of the above-mentioned melted medium into each flat-bottomed double dish, spread it evenly in the dish, and place it on a horizontal table to solidify as the bottom layer; Take another 100 ml of melted medium, cool it to 58 - 60°C, add 1 ml of the above-mentioned Bacillus subtilis bacterial suspension to make a Bacillus subtilis-containing medium; Add 5 ml of the Bacillus subtilis-containing medium to each double dish in the first group, spread it evenly on the bottom layer as the bacterial layer of the first group of double dishes; Take another 100 ml of melted medium, cool it to 48 - 50°C, add 1 ml of the above-mentioned Staphylococcus aureus bacterial suspension to make a Staphylococcus aureus-containing medium; Add 5 ml of the Staphylococcus aureus-containing medium to each double dish in the second group, spread it evenly on the bottom layer as the bacterial layer of the second group of double dishes; After placing all the double dishes on the horizontal table and cooling, place 4 Oxford cups evenly at equal distances in each double dish, cover them with a tile round cover and set aside;

[0120] Drop 0.3 ml of the test solution (U1, U2) into the Oxford cups respectively, take them out after culturing at 35 - 37°C for about 17 hours, and check whether there is an inhibition zone.

[0121] III. Experimental results

[0122] 1. There is a clear inhibition zone in the first group of double dishes (attached Figure 6 ), indicating that the test product has an inhibitory effect on Bacillus subtilis;

[0123] 2. There is a clear inhibition zone in the second group of double dishes (attached Figure 7 ), indicating that the test product has an inhibitory effect on Staphylococcus aureus.

[0124] For those skilled in the art, according to the technical solutions and concepts described above, various corresponding changes and deformations can be made, and all these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A josamycin derivative represented by Formula I, 2. The preparation method of the josamycin derivative of formula I according to claim 1, characterized in that, comprising the following steps: A. Acidify the josamycin fermentation broth with an acid, and obtain an acidified solution after solid-liquid separation; B. Adjust the pH of the acidified solution obtained in step A to alkaline, add an organic solvent for extraction, and separate out the organic phase; Back-extract the organic phase with an aqueous acid solution to obtain a back-extract solution; C. Adjust the pH of the back-extract solution obtained in step B to 7.0 - 12.0, and obtain a crude product after solid-liquid separation; D. Purify and separate the crude product by high performance liquid preparative chromatography to obtain the compound of Formula I.

3. The preparation method according to claim 2, characterized in that, In step A, the acid used in the acidification process of the josamycin fermentation broth is selected from any one or more of sulfuric acid, phosphoric acid, citric acid, and oxalic acid; The pH of the obtained acidified solution is 1.0 - 4.0; Preferably, the pH of the acidified solution is 2.0 - 3.0; More preferably, the pH of the acidified solution is 2.

3.

4. The preparation method according to claim 2, wherein, In step B, adjust the pH of the acidified solution to 7.0 - 12.0; Preferably, adjust the pH of the acidified solution to 8.0 - 10.0; More preferably, adjust the pH of the acidified solution to 9.8; The organic solvent used for extraction is selected from any one or more of ethyl acetate, butyl acetate, toluene, dichloromethane, and methyl isobutyl ketone; The aqueous acid solution is selected from any one or more of sulfuric acid aqueous solution, phosphoric acid aqueous solution, citric acid aqueous solution, and oxalic acid aqueous solution.

5. The preparation method according to claim 2, characterized in that, In step D, the packing material for purification and separation by high performance liquid preparative chromatography is C18, the mobile phase is 5 g / L sodium acetate aqueous solution - acetonitrile - tetrahydrofuran, and the volume ratio of the 5 g / L sodium acetate aqueous solution, acetonitrile, and tetrahydrofuran is 1000:1000:

25.

6. The preparation method according to any one of claims 2 to 5, characterized in that The josamycin fermentation broth in step A) is prepared by the following method: 1) Inoculate the strain into a seed medium and culture to obtain a seed solution; 2) Inoculate the seed solution into a fermentation medium and culture to obtain a fermentation broth.

7. The preparation method according to claim 6, characterized in that The formula of the seed medium is: soybean cake powder 3 - 8 g, yeast extract powder 3 - 8 g, corn steep liquor dry powder 3 - 8 g, corn protein powder 3 - 8 g, soybean oil 3 - 8 ml, calcium carbonate 2 - 5 g, drinking water 1 L, pH 6.9 - 7.5; Preferably, the culture conditions are temperature 28 - 32 °C and culture for 20 - 30 h.

8. The preparation method according to claim 6, wherein The ratio of the fermentation medium is: soybean oil 60 - 100 ml, corn protein powder 20 - 28 g, sesame cake powder 8 - 14 g, leucine 4 - 8 g, magnesium sulfate heptahydrate 0.1 - 0.5 g, ferrous sulfate heptahydrate 0.1 - 0.5 g, manganese sulfate monohydrate 0.1 - 0.5 g, calcium carbonate 1 - 5 g, drinking water 1 L, pH 6.5 - 7.0; Preferably, the culture conditions are temperature 28 - 32 °C and culture for 104 - 152 h.

9. A pharmaceutical composition, characterized in that, It contains the josamycin derivative represented by Formula I, 10. Use of the josamycin derivative according to claim 1 or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating or preventing fungal infectious diseases; Preferably, the fungus is Bacillus subtilis or Staphylococcus aureus.

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